Environmental DNA (eDNA) analysis is a rapid, non-invasive, cost-efficient biodiversity monitoring tool with enormous potential to inform aquatic conservation and management. Development is ongoing, with strong commercial interest, and new uses are continually being discovered. General applications of eDNA and guidelines for best practice in freshwater systems have been established, but habitat-specific assessments are lacking. Ponds are highly diverse, yet understudied systems that could benefit from eDNA monitoring. However, eDNA applications in ponds and methodological constraints specific to these environments remain unaddressed. Following a stakeholder workshop in 2017, researchers combined knowledge and expertise to review these applications and challenges that must be addressed for the future and consistency of eDNA monitoring in ponds. The greatest challenges for pond eDNA surveys are representative sampling, eDNA capture, and potential PCR inhibition. We provide recommendations for sampling, eDNA capture, inhibition testing, and laboratory practice, which should aid new and ongoing eDNA projects in ponds. If implemented, these recommendations will contribute towards an eventual broad standardisation of eDNA research and practice, with room to tailor workflows for optimal analysis and different applications. Such standardisation will provide more robust, comparable, and ecologically meaningful data to enable effective conservation and management of pond biodiversity.

The use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation—from field sampling to the analysis of PCR‐based results—for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Hence, the general applicability of assays, as well as associated uncertainties and limitations, often remain undetermined. The absence of clear guidelines for assay validation prevents targeted eDNA assays from being incorporated into species monitoring and policy; thus, their establishment is essential for realizing the potential of eDNA‐based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks (e.g., “in silico analysis”), and arranged on a 5‐level validation scale from “incomplete” to “operational” with defined minimum validation criteria for each level. These variables were evaluated for 546 published single‐species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. Of the 122 variables, 20% to 76% were reported; the majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterized by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The meta‐analysis demonstrates the suitability of the 5‐level validation scale for assessing targeted eDNA assays. It is a user‐friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling the appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.

Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England. Here we present data to show that in monthly water samples taken from two ponds (March 2014–February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March–August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.