The presence of bacteria and viruses in freshwater represents a global health risk. The substantial spatial and temporal variability of microbes leads to difficulties in quantifying the risks associated with their presence in freshwater. Fine particles, including bacteria and viruses are transported and accumulated into shallow streambed (i.e., benthic) sediment, delaying the downstream transmission during baseflow conditions but contributing to their resuspension and transport downstream during stormflow events. Direct measurements of pathogen accumulation in benthic sediments are rare. Until now, the dynamic role of benthic sediment as both a store and source of microbes, has not been quantified. In this study, we analyze microbial abundance in benthic sediment along a 1km reach of an intermittent Mediterranean stream receiving inputs from the effluent of a wastewater treatment plant, a known point source of microbes in streams. We sampled benthic sediment during a summer drought when the wastewater effluent constituted 100% of the stream flow, and thus, large accumulation and persistence of pathogens along the streambed was expected. We measured the abundance of total bacteria, Escherichia coli (as a fecal indicator), and presence of enteric rotavirus (RoV) and norovirus (NoV). The abundance of E. coli, based on qPCR detection, was high (4.99∙102gc /cm2) along the first 100m downstream of the wastewater effluent input and in general decreased with distance from the source, with presence of RoV and NoV along the study reach. A particle tracking model was applied, that uses stream water velocity as an input, and accounts for microbial exchange into, immobilization, degradation, and resuspension out of benthic sediment during baseflow and stormflow. Rates of exchange into benthic sediment were 3 orders of magnitude higher during stormflow, but residence times were proportionately lower, resulting in increased longitudinal connectivity from up to downstream during stormflow. Model simulations demonstrated mechanistically how the rates of exchange into and out of the benthic sediment resulted in benthic sediment to act as a store during baseflow and a source during stormflow.

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