Assessing neurological and cardiovascular effects caused by pharmaceuticals in river water: Insights from Daphnia magna and Danio rerio embryos

Pharmaceutical residues in surface waters are an emerging environmental and public health issue, yet their biological impacts on aquatic life remain poorly understood. This study presents a cost-effective bioanalytical framework using Daphnia magna juveniles and zebrafish (Danio rerio) embryos to evaluate neurotoxic and cardiotoxic effects of pharmaceutical mixtures in rivers downstream of wastewater treatment plant (WWTP) effluents. Water samples from three rivers in north-eastern Spain (Besòs, Llobregat, and Onyar) were concentrated up to 5- and 20-fold using solid-phase extraction. Bioassays were conducted over 24 h for D. magna and five days for zebrafish embryos. Eighty pharmaceutical compounds were quantified via HPLC-MS and linked with phenotypic endpoints including locomotion, feeding, heart rate, neurotransmitter profiles, and metabolomic alterations. Of the 28 concentrated extracts, four were acutely toxic to zebrafish embryos. Altered behavioral and cardiovascular responses were observed in 22.2 % (D. magna) and 35.1 % (D. rerio) of extracts, primarily at higher enrichment. Concentrations of 31 pharmaceuticals were statistically associated with observed effects. Neuroactive drugs such as topiramate, rasagiline, citalopram, and fluvoxamine showed strong correlations with altered neurotransmitter levels in zebrafish, consistent with their known mechanisms. Seven additional compounds with secondary neuroactive properties were linked to similar neurological disruptions. Seventeen pharmaceuticals were associated with disturbances in amino acid metabolism and urea cycle pathways, indicating broader metabolic dysregulation. Overall, nearly 75 % of river extracts showed no observable effect, but several samples were acutely toxic or induced sublethal neurobehavioral and metabolic responses. These findings support the utility of D. magna and zebrafish (D. rerio) embryos as sensitive and complementary biosentinels for monitoring pharmaceutical pollution and highlight zebrafish as a relevant model for studying environmentally driven neurotoxicity with potential human health implications.