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In recreational waters, pathogen pollution is a major concern for the USEPA. The USEPA is responsible for initiating the National 303(d) List of Impaired Surface Waters. Pathogen pollution from E. coli is a common reason why recreational waters are placed on the 303(d) list. E. coli O157 H:7 and other enteric pathogens can cause serious illness and even death. Sinking Creek is a part of the Watauga River watershed which runs through Carter and Washington county. Sinking Creek is currently listed on the 303(d) as impaired due to the presence of E. coli. Because of the known presence of E. coli, it is possible that other enteric pathogens may exist in the creek. The main objective of the study was to determine the presence of E. coli and Salmonella bacteria within Sinking Creek and was accomplished by using selective media to isolate the bacteria. The second goal of the study was to understand how various temperature treatments effect the growth of Salmonella and E. coli in Sinking Creek. Water samples from Sinking Creek were incubated at 4, 28, and 37°C. The third objective of the study was to determine how the survival of Salmonella and E. coli from Sinking Creek compared to samples taken from manure slurry via a meta-evaluation. It was predicted that Salmonella would not be present in water samples taken from Sinking Creek. It was further hypothesized that E. coli and any present Salmonella would have limited to no growth at 4°C and the most growth would occur at 37°C. A pilot study was conducted to determine the growth of microorganisms naturally present in Sinking Creek. The data from the pilot study was used to determine the creek’s ability to support a healthy microbiota. Samples for the main experiment were taken from September 2019 to November 2019. The main experiment found that Salmonella was present in Sinking Creek at lower amounts than E. coli. Both E. coli and Salmonella grew significantly at 4°C. On average, the least amount of growth for Salmonella and E. coli was at 37°C. Most growth on R2A peaked at 5 days of incubation. Water incubated at 37°C showed the highest growth peaks at 5 days for all three selective plates. For all three selective plates, water incubated at 4°C peaked in growth between days 5 and 7. The results of the main experiment could have been affected by factors such as contamination. Another limitation of the study was that enumeration of the colony forming units became less accurate after larger colonies had formed. Replicating the main experiment over a longer period could indicate more representative growth curves. The meta-evaluation concluded there was no difference in decay rate between samples taken from water or manure. The results of the meta-evaluation disproved the hypothesis that manure would have lower decay rates than samples taken from water. A larger sample size is recommended to yield more representative results for the meta-evolution. Further replications of the main experiment are recommended along with studies sampling the presence of Salmonella and E. coli at various distances from the below wetlands site.

Access to good quality, pathogen-free water is a necessity for human life. Pathogencontaminated water poses a threat to human health, and steps must be taken to minimize that risk using remediation techniques, such as constructed wetlands. Sinking Creek is a tributary of the Watauga River that was placed on the 2016 303(d) list published by the Tennessee Department of Environment and Conservation due to the presence of Escherichia coli. Because of this impairment, a constructed wetland was placed in Sinking Creek to decrease the downstream transport of pathogens. Knowing this, three primary goals were made for this experiment. The first goal was to establish the seasonal presence of E. coli, Salmonella spp., and other culturable bacteria in Sinking Creek. The second goal was to determine the concentration patterns of E. coli, Salmonella, and other culturable bacteria as water in Sinking Creek flows downstream. The third goal was to use the data to analyze the effectiveness of the constructed wetland in Sinking Creek and its ability to decrease bacterial concentrations downstream. To achieve these goals, water samples were collected every Wednesday from January 29th to March 11th from four sites on Sinking Creek: two upstream from the constructed wetland and two downstream from the constructed wetland. The samples were plated on mFC, XLD, and R2A agar using the micro drop technique to establish the presence of E. coli, Salmonella, and other culturable bacteria, respectively. It was hypothesized that, because of the placement of the wetland, concentrations of E. coli, Salmonella, and other culturable bacteria would be lower at Sites 3 and 4 than at Sites 1 and 2, but this hypothesis was disproved. Data analysis and statistical tests displayed that all bacterial concentrations were higher at Sites 3 and 4 than at Sites 1 and 2. From this, it was concluded that the constructed wetland is not functioning as it was intended, and the increase in bacterial concentrations at Sites 3 and 4 suggest that there is most likely a source of fecal contamination below the wetland.

Since the passage of the Clean Water Act, concern about surface water quality has increased. Reducing exposure to pathogens and adverse impacts on human health because of contact with surface waters has become the focus of many regulatory agencies. Fecal pollution is often a cause of surface water impairment. Fecal indicators, such as fecal coliforms and Escherichia coli, are used as surrogates to evaluate the presence or absence of fecal pollution. However, a growing body of research has shown that these species lack key characteristics necessary to be adequate indicators. As such, explorations into the efficacy of indicator species in predicting fecal pollution in water are necessary. Sinking Creek is a tributary of the Watauga River Watershed, located in Northeast Tennessee. Approximately ten miles of Sinking Creek have been placed on the national 303(d) list for fecal pollution, denoting the presence of fecal contamination exceeding the regulatory limit. Salmonella and Aeromonas are two enteric pathogens that would be expected to be detected in fecally contaminated waters. The primary objective of this study was to detect the presence of Salmonella and Aeromonas in Sinking Creek. The secondary objective was to evaluate their relationship with fecal coliforms, E. coli, and water quality parameters. Six study sites along Sinking Creek were sampled and standard methods were used to enumerate Salmonella and Aeromonas. Samples for Salmonella were collected for 8 months, while samples for Aeromonas were collected for seven. Salmonella and Aeromonas were present in Sinking Creek. Salmonella had the highest concentration at site 2 (the most downstream site), and was detected during all months of the study except for November. Salmonella concentrations varied by site. Aeromonas was present only during colder months, and had the highest concentration at site 2. Both Salmonella and Aeromonas show qualitative relationships with water quality parameters, such as dissolved oxygen and conductivity. However, statistically significant correlations of Salmonella and Aeromonas with water quality parameters were not observed. The lack of statistical significance is partially due to large variability and a small data set. Neither Salmonella or Aeromonas exhibited a relationship with fecal coliforms or E. coli. Therefore, fecal coliforms and E. coli may not be adequate indicator species for the presence of Salmonella, Aeromonas and possibly other waterborne pathogens. Traditional indicator species may inflate risk of pathogen exposure. Thus, many water bodies may be unnecessarily deemed as impaired. The results from this study can be used to guide further research regarding covariates influencing pathogen densities at fecally contaminated sites, as well as to guide decisions regarding impaired surface waters and management techniques.

Sinking Creek (HUC 06010103046), in the Watauga watershed of northeast Tennessee, is impaired due to Escherichia coli. To assess how E. coli and other water quality parameters fluctuated during storm events, water samples were collected with automated samplers during eight storms at two locations: Sinking Creek and a feeder spring. Turbidity and electrical conductivity data loggers were deployed in the creek, and dissolved oxygen (DO) was measured in situ. The presence of optical brighteners, used in detergents and an indicator of residential wastewater, was assessed using cotton fabric deployed at both sites and analyzed by an external laboratory. The Colilert Quanti-Tray method was used to process water samples for E. coli. Relationships between water quality parameters and lagged precipitation were assessed using cross-correlation. At the creek, E. coli and turbidity increased within 2 h of precipitation, exceeding the single sample water quality standard of 941 cfu 100 ml−1 during the storm. At the spring, E. coli became elevated more quickly than at the stream, within 30 min of precipitation, and decreased below the standard during the event. Electrical conductivity decreased within 1.5 h of the storm at the creek, and DO levels were higher at the creek than at the spring. Optical brightener analysis indicated possible presence of residential wastewater during one of two sampled storms. Targeted sampling and dye tracing are recommended to validate this hypothesis. These results may be used to inform field methods in similar storm sampling studies and will be useful in watershed restoration efforts in Sinking Creek.

Master of Science

Sinking Creek, a stream in northeast Tennessee, was added to the state 303 (d) list and a TMDL for fecal coliforms developed. The study objectives were to 1) identify areas in Sinking Creek with elevated levels of fecal coliforms and 2) compare data collected to results from watershed models used in the TMDL. Fourteen sites on Sinking Creek were monitored monthly and concentrations of total coliforms, fecal coliforms, and selected physical water quality parameters measured. Fecal coliform concentrations were >1000 CFU/100 ml at sites 1 through 4, and <400 CFU/100 ml, at all other sites indicating significant inputs between sites 4 and 5. Comparisons of results from Monte Carlo simulations and watershed models indicated geometric means listed in the TMDL were on average 64% higher than results from simulations calibrated with collected data. Proposed Best Management Practices (BMP’s) included; septic tank surveys, and the use of vegetative buffer zones.

Sinking Creek, located in upper east Tennessee, is on the 303(d) list for not meeting minimum water quality standards for recreation. A Total Maximum Daily Load (TMDL) for fecal coliforms was developed. The purpose of this study was to investigate the use of water quality parameters to identify areas that contribute to the fecal coliform loading. Concentrations of nitrate-N, orthophosphates, BOD, alkalinity, hardness, and optical brighteners (OB) were monitored at fourteen stations monthly for one year. Site 3 (agricultural region) exhibited the highest average nitrate-N loadings (627.34 mg/sec) and orthophosphate (as PO43-) loadings (84.83 mg/sec). Alkalinity loadings ranged from 10.00 mg as CaCO3/sec to 163,500.00 mgCaCO3/sec. Hardness loadings ranged from 2.00 mg as CaCO3/sec to 96,200.00 mgCaCO3/sec. The agricultural sites exhibited higher loadings for all water quality parameters measured (except OB) than the urban and forest areas. Nutrient loadings appeared to be related to agricultural land use patterns.

Fecal pollution of surface water is a significant environmental health issue. Indicator organisms are used to monitor microbial water quality, but often their presence does not coincide with the presence of pathogens. Bacterial source tracking is a term describing methods to determine the origin of fecal pollution based on bacterial traits. The objective of this research is to evaluate the use of 2 bacterial source tracking techniques, antibiotic resistance analysis (ARA) and ribotyping, to determine the sources of bacteria isolated from Sinking Creek. Based on the results of this study, ARA and ribotyping are not useful techniques for identifying sources of fecal pollution in Sinking Creek. ARA classification rates were low, and ribotype pattern generation success was 37%. The results of this study bring into question the reliability and reproducibility of these 2 source tracking methods for routine use in small watersheds.