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= WET (Whole Effluent Toxicity) = Whole Effluent Toxicity (WET) is the aggregate toxicity of effluent from sources of wastewater discharge (EPA, 2016). WET testing is used to determine the total toxic effect of all pollutants present in the effluent (SETAC, 2004). WET testing is implemented under the National Pollution Discharge Elimination System (NPDES) by the U.S. Environmental Protection Agency to regulate toxicity of surface waters (SETAC, 2004). This testing replicates the total effect of environmental exposure of aquatic life to toxic substances within effluent without the need to identify specific pollutants (EPA, 2016).

History
Whole effluent toxicity (WET) testing is an approach for detecting and identifying toxicity in surface waters as part of the U.S. Environmental Protection Agency (USEPA). Effluent toxicity and effects testing began in the 1940s to assess and limit water pollution. In the early 1980s, toxicity limits and acute monitoring started to be used on a regular basis. The USEPA formalized whole effluent toxicity testing in 1985, which have proven to be very useful tools for assessing the toxicity impacts in the environment. By 1989, the use of chronic monitoring and limits started to rise. During the early 1900s, there was an increase in chemical production causing a surge of concern among people. Early aquatic toxicologists then realized that it was not possible to test all chemicals and mixtures that may be present in the effluent. At this point in time, there were few toxicity data for the vast majority of chemicals and their potential additive or synergistic effects. These uncertainties led to the testing toxicity of effluents and streams influenced by industrial and wastewater treatment facilities (Lozano, 2017) (Chapman, 2009).

Causes of Effluent Toxicity
WET testing observes the toxicity from numerous factors that can behave independently, additively, or synergistically. These may consist of chemical factors such as inorganic chemicals, organic chemicals, and pesticides. Therefore, physical factors such as dissolved organic carbon and temperature may contribute to toxicity. Different compounds and elements in the effluent that are present in aquatic ecosystems are found to be toxic to aquatic organisms. Wastewater systems have found that the discharged chemicals retain their toxic characteristics if they are improperly disposed of (Lozano, 2017) (Chapman, 2009).

WET testing methods
WET testing methods include acute and chronic test types with sublethal endpoints. A WET test involves the testing of various concentrations of sample effluent on aquatic freshwater and marine invertebrate and vertebrate species. Test endpoints generally consist of an organism’s ability to survive, grow and reproduce. The USEPA recommends that WET testing is conducted on an aquatic vertebrate, invertebrate and plant species to determine which is the most sensitive indicator of toxicity for NPDES permit writing. The approved aquatic test species for conducting acute and chronic toxicity WET tests are surrogate species that representative or can be extrapolated to represent, the toxicological responses to organisms in an aquatic community. WET test results are used to determine effluent limitations for NPDES permits (USEPA, 2016). WET test methods are listed in full in 40 CFR 136.3, Table IA (USEPA, 2017). Selection of the test type (exposure system) depends on the NPDES permit requirements, the test objectives, resource availability, the limitations of test organisms and effluent composition (USEPA, 2002a).

The approved test procedures for WET tests for freshwater, marine/estuarine vertebrate, invertebrates and plants are documented in the three test method manuals (USEPA, 2000):
 * U.S. Environmental Protection Agency. 1993c. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 4th ed., EPA 600/4-90/027F. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, OH.
 * U.S. Environmental Protection Agency. 1994. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 3rd ed., EPA 600/4-91/002. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, OH.
 * U.S. Environmental Protection Agency. 1994. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, 2nd ed., EPA 600/4-91/003. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, OH.

Test Criteria and Quality Assurance
WET methods suggest an effluent dilution series of 6.25%, 12.5%, 25%, 50%, and 100% but it is not a requirement and should be used as a default when the toxicity of the effluent is relatively unknown (USEPA, 2000). An appropriate effluent dilution series in vital to determine the NOEC and LOEC using hypothesis testing. In addition, an appropriate dilution water to create effluent dilution series should be fitting for the objectives of the test, support adequate fitness of the test organisms with respect to survival, growth, reproduction, or other responses, has consistent quality and does not contain toxic contaminants (USEPA, 2000).

Quality assurance measures for each test type concern (USEPA, 2000a):

(1) Effluent sampling and handling

(2) The source and condition of the test organisms;

(3) Condition of equipment;

(4) Test conditions

(5) Instrument calibration;

(6) Replication

(7) Use of reference toxicant

(8) Record keeping

(9) Data evaluation

Acute Toxicity to Freshwater, Marine and Estuarine Aquatic Organisms
Acute toxicity WET tests generally involve the exposure of the test organism to effluent concentrations with a control water for a duration of 24 to 96 hours. The EPA has approved 20 freshwater and marine/estuarine organisms that can be used for acute toxicity testing (USEPA, 2017). Acute effluent toxicity is measured using a dilution series that includes a control and a minimum of five effluent concentrations. The test results provide dose-response information of the effluent concentration that induces lethality of 50% of the test organisms (LC50) for the prescribed period of time (24-96 h) or the highest effluent concentration in which survival is not statistically significantly different from the control. Tests may use static (static non-renewal or static renewal), or flow-through exposure systems (USEPA, 2002a).

Acute Toxicity Freshwater Test Organisms: Acute Toxicity Marine/Estuarine Test Organisms:
 * 1) Ceriodaphnia dubia (daphnid)
 * 2) Daphnia pulex and D. magna (daphnids)
 * 3) Pimephales promelas (fathead minnow)
 * 4) Oncorhynchus mykiss (rainbow trout) and Salvelinus fontinalis (brook trout)
 * 1) Mysidopsis bahia (mysid)
 * 2) Cyprinodon variegatus (sheepshead minnow)
 * 3) Menidia beryllina (inland silverside), M. menidia (Atlantic silverside), and M. peninsulae (tidewater silverside)

Chronic Toxicity to Freshwater Aquatic Organisms
Tests for chronic toxicity to freshwater organisms include four- to seven-day methods for three aquatic species (USEPA, 2017). Effluent chronic toxicity is measured using a dilution series that includes of a control and a minimum of five effluent concentrations. The test results provide dose-response information of the percent effluent that affects “hatchability, gross morphological abnormalities, survival, growth, and/or reproduction” for the prescribed period of time (four to seven days). The test results are represented as the highest concentration in which the observed effects are not statistically significant from the control. The test exposure method can either be static non-renewal or static renewal (USEPA, 2002b).

Chronic Toxicity Freshwater Test Organisms:
 * 1) Pimephales promelas (fathead minnow)
 * 2) Ceriodaphnia dubia (daphnid)
 * 3) Selenastrum capricornutum (green alga)

Chronic Toxicity to Marine and Estuarine Aquatic Organisms
Tests for chronic toxicity to marine/estuarine organisms includes five short-term (one-hour to nine-day) methods for four aquatic species (USEPA, 2017). Effluent chronic toxicity is measured using a dilution series that includes of a control and a minimum of five effluent concentrations. The test results provide dose-response information of the percent effluent that affects “hatchability, gross morphological abnormalities, survival, growth, and/or reproduction” for the prescribed time period (one hour and 20 minutes to nine days). The test results are represented as the highest concentration in which the observed effects are not statistically significant from the control. The test exposure method can either be static non-renewal or static renewal (USEPA, 2002c).

Chronic Toxicity Marine/Estuarine Test Organisms:
 * 1) Cyprinodon variegatus (sheepshead minnow)
 * 2) Menidia beryllina (inland silverside)
 * 3) Mysidopsis bahia (mysid)
 * 4) Arbacia punctulata (sea urchin)
 * 5) Champia parvula (red macroalga)

Enforcement and Regulations
Whole Effluent Toxicity regulation implementation is under the Clean Water Act (CWA) of the EPA and used by the National Pollutant Discharge Elimination System (NPDES) permitting authority to determine whether an entity’s permit for effluent discharge will need to include WET requirements (US EPA, 2016).

The National Pollutant Discharge Elimination System (NPDES) was created in 1972 under the CWA. The NPDES permit program integrates state, tribal, and territorial government with permitting, administrative, and enforcement of regulations. EPA retains oversight responsibilities, however, 46 states and one territory are authorized to implement the NPDES program (DOE, 2008).

The NPDES permit program regulates point sources that discharge pollutants to waters of the United States. The permit has two parts of control: technology-based limits and water quality-based limits when technology-based limits cannot provide protection of the water body. An NPDES permits are used by facilities to discharge pollutants into a receiving water in certain amounts and under certain conditions. Permits may also authorize facilities to process, incinerate, landfill, or beneficially use sewage sludge (US EPA, 2016).

An individual permit is specific to an individual facility and once it submits the appropriate application, the permitting authority develops a permit for that particular facility based on the information contained in the permit application. The authority issues the permit to the facility for a specific time period (not to exceed five years) with a requirement that the facility can reapply prior to the expiration date. A general permit covers a group of dischargers with similar qualities within a given geographical location. General permits are usually more a cost-effective option for permitting agencies.

July 1994: Final WET Control Policy Control Policy (EPA 833-B-94-002) was implemented to make nationwide requirements to control WET and help with the implementation of said requirements. Eight brackets of the policy address the steps for the EPA’s process for WET control:

1. Evaluate WET water quality criteria attainment for acute WET at the edge of the acute mixing zone and for chronic WET at the edge of the chronic mixing zone. The only exception is when state requirements are different for evaluating WET criteria. WET effluent limitations are developed by permitting authorities based on acute or chronic criterion applied at the edge of the actual or a respective mixing zone.

2. Permitting authorities to review all major dischargers for reasonable potential to cause or contribute to exceedance of WET water quality criteria.

3 Permitting authorities to evaluate whether a discharger has reasonable potential to cause or contribute to exceeding WET water quality criteria.

4. When reasonable potential to cause or contribute to the exceedance of WET water quality criteria is found, the permitting authority will impose effluent limitations to control WET.

5. The permitting authority imposes WET monitoring conditions upon dischargers that do not have effluent limitations to control WET.

6. Compliance schedules to meet WET limits allowed under state and federal law, NPDES permits may or may not contain schedules for compliance with WET effluent limitations.

7. The requirements of the water quality permitting regulations apply without regard to the pollutant(s) that may be causing toxicity, including ammonia and chlorine.

8. Whole Effluent Toxicity controls put on for Publicly Owned Treatment Works (POTWs) The requirements of the water quality permitting regulations to apply.

The EPA and Society of Environmental Toxicology and Chemistry have both had meetings to resolve important issues involving the regulatory application of WET testing and trying to implement new ways to evaluate and regulate WET.

Next Steps in Regulation
EPA wants to implement a new WET implementation strategy to develop an approach that is a weight-of-evidence approach to water quality violations. The collective results of WET testing, chemical analysis, bioassessments, and other techniques are considered for water quality standards.