Rainfall-driven dynamics of faecal pollution in river bathing sites: coupling hydrological events, and DNA-assisted tracking to improve pollution management in recreational waters

This study assesses the use of Microbial Source Tracking (MST) alongside 16S rRNA gene sequencing to overcome the limitations of Faecal Indicator Bacteria (FIB) in rivers by enabling source attribution of faecal pollution, improving microbial risk assessment, and supporting more effective bathing water management. During the 2023 bathing season, weekly samples were collected by citizen scientists at two River Wharfe sites: the Cromwheel, the Environment Agency’s (EA) bathing monitoring site upstream of the Ilkley Sewage Treatment Works (STW), and the Stepping Stones, downstream of the STW. Samples for DNA analysis were taken concurrently with the EA statutory FIB monitoring. The EA measured water physico-chemical parameters and modelled rainfall upstream in the catchment. DNA-based analyses included Microbial Source Tracking (MST) using droplet digital-PCR (ddPCR) to quantify general, human, and ruminant Bacteroidales markers, alongside 16S rRNA sequencing for bacterial characterisation. Rainfall was the main driver of FIB and Bacteroidales marker variation, with higher FIB correlated with 12 h cumulative antecedent rainfall at Cromwheel. At Cromwheel, human and ruminant sources were detected during dry weather, but wet weather E. coli peaks were dominated by human markers. At Stepping Stones, the human-associated marker was predominant, as expected for a site downstream of a STW, and correlated with elevated FIB and increased abundance of potential pathogens, including Mycobacterium, Aeromonas and Clostridium. The combined ddPCR-MST and sequencing approach enhanced the detection of low-level faecal pollution, distinguished sources, and revealed microbial community shifts associated with pollution. These results demonstrate the added value of molecular methods to inform recreational river management.