Private wells are understudied potential sources of opportunistic pathogen (OP) infections. OPs, including Legionella and Mycobacterium, are of particular concern for immunocompromised individuals and are known to proliferate in drinking water systems. Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems, which primarily use surface water sources and are always treated with primary and secondary disinfection in United States. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems will translate to private well systems with rare and infrequent exposure to chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well field surveys were conducted to document the incidence of OPs in private well systems. One survey conducted in North Carolina private wells with no history of recent flooding was focused on molecular and culture-based detection of Legionella spp. and Legionella pneumophila. The other survey was a broader molecular (i.e., DNA-based) characterization of the incidence of Legionella spp., L. pneumophila, Mycobacterium spp., Mycobacterium avium (the most commonly nontuberculous mycobacteria associated with disease), and Naegleria fowleri in private wells with recent history of flooding (i.e., Hurricanes Harvey and Irma in 2017, or the Great Louisiana Floods in 2016, extending to Texas, Florida, and Louisiana). All samples in both studies were analyzed for total bacterial 16S rRNA genes, indicator bacteria (e.g., total coliform and Escherichia coli) and inorganic constituents. Information about well system characteristics were obtained through questionnaires sent to participating residents. Widespread detection of OP DNA markers were noted in the flooded well survey. Legionella spp. (detectable in 50-100% of well waters, depending on the flood event) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly detected among the OPs targets. At the genus level, L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) were less commonly detected, but still highly variable. It is not possible to judge whether these OP levels were elevated as a result of the flooding because the sampling was carried out as an emergency response and background levels were not previously tested. Also of interest was whether well characteristics could predict OP levels, including well depth, well type, or treatment. However, none of these emerged as significant predictors of OP detection frequency or levels. Similarly, these OP DNA markers were not elevated in homes reporting submerged wellheads or system damage, suggesting that detection of these OPs is more dependent on the groundwater that supplies these private wells than influx of contaminated surface water. The incidence of DNA markers pertaining to N. fowleri, the "brain eating amoeba" that causes rare incidences of primary amebic meningoencephalitis (PAM), tended to be lower (5.0-12.7%) than that of other OPs targeted, but was more frequently detected in wells reporting submerged well heads, suggesting its occurrence was related to contamination from flood water. A positive association between total bacteria and occurrence of both Legionella spp. and Mycobacterium spp., was observed in private wells of all surveyed areas, contrary to observations in municipal drinking water systems with secondary disinfectant residuals. On the other hand, Legionella reportedly has an optimal growth range of 20-42 °C in municipal systems and recent surveys of municipal systems reported a strong association between Legionella spp. and temperature that was not observed for private wells in this study. We speculate that the essentially "infinite" water age and lack of disinfectant for well water, may contribute to these differences relative to municipal water supplies. The results presented in this work are likely an overestimation of OPs numbers in private wells, as molecular detection of OPs does not distinguish between live and dead cells. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables impacting the occurrence of OPs in private wells, given that our study shows that these pathogens are relatively common, might someday help limit the risk of infections. / Master of Science / Non-fecal pathogens that are capable of growth in drinking water systems and causing illness primarily in individuals with compromised immune systems are referred to as opportunistic pathogens (OPs). OPs of concern in drinking water systems in the United States include members of the Legionella genus (i.e., Legionella spp.) and Mycobacterium genus (i.e., Mycobacterium spp.), as well as Naegleria fowleri (also known as “the brain-eating amoeba”). Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems. Under the right conditions, OPs have the ability to grow in municipal drinking water systems and in building plumbing systems despite the use of disinfectants. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems translates to private well systems that rarely utilize chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well surveys were conducted to document the incidence of OP DNA markers and culturable OPs in private well systems. The first survey was conducted in North Carolina private wells with no history of recent flooding and focused on quantification of DNA markers for Legionella spp. and L. pneumophila, as well as culturable L. pneumophila. The second survey was conducted in flood-impacted private wells in vi Texas, Florida, and Louisiana following Hurricanes Harvey and Irma in 2017 and the Louisiana Floods of 2016, quantifying five DNA markers for OPs (Legionella spp., L. pneumophila, Mycobacterium spp., M. avium, and N. fowleri). All water samples in both studies were also analyzed for total bacterial numbers (i.e., total number of copies of a gene present in all bacteria), certain bacteria that indicate environmental and fecal contamination (e.g. total coliform bacteria and Escherichia coli), and inorganics. Information about well system characteristics was obtained through questionnaires sent to participating residents. While it was not possible to sample before the flooding events and determine the background detection rates, this survey found that detection of the target OPs in the flood-impacted wells was widespread, but highly variable. Both culture-based and DNAbased testing methods were used to verify the results because each approach has strengths and weaknesses. Detection of DNA markers indicates the genetic material of the organism being tested for is present and detects DNA of organisms that are both dead and alive. Thus, DNA detection may overestimate the presence live (and infectious) pathogens. Detection of culturable pathogens indicates the organism is alive, but only detects pathogens that readily grow on culture media. There may be other pathogens in the water that do not readily grow on the media. Thus culture may underestimate the occurrence of pathogens. DNA markers for Legionella spp. (detectable in 50-100% of well waters) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly vii detected among the targets in this study. The detection of DNA markers for L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) was less common. There were no private well characteristics, such as well depth, well type, or treatment that emerged as significant predictors of these OP detection or levels. Similarly, these OPs were not elevated in recently flooded homes reporting submerged wellheads or system damage. Thus, detection of these OPs was found to be widespread and sporadic. Detection rates of N. fowleri, which causes rare incidences of primary amebic meningoencephalitis (PAM), DNA was lower (5.0-12.7%) than other OPs, and was also not related to private well characteristics. However, N. fowleri DNA was more frequently detected in wells with wellheads that were submerged due to flooding than in wells with unsubmerged wellheads, as were total coliform bacteria (an indicator of environmental contamination) and total bacterial numbers. This demonstrates concern that submergence of wellheads during flooding is a concern for the introduction of microbial contamination in private wells. This work also explored two trends characteristic of municipal systems that were not observed in these surveys of private wells. First, positive associations between overall bacterial numbers and DNA markers for both Legionella spp. and Mycobacterium spp. were observed in private wells in all surveyed areas. This is contrary to what has been reported for overall bacterial numbers in municipal drinking water systems with a secondary disinfectant residual. Second, Legionella has been known to have an optimal growth range of 32 to 42 °C (90 to 108 °F) in municipal systems and recent surveys of municipal systems with both a free chlorine and chloramine secondary disinfectants viii showed a strong association between Legionella spp. and temperature. These associations were not observed in private wells in this work. Continuous disinfection treatment in municipal drinking water systems may eliminate certain microbes, lowering overall levels of bacteria, while OPs may persist by resisting disinfection, resulting in no correlation between total bacteria and OPs. Private wells do not generally use continuous disinfection and represent low-nutrient environments where naturally occurring OPs can grow alongside other bacteria. The results of this study are likely an overestimation of OPs infection risk associated with private wells, as infection can only be caused by live OPs, which cannot be directly determined by measuring DNA markers. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables that impact the occurrence of OPs in private wells is necessary to minimize the risk of associated infections linked to private wells. This work provides strong preliminary evidence that OP occurrence in private wells is relatively commonplace. Science-based options for at-risk (e.g., immunocompromised) individuals to decrease their exposure to OPs in private well water need to be developed.

6 pp. / 1. Drinking Water Wells; 2. Private Water Well Components; 3. Do Deeper Wells Mean Better Water; 4. Maintaining Your Private Well Water System; 5. Private Well Protection; 6. Well Water Testing and Understanding the Results; 7. Obtaining a Water Sample for Bacterial Analysis; 8. Microorganisms in Private Water Wells; 9. Lead in Private Water Wells; 10. Nitrate in Private Water Wells; 11.Arsenic in Private Water Wells; 12. Matching Drinking Water Quality Problems to Treatment Methods; 13. Commonly Available Home Water Treatment Systems; 14. Hard Water: To Soften or Not to Soften; 15. Shock Chlorination of Private Water Wells / This fact sheet is one in a series of fifteen for private water well owners. The one- to four-page fact sheets will be assembled into a two-pocket folder entitled Private Well Owners Guide. The titles will also be a part of the Changing Rural Landscapes project whose goal is to educate exurban, small acreage residents. The authors have made every effort to align the fact sheets with the proposed Arizona Cooperative Extension booklet An Arizona Well Owners Guide to Water Sources, Quality, Sources, Testing, Treatment, and Well Maintenance by Artiola and Uhlman. The private well owner project was funded by both the University of Arizonas Water Sustainability Program-Technology and Research Initiative Fund and the USDA-CSREES Region 9 Water Quality Program.

6 pp. / 1. Drinking Water Wells; 2. Private Water Well Components; 3. Do Deeper Wells Mean Better Water; 4. Maintaining Your Private Well Water System; 5. Private Well Protection; 6. Well Water Testing and Understanding the Results; 7. Obtaining a Water Sample for Bacterial Analysis; 8. Microorganisms in Private Water Wells; 9. Lead in Private Water Wells; 10. Nitrate in Private Water Wells; 11.Arsenic in Private Water Wells; 12. Matching Drinking Water Quality Problems to Treatment Methods; 13. Commonly Available Home Water Treatment Systems; 14. Hard Water: To Soften or Not to Soften; 15. Shock Chlorination of Private Water Wells / This fact sheet is one in a series of fifteen for private water well owners. The one- to four-page fact sheets will be assembled into a two-pocket folder entitled Private Well Owners Guide. The titles will also be a part of the Changing Rural Landscapes project whose goal is to educate exurban, small acreage residents. The authors have made every effort to align the fact sheets with the proposed Arizona Cooperative Extension booklet An Arizona Well Owners Guide to Water Sources, Quality, Testing, Treatment, and Well Maintenance by Artiola and Uhlman. The private well owner project was funded by both the University of Arizonas Water Sustainability Program-Technology and Research Initiative Fund and the USDA-CSREES Region 9 Water Quality Program.

Despite claims of nearly 100% access to potable drinking water in the US, issues of drinking water quality, accessibility, and equity persist in many regions of the country. Drinking water is a common health concern in rural communities, where social, geographic, and economic challenges can inhibit the provision of reliable municipal water. Households without access to municipal water often rely on private wells, which are solely the responsibility of the homeowner to test, treat, and maintain, or roadside springs. These water sources often do not employ water treatment and users can therefore be uniquely susceptible to environmental contaminants. The goal of this research was to examine point-of-use (POU) treatment options that can be used by individuals to improve their drinking water quality and reduce exposure to common contaminants prior to consumption. Two drops (~0.10 mL) of unscented, household bleach in one gallon of spring water is a simple, low-cost treatment option that successfully inactivates total coliform and E. coli and provides an appropriate free chlorine residual (> 0.5 mg/L) over a 1-month time period, without exceeding free chlorine taste thresholds (< 2 mg/L). Efforts to distribute information on this disinfection protocol to spring users in southern West Virginia and southwestern Virginia were well-received; however, only 60% of surveyed spring users report that they plan to implement the protocol. POU faucet filters have been successfully implemented in homes reliant on municipal water to reduce metal contaminant levels in drinking water. Few studies have assessed the effectiveness of these filters in improving water quality in homes reliant on private wells. Faucet-mounted POU filters distributed to homes reliant on private wells in Virginia and southern West Virginia statistically significantly lowered levels of Ba, Cd, Cr, Total Coliform, U, Cu, Pb, Al, Fe, Mn, Zn, and Sr in tap water. However, levels of many contaminants of interest still exceeded at least one Safe Drinking Water Act regulation/recommendation in several filtered samples. Additionally, less than half of study participants reported that they liked using the filters with several citing issues with flowrate. Faucet-mounted POU filters can also be a useful tool in assessing exposure to contaminants at the tap. The acid flow-through method of metals recovery has previously proven to be successful in recovering dissolved Pb from dosed filters. In this study, the acid flow-through extraction method was applied to water spiked with high or low levels of Pb, Fe, or Cu. While faucet-mounted activated carbon filters successfully removed Pb and Cu from dosed influent (>91% removal), filter behavior under influent Fe concentrations of greater than 300 ppb was extremely variable. The acid flow-through method of metals extraction provided some recovery from filters dosed with high and low concentrations of Pb (38.9-70.4%). Recovery of Cu and Fe was variable, likely in part due to Fe and Cu leaching from filter media, suggesting that alternative methods of metals extraction and recovery from POU faucet filters dosed with Fe and Cu, or other common water contaminants (e.g., As, Ba, Cd), must be explored. While POU treatment can be useful in improving drinking water quality in rural households, limitations to adoption persist and must be addressed along with efforts to protect drinking water quality in homes in a more permanent, sustainable way. / Doctor of Philosophy / Drinking water quality is a common health concern in rural communities, where social, geographic, and economic challenges can make municipal water quality unreliable. Households without access to municipal water often use private wells and sometimes roadside springs for drinking water. These water sources are often untreated which can expose users to environmental contaminants such as bacteria or metals. The goal of this research was to study point-of-use (POU) treatment options that can be used by individuals looking to improve their drinking water quality and reduce their exposure to common contaminants, perhaps while waiting for more permanent improvements and upgrades. Household bleach is a simple, low-cost way of lowering levels of bacteria in roadside spring water that is being used as drinking water. Two drops of unscented, household bleach in one gallon of spring water successfully kills total coliform and E. coli bacteria and provides an enough leftover chlorine to continue to disinfect the water for 1-month. This information was given to spring users in southern West Virginia and southwestern Virginia and, while most people who provided feedback found the information useful, only 60% of surveyed spring users report that they plan to implement this protocol. Point-of-use faucet filters have been found to successfully reduce metals contaminant levels in drinking water in homes that use municipal water. However, few studies have tested the effectiveness of POU faucet-mounted filters in lowering contaminant levels in water in homes reliant on private wells. Faucet-mounted POU filters given to homes reliant on private wells in Virginia and southern West Virginia lowered levels of many contaminants of interest in tap water, including lead, copper, iron, and total coliform bacteria. However, in some of the filtered samples, levels of many of these contaminants were higher than at least one Safe Drinking Water Act regulation. Less than half of study participants reported that they liked using the filters with several stating that they had issues with low flowrate. Faucet-mounted POU filters can also be a useful tool in better understanding exposure to contaminants at the tap. The acid flow-through method of metals recovery has previously proven to be successful in recovering lead, and other metals, that are collected inside the filters during water treatment. In this study, an extraction method using acid was tested on filters that treated water with high or low levels of lead, iron, or copper. The filters were successful in removing lead and copper from test water, but filters were not as consistently successful in removing iron from test water. The extraction method using acid provided some recovery from filters dosed with high and low concentrations of lead (38.9-70.4%). However, recovery of copper and iron was more inconsistent, suggesting that a different method of metals recovery may be necessary. While POU treatment can be useful in improving drinking water quality in rural households, there are limits to how useful it is in certain situations, such as when treating water with extreme water quality. In order to make sure rural households have access to safe drinking water, these limits need to be addressed and efforts need to be made to figure out a way to protect and supply drinking water in a more permanent way.

Emerging contaminants (ECs) are chemicals such as pharmaceuticals and personal care products that have been detected in various environmental matrices, including drinking water supplies at trace concentrations (ng/L-ug/L or ng/kg-ug/kg). Current wastewater treatment plant technology is largely ineffective at removing ECs. The objectives of this investigation were to: 1) determine the occurrence of ECs in private well water supplies in Montgomery and Roanoke County, VA 2) quantify the concentrations of three ECs in selected private water supplies; 3) examine the relationship between water quality constituents (nitrate, bacteria, pH and total dissolved solids) to EC occurrence in private water supplies; and 4) determine the ability of the MicroEvapTM, a novel wastewater treatment technology, to remove ECs from secondary wastewater effluent. In partnership with the Virginia Household Water Quality Program, 57 private water supplies were sampled and tested for the occurrence of 142 ECs and 43 other water quality constituents. Up to 73 ECs were detected in the sampled private water supplies. Higher numbers of ECs detected in the tested private water supplies were related with nitrate >1 mg/L, total dissolved solids >250 mg/L, and the presence of total coliform bacteria. Results indicate the MicroEvapTM technology had >99% removal effectiveness for all 26 tested ECs from three secondary wastewater effluent. With the increasing detection of ECs in water bodies, it is essential to understand the occurrence of ECs and environmental predictors of EC presence in different water matrices and continue to develop water treatment technology capable of treating wastewater for EC removal. / Master of Science / Emerging contaminants (ECs) are compounds intended to improve human and animal well-being, and include pharmaceuticals, personal care products, and human/veterinary antibiotics. ECs have been frequently detected in water resources worldwide including drinking water. The release of ECs from wastewater treatment plant (WWTP) effluent is their primary route into the environment. The inability of most current wastewater treatment technologies to fully remove ECs necessitates further development of technology that can effectively remove ECs. Emerging contaminants such as pharmaceuticals enter WWTPs because the human body does not fully metabolize the compound and the remainder exits in waste. Private well water is largely unregulated and often untreated and has been relatively less evaluated for EC presence in the literature. The objectives of this study were 1) determine the occurrence of ECs in private well water supplies in Montgomery and Roanoke County, VA 2) quantify the concentrations of three ECs in selected private water supplies; 3) examine the relationship between well age and depth and water quality constituents (nitrate, bacteria, pH and total dissolved solids) to EC occurrence in private water supplies; and 4) determine the ability of the MicroEvap™, a novel wastewater treatment technology, to remove ECs from secondary wastewater effluent. Emerging contaminants were detected in southwest Virginia private well water. Knowing the ECs present in private well water is necessary to allow for eventual human risk assessment of ECs for people consuming the water. The MicroEvap™ was highly effective at EC removal from wastewater with removal rates >99%. The removal of all ECs from wastewater is essential to ensure purified WWTP effluent. The continued detection of ECs and the unknown human health risks from these contaminants in drinking water means ECs are a significant pollution concern that requires continued assessment.

Opportunistic pathogens (OPs) are of special concern for immunocompromised populations and are known to grow in both drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems, with aerosol inhalation and other non-ingestion exposures that are not addressed by existing regulatory frameworks. Factors enabling the growth of OPs in water distribution and premise (i.e., building) plumbing systems distributing reclaimed and other water sources systems are poorly understood especially for the emerging OP, Naegleria fowleri (i.e. brain-eating amoeba). Three phases of investigation were carried out to identify factors that facilitate the growth of OPs in main distribution and premise plumbing systems, with particular attention on reclaimed water systems, aging water mains, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were employed to systematically examine the effects of prior granular activated carbon (GAC) biofiltration of the water; chlorine, chloramines, or no disinfectant, and water ages ranging up to 5 days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city grappling with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may unintentionally select for specific OPs, contrary to expectations and experiences with oligotrophic conditions in potable water systems. While GAC biofiltration was associated with lower total bacteria and Legionella spp. gene markers, there were no apparent benefits in terms of other OPs analyzed. Similarly, disinfectant treatments successful for controlling OPs in potable water were either ineffective or associated with increased levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water examined. In the potable water study, it was possible to recreate conditions associated with growth of N. fowleri in the aged main distribution system from where the water for the experiment was collected; including corroding iron mains, nitrification, and disinfectant decay. While the effects of nitrification could not be confirmed, there was a clear association of iron corrosion with N. fowleri proliferation. The role of iron was explored further in what, to the author's knowledge, was the first study of N. fowleri in private wells. Analysis of 40 wells found correlations between N. fowleri and stagnant iron levels, further supporting the hypothesis that iron corrosion or iron encourages the growth of N. fowleri, and, because wells are not routinely disinfected, not necessarily as a result of promoting disinfectant decay. As this study took place following a major flooding event, it provided insight not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research illuminated several factors influencing the OP growth in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems. / MS / Water borne bacteria that effect the immune systems of the sick, known as opportunistic pathogens (OPs), have become a major heath concern. These organisms are known to grow in drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems yet there are no regulations aimed at prevention. There is also limited knowledge on how premise plumbing and water sources effect the growth, population, and risk of infection by OPs, especially for Naegleria fowleri (i.e. brain-eating amoeba). An investigation was carried out in three parts to determine what influences the growth of OPs in water distribution and household plumbing systems, with particular attention to reclaimed water, municipal drinking water, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were used to examine the effects of granular activated carbon (GAC) biofiltration of the water, disinfectants (chlorine, chloramines, or no disinfectant), and water ages ranging zero to five days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may allow for the growth of specific OPs. While GAC biofiltration was associated with lower total bacteria and Legionella spp., there were no apparent benefits in reducing the presence of other OPs. Similarly, common disinfectant treatments for preventing OPs in drinking water were either ineffective or increased viii levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water. In the drinking water study, conditions were introduced to grow N. fowleri in aged drinking water distribution systems with the additions of corroding iron, nitrification (using nitrifying bacteria), and disinfectant. While the effects of nitrification could not be confirmed, there was a clear relationship between iron corrosion and N. fowleri growth. The role of iron was explored further in what, to the author’s knowledge, was the first study of N. fowleri in private wells. Forty wells were examined and the relationships between N. fowleri and stagnant iron levels supported the hypothesis that iron corrosion or iron increases the growth of N. fowleri. As this study took place following a major flooding event, it provided data not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research highlighted several variables that may allow for the growth of OPs in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems.