Environmental RNA outperforms eDNA metabarcoding in assessing impact of marine pollution: A chromium-spiked mesocosm test

Environmental (e)DNA metabarcoding holds great promise for biomonitoring and ecotoxicological applications. However, few studies have compared the performance of eDNA versus eRNA metabarcoding in assessing organismal response to marine pollution, in experimental conditions. Here, we performed a chromium (Cr)-spiked mesocosm experimental test on benthic foraminiferal community to investigate the effects on species diversity by analysing both eDNA and eRNA metabarcoding data across different Cr concentrations in the sediment. Foraminiferal diversity in the eRNA data showed a significant negative correlation with the Cr concentration in the sediment, while a positive response was observed in the eDNA data. The foraminiferal OTUs exhibited a higher turnover rate in eRNA than in the eDNA-derived community. Furthermore, in the eRNA samples, OTUs abundance was significantly affected by the Cr gradient in the sediment (Pseudo-R2 = 0.28, p = 0.05), while no significant trend was observed in the eDNA samples. The correlation between Cr concentration and foraminiferal diversity in eRNA datasets was stronger when the less abundant OTUs (<100 reads) were removed and the analyses were conducted exclusively on OTUs shared between eRNA and eDNA datasets. This indicates the importance of metabarcoding data filtering to capture ecological impacts, in addition to using the putatively active organisms in the eRNA dataset. The comparative analyses on foraminiferal diversity revealed that eRNA-based metabarcoding can better assess the response to heavy metal exposure in presence of subtle concentrations of the pollutant. Furthermore, our results suggest that to unlock the full potential for ecosystem assessment, eDNA and eRNA should be studied in parallel to control for potential sequence artifacts in routine ecosystem surveys.