Brandao-Dias, P. F.; Shaffer, M.; Guri, G.; Parsons, K. M.; Kelly, R. P.; Allan, E. A.

Environmental nucleic acids (eNA), such as DNA and RNA, are powerful tools for monitoring biodiversity. Yet, interpreting eNA detections requires understanding of their environmental persistence. We conducted a decay experiment using seawater from an open dolphin enclosure to track degradation of six eNA components derived from Tursiops truncatus: mitochondrial eDNA of varying lengths, ribosomal eRNA, and messenger eRNA. Targets were quantified over seven days via digital droplet PCR (ddPCR). Decay followed a biphasic exponential model with rapid initial loss (~48 hours at 15C), followed by slower degradation. Cytb messenger eRNA was least stable, disappearing within four hours. Ribosomal eRNA persisted longer but degraded slightly faster than its eDNA counterpart (lambda = 0.125 per hour vs. 0.105 per hour). Longer eDNA fragments decayed more rapidly (lambda = 0.121 per hour) than shorter ones (lambda = 0.083 per hour). These findings support the use of eDNA fragment length as a proxy for degradation state and reinforce that combining multiple eNA components with distinct stabilities can provide a molecular clock framework for inferring the eNA age. This approach improves the spatiotemporal resolution of eNA-based monitoring, particularly for rare species that act as point sources. We also emphasize the importance of explicitly distinguishing between RNA types (ribosomal vs. messenger) in environmental studies, given their divergent stability and interpretability.