The objective of aquatic toxicity tests with effluents or pure compounds is to estimate the 'safe' or 'no effect' concentration of these substances, which is defined as the concentration which will permit normal propagation of fish and other aquatic life in the receiving waters. The endpoints that have been considered in tests to determine the adverse effects of toxicants include death and survival, decreased reproduction and growth, locomotor activity, gill ventilation rate, heart rate, blood chemistry, histopathology, enzyme activity, olfactory function, and terata. Since it is not feasible to detect and/or measure all of these (and other possible) effects of toxic substances on a routine basis, observations in toxicity tests generally have been limited to only a few effects, such as mortality, growth, and reproduction. This manual describes chronic toxicity tests for use in the National Pollutant Discharge Elimination System (NPDES) Permits Program to identify effluents and receiving waters containing toxic materials in chronically toxic concentrations. The methods included in this manual are referenced in Table IA, 40 CFR Part 136 regulations and, therefore, constitute approved methods for chronic toxicity tests. They are also suitable for determining the toxicity of specific compounds contained in discharges. The tests may be conducted in a central laboratory or on-site, by the regulatory agency or the permittee.