Controlled Experiments Reveal Moderate, Nonlinear Relationships Between eDNA Concentration and Fish Biomass in Three Freshwater Species of Monitoring Relevance

ABSTRACT Understanding the relationship between environmental DNA (eDNA) concentration and taxa abundance is essential for the advancement of quantitative biodiversity monitoring. We experimentally manipulated biomass of three freshwater fish species of monitoring interest—the Italian‐endemic Squalius lucumonis and the exotic‐invasive Pseudorasbora parva and Lepomis gibbosus —under controlled conditions (flow‐through 310 and 1330 L tanks). Following eDNA collection (2 L water filtration) and Real‐Time PCR quantification, Generalized Additive Mixed Models (GAMMs) revealed: (i) monotonic non‐linear relationships of moderate‐to‐high magnitude (0.42 < partial‐ R 2 < 0.62), with eDNA concentrations plateauing at intermediate biomasses in smaller‐sized taxa; and (ii) a significant effect of experimental replicates (tanks) in two out of three species. These findings suggest that eDNA‐based biomass quantification should not assume linearity, and emphasize the critical role of replication to account for inherent uncertainty, likely driven by inter‐ and intra‐individual variations in eDNA shedding rates.