Water Environmental RNA Reveals Dose-Dependent Toxicant Responses in Fish

The health of aquatic ecosystems is threatened by numerous chemicals, posing a critical global challenge. This study aimed to establish a noninvasive monitoring approach based on water environmental RNA (eRNA). This method assesses aquatic ecological status and provides early warnings of chemical stress risks by capturing transcriptome-wide metabolic activity from organisms in ambient water. Zebrafish were exposed to chemicals (carbendazim, chlorpyrifos, and their mixture) with distinct concentrations and modes of action in aquariums. First, zebrafish-derived transcripts accounted for 1.77–4.81% of total water eRNA transcripts, with 3370 genes annotated, of which 13.94–28.53% could be tracked to specific tissues, predominantly the scale and testis, functionally enriched in metabolic and cellular processes. Furthermore, the water eRNA exhibited perturbation potencies of carbendazim that were ∼2 and ∼8 times higher than organismal RNA (oRNA) derived from the zebrafish whole body at the gene and pathway levels, respectively. Finally, zebrafish eRNA profiles distinguished chemical exposures via adaptive pathway perturbations, alongside the identification of 12 candidate biomarker genes, such as nccrp1, for future eRNA-based chemical discrimination. Overall, this study deciphered zebrafish transcriptional dynamics in water eRNA under varied chemical exposures, contributing to the methodological advance in ecotoxicology and advancing eRNA-based early warning applications in global water management.