Toxicometabolomic responses of Daphnia magna to pharmaceutical mixtures in municipal wastewater treatment plants of the Costa Brava (Catalonia, Spain)

The increasing scarcity of freshwater highlights the growing need to identify alternative water sources. Wastewater represents a potential resource but requires advanced treatments to remove wastewater-borne contaminants, including contaminants of emerging concern (CECs). Although wastewater treatment plants (WWTPs) can remove a large fraction of these pollutants, many persist at low concentrations, underscoring the need for improved treatment technologies and robust water quality assessment methods. This study evaluated the pharmaceutical occurrence and residual contamination patterns in effluents from 14 WWTPs located along the Costa Brava (Spain) using a multiscale approach that integrates chemical analyses, behavioural assays in Daphnia magna , and metabolomic profiling of exposed organisms. Statistical assessment together with multivariate analyses (PCA, PLS-DA) and enrichment studies (MetaboAnalyst) indicated that tertiary treatments are insufficient to remove neuroactive pharmaceuticals and that treated effluents may still pose sublethal ecological risks to aquatic organisms. Moreover, WWTPs serving larger populations were associated with higher contaminant loads, highlighting the need for site-specific treatment strategies. Overall, the findings stress the importance of stricter regulations and advanced treatment technologies to minimize pharmaceutical contamination, particularly when effluents are reused for human-related applications. Even at trace levels, pharmaceuticals may threaten ecosystem integrity and human health. Enhancing treatment processes and enforcing stricter discharge limits are essential to ensure safe water reuse and long-term environmental sustainability.