Overview of the wastewater treatment plant (WWTP) experiment. eDNA samples were collected at four WWTPs in Belgium (Ostend, Ghent, Zomergem, and Nevele) at three sampling locations per WWTP: the influent (untreated wastewater), the effluent (treated water leaving the WWTP), and the receiving river 100 m downstream. A predefined DNA spike was introduced at the influent of each WWTP, with samples collected before (T1) and after (T2) the spiking event to assess the persistence and detectability of consumption-derived fish DNA following wastewater treatment. The map shows the location of the four WWTPs within Flanders, with major waterways indicated in blue. The schematic on the right illustrates the sampling strategy within the WWTP system. Base illustration of the wastewater treatment plant adapted from NoBrokerHood (2021).