Proving a negative: Estimating species ‘confidence in absence for decision‐making’ using environmental <scp>DNA</scp> monitoring

Abstract Policy‐driven decision‐making is an important aspect of environmental management globally, often focused on protecting priority species. However, declining trends in freshwater biodiversity have resulted in uncertainty regarding the present‐day distribution of rare and elusive species. This is problematic for applied ecologists and environmental managers, since when dealing with priority species, it is generally more challenging to provide a confident assessment of absence than merely confirm their presence. Without such confident assessments, resource‐intensive management plans may be misplaced and not adequately targeted to conserve important remaining populations. We present a framework to estimate confidence in absence, referred to as “Confidence in Absence for Decision‐Making” (CIADM), based on single‐visit environmental DNA metabarcoding data obtained from water samples. It uses a case study of European eel presence/absence upstream of 44 water pumping stations. Through a high degree of biological (sample) and technical (PCR) replication, we retrospectively assigned ‘confidence in absence’ values and proposed various strategies to achieve the required confidence levels in future surveys. Seventeen out of 44 pumping stations tested positive for eel, and we were able to assign a &gt;99% confidence level that the remaining 27 sites were negative for eel DNA at the time of sampling. Increasing both biological and technical replication increased ‘confidence in absence’ values. For example, using three PCR replicates per sample, required four replicate biological samples to achieve &gt;95% and six to achieve &gt;99% confidence in eel absence given non‐detection. However, we estimate that by using seven PCR replicates per sample a &gt;99% confidence in eel absence following non‐detection could be achieved from only three replicate biological samples. Furthermore, we found that eel positive sites had significantly higher species richness, and fish communities differed between eel positive and eel negative sites. Synthesis and applications . This study highlights the importance of optimising workflow specific replication and provides an adaptable and multi‐level framework (CIADM) to produce confidence in priority species absence given non‐detection. Thereby enabling applied ecologists and decision‐makers to collaboratively design surveys and make decisions, such as policy compliance or management interventions to meet conservation objectives while effectively prioritising resources.