AbstractAssessment of the toxicity of chemicals to the respiration of activated sludge microorganisms is a relatively simple and reproducible method when applied to a standard population of bacteria. However, activated sludge is, by nature, a variable commodity, and varying results are often reported. Within‐plant variations can occur from day to day as a result of shifts in the bacterial population, probably caused by changes in the strength of components of the sewage feed. Between‐plant variations can be considerably greater. Factors such as differing operating conditions and presence of industrial waste waters in the sewage can result in differing species composition of the sludge, which, in turn, may account for differing biooxidation rates of components of the waste water and thus different respiration rates. Sludge from different sources, and/or grown under different conditions, may also vary in response to inhibitors, because of varying degrees of reaction of some inhibitors with non‐living sludge components. Therefore, in practice, it has been usual to consider EC50values from the inhibition of respiration of activated sludge test in terms of order of magnitude. Variations in EC50results of the ‘ISO’ inhibition of respiration test have been quantified for a number of chemicals, both within and between batches of sludge, and with sludges from different sources. The coefficient of variation of EC50results within batches of sludge was found to be approximately 9%. Between batch and between source variations were 28–76% depending on the te