Other Clean Water Act Test Methods: Microbiological

Not Approved Under 40 CFR Part 136

In addition to developing and approving methods for use in compliance monitoring, the EPA may develop methods to support studies of specific industries (e.g., the Effluent Guidelines program) or for use in broad national surveys. If such studies or surveys lead to regulatory limits of new contaminants, the EPA approves the applicable methods for nationwide or industry-specific use through rulemaking which includes an opportunity for public comment.

Although the following methods are not in EPA regulations, they may be of interest to regulated entities, permitting authorities, and the public. The presence of a method here does not imply a regulatory requirement for its use.

Microbial Source Tracking Methods

Methods 1696.1 and 1697.2 are used for microbial source tracking (MST) to characterize human sources of fecal pollution in recreational waters. These methods can be used to identify trends and provide additional information to support water quality management that can potentially improve public health and water quality.

Methods 1696.1 and 1697.2 include an update to the optical density at 260 nm (OD₂₆₀) example calculation in Section 7.18 (Salmon DNA Extraction Buffer) to 33 µg/mL from the original versions of these methods. It should also be noted that the automated data analysis tool has been updated to allow for the use of commercially available DNA standards.

1696.1: Characterization of Human Fecal Pollution in Water by HF183/BacR287 TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay® (pdf) (832.4 KB, January 2022)
1697.2: Characterization of Human Fecal Pollution in Water by HumM2 TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay® (pdf) (1.34 MB, September 2023, EPA 821-R-23-010)
- Automated data analysis tool for EPA Methods 1696.1 and 1697.2 (xltm) (92.33 KB, September 2023)
  Upon completion of data entry, this tool will automatically perform all calculations for method proficiency, quality assurance and estimation of human-associated genetic marker concentration in test samples. Note: Save the file to your computer and then open the file in Excel. The file is not viewable with web browsers.

Other Microbiological Methods

1696: Characterization of Human Fecal Pollution in Water by HF183/BacR287 TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (pdf) (956.65 KB, March 2019, 821-R-19-002)
1697: Characterization of Human Fecal Pollution in Water by HumM2 TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (pdf) (738.71 KB, March 2019, 821-R-19-003)

Methods 1642, 1643

1642: Male-specific (F+) and Somatic Coliphage in Recreational Waters and Wastewater by Ultrafiltration (UF) and Single Agar Layer (SAL) Procedure (Draft) (pdf) (888.43 KB, April 2018, 820-R-18-001)
Procedure to concentrate, detect and enumerate male-specific (F+) and somatic coliphages in recreational waters and advanced treatment wastewater
1643: Male-specific (F+) and Somatic Coliphage in Secondary (No Disinfection) Wastewater by the Single Agar Layer (SAL) Procedure (Draft) (pdf) (787.96 KB, April 2018, 820-R-18-003)
Procedure to detect and enumerate male-specific (F+) and somatic coliphages in secondary treatment wastewater

Methods 1609, 1611

Errata to Methods 1609.1 and 1611.1 (pdf) (151.8 KB, June 18, 2015)
1609: Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control (IAC) Assay (pdf) (1010.16 KB, March 2013, 820-R-13-005)
1609.1: Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control (IAC) Assay (pdf) (654.61 KB, April 2015, 820-R-15-099)
1611: Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (pdf) (405.72 KB, October 2012, 821-R-12-008)
1611.1: Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) (pdf) (667.83 KB, April 2015, 820-R-15-008)
- Methods 1609.1 & 1611.1 Calculation Spreadsheet (xlsx) (134.88 KB, April 2015)
  Note: This spreadsheet is completely unlocked, which allows you to edit it. Use extreme caution in changing any of the existing calculation formulas.
- Method 1609 Calculation Spreadsheet (xlsx) (122.22 KB, March 2013)
- Method 1611 Calculation Spreadsheet (xlsx) (88.45 KB, October 2012)

Method B: Bacteroidales in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (pdf) (8.76 MB, June 2010, 822-R-10-003)

qPCR Support Documents

Acceptability of the EPA qPCR Test at Your Beach (pdf) (34.9 KB, December 2013, 820-R-13-012)
Detection and Quantification Limits of EPA Enterococcus qPCR Methods (pdf) (60.67 KB, December 2013, 820-R-13-013)

Methods 1601, 1602, 1605, 1693

1601: Male-specific (F+) and Somatic Coliphage in Water by Two-step Enrichment Procedure (pdf) (332.6 KB, April 2001, 821-R-01-030)
Procedure to determine the presence or absence of male-specific (F+) and somatic coliphages in ground water
1602: Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure (pdf) (271.41 KB, April 2001, 821-R-01-029)
Procedure to detect and enumerate male-specific (F+) and somatic coliphages in ground water
1605: Aeromonas in Finished Water by Membrane Filtration using Ampicillin-Dextrin Agar with Vancomycin (ADA-V) (pdf) (143.64 KB, October 2001, 821-R-01-034)
Procedure for the detection and enumeration of Aeromonas species in finished water samples
1693: Cryptosporidium and Giardia in Disinfected Wastewater by Concentration/IMS/IFA (pdf) (1.19 MB, September 2014, 821-R-14-013)
Procedure to concentrate, detect and enumerate Cryptosporidium and Giardia in disinfected wastewater