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(a) This bio-assay procedure is intended as a non-referee batch method (Note 1) for evaluating acute toxicity of wastes and of other water pollutants to fresh-water fishes. The test provides information on the relative acute toxicity (Note 2) of the substance tested under prescribed experimental conditions. N 1.—Tests may be performed using a standard reference water and reference chemicals (Section 6, Paragraphs (b) and (c)). The reference water is recommended for obtaining basic comparative research data. Test chemicals are recommended for standardizing the lot of fish under test. N 2.—Chronic or cumulative toxicity can interfere seriously with the growth and reproduction of organisms and may eventually cause death. This method detects only acute toxicity. (b) The test is applicable to industrial waste water and to the determination of the toxicity of pure chemical compounds in water solution. Wastes having volatile or readily oxidizable constitutents (Note 3) and excessive biochemical or other oxygen demand (Note 4) may also be tested by this method. N 3.—The concentration of highly volatile and unstable toxicants may decline throughout the test. In the case of highly volatile toxicants, it may be possible to obtain more reliable data by substituting initial oxygenation of the diluent in place of the standard controlled artificial oxygenation procedure. In the case of readily oxidizable constituents, it may be advisable to use test solution renewal techniques. N 4.—A distinction is made between death due to deficiency of dissolved oxygen in polluted water and death due to toxicity. In order to detect and evaluate the direct lethality of test material, it is necessary to maintain adequate dissolved oxygen concentrations during toxicity tests. (c) The results of the test data are reported in terms of median tolerance limits (Section 2(b)). The critical concentration range may be reported, also, as supplementary information (Section 2(c)). (d) The test data is applicable for use as a guide in estimating “safe concentrations” of the discharge of industrial wastes (see Appendix III).