The hypothesis is tested that toxicity of acid mine drainage can be detected by a selection of existing macroinvertebrate community and bioindicator metrices supplemented by toxicity tests with the local mosquitofish Gambusia holbrooki Girard and the shrimp Atyaephyra desmaresti Millet. The behavioural responses of A. desmaresti to acid mine drainage were recorded in the Multispecies Freshwater Biomonitor, based on behaviour and survival as parameters. Bioassessment methods were based on community diversity, structure, function, and bioindicators and supplemented by chemical analysis (temperature, pH, metals). The Biological Monitoring Working Party adapted for the Iberian Peninsula, the number of predators (Coleoptera, Hemiptera) and the number of Ephemeroptera and Trichoptera taxa differentiated the sites well. The on-line toxicity test revealed pH-dependent acute toxicity of the acid mine drainage for the shrimp (LC(50)-48 h: pH-AMD=5.8) and a pH- dependent decrease in locomotory activity with the lowest-observed-response-times (LORTs) within 5 h of exposure. Shrimp were more sensitive to acid mine drainage than fish (LC(50)-48 h: pH-AMD=4.9). A new multimetric index combining toxicity testing and bioassessment methods is proposed.