This Reference Method was designed to provide the user with reliable techniques for the determination of certain specific biological effects of chemical contaminants. This is the first in a series of techniques which are a product of the work of the IOC/UNEP/IMO Group of Experts on the Effects of Pollution (GEEP). GEEP has tested each of these techniques in a series of experimental workshops in temperate and tropical conditions. The techniques offer the pollution scientist a sensitive tool for examining the effect of pollutants at sub-acute levels and provide a stronger scientific basis for marine pollution assessments than "traditional" experimental studies of acute toxicity.