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Clean Water Act Analytical Methods

Whole Effluent Toxicity Methods

""Whole Effluent Toxicity (WET) refers to the aggregate toxic effect to aquatic organisms from all pollutants contained in a facility's wastewater (effluent). It is one way we implement the Clean Water Act's prohibition of the discharge of toxic pollutants in toxic amounts. WET tests measure wastewater's effects on specific test organisms' ability to survive, grow and reproduce.
 
The WET methods listed below are specified at 40 CFR 136.3, Table I A. WET test methods consist of exposing living aquatic organisms (plants, vertebrates and invertebrates) to various concentrations of a sample of wastewater, usually from a facility's effluent stream. WET tests are used by the National Pollutant Discharge Elimination System (NPDES) permitting authority to determine whether a facility's permit and discharge complies with the WET requirements or limits.
 
All three manuals include guidelines on laboratory safety, quality assurance, facilities and equipment, organism culturing and handling, dilution water, effluent sampling and sample handling, test procedures, test endpoints and data analysis, and report preparation.
 

On this page:

Acute Toxicity to Freshwater and Marine Organisms

The tests in the WET Acute Methods Manual (5th edition, 2002) consist of a control and a minimum of five effluent concentrations. EPA recommends the use of ≥0.5 dilution factor and five effluent concentrations and a control. The test duration is typically 24, 48, or 96 hours. The test methods allow for permutations such as temperature, dilution water, frequency of the effluent renewal and more. The following methods listed in 40 CFR 136.3 are described in the manual.

Acute Toxicity WET Methods

No. Method Title (as listed in 40 CFR 136.3, Table I A)
Errata sheet (2016). Promulgated as part of the 2017 Methods Update Rule.
Freshwater methods:
2000.0 Fathead Minnow, Pimephales promela, and Bannerfin shiner, Cyprinella leedsi
2002.0 Daphnia, Ceriodaphnia dubia
2019.0 Rainbow trout, Oncorhynchus mykiss, and Brook trout, Salvelinus fontinalis
2021.0 Daphnia puplex and Daphnia magna
Marine/Estuarine methods:
2004.0 Sheepshead minnow, Cyprinodon variegatus
2006.0 Silverside, Menidia beryllina, Menidia menidia, and Menidia peninsulae
2007.0 Mysid, Americamysis bahia (formerly Mysidopsis bahia)

Chronic Toxicity to Freshwater Organisms

The tests in the WET Freshwater Chronic Methods Manual (4th edition, 2002) recommend the use of the ≥0.5 dilution factor and five effluent concentrations and a control for four to eight days. The test methods allow for permutations such as various dilution waters, daily or three effluent sample collections for daily test renewals, and more. The biological test endpoints include survival, growth, reproduction, or teratogenicity.

Chronic Toxicity - Freshwater WET Methods

No. Method Title (as listed in 40 CFR 136.3, Table I A)
Errata sheet (2016). Promulgated as part of the 2017 Methods Update Rule.
1000.0 Fathead minnow, Pimephales promelas, larval survival and growth
1001.0 Fathead minnow, Pimephales promelas, larval survival and teratogenicity
1002.0 Daphnia, Ceriodaphnia dubia, survival and reproduction
1003.0 Green alga, Selenastrum capricornutum, growth (species is now known as Raphidocelis subcapitata)

Chronic Toxicity to Marine and Estuarine Organisms

The tests in the WET Marine Chronic Methods Manual (3rd edition, 2002) typically involve the exposure of test organisms to five effluent concentrations and a control for one hour to nine days. The test methods allow for permutations such as various dilution waters, daily or three effluent sample collections for daily test renewals, and more. In addition to survival, sub-lethal endpoints such as growth, fecundity, teratogenicity, or fertilization are measured. These methods listed in 40 CFR Part 136.3 apply to marine and estuarine organisms of the Atlantic Ocean and the Gulf of Mexico.

Chronic Toxicity - Marine and Estuarine WET Methods

No. Method Title (as listed in 40 CFR 136.3, Table I A)
Errata sheet (2016). Promulgated as part of the 2017 Methods Update Rule.
1004.0 Sheepshead minnow, Cyprinodon variegatus, larval survival and growth
1005.0 Sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and teratogenicity
1006.0 Inland silverside, Menidia beryllina, larval survival and growth
1007.0 Mysid, Americamysis (formerly Mysidopsisbahia, survival, growth and fecundity
1008.0 Sea urchin, Arbacia punctulata, fertilization

Note: While there are additional test procedures in the marine and estuarine test method manuals, two are not listed in 40 CFR 136.3, Table I A:

  • Acute method for mysid shrimp, Holmesimysis costata, Acute Toxicity Test Method 2007.0 (Table 19). This species is specific to Pacific Coast waters and is not listed at Part 136 for nationwide use.
  • Short-term test method to estimate chronic toxicity, the Red Macroalga, Champia parvula, Reproduction Test Method 1009.0 is not listed at Part 136 for nationwide use.

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