Electronic Code of Federal Regulations:

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 §136.5, be used to perform the measurements indicated whenever the waste constituent specified is required to be measured for:

(1) An application submitted to the Administrator, or to a State having an approved NPDES program for a permit under section 402 of the Clean Water Act of 1977, as amended (CWA), and/or to reports required to be submitted under NPDES permits or other requests for quantitative or qualitative effluent data under parts 122 to 125 of title 40, 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 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.

(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.

(d) Director means the Director of the State Agency authorized to carry out an approved National Pollutant Discharge Elimination System Program under section 402 of the Act.

(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 greater than zero as determined by the procedure set forth at appendix B of this part.

(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. 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 text of the referenced test procedures are incorporated by reference into Tables IA, IB, IC, ID, IE, IF, IG, and IH. The incorporation by reference of these documents, as specified in paragraph (b) of this section, was approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR Part 51. Copies of the documents may be obtained from the sources listed in paragraph (b) of this section. Documents may be inspected at EPA's Water Docket, EPA West, 1301 Constitution Avenue, NW., Room B102, Washington, DC (Telephone: 202–566–2426); or 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: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html . These test procedures are incorporated as they exist on the day of approval and a notice of any change in these test procedures will be published in theFederal Register.The discharge parameter values for which reports are required must be determined by one of the standard analytical test procedures incorporated by reference and described in Tables IA, IB, IC, ID, IE, IF, IG, and IH or by any alternate test procedure which has been approved by the Administrator under the provisions of paragraph (d) of this section and §§136.4 and 136.5. Under certain circumstances paragraph (c) of this section, §136.5(a) through (d) or 40 CFR 401.13, other additional or alternate test procedures may be used.

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

Parameter and units	Method¹	EPA	Standard methods 18th, 19th, 20th ed.	Standard methods online	AOAC, ASTM, USGS	Other
Bacteria:
1. Coliform (fecal), number per 100 mL or number per gram dry weight	Most Probable Number (MPN),⁵tube 3 dilution, or	p. 132³ 1680^12,14 1681^12,19	9221 C E	9221 C E–99
	Membrane filter (MF)², single step	p. 124³	9222 D	9222 D–97	B–0050–85⁵
2. Coliform (fecal) in presence of chlorine, number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 132³	9221 C E	9221 C E–99
	MF², single step	p. 124³	9222 D	9222 D–97
3. Coliform (total), number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 114³	9221 B	9221 B–99
	MF², single step or two step	p. 108³	9222 B	9222 B–97	B–0025–8⁵
4. Coliform (total), in presence of chlorine, number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 114³	9221 B	9221 B–99
	MF²with enrichment	p. 111³	9222 (B+B.5c)	9222 (B+B.5c)−97
5. E. coli , number per 100 mL²⁰	MPN^7,9,15multiple tube/multiple well		9223 B¹³	9223 B–97¹³	991.15¹¹	Colilert^®^13,17 Colilert-18^®^13,16,17
	MF^2,6,7,8,9single step	1603²¹				mColiBlue-24^®¹⁸
6. Fecal streptococci, number per 100 mL	MPN, 5 tube 3 dilution,	p. 139³	9230 B	9230 B–93
	MF², or	p. 136³	9230 C	9230 C–93	B–0055–85⁵
	Plate count	p. 143³
7. Enterococci, number per 100 mL²⁰	MPN^7,9, multiple tube/multiple well				D6503–99¹⁰	Enterolert^®^13,23
	MF^2,6,7,8,9single step	1600²⁴
8. Salmonella, number per gram dry weight¹²	MPN multiple tube	1682²²
Aquatic Toxicity:
9. Toxicity, acute, fresh water organisms, LC50, percent effluent	Ceriodaphnia dubia acute	2002.0²⁵
	Daphnia puplex and Daphnia magna acute	2021.0²⁵
	Fathead Minnow, Pimephales promelas , and Bannerfin shiner , Cyprinella leedsi , acute	2000.0²⁵
	Rainbow Trout, Oncorhynchus mykiss , and brook trout, Salvelinus fontinalis , acute	2019.0²⁵
10. Toxicity, acute, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, LC50, percent effluent	Mysid, Mysidopsis bahia , acute	2007.0²⁵
	Sheepshead Minnow, Cyprinodon variegatus, acute	2004.0²⁵
	Silverside, Menidia beryllina , Menidia menidia , and Menidia peninsulae , acute	2006.0²⁵
11. Toxicity, chronic, fresh water organisms, NOEC or IC25, percent effluent	Fathead minnow, Pimephales promelas , larval survival and growth	1000.0²⁶
	Fathead minnow, Pimephales promelas , embryo-larval survival and teratogenicity	1001.0²⁶
	Daphnia, Ceriodaphnia dubia , survival and reproduction	1002.0²⁶
	Green alga, Selenastrum capricornutum , growth	1003.0²⁶
12. Toxicity, chronic, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, NOEC or IC25, percent effluent	Sheepshead minnow, Cyprinodon variegatus , larval survival and growth	1004.0²⁷
	Sheepshed minnow, Cyprinodon variegatus , embryo-larval survival and teratogenicity	1005.0²⁷
	Inland silverside, Menidia beryllina , larval survival and growth	1006.0²⁷
	Mysid, Mysidopsis bahia , survival, growth, and fecundity	1007.0²⁷
	Sea urchin, Arbacia punctulata , fertilization	1008.0²⁷

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.

³USEPA. 1978. Microbiological Methods for Monitoring the Environment, Water, and Wastes. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH, EPA/600/8–78/017.

⁴[Reserved]

⁵USGS. 1989. U.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, U.S. Geological Survey, U.S. Department of the Interior, Reston, VA.

⁶Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Most Probable Number method will be required to resolve any controversies.

⁷Tests 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.

⁸When 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.

⁹To 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.

¹⁰ASTM. 2000, 1999, 1996. Annual Book of ASTM Standards—Water and Environmental Technology. Section 11.02. ASTM International. 100 Barr Harbor Drive, West Conshohocken, PA 19428.

¹¹AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. Association of Official Analytical Chemists International. 481 North Frederick Avenue, Suite 500, Gaithersburg, MD 20877–2417.

¹²Recommended for enumeration of target organism in sewage sludge.

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 .

¹⁴USEPA. July 2006. Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation Using Lauryl-Tryptose Broth (LTB) and EC Medium. US Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–012.

¹⁵Samples 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^®Quanti-Tray^®2000, and the MPN calculated from the table provided by the manufacturer.

¹⁶Colilert-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.

¹⁷Descriptions of the Colilert^®, Colilert-18^®, Quanti-Tray^®, and Quanti-Tray^®/2000 may be obtained from IDEXX Laboratories, Inc., 1 IDEXX Drive, Westbrook, ME 04092.

¹⁸A description of the mColiBlue24^®test, Total Coliforms and E. coli , is available from Hach Company, 100 Dayton Ave., Ames, IA 50010.

¹⁹USEPA. July 2006. Method 1681: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using A–1 Medium. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–013.

²⁰Recommended for enumeration of target organism in wastewater effluent.

²¹USEPA. July 2006. Method 1603: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (modified mTEC). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–011.

²²USEPA. July 2006. Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–014.

²³A description of the Enterolert^®test may be obtained from IDEXX Laboratories, Inc., 1 IDEXX Drive, Westbrook, ME 04092.

²⁴USEPA. July 2006. Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–009.

²⁵USEPA. October 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA/821/R–02/012.

²⁶USEPA. October 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition, U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA/821/R–02/013.

²⁷USEPA. October 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. Third Edition. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA/821/R–02/014.

Table IB—List of Approved Inorganic Test Procedures

Parameter	Methodology⁵⁸	Reference (method number or page)
Parameter	Methodology⁵⁸	EPA^35,⁵²	Standard methods (18th, 19th)	Standard methods (20th)	Standard methods online	ASTM	USGS/AOAC/other
1. Acidity, as CaCO3, mg/L	Electrometric endpoint or phenolphthalein endpoint		2310 B(4a)	2310 B(4a)	2310 B(4a)–97	D1067–92, 02	I–1020–85²
2. Alkalinity, as CaCO3, mg/L	Electrometric or Colorimetric titration to pH 4.5, manual, or		2320 B	2320 B	2320 B–97	D1067–92, 02	973.43³, I–1030–85²
	automatic	310.2 (Rev. 1974)¹					I–2030–85²
3. Aluminum—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 D		3111 D–99		I–3051–85²
	AA furnace		3113 B		3113 B–99
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–9750
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	Direct Current Plasma (DCP)³⁶					D4190–94, 99	See footnote³⁴
	Colorimetric (Eriochrome cyanine R)		3500–Al D	3500–Al B	3500–Al B–01
4. Ammonia (as N), mg/L	Manual, distillation (at pH 9.5)⁶followed by:	350.1, Rev. 2.0 (1993)	4500–NH B3	4500–NH3 B	4500–NH3 B–97		973.49³
	Nesslerization		4500–NH3 C (18th only)			D1426–98, 03 (A)	973.49³, I–3520–85²
	Titration		4500–NH3 C (19th) and 4500–NH3 E (18th)	4500–NH3 C	4500–NH3 C–97
	Electrode		4500–NH3 D or E (19th) and 4500–NH3 F or G (18th)	4500–NH3 D or E	4500–NH3 D or E–97	D1426–98, 03 (B)
	Automated phenate, or	350.1⁶⁰, Rev. 2.0 (1993)	4500–NH3 G (19th) and 4500–NH3 H (18th)	4500–NH3 G	4500–NH3 G–97		I–4523–85²
	Automated electrode						See footnote 7
	Ion Chromatography					D6919–03
5. Antimony—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B		3111 B–99
	AA furnace		3113 B		3113 B–99
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
6. Arsenic—Total,⁴mg/L	Digestion⁴followed by	206.5 (Issued 1978)¹
	AA gaseous hydride		3114 B 4.d		3114 B 4.d–97	D2972–97, 03 (B)	I–3062–85²
	AA furnace		3113 B		3113 B–99	D2972–97, 03 (C)	I–4063–98⁴⁹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	Colorimetric (SDDC)		3500–As C	3500–As B	3500–As B–97	D2972–97, 03 (A)	I–3060–85
7. Barium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 D		3111 D–99		I–3084–85²
	AA furnace		3113 B		3113 B–99	D4382–95, 02
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP³⁶						See footnote³⁴
8. Beryllium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 D		3111 D–99	D3645–93 (88), 03 (A)	I–3095–85²
	AA furnace		3113 B		3113 B–99	D3645–93 (88), 03 (B)
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP, or					D4190–94, 99	See footnote³⁴
	Colorimetric (aluminon)		3500–Be D
9. Biochemical oxygen demand (BOD5), mg/L	Dissolved Oxygen Depletion		5210 B	5210 B	5210 B–01		973.44,³p. 17.⁹, I–1578–78⁸
10. Boron—Total,³⁷mg/L	Colorimetric (curcumin)		4500–B B	4500–B B	4500–B B–00		I–3112–85²
	ICP/AES, or	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B99		I–4471–97⁵⁰
	DCP					D4190–94, 99	See footnote 34
11. Bromide, mg/L	Titrimetric					D1246–95, 99 (C)	p. S44.¹⁰
							I–1125–85²
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97, 03	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
12. Cadmium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B or C		3111 B or C–99	D3557–95, 02 (A or B)	974.27,³p. 37.⁹, I–3135–85²or I–3136–85²
	AA furnace		3113 B		3113 B–99	D3557–95, 02 (D)	I–4138–89⁵¹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–1472–85²or I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP³⁶					D4190–94, 99	See footnote³⁴
	Voltametry¹¹, or					D3557–95, 02 (C)
	Colorimetric (Dithizone)		3500–Cd D
13. Calcium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99	D511–93, 03(B)	I–3152–85²
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	DCP, or						See footnote³⁴
	Titrimetric (EDTA)		3500–Ca D	3500–Ca B	3500–Ca B–97	D511–93, 03(A)
	Ion Chromatography					D6919–03
14. Carbonaceous biochemical oxygen demand (CBOD5), mg/L¹²	Dissolved Oxygen Depletion with nitrification inhibitor		5210 B	5210 B	5210 B–01
15. Chemical oxygen demand (COD), mg/L	Titrimetric	410.3 (Rev. 1978)¹	5220 C	5220 C	5220 C–97	D1252–95, 00 (A)	973.46³, p. 17⁹I–3560–85²
	Spectrophotometric, manual or automatic	410.4, Rev. 2.0 (1993)	5220 D	5220 D	5220 D–97	D1252–95, 00 (B)	See footnotes^{13, 14}. I–3561–85²
16. Chloride, mg/L	Titrimetric: (silver nitrate) or		4500–Cl–B	4500–Cl–B	4500–Cl–B–97	D512–89(99) (B)	I–1183–85²
	(Mercuric nitrate)		4500–Cl–C	4500–Cl–C	4500–Cl–C–97	D512–89 (99) (A)	973.51³, I–1184–85²
	Colorimetric: manual or						I–1187–85²
	Automated (Ferricyanide)		4500–Cl–E	4500–Cl–E	4500–Cl–E–97		I–2187–85²
	Potentiometric Titration		4500–Cl–D	4500–Cl–D	4500–Cl–D–97
	Ion Selective Electrode					D512–89(99)(C)
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97, 03	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
17. Chlorine—Total residual, mg/L; Titrimetric	Amperometric direct, or		4500–Cl D	4500–Cl D	4500–Cl D–00	D1253–86 (96), 03
	Amperometric direct (low level)		4500–Cl E	4500–Cl E	4500–Cl E–00
	Iodometric direct		4500–Cl B	4500–Cl B	4500–Cl B–00
	Back titration ether end–point¹⁵or		4500–Cl C	4500–Cl C	4500–Cl C–00
	DPD–FAS		4500–Cl F	4500–Cl F	4500–Cl F–00
	Spectrophotometric, DPD or		4500–Cl G	4500–Cl G	4500–Cl G–00
	Electrode						See footnote¹⁶
18. Chromium VI dissolved, mg/L	0.45–micron Filtration followed by:
	AA chelation–extraction or		3111 C		3111 C–99		I–1232–85
	Ion Chromatography	218.6, Rev. 3.3 (1994)	3500–Cr E	3500–Cr C	3500–Cr C–01	D5257–97	993.23
	Colorimetric (Diphenyl–carbazide)		3500–Cr D	3500–Cr B	3500–Cr B–01	D1687–92, 02 (A)	I–1230–85
19. Chromium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B		3111 B–99	D1687–92, 02 (B)	974.27³, I–3236–85²
	AA chelation–extraction		3111 C		3111 C–99
	AA furnace		3113 B		3113 B–99	D1687–92, 02 (C)	I–3233–93⁴⁶
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP,³⁶or					D4190–94, 99	See footnote³⁴
	Colorimetric (Diphenyl–carbazide)		3500–Cr D	3500–Cr B	3500–Cr B–01
20. Cobalt—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B or C		3111 B or C–99	D3558–94, 03 (A or B)	p. 37⁹, I–3239–85²
	AA furnace		3113 B		3113 B–99	D3558–94, 03 (C)	I–4243–89⁵¹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP					D4190–94, 99	See footnote³⁴
21. Color, platinum cobalt units or dominant wavelength, hue, luminance purity	Colorimetric (ADMI), or		2120 E	2120 E			See footnote¹⁸
	(Platinum cobalt), or		2120 B	2120 B	2120 B–01		I–1250–85²
	Spectrophotometric		2120 C	2120 C
22. Copper—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B or C		3111 B or C–99	D1688–95, 02 (A or B)	974.27³p. 37⁹I–3270–85²or I–3271–85²
	AA furnace		3113 B		3113 B–99	D1688–95, 02 (C)	I–4274–89⁵¹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP³⁶or					D4190–94, 99	See footnote³⁴
	Colorimetric (Neocuproine) or		3500–Cu D	3500–Cu B	3500–Cu B–99
	(Bicinchoninate)		3500–Cu E	3500–Cu C	3500–Cu C–99		See footnote¹⁹
23. Cyanide—Total, mg/L	Automated Distillation and Colorimetry, or						Kelada–01⁵⁵
	Manual distillation with MgCl2 followed by:	335.4, Rev. 1.0 (1993)⁵⁷	4500–CN^–C	4500–CN^–C		D2036–98(A)	10–204–00–1–X⁵⁶
	Titrimetric or		4500–CN^–D	4500–CN^–D	4500–CN^–D–99		p. 22⁹
	Spectrophotometric, manual or		4500–CN^–E	4500–CN^–E	4500–CN^–E–99	D2036–98(A)	I–3300–85
	Automated²⁰or	335.4, Rev. 1.0 (1993)⁵⁷					10–204–00–1–X⁵⁶, I–4302–85²
	Ion Selective Electrode		4500–CN^–F	4500–CN^–F	4500–CN^–F–99	D2036–98(A)
24. Available Cyanide, mg/L	Cyanide Amenable to Chlorination (CATC); Manual distillation with MgCl2 followed by Titrimetric or Spectrophotometric		4500–CN^–G	4500–CN^–G	4500–CN^–G–99	D2036–98(B)
	Flow injection and ligand exchange, followed by amperometry⁶¹					D6888–04	OIA–1677⁴⁴
	Automated Distillation and Colorimetry						Kelada–01⁵⁵
25. Fluoride—Total, mg/L	Manual distillation⁶followed by:		4500–F^–B	4500–F^–B	4500–F^–B–97
	Electrode, manual or		4500–F^–B	4500–F^–B	4500–F^–C–97	D1179–93, 99 (B)
	Automated						I–4327–85²
	Colorimetric, (SPADNS) or		4500–F^–D	4500–F^–D	4500–F^–D–97	D1179–93, 99 (A)
	Automated complexone		4500–F^–E	4500–F^–E	4500–F^–E–97
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97,03	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
26. Gold—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration, or		3111 B		3111 B–99
	AA furnace, or	231.2 (Rev. 1978)¹
	DCP						See footnote³⁴
27. Hardness—Total, as CaCO3, mg/L	Automated colorimetric,	130.1 (Issued 1971)¹
	Titrimetric (EDTA) or		2340 B or C	2340 B or C	2340 B or C–97	D1126–86(92), 02	973.5 2B³, I–1338–85²
	Ca plus Mg as their carbonates, by inductively coupled plasma or AA direct aspiration. (See Parameters 13 and 33).
28. Hydrogen ion (pH), pH units	Electrometric measurement or		4500–H⁺B	4500–H⁺B	4500–H⁺B–00	D1293–84 (90), 99 (A or B)	973.41.³, I–1586–85²
	Automated electrode	150.2 (Dec. 1982)¹					See footnote²¹, I–2587–85²
29. Iridium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration or		3111 B		3111 B–99
	AA furnace	235.2 (Issued 1978)¹
30. Iron—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B or C		3111 B or C–99	D1068–96, 03 (A or B)	974.27³, I–3381–85²
	AA furnace		3113 B		3113 B–99	D1068–96, 03 (C)
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	DCP³⁶or					D4190–94, 99	See footnote³⁴
	Colorimetric (Phenanthroline)		3500–Fe D	3500–Fe B	3500–Fe B–97	D1068–96, 03 (D)	See footnote²²
31. Kjeldahl Nitrogen⁵—Total, (as N), mg/L	Digestion and distillation followed by:²⁰		4500–Norg B or C and 4500–NH3 B	4500–Norg B or C and 4500–NH3 B	4500–Norg B or C–97 and 4500–NH3 B–97	D3590–89, 02 (A)
	Titration or		4500–NH3 C (19th) and 4500–NH3 E (18th)	4500–NH3 C	4500–NH3 C–97	D3590–89, 02 (A)	973.48³
	Nesslerization or		4500–NH3 C (18th Only)			D3590–89, 02 (A)
	Electrode		4500–NH3 F or G (18th) and 4500–NH3 D or E (19th)	4500–NH3 D or E	4500–NH3 D or E–97
	Automated phenate colorimetric	351.1 (Rev. 1978)¹					I–4551–78⁸
	Semi-automated block digestor colorimetric	351.2, Rev. 2.0 (1993)				D3590–89, 02 (B)	I–4515–91⁴⁵
	Manual or block digestor potentiometric					D3590–89, 02 (A)
	Block digester, followed by Auto distillation and Titration, or						See footnote³⁹
	Nesslerization, or						See footnote⁴⁰
	Flow injection gas diffusion						See footnote⁴¹
32. Lead—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B or C		3111 B or C–99	D3559–96, 03 (A or B)	974.27³, I–3399–85²
	AA furnace		3113 B		3113 B–99	D3559–96, 03 (D)	I–4403–89⁵¹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP³⁶					D4190–94, 99	See footnote³⁴
	Voltametry¹¹or					D3559–96, 03 (C)
	Colorimetric (Dithizone)		3500–Pb D	3500–Pb B	3500–Pb B–97
33. Magnesium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99	D511–93, 03(B)	974.27³, I–3447–85²
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	DCP or						See footnote³⁴
	Gravimetric		3500–Mg D
	Ion Chromatography					D6919–03
34. Manganese—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B		3111 B–99	D858–95, 02 (A or B)	974.27³, I–3454–85²
	AA furnace		3113 B		3113 B–99	D858–95, 02 (C)
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP36, or					D4190–94, 99	See footnote³⁴
	Colorimetric (Persulfate), or		3500––Mn D	3500–Mn B	3500–Mn B–99		920.203³
	(Periodate)						See footnote²³
35. Mercury—Total⁴, mg/L	Cold vapor, manual or	245.1, Rev. 3.0 (1994)	3112 B		3112 B–99	D3223–97, 02	977.22³, I–3462–85²
	Automated	245.2 (Issued 1974)
	Cold vapor atomic fluorescence spectrometry (CVAFS)	245.7 Rev. 2.0 (2005)⁵⁹
	Purge and Trap CVAFS	1631E⁴³
36. Molybdenum—Total⁴, mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 D		3111 D–99		I–3490–85²
	AA furnace		3113 B		3113 B–99		I–3492–96⁴⁷
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP						See footnote³⁴
37. Nickel—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B or C		3111 B or C–99	D1886–90, 94 (98) (A or B)	I–3499–85²
	AA furnace		3113 B		3113 B–99	D1886–90, 94 (98) (C)	I–4503–89⁵¹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP³⁶, or					D4190–94, 99	See footnote³⁴
	Colorimetric (heptoxime)		3500–Ni D (17th Edition)
38. Nitrate (as N), mg/L	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97, 03	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
	Ion Selective Electrode		4500–NO3^–D	4500–NO3^–D	4500–NO3^–D–00
	Colorimetric (Brucine sulfate), or	352.1¹					973.50³, 419D^1,⁷, p. 28⁹
	Nitrate-nitrite N minus Nitrite N (See parameters 39 and 40).
39. Nitrate-nitrite (as N), mg/L	Cadmium reduction, manual or		4500–NO3^–E	4500–NO3^–E	4500–NO3^–E–00	D3867–99(B)
	Automated, or	353.2, Rev. 2.0 (1993)	4500–NO3^–F	4500–NO3^–F	4500–NO3^–F–00	D3867–99(A)	I–4545–85²
	Automated hydrazine		4500–NO3^–H	4500–NO3^–H	4500–NO3^–H–00
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
40. Nitrite (as N), mg/L	Spectrophotometric: Manual or		4500–NO2^–B	4500–NO2^–B	4500–NO2^–B–00		See footnote²⁵
	Automated (Diazotization)						I–4540–85²
	Automated (*bypass cadmium reduction)	353.2, Rev. 2.0 (1993)	4500–NO3^–F	4500–NO3^–F	4500–NO3^–F–00	D3867–99(A)	I–4545–85²
	Manual (*bypass cadmium reduction)		4500–NO3^–E	4500–NO3^–E	4500–NO3^–E–00	D3867–99(B)
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97, 03	993.30³
	CIE/UV						D6508, Rev.2⁵⁴
41. Oil and grease—Total recoverable, mg/L	Hexane extractable material (HEM): n–Hexane extraction and gravimetry	1664A⁴²		5520 B³⁸	5520 B–01³⁸
	Silica gel treated HEM (SGT–HEM): Silica gel treatment and gravimetry.	1664A⁴²
42. Organic carbon—Total (TOC), mg/L	Combustion or oxidation		5310 B, C, or D	5310 B, C, or D	5310 B, C, or D–00	D2579–93 (A or B)	973.47,³p. 14²⁴
43. Organic nitrogen (as N), mg/L	Total Kjeldahl N (Parameter 31) minus ammonia N (Parameter 4)
44. Orthophosphate (as P), mg/L	Ascorbic acid method:
	Automated, or	365.1, Rev. 2.0 (1993)	4500–P F	4500–P F			973.56³, I–4601–85²
	Manual single reagent		4500–P E	4500–P E		D515–88(A)	973.55³
	Manual two reagent	365.3 (Issued 1978)¹
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97, 03	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
45. Osmium—Total⁴, mg/L	Digestion⁴followed by:
	AA direct aspiration, or		3111 D		3111 D–99
	AA furnace	252.2 (Issued 1978)¹
46. Oxygen, dissolved, mg/L	Winkler (Azide modification), or		4500–O C	4500–O C	4500–O C–01	D888–92, 03 (A)	973.4 5B³, I–1575–78⁸
	Electrode		4500–O G	4500–O G	4500–O G–01	D888–92, 03 (B)	I–1576–78⁸
47. Palladium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration, or		3111 B		3111 B–99		p. S27¹⁰
	AA furnace	253.2¹(Issued 1978)					p. S28¹⁰
	DCP						See footnote³⁴
48. Phenols, mg/L	Manual distillation²⁶Followed by:	420.1¹(Rev. 1978)					See footnote²⁷
	Colorimetric (4AAP) manual, or	420.1¹(Rev. 1978)					See footnote²⁷
	Automated	420.4 Rev. 1.0 (1993)
49. Phosphorus (elemental), mg/L	Gas–liquid chromatography						See footnote²⁸
50. Phosphorus—Total, mg/L	Persulfate digestion followed by:²⁰		4500–P B.5	4500–P B.5			973.55³
	Manual or	365.3¹(Issued 1978)	4500–P E	4500–P E		D515–88(A)
	Automated ascorbic acid reduction	365.1 Rev. 2.0 (1993)	4500–P F	4500–P F			973.56³, I–4600–85²
	Semi–automated block digestor	365.4¹(Issued 1974)				D515–88(B)	I–4610–91⁴⁸
51. Platinum—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99
	AA furnace	255.2¹
	DCP						See footnote³⁴
52. Potassium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99		973.53³, I–3630–85²
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	Flame photometric, or		3500–K D	3500–K B	3500–K B–97
	Colorimetric						317 B¹⁷
	Ion Chromatography					D6919–03
53. Residue—Total, mg/L	Gravimetric, 103–105°		2540 B	2540 B	2540 B–97		I–3750–85²
54. Residue—filterable, mg/L	Gravimetric, 180°		2540 C	2540 C	2540 C–97		I–1750–85²
55. Residue—non–filterable (TSS), mg/L	Gravimetric, 103–105 °C post washing of residue		2540 D	2540 D	2540 D–97		I–3765–85²
56. Residue—settleable, mg/L	Volumetric, (Imhoff cone), or gravimetric		2540 F	2540 F	2540 F–97
57. Residue—Volatile, mg/L	Gravimetric, 550 °C	160.4¹					I–3753–85²
58. Rhodium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration, or		3111 B		3111 B–99
	AA furnace	265.2¹
59. Ruthenium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration, or		3111 B		3111 B–99
	AA furnace	267.2¹
60. Selenium—Total,⁴mg/L	Digestion⁴followed by:
	AA furnace		3113 B		3113 B–99	D3859–98, 03 (B)	I–4668–98⁴⁹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	AA gaseous hydride		3114 B		3114 B–97	D3859–98, 03 (A)	I–3667–85²
61. Silica—Dissolved,³⁷mg/L	0.45 micron filtration followed by:
	Colorimetric, Manual or		4500–Si D	4500–SiO2 C	4500–SiO2C–97	D859–94, 00	I–1700–85²
	Automated (Molybdosilicate), or						I–2700–85²
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
62. Silver—Total,^{4, 31}mg/L	Digestion^{4, 29}followed by:
	AA direct aspiration		3111 B or C		3111 B or C–99		974.27³, p. 37⁹, I–3720–85²
	AA furnace		3113 B		3113 B–99		I–4724–89⁵¹
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP						See footnote³⁴
63. Sodium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99		973.54³, I–3735–85²
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	DCP, or						See footnote³⁴
	Flame photometric		3500–Na D	3500–Na B	3500–Na B–97
	Ion Chromatography					D 6919–03
64. Specific conductance, micromhos/cm at 25 °C	Wheatstone bridge	120.1¹(Rev. 1982)	2510 B	2510 B	2510 B–97	D1125–95 (99) (A)	973.40³, I–2781–85²
65. Sulfate (as SO4), mg/L	Automated colorimetric	375.2, Rev. 2.0 (1993)
	Gravimetric		4500–SO4²^–C or D	4500–SO4²^–C or D			925.54³
	Turbidimetric					D516–90, 02	426C³0
	Ion Chromatography	300.0, Rev 2.1 (1993) and 300.1, Rev 1.0 (1997)	4110 B	4110 B	4110 B–00	D4327–97, 03	993.30³
	CIE/UV						D6508, Rev. 2⁵⁴
66. Sulfide (as S), mg/L	Titrimetric (iodine), or		4500–S²^–F (19th) 4500–S²^–E (18th)	4500–S²^–F	4500–S²^–F–00		I–3840–85²
	Colorimetric (methylene blue)		4500–S²^–D	4500–S²^–D	4500–S²^–D–00
	Ion Selective Electrode		4500–S²^–G	4500–S²^–G	4500–S²^–G–00	D4658–03
67. Sulfite (as SO3), mg/L	Titrimetric (iodine-iodate)		4500–SO3²^–B	4500–SO3²^–B	4500–SO3²^–B–00
68. Surfactants, mg/L	Colorimetric (methylene blue)		5540 C	5540 C	5540 C–00	D2330–88, 02
69. Temperature, °C	Thermometric		2550 B	2550 B	2550 B–00		See footnote³²
70. Thallium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99
	AA furnace	279.2¹(Issued 1978)
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
71. Tin—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 B		3111 B–99		I–3850–78⁸
	AA furnace, or		3113 B		3113 B–99
	STGFAA	200.9, Rev. 2.2 (1994)
	ICP/AES	200.7, Rev. 4.4 (1994)
72. Titanium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 D		3111 D–99
	AA furnace	283.2¹(Issued 1978)
	DCP						See footnote³⁴
73. Turbidity, NTU⁵³	Nephelometric	180.1, Rev. 2.0 (1993)	2130 B	2130 B	2130 B–01	D1889–94, 00	I–3860–85²
74. Vanadium—Total,⁴mg/L	Digestion⁴followed by:
	AA direct aspiration		3111 D		3111 D–99
	AA furnace					D3373–93, 03
	ICP/AES	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP, or					D4190–94, 99	See footnote³⁴
	Colorimetric (Gallic Acid)		3500–V D	3500–V B	3500–V B–97
75. Zinc –Total⁴, mg/L	Digestion⁴followed by:
	AA direct aspiration³⁶		3111 B or C		3111 B or C–99	D1691–95, 02 (A or B)	974.27³, p. 37⁹, I–3900–85²
	AA furnace	289.2¹(Issued 1978)
	ICP/AES³⁶	200.7, Rev. 4.4 (1994)	3120 B	3120 B	3120 B–99⁵⁹		I–4471–97⁵⁰
	ICP/MS	200.8, Rev. 5.4 (1994)				D5673–03	993.14³
	DCP,³⁶or					D4190–94, 99	See footnote³⁴
	Colorimetric (Dithizone) or		3500–Zn E
	(Zincon)		3500–Zn F	3500–Zn B	3500–Zn B–97		See footnote³³

Table 1B Notes:

¹“Methods for Chemical Analysis of Water and Wastes,” Environmental Protection Agency, Environmental Monitoring Systems Laboratory–Cincinnati (EMSL–CI), EPA–600/4–79–020 (NTIS PB 84–128677), Revised March 1983 and 1979 where applicable.

²Fishman, M. J., et al. “Methods for Analysis of Inorganic Substances in Water and Fluvial Sediments,” U.S. Department of the Interior, Techniques of Water-Resource Investigations of the U.S. Geological Survey, Denver, CO, Revised 1989, unless otherwise stated.

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

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 the 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 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.

⁷Ammonia, Automated Electrode Method, Industrial Method Number 379–75 WE, dated February 19, 1976, Bran & Luebbe (Technicon) Auto Analyzer II, Bran & Luebbe Analyzing Technologies, Inc., Elmsford, NY 10523.

⁸The approved method is that cited in “Methods for Determination of Inorganic Substances in Water and Fluvial Sediments”, USGS TWRI, Book 5, Chapter A1 (1979).

⁹American National Standard on Photographic Processing Effluents, April 2, 1975. Available from ANSI, 25 West 43rd st., New York, NY 10036.

¹⁰“Selected Analytical Methods Approved and Cited by the United States Environmental Protection Agency,” Supplement to the Fifteenth Edition of Standard Methods for the Examination of Water and Wastewater (1981).

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 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, Oceanography International Corporation, 1978, 512 West Loop, P.O. Box 2980, College Station, TX 77840.

¹⁴Chemical Oxygen Demand, Method 8000, Hach Handbook of Water Analysis, 1979, Hach Chemical Company, P.O. Box 389, Loveland, CO 80537.

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

¹⁶Orion Research Instruction Manual, Residual Chlorine Electrode Model 97–70, 1977, Orion Research Incorporated, 840 Memorial Drive, Cambridge, MA 02138. 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.

¹⁷The approved method is that cited in Standard Methods for the Examination of Water and Wastewater, 14th Edition, 1976.

¹⁸National Council of the Paper Industry for Air and Stream Improvement, Inc., Technical Bulletin 253, December 1971.

¹⁹Copper, Biocinchoinate Method, Method 8506, Hach Handbook of Water Analysis, 1979, Hach Chemical Company, P.O. Box 389, Loveland, CO 80537.

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

²¹Hydrogen ion (pH) Automated Electrode Method, Industrial Method Number 378–75WA, October 1976, Bran & Luebbe (Technicon) Autoanalyzer II. Bran & Luebbe Analyzing Technologies, Inc., Elmsford, NY 10523.

²²Iron, 1,10-Phenanthroline Method, Method 8008, 1980, Hach Chemical Company, P.O. Box 389, Loveland, CO 80537.

²³Manganese, Periodate Oxidation Method, Method 8034, Hach Handbook of Wastewater Analysis, 1979, pages 2–113 and 2–117, Hach Chemical Company, Loveland, CO 80537.

²⁴Wershaw, R. L., et al. , “Methods for Analysis of Organic Substances in Water,” Techniques of Water-Resources Investigation of the U.S. Geological Survey, Book 5, Chapter A3, (1972 Revised 1987) p. 14.

²⁵Nitrogen, Nitrite, Method 8507, Hach Chemical Company, P.O. Box 389, Loveland, CO 80537.

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

²⁷The approved method is cited in Standard Methods for the Examination of Water and Wastewater, 14th Edition. The colorimetric reaction is conducted at a pH of 10.0±0.2. The approved methods are given on pp 576–81 of the 14th Edition: Method 510A for distillation, Method 510B for the manual colorimetric procedure, or Method 510C for the manual spectrometric procedure.

²⁸R.F. Addison and R. G. Ackman, “Direct Determination of Elemental Phosphorus by Gas–Liquid Chromatography,” Journal of Chromatography, Vol. 47, No.3, pp. 421–426, 1970.

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 Na2S2O3 and NaOH. Standards should be prepared in the same manner. For levels of silver below 1 mg/L the approved method is satisfactory.

³⁰The approved method is that cited in Standard Methods for the Examination of Water and Wastewater, 15th Edition.

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.

³²Stevens, H.H., Ficke, J. F., and Smoot, G. F., “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.

³³Zinc, Zincon Method, Method 8009, Hach Handbook of Water Analysis, 1979, pages 2–231 and 2–333, Hach Chemical Company, Loveland, CO 80537.

³⁴“Direct Current Plasma (DCP) Optical Emission Spectrometric Method for Trace Elemental Analysis of Water and Wastes, Method AES0029,” 1986—Revised 1991, Thermo Jarrell Ash Corporation, 27 Forge Parkway, Franklin, MA 02038

³⁵Precision and recovery statements for the atomic absorption direct aspiration and graphite furnace methods, and for the spectrophotometric SDDC method for arsenic are provided in Appendix D of this part titled, “Precision and Recovery Statements for Methods for Measuring Metals.”

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”, CEM Corporation, P.O. Box 200, Matthews, NC 28106–0200, April 16, 1992. Available from the 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 extraction solvent when determining Oil and Grease parameters—Hexane Extractable Material (HEM), or Silica Gel Treated HEM (analogous to EPA Method 1664A). Use of other extraction solvents (e.g., those in the 18th and 19th editions) is prohibited.

³⁹Nitrogen, Total Kjeldahl, Method PAI-DK01 (Block Digestion, Steam Distillation, Titrimetric Detection), revised 12/22/94, OI Analytical/ALPKEM, P.O. Box 9010, College Station, TX 77842.

⁴⁰Nitrogen, Total Kjeldahl, Method PAI–DK02 (Block Digestion, Steam Distillation, Colorimetric Detection), revised 12/22/94, OI Analytical/ALPKEM, P.O. Box 9010, College Station, TX 77842.

⁴¹Nitrogen, Total Kjeldahl, Method PAI–DK03 (Block Digestion, Automated FIA Gas Diffusion), revised 12/22/94, OI Analytical/ALPKEM, P.O. Box 9010, College Station, TX 77842.

⁴²Method 1664, Revision A “ 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, February 1999. Available at NTIS, PB–121949, U.S. Department of Commerce, 5285 Port Royal, Springfield, VA 22161.

⁴³USEPA. 2001. Method 1631, Revision E, “Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic Fluorescence Spectrometry” September 2002, Office of Water, U.S. Environmental Protection Agency (EPA–821–R–02–024). The application of clean techniques described in EPA's draft 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.

⁴⁴Available Cyanide, Method OIA–1677, “Available Cyanide by Flow Injection, Ligand Exchange, and Amperometry,” ALPKEM, A Division of OI Analytical, P.O. Box 9010, College Station, TX 77842–9010.

⁴⁵“Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Ammonia Plus Organic Nitrogen by a Kjeldahl Digestion Method,” Open File Report (OFR) 00–170.

⁴⁶“Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Chromium in Water by Graphite Furnace Atomic Absorption Spectrophotometry,” Open File Report (OFR) 93–449.

⁴⁷“Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Molybdenum by Graphite Furnace Atomic Absorption Spectrophotometry,” Open File Report (OFR) 97–198.

⁴⁸“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” Open File Report (OFR) 92–146.

⁴⁹“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” Open File Report (OFR) 98–639.

⁵⁰“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,” Open File Report (OFR) 98-165.

⁵¹“Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Inorganic and Organic Constituents in Water and Fluvial Sediment,” Open File Report (OFR) 93–125.

⁵²All EPA methods, excluding EPA Method 300.1, are published in “Methods for the Determination of Metals in Environmental Samples,” Supplement I, National Exposure Risk Laboratory-Cincinnati (NERL–CI), EPA/600/R–94/111, May 1994; and “Methods for the Determination of Inorganic Substances in Environmental Samples,” NERL–CI, EPA/600/R–93/100, August, 1993. EPA Method 300.1 is available from http://www.epa.gov/safewater/methods/pdfs/met300.pdf.

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

⁵⁴Method D6508, Rev. 2, “Test Method for Determination of Dissolved Inorganic Anions in Aqueous Matrices Using Capillary Ion Electrophoresis and Chromate Electrolyte,” available from Waters Corp, 34 Maple St., Milford, MA, 01757, Telephone: 508/482–2131, Fax: 508/482–3625.

⁵⁵Kelada-01, “Kelada Automated Test Methods for Total Cyanide, Acid Dissociable Cyanide, and Thiocyanate,” EPA 821–B–01–009, Revision 1.2, August 2001, National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 22161 [Order Number PB 2001–108275]. The toll free telephone number is: 800–553–6847. 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” is available from Lachat Instruments 6645 W. Mill Road, Milwaukee, WI 53218, Telephone: 414–358–4200.

⁵⁷When using sulfide removal test procedures described in Method 335.4, 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.

⁵⁹Method 245.7, Rev. 2.0, “Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry,” February 2005, EPA–821–R–05–001, available from the U.S. EPA Sample Control Center (operated by CSC), 6101 Stevenson Avenue, Alexandria, VA 22304, Telephone: 703–461–2100, Fax: 703–461–8056.

⁶⁰The use of EDTA may decrease method sensitivity in some samples. Analysts may omit EDTA provided that all method specified quality control acceptance criteria are met.

⁶¹Samples analyzed for available cyanide using Methods OIA–1677 or D6888–04 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 analysis to no more than 30 minutes to preclude settling of materials in samples.

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

Parameter¹	EPA method number^2,⁷			Other approved methods
Parameter¹	GC	GC/MS	HPLC	Standard Methods [Edition(s)]	Standard Methods Online	ASTM	Other
1. Acenaphthene	610	625, 1625B	610	6440 B [18th, 19th, 20th]		D4657–92 (99)	See footnote⁹, p. 27
2. Acenaphthylene	610	625, 1625B	610	6410 B, 6440 B, [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
3. Acrolein	603	624⁴, 1624B
4. Acrylonitrile	603	624⁴, 1624B
5. Anthracene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
6. Benzene	602	624, 1624B		6200 B [20th] and 6210 B [18th,19th], 6200 C [20th] and 6220 B [18th,19th]	6200 B and C–97
7. Benzidine		625⁵, 1625B	605				See footnote³, p.1
8. Benzo(a)anthracene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
9. Benzo(a)pyrene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
10. Benzo(b)fluoranthene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
11. Benzo(g,h,i) perylene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
12. Benzo(k) fluoranthene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
13. Benzyl chloride							See footnote³, p. 130: See footnote⁶, p. S102
14. Benzyl butyl phthalate	606	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
15. Bis(2-chloroethoxy) methane	611	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
16. Bis(2-chloroethyl) ether	611	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
17. Bis(2-ethylhexyl) phthalate	606	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
18. Bromodichloro-methane	601	624, 1624B		6200 C [20th] and 6230 B [18th, 19th], 6200 B [20th] and 6210 B [18th, 19th]	6200 B and C–97
19. Bromoform	601	624, 1624B		6200 C [20th] and 6230 B [18th, 19th], 6200 B [20th] and 6210 B [18th, 19th]	6200 B and C–97
20. Bromomethane	601	624, 1624B		6200 C [20th] and 6230 B [18th, 19th], 6200 B [20th] and 6210 B [18th, 19th]	6200 B and C–97
21. 4-Bromophenyl phenyl ether	611	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
22. Carbon tetrachloride	601	624, 1624B		6200 C [20th] and 6230 B [18th, 19th]	6200 C–97		See footnote³, p. 130
23. 4-Chloro-3-methyl phenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
24. Chlorobenzene	601, 602	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6220 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97		See footnote³, p. 130
25. Chloroethane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
26. 2-Chloroethylvinyl ether	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
27. Chloroform	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97		See footnote³, p. 130
28. Chloromethane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th] 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
29. 2-Chloronaph-thalene	612	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
30. 2-Chlorophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B(00, 6420 B–00		See footnote⁹, p. 27
31. 4-Chlorophenyl phenyl ether	611	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
32. Chrysene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
33. Dibenzo(a,h)an-thracene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
34. Dibromochloro-methane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th] 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
35. 1,2-Dichloro-benzene	601, 602	624, 1625B		6200 C [20th] and 6220 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 C–97		See footnote⁹, p. 27
36. 1,3-Dichloro-benzene	601, 602	624, 1625B		6200 C [20th] and 6220 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 C–97		See footnote⁹, p. 27
37. 1,4-Dichloro-benzene	601, 602	624, 1625B		6200 C [20th] and 6220 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 C–97		See footnote⁹, p. 27
38. 3,3-Dichloro-benzidine		625, 1625B	605	6410 B [18th, 19th, 20th]	6410 B–00
39. Dichlorodifluoro-methane	601			6200 C [20th] and 6230 B [18th, 19th]	6200 C–97
40. 1,1-Dichloroethane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
41. 1,2-Dichloroethane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
42. 1,1-Dichloroethene	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
43. trans-1,2-Dichloro-ethene	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
44. 2,4-Dichlorophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
45. 1,2-Dichloro-propane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
46. cis-1,3-Dichloro-propene	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
47. trans-1,3-Dichloro-propene	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
48. Diethyl phthalate	606	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
49. 2,4-Dimethylphenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
50. Dimethyl phthalate	606	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
51. Di-n-butyl phthalate	606	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
52. Di-n-octyl phthalate	606	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
53. 2,3-Dinitrophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00
54. 2,4-Dinitrotoluene	609	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
55. 2,6-Dinitrotoluene	609	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
56. Epichlorohydrin							See footnote³, p. 130; See footnote⁶, p. S102
57. Ethylbenzene	602	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6220 B [18th, 19th]	6200 B and C–97
58. Fluoranthene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
59. Fluorene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
60. 1,2,3,4,6,7,8-Heptachloro-dibenzofuran		1613B¹⁰
61. 1,2,3,4,7,8,9-Heptachloro-dibenzofuran		1613B¹⁰
62. 1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin		1613B¹⁰
63. Hexachlorobenzene	612	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
64. Hexachloro-butadiene	612	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
65. Hexachlorocyclo-pentadiene	612	625⁵, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
66. 1,2,3,4,7,8-Hexachlorodibenzofuran		1613B¹⁰
67. 1,2,3,6,7,8-Hexachlorodibenzofuran		1613B¹⁰
68. 1,2,3,7,8,9-Hexachlorodibenzofuran		1613B¹⁰
69. 2,3,4,6,7,8-Hexachlorodibenzofuran		1613B¹⁰
70. 1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin		1613B¹⁰
71. 1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin		1613B¹⁰
72. 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin 1613B¹⁰		1613B¹⁰
73. Hexachloroethane	612	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
74. Ideno(1,2,3-cd) pyrene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
75. Isophorone	609	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
76. Methylene chloride	601	624, 1624B		6200 C [20th] and 6230 B [18th, 19th]	6200 C–97		See footnote³, p. 130
77. 2-Methyl-4,6-dinitrophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
78. Naphthalene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
79. Nitrobenzene	609	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
80. 2-Nitrophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
81. 4-Nitrophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
82. N-Nitrosodimethylamine	607	6255, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
83. N-Nitrosodi-n-propylamine	607	6255, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
84. N-Nitrosodiphenylamine	607	6255, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote⁹, p. 27
85. Octachlorodibenzofuran		1613B¹⁰*
86. Octachlorodibenzo-p-dioxin		1613B¹⁰
87. 2,2'-Oxybis(2-chloropropane) [also known as bis(2-chloroisopropyl) ether]	611	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00
88. PCB–1016	608	625		6410 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 43; See footnote⁸
89. PCB–1221	608	625		6410 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 43; See footnote⁸
90. PCB–1232	608	625		6410 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 43; See footnote⁸
91. PCB–1242	608	625		6410 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 43; See footnote⁸
92. PCB–1248	608	625
93. PCB–1254	608	625		6410 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 43; See footnote⁸
94. PCB–1260	608	625		6410 B, 6630 B [18th, 19th, 20th]	6410 B–00		See footnote 3, p. 43; See footnote 8
95. 1,2,3,7,8-Pentachloro-dibenzofuran		1613B¹⁰
96. 2,3,4,7,8-Pentachloro-dibenzofuran		1613B¹⁰
97. 1,2,3,7,8,-Pentachlorodibenzo- p -dioxin		1613B¹⁰
98. Pentachlorophenol	604	625, 1625B		6410 B, 6630 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 140; See footnote⁹, p. 27
99. Phenanthrene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
100. Phenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
101. Pyrene	610	625, 1625B	610	6410 B, 6440 B [18th, 19th, 20th]	6410 B–00	D4657–92 (99)	See footnote⁹, p. 27
102. 2,3,7,8-Tetra-chlorodibenzofuran		1613B10
103. 2,3,7,8-Tetra-chlorodibenzo-p-dioxin		613, 625^5a, 1613B¹⁰
104. 1,1,2,2-Tetra-chloro ethane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97		See footnote³, p. 130
105. Tetrachloroethene	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97		See footnote³, p. 130
106. Toluene	602	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6220 B [18th, 19th]	6200 B and C–97
107. 1,2,4-Trichloro-benzene	612	625, 1625B		6410 B [18th, 19th, 20th]	6410 B–00		See footnote³, p. 130; See footnote⁹, p. 27
108. 1,1,1-Trichloro-ethane	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
109. 1,1,2-Trichloro-ethane	601	624, 1624B	6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97		See footnote³, p. 130
110. Trichloroethene	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
111. Trichlorofluoro-methane	601	624		6200 B [20th] and 6210 B [18th, 19th], 6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97
112. 2,4,6-Trichlorophenol	604	625, 1625B		6410 B, 6420 B [18th, 19th, 20th]	6410 B–00, 6420 B–00		See footnote⁹, p. 27
113. Vinyl chloride	601	624, 1624B		6200 B [20th] and 6210 B [18th, 19th], >6200 C [20th] and 6230 B [18th, 19th]	6200 B and C–97

¹All 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).

²The full text of Methods 601–613, 624, 625, 1624B, and 1625B, are given at Appendix A, “Test Procedures for Analysis of Organic Pollutants,” of this Part 136. The full text of Method 1613B is incorporated by reference into this Part 136 and is available from the National Technical Information Services as stock number PB95–104774. The standardized test procedure to be used to determine the method detection limit (MDL) for these test procedures is given at Appendix B, “Definition and Procedure for the Determination of the Method Detection Limit,” of this Part 136.

³“Methods for Benzidine: Chlorinated Organic Compounds, Pentachlorophenol and Pesticides in Water and Wastewater,” U.S. Environmental Protection Agency, September, 1978.

⁴Method 624 may be extended to screen samples for Acrolein and Acrylonitrile. However, when they are known to be present, the preferred method for these two compounds is Method 603 or Method 1624B.

⁵Method 625 may be extended to include benzidine, hexachlorocyclopentadiene, N-nitrosodimethylamine, 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.

^5a625, screening only.

⁶“Selected Analytical Methods Approved and Cited by the United States Environmental Protection Agency,” Supplement to the Fifteenth Edition of Standard Methods for the Examination of Water and Wastewater (1981).

⁷Each analyst must make an initial, one-time demonstration of their ability to generate acceptable precision and accuracy with Methods 601–603, 624, 625, 1624B, and 1625B (See Appendix A of this Part 136) 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 and 625 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 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.

⁸“Organochlorine Pesticides and PCBs in Wastewater Using Empore^TMDisk” 3M Corporation Revised 10/28/94.

⁹USGS Method 0–3116–87 from “Methods of Analysis by U.S. Geological Survey National Water Quality Laboratory—Determination of Inorganic and Organic Constituents in Water and Fluvial Sediments,” U.S. Geological Survey, Open File Report 93–125.

¹⁰Analysts may use Fluid Management Systems, Inc. PowerPrep system in place of manual cleanup provided that the analysis meet the requirements of Method 1613B (as specified in Section 9 of the method) and permitting authorities.

Table ID—List of Approved Test Procedures for Pesticides¹

Parameter	Method	EPA^2,⁷	Standard Methods 18th, 19th, 20th Ed.	Standard Methods Online	ASTM	Other
1. Aldrin	GC	608	6630 B & C		D3086–90, D5812-96 (2002)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
2. Ametryn	GC					See footnote³, p. 83; See footnote⁶, p S68
3. Aminocarb	TLC					See footnote³, p. 94; See footnote⁶, p. S16
4. Atraton	GC					See footnote³, p. 83; See footnote⁶, p. S68
5. Atrazine	GC					See footnote³, p. 83; See footnote⁶, p. S68; See footnote⁹
6. Azinphos methyl	GC					See footnote³, p. 25; See footnote⁶, p. S51
7. Barban	TLC					See footnote³, p. 104; See footnote⁶, p. S64
8. α–BHC	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁸
	GC/MS	625⁵	6410 B	6410 B–00
9. β–BHC	GC	608	6630 C		D3086–90, D5812–96(02)	See footnote⁸
	GC/MS	625⁵	6410 B	6410 B–00
10. δ–BHC	GC	608	6630 C		D3086–90, D5812–96(02)	See footnote⁸
	GC/MS	625⁵	6410 B	6410 B–00
11. γ-BHC (Lindane)	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00

12. Captan	GC		6630 B		D3086–90, D5812–96(02)	See footnote³, p. 7
13. Carbaryl	TLC					See footnote³, p. 94, See footnote⁶, p. S60
14. Carbo-phenothion	GC					See footnote⁴, p. 27; See footnote⁶, p. S73
15. Chlordane	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
16. Chloro-propham	TLC					See footnote³, p. 104; See footnote⁶, p. S64.
17. 2,4-D	GC		6640 B			See footnote³, p. 115; See footnote⁴, p. 40
18. 4,4'-DDD	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
19. 4,4'-DDE	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
20. 4,4'-DDT	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
21. Demeton-O	GC					See footnote³, p. 25; See footnote⁶, p. S51
22. Demeton-S	GC					See footnote³, p. 25; See footnote⁶, p. S51
23. Diazinon	GC					See footnote³, p. 25; See footnote⁴, p. 27; See footnote⁶, p. S51
24. Dicamba	GC					See footnote³, p. 115
25. Dichlofen-thion	GC					See footnote⁴, p. 27; See footnote⁶, p. S73
26. Dichloran	GC		6630 B & C			See footnote³, p. 7
27. Dicofol	GC				D3086–90, D5812–96(02)
28. Dieldrin	GC	608	6630 B & C			See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
29. Dioxathion	GC					See footnote⁴, p. 27; See footnote⁶, p. S73
30. Disulfoton	GC					See footnote³, p. 25; See footnote⁶, p. S51
31. Diuron	TLC					See footnote³, p. 104; See footnote⁶, p. S64
32. Endosulfan I	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625⁵	6410 B	6410 B–00
33. Endosulfan II	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁸
	GC/MS	625⁵	6410 B	6410 B–00
34. Endosulfan Sulfate	GC	608	6630 C			See footnote⁸
	GC/MS	625	6410 B	6410 B–00
35. Endrin	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625⁵	6410 B	6410 B–00
36. Endrin aldehyde	GC GC/MS	608 625				See footnote⁸
37. Ethion	GC					See footnote⁴, p. 27; See footnote⁶, p. S73
38. Fenuron	TLC					See footnote³, p. 104; See footnote⁶, p. S64
39. Fenuron-TCA	TLC					See footnote³, p. 104; See footnote⁶, p. S64
40. Heptachlor	GC GC/MS	608 625	6630 B & C 6410 B	6410 B–00	D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
41. Heptachlor epoxide	GC GC/MS	608 625	6630 B & C 6410 B	6410 B–00	D3086–90, D5812– 96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁶, p. S73; See footnote⁸
42. Isodrin	GC					See footnote⁴, p. 27; See footnote⁶, p. S73
43. Linuron	GC					See footnote³, p. 104; See footnote⁶, p. S64
44. Malathion	GC		6630 C			See footnote³, p. 25; See footnote⁴, p. 27; See footnote⁶, p. S51
45. Methiocarb	TLC					See footnote³, p. 94; See footnote⁶, p. S60
46. Methoxy-chlor	GC		6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
47. Mexacar-bate	TLC					See footnote³, p. 94; See footnote⁶, p. S60
48. Mirex	GC		6630 B & C			See footnote³, p. 7; See footnote⁴, p. 27
49. Monuron	TLC					See footnote³, p. 104; See footnote⁶, p. S64
50. Monuron-TCA	TLC					See footnote³, p. 104; See footnote⁶, p. S64
51. Nuburon	TLC					See footnote³, p. 104; See footnote⁶, p. S64
52. Parathion methyl	GC		6630 C			See footnote³, p. 25; See footnote⁴, p. 27
53. Parathion ethyl	GC		6630 C			See footnote³, p. 25; See footnote⁴, p. 27
54. PCNB	GC		6630 B & C			See footnote³, p. 7
55. Perthane	GC				D3086–90, D5812–96(02)	See footnote⁴, p. 27
56. Prometon	GC					See footnote³, p. 83; See footnote⁶, p. S68; See footnote⁹
57. Prometryn	GC					See footnote³, p. 83; See footnote⁶, p. S68; See footnote⁹
58. Propazine	GC					See footnote³, p. 83; See footnote⁶, p. S68; See footnote⁹
59. Propham	TLC					See footnote³, p. 104; See footnote⁶, p. S64
60. Propoxur	TLC					See footnote³, p. 94; See footnote⁶, p. S60
61. Secbumeton	TLC					See footnote³, p. 83; See footnote⁶, p. S68
62. Siduron	TLC					See footnote³, p. 104; See footnote⁶, p. S64
63. Simazine	GC					See footnote³, p. 83; See footnote⁶, p. S68; See footnote⁹
64. Strobane	GC		6630 B & C			See footnote³, p. 7
65. Swep	TLC					See footnote³, p. 104; See footnote⁶, p. S64
66. 2,4,5–T	GC		6640 B			See footnote³, p. 115; See footnote⁴, p. 40
67. 2,4,5–TP (Silvex)	GC		6640 B			See footnote³, p. 115; See footnote⁴, p. 40
68. Terbuthylazine	GC					See footnote³, p. 83; See footnote⁶, p. S68
69. Toxaphene	GC	608	6630 B & C		D3086–90, D5812–96(02)	See footnote³, p. 7; See footnote⁴, p. 27; See footnote⁸
	GC/MS	625	6410 B	6410 B–00
70. Trifluralin	GC		6630 B			See footnote³, p. 7; See footnote⁹

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

²The full text of Methods 608 and 625 are given at Appendix A, “Test Procedures for Analysis of Organic Pollutants,” of this Part 136. The standardized test procedure to be used to determine the method detection limit (MDL) for these test procedures is given at Appendix B, “Definition and Procedure for the Determination of the Method Detection Limit,” of this Part 136.

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

⁴“Methods 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 (1987).

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 is the preferred method.

⁶“Selected Analytical Methods Approved and Cited by the United States Environmental Protection Agency.” Supplement to the Fifteenth Edition of Standard Methods for the Examination of Water and Wastewater (1981).

⁷Each analyst must make an initial, one-time, demonstration of their ability to generate acceptable precision and accuracy with Methods 608 and 625 (See Appendix A of this Part 136) 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 10% of all samples analyzed with Method 608 or 5% of all samples analyzed with Method 625 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.

⁸“Organochlorine Pesticides and PCBs in Wastewater Using Empore^TMDisk”, 3M Corporation, Revised 10/28/94.

⁹USGS Method 0–3106–93 from “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” U.S. Geological Survey Open File Report 94–37.

Table IH—List of Approved Microbiological Methods for Ambient Water

Parameter and units	Method¹	EPA	Standard methods 18th, 19th, 20th Ed.	Standard methods online	AOAC, ASTM, USGS	Other
Bacteria:
1. E. coli, number per 100 mL	MPN^6,8,14multiple tube,		9221 B.1/9221 F^11,13	9221 B.1–99/9221 F^11,13
	Multiple tube/multiple well,		9223 B¹²	9223 B–97¹²	991.15¹⁰	Colilert^®^12,16Colilert-18^®^12,15,16.
	MF^2,5,6,7,8two step, or	1103.1¹⁹	9222 B/9222 G¹⁸, 9213 D	9222 B–97/9222 G¹⁸	D5392–93⁹
	Single step	1603²⁰, 1604²¹				mColiBlue-24^®¹⁷.
2. Enterococci, number per 100 mL	MPN^6,8multiple tube,		9230 B	9230 B–93
	Multiple tube/multiple well				D6503–99⁹	Enterolert^®^12,22.
	MF^2,5,6,7,8two step	1106.1²³	9230 C	9230 C–93	D5259–92⁹
	Single step, or	1600²⁴
	Plate count	p. 143³
Protozoa:
3. Cryptosporidium	Filtration/IMS/FA	1622^25,1623²⁶
4. Giardia	Filtration/IMS/FA	1623²⁶

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.

⁴[Reserved]

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 number 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.

⁹ASTM. 2000, 1999, 1996. Annual Book of ASTM Standards—Water and Environmental Technology. Section 11.02. ASTM International. 100 Barr Harbor Drive, West Conshohocken, PA 19428.

¹⁰AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. Association of Official Analytical Chemists International. 481 North Frederick Avenue, Suite 500, Gaithersburg, MD 20877–2417.

¹¹The multiple-tube fermentation test is used in 9221B.1. 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 by E. coli .

¹³After prior enrichment in a presumptive medium for total coliform using 9221B.1, 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. 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 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.

¹⁶Descriptions of the Colilert^®, Colilert-18^®, Quanti-Tray^®, and Quanti-Tray^®/2000 may be obtained from IDEXX Laboratories, Inc., 1 IDEXX Drive, Westbrook, ME 04092.

¹⁷A description of the mColiBlue24^®test, Total Coliforms and E. coli , is available from Hach Company, 100 Dayton Ave., Ames, IA 50010.

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

¹⁹USEPA. July 2006. Method 1103.1: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–010.

²⁰USEPA. July 2006. Method 1603: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–011.

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 and Escherichia coli in Water.” Appl. Environ. Microbiol. 59:3534–3544 and in USEPA. September 2002.: Method 1604: Total Coliforms and Escherichia coli ( E. coli ) in Water by Membrane Filtration by Using a Simultaneous Detection Technique (MI Medium). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA 821–R–02–024.

²²A description of the Enterolert^®test may be obtained from IDEXX Laboratories, Inc., 1 IDEXX Drive, Westbrook, ME 04092.

²³USEPA. July 2006. Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE–EIA). U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–06–008.

²⁵Method 1622 uses 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 of Cryptosporidium . USEPA. 2001. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–01–026.

²⁶Method 1623 uses 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 of Cryptosporidium and Giardia oocysts and cysts. USEPA. 2001. Method 1623. Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA–821–R–01–025.

(b) The full texts of the methods from the following references which are cited in Tables IA, IB, IC, ID, IE, IF, IG and IH are incorporated by reference into this regulation and may be obtained from the source identified. All costs cited are subject to change and must be verified from the indicated source. The full texts of all the test procedures cited are 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: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html .

(1) The full texts of Methods 601–613, 624, 625, 1613, 1624, and 1625 are printed in appendix A of this part 136. The full text for determining the method detection limit when using the test procedures is given in appendix B of this part 136. The full text of Method 200.7 is printed in appendix C of this part 136. Cited in: Table IB, Note 5; Table IC, Note 2; and Table ID, Note 2.

(2) USEPA. 1978. Microbiological Methods for Monitoring the Environment, Water, and Wastes. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. EPA/600/8–78/017. Available at http://www.epa.gov/clariton/srch.htm or from: National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, Pub. No. PB–290329/A.S. Table IA, Note 3; Table IH, Note 3.

(3) “Methods for Chemical Analysis of Water and Wastes,” U.S. Environmental Protection Agency, EPA–600/4–79–020, March 1979, or “Methods for Chemical Analysis of Water and Wastes,” U.S. Environmental Protection Agency, EPA–600/4–79–020, Revised March 1983. Available from: ORD Publications, CERI, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, Table IB, Note 1.

(4) “Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol and Pesticides in Water and Wastewater,” U.S. Environmental Protection Agency, 1978. Available from: ORD Publications, CERI, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, Table IC, Note 3; Table D, Note 3.

(5) “Prescribed Procedures for Measurement of Radioactivity in Drinking Water,” U.S. Environmental Protection Agency, EPA–600/4–80–032, 1980. Available from: ORD Publications, CERI, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, Table IE, Note 1.

(6) American Public Health Association. 1992, 1995, and 1998. Standard Methods for the Examination of Water and Wastewater. 18th, 19th, and 20th Edition (respectively). Available from: 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 ). Tables IA, IB, IC, ID, IE, and IH.

(9) “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. Available from: American Public Health Association, 1015 Fifteenth Street NW., Washington, DC 20036. Cost available from publisher. Table IB, Note 10; Table IC, Note 6; Table ID, Note 6.

(10) ASTM International. Annual Book of ASTM Standards, Water, and Environmental Technology, Section 11, Volumes 11.01 and 11.02, 1994, 1996, 1999, Volume 11.02, 2000, and individual standards published after 2000. Available from: ASTM International, 100 Barr Harbor Drive, P.O. Box C700, West Conshohocken, PA 19428–2959, or http://www.astm.org . Tables IA, IB, IC, ID, IE, and IH.

(11) USGS. 1989. 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, U.S. Geological Survey, U.S. Department of the Interior, Reston, Virginia. Available from USGS Books and Open-File Reports Section, Federal Center, Box 25425, Denver, Colorado 80225. Table IA, Note 5; Table IH.

(12) “Methods for Determination of Inorganic Substances in Water and Fluvial Sediments,” by M.J. Fishman and Linda C. Friedman, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5 Chapter A1 (1989). Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Cost: $108.75 (subject to change). Table IB, Note 2.

(13) “Methods for Determination of Inorganic Substances in Water and Fluvial Sediments,” N.W. Skougstad and others, editors. Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A1 (1979). Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Cost: $10.00 (subject to change), Table IB, Note 8.

(14) “Methods for the Determination of Organic Substances in Water and Fluvial Sediments,” Wershaw, R.L., et al, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A3 (1987). Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Cost: $0.90 (subject to change). Table IB, Note 24; Table ID, Note 4.

(15) “Water Temperature—Influential Factors, Field Measurement and Data Presentation,” by H.H. Stevens, Jr., J. Ficke, and G.F. Smoot, Techniques of Water-Resources Investigations of the U.S. Geological Survey, Book 1, Chapter D1, 1975. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Cost: $1.60 (subject to change). Table IB, Note 32.

(16) “Selected Methods of the U.S. Geological Survey of Analysis of Wastewaters,” by M.J. Fishman and Eugene Brown; U.S. Geological Survey Open File Report 76–77 (1976). Available from: U.S. Geological Survey, Branch of Distribution, 1200 South Eads Street, Arlington, VA 22202. Cost: $13.50 (subject to change). Table IE, Note 2.

(17) AOAC-International. Official Methods of Analysis of AOAC-International, 16th Edition, (1995). Available from: AOAC-International, 481 North Frederick Avenue, Suite 500, Gaithersburg, MD 20877. Table IB, See footnote 3.

(18) “American National Standard on Photographic Processing Effluents,” April 2, 1975. Available from: American National Standards Institute, 1430 Broadway, New York, New York 10018. Table IB, Note 9.

(19) “An Investigation of Improved Procedures for Measurement of Mill Effluent and Receiving Water Color,” NCASI Technical Bulletin No. 253, December 1971. Available from: National Council of the Paper Industry for Air and Stream Improvements, Inc., 260 Madison Avenue, New York, NY 10016. Cost available from publisher. Table IB, Note 18.

(20) Ammonia, Automated Electrode Method, Industrial Method Number 379–75WE, dated February 19, 1976. Technicon Auto Analyzer II. Method and price available from Technicon Industrial Systems, Tarrytown, New York 10591. Table IB, Note 7.

(21) Chemical Oxygen Demand, Method 8000, Hach Handbook of Water Analysis, 1979. Method price available from Hach Chemical Company, P.O. Box 389, Loveland, Colorado 80537. Table IB, Note 14.

(22) OIC Chemical Oxygen Demand Method, 1978. Method and price available from Oceanography International Corporation, 512 West Loop, P.O. Box 2980, College Station, Texas 77840. Table IB, Note 13.

(23) ORION Research Instruction Manual, Residual Chlorine Electrode Model 97–70, 1977. Method and price available from ORION Research Incorporation, 840 Memorial Drive, Cambridge, Massachusetts 02138. Table IB, Note 16.

(24) Bicinchoninate Method for Copper. Method 8506, Hach Handbook of Water Analysis, 1979, Method and price available from Hach Chemical Company, P.O. Box 300, Loveland, Colorado 80537. Table IB, Note 19.

(25) Hydrogen Ion (pH) Automated Electrode Method, Industrial Method Number 378–75WA. October 1976. Bran & Luebbe (Technicon) Auto Analyzer II. Method and price available from Bran & Luebbe Analyzing Technologies, Inc. Elmsford, N.Y. 10523. Table IB, Note 21.

(26) 1,10-Phenanthroline Method using FerroVer Iron Reagent for Water, Hach Method 8008, 1980. Method and price available from Hach Chemical Company, P.O. Box 389 Loveland, Colorado 80537. Table IB, Note 22.

(27) Periodate Oxidation Method for Manganese, Method 8034, Hach Handbook for Water Analysis, 1979. Method and price available from Hach Chemical Company, P.O. Box 389, Loveland, Colorado 80537. Table IB, Note 23.

(28) Nitrogen, Nitrite—Low Range, Diazotization Method for Water and Wastewater, Hach Method 8507, 1979. Method and price available from Hach Chemical Company, P.O. Box 389, Loveland, Colorado 80537. Table IB, Note 25.

(29) Zincon Method for Zinc, Method 8009. Hach Handbook for Water Analysis, 1979. Method and price available from Hach Chemical Company, P.O. Box 389, Loveland, Colorado 80537. Table IB, Note 33.

(30) “Direct Determination of Elemental Phosphorus by Gas-Liquid Chromatography,” by R.F. Addison and R.G. Ackman, Journal of Chromatography, Volume 47, No. 3, pp. 421–426, 1970. Available in most public libraries. Back volumes of the Journal of Chromatography are available from Elsevier/North-Holland, Inc., Journal Information Centre, 52 Vanderbilt Avenue, New York, NY 10164. Cost available from publisher. Table IB, Note 28.

(31) “Direct Current Plasma (DCP) Optical Emission Spectrometric Method for Trace Elemental Analysis of Water and Wastes”, Method AES 0029, 1986-Revised 1991, Fison Instruments, Inc., 32 Commerce Center, Cherry Hill Drive, Danvers, MA 01923. Table B, Note 34.

(32) “Closed Vessel Microwave Digestion of Wastewater Samples for Determination of Metals, CEM Corporation, P.O. Box 200, Matthews, North Carolina 28106–0200, April 16, 1992. Available from the CEM Corporation. Table IB, Note 36.

(33) “Organochlorine Pesticides and PCBs in Wastewater Using Empore^TM Disk” Test Method 3M 0222, Revised 10/28/94. 3M Corporation, 3M Center Building 220–9E–10, St. Paul, MN 55144–1000. Method available from 3M Corporation. Table IC, Note 8 and Table ID, Note 8.

(34) USEPA. October 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA 821–R–02–012. Available at http://www.epa.gov/epahome/index/sources.htm or from National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, Pub. No. PB2002–108488. Table IA, Note 25.

(35) “Nitrogen, Total Kjeldahl, Method PAI-DK01 (Block Digestion, Steam Distillation, Titrimetric Detection)”, revised 12/22/94. Available from Perstorp Analytical Corporation, 9445 SW Ridder Rd., Suite 310, P.O. Box 648, Wilsonville, OK 97070. Table IB, Note 39.

(36) “Nitrogen, Total Kjeldahl, Method PAI-DK02 (Block Digestion, Steam Distillation, Colorimetric Detection)”, revised 12/22/94. Available from Perstorp Analytical Corporation, 9445 SW Ridder Rd., Suite 310, P.O. Box 648, Wilsonville, OK 97070. Table IB, Note 40.

(37) “Nitrogen, Total Kjeldahl, Method PAI-DK03 (Block Digestion, Automated FIA Gas Diffusion)”, revised 12/22/94. Available from Perstorp Analytical Corporation, 9445 SW Ridder Rd., Suite 310, P.O. Box 648, Wilsonville, OK 97070. Table IB, Note 41.

(38) USEPA. October 2002. Short-Term Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA 821–R–02–013. Available at http://www.epa.gov/epahome/index/sources.htm or from National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, Pub. No. PB2002–108489. Table IA, Note 26.

(39) USEPA. October 2002. Short-Term Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. Third Edition. U.S. Environmental Protection Agency, Office of Water, Washington, DC EPA 821–R–02–014. Available at http://www.epa.gov/epahome/index/sources.htm or from National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, Pub. No. PB2002–108490. Table IA, Note 27.

(40) EPA Methods 1666, 1667, and 1671 listed in the table above are published in the compendium titled Analytical Methods for the Determination of Pollutants in Pharmaceutical Manufacturing Industry Wastewaters (EPA 821–B–98–016). EPA Methods 502.2 and 524.2 have been incorporated by reference into 40 CFR 141.24 and are in Methods for the Determination of Organic Compounds in Drinking Water, EPA–600/4–88–039, December 1988, Revised, July 1991, and Methods for the Determination of Organic Compounds in Drinking Water-Supplement II, EPA–600/R–92–129, August 1992, respectively. These EPA test method compendia are available from the National Technical Information Service, NTIS PB91–231480 and PB92–207703, U.S. Department of Commerce, 5285 Port Royal Road, Springfield, Virginia 22161. The toll-free number is 800–553–6847. ASTM test methods D3371, D3695, and D4763 are available from the American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428–2959.

(41) USEPA. 2002. Method 1631, Revision E, “Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic Fluorescence Spectrometry.” September 2002. Office of Water, U.S. Environmental Protection Agency (EPA–821–R–02–019). Available from: National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161. Publication No. PB2002–108220. Cost: $25.50 (subject to change).

(43) Method OIA–1677, Available Cyanide by Flow Injection, Ligand Exchange, and Amperometry. August 1999. ALPKEM, OI Analytical, Box 648, Wilsonville, Oregon 97070 (EPA–821–R–99–013). Available from: National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161. Publication No. PB99–132011. Cost: $22.50. Table IB, Note 44.

(44) “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”, Open File Report (OFR) 00–170. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table IB, Note 45.

(45) “Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Chromium in Water by Graphite Furnace Atomic Absorption Spectrophotometry”, Open File Report (OFR) 93–449. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table IB, Note 46.

(46) “Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Molybdenum in Water by Graphite Furnace Atomic Absorption Spectrophotometry”, Open File Report (OFR) 97–198. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table IB, Note 47.

(47) “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” Open File Report (OFR) 92–146. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table IB, Note 48.

(48) “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” Open File Report (OFR) 98–639. Table IB, Note 49.

(49) “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” , Open File Report (OFR) 98–165. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table IB, Note 50.

(50) “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” U.S.Geological Survey Open File Report 94–37. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table ID, Note 9.

(51) “Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory—Determination of Inorganic and Organic Constituents in Water and Fluvial Sediments”, Open File Report (OFR) 93–125. Available from: U.S. Geological Survey, Denver Federal Center, Box 25425, Denver, CO 80225. Table IB, Note 51; Table IC, Note 9.

(52) IDEXX Laboratories, Inc. 2002. Description of Colilert^®, Colilert-18^®, Quanti-Tray^®, Quanti-Tray^®/2000, Enterolert^®methods are available from IDEXX Laboratories, Inc., One Idexx Drive, Westbrook, Maine 04092. Table IA, Notes 17 and 23; Table IH, Notes 16 and 22.

(53) Hach Company, Inc. Revision 2, 1999. Description of m-ColiBlue24^®Method, Total Coliforms and E. coli , is available from Hach Company, 100 Dayton Ave, Ames IA 50010. Table IA, Note 18; Table IH, Note 17.

(54) USEPA. July 2006. Method 1103.1: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). U.S. Environmental Protection Agency, Office of Water, Washington DC EPA–621–R–06–010. Available at http://www.epa.gov/waterscience/methods/ . Table IH, Note 19.

(55) USEPA. July 2006. Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE–EIA). U.S. Environmental Protection Agency, Office of Water, Washington DC EPA–621–R–06–008. Available at http://www.epa.gov/waterscience/methods/ . Table IH, Note 23

(56) USEPA. July 2006. Method 1603: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC). U.S. Environmental Protection Agency, Office of Water, Washington DC EPA–821–R–06–011. Available at http://www.epa.gov/waterscience/methods/ . Table IH, Note 19; Table IH, Note 20.

(57) Brenner et al. 1993. New Medium for the Simultaneous Detection of Total Coliforms and Escherichia coli in Water. Appl. Environ. Microbiol. 59:3534–3544. Available from the American Society for Microbiology, 1752 N Street NW., Washington DC 20036. Table IH, Note 21.

(58) USEPA. September 2002. Method 1604: Total Coliforms and Escherichia coli ( E. coli ) in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium). U.S. Environmental Protection Agency, Office of Water, Washington DC EPA–821–R–02–024. Available at http://www.epa.gov/waterscience/methods/ . Table IH, Note 20.

(59) USEPA. July 2006. Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI). U.S. Environmental Protection Agency, Office of Water, Washington DC EPA–821–R–06–009. Available at http://www.epa.gov/waterscience/methods/ . Table IA, Note 24; Table IH, Note 24.

(60) USEPA. April 2001. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of Water, Washington DC EPA–821–R–01–026. Available at http://www.epa.gov/waterscience/methods/ . Table IH, Note 25.

(61) USEPA. April 2001. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA–821–R–01–025. Available at http://www.epa.gov/waterscience/methods/ . Table IH, Note 26.

(62) AOAC. 1995. Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. AOAC International, 481 North Frederick Avenue, Suite 500, Gaithersburg, Maryland 20877–2417. Table IA, Note 11; Table IH.

(63) Waters Corporation. Method D6508, Rev. 2, “Test Method for Determination of Dissolved Inorganic Anions in Aqueous Matrices Using Capillary Ion Electrophoresis and Chromate Electrolyte,” available from Waters Corp, 34 Maple Street, Milford, MA 01757, Telephone: 508/482–2131, Fax: 508/482–3625, Table IB, See footnote 54.

(64) Kelada-01, “Kelada Automated Test Methods for Total Cyanide, Acid Dissociable Cyanide, and Thiocyanate,” EPA 821–B–01–009 Revision 1.2, August 2001 is available from National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 22161 [Order Number PB 2001–108275]. Telephone: 800–553–6847. Table IB, See footnote 55.

(65) 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 is available from Lachat Instruments 6645 W. Mill Road, Milwaukee, WI 53218, Telephone: 414–358–4200. Table IB, See footnote 56.

(66) “Methods for the Determination of Metals in Environmental Samples,” Supplement I, National Exposure Risk Laboratory-Cincinnati (NERL–CI), EPA/600/R–94/111, May 1994; and “Methods for the Determination of Inorganic Substances in Environmental Samples,” NERL–CI, EPA/600/R–93/100, August, 1993 are available from National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 22161. Telephone: 800–553–6847. Table IB.

(67) “Determination of Inorganic Ions in Drinking Water by Ion Chromatography,” Rev. 1.0, 1997 is available from from http://www.epa.gov/safetwater/methods/met300.pdf . Table IB.

(68) Table IG Methods are available in “Methods For The Determination of Nonconventional Pesticides In Municipal and Industrial Wastewater, Volume I,” EPA 821–R–93–010A, August 1993 Revision I, and “Methods For The Determination of Nonconventional Pesticides In Municipal and Industrial Wastewater, Volume II,” EPA 821–R–93–010B (August 1993) are available from National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 22161. Telephone: 800–553–6847.

(69) Method 245.7, Rev. 2.0, “Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry,” February 2005, EPA–821–R–05–001, available from the U.S. EPA Sample Control Center (operated by CSC), 6101 Stevenson Avenue, Alexandria, VA 22304, Telephone: 703–461–8056. Table IB, See footnote 59.

(70) USEPA. July 2006. Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using Lauryl Tryptose Broth (LTB) and EC Medium. U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA 821–R–06–012. Available at http://www.epa.gov/waterscience/methods/ .

(71) USEPA. July 2006. Method 1681: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using A–1 Medium. U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA 821–R–06–013. Available at http://www.epa.gov/waterscience/methods/ .

(72) USEPA. July 2006. Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium. U.S. Environmental Protection Agency, Office of Water, Washington DC. EPA 821–R–06–014. Available at http://www.epa.gov/waterscience/methods/ .

(c) Under certain circumstances, the Regional Administrator or the Director in the Region or State where the discharge will occur may determine for a particular discharge that additional parameters or pollutants must be reported. Under such circumstances, additional test procedures for analysis of pollutants may be specified by the Regional Administrator, or the Director upon recommendation of the Alternate Test Procedure Program Coordinator, Washington, DC.

(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 variance from the prescribed preservation techniques, container materials, and maximum holding times applicable to samples taken from a specific discharge. Applications for variances may be made by letters to the Regional Administrator in the Region in which the discharge will occur. Sufficient data should be provided to assure such variance does not adversely affect the integrity of the sample. Such data will be forwarded by the Regional Administrator, to the Alternate Test Procedure Program Coordinator, Washington, DC, for technical review and recommendations for action on the variance application. Upon receipt of the recommendations from the Alternate Test Procedure Program Coordinator, the Regional Administrator may grant a variance applicable to the specific discharge to the applicant. A decision to approve or deny a variance will be made within 90 days of receipt of the application by the Regional Administrator.

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

Parameter No./name	Container¹	Preservation^2,3	Maximum holding time⁴
Table IA—Bacterial Tests:
1–5. Coliform, total, fecal, and E. coli	PA, G	Cool, <10 °C, 0.0008% Na2S2O3⁵	6 hours.^22,23
6. Fecal streptococci	PA, G	Cool, <10 °C, 0.0008% Na2S2O3⁵	6 hours.²²
7. Enterococci	PA, G	Cool, <10 °C, 0.0008% Na2S2O3⁵	6 hours.²²
8. Salmonella	PA, G	Cool, <10 °C, 0.0008% Na2S2O3⁵	6 hours.²²
Table IA—Aquatic Toxicity Tests:
9–11. Toxicity, acute and chronic	P, FP, G	Cool, ≤6 °C¹⁶	36 hours.
Table lB—Inorganic Tests:
1. Acidity	P, FP, G	Cool, ≤6 °C¹⁸	14 days.
2. Alkalinity	P, FP, G	Cool, ≤6 °C¹⁸	14 days.
4. Ammonia	P, FP, G	Cool, ≤6 °C¹⁸, H2SO4 to pH<2	28 days.
9. Biochemical oxygen demand	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
10. Boron	P, FP, or Quartz	HNO3 to pH<2	6 months.
11. Bromide	P, FP, G	None required	28 days.
14. Biochemical oxygen demand, carbonaceous	P, FP G	Cool, ≤6 °C¹⁸	48 hours.
15. Chemical oxygen demand	P, FP, G	Cool, ≤6 °C¹⁸, H2SO4 to pH<2	28 days.
16. Chloride	P, FP, G	None required	28 days.
17. Chlorine, total residual	P, G	None required	Analyze within 15 minutes.
21. Color	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
23–24. Cyanide, total or available (or CATC)	P, FP, G	Cool, ≤6 °C¹⁸, NaOH to pH>12⁶, reducing agent⁵	14 days.
25. Fluoride	P	None required	28 days.
27. Hardness	P, FP, G	HNO3 or H2SO4 to pH<2	6 months.
28. Hydrogen ion (pH)	P, FP, G	None required	Analyze within 15 minutes.
31, 43. Kjeldahl and organic N	P, FP, G	Cool, ≤6 °C¹⁸, H2SO4 to pH<2	28 days.
Table IB—Metals:⁷
18. Chromium VI	P, FP, G	Cool, ≤6 °C¹⁸, pH = 9.3–9.7²⁰	28 days.
35. Mercury (CVAA)	P, FP, G	HNO3 to pH<2	28 days.
35. Mercury (CVAFS)	FP, G; and FP-lined cap¹⁷	5 mL/L 12N HCl or 5 mL/L BrCl¹⁷	90 days.¹⁷
3, 5–8, 12, 13, 19, 20, 22, 26, 29, 30, 32–34, 36, 37, 45, 47, 51, 52, 58–60, 62, 63, 70–72, 74, 75	P, FP, G	HNO3 to pH<2, or at least 24 hours prior to analysis¹⁹	6 months.
Metals, except boron, chromium VI, and mercury
38. Nitrate	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
39. Nitrate-nitrite	P, FP, G	Cool, ≤6 °C¹⁸, H2SO4 to pH<2	28 days.
40. Nitrite	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
41. Oil and grease	G	Cool to ≤6 °C¹⁸, HCl or H2SO4 to pH<2	28 days.
42. Organic Carbon	P, FP, G	Cool to ≤6 °C¹⁸, HCl, H2SO4, or H3PO4 to pH<2	28 days.
44. Orthophosphate	P, FP, G	Cool, ≤6 °C¹⁸	Filter within 15 minutes; Analyze within 48 hours.
46. Oxygen, Dissolved Probe	G, Bottle and top	None required	Analyze within 15 minutes.
47. Winkler	G, Bottle and top	Fix on site and store in dark	8 hours.
48. Phenols	G	Cool, ≤6 °C¹⁸, H2SO4 to pH<2	28 days.
49. Phosphorous (elemental)	G	Cool, ≤6 °C¹⁸	48 hours.
50. Phosphorous, total	P, FP, G	Cool, ≤6 °C¹⁸, H2SO4 to pH<2	28 days.
53. Residue, total	P, FP, G	Cool, ≤6 °C¹⁸	7 days.
54. Residue, Filterable	P, FP, G	Cool, ≤6 °C¹⁸	7 days.
55. Residue, Nonfilterable (TSS)	P, FP, G	Cool, ≤6 °C¹⁸	7 days.
56. Residue, Settleable	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
57. Residue, Volatile	P, FP, G	Cool, ≤6 °C¹⁸	7 days.
61. Silica	P or Quartz	Cool, ≤6 °C¹⁸	28 days.
64. Specific conductance	P, FP, G	Cool, ≤6 °C¹⁸	28 days.
65. Sulfate	P, FP, G	Cool, ≤6 °C¹⁸	28 days.
66. Sulfide	P, FP, G	Cool, ≤6 °C¹⁸, add zinc acetate plus sodium hydroxide to pH>9	7 days.
67. Sulfite	P, FP, G	None required	Analyze within 15 minutes.
68. Surfactants	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
69. Temperature	P, FP, G	None required	Analyze.
73. Turbidity	P, FP, G	Cool, ≤6 °C¹⁸	48 hours.
Table lC—Organic Tests⁸
13, 18–20, 22, 24–28, 34–37, 39–43, 45–47, 56, 76, 104, 105, 108–111, 113. Purgeable Halocarbons	G, FP-lined septum	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵	14 days.
6, 57, 106. Purgeable aromatic hydrocarbons	G, FP-lined septum	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵, HCl to pH 2⁹	14 days.⁹
3, 4. Acrolein and acrylonitrile	G, FP-lined septum	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵, pH to 4–5¹⁰	14 days.¹⁰
23, 30, 44, 49, 53, 77, 80, 81, 98, 100, 112. Phenols¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵	7 days until extraction, 40 days after extraction.
7, 38. Benzidines^{11, 12}	G, FP-lined cap	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵	7 days until extraction.¹³
14, 17, 48, 50–52. Phthalate esters¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸	7 days until extraction, 40 days after extraction.
82–84. Nitrosamines^{11, 14}	G, FP-lined cap	Cool, ≤6 °C¹⁸, store in dark, 0.008% Na2S2O3⁵	7 days until extraction, 40 days after extraction.
88–94. PCBs¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸	1 year until extraction, 1 year after extraction.
54, 55, 75, 79. Nitroaromatics and isophorone¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸, store in dark, 0.008% Na2S2O3⁵	7 days until extraction, 40 days after extraction.
1, 2, 5, 8–12, 32, 33, 58, 59, 74, 78, 99, 101. Polynuclear aromatic hydrocarbons¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸, store in dark, 0.008% Na2S2O3⁵	7 days until extraction, 40 days after extraction.
15, 16, 21, 31, 87. Haloethers¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵	7 days until extraction, 40 days after extraction.
29, 35–37, 63–65, 107. Chlorinated hydrocarbons¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸	7 days until extraction, 40 days after extraction.
60–62, 66–72, 85, 86, 95–97, 102, 103. CDDs/CDFs¹¹
Aqueous Samples: Field and Lab Preservation	G	Cool, ≤6 °C¹⁸, 0.008% Na2S2O3⁵, pH<9	1 year.
Solids and Mixed-Phase Samples: Field Preservation	G	Cool, ≤6 °C¹⁸	7 days.
Tissue Samples: Field Preservation	G	Cool, ≤6 °C¹⁸	24 hours.
Solids, Mixed-Phase, and Tissue Samples: Lab Preservation	G	Freeze, ≤−10 °C	1 year.
Table lD—Pesticides Tests:
1–70. Pesticides¹¹	G, FP-lined cap	Cool, ≤6 °C¹⁸, pH 5–9¹⁵	7 days until extraction, 40 days after extraction.
Table IE—Radiological Tests:
1–5. Alpha, beta, and radium	P, FP, G	HNO3 to pH<2	6 months.
Table IH—Bacterial Tests:
1. E. coli	PA, G	Cool, <10 °C, 0.0008% Na2S2O3⁵	6 hours.²²
2. Enterococci	PA, G	Cool, <10 °C, 0.0008% Na2S2O3⁵	6 hours.²²
Table IH—Protozoan Tests:
8. Cryptosporidium	LDPE; field filtration	0–8 °C	96 hours.²¹
9. Giardia	LDPE; field filtration	0–8 °C	96 hours.²¹

¹“P” is 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 sterlizable 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 sampler (e.g., using a 24-hour composite sampler; 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 an aliquot split from a composite sample; otherwise, preserve the grab sample, 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 the results of analysis of individual grab samples. For examples of laboratory compositing procedures, see EPA Method 1664A (oil and grease) and the procedures at 40 CFR 141.34(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 requirements 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 (e.g., samples analyzed for fecal coliforms may be held up to 6 hours prior to commencing analysis). Samples may be held for longer periods only if the permittee or monitoring laboratory has 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 Administrator 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.

⁵Add a reducing agent only if an oxidant (e.g., chlorine) is present. Reducing agents shown to be effective are sodium thiosulfate (Na2S2O3), ascorbic acid, sodium arsenite (NaAsO2), or sodium borohydride (NaBH4). However, some of these agents have been shown to produce a positive or negative cyanide bias, depending on other substances in the sample and the analytical method used. Therefore, do not add an excess of reducing agent. Methods recommending ascorbic acid (e.g., EPA Method 335.4) specify adding ascorbic acid crystals, 0.1–0.6 g, until a drop of sample produces no color on potassium iodide (KI) starch paper, then adding 0.06 g (60 mg) for each liter of sample volume. If NaBH4 or NaAsO2 is used, 25 mg/L NaBH4 or 100 mg/L NaAsO2 will reduce more than 50 mg/L of chlorine (see method “Kelada-01” and/or Standard Method 4500–CN⁻for more information). After adding reducing agent, test the sample using KI paper, a test strip (e.g. for chlorine, SenSafe^TMTotal Chlorine Water Check 480010) moistened with acetate buffer solution (see Standard Method 4500–Cl.C.3e), or a chlorine/oxidant test method (e.g., EPA Method 330.4 or 330.5), to make sure all oxidant is removed. If oxidant remains, add more reducing agent. Whatever agent is used, it should be tested to assure that cyanide results are not affected adversely.

⁶Sample collection and preservation: Collect a volume of sample appropriate to the analytical method in a bottle of the material specified. If the sample can be analyzed within 48 hours and sulfide is not present, adjust the pH to > 12 with sodium hydroxide solution (e.g., 5% w/v), refrigerate as specified, and analyze within 48 hours. Otherwise, to extend the holding time to 14 days and mitigate interferences, treat the sample immediately using any or all of the following techniques, as necessary, followed by adjustment of the sample pH to > 12 and refrigeration as specified. There may be interferences that are not mitigated by approved procedures. Any procedure for removal or suppression of an interference may be employed, provided the laboratory demonstrates that it more accurately measures cyanide. Particulate cyanide (e.g., ferric ferrocyanide) or a strong cyanide complex (e.g., cobalt cyanide) are more accurately measured if the laboratory holds the sample at room temperature and pH > 12 for a minimum of 4 hours prior to analysis, and performs UV digestion or dissolution under alkaline (pH=12) conditions, if necessary.

(1) Sulfur: To remove elemental sulfur (S8), filter the sample immediately. If the filtration time will exceed 15 minutes, use a larger filter or a method that requires a smaller sample volume (e.g., EPA Method 335.4 or Lachat Method 01). Adjust the pH of the filtrate to > 12 with NaOH, refrigerate the filter and filtrate, and ship or transport to the laboratory. In the laboratory, extract the filter with 100 mL of 5% NaOH solution for a minimum of 2 hours. Filter the extract and discard the solids. Combine the 5% NaOH-extracted filtrate with the initial filtrate, lower the pH to approximately 12 with concentrated hydrochloric or sulfuric acid, and analyze the combined filtrate. Because the detection limit for cyanide will be increased by dilution by the filtrate from the solids, test the sample with and without the solids procedure if a low detection limit for cyanide is necessary. Do not use the solids procedure if a higher cyanide concentration is obtained without it. Alternatively, analyze the filtrates from the sample and the solids separately, add the amounts determined (in μg or mg), and divide by the original sample volume to obtain the cyanide concentration.

(2) Sulfide: If the sample contains sulfide as determined by lead acetate paper, or if sulfide is known or suspected to be present, immediately conduct one of the volatilization treatments or the precipitation treatment as follows: Volatilization—Headspace expelling. In a fume hood or well-ventilated area, transfer 0.75 liter of sample to a 4.4 L collapsible container (e.g., Cubitainer^TM). Acidify with concentrated hydrochloric acid to pH < 2. Cap the container and shake vigorously for 30 seconds. Remove the cap and expel the headspace into the fume hood or open area by collapsing the container without expelling the sample. Refill the headspace by expanding the container. Repeat expelling a total of five headspace volumes. Adjust the pH to > 12, refrigerate, and ship or transport to the laboratory. Scaling to a smaller or larger sample volume must maintain the air to sample volume ratio. A larger volume of air will result in too great a loss of cyanide (> 10%). Dynamic stripping: In a fume hood or well-ventilated area, transfer 0.75 liter of sample to a container of the material specified and acidify with concentrated hydrochloric acid to pH < 2. Using a calibrated air sampling pump or flowmeter, purge the acidified sample into the fume hood or open area through a fritted glass aerator at a flow rate of 2.25 L/min for 4 minutes. Adjust the pH to > 12, refrigerate, and ship or transport to the laboratory. Scaling to a smaller or larger sample volume must maintain the air to sample volume ratio. A larger volume of air will result in too great a loss of cyanide (> 10%). Precipitation: If the sample contains particulate matter that would be removed by filtration, filter the sample prior to treatment to assure that cyanide associated with the particulate matter is included in the measurement. Ship or transport the filter to the laboratory. In the laboratory, extract the filter with 100 mL of 5% NaOH solution for a minimum of 2 hours. Filter the extract and discard the solids. Combine the 5% NaOH-extracted filtrate with the initial filtrate, lower the pH to approximately 12 with concentrated hydrochloric or sulfuric acid, and analyze the combined filtrate. Because the detection limit for cyanide will be increased by dilution by the filtrate from the solids, test the sample with and without the solids procedure if a low detection limit for cyanide is necessary. Do not use the solids procedure if a higher cyanide concentration is obtained without it. Alternatively, analyze the filtrates from the sample and the solids separately, add the amounts determined (in μg or mg), and divide by the original sample volume to obtain the cyanide concentration. For removal of sulfide by precipitation, raise the pH of the sample to > 12 with NaOH solution, then add approximately 1 mg of powdered cadmium chloride for each mL of sample. For example, add approximately 500 mg to a 500-mL sample. Cap and shake the container to mix. Allow the precipitate to settle and test the sample with lead acetate paper. If necessary, add cadmium chloride but avoid adding an excess. Finally, filter through 0.45 micron filter. Cool the sample as specified and ship or transport the filtrate and filter to the laboratory. In the laboratory, extract the filter with 100 mL of 5% NaOH solution for a minimum of 2 hours. Filter the extract and discard the solids. Combine the 5% NaOH-extracted filtrate with the initial filtrate, lower the pH to approximately 12 with concentrated hydrochloric or sulfuric acid, and analyze the combined filtrate. Because the detection limit for cyanide will be increased by dilution by the filtrate from the solids, test the sample with and without the solids procedure if a low detection limit for cyanide is necessary. Do not use the solids procedure if a higher cyanide concentration is obtained without it. Alternatively, analyze the filtrates from the sample and the solids separately, add the amounts determined (in μg or mg), and divide by the original sample volume to obtain the cyanide concentration. If a ligand-exchange method is used (e.g., ASTM D6888), it may be necessary to increase the ligand-exchange reagent to offset any excess of cadmium chloride.

(3) Sulfite, thiosulfate, or thiocyanate: If sulfite, thiosulfate, or thiocyanate is known or suspected to be present, use UV digestion with a glass coil (Method Kelada-01) or ligand exchange (Method OIA–1677) to preclude cyanide loss or positive interference.

(4) Aldehyde: If formaldehyde, acetaldehyde, or another water-soluble aldehyde is known or suspected to be present, treat the sample with 20 mL of 3.5% ethylenediamine solution per liter of sample.

(5) Carbonate: Carbonate interference is evidenced by noticeable effervescence upon acidification in the distillation flask, a reduction in the pH of the absorber solution, and incomplete cyanide spike recovery. When significant carbonate is present, adjust the pH to ≥12 using calcium hydroxide instead of sodium hydroxide. Allow the precipitate to settle and decant or filter the sample prior to analysis (also see Standard Method 4500–CN.B.3.d).

(6) Chlorine, hypochlorite, or other oxidant: Treat a sample known or suspected to contain chlorine, hypochlorite, or other oxidant as directed in footnote 5.

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.

¹⁶Sufficient ice should be placed 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, it is necessary to 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.

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.

²²Samples analysis should begin immediately, preferably within 2 hours of collection. The maximum transport time to the laboratory is 6 hours, and samples should be processed within 2 hours of receipt at the laboratory.

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.

Editorial Note: ForFederal Registercitations affecting §136.3, see the List of CFR Sections Affected, which appears in the Finding Aids section of the printed volume and on GPO Access.

§ 136.4 Application for alternate test procedures.

(a) Any person may apply to the Regional Administrator in the Region where the discharge occurs for approval of an alternative test procedure.

(b) When the discharge for which an alternative test procedure is proposed occurs within a State having a permit program approved pursuant to section 402 of the Act, the applicant shall submit his application to the Regional Administrator through the Director of the State agency having responsibility for issuance of NPDES permits within such State.

(c) Unless and until printed application forms are made available, an application for an alternate test procedure may be made by letter in triplicate. Any application for an alternate test procedure under this paragraph (c) shall:

(1) Provide the name and address of the responsible person or firm making the discharge (if not the applicant) and the applicable ID number of the existing or pending permit, issuing agency, and type of permit for which the alternate test procedure is requested, and the discharge serial number.

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

(3) Provide justification for using testing procedures other than those specified in Table I.

(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.

(d) An application for approval of an alternate test procedure for nationwide use may be made by letter in triplicate to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303), Office of Water, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460. Any application for an alternate test procedure under this paragraph (d) 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 testing procedure is being requested.

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

(4) Provide comparability data for the performance of the proposed alternate test procedure compared to the performance of the approved test procedures.

[38 FR 28760, Oct. 16, 1973, as amended at 41 FR 52785, Dec. 1, 1976; 62 FR 30763, June 5, 1997; 72 FR 11239, Mar. 12, 2007]

(a) The Regional Administrator of the region in which the discharge will occur has final responsibility for approval of any alternate test procedure proposed by the responsible person or firm making the discharge.

(b) Within thirty days of receipt of an application, the Director will forward such application proposed by the responsible person or firm making the discharge, together with his recommendations, to the Regional Administrator. Where the Director recommends rejection of the application for scientific and technical reasons which he provides, the Regional Administrator shall deny the application and shall forward this decision to the Director of the State Permit Program and to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303), Office of Water, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460.

(c) Before approving any application for an alternate test procedure proposed by the responsible person or firm making the discharge, the Regional Administrator shall forward a copy of the application to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303), Office of Water, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460.

(d) Within ninety days of receipt by the Regional Administrator of an application for an alternate test procedure, proposed by the responsible person or firm making the discharge, the Regional Administrator shall notify the applicant and the appropriate State agency of approval or rejection, or shall specify the additional information which is required to determine whether to approve the proposed test procedure. Prior to the expiration of such ninety day period, a recommendation providing the scientific and other technical basis for acceptance or rejection will be forwarded to the Regional Administrator by the Alternate Test Procedure Program Coordinator, Washington, DC. A copy of all approval and rejection notifications will be forwarded to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303), Office of Water, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460, for the purposes of national coordination.

(e) Approval for nationwide use. (1) As expeditiously as is practicable after receipt by the Alternate Test Procedure Program Coordinator, Washington, DC, of an application for an alternate test procedure for nationwide use, the Alternate Test Procedure Program Coordinator, Washington, DC, shall notify the applicant in writing whether the application is complete. If the application is incomplete, the applicant shall be informed of the information necessary to make the application complete.

(2) As expeditiously as is practicable after receipt of a complete package, the Alternate Test Procedure Program Coordinator shall perform any analysis necessary to determine whether the alternate test procedure satisfies the applicable requirements of this part, and the Alternate Test Procedure Program Coordinator shall recommend to the Administrator that he/she approve or reject the application and shall also notify the application of the recommendation.

(3) As expeditiously as practicable, an alternate method determined by the Administrator to satisfy the applicable requirements of this part shall be proposed by EPA for incorporation in subsection 136.3 of 40 CFR part 136. EPA shall make available for review all the factual bases for its proposal, including any performance data submitted by the applicant and any available EPA analysis of those data.

(4) Following a period of public comment, EPA shall, as expeditiously as practicable, publish in theFederal Registera final decision to approve or reject the alternate method.

[38 FR 28760, Oct. 16, 1973, as amended at 41 FR 52785, Dec. 1, 1976; 55 FR 33440, Aug. 15, 1990; 62 FR 30763, June 5, 1997; 72 FR 11239, Mar. 12, 2007]

(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 the QC acceptance criteria of the approved method at this part.

(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.

(1) Allowable changes. Except as set forth in paragraph (b)(3) of this section, an analyst may modify an approved test procedure (analytical method) provided that the chemistry of the method or the determinative technique is not changed, and provided that the requirements of paragraph (b)(2) of this section are met.

(i) Potentially acceptable modifications regardless of current method performance include changes between automated and manual discrete instrumentation; changes in the calibration range (provided that the modified range covers any relevant regulatory limit); changes in equipment such as using similar equipment from a vendor other than that mentioned in the method (e.g., a purge-and-trap device from OIA rather than Tekmar), changes in equipment operating parameters such as changing the monitoring wavelength of a colorimeter or modifying the temperature program for a specific GC column; changes to chromatographic columns (treated in greater detail in paragraph (d) of this section); and increases in purge-and-trap sample volumes (provided specifications in paragraph (e) of this section are met). The changes are only allowed provided that all the requirements of paragraph (b)(2) of this section are met.

(ii) 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 using salts as specified in Guidance on Evaluation, Resolution, and Documentation of Analytical Problems Associated with Compliance Monitoring (EPA 821–B–93–001, June 1993), 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. Chlorinated samples must be dechlorinated prior to the addition of such salts.

(iii) If the characteristics of a wastewater matrix result in poor sample dispersion or reagent deposition on equipment and prevents the analyst from meeting QC requirements, the analysts may attempt to resolve the issue by adding an inert surfactant ( i.e. a surfactant that will not affect the chemistry of the method), which may include 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)(2) of this section are met. Chlorinated samples must be dechlorinated prior to the addition of such surfactant.

(2) Requirements. A modified method must produce equivalent performance to the approved methods for the analyte(s) of interest, and the equivalent performance must be documented.

(A) 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. 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., as 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 paragraph (b)(2)(i)(A) of this section, the analyst must employ QC tests specified in Protocol for EPA Approval of Alternate Test Procedures for Organic and Inorganic Analytes in Wastewater and Drinking Water (EPA–821–B–98–002, March 1999) and meet the QC provisions specified therein. In addition, the Analyst must perform on-going 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 of a discharge analyzed), and analysis of an ongoing precision and recovery sample and a blank with each batch of 20 or fewer samples.

(C) Calibration must be performed using the modified method and the modified method must be tested with every wastewater matrix to which it will be applied (up to nine distinct matrices; as described in the ATP Protocol, after validation in nine distinct matrices, the method may be applied to all wastewater matrices), in addition to any and all reagent water tests. If the performance in the wastewater matrix or reagent water does not meet the QC acceptance criteria the method modification may not be used.

(D) Analysts must test representative effluents with the modified method, and demonstrate that the results are equivalent or superior to results with the unmodified method.

(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. 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 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 (e.g., a change to a metals digestion or total cyanide distillation). An analyst may also may not modify any sample preservation and/or holding time requirements of an approved method.

(c) Analytical requirements for multi-analyte methods (Target Analytes). For the purpose of NPDES reporting, the discharger or permittee must meet QC requirements only for the analyte(s) being measured and reported under the NPDES permit.

(d) The following modifications to approved methods are authorized in the circumstances described below:

(1) Capillary column. Use of a capillary (open tubular) GC column rather than a packed column is allowed with EPA Methods 601–613, 624, 625, and 1624B in Appendix A to this part, provided that all QC tests for the approved method are performed and all QC acceptance criteria are met. When changing from a packed column to a capillary column, retention times will change. Analysts are not required to meet retention time specified in the approved method when this change is made. Instead, analysts must generate new retention time tables with capillary columns to be kept on file along with other startup test and ongoing QC data, for review by auditors.

(2) Increased sample volume in purge and trap methodology. Use of increased sample volumes, up to a maximum of 25 mL, is allowed for an approved method, provided that the height of the water column in the purge vessel is at least 5 cm. The analyst should also use one or more surrogate analytes that are chemically similar to the analytes of interest in order to demonstrate that the increased sample volume does not adversely affect the analytical results.

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.

2.1 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.

3.1 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.

4.1 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 identified^4,6 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.

5.1.1 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.

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.

6.3.1 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.

6.5 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.

6.5.2.1 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.

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).

7.3.1 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.

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, A_s/A_is, 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.

7.5.3 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.

8.1 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and Xfor 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 Xfalls 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 X; (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 (P_s) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3 Compare the percent recovery (P_s) 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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If p=90% and s_p=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).

9.1 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.

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.

10.3 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.

11.1.1 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.

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

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.

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.

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

3.2 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.

4.2 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.

5.1.1 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.

5.2.2 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.

5.2.4 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.

5.3.1 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.

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.

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

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.

6.6 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.

6.6.4 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.

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.

7.4 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.

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

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The mimimum 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.

8.1.3 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.

8.1.6 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.

8.2.2 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and Xfor 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 Xfalls 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.

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

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.

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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p=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).

8.7 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).

9.1 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.

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.

10.5 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.

10.11 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.

11.1.2 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.

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

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.

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.

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

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.

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.

3.1 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.

3.3 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.

4.1 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 identified^4,6 for the information of the analyst.

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.

5.2.1.2 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.

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.

6.3 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.

6.5.1 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.

6.7 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.

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.

7.4.1 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.

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

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.

8.1.3 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.

8.1.6 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and Xfor 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 Xfalls 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.

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

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.

8.4 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.

8.4.3 Compare the percent recovery (P_s) 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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p=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.

9.2 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.

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.

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.

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

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.

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.

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.

1.1 This method covers the determination of phenol and certain substituted phenols. The following parameters may be determined by this method:

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.

1.5 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.

2.1 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.

3.1 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.

4.1 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 identified^5,7 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.

5.1.1 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.):

5.2.2 Drying column—Chromatographic column, 400 mm long × 19 mm ID, with coarse frit filter disc.

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).

5.2.9 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.

5.6 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.

6.1 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.

6.6 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.

6.9 Pentafluorobenzyl bromide (α-Bromopentafluorotoluene)—97% minimum purity.

6.10 18-crown-6-ether (1,4,7,10,13,16-Hexaoxacyclooctadecane)—98% minimum purity.

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.

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).

7.2.1 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.

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.

8.1.1 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.

8.1.6 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and Xfor 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 Xfalls 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.

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

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 X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.⁸

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.

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.

8.5 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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p=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.

9.1 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

10.1 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.

10.4 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.

11.1 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.

11.3 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.

12.1 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.

12.5 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.

13.1 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.

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.

13.2 Determine the concentration of individual compounds in the sample analyzed by derivatization and ECDGC according to Equation 4.

A=Mass of underivatized phenol represented by area of peak in sample chromatogram, determined from calibration curve in Section 7.5.3 (ng).

V_t=Total volume of column eluate or combined fractions from which V_iwas taken (µL).

C=Volume of hexane sample solution added to cleanup column in Section 12.7 (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.

14.1 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.

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.

1.1 This method covers the determination of certain benzidines. The following parameters can be determined by this method:

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.

1.5 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.

2.1 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.

3.1 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.)

4.1 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 identified^4,6 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.

5.4 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.

5.4.3 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.

6.1 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.

6.5 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.

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).

7.2.1 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.

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.

8.1.1 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.

8.1.6 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and Xfor 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 Xfalls 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.

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

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.

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

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.

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.

8.5 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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p=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.

9.1 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.

10.1 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.

11.1 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.

12.1 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.

12.3 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.

13.1.2 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.

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

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.

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.

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).

1.1 This method covers the determination of certain phthalate esters. The following parameters can be determined by this method:

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.

1.4 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.

2.1 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.

3.1.1 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.

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).

5.2.2 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).

5.3 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.

5.6 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.

6.1 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.

6.3.1 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.

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).

7.2.1 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.

7.3.1 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.

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.

8.1.4 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.

8.1.6 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and Xfor 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 Xfalls 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.

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

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 X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.⁹

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.

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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p=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).

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

10.1 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 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.

11.3.1 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).

11.4.1 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).

12.1 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.

12.3 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.

12.6 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.

13.1.2 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.

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

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.

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.

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.

1.1 This method covers the determination of certain nitrosamines. The following parameters can be determined by this method:

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.

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.

3.1.1 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 reported6-9to 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.

4.2 These nitrosamines are known carcinogens,13-17therefore, 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.

5.1.2 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.):

5.2.2 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).

5.2.8 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.

5.6.1 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.

6.1 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.

6.4 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.

6.8.1 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.

6.11.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

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).

7.2.1 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.

7.3.1 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.

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.

8.1.4 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.

8.1.6 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 (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and Xfor 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 Xfalls 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.

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

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.

8.3.3 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 crtieria 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 X; (3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44(100 S′/T)%.¹⁸

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.

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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p=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).

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.

10.1 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.

10.4 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-

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.

11.3.1 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).

11.4.1 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).

12.1 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.

12.4 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.

13.1.2 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.

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

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.

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).

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.

1.1 This method covers the determination of certain organochlorine pesticides and PCBs. The following parameters can be determined by this method:

1.4 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 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.

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with an electron capture detector.²

2.2 The method provides a Florisil column cleanup procedure and an elemental sulfur removal procedure to aid in the elimination of interferences that may be encountered.

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 Interferences by phthalate esters can pose a major problem in pesticide analysis when using the electron capture detector. These compounds 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. These phthalates are easily 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 the use of plastics in the laboratory. Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination.^4,5 The interferences from phthalate esters can be avoided by using a microcoulometric or electrolytic conductivity detector.

4.2 The following parameters 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 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.

5.1.2 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 composting. 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.):

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

5.2.3 Chromatographic column—400 mm long × 22 mm ID, with Teflon stopcock and coarse frit filter disc (Kontes K–42054 or equivalent).

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).

5.3 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.

5.6.1 Column 1—1.8 m long × 4 mm ID glass, packed with 1.5% SP–2250/1.95% SP–2401 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2 Column 2—1.8 m long × 4 mm ID glass, packed with 3% OV–1 on Supelcoport (100/120 mesh) or equivalent.

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.

6.4 Sulfuric acid (1+1)—Slowly, add 50 mL to H₂SO₄(ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.5 Acetone, hexane, isooctane, methylene chloride—Pesticide quality or equivalent.

6.6.1 Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P1126–8, and other suppliers.)

6.6.2 Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.7 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.8 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.

6.11 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 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.

6.11.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

7.5 The cleanup procedure in Section 11 utilizes Florisil column chromatography. Florisil from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which is used, the use of lauric acid value⁹ is suggested. The referenced procedure determines the adsorption from hexane solution of lauric acid (mg) per g of Florisil. The amount of Florisil to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g.

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.

8.1.4 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.

8.1.6 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 single-component parameter of interest at the following concentrations in acetone: 4,4′–DDD, 10 µg/mL; 4,4′–DDT, 10 µg/mL; endosulfan II, 10 µg/mL; endosulfan sulfate, 10 µg/mL; endrin, 10 µg/mL; any other single-component pesticide, 2 µg/mL. If this method is only to be used to analyze for PCBs, chlordane, or toxaphene, the QC check sample concentrate should contain the most representative multicomponent parameter at a concentration of 50 µg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at the test concentrations shown in Table 3 by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X) in µg/mL; and the standard deviation of the recovery (s) in µg/mL, for each parameter using the four results.

8.2.5 For each parameter compare s and Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and Xfor 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 Xfalls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

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

8.2.6.1 Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

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

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.

8.3.3 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 5:1.¹⁰ If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 4, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.¹⁰

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

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 3 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

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

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 (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2 s_pto P+2 s_p. If P=90% and s_p=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).

9.1 Grab samples must be collected in glass containers. Conventional sampling practices¹¹ 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. If the samples will not be extracted within 72 h of collection, the sample should be adjusted to a pH range of 5.0 to 9.0 with sodium hydroxide solution or sulfuric acid. Record the volume of acid or base used. If aldrin is to be determined, add sodium thiosulfate when residual chlorine is present. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.¹² 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

10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel.

10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optium 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.

10.7 Increase the temperature of the hot water bath to about 80 °C. Momeltarily 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. 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.

11.1 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. The Florisil column allows for a select fractionation of the compounds and will eliminate polar interferences. Elemental sulfur, which interferes with the electron capture gas chromatography of certain pesticides, can be removed by the technique described in Section 11.3.

11.2.1 Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5), into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.2 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 eluate.

11.2.3 Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D concentrator tube onto the column. Rinse the tube twice with 1 to 2 mL of hexane, adding each rinse to the column.

11.2.4 Place a 500-mL K-D flask and clean concentrator tube under the chromatographic column. Drain the column into the flask until the sodium sulfate layer is nearly exposed. Elute the column with 200 mL of 6% ethyl ether in hexane (V/V) (Fraction 1) at a rate of about 5 mL/min. Remove the K-D flask and set it aside for later concentration. Elute the column again, using 200 mL of 15% ethyl ether in hexane (V/V) (Fraction 2), into a second K-D flask. Perform the third elution using 200 mL of 50% ethyl ether in hexane (V/V) (Fraction 3). The elution patterns for the pesticides and PCBs are shown in Table 2.

11.2.5 Concentrate the fractions as in Section 10.6, except use hexane to prewet the column and set the water bath at about 85 °C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of each fraction to 10 mL with hexane and analyze by gas chromatography (Section 12).

11.3 Elemental sulfur will usually elute entirely in Fraction 1 of the Florisil column cleanup. To remove sulfur interference from this fraction or the original extract, pipet 1.00 mL of the concentrated extract into a clean concentrator tube or Teflon-sealed vial. Add one to three drops of mercury and seal.¹³ Agitate the contents of the vial for 15 to 30 s. Prolonged shaking (2 h) may be required. If so, this may be accomplished with a reciprocal shaker. Alternatively, activated copper powder may be used for sulfur removal.¹⁴ Analyze by 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 to 10. 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 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.

12.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.¹⁵ Smaller (1.0 uL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, the total extract volume, and the resulting peak size in area or peak height units.

12.6 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.

13.1.2 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.

13.2 When it is apparent that two or more PCB (Aroclor) mixtures are present, the Webb and McCall procedure¹⁶ may be used to identify and quantify the Aroclors.

13.3 For multicomponent mixtures (chlordane, toxaphene, and PCBs) match retention times of peaks in the standards with peaks in the sample. Quantitate every identifiable peak unless interference with individual peaks persist after cleanup. Add peak height or peak area of each identified peak in the chromatogram. Calculate as total response in the sample versus total response in the standard.

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

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.¹⁷ 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 with the following exceptions: Chlordane recovery at 4×MDL was low (60%); Toxaphene recovery was demonstrated linear over the range of 10×MDL to 1000×MDL.¹⁷

14.3 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations.¹⁸ Concentrations used in the study ranged from 0.5 to 30 µg/L for single-component pesticides and from 8.5 to 400 µg/L for multicomponent parameters. Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 4.

2. “Determination of Pesticides and PCBs in Industrial and Municipal Wastewaters,” EPA 600/4–82–023, National Technical Information Service, PB82–214222, Springfield, Virginia 22161, April 1982.

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., 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. Mills, P.A. “Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical Chemists, 51, 29, (1968).

10. 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.)

11. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

12. “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.

13. Goerlitz, D.F., and Law, L.M. Bulletin for Environmental Contamination and Toxicology, 6, 9 (1971).

14. “Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental Samples,” EPA–600/8–80–038, U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina.

15. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

16. Webb, R.G., and McCall, A.C. “Quantitative PCB Standards for Election Capture Gas Chromatography,” Journal of Chromatographic Science, 11, 366 (1973).

17. “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.

18. “EPA Method Study 18 Method 608—Organochlorine Pesticides and PCBs,” EPA 600/4–84–061, National Technical Information Service, PB84–211358, Springfield, Virginia 22161, June 1984.

1.1 This method covers the determination of certain nitroaromatics and isophorone. The following parameters may be determined by this method:

1.4 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. Isophorone and nitrobenzene are measured by flame ionization detector gas chromatography (FIDGC). The dinitrotoluenes are measured by electron capture detector gas chromatography (ECDGC).²

2.2 The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baseliles in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.³ Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

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).

5.3 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.

5.6.1 Column 1—1.2 m long × 2 or 4 mm ID glass, packed with 1.95% QF–1/1.5% OV–17 on Gas-Chrom Q (80/100 mesh) or equivalent. This column was used to develop the method performance statements given in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2 Column 2—3.0 m long × 2 or 4 mm ID glass, packed with 3% OV–101 on Gas-Chrom Q (80/100 mesh) or equivalent.

5.6.3 Detectors—Flame ionization and electron capture detectors. The flame ionization detector (FID) is used when determining isophorone and nitrobenzene. The electron capture detector (ECD) is used when determining the dinitrotoluenes. Both detectors have proven effective in the analysis of wastewaters and were used in develop the method performance statements in Section 14. Guidelines for the use to alternate detectors are provided in Section 12.1.

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

6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3 Sulfuric acid (1+1)—Slowly, add 50 mL of H₂SO₄(ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.4 Acetone, hexane, methanol, methylene chloride—Pesticide quality or equivalent.

6.5 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.

6.6 Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 200 °C in a foil-covered glass container and allow to cool.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in hexane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 2 to 5 µL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD) linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

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.

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

8.1,5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

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

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

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest in acetone at a concentration of 20 µg/mL for each dinitrotoluene and 100 µg/mL for isophorone and nitrobenzene. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2 Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.

8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4 Calculate the average recovery (X) 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 Xwith the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and Xfor 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 Xfalls 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.

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

8.3.1.3 If it is impractical to determile background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A−B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.⁷ If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X8; (3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44 (100 S′/T)%.⁷

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.

8.5 As part of QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (s_p). Express the accuracy assessment as a percent recovery interval from P−2s_pto P+2s_p. If P=90% and s_p= 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).

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

10.1 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.

10.6 Sections 10.7 and 10.8 describe a procedure for exchanging the methylene chloride solvent to hexane while concentrating the extract volume to 1.0 mL. When it is not necessary to achieve the MDL in Table 2, the solvent exchange may be made by the addition of 50 mL of hexane and concentration to 10 mL as described in Method 606, Sections 10.7 and 10.8.

10.7 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65°C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add 1 to 2 mL of hexane and a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65°C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9 Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of hexane. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.10 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2.1 Prepare a slurry of 10 g of activated Florisil in methylene chloride/hexane (1+9)(V/V) and place the Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top. Adjust the elution rate to about 2 mL/min.

11.2.2 Just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 30 mL of methylene chloride/hexane (1 + 9)(V/V) and continue the elution of the column. Discard the eluate.

11.2.3 Next, elute the column with 30 mL of acetone/methylene chloride (1 + 9)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction as in Sections 10.6, 10.7, 10.8, and 10.9 including the solvent exchange to 1 mL of hexane. This fraction should contain the nitroaromatics and isophorone. Analyze by gas chromatography (Section 12).

12.1 Isophorone and nitrobenzene are analyzed by injection of a portion of the extract into an FIDGC. The dinitrotoluenes are analyzed by a separate injection into an ECDGC. Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3 If the internal standard calibration procedure is being used, the internal standard must be added to the same extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.⁹ Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, the total extract volume, and the resulting peak size in area or peak height units.

12.6 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.

13.1.2 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.

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

14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.¹ The MDL concentrations listed in Table 1 were obtained using reagent water.¹⁰ Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7×MDL to 1000×MDL.¹⁰

14.3 This method was tested by 18 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 515 µg/L.¹¹ Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

2. “Determination of Nitroaromatic Compounds and Isophorone in Industrial and Municipal Wastewaters,” EPA 600/ 4–82–024, National Technical Information Service, PB82–208398, Springfield, Virginia 22161, May 1982.

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.

8. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

10. “Determination of Method Detection Limit and Analytical Curve for EPA Method 609—Nitroaromatics and Isophorone,” Special letter report for EPA Contract 68–03–2624, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

11. “EPA Method Study 19, Method 609 (Nitroaromatics and Isophorone),” EPA 600/4–84–018, National Technical Information Service, PB84–176908, Springfield, Virginia 22161, March 1984.

1.1 This method covers the determination of certain polynuclear aromatic hydrocarbons (PAH). The following parameters can be determined by this method:

1.2 This is a chromatographic method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for many of the parameters listed above, using the extract produced by this method.

1.3 This method provides for both high performance liquid chromatographic (HPLC) and gas chromatographic (GC) approaches for the determination of PAHs. The gas chromatographic procedure does not adequately resolve the following four pairs of compounds: Anthracene and phenanthrene; chrysene and benzo(a)anthracene; benzo(b)fluoranthene and benzo(k)fluoranthene; and dibenzo(a,h) anthracene and indeno (1,2,3-cd)pyrene. Unless the purpose for the analysis can be served by reporting the sum of an unresolved pair, the liquid chromatographic approach must be used for these compounds. The liquid chromatographic method does resolve all 16 of the PAHs listed.

1.4 The method detection limit (MDL, defined in Section 15.1)¹ for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5 The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. Selection of the aliquots must be made prior to the solvent exchange steps of this method. The analyst is allowed the latitude, under Sections 12 and 13, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

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

1.7 This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC and GC systems and in the interpretation of liquid and gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2.1 A measured volume of sample, approximately 1–L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and concentrated to a volume of 10 mL or less. The extract is then separated by HPLC or GC. Ultraviolet (UV) and fluorescence detectors are used with HPLC to identify and measure the PAHs. A flame ionization detector is used with GC.²

2.2 The method provides a silica gel column cleanup procedure to aid in the elimination of interferences that may be encountered.

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardward that lead to discrete artifacts and/or elevated baselines in the chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1 Glassware must be scrupulously cleaned.³ Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400°C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3 The extent of interferences that may be encountered using liquid chromatographic techniques has not been fully assessed. Although the HPLC conditions described allow for a unique resolution of the specific PAH compounds covered by this method, other PAH compounds may interfere.

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

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

5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.2 Drying column—Chromatographic column, approximately 400 mm long × 19 mm ID, with coarse frit filter disc.

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).

5.2.8 Chromatographic column—250 mm long × 10 mm ID, with coarse frit filter disc at bottom and Teflon stopcock.

5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.

5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.6 High performance liquid chromatograph (HPLC)—An analytical system complete with column supplies, high pressure syringes, detectors, and compatible strip-chart recorder. A data system is recommended for measuring peak areas and retention times.

5.6.2 Reverse phase column—HC-ODS Sil-X, 5 micron particle diameter, in a 25 cm × 2.6 mm ID stainless steel column (Perkin Elmer No. 089–0716 or equivalent). This column was used to develop the method performance statements in Section 15. Guidelines for the use of alternate column packings are provided in Section 12.2.

5.6.3 Detectors—Fluorescence and/or UV detectors. The fluorescence detector is used for excitation at 280 nm and emission greater than 389 nm cutoff (Corning 3–75 or equivalent). Fluorometers should have dispersive optics for excitation and can utilize either filter or dispersive optics at the emission detector. The UV detector is used at 254 nm and should be coupled to the fluorescence detector. These detectors were used to develop the method performance statements in Section 15. Guidelines for the use of alternate detectors are provided in Section 12.2.

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

5.7.1 Column—1.8 m long × 2 mm ID glass, packed with 3% OV–17 on Chromosorb W-AW-DCMS (100/120 mesh) or equivalent. This column was used to develop the retention time data in Table 2. Guidelines for the use of alternate column packings are provided in Section 13.3.

5.7.2 Detector—Flame ionization detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), excluding the four pairs of unresolved compounds listed in Section 1.3. Guidelines for the use of alternate detectors are provided in Section 13.3.

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

6.3 Cyclohexane, methanol, acetone, methylene chloride, pentane—Pesticide quality or equivalent.

6.5 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400°C for 4 h in a shallow tray.

6.6 Silica gel—100/200 mesh, desiccant, Davison, grade-923 or equivalent. Before use, activate for at least 16 h at 130 °C in a shallow glass tray, loosely covered with foil.

6.7 Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetonitrile and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

7.1 Establish liquid or gas chromatographic operating conditions equivalent to those given in Table 1 or 2. The chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with acetonitrile. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2 Using injections of 5 to 25 µL for HPLC and 2 to 5 µL for GC, analyze each calibration standard according to Section 12 or 13, as appropriate. Tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3.1 Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with acetonitrile. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2 Using injections of 5 to 25 µL for HPLC and 2 to 5 µL for GC, analyze each calibration standard according to Section 12 or 13, as appropriate. Tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

7.4 The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

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

8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, 12.2, and 13.3) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.4 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.

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

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

8.2.1 A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetonitrile: 100 µg/mL of any of the six early-eluting PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene); 5 µg/mL of benzo(k)fluoranthene; and 10 µg/mL of any of the other PAHs. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.