Assessing the effects of urban effluent pollution on freshwater biodiversity and community networks using eDNA metabarcoding

Aquatic ecosystems provide essential services, yet they face increasing pressures from anthropogenic activities, including land-use change, eutrophication, browning, and contaminant pollution. While the ecological effects of these stressors are documented, the impacts of complex contaminant mixtures, particularly those from wastewater treatment plant (WWTP) effluents, remain poorly understood. Mixtures effects are typically assessed using traditional species-by-species toxicological approaches, which, though the gold standard, are time-intensive, require test animals, and have limited extrapolability. New Approach Methodologies (NAMs), such as environmental DNA (eDNA), offer a non-invasive alternative, enabling broader assessments of taxa responses across trophic levels. Here, we apply an eDNA approach to assess community-wide responses to effluent discharge in the St. Lawrence River, one of North America's most diverse freshwater ecosystems. We sampled water and aquatic communities along the effluent plume of the Montréal WWTP, analyzing taxa-specific responses across trophic levels using high-throughput sequencing. We evaluated the influence of water physico-chemistry and per- and polyfluoroalkyl substances (PFAS) on aquatic beta diversity and network structure. To validate our eDNA results, we compared fish-specific detections with traditional fishing surveys. Our findings highlight how wastewater-derived contaminants influence biodiversity patterns and species interactions, with taxonomic responses varying across trophic levels. Network analyses revealed shifts in ecological stability, with changes in species connectivity and modularity influenced by effluent exposure. This study demonstrates the value of eDNA for characterizing biodiversity responses to anthropogenic stressors and provides insights into the broader implications of point-source pollution for freshwater ecosystem resilience.