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= Whole effluent toxicity tests = Whole Effluent Toxicity (WET) tests are a set of standardized aquatic toxicity tests that measure the effect of wastewater on aquatic organisms. It tests an organism’s ability to survive, grow, and reproduce in an effluent mixture of chemicals. The results of these tests are significant in creating water quality regulations, setting Total Maximum Daily Load (TMDL) targets, and checking water quality permit compliance in the United States.

Background
The Clean Water Act (CWA) of 1972 gave the US Environmental Protection Agency (USEPA) the authority to implement pollution control programs such as setting wastewater standards for industries and municipalities. Section 101(a)(3) of the Act states, “it is the national policy that the discharge of toxic pollutants in toxic amounts be prohibited.” To pursue this goal, the USEPA has established water quality standards as well as the National Pollution Discharge Elimination System (NPDES) permitting program. WET tests were added along with the chemical-specific approach and the bioassessment and biosurvey approach as an approved method by the USEPA in 1995 as a way to check NPDES permit compliance.

Both the water quality standards program and the NPDES program of the USEPA have implemented water quality-based toxics control approaches, in which the WET control approach is one of them. States and Tribes are able to designate numeric and narrative criteria for water quality standards. Numeric criteria is based on data results from tests and the assessment of adverse effects. This criteria may include limits to chemical concentrations within the aquatic environment. Narrative criteria are descriptions of the water quality goals. Keeping a water source free of toxic amounts of chemicals is an example of a narrative criterion. When a discharge exceeds the numeric or narrative criteria for water quality standards, a water quality-based effluent limit will be defined in the NPDES permit to control the discharge.

WET Acute Toxicity Test
The WET acute test methods were approved under the Clean Water Act section 304(h) and specified at 40 CFR 136.3, Table I A.

The most common effluent acute toxicity test is a multi-concentration test used to assess the compliance of water quality permits. The general set-up for these tests involve a minimum of 20 healthy individual organisms of the same species being tested in 5 different effluent concentrations and a control over a period of 24-96 hours. Each effluent concentration has at least one replicate. Typical effluent concentrations tested are: 100%, 75%, 50%, 25% and 12.5%; however, these concentrations are not standardized and discretion may be used. After the designated time period, mortality of the test organisms is observed and expressed in terms of an LC50 or NOAEC. In order for the test to be considered valid, the control must have at least 90% survivability of the test organisms at the duration of the tests. If the survival rate is lower, the test is invalid and must be repeated.

Other approved WET toxicity tests are the Preliminary Toxicity Range-Finding Tests, which  are only necessary when the sample is of completely unknown quality, and the Receiving Water Tests which consists only of a control and 100% receiving water sample.

Typical test species for WET acute toxicity tests: 

Standard test procedures have been developed for the following freshwater organisms:

1.  Ceriodaphnia dubia (daphnid)

2.  Daphnia pulex and D. magna (daphnids)

3.  Pimephales promelas (fathead minnow) and Cyprinella leedsi (Bannerfin shiner)

4.  Oncorhynchus mykiss (rainbow trout) and Salvelinus fontinalis (brook trout)

Standard test procedures have been developed for the following marine organisms:

1.  Americamysis bahia (mysid)

2.  Cyprinodon variegatus (sheephead minnow)

3.  Menidia beryllina (inland silverside), M. menidia (Atlantic silverside), and M. peninsulae (tidewater silverside)

WET Chronic Toxicity Test
The methods for WET chronic toxicity tests are approved under CWA section 304(h) and are specified in Table I-A at 40 CFR 136.3.

Freshwater
Freshwater chronic toxicity tests involve effluent tests of freshwater organisms. The general set-up for freshwater chronic toxicity tests involve 10 test organisms per effluent concentration in which all test organisms are the same species. Generally there are at least five different effluent concentrations and a control concentration. Similar to acute toxicity testing, typical effluent concentrations tested are 100%, 50%, 25%, 12.5%, and 6.25%. However, effluent concentrations should be selected independently for each test studied. The test is conducted for the duration of 4 to 7 days, except when green algae is the test organism, in which the test is run for only 4 days. After the test is complete, mortality and reproduction are observed and expressed using LC50, NOEC, NOAEC, and LOEC. In addition, the estimated concentration that caused a 25% reduction in the mean young per female organisms (IC25) is determined, which can also be done for a 50% reduction (IC50).

Standard test procedures have been developed for the following freshwater organisms :


 * 1) Pimephales promelas (Fathead minnow)
 * 2) Ceriodaphnia dubia (Daphnia)
 * 3) Selenastrum capricornutum (Green Algae)

However, certain criteria has to be met for the test to be valid. For fathead minnows, there must be at least 80% survival in the control group and a dry weight of 0.25 mg or more of the surviving organisms within the control group. For daphnia, there must be at least 80% survival in the control group and 60% of the surviving females must have produced their third brood. In addition, the number of young per surviving female must be 15 or more. For green algae, the mean cell density of the control group must be at least 1 x 106 cells per mL after 96 hours and the variance between replicates has to be less than 20%. If this criteria is not met, the test has to be repeated.

Marine and Estuarine
Marine and estuarine chronic WET tests measure the chronic toxicity of effluent water to marine species. The tests can be run for the duration of 1 hour up to 9 days. However, these methods and rules do not apply to marine and estuarine organisms of the Pacific Ocean.

Standard test procedures have been developed for the following marine and estuarine organisms :


 * 1) Menidia beryllina (Inland silverside) - Larval Survival and Growth Test
 * 2) Americamysis bahia (Mysid) - Survival, Growth, and Fecundity Test
 * 3) Cyprinodon variegates (sheephead minnow) -Larval Survival and Growth Test
 * 4) Arbacia punctulata (Sea urchin) – Fecundity Test
 * 5) Champia parvula (red macroalga)

These tests involve one or more life cycles of the test species which give more observations of the effects of the wastewater being tested. More accurate estimates can be observed to create an exposure limit of the toxicant. However, the results do not include the effects of stress.

Applications
The application of WET tests are most appropriate for identifying and delineating effluents of concern. For example, in hazard ranking. In a review of the WET test approach by Peter Chapman the Water Environment Research Foundation states, “Ideally, whole effluent toxicity tests would serve as flags to indicate the need for a more formal risk-based approach…”. When used in risk assessments WET tests can be beneficial for weight of evidence approach in decision making. Properly designed toxicity tests can always be adjusted to be site specific, reactive and predictive.

Advantages
WET tests offer multiple advantages for effluent toxicity testing and assessment. The whole effluent approach allows for a limit to be set for effluent toxicity as a whole rather than individual limits for each substance found sewage discharge. WET tests can also be more cost effective than identifying toxicity for individual chemicals in the effluent. Their use to measure point sources for permitting through the NPDES lends to their favorability. As more developed effluent toxicity methods become widespread, such as the use of biomarkers and genotoxic endpoints the estimation of toxicity will only improve.

Constraints
Similar to all laboratory experiments conducted in a laboratory there are disadvantages or constraints for WET tests. For instance, there are few well-developed methods for acute toxicity and are rarely well understood. The chronic studies published are also low compared to acute toxicity tests. Once these studies are conducted the limits set from laboratory effluent toxicity on fresh effluent samples may represent real toxicity less because of fate processes and aging in streams. Although the processes mentioned above may affect toxicity of effluents, a case study performed at Scippo Creek in Circleville, Ohio found this factor to no be as applicable because permits are generally applied at the edge of the mixture zone where aging has not occurred yet.

Another constraint of WET tests is the use of test species. Species used in WET tests are generally not the same as local species affected by the results of the tests conducted. This could cause under representation or over representation of toxicity in the study results because different species have varying sensitivities to a single or combined contaminant(s).

WET tests conducted in the laboratory face many other constraints including mimicking the receiving environment. Tests must be controlled in a lab however, in the environment there is no control and this leads to many changes from abiotic and biotic factors that would not be encountered in the lab. The results from worst case WET tests is usually overprotective or conservative for a number of reasons including absence of environmental processes, adaptation or acclimation, sufficient diet, and many others.

If laboratory tests are not conducted as worst case it is still possible studies are underprotective. This could be due to the most sensitive species not being available to rear or test, or all the stresses a species faces in receiving waters are not expressed in the laboratory. Other possible explanations for a study being underprotective include absence of bioaccumulation tests and the absence of all possible endpoints. Overall WET tests contain a certain level of uncertainty in the level of protection due to biotic and abiotic factors, intermittent doses, mixtures and their various effects, adaptations, and hormesis.