Code of Federal Regulations Title 40. Protection of Environment § 40.136.3, (a) to Table IA Identification of test procedures

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

Table IA—List of Approved Biological Methods for Wastewater and Sewage Sludge
Parameter and units	Method 1	EPA	Standard methods	AOAC, ASTM, USGS	Other
Bacteria
1. Coliform (fecal), number per 100 mL or number per gram dry weight	Most Probable Number (MPN), 5 tube, 3 dilution, or	p. 132,3 1680,1115 1681 1120	9221 E-2014
	Membrane filter (MF) 25, single step	p. 124 3	9222 D-2015 29	B-0050-85 4
2. Coliform (fecal), number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 132 3	9221 E-2014; 9221 F-2014 33
	Multiple tube/multiple well, or		․		Colilert-18®.131828
	MF 25, single step 5	p. 124 3	9222 D-2015 29
3. Coliform (total), number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 114 3	9221 B-2014
	MF 25, single step or two step	p. 108 3	9222 B-2015 30	B-0025-85 4
	MF 25, with enrichment	p. 111 3	9222 B-2015 30
4. E. coli, number per 100 mL	MPN 6816 multiple tube, or		9221 B2014/9221 F-2014 121433
	multiple tube/multiple well, or		9223 B-2016 13	991.15 10	Colilert®  1318
					Colilert-18®  131718
	MF 25678, two step, or		9222 B-2015/9222 I-2015 31
	Single step	1603 21		․	m-ColiBlue24®.19
5. Fecal streptococci, number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 139 3	9230 B-2013
	MF 2, or	p. 136 3	9230 C-2013 32	B-0055-85 4
	Plate count	p. 143 3
6. Enterococci, number per 100 mL	MPN, 5 tube, 3 dilution, or	p. 139 3	9230 B-2013
	MPN 68, multiple tube/multiple well, or		9230 D-2013	D6503-99 9	Enterolert®.1323
	MF 25678 single step or	1600 24	9230 C-2013 32
	Plate count	p. 143 3
7. Salmonella, number per gram dry weight 11	MPN multiple tube	1682 22
Aquatic Toxicity
8. Toxicity, acute, fresh water organisms, LC50, percent effluent	Water flea, Cladoceran, Ceriodaphnia dubia acute	2002.0 25.
	Water fleas, Cladocerans, Daphnia pulex and Daphnia magna acute	2021.0 25.
	Fish, Fathead minnow, Pimephales promelas, and Bannerfin shiner, Cyprinella leedsi, acute	2000.0 25.
	Fish, Rainbow trout, Oncorhynchus mykiss, and brook trout, Salvelinus fontinalis, acute	2019.0 25.
9. Toxicity, acute, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, LC50, percent effluent	Mysid, Mysidopsis bahia, acute	2004.0 25.
	Fish, Sheepshead minnow, Cyprinodon variegatus, acute 2006.0 25.
	Fish, Silverside, Menidia beryllina, Menidia menidia, and Menidia peninsulae, acute 2007.0 25.
10. Toxicity, chronic, fresh water organisms, NOEC or IC25, percent effluent	Fish, Fathead minnow, Pimephales promelas, larval survival and growth	1000.0 26.
	Fish, Fathead minnow, Pimephales promelas, embryo-larval survival and teratogenicity	1001.0 26.
	Water flea, Cladoceran, Ceriodaphnia dubia, survival and reproduction	1002.0 26.
	Green alga, Selenastrum capricornutum, growth	1003.0 26.
11. Toxicity, chronic, estuarine and marine organisms of the Atlantic Ocean and Gulf of Mexico, NOEC or IC25, percent effluent	Fish, Sheepshead minnow, Cyprinodon variegatus, larval survival and growth	1004.0 27.
	Fish, Sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and teratogenicity 1005.0 27.
	Fish, Inland silverside, Menidia beryllina, larval survival and growth	1006.0 27.
	Mysid, Mysidopsis bahia, survival, growth, and fecundity	1007.0 27.
	Sea urchin, Arbacia punctulata, fertilization	1008.0 27.
Table IA notes:

¹ The method must be specified when results are reported.

² A 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.

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

⁴ 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. 1989. USGS.

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

⁶ Tests 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.

⁷ When 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.

⁸ To 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.

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

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

¹¹ Recommended for enumeration of target organism in sewage sludge.

¹² The multiple-tube fermentation test is used in 9221B.2-2014. 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.

¹³ These tests are collectively known as defined enzyme substrate tests.

¹⁴ After prior enrichment in a presumptive medium for total coliform using 9221B.2-2014, 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-2014. Commercially available EC-MUG media or EC media supplemented in the laboratory with 50 μg/mL of MUG may be used.

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

¹⁶ Samples 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.

¹⁷ Colilert-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.

¹⁸ Descriptions of the Colilert^®, Colilert-18^®, Quanti-Tray^®, and Quanti-Tray^®/2000 may be obtained from IDEXX Laboratories, Inc.

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

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

²¹ Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified Membrane-Thermotolerant Escherichia coli Agar (modified mTEC), EPA-821-R-14-010. September 2014. U.S. EPA.

²² Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, EPA-821-R-14-012. September 2014. U.S. EPA.

²³ A description of the Enterolert^® test may be obtained from IDEXX Laboratories Inc.

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

²⁵ Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, EPA-821-R-02-012. Fifth Edition, October 2002. U.S. EPA; and U.S. EPA Whole Effluent Toxicity Methods Errata Sheet, EPA 821-R-02-012-ES. December 2016.

²⁶ Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, EPA-821-R-02-013. Fourth Edition, October 2002. U.S. EPA; and U.S. EPA Whole Effluent Toxicity Methods Errata Sheet, EPA 821-R-02-012-ES. December 2016.

²⁷ Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, EPA-821-R-02-014. Third Edition, October 2002. U.S. EPA; and U.S. EPA Whole Effluent Toxicity Methods Errata Sheet, EPA 821-R-02-012-ES. December 2016.

²⁸ To use Colilert-18^® to assay for fecal coliforms, the incubation temperature is 44.5 ± 0.2 °C, and a water bath incubator is used.

²⁹ On a monthly basis, at least ten blue colonies from positive samples must be verified using Lauryl Tryptose Broth and EC broth, followed by count adjustment based on these results; and representative non-blue colonies should be verified using Lauryl Tryptose Broth. Where possible, verifications should be done from randomized sample sources.

³⁰ On a monthly basis, at least ten sheen colonies from positive samples must be verified using lauryl tryptose broth and brilliant green lactose bile broth, followed by count adjustment based on these results; and representative non-sheen colonies should be verified using lauryl tryptose broth. Where possible, verifications should be done from randomized sample sources.

³¹ Subject coliform positive samples determined by 9222 B-2015 or other membrane filter procedure to 9222 I-2015 using NA-MUG media.

³² Verification of colonies by incubation of BHI agar at 10 ± 0.5 °C for 48 ± 3 h is optional. As per the Errata to the 23rd Edition of Standard Methods for the Examination of Water and Wastewater “Growth on a BHI agar plate incubated at 10 ± 0.5 °C for 48 ± 3 h is further verification that the colony belongs to the genus Enterococcus.”

³³ 9221 F.2-2014 allows for simultaneous detection of E. coli and thermotolerant fecal coliforms by adding inverted vials to EC-MUG; the inverted vials collect gas produced by thermotolerant fecal coliforms.