Effect of Time and Temperature Before Preservation on Bacterial <scp>eDNA</scp> in Sediment Samples

ABSTRACT Environmental DNA is being increasingly used for research and regulatory purposes, but currently the lack of standardization is holding it back. There are gaps in our understanding of the eDNA analysis pipeline and the potential impacts of areas of variability within that. Standardization is necessary for all uses of eDNA to allow comparison between sample sets and ensure accuracy and reproducibility. Understanding sample stability is particularly important for planning fieldwork and writing practicable guidance for regulatory compliance monitoring. Samples collected for eDNA analysis are preserved as soon as possible for stability, but practicalities of sampling in the field can lead to delays where the sample temperature may be uncontrolled. We collected eDNA sediment samples along an organic enrichment impact gradient and incubated them at 10°C, 20°C, and 40°C for up to 48 h prior to preservation, then sequenced the bacterial 16S gene. We show that bacteria families responded differently to the incubation temperatures and times to an extent that affected ecological interpretation. Predictions of benthic health using a trained random forest machine learning model were tolerant of incubation up to 20°C, and showed sensitivity to temperature within 3 h of incubation at 40°C. We show that the influence of temperature can depend on the study aim, taxa involved, and analysis used, such that some situations may allow temporary storage up to 20°C but others will be affected by 10°C. We confirm that keeping sediment temperature low is critical for many applications, and that potential temperature deviations must be reported.