Elucidation of fecal inputs into the River Tagus catchment (Portugal) using source-specific mitochondrial DNA, HAdV, and phage markers.

Determining the source of fecal contamination in a water body is important for the application of appropriate remediation measures. However, it has been suggested in the extant literature that this can best be achieved using a 'toolbox' of molecular- and culture-based methods. In response, this study deployed three indicators (Escherichia coli (EC), intestinal enterococci (IE) and somatic coliphages (SC)), one culture-dependent human marker (Bacteroides (GB-124) bacteriophage) and five culture-independent markers (human adenovirus (HAdV), human (HMMit), cattle (CWMit), pig (PGMit) and poultry (PLMit) mitochondrial DNA markers (mtDNA)) within the River Tagus catchment (n = 105). Water samples were collected monthly over a 13-month sampling campaign at four sites (impacted by significant specific human and non-human inputs and influenced by differing degrees of marine and freshwater mixing) to determine the dominant fecal inputs and assess geographical, temporal, and meteorological (precipitation, UV, temperature) fluctuations. Our results revealed that all sampling sites were not only highly impacted by fecal contamination but that this contamination originated from human and from a range of agricultural animal sources. HMMit was present in a higher percentage (83%) and concentration (4.20 log GC/100 mL) than HAdV (32%, 2.23 log GC/100 mL) and GB-124 bacteriophage with the latter being detected once. Animal mtDNA markers were detected, with CWMit found in 73% of samples with mean concentration of 3.74 log GC/100 mL. Correlation was found between concentrations of fecal indicators (EC, IE and SC), CWMit and season. Levels of CWMit were found to be related to physico-chemical parameters, such as temperature and UV radiation, possibly as a result of the increasing presence of livestock outside in warmer months. This study provides the first evaluation of such a source-associated 'toolbox' for monitoring surface water in Portugal, and the conclusions may inform future implementation of surveillance and remediation strategies for improving water quality.