PART 136 - GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANALYSIS OF POLLUTANTS

Authority:

Secs. 301, 304(h), 307 and 501(a), Pub. L. 95-217, 91 Stat. 1566, et seq. (33 U.S.C. 1251, et seq.) (the Federal Water Pollution Control Act Amendments of 1972 as amended by the Clean Water Act of 1977).

§ 136.1 Applicability.

(a) The procedures prescribed herein shall, except as noted in §§ 136.4, 136.5, and 136.6, be used to perform the measurements indicated whenever the waste constituent specified is required to be measured for:

(1) An application submitted to the Director and/or reports required to be submitted under NPDES permits or other requests for quantitative or qualitative effluent data under parts 122 through 125 of this chapter; and

(2) Reports required to be submitted by dischargers under the NPDES established by parts 124 and 125 of this chapter; and

(3) Certifications issued by States pursuant to section 401 of the Clean Water Act (CWA), as amended.

(b) The procedure prescribed herein and in part 503 of title 40 shall be used to perform the measurements required for an application submitted to the Administrator or to a State for a sewage sludge permit under section 405(f) of the Clean Water Act and for recordkeeping and reporting requirements under part 503 of title 40.

(c) For the purposes of the NPDES program, when more than one test procedure is approved under this part for the analysis of a pollutant or pollutant parameter, the test procedure must be sufficiently sensitive as defined at 40 CFR 122.21(e)(3) and 122.44(i)(1)(iv).

[72 FR 14224, Mar. 26, 2007, as amended at 77 FR 29771, May 18, 2012; 79 FR 49013, Aug. 19, 2014; 82 FR 40846, Aug. 28, 2017]

§ 136.2 Definitions.

As used in this part, the term:

(a) Act means the Clean Water Act of 1977, Pub. L. 95–217, 91 Stat. 1566, et seq. (33 U.S.C. 1251 et seq.) (The Federal Water Pollution Control Act Amendments of 1972 as amended by the Clean Water Act of 1977).

(b) Administrator means the Administrator of the U.S. Environmental Protection Agency.

(c) Regional Administrator means one of the EPA Regional Administrators.

(d) Director means the director as defined in 40 CFR 122.2.

(e) National Pollutant Discharge Elimination System (NPDES) means the national system for the issuance of permits under section 402 of the Act and includes any State or interstate program which has been approved by the Administrator, in whole or in part, pursuant to section 402 of the Act.

(f) Detection limit means the minimum concentration of an analyte (substance) that can be measured and reported with a 99% confidence that the analyte concentration is distinguishable from the method blank results as determined by the procedure set forth at appendix B of this part.

[38 FR 28758, Oct. 16, 1973, as amended at 49 FR 43250, Oct. 26, 1984; 82 FR 40846, Aug. 28, 2017]

§ 136.3 Identification of test procedures.

(a) Parameters or pollutants, for which methods are approved, are listed together with test procedure descriptions and references in Tables IA, IB, IC, ID, IE, IF, IG, and IH of this section. The methods listed in Tables IA, IB, IC, ID, IE, IF, IG, and IH are incorporated by reference, see paragraph (b) of this section, with the exception of EPA Methods 200.7, 601–613, 624.1, 625.1, 1613, 1624, and 1625. The full texts of Methods 601–613, 624.1, 625.1, 1613, 1624, and 1625 are printed in appendix A of this part, and the full text of Method 200.7 is printed in appendix C of this part. The full text for determining the method detection limit when using the test procedures is given in appendix B of this part. In the event of a conflict between the reporting requirements of 40 CFR parts 122 and 125 and any reporting requirements associated with the methods listed in these tables, the provisions of 40 CFR parts 122 and 125 are controlling and will determine a permittee's reporting requirements. The full texts of the referenced test procedures are incorporated by reference into Tables IA, IB, IC, ID, IE, IF, IG, and IH. The date after the method number indicates the latest editorial change of the method. The discharge parameter values for which reports are required must be determined by one of the standard analytical test procedures incorporated by reference and described in Tables IA, IB, IC, ID, IE, IF, IG, and IH or by any alternate test procedure which has been approved by the Administrator under the provisions of paragraph (d) of this section and §§ 136.4 and 136.5. Under certain circumstances (paragraph (c) of this section, § 136.5(a) through (d) or 40 CFR 401.13,) other additional or alternate test procedures may be used.

Table IA—List of Approved Biological Methods for Wastewater and Sewage Sludge

Parameter and units Method1 EPA Standard methods AOAC, ASTM, USGS Other
Bacteria
1. Coliform (fecal), number per 100 mL or number per gram dry weight Most Probable Number (MPN), 5 tube, 3 dilution, or p. 132,3 1680,11 15 1681 11 20 9221 E–2014
Membrane filter (MF) 2 5, single step p. 124 3 9222 D–2015 29 B–0050–85 4
2. Coliform (fecal), number per 100 mL MPN, 5 tube, 3 dilution, or p. 132 3 9221 E–2014; 9221 F–2014 33
Multiple tube/multiple well, or Colilert-18®.13 18 28
MF 2 5, single step 5 p. 124 3 9222 D–2015 29
3. Coliform (total), number per 100 mL MPN, 5 tube, 3 dilution, or p. 114 3 9221 B–2014
MF 2 5, single step or two step p. 108 3 9222 B–2015 30 B–0025–85 4
MF 2 5, with enrichment p. 111 3 9222 B–2015 30
4. E. coli, number per 100 mL MPN 6 8 16 multiple tube, or 9221 B2014/9221 F–2014 12 14 33
multiple tube/multiple well, or 9223 B–2016 13 991.15 10 Colilert® 13 18
Colilert-18® 13 17 18
MF 2 5 6 7 8, two step, or 9222 B–2015/9222 I–2015 31
Single step 1603 21 m-ColiBlue24®.19
5. Fecal streptococci, number per 100 mL MPN, 5 tube, 3 dilution, or p. 139 3 9230 B–2013
MF 2, or p. 136 3 9230 C–2013 32 B–0055–85 4
Plate count p. 143 3
6. Enterococci, number per 100 mL MPN, 5 tube, 3 dilution, or p. 139 3 9230 B–2013
MPN 6 8, multiple tube/multiple well, or 9230 D–2013 D6503–99 9 Enterolert®.13 23
MF 2 5 6 7 8 single step or 1600 24 9230 C–2013 32
Plate count p. 143 3
7. Salmonella, number per gram dry weight 11 MPN multiple tube 1682 22
Aquatic Toxicity
8. Toxicity, acute, fresh water organisms, LC50, percent effluent Water flea, Cladoceran, Ceriodaphnia dubia acute 2002.0 25
Water fleas, Cladocerans, Daphnia pulex and Daphnia magna acute 2021.0 25
Fish, Fathead minnow, Pimephales promelas, and Bannerfin shiner, Cyprinella leedsi, acute 2000.0 25
Fish, Rainbow trout, Oncorhynchus mykiss, and brook trout, Salvelinus fontinalis, acute 2019.0 25
9. Toxicity, acute, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, LC50, percent effluent Mysid, Mysidopsis bahia, acute 2007.0 25.
Fish, Sheepshead minnow, Cyprinodon variegatus, acute 2004.0 25
Fish, Silverside, Menidia beryllina, Menidia menidia, and Menidia peninsulae, acute. 2006.0 25.
10. Toxicity, chronic, fresh water organisms, NOEC or IC25, percent effluent Fish, Fathead minnow, Pimephales promelas, larval survival and growth 1000.0 26
Fish, Fathead minnow, Pimephales promelas, embryo-larval survival and teratogenicity 1001.0 26
Water flea, Cladoceran, Ceriodaphnia dubia, survival and reproduction 1002.0 26
Green alga, Selenastrum capricornutum, growth 1003.0 26
11. Toxicity, chronic, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, NOEC or IC25, percent effluent Fish, Sheepshead minnow, Cyprinodon variegatus, larval survival and growth 1004.0 27.
Fish, Sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and teratogenicity 1005.0 27
Fish, Inland silverside, Menidia beryllina, larval survival and growth 1006.0 27
Mysid, Mysidopsis bahia, survival, growth, and fecundity 1007.0 27
Sea urchin, Arbacia punctulata, fertilization 1008.0 27

Table IB—List of Approved Inorganic Test Procedures

Parameter Methodology58 EPA52 Standard methods84 ASTM USGS/AOAC/Other
1. Acidity, as CaCO3, mg/L Electrometric endpoint or phenolphthalein endpoint 2310 B–2011 D1067–16 I–1020–85.2
2. Alkalinity, as CaCO3, mg/L Electrometric or Colorimetric titration to pH 4.5, Manual 2320 B–2011 D1067–16 973.43,3 I–1030–85.2
Automatic 310.2 (Rev. 1974)1 I–2030–85.2
3. Aluminum—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 D–2011 or 3111 E–2011 I–3051–85.2
AA furnace 3113 B–2010.
STGFAA 200.9, Rev. 2.2 (1994)/
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
Direct Current Plasma (DCP)36 D4190–15 See footnote.34
Colorimetric (Eriochrome cyanine R) 3500–Al B–2011.
4. Ammonia (as N), mg/L Manual distillation6 or gas diffusion (pH > 11), followed by any of the following: 350.1, Rev. 2.0 (1993) 4500–NH3 B–2011 973.49.3
Nesslerization D1426–15 (A) 973.49,3 I–3520–85.2
Titration 4500–NH3 C–2011.
Electrode 4500–NH3 D–2011 or E–2011 D1426–15 (B).
Manual phenate, salicylate, or other substituted phenols in Berthelot reaction-based methods 4500–NH3 F–2011 See footnote.60
Automated phenate, salicylate, or other substituted phenols in Berthelot reaction-based methods 350.1,30 Rev. 2.0 (1993) 4500–NH3 G–2011 4500–NH3 H–2011 I–4523–85,2 I–2522–90.80
Automated electrode See footnote.7
Ion Chromatography D6919–17.
Automated gas diffusion, followed by conductivity cell analysis Timberline Ammonia-001.74
Automated gas diffusion followed by fluorescence detector analysis FIAlab100.82
5. Antimony—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011.
AA furnace 3113 B–2010.
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
6. Arsenic-Total,4 mg/L Digestion,4 followed by any of the following: 206.5 (Issued 1978)1.
AA gaseous hydride 3114 B–2011 or 3114 C–2011 D2972–15 (B) I–3062–85.2
AA furnace 3113 B–2010 D2972–15 (C) I–4063–98.49
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–05.70
Colorimetric (SDDC) 3500–As B–2011 D2972–15 (A) I–3060–85.2
7. Barium-Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 D–2011 I–3084–85.2
AA furnace 3113 B–2010 D4382–18.
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP36 See footnote.34
8. Beryllium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 D–2011 or 3111 E–2011 D3645–15 (A) I–3095–85.2
AA furnace 3113 B–2010 D3645–15 (B).
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP D4190–15 See footnote.34
Colorimetric (aluminon) See footnote.61.
9. Biochemical oxygen demand (BOD5), mg/L Dissolved Oxygen Depletion 5210 B–201685 973.44,3 p. 17,9 I–1578–78,8 See footnote.10, 63
10. Boron—Total,37 mg/L Colorimetric (curcumin) 4500–B B–2011 I–3112–85.2
ICP/AES 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP D4190–15 S7ee footnote.34
11. Bromide, mg/L Electrode D1246–16 I–1125–85.2
Ion Chromatography 300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997) 4110 B–2011, C–2011, D–2011 D4327–17 993.30,3 I–2057–85.79
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
12. Cadmium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 or 3111 C–2011 D3557–17 (A or B) 974.27,3 p. 37,9 I–3135–852 or I–3136–85.2
AA furnace 3113 B–2010 D3557–17 (D) I–4138–89.51
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–1472–852 or I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP36 D4190–15 See footnote.34
Voltammetry11 D3557–17 (C).
Colorimetric (Dithizone) 3500–Cd–D–1990.
13. Calcium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011 D511–14 (B) I–3152–85.2
ICP/AES 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP See footnote.34
Titrimetric (EDTA) 3500-Ca B–2011 D511–14 (A).
Ion Chromatography D6919–17.
14. Carbonaceous biochemical oxygen demand (CBOD5), mg/L12 Dissolved Oxygen Depletion with nitrification inhibitor 5210 B–201685 See footnote.35 63
15. Chemical oxygen demand (COD), mg/L Titrimetric 410.3 (Rev. 1978)1 5220 B–2011 or C–2011 D1252–06(12) (A) 973.46,3 p. 17,9 I–3560–85.2
Spectrophotometric, manual or automatic 410.4, Rev. 2.0 (1993) 5220 D–2011 D1252–06(12) (B) See footnotes.13 14 83, I–3561–85.2
16. Chloride, mg/L Titrimetric: (silver nitrate) 4500–Cl- B–2011 D512–12 (B) I–1183–85.2
(Mercuric nitrate) 4500–Cl- C–2011 D512–12 (A) 973.51,3 I–1184–85.2
Colorimetric: manual I–1187–85.2
Automated (ferricyanide) 4500–Cl- E–2011 I–2187–85.2
Potentiometric Titration 4500–Cl- D–2011.
Ion Selective Electrode D512–12 (C).
Ion Chromatography 300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997) 4110 B–2011 or 4110 C–2011 D4327–17 993.30,3 I–2057–90.51
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
17. Chlorine-Total residual, mg/L Amperometric direct 4500–Cl D–2011 D1253–14.
Amperometric direct (low level) 4500-Cl E–2011.
Iodometric direct 4500-Cl B–2011.
Back titration ether end-point15 4500-Cl C–2011.
DPD–FAS 4500–Cl F–2011.
Spectrophotometric, DPD 4500–Cl G–2011.
Electrode See footnote.16
17A. Chlorine-Free Available, mg/L Amperometric direct 4500–Cl D–2011 D1253–14.
Amperometric direct (low level) 4500–Cl E–2011.
DPD–FAS 4500-Cl F–2011.
Spectrophotometric, DPD 4500-Cl G–2011.
18. Chromium VI dissolved, mg/L 0.45-micron filtration followed by any of the following:
AA chelation-extraction 3111 C–2011 I–1232–85.2
Ion Chromatography 218.6, Rev. 3.3 (1994) 3500-Cr C–2011 D5257–17 993.23.3
Colorimetric (diphenyl-carbazide) 3500–Cr B–2011 D1687–17 (A) I–1230–85.2
19. Chromium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 D1687–17 (B) 974.27,3 I–3236–85.2
AA chelation-extraction 3111 C–2011.
AA furnace 3113 B–2010 D1687–17 (C) I–3233–93.46
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev 4.2 (2003),68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–0570 I–4472–97.81
DCP36 D4190–15 See footnote.34
Colorimetric (diphenyl-carbazide) 3500–Cr B–2011.
20. Cobalt—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011 or 3111 C–2011 D3558–15 (A or B) p. 37,9 I–3239–85.2
AA furnace 3113 B–2010 D3558–15 (C) I–4243–89.51
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–0570 I–4472–97.81
DCP D4190–15 See footnote.34
21. Color, platinum cobalt units or dominant wavelength, hue, luminance purity Colorimetric (ADMI) 2120 F–201178.
Platinum cobalt visual comparison 2120 B–2011 I–1250–85.2
Spectrophotometric See footnote18
22. Copper—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 or 3111 C–2011 D1688–17 (A or B) 974.27,3 p. 37,9 I–3270–852 or I–3271–85.2
AA furnace 3113 B–2010 D1688–17 (C) I–4274–89.51
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–05,70, I–4472–97.81
DCP36 D4190–15 See footnote.34
Colorimetric (Neocuproine) 3500-Cu B–2011.
Colorimetric (Bathocuproine) 3500-Cu C–2011 See footnote.19
23. Cyanide—Total, mg/L Automated UV digestion/distillation and Colorimetry Kelada-01.55
Segmented Flow Injection, In-Line Ultraviolet Digestion, followed by gas diffusion amperometry D7511–12(17).
Manual distillation with MgCl2, followed by any of the following: 335.4, Rev. 1.0 (1993)57 4500–CN- B–2016 and C–2016 D2036–09(15)(A), D7284–13(17) 10–204–00–1–X.56
Flow Injection, gas diffusion amperometry D2036–09(15)(A) D7284–13(17).
Titrimetric 4500–CN- D–2016 D2036–09(15)(A) p. 22.9
Spectrophotometric, manual 4500–CN- E–2016 D2036–09(15)(A) I–3300–85.2
Semi-Automated20 335.4, Rev. 1.0 (1993)57 4500–CN- N–2016 10–204–00–1–X,56 I–4302–85.2
Ion Chromatography D2036–09(15)(A).
Ion Selective Electrode 4500–CN- F–2016 D2036–09(15)(A).
24. Cyanide-Available, mg/L Cyanide Amenable to Chlorination (CATC); Manual distillation with MgCl2, followed by Titrimetric or Spectrophotometric 4500–CN- G–2016 D2036–09(15)(B).
Flow injection and ligand exchange, followed by gas diffusion amperometry59 D6888–16 OIA–1677–09.44
Automated Distillation and Colorimetry (no UV digestion) Kelada-01.55
24.A Cyanide-Free, mg/L Flow Injection, followed by gas diffusion amperometry D7237–15 (A) OIA–1677–09.44
Manual micro-diffusion and colorimetry D4282–15.
25. Fluoride—Total, mg/L Manual distillation,6 followed by any of the following: 4500–F- B–2011 D1179–16 (A).
Electrode, manual 4500–F- C–2011 D1179–16 (B).
Electrode, automated I–4327–85.2
Colorimetric, (SPADNS) 4500–F- D–2011.
Automated complexone 4500–F- E–2011.
Ion Chromatography 300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997) 4110 B–2011 or C–2011 D4327–17 993.30.3
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
26. Gold—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011.
AA furnace 231.2 (Issued 1978)1 3113 B–2010.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP See footnote.34
27. Hardness—Total, as CaCO3, mg/L Automated colorimetric 130.1 (Issued 1971)1.
Titrimetric (EDTA) 2340 C–2011 D1126–17 973.52B3, I–1338–85.2
Ca plus Mg as their carbonates, by any approved method for Ca and Mg (See Parameters 13 and 33), provided that the sum of the lowest point of quantitation for Ca and Mg is below the NPDES permit requirement for Hardness 2340 B–2011.
28. Hydrogen ion (pH), pH units Electrometric measurement 4500–H+ B–2011 D1293–99 (A or B) 973.41,3 I–1586–85.2
Automated electrode 150.2 (Dec. 1982)1 See footnote,21 I–2587–85.2
29. Iridium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011.
AA furnace 235.2 (Issued 1978)1.
ICP/MS 3125 B–2011.
30. Iron—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 or 3111 C–2011 D1068–15 (A) 974.27,3 I–3381–85.2
AA furnace 3113 B–2010 D1068–15 (B).
STGFAA 200.9, Rev. 2.2 (1994)
ICP/AES36 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP36 D4190–15 See footnote.34
Colorimetric (Phenanthroline) 3500–Fe B–2011 D1068–15 (C) See footnote.22
31. Kjeldahl Nitrogen5—Total, (as N), mg/L Manual digestion20 and distillation or gas diffusion, followed by any of the following: 4500–Norg B–2011 or C–2011 and 4500–NH3 B–2011 D3590–17 (A) I–4515–91.45
Titration 4500–NH3 C–2011 973.48.3
Nesslerization D1426–15 (A).
Electrode 4500–NH3 D–2011 or E–2011 D1426–15 (B).
Semi-automated phenate 350.1, Rev. 2.0 (1993) 4500–NH3 G–2011 4500–NH3 H–2011
Manual phenate, salicylate, or other substituted phenols in Berthelot reaction based methods 4500–NH3 F–2011 See footnote.60
Automated gas diffusion, followed by conductivity cell analysis Timberline Ammonia-001.74
Automated gas diffusion followed by fluorescence detector analysis FIAlab 100.82
Automated Methods for TKN that do not require manual distillation
Automated phenate, salicylate, or other substituted phenols in Berthelot reaction based methods colorimetric (auto digestion and distillation) 351.1 (Rev. 1978)1 I–4551–78.8
Semi-automated block digestor colorimetric (distillation not required) 351.2, Rev. 2.0 (1993) 4500–Norg D–2011 D3590–17 (B) I–4515–9145
Block digester, followed by Auto distillation and Titration See footnote.39
Block digester, followed by Auto distillation and Nesslerization See footnote.40
Block Digester, followed by Flow injection gas diffusion (distillation not required) See footnote.41
Digestion with peroxdisulfate, followed by Spectrophotometric (2,6-dimethyl phenol) Hach 10242.76
Digestion with persulfate, followed by Colorimetric NCASI TNTP W10900.77
32. Lead—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 or 3111 C–2011 D3559–15 (A or B) 974.27,3 I–3399–85.2
AA furnace 3113 B–2010 D3559–15 (D) I–4403–89.51
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP36 D4190–15 See footnote.34
Voltammetry11 D3559–15 (C).
Colorimetric (Dithizone) 3500–Pb B–2011.
33. Magnesium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011 D511–14 (B) 974.27,3 I–3447–85.2
ICP/AES 200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP See footnote.34
Ion Chromatography D6919–17.
34. Manganese—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 D858–17 (A or B) 974.27,3 I–3454–85.2
AA furnace 3113 B–2010 D858–17 (C).
STGFAA 200.9, Rev. 2.2 (1994)
ICP/AES36 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP36 D4190–15 See footnote.34
Colorimetric (Persulfate) 3500–Mn B–2011 920.203.3
Colorimetric (Periodate) See footnote.23
35. Mercury—Total, mg/L Cold vapor, Manual 245.1, Rev. 3.0 (1994) 3112 B–2011 D3223–17 977.22,3 I–3462–85.2
Cold vapor, Automated 245.2 (Issued 1974)1.
Cold vapor atomic fluorescence spectrometry (CVAFS) 245.7 Rev. 2.0 (2005)17 I–4464–01.71
Purge and Trap CVAFS 1631E43.
36. Molybdenum—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 D–2011 I–3490–85.2
AA furnace 3113 –2010 I–3492–96.47
ICP/AES 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP See footnote.34
37. Nickel—Total,4
mg/L
Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 or 3111 C–2011 D1886–14 (A or B) I–3499–852
AA furnace 3113 B–2010 D1886–14 (C) I–4503–89.51
STGFAA 200.9, Rev. 2.2 (1994)
ICP/AES36 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–0570 I–4472–97.81
DCP36 D4190–15 See footnote.34
38. Nitrate (as N), mg/L Ion Chromatography 300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997) 4110 B–2011 or C–2011 D4327–17 993.30.3
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
Ion Selective Electrode 4500–NO3- D–2016.
Colorimetric (Brucine sulfate) 352.1 (Issued 1971)1 973.50,3 419D17, p. 28.9
Spectrophotometric (2,6-dimethylphenol) Hach 1020675
Nitrate-nitrite N minus Nitrite N (See parameters 39 and 40)
39. Nitrate-nitrite (as N), mg/L Cadmium reduction, Manual 4500–NO3- E–2016 D3867–16 (B).
Cadmium reduction, Automated 353.2, Rev. 2.0 (1993) 4500–NO3- F–2016 4500–NO3- I–2016 D3867–16 (A) I–2545–90.51
Automated hydrazine 4500–NO3- H–2016.
Reduction/Colorimetric See footnote.62
Ion Chromatography 300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997) 4110 B–2011 or C–2011 D4327–17 993.30.3
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
Enzymatic reduction, followed by automated colorimetric determination D7781–14 I–2547–11.72
I–2548–11.72
N07–0003.73
Enzymatic reduction, followed by manual colorimetric determination 4500–NO3- J–2018.
Spectrophotometric (2,6-dimethylphenol) Hach 10206.75
40. Nitrite (as N), mg/L Spectrophotometric: Manual 4500–NO2- B–2011 See footnote.25
Automated (Diazotization) I–4540–85,2 See footnote.62 I–2540–90.80
Automated (*bypass cadmium reduction) 353.2, Rev. 2.0 (1993) 4500–NO3- F–2016 4500–NO3- I–2016 D3867–16 (A) I–4545–85.2
Manual (*bypass cadmium or enzymatic reduction) 4500–NO3- E–2016, 4500–NO3- J–2018 D3867–16 (B).
Ion Chromatography 300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997) 4110 B–2011 or C–2011 D4327–17 993.30.3
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
Automated (*bypass Enzymatic reduction) D7781–14 I–2547–1172 I–2548–1172 N07–0003.73
41. Oil and grease—Total recoverable, mg/L Hexane extractable material (HEM): n-Hexane extraction and gravimetry 1664 Rev. A; 1664 Rev. B42 5520 B–201138.
Silica gel treated HEM (SGT–HEM): Silica gel treatment and gravimetry 1664 Rev. A; 1664 Rev. B42 5520 B–201138 and 5520 F–201138.
42. Organic carbon—Total (TOC), mg/L Combustion 5310 B–2014 D7573–09(17) 973.47,3 p. 14.24
Heated persulfate or UV persulfate oxidation 5310 C–2014 5310 D–2011 D4839–03(17) 973.47,3, p. 14.24
43. Organic nitrogen (as N), mg/L Total Kjeldahl N (Parameter 31) minus ammonia N (Parameter 4)
44. Ortho-phosphate (as P), mg/L Ascorbic acid method:
Automated 365.1, Rev. 2.0 (1993) 4500–P F–2011 or G–2011 973.56,3 I–4601–85,2 I–2601–90.80
Manual, single-reagent 4500–P E–2011 D515–88 (A) 973.55.3
Manual, two-reagent 365.3 (Issued 1978)1.
Ion Chromatography 300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997) 4110 B–2011 or C–2011 D4327–17 993.30.3
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
45. Osmium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 D–2011.
AA furnace 252.2 (Issued 1978)1.
46. Oxygen, dissolved, mg/L Winkler (Azide modification) 4500–O (B–F)–2016 D888–12 (A) 973.45B,3 I–1575–78.8
Electrode 4500–O G–2016 D888–12 (B) I–1576–78.8
Luminescence-Based Sensor 4500–O H–2016 D888–12 (C) See footnote.63 See footnote.64
47. Palladium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011.
AA furnace 253.2 (Issued 1978)1.
ICP/MS 3125 B–2011.
DCP See footnote.34
48. Phenols, mg/L Manual distillation,26 followed by any of the following: 420.1 (Rev. 1978)1 5530 B–2010 D1783–01(12).
Colorimetric (4AAP) manual 420.1 (Rev. 1978)1 5530 D–201027 D1783–01(12) (A or B).
Automated colorimetric (4AAP) 420.4 Rev. 1.0 (1993)
49. Phosphorus (elemental), mg/L Gas-liquid chromatography See footnote.28
50. Phosphorus—Total, mg/L Digestion,20 followed by any of the following: 4500–P B (5)–2011 973.55.3
Manual 365.3 (Issued 1978)1 4500–P E–2011 D515–88 (A).
Automated ascorbic acid reduction 365.1 Rev. 2.0 (1993) 4500–P (F–H)–2011 973.56,3 I–4600–85.2
ICP/AES 4 36 200.7, Rev. 4.4 (1994) 3120 B–2011 I–4471–97.50
Semi-automated block digestor (TKP digestion) 365.4 (Issued 1974)1 D515–88 (B) I–4610–91.48
Digestion with persulfate, followed by Colorimetric NCASI TNTP W10900.77
51. Platinum—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011.
AA furnace 255.2 (Issued 1978)1.
ICP/MS 3125 B–2011.
DCP See footnote.34
52. Potassium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011 973.53,3 I–3630–85.2
ICP/AES 200.7, Rev. 4.4 (1994) 3120 B–2011.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
Flame photometric 3500–K B–2011.
Electrode 3500–K C–2011.
Ion Chromatography D6919–17.
53. Residue—Total, mg/L Gravimetric, 103–105° 2540 B–2015 I–3750–85.2
54. Residue—filterable, mg/L Gravimetric, 180° 2540 C–2015 D5907–13 I–1750–85.2
55. Residue—non-filterable (TSS), mg/L Gravimetric, 103–105° post-washing of residue 2540 D–2015 D5907–13 I–3765–85.2
56. Residue—settleable, ml/L Volumetric (Imhoff cone), or gravimetric 2540 F–2015.
57. Residue—Volatile, mg/L Gravimetric, 550° 160.4 (Issued 1971)1 2540 E–2015 I–3753–85.2
58. Rhodium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration, or 3111 B–2011.
AA furnace 265.2 (Issued 1978)1.
ICP/MS 3125 B–2011.
59. Ruthenium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration, or 3111 B–2011.
AA furnace 267.21.
ICP/MS 3125 B–2011.
60. Selenium—Total,4 mg/L Digestion,4 followed by any of the following:
AA furnace 3113 B–2010 D3859–15 (B) I–4668–98.49
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES36 200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–0570 I–4472–97.81
AA gaseous hydride 3114 B–2011, or 3114 C–2011 D3859–15 (A) I–3667–85.2
61. Silica—Dissolved,37 mg/L 0.45-micron filtration followed by any of the following:
Colorimetric, Manual 4500-SiO2 C–2011 D859–16 I–1700–85.2
Automated (Molybdosilicate) 4500–SiO2 E–2011 or F–2011 I–2700–85.2
ICP/AES 200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994) 3120 B–2011 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
62. Silver—Total,4 31 mg/L Digestion,4 29 followed by any of the following:
AA direct aspiration 3111 B–2011 or 3111 C–2011 974.27,3 p. 37,9 I–3720–85.2
AA furnace 3113 B–2010 I–4724–89.51
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4472–97.81
DCP See footnote.34
63. Sodium—Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011 973.54,3 I–3735–85.2
ICP/AES 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP See footnote.34
Flame photometric 3500–Na B–2011.
Ion Chromatography D6919–17.
64. Specific conductance, micromhos/cm at 25 °C Wheatstone bridge 120.1 (Rev. 1982)1 2510 B–2011 D1125–95(99) (A) 973.40,3 I–2781–85.2
65. Sulfate (as SO4), mg/L Automated colorimetric 375.2, Rev. 2.0 (1993) 4500–SO42- F–2011 or G–2011
Gravimetric 4500–SO42- C–2011 or D–2011 925.54.3
Turbidimetric 4500–SO42- E–2011 D516–16.
Ion Chromatography 300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997) 4110 B–2011 or C–2011 D4327–17 993.303, I–4020–0570
CIE/UV 4140 B–2011 D6508–15 D6508, Rev. 2.54
66. Sulfide (as S), mg/L Sample Pretreatment 4500–S2- B, C–2011.
Titrimetric (iodine) 4500–S2- F–2011 I–3840–85.2
Colorimetric (methylene blue) 4500–S2- D–2011.
Ion Selective Electrode 4500–S2- G–2011 D4658–15.
67. Sulfite (as SO3), mg/L Titrimetric (iodine-iodate) 4500–SO32- B–2011.
68. Surfactants, mg/L Colorimetric (methylene blue) 5540 C–2011 D2330–02.
69. Temperature, °C Thermometric 2550 B–2010 See footnote.32
70. Thallium-Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011.
AA furnace 279.2 (Issued 1978)1 3113 B–2010.
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12.
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4471–9750 I–4472–97.81
71. Tin-Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 B–2011 I–3850–78.8
AA furnace 3113 B–2010.
STGFAA 200.9, Rev. 2.2 (1994).
ICP/AES 200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994).
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
72. Titanium-Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 D–2011.
AA furnace 283.2 (Issued 1978)1.
ICP/AES 200.7, Rev. 4.4 (1994).
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14.3
DCP See footnote.34
73. Turbidity, NTU53 Nephelometric 180.1, Rev. 2.0 (1993) 2130 B–2011 D1889–00 I–3860–852
See footnote.65
See footnote.66
See footnote.67
74. Vanadium-Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration 3111 D–2011.
AA furnace 3113 B–2010 D3373–17.
ICP/AES 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–9750
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–05.70
DCP D4190–15 See footnote.34
Colorimetric (Gallic Acid) 3500–V B–2011.
75. Zinc-Total,4 mg/L Digestion,4 followed by any of the following:
AA direct aspiration36 3111 B–2011 or 3111 C–2011 D1691–17 (A or B) 974.27,3 p. 37,9 I–3900–85.2
AA furnace 289.2 (Issued 1978)1.
ICP/AES36 200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994) 3120 B–2011 D1976–12 I–4471–97.50
ICP/MS 200.8, Rev. 5.4 (1994) 3125 B–2011 D5673–16 993.14,3 I–4020–0570 I–4472–97.81
DCP36 D4190–15 See footnote.34
Colorimetric (Zincon) 3500 Zn B–2011 See footnote.33
76. Acid Mine Drainage 162769.

Table IC—List of Approved Test Procedures for Non-Pesticide Organic Compounds

Parameter1 Method EPA27 Standard
methods
ASTM Other
1. Acenaphthene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
2. Acenaphthylene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
3. Acrolein GC 603
GC/MS 624.1,4 1624B
4. Acrylonitrile GC 603
GC/MS 624.1,4 1624B O–4127–96.13
5. Anthracene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
6. Benzene GC 602 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
7. Benzidine Spectrophoto-
metric
See footnote3, p.1.
GC/MS 625.1,5 1625B 6410 B–2000
HPLC 605
8. Benzo(a)anthracene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
9. Benzo(a)pyrene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
10. Benzo(b)fluoranthene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
11. Benzo(g,h,i)perylene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
12. Benzo(k)fluoranthene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
13. Benzyl chloride GC See footnote3, p. 130.
GC/MS See footnote6, p. S102.
14. Butyl benzyl phthalate GC 606
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
15. bis(2-Chloroethoxy) methane GC 611
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
16. bis(2-Chloroethyl) ether GC 611
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
17. bis(2-Ethylhexyl) phthalate GC 606
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
18. Bromodichloromethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
19. Bromoform GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
20. Bromomethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
21. 4-Bromophenyl phenyl ether GC 611
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
22. Carbon tetrachloride GC 601 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
23. 4-Chloro-3-methyl phenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
24. Chlorobenzene GC 601, 602 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
25. Chloroethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–96.13
26. 2-Chloroethylvinyl ether GC 601
GC/MS 624.1, 1624B
27. Chloroform GC 601 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
28. Chloromethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
29. 2-Chloronaphthalene GC 612
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
30. 2-Chlorophenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
31. 4-Chlorophenyl phenyl ether GC 611
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
32. Chrysene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
33. Dibenzo(a,h)anthracene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
34. Dibromochloromethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
35. 1,2-Dichlorobenzene GC 601, 602 6200 C–2011
GC/MS 624.1, 1625B 6200 B–2011 See footnote,9 p. 27; O–4127–96,13 O–4436–16.14
36. 1,3-Dichlorobenzene GC 601, 602 6200 C–2011
GC/MS 624.1, 1625B 6200 B–2011 See footnote9, p. 27; O–4127–96.13
37. 1,4-Dichlorobenzene GC 601, 602 6200 C–2011
GC/MS 624.1, 1625B 6200 B–2011 See footnote,9 p. 27; O–4127–96,13 O–4436–16.14
38. 3,3′-Dichlorobenzidine GC/MS 625.1, 1625B 6410 B–2000
HPLC 605
39. Dichlorodifluoromethane GC 601
GC/MS 6200 C–2011 O–4127–9613, O–4436–16.14
40. 1,1-Dichloroethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
41. 1,2-Dichloroethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
42. 1,1-Dichloroethene GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
43. trans-1,2-Dichloroethene GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
44. 2,4-Dichlorophenol GC 604 6420 B–2000.
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
45. 1,2-Dichloropropane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
46. cis-1,3-Dichloropropene GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
47. trans-1,3-Dichloropropene GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
48. Diethyl phthalate GC 606
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
49. 2,4-Dimethylphenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
50. Dimethyl phthalate GC 606
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
51. Di-n-butyl phthalate GC 606
GC/MS 625.1, 1625B 6410 B–2000 See footnote,9, p. 27.
52. Di-n-octyl phthalate GC 606
GC/MS 625.1, 1625B 6410 B–2000 See footnote,9 p. 27.
53. 2, 4-Dinitrophenol GC 604 6420 B–2000 See footnote9, p. 27.
GC/MS 625.1, 1625B 6410 B–2000
54. 2,4-Dinitrotoluene GC 609
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
55. 2,6-Dinitrotoluene GC 609
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
56. Epichlorohydrin GC See footnote3, p. 130.
GC/MS See footnote6, p. S102.
57. Ethylbenzene GC 602 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–96,13 O–4436–16.14
58. Fluoranthene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
59. Fluorene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
60. 1,2,3,4,6,7,8-Heptachloro-dibenzofuran GC/MS 1613B
61. 1,2,3,4,7,8,9-Heptachloro-dibenzofuran GC/MS 1613B
62. 1,2,3,4,6,7,8-Heptachloro-dibenzo-p-dioxin GC/MS 1613B
63. Hexachlorobenzene GC 612
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
64. Hexachlorobutadiene GC 612
GC/MS 625.1, 1625B 6410 B–2000 See footnote,9 p. 27; O–4127–96.13
65. Hexachlorocyclopentadiene GC 612
GC/MS 625.1,5 1625B 6410 B–2000 See footnote,9 p. 27; O–4127–96.13
66. 1,2,3,4,7,8-Hexachloro-dibenzofuran GC/MS 1613B
67. 1,2,3,6,7,8-Hexachloro-dibenzofuran GC/MS 1613B
68. 1,2,3,7,8,9-Hexachloro-dibenzofuran GC/MS 1613B
69. 2,3,4,6,7,8-Hexachloro-dibenzofuran GC/MS 1613B
70. 1,2,3,4,7,8-Hexachloro-dibenzo-p-dioxin GC/MS 1613B
71. 1,2,3,6,7,8-Hexachloro-dibenzo-p-dioxin GC/MS 1613B
72. 1,2,3,7,8,9-Hexachloro-dibenzo-p-dioxin GC/MS 1613B
73. Hexachloroethane GC 612
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27; O–4127–96.13
74. Indeno(1,2,3-c,d) pyrene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
75. Isophorone GC 609
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
76. Methylene chloride GC 601 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
77. 2-Methyl-4,6-dinitrophenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
78. Naphthalene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005
79. Nitrobenzene GC 609
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC D4657–92 (98)
80. 2-Nitrophenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
81. 4-Nitrophenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
82. N-Nitrosodimethylamine GC 607
GC/MS 625.1,5 1625B 6410 B–2000 See footnote,9 p. 27.
83. N-Nitrosodi-n-propylamine GC 607
GC/MS 625.1,5 1625B 6410 B–2000 See footnote,9 p. 27.
84. N-Nitrosodiphenylamine GC 607
GC/MS 625.1,5 1625B 6410 B–2000 See footnote,9 p. 27.
85. Octachlorodibenzofuran GC/MS 1613B.10
86. Octachlorodibenzo-p-dioxin GC/MS 1613B.10
87. 2,2′-oxybis(1-chloropropane)12 [also known as bis(2-Chloro-1-methylethyl) ether] GC 611
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
88. PCB–1016 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
89. PCB–1221 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
90. PCB–1232 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
91. PCB–1242 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
92. PCB–1248 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
93. PCB–1254 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
94. PCB–1260 GC 608.3 See footnote3, p. 43; See footnote.8
GC/MS 625.1 6410 B–2000
95. 1,2,3,7,8-Pentachloro-dibenzofuran GC/MS 1613B
96. 2,3,4,7,8-Pentachloro-dibenzofuran GC/MS 1613B
97. 1,2,3,7,8-Pentachloro-dibenzo-p-dioxin GC/MS 1613B
98. Pentachlorophenol GC 604 6420 B–2000 See footnote3, p. 140.
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
99. Phenanthrene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
100. Phenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
101. Pyrene GC 610
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
HPLC 610 6440 B–2005 D4657–92 (98)
102. 2,3,7,8-Tetrachloro-dibenzofuran GC/MS 1613B.10
103. 2,3,7,8-Tetrachloro-dibenzo-p-dioxin GC/MS 613, 625.1,5a 1613B
104. 1,1,2,2-Tetrachloroethane GC 601 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–96.13
105. Tetrachloroethene GC 601 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
106. Toluene GC 602 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
107. 1,2,4-Trichlorobenzene GC 612 See footnote3, p. 130.
GC/MS 625.1, 1625B 6410 B–2000 See footnote,9 p. 27; O–4127–96,13 O–4436–16.14
108. 1,1,1-Trichloroethane GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
109. 1,1,2-Trichloroethane GC 601 6200 C–2011 See footnote3, p. 130.
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
110. Trichloroethene GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
111. Trichlorofluoromethane GC 601 6200 C–2011
GC/MS 624.1 6200 B–2011 O–4127–96.13
112. 2,4,6-Trichlorophenol GC 604 6420 B–2000
GC/MS 625.1, 1625B 6410 B–2000 See footnote9, p. 27.
113. Vinyl chloride GC 601 6200 C–2011
GC/MS 624.1, 1624B 6200 B–2011 O–4127–9613, O–4436–16.14
114. Nonylphenol GC/MS D7065–17
115. Bisphenol A (BPA) GC/MS D7065–17
116. p-tert-Octylphenol (OP) GC/MS D7065–17
117. Nonylphenol Monoethoxylate (NP1EO) GC/MS D7065–17
118. Nonylphenol Diethoxylate (NP2EO) GC/MS D7065–17
119. Adsorbable Organic Halides (AOX) Adsorption and Coulometric Titration 1650.11
120. Chlorinated Phenolics In Situ Acetylation and GC/MS 1653.11

Table ID—List of Approved Test Procedures for Pesticides1

Parameter Method EPA2 7 10 Standard methods ASTM Other
1. Aldrin GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96 (02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
2. Ametryn GC 507, 619 See footnote,3 p. 83; See footnote,9 O–3106–93; See footnote,6 p. S68.
GC/MS 525.2, 625.1 See footnote,14 O–1121–91.
3. Aminocarb TLC See footnote,3 p. 94; See footnote,6 p. S60.
HPLC 632
4. Atraton GC 619 See footnote,3 p. 83; See footnote,6 p. S68.
GC/MS 625.1
5. Atrazine GC 507, 619, 608.3 See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O–3106–93.
HPLC/MS See footnote,12 O–2060–01.
GC/MS 525.1, 525.2, 625.1 See footnote,11 O–1126–95.
6. Azinphos methyl GC 614, 622, 1657 See footnote,3 p. 25; See footnote,6 p. S51.
GC/MS 625.1 See footnote,11 O–1126–95.
7. Barban TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
GC/MS 625.1
8. α-BHC GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,8 3M0222.
GC/MS 625.15 6410 B–2000 See footnote,11 O–1126–95.
9. β-BHC GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
10. δ-BHC GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
11. γ-BHC (Lindane) GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 625.15 6410 B–2000 See footnote,11 O–1126–95.
12. Captan GC 617, 608.3 6630 B–2007 D3086–90, D5812–96(02) See footnote,3 p. 7.
13. Carbaryl TLC See footnote,3 p. 94, See footnote,6 p. S60.
HPLC 531.1, 632
HPLC/MS 553 See footnote,12 O–2060–01.
GC/MS 625.1 See footnote,11 O–1126–95.
14. Carbophenothion GC 617, 608.3 6630 B–2007 See footnote,4 page 27; See footnote,6 p. S73.
GC/MS 625.1
15. Chlordane GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
16. Chloropropham TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
GC/MS 625.1
17. 2,4-D GC 615 6640 B–2006 See footnote,3 p. 115; See footnote,4 O–3105–83.
HPLC/MS See footnote,12 O–2060–01.
18. 4,4′-DDD GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3105–83; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
19. 4,4′-DDE GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000 See footnote,11 O–1126–95.
20. 4,4′-DDT GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
21. Demeton-O GC 614, 622 See footnote,3 p. 25; See footnote,6 p. S51.
GC/MS 625.1
22. Demeton-S GC 614, 622 See footnote,3 p. 25; See footnote,6 p. S51.
GC/MS 625.1
23. Diazinon GC 507, 614, 622, 1657 See footnote,3 p. 25; See footnote,4 O–3104–83; See footnote,6 p. S51.
GC/MS 525.2, 625.1 See footnote,11 O–1126–95.
24. Dicamba GC 615 See footnote,3 p. 115.
HPLC/MS See footnote,12 O–2060–01.
25. Dichlofenthion GC 622.1 See footnote,4 page 27; See footnote,6 p. S73.
26. Dichloran GC 608.2, 617, 608.3 6630 B–2007 See footnote,3 p. 7.
27. Dicofol GC 617, 608.3 See footnote,4 O–3104–83.
28. Dieldrin GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000 See footnote,11 O–1126–95.
29. Dioxathion GC 614.1, 1657 See footnote,4 page 27; See footnote,6 p. S73.
30. Disulfoton GC 507, 614, 622, 1657 See footnote,3 p. 25; See footnote,6 p. S51.
GC/MS 525.2, 625.1 See footnote,11 O–1126–95.
31. Diuron TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
HPLC/MS 553 See footnote,12 O–2060–01.
32. Endosulfan I GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222).
GC/MS 625.15 6410 B–2000 See footnote,13 O–2002–01.
33. Endosulfan II GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,8 3M0222.
GC/MS 625.15 6410 B–2000 See footnote,13 O–2002–01.
34. Endosulfan Sulfate GC 617, 608.3 6630 C–2007 See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
35. Endrin GC 505, 508, 617, 1656, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 525.1, 525.2, 625.15 6410 B–2000
36. Endrin aldehyde GC 617, 608.3 6630 C–2007 See footnote,8 3M0222.
GC/MS 625.1
37. Ethion GC 614, 614.1, 1657 See footnote,4 page 27; See footnote,6 p. S73.
GC/MS 625.1 See footnote,13 O–2002–01.
38. Fenuron TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
HPLC/MS See footnote,12 O–2060–01.
39. Fenuron-TCA TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
40. Heptachlor GC 505, 508, 617, 1656, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 525.1, 525.2, 625.1 6410 B–2000
41. Heptachlor epoxide GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,6 p. S73; See footnote,8 3M0222.
GC/MS 625.1 6410 B–2000
42. Isodrin GC 617, 608.3 6630 B–2007 & C–2007 See footnote,4 O–3104–83; See footnote,6 p. S73.
GC/MS 625.1
43. Linuron GC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
HPLC/MS 553 See footnote,12 O–2060–01.
GC/MS See footnote,11 O–1126–95.
44. Malathion GC 614, 1657 6630 B–2007 See footnote,3 p. 25; See footnote,6 p. S51.
GC/MS 625.1 See footnote,11 O–1126–95.
45. Methiocarb TLC See footnote,3 p. 94; See footnote,6 p. S60.
HPLC 632
HPLC/MS See footnote,12 O–2060–01.
46. Methoxychlor GC 505, 508, 608.2, 617, 1656, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83; See footnote,8 3M0222.
GC/MS 525.1, 525.2, 625.1 See footnote,11 O–1126–95.
47. Mexacarbate TLC See footnote,3 p. 94; See footnote,6 p. S60.
HPLC 632
GC/MS 625.1
48. Mirex GC 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote,4 O–3104–83.
GC/MS 625.1
49. Monuron TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
50. Monuron-TCA TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
51. Neburon TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
HPLC/MS See footnote,12 O–2060–01.
52. Parathion methyl GC 614, 622, 1657 6630 B–2007 See footnote,4 page 27; See footnote,3 p. 25.
GC/MS 625.1 See footnote,11 O–1126–95.
53. Parathion ethyl GC 614 6630 B–2007 See footnote,4 page 27; See footnote,3 p. 25.
GC/MS See footnote,11 O–1126–95.
54. PCNB GC 608.1, 617, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7.
55. Perthane GC 617, 608.3 D3086–90, D5812–96(02) See footnote,4 O–3104–83.
56. Prometon GC 507, 619 See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O–3106–93.
GC/MS 525.2, 625.1 See footnote,11 O–1126–95.
57. Prometryn GC 507, 619 See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O–3106–93.
GC/MS 525.1, 525.2, 625.1 See footnote,13 O–2002–01.
58. Propazine GC 507, 619, 1656, 608.3 See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O–3106–93.
GC/MS 525.1, 525.2, 625.1
59. Propham TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
HPLC/MS See footnote,12 O–2060–01.
60. Propoxur TLC See footnote,3 p. 94; See footnote,6 p. S60.
HPLC 632
61. Secbumeton TLC See footnote,3 p. 83; See footnote,6 p. S68.
GC 619
62. Siduron TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
HPLC/MS See footnote,12 O–2060–01.
63. Simazine GC 505, 507, 619, 1656, 608.3 See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O–3106–93.
GC/MS 525.1, 525.2, 625.1 See footnote,11 O–1126–95.
64. Strobane GC 617, 608.3 6630 B–2007 & C–2007 See footnote,3 p. 7.
65. Swep TLC See footnote,3 p. 104; See footnote,6 p. S64.
HPLC 632
66. 2,4,5-T GC 615 6640 B–2006 See footnote,3 p. 115; See footnote,4 O–3105–83.
67. 2,4,5-TP (Silvex) GC 615 6640 B–2006 See footnote,3 p. 115; See footnote,4 O–3105–83.
68. Terbuthylazine GC 619, 1656, 608.3 See footnote,3 p. 83; See footnote,6 p. S68.
GC/MS See footnote,13 O–2002–01.
69. Toxaphene GC 505, 508, 617, 1656, 608.3 6630 B–2007 & C–2007 D3086–90, D5812–96(02) See footnote,3 p. 7; See footnote;8 See footnote,4 O–3105–83.
GC/MS 525.1, 525.2, 625.1 6410 B–2000
70. Trifluralin GC 508, 617, 627, 1656, 608.3 6630 B–2007 See footnote,3 p. 7; See footnote,9 O–3106–93.
GC/MS 525.2, 625.1 See footnote,11 O–1126–95.

Table IE—List of Approved Radiologic Test Test Procedures

Parameter and units Method Reference (method number or page)
EPA1 Standard Methods 18th, 19th, 20th Ed. Standard Methods Online ASTM USGS2
1. Alpha-Total, pCi per liter Proportional or scintillation counter 900.0 7110 B 7110 B–00 D1943–90, 96 pp. 75 and 783
2. Alpha-Counting error, pCi per liter Proportional or scintillation counter Appendix B 7110 B 7110 B–00 D1943–90, 96 p. 79
3. Beta-Total, pCi per liter Proportional counter 900.0 7110 B 7110 B–00 D1890–90, 96 pp. 75 and 783
4. Beta-Counting error, pCi Proportional counter Appendix B 7110 B 7110 B–00 D1890–90, 96 p. 79
5. (a) Radium Total pCi per liter
(b) Ra, pCi per liter
Proportional counter 903.0 7500-Ra B 7500-Ra B–01 D2460–90, 97
Scintillation counter 903.1 7500-Ra C 7500-Ra C–01 D3454–91, 97 p. 81

Table IF—List of Approved Methods for Pharmaceutical Pollutants

Pharmaceuticals pollutants CAS registry No. Analytical method number
Acetonitrile 75–05–8 1666/1671/D3371/D3695/624.1
n-Amyl acetate 628–63–7 1666/D3695
n-Amyl alcohol 71–41–0 1666/D3695
Benzene 71–43–2 D4763/D3695/502.2/524.2/624.1
n-Butyl-acetate 123–86–4 1666/D3695
tert-Butyl alcohol 75–65–0 1666/624.1
Chlorobenzene 108–90–7 502.2/524.2/624.1
Chloroform 67–66–3 502.2/524.2/551/624.1
o-Dichlorobenzene 95–50–1 1625C/502.2/524.2/624.1
1,2-Dichloroethane 107–06–2 D3695/502.2/524.2/624.1
Diethylamine 109–89–7 1666/1671
Dimethyl sulfoxide 67–68–5 1666/1671
Ethanol 64–17–5 1666/1671/D3695/624.1
Ethyl acetate 141–78–6 1666/D3695/624.1
n-Heptane 142–82–5 1666/D3695
n-Hexane 110–54–3 1666/D3695
Isobutyraldehyde 78–84–2 1666/1667
Isopropanol 67–63–0 1666/D3695
Isopropyl acetate 108–21–4 1666/D3695
Isopropyl ether 108–20–3 1666/D3695
Methanol 67–56–1 1666/1671/D3695/624.1
Methyl Cellosolve® (2-Methoxy ethanol) 109–86–4 1666/1671
Methylene chloride 75–09–2 502.2/524.2/624.1
Methyl formate 107–31–3 1666
4-Methyl-2-pentanone (MIBK) 108–10–1 1624C/1666/D3695/D4763/524.2/624.1
Phenol 108–95–2 D4763
n-Propanol 71–23–8 1666/1671/D3695/624.1
2-Propanone (Acetone) 67–64–1 D3695/D4763/524.2/624.1
Tetrahydrofuran 109–99–9 1666/524.2/624.1
Toluene 108–88–3 D3695/D4763/502.2/524.2/624.1
Triethlyamine 121–44–8 1666/1671
Xylenes (Note 1) 1624C/1666/624.1

Table IG—Test Methods for Pesticide Active Ingredients

[40 CFR part 455]

EPA survey code Pesticide name CAS No. EPA analytical method No.(s)3
8 Triadimefon 43121–43–3 507/633/525.1/525.2/1656/625.1.
12 Dichlorvos 62–73–7 1657/507/622/525.1/525.2/625.1.
16 2,4-D; 2,4-D Salts and Esters [2,4-Dichloro-phenoxyacetic acid] 94–75–7 1658/515.1/615/515.2/555.
17 2,4-DB; 2,4-DB Salts and Esters [2,4-Dichlorophenoxybutyric acid] 94–82–6 1658/515.1/615/515.2/555.
22 Mevinphos 7786–34–7 1657/507/622/525.1/525.2/625.1.
25 Cyanazine 21725–46–2 629/507/608.3/625.1.
26 Propachlor 1918–16–7 1656/508/608.1/525.1/525.2/608.3/625.1.
27 MCPA; MCPA Salts and Esters
[2-Methyl-4-chlorophenoxyacetic acid]
94–74–6 1658/615/555.
30 Dichlorprop; Dichlorprop Salts and Esters [2-(2,4-Dichlorophenoxy) propionic acid] 120–36–5 1658/515.1/615/515.2/555.
31 MCPP; MCPP Salts and Esters [2-(2-Methyl-4-chlorophenoxy) propionic acid] 93–65–2 1658/615/555.
35 TCMTB [2-(Thiocyanomethylthio) benzo-thiazole] 21564–17–0 637.
39 Pronamide 23950–58–5 525.1/525.2/507/633.1/625.1.
41 Propanil 709–98–8 632.1/1656/608.3.
45 Metribuzin 21087–64–9 507/633/525.1/525.2/1656/608.3/625.1.
52 Acephate 30560–19–1 1656/1657/608.3.
53 Acifluorfen 50594–66–6 515.1/515.2/555.
54 Alachlor 15972–60–8 505/507/645/525.1/525.2/1656/608.3/625.1.
55 Aldicarb 116–06–3 531.1.
58 Ametryn 834–12–8 507/619/525.2/625.1.
60 Atrazine 1912–24–9 505/507/619/525.1/525.2/1656/ 608.3/625.1.
62 Benomyl 17804–35–2 631.
68 Bromacil; Bromacil Salts and Esters 314–40–9 507/633/525.1/525.2/1656/608.3/625.1.
69 Bromoxynil 1689–84–5 1625/1661/625.1.
69 Bromoxynil Octanoate 1689–99–2 1656/608.3.
70 Butachlor 23184–66–9 507/645/525.1/525.2/1656/608.3/625.1.
73 Captafol 2425–06–1 1656/608.3/625.1.
75 Carbaryl [Sevin] 63–25–2 531.1/632/553/625.1.
76 Carbofuran 1563–66–2 531.1/632/625.1.
80 Chloroneb 2675–77–6 1656/508/608.1/525.1/525.2/608.3/625.1.
82 Chlorothalonil 1897–45–6 508/608.2/525.1/525.2/1656/608.3/625.1.
84 Stirofos 961–11–5 1657/507/622/525.1/525.2/625.1.
86 Chlorpyrifos 2921–88–2 1657/508/622/625.1.
90 Fenvalerate 51630–58–1 1660.
103 Diazinon 333–41–5 1657/507/614/622/525.2/625.1.
107 Parathion methyl 298–00–0 1657/614/622/625.1.
110 DCPA [Dimethyl 2,3,5,6-tetrachloro-terephthalate] 1861–32–1 508/608.2/525.1/525.2/515.12/515.22/1656/608.3/625.1.
112 Dinoseb 88–85–7 1658/515.1/615/515.2/555/625.1.
113 Dioxathion 78–34–2 1657/614.1.
118 Nabonate [Disodium cyanodithio-imidocarbonate] 138–93–2 630.1.
119 Diuron 330–54–1 632/553.
123 Endothall 145–73–3 548/548.1.
124 Endrin 72–20–8 1656/505/508/617/525.1/525.2/608.3/625.1.
125 Ethalfluralin 55283–68–6 1656/627/608.3 See footnote 1.
126 Ethion 563–12–2 1657/614/614.1/625.1.
127 Ethoprop 13194–48–4 1657/507/622/525.1/525.2/625.1.
132 Fenarimol 60168–88–9 507/633.1/525.1/525.2/1656/608.3/625.1.
133 Fenthion 55–38–9 1657/622/625.1.
138 Glyphosate [N-(Phosphonomethyl) glycine] 1071–83–6 547.
140 Heptachlor 76–44–8 1656/505/508/617/525.1/525.2/608.3/625.1.
144 Isopropalin 33820–53–0 1656/627/608.3.
148 Linuron 330–55–2 553/632.
150 Malathion 121–75–5 1657/614/625.1.
154 Methamidophos 10265–92–6 1657.
156 Methomyl 16752–77–5 531.1/632.
158 Methoxychlor 72–43–5 1656/505/508/608.2/617/525.1/525.2/608.3/625.1.
172 Nabam 142–59–6 630/630.1.
173 Naled 300–76–5 1657/622/625.1.
175 Norflurazon 27314–13–2 507/645/525.1/525.2/1656/608.3/625.1.
178 Benfluralin 1861–40–1 1656/627/608.3 See footnote 1.
182 Fensulfothion 115–90–2 1657/622/625.1.
183 Disulfoton 298–04–4 1657/507/614/622/525.2/625.1.
185 Phosmet 732–11–6 1657/622.1/625.1.
186 Azinphos Methyl 86–50–0 1657/614/622/625.1.
192 Organo-tin pesticides 12379–54–3 Ind-01/200.7/200.9.
197 Bolstar 35400–43–2 1657/622.
203 Parathion 56–38–2 1657/614/625.1.
204 Pendimethalin 40487–42–1 1656.
205 Pentachloronitrobenzene 82–68–8 1656/608.1/617/608.3/625.1.
206 Pentachlorophenol 87–86–5 1625/515.2/555/515.1/525.1/525.2/625.1.
208 Permethrin 52645–53–1 608.2/508/525.1/525.2/1656/1660/608.34/625.14.
212 Phorate 298–02–2 1657/622/625.1.
218 Busan 85 [Potassium dimethyldithiocarbamate] 128–03–0 630/630.1.
219 Busan 40 [Potassium N-hydroxymethyl-N-methyldithiocarbamate] 51026–28–9 630/630.1.
220 KN Methyl [Potassium N-methyl-dithiocarbamate] 137–41–7 630/630.1.
223 Prometon 1610–18–0 507/619/525.2/625.1.
224 Prometryn 7287–19–6 507/619/525.1/525.2/625.1.
226 Propazine 139–40–2 507/619/525.1/525.2/1656/608.3/625.1.
230 Pyrethrin I 121–21–1 1660.
232 Pyrethrin II 121–29–9 1660.
236 DEF [S,S,S-Tributyl phosphorotrithioate] 78–48–8 1657.
239 Simazine 122–34–9 505/507/619/525.1/525.2/1656/608.3/625.1.
241 Carbam-S [Sodium dimethyldithio-carbamate] 128–04–1 630/630.1.
243 Vapam [Sodium methyldithiocarbamate] 137–42–8 630/630.1.
252 Tebuthiuron 34014–18–1 507/525.1/525.2/625.1.
254 Terbacil 5902–51–2 507/633/525.1/525.2/1656/608.3/625.1.
255 Terbufos 13071–79–9 1657/507/614.1/525.1/525.2/625.1.
256 Terbuthylazine 5915–41–3 619/1656/608.3.
257 Terbutryn 886–50–0 507/619/525.1/525.2/625.1.
259 Dazomet 533–74–4 630/630.1/1659.
262 Toxaphene 8001–35–2 1656/505/508/617/525.1/525.2/608.3/625.1.
263 Merphos [Tributyl phosphorotrithioate] 150–50–5 1657/507/525.1/525.2/622/625.1.
264 Trifluralin1 1582–09–8 1656/508/617/627/525.2/608.3/625.1.
268 Ziram [Zinc dimethyldithiocarbamate] 137–30–4 630/630.1.

Table IH—List of Approved Microbiological Methods for Ambient Water

Parameter and units Method1 EPA Standard methods AOAC, ASTM, USGS Other
Bacteria
1. Coliform (fecal), number per 100 mL Most Probable Number (MPN), 5 tube, 3 dilution, or p. 132 3 9221 E–2014, 9221 F–2014 32
Membrane filter (MF) 2, single step p. 124 3 9222 D–2015 26 B–0050–85 4
2. Coliform (total), number per 100 mL MPN, 5 tube, 3 dilution, or p. 114 3 9221 B–2014 B–0025–85 4
MF 2, single step or two step p. 108 3 9222 B–2015 27
MF 2 with enrichment p. 111 3 9222 B– 2015 27
3. E. coli, number per 100 mL MPN 5 7 13, multiple tube, or 9221 B.3–2014/9221 F–2014 10 12 32
Multiple tube/multiple well, or 9223 B–2016 11 991.15 9 Colilert® 11 15, Colilert-18®.11 14 15
MF 2 5 6 7, two step, or 1103.1 18 9222 B–2015/9222 I–2015 17, 9213 D–2007 D5392–93 8
Single step 1603 19, 1604 20 m-ColiBlue24® 16, KwikCountTM EC. 28 29
4. Fecal streptococci, number per 100 mL MPN, 5 tube, 3 dilution, or p. 139 3 9230 B–2013 B–0055–85 4
MF 2, or p. 136 3 9230 C–2013 30
Plate count p. 143 3
5. Enterococci, number per 100 mL MPN 5 7, multiple tube/multiple well, or 9230 D–2013 D6503–99 8 Enterolert®.11 21
MF 2 5 6 7 two step, or 1106.1 22 9230 C–2013 30 D5259–92 8
Single step, or 1600 23 9230 C–2013 30
Plate count p. 143 3
Protozoa
6. Cryptosporidium Filtration/IMS/FA 1622 24, 1623 25, 1623.1 25 31
7. Giardia Filtration/IMS/FA 1623 25, 1623.1 25 31

(b) Certain material is incorporated by reference into this section with the approval of the Director of the Federal Register under 5 U.S.C. 552(a) and 1 CFR part 51. All approved material may be inspected at EPA's Water Docket, EPA West, 1301 Constitution Avenue NW, Room 3334, Washington, DC 20004, (Telephone: 202–566–2426). It is also available for inspection at National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202–741–6030, or go to: http://www.archives.gov/federal-register/cfr/ibr-locations.html.

(1) Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati OH (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm or from: National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161

(i) Microbiological Methods for Monitoring the Environment, Water, and Wastes. 1978. EPA/600/8–78/017, Pub. No. PB–290329/A.S.

(A) Part III Analytical Methodology, Section B Total Coliform Methods, page 108. Table IA, Note 3; Table IH, Note 3.

(B) Part III Analytical Methodology, Section B Total Coliform Methods, 2.6.2 Two-Step Enrichment Procedure, page 111. Table IA, Note 3; Table IH, Note 3.

(C) Part III Analytical Methodology, Section B Total Coliform Methods, 4 Most Probable Number (MPN) Method, page 114. Table IA, Note 3; Table IH, Note 3.

(D) Part III Analytical Methodology, Section C Fecal Coliform Methods, 2 Direct Membrane Filter (MF) Method, page 124. Table IA, Note 3; Table IH, Note 3.

(E) Part III, Analytical Methodology, Section C Fecal Coliform Methods, 5 Most Probable Number (MPN) Method, page 132. Table IA, Note 3; Table IH, Note 3.

(F) Part III Analytical Methodology, Section D Fecal Streptococci, 2 Membrane Filter (MF) Method, page 136. Table IA, Note 3; Table IH, Note 3.

(G) Part III Analytical Methodology, Section D Fecal Streptococci, 4 Most Probable Number Method, page 139. Table IA, Note 3; Table IH, Note 3.

(H) Part III Analytical Methodology, Section D Fecal Streptococci, 5 Pour Plate Method, page 143. Table IA, Note 3; Table IH, Note 3.

(ii) [Reserved]

(2) Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati OH (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm.

(i) Method 300.1 (including Errata Cover Sheet, April 27, 1999), Determination of Inorganic Ions in Drinking Water by Ion Chromatography, Revision 1.0, 1997. Table IB, Note 52.

(ii) Method 551, Determination of Chlorination Disinfection Byproducts and Chlorinated Solvents in Drinking Water by Liquid-Liquid Extraction and Gas Chromatography With Electron-Capture Detection. 1990. Table IF.

(3) National Exposure Risk Laboratory-Cincinnati, U.S. Environmental Protection Agency, Cincinnati OH (US EPA). Available from http://water.epa.gov/scitech/methods/cwa/index.cfm or from the National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 22161. Telephone: 800–553–6847.

(i) Methods for the Determination of Inorganic Substances in Environmental Samples. August 1993. EPA/600/R–93/100, Pub. No. PB 94120821. Table IB, Note 52.

(A) Method 180.1, Determination of Turbidity by Nephelometry. Revision 2.0. Table IB, Note 52.

(B) Method 300.0, Determination of Inorganic Anions by Ion Chromatography. Revision 2.1. Table IB, Note 52.

(C) Method 335.4, Determination of Total Cyanide by Semi-Automated Colorimetry. Revision 1.0. Table IB, Notes 52 and 57.

(D) Method 350.1, Determination of Ammonium Nitrogen by Semi-Automated Colorimetry. Revision 2.0. Table IB, Notes 30 and 52.

(E) Method 351.2, Determination of Total Kjeldahl Nitrogen by Semi-Automated Colorimetry. Revision 2.0. Table IB, Note 52.

(F) Method 353.2, Determination of Nitrate-Nitrite Automated Colorimetry. Revision 2.0. Table IB, Note 52.

(G) Method 365.1, Determination of Phosphorus by Automated Colorimetry. Revision 2.0. Table IB, Note 52.

(H) Method 375.2, Determination of Sulfate by Automated Colorimetry. Revision 2.0. Table IB, Note 52.

(I) Method 410.4, Determination of Chemical Oxygen Demand by Semi-Automated Colorimetry. Revision 2.0. Table IB, Note 52.

(ii) Methods for the Determination of Metals in Environmental Samples, Supplement I. May 1994. EPA/600/R–94/111, Pub. No. PB 95125472. Table IB, Note 52.

(A) Method 200.7, Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry. Revision 4.4. Table IB, Note 52.

(B) Method 200.8, Determination of Trace Elements in Water and Wastes by Inductively Coupled Plasma Mass Spectrometry. Revision 5.3. Table IB, Note 52.

(C) Method 200.9, Determination of Trace Elements by Stabilized Temperature Graphite Furnace Atomic Absorption Spectrometry. Revision 2.2. Table IB, Note 52.

(D) Method 218.6, Determination of Dissolved Hexavalent Chromium in Drinking Water, Groundwater, and Industrial Wastewater Effluents by Ion Chromatography. Revision 3.3. Table IB, Note 52.

(E) Method 245.1, Determination of Mercury in Water by Cold Vapor Atomic Absorption Spectrometry. Revision 3.0. Table IB, Note 52.

(4) National Exposure Risk Laboratory-Cincinnati, U.S. Environmental Protection Agency, Cincinnati OH (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm.

(i) EPA Method 200.5, Determination of Trace Elements in Drinking Water by Axially Viewed Inductively Coupled Plasma-Atomic Emission Spectrometry. Revision 4.2, October 2003. EPA/600/R–06/115. Table IB, Note 68.

(ii) EPA Method 525.2, Determination of Organic Compounds in Drinking Water by Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry. Revision 2.0, 1995. Table ID, Note 10.

(5) Office of Research and Development, Cincinnati OH. U.S. Environmental Protection Agency, Cincinnati OH (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm or from ORD Publications, CERI, U.S. Environmental Protection Agency, Cincinnati OH 45268.

(i) Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol, and Pesticides in Water and Wastewater. 1978. Table IC, Note 3; Table ID, Note 3.

(ii) Methods for Chemical Analysis of Water and Wastes. March 1979. EPA–600/4–79–020. Table IB, Note 1.

(iii) Methods for Chemical Analysis of Water and Wastes. Revised March 1983. EPA–600/4–79–020. Table IB, Note 1.

(A) Method 120.1, Conductance, Specific Conductance, µmhos at 25 °C. Revision 1982. Table IB, Note 1.

(B) Method 130.1, Hardness, Total (mg/L as CaCO3), Colorimetric, Automated EDTA. Issued 1971. Table IB, Note 1.

(C) Method 150.2, pH, Continuous Monitoring (Electrometric). December 1982. Table IB, Note 1.

(D) Method 160.4, Residue, Volatile, Gravimetric, Ignition at 550 °C. Issued 1971. Table IB, Note 1.

(E) Method 206.5, Arsenic, Sample Digestion Prior to Total Arsenic Analysis by Silver Diethyldithiocarbamate or Hydride Procedures. Issued 1978. Table IB, Note 1.

(F) Method 231.2, Gold, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(G) Method 245.2, Mercury, Automated Cold Vapor Technique. Issued 1974. Table IB, Note 1.

(H) Method 252.2, Osmium, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(I) Method 253.2, Palladium, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(J) Method 255.2, Platinum, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(K) Method 265.2, Rhodium, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(L) Method 279.2, Thallium, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(M) Method 283.2, Titanium, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(N) Method 289.2, Zinc, Atomic Absorption, Furnace Technique. Issued 1978. Table IB, Note 1.

(O) Method 310.2, Alkalinity, Colorimetric, Automated, Methyl Orange. Revision 1974. Table IB, Note 1.

(P) Method 351.1, Nitrogen, Kjeldahl, Total, Colorimetric, Automated Phenate. Revision 1978. Table IB, Note 1.

(Q) Method 352.1, Nitrogen, Nitrate, Colorimetric, Brucine. Issued 1971. Table IB, Note 1.

(R) Method 365.3, Phosphorus, All Forms, Colorimetric, Ascorbic Acid, Two Reagent. Issued 1978. Table IB, Note 1.

(S) Method 365.4, Phosphorus, Total, Colorimetric, Automated, Block Digestor AA II. Issued 1974. Table IB, Note 1.

(T) Method 410.3, Chemical Oxygen Demand, Titrimetric, High Level for Saline Waters. Revision 1978. Table IB, Note 1.

(U) Method 420.1, Phenolics, Total Recoverable, Spectrophotometric, Manual 4–AAP With Distillation. Revision 1978. Table IB, Note 1.

(iv) Prescribed Procedures for Measurement of Radioactivity in Drinking Water. 1980. EPA–600/4–80–032. Table IE.

(A) Method 900.0, Gross Alpha and Gross Beta Radioactivity. Table IE.

(B) Method 903.0, Alpha-Emitting iRadio Isotopes. Table IE.

(C) Method 903.1, Radium-226, Radon Emanation Technique. Table IE.

(D) Appendix B, Error and Statistical Calculations. Table IE.

(6) Office of Science and Technology, U.S. Environmental Protection Agency, Washington DC (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm.

(i) Method 1625C, Semivolatile Organic Compounds by Isotope Dilution GCMS. 1989. Table IF.

(ii) [Reserved]

(7) Office of Water, U.S. Environmental Protection Agency, Washington DC (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm or from National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161.

(i) Method 1631, Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic Fluorescence Spectrometry. Revision E, August 2002. EPA–821–R–02–019, Pub. No. PB2002–108220. Table IB, Note 43.

(ii) Kelada-01, Kelada Automated Test Methods for Total Cyanide, Acid Dissociable Cyanide, and Thiocyanate. Revision 1.2, August 2001. EPA 821–B–01–009, Pub. No. PB 2001–108275. Table IB, Note 55.

(iii) In the compendium Analytical Methods for the Determination of Pollutants in Pharmaceutical Manufacturing Industry Wastewaters. July 1998. EPA 821–B–98–016, Pub. No. PB95201679. Table IF, Note 1.

(A) EPA Method 1666, Volatile Organic Compounds Specific to the Pharmaceutical Industry by Isotope Dilution GC/MS. Table IF, Note 1.

(B) EPA Method 1667, Formaldehyde, Isobutyraldehyde, and Furfural by Derivatization Followed by High Performance Liquid Chromatography. Table IF.

(C) Method 1671, Volatile Organic Compounds Specific to the Pharmaceutical Manufacturing Industry by GC/FID. Table IF.

(iv) Methods For The Determination of Nonconventional Pesticides In Municipal and Industrial Wastewater, Volume I. Revision I, August 1993. EPA 821–R–93–010A, Pub. No. PB 94121654. Tables ID, IG.

(A) Method 608.1, Organochlorine Pesticides. Table ID, Note 10; Table IG, Note 3.

(B) Method 608.2, Certain Organochlorine Pesticides. Table ID, Note 10; Table IG, Note 3.

(C) Method 614, Organophosphorus Pesticides. Table ID, Note 10; Table IG, Note 3.

(D) Method 614.1, Organophosphorus Pesticides. Table ID, Note 10; Table IG, Note 3.

(E) Method 615, Chlorinated Herbicides. Table ID, Note 10; Table IG, Note 3.

(F) Method 617, Organohalide Pesticides and PCBs. Table ID, Note 10; Table IG, Note 3.

(G) Method 619, Triazine Pesticides. Table ID, Note 10; Table IG, Note 3.

(H) Method 622, Organophosphorus Pesticides. Table ID, Note 10; Table IG, Note 3.

(I) Method 622.1, Thiophosphate Pesticides. Table ID, Note 10; Table IG, Note 3.

(J) Method 627, Dinitroaniline Pesticides. Table ID, Note 10; Table IG, Notes 1 and 3.

(K) Method 629, Cyanazine. Table IG, Note 3.

(L) Method 630, Dithiocarbamate Pesticides. Table IG, Note 3.

(M) Method 630.1, Dithiocarbamate Pesticides. Table IG, Note 3.

(N) Method 631, Benomyl and Carbendazim. Table IG, Note 3.

(O) Method 632, Carbamate and Urea Pesticides. Table ID, Note 10; Table IG, Note 3.

(P) Method 632.1, Carbamate and Amide Pesticides. Table IG, Note 3.

(Q) Method 633, Organonitrogen Pesticides. Table IG, Note 3.

(R) Method 633.1, Neutral Nitrogen-Containing Pesticides. Table IG, Note 3.

(S) Method 637, MBTS and TCMTB. Table IG, Note 3.

(T) Method 644, Picloram. Table IG, Note 3.

(U) Method 645, Certain Amine Pesticides and Lethane. Table IG, Note 3.

(V) Method 1656, Organohalide Pesticides. Table ID, Note 10; Table IG, Notes 1 and 3.

(W) Method 1657, Organophosphorus Pesticides. Table ID, Note 10; Table IG, Note 3.

(X) Method 1658, Phenoxy-Acid Herbicides. Table IG, Note 3.

(Y) Method 1659, Dazomet. Table IG, Note 3.

(Z) Method 1660, Pyrethrins and Pyrethroids. Table IG, Note 3.

(AA) Method 1661, Bromoxynil. Table IG, Note 3.

(BB) Ind-01. Methods EV–024 and EV–025, Analytical Procedures for Determining Total Tin and Triorganotin in Wastewater. Table IG, Note 3.

(v) Methods For The Determination of Nonconventional Pesticides In Municipal and Industrial Wastewater, Volume II. August 1993. EPA 821–R–93–010B, Pub. No. PB 94166311. Table IG.

(A) Method 200.9, Determination of Trace Elements by Stabilized Temperature Graphite Furnace Atomic Absorption Spectrometry. Table IG, Note 3.

(B) Method 505, Analysis of Organohalide Pesticides and Commercial Polychlorinated Biphenyl (PCB) Products in Water by Microextraction and Gas Chromatography. Table ID, Note 10; Table IG, Note 3.

(C) Method 507, The Determination of Nitrogen- and Phosphorus-Containing Pesticides in Water by Gas Chromatography with a Nitrogen-Phosphorus Detector. Table ID, Note 10; Table IG, Note 3.

(D) Method 508, Determination of Chlorinated Pesticides in Water by Gas Chromatography with an Electron Capture Detector. Table ID, Note 10; Table IG, Note 3.

(E) Method 515.1, Determination of Chlorinated Acids in Water by Gas Chromatography with an Electron Capture Detector. Table IG, Notes 2 and 3.

(F) Method 515.2, Determination of Chlorinated Acids in Water Using Liquid-Solid Extraction and Gas Chromatography with an Electron Capture Detector. Table IG, Notes 2 and 3.

(G) Method 525.1, Determination of Organic Compounds in Drinking Water by Liquids-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry. Table ID, Note 10; Table IG, Note 3.

(H) Method 531.1, Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates in Water by Direct Aqueous Injection HPLC with Post-Column Derivatization. Table ID, Note 10; Table IG, Note 3.

(I) Method 547, Determination of Glyphosate in Drinking Water by Direct-Aqueous-Injection HPLC, Post-Column Derivatization, and Fluorescence Detection. Table IG, Note 3.

(J) Method 548, Determination of Endothall in Drinking Water by Aqueous Derivatization, Liquid-Solid Extraction, and Gas Chromatography with Electron-Capture Detector. Table IG, Note 3.

(K) Method 548.1, Determination of Endothall in Drinking Water by Ion-Exchange Extraction, Acidic Methanol Methylation and Gas Chromatography/Mass Spectrometry. Table IG, Note 3.

(L) Method 553, Determination of Benzidines and Nitrogen-Containing Pesticides in Water by Liquid-Liquid Extraction or Liquid-Solid Extraction and Reverse Phase High Performance Liquid Chromatography/Particle Beam/Mass Spectrometry Table ID, Note 10; Table IG, Note 3.

(M) Method 555, Determination of Chlorinated Acids in Water by High Performance Liquid Chromatography With a Photodiode Array Ultraviolet Detector. Table IG, Note 3.

(vi) In the compendium Methods for the Determination of Organic Compounds in Drinking Water. Revised July 1991, December 1998. EPA–600/4–88–039, Pub. No. PB92–207703. Table IF.

(A) EPA Method 502.2, Volatile Organic Compounds in Water by Purge and Trap Capillary Column Gas Chromatography with Photoionization and Electrolytic Conductivity Detectors in Series. Table IF.

(B) [Reserved]

(vii) In the compendium Methods for the Determination of Organic Compounds in Drinking Water-Supplement II. August 1992. EPA–600/R–92–129, Pub. No. PB92–207703. Table IF.

(A) EPA Method 524.2, Measurement of Purgeable Organic Compounds in Water by Capillary Column Gas Chromatography/Mass Spectrometry. Table IF.

(B) [Reserved]

(viii) Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. October 2002. EPA 821–R–02–012, Pub. No. PB2002–108488. Table IA, Note 26.

(ix) Short-Term Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. October 2002. EPA 821–R–02–013, Pub. No. PB2002–108489. Table IA, Note 27.

(x) Short-Term Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. October 2002. EPA 821–R–02–014, Pub. No. PB2002–108490. Table IA, Note 28.

(8) Office of Water, U.S. Environmental Protection Agency, Washington, DC (U.S. EPA). Available at https://www.epa.gov/cwa-methods.

(i) Method 245.7, Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry. Revision 2.0, February 2005. EPA–821–R–05–001. Table IB, Note 17.

(ii) Method 1103.1: Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). March 2010. EPA–621–R–10–002. Table IH, Note 19.

(iii) Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE–EIA). December 2009. EPA–621–R–09–015. Table IH, Note 23.

(iv) Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI). September 2014. EPA–821–R–14–011. Table IA, Note 25; Table IH, Note 24.

(v) Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC). September 2014. EPA–821–R–14–010. Table IA, Note 22; Table IH, Note 20.

(vi) Method 1604: Total Coliforms and Escherichia coli (E. coli) in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium). September 2002. EPA–821–R–02–024. Table IH, Note 21.

(vii) Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. December 2005. EPA–821–R–05–001. Table IH, Note 25.

(viii) Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. December 2005. EPA–821–R–05–002. Table IH, Note 26.

(ix) Method 1623.1: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. EPA 816–R–12–001. January 2012. U.S. EPA, Table IH, Note 25.

(x) Method 1627, Kinetic Test Method for the Prediction of Mine Drainage Quality. December 2011. EPA–821–R–09–002. Table IB, Note 69.

(xi) Method 1664, n-Hexane Extractable Material (HEM; Oil and Grease) and Silica Gel Treated n-Hexane Extractable Material (SGT–HEM; Nonpolar Material) by Extraction and Gravimetry. Revision A, February 1999. EPA–821–R–98–002. Table IB, Notes 38 and 42.

(xii) Method 1664, n-Hexane Extractable Material (HEM; Oil and Grease) and Silica Gel Treated n-Hexane Extractable Material (SGT–HEM; Nonpolar Material) by Extraction and Gravimetry, Revision B, February 2010. EPA–821–R–10–001. Table IB, Notes 38 and 42.

(xiii) Method 1669, Sampling Ambient Water for Trace Metals at EPA Water Quality Criteria Levels. July 1996. Table IB, Note 43.

(xiv) Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using Lauryl Tryptose Broth (LTB) and EC Medium. September 2014. EPA–821–R–14–009.Table IA, Note 15.

(xv) Method 1681: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using A–1 Medium. July 2006. EPA 821–R–06–013. Table IA, Note 20.

(xvi) Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium. September 2014. EPA 821–R–14–012. Table IA, Note 23.

(9) American National Standards Institute, 1430 Broadway, New York NY 10018.

(i) ANSI. American National Standard on Photographic Processing Effluents. April 2, 1975. Table IB, Note 9.

(ii) [Reserved]

(10) American Public Health Association, 1015 15th Street NW., Washington, DC 20005. Standard Methods Online is available through the Standard Methods Web site (http://www.standardmethods.org).

(i) Standard Methods for the Examination of Water and Wastewater. 14th Edition, 1975. Table IB, Notes 17 and 27.

(ii) Standard Methods for the Examination of Water and Wastewater. 15th Edition, 1980, Table IB, Note 30; Table ID.

(iii) Selected Analytical Methods Approved and Cited by the United States Environmental Protection Agency, Supplement to the 15th Edition of Standard Methods for the Examination of Water and Wastewater. 1981. Table IC, Note 6; Table ID, Note 6.

(iv) Standard Methods for the Examination of Water and Wastewater. 18th Edition, 1992. Tables IA, IB, IC, ID, IE, and IH.

(v) Standard Methods for the Examination of Water and Wastewater. 19th Edition, 1995. Tables IA, IB, IC, ID, IE, and IH.

(vi) Standard Methods for the Examination of Water and Wastewater. 20th Edition, 1998. Tables IA, IB, IC, ID, IE, and IH.

(vii) Standard Methods for the Examination of Water and Wastewater. 21st Edition, 2005. Table IB, Notes 17 and 27.

(viii) 2120, Color. 2011. Table IB.

(ix) 2130, Turbidity. 2011. Table IB.

(x) 2310, Acidity. 2011. Table IB.

(xi) 2320, Alkalinity. 2011. Table IB.

(xii) 2340, Hardness. 2011. Table IB.

(xiii) 2510, Conductivity. 2011. Table IB.

(xiv) 2540, solids. 2015. Table IB.

(xv) 2550, Temperature. 2010. Table IB.

(xvi) 3111, Metals by Flame Atomic Absorption Spectrometry. 2011. Table IB.

(xvii) 3112, Metals by Cold-Vapor Atomic Absorption Spectrometry. 2011. Table IB.

(xviii) 3113, Metals by Electrothermal Atomic Absorption Spectrometry. 2010. Table IB.

(xix) 3114, Arsenic and Selenium by Hydride Generation/Atomic Absorption Spectrometry. 2011. Table IB.

(xx) 3120, Metals by Plasma Emission Spectroscopy. 2011. Table IB.

(xxi) 3125, Metals by Inductively Coupled Plasma-Mass Spectrometry. 2011. Table IB.

(xxii) 3500-Al, Aluminum. 2011. Table IB.

(xxiii) 3500-As, Arsenic. 2011. Table IB.

(xxiv) 3500-Ca, Calcium. 2011. Table IB.

(xxv) 3500-Cr, Chromium. 2011. Table IB.

(xxvi) 3500-Cu, Copper. 2011. Table IB.

(xxvii) 3500-Fe, Iron. 2011. Table IB.

(xxviii) 3500-Pb, Lead. 2011. Table IB.

(xxix) 3500-Mn, Manganese. 2011. Table IB.

(xxx) 3500-K, Potassium. 2011. Table IB.

(xxxi) 3500-Na, Sodium. 2011. Table IB.

(xxxii) 3500-V, Vanadium. 2011. Table IB.

(xxxiii) 3500-Zn, Zinc. 2011. Table IB.

(xxxiv) 4110, Determination of Anions by Ion Chromatography. 2011. Table IB.

(xxxv) 4140, Inorganic Anions by Capillary Ion Electrophoresis. 2011. Table IB.

(xxxvi) 4500-B, Boron. 2011. Table IB.

(xxxvii) 4500-Cl, Chloride. 2011. Table IB.

(xxxviii) 4500-Cl, Chlorine (Residual). 2011. Table IB.

(xxxix) 4500–CN, Cyanide. 2016. Table IB.

(xl) 4500-F, Fluoride. 2011. Table IB.

(xli) 4500-H+, pH Value. 2011. Table IB.

(xlii) 4500-NH3, Nitrogen (Ammonia). 2011. Table IB.

(xliii) 4500-NO2, Nitrogen (Nitrite). 2011. Table IB.

(xliv) 4500–NO3-, Nitrogen (Nitrate). 2016. Table IB.

(xlv) 4500–Norg, Nitrogen (Organic). 2011. Table IB.

(xlvi) 4500–O, Oxygen (Dissolved). 2016. Table IB.

(xlvii) 4500–P, Phosphorus. 2011. Table IB.

(xlviii) 4500–SiO2, Silica. 2011. Table IB.

(xlix) 4500–S2−, Sulfide. 2011. Table IB.

(l) 4500–SO32−, Sulfite. 2011. Table IB.

(li) 4500–SO42−, Sulfate. 2011. Table IB.

(lii) 5210, Biochemical Oxygen Demand (BOD). 2016. Table IB.

(liii) 5220, Chemical Oxygen Demand (COD). 2011. Table IB.

(liv) 5310, Total Organic Carbon (TOC). 2014. Table IB.

(lv) 5520, Oil and Grease. 2011. Table IB.

(lvi) 5530, Phenols. 2010. Table IB.

(lvii) 5540, Surfactants. 2011. Table IB.

(lviii) 6200, Volatile Organic Compounds. 2011. Table IC.

(lix) 6410, Extractable Base/Neutrals and Acids. 2000. Tables IC, ID.

(lx) 6420, Phenols. 2000. Table IC.

(lxi) 6440, Polynuclear Aromatic Hydrocarbons. 2005. Table IC.

(lxii) 6630, Organochlorine Pesticides. 2007. Table ID.

(lxiii) 6640, Acidic Herbicide Compounds. 2006. Table ID.

(lxiv) 7110, Gross Alpha and Gross Beta Radioactivity (Total, Suspended, and Dissolved). 2000. Table IE.

(lxv) 7500, Radium. 2001. Table IE.

(lxvi) 9213, Recreational Waters. 2007. Table IH.

(lxvii) 9221 Multiple-Tube Fermentation Technique for Members of the Coliform Group. 2014. Table IA, Notes 12 and 14; Table IH, Notes 10 and 12.

(lxviii) 9222, Membrane Filter Technique for Members of the Coliform Group. 2015. Table IA; Table IH, Note 17.

(lxix) 9223 Enzyme Substrate Coliform Test. 2016. Table IA; Table IH.

(lxx) 9230 Fecal Enterococcus/Streptococcus Groups. 2013. Table IA; Table IH.

(11) The Analyst, The Royal Society of Chemistry, RSC Publishing, Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 0WF, United Kingdom. (Also available from most public libraries.)

(i) Spectrophotometric Determination of Ammonia: A Study of a Modified Berthelot Reaction Using Salicylate and Dichloroisocyanurate. Krom, M.D. 105:305–316, April 1980. Table IB, Note 60.

(ii) [Reserved]

(12) Analytical Chemistry, ACS Publications, 1155 Sixteenth St. NW., Washington DC 20036. (Also available from most public libraries.)

(i) Spectrophotometric and Kinetics Investigation of the Berthelot Reaction for the Determination of Ammonia. Patton, C.J. and S.R. Crouch. 49(3):464–469, March 1977. Table IB, Note 60.

(ii) [Reserved]

(13) AOAC International, 481 North Frederick Avenue, Suite 500, Gaithersburg, MD 20877–2417.

(i) Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision, 1998.

(A) 920.203, Manganese in Water, Persulfate Method. Table IB, Note 3.

(B) 925.54, Sulfate in Water, Gravimetric Method. Table IB, Note 3.

(C) 973.40, Specific Conductance of Water. Table IB, Note 3.

(D) 973.41, pH of Water. Table IB, Note 3.

(E) 973.43, Alkalinity of Water, Titrimetric Method. Table IB, Note 3.

(F) 973.44, Biochemical Oxygen Demand (BOD) of Water, Incubation Method. Table IB, Note 3.

(G) 973.45, Oxygen (Dissolved) in Water, Titrimetric Methods. Table IB, Note 3.

(H) 973.46, Chemical Oxygen Demand (COD) of Water, Titrimetric Methods. Table IB, Note 3.

(I) 973.47, Organic Carbon in Water, Infrared Analyzer Method. Table IB, Note 3.

(J) 973.48, Nitrogen (Total) in Water, Kjeldahl Method. Table IB, Note 3.

(K) 973.49, Nitrogen (Ammonia) in Water, Colorimetric Method. Table IB, Note 3.

(L) 973.50, Nitrogen (Nitrate) in Water, Brucine Colorimetric Method. Table IB, Note 3.

(M) 973.51, Chloride in Water, Mercuric Nitrate Method. Table IB, Note 3.

(N) 973.52, Hardness of Water. Table IB, Note 3.

(O) 973.53, Potassium in Water, Atomic Absorption Spectrophotometric Method. Table IB, Note 3.

(P) 973.54, Sodium in Water, Atomic Absorption Spectrophotometric Method. Table IB, Note 3.

(Q) 973.55, Phosphorus in Water, Photometric Method. Table IB, Note 3.

(R) 973.56, Phosphorus in Water, Automated Method. Table IB, Note 3.

(S) 974.27, Cadmium, Chromium, Copper, Iron, Lead, Magnesium, Manganese, Silver, Zinc in Water, Atomic Absorption Spectrophotometric Method. Table IB, Note 3.

(T) 977.22, Mercury in Water, Flameless Atomic Absorption Spectrophotometric Method. Table IB, Note 3.

(U) 991.15. Total Coliforms and Escherichia coli in Water Defined Substrate Technology (Colilert) Method. Table IA, Note 10; Table IH, Note 10.

(V) 993.14, Trace Elements in Waters and Wastewaters, Inductively Coupled Plasma-Mass Spectrometric Method. Table IB, Note 3.

(W) 993.23, Dissolved Hexavalent Chromium in Drinking Water, Ground Water, and Industrial Wastewater Effluents, Ion Chromatographic Method. Table IB, Note 3.

(X) 993.30, Inorganic Anions in Water, Ion Chromatographic Method. Table IB, Note 3.

(ii) [Reserved]

(14) Applied and Environmental Microbiology, American Society for Microbiology, 1752 N Street NW., Washington DC 20036. (Also available from most public libraries.)

(i) New Medium for the Simultaneous Detection of Total Coliforms and Escherichia coli in Water. Brenner, K.P., C.C. Rankin, Y.R. Roybal, G.N. Stelma, Jr., P.V. Scarpino, and A.P. Dufour. 59:3534–3544, November 1993. Table IH, Note 21.

(ii) [Reserved]

(15) ASTM International, 100 Barr Harbor Drive, P.O. Box C700, West Conshohocken, PA 19428–2959, or online at http://www.astm.org.

(i) Annual Book of ASTM Standards, Water, and Environmental Technology, Section 11, Volumes 11.01 and 11.02. 1994. Tables IA, IB, IC, ID, IE, and IH.

(ii) Annual Book of ASTM Standards, Water, and Environmental Technology, Section 11, Volumes 11.01 and 11.02. 1996. Tables IA, IB, IC, ID, IE, and IH.

(iii) Annual Book of ASTM Standards, Water, and Environmental Technology, Section 11, Volumes 11.01 and 11.02. 1999. Tables IA, IB, IC, ID, IE, and IH.

(iv) Annual Book of ASTM Standards, Water, and Environmental Technology, Section 11, Volumes 11.01 and 11.02. 2000. Tables IA, IB, IC, ID, IE, and IH.

(v) ASTM D511–14, Standard Test Methods for Calcium and Magnesium in Water. November 2014. Table IB.

(vi) ASTM D512–12, Standard Test Methods for Chloride Ion in Water. July 2012. Table IB.

(vii) ASTM D515–88, Test Methods for Phosphorus in Water, March 1989. Table IB.

(viii) ASTM D516–16, Standard Test Method for Sulfate Ion in Water, June 2016. Table IB.

(ix) ASTM D858–17, Standard Test Methods for Manganese in Water. June 2017. Table IB.

(x) ASTM D859–16, Standard Test Method for Silica in Water. June 2016. Table IB.

(xi) ASTM D888–12, Standard Test Methods for Dissolved Oxygen in Water. March 2012. Table IB.

(xii) ASTM D1067–16, Standard Test Methods for Acidity or Alkalinity of Water. June 2016. Table IB.

(xiii) ASTM D1068–15, Standard Test Methods for Iron in Water. October 2015. Table IB.

(xiv) ASTM D1125–95 (Reapproved 1999), Standard Test Methods for Electrical Conductivity and Resistivity of Water. December 1995. Table IB.

(xv) ASTM D1126–17, Standard Test Method for Hardness in Water. December 2017. Table IB.

(xvi) ASTM D1179–16, Standard Test Methods for Fluoride Ion in Water. June 2016. Table IB.

(xvii) ASTM D1246–16, Standard Test Method for Bromide Ion in Water. June 2016. Table IB.

(xviii) ASTM D1252–06 (Reapproved 2012), Standard Test Methods for Chemical Oxygen Demand (Dichromate Oxygen Demand) of Water. June 2012. Table IB.

(xix) ASTM D1253–14, Standard Test Method for Residual Chlorine in Water. February 2014. Table IB.

(xx) ASTM D1293–99, Standard Test Methods for pH of Water. March 2000. Table IB.

(xxi) ASTM D1426–15, Standard Test Methods for Ammonia Nitrogen in Water. April 2015. Table IB.

(xxii) ASTM D1687–17, Standard Test Methods for Chromium in Water. July 2017. Table IB.

(xxiii) ASTM D1688–17, Standard Test Methods for Copper in Water. July 2017. Table IB.

(xxiv) ASTM D1691–17, Standard Test Methods for Zinc in Water. June 2017. Table IB.

(xxv) ASTM D1783–01 (Reapproved 2012), Standard Test Methods for Phenolic Compounds in Water. August 2012. Table IB.

(xxvi) ASTM D1886–14, Standard Test Methods for Nickel in Water. November 2014. Table IB.

(xxvii) ASTM D1889–00, Standard Test Method for Turbidity of Water. October 2000. Table IB.

(xxviii) ASTM D1890–96, Standard Test Method for Beta Particle Radioactivity of Water. April 1996. Table IE.

(xxix) ASTM D1943–96, Standard Test Method for Alpha Particle Radioactivity of Water. April 1996. Table IE.

(xxx) ASTM D1976–12, Standard Test Method for Elements in Water by Inductively-Coupled Argon Plasma Atomic Emission Spectroscopy. March 2012. Table IB.

(xxxi) ASTM D2036–09 (Reapproved 2015), Standard Test Methods for Cyanides in Water. July 2015. Table IB.

(xxxii) ASTM D2330–02, Standard Test Method for Methylene Blue Active Substances. August 2002. Table IB.

(xxxiii) ASTM D2460–97, Standard Test Method for Alpha-Particle-Emitting Isotopes of Radium in Water. October 1997. Table IE.

(xxxiv) ASTM D2972–15, Standard Tests Method for Arsenic in Water. March 2015. Table IB.

(xxxv) ASTM D3223–17, Standard Test Method for Total Mercury in Water. June 2017. Table IB.

(xxxvi) ASTM D3371–95, Standard Test Method for Nitriles in Aqueous Solution by Gas-Liquid Chromatography, February 1996. Table IF.

(xxxvii) ASTM D3373–17, Standard Test Method for Vanadium in Water. June 2017. Table IB.

(xxxviii) ASTM D3454–97, Standard Test Method for Radium-226 in Water. February 1998. Table IE.

(xxxix) ASTM D3557–17, Standard Test Method for Cadmium in Water. June 2017. Table IB.

(xl) ASTM D3558–15, Standard Test Method for Cobalt in Water. March 2015. Table IB.

(xli) ASTM D3559–15, Standard Test Methods for Lead in Water. October 2015. Table IB.

(xlii) ASTM D3590–17, Standard Test Methods for Total Kjeldahl Nitrogen in Water. June 2017. Table IB.

(xliii) ASTM D3645–15, Standard Test Methods for Beryllium in Water. March 2015. Table IB.

(xliv) ASTM D3695–95, Standard Test Method for Volatile Alcohols in Water by Direct Aqueous-Injection Gas Chromatography. April 1995. Table IF.

(xlv) ASTM D3859–15, Standard Test Methods for Selenium in Water. April 2015. Table IB.

(xlvi) ASTM D3867–16, Standard Test Method for Nitrite-Nitrate in Water. June 2016. Table IB.

(xlvii) ASTM D4190–15, Standard Test Method for Elements in Water by Direct- Current Plasma Atomic Emission Spectroscopy. March 2015. Table IB.

(xlviii) ASTM D4282–15, Standard Test Method for Determination of Free Cyanide in Water and Wastewater by Microdiffusion. July 2015. Table IB.

(xlix) ASTM D4327–17, Standard Test Method for Anions in Water by Suppressed Ion Chromatography. December 2017. Table IB.

(l) ASTM D4382–18, Standard Test Method for Barium in Water, Atomic Absorption Spectrophotometry, Graphite Furnace. May 2018. Table IB.

(li) ASTM D4657–92 (Reapproved 1998), Standard Test Method for Polynuclear Aromatic Hydrocarbons in Water. January 1993. Table IC.

(lii) ASTM D4658–15, Standard Test Method for Sulfide Ion in Water. April 2015. Table IB.

(liii) ASTM D4763–88 (Reapproved 2001), Standard Practice for Identification of Chemicals in Water by Fluorescence Spectroscopy. September 1988. Table IF.

(liv) ASTM D4839–03 (Reapproved 2017), Standard Test Method for Total Carbon and Organic Carbon in Water by Ultraviolet, or Persulfate Oxidation, or Both, and Infrared Detection. December 2017. Table IB.

(lv) ASTM D5257–17, Standard Test Method for Dissolved Hexavalent Chromium in Water by Ion Chromatography. December 2017. Table IB.

(lvi) ASTM D5259–92, Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure. October 1992. Table IH, Note 9.

(lvii) ASTM D5392–93, Standard Test Method for Isolation and Enumeration of Escherichia coli in Water by the Two-Step Membrane Filter Procedure. September 1993. Table IH, Note 9.

(lviii) ASTM D5673–16, Standard Test Method for Elements in Water by Inductively Coupled Plasma—Mass Spectrometry. February 2016. Table IB.

(lix) ASTM D5(19)907–13, Standard Test Method for Filterable Matter (Total Dissolved Solids) and Nonfilterable Matter (Total Suspended Solids) in Water. July 2013. Table IB.

(lx) ASTM D6503–99, Standard Test Method for Enterococci in Water Using Enterolert. April 2000. Table IA Note 9, Table IH, Note 9.

(lxi) ASTM. D6508–15, Standard Test Method for Determination of Dissolved Inorganic Anions in Aqueous Matrices Using Capillary Ion Electrophoresis and Chromate Electrolyte. October 2015. Table IB, Note 54.

(lxii) ASTM. D6888–16, Standard Test Method for Available Cyanide with Ligand Displacement and Flow Injection Analysis (FIA) Utilizing Gas Diffusion Separation and Amperometric Detection. June 2016. Table IB, Note 59.

(lxiii) ASTM. D6919–17, Standard Test Method for Determination of Dissolved Alkali and Alkaline Earth Cations and Ammonium in Water and Wastewater by Ion Chromatography. June 2017. Table IB.

(lxiv) ASTM. D7065–17, Standard Test Method for Determination of Nonylphenol, Bisphenol A, p-tert-Octylphenol, Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate in Environmental Waters by Gas Chromatography Mass Spectrometry. January 2018. Table IC.

(lxv) ASTM. D7237–15a, Standard Test Method for Free Cyanide with Flow Injection Analysis (FIA) Utilizing Gas Diffusion Separation and Amperometric Detection. June 2015. Table IB.

(lxvi) ASTM. D7284–13 (Reapproved 2017), Standard Test Method for Total Cyanide in Water by Micro Distillation followed by Flow Injection Analysis with Gas Diffusion Separation and Amperometric Detection. July 2017. Table IB.

(lxvii) ASTM. D7365–09a, Standard Practice for Sampling, Preservation, and Mitigating Interferences in Water Samples for Analysis of Cyanide. October 2009. Table II, Notes 5 and 6.

(lxviii) ASTM. D7511–12 (Reapproved 2017), Standard Test Method for Total Cyanide by Segmented Flow Injection Analysis, In-Line Ultraviolet Digestion and Amperometric Detection. July 2017. Table IB.

(lxix) ASTM. D7573–09 (Reapproved 2017), Standard Test Method for Total Carbon and Organic Carbon in Water by High Temperature Catalytic Combustion and Infrared Detection, February 2017. Table IB.

(lxx) ASTM D7781–14 Standard Test Method for Nitrate-Nitrite in Water by Nitrate Reductase, May 2014. Table IB.

(16) Bran & Luebbe Analyzing Technologies, Inc., Elmsford NY 10523.

(i) Industrial Method Number 378–75WA, Hydrogen Ion (pH) Automated Electrode Method, Bran & Luebbe (Technicon) Auto Analyzer II. October 1976. Table IB, Note 21.

(ii) [Reserved]

(17) CEM Corporation, P.O. Box 200, Matthews NC 28106–0200.

(i) Closed Vessel Microwave Digestion of Wastewater Samples for Determination of Metals. April 16, 1992. Table IB, Note 36.

(ii) [Reserved]

(18) Craig R. Chinchilla, 900 Jorie Blvd., Suite 35, Oak Brook IL 60523. Telephone: 630–645–0600.

(i) Nitrate by Discrete Analysis Easy (1-Reagent) Nitrate Method, (Colorimetric, Automated, 1 Reagent). Revision 1, November 12, 2011. Table IB, Note 62.

(ii) [Reserved]

(19) FIAlab Instruments, Inc., 2151 N. Northlake Way, Seattle, WA 98103. Telephone: 425–376–0450.

(i) Method 100, Determination of Inorganic Ammonia by Continuous Flow Gas Diffusion and Fluorescence Detector Analysis, April 4, 2018. Table IB, Note 82.

(ii) [Reserved]

(20) Hach Company, P.O. Box 389, Loveland CO 80537.

(i) Method 8000, Chemical Oxygen Demand. Hach Handbook of Water Analysis. 1979. Table IB, Note 14.

(ii) Method 8008, 1,10-Phenanthroline Method using FerroVer Iron Reagent for Water. 1980. Table IB, Note 22.

(iii) Method 8009, Zincon Method for Zinc. Hach Handbook for Water Analysis. 1979. Table IB, Note 33.

(iv) Method 8034, Periodate Oxidation Method for Manganese. Hach Handbook for Water Analysis. 1979. Table IB, Note 23.

(v) Method 8506, Bicinchoninate Method for Copper. Hach Handbook of Water Analysis. 1979. Table IB, Note 19.

(vi) Method 8507, Nitrogen, Nitrite—Low Range, Diazotization Method for Water and Wastewater. 1979. Table IB, Note 25.

(vii) Method 10206, Hach Company TNTplus 835/836 Nitrate Method 10206, Spectrophotometric Measurement of Nitrate in Water and Wastewater. Revision 2.1, January 10, 2013. Table IB, Note 75.

(viii) Method 10242, Hach Company TNTplus 880 Total Kjeldahl Nitrogen Method 10242, Simplified Spectrophotometric Measurement of Total Kjeldahl Nitrogen in Water and Wastewater. Revision 1.1, January 10, 2013. Table IB, Note 76.

(ix) Hach Method 10360, Luminescence Measurement of Dissolved Oxygen in Water and Wastewater and for Use in the Determination of BOD5 and cBOD5. Revision 1.2, October 2011. Table IB, Note 63.

(x) m-ColiBlue24® Method, for total Coliforms and E. coli. Revision 2, 1999. Table IA, Note 18; Table IH, Note 17.

(21) IDEXX Laboratories Inc., One Idexx Drive, Westbrook ME 04092.

(i) Colilert. 2013. Table IA, Notes 17 and 18; Table IH, Notes 14, 15 and 16.

(ii) Colilert-18. 2013. Table IA, Notes 17 and 18; Table IH, Notes 14, 15 and 16.

(iii) Enterolert. 2013. Table IA, Note 24; Table IH, Note 12.

(iv) Quanti-Tray Insert and Most Probable Number (MPN) Table. 2013. Table IA, Note 18; Table IH, Notes 14 and 16.

(22) In-Situ Incorporated, 221 E. Lincoln Ave., Ft. Collins CO 80524. Telephone: 970–498–1500.

(i) In-Situ Inc. Method 1002–8–2009, Dissolved Oxygen Measurement by Optical Probe. 2009. Table IB, Note 64.

(ii) In-Situ Inc. Method 1003–8–2009, Biochemical Oxygen Demand (BOD) Measurement by Optical Probe. 2009. Table IB, Note 10.

(iii) In-Situ Inc. Method 1004–8–2009, Carbonaceous Biochemical Oxygen Demand (CBOD) Measurement by Optical Probe. 2009. Table IB, Note 35.

(23) Journal of Chromatography, Elsevier/North-Holland, Inc., Journal Information Centre, 52 Vanderbilt Avenue, New York NY 10164. (Also available from most public libraries.

(i) Direct Determination of Elemental Phosphorus by Gas-Liquid Chromatography. Addison, R.F. and R.G. Ackman. 47(3): 421–426, 1970. Table IB, Note 28.

(ii) [Reserved]

(24) Lachat Instruments, 6645 W. Mill Road, Milwaukee WI 53218, Telephone: 414–358–4200.

(i) QuikChem Method 10–204–00–1–X, Digestion and Distillation of Total Cyanide in Drinking and Wastewaters using MICRO DIST and Determination of Cyanide by Flow Injection Analysis. Revision 2.2, March 2005. Table IB, Note 56.

(ii) [Reserved]

(25) Leck Mitchell, Ph.D., P.E., 656 Independence Valley Dr., Grand Junction CO 81507. Telephone: 970–244–8661.

(i) Mitchell Method M5271, Determination of Turbidity by Nephelometry. Revision 1.0, July 31, 2008. Table IB, Note 66.

(ii) Mitchell Method M5331, Determination of Turbidity by Nephelometry. Revision 1.0, July 31, 2008. Table IB, Note 65.

(26) MACHEREY-NAGEL GmbH and Co., 2850 Emrick Blvd. Bethlehem, PA 18020. Telephone: 888–321–6224.

(i) Method 036/038 NANOCOLOR® COD LR/HR, Spectrophotometric Measurement of Chemical Oxygen Demand in Water and Wastewater, Revision 1.5, May 2018. Table IB, Note 83.

(ii) [Reserved]

(27) Micrology Laboratories, LLC, 1303 Eisenhower Drive, Goshen, IN 46526. Telephone: 574–533–3351.

(i) KwikCountTM EC Medium E. coli enzyme substrate test, Rapid Detection of E. coli in Beach Water By KwikCountTM EC Membrane Filtration. 2014. Table IH, Notes 28 and 29.

(ii) [Reserved]

(28) National Council of the Paper Industry for Air and Stream Improvements, Inc. (NCASI), 260 Madison Avenue, New York NY 10016.

(i) NCASI Method TNTP–W10900, Total Nitrogen and Total Phophorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion. June 2011. Table IB, Note 77.

(ii) NCASI Technical Bulletin No. 253, An Investigation of Improved Procedures for Measurement of Mill Effluent and Receiving Water Color. December 1971. Table IB, Note 18.

(iii) NCASI Technical Bulletin No. 803, An Update of Procedures for the Measurement of Color in Pulp Mill Wastewaters. May 2000. Table IB, Note 18.

(29) The Nitrate Elimination Co., Inc. (NECi), 334 Hecla St., Lake Linden NI 49945.

(i) NECi Method N07–0003, Method for Nitrate Reductase Nitrate-Nitrogen Analysis. Revision 9.0. March 2014. Table IB, Note 73.

(ii) [Reserved]

(30) Oceanography International Corporation, 512 West Loop, P.O. Box 2980, College Station TX 77840.

(i) OIC Chemical Oxygen Demand Method. 1978. Table IB, Note 13.

(ii) [Reserved]

(31) OI Analytical, Box 9010, College Station TX 77820–9010.

(i) Method OIA–1677–09, Available Cyanide by Ligand Exchange and Flow Injection Analysis (FIA). Copyright 2010. Table IB, Note 59.

(ii) Method PAI–DK01, Nitrogen, Total Kjeldahl, Block Digestion, Steam Distillation, Titrimetric Detection. Revised December 22, 1994. Table IB, Note 39.

(iii) Method PAI–DK02, Nitrogen, Total Kjeldahl, Block Digestion, Steam Distillation, Colorimetric Detection. Revised December 22, 1994. Table IB, Note 40.

(iv) Method PAI–DK03, Nitrogen, Total Kjeldahl, Block Digestion, Automated FIA Gas Diffusion. Revised December 22, 1994. Table IB, Note 41.

(32) ORION Research Corporation, 840 Memorial Drive, Cambridge, Massachusetts 02138.

(i) ORION Research Instruction Manual, Residual Chlorine Electrode Model 97–70. 1977. Table IB, Note 16.

(ii) [Reserved]

(33) Technicon Industrial Systems, Tarrytown NY 10591.

(i) Industrial Method Number 379–75WE Ammonia, Automated Electrode Method, Technicon Auto Analyzer II. February 19, 1976. Table IB, Note 7.

(ii) [Reserved]

(34) Thermo Jarrell Ash Corporation, 27 Forge Parkway, Franklin MA 02038.

(i) Method AES0029. Direct Current Plasma (DCP) Optical Emission Spectrometric Method for Trace Elemental Analysis of Water and Wastes. 1986, Revised 1991. Table IB, Note 34.

(ii) [Reserved]

(35) Thermo Scientific, 166 Cummings Center, Beverly MA 01915. Telephone: 1–800–225–1480. www.thermoscientific.com.

(i) Thermo Scientific Orion Method AQ4500, Determination of Turbidity by Nephelometry. Revision 5, March 12, 2009. Table IB, Note 67.

(ii) [Reserved]

(36) 3M Corporation, 3M Center Building 220–9E–10, St. Paul MN 55144–1000.

(i) Organochlorine Pesticides and PCBs in Wastewater Using EmporeTM Disk” Test Method 3M 0222. Revised October 28, 1994. Table IC, Note 8; Table ID, Note 8.

(ii) [Reserved]

(37) Timberline Instruments, LLC, 1880 South Flatiron Ct., Unit I, Boulder CO 80301.

(i) Timberline Amonia-001, Determination of Inorganic Ammonia by Continuous Flow Gas Diffusion and Conductivity Cell Analysis. June 24, 2011. Table IB, Note 74.

(ii) [Reserved]

(38) U.S. Geological Survey (USGS), U.S. Department of the Interior, Reston, Virginia. Available from USGS Books and Open-File Reports (OFR) Section, Federal Center, Box 25425, Denver, CO 80225.

(i) Colorimetric determination of nitrate plus nitrite in water by enzymatic reduction, automated discrete analyzer methods. U.S. Geological Survey Techniques and Methods, Book 5—Laboratory Analysis, Section B—Methods of the National Water Quality Laboratory, Chapter 8. 2011. Table IB, Note 72.

(ii) Determination of Heat Purgeable and Ambient Purgeable Volatile Organic Compounds in Water by Gas Chromatography/Mass Spectrometry. Chapter 12 of Section B, Methods of the National Water Quality Laboratory, of Book 5, Laboratory Analysis. 2016.

(iii) Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, editors, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A1. 1979. Table IB, Note 8.

(iv) Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A1. 1989. Table IB, Notes 2 and 79.

(v) Methods for the Determination of Organic Substances in Water and Fluvial Sediments. Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A3. 1987. Table IB, Note 24; Table ID, Note 4.

(vi) OFR 76–177, Selected Methods of the U.S. Geological Survey of Analysis of Wastewaters. 1976. Table IE, Note 2.

(vii) OFR 91–519, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Organonitrogen Herbicides in Water by Solid-Phase Extraction and Capillary-Column Gas Chromatography/Mass Spectrometry With Selected-Ion Monitoring. 1992. Table ID, Note 14.

(viii) OFR 92–146, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Total Phosphorus by a Kjeldahl Digestion Method and an Automated Colorimetric Finish That Includes Dialysis. 1992. Table IB, Note 48.

(ix) OFR 93–125, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Inorganic and Organic Constituents in Water and Fluvial Sediments. 1993. Table IB, Note 51 and 80; Table IC, Note 9.

(x) OFR 93–449, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Chromium in Water by Graphite Furnace Atomic Absorption Spectrophotometry. 1993. Table IB, Note 46.

(xi) OFR 94–37, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Triazine and Other Nitrogen-containing Compounds by Gas Chromatography with Nitrogen Phosphorus Detectors. 1994. Table ID, Note 9.

(xii) OFR 95–181, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Pesticides in Water by C–18 Solid-Phase Extraction and Capillary-Column Gas Chromatography/Mass Spectrometry With Selected-Ion Monitoring. 1995. Table ID, Note 11.

(xiii) OFR 97–198, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Molybdenum in Water by Graphite Furnace Atomic Absorption Spectrophotometry. 1997. Table IB, Note 47.

(xiv) OFR 97–829, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory— Determination of 86 Volatile Organic Compounds in Water by Gas Chromatography/Mass Spectrometry, Including Detections Less Than Reporting Limits. 1999. Table IC, Note 13.

(xv) OFR 98–165, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Elements in Whole-Water Digests Using Inductively Coupled Plasma-Optical Emission Spectrometry and Inductively Coupled Plasma-Mass Spectrometry. 1998. Table IB, Notes 50 and 81.

(xvi) OFR 98–639, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Arsenic and Selenium in Water and Sediment by Graphite Furnace—Atomic Absorption Spectrometry. 1999. Table IB, Note 49.

(xvii) OFR 00–170, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Ammonium Plus Organic Nitrogen by a Kjeldahl Digestion Method and an Automated Photometric Finish that Includes Digest Cleanup by Gas Diffusion. 2000. Table IB, Note 45.

(xviii) Techniques and Methods Book 5–B1, Determination of Elements in Natural-Water, Biota, Sediment and Soil Samples Using Collision/Reaction Cell Inductively Coupled Plasma-Mass Spectrometry. Chapter 1, Section B, Methods of the National Water Quality Laboratory, Book 5, Laboratory Analysis. 2006. Table IB, Note 70.

(xix) U.S. Geological Survey Techniques of Water-Resources Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples. 1989. Table IA, Note 4; Table IH, Note 4.

(xx) Water-Resources Investigation Report 01–4098, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Moderate-Use Pesticides and Selected Degradates in Water by C–18 Solid-Phase Extraction and Gas Chromatography/Mass Spectrometry. 2001. Table ID, Note 13.

(xxi) Water-Resources Investigations Report 01–4132, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Organic Plus Inorganic Mercury in Filtered and Unfiltered Natural Water With Cold Vapor-Atomic Fluorescence Spectrometry. 2001. Table IB, Note 71.

(xxii) Water-Resources Investigation Report 01–4134, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Pesticides in Water by Graphitized Carbon-Based Solid-Phase Extraction and High-Performance Liquid Chromatography/Mass Spectrometry. 2001. Table ID, Note 12.

(xxiii) Water Temperature—Influential Factors, Field Measurement and Data Presentation, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 1, Chapter D1. 1975. Table IB, Note 32.

(39) Waters Corporation, 34 Maple Street, Milford MA 01757, Telephone: 508–482–2131, Fax: 508–482–3625.

(i) Method D6508, Test Method for Determination of Dissolved Inorganic Anions in Aqueous Matrices Using Capillary Ion Electrophoresis and Chromate Electrolyte. Revision 2, December 2000. Table IB, Note 54.

(ii) [Reserved]

(c) Under certain circumstances, the Director may establish limitations on the discharge of a parameter for which there is no test procedure in this part or in 40 CFR parts 405 through 499. In these instances the test procedure shall be specified by the Director.

(d) Under certain circumstances, the Administrator may approve additional alternate test procedures for nationwide use, upon recommendation by the Alternate Test Procedure Program Coordinator, Washington, DC.

(e) Sample preservation procedures, container materials, and maximum allowable holding times for parameters are cited in Tables IA, IB, IC, ID, IE, IF, IG, and IH are prescribed in Table II. Information in the table takes precedence over information in specific methods or elsewhere. Any person may apply for a change from the prescribed preservation techniques, container materials, and maximum holding times applicable to samples taken from a specific discharge. Applications for such limited use changes may be made by letters to the Regional Alternative Test Procedure (ATP) Program Coordinator or the permitting authority in the Region in which the discharge will occur. Sufficient data should be provided to assure such changes in sample preservation, containers or holding times do not adversely affect the integrity of the sample. The Regional ATP Coordinator or permitting authority will review the application and then notify the applicant and the appropriate State agency of approval or rejection of the use of the alternate test procedure. A decision to approve or deny any request on deviations from the prescribed Table II requirements will be made within 90 days of receipt of the application by the Regional Administrator. An analyst may not modify any sample preservation and/or holding time requirements of an approved method unless the requirements of this section are met.

Table II—Required Containers, Preservation Techniques, and Holding Times

Parameter number/name Container 1 Preservation 2 3 Maximum holding time 4
Table IA—Bacterial Tests
1–4. Coliform, total, fecal, and E. coli PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22 23
5. Fecal streptococci PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
6. Enterococci PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
7. Salmonella PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
Table IA—Aquatic Toxicity Tests
8–11. Toxicity, acute and chronic P, FP, G Cool, ≤6 °C 16 36 hours.
Table IB—Inorganic Tests
1. Acidity P, FP, G Cool, ≤6 °C 18 14 days.
2. Alkalinity P, FP, G Cool, ≤6 °C 18 14 days.
4. Ammonia P, FP, G Cool, ≤6 °C 18, H2SO4 to pH <2 28 days.
9. Biochemical oxygen demand P, FP, G Cool, ≤6 °C 18 48 hours.
10. Boron P, FP, or Quartz HNO3 to pH <2 6 months.
11. Bromide P, FP, G None required 28 days.
14. Biochemical oxygen demand, carbonaceous P, FP G Cool, ≤6 °C 18 48 hours.
15. Chemical oxygen demand P, FP, G Cool, ≤6 °C 18, H2SO4 to pH <2 28 days.
16. Chloride P, FP, G None required 28 days.
17. Chlorine, total residual P, G None required Analyze within 15 minutes.
21. Color P, FP, G Cool, ≤6 °C 18 48 hours.
23–24. Cyanide, total or available (or CATC) and free P, FP, G Cool, ≤6 °C 18, NaOH to pH >10 5 6, reducing agent if oxidizer present 14 days.
25. Fluoride P None required 28 days.
27. Hardness P, FP, G HNO3 or H2SO4 to pH <2 6 months.
28. Hydrogen ion (pH) P, FP, G None required Analyze within 15 minutes.
31, 43. Kjeldahl and organic N P, FP, G Cool, ≤6 °C 18, H2SO4 to pH <2 28 days.
Table IB—Metals 7
18. Chromium VI P, FP, G Cool, ≤6 °C 18, pH = 9.3–9.7 20 28 days.
35. Mercury (CVAA) P, FP, G HNO3 to pH <2 28 days.
35. Mercury (CVAFS) FP, G; and FP-lined cap 17 5 mL/L 12N HCl or 5 mL/L BrCl 17 90 days.17
3, 5–8, 12, 13, 19, 20, 22, 26, 29, 30, 32–34, 36, 37, 45, 47, 51, 52, 58–60, 62, 63, 70–72, 74, 75. Metals, except boron, chromium VI, and mercury P, FP, G HNO3 to pH <2, or at least 24 hours prior to analysis 19 6 months.
38. Nitrate P, FP, G Cool, ≤6 °C 18 48 hours.
39. Nitrate-nitrite P, FP, G Cool, ≤6 °C 18, H2SO4 to pH <2 28 days.
40. Nitrite P, FP, G Cool, ≤6 °C 18 48 hours.
41. Oil and grease G Cool to ≤6 °C 18, HCl or H2SO4 to pH <2 28 days.
42. Organic Carbon P, FP, G Cool to ≤6 °C 18, HCl, H2SO4, or H3PO4 to pH <2 28 days.
44. Orthophosphate P, FP, G Cool, to ≤6 °C 18 24 Filter within 15 minutes; Analyze within 48 hours.
46. Oxygen, Dissolved Probe G, Bottle and top None required Analyze within 15 minutes.
47. Winkler G, Bottle and top Fix on site and store in dark 8 hours.
48. Phenols G Cool, ≤6 °C 18, H2SO4 to pH <2 28 days.
49. Phosphorus (elemental) G Cool, ≤6 °C 18 48 hours.
50. Phosphorus, total P, FP, G Cool, ≤6 °C 18, H2SO4 to pH <2 28 days.
53. Residue, total P, FP, G Cool, ≤6 °C 18 7 days.
54. Residue, Filterable (TDS) P, FP, G Cool, ≤6 °C 18 7 days.
55. Residue, Nonfilterable (TSS) P, FP, G Cool, ≤6 °C 18 7 days.
56. Residue, Settleable P, FP, G Cool, ≤6 °C 18 48 hours.
57. Residue, Volatile P, FP, G Cool, ≤6 °C 18 7 days.
61. Silica P or Quartz Cool, ≤6 °C 18 28 days.
64. Specific conductance P, FP, G Cool, ≤6 °C 18 28 days.
65. Sulfate P, FP, G Cool, ≤6 °C 18 28 days.
66. Sulfide P, FP, G Cool, ≤6 °C 18, add zinc acetate plus sodium hydroxide to pH >9 7 days.
67. Sulfite P, FP, G None required Analyze within 15 minutes.
68. Surfactants P, FP, G Cool, ≤6 °C 18 48 hours.
69. Temperature P, FP, G None required Analyze within 15 minutes.
73. Turbidity P, FP, G Cool, ≤6 °C 18 48 hours.
Table IC—Organic Tests 8
13, 18–20, 22, 24, 25, 27, 28, 34–37, 39–43, 45–47, 56, 76, 104, 105, 108–111, 113. Purgeable Halocarbons G, FP-lined septum Cool, ≤6 °C 18, 0.008% Na2S2O3 5, HCl to pH 2 9 14 days.9
26. 2-Chloroethylvinyl ether G, FP-lined septum Cool, ≤6 °C 18, 0.008% Na2S2O3 5 14 days.
6, 57, 106. Purgeable aromatic hydrocarbons G, FP-lined septum Cool, ≤6 °C 18, 0.008% Na2S2O3 5, HCl to pH 2 9 14 days.9
3, 4. Acrolein and acrylonitrile G, FP-lined septum Cool, ≤6 °C 18, 0.008% Na2S2O3, pH to 4–5 10 14 days.10
23, 30, 44, 49, 53, 77, 80, 81, 98, 100, 112. Phenols 11 G, FP-lined cap Cool, ≤6 °C 18, 0.008% Na2S2O3 7 days until extraction, 40 days after extraction.
7, 38. Benzidines 11 12 G, FP-lined cap Cool, ≤6 °C 18, 0.008% Na2S2O3 5 7 days until extraction.13
14, 17, 48, 50–52. Phthalate esters 11 G, FP-lined cap Cool, ≤6 °C 18 7 days until extraction, 40 days after extraction.
82–84. Nitrosamines 11 14 G, FP-lined cap Cool, ≤6 °C 18, store in dark, 0.008% Na2S2O3 5 7 days until extraction, 40 days after extraction.
88–94. PCBs 11 G, FP-lined cap Cool, ≤6 °C 18 1 year until extraction, 1 year after extraction.
54, 55, 75, 79. Nitroaromatics and isophorone 11 G, FP-lined cap Cool, ≤6 °C 18, store in dark, 0.008% Na2S2O3 5 7 days until extraction, 40 days after extraction.
1, 2, 5, 8–12, 32, 33, 58, 59, 74, 78, 99, 101. Polynuclear aromatic hydrocarbons 11 G, FP-lined cap Cool, ≤6 °C 18, store in dark, 0.008% Na2S2O3 5 7 days until extraction, 40 days after extraction.
15, 16, 21, 31, 87. Haloethers 11 G, FP-lined cap Cool, ≤6 °C 18, 0.008% Na2S2O3 5 7 days until extraction, 40 days after extraction.
29, 35–37, 63–65, 73, 107. Chlorinated hydrocarbons 11 G, FP-lined cap Cool, ≤6 °C 18 7 days until extraction, 40 days after extraction.
60–62, 66–72, 85, 86, 95–97, 102, 103. CDDs/CDFs 11 G See footnote 11 See footnote 11.
Aqueous Samples: Field and Lab Preservation G Cool, ≤6 °C 18, 0.008% Na2S2O3 5, pH <9 1 year.
Solids and Mixed-Phase Samples: Field Preservation G Cool, ≤6 °C 18 7 days.
Tissue Samples: Field Preservation G Cool, ≤6 °C 18 24 hours.
Solids, Mixed-Phase, and Tissue Samples: Lab Preservation G Freeze, ≤−10 °C 1 year.
114–118. Alkylated phenols G Cool, <6 °C, H2SO4 to pH <2 28 days until extraction, 40 days after extraction.
119. Adsorbable Organic Halides (AOX) G Cool, <6 °C, 0.008% Na2S2O3, HNO3 to pH <2 Hold at least 3 days, but not more than 6 months.
120. Chlorinated Phenolics G, FP-lined cap Cool, <6 °C, 0.008% Na2S2O3, H2SO4 to pH <2 30 days until acetylation, 30 days after acetylation.
Table ID—Pesticides Tests
1–70. Pesticides 11 G, FP-lined cap Cool, ≤6 °C 18, pH 5–9 15 7 days until extraction, 40 days after extraction.
Table IE—Radiological Tests
1–5. Alpha, beta, and radium P, FP, G HNO3 to pH <2 6 months.
Table IH—Bacterial Tests
1, 2. Coliform, total, fecal PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
3.E. coli PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
4. Fecal streptococci PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
5. Enterococci PA, G Cool, <10 °C, 0.008% Na2S2O3 5 8 hours.22
Table IH—Protozoan Tests
6. Cryptosporidium LDPE; field filtration 1–10 °C 96 hours.21
7. Giardia LDPE; field filtration 1–10 °C 96 hours.21

[38 FR 28758, Oct. 16, 1973]

§ 136.4 Application for and approval of alternate test procedures for nationwide use.

(a) A written application for review of an alternate test procedure (alternate method) for nationwide use may be made by letter via email or by hard copy in triplicate to the National Alternate Test Procedure (ATP) Program Coordinator (National Coordinator), Office of Science and Technology (4303T), Office of Water, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave. NW., Washington, DC 20460. Any application for an ATP under this paragraph (a) shall:

(1) Provide the name and address of the responsible person or firm making the application.

(2) Identify the pollutant(s) or parameter(s) for which nationwide approval of an alternate test procedure is being requested.

(3) Provide a detailed description of the proposed alternate test procedure, together with references to published or other studies confirming the general applicability of the alternate test procedure for the analysis of the pollutant(s) or parameter(s) in wastewater discharges from representative and specified industrial or other categories.

(4) Provide comparability data for the performance of the proposed alternative test procedure compared to the performance of the reference method.

(b) The National Coordinator may request additional information and analyses from the applicant in order to evaluate whether the alternate test procedure satisfies the applicable requirements of this part.

(c) Approval for nationwide use.

(1) After a review of the application and any additional analyses requested from the applicant, the National Coordinator will notify the applicant, in writing, of whether the National Coordinator will recommend approval or disapproval of the alternate test procedure for nationwide use in CWA programs. If the application is not recommended for approval, the National Coordinator may specify what additional information might lead to a reconsideration of the application and notify the Regional Alternate Test Procedure Coordinators of the disapproval recommendation. Based on the National Coordinator's recommended disapproval of a proposed alternate test procedure and an assessment of any current approvals for limited uses for the unapproved method, the Regional ATP Coordinator may decide to withdraw approval of the method for limited use in the Region.

(2) Where the National Coordinator has recommended approval of an applicant's request for nationwide use of an alternate test procedure, the National Coordinator will notify the applicant. The National Coordinator will also notify the Regional ATP Coordinators that they may consider approval of this alternate test procedure for limited use in their Regions based on the information and data provided in the application until the alternate test procedure is approved by publication in a final rule in the Federal Register.

(3) EPA will propose to amend this part to include the alternate test procedure in § 136.3. EPA shall make available for review all the factual bases for its proposal, including the method, any performance data submitted by the applicant and any available EPA analysis of those data.

(4) Following public comment, EPA shall publish in the Federal Register a final decision on whether to amend this part to include the alternate test procedure as an approved analytical method for nationwide use.

(5) Whenever the National Coordinator has recommended approval of an applicant's ATP request for nationwide use, any person may request an approval of the method for limited use under § 136.5 from the EPA Region.

[77 FR 29809, May 18, 2012, as amended at 82 FR 40874, Aug. 28, 2017]

§ 136.5 Approval of alternate test procedures for limited use.

(a) Any person may request the Regional ATP Coordinator to approve the use of an alternate test procedure in the Region.

(b) When the request for the use of an alternate test procedure concerns use in a State with an NPDES permit program approved pursuant to section 402 of the Act, the requestor shall first submit an application for limited use to the Director of the State agency having responsibility for issuance of NPDES permits within such State (i.e., permitting authority). The Director will forward the application to the Regional ATP Coordinator with a recommendation for or against approval.

(c) Any application for approval of an alternate test procedure for limited use may be made by letter, email or by hard copy. The application shall include the following:

(1) Provide the name and address of the applicant and the applicable ID number of the existing or pending permit(s) and issuing agency for which use of the alternate test procedure is requested, and the discharge serial number.

(2) Identify the pollutant or parameter for which approval of an alternate test procedure is being requested.

(3) Provide justification for using testing procedures other than those specified in Tables IA through IH of § 136.3, or in the NPDES permit.

(4) Provide a detailed description of the proposed alternate test procedure, together with references to published studies of the applicability of the alternate test procedure to the effluents in question.

(5) Provide comparability data for the performance of the proposed alternate test procedure compared to the performance of the reference method.

(d) Approval for limited use.

(1) The Regional ATP Coordinator will review the application and notify the applicant and the appropriate State agency of approval or rejection of the use of the alternate test procedure. The approval may be restricted to use only with respect to a specific discharge or facility (and its laboratory) or, at the discretion of the Regional ATP Coordinator, to all dischargers or facilities (and their associated laboratories) specified in the approval for the Region. If the application is not approved, the Regional ATP Coordinator shall specify what additional information might lead to a reconsideration of the application.

(2) The Regional ATP Coordinator will forward a copy of every approval and rejection notification to the National Alternate Test Procedure Coordinator.

[77 FR 29809, May 18, 2012, as amended at 82 FR 40875, Aug. 28, 2017]

§ 136.6 Method modifications and analytical requirements.

(a) Definitions of terms used in this section

(1) Analyst means the person or laboratory using a test procedure (analytical method) in this part.

(2) Chemistry of the method means the reagents and reactions used in a test procedure that allow determination of the analyte(s) of interest in an environmental sample.

(3) Determinative technique means the way in which an analyte is identified and quantified (e.g., colorimetry, mass spectrometry).

(4) Equivalent performance means that the modified method produces results that meet or exceed the QC acceptance criteria of the approved method.

(5) Method-defined analyte means an analyte defined solely by the method used to determine the analyte. Such an analyte may be a physical parameter, a parameter that is not a specific chemical, or a parameter that may be comprised of a number of substances. Examples of such analytes include temperature, oil and grease, total suspended solids, total phenolics, turbidity, chemical oxygen demand, and biochemical oxygen demand.

(6) QC means “quality control.”

(b) Method modifications.

(1) If the underlying chemistry and determinative technique in a modified method are essentially the same as an approved Part 136 method, then the modified method is an equivalent and acceptable alternative to the approved method provided the requirements of this section are met. However, those who develop or use a modification to an approved (Part 136) method must document that the performance of the modified method, in the matrix to which the modified method will be applied, is equivalent to the performance of the approved method. If such a demonstration cannot be made and documented, then the modified method is not an acceptable alternative to the approved method. Supporting documentation must, if applicable, include the routine initial demonstration of capability and ongoing QC including determination of precision and accuracy, detection limits, and matrix spike recoveries. Initial demonstration of capability typically includes analysis of four replicates of a mid-level standard and a method detection limit study. Ongoing quality control typically includes method blanks, mid-level laboratory control samples, and matrix spikes (QC is as specified in the method). The method is considered equivalent if the quality control requirements in the reference method are achieved. Where the laboratory is using a vendor-supplied method, it is the QC criteria in the reference method, not the vendor's method, that must be met to show equivalency. Where a sample preparation step is required (i.e., digestion, distillation), QC tests are to be run using standards treated in the same way as the samples. The method user's Standard Operating Procedure (SOP) must clearly document the modifications made to the reference method. Examples of allowed method modifications are listed in this section. If the method user is uncertain whether a method modification is allowed, the Regional ATP Coordinator or Director should be contacted for approval prior to implementing the modification. The method user should also complete necessary performance checks to verify that acceptable performance is achieved with the method modification prior to analyses of compliance samples.

(2) Requirements. The modified method must meet or exceed performance of the approved method(s) for the analyte(s) of interest, as documented by meeting the initial and ongoing quality control requirements in the method.

(i) Requirements for establishing equivalent performance. If the approved method contains QC tests and QC acceptance criteria, the modified method must use these QC tests and the modified method must meet the QC acceptance criteria with the following conditions:

(A) The analyst may only rely on QC tests and QC acceptance criteria in a method if it includes wastewater matrix QC tests and QC acceptance criteria (e.g., matrix spikes) and both initial (start-up) and ongoing QC tests and QC acceptance criteria.

(B) If the approved method does not contain QC tests and QC acceptance criteria or if the QC tests and QC acceptance criteria in the method do not meet the requirements of this section, then the analyst must employ QC tests published in the “equivalent” of a Part 136 method that has such QC, or the essential QC requirements specified at 136.7, as applicable. If the approved method is from a compendium or VCSB and the QA/QC requirements are published in other parts of that organization's compendium rather than within the Part 136 method then that part of the organization's compendium must be used for the QC tests.

(C) In addition, the analyst must perform ongoing QC tests, including assessment of performance of the modified method on the sample matrix (e.g., analysis of a matrix spike/matrix spike duplicate pair for every twenty samples), and analysis of an ongoing precision and recovery sample (e.g., laboratory fortified blank or blank spike) and a blank with each batch of 20 or fewer samples.

(D) If the performance of the modified method in the wastewater matrix or reagent water does not meet or exceed the QC acceptance criteria, the method modification may not be used.

(ii) Requirements for documentation. The modified method must be documented in a method write-up or an addendum that describes the modification(s) to the approved method prior to the use of the method for compliance purposes. The write-up or addendum must include a reference number (e.g., method number), revision number, and revision date so that it may be referenced accurately. In addition, the organization that uses the modified method must document the results of QC tests and keep these records, along with a copy of the method write-up or addendum, for review by an auditor.

(3) Restrictions. An analyst may not modify an approved Clean Water Act analytical method for a method-defined analyte. In addition, an analyst may not modify an approved method if the modification would result in measurement of a different form or species of an analyte. Changes in method procedures are not allowed if such changes would alter the defined chemistry (i.e., method principle) of the unmodified method. For example, phenol method 420.1 or 420.4 defines phenolics as ferric iron oxidized compounds that react with 4-aminoantipyrine (4-AAP) at pH 10 after being distilled from acid solution. Because total phenolics represents a group of compounds that all react at different efficiencies with 4-AAP, changing test conditions likely would change the behavior of these different phenolic compounds. An analyst may not modify any sample collection, preservation, or holding time requirements of an approved method. Such modifications to sample collection, preservation, and holding time requirements do not fall within the scope of the flexibility allowed at § 136.6. Method flexibility refers to modifications of the analytical procedures used for identification and measurement of the analyte only and does not apply to sample collection, preservation, or holding time procedures, which may only be modified as specified in § 136.3(e).

(4) Allowable changes. Except as noted under paragraph (b)(3) of this section, an analyst may modify an approved test procedure (analytical method) provided that the underlying reactions and principles used in the approved method remain essentially the same, and provided that the requirements of this section are met. If equal or better performance can be obtained with an alternative reagent, then it is allowed. A laboratory wishing to use these modifications must demonstrate acceptable method performance by performing and documenting all applicable initial demonstration of capability and ongoing QC tests and meeting all applicable QC acceptance criteria as described in § 136.7. Some examples of the allowed types of changes, provided the requirements of this section are met include:

(i) Changes between manual method, flow analyzer, and discrete instrumentation.

(ii) Changes in chromatographic columns or temperature programs.

(iii) Changes between automated and manual sample preparation, such as digestions, distillations, and extractions; in-line sample preparation is an acceptable form of automated sample preparation for CWA methods.

(iv) In general, ICP–MS is a sensitive and selective detector for metal analysis; however isobaric interference can cause problems for quantitative determination, as well as identification based on the isotope pattern. Interference reduction technologies, such as collision cells or reaction cells, are designed to reduce the effect of spectroscopic interferences that may bias results for the element of interest. The use of interference reduction technologies is allowed, provided the method performance specifications relevant to ICP–MS measurements are met.

(v) The use of EPA Method 200.2 or the sample preparation steps from EPA Method 1638, including the use of closed-vessel digestion, is allowed for EPA Method 200.8, provided the method performance specifications relevant to the ICP–MS are met.

(vi) Changes in pH adjustment reagents. Changes in compounds used to adjust pH are acceptable as long as they do not produce interference. For example, using a different acid to adjust pH in colorimetric methods.

(vii) Changes in buffer reagents are acceptable provided that the changes do not produce interferences.

(viii) Changes in the order of reagent addition are acceptable provided that the change does not alter the chemistry and does not produce an interference. For example, using the same reagents, but adding them in different order, or preparing them in combined or separate solutions (so they can be added separately), is allowed, provided reagent stability or method performance is equivalent or improved.

(ix) Changes in calibration range (provided that the modified range covers any relevant regulatory limit and the method performance specifications for calibration are met).

(x) Changes in calibration model.

(A) Linear calibration models do not adequately fit calibration data with one or two inflection points. For example, vendor-supplied data acquisition and processing software on some instruments may provide quadratic fitting functions to handle such situations. If the calibration data for a particular analytical method routinely display quadratic character, using quadratic fitting functions may be acceptable. In such cases, the minimum number of calibrators for second order fits should be six, and in no case should concentrations be extrapolated for instrument responses that exceed that of the most concentrated calibrator. Examples of methods with nonlinear calibration functions include chloride by SM4500–Cl–E–1997, hardness by EPA Method 130.1, cyanide by ASTM D6888 or OIA1677, Kjeldahl nitrogen by PAI–DK03, and anions by EPA Method 300.0.

(B) As an alternative to using the average response factor, the quality of the calibration may be evaluated using the Relative Standard Error (RSE). The acceptance criterion for the RSE is the same as the acceptance criterion for Relative Standard Deviation (RSD), in the method. RSE is calculated as:

Where:

x′i = Calculated concentration at level i

xi = Actual concentration of the calibration level i

n = Number of calibration points

p = Number of terms in the fitting equation (average = 1, linear = 2, quadratic = 3)

(C) Using the RSE as a metric has the added advantage of allowing the same numerical standard to be applied to the calibration model, regardless of the form of the model. Thus, if a method states that the RSD should be ≤20% for the traditional linear model through the origin, then the RSE acceptance limit can remain ≤20% as well. Similarly, if a method provides an RSD acceptance limit of ≤15%, then that same figure can be used as the acceptance limit for the RSE. The RSE may be used as an alternative to correlation coefficients and coefficients of determination for evaluating calibration curves for any of the methods at part 136. If the method includes a numerical criterion for the RSD, then the same numerical value is used for the RSE. Some older methods do not include any criterion for the calibration curve—for these methods, if RSE is used the value should be ≤20%. Note that the use of the RSE is included as an alternative to the use of the correlation coefficient as a measure of the suitability of a calibration curve. It is not necessary to evaluate both the RSE and the correlation coefficient.

(xi) Changes in equipment such as equipment from a vendor different from the one specified in the method.

(xii) The use of micro or midi distillation apparatus in place of macro distillation apparatus.

(xiii) The use of prepackaged reagents.

(xiv) The use of digital titrators and methods where the underlying chemistry used for the determination is similar to that used in the approved method.

(xv) Use of selected ion monitoring (SIM) mode for analytes that cannot be effectively analyzed in full-scan mode and reach the required sensitivity. False positives are more of a concern when using SIM analysis, so at a minimum, one quantitation and two qualifying ions must be monitored for each analyte (unless fewer than three ions with intensity greater than 15% of the base peak are available). The ratio of each of the two qualifying ions to the quantitation ion must be evaluated and should agree with the ratio observed in an authentic standard within ±20 percent. Analyst judgment must be applied to the evaluation of ion ratios because the ratios can be affected by co-eluting compounds present in the sample matrix. The signal-to-noise ratio of the least sensitive ion should be at least 3:1. Retention time in the sample should match within 0.05 minute of an authentic standard analyzed under identical conditions. Matrix interferences can cause minor shifts in retention time and may be evident as shifts in the retention times of the internal standards. The total scan time should be such that a minimum of eight scans are obtained per chromatographic peak.

(xvi) Changes are allowed in purge-and-trap sample volumes or operating conditions. Some examples are:

(A) Changes in purge time and purge-gas flow rate. A change in purge time and purge-gas flow rate is allowed provided that sufficient total purge volume is used to achieve the required minimum detectible concentration and calibration range for all compounds. In general, a purge rate in the range 20–200 mL/min and a total purge volume in the range 240–880 mL are recommended.

(B) Use of nitrogen or helium as a purge gas, provided that the required sensitivities for all compounds are met.

(C) Sample temperature during the purge state. Gentle heating of the sample during purging (e.g., 40 °C) increases purging efficiency of hydrophilic compounds and may improve sample-to-sample repeatability because all samples are purged under precisely the same conditions.

(D) Trap sorbent. Any trap design is acceptable, provided that the data acquired meet all QC criteria.

(E) Changes to the desorb time. Shortening the desorb time (e.g., from4 minutes to 1 minute) may not affect compound recoveries, and can shorten overall cycle time and significantly reduce the amount of water introduced to the analytical system, thus improving the precision of analysis, especially for water-soluble analytes. A desorb time of four minutes is recommended, however a shorter desorb time may be used, provided that all QC specifications in the method are met.

(F) Use of water management techniques is allowed. Water is always collected on the trap along with the analytes and is a significant interference for analytical systems (GC and GC/MS). Modern water management techniques (e.g., dry purge or condensation points) can remove moisture from the sample stream and improve analytical performance.

(xvii) If the characteristics of a wastewater matrix prevent efficient recovery of organic pollutants and prevent the method from meeting QC requirements, the analyst may attempt to resolve the issue by adding salts to the sample, provided that such salts do not react with or introduce the target pollutant into the sample (as evidenced by the analysis of method blanks, laboratory control samples, and spiked samples that also contain such salts), and that all requirements of paragraph (b)(2) of this section are met. Samples having residual chlorine or other halogen must be dechlorinated prior to the addition of such salts.

(xviii) If the characteristics of a wastewater matrix result in poor sample dispersion or reagent deposition on equipment and prevent the analyst from meeting QC requirements, the analyst may attempt to resolve the issue by adding a inert surfactant that does not affect the chemistry of the method, such as Brij-35 or sodium dodecyl sulfate (SDS), provided that such surfactant does not react with or introduce the target pollutant into the sample (as evidenced by the analysis of method blanks, laboratory control samples, and spiked samples that also contain such surfactant) and that all requirements of paragraph (b)(1) and (b)(2) of this section are met. Samples having residual chlorine or other halogen must be dechlorinated prior to the addition of such surfactant.

(xix) The use of gas diffusion (using pH change to convert the analyte to gaseous form and/or heat to separate an analyte contained in steam from the sample matrix) across a hydrophobic semi-permeable membrane to separate the analyte of interest from the sample matrix may be used in place of manual or automated distillation in methods for analysis such as ammonia, total cyanide, total Kjeldahl nitrogen, and total phenols. These procedures do not replace the digestion procedures specified in the approved methods and must be used in conjunction with those procedures.

(xx) Changes in equipment operating parameters such as the monitoring wavelength of a colorimeter or the reaction time and temperature as needed to achieve the chemical reactions defined in the unmodified CWA method. For example, molybdenum blue phosphate methods have two absorbance maxima, one at about 660 nm and another at about 880 nm. The former is about 2.5 times less sensitive than the latter. Wavelength choice provides a cost-effective, dilution-free means to increase sensitivity of molybdenum blue phosphate methods.

(xxi) Interchange of oxidants, such as the use of titanium oxide in UV-assisted automated digestion of TOC and total phosphorus, as long as complete oxidation can be demonstrated.

(xxii) Use of an axially viewed torch with Method 200.7.

(xxiii) When analyzing metals by inductively coupled plasma-atomic emission spectroscopy, inductively coupled plasma-mass spectrometry, and stabilized temperature graphite furnace atomic absorption, closed-vessel microwave digestion of wastewater samples is allowed as alternative heating source for Method 200.2—“Sample Preparation Procedure for Spectrochemical Determination of Total Recoverable Elements” for the following elements: Aluminum, antimony, arsenic, barium, beryllium, boron, cadmium, calcium, chromium, cobalt, copper, iron, lead, magnesium, manganese, molybdenum, nickel, potassium, selenium, silver, sodium, thallium, tin, titanium, vanadium, zinc, provided the performance specifications in the relevant determinative method are met. (Note that this list does not include Mercury.) Each laboratory determining total recoverable metals is required to operate a formal quality control (QC) program. The minimum requirements include initial demonstration of capability, method detection limit (MDL), analysis of reagent blanks, fortified blanks, matrix spike samples, and blind proficiency testing samples, as continuing quality control checks on performance. The laboratory is required to maintain performance records on file that define the quality of the data generated.

(c) The permittee must notify their permitting authority of the intent to use a modified method. Such notification should be of the form “Method xxx has been modified within the flexibility allowed in 40 CFR 136.6.” The permittee may indicate the specific paragraph of § 136.6 allowing the method modification. Specific details of the modification need not be provided, but must be documented in the Standard Operating Procedure (SOP) and maintained by the analytical laboratory that performs the analysis.

[77 FR 29810, May 18, 2012, as amended at 82 FR 40875, Aug. 28, 2017; 86 FR 27260, May 19, 2021]

§ 136.7 Quality assurance and quality control.

The permittee/laboratory shall use suitable QA/QC procedures when conducting compliance analyses with any part 136 chemical method or an alternative method specified by the permitting authority. These QA/QC procedures are generally included in the analytical method or may be part of the methods compendium for approved Part 136 methods from a consensus organization. For example, Standard Methods contains QA/QC procedures in the Part 1000 section of the Standard Methods Compendium. The permittee/laboratory shall follow these QA/QC procedures, as described in the method or methods compendium. If the method lacks QA/QC procedures, the permittee/laboratory has the following options to comply with the QA/QC requirements:

(a) Refer to and follow the QA/QC published in the “equivalent” EPA method for that parameter that has such QA/QC procedures;

(b) Refer to the appropriate QA/QC section(s) of an approved part 136 method from a consensus organization compendium;

(c)

(1) Incorporate the following twelve quality control elements, where applicable, into the laboratory's documented standard operating procedure (SOP) for performing compliance analyses when using an approved part 136 method when the method lacks such QA/QC procedures. One or more of the twelve QC elements may not apply to a given method and may be omitted if a written rationale is provided indicating why the element(s) is/are inappropriate for a specific method.

(i) Demonstration of Capability (DOC);

(ii) Method Detection Limit (MDL);

(iii) Laboratory reagent blank (LRB), also referred to as method blank (MB);

(iv) Laboratory fortified blank (LFB), also referred to as a spiked blank, or laboratory control sample (LCS);

(v) Matrix spike (MS) and matrix spike duplicate (MSD), or laboratory fortified matrix (LFM) and LFM duplicate, may be used for suspected matrix interference problems to assess precision;

(vi) Internal standards (for GC/MS analyses), surrogate standards (for organic analysis) or tracers (for radiochemistry);

(vii) Calibration (initial and continuing), also referred to as initial calibration verification (ICV) and continuing calibration verification (CCV);

(viii) Control charts (or other trend analyses of quality control results);

(ix) Corrective action (root cause analysis);

(x) QC acceptance criteria;

(xi) Definitions of preparation and analytical batches that may drive QC frequencies; and

(xii) Minimum frequency for conducting all QC elements.

(2) These twelve quality control elements must be clearly documented in the written standard operating procedure for each analytical method not containing QA/QC procedures, where applicable.

[77 FR 29813, May 18, 2012]

Appendix A to Part 136—Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater

Method 601—Purgeable Halocarbons

1. Scope and Application

1.1 This method covers the determination of 29 purgeable halocarbons.

The following parameters may be determined by this method:

Parameter STORET No. CAS No.
Bromodichloromethane 32101 75–27–4
Bromoform 32104 75–25–2
Bromomethane 34413 74–83–9
Carbon tetrachloride 32102 56–23–5
Chlorobenzene 34301 108–90–7
Chloroethane 34311 75–00–3
2-Chloroethylvinyl ether 34576 100–75–8
Chloroform 32106 67–66–3
Chloromethane 34418 74–87–3
Dibromochloromethane 32105 124–48–1
1,2-Dichlorobenzene 34536 95–50–1
1,3-Dichlorobenzene 34566 541–73–1
1,4-Dichlorobenzene 34571 106–46–7
Dichlorodifluoromethane 34668 75–71–8
1,1-Dichloroethane 34496 75–34–3
1,2-Dichloroethane 34531 107–06–2
1,1-Dichloroethane 34501 75–35–4
trans-1,2-Dichloroethene 34546 156–60–5
1,2-Dichloropropane 34541 78–87–5
cis-1,3-Dichloropropene 34704 10061–01–5
trans-1,3-Dichloropropene 34699 10061–02–6
Methylene chloride 34423 75–09–2
1,1,2,2-Tetrachloroethane 34516 79–34–5
Tetrachloroethene 34475 127–18–4
1,1,1-Trichloroethane 34506 71–55–6
1,1,2-Trichloroethane 34511 79–00–5
Tetrachloroethene 39180 79–01–6
Trichlorofluoromethane 34488 75–69–4
Vinyl chloride 39715 75–01–4

1.2 This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for most of the parameters listed above.

1.3 The method detection limit (MDL, defined in Section 12.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5 This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The halocarbons are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the halocarbons are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the halocarbons onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the halocarbons which are then detected with a halide-specific detector.2 3

2.2 The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from interferences that may occur.

3. Interferences

3.1 Impurities in the purge gas and organic compounds outgassing from the plumbing ahead of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2 Samples can be contaminated by diffusion of volatile organics (particularly fluorocarbons and methylene chloride) through the septum seal ilto the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3 Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high organohalide levels, it may be necessary to wash out the purging device with a detergent solution, rinse it with distilled water, and then dry it in a 105 °C oven between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4 6 for the information of the analyst.

4.2 The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl chloride. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete sampling.

5.1.1 Vial—25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.1.2 Septum—Teflon-faced silicone (Pierce #12722 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C for 1 h before use.

5.2 Purge and trap system—The purge and trap system consists of three separate pieces of equipment: a purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1 The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2 The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain the following minimum lengths of adsorbents: 1.0 cm of methyl silicone coated packing (Section 6.3.3), 7.7 cm of 2,6-diphenylene oxide polymer (Section 6.3.2), 7.7 cm of silica gel (Section 6.3.4), 7.7 cm of coconut charcoal (Section 6.3.1). If it is not necessary to analyze for dichlorodifluoromethane, the charcoal can be eliminated, and the polymer section lengthened to 15 cm. The minimum specifications for the trap are illustrated in Figure 2.

5.2.3 The desorber must be capable of rapidly heating the trap to 180 °C. The polymer section of the trap should not be heated higher than 180 °C and the remaining sections should not exceed 200 °C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4 The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3 and 4.

5.3 Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.3.1 Column 1—8 ft long × 0.1 in. ID stainless steel or glass, packed with 1% SP–1000 on Carbopack B (60/80 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.3.2 Column 2—6 ft long × 0.1 in. ID stainless steel or glass, packed with chemically bonded n-octane on Porasil-C (100/120 mesh) or equivalent.

5.3.3 Detector—Electrolytic conductivity or microcoulometric detector. These types of detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). The electrolytic conductivity detector was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.4 Syringes—5-mL glass hypodermic with Luerlok tip (two each), if applicable to the purging device.

5.5 Micro syringes—25-µL, 0.006 in. ID needle.

5.6 Syringe valve—2-way, with Luer ends (three each).

5.7 Syringe—5-mL, gas-tight with shut-off valve.

5.8 Bottle—15-mL, screw-cap, with Teflon cap liner.

5.9 Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1 Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2 A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3 Reagent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90 °C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2 Sodium thiosulfate—(ACS) Granular.

6.3 Trap Materials:

6.3.1 Coconut charcoal—6/10 mesh sieved to 26 mesh, Barnabey Cheney, CA–580–26 lot # M–2649 or equivalent.

6.3.2 2,6-Diphenylene oxide polymer—Tenax, (60/80 mesh), chromatographic grade or equivalent.

6.3.3 Methyl silicone packing—3% OV–1 on Chromosorb-W (60/80 mesh) or equivalent.

6.3.4 Silica gel—35/60 mesh, Davison, grade-15 or equivalent.

6.4 Methanol—Pesticide quality or equivalent.

6.5 Stock standard solutions—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in methanol using assayed liquids or gases as appropriate. Because of the toxicity of some of the organohalides, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.5.1 Place about 9.8 mL of methanol into a 10-mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the learest 0.1 mg.

6.5.2 Add the assayed reference material:

6.5.2.1 Liquid—Using a 100 µL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.5.2.2 Gases—To prepare standards for any of the six halocarbons that boil below 30 °C (bromomethane, chloroethane, chloromethane, dichlorodifluoromethane, trichlorofluoromethane, vinyl chloride), fill a 5-mL valved gas-tight syringe with the reference standard to the 5.0-mL mark. Lower the needle to 5 mm above the methanol meniscus. Slowly introduce the reference standard above the surface of the liquid (the heavy gas will rapidly dissolve into the methanol).

6.5.3 Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in µg/µL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the malufacturer or by an independent source.

6.5.4 Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store, with minimal headspace, at −10 to −20 °C and protect from light.

6.5.5 Prepare fresh standards weekly for the six gases and 2-chloroethylvinyl ether. All other standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.6 Secondary dilution standards—Using stock standard solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary dilution standards should be stored with minimal headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2 Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3 External standard calibration procedure:

7.3.1 Prepare calibration standards at a miminum of three concentration levels for each parameter by carefully adding 20.0 µL of one or more secondary dilution standards to 100, 500, or 1000 µL of reagent water. A 25-µL syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These aqueous standards can be stored up to 24 h, if held in sealed vials with zero headspace as described in Section 9.2. If not so stored, they must be discarded after 1 h.

7.3.2 Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration in the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.4 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples. The compounds recommended for use as surrogate spikes in Section 8.7 have been used successfully as internal standards, because of their generally unique retention times.

7.4.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2 Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.5 and 6.6. It is recommended that the secondary dilution standard be prepared at a concentration of 15 µg/mL of each internal standard compound. The addition of 10 µL of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 µg/L.

7.4.3 Analyze each calibration standard according to Section 10, adding 10 µL of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard.

Cs = Concentration of the parameter to be measured.

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.5 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1 Prepare the QC check sample as described in Section 8.2.2.

7.5.2 Analyze the QC check sample according to Section 10.

7.5.3 For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, proceed according to Section 7.5.4.

Note:

The large number of parameters in Table 2 present a substantial probability that one or more will not meet the calibration acceptance criteria when all parameters are analyzed.

7.5.4 Repeat the test only for those parameters that failed to meet the calibration acceptance criteria. If the response for a parameter does not fall within the range in this second test, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 µg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Prepare a QC check sample to contain 20 µg/L of each parameter by adding 200 µL of QC check sample concentrate to 100 mL of reagent water.

8.2.3 Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter of interest using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, then the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6 When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1 Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2 Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2 Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 µL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 µg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 2 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spiked sample.

8.4.1 Prepare the QC check standard by adding 10 µL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If p̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

8.7 The analyst should monitor both the performance of the analytical system and the effectiveness of the method in dealing with each sample matrix by spiking each sample, standard, and reagent water blank with surrogate halocarbons. A combination of bromochloromethane, 2-bromo-1-chloropropane, and 1,4-dichlorobutane is recommended to encompass the range of the temperature program used in this method. From stock standard solutions prepared as in Section 6.5, add a volume to give 750 µg of each surrogate to 45 mL of reagent water contained in a 50-mL volumetric flask, mix and dilute to volume for a concentration of 15 ng/µL. Add 10 µL of this surrogate spiking solution directly into the 5-mL syringe with every sample and reference standard analyzed. Prepare a fresh surrogate spiking solution on a weekly basis. If the internal standard calibration procedure is being used, the surrogate compounds may be added directly to the internal standard spiking solution (Section 7.4.2).

9. Sample Collection, Preservation, and Handling

9.1 All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2 Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3 All samples must be analyzed within 14 days of collection.3

10. Procedure

10.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 5. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2 Calibrate the system daily as described in Section 7.

10.3 Adjust the purge gas (nitrogen or helium) flow rate to 40 mL/min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4 Allow the sample to come to ambient temperature prior to introducing it to the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 µL of the surrogate spiking solution (Section 8.7) and 10.0 µL of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore, then close the valve.

10.5 Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6 Close both valves and purge the sample for 11.0 ±0.1 min at ambient temperature.

10.7 After the 11-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be used as a secondary trap by cooling it to 30 °C (subambient temperature, if poor peak geometry or random retention time problems persist) instead of the initial program temperature of 45 °C

10.8 While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9 After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 180 °C After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

10.11 If the response for a peak exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

11. Calculations

11.1 Determine the concentration of individual compounds in the sample.

11.1.1 If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2.

Equation 2

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard.

11.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance

12.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentration listed in Table 1 were obtained using reagent water.11. Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2 This method is recommended for use in the concentration range from the MDL to 1000 × MDL. Direct aqueous injection techniques should be used to measure concentration levels above 1000 × MDL.

12.3 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 8.0 to 500 µg/L.9 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References

1. 40 CFR part 136, appendix B.

2. Bellar, T.A., and Lichtenberg, J.J. “Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography,” Journal of the American Water Works Association, 66, 739 (1974).

3. Bellar, T.A., and Lichtenberg, J.J. “Semi-Automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds,” Proceedings from Symposium on Measurement of Organic Pollutants in Water and Wastewater, American Society for Testing and Materials, STP 686, C.E. Van Hall, editor, 1978.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA 600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

9. “EPA Method Study 24, Method 601—Purgeable Halocarbons by the Purge and Trap Method,” EPA 600/4–84–064, National Technical Information Service, PB84–212448, Springfield, Virginia 22161, July 1984.

10. “Method Validation Data for EPA Method 601,” Memorandum from B. Potter, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, November 10, 1983.

11. Bellar, T. A., Unpublished data, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, 1981.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Column 1 Column 2
Chloromethane 1.50 5.28 0.08
Bromomethane 2.17 7.05 1.18
Dichlorodifluoromethane 2.62 nd 1.81
Vinyl chloride 2.67 5.28 0.18
Chloroethane 3.33 8.68 0.52
Methylene chloride 5.25 10.1 0.25
Trichlorofluoromethane 7.18 nd nd
1,1-Dichloroethene 7.93 7.72 0.13
1,1-Dichloroethane 9.30 12.6 0.07
trans-1,2-Dichloroethene 10.1 9.38 0.10
Chloroform 10.7 12.1 0.05
1,2-Dichloroethane 11.4 15.4 0.03
1,1,1-Trichloroethane 12.6 13.1 0.03
Carbon tetrachloride 13.0 14.4 0.12
Bromodichloromethane 13.7 14.6 0.10
1,2-Dichloropropane 14.9 16.6 0.04
cis-1,3-Dichloropropene 15.2 16.6 0.34
Trichloroethene 15.8 13.1 0.12
Dibromochloromethane 16.5 16.6 0.09
1,1,2-Trichloroethane 16.5 18.1 0.02
trans-1,3-Dichloropropene 16.5 18.0 0.20
2-Chloroethylvinyl ether 18.0 nd 0.13
Bromoform 19.2 19.2 0.20
1,1,2,2-Tetrachloroethane 21.6 nd 0.03
Tetrachloroethene 21.7 15.0 0.03
Chlorobenzene 24.2 18.8 0.25
1,3-Dichlorobenzene 34.0 22.4 0.32
1,2-Dichlorobenzene 34.9 23.5 0.15
1,4-Dichlorobenzene 35.4 22.3 0.24

Table 2—Calibration and QC Acceptance Criteria—Method 601 a

Parameter Range for Q (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range P, Ps (%)
Bromodichloromethane 15.2–24.8 4.3 10.7–32.0 42–172
Bromoform 14.7–25.3 4.7 5.0–29.3 13–159
Bromomethane 11.7–28.3 7.6 3.4–24.5 D–144
Carbon tetrachloride 13.7–26.3 5.6 11.8–25.3 43–143
Chlorobenzene 14.4–25.6 5.0 10.2–27.4 38–150
Chloroethane 15.4–24.6 4.4 11.3–25.2 46–137
2-Chloroethylvinyl ether 12.0–28.0 8.3 4.5–35.5 14–186
Chloroform 15.0–25.0 4.5 12.4–24.0 49–133
Chloromethane 11.9–28.1 7.4 D–34.9 D–193
Dibromochloromethane 13.1–26.9 6.3 7.9–35.1 24–191
1,2-Dichlorobenzene 14.0–26.0 5.5 1.7–38.9 D–208
1,3-Dichlorobenzene 9.9–30.1 9.1 6.2–32.6 7–187
1,4-Dichlorobenzene 13.9–26.1 5.5 11.5–25.5 42–143
1,1-Dichloroethane 16.8–23.2 3.2 11.2–24.6 47–132
1,2-Dichloroethane 14.3–25.7 5.2 13.0–26.5 51–147
1,1-Dichloroethene 12.6–27.4 6.6 10.2–27.3 28–167
trans-1,2-Dichloroethene 12.8–27.2 6.4 11.4–27.1 38–155
1,2-Dichloropropane 14.8–25.2 5.2 10.1–29.9 44–156
cis-1,3-Dichloropropene 12.8–27.2 7.3 6.2–33.8 22–178
trans-1,3-Dichloropropene 12.8–27.2 7.3 6.2–33.8 22–178
Methylene chloride 15.5–24.5 4.0 7.0–27.6 25–162
1,1,2,2-Tetrachloroethane 9.8–30.2 9.2 6.6–31.8 8–184
Tetrachloroethene 14.0–26.0 5.4 8.1–29.6 26–162
1,1,1-Trichloroethane 14.2–25.8 4.9 10.8–24.8 41–138
1,1,2-Trichloroethane 15.7–24.3 3.9 9.6–25.4 39–136
Trichloroethene 15.4–24.6 4.2 9.2–26.6 35–146
Trichlorofluoromethane 13.3–26.7 6.0 7.4–28.1 21–156
Vinyl chloride 13.7–26.3 5.7 8.2–29.9 28–163

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 601

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
Bromodichloromethane 1.12C−1.02 0.11X̄ + 0.04 0.20X̄ + 1.00
Bromoform 0.96C−2.05 0.12X̄ + 0.58 0.21X̄ + 2.41
Bromomethane 0.76C−1.27 0.28X̄ + 0.27 0.36X̄ + 0.94
Carbon tetrachloride 0.98C−1.04 0.15X̄ + 0.38 0.20X̄ + 0.39
Chlorobenzene 1.00C−1.23 0.15X̄−0.02 0.18X̄ + 1.21
Choroethane 0.99C−1.53 0.14X̄−0.13 0.17X̄ + 0.63
2-Chloroethylvinyl ethera 1.00C 0.20X̄ 0.35X̄
Chloroform 0.93C−0.39 0.13X̄ + 0.15 0.19X̄−0.02
Chloromethane 0.77C + 0.18 0.28X̄−0.31 0.52X̄ + 1.31
Dibromochloromethane 0.94C + 2.72 0.11X̄ + 1.10 0.24X̄ + 1.68
1,2-Dichlorobenzene 0.93C + 1.70 0.20X̄ + 0.97 0.13X̄ + 6.13
1,3-Dichlorobenzene 0.95C + 0.43 0.14X̄ + 2.33 0.26X̄ + 2.34
1,4-Dichlorobenzene 0.93C−0.09 0.15X̄ + 0.29 0.20X̄ + 0.41
1,1-Dichloroethane 0.95C−1.08 0.09X̄ + 0.17 0.14X̄ + 0.94
1,2-Dichloroethane 1.04C−1.06 0.11X̄ + 0.70 0.15X̄ + 0.94
1,1-Dichloroethene 0.98C−0.87 0.21X̄−0.23 0.29X̄−0.40
trans-1,2-Dichloroethene 0.97C−0.16 0.11X̄ + 1.46 0.17X̄ + 1.46
1,2-Dichloropropanea 1.00C 0.13X̄ 0.23X̄
cis-1,3-Dichloropropenea 1.00C 0.18X̄ 0.32X̄
trans-1,3-Dichloropropenea 1.00C 0.18X̄ 0.32X̄
Methylene chloride 0.91C−0.93 0.11X̄ + 0.33 0.21X̄ + 1.43
1,1,2,2-Tetrachloroethene 0.95C + 0.19 0.14X̄ + 2.41 0.23X̄ + 2.79
Tetrachloroethene 0.94C + 0.06 0.14X̄ + 0.38 0.18X̄ + 2.21
1,1,1-Trichloroethane 0.90C−0.16 0.15X̄ + 0.04 0.20X̄ + 0.37
1,1,2-Trichloroethane 0.86C + 0.30 0.13X̄−0.14 0.19X̄ + 0.67
Trichloroethene 0.87C + 0.48 0.13X̄−0.03 0.23X̄ + 0.30
Trichlorofluoromethane 0.89C−0.07 0.15X̄ + 0.67 0.26X̄ + 0.91
Vinyl chloride 0.97C−0.36 0.13X̄ + 0.65 0.27X̄ + 0.40

Method 602—Purgeable Aromatics

1. Scope and Application

1.1 This method covers the determination of various purgeable aromatics. The following parameters may be determined by this method:

Parameter STORET No. CAS No.
Benzene 34030 71–43–2
Chlorobenzene 34301 108–90–7
1,2-Dichlorobenzene 34536 95–50–1
1,3-Dichlorobenzene 34566 541–73–1
1,4-Dichlorobenzene 34571 106–46–7
Ethylbenzene 34371 100–41–4
Toluene 34010 108–88–3

1.2 This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above.

1.3 The method detection limit (MDL, defined in Section 12.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5 This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The aromatics are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the aromatics are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the aromatics onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the aromatics which are then detected with a photoionization detector.2 3

2.2 The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from interferences that may occur.

3. Interferences

3.1 Impurities in the purge gas and organic compounds outgassing from the plumbing ahead of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2 Samples can be contaminated by diffusion of volatile organics through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3 Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high aromatic levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in an oven at 105 °C between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4 6 for the information of the analyst.

4.2 The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzene and 1,4-dichlorobenzene. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete sampling.

5.1.1 Vial]25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.1.2 Septum—Teflon-faced silicone (Pierce #12722 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C for 1 h before use.

5.2 Purge and trap system—The purge and trap system consists of three separate pieces of equipment: A purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1 The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2 The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in.

5.2.2.1 The trap is packed with 1 cm of methyl silicone coated packing (Section 6.4.2) and 23 cm of 2,6-diphenylene oxide polymer (Section 6.4.1) as shown in Figure 2. This trap was used to develop the method performance statements in Section 12.

5.2.2.2 Alternatively, either of the two traps described in Method 601 may be used, although water vapor will preclude the measurement of low concentrations of benzene.

5.2.3 The desorber must be capable of rapidly heating the trap to 180 °C. The polymer section of the trap should not be heated higher than 180 °C and the remaining sections should not exceed 200 °C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4 The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3, 4, and 5.

5.3 Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.3.1 Column 1—6 ft long × 0.082 in. ID stainless steel or glass, packed with 5% SP–1200 and 1.75% Bentone-34 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.3.2 Column 2—8 ft long × 0.1 in ID stainless steel or glass, packed with 5% 1,2,3-Tris(2-cyanoethoxy)propane on Chromosorb W-AW (60/80 mesh) or equivalent.

5.3.3 Detector—Photoionization detector (h-Nu Systems, Inc. Model PI–51–02 or equivalent). This type of detector has been proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.4 Syringes—5-mL glass hypodermic with Luerlok tip (two each), if applicable to the purging device.

5.5 Micro syringes—25-µL, 0.006 in. ID needle.

5.6 Syringe valve—2-way, with Luer ends (three each).

5.7 Bottle—15-mL, screw-cap, with Teflon cap liner.

5.8 Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1 Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2 A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3 Reagent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90 °C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2 Sodium thiosulfate—(ACS) Granular.

6.3 Hydrochloric acid (1 + 1)—Add 50 mL of concentrated HCl (ACS) to 50 mL of reagent water.

6.4 Trap Materials:

6.4.1 2,6-Diphenylene oxide polymer—Tenax, (60/80 mesh), chromatographic grade or equivalent.

6.4.2 Methyl silicone packing—3% OV–1 on Chromosorb-W (60/80 mesh) or equivalent.

6.5 Methanol—Pesticide quality or equivalent.

6.6 Stock standard solutions—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in methanol using assayed liquids. Because of the toxicity of benzene and 1,4-dichlorobenzene, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.6.1 Place about 9.8 mL of methanol into a 10–mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg.

6.6.2 Using a 100–µL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.6.3 Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in µg/µL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.6.4 Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store at 4 °C and protect from light.

6.6.5 All standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.7 Secondary dilution standards—Using stock standard solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary solution standards must be stored with zero headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.8 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2 Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3 External standard calibration procedure:

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 µL of one or more secondary dilution standards to 100, 500, or 1000 mL of reagent water. A 25-µL syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These aqueous standards must be prepared fresh daily.

7.3.2 Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration in the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.4 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples. The compound, α,α,α,-trifluorotoluene, recommended as a surrogate spiking compound in Section 8.7 has been used successfully as an internal standard.

7.4.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2 Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.6 and 6.7. It is recommended that the secondary dilution standard be prepared at a concentration of 15 µg/mL of each internal standard compound. The addition of 10 µl of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 µg/L.

7.4.3 Analyze each calibration standard according to Section 10, adding 10 µL of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

RF = (As)(Cis (Ais)(Cs)

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard

Cs = Concentration of the parameter to be measured.

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.5 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1 Prepare the QC check sample as described in Section 8.2.2.

7.5.2 Analyze the QC check sample according to Section 10.

7.5.3 For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 µg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Prepare a QC check sample to contain 20 µg/L of each parameter by adding 200 µL of QC check sample concentrate to 100 mL of reagant water.

8.2.3 Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter of interest using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6 When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1 Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2 Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2 Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 µL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 µg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44(100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 10 µL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

8.7 The analyst should monitor both the performance of the analytical system and the effectiveness of the method in dealing with each sample matrix by spiking each sample, standard, and reagent water blank with surrogate compounds (e.g. α, α, α,-trifluorotoluene) that encompass the range of the temperature program used in this method. From stock standard solutions prepared as in Section 6.6, add a volume to give 750 µg of each surrogate to 45 mL of reagent water contained in a 50-mL volumetric flask, mix and dilute to volume for a concentration of 15 mg/µL. Add 10 µL of this surrogate spiking solution directly into the 5-mL syringe with every sample and reference standard analyzed. Prepare a fresh surrogate spiking solution on a weekly basis. If the internal standard calibration procedure is being used, the surrogate compounds may be added directly to the internal standard spiking solution (Section 7.4.2).

9. Sample Collection, Preservation, and Handling

9.1 The samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Method 330.4 or 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2 Collect about 500 mL of sample in a clean container. Adjust the pH of the sample to about 2 by adding 1 + 1 HCl while stirring. Fill the sample bottle in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3 All samples must be analyzed within 14 days of collection.3

10. Procedure

10.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 6. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2 Calibrate the system daily as described in Section 7.

10.3 Adjust the purge gas (nitrogen or helium) flow rate to 40 mL/min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4 Allow the sample to come to ambient temperature prior to introducing it to the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 µL of the surrogate spiking solution (Section 8.7) and 10.0 µL of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore, then close the valve.

10.5 Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6 Close both valves and purge the sample for 12.0 ±0.1 min at ambient temperature.

10.7 After the 12-min purge time, disconnect the purging device from the trap. Dry the trap by maintaining a flow of 40 mL/min of dry purge gas through it for 6 min (Figure 4). If the purging device has no provision for bypassing the purger for this step, a dry purger should be inserted into the device to minimize moisture in the gas. Attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 5), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be used as a secondary trap by cooling it to 30 °C (subambient temperature, if poor peak geometry and random retention time problems persist) instead of the initial program temperature of 50 °C.

10.8 While the trap is being desorbed into the gas chromatograph column, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9 After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s, then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 180 °C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

10.11 If the response for a peak exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

11. Calculations

11.1 Determine the concentration of individual compounds in the sample.

11.1.1 If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2.

Equation 2

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard.

11.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance

12.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.9 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2 This method has been demonstrated to be applicable for the concentration range from the MDL to 100 × MDL.9 Direct aqueous injection techniques should be used to measure concentration levels above 1000 × MDL.

12.3 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 2.1 to 550 µg/L.9 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References

1. 40 CFR part 136, appendix B.

2. Lichtenberg, J.J. “Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography,” Journal American Water Works Association, 66, 739 (1974).

3. Bellar, T.A., and Lichtenberg, J.J. “Semi-Automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds,” Proceedings of Symposium on Measurement of Organic Pollutants in Water and Wastewater. American Society for Testing and Materials, STP 686, C.E. Van Hall, editor, 1978.

4. “Carcinogens—Working with Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health. Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3. is two times the value 1.22 derived in this report.)

8.“Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Office of Research and Development, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268. March 1979.

9. “EPA Method Study 25, Method 602, Purgeable Aromatics,” EPA 600/4–84–042, National Technical Information Service, PB84–196682, Springfield, Virginia 22161, May 1984.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Column 1 Column 2
Benzene 3.33 2.75 0.2
Toluene 5.75 4.25 0.2
Ethylbenzene 8.25 6.25 0.2
Chlorobenzene 9.17 8.02 0.2
1,4-Dichlorobenzene 16.8 16.2 0.3
1,3-Dichlorobenzene 18.2 15.0 0.4
1,2-Dichlorobenzene 25.9 19.4 0.4

Table 2—Calibration and QC Acceptance Criteria—Method 602 a

Parameter Range for Q (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps(%)
Benzene 15.4–24.6 4.1 10.0–27.9 39–150
Chlorobenzene 16.1–23.9 3.5 12.7–25.4 55–135
1,2-Dichlorobenzene 13.6–26.4 5.8 10.6–27.6 37–154
1,3-Dichlorobenzene 14.5–25.5 5.0 12.8–25.5 50–141
1,4-Dichlorobenzene 13.9–26.1 5.5 11.6–25.5 42–143
Ethylbenzene 12.6–27.4 6.7 10.0–28.2 32–160
Toluene 15.5–24.5 4.0 11.2–27.7 46–148

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 602

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, s′ (µg/L) Overall precision, S′ (µg/L)
Benzene 0.92C + 0.57 0.09X̄ + 0.59 0.21X̄ + 0.56
Chlorobenzene 0.95C + 0.02 0.09X̄ + 0.23 0.17X̄ + 0.10
1,2-Dichlorobenzene 0.93C + 0.52 0.17X̄−0.04 0.22X̄ + 0.53
1,3-Dichlorobenzene 0.96C−0.05 0.15X̄−0.10 0.19X̄ + 0.09
1,4-Dichlorobenzene 0.93C−0.09 0.15X̄ + 0.28 0.20X̄ + 0.41
Ethylbenzene 0.94C + 0.31 0.17X̄ + 0.46 0.26X̄ + 0.23
Toluene 0.94C + 0.65 0.09X̄ + 0.48 0.18X̄ + 0.71

Method 603—Acrolein and Acrylonitrile

1. Scope and Application

1.1 This method covers the determination of acrolein and acrylonitrile. The following parameters may be determined by this method:

Parameter STORET No. CAS No.
Acrolein 34210 107–02–8
Acrylonitrile 34215 107–13–1

1.2 This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for either or both of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for the parameters listed above, if used with the purge and trap conditions described in this method.

1.3 The method detection limit (MDL, defined in Section 12.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5 This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 An inert gas is bubbled through a 5-mL water sample contained in a heated purging chamber. Acrolein and acrylonitrile are transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the analytes are trapped. After the purge is completed, the trap is heated and backflushed with the inert gas to desorb the compound onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the analytes which are then detected with a flame ionization detector.2 3

2.2 The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from the interferences that may occur.

3. Interferences

3.1 Impurities in the purge gas and organic compound outgassing from the plumbing of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2 Samples can be contaminated by diffusion of volatile organics through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3 Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed between samples with reagent water. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high analyte levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in an oven at 105 °C between analyses. The trap and other parts of the system are also subject to contamination, therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this view point, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4 6 for the information of the analyst.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete sampling.

5.1.1 Vial—25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.1.2 Septum—Teflon-faced silicone (Pierce #12722 or equivalent). Detergent wash, rinse with tap and distilled water and dry at 105 °C for 1 h before use.

5.2 Purge and trap system—The purge and trap system consists of three separate pieces of equipment: a purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1 The purging device must be designed to accept 5-mL, samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device must be capable of being heated to 85 °C within 3.0 min after transfer of the sample to the purging device and being held at 85 ±2 °C during the purge cycle. The entire water column in the purging device must be heated. Design of this modification to the standard purging device is optional, however, use of a water bath is suggested.

5.2.1.1 Heating mantle—To be used to heat water bath.

5.2.1.2 Temperature controller—Equipped with thermocouple/sensor to accurately control water bath temperature to ±2 °C. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2 The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain 1.0 cm of methyl silicone coated packing (Section 6.5.2) and 23 cm of 2,6-diphenylene oxide polymer (Section 6.5.1). The minimum specifications for the trap are illustrated in Figure 2.

5.2.3 The desorber must be capable of rapidly heating the trap to 180 °C, The desorber illustrated in Figure 2 meets these design criteria.

5.2.4 The purge and trap system may be assembled as a separate unit as illustrated in Figure 3 or be coupled to a gas chromatograph.

5.3 pH paper—Narrow pH range, about 3.5 to 5.5 (Fisher Scientific Short Range Alkacid No. 2, #14–837–2 or equivalent).

5.4 Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.4.1 Column 1—10 ft long × 2 mm ID glass or stainless steel, packed with Porapak-QS (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.4.2 Column 2—6 ft long × 0.1 in. ID glass or stainless steel, packed with Chromosorb 101 (60/80 mesh) or equivalent.

5.4.3 Detector—Flame ionization detector. This type of detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.5 Syringes—5-mL, glass hypodermic with Luerlok tip (two each).

5.6 Micro syringes—25-µL, 0.006 in. ID needle.

5.7 Syringe valve—2-way, with Luer ends (three each).

5.8 Bottle—15-mL, screw-cap, with Teflon cap liner.

5.9 Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1 Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2 A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3 Regent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90 °C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2 Sodium thiosulfate—(ACS) Granular.

6.3 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.4 Hydrochloric acid (1 + 1)—Slowly, add 50 mL of concentrated HCl (ACS) to 50 mL of reagent water.

6.5 Trap Materials:

6.5.1 2,6-Diphenylene oxide polymer—Tenax (60/80 mesh), chromatographic grade or equivalent.

6.5.2 Methyl silicone packing—3% OV–1 on Chromosorb-W (60/80 mesh) or equivalent.

6.6 Stock standard solutions—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in reagent water using assayed liquids. Since acrolein and acrylonitrile are lachrymators, primary dilutions of these compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.6.1 Place about 9.8 mL of reagent water into a 10-mL ground glass stoppered volumetric flask. For acrolein standards the reagent water must be adjusted to pH 4 to 5. Weight the flask to the nearest 0.1 mg.

6.6.2 Using a 100-µL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the water without contacting the neck of the flask.

6.6.3 Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in µg/µL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock staldard. Optionally, stock standard solutions may be prepared using the pure standard material by volumetrically measuring the appropriate amounts and determining the weight of the material using the density of the material. Commercially prepared stock standards may be used at any concentration if they are certified by the manufactaurer or by an independent source.

6.6.4 Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store at 4 °C and protect from light.

6.6.5 Prepare fresh standards daily.

6.7 Secondary dilution standards—Using stock standard solutions, prepare secondary dilution standards in reagent water that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary dilution standards should be prepared daily and stored at 4 °C.

6.8 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2 Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3 External standard calibration procedure:

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 µL of one or more secondary dilution standards to 100, 500, or 1000 mL of reagent water. A 25-µL syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These standards must be prepared fresh daily.

7.3.2 Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration of the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.4 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.4.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2 Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.6 and 6.7. It is recommended that the secondary dilution standard be prepared at a concentration of 15 µg/mL of each internal standard compound. The addition of 10 µL of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 µg/L.

7.4.3 Analyze each calibration standard according to Section 10, adding 10 µL of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

RF = (As)(Cis (Ais)(Cs)

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard.

Cs = Concentration of the parameter to be measured.

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.5 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1 Prepare the QC check sample as described in Section 8.2.2.

7.5.2 Analyze the QC check sample according to Section 10.

7.5.3 For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 25 µg/mL in reagent water. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Prepare a QC check sample to contain 50 µg/L of each parameter by adding 200 µL of QC check sample concentrate to 100 mL of reagent water.

8.2.3 Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If either s exceeds the precision limit or X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for each compound of interest.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 50 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2 Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 µL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 10 µL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2 If acrolein is to be analyzed, collect about 500 mL of sample in a clean glass container. Adjust the pH of the sample to 4 to 5 using acid or base, measuring with narrow range pH paper. Samples for acrolein analysis receiving no pH adjustment must be analyzed within 3 days of sampling.

9.3 Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.4 All samples must be analyzed within 14 days of collection.3

10. Procedure

10.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 5. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2 Calibrate the system daily as described in Section 7.

10.3 Adjust the purge gas (nitrogen or helium) flow rate to 20 mL-min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4 Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 µL of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore then close the valve.

10.5 Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6 Close both valves and purge the sample for 15.0 ±0.1 min while heating at 85 ±2 °C.

10.7 After the 15-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 1.5 min.

10.8 While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9 After desorbing the sample for 1.5 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 210 °C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

11. Calculations

11.1 Determine the concentration of individual compounds in the sample.

11.1.1 If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2.

Equation 2

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard.

11.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance

12.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.9 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2 This method is recommended for the concentration range from the MDL to 1,000 × MDL. Direct aqueous injection techniques should be used to measure concentration levels above 1,000 × MDL.

12.3 In a single laboratory (Battelle-Columbus), the average recoveries and standard deviations presented in Table 2 were obtained.9 Seven replicate samples were analyzed at each spike level.

References

1. 40 CFR part 136, appendix B.

2. Bellar, T.A., and Lichtenberg, J.J. “Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography,” Journal American Water Works Association, 66, 739 (1974).

3. “Evaluate Test Procedures for Acrolein and Acrylonitrile,” Special letter report for EPA Project 4719–A, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, 27 June 1979.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983).

8. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

9. “Evaluation of Method 603 (Modified),” EPA–600/4–84–ABC, National Technical Information Service, PB84–, Springfield, Virginia 22161, Nov. 1984.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Column 1 Column 2
Acrolein 10.6 8.2 0.7
Acrylonitrile 12.7 9.8 0.5

Table 2—Single Laboratory Accuracy and Precision—Method 603

Parameter Sample matrix Spike conc. (µg/L) Average recovery (µg/L) Standard deviation (µg/L) Average percent recovery
Acrolein RW 5.0 5.2 0.2 104
RW 50.0 51.4 0.7 103
POTW 5.0 4.0 0.2 80
POTW 50.0 44.4 0.8 89
IW 5.0 0.1 0.1 2
IW 100.0 9.3 1.1 9
Acrylonitrile RW 5.0 4.2 0.2 84
RW 50.0 51.4 1.5 103
POTW 20.0 20.1 0.8 100
POTW 100.0 101.3 1.5 101
IW 10.0 9.1 0.8 91
IW 100.0 104.0 3.2 104

Table 3—Calibration and QC Acceptance Criteria—Method 603 a

Parameter Range for Q (µg/L) Limit for S (µg/L) Range for X (µg/L) Range for P, Ps (%)
Acrolein 45.9–54.1 4.6 42.9–60.1 88–118
Acrylonitrile 41.2–58.8 9.9 33.1–69.9 71–135

Method 604—Phenols

1. Scope and Application

1.1 This method covers the determination of phenol and certain substituted phenols. The following parameters may be determined by this method:

Parameter STORET No. CAS No.
4-Chloro-3-methylphenol 34452 59–50–7
2–-Chlorophenol 34586 95–57–8
2,4-Dichlorophenol 34601 120–83–2
2,4-Dimethylphenol 34606 105–67–9
2,4-Dinitrophenol 34616 51–28–5
2-Methyl-4,6-dinitrophenol 34657 534–52–1
2-Nitrophenol 34591 88–75–5
4-Nitrophenol 34646 100–02–7
Pentachlorophenol 39032 87–86–5
Phenol 34694 108–95–2
2,4,6-Trichlorophenol 34621 88–06–2

1.2 This is a flame ionization detector gas chromatographic (FIDGC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for derivatization, cleanup, and electron capture detector gas chromatography (ECDGC) that can be used to confirm measurements made by FIDGC. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix. The MDL listed in Table 1 for each parameter was achieved with a flame ionization detector (FID). The MDLs that were achieved when the derivatization cleanup and electron capture detector (ECD) were employed are presented in Table 2.

1.4 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1-L, is acidified and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to 2-propanol during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the phenols are then measured with an FID.2

2.2 A preliminary sample wash under basic conditions can be employed for samples having high general organic and organic base interferences.

2.3 The method also provides for a derivatization and column chromatography cleanup procedure to aid in the elimination of interferences.2 3 The derivatives are analyzed by ECDGC.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The derivatization cleanup procedure in Section 12 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Tables 1 and 2.

3.3 The basic sample wash (Section 10.2) may cause significantly reduced recovery of phenol and 2,4-dimethylphenol. The analyst must recognize that results obtained under these conditions are minimum concentrations.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this mothod has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified5 7 for the information of analyst.

4.2 Special care should be taken in handling pentafluorobenzyl bromide, which is a lachrymator, and 18-crown-6-ether, which is highly toxic.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnel—2–L, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Chromatographic column—100 mm long × 10 mm ID, with Teflon stopcock.

5.2.4 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5 Evaporative flask, Kuderna-Danish—500-mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.6 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.7 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).

5.2.8 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.9 Reaction flask—15 to 25-mL round bottom flask, with standard tapered joint, fitted with a water-cooled condenser and U-shaped drying tube containing granular calcium chloride.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighting 0.0001 g.

5.6 Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1 Column for underivatized phenols—1.8 m long × 2 mm ID glass, packed with 1% SP–1240DA on Supelcoport (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 11.1.

5.6.2 Column for derivatized phenols—1.8 m long × 2 mm ID glass, packed with 5% OV–17 on Chromosorb W-AW-DMCS (80/100 mesh) or equivalent. This column has proven effective in the analysis of wastewaters for derivatization products of the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 11.1.

5.6.3 Detectors—Flame ionization and electron capture detectors. The FID is used when determining the parent phenols. The ECD is used when determining the derivatized phenols. Guidelines for the use of alternatve detectors are provided in Section 11.1.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3 Sodium hydroxide solution (1 N)—Dissolve 4 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.4 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.5 Sodium thiosulfate—(ACS) Granular.

6.6 Sulfuric acid (1 + 1)—Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.7 Sulfuric acid (1 N)—Slowly, add 58 mL of H2SO4 (ACS, sp. gr. 1.84) to reagent water and dilute to 1 L.

6.8 Potassium carbonate—(ACS) Powdered.

6.9 Pentafluorobenzyl bromide (α-Bromopentafluorotoluene)—97% minimum purity.

Note:

This chemical is a lachrymator. (See Section 4.2.)

6.10 18-crown-6-ether (1,4,7,10,13,16-Hexaoxacyclooctadecane)—98% minimum purity.

Note:

This chemical is highly toxic.

6.11 Derivatization reagent—Add 1 mL of pentafluorobenzyl bromide and 1 g of 18-crown-6-ether to a 50-mL volumetric flask and dilute to volume with 2-propanol. Prepare fresh weekly. This operation should be carried out in a hood. Store at 4 °C and protect from light.

6.12 Acetone, hexane, methanol, methylene chloride, 2-propanol, toluene—Pesticide quality or equivalent.

6.13 Silica gel—100/200 mesh, Davison, grade-923 or equivalent. Activate at 130 °C overnight and store in a desiccator.

6.14 Stock standard solutions (1.00 µg/µL)—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions.

6.14.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in 2-propanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.14.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.14.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.15 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 To calibrate the FIDGC for the anaylsis of underivatized phenols, establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure for FIDGC:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with 2-propanol. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µl, analyze each calibration standard according to Section 11 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure for FIDGC—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with 2-propanol. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 11 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF = (As)(Cis (Ais)(Cs)

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5 To calibrate the ECDGC for the analysis of phenol derivatives, establish gas chromatographic operating conditions equivalent to those given in Table 2.

7.5.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with 2-propanol. One of the external standards should be at a concentration near, but above, the MDL (Table 2) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.5.2 Each time samples are to be derivatized, simultaneously treat a 1-mL aliquot of each calibration standard as described in Section 12.

7.5.3 After derivatization, analyze 2 to 5 µL of each column eluate collected according to the method beginning in Section 12.8 and tabulate peak height or area responses against the calculated equivalent mass of underivatized phenol injected. The results can be used to prepare a calibration curve for each compound.

7.6 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.6 and 11.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 µg/mL in 2-propanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at a concentration of 100 µg/L by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Talbe 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6 When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1 Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2 Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any, or, if none,

(2) the larger of either 5 times higher than the expected background concentration or 100 µg/L.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.8 If spiking was performed at a concentration lower than 100 µg/L, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 4, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 4, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.8

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6. It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices9 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.10 Field test kits are available for this purpose.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1 Mark the water meniscus on the side of sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2 For samples high in organic content, the analyst may solvent wash the sample at basic pH as prescribed in Sections 10.2.1 and 10.2.2 to remove potential method interferences. Prolonged or exhaustive contact with solvent during the wash may result in low recovery of some of the phenols, notably phenol and 2,4-dimethylphenol. For relatively clean samples, the wash should be omitted and the extraction, beginning with Section 10.3, should be followed.

10.2.1 Adjust the pH of the sample to 12.0 or greater with sodium hydroxide solution.

10.2.2 Add 60 mL of methylene chloride to the sample by shaking the funnel for 1 min with periodic venting to release excess pressure. Discard the solvent layer. The wash can be repeated up to two additional times if significant color is being removed.

10.3 Adjust the sample to a pH of 1 to 2 with sulfuric acid.

10.4 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.5 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.6 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.7 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.8 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9 Increase the temperature of the hot water bath to 95 to 100 °C. Remove the Synder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of 2-propanol. A 5-mL syringe is recommended for this operation. Attach a two-ball micro-Snyder column to the concentrator tube and prewet the column by adding about 0.5 mL of 2-propanol to the top. Place the micro-K-D apparatus on the water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 2.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Add an additional 2 mL of 2-propanol through the top of the micro-Snyder column and resume concentrating as before. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.10 Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of 2-propanol. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated at 4 °C if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with FIDGC analysis (Section 11). If the sample requires further cleanup, proceed to Section 12.

10.11 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Flame Ionization Detector Gas Chromatography

11.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 1. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

11.2 Calibrate the system daily as described in Section 7.

11.3 If the internal standard calibration procedure is used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

11.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.11 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, and the resulting peak size in area or peak height units.

11.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound may be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

11.6 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

11.7 If the measurement of the peak response is prevented by the presence of interferences, an alternative gas chromatographic procedure is required. Section 12 describes a derivatization and column chromatographic procedure which has been tested and found to be a practical means of analyzing phenols in complex extracts.

12. Derivatization and Electron Capture Detector Gas Chromatography

12.1 Pipet a 1.0-mL aliquot of the 2-propanol solution of standard or sample extract into a glass reaction vial. Add 1.0 mL of derivatizing reagent (Section 6.11). This amount of reagent is sufficient to derivatize a solution whose total phenolic content does not exceed 0.3 mg/mL.

12.2 Add about 3 mg of potassium carbonate to the solution and shake gently.

12.3 Cap the mixture and heat it for 4 h at 80 °C in a hot water bath.

12.4 Remove the solution from the hot water bath and allow it to cool.

12.5 Add 10 mL of hexane to the reaction flask and shake vigorously for 1 min. Add 3.0 mL of distilled, deionized water to the reaction flask and shake for 2 min. Decant a portion of the organic layer into a concentrator tube and cap with a glass stopper.

12.6 Place 4.0 g of silica gel into a chromatographic column. Tap the column to settle the silica gel and add about 2 g of anhydrous sodium sulfate to the top.

12.7 Preelute the column with 6 mL of hexane. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, pipet onto the column 2.0 mL of the hexane solution (Section 12.5) that contains the derivatized sample or standard. Elute the column with 10.0 mL of hexane and discard the eluate. Elute the column, in order, with: 10.0 mL of 15% toluene in hexane (Fraction 1); 10.0 mL of 40% toluene in hexane (Fraction 2); 10.0 mL of 75% toluene in hexane (Fraction 3); and 10.0 mL of 15% 2-propanol in toluene (Fraction 4). All elution mixtures are prepared on a volume: volume basis. Elution patterns for the phenolic derivatives are shown in Table 2. Fractions may be combined as desired, depending upon the specific phenols of interest or level of interferences.

12.8 Analyze the fractions by ECDGC. Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 2.

12.9 Calibrate the system daily with a minimum of three aliquots of calibration standards, containing each of the phenols of interest that are derivatized according to Section 7.5.

12.10 Inject 2 to 5 µL of the column fractions into the gas chromatograph using the solvent-flush technique. Smaller (1.0 µL) volumes can be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, and the resulting peak size in area or peak height units. If the peak response exceeds the linear range of the system, dilute the extract and reanalyze.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample analyzed by FIDGC (without derivatization) as indicated below.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Determine the concentration of individual compounds in the sample analyzed by derivatization and ECDGC according to Equation 4.

Equation 4

where:

A = Mass of underivatized phenol represented by area of peak in sample chromatogram, determined from calibration curve in Section 7.5.3 (ng).

Vi = Volume of eluate injected (µL).

Vt = Total volume of column eluate or combined fractions from which Vi was taken (µL).

Vs = Volume of water extracted in Section 10.10 (mL).

B = Total volume of hexane added in Section 12.5 (mL).

C = Volume of hexane sample solution added to cleanup column in Section 12.7 (mL).

D = Total volume of 2-propanol extract prior to derivatization (mL).

E = Volume of 2-propanol extract carried through derivatization in Section 12.1 (mL).

13.3 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Tables 1 and 2 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked as six concentrations over the range 12 to 450 µg/L.13 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships for a flame ionization detector are presented in Table 4.

References

1. 40 CFR part 136, appendix B.

2. “Determination of Phenols in Industrial and Municipal Wastewaters,” EPA 600/4–84–ABC, National Technical Information Service, PBXYZ, Springfield, Virginia 22161, November 1984.

3. Kawahara, F. K. “Microdetermination of Derivatives of Phenols and Mercaptans by Means of Electron Capture Gas Chromatography,” Analytical Chemistry, 40, 1009 (1968).

4. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

5. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

6. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

7. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

8. Provost, L. P., and Elder, R. S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

9. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

10. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methmds for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

11. Burke, J. A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

12. “Development of Detection Limits, EPA Method 604, Phenols,” Special letter report for EPA Contract 68–03–2625, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.

13. “EPA Method Study 14 Method 604-Phenols,” EPA 600/4–84–044, National Technical Information Service, PB84–196211, Springfield, Virginia 22161, May 1984.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
2-Chlorophenol 1.70 0.31
2-Nitrophenol 2.00 0.45
Phenol 3.01 0.14
2,4-Dimethylphenol 4.03 0.32
2,4-Dichlorophenol 4.30 0.39
2,4,6-Trichlorophenol 6.05 0.64
4-Chloro-3-methylphenol 7.50 0.36
2,4-Dinitrophenol 10.00 13.0
2-Methyl-4,6-dinitrophenol 10.24 16.0
Pentachlorophenol 12.42 7.4
4-Nitrophenol 24.25 2.8

Table 2—Silica Gel Fractionation and Electron Capture Gas Chromatography of PFBB Derivatives

Parent compound Percent recovery by fractiona Retention time (min) Method detection limit (µg/L)
1 2 3 4
2-Chlorophenol 90 1 3.3 0.58
2-Nitrophenol 9 90 9.1 0.77
Phenol 90 10 1.8 2.2
2,4-Dimethylphenol 95 7 2.9 0.63
2,4-Dichlorophenol 95 1 5.8 0.68
2,4,6-Trichlorophenol 50 50 7.0 0.58
4-Chloro-3-methylphenol 84 14 4.8 1.8
Pentachlorophenol 75 20 28.8 0.59
4-Nitrophenol 1 90 14.0 0.70

Table 3—QC Acceptance Criteria—Method 604

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (percent)
4-Chloro-3-methylphenol 100 16.6 56.7–113.4 49–122
2-Chlorophenol 100 27.0 54.1–110.2 38–126
2,4-Dichlorophenol 100 25.1 59.7–103.3 44–119
2,4-Dimethylphenol 100 33.3 50.4–100.0 24–118
4,6-Dinitro-2-methylphenol 100 25.0 42.4–123.6 30–136
2,4-Dinitrophenol 100 36.0 31.7–125.1 12–145
2-Nitrophenol 100 22.5 56.6–103.8 43–117
4-Nitrophenol 100 19.0 22.7–100.0 13–110
Pentachlorophenol 100 32.4 56.7–113.5 36–134
Phenol 100 14.1 32.4–100.0 23–108
2,4,6-Trichlorophenol 100 16.6 60.8–110.4 53–119

Table 4—Method Accuracy and Precision as Functions of Concentration—Method 604

Parameter Accuracy, as recovery, X′ (µg/L) Single Analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
4-Chloro-3-methylphenol 0.87C–1.97 0.11X̄–0.21 0.16X̄ + 1.41
2-Chlorophenol 0.83C–0.84 0.18X̄ + 0.20 0.21X̄ + 0.75
2,4-Dichlorophenol 0.81C + 0.48 0.17X̄–0.02 0.18X̄ + 0.62
2,4-Dimethylphenol 0.62C–1.64 0.30X̄–0.89 0.25X̄ + 0.48
4,6-Dinitro-2-methylphenol 0.84C–1.01 0.15X̄ + 1.25 0.19X̄ + 5.85
2,4-Dinitrophenol 0.80C–1.58 0.27X̄–1.15 0.29X̄ + 4.51
2-Nitrophenol 0.81C–0.76 0.15X̄ + 0.44 0.14X̄ + 3.84
4-Nitrophenol 0.46C + 0.18 0.17X̄ + 2.43 0.19X̄ + 4.79
Pentachlorophenol 0.83C + 2.07 0.22X̄–0.58 0.23X̄ + 0.57
Phenol 0.43C + 0.11 0.20X̄–0.88 0.17X̄ + 0.77
2,4,6-Trichlorophenol 0.86C–0.40 0.10X̄ + 0.53 0.13X̄ + 2.40

Method 605—Benzidines

1. Scope and Application

1.1 This method covers the determination of certain benzidines. The following parameters can be determined by this method:

Parameter Storet No CAS No.
Benzidine 39120 92–87–5
3,3′-Dichlorobenzidine 34631 91–94–1

1.2 This is a high performance liquid chromatography (HPLC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for the compounds above, identifications should be supported by at least one additional qualitative technique. This method describes electrochemical conditions at a second potential which can be used to confirm measurements made with this method. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of the interferences in the sample matrix.

1.4 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC instrumentation and in the interpretation of liquid chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with chloroform using liquid-liquid extractions in a separatory funnel. The chloroform extract is extracted with acid. The acid extract is then neutralized and extracted with chloroform. The final chloroform extract is exchanged to methanol while being concentrated using a rotary evaporator. The extract is mixed with buffer and separated by HPLC. The benzidine compounds are measured with an electrochemical detector.2

2.2 The acid back-extraction acts as a general purpose cleanup to aid in the elimination of interferences.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures that are inherent in the extraction step are used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3 Some dye plant effluents contain large amounts of components with retention times closed to benzidine. In these cases, it has been found useful to reduce the electrode potential in order to eliminate interferences and still detect benzidine. (See Section 12.7.)

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health harzard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4 6 for the information of the analyst.

4.2 The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzidine and 3,3′-dichlorobenzidine. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

4.3 Exposure to chloroform should be minimized by performing all extractions and extract concentrations in a hood or other well-ventiliated area.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested):

5.2.1 Separatory funnels—2000, 1000, and 250-mL, with Teflon stopcock.

5.2.2 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.3 Rotary evaporator.

5.2.4 Flasks—Round bottom, 100–mL, with 24/40 joints.

5.2.5 Centrifuge tubes—Conical, graduated, with Teflon-lined screw caps.

5.2.6 Pipettes—Pasteur, with bulbs.

5.3 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.4 High performance liquid chromatograph (HPLC)—An analytical system complete with column supplies, high pressure syringes, detector, and compatible recorder. A data system is recommended for measuring peak areas and retention times.

5.4.1 Solvent delivery system—With pulse damper, Altex 110A or equivalent.

5.4.2 Injection valve (optional)—Waters U6K or equivalent.

5.4.3 Electrochemical detector—Bioanalytical Systems LC–2A with glassy carbon electrode, or equivalent. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

5.4.4 Electrode polishing kit—Princeton Applied Research Model 9320 or equivalent.

5.4.5 Column—Lichrosorb RP–2, 5 micron particle diameter, in a 25 cm × 4.6 mm ID stainless steel column. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Sodium hydroxide solution (5 N)—Dissolve 20 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3 Sodium hydroxide solution (1 M)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 1 L.

6.4 Sodium thiosulfate—(ACS) Granular.

6.5 Sodium tribasic phosphate (0.4 M)—Dissolve 160 g of trisodium phosphate decahydrate (ACS) in reagent water and dilute to 1 L.

6.6 Sulfuric acid (1 + 1)—Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.7 Sulfuric acid (1 M)—Slowly, add 58 mL of H2SO4 (ACS, sp. gr. 1.84) to reagent water and dilute to 1 L.

6.8 Acetate buffer (0.1 M, pH 4.7)—Dissolve 5.8 mL of glacial acetic acid (ACS) and 13.6 g of sodium acetate trihydrate (ACS) in reagent water which has been purified by filtration through a RO–4 Millipore System or equivalent and dilute to 1 L.

6.9 Acetonitrile, chloroform (preserved with 1% ethanol), methanol—Pesticide quality or equivalent.

6.10 Mobile phase—Place equal volumes of filtered acetonitrile (Millipore type FH filter or equivalent) and filtered acetate buffer (Millipore type GS filter or equivalent) in a narrow-mouth, glass container and mix thoroughly. Prepare fresh weekly. Degas daily by sonicating under vacuum, by heating and stirring, or by purging with helium.

6.11 Stock standard solutions (1.00 µg/µL)—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions.

6.11.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in methanol and dilute to volume in a 10–mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish chromatographic operating conditions equivalent to those given in Table 1. The HPLC system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with mobile phase. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using syringe injections of 5 to 25 µL or a constant volume injection loop, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with mobile phase. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using syringe injections of 5 to 25 µL or a constant volume injection loop, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF = (As)(Cis (Ais)(Cs)

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound. If serious loss of response occurs, polish the electrode and recalibrate.

7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.9, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing benzidine and/or 3,3′-dichlorobenzidine at a concentration of 50 µg/mL each in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at a concentration of 50 µg/L by adding 1.00 mL of QC check sample concentrate to each of four 1–L-L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 50 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none

(2) the larger of either 5 times higher than the expected background concentration or 50 µg/L.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 50 µg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as HPLC with a dissimilar column, gas chromatography, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C and stored in the dark from the time of collection until extraction. Both benzidine and 3,3′-dichlorobenzidine are easily oxidized. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.9 Field test kits are available for this purpose. After mixing, adjust the pH of the sample to a range of 2 to 7 with sulfuric acid.

9.3 If 1,2-diphenylhydrazine is likely to be present, adjust the pH of the sample to 4.0 ±0.2 to prevent rearrangement to benzidine.

9.4 All samples must be extracted within 7 days of collection. Extracts may be held up to 7 days before analysis, if stored under an inert (oxidant free) atmosphere.2 The extract should be protected from light.

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 6.5 to 7.5 with sodium hydroxide solution or sulfuric acid.

10.2 Add 100 mL of chloroform to the sample bottle, seal, and shake 30 s to rinse the inner surface. (Caution: Handle chloroform in a well ventilated area.) Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the chloroform extract in a 250-mL separatory funnel.

10.3 Add a 50-mL volume of chloroform to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the separatory funnel. Perform a third extraction in the same manner.

10.4 Separate and discard any aqueous layer remaining in the 250-mL separatory funnel after combining the organic extracts. Add 25 mL of 1 M sulfuric acid and extract the sample by shaking the funnel for 2 min. Transfer the aqueous layer to a 250-mL beaker. Extract with two additional 25-mL portions of 1 M sulfuric acid and combine the acid extracts in the beaker.

10.5 Place a stirbar in the 250-mL beaker and stir the acid extract while carefully adding 5 mL of 0.4 M sodium tribasic phosphate. While monitoring with a pH meter, neutralize the extract to a pH between 6 and 7 by dropwise addition of 5 N sodium hydroxide solution while stirring the solution vigorously. Approximately 25 to 30 mL of 5 N sodium hydroxide solution will be required and it should be added over at least a 2-min period. Do not allow the sample pH to exceed 8.

10.6 Transfer the neutralized extract into a 250-mL separatory funnel. Add 30 mL of chloroform and shake the funnel for 2 min. Allow the phases to separate, and transfer the organic layer to a second 250-mL separatory funnel.

10.7 Extract the aqueous layer with two additional 20-mL aliquots of chloroform as before. Combine the extracts in the 250-mL separatory funnel.

10.8 Add 20 mL of reagent water to the combined organic layers and shake for 30 s.

10.9 Transfer the organic extract into a 100-mL round bottom flask. Add 20 mL of methanol and concentrate to 5 mL with a rotary evaporator at reduced pressure and 35 °C. An aspirator is recommended for use as the source of vacuum. Chill the receiver with ice. This operation requires approximately 10 min. Other concentration techniques may be used if the requirements of Section 8.2 are met.

10.10 Using a 9-in. Pasteur pipette, transfer the extract to a 15-mL, conical, screw-cap centrifuge tube. Rinse the flask, including the entire side wall, with 2-mL portions of methanol and combine with the original extract.

10.11 Carefully concentrate the extract to 0.5 mL using a gentle stream of nitrogen while heating in a 30 °C water bath. Dilute to 2 mL with methanol, reconcentrate to 1 mL, and dilute to 5 mL with acetate buffer. Mix the extract thoroughly. Cap the centrifuge tube and store refrigerated and protected from light if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with HPLC analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.12 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1,000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

12. High Performance Liquid Chromatography

12.1 Table 1 summarizes the recommended operating conditions for the HPLC. Included in this table are retention times, capacity factors, and MDL that can be achieved under these conditions. An example of the separations achieved by this HPLC column is shown in Figure 1. Other HPLC columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met. When the HPLC is idle, it is advisable to maintain a 0.1 mL/min flow through the column to prolong column life.

12.2 Calibrate the system daily as described in Section 7.

12.3 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the instrument.

12.4 Inject 5 to 25 µL of the sample extract or standard into the HPLC. If constant volume injection loops are not used, record the volume injected to the nearest 0.05 µL, and the resulting peak size in area or peak height units.

12.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6 If the response for a peak exceeds the working range of the system, dilute the extract with mobile phase and reanalyze.

12.7 If the measurement of the peak response for benzidine is prevented by the presence of interferences, reduce the electrode potential to + 0.6 V and reanalyze. If the benzidine peak is still obscured by interferences, further cleanup is required.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7 × MDL to 3000 × MDL.10

14.3 This method was tested by 17 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 70 µg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References

1. 40 CFR part 136, appendix B.

2. “Determination of Benzidines in Industrial and Muncipal Wastewaters,” EPA 600/4–82–022, National Technical Information Service, PB82–196320, Springfield, Virginia 22161, April 1982.

3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

10. “EPA Method Study 15, Method 605 (Benzidines),” EPA 600/4–84–062, National Technical Information Service, PB84–211176, Springfield, Virginia 22161, June 1984.

11. “EPA Method Validation Study 15, Method 605 (Benzidines),” Report for EPA Contract 68–03–2624 (In preparation).

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Column capacity factor (k′) Method detection limit (µg/L)
Benzidine 6.1 1.44 0.08
3,3′-Dichlorobenzidine 12.1 3.84 0.13

Table 2—QC Acceptance Criteria—Method 605

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (percent)
Benzidine 50 18.7 9.1–61.0 D–140
3.3′-Dichlorobenzidine 50 23.6 18.7–50.0 5–128

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 605

Parameter Accuracy, as recovery, X′(µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
Benzidine 0.70C + 0.06 0.28X̄ + 0.19 0.40X̄ + 0.18
3,3′-Dichlorobenzidine 0.66C + 0.23 0.39X̄−0.05 0.38X̄ + 0.02

Method 606—Phthalate Ester

1. Scope and Application

1.1 This method covers the determination of certain phthalate esters. The following parameters can be determined by this method:

Parameter STORET No. CAS No.
Bis(2-ethylhexyl) phthalate 39100 117–81–7
Butyl benzyl phthalate 34292 85–68–7
Di-n-butyl phthalate 39110 84–74–2
Diethyl phthalate 34336 84–66–2
Dimethyl phthalate 34341 131–11–3
Di-n-octyl phthalate 34596 117–84–0

1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 The sample extraction and concentration steps in this method are essentially the same as in Methods 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the phthalate esters are then measured with an electron capture detector.2

2.2 Analysis for phthalates is especially complicated by their ubiquitous occurrence in the environment. The method provides Florisil and alumina column cleanup procedures to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Phthalate esters are contaminants in many products commonly found in the laboratory. It is particularly important to avoid the use of plastics because phthalates are commonly used as plasticizers and are easily extracted from plastic materials. Serious phthalate contamination can result at any time, if consistent quality control is not practiced. Great care must be experienced to prevent such contamination. Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination.4 5

3.3 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified6 8 for the information of the analyst.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1–qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only).

5.2.1 Separatory funnel—2–L, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Chromatographic column—300 mm long × 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K–420540–0213 or equivalent).

5.2.4 Concentrator tube, Kuderna-Danish—10–mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5 Evaporative flask, Kuderna-Danish—500–mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.6 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.7 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).

5.2.8 Vials—10 to 15–mL, amber glass, with Teflon-lined screw cap.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6 Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1 Column 1—1.8 m long × 4 mm ID glass, packed with 1.5% SP–2250/1.95% SP–2401 Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statemelts in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2 Column 2—1.8 m long × 4 mm ID glass, packed with 3% OV–1 on Supelcoport (100/120 mesh) or equivalent.

5.6.3 Detector—Electron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Acetone, hexane, isooctane, methylene chloride, methanol—Pesticide quality or equivalent.

6.3 Ethyl ether—nanograde, redistilled in glass if necessary.

6.3.1 Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P1126–8, and other suppliers.)

6.3.2 Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.4 Sodium sulfate—(ACS) Granular, anhydrous. Several levels of purification may be required in order to reduce background phthalate levels to an acceptable level: 1) Heat 4 h at 400 °C in a shallow tray, 2) Heat 16 h at 450 to 500 °C in a shallow tray, 3) Soxhlet extract with methylene chloride for 48 h.

6.5 Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. To prepare for use, place 100 g of Florisil into a 500-mL beaker and heat for approximately 16 h at 40 °C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room temperature. When cool add 3 mL of reagent water. Mix thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. Keep the bottle sealed tightly.

6.6 Alumina—Neutral activity Super I, W200 series (ICN Life Sciences Group, No. 404583). To prepare for use, place 100 g of alumina into a 500-mL beaker and heat for approximately 16 h at 400 °C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room temperature. When cool add 3 mL of reagent water. Mix thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. Keep the bottle sealed tightly.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish gas chromatograph operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepared calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF = (As)(Cis (Ais)(Cs)

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality contrml (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetone: butyl benzyl phthalate, 10 µg/mL; bis(2-ethylhexyl) phthalate, 50 µg/mL; di-n-octyl phthalate, 50 µg/mL; any other phthlate, 25 µg/mL. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agancy, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none

(2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.9 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.9

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices10 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel.

10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phrase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentrator devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7 Increase the temperature of the hot water bath to about 80 °C. Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Adjust the extract volume to 10 mL. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11. Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2 If the entire extract is to be cleaned up by one of the following procedures, it must be concentrated to 2.0 mL. To the concentrator tube in Section 10.8, add a clean boiling chip and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (80 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of hexane. Adjust the final volume to 2.0 mL and proceed with one of the following cleanup procedures.

11.3 Florisil column cleanup for phthalate esters:

11.3.1 Place 10 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top.

11.3.2 Preelute the column with 40 mL of hexane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 40 mL of hexane and continue the elution of the column. Discard this hexane eluate.

11.3.3 Next, elute the column with 100 mL of 20% ethyl ether in hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction as in Section 10.6. No solvent exchange is necessary. Adjust the volume of the cleaned up extract to 10 mL in the concentrator tube and analyze by gas chromatography (Section 12).

11.4 Alumina column cleanup for phthalate esters:

11.4.1 Place 10 g of alumina into a chromatographic column. Tap the column to settle the alumina and add 1 cm of anhydrous sodium sulfate to the top.

11.4.2 Preelute the column with 40 mL of hexane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 35 mL of hexane and continue the elution of the column. Discard this hexane eluate.

11.4.3 Next, elute the column with 140 mL of 20% ethyl ether in hexane (V/V) into a 500-mL K-D flask equipped with a 10–mL concentrator type. Concentrate the collected fraction as in Section 10.6. No solvent exchange is necessary. Adjust the volume of the cleaned up extract to 10 mL in the concentrator tube and analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2 Calibrate the system daily as described in Section 7.

12.3 If the internal standard calibration procedure is being used, the internal staldard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4 Inject 2 to 5 µL of the sample extract or standard into the gas-chromatograph using the solvent-flush technique.11 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, and the resulting peak size in area or peak height units.

12.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 5 × MDL to 1000 × MDL with the following exceptions: dimethyl and diethyl phthalate recoveries at 1000 × MDL were low (70%); bis-2-ethylhexyl and di-n-octyl phthalate recoveries at 5 × MDL were low (60%).12

14.3 This method was tested by 16 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.7 to 106 µg/L.13 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References

1. 40 CFR part 136, appendix B.

2. “Determination of Phthalates in Industrial and Muncipal Wastewaters,” EPA 600/4–81–063, National Technical Information Service, PB81–232167, Springfield, Virginia 22161, July 1981.

3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. Giam, C.S., Chan, H.S., and Nef, G.S. “Sensitive Method for Determination of Phthalate Ester Plasticizers in Open-Ocean Biota Samples,” Analytical Chemistry, 47, 2225 (1975).

5. Giam, C.S., and Chan, H.S. “Control of Blanks in the Analysis of Phthalates in Air and Ocean Biota Samples,” U.S. National Bureau of Standards, Special Publication 442, pp. 701–708, 1976.

6. “Carcinogens—Working with Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

7. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

8. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

9. Provost L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

10. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

11. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

12. “Method Detection Limit and Analytical Curve Studies, EPA Methods 606, 607, and 608,” Special letter report for EPA Contract 68–03–2606, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

13. “EPA Method Study 16 Method 606 (Phthalate Esters),” EPA 600/4–84–056, National Technical Information Service, PB84–211275, Springfield, Virginia 22161, June 1984.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Column 1 Column 2
Dimethyl phthalate 2.03 0.95 0.29
Diethyl phthalate 2.82 1.27 0.49
Di-n-butyl phthalate 8.65 3.50 0.36
Butyl benzyl phthalate a 6.94 a 5.11 0.34
Bis(2-ethylhexyl) phthalate a 8.92 a 10.5 2.0
Di-n-octyl phthalate a 16.2 a 18.0 3.0

Table 2—QC Acceptance Criteria—Method 606

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (percent)
Bis(2-ethylhexyl) phthalate 50 38.4 1.2–55.9 D–158
Butyl benzyl phthalate 10 4.2 5.7–11.0 30–136
Di-n-butyl phthalate 25 8.9 10.3–29.6 23–136
Diethyl phthalate 25 9.0 1.9–33.4 D–149
Dimethyl phathalate 25 9.5 1.3–35.5 D–156
Di-n-octyl phthalate 50 13.4 D–50.0 D–114

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 606

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
Bis(2-ethylhexyl) phthalate 0.53C + 2.02 0.80X̄−2.54 0.73X̄−0.17
Butyl benzyl phthalate 0.82C + 0.13 0.26X̄ + 0.04 0.25X̄ + 0.07
Di-n-butyl phthalate 0.79C + 0.17 0.23X̄ + 0.20 0.29X̄ + 0.06
Diethyl phthalate 0.70C + 0.13 0.27X̄ + 0.05 0.45X̄ + 0.11
Dimethyl phthalate 0.73C + 0.17 0.26X̄ + 0.14 0.44X̄ + 0.31
Di-n-octyl phthalate 0.35C−0.71 0.38X̄ + 0.71 0.62X̄ + 0.34

Method 607—Nitrosamines

1. Scope and Application

1.1 This method covers the determination of certain nitrosamines. The following parameters can be determined by this method:

Parameter Storet No. CAS No.
N-Nitrosodimethylamine 34438 62–75–9
N-Nitrosodiphenylamine 34433 86–30–6
N-Nitrosodi-n-propylamine 34428 621–64–7

1.2 This is a gas chromatographic (GC) method applicable to the determination of the parameters listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compmunds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditimns for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for N-nitrosodi-n-propylamine. In order to confirm the presence of N-nitrosodiphenylamine, the cleanup procedure specified in Section 11.3 or 11.4 must be used. In order to confirm the presence of N-nitrosodimethylamine by GC/MS, Column 1 of this method must be substituted for the column recommended in Method 625. Confirmation of these parameters using GC-high resolution mass spectrometry or a Thermal Energy Analyzer is also recommended.1 2

1.3 The method detection limit (MDL, defined in Section 14.1)3 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is washed with dilute hydrochloric acid to remove free amines, dried, and concentrated to a volume of 10 mL or less. After the extract has been exchanged to methanol, it is separated by gas chromatography and the parameters are then measured with a nitrogen-phosphorus detector.4

2.2 The method provides Florisil and alumina column cleanup procedures to separate diphenylamine from the nitrosamines and to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.5 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3 N-Nitrosodiphenylamine is reported6-9 to undergo transnitrosation reactions. Care must be exercised in the heating or concentrating of solutions containing this compound in the presence of reactive amines.

3.4 The sensitive and selective Thermal Energy Analyzer and the reductive Hall detector may be used in place of the nitrogen-phosphorus detector when interferences are encountered. The Thermal Energy Analyzer offers the highest selectivity of the non-MS detectors.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified10-12 for the information of the analyst.

4.2 These nitrosamines are known carcinogens,13-17 therefore, utmost care must be exercised in the handling of these materials. Nitrosamine reference standards and standard solutions should be handled and prepared in a ventilated glove box within a properly ventilated room.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flowmeter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnels—2–L and 250–mL, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.4 Evaporative flask, Kuderna-Danish—500-mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.5 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.6 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).

5.2.7 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.8 Chromatographic column—Approximately 400 mm long × 22 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K–420540–0234 or equivalent), for use in Florisil column cleanup procedure.

5.2.9 Chromatographic column—Approximately 300 mm long × 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K–420540–0213 or equivalent), for use in alumina column cleanup procedure.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6 Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1 Column 1—1.8 m long × 4 mm ID glass, packed with 10% Carbowax 20 M/2% KOH on Chromosorb W-AW (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.2.

5.6.2 Column 2—1.8 m long × 4 mm ID glass, packed with 10% SP–2250 on Supel-coport (100/120 mesh) or equivalent.

5.6.3 Detector—Nitrogen-phosphorus, reductive Hall, or Thermal Energy Analyzer detector.1 2 These detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). A nitrogen-phosphorus detector was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.2.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 ml.

6.3 Sodium thiosulfate—(ACS) Granular.

6.4 Sulfuric acid (1 + 1)—Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.5 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.6 Hydrochloric acid (1 + 9)—Add one volume of concentrated HCl (ACS) to nine volumes of reagent water.

6.7 Acetone, methanol, methylene chloride, pentane—Pesticide quality or equivalent.

6.8 Ethyl ether—Nanograde, redistilled in glass if necessary.

6.8.1 Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat No. P1126–8, and other suppliers.)

6.8.2 Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.9 Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.10 Alumina—Basic activity Super I, W200 series (ICN Life Sciences Group, No. 404571, or equivalent). To prepare for use, place 100 g of alumina into a 500-mL reagent bottle and add 2 mL of reagent water. Mix the alumina preparation thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. The preparation should be homogeneous before use. Keep the bottle sealed tightly to ensure proper activity.

6.11 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.11.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in methanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with methanol. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with methanol. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF = (As)(Cis (Ais)(Cs)

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.2) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 20 µg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at a concentration of 20 µg/L by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none

(2) the larger of either 5 times higher than the expected background concentration or 20 µg/L.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were caluclated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.18 If spiking was performed at a concentration lower than 20 µg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria caluclated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44(100 S′/T)%.18

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices19 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.20 Field test kits are available for this purpose. If N-nitrosodiphenylamine is to be determined, adjust the sample pH to 7 to 10 with sodium hydroxide solution or sulfuric acid.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.4

9.4 Nitrosamines are known to be light sensitive.7 Samples should be stored in amber or foil-wrapped bottles in order to minimize photolytic decomposition.

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with sodium hydroxide solution or sulfuric acid.

10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5 Add 10 mL of hydrochloric acid to the combined extracts and shake for 2 min. Allow the layers to separate. Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If N-nitrosodiphenylamine is to be measured by gas chromatography, the analyst must first use a cleanup column to eliminate diphenylamine interference (Section 11). If N-nitrosodiphenylamine is of no interest, the analyst may proceed directly with gas chromatographic analysis (Section 12).

10.8 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-

mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. Diphenylamine, if present in the original sample extract, must be separated from the nitrosamines if N-nitrosodiphenylamine is to be determined by this method.

11.2 If the entire extract is to be cleaned up by one of the following procedures, it must be concentrated to 2.0 mL. To the concentrator tube in Section 10.7, add a clean boiling chip and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of methylene chloride to the top. Place the micr-K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of methylene chloride. Adjust the final volume to 2.0 mL and proceed with one of the following cleanup procedures.

11.3 Florisil column cleanup for nitrosamines:

11.3.1 Place 22 g of activated Florisil into a 22-mm ID chromatographic column. Tap the column to settle the Florisil and add about 5 mm of anhydrous sodium sulfate to the top.

11.3.2 Preelute the column with 40 mL of ethyl ether/pentane (15 + 85)(V/V). Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of pentane to complete the transfer.

11.3.3 Elute the column with 90 mL of ethyl ether/pentane (15 + 85)(V/V) and discard the eluate. This fraction will contain the diphenylamine, if it is present in the extract.

11.3.4 Next, elute the column with 100 mL of acetone/ethyl ether (5 + 95)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction will contain all of the nitrosamines listed in the scope of the method.

11.3.5 Add 15 mL of methanol to the collected fraction and concentrate as in Section 10.6, except use pentane to prewet the column and set the water bath at 70 to 75 °C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of pentane. Analyze by gas chromatography (Section 12).

11.4 Alumina column cleanup for nitrosamines:

11.4.1 Place 12 g of the alumina preparation (Section 6.10) into a 10-mm ID chromatographic column. Tap the column to settle the alumina and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.4.2 Preelute the column with 10 mL of ethyl ether/pentane (3 + 7)(V/V). Discard the eluate (about 2 mL) and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2 mL sample extract onto the column using an additional 2 mL of pentane to complete the transfer.

11.4.3 Just prior to exposure of the sodium sulfate layer to the air, add 70 mL of ethyl ether/pentane (3 + 7)(V/V). Discard the first 10 mL of eluate. Collect the remainder of the eluate in a 500–mL K-D flask equipped with a 10 mL concentrator tube. This fraction contains N-nitrosodiphenylamine and probably a small amount of N-nitrosodi-n-propylamine.

11.4.4 Next, elute the column with 60 mL of ethyl ether/pentane (1 + 1)(V/V), collecting the eluate in a second K-D flask equipped with a 10–mL concentrator tube. Add 15 mL of methanol to the K-D flask. This fraction will contain N-nitrosodimethylamine, most of the N-nitrosodi-n-propylamine and any diphenylamine that is present.

11.4.5 Concentrate both fractions as in Section 10.6, except use pentane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of pentane. Analyze the fractions by gas chromatography (Section 12).

12. Gas Chromatography

12.1 N-nitrosodiphenylamine completely reacts to form diphenylamine at the normal operating temperatures of a GC injection port (200 to 250 °C). Thus, N-nitrosodiphenylamine is chromatographed and detected as diphenylamine. Accurate determination depends on removal of diphenylamine that may be present in the original extract prior to GC analysis (See Section 11).

12.2 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3 Calibrate the system daily as described in Section 7.

12.4 If the extract has not been subjected to one of the cleanup procedures in Section 11, it is necessary to exchange the solvent from methylene chloride to methanol before the thermionic detector can be used. To a 1 to 10-mL volume of methylene chloride extract in a concentrator tube, add 2 mL of methanol and a clean boiling chip. Attach a two-ball micro-Snyder column to the concentrator tube. Prewet the column by adding about 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a boiling (100 °C) water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of methanol. Adjust the final volume to 2.0 mL.

12.5 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.6 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.21 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, and the resulting peak size in area or peak height units.

12.7 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.8 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.9 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.3 The MDL concentrations listed in Table 1 were obtained using reagent water.22 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 × MDL to 1000 × MDL.22

14.3 This method was tested by 17 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.8 to 55 µg/L.23 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References

1. Fine, D.H., Lieb, D., and Rufeh, R. “Principle of Operation of the Thermal Energy Analyzer for the Trace Analysis of Volatile and Non-volatile N-nitroso Compounds,” Journal of Chromatography, 107, 351 (1975).

2. Fine, D.H., Hoffman, F., Rounbehler, D.P., and Belcher, N.M. “Analysis of N-nitroso Compounds by Combined High Performance Liquid Chromatography and Thermal Energy Analysis,” Walker, E.A., Bogovski, P. and Griciute, L., Editors, N-nitroso Compounds—Analysis and Formation, Lyon, International Agency for Research on Cancer (IARC Scientific Publications No. 14), pp. 43–50 (1976).

3. 40 CFR part 136, appendix B.

4. “Determination of Nitrosamines in Industrial and Municipal Wastewaters,” EPA 600/4–82–016, National Technical Information Service, PB82–199621, Springfield, Virginia 22161, April 1982.

5. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

6. Buglass,

A. J., Challis,

B. C., and Osborn, M.R. “Transnitrosation and Decomposition of Nitrosamines,” Bogovski, P. and Walker, E.A., Editors, N-nitroso Compounds in the Environment, Lyon, International Agency for Research on Cancer (IARC Scientific Publication No. 9), pp. 94–100 (1974).

7. Burgess, E.M., and Lavanish, J.M. “Photochemical Decomposition of N-nitrosamines,” Tetrahedon Letters, 1221 (1964)

8. Druckrey, H., Preussmann, R., Ivankovic, S., and Schmahl, D. “Organotrope Carcinogene Wirkungen bei 65 Verschiedenen N-NitrosoVerbindungen an BD-Ratten,” Z. Krebsforsch., 69, 103 (1967).

9. Fiddler, W. “The Occurrence and Determination of N-nitroso Compounds,” Toxicol. Appl. Pharmacol., 31, 352 (1975).

10. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

11. “OSHA Safety and Health Standards, General Industry,” (29 CFR Part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

12. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

13. Lijinsky, W. “How Nitrosamines Cause Cancer,” New Scientist, 73, 216 (1977).

14. Mirvish, S.S. “N-Nitroso compounds: Their Chemical and in vivo Formation and Possible Importance as Environmental Carcinogens,” J. Toxicol. Environ. Health, 3, 1267 (1977).

15. “Reconnaissance of Environmental Levels of Nitrosamines in the Central United States,” EPA–330/1–77–001, National Enforcement Investigations Center, U.S. Environmental Protection Agency (1977).

16. “Atmospheric Nitrosamine Assessment Report,” Office of Air Quality Planning and Standards, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (1976).

17. “Scientific and Technical Assessment Report on Nitrosamines,” EPA–660/6–7–001, Office of Research and Development, U.S. Environmental Protection Agency (1976).

18. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value of 1.22 derived in this report.)

19. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

20. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

21. Burke, J. A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

22. “Method Detection Limit and Analytical Curve Studies EPA Methods 606, 607, and 608,” Special letter report for EPA Contract 68–03–2606, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

23. “EPA Method Study 17 Method 607—Nitrosamines,” EPA 600/4–84–051, National Technical Information Service, PB84–207646, Springfield, Virginia 22161, June 1984.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Column 1 Column 2
N-Nitrosodimethylamine 4.1 0.88 0.15
N-Nitrosodi-n-propylamine 12.1 4.2 .46
N-Nitrosodiphenylaminea b 12.8 c 6.4 .81

Table 2—QC Acceptance Criteria—Method 607

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (percent)
N-Nitrosodimethylamine 20 3.4 4.6–20.0 13–109
N-Nitrosodiphenyl 20 6.1 2.1–24.5 D–139
N-Nitrosodi-n-propylamine 20 5.7 11.5–26.8 45–146

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 607

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
N-Nitrosodimethylamine 0.37C + 0.06 0.25X̄−0.04 0.25X̄ + 0.11
N-Nitrosodiphenylamine 0.64C + 0.52 0.36X̄−1.53 0.46X̄−0.47
N-Nitrosodi-n-propylamine 0.96C−0.07 0.15X̄ + 0.13 0.21X̄ + 0.15

Method 608.3—Organochlorine Pesticides And PCBs By GC/HSD

1. Scope and Application

1.1 This method is for determination of organochlorine pesticides and polychlorinated biphenyls (PCBs) in industrial discharges and other environmental samples by gas chromatography (GC) combined with a halogen-specific detector (HSD; e.g., electron capture, electrolytic conductivity), as provided under 40 CFR 136.1. This revision is based on a previous protocol (Reference 1), on the revision promulgated October 26, 1984, on an inter-laboratory method validation study (Reference 2), and on EPA Method 1656 (Reference 16). The analytes that may be qualitatively and quantitatively determined using this method and their CAS Registry numbers are listed in Table 1.

1.2 This method may be extended to determine the analytes listed in Table 2. However, extraction or gas chromatography challenges for some of these analytes may make quantitative determination difficult.

1.3 When this method is used to analyze unfamiliar samples for an analyte listed in Table 1 or Table 2, analyte identification must be supported by at least one additional qualitative technique. This method gives analytical conditions for a second GC column that can be used to confirm and quantify measurements. Additionally, Method 625.1 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative confirmation of results for the analytes listed in Tables 1 and 2 using the extract produced by this method, and Method 1699 (Reference 18) provides high resolution GC/MS conditions for qualitative confirmation of results using the original sample. When such methods are used to confirm the identifications of the target analytes, the quantitative results should be derived from the procedure with the calibration range and sensitivity that are most appropriate for the intended application.

1.4 The large number of analytes in Tables 1 and 2 makes testing difficult if all analytes are determined simultaneously. Therefore, it is necessary to determine and perform quality control (QC) tests for the “analytes of interest” only. The analytes of interest are those required to be determined by a regulatory/control authority or in a permit, or by a client. If a list of analytes is not specified, the analytes in Table 1 must be determined, at a minimum, and QC testing must be performed for these analytes. The analytes in Table 1 and some of the analytes in Table 2 have been identified as Toxic Pollutants (40 CFR 401.15), expanded to a list of Priority Pollutants (40 CFR part 423, appendix A).

1.5 In this revision to Method 608, Chlordane has been listed as the alpha- and gamma- isomers in Table 1. Reporting may be by the individual isomers, or as the sum of the concentrations of these isomers, as requested or required by a regulatory/control authority or in a permit. Technical Chlordane is listed in Table 2 and may be used in cases where historical reporting has only been the Technical Chlordane. Toxaphene and the PCBs have been moved from Table 1 to Table 2 (Additional Analytes) to distinguish these analytes from the analytes required in quality control tests (Table 1). QC acceptance criteria for Toxaphene and the PCBs have been retained in Table 4 and may continue to be applied if desired, or if these analytes are requested or required by a regulatory/control authority or in a permit. Method 1668C (Reference 17) may be useful for determination of PCBs as individual chlorinated biphenyl congeners, and Method 1699 (Reference 18) may be useful for determination of the pesticides listed in this method. However, at the time of writing of this revision, Methods 1668C and 1699 had not been approved for use at 40 CFR part 136.

1.6 Method detection limits (MDLs; Reference 3) for the analytes in Tables 1 and some of the analytes in Table 2 are listed in those tables. These MDLs were determined in reagent water (Reference 3). Advances in analytical technology, particularly the use of capillary (open-tubular) columns, allowed laboratories to routinely achieve MDLs for the analytes in this method that are 2–10 times lower than those in the version promulgated in 1984. The MDL for an analyte in a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.6.1 EPA has promulgated this method at 40 CFR part 136 for use in wastewater compliance monitoring under the National Pollutant Discharge Elimination System (NPDES). The data reporting practices described in section 15.6 are focused on such monitoring needs and may not be relevant to other uses of the method.

1.6.2 This method includes “reporting limits” based on EPA's “minimum level” (ML) concept (see the glossary in section 23). Tables 1 and 2 contain MDL values and ML values for many of the analytes.

1.7 The separatory funnel and continuous liquid-liquid sample extraction and concentration steps in this method are essentially the same as those steps in Methods 606, 609, 611, and 612. Thus, a single sample may be extracted to measure the analytes included in the scope of each of these methods. Samples may also be extracted using a disk-based solid-phase extraction (SPE) procedure developed by the 3M Corporation and approved by EPA as an Alternate Test Procedure (ATP) for wastewater analyses in 1995 (Reference 20).

1.8 This method is performance-based. It may be modified to improve performance (e.g., to overcome interferences or improve the accuracy of results) provided all performance requirements are met.

1.8.1 Examples of allowed method modifications are described at 40 CFR 136.6. Other examples of allowed modifications specific to this method are described in section 8.1.2.

1.8.2 Any modification beyond those expressly permitted at 40 CFR 136.6 or in section 8.1.2 of this method shall be considered a major modification subject to application and approval of an alternate test procedure under 40 CFR 136.4 and 136.5.

1.8.3 For regulatory compliance, any modification must be demonstrated to produce results equivalent or superior to results produced by this method when applied to relevant wastewaters (section 8.1.2).

1.9 This method is restricted to use by or under the supervision of analysts experienced in the use of GC/HSD. The laboratory must demonstrate the ability to generate acceptable results with this method using the procedure in section 8.2.

1.10 Terms and units of measure used in this method are given in the glossary at the end of the method.

2. Summary of Method

2.1 A measured volume of sample, the amount required to meet an MDL or reporting limit (nominally 1–L), is extracted with methylene chloride using a separatory funnel, a continuous liquid/liquid extractor, or disk-based solid-phase extraction equipment. The extract is dried and concentrated for cleanup, if required. After cleanup, or if cleanup is not required, the extract is exchanged into an appropriate solvent and concentrated to the volume necessary to meet the required compliance or detection limit, and analyzed by GC/HSD.

2.2 Qualitative identification of an analyte in the extract is performed using the retention times on dissimilar GC columns. Quantitative analysis is performed using the peak areas or peak heights for the analyte on the dissimilar columns with either the external or internal standard technique.

2.3 Florisil®, alumina, a C18 solid-phase cleanup, and an elemental sulfur cleanup procedure are provided to aid in elimination of interferences that may be encountered. Other cleanup procedures may be used if demonstrated to be effective for the analytes in a wastewater matrix.

3. Contamination and Interferences

3.1 Solvents, reagents, glassware, and other sample processing lab ware may yield artifacts, elevated baselines, or matrix interferences causing misinterpretation of chromatograms. All materials used in the analysis must be demonstrated free from contamination and interferences by running blanks initially and with each extraction batch (samples started through the extraction process in a given 24-hour period, to a maximum of 20 samples—see Glossary for detailed definition), as described in section 8.5. Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Where possible, labware is cleaned by extraction or solvent rinse, or baking in a kiln or oven.

3.2 Glassware must be scrupulously cleaned (Reference 4). Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and reagent water. The glassware should then be drained dry, and heated at 400 °C for 15–30 minutes. Some thermally stable materials, such as PCBs, may require higher temperatures and longer baking times for removal. Solvent rinses with pesticide quality acetone, hexane, or other solvents may be substituted for heating. Do not heat volumetric labware above 90 °C. After drying and cooling, store inverted or capped with solvent-rinsed or baked aluminum foil in a clean environment to prevent accumulation of dust or other contaminants.

3.3 Interferences by phthalate esters can pose a major problem in pesticide analysis when using the electron capture detector. The phthalate esters generally appear in the chromatogram as large late eluting peaks, especially in the 15 and 50% fractions from Florisil®. Common flexible plastics contain varying amounts of phthalates that may be extracted or leached from such materials during laboratory operations. Cross contamination of clean glassware routinely occurs when plastics are handled during extraction steps, especially when solvent-wetted surfaces are handled. Interferences from phthalates can best be minimized by avoiding use of non-fluoropolymer plastics in the laboratory. Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination (References 5 and 6). Interferences from phthalate esters can be avoided by using a microcoulometric or electrolytic conductivity detector.

3.4 Matrix interferences may be caused by contaminants co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. Interferences extracted from samples high in total organic carbon (TOC) may result in elevated baselines, or by enhancing or suppressing a signal at or near the retention time of an analyte of interest. Analyses of the matrix spike and matrix spike duplicate (Section 8.3) may be useful in identifying matrix interferences, and the cleanup procedures in Section 11 may aid in eliminating these interferences. EPA has provided guidance that may aid in overcoming matrix interferences (Reference 7); however, unique samples may require additional cleanup approaches to achieve the MDLs listed in Tables 1 and 2.

4. Safety

4.1 Hazards associated with each reagent used in this method have not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of safety data sheets (SDSs, OSHA, 29 CFR 1910.12009(g)) should also be made available to all personnel involved in sample handling and chemical analysis. Additional references to laboratory safety are available and have been identified (References 8 and 9) for the information of the analyst.

4.2 The following analytes covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: 4,4′-DDT, 4,4′-DDD, the BHCs, and the PCBs. Primary standards of these toxic analytes should be prepared in a chemical fume hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations are handled.

4.3 This method allows the use of hydrogen as a carrier gas in place of helium (section 5.8.2). The laboratory should take the necessary precautions in dealing with hydrogen, and should limit hydrogen flow at the source to prevent buildup of an explosive mixture of hydrogen in air.

5. Apparatus and Materials

Note:

Brand names and suppliers are for illustration purposes only. No endorsement is implied. Equivalent performance may be achieved using equipment and materials other than those specified here. Demonstrating that the equipment and supplies used in the laboratory achieve the required performance is the responsibility of the laboratory. Suppliers for equipment and materials in this method may be found through an on-line search. Please do not contact EPA for supplier information.

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—Amber glass bottle large enough to contain the necessary sample volume (nominally 1 L), fitted with a fluoropolymer-lined screw cap. Foil may be substituted for fluoropolymer if the sample is not corrosive. If amber bottles are not available, protect samples from light. Unless pre-cleaned, the bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must use a glass or fluoropolymer container and tubing for sample collection. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, rinse the compressible tubing thoroughly with methanol, followed by repeated rinsing with reagent water to minimize the potential for sample contamination. An integrating flow meter is required to collect flow proportional composites. The sample container must be kept refrigerated at ≤6 °C and protected from light during compositing.

5.2. Lab ware.

5.2.1 Extraction.

5.2.1.1 pH measurement.

5.2.1.1.1 pH meter, with combination glass electrode.

5.2.1.1.2 pH paper, wide range (Hydrion Papers, or equivalent).

5.2.1.2 Separatory funnel—Size appropriate to hold the sample and extraction solvent volumes, equipped with fluoropolymer stopcock.

5.2.1.3 Continuous liquid-liquid extractor—Equipped with fluoropolymer or glass connecting joints and stopcocks requiring no lubrication. (Hershberg-Wolf Extractor, Ace Glass Company, Vineland, NJ, or equivalent.)

5.2.1.3.1 Round-bottom flask, 500-mL, with heating mantle.

5.2.1.3.2 Condenser, Graham, to fit extractor.

5.2.1.4 Solid-phase extractor—90-mm filter apparatus (Figure 2) or multi-position manifold.

Note:

The approved ATP for solid-phase extraction is limited to disk-based extraction media and associated peripheral equipment.

5.2.1.4.1 Vacuum system—Capable of achieving 0.1 bar (25 inch) Hg (house vacuum, vacuum pump, or water aspirator), equipped with shutoff valve and vacuum gauge.

5.2.1.4.2 Vacuum trap—Made from 500-mL sidearm flask fitted with single-hole rubber stopper and glass tubing.

5.2.2 Filtration.

5.2.2.1 Glass powder funnel, 125- to 250-mL.

5.2.2.2 Filter paper for above, Whatman 41, or equivalent.

5.2.2.3 Prefiltering aids—90-mm 1-µm glass fiber filter or Empore® Filter Aid 400.

5.2.3 Drying column.

5.2.3.1 Chromatographic column—Approximately 400 mm long x 15 mm ID, with fluoropolymer stopcock and coarse frit filter disc (Kontes or equivalent).

5.2.3.2 Glass wool—Pyrex, extracted with methylene chloride or baked at 450 °C for 1 hour minimum.

5.2.4 Column for Florisil® or alumina cleanup—Approximately 300 mm long x 10 mm ID, with fluoropolymer stopcock. (This column is not required if cartridges containing Florisil® are used.)

5.2.5 Concentration/evaporation.

Note:

Use of a solvent recovery system with the K–D or other solvent evaporation apparatus is strongly recommended.

5.2.5.1 Kuderna-Danish concentrator.

5.2.5.1.1 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes or equivalent). Calibration must be checked at the volumes employed for extract volume measurement. A ground-glass stopper is used to prevent evaporation of extracts.

5.2.5.1.2 Evaporative flask, Kuderna-Danish—500-mL (Kontes or equivalent). Attach to concentrator tube with connectors.

5.2.5.1.3 Snyder column, Kuderna/Danish—Three-ball macro (Kontes or equivalent).

5.2.5.1.4 Snyder column—Two-ball micro (Kontes or equivalent).

5.2.5.1.5 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C), installed in a hood using appropriate engineering controls to limit exposure to solvent vapors.

5.2.5.2 Nitrogen evaporation device—Equipped with heated bath that can be maintained at an appropriate temperature for the solvent and analytes. (N-Evap, Organomation Associates, Inc., or equivalent).

5.2.5.3 Rotary evaporator—Buchi/Brinkman-American Scientific or equivalent, equipped with a variable temperature water bath, vacuum source with shutoff valve at the evaporator, and vacuum gauge.

5.2.5.3.1 A recirculating water pump and chiller are recommended, as use of tap water for cooling the evaporator wastes large volumes of water and can lead to inconsistent performance as water temperatures and pressures vary.

5.2.5.3.2 Round-bottom flask—100-mL and 500-mL or larger, with ground-glass fitting compatible with the rotary evaporator

Note:

This equipment is used to prepare copper foil or copper powder for removing sulfur from sample extracts (see Section 6.7.4).

5.2.5.4 Automated concentrator—Equipped with glassware sufficient to concentrate 3–400 mL extract to a final volume of 1–10 mL under controlled conditions of temperature and nitrogen flow (Turbovap, or equivalent). Follow manufacturer's directions and requirements.

5.2.5.5 Boiling chips—Glass, silicon carbide, or equivalent, approximately 10/40 mesh. Heat at 400 °C for 30 minutes, or solvent rinse or Soxhlet extract with methylene chloride.

5.2.6 Solid-phase extraction disks—90-mm extraction disks containing 2 g of 8-µm octadecyl (C18) bonded silica uniformly enmeshed in a matrix of inert PTFE fibrils (3M Empore® or equivalent). The disks should not contain any organic compounds, either from the PTFE or the bonded silica, which will leach into the methylene chloride eluant. One liter of reagent water should pass through the disks in 2–5 minutes, using a vacuum of at least 25 inches of mercury.

Note:

Extraction disks from other manufacturers may be used in this procedure, provided that they use the same solid-phase materials (i.e., octadecyl bonded silica). Disks of other diameters also may be used, but may adversely affect the flow rate of the sample through the disk.

5.3 Vials.

5.3.1 Extract storage—10- to 15-mL, amber glass, with fluoropolymer-lined screw cap.

5.3.2 GC autosampler—1- to 5-mL, amber glass, with fluoropolymer-lined screw- or crimp-cap, to fit GC autosampler.

5.4 Balances.

5.4.1 Analytical—Capable of accurately weighing 0.1 mg.

5.4.2 Top loading—Capable of weighing 10 mg.

5.5 Sample cleanup.

5.5.1 Oven—For baking and storage of adsorbents, capable of maintaining a constant temperature (±5 °C) in the range of 105–250 °C.

5.5.2 Muffle furnace—Capable of cleaning glassware or baking sodium sulfate in the range of 400–450 °C.

5.5.3 Vacuum system and cartridges for solid-phase cleanup (see Section 11.2).

5.5.3.1 Vacuum system—Capable of achieving 0.1 bar (25 in.) Hg (house vacuum, vacuum pump, or water aspirator), equipped with shutoff valve and vacuum gauge.

5.5.3.2 VacElute Manifold (Analytichem International, or equivalent).

5.5.3.3 Vacuum trap—Made from 500-mL sidearm flask fitted with single-hole rubber stopper and glass tubing.

5.5.3.4 Rack for holding 50-mL volumetric flasks in the manifold.

5.5.3.5 Cartridge—Mega Bond Elute, Non-polar, C18 Octadecyl, 10 g/60 mL (Analytichem International or equivalent), used for solid-phase cleanup of sample extracts (see Section 11.2).

5.5.4 Sulfur removal tube—40- to 50-mL bottle, test tube, or Erlenmeyer flask with fluoropolymer-lined screw cap.

5.6 Centrifuge apparatus.

5.6.1 Centrifuge—Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes at 5,000 rpm minimum.

5.6.2 Centrifuge bottle—500-mL, with screw cap, to fit centrifuge.

5.6.3 Centrifuge tube—15-mL, with screw cap, to fit centrifuge.

5.7 Miscellaneous lab ware—Graduated cylinders, pipettes, beakers, volumetric flasks, vials, syringes, and other lab ware necessary to support the operations in this method.

5.8 Gas chromatograph—Dual-column with simultaneous split/splitless, temperature programmable split/splitless (PTV), or on-column injection; temperature program with isothermal holds, and all required accessories including syringes, analytical columns, gases, and detectors. An autosampler is highly recommended because it injects volumes more reproducibly than manual injection techniques. Alternatively, two separate single-column gas chromatographic systems may be employed.

5.8.1 Example columns and operating conditions.

5.8.1.1 DB–608 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 0.83-µm film thickness.

5.8.1.2 DB–1701 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 1.0-µm film thickness.

5.8.1.3 Suggested operating conditions used to meet the retention times shown in Table 3 are:

(a) Carrier gas flow rate: Approximately 7 mL/min,

(b) Initial temperature: 150 °C for 0.5 minute,

(c) Temperature program: 150–270 °C at 5 °C/min, and

(d) Final temperature: 270 °C, until trans-Permethrin elutes.

Note:

Other columns, internal diameters, film thicknesses, and operating conditions may be used, provided that the performance requirements in this method are met. However, the column pair chosen must have dissimilar phases/chemical properties in order to separate the compounds of interest in different retention time order. Columns that only differ in the length, ID, or film thickness, but use the same stationary phase do not qualify as “dissimilar.”

5.8.2 Carrier gas—Helium or hydrogen. Data in the tables in this method were obtained using helium carrier gas. If hydrogen is used, analytical conditions may need to be adjusted for optimum performance, and calibration and all QC tests must be performed with hydrogen carrier gas. See Section 4.3 for precautions regarding the use of hydrogen as a carrier gas.

5.8.3 Detector—Halogen-specific detector (electron capture detector [ECD], electrolytic conductivity detector [ELCD], or equivalent). The ECD has proven effective in the analysis of wastewaters for the analytes listed in Tables 1 and 2, and was used to develop the method performance data in Section 17 and Tables 4 and 5.

5.8.4 Data system—A computer system must be interfaced to the GC that allows continuous acquisition and storage of data from the detectors throughout the chromatographic program. The computer must have software that allows searching GC data for specific analytes, and for plotting responses versus time. Software must also be available that allows integrating peak areas or peak heights in selected retention time windows and calculating concentrations of the analytes.

6. Reagents and Standards

6.1 pH adjustment.

6.1.1 Sodium hydroxide solutions.

6.1.1.1 Concentrated (10 M)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.1.1.2 Dilute (1 M)—Dissolve 40 g NaOH in 1 L of reagent water.

6.1.2 Sulfuric acid (1+1)—Slowly add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.1.3 Hydrochloric acid—Reagent grade, 6 N.

6.2 Sodium thiosulfate—(ACS) granular.

6.3 Sodium sulfate—Sodium sulfate, reagent grade, granular anhydrous (Baker or equivalent), rinsed with methylene chloride, baked in a shallow tray at 450 °C for 1 hour minimum, cooled in a desiccator, and stored in a pre-cleaned glass bottle with screw cap which prevents moisture from entering. If, after heating, the sodium sulfate develops a noticeable grayish cast (due to the presence of carbon in the crystal matrix), that batch of reagent is not suitable for use and should be discarded. Extraction with methylene chloride (as opposed to simple rinsing) and baking at a lower temperature may produce sodium sulfate suitable for use.

6.4 Reagent water—Reagent water is defined as water in which the analytes of interest and interfering compounds are not observed at the MDLs of the analytes in this method.

6.5 Solvents—Methylene chloride, acetone, methanol, hexane, acetonitrile, and isooctane, high purity pesticide quality, or equivalent, demonstrated to be free of the analytes and interferences (section 3). Purification of solvents by distillation in all-glass systems may be required.

Note:

The standards and final sample extracts must be prepared in the same final solvent.

6.6 Ethyl ether—Nanograde, redistilled in glass if necessary. Ethyl ether must be shown to be free of peroxides before use, as indicated by EM Laboratories Quant test strips (available from Scientific Products Co. and other suppliers). Procedures recommended for removal of peroxides are provided with the test strips. After removal of peroxides, add 20 mL of ethyl alcohol preservative to each liter of ether.

6.7 Materials for sample cleanup.

6.7.1 Florisil®—PR grade (60/100 mesh), activated at 650–700 °C, stored in the dark in a glass container with fluoropolymer-lined screw cap. Activate each batch immediately prior to use for 16 hours minimum at 130 °C in a foil-covered glass container and allow to cool. Alternatively, 500 mg cartridges (J.T. Baker, or equivalent) may be used.

6.7.1.1 Cartridge certification—Each cartridge lot must be certified to ensure recovery of the analytes of interest and removal of 2,4,6-trichlorophenol. To make the test mixture, add the trichlorophenol solution (section 6.7.1.3) to the same standard used to prepare the Quality Control Check Sample (section 6.8.3). Transfer the mixture to the column and dry the column. Pre-elute with three 10-mL portions of elution solvent, drying the column between elutions. Elute the cartridge with 10 mL each of methanol and water, as in section 11.2.3.3.

6.7.1.2 Concentrate the eluant to per section 10.3.3, exchange to isooctane or hexane per section 10.3.3, and inject 1.0 µL of the concentrated eluant into the GC using the procedure in section 12. The recovery of all analytes (including the unresolved GC peaks) shall be within the ranges for calibration verification (section 13.6 and Table 4), the recovery of trichlorophenol shall be less than 5%, and no peaks interfering with the target analytes shall be detected. Otherwise the Florisil cartridge is not performing properly and the cartridge lot shall be rejected.

6.7.1.3 Florisil cartridge calibration solution—2,4,6-Trichlorophenol, 0.1 µg/mL in acetone.

6.7.2 SPE elution solvent—Methylene chloride:acetonitrile:hexane (50:3:47).

6.7.3 Alumina, neutral, Brockman Activity I, 80–200 mesh (Fisher Scientific certified, or equivalent). Heat in a glass bottle for 16 hours at 400 to 450 °C. Seal and cool to room temperature. Add 7% (w/w) reagent water and mix for 10 to 12 hours. Keep bottle tightly sealed.

6.7.4 Sulfur removal.

6.7.4.1 Copper foil or powder—Fisher, Alfa Aesar, or equivalent. Cut copper foil into approximately 1-cm squares. Copper must be activated before it may be used, as described below.

6.7.4.1.1 Place the quantity of copper needed for sulfur removal (section 11.5.1.3) in a ground-glass-stoppered Erlenmeyer flask or bottle. Cover the foil or powder with methanol.

6.7.4.1.2 Add HCl dropwise (0.5–1.0 mL) while swirling, until the copper brightens.

6.7.4.1.3 Pour off the methanol/HCl and rinse 3 times with reagent water to remove all traces of acid, then 3 times with acetone, then 3 times with hexane.

6.7.4.1.4 For copper foil, cover with hexane after the final rinse. Store in a stoppered flask under nitrogen until used. For the powder, dry on a rotary evaporator. Store in a stoppered flask under nitrogen until used. Inspect the copper foil or powder before each use. It must have a bright, non-oxidized appearance to be effective. Copper foil or powder that has oxidized may be reactivated using the procedure described above.

6.7.4.2 Tetrabutylammonium sulfite (TBA sulfite)—Prepare as described below.

6.7.4.2.1 Tetrabutylammonium hydrogen sulfate, [CH3(CH2)3]4NHSO4.

6.7.4.2.2 Sodium sulfite, Na2SO3.

6.7.4.2.3 Dissolve approximately 3 g tetrabutylammonium hydrogen sulfate in 100 mL of reagent water in an amber bottle with fluoropolymer-lined screw cap. Extract with three 20-mL portions of hexane and discard the hexane extracts.

6.7.4.2.4 Add 25 g sodium sulfite to produce a saturated solution. Store at room temperature. Replace after 1 month.

6.7.5 Sodium chloride—Reagent grade, prepare at 5% (w/v) solution in reagent water.

6.8 Stock standard solutions—Stock standard solutions may be prepared from pure materials, or purchased as certified solutions. Traceability must be to the National Institute of Standards and Technology (NIST) or other national or international standard, when available. Stock solution concentrations alternative to those below may be used. Because of the toxicity of some of the compounds, primary dilutions should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations of neat materials are handled. The following procedure may be used to prepare standards from neat materials.

6.8.1 Accurately weigh about 0.0100 g of pure material in a 10-mL volumetric flask. Dilute to volume in pesticide quality hexane, isooctane, or other suitable solvent. Larger volumes may be used at the convenience of the laboratory. When compound purity is assayed to be 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.

6.8.1.1 Unless stated otherwise in this method, store non-aqueous standards in fluoropolymer-lined screw-cap, or heat-sealed, glass containers, in the dark at −20 to −10 °C. Store aqueous standards; e.g., the aqueous LCS (section 8.4), in the dark at ≤6 °C, but do not freeze.

6.8.1.2 Standards prepared by the laboratory may be stored for up to one year, except when comparison with QC check standards indicates that a standard has degraded or become more concentrated due to evaporation, or unless the laboratory has data on file to prove stability for a longer period. Commercially prepared standards may be stored until the expiration date provided by the vendor, except when comparison with QC check standards indicates that a standard has degraded or become more concentrated due to evaporation, or unless the laboratory has data from the vendor on file to prove stability for a longer period.

6.8.2 Calibration solutions—It is necessary to prepare calibration solutions for the analytes of interest (section 1.4) only using an appropriate solvent (isooctane or hexane may be used). Whatever solvent is used, both the calibration standards and the final sample extracts must use the same solvent. Other analytes may be included as desired.

6.8.2.1 Prepare calibration standards for the single-component analytes of interest and surrogates at a minimum of three concentration levels (five are suggested) by adding appropriate volumes of one or more stock standards to volumetric flasks. One of the calibration standards should be at a concentration at or below the ML specified in Table 1, or 2, or as specified by a regulatory/control authority or in a permit. The ML value may be rounded to a whole number that is more convenient for preparing the standard, but must not exceed the ML value listed in Tables 1 or 2 for those analytes which list ML values. Alternatively, the laboratory may establish an ML for each analyte based on the concentration of the lowest calibration standard in a series of standards produced by the laboratory or obtained from a commercial vendor, again, provided that the ML does not exceed the ML in Table 1 and 2, and provided that the resulting calibration meets the acceptance criteria in section 7.5.2 based on the RSD, RSE, or R2.

(a) The other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC system. A minimum of six concentration levels is required for a second order, non-linear (e.g., quadratic; ax2 + bx + c = 0) calibration (section 7.5.2 or 7.6.2). Calibrations higher than second order are not allowed. A separate standard near the MDL may be analyzed as a check on sensitivity, but should not be included in the linearity assessment. The solvent for the standards must match the final solvent for the sample extracts (e.g., isooctane or hexane).

Note:

The option for non-linear calibration may be necessary to address specific instrumental techniques. However, it is not EPA's intent to allow non-linear calibration to be used to compensate for detector saturation or to avoid proper instrument maintenance.

(b) Given the number of analytes included in this method, it is highly likely that some will coelute on one or both of the GC columns used for the analysis. Divide the analytes into two or more groups and prepare separate calibration standards for each group, at multiple concentrations (e.g., a five-point calibration will require ten solutions to cover two groups of analytes). Table 7 provides information on dividing the target analytes into separate calibration mixtures that should minimize or eliminate co-elutions. This table is provided solely as guidance, based on the GC columns suggested in this method. If an analyte listed in Table 7 is not an analyte of interest in a given laboratory setting, then it need not be included in a calibration mixture.

Note:

Many commercially available standards are divided into separate mixtures to address this issue.

(c) If co-elutions occur in analysis of a sample, a co-elution on one column is acceptable so long as effective separation of the co-eluting compounds can be achieved on the second column.

6.8.2.2 Multi-component analytes (e.g., PCBs as Aroclors, and Toxaphene).

6.8.2.2.1 A standard containing a mixture of Aroclor 1016 and Aroclor 1260 will include many of the peaks represented in the other Aroclor mixtures. As a result, a multi-point initial calibration employing a mixture of Aroclors 1016 and 1260 at three to five concentrations should be sufficient to demonstrate the linearity of the detector response without the necessity of performing multi-point initial calibrations for each of the seven Aroclors. In addition, such a mixture can be used as a standard to demonstrate that a sample does not contain peaks that represent any one of the Aroclors. This standard can also be used to determine the concentrations of either Aroclor 1016 or Aroclor 1260, should they be present in a sample. Therefore, prepare a minimum of three calibration standards containing equal concentrations of both Aroclor 1016 and Aroclor 1260 by dilution of the stock standard with isooctane or hexane. The concentrations should correspond to the expected range of concentrations found in real samples and should bracket the linear range of the detector.

6.8.2.2.2 Single standards of each of the other five Aroclors are required to aid the analyst in pattern recognition. Assuming that the Aroclor 1016/1260 standards described in Section 6.8.2.2.1 have been used to demonstrate the linearity of the detector, these single standards of the remaining five Aroclors also may be used to determine the calibration factor for each Aroclor. Prepare a standard for each of the other Aroclors. The concentrations should generally correspond to the mid-point of the linear range of the detector, but lower concentrations may be employed at the discretion of the analyst based on project requirements.

6.8.2.2.3 For Toxaphene, prepare a minimum of three calibration standards containing Toxaphene by dilution of the stock standard with isooctane or hexane. The concentrations should correspond to the expected range of concentrations found in real samples and should bracket the linear range of the detector.

6.8.3 Quality Control (QC) Check Sample Concentrate—Prepare one or more mid-level standard mixtures (concentrates) in acetone (or other water miscible solvent). The concentrate is used as the spiking solution with which to prepare the Demonstration of Capabilities (DOC) samples, the Laboratory Control Sample (LCS), and Matrix Spike (MS) and Matrix Spike Duplicate (MSD) samples described in section 8. If prepared by the laboratory (as opposed the purchasing it from a commercial supplier), the concentrate must be prepared independently from the standards used for calibration, but may be prepared from the same source as the second-source standard used for calibration verification (section 7.7). Regardless of the source, the concentrate must be in a water-miscible solvent, as noted above. The concentrate is used to prepare the DOC and LCS (sections 8.2.1 and 8.4) and MS/MSD samples (section 8.3). Depending on the analytes of interest for a given sample (see Section 1.4), multiple solutions and multiple LCS or MS/MSD samples may be required to account for co-eluting analytes. However, a co-elution on one column is acceptable so long as effective separation of the co-eluting compounds can be achieved on the second column. In addition, the concentrations of the MS/MSD samples should reflect any relevant compliance limits for the analytes of interest, as described in section 8.3.1. If a custom spiking solution is required for a specific discharge (section 8.3.1), prepare it separately from the DOC and LCS solution.

Note:

Some commercially available standards are divided into separate mixtures to address the co-elution issue.

6.8.4 Calibration Verification Standards—In order to verify the results of the initial calibration standards, prepare one or more mid-level standard mixtures in isooctane or hexane, using standards obtained from a second source (different manufacturer or different certified lot from the calibration standards). These standards will be analyzed to verify the accuracy of the calibration (sections 7.7 and 13.6.2). As with the QC sample concentrate in section 6.8.3, multiple solutions may be required to address co-elutions among all of the analytes.

6.8.5 Internal standard solution—If the internal standard calibration technique is to be used, prepare pentachloronitrobenzene (PCNB) at a concentration of 10 µg/mL in ethyl acetate. Alternative and multiple internal standards; e.g., tetrachloro-m-xylene, 4,4′-dibromobiphenyl, and/or decachlorobiphenyl may be used provided that the laboratory performs all QC tests and meets all QC acceptance criteria with the alternative or additional internal standard(s) as an integral part of this method.

6.8.6 Surrogate solution—Prepare a solution containing one or more surrogates at a concentration of 2 µg/mL in acetone. Potential surrogates include: dibutyl chlorendate (DBC), tetrachloro-m-xylene (TCMX), 4,4′-dibromobiphenyl, or decachlorobiphenyl. Alternative surrogates and concentrations may be used, provided the laboratory performs all QC tests and meets all QC acceptance criteria with the alternative surrogate(s) as an integral part of this method. If the internal standard calibration technique is used, do not use the internal standard as a surrogate.

6.8.7 DDT and endrin decomposition (breakdown) solution—Prepare a solution containing endrin at a concentration of 50 ng/mL and 4,4'-DDT at a concentration of 100 ng/mL, in isooctane or hexane. A 1-µL injection of this standard will contain 50 picograms (pg) of endrin and 100 pg of DDT. The concentration of the solution may be adjusted by the laboratory to accommodate other injection volumes such that the same masses of the two analytes are introduced into the instrument.

7. Calibration

7.1 Establish gas chromatographic operating conditions equivalent to those in Section 5.8.1 and Footnote 2 to Table 3. Alternative temperature program and flow rate conditions may be used. The system may be calibrated using the external standard technique (section 7.5) or the internal standard technique (section 7.6). It is necessary to calibrate the system for the analytes of interest (section 1.4) only.

7.2 Separately inject the mid-level calibration standard for each calibration mixture. Store the retention time on each GC column.

7.3 Injection of calibration solutions—Inject a constant volume in the range of 0.5 to 2.0 µL of each calibration solution into the GC column/detector pairs. An alternative volume (see Section 12.3) may be used provided all requirements in this method are met. Beginning with the lowest level mixture and proceeding to the highest level mixture may limit the risk of carryover from one standard to the next, but other sequences may be used. An instrument blank should be analyzed after the highest standard to demonstrate that there is no carry-over within the system for this calibration range.

7.4 For each analyte, compute, record, and store, as a function of the concentration injected, the retention time and peak area on each column/detector system. If multi-component analytes are to be analyzed, store the retention time and peak area for the three to five exclusive (unique large) peaks for each PCB or technical chlordane. Use four to six peaks for toxaphene.

7.5 External standard calibration.

7.5.1 From the calibration data (Section 7.4), calculate the calibration factor (CF) for each analyte at each concentration according to the following equation:

Where:

Cs = Concentration of the analyte in the standard (ng/mL)

As = Peak height or area

For multi-component analytes, choose a series of characteristic peaks for each analyte (3 to 5 for each Aroclor, 4 to 6 for toxaphene) and calculate individual calibration factors for each peak. Alternatively, for toxaphene, sum the areas of all of the peaks in the standard chromatogram and use the summed area to determine the calibration factor. (If this alternative is used, the same approach must be used to quantitate the analyte in the samples.)

7.5.2 Calculate the mean (average) and relative standard deviation (RSD) of the calibration factors. If the RSD is less than 20%, linearity through the origin can be assumed and the average CF can be used for calculations. Alternatively, the results can be used to fit a linear or quadratic regression of response, As, vs. concentration Cs. If used, the regression must be weighted inversely proportional to concentration. The coefficient of determination (R2) of the weighted regression must be greater than 0.920. Alternatively, the relative standard error (Reference 10) may be used as an acceptance criterion. As with the RSD, the RSE must be less than 20%. If an RSE less than 20% cannot be achieved for a quadratic regression, system performance is unacceptable and the system must be adjusted and re-calibrated.

Note:

Regression calculations are not included in this method because the calculations are cumbersome and because many GC/ECD data systems allow selection of weighted regression for calibration and calculation of analyte concentrations.

7.6 Internal standard calibration.

7.6.1 From the calibration data (Section 7.4), calculate the response factor (RF) for each analyte at each concentration according to the following equation:

Where:

As = Response for the analyte to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (ng/mL)

Cs = Concentration of the analyte to be measured (ng/mL).

7.6.2 Calculate the mean (average) and relative standard deviation (RSD) of the response factors. If the RSD is less than 15%, linearity through the origin can be assumed and the average RF can be used for calculations. Alternatively, the results can be used to prepare a calibration curve of response ratios, As/Ais, vs. concentration ratios, Cs/Cis, for the analyte. A minimum of six concentration levels is required for a non-linear (e.g., quadratic) regression. If used, the regression must be weighted inversely proportional to concentration, and the coefficient of determination of the weighted regression must be greater than 0.920. Alternatively, the relative standard error (Reference 10) may be used as an acceptance criterion. As with the RSD, the RSE must be less than 15%. If an RSE less than 15% cannot be achieved for a quadratic regression, system performance is unacceptable and the system must be adjusted and re-calibrated.

7.7 The working calibration curve, CF, or RF must be verified immediately after calibration and at the beginning and end of each 24-hour shift by the analysis of a mid-level calibration standard. The calibration verification standard(s) must be obtained from a second manufacturer or a manufacturer's batch prepared independently from the batch used for calibration (Section 6.8.4). Requirements for calibration verification are given in Section 13.6 and Table 4. Alternatively, calibration verification may be performed after a set number of injections (e.g., every 20 injections), to include injection of extracts of field samples, QC samples, instrument blanks, etc. (i.e., it is based on the number of injections performed, not sample extracts). The time for the injections may not exceed 24 hours.

Note:

The 24-hour shift begins after analysis of the combined QC standard (calibration verification) and ends 24 hours later. The ending calibration verification standard is run immediately after the last sample run during the 24-hour shift, so the beginning and ending calibration verifications are outside of the 24-hour shift. If calibration verification is based on the number of injections instead of time, then the ending verification standard for one group of injections may be used as the beginning verification for the next group of injections.

7.8 Florisil® calibration—The column cleanup procedure in Section 11.3 utilizes Florisil column chromatography. Florisil® from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil® which is used, use of the lauric acid value (Reference 11) is suggested. The referenced procedure determines the adsorption from a hexane solution of lauric acid (mg) per g of Florisil®. The amount of Florisil® to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g. If cartridges containing Florisil® are used, then this step is not necessary.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and ongoing analysis of spiked samples and blanks to evaluate and document data quality. The laboratory must maintain records to document the quality of data generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet performance requirements of this method. A quality control check standard (LCS, section 8.4) must be prepared and analyzed with each batch of samples to confirm that the measurements were performed in an in-control mode of operation. A laboratory may develop its own performance criteria (as QC acceptance criteria), provided such criteria are as or more restrictive than the criteria in this method.

8.1.1 The laboratory must make an initial demonstration of the capability (IDC) to generate acceptable precision and recovery with this method. This demonstration is detailed in Section 8.2. On a continuing basis, the laboratory must repeat demonstration of capability (DOC) at least annually.

8.1.2 In recognition of advances that are occurring in analytical technology, and to overcome matrix interferences, the laboratory is permitted certain options (section 1.8 and 40 CFR 136.6(b) [Reference 12]) to improve separations or lower the costs of measurements. These options may include alternative extraction (e.g., other solid-phase extraction materials and formats), concentration, and cleanup procedures, and changes in GC columns (Reference 12). Alternative determinative techniques, such as the substitution of spectroscopic or immunoassay techniques, and changes that degrade method performance, are not allowed. If an analytical technique other than the techniques specified in this method is used, that technique must have a specificity equal to or greater than the specificity of the techniques in this method for the analytes of interest. The laboratory is also encouraged to participate in performance evaluation studies (see section 8.8).

8.1.2.1 Each time a modification listed above is made to this method, the laboratory is required to repeat the procedure in section 8.2. If the detection limit of the method will be affected by the change, the laboratory is required to demonstrate that the MDLs (40 CFR part 136, appendix B) are lower than one-third the regulatory compliance limit or as low as the MDLs in this method, whichever are greater. If calibration will be affected by the change, the instrument must be recalibrated per section 7. Once the modification is demonstrated to produce results equivalent or superior to results produced by this method as written, that modification may be used routinely thereafter, so long as the other requirements in this method are met (e.g., matrix spike/matrix spike duplicate recovery and relative percent difference).

8.1.2.1.1 If an allowed method modification, is to be applied to a specific discharge, the laboratory must prepare and analyze matrix spike/matrix spike duplicate (MS/MSD) samples (section 8.3) and LCS samples (section 8.4). The laboratory must include surrogates (Section 8.7) in each of the samples. The MS/MSD and LCS samples must be fortified with the analytes of interest (section 1.4). If the modification is for nationwide use, MS/MSD samples must be prepared from a minimum of nine different discharges (See section 8.1.2.1.2), and all QC acceptance criteria in this method must be met. This evaluation only needs to be performed once other than for the routine QC required by this method (for example it could be performed by the vendor of an alternative material) but any laboratory using that specific material must have the results of the study available. This includes a full data package with the raw data that will allow an independent reviewer to verify each determination and calculation performed by the laboratory (see section 8.1.2.2.5, items (a)–(q)).

8.1.2.1.2 Sample matrices on which MS/MSD tests must be performed for nationwide use of an allowed modification:

(a) Effluent from a publicly owned treatment works (POTW).

(b) ASTM D5905 Standard Specification for Substitute Wastewater.

(c) Sewage sludge, if sewage sludge will be in the permit.

(d) ASTM D1141 Standard Specification for Substitute Ocean Water, if ocean water will be in the permit.

(e) Untreated and treated wastewaters up to a total of nine matrix types (see https://www.epa.gov/eg/industrial-effluent-guidelines for a list of industrial categories with existing effluent guidelines).

(i) At least one of the above wastewater matrix types must have at least one of the following characteristics:

(A) Total suspended solids greater than 40 mg/L.

(B) Total dissolved solids greater than 100 mg/L.

(C) Oil and grease greater than 20 mg/L.

(D) NaCl greater than 120 mg/L.

(E) CaCO3 greater than 140 mg/L.

(ii) The interim acceptance criteria for MS, MSD recoveries that do not have recovery limits in Table 4 or developed in section 8.3.3, and for surrogates that do not have recovery limits developed in section 8.6, must be no wider than 60–140%, and the relative percent difference (RPD) of the concentrations in the MS and MSD that do not have RPD limits in Table 4 or developed in section 8.3.3, must be less than 30%. Alternatively, the laboratory may use the laboratory's in-house limits if they are tighter.

(f) A proficiency testing (PT) sample from a recognized provider, in addition to tests of the nine matrices (section 8.1.2.1.1).

8.1.2.2 The laboratory must maintain records of modifications made to this method. These records include the following, at a minimum:

8.1.2.2.1 The names, titles, and business street addresses, telephone numbers, and email addresses, of the analyst(s) that performed the analyses and modification, and of the quality control officer that witnessed and will verify the analyses and modifications.

8.1.2.2.2 A list of analytes, by name and CAS Registry number.

8.1.2.2.3 A narrative stating reason(s) for the modifications.

8.1.2.2.4 Results from all quality control (QC) tests comparing the modified method to this method, including:

(a) Calibration (section 7).

(b) Calibration verification (section 13.6).

(c) Initial demonstration of capability (section 8.2).

(d) Analysis of blanks (section 8.5).

(e) Matrix spike/matrix spike duplicate analysis (section 8.3).

(f) Laboratory control sample analysis (section 8.4).

8.1.2.2.5 Data that will allow an independent reviewer to validate each determination by tracing the instrument output (peak height, area, or other signal) to the final result. These data are to include:

(a) Sample numbers and other identifiers.

(b) Extraction dates.

(c) Analysis dates and times.

(d) Analysis sequence/run chronology.

(e) Sample weight or volume (section 10).

(f) Extract volume prior to each cleanup step (sections 10 and 11).

(g) Extract volume after each cleanup step (section 11).

(h) Final extract volume prior to injection (sections 10 and 12).

(i) Injection volume (sections 12.3 and 13.2).

(j) Sample or extract dilution (section 15.4).

(k) Instrument and operating conditions.

(l) Column (dimensions, material, etc.).

(m) Operating conditions (temperatures, flow rates, etc.).

(n) Detector (type, operating conditions, etc.).

(o) Chromatograms and other recordings of raw data.

(p) Quantitation reports, data system outputs, and other data to link the raw data to the results reported.

(q) A written Standard Operating Procedure (SOP).

8.1.2.2.6 Each individual laboratory wishing to use a given modification must perform the start-up tests in section 8.1.2 (e.g., DOC, MDL), with the modification as an integral part of this method prior to applying the modification to specific discharges. Results of the DOC must meet the QC acceptance criteria in Table 5 for the analytes of interest (section 1.4), and the MDLs must be equal to or lower than the MDLs in Tables 1 and 2 for the analytes of interest.

8.1.3 Before analyzing samples, the laboratory must analyze a blank to demonstrate that interferences from the analytical system, lab ware, and reagents, are under control. Each time a batch of samples is extracted or reagents are changed, a blank must be extracted and analyzed as a safeguard against laboratory contamination. Requirements for the blank are given in section 8.5.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze samples to monitor and evaluate method and laboratory performance on the sample matrix. The procedure for spiking and analysis is given in section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through analysis of a quality control check sample (laboratory control sample, LCS; on-going precision and recovery sample, OPR) that the measurement system is in control. This procedure is described in Section 8.4.

8.1.6 The laboratory should maintain performance records to document the quality of data that is generated. This procedure is given in section 8.7.

8.1.7 The large number of analytes tested in performance tests in this method present a substantial probability that one or more will fail acceptance criteria when all analytes are tested simultaneously, and a re-test (reanalysis) is allowed if this situation should occur. If, however, continued re-testing results in further repeated failures, the laboratory should document the failures and either avoid reporting results for the analytes that failed or report the problem and failures with the data. A QC failure does not relieve a discharger or permittee of reporting timely results.

8.2 Demonstration of capability (DOC)—To establish the ability to generate acceptable recovery and precision, the laboratory must perform the DOC in sections 8.2.1 through 8.2.6 for the analytes of interest initially and in an on-going manner at least annually. The laboratory must also establish MDLs for the analytes of interest using the MDL procedure at 40 CFR part 136, appendix B. The laboratory's MDLs must be equal to or lower than those listed in Tables 1 or 2, or lower than one-third the regulatory compliance limit, whichever is greater. For MDLs not listed in Tables 1 or 2, the laboratory must determine the MDLs using the MDL procedure at 40 CFR part 136, appendix B under the same conditions used to determine the MDLs for the analytes listed in Tables 1 and 2. When analyzing the PCBs as Aroclors, it is only necessary to establish an MDL for one of the multi-component analytes (e.g., PCB 1254), or the mixture of Aroclors 1016 and 1260 may be used to establish MDLs for all of the Aroclors. Similarly, MDLs for other multi-component analytes (e.g., Chlordanes) may be determined using only one of the major components. All procedures used in the analysis, including cleanup procedures, must be included in the DOC.

8.2.1 For the DOC, a QC check sample concentrate containing each analyte of interest (section 1.4) is prepared in a water-miscible solvent using the solution in section 6.8.3.

Note:

QC check sample concentrates are no longer available from EPA.

8.2.2 Using a pipet or syringe, prepare four QC check samples by adding an appropriate volume of the concentrate and of the surrogate(s) to each of four 1–L aliquots of reagent water. Swirl or stir to mix.

8.2.3 Extract and analyze the well-mixed QC check samples according to the method beginning in section 10.

8.2.4 Calculate the average percent recovery (X) and the standard deviation (s) of the percent recovery for each analyte using the four results.

8.2.5 For each analyte, compare s and X with the corresponding acceptance criteria for precision and recovery in Table 4. For analytes in Table 2 that are not listed in Table 4, QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 12 and 13). If s and X for all analytes of interest meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for recovery, system performance is unacceptable for that analyte.

Note:

The large number of analytes in Tables 1 and 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when many or all analytes are determined simultaneously.

8.2.6 When one or more of the analytes tested fail at least one of the acceptance criteria, repeat the test for only the analytes that failed. If results for these analytes pass, system performance is acceptable and analysis of samples and blanks may proceed. If one or more of the analytes again fail, system performance is unacceptable for the analytes that failed the acceptance criteria. Correct the problem and repeat the test (section 8.2). See section 8.1.7 for disposition of repeated failures.

Note:

To maintain the validity of the test and re-test, system maintenance and/or adjustment is not permitted between this pair of tests.

8.3 Matrix spike and matrix spike duplicate (MS/MSD)—The purpose of the MS/MSD requirement is to provide data that demonstrate the effectiveness of the method as applied to the samples in question by a given laboratory, and both the data user (discharger, permittee, regulated entity, regulatory/control authority, customer, other) and the laboratory share responsibility for provision of such data. The data user should identify the sample and the analytes of interest (section 1.4) to be spiked and provide sufficient sample volume to perform MS/MSD analyses. The laboratory must, on an ongoing basis, spike at least 5% of the samples in duplicate from each discharge being monitored to assess accuracy (recovery and precision). If direction cannot be obtained from the data user, the laboratory must spike at least one sample in duplicate per extraction batch of up to 20 samples with the analytes in Table 1. Spiked sample results should be reported only to the data user whose sample was spiked, or as requested or required by a regulatory/control authority, or in a permit.

8.3.1. If, as in compliance monitoring, the concentration of a specific analyte will be checked against a regulatory concentration limit, the concentration of the spike should be at that limit; otherwise, the concentration of the spike should be one to five times higher than the background concentration determined in section 8.3.2, at or near the midpoint of the calibration range, or at the concentration in the LCS (section 8.4) whichever concentration would be larger. When no information is available, the mid-point of the calibration may be used.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the each analyte of interest. If necessary to meet the requirement in section 8.3.1, prepare a new check sample concentrate (section 8.2.1) appropriate for the background concentration. Spike and analyze two additional sample aliquots of the same volume as the original sample, and determine the concentrations after spiking (A1 and A2) of each analyte. Calculate the percent recoveries (P1 and P2) as:

where T is the known true value of the spike.

Also calculate the relative percent difference (RPD) between the concentrations (A1 and A2):

8.3.3 Compare the percent recoveries (P1 and P2) and the RPD for each analyte in the MS/MSD aliquots with the corresponding QC acceptance criteria for recovery (P) and RPD in Table 4.

(a) If any individual P falls outside the designated range for recovery in either aliquot, or the RPD limit is exceeded, the result for the analyte in the unspiked sample is suspect and may not be reported or used for permitting or regulatory compliance. See section 8.1.7 for disposition of failures.

(b) For analytes in Table 2 not listed in Table 4, QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 12 and 13).

8.3.4 After analysis of a minimum of 20 MS/MSD samples for each target analyte and surrogate, and if the laboratory chooses to develop and apply optional in-house QC limits, the laboratory should calculate and apply the optional in-house QC limits for recovery and RPD of future MS/MSD samples (Section 8.3). The optional in-house QC limits for recovery are calculated as the mean observed recovery ±3 standard deviations, and the upper QC limit for RPD is calculated as the mean RPD plus 3 standard deviations of the RPDs. The in-house QC limits must be updated at least every two years and re-established after any major change in the analytical instrumentation or process. At least 80% of the analytes tested in the MS/MSD must have in-house QC acceptance criteria that are tighter than those in Table 4 and the remaining analytes (those not included in the 80%) must meet the acceptance criteria in Table 4. If an in-house QC limit for the RPD is greater than the limit in Table 4, then the limit in Table 4 must be used. Similarly, if an in-house lower limit for recovery is below the lower limit in Table 4, then the lower limit in Table 4 must be used, and if an in-house upper limit for recovery is above the upper limit in Table 4, then the upper limit in Table 4 must be used. The laboratory must evaluate surrogate recovery data in each sample against its in-house surrogate recovery limits. The laboratory may use 60 -140% as interim acceptance criteria for surrogate recoveries until in-house limits are developed. Alternatively, surrogate recovery limits may be developed from laboratory control charts. In-house QC acceptance criteria must be updated at least every two years.

8.4 Laboratory control sample (LCS)—A QC check sample (laboratory control sample, LCS; on-going precision and recovery sample, OPR) containing each single-component analyte of interest (section 1.4) must be extracted, concentrated, and analyzed with each extraction batch of up to 20 samples (section 3.1) to demonstrate acceptable recovery of the analytes of interest from a clean sample matrix. If multi-peak analytes are required, extract and prepare at least one as an LCS for each batch. Alternatively, the laboratory may set up a program where multi-peak LCS is rotated with a single-peak LCS.

8.4.1 Prepare the LCS by adding QC check sample concentrate (sections 6.8.3 and 8.2.1) to reagent water. Include all analytes of interest (section 1.4) in the LCS. The volume of reagent water must be the same as the nominal volume used for the sample, the DOC (Section 8.2), the blank (section 8.5), and the MS/MSD (section 8.3). Also add a volume of the surrogate solution (section 6.8.6).

8.4.2 Analyze the LCS prior to analysis of samples in the extraction batch (Section 3.1). Determine the concentration

(A) of each analyte. Calculate the percent recovery as:

where T is the true value of the concentration in the LCS.

8.4.3 For each analyte, compare the percent recovery (P) with its corresponding QC acceptance criterion in Table 4. For analytes of interest in Table 2 not listed in Table 4, use the QC acceptance criteria developed for the MS/MSD (section 8.3.3.2), or limits based on laboratory control charts. If the recoveries for all analytes of interest fall within the designated ranges, analysis of blanks and field samples may proceed. If any individual recovery falls outside the range, proceed according to section 8.4.4.

Note:

The large number of analytes in Tables 1 and 2 present a substantial probability that one or more will fail the acceptance criteria when all analytes are tested simultaneously. Because a re-test is allowed in event of failure (sections 8.1.7 and 8.4.4), it may be prudent to extract and analyze two LCSs together and evaluate results of the second analysis against the QC acceptance criteria only if an analyte fails the first test.

8.4.4 Repeat the test only for those analytes that failed to meet the acceptance criteria (P). If these analytes now pass, system performance is acceptable and analysis of blanks and samples may proceed. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, repeat the test using a fresh LCS (section 8.2.1) or an LCS prepared with a fresh QC check sample concentrate (section 8.2.1), or perform and document system repair. Subsequent to analysis of the LCS prepared with a fresh sample concentrate, or to system repair, repeat the LCS test (Section 8.4). If failure of the LCS indicates a systemic problem with samples in the batch, re-extract and re-analyze the samples in the batch. See Section 8.1.7 for disposition of repeated failures.

8.4.5 After analysis of 20 LCS samples, and if the laboratory chooses to develop and apply optional in-house QC limits, the laboratory should calculate and apply the optional in-house QC limits for recovery of future LCS samples (section 8.4). Limits for recovery in the LCS should be calculated as the mean recovery ±3 standard deviations. A minimum of 80% of the analytes tested for in the LCS must have QC acceptance criteria tighter than those in Table 4, and the remaining analytes (those not included in the 80%) must meet the acceptance criteria in Table 4. If an in-house lower limit for recovery is lower than the lower limit in Table 4, the lower limit in Table 4 must be used, and if an in-house upper limit for recovery is higher than the upper limit in Table 4, the upper limit in Table 4 must be used. Many of the analytes and surrogates do not contain acceptance criteria. The laboratory should use 60–140% as interim acceptance criteria for recoveries of spiked analytes and surrogates that do not have recovery limits specified in Table 4, and at least 80% of the surrogates must meet the 60–140% interim criteria until in-house LCS and surrogate limits are developed. Alternatively, acceptance criteria for analytes that do not have recovery limits in Table 4 may be based on laboratory control charts. In-house QC acceptance criteria must be updated at least every two years.

8.5 Blank—Extract and analyze a blank with each extraction batch (section 3.1) to demonstrate that the reagents and equipment used for preparation and analysis are free from contamination.

8.5.1 Prepare the blank from reagent water and spike it with the surrogates. The volume of reagent water must be the same as the volume used for samples, the DOC (section 8.2), the LCS (section 8.4), and the MS/MSD (section 8.3). Extract, concentrate, and analyze the blank using the same procedures and reagents used for the samples, LCS, and MS/MSD in the batch. Analyze the blank immediately after analysis of the LCS (section 8.4) and prior to analysis of the MS/MSD and samples to demonstrate freedom from contamination.

8.5.2 If any analyte of interest is found in the blank at a concentration greater than the MDL for the analyte, at a concentration greater than one-third the regulatory compliance limit, or at a concentration greater than one-tenth the concentration in a sample in the batch (section 3.1), whichever is greatest, analysis of samples must be halted and samples in the batch must be re-extracted and the extracts reanalyzed. Samples in a batch must be associated with an uncontaminated blank before the results for those samples may be reported or used for permitting or regulatory compliance purposes. If re-testing of blanks results in repeated failures, the laboratory should document the failures and report the problem and failures with the data.

8.6 Surrogate recovery—The laboratory must spike all samples with the surrogate standard spiking solution (section 6.8.6) per section 10.2.2 or 10.4.2, analyze the samples, and calculate the percent recovery of each surrogate. QC acceptance criteria for surrogates must be developed by the laboratory (section 8.4). If any recovery fails its criterion, attempt to find and correct the cause of the failure, and if sufficient volume is available, re-extract another aliquot of the affected sample; otherwise, see section 8.1.7 for disposition of repeated failures.

8.7 As part of the QC program for the laboratory, it is suggested but not required that method accuracy for wastewater samples be assessed and records maintained. After analysis of five or more spiked wastewater samples as in Section 8.3, calculate the average percent recovery (X) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent interval from X−2sp to X+2sp. For example, if X = 90% and sp = 10%, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each analyte on a regular basis to ensure process control (e.g., after each 5–10 new accuracy measurements). If desired, statements of accuracy for laboratory performance, independent of performance on samples, may be developed using LCSs.

8.8 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with another dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Collect samples as grab samples in glass bottles, or in refrigerated bottles using automatic sampling equipment. Collect 1-L of ambient waters, effluents, and other aqueous samples. If high concentrations of the analytes of interest are expected (e.g., for untreated effluents or in-process waters), collect a smaller volume (e.g., 250 mL), but not less than 100 mL, in addition to the 1-L sample. Follow conventional sampling practices, except do not pre-rinse the bottle with sample before collection. Automatic sampling equipment must be as free as possible of polyvinyl chloride or other tubing or other potential sources of contamination. If needed, collect additional sample(s) for the MS/MSD (section 8.3).

9.2 Ice or refrigerate the sample at ≤6 °C from the time of collection until extraction, but do not freeze. If aldrin is to be determined and residual chlorine is present, add 80 mg/L of sodium thiosulfate but do not add excess. Any method suitable for field use may be employed to test for residual chlorine (Reference 14). If sodium thiosulfate interferes in the determination of the analytes, an alternative preservative (e.g., ascorbic acid or sodium sulfite) may be used.

9.3 Extract all samples within seven days of collection and completely analyze within 40 days of extraction (Reference 1). If the sample will not be extracted within 72 hours of collection, adjust the sample pH to a range of 5.0–9.0 with sodium hydroxide solution or sulfuric acid. Record the volume of acid or base used.

10. Sample Extraction

10.1 This section contains procedures for separatory funnel liquid-liquid extraction (SFLLE, section 10.2), continuous liquid-liquid extraction (CLLE, section 10.4), and disk-based solid-phase extraction (SPE, section 10.5). SFLLE is faster, but may not be as effective as CLLE for extracting polar analytes. SFLLE is labor intensive and may result in formation of emulsions that are difficult to break. CLLE is less labor intensive, avoids emulsion formation, but requires more time (18–24 hours), more hood space, and may require more solvent. SPE can be faster, unless the particulate load in an aqueous sample is so high that it slows the filtration process. If an alternative extraction scheme to those detailed in this method is used, all QC tests must be performed and all QC acceptance criteria must be met with that extraction scheme as an integral part of this method.

10.2 Separatory funnel liquid-liquid extraction (SFLLE).

10.2.1 The SFLLE procedure below assumes a sample volume of 1 L. When a different sample volume is extracted, adjust the volume of methylene chloride accordingly.

10.2.2 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into the separatory funnel. Pipet the surrogate standard spiking solution (section 6.8.6) into the separatory funnel. If the sample will be used for the LCS or MS or MSD, pipet the appropriate QC check sample concentrate (section 8.3 or 8.4) into the separatory funnel. Mix well. If the sample arrives in a larger sample bottle, 1 L may be measured in a graduated cylinder, then added to the separatory funnel.

Note:

Instances in which the sample is collected in an oversized bottle should be reported by the laboratory to the data user. Of particular concern is that fact that this practice precludes rinsing the empty bottle with solvent as described below, which could leave hydrophobic pesticides on the wall of the bottle, and underestimate the actual sample concentrations.

10.2.3 Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 seconds to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for two minutes with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If an emulsion forms and the emulsion interface between the layers is more than one-third the volume of the solvent layer, employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, use of phase-separation paper, centrifugation, salting, freezing, or other physical methods. Collect the methylene chloride extract in a flask. If the emulsion cannot be broken (recovery of less than 80% of the methylene chloride, corrected for the water solubility of methylene chloride), transfer the sample, solvent, and emulsion into the extraction chamber of a continuous extractor and proceed as described in section 10.4.

10.2.4 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the flask. Perform a third extraction in the same manner. Proceed to macro-concentration (section 10.3.1).

10.2.5 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to an appropriately sized graduated cylinder. Record the sample volume to the nearest 5 mL. Sample volumes may also be determined by weighing the container before and after extraction or filling to the mark with water.

10.3 Concentration.

10.3.1 Macro concentration.

10.3.1.1 Assemble a Kuderna-Danish (K–D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K–D concentrator so long as the requirements of section 8.2 are met.

10.3.1.2 Pour the extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K–D concentrator. Rinse the flask and column with 20–30 mL of methylene chloride to complete the quantitative transfer.

10.3.1.3 If no cleanup is to be performed on the sample, add 500 µL (0.5 mL) of isooctane to the extract to act as a keeper during concentration.

10.3.1.4 Add one or two clean boiling chips and attach a three-ball Snyder column to the K–D evaporative flask. Pre-wet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K–D apparatus on a hot water bath (60–65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15–20 minutes. At the proper rate of evaporation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL or other determined amount, remove the K–D apparatus from the water bath and allow it to drain and cool for at least 10 minutes.

10.3.1.5 If the extract is to be cleaned up by sulfur removal or acid back extraction, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Adjust the final volume to 10 mL in methylene chloride and proceed to sulfur removal (section 11.5) or acid back extraction (section 11.6). If the extract is to cleaned up using one of the other cleanup procedures or is to be injected into the GC, proceed to Kuderna-Danish micro-concentration (section 10.3.2) or nitrogen evaporation and solvent exchange (section 10.3.3).

10.3.2 Kuderna-Danish micro concentration—Add another one or two clean boiling chips to the concentrator tube and attach a two-ball micro-Snyder column. Pre-wet the Snyder column by adding about 0.5 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60–65 °C) so that the concentrator tube is partially immersed in hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5–10 minutes. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches approximately 1 mL or other required amount, remove the K–D apparatus from the water bath and allow it to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with approximately 0.2 mL of methylene chloride, and proceed to section 10.3.3 for nitrogen evaporation and solvent exchange.

10.3.3 Nitrogen evaporation and solvent exchange—Extracts to be subjected to solid-phase cleanup (SPE) are exchanged into 1.0 mL of the SPE elution solvent (section 6.7.2.2). Extracts to be subjected to Florisil® or alumina cleanups are exchanged into hexane. Extracts that have been cleaned up and are ready for analysis are exchanged into isooctane or hexane, to match the solvent used for the calibration standards.

10.3.3.1 Transfer the vial containing the sample extract to the nitrogen evaporation (blowdown) device (section 5.2.5.2). Lower the vial into a 50–55 °C water bath and begin concentrating. During the solvent evaporation process, do not allow the extract to become dry. Adjust the flow of nitrogen so that the surface of the solvent is just visibly disturbed. A large vortex in the solvent may cause analyte loss.

10.3.3.2 Solvent exchange.

10.3.3.2.1 When the volume of the liquid is approximately 500 µL, add 2 to 3 mL of the desired solvent (SPE elution solvent for SPE cleanup, hexane for Florisil or alumina, or isooctane for final injection into the GC) and continue concentrating to approximately 500 µL. Repeat the addition of solvent and concentrate once more.

10.3.3.3.2 Adjust the volume of an extract to be cleaned up by SPE, Florisil®, or alumina to 1.0 mL. Proceed to extract cleanup (section 11).

10.3.3.3 Extracts that have been cleaned up and are ready for analysis—Adjust the final extract volume to be consistent with the volume extracted and the sensitivity desired. The goal is for a full-volume sample (e.g., 1-L) to have a final extract volume of 10 mL, but other volumes may be used.

10.3.4 Transfer the concentrated extract to a vial with fluoropolymer-lined cap. Seal the vial and label with the sample number. Store in the dark at room temperature until ready for GC analysis. If GC analysis will not be performed on the same day, store the vial in the dark at ≤6 °C. Analyze the extract by GC per the procedure in section 12.

10.4 Continuous liquid/liquid extraction (CLLE).

10.4.1 Use CLLE when experience with a sample from a given source indicates an emulsion problem, or when an emulsion is encountered using SFLLE. CLLE may be used for all samples, if desired.

10.4.2 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Transfer the sample to the continuous extractor and, using a pipet, add surrogate standard spiking solution. If the sample will be used for the LCS, MS, or MSD, pipet the appropriate check sample concentrate (section 8.2.1 or 8.3.2) into the separatory funnel. Mix well. Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 seconds to rinse the inner surface. Transfer the solvent to the extractor.

10.4.3 Repeat the sample bottle rinse with two additional 50–100 mL portions of methylene chloride and add the rinses to the extractor.

10.4.4 Add a suitable volume of methylene chloride to the distilling flask (generally 200–500 mL) and sufficient reagent water to ensure proper operation of the extractor, and extract the sample for 18–24 hours. A shorter or longer extraction time may be used if all QC acceptance criteria are met. Test and, if necessary, adjust the pH of the water to a range of 5.0–9.0 during the second or third hour of the extraction. After extraction, allow the apparatus to cool, then detach the distilling flask. Dry, concentrate, solvent exchange, and transfer the extract to a vial with fluoropolymer-lined cap, per Section 10.3.

10.4.5 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to an appropriately sized graduated cylinder. Record the sample volume to the nearest 5 mL. Sample volumes may also be determined by weighing the container before and after extraction or filling to the mark with water.

10.5 Solid-phase extraction of aqueous samples. The steps in this section address the extraction of aqueous field samples using disk-based solid-phase extraction (SPE) media, based on an ATP approved by EPA in 1995 (Reference 20). This application of SPE is distinct from that used in this method for the cleanup of sample extracts in section 11.2. Analysts must be careful not to confuse the equipment, supplies, or the procedural steps from these two different uses of SPE.

Note:

Changes to the extraction conditions described below may be made by the laboratory under the allowance for method flexibility described in section 8.1, provided that the performance requirements in section 8.2 are met. However, changes in SPE materials, formats, and solvents must meet the requirements in section 8.1.2 and its subsections.

10.5.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. If the sample contains particulates, let stand to settle out the particulates before extraction.

10.5.2 Extract the sample as follows:

10.5.2.1 Place a 90-mm standard filter apparatus on a vacuum filtration flask or manifold and attach to a vacuum source. The vacuum gauge must read at least 25 in. of mercury when all valves are closed. Position a 90-mm C18 extraction disk onto the filter screen. Wet the entire disk with methanol. To aid in filtering samples with particulates, a 1-µm glass fiber filter or Empore® Filter Aid 400 can be placed on the top of the disk and wetted with methanol. Install the reservoir and clamp. Resume vacuum to dry the disk. Interrupt the vacuum. Wash the disk and reservoir with 20 mL of methylene chloride. Resume the vacuum briefly to pull methylene chloride through the disk. Interrupt the vacuum and allow the disk to soak for about a minute. Resume vacuum and completely dry the disk.

10.5.2.2 Condition the disk with 20 mL of methanol. Apply vacuum until nearly all the solvent has passed through the disk, interrupting it while solvent remains on the disk. Allow the disk to soak for about a minute. Resume vacuum to pull most of the methanol through, but interrupting it to leave a layer of methanol on the surface of the disk. Do not allow disk to dry. For uniform flow and good recovery, it is critical the disk not be allowed to dry from now until the end of the extraction. Discard waste solvent. Rinse the disk with 20 mL of deionized water. Resume vacuum to pull most of the water through, but interrupt it to leave a layer of water on the surface of the disk. Do not allow the disk to dry. If disk does dry, recondition with methanol as above.

10.5.2.3 Add the water sample to the reservoir and immediately apply the vacuum. If particulates have settled in the sample, gently decant the clear layer into the apparatus until most of the sample has been processed. Then pour the remainder including the particulates into the reservoir. Empty the sample bottle completely. When the filtration is complete, dry the disk for three minutes. Turn off the vacuum.

10.5.3 Discard sample filtrate. Insert tube to collect the eluant. The tube should fit around the drip tip of the base. Reassemble the apparatus. Add 5.0 mL of acetone to the center of the disk, allowing it to spread evenly over the disk. Turn the vacuum on and quickly off when the filter surface nears dryness but still remains wet. Allow to soak for 15 seconds. Add 20 mL of methylene chloride to the sample bottle, seal and shake to rinse the inside of the bottle. Transfer the methylene chloride from the bottle to the filter. Resume the vacuum slowly so as to avoid splashing.

Interrupt the vacuum when the filter surface nears dryness but still remains wet. Allow disk to soak in solvent for 20 seconds. Rinse the reservoir glass and disk with 10 mL of methylene chloride. Resume vacuum slowly. Interrupt vacuum when disk is covered with solvent. Allow to soak for 20 seconds. Resume vacuum to dry the disk. Remove the sample tube.

10.5.4 Dry, concentrate, solvent exchange, and transfer the extract to a vial with fluoropolymer-lined cap, per section 10.3.

10.5.5 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to an appropriately sized graduated cylinder. Record the sample volume to the nearest 5 mL. Sample volumes may also be determined by weighing the container before and after extraction or filling to the mark with water.

11. Extract Cleanup

11.1 Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater, drinking water). If particular circumstances require the use of a cleanup procedure, the laboratory may use any or all of the procedures below or any other appropriate procedure (e.g., gel permeation chromatography). However, the laboratory must first repeat the tests in sections 8.2, 8.3, and 8.4 to demonstrate that the requirements of those sections can be met using the cleanup procedure(s) as an integral part of this method. This is particularly important when the target analytes for the analysis include any of the single component pesticides in Table 2, because some cleanups have not been optimized for all of those analytes.

11.1.1 The solid-phase cartridge (section 11.2) removes polar organic compounds such as phenols.

11.1.2 The Florisil® column (section 11.3) allows for selected fractionation of the organochlorine analytes and will also eliminate polar interferences.

11.1.3 Alumina column cleanup (section 11.4) also removes polar materials.

11.1.4 Elemental sulfur, which interferes with the electron capture gas chromatography of some of the pesticides, may be removed using activated copper, or TBA sulfite. Sulfur removal (section 11.5) is required when sulfur is known or suspected to be present. Some chlorinated pesticides which also contain sulfur may be removed by this cleanup.

11.1.5 Acid back extraction (section 11.6) may be useful for cleanup of PCBs and other compounds not adversely affected by sulfuric acid.

11.2 Solid-phase extraction (SPE) as a cleanup. In order to use the C18 SPE cartridge in section 5.5.3.5 as a cleanup procedure, the sample extract must be exchanged from methylene chloride to methylene chloride:acetonitrile:hexane (50:3:47). Follow the solvent exchange steps in section 10.3.3.2 prior to attempting solid-phase cleanup.

Note:

This application of SPE is distinct from that used in this method for the extraction of aqueous samples in section 10.5. Analysts must be careful not to confuse the equipment, supplies, or procedural steps from these two different uses of SPE.

11.2.1 Setup.

11.2.1.1 Attach the VacElute Manifold (section 5.5.3.2) to a water aspirator or vacuum pump with the trap and gauge installed between the manifold and vacuum source.

11.2.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and adjust the vacuum to 5 to 10 psi.

11.2.2 Cartridge washing—Pre-elute each cartridge prior to use sequentially with 10-mL portions each of hexane, methanol, and water using vacuum for 30 seconds after each eluting solvent. Follow this pre-elution with 1 mL methylene chloride and three 10-mL portions of the elution solvent (section 6.7.2.2) using vacuum for 5 minutes after each eluting solvent. Tap the cartridge lightly while under vacuum to dry between solvent rinses. The three portions of elution solvent may be collected and used as a cartridge blank, if desired. Finally, elute the cartridge with 10 mL each of methanol and water, using the vacuum for 30 seconds after each eluant.

11.2.3 Extract cleanup.

11.2.3.1 After cartridge washing (section 11.2.2), release the vacuum and place the rack containing the 50-mL volumetric flasks (section 5.5.3.4) in the vacuum manifold. Re-establish the vacuum at 5 to 10 psi.

11.2.3.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE cartridge. Apply vacuum for five minutes to dry the cartridge. Tap gently to aid in drying.

11.2.3.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL portions of the methylene chloride:acetonitrile:hexane (50:3:47) elution solvent (section 6.7.2.2), using vacuum for five minutes after each portion. Collect the eluants in the 50-mL volumetric flasks.

11.2.3.4 Release the vacuum and remove the 50-mL volumetric flasks.

11.2.3.5 Concentrate the eluted extracts per Section 10.3.

11.3 Florisil®. In order to use Florisil cleanup, the sample extract must be exchanged from methylene chloride to hexane. Follow the solvent exchange steps in section 10.3.3.2 prior to attempting Florisil® cleanup.

Note:

Alternative formats for this cleanup may be used by the laboratory, including cartridges containing Florisil®. If an alternative format is used, consult the manufacturer's instructions and develop a formal documented procedure to replace the steps in section 11.3 of this method and demonstrate that the alternative meets the relevant quality control requirements of this method.

11.3.1 If the chromatographic column does not contain a frit at the bottom, place a small plug of pre-cleaned glass wool in the column (section 5.2.4) to retain the Florisil®. Place the mass of Florisil® (nominally 20 g) predetermined by calibration (section 7.8 and Table 6) in a chromatographic column. Tap the column to settle the Florisil® and add 1 to 2 cm of granular anhydrous sodium sulfate to the top.

11.3.2 Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil®. Just prior to exposure of the sodium sulfate layer to the air, stop the elution of the hexane by closing the stopcock on the chromatographic column. Discard the eluant.

11.3.3 Transfer the concentrated extract (section 10.3.3) onto the column. Complete the transfer with two 1-mL hexane rinses, drawing the extract and rinses down to the level of the sodium sulfate.

11.3.4 Place a clean 500-mL K–D flask and concentrator tube under the column. Elute Fraction 1 with 200 mL of 6% (v/v) ethyl ether in hexane at a rate of approximately 5 mL/min. Remove the K–D flask and set it aside for later concentration. Elute Fraction 2 with 200 mL of 15% (v/v) ethyl ether in hexane into a second K–D flask. Elute Fraction 3 with 200 mL of 50% (v/v) ethyl ether in hexane into a third K–D flask. The elution patterns for the pesticides and PCBs are shown in Table 6.

11.3.5 Concentrate the fractions as in Section 10.3, except use hexane to prewet the column and set the water bath at about 85 °C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of Fraction 1 to approximately 10 mL for sulfur removal (Section 11.5), if required; otherwise, adjust the volume of the fractions to 10 mL, 1.0 mL, or other volume needed for the sensitivity desired. Analyze the concentrated extract by gas chromatography (Section 12).

11.4 Alumina. The sample extract must be exchanged from methylene chloride to hexane. Follow the solvent exchange steps in section 10.3.3.2 prior to attempting alumina cleanup.

11.4.1 If the chromatographic column does not contain a frit at the bottom, place a small plug of pre-cleaned glass wool in the chromatographic column (section 5.2.4) to retain the alumina. Add 10 g of alumina (section 6.7.3) on top of the plug. Tap the column to settle the alumina. Place 1–2 g of anhydrous sodium sulfate on top of the alumina.

11.4.2 Close the stopcock and fill the column to just above the sodium sulfate with hexane. Add 25 mL of hexane. Open the stopcock and adjust the flow rate of hexane to approximately 2 mL/min. Do not allow the column to go dry throughout the elutions.

11.4.3 When the level of the hexane is at the top of the column, quantitatively transfer the extract to the column. When the level of the extract is at the top of the column, slowly add 25 mL of hexane and elute the column to the level of the sodium sulfate. Discard the hexane.

11.4.4 Place a K–D flask (section 5.2.5.1.2) under the column and elute the pesticides with approximately 150 mL of hexane:ethyl ether (80:20 v/v). It may be necessary to adjust the volume of elution solvent for slightly different alumina activities.

11.4.5 Concentrate the extract per section 10.3.

11.5 Sulfur removal—Elemental sulfur will usually elute in Fraction 1 of the Florisil® column cleanup. If Florisil® cleanup is not used, or to remove sulfur from any of the Florisil® fractions, use one of the sulfur removal procedures below. These procedures may be applied to extracts in hexane, ethyl ether, or methylene chloride.

Note:

Separate procedures using copper or TBA sulfite are provided in this section for sulfur removal. They may be used separately or in combination, if desired.

11.5.1 Removal with copper (Reference 15).

Note:

Some of the analytes in Table 2 are not amenable to sulfur removal with copper (e.g., atrazine and diazinon). Therefore, before using copper to remove sulfur from an extract that will be analyzed for any of the non-PCB analytes in Table 2, the laboratory must demonstrate that the analytes can be extracted from an aqueous sample matrix that contains sulfur and recovered from an extract treated with copper. Acceptable performance can be demonstrated through the preparation and analysis of a matrix spike sample that meets the QC requirements for recovery.

11.5.1.1 Quantitatively transfer the extract to a 40- to 50-mL flask or bottle. If there is evidence of water in the K–D or round-bottom flask after the transfer, rinse the flask with small portions of hexane:acetone (40:60) and add to the flask or bottle. Mark and set aside the concentration flask for future use.

11.5.1.2 Add 10–20 g of granular anhydrous sodium sulfate to the flask. Swirl to dry the extract.

11.5.1.3 Add activated copper (section 6.7.4.1.4) and allow to stand for 30–60 minutes, swirling occasionally. If the copper does not remain bright, add more and swirl occasionally for another 30–60 minutes.

11.5.1.4 After drying and sulfur removal, quantitatively transfer the extract to a nitrogen-evaporation vial or tube and proceed to section 10.3.3 for nitrogen evaporation and solvent exchange, taking care to leave the sodium sulfate and copper foil in the flask.

11.5.2 Removal with TBA sulfite.

11.5.2.1 Using small volumes of hexane, quantitatively transfer the extract to a 40- to 50-mL centrifuge tube with fluoropolymer-lined screw cap.

11.5.2.2 Add 1–2 mL of TBA sulfite reagent (section 6.7.4.2.4), 2–3 mL of 2-propanol, and approximately 0.7 g of sodium sulfite (section 6.7.4.2.2) crystals to the tube. Cap and shake for 1–2 minutes. If the sample is colorless or if the initial color is unchanged, and if clear crystals (precipitated sodium sulfite) are observed, sufficient sodium sulfite is present. If the precipitated sodium sulfite disappears, add more crystalline sodium sulfite in approximately 0.5-g portions until a solid residue remains after repeated shaking.

11.5.2.3 Add 5–10 mL of reagent water and shake for 1–2 minutes. Centrifuge to settle the solids.

11.5.2.4 Quantitatively transfer the hexane (top) layer through a small funnel containing a few grams of granular anhydrous sodium sulfate to a nitrogen-evaporation vial or tube and proceed to section 10.3.3 for micro-concentration and solvent exchange.

11.6 Acid back extraction (section 6.1.2).

11.6.1 Quantitatively transfer the extract (section 10.3.1.5) to a 250-mL separatory funnel.

11.6.2 Partition the extract against 50 mL of sulfuric acid solution (section 6.1.2). Discard the aqueous layer. Repeat the acid washing until no color is visible in the aqueous layer, to a maximum of four washings.

11.6.3 Partition the extract against 50 mL of sodium chloride solution (section 6.7.5). Discard the aqueous layer.

11.6.4 Proceed to section 10.3.3 for micro-concentration and solvent exchange.

12. Gas Chromatography

12.1 Establish the same operating conditions used in section 7.1 for instrument calibration.

12.2 If the internal standard calibration procedure is used, add the internal standard solution (section 6.9.3) to the extract as close as possible to the time of injection to minimize the possibility of loss by evaporation, adsorption, or reaction. For example, add 1 µL of 10 µg/mL internal standard solution into the extract, assuming no dilutions. Mix thoroughly.

12.3 Simultaneously inject an appropriate volume of the sample extract or standard solution onto both columns, using split, splitless, solvent purge, large-volume, or on-column injection. Alternatively, if using a single-column GC configuration, inject an appropriate volume of the sample extract or standard solution onto each GC column independently. If the sample is injected manually, the solvent-flush technique should be used. The injection volume depends upon the technique used and the sensitivity needed to meet MDLs or reporting limits for regulatory compliance. Injection volumes must be the same for all extracts. Record the volume injected to the nearest 0.05 µL.

12.4 Set the data system or GC control to start the temperature program upon sample injection, and begin data collection after the solvent peak elutes. Set the data system to stop data collection after the last analyte is expected to elute and to return the column to the initial temperature.

12.5 Perform all qualitative and quantitative measurements as described in Sections 14 and 15. When standards and extracts are not being used for analyses, store them refrigerated at <6 °C, protected from light, in screw-cap vials equipped with un-pierced fluoropolymer-lined septa.

13. System and Laboratory Performance

13.1 At the beginning of each shift during which standards or extracts are analyzed, GC system performance and calibration must be verified for all analytes and surrogates on both column/detector systems. Adjustment and/or recalibration (per section 7) are performed until all performance criteria are met. Only after all performance criteria are met may samples, blanks and other QC samples, and standards be analyzed.

13.2 Inject an aliquot of the calibration verification standard (section 6.8.4) on both columns. Inject an aliquot of each of the multi-component standards.

13.3 Retention times—The absolute retention times of the peak maxima shall be within ±2 seconds of the retention times in the calibration verification (section 7.8).

13.4 GC resolution—Resolution is acceptable if the valley height between two peaks (as measured from the baseline) is less than 40% of the shorter of the two peaks.

13.4.1 DB–608 column—DDT and endrin aldehyde

13.4.2 DB–1701 column—alpha and gamma chlordane

Note:

If using other GC columns or stationary phases, these resolution criteria apply to these four target analytes and any other closely eluting analytes on those other GC columns.

13.5 Decomposition of DDT and endrin—If DDT, endrin, or their breakdown products are to be determined, this test must be performed prior to calibration verification (section 13.6). DDT decomposes to DDE and DDD. Endrin decomposes to endrin aldehyde and endrin ketone.

13.5.1 Inject 1 µL of the DDT and endrin decomposition solution (section 6.8.7). As noted in section 6.8.7, other injection volumes may be used as long as the concentrations of DDT and endrin in the solution are adjusted to introduce the masses of the two analytes into the instrument that are listed in section 6.8.7.

13.5.2 Measure the areas of the peaks for DDT, DDE, DDD, endrin, endrin aldehyde, and endrin ketone in the chromatogram and calculate the percent breakdown as shown in the equations below:

13.5.3 Both the % breakdown of DDT and of endrin must be less than 20%, otherwise the system is not performing acceptably for DDT and endrin. In this case, repair the GC column system that failed and repeat the performance tests (sections 13.2 to 13.6) until the specification is met.

Note:

DDT and endrin decomposition are usually caused by accumulations of particulates in the injector and in the front end of the column. Cleaning and silanizing the injection port liner, and breaking off a short section of the front end of the column will usually eliminate the decomposition problem. Either of these corrective actions may affect retention times, GC resolution, and calibration linearity.

13.6 Calibration verification.

13.6.1 Compute the percent recovery of each analyte and of the coeluting analytes, based on the initial calibration data (section 7.5 or 7.6).

13.6.2 For each analyte or for coeluting analytes, compare the concentration with the limits for calibration verification in Table 4. For coeluting analytes, use the coeluting analyte with the least restrictive specification (the widest range). For analytes in Table 2 not listed in Table 4, QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 13 and 14). If the recoveries for all analytes meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may continue. If, however, any recovery falls outside the calibration verification range, system performance is unacceptable for that analyte. If this occurs, repair the system and repeat the test (section 13.6), or prepare a fresh calibration standard and repeat the test, or recalibrate (section 7). See Section 8.1.7 for information on repeated test failures.

13.7 Laboratory control sample.

13.7.1 Analyze the extract of the LCS (section 6.8.3) extracted with each sample batch (Section 8.4). See Section 8.4 for criteria acceptance of the LCS.

13.7.2 It is suggested, but not required, that the laboratory update statements of data quality. Add results that pass the specifications in section 13.7.3 to initial (section 8.7) and previous ongoing data. Update QC charts to form a graphic representation of continued laboratory performance. Develop a statement of laboratory data quality for each analyte by calculating the average percent recovery (R) and the standard deviation of percent recovery, sr. Express the accuracy as a recovery interval from R − 2sr to R + 2sr. For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.

13.8 Internal standard response—If internal standard calibration is used, verify that detector sensitivity has not changed by comparing the response (area or height) of each internal standard in the sample, blank, LCS, MS, and MSD to the response in calibration verification (section 6.8.3). The peak area or height of the internal standard should be within 50% to 200% (12 to 2x) of its respective peak area or height in the verification standard. If the area or height is not within this range, compute the concentration of the analytes using the external standard method (section 7.5). If the analytes are affected, re-prepare and reanalyze the sample, blank, LCS, MS, or MSD, and repeat the pertinent test.

14. Qualitative Identification

14.1 Identification is accomplished by comparison of data from analysis of a sample, blank, or other QC sample with data from calibration verification (section 7.7.1 or 13.5), and with data stored in the retention-time and calibration libraries (section 7.7). The retention time window is determined as described in section 14.2. Identification is confirmed when retention time agrees on both GC columns, as described below. Alternatively, GC/MS identification may be used to provide another means of identification.

14.2 Establishing retention time windows.

14.2.1 Using the data from the multi-point initial calibration (section 7.4), determine the retention time in decimal minutes (not minutes:seconds) of each peak representing a single-component target analyte on each column/detector system. For the multi-component analytes, use the retention times of the five largest peaks in the chromatograms on each column/detector system.

14.2.2 Calculate the standard deviation of the retention times for each single-component analyte on each column/detector system and for the three to five exclusive (unique large) peaks for each multi-component analyte.

14.2.3 Define the width of the retention time window as three times that standard deviation. Establish the center of the retention time window for each analyte by using the absolute retention time for each analyte from the calibration verification standard at the beginning of the analytical shift. For samples run during the same shift as an initial calibration, use the retention time of the mid-point standard of the initial calibration. If the calculated RT window is less than 0.02 minutes, then use 0.02 minutes as the window.

Note:

Procedures for establishing retention time windows from other sources may be employed provided that they are clearly documented and provide acceptable performance. Such performance may be evaluated using the results for the spiked QC samples described in this method, such as laboratory control samples and matrix spike samples.

14.2.4 The retention time windows must be recentered when a new GC column is installed or if a GC column has been shortened during maintenance to a degree that the retention times of analytes in the calibration verification standard have shifted close to the lower limits of the established retention time windows.

14.2.5 RT windows should be checked periodically by examining the peaks in spiked samples such as the LCS or MS/MSD to confirm that peaks for known analytes are properly identified.

14.2.6 If the retention time of an analyte in the calibration (Section 7.4) varies by more than 5 seconds across the calibration range as a function of the concentration of the standard, using the standard deviation of the retention times (section 14.2.3) to set the width of the retention time window may not adequately serve to identify the analyte in question under routine conditions. In such cases, data from additional analyses of standards may be required to adequately model the chromatographic behavior of the analyte.

14.3 Identifying the analyte in a sample.

14.3.1 In order to identify a single-component analyte from analysis of a sample, blank, or other QC sample, the peak representing the analyte must fall within its respective retention time windows on both column/detector systems (as defined in section 14.2). That identification is further supported by the comparison of the numerical results on both columns, as described in section 15.7.

14.3.2 In order to identify a multi-component analyte, pattern matching (fingerprinting) may be used, or the three to five exclusive (unique and largest) peaks for that analyte must fall within their respective retention time windows on both column/detector systems (as defined in section 14.2). That identification is further supported by the comparison of the numerical results on both columns, as described in section 15.7. Alternatively, GC/MS identification may be used. Differentiation among some of the Aroclors may require evaluation of more than five peaks to ensure correct identification.

14.4 GC/MS confirmation. When the concentration of an analyte is sufficient and the presence or identity is suspect, its presence should be confirmed by GC/MS. In order to match the sensitivity of the GC/ECD, confirmation would need to be by GC/MS–SIM, or the estimated concentration would need to be 100 times higher than the GC/ECD calibration range. The extract may be concentrated by an additional amount to allow a further attempt at GC/MS confirmation.

14.5 Additional information that may aid the laboratory in the identification of an analyte. The occurrence of peaks eluting near the retention time of an analyte of interest increases the probability of a false positive for the analyte. If the concentration is insufficient for confirmation by GC/MS, the laboratory may use the cleanup procedures in this method (section 11) on a new sample aliquot to attempt to remove the interferent. After attempts at cleanup are exhausted, the following steps may be helpful to assure that the substance that appears in the RT windows on both columns is the analyte of interest.

14.5.1 Determine the consistency of the RT data for the analyte on each column. For example, if the RT is very stable (i.e., varies by no more than a few seconds) for the calibration, calibration verification, blank, LCS, and MS/MSD, the RT for the analyte of interest in the sample should be within this variation regardless of the window established in Section 14.2. If the analyte is not within this variation on both columns, it is likely not present.

14.5.2 The possibility exists that the RT for the analyte in a sample could shift if extraneous materials are present. This possibility may be able to be confirmed or refuted by the behavior of the surrogates in the sample. If multiple surrogates are used that span the length of the chromatographic run, the RTs for the surrogates on both columns are consistent with their RTs in calibration, calibration verification, blank, LCS, and MS/MSD, it is unlikely that the RT for the analyte of interest has shifted.

14.5.3 If the RT for the analyte is shifted slightly later on one column and earlier on the other, and the surrogates have not shifted, it is highly unlikely that the analyte is present, because shifts nearly always occur in the same direction on both columns.

15. Quantitative Determination

15.1 External standard quantitation—Calculate the concentration of the analyte in the extract using the calibration curve or average calibration factor determined in calibration (section 7.5.2) and the following equation:

where:

Cex = Concentration of the analyte in the extract (ng/mL)

As = Peak height or area for the analyte in the standard or sample

CF = Calibration factor, as defined in Section 7.5.1

15.2 Internal standard quantitation—Calculate the concentration of the analyte in the extract using the calibration curve or average response factor determined in calibration (section 7.6.2) and the following equation:

where:

Cex = Concentration of the analyte in the extract (ng/mL)

As = Peak height or area for the analyte in the standard or sample

Cis = Concentration of the internal standard (ng/mL)

Ais = Area of the internal standard

RF = Response factor, as defined in section 7.6.1

15.3 Calculate the concentration of the analyte in the sample using the concentration in the extract, the extract volume, the sample volume, and the dilution factor, per the following equation:

where:

Cs = Concentration of the analyte in the sample (µg/L)

Vex = Final extract volume (mL)

Cex = Concentration in the extract (ng/mL)

Vs = Volume of sample (L)

DF = Dilution factor

and the factor of 1,000 in the denominator converts the final units from ng/L to µg/L

15.4 If the concentration of any target analyte exceeds the calibration range, either extract and analyze a smaller sample volume, or dilute and analyze the diluted extract.

15.5 Quantitation of multi-component analytes.

15.5.1 PCBs as Aroclors. Quantify an Aroclor by comparing the sample chromatogram to that of the most similar Aroclor standard as indicated in section 14.3.2. Compare the responses of 3 to 5 major peaks in the calibration standard for that Aroclor with the peaks observed in the sample extract. The amount of Aroclor is calculated using the individual calibration factor for each of the 3 to 5 characteristic peaks chosen in section 7.5.1. Determine the concentration of each of the characteristic peaks, using the average calibration factor calculated for that peak in section 7.5.2, and then those 3 to 5 concentrations are averaged to determine the concentration of that Aroclor.

15.5.2 Other multi-component analytes. Quantify any other multi-component analytes (technical chlordane or toxaphene) using the same peaks used to develop the average calibration factors in section 7.5.2. Determine the concentration of each of the characteristic peaks, and then the concentrations represented by those characteristic peaks are averaged to determine the concentration of the analyte. Alternatively, for toxaphene, the analyst may determine the calibration factor in section 7.5.2 by summing the areas of all of the peaks for the analyte and using the summed of the peak areas in the sample chromatogram to determine the concentration. However, the approach used for toxaphene must be the same for the calibration and the sample analyses.

15.6 Reporting of results. As noted in section 1.6.1, EPA has promulgated this method at 40 CFR part 136 for use in wastewater compliance monitoring under the National Pollutant Discharge Elimination System (NPDES). The data reporting practices described here are focused on such monitoring needs and may not be relevant to other uses of the method.

15.6.1 Report results for wastewater samples in µg/L without correction for recovery. (Other units may be used if required by in a permit.) Report all QC data with the sample results.

15.6.2 Reporting level. Unless specified otherwise by a regulatory authority or in a discharge permit, results for analytes that meet the identification criteria are reported down to the concentration of the ML established by the laboratory through calibration of the instrument (see section 7.5 or 7.6 and the glossary for the derivation of the ML). EPA considers the terms “reporting limit,” “quantitation limit,” and “minimum level” to be synonymous.

15.6.2.1 Report the lower result from the two columns (see section 15.7 below) for each analyte in each sample or QC standard at or above the ML to 3 significant figures. Report a result for each analyte in each sample or QC standard below the ML as “<ML,” where “ML” is the concentration of the analyte at the ML (e.g., if the ML is 10 µg/L, then report the result as <10 µg/L), or as required by the regulatory authority or permit. Report a result for each analyte in a blank at or above the MDL to 2 significant figures. Report a result for each analyte found in a blank below the MDL as “<MDL,” where MDL is the concentration of the analyte at the MDL, or as required by the regulatory/control authority or permit.

15.6.2.2 In addition to reporting results for samples and blank(s) separately, the concentration of each analyte in a blank or field blank associated with that sample may be subtracted from the result for that sample, but only if requested or required by a regulatory authority or in a permit. In this case, both the sample result and the blank results must be reported together.

15.6.2.3 Report the result for an analyte in a sample or extract that has been diluted at the least dilute level at which the peak area is within the calibration range (i.e., above the ML for the analyte) and the MS/MSD recovery and RPD are within their respective QC acceptance criteria (Table 4). This may require reporting results for some analytes from different analyses. Results for each analyte in MS/MSD samples should be reported from the same GC column as used to report the results for that analyte in the unspiked sample. If the MS/MSD recoveries and RPDs calculated in this manner do not meet the acceptance criteria in Table 4, the analyst may use the results from the other GC column to determine if the MS/MSD results meet the acceptance criteria. If such a situation occurs, the results for the sample should be recalculated using the same GC column data as used for the MS/MSD samples, and reported with appropriate annotations that alert the data user of the issue.

15.6.2.4 Results from tests performed with an analytical system that is not in control (i.e., that does not meet acceptance criteria for all of QC tests in this method) must not be reported or otherwise used for permitting or regulatory compliance purposes, but do not relieve a discharger or permittee of reporting timely results. See section 8.1.7 for dispositions of failures. If the holding time would be exceeded for a re-analysis of the sample, the regulatory/control authority should be consulted for disposition.

15.6.3 Analyze the sample by GC/MS or on a third column when analytes have co-eluted or interfere with determination on both columns.

Note:

Dichlone and kepone do not elute from the DB–1701 column and must be confirmed on a DB–5 column, or by GC/MS.

15.7 Quantitative information that may aid in the confirmation of the presence of an analyte.

15.7.1 As noted in Section 14.3, the relative agreement between the numerical results from the two GC columns may be used to support the identification of the target analyte by providing evidence that co-eluting interferences are not present at the retention time of the target analyte. Calculate the percent difference (%D) between the results for the analyte from both columns, as follows:

In general, if the %D of the two results is less than 50% (e.g., a factor of 2), then the pesticide is present. This %D is generous and allows for the pesticide that has the largest measurement error.

Note:

Laboratories may employ metrics less than 50% for this comparison, including those specified in other analytical methods for these pesticides (e.g., CLP or SW–846).

15.7.2 If the amounts do not agree, and the RT data indicate the presence of the analyte (per Section 14), it is likely that a positive interference is present on the column that yielded the higher result. That interferent may be represented by a separate peak on the other column that does not coincide with the retention time of any of the target analytes. If the interfering peak is evident on the other column, report the result from that column and advise the data user that the interference resulted in a %D value greater than 50%. If an interferent is not identifiable on the second column, then the results must be reported as “not detected” at the lower concentration. In this event, the pesticide is not confirmed and the reporting limit is elevated. See section 8.1.7 for disposition of problem results.

Note:

The resulting elevation of the reporting limit may not meet the requirements for compliance monitoring and the use of additional cleanup procedures may be required.

16. Analysis of Complex Samples

16.1 Some samples may contain high levels (greater than 1 µg/L) of the analytes of interest, interfering analytes, and/or polymeric materials. Some samples may not concentrate to 1.0 mL (section 10.3.3.3.2); others may overload the GC column and/or detector.

16.2 When an interference is known or suspected to be present, the laboratory should attempt to clean up the sample extract using the SPE cartridge (section 11.2), by Florisil® (Section 11.3), Alumina (Section 11.4), sulfur removal (section 11.5), or another clean up procedure appropriate to the analytes of interest. If these techniques do not remove the interference, the extract is diluted by a known factor and reanalyzed (section 12). Dilution until the extract is lightly colored is preferable. Typical dilution factors are 2, 5, and 10.

16.3 Recovery of surrogate(s)—In most samples, surrogate recoveries will be similar to those from reagent water. If surrogate recovery is outside the limits developed in Section 8.6, re-extract and reanalyze the sample if there is sufficient sample and if it is within the 7-day extraction holding time. If surrogate recovery is still outside this range, extract and analyze one-tenth the volume of sample to overcome any matrix interference problems. If a sample is highly colored or suspected to be high in concentration, a 1–L sample aliquot and a 100-mL sample aliquot could be extracted simultaneously and still meet the holding time criteria, while providing information about a complex matrix.

16.4 Recovery of the matrix spike and matrix spike duplicate (MS/MSD)—In most samples, MS/MSD recoveries will be similar to those from reagent water. If either the MS or MSD recovery is outside the range specified in Section 8.3.3, one-tenth the volume of sample is spiked and analyzed. If the matrix spike recovery is still outside the range, the result for the unspiked sample may not be reported or used for permitting or regulatory compliance purposes. See Section 8.1.7 for dispositions of failures. Poor matrix spike recovery does not relieve a discharger or permittee of reporting timely results.

17. Method Performance

17.1 This method was tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4x MDL to 1000x MDL with the following exceptions: Chlordane recovery at 4x MDL was low (60%); Toxaphene recovery was demonstrated linear over the range of 10x MDL to 1000x MDL (Reference 3).

17.2 The 1984 version of this method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations (Reference 2). Concentrations used in the study ranged from 0.5 to 30 µg/L for single-component pesticides and from 8.5 to 400 µg/L for multi-component analytes. These data are for a subset of analytes described in the current version of the method.

17.3 During the development of Method 1656, a similar EPA procedure for the organochlorine pesticides, single-operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the analyte and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 5.

18. Pollution Prevention

18.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory operations. EPA has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, the laboratory should use pollution prevention techniques to address waste generation. When wastes cannot be reduced at the source, the Agency recommends recycling as the next best option.

18.2 The analytes in this method are used in extremely small amounts and pose little threat to the environment when managed properly. Standards should be prepared in volumes consistent with laboratory use to minimize the disposal of excess volumes of expired standards. This method utilizes significant quantities of methylene chloride. Laboratories are encouraged to recover and recycle this and other solvents during extract concentration.

18.3 For information about pollution prevention that may be applied to laboratories and research institutions, consult “Less is Better: Laboratory Chemical Management for Waste Reduction” (Reference 19), available from the American Chemical Society's Department of Governmental Relations and Science Policy, 1155 16th Street NW., Washington DC 20036, 202–872–4477.

19. Waste Management

19.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations. An overview of requirements can be found in Environmental Management Guide for Small Laboratories (EPA 233–B–98–001).

19.2 Samples at pH <2, or pH >12, are hazardous and must be handled and disposed of as hazardous waste, or neutralized and disposed of in accordance with all federal, state, and local regulations. It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions. The laboratory using this method has the responsibility to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations. For further information on waste management, see “The Waste Management Manual for Laboratory Personnel,” also available from the American Chemical Society at the address in section 18.3.

19.3 Many analytes in this method decompose above 500 °C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in an appropriate incinerator. Gross quantities of neat or highly concentrated solutions of toxic or hazardous chemicals should be packaged securely and disposed of through commercial or governmental channels that are capable of handling toxic wastes.

19.4 For further information on waste management, consult The Waste Management Manual for Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street NW., Washington, DC 20036, 202–872–4477.

20. References

1. “Determination of Pesticides and PCBs in Industrial and Municipal Wastewaters,” EPA 600/4–82–023, National Technical Information Service, PB82–214222, Springfield, Virginia 22161, April 1982.

2. “EPA Method Study 18 Method 608-Organochlorine Pesticides and PCBs,” EPA 600/4–84–061, National Technical Information Service, PB84–211358, Springfield, Virginia 22161, June 1984.

3. “Method Detection Limit and Analytical Curve Studies, EPA Methods 606, 607, and 608,” Special letter report for EPA Contract 68–03–2606, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

4. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practice for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

5. Giam, C.S., Chan, H.S., and Nef, G.S. “Sensitive Method for Determination of Phthalate Ester Plasticizers in Open-Ocean Biota Samples,” Analytical Chemistry, 47:2225 (1975).

6. Giam, C.S. and Chan, H.S. “Control of Blanks in the Analysis of Phthalates in Air and Ocean Biota Samples,” U.S. National Bureau of Standards, Special Publication 442, pp. 701–708, 1976.

7. Solutions to Analytical Chemistry Problems with Clean Water Act Methods, EPA 821–R–07–002, March 2007.

8. “Carcinogens-Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

9. “Occupational Exposure to Hazardous Chemicals in Laboratories,” (29 CFR 1910.1450), Occupational Safety and Health Administration, OSHA.

10. 40 CFR 136.6(b)(4)(j).

11. Mills, P.A. “Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical Chemists, 51:29, (1968).

12. 40 CFR 136.6(b)(2)(i).

13. Protocol for EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater and Drinking Water (EPA–821–B–98–003) March 1999.

14. Methods 4500 Cl F and 4500 Cl G, Standard Methods for the Examination of Water and Wastewater, published jointly by the American Public Health Association, American Water Works Association, and Water Environment Federation, 1015 Fifteenth St., Washington, DC 20005, 20th Edition, 2000.

15. “Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental Samples,” EPA–600/8–80–038, U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina.

16. USEPA, 2000, Method 1656 Organo-Halide Pesticides In Wastewater, Soil, Sludge, Sediment, and Tissue by GC/HSD, EPA–821–R–00–017, September 2000.

17. USEPA, 2010, Method 1668C Chlorinated Biphenyl Congeners in Water, Soil, Sediment, Biosolids, and Tissue by HRGC/HRMS, EPA–820–R–10–005, April 2010.

18. USEPA, 2007, Method 1699: Pesticides in Water, Soil, Sediment, Biosolids, and Tissue by HRGC/HRMS, EPA–821–R–08–001, December 2007.

19. “Less is Better,” American Chemical Society on-line publication, http://www.acs.org/content/dam/acsorg/about/governance/committees/chemicalsafety/publications/less-is-better.pdf.

20. EPA Method 608 ATP 3M0222, An alternative test procedure for the measurement of organochlorine pesticides and polychlorinated biphenyls in waste water. Federal Register, Vol. 60, No. 148 August 2, 1995.

21. Tables

Table 1—Pesticides1

Analyte CAS No. MDL2
(ng/L)
ML3
(ng/L)
Aldrin 309–00–2 4 12
alpha-BHC 319–84–6 3 9
beta-BHC 319–85–7 6 18
delta-BHC 319–86–8 9 27
gamma-BHC (Lindane) 58–89–9 4 12
alpha-Chlordane4 5103–71–9 14 42
gamma-Chlordane4 5103–74–2 14 42
4,4′-DDD 72–54–8 11 33
4,4′-DDE 72–55–9 4 12
4,4′-DDT 50–29–3 12 36
Dieldrin 60–57–1 2 6
Endosulfan I 959–98–8 14 42
Endosulfan II 33213–65–9 4 12
Endosulfan sulfate 1031–07–8 66 198
Endrin 72–20–8 6 18
Endrin aldehyde 7421–93–4 23 70
Heptachlor 76–44–8 3 9
Heptachlor epoxide 1024–57–3 83 249

Table 2—Additional Analytes

Analyte CAS No. MDL3
(ng/L)
ML4
(ng/L)
Acephate 30560–19–1
Alachlor 15972–60–8
Atrazine 1912–24–9
Benfluralin (Benefin) 1861–40–1
Bromacil 314–40–9
Bromoxynil octanoate 1689–99–2
Butachlor 23184–66–9
Captafol 2425–06–1
Captan 133–06–2
Carbophenothion (Trithion) 786–19–6
Chlorobenzilate 510–15–6
Chloroneb (Terraneb) 2675–77–6
Chloropropylate (Acaralate) 5836–10–2
Chlorothalonil 1897–45–6
Cyanazine 21725–46–2
DCPA (Dacthal) 1861–32–1
2,4′-DDD 53–19–0
2,4′-DDE 3424–82–6
2,4′-DDT 789–02–6
Diallate (Avadex) 2303–16–4
1,2-Dibromo-3-chloropropane (DBCP) 96–12–8
Dichlone 117–80–6
Dichloran 99–30–9
Dicofol 115–32–2
Endrin ketone 53494–70–5
Ethalfluralin (Sonalan) 55283–68–6
Etridiazole 2593–15–9
Fenarimol (Rubigan) 60168–88–9
Hexachlorobenzene1 118–74–1
Hexachlorocyclopentadiene1 77–47–4
Isodrin 465–73–6
Isopropalin (Paarlan) 33820–53–0
Kepone 143–50–0
Methoxychlor 72–43–5
Metolachlor 51218–45–2
Metribuzin 21087–64–9
Mirex 2385–85–5
Nitrofen (TOK) 1836–75–5
cis-Nonachlor 5103–73–1
trans-Nonachlor 39765–80–5
Norfluorazon 27314–13–2
Octachlorostyrene 29082–74–4
Oxychlordane 27304–13–8
PCNB (Pentachloronitrobenzene) 82–68–8
Pendamethalin (Prowl) 40487–42–1
cis-Permethrin 61949–76–6
trans-Permethrin 61949–77–7
Perthane (Ethylan) 72–56–0
Propachlor 1918–16–7
Propanil 709–98–8
Propazine 139–40–2
Quintozene 82–68–8
Simazine 122–34–9
Strobane 8001–50–1
Technazene 117–18–0
Technical Chlordane2
Terbacil 5902–51–2
Terbuthylazine 5915–41–3
Toxaphene1 8001–35–2 240 720
Trifluralin 1582–09–8
PCB–10161 12674–11–2
PCB–12211 11104–28–2
PCB–12321 11141–16–5
PCB–12421 53469–21–9 65 95
PCB–12481 12672–29–6
PCB–12541 11097–69–1
PCB–12601 11096–82–5
PCB–1268 11100–14–4

Table 3—Example Retention Times1

Analyte Retention time
(min)2
DB–608 DB–1701
Acephate 5.03 (3)
Trifluralin 5.16 6.79
Ethalfluralin 5.28 6.49
Benfluralin 5.53 6.87
Diallate-A 7.15 6.23
Diallate-B 7.42 6.77
alpha-BHC 8.14 7.44
PCNB 9.03 7.58
Simazine 9.06 9.29
Atrazine 9.12 9.12
Terbuthylazine 9.17 9.46
gamma-BHC (Lindane) 9.52 9.91
beta-BHC 9.86 11.90
Heptachlor 10.66 10.55
Chlorothalonil 10.66 10.96
Dichlone 10.80 (4)
Terbacil 11.11 12.63
delta-BHC 11.20 12.98
Alachlor 11.57 11.06
Propanil 11.60 14.10
Aldrin 11.84 11.46
DCPA 12.18 12.09
Metribuzin 12.80 11.68
Triadimefon 12.99 13.57
Isopropalin 13.06 13.37
Isodrin 13.47 11.12
Heptachlor epoxide 13.97 12.56
Pendamethalin 14.21 13.46
Bromacil 14.39 (3)
alpha-Chlordane 14.63 14.20
Butachlor 15.03 15.69
gamma-Chlordane 15.24 14.36
Endosulfan I 15.25 13.87
4,4′-DDE 16.34 14.84
Dieldrin 16.41 15.25
Captan 16.83 15.43
Chlorobenzilate 17.58 17.28
Endrin 17.80 15.86
Nitrofen (TOK) 17.86 17.47
Kepone 17.92 (3 5)
4,4′-DDD 18.43 17.77
Endosulfan II 18.45 18.57
Bromoxynil octanoate 18.85 18.57
4,4′-DDT 19.48 18.32
Carbophenothion 19.65 18.21
Endrin aldehyde 19.72 19.18
Endosulfan sulfate 20.21 20.37
Captafol 22.51 21.22
Norfluorazon 20.68 22.01
Mirex 22.75 19.79
Methoxychlor 22.80 20.68
Endrin ketone 23.00 21.79
Fenarimol 24.53 23.79
cis-Permethrin 25.00 23.59
trans-Permethrin 25.62 23.92
PCB–1016
PCB–1221
PCB–1232
PCB–1242
PCB–1248
PCB–1254
PCB–1260 (5 peaks) 15.44 14.64
15.73 15.36
16.94 16.53
17.28 18.70
19.17 19.92
Toxaphene (5 peaks) 16.60 16.60
17.37 17.52
18.11 17.92
19.46 18.73
19.69 19.00

Table 4—QC Acceptance Criteria

Analyte Calibration verification
(%)
Test
concentration
(µg/L)
Limit for s
(% SD)
Range for X
(%)
Range for P
(%)
Maximum
MS/MSD
RPD
(%)
Aldrin 75–125 2.0 25 54–130 42–140 35
alpha-BHC 69–125 2.0 28 49–130 37–140 36
beta-BHC 75–125 2.0 38 39–130 17–147 44
delta-BHC 75–125 2.0 43 51–130 19–140 52
gamma-BHC 75–125 2.0 29 43–130 32–140 39
alpha-Chlordane 73–125 50.0 24 55–130 45–140 35
gamma-Chlordane 75–125 50.0 24 55–130 45–140 35
4,4′-DDD 75–125 10.0 32 48–130 31–141 39
4,4′-DDE 75–125 2.0 30 54–130 30–145 35
4,4′-DDT 75–125 10.0 39 46–137 25–160 42
Dieldrin 48–125 2.0 42 58–130 36–146 49
Endosulfan I 75–125 2.0 25 57–141 45–153 28
Endosulfan II 75–125 10.0 63 22–171 D–202 53
Endosulfan sulfate 70–125 10.0 32 38–132 26–144 38
Endrin 5–125 10.0 42 51–130 30–147 48
Heptachlor 75–125 2.0 28 43–130 34–140 43
Heptachlor epoxide 75–125 2.0 22 57–132 37–142 26
Toxaphene 68–134 50.0 30 56–130 41–140 41
PCB–1016 75–125 50.0 24 61–103 50–140 36
PCB–1221 75–125 50.0 50 44–150 15–178 48
PCB–1232 75–125 50.0 32 28–197 10–215 25
PCB–1242 75–125 50.0 26 50–139 39–150 29
PCB–1248 75–125 50.0 32 58–140 38–158 35
PCB–1254 75–125 50.0 34 44–130 29–140 45
PCB–1260 75–125 50.0 28 37–130 8–140 38

Table 5—Precision and Recovery as Functions of Concentration

Analyte Recovery, X′
(µg/L)
Single analyst
precision, sr
(µg/L)
Overall
precision, S′
(µg/L)
Aldrin 0.81C + 0.04 0.16(X) − 0.04 0.20(X) − 0.01
alpha-BHC 0.84C + 0.03 0.13(X) + 0.04 0.23(X) − 0.00
beta-BHC 0.81C + 0.07 0.22(X) − 0.02 0.33(X) − 0.05
delta-BHC 0.81C + 0.07 0.18(X) + 0.09 0.25(X) + 0.03
gamma-BHC (Lindane) 0.82C − 0.05 0.12(X) + 0.06 0.22(X) + 0.04
Chlordane 0.82C − 0.04 0.13(X) + 0.13 0.18(X) + 0.18
4,4′-DDD 0.84C + 0.30 0.20(X) − 0.18 0.27(X) − 0.14
4,4′-DDE 0.85C + 0.14 0.13(X) + 0.06 0.28(X) − 0.09
4,4′-DDT 0.93C − 0.13 0.17(X) + 0.39 0.31(X) − 0.21
Dieldrin 0.90C + 0.02 0.12(X) + 0.19 0.16(X) + 0.16
Endosulfan I 0.97C + 0.04 0.10(X) + 0.07 0.18(X) + 0.08
Endosulfan II 0.93C + 0.34 0.41(X) − 0.65 0.47(X) − 0.20
Endosulfan sulfate 0.89C − 0.37 0.13(X) + 0.33 0.24(X) + 0.35
Endrin 0.89C − 0.04 0.20(X) + 0.25 0.24(X) + 0.25
Heptachlor 0.69C + 0.04 0.06(X) + 0.13 0.16(X) + 0.08
Heptachlor epoxide 0.89C + 0.10 0.18(X) − 0.11 0.25(X) − 0.08
Toxaphene 0.80C + 1.74 0.09(X) + 3.20 0.20(X) + 0.22
PCB–1016 0.81C + 0.50 0.13(X) + 0.15 0.15(X) + 0.45
PCB–1221 0.96C + 0.65 0.29(X) − 0.76 0.35(X) − 0.62
PCB–1232 0.91C + 10.8 0.21(X) − 1.93 0.31(X) + 3.50
PCB–1242 0.93C + 0.70 0.11(X) + 1.40 0.21(X) + 1.52
PCB–1248 0.97C + 1.06 0.17(X) + 0.41 0.25(X) − 0.37
PCB–1254 0.76C + 2.07 0.15(X) + 1.66 0.17(X) + 3.62
PCB–1260 0.66C + 3.76 0.22(X) − 2.37 0.39(X) − 4.86

Table 6—Distribution of Chlorinated Pesticides and PCBs Into Florisil® Column Fractions

Analyte Percent Recovery by Fraction1
1 2 3
Aldrin 100
alpha-BHC 100
beta-BHC 97
delta-BHC 98
gamma-BHC (Lindane) 100
Chlordane 100
4,4′-DDD 99
4,4′-DDE 98
4,4′-DDT 100
Dieldrin 0 100
Endosulfan I 37 64
Endosulfan II 0 7 91
Endosulfan sulfate 0 0 106
Endrin 4 96
Endrin aldehyde 0 68 26
Heptachlor 100
Heptachlor epoxide 100
Toxaphene 96
PCB–1016 97
PCB–1221 97
PCB–1232 95 4
PCB–1242 97
PCB–1248 103
PCB–1254 90
PCB–1260

Table 7—Suggested Calibration Groups1

Analyte
Calibration Group 1:
Acephate
Alachlor
Atrazine
beta-BHC
Bromoxynil octanoate
Captafol
Diallate
Endosulfan sulfate
Endrin
Isodrin
Pendimethalin (Prowl)
trans-Permethrin
Calibration Group 2:
alpha-BHC
DCPA
4,4′-DDE
4,4′-DDT
Dichlone
Ethalfluralin
Fenarimol
Methoxychlor
Metribuzin
Calibration Group 3:
gamma-BHC (Lindane)
gamma-Chlordane
Endrin ketone
Heptachlor epoxide
Isopropalin
Nitrofen (TOK)
PCNB
cis-Permethrin
Trifluralin
Callibration Group 4:
Benfluralin
Chlorobenzilate
Dieldrin
Endosulfan I
Mirex
Terbacil
Terbuthylazine
Triadimefon
Calibration Group 5:
alpha-Chlordane
Captan
Chlorothalonil
4,4′-DDD
Norfluorazon
Simazine
Calibration Group 6:
Aldrin
delta-BHC
Bromacil
Butachlor
Endosulfan II
Heptachlor
Kepone
Calibration Group 7:
Carbophenothion
Chloroneb
Chloropropylate
DBCP
Dicofol
Endrin aldehyde
Etridiazone
Perthane
Propachlor
Propanil
Propazine

22. Figures

23. Glossary

These definitions and purposes are specific to this method but have been conformed to common usage to the extent possible.

23.1 Units of weight and measure and their abbreviations.

23.1.1 Symbols.

°C degrees Celsius

µg microgram

µL microliter

< less than

≤ less than or equal to

> greater than

% percent

23.1.2 Abbreviations (in alphabetical order).

cm centimeter

g gram

hr hour

ID inside diameter

in. inch

L liter

M molar solution—one mole or gram molecular weight of solute in one liter of solution

mg milligram

min minute

mL milliliter

mm millimeter

N Normality—one equivalent of solute in one liter of solution

ng nanogram

psia pounds-per-square inch absolute

psig pounds-per-square inch gauge

v/v volume per unit volume

w/v weight per unit volume

23.2 Definitions and acronyms (in alphabetical order)

Analyte—A compound or mixture of compounds (e.g., PCBs) tested for by this method. The analytes are listed in Tables 1 and 2.

Analytical batch—The set of samples analyzed on a given instrument during a 24-hour period that begins and ends with calibration verification (sections 7.8 and 13). See also “Extraction batch.”

Blank (method blank; laboratory blank)—An aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples. The blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus.

Calibration factor (CF)—See section 7.5.1.

Calibration standard—A solution prepared from stock solutions and/or a secondary standards and containing the analytes of interest, surrogates, and internal standards. This standard is used to model the response of the GC instrument against analyte concentration.

Calibration verification—The process of confirming that the response of the analytical system remains within specified limits of the calibration.

Calibration verification standard—The standard (section 6.8.4) used to verify calibration (sections 7.8 and 13.6).

Extraction Batch—A set of up to 20 field samples (not including QC samples) started through the extraction process in a given 24-hour shift. Each extraction batch of 20 or fewer samples must be accompanied by a blank (section 8.5), a laboratory control sample (LCS, section 8.4), a matrix spike and duplicate (MS/MSD; section 8.3), resulting in a minimum of five samples (1 field sample, 1 blank, 1 LCS, 1 MS, and 1 MSD) and a maximum of 24 samples (20 field samples, 1 blank, 1 LCS, 1 MS, and 1 MSD) for the batch. If greater than 20 samples are to be extracted in a 24-hour shift, the samples must be separated into extraction batches of 20 or fewer samples.

Field Duplicates—Two samples collected at the same time and place under identical conditions, and treated identically throughout field and laboratory procedures. Results of analyses the field duplicates provide an estimate of the precision associated with sample collection, preservation, and storage, as well as with laboratory procedures.

Field blank—An aliquot of reagent water or other reference matrix that is placed in a sample container in the field, and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to determine if the field or sample transporting procedures and environments have contaminated the sample. See also “Blank.”

GC—Gas chromatograph or gas chromatography.

Gel-permeation chromatography (GPC)—A form of liquid chromatography in which the analytes are separated based on exclusion from the solid phase by size.

Internal standard—A compound added to an extract or standard solution in a known amount and used as a reference for quantitation of the analytes of interest and surrogates. Also see Internal standard quantitation.

Internal standard quantitation—A means of determining the concentration of an analyte of interest (Tables 1 and 2) by reference to a compound not expected to be found in a sample.

IDC—Initial Demonstration of Capability (section 8.2); four aliquots of a reference matrix spiked with the analytes of interest and analyzed to establish the ability of the laboratory to generate acceptable precision and recovery. An IDC is performed prior to the first time this method is used and any time the method or instrumentation is modified.

Laboratory Control Sample (LCS; laboratory fortified blank; section 8.4)—An aliquot of reagent water spiked with known quantities of the analytes of interest and surrogates. The LCS is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified in this method for precision and recovery.

Laboratory Fortified Sample Matrix—See Matrix spike.

Laboratory reagent blank—See blank.

Matrix spike (MS) and matrix spike duplicate (MSD) (laboratory fortified sample matrix and duplicate)—Two aliquots of an environmental sample to which known quantities of the analytes of interest and surrogates are added in the laboratory. The MS/MSD are prepared and analyzed exactly like a field sample. Their purpose is to quantify any additional bias and imprecision caused by the sample matrix. The background concentrations of the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the MS/MSD corrected for background concentrations.

May—This action, activity, or procedural step is neither required nor prohibited.

May not—This action, activity, or procedural step is prohibited.

Method detection limit (MDL)—A detection limit determined by the procedure at 40 CFR part 136, appendix B. The MDLs determined by EPA are listed in Tables 1 and 2. As noted in section 1.6, use the MDLs in Tables 1 and 2 in conjunction with current MDL data from the laboratory actually analyzing samples to assess the sensitivity of this procedure relative to project objectives and regulatory requirements (where applicable).

Minimum level (ML)—The term “minimum level” refers to either the sample concentration equivalent to the lowest calibration point in a method or a multiple of the method detection limit (MDL), whichever is higher. Minimum levels may be obtained in several ways: They may be published in a method; they may be based on the lowest acceptable calibration point used by a laboratory; or they may be calculated by multiplying the MDL in a method, or the MDL determined by a laboratory, by a factor of 3. For the purposes of NPDES compliance monitoring, EPA considers the following terms to be synonymous: “quantitation limit,” “reporting limit,” and “minimum level.”

MS—Mass spectrometer or mass spectrometry.

Must—This action, activity, or procedural step is required.

Preparation blank—See blank.

Reagent water—Water demonstrated to be free from the analytes of interest and potentially interfering substances at the MDLs for the analytes in this method.

Regulatory compliance limit—A limit on the concentration or amount of a pollutant or contaminant specified in a nationwide standard, in a permit, or otherwise established by a regulatory/control authority.

Relative standard deviation (RSD)—The standard deviation times 100 divided by the mean. Also termed “coefficient of variation.”

RF—Response factor. See section 7.6.2.

RPD—Relative percent difference.

RSD—See relative standard deviation.

Safety Data Sheet (SDS)—Written information on a chemical's toxicity, health hazards, physical properties, fire, and reactivity, including storage, spill, and handling precautions that meet the requirements of OSHA, 29 CFR 1910.1200(g) and appendix D to § 1910.1200. United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), third revised edition, United Nations, 2009.

Should—This action, activity, or procedural step is suggested but not required.

SPE—Solid-phase extraction; a sample extraction or extract cleanup technique in which an analyte is selectively removed from a sample or extract by passage over or through a material capable of reversibly adsorbing the analyte.

Stock solution—A solution containing an analyte that is prepared using a reference material traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will attest to the purity and authenticity of the reference material.

Surrogate—A compound unlikely to be found in a sample, which is spiked into the sample in a known amount before extraction, and which is quantified with the same procedures used to quantify other sample components. The purpose of the surrogate is to monitor method performance with each sample.

Method 609—Nitroaromatics and Isophorone

1. Scope and Application

1.1 This method covers the determination of certain nitroaromatics and isophorone. The following parameters may be determined by this method:

Parameter STORET No. CAS No.
2,4-Dinitrotoluene 34611 121–14–2
2,6-Dinitrotoluene 34626 606–20–2
Isophorone 34408 78–59–1
Nitrobenzene 34447 98–95–3

1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. Isophorone and nitrobenzene are measured by flame ionization detector gas chromatography (FIDGC). The dinitrotoluenes are measured by electron capture detector gas chromatography (ECDGC).2

2.2 The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baseliles in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4-6 for the information of the analyst.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnel—2–L, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Chromatographic column—100 mm long × 10 mm ID, with Teflon stopcock.

5.2.4 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5 Evaporative flask, Kuderna-Danish—500-mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.6 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.7 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).

5.2.8 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6 Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1 Column 1—1.2 m long × 2 or 4 mm ID glass, packed with 1.95% QF–1/1.5% OV–17 on Gas-Chrom Q (80/100 mesh) or equivalent. This column was used to develop the method performance statements given in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2 Column 2—3.0 m long × 2 or 4 mm ID glass, packed with 3% OV–101 on Gas-Chrom Q (80/100 mesh) or equivalent.

5.6.3 Detectors—Flame ionization and electron capture detectors. The flame ionization detector (FID) is used when determining isophorone and nitrobenzene. The electron capture detector (ECD) is used when determining the dinitrotoluenes. Both detectors have proven effective in the analysis of wastewaters and were used in develop the method performance statements in Section 14. Guidelines for the use to alternate detectors are provided in Section 12.1.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3 Sulfuric acid (1 + 1)—Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.4 Acetone, hexane, methanol, methylene chloride—Pesticide quality or equivalent.

6.5 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.6 Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 200 °C in a foil-covered glass container and allow to cool.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in hexane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD) linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

Equation 1.

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1,5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest in acetone at a concentration of 20 µg/mL for each dinitrotoluene and 100 µg/mL for isophorone and nitrobenzene. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determile background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none

(2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄8;

(3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44 (100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4. If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with sodium hydroxide solution or sulfuric acid.

10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6 Sections 10.7 and 10.8 describe a procedure for exchanging the methylene chloride solvent to hexane while concentrating the extract volume to 1.0 mL. When it is not necessary to achieve the MDL in Table 2, the solvent exchange may be made by the addition of 50 mL of hexane and concentration to 10 mL as described in Method 606, Sections 10.7 and 10.8.

10.7 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add 1 to 2 mL of hexane and a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9 Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of hexane. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.10 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2 Florisil column cleanup:

11.2.1 Prepare a slurry of 10 g of activated Florisil in methylene chloride/hexane (1 + 9)(V/V) and place the Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top. Adjust the elution rate to about 2 mL/min.

11.2.2 Just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 30 mL of methylene chloride/hexane (1 + 9)(V/V) and continue the elution of the column. Discard the eluate.

11.2.3 Next, elute the column with 30 mL of acetone/methylene chloride (1 + 9)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction as in Sections 10.6, 10.7, 10.8, and 10.9 including the solvent exchange to 1 mL of hexane. This fraction should contain the nitroaromatics and isophorone. Analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1 Isophorone and nitrobenzene are analyzed by injection of a portion of the extract into an FIDGC. The dinitrotoluenes are analyzed by a separate injection into an ECDGC. Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2 Calibrate the system daily as described in Section 7.

12.3 If the internal standard calibration procedure is being used, the internal standard must be added to the same extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.9 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, the total extract volume, and the resulting peak size in area or peak height units.

12.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7 × MDL to 1000 × MDL.10

14.3 This method was tested by 18 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 515 µg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

2. “Determination of Nitroaromatic Compounds and Isophorone in Industrial and Municipal Wastewaters,” EPA 600/ 4–82–024, National Technical Information Service, PB82–208398, Springfield, Virginia 22161, May 1982.

3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

10. “Determination of Method Detection Limit and Analytical Curve for EPA Method 609—Nitroaromatics and Isophorone,” Special letter report for EPA Contract 68–03–2624, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

11. “EPA Method Study 19, Method 609 (Nitroaromatics and Isophorone),” EPA 600/4–84–018, National Technical Information Service, PB84–176908, Springfield, Virginia 22161, March 1984.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Col. 1 Col. 2 ECDGC FIDGC
Nitrobenzene 3.31 4.31 13.7 3.6
2,6-Dinitrotoluene 3.52 4.75 0.01
Isophorone 4.49 5.72 15.7 5.7
2,4-Dinitrotoluene 5.35 6.54 0.02

Table 2—QC Acceptance Criteria—Method 609

Parameter Test Conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (%)
2,4-Dinitrotoluene 20 5.1 3.6–22.8 6–125
2,6-Dinitrotoluene 20 4.8 3.8–23.0 8–126
Isophorone 100 32.3 8.0–100.0 D–117
Nitrobenzene 100 33.3 25.7–100.0 6–118

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 609

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
2,4-Dinitro-
toluene 0.65C + 0.22 0.20X̄ + 0.08 0.37X̄−0.07
2,6-Dinitro-
toluene 0.66C + 0.20 0.19X̄ + 0.06 0.36X̄−0.00
Isophorone 0.49C + 2.93 0.28X̄ + 2.77 0.46X̄ + 0.31
Nitrobenzene 0.60C + 2.00 0.25X̄ + 2.53 0.37X̄−0.78

Method 610—Polynuclear Aromatic Hydrocarbons

1. Scope and Application

1.1 This method covers the determination of certain polynuclear aromatic hydrocarbons (PAH). The following parameters can be determined by this method:

Parameter STORET No. CAS No.
Acenaphthene 34205 83–32–9
Acenaphthylene 34200 208–96–8
Anthracene 34220 120–12–7
Benzo(a)anthracene 34526 56–55–3
Benzo(a)pyrene 34247 50–32–8
Benzo(b)fluoranthene 34230 205–99–2
Benzo(ghi)perylene 34521 191–24–2
Benzo(k)fluoranthene 34242 207–08–9
Chrysene 34320 218–01–9
Dibenzo(a,h)anthracene 34556 53–70–3
Fluoranthene 34376 206–44–0
Fluorene 34381 86–73–7
Indeno(1,2,3-cd)pyrene 34403 193–39–5
Naphthalene 34696 91–20–3
Phenanthrene 34461 85–01–8
Pyrene 34469 129–00–0

1.2 This is a chromatographic method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for many of the parameters listed above, using the extract produced by this method.

1.3 This method provides for both high performance liquid chromatographic (HPLC) and gas chromatographic (GC) approaches for the determination of PAHs. The gas chromatographic procedure does not adequately resolve the following four pairs of compounds: Anthracene and phenanthrene; chrysene and benzo

(a) anthracene; benzo

(b) fluoranthene and benzo(k)fluoranthene; and dibenzo(a,h) anthracene and indeno (1,2,3-cd)pyrene. Unless the purpose for the analysis can be served by reporting the sum of an unresolved pair, the liquid chromatographic approach must be used for these compounds. The liquid chromatographic method does resolve all 16 of the PAHs listed.

1.4 The method detection limit (MDL, defined in Section 15.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. Selection of the aliquots must be made prior to the solvent exchange steps of this method. The analyst is allowed the latitude, under Sections 12 and 13, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.6 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.7 This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC and GC systems and in the interpretation of liquid and gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and concentrated to a volume of 10 mL or less. The extract is then separated by HPLC or GC. Ultraviolet (UV) and fluorescence detectors are used with HPLC to identify and measure the PAHs. A flame ionization detector is used with GC.2

2.2 The method provides a silica gel column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardward that lead to discrete artifacts and/or elevated baselines in the chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3 The extent of interferences that may be encountered using liquid chromatographic techniques has not been fully assessed. Although the HPLC conditions described allow for a unique resolution of the specific PAH compounds covered by this method, other PAH compounds may interfere.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method have not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4-6 for the information of the analyst.

4.2 The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)-anthracene. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnel—2–L, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.4 Evaporative flask, Kuderna-Danish—500-mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.5 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.6 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).

5.2.7 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.8 Chromatographic column—250 mm long × 10 mm ID, with coarse frit filter disc at bottom and Teflon stopcock.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6 High performance liquid chromatograph (HPLC)—An analytical system complete with column supplies, high pressure syringes, detectors, and compatible strip-chart recorder. A data system is recommended for measuring peak areas and retention times.

5.6.1 Gradient pumping system—Constant flow.

5.6.2 Reverse phase column—HC-ODS Sil-X, 5 micron particle diameter, in a 25 cm × 2.6 mm ID stainless steel column (Perkin Elmer No. 089–0716 or equivalent). This column was used to develop the method performance statements in Section 15. Guidelines for the use of alternate column packings are provided in Section 12.2.

5.6.3 Detectors—Fluorescence and/or UV detectors. The fluorescence detector is used for excitation at 280 nm and emission greater than 389 nm cutoff (Corning 3–75 or equivalent). Fluorometers should have dispersive optics for excitation and can utilize either filter or dispersive optics at the emission detector. The UV detector is used at 254 nm and should be coupled to the fluorescence detector. These detectors were used to develop the method performance statements in Section 15. Guidelines for the use of alternate detectors are provided in Section 12.2.

5.7 Gas chromatograph—An analytical system complete with temperature programmable gas chromatograph suitable for on-column or splitless injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.7.1 Column—1.8 m long × 2 mm ID glass, packed with 3% OV–17 on Chromosorb W-AW-DCMS (100/120 mesh) or equivalent. This column was used to develop the retention time data in Table 2. Guidelines for the use of alternate column packings are provided in Section 13.3.

5.7.2 Detector—Flame ionization detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), excluding the four pairs of unresolved compounds listed in Section 1.3. Guidelines for the use of alternate detectors are provided in Section 13.3.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Sodium thiosulfate—(ACS) Granular.

6.3 Cyclohexane, methanol, acetone, methylene chloride, pentane—Pesticide quality or equivalent.

6.4 Acetonitrile—HPLC quality, distilled in glass.

6.5 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.6 Silica gel—100/200 mesh, desiccant, Davison, grade-923 or equivalent. Before use, activate for at least 16 h at 130 °C in a shallow glass tray, loosely covered with foil.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetonitrile and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish liquid or gas chromatographic operating conditions equivalent to those given in Table 1 or 2. The chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with acetonitrile. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 5 to 25 µL for HPLC and 2 to 5 µL for GC, analyze each calibration standard according to Section 12 or 13, as appropriate. Tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with acetonitrile. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 5 to 25 µL for HPLC and 2 to 5 µL for GC, analyze each calibration standard according to Section 12 or 13, as appropriate. Tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, 12.2, and 13.3) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetonitrile: 100 µg/mL of any of the six early-eluting PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene); 5 µg/mL of benzo(k)fluoranthene; and 10 µg/mL of any of the other PAHs. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at the test concentrations shown in Table 3 by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6 When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1 Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2 Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none,

(2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 4, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 4, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the critiera must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 3 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄–2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. PAHs are known to be light sensitive; therefore, samples, extracts, and standards should be stored in amber or foil-wrapped bottles in order to minimize photolytic decomposition. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.9 Field test kits are available for this purpose.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel.

10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250–mL Erlenmeyer flask.

10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial and protected from light. If the sample extract requires no further cleanup, proceed with gas or liquid chromatographic analysis (Section 12 or 13). If the sample requires further cleanup, proceed to Section 11.

10.8 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the methods as revised to incorporate the cleanup procedure.

11.2 Before the silica gel cleanup technique can be utilized, the extract solvent must be exchanged to cyclohexane. Add 1 to 10 mL of the sample extract (in methylene chloride) and a boiling chip to a clean K-D concentrator tube. Add 4 mL of cyclohexane and attach a two-ball micro-Snyder column. Prewet the column by adding 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a boiling (100 °C) water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of the liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of cyclohexane. Adjust the extract volume to about 2 mL.

11.3 Silica gel column cleanup for PAHs:

11.3.1 Prepare a slurry of 10 g of activiated silica gel in methylene chloride and place this into a 10-mm ID chromatographic column. Tap the column to settle the silica gel and elute the methylene chloride. Add 1 to 2 cm of anhydrous sodium sulfate to the top of the silica gel.

11.3.2 Preelute the column with 40 mL of pentane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, transfer the 2-mL cyclohexane sample extract onto the column using an additional 2 mL cyclohexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 25 mL of pentane and continue the elution of the column. Discard this pentane eluate.

11.3.3 Next, elute the column with 25 mL of methylene chloride/pentane (4 + 6)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to less than 10 mL as in Section 10.6. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint with pentane. Proceed with HPLC or GC analysis.

12. High Performance Liquid Chromatography

12.1 To the extract in the concentrator tube, add 4 mL of acetonitrile and a new boiling chip, then attach a two-ball micro-Snyder column. Concentrate the solvent as in Section 10.6, except set the water bath at 95 to 100 °C. When the apparatus is cool, remove the micro-Snyder column and rinse its lower joint into the concentrator tube with about 0.2 mL of acetonitrile. Adjust the extract volume to 1.0 mL.

12.2 Table 1 summarizes the recommended operating conditions for the HPLC. Included in this table are retention times, capacity factors, and MDL that can be achieved under these conditions. The UV detector is recommended for the determination of naphthalene, acenaphthylene, acenapthene, and fluorene and the fluorescence detector is recommended for the remaining PAHs. Examples of the separations achieved by this HPLC column are shown in Figures 1 and 2. Other HPLC columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3 Calibrate the system daily as described in Section 7.

12.4 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the instrument.

12.5 Inject 5 to 25 µL of the sample extract or standard into the HPLC using a high pressure syringe or a constant volume sample injection loop. Record the volume injected to the nearest 0.1 µL, and the resulting peak size in area or peak height units. Re-equilibrate the HPLC column at the initial gradient conditions for at least 10 min between injections.

12.6 Identify the parameters in the sample by comparing the retention time of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.7 If the response for a peak exceeds the working range of the system, dilute the extract with acetonitrile and reanalyze.

12.8 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Gas Chromatography

13.1 The packed column GC procedure will not resolve certain isomeric pairs as indicated in Section 1.3 and Table 2. The liquid chromatographic procedure (Section 12) must be used for these parameters.

13.2 To achieve maximum sensitivity with this method, the extract must be concentrated to 1.0 mL. Add a clean boiling chip to the methylene chloride extract in the concentrator tube. Attach a two-ball micro-Snyder column. Prewet the micro-Snyder column by adding about 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of methylene chloride. Adjust the final volume to 1.0 mL and stopper the concentrator tube.

13.3 Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times that were obtained under these conditions. An example of the separations achieved by this column is shown in Figure 3. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

13.4 Calibrate the gas chromatographic system daily as described in Section 7.

13.5 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

13.6 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.10 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, and the resulting peak size in area or peak height units.

13.7 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

13.8 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

13.9 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

14. Calculations

14.1 Determine the concentration of individual compounds in the sample.

14.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

14.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

15. Method Performance

15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.11 Similar results were achieved using representative wastewaters. MDL for the GC approach were not determined. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

15.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 8 × MDL to 800 × MDL11 with the following exception: benzo(ghi)perylene recovery at 80 × and 800 × MDL were low (35% and 45%, respectively).

15.3 This method was tested by 16 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.1 to 425 µg/L.12 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 4.

2. “Determination of Polynuclear Aromatic Hydrocarbons in Industrial and Municipal Wastewaters,” EPA 600/4–82–025, National Technical Information Service, PB82–258799, Springfield, Virginia 22161, June 1982.

3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

10. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

11. Cole, T., Riggin, R., and Glaser, J. “Evaluation of Method Detection Limits and Analytical Curve for EPA Method 610—PNAs,” International Symposium on Polynuclear Aromatic Hydrocarbons, 5th, Battelle's Columbus Laboratories, Columbus, Ohio (1980).

12. “EPA Method Study 20, Method 610 (PNA's),” EPA 600/4–84–063, National Technical Information Service, PB84–211614, Springfield, Virginia 22161, June 1984.

Table 1—High Performance Liquid Chromatography Conditions and Method Detection Limits

Parameter Retention time (min) Column capacity factor (k′) Method detection limit (µg/L)a
Naphthalene 16.6 12.2 1.8
Acenaphthylene 18.5 13.7 2.3
Acenaphthene 20.5 15.2 1.8
Fluorene 21.2 15.8 0.21
Phenanthrene 22.1 16.6 0.64
Anthracene 23.4 17.6 0.66
Fluoranthene 24.5 18.5 0.21
Pyrene 25.4 19.1 0.27
Benzo(a)anthracene 28.5 21.6 0.013
Chrysene 29.3 22.2 0.15
Benzo(b)fluoranthene 31.6 24.0 0.018
Benzo(k)fluoranthene 32.9 25.1 0.017
Benzo(a)pyrene 33.9 25.9 0.023
Dibenzo(a,h)anthracene 35.7 27.4 0.030
Benzo(ghi)perylene 36.3 27.8 0.076
Indeno(1,2,3-cd)pyrene 37.4 28.7 0.043

Table 2—Gas Chromatographic Conditions and Retention Times

Parameter Retention time (min)
Naphthalene 4.5
Acenaphthylene 10.4
Acenaphthene 10.8
Fluorene 12.6
Phenanthrene 15.9
Anthracene 15.9
Fluoranthene 19.8
Pyrene 20.6
Benzo(a)anthracene 24.7
Chrysene 24.7
Benzo(b)fluoranthene 28.0
Benzo(k)fluoranthene 28.0
Benzo(a)pyrene 29.4
Dibenzo(a,h)anthracene 36.2
Indeno(1,2,3-cd)pyrene 36.2
Benzo(ghi)perylene 38.6

Table 3—QC Acceptance Criteria—Method 610

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (%)
Acenaphthene 100 40.3 D–105.7 D–124
Acenaphthylene 100 45.1 22.1–112.1 D–139
Anthracene 100 28.7 11.2–112.3 D–126
Benzo(a)anthracene 10 4.0 3.1–11.6 12–135
Benzo(a)pyrene 10 4.0 0.2–11.0 D–128
Benzo(b)fluor-anthene 10 3.1 1.8–13.8 6–150
Benzo(ghi)perylene 10 2.3 D–10.7 D–116
Benzo(k)fluo-ranthene 5 2.5 D–7.0 D–159
Chrysene 10 4.2 D–17.5 D–199
Dibenzo(a,h)an-thracene 10 2.0 0.3–10.0 D–110
Fluoranthene 10 3.0 2.7–11.1 14–123
Fluorene 100 43.0 D–119 D–142
Indeno(1,2,3–cd)pyrene 10 3.0 1.2–10.0 D–116
Naphthalene 100 40.7 21.5–100.0 D–122
Phenanthrene 100 37.7 8.4–133.7 D–155
Pyrene 10 3.4 1.4–12.1 D–140

Table 4—Method Accuracy and Precision as Functions of Concentration—Method 610

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′
(µg/L)
Acenaphthene 0.52C + 0.54 0.39X̄ + 0.76 0.53X̄ + 1.32
Acenaphthylene 0.69C − 1.89 0.36X̄ + 0.29 0.42X̄ + 0.52
Anthracene 0.63C − 1.26 0.23X̄ + 1.16 0.41X̄ + 0.45
Benzo(a)anthracene 0.73C + 0.05 0.28X̄ + 0.04 0.34X̄ + 0.02
Benzo(a)pyrene 0.56C + 0.01 0.38X̄ − 0.01 0.53X̄ − 0.01
Benzo(b)fluoranthene 0.78C + 0.01 0.21X̄ + 0.01 0.38X̄ − 0.00
Benzo(ghi)perylene 0.44C + 0.30 0.25X̄ + 0.04 0.58X̄ + 0.10
Benzo(k)fluoranthene 0.59C + 0.00 0.44X̄ − 0.00 0.69X̄ + 0.01
Chrysene 0.77C − 0.18 0.32X̄ − 0.18 0.66X̄ − 0.22
Dibenzo(a,h)anthracene 0.41C + 0.11 0.24X̄ + 0.02 0.45X̄ + 0.03
Fluoranthene 0.68C + 0.07 0.22X̄ + 0.06 0.32X̄ + 0.03
Fluorene 0.56C − 0.52 0.44X̄ − 1.12 0.63X̄ − 0.65
Indeno(1,2,3–cd)pyrene 0.54C + 0.06 0.29X̄ + 0.02 0.42X̄ + 0.01
Naphthalene 0.57C − 0.70 0.39X̄ − 0.18 0.41X̄ + 0.74
Phenanthrene 0.72C − 0.95 0.29X̄ + 0.05 0.47X̄ − 0.25
Pyrene 0.69C − 0.12 0.25X̄ + 0.14 0.42X̄ − 0.00

Method 611—Haloethers

1. Scope and Application

1.1 This method covers the determination of certain haloethers. The following parameters can be determined by this method:

Parameter STORET No. CAS No.
Bis(2-chloroethyl) ether 34273 111–44–4
Bis(2-chloroethoxy) methane 34278 111–91–1
2, 2′-oxybis (1-chloropropane) 34283 108–60–1
4-Bromophenyl phenyl ether 34636 101–55–3
4-Chlorophenyl phenyl ether 34641 7005–72–3

1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with a halide specific detector.2

2.2 The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed be detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such a PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3 Dichlorobenzenes are known to coelute with haloethers under some gas chromatographic conditions. If these materials are present together in a sample, it may be necessary to analyze the extract with two different column packings to completely resolve all of the compounds.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4-6 for the information of the analyst.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnel—2-L, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Chromatographic column—400 mm long × 19 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K–420540–0224 or equivalent).

5.2.4 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5 Evaporative flask, Kuderna-Danish—500-mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.6 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.7 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6 Gas chromatograph—An analytical system complete with temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1 Column 1—1.8 m long × 2 mm ID glass, packed with 3% SP–1000 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2 Column 2—1.8 m long × 2 mm ID glass, packed with 2,6-diphenylene oxide polymer (60/80 mesh), Tenax, or equivalent.

5.6.3 Detector—Halide specific detector: electrolytic conductivity or microcoulometric. These detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). The Hall conductivity detector was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1. Although less selective, an electron capture detector is an acceptable alternative.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Sodium thiosulfate—(ACS) Granular.

6.3 Acetone, hexane, methanol, methylene chloride, petroleum ether (boiling range 30–60 °C)—Pesticide quality or equivalent.

6.4 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.5 Florisil—PR Grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.6 Ethyl ether—Nanograde, redistilled in glass if necessary.

6.6.1 Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P1126–8, and other suppliers.)

6.6.2 Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetone and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5 The cleanup procedure in Section 11 utilizes Florisil column chromatography. Florisil from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which is used, the use of lauric acid value7 is suggested. The referenced procedure determines the adsorption from hexane solution of lauric acid (mg) per g of Florisil. The amount of Florisil to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g.

7.6 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 µg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at a concentration of 100 µg/L by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1. The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none

(2) the larger of either 5 times higher than the expected background concentration or 100 µg/L.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.8 If spiking was performed at a concentration lower than 100 µg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.8

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4 If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 m/L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄–2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices9 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.10 Field test kits are available for this purpose.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

Note:

Some of the haloethers are very volatile and significant losses will occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.

10.7 Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the temperature of the water bath to 85 to 90 °C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2 Florisil column cleanup for haloethers:

11.2.1 Adjust the sample extract volume to 10 mL.

11.2.2 Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5), into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.3 Preelute the column with 50 to 60 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate. Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 300 mL of ethyl ether/petroleum ether (6 + 94) (V/V). Adjust the elution rate to approximately 5 mL/min and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction should contain all of the haloethers.

11.2.4 Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of the cleaned up extract to 10 mL with hexane and analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columns 1 and 2 are shown in Figures 1 and 2, respectively. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2 Calibrate the system daily as described in Section 7.

12.3 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatrograph.

12.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.11 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, the total extract volume, and the resulting peak size in area or peak height units.

12.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weight heavily in the interpretation of chromatograms.

12.6 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 × MDL to 1000 × MDL.12

14.3 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 626 µ/L.12 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

2. “Determination of Haloethers in Industrial and Municipal Wastewaters,” EPA 600/4–81–062, National Technical Information Service, PB81–232290, Springfield, Virginia 22161, July 1981.

3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constitutents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working Carcinogens, ” Department of Health, Education, and Welfare, Public Health Services, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Mills., P.A. “Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical Chemists, 51, 29 (1968).

8. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

9. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

10. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

11. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

12. “EPA Method Study 21, Method 611, Haloethers,” EPA 600/4–84–052, National Technical Information Service, PB84–205939, Springfield, Virginia 22161, June 1984.

Table 1—Chromatographic Conditions and Methods Detection Limits

Parameters Retention time (min) Method detection limit (µ/L)
Column 1 Column 2
Bis(2-chloroisopropyl) ether 8.4 9.7 0.8
Bis(2-chloroethyl) ether 9.3 9.1 0.3
Bis(2-chloroethoxy) methane 13.1 10.0 0.5
4-Chlorophenyl ether 19.4 15.0 3.9
4-Bromophenyl phenyl ether 21.2 16.2 2.3

Table 2—QC Acceptance Criteria—Method 611

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps percent
Bis (2-chloroethyl)ether 100 26.3 26.3–136.8 11–152
Bis (2-chloroethoxy)methane 100 25.7 27.3–115.0 12–128
Bis (2-chloroisopropyl)ether 100 32.7 26.4–147.0 9–165
4-Bromophenyl phenyl ether 100 39.3 7.6–167.5 D–189
4-Chlorophenyl phenyl ether 100 30.7 15.4–152.5 D–170

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 611

Parameter Accuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
Bis(2-chloroethyl) ether 0.81C + 0.54 0.19X̄ + 0.28 0.35X̄ + 0,36
Bis(2-chloroethoxy) methane 0.71C + 0.13 0.20X̄ + 0.15 0.33X̄ + 0.11
Bis(2-chloroisopropyl) ether 0.85C + 1.67 0.20X̄ + 1.05 0.36X̄ + 0.79
4–Bromophenyl phenyl ether 0.85C + 2.55 0.25X̄ + 0.21 0.47X̄ + 0.37
4–Chlorophenyl phenyl ether 0.82C + 1.97 0.18X̄ + 2.13 0.41X̄ + 0.55

Method 612—Chlorinated Hydrocarbons

1. Scope and Application

1.1 This method covers the determination of certain chlorinated hydrocarbons. The following parameters can be determined by this method:

Parameter STORET No. CAS No.
2-Chloronaphthalene 34581 91–58–7
1,2-Dichlorobenzene 34536 95–50–1
1,3-Dichlorobenzene 34566 541–73–1
1,4-Dichlorobenzene 34571 106–46–7
Hexachlorobenzene 39700 118–74–1
Hexachlorobutadiene 34391 87–68–3
Hexachlorocyclopentadiene 34386 77–47–4
Hexachloroethane 34396 67–72–1
1,2,4-Trichlorobenzene 34551 120–82–1

1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, and 611. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with an electron capture detector.2

2.2 The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4-6 for the information of the analyst.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1cL or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnel—2–L, with Teflon stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Chromatographic column—300 long × 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.

5.2.4 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5 Evaporative flask, Kuderna-Danish—500-mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.6 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.7 Vials—10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6 Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1 Column 1—1.8 m long × 2 mm ID glass, packed with 1% SP–1000 on Supelcoport (100/120 mesh) or equivalent. Guidelines for the use of alternate column packings are provide in Section 12.1.

5.6.2 Column 2—1.8 m long × 2 mm ID glass, packed with 1.5% OV–1/2.4% OV–225 on Supelcoport (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14.

5.6.3 Detector—Electron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2 Acetone, hexane, isooctane, methanol, methylene chloride, petroleum ether (boiling range 30 to 60 °C)—Pesticide quality or equivalent.

6.3 Sodium sulfate—(ACS) Granular, anhydrous. Purify heating at 400 °C for 4 h in a shallow tray.

6.4 Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.5 Stock standard solution (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.5.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 120-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.5.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.5.3 Stock standard solutions must be replaced after six months, or sooner if comparision with check standards indicates a problem.

6.6 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2 External standard calibration procedure:

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3 Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

Equation 1

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When the results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetone: Hexachloro-substituted parameters, 10 µg/mL; any other chlorinated hydrocarbon, 100 µg/mL. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for each parameter using the four results.

8.2.5 For each parameter compare s and X̄ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 2 presents a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6 When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1 Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2 Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spike sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none by

(2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. In necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X̄;

(3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44 (100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4. If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P + 2sp. If P = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevent performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C from the time of collection until extraction.

9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel.

10.2 Add 60 mL of methylele chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 to 2 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

Note:

The dichloribenzenes have a sufficiently high volatility that significant losses may occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.

10.7 Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the tempeature of the water bath to 85 to 90 °C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8 Romove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2 Florisil column cleanup for chlorinated hydrocarbons:

11.2.1 Adjust the sample extract to 10 mL with hexane.

11.2.2 Place 12 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.3 Preelute the column with 100 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate. Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 200 mL of petroleum ether and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction should contain all of the chlorinated hydrocarbons.

11.2.4 Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columl 2 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2 Calibrate the system daily as described in Section 7.

12.3 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed throughly immediately before injection into the gas chromatograph.

12.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush techlique.9 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, the total extract volume, and the resulting peak size in area or peak height units.

12.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1 Determine the concentration of individual compounds in the sample.

13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

Equation 2

where:

A = Amount of material injected (ng).

Vi = Volume of extract injected (µL).

Vt = Volume of total extract (µL).

Vs = Volume of water extracted (mL).

13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 × MDL to 1000 × MDL.10

14.3 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 356 µg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

2. “Determination of Chlorinated Hydrocarbons In Industrial and Municipal Wastewaters, “EPA 6090/4–84–ABC, National Technical Information Service, PBXYZ, Springfield, Virginia, 22161 November 1984.

3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,”American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

10. “Development of Detection Limits, EPA Method 612, Chlorinated Hydrocarbons,” Special letter report for EPA Contract 68–03–2625, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.

11. “EPA Method Study Method 612—Chlorinated Hydrocarbons,” EPA 600/4–84–039, National Technical Information Service, PB84–187772, Springfield, Virginia 22161, May 1984.

12. “Method Performance for Hexachlorocyclopentadiene by Method 612,” Memorandum from R. Slater, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, December 7, 1983.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter Retention time (min) Method detection limit (µg/L)
Column 1 Column 2
1,3-Dichlorobenzene 4.5 6.8 1.19
Hexachloroethane 4.9 8.3 0.03
1,4-Dichlorobenzene 5.2 7.6 1.34
1,2-Dichlorobenzene 6.6 9.3 1.14
Hexachlorobutadiene 7.7 20.0 0.34
1,2,4-Trichlorobenzene 15.5 22.3 0.05
Hexachlorocyclopentadiene nd c 16.5 0.40
2-Chloronaphthalene a 2.7 b 3.6 0.94
Hexachlorobenzene a 5.6 b 10.1 0.05

Table 2—QC Acceptance Criteria—Method 612

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X̄ (µg/L) Range for P, Ps (percent)
2-Chloronaphthalene 100 37.3 29.5–126.9 9–148
1,2-Dichlorobenzene 100 28.3 23.5–145.1 9–160
1,3-Dichlorobenzene 100 26.4 7.2–138.6 D–150
1,4-Dichlorobenzene 100 20.8 22.7–126.9 13–137
Hexachlorobenzene 10 2.4 2.6–14.8 15–159
Hexachlorobutadiene 10 2.2 D–12.7 D–139
Hexachlorocyclopentadiene 10 2.5 D–10.4 D–111
Hexachloroethane 10 3.3 2.4–12.3 8–139
1,2,4-Trichlorobenzene 100 31.6 20.2–133.7 5–149

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 612

Parameter Acccuracy, as recovery, X′ (µg/L) Single analyst precision, sr′ (µg/L) Overall precision, S′ (µg/L)
2-Chloronaphthalene 0.75C + 3.21 0.28X̄−1.17 0.38X̄−1.39
1,2-Dichlorobenzene 0.85C−0.70 0.22X̄−2.95 0.41X̄−3.92
1,3-Dichlorobenzene 0.72C + 0.87 0.21X̄−1.03 0.49X̄−3.98
1,4-Dichlorobenzene 0.72C + 2.80 0.16X̄−0.48 0.35X̄−0.57
Hexachlorobenzene 0.87C−0.02 0.14X̄ + 0.07 0.36X̄−0.19
Hexachlorobutadiene 0.61C + 0.03 0.18X̄ + 0.08 0.53X̄−0.12
Hexachlorocyclopentadienea 0.47C 0.24X̄ 0.50X̄
Hexachloroethane 0.74C−0.02 0.23X̄ + 0.07 0.36X̄−0.00
1,2,4-Trichlorobenzene 0.76C + 0.98 0.23X̄−0.44 0.40X̄−1.37

Method 613—2,3,7,8-Tetrachlorodibenzo-p-Dioxin

1. Scope and Application

1.1 This method covers the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The following parameter may be determined by this method:

Parameter STORET No. GAS No.
2,3,7,8-TCDD 34675 1746–01–6

1.2 This is a gas chromatographic/mass spectrometer (GC/MS) method applicable to the determination of 2,3,7,8–TCDD in municipal and industrial discharges as provided under 40 CFR 136.1. Method 625 may be used to screen samples for 2,3,7,8–TCDD. When the screening test is positive, the final qualitative confirmation and quantification must be made using Method 613.

1.3 The method detection limit (MDL, defined in Section 14.1)1 for 2,3,7,8–TCDD is listed in Table 1. The MDL for a specific wastewater may be different from that listed, depending upon the nature of interferences in the sample matrix.

1.4 Because of the extreme toxicity of this compound, the analyst must prevent exposure to himself, of to others, by materials knows or believed to contain 2,3,7,8–TCDD. Section 4 of this method contains guidelines and protocols that serve as minimum safe-handling standards in a limited-access laboratory.

1.5 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1 A measured volume of sample, approximately 1–L, is spiked with an internal standard of labeled 2,3,7,8–TCDD and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is exchanged to hexane during concentration to a volume of 1.0 mL or less. The extract is then analyzed by capillary column GC/MS to separate and measure 2,3,7,8–TCDD.2 3

2.2 The method provides selected column chromatographic cleanup proceudres to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated backgrounds at the masses (m/z) monitored. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by the treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to mininmize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. 2,3,7,8–TCDD is often associated with other interfering chlorinated compounds which are at concentrations several magnitudes higher than that of 2,3,7,8–TCDD. The cleanup producers in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches1 5-7 to eliminate false positives and achieve the MDL listed in Table 1.

3.3 The primary column, SP–2330 or equivalent, resolves 2,3,7,8–TCDD from the other 21 TCDD insomers. Positive results using any other gas chromatographic column must be confirmed using the primary column.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified8-10 for the information of the analyst. Benzene and 2,3,7,8–TCDD have been identified as suspected human or mammalian carcinogens.

4.2 Each laboratory must develop a strict safety program for handling 2,3,7,8–TCDD. The following laboratory practices are recommended:

4.2.1 Contamination of the laboratory will be minimized by conducting all manipulations in a hood.

4.2.2 The effluents of sample splitters for the gas chromatograph and roughing pumps on the GC/MS should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols.

4.2.3 Liquid waste should be dissolved in methanol or ethanol and irradiated with ultraviolet light with a wavelength greater than 290 nm for several days. (Use F 40 BL lamps or equivalent). Analyze liquid wastes and dispose of the solutions when 2,3,7,8–TCDD can no longer be detected.

4.3 Dow Chemical U.S.A. has issued the following precautimns (revised November 1978) for safe handling of 2,3,7,8–TCDD in the laboratory:

4.3.1 The following statements on safe handling are as complete as possible on the basis of available toxicological information. The precautions for safe handling and use are necessarily general in nature since detailed, specific recommendations can be made only for the particular exposure and circumstances of each individual use. Inquiries about specific operations or uses may be addressed to the Dow Chemical Company. Assistance in evaluating the health hazards of particular plant conditions may be obtained from certain consulting laboratories and from State Departments of Health or of Labor, many of which have an industrial health service. 2,3,7,8–TCDD is extremely toxic to laboratory animals. However, it has been handled for years without injury in analytical and biological laboratories. Techniques used in handling radioactive and infectious materials are applicable to 2,3,7,8,–TCDD.

4.3.1.1 Protective equipment—Throw-away plastic gloves, apron or lab coat, safety glasses, and a lab hood adequate for radioactive work.

4.3.1.2 Training—Workers must be trained in the proper method of removing contaminated gloves and clothing without contacting the exterior surfaces.

4.3.1.3 Personal hygiene—Thorough washing of hands and forearms after each manipulation and before breaks (coffee, lunch, and shift).

4.3.1.4 Confinement—Isolated work area, posted with signs, segregated glassware and tools, plastic-backed absorbent paper on benchtops.

4.3.1.5 Waste—Good technique includes minimizing contaminated waste. Plastic bag liners should be used in waste cans. Janitors must be trained in the safe handling of waste.

4.3.1.6 Disposal of wastes—2,3,7,8–TCDD decomposes above 800 °C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in a good incinerator. Gross quantities (milligrams) should be packaged securely and disposed through commercial or governmental channels which are capable of handling high-level radioactive wastes or extremely toxic wastes. Liquids should be allowed to evaporate in a good hood and in a disposable container. Residues may then be handled as above.

4.3.1.7 Decontamination—For personal decontamination, use any mild soap with plenty of scrubbing action. For decontamination of glassware, tools, and surfaces, Chlorothene NU Solvent (Trademark of the Dow Chemical Company) is the least toxic solvent shown to be effective. Satisfactory cleaning may be accomplished by rinsing with Chlorothene, then washing with any detergent and water. Dishwater may be disposed to the sewer. It is prudent to minimize solvent wastes because they may require special disposal through commercial sources which are expensive.

4.3.1.8 Laundry—Clothing known to be contaminated should be disposed with the precautions described under Section 4.3.1.6. Lab coats or other clothing worn in 2,3,7,8–TCDD work areas may be laundered.

Clothing should be collected in plastic bags. Persons who convey the bags and launder the clothing should be advised of the hazard and trained in proper handling. The clothing may be put into a washer without contact if the launderer knows the problem. The washer should be run through a cycle before being used again for other clothing.

4.3.1.9 Wipe tests—A useful method of determining cleanliness of work surfaces and tools is to wipe the surface with a piece of filter paper. Extraction and analysis by gas chromatography can achieve a limit of sensitivity of 0.1 µg per wipe. Less than 1 µg of 2,3,7,8–TCDD per sample indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 µg on a wipe sample constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space. A high (>10 µg) 2,3,7,8–TCDD level indicates that unacceptable work practices have been employed in the past.

4.3.1.10 Inhalation—Any procedure that may produce airborne contamination must be done with good ventilation. Gross losses to a ventilation system must not be allowed. Handling of the dilute solutions normally used in analytical and animal work presents no inhalation hazards except in the case of an accident.

4.3.1.11 Accidents—Remove contaminated clothing immediately, taking precautions not to contaminate skin or other articles. Wash exposed skin vigorously and repeatedly until medical attention is obtained.

5. Apparatus and Materials

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.1.3 Clearly label all samples as “POISON” and ship according to U.S. Department of Transportation regulations.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1 Separatory funnels—2–L and 125-mL, with Teflon stopcock.

5.2.2 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.3 Evaporative flask, Kuderna-Danish—500–mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.

5.2.4 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).

5.2.5 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).

5.2.6 Vials—10 to 15–mL, amber glass, with Teflon-lined screw cap.

5.2.7 Chromatographic column—300 mm long × 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.

5.2.8 Chromatographic column—400 mm long × 11 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 GC/MS system:

5.5.1 Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph and all required accessories including syringes, analytical columns, and gases. The injection port must be designed for capillary columns. Either split, splitless, or on-column injection techniques may be employed, as long as the requirements of Section 7.1.1 are achieved.

5.5.2 Column—60 m long × 0.25 mm ID glass or fused silica, coated with SP–2330 (or equivalent) with a film thickness of 0.2 µm. Any equivalent column must resolve 2, 3, 7, 8–TCDD from the other 21 TCDD isomers.16

5.5.3 Mass spectrometer—Either a low resolution mass spectrometer (LRMS) or a high resolution mass spectrometer (HRMS) may be used. The mass spectrometer must be equipped with a 70 V (nominal) ion source and be capable of aquiring m/z abundance data in real time selected ion monitoring (SIM) for groups of four or more masses.

5.5.4 GC/MS interface—Any GC to MS interface can be used that achieves the requirements of Section 7.1.1. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass surfaces can be deactivated by silanizing with dichlorodimethylsilane. To achieve maximum sensitivity, the exit end of the capillary column should be placed in the ion source. A short piece of fused silica capillary can be used as the interface to overcome problems associated with straightening the exit end of glass capillary columns.

5.5.5 The SIM data acquired during the chromatographic program is defined as the Selected Ion Current Profile (SICP). The SICP can be acquired under computer control or as a real time analog output. If computer control is used, there must be software available to plot the SICP and report peak height or area data for any m/z in the SICP between specified time or scan number limits.

5.6 Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of 2, 3, 7, 8–TCDD.

6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL. Wash the solution with methylene chloride and hexane before use.

6.3 Sodium thiosulfate—(ACS) Granular.

6.4 Sulfuric acid—Concentrated (ACS, sp. gr. 1.84).

6.5 Acetone, methylene chloride, hexane, benzene, ortho-xylene, tetradecane—Pesticide quality or equivalent.

6.6 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.7 Alumina—Neutral, 80/200 mesh (Fisher Scientific Co., No. A–540 or equivalent). Before use, activate for 24 h at 130 °C in a foil-covered glass container.

6.8 Silica gel—High purity grade, 100/120 mesh (Fisher Scientific Co., No. S–679 or equivalent).

6.9 Stock standard solutions (1.00 µg/µL)—Stock standard solutimns can be prepared from pure standard materials or purchased as certified solutions. Acetone should be used as the solvent for spiking solutions; ortho-xylene is recommended for calibration standards for split injectors; and tetradecane is recommended for splitless or on-colum injectors. Analyze stock internal standards to verify the absence of native 2,3,7,8–TCDD.

6.9.1 Prepare stock standard solutions of 2,3,7,8–TCDD (mol wt 320) and either37C14 2,3,7,8–TCDD (mol wt 328) or13C112 2,3,7,8–TCDD (mol wt 332) in an isolated area by accurately weighing about 0.0100 g of pure material. Dissolve the material in pesticide quality solvent and dilute to volume in a 10-mL volumetric flask. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.9.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store in an isolated refrigerator protected from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards or spiking solutions from them.

6.9.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.10 Internal standard spiking solution (25 ng/mL)—Using stock standard solution, prepare a spiking solution in acetone of either13 Cl12 or37 Cl4 2,3,7,8–TCDD at a concentration of 25 ng/mL. (See Section 10.2)

6.11 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Establish gas chromatograhic operating conditions equivalent to those given in Table 1 and SIM conditions for the mass spectrometer as described in Section 12.2 The GC/MS system must be calibrated using the internal standard technique.

7.1.1 Using stock standards, prepare calibration standards that will allow measurement of relative response factors of at least three concentration ratios of 2,3,7,8–TCDD to internal standard. Each calibration standard must be prepared to contain the internal standard at a concentration of 25 ng/mL. If any interferences are contributed by the internal standard at m/z 320 and 322, its concentration may be reduced in the calibration standards and in the internal standard spiking solution (Section 6.10). One of the calibration standards should contain 2,3,7,8–TCDD at a concentration near, but above, the MDL and the other 2,3,7,8–TCDD concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system.

7.1.2 Using injections of 2 to 5 µL, analyze each calibration standardaccording to Section 12 and tabulate peak height or area response against the concentration of 2,3,7,8–TCDD and internal standard. Calculate response factors (RF) for 2,3,7,8–TCDD using Equation 1.

Equation 1

where:

As = SIM response for 2,3,7,8–TCDD m/z 320.

Ais = SIM response for the internal standard, m/z 332 for13 C12 2,3,7,8–TCDD m/z 328 for37 Cl4 2,3,7,8–TCDD.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of 2,3,7,8–TCDD (µg/L).

If the RF value over the working range is a constant (<10% relative standard deviation, RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.1.3 The working calibration curve or RF must be verified on each working day by the measurement of one or more 2,3,7,8–TCDD calibration standards. If the response for 2,3,7,8–TCDD varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared.

7.2 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.5, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2

8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples with native 2,3,7,8–TCDD to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1 A quality control (QC) check sample concentrate is required containing 2,3,7,8–TCDD at a concentration of 0.100 µg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at a concentration of 0.100 µg/L (100 ng/L) by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X̄) in µg/L, and the standard deviation of the recovery (s) in µg/L, for 2,3,7,8–TCDD using the four results.

8.2.5 Compare s and (X̄) with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̄ meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If s exceeds the precision limit or X̄ falls outside the range for accuracy, the system performance is unacceptable for 2,3,7,8–TCDD. Locate and correct the source of the problem and repeat the test beginning with Section 8.2.2.

8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1 The concentration of the spike in the sample should be determined as follows:

8.3.1.1 If, as in compliance monitoring, the concentration of 2,3,7,8–TCDD in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2 If the concentration of 2,3,7,8–TCDD in the sample is not being checked against a limit specific to that parameter, the spike should be at 0.100 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be

(1) the regulatory concentration limit, if any; or, if none

(2) the larger of either 5 times higher than the expected background concentration or 0.100 µg/L.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of 2,3,7,8–TCDD. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentration in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of 2,3,7,8–TCDD. Calculate percent recovery (P) as 100(A−B)%T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for 2,3,7,8–TCDD with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.11 If spiking was performed at a concentration lower than 0.100 µg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of 2,3,7,8–TCDD:

(1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C;

(2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X;

(3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.11

8.3.4 If the recovery of 2,3,7,8–TCDD falls outside the designated range for recovery, a check standard must be analyzed as described in Section 8.4.

8.4 If the recovery of 2,3,7,8–TCDD fails the acceptance criteria for recovery in Section 8.3, a QC check standard must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the complexity of the sample matrix and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water.

8.4.2 Analyze the QC check standard to determine the concentration measured (A) of 2,3,7,8–TCDD. Calculate the percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (Ps) with the corresponding QC acceptance criteria found in Table 2. If the recovery of 2,3,7,8–TCDD falls outside the designated range, the laboratory performance is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for 2,3,7,8–TCDD in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P̄) and the spandard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̄−2sp to P̄ + 2sp. If P̄ = 90% and sp = 10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples must be collected in glass containers. Conventional sampling practices12 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2 All samples must be iced or refrigerated at 4 °C and protected from light from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.13 Field test kits are available for this purpose.

9.3 Label all samples and containers “POISON” and ship according to applicable U.S. Department of Transportation regulations.

9.4 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

Caution: When using this method to analyze for 2,3,7,8–TCDD, all of the following operations must be performed in a limited-access laboratory with the analyst wearing full protective covering for all exposed skin surfaces. See Section 4.2.

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel.

10.2 Add 1.00 mL of internal standard spiking solution to the sample in the separatory funnel. If the final extract will be concentrated to a fixed volume below 1.00 mL (Section 12.3), only that volume of spiking solution should be added to the sample so that the final extract will contain 25 ng/mL of internal standard at the time of analysis.

10.3 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the vmlume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.4 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.5 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.6 Pour the combined extract into the K-D concentrator. Rinse the Erlenmeyer flask with 20 to 30 mL of methylele chloride to complete the quantitative transfer.

10.7 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8 Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the temperature of the water bath to 85 to 90 °C. Concentrate the extract as in Section 10.7, except use hexane to prewet the column. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Set aside the K-D glassware for reuse in Section 10.14.

10.9 Pour the hexane extract from the concentrator tube into a 125-mL separatory funnel. Rinse the concentrator tube four times with 10-mL aliquots of hexane. Combine all rinses in the 125-mL separatory funnel.

10.10 Add 50 mL of sodium hydroxide solution to the funnel and shake for 30 to 60 s. Discard the aqueous phase.

10.11 Perform a second wash of the organic layer with 50 mL of reagent water. Discard the aqueous phase.

10.12 Wash the hexane layer with a least two 50-mL aliquots of concentrated sulfuric acid. Continue washing the hexane layer with 50-mL aliquots of concentrated sulfuric acid until the acid layer remains colorless. Discard all acid fractions.

10.13 Wash the hexane layer with two 50-mL aliquots of reagent water. Discard the aqueous phases.

10.14 Transfer the hexane extract into a 125-mL Erlenmeyer flask containing 1 to 2 g of anhydrous sodium sulfate. Swirl the flask for 30 s and decant the hexane extract into the reassembled K-D apparatus. Complete the quantitative transfer with two 10-mL hexane rinses of the Erlenmeyer flask.

10.15 Replace the one or two clean boiling chips and concentrate the extract to 6 to 10 mL as in Section 10.8.

10.16 Add a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 1 mL of hexane to the top. Place the micro-K-D apparatus on the water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of hexane.

Adjust the extract volume to 1.0 mL with hexane. Stopper the concentrator tube and store refrigerated and protected from light if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with GC/MS analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.17 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure.1 5-7 However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. Two cleanup column options are offered to the analyst in this section. The alumina column should be used first to overcome interferences. If background problems are still encountered, the silica gel column may be helpful.

11.2 Alumina column cleanup for 2,3,7,8–TCDD:

11.2.1 Fill a 300 mm long × 10 mm ID chromatographic column with activated alumina to the 150 mm level. Tap the column gently to settle the alumina and add 10 mm of anhydrous sodium sulfate to the top.

11.2.2 Preelute the column with 50 mL of hexane. Adjust the elution rate to 1 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 1.0-mL sample extract onto the column using two 2-mL portions of hexane to complete the transfer.

11.2.3 Just prior to exposure of the sodium sulfate layer to the air, add 50 mL of 3% methylene chloride/95% hexane (V/V) and continue the elution of the column. Discard the eluate.

11.2.4 Next, elute the column with 50 mL of 20% methylene chloride/80% hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to 1.0 mL as in Section 10.16 and analyze by GC/MS (Section 12).

11.3 Silica gel column cleanup for 2,3,7,8–TCDD:

11.3.1 Fill a 400 mm long × 11 mm ID chromatmgraphic column with silica gel to the 300 mm level. Tap the column gently to settle the silica gel and add 10 mm of anhydrous sodium sulfate to the top.

11.3.2 Preelute the column with 50 mL of 20% benzene/80% hexane (V/V). Adjust the elution rate to 1 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 1.0-mL sample extract onto the column using two 2-mL portions of 20% benzene/80% hexane to complete the transfer.

11.3.3 Just prior to exposure of the sodium sulfate layer to the air, add 40 mL of 20% benzene/80% hexane to the column. Collect the eluate in a clean 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to 1.0 mL as in Section 10.16 and analyze by GC/MS.

12. GC/MS Analysis

12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Other capillary columns or chromatographic conditions may be used if the requirements of Sections 5.5.2 and 8.2 are met.

12.2 Analyze standards and samples with the mass spectrometer operating in the selected ion monitoring (SIM) mode using a dwell time to give at least seven points per peak. For LRMS, use masses at m/z 320, 322, and 257 for 2,3,7,8–TCDD and either m/z 328 for37Cl4 2,3,7,8–TCDD or m/z 332 for13C12 2,3,7,8–TCDD. For HRMS, use masses at m/z 319.8965 and 321.8936 for 2,3,7,8–TCDD and either m/z 327.8847 for37Cl4 2,3,7,8–TCDD or m/z 331.9367 for13C12 2,3,7,8–TCDD.

12.3 If lower detection limits are required, the extract may be carefully evaporated to dryness under a gentle stream of nitrogen with the concentrator tube in a water bath at about 40 °C. Conduct this operation immediately before GC/MS analysis. Redissolve the extract in the desired final volume of ortho-xylene or tetradecane.

12.4 Calibrate the system daily as described in Section 7.

12.5 Inject 2 to 5 µL of the sample extract into the gas chromatograph. The volume of calibration standard injected must be measured, or be the same as all sample injection volumes.

12.6 The presence of 2,3,7,8–TCDD is qualitatively confirmed if all of the following criteria are achieved:

12.6.1 The gas chromatographic column must resolve 2,3,7,8–TCDD from the other 21 TCDD isomers.

12.6.2 The masses for native 2,3,7,8–TCDD (LRMS-m/z 320, 322, and 257 and HRMS-m/z 320 and 322) and labeled 2,3,7,8–TCDD (m/z 328 or 332) must exhibit a simultaneous maximum at a retention time that matches that of native 2,3,7,8–TCDD in the calibration standard, with the performance specifications of the analytical system.

12.6.3 The chlorine isotope ratio at m/z 320 and m/z 322 must agree to within±10% of that in the calibration standard.

12.6.4 The signal of all peaks must be greater than 2.5 times the noise level.

12.7 For quantitation, measure the response of the m/z 320 peak for 2,3,7,8–TCDD and the m/z 332 peak for13C12 2,3,7,8–TCDD or the m/z 328 peak for37Cl4 2,3,7,8–TCDD.

12.8 Co-eluting impurities are suspected if all criteria are achieved except those in Section 12.6.3. In this case, another SIM analysis using masses at m/z 257, 259, 320 and either m/a 328 or m/z 322 can be performed. The masses at m/z 257 and m/z 259 are indicative of the loss of one chlorine and one carbonyl group from 2,3,7,8–TCDD. If masses m/z 257 and m/z 259 give a chlorine isotope ratio that agrees to within ±10% of the same cluster in the calibration standards, then the presence of TCDD can be confirmed. Co-eluting DDD, DDE, and PCB residues can be confirmed, but will require another injection using the appropriate SIM masses or full repetitive mass scans. If the response for37Cl4 2,3,7,8–TCDD at m/z 328 is too large, PCB contamination is suspected and can be confirmed by examining the response at both m/z 326 and m/z 328. The37Cl4 2,3,7,8–TCDD internal standard gives negligible response at m/z 326. These pesticide residues can be removed using the alumina column cleanup procedure.

12.9 If broad background interference restricts the sensitivity of the GC/MS analysis, the analyst should employ additional cleanup procedures and reanalyze by GC/MS.

12.10 In those circumstances where these procedures do not yield a definitive conclusion, the use of high resolution mass spectrometry is suggested.5

13. Calculations

13.1 Calculate the concentration of 2,3,7,8–TCDD in the sample using the response factor (RF) determined in Section 7.1.2 and Equation 2.

Equation 2

where:

As = SIM response for 2,3,7,8–TCDD at m/z 320.

Ais = SIM response for the internal standard at m/z 328 or 332.

Is = Amount of internal standard added to each extract (µg).

Vo = Volume of water extracted (L).

13.2 For each sample, calculate the percent recovery of the internal standard by comparing the area of the m/z peak measured in the sample to the area of the same peak in the calibration standard. If the recovery is below 50%, the analyst should review all aspects of his analytical technique.

13.3 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentration listed in Table 1 was obtained using reagent water.14 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method was tested by 11 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.02 to 0.20 µg/L.15 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

2. “Determination of TCDD in Industrial and Municipal Wastewaters,” EPA 600/4–82–028, National Technical Information Service, PB82–196882, Springfield, Virginia 22161, April 1982.

3. Buser, H.R., and Rappe, C. “High Resolution Gas Chromatography of the 22 Tetrachlorodibenzo-p-dioxin Isomers,” Analytical Chemistry, 52, 2257 (1980).

4. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

5. Harless, R. L., Oswald, E. O., and Wilkinson, M. K. “Sample Preparation and Gas Chromatography/Mass Spectrometry Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin,” Analytical Chemistry, 52, 1239 (1980).

6. Lamparski, L. L., and Nestrick, T. J. “Determination of Tetra-, Hepta-, and Octachlorodibenzo-p-dioxin Isomers in Particulate Samples at Parts per Trillion Levels,” Analytical Chemistry, 52, 2045 (1980).

7. Longhorst, M. L., and Shadoff, L. A. “Determination of Parts-per-Trillion Concentrations of Tetra-, Hexa-, and Octachlorodibenzo-p-dioxins in Human Milk,” Analytical Chemistry, 52, 2037 (1980).

8. “Carcinogens—Working with Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

9. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occuptional Safety and Health Administration, OSHA 2206 (Revised, January 1976).

10. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

11. Provost, L. P., and Elder, R. S., “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

12. ASTM Annual Book of Standards, Part 31, D3370–76, “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

13. “Methods, 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA–600/4–79–020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

14. Wong, A.S. et al. “The Determination of 2,3,7,8–TCDD in Industrial and Municipal Wastewaters, Method 613, Part 1—Development and Detection Limits,” G. Choudhay, L. Keith, and C. Ruppe, ed., Butterworth Inc., (1983).

15. “EPA Method Study 26, Method 613: 2,3,7,8–Tetrachlorodibenzo-p-dioxin,” EPA 600/4–84–037, National Technical Information Service, PB84–188879, Springfield, Virginia 22161, May 1984.

Table 1—Chromatographic Conditions and Method Detection Limit

Parameter Retention time (min) Method detection limit (µg/L)
2,3,7,8–TCDD 13.1 0.002

Table 2—QC Acceptance Criteria—Method 613

Parameter Test conc. (µg/L) Limit for s (µg/L) Range for X (µg/L) Range for P, Ps (%)
2,3,7,8–TCDD 0.100 0.0276 0.0523–0.1226 45–129

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 613

Parameter Accuracy, as recovery, X″ (µg/L) Single analyst, precision, sr″ (µ/L) Overall precision, S″ (µ/g/L)
2,3,7,8-TCDD 0.86C + 0.00145 0.13X̄ + 0.00129 0.19X̄ + 0.00028

Method 624.1—Purgeables by GC/MS

1. Scope and Application

1.1 This method is for determination of purgeable organic pollutants in industrial discharges and other environmental samples by gas chromatography combined with mass spectrometry (GC/MS), as provided under 40 CFR 136.1. This revision is based on previous protocols (References 1—3), on the revision promulgated October 26, 1984, and on an interlaboratory method validation study (Reference 4). Although this method was validated through an interlaboratory study conducted in the early 1980s, the fundamental chemistry principles used in this method remain sound and continue to apply.

1.2 The analytes that may be qualitatively and quantitatively determined using this method and their CAS Registry numbers are listed in Table 1. The method may be extended to determine the analytes listed in Table 2; however, poor purging efficiency or gas chromatography of some of these analytes may make quantitative determination difficult. For example, an elevated temperature may be required to purge some analytes from water. If an elevated temperature is used, calibration and all quality control (QC) tests must be performed at the elevated temperature. EPA encourages the use of this method to determine additional compounds amenable to purge-and-trap GC/MS.

1.3 The large number of analytes in Tables 1 and 2 of this method makes testing difficult if all analytes are determined simultaneously. Therefore, it is necessary to determine and perform QC tests for “analytes of interest” only. Analytes of interest are those required to be determined by a regulatory/control authority or in a permit, or by a client. If a list of analytes is not specified, the analytes in Table 1 must be determined, at a minimum, and QC testing must be performed for these analytes. The analytes in Table 1 and some of the analytes in Table 2 have been identified as Toxic Pollutants (40 CFR 401.15), expanded to a list of Priority Pollutants (40 CFR part 423, appendix A).

1.4 Method detection limits (MDLs; Reference 5) for the analytes in Table 1 are listed in that table. These MDLs were determined in reagent water (Reference 6). Advances in analytical technology, particularly the use of capillary (open-tubular) columns, allowed laboratories to routinely achieve MDLs for the analytes in this method that are 2–10 times lower than those in the version promulgated in 1984. The MDL for a specific wastewater may differ from those listed, depending on the nature of interferences in the sample matrix.

1.4.1 EPA has promulgated this method at 40 CFR part 136 for use in wastewater compliance monitoring under the National Pollutant Discharge Elimination System (NPDES). The data reporting practices described in section 13.2 are focused on such monitoring needs and may not be relevant to other uses of the method.

1.4.2 This method includes “reporting limits” based on EPA's “minimum level” (ML) concept (see the glossary in section 20). Table 1 contains MDL values and ML values for many of the analytes. The MDL for an analyte in a specific wastewater may differ from that listed in Table 1, depending upon the nature of interferences in the sample matrix.

1.5 This method is performance-based. It may be modified to improve performance (e.g., to overcome interferences or improve the accuracy of results) provided all performance requirements are met.

1.5.1 Examples of allowed method modifications are described at 40 CFR 136.6. Other examples of allowed modifications specific to this method are described in section 8.1.2.

1.5.2 Any modification beyond those expressly allowed at 40 CFR 136.6 or in section 8.1.2 of this method shall be considered a major modification that is subject to application and approval of an alternate test procedure under 40 CFR 136.4 and 136.5.

1.5.3 For regulatory compliance, any modification must be demonstrated to produce results equivalent or superior to results produced by this method when applied to relevant wastewaters (section 8.3).

1.6 This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge-and-trap system and a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure in section 8.2.

1.7 Terms and units of measure used in this method are given in the glossary at the end of the method.

2. Summary of Method

2.1 A gas is bubbled through a measured volume of water in a specially-designed purging chamber. The purgeables are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the purgeables are trapped. After purging is completed, the trap is heated and backflushed with the gas to desorb the purgeables onto a gas chromatographic column. The column is temperature programmed to separate the purgeables which are then detected with a mass spectrometer.

2.2 Different sample sizes in the range of 5–25 mL are allowed in order to meet differing sensitivity requirements. Calibration and QC samples must have the same volume as field samples.

3. Interferences

3.1 Impurities in the purge gas, organic compounds outgassing from the plumbing ahead of the trap, and solvent vapors in the laboratory account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by analyzing blanks initially and with each analytical batch (samples analyzed on a given 12-hour shift, to a maximum of 20 samples), as described in Section 8.5. Fluoropolymer tubing, fittings, and thread sealant should be used to avoid contamination.

3.2 Samples can be contaminated by diffusion of volatile organics (particularly fluorocarbons and methylene chloride) through the septum seal into the sample during shipment and storage. Protect samples from sources of volatiles during collection, shipment, and storage. A reagent water field blank carried through sampling and analysis can serve as a check on such contamination.

3.3 Contamination by carry-over can occur whenever high level and low level samples are analyzed sequentially. To reduce the potential for carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of a blank to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high purgeable levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in a 105 °C oven between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required. Screening samples at high dilution may prevent introduction of contaminants into the system.

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of safety data sheets (SDSs, OSHA, 29 CFR 1910.1200(g)) should also be made available to all personnel involved in sample handling and chemical analysis. Additional references to laboratory safety are available and have been identified (References 7–9) for the information of the analyst.

4.2. The following analytes covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: Benzene; carbon tetrachloride; chloroform; 1,4-dichlorobenzene; 1,2-dichloroethane; 1,2-dichloropropane; methylene chloride; tetrachloroethylene; trichloroethylene; and vinyl chloride. Primary standards of these toxic compounds should be prepared in a chemical fume hood, and a NIOSH/MESA approved toxic gas respirator should be worn when handling high concentrations of these compounds.

4.3 This method allows the use of hydrogen as a carrier gas in place of helium (Section 5.3.1.2). The laboratory should take the necessary precautions in dealing with hydrogen, and should limit hydrogen flow at the source to prevent buildup of an explosive mixture of hydrogen in air.

5. Apparatus and Materials

Note:

Brand names, suppliers, and part numbers are cited for illustration purposes only. No endorsement is implied. Equivalent performance may be achieved using equipment and materials other than those specified here. Demonstration of equivalent performance that meets the requirements of this method is the responsibility of the laboratory. Suppliers for equipment and materials in this method may be found through an on-line search.

5.1 Sampling equipment for discrete sampling.

5.1.1 Vial—25- or 40-mL capacity, or larger, with screw cap with a hole in the center (Fisher #13075 or equivalent). Unless pre-cleaned, detergent wash, rinse with tap and reagent water, and dry at 105 ± 5 °C before use.

5.1.2 Septum—Fluoropolymer-faced silicone (Fisher #12722 or equivalent). Unless pre-cleaned, detergent wash, rinse with tap and reagent water, and dry at 105 ± 5 °C for one hour before use.

5.2 Purge-and-trap system—The purge-and-trap system consists of three separate pieces of equipment: A purging device, trap, and desorber. Several complete systems are commercially available with autosamplers. Any system that meets the performance requirements in this method may be used.

5.2.1 The purging device should accept 5- to 25-mL samples with a water column at least 3 cm deep. The purge gas must pass though the water column as finely divided bubbles. The purge gas must be introduced no more than 5 mm from the base of the water column. Purge devices of a different volume may be used so long as the performance requirements in this method are met.

5.2.2 The trap should be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap should be packed to contain the following minimum lengths of adsorbents: 1.0 cm of methyl silicone coated packing (section 6.3.2), 15 cm of 2,6-diphenylene oxide polymer (section 6.3.1), and 8 cm of silica gel (section 6.3.3). A trap with different dimensions and packing materials is acceptable so long as the performance requirements in this method are met.

5.2.3 The desorber should be capable of rapidly heating the trap to the temperature necessary to desorb the analytes of interest, and of maintaining this temperature during desorption. The trap should not be heated higher than the maximum temperature recommended by the manufacturer.

5.2.4 The purge-and-trap system may be assembled as a separate unit or coupled to a gas chromatograph.

5.3 GC/MS system.

5.3.1 Gas chromatograph (GC)—An analytical system complete with a temperature programmable gas chromatograph and all required accessories, including syringes and analytical columns. Autosamplers designed for purge-and-trap analysis of volatiles also may be used.

5.3.1.1 Injection port—Volatiles interface, split, splitless, temperature programmable split/splitless (PTV), large volume, on-column, backflushed, or other.

5.3.1.2 Carrier gas—Data in the tables in this method were obtained using helium carrier gas. If another carrier gas is used, analytical conditions may need to be adjusted for optimum performance, and calibration and all QC tests must be performed with the alternative carrier gas. See Section 4.3 for precautions regarding the use of hydrogen as a carrier gas.

5.3.2 GC column—See the footnote to Table 3. Other columns or column systems may be used provided all requirements in this method are met.

5.3.3 Mass spectrometer—Capable of repetitively scanning from 35–260 Daltons (amu) every 2 seconds or less, utilizing a 70 eV (nominal) electron energy in the electron impact ionization mode, and producing a mass spectrum which meets all criteria in Table 4 when 50 ng or less of 4-bromofluorobenzene (BFB) is injected through the GC inlet. If acrolein, acrylonitrile, chloromethane, and vinyl chloride are to be determined, it may be necessary to scan from below 25 Daltons to measure the peaks in the 26–35 Dalton range for reliable identification.

5.3.4 GC/MS interface—Any GC to MS interface that meets all performance requirements in this method may be used.

5.3.5 Data system—A computer system must be interfaced to the mass spectrometer that allows continuous acquisition and storage of mass spectra throughout the chromatographic program. The computer must have software that allows searching any GC/MS data file for specific m/z's (masses) and plotting m/z abundances versus time or scan number. This type of plot is defined as an extracted ion current profile (EICP). Software must also be available that allows integrating the abundance at any EICP between specified time or scan number limits.

5.4 Syringes—Graduated, 5–25 mL, glass hypodermic with Luerlok tip, compatible with the purging device.

5.5 Micro syringes—Graduated, 25–1000 µL, with 0.006 in. ID needle.

5.6 Syringe valve—Two-way, with Luer ends.

5.7 Syringe—5 mL, gas-tight with shut-off valve.

5.8 Bottle—15 mL, screw-cap, with Teflon cap liner.

5.9 Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1 Reagent water—Reagent water is defined as water in which the analytes of interest and interfering compounds are not detected at the MDLs of the analytes of interest. It may be generated by passing deionized water, distilled water, or tap water through a carbon bed, passing the water through a water purifier, or heating the water to between 90 and 100 °C while bubbling contaminant-free gas through it for approximately 1 hour. While still hot, transfer the water to screw-cap bottles and seal with a fluoropolymer-lined cap.

6.2 Sodium thiosulfate—(ACS) Granular.

6.3 Trap materials.

6.3.1 2,6-Diphenylene oxide polymer—Tenax, 60/80 mesh, chromatographic grade, or equivalent.

6.3.2 Methyl silicone packing—3% OV–1 on Chromosorb-W, 60/80 mesh, or equivalent.

6.3.3 Silica gel—35/60 mesh, Davison, Grade-15 or equivalent.

6.3.4 Other trap materials are acceptable if performance requirements in this method are met.

6.4 Methanol—Demonstrated to be free from the target analytes and potentially interfering compounds.

6.5 Stock standard solutions—Stock standard solutions may be prepared from pure materials, or purchased as certified solutions. Traceability must be to the National Institute of Standards and Technology (NIST) or other national or international standard, when available. Stock solution concentrations alternative to those below may be used. Prepare stock standard solutions in methanol using assayed liquids or gases as appropriate. Because some of the compounds in this method are known to be toxic, primary dilutions should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations of neat materials are handled. The following procedure may be used to prepare standards from neat materials:

6.5.1 Place about 9.8 mL of methanol in a 10-mL ground-glass-stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 minutes or until all alcohol wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg.

6.5.2 Add the assayed reference material.

6.5.2.1 Liquids—Using a 100 µL syringe, immediately add two or more drops of assayed reference material to the flask. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask. Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in µg/µL from the net gain in weight.

6.5.2.2 Gases—To prepare standards for any of compounds that boil below 30 °C, fill a 5-mL valved gas-tight syringe with reference standard vapor to the 5.0 mL mark. Lower the needle to 5 mm above the methanol meniscus. Slowly introduce the vapor above the surface of the liquid (the vapor will rapidly dissolve in the methanol). Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in µg/µL from the net gain in weight.

6.5.3 When compound purity is assayed to be 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.

6.5.4 Prepare fresh standards weekly for the gases and 2-chloroethylvinyl ether. Unless stated otherwise in this method, store non-aqueous standards in fluoropolymer-lined screw-cap, or heat-sealed, glass containers, in the dark at −20 to −10 °C. Store aqueous standards; e.g., the aqueous LCS (section 8.4.1) in the dark at ≤6 °C (but do not freeze) with zero headspace; e.g., in VOA vials (section 5.1.1). Standards prepared by the laboratory may be stored for up to one month, except when comparison with QC check standards indicates that a standard has degraded or become more concentrated due to evaporation, or unless the laboratory has data on file to prove stability for a longer period. Commercially prepared standards may be stored until the expiration date provided by the vendor, except when comparison with QC check standards indicates that a standard has degraded or become more concentrated due to evaporation, or unless the laboratory has data from the vendor on file to prove stability for a longer period.

Note:

2-Chloroethylvinyl ether has been shown to be stable for as long as one month if prepared as a separate standard, and the other analytes have been shown to be stable for as long as 2 months if stored at less than −10 °C with minimal headspace in sealed, miniature inert-valved vials.

6.6 Secondary dilution standards—Using stock solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed. Secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in section 7.3.2 will bracket the working range of the analytical system.

6.7 Surrogate standard spiking solution—Select a minimum of three surrogate compounds from Table 5. The surrogates selected should match the purging characteristics of the analytes of interest as closely as possible. Prepare a stock standard solution for each surrogate in methanol as described in section 6.5, and prepare a solution for spiking the surrogates into all blanks, LCSs, and MS/MSDs. Prepare the spiking solution such that spiking a small volume will result in a constant concentration of the surrogates. For example, add 10 µL of a spiking solution containing the surrogates at a concentration of 15 µg/mL in methanol to a 5-mL aliquot of water to produce a concentration of 30 µg/L for each surrogate. Other surrogate concentrations may be used. Store per section 6.5.4.

6.8 BFB standard—Prepare a solution of BFB in methanol as described in Sections 6.5 and 6.6. The solution should be prepared such that an injection or purging from water will result in introduction of ≤ 50 ng into the GC. BFB may be included in a mixture with the internal standards and/or surrogates.

6.9 Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1 Assemble a purge-and-trap system that meets the specifications in Section 5.2. Prior to first use, condition the trap overnight at 180 °C by backflushing with gas at a flow rate of at least 20 mL/min. Condition the trap after each analysis at a temperature and time sufficient to prevent detectable concentrations of the analytes or contaminants in successive analyses.

7.2 Connect the purge-and-trap system to the gas chromatograph. The gas chromatograph should be operated using temperature and flow rate conditions equivalent to those given in the footnotes to Table 3. Alternative temperature and flow rate conditions may be used provided that performance requirements in this method are met.

7.3 Internal standard calibration.

7.3.1 Internal standards.

7.3.1.1 Select three or more internal standards similar in chromatographic behavior to the compounds of interest. Suggested internal standards are listed in Table 5. Use the base peak m/z as the primary m/z for quantification of the standards. If interferences are found at the base peak, use one of the next two most intense m/z's for quantitation. Demonstrate that measurements of the internal standards are not affected by method or matrix interferences.

7.3.1.2 To assure accurate analyte identification, particularly when selected ion monitoring (SIM) is used, it may be advantageous to include more internal standards than those suggested in Section 7.3.1.1. An analyte will be located most accurately if its retention time relative to an internal standard is in the range of 0.8 to 1.2.

7.3.1.3 Prepare a stock standard solution for each internal standard in methanol as described in Section 6.5, and prepare a solution for spiking the internal standards into all blanks, LCSs, and MS/MSDs. Prepare the spiking solution such that spiking a small volume will result in a constant concentration of the internal standards. For example, add 10 µL of a spiking solution containing the internal standards at a concentration of 15 µg/mL in methanol to a 5-mL aliquot of water to produce a concentration of 30 µg/L for each internal standard. Other concentrations may be used. The internal standard solution and the surrogate standard spiking solution (Section 6.7) may be combined, if desired. Store per section 6.5.4.

7.3.2 Calibration.

7.3.2.1 Calibration standards.

7.3.2.1.1 Prepare calibration standards at a minimum of five concentration levels for each analyte of interest by adding appropriate volumes of one or more stock standards to a fixed volume (e.g., 40 mL) of reagent water in volumetric glassware. Fewer levels may be necessary for some analytes based on the sensitivity of the MS, but no fewer than 3 levels may be used, and only the highest or lowest point(s) may be dropped from the calibration. One of the calibration standards should be at a concentration at or below the ML or as specified by a regulatory/control authority or in a permit. The ML value may be rounded to a whole number that is more convenient for preparing the standard, but must not exceed the ML values listed in Table 1 for those analytes which list ML values. Alternatively, the laboratory may establish the ML for each analyte based on the concentration of the lowest calibration standard in a series of standards produced in the laboratory or obtained from a commercial vendor, again, provided that the ML value does not exceed the MLs in Table 1, and provided that the resulting calibration meets the acceptance criteria in Section 7.3.4, based on the RSD, RSE, or R2. The concentrations of the higher standards should correspond to the expected range of concentrations found in real samples, or should define the working range of the GC/MS system for full-scan and/or SIM operation, as appropriate. A minimum of six concentration levels is required for a second order, non-linear (e.g., quadratic; ax2 + bx + c = 0) calibration. Calibrations higher than second order are not allowed.

7.3.2.1.2 To each calibration standard or standard mixture, add a known constant volume of the internal standard spiking solution (section 7.3.1.3) and surrogate standard spiking solution (section 6.7) or the combined internal standard solution and surrogate spiking solution (section 7.3.1.3). Aqueous standards may be stored up to 24 hours, if held in sealed vials with zero headspace. If not so stored, they must be discarded after one hour.

7.3.2.2 Prior to analysis of the calibration standards, analyze the BFB standard (section 6.8) and adjust the scan rate of the MS to produce a minimum of 5 mass spectra across the BFB GC peak, but do not exceed 2 seconds per scan. Adjust instrument conditions until the BFB criteria in Table 4 are met. Once the scan conditions are established, they must be used for analyses of all standards, blanks, and samples.

Note:

The BFB spectrum may be evaluated by summing the intensities of the m/z's across the GC peak, subtracting the background at each m/z in a region of the chromatogram within 20 scans of but not including any part of the BFB peak. The BFB spectrum may also be evaluated by fitting a Gaussian to each m/z and using the intensity at the maximum for each Gaussian, or by integrating the area at each m/z and using the integrated areas. Other means may be used for evaluation of the BFB spectrum so long as the spectrum is not distorted to meet the criteria in Table 4.

7.3.2.3 Analyze the mid-point standard and enter or review the retention time, relative retention time, mass spectrum, and quantitation m/z in the data system for each analyte of interest, surrogate, and internal standard. If additional analytes (Table 2) are to be quantified, include these analytes in the standard. The mass spectrum for each analyte must be comprised of a minimum of 2 m/z's; 3 to 5 m/z's assure more reliable analyte identification. Suggested quantitation m/z's are shown in Table 6 as the primary m/z. For analytes in Table 6 that do not have a secondary m/z, acquire a mass spectrum and enter one or more secondary m/z's for more reliable identification. If an interference occurs at the primary m/z, use one of the secondary m/z's or an alternative m/z. A single m/z only is required for quantitation.

7.3.2.4 For SIM operation, determine the analytes in each descriptor, the quantitation m/z for each analyte (the quantitation m/z can be the same as for full-scan operation; Section 7.3.2.3), the dwell time on each m/z for each analyte, and the beginning and ending retention time for each descriptor. Analyze the verification standard in scan mode to verify m/z's and establish retention times for the analytes. There must be a minimum of two m/z's for each analyte to assure analyte identification. To maintain sensitivity, the number of m/z's in a descriptor should be limited. For example, for a descriptor with 10 m/z's and a chromatographic peak width of 5 sec, a dwell time of 100 ms at each m/z would result in a scan time of 1 second and provide 5 scans across the GC peak. The quantitation m/z will usually be the most intense peak in the mass spectrum. The quantitation m/z and dwell time may be optimized for each analyte. The acquisition table used for SIM must take into account the mass defect (usually less than 0.2 Dalton) that can occur at each m/z monitored. Refer to the footnotes to Table 3 for establishing operating conditions and to section 7.3.2.2 for establishing scan conditions.

7.3.2.5 For combined scan and SIM operation, set up the scan segments and descriptors to meet requirements in sections 7.3.2.2–7.3.2.4. Analyze unfamiliar samples in the scan mode to assure that the analytes of interest are determined.

7.3.3 Analyze each calibration standard according to Section 10 and tabulate the area at the quantitation m/z against concentration for each analyte of interest, surrogate, and internal standard. Calculate the response factor (RF) for each compound at each concentration using Equation 1.

Where:

As = Area of the characteristic m/z for the analyte to be measured.

Ais = Area of the characteristic m/z for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the analyte to be measured (µg/L).

7.3.4 Calculate the mean (average) and relative standard deviation (RSD) of the response factors. If the RSD is less than 35%, the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to fit a linear or quadratic regression of response ratios, As/Ais, vs. concentration ratios Cs/Cis. If used, the regression must be weighted inversely proportional to concentration (1/C). The coefficient of determination (R2) of the weighted regression must be greater than 0.920 (this value roughly corresponds to the RSD limit of 35%). Alternatively, the relative standard error (Reference 10) may be used as an acceptance criterion. As with the RSD, the RSE must be less than 35%. If an RSE less than 35% cannot be achieved for a quadratic regression, system performance is unacceptable, and the system must be adjusted and re-calibrated.

Note:

Using capillary columns and current instrumentation, it is quite likely that a laboratory can calibrate the target analytes in this method and achieve a linearity metric (either RSD or RSE) well below 35%. Therefore, laboratories are permitted to use more stringent acceptance criteria for calibration than described here, for example, to harmonize their application of this method with those from other sources.

7.4 Calibration verification—Because the analytical system is calibrated by purge of the analytes from water, calibration verification is performed using the laboratory control sample (LCS). See section 8.4 for requirements for calibration verification using the LCS, and the Glossary for further definition.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and ongoing analysis of spiked samples and blanks to evaluate and document data quality (40 CFR 136.7). The laboratory must maintain records to document the quality of data generated. Results of ongoing performance tests are compared with established QC acceptance criteria to determine if the results of analyses meet performance requirements of this method. When results of spiked samples do not meet the QC acceptance criteria in this method, a quality control check sample (laboratory control sample; LCS) must be analyzed to confirm that the measurements were performed in an in-control mode of operation. A laboratory may develop its own performance criteria (as QC acceptance criteria), provided such criteria are as or more restrictive than the criteria in this method.

8.1.1 The laboratory must make an initial demonstration of capability (DOC) to generate acceptable precision and recovery with this method. This demonstration is detailed in Section 8.2. On a continuing basis, the laboratory must repeat demonstration of capability (DOC) at least annually.

8.1.2 In recognition of advances that are occurring in analytical technology, and to overcome matrix interferences, the laboratory is permitted certain options (section 1.5 and 40 CFR 136.6(b)) to improve separations or lower the costs of measurements. These options may include an alternative purge-and-trap device, and changes in both column and type of mass spectrometer (see 40 CFR 136.6(b)(4)(xvi)). Alternative determinative techniques, such as substitution of spectroscopic or immunoassay techniques, and changes that degrade method performance, are not allowed. If an analytical technique other than GC/MS is used, that technique must have a specificity equal to or greater than the specificity of GC/MS for the analytes of interest. The laboratory is also encouraged to participate in inter-comparison and performance evaluation studies (see section 8.8).

8.1.2.1 Each time a modification is made to this method, the laboratory is required to repeat the procedure in section 8.2. If the detection limit of the method will be affected by the change, the laboratory must demonstrate that the MDLs (40 CFR part 136, appendix B) are lower than one-third the regulatory compliance limit or the MDLs in this method, whichever are greater. If calibration will be affected by the change, the instrument must be recalibrated per section 7. Once the modification is demonstrated to produce results equivalent or superior to results produced by this method, that modification may be used routinely thereafter, so long as the other requirements in this method are met (e.g., matrix spike/matrix spike duplicate recovery and relative percent difference).

8.1.2.1.1 If a modification is to be applied to a specific discharge, the laboratory must prepare and analyze matrix spike/matrix spike duplicate (MS/MSD) samples (Section 8.3) and LCS samples (section 8.4). The laboratory must include internal standards and surrogates (section 8.7) in each of the samples. The MS/MSD and LCS samples must be fortified with the analytes of interest (section 1.3.). If the modification is for nationwide use, MS/MSD samples must be prepared from a minimum of nine different discharges (See section 8.1.2.1.2), and all QC acceptance criteria in this method must be met. This evaluation only needs to be performed once, other than for the routine QC required by this method (for example it could be performed by the vendor of the alternative materials) but any laboratory using that specific material must have the results of the study available. This includes a full data package with the raw data that will allow an independent reviewer to verify each determination and calculation performed by the laboratory (see section 8.1.2.2.5, items (a)–(l)).

8.1.2.1.2 Sample matrices on which MS/MSD tests must be performed for nationwide use of an allowed modification:

(a) Effluent from a publicly owned treatment works (POTW).

(b) ASTM D5905 Standard Specification for Substitute Wastewater.

(c) Sewage sludge, if sewage sludge will be in the permit.

(d) ASTM D1141 Standard Specification for Substitute Ocean Water, if ocean water will be in the permit.

(e) Untreated and treated wastewaters up to a total of nine matrix types (see https://www.epa.gov/eg/industrial-effluent-guidelines for a list of industrial categories with existing effluent guidelines).

(i) At least one of the above wastewater matrix types must have at least one of the following characteristics:

(A) Total suspended solids greater than 40 mg/L.

(B) Total dissolved solids greater than 100 mg/L.

(C) Oil and grease greater than 20 mg/L.

(D) NaCl greater than 120 mg/L.

(E) CaCO3 greater than 140 mg/L.

(ii) Results of MS/MSD tests must meet QC acceptance criteria in section 8.3.

(f) A proficiency testing (PT) sample from a recognized provider, in addition to tests of the nine matrices (section 8.1.2.1.1).

8.1.2.2 The laboratory is required to maintain records of modifications made to this method. These records include the following, at a minimum:

8.1.2.2.1 The names, titles, and business street addresses, telephone numbers, and email addresses of the analyst(s) that performed the analyses and modification, and of the quality control officer that witnessed and will verify the analyses and modifications.

8.1.2.2.2 A list of analytes, by name and CAS Registry Number.

8.1.2.2.3 A narrative stating reason(s) for the modifications.

8.1.2.2.4 Results from all quality control (QC) tests comparing the modified method to this method, including:

(a) Calibration (section 7).

(b) Calibration verification/LCS (section 8.4).

(c) Initial demonstration of capability (section 8.2).

(d) Analysis of blanks (section 8.5).

(e) Matrix spike/matrix spike duplicate analysis (section 8.3).

(f) Laboratory control sample analysis (section 8.4).

8.1.2.2.5 Data that will allow an independent reviewer to validate each determination by tracing the instrument output (peak height, area, or other signal) to the final result. These data are to include:

(a) Sample numbers and other identifiers.

(b) Analysis dates and times.

(c) Analysis sequence/run chronology.

(d) Sample volume (Section 10).

(e) Sample dilution (Section 13.2).

(f) Instrument and operating conditions.

(g) Column (dimensions, material, etc).

(h) Operating conditions (temperature program, flow rate, etc).

(i) Detector (type, operating conditions, etc).

(j) Chromatograms, mass spectra, and other recordings of raw data.

(k) Quantitation reports, data system outputs, and other data to link the raw data to the results reported.

(l) A written Standard Operating Procedure (SOP).

8.1.2.2.6 Each individual laboratory wishing to use a given modification must perform the start-up tests in section 8.1.2 (e.g., DOC, MDL), with the modification as an integral part of this method prior to applying the modification to specific discharges. Results of the DOC must meet the QC acceptance criteria in Table 7 for the analytes of interest (section 1.3), and the MDLs must be equal to or lower than the MDLs in Table3 for the analytes of interest

8.1.3 Before analyzing samples, the laboratory must analyze a blank to demonstrate that interferences from the analytical system, labware, and reagents are under control. Each time a batch of samples is analyzed or reagents are changed, a blank must be analyzed as a safeguard against laboratory contamination. Requirements for the blank are given in section 8.5.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze samples to monitor and evaluate method and laboratory performance on the sample matrix. The procedure for spiking and analysis is given in section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through analysis of a quality control check sample (laboratory control sample, LCS; on-going precision and recovery sample, OPR) that the measurement system is in control. This procedure is given in section 8.4.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is given in section 8.8.

8.1.7 The large number of analytes tested in performance tests in this method present a substantial probability that one or more will fail acceptance criteria when many analytes are tested simultaneously, and a re-test is allowed if this situation should occur. If, however, continued re-testing results in further repeated failures, the laboratory must document and report the failures (e.g., as qualifiers on results), unless the failures are not required to be reported as determined by the regulatory/control authority. Results associated with a QC failure for an analyte regulated in a discharge cannot be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee of reporting timely results.

8.2 Initial demonstration of capability (DOC)—To establish the ability to generate acceptable recovery and precision, the laboratory must perform the DOC in sections 8.2.1 through 8.2.6 for the analytes of interest. The laboratory must also establish MDLs for the analytes of interest using the MDL procedure at 40 CFR part 136, appendix B. The laboratory's MDLs must be equal to or lower than those listed in Table 1 for those analytes which list MDL values, or lower than one-third the regulatory compliance limit, whichever is greater. For MDLs not listed in Table 1, the laboratory must determine the MDLs using the MDL procedure at 40 CFR part 136, appendix B under the same conditions used to determine the MDLs for the analytes listed in Table 1. All procedures used in the analysis must be included in the DOC.

8.2.1 For the DOC, a QC check sample concentrate (LCS concentrate) containing each analyte of interest (section 1.3) is prepared in methanol. The QC check sample concentrate must be prepared independently from those used for calibration, but may be from the same source as the second-source standard used for calibration verification/LCS (sections 7.4 and 8.4). The concentrate should produce concentrations of the analytes of interest in water at the mid-point of the calibration range, and may be at the same concentration as the LCS (section 8.4).

Note:

QC check sample concentrates are no longer available from EPA.

8.2.2 Using a pipet or micro-syringe, prepare four LCSs by adding an appropriate volume of the concentrate to each of four aliquots of reagent water. The volume of reagent water must be the same as the volume that will be used for the sample, blank (section 8.5), and MS/MSD (section 8.3). A volume of 5 mL and a concentration of 20 µg/L were used to develop the QC acceptance criteria in Table 7. An alternative volume and sample concentration may be used, provided that all QC tests are performed and all QC acceptance criteria in this method are met. Also add an aliquot of the surrogate spiking solution (section 6.7) and internal standard spiking solution (section 7.3.1.3) to the reagent-water aliquots.

8.2.3 Analyze the four LCSs according to the method beginning in section 10.

8.2.4 Calculate the average percent recovery (X) and the standard deviation of the percent recovery (s) for each analyte using the four results.

8.2.5 For each analyte, compare s and X with the corresponding acceptance criteria for precision and recovery in Table 7. For analytes in Tables 1 and 2 not listed in Table 7, DOC QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 11 and 12). Alternatively, acceptance criteria for analytes not listed in Table 7 may be based on laboratory control charts. If s and X for all analytes of interest meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If any individual s exceeds the precision limit or any individual X falls outside the range for recovery, system performance is unacceptable for that analyte.

Note:

The large number of analytes in Tables 1 and 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when many or all analytes are determined simultaneously. Therefore, the analyst is permitted to conduct a “re-test” as described in section 8.2.6.

8.2.6 When one or more of the analytes tested fail at least one of the acceptance criteria, repeat the test for only the analytes that failed. If results for these analytes pass, system performance is acceptable and analysis of samples and blanks may proceed. If one or more of the analytes again fail, system performance is unacceptable for the analytes that failed the acceptance criteria. Correct the problem and repeat the test (section 8.2). See section 8.1.7 for disposition of repeated failures.

Note:

To maintain the validity of the test and re-test, system maintenance and/or adjustment is not permitted between this pair of tests.

8.3 Matrix spike and matrix spike duplicate (MS/MSD)—The purpose of the MS/MSD requirement is to provide data that demonstrate the effectiveness of the method as applied to the samples in question by a given laboratory, and both the data user (discharger, permittee, regulated entity, regulatory/control authority, customer, other) and the laboratory share responsibility for provision of such data. The data user should identify the sample and the analytes of interest (section 1.3) to be spiked and provide sufficient sample volume to perform MS/MSD analyses. The laboratory must, on an ongoing basis, spike at least 5% of the samples in duplicate from each discharge being monitored to assess accuracy (recovery and precision). If direction cannot be obtained from the data user, the laboratory must spike at least one sample in duplicate per extraction batch of up to 20 samples with the analytes in Table 1. Spiked sample results should be reported only to the data user whose sample was spiked, or as requested or required by a regulatory/control authority, or in a permit.

8.3.1 If, as in compliance monitoring, the concentration of a specific analyte will be checked against a regulatory concentration limit, the concentration of the spike should be at that limit; otherwise, the concentration of the spike should be one to five times higher than the background concentration determined in section 8.3.2, at or near the mid-point of the calibration range, or at the concentration in the LCS (section 8.4) whichever concentration would be larger.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the each analyte of interest. If necessary, prepare a new check sample concentrate (section 8.2.1) appropriate for the background concentration. Spike and analyze two additional sample aliquots, and determine the concentration after spiking (A1 and A2) of each analyte. Calculate the percent recoveries (P1 and P2) as 100 (A1−B)/T and 100 (A2−B)/T, where T is the known true value of the spike. Also calculate the relative percent difference (RPD) between the concentrations (A1 and A2) as 200 |A1−A2|/(A1 + A2). If necessary, adjust the concentrations used to calculate the RPD to account for differences in the volumes of the spiked aliquots.

8.3.3 Compare the percent recoveries (P1 and P2) and the RPD for each analyte in the MS/MSD aliquots with the corresponding QC acceptance criteria in Table 7. A laboratory may develop and apply QC acceptance criteria more restrictive than the criteria in Table 7, if desired.

8.3.3.1 If any individual P falls outside the designated range for recovery in either aliquot, or the RPD limit is exceeded, the result for the analyte in the unspiked sample is suspect. See Section 8.1.7 for disposition of failures.

8.3.3.2 The acceptance criteria in Table 7 were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the spike to background ratio approaches 5:1 (Reference 13) and is applied to spike concentrations of 20 µg/L and higher. If spiking is performed at a concentration lower than 20 µg/L, the laboratory must use the QC acceptance criteria in Table 7, the optional QC acceptance criteria calculated for the specific spike concentration in Table 8, or optional in-house criteria (Section 8.3.4). To use the acceptance criteria in Table 8:

(1) Calculate recovery (X') using the equation in Table 8, substituting the spike concentration (T) for C;

(2) Calculate overall precision (S') using the equation in Table 8, substituting X' for X;

(3) Calculate the range for recovery at the spike concentration as (100 X'/T) ± 2.44(100 S'/T)% (Reference 4). For analytes of interest in Tables 1 and 2 not listed in Table 7, QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 11 and 12). Alternatively, acceptance criteria may be based on laboratory control charts. In-house LCS QC acceptance criteria must be updated at least every two years.

8.3.4 After analysis of a minimum of 20 MS/MSD samples for each target analyte and surrogate, and if the laboratory chooses to develop and apply in-house QC limits, the laboratory should calculate and apply in-house QC limits for recovery and RPD of future MS/MSD samples (section 8.3). The QC limits for recovery are calculated as the mean observed recovery ± 3 standard deviations, and the upper QC limit for RPD is calculated as the mean RPD plus 3 standard deviations of the RPDs. The in-house QC limits must be updated at least every two years and re-established after any major change in the analytical instrumentation or process. If in-house QC limits are developed, at least 80% of the analytes tested in the MS/MSD must have in-house QC acceptance criteria that are tighter than those in Table 7 and the remaining analytes (those other than the analytes included in the 80%) must meet the acceptance criteria in Table 7. If an in-house QC limit for the RPD is greater than the limit in Table 7, then the limit in Table 7 must be used. Similarly, if an in-house lower limit for recovery is below the lower limit in Table 7, then the lower limit in Table 7 must be used, and if an in-house upper limit for recovery is above the upper limit in Table 7, then the upper limit in Table 7 must be used.

8.4 Calibration verification/laboratory control sample (LCS)—The working calibration curve or RF must be verified immediately after calibration and at the beginning of each 12-hour shift by the measurement of an LCS. The LCS must be from a source different from the source used for calibration (section 7.3.2.1), but may be the same as the sample prepared for the DOC (section 8.2.1).

Note:

The 12-hour shift begins after analysis of BFB, the LCS, and the blank, and ends 12 hours later. BFB, the LCS, and blank are outside of the 12-hour shift (Section 11.4). The MS and MSD are treated as samples and are analyzed within the 12-hour shift.

8.4.1 Prepare the LCS by adding QC check sample concentrate (section 8.2.1) to reagent water. Include all analytes of interest (Section 1.3) in the LCS. The volume of reagent water must be the same as the volume used for the sample, blank (Section 8.5), and MS/MSD (section 8.3). Also add an aliquot of the surrogate solution (Section 6.7) and internal standard solution (section 7.3.1.3). The concentration of the analytes in reagent water should be the same as the concentration in the DOC (section 8.2.2).

8.4.2 Analyze the LCS prior to analysis of field samples in the batch of samples analyzed during the 12-hour shift (see the Note at section 8.4). Determine the concentration (A) of each analyte. Calculate the percent recovery (Q) as 100 (A/T) %, where T is the true value of the concentration in the LCS.

8.4.3 Compare the percent recovery (Q) for each analyte with its corresponding QC acceptance criterion in Table 7. For analytes of interest in Tables 1 and 2 not listed in Table 7, use the QC acceptance criteria developed for the LCS (section 8.4.5). If the recoveries for all analytes of interest fall within their respective QC acceptance criteria, analysis of blanks and field samples may proceed. If any individual Q falls outside the range, proceed according to section 8.4.4.

Note:

The large number of analytes in Tables 1—2 present a substantial probability that one or more will fail the acceptance criteria when all analytes are tested simultaneously. Because a re-test is allowed in event of failure (sections 8.1.7 and 8.4.3), it may be prudent to analyze two LCSs together and evaluate results of the second analysis against the QC acceptance criteria only if an analyte fails the first test.

8.4.4 Repeat the test only for those analytes that failed to meet the acceptance criteria (Q). If these analytes now pass, system performance is acceptable and analysis of blanks and samples may proceed. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, repeat the test (section 8.4.2). using a fresh LCS (section 8.2.2) or an LCS prepared with a fresh QC check sample concentrate (section 8.2.1), or perform and document system repair. Subsequent to repair, repeat the calibration verification/LCS test (section 8.4). If the acceptance criteria for Q cannot be met, re-calibrate the instrument (section 7). See section 8.1.7 for disposition of repeated failures.

Note:

To maintain the validity of the test and re-test, system maintenance and/or adjustment is not permitted between the pair of tests.

8.4.5 After analysis of 20 LCS samples, and if the laboratory chooses to develop and apply in-house QC limits, the laboratory should calculate and apply in-house QC limits for recovery to future LCS samples (section 8.4). Limits for recovery in the LCS calculated as the mean recovery ±3 standard deviations. A minimum of 80% of the analytes tested for in the LCS must have QC acceptance criteria tighter than those in Table 7, and the remaining analytes (those other than the analytes included in the 80%) must meet the acceptance criteria in Table 7. If an in-house lower limit for recovery is lower than the lower limit in Table 7, the lower limit in Table 7 must be used, and if an in-house upper limit for recovery is higher than the upper limit in Table 7, the upper limit in Table 7 must be used. Many of the analytes and surrogates do not have acceptance criteria. The laboratory should use 60–140% as interim acceptance criteria for recoveries of spiked analytes that do not have recovery limits specified in Table 7, and least 80% of the analytes should meet the 60–140% interim criteria until in-house LCS limits are developed. Alternatively, acceptance criteria for analytes that do not have recovery limits in Table 7 may be based on laboratory control charts. In-house QC acceptance criteria must be updated at least every two years.

8.5 Blank—A blank must be analyzed prior to each 12-hour shift to demonstrate freedom from contamination. A blank must also be analyzed after a sample containing a high concentration of an analyte or potentially interfering compound to demonstrate freedom from carry-over.

8.5.1 Spike the internal standards and surrogates into the blank. Analyze the blank immediately after analysis of the LCS (Section 8.4) and prior to analysis of the MS/MSD and samples to demonstrate freedom from contamination.

8.5.2 If any analyte of interest is found in the blank: At a concentration greater than the MDL for the analyte, at a concentration greater than one-third the regulatory compliance limit, or at a concentration greater than one-tenth the concentration in a sample analyzed during the 12-hour shift (section 8.4), whichever is greater; analysis of samples must be halted and samples affected by the blank must be re-analyzed. If, however, continued re-testing results in repeated blank contamination, the laboratory must document and report the failures (e.g., as qualifiers on results), unless the failures are not required to be reported as determined by the regulatory/control authority. Results associated with blank contamination for an analyte regulated in a discharge cannot be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee of reporting timely results.

8.6 Surrogate recoveries—The laboratory must evaluate surrogate recovery data in each sample against its in-house surrogate recovery limits for surrogates that do not have acceptance criteria in Table 7. The laboratory may use 60–140% as interim acceptance criteria for recoveries for surrogates not listed in Table 5. At least 80% of the surrogates must meet the 60–140% interim criteria until in-house limits are developed. Alternatively, surrogate recovery limits may be developed from laboratory control charts.

8.6.1 Spike the surrogates into all samples, blanks, LCSs, and MS/MSDs. Compare surrogate recoveries against the QC acceptance criteria in Table 7. For surrogates in Table 5 without QC acceptance criteria in Table 7, and for other surrogates that may be used by the laboratory, limits must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 11 and 12). Alternatively, surrogate recovery limits may be developed from laboratory control charts. In-house QC acceptance criteria must be updated at least every two years.

8.6.2 If any recovery fails its criteria, attempt to find and correct the cause of the failure. See section 8.1.7 for disposition of failures.

8.7 Internal standard responses.

8.7.1 Calibration verification/LCS—The responses (GC peak heights or areas) of the internal standards in the calibration verification/LCS must be within 50% to 200% (1/2 to 2×) of their respective responses in the mid-point calibration standard. If they are not, repeat the LCS test using a fresh QC check sample (section 8.4.1) or perform and document system repair. Subsequent to repair, repeat the calibration verification/LCS test (section 8.4). If the responses are still not within 50% to 200%, re-calibrate the instrument (section 7) and repeat the calibration verification/LCS test.

8.7.2 Samples, blanks, and MS/MSDs—The responses (GC peak heights or areas) of each internal standard in each sample, blank, and MS/MSD must be within 50% to 200% (1/2 to 2×) of its respective response in the mid-point calibration standard. If, as a group, all internal standards are not within this range, perform and document system repair, repeat the calibration verification/LCS test (section 8.4), and re-analyze the affected samples. If a single internal standard is not within the 50% to 200% range, use an alternative internal standard for quantitation of the analyte referenced to the affected internal standard. It may be necessary to use the data system to calculate a new response factor from calibration data for the alternative internal standard/analyte pair. If an internal standard fails the 50–200% criteria and no analytes are detected in the sample, ignore the failure or report it if required by the regulatory/control authority.

8.8 As part of the QC program for the laboratory, control charts or statements of accuracy for wastewater samples must be assessed and records maintained periodically (see 40 CFR 136.7(c)(1)(viii)). After analysis of five or more spiked wastewater samples as in section 8.3, calculate the average percent recovery (PX) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent interval from PX−2sp to PX + 2sp. For example, if PX = 90% and sp = 10%, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each analyte on a regular basis (e.g., after each 5–10 new accuracy measurements). If desired, statements of accuracy for laboratory performance, independent of performance on samples, may be developed using LCSs.

8.9 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Collect the sample as a grab sample in a glass container having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If needed, collect additional sample(s) for the MS/MSD (section 8.3).

9.2 Ice or refrigerate samples at ≤6 °C from the time of collection until analysis, but do not freeze. If residual chlorine is present, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. Any method suitable for field use may be employed to test for residual chlorine (Reference 14). Field test kits are also available for this purpose. If sodium thiosulfate interferes in the determination of the analytes, an alternative preservative (e.g., ascorbic acid or sodium sulfite) may be used. If preservative has been added, shake the sample vigorously for one minute. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3 If acrolein is to be determined, analyze the sample within 3 days. To extend the holding time to 14 days, acidify a separate sample to pH 4–5 with HCl using the procedure in section 9.7.

9.4 Experimental evidence indicates that some aromatic compounds, notably benzene, toluene, and ethyl benzene are susceptible to rapid biological degradation under certain environmental conditions (Reference 3). Refrigeration alone may not be adequate to preserve these compounds in wastewaters for more than seven days. To extend the holding time for aromatic compounds to 14 days, acidify the sample to approximately pH 2 using the procedure in section 9.7.

9.5 If halocarbons are to be determined, either use the acidified aromatics sample in section 9.4 or acidify a separate sample to a pH of about 2 using the procedure in section 9.7.

9.6 The ethers listed in Table 2 are prone to hydrolysis at pH 2 when a heated purge is used. Aqueous samples should not be acid preserved if these ethers are of interest, or if the alcohols they would form upon hydrolysis are of interest and the ethers are anticipated to present.

9.7 Sample acidification—Collect about 500 mL of sample in a clean container and adjust the pH of the sample to 4–5 for acrolein (section 9.3), or to about 2 for the aromatic compounds (section 9.4) by adding 1+1 HCl while swirling or stirring. Check the pH with narrow range pH paper. Fill a sample container as described in section 9.1. Alternatively, fill a precleaned vial (section 5.1.1) that contains approximately 0.25 mL of 1+1 HCl with sample as in section 9.1. If preserved using this alternative procedure, the pH of the sample can be verified to be <2 after some of the sample is removed for analysis. Acidification will destroy 2-chloroethylvinyl ether; therefore, determine 2-chloroethylvinyl ether from the unacidified sample.

9.8 All samples must be analyzed within 14 days of collection (Reference 3), unless specified otherwise in sections 9.3–9.7.

10. Sample Purging and Gas Chromatography

10.1 The footnote to Table 3 gives the suggested GC column and operating conditions MDLs and MLs for many of the analytes are given in Table 1. Retention times for many of the analytes are given in Table 3. Sections 10.2 through 10.7 suggest procedures that may be used with a manual purge-and-trap system. Auto-samplers and other columns or chromatographic conditions may be used if requirements in this method are met. Prior to performing analyses, and between analyses, it may be necessary to bake the purge-and-trap and GC systems (section 3.3).

10.2 Attach the trap inlet to the purging device, and set the purge-and-trap system to purge. Open the syringe valve located on the purging device sample introduction needle.

10.3 Allow the sample to come to ambient temperature prior to pouring an aliquot into the syringe. Remove the plunger from a syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add the surrogate spiking solution (section 6.7) and internal standard spiking solution (section 7.3.1.3) through the valve bore, then close the valve. The surrogate and internal standards may be mixed and added as a single spiking solution. Autosamplers designed for purge-and-trap analysis of volatiles also may be used.

10.4 Attach the syringe valve assembly to the syringe valve on the purging device. Open the syringe valve and inject the sample into the purging chamber.

10.5 Close both valves and purge the sample at a temperature, flow rate, and duration sufficient to purge the less-volatile analytes onto the trap, yet short enough to prevent blowing the more-volatile analytes through the trap. The temperature, flow rate, and time should be determined by test. The same purge temperature, flow rate, and purge time must be used for all calibration, QC, and field samples.

10.6 After the purge, set the purge-and-trap system to the desorb mode, and begin the temperature program of the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to the desorb temperature while backflushing the trap with carrier gas at the flow rate and for the time necessary to desorb the analytes of interest. The optimum temperature, flow rate, and time should be determined by test. The same temperature, desorb time, and flow rate must be used for all calibration, QC, and field samples. If heating of the trap does not result in sharp peaks for the early eluting analytes, the GC column may be used as a secondary trap by cooling to an ambient or subambient temperature. To avoid carry-over and interferences, maintain the trap at the desorb temperature and flow rate until the analytes, interfering compounds, and excess water are desorbed. The optimum conditions should be determined by test.

10.7 Start MS data acquisition at the start of the desorb cycle and stop data collection when the analytes of interest, potentially interfering compounds, and water have eluted (see the footnote to Table 3 for conditions).

10.8 Cool the trap to the purge temperature and return the trap to the purge mode. When the trap is cool, the next sample can be analyzed.

11. Performance Tests

11.1 At the beginning of each 12-hour shift during which standards or samples will be analyzed, perform the tests in sections 11.2–11.3 to verify system performance. Use the instrument operating conditions in the footnotes to Table 3 for these performance tests. Alternative conditions may be used so as long as all QC requirements are met.

11.2 BFB—Inject 50 ng of BFB solution directly on the column. Alternatively, add BFB to reagent water or an aqueous standard such that 50 ng or less of BFB will be introduced into the GC. Analyze according to section 10. Confirm that all criteria in section 7.3.2.2 and Table 4 are met. If all criteria are not met, perform system repair, retune the mass spectrometer, and repeat the test until all criteria are met.

11.3 Verify calibration with the LCS (section 8.4) after the criteria for BFB are met (Reference 15) and prior to analysis of a blank or sample. After verification, analyze a blank (section 8.5) to demonstrate freedom from contamination and carry-over at the MDL. Tests for BFB, the LCS, and the blank are outside of the 12-hour shift, and the 12-hour shift includes samples and matrix spikes and matrix spike duplicates (section 8.4). The total time for analysis of BFB, the LCS, the blank, and the 12-hour shift must not exceed 14 hours.

12. Qualitative Identification

12.1 Identification is accomplished by comparison of results from analysis of a sample or blank with data stored in the GC/MS data system (section 7.3.2.3). Identification of an analyte is confirmed per sections 12.1.1 through 12.1.4.

12.1.1 The signals for the quantitation and secondary m/z's stored in the data system (section 7.3.2.3) for each analyte of interest must be present and must maximize within the same two consecutive scans.

12.1.2 The retention time for the analyte should be within ± 10 seconds of the analyte in the LCS run at the beginning of the shift (section 8.4).

Note:

Retention time windows other than ± 10 seconds may be appropriate depending on the performance of the gas chromatograph or observed retention time drifts due to certain types of matrix effects. Relative retention time (RRT) may be used as an alternative to absolute retention times if retention time drift is a concern. RRT is a unitless quantity (see section 20.2), although some procedures refer to “RRT units” in providing the specification for the agreement between the RRT values in the sample and the LCS or other standard. When significant retention time drifts are observed, dilutions or spiked samples may help the analyst determine the effects of the matrix on elution of the target analytes and to assist in qualitative identification.

12.1.3 Either the background corrected EICP areas, or the corrected relative intensities of the mass spectral peaks at the GC peak maximum, must agree within 50% to 200% (12 to 2 times) for the quantitation and secondary m/z's in the reference mass spectrum stored in the data system (section 7.3.2.3), or from a reference library. For example, if a peak has an intensity of 20% relative to the base peak, the analyte is identified if the intensity of the peak in the sample is in the range of 10% to 40% of the base peak.

12.1.4 If the acquired mass spectrum is contaminated, or if identification is ambiguous, an experienced spectrometrist (section 1.6) must determine the presence or absence of the compound.

12.2 Structural isomers that produce very similar mass spectra should be identified as individual isomers if they have sufficiently different gas chromatographic retention times. Sufficient gas chromatographic resolution is achieved if the height of the valley between two isomer peaks is less than 50% of the average of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs. The resolution should be verified on the mid-point concentration of the initial calibration as well as the laboratory designated continuing calibration verification level if closely eluting isomers are to be reported.

13. Calculations

13.1 When an analyte has been identified, quantitation of that analyte is based on the integrated abundance from the EICP of the primary characteristic m/z in Table 5 or 6. Calculate the concentration using the response factor (RF) determined in section 7.3.3 and Equation 2. If a calibration curve was used, calculate the concentration using the regression equation for the curve. If the concentration of an analyte exceeds the calibration range, dilute the sample by the minimum amount to bring the concentration into the calibration range, and re-analyze. Determine a dilution factor (DF) from the amount of the dilution. For example, if the extract is diluted by a factor of 2, DF = 2.

Where:

Cs = Concentration of the analyte in the sample, and the other terms are as defined in Section 7.3.3.

13.2 Reporting of results

As noted in section 1.4.1, EPA has promulgated this method at 40 CFR part 136 for use in wastewater compliance monitoring under the National Pollutant Discharge Elimination System (NPDES). The data reporting practices described here are focused on such monitoring needs and may not be relevant to other uses of this method.

13.2.1 Report results for wastewater samples in µg/L without correction for recovery. (Other units may be used if required by a permit.) Report all QC data with the sample results.

13.2.2 Reporting level. Unless otherwise specified in by a regulatory authority or in a discharge permit, results for analytes that meet the identification criteria are reported down to the concentration of the ML established by the laboratory through calibration of the instrument (see section 7.3.2 and the glossary for the derivation of the ML). EPA considers the terms “reporting limit,” “limit of quantitation,” “quantitation limit,” and “minimum level” to be synonymous.

13.2.2.1 Report a result for each analyte in each field sample or QC standard at or above the ML to 3 significant figures. Report a result for each analyte found in each field sample or QC standard below the ML as “<ML,” where ML is the concentration of the analyte at the ML, or as required by the regulatory/control authority or permit. Report a result for each analyte in a blank at or above the MDL to 2 significant figures. Report a result for each analyte found in a blank below the MDL as “<MDL,” where MDL is the concentration of the analyte at the MDL, or as required by the regulatory/control authority or permit.

13.2.2.2 In addition to reporting results for samples and blanks separately, the concentration of each analyte in a blank associated with the sample may be subtracted from the result for that sample, but only if requested or required by a regulatory authority or in a permit. In this case, both the sample result and the blank result must be reported together.

13.2.2.3 Report a result for an analyte found in a sample that has been diluted at the least dilute level at which the area at the quantitation m/z is within the calibration range (i.e., above the ML for the analyte) and the MS/MSD recovery and RPD are within their respective QC acceptance criteria (Table 7). This may require reporting results for some analytes from different analyses.

13.2.3 Results from tests performed with an analytical system that is not in control (i.e., that does not meet acceptance criteria for any of the QC test in this method) must be documented and reported (e.g., as a qualifier on results), unless the failure is not required to be reported as determined by the regulatory/control authority. Results associated with a QC failure cannot be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee of reporting timely results. If the holding time would be exceeded for a re-analysis of the sample, the regulatory/control authority should be consulted for disposition.

14. Method Performance

14.1 This method was tested by 15 laboratories using reagent water, drinking water, surface water, and industrial wastewaters spiked at six concentrations over the range 5–600 µg/L (References 4 and 16). Single-operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the analyte and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 8.

14.2 As noted in section 1.1, this method was validated through an interlaboratory study conducted in the early 1980s. However, the fundamental chemistry principles used in this method remain sound and continue to apply.

15. Pollution Prevention

15.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory operations. EPA has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, the laboratory should use pollution prevention techniques to address waste generation. When wastes cannot be reduced at the source, the Agency recommends recycling as the next best option.

15.2 The analytes in this method are used in extremely small amounts and pose little threat to the environment when managed properly. Standards should be prepared in volumes consistent with laboratory use to minimize the disposal of excess volumes of expired standards.

15.3 For information about pollution prevention that may be applied to laboratories and research institutions, consult “Less is Better: Laboratory Chemical Management for Waste Reduction,” available from the American Chemical Society's Department of Governmental Relations and Science Policy, 1155 16th Street NW., Washington, DC 20036, 202–872–4477.

16. Waste Management

16.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations. An overview of requirements can be found in Environmental Management Guide for Small Laboratories (EPA 233–B–98–001).

16.2 Samples at pH <2, or pH >12, are hazardous and must be handled and disposed of as hazardous waste, or neutralized and disposed of in accordance with all federal, state, and local regulations. It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions. The laboratory using this method has the responsibility to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations. For further information on waste management, see “The Waste Management Manual for Laboratory Personnel,” also available from the American Chemical Society at the address in Section 15.3.

16.3 Many analytes in this method decompose above 500 °C. Low-level waste such as absorbent paper, tissues, and plastic gloves may be burned in an appropriate incinerator. Gross quantities of neat or highly concentrated solutions of toxic or hazardous chemicals should be packaged securely and disposed of through commercial or governmental channels that are capable of handling these types of wastes.

16.4 For further information on waste management, consult “Waste Management Manual for Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste Reduction,” available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street NW., Washington, DC 20036, 202–872–4477.

17. References

1. Bellar, T.A. and Lichtenberg, J.J. “Determining Volatile Organics at Microgram-per-Litre Levels by Gas Chromatography,” Journal American Water Works Association, 66: 739 (1974).

2. “Sampling and Analysis Procedures for Screening of Industrial Effluents for Priority Pollutants,” U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1977, Revised April 1977.

3. Bellar, T.A. and Lichtenberg, J.J. “Semi-Automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds,” Measurement of Organic Pollutants in Water and Wastewater, C.E. Van Hall, editor, American Society for Testing and Materials, Philadelphia, PA. Special Technical Publication 686, 1978.

4. “EPA Method Study 29 EPA Method 624-Purgeables,” EPA 600/4–84–054, National Technical Information Service, PB84–209915, Springfield, Virginia 22161, June 1984.

5. 40 CFR part 136, appendix B.

6. “Method Detection Limit for Methods 624 and 625,” Olynyk, P., Budde, W.L., and Eichelberger, J.W. Unpublished report, May 14, 1980.

7. “Carcinogens-Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

8. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

9. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 7th Edition, 2003.

10. 40 CFR 136.6(b)(5)(x).

11. 40 CFR 136.6(b)(2)(i).

12. Protocol for EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater and Drinking Water (EPA–821–B–98–003) March 1999.

13. Provost, L.P. and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983).

14. 40 CFR 136.3(a), Table IB, Chlorine—Total residual.

15. Budde, W.L. and Eichelberger, J.W. “Performance Tests for the Evaluation of Computerized Gas Chromatography/Mass Spectrometry Equipment and Laboratories,” EPA–600/4–80–025, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, April 1980.

16. “Method Performance Data for Method 624,” Memorandum from R. Slater and T. Pressley, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, January 17, 1984.

18. Tables

Table 1—Purgeables1

Analyte CAS Registry No. MDL (µg/L)2 ML (µg/L)3
Acrolein 107–02–8
Acrylonitrile 107–13–1
Benzene 71–43–2 4.4 13.2
Bromodichloromethane 75–27–4 2.2 6.6
Bromoform 75–25–2 4.7 14.1
Bromomethane 74–83–9
Carbon tetrachloride 56–23–5 2.8 8.4
Chlorobenzene 108–90–7 6.0 18.0
Chloroethane 75–00–3
2-Chloroethylvinyl ether 110–75–8
Chloroform 67–66–3 1.6 4.8
Chloromethane 74–87–3
Dibromochloromethane 124–48–1 3.1 9.3
1,2-Dichlorobenzene 95–50–1
1,3-Dichlorobenzene 541–73–1
1,4-Dichlorobenzene 106–46–7
1,1-Dichloroethane 75–34–3 4.7 14.1
1,2-Dichloroethane 107–06–2 2.8 8.4
1,1-Dichloroethene 75–35–4 2.8 8.4
trans-1,2-Dichloroethene 156–60–5 1.6 4.8
1,2-Dichloropropane 78–87–5 6.0 18.0
cis-1,3-Dichloropropene 10061–01–5 5.0 15.0
trans-1,3-Dichloropropene 10061–02–6
Ethyl benzene 100–41–4 7.2 21.6
Methylene chloride 75–09–2 2.8 8.4
1,1,2,2-Tetrachloroethane 79–34–5 6.9 20.7
Tetrachloroethene 127–18–4 4.1 12.3
Toluene 108–88–3 6.0 18.0
1,1,1-Trichloroethane 71–55–6 3.8 11.4
1,1,2-Trichloroethane 79–00–5 5.0 15.0
Trichloroethene 79–01–6 1.9 5.7
Vinyl chloride 75–01–4

Table 2—Additional Purgeables

Analyte CAS Registry
Acetone1 67–64–1
Acetonitrile2 75–05–8
Acrolein 107–02–8
Acrylonitrile 107–13–1
Allyl alcohol1 107–18–6
Allyl chloride 107–05–1
t-Amyl ethyl ether (TAEE) 919–94–8
t-Amyl methyl ether (TAME) 994–058
Benzyl chloride 100–44–7
Bromoacetone2 598–31–2
Bromobenzene 108–86–1
Bromochloromethane 74–97–5
1,3-Butadiene 106–99–0
n-Butanol1 71–36–3
2-Butanone (MEK)1 2 78–93–3
t-Butyl alcohol (TBA) 75–65–0
n-Butylbenzene 104–51–8
sec-Butylbenzene 135–98–8
t-Butylbenzene 98–06–6
t-Butyl ethyl ether (ETBE) 637–92–3
Carbon disulfide 75–15–0
Chloral hydrate2 302–17–0
Chloroacetonitrile1 107–14–2
1-Chlorobutane 109–69–3
Chlorodifluoromethane 75–45–6
2-Chloroethanol2 107–07–3
bis (2-Chloroethyl) sulfide2 505–60–2
1-Chlorohexanone 20261–68–1
Chloroprene (2-chloro-1,3-butadiene) 126–99–8
3-Chloropropene 107–05–1
3-Chloropropionitrile 542–76–7
2-Chlorotoluene 95–49–8
4-Chlorotoluene 106–43–4
Crotonaldehyde1 2 123–73–9
Cyclohexanone 108–94–1
1,2-Dibromo-3-chloropropane 96–12–8
1,2-Dibromoethane 106–93–4
Dibromomethane 74–95–3
cis-1,4-Dichloro-2-butene 1476–11–5
trans-1,4-Dichloro-2-butene 110–57–6
cis-1,2-Dichloroethene 156–59–2
Dichlorodifluoromethane 75–71–8
1,3-Dichloropropane 142–28–9
2,2-Dichloropropane 590–20–7
1,3-Dichloro-2-propanol2 96–23–1
1,1-Dichloropropene 563–58–6
cis-1,3-Dichloropropene 10061–01–5
1:2,3:4-Diepoxybutane 1464–53–5
Diethyl ether 60–29–7
Diisopropyl ether (DIPE) 108–20–3
1,4-Dioxane2 123–91–1
Epichlorohydrin2 106–89–8
Ethanol2 64–17–5
Ethyl acetate2 141–78–6
Ethyl methacrylate 97–63–2
Ethylene oxide2 75–21–8
Hexachlorobutadiene 87–63–3
Hexachloroethane 67–72–1
2-Hexanone2 591–78–6
Iodomethane 74–88–4
Isobutyl alcohol1 78–83–1
Isopropylbenzene 98–82–8
p-Isopropyltoluene 99–87–6
Methacrylonitrile2 126–98–7
Methanol2 67–56–1
Malonitrile2 109–77–3
Methyl acetate 79–20–9
Methyl acrylate 96–33–3
Methyl cyclohexane 108–87–2
Methyl iodide 74–88–4
Methyl methacrylate 78–83–1
4-Methyl-2-pentanone (MIBK)2 108–10–1
Methyl-t-butyl ether (MTBE) 1634–04–4
Naphthalene 91–20–3
Nitrobenzene 98–95–3
N-Nitroso-di-n-butylamine2 924–16–3
2-Nitropropane 79–46–9
Paraldehyde2 123–63–7
Pentachloroethane2 76–01–7
Pentafluorobenzene 363–72–4
2-Pentanone2 107–19–7
2-Picoline2 109–06–8
1-Propanol1 71–23–8
2-Propanol1 67–63–0
Propargyl alcohol2 107–19–7
beta-Propiolactone2 57–58–8
Propionitrile (ethyl cyanide)1 107–12–0
n-Propylamine 107–10–8
n-Propylbenzene 103–65–1
Pyridine2 110–86–1
Styrene 100–42–5
1,1,1,2-Tetrachloroethane 630–20–6
Tetrahydrofuran 109–99–9
o-Toluidine2 95–53–4
1,2,3-Trichlorobenzene 87–61–6
Trichlorofluoromethane 75–69–4
1,2,3-Trichloropropane 96–18–4
1,2,3-Trimethylbenzene 526–73–8
1,2,4-Trimethylbenzene 95–63–6
1,3,5-Trimethylbenzene 108–67–8
Vinyl acetate 108–05–4
m-Xylene3 108–38–3
o-Xylene3 95–47–6
p-Xylene3 106–42–3
m+o-Xylene3 179601–22–0
m+p-Xylene3 179601–23–1
o+p-Xylene3 136777–61–2

Table 3—Example Retention Times

Analyte Retention time
(min)
Chloromethane 3.68
Vinyl chloride 3.92
Bromomethane 4.50
Chloroethane 4.65
Trichlorofluoromethane 5.25
Diethyl ether 5.88
Acrolein 6.12
1,1-Dichloroethene 6.30
Acetone 6.40
Iodomethane 6.58
Carbon disulfide 6.72
3-Chloropropene 6.98
Methylene chloride 7.22
Acrylonitrile 7.63
trans-1,2-Dichloroethene 7.73
1,1-Dichloroethane 8.45
Vinyl acetate 8.55
Allyl alcohol 8.58
2-Chloro-1,3-butadiene 8.65
Methyl ethyl ketone 9.50
cis-1,2-Dichloroethene 9.50
Ethyl cyanide 9.57
Methacrylonitrile 9.83
Chloroform 10.05
1,1,1-Trichloroethane 10.37
Carbon tetrachloride 10.70
Isobutanol 10.77
Benzene 10.98
1,2-Dichloroethane 11.00
Crotonaldehyde 11.45
Trichloroethene 12.08
1,2-Dichloropropane 12.37
Methyl methacrylate 12.55
p-Dioxane 12.63
Dibromomethane 12.65
Bromodichloromethane 12.95
Chloroacetonitrile 13.27
2-Chloroethylvinyl ether 13.45
cis-1,3-Dichloropropene 13.65
4-Methyl-2-pentanone 13.83
Toluene 14.18
trans-1,3-Dichloropropene 14.57
Ethyl methacrylate 14.70
1,1,2-Trichloroethane 14.93
1,3-Dichloropropane 15.18
Tetrachloroethene 15.22
2-Hexanone 15.30
Dibromochloromethane 15.68
1,2-Dibromoethane 15.90
Chlorobenzene 16.78
Ethylbenzene 16.82
1,1,1,2-Tetrachloroethane 16.87
m+p-Xylene 17.08
o-Xylene 17.82
Bromoform 18.27
Bromofluorobenzene 18.80
1,1,2,2-Tetrachloroethane 18.98
1,2,3-Trichloropropane 19.08
trans-1,4-Dichloro-2-butene 19.12

Table 4—BFB Key m/z Abundance Criteria1

m/z Abundance criteria
50 15–40% of m/z 95.
75 30–60% of m/z 95.
95 Base Peak, 100% Relative Abundance.
96 5–9% of m/z 95.
173 <2% of m/z 174.
174 >50% of m/z 95.
175 5–9% of m/z 174.
176 >95% but <101% of m/z 174.
177 5–9% of m/z 176.

Table 5—Suggested Surrogate and Internal Standards

Analyte Retention time (min)1 Primary m/z Secondary
m/z's
Benzene-d6 10.95 84
4-Bromofluorobenzene 18.80 95 174, 176
Bromochloromethane 9.88 128 49, 130, 51
2-Bromo-1-chloropropane 14.80 77 79, 156
2-Butanone-d5 9.33 77
Chloroethane-d5 4.63 71
Chloroform-13C 10.00 86
1,2-Dichlorobenzene-d4 152
1,4-Dichlorobutane 18.57 55 90, 92
1,2-Dichloroethane-d4 10.88 102
1,1-Dichloroethene-d2 6.30 65
1,2-Dichloropropane-d6 12.27 67
trans-1,3-Dichloropropene-d4 14.50 79
1,4-Difluorobenzene 114 63, 88
Ethylbenzene-d10 16.77 98
Fluorobenzene 96 70
2-Hexanone-d5 15.30 63
Pentafluorobenzene 168
1,1,2,2-Tetrachloroethane-d2 18.93 84
Toluene-d8 14.13 100
Vinyl chloride-d3 3.87 65

Table 6—Characteristic m/z's for Purgeable Organics

Analyte Primary m/z Secondary m/z's
Acrolein 56 55 and 58.
Acrylonitrile 53 52 and 51.
Chloromethane 50 52.
Bromomethane 94 96.
Vinyl chloride 62 64.
Chloroethane 64 66.
Methylene chloride 84 49, 51, and 86.
Trichlorofluoromethane 101 103.
1,1-Dichloroethene 96 61 and 98.
1,1-Dichloroethane 63 65, 83, 85, 98, and 100.
trans-1,2-Dichloroethene 96 61 and 98.
Chloroform 83 85.
1,2-Dichloroethane 98 62, 64, and 100.
1,1,1-Trichloroethane 97 99, 117, and 119.
Carbon tetrachloride 117 119 and 121.
Bromodichloromethane 83 127, 85, and 129.
1,2-Dichloropropane 63 112, 65, and 114.
trans-1,3-Dichloropropene 75 77.
Trichloroethene 130 95, 97, and 132.
Benzene 78
Dibromochloromethane 127 129, 208, and 206.
1,1,2-Trichloroethane 97 83, 85, 99, 132, and 134.
cis-1,3-Dichloropropene 75 77.
2-Chloroethylvinyl ether 106 63 and 65.
Bromoform 173 171, 175, 250, 252, 254, and 256.
1,1,2,2-Tetrachloroethane 168 83, 85, 131, 133, and 166.
Tetrachloroethene 164 129, 131, and 166.
Toluene 92 91.
Chlorobenzene 112 114.
Ethyl benzene 106 91.
1,3-Dichlorobenzene 146 148 and 111.
1,2-Dichlorobenzene 146 148 and 111.
1,4-Dichlorobenzene 146 148 and 111.

Table 7—LCS (Q), DOC (s and X), and MS/MSD (P and RPD) Acceptance Criteria1

Analyte Range for Q
(%)
Limit for s
(%)
Range for X
(%)
Range for P1, P2
(%)
Limit for RPD
Acrolein 60–140 30 50–150 40–160 60
Acrylonitrile 60–140 30 50–150 40–160 60
Benzene 65–135 33 75–125 37–151 61
Benzene-d6
Bromodichloromethane 65–135 34 50–140 35–155 56
Bromoform 70–130 25 57–156 45–169 42
Bromomethane 15–185 90 D–206 D–242 61
2-Butanone-d5
Carbon tetrachloride 70–130 26 65–125 70–140 41
Chlorobenzene 65–135 29 82–137 37–160 53
Chloroethane 40–160 47 42–202 14–230 78
Chloroethane-d5
2-Chloroethylvinyl ether D–225 130 D–252 D–305 71
Chloroform 70–135 32 68–121 51–138 54
Chloroform-13C
Chloromethane D–205 472 D–230 D–273 60
Dibromochloromethane 70–135 30 69–133 53–149 50
1,2-Dichlorobenzene 65–135 31 59–174 18–190 57
1,2-Dichlorobenzene-d4
1,3-Dichlorobenzene 70–130 24 75–144 59–156 43
1,4-Dichlorobenzene 65–135 31 59–174 18–190 57
1,1-Dichloroethane 70–130 24 71–143 59–155 40
1,2-Dichloroethane 70–130 29 72–137 49–155 49
1,2-Dichloroethane-d4
1,1-Dichloroethene 50–150 40 19–212 D–234 32
1,1-Dichloroethene-d2
trans-1,2-Dichloroethene 70–130 27 68–143 54–156 45
1,2-Dichloropropane 35–165 69 19–181 D–210 55
1,2-Dichloropropane-d6
cis-1,3-Dichloropropene 25–175 79 5–195 D–227 58
trans-1,3-Dichloropropene 50–150 52 38–162 17–183 86
trans-1,3-Dichloropropene-d4
Ethyl benzene 60–140 34 75–134 37–162 63
2-Hexanone-d5
Methylene chloride 60–140 192 D–205 D–221 28
1,1,2,2-Tetrachloroethane 60–140 36 68–136 46–157 61
1,1,2,2-Tetrachloroethane-d2
Tetrachloroethene 70–130 23 65–133 64–148 39
Toluene 70–130 22 75–134 47–150 41
Toluene-d8
1,1,1-Trichloroethane 70–130 21 69–151 52–162 36
1,1,2-Trichloroethane 70–130 27 75–136 52–150 45
Trichloroethene 65–135 29 75–138 70–157 48
Trichlorofluoromethane 50–150 50 45–158 17–181 84
Vinyl chloride 5–195 100 D–218 D–251 66
Vinyl chloride-d3

Table 8—Recovery and Precision as Functions of Concentration

Analyte Recovery, X′
(µg/L)
Single analyst precision, sr
(µg/L)
Overall
precision, S′
(µg/L)
Benzene 0.93C+2.00 20.26 X−1.74 0.25 X−1.33
Bromodichloromethane 1.03C−1.58 0.15 X+0.59 0.20 X+1.13
Bromoform 1.18C−2.35 0.12 X+0.36 0.17 X+1.38
Bromomethanea 1.00C 0.43 X 0.58 X
Carbon tetrachloride 1.10C−1.68 0.12 X+0.25 0.11 X+0.37
Chlorobenzene 0.98C+2.28 0.16 X−0.09 0.26 X−1.92
Chloroethane 1.18C+0.81 0.14 X+2.78 0.29 X+1.75
2-Chloroethylvinyl ethera 1.00C 0.62 X 0.84 X
Chloroform 0.93C+0.33 0.16 X+0.22 0.18 X+0.16
Chloromethane 1.03C+0.81 0.37 X+2.14 0.58 X+0.43
Dibromochloromethane 1.01C−0.03 0.17 X−0.18 0.17 X+0.49
1,2-Dichlorobenzeneb 0.94C+4.47 0.22 X−1.45 0.30 X−1.20
1,3-Dichlorobenzene 1.06C+1.68 0.14 X−0.48 0.18 X−0.82
1,4-Dichlorobenzeneb 0.94C+4.47 0.22 X−1.45 0.30 X−1.20
1,1-Dichloroethane 1.05C+0.36 0.13 X−0.05 0.16 X+0.47
1,2-Dichloroethane 1.02C+0.45 0.17 X−0.32 0.21 X−0.38
1,1-Dichloroethene 1.12C+0.61 0.17 X+1.06 0.43 X−0.22
trans-1,2,-Dichloroethene 1.05C+0.03 0.14 X−+0.09 0.19 X−+0.17
1,2-Dichloropropanea 1.00C 0.33 X 0.45 X
cis-1,3-Dichloropropenea 1.00C 0.38 X 0.52 X
trans-1,3-Dichloropropenea 1.00C 0.25 X 0.34 X
Ethyl benzene 0.98C+2.48 0.14 X+1.00 0.26 X−1.72
Methylene chloride 0.87C+1.88 0.15 X+1.07 0.32 X+4.00
1,1,2,2-Tetrachloroethane 0.93C+1.76 0.16 X+0.69 0.20 X+0.41
Tetrachloroethene 1.06C+0.60 0.13 X−0.18 0.16 X−0.45
Toluene 0.98C+2.03 0.15 X−0.71 0.22 X−1.71
1,1,1-Trichloroethane 1.06C+0.73 0.12 X−0.15 0.21 X−0.39
1,1,2-Trichloroethane 0.95C+1.71 0.14 X+0.02 0.18 X+0.00
Trichloroethene 1.04C+2.27 0.13 X+0.36 0.12 X+0.59
Trichlorofluoromethane 0.99C+0.39 0.33 X−1.48 0.34 X−0.39
Vinyl chloride 1.00C 0.48 X 0.65 X

19. Glossary

These definitions and purposes are specific to this method, but have been conformed to common usage to the extent possible.

19.1 Units of weight and measure and their abbreviations.

19.1.1 Symbols.

°C degrees Celsius

µg microgram

µL microliter

< less than

> greater than

% percent

19.1.2 Abbreviations (in alphabetical order).

cm centimeter

g gram

h hour

ID inside diameter

in. inch

L liter

m mass

mg milligram

min minute

mL milliliter

mm millimeter

ms millisecond

m/z mass-to-charge ratio

N normal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of solution

ng nanogram

pg picogram

ppb part-per-billion

ppm part-per-million

ppt part-per-trillion

psig pounds-per-square inch gauge

v/v volume per unit volume

w/v weight per unit volume

19.2 Definitions and acronyms (in alphabetical order).

Analyte—A compound tested for by this method. The analytes are listed in Tables 1 and 2.

Analyte of interest—An analyte of interest is an analyte required to be determined by a regulatory/control authority or in a permit, or by a client.

Analytical batch—The set of samples analyzed on a given instrument during a 12-hour period that begins with analysis of a calibration verification/LCS. See section 8.4.

Blank—An aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples. The blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus. See section 8.5.

Calibration—The process of determining the relationship between the output or response of a measuring instrument and the value of an input standard. Historically, EPA has referred to a multi-point calibration as the “initial calibration,” to differentiate it from a single-point calibration verification.

Calibration standard—A solution prepared from stock solutions and/or a secondary standards and containing the analytes of interest, surrogates, and internal standards. The calibration standard is used to calibrate the response of the GC/MS instrument against analyte concentration.

Calibration verification standard—The laboratory control sample (LCS) used to verify calibration. See Section 8.4.

Descriptor—In SIM, the beginning and ending retention times for the RT window, the m/z's sampled in the RT window, and the dwell time at each m/z.

Extracted ion current profile (EICP)—The line described by the signal at a given m/z.

Field duplicates—Two samples collected at the same time and place under identical conditions, and treated identically throughout field and laboratory procedures. Results of analyses of field duplicates provide an estimate of the precision associated with sample collection, preservation, and storage, as well as with laboratory procedures.

Field blank—An aliquot of reagent water or other reference matrix that is placed in a sample container in the field, and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to determine if the field or sample transporting procedures and environments have contaminated the sample.

GC—Gas chromatograph or gas chromatography.

Internal standard—A compound added to a sample in a known amount and used as a reference for quantitation of the analytes of interest and surrogates. Internal standards are listed in Table 5. Also see Internal standard quantitation.

Internal standard quantitation—A means of determining the concentration of an analyte of interest (Tables 1 and 2) by reference to a compound added to a sample and not expected to be found in the sample.

DOC—Initial demonstration of capability (DOC; section 8.2); four aliquots of reagent water spiked with the analytes of interest and analyzed to establish the ability of the laboratory to generate acceptable precision and recovery. A DOC is performed prior to the first time this method is used and any time the method or instrumentation is modified.

Laboratory control sample (LCS; laboratory fortified blank (LFB); on-going precision and recovery sample; OPR)—An aliquot of reagent water spiked with known quantities of the analytes of interest and surrogates. The LCS is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified in this method for precision and recovery. In this method, the LCS is synonymous with a calibration verification sample (See sections 7.4 and 8.4).

Laboratory fortified sample matrix—See Matrix spike.

Laboratory reagent blank—See Blank.

Matrix spike (MS) and matrix spike duplicate (MSD) (laboratory fortified sample matrix and duplicate)—Two aliquots of an environmental sample to which known quantities of the analytes of interest and surrogates are added in the laboratory. The MS/MSD are prepared and analyzed exactly like a field sample. Their purpose is to quantify any additional bias and imprecision caused by the sample matrix. The background concentrations of the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the MS/MSD corrected for background concentrations.

May—This action, activity, or procedural step is neither required nor prohibited.

May not—This action, activity, or procedural step is prohibited.

Method blank (laboratory reagent blank)—See Blank.

Method detection limit (MDL)—A detection limit determined by the procedure at 40 CFR part 136, appendix B. The MDLs determined by EPA in the original version of the method are listed in Table 1. As noted in Sec. 1.4, use the MDLs in Table 1 in conjunction with current MDL data from the laboratory actually analyzing samples to assess the sensitivity of this procedure relative to project objectives and regulatory requirements (where applicable).

Minimum level (ML)—The term “minimum level” refers to either the sample concentration equivalent to the lowest calibration point in a method or a multiple of the method detection limit (MDL), whichever is higher. Minimum levels may be obtained in several ways: They may be published in a method; they may be based on the lowest acceptable calibration point used by a laboratory; or they may be calculated by multiplying the MDL in a method, or the MDL determined by a laboratory, by a factor of 3. For the purposes of NPDES compliance monitoring, EPA considers the following terms to be synonymous: “quantitation limit,” “reporting limit,” and “minimum level.”

MS—Mass spectrometer or mass spectrometry.

Must—This action, activity, or procedural step is required.

m/z—The ratio of the mass of an ion (m) detected in the mass spectrometer to the charge (z) of that ion.

Quality control sample (QCS)—A sample containing analytes of interest at known concentrations. The QCS is obtained from a source external to the laboratory or is prepared from standards obtained from a different source than the calibration standards.

The purpose is to check laboratory performance using test materials that have been prepared independent of the normal preparation process.

Reagent water—Water demonstrated to be free from the analytes of interest and potentially interfering substances at the MDLs for the analytes in this method.

Regulatory compliance limit (or regulatory concentration limit)—A limit on the concentration or amount of a pollutant or contaminant specified in a nationwide standard, in a permit, or otherwise established by a regulatory/control authority.

Relative retention time (RRT)—The ratio of the retention time of an analyte to the retention time of its associated internal standard. RRT compensates for small changes in the GC temperature program that can affect the absolute retention times of the analyte and internal standard. RRT is a unitless quantity.

Relative standard deviation (RSD)—The standard deviation times 100 divided by the mean. Also termed “coefficient of variation.”

RF—Response factor. See section 7.3.3.

RSD—See relative standard deviation.

Safety Data Sheet (SDS)—Written information on a chemical's toxicity, health hazards, physical properties, fire, and reactivity, including storage, spill, and handling precautions that meet the requirements of OSHA, 29 CFR 1910.1200(g) and appendix D to § 1910.1200. United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), third revised edition, United Nations, 2009.

Selected Ion Monitoring (SIM)—An MS technique in which a few m/z's are monitored. When used with gas chromatography, the m/z's monitored are usually changed periodically throughout the chromatographic run to correlate with the characteristic m/z's for the analytes, surrogates, and internal standards as they elute from the chromatographic column. The technique is often used to increase sensitivity and minimize interferences.

Signal-to-noise ratio (S/N)—The height of the signal as measured from the mean (average) of the noise to the peak maximum divided by the width of the noise.

SIM—See Selection Ion Monitoring.

Should—This action, activity, or procedural step is suggested but not required.

Stock solution—A solution containing an analyte that is prepared using a reference material traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will attest to the purity and authenticity of the reference material.

Surrogate—A compound unlikely to be found in a sample, and which is spiked into sample in a known amount before purge-and-trap. The surrogate is quantitated with the same procedures used to quantitate the analytes of interest. The purpose of the surrogate is to monitor method performance with each sample.

VOA—Volatile organic analysis: e.g., the analysis performed by this method.

Method 625.1—Base/Neutrals and Acids by GC/MS

1. Scope and Application

1.1 This method is for determination of semivolatile organic pollutants in industrial discharges and other environmental samples by gas chromatography combined with mass spectrometry (GC/MS), as provided under 40 CFR 136.1. This revision is based on a previous protocol (Reference 1), on the basic revision promulgated October 26, 1984, and on an interlaboratory method validation study (Reference 2). Although this method was validated through an interlaboratory study conducted in the early 1980s, the fundamental chemistry principles used in this method remain sound and continue to apply.

1.2 The analytes that may be qualitatively and quantitatively determined using this method and their CAS Registry numbers are listed in Tables 1 and 2. The method may be extended to determine the analytes listed in Table 3; however, extraction or gas chromatography of some of these analytes may make quantitative determination difficult. For example, benzidine is subject to oxidative losses during extraction and/or solvent concentration. Under the alkaline conditions of the extraction, alpha-BHC, gamma-BHC, endosulfan I and II, and endrin are subject to decomposition. Hexachlorocyclopentadiene is subject to thermal decomposition in the inlet of the gas chromatograph, chemical reaction in acetone solution, and photochemical decomposition. N-nitrosodiphenylamine and other nitrosoamines may decompose in the gas chromatographic inlet. The sample may be extracted at neutral pH if necessary to overcome these or other decomposition problems that could occur at alkaline or acidic pH. EPA also has provided other methods (e.g., Method 607—Nitrosamines) that may be used for determination of some of these analytes. EPA encourages use of Method 625.1 to determine additional compounds amenable to extraction and GC/MS.

1.3 The large number of analytes in Tables 1–3 of this method makes testing difficult if all analytes are determined simultaneously. Therefore, it is necessary to determine and perform quality control (QC) tests for the “analytes of interest” only. Analytes of interest are those required to be determined by a regulatory/control authority or in a permit, or by a client. If a list of analytes is not specified, the analytes in Tables 1 and 2 must be determined, at a minimum, and QC testing must be performed for these analytes. The analytes in Tables 1 and 2, and some of the analytes in Table 3 have been identified as Toxic Pollutants (40 CFR 401.15), expanded to a list of Priority Pollutants (40 CFR part 423, appendix A).

1.4 In this revision to Method 625, the pesticides and polychlorinated biphenyls (PCBs) have been moved from Table 1 to Table 3 (Additional Analytes) to distinguish these analytes from the analytes required in quality control tests (Tables 1 and 2). QC acceptance criteria for pesticides and PCBs have been retained in Table 6 and may continue to be applied if desired, or if requested or required by a regulatory/control authority or in a permit. Method 608.3 should be used for determination of pesticides and PCBs. However, if pesticides and/or PCBs are to be determined, an additional sample must be collected and extracted using the pH adjustment and extraction procedures specified in Method 608.3. Method 1668C may be useful for determination of PCBs as individual chlorinated biphenyl congeners, and Method 1699 may be useful for determination of pesticides. At the time of writing of this revision, Methods 1668C and 1699 had not been approved for use at 40 CFR part 136. The screening procedure for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) contained in the version of Method 625 promulgated October 26, 1984 has been replaced with procedures for selected ion monitoring (SIM), and 2,3,7,8-TCDD may be determined using the SIM procedures. However, EPA Method 613 or 1613B should be used for analyte-specific determination of 2,3,7,8-TCDD because of the focus of these methods on this compound. Methods 613 and 1613B are approved for use at 40 CFR part 136.

1.5 Method detection limits (MDLs; Reference 3) for the analytes in Tables 1, 2, and 3 are listed in those tables. These MDLs were determined in reagent water (Reference 4). Advances in analytical technology, particularly the use of capillary (open-tubular) columns, allowed laboratories to routinely achieve MDLs for the analytes in this method that are 2–10 times lower than those in the version promulgated in 1984. The MDL for an analyte in a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5.1 EPA has promulgated this method at 40 CFR part 136 for use in wastewater compliance monitoring under the National Pollutant Discharge Elimination System (NPDES). The data reporting practices described in section 15.2 are focused on such monitoring needs and may not be relevant to other uses of the method.

1.5.2 This method includes “reporting limits” based on EPA's “minimum level” (ML) concept (see the glossary in section 22). Tables 1, 2, and 3 contain MDL values and ML values for many of the analytes.

1.6 This method is performance-based. It may be modified to improve performance (e.g., to overcome interferences or improve the accuracy of results) provided all performance requirements are met.

1.6.1 Examples of allowed method modifications are described at 40 CFR 136.6. Other examples of allowed modifications specific to this method, including solid-phase extraction (SPE) are described in section 8.1.2.

1.6.2 Any modification beyond those expressly permitted at 40 CFR 136.6 or in section 8.1.2 of this method shall be considered a major modification subject to application and approval of an alternate test procedure under 40 CFR 136.4 and 136.5.

1.6.3 For regulatory compliance, any modification must be demonstrated to produce results equivalent or superior to results produced by this method when applied to relevant wastewaters (section 8.3).

1.7 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each laboratory that uses this method must demonstrate the ability to generate acceptable results using the procedure in Section 8.2.

1.8 Terms and units of measure used in this method are given in the glossary at the end of the method.

2. Summary of Method

2.1 A measured volume of sample, sufficient to meet an MDL or reporting limit, is serially extracted with methylene chloride at pH 11–13 and again at a pH less than 2 using a separatory funnel or continuous liquid/liquid extractor.

2.2 The extract is concentrated to a volume necessary to meet the required compliance or detection limit, and analyzed by GC/MS. Qualitative identification of an analyte in the extract is performed using the retention time and the relative abundance of two or more characteristic masses (m/z's). Quantitative analysis is performed using the internal standard technique with a single characteristic m/z.

3. Contamination and Interferences

3.1 Solvents, reagents, glassware, and other sample processing labware may yield artifacts, elevated baselines, or matrix interferences causing misinterpretation of chromatograms and mass spectra. All materials used in the analysis must be demonstrated to be free from contamination and interferences by analyzing blanks initially and with each extraction batch (samples started through the extraction process in a given 24-hour period, to a maximum of 20 samples—see Glossary for detailed definition), as described in Section 8.5. Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Where possible, labware is cleaned by extraction or solvent rinse, or baking in a kiln or oven.

3.2 Glassware must be scrupulously cleaned (Reference 5). Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and reagent water. The glassware should then be drained dry, and heated at 400 °C for 15–30 minutes. Some thermally stable materials, such as PCBs, may require higher temperatures and longer baking times for removal. Solvent rinses with pesticide quality acetone, hexane, or other solvents may be substituted for heating. Do not heat volumetric labware above 90 °C. After drying and cooling, store inverted or capped with solvent-rinsed or baked aluminum foil in a clean environment to prevent accumulation of dust or other contaminants.

3.3 Matrix interferences may be caused by contaminants co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. Interferences extracted from samples high in total organic carbon (TOC) may result in elevated baselines, or by enhancing or suppressing a signal at or near the retention time of an analyte of interest. Analyses of the matrix spike and duplicate (section 8.3) may be useful in identifying matrix interferences, and gel permeation chromatography (GPC; Section 11.1) and sulfur removal (section 11.2) may aid in eliminating these interferences. EPA has provided guidance that may aid in overcoming matrix interferences (Reference 6).

3.4 In samples that contain an inordinate number of interferences, the use of chemical ionization (CI) or triple quadrupole (MRM) mass spectrometry may make identification easier. Tables 4 and 5 give characteristic CI and MRM m/z's for many of the analytes covered by this method. The use of CI or MRM mass spectrometry may be utilized to support electron ionization (EI) mass spectrometry or as a primary method for identification and quantification. While the use of these enhanced techniques is encouraged, it is not required.

4. Safety

4.1 Hazards associated with each reagent used in this method have not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of safety data sheets (SDSs, OSHA, 29 CFR 1910.1200(g)) should also be made available to all personnel involved in sample handling and chemical analysis. Additional references to laboratory safety are available and have been identified (References 7–9) for the information of the analyst.

4.2 The following analytes covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: Benzo(a)anthracene, benzidine, 3,3′-dichlorobenzidine, benzo(a)pyrene, alpha-BHC, beta-BHC, delta-BHC, gamma-BHC, Dibenz(a,h)-anthracene, N-nitrosodimethylamine, 4,4′-DDT, and PCBs. Other compounds in Table 3 may also be toxic. Primary standards of toxic compounds should be prepared in a chemical fume hood, and a NIOSH/MESA approved toxic gas respirator should be worn when handling high concentrations of these compounds.

4.3 This method allows the use of hydrogen as a carrier gas in place of helium (section 5.6.1.2). The laboratory should take the necessary precautions in dealing with hydrogen, and should limit hydrogen flow at the source to prevent buildup of an explosive mixture of hydrogen in air.

5. Apparatus and Materials

Note:

Brand names, suppliers, and part numbers are for illustration purposes only. No endorsement is implied. Equivalent performance may be achieved using equipment and materials other than those specified here. Demonstrating that the equipment and supplies used in the laboratory achieves the required performance is the responsibility of the laboratory. Suppliers for equipment and materials in this method may be found through an on-line search. Please do not contact EPA for supplier information.

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab sample bottle—amber glass bottle large enough to contain the necessary sample volume, fitted with a fluoropolymer-lined screw cap. Foil may be substituted for fluoropolymer if the sample is not corrosive. If amber bottles are not available, protect samples from light. Unless pre-cleaned, the bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2 Automatic sampler (optional)—the sampler must incorporate a pre-cleaned glass sample container. Samples must be kept refrigerated at ≤6 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, rinse the compressible tubing with methanol, followed by repeated rinsing with reagent water, to minimize the potential for sample contamination. An integrating flow meter is required to collect flow-proportioned composites.

5.2 Glassware.

5.2.1 Separatory funnel—Size appropriate to hold sample volume and extraction solvent volume, and equipped with fluoropolymer stopcock.

5.2.2 Drying column—Chromatographic column, approximately 400 mm long by 19 mm ID, with coarse frit, or equivalent, sufficient to hold 15 g of anhydrous sodium sulfate.

5.2.3 Concentrator tube, Kuderna-Danish—10 mL, graduated (Kontes 570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. A ground glass stopper is used to prevent evaporation of extracts.

5.2.4 Evaporative flask, Kuderna-Danish—500 mL (Kontes 57001–0500 or equivalent). Attach to concentrator tube with springs.

Note:

Use of a solvent recovery system with the K–D or other solvent evaporation apparatus is strongly recommended.

5.2.5 Snyder column, Kuderna-Danish—Three-ball macro (Kontes 503000–0121 or equivalent).

5.2.6 Snyder column, Kuderna-Danish—Two-ball micro (Kontes 569001–0219 or equivalent).

5.2.7 Vials—10–15 mL, amber glass, with Teflon-lined screw cap.

5.2.8 Continuous liquid-liquid extractor—Equipped with fluoropolymer or glass connecting joints and stopcocks requiring no lubrication. (Hershberg-Wolf Extractor, Ace Glass Company, Vineland, NJ, P/N 6848–20, or equivalent.)

5.2.9 In addition to the glassware listed above, the laboratory should be equipped with all necessary pipets, volumetric flasks, beakers, and other glassware listed in this method and necessary to perform analyses successfully.

5.3 Boiling chips—Approximately 10/40 mesh, glass, silicon carbide, or equivalent. Heat to 400 °C for 30 minutes, or solvent rinse or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5 Balances.

5.5.1 Analytical, capable of accurately weighing 0.1 mg.

5.5.2 Top loading, capable of accurately weighing 10 mg.

5.6 GC/MS system.

5.6.1 Gas chromatograph (GC)—An analytical system complete with a temperature programmable gas chromatograph and all required accessories, including syringes and analytical columns.

5.6.1.1 Injection port—Can be split, splitless, temperature programmable vaporization split/splitless (PTV), solvent-purge, large-volume, on-column, backflushed, or other. An autosampler is highly recommended because it injects volumes more precisely than volumes injected manually.

5.6.1.2 Carrier gas—Helium or hydrogen. Data in the tables in this method were obtained using helium carrier gas. If hydrogen is used, analytical conditions may need to be adjusted for optimum performance, and calibration and all QC tests must be performed with hydrogen carrier gas. See Section 4.3 for precautions regarding the use of hydrogen as a carrier gas.

5.6.2 GC column—See the footnotes to Tables 4 and 5. Other columns or column systems may be used provided all requirements in this method are met.

5.6.3 Mass spectrometer—Capable of repetitively scanning from 35–450 Daltons (amu) every two seconds or less, utilizing a 70 eV (nominal) electron energy in the electron impact ionization mode, and producing a mass spectrum which meets all the criteria in Table 9A or 9B when 50 ng or less of decafluorotriphenyl phosphine (DFTPP; CAS 5074–71–5; bis(pentafluorophenyl) phenyl phosphine) is injected into the GC.

5.6.4 GC/MS interface—Any GC to MS interface that meets all performance requirements in this method may be used.

5.6.5 Data system—A computer system must be interfaced to the mass spectrometer that allows the continuous acquisition and storage of mass spectra acquired throughout the chromatographic program. The computer must have software that allows searching any GC/MS data file for specific m/z's (masses) and plotting m/z abundances versus time or scan number. This type of plot is defined as an extracted ion current profile (EICP). Software must also be available that allows integrating the abundance at any EICP between specified time or scan number limits.

5.7 Automated gel permeation chromatograph (GPC).

5.7.1 GPC column—150–700 mm long × 21–25 mm ID, packed with 70 g of SX–3 Biobeads; Bio-Rad Labs, or equivalent.

5.7.2 Pump, injection valve, UV detector, and other apparatus necessary to meet the requirements in this method.

5.8 Nitrogen evaporation device—Equipped with a water bath than can be maintained at 30–45 °C; N-Evap, Organomation Associates, or equivalent.

5.9 Muffle furnace or kiln—Capable of baking glassware or sodium sulfate in the range of 400–450 °C.

6. Reagents

6.1 Reagent water—Reagent water is defined as water in which the analytes of interest and interfering compounds are not detected at the MDLs of the analytes of interest.

6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3 Sodium thiosulfate—(ACS) granular.

6.4 Sulfuric acid (1+1)—Slowly add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.5 Acetone, methanol, methylene chloride, 2-propanol—High purity pesticide quality, or equivalent, demonstrated to be free of the analytes of interest and interferences (Section 3). Purification of solvents by distillation in all-glass systems may be required.

6.6 Sodium sulfate—(ACS) granular, anhydrous, rinsed or Soxhlet extracted with methylene chloride (20 mL/g), baked in a shallow tray at 450 °C for one hour minimum, cooled in a desiccator, and stored in a pre-cleaned glass bottle with screw cap that prevents moisture from entering.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions may be prepared from pure materials, or purchased as certified solutions. Traceability must be to the National Institute of Standards and Technology (NIST) or other national or international standard, when available. Stock solution concentrations alternate to those below may be used. Because of the toxicity of some of the compounds, primary dilutions should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations of neat materials are handled. The following procedure may be used to prepare standards from neat materials.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in pesticide quality methanol or other suitable solvent and dilute to volume in a 10-mL volumetric flask. Larger volumes may be used at the convenience of the laboratory. When compound purity is assayed to be 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2 Unless stated otherwise in this method, store non-aqueous standards in fluoropolymer-lined screw-cap, or heat-sealed, glass containers, in the dark at −20 to −10 °C. Store aqueous standards; e.g., the aqueous LCS (section 8.4.1), in the dark at ≤ 6 °C, but do not freeze. Standards prepared by the laboratory may be stored for up to one year, except when comparison with QC check standards indicates that a standard has degraded or become more concentrated due to evaporation, or unless the laboratory has data on file to prove stability for a longer period. Commercially prepared standards may be stored until the expiration date provided by the vendor, except when comparison with QC check standards indicates that a standard has degraded or become more concentrated due to evaporation, or unless the laboratory has data from the vendor on file to prove stability for a longer period.

6.8 Surrogate standard spiking solution.

6.8.1 Select a minimum of three surrogate compounds from Table 8 that most closely match the recovery of the analytes of interest. For example, if all analytes tested are considered acids, use surrogates that have similar chemical attributes. Other compounds may be used as surrogates so long as they do not interfere in the analysis. If only one or two analytes are determined, one or two surrogates may be used.

6.8.2 Prepare a solution containing each selected surrogate such that the concentration in the sample would match the concentration in the mid-point calibration standard. For example, if the midpoint of the calibration is 100 µg/L, prepare the spiking solution at a concentration of 100 µg/mL in methanol. Addition of 1.00 mL of this solution to 1000 mL of sample will produce a concentration of 100 µg/L of the surrogate. Alternate volumes and concentrations appropriate to the response of the GC/MS instrument or for selective ion monitoring (SIM) may be used, if desired. Store per section 6.7.2.

6.9 Internal standard spiking solution.

6.9.1 Select three or more internal standards similar in chromatographic behavior to the analytes of interest. Internal standards are listed in Table 8. Suggested internal standards are: 1,4-dichlorobenzene-d4; naphthalene-d8; acenaphthene-d10; phenanthrene-d10; chrysene-d12; and perylene-d12. The laboratory must demonstrate that measurement of the internal standards is not affected by method or matrix interferences (see also section 7.3.4).

6.9.2 Prepare the internal standards at a concentration of 10 mg/mL in methylene chloride or other suitable solvent. When 10 µL of this solution is spiked into a 1-mL extract, the concentration of the internal standards will be 100 µg/mL. A lower concentration appropriate to the response of the GC/MS instrument or for SIM may be used, if desired. Store per section 6.7.3.

6.9.3 To assure accurate analyte identification, particularly when SIM is used, it may be advantageous to include more internal standards than those suggested in section 6.9.1. An analyte will be located most accurately if its retention time relative to an internal standard is in the range of 0.8 to 1.2.

6.10 DFTPP standard—Prepare a solution of DFTPP in methanol or other suitable solvent such that 50 ng or less will be injected (see section 13.2). An alternative concentration may be used to compensate for specific injection volumes or to assure that the operating range of the instrument is not exceeded, so long as the total injected is 50 ng or less. Include benzidine and pentachlorophenol in this solution such that ≤100 ng of benzidine and ≤50 ng of pentachlorophenol will be injected.

6.11 Quality control check sample concentrate—See section 8.2.1.

6.12 GPC calibration solution.

6.12.1 Prepare a methylene chloride solution to contain corn oil, bis(2-ethylhexyl) phthalate (BEHP), perylene, and sulfur at the concentrations in section 6.12.2, or at concentrations appropriate to the response of the detector.

Note:

Sulfur does not readily dissolve in methylene chloride, but is soluble in warm corn oil. The following procedure is suggested for preparation of the solution.

6.12.2 Weigh 8 mg sulfur and 2.5 g corn oil into a 100-mL volumetric flask and warm to dissolve the sulfur. Separately weigh 100 mg BEHP, 20 mg pentachlorophenol, and 2 mg perylene and add to flask. Bring to volume with methylene chloride and mix thoroughly.

6.12.3 Store the solution in an amber glass bottle with a fluoropolymer-lined screw cap at 0–6 °C. Protect from light. Refrigeration may cause the corn oil to precipitate. Before use, allow the solution to stand at room temperature until the corn oil dissolves, or warm slightly to aid in dissolution. Replace the solution every year, or more frequently if the response of a component changes.

6.13 Sulfur removal—Copper foil or powder (bright, non-oxidized), or tetrabutylammonium sulfite (TBA sulfite).

6.13.1 Copper foil, or powder—Fisher, Alfa Aesar 42455–18, 625 mesh, or equivalent. Cut copper foil into approximately 1-cm squares. Copper must be activated before it may be used, as described below:

6.13.1.1 Place the quantity of copper needed for sulfur removal (section 11.2.1.3) in a ground-glass-stoppered Erlenmeyer flask or bottle. Cover the foil or powder with methanol.

6.13.1.2 Add HCl dropwise (0.5–1.0 mL) while swirling, until the copper brightens.

6.13.1.3 Pour off the methanol/HCl and rinse 3 times with reagent water to remove all traces of acid, then 3 times with acetone, then 3 times with hexane.

6.13.1.4 For copper foil, cover with hexane after the final rinse. Store in a stoppered flask under nitrogen until used. For the powder, dry on a rotary evaporator or under a stream of nitrogen. Store in a stoppered flask under nitrogen until used. Inspect the copper foil or powder before each use. It must have a bright, non-oxidized appearance to be effective. Copper foil or powder that has oxidized may be reactivated using the procedure described above.

6.13.2 Tetrabutylammonium sodium sulfite (TBA sodium sulfite).

6.13.2.1 Tetrabutylammonium hydrogen sulfate, [CH3(CH2)3]4NHSO4.

6.13.2.2 Sodium sulfite, Na2SO3.

6.13.2.3 Dissolve approximately 3 g tetrabutylammonium hydrogen sulfate in 100 mL of reagent water in an amber bottle with fluoropolymer-lined screw cap. Extract with three 20-mL portions of hexane and discard the hexane extracts.

6.13.2.4 Add 25 g sodium sulfite to produce a saturated solution. Store at room temperature. Replace after 1 month.

6.14 DDT and endrin decomposition (breakdown) solution—Prepare a solution containing endrin at a concentration of 1 µg/mL and 4,4′-DDT at a concentration of 2 µg/mL, in isooctane or hexane. A 1-µL injection of this standard will contain 1 nanogram (ng) of endrin and 2 ng of DDT. The concentration of the solution may be adjusted by the laboratory to accommodate other injection volumes such that the same masses of the two analytes are introduced into the instrument.

7. Calibration

7.1 Establish operating conditions equivalent to those in the footnote to Table 4 or 5 for the base/neutral or acid fraction, respectively. If a combined base/neutral/acid fraction will be analyzed, use the conditions in the footnote to Table 4. Alternative temperature program and flow rate conditions may be used. It is necessary to calibrate the GC/MS for the analytes of interest (Section 1.3) only.

7.2 Internal standard calibration.

7.2.1 Prepare calibration standards for the analytes of interest and surrogates at a minimum of five concentration levels by adding appropriate volumes of one or more stock standards to volumetric flasks. One of the calibration standards should be at a concentration at or below the ML specified in Table 1, 2, or 3, or as specified by a regulatory/control authority or in a permit. The ML value may be rounded to a whole number that is more convenient for preparing the standard, but must not exceed the ML in Table 1, 2, or 3 for those analytes which list ML values. Alternatively, the laboratory may establish a laboratory ML for each analyte based on the concentration in a nominal whole-volume sample that is equivalent to the concentration of the lowest calibration standard in a series of standards produced in the laboratory or obtained from a commercial vendor. The laboratory's ML must not exceed the ML in Table 1, 2, or 3, and the resulting calibration must meet the acceptance criteria in Section 7.2.3, based on the RSD, RSE, or R2. The concentrations of the other calibration standards should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system for full-scan and/or SIM operation, as appropriate. A minimum of six concentration levels is required for a second order, non-linear (e.g., quadratic; ax2 + bx + c = 0) calibration (section 7.2.3). Calibrations higher than second order are not allowed. To each calibration standard or standard mixture, add a known constant volume of the internal standard solution (section 6.9), and dilute to volume with methylene chloride.

Note:

The large number of analytes in Tables 1 through 3 may not be soluble or stable in a single solution; multiple solutions may be required if a large number of analytes are to be determined simultaneously.

7.2.1.1 Prior to analysis of the calibration standards, inject the DFTPP standard (Section 6.10) and adjust the scan rate of the mass spectrometer to produce a minimum of 5 mass spectra across the DFTPP GC peak. Adjust instrument conditions until the DFTPP criteria in Table 9A or 9B are met. Calculate peak tailing factors for benzidine and pentachlorophenol. Calculation of the tailing factor is illustrated in Figure 1. The tailing factor for benzidine and pentachlorophenol must be <2; otherwise, adjust instrument conditions and either replace the column or break off a short section of the front end of the column, and repeat the test. Once the scan conditions are established, they must be used for analyses of all standards, blanks, and samples.

Note:

The DFTPP spectrum may be evaluated by summing the intensities of the m/z's across the GC peak, subtracting the background at each m/z in a region of the chromatogram within 20 scans of but not including any part of, the DFTPP peak. The DFTPP spectrum may also be evaluated by fitting a Gaussian to each m/z and using the intensity at the maximum for each Gaussian or by integrating the area at each m/z and using the integrated areas. Other means may be used for evaluation of the DFTPP spectrum so long as the spectrum is not distorted to meet the criteria in Table 9A or 9B.

7.2.1.2 Analyze the mid-point combined base/neutral and acid calibration standard and enter or review the retention time, relative retention time, mass spectrum, and quantitation m/z in the data system for each analyte of interest, surrogate, and internal standard. If additional analytes (Table 3) are to be quantified, include these analytes in the standard. The mass spectrum for each analyte must be comprised of a minimum of 2 m/z's (Tables 4 and 5); 3 to 5 m/z's assure more reliable analyte identification. Suggested quantitation m/z's are shown in Tables 4 and 5 as the primary m/z. If an interference occurs at the primary m/z, use one of the secondary m/z's or an alternate m/z. A single m/z only is required for quantitation.

7.2.1.3 For SIM operation, determine the analytes in each descriptor, the quantitation m/z for each analyte (the quantitation m/z can be the same as for full-scan operation; section 7.2.1.2), the dwell time on each m/z for each analyte, and the beginning and ending retention time for each descriptor. Analyze the verification standard in scan mode to verify m/z's and establish retention times for the analytes. There must be a minimum of two m/z's for each analyte to assure analyte identification. To maintain sensitivity, the number of m/z's in a descriptor should be limited. For example, for a descriptor with 10 m/z's and a chromatographic peak width of 5 sec, a dwell time of 100 ms at each m/z would result in a scan time of 1 second and provide 5 scans across the GC peak. The quantitation m/z will usually be the most intense peak in the mass spectrum. The quantitation m/z and dwell time may be optimized for each analyte. The acquisition table used for SIM must take into account the mass defect (usually less than 0.2 Dalton) that can occur at each m/z monitored. Refer to the footnotes to Table 4 or 5 for establishing operating conditions and to section 7.2.1.1 for establishing scan conditions.

7.2.1.4 For combined scan and SIM operation, set up the scan segments and descriptors to meet requirements in sections 7.2.1.1–7.2.1.3. Analyze unfamiliar samples in the scan mode to assure that the analytes of interest are determined.

7.2.2 Analyze each calibration standard according to section 12 and tabulate the area at the quantitation m/z against concentration for each analyte of interest, surrogate, and internal standard. If an interference is encountered, use a secondary m/z (Table 4 or 5) for quantitation. Calculate a response factor (RF) for each analyte of interest at each concentration using Equation 1.

where:

As = Area of the characteristic m/z for the analyte of interest or surrogate.

Ais = Area of the characteristic m/z for the internal standard.

Cis = Concentration of the internal standard (µg/mL).

Cs = Concentration of the analyte of interest or surrogate (µg/mL).

7.2. Calculate the mean (average) and relative standard deviation (RSD) of the responses factors. If the RSD is less than 35%, the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to fit a linear or quadratic regression of response ratios, As/Ais, vs. concentration ratios Cs/Cis. If used, the regression must be weighted inversely proportional to concentration. The coefficient of determination (R2; Reference 10) of the weighted regression must be greater than 0.920 (this value roughly corresponds to the RSD limit of 35%). Alternatively, the relative standard error (Reference 11) may be used as an acceptance criterion. As with the RSD, the RSE must be less than 35%. If an RSE less than 35% cannot be achieved for a quadratic regression, system performance is unacceptable and the system must be adjusted and re-calibrated.

Note:

Using capillary columns and current instrumentation, it is quite likely that a laboratory can calibrate the target analytes in this method and achieve a linearity metric (either RSD or RSE) well below 35%. Therefore, laboratories are permitted to use more stringent acceptance criteria for calibration than described here, for example, to harmonize their application of this method with those from other sources.

7.3 Calibration verification—The RF or calibration curve must be verified immediately after calibration and at the beginning of each 12-hour shift, by analysis of a standard at or near the concentration of the mid-point calibration standard (section 7.2.1). The standard(s) must be obtained from a second manufacturer or a manufacturer's batch prepared independently from the batch used for calibration. Traceability must be to a national standard, when available. Include the surrogates (section 6.8) in this solution. It is necessary to verify calibration for the analytes of interest (section 1.3) only.

Note:

The 12-hour shift begins after the DFTPP (section 13.1) and DDT/endrin tests (if DDT and endrin are to be determined), and after analysis of the calibration verification standard. The 12-hour shift ends 12 hours later. The DFTPP, DDT/endrin, and calibration verification tests are outside of the 12-hour shift.

7.3.1 Analyze the calibration verification standard(s) beginning in section 12. Calculate the percent recovery of each analyte. Compare the recoveries for the analytes of interest against the acceptance criteria for recovery (Q) in Table 6, and the recoveries for the surrogates against the acceptance criteria in Table 8. If recovery of the analytes of interest and surrogates meet acceptance criteria, system performance is acceptable and analysis of samples may continue. If any individual recovery is outside its limit, system performance is unacceptable for that analyte.

Note:

The large number of analytes in Tables 6 and 8 present a substantial probability that one or more will fail acceptance criteria when all analytes are tested simultaneously.

7.3.2 When one or more analytes fail acceptance criteria, analyze a second aliquot of the calibration verification standard and compare ONLY those analytes that failed the first test (section 7.3.1) with their respective acceptance criteria. If these analytes now pass, system performance is acceptable and analysis of samples may continue. A repeat failure of any analyte that failed the first test, however, will confirm a general problem with the measurement system. If this occurs, repair the system (section 7.2.1.1) and repeat the test (section 7.3.1), or prepare a fresh calibration standard and repeat the test. If calibration cannot be verified after maintenance or injection of the fresh calibration standard, re-calibrate the instrument.

Note:

If it is necessary to perform a repeat verification test frequently; i.e., perform two tests in order to pass, it may be prudent to perform two injections in succession and review the results, rather than perform one injection, review the results, then perform the second injection if results from the first injection fail. To maintain the validity of the test and re-test, system maintenance and/or adjustment is not permitted between the injections.

7.3.3 Many of the analytes in Table 3 do not have QC acceptance criteria in Table 6, and some of the surrogates in Table 8 do not have acceptance criteria. If calibration is to be verified and other QC tests are to be performed for these analytes, acceptance criteria must be developed and applied. EPA has provided guidance for development of QC acceptance criteria (References 12 and 13). Alternatively, analytes that do not have acceptance criteria in Table 6 or Table 8 may be based on laboratory control charts, or 60 to 140% may be used.

7.3.4 Internal standard responses—Verify that detector sensitivity has not changed by comparing the response of each internal standard in the calibration verification standard (section 7.3) to the response of the respective internal standard in the midpoint calibration standard (section 7.2.1). The peak areas or heights of the internal standards in the calibration verification standard must be within 50% to 200% (1/2 to 2x) of their respective peak areas or heights in the mid-point calibration standard. If not, repeat the calibration verification test using a fresh calibration verification standard (7.3), or perform and document system repair. Subsequent to repair, repeat the calibration verification test (section 7.3.1). If the responses are still not within 50% to 200%, re-calibrate the instrument (section 7.2.2) and repeat the calibration verification test.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and ongoing analysis of spiked samples and blanks to evaluate and document data quality (40 CFR 136.7). The laboratory must maintain records to document the quality of data generated. Results of ongoing performance tests are compared with established QC acceptance criteria to determine if the results of analyses meet performance requirements of this method. When results of spiked samples do not meet the QC acceptance criteria in this method, a quality control check sample (laboratory control sample; LCS) must be analyzed to confirm that the measurements were performed in an in-control mode of operation. A laboratory may develop its own performance criteria (as QC acceptance criteria), provided such criteria are as or more restrictive than the criteria in this method.

8.1.1 The laboratory must make an initial demonstration of capability (DOC) to generate acceptable precision and recovery with this method. This demonstration is detailed in Section 8.2.

8.1.2 In recognition of advances that are occurring in analytical technology, and to overcome matrix interferences, the laboratory is permitted certain options (section 1.6 and 40 CFR 136.6(b)) to improve separations or lower the costs of measurements. These options may include alternate extraction, concentration, and cleanup procedures (e.g., solid-phase extraction; rotary-evaporator concentration; column chromatography cleanup), changes in column and type of mass spectrometer (40 CFR 136.6(b)(4)(xvi)). Alternate determinative techniques, such as substitution of spectroscopic or immunoassay techniques, and changes that degrade method performance, are not allowed. If an analytical technique other than GC/MS is used, that technique must have a specificity equal to or greater than the specificity of GC/MS for the analytes of interest. The laboratory is also encouraged to participate in inter-comparison and performance evaluation studies (see section 8.10).

8.1.2.1 Each time a modification is made to this method, the laboratory is required to repeat the procedure in section 8.2. If the detection limit of the method will be affected by the change, the laboratory must demonstrate that the MDLs (40 CFR part 136, appendix B) are lower than one-third the regulatory compliance limit or the MDLs in this method, whichever are greater. If calibration will be affected by the change, the instrument must be recalibrated per section 7. Once the modification is demonstrated to produce results equivalent or superior to results produced by this method, that modification may be used routinely thereafter, so long as the other requirements in this method are met (e.g., matrix spike/matrix spike duplicate recovery and relative percent difference).

8.1.2.1.1 If SPE, or another allowed method modification, is to be applied to a specific discharge, the laboratory must prepare and analyze matrix spike/matrix spike duplicate (MS/MSD) samples (section 8.3) and LCS samples (section 8.4). The laboratory must include surrogates (section 8.7) in each of the samples. The MS/MSD and LCS samples must be fortified with the analytes of interest (Section 1.3). If the modification is for nationwide use, MS/MSD samples must be prepared from a minimum of nine different discharges (See section 8.1.2.1.2), and all QC acceptance criteria in this method must be met. This evaluation only needs to be performed once other than for the routine QC required by this method (for example it could be performed by the vendor of the SPE materials) but any laboratory using that specific material must have the results of the study available. This includes a full data package with the raw data that will allow an independent reviewer to verify each determination and calculation performed by the laboratory (see section 8.1.2.2.5, items (a)–(q)).

8.1.2.1.2 Sample matrices on which MS/MSD tests must be performed for nationwide use of an allowed modification:

(a) Effluent from a POTW.

(b) ASTM D5905 Standard Specification for Substitute Wastewater.

(c) Sewage sludge, if sewage sludge will be in the permit.

(d) ASTM D1141 Standard Specification for Substitute Ocean Water, if ocean water will be in the permit.

(e) Untreated and treated wastewaters up to a total of nine matrix types (see https://www.epa.gov/eg/industrial-effluent-guidelines for a list of industrial categories with existing effluent guidelines).

(i) At least one of the above wastewater matrix types must have at least one of the following characteristics:

(A) Total suspended solids greater than 40 mg/L.

(B) Total dissolved solids greater than 100 mg/L.

(C) Oil and grease greater than 20 mg/L.

(D) NaCl greater than 120 mg/L.

(E) CaCO3 greater than 140 mg/L.

(ii) Results of MS/MSD tests must meet QC acceptance criteria in Section 8.3.

(f) A proficiency testing (PT) sample from a recognized provider, in addition to tests of the nine matrices (section 8.1.2.1.1).

8.1.2.2 The laboratory is required to maintain records of modifications made to this method. These records include the following, at a minimum:

8.1.2.2.1 The names, titles, and business street addresses, telephone numbers, and email addresses, of the analyst(s) that performed the analyses and modification, and of the quality control officer that witnessed and will verify the analyses and modifications.

8.1.2.2.2 A list of analytes, by name and CAS Registry Number.

8.1.2.2.3 A narrative stating reason(s) for the modifications.

8.1.2.2.4 Results from all quality control (QC) tests comparing the modified method to this method, including:

(a) Calibration (section 7).

(b) Calibration verification (section 7).

(c) Initial demonstration of capability (section 8.2).

(d) Analysis of blanks (section 8.5).

(e) Matrix spike/matrix spike duplicate analysis (section 8.3).

(f) Laboratory control sample analysis (section 8.4).

8.1.2.2.5 Data that will allow an independent reviewer to validate each determination by tracing the instrument output (peak height, area, or other signal) to the final result. These data are to include:

(a) Sample numbers and other identifiers.

(b) Extraction dates.

(c) Analysis dates and times.

(d) Analysis sequence/run chronology.

(e) Sample weight or volume (ssection 10).

(f) Extract volume prior to each cleanup step (sections 10 and 11).

(g) Extract volume after each cleanup step (section 11).

(h) Final extract volume prior to injection (sections 10 and 12).

(i) Injection volume (section 12.2.3).

(j) Sample or extract dilution (section 12.2.3.2).

(k) Instrument and operating conditions.

(l) Column (dimensions, material, etc).

(m) Operating conditions (temperature program, flow rate, etc).

(n) Detector (type, operating conditions, etc).

(o) Chromatograms, mass spectra, and other recordings of raw data.

(p) Quantitation reports, data system outputs, and other data to link the raw data to the results reported.

(q) A written Standard Operating Procedure (SOP).

8.1.2.2.6 Each individual laboratory wishing to use a given modification must perform the start-up tests in section 8.1.2 (e.g., DOC, MDL), with the modification as an integral part of this method prior to applying the modification to specific discharges. Results of the DOC must meet the QC acceptance criteria in Table 6 for the analytes of interest (section 1.3), and the MDLs must be equal to or lower than the MDLs in Tables 1, 2, or 3 for the analytes of interest.

8.1.3 Before analyzing samples, the laboratory must analyze a blank to demonstrate that interferences from the analytical system, labware, and reagents, are under control. Each time a batch of samples is extracted or reagents are changed, a blank must be extracted and analyzed as a safeguard against laboratory contamination. Requirements for the blank are given in section 8.5.

8.1.4 The laboratory must, on an ongoing basis, spike and analyze to monitor and evaluate method and laboratory performance on the sample matrix. The procedure for spiking and analysis is given in section 8.3.

8.1.5 The laboratory must, on an ongoing basis, demonstrate through analysis of a quality control check sample (laboratory control sample, LCS; on-going precision and recovery sample, OPR) that the measurement system is in control. This procedure is given in section 8.4.

8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is given in section 8.9.

8.1.7 The large number of analytes tested in performance tests in this method present a substantial probability that one or more will fail acceptance criteria when many analytes are tested simultaneously, and a re-test is allowed if this situation should occur. If, however, continued re-testing results in further repeated failures, the laboratory must document and report the failures (e.g., as qualifiers on results), unless the failures are not required to be reported as determined by the regulatory/control authority. Results associated with a QC failure for an analyte regulated in a discharge cannot be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee of reporting timely results.

8.2 Initial demonstration of capability (DOC)—To establish the ability to generate acceptable recovery and precision, the laboratory must perform the DOC in sections 8.2.1 through 8.2.6 for the analytes of interest. The laboratory must also establish MDLs for the analytes of interest using the MDL procedure at 40 CFR part 136, appendix B. The laboratory's MDLs must be equal to or lower than those listed in Tables 1, 2, or 3 or lower than one third the regulatory compliance limit, whichever is greater. For MDLs not listed in Tables 4 and 5, the laboratory must determine the MDLs using the MDL procedure at 40 CFR part 136, appendix B under the same conditions used to determine the MDLs for the analytes listed in Tables 1, 2, and 3. All procedures used in the analysis, including cleanup procedures, must be included in the DOC.

8.2.1 For the DOC, a QC check sample concentrate (LCS concentrate) containing each analyte of interest (section 1.3) is prepared in a water-miscible solvent. The QC check sample concentrate must be prepared independently from those used for calibration, but may be from the same source as the second-source standard used for calibration verification (Section 7.3). The concentrate should produce concentrations of the analytes of interest in water at the mid-point of the calibration range, and may be at the same concentration as the LCS (section 8.4). Multiple solutions may be required.

Note:

QC check sample concentrates are no longer available from EPA.

8.2.2 Using a pipet or micro-syringe, prepare four LCSs by adding an appropriate volume of the concentrate to each of four aliquots of reagent water, and mix well. The volume of reagent water must be the same as the volume that will be used for the sample, blank (section 8.5), and MS/MSD (section 8.3). A volume of 1–L and a concentration of 100 µg/L were used to develop the QC acceptance criteria in Table 6. Also add an aliquot of the surrogate spiking solution (section 6.8) to the reagent-water aliquots.

8.2.3 Extract and analyze the four LCSs according to the method beginning in Section 10.

8.2.4 Calculate the average percent recovery (X) and the standard deviation of the percent recovery (s) for each analyte using the four results.

8.2.5 For each analyte, compare s and (X) with the corresponding acceptance criteria for precision and recovery in Table 6. For analytes in Table 3 not listed in Table 6, DOC QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 12 and 13). Alternatively, acceptance criteria for analytes not listed in Table 6 may be based on laboratory control charts. If s and (X) for all analytes of interest meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If any individual s exceeds the precision limit or any individual (X) falls outside the range for recovery, system performance is unacceptable for that analyte.

Note:

The large number of analytes in Tables 1–3 present a substantial probability that one or more will fail at least one of the acceptance criteria when many or all analytes are determined simultaneously. Therefore, the analyst is permitted to conduct a “re-test” as described in section 8.2.6.

8.2.6 When one or more of the analytes tested fail at least one of the acceptance criteria, repeat the test for only the analytes that failed. If results for these analytes pass, system performance is acceptable and analysis of samples and blanks may proceed. If one or more of the analytes again fail, system performance is unacceptable for the analytes that failed the acceptance criteria. Correct the problem and repeat the test (section 8.2). See section 8.1.7 for disposition of repeated failures.

Note:

To maintain the validity of the test and re-test, system maintenance and/or adjustment is not permitted between this pair of tests.

8.3 Matrix spike and matrix spike duplicate (MS/MSD)—The purpose of the MS/MSD requirement is to provide data that demonstrate the effectiveness of the method as applied to the samples in question by a given laboratory, and both the data user (discharger, permittee, regulated entity, regulatory/control authority, customer, other) and the laboratory share responsibility for provision of such data. The data user should identify the sample and the analytes of interest (section 1.3) to be spiked and provide sufficient sample volume to perform MS/MSD analyses. The laboratory must, on an ongoing basis, spike at least 5% of the samples in duplicate from each discharge being monitored to assess accuracy (recovery and precision). If direction cannot be obtained from the data user, the laboratory must spike at least one sample in duplicate per extraction batch of up to 20 samples with the analytes in Table 1. Spiked sample results should be reported only to the data user whose sample was spiked, or as requested or required by a regulatory/control authority, or in a permit.

8.3.1 If, as in compliance monitoring, the concentration of a specific analyte will be checked against a regulatory concentration limit, the concentration of the spike should be at that limit; otherwise, the concentration of the spike should be one to five times higher than the background concentration determined in section 8.3.2, at or near the midpoint of the calibration range, or at the concentration in the LCS (section 8.4) whichever concentration would be larger.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the each analyte of interest. If necessary, prepare a new check sample concentrate (section 8.2.1) appropriate for the background concentration. Spike and analyze two additional sample aliquots, and determine the concentration after spiking (A1 and A2) of each analyte. Calculate the percent recoveries (P1 and P2) as 100 (A1 − B)/T and 100 (A2 − B)/T, where T is the known true value of the spike. Also calculate the relative percent difference (RPD) between the concentrations (A1 and A2) as 200 |A1 − A2|/(A1 + A2). If necessary, adjust the concentrations used to calculate the RPD to account for differences in the volumes of the spiked aliquots.

8.3.3 Compare the percent recoveries (P1 and P2) and the RPD for each analyte in the MS/MSD aliquots with the corresponding QC acceptance criteria in Table 6. A laboratory may develop and apply QC acceptance criteria more restrictive than the criteria in Table 6, if desired.

8.3.3.1 If any individual P falls outside the designated range for recovery in either aliquot, or the RPD limit is exceeded, the result for the analyte in the unspiked sample is suspect. See Section 8.1.7 for disposition of failures.

8.3.3.2 The acceptance criteria in Table 6 were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the spike to background ratio approaches 5:1 (Reference 14) and is applied to spike concentrations of 100 µg/L and higher. If spiking is performed at a concentration lower than 100 µg/L, the laboratory must use the QC acceptance criteria in Table 6, the optional QC acceptance criteria calculated for the specific spike concentration in Table 7, or optional in-house criteria (section 8.3.4). To use the acceptance criteria in Table 7:

(1) Calculate recovery (X′) using the equation in Table 7, substituting the spike concentration (T) for C;

(2) Calculate overall precision (S′) using the equation in Table 7, substituting X′ for X;

(3) Calculate the range for recovery at the spike concentration as (100 X′/T) ± 2.44(100 S′/T)% (Reference 14). For analytes in Table 3 not listed in Table 6, QC acceptance criteria must be developed by the laboratory. EPA has provided guidance for development of QC acceptance criteria (References 12 and 13). Alternatively, acceptance criteria may be based on laboratory control charts.

8.3.4 After analysis of a minimum of 20 MS/MSD samples for each target analyte and surrogate, and if the laboratory chooses to develop and apply the optional in-house QC limits (Section 8.3.3), the laboratory should calculate and apply the optional in-house QC limits for recovery and RPD of future MS/MSD samples (Section 8.3). The QC limits for recovery are calculated as the mean observed recovery ±3 standard deviations, and the upper QC limit for RPD is calculated as the mean RPD plus 3 standard deviations of the RPDs. The in-house QC limits must be updated at least every two years and re-established after any major change in the analytical instrumentation or process. If in-house QC limits are developed, at least 80% of the analytes tested in the MS/MSD must have in-house QC acceptance criteria that are tighter than those in Table 6, and the remaining analytes (those other than the analytes included in the 80%) must meet the acceptance criteria in Table 6. If an in-house QC limit for the RPD is greater than the limit in Table 6, then the limit in Table 6 must be used. Similarly, if an in-house lower limit for recovery is below the lower limit in Table 6, then the lower limit in Table 6 must be used, and if an in-house upper limit for recovery is above the upper limit in Table 6, then the upper limit in Table 6 must be used.

8.4 Laboratory control sample (LCS)—A QC check sample (laboratory control sample, LCS; on-going precision and recovery sample, OPR) containing each analyte of interest (Section 1.3) and surrogate must be prepared and analyzed with each extraction batch of up to 20 samples to demonstrate acceptable recovery of the analytes of interest from a clean sample matrix.

8.4.1 Prepare the LCS by adding QC check sample concentrate (section 8.2.1) to reagent water. Include all analytes of interest (section 1.3) in the LCS. The LCS may be the same sample prepared for the DOC (section 8.2.1). The volume of reagent water must be the same as the volume used for the sample, blank (section 8.5), and MS/MSD (Section 8.3). Also add an aliquot of the surrogate spiking solution (section 6.8). The concentration of the analytes in reagent water should be the same as the concentration in the DOC (section 8.2.2).

8.4.2 Analyze the LCS prior to analysis of field samples in the extraction batch. Determine the concentration (A) of each analyte. Calculate the percent recovery (PS) as 100 (A/T)%, where T is the true value of the concentration in the LCS.

8.4.3 Compare the percent recovery (PS) for each analyte with its corresponding QC acceptance criterion in Table 6. For analytes of interest in Table 3 not listed in Table 6, use the QC acceptance criteria developed for the LCS (section 8.4.5), or limits based on laboratory control charts. If the recoveries for all analytes of interest fall within their respective QC acceptance criteria, analysis of blanks and field samples may proceed. If any individual PS falls outside the range, proceed according to section 8.4.4.

Note:

The large number of analytes in Tables 1–3 present a substantial probability that one or more will fail the acceptance criteria when all analytes are tested simultaneously. Because a re-test is allowed in event of failure (sections 8.1.7 and 8.4.3), it may be prudent to extract and analyze two LCSs together and evaluate results of the second analysis against the QC acceptance criteria only if an analyte fails the first test.

8.4.4 Repeat the test only for those analytes that failed to meet the acceptance criteria (PS). If these analytes now pass, system performance is acceptable and analysis of blanks and samples may proceed. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, repeat the test using a fresh LCS (section 8.2.2) or an LCS prepared with a fresh QC check sample concentrate (section 8.2.1), or perform and document system repair. Subsequent to analysis of the LCS prepared with a fresh sample concentrate, or to system repair, repeat the LCS test (section 8.4). If failure of the LCS indicates a systemic problem with samples in the batch, re-extract and re-analyze the samples in the batch. See section 8.1.7 for disposition of repeated failures.

Note:

To maintain the validity of the test and re-test, system maintenance and/or adjustment is not permitted between the pair of tests.

8.4.5 After analysis of 20 LCS samples, and if the laboratory chooses to develop and apply in-house QC limits, the laboratory should calculate and apply in-house QC limits for recovery to future LCS samples (section 8.4). Limits for recovery in the LCS should be calculated as the mean recovery ±3 standard deviations. A minimum of 80% of the analytes tested for in the LCS must have QC acceptance criteria tighter than those in Table 6, and the remaining analytes (those other than the analytes included in the 80%) must meet the acceptance criteria in Table 6. If an in-house lower limit for recovery is lower than the lower limit in Table 6, the lower limit in Table 6 must be used, and if an in-house upper limit for recovery is higher than the upper limit in Table 6, the upper limit in Table 6 must be used. Many of the analytes and surrogates do not contain acceptance criteria. The laboratory should use 60–140% as interim acceptance criteria for recoveries of spiked analytes and surrogates that do not have recovery limits specified in Table 8, and at least 80% of the surrogates must meet the 60–140% interim criteria until in-house LCS and surrogate limits are developed. Alternatively, acceptance criteria for analytes that do not have recovery limits in Table 6 may be based on laboratory control charts. In-house QC acceptance criteria must be updated at least every two years.

8.5 Blank—A blank must be extracted and analyzed with each extraction batch to demonstrate that the reagents and equipment used for preparation and analysis are free from contamination.

8.5.1 Spike the surrogates into the blank. Extract and concentrate the blank using the same procedures and reagents used for the samples, LCS, and MS/MSD in the batch. Analyze the blank immediately after analysis of the LCS (section 8.4) and prior to analysis of the MS/MSD and samples to demonstrate freedom from contamination.

8.5.2 If an analyte of interest is found in the blank: At a concentration greater than the MDL for the analyte, at a concentration greater than one-third the regulatory compliance limit, or at a concentration greater than one-tenth the concentration in a sample in the extraction batch, whichever is greater, analysis of samples must be halted, and the problem corrected. If the contamination is traceable to the extraction batch, samples affected by the blank must be re-extracted and the extracts re-analyzed. If, however, continued re-testing results in repeated blank contamination, the laboratory must document and report the failures (e.g., as qualifiers on results), unless the failures are not required to be reported as determined by the regulatory/control authority. Results associated with blank contamination for an analyte regulated in a discharge cannot be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee of reporting timely results.

8.6 Internal standards responses.

8.6.1 Calibration verification—The responses (GC peak heights or areas) of the internal standards in the calibration verification must be within 50% to 200% (1/2 to 2x) of their respective responses in the mid-point calibration standard. If they are not, repeat the calibration verification (Section 7.4) test or perform and document system repair. Subsequent to repair, repeat the calibration verification. If the responses are still not within 50% to 200%, re-calibrate the instrument (Section 7) and repeat the calibration verification test.

8.6.2 Samples, blanks, LCSs, and MS/MSDs—The responses (GC peak heights or areas) of each internal standard in each sample, blank, and MS/MSD must be within 50% to 200% (1/2 to 2x) of its respective response in the LCS for the extraction batch. If, as a group, all internal standards are not within this range, perform and document system repair, repeat the calibration verification (section 8.4), and re-analyze the affected samples. If a single internal standard is not within the 50% to 200% range, use an alternate internal standard for quantitation of the analyte referenced to the affected internal standard. It may be necessary to use the data system to calculate a new response factor from calibration data for the alternate internal standard/analyte pair. If an internal standard fails the 50–200% criteria and no analytes are detected in the sample, ignore the failure or report it if required by the regulatory/control authority.

8.7 Surrogate recoveries—The laboratory must evaluate surrogate recovery data in each sample against its in-house surrogate recovery limits. The laboratory may use 60–140% as interim acceptance criteria for recoveries for surrogates not listed in Table 8. At least 80% of the surrogates must meet the 60–140% interim criteria until in-house limits are developed. Alternatively, surrogate recovery limits may be developed from laboratory control charts, but such limits must be at least as restrictive as those in Table 8. Spike the surrogates into all samples, blanks, LCSs, and MS/MSDs. Compare surrogate recoveries against the QC acceptance criteria in Table 8 and/or those developed in section 7.3.3 or 8.4.5. If any recovery fails its criteria, attempt to find and correct the cause of the failure. See section 8.1.7 for disposition of failures.

8.8 DDT and endrin decomposition (breakdown)—If DDT and/or endrin are to be analyzed using this method, the DDT/endrin decomposition test in section 13.8 must be performed to reliably quantify these two pesticides.

8.9 As part of the QC program for the laboratory, control charts or statements of accuracy for wastewater samples must be assessed and records maintained (40 CFR 136.7(c)(1)(viii)). After analysis of five or more spiked wastewater samples as in section 8.3, calculate the average percent recovery (PX) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent interval from PX −2sp to PX +2sp. For example, if PX = 90% and sp = 10%, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each analyte on a regular basis (e.g., after each 5–10 new accuracy measurements). If desired, statements of accuracy for laboratory performance, independent of performance on samples, may be developed using LCSs.

8.10 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1 Collect samples as grab samples in amber or clear glass bottles, or in refrigerated bottles using automatic sampling equipment. If clear glass is used, protect samples from light. Collect 1–L of ambient waters, effluents, and other aqueous samples. If the sensitivity of the analytical system is sufficient, a smaller volume (e.g., 250 mL), but no less than 100 mL, may be used. Conventional sampling practices (Reference 15) should be followed, except that the bottle must not be pre-rinsed with sample before collection. Automatic sampling equipment must be as free as possible of polyvinyl chloride or other tubing or other potential sources of contamination. If needed, collect additional sample(s) for the MS/MSD (section 8.3).

9.2 Ice or refrigerate samples at ≤6 °C from the time of collection until extraction, but do not freeze. If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. Any method suitable for field use may be employed to test for residual chlorine (Reference 16). Add more sodium sulfate if 80 mg/L is insufficient but do not add excess sodium thiosulfate. If sodium thiosulfate interferes in the determination of the analytes, an alternate preservative (e.g., ascorbic acid or sodium sulfite) may be used. If preservative has been added, shake the sample vigorously for one minute. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3 All samples must be extracted within 7 days of collection and sample extracts must be analyzed within 40 days of extraction.

10. Extraction

10.1 This section contains procedures for separatory funnel liquid-liquid extraction (SFLLE) and continuous liquid-liquid extraction (CLLE). SFLLE is faster, but may not be as effective as CLLE for recovery of polar analytes such as phenol. SFLLE is labor intensive and may result in formation of emulsions that are difficult to break. CLLE is less labor intensive, avoids emulsion formation, but requires more time (18–24 hours) and more hood space, and may require more solvent. The procedures assume base-neutral extraction followed by acid extraction. For some matrices and analytes of interest, improved results may be obtained by acid-neutral extraction followed by base extraction. A single acid or base extraction may also be performed. If an extraction scheme alternate to base-neutral followed by acid extraction is used, all QC tests must be performed and all QC acceptance criteria must be met with that extraction scheme as an integral part of this method. Solid-phase extraction (SPE) may be used provided requirements in section 8.1.2 are met.

10.2 Separatory funnel liquid-liquid extraction (SFLLE) and extract concentration.

10.2.1 The SFLLE procedure below assumes a sample volume of 1 L. When a different sample volume is extracted, adjust the volume of methylene chloride accordingly.

10.2.2 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into the separatory funnel. Pipet the surrogate standard spiking solution (section 6.8) into the separatory funnel. If the sample will be used for the LCS or MS or MSD, pipet the appropriate check sample concentrate (section 8.2.1 or 8.3.2) into the separatory funnel. Mix well. Check the pH of the sample with wide-range pH paper and adjust to pH 11–13 with sodium hydroxide solution.

10.2.3 Add 60 mL of methylene chloride to the sample bottle, seal, and shake for approximately 30 seconds to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for two minutes with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool or phase-separation paper, salting, centrifugation, or other physical methods. Collect the methylene chloride extract in a flask. If the emulsion cannot be broken (recovery of <80% of the methylene chloride), transfer the sample, solvent, and emulsion into a continuous extractor and proceed as described in section 10.3.

10.2.4 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.2.5 Adjust the pH of the aqueous phase to less than 2 using sulfuric acid. Serially extract the acidified aqueous phase three times with 60 mL aliquots of methylene chloride. Collect and combine the extracts in a flask in the same manner as the base/neutral extracts.

Note:

Base/neutral and acid extracts may be combined for concentration and analysis provided all QC tests are performed and all QC acceptance criteria met for the analytes of interest with the combined extract as an integral part of this method, and provided that the analytes of interest are as reliably identified and quantified as when the extracts are analyzed separately. If doubt exists as to whether identification and quantitation will be affected by use of a combined extract, the fractions must be analyzed separately.

10.2.6 For each fraction or the combined fractions, assemble a Kuderna-Danish (K–D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K–D concentrator so long as the requirements in section 8.2 are met.

10.2.7 For each fraction or the combined fractions, pour the extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K–D concentrator. Rinse the Erlenmeyer flask and column with 20–30 mL of methylene chloride to complete the quantitative transfer.

10.2.8 Add one or two clean boiling chips and attach a three-ball Snyder column to the evaporative flask for each fraction (section 10.2.7). Pre-wet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K–D apparatus on a hot water bath (60–65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15–20 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL or other determined amount, remove the K–D apparatus from the water bath and allow to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1–2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. If the sample will be cleaned up, reserve the K–D apparatus for concentration of the cleaned up extract. Adjust the volume to 5 mL with methylene chloride and proceed to section 11 for cleanup; otherwise, further concentrate the extract for GC/MS analysis per section 10.2.9 or 10.2.10.

10.2.9 Micro Kuderna-Danish concentration—Add another one or two clean boiling chips to the concentrator tube for each fraction and attach a two-ball micro-Snyder column. Pre-wet the Snyder column by adding about 0.5 mL of methylene chloride to the top. Place the K–D apparatus on a hot water bath (60–65 °C) so that the concentrator tube is partially immersed in hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5–10 minutes. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches about 1 mL or other determined amount, remove the K–D apparatus from the water bath and allow it to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with approximately 0.2 mL of or methylene chloride. Adjust the final volume to 1.0 mL or a volume appropriate to the sensitivity desired (e.g., to meet lower MDLs or for selected ion monitoring). Record the volume, stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extracts will be stored longer than two days, they should be transferred to fluoropolymer-lined screw-cap vials and labeled base/neutral or acid fraction as appropriate. Mark the level of the extract on the vial so that solvent loss can be detected.

10.2.10 Nitrogen evaporation and solvent exchange—Extracts may be concentrated for analysis using nitrogen evaporation in place of micro K–D concentration (section 10.2.9). Extracts that have been cleaned up using sulfur removal (section 11.2) and are ready for analysis are exchanged into methylene chloride.

10.2.10.1 Transfer the vial containing the sample extract to the nitrogen evaporation (blowdown) device (section 5.8). Lower the vial into the water bath and begin concentrating. If the more volatile analytes (section 1.2) are to be concentrated, use room temperature for concentration; otherwise, a slightly elevated (e.g., 30–45 °C) may be used. During the solvent evaporation process, keep the solvent level below the water level of the bath and do not allow the extract to become dry. Adjust the flow of nitrogen so that the surface of the solvent is just visibly disturbed. A large vortex in the solvent may cause analyte loss.

10.2.10.2 Extracts to be solvent exchanged—When the volume of the liquid is approximately 200 µL, add 2 to 3 mL of methylene chloride and continue concentrating to approximately 100 µL. Repeat the addition of solvent and concentrate once more. Adjust the final extract volume to be consistent with the volume extracted and the sensitivity desired.

10.2.10.3 For extracts that have been cleaned up by GPC and that are to be concentrated to a nominal volume of 1 mL, adjust the final volume to compensate the GPC loss. For a 50% GPC loss, concentrate the extract to 1/2000 of the volume extracted. For example, if the volume extracted is 950 mL, adjust the final volume to 0.48 mL. For extracts that have not been cleaned up by GPC and are to be concentrated to a nominal volume of 1.0 mL, adjust the final extract volume to 1/1000 of the volume extracted. For example, if the volume extracted is 950 mL, adjust the final extract volume to 0.95 mL. Alternative means of compensating the loss during GPC are acceptable so long as they produce results as accurate as results produced using the procedure detailed in this Section. An alternative final volume may be used, if desired, and the calculations adjusted accordingly.

Note:

The difference in the volume fraction for an extract cleaned up by GPC accounts for the loss in GPC cleanup. Also, by preserving the ratio between the volume extracted and the final extract volume, the concentrations and detection limits do not need to be adjusted for differences in the volume extracted and the extract volume.

10.2.11 Transfer the concentrated extract to a vial with fluoropolymer-lined cap. Seal the vial and label with the sample number. Store in the dark at room temperature until ready for GC analysis. If GC analysis will not be performed on the same day, store the vial in the dark at ≤6 °C. Analyze the extract by GC/MS per the procedure in section 12.

10.2.12 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to an appropriately sized graduated cylinder. For sample volumes on the order of 1000 mL, record the sample volume to the nearest 10 mL; for sample volumes on the order of 100 mL, record the volume to the nearest 1 mL. Sample volumes may also be determined by weighing the container before and after filling to the mark with water.

10.3 Continuous liquid/liquid extraction (CLLE).

Note:

With CLLE, phenol, 2,4-dimethyl phenol, and some other analytes may be preferentially extracted into the base-neutral fraction. Determine an analyte in the fraction in which it is identified and quantified most reliably. Also, the short-chain phthalate esters (e.g., dimethyl phthalate, diethyl phthalate) and some other compounds may hydrolyze during prolonged exposure to basic conditions required for continuous extraction, resulting in low recovery of these analytes. When these analytes are of interest, their recovery may be improved by performing the acid extraction first.

10.3.1 Use CLLE when experience with a sample from a given source indicates an emulsion problem, or when an emulsion is encountered during SFLLE. CLLE may be used for all samples, if desired.

10.3.2 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Check the pH of the sample with wide-range pH paper and adjust to pH 11–13 with sodium hydroxide solution. Transfer the sample to the continuous extractor. Pipet surrogate standard spiking solution (section 6.8) into the sample. If the sample will be used for the LCS or MS or MSD, pipet the appropriate check sample concentrate (section 8.2.1 or 8.3.2) into the extractor. Mix well. Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 seconds to rinse the inner surface. Transfer the solvent to the extractor.

10.3.3 Repeat the sample bottle rinse with an additional 50–100 mL portion of methylene chloride and add the rinse to the extractor.

10.3.4 Add a suitable volume of methylene chloride to the distilling flask (generally 200–500 mL), add sufficient reagent water to ensure proper operation, and extract for 18–24 hours. A shorter or longer extraction time may be used if all QC acceptance criteria are met. Test and, if necessary, adjust the pH of the water during the second or third hour of the extraction. After extraction, allow the apparatus to cool, then detach the distilling flask. Dry, concentrate, and seal the extract per sections 10.2.6 through 10.2.11. See the note at section 10.2.5 regarding combining extracts of the base/neutral and acid fractions.

10.3.5 Charge the distilling flask with methylene chloride and attach it to the continuous extractor. Carefully, while stirring, adjust the pH of the aqueous phase to less than 2 using sulfuric acid. Extract for 18–24 hours. A shorter or longer extraction time may be used if all QC acceptance criteria are met. Test and, if necessary, adjust the pH of the water during the second or third hour of the extraction. After extraction, allow the apparatus to cool, then detach the distilling flask. Dry, concentrate, and seal the extract per sections 10.2.6 through 10.2.11. Determine the sample volume per section 10.2.12.

11. Extract Cleanup

Note:

Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater, drinking water). If particular circumstances require the use of a cleanup procedure, the laboratory may use any or all of the procedures below or any other appropriate procedure. Before using a cleanup procedure, the laboratory must demonstrate that the requirements of section 8.1.2 can be met using the cleanup procedure as an integral part of this method.

11.1 Gel permeation chromatography (GPC).

11.1.1 Calibration.

11.1.1.1 Load the calibration solution (section 6.12) into the sample loop.

11.1.1.2 Inject the calibration solution and record the signal from the detector. The elution pattern will be corn oil, bis(2-ethylhexyl) phthalate, pentachlorophenol, perylene, and sulfur.

11.1.1.3 Set the “dump time” to allow >85% removal of the corn oil and >85% collection of the phthalate.

11.1.1.4 Set the “collect time” to the peak minimum between perylene and sulfur.

11.1.1.5 Verify calibration with the calibration solution after every 20 or fewer extracts. Calibration is verified if the recovery of the pentachlorophenol is greater than 85%. If calibration is not verified, recalibrate using the calibration solution, and re-extract and clean up the preceding extracts using the calibrated GPC system.

11.1.2 Extract cleanup—GPC requires that the column not be overloaded. The column specified in this method is designed to handle a maximum of 0.5 g of high molecular weight material in a 5-mL extract. If the extract is known or expected to contain more than 0.5 g, the extract is split into fractions for GPC and the fractions are combined after elution from the column. The solids content of the extract may be obtained gravimetrically by evaporating the solvent from a 50-µL aliquot.

11.1.2.1 Filter the extract or load through the filter holder to remove particulates. Load the extract into the sample loop. The maximum capacity of the column is 0.5–1.0 g. If necessary, split the extract into multiple aliquots to prevent column overload.

11.1.2.2 Elute the extract using the calibration data determined in Section 11.1.1. Collect the eluate in the K–D apparatus reserved in section 10.2.8.

11.1.3 Concentrate the cleaned up extract per sections 10.2.8 and 10.2.9 or 10.2.10.

11.1.4 Rinse the sample loading tube thoroughly with methylene chloride between extracts to prepare for the next sample.

11.1.5 If a particularly dirty extract is encountered, run a methylene chloride blank through the system to check for carry-over.

11.2 Sulfur removal.

Note:

Separate procedures using copper or TBA sulfite are provided in this section for sulfur removal. They may be used separately or in combination, if desired.

11.2.1 Removal with copper (Reference 17).

Note:

If an additional compound (Table 3) is to be determined; sulfur is to be removed; copper will be used for sulfur removal; and a sulfur matrix is known or suspected to be present, the laboratory must demonstrate that the additional compound can be successfully extracted and treated with copper in the sulfur matrix. Some of the additional compounds (Table 3) are known not to be amenable to sulfur removal with copper (e.g., Atrazine and Diazinon).

11.2.1.1 Quantitatively transfer the extract from section 10.2.8 to a 40- to 50-mL flask or bottle. If there is evidence of water in the concentrator tube after the transfer, rinse the tube with small portions of hexane:acetone (40:60) and add to the flask or bottle. Mark and set aside the concentrator tube for use in re-concentrating the extract.

11.2.1.2 Add 10–20 g of granular anhydrous sodium sulfate to the flask. Swirl to dry the extract.

11.2.1.3 Add activated copper (section 6.13.1.4) and allow to stand for 30—60 minutes, swirling occasionally. If the copper does not remain bright, add more and swirl occasionally for another 30–60 minutes.

11.2.1.4 After drying and sulfur removal, quantitatively transfer the extract to a nitrogen-evaporation vial or tube and proceed to section 10.2.10 for nitrogen evaporation and solvent exchange, taking care to leave the sodium sulfate and copper in the flask.

11.2.2 Removal with TBA sulfite.

11.2.2.1 Using small volumes of hexane, quantitatively transfer the extract to a 40- to 50-mL centrifuge tube with fluoropolymer-lined screw cap.

11.2.2.2 Add 1–2 mL of TBA sulfite reagent (section 6.13.2.4), 2–3 mL of 2-propanol, and approximately 0.7 g of sodium sulfite (section 6.13.2.2) crystals to the tube. Cap and shake for 1–2 minutes. If the sample is colorless or if the initial color is unchanged, and if clear crystals (precipitated sodium sulfite) are observed, sufficient sodium sulfite is present. If the precipitated sodium sulfite disappears, add more crystalline sodium sulfite in approximately 0.5 g portions until a solid residue remains after repeated shaking.

11.2.2.3 Add 5–10 mL of reagent water and shake for 1–2 minutes. Centrifuge to settle the solids.

11.2.2.4 Quantitatively transfer the hexane (top) layer through a small funnel containing a few grams of granular anhydrous sodium sulfate to a nitrogen-evaporation vial or tube and proceed to section 10.2.10 for nitrogen evaporation and solvent exchange.

12. Gas Chromatography/Mass Spectrometry

12.1 Establish the operating conditions in Table 4 or 5 for analysis of a base/neutral or acid extract, respectively. For analysis of a combined extract (section 10.2.5, note), use the operating conditions in Table 4 MDLs and MLs for the analytes are given in Tables 1, 2, and 3. Retention times for many of the analytes are given in Tables 4 and 5. Examples of the separations achieved are shown in Figure 2 for the combined extract. Alternative columns or chromatographic conditions may be used if the requirements of section 8.2 are met. Verify system performance per section 13.

12.2 Analysis of a standard or extract.

12.2.1 Bring the standard or concentrated extract (section 10.2.9 or 10.2.11) to room temperature and verify that any precipitate has redissolved. Verify the level on the extract and bring to the mark with solvent if required.

12.2.2 Add the internal standard solution (section 6.9) to the extract. Mix thoroughly.

12.2.3 Inject an appropriate volume of the sample extract or standard solution using split, splitless, solvent purge, large-volume, or on-column injection. If the sample is injected manually the solvent-flush technique should be used. The injection volume depends upon the technique used and the ability to meet MDLs or reporting limits for regulatory compliance. Injected volumes must be the same for standards and sample extracts. Record the volume injected to two significant figures.

12.2.3.1 Start the GC column oven program upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after benzo(ghi)perylene elutes for the base/neutral or combined fractions, or after pentachlorophenol elutes for the acid fraction. Return the column to the initial temperature for analysis of the next standard solution or extract.

12.2.3.2 If the concentration of any analyte of interest exceeds the calibration range, either extract and analyze a smaller sample volume, or dilute and analyze the diluted extract after bringing the concentrations of the internal standards to the levels in the undiluted extract.

12.2.4 Perform all qualitative and quantitative measurements as described in Sections 14 and 15. When standards and extracts are not being used for analyses, store them refrigerated at ≤6 °C protected from light in screw-cap vials equipped with un-pierced fluoropolymer-lined septa.

13. Performance Tests

13.1 At the beginning of each 12-hour shift during which standards or extracts will be analyzed, perform the tests in sections 13.2–13.4 to verify system performance. If an extract is concentrated for greater sensitivity (e.g., by SIM), all tests must be performed at levels consistent with the reduced extract volume.

13.2 DFTPP—Inject the DFTPP standard (section 6.10) and verify that the criteria for DFTPP in section 7.2.1.1 and Table 9A (Reference 18) for a quadrupole MS, or Table 9B (Reference 19) for a time-of-flight MS, are met.

13.3 GC resolution—The resolution should be verified on the mid-point concentration of the initial calibration as well as the laboratory designated continuing calibration verification level if closely eluting isomers are to be reported (e.g., benzo(b)fluoranthene and benzo(k)fluoranthene). Sufficient gas chromatographic resolution is achieved if the height of the valley between two isomer peaks is less than 50% of the average of the two peak heights.

13.4 Calibration verification—Verify calibration per sections 7.3 and Table 6.

13.5 Peak tailing—Verify the tailing factor specifications are met per Section 7.2.1.1.

13.6 Laboratory control sample and blank—Analyze the extracts of the LCS and blank at the beginning of analyses of samples in the extraction batch (section 3.1). The LCS must meet the requirements in section 8.4, and the blank must meet the requirements in section 8.5 before sample extracts may be analyzed.

13.7 Analysis of DFTPP, the DDT/Endrin decomposition test (if used), the LCS, and the blank are outside of the 12-hour analysis shift (section 3.1). The total time for DFTPP, DDT/Endrin, the LCS, the blank, and the 12-hour shift must not exceed 15 hours.

13.8 Decomposition of DDT and endrin—If DDT and/or endrin are to be determined, this test must be performed prior to calibration verification (section 13.4). The QC acceptance criteria (section 13.8.3) must be met before analyzing samples for DDE and/or Endrin. DDT decomposes to DDE and DDD. Endrin decomposes to endrin aldehyde and endrin ketone.

13.8.1 Inject 1 µL of the DDT and endrin decomposition solution (section 6.14). As noted in section 6.14, other injection volumes may be used as long as the concentrations of DDT and endrin in the solution are adjusted to introduce the masses of the two analytes into the instrument that are listed in section 6.14.

13.8.2 Measure the areas of the peaks for DDT, DDE, DDD, Endrin, Endrin aldehyde, and Endrin ketone. Calculate the percent breakdown as shown in the equations below:

13.8.3 Both the % breakdown of DDT and of Endrin must be less than 20%, otherwise the system is not performing acceptably for DDT and endrin. In this case, repair the GC column system that failed and repeat the performance tests (sections 13.2 to 13.6) until the specification is met.

Note:

DDT and endrin decomposition are usually caused by accumulation of particulates in the injector and in the front end of the column. Cleaning and silanizing the injection port liner, and breaking off a short section of the front end of the column will usually eliminate the decomposition problem. Either of these corrective actions may affect retention times, GC resolution, and calibration linearity.

14. Qualitative Identification

14.1 Identification is accomplished by comparison of data from analysis of a sample or blank with data stored in the GC/MS data system (sections 5.6.5 and 7.2.1.2). Identification of an analyte is confirmed per sections 14.1.1 through 14.1.4.

14.1.1 The signals for the quantitation and secondary m/z's stored in the data system for each analyte of interest must be present and must maximize within the same two consecutive scans.

14.1.2 The retention time for the analyte should be within ± 10 seconds of the analyte in the calibration verification run at the beginning of the shift (section 7.3 or 13.4).

Note:

Retention time windows other than ± 10 seconds may be appropriate depending on the performance of the gas chromatograph or observed retention time drifts due to certain types of matrix effects. Relative retention time (RRT) may be used as an alternative to absolute retention times if retention time drift is a concern. RRT is a unitless quantity (see Sec. 22.2), although some procedures refer to “RRT units” in providing the specification for the agreement between the RRT values in the sample and the calibration verification or other standard. When significant retention time drifts are observed, dilutions or spiked samples may help the analyst determine the effects of the matrix on elution of the target analytes and to assist in qualitative identification.

14.1.3 Either the background corrected EICP areas, or the corrected relative intensities of the mass spectral peaks at the GC peak maximum, must agree within 50% to 200% (1/2 to 2 times) for the quantitation and secondary m/z's in the reference mass spectrum stored in the data system (section 7.2.1.2), or from a reference library. For example, if a peak has an intensity of 20% relative to the base peak, the analyte is identified if the intensity of the peak in the sample is in the range of 10% to 40% of the base peak. If identification is ambiguous, an experienced spectrometrist (section 1.7) must determine the presence or absence of the compound.

14.2 Structural isomers that produce very similar mass spectra should be identified as individual isomers if they have sufficiently different gas chromatographic retention times. Sufficient gas chromatographic resolution is achieved if the height of the valley between two isomer peaks is less than 50% of the average of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs.

15. Calculations

15.1 When an analyte has been identified, quantitation of that analyte is based on the integrated abundance from the EICP of the primary characteristic m/z in Table 4 or 5. Calculate the concentration in the extract using the response factor (RF) determined in Section 7.2.2 and Equation 2. If the concentration of an analyte exceeds the calibration range, dilute the extract by the minimum amount to bring the concentration into the calibration range, and re-analyze the extract. Determine a dilution factor (DF) from the amount of the dilution. For example, if the extract is diluted by a factor of 2, DF = 2.

where:

Cex = Concentration of the analyte in the extract, in µg/mL, and the other terms are as defined in section 7.2.2.

Calculate the concentration of the analyte in the sample using the concentration in the extract, the extract volume, the sample volume, and the dilution factor, per Equation 3:

where:

Csamp = Concentration of the analyte in the sample

Cex = Concentration of the analyte in the extract, in µg/mL

Vex = Volume of extract (mL)

Vs = Volume of sample (L)

DF = Dilution factor

15.2 Reporting of results. As noted in section 1.4.1, EPA has promulgated this method at 40 CFR part 136 for use in wastewater compliance monitoring under the National Pollutant Discharge Elimination System (NPDES). The data reporting practices described here are focused on such monitoring needs and may not be relevant to other uses of the method.

15.2.1 Report results for wastewater samples in µg/L without correction for recovery. (Other units may be used if required by in a permit.) Report all QC data with the sample results.

15.2.2 Reporting level. Unless specified otherwise by a regulatory authority or in a discharge permit, results for analytes that meet the identification criteria are reported down to the concentration of the ML established by the laboratory through calibration of the instrument (see section 7.3.2 and the glossary for the derivation of the ML). EPA considers the terms “reporting limit,” “quantitation limit,” “limit of quantitation,” and “minimum level” to be synonymous.

15.2.2.1 Report a result for each analyte in each field sample or QC standard at or above the ML to 3 significant figures. Report a result for each analyte found in each field sample or QC standard below the ML as “ML” where ML is the concentration of the analyte at the ML, or as required by the regulatory/control authority or permit. Report a result for each analyte in a blank at or above the MDL to 2 significant figures. Report a result for each analyte found in a blank below the MDL as “MDL,” where MDL is the concentration of the analyte at the MDL, or as required by the regulatory/control authority or permit.

15.2.2.2 In addition to reporting results for samples and blanks separately, the concentration of each analyte in a blank associated with the sample may be subtracted from the result for that sample, but only if requested or required by a regulatory authority or in a permit. In this case, both the sample result and the blank results must be reported together.

15.2.2.3 Report a result for an analyte found in a sample or extract that has been diluted at the least dilute level at which the area at the quantitation m/z is within the calibration range (i.e., above the ML for the analyte) and the MS/MSD recovery and RPD are within their respective QC acceptance criteria (Table 6). This may require reporting results for some analytes from different analyses.

15.2.3 Results from tests performed with an analytical system that is not in control (i.e., that does not meet acceptance criteria for any QC test in this method) must be documented and reported (e.g., as a qualifier on results), unless the failure is not required to be reported as determined by the regulatory/control authority. Results associated with a QC failure cannot be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee of reporting timely results. If the holding time would be exceeded for a re-analysis of the sample, the regulatory/control authority should be consulted for disposition.

16. Method Performance

16.1 The basic version of this method was tested by 15 laboratories using reagent water, drinking water, surface water, and industrial wastewaters spiked at six concentrations over the range 5–1300 µg/L (Reference 2). Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the analyte and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 7.

16.2 As noted in section 1.1, this method was validated through an interlaboratory study in the early 1980s. However, the fundamental chemistry principles used in this method remain sound and continue to apply.

16.3 A chromatogram of the combined acid/base/neutral calibration standard is shown in Figure 2.

17. Pollution Prevention

17.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory operations. EPA has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, the laboratory should use pollution prevention techniques to address waste generation. When wastes cannot be reduced at the source, the Agency recommends recycling as the next best option.

17.2 The analytes in this method are used in extremely small amounts and pose little threat to the environment when managed properly. Standards should be prepared in volumes consistent with laboratory use to minimize the disposal of excess volumes of expired standards. This method utilizes significant quantities of methylene chloride. Laboratories are encouraged to recover and recycle this and other solvents during extract concentration.

17.3 For information about pollution prevention that may be applied to laboratories and research institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Governmental Relations and Science Policy, 1155 16th Street NW., Washington DC 20036, 202–872–4477.

18. Waste Management

18.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations. An overview of requirements can be found in Environmental Management Guide for Small Laboratories (EPA 233–B–98–001).

18.2 Samples at pH <2, or pH >12, are hazardous and must be handled and disposed of as hazardous waste, or neutralized and disposed of in accordance with all federal, state, and local regulations. It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions. The laboratory using this method has the responsibility to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations. For further information on waste management, see “The Waste Management Manual for Laboratory Personnel,” also available from the American Chemical Society at the address in section 17.3.

18.3 Many analytes in this method decompose above 500 ºC. Low-level waste such as absorbent paper, tissues, and plastic gloves may be burned in an appropriate incinerator. Gross quantities of neat or highly concentrated solutions of toxic or hazardous chemicals should be packaged securely and disposed of through commercial or governmental channels that are capable of handling these types of wastes.

18.4 For further information on waste management, consult The Waste Management Manual for Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street NW., Washington, DC 20036, 202–872–4477.

19. References

1. “Sampling and Analysis Procedures for Screening of Industrial Effluents for Priority Pollutants,” U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1977, Revised April 1977.

2. “EPA Method Study 30, Method 625, Base/Neutrals, Acids, and Pesticides,” EPA 600/4–84–053, National Technical Information Service, PB84–206572, Springfield, Virginia 22161, June 1984.

3. 40 CFR part 136, appendix B.

4. Olynyk, P., Budde, W.L. and Eichelberger, J.W. “Method Detection Limit for Methods 624 and 625,” Unpublished report, May 14, 1980.

5. Annual Book of ASTM Standards, Volume 11.02, D3694–96, “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

6. Solutions to Analytical Chemistry Problems with Clean Water Act Methods, EPA 821–R–07–002, March 2007.

7. “Carcinogens-Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.

8. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

9. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 7th Edition, 2003.

10. Johnson, R.A., and Wichern, D.W., “Applied Multivariate Statistical Analysis,” 3rd edition, Prentice Hall, Englewood Cliffs, NJ, 1992.

11. 40 CFR 136.6(b)(4)(x).

12. 40 CFR 136.6(b)(2)(i).

13. Protocol for EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater and Drinking Water (EPA–821–B–98–003) March 1999.

14. Provost, L.P. and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in section 8.3.3 is two times the value 1.22 derived in this report.)

15. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

16. 40 CFR 136.3(a), Table IB, Chlorine—Total Residual.

17. “Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental Samples,” EPA–600/8–80–038, U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina.

18. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chemistry, 47, 995 (1975).

19. Letter of approval of acceptance criteria for DFTPP for time-of-flight mass spectrometers from William A. Telliard and Herb Brass of EPA to Jack Cochran of LECO Corporation, February 9, 2005.

20. Tables

Table 1—Non Pesticide/PCB Base/Neutral Extractables1

Analyte CAS registry MDL4
(ug/L)
ML5
(ug/L)
Acenaphthene 83–32–9 1.9 5.7
Acenaphthylene 208–96–8 3.5 10.5
Anthracene 120–12–7 1.9 5.7
Benzidine2 92–87–5 44 132
Benzo(a)anthracene 56–55–3 7.8 23.4
Benzo(a)pyrene 50–32–8 2.5 7.5
Benzo(b)fluoranthene 205–99–2 4.8 14.4
Benzo(k)fluoranthene 207–08–9 2.5 7.5
Benzo(ghi)perylene 191–24–2 4.1 12.3
Benzyl butyl phthalate 85–68–7 2.5 7.5
bis(2-Chloroethoxy)methane 111–91–1 5.3 15.9
bis(2-Ethylhexyl)phthalate 117–81–7 2.5 7.5
bis(2-Chloroisopropyl) ether (2,2'-Oxybis[1-chloropropane]) 108–60–1 5.7 17.1
4-Bromophenyl phenyl ether 101–55–3 1.9 5.7
2-Chloronaphthalene 91–58–7 1.9 5.7
4-Chlorophenyl phenyl ether 7005–72–3 4.2 12.6
Chrysene 218–01–9 2.5 7.5
Dibenz(a,h)anthracene 53–70–3 2.5 7.5
Di-n-butylphthalate 84–74–2 2.5 7.5
3,3'-Dichlorobenzidine 91–94–1 16.5 49.5
Diethyl phthalate 84–66–2 1.9 5.7
Dimethyl phthalate 131–11–3 1.6 4.8
2,4-Dinitrotoluene 121–14–2 5.7 17.1
2,6-Dinitrotoluene 606–20–2 1.9 5.7
Di-n-octylphthalate 117–84–0 2.5 7.5
Fluoranthene 206–44–0 2.2 6.6
Fluorene 86–73–7 1.9 5.7
Hexachlorobenzene 118–74–1 1.9 5.7
Hexachlorobutadiene 87–68–3 0.9 2.7
Hexachloroethane 67–72–1 1.6 4.8
Indeno(1,2,3-cd)pyrene 193–39–5 3.7 11.1
Isophorone 78–59–1 2.2 6.6
Naphthalene 91–20–3 1.6 4.8
Nitrobenzene 98–95–3 1.9 5.7
N-Nitrosodi-n-propylamine3 621–64–7
Phenanthrene 85–01–8 5.4 16.2
Pyrene 129–00–0 1.9 5.7
1,2,4-Trichlorobenzene 120–82–1 1.9 5.7

Table 2—Acid Extractables1

Analyte CAS registry MDL3
(ug/L)
ML4
(ug/L)
4-Chloro-3-methylphenol 59–50–7 3.0 9.0
2-Chlorophenol 95–57–8 3.3 9.9
2,4-Dichlorophenol 120–83–2 2.7 8.1
2,4-Dimethylphenol 105–67–9 2.7 8.1
2,4-Dinitrophenol 51–28–5 42 126
2-Methyl-4,6-dinitrophenol 534–52–1 24 72
2-Nitrophenol 88–75–5 3.6 10.8
4-Nitrophenol 100–02–7 2.4 7.2
Pentachlorophenol2 87–86–5 3.6 10.8
Phenol 108–95–2 1.5 4.5
2,4,6-Trichlorophenol 88–06–2 2.7 8.1

Table 3—Additional Extractable Analytes1 2

Analyte CAS registry MDL7
(ug/L)
ML8
(ug/L)
Acetophenone 98–86–2
2-Acetylaminofluorene 53–96–3
1-Acetyl-2-thiourea 591–08–2
Alachlor 15972–60–8
Aldrin3 309–00–2 1.9 5.7
Ametryn 834–12–8
2-Aminoanthraquinone 117–79–3
Aminoazobenzene 60–09–3
4-Aminobiphenyl 92–67–1
3-Amino-9-ethylcarbazole 132–32–1
Anilazine 101–05–3
Aniline 62–53–3
o-Anisidine 90–04–0
Aramite 140–57–8
Atraton 1610–17–9
Atrazine 1912–24–9
Azinphos-methyl 86–50–0
Barban 101–27–9
Benzanthrone 82–05–3
Benzenethiol 108–98–5
Benzoic acid 65–85–0
2,3-Benzofluorene 243–17–4
p-Benzoquinone 106–51–4
Benzyl alcohol 100–51–6
alpha-BHC3 4 319–84–6
beta-BHC3 319–85–7 3.1 9.3
gamma-BHC (Lindane)3 4 58–89–8 4.2 12.6
delta-BHC3 319–86–8
Biphenyl 92–52–4
Bromacil 314–40–9
2-Bromochlorobenzene 694–80–4
3-Bromochlorobenzene 108–39–2
Bromoxynil 1689–84–5
Butachlor 2318–4669
Butylate 2008–41–5
n-C10 (n-decane) 124–18–5
n-C12 (n-undecane) 112–40–2
n-C14 (n-tetradecane) 629–59–4
n-C16 (n-hexadecane) 544–76–3
n-C18 (n-octadecane) 593–45–3
n-C20 (n-eicosane) 112–95–8
n-C22 (n-docosane) 629–97–0
n-C24 (n-tetracosane) 646–31–1
n-C26 (n-hexacosane) 630–01–3
n-C28 (n-octacosane) 630–02–4
n-C30 (n-triacontane) 638–68–6
Captafol 2425–06–1
Captan 133–06–2
Carbaryl 63–25–2
Carbazole 86–74–8
Carbofuran 1563–66–2
Carboxin 5234–68 –4
Carbophenothion 786–19–6
Chlordane3 5 57–74–9
bis(2-Chloroethyl) ether3 4 111–44–4 5.7 17.1
Chloroneb 2675–77–6
4-Chloroaniline 106–47–8
Chlorobenzilate 510–15–6
Chlorfenvinphos 470–90–6
4-Chloro-2-methylaniline 95–69–2
3-(Chloromethyl)pyridine hydrochloride 6959–48–4
4-Chloro-2-nitroaniline 89–63–4
Chlorpropham 101–21–3
Chlorothalonil 1897–45–6
1-Chloronaphthalene 90–13–1
3-Chloronitrobenzene 121–73–3
4-Chloro-1,2-phenylenediamine 95–83–0
4-Chloro-1,3-phenylenediamine 5131–60–2
2-Chlorobiphenyl 2051–60–7
Chlorpyrifos 2921–88–2
Coumaphos 56–72–4
m + p-Cresol 65794–96–9
o-Cresol 95–48–7
p-Cresidine 120–71–8
Crotoxyphos 7700–17–6
2-Cyclohexyl-4,6-dinitro-phenol 131–89–5
Cyanazine 21725–46–2
Cycloate 1134–23–2
p-Cymene 99–87–6
Dacthal (DCPA) 1861–32–1
4,4'-DDD3 72–54–8 2.8 8.4
4,4'-DDE3 72–55–9 5.6 16.8
4,4'-DDT3 50–29–3 4.7 14.1
Demeton-O 298–03–3
Demeton-S 126–75–0
Diallate (cis or trans) 2303–16–4
2,4-Diaminotoluene 95–80–7
Diazinon 333–41–5
Dibenz(a,j)acridine 224–42–0
Dibenzofuran 132–64–9
Dibenzo(a,e)pyrene 192–65–4
Dibenzothiophene 132–65–0
1,2-Dibromo-3-chloropropane 96–12–8
3,5-Dibromo-4-hydroxybenzonitrile 1689–84–5
2,6-Di-tert-butyl-p-benzoquinone 719–22–2
Dichlone 117–80–6
2,3-Dichloroaniline 608–27–5
2,3-Dichlorobiphenyl 16605–91–7
2,6-Dichloro-4-nitroaniline 99–30–9
2,3-Dichloronitrobenzene 3209–22–1
1,3-Dichloro-2-propanol 96–23–1
2,6-Dichlorophenol 120–83–2
Dichlorvos 62–73–7
Dicrotophos 141–66–2
Dieldrin3 60–57–1 2.5 7.5
1,2:3,4-Diepoxybutane 1464–53–5
Di(2-ethylhexyl) adipate 103–23–1
Diethylstilbestrol 56–53–1
Diethyl sulfate 64–67–5
Dilantin (5,5-Diphenylhydantoin) 57–41–0
Dimethoate 60–51–5
3,3′-Dimethoxybenzidine 119–90–4
Dimethylaminoazobenzene 60–11–7
7,12-Dimethylbenz(a)anthracene 57–97–6
3,3′-Dimethylbenzidine 119–93–7
N,N-Dimethylformamide 68–12–2
3,6-Dimethylphenathrene 1576–67–6
alpha, alpha-Dimethylphenethylamine 122–09–8
Dimethyl sulfone 67–71–0
1,2-Dinitrobenzene 528–29–0
1,3-Dinitrobenzene 99–65–0
1,4-Dinitrobenzene 100–25–4
Dinocap 39300–45–3
Dinoseb 88–85–7
Diphenylamine 122–39–4
Diphenyl ether 101–84–8
1,2-Diphenylhydrazine 122–66–7
Diphenamid 957–51–7
Diphenyldisulfide 882–33–7
Disulfoton 298–04–4
Disulfoton sulfoxide 2497–07–6
Disulfoton sulfone 2497–06–5
Endosulfan I4 959–98–8
Endosulfan II3 4 33213–65–9
Endosulfan sulfate3 1031–07–8 5.6 16.8
Endrin3 4 72–20–8
Endrin aldehyde3 4 7421–93–4
Endrin ketone3 4 53494–70–5
EPN 2104–64–5
EPTC 759–94–4
Ethion 563–12–2
Ethoprop 13194–48–4
Ethyl carbamate 51–79–6
Ethyl methanesulfonate 65–50–0
Ethylenethiourea 96–45–7
Etridiazole 2593–15–9
Ethynylestradiol-3-methyl ether 72–33–3
Famphur 52–85–7
Fenamiphos 22224–92–6
Fenarimol 60168–88–9
Fensulfothion 115–90–2
Fenthion 55–38–9
Fluchloralin 33245–39–5
Fluridone 59756–60–4
Heptachlor3 76–44–8 1.9 5.7
Heptachlor epoxide3 1024–57–3 2.2 6.6
2,2′,3,3′,4,4′,6-Heptachlorobiphenyl 52663–71–5
2,2′,4,4′,5′,6-Hexachlorobiphenyl 60145–22–4
Hexachlorocyclopentadiene3 4 77–47–4
Hexachlorophene 70–30–4
Hexachloropropene 1888–71–7
Hexamethylphosphoramide 680–31–9
Hexanoic acid 142–62–1
Hexazinone 51235–04–2
Hydroquinone 123–31–9
Isodrin 465–73–6
2-Isopropylnaphthalene 2027–17–0
Isosafrole 120–58–1
Kepone 143–50–0
Leptophos 21609–90–5
Longifolene 475–20–7
Malachite green 569–64–2
Malathion 121–75–5
Maleic anhydride 108–31–6
Merphos 150–50–5
Mestranol 72–33–3
Methapyrilene 91–80–5
Methoxychlor 72–43–5
2-Methylbenzothioazole 120–75–2
3-Methylcholanthrene 56–49–5
4,4′-Methylenebis(2-chloroaniline) 101–14–4
4,4′-Methylenebis(N,N-dimethylaniline) 101–61–1
4,5-Methylenephenanthrene 203–64–5
1-Methylfluorene 1730–37–6
Methyl methanesulfonate 66–27–3
2-Methylnaphthalene 91–57–6
Methylparaoxon 950–35–6
Methyl parathion 298–00–0
1-Methylphenanthrene 832–69–9
2-(Methylthio)benzothiazole 615–22–5
Metolachlor 5218–45–2
Metribuzin 21087–64–9
Mevinphos 7786–34–7
Mexacarbate 315–18–4
MGK 264 113–48–4
Mirex 2385–85–5
Molinate 2212–67–1
Monocrotophos 6923–22–4
Naled 300–76–5
Napropamide 15299–99–7
1,4-Naphthoquinone 130–15–4
1-Naphthylamine 134–32–7
2-Naphthylamine 91–59–8
1,5-Naphthalenediamine 2243–62–1
Nicotine 54–11–5
5-Nitroacenaphthene 602–87–9
2-Nitroaniline 88–74–4
3-Nitroaniline 99–09–2
4-Nitroaniline 100–01–6
5-Nitro-o-anisidine 99–59–2
4-Nitrobiphenyl 92–93–3
Nitrofen 1836–75–5
5-Nitro-o-toluidine 99–55–8
Nitroquinoline-1-oxide 56–57–5
N-Nitrosodi-n-butylamine4 924–16–3
N-Nitrosodiethylamine4 55–18–5
N-Nitrosodimethylamine3 4 62–75–9
N-Nitrosodiphenylamine3 4 86–30–6
N-Nitrosomethylethylamine4 10595–95–6
N-Nitrosomethylphenylamine4 614–00–6
N-Nitrosomorpholine4 59–89–2
N-Nitrosopiperidine4 100–75–5
N-Nitrosopyrrolidine4 930–55–2
trans-Nonachlor 39765–80–5
Norflurazon 27314–13–2
2,2′,3,3′,4,5′,6,6′-Octachlorobiphenyl 40186–71–8
Octamethyl pyrophosphoramide 152–16–9
4,4'-Oxydianiline 101–80–4
Parathion 56–38–2
PCB–10163 5 12674–11–2
PCB–12213 5 11104–28–2 30 90
PCB–12323 5 11141–16–5
PCB–12423 5 53469–21–9
PCB–12483 5 12672–29–6
PCB–12543 5 11097–69–1 36 108
PCB–12603 5 11098–82–5
PCB–12683 5 11100–14–4
Pebulate 1114–71–2
Pentachlorobenzene 608–93–5
Pentachloronitrobenzene 82–68–8
2,2′,3,4′,6-Pentachlorobiphenyl 68194–05–8
Pentachloroethane 76–01–7
Pentamethylbenzene 700–12–9
Perylene 198–55–0
Phenacetin 62–44–2
cis-Permethrin 61949–76–6
trans-Permethrin 61949–77–7
Phenobarbital 50–06–6
Phenothiazene 92–84–2
1,4-Phenylenediamine 624–18–0
1-Phenylnaphthalene 605–02–7
2-Phenylnaphthalene 612–94–2
Phorate 298–02–2
Phosalone 2310–18–0
Phosmet 732–11–6
Phosphamidon 13171–21–6
Phthalic anhydride 85–44–9
alpha-Picoline (2-Methylpyridine) 109–06–8
Piperonyl sulfoxide 120–62–7
Prometon 1610–18–0
Prometryn 7287–19–6
Pronamide 23950–58–5
Propachlor 1918–16–7
Propazine 139–40–2
Propylthiouracil 51–52–5
Pyridine 110–86–1
Resorcinol (1,3-Benzenediol) 108–46–3
Safrole 94–59–7
Simazine 122–34–9
Simetryn 1014–70–6
Squalene 7683–64–9
Stirofos 22248–79–9
Strychnine 57–24–9
Styrene9 100–42–5
Sulfallate 95–06–7
Tebuthiuron 34014–18–1
Terbacil 5902–51–2
Terbufos 13071–79–9
Terbutryn 886–50–0
alpha-Terpineol 98–55–5
1,2,4,5-Tetrachlorobenzene 95–94–3
2,2′,4,4′-Tetrachlorobiphenyl 2437–79–8
2,3,7,8-Tetrachlorodibenzo-p-dioxin 1746–01–6
2,3,4,6-Tetrachlorophenol 58–90–2
Tetrachlorvinphos 22248–79–9
Tetraethyl dithiopyrophosphate 3689–24–5
Tetraethyl pyrophosphate 107–49–3
Thianaphthene (2,3-Benzothiophene) 95–15–8
Thioacetamide 62–55–5
Thionazin 297–97–2
Thiophenol (Benzenethiol) 108–98–5
Thioxanthone 492–22–8
Toluene-1,3-diisocyanate 26471–62–5
Toluene-2,4-diisocyanate 584–84–9
o-Toluidine 95–53–4
Toxaphene3 5 8001–35–2
Triadimefon 43121–43–3
1,2,3-Trichlorobenzene 87–61–6
2,4,5-Trichlorobiphenyl 15862–07–4
2,3,6-Trichlorophenol 933–75–5
2,4,5-Trichlorophenol 95–95–4
Tricyclazole 41814–78–2
Trifluralin 1582–09–8
1,2,3-Trimethoxybenzene 634–36–6
2,4,5-Trimethylaniline 137–17–7
Trimethyl phosphate 512–56–1
Triphenylene 217–59–4
Tripropyleneglycolmethyl ether 20324–33–8
1,3,5-Trinitrobenzene 99–35–4
Tris(2,3-dibromopropyl) phosphate 126–72–7
Tri-p-tolyl phosphate 78–32–0
O,O,O-Triethyl phosphorothioate 126–68–1
Trithiane 291–29–4
Vernolate 1929–77–7

Table 4—Chromatographic Conditions and Characteristic m/z's for Base/Neutral Extractables

Analyte Retention time
(sec)1
Characteristic m/z's
Electron impact ionization Chemical ionization
Primary Second Second Methane Methane Methane
N-Nitrosodimethylamine 385 42 74 44
bis(2-Chloroethyl) ether 704 93 63 95 63 107 109
bis(2-Chloroisopropyl) ether 799 45 77 79 77 135 137
Hexachloroethane 823 117 201 199 199 201 203
N-Nitrosodi-n-propylamine 830 130 42 101
Nitrobenzene 849 77 123 65 124 152 164
Isophorone 889 82 95 138 139 167 178
bis(2-Chloroethoxy) methane 939 93 95 123 65 107 137
1,2,4-Trichlorobenzene 958 180 182 145 181 183 209
Naphthalene 967 128 129 127 129 157 169
Hexachlorobutadiene 1006 225 223 227 223 225 227
Hexachlorocyclopentadiene 1142 237 235 272 235 237 239
2-Chloronaphthalene 1200 162 164 127 163 191 203
Acenaphthylene 1247 152 151 153 152 153 181
Dimethyl phthalate 1273 163 194 164 151 163 164
2,6-Dinitrotoluene 1300 165 89 121 183 211 223
Acenaphthene 1304 154 153 152 154 155 183
2,4-Dinitrotoluene 1364 165 63 182 183 211 223
Fluorene 1401 166 165 167 166 167 195
4-Chlorophenyl phenyl ether 1409 204 206 141
Diethyl phthalate 1414 149 177 150 177 223 251
N-Nitrosodiphenylamine 1464 169 168 167 169 170 198
4-Bromophenyl phenyl ether 1498 248 250 141 249 251 277
alpha-BHC 1514 183 181 109
Hexachlorobenzene 1522 284 142 249 284 286 288
beta-BHC 1544 183 181 109
gamma-BHC 1557 181 183 109
Phenanthrene 1583 178 179 176 178 179 207
Anthracene 1592 178 179 176 178 179 207
delta-BHC 1599 183 109 181
Heptachlor 1683 100 272 274
Di-n-butyl phthalate 1723 149 150 104 149 205 279
Aldrin 1753 66 263 220
Fluoranthene 1817 202 101 100 203 231 243
Heptachlor epoxide 1820 353 355 351
gamma-Chlordane 1834 373 375 377
Pyrene 1852 202 101 100 203 231 243
Benzidine2 1853 184 92 185 185 213 225
alpha-Chlordane 1854 373 375 377
Endosulfan I 1855 237 339 341
4,4′-DDE 1892 246 248 176
Dieldrin 1907 79 263 279
Endrin 1935 81 263 82
Endosulfan II 2014 237 339 341
4,4′-DDD 2019 235 237 165
Endrin aldehyde 2031 67 345 250
Butyl benzyl phthalate 2060 149 91 206 149 299 327
Endosulfan sulfate 2068 272 387 422
4,4′-DDT 2073 235 237 165
Chrysene 2083 228 226 229 228 229 257
3,3′-Dichlorobenzidine 2086 252 254 126
Benzo(a)anthracene 2090 228 229 226 228 229 257
bis(2-Ethylhexyl) phthalate 2124 149 167 279 149
Di-n-octyl phthalate 2240 149 43 57
Benzo(b)fluoranthene 2286 252 253 125 252 253 281
Benzo(k)fluoranthene 2293 252 253 125 252 253 281
Benzo(a)pyrene 2350 252 253 125 252 253 281
Indeno(1,2,3-cd) pyrene 2650 276 138 277 276 277 305
Dibenz(a,h)anthracene 2660 278 139 279 278 279 307
Benzo(ghi)perylene 2750 276 138 277 276 277 305
Toxaphene 159 231 233
PCB 1016 224 260 294
PCB 1221 190 224 260
PCB 1232 190 224 260
PCB 1242 224 260 294
PCB 1248 294 330 262
PCB 1254 294 330 362
PCB 1260 330 362 394

Table 5—Chromatographic Conditions and Characteristic m/z's for Acid Extractables

Analyte Retention Time
(sec)1
Characteristic m/z's
Electron impact ionization Chemical ionization
Prime Second Second Methane Methane Methane
2-Chlorophenol 705 128 64 130 129 131 157
Phenol 700 94 65 66 95 123 135
2-Nitrophenol 900 139 65 109 140 168 122
2,4-Dimethylphenol 924 122 107 121 123 151 163
2,4-Dichlorophenol 947 162 164 98 163 165 167
4-Chloro-3-methylphenol 1091 142 107 144 143 171 183
2,4,6-Trichlorophenol 1165 196 198 200 197 199 201
2,4-Dinitrophenol 1325 184 63 154 185 213 225
4-Nitrophenol 1354 65 139 109 140 168 122
2-Methyl-4,6-dinitrophenol 1435 198 182 77 199 227 239
Pentachlorophenol 1561 266 264 268 267 265 269

Table 6—QC Acceptance Criteria—Method 6251

Analyte Range for Q
(%)2
Limit for s
(%)3
Range for
X (%)3
Range for
P1, P2(%)3
Limit for
RPD (%)
Acenaphthene 70–130 29 60–132 47–145 48
Acenaphthylene 60–130 45 54–126 33–145 74
Aldrin 7–152 39 7–152 D–166 81
Anthracene 58–130 40 43–120 27–133 66
Benzo(a)anthracene 42–133 32 42–133 33–143 53
Benzo(b)fluoranthene 42–140 43 42–140 24–159 71
Benzo(k)fluoranthene 25–146 38 25–146 11–162 63
Benzo(a)pyrene 32–148 43 32–148 17–163 72
Benzo(ghi)perylene 13–195 61 D–195 D–219 97
Benzyl butyl phthalate 43–140 36 D–140 D–152 60
beta-BHC 42–131 37 42–131 24–149 61
delta-BHC D–130 77 D–120 D–120 129
bis(2-Chloroethyl)ether 52–130 65 43–126 12–158 108
bis(2-Chloroethoxy)methane 52–164 32 49–165 33–184 54
bis(2-Chloroisopropyl) ether 63–139 46 63–139 36–166 76
bis(2-Ethylhexyl) phthalate 43–137 50 29–137 8–158 82
4-Bromophenyl phenyl ether 70–130 26 65–120 53–127 43
2-Chloronaphthalene 70–130 15 65–120 60–120 24
4-Chlorophenyl phenyl ether 57–145 36 38–145 25–158 61
Chrysene 44–140 53 44–140 17–168 87
4,4′-DDD D–135 56 D–135 D–145 93
4,4′-DDE 19–130 46 19–120 4–136 77
4,4′-DDT D–171 81 D–171 D–203 135
Dibenz(a,h)anthracene 13–200 75 D–200 D–227 126
Di-n-butyl phthalate 52–130 28 8–120 1–120 47
3,3′-Dichlorobenzidine 18–213 65 8–213 D–262 108
Dieldrin 70–130 38 44–119 29–136 62
Diethyl phthalate 47–130 60 D–120 D–120 100
Dimethyl phthalate 50–130 110 D–120 D–120 183
2,4-Dinitrotoluene 53–130 25 48–127 39–139 42
2,6-Dinitrotoluene 68–137 29 68–137 50–158 48
Di-n-octyl phthalate 21–132 42 19–132 4–146 69
Endosulfan sulfate D–130 42 D–120 D–120 70
Endrin aldehyde D–189 45 D–189 D–209 75
Fluoranthene 47–130 40 43–121 26–137 66
Fluorene 70–130 23 70–120 59–121 38
Heptachlor D–172 44 D–172 D–192 74
Heptachlor epoxide 70–130 61 71–120 26–155 101
Hexachlorobenzene 38–142 33 8–142 D–152 55
Hexachlorobutadiene 68–130 38 38–120 24–120 62
Hexachloroethane 55–130 32 55–120 40–120 52
Indeno(1,2,3-cd)pyrene 13–151 60 D–151 D–171 99
Isophorone 52–180 56 47–180 21–196 93
Naphthalene 70–130 39 36–120 21–133 65
Nitrobenzene 54–158 37 54–158 35–180 62
N-Nitrosodi-n-propylamine 59–170 52 14–198 D–230 87
PCB–1260 19–130 77 19–130 D–164 128
Phenanthrene 67–130 24 65–120 54–120 39
Pyrene 70–130 30 70–120 52–120 49
1,2,4-Trichlorobenzene 61–130 30 57–130 44–142 50
4-Chloro-3-methylphenol 68–130 44 41–128 22–147 73
2-Chlorophenol 55–130 37 36–120 23–134 61
2,4-Dichlorophenol 64–130 30 53–122 39–135 50
2,4-Dimethylphenol 58–130 35 42–120 32–120 58
2,4-Dinitrophenol 39–173 79 D–173 D–191 132
2-Methyl-4,6-dinitrophenol 56–130 122 53–130 D–181 203
2-Nitrophenol 61–163 33 45–167 29–182 55
4-Nitrophenol 35–130 79 13–129 D–132 131
Pentachlorophenol 42–152 52 38–152 14–176 86
Phenol 48–130 39 17–120 5–120 64
2,4,6-Trichlorophenol 69–130 35 52–129 37–144 58

Table 7—Precision and Recovery as Functions of Concentration—Method 6251

Analyte Recovery, X′
(µg/L)
Single analyst
precision, sr
(µg/L)
Overall
precision, S′
(µg/L)
Acenaphthene 0.96C + 0.19 0.15 X−0.12 0.21 X−0.67
Acenaphthylene 0.89C + 0.74 0.24 X−1.06 0.26 X−0.54
Aldrin 0.78C + 1.66 0.27 X−1.28 0.43 X + 1.13
Anthracene 0.80C + 0.68 0.21 X−0.32 0.27 X−0.64
Benzo(a)anthracene 0.88C−0.60 0.15 X + 0.93 0.26 X−0.28
Benzo(b)fluoranthene 0.93C−1.80 0.22 X + 0.43 0.29 X + 0.96
Benzo(k)fluoranthene 0.87C−1.56 0.19 X + 1.03 0.35 X + 0.40
Benzo(a)pyrene 0.90C−0.13 0.22 X + 0.48 0.32 X + 1.35
Benzo(ghi)perylene 0.98C−0.86 0.29 X + 2.40 0.51 X−0.44
Benzyl butyl phthalate 0.66C−1.68 0.18 X + 0.94 0.53 X + 0.92
beta-BHC 0.87C−0.94 0.20 X−0.58 0.30 X−1.94
delta-BHC 0.29C−1.09 0.34 X + 0.86 0.93 X−0.17
bis(2-Chloroethyl) ether 0.86C−1.54 0.35 X−0.99 0.35 X + 0.10
bis(2-Chloroethoxy) methane 1.12C−5.04 0.16 X + 1.34 0.26 X + 2.01
bis(2-Chloroisopropyl) ether 1.03C−2.31 0.24 X + 0.28 0.25 X + 1.04
bis(2-Ethylhexyl) phthalate 0.84C−1.18 0.26 X + 0.73 0.36 X + 0.67
4-Bromophenyl phenyl ether 0.91C−1.34 0.13 X + 0.66 0.16 X + 0.66
2-Chloronaphthalene 0.89C + 0.01 0.07 X + 0.52 0.13 X + 0.34
4-Chlorophenyl phenyl ether 0.91C + 0.53 0.20 X−0.94 0.30 X−0.46
Chrysene 0.93C−1.00 0.28 X + 0.13 0.33 X−0.09
4,4′-DDD 0.56C−0.40 0.29 X−0.32 0.66 X−0.96
4,4′-DDE 0.70C−0.54 0.26 X−1.17 0.39 X−1.04
4,4′-DDT 0.79C−3.28 0.42 X + 0.19 0.65 X−0.58
Dibenz(a,h)anthracene 0.88C + 4.72 0.30 X + 8.51 0.59 X + 0.25
Di-n-butyl phthalate 0.59C + 0.71 0.13 X + 1.16 0.39 X + 0.60
3,3'-Dichlorobenzidine 1.23C−12.65 0.28 X + 7.33 0.47 X + 3.45
Dieldrin 0.82C−0.16 0.20 X−0.16 0.26 X−0.07
Diethyl phthalate 0.43C + 1.00 0.28 X + 1.44 0.52 X + 0.22
Dimethyl phthalate 0.20C + 1.03 0.54 X + 0.19 1.05 X−0.92
2,4-Dinitrotoluene 0.92C−4.81 0.12 X + 1.06 0.21 X + 1.50
2,6-Dinitrotoluene 1.06C−3.60 0.14 X + 1.26 0.19 X + 0.35
Di-n-octyl phthalate 0.76C−0.79 0.21 X + 1.19 0.37 X + 1.19
Endosulfan sulfate 0.39C + 0.41 0.12 X + 2.47 0.63 X−1.03
Endrin aldehyde 0.76C−3.86 0.18 X + 3.91 0.73 X−0.62
Fluoranthene 0.81C + 1.10 0.22 X + 0.73 0.28 X−0.60
Fluorene 0.90C−0.00 0.12 X + 0.26 0.13 X + 0.61
Heptachlor 0.87C−2.97 0.24 X−0.56 0.50 X−0.23
Heptachlor epoxide 0.92C−1.87 0.33 X−0.46 0.28 X + 0.64
Hexachlorobenzene 0.74C + 0.66 0.18 X−0.10 0.43 X−0.52
Hexachlorobutadiene 0.71C−1.01 0.19 X + 0.92 0.26 X + 0.49
Hexachloroethane 0.73C−0.83 0.17 X + 0.67 0.17 X + 0.80
Indeno(1,2,3-cd)pyrene 0.78C−3.10 0.29 X + 1.46 0.50 X + 0.44
Isophorone 1.12C + 1.41 0.27 X + 0.77 0.33 X + 0.26
Naphthalene 0.76C + 1.58 0.21 X−0.41 0.30 X−0.68
Nitrobenzene 1.09C−3.05 0.19 X + 0.92 0.27 X + 0.21
N-Nitrosodi-n-propylamine 1.12C−6.22 0.27 X + 0.68 0.44 X + 0.47
PCB–1260 0.81C−10.86 0.35 X + 3.61 0.43 X + 1.82
Phenanthrene 0.87C−0.06 0.12 X + 0.57 0.15 X + 0.25
Pyrene 0.84C−0.16 0.16 X + 0.06 0.15 X + 0.31
1,2,4-Trichlorobenzene 0.94C−0.79 0.15 X + 0.85 0.21 X + 0.39
4-Chloro-3-methylphenol 0.84C + 0.35 0.23 X + 0.75 0.29 X + 1.31
2-Chlorophenol 0.78C + 0.29 0.18 X + 1.46 0.28 X + 0.97
2,4-Dichlorophenol 0.87C + 0.13 0.15 X + 1.25 0.21 X + 1.28
2,4-Dimethylphenol 0.71C + 4.41 0.16 X + 1.21 0.22 X + 1.31
2,4-Dinitrophenol 0.81C−18.04 0.38 X + 2.36 0.42 X + 26.29
2-Methyl-4,6-Dinitrophenol 1.04C−28.04 0.05 X + 42.29 0.26 X + 23.10
2-Nitrophenol 1.07C−1.15 0.16 X + 1.94 0.27 X + 2.60
4-Nitrophenol 0.61C−1.22 0.38 X + 2.57 0.44 X + 3.24
Pentachlorophenol 0.93C + 1.99 0.24 X + 3.03 0.30 X + 4.33
Phenol 0.43C + 1.26 0.26 X + 0.73 0.35 X + 0.58
2,4,6-Trichlorophenol 0.91C−0.18 0.16 X + 2.22 0.22 X + 1.81

Table 8—Suggested Internal and Surrogate Standards

Base/neutral fraction Range for surrogate recovery (%)1
Calibration verification Recovery from samples
Acenaphthalene-d8 66–152 33–168
Acenaphthene-d10 71–141 30–180
Aniline-d5
Anthracene-d10 58–171 23–142
Benzo(a)anthracene-d12 28–357 22–329
Benzo(a)pyrene-d12 32–194 32–194
4-Chloroaniline-d4 1–145 1–145
bis(2-Chloroethyl) ether-d8 52–194 25–222
Chrysene-d12 23–290 23–290
Decafluorobiphenyl
4,4′-Dibromobiphenyl
4,4′-Dibromooctafluorobiphenyl
1,4-Dichlorobenzene-d4 65–153 11–245
2,2′-Difluorobiphenyl
Dimethyl phthalate-d6 47–211 1–500
Fluoranthene-d10 47–215 30–187
Fluorene-d10 61–164 38–172
4-Fluoroaniline
1-Fluoronaphthalene
2-Fluoronaphthalene
2-Methylnaphthalene-d10 50–150 50–150
Naphthalene-d8 71–141 22–192
Nitrobenzene-d5 46–219 15–314
2,3,4,5,6-Pentafluorobiphenyl
Perylene-d12
Phenanthrene-d10 67–149 34–168
Pyrene-d10 48–210 28–196
Pyridine-d5
Acid fraction
2-Chlorophenol-d4 55–180 33–180
2,4-Dichlorophenol-d3 64–157 34–182
4,6-Dinitro-2-methylphenol-d2 56–177 22–307
2-Fluorophenol
4-Methylphenol-d8 25–111 25–111
2-Nitrophenol-d4 61–163 37–163
4-Nitrophenol-d4 35–287 6–500
Pentafluorophenol
2-Perfluoromethylphenol
Phenol-d5 48–208 8–424

Table 9A—DFTPP Key m/z's and Abundance Criteria for Quadrupole Instruments1

m/z Abundance criteria
51 30–60 percent of m/z 198.
68 Less than 2 percent of m/z 69.
70 Less than 2 percent of m/z 69.
127 40–60 percent of base peak m/z 198.
197 Less than 1 percent of m/z 198.
198 Base peak, 100 percent relative abundance.
199 5–9 percent of m/z 198.
275 10–30 percent of m/z 198.
365 Greater than 1 percent of m/z 198.
441 Present but less than m/z 443.
442 40–100 percent of m/z 198.
443 17–23 percent of m/z 442.

Table 9B—DFTPP Key m/z's and Abundance Criteria for Time-of-flight Instruments1

m/z Abundance criteria
51 10–85 percent of the base peak.
68 Less than 2 percent of m/z 69.
70 Less than 2 percent of m/z 69.
127 10–80 percent of the base peak.
197 Less than 2 percent of Mass 198.
198 Base peak, or greater than 50% of m/z 442.
199 5–9 percent of m/z 198.
275 10–60 percent of the base peak.
365 Greater than 0.5 percent of m/z 198.
441 Less than 150 percent of m/z 443.
442 Base peak or greater than 30 percent of m/z 198.
443 15–24 percent of m/z 442.

21. Figures

22. Glossary

These definitions and purposes are specific to this method but have been conformed to common usage to the extent possible.

22.1 Units of weight and measure and their abbreviations.

22.1.1 Symbols.

°C degrees Celsius

µg microgram

µL microliter

< less than

> greater than

≤ less than or equal to

% percent

22.1.2 Abbreviations (in alphabetical order).

cm centimeter

g gram

h hour

ID inside diameter

in. inch

L liter

m mass or meter

mg milligram

min minute

mL milliliter

mm millimeter

ms millisecond

m/z mass-to-charge ratio

N normal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of solution

ng nanogram

pg picogram

ppb part-per-billion

ppm part-per-million

ppt part-per-trillion

psig pounds-per-square inch gauge

22.2 Definitions and acronyms (in alphabetical order).

Analyte—A compound or mixture of compounds (e.g., PCBs) tested for by this method. The analytes are listed in Tables 1–3.

Batch—See Extraction.

Blank—An aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples. The blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus.

Calibration—The process of determining the relationship between the output or response of a measuring instrument and the value of an input standard. Historically, EPA has referred to a multi-point calibration as the “initial calibration,” to differentiate it from a single-point calibration verification.

Calibration standard—A solution prepared from stock solutions and/or a secondary standards and containing the analytes of interest, surrogates, and internal standards. The calibration standard is used to calibrate the response of the GC/MS instrument against analyte concentration.

Calibration verification standard—The mid-point calibration standard used to verify calibration. See sections 7.3 and 13.4.

Descriptor—In SIM, the beginning and ending retention times for the RT window, the m/z's sampled in the RT window, and the dwell time at each m/z.

Extracted ion current profile (EICP)—The line described by the signal at a given m/z.

Extraction Batch—A set of up to 20 field samples (not including QC samples) started through the extraction process on a given 24-hour shift (section 3.1). Each extraction batch must be accompanied by a blank (section 8.5), a laboratory control sample (LCS, section 8.4), and a matrix spike and duplicate (MS/MSD; Section 8.3), resulting in a minimum of five analyses (1 sample, 1 blank, 1 LCS, 1 MS, and 1 MSD) and a maximum of 24 analyses (20 field samples, 1 blank, 1 LCS, 1 MS, and 1 MSD) for the batch. If greater than 20 samples are to be extracted in a 24-hour shift, the samples must be separated into extraction batches of 20 or fewer samples.

Field Duplicates—Two samples collected at the same time and placed under identical conditions, and treated identically throughout field and laboratory procedures. Results of analyses of the field duplicates provide an estimate of the precision associated with sample collection, preservation, and storage, as well as with laboratory procedures.

Field blank—An aliquot of reagent water or other reference matrix that is placed in a sample container in the field, and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to determine if the field or sample transporting procedures and environments have contaminated the sample.

GC—Gas chromatograph or gas chromatography.

Internal standard—A compound added to an extract or standard solution in a known amount and used as a reference for quantitation of the analytes of interest and surrogates. In this method the internal standards are stable isotopically labeled analogs of selected method analytes (Table 8). Also see Internal standard quantitation.

Internal standard quantitation—A means of determining the concentration of an analyte of interest (Tables 1–3) by reference to a compound not expected to be found in a sample.

DOC—Initial demonstration of capability (section 8.2); four aliquots of reagent water spiked with the analytes of interest and analyzed to establish the ability of the laboratory to generate acceptable precision and recovery. A DOC is performed prior to the first time this method is used and any time the method or instrumentation is modified.

Laboratory Control Sample (LCS; laboratory fortified blank; section 8.4)—An aliquot of reagent water spiked with known quantities of the analytes of interest and surrogates. The LCS is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified in this method for precision and recovery.

Laboratory fortified sample matrix—See Matrix spike.

Laboratory reagent blank—A blank run on laboratory reagents; e.g., methylene chloride (section 11.1.5).

Matrix spike (MS) and matrix spike duplicate (MSD) (laboratory fortified sample matrix and duplicate)—Two aliquots of an environmental sample to which known quantities of the analytes of interest and surrogates are added in the laboratory. The MS/MSD are prepared and analyzed exactly like a field sample. Their purpose is to quantify any additional bias and imprecision caused by the sample matrix. The background concentrations of the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the MS/MSD corrected for background concentrations.

May—This action, activity, or procedural step is neither required nor prohibited.

May not—This action, activity, or procedural step is prohibited.

Method blank—See blank.

Method detection limit (MDL)—A detection limit determined by the procedure at 40 CFR part 136, appendix B. The MDLs determined by EPA in the original version of the method are listed in Tables 1, 2 and 3. As noted in section 1.5, use the MDLs in Tables 1, 2, and 3 in conjunction with current MDL data from the laboratory actually analyzing samples to assess the sensitivity of this procedure relative to project objectives and regulatory requirements (where applicable).

Minimum level (ML)—The term “minimum level” refers to either the sample concentration equivalent to the lowest calibration point in a method or a multiple of the method detection limit (MDL), whichever is higher. Minimum levels may be obtained in several ways: They may be published in a method; they may be based on the lowest acceptable calibration point used by a laboratory; or they may be calculated by multiplying the MDL in a method, or the MDL determined by a laboratory, by a factor of 3. For the purposes of NPDES compliance monitoring, EPA considers the following terms to be synonymous: “quantitation limit,” “reporting limit,” and “minimum level.”

MS—Mass spectrometer or mass spectrometry, or matrix spike (a QC sample type).

MSD—Matrix spike duplicate (a QC sample type).

Must—This action, activity, or procedural step is required.

m/z—The ratio of the mass of an ion (m) detected in the mass spectrometer to the charge (z) of that ion.

Preparation blank—See blank.

Quality control check sample (QCS)—See Laboratory Control Sample.

Reagent water—Water demonstrated to be free from the analytes of interest and potentially interfering substances at the MDLs for the analytes in this method.

Regulatory compliance limit (or regulatory concentration limit)—A limit on the concentration or amount of a pollutant or contaminant specified in a nationwide standard, in a permit, or otherwise established by a regulatory/control authority.

Relative retention time (RRT)—The ratio of the retention time of an analyte to the retention time of its associated internal standard. RRT compensates for small changes in the GC temperature program that can affect the absolute retention times of the analyte and internal standard. RRT is a unitless quantity.

Relative standard deviation (RSD)—The standard deviation times 100 divided by the mean. Also termed “coefficient of variation.”

RF—Response factor. See section 7.2.2.

RSD—See relative standard deviation.

Safety Data Sheet (SDS)—Written information on a chemical's toxicity, health hazards, physical properties, fire, and reactivity, including storage, spill, and handling precautions that meet the requirements of OSHA, 29 CFR 1910.1200(g) and appendix D to § 1910.1200. United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), third revised edition, United Nations, 2009.

Selected Ion Monitoring (SIM)—An MS technique in which a few m/z's are monitored. When used with gas chromatography, the m/z's monitored are usually changed periodically throughout the chromatographic run, to correlate with the characteristic m/z's of the analytes, surrogates, and internal standards as they elute from the chromatographic column. The technique is often used to increase sensitivity and minimize interferences.

Signal-to-noise ratio (S/N)—The height of the signal as measured from the mean (average) of the noise to the peak maximum divided by the width of the noise.

Should—This action, activity, or procedural step is suggested but not required.

SPE—Solid-phase extraction; an extraction technique in which an analyte is extracted from an aqueous solution by passage over or through a material capable of reversibly adsorbing the analyte. Also termed liquid-solid extraction.

Stock solution—A solution containing an analyte that is prepared using a reference material traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will attest to the purity, authenticity, and concentration of the standard.

Surrogate—A compound unlikely to be found in a sample, and which is spiked into sample in a known amount before extraction or other processing, and is quantitated with the same procedures used to quantify other sample components. The purpose of the surrogate is to monitor method performance with each sample.

Method 1613, Revision B

Tetra- Through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS

1.0 Scope and Application

1.1 This method is for determination of tetra- through octa-chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) in water, soil, sediment, sludge, tissue, and other sample matrices by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The method is for use in EPA's data gathering and monitoring programs associated with the Clean Water Act, the Resource Conservation and Recovery Act, the Comprehensive Environmental Response, Compensation and Liability Act, and the Safe Drinking Water Act. The method is based on a compilation of EPA, industry, commercial laboratory, and academic methods (References 1–6).

1.2 The seventeen 2,3,7,8-substituted CDDs/CDFs listed in Table 1 may be determined by this method. Specifications are also provided for separate determination of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD) and 2,3,7,8-tetrachloro-dibenzofuran (2,3,7,8-TCDF).

1.3 The detection limits and quantitation levels in this method are usually dependent on the level of interferences rather than instrumental limitations. The minimum levels (MLs) in Table 2 are the levels at which the CDDs/CDFs can be determined with no interferences present. The Method Detection Limit (MDL) for 2,3,7,8-TCDD has been determined as 4.4 pg/L (parts-per-quadrillion) using this method.

1.4 The GC/MS portions of this method are for use only by analysts experienced with HRGC/HRMS or under the close supervision of such qualified persons. Each laboratory that uses this method must demonstrate the ability to generate acceptable results using the procedure in Section 9.2.

1.5 This method is “performance-based”. The analyst is permitted to modify the method to overcome interferences or lower the cost of measurements, provided that all performance criteria in this method are met. The requirements for establishing method equivalency are given in Section 9.1.2.

1.6 Any modification of this method, beyond those expressly permitted, shall be considered a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

2.0 Summary of Method

Flow charts that summarize procedures for sample preparation, extraction, and analysis are given in Figure 1 for aqueous and solid samples, Figure 2 for multi-phase samples, and Figure 3 for tissue samples.

2.1 Extraction.

2.1.1 Aqueous samples (samples containing less than 1% solids)—Stable isotopically labeled analogs of 15 of the 2,3,7,8-substituted CDDs/CDFs are spiked into a 1 L sample, and the sample is extracted by one of three procedures:

2.1.1.1 Samples containing no visible particles are extracted with methylene chloride in a separatory funnel or by the solid-phase extraction technique summarized in Section 2.1.1.3. The extract is concentrated for cleanup.

2.1.1.2 Samples containing visible particles are vacuum filtered through a glass-fiber filter. The filter is extracted in a Soxhlet/Dean-Stark (SDS) extractor (Reference 7), and the filtrate is extracted with methylene chloride in a separatory funnel. The methylene chloride extract is concentrated and combined with the SDS extract prior to cleanup.

2.1.1.3 The sample is vacuum filtered through a glass-fiber filter on top of a solid-phase extraction (SPE) disk. The filter and disk are extracted in an SDS extractor, and the extract is concentrated for cleanup.

2.1.2 Solid, semi-solid, and multi-phase samples (but not tissue)—The labeled compounds are spiked into a sample containing 10 g (dry weight) of solids. Samples containing multiple phases are pressure filtered and any aqueous liquid is discarded. Coarse solids are ground or homogenized. Any non-aqueous liquid from multi-phase samples is combined with the solids and extracted in an SDS extractor. The extract is concentrated for cleanup.

2.1.3 Fish and other tissue—The sample is extracted by one of two procedures:

2.1.3.1 Soxhlet or SDS extraction—A 20 g aliquot of sample is homogenized, and a 10 g aliquot is spiked with the labeled compounds. The sample is mixed with sodium sulfate, allowed to dry for 12–24 hours, and extracted for 18–24 hours using methylene chloride:hexane (1:1) in a Soxhlet extractor. The extract is evaporated to dryness, and the lipid content is determined.

2.1.3.2 HCl digestion—A 20 g aliquot is homogenized, and a 10 g aliquot is placed in a bottle and spiked with the labeled compounds. After equilibration, 200 mL of hydrochloric acid and 200 mL of methylene chloride:hexane (1:1) are added, and the bottle is agitated for 12–24 hours. The extract is evaporated to dryness, and the lipid content is determined.

2.2 After extraction,37Cl4-labeled 2,3,7,8-TCDD is added to each extract to measure the efficiency of the cleanup process. Sample cleanups may include back-extraction with acid and/or base, and gel permeation, alumina, silica gel, Florisil and activated carbon chromatography. High-performance liquid chromatography (HPLC) can be used for further isolation of the 2,3,7,8-isomers or other specific isomers or congeners. Prior to the cleanup procedures cited above, tissue extracts are cleaned up using an anthropogenic isolation column, a batch silica gel adsorption, or sulfuric acid and base back-extraction, depending on the tissue extraction procedure used.

2.3 After cleanup, the extract is concentrated to near dryness. Immediately prior to injection, internal standards are added to each extract, and an aliquot of the extract is injected into the gas chromatograph. The analytes are separated by the GC and detected by a high-resolution (≥10,000) mass spectrometer. Two exact m/z's are monitored for each analyte.

2.4 An individual CDD/CDF is identified by comparing the GC retention time and ion-abundance ratio of two exact m/z's with the corresponding retention time of an authentic standard and the theoretical or acquired ion-abundance ratio of the two exact m/z's. The non-2,3,7,8 substituted isomers and congeners are identified when retention times and ion-abundance ratios agree within predefined limits. Isomer specificity for 2,3,7,8-TCDD and 2,3,7,8-TCDF is achieved using GC columns that resolve these isomers from the other tetra-isomers.

2.5 Quantitative analysis is performed using selected ion current profile (SICP) areas, in one of three ways:

2.5.1 For the 15 2,3,7,8-substituted CDDs/CDFs with labeled analogs (see Table 1), the GC/MS system is calibrated, and the concentration of each compound is determined using the isotope dilution technique.

2.5.2 For 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds, the GC/MS system is calibrated and the concentration of each compound is determined using the internal standard technique.

2.5.3 For non-2,3,7,8-substituted isomers and for all isomers at a given level of chlorination (i.e., total TCDD), concentrations are determined using response factors from calibration of the CDDs/CDFs at the same level of chlorination.

2.6 The quality of the analysis is assured through reproducible calibration and testing of the extraction, cleanup, and GC/MS systems.

3.0 Definitions

Definitions are given in the glossary at the end of this method.

4.0 Contamination and Interferences

4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines causing misinterpretation of chromatograms (References 8–9). Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Where possible, reagents are cleaned by extraction or solvent rinse.

4.2 Proper cleaning of glassware is extremely important, because glassware may not only contaminate the samples but may also remove the analytes of interest by adsorption on the glass surface.

4.2.1 Glassware should be rinsed with solvent and washed with a detergent solution as soon after use as is practical. Sonication of glassware containing a detergent solution for approximately 30 seconds may aid in cleaning. Glassware with removable parts, particularly separatory funnels with fluoropolymer stopcocks, must be disassembled prior to detergent washing.

4.2.2 After detergent washing, glassware should be rinsed immediately, first with methanol, then with hot tap water. The tap water rinse is followed by another methanol rinse, then acetone, and then methylene chloride.

4.2.3 Do not bake reusable glassware in an oven as a routine part of cleaning. Baking may be warranted after particularly dirty samples are encountered but should be minimized, as repeated baking of glassware may cause active sites on the glass surface that will irreversibly adsorb CDDs/CDFs.

4.2.4 Immediately prior to use, the Soxhlet apparatus should be pre-extracted with toluene for approximately three hours (see Sections 12.3.1 through 12.3.3). Separatory funnels should be shaken with methylene chloride/toluene (80/20 mixture) for two minutes, drained, and then shaken with pure methylene chloride for two minutes.

4.3 All materials used in the analysis shall be demonstrated to be free from interferences by running reference matrix method blanks initially and with each sample batch (samples started through the extraction process on a given 12-hour shift, to a maximum of 20 samples).

4.3.1 The reference matrix must simulate, as closely as possible, the sample matrix under test. Ideally, the reference matrix should not contain the CDDs/CDFs in detectable amounts, but should contain potential interferents in the concentrations expected to be found in the samples to be analyzed. For example, a reference sample of human adipose tissue containing pentachloronaphthalene can be used to exercise the cleanup systems when samples containing pentachloronaphthalene are expected.

4.3.2 When a reference matrix that simulates the sample matrix under test is not available, reagent water (Section 7.6.1) can be used to simulate water samples; playground sand (Section 7.6.2) or white quartz sand (Section 7.3.2) can be used to simulate soils; filter paper (Section 7.6.3) can be used to simulate papers and similar materials; and corn oil (Section 7.6.4) can be used to simulate tissues.

4.4 Interferences coextracted from samples will vary considerably from source to source, depending on the diversity of the site being sampled. Interfering compounds may be present at concentrations several orders of magnitude higher than the CDDs/CDFs. The most frequently encountered interferences are chlorinated biphenyls, methoxy biphenyls, hydroxydiphenyl ethers, benzylphenyl ethers, polynuclear aromatics, and pesticides. Because very low levels of CDDs/CDFs are measured by this method, the elimination of interferences is essential. The cleanup steps given in Section 13 can be used to reduce or eliminate these interferences and thereby permit reliable determination of the CDDs/CDFs at the levels shown in Table 2.

4.5 Each piece of reusable glassware should be numbered to associate that glassware with the processing of a particular sample. This will assist the laboratory in tracking possible sources of contamination for individual samples, identifying glassware associated with highly contaminated samples that may require extra cleaning, and determining when glassware should be discarded.

4.6 Cleanup of tissue—The natural lipid content of tissue can interfere in the analysis of tissue samples for the CDDs/CDFs. The lipid contents of different species and portions of tissue can vary widely. Lipids are soluble to varying degrees in various organic solvents and may be present in sufficient quantity to overwhelm the column chromatographic cleanup procedures used for cleanup of sample extracts. Lipids must be removed by the lipid removal procedures in Section 13.7, followed by alumina (Section 13.4) or Florisil (Section 13.8), and carbon (Section 13.5) as minimum additional cleanup steps. If chlorodiphenyl ethers are detected, as indicated by the presence of peaks at the exact m/z's monitored for these interferents, alumina and/or Florisil cleanup must be employed to eliminate these interferences.

5.0 Safety

5.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely determined; however, each chemical compound should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level.

5.1.1 The 2,3,7,8-TCDD isomer has been found to be acnegenic, carcinogenic, and teratogenic in laboratory animal studies. It is soluble in water to approximately 200 ppt and in organic solvents to 0.14%. On the basis of the available toxicological and physical properties of 2,3,7,8-TCDD, all of the CDDs/CDFs should be handled only by highly trained personnel thoroughly familiar with handling and cautionary procedures and the associated risks.

5.1.2 It is recommended that the laboratory purchase dilute standard solutions of the analytes in this method. However, if primary solutions are prepared, they shall be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator shall be worn when high concentrations are handled.

5.2 The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material safety data sheets (MSDSs) should also be made available to all personnel involved in these analyses. It is also suggested that the laboratory perform personal hygiene monitoring of each analyst who uses this method and that the results of this monitoring be made available to the analyst. Additional information on laboratory safety can be found in References 10–13. The references and bibliography at the end of Reference 13 are particularly comprehensive in dealing with the general subject of laboratory safety.

5.3 The CDDs/CDFs and samples suspected to contain these compounds are handled using essentially the same techniques employed in handling radioactive or infectious materials. Well-ventilated, controlled access laboratories are required. Assistance in evaluating the health hazards of particular laboratory conditions may be obtained from certain consulting laboratories and from State Departments of Health or Labor, many of which have an industrial health service. The CDDs/CDFs are extremely toxic to laboratory animals. Each laboratory must develop a strict safety program for handling these compounds. The practices in References 2 and 14 are highly recommended.

5.3.1 Facility—When finely divided samples (dusts, soils, dry chemicals) are handled, all operations (including removal of samples from sample containers, weighing, transferring, and mixing) should be performed in a glove box demonstrated to be leak tight or in a fume hood demonstrated to have adequate air flow. Gross losses to the laboratory ventilation system must not be allowed. Handling of the dilute solutions normally used in analytical and animal work presents no inhalation hazards except in the case of an accident.

5.3.2 Protective equipment—Disposable plastic gloves, apron or lab coat, safety glasses or mask, and a glove box or fume hood adequate for radioactive work should be used. During analytical operations that may give rise to aerosols or dusts, personnel should wear respirators equipped with activated carbon filters. Eye protection equipment (preferably full face shields) must be worn while working with exposed samples or pure analytical standards. Latex gloves are commonly used to reduce exposure of the hands. When handling samples suspected or known to contain high concentrations of the CDDs/CDFs, an additional set of gloves can also be worn beneath the latex gloves.

5.3.3 Training—Workers must be trained in the proper method of removing contaminated gloves and clothing without contacting the exterior surfaces.

5.3.4 Personal hygiene—Hands and forearms should be washed thoroughly after each manipulation and before breaks (coffee, lunch, and shift).

5.3.5 Confinement—Isolated work areas posted with signs, segregated glassware and tools, and plastic absorbent paper on bench tops will aid in confining contamination.

5.3.6 Effluent vapors—The effluents of sample splitters from the gas chromatograph (GC) and from roughing pumps on the mass spectrometer (MS) should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols to condense CDD/CDF vapors.

5.3.7 Waste Handling—Good technique includes minimizing contaminated waste. Plastic bag liners should be used in waste cans. Janitors and other personnel must be trained in the safe handling of waste.

5.3.8 Decontamination

5.3.8.1 Decontamination of personnel—Use any mild soap with plenty of scrubbing action.

5.3.8.2 Glassware, tools, and surfaces—Chlorothene NU Solvent is the least toxic solvent shown to be effective. Satisfactory cleaning may be accomplished by rinsing with Chlorothene, then washing with any detergent and water. If glassware is first rinsed with solvent, then the dish water may be disposed of in the sewer. Given the cost of disposal, it is prudent to minimize solvent wastes.

5.3.9 Laundry—Clothing known to be contaminated should be collected in plastic bags. Persons who convey the bags and launder the clothing should be advised of the hazard and trained in proper handling. The clothing may be put into a washer without contact if the launderer knows of the potential problem. The washer should be run through a cycle before being used again for other clothing.

5.3.10 Wipe tests—A useful method of determining cleanliness of work surfaces and tools is to wipe the surface with a piece of filter paper. Extraction and analysis by GC with an electron capture detector (ECD) can achieve a limit of detection of 0.1 µg per wipe; analysis using this method can achieve an even lower detection limit. Less than 0.1 µg per wipe indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 µg on a wipe constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space, and indicates that unacceptable work practices have been employed.

5.3.11 Table or wrist-action shaker—The use of a table or wrist-action shaker for extraction of tissues presents the possibility of breakage of the extraction bottle and spillage of acid and flammable organic solvent. A secondary containment system around the shaker is suggested to prevent the spread of acid and solvents in the event of such a breakage. The speed and intensity of shaking action should also be adjusted to minimize the possibility of breakage.

6.0 Apparatus and Materials

Note:

Brand names, suppliers, and part numbers are for illustration purposes only and no endorsement is implied. Equivalent performance may be achieved using apparatus and materials other than those specified here. Meeting the performance requirements of this method is the responsibility of the laboratory.

6.1 Sampling Equipment for Discrete or Composite Sampling

6.1.1 Sample bottles and caps

6.1.1.1 Liquid samples (waters, sludges and similar materials containing 5% solids or less)—Sample bottle, amber glass, 1.1 L minimum, with screw cap.

6.1.1.2 Solid samples (soils, sediments, sludges, paper pulps, filter cake, compost, and similar materials that contain more than 5% solids)—Sample bottle, wide mouth, amber glass, 500 mL minimum.

6.1.1.3 If amber bottles are not available, samples shall be protected from light.

6.1.1.4 Bottle caps—Threaded to fit sample bottles. Caps shall be lined with fluoropolymer.

6.1.1.5 Cleaning

6.1.1.5.1 Bottles are detergent water washed, then solvent rinsed before use.

6.1.1.5.2 Liners are detergent water washed, rinsed with reagent water (Section 7.6.1) followed by solvent, and baked at approximately 200 °C for a minimum of 1 hour prior to use.

6.1.2 Compositing equipment—Automatic or manual compositing system incorporating glass containers cleaned per bottle cleaning procedure above. Only glass or fluoropolymer tubing shall be used. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used in the pump only. Before use, the tubing shall be thoroughly rinsed with methanol, followed by repeated rinsing with reagent water to minimize sample contamination. An integrating flow meter is used to collect proportional composite samples.

6.2 Equipment for Glassware Cleaning—Laboratory sink with overhead fume hood.

6.3 Equipment for Sample Preparation

6.3.1 Laboratory fume hood of sufficient size to contain the sample preparation equipment listed below.

6.3.2 Glove box (optional).

6.3.3 Tissue homogenizer—VirTis Model 45 Macro homogenizer (American Scientific Products H–3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear blade.

6.3.4 Meat grinder—Hobart, or equivalent, with 3–5 mm holes in inner plate.

6.3.5 Equipment for determining percent moisture

6.3.5.1 Oven—Capable of maintaining a temperature of 110 ±5 °C.

6.3.5.2 Dessicator.

6.3.6 Balances

6.3.6.1 Analytical—Capable of weighing 0.1 mg.

6.3.6.2 Top loading—Capable of weighing 10 mg.

6.4 Extraction Apparatus

6.4.1 Water samples

6.4.1.1 pH meter, with combination glass electrode.

6.4.1.2 pH paper, wide range (Hydrion Papers, or equivalent).

6.4.1.3 Graduated cylinder, 1 L capacity.

6.4.1.4 Liquid/liquid extraction—Separatory funnels, 250 mL, 500 mL, and 2000 mL, with fluoropolymer stopcocks.

6.4.1.5 Solid-phase extraction

6.4.1.5.1 One liter filtration apparatus, including glass funnel, glass frit support, clamp, adapter, stopper, filtration flask, and vacuum tubing (Figure 4). For wastewater samples, the apparatus should accept 90 or 144 mm disks. For drinking water or other samples containing low solids, smaller disks may be used.

6.4.1.5.2 Vacuum source capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum gauge.

6.4.1.5.3 Glass-fiber filter—Whatman GMF 150 (or equivalent), 1 micron pore size, to fit filtration apparatus in Section 6.4.1.5.1.

6.4.1.5.4 Solid-phase extraction disk containing octadecyl (C18) bonded silica uniformly enmeshed in an inert matrix—Fisher Scientific 14–378F (or equivalent), to fit filtration apparatus in Section 6.4.1.5.1.

6.4.2 Soxhlet/Dean-Stark (SDS) extractor (Figure 5)—For filters and solid/sludge samples.

6.4.2.1 Soxhlet—50 mm ID, 200 mL capacity with 500 mL flask (Cal-Glass LG–6900, or equivalent, except substitute 500 mL round-bottom flask for 300 mL flat-bottom flask).

6.4.2.2 Thimble—43 × 123 to fit Soxhlet (Cal-Glass LG–6901–122, or equivalent).

6.4.2.3 Moisture trap—Dean Stark or Barret with fluoropolymer stopcock, to fit Soxhlet.

6.4.2.4 Heating mantle—Hemispherical, to fit 500 mL round-bottom flask (Cal-Glass LG–8801–112, or equivalent).

6.4.2.5 Variable transformer—Powerstat (or equivalent), 110 volt, 10 amp.

6.4.3 Apparatus for extraction of tissue.

6.4.3.1 Bottle for extraction (if digestion/extraction using HCl is used)” 500–600 mL wide-mouth clear glass, with fluoropolymer-lined cap.

6.4.3.2 Bottle for back-extraction—100–200 mL narrow-mouth clear glass with fluoropolymer-lined cap.

6.4.3.3 Mechanical shaker—Wrist-action or platform-type rotary shaker that produces vigorous agitation (Sybron Thermolyne Model LE “Big Bill” rotator/shaker, or equivalent).

6.4.3.4 Rack attached to shaker table to permit agitation of four to nine samples simultaneously.

6.4.4 Beakers—400–500 mL.

6.4.5 Spatulas—Stainless steel.

6.5 Filtration Apparatus.

6.5.1 Pyrex glass wool—Solvent-extracted by SDS for three hours minimum.

Note:

Baking glass wool may cause active sites that will irreversibly adsorb CDDs/CDFs.

6.5.2 Glass funnel—125–250 mL.

6.5.3 Glass-fiber filter paper—Whatman GF/D (or equivalent), to fit glass funnel in Section 6.5.2.

6.5.4 Drying column—15–20 mm ID Pyrex chromatographic column equipped with coarse-glass frit or glass-wool plug.

6.5.5 Buchner funnel—15 cm.

6.5.6 Glass-fiber filter paper—to fit Buchner funnel in Section 6.5.5.

6.5.7 Filtration flasks—1.5–2.0 L, with side arm.

6.5.8 Pressure filtration apparatus—Millipore YT30 142 HW, or equivalent.

6.6 Centrifuge Apparatus.

6.6.1 Centrifuge—Capable of rotating 500 mL centrifuge bottles or 15 mL centrifuge tubes at 5,000 rpm minimum.

6.6.2 Centrifuge bottles—500 mL, with screw-caps, to fit centrifuge.

6.6.3 Centrifuge tubes—12–15 mL, with screw-caps, to fit centrifuge.

6.7 Cleanup Apparatus.

6.7.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc, Columbia, MO, Model GPC Autoprep 1002, or equivalent).

6.7.1.1 Column—600–700 mm long × 25 mm ID, packed with 70 g of

SX–3 Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).

6.7.1.2 Syringe—10 mL, with Luer fitting.

6.7.1.3 Syringe filter holder—stainless steel, and glass-fiber or fluoropolymer filters (Gelman 4310, or equivalent).

6.7.1.4 UV detectors—254 nm, preparative or semi-preparative flow cell (Isco, Inc., Type 6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8 µL micro-prep flow cell, 2 mm path; Pharmacia UV–1, 3 mm flow cell; LDC Milton-Roy UV–3, monitor #1203; or equivalent).

6.7.2 Reverse-phase high-performance liquid chromatograph.

6.7.2.1 Column oven and detector—Perkin-Elmer Model LC–65T (or equivalent) operated at 0.02 AUFS at 235 nm.

6.7.2.2 Injector—Rheodyne 7120 (or equivalent) with 50 µL sample loop.

6.7.2.3 Column—Two 6.2 mm × 250 mm Zorbax-ODS columns in series (DuPont Instruments Division, Wilmington, DE, or equivalent), operated at 50 °C with 2.0 mL/min methanol isocratic effluent.

6.7.2.4 Pump—Altex 110A (or equivalent).

6.7.3 Pipets.

6.7.3.1 Disposable, pasteur—150 mm long × 5-mm ID (Fisher Scientific 13–678–6A, or equivalent).

6.7.3.2 Disposable, serological—10 mL (6 mm ID).

6.7.4 Glass chromatographic columns.

6.7.4.1 150 mm long × 8-mm ID, (Kontes K–420155, or equivalent) with coarse-glass frit or glass-wool plug and 250 mL reservoir.

6.7.4.2 200 mm long × 15 mm ID, with coarse-glass frit or glass-wool plug and 250 mL reservoir.

6.7.4.3 300 mm long × 25 mm ID, with 300 mL reservoir and glass or fluoropolymer stopcock.

6.7.5 Stirring apparatus for batch silica cleanup of tissue extracts.

6.7.5.1 Mechanical stirrer—Corning Model 320, or equivalent.

6.7.5.2 Bottle—500–600 mL wide-mouth clear glass.

6.7.6 Oven—For baking and storage of adsorbents, capable of maintaining a constant temperature (±5 °C) in the range of 105–250 °C.

6.8 Concentration Apparatus.

6.8.1 Rotary evaporator—Buchi/Brinkman-American Scientific No. E5045–10 or equivalent, equipped with a variable temperature water bath.

6.8.1.1 Vacuum source for rotary evaporator equipped with shutoff valve at the evaporator and vacuum gauge.

6.8.1.2 A recirculating water pump and chiller are recommended, as use of tap water for cooling the evaporator wastes large volumes of water and can lead to inconsistent performance as water temperatures and pressures vary.

6.8.1.3 Round-bottom flask—100 mL and 500 mL or larger, with ground-glass fitting compatible with the rotary evaporator.

6.8.2 Kuderna-Danish (K-D) Concentrator.

6.8.2.1 Concentrator tube—10 mL, graduated (Kontes K–570050–1025, or equivalent) with calibration verified. Ground-glass stopper (size 19/22 joint) is used to prevent evaporation of extracts.

6.8.2.2 Evaporation flask—500 mL (Kontes K–570001–0500, or equivalent), attached to concentrator tube with springs (Kontes K–662750–0012 or equivalent).

6.8.2.3 Snyder column—Three-ball macro (Kontes K–503000–0232, or equivalent).

6.8.2.4 Boiling chips.

6.8.2.4.1 Glass or silicon carbide—Approximately 10/40 mesh, extracted with methylene chloride and baked at 450 °C for one hour minimum.

6.8.2.4.2 Fluoropolymer (optional)—Extracted with methylene chloride.

6.8.2.5 Water bath—Heated, with concentric ring cover, capable of maintaining a temperature within ±2 °C, installed in a fume hood.

6.8.3 Nitrogen blowdown apparatus—Equipped with water bath controlled in the range of 30–60 °C (N-Evap, Organomation Associates, Inc., South Berlin, MA, or equivalent), installed in a fume hood.

6.8.4 Sample vials.

6.8.4.1 Amber glass—2–5 mL with fluoropolymer-lined screw-cap.

6.8.4.2 Glass—0.3 mL, conical, with fluoropolymer-lined screw or crimp cap.

6.9 Gas Chromatograph—Shall have splitless or on-column injection port for capillary column, temperature program with isothermal hold, and shall meet all of the performance specifications in Section 10.

6.9.1 GC column for CDDs/CDFs and for isomer specificity for 2,3,7,8-TCDD—60 ±5 m long × 0.32 ±0.02 mm ID; 0.25 µm 5% phenyl, 94% methyl, 1% vinyl silicone bonded-phase fused-silica capillary column (J&W DB–5, or equivalent).

6.9.2 GC column for isomer specificity for 2,3,7,8-TCDF—30 ±5 m long × 0.32 ±0.02 mm ID; 0.25 µm bonded-phase fused-silica capillary column (J&W DB–225, or equivalent).

6.10 Mass Spectrometer—28–40 eV electron impact ionization, shall be capable of repetitively selectively monitoring 12 exact m/z's minimum at high resolution (≥10,000) during a period of approximately one second, and shall meet all of the performance specifications in Section 10.

6.11 GC/MS Interface—The mass spectrometer (MS) shall be interfaced to the GC such that the end of the capillary column terminates within 1 cm of the ion source but does not intercept the electron or ion beams.

6.12 Data System—Capable of collecting, recording, and storing MS data.

7.0 Reagents and Standards

7.1 pH Adjustment and Back-Extraction.

7.1.1 Potassium hydroxide—Dissolve 20 g reagent grade KOH in 100 mL reagent water.

7.1.2 Sulfuric acid—Reagent grade (specific gravity 1.84).

7.1.3 Hydrochloric acid—Reagent grade, 6N.

7.1.4 Sodium chloride—Reagent grade, prepare at 5% (w/v) solution in reagent water.

7.2 Solution Drying and Evaporation.

7.2.1 Solution drying—Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 °C for one hour minimum, cooled in a dessicator, and stored in a pre-cleaned glass bottle with screw-cap that prevents moisture from entering. If, after heating, the sodium sulfate develops a noticeable grayish cast (due to the presence of carbon in the crystal matrix), that batch of reagent is not suitable for use and should be discarded. Extraction with methylene chloride (as opposed to simple rinsing) and baking at a lower temperature may produce sodium sulfate that is suitable for use.

7.2.2 Tissue drying—Sodium sulfate, reagent grade, powdered, treated and stored as above.

7.2.3 Prepurified nitrogen.

7.3 Extraction.

7.3.1 Solvents—Acetone, toluene, cyclohexane, hexane, methanol, methylene chloride, and nonane; distilled in glass, pesticide quality, lot-certified to be free of interferences.

7.3.2 White quartz sand, 60/70 mesh—For Soxhlet/Dean-Stark extraction (Aldrich Chemical, Cat. No. 27–437–9, or equivalent). Bake at 450 °C for four hours minimum.

7.4 GPC Calibration Solution—Prepare a solution containing 300 mg/mL corn oil, 15 mg/mL bis(2-ethylhexyl) phthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.

7.5 Adsorbents for Sample Cleanup.

7.5.1 Silica gel.

7.5.1.1 Activated silica gel—100–200 mesh, Supelco 1–3651 (or equivalent), rinsed with methylene chloride, baked at 180 °C for a minimum of one hour, cooled in a dessicator, and stored in a precleaned glass bottle with screw-cap that prevents moisture from entering.

7.5.1.2 Acid silica gel (30% w/w)—Thoroughly mix 44.0 g of concentrated sulfuric acid with 100.0 g of activated silica gel in a clean container. Break up aggregates with a stirring rod until a uniform mixture is obtained. Store in a bottle with a fluoropolymer-lined screw-cap.

7.5.1.3 Basic silica gel—Thoroughly mix 30 g of 1N sodium hydroxide with 100 g of activated silica gel in a clean container. Break up aggregates with a stirring rod until a uniform mixture is obtained. Store in a bottle with a fluoropolymer-lined screw-cap.

7.5.1.4 Potassium silicate.

7.5.1.4.1 Dissolve 56 g of high purity potassium hydroxide (Aldrich, or equivalent) in 300 mL of methanol in a 750–1000 mL flat-bottom flask.

7.5.1.4.2 Add 100 g of silica gel and a stirring bar, and stir on a hot plate at 60–70 °C for one to two hours.

7.5.1.4.3 Decant the liquid and rinse the potassium silicate twice with 100 mL portions of methanol, followed by a single rinse with 100 mL of methylene chloride.

7.5.1.4.4 Spread the potassium silicate on solvent-rinsed aluminum foil and dry for two to four hours in a hood.

7.5.1.4.5 Activate overnight at 200–250 °C.

7.5.2 Alumina—Either one of two types of alumina, acid or basic, may be used in the cleanup of sample extracts, provided that the laboratory can meet the performance specifications for the recovery of labeled compounds described in Section 9.3. The same type of alumina must be used for all samples, including those used to demonstrate initial precision and recovery (Section 9.2) and ongoing precision and recovery (Section 15.5).

7.5.2.1 Acid alumina—Supelco 19996–6C (or equivalent). Activate by heating to 130 °C for a minimum of 12 hours.

7.5.2.2 Basic alumina—Supelco 19944–6C (or equivalent). Activate by heating to 600 °C for a minimum of 24 hours. Alternatively, activate by heating in a tube furnace at 650–700 °C under an air flow rate of approximately 400 cc/minute. Do not heat over 700 °C, as this can lead to reduced capacity for retaining the analytes. Store at 130 °C in a covered flask. Use within five days of baking.

7.5.3 Carbon.

7.5.3.1 Carbopak C—(Supelco 1–0258, or equivalent).

7.5.3.2 Celite 545—(Supelco 2–0199, or equivalent).

7.5.3.3 Thoroughly mix 9.0 g Carbopak C and 41.0 g Celite 545 to produce an 18% w/w mixture. Activate the mixture at 130 °C for a minimum of six hours. Store in a dessicator.

7.5.4 Anthropogenic isolation column—Pack the column in Section 6.7.4.3 from bottom to top with the following:

7.5.4.1 2 g silica gel (Section 7.5.1.1).

7.5.4.2 2 g potassium silicate (Section 7.5.1.4).

7.5.4.3 2 g granular anhydrous sodium sulfate (Section 7.2.1).

7.5.4.4 10 g acid silica gel (Section 7.5.1.2).

7.5.4.5 2 g granular anhydrous sodium sulfate.

7.5.5 Florisil column.

7.5.5.1 Florisil—60–100 mesh, Floridin Corp (or equivalent). Soxhlet extract in 500 g portions for 24 hours.

7.5.5.2 Insert a glass wool plug into the tapered end of a graduated serological pipet (Section 6.7.3.2). Pack with 1.5 g (approx 2 mL) of Florisil topped with approx 1 mL of sodium sulfate (Section 7.2.1) and a glass wool plug.

7.5.5.3 Activate in an oven at 130–150 °C for a minimum of 24 hours and cool for 30 minutes. Use within 90 minutes of cooling.

7.6 Reference Matrices—Matrices in which the CDDs/CDFs and interfering compounds are not detected by this method.

7.6.1 Reagent water—Bottled water purchased locally, or prepared by passage through activated carbon.

7.6.2 High-solids reference matrix—Playground sand or similar material. Prepared by extraction with methylene chloride and/or baking at 450 °C for a minimum of four hours.

7.6.3 Paper reference matrix—Glass-fiber filter, Gelman Type A, or equivalent. Cut paper to simulate the surface area of the paper sample being tested.

7.6.4 Tissue reference matrix—Corn or other vegetable oil. May be prepared by extraction with methylene chloride.

7.6.5 Other matrices—This method may be verified on any reference matrix by performing the tests given in Section 9.2. Ideally, the matrix should be free of the CDDs/CDFs, but in no case shall the background level of the CDDs/CDFs in the reference matrix exceed three times the minimum levels in Table 2. If low background levels of the CDDs/CDFs are present in the reference matrix, the spike level of the analytes used in Section 9.2 should be increased to provide a spike-to-background ratio in the range of 1:1 to 5:1 (Reference 15).

7.7 Standard Solutions—Purchased as solutions or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If the chemical purity is 98% or greater, the weight may be used without correction to compute the concentration of the standard. When not being used, standards are stored in the dark at room temperature in screw-capped vials with fluoropolymer-lined caps. A mark is placed on the vial at the level of the solution so that solvent loss by evaporation can be detected. If solvent loss has occurred, the solution should be replaced.

7.8 Stock Solutions.

7.8.1 Preparation—Prepare in nonane per the steps below or purchase as dilute solutions (Cambridge Isotope Laboratories (CIL), Woburn, MA, or equivalent). Observe the safety precautions in Section 5, and the recommendation in Section 5.1.2.

7.8.2 Dissolve an appropriate amount of assayed reference material in solvent. For example, weigh 1–2 mg of 2,3,7,8-TCDD to three significant figures in a 10 mL ground-glass-stoppered volumetric flask and fill to the mark with nonane. After the TCDD is completely dissolved, transfer the solution to a clean 15 mL vial with fluoropolymer-lined cap.

7.8.3 Stock standard solutions should be checked for signs of degradation prior to the preparation of calibration or performance test standards. Reference standards that can be used to determine the accuracy of calibration standards are available from CIL and may be available from other vendors.

7.9 PAR Stock Solution

7.9.1 All CDDs/CDFs—Using the solutions in Section 7.8, prepare the PAR stock solution to contain the CDDs/CDFs at the concentrations shown in Table 3. When diluted, the solution will become the PAR (Section 7.14).

7.9.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the PAR stock solution to contain these compounds only.

7.10 Labeled-Compound Spiking Solution.

7.10.1 All CDDs/CDFs—From stock solutions, or from purchased mixtures, prepare this solution to contain the labeled compounds in nonane at the concentrations shown in Table 3. This solution is diluted with acetone prior to use (Section 7.10.3).

7.10.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the labeled-compound solution to contain these compounds only. This solution is diluted with acetone prior to use (Section 7.10.3).

7.10.3 Dilute a sufficient volume of the labeled compound solution (Section 7.10.1 or 7.10.2) by a factor of 50 with acetone to prepare a diluted spiking solution. Each sample requires 1.0 mL of the diluted solution, but no more solution should be prepared than can be used in one day.

7.11 Cleanup Standard—Prepare37Cl4-2,3,7,8-TCDD in nonane at the concentration shown in Table 3. The cleanup standard is added to all extracts prior to cleanup to measure the efficiency of the cleanup process.

7.12 Internal Standard(s).

7.12.1 All CDDs/CDFs—Prepare the internal standard solution to contain13C12-1,2,3,4-TCDD and13C2-1,2,3,7,8,9-HxCDD in nonane at the concentration shown in Table 3.

7.12.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the internal standard solution to contain13C12-1,2,3,4-TCDD only.

7.13 Calibration Standards (CS1 through CS5)—Combine the solutions in Sections 7.9 through 7.12 to produce the five calibration solutions shown in Table 4 in nonane. These solutions permit the relative response (labeled to native) and response factor to be measured as a function of concentration. The CS3 standard is used for calibration verification (VER). If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, combine the solutions appropriate to these compounds.

7.14 Precision and Recovery (PAR) Standard—Used for determination of initial (Section 9.2) and ongoing (Section 15.5) precision and recovery. Dilute 10 µL of the precision and recovery standard (Section 7.9.1 or 7.9.2) to 2.0 mL with acetone for each sample matrix for each sample batch. One mL each are required for the blank and OPR with each matrix in each batch.

7.15 GC Retention Time Window Defining Solution and Isomer Specificity Test Standard—Used to define the beginning and ending retention times for the dioxin and furan isomers and to demonstrate isomer specificity of the GC columns employed for determination of 2,3,7,8-TCDD and 2,3,7,8-TCDF. The standard must contain the compounds listed in Table 5 (CIL EDF—4006, or equivalent), at a minimum. It is not necessary to monitor the window-defining compounds if only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined. In this case, an isomer-specificity test standard containing the most closely eluted isomers listed in Table 5 (CIL EDF-4033, or equivalent) may be used.

7.16 QC Check Sample—A QC Check Sample should be obtained from a source independent of the calibration standards. Ideally, this check sample would be a certified reference material containing the CDDs/CDFs in known concentrations in a sample matrix similar to the matrix under test.

7.17 Stability of Solutions—Standard solutions used for quantitative purposes (Sections 7.9 through 7.15) should be analyzed periodically, and should be assayed against reference standards (Section 7.8.3) before further use.

8.0 Sample Collection, Preservation, Storage, and Holding Times

8.1 Collect samples in amber glass containers following conventional sampling practices (Reference 16). Aqueous samples that flow freely are collected in refrigerated bottles using automatic sampling equipment. Solid samples are collected as grab samples using wide-mouth jars.

8.2 Maintain aqueous samples in the dark at 0–4 °C from the time of collection until receipt at the laboratory. If residual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 17). If sample pH is greater than 9, adjust to pH 7–9 with sulfuric acid.

Maintain solid, semi-solid, oily, and mixed-phase samples in the dark at <4 °C from the time of collection until receipt at the laboratory.

Store aqueous samples in the dark at 0–4 °C. Store solid, semi-solid, oily, mixed-phase, and tissue samples in the dark at <−10 °C.

8.3 Fish and Tissue Samples.

8.3.1 Fish may be cleaned, filleted, or processed in other ways in the field, such that the laboratory may expect to receive whole fish, fish fillets, or other tissues for analysis.

8.3.2 Fish collected in the field should be wrapped in aluminum foil, and must be maintained at a temperature less than 4 °C from the time of collection until receipt at the laboratory.

8.3.3 Samples must be frozen upon receipt at the laboratory and maintained in the dark at <−10 °C until prepared. Maintain unused sample in the dark at <−10 °C.

8.4 Holding Times.

8.4.1 There are no demonstrated maximum holding times associated with CDDs/CDFs in aqueous, solid, semi-solid, tissues, or other sample matrices. If stored in the dark at 0–4 °C and preserved as given above (if required), aqueous samples may be stored for up to one year. Similarly, if stored in the dark at <−10 °C, solid, semi-solid, multi-phase, and tissue samples may be stored for up to one year.

8.4.2 Store sample extracts in the dark at <−10 °C until analyzed. If stored in the dark at <−10 °C, sample extracts may be stored for up to one year.

9.0 Quality Assurance/Quality Control

9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Reference 18). The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

If the method is to be applied to sample matrix other than water (e.g., soils, filter cake, compost, tissue) the most appropriate alternate matrix (Sections 7.6.2 through 7.6.5) is substituted for the reagent water matrix (Section 7.6.1) in all performance tests.

9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 9.2.

9.1.2 In recognition of advances that are occurring in analytical technology, and to allow the analyst to overcome sample matrix interferences, the analyst is permitted certain options to improve separations or lower the costs of measurements. These options include alternate extraction, concentration, cleanup procedures, and changes in columns and detectors. Alternate determinative techniques, such as the substitution of spectroscopic or immuno-assay techniques, and changes that degrade method performance, are not allowed. If an analytical technique other than the techniques specified in this method is used, that technique must have a specificity equal to or better than the specificity of the techniques in this method for the analytes of interest.

9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat the procedure in Section 9.2. If the detection limit of the method will be affected by the change, the laboratory is required to demonstrate that the MDL (40 CFR part 136, appendix B) is lower than one-third the regulatory compliance level or one-third the ML in this method, whichever is higher. If calibration will be affected by the change, the analyst must recalibrate the instrument per Section 10.

9.1.2.2 The laboratory is required to maintain records of modifications made to this method. These records include the following, at a minimum:

9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s) who performed the analyses and modification, and of the quality control officer who witnessed and will verify the analyses and modifications.

9.1.2.2.2 A listing of pollutant(s) measured, by name and CAS Registry number.

9.1.2.2.3 A narrative stating reason(s) for the modifications.

9.1.2.2.4 Results from all quality control (QC) tests comparing the modified method to this method, including:

(a) Calibration (Section 10.5 through 10.7).

(b) Calibration verification (Section 15.3).

(c) Initial precision and recovery (Section 9.2).

(d) Labeled compound recovery (Section 9.3).

(e) Analysis of blanks (Section 9.5).

(f) Accuracy assessment (Section 9.4).

9.1.2.2.5 Data that will allow an independent reviewer to validate each determination by tracing the instrument output (peak height, area, or other signal) to the final result. These data are to include:

(a) Sample numbers and other identifiers.

(b) Extraction dates.

(c) Analysis dates and times.

(d) Analysis sequence/run chronology.

(e) Sample weight or volume (Section 11).

(f) Extract volume prior to each cleanup step (Section 13).

(g) Extract volume after each cleanup step (Section 13).

(h) Final extract volume prior to injection (Section 14).

(i) Injection volume (Section 14.3).

(j) Dilution data, differentiating between dilution of a sample or extract (Section 17.5).

(k) Instrument and operating conditions.

(l) Column (dimensions, liquid phase, solid support, film thickness, etc).

(m) Operating conditions (temperatures, temperature program, flow rates).

(n) Detector (type, operating conditions, etc).

(o) Chromatograms, printer tapes, and other recordings of raw data.

(p) Quantitation reports, data system outputs, and other data to link the raw data to the results reported.

9.1.3 Analyses of method blanks are required to demonstrate freedom from contamination (Section 4.3). The procedures and criteria for analysis of a method blank are described in Sections 9.5 and 15.6.

9.1.4 The laboratory shall spike all samples with labeled compounds to monitor method performance. This test is described in Section 9.3. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits. Procedures for dilution are given in Section 17.5.

9.1.5 The laboratory shall, on an ongoing basis, demonstrate through calibration verification and the analysis of the ongoing precision and recovery aliquot that the analytical system is in control. These procedures are described in Sections 15.1 through 15.5.

9.1.6 The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Section 9.4.

9.2 Initial Precision and Recovery (IPR)—To establish the ability to generate acceptable precision and recovery, the analyst shall perform the following operations.

9.2.1 For low solids (aqueous) samples, extract, concentrate, and analyze four 1 L aliquots of reagent water spiked with the diluted labeled compound spiking solution (Section 7.10.3) and the precision and recovery standard (Section 7.14) according to the procedures in Sections 11 through 18. For an alternative sample matrix, four aliquots of the alternative reference matrix (Section 7.6) are used. All sample processing steps that are to be used for processing samples, including preparation (Section 11), extraction (Section 12), and cleanup (Section 13), shall be included in this test.

9.2.2 Using results of the set of four analyses, compute the average concentration (X) of the extracts in ng/mL and the standard deviation of the concentration (s) in ng/mL for each compound, by isotope dilution for CDDs/CDFs with a labeled analog, and by internal standard for 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds.

9.2.3 For each CDD/CDF and labeled compound, compare s and X with the corresponding limits for initial precision and recovery in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare s and X with the corresponding limits for initial precision and recovery in Table 6a. If s and X for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If, however, any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, system performance is unacceptable for that compound. Correct the problem and repeat the test (Section 9.2).

9.3 The laboratory shall spike all samples with the diluted labeled compound spiking solution (Section 7.10.3) to assess method performance on the sample matrix.

9.3.1 Analyze each sample according to the procedures in Sections 11 through 18.

9.3.2 Compute the percent recovery of the labeled compounds and the cleanup standard using the internal standard method (Section 17.2).

9.3.3 The recovery of each labeled compound must be within the limits in Table 7 when all 2,3,7,8-substituted CDDs/CDFs are determined, and within the limits in Table 7a when only 2,3,7,8-TCDD and 2,3,7,8-TCDF are determined. If the recovery of any compound falls outside of these limits, method performance is unacceptable for that compound in that sample. To overcome such difficulties, water samples are diluted and smaller amounts of soils, sludges, sediments, and other matrices are reanalyzed per Section 18.4.

9.4 Recovery of labeled compounds from samples should be assessed and records should be maintained.

9.4.1 After the analysis of five samples of a given matrix type (water, soil, sludge, pulp, etc.) for which the labeled compounds pass the tests in Section 9.3, compute the average percent recovery (R) and the standard deviation of the percent recovery (SR) for the labeled compounds only. Express the assessment as a percent recovery interval from R−2SR to R = 2SR for each matrix. For example, if R = 90% and SR = 10% for five analyses of pulp, the recovery interval is expressed as 70–110%.

9.4.2 Update the accuracy assessment for each labeled compound in each matrix on a regular basis (e.g., after each 5–10 new measurements).

9.5 Method Blanks—Reference matrix method blanks are analyzed to demonstrate freedom from contamination (Section 4.3).

9.5.1 Prepare, extract, clean up, and concentrate a method blank with each sample batch (samples of the same matrix started through the extraction process on the same 12-hour shift, to a maximum of 20 samples). The matrix for the method blank shall be similar to sample matrix for the batch, e.g., a 1 L reagent water blank (Section 7.6.1), high-solids reference matrix blank (Section 7.6.2), paper matrix blank (Section 7.6.3); tissue blank (Section 7.6.4) or alternative reference matrix blank (Section 7.6.5). Analyze the blank immediately after analysis of the OPR (Section 15.5) to demonstrate freedom from contamination.

9.5.2 If any 2,3,7,8-substituted CDD/CDF (Table 1) is found in the blank at greater than the minimum level (Table 2) or one-third the regulatory compliance level, whichever is greater; or if any potentially interfering compound is found in the blank at the minimum level for each level of chlorination given in Table 2 (assuming a response factor of 1 relative to the13C12-1,2,3,4-TCDD internal standard for compounds not listed in Table 1), analysis of samples is halted until the blank associated with the sample batch shows no evidence of contamination at this level. All samples must be associated with an uncontaminated method blank before the results for those samples may be reported for regulatory compliance purposes.

9.6 QC Check Sample—Analyze the QC Check Sample (Section 7.16) periodically to assure the accuracy of calibration standards and the overall reliability of the analytical process. It is suggested that the QC Check Sample be analyzed at least quarterly.

9.7 The specifications contained in this method can be met if the apparatus used is calibrated properly and then maintained in a calibrated state. The standards used for calibration (Section 10), calibration verification (Section 15.3), and for initial (Section 9.2) and ongoing (Section 15.5) precision and recovery should be identical, so that the most precise results will be obtained. A GC/MS instrument will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of CDDs/CDFs by this method.

9.8 Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when the internal standard method is used.

10.0 Calibration

10.1 Establish the operating conditions necessary to meet the minimum retention times for the internal standards in Section 10.2.4 and the relative retention times for the CDDs/CDFs in Table 2.

10.1.1 Suggested GC operating conditions:

Injector temperature: 270 °C

Interface temperature: 290 °C

Initial temperature: 200 °C

Initial time: Two minutes

Temperature program:

200–220 °C, at 5 °C/minute

220 °C for 16 minutes

220–235 °C, at 5 °C/minute

235 °C for seven minutes

235–330 °C, at 5 °C/minute

Note:

All portions of the column that connect the GC to the ion source shall remain at or above the interface temperature specified above during analysis to preclude condensation of less volatile compounds.

Optimize GC conditions for compound separation and sensitivity. Once optimized, the same GC conditions must be used for the analysis of all standards, blanks, IPR and OPR aliquots, and samples.

10.1.2 Mass spectrometer (MS) resolution—Obtain a selected ion current profile (SICP) of each analyte in Table 3 at the two exact m/z's specified in Table 8 and at ≥10,000 resolving power by injecting an authentic standard of the CDDs/CDFs either singly or as part of a mixture in which there is no interference between closely eluted components.

10.1.2.1 The analysis time for CDDs/CDFs may exceed the long-term mass stability of the mass spectrometer. Because the instrument is operated in the high-resolution mode, mass drifts of a few ppm (e.g., 5 ppm in mass) can have serious adverse effects on instrument performance. Therefore, a mass-drift correction is mandatory and a lock-mass m/z from PFK is used for drift correction. The lock-mass m/z is dependent on the exact m/z's monitored within each descriptor, as shown in Table 8. The level of PFK metered into the HRMS during analyses should be adjusted so that the amplitude of the most intense selected lock-mass m/z signal (regardless of the descriptor number) does not exceed 10% of the full-scale deflection for a given set of detector parameters. Under those conditions, sensitivity changes that might occur during the analysis can be more effectively monitored.

Note:

Excessive PFK (or any other reference substance) may cause noise problems and contamination of the ion source necessitating increased frequency of source cleaning.

10.1.2.2 If the HRMS has the capability to monitor resolution during the analysis, it is acceptable to terminate the analysis when the resolution falls below 10,000 to save reanalysis time.

10.1.2.3 Using a PFK molecular leak, tune the instrument to meet the minimum required resolving power of 10,000 (10% valley) at m/z 304.9824 (PFK) or any other reference signal close to m/z 304 (from TCDF). For each descriptor (Table 8), monitor and record the resolution and exact m/z's of three to five reference peaks covering the mass range of the descriptor. The resolution must be greater than or equal to 10,000, and the deviation between the exact m/z and the theoretical m/z (Table 8) for each exact m/z monitored must be less than 5 ppm.

10.2 Ion Abundance Ratios, Minimum Levels, Signal-to-Noise Ratios, and Absolute Retention Times—Choose an injection volume of either 1 µL or 2 µL, consistent with the capability of the HRGC/HRMS instrument. Inject a 1 µL or 2 µL aliquot of the CS1 calibration solution (Table 4) using the GC conditions from Section 10.1.1. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the operating conditions and specifications below apply to analysis of those compounds only.

10.2.1 Measure the SICP areas for each analyte, and compute the ion abundance ratios at the exact m/z's specified in Table 8. Compare the computed ratio to the theoretical ratio given in Table 9.

10.2.1.1 The exact m/z's to be monitored in each descriptor are shown in Table 8. Each group or descriptor shall be monitored in succession as a function of GC retention time to ensure that all CDDs/CDFs are detected. Additional m/z's may be monitored in each descriptor, and the m/z's may be divided among more than the five descriptors listed in Table 8, provided that the laboratory is able to monitor the m/z's of all the CDDs/CDFs that may elute from the GC in a given retention-time window. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the descriptors may be modified to include only the exact m/z's for the tetra-and penta-isomers, the diphenyl ethers, and the lock m/z's.

10.2.1.2 The mass spectrometer shall be operated in a mass-drift correction mode, using perfluorokerosene (PFK) to provide lock m/z's. The lock-mass for each group of m/z's is shown in Table 8. Each lock mass shall be monitored and shall not vary by more than ±20% throughout its respective retention time window. Variations of the lock mass by more than 20% indicate the presence of coeluting interferences that may significantly reduce the sensitivity of the mass spectrometer. Reinjection of another aliquot of the sample extract will not resolve the problem. Additional cleanup of the extract may be required to remove the interferences.

10.2.2 All CDDs/CDFs and labeled compounds in the CS1 standard shall be within the QC limits in Table 9 for their respective ion abundance ratios; otherwise, the mass spectrometer shall be adjusted and this test repeated until the m/z ratios fall within the limits specified. If the adjustment alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2) prior to repeat of the test.

10.2.3 Verify that the HRGC/HRMS instrument meets the minimum levels in Table 2. The peaks representing the CDDs/CDFs and labeled compounds in the CS1 calibration standard must have signal-to-noise ratios (S/N) greater than or equal to 10.0. Otherwise, the mass spectrometer shall be adjusted and this test repeated until the minimum levels in Table 2 are met.

10.2.4 The absolute retention time of13C12-1,2,3,4–TCDD (Section 7.12) shall exceed 25.0 minutes on the DB–5 column, and the retention time of13C12-1,2,3,4–TCDD shall exceed 15.0 minutes on the DB–225 column; otherwise, the GC temperature program shall be adjusted and this test repeated until the above-stated minimum retention time criteria are met.

2010.3 Retention-Time Windows—Analyze the window defining mixtures (Section 7.15) using the optimized temperature program in Section 10.1. Table 5 gives the elution order (first/last) of the window-defining compounds. If 2,3,7,8-TCDD and 2,3,7,8-TCDF only are to be analyzed, this test is not required.

10.4 Isomer Specificity.

10.4.1 Analyze the isomer specificity test standards (Section 7.15) using the procedure in Section 14 and the optimized conditions for sample analysis (Section 10.1.1).

10.4.2 Compute the percent valley between the GC peaks that elute most closely to the 2,3,7,8-TCDD and TCDF isomers, on their respective columns, per Figures 6 and 7.

10.4.3 Verify that the height of the valley between the most closely eluted isomers and the 2,3,7,8-substituted isomers is less than 25% (computed as 100 x/y in Figures 6 and 7). If the valley exceeds 25%, adjust the analytical conditions and repeat the test or replace the GC column and recalibrate (Sections 10.1.2 through 10.7).

10.5 Calibration by Isotope Dilution—Isotope dilution calibration is used for the 15 2,3,7,8-substituted CDDs/CDFs for which labeled compounds are added to samples prior to extraction. The reference compound for each CDD/CDF compound is shown in Table 2.

10.5.1 A calibration curve encompassing the concentration range is prepared for each compound to be determined. The relative response (RR) (labeled to native) vs. concentration in standard solutions is plotted or computed using a linear regression. Relative response is determined according to the procedures described below. Five calibration points are employed.

10.5.2 The response of each CDD/CDF relative to its labeled analog is determined using the area responses of both the primary and secondary exact m/z's specified in Table 8, for each calibration standard, as follows:

where:

A1n and A2n = The areas of the primary and secondary m/z's for the CDD/CDF.

A1l and A2l = The areas of the primary and secondary m/z's for the labeled compound.

Cl = The concentration of the labeled compound in the calibration standard (Table 4).

Cn = The concentration of the native compound in the calibration standard (Table 4).

10.5.3 To calibrate the analytical system by isotope dilution, inject a volume of calibration standards CS1 through CS5 (Section 7.13 and Table 4) identical to the volume chosen in Section 10.2, using the procedure in Section 14 and the conditions in Section 10.1.1 and Table 2. Compute the relative response (RR) at each concentration.

10.5.4 Linearity—If the relative response for any compound is constant (less than 20% coefficient of variation) over the five-point calibration range, an averaged relative response may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the five-point calibration range.

10.6 Calibration by Internal Standard—The internal standard method is applied to determination of 1,2,3,7,8,9-HxCDD (Section 17.1.2), OCDF (Section 17.1.1), the non 2,3,7,8-substituted compounds, and to the determination of labeled compounds for intralaboratory statistics (Sections 9.4 and 15.5.4).

10.6.1 Response factors—Calibration requires the determination of response factors (RF) defined by the following equation:

where:

A1s and A2s = The areas of the primary and secondary m/z's for the CDD/CDF.

A1is and A2is = The areas of the primary and secondary m/z's for the internal standard.

Cis = The concentration of the internal standard (Table 4).

Cs = The concentration of the compound in the calibration standard (Table 4).

Note:

There is only one m/z for37Cl4-2,3,7,8-TCDD. See Table 8.

10.6.2 To calibrate the analytical system by internal standard, inject 1.0 µL or 2.0 µL of calibration standards CS1 through CS5 (Section 7.13 and Table 4) using the procedure in Section 14 and the conditions in Section 10.1.1 and Table 2. Compute the response factor (RF) at each concentration.

10.6.3 Linearity—If the response factor (RF) for any compound is constant (less than 35% coefficient of variation) over the five-point calibration range, an averaged response factor may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the five-point range.

10.7 Combined Calibration—By using calibration solutions (Section 7.13 and Table 4) containing the CDDs/CDFs and labeled compounds and the internal standards, a single set of analyses can be used to produce calibration curves for the isotope dilution and internal standard methods. These curves are verified each shift (Section 15.3) by analyzing the calibration verification standard (VER, Table 4). Recalibration is required if any of the calibration verification criteria (Section 15.3) cannot be met.

10.8 Data Storage—MS data shall be collected, recorded, and stored.

10.8.1 Data acquisition—The signal at each exact m/z shall be collected repetitively throughout the monitoring period and stored on a mass storage device.

10.8.2 Response factors and multipoint calibrations—The data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and multipoint calibration curves. Computations of relative standard deviation (coefficient of variation) shall be used to test calibration linearity. Statistics on initial performance (Section 9.2) and ongoing performance (Section 15.5) should be computed and maintained, either on the instrument data system, or on a separate computer system.

11.0 Sample Preparation

11.1 Sample preparation involves modifying the physical form of the sample so that the CDDs/CDFs can be extracted efficiently. In general, the samples must be in a liquid form or in the form of finely divided solids in order for efficient extraction to take place. Table 10 lists the phases and suggested quantities for extraction of various sample matrices.

For samples known or expected to contain high levels of the CDDs/CDFs, the smallest sample size representative of the entire sample should be used (see Section 17.5).

For all samples, the blank and IPR/OPR aliquots must be processed through the same steps as the sample to check for contamination and losses in the preparation processes.

11.1.1 For samples that contain particles, percent solids and particle size are determined using the procedures in Sections 11.2 and 11.3, respectively.

11.1.2 Aqueous samples—Because CDDs/CDFs may be bound to suspended particles, the preparation of aqueous samples is dependent on the solids content of the sample.

11.1.2.1 Aqueous samples visibly absent particles are prepared per Section 11.4 and extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2, respectively.

11.1.2.2 Aqueous samples containing visible particles and containing one percent suspended solids or less are prepared using the procedure in Section 11.4. After preparation, the sample is extracted directly using the SPE technique in 12.2 or filtered per Section 11.4.3. After filtration, the particles and filter are extracted using the SDS procedure in Section 12.3 and the filtrate is extracted using the separatory funnel procedure in Section 12.1.

11.1.2.3 For aqueous samples containing greater than one percent solids, a sample aliquot sufficient to provide 10 g of dry solids is used, as described in Section 11.5.

11.1.3 Solid samples are prepared using the procedure described in Section 11.5 followed by extraction via the SDS procedure in Section 12.3.

11.1.4 Multiphase samples—The phase(s) containing the CDDs/CDFs is separated from the non-CDD/CDF phase using pressure filtration and centrifugation, as described in Section 11.6. The CDDs/CDFs will be in the organic phase in a multiphase sample in which an organic phase exists.

11.1.5 Procedures for grinding, homogenization, and blending of various sample phases are given in Section 11.7.

11.1.6 Tissue samples—Preparation procedures for fish and other tissues are given in Section 11.8.

11.2 Determination of Percent Suspended Solids.

Note:

This aliquot is used for determining the solids content of the sample, not for determination of CDDs/CDFs.

11.2.1 Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.

11.2.1.1 Dessicate and weigh a GF/D filter (Section 6.5.3) to three significant figures.

11.2.1.2 Filter 10.0 ±0.02 mL of well-mixed sample through the filter.

11.2.1.3 Dry the filter a minimum of 12 hours at 110 ±5 °C and cool in a dessicator.

11.2.1.4 Calculate percent solids as follows:

11.2.2 Non-aqueous liquids, solids, semi-solid samples, and multi-phase samples in which the main phase is not aqueous; but not tissues.

11.2.2.1 Weigh 5–10 g of sample to three significant figures in a tared beaker.

11.2.2.2 Dry a minimum of 12 hours at 110 ±5 °C, and cool in a dessicator.

11.2.2.3 Calculate percent solids as follows:

11.3 Determination of Particle Size.

11.3.1 Spread the dried sample from Section 11.2.2.2 on a piece of filter paper or aluminum foil in a fume hood or glove box.

11.3.2 Estimate the size of the particles in the sample. If the size of the largest particles is greater than 1 mm, the particle size must be reduced to 1 mm or less prior to extraction using the procedures in Section 11.7.

11.4 Preparation of Aqueous Samples Containing 1% Suspended Solids or Less.

11.4.1 Aqueous samples visibly absent particles are prepared per the procedure below and extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2, respectively. Aqueous samples containing visible particles and one percent suspended solids or less are prepared using the procedure below and extracted using either the SPE technique in Section 12.2 or further prepared using the filtration procedure in Section 11.4.3. The filtration procedure is followed by SDS extraction of the filter and particles (Section 12.3) and separatory funnel extraction of the filtrate (Section 12.1). The SPE procedure is followed by SDS extraction of the filter and disk.

11.4.2 Preparation of sample and QC aliquots.

11.4.2.1 Mark the original level of the sample on the sample bottle for reference. Weigh the sample plus bottle to ±1.

11.4.2.2 Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3) into the sample bottle. Cap the bottle and mix the sample by careful shaking. Allow the sample to equilibrate for one to two hours, with occasional shaking.

11.4.2.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during the same 12-hour shift, place two 1.0 L aliquots of reagent water in clean sample bottles or flasks.

11.4.2.4 Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3) into both reagent water aliquots. One of these aliquots will serve as the method blank.

11.4.2.5 Spike 1.0 mL of the PAR standard (Section 7.14) into the remaining reagent water aliquot. This aliquot will serve as the OPR (Section 15.5).

11.4.2.6 If SPE is to be used, add 5 mL of methanol to the sample, cap and shake the sample to mix thoroughly, and proceed to Section 12.2 for extraction. If SPE is not to be used, and the sample is visibly absent particles, proceed to Section 12.1 for extraction. If SPE is not to be used and the sample contains visible particles, proceed to the following section for filtration of particles.

11.4.3 Filtration of particles.

11.4.3.1 Assemble a Buchner funnel (Section 6.5.5) on top of a clean filtration flask. Apply vacuum to the flask, and pour the entire contents of the sample bottle through a glass-fiber filter (Section 6.5.6) in the Buchner funnel, swirling the sample remaining in the bottle to suspend any particles.

11.4.3.2 Rinse the sample bottle twice with approximately 5 mL portions of reagent water to transfer any remaining particles onto the filter.

11.4.3.3 Rinse any particles off the sides of the Buchner funnel with small quantities of reagent water.

11.4.3.4 Weigh the empty sample bottle to ±1 g. Determine the weight of the sample by difference. Save the bottle for further use.

11.4.3.5 Extract the filtrate using the separatory funnel procedure in Section 12.1.

11.4.3.6 Extract the filter containing the particles using the SDS procedure in Section 12.3.

11.5 Preparation of Samples Containing Greater Than 1% Solids.

11.5.1 Weigh a well-mixed aliquot of each sample (of the same matrix type) sufficient to provide 10 g of dry solids (based on the solids determination in Section 11.2) into a clean beaker or glass jar.

11.5.2 Spike 1.0 mL of the diluted labeled compound spiking solution (Section 7.10.3) into the sample.

11.5.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during the same 12-hour shift, weigh two 10 g aliquots of the appropriate reference matrix (Section 7.6) into clean beakers or glass jars.

11.5.4 Spike 1.0 mL of the diluted labeled compound spiking solution (Section 7.10.3) into each reference matrix aliquot. One aliquot will serve as the method blank. Spike 1.0 mL of the PAR standard (Section 7.14) into the other reference matrix aliquot. This aliquot will serve as the OPR (Section 15.5).

11.5.5 Stir or tumble and equilibrate the aliquots for one to two hours.

11.5.6 Decant excess water. If necessary to remove water, filter the sample through a glass-fiber filter and discard the aqueous liquid.

11.5.7 If particles >1mm are present in the sample (as determined in Section 11.3.2), spread the sample on clean aluminum foil in a hood. After the sample is dry, grind to reduce the particle size (Section 11.7).

11.5.8 Extract the sample and QC aliquots using the SDS procedure in Section 12.3.

11.6 Multiphase Samples.

11.6.1 Using the percent solids determined in Section 11.2.1 or 11.2.2, determine the volume of sample that will provide 10 g of solids, up to 1 L of sample.

11.6.2 Pressure filter the amount of sample determined in Section 11.6.1 through Whatman GF/D glass-fiber filter paper (Section 6.5.3). Pressure filter the blank and OPR aliquots through GF/D papers also. If necessary to separate the phases and/or settle the solids, centrifuge these aliquots prior to filtration.

11.6.3 Discard any aqueous phase (if present). Remove any non-aqueous liquid present and reserve the maximum amount filtered from the sample (Section 11.6.1) or 10 g, whichever is less, for combination with the solid phase (Section 12.3.5).

11.6.4 If particles >1mm are present in the sample (as determined in Section 11.3.2) and the sample is capable of being dried, spread the sample and QC aliquots on clean aluminum foil in a hood. After the aliquots are dry or if the sample cannot be dried, reduce the particle size using the procedures in Section 11.7 and extract the reduced particles using the SDS procedure in Section 12.3. If particles >1mm are not present, extract the particles and filter in the sample and QC aliquots directly using the SDS procedure in Section 12.3.

11.7 Sample grinding, homogenization, or blending—Samples with particle sizes greater than 1 mm (as determined in Section 11.3.2) are subjected to grinding, homogenization, or blending. The method of reducing particle size to less than 1 mm is matrix-dependent. In general, hard particles can be reduced by grinding with a mortar and pestle. Softer particles can be reduced by grinding in a Wiley mill or meat grinder, by homogenization, or in a blender.

11.7.1 Each size-reducing preparation procedure on each matrix shall be verified by running the tests in Section 9.2 before the procedure is employed routinely.

11.7.2 The grinding, homogenization, or blending procedures shall be carried out in a glove box or fume hood to prevent particles from contaminating the work environment.

11.7.3 Grinding—Certain papers and pulps, slurries, and amorphous solids can be ground in a Wiley mill or heavy duty meat grinder. In some cases, reducing the temperature of the sample to freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding process. Grind the sample aliquots from Section 11.5.7 or 11.6.4 in a clean grinder. Do not allow the sample temperature to exceed 50 °C. Grind the blank and reference matrix aliquots using a clean grinder.

11.7.4 Homogenization or blending—Particles that are not ground effectively, or particles greater than 1 mm in size after grinding, can often be reduced in size by high speed homogenization or blending. Homogenize and/or blend the particles or filter from Section 11.5.7 or 11.6.4 for the sample, blank, and OPR aliquots.

11.7.5 Extract the aliquots using the SDS procedure in Section 12.3.

11.8 Fish and Other Tissues—Prior to processing tissue samples, the laboratory must determine the exact tissue to be analyzed. Common requests for analysis of fish tissue include whole fish—skin on, whole fish—skin removed, edible fish fillets (filleted in the field or by the laboratory), specific organs, and other portions. Once the appropriate tissue has been determined, the sample must be homogenized.

11.8.1 Homogenization.

11.8.1.1 Samples are homogenized while still frozen, where practical. If the laboratory must dissect the whole fish to obtain the appropriate tissue for analysis, the unused tissues may be rapidly refrozen and stored in a clean glass jar for subsequent use.

11.8.1.2 Each analysis requires 10 g of tissue (wet weight). Therefore, the laboratory should homogenize at least 20 g of tissue to allow for re-extraction of a second aliquot of the same homogenized sample, if re-analysis is required. When whole fish analysis is necessary, the entire fish is homogenized.

11.8.1.3 Homogenize the sample in a tissue homogenizer (Section 6.3.3) or grind in a meat grinder (Section 6.3.4). Cut tissue too large to feed into the grinder into smaller pieces. To assure homogeneity, grind three times.

11.8.1.4 Transfer approximately 10 g (wet weight) of homogenized tissue to a clean, tared, 400–500 mL beaker. For the alternate HCl digestion/extraction, transfer the tissue to a clean, tared 500–600 mL wide-mouth bottle. Record the weight to the nearest 10 mg.

11.8.1.5 Transfer the remaining homogenized tissue to a clean jar with a fluoropolymer-lined lid. Seal the jar and store the tissue at <−10 °C. Return any tissue that was not homogenized to its original container and store at <−10 °C.

11.8.2 QC aliquots.

11.8.2.1 Prepare a method blank by adding approximately 10 g of the oily liquid reference matrix (Section 7.6.4) to a 400–500 mL beaker. For the alternate HCl digestion/extraction, add the reference matrix to a 500–600 mL wide-mouth bottle. Record the weight to the nearest 10 mg.

11.8.2.2 Prepare a precision and recovery aliquot by adding approximately 10 g of the oily liquid reference matrix (Section 7.6.4) to a separate 400–500 mL beaker or wide-mouth bottle, depending on the extraction procedure to be used. Record the weight to the nearest 10 mg. If the initial precision and recovery test is to be performed, use four aliquots; if the ongoing precision and recovery test is to be performed, use a single aliquot.

11.8.3 Spiking

11.8.3.1 Spike 1.0 mL of the labeled compound spiking solution (Section 7.10.3) into the sample, blank, and OPR aliquot.

11.8.3.2 Spike 1.0 mL of the PAR standard (Section 7.14) into the OPR aliquot.

11.8.4 Extract the aliquots using the procedures in Section 12.4.

12.0 Extraction and Concentration

Extraction procedures include separatory funnel (Section 12.1) and solid phase (Section 12.2) for aqueous liquids; Soxhlet/Dean-Stark (Section 12.3) for solids, filters, and SPE disks; and Soxhlet extraction (Section 12.4.1) and HCl digestion (Section 12.4.2) for tissues. Acid/base back-extraction (Section 12.5) is used for initial cleanup of extracts.

Macro-concentration procedures include rotary evaporation (Section 12.6.1), heating mantle (Section 12.6.2), and Kuderna-Danish (K-D) evaporation (Section 12.6.3). Micro-concentration uses nitrogen blowdown (Section 12.7).

12.1 Separatory funnel extraction of filtrates and of aqueous samples visibly absent particles.

12.1.1 Pour the spiked sample (Section 11.4.2.2) or filtrate (Section 11.4.3.5) into a 2 L separatory funnel. Rinse the bottle or flask twice with 5 mL of reagent water and add these rinses to the separatory funnel.

12.1.2 Add 60 mL methylene chloride to the empty sample bottle (Section 12.1.1), seal, and shake 60 seconds to rinse the inner surface. Transfer the solvent to the separatory funnel, and extract the sample by shaking the funnel for two minutes with periodic venting. Allow the organic layer to separate from the aqueous phase for a minimum of 10 minutes. If an emulsion forms and is more than one-third the volume of the solvent layer, employ mechanical techniques to complete the phase separation (see note below). Drain the methylene chloride extract through a solvent-rinsed glass funnel approximately one-half full of granular anhydrous sodium sulfate (Section 7.2.1) supported on clean glass-fiber paper into a solvent-rinsed concentration device (Section 12.6).

Note:

If an emulsion forms, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration through glass wool, use of phase separation paper, centrifugation, use of an ultrasonic bath with ice, addition of NaCl, or other physical methods. Alternatively, solid-phase or other extraction techniques may be used to prevent emulsion formation. Any alternative technique is acceptable so long as the requirements in Section 9 are met.

Experience with aqueous samples high in dissolved organic materials (e.g., paper mill effluents) has shown that acidification of the sample prior to extraction may reduce the formation of emulsions. Paper industry methods suggest that the addition of up to 400 mL of ethanol to a 1 L effluent sample may also reduce emulsion formation. However, studies by EPA suggest that the effect may be a result of sample dilution, and that the addition of reagent water may serve the same function. Mechanical techniques may still be necessary to complete the phase separation. If either acidification or addition of ethanol is utilized, the laboratory must perform the startup tests described in Section 9.2 using the same techniques.

12.1.3 Extract the water sample two more times with 60 mL portions of methylene chloride. Drain each portion through the sodium sulfate into the concentrator. After the third extraction, rinse the separatory funnel with at least 20 mL of methylene chloride, and drain this rinse through the sodium sulfate into the concentrator. Repeat this rinse at least twice. Set aside the funnel with sodium sulfate if the extract is to be combined with the extract from the particles.

12.1.4 Concentrate the extract using one of the macro-concentration procedures in Section 12.6.

12.1.4.1 If the extract is from a sample visibly absent particles (Section 11.1.2.1), adjust the final volume of the concentrated extract to approximately 10 mL with hexane, transfer to a 250 mL separatory funnel, and back-extract using the procedure in Section 12.5.

12.1.4.2 If the extract is from the aqueous filtrate (Section 11.4.3.5), set aside the concentration apparatus for addition of the SDS extract from the particles (Section 12.3.9.1.2).

12.2 SPE of Samples Containing Less Than 1% Solids (References 19–20).

12.2.1 Disk preparation.

12.2.1.1 Place an SPE disk on the base of the filter holder (Figure 4) and wet with toluene. While holding a GMF 150 filter above the SPE disk with tweezers, wet the filter with toluene and lay the filter on the SPE disk, making sure that air is not trapped between the filter and disk. Clamp the filter and SPE disk between the 1 L glass reservoir and the vacuum filtration flask.

12.2.1.2 Rinse the sides of the filtration flask with approx 15 mL of toluene using a squeeze bottle or syringe. Apply vacuum momentarily until a few drops appear at the drip tip. Release the vacuum and allow the filter/disk to soak for approx one minute. Apply vacuum and draw all of the toluene through the filter/disk. Repeat the wash step with approx 15 mL of acetone and allow the filter/disk to air dry.

12.2.1.3 Re-wet the filter/disk with approximately 15 mL of methanol, allowing the filter/disk to soak for approximately one minute. Pull the methanol through the filter/disk using the vacuum, but retain a layer of methanol approximately 1 mm thick on the filter. Do not allow the disk to go dry from this point until the end of the extraction.

12.2.1.4 Rinse the filter/disk with two 50-mL portions of reagent water by adding the water to the reservoir and pulling most through, leaving a layer of water on the surface of the filter.

12.2.2 Extraction.

12.2.2.1 Pour the spiked sample (Section 11.4.2.2), blank (Section 11.4.2.4), or IPR/OPR aliquot (Section 11.4.2.5) into the reservoir and turn on the vacuum to begin the extraction. Adjust the vacuum to complete the extraction in no less than 10 minutes. For samples containing a high concentration of particles (suspended solids), filtration times may be eight hours or longer.

12.2.2.2 Before all of the sample has been pulled through the filter/disk, rinse the sample bottle with approximately 50 mL of reagent water to remove any solids, and pour into the reservoir. Pull through the filter/disk. Use additional reagent water rinses until all visible solids are removed.

12.2.2.3 Before all of the sample and rinses have been pulled through the filter/disk, rinse the sides of the reservoir with small portions of reagent water.

12.2.2.4 Allow the filter/disk to dry, then remove the filter and disk and place in a glass Petri dish. Extract the filter and disk per Section 12.3.

12.3 SDS Extraction of Samples Containing Particles, and of Filters and/or Disks.

12.3.1 Charge a clean extraction thimble (Section 6.4.2.2) with 5.0 g of 100/200 mesh silica (Section 7.5.1.1) topped with 100 g of quartz sand (Section 7.3.2).

Note:

Do not disturb the silica layer throughout the extraction process.

12.3.2 Place the thimble in a clean extractor. Place 30–40 mL of toluene in the receiver and 200–250 mL of toluene in the flask.

12.3.3 Pre-extract the glassware by heating the flask until the toluene is boiling. When properly adjusted, one to two drops of toluene will fall per second from the condenser tip into the receiver. Extract the apparatus for a minimum of three hours.

12.3.4 After pre-extraction, cool and disassemble the apparatus. Rinse the thimble with toluene and allow to air dry.

12.3.5 Load the wet sample, filter, and/or disk from Section 11.4.3.6, 11.5.8, 11.6.4, 11.7.3, 11.7.4, or 12.2.2.4 and any nonaqueous liquid from Section 11.6.3 into the thimble and manually mix into the sand layer with a clean metal spatula, carefully breaking up any large lumps of sample.

12.3.6 Reassemble the pre-extracted SDS apparatus, and add a fresh charge of toluene to the receiver and reflux flask. Apply power to the heating mantle to begin refluxing. Adjust the reflux rate to match the rate of percolation through the sand and silica beds until water removal lessens the restriction to toluene flow. Frequently check the apparatus for foaming during the first two hours of extraction. If foaming occurs, reduce the reflux rate until foaming subsides.

12.3.7 Drain the water from the receiver at one to two hours and eight to nine hours, or sooner if the receiver fills with water. Reflux the sample for a total of 16–24 hours. Cool and disassemble the apparatus. Record the total volume of water collected.

12.3.8 Remove the distilling flask. Drain the water from the Dean-Stark receiver and add any toluene in the receiver to the extract in the flask.

12.3.9 Concentrate the extract using one of the macro-concentration procedures in Section 12.6 per the following:

12.3.9.1 Extracts from the particles in an aqueous sample containing less than 1% solids (Section 11.4.3.6).

12.3.9.1.1 Concentrate the extract to approximately 5 mL using the rotary evaporator or heating mantle procedures in Section 12.6.1 or 12.6.2.

12.3.9.1.2 Quantitatively transfer the extract through the sodium sulfate (Section 12.1.3) into the apparatus that was set aside (Section 12.1.4.2) and reconcentrate to the level of the toluene.

12.3.9.1.3 Adjust to approximately 10 mL with hexane, transfer to a 250 mL separatory funnel, and proceed with back-extraction (Section 12.5).

12.3.9.2 Extracts from particles (Sections 11.5 through 11.6) or from the SPE filter and disk (Section 12.2.2.4)—Concentrate to approximately 10 mL using the rotary evaporator or heating mantle (Section 12.6.1 or 12.6.2), transfer to a 250 mL separatory funnel, and proceed with back-extraction (Section 12.5).

12.4 Extraction of Tissue—Two procedures are provided for tissue extraction.

12.4.1 Soxhlet extraction (Reference 21).

12.4.1.1 Add 30–40 g of powdered anhydrous sodium sulfate to each of the beakers (Section 11.8.4) and mix thoroughly. Cover the beakers with aluminum foil and allow to equilibrate for 12–24 hours. Remix prior to extraction to prevent clumping.

12.4.1.2 Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1 through 12.3.4, except use the methylene chloride:hexane (1:1) mixture for the pre-extraction and rinsing and omit the quartz sand. The Dean-Stark moisture trap may also be omitted, if desired.

12.4.1.3 Reassemble the pre-extracted Soxhlet apparatus and add a fresh charge of methylene chloride:hexane to the reflux flask.

12.4.1.4 Transfer the sample/sodium sulfate mixture (Section 12.4.1.1) to the Soxhlet thimble, and install the thimble in the Soxhlet apparatus.

12.4.1.5 Rinse the beaker with several portions of solvent mixture and add to the thimble. Fill the thimble/receiver with solvent. Extract for 18–24 hours.

12.4.1.6 After extraction, cool and disassemble the apparatus.

12.4.1.7 Quantitatively transfer the extract to a macro-concentration device (Section 12.6), and concentrate to near dryness. Set aside the concentration apparatus for re-use.

12.4.1.8 Complete the removal of the solvent using the nitrogen blowdown procedure (Section 12.7) and a water bath temperature of 60 °C. Weigh the receiver, record the weight, and return the receiver to the blowdown apparatus, concentrating the residue until a constant weight is obtained.

12.4.1.9 Percent lipid determination—The lipid content is determined by extraction of tissue with the same solvent system (methylene chloride:hexane) that was used in EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent with that study.

12.4.1.9.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of the cleanup standard (Section 7.11) into the solution.

12.4.1.9.2 Transfer the residue/hexane to the anthropogenic isolation column (Section 13.7.1) or bottle for the acidified silica gel batch cleanup (Section 13.7.2), retaining the boiling chips in the concentration apparatus. Use several rinses to assure that all material is transferred. If necessary, sonicate or heat the receiver slightly to assure that all material is re-dissolved. Allow the receiver to dry. Weigh the receiver and boiling chips.

12.4.1.9.3 Calculate the lipid content to the nearest three significant figures as follows:

12.4.1.9.4 It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.

12.4.2 HCl digestion/extraction and concentration (References 23–26).

12.4.2.1 Add 200 mL of 6 N HCl and 200 mL of methylene chloride:hexane (1:1) to the sample and QC aliquots (Section 11.8.4).

12.4.2.2 Cap and shake each bottle one to three times. Loosen the cap in a hood to vent excess pressure. Shake each bottle for 10–30 seconds and vent.

12.4.2.3 Tightly cap and place on shaker. Adjust the shaker action and speed so that the acid, solvent, and tissue are in constant motion. However, take care to avoid such violent action that the bottle may be dislodged from the shaker. Shake for 12–24 hours.

12.4.2.4 After digestion, remove the bottles from the shaker. Allow the bottles to stand so that the solvent and acid layers separate.

12.4.2.5 Decant the solvent through a glass funnel with glass-fiber filter (Sections 6.5.2 through 6.5.3) containing approximately 10 g of granular anhydrous sodium sulfate (Section 7.2.1) into a macro-concentration apparatus (Section 12.6). Rinse the contents of the bottle with two 25 mL portions of hexane and pour through the sodium sulfate into the apparatus.

12.4.2.6 Concentrate the solvent to near dryness using a macro-concentration procedure (Section 12.6).

12.4.2.7 Complete the removal of the solvent using the nitrogen blowdown apparatus (Section 12.7) and a water bath temperature of 60 °C. Weigh the receiver, record the weight, and return the receiver to the blowdown apparatus, concentrating the residue until a constant weight is obtained.

12.4.2.8 Percent lipid determination—The lipid content is determined in the same solvent system [methylene chloride:hexane (1:1)] that was used in EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent with that study.

12.4.2.8.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of the cleanup standard (Section 7.11) into the solution.

12.4.2.8.2 Transfer the residue/hexane to the narrow-mouth 100–200 mL bottle retaining the boiling chips in the receiver. Use several rinses to assure that all material is transferred, to a maximum hexane volume of approximately 70 mL. Allow the receiver to dry. Weigh the receiver and boiling chips.

12.4.2.8.3 Calculate the percent lipid per Section 12.4.1.9.3. It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.

12.4.2.9 Clean up the extract per Section 13.7.3.

12.5 Back-Extraction with Base and Acid.

12.5.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into the separatory funnels containing the sample and QC extracts from Section 12.1.4.1, 12.3.9.1.3, or 12.3.9.2.

12.5.2 Partition the extract against 50 mL of potassium hydroxide solution (Section 7.1.1). Shake for two minutes with periodic venting into a hood. Remove and discard the aqueous layer. Repeat the base washing until no color is visible in the aqueous layer, to a maximum of four washings. Minimize contact time between the extract and the base to prevent degradation of the CDDs/CDFs. Stronger potassium hydroxide solutions may be employed for back-extraction, provided that the laboratory meets the specifications for labeled compound recovery and demonstrates acceptable performance using the procedure in Section 9.2.

12.5.3 Partition the extract against 50 mL of sodium chloride solution (Section 7.1.4) in the same way as with base. Discard the aqueous layer.

12.5.4 Partition the extract against 50 mL of sulfuric acid (Section 7.1.2) in the same way as with base. Repeat the acid washing until no color is visible in the aqueous layer, to a maximum of four washings.

12.5.5 Repeat the partitioning against sodium chloride solution and discard the aqueous layer.

12.5.6 Pour each extract through a drying column containing 7–10 cm of granular anhydrous sodium sulfate (Section 7.2.1). Rinse the separatory funnel with 30–50 mL of solvent, and pour through the drying column. Collect each extract in a round-bottom flask. Re-concentrate the sample and QC aliquots per Sections 12.6 through 12.7, and clean up the samples and QC aliquots per Section 13.

12.6 Macro-Concentration—Extracts in toluene are concentrated using a rotary evaporator or a heating mantle; extracts in methylene chloride or hexane are concentrated using a rotary evaporator, heating mantle, or Kuderna-Danish apparatus.

12.6.1 Rotary evaporation—Concentrate the extracts in separate round-bottom flasks.

12.6.1.1 Assemble the rotary evaporator according to manufacturer's instructions, and warm the water bath to 45 °C. On a daily basis, preclean the rotary evaporator by concentrating 100 mL of clean extraction solvent through the system. Archive both the concentrated solvent and the solvent in the catch flask for a contamination check if necessary. Between samples, three 2–3 mL aliquots of solvent should be rinsed down the feed tube into a waste beaker.

12.6.1.2 Attach the round-bottom flask containing the sample extract to the rotary evaporator. Slowly apply vacuum to the system, and begin rotating the sample flask.

12.6.1.3 Lower the flask into the water bath, and adjust the speed of rotation and the temperature as required to complete concentration in 15–20 minutes. At the proper rate of concentration, the flow of solvent into the receiving flask will be steady, but no bumping or visible boiling of the extract will occur.

Note:

If the rate of concentration is too fast, analyte loss may occur.

12.6.1.4 When the liquid in the concentration flask has reached an apparent volume of approximately 2 mL, remove the flask from the water bath and stop the rotation. Slowly and carefully admit air into the system. Be sure not to open the valve so quickly that the sample is blown out of the flask. Rinse the feed tube with approximately 2 mL of solvent.

12.6.1.5 Proceed to Section 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.2 Heating mantle—Concentrate the extracts in separate round-bottom flasks.

12.6.2.1 Add one or two clean boiling chips to the round-bottom flask, and attach a three-ball macro Snyder column. Prewet the column by adding approximately 1 mL of solvent through the top. Place the round-bottom flask in a heating mantle, and apply heat as required to complete the concentration in 15–20 minutes. At the proper rate of distillation, the balls of the column will actively chatter, but the chambers will not flood.

12.6.2.2 When the liquid has reached an apparent volume of approximately 10 mL, remove the round-bottom flask from the heating mantle and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the glass joint into the receiver with small portions of solvent.

12.6.2.3 Proceed to Section 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.3 Kuderna-Danish (K-D)—Concentrate the extracts in separate 500 mL K-D flasks equipped with 10 mL concentrator tubes. The K-D technique is used for solvents such as methylene chloride and hexane. Toluene is difficult to concentrate using the K-D technique unless a water bath fed by a steam generator is used.

12.6.3.1 Add one to two clean boiling chips to the receiver. Attach a three-ball macro Snyder column. Prewet the column by adding approximately 1 mL of solvent through the top. Place the K-D apparatus in a hot water bath so that the entire lower rounded surface of the flask is bathed with steam.

12.6.3.2 Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15–20 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood.

12.6.3.3 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus from the bath and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1–2 mL of solvent. A 5 mL syringe is recommended for this operation.

12.6.3.4 Remove the three-ball Snyder column, add a fresh boiling chip, and attach a two-ball micro Snyder column to the concentrator tube. Prewet the column by adding approximately 0.5 mL of solvent through the top. Place the apparatus in the hot water bath.

12.6.3.5 Adjust the vertical position and the water temperature as required to complete the concentration in 5–10 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood.

12.6.3.6 When the liquid reaches an apparent volume of 0.5 mL, remove the apparatus from the water bath and allow to drain and cool for at least 10 minutes.

12.6.3.7 Proceed to 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.4 Preparation for back-extraction or micro-concentration and solvent exchange.

12.6.4.1 For back-extraction (Section 12.5), transfer the extract to a 250 mL separatory funnel. Rinse the concentration vessel with small portions of hexane, adjust the hexane volume in the separatory funnel to 10–20 mL, and proceed to back-extraction (Section 12.5).

12.6.4.2 For determination of the weight of residue in the extract, or for clean-up procedures other than back-extraction, transfer the extract to a blowdown vial using two to three rinses of solvent. Proceed with micro-concentration and solvent exchange (Section 12.7).

12.7 Micro-Concentration and Solvent Exchange.

12.7.1 Extracts to be subjected to GPC or HPLC cleanup are exchanged into methylene chloride. Extracts to be cleaned up using silica gel, alumina, carbon, and/or Florisil are exchanged into hexane.

12.7.2 Transfer the vial containing the sample extract to a nitrogen blowdown device. Adjust the flow of nitrogen so that the surface of the solvent is just visibly disturbed.

Note:

A large vortex in the solvent may cause analyte loss.

12.7.3 Lower the vial into a 45 °C water bath and continue concentrating.

12.7.3.1 If the extract is to be concentrated to dryness for weight determination (Sections 12.4.1.8, 12.4.2.7, and 13.7.1.4), blow dry until a constant weight is obtained.

12.7.3.2 If the extract is to be concentrated for injection into the GC/MS or the solvent is to be exchanged for extract cleanup, proceed as follows:

12.7.4 When the volume of the liquid is approximately 100 L, add 2–3 mL of the desired solvent (methylene chloride for GPC and HPLC, or hexane for the other cleanups) and continue concentration to approximately 100 µL. Repeat the addition of solvent and concentrate once more.

12.7.5 If the extract is to be cleaned up by GPC, adjust the volume of the extract to 5.0 mL with methylene chloride. If the extract is to be cleaned up by HPLC, further concentrate the extract to 30 µL. Proceed with GPC or HPLC cleanup (Section 13.2 or 13.6, respectively).

12.7.6 If the extract is to be cleaned up by column chromatography (alumina, silica gel, Carbopak/Celite, or Florisil), bring the final volume to 1.0 mL with hexane. Proceed with column cleanups (Sections 13.3 through 13.5 and 13.8).

12.7.7 If the extract is to be concentrated for injection into the GC/MS (Section 14), quantitatively transfer the extract to a 0.3 mL conical vial for final concentration, rinsing the larger vial with hexane and adding the rinse to the conical vial. Reduce the volume to approximately 100 µL. Add 10 µL of nonane to the vial, and evaporate the solvent to the level of the nonane. Seal the vial and label with the sample number. Store in the dark at room temperature until ready for GC/MS analysis. If GC/MS analysis will not be performed on the same day, store the vial at <−10 °C.

13.0 Extract Cleanup

13.1 Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater, drinking water). If particular circumstances require the use of a cleanup procedure, the analyst may use any or all of the procedures below or any other appropriate procedure. Before using a cleanup procedure, the analyst must demonstrate that the requirements of Section 9.2 can be met using the cleanup procedure. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the cleanup procedures may be optimized for isolation of these two compounds.

13.1.1 Gel permeation chromatography (Section 13.2) removes high molecular weight interferences that cause GC column performance to degrade. It should be used for all soil and sediment extracts and may be used for water extracts that are expected to contain high molecular weight organic compounds (e.g., polymeric materials, humic acids).

13.1.2 Acid, neutral, and basic silica gel (Section 13.3), alumina (Section 13.4), and Florisil (Section 13.8) are used to remove nonpolar and polar interferences. Alumina and Florisil are used to remove chlorodiphenyl ethers.

13.1.3 Carbopak/Celite (Section 13.5) is used to remove nonpolar interferences.

13.1.4 HPLC (Section 13.6) is used to provide specificity for the 2,3,7,8-substituted and other CDD and CDF isomers.

13.1.5 The anthropogenic isolation column (Section 13.7.1), acidified silica gel batch adsorption procedure (Section 13.7.2), and sulfuric acid and base back-extraction (Section 13.7.3) are used for removal of lipids from tissue samples.

13.2 Gel Permeation Chromatography (GPC).

13.2.1 Column packing.

13.2.1.1 Place 70–75 g of SX–3 Bio-beads (Section 6.7.1.1) in a 400–500 mL beaker.

13.2.1.2 Cover the beads with methylene chloride and allow to swell overnight (a minimum of 12 hours).

13.2.1.3 Transfer the swelled beads to the column (Section 6.7.1.1) and pump solvent through the column, from bottom to top, at 4.5–5.5 mL/minute prior to connecting the column to the detector.

13.2.1.4 After purging the column with solvent for one to two hours, adjust the column head pressure to 7–10 psig and purge for four to five hours to remove air. Maintain a head pressure of 7–10 psig. Connect the column to the detector (Section 6.7.1.4).

13.2.2 Column calibration.

13.2.2.1 Load 5 mL of the calibration solution (Section 7.4) into the sample loop.

13.2.2.2 Inject the calibration solution and record the signal from the detector. The elution pattern will be corn oil, bis(2-ethyl hexyl)phthalate, pentachlorophenol, perylene, and sulfur.

13.2.2.3 Set the “dump time” to allow >85% removal of the corn oil and >85% collection of the phthalate.

13.2.2.4 Set the “collect time” to the peak minimum between perylene and sulfur.

13.2.2.5 Verify the calibration with the calibration solution after every 20 extracts. Calibration is verified if the recovery of the pentachlorophenol is greater than 85%. If calibration is not verified, the system shall be recalibrated using the calibration solution, and the previous 20 samples shall be re-extracted and cleaned up using the calibrated GPC system.

13.2.3 Extract cleanup—GPC requires that the column not be overloaded. The column specified in this method is designed to handle a maximum of 0.5 g of high molecular weight material in a 5 mL extract. If the extract is known or expected to contain more than 0.5 g, the extract is split into aliquots for GPC, and the aliquots are combined after elution from the column. The residue content of the extract may be obtained gravimetrically by evaporating the solvent from a 50 µL aliquot.

13.2.3.1 Filter the extract or load through the filter holder (Section 6.7.1.3) to remove the particles. Load the 5.0 mL extract onto the column.

13.2.3.2 Elute the extract using the calibration data determined in Section 13.2.2. Collect the eluate in a clean 400–500 mL beaker.

13.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between extracts to prepare for the next sample.

13.2.3.4 If a particularly dirty extract is encountered, a 5.0 mL methylene chloride blank shall be run through the system to check for carry-over.

13.2.3.5 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the GC/MS.

13.3 Silica Gel Cleanup.

13.3.1 Place a glass-wool plug in a 15 mm ID chromatography column (Section 6.7.4.2). Pack the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel (Section 7.5.1.3), 1 g silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g granular anhydrous sodium sulfate (Section 7.2.1). Tap the column to settle the adsorbents.

13.3.2 Pre-elute the column with 50–100 mL of hexane. Close the stopcock when the hexane is within 1 mm of the sodium sulfate. Discard the eluate. Check the column for channeling. If channeling is present, discard the column and prepare another.

13.3.3 Apply the concentrated extract to the column. Open the stopcock until the extract is within 1 mm of the sodium sulfate.

13.3.4 Rinse the receiver twice with 1 mL portions of hexane, and apply separately to the column. Elute the CDDs/CDFs with 100 mL hexane, and collect the eluate.

13.3.5 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.3.6 For extracts of samples known to contain large quantities of other organic compounds (such as paper mill effluents), it may be advisable to increase the capacity of the silica gel column. This may be accomplished by increasing the strengths of the acid and basic silica gels. The acid silica gel (Section 7.5.1.2) may be increased in strength to as much as 44% w/w (7.9 g sulfuric acid added to 10 g silica gel). The basic silica gel (Section 7.5.1.3) may be increased in strength to as much as 33% w/w (50 mL 1N NaOH added to 100 g silica gel), or the potassium silicate (Section 7.5.1.4) may be used.

Note:

The use of stronger acid silica gel (44% w/w) may lead to charring of organic compounds in some extracts. The charred material may retain some of the analytes and lead to lower recoveries of CDDs/CDFs. Increasing the strengths of the acid and basic silica gel may also require different volumes of hexane than those specified above to elute the analytes off the column. Therefore, the performance of the method after such modifications must be verified by the procedure in Section 9.2.

13.4 Alumina Cleanup.

13.4.1 Place a glass-wool plug in a 15 mm ID chromatography column (Section 6.7.4.2).

13.4.2 If using acid alumina, pack the column by adding 6 g acid alumina (Section 7.5.2.1). If using basic alumina, substitute 6 g basic alumina (Section 7.5.2.2). Tap the column to settle the adsorbents.

13.4.3 Pre-elute the column with 50–100 mL of hexane. Close the stopcock when the hexane is within 1 mm of the alumina.

13.4.4 Discard the eluate. Check the column for channeling. If channeling is present, discard the column and prepare another.

13.4.5 Apply the concentrated extract to the column. Open the stopcock until the extract is within 1 mm of the alumina.

13.4.6 Rinse the receiver twice with 1 mL portions of hexane and apply separately to the column. Elute the interfering compounds with 100 mL hexane and discard the eluate.

13.4.7 The choice of eluting solvents will depend on the choice of alumina (acid or basic) made in Section 13.4.2.

13.4.7.1 If using acid alumina, elute the CDDs/CDFs from the column with 20 mL methylene chloride:hexane (20:80 v/v). Collect the eluate.

13.4.7.2 If using basic alumina, elute the CDDs/CDFs from the column with 20 mL methylene chloride:hexane (50:50 v/v). Collect the eluate.

13.4.8 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.5 Carbon Column.

13.5.1 Cut both ends from a 10 mL disposable serological pipet (Section 6.7.3.2) to produce a 10 cm column. Fire-polish both ends and flare both ends if desired. Insert a glass-wool plug at one end, and pack the column with 0.55 g of Carbopak/Celite (Section 7.5.3.3) to form an adsorbent bed approximately 2 cm long. Insert a glass-wool plug on top of the bed to hold the adsorbent in place.

13.5.2 Pre-elute the column with 5 mL of toluene followed by 2 mL of methylene chloride: methanol:toluene (15:4:1 v/v), 1 mL of methylene chloride:cyclohexane (1:1 v/v), and 5 mL of hexane. If the flow rate of eluate exceeds 0.5 mL/minute, discard the column.

13.5.3 When the solvent is within 1 mm of the column packing, apply the sample extract to the column. Rinse the sample container twice with 1 mL portions of hexane and apply separately to the column. Apply 2 mL of hexane to complete the transfer.

13.5.4 Elute the interfering compounds with two 3 mL portions of hexane, 2 mL of methylene chloride:cyclohexane (1:1 v/v), and 2 mL of methylene chloride:methanol:toluene (15:4:1 v/v). Discard the eluate.

13.5.5 Invert the column, and elute the CDDs/CDFs with 20 mL of toluene. If carbon particles are present in the eluate, filter through glass-fiber filter paper.

13.5.6 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.6 HPLC (Reference 6).

13.6.1 Column calibration.

13.6.1.1 Prepare a calibration standard containing the 2,3,7,8-substituted isomers and/or other isomers of interest at a concentration of approximately 500 pg/µL in methylene chloride.

13.6.1.2 Inject 30 µL of the calibration solution into the HPLC and record the signal from the detector. Collect the eluant for reuse. The elution order will be the tetra- through octa-isomers.

13.6.1.3 Establish the collection time for the tetra-isomers and for the other isomers of interest. Following calibration, flush the injection system with copious quantities of methylene chloride, including a minimum of five 50 µL injections while the detector is monitored, to ensure that residual CDDs/CDFs are removed from the system.

13.6.1.4 Verify the calibration with the calibration solution after every 20 extracts. Calibration is verified if the recovery of the CDDs/CDFs from the calibration standard (Section 13.6.1.1) is 75–125% compared to the calibration (Section 13.6.1.2). If calibration is not verified, the system shall be recalibrated using the calibration solution, and the previous 20 samples shall be re-extracted and cleaned up using the calibrated system.

13.6.2 Extract cleanup—HPLC requires that the column not be overloaded. The column specified in this method is designed to handle a maximum of 30 µL of extract. If the extract cannot be concentrated to less than 30 µL, it is split into fractions and the fractions are combined after elution from the column.

13.6.2.1 Rinse the sides of the vial twice with 30 µL of methylene chloride and reduce to 30 µL with the evaporation apparatus (Section 12.7).

13.6.2.2 Inject the 30 µL extract into the HPLC.

13.6.2.3 Elute the extract using the calibration data determined in Section 13.6.1. Collect the fraction(s) in a clean 20 mL concentrator tube containing 5 mL of hexane:acetone (1:1 v/v).

13.6.2.4 If an extract containing greater than 100 ng/mL of total CDD or CDF is encountered, a 30 µL methylene chloride blank shall be run through the system to check for carry-over.

13.6.2.5 Concentrate the eluate per Section 12.7 for injection into the GC/MS.

13.7 Cleanup of Tissue Lipids—Lipids are removed from the Soxhlet extract using either the anthropogenic isolation column (Section 13.7.1) or acidified silica gel (Section 13.7.2), or are removed from the HCl digested extract using sulfuric acid and base back-extraction (Section 13.7.3).

13.7.1 Anthropogenic isolation column (References 22 and 27)—Used for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).

13.7.1.1 Prepare the column as given in Section 7.5.4.

13.7.1.2 Pre-elute the column with 100 mL of hexane. Drain the hexane layer to the top of the column, but do not expose the sodium sulfate.

13.7.1.3 Load the sample and rinses (Section 12.4.1.9.2) onto the column by draining each portion to the top of the bed. Elute the CDDs/CDFs from the column into the apparatus used for concentration (Section 12.4.1.7) using 200 mL of hexane.

13.7.1.4 Concentrate the cleaned up extract (Sections 12.6 through 12.7) to constant weight per Section 12.7.3.1. If more than 500 mg of material remains, repeat the cleanup using a fresh anthropogenic isolation column.

13.7.1.5 Redissolve the extract in a solvent suitable for the additional cleanups to be used (Sections 13.2 through 13.6 and 13.8).

13.7.1.6 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.

13.7.1.7 Clean up the extract using the procedures in Sections 13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.1.8 Following cleanup, concentrate the extract to 10 µL as described in Section 12.7 and proceed with the analysis in Section 14.

13.7.2 Acidified silica gel (Reference 28)—Procedure alternate to the anthropogenic isolation column (Section 13.7.1) that is used for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).

13.7.2.1 Adjust the volume of hexane in the bottle (Section 12.4.1.9.2) to approximately 200 mL.

13.7.2.2 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.

13.7.2.3 Drop the stirring bar into the bottle, place the bottle on the stirring plate, and begin stirring.

13.7.2.4 Add 30–100 g of acid silica gel (Section 7.5.1.2) to the bottle while stirring, keeping the silica gel in motion. Stir for two to three hours.

Note:

30 grams of silica gel should be adequate for most samples and will minimize contamination from this source.

13.7.2.5 After stirring, pour the extract through approximately 10 g of granular anhydrous sodium sulfate (Section 7.2.1) contained in a funnel with glass-fiber filter into a macro contration device (Section 12.6). Rinse the bottle and sodium sulfate with hexane to complete the transfer.

13.7.2.6 Concentrate the extract per Sections 12.6 through 12.7 and clean up the extract using the procedures in Sections 13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.3 Sulfuric acid and base back-extraction. Used with HCl digested extracts (Section 12.4.2).

13.7.3.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent (Section 12.4.2.8.2).

13.7.3.2 Add 10 mL of concentrated sulfuric acid to the bottle. Immediately cap and shake one to three times. Loosen cap in a hood to vent excess pressure. Cap and shake the bottle so that the residue/solvent is exposed to the acid for a total time of approximately 45 seconds.

13.7.3.3 Decant the hexane into a 250 mL separatory funnel making sure that no acid is transferred. Complete the quantitative transfer with several hexane rinses.

13.7.3.4 Back extract the solvent/residue with 50 mL of potassium hydroxide solution per Section 12.5.2, followed by two reagent water rinses.

13.7.3.5 Drain the extract through a filter funnel containing approximately 10 g of granular anhydrous sodium sulfate in a glass-fiber filter into a macro concentration device (Section 12.6).

13.7.3.6 Concentrate the cleaned up extract to a volume suitable for the additional cleanups given in Sections 13.2 through 13.6 and 13.8. Gel permeation chromatography (Section 13.2), alumina (Section 13.4) or Florisil (Section 13.8), and Carbopak/Celite (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.3.7 Following cleanup, concentrate the extract to 10 L as described in Section 12.7 and proceed with analysis per Section 14.

13.8 Florisil Cleanup (Reference 29).

13.8.1 Pre-elute the activated Florisil column (Section 7.5.3) with 10 mL of methylene chloride followed by 10 mL of hexane:methylene chloride (98:2 v/v) and discard the solvents.

13.8.2 When the solvent is within 1 mm of the packing, apply the sample extract (in hexane) to the column. Rinse the sample container twice with 1 mL portions of hexane and apply to the column.

13.8.3 Elute the interfering compounds with 20 mL of hexane:methylene chloride (98:2) and discard the eluate.

13.8.4 Elute the CDDs/CDFs with 35 mL of methylene chloride and collect the eluate. Concentrate the eluate per Sections 12.6 through 12.7 for further cleanup or for injection into the HPLC or GC/MS.

14.0 HRGC/HRMS Analysis

14.1 Establish the operating conditions given in Section 10.1.

14.2 Add 10 uL of the appropriate internal standard solution (Section 7.12) to the sample extract immediately prior to injection to minimize the possibility of loss by evaporation, adsorption, or reaction. If an extract is to be reanalyzed and evaporation has occurred, do not add more instrument internal standard solution. Rather, bring the extract back to its previous volume (e.g., 19 L) with pure nonane only (18 L if 2 L injections are used).

14.3 Inject 1.0 µL or 2.0 µL of the concentrated extract containing the internal standard solution, using on-column or splitless injection. The volume injected must be identical to the volume used for calibration (Section 10). Start the GC column initial isothermal hold upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after the OCDD and OCDF have eluted. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, stop data collection after elution of these compounds. Return the column to the initial temperature for analysis of the next extract or standard.

15.0 System and Laboratory Performance

15.1 At the beginning of each 12-hour shift during which analyses are performed, GC/MS system performance and calibration are verified for all CDDs/CDFs and labeled compounds. For these tests, analysis of the CS3 calibration verification (VER) standard (Section 7.13 and Table 4) and the isomer specificity test standards (Section 7.15 and Table 5) shall be used to verify all performance criteria. Adjustment and/or recalibration (Section 10) shall be performed until all performance criteria are met. Only after all performance criteria are met may samples, blanks, IPRs, and OPRs be analyzed.

15.2 MS Resolution—A static resolving power of at least 10,000 (10% valley definition) must be demonstrated at the appropriate m/z before any analysis is performed. Static resolving power checks must be performed at the beginning and at the end of each 12-hour shift according to procedures in Section 10.1.2. Corrective actions must be implemented whenever the resolving power does not meet the requirement.

15.3 Calibration Verification.

15.3.1 Inject the VER standard using the procedure in Section 14.

15.3.2 The m/z abundance ratios for all CDDs/CDFs shall be within the limits in Table 9; otherwise, the mass spectrometer shall be adjusted until the m/z abundance ratios fall within the limits specified, and the verification test shall be repeated. If the adjustment alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2) prior to repeat of the verification test.

15.3.3 The peaks representing each CDD/CDF and labeled compound in the VER standard must be present with S/N of at least 10; otherwise, the mass spectrometer shall be adjusted and the verification test repeated.

15.3.4 Compute the concentration of each CDD/CDF compound by isotope dilution (Section 10.5) for those compounds that have labeled analogs (Table 1). Compute the concentration of the labeled compounds by the internal standard method (Section 10.6). These concentrations are computed based on the calibration data in Section 10.

15.3.5 For each compound, compare the concentration with the calibration verification limit in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the concentration to the limit in Table 6a. If all compounds meet the acceptance criteria, calibration has been verified and analysis of standards and sample extracts may proceed. If, however, any compound fails its respective limit, the measurement system is not performing properly for that compound. In this event, prepare a fresh calibration standard or correct the problem causing the failure and repeat the resolution (Section 15.2) and verification (Section 15.3) tests, or recalibrate (Section 10).

15.4 Retention Times and GC Resolution.

15.4.1 Retention times.

15.4.1.1 Absolute—The absolute retention times of the13C12-1,2,3,4–TCDD and13C12-1,2,3,7,8,9-HxCDD GCMS internal standards in the verification test (Section 15.3) shall be within ±15 seconds of the retention times obtained during calibration (Sections 10.2.1 and 10.2.4).

15.4.1.2 Relative—The relative retention times of CDDs/CDFs and labeled compounds in the verification test (Section 15.3) shall be within the limits given in Table 2.

15.4.2 GC resolution.

15.4.2.1 Inject the isomer specificity standards (Section 7.15) on their respective columns.

15.4.2.2 The valley height between 2,3,7,8-TCDD and the other tetra-dioxin isomers at m/z 319.8965, and between 2,3,7,8-TCDF and the other tetra-furan isomers at m/z 303.9016 shall not exceed 25% on their respective columns (Figures 6 and 7).

15.4.3 If the absolute retention time of any compound is not within the limits specified or if the 2,3,7,8-isomers are not resolved, the GC is not performing properly. In this event, adjust the GC and repeat the verification test (Section 15.3) or recalibrate (Section 10), or replace the GC column and either verify calibration or recalibrate.

15.5 Ongoing Precision and Recovery.

15.5.1 Analyze the extract of the ongoing precision and recovery (OPR) aliquot (Section 11.4.2.5, 11.5.4, 11.6.2, 11.7.4, or 11.8.3.2) prior to analysis of samples from the same batch.

15.5.2 Compute the concentration of each CDD/CDF by isotope dilution for those compounds that have labeled analogs (Section 10.5). Compute the concentration of 1,2,3,7,8,9-HxCDD, OCDF, and each labeled compound by the internal standard method (Section 10.6).

15.5.3 For each CDD/CDF and labeled compound, compare the concentration to the OPR limits given in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the concentration to the limits in Table 6a. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual concentration falls outside of the range given, the extraction/concentration processes are not being performed properly for that compound. In this event, correct the problem, re-prepare, extract, and clean up the sample batch and repeat the ongoing precision and recovery test (Section 15.5).

15.5.4 Add results that pass the specifications in Section 15.5.3 to initial and previous ongoing data for each compound in each matrix. Update QC charts to form a graphic representation of continued laboratory performance. Develop a statement of laboratory accuracy for each CDD/CDF in each matrix type by calculating the average percent recovery (R) and the standard deviation of percent recovery (SR). Express the accuracy as a recovery interval from R−2SR to R = 2SR. For example, if R = 95% and SR = 5%, the accuracy is 85–105%.

15.6 Blank—Analyze the method blank extracted with each sample batch immediately following analysis of the OPR aliquot to demonstrate freedom from contamination and freedom from carryover from the OPR analysis. The results of the analysis of the blank must meet the specifications in Section 9.5.2 before sample analyses may proceed.

16.0 Qualitative Determination

A CDD, CDF, or labeled compound is identified in a standard, blank, or sample when all of the criteria in Sections 16.1 through 16.4 are met.

16.1 The signals for the two exact m/z's in Table 8 must be present and must maximize within the same two seconds.

16.2 The signal-to-noise ratio (S/N) for the GC peak at each exact m/z must be greater than or equal to 2.5 for each CDD or CDF detected in a sample extract, and greater than or equal to 10 for all CDDs/CDFs in the calibration standard (Sections 10.2.3 and 15.3.3).

16.3 The ratio of the integrated areas of the two exact m/z's specified in Table 8 must be within the limit in Table 9, or within ±10% of the ratio in the midpoint (CS3) calibration or calibration verification (VER), whichever is most recent.

16.4 The relative retention time of the peak for a 2,3,7,8-substituted CDD or CDF must be within the limit in Table 2. The retention time of peaks representing non-2,3,7,8-substituted CDDs/CDFs must be within the retention time windows established in Section 10.3.

16.5 Confirmatory Analysis—Isomer specificity for 2,3,7,8-TCDF cannot be achieved on the DB–5 column. Therefore, any sample in which 2,3,7,8-TCDF is identified by analysis on a DB–5 column must have a confirmatory analysis performed on a DB–225, SP–2330, or equivalent GC column. The operating conditions in Section 10.1.1 may be adjusted to optimize the analysis on the second GC column, but the GC/MS must meet the mass resolution and calibration specifications in Section 10.

16.6 If the criteria for identification in Sections 16.1 through 16.5 are not met, the CDD or CDF has not been identified and the results may not be reported for regulatory compliance purposes. If interferences preclude identification, a new aliquot of sample must be extracted, further cleaned up, and analyzed.

17.0 Quantitative Determination

17.1 Isotope Dilution Quantitation—By adding a known amount of a labeled compound to every sample prior to extraction, correction for recovery of the CDD/CDF can be made because the CDD/CDF and its labeled analog exhibit similar effects upon extraction, concentration, and gas chromatography. Relative response (RR) values are used in conjunction with the initial calibration data described in Section 10.5 to determine concentrations directly, so long as labeled compound spiking levels are constant, using the following equation:

where:

Cex = The concentration of the CDD/CDF in the extract, and the other terms are as defined in Section 10.5.2.

17.1.1 Because of a potential interference, the labeled analog of OCDF is not added to the sample. Therefore, OCDF is quantitated against labeled OCDD. As a result, the concentration of OCDF is corrected for the recovery of the labeled OCDD. In instances where OCDD and OCDF behave differently during sample extraction, concentration, and cleanup procedures, this may decrease the accuracy of the OCDF results. However, given the low toxicity of this compound relative to the other dioxins and furans, the potential decrease in accuracy is not considered significant.

17.1.2 Because13C12-1,2,3,7,8,9-HxCDD is used as an instrument internal standard (i.e., not added before extraction of the sample), it cannot be used to quantitate the 1,2,3,7,8,9-HxCDD by strict isotope dilution procedures. Therefore, 1,2,3,7,8,9-HxCDD is quantitated using the averaged response of the labeled analogs of the other two 2,3,7,8-substituted HxCDD's: 1,2,3,4,7,8-HxCDD and 1,2,3,6,7,8-HxCDD. As a result, the concentration of 1,2,3,7,8,9-HxCDD is corrected for the average recovery of the other two HxCDD's.

17.1.3 Any peaks representing non-2,3,7,8-substituted CDDs/CDFs are quantitated using an average of the response factors from all of the labeled 2,3,7,8-isomers at the same level of chlorination.

17.2 Internal Standard Quantitation and Labeled Compound Recovery.

17.2.1 Compute the concentrations of 1,2,3,7,8,9-HxCDD, OCDF, the13C-labeled analogs and the37C-labeled cleanup standard in the extract using the response factors determined from the initial calibration data (Section 10.6) and the following equation:

where:

Cex = The concentration of the CDD/CDF in the extract, and the other terms are as defined in Section 10.6.1.

Note:

There is only one m/z for the37Cl-labeled standard.

17.2.2 Using the concentration in the extract determined above, compute the percent recovery of the13C-labeled compounds and the37C-labeled cleanup standard using the following equation:

17.3 The concentration of a CDD/CDF in the solid phase of the sample is computed using the concentration of the compound in the extract and the weight of the solids (Section 11.5.1), as follows:

where:

Cex = The concentration of the compound in the extract.

Vex = The extract volume in mL.

Ws = The sample weight (dry weight) in kg.

17.4 The concentration of a CDD/CDF in the aqueous phase of the sample is computed using the concentration of the compound in the extract and the volume of water extracted (Section 11.4 or 11.5), as follows:

where:

Cex = The concentration of the compound in the extract.

Vex = The extract volume in mL.

Vs = The sample volume in liters.

17.5 If the SICP area at either quantitation m/z for any compound exceeds the calibration range of the system, a smaller sample aliquot is extracted.

17.5.1 For aqueous samples containing 1% solids or less, dilute 100 mL, 10 mL, etc., of sample to 1 L with reagent water and re-prepare, extract, clean up, and analyze per Sections 11 through 14.

17.5.2 For samples containing greater than 1% solids, extract an amount of sample equal to 110, 1100, etc., of the amount used in Section 11.5.1. Re-prepare, extract, clean up, and analyze per Sections 11 through 14.

17.5.3 If a smaller sample size will not be representative of the entire sample, dilute the sample extract by a factor of 10, adjust the concentration of the instrument internal standard to 100 pg/µL in the extract, and analyze an aliquot of this diluted extract by the internal standard method.

17.6 Results are reported to three significant figures for the CDDs/CDFs and labeled compounds found in all standards, blanks, and samples.

17.6.1 Reporting units and levels.

17.6.1.1 Aqueous samples—Report results in pg/L (parts-per-quadrillion).

17.6.1.2 Samples containing greater than 1% solids (soils, sediments, filter cake, compost)—Report results in ng/kg based on the dry weight of the sample. Report the percent solids so that the result may be corrected.

17.6.1.3 Tissues—Report results in ng/kg of wet tissue, not on the basis of the lipid content of the sample. Report the percent lipid content, so that the data user can calculate the concentration on a lipid basis if desired.

17.6.1.4 Reporting level.

17.6.1.4.1 Standards (VER, IPR, OPR) and samples—Report results at or above the minimum level (Table 2). Report results below the minimum level as not detected or as required by the regulatory authority.

17.6.1.4.2 Blanks—Report results above one-third the ML.

17.6.2 Results for CDDs/CDFs in samples that have been diluted are reported at the least dilute level at which the areas at the quantitation m/z's are within the calibration range (Section 17.5).

17.6.3 For CDDs/CDFs having a labeled analog, results are reported at the least dilute level at which the area at the quantitation m/z is within the calibration range (Section 17.5) and the labeled compound recovery is within the normal range for the method (Section 9.3 and Tables 6, 6a, 7, and 7a).

17.6.4 Additionally, if requested, the total concentration of all isomers in an individual level of chlorination (i.e., total TCDD, total TCDF, total Paced, etc.) may be reported by summing the concentrations of all isomers identified in that level of chlorination, including both 2,3,7,8-substituted and non-2,3,7,8-substituted isomers.

18.0 Analysis of Complex Samples

18.1 Some samples may contain high levels (>10 ng/L; >1000 ng/kg) of the compounds of interest, interfering compounds, and/or polymeric materials. Some extracts will not concentrate to 10 µL (Section 12.7); others may overload the GC column and/or mass spectrometer.

18.2 Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not concentrate to 10 µL after all cleanup procedures have been exhausted.

18.3 Chlorodiphenyl Ethers—If chromatographic peaks are detected at the retention time of any CDDs/CDFs in any of the m/z channels being monitored for the chlorodiphenyl ethers (Table 8), cleanup procedures must be employed until these interferences are removed. Alumina (Section 13.4) and Florisil (Section 13.8) are recommended for removal of chlorodiphenyl ethers.

18.4 Recovery of Labeled Compounds—In most samples, recoveries of the labeled compounds will be similar to those from reagent water or from the alternate matrix (Section 7.6).

18.4.1 If the recovery of any of the labeled compounds is outside of the normal range (Table 7), a diluted sample shall be analyzed (Section 17.5).

18.4.2 If the recovery of any of the labeled compounds in the diluted sample is outside of normal range, the calibration verification standard (Section 7.13) shall be analyzed and calibration verified (Section 15.3).

18.4.3 If the calibration cannot be verified, a new calibration must be performed and the original sample extract reanalyzed.

18.4.4 If the calibration is verified and the diluted sample does not meet the limits for labeled compound recovery, the method does not apply to the sample being analyzed and the result may not be reported for regulatory compliance purposes. In this case, alternate extraction and cleanup procedures in this method must be employed to resolve the interference. If all cleanup procedures in this method have been employed and labeled compound recovery remains outside of the normal range, extraction and/or cleanup procedures that are beyond this scope of this method will be required to analyze these samples.

19.0 Pollution Prevention

19.1 The solvents used in this method pose little threat to the environment when managed properly. The solvent evaporation techniques used in this method are amenable to solvent recovery, and it is recommended that the laboratory recover solvents wherever feasible.

19.2 Standards should be prepared in volumes consistent with laboratory use to minimize disposal of standards.

20.0 Waste Management

20.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations.

20.2 Samples containing HCl to pH <2 are hazardous and must be neutralized before being poured down a drain or must be handled as hazardous waste.

20.3 The CDDs/CDFs decompose above 800 °C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in an appropriate incinerator. Gross quantities (milligrams) should be packaged securely and disposed of through commercial or governmental channels that are capable of handling extremely toxic wastes.

20.4 Liquid or soluble waste should be dissolved in methanol or ethanol and irradiated with ultraviolet light with a wavelength shorter than 290 nm for several days. Use F40 BL or equivalent lamps. Analyze liquid wastes, and dispose of the solutions when the CDDs/CDFs can no longer be detected.

20.5 For further information on waste management, consult “The Waste Management Manual for Laboratory Personnel” and “Less is Better—Laboratory Chemical Management for Waste Reduction,” available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.

21.0 Method Performance

Method performance was validated and performance specifications were developed using data from EPA's international interlaboratory validation study (References 30–31) and the EPA/paper industry Long-Term Variability Study of discharges from the pulp and paper industry (58 FR 66078).

22.0 References

1. Tondeur, Yves. “Method 8290: Analytical Procedures and Quality Assurance for Multimedia Analysis of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans by High Resolution Gas Chromatography/High Resolution Mass Spectrometry,” USEPA EMSL, Las Vegas, Nevada, June 1987.

2. “Measurement of 2,3,7,8-Tetrachlorinated Dibenzo-p -dioxin (TCDD) and 2,3,7,8-Tetrachlorinated Dibenzofuran (TCDF) in Pulp, Sludges, Process Samples and Wastewaters from Pulp and Paper Mills,” Wright State University, Dayton, OH 45435, June 1988.

3. “NCASI Procedures for the Preparation and Isomer Specific Analysis of Pulp and Paper Industry Samples for 2,3,7,8-TCDD and 2,3,7,8-TCDF,” National Council of the Paper Industry for Air and Stream Improvement Inc., 260 Madison Avenue, New York, NY 10016, Technical Bulletin No. 551, Pre-Release Copy, July 1988.

4. “Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish,” USEPA, Environmental Research Laboratory, 6201 Congdon Boulevard, Duluth, MN 55804, April 1988.

5. Tondeur, Yves. “Proposed GC/MS Methodology for the Analysis of PCDDs and PCDFs in Special Analytical Services Samples,” Triangle Laboratories, Inc., 801–10 Capitola Dr, Research Triangle Park, NC 27713, January 1988; updated by personal communication September 1988.

6. Lamparski, L.L. and Nestrick, T.J. “Determination of Tetra-, Hexa-, Hepta-, and Octachlorodibenzo-p-dioxin Isomers in Particulate Samples at Parts per Trillion Levels,” Analytical Chemistry, 52: 2045–2054, 1980.

7. Lamparski, L.L. and Nestrick, T.J. “Novel Extraction Device for the Determination of Chlorinated Dibenzo-p-dioxins (PCDDs) and Dibenzofurans (PCDFs) in Matrices Containing Water,” Chemosphere, 19:27–31, 1989.

8. Patterson, D.G., et. al. “Control of Interferences in the Analysis of Human Adipose Tissue for 2,3,7,8-Tetrachlorodibenzo-p-dioxin,” Environmental Toxicological Chemistry, 5:355–360, 1986.

9. Stanley, John S. and Sack, Thomas M. “Protocol for the Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin by High Resolution Gas Chromatography/High Resolution Mass Spectrometry,” USEPA EMSL, Las Vegas, Nevada 89114, EPA 600/4–86–004, January 1986.

10. “Working with Carcinogens,” Department of Health, Education, & Welfare, Public Health Service, Centers for Disease Control, NIOSH, Publication 77–206, August 1977, NTIS PB–277256.

11. “OSHA Safety and Health Standards, General Industry,” OSHA 2206, 29 CFR 1910.

12. “Safety in Academic Chemistry Laboratories,” ACS Committee on Chemical Safety, 1979.

13. “Standard Methods for the Examination of Water and Wastewater,” 18th edition and later revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC 20005, 1–35: Section 1090 (Safety), 1992.

14. “Method 613—2,3,7,8-Tetrachlorodibenzo-p-dioxin,” 40 CFR 136 (49 FR 43234), October 26, 1984, Section 4.1.

15. Provost, L.P. and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15: 56–83, 1983.

16. “Standard Practice for Sampling Water,” ASTM Annual Book of Standards, ASTM, 1916 Race Street, Philadelphia, PA 19103–1187, 1980.

17. “Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA, EMSL, Cincinnati, OH 45268, EPA 600/4–79–020, March 1979.

18. “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA EMSL, Cincinnati, OH 45268, EPA–600/4–79–019, March 1979.

19. Williams, Rick. Letter to Bill Telliard, June 4, 1993, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

20. Barkowski, Sarah. Fax to Sue Price, August 6, 1992, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria VA 22314, 703–519–1140.

21. “Analysis of Multi-media, Multi-concentration Samples for Dioxins and Furans, PCDD/PCDF Analyses Data Package” , Narrative for Episode 4419, MRI Project No. 3091-A, op.cit. February 12, 1993, Available from the EPA Sample Control Center operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314 (703–519–1140).

22. “Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish” , U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, MN 55804, EPA/600/3–90/022, March 1990.

23. Afghan, B.K., Carron, J., Goulden, P.D., Lawrence, J., Leger, D., Onuska, F., Sherry, J., and Wilkenson, R.J., “Recent Advances in Ultratrace Analysis of Dioxins and Related Halogenated Hydrocarbons”, Can J. Chem., 65: 1086–1097, 1987.

24. Sherry, J.P. and Tse, H. “A Procedure for the Determination of Polychlorinated Dibenzo-p-dioxins in Fish”, Chemosphere, 20: 865–872, 1990.

25. “Preliminary Fish Tissue Study” , Results of Episode 4419, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

26. Nestrick, Terry L. DOW Chemical Co., personal communication with D.R. Rushneck, April 8, 1993. Details available from the U.S. Environmental Protection Agency Sample Control Center operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

27. Barnstadt, Michael. “Big Fish Column”, Triangle Laboratories of RTP, Inc., SOP 129–90, 27 March 27, 1992.

28. “Determination of Polychlorinated Dibenzo-p-Dioxins (PCDD) and Dibenzofurans (PCDF) in Environmental Samples Using EPA Method 1613” , Chemical Sciences Department, Midwest Research Institute, 425 Volker Boulevard, Kansas City, MO 44110–2299, Standard Operating Procedure No. CS–153, January 15, 1992.

29. Ryan, John J. Raymonde Lizotte and William H. Newsome, J. Chromatog. 303 (1984) 351-360.

30. Telliard, William A., McCarty, Harry B., and Riddick, Lynn S. “Results of the Interlaboratory Validation Study of USEPA Method 1613 for the Analysis of Tetra-through Octachlorinated Dioxins and Furans by Isotope Dilution GC/MS,” Chemosphere, 27, 41–46 (1993).

31. “Results of the International Interlaboratory Validation Study of USEPA Method 1613” , October 1994, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

23.0 Tables and Figures

Table 1—Chlorinated Dibenzo-p-Dioxins and Furans Determined by Isotope Dilution and Internal Standard High Resolution Gas Chromatography (HRGC)/High Resolution Mass Spectrometry (HRMS)

CDDs/CDFs1 CAS registry Labeled analog CAS registry
2,3,7,8-TCDD 1746–01–6 13C12-2,3,7,8-TCDD
37Cl4-2,3,7,8-TCDD
76523–40–5
85508–50–5
Total TCDD 41903–57–5
2,3,7,8-TCDF 51207–31–9 13C12-2,3,7,8-TCDF 89059–46–1
Total-TCDF 55722–27–5
1,2,3,7,8-PeCDD 40321–76–4 13C12-1,2,3,7,8-PeCDD 109719–79–1
Total-PeCDD 36088–22–9
1,2,3,7,8-PeCDF 57117–41–6 13C12-1,2,3,7,8-PeCDF 109719–77–9
2,3,4,7,8-PeCDF 57117–31–4 13C12-2,3,4,7,8-PeCDF 116843–02–8
Total-PeCDF 30402–15–4
1,2,3,4,7,8-HxCDD 39227–28–6 13C12-1,2,3,4,7,8-HxCDD 109719–80–4
1,2,3,6,7,8-HxCDD 57653–85–7 13C12-1,2,3,6,7,8-HxCDD 109719–81–5
1,2,3,7,8,9-HxCDD 19408–74–3 13C12-1,2,3,7,8,9-HxCDD 109719–82–6
Total-HxCDD 34465–46–8
1,2,3,4,7,8-HxCDF 70648–26–9 13C12-1,2,3,4,7,8-HxCDF 114423–98–2
1,2,3,6,7,8-HxCDF 57117–44–9 13C12-1,2,3,6,7,8-HxCDF 116843–03–9
1,2,3,7,8,9-HxCDF 72918–21–9 13C12-1,2,3,7,8,9-HxCDF 116843–04–0
2,3,4,6,7,8-HxCDF 60851–34–5 13C12-2,3,4,6,7,8-HxCDF 116843–05–1
Total-HxCDF 55684–94–1
1,2,3,4,6,7,8-HpCDD 35822–46–9 13C12-1,2,3,4,6,7,8-HpCDD 109719–83–7
Total-HpCDD 37871–00–4
1,2,3,4,6,7,8-HpCDF 67562–39–4 13C12-1,2,3,4,6,7,8-HpCDF 109719–84–8
1,2,3,4,7,8,9-HpCDF 55673–89–7 13C12-1,2,3,4,7,8,9-HpCDF 109719–94–0
Total-HpCDF 38998–75–3
OCDD 3268–87–9 13C12-OCDD 114423–97–1
OCDF 39001–02–0 Not used

Table 2—Retention Time References, Quantitation References, Relative Retention Times, and Minimum Levels for CDDS and DCFS

CDD/CDF Retention time and quantitation
reference
Relative retention time Minimum level1
Water (pg/L; ppq) Solid (ng/kg; ppt) Extract (pg/µL; ppb)
Compounds using13 C12–1,2,3,4-TCDD as the Injection Internal Standard
2,3,7,8-TCDF 13 C12-2,3,7,8-TCDF 0.999–1.003 10 1 0.5
2,3,7,8-TCDD 13 C12-2,3,7,8-TCDD 0.999–1.002 10 1 0.5
1,2,3,7,8-Pe 13 C12-1,2,3,7,8-PeCDF 0.999–1.002 50 5 2.5
2,3,4,7,8-PeCDF 13 C12-2,3,4,7,8-PeCDF 0.999–1.002 50 5 2.5
1,2,3,7,8-PeCDD 13 C12-1,2,3,7,8-PeCDD 0.999–1.002 50 5 2.5
13 C12-2,3,7,8-TCDF 13 C12-1,2,3,4-TCDD 0.923–1.103
13 C12-2,3,7,8-TCDD 13 C12-1,2,3,4-TCDD 0.976–1.043
13 C12-2,3,7,8-TCDD 13 C12-1,2,3,4-TCDD 0.989–1.052
13 C12-1,2,3,7,8-PeCDF 13 C12-1,2,3,4-TCDD 1.000–1.425
13 C12-2,3,4,7,8-PeCDF 13 C12-1,2,3,4-TCDD 1.001–1.526
13 C12-1,2,3,7,8-PeCDF 13 C12-1,2,3,4-TCDD 1.000–1.567
Compounds using13 C12–1,2,3,7,8,9-HxCDD as the Injection Internal Standard
1,2,3,4,7,8-HxCDF 13 C12-1,2,3,4,7,8-HxCDF 0.999–1.001 50 5 2.5
1,2,3,6,7,8-HxCDF 13 C12-1,2,3,6,7,8-HxCDF 0.997–1.005 50 5 2.5
1,2,3,7,8,9-HxCDF 13 C12-1,2,3,7,8,9-HxCDF 0.999–1.001 50 5 2.5
2,3,4,6,7,8-HxCDF 13 C12-2,3,4,6,7,8-HxCDF 0.999–1.001 50 5 2.5
1,2,3,4,7,8-HxCDD 13 C12-1,2,3,4,7,8-HxCDD 0.999–1.001 50 5 2.5
1,2,3,6,7,8-HxCDD 13 C12-1,2,3,6,7,8-HxCDD 0.998–1.004 50 5 2.5
1,2,3,7,8,9-HxCDD (2) 1.000–1.019 50 5 2.5
1,2,3,4,6,7,8-HpCDF 13 C12-1,2,3,4,6,7,8-HpCDF 0.999–1.001 50 5 2.5
1,2,3,4,7,8,9-HpCDF 13 C12-1,2,3,4,7,8,9-HpCDF 0.999–1.001 50 5 2.5
1,2,3,4,6,7,8-HpCDD 13 C12-1,2,3,4,6,7,8-HpCDD 0.999–1.001 50 5 2.5
OCDF 13 C12-OCDD 0.999–1.001 100 10 5.0
OCDD 13 C12-OCDD 0.999–1.001 100 10 5.0
1,2,3,4,6,7,8,-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 0.949–0.975
13 C121,2,3,7,8,9-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 0.977–1.047
13 C122,3,4,6,7,8,-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 0.959–1.021
13 C121,2,3,4,7,8,-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 0.977–1.000
13 C121,2,3,6,7,8,-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 0.981–1.003
13 C121,2,3,4,6,7,8-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 1.043–1.085
13 C121,2,3,4,7,8,9-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 1.057–1.151
13 C121,2,3,4,6,7,8-HxCDF 13 C12-1,2,3,7,8,9-HpCDD 1.086–1.110
13 C12OCDD 13 C12-1,2,3,7,8,9-HpCDD 1.032–1.311

Table 3—Concentration of Stock and Spiking Solutions Containing CDDS/CDFS and Labeled Compounds

CDD/CDF Labeled compound stock solution1
(ng/mL)
Labeled compound spiking solution2
(ng/mL)
PAR stock solution3 (ng/mL) PAR spiking solution4
(ng/mL)
2,3,7,8-TCDD 40 0.8
2,3,7,8-TCDF 40 0.8
1,2,3,7,8-PeCDD 200 4
1,2,3,7,8-PeCDF 200 4
2,3,4,7,8-PeCDF 200 4
1,2,3,4,7,8-HxCDD 200 4
1,2,3,6,7,8-HxCDD 200 4
1,2,3,7,8,9-HxCDD 200 4
1,2,3,4,7,8-HxCDF 200 4
1,2,3,6,7,8-HxCDF 200 4
1,2,3,7,8,9-HxCDF 200 4
2,3,4,6,7,8-HxCDF 200 4
1,2,3,4,6,7,8-HpCDD 200 4
1,2,3,4,6,7,8-HpCDF 200 4
1,2,3,4,7,8,9-HpCDF 200 4
OCDD 400 8
OCDF 400 8
13C12-2,3,7,8-TCDD 100 2
13C12-2,3,7,8-TCDF 100 2
13C12-1,2,3,7,8-PeCDD 100 2
13C12-1,2,3,7,8-PeCDF 100 2
13C12-2,3,4,7,8-PeCDF 100 2
13C12-1,2,3,4,7,8-HxCDD 100 2
13C12-1,2,3,6,7,8-HxCDD 100 2
13C12-1,2,3,4,7,8-HxCDF 100 2
13C12-1,2,3,6,7,8-HxCDF 100 2
13C12-1,2,3,7,8,9-HxCDF 100 2
13C12-2,3,4,6,7,8-HxCDF 100 2
13C12-1,2,3,4,6,7,8-HpCDD 100 2
13C12-1,2,3,4,6,7,8-HpCDF 100 2
13C12-1,2,3,4,7,8,9-HpCDF 100 2
13C12-OCDD 200 4
Cleanup Standard5
37Cl4-2,3,7,8-TCDD 0.8
Internal Standards6
13C12-1,2,3,4-TCDD 200
13C12-1,2,3,7,8,9-HxCDD 200

Table 4—Concentration of CDDS/CDFS in Calibration and Calibration Verification Solutions1 (Section 15.3)

CDD/CDF CS2
(ng/mL)
CS3
(ng/mL)
CS4
(ng/mL)
CS5
(ng/mL)
2,3,7,8-TCDD 0.5 2 10 40 200
2,3,7,8-TCDF 0.5 2 10 40 200
1,2,3,7,8-PeCDD 2.5 10 50 200 1000
1,2,3,7,8-PeCDF 2.5 10 50 200 1000
2,3,4,7,8-PeCDF 2.5 10 50 200 1000
1,2,3,4,7,8-HxCDD 2.5 10 50 200 1000
1,2,3,6,7,8-HxCDD 2.5 10 50 200 1000
1,2,3,7,8,9-HxCDD 2.5 10 50 200 1000
1,2,3,4,7,8-HxCDF 2.5 10 50 200 1000
1,2,3,6,7,8-HxCDF 2.5 10 50 200 1000
1,2,3,7,8,9-HxCDF 2.5 10 50 200 1000
2,3,4,6,7,8-HxCDF 2.5 10 50 200 1000
1,2,3,4,6,7,8-HpCDD 2.5 10 50 200 1000
1,2,3,4,6,7,8-HpCDF 2.5 10 50 200 1000
1,2,3,4,7,8,9-HpCDF 2.5 10 50 200 1000
OCDD 5.0 20 100 400 2000
OCDF 5.0 20 100 400 2000
13 C12-2,3,7,8-TCDD 100 100 100 100 100
13 C12-2,3,7,8-TCDF 100 100 100 100 100
13 C12-1,2,3,7,8-PeCDD 100 100 100 100 100
13 C12-PeCDF 100 100 100 100 100
13 C12-2,3,4,7,8-PeCDF 100 100 100 100 100
13 C12-1,2,3,4,7,8-HxCDD 100 100 100 100 100
13 C12-1,2,3,6,7,8-HxCDD 100 100 100 100 100
13 C12-1,2,3,4,7,8-HxCDF 100 100 100 100 100
13 C12-1,2,3,6,7,8-HxCDF 100 100 100 100 100
13 C12-1,2,3,7,8,9-HxCDF 100 100 100 100 100
13 C12-1,2,3,4,6,7,8-HpCDD 100 100 100 100 100
13 C12-1,2,3,4,6,7,8-HpCDF 100 100 100 100 100
13 C12-1,2,3,4,7,8,9-Hp CDF 100 100 100 100 100
13 C12-OCDD 200 200 200 200 200
Cleanup Standard:
37 C14-2,3,7,8-TCDD 0.5 2 10 40 200
Internal Standards:
13 C12-1,2,3,4-TCDD 100 100 100 100 100
13 C12-1,2,3,7,8,9-HxCDD 100 100 100 100 100

Table 5—GC Retention Time Window Defining Solution and Isomer Specificity Test Standard (Section 7.15)

DB–5 column GC retention-time window defining solution
CDD/CDF First eluted Last eluted
TCDF 1,3,6,8- 1,2,8,9-
TCDD 1,3,6,8- 1,2,8,9-
PeCDF 1,3,4,6,8- 1,2,3,8,9-
PeCDD 1,2,4,7,9- 1,2,3,8,9-
HxCDF 1,2,3,4,6,8- 1,2,3,4,8,9-
HxCDD 1,2,4,6,7,9- 1,2,3,4,6,7-
HpCDF 1,2,3,4,6,7,8- 1,2,3,4,7,8,9-
HpCDD 1,2,3,4,6,7,9- 1,2,3,4,6,7,8-
DB–5 Column TCDD Specificity Test Standard
1,2,3,7 = 1,2,3,8-TCDD
2,3,7,8-TCDD
1,2,3,9-TCDD
DB–225 Column TCDF Isomer Specificity Test Standard
2,3,4,7-TCDF
2,3,7,8-TCDF
1,2,3,9-TCDF

Table 6—Acceptance Criteria for Performance Tests When All CDDS/CDFS Are Tested1

CDD/CDF Test conc. (ng/mL) IPR2 3 OPR
(ng/mL)
VER
(ng/mL)
s
(ng/mL)
X
(ng/mL)
2,3,7,8-TCDD 10 2.8 8.3–12.9 6.7–15.8 7.8–12.9
2,3,7,8-TCDF 10 2.0 8.7–13.7 7.5–15.8 8.4–12.0
1,2,3,7,8-PeCDD 50 7.5 38–66 35–71 39–65
1,2,3,7,8-PeCDF 50 7.5 43–62 40–67 41–60
2,3,4,7,8-PeCDF 50 8.6 36–75 34–80 41–61
1,2,3,4,7,8-HxCDD 50 9.4 39–76 35–82 39–64
1,2,3,6,7,8-HxCDD 50 7.7 42–62 38–67 39–64
1,2,3,7,8,9-HxCDD 50 11.1 37–71 32–81 41–61
1,2,3,4,7,8-HxCDF 50 8.7 41–59 36–67 45–56
1,2,3,6,7,8-HxCDF 50 6.7 46–60 42–65 44–57
1,2,3,7,8,9-HxCDF 50 6.4 42–61 39–65 45–56
2,3,4,6,7,8-HxCDF 50 7.4 37–74 35–78 44–57
1,2,3,4,6,7,8-HpCDD 50 7.7 38–65 35–70 43–58
1,2,3,4,6,7,8-HpCDF 50 6.3 45–56 41–61 45–55
1,2,3,4,7,8,9-HpCDF 50 8.1 43–63 39–69 43–58
OCDD 100 19 89–127 78–144 79–126
OCDF 100 27 74–146 63–170 63–159
13C12-2,3,7,8-TCDD 100 37 28–134 20–175 82–121
13C12-2,3,7,8-TCDF 100 35 31–113 22–152 71–140
13C12-1,2,3,7,8-PeCDD 100 39 27–184 21–227 62–160
13C12-1,2,3,7,8-PeCDF 100 34 27–156 21–192 76–130
13C12-2,3,4,7,8-PeCDF 100 38 16–279 13–328 77–130
13C12-1,2,3,4,7,8-HxCDD 100 41 29–147 21–193 85–117
13C12-1,2,3,6,7,8-HxCDD 100 38 34–122 25–163 85–118
13C12-1,2,3,4,7,8-HxCDF 100 43 27–152 19–202 76–131
13C12-1,2,3,6,7,8-HxCDF 100 35 30–122 21–159 70–143
13C12-1,2,3,7,8,9-HxCDF 100 40 24–157 17–205 74–135
13C12-2,3,4,6,7,8,-HxCDF 100 37 29–136 22–176 73–137
13C12-1,2,3,4,6,7,8-HpCDD 100 35 34–129 26–166 72–138
13C12-1,2,3,4,6,7,8-HpCDF 100 41 32–110 21–158 78–129
13C12-1,2,3,4,7,8,9-HpCDF 100 40 28–141 20–186 77–129
13C12-OCDD 200 95 41–276 26–397 96–415
37Cl4-2,3,7,8-TCDD 10 3.6 3.9–15.4 3.1–19.1 7.9–12.7

Table 6a—Acceptance Criteria for Performance Tests When Only Tetra Compounds are Tested1

CDD/CDF Test Conc. (ng/mL) IPR2 3 OPR
(ng/mL)
VER
(ng/mL)
s (ng/mL) X (ng/mL)
2,3,7,8-TCDD 10 2.7 8.7–12.4 7.314.6 8.2–12.3
2,3,7,8-TCDF 10 2.0 9.1–13.1 8.0–14.7 8.6–11.6
13C12-2,3,7,8-TCDD 100 35 32–115 25–141 85–117
13C12-2,3,7,8-TCDF 100 34 35–99 26–126 76–131
37Cl4-2,3,7,8-TCDD 10 3.4 4.5–13.4 3.7–15.8 8.3–12.1

Table 7—Labeled Compounds Recovery in Samples When all CDDS/CDFS are Tested

Compound Test conc. (ng/mL) Labeled compound
recovery
(ng/mL)1 (%)
13C12-2,3,7,8-TCDD 100 25–164 25–164
13C12-2,3,7,8-TCDF 100 24–169 24–169
13C12-1,2,3,7,8-PeCDD 100 25–181 25–181
13C12-1,2,3,7,8-PeCDF 100 24–185 24–185
13C12-2,3,4,7,8-PeCDF 100 21–178 21–178
13C12-1,2,3,4,7,8-HxCDD 100 32–141 32–141
13C12-1,2,3,6,7,8-HxCDD 100 28–130 28–130
13C12-1,2,3,4,7,8-HxCDF 100 26–152 26–152
13C12-1,2,3,6,7,8-HxCDF 100 26–123 26–123
13C12-1,2,3,7,8,9-HxCDF 100 29–147 29–147
13C12-2,3,4,6,7,8-HxCDF 100 28–136 28–136
13C12-1,2,3,4,6,7,8-HpCDD 100 23–140 23–140
13C12-1,2,3,4,6,7,8-HpCDF 100 28–143 28–143
13C12-1,2,3,4,7,8,9-HpCDF 100 26–138 26–138
13C12-OCDD 200 34-313 17–157
37Cl4-2,3,7,8-TCDD 10 3.5–19.7 35–197

Table 7a—Labeled Compound Recovery in Samples When Only Tetra Compounds are Tested

Compound Test conc. (ng/mL) Labeled compound
recovery
(ng/mL)1 (%)
13C12-2,3,7,8-TCDD 100 31–137 31–137
13C12-2,3,7,8-TCDF 100 29–140 29–140
37Cl4-2,3,7,8-TCDD 10 4.2–16.4 42–164

Table 8—Descriptors, Exact M/Z's, M/Z Types, and Elemental Compositions of the CDDs and CDFs

Descriptor Exact M/Z1 M/Z type Elemental composition Substance2
1 292.9825 Lock C7F11 PFK
303.9016 M C12H435Cl4O TCDF
305.8987 M = 2 C12H435Cl337ClO TCDF
315.9419 M 13C12H435Cl4O TCDF3
317.9389 M = 2 13C12H435Cl337ClO TCDF3
319.8965 M C12H435Cl4O2 TCDD
321.8936 M = 2 C12H435Cl337ClO2 TCDD
327.8847 M C12H437Cl4O2 TCDD4
330.9792 QC C7F13 PFK
331.9368 M 13C12H435Cl4O2 TCDD3
333.9339 M = 2 13C12H435Cl337ClO2 TCDD3
375.8364 M = 2 C12H435Cl537ClO HxCDPE
2 339.8597 M = 2 C12H335Cl437ClO PeCDF
341.8567 M = 4 C12H335Cl337Cl2O PeCDF
351.9000 M = 2 13C12H335Cl437ClO PeCDF
353.8970 M = 4 13C12H335Cl337Cl2O PeCDF3
354.9792 Lock C9F13 PFK
355.8546 M = 2 C12H335Cl437ClO2 PeCDD
357.8516 M = 4 C12H335Cl337Cl2O2 PeCDD
367.8949 M = 2 13C12H335Cl437ClO2 PeCDD3
369.8919 M = 4 13C12H335Cl337Cl2O2 PeCDD3
409.7974 M = 2 C12H335Cl637ClO HpCDPE
3 373.8208 M = 2 C12H235Cl537ClO HxCDF
375.8178 M = 4 C12H235Cl437Cl2O HxCDF
383.8639 M 13C12H235Cl6O HxCDF3
385.8610 M = 2 13C12H235Cl537ClO HxCDF3
389.8157 M = 2 C12H235Cl537ClO2 HxCDD
391.8127 M = 4 C12H235Cl437Cl2O2 HxCDD
392.9760 Lock C9F15 PFK
401.8559 M = 2 13C12H235Cl537ClO2 HxCDD3
403.8529 M = 4 13C12H235Cl437Cl2O2 HxCDD3
430.9729 QC C9F17 PFK
445.7555 M = 4 C12H235Cl637Cl2O OCDPE
4 407.7818 M = 2 C12H35Cl637ClO HpCDF
409.7789 M = 4 C12H35Cl537Cl2O HpCDF
417.8253 M 13C12H35Cl7O HpCDF3
419.8220 M = 2 13C12H35Cl637ClO HpCDF3
423.7766 M = 2 C12H35Cl637ClO2 HpCDD
425.7737 M = 4 C12H35Cl537Cl2O2 HpCDD
430.9729 Lock C9F17 PFK
435.8169 M = 2 13C12H35Cl637ClO2 HpCDD3
437.8140 M = 4 13C12H35Cl537Cl2O2 HpCDD3
479.7165 M = 4 C12H35Cl737Cl2O NCDPE
5 441.7428 M = 2 C1235Cl737ClO OCDF
442.9728 Lock C10F17 PFK
443.7399 M = 4 C1235Cl637Cl2O OCDF
457.7377 M = 2 C1235Cl737ClO2 OCDD
459.7348 M = 4 C1235Cl637Cl2O2 OCDD
469.7779 M = 2 13C1235Cl737ClO2 OCDD3
471.7750 M = 4 13C1235Cl637Cl2O2 OCDD3
513.6775 M = 4 C1235Cl837Cl2O DCDPE

Table 9—Theoretical Ion Abundance Ratios and QC Limits

Number of chlorine atoms M/Z's forming ratio Theoretical ratio QC limit1
Lower Upper
42 M/(M = 2) 0.77 0.65 0.89
5 (M = 2)/(M = 4) 1.55 1.32 1.78
6 (M = 2)/(M = 4) 1.24 1.05 1.43
63 M/(M = 2) 0.51 0.43 0.59
7 (M = 2)/(M = 4) 1.05 0.88 1.20
74 M/(M = 2) 0.44 0.37 0.51
8 (M = 2)/(M = 4) 0.89 0.76 1.02

Table 10—Suggested Sample Quantities To Be Extracted for Various Matrices1

Sample Matrix2 Example Percent solids Phase Quantity extracted
Single-phase:
Aqueous Drinking water <1 (3) 1000 mL.
Groundwater
Treated wastewater
Solid Dry soil >20 Solid 10 g.
Compost
Ash
Organic Waste solvent <1 Organic 10 g.
Waste oil
Organic polymer
Tissue Fish Organic 10 g.
Human adipose
Multi-phase:
Liquid/Solid:
Aqueous/Solid Wet soil 1–30 Solid 10 g.
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Organic/solid Industrial sludge 1–100 Both 10 g.
Oily waste
Liquid/Liquid:
Aqueous/organic In-process effluent <1 Organic 10 g.
Untreated effluent
Drum waste
Aqueous/organic/solid Untreated effluent >1 Organic and solid 10 g.
Drum waste

24.0 Glossary of Definitions and Purposes

These definitions and purposes are specific to this method but have been conformed to common usage as much as possible.

24.1 Units of weight and Measure and Their Abbreviations.

24.1.1 Symbols:

°C—degrees Celsius

µL—microliter

µm—micrometer

<—less than

>—greater than

%—percent

24.1.2 Alphabetical abbreviations:

amp—ampere

cm—centimeter

g—gram

h—hour

D—inside diameter

in.—inch

L—liter

M—Molecular ion

m—meter

mg—milligram

min—minute

mL—milliliter

mm—millimeter

m/z—mass-to-charge ratio

N—normal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of solution

OD—outside diameter

pg—picogram

ppb—part-per-billion

ppm—part-per-million

ppq—part-per-quadrillion

ppt—part-per-trillion

psig—pounds-per-square inch gauge

v/v—volume per unit volume

w/v—weight per unit volume

24.2 Definitions and Acronyms (in Alphabetical Order).

Analyte—A CDD or CDF tested for by this method. The analytes are listed in Table 1.

Calibration Standard (CAL)—A solution prepared from a secondary standard and/or stock solutions and used to calibrate the response of the instrument with respect to analyte concentration.

Calibration Verification Standard (VER)—The mid-point calibration standard (CS3) that is used in to verify calibration. See Table 4.

CDD—Chlorinated Dibenzo-p-ioxin—The isomers and congeners of tetra-through octa-chlorodibenzo-p-dioxin.

CDF—Chlorinated Dibenzofuran—The isomers and congeners of tetra-through octa-chlorodibenzofuran.

CS1, CS2, CS3, CS4, CS5—See Calibration standards and Table 4.

Field Blank—An aliquot of reagent water or other reference matrix that is placed in a sample container in the laboratory or the field, and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to determine if the field or sample transporting procedures and environments have contaminated the sample.

GC—Gas chromatograph or gas chromatography.

GPC—Gel permeation chromatograph or gel permeation chromatography.

HPLC—High performance liquid chromatograph or high performance liquid chromatography.

HRGC—High resolution GC.

HRMS—High resolution MS.

IPR—Initial precision and recovery; four aliquots of the diluted PAR standard analyzed to establish the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first time this method is used and any time the method or instrumentation is modified.

K-D—Kuderna-Danish concentrator; a device used to concentrate the analytes in a solvent.

Laboratory Blank—See method blank.

Laboratory Control sample (LCS)—See ongoing precision and recovery standard (OPR).

Laboratory Reagent Blank—See method blank.

May—This action, activity, or procedural step is neither required nor prohibited.

May Not—This action, activity, or procedural step is prohibited.

Method Blank—An aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples. The method blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus.

Minimum Level (ML)—The level at which the entire analytical system must give a recognizable signal and acceptable calibration point for the analyte. It is equivalent to the concentration of the lowest calibration standard, assuming that all method-specified sample weights, volumes, and cleanup procedures have been employed.

MS—Mass spectrometer or mass spectrometry.

Must—This action, activity, or procedural step is required.

OPR—Ongoing precision and recovery standard (OPR); a laboratory blank spiked with known quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified in this method for precision and recovery.

PAR—Precision and recovery standard; secondary standard that is diluted and spiked to form the IPR and OPR.

PFK—Perfluorokerosene; the mixture of compounds used to calibrate the exact m/z scale in the HRMS.

Preparation Blank—See method blank.

Primary Dilution Standard—A solution containing the specified analytes that is purchased or prepared from stock solutions and diluted as needed to prepare calibration solutions and other solutions.

Quality Control Check Sample (QCS)—A sample containing all or a subset of the analytes at known concentrations. The QCS is obtained from a source external to the laboratory or is prepared from a source of standards different from the source of calibration standards. It is used to check laboratory performance with test materials prepared external to the normal preparation process.

Reagent Water—Water demonstrated to be free from the analytes of interest and potentially interfering substances at the method detection limit for the analyte.

Relative Standard Deviation (RSD)—The standard deviation times 100 divided by the mean. Also termed “coefficient of variation.”

RF—Response factor. See Section 10.6.1.

RR—Relative response. See Section 10.5.2.

RSD—See relative standard deviation.

SDS—Soxhlet/Dean-Stark extractor; an extraction device applied to the extraction of solid and semi-solid materials (Reference 7).

Should—This action, activity, or procedural step is suggested but not required.

SICP—Selected ion current profile; the line described by the signal at an exact m/z.

SPE—Solid-phase extraction; an extraction technique in which an analyte is extracted from an aqueous sample by passage over or through a material capable of reversibly adsorbing the analyte. Also termed liquid-solid extraction.

Stock Solution—A solution containing an analyte that is prepared using a reference material traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will attest to the purity and authenticity of the reference material.

TCDD—Tetrachlorodibenzo-p-dioxin.

TCDF—Tetrachlorodibenzofuran.

VER—See calibration verification standard.

Method 1624 Revision B—Volatile Organic Compounds by Isotope Dilution GC/MS

1. Scope and Application

1.1 This method is designed to determine the volatile toxic organic pollutants associated with the 1976 Consent Decree and additional compounds amenable to purge and trap gas chromatography-mass spectrometry (GC/MS).

1.2 The chemical compounds listed in table 1 may be determined in municipal and industrial discharges by this method. The methmd is designed to meet the survey requirements of Effluent Guidelines Division (EGD) and the National Pollutants Discharge Elimination System (NPDES) under 40 CFR 136.1 and 136.5. Any modifications of this method, beyond those expressly permitted, shall be considered as major modifications subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.3 The detection limit of this method is usually dependent on the level of interferences rather than instrumental limitations. The limits in table 2 represent the minimum quantity that can be detected with no interferences present.

1.4 The GC/MS portions of this method are for use only by analysts experienced with GC/MS or under the close supervision of such qualified persons. Laboratories unfamiliar with the analyses of environmental samples by GC/MS should run the performance tests in reference 1 before beginning.

2. Summary of Method

2.1 Stable isotopically labeled analogs of the compounds of interest are added to a 5 mL water sample. The sample is purged at 20–25 °C with an inert gas in a specially designed chamber. The volatile organic compounds are transferred from the aqueous phase into the gaseous phase where they are passed into a sorbent column and trapped. After purging is completed, the trap is backflushed and heated rapidly to desorb the compounds into a gas chromatograph (GC). The compounds are separated by the GC and detected by a mass spectrometer (MS) (references 2 and 3). The labeled compounds serve to correct the variability of the analytical technique.

2.2 Identification of a compound (qualitative analysis) is performed by comparing the GC retention time and the background corrected characteristic spectral masses with those of authentic standards.

2.3 Quantitative analysis is performed by GC/MS using extracted ion current profile (EICP) areas. Isotope dilution is used when labeled compounds are available; otherwise, an internal standard method is used.

2.4 Quality is assured through reproducible calibration and testing of the purge and trap and GC/MS systems.

3. Contamination and Interferences

3.1 Impurities in the purge gas, organic compounds out-gassing from the plumbing upstream of the trap, and solvent vapors in the laboratory account for the majority of contamination problems. The analytical system is demonstrated to be free from interferences under conditions of the analysis by analyzing blanks initially and with each sample lot (samples analyzed on the same 8 hr shift), as described in Section 8.5.

3.2 Samples can be contaminated by diffusion of volatile organic compounds (particularly methylene chloride) through the bottle seal during shipment and storage. A field blank prepared from reagent water and carried through the sampling and handling protocol serves as a check on such contamination.

3.3 Contamination by carry-over can occur when high level and low level samples are analyzed sequentially. To reduce carry-over, the purging device and sample syringe are rinsed between samples with reagent water. When an unusually concentrated sample is encountered, it is followed by analysis of a reagent water blank to check for carry-over. For samples containing large amounts of water soluble materials, suspended solids, high boiling compounds, or high levels or purgeable compounds, the purge device is washed with soap solution, rinsed with tap and distilled water, and dried in an oven at 100–125 °C. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

3.4 Interferences resulting from samples will vary considerably from source to source, depending on the diversity of the industrial complex or municipality being sampled.

4. Safety

4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely determined; however, each chemical compound should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of data handling sheets should also be made available to all personnel involved in these analyses. Additional information on laboratory safety can be found in references 4–6.

4.2 The following compounds covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: benzene, carbon tetrachloride, chloroform, and vinyl chloride. Primary standards of these toxic compounds should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations are handled.

5. Apparatus and Materials

5.1 Sample bottles for discrete sampling.

5.1.1 Bottle—25 to 40 mL with screw cap (Pierce 13075, or equivalent). Detergent wash, rinse with tap and distilled water, and dry at >105 °C for one hr minimum before use.

5.1.2 Septum—Teflon-faced silicone (Pierce 12722, or equivalent), cleaned as above and baked at 100–200 °C, for one hour minimum.

5.2 Purge and trap device—consists of purging device, trap, and desorber. Complete devices are commercially available.

5.2.1 Purging device—designed to accept 5 mL samples with water column at least 3 cm deep. The volume of the gaseous head space between the water and trap shall be less than 15 mL. The purge gas shall be introduced less than 5 mm from the base of the water column and shall pass through the water as bubbles with a diameter less than 3 mm. The purging device shown in Figure 1 meets these criteria.

5.2.2 Trap—25 to 30 cm × 2.5 mm i.d. minimum, containing the following:

5.2.2.1 Methyl silicone packing—one ±0.2 cm, 3 percent OV–1 on 60/80 mesh Chromosorb W, or equivalent.

5.2.2.2 Porous polymer—15 ±1.0 cm, Tenax GC (2,6-diphenylene oxide polymer), 60/80 mesh, chromatographic grade, or equivalent.

5.2.2.3 Silica gel—8 ±1.0 cm, Davison Chemical, 35/60 mesh, grade 15, or equivalent. The trap shown in Figure 2 meets these specifications.

5.2.3 Desorber—shall heat the trap to 175 ±5 °C in 45 seconds or less. The polymer section of the trap shall not exceed 180 °C, and the remaining sections shall not exceed 220 °C. The desorber shown in Figure 2 meets these specifications.

5.2.4 The purge and trap device may be a separate unit or coupled to a GC as shown in Figures 3 and 4.

5.3 Gas chromatograph—shall be linearly temperature programmable with initial and final holds, shall contain a glass jet separator as the MS interface, and shall produce results which meet the calibration (Section 7), quality assurance (Section 8), and performance tests (Section 11) of this method.

5.3.1 Column—2.8 ±0.4 m × 2 ±0.5 mm i. d. glass, packekd with one percent SP–1000 on Carbopak B, 60/80 mesh, or equivalent.

5.4 Mass spectrometer—70 eV electron impact ionization; shall repetitively scan from 20 to 250 amu every 2–3 seconds, and produce a unit resolution (valleys between m/z 174–176 less than 10 percent of the height of the m/z 175 peak), background corrected mass spectrum from 50 ng 4-bromo-fluorobenzene (BFB) injected into the GC. The BFB spectrum shall meet the mass-intensity criteria in Table 3. All portions of the GC column, transfer lines, and separator which connect the GC column to the ion source shall remain at or above the column temperature during analysis to preclude condensation of less volatile compounds.

5.5 Data system—shall collect and record MS data, store mass intensity data in spectral libraries, process GC/MS data and generate reports, and shall calculate and record response factors.

5.5.1 Data acquisition—mass spectra shall be collected continuously throughout the analysis and stored on a mass storage device.

5.5.2 Mass spectral libraries—user created libraries containing mass spectra obtained from analysis of authentic standards shall be employed to reverse search GC/MS runs for the compounds of interest (Section 7.2).

5.5.3 Data processing—the data system shall be used to search, locate, identify, and quantify the compounds of interest in each GC/MS analysis. Software routines shall be employed to compute retention times and EICP areas. Displays of spectra, mass chromatograms, and library comparisons are required to verify results.

5.5.4 Response factors and multipoint calibrations—the data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and generate multi-point calibration curves (Section 7). Computations of relative standard deviation (coefficient of variation) are useful for testing calibration linearity. Statistics on initial and on-going performance shall be maintained (Sections 8 and 11).

5.6 Syringes—5 mL glass hypodermic, with Luer-lok tips.

5.7 Micro syringes—10, 25, and 100 uL.

5.8 Syringe valves—2-way, with Luer ends (Telfon or Kel-F).

5.9 Syringe—5 mL, gas-tight, with shut-off valve.

5.10 Bottles—15 mL., screw-cap with Telfon liner.

5.11 Balance—analytical, capable of weighing 0.1 mg.

6. Reagents and Standards

6.1 Reagent water—water in which the compounds of interest and interfering compounds are not detected by this method (Section 11.7). It may be generated by any of the following methods:

6.1.1 Activated carbon—pass tap water through a carbon bed (Calgon Filtrasorb-300, or equivalent).

6.1.2 Water purifier—pass tap water through a purifier (Millipore Super Q, or equivalent).

6.1.3 Boil and purge—heat tap water to 90–100 °C and bubble contaminant free inert gas through it for approx one hour. While still hot, transfer the water to screw-cap bottles and seal with a Teflon-lined cap.

6.2 Sodium thiosulfate—ACS granular.

6.3 Methanol—pesticide quality or equivalent.

6.4 Standard solutions—purchased as solution or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If compound purity is 96 percent or greater, the weight may be used without correction to calculate the concentration of the standard.

6.5 Preparation of stock solutions—prepare in methanol using liquid or gaseous standards per the steps below. Observe the safety precautions given in Section 4.

6.5.1 Place approx 9.8 mL of methanol in a 10 mL ground glass stoppered volumetric flask. Allow the flask to stand unstoppered for approximately 10 minutes or until all methanol wetted surfaces have dried. In each case, weigh the flask, immediately add the compound, then immediately reweigh to prevent evaporation losses from affecting the measurement.

6.5.1.1 Liquids—using a 100 µL syringe, permit 2 drops of liquid to fall into the methanol without contacting the leck of the flask. Alternatively, inject a known volume of the compound into the methanol in the flask using a micro-syringe.

6.5.1.2 Gases (chloromethane, bromomethane, chloroethane, vinyl chloride)—fill a valved 5 mL gas-tight syringe with the compound. Lower the needle to approximately 5 mm above the methanol meniscus. Slowly introduce the compound above the surface of the meniscus. The gas will dissolve rapidly in the methanol.

6.5.2 Fill the flask to volume, stopper, then mix by inverting several times. Calculate the concentration in mg/mL (µg/µL) from the weight gain (or density if a known volume was injected).

6.5.3 Transfer the stock solution to a Teflon sealed screw-cap-bottle. Store, with minimal headspace, in the dark at −10 to −20 °C.

6.5.4 Prepare fresh standards weekly for the gases and 2-chloroethylvinyl ether. All other standards are replaced after one month, or sooner if comparison with check standards indicate a change in concentration. Quality control check standards that can be used to determine the accuracy of calibration standards are available from the US Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.

6.6 Labeled compound spiking solution—from stock standard solutions prepared as above, or from mixtures, prepare the spiking solution to contain a concentration such that a 5–10 µL spike into each 5 mL sample, blank, or aqueous standard analyzed will result in a concentration of 20 µg/L of each labeled compound. For the gases and for the water soluble compounds (acrolein, acrylonitrile, acetone, diethyl ether, and MEK), a concentration of 100 µg/L may be used. Include the internal standards (Section 7.5) in this solution so that a concentration of 20 µg/L in each sample, blank, or aqueous standard will be produced.

6.7 Secondary standards—using stock solutions, prepare a secondary standard in methanol to contain each pollutant at a concentration of 500 µg/mL For the gases and water soluble compounds (Section 6.6), a concentration of 2.5 mg/mL may be used.

6.7.1 Aqueous calibration standards—using a 25 µL syringe, add 20 µL of the secondary standard (Section 6.7) to 50, 100, 200, 500, and 1000 mL of reagent water to produce concentrations of 200, 100, 50, 20, and 10 µg/L, respectively. If the higher concentration standard for the gases and water soluble compounds was chosen (Section 6.6), these compounds will be at concentrations of 1000, 500, 250, 100, and 50 µg/L in the aqueous calibration standards.

6.7.2 Aqueous performance standard—an aqueous standard containing all pollutants, internal standards, labeled compounds, and BFB is prepared daily, and analyzed each shift to demonstrate performance (Section 11). This standard shall contain either 20 or 100 µg/L of the labeled and pollutant gases and water soluble compounds, 10 µg/L BFB, and 20 µg/L of all other pollutants, labeled compounds, and internal standards. It may be the nominal 20 µg/L aqueous calibration standard (Section 6.7.1).

6.7.3 A methanolic standard containing all pollutants and internal standards is prepared to demonstrate recovery of these compounds when syringe injection and purge and trap analyses are compared. This standard shall contain either 100 µg/mL or 500 µg/mL of the gases and water soluble compounds, and 100 µg/mL of the remaining pollutants and internal standards (consistent with the amounts in the aqueous performance standard in 6.7.2).

6.7.4 Other standards which may be needed are those for test of BFB performance (Section 7.1) and for collection of mass spectra for storage in spectral libraries (Section 7.2).

7. Calibration

7.1 Assemble the gas chromatographic apparatus and establish operating conditions given in table 2. By injecting standards into the GC, demonstrate that the analytical system meets the detection limits in table 2 and the mass-intensity criteria in table 3 for 50 ng BFB.

7.2 Mass spectral libraries—detection and identification of the compound of interest are dependent upon the spectra stored in user created libraries.

7.2.1 Obtain a mass spectrum of each pollutant and labeled compound and each internal standard by analyzing an authentic standard either singly or as part of a mixture in which there is no interference between closely eluted components. That only a single compound is present is determined by examination of the spectrum. Fragments not attributable to the compound under study indicate the presence of an interfering compound. Adjust the analytical conditions and scan rate (for this test only) to produce an undistorted spectrum at the GC peak maximum. An undistorted spectrum will usually be obtained if five complete spectra are collected across the upper half of the GC peak. Software algorithms designed to “enhance” the spectrum may eliminate distortion, but may also eliminate authentic m/z's or introduce other distortion.

7.2.3 The authentic reference spectrum is obtained under BFB tuning conditions (Section 7.1 and table 3) to normalize it to spectra from other instruments.

7.2.4 The spectrum is edited by saving the 5 most intense mass spectral peaks and all other mass spectral peaks greater than 10 percent of the base peak. This spectrum is stored for reverse search and for compound confirmation.

7.3 Assemble the purge and trap device. Pack the trap as shown in Figure 2 and condition overnight at 170–180 °C by backflushing with an inert gas at a flow rate of 20–30 mL/min. Condition traps daily for a minimum of 10 minutes prior to use.

7.3.1 Analyze the aqueous performance standard (Section 6.7.2) according to the purge and trap procedure in Section 10. Compute the area at the primary m/z (table 4) for each compound. Compare these areas to those obtained by injecting one µL of the methanolic standard (Section 6.7.3) to determine compound recovery. The recovery shall be greater than 20 percent for the water soluble compounds, and 60–110 percent for all other compounds. This recovery is demonstrated initially for each purge and trap GC/MS system. The test is repeated only if the purge and trap or GC/MS systems are modified in any way that might result in a change in recovery.

7.3.2 Demonstrate that 100 ng toluene (or toluene-d8) produces an area at m/z 91 (or 99) approx one-tenth that required to exceed the linear range of the system. The exact value must be determined by experience for each instrument. It is used to match the calibration range of the instrument to the analytical range and detection limits required.

7.4 Calibration by isotope dilution—the isotope dilution approach is used for the purgeable organic compounds when appropriate labeled compounds are available and when interferences do not preclude the analysis. If labeled compounds are not available, or interferences are present, internal standard methods (Section 7.5 or 7.6) are used. A calibration curve encompassing the concentration range of interest is prepared for each compound determined. The relative response (RR) vs concentration (µg/L) is plotted or computed using a linear regression. An example of a calibration curve for toluene using toluene-d8 is given in figure 5. Also shown are the ±10 percent error limits (dotted lines). Relative response is determined according to the procedures described below. A minimum of five data points are required for calibration (Section 7.4.4).

7.4.1 The relative response (RR) of pollutant to labeled compound is determined from isotope ratio values calculated from acquired data. Three isotope ratios are used in this process:

RX = the isotope ratio measured in the pure pollutant (figure 6A).

Ry = the isotope ratio of pure labeled compound (figure 6B).

Rm = the isotope ratio measured in the analytical mixture of the pollutant and labeled compounds (figure 6C).

The correct way to calculate RR is: RR = (Ry−Rm) (RX + 1)/(Rm−RX)(Ry + 1) If Rm is not between 2Ry and 0.5RX, the method does not apply and the sample is analyzed by internal or external standard methods (Section 7.5 or 7.6).

7.4.2 In most cases, the retention times of the pollutant and labeled compound are the same and isotope ratios (R's) can be calculated from the EICP areas, where: R = (area at m1/z)/(area at m2/z) If either of the areas is zero, it is assigned a value of one in the calculations; that is, if: area of m1/z = 50721, and area of m2/z = 0, then R = 50721/1 = 50720. The m/z's are always selected such that RX>Ry. When there is a difference in retention times (RT) between the pollutant and labeled compounds, special precautions are required to determine the isotope ratios.

RX, Ry, and Rm are defined as follows:

RX=[area m1/z (at RT1)]/1

Ry = 1/[area m2/z (at RT2)]

Rm=[area m1/z (at RT1)]/[area m2/z (at RT2)]

7.4.3 An example of the above calculations can be taken from the data plotted in figure 6 for toluene and toluene-d8. For these data, RX = 168920/1 = 168900, Ry = 1/60960 = 0.00001640, and Rm = 96868/82508 = 1.174. The RR for the above data is then calculated using the equation given in Section 7.4.1. For the example, RR = 1.174.

Note:

Not all labeled compounds elute before their pollutant analogs.

7.4.4 To calibrate the analytical system by isotope dilution, analyze a 5 mL aliquot of each of the aqueous calibration standards (Section 6.7.1) spiked with an appropriate constant amount of the labeled compound spiking solution (Section 6.6), using the purge and trap procedure in section 10. Compute the RR at each concentration.

7.4.5 Linearity—if the ratio of relative response to concentration for any compound is constant (less than 20 percent coefficient of variation) over the 5 point calibration range, an averaged relative response/concentration ratio may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point calibration range.

7.5 Calibration by internal standard—used when criteria for isotope dilution (Section 7.4) cannot be met. The method is applied to pollutants having no labeled analog and to the labeled compounds. The internal standards used for volatiles analyses are bromochloromethane, 2-bromo-1-chloropropane, and 1,4-dichlorobutane. Concentrations of the labeled compounds and pollutants without labeled analogs are computed relative to the nearest eluted internal standard, as shown in table 2.

7.5.1 Response factors—calibration requires the determination of response factors (RF) which are defined by the following equation:

RF = (AsxCis)/(AisxCs), where As is the EICP area at the characteristic m/z for the compound in the daily standard. Ais is the EICP area at the characteristic m/z for the internal standard.

Cis is the concentration (ug/L) of the internal standard

Cs is the concentration of the pollutant in the daily standard.

7.5.2 The response factor is determined at 10, 20, 50, 100, and 200 ug/L for the pollutants (optionally at five times these concentrations for gases and water soluble pollutants—see Section 6.7), in a way analogous to that for calibration by isotope dilution (Section 7.4.4). The RF is plotted against concentration for each compound in the standard (Cs) to produce a calibration curve.

7.5.3 Linearity—if the response factor (RF) for any compound is constant (less than 35 percent coefficient of variation) over the 5 point calibration range, an averaged response factor may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point range.

7.6 Combined calibration—by adding the isotopically labeled compounds and internal standards (Section 6.6) to the aqueous calibration standards (Section 6.7.1), a single set of analyses can be used to produce calibration curves for the isotope dilution and internal standard methods. These curves are verified each shift (Section 11.5) by purging the aqueous performance standard (Section 6.7.2). Recalibration is required only if calibration and on-going performance (Section 11.5) criteria cannot be met.

8. Quality Assurance/Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs of measurements, provided all performance specifications are met. Each time a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2 to demonstrate method performance.

8.1.3 Analyses of blanks are required to demonstrate freedom from contamination and that the compounds of interest and interfering compounds have not been carried over from a previous analysis (Section 3). The procedures and criteria for analysis of a blank are described in Sections 8.5 and 11.7.

8.1.4 The laboratory shall spike all samples with labeled compounds to monitor method performance. This test is described in Section 8.3. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits (Section 14.2).

8.1.5 The laboratory shall, on an on-going basis, demonstrate through the analysis of the aqueous performance standard (Section 6.7.2) that the analysis system is in control. This procedure is described in Sections 11.1 and 11.5.

8.1.6 The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Sections 8.4 and 11.5.2.

8.2 Initial precision and accuracy—to establish the ability to generate acceptable precision and accuracy, the analyst shall perform the following operations:

8.2.1 Analyze two sets of four 5–mL aliquots (8 aliquots total) of the aqueous performance standard (Section 6.7.2) according to the method beginning in Section 10.

8.2.2 Using results of the first set of four analyses in Section 8.2.1, compute the average recovery (X̄) in µg/L and the standard deviation of the recovery (s) in µg/L for each compound, by isotope dilution for polluitants with a labeled analog, and by internal standard for labeled compounds and pollutants with no labeled analog.

8.2.3 For each compound, compare s and X̄ with the corresponding limits for initial precision and accuracy found in table 5. If s and X̄ for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If individual X̄ falls outside the range for accuracy, system performance is unacceptable for that compound.

Note:

The large number of compounds in table 5 present a substantial probability that one or more will fail one of the acceptance criteria when all compoulds are analyzed. To determine if the analytical system is out of control, or if the failure can be attributed to probability, proceed as follows:

8.2.4 Using the results of the second set of four analyses, compute s and X̄ for only those compounds which failed the test of the first set of four analyses (Section 8.2.3). If these compounds now pass, system performance is acceptable for all compounds and analysis of blanks and samples may begin. If, however, any of the same compounds fail again, the analysis system is not performing properly for the compound(s) in question. In this event, correct the problem and repeat the entire test (Section 8.2.1).

8.3 The laboratory shall spike all samples with labeled compounds to assess method performance on the sample matrix.

8.3.1 Spike and analyze each sample according to the method beginning in Section 10.

8.3.2 Compute the percent recovery (P) of the labeled compounds using the internal standard method (Section 7.5).

8.3.3 Compare the percent recovery for each compound with the corresponding labeled compound recovery limit in table 5. If the recovery of any compound falls outside its warning limit, method performance is unacceptable for that compound in that sample. Therefore, the sample matrix is complex and the sample is to be diluted and reanalyzed, per Section 14.2.

8.4 As part of the QA program for the laboratory, method accuracy for wastewater samples shall be assessed and records shall be maintained. After the analysis of five wastewater samples for which the labeled compounds pass the tests in Section 8.3.3, compute the average percent recovery (P) and the standard deviation of the percent recovery (sp) for the labeled compounds only. Express the accuracy assessment as a percent recovery interval from P−2sp to P + 2sp. For example, if P = 90% and sp = 10%, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each compound on a regular basis (e.g. after each 5–10 new accuracy measurements).

8.5 Blanks—reagent water blanks are analyzed to demonstrate freedom from carry-over (Section 3) and contamination.

8.5.1 The level at which the purge and trap system will carry greater than 5 µg/L of a pollutant of interest (table 1) into a succeeding blank shall be determined by analyzing successively larger concentrations of these compounds. When a sample contains this concentration or more, a blank shall be analyzed immediately following this sample to demonstrate no carry-over at the 5 µg/L level.

8.5.2 With each sample lot (samples analyzed on the same 8 hr shift), a blank shall be analyzed immediately after analysis of the aqueous performance standard (Section 11.1) to demonstrate freedom from contamination. If any of the compounds of interest (table 1) or any potentially interfering compound is found in a blank at greater than 10 µg/L (assuming a response factor of 1 relative to the nearest eluted internal standard for compounds not listed in table 1), analysis of samples is halted until the source of contamination is eliminated and a blank shows no evidence of contamination at this level.

8.6 The specifications contained in this method can be met if the apparatus used is calibrated properly, then maintained in a calibrated state.

The standards used for calibration (Section 7), calibration verification (Section 11.5) and for initial (Section 8.2) and on-going (Section 11.5) precision and accuracy should be identical, so that the most precise results will be obtained. The GC/MS instrument in particular will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of volatiles by this method.

8.7 Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when internal or external standard methods are used.

9. Sample Collection, Preservation, and Handling

9.1 Grab samples are collected in glass containers having a total volume greater than 20 mL. Fill sample bottles so that no air bubbles pass through the sample as the bottle is filled. Seal each bottle so that no air bubbles are entrapped. Maintain the hermetic seal on the sample bottle until time of analysis.

9.2 Samples are maintained at 0–4 °C from the time of collection until analysis. If the sample contains residual chlorine, add sodium thiosulfate preservative (10 mg/40 mL) to the empty sample bottles just prior to shipment to the sample site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine (Reference 8). If preservative has been added, shake bottle vigorously for one minute immediately after filling.

9.3 Experimental evidence indicates that some aromatic compounds, notably benzene, toluene, and ethyl benzene are susceptible to rapid biological degradation under certain environmental conditions. Refrigeration alone may not be adequate to preserve these compounds in wastewaters for more than seven days. For this reason, a separate sample should be collected, acidified, and analyzed when these aromatics are to be determined. Collect about 500 mL of sample in a clean container.

Adjust the pH of the sample to about 2 by adding HCl (1 + 1) while stirring. Check pH with narrow range (1.4 to 2.8) pH paper. Fill a sample container as described in Section 9.1. If residual chlorine is present, add sodium thiosulfate to a separate sample container and fill as in Section 9.1.

9.4 All samples shall be analyzed within 14 days of collection.

10. Purge, Trap, and GC/MS Analysis

10.1 Remove standards and samples from cold storage and bring to 20–25 °.

10.2 Adjust the purge gas flow rate to 40 ±4 mL/min. Attach the trap inlet to the purging device and set the valve to the purge mode (figure 3). Open the syringe valve located on the purging device sample introduction needle (figure 1).

10.3 Remove the plunger from a 5–mL syringe and attach a closed syringe valve. Open the sample bottle and carefully pour the sample into the syringe barrel until it overflows. Replace the plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Because this process of taking an aliquot destroys the validity of the sample for future analysis, fill a second syringe at this time to protect against possible loss of data. Add an appropriate amount of the labeled compound spiking solution (Section 6.6) through the valve bore, then close the valve.

10.4 Attach the syringe valve assembly to the syringe valve on the purging device. Open both syringe valves and inject the sample into the purging chamber.

10.5 Close both valves and purge the sample for 11.0 ±0.1 minutes at 20–25 °C.

10.6 After the 11 minute purge time, attach the trap to the chromatograph and set the purge and trap apparatus to the desorb mode (figure 4). Desorb the trapped compounds into the GC column by heating the trap to 170–180 °C while backflushing with carrier gas at 20–60 mL/min for four minutes. Start MS data acquisition upon start of the desorb cycle, and start the GC column temperature program 3 minutes later. Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and detection limits that were achieved under these conditions. Other columns may be used provided the requirements in Section 8 can be met. If the priority pollutant gases produce GC peaks so broad that the precision and recovery specifications (Section 8.2) cannot be met, the column may be cooled to ambient or sub-ambient temperatures to sharpen these peaks.

10.7 While analysis of the desorbed compounds proceeds, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL portions of reagent water. After the purging device has been emptied, allow the purge gas to vent through the chamber until the frit is dry, so that it is ready for the next sample.

10.8 After desorbing the sample for four minutes, recondition the trap by returning to the purge mode. Wait 15 seconds, then close the syringe valve on the purging device to begin gas flow through the trap. Maintain the trap temperature at 170–180 °C. After approximately seven minutes, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When cool, the trap is ready for the next sample.

11. System Performance

11.1 At the beginning of each 8 hr shift during which analyses are performed, system calibration and performance shall be verified for all pollutants and labeled compounds. For these tests, analysis of the aqueous performance standard (Section 6.7.2) shall be used to verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed until all performance criteria are met. Only after all performance criteria are met may blanks and samples be analyzed.

11.2 BFB spectrum validity—the criteria in table 3 shall be met.

11.3 Retention times—the absolute retention times of all compounds shall approximate those given in Table 2.

11.4 GC resolution—the valley height between toluene and toluene-d8 (at m/z 91 and 99 plotted on the same graph) shall be less than 10 percent of the taller of the two peaks.

11.5 Calibration verification and on-going precision and accuracy—compute the concentration of each polutant (Table 1) by isotope dilution (Section 7.4) for those compmunds which have labeled analogs. Compute the concentration of each pollutant (Table 1) which has no labeled analog by the internal standard method (Section 7.5). Compute the concentration of the labeled compounds by the internal standard method. These concentrations are computed based on the calibration data determined in Section 7.

11.5.1 For each pollutant and labeled compound, compare the concentration with the corresponding limit for on-going accuracy in Table 5. If all compmunds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may continue. If any individual value falls outside the range given, system performance is unacceptable for that compound.

Note:

The large number of compounds in Table 5 present a substantial probability that one or more will fail the acceptance criteria when all compounds are analyzed. To determine if the analytical system is out of control, or if the failure may be attributed to probability, proceed as follows:

11.5.1.1 Analyze a second aliquot of the aqueous performance standard (Section 6.7.2).

11.5.1.2 Compute the concentration for only those compounds which failed the first test (Section 11.5.1). If these compounds now pass, system performance is acceptable for all compounds and analyses of blanks and samples may proceed. If, however, any of the compounds fail again, the measurement system is not performing properly for these compounds. In this event, locate and correct the problem or recalibrate the system (Section 7), and repeat the entire test (Section 11.1) for all compounds.

11.5.2 Add results which pass the specification in 11.5.1.2 to initial (Section 8.2) and previous on-going data. Update QC charts to form a graphic representation of laboratory performance (Figure 7). Develop a statement of accuracy for each pollutant and labeled compound by calculating the average percentage recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as a recovery interval from R−2sr to R + 2sr. For example, if R = 95% and sr = 5%, the accuracy is 85–105 percent.

12. Qualitative Determination—Accomplished by Comparison of Data from Analysis of a Sample or Blank with Data from Analysis of the Shift Standard (Section 11.1). Identification is Confirmed When Spectra and Retention Times Agree Per the Criteria Below

12.1 Labeled compounds and pollutants having no labeled analog:

12.1.1 The signals for all characteristic masses stored in the spectral library (Section 7.2.4) shall be present and shall maximize within the same two consecutive scans.

12.1.2 Either

(1) the background corrected EICP areas, or

(2) the corrected relative intensities of the mass spectral peaks at the GC peak maximum shall agree within a factor of two (0.5 to 2 times) for all masses stored in the library.

12.1.3 The retention time relative to the nearest eluted internal standard shall be within ±7 scans or ±20 seconds, whichever is greater.

12.2 Pollutants having a labeled analog:

12.2.1 The signals for all characteristic masses stored in the spectral library (Section 7.2.4) shall be present and shall maximize within the same two consecutive scans.

12.2.2 Either

(1) the background corrected EICP areas, or

(2) the corrected relative intensities of the mass spectral peaks at the GC peak maximum shall agree within a factor of two for all masses stored in the spectral library.

12.2.3 The retention time difference between the pollutant and its labeled analog shall agree within ±2 scans or ±6 seconds (whichever is greater) of this difference in the shift standard (Section 11.1).

12.3 Masses present in the experimental mass spectrum that are not present in the reference mass spectrum shall be accounted for by contaminant or background ions. If the experimental mass spectrum is contaminated, an experienced spectrometrist (Section 1.4) is to determine the presence or absence of the compound.

13. Quantitative Determination

13.1 Isotope dilution—by adding a known amount of a labeled compound to every sample prior to purging, correction for recovery of the pollutant can be made because the pollutant and its labeled analog exhibit the same effects upon purging, desorption, and gas chromatography. Relative response (RR) values for sample mixtures are used in conjunction with calibration curves described in Section 7.4 to determine concentrations directly, so long as labeled compound spiking levels are constant. For the toluene example given in Figure 6 (Section 7.4.3), RR would be equal to 1.174. For this RR value, the toluene calibration curve given in Figure 5 indicates a concentration of 31.8 µg/L.

13.2 Internal standard—calculate the concentration using the response factor determined from calibration data (Section 7.5) and the following equation:

Concentration = (As × Cis)/(Ais × RF) where the terms are as defined in Section 7.5.1.

13.3 If the EICP area at the quantitation mass for any compound exceeds the calibration range of the system, the sample is diluted by successive factors of 10 and these dilutions are analyzed until the area is within the calibration range.

13.4 Report results for all pollutants and labeled compounds (Table 1) found in all standards, blanks, and samples, in µg/L to three significant figures. Results for samples which have been diluted are reported at the least dilute level at which the area at the quantitation mass is within the calibration range (Section 13.3) and the labeled compound recovery is within the normal range for the Method (Section 14.2).

14. Analysis of Complex Samples

14.1 Untreated effluents and other samples frequently contain high levels (>1000 µg/L) of the compounds of interest and of interfering compounds. Some samples will foam excessively when purged; others will overload the trap/or GC column.

14.2 Dilute 0.5 mL of sample with 4.5 mL of reagent water and analyze this diluted sample when labeled compound recovery is outside the range given in Table 5. If the recovery remains outside of the range for this diluted sample, the aqueous performance standard shall be analyzed (Section 11) and calibration verified (Section 11.5). If the recovery for the labeled compmund in the aqueous performance standard is outside the range given in Table 5, the analytical system is out of control. In this case, the instrumelt shall be repaired, the performance specifications in Section 11 shall be met, and the analysis of the undiluted sample shall be repeated. If the recovery for the aqueous performance standard is within the range given in Table 5, the method does not work on the sample being analyzed and the result may not be reported for regulatory compliance purposes.

14.3 Reverse search computer programs can misinterpret the spectrum of chromatographically unresolved pollutant and labeled compound pairs with overlapping spectra when a high level of the pollutant is present. Examine each chromatogram for peaks greater than the height of the internal standard peaks. These peaks can obscure the compounds of interest.

15. Method Performance

15.1 The specifications for this method were taken from the inter-laboratory validation of EPA Method 624 (reference 9). Method 1624 has been shown to yield slightly better performance on treated effluents than Method 624. Additional method performance data can be found in Reference 10.

References

1. “Performance Tests for the Evaluation of Computerized Gas Chromatography/Mass Spectrometry Equipment and Laboratories,” USEPA, EMSL/Cincinnati, OH 45268, EPA–600/4–80–025 (April 1980).

2. Bellar, T.A. and Lichtenberg, J.J., “Journal American Water Works Association,” 66, 739 (1974).

3. Bellar, T.A. and Lichtenberg, J.J., “Semi-automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds,” in Measurement of Organic Pollutants Water and Wastewater, C.E. VanHall, ed., American Society for Testing Materials, Philadelphia, PA, Special Technical Publication 686, (1978).

4. “Working with Carcinogens,” DHEW, PHS, NIOSH, Publication 77–206 (1977).

5. “OSHA Safety and Health Standards, General Industry,” 29 CFR part 1910, OSHA 2206, (1976).

6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety (1979).

7. “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA, EMSL/Cincinnati, OH 45268, EPA–4–79–019 (March 1979).

8. “Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA, EMSL/Cincinnati, OH 45268, EPA–4–79–020 (March 1979).

9. “EPA Method Study 29 EPA Method 624—Purgeables,” EPA 600/4–84–054, National Technical Information Service, PB84–209915, Springfield, Virginia 22161, June 1984.

10. “Colby, B.N., Beimer, R.G., Rushneck, D.R., and Telliard, W.A., “Isotope Dilution Gas Chromatography-Mass Spectrometry for the Determination of Priority Pollutants in Industrial Effluents,” USEPA, Effluent Guidelines Division, Washington, DC 20460 (1980).

Table 1—Volatile Organic Compounds Analyzed by Isotope Dilution Gc/MS

Compound Storet CAS registry EPA-EGD NPDES
Acetone 81552 67–64–1 516 V
Acrolein 34210 107–02–8 002 V 001 V
Acrylonitrile 34215 107–13–1 003 V 002 V
Benzene 34030 71–43–2 004 V 003 V
Bromodichloromethane 32101 75–27–4 048 V 012 V
Bromoform 32104 75–25–2 047 V 005 V
Bromomethane 34413 74–83–9 046 V 020 V
Carbon tetrachloride 32102 56–23–5 006 V 006 V
Chlorobenzene 34301 108–90–7 007 V 007 V
Chloroethane 34311 75–00–3 016 V 009 V
2-chloroethylvinyl ether 34576 110–75–8 019 V 010 V
Chloroform 32106 67–66–1 023 V 011 V
Chloromethane 34418 74–87–3 045 V 021 V
Dibromochloromethane 32105 124–48–1 051 V 008 V
1,1-dichloroethane 34496 75–34–3 013 V 014 V
1,2-dichloroethane 34536 107–06–2 010 V 015 V
1,1-dichloroethene 34501 75–35–4 029 V 016 V
Trans-1,2-dichloroethane 34546 156–60–5 030 V 026 V
1,2-dichloropropane 34541 78–87–5 032 V 017 V
Cis-1,3-dichloropropene 34704 10061–01–5
Trans-1,3-dichloropropene 34699 10061–02–6 033 V
Diethyl ether 81576 60–29–7 515 V
P-dioxane 81582 123–91–1 527 V
Ethylbenzene 34371 100–41–4 038 V 019 V
Methylene chloride 34423 75–09–2 044 V 022 V
Methyl ethyl ketone 81595 78–93–3 514 V
1,1,2,2-tetrachloroethane 34516 79–34–5 015 V 023 V
Tetrachlorethene 34475 127–18–4 085 V 024 V
Toluene 34010 108–88–3 086 V 025 V
1,1,1-trichloroethane 34506 71–55–6 011 V 027 V
1,1,2-trichloroethane 34511 79–00–5 014 V 028 V
Trichloroethene 39180 79–01–6 087 V 029 V
Vinyl chloride 39175 75–01–4 088 V 031 V

Table 2—Gas Chromatography of Purgeable Organic Compounds by Isotope Dilution GC/MS

EGD No. (1) Compound Ref EGD No. Mean retention time (sec) Minimum level (2) (µg/L)
181 Bromochloromethane (I.S.) 181 730 10
245 Chloromethane-d3 181 147 50
345 Chloromethane 245 148 50
246 Bromomethane-d3 181 243 50
346 Bromomethane 246 246 50
288 Vinyl chloride-d3 181 301 50
388 Vinyl chloride 288 304 10
216 Chloroethane-d5 181 378 50
316 Chloroethane 216 386 50
244 Methylene chloride-d2 181 512 10
344 Methylene chloride 244 517 10
616 Acetone-d6 181 554 50
716 Acetone 616 565 50
002 Acrolein 181 566 50
203 Acrylonitrile-d3 181 606 50
303 Acrylonitrile 203 612 50
229 1,1-dichloroethene-d2 181 696 10
329 1,1-dichloroethene 229 696 10
213 1,1-dichloroethane-d3 181 778 10
313 1,1-dichloroethane 213 786 10
615 Diethyl ether-d10 181 804 50
715 Diethyl ether 615 820 50
230 Trans-1,2-dichloroethene-d2 181 821 10
330 Trans-1,2-dichloroethene 230 821 10
614 Methyl ethyl ketone-d3 181 840 50
714 Methyl ethyl ketone 614 848 50
223 Chloroform-13C1 181 861 10
323 Chloroform 223 861 10
210 1,2-dichloroethane-d4 181 901 10
310 1,2-dichloroethane 210 910 10
211 1,1,1-trichloroethane-13C2 181 989 10
311 1,1,1-trichloroethane 211 999 10
527 p-dioxane 181 1001 10
206 Carbon tetrachloride-13C1 182 1018 10
306 Carbon tetrachloride 206 1018 10
248 Bromodichloromethane-13C1 182 1045 10
348 Bromodichloromethane 248 1045 10
232 1,2-dichloropropane-d6 182 1123 10
332 1.2-dichloropropane 232 1134 10
233 Trans-1,3-dichloropropene-d4 182 1138 10
333 Trans-1,3-dichloropropene 233 1138 10
287 Trichloroethene-13C1 182 1172 10
387 Trichloroethene 287 1187 10
204 Benzene-d6 182 1200 10
304 Benzene 204 1212 10
251 Chlorodibromemethane-13C1 182 1222 10
351 Chlorodibromomethane 251 1222 10
214 1,1,2-trichloroethane-13C2 182 1224 10
314 1,1,2-trichloroethane 214 1224 10
019 2-chloroethylvinyl ether 182 1278 10
182 2-bromo-1-chloropropane (I.S.) 182 1306 10
247 Bromoform-13C1 182 1386 10
347 Bromoform 247 1386 10
215 1,1,2,2-tetrachloroethane-d2 183 1525 10
315 1,1,2,2-tetrachloroethane 215 1525 10
285 Tetrachloroethene-13C2 183 1528 10
385 Tetrachloroethene 285 1528 10
183 1,4-dichlorobutale (int std) 183 1555 10
286 Toluene-d8 183 1603 10
386 Toluene 286 1619 10
207 Chlorobenzene-d5 183 1679 10
307 Chlorobenzene 207 1679 10
238 Ethylbenzene-d10 183 1802 10
338 Ethylbenzene 238 1820 10
185 Bromofluorobenzene 183 1985 10

Table 3—BFB Mass-Intensity Specifications

Mass Intensity required
50 15 to 40 percent of mass 95.
75 30 to 60 percent of mass 95.
95 base peak, 100 percent.
96 5 to 9 percent of mass 95.
173 <2 percent of mass 174.
174 >50 percent of mass 95.
175 5 to 9 percent of mass 174
176 95 to 101 percent of mass 174
177 5 to 9 percent of mass 176.

Table 4—Volatile Organic Compound Characteristic Masses

Labeled compound Analog Primary m/z's
Acetone d6 58/64
Acrolein d2 56/58
Acrylonitrile d3 53/56
Benzene d6 78/84
Bromodichloromethane 13C 83/86
Bromoform 13C 173/176
Bromomethale d3 96/99
Carbon tetrachloride 13C 47/48
Chlorobenzene d5 112/117
Chloroethane d5 64/71
2-chloroethylvinyl ether d7 106/113
Chloroform 13C 85/86
Chloromethane d3 50/53
Dibromochloromethane 13C 129/130
1,1-dichloroethane d3 63/66
1,2-dichloroethane d4 62/67
1,1-dichloroethene d2 61/65
Trans-1,2-dichloroethene d2 61/65
1,2-dichloropropane d6 63/67
Cis-1,3-dichloropropene d4 75/79
Trans-1,3-dichloropropene d4 75/79
Diethyl ether d10 74/84
p-dioxane d8 88/96
Ethylbenzene d10 106/116
Methylene chloride d2 84/88
Methyl ethyl ketone d3 72/75
1,1,2,2-tetrachloroethane d2 83/84
Tetrachloroethene 13C2 166/172
Toluene d8 92/99
1,1,1-trichloroethane d3 97/102
1,1,2-trichloroethane 13C2 83/84
Trichloroethene 13C 95/133
Vinyl chloride d3 62/65

Table 5—Acceptance Criteria for Performance Tests

Compound Acceptance criteria at 20 µg/L
Initial precision and accuracy section 8.2.3 Labeled compound recovery sec. 8.3 and 14.2 On-going accuracy sec. 11.5
s (µg/L) X̄ (µg/L) P (percent) R (µg/L)
Acetone Note 1
Acrolein Note 2
Acrylonitrile Note 2
Benzene 9.0 13.0–28.2 ns–196 4–33
Bromodichloromethane 8.2 6.5–31.5 ns–199 4–34
Bromoform 7.0 7.4–35.1 ns–214 6–36
Bromomethane 25.0 d–54.3 ns–414 d–61
Carbon tetrachloride 6.9 15.9–24.8 42–165 12–30
Chlorobenzene 8.2 14.2–29.6 ns–205 4–35
Chloroethane 14.8 2.1–46.7 ns–308 d–51
2–chloroethylvinyl ether 36.0 d–69.8 ns–554 d–79
Chloroform 7.9 11.6–26.3 18–172 8–30
Chloromethane 26.0 d–55.5 ns–410 d–64
Dibromochloromethane 7.9 11.2–29.1 16–185 8–32
1,1-dichloroethane 6.7 11.4–31.4 23–191 9–33
1,2-dichloroethane 7.7 11.6–30.1 12–192 8–33
1,1-dichloroethene 11.7 d–49.8 ns–315 d–52
Trans-1,2–dichloroethene 7.4 10.5–31.5 15–195 8–34
1,2-dichloropropane 19.2 d–46.8 ns–343 d–51
Cis–1,3–dichloropropene 22.1 d–51.0 ns–381 d–56
Trans–1,3–dichloropropene 14.5 d–40.2 ns–284 d–44
Diethyl ether Note 1
P-dioxane Note 1
Ethyl benzene 9.6 15.6–28.5 ns–203 5–35
Methylene chloride 9.7 d–49.8 ns–316 d–50
Methyl ethyl ketone Note 1
1,1,2,2-tetrachloroethane 9.6 10.7–30.0 5–199 7–34
Tetrachloroethene 6.6 15.1–28.5 31–181 11–32
Toluene 6.3 14.5–28.7 4–193 6–33
1,1,1-trichloroethane 5.9 10.5–33.4 12–200 8–35
1,1,2-trichloroethane 7.1 11.8–29.7 21–184 9–32
Trichloroethene 8.9 16.6–29.5 35–196 12–34
Vinyl chloride 27.9 d–58.5 ns–452 d–65

Method 1625 Revision B—Semivolatile Organic Compounds by Isotope Dilution GC/MS

1. Scope and Application

1.1 This method is designed to determine the semivolatile toxic organic pollutants associated with the 1976 Consent Decree and additional compounds amenable to extraction and analysis by capillary column gas chromatography-mass spectrometry (GC/MS).

1.2 The chemical compounds listed in Tables 1 and 2 may be determined in municipal and industrial discharges by this method. The method is designed to meet the survey requirements of Effluent Guidelines Division (EGD) and the National Pollutants Discharge Elimination System (NPDES) under 40 CFR 136.1. Any modifications of this method, beyond those expressly permitted, shall be considered as major modifications subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.3 The detection limit of this method is usually dependent on the level of interferences rather than instrumental limitations. The limits listed in Tables 3 and 4 represent the minimum quantity that can be detected with no interferences present.

1.4 The GC/MS portions of this method are for use only by analysts experienced with GC/MS or under the close supervision of such qualified persons. Laboratories unfamiliar with analyses of environmental samples by GC/MS should run the performance tests in reference 1 before beginning.

2. Summary of Method

2.1 Stable isotopically labeled analogs of the compounds of interest are added to a one liter wastewater sample. The sample is extracted at pH 12–13, then at pH <2 with methylene chloride using continuous extraction techniques. The extract is dried over sodium sulfate and concentrated to a volume of one mL. An internal standard is added to the extract, and the extract is injected into the gas chromatograph (GC). The compounds are separated by GC and detected by a mass spectrometer (MS). The labeled compounds serve to correct the variability of the analytical technique.

2.2 Identification of a compound (qualitative analysis) is performed by comparing the GC retention time and background corrected characteristic spectral masses with those of authentic standards.

2.3 Quantitative analysis is performed by GC/MS using extracted ion current profile (EICP) areas. Isotope dilution is used when labeled compounds are available; otherwise, an internal standard method is used.

2.4 Quality is assured through reproducible calibration and testing of the extraction and GC/MS systems.

3. Contamination and Interferences

3.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines causing misinterpretation of chromatograms and spectra. All materials shall be demonstrated to be free from interferences under the conditions of analysis by running method blanks initially and with each sample lot (samples started through the extraction process on a given 8 hr shift, to a maximum of 20). Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Glassware and, where possible, reagents are cleaned by solvent rinse and baking at 450 °C for one hour minimum.

3.2 Interferences coextracted from samples will vary considerably from source to source, depending on the diversity of the industrial complex or municipality being samples.

4. Safety

4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely determined; however, each chemical compound should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of data handling sheets should also be made available to all personnel involved in these analyses. Additional information on laboratory safety can be found in references 2–4.

4.2 The following compounds covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: benzidine benzo(a)anthracene, 3,3′-dichlorobenzidine, benzo(a)pyrene, dibenzo(a,h)anthracene, N-nitrosodimethylamine, and β-naphtylamine. Primary standards of these compounds shall be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations are handled.

5. Apparatus and Materials

5.1 Sampling equipment for discrete or composite sampling.

5.1.1 Sample bottle, amber glass, 1.1 liters minimum. If amber bottles are not available, samples shall be protected from light. Bottles are detergent water washed, then solvent rinsed or baked at 450 °C for one hour minimum before use.

5.1.2 Bottle caps—threaded to fit sample bottles. Caps are lined with Teflon. Aluminum foil may be substituted if the sample is not corrosive. Liners are detergent water washed, then reagent water (Section 6.5) and solvent rinsed, and baked at approximately 200 °C for one hour minimum before use.

5.1.3 Compositing equipment—automatic or manual compositing system incorporating glass containers for collection of a minimum 1.1 liters. Sample containers are kept at 0 to 4 °C during sampling. Glass or Teflon tubing only shall be used. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used in the pump only. Before use, the tubing is thoroughly rinsed with methanol, followed by repeated rinsings with reagent water (Section 6.5) to minimize sample contamination. An integrating flow meter is used to collect proportional composite samples.

5.2 Continuous liquid-liquid extractor—Teflon or glass conncecting joints and stopcocks without lubrication (Hershberg-Wolf Extractor) one liter capacity, Ace Glass 6841–10, or equivalent.

5.3 Drying column—15 to 20 mm i.d. Pyrex chromatographic column equipped with coarse glass frit or glass wool plug.

5.4 Kuderna-Danish (K-D) apparatus

5.4.1 Concentrator tube—10mL, graduated (Kontes K–570050–1025, or equivalent) with calibration verified. Ground glass stopper (size 19/22 joint) is used to prevent evaporation of extracts.

5.4.2 Evaporation flask—500 mL (Kontes K–570001–0500, or equivalent), attached to concentrator tube with springs (Kontes K–662750–0012).

5.4.3 Snyder column—three ball macro (Kontes K–503000–0232, or equivalent).

5.4.4 Snyder column—two ball micro (Kontes K–469002–0219, or equivalent).

5.4.5 Boiling chips—approx 10/40 mesh, extracted with methylene chloride and baked at 450 °C for one hr minimum.

5.5 Water bath—heated, with concentric ring cover, capable of temperature control ±2 °C, installed in a fume hood.

5.6 Sample vials—amber glass, 2–5 mL with Teflon-lined screw cap.

5.7 Analytical balance—capable of weighing 0.1 mg.

5.8 Gas chromatograph—shall have splitless or on-column injection port for capillary column, temperature program with 30 °C hold, and shall meet all of the performance specifications in Section 12.

5.8.1 Column—30 ±5 m × 0.25 ±0.02 mm i.d. 5% phenyl, 94% methyl, 1% vinyl silicone bonded phase fused silica capillary column (J & W DB–5, or equivalent).

5.9 Mass spectrometer—70 eV electron impact ionization, shall repetitively scan from 35 to 450 amu in 0.95 to 1.00 second, and shall produce a unit resolution (valleys between m/z 441–442 less than 10 percent of the height of the 441 peak), backgound corrected mass spectrum from 50 ng decafluorotriphenylphosphine (DFTPP) introduced through the GC inlet. The spectrum shall meet the mass-intensity criteria in Table 5 (reference 5). The mass spectrometer shall be interfaced to the GC such that the end of the capillary column terminates within one centimeter of the ion source but does not intercept the electron or ion beams. All portions of the column which connect the GC to the ion source shall remain at or above the column temperature during analysis to preclude condensation of less volatile compounds.

5.10 Data system—shall collect and record MS data, store mass-intensity data in spectral libraries, process GC/MS data, generate reports, and shall compute and record response factors.

5.10.1 Data acquisition—mass spectra shall be collected continuously throughout the analysis and stored on a mass storage device.

5.10.2 Mass spectral libraries—user created libraries containing mass spectra obtained from analysis of authentic standards shall be employed to reverse search GC/MS runs for the compounds of interest (Section 7.2).

5.10.3 Data processing—the data system shall be used to search, locate, identify, and quantify the compounds of interest in each GC/MS analysis. Software routines shall be employed to compute retention times and peak areas. Displays of spectra, mass chromatograms, and library comparisons are required to verify results.

5.10.4 Response factors and multipoint calibrations—the data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and multipoint calibration curves (Section 7). Computations of relative standard deviation (coefficient of variation) are useful for testing calibration linearity. Statistics on initial (Section 8.2) and on-going (Section 12.7) performance shall be computed and maintained.

6. Reagents and Standards

6.1 Sodium hydroxide—reagent grade, 6N in reagent water.

6.2 Sulfuric acid—reagent grade, 6N in reagent water.

6.3 Sodium sulfate—reagent grade, granular anhydrous, rinsed with methylene chloride (20 mL/g) and conditioned at 450 °C for one hour minimum.

6.4 Methylene chloride—distilled in glass (Burdick and Jackson, or equivalent).

6.5 Reagent water—water in which the compounds of interest and interfering compounds are not detected by this method.

6.6 Standard solutions—purchased as solutions or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If compound purity is 96 percent or greater, the weight may be used without correction to compute the concentration of the standard. When not being used, standards are stored in the dark at −20 to −10 °C in screw-capped vials with Teflon-lined lids. A mark is placed on the vial at the level of the solution so that solvent evaporation loss can be detected. The vials are brought to room temperature prior to use. Any precipitate is redissolved and solvent is added if solvent loss has occurred.

6.7 Preparation of stock solutions—prepare in methylene chloride, benzene, p-dioxane, or a mixture of these solvents per the steps below. Observe the safety precautions in Section 4. The large number of labeled and unlabeled acid, base/neutral, and Appendix C compounds used for combined calibration (Section 7) and calibration verification (12.5) require high concentratimns (approx 40 mg/mL) when individual stock solutions are prepared, so that dilutions of mixtures will permit calibration with all compounds in a single set of solutions. The working range for most compounds is 10–200 µg/mL. Compounds with a reduced MS response may be prepared at higher concentrations.

6.7.1 Dissolve an appropriate amount of assayed reference material in a suitable solvent. For example, weigh 400 mg naphthalene in a 10 mL ground glass stoppered volumetric flask and fill to the mark with benzene. After the naphthalene is completely dissolved, transfer the solution to a 15 mL vial with Teflon-lined cap.

6.7.2 Stock standard solutions should be checked for signs of degradation prior to the preparation of calibration or performance test standards. Quality control check samples that can be used to determine the accuracy of calibration standards are available from the US Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.

6.7.3 Stock standard solutions shall be replaced after six months, or sooner if comparison with quality control check samples indicates a change in concentration.

6.8 Labeled compound spiking solution—from stock standard solutions prepared as above, or from mixtures, prepare the spiking solution at a concentration of 200 µg/mL, or at a concentration appropriate to the MS response of each compound.

6.9 Secondary standard—using stock solutions (Section 6.7), prepare a secondary standard containing all of the compounds in Tables 1 and 2 at a concentration of 400 µg/mL, or higher concentration appropriate to the MS response of the compound.

6.10 Internal standard solution—prepare 2,2′-difluorobiphenyl (DFB) at a concentration of 10 mg/mL in benzene.

6.11 DFTPP solution—prepare at 50 µg/mL in acetone.

6.12 Solutions for obtaining authentic mass spectra (Section 7.2)—prepare mixtures of compounds at concentrations which will assure authentic spectra are obtained for storage in libraries.

6.13 Calibration solutions—combine 0.5 mL of the solution in Section 6.8 with 25, 50, 125, 250, and 500 uL of the solution in section 6.9 and bring to 1.00 mL total volume each. This will produce calibration solutions of nominal 10, 20, 50, 100, and 200 µg/mL of the pollutants and a constant nominal 100 µg/mL of the labeled compounds. Spike each solution with 10 µL of the internal standard solution (Section 6.10). These solutions permit the relative response (labeled to unlabeled) to be measured as a function of concentration (Section 7.4).

6.14 Precision and recovery standard—used for determination of initial (Section 8.2) and on-going (Section 12.7) precision and recovery. This solution shall contain the pollutants and labeled compounds at a nominal concentration of 100 µg/mL.

6.15 Stability of solutions—all standard solutions (Sections 6.8–6.14) shall be analyzed within 48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will remain acceptable if the peak area at the quantitation mass relative to the DFB internal standard remains within ±15 percent of the area obtained in the initial analysis of the standard.

7. Calibration

7.1 Assemble the GC/MS and establish the operating conditions in Table 3. Analyze standards per the procedure in Section 11 to demonstrate that the analytical system meets the detection limits in Tables 3 and 4, and the mass-intensity criteria in Table 5 for 50 ng DFTPP.

7.2 Mass spectral libraries—detection and identification of compounds of interest are dependent upon spectra stored in user created libraries.

7.2.1 Obtain a mass spectrum of each pollutant, labeled compound, and the internal standard by analyzing an authentic standard either singly or as part of a mixture in which there is no interference between closely eluted components. That only a single compound is present is determined by examination of the spectrum. Fragments not attributable to the compound under study indicate the presence of an interfering compound.

7.2.2 Adjust the analytical conditions and scan rate (for this test only) to produce an undistorted spectrum at the GC peak maximum. An undistorted spectrum will usually be obtained if five complete spectra are collected across the upper half of the GC peak. Software algorithms designed to “enhance” the spectrum may eliminate distortion, but may also eliminate authentic masses or introduce other distortion.

7.2.3 The authentic reference spectrum is obtained under DFTPP tuning conditions (Section 7.1 and Table 5) to normalize it to spectra from other instruments.

7.2.4 The spectrum is edited by saving the 5 most intense mass spectral peaks and all other mass spectral peaks greater than 10 percent of the base peak. This edited spectrum is stored for reverse search and for compound confirmation.

7.3 Analytical range—demonstrate that 20 ng anthracene or phenanthrene produces an area at m/z 178 approx one-tenth that required to exceed the linear range of the system. The exact value must be determined by experience for each instrument. It is used to match the calibration range of the instrument to the analytical range and detection limits required, and to diagnose instrument sensitivity problems (Section 15.4). The 20 ug/mL calibration standard (Section 6.13) can be used to demonstrate this performance.

7.3.1 Polar compound detection—demonstrate that unlabeled pentachlorophenol and benzidine are detectable at the 50 µg/mL level (per all criteria in Section 13). The 50 µg/mL calibration standard (Section 6.13) can be used to demonstrate this performance.

7.4 Calibration with isotope dilution—isotope dilution is used when (1) labeled compounds are available, (2) interferences do not preclude its use, and (3) the quantitation mass extracted ion current profile (EICP) area for the compound is in the calibration range. If any of these conditions preclude isotope dilution, internal standard methods (Section 7.5 or 7.6) are used.

7.4.1 A calibration curve encompassing the concentration range is prepared for each compound to be determined. The relative response (pollutant to labeled) vs concentration in standard solutions is plotted or computed using a linear regression. The example in Figure 1 shows a calibration curve for phenol using phenol-d5 as the isotopic diluent. Also shown are the ±10 percent error limits (dotted lines). Relative Reponse (RR) is determined according to the procedures described below. A minimum of five data points are employed for calibration.

7.4.2 The relative response of a pollutant to its labeled analog is determined from isotope ratio values computed from acquired data. Three isotope ratios are used in this process:

RX = the isotope ratio measured for the pure pollutant.

Ry = the isotope ratio measured for the labeled compound.

Rm = the isotope ratio of an analytical mixture of pollutant and labeled compounds.

The m/z's are selected such that RX>Ry. If Rm is not between 2Ry and 0.5RX, the method does not apply and the sample is analyzed by internal or external standard methods.

7.4.3 Capillary columns usually separate the pollutant-labeled pair, with the labeled compound eluted first (Figure 2). For this case, RX = [area m1/z]/1, at the retention time of the pollutant (RT2). Ry = 1/[area m2/z, at the retention time of the labeled compound RT1). Rm = [area at m1/z (at RT2)]/[area at RT1)], as measured in the mixture of the pollutant and labeled compounds (Figure 2), and RR = Rm.

7.4.4 Special precautions are taken when the pollutant-labeled pair is not separated, or when another labeled compound with interfering spectral masses overlaps the pollutant (a case which can occur with isomeric compounds). In this case, it is necessary to determine the respective contributions of the pollutant and labeled compounds to the respective EICP areas. If the peaks are separated well enough to permit the data system or operator to remove the contributions of the compounds to each other, the equations in Section 7.4.3 apply. This usually occurs when the height of the valley between the two GC peaks at the same m/z is less than 10 percent of the height of the shorter of the two peaks. If significant GC and spectral overlap occur, RR is computed using the following equation:

RR = (Ry − Rm) (RX + 1)/(Rm − RX) (Ry + 1), where RX is measured as shown in Figure 3A, Ry is measured as shown in Figure 3B, and Rm is measured as shown in Figure 3C. For example, RX = 46100/4780 = 9.644, Ry = 2650/43600 = 0.0608, Rm = 49200/48300 = 1.019. amd RR = 1.114.

7.4.5 To calibrate the analytical system by isotope dilution, analyze a 1.0 µL aliquot of each of the calibration standards (Section 6.13) using the procedure in Section 11. Compute the RR at each concentration.

7.4.6 Linearity—if the ratio of relative response to concentration for any compound is constant (less than 20 percent coefficient of variation) over the 5 point calibration range, and averaged relative response/concentration ratio may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point calibration range.

7.5 Calibration by internal standard—used when criteria for istope dilution (Section 7.4) cannot be met. The internal standard to be used for both acid and base/neutral analyses is 2,2′–difluorobiphenyl. The internal standard method is also applied to determination of compounds having no labeled analog, and to measurement of labeled compounds for intra-laboratory statistics (Sections 8.4 and 12.7.4).

7.5.1 Response factors—calibration requires the determination of response factors (RF) which are defined by the following equation:

RF = (As × Cis)/(Ais × Cs), where

As is the area of the characteristic mass for the compmund in the daily standard

Ais is the area of the characteristic mass for the internal standard

Cis is the concentration of the internal standard (µg/mL)

Cs is the concentration of the compound in the daily standard (µg/mL)

7.5.1.1 The response factor is determined for at least five concentrations appropriate to the response of each compound (Section 6.13); nominally, 10, 20, 50, 100, and 200 µg/mL. The amount of internal standard added to each extract is the same (100 µg/mL) so that Cis remains constant. The RF is plotted vs concentration for each compound in the standard (Cs) to produce a calibration curve.

7.5.1.2 Linearity—if the response factor (RF) for any compound is constant (less than 35 percent coefficient of variation) over the 5 point calibration range, an averaged response factor may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point range.

7.6 Combined calibration—by using calibration solutions (Section 6.13) containing the pollutants, labeled compounds, and the internal standard, a single set of analyses can be used to produce calibration curves for the isotope dilution and internal standard methods. These curves are verified each shift (Section 12.5) by analyzing the 100 µg/mL calibration standard (Section 6.13). Recalibration is required only if calibration verification (Section 12.5) criteria cannot be met.

8. Quality Assurance/Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs of measurements, provided all performance specifications are met. Each time a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2 to demonstrate method performance.

8.1.3 Analyses of blanks are required to demonstrate freedom from contamination. The procedures and criteria for analysis of a blank are described in Section 8.5.

8.1.4 The laboratory shall spike all samples with labeled compounds to monitor method performance. This test is described in Section 8.3. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits (Section 15).

8.1.5 The laboratory shall, on an on-going basis, demonstrate through calibration verification and the analysis of the precision and recovery standard (Section 6.14) that the analysis system is in control. These procedures are described in Sections 12.1, 12.5, and 12.7.

8.1.6 The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Section 8.4.

8.2 Initial precision and accuracy—to establish the ability to generate acceptable precision and accuracy, the analyst shall perform the following operations:

8.2.1 Extract, concentrate, and analyze two sets of four one-liter aliquots (8 aliquots total) of the precision and recovery standard (Section 6.14) according to the procedure in Section 10.

8.2.2 Using results of the first set of four analyses, compute the average recovery (X̄) in µg/mL and the standard deviation of the recovery (s) in θg/µL for each compound, by isotope dilution for pollutants with a labeled analog, and by internal standard for labeled compounds and pollutants with no labeled analog.

8.2.3 For each compound, compare s and X̄ with the corresponding limits for initial precision and accuracy in Table 8. If s and X̄ for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If, however, any individual s exceeds the precision limit or any individual X̄ falls outside the range for accuracy, system performance is unacceptable for that compound.

Note:

The large number of compounds in Table 8 present a substantial probability that one or more will fail the acceptance criteria when all compounds are analyzed. To determine if the analytical system is out of control, or if the failure can be attributed to probability, proceed as follows:

8.2.4 Using the results of the second set of four analyses, compute s and X̄ for only those compounds which failed the test of the first set of four analyses (Section 8.2.3). If these compounds now pass, system performance is acceptable for all compounds and analysis of blanks and samples may begin. If, however, any of the same compoulds fail again, the analysis system is not performing properly for these compounds. In this event, correct the problem and repeat the entire test (Section 8.2.1).

8.3 The laboratory shall spike all samples with labeled compounds to assess method performance on the sample matrix.

8.3.1 Analyze each sample according to the method in Section 10.

8.3.2 Compute the percent recovery (P) of the labeled compounds using the internal standard methmd (Section 7.5).

8.3.3 Compare the labeled compound recovery for each compound with the corresponding limits in Table 8. If the recovery of any compounds falls outside its warning limit, method performance is unacceptable for that compound in that sample, Therefore, the sample is complex and is to be diluted and reanalyzed per Section 15.4.

8.4 As part of the QA program for the laboratory, method accuracy for wastewater samples shall be assessed and records shall be maintained. After the analysis of five wastewater samples for which the labeled compounds pass the tests in Section 8.3, compute the average percent recovery (P) and the standard deviation of the percent recovery (sp) for the labeled compounds only. Express the accuracy assessment as a percent recovery interval from P—2sp to P + 2sp. For example, if P = 90% and sp = 10%, the accuracy interval is expressed as 70–100%. Update the accuracy assessment for each compound on a regular basis (e.g. after each 5–10 new accuracy measurements).

8.5 Blanks—reagent water blanks are analyzed to demonstrate freedom from contamination.

8.5.1 Extract and concentrate a blank with each sample lot (samples started through the extraction process on the same 8 hr shift, to a maximum of 20 samples). Analyze the blank immediately after analysis of the precision and recovery standard (Section 6.14) to demonstrate freedom from contamination.

8.5.2 If any of the compounds of interest (Tables 1 and 2) or any potentially interfering compound is found in a blank at greater than 10 µg/L (assuming a response factor of 1 relative to the internal standard for compounds not listed in Tables 1 and 2), analysis of samples is halted until the source of contamination is eliminated and a blank shows no evidence of contamination at this level.

8.6 The specifications contained in this method can be met if the apparatus used is calibrated properly, then maintained in a calibrated state. The standards used for calibration (Section 7), calibration verification (Section 12.5), and for initial (Section 8.2) and on-going (Section 12.7) precision and recovery should be identical, so that the most precise results will be obtained. The GC/MS instrument in particular will provide the most reproducible results if dedicated to the settings and conditions required for the analysis of semi-volatiles by this method.

8.7 Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when internal or external standard methods are used.

9. Sample Collection, Preservation, and Handling

9.1 Collect samples in glass containers following conventional sampling practices (Reference 7). Composite samples are collected in refrigerated glass containers (Section 5.1.3) in accordance with the requirements of the sampling program.

9.2 Maintain samples at 0–4 °C from the time collectimn until extraction. If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 8).

9.3 Begin sample extraction within seven days of collection, and analyze all extracts within 40 days of extraction.

10. Sample Extraction and Concentration (See Figure 4)

10.1 Labeled compound spiking—measure 1.00 ±0.01 liter of sample into a glass container. For untreated effluents, and samples which are expected to be difficult to extract and/or concentrate, measure an additional 10.0 ±0.1 mL and dilute to a final volume of 1.00 ±0.01 liter with reagent water in a glass container.

10.1.1 For each sample or sample lot (to a maximum of 20) to be extracted at the same time, place three 1.00 ±0.10 liter aliquots of reagent water in glass containers.

10.1.2 Spike 0.5 mL of the labeled compound spiking solution (Section 6.8) into all samples and one reagant water aliquot.

10.1.3 Spike 1.0 mL of the precision and recovery standard (Section 6.14) into the two remaining reagent water aliquots.

10.1.4 Stir and equilibrate all solutions for 1–2 hr.

10.2 Base/neutral extraction—place 100–150 mL methylene chloride in each continuous extractor and 200–300 in each distilling flask.

10.2.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass containers with 50–100 mL methylene chloride and add to the respective extractor.

10.2.2 Adjust the pH of the waters in the extractors to 12–13 with 6N NaOH while monitoring with a pH meter. Begin the extraction by heating the flask until the methylene chloride is boiling. When properly adjusted, 1–2 drops of methylene chloride per second will fall from the condensor tip into the water. After 1–2 hours of extraction, test the pH and readjust to 12–13 if required. Extract for 18–24 hours.

10.2.3 Remove the distilling flask, estimate and record the volume of extract (to the nearest 100 mL), and pour the contents through a drying column containing 7 to 10 cm anhydrous sodium sulfate. Rinse the distilling flask with 30–50 mL of methylene chloride and pour through the drying column. Collect the solution in a 500 mL K-D evaporator flask equipped with a 10 mL concentrator tube. Seal, label as the base/neutral fraction, and concentrate per Sections 10.4 to 10.5.

10.3 Acid extraction—adjust the pH of the waters in the extractors to 2 or less using 6N sulfuric acid. Charge clean distilling flasks with 300–400 mL of methylene chloride. Test and adjust the pH of the waters after the first 1–2 hr of extraction. Extract for 18–24 hours.

10.3.1 Repeat Section 10.2.3, except label as the acid fraction.

10.4 Concentration—concentrate the extracts in separate 500 mL K-D flasks equipped with 10 mL concentrator tubes.

10.4.1 Add 1 to 2 clean boiling chips to the flask and attach a three-ball macro Snyder column. Prewet the column by adding approximately one mL of methylene chloride through the top. Place the K-D apparatus in a hot water bath so that the entire lower rounded surface of the flask is bathed with steam. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood. When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus from the bath and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1–2 mL of methylene chloride. A 5-mL syringe is recommended for this operation.

10.4.2 For performance standards (Sections 8.2 and 12.7) and for blanks (Section 8.5), combine the acid and base/neutral extracts for each at this point. Do not combine the acid and base/neutral extracts for samples.

10.5 Add a clean boiling chip and attach a two ball micro Snyder column to the concentrator tube. Prewet the column by adding approx 0.5 mL methylene chloride through the top. Place the apparatus in the hot water bath. Adjust the vertical position and the water temperature as required to complete the concentration in 5–10 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood. When the liquid reaches an apparent volume of approx 0.5 mL, remove the apparatus from the water bath and allow to drain and cool for at least 10 minutes. Remove the micro Snyder column and rinse its lower joint into the concentrator tube with approx 0.2 mL of methylene chloride. Adjust the final volume to 1.0 mL.

10.6 Transfer the concentrated extract to a clean screw-cap vial. Seal the vial with a Teflon-lined lid, and mark the level on the vial. Label with the sample number and fraction, and store in the dark at −20 to −10 °C until ready for analysis.

11. GC/MS Analysis

11.1 Establish the operating conditions given in Table 3 or 4 for analysis of the base/neutral or acid extracts, respectively. For analysis of combined extracts (Section 10.4.2), use the operating conditions in Table 3.

11.2 Bring the concentrated extract (Section 10.6) or standard (Sections 6.13 through 6.14) to room temperature and verify that any precipitate has redissolved. Verify the level on the extract (Sections 6.6 and 10.6) and bring to the mark with solvent if required.

11.3 Add the internal standard solution (Section 6.10) to the extract (use 1.0 uL of solution per 0.1 mL of extract) immediately prior to injection to minimize the possibility of loss by evaporation, adsorption, or reaction. Mix thoroughly.

11.4 Inject a volume of the standard solution or extract such that 100 ng of the internal standard will be injected, using on-column or splitless injection. For 1 mL extracts, this volume will be 1.0 uL. Start the GC column initial isothermal hold upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after the benzo (ghi) perylene or pentachlorophenol peak elutes for the base/neutral or acid fraction, respectively. Return the column to the initial temperature for analysis of the next sample.

12. System and Laboratory Performance

12.1 At the beginning of each 8 hr shift during which analyses are performed, GC/MS system performance and calibration are verified for all pollutants and labeled compounds. For these tests, analysis of the 100 µg/mL calibration standard (Section 6.13) shall be used to verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed until all performance criteria are met. Only after all performance criteria are met may samples, blanks, and precision and recovery standards be analyzed.

12.2 DFTPP spectrum validity—inject 1 µL of the DFTPP solution (Section 6.11) either separately or within a few seconds of injection of the standard (Section 12.1) analyzed at the beginning of each shift. The criteria in Table 5 shall be met.

12.3 Retention times—the absolute retention time of 2,2′-difluorobiphenyl shall be within the range of 1078 to 1248 seconds and the relative retention times of all pollutants and labeled compounds shall fall within the limits given in Tables 3 and 4.

12.4 GC resolution—the valley height between anthracene and phenanthrene at m/z 178 (or the analogs at m/z 188) shall not exceed 10 percent of the taller of the two peaks.

12.5 Calibration verification—compute the concentration of each pollutant (Tables 1 and 2) by isotope dilution (Section 7.4) for those compounds which have labeled analogs. Compute the concentration of each pollutant which has no labeled analog by the internal standard method (Section 7.5). Compute the concentration of the labeled compounds by the internal standard method. These concentrations are computed based on the calibration data determined in Section 7.

12.5.1 For each pollutant and labeled compound being tested, compare the concentration with the calibration verification limit in Table 8. If all compounds meet the acceptance criteria, calibration has been verified and analysis of blanks, samples, and precision and recovery standards may proceed. If, however, any compound fails, the measurement system is not performing properly for that compound. In this event, prepare a fresh calibration standard or correct the problem causing the failure and repeat the test (Section 12.1), or recalibrate (Section 7).

12.6 Multiple peaks—each compound injected shall give a single, distinct GC peak.

12.7 On-going precision and accuracy.

12.7.1 Analyze the extract of one of the pair of precision and recovery standards (Section 10.1.3) prior to analysis of samples from the same lot.

12.7.2 Compute the concentration of each pollutant (Tables 1 and 2) by isotope dilution (Section 7.4) for those compounds which have labeled analogs. Compute the concentration of each pollutant which has no labeled analog by the internal standard method (Section 7.5). Compute the concentration of the labeled compounds by the internal standard method.

12.7.3 For each pollutant and labeled compound, compare the concentration with the limits for on-going accuracy in Table 8. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual concentration falls outside of the range given, system performance is unacceptable for that compound.

Note:

The large number of compounds in Table 8 present a substantial probability that one or more will fail when all compounds are analyzed. To determine if the extraction/concentration system is out of control or if the failure is caused by probability, proceed as follows:

12.7.3.1 Analyze the second aliquot of the pair of precision and recovery standard (Section 10.1.3).

12.7.3.2 Compute the concentration of only those pollutants or labeled compounds that failed the previous test (Section 12.7.3). If these compounds now pass, the extraction/concentration processes are in control and analysis of blanks and samples may proceed. If, however, any of the same compounds fail again, the extraction/concentration processes are not being performed properly for these compounds. In this event, correct the problem, re-extract the sample lot (Section 10) and repeat the on-going precision and recovery test (Section 12.7).

12.7.4 Add results which pass the specifications in Section 12.7.2 to initial and previous on-going data. Update QC charts to perform a graphic representation of continued laboratory performance (Figure 5). Develop a statement of laboratory accuracy for each pollutant and labeled compound by calculating the average percent recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as a recovery interval from R−2sr to R + 2sr. For example, if R = 95% and sr = 5%, the accuracy is 85−105%.

13. Qualitative Determination

13.1 Qualititative determination is accomplished by comparison of data from analysis of a sample or blank with data from analysis of the shift standard (Section 12.1) and with data stored in the spectral libraries (Section 7.2.4). Identification is confirmed when spectra and retention times agree per the criteria below.

13.2 Labeled compounds and pollutants having no labeled analog:

13.2.1 The signals for all characteristic masses stored in the spectral library (Section 7.2.4) shall be present and shall maximize within the same two consecutive scans.

13.2.2 Either

(1) the background corrected EICP areas, or

(2) the corrected relative intensities of the mass spectral peaks at the GC peak maximum shall agree within a factor of two (0.5 to 2 times) for all masses stored in the library.

13.2.3 The retention time relative to the nearest eluted internal standard shall be within ±15 scans or ±15 seconds, whichever is greater of this difference in the shift standard (Section 12.1).

13.3 Pollutants having a labled analog:

13.3.1 The signals for all characteristic masses stored in the spectral library (Section 7.2.4) shall be present and shall maximize within the same two consecutive scans.

13.3.2. Either

(1) the background corrected EICP areas, or

(2) the corrected relative intensities of the mass spectral peaks at the GC peak maximum shall agree within a factor of two for all masses stored in the spectral library.

13.3.3. The retention time difference between the pollutant and its labeled analog shall agree within ±6 scans or ±6 seconds (whichever is greater) of this difference in the shift standard (Section 12.1).

13.4 Masses present in the experimental mass spectrum that are not present in the reference mass spectrum shall be accounted for by contaminant or background ions. If the experimental mass spectrum is contaminated, an experienced spectrometrist (Section 1.4) is to determine the presence or absence of the cmmpound.

14. Quantitative Determination

14.1 Isotope dilution—by adding a known amount of a labeled compound to every sample prior to extraction, correction for recovery of the pollutant can be made because the pollutant and its labeled analog exhibit the same effects upon extraction, concentration, and gas chromatography. Relative response (RR) values for mixtures are used in conjunction with calibration curves described in Section 7.4 to determine concentrations directly, so long as labeled compound spiking levels are constant. For the phenml example given in Figure 1 (Section 7.4.1), RR would be equal to 1.114. For this RR value, the phenol calibration curve given in Figure 1 indicates a concentration of 27 µg/mL in the sample extract (Cex).

14.2 Internal standard—compute the concentration in the extract using the response factor determined from calibration data (Section 7.5) and the following equation: Cex(µg/mL) = (As × Cis/(Ais × RF) where Cex is the concentration of the compound in the extract, and the other terms are as defined in Section 7.5.1.

14.3 The concentration of the pollutant in water is computed using the volumes of the original water sample (Section 10.1) and the final extract volume (Section 10.5), as follows: Concentration in water (µg/L) = (Cex × Vex)/Vs where Vex is the extract volume in mL, and Vs is the sample volume in liters.

14.4 If the EICP area at the quantitiation mass for any compound exceeds the calibration range of the system, the extract of the dilute aliquot (Section 10.1) is analyzed by isotope dilution; otherwise, the extract is diluted by a factor of 10, 9 µL of internal standard solution (Section 6.10) are added to a 1.0 mL aliquot, and this diluted extract is analyzed by the internal standard method (Section 14.2). Quantify each compound at the highest concentration level within the calibration range.

14.5 Report results for all pollutants and labeled compounds (Tables 1 and 2) found in all standards, blanks, and samples in µg/L, to three significant figures. Results for samples which have been diluted are reported at the least dilute level at which the area at the quantitation mass is within the calibration range (Section 14.4) and the labeled compound recovery is within the normal range for the method (Section 15.4).

15. Analysis of Complex Samples

15.1 Untreated effluents and other samples frequently contain high levels (>1000 µg/L) of the compounds of interest, interfering compounds, and/or polymeric materials. Some samples will not concentrate to one mL (Section 10.5); others will overload the GC column and/or mass spectrometer.

15.2 Analyze the dilute aliquot (Section 10.1) when the sample will not concentrate to 1.0 mL. If a dilute aliquot was not extracted, and the sample holding time (Section 9.3) has not been exceeded, dilute an aliquot of the sample with reagent water and re-extract (Section 10.1); otherwise, dilute the extract (Section 14.4) and analyze by the internal standard method (Section 14.2).

15.3 Recovery of internal standard—the EICP area of the internal standard should be within a factor of two of the area in the shift standard (Section 12.1). If the absolute areas of the labeled compounds are within a factor of two of the respective areas in the shift standard, and the internal standard area is less than one-half of its respective area, then internal standard loss in the extract has occurred. In this case, use one of the labeled compounds (perferably a polynuclear aromatic hydrocarbon) to compute the concentration of a pollutant with no labeled analog.

15.4 Recovery of labeled compounds—in most samples, labeled compound recoveries will be similar to those from reagent water (Section 12.7). If the labeled compound recovery is outside the limits given in Table 8, the dilute extract (Section 10.1) is analyzed as in Section 14.4. If the recoveries of all labeled compounds and the internal staldard are low (per the criteria above), then a loss in instrument sensitivity is the most likely cause. In this case, the 100 µg/mL calibration standard (Section 12.1) shall be analyzed and calibration verified (Section 12.5). If a loss in sensitivity has occurred, the instrument shall be repaired, the performance specifications in Section 12 shall be met, and the extract reanalyzed. If a loss in instrument sensitivity has not occurred, the method does not work on the sample being analyzed and the result may not be reported for regulatory compliance purposes.

16. Method Performance

16.1 Interlaboratory performance for this method is detailed in references 9 and 10.

16.2 A chromatogram of the 100 µg/mL acid/base/neutral calibration standard (Section 6.13) is shown in Figure 6.

References

1. “Performance Tests for the Evaluation of Computerized Gas Chromatography/Mass Spectrometry Equipment and Laboratories” USEPA, EMSL/Cincinnati, OH 45268, EPA–600/4–80–025 (April 1980).

2. “Working with Carcinogens,” DHEW, PHS, CDC, NIOSH, Publication 77–206, (August 1977).

3. “OSHA Safety and Health Standards, General Industry” OSHA 2206, 29 CFR part 1910 (January 1976).

4. “Safety in Academic Chemistry Laboratories, ” ACS Committee on Chemical Safety (1979).

5. “Reference Compound to Calibrate Ion Abundance Measurement in Gas Chromatography-Mass Spectrometry Systems,” J.W. Eichelberger, L.E. Harris, and W.L. Budde, Anal. Chem., 47, 955 (1975).

6. “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA, EMSL/Cincinnati, OH 45268, EPA–600/4–79–019 (March 1979).

7. “Standard Practice for Sampling Water,” ASTM Annual Book of Standards, ASTM, Philadelphia, PA, 76 (1980).

8. “Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA, EMSL/ Cincinnati, OH 45268, EPA 600/4–70–020 (March 1979).

9. Colby, B.N., Beimer, R.G., Rushneck, D.R., and Telliard, W.A., “Isotope Dilution Gas Chromatography-Mass Spectrometry for the determination of Priority Pollutants in Industrial Effluents.” USEPA, Effluent Guidelines Division, Washington, DC 20460 (1980).

10. “Inter-laboratory Validation of US Environmental Protection Agency Method 1625,” USEPA, Effluent Guidelines Division, Washington, DC 20460 (June 15, 1984).

Table 1—Base/Neutral Extractable Compounds

Compound STORET CAS registry EPA-EGD NPDES
Acenaphthene 34205 83–32–9 001 B 001 B
Acenaphthylene 34200 208–96–8 077 B 002 B
Anthracene 34220 120–12–7 078 B 003 B
Benzidine 39120 92–87–5 005 B 004 B
Benzo(a)anthracene 34526 56–55–3 072 B 005 B
Benzo(b)fluoranthene 34230 205–99–2 074 B 007 B
Benzo(k)fluoranthene 34242 207–08–9 075 B 009 B
Benzo(a)pyrene 34247 50–32–8 073 B 006 B
Benzo(ghi)perylene 34521 191–24–2 079 B 008 B
Biphenyl (Appendix C) 81513 92–52–4 512 B
Bis(2-chloroethyl) ether 34273 111–44–4 018 B 011 B
Bis(2-chloroethyoxy)methane 34278 111–91–1 043 B 010 B
Bis(2-chloroisopropyl) ether 34283 108–60–1 042 B 012 B
Bis(2-ethylhexyl) phthalate 39100 117–81–7 066 B 013 B
4-bromophenyl phenyl ether 34636 101–55–3 041 B 014 B
Butyl benzyl phthalate 34292 85–68–7 067 B 015 B
n-C10 (Appendix C) 77427 124–18–5 517 B
n-C12 (Appendix C) 77588 112–40–2 506 B
n-C14 (Appendix C) 77691 629–59–4 518 B
n-C16 (Appendix C) 77757 544–76–3 519 B
n-C18 (Appendix C) 77804 593–45–3 520 B
n-C20 (Appendix C) 77830 112–95–8 521 B
n-C22 (Appendix C) 77859 629–97–0 522 B
n-C24 (Appendix C) 77886 646–31–1 523 B
n-C26 (Appendix C) 77901 630–01–3 524 B
n-C28 (Appendix C) 78116 630–02–4 525 B
n-C30 (Appendix C) 78117 638–68–6 526 B
Carbazole (4c) 77571 86–74–8 528 B
2-chloronaphthalene 34581 91–58–7 020 B 016 B
4-chlorophenyl phenyl ether 34641 7005–72–3 040 B 017 B
Chrysene 34320 218–01–9 076 B 018 B
P-cymene (Appendix C) 77356 99–87–6 513 B
Dibenzo(a,h)anthracene 34556 53–70–3 082 B 019 B
Dibenzofuran (Appendix C and 4c) 81302 132–64–9 505 B
Dibenzothiophene (Synfuel) 77639 132–65–0 504 B
Di-n-butyl phthalate 39110 84–74–2 068 B 026 B
1,2-dichlorobenzene 34536 95–50–1 025 B 020 B
1,3-dichlorobenzene 34566 541–73–1 026 B 021 B
1,4-dichlorobenzene 34571 106–46–7 027 B 022 B
3,3′-dichlorobenzidine 34631 91–94–1 028 B 023 B
Diethyl phthalate 34336 84–66–2 070 B 024 B
2,4-dimethylphenol 34606 105–67–9 034 A 003 A
Dimethyl phthalate 34341 131–11–3 071 B 025 B
2,4-dinitrotoluene 34611 121–14–2 035 B 027 B
2,6-dinitrotoluene 34626 606–20–2 036 B 028 B
Di-n-octyl phthalate 34596 117–84–0 069 B 029 B
Diphenylamine (Appendix C) 77579 122–39–4 507 B
Diphenyl ether (Appendix C) 77587 101–84–8 508 B
1,2-diphenylhydrazine 34346 122–66–7 037 B 030 B
Fluoranthene 34376 206–44–0 039 B 031 B
Fluorene 34381 86–73–7 080 B 032 B
Hexachlorobenzene 39700 118–74–1 009 B 033 B
Hexachlorobutadiene 34391 87–68–3 052 B 034 B
Hexachloroethane 34396 67–72–1 012 B 036 B
Hexachlorocyclopentadiene 34386 77–47–4 053 B 035 B
Indeno(1,2,3-cd)pyrene 34403 193–39–5 083 B 037 B
Isophorone 34408 78–59–1 054 B 038 B
Naphthalene 34696 91–20–3 055 B 039 B
B-naphthylamine (Appendix C) 82553 91–59–8 502 B
Nitrobenzene 34447 98–95–3 056 B 040 B
N-nitrosodimethylamine 34438 62–75–9 061 B 041 B
N-nitrosodi-n-propylamine 34428 621–64–7 063 B 042 B
N-nitrosodiphenylamine 34433 86–30–3 062 B 043 B
Phenanthrene 34461 85–01–8 081 B 044 B
Phenol 34694 108–95–2 065 A 010 A
a-Picoline (Synfuel) 77088 109–06–89 503 B
Pyrene 34469 129–00–0 084 B 045 B
styrene (Appendix C) 77128 100–42–5 510 B
a-terpineol (Appendix C) 77493 98–55–5 509 B
1,2,3-trichlorobenzene (4c) 77613 87–61–6 529 B
1,2,4-trichlorobenzene 34551 120–82–1 008 B 046 B

Table 2—Acid Extractable Compounds

Compound STORET CAS registry EPA-EGD NPDES
4-chloro-3-methylphenol 34452 59–50–7 022 A 008 A
2-chlorophenol 34586 95–57–8 024 A 001 A
2,4-dichlorophenol 34601 120–83–2 031 A 002 A
2,4-dinitrophenol 34616 51–28–5 059 A 005 A
2-methyl-4,6-dinitrophenol 34657 534–52–1 060 A 004 A
2-nitrophenol 34591 88–75–5 057 A 006 A
4-nitrophenol 34646 100–02–7 058 A 007 A
Pentachlorophenol 39032 87–86–5 064 A 009 A
2,3,6-trichlorophenol (4c) 77688 93–37–55 530 A
2,4,5-trichlorophenol (4c) 95–95–4 531 A
2,4,6-trichlorophenol 34621 88–06–2 021 A 011 A

Table 3—Gas Chromatography of Base/Neutral Extractable Compounds

EGD No.1 Compound Retention time Detection limit2 (µg/L)
Mean (sec) EGD Ref Relative
164 2,2′-difluorobiphenyl (int std) 1163 164 1.000–1.000 10
061 N-nitrosodimethylamine 385 164 ns 50
603 alpha picoline-d7 417 164 0.326–0.393 50
703 alpha picoline 426 603 1.006–1.028 50
610 styrene-d5 546 164 0.450–0.488 10
710 styrene 549 610 1.002–1.009 10
613 p-cymene-d14 742 164 0.624–0.652 10
713 p-cymene 755 613 1.008–1.023 10
265 phenol-d5 696 164 0.584–0.613 10
365 phenol 700 265 0.995–1.010 10
218 bis(2-chloroethyl) ether-d8 696 164 0.584–0.607 10
318 bis(2-chloroethyl) ether 704 218 1.007–1.016 10
617 n-decane-d22 698 164 0.585–0.615 10
717 n-decane 720 617 1.022–1.038 10
226 1,3-dichlorobenzene-d4 722 164 0.605–0.636 10
326 1,3-dichlorobenzene 724 226 0.998–1.008 10
227 1,4-dichlorobenzene-d4 737 164 0.601–0.666 10
327 1,4-dichlorobenzene 740 227 0.997–1.009 10
225 1,2-dichlorobenzene-d4 758 164 0.632–0.667 10
325 1,2-dichlorobenzene 760 225 0.995–1.008 10
242 bis(2-chloroisopropyl) ether-d12 788 164 0.664–0.691 10
342 bis(2-chloroisopropyl) ether 799 242 1.010–1.016 10
212 hexachloroethane-13C 819 164 0.690–0.717 10
312 hexachloroethane 823 212 0.999–1.001 10
063 N-nitrosodi-n-propylamine 830 164 ns 20
256 nitrobenzene-d5 845 164 0.706–0.727 10
356 nitrobenzene 849 256 1.002–1.007 10
254 isophorone-d8 881 164 0.747–0.767 10
354 isophorone 889 254 0.999–1.017 10
234 2,4-dimethyl phenol-d3 921 164 0.781–0.803 10
334 2,4-dimethylphenol 924 234 0.999–1.003 10
043 bis(2-chloroethoxy) methane 939 164 ns 10
208 1,2,4-trichlorobenzene-d3 955 164 0.813–0.830 10
308 1,2,4-trichlorobenzene 958 208 1.000–1.005 10
255 naphthalene-d8 963 164 0.819–0.836 10
355 naphthalene 967 255 1.001–1.006 10
609 alpha-terpineol-d3 973 164 0.829–0.844 10
709 alpha-terpineol 975 609 0.998–1.008 10
606 n-dodecane-d26 953 164 0.730–0.908 10
706 n-dodecane 981 606 0.986–1.051 10
529 1,2,3-trichlorobenzene 1003 164 ns 10
252 hexachlorobutadiene-13C4 1005 164 0.856–0.871 10
352 hexachlorobutadiene 1006 252 0.999–1.002 10
253 hexachlorocyclopentadiene-13C4 1147 164 0.976–0.986 10
353 hexachlorocyclopentadiene 1142 253 0.999–1.001 10
220 2-chloronaphthalene-d7 1185 164 1.014–1.024 10
320 2-chloronaphthalene 1200 220 0.997–1.007 10
518 n-tetradecane 1203 164 ns 10
612 Biphenyl-d10 1205 164 1.016–1.027 10
712 Biphenyl 1195 612 1.001–1.006 10
608 Diphenyl ether-d10 1211 164 1.036–1.047 10
708 Diphenyl ether 1216 608 0.997–1.009 10
277 Acenaphthylene-d8 1265 164 1.080–1.095 10
377 Acenaphthylene 1247 277 1.000–1.004 10
271 Dimethyl phthalate-d4 1269 164 1.083–1.102 10
371 Dimethyl phthalate 1273 271 0.998–1.005 10
236 2,6-dinitrotoluene-d3 1283 164 1.090–1.112 10
336 2,6-dinitrotoluene 1300 236 1.001–1.005 10
201 Acenaphthene-d10 1298 164 1.107–1.125 10
301 Acenaphthene 1304 201 0.999–1.009 10
605 Dibenzofuran-d8 1331 164 1.134–1.155 10
705 Dibenzofuran 1335 605 0.998–1.007 10
602 Beta-naphthylamine-d7 1368 164 1.163–1.189 50
702 Beta-naphthylamine 1371 602 0.996–1.007 50
280 Fluorene-d10 1395 164 1.185–1.214 10
380 Fluorene 1401 281 0.999–1.008 10
240 4-chlorophenyl phenyl ether-d5 1406 164 1.194–1.223 10
340 4-chlorophenyl phenyl ether 1409 240 0.990–1.015 10
270 Diethyl phthalate-d4 1409 164 1.197–1.229 10
370 Diethyl phthalate 1414 270 0.996–1.006 10
619 n-hexadecane-d34 1447 164 1.010–1.478 10
719 n-hexadecane 1469 619 1.013–1.020 10
235 2,4-dinitrotoluene-d3 1359 164 1.152–1.181 10
335 2,4-dinitrotoluene 1344 235 1.000–1.002 10
237 1,2-diphenylhydrazine-d8 1433 164 1.216–1.248 20
337 1,2-diphenylhydrazine (3) 1439 237 0.999–1.009 20
607 Diphenylamine-d10 1437 164 1.213–1.249 20
707 Diphenylamine 1439 607 1.000–1.007 20
262 N-nitrosodiphenylamine-d6 1447 164 1.225–1.252 20
362 N-nitrosodiphenylamine (4) 1464 262 1.000–1.002 20
041 4-bromophenyl phenyl ether 1498 164 1.271–1.307 10
209 Hexachlorobenzene-13C6 1521 164 1.288–1.327 10
309 Hexachlorobenzene 1522 209 0.999–1.001 10
281 Phenanthrene-d10 1578 164 1.334–1.380 10
520 n-octadecane 1580 164 ns 10
381 Phenanthrene 1583 281 1.000–1.005 10
278 Anthracene-d10 1588 164 1.342–1.388 10
378 Anthracene 1592 278 0.998–1.006 10
604 Dibenzothiophene-d8 1559 164 1.314–1.361 10
704 Dibenzothiophene 1564 604 1.000–1.006 10
528 Carbazole 1650 164 ns 20
621 n-eicosane-d42 1655 164 1.184–1.662 10
721 n-eicosane 1677 621 1.010–1.021 10
268 Di-n-butyl phthalate-d4 1719 164 1.446–1.510 10
368 Di-n-butyl phthalate 1723 268 1.000–1.003 10
239 Fluoranthene-d10 1813 164 1.522–1.596 10
339 Fluoranthene 1817 239 1.000–1.004 10
284 Pyrene-d10 1844 164 1.523–1.644 10
384 Pyrene 1852 284 1.001–1.003 10
205 Benzidine-d8 1854 164 1.549–1.632 50
305 Benzidine 1853 205 1.000–1.002 50
522 n-docosane 1889 164 ns 10
623 n-tetracosane-d50 1997 164 1.671–1.764 10
723 n-tetracosane 2025 612 1.012–1.015 10
067 Butylbenzyl phthalate 2060 164 ns 10
276 Chrysene-d12 2081 164 1.743–1.837 10
376 Chrysene 2083 276 1.000–1.004 10
272 Benzo(a)anthracene-d12 2082 164 1.735–1.846 10
372 Benzo(a)anthracene 2090 272 0.999–1.007 10
228 3,3′-dichlorobenzidine-d6 2088 164 1.744–1.848 50
328 3,3′-dichlorobenzidine 2086 228 1.000–1.001 50
266 Bis(2-ethylhexyl) phthalate-d4 2123 164 1.771–1.880 10
366 Bis(2-ethylhexyl) phthalate 2124 266 1.000–1.002 10
524 n-hexacosane 2147 164 ns 10
269 di-n-octyl phthalate-d4 2239 164 1.867–1.982 10
369 di-n-octyl phthalate 2240 269 1.000–1.002 10
525 n-octacosane 2272 164 ns 10
274 Benzo(b)fluoranthene-d12 2281 164 1.902–2.025 10
354 Benzo(b)fluoranthene 2293 274 1.000–1.005 10
275 Benzo(k)fluoranthene-d12 2287 164 1.906–2.033 10
375 Benzo(k)fluoranthene 2293 275 1.000–1.005 10
273 Benzo(a)pyrene-d12 2351 164 1.954–2.088 10
373 Benzo(a)pyrene 2350 273 1.000–1.004 10
626 N-triacontane-d62 2384 164 1.972–2.127 10
726 N-triacontane 2429 626 1.011–1.028 10
083 Indeno(1,2,3-cd)pyrene 2650 164 ns 20
082 Dibenzo(a,h)anthracene 2660 164 ns 20
279 Benzo(ghi)perylene-d12 2741 164 2.187–2.524 20
379 Benzo(ghi)perylene 2750 279 1.001–1.006 20

Table 4—Gas Chromatography of Acid Extractable Compounds

EGD No.1 Compound Retention time Detection limit2 (µg/L)
Mean (sec) EGD Ref Relative
164 2,2′-difluorobiphenyl (int std) 1163 164 1.000–1.000 10
224 2-chlorophenol-d4 701 164 0.587–0.618 10
324 2-chlorophenol 705 224 0.997–1.010 10
257 2-nitrophenol-d4 898 164 0.761–0.783 20
357 2-nitrophenol 900 257 0.994–1.009 20
231 2,4-dichlorophenol-d3 944 164 0.802–0.822 10
331 2,4-dichlorophenol 947 231 0.997–1.006 10
222 4-chloro-3-methylphenol-d2 1086 164 0.930–0.943 10
322 4-chloro-3-methylphenol 1091 222 0.998–1.003 10
221 2,4,6-trichlorophenol-d2 1162 164 0.994–1.005 10
321 2,4,6-trichlorophenol 1165 221 0.998–1.004 10
531 2,4,5-trichlorophenol 1170 164 ns 10
530 2,3,6-trichlorophenol 1195 164 ns 10
259 2,4-dinitrophenol-d3 1323 164 1.127–1.149 50
359 2,4-dinitrophenol 1325 259 1.000–1.005 50
258 4-nitrophenol-d4 1349 164 1.147–1.175 50
358 4-nitrophenol 1354 258 0.997–1.006 50
260 2-methyl-4,6-dinitrophenol-d2 1433 164 1.216–1.249 20
360 2-methyl-4,6-dinitrophenol 1435 260 1.000–1.002 20
264 Pentachlorophenol-13C6 1559 164 1.320–1.363 50
364 Pentachlorophenol 1561 264 0.998–1.002 50

Table 5—DFTPP Mass Intensity Specifications

Mass Intensity required
51 30–60 percent of mass 198.
68 Less than 2 percent of mass 69.
70 Less than 2 percent of mass 69.
127 40–60 percent of mass 198.
197 Less than 1 percent of mass 198.
199 5–9 percent of mass 198.
275 10–30 percent of mass 198.
365 greater than 1 percent of mass 198
441 present and less than mass 443
442 40–100 percent of mass 198.
443 17–23 percent of mass 442.

Table 6—Base/Neutral Extractable Compound Characteristic Masses

Compound Labeled analog Primary m/z
Acenaphthene d10 154/164
Acenaphthylene d8 152/160
Anthracene d10 178/188
Benzidine d8 184/192
Benzo(a)anthracene d12 228/240
Benzo(b)fluoranthene d12 252/264
Benzo(k)fluoranthene d12 252/264
Benzo(a)pyrene d12 252/264
Benzo(ghi)perylene d12 276/288
Biphenyl d10 154/164
Bis(2-chloroethyl) ether d8 93/101
Bis(2-chloroethoxy)methane 93
Bis(2-chloroisopropyl) ether d12 121/131
Bis(2-ethylhexyl) phthalate d4 149/153
4-bromophenyl phenyl ether 248
Butyl benzyl phthalate 149
n-C10 d22 55/66
n-C12 d26 55/66
n-C14 55
n-C16 d34 55/66
n-C18 55
n-C20 d42 55/66
n-C22 55
n-C24 d50 55/66
n-C26 55
n-C28 55
n-C30 d62 55/66
Carbazole d8 167/175
2-chloronaphthalene d7 162/169
4-chlorophenyl phenyl ether d5 204/209
Chrysene d12 228/240
p-cymene d14 114/130
Dibenzo(a,h)anthracene 278
Dibenzofuran d8 168/176
Dibenzothiophene d8 184/192
Di-n-butyl phthalate d4 149/153
1,2-dichlorobenzene d4 146/152
1,3-dichlorobenzene d4 146/152
1,4-dichlorobenzene d4 146/152
3,3′-dichlorobenzidine d6 252/258
Diethyl phthalate d4 149/153
2,4-dimethylphenol d3 122/125
Dimethyl phthalate d4 163/167
2,4-dinitrotoluene d3 164/168
2,6-dinitrotoluene d3 165/167
Di-n-octyl phthalate d4 149/153
Diphenylamine d10 169/179
Diphenyl ether d10 170/180
1,2-diphenylhydrazine1 d10 77/82
Fluoranthene d10 202/212
Fluorene d10 166/176
Hexachlorobenzene 13C6 284/292
Hexachlorobutadiene 13C4 225/231
Hexachloroethane 13C 201/204
Hexachlorocyclopentadiene 13C4 237/241
Ideno(1,2,3-cd)pyrene 276
Isophorone d8 82/88
Naphthalene d8 128/136
B-naphthylamine d7 143/150
Nitrobenzene d5 123/128
N-nitrosodimethylamine 74
N-nitrosodi-n-propylamine 70
N-nitrosodiphenylamile2 d6 169/175
Phenanthrene d10 178/188
Phenol d5 94/71
a-picoline d7 93/100
Pyrene d10 202/212
Styrene d5 104/109
a-terpineol d3 59/62
1,2,3-trichlorobenzene d3 180/183
1,2,4-trichlorobenzene d3 180/183

Table 7—Acid Extractable Compound Characteristic Masses

Compound Labeled analog Primary m/z
4-chloro-3-methylphenol d2 107/109
2-chlorophenol d4 128/132
2,4-dichlorophenol d3 162/167
2,4-dinitrophenol d3 184/187
2-methyl-4,6-dinitrophenol d2 198/200
2-nitrophenol d4 139/143
4-nitrophenol d4 139/143
Pentachlorophenol 13C6 266/272
2,3,6-trichlorophenol d2 196/200
2,4,5-trichlorophenol d2 196/200
2,4,6-trichlorophenol d2 196/200

Table 8—Acceptance Criteria for Performance Tests

EGD No.1 Compound Acceptance criteria
Initial precision and accuracy section 8.2.3 (µg/L) Labeled compound recovery sec. 8.3 and 14.2 P (percent) Calibration verification sec. 12.5 (µg/mL) On-going accuracy sec. 11.6 R (µg/L)
s X
301 Acenaphthene 21 79–134 80–125 72–144
201 Acenaphthene-d10 38 38–147 20–270 71–141 30–180
377 Acenaphtylene 38 69–186 60–166 61–207
277 Acenaphthylene-d8 31 38–146 23–239 66–152 33–168
378 Anthracene 41 58–174 60–168 50–199
278 Anthracene-d10 49 31–194 14–419 58–171 23–242
305 Benzidine 119 16–518 34–296 11–672
205 Benzidine-d8 269 ns-ns ns-ns ns-ns ns-ns
372 Benzo(a)anthracene 20 65–168 70–142 62–176
272 Benzo(a)anthracene-d12 41 25–298 12–605 28–357 22–329
374 Benzo(b)fluoranthene 183 32–545 61–164 20–ns
274 Benzo(b)fluoranthene-d12 168 11–577 ns-ns 14–ns ns-ns
375 Benzo(k)fluoranthene 26 59–143 13–ns 53–155
275 Benzo(k)fluoranthene-d12 114 15–514 ns-ns 13–ns ns–685
373 Benzo(a)pyrene 26 62–195 78–129 59–206
273 Benzo(a)pyrene-d12 24 35–181 21–290 12–ns 32–194
379 Benzo(ghi)perylene 21 72–160 69–145 58–168
279 Benzo(ghi)perylene-d12 45 29–268 14–529 13–ns 25–303
712 Biphenyl (Appendix C) 41 75–148 58–171 62–176
612 Biphenyl-d12 43 28–165 ns-ns 52–192 17–267
318 Bis(2-chloroethyl) ether 34 55–196 61–164 50–213
218 Bis(2-chloroethyl) ether-d8 33 29–196 15–372 52–194 25–222
043 Bis(2-chloroethoxy)methane* 27 43–153 44–228 39–166
342 Bis(2-chloroisopropyl) ether 17 81–138 67–148 77–145
242 Bis(2-chloroisopropyl)ether-d12 27 35–149 20–260 44–229 30–169
366 Bis(2-ethylhexyl) phthalate 31 69–220 76–131 64–232
266 Bis(2-ethylhexyl) phthalate-d4 29 32–205 18–364 43–232 28–224
041 4-bromophenyl phenyl ether* 44 44–140 52–193 35–172
067 Butyl benzyl phthalate* 31 19–233 22–450 35–170
717 n-C10 (Appendix C) 51 24–195 42–235 19–237
617 n-C10-d22 70 ns–298 ns-ns 44–227 ns–504
706 n-C12 (Appendix C) 74 35–369 60–166 29–424
606 n-C12-d26 53 ns–331 ns-ns 41–242 ns–408
518 n-C14 (Appendix C)* 109 ns–985 37–268 ns-ns
719 n-C16 (Appendix C) 33 80–162 72–138 71–181
619 n-C16-d34 46 37–162 18–308 54–186 28–202
520 n-C18 (Appendix C)* 39 42–131 40–249 35–167
721 n-C20 (Appendix C) 59 53–263 54–184 46–301
621 n-C20-d42 34 34–172 19–306 62–162 29–198
522 n-C22 (Appendix C)* 31 45–152 40–249 39–195
723 n-C24 (Appendix C) 11 80–139 65–154 78–142
623 n-C24-d50 28 27–211 15–376 50–199 25–229
524 n-C26 (Appendix C)* 35 35–193 26–392 31–212
525 n-C28 (Appendix C)* 35 35–193 26–392 31–212
726 n-C30 (Appendix C) 32 61–200 66–152 56–215
626 n-C30-d62 41 27–242 13–479 24–423 23–274
528 Carbazole (4c)* 38 36–165 44–227 31–188
320 2-chloronaphthalene 100 46–357 58–171 35–442
220 2-chloronaphthalene-d7 41 30–168 15–324 72–139 24–204
322 4-chloro-3-methylphenol 37 76–131 85–115 62–159
222 4-chloro-3-methylphenol-d2 111 30–174 ns–613 68–147 14–314
324 2-chlorophenol 13 79–135 78–129 76–138
224 2-chlorophenol-d4 24 36–162 23–255 55–180 33–176
340 4-chlorophenyl phenyl ether 42 75–166 71–142 63–194
240 4-chlorophenyl phenyl ether-d5 52 40–161 19–325 57–175 29–212
376 Chrysene 51 59–186 70–142 48–221
276 Chrysene-d12 69 33–219 13–512 24–411 23–290
713 p-cymene (Appendix C) 18 76–140 79–127 72–147
613 p-cymene-d14 67 ns–359 ns-ns 66–152 ns–468
082 Dibenzo(a,h)anthracene* 55 23–299 13–761 19–340
705 Dibenzofuran (Appendix C) 20 85–136 73–136 79–146
605 Dibenzofuran-d8 31 47–136 28–220 66–150 39–160
704 Dibenzothiophene (Synfuel) 31 79–150 72–140 70–168
604 Dibenzothiophene-d8 31 48–130 29–215 69–145 40–156
368 Di-n-butyl phthalate 15 76–165 71–142 74–169
268 Di-n-butyl phthalate-d4 23 23–195 13–346 52–192 22–209
325 1,2-dichlorobenzene 17 73–146 74–135 70–152
225 1,2-dichlorobenzene-d4 35 14–212 ns–494 61–164 11–247
326 1,3-dichlorobenzene 43 63–201 65–154 55–225
226 1,3-dichlorobenzene-d4 48 13–203 ns–550 52–192 ns–260
327 1,4-dichlorobenzene 42 61–194 62–161 53–219
227 1,4-dichlorobenzene-d4 48 15–193 ns–474 65–153 11–245
328 3,3′-dichlorobenzidine 26 68–174 77–130 64–185
228 3,3′-dichlorobenzidine-d6 80 ns–562 ns-ns 18–558 ns-ns
331 2,4-dichlorophenol 12 85–131 67–149 83–135
231 2,4-dichlorophenol-d3 28 38–164 24–260 64–157 34–182
370 Diethyl phthalate 44 75–196 74–135 65–222
270 Diethyl phthalate-d4 78 ns–260 ns-ns 47–211 ns-ns
334 2,4-dimethylphenol 13 62–153 67–150 60–156
234 2,4-dimethylphenol-d3 22 15–228 ns–449 58–172 14–242
371 Dimethyl phthalate 36 74–188 73–137 67–207
271 Dimethyl phthalate-d4 108 ns–640 ns-ns 50–201 ns-ns
359 2,4-dinitrophenol 18 72–134 75–133 68–141
259 2,4-dinitrophenol-d3 66 22–308 ns-ns 39–256 17–378
335 2,4-dinitrotoluene 18 75–158 79–127 72–164
235 2,4-dinitrotoluene-d3 37 22–245 10–514 53–187 19–275
336 2,6-dinitrotoluene 30 80–141 55–183 70–159
236 2,6-dinitrotoluene-d3 59 44–184 17–442 36–278 31–250
369 Di-n-octyl phthalate 16 77–161 71–140 74–166
269 Di-n-octyl phthalate-d4 46 12–383 ns-ns 21–467 10–433
707 Diphenylamine (Appendix C) 45 58–205 57–176 51–231
607 Diphenylamine-d10 42 27–206 11–488 59–169 21–249
708 Diphenyl ether (Appendix C) 19 82–136 83–120 77–144
608 Diphenyl ether-d10 37 36–155 19–281 77–129 29–186
337 1,2-diphenylhydrazine 73 49–308 75–134 40–360
237 1,2-diphenylhydrazine-d10 35 31–173 17–316 58–174 26–200
339 Fluoranthene 33 71–177 67–149 64–194
239 Fluoranthene-d10 35 36–161 20–278 47–215 30–187
380 Fluorene 29 81–132 74–135 70–151
280 Fluorene-d10 43 51–131 27–238 61–164 38–172
309 Hexachlorobenzene 16 90–124 78–128 85–132
209 Hexachlorobenzene-13C6 81 36–228 13–595 38–265 23–321
352 hexachlorobutadiene 56 51–251 74–135 43–287
252 hexachlorobutadiene-13C4 63 ns–316 ns-ns 68–148 ns–413
312 hexachloroethane 227 21–ns 71–141 13–ns
212 hexachloroethane-13C1 77 ns–400 ns-ns 47–212 ns–563
353 hexachlorocyclopentadiene 15 69–144 77–129 67–148
253 hexachlorocyclopentadiene-13C4 60 ns-ns ns-ns 47–211 ns-ns
083 ideno(1,2,3-cd)pyrene* 55 23–299 13–761 19–340
354 isophorone 25 76–156 70–142 70–168
254 isophorone-d8 23 49–133 33–193 52–194 44–147
360 2-methyl-4,6-dinitrophenol 19 77–133 69–145 72–142
260 2-methyl-4,6-dinitrophenol-d2 64 36–247 16–527 56–177 28–307
355 naphthalene 20 80–139 73–137 75–149
255 naphthalene-d8 39 28–157 14–305 71–141 22–192
702 B-naphthylamine (Appendix C) 49 10–ns 39–256 ns-ns
602 B-naphthylamine-d7 33 ns-ns ns-ns 44–230 ns-ns
356 nitrobenzene 25 69–161 85–115 65–169
256 nitrobenzene-d5 28 18–265 ns-ns 46–219 15–314
357 2-nitrophenol 15 78–140 77–129 75–145
257 2-nitrophenol-d4 23 41–145 27–217 61–163 37–158
358 4-nitrophenol 42 62–146 55–183 51–175
258 4-nitrophenol-d4 188 14–398 ns-ns 35–287 ns-ns
061 N-nitrosodimethylamile* 198 21–472 40–249 12–807
063 N-nitrosodi-n-proplyamine* 198 21–472 40–249 12–807
362 N-nitrosodiphenylamine 45 65–142 68–148 53–173
262 N-nitrosodiphenylamine-d6 37 54–126 26–256 59–170 40–166
364 pentachlorophenol 21 76–140 77–130 71–150
264 pentachlorophenol-13C6 49 37–212 18–412 42–237 29–254
381 phenanthrene 13 93–119 75–133 87–126
281 phenanthrene-d10 40 45–130 24–241 67–149 34–168
365 phenol 36 77–127 65–155 62–154
265 phenol-d5 161 21–210 ns-ns 48–208 ns-ns
703 a-picoline (Synfuel) 38 59–149 60–165 50–174
603 a-picoline-d7 138 11–380 ns-ns 31–324 ns–608
384 pyrene 19 76–152 76–132 72–159
284 pyrene-d10 29 32–176 18–303 48–210 28–196
710 styrene (Appendix C) 42 53–221 65–153 48–244
610 styrene-d5 49 ns–281 ns-ns 44–228 ns–348
709 a-terpineol (Appendix C) 44 42–234 54–186 38–258
609 a-terpineol-d3 48 22–292 ns–672 20–502 18–339
529 1,2,3-trichlorobenzene (4c)* 69 15–229 60–167 11–297
308 1,2,4-trichlorobenzene 19 82–136 78–128 77–144
208 1,2,4-trichlorobenzene-d3 57 15–212 ns–592 61–163 10–282
530 2,3,6-trichlorophenol (4c)* 30 58–137 56–180 51–153
531 2,4,5-trichlorophenol (4c)* 30 58–137 56–180 51–153
321 2,4,6-trichlorophenol 57 59–205 81–123 48–244
221 2,4,6-trichlorophenol-d2 47 43–183 21–363 69–144 34–226

Attachment 1 to Method 1625

Introduction

To support measurement of several semivolatile pollutants, EPA has developed this attachment to EPA Method 1625B.[1] The modifications listed in this attachment are approved only for monitoring wastestreams from the Centralized Waste Treatment Point Source Category (40 CFR part 437) and the Landfills Point Source Category (40 CFR part 445). EPA Method 1625B (the Method) employs sample extraction with methylene chloride followed by analysis of the extract using capillary column gas chromatography-mass spectrometry (GC/MS). This attachment addresses the addition of the semivolatile pollutants listed in Tables 1 and 2 to all applicable standard, stock, and spiking solutions utilized for the determination of semivolatile organic compounds by EPA Method 1625B.

1.0 EPA METHOD 1625 REVISION B MODIFICATION SUMMARY

The additional semivolatile organic compounds listed in Tables 1 and 2 are added to all applicable calibration, spiking, and other solutions utilized in the determination of semivolatile compounds by EPA Method 1625. The instrument is to be calibrated with these compounds, and all procedures and quality control tests described in the Method must be performed.

2.0 SECTION MODIFICATIONS

Note:

All section and figure numbers in this Attachment reference section and figure numbers in EPA Method 1625 Revision B unless noted otherwise. Sections not listed here remain unchanged.

Section 6.7 The stock standard solutions described in this section are modified such that the analytes in Tables 1 and 2 of this attachment are required in addition to those specified in the Method.

Section 6.8 The labeled compound spiking solution in this section is modified to include the labeled compounds listed in Tables 5 and 6 of this attachment.

Section 6.9 The secondary standard is modified to include the additional analytes listed in Tables 1 and 2 of this attachment.

Section 6.12 The solutions for obtaining authentic mass spectra are to include all additional analytes listed in Tables 1 and 2 of this attachment.

Section 6.13 The calibration solutions are modified to include the analytes listed in Tables 1 and 2 and the labeled compounds listed in Tables 5 and 6 of this attachment.

Section 6.14 The precision and recovery standard is modified to include the analytes listed in Tables 1 and 2 and the labeled compounds listed in Tables 5 and 6 of this attachment.

Section 6.15 The solutions containing the additional analytes listed in Tables 1 and 2 of this attachment are to be analyzed for stability.

Section 7.2.1 This section is modified to include the analytes listed in Tables 1 and 2 and the labeled compounds listed in Tables 5 and 6 of this attachment.

Section 7.4.5 This section is modified to include the analytes listed in Tables 1 and 2 and the labeled compounds listed in Tables 5 and 6 in the calibration.

Section 8.2 The initial precision and recovery (IPR) requirements are modified to include the analytes listed in Tables 1 and 2 and the labeled compounds listed in Tables 5 and 6 of this attachment. Additional IPR performance criteria are supplied in Table 7 of this attachment.

Section 8.3 The labeled compounds listed in Tables 3 and 4 of this attachment are to be included in the method performance tests. Additional method performance criteria are supplied in Table 7 of this attachment.

Section 8.5.2 The acceptance criteria for blanks includes the analytes listed in Tables 1 and 2 of this attachment.

Section 10.1.2 The labeled compound solution must include the labeled compounds listed in Tables 5 and 6 of this attachment.

Section 10.1.3 The precision and recovery standard must include the analytes listed in Tables 1 and 2 and the labeled compounds listed in Tables 5 and 6 of this attachment.

Section 12.5 Additional QC requirements for calibration verification are supplied in Table 7 of this attachment.

Section 12.7 Additional QC requirements for ongoing precision and recovery are supplied in Table 7 of this attachment.

Table 1—Base/Neutral Extractable Compounds

Compound Pollutant
CAS
Registry
EPA-EGD
acetophenone1 98–86–2 758
aniline2 62–53–3 757
-2,3-dichloroaniline1 608–27–5 578
-o-cresol1 95–48–7 771
pyridine2 110–86–1 1330

Table 2—Acid Extractable Compounds

Compound Pollutant
CAS
Registry
EPA-EGD
p-cresol1 106–44–5 1744

Table 3—Gas Chromatography1 of Base/Neutral Extractable Compounds

EGD No. Compound Retention time2 Minimum
level3
(µg/L)
EGD No. Compound Retention time2 Minimum level3
(µg/L)
Mean
(sec)
EGD Ref Relative Mean
(sec)
EGD Ref Relative
758 acetophenone4 818 658 1.003–1.005 10
757 aniline5 694 657 0.994–1.023 10
578 2,3-dichloroaniline4 1160 164 1.003–1.007 10
771 o-cresol4 814 671 1.005–1.009 10
1330 pyridine5 378 1230 1.005–1.011 10

Table 4—Gas Chromatography1 of Acid Extractable Compounds

EGD No. Compound Retention time2 Minimum
level
(µ/L)3
Mean
(sec)
EGD Ref Relative
1744 p-cresol4 834 1644 1.004–1.008 20

Table 5—Base/Neutral Extractable Compound Characteristic m/z's

Compound Labeled Analog Primary
m/z1
acetophenone2 d5 105/110
aniline3 d7 93/100
o-cresol2 d7 108/116
2,3-dichloroaniline2 n/a 161
pyridine3 d5 79/84

Table 6—Acid Extractable Compound Characteristic m/z's

Compound Labeled Analog Primary
m/z1
p-cresol2 d7 108/116

Table 7—Acceptance Criteria for Performance Tests

EGD No. Compound Acceptance criteria Calibration verification sec. 12.5
µg/mL)
On-going accuracy sec. 12.7 R
(µg/L)
Initial precision and accuracy section 8.2
(µg/L)
Labeled compound recovery sec. 8.3 and 14.2 P
(percent)
s
(µg/L)
X
758 acetophenone1 34 44–167 85–115 45–162
658 acetophenone-d51 51 23–254 45–162 85–115 22–264
757 aniline2 32 30–171 85–115 33–154
657 aniline-d72 71 15–278 33–154 85–115 12–344
771 o-cresol1 40 31–226 85–115 35–196
671 o-cresol-d71 23 30–146 35–196 85–115 31–142
1744 p-cresol2 59 54–140 85–115 37–203
1644 p-cresol-d72 22 11–618 37–203 85–115 16–415
578 2,3-dichloroaniline1 13 40–160 85–115 44–144
1330 pyridine2 28 10–421 83–117 18–238
1230 pyridine-d52 ns 7–392 19–238 85–115 4–621

[49 FR 43261, Oct. 26, 1984; 50 FR 692, 695, Jan. 4, 1985, as amended at 51 FR 23702, June 30, 1986; 62 FR 48405, Sept. 15, 1997; 65 FR 3044, Jan. 19, 2000; 65 FR 81295, 81298, Dec. 22, 2000; 82 FR 40875, Aug. 28, 2017]

Appendix B to Part 136—Definition and Procedure for the Determination of the Method Detection Limit—Revision 2

Definition

The method detection limit (MDL) is defined as the minimum measured concentration of a substance that can be reported with 99% confidence that the measured concentration is distinguishable from method blank results.

I. Scope and Application

(1) The MDL procedure is designed to be a straightforward technique for estimation of the detection limit for a broad variety of physical and chemical methods. The procedure requires a complete, specific, and well-defined analytical method. It is essential that all sample processing steps used by the laboratory be included in the determination of the method detection limit.

(2) The MDL procedure is not applicable to methods that do not produce results with a continuous distribution, such as, but not limited to, methods for whole effluent toxicity, presence/absence methods, and microbiological methods that involve counting colonies. The MDL procedure also is not applicable to measurements such as, but not limited to, biochemical oxygen demand, color, pH, specific conductance, many titration methods, and any method where low-level spiked samples cannot be prepared. Except as described in the addendum, for the purposes of this procedure, “spiked samples” are prepared from a clean reference matrix, such as reagent water, spiked with a known and consistent quantity of the analyte. MDL determinations using spiked samples may not be appropriate for all gravimetric methods (e.g., residue or total suspended solids), but an MDL based on method blanks can be determined in such instances.

II. Procedure

(1) Estimate the initial MDL using one or more of the following:

(a) The mean determined concentration plus three times the standard deviation of a set of method blanks.

(b) The concentration value that corresponds to an instrument signal-to-noise ratio in the range of 3 to 5.

(c) The concentration equivalent to three times the standard deviation of replicate instrumental measurements of spiked blanks.

(d) That region of the calibration where there is a significant change in sensitivity, i.e., a break in the slope of the calibration.

(e) Instrumental limitations.

(f) Previously determined MDL.

Note:

It is recognized that the experience of the analyst is important to this process. However, the analyst should include some or all of the above considerations in the initial estimate of the MDL.

(2) Determine the initial MDL.

Note:

The Initial MDL is used when the laboratory does not have adequate data to perform the Ongoing Annual Verification specified in Section (4), typically when a new method is implemented or if a method was rarely used in the last 24 months.

(a) Select a spiking level, typically 2—10 times the estimated MDL in Section 1. Spiking levels in excess of 10 times the estimated detection limit may be required for analytes with very poor recovery (e.g., for an analyte with 10% recovery, spiked at 100 micrograms/L, with mean recovery of 10 micrograms/L; the calculated MDL may be around 3 micrograms/L. Therefore, in this example, the spiking level would be 33 times the MDL, but spiking lower may result in no recovery at all).

(b) Process a minimum of seven spiked samples and seven method blank samples through all steps of the method. The samples used for the MDL must be prepared in at least three batches on three separate calendar dates and analyzed on three separate calendar dates. (Preparation and analysis may be on the same day.) Existing data may be used, if compliant with the requirements for at least three batches, and generated within the last twenty four months. The most recent available data for method blanks and spiked samples must be used. Statistical outlier removal procedures should not be used to remove data for the initial MDL determination, since the total number of observations is small and the purpose of the MDL procedure is to capture routine method variability. However, documented instances of gross failures (e.g., instrument malfunctions, mislabeled samples, cracked vials) may be excluded from the calculations, provided that at least seven spiked samples and seven method blanks are available. (The rationale for removal of specific outliers must be documented and maintained on file with the results of the MDL determination.)

(i) If there are multiple instruments that will be assigned the same MDL, then the sample analyses must be distributed across all of the instruments.

(ii) A minimum of two spiked samples and two method blank samples prepared and analyzed on different calendar dates is required for each instrument. Each analytical batch may contain one spiked sample and one method blank sample run together. A spiked sample and a method blank sample may be analyzed in the same batch, but are not required to be.

(iii) The same prepared extract may be analyzed on multiple instruments so long as the minimum requirement of seven preparations in at least three separate batches is maintained.

(c) Evaluate the spiking level: If any result for any individual analyte from the spiked samples does not meet the method qualitative identification criteria or does not provide a numerical result greater than zero, then repeat the spiked samples at a higher concentration. (Qualitative identification criteria are a set of rules or guidelines for establishing the identification or presence of an analyte using a measurement system. Qualitative identification does not ensure that quantitative results for the analyte can be obtained.)

(d) Make all computations as specified in the analytical method and express the final results in the method-specified reporting units.

(i) Calculate the sample standard deviation (S) of the replicate spiked sample measurements and the sample standard deviation of the replicate method blank measurements from all instruments to which the MDL will be applied.

(ii) Compute the MDLs (the MDL based on spiked samples) as follows:

MDLS = t(n −1, 1−α= 0.99)Ss

Where:

MDLs = the method detection limit based on spiked samples

t(n-1, 1−α= 0.99) = the Student's t-value appropriate for a single-tailed 99th percentile t statistic and a standard deviation estimate with n-1 degrees of freedom. See Addendum Table 1.

Ss = sample standard deviation of the replicate spiked sample analyses.

(iii) Compute the MDLb (the MDL based on method blanks) as follows:

(A) If none of the method blanks give numerical results for an individual analyte, the MDLb does not apply. A numerical result includes both positive and negative results, including results below the current MDL, but not results of “ND” (not detected) commonly observed when a peak is not present in chromatographic analysis.

(B) If some (but not all) of the method blanks for an individual analyte give numerical results, set the MDLb equal to the highest method blank result. If more than 100 method blanks are available, set MDLb to the level that is no less than the 99th percentile of the method blank results. For “n” method blanks where n ≥ 100, sort the method blanks in rank order. The (n * 0.99) ranked method blank result (round to the nearest whole number) is the MDLb. For example, to find MDLb from a set of 164 method blanks where the highest ranked method blank results are . . . 1.5, 1.7, 1.9, 5.0, and 10, then 164 × 0.99 = 162.36 which rounds to the 162nd method blank result. Therefore, MDLb is 1.9 for n = 164 (10 is the 164th result, 5.0 is the 163rd result, and 1.9 is the 162nd result). Alternatively, you may use spreadsheet algorithms to calculate the 99th percentile to interpolate between the ranks more precisely.

(C) If all of the method blanks for an individual analyte give numerical results, then calculate the MDLb as:

MDLb = X + tn−1,1−α= (0.99)Sb

Where:

MDLb = the MDL based on method blanks

X = mean of the method blank results (use zero in place of the mean if the mean is negative)

t(n−1, 1α= 0.99) = the Student's t-value appropriate for the single-tailed 99th percentile t statistic and a standard deviation estimate with n−1 degrees of freedom. See Addendum Table 1.

Sb = sample standard deviation of the replicate method blank sample analyses.

Note:

If 100 or more method blanks are available, as an option, MDLb may be set to the concentration that is greater than or equal to the 99th percentile of the method blank results, as described in Section (2)(d)(iii)(B).

(e) Select the greater of MDLs or MDLb as the initial MDL.

(3) Ongoing Data Collection.

(a) During any quarter in which samples are being analyzed, prepare and analyze a minimum of two spiked samples on each instrument, in separate batches, using the same spiking concentration used in Section 2. If any analytes are repeatedly not detected in the quarterly spiked sample analyses, or do not meet the qualitative identification criteria of the method (see section 2(c) of this procedure), then this is an indication that the spiking level is not high enough and should be adjusted upward. Note that it is not necessary to analyze additional method blanks together with the spiked samples, the method blank population should include all of the routine method blanks analyzed with each batch during the course of sample analysis.

(b) Ensure that at least seven spiked samples and seven method blanks are completed for the annual verification. If only one instrument is in use, a minimum of seven spikes are still required, but they may be drawn from the last two years of data collection.

(c) At least once per year, re-evaluate the spiking level.

(i) If more than 5% of the spiked samples do not return positive numerical results that meet all method qualitative identification criteria, then the spiking level must be increased and the initial MDL re-determined following the procedure in section 2.

(ii) [Reserved]

(d) If the method is altered in a way that can be reasonably expected to change its sensitivity, then re-determine the initial MDL according to section 2, and the restart the ongoing data collection.

(e) If a new instrument is added to a group of instruments whose data are being pooled to create a single MDL, analyze a minimum of two spiked replicates and two method blank replicates on the new instrument. If both method blank results are below the existing MDL, then the existing MDLb is validated. Combine the new spiked sample results to the existing spiked sample results and recalculate the MDLs as in Section 4. If the recalculated MDLs does not vary by more than the factor specified in section 4(f) of this procedure, then the existing MDLs is validated. If either of these two conditions is not met, then calculate a new MDL following the instructions in section 2.

(4) Ongoing Annual Verification.

(a) At least once every thirteen months, re-calculate MDLs and MDLb from the collected spiked samples and method blank results using the equations in section 2.

(b) Include data generated within the last twenty four months, but only data with the same spiking level. Only documented instances of gross failures (e.g., instrument malfunctions, mislabeled samples, cracked vials) may be excluded from the calculations. (The rationale for removal of specific outliers must be documented and maintained on file with the results of the MDL determination.) If the laboratory believes the sensitivity of the method has changed significantly, then the most recent data available may be used, maintaining compliance with the requirement for at least seven replicates in three separate batches on three separate days (see section 2b).

(c) Include the initial MDL spiked samples, if the data were generated within twenty four months.

(d) Only use data associated with acceptable calibrations and batch QC. Include all routine data, with the exception of batches that are rejected and the associated samples reanalyzed. If the method has been altered in a way that can be reasonably expected to change its sensitivity, then use only data collected after the change.

(e) Ideally, use all method blank results from the last 24 months for the MDLb calculation. The laboratory has the option to use only the last six months of method blank data or the fifty most recent method blanks, whichever criteria yields the greater number of method blanks.

(f) The verified MDL is the greater of the MDLs or MDLb. If the verified MDL is within 0.5 to 2.0 times the existing MDL, and fewer than 3% of the method blank results (for the individual analyte) have numerical results above the existing MDL, then the existing MDL may optionally be left unchanged. Otherwise, adjust the MDL to the new verification MDL. (The range of 0.5 to 2.0 approximates the 95th percentile confidence interval for the initial MDL determination with six degrees of freedom.)

Addendum to Section II: Determination of the MDL for a Specific Matrix

The MDL may be determined in a specific sample matrix as well as in reagent water.

(1) Analyze the sample matrix to determine the native (background) concentration of the analyte(s) of interest.

(2) If the response for the native concentration is at a signal-to-noise ratio of approximately 5–20, determine the matrix-specific MDL according to Section 2 but without spiking additional analyte.

(3) Calculate MDLb using the method blanks, not the sample matrix.

(4) If the signal-to-noise ratio is less than 5, then the analyte(s) should be spiked into the sample matrix to obtain a concentration that will give results with a signal-to-noise ratio of approximately 10–20.

(5) If the analytes(s) of interest have signal-to-noise ratio(s) greater than approximately 20, then the resulting MDL is likely to be biased high.

Table 1—Single-Tailed 99th Percentile t Statistic

Number of replicates Degrees of freedom
(n−1)
t(n−1, 0.99)
7 6 3.143
8 7 2.998
9 8 2.896
10 9 2.821
11 10 2.764
16 15 2.602
21 20 2.528
26 25 2.485
31 30 2.457
32 31 2.453
48 47 2.408
50 49 2.405
61 60 2.390
64 63 2.387
80 79 2.374
96 95 2.366
100 99 2.365

III. Documentation

The analytical method used must be specifically identified by number or title and the MDL for each analyte expressed in the appropriate method reporting units. Data and calculations used to establish the MDL must be able to be reconstructed upon request. The sample matrix used to determine the MDL must also be identified with MDL value. Document the mean spiked and recovered analyte levels with the MDL. The rationale for removal of outlier results, if any, must be documented and maintained on file with the results of the MDL determination.

[82 FR 40939, Aug. 28, 2017]

Appendix C to Part 136—Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry Method 200.7

1.0 Scope and Application

1.1 Inductively coupled plasma-atomic emission spectrometry (ICP–AES) is used to determine metals and some nonmetals in solution. This method is a consolidation of existing methods for water, wastewater, and solid wastes.1–4 (For analysis of petroleum products see References 5 and 6, Section 16.0). This method is applicable to the following analytes:

Analyte Chemical abstract services registry
number (CASRN)
Aluminum (Al) 7429–90–5
Antimony (Sb) 7440–36–0
Arsenic (As) 7440–38–2
Barium (Ba) 7440–39–3
Beryllium (Be) 7440–41–7
Boron (B) 7440–42–8
Cadmium (Cd) 7440–43–9
Calcium (Ca) 7440–70–2
Ceriuma (Cr) 7440–45–1
Chromium (Cr) 7440–47–3
Cobalt (Co) 7440–48–4
Copper (Cu) 7440–50–8
Iron (Fe) 7439–89–6
Lead (Pb) 7439–92–1
Lithium (Li) 7439–93–2
Magnesium (Mg) 7439–95–4
Manganese (Mn) 7439–96–5
Mercury (Hg) 7439–97–6
Molybdenum (Mo) 7439–98–7
Nickel (Ni) 7440–02–0
Phosphorus (P) 7723–14–0
Potassium (K) 7440–09–7
Selenium (Se) 7782–49–2
Silicab (Si02) 7631–86–9
Silver (Ag) 7440–22–4
Sodium (Na) 7440–23–5
Strontium (Sr) 7440–24–6
Thallium (Tl) 7440–28–0
Tin (Sn) 7440–31–5
Titanium (Ti) 7440–32–6
Vanadium (V) 7440–62–2
Zinc (Zn) 7440–66–6

1.2 For reference where this method is approved for use in compliance monitoring programs [e.g., Clean Water Act (NPDES) or Safe Drinking Water Act (SDWA)] consult both the appropriate sections of the Code of Federal Regulation (40 CFR Part 136 Table 1B for NPDES, and Part 141 § 141.23 for drinking water), and the latest Federal Register announcements.

1.3 ICP–AES can be used to determine dissolved analytes in aqueous samples after suitable filtration and acid preservation. To reduce potential interferences, dissolved solids should be <0.2% (w/v) (Section 4.2).

1.4 With the exception of silver, where this method is approved for the determination of certain metal and metalloid contaminants in drinking water, samples may be analyzed directly by pneumatic nebulization without acid digestion if the sample has been properly preserved with acid and has turbidity of <1 NTU at the time of analysis. This total recoverable determination procedure is referred to as “direct analysis”. However, in the determination of some primary drinking water metal contaminants, preconcentration of the sample may be required prior to analysis in order to meet drinking water acceptance performance criteria (Sections 11.2.2 through 11.2.7).

1.5 For the determination of total recoverable analytes in aqueous and solid samples a digestion/extraction is required prior to analysis when the elements are not in solution (e.g., soils, sludges, sediments and aqueous samples that may contain particulate and suspended solids). Aqueous samples containing suspended or particulate material 1% (w/v) should be extracted as a solid type sample.

1.6 When determining boron and silica in aqueous samples, only plastic, PTFE or quartz labware should be used from time of sample collection to completion of analysis. For accurate determination of boron in solid samples only quartz or PTFE beakers should be used during acid extraction with immediate transfer of an extract aliquot to a plastic centrifuge tube following dilution of the extract to volume. When possible, borosilicate glass should be avoided to prevent contamination of these analytes.

1.7 Silver is only slightly soluble in the presence of chloride unless there is a sufficient chloride concentration to form the soluble chloride complex. Therefore, low recoveries of silver may occur in samples, fortified sample matrices and even fortified blanks if determined as a dissolved analyte or by “direct analysis” where the sample has not been processed using the total recoverable mixed acid digestion. For this reason it is recommended that samples be digested prior to the determination of silver. The total recoverable sample digestion procedure given in this method is suitable for the determination of silver in aqueous samples containing concentrations up to 0.1 mg/L. For the analysis of wastewater samples containing higher concentrations of silver, succeeding smaller volume, well mixed aliquots should be prepared until the analysis solution contains <0.1 mg/L silver. The extraction of solid samples containing concentrations of silver >50 mg/kg should be treated in a similar manner. Also, the extraction of tin from solid samples should be prepared again using aliquots <1 g when determined sample concentrations exceed 1%.

1.8 The total recoverable sample digestion procedure given in this method will solubilize and hold in solution only minimal concentrations of barium in the presence of free sulfate. For the analysis of barium in samples having varying and unknown concentrations of sulfate, analysis should be completed as soon as possible after sample preparation.

1.9 The total recoverable sample digestion procedure given in this method is not suitable for the determination of volatile organo-mercury compounds. However, if digestion is not required (turbidity <1 NTU), the combined concentrations of inorganic and organo-mercury in solution can be determined by “direct analysis” pneumatic nebulization provided the sample solution is adjusted to contain the same mixed acid (HNO3 + HCl) matrix as the total recoverable calibration standards and blank solutions.

1.10 Detection limits and linear ranges for the elements will vary with the wavelength selected, the spectrometer, and the matrices. Table 1 provides estimated instrument detection limits for the listed wavelengths.7 However, actual method detection limits and linear working ranges will be dependent on the sample matrix, instrumentation, and selected operating conditions.

1.11 Users of the method data should state the data-quality objectives prior to analysis. Users of the method must document and have on file the required initial demonstration performance data described in Section 9.2 prior to using the method for analysis.

2.0 Summary of Method

2.1 An aliquot of a well mixed, homogeneous aqueous or solid sample is accurately weighed or measured for sample processing. For total recoverable analysis of a solid or an aqueous sample containing undissolved material, analytes are first solubilized by gentle refluxing with nitric and hydrochloric acids. After cooling, the sample is made up to volume, is mixed and centrifuged or allowed to settle overnight prior to analysis. For the determination of dissolved analytes in a filtered aqueous sample aliquot, or for the “direct analysis” total recoverable determination of analytes in drinking water where sample turbidity is <1 NTU, the sample is made ready for analysis by the appropriate addition of nitric acid, and then diluted to a predetermined volume and mixed before analysis.

2.2 The analysis described in this method involves multielemental determinations by ICP–AES using sequential or simultaneous instruments. The instruments measure characteristic atomic-line emission spectra by optical spectrometry. Samples are nebulized and the resulting aerosol is transported to the plasma torch. Element specific emission spectra are produced by a radio-frequency inductively coupled plasma. The spectra are dispersed by a grating spectrometer, and the intensities of the line spectra are monitored at specific wavelengths by a photosensitive device. Photocurrents from the photosensitive device are processed and controlled by a computer system. A background correction technique is required to compensate for variable background contribution to the determination of the analytes. Background must be measured adjacent to the analyte wavelength during analysis. Various interferences must be considered and addressed appropriately as discussed in Sections 4.0, 7.0, 9.0, 10.0, and 11.0.

3.0 Definitions

3.1 Calibration Blank—A volume of reagent water acidified with the same acid matrix as in the calibration standards. The calibration blank is a zero standard and is used to calibrate the ICP instrument (Section 7.10.1).

3.2 Calibration Standard (CAL)—A solution prepared from the dilution of stock standard solutions. The CAL solutions are used to calibrate the instrument response with respect to analyte concentration (Section 7.9).

3.3 Dissolved Analyte—The concentration of analyte in an aqueous sample that will pass through a 0.45 µm membrane filter assembly prior to sample acidification (Section 11.1).

3.4 Field Reagent Blank (FRB)—An aliquot of reagent water or other blank matrix that is placed in a sample container in the laboratory and treated as a sample in all respects, including shipment to the sampling site, exposure to the sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the FRB is to determine if method analytes or other interferences are present in the field environment (Section 8.5).

3.5 Instrument Detection Limit (IDL)—The concentration equivalent to the analyte signal which is equal to three times the standard deviation of a series of 10 replicate measurements of the calibration blank signal at the same wavelength (Table 1.).

3.6 Instrument Performance Check (IPC) Solution—A solution of method analytes, used to evaluate the performance of the instrument system with respect to a defined set of method criteria (Sections 7.11 and 9.3.4).

3.7 Internal Standard—Pure analyte(s) added to a sample, extract, or standard solution in known amount(s) and used to measure the relative responses of other method analytes that are components of the same sample or solution. The internal standard must be an analyte that is not a sample component (Section 11.5).

3.8 Laboratory Duplicates (LD1 and LD2)—Two aliquots of the same sample taken in the laboratory and analyzed separately with identical procedures. Analyses of LD1 and LD2 indicate precision associated with laboratory procedures, but not with sample collection, preservation, or storage procedures.

3.9 Laboratory Fortified Blank (LFB)—An aliquot of LRB to which known quantities of the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine whether the methodology is in control and whether the laboratory is capable of making accurate and precise measurements (Sections 7.10.3 and 9.3.2).

3.10 Laboratory Fortified Sample Matrix (LFM)—An aliquot of an environmental sample to which known quantities of the method analytes are added in the laboratory. The LFM is analyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the LFM corrected for background concentrations (Section 9.4).

3.11 Laboratory Reagent Blank (LRB)—An aliquot of reagent water or other blank matrices that are treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, and internal standards that are used with other samples. The LRB is used to determine if method analytes or other interferences are present in the laboratory environment, reagents, or apparatus (Sections 7.10.2 and 9.3.1).

3.12 Linear Dynamic Range (LDR)—The concentration range over which the instrument response to an analyte is linear (Section 9.2.2).

3.13 Method Detection Limit (MDL)—The minimum concentration of an analyte that can be identified, measured, and reported with 99% confidence that the analyte concentration is greater than zero (Section 9.2.4 and Table 4.).

3.14 Plasma Solution—A solution that is used to determine the optimum height above the work coil for viewing the plasma (Sections 7.15 and 10.2.3).

3.15 Quality Control Sample (QCS)—A solution of method analytes of known concentrations which is used to fortify an aliquot of LRB or sample matrix. The QCS is obtained from a source external to the laboratory and different from the source of calibration standards. It is used to check either laboratory or instrument performance (Sections 7.12 and 9.2.3).

3.16 Solid Sample—For the purpose of this method, a sample taken from material classified as soil, sediment or sludge.

3.17 Spectral Interference Check (SIC) Solution—A solution of selected method analytes of higher concentrations which is used to evaluate the procedural routine for correcting known interelement spectral interferences with respect to a defined set of method criteria (Sections 7.13, 7.14 and 9.3.5).

3.18 Standard Addition—The addition of a known amount of analyte to the sample in order to determine the relative response of the detector to an analyte within the sample matrix. The relative response is then used to assess either an operative matrix effect or the sample analyte concentration (Sections 9.5.1 and 11.5).

3.19 Stock Standard Solution—A concentrated solution containing one or more method analytes prepared in the laboratory using assayed reference materials or purchased from a reputable commercial source (Section 7.8).

3.20 Total Recoverable Analyte—The concentration of analyte determined either by “direct analysis” of an unfiltered acid preserved drinking water sample with turbidity of <1 NTU (Section 11.2.1), or by analysis of the solution extract of a solid sample or an unfiltered aqueous sample following digestion by refluxing with hot dilute mineral acid(s) as specified in the method (Sections 11.2 and 11.3).

3.21 Water Sample—For the purpose of this method, a sample taken from one of the following sources: drinking, surface, ground, storm runoff, industrial or domestic wastewater.

4.0 Interferences

4.1 Spectral interferences are caused by background emission from continuous or recombination phenomena, stray light from the line emission of high concentration elements, overlap of a spectral line from another element, or unresolved overlap of molecular band spectra.

4.1.1 Background emission and stray light can usually be compensated for by subtracting the background emission determined by measurement(s) adjacent to the analyte wavelength peak. Spectral scans of samples or single element solutions in the analyte regions may indicate not only when alternate wavelengths are desirable because of severe spectral interference, but also will show whether the most appropriate estimate of the background emission is provided by an interpolation from measurements on both sides of the wavelength peak or by the measured emission on one side or the other. The location(s) selected for the measurement of background intensity will be determined by the complexity of the spectrum adjacent to the wavelength peak. The location(s) used for routine measurement must be free of off-line spectral interference (interelement or molecular) or adequately corrected to reflect the same change in background intensity as occurs at the wavelength peak.

4.1.2 Spectral overlaps may be avoided by using an alternate wavelength or can be compensated for by equations that correct for interelement contributions, which involves measuring the interfering elements. Some potential on-line spectral interferences observed for the recommended wavelengths are given in Table 2. When operative and uncorrected, these interferences will produce false-positive determinations and be reported as analyte concentrations. The interferences listed are only those that occur between method analytes. Only interferences of a direct overlap nature that were observed with a single instrument having a working resolution of 0.035 nm are listed. More extensive information on interferant effects at various wavelengths and resolutions is available in Boumans' Tables.8 Users may apply interelement correction factors determined on their instruments within tested concentration ranges to compensate (off-line or on-line) for the effects of interfering elements.

4.1.3 When interelement corrections are applied, there is a need to verify their accuracy by analyzing spectral interference check solutions as described in Section 7.13. Interelement corrections will vary for the same emission line among instruments because of differences in resolution, as determined by the grating plus the entrance and exit slit widths, and by the order of dispersion. Interelement corrections will also vary depending upon the choice of background correction points. Selecting a background correction point where an interfering emission line may appear should be avoided when practical. Interelement corrections that constitute a major portion of an emission signal may not yield accurate data. Users should not forget that some samples may contain uncommon elements that could contribute spectral interferences.7 8

4.1.4 The interference effects must be evaluated for each individual instrument whether configured as a sequential or simultaneous instrument. For each instrument, intensities will vary not only with optical resolution but also with operating conditions (such as power, viewing height and argon flow rate). When using the recommended wavelengths given in Table 1, the analyst is required to determine and document for each wavelength the effect from the known interferences given in Table 2, and to utilize a computer routine for their automatic correction on all analyses. To determine the appropriate location for off-line background correction, the user must scan the area on either side adjacent to the wavelength and record the apparent emission intensity from all other method analytes. This spectral information must be documented and kept on file. The location selected for background correction must be either free of off-line interelement spectral interference or a computer routine must be used for their automatic correction on all determinations. If a wavelength other than the recommended wavelength is used, the user must determine and document both the on-line and off-line spectral interference effect from all method analytes and provide for their automatic correction on all analyses. Tests to determine the spectral interference must be done using analyte concentrations that will adequately describe the interference. Normally, 100 mg/L single element solutions are sufficient, however, for analytes such as iron that may be found at high concentration a more appropriate test would be to use a concentration near the upper LDR limit. See Section 10.4 for required spectral interference test criteria.

4.1.5 When interelement corrections are not used, either on-going SIC solutions (Section 7.14) must be analyzed to verify the absence of interelement spectral interference or a computer software routine must be employed for comparing the determinative data to limits files for notifying the analyst when an interfering element is detected in the sample at a concentration that will produce either an apparent false positive concentration, greater than the analyte IDL, or false negative analyte concentration, less than the 99% lower control limit of the calibration blank. When the interference accounts for 10% or more of the analyte concentration, either an alternate wavelength free of interference or another approved test procedure must be used to complete the analysis. For example, the copper peak at 213.853 nm could be mistaken for the zinc peak at 213.856 nm in solutions with high copper and low zinc concentrations. For this example, a spectral scan in the 213.8 nm region would not reveal the misidentification because a single peak near the zinc location would be observed. The possibility of this misidentification of copper for the zinc peak at 213.856 nm can be identified by measuring the copper at another emission line, e.g., 324.754 nm. Users should be aware that, depending upon the instrumental resolution, alternate wavelengths with adequate sensitivity and freedom from interference may not be available for all matrices. In these circumstances the analyte must be determined using another approved test procedure.

4.2 Physical interferences are effects associated with the sample nebulization and transport processes. Changes in viscosity and surface tension can cause significant inaccuracies, especially in samples containing high dissolved solids or high acid concentrations. If physical interferences are present, they must be reduced by such means as a high-solids nebulizer, diluting the sample, using a peristaltic pump, or using an appropriate internal standard element. Another problem that can occur with high dissolved solids is salt buildup at the tip of the nebulizer, which affects aerosol flow rate and causes instrumental drift. This problem can be controlled by a high-solids nebulizer, wetting the argon prior to nebulization, using a tip washer, or diluting the sample. Also, it has been reported that better control of the argon flow rates, especially for the nebulizer, improves instrument stability and precision; this is accomplished with the use of mass flow controllers.

4.3 Chemical interferences include molecular-compound formation, ionization effects, and solute-vaporization effects. Normally, these effects are not significant with the ICP–AES technique. If observed, they can be minimized by careful selection of operating conditions (such as incident power and observation height), by buffering of the sample, by matrix matching, and by standard-addition procedures. Chemical interferences are highly dependent on matrix type and the specific analyte element.

4.4 Memory interferences result when analytes in a previous sample contribute to the signals measured in a new sample. Memory effects can result from sample deposition on the uptake tubing to the nebulizer, and from the buildup of sample material in the plasma torch and spray chamber. The site where these effects occur is dependent on the element and can be minimized by flushing the system with a rinse blank between samples (Section 7.10.4). The possibility of memory interferences should be recognized within an analytical run and suitable rinse times should be used to reduce them. The rinse times necessary for a particular element must be estimated prior to analysis. This may be achieved by aspirating a standard containing elements corresponding to either their LDR or a concentration ten times those usually encountered. The aspiration time should be the same as a normal sample analysis period, followed by analysis of the rinse blank at designated intervals. The length of time required to reduce analyte signals to within a factor of two of the method detection limit, should be noted. Until the required rinse time is established, this method requires a rinse period of at least 60 seconds between samples and standards. If a memory interference is suspected, the sample must be re-analyzed after a long rinse period.

5.0 Safety

5.1 The toxicity or carcinogenicity of each reagent used in this method have not been fully established. Each chemical should be regarded as a potential health hazard and exposure to these compounds should be as low as reasonably achievable. Each laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method.9–12 A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Specifically, concentrated nitric and hydrochloric acids present various hazards and are moderately toxic and extremely irritating to skin and mucus membranes. Use these reagents in a fume hood whenever possible and if eye or skin contact occurs, flush with large volumes of water. Always wear safety glasses or a shield for eye protection, protective clothing and observe proper mixing when working with these reagents.

5.2 The acidification of samples containing reactive materials may result in the release of toxic gases, such as cyanides or sulfides. Acidification of samples should be done in a fume hood.

5.3 All personnel handling environmental samples known to contain or to have been in contact with human waste should be immunized against known disease causative agents.

5.4 The inductively coupled plasma should only be viewed with proper eye protection from the ultraviolet emissions.

5.5 It is the responsibility of the user of this method to comply with relevant disposal and waste regulations. For guidance see Sections 14.0 and 15.0.

6.0 Equipment and Supplies

6.1 Inductively coupled plasma emission spectrometer:

6.1.1 Computer-controlled emission spectrometer with background-correction capability.

The spectrometer must be capable of meeting and complying with the requirements described and referenced in Section 2.2.

6.1.2 Radio-frequency generator compliant with FCC regulations.

6.1.3 Argon gas supply—High purity grade (99.99%). When analyses are conducted frequently, liquid argon is more economical and requires less frequent replacement of tanks than compressed argon in conventional cylinders.

6.1.4 A variable speed peristaltic pump is required to deliver both standard and sample solutions to the nebulizer.

6.1.5 (Optional) Mass flow controllers to regulate the argon flow rates, especially the aerosol transport gas, are highly recommended. Their use will provide more exacting control of reproducible plasma conditions.

6.2 Analytical balance, with capability to measure to 0.1 mg, for use in weighing solids, for preparing standards, and for determining dissolved solids in digests or extracts.

6.3 A temperature adjustable hot plate capable of maintaining a temperature of 95 °C.

6.4 (Optional) A temperature adjustable block digester capable of maintaining a temperature of 95 °C and equipped with 250 mL constricted digestion tubes.

6.5 (Optional) A steel cabinet centrifuge with guard bowl, electric timer and brake.

6.6 A gravity convection drying oven with thermostatic control capable of maintaining 180 °C ±5 °C.

6.7 (Optional) An air displacement pipetter capable of delivering volumes ranging from 0.1–2500 µL with an assortment of high quality disposable pipet tips.

6.8 Mortar and pestle, ceramic or nonmetallic material.

6.9 Polypropylene sieve, 5-mesh (4 mm opening).

6.10 Labware—For determination of trace levels of elements, contamination and loss are of prime consideration. Potential contamination sources include improperly cleaned laboratory apparatus and general contamination within the laboratory environment from dust, etc. A clean laboratory work area designated for trace element sample handling must be used. Sample containers can introduce positive and negative errors in the determination of trace elements by contributing contaminants through surface desorption or leaching, or depleting element concentrations through adsorption processes. All reusable labware (glass, quartz, polyethylene, PTFE, FEP, etc.) should be sufficiently clean for the task objectives. Several procedures found to provide clean labware include washing with a detergent solution, rinsing with tap water, soaking for four hours or more in 20% (v/v) nitric acid or a mixture of HNO3 and HCl (1 + 2 + 9), rinsing with reagent water and storing clean.2 3 Chromic acid cleaning solutions must be avoided because chromium is an analyte.

6.10.1 Glassware—Volumetric flasks, graduated cylinders, funnels and centrifuge tubes (glass and/or metal-free plastic).

6.10.2 Assorted calibrated pipettes.

6.10.3 Conical Phillips beakers (Corning 1080–250 or equivalent), 250 mL with 50 mm watch glasses.

6.10.4 Griffin beakers, 250 mL with 75 mm watch glasses and (optional) 75 mm ribbed watch glasses.

6.10.5 (Optional) PTFE and/or quartz Griffin beakers, 250 mL with PTFE covers.

6.10.6 Evaporating dishes or high-form crucibles, porcelain, 100 mL capacity.

6.10.7 Narrow-mouth storage bottles, FEP (fluorinated ethylene propylene) with screw closure, 125 mL to 1 L capacities.

6.10.8 One-piece stem FEP wash bottle with screw closure, 125 mL capacity.

7.0 Reagents and Standards

7.1 Reagents may contain elemental impurities which might affect analytical data. Only high-purity reagents that conform to the American Chemical Society specifications13 should be used whenever possible. If the purity of a reagent is in question, analyze for contamination. All acids used for this method must be of ultra high-purity grade or equivalent. Suitable acids are available from a number of manufacturers. Redistilled acids prepared by sub-boiling distillation are acceptable.

7.2 Hydrochloric acid, concentrated (sp.gr. 1.19)—HCl.

7.2.1 Hydrochloric acid (1 + 1)—Add 500 mL concentrated HCl to 400 mL reagent water and dilute to 1 L.

7.2.2 Hydrochloric acid (1 + 4)—Add 200 mL concentrated HCl to 400 mL reagent water and dilute to 1 L.

7.2.3 Hydrochloric acid (1 + 20)—Add 10 mL concentrated HCl to 200 mL reagent water.

7.3 Nitric acid, concentrated (sp.gr. 1.41)—HNO3.

7.3.1 Nitric acid (1 + 1)—Add 500 mL concentrated HNO3 to 400 mL reagent water and dilute to 1 L.

7.3.2 Nitric acid (1 + 2)—Add 100 mL concentrated HNO3 to 200 mL reagent water.

7.3.3 Nitric acid (1 + 5)—Add 50 mL concentrated HNO3 to 250 mL reagent water.

7.3.4 Nitric acid (1 + 9)—Add 10 mL concentrated HNO3 to 90 mL reagent water.

7.4 Reagent water. All references to water in this method refer to ASTM Type I grade water.14

7.5 Ammonium hydroxide, concentrated (sp.gr. 0.902).

7.6 Tartaric acid, ACS reagent grade.

7.7 Hydrogen peroxide, 50%, stabilized certified reagent grade.

7.8 Standard Stock Solutions—Stock standards may be purchased or prepared from ultra-high purity grade chemicals (99.99–99.999% pure). All compounds must be dried for one hour at 105 °C, unless otherwise specified. It is recommended that stock solutions be stored in FEP bottles. Replace stock standards when succeeding dilutions for preparation of calibration standards cannot be verified.

CAUTION: Many of these chemicals are extremely toxic if inhaled or swallowed (Section 5.1). Wash hands thoroughly after handling.

Typical stock solution preparation procedures follow for 1 L quantities, but for the purpose of pollution prevention, the analyst is encouraged to prepare smaller quantities when possible. Concentrations are calculated based upon the weight of the pure element or upon the weight of the compound multiplied by the fraction of the analyte in the compound

From pure element,

where: gravimetric factor = the weight fraction of the analyte in the compound

7.8.1 Aluminum solution, stock, 1 mL = 1000 µg Al: Dissolve 1.000 g of aluminum metal, weighed accurately to at least four significant figures, in an acid mixture of 4.0 mL of (1 + 1) HCl and 1 mL of concentrated HNO3 in a beaker. Warm beaker slowly to effect solution. When dissolution is complete, transfer solution quantitatively to a 1 L flask, add an additional 10.0 mL of (1 + 1) HCl and dilute to volume with reagent water.

7.8.2 Antimony solution, stock, 1 mL = 1000 µg Sb: Dissolve 1.000 g of antimony powder, weighed accurately to at least four significant figures, in 20.0 mL (1 + 1) HNO3 and 10.0 mL concentrated HCl. Add 100 mL reagent water and 1.50 g tartaric acid. Warm solution slightly to effect complete dissolution. Cool solution and add reagent water to volume in a 1 L volumetric flask.

7.8.3 Arsenic solution, stock, 1 mL = 1000 µg As: Dissolve 1.320 g of As2O3 (As fraction = 0.7574), weighed accurately to at least four significant figures, in 100 mL of reagent water containing 10.0 mL concentrated NH4OH. Warm the solution gently to effect dissolution. Acidify the solution with 20.0 mL concentrated HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.4 Barium solution, stock, 1 mL = 1000 µg Ba: Dissolve 1.437 g BaCO3 (Ba fraction = 0.6960), weighed accurately to at least four significant figures, in 150 mL (1 + 2) HNO3 with heating and stirring to degas and dissolve compound. Let solution cool and dilute with reagent water in 1 L volumetric flask.

7.8.5 Beryllium solution, stock, 1 mL = 1000 µg Be: DO NOT DRY. Dissolve 19.66 g BeSO4•4H2O (Be fraction = 0.0509), weighed accurately to at least four significant figures, in reagent water, add 10.0 mL concentrated HNO3, and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.6 Boron solution, stock, 1 mL = 1000 µg B: DO NOT DRY. Dissolve 5.716 g anhydrous H3BO3 (B fraction = 0.1749), weighed accurately to at least four significant figures, in reagent water and dilute in a 1 L volumetric flask with reagent water. Transfer immediately after mixing to a clean FEP bottle to minimize any leaching of boron from the glass volumetric container. Use of a nonglass volumetric flask is recommended to avoid boron contamination from glassware.

7.8.7 Cadmium solution, stock, 1 mL = 1000 µg Cd: Dissolve 1.000 g Cd metal, acid cleaned with (1 + 9) HNO3, weighed accurately to at least four significant figures, in 50 mL (1 + 1) HNO3 with heating to effect dissolution. Let solution cool and dilute with reagent water in a 1 L volumetric flask.

7.8.8 Calcium solution, stock, 1 mL = 1000 µg Ca: Suspend 2.498 g CaCO3 (Ca fraction = 0.4005), dried at 180 °C for one hour before weighing, weighed accurately to at least four significant figures, in reagent water and dissolve cautiously with a minimum amount of (1 + 1) HNO3. Add 10.0 mL concentrated HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.9 Cerium solution, stock, 1 mL = 1000 µg Ce: Slurry 1.228 g CeO2 (Ce fraction = 0.8141), weighed accurately to at least four significant figures, in 100 mL concentrated HNO3 and evaporate to dryness. Slurry the residue in 20 mL H2O, add 50 mL concentrated HNO3, with heat and stirring add 60 mL 50% H2O2 dropwise in 1 mL increments allowing periods of stirring between the 1 mL additions. Boil off excess H2O2 before diluting to volume in a 1 L volumetric flask with reagent water.

7.8.10 Chromium solution, stock, 1 mL = 1000 µg Cr: Dissolve 1.923 g CrO3 (Cr fraction = 0.5200), weighed accurately to at least four significant figures, in 120 mL (1 + 5) HNO3. When solution is complete, dilute to volume in a 1 L volumetric flask with reagent water.

7.8.11 Cobalt solution, stock, 1 mL = 1000 µg Co: Dissolve 1.000 g Co metal, acid cleaned with (1 + 9) HNO3, weighed accurately to at least four significant figures, in 50.0 mL (1 + 1) HNO3. Let solution cool and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.12 Copper solution, stock, 1 mL = 1000 µg Cu: Dissolve 1.000 g Cu metal, acid cleaned with (1 + 9) HNO3, weighed accurately to at least four significant figures, in 50.0 mL (1 + 1) HNO3 with heating to effect dissolution. Let solution cool and dilute in a 1 L volumetric flask with reagent water.

7.8.13 Iron solution, stock, 1 mL = 1000 µg Fe: Dissolve 1.000 g Fe metal, acid cleaned with (1 + 1) HCl, weighed accurately to four significant figures, in 100 mL (1 + 1) HCl with heating to effect dissolution. Let solution cool and dilute with reagent water in a 1 L volumetric flask.

7.8.14 Lead solution, stock, 1 mL = 1000 µg Pb: Dissolve 1.599 g Pb(NO3)2 (Pb fraction = 0.6256), weighed accurately to at least four significant figures, in a minimum amount of (1 + 1) HNO3. Add 20.0 mL (1 + 1) HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.15 Lithium solution, stock, 1 mL = 1000 µg Li: Dissolve 5.324 g Li2CO3 (Li fraction = 0.1878), weighed accurately to at least four significant figures, in a minimum amount of (1 + 1) HCl and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.16 Magnesium solution, stock, 1 mL = 1000 µg Mg: Dissolve 1.000 g cleanly polished Mg ribbon, accurately weighed to at least four significant figures, in slowly added 5.0 mL (1 + 1) HCl (CAUTION: reaction is vigorous). Add 20.0 mL (1 + 1) HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.17 Manganese solution, stock, 1 mL = 1000 µg Mn: Dissolve 1.000 g of manganese metal, weighed accurately to at least four significant figures, in 50 mL (1 + 1) HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.18 Mercury solution, stock, 1 mL = 1000 µg Hg: DO NOT DRY. CAUTION: highly toxic element. Dissolve 1.354 g HgCl2 (Hg fraction = 0.7388) in reagent water. Add 50.0 mL concentrated HNO3 and dilute to volume in 1 L volumetric flask with reagent water.

7.8.19 Molybdenum solution, stock, 1 mL = 1000 µg Mo: Dissolve 1.500 g MoO3 (Mo fraction = 0.6666), weighed accurately to at least four significant figures, in a mixture of 100 mL reagent water and 10.0 mL concentrated NH4OH, heating to effect dissolution. Let solution cool and dilute with reagent water in a 1 L volumetric flask.

7.8.20 Nickel solution, stock, 1 mL = 1000 µg Ni: Dissolve 1.000 g of nickel metal, weighed accurately to at least four significant figures, in 20.0 mL hot concentrated HNO3, cool, and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.21 Phosphorus solution, stock, 1 mL = 1000 µg P: Dissolve 3.745 g NH4H2PO4 (P fraction = 0.2696), weighed accurately to at least four significant figures, in 200 mL reagent water and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.22 Potassium solution, stock, 1 mL = 1000 µg K: Dissolve 1.907 g KCl (K fraction = 0.5244) dried at 110 °C, weighed accurately to at least four significant figures, in reagent water, add 20 mL (1 + 1) HCl and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.23 Selenium solution, stock, 1 mL = 1000 µg Se: Dissolve 1.405 g SeO2 (Se fraction = 0.7116), weighed accurately to at least four significant figures, in 200 mL reagent water and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.24 Silica solution, stock, 1 mL = 1000 µg SiO2: DO NOT DRY. Dissolve 2.964 g (NH4)2SiF6, weighed accurately to at least four significant figures, in 200 mL (1 + 20) HCl with heating at 85 °C to effect dissolution. Let solution cool and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.25 Silver solution, stock, 1 mL = 1000 µg Ag: Dissolve 1.000 g Ag metal, weighed accurately to at least four significant figures, in 80 mL (1 + 1) HNO3 with heating to effect dissolution. Let solution cool and dilute with reagent water in a 1 L volumetric flask. Store solution in amber bottle or wrap bottle completely with aluminum foil to protect solution from light.

7.8.26 Sodium solution, stock, 1 mL = 1000 µg Na: Dissolve 2.542 g NaCl (Na fraction = 0.3934), weighed accurately to at least four significant figures, in reagent water. Add 10.0 mL concentrated HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.27 Strontium solution, stock, 1 mL = 1000 µg Sr: Dissolve 1.685 g SrCO3 (Sr fraction = 0.5935), weighed accurately to at least four significant figures, in 200 mL reagent water with dropwise addition of 100 mL (1 + 1) HCl. Dilute to volume in a 1 L volumetric flask with reagent water.

7.8.28 Thallium solution, stock, 1 mL = 1000 µg Tl: Dissolve 1.303 g TlNO3 (Tl fraction = 0.7672), weighed accurately to at least four significant figures, in reagent water. Add 10.0 mL concentrated HNO3 and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.29 Tin solution, stock, 1 mL = 1000 µg Sn: Dissolve 1.000 g Sn shot, weighed accurately to at least four significant figures, in an acid mixture of 10.0 mL concentrated HCl and 2.0 mL (1 + 1) HNO3 with heating to effect dissolution. Let solution cool, add 200 mL concentrated HCl, and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.30 Titanium solution, stock, 1 mL = 1000 µg Ti: DO NOT DRY. Dissolve 6.138 g (NH4)2TiO(C2O4)2•H2O (Ti fraction = 0.1629), weighed accurately to at least four significant figures, in 100 mL reagent water. Dilute to volume in a 1 L volumetric flask with reagent water.

7.8.31 Vanadium solution, stock, 1 mL = 1000 µg V: Dissolve 1.000 g V metal, acid cleaned with (1 + 9) HNO3, weighed accurately to at least four significant figures, in 50 mL (1 + 1) HNO3 with heating to effect dissolution. Let solution cool and dilute with reagent water to volume in a 1 L volumetric flask.

7.8.32 Yttrium solution, stock 1 mL = 1000 µg Y: Dissolve 1.270 g Y2O3 (Y fraction = 0.7875), weighed accurately to at least four significant figures, in 50 mL (1 + 1) HNO3, heating to effect dissolution. Cool and dilute to volume in a 1 L volumetric flask with reagent water.

7.8.33 Zinc solution, stock, 1 mL = 1000 µg Zn: Dissolve 1.000 g Zn metal, acid cleaned with (1 + 9) HNO3, weighed accurately to at least four significant figures, in 50 mL (1 + 1) HNO3 with heating to effect dissolution. Let solution cool and dilute with reagent water to volume in a 1 L volumetric flask.

7.9 Mixed Calibration Standard Solutions—For the analysis of total recoverable digested samples prepare mixed calibration standard solutions (see Table 3) by combining appropriate volumes of the stock solutions in 500 mL volumetric flasks containing 20 mL (1 + 1) HNO3 and 20 mL (1 + 1) HCl and dilute to volume with reagent water. Prior to preparing the mixed standards, each stock solution should be analyzed separately to determine possible spectral interferences or the presence of impurities. Care should be taken when preparing the mixed standards to ensure that the elements are compatible and stable together. To minimize the opportunity for contamination by the containers, it is recommended to transfer the mixed-standard solutions to acid-cleaned, never-used FEP fluorocarbon (FEP) bottles for storage. Fresh mixed standards should be prepared, as needed, with the realization that concentrations can change on aging. Calibration standards not prepared from primary standards must be initially verified using a certified reference solution. For the recommended wavelengths listed in Table 1 some typical calibration standard combinations are given in Table 3.

Note: If the addition of silver to the recommended mixed-acid calibration standard results in an initial precipitation, add 15 mL of reagent water and warm the flask until the solution clears. For this acid combination, the silver concentration should be limited to 0.5 mg/L.

7.10 Blanks—Four types of blanks are required for the analysis. The calibration blank is used in establishing the analytical curve, the laboratory reagent blank is used to assess possible contamination from the sample preparation procedure, the laboratory fortified blank is used to assess routine laboratory performance and a rinse blank is used to flush the instrument uptake system and nebulizer between standards, check solutions, and samples to reduce memory interferences.

7.10.1 The calibration blank for aqueous samples and extracts is prepared by acidifying reagent water to the same concentrations of the acids as used for the standards. The calibration blank should be stored in a FEP bottle.

7.10.2 The laboratory reagent blank (LRB) must contain all the reagents in the same volumes as used in the processing of the samples. The LRB must be carried through the same entire preparation scheme as the samples including sample digestion, when applicable.

7.10.3 The laboratory fortified blank (LFB) is prepared by fortifying an aliquot of the laboratory reagent blank with all analytes to a suitable concentration using the following recommended criteria: Ag 0.1 mg/L, K 5.0 mg/L and all other analytes 0.2 mg/L or a concentration approximately 100 times their respective MDL, whichever is greater. The LFB must be carried through the same entire preparation scheme as the samples including sample digestion, when applicable.

7.10.4 The rinse blank is prepared by acidifying reagent water to the same concentrations of acids as used in the calibration blank and stored in a convenient manner.

7.11 Instrument Performance Check (IPC) Solution—The IPC solution is used to periodically verify instrument performance during analysis. It should be prepared in the same acid mixture as the calibration standards by combining method analytes at appropriate concentrations. Silver must be limited to <0.5 mg/L; while potassium and phosphorus because of higher MDLs and silica because of potential contamination should be at concentrations of 10 mg/L. For other analytes a concentration of 2 mg/L is recommended. The IPC solution should be prepared from the same standard stock solutions used to prepare the calibration standards and stored in an FEP bottle. Agency programs may specify or request that additional instrument performance check solutions be prepared at specified concentrations in order to meet particular program needs.

7.12 Quality Control Sample (QCS)—Analysis of a QCS is required for initial and periodic verification of calibration standards or stock standard solutions in order to verify instrument performance. The QCS must be obtained from an outside source different from the standard stock solutions and prepared in the same acid mixture as the calibration standards. The concentration of the analytes in the QCS solution should be 1 mg/L, except silver, which must be limited to a concentration of 0.5 mg/L for solution stability. The QCS solution should be stored in a FEP bottle and analyzed as needed to meet data-quality needs. A fresh solution should be prepared quarterly or more frequently as needed.

7.13 Spectral Interference Check (SIC) Solutions—When interelement corrections are applied, SIC solutions are needed containing concentrations of the interfering elements at levels that will provide an adequate test of the correction factors.

7.13.1 SIC solutions containing (a) 300 mg/L Fe; (b) 200 mg/L AL; (c) 50 mg/L Ba; (d) 50 mg/L Be; (e) 50 mg/L Cd; (f) 50 mg/L Ce; (g) 50 mg/L Co; (h) 50 mg/L Cr; (i) 50 mg/L Cu; (j) 50 mg/L Mn; (k) 50 mg/L Mo; (l) 50 mg/L Ni; (m) 50 mg/L Sn; (n) 50 mg/L SiO2; (o) 50 mg/L Ti; (p) 50 mg/L Tl and (q) 50 mg/L V should be prepared in the same acid mixture as the calibration standards and stored in FEP bottles. These solutions can be used to periodically verify a partial list of the on-line (and possible off-line) interelement spectral correction factors for the recommended wavelengths given in Table 1. Other solutions could achieve the same objective as well. (Multielement SIC solutions3 may be prepared and substituted for the single element solutions provided an analyte is not subject to interference from more than one interferant in the solution.)

Note: If wavelengths other than those recommended in Table 1 are used, other solutions different from those above (a through q) may be required.

7.13.2 For interferences from iron and aluminum, only those correction factors (positive or negative) when multiplied by 100 to calculate apparent analyte concentrations that exceed the determined analyte IDL or fall below the lower 3-sigma control limit of the calibration blank need be tested on a daily basis.

7.13.3 For the other interfering elements, only those correction factors (positive or negative) when multiplied by 10 to calculate apparent analyte concentrations that exceed the determined analyte IDL or fall below the lower 3-sigma control limit of the calibration blank need be tested on a daily basis.

7.13.4 If the correction routine is operating properly, the determined apparent analyte(s) concentration from analysis of each interference solution (a through q) should fall within a specific concentration range bracketing the calibration blank. This concentration range is calculated by multiplying the concentration of the interfering element by the value of the correction factor being tested and dividing by 10. If after subtraction of the calibration blank the apparent analyte concentration is outside (above or below) this range, a change in the correction factor of more than 10% should be suspected. The cause of the change should be determined and corrected and the correction factor should be updated.

Note: The SIC solution should be analyzed more than once to confirm a change has occurred with adequate rinse time between solutions and before subsequent analysis of the calibration blank.

7.13.5 If the correction factors tested on a daily basis are found to be within the 10% criteria for five consecutive days, the required verification frequency of those factors in compliance may be extended to a weekly basis. Also, if the nature of the samples analyzed is such (e.g., finished drinking water) that they do not contain concentrations of the interfering elements at the 10 mg/L level, daily verification is not required; however, all interelement spectral correction factors must be verified annually and updated, if necessary.

7.13.6 If the instrument does not display negative concentration values, fortify the SIC solutions with the elements of interest at 1 mg/L and test for analyte recoveries that are below 95%. In the absence of measurable analyte, over-correction could go undetected because a negative value could be reported as zero.

7.14 For instruments without interelement correction capability or when interelement corrections are not used, SIC solutions (containing similar concentrations of the major components in the samples, e.g., 10 mg/L) can serve to verify the absence of effects at the wavelengths selected. These data must be kept on file with the sample analysis data. If the SIC solution confirms an operative interference that is 10% of the analyte concentration, the analyte must be determined using a wavelength and background correction location free of the interference or by another approved test procedure. Users are advised that high salt concentrations can cause analyte signal suppressions and confuse interference tests.

7.15 Plasma Solution—The plasma solution is used for determining the optimum viewing height of the plasma above the work coil prior to using the method (Section 10.2). The solution is prepared by adding a 5 mL aliquot from each of the stock standard solutions of arsenic, lead, selenium, and thallium to a mixture of 20 mL (1 + 1) nitric acid and 20 mL (1 + 1) hydrochloric acid and diluting to 500 mL with reagent water. Store in a FEP bottle.

8.0 Sample Collection, Preservation, and Storage

8.1 Prior to the collection of an aqueous sample, consideration should be given to the type of data required, (i.e., dissolved or total recoverable), so that appropriate preservation and pretreatment steps can be taken. The pH of all aqueous samples must be tested immediately prior to aliquoting for processing or “direct analysis” to ensure the sample has been properly preserved. If properly acid preserved, the sample can be held up to six months before analysis.

8.2 For the determination of the dissolved elements, the sample must be filtered through a 0.45 µm pore diameter membrane filter at the time of collection or as soon thereafter as practically possible. (Glass or plastic filtering apparatus are recommended to avoid possible contamination. Only plastic apparatus should be used when the determinations of boron and silica are critical.) Use a portion of the filtered sample to rinse the filter flask, discard this portion and collect the required volume of filtrate. Acidify the filtrate with (1 + 1) nitric acid immediately following filtration to pH <2.

8.3 For the determination of total recoverable elements in aqueous samples, samples are not filtered, but acidified with (1 + 1) nitric acid to pH <2 (normally, 3 mL of (1 + 1) acid per liter of sample is sufficient for most ambient and drinking water samples). Preservation may be done at the time of collection, however, to avoid the hazards of strong acids in the field, transport restrictions, and possible contamination it is recommended that the samples be returned to the laboratory within two weeks of collection and acid preserved upon receipt in the laboratory. Following acidification, the sample should be mixed, held for 16 hours, and then verified to be pH <2 just prior withdrawing an aliquot for processing or “direct analysis”. If for some reason such as high alkalinity the sample pH is verified to be >2, more acid must be added and the sample held for 16 hours until verified to be pH <2. See Section 8.1.

Note: When the nature of the sample is either unknown or is known to be hazardous, acidification should be done in a fume hood. See Section 5.2.

8.4 Solid samples require no preservation prior to analysis other than storage at 4 °C. There is no established holding time limitation for solid samples.

8.5 For aqueous samples, a field blank should be prepared and analyzed as required by the data user. Use the same container and acid as used in sample collection.

9.0 Quality Control

9.1 Each laboratory using this method is required to operate a formal quality control (QC) program. The minimum requirements of this program consist of an initial demonstration of laboratory capability, and the periodic analysis of laboratory reagent blanks, fortified blanks and other laboratory solutions as a continuing check on performance. The laboratory is required to maintain performance records that define the quality of the data thus generated.

9.2 Initial Demonstration of Performance (mandatory).

9.2.1 The initial demonstration of performance is used to characterize instrument performance (determination of linear dynamic ranges and analysis of quality control samples) and laboratory performance (determination of method detection limits) prior to analyses conducted by this method.

9.2.2 Linear dynamic range (LDR)—The upper limit of the LDR must be established for each wavelength utilized. It must be determined from a linear calibration prepared in the normal manner using the established analytical operating procedure for the instrument. The LDR should be determined by analyzing succeedingly higher standard concentrations of the analyte until the observed analyte concentration is no more than 10% below the stated concentration of the standard. Determined LDRs must be documented and kept on file. The LDR which may be used for the analysis of samples should be judged by the analyst from the resulting data. Determined sample analyte concentrations that are greater than 90% of the determined upper LDR limit must be diluted and reanalyzed. The LDRs should be verified annually or whenever, in the judgment of the analyst, a change in analytical performance caused by either a change in instrument hardware or operating conditions would dictate they be redetermined.

9.2.3 Quality control sample (QCS)—When beginning the use of this method, on a quarterly basis, after the preparation of stock or calibration standard solutions or as required to meet data-quality needs, verify the calibration standards and acceptable instrument performance with the preparation and analyses of a QCS (Section 7.12). To verify the calibration standards the determined mean concentrations from three analyses of the QCS must be within 5% of the stated values. If the calibration standard cannot be verified, performance of the determinative step of the method is unacceptable. The source of the problem must be identified and corrected before either proceeding on with the initial determination of method detection limits or continuing with on-going analyses.

9.2.4 Method detection limit (MDL)—MDLs must be established for all wavelengths utilized, using reagent water (blank) fortified at a concentration of two to three times the estimated instrument detection limit.15 To determine MDL values, take seven replicate aliquots of the fortified reagent water and process through the entire analytical method. Perform all calculations defined in the method and report the concentration values in the appropriate units. Calculate the MDL as follows:

MDL = (t) × (S)

where:

t = students' t value for a 99% confidence level and a standard deviation estimate with n-1 degrees of freedom [t = 3.14 for seven replicates]

S = standard deviation of the replicate analyses

Note: If additional confirmation is desired, reanalyze the seven replicate aliquots on two more nonconsecutive days and again calculate the MDL values for each day. An average of the three MDL values for each analyte may provide for a more appropriate MDL estimate. If the relative standard deviation (RSD) from the analyses of the seven aliquots is <10%, the concentration used to determine the analyte MDL may have been inappropriately high for the determination. If so, this could result in the calculation of an unrealistically low MDL. Concurrently, determination of MDL in reagent water represents a best case situation and does not reflect possible matrix effects of real world samples. However, successful analyses of LFMs (Section 9.4) and the analyte addition test described in Section 9.5.1 can give confidence to the MDL value determined in reagent water. Typical single laboratory MDL values using this method are given in Table 4.

The MDLs must be sufficient to detect analytes at the required levels according to compliance monitoring regulation (Section 1.2). MDLs should be determined annually, when a new operator begins work or whenever, in the judgment of the analyst, a change in analytical performance caused by either a change in instrument hardware or operating conditions would dictate they be redetermined.

9.3 Assessing Laboratory Performance (mandatory)

9.3.1 Laboratory reagent blank (LRB)—The laboratory must analyze at least one LRB (Section 7.10.2) with each batch of 20 or fewer samples of the same matrix. LRB data are used to assess contamination from the laboratory environment. LRB values that exceed the MDL indicate laboratory or reagent contamination should be suspected. When LRB values constitute 10% or more of the analyte level determined for a sample or is 2.2 times the analyte MDL whichever is greater, fresh aliquots of the samples must be prepared and analyzed again for the affected analytes after the source of contamination has been corrected and acceptable LRB values have been obtained.

9.3.2 Laboratory fortified blank (LFB)—The laboratory must analyze at least one LFB (Section 7.10.3) with each batch of samples. Calculate accuracy as percent recovery using the following equation:

where:

R = percent recovery

LFB = laboratory fortified blank

LRB = laboratory reagent blank

s = concentration equivalent of analyte added to fortify the LBR solution

If the recovery of any analyte falls outside the required control limits of 85–115%, that analyte is judged out of control, and the source of the problem should be identified and resolved before continuing analyses.

9.3.3 The laboratory must use LFB analyses data to assess laboratory performance against the required control limits of 85–115% (Section 9.3.2). When sufficient internal performance data become available (usually a minimum of 20–30 analyses), optional control limits can be developed from the mean percent recovery (x) and the standard deviation (S) of the mean percent recovery. These data can be used to establish the upper and lower control limits as follows:

UPPER CONTROL LIMIT = x + 3S

LOWER CONTROL LIMIT = x − 3S

The optional control limits must be equal to or better than the required control limits of 85–115%. After each five to 10 new recovery measurements, new control limits can be calculated using only the most recent 20–30 data points. Also, the standard deviation (S) data should be used to establish an on-going precision statement for the level of concentrations included in the LFB. These data must be kept on file and be available for review.

9.3.4 Instrument performance check (IPC) solution—For all determinations the laboratory must analyze the IPC solution (Section 7.11) and a calibration blank immediately following daily calibration, after every 10th sample (or more frequently, if required) and at the end of the sample run. Analysis of the calibration blank should always be <the analyte IDL, but greater than the lower 3-sigma control limit of the calibration blank. Analysis of the IPC solution immediately following calibration must verify that the instrument is within 5% of calibration with a relative standard deviation <3% from replicate integrations 4. Subsequent analyses of the IPC solution must be within 10% of calibration. If the calibration cannot be verified within the specified limits, reanalyze either or both the IPC solution and the calibration blank. If the second analysis of the IPC solution or the calibration blank confirm calibration to be outside the limits, sample analysis must be discontinued, the cause determined, corrected and/or the instrument recalibrated. All samples following the last acceptable IPC solution must be reanalyzed. The analysis data of the calibration blank and IPC solution must be kept on file with the sample analyses data.

9.3.5 Spectral interference check (SIC) solution—For all determinations the laboratory must periodically verify the interelement spectral interference correction routine by analyzing SIC solutions. The preparation and required periodic analysis of SIC solutions and test criteria for verifying the interelement interference correction routine are given in Section 7.13. Special cases where on-going verification is required are described in Section 7.14.

9.4 Assessing Analyte Recovery and Data Quality.

9.4.1 Sample homogeneity and the chemical nature of the sample matrix can affect analyte recovery and the quality of the data. Taking separate aliquots from the sample for replicate and fortified analyses can in some cases assess the effect. Unless otherwise specified by the data user, laboratory or program, the following laboratory fortified matrix (LFM) procedure (Section 9.4.2) is required. Also, other tests such as the analyte addition test (Section 9.5.1) and sample dilution test (Section 9.5.2) can indicate if matrix effects are operative.

9.4.2 The laboratory must add a known amount of each analyte to a minimum of 10% of the routine samples. In each case the LFM aliquot must be a duplicate of the aliquot used for sample analysis and for total recoverable determinations added prior to sample preparation. For water samples, the added analyte concentration must be the same as that used in the laboratory fortified blank (Section 7.10.3). For solid samples, however, the concentration added should be expressed as mg/kg and is calculated for a one gram aliquot by multiplying the added analyte concentration (mg/L) in solution by the conversion factor 100 (mg/L × 0.1L/0.001kg = 100, Section 12.5). (For notes on Ag, Ba, and Sn see Sections 1.7 and 1.8.) Over time, samples from all routine sample sources should be fortified.

Note: The concentration of calcium, magnesium, sodium and strontium in environmental waters, along with iron and aluminum in solids can vary greatly and are not necessarily predictable. Fortifying these analytes in routine samples at the same concentration used for the LFB may prove to be of little use in assessing data quality for these analytes. For these analytes sample dilution and reanalysis using the criteria given in Section 9.5.2 is recommended. Also, if specified by the data user, laboratory or program, samples can be fortified at higher concentrations, but even major constituents should be limited to <25 mg/L so as not to alter the sample matrix and affect the analysis.

9.4.3 Calculate the percent recovery for each analyte, corrected for background concentrations measured in the unfortified sample, and compare these values to the designated LFM recovery range of 70–130% or a 3-sigma recovery range calculated from the regression equations given in Table 9.16 Recovery calculations are not required if the concentration added is less than 30% of the sample background concentration. Percent recovery may be calculated in units appropriate to the matrix, using the following equation:

where:

R = percent recovery

Cs = fortified sample concentration

C = sample background concentration

s = concentration equivalent of analyte added to fortify the sample

9.4.4 If the recovery of any analyte falls outside the designated LFM recovery range, and the laboratory performance for that analyte is shown to be in control (Section 9.3), the recovery problem encountered with the fortified sample is judged to be matrix related, not system related. The data user should be informed that the result for that analyte in the unfortified sample is suspect due to either the heterogeneous nature of the sample or matrix effects and analysis by method of standard addition or the use of an internal standard(s) (Section 11.5) should be considered.

9.4.5 Where reference materials are available, they should be analyzed to provide additional performance data. The analysis of reference samples is a valuable tool for demonstrating the ability to perform the method acceptably. Reference materials containing high concentrations of analytes can provide additional information on the performance of the spectral interference correction routine.

9.5 Assess the possible need for the method of standard additions (MSA) or internal standard elements by the following tests. Directions for using MSA or internal standard(s) are given in Section 11.5.

9.5.1 Analyte addition test: An analyte(s) standard added to a portion of a prepared sample, or its dilution, should be recovered to within 85% to 115% of the known value. The analyte(s) addition should produce a minimum level of 20 times and a maximum of 100 times the method detection limit. If the analyte addition is <20% of the sample analyte concentration, the following dilution test should be used. If recovery of the analyte(s) is not within the specified limits, a matrix effect should be suspected, and the associated data flagged accordingly. The method of additions or the use of an appropriate internal standard element may provide more accurate data.

9.5.2 Dilution test: If the analyte concentration is sufficiently high (minimally, a factor of 50 above the instrument detection limit in the original solution but <90% of the linear limit), an analysis of a 1 + 4 dilution should agree (after correction for the fivefold dilution) within 10% of the original determination. If not, a chemical or physical interference effect should be suspected and the associated data flagged accordingly. The method of standard additions or the use of an internal-standard element may provide more accurate data for samples failing this test.

10.0 Calibration and Standardization

10.1 Specific wavelengths are listed in Table 1. Other wavelengths may be substituted if they can provide the needed sensitivity and are corrected for spectral interference. However, because of the difference among various makes and models of spectrometers, specific instrument operating conditions cannot be given. The instrument and operating conditions utilized for determination must be capable of providing data of acceptable quality to the program and data user. The analyst should follow the instructions provided by the instrument manufacturer unless other conditions provide similar or better performance for a task. Operating conditions for aqueous solutions usually vary from 1100–1200 watts forward power, 15–16 mm viewing height, 15–19 L/min. argon coolant flow, 0.6–1 L/min. argon aerosol flow, 1–1.8 mL/min. sample pumping rate with a one minute preflush time and measurement time near 1 s per wavelength peak (for sequential instruments) and near 10 s per sample (for simultaneous instruments). Use of the Cu/Mn intensity ratio at 324.754 nm and 257.610 nm (by adjusting the argon aerosol flow) has been recommended as a way to achieve repeatable interference correction factors.17

10.2 Prior to using this method optimize the plasma operating conditions. The following procedure is recommended for vertically configured plasmas. The purpose of plasma optimization is to provide a maximum signal-to-background ratio for the least sensitive element in the analytical array. The use of a mass flow controller to regulate the nebulizer gas flow rate greatly facilitates the procedure.

10.2.1 Ignite the plasma and select an appropriate incident rf power with minimum reflected power. Allow the instrument to become thermally stable before beginning. This usually requires at least 30 to 60 minutes of operation. While aspirating the 1000 µg/mL solution of yttrium (Section 7.8.32), follow the instrument manufacturer's instructions and adjust the aerosol carrier gas flow rate through the nebulizer so a definitive blue emission region of the plasma extends approximately from 5–20 mm above the top of the work coil.18 Record the nebulizer gas flow rate or pressure setting for future reference.

10.2.2 After establishing the nebulizer gas flow rate, determine the solution uptake rate of the nebulizer in mL/min. by aspirating a known volume calibration blank for a period of at least three minutes. Divide the spent volume by the aspiration time (in minutes) and record the uptake rate. Set the peristaltic pump to deliver the uptake rate in a steady even flow.

10.2.3 After horizontally aligning the plasma and/or optically profiling the spectrometer, use the selected instrument conditions from Sections 10.2.1 and 10.2.2, and aspirate the plasma solution (Section 7.15), containing 10 µg/mL each of As, Pb, Se and Tl. Collect intensity data at the wavelength peak for each analyte at 1 mm intervals from 14–18 mm above the top of the work coil. (This region of the plasma is commonly referred to as the analytical zone.)19 Repeat the process using the calibration blank. Determine the net signal to blank intensity ratio for each analyte for each viewing height setting. Choose the height for viewing the plasma that provides the largest intensity ratio for the least sensitive element of the four analytes. If more than one position provides the same ratio, select the position that provides the highest net intensity counts for the least sensitive element or accept a compromise position of the intensity ratios of all four analytes.

10.2.4 The instrument operating condition finally selected as being optimum should provide the lowest reliable instrument detection limits and method detection limits. Refer to Tables 1 and 4 for comparison of IDLs and MDLs, respectively.

10.2.5 If either the instrument operating conditions, such as incident power and/or nebulizer gas flow rate are changed, or a new torch injector tube having a different orifice i.d. is installed, the plasma and plasma viewing height should be reoptimized.

10.2.6 Before daily calibration and after the instrument warmup period, the nebulizer gas flow must be reset to the determined optimized flow. If a mass flow controller is being used, it should be reset to the recorded optimized flow rate. In order to maintain valid spectral interelement correction routines the nebulizer gas flow rate should be the same from day-to-day (<2% change). The change in signal intensity with a change in nebulizer gas flow rate for both “hard” (Pb 220.353 nm) and “soft” (Cu 324.754) lines is illustrated in Figure 1.

10.3 Before using the procedure (Section 11.0) to analyze samples, there must be data available documenting initial demonstration of performance. The required data and procedure is described in Section 9.2. This data must be generated using the same instrument operating conditions and calibration routine (Section 11.4) to be used for sample analysis. These documented data must be kept on file and be available for review by the data user.

10.4 After completing the initial demonstration of performance, but before analyzing samples, the laboratory must establish and initially verify an interelement spectral interference correction routine to be used during sample analysis. A general description concerning spectral interference and the analytical requirements for background correction and for correction of interelement spectral interference in particular are given in Section 4.1. To determine the appropriate location for background correction and to establish the interelement interference correction routine, repeated spectral scan about the analyte wavelength and repeated analyses of the single element solutions may be required. Criteria for determining an interelement spectral interference is an apparent positive or negative concentration on the analyte that is outside the 3-sigma control limits of the calibration blank for the analyte. (The upper-control limit is the analyte IDL.) Once established, the entire routine must be initially and periodically verified annually, or whenever there is a change in instrument operating conditions (Section 10.2.5). Only a portion of the correction routine must be verified more frequently or on a daily basis. Test criteria and required solutions are described in Section 7.13. Initial and periodic verification data of the routine should be kept on file. Special cases where on-going verification are required is described in Section 7.14.

11.0 Procedure

11.1 Aqueous Sample Preparation—Dissolved Analytes

11.1.1 For the determination of dissolved analytes in ground and surface waters, pipet an aliquot (20 mL) of the filtered, acid preserved sample into a 50 mL polypropylene centrifuge tube. Add an appropriate volume of (1 + 1) nitric acid to adjust the acid concentration of the aliquot to approximate a 1% (v/v) nitric acid solution (e.g., add 0.4 mL (1 + 1) HNO3 to a 20 mL aliquot of sample). Cap the tube and mix. The sample is now ready for analysis (Section 1.3). Allowance for sample dilution should be made in the calculations. (If mercury is to be determined, a separate aliquot must be additionally acidified to contain 1% (v/v) HCl to match the signal response of mercury in the calibration standard and reduce memory interference effects. Section 1.9).

Note:

If a precipitate is formed during acidification, transport, or storage, the sample aliquot must be treated using the procedure described in Sections 11.2.2 through 11.2.7 prior to analysis.

11.2 Aqueous Sample Preparation—Total Recoverable Analytes

11.2.1 For the “direct analysis” of total recoverable analytes in drinking water samples containing turbidity <1 NTU, treat an unfiltered acid preserved sample aliquot using the sample preparation procedure described in Section 11.1.1 while making allowance for sample dilution in the data calculation (Section 1.2). For the determination of total recoverable analytes in all other aqueous samples or for preconcentrating drinking water samples prior to analysis follow the procedure given in Sections 11.2.2 through 11.2.7.

11.2.2 For the determination of total recoverable analytes in aqueous samples (other than drinking water with <1 NTU turbidity), transfer a 100 mL (1 mL) aliquot from a well mixed, acid preserved sample to a 250 mL Griffin beaker (Sections 1.2, 1.3, 1.6, 1.7, 1.8, and 1.9). (When necessary, smaller sample aliquot volumes may be used.)

Note:

If the sample contains undissolved solids >1%, a well mixed, acid preserved aliquot containing no more than 1 g particulate material should be cautiously evaporated to near 10 mL and extracted using the acid-mixture procedure described in Sections 11.3.3 through 11.3.6.

11.2.3 Add 2 mL (1 + 1) nitric acid and 1.0 mL of (1 + 1) hydrochloric acid to the beaker containing the measured volume of sample. Place the beaker on the hot plate for solution evaporation. The hot plate should be located in a fume hood and previously adjusted to provide evaporation at a temperature of approximately but no higher than 85 °C. (See the following note.) The beaker should be covered with an elevated watch glass or other necessary steps should be taken to prevent sample contamination from the fume hood environment.

Note:

For proper heating adjust the temperature control of the hot plate such that an uncovered Griffin beaker containing 50 mL of water placed in the center of the hot plate can be maintained at a temperature approximately but no higher than 85 °C. (Once the beaker is covered with a watch glass the temperature of the water will rise to approximately 95 °C.)

11.2.4 Reduce the volume of the sample aliquot to about 20 mL by gentle heating at 85 °C. DO NOT BOIL. This step takes about two hours for a 100 mL aliquot with the rate of evaporation rapidly increasing as the sample volume approaches 20 mL. (A spare beaker containing 20 mL of water can be used as a gauge.)

11.2.5 Cover the lip of the beaker with a watch glass to reduce additional evaporation and gently reflux the sample for 30 minutes. (Slight boiling may occur, but vigorous boiling must be avoided to prevent loss of the HCl-H2O azeotrope.)

11.2.6 Allow the beaker to cool. Quantitatively transfer the sample solution to a 50 mL volumetric flask, make to volume with reagent water, stopper and mix.

11.2.7 Allow any undissolved material to settle overnight, or centrifuge a portion of the prepared sample until clear. (If after centrifuging or standing overnight the sample contains suspended solids that would clog the nebulizer, a portion of the sample may be filtered for their removal prior to analysis. However, care should be exercised to avoid potential contamination from filtration.) The sample is now ready for analysis. Because the effects of various matrices on the stability of diluted samples cannot be characterized, all analyses should be performed as soon as possible after the completed preparation.

11.3 Solid Sample Preparation—Total Recoverable Analytes

11.3.1 For the determination of total recoverable analytes in solid samples, mix the sample thoroughly and transfer a portion (>20 g) to tared weighing dish, weigh the sample and record the wet weight (WW). (For samples with <35% moisture a 20 g portion is sufficient. For samples with moisture >35% a larger aliquot 50–100 g is required.) Dry the sample to a constant weight at 60 °C and record the dry weight (DW) for calculation of percent solids (Section 12.6). (The sample is dried at 60 °C to prevent the loss of mercury and other possible volatile metallic compounds, to facilitate sieving, and to ready the sample for grinding.)

11.3.2 To achieve homogeneity, sieve the dried sample using a 5-mesh polypropylene sieve and grind in a mortar and pestle. (The sieve, mortar and pestle should be cleaned between samples.) From the dried, ground material weigh accurately a representative 1.0 ±0.01 g aliquot (W) of the sample and transfer to a 250 mL Phillips beaker for acid extraction (Sections 1.6, 1.7, 1.8, and 1.9).

11.3.3 To the beaker add 4 mL of (1 + 1) HNO3 and 10 mL of (1 + 4) HCl. Cover the lip of the beaker with a watch glass. Place the beaker on a hot plate for reflux extraction of the analytes. The hot plate should be located in a fume hood and previously adjusted to provide a reflux temperature of approximately 95 °C. (See the following note.)

Note:

For proper heating adjust the temperature control of the hot plate such that an uncovered Griffin beaker containing 50 mL of water placed in the center of the hot plate can be maintained at a temperature approximately but no higher than 85 °C. (Once the beaker is covered with a watch glass the temperature of the water will rise to approximately 95 °C.) Also, a block digester capable of maintaining a temperature of 95 °C and equipped with 250 mL constricted volumetric digestion tubes may be substituted for the hot plate and conical beakers in the extraction step.

11.3.4 Heat the sample and gently reflux for 30 minutes. Very slight boiling may occur, however vigorous boiling must be avoided to prevent loss of the HCl-H2O azeotrope. Some solution evaporation will occur (3–4 mL).

11.3.5 Allow the sample to cool and quantitatively transfer the extract to a 100 mL volumetric flask. Dilute to volume with reagent water, stopper and mix.

11.3.6 Allow the sample extract solution to stand overnight to separate insoluble material or centrifuge a portion of the sample solution until clear. (If after centrifuging or standing overnight the extract solution contains suspended solids that would clog the nebulizer, a portion of the extract solution may be filtered for their removal prior to analysis. However, care should be exercised to avoid potential contamination from filtration.) The sample extract is now ready for analysis. Because the effects of various matrices on the stability of diluted samples cannot be characterized, all analyses should be performed as soon as possible after the completed preparation.

11.4 Sample Analysis

11.4.1 Prior to daily calibration of the instrument inspect the sample introduction system including the nebulizer, torch, injector tube and uptake tubing for salt deposits, dirt and debris that would restrict solution flow and affect instrument performance. Clean the system when needed or on a daily basis.

11.4.2 Configure the instrument system to the selected power and operating conditions as determined in Sections 10.1 and 10.2.

11.4.3 The instrument must be allowed to become thermally stable before calibration and analyses. This usually requires at least 30 to 60 minutes of operation. After instrument warmup, complete any required optical profiling or alignment particular to the instrument.

11.4.4 For initial and daily operation calibrate the instrument according to the instrument manufacturer's recommended procedures, using mixed calibration standard solutions (Section 7.9) and the calibration blank (Section 7.10.1). A peristaltic pump must be used to introduce all solutions to the nebulizer. To allow equilibrium to be reached in the plasma, aspirate all solutions for 30 seconds after reaching the plasma before beginning integration of the background corrected signal to accumulate data. When possible, use the average value of replicate integration periods of the signal to be correlated to the analyte concentration. Flush the system with the rinse blank (Section 7.10.4) for a minimum of 60 seconds (Section 4.4) between each standard. The calibration line should consist of a minimum of a calibration blank and a high standard. Replicates of the blank and highest standard provide an optimal distribution of calibration standards to minimize the confidence band for a straight-line calibration in a response region with uniform variance.20

11.4.5 After completion of the initial requirements of this method (Sections 10.3 and 10.4), samples should be analyzed in the same operational manner used in the calibration routine with the rinse blank also being used between all sample solutions, LFBs, LFMs, and check solutions (Section 7.10.4).

11.4.6 During the analysis of samples, the laboratory must comply with the required quality control described in Sections 9.3 and 9.4. Only for the determination of dissolved analytes or the “direct analysis” of drinking water with turbidity of <1 NTU is the sample digestion step of the LRB, LFB, and LFM not required.

11.4.7 Determined sample analyte concentrations that are 90% or more of the upper limit of the analyte LDR must be diluted with reagent water that has been acidified in the same manner as calibration blank and reanalyzed (see Section 11.4.8). Also, for the interelement spectral interference correction routines to remain valid during sample analysis, the interferant concentration must not exceed its LDR. If the interferant LDR is exceeded, sample dilution with acidified reagent water and reanalysis is required. In these circumstances analyte detection limits are raised and determination by another approved test procedure that is either more sensitive and/or interference free is recommended.

11.4.8 When it is necessary to assess an operative matrix interference (e.g., signal reduction due to high dissolved solids), the tests described in Section 9.5 are recommended.

11.4.9 Report data as directed in Section 12.0.

11.5 If the method of standard additions (MSA) is used, standards are added at one or more levels to portions of a prepared sample. This technique21 compensates for enhancement or depression of an analyte signal by a matrix. It will not correct for additive interferences such as contamination, interelement interferences, or baseline shifts. This technique is valid in the linear range when the interference effect is constant over the range, the added analyte responds the same as the endogenous analyte, and the signal is corrected for additive interferences. The simplest version of this technique is the single-addition method. This procedure calls for two identical aliquots of the sample solution to be taken. To the first aliquot, a small volume of standard is added; while to the second aliquot, a volume of acid blank is added equal to the standard addition. The sample concentration is calculated by the following:

where:

C = Concentration of the standard solution (mg/L)

S1 = Signal for fortified aliquot

S2 = Signal for unfortified aliquot

V1 = Volume of the standard addition (L)

V2 = Volume of the sample aliquot (L) used for MSA

For more than one fortified portion of the prepared sample, linear regression analysis can be applied using a computer or calculator program to obtain the concentration of the sample solution. An alternative to using the method of standard additions is use of the internal standard technique by adding one or more elements (not in the samples and verified not to cause an uncorrected interelement spectral interference) at the same concentration (which is sufficient for optimum precision) to the prepared samples (blanks and standards) that are affected the same as the analytes by the sample matrix. Use the ratio of analyte signal to the internal standard signal for calibration and quantitation.

12.0 Data Analysis and Calculations

12.1 Sample data should be reported in units of mg/L for aqueous samples and mg/kg dry weight for solid samples.

12.2 For dissolved aqueous analytes (Section 11.1) report the data generated directly from the instrument with allowance for sample dilution. Do not report analyte concentrations below the IDL.

12.3 For total recoverable aqueous analytes (Section 11.2), multiply solution analyte concentrations by the dilution factor 0.5, when 100 mL aliquot is used to produce the 50 mL final solution, and report data as instructed in Section 12.4. If a different aliquot volume other than 100 mL is used for sample preparation, adjust the dilution factor accordingly. Also, account for any additional dilution of the prepared sample solution needed to complete the determination of analytes exceeding 90% or more of the LDR upper limit. Do not report data below the determined analyte MDL concentration or below an adjusted detection limit reflecting smaller sample aliquots used in processing or additional dilutions required to complete the analysis.

12.4 For analytes with MDLs <0.01 mg/L, round the data values to the thousandth place and report analyte concentrations up to three significant figures. For analytes with MDLs <0.01 mg/L round the data values to the 100th place and report analyte concentrations up to three significant figures. Extract concentrations for solids data should be rounded in a similar manner before calculations in Section 12.5 are performed.

12.5 For total recoverable analytes in solid samples (Section 11.3), round the solution analyte concentrations (mg/L) as instructed in Section 12.4. Report the data up to three significant figures as mg/kg dry-weight basis unless specified otherwise by the program or data user. Calculate the concentration using the equation below:

where:

C = Concentration in extract (mg/L)

V = Volume of extract (L, 100 mL = 0.1L)

D = Dilution factor (undiluted = 1)

W = Weight of sample aliquot extracted (g × 0.001 = kg)

Do not report analyte data below the estimated solids MDL or an adjusted MDL because of additional dilutions required to complete the analysis.

12.6 To report percent solids in solid samples (Section 11.3) calculate as follows:

where:

DW = Sample weight (g) dried at 60 ºC

WW = Sample weight (g) before drying

Note: If the data user, program or laboratory requires that the reported percent solids be determined by drying at 105 °C, repeat the procedure given in Section 11.3 using a separate portion (>20 g) of the sample and dry to constant weight at 103–105 °C.

12.7 The QC data obtained during the analyses provide an indication of the quality of the sample data and should be provided with the sample results.

13.0 Method Performance

13.1 Listed in Table 4 are typical single laboratory total recoverable MDLs determined for the recommended wavelengths using simultaneous ICP–AES and the operating conditions given in Table 5. The MDLs were determined in reagent blank matrix (best case situation). PTFE beakers were used to avoid boron and silica contamination from glassware with the final dilution to 50 mL completed in polypropylene centrifuged tubes. The listed MDLs for solids are estimates and were calculated from the aqueous MDL determinations.

13.2 Data obtained from single laboratory method testing are summarized in Table 6 for five types of water samples consisting of drinking water, surface water, ground water, and two wastewater effluents. The data presented cover all analytes except cerium and titanium. Samples were prepared using the procedure described in Section 11.2. For each matrix, five replicate aliquots were prepared, analyzed and the average of the five determinations used to define the sample background concentration of each analyte. In addition, two pairs of duplicates were fortified at different concentration levels. For each method analyte, the sample background concentration, mean percent recovery, standard deviation of the percent recovery, and relative percent difference between the duplicate fortified samples are listed in Table 6. The variance of the five replicate sample background determinations is included in the calculated standard deviation of the percent recovery when the analyte concentration in the sample was greater than the MDL. The tap and well waters were processed in Teflon and quartz beakers and diluted in polypropylene centrifuged tubes. The nonuse of borosilicate glassware is reflected in the precision and recovery data for boron and silica in those two sample types.

13.3 Data obtained from single laboratory method testing are summarized in Table 7 for three solid samples consisting of EPA 884 Hazardous Soil, SRM 1645 River Sediment, and EPA 286 Electroplating Sludge. Samples were prepared using the procedure described in Section 11.3. For each method analyte, the sample background concentration, mean percent recovery of the fortified additions, the standard deviation of the percent recovery, and relative percent difference between duplicate additions were determined as described in Section 13.2. Data presented are for all analytes except cerium, silica, and titanium. Limited comparative data to other methods and SRM materials are presented in Reference 23 of Section 16.0.

13.4 Performance data for aqueous solutions independent of sample preparation from a multilaboratory study are provided in Table 8.22

13.5 Listed in Table 9 are regression equations for precision and bias for 25 analytes abstracted from EPA Method Study 27, a multilaboratory validation study of Method 200.7.1 These equations were developed from data received from 12 laboratories using the total recoverable sample preparation procedure on reagent water, drinking water, surface water and three industrial effluents. For a complete review and description of the study, see Reference 16 of Section 16.0.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Numerous opportunities for pollution prevention exist in laboratory operation. The EPA has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, laboratory personnel should use pollution prevention techniques to address their waste generation (e.g., Section 7.8). When wastes cannot be feasibly reduced at the source, the Agency recommends recycling as the next best option.

14.2 For information about pollution prevention that may be applicable to laboratories and research institutions, consult “Less is Better: Laboratory Chemical Management for Waste Reduction”, available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street NW., Washington, DC 20036, (202) 872–4477.

15.0 Waste Management

15.1 The Environmental Protection Agency requires that laboratory waste management practices be conducted consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the letter and spirit of any sewer discharge permits and regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste identification rules and land disposal restrictions. For further information on waste management consult “The Waste Management Manual for Laboratory Personnel”, available from the American Chemical Society at the address listed in the Section 14.2.

16.0 References

1. U.S. Environmental Protection Agency. Inductively Coupled Plasma—Atomic Emission Spectrometric Method for Trace Element Analysis of Water and Wastes—Method 200.7, Dec. 1982. EPA–600/4–79–020, revised March 1983.

2. U.S. Environmental Protection Agency. Inductively Coupled Plasma Atomic Emission Spectroscopy Method 6010, SW–846 Test Methods for Evaluating Solid Waste, 3rd Edition, 1986.

3. U.S. Environmental Protection Agency. Method 200.7: Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma—Atomic Emission Spectrometry, revision 3.3, EPA 600 4–91/010, June 1991.

4. U.S. Environmental Protection Agency. Inductively Coupled Plasma—Atomic Emission Spectrometry Method for the Analysis of Waters and Solids, EMMC, July 1992.

5. Fassel, V.A. et al. Simultaneous Determination of Wear Metals in Lubricating Oils by Inductively-Coupled Plasma Atomic Emission Spectrometry. Anal. Chem. 48:516–519, 1976.

6. Merryfield, R.N. and R.C. Loyd. Simultaneous Determination of Metals in Oil by Inductively Coupled Plasma Emission Spectrometry. Anal. Chem. 51:1965–1968, 1979.

7. Winge, R.K. et al. Inductively Coupled Plasma—Atomic Emission Spectroscopy: An Atlas of Spectral Information, Physical Science Data 20. Elsevier Science Publishing, New York, New York, 1985.

8. Boumans, P.W.J.M. Line Coincidence Tables for Inductively Coupled Plasma Atomic Emission Spectrometry, 2nd edition. Pergamon Press, Oxford, United Kingdom, 1984.

9. Carcinogens—Working With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, Aug. 1977. Available from the National Technical Information Service (NTIS) as PB–277256.

10. OSHA Safety and Health Standards, General Industry, (29 CFR 1910), Occupational Safety and Health Administration, OSHA 2206, (Revised, January 1976).

11. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

12. Proposed OSHA Safety and Health Standards, Laboratories, Occupational Safety and Health Administration, Federal Register, July 24, 1986.

13. Rohrbough, W.G. et al. Reagent Chemicals, American Chemical Society Specifications, 7th edition. American Chemical Society, Washington, DC, 1986.

14. American Society for Testing and Materials. Standard Specification for Reagent Water, D1193–77. Annual Book of ASTM Standards, Vol. 11.01. Philadelphia, PA, 1991.

15. Code of Federal Regulations 40, Ch. 1, Pt. 136 Appendix B.

16. Maxfield, R. and B. Mindak. EPA Method Study 27, Method 200.7 Trace Metals by ICP, Nov. 1983. Available from National Technical Information Service (NTIS) as PB 85–248–656.

17. Botto, R.I. Quality Assurance in Operating a Multielement ICP Emission Spectrometer. Spectrochim. Acta, 39B(1):95–113, 1984.

18. Wallace, G.F., Some Factors Affecting the Performance of an ICP Sample Introduction System. Atomic Spectroscopy, Vol. 4, p. 188–192, 1983.

19. Koirtyohann, S.R. et al. Nomenclature System for the Low-Power Argon Inductively Coupled Plasma, Anal. Chem. 52:1965, 1980.

20. Deming, S.N. and S.L. Morgan. Experimental Design for Quality and Productivity in Research, Development, and Manufacturing, Part III, pp. 119–123. Short course publication by Statistical Designs, 9941 Rowlett, Suite 6, Houston, TX 77075, 1989.

21. Winefordner, J.D., Trace Analysis: Spectroscopic Methods for Elements, Chemical Analysis, Vol. 46, pp. 41–42.

22. Jones, C.L. et al. An Interlaboratory Study of Inductively Coupled Plasma Atomic Emission Spectroscopy Method 6010 and Digestion Method 3050. EPA–600/4–87–032, U.S. Environmental Protection Agency, Las Vegas, Nevada, 1987.

23. Martin, T.D., E.R. Martin and SE. Long. Method 200.2: Sample Preparation Procedure for Spectrochemical Analyses of Total Recoverable Elements, EMSL ORD, USEPA, 1989.

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1—Wavelengths, Estimated Instrument Detection Limits, and Recommended Calibration

Analyte Wavelengtha
(nm)
Estimated
detection
limitb (µg/L)
Calibratec
to (mg/L)
Aluminum 308.215 45 10
Antimony 206.833 32 5
Arsenic 193.759 53 10
Barium 493.409 2.3 1
Beryllium 313.042 0.27 1
Boron 249.678 5.7 1
Cadmium 226.502 3.4 2
Calcium 315.887 30 10
Cerium 413.765 48 2
Chromium 205.552 6.1 5
Cobalt 228.616 7.0 2
Copper 324.754 5.4 2
Iron 259.940 6.2 10
Lead 220.353 42 10
Lithium 670.784 d 3.7 5
Magnesium 279.079 30 10
Manganese 257.610 1.4 2
Mercury 194.227 2.5 2
Molybdenum 203.844 12 10
Nickel 231.604 15 2
Phosphorus 214.914 76 10
Potassium 766.491 e 700 20
Selenium 196.090 75 5
Silica (SiO2) 251.611 d 26 (SiO2) 10
Silver 328.068 7.0 0.5
Sodium 588.995 29 10
Strontium 421.552 0.77 1
Thallium 190.864 40 5
Tin 189.980 25 4
Titanium 334.941 3.8 10
Vanadium 292.402 7.5 2
Zinc 213.856 1.8 5

TABLE 2—On-Line Method Interelement Spectral Interferances Arising From Interferants at the 100 mg/L Level

Analyte Wavelength (nm) Interferant*
Ag
Al
As
B
Ba
Be
Ca
Cd
Ce
Co
Cr
Cu
Fe
Hg
K
Li
Mg
Mn
Mo
Na
Ni
P
Pb
Sb
Se
SiO2
Sn
Sr
Tl
Ti
V
Zn
328.068
308.215
193.759
249.678
493.409
313.042
315.887
226.502
413.765
228.616
205.552
324.754
259.940
194.227
766.491
670.784
279.079
257.610
203.844
588.995
231.604
214.914
220.353
206.833
196.099
251.611
189.980
421.552
190.864
334.941
292.402
213.856
Ce, Ti, Mn
V, Mo, Ce, Mn
V, Al, Co, Fe, Ni
None
None
V, Ce
Co, Mo, Ce
Ni, Ti, Fe, Ce
None
Ti, Ba, Cd, Ni, Cr, Mo, Ce
Be, Mo, Ni
Mo, Ti
None
V, Mo
None
None
Ce
Ce
Ce
None
Co, Tl
Cu, Mo
Co, Al, Ce, Cu, Ni, Ti, Fe
Cr, Mo, Sn, Ti, Ce, Fe
Fe
None
Mo, Ti, Fe, Mn, Si
None
Ti, Mo, Co, Ce, Al, V, Mn
None
Mo, Ti, Cr, Fe, Ce
Ni, Cu, Fe

TABLE 3—Mixed Standard Solutions

Solution Analytes
I
II
III
IV
V
Ag, As, B, Ba, Ca, Cd, Cu, Mn, Sb, and Se
K, Li, Mo, Na, Sr, and Ti
Co, P, V, and Ce
Al, Cr, Hg, SiO2, Sn, and Zn
Be, Fe, Mg, Ni, Pb, and Tl

TABLE 4—Total Recoverable Method Detection Limits (MDL)

Analyte MDLs
Aqueous, mg/L(1)
Solids, mg/kg(2)
Ag 0.002 0.3
Al 0.02 3
As 0.008 2
B 0.003
Ba 0.001 0.2
Be 0.0003 0.1
Ca 0.01 2
Cd 0.001 0.2
Ce 0.02 3
Co 0.002 0.4
Cr 0.004 0.8
Cu 0.003 0.5
Fe *0.03 6
Hg 0.007 2
K 0.3 60
Li 0.001 0.2
Mg 0.02 3
Mn 0.001 0.2
Mo 0.004 1
Na 0.03 6
Ni 0.005 1
P 0.06 12
Pb 0.01 2
Sb 0.008 2
Se 0.02 5
SiO2 0.02
Sn 0.007 2
Sr 0.0003 0.1
Tl 0.001 0.2
Ti 0.02 3
V 0.003 1
Zn 0.002 0.3

TABLE 5—Inductively Coupled Plasma Instrument Operating Conditions

Incident rf power 1100 watts
Reflected rf power <5 watts
Viewing height above work coil 15 mm
Injector tube orifice i.d. 1 mm
Argon supply liquid argon
Argon pressure 40 psi
Coolant argon flow rate 19 L/min.
Aerosol carrier argon flow rate 620 mL/min.
Auxiliary (plasma) argon flow rate 300 mL/min.
Sample uptake rate controlled to 1.2 mL/min.

Table 6—Precision and Recovery Data in Aqueous Matrices

Analyte Sample conc.
mg/L
Low spike
mg/L
Average
recovery
R (%)
S (R) RPD High spike
mg/L
Average
recovery
R (%)
S (R) RPD
Tap Water
Ag <0.002 0.05 95 0.7 2.1 0.2 96 0.0 0.0
Al 0.185 0.05 98 8.8 1.7 0.2 105 3.0 3.1
As <0.008 0.05 108 1.4 3.7 0.2 101 0.7 2.0
B 0.023 0.1 98 0.2 0.0 0.4 98 0.2 0.5
Ba 0.042 0.05 102 1.6 2.2 0.2 98 0.4 0.8
Be <0.0003 0.01 100 0.0 0.0 0.1 99 0.0 0.0
Ca 35.2 5.0 101 8.8 1.7 20.0 103 2.0 0.9
Cd <0.001 0.01 105 3.5 9.5 0.1 98 0.0 0.0
Co <0.002 0.02 100 0.0 0.0 0.2 99 0.5 1.5
Cr <0.004 0.01 110 0.0 0.0 0.1 102 0.0 0.0
Cu <0.003 0.02 103 1.8 4.9 0.2 101 1.2 3.5
Fe 0.008 0.1 106 1.0 1.8 0.4 105 0.3 0.5
Hg <0.007 0.05 103 0.7 1.9 0.2 100 0.4 1.0
K 1.98 5.0 109 1.4 2.3 20. 107 0.7 1.7
Li 0.006 0.02 103 6.9 3.8 0.2 110 1.9 4.4
Mg 8.08 5.0 104 2.2 1.5 20.0 100 0.7 1.1
Mn <0.001 0.01 100 0.0 0.0 0.1 99 0.0 0.0
Mo <0.004 0.02 95 3.5 10.5 0.2 108 0.5 1.4
Na 10.3 5.0 99 3.0 2.0 20.0 106 1.0 1.6
Ni <0.005 0.02 108 1.8 4.7 0.2 104 1.1 2.9
P 0.045 0.1 102 13.1 9.4 0.4 104 3.2 1.3
Pb <0.01 0.05 95 0.7 2.1 0.2 100 0.2 0.5
Sb <0.008 0.05 99 0.7 2.0 0.2 102 0.7 2.0
Se <0.02 0.1 87 1.1 3.5 0.4 99 0.8 2.3
SiO2 6.5 5.0 104 3.3 3.4 20.0 96 1.1 2.3
Sn <0.007 0.05 103 2.1 5.8 0.2 101 1.8 5.0
Sr 0.181 0.1 102 3.3 2.1 0.4 105 0.8 1.0
Tl <0.02 0.1 101 3.9 10.9 0.4 101 0.1 0.3
V <0.003 0.05 101 0.7 2.0 0.2 99 0.2 0.5
Zn 0.005 0.05 101 3.7 9.0 0.2 98 0.9 2.5
Pond Water
Ag <0.002 0.05 92 0.0 0.0 0.2 94 0.0 0.0
Al 0.819 0.2 88 10.0 5.0 0.8 100 2.9 3.7
As <0.008 0.05 102 0.0 0.0 0.2 98 1.4 4.1
B 0.034 0.1 111 8.9 6.9 0.4 103 2.0 0.0
Ba 0.029 0.05 96 0.9 0.0 0.2 97 0.3 0.5
Be <0.0003 0.01 95 0.4 1.1 0.2 95 0.0 0.0
Ca 53.9 5.0 * * 0.7 20.0 100 2.0 1.5
Cd <0.001 0.01 107 0.0 0.0 0.1 97 0.0 0.0
Co <0.002 0.02 100 2.7 7.5 0.2 97 0.7 2.1
Cr <0.004 0.01 105 3.5 9.5 0.1 103 1.1 2.9
Cu <0.003 0.02 98 2.1 4.4 0.2 100 0.5 1.5
Fe 0.875 0.2 95 8.9 2.8 0.8 97 3.2 3.6
Hg <0.007 0.05 97 3.5 10.3 0.2 98 0.0 0.0
K 2.48 5.0 106 0.3 0.1 20.0 103 0.2 0.4
Li <0.001 0.02 110 0.0 0.0 0.2 106 0.2 0.5
Mg 10.8 5.0 102 0.5 0.0 20.0 96 0.7 1.3
Mn 0.632 0.01 * * 0.2 0.1 97 2.3 0.3
Mo <0.004 0.02 105 3.5 9.5 0.2 103 0.4 1.0
Na 17.8 5.0 103 1.3 0.4 20.0 94 0.3 0.0
Ni <0.005 0.02 96 5.6 9.1 0.2 100 0.7 1.5
P 0.196 0.1 91 14.7 0.3 0.4 108 3.9 1.3
Pb <0.01 0.05 96 2.6 7.8 0.2 100 0.7 2.0
Sb <0.008 0.05 102 2.8 7.8 0.2 104 0.4 1.0
Se <0.02 0.1 104 2.1 5.8 0.4 103 1.6 4.4
SiO2 7.83 5.0 151 1.6 1.3 20.0 117 0.4 0.6
Sn <0.007 0.05 98 0.0 0.0 0.2 99 1.1 3.0
Sr 0.129 0.1 105 0.4 0.0 0.4 99 0.1 0.2
Tl <0.02 0.1 103 1.1 2.9 0.4 97 1.3 3.9
V 0.003 0.05 94 0.4 0.0 0.2 98 0.1 0.0
Zn 0.006 0.05 97 1.6 1.8 0.2 94 0.4 0.0
Well Water
Ag <0.002 0.05 97 0.7 2.1 0.2 96 0.2 0.5
Al 0.036 0.05 107 7.6 10.1 0.2 101 1.1 0.8
As <0.008 0.05 107 0.7 1.9 0.2 104 0.4 1.0
B 0.063 0.1 97 0.6 0.7 0.4 98 0.8 2.1
Ba 0.102 0.05 102 3.0 0.0 0.2 99 0.9 1.0
Be <0.0003 0.01 100 0.0 0.0 0.1 100 0.0 0.0
Ca 93.8 5.0 * * 2.1 20.0 100 4.1 0.1
Cd 0.002 0.01 90 0.0 0.0 0.1 96 0.0 0.0
Co <0.002 0.02 94 0.4 1.1 0.2 94 0.4 1.1
Cr <0.004 0.01 100 7.1 20.0 0.1 100 0.4 1.0
Cu <0.005 0.02 100 1.1 0.4 0.2 96 0.5 1.5
Fe 0.042 0.1 99 2.3 1.4 0.4 97 1.4 3.3
Hg <0.007 0.05 94 2.8 8.5 0.2 93 1.2 3.8
K 6.21 5.0 96 3.4 3.6 20.0 101 1.2 2.3
Li 0.001 0.02 100 7.6 9.5 0.2 104 1.0 1.9
Mg 24.5 5.0 95 5.6 0.3 20.0 93 1.6 1.2
Mn 2.76 0.01 * * 0.4 0.1 * * 0.7
Mo <0.004 0.02 108 1.8 4.7 0.2 101 0.2 0.5
Na 35.0 5.0 101 11.4 0.8 20.0 100 3.1 1.5
Ni <0.005 0.02 112 1.8 4.4 0.2 96 0.2 0.5
P 0.197 0.1 95 12.7 1.9 0.4 98 3.4 0.9
Pb <0.01 0.05 87 4.9 16.1 0.2 95 0.2 0.5
Sb <0.008 0.05 98 2.8 8.2 0.2 99 1.4 4.0
Se <0.02 0.1 102 0.4 1.0 0.4 94 1.1 3.4
SiO2 13.1 5.0 93 4.8 2.8 20.0 99 0.8 0.0
Sn <0.007 0.05 98 2.8 8.2 0.2 94 0.2 0.5
Sr 0.274 0.1 94 5.7 2.7 0.4 95 1.7 2.2
Tl <0.02 0.1 92 0.4 1.1 0.4 95 1.1 3.2
V <0.003 0.05 98 0.0 0.0 0.2 99 0.4 1.0
Zn 0.538 0.05 * * 0.7 0.2 99 2.5 1.1
Sewage Treatment Effluent
Ag 0.009 0.05 92 1.5 3.6 0.2 95 0.1 0.0
Al 1.19 0.05 * * 0.9 0.2 113 12.4 2.1
As <0.008 0.05 99 2.1 6.1 0.2 93 2.1 6.5
B 0.226 0.1 217 16.3 9.5 0.4 119 13.1 20.9
Ba 0.189 0.05 90 6.8 1.7 0.2 99 1.6 0.5
Be <0.0003 0.01 94 0.4 1.1 0.1 100 0.4 1.0
Ca 87.9 5.0 * * 0.6 20.0 101 3.7 0.0
Cd 0.009 0.01 89 2.6 2.3 0.1 97 0.4 1.0
Co 0.016 0.02 95 3.1 0.0 0.2 93 0.4 0.5
Cr 0.128 0.01 * * 1.5 0.1 97 2.4 2.7
Cu 0.174 0.02 98 33.1 4.7 0.2 98 3.0 1.4
Fe 1.28 0.1 * * 2.8 0.4 111 7.0 0.6
Hg <0.007 0.05 102 1.4 3.9 0.2 98 0.5 1.5
K 10.6 5.0 104 2.8 1.3 20.0 101 0.6 0.0
Li 0.011 0.02 103 8.5 3.2 0.2 105 0.8 0.5
Mg 22.7 5.0 100 4.4 0.0 20.0 92 1.1 0.2
Mn 0.199 0.01 * * 2.0 0.1 104 1.9 0.3
Mo 0.125 0.02 110 21.2 6.8 0.2 102 1.3 0.9
Na 0.236 5.0 * * 0.0 20.0 * * 0.4
Ni 0.087 0.02 122 10.7 4.5 0.2 98 0.8 1.1
P 4.71 0.1 * * 2.6 0.4 * * 1.4
Pb 0.015 0.05 91 3.5 5.0 0.2 96 1.3 2.9
Sb <0.008 0.05 97 0.7 2.1 0.2 103 1.1 2.9
Se <0.02 0.1 108 3.9 10.0 0.4 101 2.6 7.2
SiO2 16.7 5.0 124 4.0 0.9 20.0 108 1.1 0.8
Sn 0.016 0.05 90 3.8 0.0 0.2 95 1.0 0.0
Sr 0.515 0.1 103 6.4 0.5 0.4 96 1.6 0.2
Tl <0.02 0.1 105 0.4 1.0 0.4 95 0.0 0.0
V 0.003 0.05 93 0.9 2.0 0.2 97 0.2 0.5
Zn 0.160 0.05 98 3.3 1.9 0.2 101 1.0 1.4
Industrial Effluent
Ag <0.0003 0.05 88 0.0 0.0 0.2 84 0.9 3.0
Al 0.054 0.05 88 11.7 12.2 0.2 90 3.9 8.1
As <0.02 0.05 82 2.8 9.8 0.2 88 0.5 1.7
B 0.17 0.1 162 17.6 13.9 0.4 92 4.7 9.3
Ba 0.083 0.05 86 8.2 1.6 0.2 85 2.3 2.4
Be <0.0006 0.01 94 0.4 1.1 0.1 82 1.4 4.9
Ca 500 5.0 * * 2.8 20.0 * * 2.3
Cd 0.008 0.01 85 4.7 6.1 0.1 82 1.4 4.4
Co <0.004 0.02 93 1.8 5.4 0.2 83 0.4 1.2
Cr 0.165 0.01 * * 4.5 0.1 106 6.6 5.6
Cu 0.095 0.02 93 23.3 0.9 0.2 95 2.7 2.8
Fe 0.315 0.1 88 16.4 1.0 0.4 99 6.5 8.0
Hg <0.01 0.05 87 0.7 2.3 0.2 86 0.4 1.2
K 2.87 5.0 101 3.4 2.4 20.0 100 0.8 0.4
Li 0.069 0.02 103 24.7 5.6 0.2 104 2.5 2.2
Mg 6.84 5.0 87 3.1 0.0 20.0 87 0.9 1.2
Mn 0.141 0.01 * * 1.2 0.1 89 6.6 4.8
Mo 1.27 0.02 * * 0.0 0.2 100 15.0 2.7
Na 1500 5.0 * * 2.7 20.0 * * 2.0
Ni 0.014 0.02 98 4.4 3.0 0.2 87 0.5 1.1
P 0.326 0.1 105 16.0 4.7 0.4 97 3.9 1.4
Pb 0.251 0.05 80 19.9 1.4 0.2 88 5.0 0.9
Sb 2.81 0.05 * * 0.4 0.2 * * 2.0
Se 0.021 0.1 106 2.6 3.2 0.4 105 1.9 4.6
SiO2 6.83 5.0 99 6.8 1.7 20.0 100 2.2 3.0
Sn <0.01 0.05 87 0.7 2.3 0.2 86 0.4 1.2
Sr 6.54 0.1 * * 2.0 0.4 * * 2.7
Tl <0.03 0.1 87 1.8 5.8 0.4 84 1.1 3.6
V <0.005 0.05 90 1.4 4.4 0.2 84 1.1 3.6
Zn 0.024 0.05 89 6.0 4.4 0.2 91 3.5 8.9

Table 7—Precision and Recovery Data in Solid Matrices

Analyte Sample conc.
mg/kg
Low + spike
mg/kg
Average
recovery R (%)
S (R) RPD High + spike
mg/kg
Average
recovery R (%)
S (R) RPD
EPA Hazardous Soil #884
Ag 1.1 20 98 0.7 1.0 100 96 0.2 0.6
Al 5080 20 * * 7.2 100 * * 5.4
As 5.7 20 95 5.4 10.6 100 96 1.4 3.6
B 20.4 100 93 2.7 5.3 400 100 2.1 5.5
Ba 111 20 98 71.4 22.2 100 97 10.0 1.0
Be 0.66 20 97 0.7 2.3 100 99 0.1 0.2
Ca 85200
Cd 2 20 93 0.7 1.0 100 94 0.2 0.4
Co 5.5 20 96 3.5 7.7 100 93 0.8 2.1
Cr 79.7 20 87 28.8 16.5 100 104 1.3 1.1
Cu 113 20 110 16.2 4.4 100 104 4.0 4.2
Fe 16500
Hg <1.4 10 92 2.5 7.7 40 98 0.0 0.0
K 621 500 121 1.3 0.0 2000 107 0.9 1.8
Li 6.7 10 113 3.5 4.4 40 106 0.6 0.6
Mg 24400 500 * * 8.4 2000 * * 10.1
Mn 343 20 * * 8.5 100 95 11.0 1.6
Mo 5.3 20 88 5.3 13.2 100 91 1.4 4.1
Na 195 500 102 2.2 2.4 2000 100 1.5 3.7
Ni 15.6 20 100 1.8 0.0 100 94 1.5 3.6
P 595 500 106 13.4 8.0 2000 103 3.2 2.7
Pb 145 20 88 51.8 17.9 100 108 15.6 17.4
Sb 6.1 20 83 3.9 7.5 100 81 1.9 5.9
Se <5 20 79 14.7 52.4 100 99 0.7 2.1
Sn 16.6 20 91 34.6 5.8 80 112 8.7 2.8
Sr 102 100 84 9.6 10.8 400 94 2.5 4.6
Tl <4 20 92 4.8 14.6 100 91 1.5 4.6
V 16.7 20 104 4.2 5.4 100 99 0.8 1.7
Zn 131 20 103 31.2 7.3 100 104 7.2 6.4
EPA Electroplating Sludge #286
Ag 6 20 96 0.2 0.4 100 93 0.1 0.4
Al 4980 20 * * 4.4 100 * * 5.6
As 32 20 94 1.3 0.8 100 97 0.7 1.6
B 210 100 113 2.0 1.6 400 98 1.9 3.5
Ba 39.8 20 0 6.8 0.3 100 0 1.6 5.7
Be 0.32 20 96 0.2 0.5 100 101 0.7 2.0
Ca 48500
Cd 108 20 98 2.5 0.8 100 96 0.5 0.5
Co 5.9 20 93 2.9 5.7 100 93 0.6 1.5
Cr 7580 20 * * 0.7 100 * * 1.3
Cu 806 20 * * 1.5 100 94 8.3 0.7
Fe 31100
Hg 6.1 10 90 2.5 4.0 40 97 1.7 4.3
K 2390 500 75 8.3 4.0 2000 94 2.9 3.8
Li 9.1 10 101 2.8 0.5 40 106 1.6 3.1
Mg 1950 500 110 2.0 0.8 2000 108 2.3 3.2
Mn 262 20 * * 1.8 100 91 1.2 0.9
Mo 13.2 20 92 2.1 2.9 100 92 0.3 0.0
Na 73400 500 * * 1.7 2000 * * 1.4
Ni 456 20 * * 0.4 100 88 2.7 0.9
P 9610 500 * * 2.9 2000 114 7.4 3.4
Pb 1420 20 * * 2.1 100 * * 1.3
Sb <2 20 76 0.9 3.3 100 75 2.8 10.7
Se 6.3 20 86 9.0 16.6 100 103 1.6 2.7
Sn 24.0 20 87 4.0 2.7 80 92 0.7 0.0
Sr 145 100 90 8.1 8.1 400 93 2.4 4.6
Tl 16 20 89 4.6 5.3 100 92 0.8 0.9
V 21.7 20 95 1.2 1.0 100 96 0.4 0.9
Zn 12500 20 * * 0.8 100 * * 0.8
NBS 1645 River Sediment
Ag 1.6 20 92 0.4 1.0 100 96 0.3 0.9
Al 5160 20 * * 8.4 100 * * 2.4
As 62.8 20 89 14.4 9.7 100 97 2.9 5.0
B 31.9 100 116 7.1 13.5 400 95 0.6 1.5
Ba 54.8 20 95 6.1 2.8 100 98 1.2 1.3
Be 0.72 20 101 0.4 1.0 100 103 1.4 3.9
Ca 28000
Cd 9.7 20 100 1.1 0.0 100 101 0.7 1.8
Co 9.4 20 98 3.8 4.8 100 98 0.9 1.8
Cr 28500 20 * * 0.4 100 * * 0.7
Cu 109 20 115 8.5 0.0 100 102 1.8 1.0
Fe 84800
Hg 3.1 10 99 4.3 7.7 40 96 0.7 1.0
K 452 500 98 4.1 2.0 2000 106 1.4 2.3
Li 3.7 10 101 2.0 0.7 40 108 1.3 3.0
Mg 6360 500 * * 1.8 2000 93 2.7 1.0
Mn 728 20 * * 3.5 100 97 12.4 2.2
Mo 17.9 20 97 12.5 18.5 100 98 0.6 0.0
Na 1020 500 92 2.6 0.0 2000 97 1.1 1.7
Ni 36.2 20 94 5.9 4.0 100 100 1.1 1.5
P 553 500 102 1.4 0.9 2000 100 0.8 1.6
Pb 707 20 * * 0.8 100 103 5.9 0.4
Sb 22.8 20 86 2.3 0.0 100 88 0.6 0.9
Se 6.7 20 103 14.3 27.1 100 98 3.1 7.6
Sn 309 20 * * 1.0 80 101 7.9 2.7
Sr 782 100 91 12.3 3.0 400 96 3.3 2.6
Tl <4 20 90 0.0 0.0 100 95 1.3 4.0
V 20.1 20 89 5.4 5.8 100 98 0.7 0.0
Zn 1640 20 * * 1.8 100 * * 1.1

Table 8—ICP–AES Instrumental Precision and Accuracy for Aqueous Solutions a

Element Mean conc.
(mg/L)
Nb RSD (%) Accuracec
(% of Nominal)
Al 14.8 8 6.3 100
Sb 15.1 8 7.7 102
As 14.7 7 6.4 99
Ba 3.66 7 3.1 99
Be 3.78 8 5.8 102
Cd 3.61 8 7.0 97
Ca 15.0 8 7.4 101
Cr 3.75 8 8.2 101
Co 3.52 8 5.9 95
Cu 3.58 8 5.6 97
Fe 14.8 8 5.9 100
Pb 14.4 7 5.9 97
Mg 14.1 8 6.5 96
Mn 3.70 8 4.3 100
Mo 3.70 8 6.9 100
Ni 3.70 7 5.7 100
K 14.1 8 6.6 95
Se 15.3 8 7.5 104
Na 14.0 8 4.2 95
Tl 15.1 7 8.5 102
V 3.51 8 6.6 95
Zn 3.57 8 8.3 96

Table 9—Multilaboratory ICP Precision and Accuracy Data*

Analyte Concentration
µg/L
Total recoverable digestion
µ/L
Aluminum 69–4792 X = 0.9380 (C) + 22.1
SR = 0.0481 (X) + 18.8
Antimony 77–1406 0.8908 (C) + 0.9
SR = 0.0682 (X) + 2.5
Arsenic 69–1887 X = 1.0175 (C) + 3.9
SR = 0.0643 (X) + 10.3
Barium 9–377 X = 0.8.80 (C) + 1.68
SR = 0.0826 (X) + 3.54
Beryllium 3–1906 X = 1.0177 (C) − 0.55
SR = 0.0445 (X) − 0.10
Boron 19–5189 X = 0.9676 (C) + 18.7
SR = 0.0743 (X) + 21.1
Cadmium 9–1943 X = 1.0137 (C) − 0.65
SR = 0.0332 (X) + 0.90
Calcium 17–47170 X = 0.9658 (C) + 0.8
SR = 0.0327 (X) + 10.1
Chromium 13–1406 X = 1.0049 (C) − 1.2
SR = 0.0571 (X) + 1.0
Cobalt 17–2340 X = 0.9278 (C) + 1.5
SR = 0.0407 (X) + 0.4
Copper 8–1887 X = 0.9647 (C) − 3.64
SR = 0.0406 (X) + 0.96
Iron 13–9359 X = 0.9830 (C) + 5.7
SR = 0.0790 (X) + 11.5
Lead 42–4717 X = 1.0056 (C) + 4.1
SR = 0.0448 (X) + 3.5
Magnesium 34–13868 X = 0.9879 (C) + 2.2
SR = 0.0268 (X) + 8.1
Manganese 4–1887 X = 0.9725 (C) + 0.07
SR = 0.0400 (X) + 0.82
Molybdenum 17–1830 X = 0.9707 (C) − 2.3
SR = 0.0529 (X) + 2.1
Nickel 17–47170 X = 0.9869 (C) + 1.5
SR = 0.0393 (X) + 2.2
Potassium 347–14151 X = 0.9355 (C) − 183.1
SR = 0.0329 (X) + 60.9
Selenium 69–1415 X = 0.9737 (C) − 1.0
SR = 0.0443 (X) + 6.6
Silicon 189–9434 X = 0.9737 (C) − 22.6
SR = 0.2133 (X) + 22.6
Silver 8–189 X = 0.3987 (C) + 8.25
SR = 0.1836 (X) − 0.27
Sodium 35–47170 X = 1.0526 (C) + 26.7
SR = 0.0884 (X) + 50.5
Thallium 79–1434 X = 0.9238 (C) + 5.5
SR = 0.0106 (X) + 48.0
Vanadium 13–4698 X = 0.9551 (C) + 0.4
SR = 0.0472 (X) + 0.5
Zinc 7–7076 X = 0.9500 (C) + 1.82
SR = 0.0153 (X) + 7.78

[77 FR 29813, May 18, 2012]

Appendix D to Part 136—Precision and Recovery Statements for Methods for Measuring Metals

Two selected methods from “Methods for Chemical Analysis of Water and Wastes,” EPA–600/4–79–020 (1979) have been subjected to interlaboratory method validation studies. The two selected methods are for Thallium and Zinc. The following precision and recovery statements are presented in this appendix and incorporated into Part 136:

Method 279.2

For Thallium, Method 279.2 (Atomic Absorption, Furnace Technique) replace the Precision and Accuracy Section statement with the following:

Precision and Accuracy

An interlaboratory study on metal analyses by this method was conducted by the Quality Assurance Branch (QAB) of the Environmental Monitoring Systems Laboratory—Cincinnati (EMSL–CI). Synthetic concentrates containing various levels of this element were added to reagent water, surface water, drinking water and three effluents. These samples were digested by the total digestion procedure, 4.1.3 in this manual. Results for the reagent water are given below. Results for other water types and study details are found in “EPA Method Study 31, Trace Metals by Atomic Absorption (Furnace Techniques),” National Technical Information Service, 5285 Port Royal Road, Springfield, VA 22161 Order No. PB 86–121 704/AS, by Copeland, F.R. and Maney, J.P., January 1986.

For a concentration range of 10.00–252 µg/L

X = 0.8781(C) − 0.715

S = 0.1112(X) + 0.669

SR = 0.1005(X) + 0.241

Where:

C = True Value for the Concentration, µg/L

X = Mean Recovery, µg/L

S = Multi-laboratory Standard Deviation, µg/L

SR = Single-analyst Standard Deviation, µg/L

Method 289.2

For Zinc, Method 289.2 (Atomic Absorption, Furnace Technique) replace the Precision and Accuracy Section statement with the following:

Precision and Accuracy

An interlaboratory study on metal analyses by this method was conducted by the Quality Assurance Branch (QAB) of the Environmental Monitoring Systems Laboratory—Cincinnati (EMSL–CI). Synthetic concentrates containing various levels of this element were added to reagent water, surface water, drinking water and three effluents. These samples were digested by the total digestion procedure, 4.1.3 in this manual. Results for the reagent water are given below. Results for other water types and study details are found in “EPA Method Study 31, Trace Metals by Atomic Absorption (Furnace Techniques),” National Technical Information Service, 5285 Port Royal Road, Springfield, VA 22161 Order No. PB 86–121 704/AS, by Copeland, F.R. and Maney, J.P., January 1986.

For a concentration range of 0.51–189 µg/L

X = 1.6710(C) + 1.485

S = 0.6740(X) − 0.342

SR = 0.3895(X)− 0.384

Where:

C = True Value for the Concentration, µg/L

X = Mean Recovery, µg/L

S = Multi-laboratory Standard Deviation, µg/L

SR = Single-analyst Standard Deviation, µg/L

[77 FR 29833, May 18, 2012]