Evaluation and further development of the activated sludge respiration inhibition test

The activated sludge respiration inhibition test is an important bacterial test system for the determination of bacterial toxicity of chemical compounds. The exposure period recommended by OECD 209 and ISO 8192 protocols is 30 and 180 min. A modified version of the test was developed which allowed a prolonged incubation period of 27 h to enhance the possibilities of the test system. The test system with the prolonged incubation time was evaluated by the recommended reference compound 3.5-dichlorophenol and showed an EC50 of 6.3 mg l−1 with a coefficient of variation of 12.7%. Furthermore, the use of an open test system was evaluated showing a comparable toxicity but a higher coefficient of variation than the closed test system. A storage of activated sludge for several days accompanied by a daily feed with OECD nutrient solution should be avoided, for it can cause a decreased sensitivity of the inoculum. Different statistical fit procedures were tested indicating that Weibull fit procedures were superior for extended data sets covering a wide range of concentrations whereas Gamma and Probit fits were appropriate for smaller data sets mainly restricted to the linear part of the dose response curve.