Integrating eDNA metabarcoding into fish-based ecological indices for lowland rivers under the EU Water Framework Directive

Abstract Environmental DNA (eDNA) metabarcoding is widely used as a non-invasive alternative to catch-based methods for fish monitoring in general, with strong potential to support the EU Water Framework Directive (WFD) in particular. However, its suitability for ecological index calculation and intercalibration under the WFD remains largely unknown. We therefore compare eDNA metabarcoding and electrofishing at 91 sites of lowland rivers across a habitat pressure gradient in Flanders (Belgium), and evaluate the integration of eDNA-derived fish community data into the fish-based lowland Index of Biotic Integrity (IBI). Specifically, we compared (i) fish species richness and (ii) Ecological Quality Ratios (EQRs) between eDNA metabarcoding and electrofishing across all sites, and for a subset of sites across a seasonal gradient. eDNA metabarcoding consistently detected higher species richness than electrofishing, and showed high within-site replicability. Additionally, eDNA-derived species richness showed an asymptotic relationship with electrofishing-based richness, reflecting differing variation in detection sensitivity across the richness gradient. Despite pronounced metric-level differences, eDNA-derived EQRs did not differ significantly from those obtained using the fish-based index. Agreement between eDNA- and fish-based EQRs showed no evidence of systematic bias. Both methods yielded seasonally stable EQR values and showed consistent negative responses to increasing habitat pressure. Moreover, our results show that eDNA metabarcoding yields Ecological Quality Classes (EQCs) comparable to conventional fish-based assessments. These findings support a hybrid WFD monitoring approach in lowland rivers, in which eDNA enables cost-effective, broad-scale status assessment combined with targeted electrofishing providing more detailed information about recruitment and size-structure of the fish populations. Development of eDNA-native indices and typology-specific calibration as next critical steps toward broader regulatory implementation are discussed.