Universal performance of benzalkonium chloride for the preservation of environmental DNA in seawater samples

Environmental DNA (eDNA) analysis allows noninvasive and cost-effective monitoring of macroorganisms' distribution and composition in aquatic ecosystems. Benzalkonium chloride (BAC) is an inexpensive and simple preservative for eDNA in water samples and has been used in many eDNA studies for preventing its degradation during transportation. However, its preservation performance has limitedly been evaluated by species-specific assays, targeting short fragments of mitochondrial DNA in freshwater and brackish ecosystems. Here, we examined the performance of BAC in preserving eDNA in seawater samples, targeting different fragment lengths of mitochondrial and nuclear eDNA, and community information inferred by eDNA metabarcoding. We quantified the time series changes of Japanese jack mackerel (Trachurus japonicus) eDNA concentrations in experimental tanks and inshore seawater to compare the yields and decay rates of eDNA between BAC treatments. For both tank and field samples, BAC treatment substantially suppressed the degradation of all types of target eDNA and increased the eDNA yields at the start of the experiment. In addition, we performed eDNA metabarcoding targeting fish community to compare the species richness and composition in seawater samples between BAC treatments, showing that the number of fish species in field samples hardly varied throughout a day by BAC treatment. Our findings indicate high versatility of BAC in preserving both the quantitative (copy number) and the qualitative (species richness) information on various types of aqueous eDNA in various environmental conditions. BAC should therefore be used to minimize the false-negative detection of eDNA, regardless of target genetic regions, fragment sizes, environmental conditions, and detection strategies.