The particle size distribution of environmental DNA varies with species and degradation.

Environmental DNA (eDNA) analysis is frequently used as a non-invasive method to investigate species and biodiversity in ecosystems. However, such eDNA may represent both organisms currently present as well as species that released their DNA some point in the past, thereby representing a mix of current and historic biodiversity. This may lead to a false-positive detection of organisms' presence. As the eDNA particle size distribution (PSD) changes along with the decay process, it may facilitate solving the above problem. Here, we set up tank experiments with snails, zebrafish and daphnids, respectively, to monitor the change in eDNA PSD and eDNA degradation through time after removing organisms. We found that zebrafish eDNA decays more slowly for larger particle sizes. Across all species tested, the percentage of large size ranges tended to increase over time while the smaller sizes showed relatively fast decay rates. As a result, PSD changed consistently with eDNA decay, although initial PSD varied between species. In combination, we propose that eDNA PSD can be used to assess the current prevalence of organisms at an eDNA sampling location while avoiding false-positives on the presence of species. Our findings expand the applicability of eDNA for monitoring target species in freshwater ecosystems.