Neurotoxic effects of representative pharmaceutical mixtures from Mediterranean rivers impacted by waste water treated effluents

Mixture toxicity studies on neuroactive chemicals remain scarce and often rely on simplified combinations of compounds with known modes of action (MoA), despite the frequent non-monotonicity of neuroactive responses. Furthermore, real environmental mixtures are far more complex, containing chemicals with diverse MoAs, concentrations, and effect directions. This study identified environmentally relevant neuroactive mixtures in Mediterranean rivers by integrating behavioural and cardiovascular assays in Daphnia magna and zebrafish embryos with mixture-based laboratory tests and field sample comparisons. Lowest effect concentrations (LOECs) were determined for 19 of 24 pharmaceuticals across ten behavioural and physiological endpoints, with 78% of compounds eliciting significant bioactivity and 35% producing non-monotonic responses. Nine artificial mixtures—some containing up to 16 compounds—were designed using equitoxic ratios. Most joint effects were additive or less-than-additive and conservatively predictable using concentration addition. Comparisons with solid-phase extracts from two Mediterranean rivers showed that approximately half (3 out of 6) of the tested mixtures partially or fully explained field toxicity patterns on D. magna antipredator responses (Max and visual motor activity and phototaxis) and Basal activity in zebrafish embryos, while the remaining mixtures failed to reproduce effects—particularly D. magna feeding inhibition and cardiac effects in both species—indicating additional, unmeasured contaminants contributed to joint toxicity. Metabolomic analyses revealed that artificial and field mixtures shared 36–70% of enriched neurological KEGG pathways and up to 84% of deregulated metabolites in D. magna, highlighting convergent disruption of cholinergic, catecholaminergic, serotonergic, GABAergic and related pathways. Overall, the study demonstrates that a subset of pharmaceuticals can account for observed neurobehavioral impairments in river water, but additional unknown mixture components likely contribute to excess toxicity, supporting the need for broader chemical screening and non-target approaches in environmental monitoring.