Jay Levine

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, 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.

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.

Freshwater mussel populations throughout North America have declined precipitously during the last three decades. Captive propagation and release of the captive reared stock has become an integral component of efforts to mitigate the decline and augment the reproductive capacity of remaining populations. Freshwater mussels are reared in captivity using two alternative approaches that focus on different approaches to facilitating the metamorphosis of juveniles. Either host-fish are used to support the metamorphosis of the larval stage or the larvae are reared in vitro in petri dishes and the metamorphosis is nutritionally supported with culture media. The initial survival of in vitro reared mussels is poor and their initial growth lags behind that of host-fish reared animals. Our limited understanding of the nutritional needs of freshwater mussels and how specific components of their diet contribute to their nutritional health impedes our ability to sustain them in captivity. In addition, declines noted in some free-ranging populations appear to be associated with poor nutrition. We propose studies to further inform our understanding of the role of different food-web resources in the diets of freshwater mussels. Specific objectives include: 1) Examining the role of pollen in the diet of freshwater mussels; 2) Assessing the role of detritus in freshwater mussel diets; 3) Quantifying the filtration ability of selected freshwater mussels species; 4) Preliminary studies of diet and freshwater mussel nutritional health; and 5) Refining procedures for characterizing the chemical composition of freshwater mussel shells.

This project builds upon and expands the Forest Service’s Partnership Outreach and Capacity Building, and the Multicultural Workforce Strategic Initiative Programs, and is an initiative that represents an opportunity for substantial collaboration between Hispanic-Serving Institutions (HSI), the 1862 land grant institutions, the Puerto Rico Commonwealth environmental and educational agencies, and the public served by the consortium/partnership. The Forest Service has utilized similar projects to implement its’ Mission, build capacity at the academic institutions, and to increase its engagement with underserved communities (i.e. American Indian and Alaska Native, Asian, African American, Hispanic, Native Hawaiian/Pacific Islander, Persons with Disabilities, and low income and socially disadvantaged groups). This project enhances the Forest Service’s commitment to capacity-building at UPR and NC State University by providing for education, advocating student mentoring/employment in the institutions research programs, and technical assistance and other outreach engagement to communities.