Although the use and development of molecular biomonitoring tools based on environmental nucleic acids (eDNA and eRNA; collectively known as eNAs) have gained broad interest for the quantification of biodiversity in natural ecosystems, studies investigating the impact of site‐specific physicochemical parameters on eNA‐based detection methods (particularly eRNA) remain scarce. Here, we used a controlled laboratory microcosm experiment to comparatively assess the environmental degradation of eDNA and eRNA across an acid–base gradient following complete removal of the progenitor organism (Daphnia pulex). Using water samples collected over a 30‐day period, eDNA and eRNA copy numbers were quantified using a droplet digital PCR (ddPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene of D. pulex. We found that eRNA decayed more rapidly than eDNA at all pH conditions tested, with detectability—predicted by an exponential decay model—for up to 57 h (eRNA; neutral pH) and 143 days (eDNA; acidic pH) post organismal removal. Decay rates for eDNA were significantly higher in neutral and alkaline conditions than in acidic conditions, while decay rates for eRNA did not differ significantly among pH levels. Collectively, our findings provide the basis for a predictive framework assessing the persistence and degradation dynamics of eRNA and eDNA across a range of ecologically relevant pH conditions, establish the potential for eRNA to be used in spatially and temporally sensitive biomonitoring studies (as it is detectable across a range of pH levels), and may be used to inform future sampling strategies in aquatic habitats.

Although the use and development of molecular biomonitoring tools based on eNAs (environmental nucleic acids; eDNA and eRNA) have gained broad interest for the quantification of biodiversity in natural ecosystems, studies investigating the impact of site-specific physicochemical parameters on eNA-based detection methods (particularly eRNA) remain scarce. Here, we used a controlled laboratory microcosm experiment to comparatively assess the environmental degradation of eDNA and eRNA across an acid-base gradient following complete removal of the progenitor organism (Daphnia pulex). Using water samples collected over a 30-day period, eDNA and eRNA copy numbers were quantified using a droplet digital PCR (ddPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene of D. pulex. We found that eRNA decayed more rapidly than eDNA at all pH conditions tested, with detectability—predicted by an exponential decay model—for up to 57 hours (eRNA; neutral pH) and 143 days (eDNA; acidic pH) post organismal removal. Decay rates for eDNA were significantly higher in neutral and alkaline conditions than in acidic conditions, while decay rates for eRNA did not differ significantly among pH levels. Collectively, our findings provide the basis for a predictive framework assessing the persistence and degradation dynamics of eRNA and eDNA across a range of ecologically relevant pH conditions, establish the potential for eRNA to be used in spatially and temporally sensitive biomonitoring studies (as it is detectable across a range of pH levels), and may be used to inform future sampling strategies in aquatic habitats.

Molecular tools of species identification based on eNAs (environmental nucleic acids; environmental DNA [eDNA] and environmental RNA [eRNA]) have the potential to greatly transform biodiversity science. However, the ability of eNAs to obtain “real‐time” biodiversity estimates may be complicated by the differential persistence and degradation dynamics of the molecular template (eDNA or eRNA) and the barcode marker used. Here, we collected water samples over a 28‐day period to comparatively assess species detection using eDNA and eRNA metabarcoding of two distinct barcode markers—a mitochondrial mRNA marker (COI) and a nuclear rRNA marker (18S)—following complete removal of Arthropoda taxa in a semi‐natural freshwater system. Our findings demonstrate that Arthropoda community composition was largely influenced by marker choice, rather than molecular template, individual microcosm, or sampling time point. Furthermore, although eRNA may capture similar species diversity as the established eDNA method, this finding may be marker‐dependent. Although we found little to no difference in decay rates observed among sample groups (COI eDNA, COI eRNA, 18S eDNA, 18S eRNA), this result is likely due to limitations in the ability of eNA‐based metabarcoding to provide a strong correlation between true eNA copy numbers present in the environment and final read counts obtained (following the metabarcoding workflow). Collectively, our findings provide further support for the use of multi‐marker assessments in metabarcoding surveys to unravel the broadest taxonomic diversity possible, highlight the limitations of eNA metabarcoding methods in providing accurate decay rate estimates, as well as establish the need for further comparative studies using both metabarcoding and single‐species detection methods to assess the persistence and degradation dynamics of eNAs for a diverse range of taxa.