The Santa Cruz River, in southern Arizona, receives steady inputs of nutrient-enriched treated wastewater (effluent). Previous studies have documented reduced infiltration of surface water in the river. This disruption of hydrologic connectivity, or clogging, can have consequences for groundwater recharge, flows of wastewater in unwanted locations, and potentially even survivorship of floodplain riparian vegetation. Clogging can result from biotic processes (microbial or algal growth), abiotic processes (siltation of interstitial spaces), or both. Little is known about clogging in rivers and the environmental factors that regulate their dynamics, so natural field experiments along the Santa Cruz and San Pedro Rivers were used to answer: 1) Are there spatial patterns of hydraulic conductivity in the riverbed downstream from the effluent point-source? 2) Is there temporal variability in hydraulic conductivity and microbial abundance associated with flooding? 3) Are there environmental variables, such as nutrients or stream flow, related to differences in hydraulic conductivity and microbial abundance? To address these questions, a series of sites at increasing distance from two municipal effluent discharge points with differing water quality were selected on the Santa Cruz River and compared with non-effluent control reaches of the San Pedro River. Physical, chemical, and biological parameters were monitored over one year to capture seasonal changes and flood cycles.

Water quality in surface water is frequently degraded by fecal contamination from human and animal sources, imposing negative implications for recreational water use and public safety. For this reason it is critical to identify the source of fecal contamination in bodies of water in order to take proper corrective actions for controlling fecal pollution. Bacteroides genetic markers have been widely used to differentiate human from other sources of fecal bacteria in water. The results of this study indicate that many assays currently used to detect human-specific Bacteroides produce false positive results in the presence of freshwater fish. To further characterize Bacteroides from fish and human, the fecal samples were cultured, speciated, and identified. As a result, forty six new Bacteroides 16S rRNA gene sequences have been deposited to the NCBI database. These sequences, along with selected animal fecal sample Bacteroides, were aligned against human B. volgatus, B. fragilis, and B. dorei to identify multi-segmented variable regions within the 16S rRNA gene sequence. The collected sequences were truncated and used to construct a cladogram, showing a clear separation between human B. dorei and Bacteroides from other sources. A proposed strategy for source tracking was field tested by collecting water samples from central AZ source water and three different recreational ponds. PCR using HF134 and HF183 primer sets were performed and sequences for positive reactions were then aligned against human Bacteroides to identify the source of contamination. For the samples testing positive using the HF183 primer set (8/13), fecal contamination was determined to be from human sources. To confirm the results, PCR products were sequenced and aligned against the four variable regions and incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped within the human clade. As an outcome of this study, a tool box strategy for Bacteroides source identification relying on PCR amplification, variable region analysis, human-specific Bacteroides PCR assays, and subsequent truncated cladogram grouping analysis has been developed. The proposed strategy offers a new method for microbial source tracking and provides step-wise methodology essential for identifying sources of fecal pollution.