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Widespread wastewater pollution is one of the greatest challenges threatening the sustainable management of rivers globally. Understanding microbial responses to gradients in environmental stressors, such as wastewater pollution, is crucial to identify thresholds of community change and to develop management strategies that protect ecosystem integrity. This study used multiple lines of empirical evidence, including a novel combination of microbial ecotoxicology methods in the laboratory and field to link pressure-stressor-response relationships. Specifically, community-based whole effluent toxicity (WET) testing and environmental genomics were integrated to determine real-world community interactions, shifts and functional change in response to wastewater pollution. Here we show that wastewater effluents above moderate (>10%) concentrations caused consistent significant shifts in bacterial community …