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Electronic Code of Federal Regulations

e-CFR data is current as of November 7, 2019

Title 40Chapter ISubchapter D → Part 136


Title 40: Protection of Environment


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


Contents
§136.1   Applicability.
§136.2   Definitions.
§136.3   Identification of test procedures.
§136.4   Application for and approval of alternate test procedures for nationwide use.
§136.5   Approval of alternate test procedures for limited use.
§136.6   Method modifications and analytical requirements.
§136.7   Quality assurance and quality control.
Appendix A to Part 136—Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater
Appendix B to Part 136—Definition and Procedure for the Determination of the Method Detection Limit—Revision 2
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
Appendix D to Part 136—Precision and Recovery Statements for Methods for Measuring Metals

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 year 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, in §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 unitsMethod1EPAStandard methodsAOAC, ASTM, USGSOther
Bacteria
1. Coliform (fecal), number per 100 mL or number per gram dry weightMost Probable Number (MPN), 5 tube, 3 dilution, orp. 132,3 1680,11 15 168111 209221 C E-2006
   Multiple tube/multiple well, orColilert-18®13 18 21 29.
Membrane filter (MF)2, single stepp. 12439222 D-200630B-0050-854
2. Coliform (fecal) in presence of chlorine, number per 100 mLMPN, 5 tube, 3 dilution, orp. 13239221 C E-2006
MF2, single step5p. 12439222 D-200630
3. Coliform (total), number per 100 mLMPN, 5 tube, 3 dilution, orp. 11439221 B-2006
MF2, single step or two stepp. 10839222 B-2006B-0025-854
4. Coliform (total), in presence of chlorine, number per 100 mLMPN, 5 tube, 3 dilution, orp. 11439221 B-2006
MF2 with enrichment5p. 11139222 B-2006
5. E. coli, number per 100 mL21MPN6 8 16 multiple tube, or9221B.2-2006/9221F-200612 14
multiple tube/multiple well, or9223 B-200413991.1510Colilert® 13 18.
Colilert-18® 13 17 18
MF2 6 7 8 single step160322mColiBlue-24®19.
6. Fecal streptococci, number per 100 mLMPN, 5 tube, 3 dilution, orp. 13939230 B-2007
MF2, orp. 13639230 C-2007B-0055-854
Plate countp. 1433
7. Enterococci, number per 100 mL21MPN, 5 tube, 3 dilution, orp. 13939230 B-2007
MPN6 8, multiple tube/multiple well, or9230 D-2007D6503-999Enterolert® 13 24.
MF2 6 7 8 single step or1600259230 C-2007
Plate countp. 1433
8.Salmonella number per gram dry weight11MPN multiple tube168223
Aquatic Toxicity
9. Toxicity, acute, fresh water organisms, LC50, percent effluentCeriodaphnia dubia acute2002.026
Daphnia puplex and Daphnia magna acute2021.026
Fathead Minnow, Pimephales promelas, and Bannerfin shiner, Cyprinella leedsi, acute2000.026
Rainbow Trout, Oncorhynchus mykiss, and brook trout, Salvelinus fontinalis, acute2019.026
10. Toxicity, acute, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, LC50, percent effluentMysid, Mysidopsis bahia, acute2007.026
Sheepshead Minnow, Cyprinodon variegatus, acute2004.026
Silverside, Menidia beryllina, Menidia menidia, and Menidia peninsulae, acute2006.026
11. Toxicity, chronic, fresh water organisms, NOEC or IC25, percent effluentFathead minnow, Pimephales promelas, larval survival and growth1000.027
Fathead minnow, Pimephales promelas, embryo-larval survival and teratogenicity1001.027
Daphnia, Ceriodaphnia dubia, survival and reproduction1002.027
Green alga, Selenastrum capricornutum, growth1003.027
12. Toxicity, chronic, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, NOEC or IC25, percent effluentSheepshead minnow, Cyprinodon variegatus, larval survival and growth1004.028
Sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and teratogenicity1005.028
Inland silverside, Menidia beryllina, larval survival and growth1006.028
Mysid, Mysidopsis bahia, survival, growth, and fecundity1007.028
Sea urchin, Arbacia punctulata, fertilization1008.028

Table IA notes:

1The method must be specified when results are reported.

2A 0.45-µm membrane filter (MF) or other pore size certified by the manufacturer to fully retain organisms to be cultivated and to be free of extractables which could interfere with their growth.

3Microbiological Methods for Monitoring the Environment, Water, and Wastes, EPA/600/8-78/017. 1978. U.S. EPA.

4U.S. Geological Survey Techniques of Water-Resource Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples. 1989. USGS.

5Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Most Probable Number method will be required to resolve any controversies.

6Tests must be conducted to provide organism enumeration (density). Select the appropriate configuration of tubes/filtrations and dilutions/volumes to account for the quality, character, consistency, and anticipated organism density of the water sample.

7When the MF method has been used previously to test waters with high turbidity, large numbers of noncoliform bacteria, or samples that may contain organisms stressed by chlorine, a parallel test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of results.

8To assess the comparability of results obtained with individual methods, it is suggested that side-by-side tests be conducted across seasons of the year with the water samples routinely tested in accordance with the most current Standard Methods for the Examination of Water and Wastewater or EPA alternate test procedure (ATP) guidelines.

9Annual Book of ASTM Standards-Water and Environmental Technology, Section 11.02. 2000, 1999, 1996. ASTM International.

10Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision, 1998. AOAC International.

11Approved for enumeration of target organism in sewage sludge.

12The multiple-tube fermentation test is used in 9221B.2-2006. Lactose broth may be used in lieu of lauryl tryptose broth (LTB), if at least 25 parallel tests are conducted between this broth and LTB using the water samples normally tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total coliform using lactose broth is less than 10 percent. No requirement exists to run the completed phase on 10 percent of all total coliform-positive tubes on a seasonal basis.

13These tests are collectively known as defined enzyme substrate tests, where, for example, a substrate is used to detect the enzyme β-glucuronidase produced by E. coli.

14After prior enrichment in a presumptive medium for total coliform using 9221B.2-2006, all presumptive tubes or bottles showing any amount of gas, growth or acidity within 48 h ± 3 h of incubation shall be submitted to 9221F-2006. Commercially available EC-MUG media or EC media supplemented in the laboratory with 50 µg/mL of MUG may be used.

15Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation Using Lauryl-Tryptose Broth (LTB) and EC Medium, EPA-821-R-14-009. September 2014. U.S. EPA.

16Samples shall be enumerated by the multiple-tube or multiple-well procedure. Using multiple-tube procedures, employ an appropriate tube and dilution configuration of the sample as needed and report the Most Probable Number (MPN). Samples tested with Colilert® may be enumerated with the multiple-well procedures, Quanti-Tray® and the MPN calculated from the table provided by the manufacturer.

17Colilert-18® is an optimized formulation of the Colilert® for the determination of total coliforms and E. coli that provides results within 18 h of incubation at 35 °C rather than the 24 h required for the Colilert® test and is recommended for marine water samples.

18Descriptions of the Colilert®, Colilert-18®, and Quanti-Tray® may be obtained from IDEXX Laboratories, Inc.

19A description of the mColiBlue24® test, is available from Hach Company.

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

21Approved for enumeration of target organism in wastewater effluent.

22Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (modified mTEC), EPA-821-R-14-010. September 2014. U.S. EPA.

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

24A description of the Enterolert® test may be obtained from IDEXX Laboratories Inc.

25 Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI), EPA-821-R-14-011. September 2014. U.S. EPA.

26Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, EPA-821-R-02-012. Fifth Edition, October 2002. U.S. EPA.

27Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, EPA-821-R-02-013. Fourth Edition, October 2002. U.S. EPA.

28Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, EPA-821-R-02-014. Third Edition, October 2002. U.S. EPA.

29To use Colilert-18® to assay for fecal coliforms, the incubation temperature is 44.5 ± 0.2 °C, and a water bath incubator is used.

30On a monthly basis, at least ten blue colonies from the medium must be verified using Lauryl Tryptose Broth and EC broth, followed by count adjustment based on these results; and representative non-blue colonies should be verified using Lauryl Tryptose Broth. Where possible, verifications should be done from randomized sample sources.

Table IB—List of Approved Inorganic Test Procedures

ParameterMethodology58EPA52Standard methodsASTMUSGS/AOAC/other
1. Acidity, as CaCO3, mg/LElectrometric endpoint or phenolphthalein endpoint2310 B-2011D1067-11I-1020-85.2
2. Alkalinity, as CaCO3, mg/LElectrometric or Colorimetric titration to pH 4.5, Manual2320 B-2011D1067-11973.43,3 I-1030-85.2
   Automatic310.2 (Rev. 1974)1I-2030-85.2
3. Aluminum—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 D-2011 or 3111 E-2011I-3051-85.2
   AA furnace3113 B-2010.
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   Direct Current Plasma (DCP)36D4190-08See footnote.34
   Colorimetric (Eriochrome cyanine R)3500-Al B-2011
4. Ammonia (as N), mg/LManual distillation6 or gas diffusion (pH > 11), followed by any of the following:350.1, Rev. 2.0 (1993)4500-NH3 B-2011973.49.3
   NesslerizationD1426-08 (A)973.49,3 I-3520-85.2
   Titration4500-NH3 C-2011
   Electrode4500-NH3 D-2011 or E-2011D1426-08 (B)
   Manual phenate, salicylate, or other substituted phenols in Berthelot reaction based methods4500-NH3 F-2011See footnote.60
   Automated phenate, salicylate, or other substituted phenols in Berthelot reaction based methods350.1,30 Rev. 2.0 (1993)4500-NH3 G-2011, 4500-NH3 H-2011I-4523-85.2
   Automated electrodeSee footnote.7
   Ion ChromatographyD6919-09
   Automated gas diffusion, followed by conductivity cell analysisTimberline Ammonia-001.74
5. Antimony—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011
   AA furnace3113 B-2010
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
6. Arsenic-Total,4 mg/LDigestion,4 followed by any of the following:206.5 (Issued 1978)1
   AA gaseous hydride3114 B-2011 or 3114 C-2011D2972-08 (B)I-3062-85.2
   AA furnace3113 B-2010D2972-08 (C)I-4063-98.49
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   Colorimetric (SDDC)3500-As B-2011D2972-08 (A)I-3060-85.2
7. Barium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 D-2011I-3084-85.2
   AA furnace3113 B-2010D4382-12
   ICP/AES36200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCP36See footnote.34
8. Beryllium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 D-2011 or 3111 E-2011D3645-08 (A)I-3095-85.2
   AA furnace3113 B-2010D3645-08 (B)
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCPD4190-08See footnote.34
   Colorimetric (aluminon)See footnote.61
9. Biochemical oxygen demand (BOD5), mg/LDissolved Oxygen Depletion5210 B-2011973.44,3 p. 17,9 I-1578-78,8 See footnote.10 63
10. Boron—Total,37 mg/LColorimetric (curcumin)4500-B B-2011I-3112-85.2
   ICP/AES200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCPD4190-08See footnote.34
11. Bromide, mg/LElectrodeD1246-10I-1125-85.2
   Ion Chromatography300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)4110 B-2011, C-2011, D-2011D4327-03993.30.3
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
12. Cadmium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011 or 3111 C-2011D3557-12 (A or B)974.27,3 p. 37,9 I-3135-852 or I-3136-85.2
   AA furnace3113 B-2010D3557-12 (D)I-4138-89.51
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-1472-852 or I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCP36D4190-08See footnote.34
   Voltametry11D3557-12 (C)
   Colorimetric (Dithizone)3500-Cd-D-1990
13. Calcium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011D511-09(B)I-3152-85.2
   ICP/AES200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   DCPSee footnote.34
   Titrimetric (EDTA)3500-Ca B-2011D511-09 (A)
   Ion ChromatographyD6919-09
14. Carbonaceous biochemical oxygen demand (CBOD5), mg/L12Dissolved Oxygen Depletion with nitrification inhibitor5210 B-2011See footnote.35 63
15. Chemical oxygen demand (COD), mg/LTitrimetric410.3 (Rev. 1978)15220 B-2011 or C-2011D1252-06 (A)973.46,3 p. 17,9 I-3560-85.2
   Spectrophotometric, manual or automatic410.4, Rev. 2.0 (1993)5220 D-2011D1252-06 (B)See footnotes.13 14, I-3561-85.2
16. Chloride, mg/LTitrimetric: (silver nitrate)4500-Cl B-2011D512-04 (B)I-1183-85.2
   (Mercuric nitrate)4500-Cl C-2011D512-04 (A)973.51,3 I-1184-85.2
   Colorimetric: ManualI-1187-85.2
   Automated (ferricyanide)4500-Cl E-2011I-2187-85.2
   Potentiometric Titration4500-Cl D-2011
   Ion Selective ElectrodeD512-04 (C)
   Ion Chromatography300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)4110 B-2011 or 4110 C-2011D4327-03993.30,3 I-2057-90.51
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
17. Chlorine-Total residual, mg/LAmperometric direct4500-Cl D-2011D1253-08
   Amperometric direct (low level)4500-Cl E-2011
   Iodometric direct4500-Cl B-2011
   Back titration ether end-point154500-Cl C-2011
   DPD-FAS4500-Cl F-2011
   Spectrophotometric, DPD4500-Cl G-2011
   ElectrodeSee footnote.16
17A. Chlorine-Free Available, mg/LAmperometric direct4500-Cl D-2011D1253-08
   Amperometric direct (low level)4500-Cl E-2011
   DPD-FAS4500-Cl F-2011
   Spectrophotometric, DPD4500-Cl G-2011
18. Chromium VI dissolved, mg/L0.45-micron filtration followed by any of the following:
   AA chelation-extraction3111 C-2011I-1232-85.2
   Ion Chromatography218.6, Rev. 3.3 (1994)3500-Cr C-2011D5257-11993.23.3
   Colorimetric (diphenyl-carbazide)3500-Cr B-2011D1687-12 (A)I-1230-85.2
19. Chromium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011D1687-12 (B)974.27,3 I-3236-85.2
   AA chelation-extraction3111 C-2011
   AA furnace3113 B-2010D1687-12 (C)I-3233-93.46
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003),68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   DCP36D4190-08See footnote.34
   Colorimetric (diphenyl-carbazide)3500-Cr B-2011
20. Cobalt—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011 or 3111 C-2011D3558-08 (A or B)p. 37,9 I-3239-85.2
   AA furnace3113 B-2010D3558-08 (C)I-4243-89.51
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   DCPD4190-08See footnote.34
21. Color, platinum cobalt units or dominant wavelength, hue, luminance purityColorimetric (ADMI)2120 F-201178
   Platinum cobalt visual comparison2120 B-2011I-1250-85.2
   SpectrophotometricSee footnote.18
22. Copper—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011 or 3111 C-2011D1688-12 (A or B)974.27,3 p. 37,9 I-3270-852 or I-3271-85.2
   AA furnace3113 B-2010D1688-12 (C)I-4274-89.51
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   DCP36D4190-08See footnote.34
   Colorimetric (Neocuproine)3500-Cu B-2011
   Colorimetric (Bathocuproine)3500-Cu C-2011See footnote.19
23. Cyanide—Total, mg/LAutomated UV digestion/distillation and ColorimetryKelada-01.55
   Segmented Flow Injection, In-Line Ultraviolet Digestion, followed by gas diffusion amperometryD7511-12
   Manual distillation with MgCl2, followed by any of the following:335.4, Rev. 1.0 (1993)574500-CN B-2011 and C-2011D2036-09(A), D7284-1310-204-00-1-X.56
   Flow Injection, gas diffusion amperometryD2036-09(A) D7284-13
   Titrimetric4500-CN D-2011D2036-09(A)p. 22.9
   Spectrophotometric, manual4500-CN E-2011D2036-09(A)I-3300-85.2
   Semi-Automated20335.4, Rev. 1.0 (1993)5710-204-00-1-X,56 I-4302-85.2
   Ion ChromatographyD2036-09(A)
   Ion Selective Electrode4500-CN F-2011D2036-09(A)
24. Cyanide—Available, mg/LCyanide Amenable to Chlorination (CATC); Manual distillation with MgCl2, followed by Titrimetric or Spectrophotometric4500-CN G-2011D2036-09(B)
   Flow injection and ligand exchange, followed by gas diffusion amperometry59D6888-09OIA-1677-09.44
   Automated Distillation and Colorimetry (no UV digestion)Kelada-01.55
24.A Cyanide—Free, mg/LFlow Injection, followed by gas diffusion amperometryD7237-10OIA-1677-09.44
   Manual micro-diffusion and colorimetryD4282-02
25. Fluoride—Total, mg/LManual distillation,6 followed by any of the following:4500-F B-2011
   Electrode, manual4500-F C-2011D1179-10 (B)
   Electrode, automatedI-4327-85.2
   Colorimetric, (SPADNS)4500-F D-2011D1179-10 (A)
   Automated complexone4500-F E-2011
   Ion Chromatography300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)4110 B-2011 or C-2011D4327-03993.30.3
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
26. Gold—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011
   AA furnace231.2 (Issued 1978)13113 B-2010
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   DCPSee footnote.34
27. Hardness—Total, as CaCO3, mg/LAutomated colorimetric130.1 (Issued 1971)1
   Titrimetric (EDTA)2340 C-2011D1126-12973.52B,3 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 unitsElectrometric measurement4500-H+ B-2011D1293-99 (A or B)973.41,3 I-1586-85.2
   Automated electrode150.2 (Dec. 1982)1See footnote,21 I-2587-85.2
29. Iridium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011
   AA furnace235.2 (Issued 1978)1
   ICP/MS3125 B-2011
30. Iron—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011 or 3111 C-2011D1068-10 (A)974.27,3 I-3381-85.2
   AA furnace3113 B-2010D1068-10 (B)
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   DCP36D4190-08See footnote.34
   Colorimetric (Phenanthroline)3500-Fe B-2011D1068-10 (C)See footnote.22
31. Kjeldahl Nitrogen5—Total, (as N), mg/LManual digestion20 and distillation or gas diffusion, followed by any of the following:4500-Norg B-2011 or C-2011 and 4500-NH3 B-2011D3590-11 (A)I-4515-91.45
   Titration4500-NH3 C-2011973.48.3
   NesslerizationD1426-08 (A)
   Electrode4500-NH3 D-2011 or E-2011D1426-08 (B)
   Semi-automated phenate350.1, Rev. 2.0 (1993)4500-NH3 G-2011 4500-NH3 H-2011
   Manual phenate, salicylate, or other substituted phenols in Berthelot reaction based methods4500-NH3 F-2011See footnote.60
   Automated gas diffusion, followed by conductivity cell analysisTimberline Ammonia-001.74
   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)1I-4551-78.8
   Semi-automated block digestor colorimetric (distillation not required)351.2, Rev. 2.0 (1993)4500-Norg D-2011D3590-11 (B)I-4515-91.45
   Block digester, followed by Auto distillation and TitrationSee footnote.39
   Block digester, followed by Auto distillation and NesslerizationSee 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 ColorimetricNCASI TNTP W10900.77
32. Lead—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011 or 3111 C-2011D3559-08 (A or B)974.27,3 I-3399-85.2
   AA furnace3113 B-2010D3559-08 (D)I-4403-89.51
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCP36D4190-08See footnote.34
   Voltametry11D3559-08 (C)
   Colorimetric (Dithizone)3500-Pb B-2011
33. Magnesium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011D511-09 (B)974.27,3 I-3447-85.2
   ICP/AES200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   DCPSee footnote.34
Ion ChromatographyD6919-09
34. Manganese—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011D858-12 (A or B)974.27,3 I-3454-85.2
   AA furnace3113 B-2010D858-12 (C)
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCP36D4190-08See footnote.34
   Colorimetric (Persulfate)3500-Mn B-2011920.203.3
   Colorimetric (Periodate)See footnote.23
35. Mercury—Total,4 mg/LCold vapor, Manual245.1, Rev. 3.0 (1994)3112 B-2011D3223-12977.22,3 I-3462-85.2
   Cold vapor, Automated245.2 (Issued 1974)1
   Cold vapor atomic fluorescence spectrometry (CVAFS)245.7 Rev. 2.0 (2005)17I-4464-01.71
   Purge and Trap CVAFS1631E43
36. Molybdenum—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 D-2011I-3490-85.2
   AA furnace3113 B-2010I-3492-96.47
   ICP/AES36200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCPSee footnote.34
37. Nickel—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011 or 3111 C-2011D1886-08 (A or B)I-3499-85.2
   AA furnace3113 B-2010D1886-08 (C)I-4503-89.51
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   DCP36D4190-08See footnote.34
38. Nitrate (as N), mg/LIon Chromatography300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997)4110 B-2011 or C-2011D4327-03993.30.3
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
   Ion Selective Electrode4500-NO3 D-2011
   Colorimetric (Brucine sulfate)352.1 (Issued 1971)1973.50,3 419D1 7 p. 28.9
   Spectrophotometric (2,6-dimethylphenol)Hach 10206.75
   Nitrate-nitrite N minus Nitrite N (See parameters 39 and 40)
39. Nitrate-nitrite (as N), mg/LCadmium reduction, Manual4500-NO3 E-2011D3867-04 (B)
   Cadmium reduction, Automated353.2, Rev. 2.0 (1993)4500-NO3 F-2011D3867-04 (A)I-2545-90.51
   Automated hydrazine4500-NO3 H-2011
   Reduction/ColorimetricSee footnote.62
   Ion Chromatography300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997)4110 B-2011 or C-2011D4327-03993.30.3
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
   Enzymatic reduction, followed by automated colorimetric determinationI-2547-11,72 I-2548-11,72 N07-0003.73
   Spectrophotometric (2,6-dimethylphenol)Hach 10206.75
40. Nitrite (as N), mg/LSpectrophotometric: Manual4500-NO2 B-2011See footnote.25
   Automated (Diazotization)I-4540-85,2 See footnote.62
   Automated (*bypass cadmium reduction)353.2, Rev. 2.0 (1993)4500-NO3 F-2011D3867-04 (A)I-4545-85.2
   Manual (*bypass cadmium reduction)4500-NO3 E-2011D3867-04 (B)
   Ion Chromatography300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997)4110 B-2011 or C-2011D4327-03993.30.3
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
   Automated (*bypass Enzymatic reduction)I-2547-11,72 I-2548-11,72 N07-0003.73
41. Oil and grease—Total recoverable, mg/LHexane extractable material (HEM): n-Hexane extraction and gravimetry1664 Rev. A; 1664 Rev. B425520 B-201138
   Silica gel treated HEM (SGT-HEM): Silica gel treatment and gravimetry1664 Rev. A; 1664 Rev. B425520 B-201138 and 5520 F-201138
42. Organic carbon—Total (TOC), mg/LCombustion5310 B-2011D7573-09973.47,3 p. 14.24
   Heated persulfate or UV persulfate oxidation5310 C-2011, 5310 D-2011D4839-03973.47,3 p. 14.24
43. Organic nitrogen (as N), mg/LTotal Kjeldahl N (Parameter 31) minus ammonia N (Parameter 4).
44. Ortho-phosphate (as P), mg/LAscorbic acid method:
   Automated365.1, Rev. 2.0 (1993)4500-P F-2011 or G-2011973.56,3 I-4601-85.2
   Manual single reagent4500-P E-2011D515-88 (A)973.55.3
   Manual two reagent365.3 (Issued 1978)1
   Ion Chromatography300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997)4110 B-2011 or C-2011D4327-03993.30.3
   CIE/UV4140 B-2011D6508-10D6508, Rev. 2.54
45. Osmium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 D-2011
   AA furnace252.2 (Issued 1978)1
46. Oxygen, dissolved, mg/LWinkler (Azide modification)4500-O (B-F)-2011D888-09 (A)973.45B,3 I-1575-78.8
   Electrode4500-O G-2011D888-09 (B)I-1576-78.8
   Luminescence Based SensorD888-09 (C)See footnote.63 See footnote.64
47. Palladium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011
   AA furnace253.2 (Issued 1978)1
   ICP/MS3125 B-2011
   DCPSee footnote.34
48. Phenols, mg/LManual distillation,26 followed by any of the following:420.1 (Rev. 1978)15530 B-2010D1783-01
   Colorimetric (4AAP) manual420.1 (Rev. 1978)15530 D-201027D1783-01 (A or B)
   Automated colorimetric (4AAP)420.4 Rev. 1.0 (1993)
49. Phosphorus (elemental), mg/LGas-liquid chromatographySee footnote.28
50. Phosphorus—Total, mg/LDigestion,20 followed by any of the following:4500-P B(5)-2011973.55.3
   Manual365.3 (Issued 1978)14500-P E-2011D515-88 (A)
   Automated ascorbic acid reduction365.1 Rev. 2.0 (1993)4500-P (F-H)-2011973.56,3 I-4600-85.2
   ICP/AES4 36200.7, Rev. 4.4 (1994)3120 B-2011I-4471-97.50
   Semi-automated block digestor (TKP digestion)365.4 (Issued 1974)1D515-88 (B)I-4610-91.48
   Digestion with persulfate, followed by ColorimetricNCASI TNTP W10900.77
51. Platinum—Total4, mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011
   AA furnace255.2 (Issued 1978)1
   ICP/MS3125 B-2011
   DCPSee footnote.34
52. Potassium—Total4, mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011973.53,3 I-3630-85.2
   ICP/AES200.7, Rev. 4.4 (1994)3120 B-2011
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   Flame photometric3500-K B-2011
   Electrode3500-K C-2011
   Ion ChromatographyD6919-09
53. Residue—Total, mg/LGravimetric, 103-105°2540 B-2011I-3750-85.2
54. Residue—filterable, mg/LGravimetric, 180°2540 C-2011D5907-13I-1750-85.2
55. Residue—non-filterable (TSS), mg/LGravimetric, 103-105° post washing of residue2540 D-2011D5907-13I-3765-85.2
56. Residue—settleable, mg/LVolumetric, (Imhoff cone), or gravimetric2540 F-2011
57. Residue—Volatile, mg/LGravimetric, 550°160.4 (Issued 1971)12540-E-2011I-3753-85.2
58. Rhodium—Total4, mg/LDigestion,4 followed by any of the following:
   AA direct aspiration, or3111 B-2011
   AA furnace265.2 (Issued 1978)1
   ICP/MS3125 B-2011
59. Ruthenium—Total4, mg/LDigestion,4 followed by any of the following:
   AA direct aspiration, or3111 B-2011
   AA furnace267.21
   ICP/MS3125 B-2011
60. Selenium—Total4, mg/LDigestion,4 followed by any of the following:
   AA furnace3113 B-2010D3859-08 (B)I-4668-98.49
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES36200.5, Rev 4.2 (2003)68; 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   AA gaseous hydride3114 B-2011, or 3114 C-2011D3859-08 (A)I-3667-85.2
61. Silica—Dissolved,37 mg/L0.45-micron filtration followed by any of the following:
   Colorimetric, Manual4500-SiO2 C-2011D859-10I-1700-85.2
   Automated (Molybdosilicate)4500-SiO2 E-2011 or F-2011I-2700-85.2
   ICP/AES200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994)3120 B-2011I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
62. Silver—Total,4 31 mg/LDigestion,4 29 followed by any of the following:
   AA direct aspiration3111 B-2011 or 3111 C-2011974.27,3 p. 37,9 I-3720-85.2
   AA furnace3113 B-2010I-4724-89.51
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
   DCPSee footnote.34
63. Sodium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011973.54,3 I-3735-85.2
   ICP/AES200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994)3120 B-2011I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   DCPSee footnote.34
   Flame photometric3500-Na B-2011
   Ion ChromatographyD6919-09
64. Specific conductance, micromhos/cm at 25 °CWheatstone bridge120.1 (Rev. 1982)12510 B-2011D1125-95(99) (A)973.40,3 I-2781-85.2
65. Sulfate (as SO4), mg/LAutomated colorimetric375.2, Rev. 2.0 (1993)4500-SO42− F-2011 or G-2011
   Gravimetric4500-SO42− C-2011 or D-2011925.54.3
Turbidimetric4500-SO42− E-2011D516-11
   Ion Chromatography300.0, Rev. 2.1 (1993) and 300.1, Rev. 1.0 (1997)4110 B-2011 or C-2011D4327-03993.30,3 I-4020-05.70
   CIE/UV4140 B-2011D6508-1010D6508, Rev. 2.54
66. Sulfide (as S), mg/LSample Pretreatment4500-S2− B, C-2011
   Titrimetric (iodine)4500-S2− F-2011I-3840-85.2
   Colorimetric (methylene blue)4500-S2− D-2011
   Ion Selective Electrode4500-S2− G-2011D4658-09
67. Sulfite (as SO3), mg/LTitrimetric (iodine-iodate)4500-SO32− B-2011
68. Surfactants, mg/LColorimetric (methylene blue)5540 C-2011D2330-02
69. Temperature, °CThermometric2550 B-2010See footnote.32
70. Thallium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011
   AA furnace279.2 (Issued 1978)13113 B-2010
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES200.7, Rev. 4.4 (1994)3120 B-2011D1976-12
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4471-97.50
71. Tin—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 B-2011I-3850-78.8
   AA furnace3113 B-2010
   STGFAA200.9, Rev. 2.2 (1994)
   ICP/AES200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994)
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
72. Titanium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 D-2011
   AA furnace283.2 (Issued 1978)1
   ICP/AES200.7, Rev. 4.4 (1994)
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14.3
   DCPSee footnote.34
73. Turbidity, NTU53Nephelometric180.1, Rev. 2.0 (1993)2130 B-2011D1889-00I-3860-85.2 See footnote.65 See footnote.66 See footnote.67
74. Vanadium—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration3111 D-2011
   AA furnace3113 B-2010D3373-12
   ICP/AES200.5, Rev. 4.2 (2003);68 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   DCPD4190-08See footnote.34
   Colorimetric (Gallic Acid)3500-V B-2011
75. Zinc—Total,4 mg/LDigestion,4 followed by any of the following:
   AA direct aspiration363111 B-2011 or 3111 C-2011D1691-12 (A or B)974.27,3 p. 37,9 I-3900-85.2
   AA furnace289.2 (Issued 1978)1
   ICP/AES36200.5, Rev. 4.2 (2003)68; 200.7, Rev. 4.4 (1994)3120 B-2011D1976-12I-4471-97.50
   ICP/MS200.8, Rev. 5.4 (1994)3125 B-2011D5673-10993.14,3 I-4020-05.70
   DCP36D4190-08See footnote.34
   Colorimetric (Zincon)3500 Zn B-2011See footnote.33
76. Acid Mine Drainage162769

Table IB Notes:

1Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020. Revised March 1983 and 1979, where applicable. U.S. EPA.

2Methods for Analysis of Inorganic Substances in Water and Fluvial Sediments, Techniques of Water-Resource Investigations of the U.S. Geological Survey, Book 5, Chapter A1., unless otherwise stated. 1989. USGS.

3Official Methods of Analysis of the Association of Official Analytical Chemists, Methods Manual, Sixteenth Edition, 4th Revision, 1998. AOAC International.

4For the determination of total metals (which are equivalent to total recoverable metals) the sample is not filtered before processing. A digestion procedure is required to solubilize analytes in suspended material and to break down organic-metal complexes (to convert the analyte to a detectable form for colorimetric analysis). For non-platform graphite furnace atomic absorption determinations, a digestion using nitric acid (as specified in Section 4.1.3 of Methods for the Chemical Analysis of Water and Wastes) is required prior to analysis. The procedure used should subject the sample to gentle, acid refluxing and at no time should the sample be taken to dryness. For direct aspiration flame atomic absorption determinations (FLAA) a combination acid (nitric and hydrochloric acids) digestion is preferred prior to analysis. The approved total recoverable digestion is described as Method 200.2 in Supplement I of “Methods for the Determination of Metals in Environmental Samples” EPA/600R-94/111, May, 1994, and is reproduced in EPA Methods 200.7, 200.8, and 200.9 from the same Supplement. However, when using the gaseous hydride technique or for the determination of certain elements such as antimony, arsenic, selenium, silver, and tin by non-EPA graphite furnace atomic absorption methods, mercury by cold vapor atomic absorption, the noble metals and titanium by FLAA, a specific or modified sample digestion procedure may be required and in all cases the referenced method write-up should be consulted for specific instruction and/or cautions. For analyses using inductively coupled plasma-atomic emission spectrometry (ICP-AES), the direct current plasma (DCP) technique or EPA spectrochemical techniques (platform furnace AA, ICP-AES, and ICP-MS) use EPA Method 200.2 or an approved alternate procedure (e.g., CEM microwave digestion, which may be used with certain analytes as indicated in Table IB); the total recoverable digestion procedures in EPA Methods 200.7, 200.8, and 200.9 may be used for those respective methods. Regardless of the digestion procedure, the results of the analysis after digestion procedure are reported as “total” metals.

5Copper sulfate or other catalysts that have been found suitable may be used in place of mercuric sulfate.

6Manual distillation is not required if comparability data on representative effluent samples are on file to show that this preliminary distillation step is not necessary: However, manual distillation will be required to resolve any controversies. In general, the analytical method should be consulted regarding the need for distillation. If the method is not clear, the laboratory may compare a minimum of 9 different sample matrices to evaluate the need for distillation. For each matrix, a matrix spike and matrix spike duplicate are analyzed both with and without the distillation step. (A total of 36 samples, assuming 9 matrices). If results are comparable, the laboratory may dispense with the distillation step for future analysis. Comparable is defined as <20% RPD for all tested matrices). Alternatively the two populations of spike recovery percentages may be compared using a recognized statistical test.

7Industrial Method Number 379-75 WE Ammonia, Automated Electrode Method, Technicon Auto Analyzer II. February 19, 1976. Bran & Luebbe Analyzing Technologies Inc.

8The approved method is that cited in 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. 1979. USGS.

9American National Standard on Photographic Processing Effluents. April 2, 1975. American National Standards Institute.

10In-Situ Method 1003-8-2009, Biochemical Oxygen Demand (BOD) Measurement by Optical Probe. 2009. In-Situ Incorporated.

11The use of normal and differential pulse voltage ramps to increase sensitivity and resolution is acceptable.

12Carbonaceous biochemical oxygen demand (CBOD5) must not be confused with the traditional BOD5 test method which measures “total 5-day BOD.” The addition of the nitrification inhibitor is not a procedural option, but must be included to report the CBOD5 parameter. A discharger whose permit requires reporting the traditional BOD5 may not use a nitrification inhibitor in the procedure for reporting the results. Only when a discharger's permit specifically states CBOD5 is required can the permittee report data using a nitrification inhibitor.

13OIC Chemical Oxygen Demand Method. 1978. Oceanography International Corporation.

14Method 8000, Chemical Oxygen Demand, Hach Handbook of Water Analysis, 1979. Hach Company.

15The back titration method will be used to resolve controversy.

16Orion Research Instruction Manual, Residual Chlorine Electrode Model 97-70. 1977. Orion Research Incorporated. The calibration graph for the Orion residual chlorine method must be derived using a reagent blank and three standard solutions, containing 0.2, 1.0, and 5.0 mL 0.00281 N potassium iodate/100 mL solution, respectively.

17Method 245.7, Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, EPA-821-R-05-001. Revision 2.0, February 2005. US EPA.

18National Council of the Paper Industry for Air and Stream Improvement (NCASI) Technical Bulletin 253 (1971) and Technical Bulletin 803, May 2000.

19Method 8506, Bicinchoninate Method for Copper, Hach Handbook of Water Analysis. 1979. Hach Company.

20When using a method with block digestion, this treatment is not required.

21Industrial Method Number 378-75WA, Hydrogen ion (pH) Automated Electrode Method, Bran & Luebbe (Technicon) Autoanalyzer II. October 1976. Bran & Luebbe Analyzing Technologies.

22Method 8008, 1,10-Phenanthroline Method using FerroVer Iron Reagent for Water. 1980. Hach Company.

23Method 8034, Periodate Oxidation Method for Manganese, Hach Handbook of Wastewater Analysis. 1979. Hach Company.

24Methods for Analysis of Organic Substances in Water and Fluvial Sediments, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A3, (1972 Revised 1987). 1987. USGS.

25Method 8507, Nitrogen, Nitrite-Low Range, Diazotization Method for Water and Wastewater. 1979. Hach Company.

26Just prior to distillation, adjust the sulfuric-acid-preserved sample to pH 4 with 1 + 9 NaOH.

27The colorimetric reaction must be conducted at a pH of 10.0 ± 0.2.

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

29Approved methods for the analysis of silver in industrial wastewaters at concentrations of 1 mg/L and above are inadequate where silver exists as an inorganic halide. Silver halides such as the bromide and chloride are relatively insoluble in reagents such as nitric acid but are readily soluble in an aqueous buffer of sodium thiosulfate and sodium hydroxide to pH of 12. Therefore, for levels of silver above 1 mg/L, 20 mL of sample should be diluted to 100 mL by adding 40 mL each of 2 M Na2S2O3and NaOH. Standards should be prepared in the same manner. For levels of silver below 1 mg/L the approved method is satisfactory.

30The use of EDTA decreases method sensitivity. Analysts may omit EDTA or replace with another suitable complexing reagent provided that all method specified quality control acceptance criteria are met.

31For samples known or suspected to contain high levels of silver (e.g., in excess of 4 mg/L), cyanogen iodide should be used to keep the silver in solution for analysis. Prepare a cyanogen iodide solution by adding 4.0 mL of concentrated NH4OH, 6.5 g of KCN, and 5.0 mL of a 1.0 N solution of I2 to 50 mL of reagent water in a volumetric flask and dilute to 100.0 mL. After digestion of the sample, adjust the pH of the digestate to >7 to prevent the formation of HCN under acidic conditions. Add 1 mL of the cyanogen iodide solution to the sample digestate and adjust the volume to 100 mL with reagent water (NOT acid). If cyanogen iodide is added to sample digestates, then silver standards must be prepared that contain cyanogen iodide as well. Prepare working standards by diluting a small volume of a silver stock solution with water and adjusting the pH>7 with NH4OH. Add 1 mL of the cyanogen iodide solution and let stand 1 hour. Transfer to a 100-mL volumetric flask and dilute to volume with water.

32“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. USGS.

33Method 8009, Zincon Method for Zinc, Hach Handbook of Water Analysis, 1979. Hach Company.

34Method AES0029, Direct Current Plasma (DCP) Optical Emission Spectrometric Method for Trace Elemental Analysis of Water and Wastes. 1986-Revised 1991. Thermo Jarrell Ash Corporation.

35In-Situ Method 1004-8-2009, Carbonaceous Biochemical Oxygen Demand (CBOD) Measurement by Optical Probe. 2009. In-Situ Incorporated.

36Microwave-assisted digestion may be employed for this metal, when analyzed by this methodology. Closed Vessel Microwave Digestion of Wastewater Samples for Determination of Metals. April 16, 1992. CEM Corporation.

37When determining boron and silica, only plastic, PTFE, or quartz laboratory ware may be used from start until completion of analysis.

38Only use n-hexane (n-Hexane—85% minimum purity, 99.0% min. saturated C6 isomers, residue less than 1 mg/L) extraction solvent when determining Oil and Grease parameters—Hexane Extractable Material (HEM), or Silica Gel Treated HEM (analogous to EPA Methods 1664 Rev. A and 1664 Rev. B). Use of other extraction solvents is prohibited.

39Method PAI-DK01, Nitrogen, Total Kjeldahl, Block Digestion, Steam Distillation, Titrimetric Detection. Revised December 22, 1994. OI Analytical.

40Method PAI-DK02, Nitrogen, Total Kjeldahl, Block Digestion, Steam Distillation, Colorimetric Detection. Revised December 22, 1994. OI Analytical.

41Method PAI-DK03, Nitrogen, Total Kjeldahl, Block Digestion, Automated FIA Gas Diffusion. Revised December 22, 1994. OI Analytical.

42Method 1664 Rev. B is the revised version of EPA Method 1664 Rev. A. U.S. EPA. February 1999, Revision A. Method 1664, n-Hexane Extractable Material (HEM; Oil and Grease) and Silica Gel Treated n-Hexane Extractable Material (SGT-HEM; Non-polar Material) by Extraction and Gravimetry. EPA-821-R-98-002. U.S. EPA. February 2010, Revision B. Method 1664, n-Hexane Extractable Material (HEM; Oil and Grease) and Silica Gel Treated n-Hexane Extractable Material (SGT-HEM; Non-polar Material) by Extraction and Gravimetry. EPA-821-R-10-001.

43Method 1631, Revision E, Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic Fluorescence Spectrometry, EPA-821-R-02-019. Revision E. August 2002, U.S. EPA. The application of clean techniques described in EPA's Method 1669: Sampling Ambient Water for Trace Metals at EPA Water Quality Criteria Levels, EPA-821-R-96-011, are recommended to preclude contamination at low-level, trace metal determinations.

44Method OIA-1677-09, Available Cyanide by Ligand Exchange and Flow Injection Analysis (FIA). 2010. OI Analytical.

45Open File Report 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. USGS.

46Open File Report 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. USGS.

47Open File Report 97-198, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Molybdenum by Graphite Furnace Atomic Absorption Spectrophotometry. 1997. USGS.

48Open File Report 92-146, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Total Phosphorus by Kjeldahl Digestion Method and an Automated Colorimetric Finish That Includes Dialysis. 1992. USGS.

49Open File Report 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. USGS.

50Open File Report 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. USGS.

51Open File Report 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. USGS.

52Unless otherwise indicated, all EPA methods, excluding EPA Method 300.1, are published in U.S. EPA. May 1994. Methods for the Determination of Metals in Environmental Samples, Supplement I, EPA/600/R-94/111; or U.S. EPA. August 1993. Methods for the Determination of Inorganic Substances in Environmental Samples, EPA/600/R-93/100. EPA Method 300.1 is US EPA. Revision 1.0, 1997, including errata cover sheet April 27, 1999. Determination of Inorganic Ions in Drinking Water by Ion Chromatography.

53Styrene divinyl benzene beads (e.g., AMCO-AEPA-1 or equivalent) and stabilized formazin (e.g., Hach StablCalTMor equivalent) are acceptable substitutes for formazin.

54Method D6508-10, Test Method for Determination of Dissolved Inorganic Anions in Aqueous Matrices Using Capillary Ion Electrophoresis and Chromate Electrolyte. 2010. ASTM.

55Kelada-01, Kelada Automated Test Methods for Total Cyanide, Acid Dissociable Cyanide, and Thiocyanate, EPA 821-B-01-009, Revision 1.2, August 2001. US EPA. Note: A 450-W UV lamp may be used in this method instead of the 550-W lamp specified if it provides performance within the quality control (QC) acceptance criteria of the method in a given instrument. Similarly, modified flow cell configurations and flow conditions may be used in the method, provided that the QC acceptance criteria are met.

56QuikChem 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. Lachat Instruments.

57When using sulfide removal test procedures described in EPA Method 335.4-1, reconstitute particulate that is filtered with the sample prior to distillation.

58Unless otherwise stated, if the language of this table specifies a sample digestion and/or distillation “followed by” analysis with a method, approved digestion and/or distillation are required prior to analysis.

59Samples analyzed for available cyanide using OI Analytical method OIA-1677-09 or ASTM method D6888-09 that contain particulate matter may be filtered only after the ligand exchange reagents have been added to the samples, because the ligand exchange process converts complexes containing available cyanide to free cyanide, which is not removed by filtration. Analysts are further cautioned to limit the time between the addition of the ligand exchange reagents and sample filtration to no more than 30 minutes to preclude settling of materials in samples.

60Analysts should be aware that pH optima and chromophore absorption maxima might differ when phenol is replaced by a substituted phenol as the color reagent in Berthelot Reaction (“phenol-hypochlorite reaction”) colorimetric ammonium determination methods. For example when phenol is used as the color reagent, pH optimum and wavelength of maximum absorbance are about 11.5 and 635 nm, respectively—see, Patton, C.J. and S.R. Crouch. March 1977. Anal. Chem. 49:464-469. These reaction parameters increase to pH > 12.6 and 665 nm when salicylate is used as the color reagent—see, Krom, M.D. April 1980. The Analyst 105:305-316.

61If atomic absorption or ICP instrumentation is not available, the aluminon colorimetric method detailed in the 19th Edition of Standard Methods may be used. This method has poorer precision and bias than the methods of choice.

62Easy (1-Reagent) Nitrate Method, Revision November 12, 2011. Craig Chinchilla.

63Hach 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. Hach Company. This method may be used to measure dissolved oxygen when performing the methods approved in Table IB for measurement of biochemical oxygen demand (BOD) and carbonaceous biochemical oxygen demand (CBOD).

64In-Situ Method 1002-8-2009, Dissolved Oxygen (DO) Measurement by Optical Probe. 2009. In-Situ Incorporated.

65Mitchell Method M5331, Determination of Turbidity by Nephelometry. Revision 1.0, July 31, 2008. Leck Mitchell.

66Mitchell Method M5271, Determination of Turbidity by Nephelometry. Revision 1.0, July 31, 2008. Leck Mitchell.

67Orion Method AQ4500, Determination of Turbidity by Nephelometry. Revision 5, March 12, 2009. Thermo Scientific.

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

69Method 1627, Kinetic Test Method for the Prediction of Mine Drainage Quality, EPA-821-R-09-002. December 2011. US EPA.

70Techniques 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. USGS.

71Water-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. USGS.

72USGS Techniques and Methods 5-B8, Chapter 8, Section B, Methods of the National Water Quality Laboratory Book 5, Laboratory Analysis, 2011 USGS.

73NECi Method N07-0003, ”Nitrate Reductase Nitrate-Nitrogen Analysis,” Revision 9.0, March 2014, The Nitrate Elimination Co., Inc.

74Timberline Instruments, LLC Method Ammonia-001, “Determination of Inorganic Ammonia by Continuous Flow Gas Diffusion and Conductivity Cell Analysis,” June 2011, Timberline Instruments, LLC.

75Hach Company Method 10206, “Spectrophotometric Measurement of Nitrate in Water and Wastewater,” Revision 2.1, January 2013, Hach Company.

76Hach Company Method 10242, “Simplified Spectrophotometric Measurement of Total Kjeldahl Nitrogen in Water and Wastewater,” Revision 1.1, January 2013, Hach Company.

77National Council for Air and Stream Improvement (NCASI) Method TNTP-W10900, “Total (Kjeldahl) Nitrogen and Total Phosphorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion,” June 2011, National Council for Air and Stream Improvement, Inc.

78The pH adjusted sample is to be adjusted to 7.6 for NPDES reporting purposes.

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

Parameter1MethodEPA2 7Standard methodsASTMOther
1. AcenaphtheneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
2. AcenaphthyleneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
3. AcroleinGC603
GC/MS624.1,4 1624B
4. AcrylonitrileGC603
GC/MS624.1,4 1624B
5. AnthraceneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
6. BenzeneGC6026200 C-2011
GC/MS624.1, 1624B6200 B-2011
7. BenzidineSpectro-photometricSee footnote,3 p.1.
GC/MS625.15, 1625B6410 B-2000
HPLC605
8. Benzo(a)anthraceneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
9. Benzo(a)pyreneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
10. Benzo(b)fluorantheneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
11. Benzo(g,h,i)peryleneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
12. Benzo(k)fluorantheneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
13. Benzyl chlorideGCSee footnote,3 p. 130.
GC/MSSee footnote,6 p. S102.
14. Butyl benzyl phthalateGC606
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
15. bis(2-Chloroethoxy) methaneGC611
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
16. bis(2-Chloroethyl) etherGC611
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
17. bis(2-Ethylhexyl) phthalateGC606
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
18. BromodichloromethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
19. BromoformGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
20. BromomethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
21. 4-Bromophenyl phenyl etherGC611
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
22. Carbon tetrachlorideGC6016200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
23. 4-Chloro-3-methyl phenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
24. ChlorobenzeneGC601, 6026200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
25. ChloroethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
26. 2-Chloroethylvinyl etherGC601
GC/MS624.1, 1624B
27. ChloroformGC6016200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
28. ChloromethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
29. 2-ChloronaphthaleneGC612
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
30. 2-ChlorophenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
31. 4-Chlorophenyl phenyl etherGC611
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
32. ChryseneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
33. Dibenzo(a,h)anthraceneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
34. DibromochloromethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
35. 1,2-DichlorobenzeneGC601, 6026200 C-2011
GC/MS624.1, 1625B6200 B-2011See footnote,9 p. 27.
36. 1,3-DichlorobenzeneGC601, 6026200 C-2011
GC/MS624.1, 1625B6200 B-2011See footnote,9 p. 27.
37. 1,4-DichlorobenzeneGC601, 6026200 C-2011
GC/MS624.1, 1625B6200 B-2011See footnote,9 p. 27.
38. 3,3'-DichlorobenzidineGC/MS625.1, 1625B6410 B-2000
HPLC605
39. DichlorodifluoromethaneGC601
GC/MS6200 C-2011
40. 1,1-DichloroethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
41. 1,2-DichloroethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
42. 1,1-DichloroetheneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
43. trans-1,2-DichloroetheneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
44. 2,4-DichlorophenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
45. 1,2-DichloropropaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
46. cis-1,3-DichloropropeneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
47. trans-1,3-DichloropropeneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
48. Diethyl phthalateGC606
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
49. 2,4-DimethylphenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
50. Dimethyl phthalateGC606
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
51. Di-n-butyl phthalateGC606
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
52. Di-n-octyl phthalateGC606
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
53. 2, 4-DinitrophenolGC6046420 B-2000See footnote,9 p. 27.
GC/MS625.1, 1625B6410 B-2000
54. 2,4-DinitrotolueneGC609
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
55. 2,6-DinitrotolueneGC609
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
56. EpichlorohydrinGCSee footnote,3 p. 130.
GC/MSSee footnote,6 p. S102.
57. EthylbenzeneGC6026200 C-2011
GC/MS624.1, 1624B6200 B-2011
58. FluorantheneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
59. FluoreneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
60. 1,2,3,4,6,7,8-Heptachloro-dibenzofuranGC/MS1613B
61. 1,2,3,4,7,8,9-Heptachloro-dibenzofuranGC/MS1613B
62. 1,2,3,4,6,7,8- Heptachloro-dibenzo-p-dioxinGC/MS1613B
63. HexachlorobenzeneGC612
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
64. HexachlorobutadieneGC612
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
65. HexachlorocyclopentadieneGC612
GC/MS625.1,5 1625B6410 B-2000See footnote,9 p. 27.
66. 1,2,3,4,7,8-Hexachloro-dibenzofuranGC/MS1613B
67. 1,2,3,6,7,8-Hexachloro-dibenzofuranGC/MS1613B
68. 1,2,3,7,8,9-Hexachloro-dibenzofuranGC/MS1613B
69. 2,3,4,6,7,8-Hexachloro-dibenzofuranGC/MS1613B
70. 1,2,3,4,7,8-Hexachloro-dibenzo-p-dioxinGC/MS1613B
71. 1,2,3,6,7,8-Hexachloro-dibenzo-p-dioxinGC/MS1613B
72. 1,2,3,7,8,9-Hexachloro-dibenzo-p-dioxinGC/MS1613B
73. HexachloroethaneGC612
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
74. Indeno(1,2,3-c,d) pyreneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
75. IsophoroneGC609
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
76. Methylene chlorideGC6016200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
77. 2-Methyl-4,6-dinitrophenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
78. NaphthaleneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005
79. NitrobenzeneGC609
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLCD4657-92 (98)
80. 2-NitrophenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
81. 4-NitrophenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
82. N-NitrosodimethylamineGC607
GC/MS625.1,5 1625B6410 B-2000See footnote,9 p. 27.
83. N-Nitrosodi-n-propylamineGC607
GC/MS625.1,5 1625B6410 B-2000See footnote,9 p. 27.
84. N-NitrosodiphenylamineGC607
GC/MS625.1,5 1625B6410 B-2000See footnote,9 p. 27.
85. OctachlorodibenzofuranGC/MS1613B10
86. Octachlorodibenzo-p-dioxinGC/MS1613B10
87. 2,2'-oxybis(1-chloropropane)12 [also known as bis(2-Chloro-1-methylethyl) ether]GC611
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
88. PCB-1016GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
89. PCB-1221GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
90. PCB-1232GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
91. PCB-1242GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
92. PCB-1248GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
93. PCB-1254GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
94. PCB-1260GC608.3See footnote,3 p. 43; See footnote.8
GC/MS625.16410 B-2000
95. 1,2,3,7,8-Pentachloro-dibenzofuranGC/MS1613B
96. 2,3,4,7,8-Pentachloro-dibenzofuranGC/MS1613B
97. 1,2,3,7,8,-Pentachloro-dibenzo-p-dioxinGC/MS1613B
98. PentachlorophenolGC6046420 B-2000See footnote,3 p. 140.
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
99. PhenanthreneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
100. PhenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
101. PyreneGC610
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
HPLC6106440 B-2005D4657-92 (98)
102. 2,3,7,8-Tetrachloro-dibenzofuranGC/MS1613B10
103. 2,3,7,8-Tetrachloro-dibenzo-p-dioxinGC/MS613, 625.1,5a 1613B
104. 1,1,2,2-TetrachloroethaneGC6016200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
105. TetrachloroetheneGC6016200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
106. TolueneGC6026200 C-2011
GC/MS624.1, 1624B6200 B-2011
107. 1,2,4-TrichlorobenzeneGC612See footnote,3 p. 130.
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
108. 1,1,1-TrichloroethaneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
109. 1,1,2-TrichloroethaneGC6016200 C-2011See footnote,3 p. 130.
GC/MS624.1, 1624B6200 B-2011
110. TrichloroetheneGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
111. TrichlorofluoromethaneGC6016200 C-2011
GC/MS624.16200 B-2011
112. 2,4,6-TrichlorophenolGC6046420 B-2000
GC/MS625.1, 1625B6410 B-2000See footnote,9 p. 27.
113. Vinyl chlorideGC6016200 C-2011
GC/MS624.1, 1624B6200 B-2011
114. NonylphenolGC/MSD7065-11
115. Bisphenol A (BPA)GC/MSD7065-11
116. p-tert-Octylphenol (OP)GC/MSD7065-11
117. Nonylphenol Monoethoxylate (NP1EO)GC/MSD7065-11
118. Nonylphenol Diethoxylate (NP2EO)GC/MSD7065-11
119. Adsorbable Organic Halides (AOX)Adsorption and Coulometric Titration165011
120. Chlorinated PhenolicsIn Situ Acetylation and GC/MS165311

Table IC notes:

1 All parameters are expressed in micrograms per liter (µg/L) except for Method 1613B, in which the parameters are expressed in picograms per liter (pg/L).

2 The full text of Methods 601-613, 1613B, 1624B, and 1625B are provided at appendix A, Test Procedures for Analysis of Organic Pollutants. The standardized test procedure to be used to determine the method detection limit (MDL) for these test procedures is given at appendix B of this part, Definition and Procedure for the Determination of the Method Detection Limit. These methods are available at: https://www.epa.gov/cwa-methods as individual PDF files.

3 Methods for Benzidine: Chlorinated Organic Compounds, Pentachlorophenol and Pesticides in Water and Wastewater. September 1978. U.S. EPA.

4 Method 624.1 may be used for quantitative determination of acrolein and acrylonitrile, provided that the laboratory has documentation to substantiate the ability to detect and quantify these analytes at levels necessary to comply with any associated regulations. In addition, the use of sample introduction techniques other than simple purge-and-trap may be required. QC acceptance criteria from Method 603 should be used when analyzing samples for acrolein and acrylonitrile in the absence of such criteria in Method 624.1.

5 Method 625.1 may be extended to include benzidine, hexachlorocyclopentadiene, N-nitrosodimethylamine, N-nitrosodi-n-propylamine, and N-nitrosodiphenylamine. However, when they are known to be present, Methods 605, 607, and 612, or Method 1625B, are preferred methods for these compounds.

5a Method 625.1 screening only.

6 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. American Public Health Association (APHA).

7 Each analyst must make an initial, one-time demonstration of their ability to generate acceptable precision and accuracy with Methods 601-603, 1624B, and 1625B in accordance with procedures each in Section 8.2 of each of these Methods. Additionally, each laboratory, on an on-going basis must spike and analyze 10% (5% for Methods 624.1 and 625.1 and 100% for methods 1624B and 1625B) of all samples to monitor and evaluate laboratory data quality in accordance with Sections 8.3 and 8.4 of these methods. When the recovery of any parameter falls outside the quality control (QC) acceptance criteria in the pertinent method, analytical results for that parameter in the unspiked sample are suspect. The results should be reported but cannot be used to demonstrate regulatory compliance. If the method does not contain QC acceptance criteria, control limits of ± three standard deviations around the mean of a minimum of five replicate measurements must be used. These quality control requirements also apply to the Standard Methods, ASTM Methods, and other methods cited.

8 Organochlorine Pesticides and PCBs in Wastewater Using EmporeTM Disk. Revised October 28, 1994. 3M Corporation.

9 Method O-3116-87 is in Open File Report 93-125, Methods of Analysis by U.S. Geological Survey National Water Quality Laboratory—Determination of Inorganic and Organic Constituents in Water and Fluvial Sediments. 1993. USGS.

10 Analysts may use Fluid Management Systems, Inc. Power-Prep system in place of manual cleanup provided the analyst meets the requirements of Method 1613B (as specified in Section 9 of the method) and permitting authorities. Method 1613, Revision B, Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS. Revision B, 1994. U.S. EPA. The full text of this method is provided in appendix A to this part and at https://www.epa.gov/cwa-methods/approved-cwa-methods-organic-compounds.

11 Method 1650, Adsorbable Organic Halides by Adsorption and Coulometric Titration. Revision C, 1997 U.S. EPA. Method 1653, Chlorinated Phenolics in Wastewater by In Situ Acetylation and GCMS. Revision A, 1997 U.S. EPA. The full text for both of these methods is provided at appendix A in part 430 of this chapter, The Pulp, Paper, and Paperboard Point Source Category.

12 The compound was formerly inaccurately labeled as 2,2'-oxybis(2-chloropropane) and bis(2-chloroisopropyl) ether. Some versions of Methods 611, and 1625 inaccurately list the analyte as “bis(2-chloroisopropyl)ether,” but use the correct CAS number of 108-60-1.

Table ID—List of Approved Test Procedures for Pesticides1

ParameterMethodEPA2 7 10Standard methodsASTMOther
1. AldrinGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96 (02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS625.16410 B-2000
2. AmetrynGC507, 619See footnote,3 p. 83; See footnote,9 O-3106-93; See footnote,6 p. S68.
   GC/MS525.2, 625.1See footnote,14 O-1121-91.
3. AminocarbTLCSee footnote,3 p. 94; See footnote,6 p. S60.
   HPLC632
4. AtratonGC619See footnote,3 p. 83; See footnote,6 p. S68.
   GC/MS625.1
5. AtrazineGC507, 619, 608.3See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O-3106-93.
   HPLC/MSSee footnote,12 O-2060-01.
   GC/MS525.1, 525.2, 625.1See footnote,11 O-1126-95.
6. Azinphos methylGC614, 622, 1657See footnote,3 p. 25; See footnote,6 p. S51.
   GC/MS625.1See footnote,11 O-1126-95.
7. BarbanTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   GC/MS625.1
8. α-BHCGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,8 3M0222.
   GC/MS625.156410 B-2000See footnote,11 O-1126-95.
9. β-BHCGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,8 3M0222.
   GC/MS625.16410 B-2000
10. δ-BHCGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,8 3M0222.
   GC/MS625.16410 B-2000
11. γ-BHC (Lindane)GC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS625.156410 B-2000See footnote,11 O-1126-95.
12. CaptanGC617, 608.36630 B-2007D3086-90, D5812-96(02)See footnote,3 p. 7.
13. CarbarylTLCSee footnote,3 p. 94, See footnote,6 p. S60.
   HPLC531.1, 632
   HPLC/MS553See footnote,12 O-2060-01.
   GC/MS625.1See footnote,11 O-1126-95.
14. CarbophenothionGC617, 608.36630 B-2007See footnote,4 page 27; See footnote,6 p. S73.
   GC/MS625.1
15. ChlordaneGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS625.16410 B-2000
16. ChloroprophamTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   GC/MS625.1
17. 2,4-DGC6156640 B-2006See footnote,3 p. 115; See footnote,4 O-3105-83.
   HPLC/MSSee footnote,12 O-2060-01.
18. 4,4-DDDGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3105-83; See footnote,8 3M0222.
   GC/MS625.16410 B-2000
19. 4,4-DDEGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS625.16410 B-2000See footnote,11 O-1126-95.
20. 4,4-DDTGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS625.16410 B-2000
21. Demeton-OGC614, 622See footnote,3 p. 25; See footnote,6 p. S51.
   GC/MS625.1
22. Demeton-SGC614, 622See footnote,3 p. 25; See footnote,6 p. S51.
   GC/MS625.1
23. DiazinonGC507, 614, 622, 1657See footnote,3 p. 25; See footnote,4 O-3104-83; See footnote,6 p. S51.
   GC/MS525.2, 625.1See footnote,11 O-1126-95.
24. DicambaGC615See footnote,3 p. 115.
   HPLC/MSSee footnote,12 O-2060-01.
25. DichlofenthionGC622.1See footnote,4 page 27; See footnote,6 p. S73.
26. DichloranGC608.2, 617, 608.36630 B-2007See footnote,3 p. 7.
27. DicofolGC617, 608.3See footnote,4 O-3104-83.
28. DieldrinGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS625.16410 B-2000See footnote,11 O-1126-95.
29. DioxathionGC614.1, 1657See footnote,4 page 27; See footnote,6 p. S73.
30. DisulfotonGC507, 614, 622, 1657See footnote,3 p. 25; See footnote,6 p. S51.
   GC/MS525.2, 625.1See footnote,11 O-1126-95.
31. DiuronTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   HPLC/MS553See footnote,12 O-2060-01.
32. Endosulfan IGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222).
   GC/MS625.156410 B-2000See footnote,13 O-2002-01.
33. Endosulfan IIGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,8 3M0222.
   GC/MS625.156410 B-2000See footnote,13 O-2002-01.
34. Endosulfan SulfateGC617, 608.36630 C-2007See footnote,8 3M0222.
   GC/MS625.16410 B-2000
35. EndrinGC505, 508, 617, 1656, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS525.1, 525.2, 625.156410 B-2000
36. Endrin aldehydeGC617, 608.36630 C-2007See footnote,8 3M0222.
   GC/MS625.1
37. EthionGC614, 614.1, 1657See footnote,4 page 27; See footnote,6 p. S73.
   GC/MS625.1See footnote,13 O-2002-01.
38. FenuronTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   HPLC/MSSee footnote,12 O-2060-01.
39. Fenuron-TCATLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
40. HeptachlorGC505, 508, 617, 1656, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS525.1, 525.2, 625.16410 B-2000
41. Heptachlor epoxideGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,6 p. S73; See footnote,8 3M0222.
   GC/MS625.16410 B-2000
42. IsodrinGC617, 608.36630 B-2007 & C-2007See footnote,4 O-3104-83; See footnote,6 p. S73.
   GC/MS625.1
43. LinuronGCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   HPLC/MS553See footnote,12 O-2060-01.
   GC/MSSee footnote,11 O-1126-95.
44. MalathionGC614, 16576630 B-2007See footnote,3 p. 25; See footnote,6 p. S51.
   GC/MS625.1See footnote,11 O-1126-95.
45. MethiocarbTLCSee footnote,3 p. 94; See footnote,6 p. S60.
   HPLC632
   HPLC/MSSee footnote,12 O-2060-01.
46. MethoxychlorGC505, 508, 608.2, 617, 1656, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83; See footnote,8 3M0222.
   GC/MS525.1, 525.2, 625.1See footnote,11 O-1126-95.
47. MexacarbateTLCSee footnote,3 p. 94; See footnote,6 p. S60.
   HPLC632
   GC/MS625.1
48. MirexGC617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote,4 O-3104-83.
   GC/MS625.1
49. MonuronTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
50. Monuron-TCATLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
51. NeburonTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   HPLC/MSSee footnote,12 O-2060-01.
52. Parathion methylGC614, 622, 16576630 B-2007See footnote,4 page 27; See footnote,3 p. 25.
   GC/MS625.1See footnote,11 O-1126-95.
53. Parathion ethylGC6146630 B-2007See footnote,4 page 27; See footnote,3 p. 25.
   GC/MSSee footnote,11 O-1126-95.
54. PCNBGC608.1, 617, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7.
55. PerthaneGC617, 608.3D3086-90, D5812-96(02)See footnote,4 O-3104-83.
56. PrometonGC507, 619See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O-3106-93.
   GC/MS525.2, 625.1See footnote,11 O-1126-95.
57. PrometrynGC507, 619See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O-3106-93.
   GC/MS525.1, 525.2, 625.1See footnote,13 O-2002-01.
58. PropazineGC507, 619, 1656, 608.3See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O-3106-93.
   GC/MS525.1, 525.2, 625.1
59. ProphamTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   HPLC/MSSee footnote,12 O-2060-01.
60. PropoxurTLCSee footnote,3 p. 94; See footnote,6 p. S60.
   HPLC632
61. SecbumetonTLCSee footnote,3 p. 83; See footnote,6 p. S68.
   GC619
62. SiduronTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
   HPLC/MSSee footnote,12 O-2060-01.
63. SimazineGC505, 507, 619, 1656, 608.3See footnote,3 p. 83; See footnote,6 p. S68; See footnote,9 O-3106-93.
   GC/MS525.1, 525.2, 625.1See footnote,11 O-1126-95.
64. StrobaneGC617, 608.36630 B-2007 & C-2007See footnote,3 p. 7.
65. SwepTLCSee footnote,3 p. 104; See footnote,6 p. S64.
   HPLC632
66. 2,4,5-TGC6156640 B-2006See footnote,3 p. 115; See footnote,4 O-3105-83.
67. 2,4,5-TP (Silvex)GC6156640 B-2006See footnote,3 p. 115; See footnote,4 O-3105-83.
68. TerbuthylazineGC619, 1656, 608.3See footnote,3 p. 83; See footnote,6 p. S68.
   GC/MSSee footnote,13 O-2002-01.
69. ToxapheneGC505, 508, 617, 1656, 608.36630 B-2007 & C-2007D3086-90, D5812-96(02)See footnote,3 p. 7; See footnote;8 See footnote,4 O-3105-83.
   GC/MS525.1, 525.2, 625.16410 B-2000
70. TrifluralinGC508, 617, 627, 1656, 608.36630 B-2007See footnote,3 p. 7; See footnote,9 O-3106-93.
   GC/MS525.2, 625.1See footnote,11 O-1126-95.

Table ID notes:

1Pesticides are listed in this table by common name for the convenience of the reader. Additional pesticides may be found under Table IC of this section, where entries are listed by chemical name.

2The standardized test procedure to be used to determine the method detection limit (MDL) for these test procedures is given at appendix B of this part, Definition and Procedure for the Determination of the Method Detection Limit.

3Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol and Pesticides in Water and Wastewater. September 1978. U.S. EPA. This EPA publication includes thin-layer chromatography (TLC) methods.

4Methods 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. USGS.

5The method may be extended to include α-BHC, γ-BHC, endosulfan I, endosulfan II, and endrin. However, when they are known to exist, Method 608.3 is the preferred method.

6Selected 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. American Public Health Association (APHA).

7Each analyst must make an initial, one-time, demonstration of their ability to generate acceptable precision and accuracy with Methods 608.3 and 625.1 in accordance with procedures given in Section 8.2 of each of these methods. Additionally, each laboratory, on an on-going basis, must spike and analyze 5% of all samples analyzed with Method 608.3 or 5% of all samples analyzed with Method 625.1 to monitor and evaluate laboratory data quality in accordance with Sections 8.3 and 8.4 of these methods. When the recovery of any parameter falls outside the warning limits, the analytical results for that parameter in the unspiked sample are suspect. The results should be reported, but cannot be used to demonstrate regulatory compliance. These quality control requirements also apply to the Standard Methods, ASTM Methods, and other methods cited.

8Organochlorine Pesticides and PCBs in Wastewater Using EmporeTM Disk. Revised October 28, 1994. 3M Corporation.

9Method O-3106-93 is in Open File Report 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. USGS.

10EPA Methods 608.1, 608.2, 614, 614.1, 615, 617, 619, 622, 622.1, 627, and 632 are found in Methods for the Determination of Nonconventional Pesticides in Municipal and Industrial Wastewater, EPA 821-R-92-002, April 1992, U.S. EPA. EPA Methods 505, 507, 508, 525.1, 531.1 and 553 are in Methods for the Determination of Nonconventional Pesticides in Municipal and Industrial Wastewater, Volume II, EPA 821-R-93-010B, 1993, U.S. EPA. EPA Method 525.2 is in Determination of Organic Compounds in Drinking Water by Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry, Revision 2.0, 1995, U.S. EPA. EPA methods 1656 and 1657 are in Methods for the Determination of Nonconventional Pesticides in Municipal and Industrial Wastewater, Volume I, EPA 821-R-93-010A, 1993, U.S. EPA. Methods 608.3 and 625.1 are available at https://www.epa.gov/cwa-methods/approved-cwa-test-methods-organic-compounds.

11Method O-1126-95 is in Open-File Report 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. USGS.

12Method O-2060-01 is in Water-Resources Investigations 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. USGS.

13Method O-2002-01 is in Water-Resources Investigations Report 01-4098, Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of moderate-use pesticides in water by C-18 solid-phase extraction and capillary-column gas chromatography/mass spectrometry. 2001. USGS.

14Method O-1121-91 is in Open-File Report 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. USGS.

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 literProportional or scintillation counter900.07110 B7110 B-00D1943-90, 96pp. 75 and 783
2. Alpha-Counting error, pCi per literProportional or scintillation counterAppendix B7110 B7110 B-00D1943-90, 96p. 79
3. Beta-Total, pCi per literProportional counter900.07110 B7110 B-00D1890-90, 96pp. 75 and 783
4. Beta-Counting error, pCiProportional counterAppendix B7110 B7110 B-00D1890-90, 96p. 79
5. (a) Radium Total pCi per liter
(b) Ra, pCi per liter
Proportional counter903.07500-Ra B7500-Ra B-01D2460-90, 97
   Scintillation counter903.17500-Ra C7500-Ra C-01D3454-91, 97p. 81

1Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA-600/4-80-032 (1980), U.S. Environmental Protection Agency, August 1980.

2Fishman, M. J. and Brown, Eugene, “Selected Methods of the U.S. Geological Survey of Analysis of Wastewaters,” U.S. Geological Survey, Open-File Report 76-177 (1976).

3The method found on p. 75 measures only the dissolved portion while the method on p. 78 measures only the suspended portion. Therefore, the two results must be added to obtain the “total.”

Table IF—List of Approved Methods for Pharmaceutical Pollutants

Pharmaceuticals pollutantsCAS registry No.Analytical method number
Acetonitrile75-05-81666/1671/D3371/D3695/624.1
n-Amyl acetate628-63-71666/D3695
n-Amyl alcohol71-41-01666/D3695
Benzene71-43-2D4763/D3695/502.2/524.2/624.1
n-Butyl-acetate123-86-41666/D3695
tert-Butyl alcohol75-65-01666/624.1
Chlorobenzene108-90-7502.2/524.2/624.1
Chloroform67-66-3502.2/524.2/551/624.1
o-Dichlorobenzene95-50-11625C/502.2/524.2/624.1
1,2-Dichloroethane107-06-2D3695/502.2/524.2/624.1
Diethylamine109-89-71666/1671
Dimethyl sulfoxide67-68-51666/1671
Ethanol64-17-51666/1671/D3695/624.1
Ethyl acetate141-78-61666/D3695/624.1
n-Heptane142-82-51666/D3695
n-Hexane110-54-31666/D3695
Isobutyraldehyde78-84-21666/1667
Isopropanol67-63-01666/D3695
Isopropyl acetate108-21-41666/D3695
Isopropyl ether108-20-31666/D3695
Methanol67-56-11666/1671/D3695/624.1
Methyl Cellosolve® (2-Methoxy ethanol)109-86-41666/1671
Methylene chloride75-09-2502.2/524.2/624.1
Methyl formate107-31-31666
4-Methyl-2-pentanone (MIBK)108-10-11624C/1666/D3695/D4763/524.2/624.1
Phenol108-95-2D4763
n-Propanol71-23-81666/1671/D3695/624.1
2-Propanone (Acetone)67-64-1D3695/D4763/524.2/624.1
Tetrahydrofuran109-99-91666/524.2/624.1
Toluene108-88-3D3695/D4763/502.2/524.2/624.1
Triethlyamine121-44-81666/1671
Xylenes(Note 1)1624C/1666/624.1

Table IF note:

11624C: m-xylene 108-38-3, o,p-xylene, E-14095 (Not a CAS number; this is the number provided in the Environmental Monitoring Methods Index [EMMI] database.); 1666: m,p-xylene 136777-61-2, o-xylene 95-47-6.

Table IG—Test Methods for Pesticide Active Ingredients

[40 CFR part 455]

EPA survey codePesticide nameCAS No.EPA analytical method No.(s)3
8Triadimefon43121-43-3507/633/525.1/525.2/1656/625.1.
12Dichlorvos62-73-71657/507/622/525.1/525.2/625.1.
162,4-D; 2,4-D Salts and Esters [2,4-Dichloro-phenoxyacetic acid]94-75-71658/515.1/615/515.2/555.
172,4-DB; 2,4-DB Salts and Esters [2,4-Dichlorophenoxybutyric acid]94-82-61658/515.1/615/515.2/555.
22Mevinphos7786-34-71657/507/622/525.1/525.2/625.1.
25Cyanazine21725-46-2629/507/608.3/625.1.
26Propachlor1918-16-71656/508/608.1/525.1/525.2/608.3/625.1.
27MCPA; MCPA Salts and Esters
[2-Methyl-4-chlorophenoxyacetic acid]
94-74-61658/615/555.
30Dichlorprop; Dichlorprop Salts and Esters [2-(2,4-Dichlorophenoxy) propionic acid]120-36-51658/515.1/615/515.2/555.
31MCPP; MCPP Salts and Esters [2-(2-Methyl-4-chlorophenoxy) propionic acid]93-65-21658/615/555.
35TCMTB [2-(Thiocyanomethylthio) benzo-thiazole]21564-17-0637.
39Pronamide23950-58-5525.1/525.2/507/633.1/625.1.
41Propanil709-98-8632.1/1656/608.3.
45Metribuzin21087-64-9507/633/525.1/525.2/1656/608.3/625.1.
52Acephate30560-19-11656/1657/608.3.
53Acifluorfen50594-66-6515.1/515.2/555.
54Alachlor15972-60-8505/507/645/525.1/525.2/1656/608.3/625.1.
55Aldicarb116-06-3531.1.
58Ametryn834-12-8507/619/525.2/625.1.
60Atrazine1912-24-9505/507/619/525.1/525.2/1656/ 608.3/625.1.
62Benomyl17804-35-2631.
68Bromacil; Bromacil Salts and Esters314-40-9507/633/525.1/525.2/1656/608.3/625.1.
69Bromoxynil1689-84-51625/1661/625.1.
69Bromoxynil Octanoate1689-99-21656/608.3.
70Butachlor23184-66-9507/645/525.1/525.2/1656/608.3/625.1.
73Captafol2425-06-11656/608.3/625.1.
75Carbaryl [Sevin]63-25-2531.1/632/553/625.1.
76Carbofuran1563-66-2531.1/632/625.1.
80Chloroneb2675-77-61656/508/608.1/525.1/525.2/608.3/625.1.
82Chlorothalonil1897-45-6508/608.2/525.1/525.2/1656/608.3/625.1.
84Stirofos961-11-51657/507/622/525.1/525.2/625.1.
86Chlorpyrifos2921-88-21657/508/622/625.1.
90Fenvalerate51630-58-11660.
103Diazinon333-41-51657/507/614/622/525.2/625.1.
107Parathion methyl298-00-01657/614/622/625.1.
110DCPA [Dimethyl 2,3,5,6-tetrachloro-terephthalate]1861-32-1508/608.2/525.1/525.2/515.12/515.22/1656/608.3/625.1.
112Dinoseb88-85-71658/515.1/615/515.2/555/625.1.
113Dioxathion78-34-21657/614.1.
118Nabonate [Disodium cyanodithio-imidocarbonate]138-93-2630.1.
119Diuron330-54-1632/553.
123Endothall145-73-3548/548.1.
124Endrin72-20-81656/505/508/617/525.1/525.2/608.3/625.1.
125Ethalfluralin55283-68-61656/627/608.3 See footnote 1.
126Ethion563-12-21657/614/614.1/625.1.
127Ethoprop13194-48-41657/507/622/525.1/525.2/625.1.
132Fenarimol60168-88-9507/633.1/525.1/525.2/1656/608.3/625.1.
133Fenthion55-38-91657/622/625.1.
138Glyphosate [N-(Phosphonomethyl) glycine]1071-83-6547.
140Heptachlor76-44-81656/505/508/617/525.1/525.2/608.3/625.1.
144Isopropalin33820-53-01656/627/608.3.
148Linuron330-55-2553/632.
150Malathion121-75-51657/614/625.1.
154Methamidophos10265-92-61657.
156Methomyl16752-77-5531.1/632.
158Methoxychlor72-43-51656/505/508/608.2/617/525.1/525.2/608.3/625.1.
172Nabam142-59-6630/630.1.
173Naled300-76-51657/622/625.1.
175Norflurazon27314-13-2507/645/525.1/525.2/1656/608.3/625.1.
178Benfluralin1861-40-11656/627/608.3 See footnote 1.
182Fensulfothion115-90-21657/622/625.1.
183Disulfoton298-04-41657/507/614/622/525.2/625.1.
185Phosmet732-11-61657/622.1/625.1.
186Azinphos Methyl86-50-01657/614/622/625.1.
192Organo-tin pesticides12379-54-3Ind-01/200.7/200.9.
197Bolstar35400-43-21657/622.
203Parathion56-38-21657/614/625.1.
204Pendimethalin40487-42-11656.
205Pentachloronitrobenzene82-68-81656/608.1/617/608.3/625.1.
206Pentachlorophenol87-86-51625/515.2/555/515.1/525.1/525.2/625.1.
208Permethrin52645-53-1608.2/508/525.1/525.2/1656/1660/608.34/625.14.
212Phorate298-02-21657/622/625.1.
218Busan 85 [Potassium dimethyldithiocarbamate]128-03-0630/630.1.
219Busan 40 [Potassium N-hydroxymethyl-N-methyldithiocarbamate]51026-28-9630/630.1.
220KN Methyl [Potassium N-methyl-dithiocarbamate]137-41-7630/630.1.
223Prometon1610-18-0507/619/525.2/625.1.
224Prometryn7287-19-6507/619/525.1/525.2/625.1.
226Propazine139-40-2507/619/525.1/525.2/1656/608.3/625.1.
230Pyrethrin I121-21-11660.
232Pyrethrin II121-29-91660.
236DEF [S,S,S-Tributyl phosphorotrithioate]78-48-81657.
239Simazine122-34-9505/507/619/525.1/525.2/1656/608.3/625.1.
241Carbam-S [Sodium dimethyldithio-carbamate]128-04-1630/630.1.
243Vapam [Sodium methyldithiocarbamate]137-42-8630/630.1.
252Tebuthiuron34014-18-1507/525.1/525.2/625.1.
254Terbacil5902-51-2507/633/525.1/525.2/1656/608.3/625.1.
255Terbufos13071-79-91657/507/614.1/525.1/525.2/625.1.
256Terbuthylazine5915-41-3619/1656/608.3.
257Terbutryn886-50-0507/619/525.1/525.2/625.1.
259Dazomet533-74-4630/630.1/1659.
262Toxaphene8001-35-21656/505/508/617/525.1/525.2/608.3/625.1.
263Merphos [Tributyl phosphorotrithioate]150-50-51657/507/525.1/525.2/622/625.1.
264Trifluralin11582-09-81656/508/617/627/525.2/608.3/625.1.
268Ziram [Zinc dimethyldithiocarbamate]137-30-4630/630.1.

Table IG notes:

1Monitor and report as total Trifluralin.

2Applicable to the analysis of DCPA degradates.

3EPA Methods 608.1 through 645, 1645 through 1661, and Ind-01 are available in Methods for the Determination of Nonconventional Pesticides in Municipal and Industrial Wastewater, Volume I, EPA 821-R-93-010A, Revision I, August 1993, U.S. EPA. EPA Methods 200.9 and 505 through 555 are available in Methods for the Determination of Nonconventional Pesticides in Municipal and Industrial Wastewater, Volume II, EPA 821-R-93-010B, August 1993, U.S. EPA. The full text of Methods 608.3, 625.1, and 1625 are provided at appendix A of this part. The full text of Method 200.7 is provided at appendix C of this part. Methods 608.3 and 625.1 are available at https://www.epa.gov/cwa-methods/approved-cwa-test-methods-organic-compounds.

4Permethrin is not listed within methods 608.3 and 625.1; however, cis-permethrin and trans-permethrin are listed. Permethrin can be calculated by adding the results of cis- and trans-permethrin.

Table IH—List of Approved Microbiological Methods for Ambient Water

Parameter and unitsMethod1EPAStandard methodsAOAC, ASTM, USGSOther
Bacteria
1. Coliform (fecal), number per 100 mL or number per gram dry weightMost Probable Number (MPN), 5 tube, 3 dilution, orp. 13239221 C E-2006.
   Membrane filter (MF),2 single stepp. 12439222 D-200627B-0050-85.4
2. Coliform (fecal) in presence of chlorine, number per 100 mLMPN, 5 tube, 3 dilution, orp. 13239221 C E-2006.
   MF,2 single step5p. 12439222 D-2006.27
3. Coliform (total), number per 100 mLMPN, 5 tube, 3 dilution, orp. 11439221 B-2006.
   MF,2 single step or two stepp. 10839222 B-2006B-0025-85.4
4. Coliform (total), in presence of chlorine, number per 100 mLMPN, 5 tube, 3 dilution, orp. 11439221 B-2006.
   MF2 with enrichmentp. 11139222 B-2006.
5. E. coli, number per 100 mLMPN,6 8 14 multiple tube, or9221 B.2-2006/9221 F-200611 13
   Multiple tube/multiple well, or9223 B-200412991.1510Colilert®,12 16 Colilert-18®.12 15 16
   MF,2 5 6 7 8 two step, or1103.1199222 B-2006/9222 G-2006,18 9213 D-2007D-5392-93.9
   Single step1603,20 160421mColiBlue-24®.17
6. Fecal streptococci, number per 100 mLMPN, 5 tube, 3 dilution, orp. 13939230 B-2007.
   MF2, orp. 13639230 C-2007B-0055-854
   Plate countp. 143.3
7. Enterococci, number per 100 mLMPN,6 8 multiple tube/multiple well, or9230 D-2007D6503-999Enterolert®.12 22
   MF2 5 6 7 8 two step, or1106.1239230 C-2007D5259-92.9
   Single step, or1600249230 C-2007.
   Plate countp. 143.3
Protozoa
8. CryptosporidiumFiltration/IMS/FA1622,25 1623.26
9. GiardiaFiltration/IMS/FA1623.26

Table IH notes:

1The method must be specified when results are reported.

2A 0.45-µm membrane filter (MF) or other pore size certified by the manufacturer to fully retain organisms to be cultivated and to be free of extractables which could interfere with their growth.

3Microbiological Methods for Monitoring the Environment, Water, and Wastes. EPA/600/8-78/017. 1978. U.S. EPA.

4U.S. Geological Survey Techniques of Water-Resource Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples. 1989. USGS.

5Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Most Probable Number method will be required to resolve any controversies.

6Tests must be conducted to provide organism enumeration (density). Select the appropriate configuration of tubes/filtrations and dilutions/volumes to account for the quality, character, consistency, and anticipated organism density of the water sample.

7When the MF method has not been used previously to test waters with high turbidity, large numbers of noncoliform bacteria, or samples that may contain organisms stressed by chlorine, a parallel test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of results.

8To assess the comparability of results obtained with individual methods, it is suggested that side-by-side tests be conducted across seasons of the year with the water samples routinely tested in accordance with the most current Standard Methods for the Examination of Water and Wastewater or EPA alternate test procedure (ATP) guidelines.

9Annual Book of ASTM Standards—Water and Environmental Technology. Section 11.02. 2000, 1999, 1996. ASTM International.

10Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. 1995. AOAC International.

11The multiple-tube fermentation test is used in 9221B.2-2006. Lactose broth may be used in lieu of lauryl tryptose broth (LTB), if at least 25 parallel tests are conducted between this broth and LTB using the water samples normally tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total coliform using lactose broth is less than 10 percent. No requirement exists to run the completed phase on 10 percent of all total coliform-positive tubes on a seasonal basis.

12These tests are collectively known as defined enzyme substrate tests, where, for example, a substrate is used to detect the enzyme β-glucuronidase produced byE. coli.

13After prior enrichment in a presumptive medium for total coliform using 9221B.2-2006, all presumptive tubes or bottles showing any amount of gas, growth or acidity within 48 h ± 3 h of incubation shall be submitted to 9221F-2006. Commercially available EC-MUG media or EC media supplemented in the laboratory with 50 µg/mL of MUG may be used.

14Samples shall be enumerated by the multiple-tube or multiple-well procedure. Using multiple-tube procedures, employ an appropriate tube and dilution configuration of the sample as needed and report the Most Probable Number (MPN). Samples tested with Colilert® may be enumerated with the multiple-well procedures, Quanti-Tray® or Quanti-Tray®/2000, and the MPN calculated from the table provided by the manufacturer.

15Colilert-18® is an optimized formulation of the Colilert® for the determination of total coliforms andE. coli that provides results within 18 h of incubation at 35 °C, rather than the 24 h required for the Colilert® test, and is recommended for marine water samples.

16Descriptions of the Colilert®, Colilert-18®, and Quanti-Tray® may be obtained from IDEXX Laboratories Inc.

17A description of the mColiBlue24® test may be obtained from Hach Company.

18Subject total coliform positive samples determined by 9222B-2006 or other membrane filter procedure to 9222G-2006 using NA-MUG media.

19Method 1103.1:Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-ThermotolerantEscherichia coli Agar (mTEC), EPA-821-R-10-002. March 2010. U.S. EPA.

20Method 1603:Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-ThermotolerantEscherichia coli Agar (Modified mTEC), EPA-821-R-14-010. September 2014. U.S. EPA.

21Preparation and use of MI agar with a standard membrane filter procedure is set forth in the article, Brenner et al. 1993. New Medium for the Simultaneous Detection of Total Coliform andEscherichia coli in Water. Appl. Environ. Microbiol. 59:3534-3544 and in Method 1604: Total Coliforms andEscherichia coli (E. coli) in Water by Membrane Filtration by Using a Simultaneous Detection Technique (MI Medium), EPA 821-R-02-024, September 2002, U.S. EPA.

22A description of the Enterolert® test may be obtained from IDEXX Laboratories Inc.

23Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA), EPA-821-R-09-015. December 2009. U.S. EPA.

24Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI), EPA-821-R-14-011. September 2014. U.S. EPA.

25Method 1622 uses a filtration, concentration, immunomagnetic separation of oocysts from captured material, immunofluorescence assay to determine concentrations, and confirmation through vital dye staining and differential interference contrast microscopy for the detection ofCryptosporidium. Method 1622:Cryptosporidium in Water by Filtration/IMS/FA, EPA-821-R-05-001. December 2005. U.S. EPA.

26Method 1623 uses a filtration, concentration, immunomagnetic separation of oocysts and cysts from captured material, immunofluorescence assay to determine concentrations, and confirmation through vital dye staining and differential interference contrast microscopy for the simultaneous detection ofCryptosporidium andGiardia oocysts and cysts. Method 1623:Cryptosporidium andGiardia in Water by Filtration/IMS/FA. EPA-821-R-05-002. December 2005. U.S. EPA.

27On a monthly basis, at least ten blue colonies from the medium must be verified using Lauryl Tryptose Broth and EC broth, followed by count adjustment based on these results; and representative non-blue colonies should be verified using Lauryl Tryptose Broth. Where possible, verifications should be done from randomized sample sources.

(b) Certain material is incorporated by reference into this part 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 is available for inspection at EPA's Water Docket, EPA West, 1301 Constitution Avenue NW., Room 3334, Washington, DC 20004, Telephone: 202-566-2426, and is available from the sources listed below. It is also available for inspection at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: https://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 (US EPA). Available at http://water.epa.gov/scitech/methods/cwa/index.cfm.

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

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

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

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

(xiii) 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.

(xiv) 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.

(xv) 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. 2011. 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. 2011. 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). 2011. Table IB.

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

(xlvi) 4500-O, Oxygen (Dissolved). 2011. 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). 2011. Table IB.

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

(liv) 5310, Total Organic Carbon (TOC). 2011. 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. 2006. Table IA, Notes 12 and 14; Table IH, Notes 11 and 13.

(lxviii) 9222, Membrane Filter Technique for Members of the Coliform Group. 2006. Table IA; Table IH, Note 18.

(lxix) 9223, Enzyme Substrate Coliform Test. 2004. Table IA; Table IH.

(lxx) 9230, Fecal Enterococcus/Streptococcus Groups. 2007. 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 D511-09, Standard Test Methods for Calcium and Magnesium in Water. May 2009. Table IB.

(vi) ASTM D512-04, Standard Test Methods for Chloride Ion in Water. July 2004. Table IB.

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

(viii) ASTM D516-11, Standard Test Method for Sulfate Ion in Water, September 2011. Table IB.

(ix) ASTM D858-12, Standard Test Methods for Manganese in Water. September 2012. Table IB.

(x) ASTM D859-10, Standard Test Method for Silica in Water. July 2010. Table IB.

(xi) ASTM D888-09, Standard Test Methods for Dissolved Oxygen in Water. December 2009. Table IB.

(xii) ASTM D1067-11, Standard Test Methods for Acidity or Alkalinity of Water. April 2011. Table IB.

(xiii) ASTM D1068-10, Standard Test Methods for Iron in Water. October 2010. Table IB.

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

(xv) ASTM D1126-12, Standard Test Method for Hardness in Water. March 2012. Table IB.

(xvi) ASTM D1179-10, Standard Test Methods for Fluoride Ion in Water. July 2010. Table IB.

(xvii) ASTM D1246-10, Standard Test Method for Bromide Ion in Water. July 2010. Table IB.

(xviii) ASTM D1252-06, Standard Test Methods for Chemical Oxygen Demand (Dichromate Oxygen Demand) of Water. February 2006. Table IB.

(xix) ASTM D1253-08, Standard Test Method for Residual Chlorine in Water. October 2008. Table IB.

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

(xxi) ASTM D1426-08, Standard Test Methods for Ammonia Nitrogen in Water. September 2008. Table IB.

(xxii) ASTM D1687-12 (Approved September 1, 2012), Standard Test Methods for Chromium in Water. August 2007. Table IB.

(xxiii) ASTM D1688-12, Standard Test Methods for Copper in Water. September 2012. Table IB.

(xxiv) ASTM D1691-12, Standard Test Methods for Zinc in Water. September 2012. Table IB.

(xxv) ASTM D1783-01 (Reapproved 2007), Standard Test Methods for Phenolic Compounds in Water. January 2008). Table IB.

(xxvi) ASTM D1886-08, Standard Test Methods for Nickel in Water. October 2008. 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, Standard Test Methods for Cyanides in Water. October 2009. 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 D2972-08, Standard Tests Method for Arsenic in Water. October 2008. Table IB.

(xxxv) ASTM D3223-12, Standard Test Method for Total Mercury in Water. September 2012. Table IB.

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

(xxxvii) ASTM D3373-12, Standard Test Method for Vanadium in Water. September 2012. Table IB.

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

(xxxix) ASTM D3557-12, Standard Test Method for Cadmium in Water. September 2012. Table IB.

(xl) ASTM D3558-08, Standard Test Method for Cobalt in Water. November 2008. Table IB.

(xli) ASTM D3559-08, Standard Test Methods for Lead in Water. October 2008. Table IB.

(xlii) ASTM D3590-11, Standard Test Methods for Total Kjeldahl Nitrogen in Water. April 2011. Table IB.

(xliii) ASTM D3645-08, Standard Test Methods for Beryllium in Water. October 2008. 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 D3859-08, Standard Test Methods for Selenium in Water. October 2008. Table IB.

(xlvi) ASTM D3867-04, Standard Test Method for Nitrite-Nitrate in Water. July 2004. Table IB.

(xlvii) ASTM D4190-08, Standard Test Method for Elements in Water by Direct-Current Plasma Atomic Emission Spectroscopy. October 2008. Table IB.

(xlviii) ASTM D4282-02, Standard Test Method for Determination of Free Cyanide in Water and Wastewater by Microdiffusion. August 2002. Table IB.

(xlix) ASTM D4327-03, Standard Test Method for Anions in Water by Chemically Suppressed Ion Chromatography. January 2003. Table IB.

(l) ASTM D4382-12, Standard Test Method for Barium in Water, Atomic Absorption Spectrophotometry, Graphite Furnace. September 2012. Table IB.

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

(lii) ASTM D4658-09, Standard Test Method for Sulfide Ion in Water. May 2009. 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, Standard Test Method for Total Carbon and Organic Carbon in Water by Ultraviolet, or Persulfate Oxidation, or Both, and Infrared Detection. January 2003. Table IB.

(lv) ASTM D5257-11, Standard Test Method for Dissolved Hexavalent Chromium in Water by Ion Chromatography. April 2011. 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 D5673-10, Standard Test Method for Elements in Water by Inductively Coupled Plasma—Mass Spectrometry. September 2010. 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. D6508-10, Standard Test Method for Determination of Dissolved Inorganic Anions in Aqueous Matrices Using Capillary Ion Electrophoresis and Chromate Electrolyte. October 2010. Table IB, Note 54.

(lxii) ASTM. D6888-09, Standard Test Method for Available Cyanide with Ligand Displacement and Flow Injection Analysis (FIA) Utilizing Gas Diffusion Separation and Amperometric Detection. October 2009. Table IB, Note 59.

(lxiii) ASTM. D6919-09, Standard Test Method for Determination of Dissolved Alkali and Alkaline Earth Cations and Ammonium in Water and Wastewater by Ion Chromatography. May 2009. Table IB.

(lxiv) ASTM. D7065-11, 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. July 2011. Table IB.

(lxv) ASTM. D7237-10, Standard Test Method for Free Cyanide with Flow Injection Analysis (FIA) Utilizing Gas Diffusion Separation and Amperometric Detection. June 2010. Table IB.

(lxvi) ASTM. D7284-13, Standard Test Method for Total Cyanide in Water by Micro Distillation followed by Flow Injection Analysis with Gas Diffusion Separation and Amperometric Detection. July 2013. 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, Standard Test Method for Total Cyanide by Segmented Flow Injection Analysis, In-Line Ultraviolet Digestion and Amperometric Detection. January 2012. Table IB.

(lxix) ASTM. D7573-09, Standard Test Method for Total Carbon and Organic Carbon in Water by High Temperature Catalytic Combustion and Infrared Detection. November 2009. 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) 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.

(20) 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.

(21) 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.

(22) 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]

(23) 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]

(24) 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.

(25) 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.

(26) 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]

(27) 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]

(28) 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.

(29) 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]

(30) 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]

(31) 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]

(32) 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]

(33) 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]

(34) 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]

(35) 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) 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.

(iii) 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, Note 2.

(iv) 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.

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

(vi) 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.

(vii) 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.

(viii) 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; Table IC, Note 9.

(ix) 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.

(x) 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.

(xi) 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.

(xii) 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.

(xiii) 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, Note 50.

(xiv) 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.

(xv) 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.

(xvi) 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.

(xvii) 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.

(xviii) 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.

(xix) 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.

(xx) 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 Chormatography/Mass Spectrometry. 2001. Table ID, Note 12.

(xxi) 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.

(36) 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/nameContainer1Preservation2 3Maximum holding time4
Table IA—Bacterial Tests
1-5. Coliform, total, fecal, and E. coliPA, GCool, <10 °C, 0.008% Na2S2O358 hours.22 23
6. Fecal streptococciPA, GCool, <10 °C, 0.008% Na2S2O358 hours.22
7. EnterococciPA, GCool, <10 °C, 0.008% Na2S2O358 hours.22
8. SalmonellaPA, GCool, <10 °C, 0.008% Na2S2O358 hours.22
Table IA—Aquatic Toxicity Tests
9-12. Toxicity, acute and chronicP, FP, GCool, ≤6 °C1636 hours.
Table IB—Inorganic Tests
1. AcidityP, FP, GCool, ≤6 °C1814 days.
2. AlkalinityP, FP, GCool, ≤6 °C1814 days.
4. AmmoniaP, FP, GCool, ≤6 °C,18 H2SO4 to pH <228 days.
9. Biochemical oxygen demandP, FP, GCool, ≤6 °C1848 hours.
10. BoronP, FP, or QuartzHNO3 to pH <26 months.
11. BromideP, FP, GNone required28 days.
14. Biochemical oxygen demand, carbonaceousP, FP GCool, ≤6 °C1848 hours.
15. Chemical oxygen demandP, FP, GCool, ≤6 °C,18 H2SO4 to pH <228 days.
16. ChlorideP, FP, GNone required28 days.
17. Chlorine, total residualP, GNone requiredAnalyze within 15 minutes.
21. ColorP, FP, GCool, ≤6 °C1848 hours.
23-24. Cyanide, total or available (or CATC) and freeP, FP, GCool, ≤6 °C,18 NaOH to pH >10,5 6 reducing agent if oxidizer present14 days.
25. FluoridePNone required28 days.
27. HardnessP, FP, GHNO3 or H2SO4 to pH <26 months.
28. Hydrogen ion (pH)P, FP, GNone requiredAnalyze within 15 minutes.
31, 43. Kjeldahl and organic NP, FP, GCool, ≤6 °C,18 H2SO4 to pH <228 days.
Table IB—Metals7
18. Chromium VIP, FP, GCool, ≤6 °C,18 pH = 9.3-9.72028 days.
35. Mercury (CVAA)P, FP, GHNO3 to pH <228 days.
35. Mercury (CVAFS)FP, G; and FP-lined cap175 mL/L 12N HCl or 5 mL/L BrCl1790 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 mercuryP, FP, GHNO3 to pH <2, or at least 24 hours prior to analysis196 months.
38. NitrateP, FP, GCool, ≤6 °C1848 hours.
39. Nitrate-nitriteP, FP, GCool, ≤6 °C,18H2SO4 to pH <228 days.
40. NitriteP, FP, GCool, ≤6 °C1848 hours.
41. Oil and greaseGCool to ≤6 °C,18 HCl or H2SO4 to pH <228 days.
42. Organic CarbonP, FP, GCool to ≤6 °C,18 HCl, H2SO4, or H3PO4 to pH <228 days.
44. OrthophosphateP, FP, GCool, to ≤6 °C18 24Filter within 15 minutes; Analyze within 48 hours.
46. Oxygen, Dissolved ProbeG, Bottle and topNone requiredAnalyze within 15 minutes.
47. WinklerG, Bottle and topFix on site and store in dark8 hours.
48. PhenolsGCool, ≤6 °C,18 H2SO4 to pH <228 days.
49. Phosphorous (elemental)GCool, ≤6 °C1848 hours.
50. Phosphorous, totalP, FP, GCool, ≤6 °C,18 H2SO4 to pH <228 days.
53. Residue, totalP, FP, GCool, ≤6 °C187 days.
54. Residue, FilterableP, FP, GCool, ≤6 °C187 days.
55. Residue, Nonfilterable (TSS)P, FP, GCool, ≤6 °C187 days.
56. Residue, SettleableP, FP, GCool, ≤6 °C1848 hours.
57. Residue, VolatileP, FP, GCool, ≤6 °C187 days.
61. SilicaP or QuartzCool, ≤6 °C1828 days.
64. Specific conductanceP, FP, GCool, ≤6 °C1828 days.
65. SulfateP, FP, GCool, ≤6 °C1828 days.
66. SulfideP, FP, GCool, ≤6 °C,18 add zinc acetate plus sodium hydroxide to pH >97 days.
67. SulfiteP, FP, GNone requiredAnalyze within 15 minutes.
68. SurfactantsP, FP, GCool, ≤6 °C1848 hours.
69. TemperatureP, FP, GNone requiredAnalyze within 15 minutes.
73. TurbidityP, FP, GCool, ≤6 °C1848 hours.
Table IC—Organic Tests8
13, 18-20, 22, 24, 25, 27,28, 34-37, 39-43, 45-47, 56, 76, 104, 105, 108-111, 113. Purgeable HalocarbonsG, FP-lined septumCool, ≤6 °C,18 0.008% Na2S2O3,5 HCl to pH 214 days.
26. 2-Chloroethylvinyl etherG, FP-lined septumCool, ≤6 °C,18 0.008% Na2S2O3514 days.
6, 57, 106. Purgeable aromatic hydrocarbonsG, FP-lined septumCool, ≤6 °C,18 0.008% Na2S2O3,5 HCl to pH 2914 days.9
3, 4. Acrolein and acrylonitrileG, FP-lined septumCool, ≤6 °C,18 0.008% Na2S2O3, pH to 4-51014 days.10
23, 30, 44, 49, 53, 77, 80, 81, 98, 100, 112. Phenols11G, FP-lined capCool, ≤6 °C,18 0.008% Na2S2O37 days until extraction, 40 days after extraction.
7, 38. Benzidines11 12G, FP-lined capCool, ≤6 °C,18 0.008% Na2S2O357 days until extraction.13
14, 17, 48, 50-52. Phthalate esters11G, FP-lined capCool, ≤6 °C187 days until extraction, 40 days after extraction.
82-84. Nitrosamines11 14G, FP-lined capCool, ≤6 °C,18 store in dark, 0.008% Na2S2O357 days until extraction, 40 days after extraction.
88-94. PCBs11G, FP-lined capCool, ≤6 °C181 year until extraction, 1 year after extraction.
54, 55, 75, 79. Nitroaromatics and isophorone11G, FP-lined capCool, ≤6 °C,18 store in dark, 0.008% Na2S2O357 days until extraction, 40 days after extraction.
1, 2, 5, 8-12, 32, 33, 58, 59, 74, 78, 99, 101. Polynuclear aromatic hydrocarbons11G, FP-lined capCool, ≤6 °C,18 store in dark, 0.008% Na2S2O357 days until extraction, 40 days after extraction.
15, 16, 21, 31, 87. Haloethers11G, FP-lined capCool, ≤6 °C,18 0.008% Na2S2O357 days until extraction, 40 days after extraction.
29, 35-37, 63-65, 107. Chlorinated hydrocarbons11G, FP-lined capCool, ≤6 °C187 days until extraction, 40 days after extraction.
60-62, 66-72, 85, 86, 95-97, 102, 103. CDDs/CDFs11GSee footnote 11See footnote 11.
Aqueous Samples: Field and Lab PreservationGCool, ≤6 °C,18 0.008% Na2S2O3,5 pH <91 year.
Solids and Mixed-Phase Samples: Field PreservationGCool, ≤6 °C187 days.
Tissue Samples: Field PreservationGCool, ≤6 °C1824 hours.
Solids, Mixed-Phase, and Tissue Samples: Lab PreservationGFreeze, ≤−10 °C1 year.
114-118. Alkylated phenolsGCool, <6 °C, H2SO4 to pH <228 days until extraction, 40 days after extraction.
119. Adsorbable Organic Halides (AOX)GCool, <6 °C, 0.008% Na2S2O3, HNO3 to pH <2Hold at least 3 days, but not more than 6 months.
120. Chlorinated PhenolicsG, FP-lined capCool, <6 °C, 0.008% Na2S2O3, H2SO4 to pH <230 days until acetylation, 30 days after acetylation.
Table ID—Pesticides Tests
1-70. Pesticides11G, FP-lined capCool, ≤6 °C,18 pH 5-9157 days until extraction, 40 days after extraction.
Table IE—Radiological Tests
1-5. Alpha, beta, and radiumP, FP, GHNO3 to pH <26 months.
Table IH—Bacterial Tests
1-4. Coliform, total, fecalPA, GCool, <10 °C, 0.008% Na2S2O358 hours.22 23
5. E. coliPA, GCool, <10 °C, 0. 008% Na2S2O358 hours.22
6. Fecal streptococciPA, GCool, <10 °C, 0.008% Na2S2O358 hours.22
7. EnterococciPA, GCool, <10 °C, 0. 008% Na2S2O358 hours.22
Table IH—Protozoan Tests
8. CryptosporidiumLDPE; field filtration1-10 °C96 hours.21
9. GiardiaLDPE; field filtration1-10 °C96 hours.21

1“P” is for polyethylene; “FP” is fluoropolymer (polytetrafluoroethylene (PTFE); Teflon®), or other fluoropolymer, unless stated otherwise in this Table II; “G” is glass; “PA” is any plastic that is made of a sterilizable material (polypropylene or other autoclavable plastic); “LDPE” is low density polyethylene.

2Except where noted in this Table II and the method for the parameter, preserve each grab sample within 15 minutes of collection. For a composite sample collected with an automated sample (e.g., using a 24-hour composite sample; see 40 CFR 122.21(g)(7)(i) or 40 CFR part 403, appendix E), refrigerate the sample at ≤6 °C during collection unless specified otherwise in this Table II or in the method(s). For a composite sample to be split into separate aliquots for preservation and/or analysis, maintain the sample at ≤6 °C, unless specified otherwise in this Table II or in the method(s), until collection, splitting, and preservation is completed. Add the preservative to the sample container prior to sample collection when the preservative will not compromise the integrity of a grab sample, a composite sample, or aliquot split from a composite sample within 15 minutes of collection. If a composite measurement is required but a composite sample would compromise sample integrity, individual grab samples must be collected at prescribed time intervals (e.g., 4 samples over the course of a day, at 6-hour intervals). Grab samples must be analyzed separately and the concentrations averaged. Alternatively, grab samples may be collected in the field and composited in the laboratory if the compositing procedure produces results equivalent to results produced by arithmetic averaging of results of analysis of individual grab samples. For examples of laboratory compositing procedures, see EPA Method 1664 Rev. A (oil and grease) and the procedures at 40 CFR 141.24(f)(14)(iv) and (v) (volatile organics).

3When any sample is to be shipped by common carrier or sent via the U.S. Postal Service, it must comply with the Department of Transportation Hazardous Materials Regulations (49 CFR part 172). The person offering such material for transportation is responsible for ensuring such compliance. For the preservation requirement of Table II, the Office of Hazardous Materials, Materials Transportation Bureau, Department of Transportation has determined that the Hazardous Materials Regulations do not apply to the following materials: Hydrochloric acid (HCl) in water solutions at concentrations of 0.04% by weight or less (pH about 1.96 or greater; Nitric acid (HNO3) in water solutions at concentrations of 0.15% by weight or less (pH about 1.62 or greater); Sulfuric acid (H2SO4) in water solutions at concentrations of 0.35% by weight or less (pH about 1.15 or greater); and Sodium hydroxide (NaOH) in water solutions at concentrations of 0.080% by weight or less (pH about 12.30 or less).

4Samples should be analyzed as soon as possible after collection. The times listed are the maximum times that samples may be held before the start of analysis and still be considered valid. Samples may be held for longer periods only if the permittee or monitoring laboratory have data on file to show that, for the specific types of samples under study, the analytes are stable for the longer time, and has received a variance from the Regional ATP Coordinator under §136.3(e). For a grab sample, the holding time begins at the time of collection. For a composite sample collected with an automated sampler (e.g., using a 24-hour composite sampler; see 40 CFR 122.21(g)(7)(i) or 40 CFR part 403, appendix E), the holding time begins at the time of the end of collection of the composite sample. For a set of grab samples composited in the field or laboratory, the holding time begins at the time of collection of the last grab sample in the set. Some samples may not be stable for the maximum time period given in the table. A permittee or monitoring laboratory is obligated to hold the sample for a shorter time if it knows that a shorter time is necessary to maintain sample stability. See §136.3(e) for details. The date and time of collection of an individual grab sample is the date and time at which the sample is collected. For a set of grab samples to be composited, and that are all collected on the same calendar date, the date of collection is the date on which the samples are collected. For a set of grab samples to be composited, and that are collected across two calendar dates, the date of collection is the dates of the two days; e.g., November 14-15. For a composite sample collected automatically on a given date, the date of collection is the date on which the sample is collected. For a composite sample collected automatically, and that is collected across two calendar dates, the date of collection is the dates of the two days; e.g., November 14-15. For static-renewal toxicity tests, each grab or composite sample may also be used to prepare test solutions for renewal at 24 h, 48 h, and/or 72 h after first use, if stored at 0-6 °C, with minimum head space.

5ASTM D7365-09a specifies treatment options for samples containing oxidants (e.g., chlorine) for cyanide analyses. Also, Section 9060A of Standard Methods for the Examination of Water and Wastewater (20th and 21st editions) addresses dechlorination procedures for microbiological analyses.

6Sampling, preservation and mitigating interferences in water samples for analysis of cyanide are described in ASTM D7365-09a. There may be interferences that are not mitigated by the analytical test methods or D7365-09a. Any technique for removal or suppression of interference may be employed, provided the laboratory demonstrates that it more accurately measures cyanide through quality control measures described in the analytical test method. Any removal or suppression technique not described in D7365-09a or the analytical test method must be documented along with supporting data.

7For dissolved metals, filter grab samples within 15 minutes of collection and before adding preservatives. For a composite sample collected with an automated sampler (e.g., using a 24-hour composite sampler; see 40 CFR 122.21(g)(7)(i) or 40 CFR part 403, appendix E), filter the sample within 15 minutes after completion of collection and before adding preservatives. If it is known or suspected that dissolved sample integrity will be compromised during collection of a composite sample collected automatically over time (e.g., by interchange of a metal between dissolved and suspended forms), collect and filter grab samples to be composited (footnote 2) in place of a composite sample collected automatically.

8Guidance applies to samples to be analyzed by GC, LC, or GC/MS for specific compounds.

9If the sample is not adjusted to pH 2, then the sample must be analyzed within seven days of sampling.

10The pH adjustment is not required if acrolein will not be measured. Samples for acrolein receiving no pH adjustment must be analyzed within 3 days of sampling.

11When the extractable analytes of concern fall within a single chemical category, the specified preservative and maximum holding times should be observed for optimum safeguard of sample integrity (i.e., use all necessary preservatives and hold for the shortest time listed). When the analytes of concern fall within two or more chemical categories, the sample may be preserved by cooling to ≤6 °C, reducing residual chlorine with 0.008% sodium thiosulfate, storing in the dark, and adjusting the pH to 6-9; samples preserved in this manner may be held for seven days before extraction and for forty days after extraction. Exceptions to this optional preservation and holding time procedure are noted in footnote 5 (regarding the requirement for thiosulfate reduction), and footnotes 12, 13 (regarding the analysis of benzidine).

12If 1,2-diphenylhydrazine is likely to be present, adjust the pH of the sample to 4.0 ± 0.2 to prevent rearrangement to benzidine.

13Extracts may be stored up to 30 days at <0 °C.

14For the analysis of diphenylnitrosamine, add 0.008% Na2S2O3 and adjust pH to 7-10 with NaOH within 24 hours of sampling.

15The pH adjustment may be performed upon receipt at the laboratory and may be omitted if the samples are extracted within 72 hours of collection. For the analysis of aldrin, add 0.008% Na2S2O3.

16Place sufficient ice with the samples in the shipping container to ensure that ice is still present when the samples arrive at the laboratory. However, even if ice is present when the samples arrive, immediately measure the temperature of the samples and confirm that the preservation temperature maximum has not been exceeded. In the isolated cases where it can be documented that this holding temperature cannot be met, the permittee can be given the option of on-site testing or can request a variance. The request for a variance should include supportive data which show that the toxicity of the effluent samples is not reduced because of the increased holding temperature. Aqueous samples must not be frozen. Hand-delivered samples used on the day of collection do not need to be cooled to 0 to 6 °C prior to test initiation.

17Samples collected for the determination of trace level mercury (<100 ng/L) using EPA Method 1631 must be collected in tightly-capped fluoropolymer or glass bottles and preserved with BrCl or HCl solution within 48 hours of sample collection. The time to preservation may be extended to 28 days if a sample is oxidized in the sample bottle. A sample collected for dissolved trace level mercury should be filtered in the laboratory within 24 hours of the time of collection. However, if circumstances preclude overnight shipment, the sample should be filtered in a designated clean area in the field in accordance with procedures given in Method 1669. If sample integrity will not be maintained by shipment to and filtration in the laboratory, the sample must be filtered in a designated clean area in the field within the time period necessary to maintain sample integrity. A sample that has been collected for determination of total or dissolved trace level mercury must be analyzed within 90 days of sample collection.

18Aqueous samples must be preserved at ≤6 °C, and should not be frozen unless data demonstrating that sample freezing does not adversely impact sample integrity is maintained on file and accepted as valid by the regulatory authority. Also, for purposes of NPDES monitoring, the specification of “≤ °C” is used in place of the “4 °C” and “<4 °C” sample temperature requirements listed in some methods. It is not necessary to measure the sample temperature to three significant figures (1/100th of 1 degree); rather, three significant figures are specified so that rounding down to 6 °C may not be used to meet the ≤6 °C requirement. The preservation temperature does not apply to samples that are analyzed immediately (less than 15 minutes).

19An aqueous sample may be collected and shipped without acid preservation. However, acid must be added at least 24 hours before analysis to dissolve any metals that adsorb to the container walls. If the sample must be analyzed within 24 hours of collection, add the acid immediately (see footnote 2). Soil and sediment samples do not need to be preserved with acid. The allowances in this footnote supersede the preservation and holding time requirements in the approved metals methods.

20To achieve the 28-day holding time, use the ammonium sulfate buffer solution specified in EPA Method 218.6. The allowance in this footnote supersedes preservation and holding time requirements in the approved hexavalent chromium methods, unless this supersession would compromise the measurement, in which case requirements in the method must be followed.

21Holding time is calculated from time of sample collection to elution for samples shipped to the laboratory in bulk and calculated from the time of sample filtration to elution for samples filtered in the field.

22Sample analysis should begin as soon as possible after receipt; sample incubation must be started no later than 8 hours from time of collection.

23For fecal coliform samples for sewage sludge (biosolids) only, the holding time is extended to 24 hours for the following sample types using either EPA Method 1680 (LTB-EC) or 1681 (A-1): Class A composted, Class B aerobically digested, and Class B anaerobically digested.

24The immediate filtration requirement in orthophosphate measurement is to assess the dissolved or bio-available form of orthophosphorus (i.e., that which passes through a 0.45-micron filter), hence the requirement to filter the sample immediately upon collection (i.e., within 15 minutes of collection).

[38 FR 28758, Oct. 16, 1973]

Editorial Note: For Federal Register citations affecting §136.3, see the List of CFR Sections Affected, which appears in the Finding Aids section of the printed volume and at www.govinfo.gov.

§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:

eCFR graphic er18my12.000.gif

View or download PDF

Where:

xi = 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.

(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]

§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:

ParameterSTORET No.CAS No.
Bromodichloromethane3210175-27-4
Bromoform3210475-25-2
Bromomethane3441374-83-9
Carbon tetrachloride3210256-23-5
Chlorobenzene34301108-90-7
Chloroethane3431175-00-3
2-Chloroethylvinyl ether34576100-75-8
Chloroform3210667-66-3
Chloromethane3441874-87-3
Dibromochloromethane32105124-48-1
1,2-Dichlorobenzene3453695-50-1
1,3-Dichlorobenzene34566541-73-1
1,4-Dichlorobenzene34571106-46-7
Dichlorodifluoromethane3466875-71-8
1,1-Dichloroethane3449675-34-3
1,2-Dichloroethane34531107-06-2
1,1-Dichloroethane3450175-35-4
trans-1,2-Dichloroethene34546156-60-5
1,2-Dichloropropane3454178-87-5
cis-1,3-Dichloropropene3470410061-01-5
trans-1,3-Dichloropropene3469910061-02-6
Methylene chloride3442375-09-2
1,1,2,2-Tetrachloroethane3451679-34-5
Tetrachloroethene34475127-18-4
1,1,1-Trichloroethane3450671-55-6
1,1,2-Trichloroethane3451179-00-5
Tetrachloroethene3918079-01-6
Trichlorofluoromethane3448875-69-4
Vinyl chloride3971575-01-4

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.

eCFR graphic ec15no91.094.gif

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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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̅ 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 X̅ 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 X̅; (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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If p̅ = 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

eCFR graphic ec15no91.095.gif

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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
Chloromethane1.505.280.08
Bromomethane2.177.051.18
Dichlorodifluoromethane2.62nd1.81
Vinyl chloride2.675.280.18
Chloroethane3.338.680.52
Methylene chloride5.2510.10.25
Trichlorofluoromethane7.18ndnd
1,1-Dichloroethene7.937.720.13
1,1-Dichloroethane9.3012.60.07
trans-1,2-Dichloroethene10.19.380.10
Chloroform10.712.10.05
1,2-Dichloroethane11.415.40.03
1,1,1-Trichloroethane12.613.10.03
Carbon tetrachloride13.014.40.12
Bromodichloromethane13.714.60.10
1,2-Dichloropropane14.916.60.04
cis-1,3-Dichloropropene15.216.60.34
Trichloroethene15.813.10.12
Dibromochloromethane16.516.60.09
1,1,2-Trichloroethane16.518.10.02
trans-1,3-Dichloropropene16.518.00.20
2-Chloroethylvinyl ether18.0nd0.13
Bromoform19.219.20.20
1,1,2,2-Tetrachloroethane21.6nd0.03
Tetrachloroethene21.715.00.03
Chlorobenzene24.218.80.25
1,3-Dichlorobenzene34.022.40.32
1,2-Dichlorobenzene34.923.50.15
1,4-Dichlorobenzene35.422.30.24

Column 1 conditions: Carbopack B (60/80 mesh) coated with 1% SP-1000 packed in an 8 ft × 0.1 in. ID stainless steel or glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held at 45 °C for 3 min then programmed at 8 °C/min to 220 °C and held for 15 min.

Column 2 conditions: Porisil-C (100/120 mesh) coated with n-octane packed in a 6 ft × 0.1 in. ID stainless steel or glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held at 50 °C for 3 min then programmed at 6 °C/min to 170 °C and held for 4 min.

nd = not determined.

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

Parameter Range for Q (µg/L) Limit for s (µg/L)Range for X̅ (µg/L) Range P, Ps (%)
Bromodichloromethane15.2-24.84.310.7-32.042-172
Bromoform14.7-25.34.75.0-29.313-159
Bromomethane11.7-28.37.63.4-24.5D-144
Carbon tetrachloride13.7-26.35.611.8-25.343-143
Chlorobenzene14.4-25.65.010.2-27.438-150
Chloroethane15.4-24.64.411.3-25.246-137
2-Chloroethylvinyl ether12.0-28.08.34.5-35.514-186
Chloroform15.0-25.04.512.4-24.049-133
Chloromethane11.9-28.17.4D-34.9D-193
Dibromochloromethane13.1-26.96.37.9-35.124-191
1,2-Dichlorobenzene14.0-26.05.51.7-38.9D-208
1,3-Dichlorobenzene9.9-30.19.16.2-32.67-187
1,4-Dichlorobenzene13.9-26.15.511.5-25.542-143
1,1-Dichloroethane16.8-23.23.211.2-24.647-132
1,2-Dichloroethane14.3-25.75.213.0-26.551-147
1,1-Dichloroethene12.6-27.46.610.2-27.328-167
trans-1,2-Dichloroethene12.8-27.26.411.4-27.138-155
1,2-Dichloropropane14.8-25.25.210.1-29.944-156
cis-1,3-Dichloropropene12.8-27.27.36.2-33.822-178
trans-1,3-Dichloropropene12.8-27.27.36.2-33.822-178
Methylene chloride15.5-24.54.07.0-27.625-162
1,1,2,2-Tetrachloroethane9.8-30.29.26.6-31.88-184
Tetrachloroethene14.0-26.05.48.1-29.626-162
1,1,1-Trichloroethane14.2-25.84.910.8-24.841-138
1,1,2-Trichloroethane15.7-24.33.99.6-25.439-136
Trichloroethene15.4-24.64.29.2-26.635-146
Trichlorofluoromethane13.3-26.76.07.4-28.121-156
Vinyl chloride13.7-26.35.78.2-29.928-163

aCriteria were calculated assuming a QC check sample concentration of 20 µg/L.

Q = Concentration measured in QC check sample, in µg/L (Section 7.5.3).

s = Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).

X̅ = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

P, Ps = Percent recovery measured (Section 8.3.2, Section 8.4.2).

D = Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

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

ParameterAccuracy, as recovery, X′ (µg/L)Single analyst precision, sr′ (µg/L)Overall precision, S′ (µg/L)
Bromodichloromethane1.12C−1.020.11X̅ + 0.040.20X̅ + 1.00
Bromoform0.96C−2.050.12X̅ + 0.580.21X̅ + 2.41
Bromomethane0.76C−1.270.28X̅ + 0.270.36X̅ + 0.94
Carbon tetrachloride0.98C−1.040.15X̅ + 0.380.20X̅ + 0.39
Chlorobenzene1.00C−1.230.15X̅−0.020.18X̅ + 1.21
Choroethane0.99C−1.530.14X̅−0.130.17X̅ + 0.63
2-Chloroethylvinyl ethera1.00C0.20X̅0.35X̅
Chloroform0.93C−0.390.13X̅ + 0.150.19X̅−0.02
Chloromethane0.77C + 0.180.28X̅−0.310.52X̅ + 1.31
Dibromochloromethane0.94C + 2.720.11X̅ + 1.100.24X̅ + 1.68
1,2-Dichlorobenzene0.93C + 1.700.20X̅ + 0.970.13X̅ + 6.13
1,3-Dichlorobenzene0.95C + 0.430.14X̅ + 2.330.26X̅ + 2.34
1,4-Dichlorobenzene0.93C−0.090.15X̅ + 0.290.20X̅ + 0.41
1,1-Dichloroethane0.95C−1.080.09X̅ + 0.170.14X̅ + 0.94
1,2-Dichloroethane1.04C−1.060.11X̅ + 0.700.15X̅ + 0.94
1,1-Dichloroethene0.98C−0.870.21X̅−0.230.29X̅−0.40
trans-1,2-Dichloroethene0.97C−0.160.11X̅ + 1.460.17X̅ + 1.46
1,2-Dichloropropanea1.00C0.13X̅0.23X̅
cis-1,3-Dichloropropenea1.00C0.18X̅0.32X̅
trans-1,3-Dichloropropenea1.00C0.18X̅0.32X̅
Methylene chloride0.91C−0.930.11X̅ + 0.330.21X̅ + 1.43
1,1,2,2-Tetrachloroethene0.95C + 0.190.14X̅ + 2.410.23X̅ + 2.79
Tetrachloroethene0.94C + 0.060.14X̅ + 0.380.18X̅ + 2.21
1,1,1-Trichloroethane0.90C−0.160.15X̅ + 0.040.20X̅ + 0.37
1,1,2-Trichloroethane0.86C + 0.300.13X̅−0.140.19X̅ + 0.67
Trichloroethene0.87C + 0.480.13X̅−0.030.23X̅ + 0.30
Trichlorofluoromethane0.89C−0.070.15X̅ + 0.670.26X̅ + 0.91
Vinyl chloride0.97C−0.360.13X̅ + 0.650.27X̅ + 0.40

X̅′ = Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/L.

sn′ = Expected single analyst standard deviation of measurements at an average concentration found of X̅, in µg/L.

S1 = Expected interlaboratory standard deviation of measurements at an average concentration found of X̅, in µg/L.

C = True value for the concentration, in µg/L.

X̅ = Average recovery found for measurements of samples containing a concentration of C, in µg/L.

aEstimates based upon the performance in a single laboratory.10

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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.
Benzene3403071-43-2
Chlorobenzene34301108-90-7
1,2-Dichlorobenzene3453695-50-1
1,3-Dichlorobenzene34566541-73-1
1,4-Dichlorobenzene34571106-46-7
Ethylbenzene34371100-41-4
Toluene34010108-88-3

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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̅ 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 X̅ 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 X̅; (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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If P̅ = 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.

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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
Benzene3.332.750.2
Toluene5.754.250.2
Ethylbenzene8.256.250.2
Chlorobenzene9.178.020.2
1,4-Dichlorobenzene16.816.20.3
1,3-Dichlorobenzene18.215.00.4
1,2-Dichlorobenzene25.919.40.4

Column 1 conditions: Supelcoport (100/120 mesh) coated with 5% SP-1200/1.75% Bentone-34 packed in a 6 ft × 0.085 in. ID stainless steel column with helium carrier gas at 36 mL/min flow rate. Column temperature held at 50 °C for 2 min then programmed at 6 °C/min to 90 °C for a final hold.

Column 2 conditions: Chromosorb W-AW (60/80 mesh) coated with 5% 1,2,3-Tris(2-cyanoethyoxy)propane packed in a 6 ft × 0.085 in. ID stainless steel column with helium carrier gas at 30 mL/min flow rate. Column temperature held at 40 °C for 2 min then programmed at 2 °C/min to 100 °C for a final hold.

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

Parameter Range for Q (µg/L)Limit for s (µg/L)Range for X̅ (µg/L)Range for P, Ps(%)
Benzene15.4-24.64.110.0-27.939-150
Chlorobenzene16.1-23.93.512.7-25.455-135
1,2-Dichlorobenzene13.6-26.45.810.6-27.637-154
1,3-Dichlorobenzene14.5-25.55.012.8-25.550-141
1,4-Dichlorobenzene13.9-26.15.511.6-25.542-143
Ethylbenzene12.6-27.46.710.0-28.232-160
Toluene15.5-24.54.011.2-27.746-148

Q = Concentration measured in QC check sample, in µg/L (Section 7.5.3).

s = Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).

X̅ = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

Ps, P = Percent recovery measured (Section 8.3.2, Section 8.4.2).

aCriteria were calculated assuming a QC check sample concentration of 20 µg/L.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

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)
Benzene0.92C + 0.570.09X̅ + 0.590.21X̅ + 0.56
Chlorobenzene0.95C + 0.020.09X̅ + 0.230.17X̅ + 0.10
1,2-Dichlorobenzene0.93C + 0.520.17X̅−0.040.22X̅ + 0.53
1,3-Dichlorobenzene0.96C−0.050.15X̅−0.100.19X̅ + 0.09
1,4-Dichlorobenzene0.93C−0.090.15X̅ + 0.280.20X̅ + 0.41
Ethylbenzene0.94C + 0.310.17X̅ + 0.460.26X̅ + 0.23
Toluene0.94C + 0.650.09X̅ + 0.480.18X̅ + 0.71

X′ = Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/L.

S′ = Expected single analyst standard deviation of measurements at an average concentration found of X̅, in X µg/L.

S′ = Expected interlaboratory standard deviation of measurements at an average concentration found of X̅, in µg/L.

C = True value for the Concentration, in µg/L.

X̅ = Average recovery found for measurements of samples containing a concentration of C, in µg/L.

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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:

ParameterSTORET No.CAS No.
Acrolein34210107-02-8
Acrylonitrile34215107-13-1

   

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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X̅ 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 X̅ 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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If P̅ = 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.

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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
Acrolein10.68.20.7
Acrylonitrile12.79.80.5

Column 1 conditions: Porapak-QS (80/100 mesh) packed in a 10 ft × 2 mm ID glass or stainless steel column with helium carrier gas at 30 mL/min flow rate. Column temperature held isothermal at 110 °C for 1.5 min (during desorption), then heated as rapidly as possible to 150 °C and held for 20 min; column bakeout at 190 °C for 10 min.9

Column 2 conditions: Chromosorb 101 (60/80 mesh) packed in a 6 ft. × 0.1 in. ID glass or stainless steel column with helium carrier gas at 40 mL/min flow rate. Column temperature held isothermal at 80 °C for 4 min, then programmed at 50 °C/min to 120 °C and held for 12 min.

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
AcroleinRW5.05.20.2104
   RW50.051.40.7103
   POTW5.04.00.280
   POTW50.044.40.889
   IW5.00.10.12
   IW100.09.31.19
AcrylonitrileRW5.04.20.284
   RW50.051.41.5103
   POTW20.020.10.8100
   POTW100.0101.31.5101
   IW10.09.10.891
   IW100.0104.03.2104

RW = Reagent water.

POTW = Prechlorination secondary effluent from a municipal sewage treatment plant.

IW = Industrial wastewater containing an unidentified acrolein reactant.

Table 3—Calibration and QC Acceptance Criteria—Method 603a

ParameterRange for Q (µg/L)Limit for S (µg/L)Range for X (µg/L)Range for P, Ps (%)
Acrolein45.9-54.14.642.9-60.188-118
Acrylonitrile41.2-58.89.933.1-69.971-135

a = Criteria were calculated assuming a QC check sample concentration of 50 µg/L.9

Q = Concentration measured in QC check sample, in µg/L (Section 7.5.3).

s = Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).

X = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

P, Ps = Percent recovery measured (Section 8.3.2, Section 8.4.2).

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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-methylphenol3445259-50-7
2--Chlorophenol3458695-57-8
2,4-Dichlorophenol34601120-83-2
2,4-Dimethylphenol34606105-67-9
2,4-Dinitrophenol3461651-28-5
2-Methyl-4,6-dinitrophenol34657534-52-1
2-Nitrophenol3459188-75-5
4-Nitrophenol34646100-02-7
Pentachlorophenol3903287-86-5
Phenol34694108-95-2
2,4,6-Trichlorophenol3462188-06-2

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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X̅ 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 X̅ 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 X̅; (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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If P̅ = 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.

eCFR graphic ec15no91.098.gif

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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.

eCFR graphic ec15no91.099.gif

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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.

eCFR graphic ec15no91.100.gif

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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

ParameterRetention time (min) Method detection limit (µg/L)
2-Chlorophenol1.700.31
2-Nitrophenol2.000.45
Phenol3.010.14
2,4-Dimethylphenol4.030.32
2,4-Dichlorophenol4.300.39
2,4,6-Trichlorophenol6.050.64
4-Chloro-3-methylphenol7.500.36
2,4-Dinitrophenol10.0013.0
2-Methyl-4,6-dinitrophenol10.2416.0
Pentachlorophenol12.427.4
4-Nitrophenol24.252.8

Column conditions: Supelcoport (80/100 mesh) coated with 1% SP-1240DA packed in a 1.8 m long × 2 mm ID glass column with nitrogen carrier gas at 30 mL/min flow rate. Column temperature was 80 °C at injection, programmed immediately at 8 °C/min to 150 °C final temperature. MDL were determined with an FID.

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-Chlorophenol9013.30.58
2-Nitrophenol9909.10.77
Phenol90101.82.2
2,4-Dimethylphenol9572.90.63
2,4-Dichlorophenol9515.80.68
2,4,6-Trichlorophenol50507.00.58
4-Chloro-3-methylphenol84144.81.8
Pentachlorophenol752028.80.59
4-Nitrophenol19014.00.70

Column conditions: Chromosorb W-AW-DMCS (80/100 mesh) coated with 5% OV-17 packed in a 1.8 m long × 2.0 mm ID glass column with 5% methane/95% argon carrier gas at 30 mL/min flow rate. Column temperature held isothermal at 200 °C. MDL were determined with an ECD.

aEluant composition:

      Fraction 1—15% toluene in hexane.

      Fraction 2—40% toluene in hexane.

      Fraction 3—75% toluene in hexane.

      Fraction 4—15% 2-propanol in toluene.

Table 3—QC Acceptance Criteria—Method 604

ParameterTest conc. (µg/L)Limit for s (µg/L)Range for X̅ (µg/L)Range for P, Ps (percent)
4-Chloro-3-methylphenol10016.656.7-113.449-122
2-Chlorophenol10027.054.1-110.238-126
2,4-Dichlorophenol10025.159.7-103.344-119
2,4-Dimethylphenol10033.350.4-100.024-118
4,6-Dinitro-2-methylphenol10025.042.4-123.630-136
2,4-Dinitrophenol10036.031.7-125.112-145
2-Nitrophenol10022.556.6-103.843-117
4-Nitrophenol10019.022.7-100.013-110
Pentachlorophenol10032.456.7-113.536-134
Phenol10014.132.4-100.023-108
2,4,6-Trichlorophenol10016.660.8-110.453-119

s—Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).

X̅—Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

P, Ps—Percent recovery measured (Section 8.3.2, Section 8.4.2).

Note: These criteria are based directly upon the method performance data in Table 4. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 4.

Table 4—Method Accuracy and Precision as Functions of Concentration—Method 604

ParameterAccuracy, as recovery, X′ (µg/L)Single Analyst precision, sr′ (µg/L)Overall precision, S′ (µg/L)
4-Chloro-3-methylphenol0.87C-1.970.11X̅-0.210.16X̅ + 1.41
2-Chlorophenol0.83C-0.840.18X̅ + 0.200.21X̅ + 0.75
2,4-Dichlorophenol0.81C + 0.480.17X̅-0.020.18X̅ + 0.62
2,4-Dimethylphenol0.62C-1.640.30X̅-0.890.25X̅ + 0.48
4,6-Dinitro-2-methylphenol0.84C-1.010.15X̅ + 1.250.19X̅ + 5.85
2,4-Dinitrophenol0.80C-1.580.27X̅-1.150.29X̅ + 4.51
2-Nitrophenol0.81C-0.760.15X̅ + 0.440.14X̅ + 3.84
4-Nitrophenol0.46C + 0.180.17X̅ + 2.430.19X̅ + 4.79
Pentachlorophenol0.83C + 2.070.22X̅-0.580.23X̅ + 0.57
Phenol0.43C + 0.110.20X̅-0.880.17X̅ + 0.77
2,4,6-Trichlorophenol0.86C-0.400.10X̅ + 0.530.13X̅ + 2.40

X′ = Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/L.

sr′ = Expected single analyst standard deviation of measurements at an average concentration found of X̅, in µg/L.

S′ = Expected interlaboratory standard deviation of measurements at an average concentration found of X̅, in µg/L.

C = True value for the concentration, in µg/L.

X̅ = Average recovery found for measurements of samples containing a concentration of C, in µg/L.

eCFR graphic ec02jy92.012.gif

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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:

ParameterStoret NoCAS No.
Benzidine3912092-87-5
3,3′-Dichlorobenzidine3463191-94-1

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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̅ 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 X̅ 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 X̅; (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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If P̅ = 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.

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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.

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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)
Benzidine6.11.440.08
3,3′-Dichlorobenzidine12.13.840.13

HPLC Column conditions: Lichrosorb RP-2, 5 micron particle size, in a 25 cm × 4.6 mm ID stainless steel column. Mobile Phase: 0.8 mL/min of 50% acetonitrile/50% 0.1M pH 4.7 acetate buffer. The MDL were determined using an electrochemical detector operated at + 0.8 V.

Table 2—QC Acceptance Criteria—Method 605

Parameter Test conc. (µg/L)Limit for s (µg/L) Range for X̅ (µg/L) Range for P, Ps (percent)
Benzidine5018.79.1-61.0D-140
3.3′-Dichlorobenzidine5023.618.7-50.05-128

s = Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).

X̅ = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

P, Ps = Percent recovery measured (Section 8.3.2, Section 8.4.2).

D = Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 605

ParameterAccuracy, as recovery, X′(µg/L)Single analyst precision, sr′ (µg/L)Overall precision, S′ (µg/L)
Benzidine0.70C + 0.060.28X̅ + 0.190.40X̅ + 0.18
3,3′-Dichlorobenzidine0.66C + 0.230.39X̅−0.050.38X̅ + 0.02

X′ = Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/L.

sr′ = Expected single analyst standard deviation of measurements at an average concentration found of X̅, in µg/L.

S′ = Expected interlaboratory standard deviation of measurements at an average concentration found of X̅, in µg/L.

C = True value for the concentration, in µg/L.

X̅ = Average recovery found for measurements of samples containing a concentration of C, in µg/L.

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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:

ParameterSTORET No.CAS No.
Bis(2-ethylhexyl) phthalate39100117-81-7
Butyl benzyl phthalate3429285-68-7
Di-n-butyl phthalate3911084-74-2
Diethyl phthalate3433684-66-2
Dimethyl phthalate34341131-11-3
Di-n-octyl phthalate34596117-84-0

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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̅ 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 X̅ 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 X̅; (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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If P̅ = 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.

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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.

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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

ParameterRetention time (min)Method detection limit (µg/L)
Column 1Column 2
Dimethyl phthalate2.030.950.29
Diethyl phthalate2.821.270.49
Di-n-butyl phthalate8.653.500.36
Butyl benzyl phthalatea6.94a5.110.34
Bis(2-ethylhexyl) phthalatea8.92a10.52.0
Di-n-octyl phthalatea16.2a18.03.0

Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/1.95% SP-2401 packed in a 1.8 m long × 4 mm ID glass column with 5% methane/95% argon carrier gas at 60 mL/min flow rate. Column temperature held isothermal at 180 °C, except where otherwise indicated.

Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% OV-1 packed in a 1.8 m long × 4 mm ID glass column with 5% methane/95% argon carrier gas at 60 mL/min flow rate. Column temperature held isothermal at 200 °C, except where otherwise indicated.

a220 °C column temperature.

Table 2—QC Acceptance Criteria—Method 606

ParameterTest conc. (µg/L)Limit for s (µg/L)Range for X̅ (µg/L)Range for P, Ps (percent)
Bis(2-ethylhexyl) phthalate5038.41.2-55.9D-158
Butyl benzyl phthalate104.25.7-11.030-136
Di-n-butyl phthalate258.910.3-29.623-136
Diethyl phthalate259.01.9-33.4D-149
Dimethyl phathalate259.51.3-35.5D-156
Di-n-octyl phthalate5013.4D-50.0D-114

s = Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).

X̅ = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

P, Ps = Percent recovery measured (Section 8.3.2, Section 8.4.2).

D = Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 606

ParameterAccuracy, as recovery, X′ (µg/L)Single analyst precision, sr′ (µg/L)Overall precision, S′ (µg/L)
Bis(2-ethylhexyl) phthalate0.53C + 2.020.80X̅−2.540.73X̅−0.17
Butyl benzyl phthalate0.82C + 0.130.26X̅ + 0.040.25X̅ + 0.07
Di-n-butyl phthalate0.79C + 0.170.23X̅ + 0.200.29X̅ + 0.06
Diethyl phthalate0.70C + 0.130.27X̅ + 0.050.45X̅ + 0.11
Dimethyl phthalate0.73C + 0.170.26X̅ + 0.140.44X̅ + 0.31
Di-n-octyl phthalate0.35C−0.710.38X̅ + 0.710.62X̅ + 0.34

X̅′ = Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/L.

sr′ = Expected single analyst standard deviation of measurements at an average concentration found of X̅, in µg/L.

S′ = Expected interlaboratory standard deviation of measurements at an average concentration found of X̅, in µg/L.

C = True value for the concentration, in µg/L.

X̅ = Average recovery found for measurements of samples containing a concentration of C, in µg/L.

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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:

ParameterStoret No.CAS No.
N-Nitrosodimethylamine3443862-75-9
N-Nitrosodiphenylamine3443386-30-6
N-Nitrosodi-n-propylamine34428621-64-7

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 (X̅) 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 X̅ with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X̅ 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 X̅ 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 X̅; (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 (P̅) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P̅−2sp to P̅ + 2sp. If P̅ = 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.

eCFR graphic ec15no91.105.gif

View or download PDF

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.

eCFR graphic ec15no91.106.gif

View or download PDF

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

ParameterRetention time (min)Method detection limit (µg/L)
Column 1Column 2
N-Nitrosodimethylamine4.10.880.15
N-Nitrosodi-n-propylamine12.14.2.46
N-Nitrosodiphenylamineab12.8c6.4.81

Column 1 conditions: Chromosorb W-AW (80/100 mesh) coated with 10% Carbowax 20 M/2% KOH packed in a 1.8 m long × 4mm ID glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held isothermal at 110 °C, except where otherwise indicated.

Column 2 conditions: Supelcoport (100/120 mesh) coated with 10% SP-2250 packed in a 1.8 m long × 4 mm ID glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held isothermal at 120 °C, except where otherwise indicated.

aMeasured as diphenylamine.

b220 °C column temperature.

c210 °C column temperature.

Table 2—QC Acceptance Criteria—Method 607

ParameterTest conc. (µg/L)Limit for s (µg/L)Range for X̅ (µg/L)Range for P, Ps (percent)
N-Nitrosodimethylamine203.44.6-20.013-109
N-Nitrosodiphenyl206.12.1-24.5D-139
N-Nitrosodi-n-propylamine205.711.5-26.845-146

s = Standard deviation for four recovery measurements, in µg/L (Section 8.2.4).

X̅ = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).

P, Ps = Percent recovery measured (Section 8.3.2, Section 8.4.2).

D = Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

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-Nitrosodimethylamine0.37C + 0.060.25X̅−0.040.25X̅ + 0.11
N-Nitrosodiphenylamine0.64C + 0.520.36X̅−1.530.46X̅−0.47
N-Nitrosodi-n-propylamine0.96C−0.070.15X̅ + 0.130.21X̅ + 0.15

X′ = Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/L.

sr′ = Expected single analyst standard deviation of measurements at an average concentration found of X̅, in µg/L.

S′ = Expected interlaboratory standard deviation of measurements at an average concentration found of X̅, in µg/L.

C = True value for the concentration, in µg/L.

X̅ = Average recovery found for measurements of samples containing a concentration of C, in µg/L.

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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:

eCFR graphic er28au17.000.gif

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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:

eCFR graphic er28au17.001.gif

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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:

eCFR graphic er28au17.002.gif

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where T is the known true value of the spike.

Also calculate the relative percent difference (RPD) between the concentrations (A1 and A2):

eCFR graphic er28au17.003.gif

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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:

eCFR graphic er28au17.004.gif

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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 Tab