Enumeration of Faecal Indicator Bacteria

Faecal indicator bacteria were enumerated for samples M1–M3, G1, and GR3 by the Dublin City Council Central Laboratory. E. coli were enumerated for these samples using the IDEXX Colilert-18 assay according to ISO 9308-2:2012. Briefly, 10 and 100 ml of sample were mixed with dehydrated Colilert medium and incubated in Quanti-Trays at 36.0 ± 2.0°C. E. coli concentrations for these samples are determined as most probable number per 100 ml (MPN/100 ml). E. coli concentrations for samples GR1–2, EP2–3, RK1, and RV1 were determined using standard filtration methods in University College Dublin according to ISO 16649-1:2018. Samples were passed through 0.45 μm nitrocellulose filter membranes (Nalgene, Thermo Scientific) and then incubated on Tryptone Bile X-Glucuronide agar (TBX; Sigma-Aldrich) for 4 h at 37°C followed by incubation for 18 h at 44°C to enumerate E. coli as colony forming units per 100 ml (CFU/100 ml). Previously, split sampling of stations in the Elm Park stream catchment and subsequent analysis by both laboratories demonstrated these protocols to be comparable, therefore E. coli concentrations are referred to as CFU/100 ml throughout (Supplementary Figure 1). For all samples, intestinal enterococci were enumerated by incubating filter membranes on Slanetz and Bartley agar (Oxoid) at 37°C for 44 h. Filter membranes were then transferred to Bile Aesculin Agar (Sigma-Aldrich) and incubated at 44°C for 2 h to enumerate intestinal enterococci as CFU/100 ml according to ISO 7899-2:2000. For sites M1–M3, G1, and GR3 the upper limit of quantification was 20,000 MPN/100 ml.