: Wastewater, especially containing hospital effluents, exhibits high chemical complexity and specificity since it includes various chemicals, biocides, pharmaceuticals, surfactants, radionuclides, disinfectants and pathogens. Biological tests provide true evidence of the wastewater quality and unlike chemical analytical tests show comprehensive pollution effects on the environment and human health. Normalized conventional bioassays are not sensitive enough for ecotoxicological evaluation of wastewater and there is a great need for the development of suitable sensitive bioassays in order to characterize properly the residual toxicity of treated effluents. Provisions of binding EU legislation regarding protection of animals used for scientific purposes and legislation dealing with test methods for identification and classification of health hazard of chemicals, pharmaceuticals, biocides, medical devices and consumer products such as cosmetics for environmental ecosystems and for man require to employ alternative toxicological methods respecting the 3Rs concept with priority given to methods in vitro. The Fish Embryo Test (FET) is identified as a relevant, reliable and efficient alternative test method in vitro for determination of acute toxicity for fish. Using the FET, additional toxicological endpoints may be investigated to assess organ specific bioaccumulation, genotoxicity and mutagenicity, developmental toxicity, teratogenicity, various forms of neurotoxicity or endocrine disruptivity. The addition of multiparametric sensitive endpoints makes the FET a true alternative in vitro assay and a powerful tool in toxicology.