Advancing ecological assessment: towards the integration of eDNA metabarcoding into an estuarine fish index

• Surface water eDNA and bottom trawling were used to assess estuarine fish diversity. • eDNA, sampling organismal traces in the surface, produced higher species richness. • Bottom trawling, sampling fish, was more effective in detecting demersal species. • Due to different catchability, ecological status assessed with both methods differ. • Method-specific reference conditions are needed for ecological status classification. In the face of increasing anthropogenic pressures on estuarine ecosystems, methods to efficiently and reliably assess their ecological status are essential. This study explores the integration of environmental DNA analysis into the AZTI’s Fish Index (AFI) to assess ecological status of estuarine ecosystems. Surface water eDNA sampling and bottom trawl surveys were performed across multiple estuaries in the Basque Country, Spain, and resulting species data were used to calculate AFI scores. eDNA metabarcoding consistently detected higher fish species richness than bottom trawling, while the latter remained more effective at capturing demersal species. In general, ecological classifications from eDNA- and bottom trawl derived data displayed low concordance, largely due to differing species assemblages and metric contributions. These results emphasize the respective strengths and weaknesses of each methodology and the necessity for method-specific calibration. Considering that the AFI is calibrated using bottom trawl data, its direct application to eDNA-derived species lists may lead to some inconsistencies. This study underscores the critical necessity to establish eDNA-specific reference conditions and to recalibrate index thresholds accordingly. While eDNA approach may not entirely replace traditional methods, its scalability, sensitivity, and minimal ecological disturbances establish it as an essential complementary application within monitoring programs. This research strongly supports the urgent advancement of eDNA-based indices and the critical enhancement of reference conditions for their effective incorporation into ecological assessment frameworks under the Water Framework Directive.