Wastewater treatment effectively eliminates exogenous eDNA, preventing downstream false-positive detections

Environmental DNA (eDNA) is widely applied to monitor aquatic biodiversity, but false-positive detections may arise when DNA from consumed or processed organisms is discharged to waterways through domestic wastewater. We experimentally tested whether such wastewater-derived eDNA persists through municipal treatment and contributes to downstream false-positive detections. Controlled spikes of homogenized tissue from six target taxa (two marine fishes and four freshwater fish/amphibians) were introduced into the influent of four wastewater treatment plants (WWTPs) in Flanders, Belgium. Water samples were collected at three points: influent, treated effluent, and the receiving river. Sampling was performed before and up to 24 h after spiking, and eDNA concentrations of each of the six target taxa were quantified using droplet digital PCR. All species showed post-spike increases in influent eDNA concentrations, although individual taxa were not detected in every influent sample, particularly at the largest facilities where hydraulic dilution reduced short-term detectability. In contrast, effluent and downstream concentrations remained zero across all WWTPs, indicating that no spiked eDNA persisted beyond the treatment process. One WWTP located near a marine harbor showed background detection of marine fish species even before spiking, likely originating from seafood processing or domestic discharge. However, these signals were also fully removed during treatment. Overall, modern secondary treatment effectively eliminates exogenous eDNA, preventing its release into receiving waters. While systems with incomplete sewer connectivity or combined sewer overflows may still discharge untreated domestic wastewater, under standard operating conditions riverine eDNA detections can be interpreted as reflecting local biota rather than wastewater contamination.