Composition of the eDNA spike introduced at the influent of each WWTP. Each spike contained a predefined mixture of six species and their biomasses, including two commonly consumed flatfish species (Solea solea and Pleuronectes platessa) as well as the rare Misgurnus fossilis and three non-native species (i.e., Misgurnus bipartitus, Xenopus laevis, and Lithobates catesbeianus) that are unlikely to occur naturally in the river or to be present in human diets.