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General Information

Acute biomonitoring tests specified by NPDES permits are usually definitive (multi-concentration) tests.  Typically these tests are carried out for 24 hours or 48 hours.  If a renewal test is specified, that means that the test chambers must be renewed with dilutions prepared from a second effluent sample collected the next day.  The detailed methods manual  for conducting acute WET tests is available at the EPA web site at http://www.epa.gov/OST/WET/disk2/

Dilutions and Replicates

Definitive acute toxicity tests typically require a series of five dilutions of the effluent, and a dilution water control, for a total of six treatments.  The dilutions, which are specified in the permit, are often 100%, 50%, 25%, 12.5%, 6.25%, and the control. 

Usually a minimum of four replicates are required for each treatment.   Each replicate normally contains ten organisms for a total of at least 40 organisms per treatment. At least 90% of the organisms must survive in the controls for the test to be valid.

Endpoints

Endpoints for definitive acute toxicity tests are determination of LC50, the LOEC, and the NOEC.  These endpoints are determined through statistical analyses, using appropriate statistical tests as outlined in the EPA biomonitoring manuals.

Test Conditions

Conditions under which acute tests are performed include a temperature of either 20 or 25 degrees C, and a photoperiod of 16 hours of light and 8 hours of darkness per day.  The following test conditions vary according the particular species being tested...

Minimum test solution volume per replicate
Cladocerans:  25 ml
Fathead minnows, sheepshead minnows, mysid shrimp: 200 ml

Age of Organisms
Cladocerans:  less than 24 hours old
Fathead minnows, sheepshead minnows: 1 - 14 days old
Mysid Shrimp: 1 - 5 days old

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