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Detecting environmental DNA (eDNA) in water samples is a powerful tool in determining the presence of rare aquatic species. However, many open questions remain as to how biological and physical conditions in flowing waters influence eDNA. Motivated by what one might find in a stream/river benthos we conducted experiments in continuous flow columns packed with porous substrates to explore eDNA transport and ask whether substrate type and the presence of colonized biofilms plays an important role for eDNA retention. To interpret our data, and for modelling purposes, we began with the assumption that eDNA could be treated as a classical tracer. Comparing our experimental data with traditional transport models, we found that eDNA behaves anomalously, displaying characteristics of a heterogeneous, polydisperse substance with particle-like behaviour that can be filtered by the substrate. Columns …