EPA Method 2000

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EPA Method 2000:
Acute Toxicity Test for Waters Using Fathead Minnow. Official Name: EPA Test Method 2000.0: Fathead Minnow, Pimephales promelas, Acute Toxicity Tests with Effluents and Receiving Waters

Larval Pimephales promelas are exposed to varying concentrations of effluent or receiving water in a static non-renewal, static-renewal, or flow-through system for a pre-determined amount of time (24, 48, or 96 h). The survival in each concentration is compared to the survival in the control to determine the concentration of effluent or receiving water lethal to 50% of test organisms.

This method describes acute toxicity tests for use in the National Pollutant Discharge Elimination System (NPDES) Permits Program to identify effluents and receiving waters containing toxic materials in acutely toxic concentrations. This method is also suitable for determining the toxicity of specific compounds contained in discharges. This test may be performed as part of a self-monitoring permit requirement, compliance biomonitoring inspection, toxics sampling inspection, and other special investigations.

Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition, October 2002. EPA Volume 821-R-02-012

1) Toxic substances may be introduced by contaminants in dilution water, glassware, sample hardware, and testing equipment. 2) Improper effluent sampling and handling may adversely affect test results. 3) Pathogenic and/or predatory organisms in the dilution water and effluent may affect test organism survival and confound test results.

QC Requirements:
Pimephales promelas controls must have 90% or greater survival

Maximum Holding Time:
Maximum holding time of 36 h before first use.


Acute Toxicity

Test Specific

Sample Prep:
Warm to test temperature in water bath.

Precision can be estimated by using the same strain of organisms under the same test conditions, and employing a known toxicant, such as a reference toxicant. Single laboratory and multi-laboratory precision data described by the mean, standard deviation, and relative standard deviation (CV).


Revision Number:
Fifth Edition, October 2002.