NPDES

Overview
This toxicity test is conducted on municipal wastewaters that are directly discharged into receiving waters (streams, rivers, ocean, etc.) or on industrial dischargers who may or may not have their own outfall.

EPA Acute Testing Manual
There are several editions of the EPA's testing manual, each having minor differences which may significantly influence the outcome of the test. The most recent edition, the fifth (EPA-821-R-02-012) calls for using fish of one to 14 days in age and a minimum renewal at 48 hours. Both of these requirements tend to make this test more sensitive than previous editions of the test method. A detailed test protocol of this method is given along with the entire acute manual is available directly from EPA's homepage. You may access it by clicking here. The choice of which protocol is often directed by regional water quality control boards and should be specified in the NPDES permit. Any protocol modifications are also usually listed in the NPDES permit.

Description
The basic acute test uses a series of five test chambers each containing a specific concentration of the effluent diluted with standard laboratory water plus a control. A common dilution series such as: 100%, 50%, 25%, 12.5%, and 6.25% may be used. The dilution series may be specified in the NPDES permit and may differ between permits to reflect individual regulatory concerns. Ten test fish are placed into each chamber and their survival and water quality of the test chambers are monitored for 96 hours. All tests are conducted in replicate (2, 4, or 5 replicates, depending upon species). The number of fish surviving at each test concentration and in the control chamber at the end of the test are used to mathematically calculate the sample concentration toxic to 50% of the test fish. This known as the LC50. Toxicity units can then be calculated by dividing 100 by the LC50 value. For example, an LC50 of 75% would yield a TU of 1.3 TUa (100/75 Toxic Units acute). This test protocol can also be conducted as a percent survival bioassay where 100% sample is divided into two or more test tanks, fish are added, and water quality and survival are monitored for 96 hours. Results are then reported as percent survival in 100% effluent.

Other important considerations:

1. Test organism - there are over 45 different species listed in the EPA manual, ranging from water fleas to rainbow trout, however fathead minnows are by far the most commonly used organism; rainbow trout are known to be one of the most sensitive species;
2. Length of test - tests can range from 24 to 96 hours in length with 48 and 96 hour tests the most common;
3. Static or static renewal - static test solutions are mixed at the beginning of the test with test organisms exposed to the same solution throughout the test, while in static renewal tests solutions are mixed and changed daily or at a minimum every 48 hours (these are much more labor intensive and therefore more expensive).

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