Single and mixture toxicity of four pharmaceuticals of environmental concern to aquatic organisms, including a behavioral assessment.  Pharmaceuticals are frequently detected in aquatic environments as mixtures and can cause toxic effects to non-target organisms. We aimed to evaluate the single and mixture effects of the pharmaceuticals metformin, bisoprolol, ranitidine and sotalol using Daphnia similis and Danio rerio. In addition, we aimed to test the predictive accuracy of the mathematical models concentration addition and independent action and to evaluate the nature of the possible toxicological interactions among these pharmaceuticals using the combination index-isobologram model. The acute toxicity of these four pharmaceuticals individually and of their binary mixtures were evaluated using the D. similis tests. Developmental and behavioral effects induced by the pharmaceuticals in quaternary mixtures were evaluated using D. rerio embryos. We observed that most of the binary mixture effects were in the zone between the effects predicted by the concentration addition and the independent action model. The combination index-isobologram model showed to be adequate to describe the nature of possible interactions occurring between the combined pharmaceuticals. Developmental and behavioral acute adverse effects seem not to be induced by the joint action of the quaternary mixture of the evaluated pharmaceuticals on D. rerio embryos, at the concentrations at which they are usually found in surface fresh waters. However, from the results obtained with D. similis, we can conclude that assessing the ecological risk based on the effects of individual pharmaceuticals can underestimate the risk level posed by these environmental contaminants.