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​Whole Effluent Toxicity Testing

The Whole Effluent Toxicity (WET) program is managed by the Surface Water Permits Branch of the Division of Water.  Staff provide technical review of all self-monitoring reports conducted by the regulated community for compliance, as well as Toxicity Reduction Evaluations (TREs).

In 1988 the Department for Environmental Protection adopted a strategy to control toxic discharges into waters of the Commonwealth.  This toxics control strategy was implemented by including both chemical-specific and WET limits in Kentucky Pollutant Discharge Elimination System (KPDES) permits issued to major industrial dischargers and major municipal wastewater treatment plants (WWTPs) as well as minor WWTPs with an approved pretreatment program. In 2014, WET testing was added to coal facilities. The WET limits include both acute and chronic levels based on the instream waste concentration. Acute Wet tests are conducted using the water flea (Ceriodaphnia dubia, Daphnia magna or Daphnia pulex) and the fathead minnow (Pimephales promelas). Chronic WET tests are conducted using the water flea (Ceriodaphnia dubia) and the fathead minnow (Pimephales promelas).

The toxics control strategy consists of a two-tiered approach of chemical-specific and WET limits that are implemented through the KPDES program.  The chemical-specific tier is generally well known.  The WET tier was added by the agency because it directly measures toxicity and takes into account the aggregate toxicity in complex effluents and the chemical and physical interactions that can occur.

The DOW currently has approximately 800 industrial facilities, municipal facilities and coal facilities whose permit includes the WET limit.  All these facilities are required to perform self-monitoring for the WET permit condition (usually performed by a consulting laboratory). Our staff reviews all toxicity data reported on discharge monitoring reports (DMRs).  Our job is to verify the quality of the data, ensure proper methods were followed, track compliance and determine enforcement for noncompliance.  The staff has reviewed more than 11,000 toxicity test reports since 1988.  We now average approximately 800  reports per calendar quarter.

In addition to DMR reports, staff will review and make recommendations on TRE plans and quarterly progress reports.  All WET reports should be submitted via the ePortal using the Whole Effluent Toxicity Reporting Form.  

When submitting WET reports, please use the following naming convention: 

Example:  SmithWWTP_KY8675309_11-18-15

WET Testing Labs

A list of approved laboratories that currently perform WET testing for discharge operations under a Kentucky Pollution Discharge Elimination System (KPDES) permit can be found by emailing and requesting the information.

Approval by the state includes certain requirements that must be met including submittal of standard operating procedures and results of tests performed on aquatic organisms. For more information, contact program staff.

This list does not constitute an endorsement for services by the Commonwealth of Kentucky. The list is provided for information only.


WET Permits

Whole Effluent Toxicity (WET) limits have been added to Kentucky Pollutant Discharge Elimination System (KPDES) permits since 1988, with the exception of coal which was added in 2014. The program has grown to the point that now approximately 800 facilities have WET as a permit condition requiring self-monitoring. In the permit, the limit is expressed on the limits page as a toxicity unit (TU), either acute or chronic.

Almost all major discharge facilities are required to perform WET, as well as some minor industries or municipalities with pretreatment requirements. Both acute and chronic limits based on stream flow versus volume of discharge are used for testing.

When a KPDES permit is issued, the facility is required to test using both the fathead minnow, Pimephales promelas, and the water flea (Ceriodaphnia dubia, Daphnia magna or Daphnia pulex) Most facilities begin with quarterly WET testing. A permittee may request a reduction in frequency from quarterly to annual upon demonstration that no test failures, incomplete tests, or invalid tests occurred during the following specified timeframes:

1) Existing facilities: four (4) consecutive quarters

2) New or expanded facilities: eight (8) consecutive quarters.

A permittee may request a reduction in species after at least six (6) consecutive passing toxicity tests using both the water flea and the fathead minnow.

Upon permit renewal, the facility is required to return to testing both species for four tests to re-establish the most sensitive species. If the facility is currently testing monthly, then both species may be tested monthly. If quarterly testing was in effect at renewal, both species are tested quarterly.

The most recent minor modifications were made to the generic permit language for biomonitoring in Kentucky in 1999. The changes cleared up confusion that had arisen over the years concerning interpreting specific language. To get a copy of a specific permit, please submit a written Freedom of Information request to the Surface Water Permits Branch, email to swpbsupport@kygov.

​Following is a list of common questions about Whole Effluent Toxicity (WET) testing.

What does LC50 mean?

LC stands for lethal concentration. The LC50 is a calculated percentage of effluent at which 50 percent of the organisms die in the test period. It is calculated statistically and used in acute toxicity testing.

What is an IC25?

IC stands for inhibition concentration. The IC25 is also a calculated percentage of effluent. It is the level at which the organisms exhibit 25 percent reduction in a biological measurement such as reproduction or growth. It is calculated statistically and used in chronic toxicity testing.

What is a toxicity unit (TU)?

A toxicity unit is a unit of measure for effluent toxicity. It is not a percentage. The major advantage of using toxicity units to express toxicity test results is that they increase as the toxicity of the effluent increases. A TU of 4.00 is twice as toxic as a TU of 2.00.  The LC50 and IC25 are just the opposite. The lower the toxicity, the higher its percentage. TUs are also used as permit limits. Like metal concentrations and other permit parameters, increasing values reflect higher impacts.

How do the LC50 and IC25 translate into toxicity units?

To convert a percentage into a toxicity unit (TU), the percentage is divided into 100. For example, if an acute LC50 equals 100 percent effluent, the test results would be equivalent to a TUa of 1.00 (100/100=1.00). Or, if a chronic IC25 equals 80 percent effluent, the result would be 100/80=1.25 TUc.

How do I report test results from two grab acute tests on the Discharge Monitoring Report (DMR)?

For acute tests, do not average the results from the two grabs for the same test organism. The permit limit is the maximum; therefore, the maximum (highest) test result should be reported. If you test both species, each maximum is to be reported.

What is a major and minor facility?

A major facility is defined as one that discharges greater than 1 million gallons of effluent per day. A minor facility discharges less than that.

Why are water fleas and fathead minnows used as the test organisms for WET?

The species Ceriodaphnia dubia belongs to a group of freshwater microcrustaceans commonly referred to as water fleas. These invertebrates are a major component of the freshwater zooplankton found in lakes, streams, ponds and rivers throughout North America. The selection of Ceriodaphnia for toxicity testing is appropriate for a number of reasons:

  • They are broadly distributed in fresh water and are present throughout a wide range of habitat.
  • They are an important link in aquatic food chains and are a significant source of food for small fish.
  • They have a short life cycle and are easy to culture in the laboratory.
  • They are sensitive to a broad range of aquatic contaminants.
  • Their small size requires small volumes of test water leading to ease of sampling.

Fathead minnows, Pimephales promelas, belong to the fish family Cyprinidae, or carps and minnows, the dominant freshwater family in terms of number of species. Fathead minnows are native to North America and thrive in ponds, lakes, ditches and slow muddy streams. They are easy to culture in the laboratory, adapting well to the dry commercial fish food and brine shrimp necessary for culturing in the laboratory. These species have been used for acute and chronic tests for many years. Their life cycles allow for tests that run from two to seven days, thus reducing testing costs and sample volumes considerably.

​For more information contact:

Environmental Scientist IV