Soupir, Michelle Lynn2014-03-142014-03-142007-12-10etd-04012008-162954http://hdl.handle.net/10919/26581The U.S. EPA has identified pathogen indicators as a leading cause of impairments in rivers and streams in the U.S. Elevated levels of bacteria in streams draining the agricultural watersheds cause concern because they indicate the potential presence of pathogenic organisms. Limited understanding of how bacteria survive in the environment and are released from fecal matter and transported along overland flow pathways results in high uncertainty in the design and selection of appropriate best management practices (BMPs) and in the bacterial fate and transport models used to identify sources of pathogens. The overall goal of this study was to improve understanding of the fate and transport mechanisms of two pathogen indicators, E. coli and enterococci, from grazed pasturelands. This goal was addressed by monitoring pathogen indicator concentrations in fresh fecal deposits for an extended period of time. Transport mechanisms of pathogen indicators were examined by developing a method to partition between the attached and unattached phases and then applying this method to analyze runoff samples collected from small box plots and large transport plots. The box plot experiments examined the partitioning of pathogen indicators in runoff from three different soil types while the transport plot experiments examined partitioning at the edge-of-the-field from well-managed and poorly-managed pasturelands. A variety of techniques have been previously used to assess bacterial attachment to particulates including filtration, fractional filtration and centrifugation. In addition, a variety of chemical and physical dispersion techniques are employed to release attached and bioflocculated cells from particulates. This research developed and validated an easy-to-replicate laboratory procedure for separation of unattached from attached E. coli with the ability to identify particle sizes to which indicators preferentially attach. Testing of physical and chemical dispersion techniques identified a hand shaker treatment for 10 minutes followed by dilutions in 1,000 mg L-1 of Tween-85 as increasing total E. coli concentrations by 31% (P value = 0.0028) and enterococci concentrations by 17% (P value = 0.3425) when compared to a control. Separation of the unattached and attached fractions was achieved by fractional filtration followed by centrifugation. Samples receiving the filtration and centrifugation treatments did not produce statistically different E. coli (P value = 0.97) or enterococci (P value = 0.83) concentrations when compared to a control, indicating that damage was not inflicted upon the cells during the separation procedure. In-field monitoring of E. coli and enterococci re-growth and decay patterns in cowpats applied to pasturelands was conducted during the spring, summer, fall and winter seasons. First order approximations were used to determine die-off rate coefficients and decimal reduction times (D-values). Higher order approximations and weather parameters were evaluated by multiple regression analysis to identify environmental parameters impacting in-field E. coli and enterococci decay. First order kinetics approximated E. coli and enterococci decay rates with regression coefficients ranging from 0.70 to 0.90. Die-off rate constants were greatest in cowpats applied to pasture during late winter and monitored into summer months for E. coli (k = 0.0995 d-1) and applied to the field during the summer and monitored until December for enterococci (k = 0.0978 d-1). Decay rates were lowest in cowpats applied to the pasture during the fall and monitored over the winter (k = 0.0581 d-1 for E. coli and k = 0.0557 d-1 for enterococci). Higher order approximations and the addition of weather variables improved regression coefficients (R2) to values ranging from 0.81 to 0.97. Statistically significant variables used in the models for predicting bacterial decay included temperature, solar radiation, rainfall and relative humidity. Attachment of E. coli and enterococci to particulates present in runoff from highly erodible soils was evaluated through the application of rainfall to small box plots containing different soil types. Partitioning varied by indicator and by soil type. In general, enterococci had a higher percent attached to the silty loam (49%) and silty clay loam (43%) soils while E. coli had a higher percent attached to the loamy fine sand soils (43%). At least 50% of all attached E. coli and enterococci were associated with sediment and organic particles ranging from 8 – 62 μm in diameter. Much lower attachment rates were observed from runoff samples collected at the edge-of-the-field, regardless of pastureland management strategy. On average, 4.8% of E. coli and 13% of enterococci were attached to particulates in runoff from well-managed pasturelands. A second transport plot study found that on average only 0.06% of E. coli PC and 0.98% of enterococci were attached to particulates in runoff from well-managed pasturelands, but percent attachment increased slightly in runoff from poorly-managed pasture with 2.8% of E. coli and 1.23% of enterococci attached to particulates. Equations to predict E. coli and enterococci loading rates in the attached and unattached forms as a function of total suspended solids (TSS), phosphorous and organic carbon loading rates appeared to be a promising tool for improving prediction of bacterial loading rates from grazed pasturelands (R2 values ranged from 0.61 to 0.99). This study provides field-based seasonal die-off rate coefficients and higher order approximations to improve predictions of indicator re-growth and decay patterns. The transport studies provide partitioning coefficients that can be implemented into NPS models to improve predictions of bacterial concentrations in surface waters and regression equations to predict bacterial partitioning and loading based on TSS and nutrient data. Best management practices to reduce bacterial loadings to the edge-of-the-field from pasturelands (regardless of management strategy) should focus on retention of pathogen indicators moving through overland flow pathways in the unattached state. Settling of particulates prior to release of runoff to surface waters might be an appropriate method of reducing bacterial loadings by as much as 50% from highly erodible soils.In CopyrightnutrientsWater qualitydie-offpasturelandmicrobial partitioningenterococciE. coliDissertationhttp://scholar.lib.vt.edu/theses/available/etd-04012008-162954/