Understanding of the ecology of environmental DNA (eDNA)-its origin, state, transport, and fate- is critical for interpreting the results of eDNA applications ranging from single-species presence/absence monitoring to whole-community analysis via metabarcoding and metagenomics. Accumulation and degradation of eDNA can be most accurately measured by conducting systematic manipulative experiments under controlled conditions. We compared eDNA accumulation and degradation in a laboratory setting for two different taxa (Daphnia magna and Egeria densa) across two different substrates (water and sediment). In accumulation experiments, both D. magna and E. densa showed increased eDNA levels in both water and sediment over 24 h. Daphnia magna eDNA concentrations were twice as high in sediment compared to water, whereas E. densa eDNA remained consistent between the two substrates. Degradation kinetic models were applied to our data for the two model organisms to characterize eDNA decay rates, inadvertently introducing an additional nutrient treatment with three different levels. The degradation of D. magna and E. densa eDNA varied based on nutrient concentrations and substrate types, with different models providing the best fit for different scenarios. Overall, we have demonstrated the importance of taxonomic, sample type, and environmental differences in eDNA ecological processes such as accumulation and degradation. These and similar processes require further study to enhance the interpretation of eDNA results in both research and management contexts.