Jay Levine

The main objective of the proposed work will be to answer the following research questions: 1-How community-based research can inform best practices for community aqueducts that are impacted by climate change? 2-What are the best science-based indicators communities (rainfall, presence of certain contaminants, etc.) can be trained to implement to identify climate change impacts in their aqueducts? 3-What are the cumulative and short-term health impacts from water pollution on households connected to small water systems in PR? and 4-our main hypothesis is: The negative impact of drinking water service from droughts in community-owned aqueducts contributes to increased bacterial contaminant presence in the home water distribution system and has cumulative health impacts. The expected results are based upon pilot work conducted in participating communities where drought has affected drinking water sources and quality. Through community engagement methods, data collection on rainfall, climate, water quality, and presence of bacteria will be conducted. A survey instrument with questions on human health, mental health, water surface and system infrastructure will be conducted with participants. Results will be presented and discussed with water operators, community leaders, members and other stakeholders to determine the best community-led, but science-based solutions appropriate for each community to mitigate health impacts of drought. It is expected that factors such as number of people served, financial status, level of rainfall, climate modeling projections, source of water, treatment of water and source of power (electricity vs. gravity) among others will impact the solutions developed. Expected outputs and outcomes are educational materials for homeowners, resources developed for water operator, science guided, community generated solutions to assess risk and reduce contamination, increased regional coordination of water operators to obtain more affordable pricing on supplies and infrastructure repairs. Improved compliance with EPA regulations on safe drinking water is an expected outcome.

Project Summary: Effective riparian vegetative buffers and wetlands are carbon sinks, minimize nutrient input, soil erosion and related runoff into adjacent surface waters. They are an essential component of livestock environmental resource management and mitigate the movement of nitrogenous and fecal waste from livestock operations and manure management fields into waterways. Watersheds in the coastal plain of North Carolina include a mixture of homes, businesses, livestock operations and other forms of agriculture. Each is a potential source of nutrient and fecal waste in surface waters. Watershed contamination with nutrients or fecal waste are traditionally considered to be non-point sources of contamination. However, all fecal waste has a vertebrate animal origin, and the species of origin varies with adjacent land-use practices. All vertebrates release cells from their gastrointestinal tract in their feces. These cells contain mitochondrial DNA (mtDNA), a routine aspect of forensic investigation that can be applied to identify the animal hosts associated with fecal waste. An mtDNA-based assay we have developed can now specifically attribute the source of fecal waste to humans, livestock (cattle, pigs, poultry, goats), companion animals (dog, cat) and wildlife (white-tailed deer and Canada goose). We propose a comprehensive cross-sectional study to identify locations contaminated by nitrate and fecal waste in mixed use livestock intensive areas of the lower Neuse and Cape Fear watersheds. The study will be conducted with the aim of identifying the presence of fecal contamination and attributing the source of fecal contamination to their species of origin. Water grab samples will be screened for Enterococcus spp. as an indicator of fecal contamination. Samples from positive locations will then be tested for vertebrate mtDNA to attribute the contamination to specific species. Additional sampling at positive sites will assess fecal waste input during storm events. Riparian areas adjacent to waterways testing positive will be visualized using satellite imagery to identify proximity to potential sources of contamination with the identified species. Samples will be assessed for the potential correlation of fecal sources with ammonium, nitrate, chloride, silicate, phosphate, dissolved organic nitrogen and dissolved organic nitrogen. We will conduct community design charettes with cooperating town environmental management personnel and residents, in cooperation with local extension agents and resource managers to identify opportunities for riparian vegetative buffer or wetlands enhancement. Specific objectives include: 1) Identifying locations in agricultural livestock intensive areas where water quality is being degraded by nitrogenous and fecal waste; 2) Identifying riparian locations that will benefit from buffer or wetlands development or refinement; and 3) Working with community stakeholders to develop a plan for buffer and wetland development or refinement. The studies reflect the ecosystem health-oriented objectives of the Environmental Enhancement Grant (EEG) program by facilitating efforts to identify locations that would benefit from either the introduction of vegetative buffers or wetlands or the potential refinement or restoration of existing buffers or wetlands.

The overall goal of the proposed project is to increase awareness of the social determinants of health and how the built environment, environmental health, and equity are linked to public health and the quality of life. The headwaters of the Richland Creek watershed are located within US Census tract 143. (Map Sent to Email.) The Southwest Renewal Foundation pledges a $50,000 cash match for a $50,000 EJDF grant to implement a project with two specific objectives: 1) Identifying the source of fecal waste entering Richland Creek; and 2) Increasing community awareness about the interconnectivity between environmental health, the quality of life, human health and well-being. Elevated levels of fecal coliforms above the EPA bacterial TMDL standard have consistently been detected in the headwaters of Richland Creek. The creek is at the heart of the urban West Green Drive community. There are no agricultural livestock operations in the area, and we believe that the fecal waste is potentially associated with aging municipal sewer infrastructure that was constructed during the 1920s. Also, inadequate stormwater management on W. Green causes severe flooding that flushes surface runoff and trash into the creek. In support of the first objective, we will work with our partners at NC State University to document the presence of the contamination and attribute the source of the fecal waste to either a human, companion animal or wildlife origin. Initial sampling will be conducted with grab samples collected at 12 locations within the tributaries of Richland Creek (Figure 1). An Enterococcus spp. assay will be used to identify the presence or absence of fecal waste. If fecal waste is detected a second assay will be used to attribute the source of the fecal waste to a human, companion animal or wildlife origin. At two locations where fecal waste is detected we will place an automated sampling device prior to anticipated storm events. Water samples will be collected at two- hour intervals throughout two storms. These samples will be tested for fecal waste to attribute the waste to a specific species of origin.

The southeastern United States is the richest region of global diversity for freshwater mussels, snails, fish, and crayfish, and is, therefore, a region of high conservation priority. However, this high regional biodiversity intersects with intense pressures of urban and suburban development and sprawl, increasingly intensive agricultural practices, and growing demands on water and other natural resources for human use. Nestled within this complex landscape, and falling within this rich faunal province, North Carolina contains streams that drain to the Interior Basin (Tennessee ?????????????????? Cumberland) in the west and to the Atlantic Ocean (Atlantic Slope) in the Piedmont and East. The species of freshwater mussels (Unionoida), snails, and fish vary among these regions of the state, face differing landscape and water quality challenges, and, therefore, have differing statuses of conservation concern. For example, the North Carolina Wildlife Resources Commission and the North Carolina Natural Heritage Program recognize approximately 50 species of freshwater mussels native to North Carolina and identifies 31 (62%) as Species of Greatest Conservation Need in the Wildlife Action Plan. The proposed research will specifically benefit these species and contains tasks related to their captive propagation and culture for restoration and recovery.

Livestock operations generate fecal waste and manure management is an essential aspect of livestock production. Local and state regulations mandate permitting, training, design specifications, and stream vegetation buffers between livestock operations and surface waters. The scale of pork production has increased to meet consumer demand and as production facilities have grown, communities have heightened their concern about the environmental impact of pork operations. Pork producers have worked effectively to reduce their overall water usage, land use, and the carbon footprint of farming operations (National Pork Board, 2018, Thoma et al. 2011). Responsible environmental farm management has become a business necessity for pork producers in the US and producers have affirmed their obligation to manage pork production operations in a manner that protects natural ecosystems and public health. Watersheds, however, generally support multiple types of livestock operations and human dwellings. Each livestock enterprise and residential community is a potential source of fecal waste in surface waters. Fecal waste in surface waters is generally referred to as non-point source contamination. In reality, all fecal waste has a vertebrate animal origin and the species of origin varies with adjacent land-use practices. Monitoring programs established to protect public health have traditionally relied on the culture or detection of fecal coliforms, total coliforms or Enterococcus bacteria in water samples as indicators of fecal contamination in surface waters. These enteric organisms are non-specific indicators of the presence of fecal waste but do not attribute contamination to specific animal hosts. The detection of host-specific enteric organisms, such as Bacteroidales spp. and genetic assays focused on detecting these microbial species have been developed as alternatives to non-host specific indicator organism detection methods (Harwood et al. 2009). All vertebrates release cells from their gastrointestinal tract in their feces. These cells contain mitochondrial DNA (mtDNA), a routine aspect of forensic investigation that can be applied to identify the animal hosts associated with fecal waste (Caldwell et al. 2007). The detection of mtDNA is highly host specific. If mtDNA is detected in a water sample, the vertebrate animal associated with that mtDNA can be determined. Initial studies, however, lacked sensitivity (Caldwell et al. 2009). When we initially developed and tested these assays, at times, fecal waste was present in a stream but not detected. In studies supported by the National Pork Board, we refined these initial mtDNA assays by adapting the use of new Droplet digital PCR technology (BioRad Inc., California, USA), which markedly enhanced the sensitivity of the assay for identifying the presence of host mtDNA in surface waters. New primers and probes were designed, and the assay proved both sensitive and specific. Our studies confirmed the presence of fecal contamination in Stockinghead Creek in Duplin County, NC and documented that the fecal contamination in the creek originates from at least four species, cattle, humans, poultry and swine. This proposal focuses on addressing concerns about the origin of fecal waste in surface waters in other North Carolina livestock intensive watersheds. Specific objectives include: 1) Attributing the source of fecal contamination in NC surface waters in Duplin and Sampson County, NC watersheds ; and 2) Responding to concerns about fecal contamination.

The southeastern United States (US) is the richest region of global diversity for freshwater mussel, snail, fish, and crayfish, and is, therefore, a region of high conservation priority. However, this high regional biodiversity intersects with intense pressures of energy mining and development, urbanization and sprawl, increasingly intensive agricultural practices, and growing demands on water and other natural resources for human use. Nestled within this complex landscape, and falling within this rich faunal province, North Carolina contains streams that drain to the Interior Basin (Tennessee ?????????????????? Cumberland) in the west and to the Atlantic Ocean (Atlantic Slope) in the Piedmont and East. The species of freshwater mussels (Unionoida), snails, and fish vary among these regions of the state, face differing landscape and water quality challenges, and, therefore, have differing statues of conservation concern. For example, North Carolina once supported more than 60 species of freshwater mussels, but unfortunately, 50% of these species are now designated as Endangered, Threatened, or of Special Concern and the state??????????????????s 161 freshwater fish of conservation concern are also likely integral to the unique unionoid mussel life cycle, serving as obligate hosts during the mussels?????????????????? parasitic larval stage. Because of these declines and degree of imperilment, protection, restoration, and conservation of these irreplaceable aquatic organisms are paramount. The proposed research will specifically benefit these imperiled mollusks and non-game fishes and contains objectives related to their captive propagation and culture, improvement of their water quality and riparian environment, and better understanding of their ecosystem function and services. The specific tasks include: (1) propagation and culture of the federally endangered Dwarf Wedgemussel, the at-risk Yellow Lance mussel, and the at-risk Magnificent Ramshorn snail; (2) understanding the ecosystem functions and services provided by native freshwater mussels and their associated economic and social benefits to humans and other wildlife; (3) determining the effects of transportation and energy production stressors on the survival, health and well-being of native freshwater mussels; and (4) assessing the integrated risk, ecology, and control of Giant Lyngbya (an invasive Cyanobacteria species) on native mussels. These collective projects will provide natural resource managers and other decision makers with the tools, organisms, and science-based information needed to restore, improve, and conserve these important faunal resources.

All livestock operations generate fecal waste and manure management is an essential aspect of pork production. Regulations mandate permitting, training, design specifications, and stream vegetation buffers. As the scale of pork production has increased to meet consumer demand communities have heightened their concern about the environmental impact of pork operations. Pork producers are working to reduce their overall water usage, land use and carbon footprint of farming operations (National Pork Board, 2018). Responsible environmental farm management has become a necessity and pork producers in the US have affirmed their obligation to manage pork production operations in a manner that protects natural ecosystems and public health. Watersheds generally support multiple types of livestock operations and human dwellings. Each is a potential source of fecal waste in surface waters. However, when fecal material is detected it is often swine farms that are the target of concern. Fecal waste in surface waters is generally referred to as non-point source contamination. In reality, all fecal waste has a vertebrate animal origin and the species of origin varies with adjacent land-use practices. Monitoring programs established to protect public health have traditionally relied on the culture or detection of enteric bacteria in water samples, which do not differentiate contamination from animals hosts. Genetic assays focused on detecting microbial species have also been developed as an alternative to these culture-based methods. Prior studies by the NC Division of Environmental Quality (DEQ) and our laboratories have noted the presence of fecal coliform contamination in Stockinghead Creek, Duplin County, NC. Additional microbial-based studies conducted by other laboratories suggested pigs were the origin of the fecal waste and public reaction targeted the swine operations in the watershed. All vertebrates release cells from their gastrointestinal tract in their feces. These cells contain mitochondrial DNA (mtDNA), a routine aspect of forensic investigation that can be applied to identify the animal hosts associated with fecal waste. Unlike the majority of molecular microbial-based approaches noted above, mtDNA detection is highly specific. If mtDNA is detected in a water sample, the vertebrate animal associated with that mtDNA can be determined. However, an initial real-time polymerase chain reaction assay we developed lacked sensitivity. In our initial Pork Board funded studies we refined the mtDNA assay for classifying the species of origin of fecal contamination in surface waters by adapting the use of new droplet digital PCR technology. New primers and probes were designed, and the assay proved both sensitive and specific. Our studies confirmed the presence of fecal contamination in Stockinghead Creek and documented that the fecal contamination originates from at least three species, cattle, humans and swine. This renewal is requested to continue source tracking for fecal waste in the Stockinghead Creek Watershed. In addition, we will work with DEQ and test samples they collect in the Stockinghead Creek Watershed. Specific objectives include: 1) Broadening the species detected with the Droplet Digital mtDNA assay; 2) Conducting parallel sampling with DEQ to develop a more comprehensive assessment of fecal contamination in the watershed; and 3) Comparing and contrasting results obtained with the mtDNA assays with two bacterial-based assays for identifying the animal hosts contributing to fecal contamination. Water is one of our most limiting resources. The proposed studies address the environmental mitigation technology and practice objectives stated by the National Pork Board Sustainability Committee. By facilitating accurate detection of fecal waste in rivers and streams the proposed studies support the objective of the Producer/public health Workplace Safety Committee focused on reducing exposure to zoonotic pathogens. The regulatory necessity of ensuring that water resources are free of fecal waste sometimes pl

Fort Bragg military base in the Sandhills of North Carolina??????????????????s Piedmont is situated on more than 150,000 acres. Sections of the base are used for military training, others serve as refugia for endangered and threatened species. Recent stream surveys conducted at Fort Bragg documented the presence, abundance and distribution of freshwater mussels on Post. Villosa delumbis, a species listed as state endangered was found in the Little River, which is part of the Cape Fear River basin. Ellipitio complanata, and Uniomerus caroliniana were found in both the Little River and in Drowning Creek, which is part of the Lumber River basin. Stream channel substrate size, availability, and stability were the primary factors contributing to habitat suitability for freshwater mussel species. Measurements of stream channel grain size distributions from study reaches were used to calibrate a sediment transport model. The model serves as a predictive tool for identifying areas with greater potential for future in-channel mussel augmentation and enhancement efforts. Catchment-average erosion rates, measured from in-situ cosmogenic nuclide 10Be extracted from quartz-bearing stream sediments indicates that the Little River basin is eroding at about 25 m/Ma (0.025 cm/yr) over timescales of ~104 years. These first 10Be results from the Sandhills region of North Carolina provided baseline reference frame estimates of the upland erosion and sediment transport rate through the Little River basin prior to anthropogenic modifications of the landscape. This proposal builds on these prior studies and the recommendations contained within the Levine et al. (2015) final report for continued study of Fort Bragg??????????????????s freshwater mussel populations and river and hillslope landscape factors important to their present distribution and future fate. In this document we propose to: 1) Establish a routine monitoring program to document the presence of freshwater mussel fauna in Fort Bragg streams; 2) Survey streams upriver of Fort Bragg to determine if they can serve as sources of freshwater mussel stock for population augmentation; 3)Determine the value of using freshwater mussels as environmental monitors; 4) Develop a dynamic model of upland soil erosion potential paired with tributary stream sediment transport and delivery to the Little River and Drowning Creek trunk channels, which can be used to predict the potential viability of stream sites for sustainable restoration; 5) Develop an environmental education poster that demonstrates the importance of preserving Fort Bragg??????????????????s aquatic fauna; and 6) Develop a demonstration of a Tangible Landscape system as a collaborative environment for communication of spatial patterns and sediment transport. ????????????????

All livestock operations generate fecal waste and manure management is an essential aspect of pork production. Regulations mandate permitting, training, design specifications, soil testing and livestock operation stream vegetation buffers. As the scale of pork production has increased to meet consumer demand communities have heightened their concern about the environmental impact of pork operations. Pork producers are actively working to reduce their overall water usage, land use and the carbon foot-print of their farming operations. Responsible environmental farm management has become an inherent necessity to maintain the sustainability of the pork industry and pork producers in the US have affirmed their obligation to safeguard our natural resources and manage pork production operations in a manner that protects natural environments and public health. The proposed studies support environmental management of pork production and address community concerns by facilitating accurate detection and effective attribution of the origin of fecal waste in surface waters and groundwater.

The Dwarf wedgemussel, Alasmidonta heterodon, is a federally listed endangered species of freshwater mussel that historically ranged from New Brunswick in Canada to locations in North Carolina. The species is considered extripated from Canada and in the last decade has been only found in limited numbers at ten geographically fragmented locations. In North Carolina, it still occurs in the Neuse and Tar River basins. Alasmidonta heterodon is a long-term brooder, spawning in late summer, gravid in the fall and releasing its glochidia in late winter and the spring. The recovery plan for A. heterodon includes captive propagation and augmentation of remaining populations. Etheostoma flabellare and other darters have proven useful as fish-hosts for rearing juveniles in captivity. However, comparatively low numbers of juveniles have been successfully reared using these relatively small fish-hosts. Captive propagation using fish-hosts is labor intensive and expensive. In vitro culture of larvae and their subsequent metamorphosis to juveniles in culture media has proven to be a less expensive viable alternative to captive propagation using host fish. North Carolina State University investigators have successfully reared 13 species using in vitro techniques, raised them to maturity and produced second generation juveniles from captive reared stock. Glochidia derived from these in vitro reared adults have the same host attachment preference of naturally reared adults indicating the viability of employing in vitro culture as a less expensive alternative for captive propagation of imperiled species. Although Alasmidonta raveneliana and other endangered species have been successfully reared using in vitro techniques, similar work has not been conducted with Alasmidonta heterodon. We propose studies to attempt the rearing of A. heterodon in vitro and optimizing the nutritional health of recently transformed juveniles. Adult female brood stock will be obtained from streams in the Neuse River Basin. Glochidia will be harvested from the gravid females. The glochidia will be reared in cell culture media and the survival and growth of metamorphosed juveniles will be measured.