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

## TL;DR

This study shows that eDNA concentration and fish biomass have moderate, nonlinear relationships, highlighting the need for replication in eDNA-based monitoring.

## Contribution

The study experimentally reveals nonlinear eDNA-biomass relationships and emphasizes the importance of replication in freshwater fish monitoring.

## Key findings

- Nonlinear relationships between eDNA concentration and biomass were observed in three freshwater fish species.
- eDNA concentrations plateaued at intermediate biomasses in smaller-sized species.
- Replicates significantly affected results in two out of three species.

## 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.

Environmental DNA (eDNA) is a sensitive and non‐invasive tool to assist biodiversity monitoring. However, quantitative monitoring is limited by the often uncertain association between eDNA amount and fish abundance. Here we assessed the relationship between eDNA concentration (quantified by qPCR) and biomass of three freshwater fish of monitoring interest (Squalius lucumonis,
Lepomis gibbosus
, and
Pseudorasbora parva
) in controlled conditions, revealing moderate and non‐linear relationships, with plateauing curves in two out of the three examined species. Replicates' variability also emerged as a source of uncertainty. This contributes to improving eDNA‐based monitoring and understanding patterns of eDNA shedding.