AbstractEcotoxicology is concerned with the effects of chemicals on biological systems Identifying components of complex aqueous effluents poses special problems, and can be useless if there is a lack of information on the biological effects of the identified chemicals Toxicity-based (bioassay directed) sample fractionation can be very useful, but the small amount of fractioned material is a constraint that can be solved by using in vitro tests The RTG 2 in vitro cytotoxicity test has been used to assess (a) the efficacy of a treatment plant in the aeronautics industry and (b) the exposure of fish and molluses cultured in Esteiro Bay to the effluent of a fish processing factory Ecotoxicological assessments could be done without identifying the responsible chemicals The RTG-2 test was used in combination with concentration/fractionation procedures It proved that the toxicity of the liquid wastes from the aeronautics industry was eliminated by the treatment, and that molluses and fish reared in Esteiro Bay had accumulated toxic chemicals dumped by the fish-processing factory A combination of the RTG-2 cytotoxicity test and HPLC proved to give useful information even for chemicals not identified by GC MS