ASSESSMENT OF THE MICROBIAL AND CHEMICAL WATER QUALITY OF INDIVIDUAL AND SMALL SYSTEM GROUNDWATER SUPPLIES IN ARIZONA Roberto Marrero-Ortiz, Ph.D. ABSTRACT Arizona has more non-disinfected drinking water supply systems than any other state in the U.S. All of these systems depend on groundwater that is subject to contamination by waterborne and waterbased pathogens. The goal of this project was to assess the microbial quality of non-disinfected individual and small groundwater drinking water systems in Arizona. In addition, data was collected on the occurrence of heavy metals. The microbiological parameters tested included total and fecal coliforms, enterococci, heterotrophic bacteria, Helicobacter pylori, Aeromonas hydrophila, enteric viruses, and somatic and male specific coliphages. Physical parameters tested included pH, turbidity, total dissolved solids (TDS) and temperature. Chemical parameters tested included nitrate/nitrogen, total organic carbon (TOC) and metals. Forty-nine groundwater sources were tested in seven Arizona counties. A total of 70 samples were collected over 14 months. Forty-three percent of the systems were positive for total coliforms, 16% for fecal coliforms and 4% for E. coli. Twenty-nine percent of the wells were positive for enterococci, and 57% were positive for Aeromonas hydrophila. H. pylori, norovirus and enterovirus were not detected by direct PCR in any samples. At least one primary and/or secondary Drinking Water Standard (DWS) was exceeded for heavy metals in all samples. Metals which exceeded standards included arsenic, aluminum, iron, lead, manganese, molybdenum, selenium, and uranium. Seventy-four percent of the sites sampled exceeded at least one of the primary DWS, 80% exceeded at least one secondary DWS, and 95% exceeded one primary and/or one secondary DWS.

In Mexico, wastewater recycling is an important alternative source of water, particularly in arid regions like the state of Sonora, Mexico. In El Valle del Yaqui, Sonora, Mexico, where 500 million m3 per year of wastewater is available for recycling in agriculture activities . The main objective of the three studies presented in this dissertation was to assess the microbial water quality of surface water, untreated wastewater, and treated wastewater for produce irrigation, as well as the impact of microbial water quality on vegetable contamination. The results suggest that the three sources of water meet Mexican and international guidelines for use in production of food crops.Despite wide differences in the concentration of bacterial indicators (Escherichia coli, Clostridium perfringens) and enteric pathogens (Cryptosporidium, Giardia, and enteroviruses) in the various types of water studied there was little impact on contamination of the produce studied (carrots, lettuce, tomatoes, and peppers) at harvest. Apparently, the time between the last irrigation event and harvesting was sufficient to allow for die-off of enteric organisms that may have contaminated the produce.

Escherichia coli is a bacterial species that lives in the gut of all warm-blooded animals, fish, birds as well as reptiles and is commonly used as an indicator of fecal contamination in water. This project assessed currently used culture based media for the detection of E. coli in irrigation waters used in Arizona and California, and will present recommendations towards the most reliable media for the evaluation of irrigation waters used for produce. Currently, no microbial indicator standards exist for irrigation waters used for produce production in the United States. The produce industry suggests that the recreational water standard guideline (126 E. coli/100 ml) established by the United States Environmental Protection Agency (USEPA) be used. There is concern that the false positive rate of E. coli detection may be high in these waters giving false indications of the level of risk from enteric pathogens. This project evaluated three commercially available media for E. coli detection to test irrigation waters from three agricultural areas (Yuma and Maricopa, AZ and Imperial Valley, CA) and then assessed false positive rates by utilizing Polymerase Chain Reaction (PCR) and DNA sequencing of the bacterial isolates. The media that were chosen for evaluation were (1) MI Agar, (2) IDEXX Colilert Quanti-Tray® and (3) m-ColiBlue24® broth, all evaluation media accepted by the USEPA and widely used in the monitoring of irrigation water quality by the produce industry. Four hundred and fifty 1-L irrigation water grab samples were collected between March 2012 and November 2012. The samples were analyzed for both cultural counts and water quality parameters including temperature, salinity and pH. Isolates positive and negative for E. coli were then selected and assessed utilizing PCR and DNA sequencing. The false positive rate of each method was found to be high, with MI Agar, m-ColiBlue24® broth and the IDEXX Colilert Quanti-Tray® at an accuracy of 67%, 72%, and 51% respectively. A false positive result is reported when presumptive E. coli sub cultured from the media is found to be non-E. coli through molecular analysis. Overall the IDEXX Colilert Quanti-Tray® performed at a greater rate of accuracy than the other two media evaluated, however, high false positive rates may lead to inaccurate assessment of water quality.

As aquaculture has become a significant provider of the human diet, the interest to have better quality of sea and fresh products has been increasing. However the potential hazards associated with pathogenic agents resulting in losses to the industry are major concerns that provided the motivation for this study.The use of ultraviolet irradiation is an alternative to disinfect water in inlet and outlet water sources. However the ultraviolet disinfection method has some drawbacks including no disinfectant residuals and high cost of lamp fouling and replacement. The ultraviolet system needs to be calibrated according with the life time of the ultraviolet lamps.The MS-2 coliphage in this study is an approach to determine a good indicator for determining if an ultraviolet system can be effective in an aquaculture recirculation system. The susceptibility of this system can provide an indication if WSSV can be inactivated and possible other pathogenic agents.The WSSV experiment was successful in reducing mortality. Further studies have to be completed and analyzed before recommending for control of other pathogens.

Mining activities and their resulting wastes, mine tailings, have created a sizable problem globally. Semiarid lands have been particularly impacted due to intense mining activities in these areas. Growing concerns regarding human health risks and environmental consequences associated with these tailings has created a need for efficient and effective remediation strategies. Phytostabilization, the establishment of a vegetative cover on mine tailings to reduce erosion and dispersion of material, is emerging as a cost-effective remediation technology. However, due to elevated levels of metal contaminants, acidic pH values and poor substrate quality many tailings sites are inhospitable to plant growth. The addition of compost amendments can mitigate the toxic effects of tailings material and facilitate plant growth; however, in many instances the necessary compost amendments may be cost prohibitive. The use of specialized bacterial isolates, known as plant growth-promoting bacteria (PGPB), to enhance plant growth is a developing technology that has a broad range of applications. The use of PGPB to enhance one or more aspect of plant establishment and growth has been demonstrated to be effective in hundreds of previous studies conducted primarily under agricultural settings. To date, very few studies have utilized PGPB in attempts to enhance plant growth in mine tailings. The current study is an investigation into the potential for utilizing PGPB to enhance plant growth during the phytostabilization of semiarid mine tailings. During this investigation a large collection of bacterial isolates was screened for common plant growth-promoting mechanisms such as siderophore and indole-3-acetic acid production, phosphate solubilization and ACC-deaminase activity. Isolates possessing beneficial qualities were utilized in a series of greenhouse screening studies to evaluate their abilities to enhance the growth of native desert plants in various tailings materials. A number of isolates tested have demonstrated the ability to enhance plant growth in composted and non-composted tailings material. Optimization of this technology has now indicated that alginate-encapsulated inoculation of target plants is a beneficial and practical technology.

It has been demonstrated the technical feasibility of using seawater and other saline water for irrigation. Through the use of saline water for irrigation, highly salt-tolerant crops could greatly increase global agriculture. Brackish water and seawater from different sources are available in areas suitable for production of salt-tolerant crops. Dwarf glasswort Salicornia bigelovii Torr. (Chenopodiaceae), is a leafless, succulent, small-seeded, annual saltmarsh plant, with potential as a saline water crop. It is also a potential oilseed, forage, biomass crop, and a promising carbon sequestration plant. In the first chapter of this document we describe a study where we grew Salicornia bigelovii from seedlings, in saline, drying soils in a greenhouse experiment. The effects of drought and salinity stress were additive. Optimal growth and water use efficiency coincided at 0.35-0.53 M NaCl. The plants were tolerant of high salinity but exhibited little drought tolerance. Salicornia bigelovii plants varied little in their uptake of Na+ for osmotic adjustment, with final Na+ contents of 18% on a dry mass basis. Both growth and water use efficiency of Salicornia bigelovii were affected by salinity. Also, Na+, the primary cation involved in osmotic adjustment of this species, apparently stimulates growth by mechanisms apart from its role as an osmoticum. In the second chapter of this dissertation we developed a research study where we evaluated the production and osmotic adjustment of two S. bigelovii lines (Texas and Florida), plants were grown in pot in a green house and irrigated with water treated with three different levels of NaCl (5 ppt, 15 ppt and 30 ppt) combined with inorganic fertilizer. At the end of the experiment sixty plants from each line were measured for height, biomass, seed yield, seed size, dry matter yield, and tissue osmolarity. There was no significant difference among groups in plant height, or final biomass either in salinity irrigation treatments, or S. bigelovii lines. Tissue osmolarity differed among salinity treatments but not among S. bigelovii lines. The highest tissue osmolarity value was 1192 mM kg-1 found at the treatment 30ppt in the Florida line. Total biomass production was 12 000 kg/Ha.

As of 2010, there are approximately twenty one surface water locations classified as impaired for Escherichia coli (E. coli) contamination in the State of Arizona. Of note is the San Francisco River (SFR) which is currently listed on the US EPA 303d list of impaired waters due to E. coli bacteria present at higher concentrations than the US EPA standards for partial- and full-body contact. In 2010-2011 surface water samples were collected at sites within the impaired region to monitor E. coli and areas known for heavy recreational uses. Of 70 samples collected over 1 year, 81% were positive for universal Bacteroides marker (Allbac). Of the 57 Allbac-positive samples, 68% show contributions of the human-specific marker and 60% were positive for bovine-specific marker. While 28% of the total samples assayed showed elevated levels of E. coli (>235 MPN/100mL), there were minimal significant correlations between Bacteroides and generic E. coli across all samples. While this information is significant, past research has suggested that successfully distinguishing the sources of fecal contamination will not alone reduce or eliminate disease associated with contaminated water unless these investigations are coupled with public outreach and education. With this in mind a survey was developed to gather information about water quality perceptions, water use, peoples' attitudes, knowledge, and behaviors related to the water resources in Clifton, AZ. Survey questions consisted of multiple choice and Likert scales questions and were provided in both English and Spanish and were conducted during the summer of 2012 and winter of 2013. A total of 150 surveys were deployed with 38 surveys completed for a response rate of 25%. Our study findings indicate mixed attitudes on water quality with 80% reporting the SFR has poor water quality for drinking and 39% agree the SFR has poor water quality for swimming. Yet, 84% consider the river safe enough for picnics and activities near the water. Also, it was interesting to note participants' opinions regarding consequences of poor water quality with 66% of respondents indicating that they are concerned with poor water quality and their health. Clifton is a very tight knit community so it was not unexpected that the majority of the respondents (61%) get water quality information by having conversations with other people and 68% from newspapers, factsheets and brochures. Based on the survey responses, our team worked to develop two peer reviewed Extension publications entitled; Microbial Source Tracking: Watershed Characterization and Source Identification (Arizona Cooperative Extension, #AZ1547) and Water Quality, E. coli, and Your Health (#AZ1624). Publications have been developed in both English and Spanish and will be part of future outreach to this and other Arizona communities. It is our goal that these survey findings can be used to better tailor outputs appropriate for the targeted audience, namely the local Hispanic population. These results are important because they add to understanding perceptions of water quality and health risks in this rural community; and can lend towards enhanced outreach practices in other similar communities.

Utilization of areas adjacent to rivers for agricultural and industrial purposes can have detrimental effects on water quality and can potentially impact human and ecosystem health downstream. In this study we tested water quality along a stretch of the effluent-dependent Santa Cruz River near Nogales, AZ. This stretch of river has historically been impaired, but prior to upgrades to the Nogales International Wastewater Treatment Plant (NIWTP) in 2009. Our work endeavored to assess water quality according to the Arizona Department of Environmental Quality (ADEQ) standards, and identify sources of pollution entering the river system. Heavy metals were analyzed via ICP. Three IDEXX quantification systems were used to detect E. coli, Enterococcus, and P. aeruginosa as fecal indicators or potential threats to public health. Potential fecal sources were analyzed using quantitative PCR targeting Bacteroides DNA markers for total, human, and bovine feces (Allbac, HF183, and CowM2, respectively). The NIWTP effectively removed chemical and biological contaminants. The lowest E. coli counts occurred at the site located at the NIWTP outfall (mean = 5 MPN/100ml) while the highest counts (mean = 348 MPN/100 ml) came from Nogales Wash, a tributary receiving untreated flow from Nogales, Mexico. The Allbac marker was detected in all samples, while approximately 97% of samples tested positive for HF183 and 33% tested positive for the CowM2 marker. Continued monitoring of effluent effects on river quality and evaluation of water quality degradation will lead to improvements in the management of Arizona's riparian areas and will ultimately contribute to healthy water bodies.

Annual application of N at rates of 118 (118N), 236 (236N), and 354 (354N) kg•ha⁻¹ on 35 year old 'Western Schley' pecan trees during four years had little effect on mineral composition and foliar N. No differences in yield, nut quality, and reproductive characteristics were found. Alternate bearing intensity in four consecutive years was 37, 33 and 28% in 118N, 236N, and 354N, respectively, with a significant linear response. Rates from 118 to 236 kg N•ha⁻¹ satisfy N needs for pecan in irrigated pecan orchard of the southwest of United States. The effect of one-time banding of zinc sulfate (74 kg Zn•ha⁻¹) and zinc-EDTA (19 kg Zn•ha⁻¹) was evaluated over a period of four years on ‘Wichita’ pecans growing in alkaline soil. Significant differences in foliar Zn levels were found one month after application of Zinc-EDTA. Differences also were noted during the next three years on approximately 25% of the sampling dates. Yield, leaflet area, and trunk cross sectional area were not affected. Zinc-EDTA increased Zn uptake by 'Wichita' pecan trees in alkaline conditions during three years. A field study indicated that manure or manure plus Zn increased foliar Zn levels in pecans after two years of annual applications. Manure (24 ton ha⁻¹) plus zinc sulfate (258 kg Zn•ha⁻¹ as zinc sulfate) treatment had the highest foliar Zn levels. No differences were observed in trunk growth, leaf area, leaf weight, nut filling, and yield. Manganese toxicity symptoms are exhibited when leaf Mn levels are higher than 1700 μg•g⁻¹ during the standard date sampling of July and affected reproductive characteristics and leaf and shoot growth. More severe visible symptoms include delayed budbreak and die-back of young shoots. In potted pecan trees, zinc EDTA treated trees had a foliar Zn of 244 μg•g⁻¹, in foliar sprayed trees (eight foliar sprays of a combination of zinc sulfate and UAN32) had 140 μg•g⁻¹, and in control trees had 33 μg•g⁻¹. Soil adsorption isotherms showed that of the three fertilizers evaluated Zn sulfate was adsorbed most strongly by the soil (1.5 mg Zn•g⁻¹ of soil). Soil adsorption from Zn EDTA solutions was insignificant.

Waterborne enteric viruses are transmitted via the fecal-oral route and have been isolated from various types of water ranging from sewage to tap water. Water matrices characterized by low levels of organic material (e.g. clean surface water and tap water) contain fewer numbers of viruses than sewage and wastewater effluents. A number of methods have been developed to concentrate, elute (recover), and re-concentrate viruses from water. The goal of this dissertation is two-fold. An extensive review of the literature is provided in Appendix A that focuses on method development in the three aforementioned areas. A review of this detail has not been conducted in over two decades, and as such will contribute to the fields of water quality and environmental virology. Second, a novel and inexpensive method for the concentration of viruses (MS2 coliphage, poliovirus 1, echovirus 1, Coxsackievirus B5, and adenovirus 2) is presented in Appendix B. The method uses a new electropositive filter (comprised of nanoalumina fibers) for the capture of viruses from 20-L volumes of dechlorinated tap water. Average filter retention efficiencies for each of the viruses was ≥ 99%. Viruses that are adsorbed to filters must then be recovered (eluted). A number of inorganic solutions were evaluated for this purpose, the most effective being a moderately alkaline (pH 9.3) glycine buffered-polyphosphate solution. Secondary reconcetration of the eluates was performed using an optimized ultrafiltration method (Centricon Plus-70, Millipore, Billerica, MA), and achieved final concentrates volumes of 3.3 ± 0.3 mL. Total method efficiencies meeting the project recovery goal of ≥ 50% were obtained for each of the tested viruses except for MS2 coliphage at high input titers (45 ± 15%) and adenovirus 2 (14 ± 4%). Appendix C provides the Standard Operating Procedures, sample calculations, and detailed data for the experiments conducted. Appendix D details the steps taken towards optimizing the secondary concentration procedure in effort to meet the 50% recovery goal.