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Stream ecosystems are threatened by chemical contamination and altered vertical connectivity between surface water and groundwater. Vertical hydrological exchanges occur through the porous matrix—comprising coarse surface sediments and the hyporheic zone—which acts as a critical interface for pollutant storage. The Functional Trait (FTR) method, based on oligochaete communities in these two compartments, allows for simultaneous assessment of toxic stress and vertical hydrological exchange dynamics. We applied this method upstream and downstream of four wastewater treatment plants as well as within an impacted tributary and around its confluence zone. Our results, supported by physicochemical analyses confirming effluent-driven pollution, demonstrate marked degradation of biological quality and ecosystem functioning downstream of all discharge points. While coarse surface sediments provided a reliable diagnostic of point-source pollution impacts, investigating the hyporheic zone refined the assessment by identifying site-specific hydrological trends. In one case, it revealed enhanced infiltration dynamics and pollutant accumulation in deep interstitial spaces, while in another, it highlighted groundwater exfiltration as a driver of ecosystem resilience. These findings highlight the FTR method as a robust and flexible tool for water managers. While integrating the hyporheic zone allows for a comprehensive analysis of vertical exchange dynamics and ecosystem vulnerability, focusing on coarse surface sediments alone provides a cost-effective strategy for routine impact assessment and the detection of major exfiltrations reaching the surface. Future research should integrate this biological approach with high-resolution physicochemical monitoring and direct physical measurements of vertical hydrological exchanges.