Overall treatment barrier effect: Mean eDNA detection probability (±95% CI) of the six spiked species across WWTPs, based on the proportion of positive ddPCR detections before (pre-spike) and after (post-spike) the controlled introduction of the homogenized tissue mixture at the influent. Bars show mean detection probabilities aggregated across all taxa and facilities. The X-axis represents the sampling locations per WWTP, i.e., the influent (incoming untreated wastewater), the effluent (treated discharge water), and the downstream river site (100 m from the effluent outflow).