Bioretention cells (BRCs) are low-impact development stormwater management structures that integrate water quantity and quality management. Although BRCs have a predicted design life of about 25 years, most current research focuses on performance of cells less than two years old. This project evaluated the effectiveness of a BRC installed in 2007 to treat a 0.16-ha parking lot in Blacksburg, VA. After installation, this BRC was monitored for five months to determine initial flow reduction and total suspended solids, and nutrient removal. By monitoring for the same parameters, changes in cell performance since installation were quantified. ISCO automated stormwater samplers collected inflow and outflow composite samples from the cell, which were then analyzed for fecal indicator bacteria (total coliforms, E. coli, and enterococci), total suspended solids (TSS), total nitrogen (TN), and total phosphorus (TP). To determine if denitrification is occurring within the BRC, media samples taken throughout the cell were analyzed using qPCR. The bioretention media was also sampled to quantify changes in media nutrient content and particle size over the past seven years. Results indicate the bioretention media has not accumulated nitrogen and phosphorus since installation, and that the BRC remains effective at reducing flow volume and peak flow rates, as well as TSS, TN, TP, total coliforms, E. coli, and enterococci loads. Bacterial analysis of the media show most of the denitrifiers are present in the top layers of the bioretention media, despite an internal water storage layer and the bottom of the cell designed specifically for denitrification. / Master of Science

The contamination of inland surface water by point and nonpoint sources is a widespread human health concern. To address this problem, a thorough understanding of the sources and persistence of this pollution is necessary. Using fecal indicator bacteria (FIB) as a surrogate for enteric pathogens, the Environmental Protection Agency (EPA) and state agencies have developed surface water quality standards. If a segment does not meet these standards, a Total Maximum Daily Load (TMDL) must be developed for the watershed to identify sources of bacterial pollution. Currently, FIB pollution is the leading pollution type addressed by TMDLs in the country. One source of FIB identified in TMDLs is colonies of birds roosting under bridges. It has been proposed that the birds’ feces can augment the FIB concentrations downstream of bridges. In this year-long study of Bull Creek in Austin, Texas, it was determined that the concentrations of Escherichia coli and fecal coliform downstream of a bridge were significantly greater when migratory cliff swallows were nesting under the bridge. The downstream concentrations of both FIB exceeded contact recreation standards. Data also suggest that FIB from the feces could be swept into the stream by runoff from storm events. No enterococci loading was observed in conjunction with the swallows. This study also investigated the affects of sediment resuspension on surface water FIB concentrations and the persistence of FIB in shady, inland streams. The resuspension of sediments with attached FIB could also increase the FIB concentration in the water column and increase its persistence. The results of reactor-based experiments demonstrated that the concentration of E. coli in water from an effluent-dominated stream increased by a factor of 3 when riverine sediments were resuspended and exceeded single sample standards for contact recreation, suggesting sediments as a reservoir of E. coli. Additionally, concentrations of E. coli decreased by approximately 90% and 70% over 2 days in reactors containing stream water and sediment-laden stream water, respectively. / text

Fecal indicator bacteria (FIB) such as Escherichia coli and enterococci are used to assess microbiological water quality in recreational waters worldwide. FIB are used with the assumption that their presence correlates with that of fecal-associated pathogens in recreational waters. In aquatic habitats, several factors can interfere with the predictive relationship between FIB and pathogens including extended survival of FIB in secondary habitats such as sediment, vegetation and sand. Furthermore, many biotic (e.g. predation from bacterivorous protozoa and competition from indigenous bacteria) and abiotic factors (e.g. temperature, salinity, ultraviolet (UV) light irradiation, and nutrient availability) can influence the fate of FIB and pathogens associated with gastrointestinal tracts of animals (enteric pathogens) in secondary habitats. The relative importance of these factors is not well characterized, thus limiting our knowledge on the efficacy of FIB as indicators of fecal contamination and microbial pathogens in water. The studies presented in this dissertation investigated the influence of biotic (predation from bacterivorous protozoa and competition from indigenous bacteria) and abiotic factors (e.g. nutrient availability) on the survival of FIB (E. coli and Enterococcus faecalis) and pathogens (E. coli O157 and Salmonella enterica) in aquatic habitats. Water and sediment samples were collected from a fresh water river source (Hillsborough River, Tampa, FL) and used to prepare a series of outdoor mesocosm experiments. In each experiment, biota treatments were varied to include various combinations of predation and competition, both or neither. Manipulation of biota treatments involved disinfection of water and baking of sediments to remove indigenous microbiota, or addition of cycloheximide or kanamycin to diminish the effect of predation from natural protozoa or competition from indigenous bacteria respectively. Bacterial levels in all experiments were monitored over a five day period. In the mesocosms investigating the effect of predation and competition on FIB (E. coli and Ent. faecalis) and a pathogen (E. coli O157:H7), predation had a detrimental effect on the survival of the FIB and pathogen in the water column but only influenced the survival of the FIB in the sediment. Unlike predation, competition from indigenous bacteria influenced the survival of E. coli but not Ent. faecalis in both water and sediment. The second set of mesocosms investigated the effect of predation on two motile and non-motile enteric bacteria types (E. coli O157 and S. enterica), each with a motile and non-motile counterpart. An allochthonous predation source (Tetrahymena pyriformis) was added into the mesocosms to supply a consistent level of predation. Motility had a significant positive effect on the survival of S. enterica in the water and sediment but had negative significant effect for E. coli O157 in sediment only. Motility also played a more important role in the sediment compared to predation while predation played a more important role in the water column for both bacteria types. The third study compared the relative effects of predation, competition and nutrients on the survival of E. coli. Natural waters (not amended with nutrients) served as a baseline condition to which organic nutrients were added in two increments. Significant interactions among predation, nutrients and competition (all possible combinations) were observed. Interactions between predation and nutrients as well as competition and predation also accounted for the greatest effects (10% and 8% respectively). The interaction between predation and competition was particularly pronounced at the highest nutrient level. These studies reveal that predation, competition and nutrients are all important factors in the survival of FIB and enteric bacteria in water and sediment, and provide new observations on the relative magnitude of these effects. I show that survival characteristics of FIB and enteric bacteria in secondary habitats can vary depending on bacteria type (FIB or pathogen), location (water or sediment), prey characteristics (motile or non-motile) and specific environmental stressor present (predation, competition or nutrients). The findings of this dissertation provide new insights on the ecology of FIB and enteric bacteria in secondary habitats and underscore the importance of biotic and abiotic factors as determinants of the fate of FIB and enteric bacteria in secondary habitats.

The reduced capacity of antibiotics to treat infections is one of the greatest health concerns that society faces. There is substantial evidence that links this reduced capacity with the widespread use of antibiotics in livestock production. Livestock can act as reservoirs of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria, which can pass resistance on in the livestock's manure. It is important to understand the fate of antibiotic resistance genes and resistant bacteria in the environment after land-application of manure-based amendments. The goal of this field-scale study was to identify the effects of soil amendments (inorganic fertilizer, compost, or raw manure) and crop cover (lettuce or radish) on sediment transfer, fecal indicator bacteria (FIB), and release of ARGs in runoff over six storm events. Two FIB (Escherichia coli and enterococci) and two ARGs (sulI and ermB) were quantified in runoff from each of the constructed plots throughout the growing season. FIB and ARGs were recovered from all plots, including control plots indicating a background level within the soil. Additionally, only the effects of variability among individual storms had an impact on the concentration of FIB in runoff. Vegetative cover and storm variability affected sediment release. A trend of higher sul1 and ermB in runoff from compost and raw manure-amended plots for at least 2 months after planting crops was observed. Only one of these ARGs (ermB) is associated with the class of drugs given to the dairy cows used for the manure and compost, indicating inherent carriage of some ARGs independent of the type of antibiotic administered, and such genes can persist in the environment. These results suggest that there is a risk of ARGs being carried into areas downgradient from agricultural plots that have been amended with compost or manure. / MS

Foodborne outbreaks have continued to be associated with produce contamination originating from on-farm sources, such as soil or agricultural water. Additionally, the heterogeneity of the pre-harvest environment complicates the development of universal strategies for managing produce safety risks. Understanding the ecology and epidemiology of foodborne pathogens and fecal indicator bacteria (FIB) by growing regions, sample types, scale of analysis, and detection method is essential for developing targeted mitigation strategies. This dissertation utilized quantitative research methods and statistical modeling to examine the impact of sampling method, spatial, temporal, meteorological, and physicochemical factors on pathogen prevalence and FIB levels. Key findings highlight that the drivers of prevalence differ between pathogens and were influenced by sample type, scale, and region.. The variations in associations emphasize that risk varies by space and time. Therefore, results support regional and scale-dependent food safety standards and guidance documents for controlling hazards to minimize risk. Additionally, the method used for pathogen detection influences prevalence highlighting the need for standard methods since methodological differences confound comparisons across studies. Furthermore, since agricultural water quality is an important food safety priority, this dissertation aimed to determine the efficacy of chemical antimicrobial sanitizers against Salmonella in pre-harvest agricultural water. Results demonstrated that certain sanitizer treatments and conditions can significantly reduce Salmonella populations in preharvest agricultural water sources and thus may serve as a risk reduction option when used correctly. / Doctor of Philosophy / Fresh fruits and vegetables are continually implicated in foodborne outbreaks. Additionally, the source of the pathogen that causes illness in these outbreaks is often due to contact with contaminated soil or water on the farm. Since the environment is extremely diverse, the risk of foodborne pathogens is not uniform across a farm and between farms. Therefore, the development of a one-size-fits-all plan to reduce the risk of foodborne pathogens from contaminating produce on a farm is difficult. Understanding the incidence and distribution of foodborne pathogens and fecal bacteria and how these microorganisms interact with the environment is important to develop strategies to manage risk. Additionally, understanding how the prevalence of bacteria varies by state, medium (water vs soil), and farm is needed to develop targeted mitigation plans. This dissertation utilized laboratory and field-based experiments to understand how space, time, weather, and physical properties impact the occurrence of foodborne pathogens and fecal bacteria. The primary results show factors that impact prevalence are different between pathogens (Salmonella vs Listeria vs E. coli). Furthermore, the occurrence differed by sampling method (molecular vs culture), sample type (water vs soil), scale (within a farm vs between multiple farms), and region emphasizing that the risk from foodborne pathogens varies over space and time. Overall, this dissertation's results suggest that both regional and scale-specific guidelines are needed to reduce foodborne pathogen risks in the farm environment. Lastly, since the quality of the water used in growing fresh produce is an important food safety priority, the effectiveness of chemical antimicrobial sanitizers against Salmonella in agricultural water was evaluated. Results demonstrated that certain sanitizer treatments and conditions (sanitizer concentrations, water temperatures) can significantly reduce Salmonella populations in pre-harvest water sources and may serve as a risk reduction option when sanitizers are used correctly.

E. coli and Enterococcus were used to determine the fate and survival of fecal indicator bacteria (FIB) in sand and sediments. The microbial source tracking (MST) methods antibiotic resistance analysis (ARA), Bacteroides human-specific primer test, and fluorometry were compared against the FIBs to determine how reliable each method was in detecting the presence of human fecal contamination. Two phases (Summer 2009 and 2010) were evaluated based on the type of contamination event. A combined sewage overflow (CSO) event was simulated in Phase I, where large amounts of influent were added to sand and bay water columns over 1 to 4 days. In 2010, a low volume sewage leak was simulated in which smaller doses of influent were added to sand and bay water columns over a period of 5 to 15 days. Within each of the phases, both non- and re-circulated columns were also evaluated. Evaluation of FIB survival indicated that Enterococcus was able to stabilize and re-grow in the water and at the sediment/water interface within the Phase I non-circulated columns. E. coli was unable to re-grow and/or stabilize within any environment. Comparisons between the ARA and the FIBs revealed a large majority of isolates identified as coming from either bird or wildlife sources. Human sources were identified but at much lower concentrations than expected. Bacteroides results indicated strong relationships between the increase of FIB concentrations and the presence of the human-specific Bacteroides. Fluorometry results did not indicate any relationship with the FIBs. Unexpectedly, fluorometry readings increased as time progressed indicating that another compound was present that fluoresced at the same wavelength as optical brighteners (OBs). This project was one of the first to study the differences related to two different pollution events (CSO vs. sewage leak) while also evaluating what happens to pollution as it settles into the sediment. It was also unique because it compared bacterial (ARA), molecular (Bacteroides), and chemical (fluorometry) MST methods. / Master of Science

Water quality worldwide is assessed by enumeration of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, and enterococci) intended to act as surrogates for human enteric pathogens. In environmental waters, this predictive relationship is confounded by many possible sources of FIB with varying implications for human health. Many physico-chemical and biological factors influence the fate of enteric pathogens and FIB in aquatic habitats, but are poorly understood, thus limiting our understanding of the usefulness of FIB as fecal pollution indicators. These studies explored the field application of a “toolbox” approach to microbial source tracking (MST) intended to discriminate between human and non-human fecal pollution: a) in a Florida estuary used for shellfishing and recreational activities and b) at public beaches before and after remediation of wastewater infrastructure. Lastly, the effects of environmental factors (sediments, protozoa, sunlight) on survival of culturable E. coli were investigated in freshwater and seawater mesocosms simulating environmental conditions. Detection of a human- associated MST marker (the esp gene of Enterococcus faecium) at sites with suspected sewage contamination indicated that human fecal pollution is impacting water quality in Wakulla County, while Lagrangian drifters designed to follow current and tidal movement suggested that local hydrology plays an important role in bacterial transport and deposition pathways. Elevated FIB concentrations and frequent detection of human-associated MST markers (esp and human polyomaviruses) identified human sewage pollution at a public beach, facilitating remediation efforts (sewage main repair, removal of portable/abandoned restrooms), followed by significant decreases in FIB concentrations and MST marker detection. These studies show that comprehensive microbial water quality assessment can reliably identify contamination sources, thereby improving pollution mitigation and restoring recreational water quality. Protozoan predation, freshwater vs. seawater habitat and sediment vs. water column location affected the concentration of culturable E. coli in outdoor mesocosms. Sediments offered a refuge from predation where freshwater vs. seawater habitat was amore important determinant of survival. These findings provide important insight into the ecology of E. coli and their natural predators in aquatic habitats and underscore the inherent effect different habitats play in their survival.

Between 1970 and 2010 almost one-third of drinking water related waterborne disease outbreaks reported to the US Centers for Disease Control and Prevention were associated with systems dependent on untreated groundwater (i.e., most commonly, household wells). This is unsurprising, given that numerous past efforts to monitor household well water quality have indicated a high prevalence of fecal coliforms and/or E. coli at the point of use. Non-point sources of pollution, including septic tank leakages and poorly constructed drain fields, have been identified as the leading risk factors associated with outbreaks in households dependent on groundwater. Ideally, the integration of emerging source tracking (ST) analyses in well monitoring programs could be used to identify whether the presence of fecal indicator bacteria (FIB) is associated with human or non-human sources in order to inform remediation strategies. However, the application of ST to groundwater has been limited, and the interpretation of data is consequently difficult. This research compares the fate and transport of FIB (E. coli and enterococci) with a chemical (optical brighteners, OB) and a molecular (Bacteroides HF183) ST target in order to evaluate their potential use as indicators of water quality issues in private drinking water systems. Eighteen PVC soil columns were constructed in an outdoor soil column facility to represent small-scale septic drainfield models; they received synchronized doses of primary-treated wastewater twice daily and were monitored bi-weekly over a 7-month period. Columns were subject to variable influent loading rates of wastewater effluent, and differing degrees of soil compromisation (i.e. synthetic solution channels). Results show that while column effluent volume and constituent levels were related to dosage, they were not always related to soil compromisation (ANOVA, p < 0.05). E. coli and enterococci concentrations were associated with effluent volume and OB levels (Spearman's rank, p < 0.05). The presence of Bacteroides HF183 was not strongly associated with the other measured ST target levels (Point-biserial correlation, p < 0.05). Findings from this study suggest surface water ST methodologies may have a role in groundwater quality monitoring efforts. Quantifying the relative recovery of ST targets and FIB from controlled groundwater simulations will assist in the development of strategies to identify non-point sources of human wastewater pollution efficiently and effectively to inform remediation. / Master of Science

Pathogen contamination, typically quantified by elevated levels of fecal indicator bacteria (FIB), remains the leading cause of surface water-quality impairments in the United States. Continuous watershed-scale models are typically employed to facilitate Total Maximum Daily Load (TMDL) restoration efforts. Due to limited understanding of microbial fate and transport, predictions of FIB concentrations are associated with considerable uncertainty relative to other water-quality contaminants. By focusing on a data-rich instrumented urban watershed, this study aims to improve understanding of microbial fate and transport processes. Weekly FIB concentrations in both the water column and streambed sediments were monitored for one year, and statistical correlations with hydrometeorological and physicochemical variables were identified. An intensive six storm intra-sampling campaign quantified and contrasted loading trends of both traditional regulatory FIB and emerging Microbial Source Tracking (MST) markers. Together, these intensive monitoring efforts facilitated evaluation of the impacts of bacteria-sediment interactions on the predictions of daily FIB concentrations in Hydrological Simulation Program-Fortran (HSPF) over multiple years. While superior overall model performance was demonstrated as compared to earlier efforts, the inclusion of bacteria-sediment interactions did not improve performance. Large wet-weather microbial loading appears to have dwarfed the effects of FIB release and resuspension from sediment. Although wet-weather loading is generally considered as a primary source of waterbody microbial loads, dry-weather periods are more directly associated with public health concern, which may be a more suitable area for future model-refinement efforts. Site evaluation is critical to determine whether the added model complexity and effort associated with partitioning phases of FIB can be sufficiently offset by gains in predictive capacity. Finally, a stochastic framework to translate simulated daily FIB concentrations into estimates of human illness risks is presented that can be can be readily integrated into existing TMDLs. As even small concentrations of FIB from human sources are associated with great risk, and monitoring efforts indicated moderate/high levels of human-associated MST marker in this watershed, remediation efforts to protect public health would be best directed toward infrastructure improvements. Uncertainty analysis indicates more site-specific knowledge of pathogen presence and densities would best improve the estimation of illness risks. / Ph. D.

Master of Science / Geology / Saugata Datta / The province (upazila) of Matlab in SE Bangladesh is highly affected with elevated concentrations of dissolved As content and widespread fecal contamination in untreated drinking waters. The study area is sedimentologically composed of thick floodplain deposits of Holocene age overlying Plio-Pleistocene grey fine to coarse sands with considerable clays (consisting of Dupi Tila formation). The goal of the current study is to understand the possible impact of co-occurrence of dissolved organic carbon (along with As release) and fecal indicator bacteria (e.g., E.coli) in aquifers from shallow to deep groundwater quality in this area. Nineteen groundwater (spanning a depth range of 14 to 240 m) samples and nine surface water samples (eight ponds and one canal in proximity to the piezometer nests) were collected from four different piezometric nests within north and south Matlab Upazila in Bangladesh during the monsoonal season (Jun-Jul 2014). The analyses of dissolved organic carbon (DOC) and its fluorescence properties indicate that the chemical character of DOC from shallow to intermediate groundwaters (<150 m) and surface water is dominated by more aromatic and humic materials than deeper groundwaters. Both groundwaters and surface waters may receive humic substances leached from soil and/or from the cellular constituents and exudates of indigenous aquatic organisms. Dissolved organic carbons in groundwater and surface waters are composed of predominantly UVA and UVC-humic like along with tryptophan like components. Only 15% of total C is modern carbon at shallowest depths (<30 m) in groundwaters. The recharge source of groundwaters is from local precipitation, with or without some evaporation before infiltration as depicted by the δ2H and δ¹⁸O variations and the water is infiltrating through mostly terrestrially derived weathered sediments into the aquifers. The type of water in the study area is Ca-Na-HCO₃⁻ type. More toxic and soluble As (III) is present in shallow groundwaters (<30 m). High concentrations of As (V) and As[subscript (t)] are observed high in shallow and intermediate depth wells (<150 m). The most probable number based on the Colilert test and qPCR result for E.coli suggest that unprotected surface waters are harbingers for high microbial population compared to hand pumped wells. However, the very low observed concentrations of cultured E. coli (<1-10 MPN/100 mL) and E. coli DNA (<40 Copies/100 mL) in the wells indicates that the abundance of E.coli cells decrease rapidly with residence time in oligotrophic aquifers. Thus, it may be suggested that more humic DOC in shallow and intermediate groundwaters may be involved in complexation or other biogeochemical reactions that may mobilize As in groundwater. The non-indigenous bacteria can be the primary producers of DOC in the aquifers which can be utilizing surface derived DOC.