Biomonitoring emerging hazards of pharmaceuticals in river water using gut microbiome and behavioural Daphnia magna responses

A cost-effective Daphnia magna testing framework was applied to identify emerging hazards such as neurological and cardiovascular defects as well as antibiotic resistant genes (ARGs), related to pharmaceuticals present in waste water treated (WWTP) effluent discharged into rivers. D. magna juveniles were exposed during 48 h to water samples from three rivers in the vicinity of Barcelona (NE Spain), Besós, Llobregat and Onyar, upstream and downstream of WWTP discharging points. The analyses included measuring levels of 80 pharmaceutical residues in water samples by HPLC-MS, determination of the loads of different clinically relevant antibiotic resistant genes (ARGs) in both water samples and exposed animals, and assessment of toxic effects in feeding, heartbeat responses, and behavioural indicators. ARG prevalence in water, but not in gut microbiomes, was associated with the presence of bactericides in water. These results suggest that their levels were high enough to put a selective pressure over river microbial populations, but that Daphnia guts were not easily populated by environmental bacteria. Toxic effects were found in 20-43% of water samples, depending on the river, and related to water quality parameters and to pollutant levels. For example, heartbeats were correlated with salinity, whereas feeding impairment did so with high loads of suspended solids. In contrast, behavioural alterations were associated to the concentration of neuroactive chemicals. Accordingly, we hypothesize that measured neuroactive chemicals have caused the observed effects. If this also applies to local invertebrate populations, the environmental consequences may be severe and unpredictable.