Parameter and units | Analytical Technology 1 | EPA | Standard Methods 27 | AOAC, ASTM, USGS | Other |
1. Coliform (fecal), number per 100 mL or number per gram dry weight | Most Probable Number (MPN), 5 tube, 3 dilution, or | p. 132,3 1680,11,15 168111,20 | 9221 C E-2006 | | |
| Multiple tube/multiple well, or | | | | Colilert-18®13,18,21,29 |
| Membrane filter (MF)2, single step | p. 1243 | 9222 D-200630 | B-0050-854 | |
2. Coliform (fecal) in presence of chlorine, number per 100 mL | MPN, 5 tube, 3 dilution, or | p. 1323 | 9221 C E-2006 | | |
| MF2, single step5 | p. 1243 | 9222 D-200630 | | |
3. Coliform (total), number per 100 mL | MPN, 5 tube, 3 dilution, or | p. 1143 | 9221 B-2006 | | |
| MF2, single step or two step | p. 1083 | 9222 B-2006 | B-0025-854 | |
4. Coliform (total), in presence of chlorine, number per 100 mL | MPN, 5 tube, 3 dilution, or | p. 1143 | 9221 B-2006 | | |
| MF2 with enrichment5 | p. 1113 | 9222 B-2006 | | |
5. E. coli, number per 100 mL21 | MPN6,8,16 multiple tube, or | | 9221B.2-2006/9221F-200612 14 | | |
| multiple tube/multiple well, or | | 9223 B-200413 | 991.1510 | Colilert® 13 18
Colilert-18® 13,17,18 |
| MF2,6,7,8 single step | 160322 | | | mColiBlue-24®19 |
6. Fecal streptococci, number per 100 mL | MPN, 5 tube, 3 dilution, or | p. 1393 | 9230 B-2007 | | |
| MF2, or | p. 1363 | 9230 C-2007 | B-0055-854 | |
7. Enterococci, number per 100 mL21 | MPN, 5 tube, 3 dilution, or | p. 1393 | 9230 B-2007 | | |
| MPN6,8, multiple tube/multiple well, or | | 9230 D-2007 | D6503-999 | Enterolert® 13,24 |
| MF2,6,7,8 single step or | 160025 | 9230 C-2007 | | |
8. Salmonella number per gram dry weight11 | MPN multiple tube | 168223 | | | |
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 Approved for enumeration of target organism in sewage sludge.
12 The multiple-tube fermentation test is used in 9221B.2-2006. Lactose broth may be used in lieu of lauryl tryptose broth (LTB), if at least 25 parallel tests are conducted between this broth and LTB using the water samples normally tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total coliform using lactose broth is less than 10 percent. No requirement exists to run the completed phase on 10 percent of all total coliform-positive tubes on a seasonal basis.
13 These 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.
14 After prior enrichment in a presumptive medium for total coliform using 9221B.2-2006, all presumptive tubes or bottles showing any amount of gas, growth or acidity within 48 h ± 3 h of incubation shall be submitted to 9221F-2006. Commercially available EC-MUG media or EC media supplemented in the laboratory with 50 µg/mL of MUG may be used.
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® 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®, and Quanti-Tray® 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 Approved for enumeration of target organism in wastewater effluent.
22 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.
23 Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, EPA-821-R-14-012. September 2014. U.S. EPA.
24 A description of the Enterolert® test may be obtained from IDEXX Laboratories Inc.
25 Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI), EPA-821-R-14-011. September 2014. U.S. EPA.
26 Standard Methods for the Analysis of Water and Wastewater. With the promulgation of Federal Register /Vol. 77, No. 97 / Friday, May 18, 2012, the EPA lists only the most recently EPA−approved version of a Standard Method (regardless of the printed or online edition) in 40 CFR Part 136, with few exceptions, to identify the method with the year of Standard Methods approval or adoption designated by the last four digits in the method number (e.g., Standard Method 3113B–2004). This approach clearly identifies the version of the standard method approved under Part 136 and no longer ties it to a particular compendium printing or edition of Standard Methods. Methods can be purchased at www.standardmethods.org/. 27 Compliance monitoring must be performed in accordance with the specifications in the “State of Wisconsin Aquatic Life Toxicity Testing Methods Manual, 2nd Edition,” Wisconsin Department of Natural Resources, 2004. This publication is available for inspection at the offices of the Department of Natural Resources and the Legislative Reference Bureau. Copies are available from the Department of Natural Resources, Bureau of Science Services, P.O. Box 7921, Madison, WI 53707.
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 the medium must be verified using Lauryl Tryptose Broth and EC broth, followed by count adjustment based on these results; and representative non-blue colonies should be verified using Lauryl Tryptose Broth. Where possible, verifications should be done from randomized sample sources.
Section 16. NR 219.04 Table EM header row 2, Parameters “Fecal Coliform” and “Salmonella” (including sub-rows), and table note 11 are amended to read:
Table EM
List of Approved Analytical Methods for Sludge
| | Sample Preparation | Determinative Method |
Parameter | Analytical Technology | SW−8461 SW−8461 | EPA4 | SW−8461 | EPA 2,3 | Standard Methods [ed.] 8,9 | Other |
Fecal Coliform | Most Probable Number Membrane Filter | | 1680 16 1681 17 | | 1680 16 1681 17 | 9221 E [18,19, 20,21], 9221 E−99, 9222 D, 9222 D−97 | Appendix F10 |
Salmonella | Most Probable Number | | 1682 18 | | 1682 18 | | 9260 D.18 |
| Selective Media Culture | | | | | | Appendix G10 |
11 “Environmental Regulations and Technology – Control of Pathogens and Vectors Attraction in Sewage Sludge”, EPA–625/R–92/013, Revised October 1999,July 2003, Environmental Protection Agency, Cincinnati, OH, 1999. Available from: the National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161. 2003.
Section 17. NR 219.04 Table EM table notes 16, 17, and 18 are created to read:
16 “Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple Tube Fermentation using Lauryl Tryptose Broth (LTB) and EC Medium" April 2010, EPA-821-R-10-003, U.S. Environmental Protection Agency, Office of Water (4303T), 1200 Pennsylvania Avenue, NW, Washington, DC 20460.
17 “Method 1681: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using A-1 medium” July 2006, EPA-821-R-06-013, U.S. Environmental Protection Agency, Office of Water (4303T), 1200 Pennsylvania Avenue, NW, Washington, DC 20460.
18 “Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium” July 2016, EPA-821-R-06-14, U.S. Environmental Protection Agency, Office of Water (4303T), 1200 Pennsylvania Avenue, NW, Washington, DC 20460.
Section 18. Effective Date. This rule takes effect on the first day of the month following publication in the Wisconsin Administrative Register as provided in s. 227.22 (2) (intro.), Stats. Section 19. Board adoption. This rule was approved and adopted by the State of Wisconsin Natural Resources Board on ________________.
Dated at Madison, Wisconsin _____________________________.
STATE OF Wisconsin DNR
DEPARTMENT OF NATURAL RESOURCES
BY ______________________________________
Preston D. Cole, Secretary
