Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

Environmental nucleic acids (eDNA and eRNA) metabarcoding analyses have attracted considerable research attention as they non-invasively and cost-effectively monitor ecosystems. For heretofore unknown reasons, however, they often detect false positives. Here, we focused on domestic wastewater as a possible source of false positives and characterized fish nucleic acids in the influent and effluent of a wastewater treatment plant (WWTP). The metabarcoding analysis revealed that DNA and RNA detected in the influent wastewater originated from over 120 fish species, and most of these were consumed fish. The nucleic acid content was significantly higher in the influent than in the effluent, and the copy numbers of DNA in the influent were significantly higher than those of RNA. These findings were corroborated by the current literature and indicated that eRNA could effectively mitigate false positives. The present study demonstrated that rivers may be extensively contaminated by fish nucleic acids, and WWTPs substantially alter the quantities and ratios of fish DNA and RNA. Therefore, analyses of the wastewater near sampling points and coverage of the sewage system enable us to select and use the appropriate nucleic acid type (DNA or RNA) for metabarcoding analysis and improve the accuracy of fish species detection and identification.