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Special Research Grant Program
Cooperative State Research, Education, and Extension Service
U. S. Department of Agriculture

West Virginia Agricultural and Forestry Experiment Station
West Virginia University

Gerard E. D'Souza
P. Brett Kenney
Cyril M. Logar
Patricia M. Mazik
Thomas G. Ponzurick
Kenneth J. Semmens
Dennis K. Smith

This project builds on research initiated under the FY 98 Aquaculture Food and Marketing Development Project of West Virginia University. It strengthens the base of information needed for a viable and competitive aquaculture industry in Appalachia. The mission is to conduct research and disseminate information that will facilitate the development and growth of a sustainable aquaculture industry in a multi-state area of the Appalachian Region. The Appalachian aquaculture industry possesses a limited infrastructure and is not well organized or influential. Thus, specific objectives are to: implement the marketing strategies developed in the FY I998 project and assess further opportunities for developing a sustainable aquaculture industry in the region; to determine the expected costs and returns of producing and processing aquaculture products and examine other issues including identification of suitable water supply sources, waste management, and economic development; improve the consistency and quality of fresh trout fillets through the use of feeding and harvesting techniques; implementation of technology transfer activities; characterization of effluents from West Virginia trout production facilities and assessment of the feasibility for developing impaired waters for aquaculture production; and to conduct farm level research focusing on production efficiency and fish health. A variety of approaches will be used by an interdisciplinary and inter-institutional team that includes aquaculturalists, animal scientists, economists, engineers, and marketing specialists, as well as public and private institutions in a multi-state area. These include studies and strategies to expand markets, economic modeling to develop production and processing efficiencies, feeding and handling experiments to improve production and product quality, engineering studies to enhance water quality and waste handling, and on-farm research to increase efficiency, improve and certify fish health, and to improve technology transfer procedures. Expected outcomes include increased knowledge about and strategies for improved production and marketing of aquacultural products, improved quality and health of such products produced in Appalachia, decreased environmental damage due to improved waste management practices, an expansion of knowledge on available water resources, and development of technologies to treat impaired water to expand the potential for aquaculture production.

This project builds on research initiated in the first (FY 98) Special Grant on Aquaculture Food and Marketing Development (awarded 4/28/99) and strengthens the base of information needed for a viable, competitive, cold-water aquaculture industry in Appalachia with a focus on West Virginia. The mission of this current proposal is to conduct research and disseminate information that will facilitate the development and growth of a sustainable aquaculture industry in a multi-state area of the Appalachian Region.

Aquaculture production in the Central Appalachian Mountains, including West Virginia, can be characterized primarily as small-scale businesses producing rainbow trout for local and recreational markets. Water resources available for aquaculture development in West Virginia range from low-flow cold water springs and streams (less than 200 gallons per minute [gpm]) to large springs and discharges from active and abandoned mine sites (1,000 to 5,000 gpm). The aquaculture industry possesses a limited infrastructure and is not well organized or influential. There are a variety of constraints to aquaculture industry growth and enterprise establishment.

To accomplish this, implementation of a technology transfer program targeted at cooperators from the private sector as well as state government and federal government agencies will be used to promote state, regional, and national adoption of the research results from the project.

The Appalachian Region consists of parts of thirteen states: Alabama, Georgia, Kentucky, Maryland, Mississippi, New York, North Carolina, South Carolina, Ohio, Pennsylvania, Tennessee, Virginia, and West Virginia-West Virginia is the only state wholly within the Appalachian region. However, the scope of this research is limited to cool- and cold-water species. Thus, only states with significant cool- and cold-water conditions are included in this research project, although some aspects of the project will include information and data from states other than the targeted area. These findings will also be useful outside of the region. While the project will concentrate on organizations operating within the Appalachian region, the working arrangements for research are centered at West Virginia University and will concentrate on the state, as in Phase One of the project.

Work initiated under the first Special Grant (Phase 1) to West Virginia University addresses the following objectives:

The current project builds upon and adds to the research already initiated with the first Special Grant through modifications of the original four and the addition of two more objectives. These activities directly address the mission of the aquaculture program. The specific objectives of this project address state and regional needs through improvement of short-term viability and long-term sustainability of aquaculture production and processing firms in West Virginia and similar areas of Appalachia. The increased contributions of regional aquaculture products to regional, national, and export markets will add support to global competitiveness of U.S. aquaculture and to meeting regional and national consumer demands for safe, affordable, food.

The general planning and review processes used to develop the activities and research plan for this second grant, including the revised objectives, have involved:

OBJECTIVE: Implement the marketing strategies developed in the FY1998 project for aquaculture producers and processors and assess further opportunities for developing a sustainable aquaculture industry in a multi-state area including the Appalachian region.

Expected Outcome: The expected outcome of this objective is a viable marketing system for aquacultural products produced in the region and, hence, a more economically viable industry that contributes to improvements in the region's economic base.

Personnel: Cyril M. Logar and Thomas G. Ponzurick, Professors, College of Business and Economics, West Virginia University.

Background: The primary purpose of this objective is to implement a strategic marketing program that is designed not to replace the marketing strategies that may or may not be currently in place and utilized by Appalachian aquaculture operators but rather to assist them in sharpening the focus of their marketing strategies. Those operators without any current or formal marketing program will be offered the opportunity to utilize the information provided by the researchers to develop and implement a marketing initiative.

In Objective One of the Phase 1 "Aquaculture Food and Marketing Development Project" the task was to develop marketing strategies for aquaculture (primarily trout) producers and processors. The data obtained from Objective One of Phase One of the project serves as the foundation for Tasks 1 -1 and 1-2 of this grant. Task 1-3 below is a proposed new direction of opportunity for the industry. The information collected for Task 1 - 1, Task 1-2 and Task 1-3 will be disseminated to aquaculture policymakers and industry operators via conferences, business counseling and through West Virginia University Extension Service.

The proposed research for Task 1 -1 will develop and initiate the implementation of a market-driven network for marketing Appalachian aquaculture products to the identified segments of the recreational fee fishing market. This network will be both vertical (between recreational fishing operations and consumers) as well as horizontal (grouping recreational operations together) so as to improve the impact these small to medium operations have on meeting needs and potential demands of the marketplace.

The preliminary research results from Phase One have begun to identify the preferences of recreational fee fishermen when selecting fishing locations to patronize. This research shows that recreational fee fishermen would like to have options that a network would provide. These options will be developed further and thus provide the edge (i.e., competitive advantage) that would make such an Appalachian aquaculture network economically viable. In order to be successful and market-driven, the network must focus on delivering a service that these potential buyers (recreational fee fishermen) would patronize.

The research effort associated with Task 1-1 will focus on collecting data from three distinct entities: 1. Appalachian aquaculture operators with the ability to provide fee fishing facilities; 2. fishermen interested in patronizing Appalachian fee fishing operations; and 3. Organizations (West Virginia resorts and parks) with the ability to attract prospective fishermen to the geographic region. The horizontal and vertical nature of this recreational fee-fishing network is outlined in Paradigm I in Appendix I. The results of Task 1-1 will be used in conjunction with those of Task 1-2.

West Virginia aquaculture operators who were surveyed in Phase One of the project and who have expressed an interest in being associated with a network of similar operators to attract fee fishing patrons will be surveyed again as part of Task 1-1. The purpose of this survey is to determine their expectations and requirements for participating in this type of network. Niche opportunities (i.e., bait fish) for supplying these operators will be examined here, as well. The data will be collected via personal interviews.

Fishermen were also surveyed in Phase One of the project to determine their level of interest toward patronizing a proposed fee-fishing network. Those fishermen indicating an interest in patronizing a network will be surveyed again as part of Task 1-1 to determine their expectations and requirements for patronizing this type of network. That is, the fishing and venue expectations as well as the requirements of the fishermen will be assessed to determine not only their level of interest but what it would take to attract the fishermen to the proposed network. This sample will be stratified to include in-state (WV) fishing license holders, lifetime in-state (WV) fishing license holders, and out-of-state fishermen holding West Virginia fishing licenses. The data will be collected via a mail survey.

Data will also be collected from a third entity--organizations with the ability to attract fishermen to a geographic area for a recreational fishing experience. It is hypothesized that these organizations consist of both public and private recreational parks and resorts which would be responsible for marketing the fee fishing experience as a recreational attraction for visiting their respective geographic areas. These organizations will be identified and surveyed to assess the level of interest and degree of expectations they would have in marketing such a program to potential fee fishing visitors. Data will be collected via a telephone interview with these organizations. West Virginia private resorts and state parks with lodging facilities will comprise the sample of recreational facilities to be surveyed. All identifiable West Virginia organizations will be included in the survey sample frame.

The proposed research for Task 1-2 will develop and initiate the implementation of a horizontal market driven cooperative approach among small (both fee and food) producers for meeting the processing capabilities of West Virginia aquaculture operations and aquaculture resellers in the identified market area. The horizontal and vertical nature of this processed food-fishing network is outlined in Paradigm II in Appendix I.

The research effort associated with Task 1-2 will focus on collecting data from two distinct entities: 1) Appalachian aquaculture operators with the ability to provide products to West Virginia processors (i.e., High Appalachian, LLC and Mountain Aquaculture and Producers Association [generally referred to as Ma & Pa]) and 2) wholesalers, retailers and restaurants with the ability and interest in purchasing processed products from West Virginia processors.

West Virginia aquaculture operators were surveyed in Phase One to determine their capability to provide fish for processing purposes. Those who have expressed an interest in providing fish for processing purposes will be surveyed again under Task 1-2 to determine the level of interest they have toward being associated with a cooperative network of similar operators desiring to sell their product output to the West Virginia processors. In addition to their interest, the survey will also determine their expectations and requirements for participating in this type of cooperative network. The data will be collected via personal interviews in conjunction with the information sought in Task 1-1.

Resellers (wholesalers, retailers and restaurants) were surveyed in Phase One of the project to determine their level of interest in purchasing processed fish products from the proposed cooperative network. Those resellers who indicate an interest in patronizing such a network will be interviewed again in Task 1-2 to determine their expectations and requirements for patronizing this type of network and more importantly what it would take to attract resellers to purchase from the proposed cooperative network. This sample will be stratified to include regionally accessible (by geographic distance) and interested wholesalers, retailers and restaurants. Data will be collected via a telephone interview.

The purpose of Task 1-3 is to assess the market potential for recreational fee fishing utilizing streams rather than ponds in West Virginia. The research effort here is designed to determine the level of interest and potential for stream versus pond recreational fee fishing in West Virginia.

Fishermen who were surveyed in Phase One of the project, will be asked about their interest in fishing streams rather than ponds. From these results, those fishermen who express a preference for or interest in fishing in streams rather than ponds will be surveyed again to determine their expectations and requirements of a stream fishing experience in West Virginia. More importantly to what extent would they be interested in patronizing fee fishing opportunities in West Virginia streams (rather than ponds) and what it would take to attract these fishermen to stream fee fishing venues. Those surveyed will be stratified to include in-state (WV) fishing license holders, lifetime in-state (WV) fishing license holders, and out-of-state fishermen with West Virginia fishing licenses. The data will be collected via a mail survey.

OBJECTIVE: To determine the expected costs and returns of producing and processing aquaculture products suitable to hill country such as that in West Virginia, and to examine other issues relating to the development of a sustainable aquaculture industry in the study area, including identification of suitable water supply sources, waste management practices, and economic development impacts of aquaculture.

Expected Outcome: The primary outcome will be to provide baseline information that can increase efficiency and profitability of production and processing in the short run and the development of a sustainable aquaculture industry in the study area in the long run.

Personnel: Gerard D'Souza and Dennis Smith, Professors; Nu Nu San, Post Doctoral Fellow; Daniel Miller, Research Assistant; Division of Resource Management, College of Agriculture, Forestry and Consumer Sciences, West Virginia University.

Background: The economic analysis component of the FY 1998 project included the following activities: (a) compiling information from primary and secondary sources on aquaculture production and processing in West Virginia and surrounding states using a combination of site visits and attendance at workshops; (b) developing, pre-testing, and utilizing a questionnaire to collect data from West Virginia producers; (c) conducting a feasibility case study including complete documentation of one major aquaculture processor in West Virginia; (d) initiation of a case study of the other major aquaculture processor in state; (e) development of farm-level aquaculture enterprise budgets; and (f) undertaking a preliminary GIS analyses of aquaculture production sites.

Procedures: In Phase II of the project, additional aspects of the industry will be evaluated using the data from and building on the knowledge acquired in Phase I, as well as by collecting additional data from secondary and primary sources. These activities will include modeling farm-level and processor-level operations to assist producers and processors with decision-making; identification of suitable water supply sources, identified as a key constraint to the development of a viable aquaculture industry, examining economic development impacts, and analyzing the economics of waste disposal options.

Two sets of analyses, for producers and processors, will be undertaken concurrently to help determine the conditions under which production and processing, respectively, can be profitable.

(A) Farm-level (Producer's) model: Using a combination of primary and secondary data, a farm-level, multi-period optimization model, developed in Phase I, will be refined and estimated during Phase II. This model will be specified to estimate various levels of net return associated with alternative firm sizes, alternative production systems (pond, tank and/or raceway), different types of enterprises (food fishing and fee fishing), and different species, e.g., trout, arctic char, hybrid striped bass. This model will incorporate information and assumptions regarding feed quality, survival rate, feed conversion ratio and the environment. Sensitivity analyses to determine the impacts of changing economic and biological production parameters on profitability will be conducted. Data will come from the enterprise budgets that are in development and from published secondary sources.

This modeling approach will allow better understanding of the factors determining the viability of a typical aquaculture farm in West Virginia, with implications for producers across Appalachia. One advantage of the multi-period approach to be employed is that it could provide the capital recovery rate of investment on enterprises. With appropriate modifications, economic and production risk impacts will be analyzed using this framework.

(B) Processor-level model: The organizational structure and operations of the High Appalachian trout production and processing plant in Raleigh County, West Virginia will be reviewed and evaluated as part of a case study (similar to the case study of the Ma & Pa operation carried out in the FY 1998 project). Based on data provided by the firm, the product distribution aspect of the operation will be analyzed with a view to minimizing the cost of product distribution. A transshipment model will be used to minimize distribution costs subject to fixed supplies and given various market demands. The linear programming framework will be used for this purpose. Sensitivity analyses will be conducted to determine the potential impacts of price, demand, and supply fluctuations. The results will be provided in user friendly spreadsheets for different scenarios. This information should be useful to processors in other aquaculture production areas.

A preliminary analysis of statewide economic development impacts will be conducted using IMPLAN or related input-output modeling software to measure direct and indirect effects of aquacultural production and processing activities. This will enable a more complete evaluation of local economic development impacts, including the computation of output, income and employment multipliers, as a result of increased production, processing and fee-fishing or recreational activities. An input-output model of the state agricultural sector, estimated by D'Souza et al. (1988) in the mid-1980s, will provide the framework for developing this analysis.

Development of a sustainable aquaculture industry also requires an assessment of waste management options. To initiate this, a detailed review of literature will be carried out to collect information on the efficacy of alternative waste disposal options suitable for hill-land producers. Laws and regulations pertaining to waste disposal were reviewed in an earlier analysis (Smearman, D'Souza, and Norton, 1997), and this review will be updated. The initial economic assessment will focus on settling ponds, aquatic plants, and filtration units. The results of the technical studies (Objective 5) on treatment of mine waters will be utilized in making the economic assessment to determine the more cost effective methods of handling and treating waste from aquaculture operations.

Task 2-3: Assess Mine Water Sources Suitable for Economical Production of Food Size Trout in West Virginia.

An economic assessment of aquaculture has limited use unless water supply sources are available for use by potential producers and existing producers considering expansion. Thus, the major purpose of this task is to identify and evaluate potential water sources that can be used for aquaculture in West Virginia. This task will assess information from secondary sources as well as from the research conducted under this objective on quantity (i.e., flow rates) and quality of water resources of potential production sites. These will be used to develop a data base will provide information on potential aquaculture production sites.

Developing mine water resources will require specific, reliable information regarding the locations and quantities of water available. It will also require information on the risk associated with developing the site and a variety of site specific data. Coupling this information with enterprise budgets and markets developed in grant 1, firm-level optimization models (Task 2-1), proven protocols for production (Objective 6), and methods to address the impact of effluents (Task 2- and Objective 5) will provide many of the tools necessary to develop these resources. Mine water resources suitable for production of food size rainbow trout at costs competitive within the processing plant price structure (i.e., about $1/lb.) will be cataloged and made available to potential producers. Experience within the North Carolina aquaculture industry indicates that a flow rate of 1,000 gpm or greater are required to generate a source adequate for a primary income enterprise. This will be evaluated for conditions in West Virginia.

Based on National Pollution Discharge Elimination System (NPDES) data and field observations reported by West Virginia Division of Environmental Protection (DEP) staff, Jenkins et al. identified 33 sites with flow rates greater than 1,000 gpm. A state-wide assessment of mine water resources producing 1,000 gpm or greater will be undertaken to determine the suitability of each site for the development of a food-size trout production facility utilizing traditional flowing water system technology. Each site will be visited, water flow rates will be estimated, and the area will be catalogued using digital photography. If reliable water quality data are not available, samples will be collected and tested for the presence of metals (especially iron, aluminum, and manganese); pH; temperature; and alkalinity. A database consisting of the following parameters will be constructed for each water resource:

Each site will then be assigned to one of four risk-based categories, where "1" is the lowest risk and "4" is the highest:

Data will be summarized by site and on a statewide basis for each of the groups listed above. Once a hard copy is completed, it will be distributed among individuals working in the field of economic development, such as WW DNR, WV DEP, WV Department of Agriculture, and the mining industry. In addition, it will be made available for posting on the Extension aquaculture program web site.

OBJECTIVE: Improve the consistency and quality of fresh trout fillets through improved feeding and harvesting techniques.

Expected Outcome: The pre- and post-harvest conditions needed to ensure high quality attributes of trout fillets, including color, flavor, texture, and composition, will be determined through experimental manipulation of feeding and pre- and post-harvest handling practices. Improvements in quality will result in a more economically viable industry.

Personnel: Scientists with primary responsibility for Objective 3 are P. Brett Kenney and Patricia Mazlk. Joseph Hankins of the Freshwater Institute, Keameysville, West Virginia, will provide aquaculture facilities and office facilities for one Post-doctoral Fellow, and will collaborate on the feeding, stress and harvesting trials. Dr. Joseph Regenstein, Cornell University, will collaborate on sensory evaluations and selected protein functionality attributes.

Background: Smaller scattered farms providing products through distributed channels to modest scale processors characterize most of Appalachian aquaculture. Even under ideal conditions, fresh fish products have a relatively short shelf life when compared to other meat products. Fish flesh has a higher pH, contains relatively high levels of unsaturated fats that are easily oxidized, causing off-flavors and more active proteases that can adversely affect fillet texture, if uncontrolled. These muscle-food characteristics challenge producers and processors in ensuring consistently optimal quality. Appalachian fish producers and processors must develop products and procedures that will enhance values for regionally produced products and provide better quality control. Antioxidants in the finishing nutrition plan, nutrition that maximizes growth potential and feed efficiency, modifications in harvest and conditioning practices, and improved post-harvest handling practices may significantly improve the ability of small producers to compete and maintain market share. Objective 3 of the Phase 1 of the Special Aquaculture grant examined the effects of handling practices and water quality in production systems prior to and at harvest on the quality of fresh trout fillets and value-added smoked trout products. First-year research findings revealed significant variation in fillet yield and quality on the farms sampled. They also support the need for research and education to define the sources of variation and communicate recommendations to producers to enable them to guarantee a more consistent and higher quality fillet. An evaluation of the links among fillet data, water characteristics, and management practices is forthcoming.

Procedures: Achieving Objective 3 will be met by determining the fish's stress response to handling and harvest, and evaluating the fillet for flavor (sensory) and texture (sensory and instrumental) differences after being fed alternative finishing feeds, as well as by evaluating harvesting practices and determining which cause the least impact to the fish's stress response and resulting fillet sensory flavor and texture while remaining efficient and applicable to the small farmer. The impacts of standardized alternative production and handling techniques during the last stage of the production cycle on reducing product variability, improving overall product quality, and improving producer reputation will be examined. Alternative finishing feeds will be examined to determine those that have positive benefits for flavor or sensory characteristics, reduce oxidation, extend shelf life, improve smoking or value added opportunities, improve overall carcass appearance, improve flesh color (whiteness), and improve flesh texture.

Production Facilities Research for Objective 3 will be conducted at the Freshwater Institute's research farm in Shepherdstown, West Virginia, where the facilities include sixteen 500-liter and twelve 1,400-liter fiberglass circular tanks supplied with single-pass, air-stripped and oxygenated spring water. The tank pad is enclosed in a weatherproof structure to maintain bio-secure access and contains telephone and state-of-the-art data acquisition and alarm capacity. The tanks can be configured with self-feeding demand (Babington) or mechanically timed (Sterner) feeders. The Freshwater Institute's facility also includes an environmental water chemistry laboratory, a fish health diagnostic laboratory, a walk-in cooler (O-5�C), and walk-in freezer (-20�C). The Institute will also provide office space and office support services, including telephone, fax and Internet access for WVU personnel stationed on-site. Experimental fish utilized in this work will be from certified SPF stock hatched and reared at the Freshwater Institute. It is anticipated that rainbow trout (Kamloop strain, Trout Lodge, Inc.) and arctic char (Yukon Gold TM strain, Icy Waters Ltd.) will be the focus of study objectives.

Task 3.1: Assess Culture Conditions and Post Harvest Handling on Product Quality

Experimental Design: Fish (100 g and 25 cm) will be stocked in sixteen 500-liter tanks at a density not to exceed 0.225 kilograms per liter of water. Fish will have pit tags to allow identification of individual fish. Oxygen will be above 80% saturation with a water exchange of at least two per hour. Fish will be grown to approximately 350 grams and 25 to 30 cm that is considered market size. Two feeding rates (full ration to satiation, restricted ration), two water velocity rates (high and low; exact rates determined during preliminary tests), and multiple on-farm harvest practices (ice slurry chill kill, ice slurry chill kill and bleeding, carbon dioxide stunning and ice slurry, live haul transport) will be replicated four times. The experimental treatments and design will be staggered to minimize volume of sampling required at any one point.

Experimental Conditions: Water Quality Dissolved oxygen, total gas pressure, temperature and pH will be measured daily. Total ammonia nitrogen, free C0₂, total hardness (as CaCO₃) and alkalinity (as CaCO₃) will be measured weekly throughout the experiment.

Water Velocity: Water velocity will be maintained by adjusting the orifice size on the inlet manifold to the rearing tanks and by adjusting the orientation of the manifold. Velocity will be measured weekly by using a Teledyne Gurley Pygmy meter. In all cases, water flow to the rearing tanks will be equivalent, and loading will be adjusted to maintain 80% DO saturation to the heaviest experimental tank.

Feed: Feeding rates will be calculated by the method of Cho (1992). Feed utilized will be typical of feeds in use in the region (42% protein, 15% fat as Zeigler #38-5315). Basal feed rates will be adjusted every 14 days; the ration will auto-increment daily by a fixed percentage to allow for growth. All feed will be purchased fresh in a single production lot and stored at 40� F until used.

Growth and conversion: All fish in each tank will be weighed initially and every 28 days, all feed applied will be pre-weighed and accumulated as weight of feed fed. Feed conversions will be calculated as weight of feed fed:biomass gain over period of interest.

Stress Parameter Assessment: Fish will be bled using heparinized syringes from arteries in the caudal peduncle. Fish will be stunned using clove oil. All fish will be bled within five minutes of initial disturbance, and each fish will be sampled only once. Blood will be centrifuged and plasma stored at -55'C until analyzed. Plasma concentrations of cortisol will be determined by radioimmunoassay (RIA) with a commercially prepared kit (Ciba-Coming Diagnostics Corporation, Medfield, MA). Plasma glucose will be determined using a clinical diagnostic kit (Sigrna Chemical Company, St. Louis, MO), and plasma chloride levels will be determined using a chloridometer (Buchler Instruments, Lenexa, KS). In addition to the water quality parameters, mineral content (Fe, Mn, Al, Ca, and Mg) will be determined. Minerals also will be quantified for the feed. At the end of the finishing phase of production, fish will be held off feed for 72 hours prior to harvest. Fish will be bled for stress parameters at 0 hour (when fish are placed in tanks), when fish are removed from tanks for harvesting, and after transport (when fish arrive at the processing plants). At each sample time and after bleeding, fish (fillets) will be processed for fresh and value-added products (see below).

Fish will be fed and acclimated for eight weeks on the commercially available (basal) diet.Fifty-six days prior to harvest the diet will be shifted to a formulation supplemented with two levels of anti-oxidant (Vitamin E) amendment (the control basal diet will continue and two treatment levels will be substituted with replication). At harvest, individual fish will be sampled for level and variability of product quality characteristics, including lipid oxidation, and level and variability of anti-oxidant tissue level.

Experimental Design: Fish (250-300 g and 11 inches) will be stocked in ten 350-gallon tanks at a density not to exceed 1 pound/gallon. Oxygen will be above 90% saturation with a water exchange of at least two per hour. Fish will be grown to approximately 350 grams and 11 to 12 inches, which is considered market size. Dietary Vitamin E levels will be replicated at least three times at 250-400 mg/kg feed (basal or control diet), 1000 mg/kg feed (level 2) and 2000 mg/kg feed (level 3). All tanks will be sub-sampled without replacement every 14 days for at least eight weeks. Muscle tissue (fillets) will be sampled for lipid content and evidence of lipid oxidation at fresh harvest, and periodically in storage as fresh and frozen products. Sensory assessment of fillet quality will be made on selected Vitamin E and storage time treatments.

Water Quality: Dissolved oxygen, total gas pressure, temperature and pH will be measured daily. Total ammonia nitrogen, free C02, total hardness (as CaCO3) and alkalinity (as CaCO3) will be measured weekly throughout the experiment.

Water Velocity: Water velocity will be maintained by adjusting the orifice size on the inlet manifold to the rearing tanks and by adjusting the orientation of the manifold. Velocity will be measured weekly by using a Teledyne Gurley Pygmy meter. In all cases, water flow to the rearing tanks will remain equivalent and loading will be adjusted to maintain 80% DO saturation to the heaviest experimental tank.

Feed: Feeding rates will be calculated by the method of Cho (1992). Feed utilized will be typical of feeds in use in the region (typical 42% protein, 15% fat as Zeigler #38-5315). Basal feed rates will be adjusted every 14 days, ration will auto-increment daily by fixed percentage to allow for growth. All feed will be purchased fresh in a single production lot and stored at 40�F until used.

Growth and conversion: All fish in each tank will be weighed initially and every 14 days; all feed applied will be pre-weighed and accumulated as weight of feed fed. Feed conversions will be calculated as weight of feed fed:biomass gain over period of interest.

Fresh and Value-Added Product Manufacture, Quality, and Functionality Assessments.

Fresh Fillet Evaluations. Fish will be filleted within six hours of harvest for each of the treatment combinations applied. Fillet yield will be calculated for each fish. Fresh fillets will be graded according to the Code of Federal Register (50 CFR, Ch. I 1, Part 260) entitled, "Regulations Governing the Processed Fishery Products and US Standards for Grades of Fishery Products." Fresh fillet color will be measured using a Minolta chronometer when graded following 0, 24, 48, and 72 h of aerobic storage at 2^�C. Thiobarbitunic reactive substances will be measured as an indication of lipid oxidation (McDonald and Hultin, 1987) following each storage period. Mineral content of the dorsal musculature (Ca, Mn, Zn, Mg, Cu, Fe, Fe") will be determined as well.

Expressible moisture of fresh fillets will be measured according to procedures of Jauregui et al. (1981). This trait will indicate the water retention ability of post-harvest trout muscle. Color, cook yields, instrumental texture, protein-water interactions (water binding potential, expressible moisture, protein solubility) and proximate analyses (moisture, protein, fat, and ash) will be evaluated in fresh and value-added products. In addition, fresh fillets will be shipped overnight to Cornell University (Joe Regenstein) for sensory texture and flavor evaluations.

Postmortem Metabolism: Postmortem metabolism will be assessed at the time fish are sampled. Muscle pH will be monitored in five fish per tank with a pH/ion analyzer (Model 350, Coming Inc., Coming, NY) equipped with a needle probe. The pH probe will be placed in the dorsal musculature, just caudal to the head. Temperature will be recorded with a Doric data logger (Model 205, Beckman Industrial, Carlco, Inc.; Overland Park, KS) at a position in the musculature next to the pH probe. Placement of pH and temperature probes will alternate from left to right sides of the musculature. Temperature and pH measurements will be taken every five minutes for a 1.5 h period.

Rate and extent of pH changes, as a function of temperature, will be determined for use in evaluating the significance of these traits to processing quality of smoked trout fillets and restructured trout steaks. Because muscles attempt to maintain ATP levels and thus cell viability modulates postmortem changes, ATP/IMP ratios will be determined according to the procedure of Khan and Frey (1971). ATP/INIP measurements will be taken every 0.25 h for 1.5 h and then at 6, 12, and 24 h post-sampling. Sampling will be conducted as described by Korhonen et al. (I 990); a 1 0 gram sample of dorsal musculature will be excised from five separate fish collected at the same time as those used for pH and temperature decline studies. Tissue will be collected from an anatomical location similar to where the pH and temperature were measured. Following sample preparation, absorbency will be measured using a Shimadzu UV-1201 spectrophotometer at 250 and 280 nm. The samples will be blended with perchloric acid and then analyzed. K- value (Lowe et al., 1993) will be calculated following determination of ATP and its catabolites. These data will be related to sensory flavor evaluations conducted at Cornell University.

Smoked Trout Evaluations: Investigations of the impact of fillet quality on smoked trout will be determined using a two-stage brining protocol. Stage One will consist of subjecting fish to brine containing 8.7% NaCl for 150 min at 3� C. Stage Two will consist of removing fillets from brine and allowing the salt to equilibrate for 48 h. Following removal from the brine and prior to storage for equilibration, fish fillets will be weighed and weights will be recorded in order to determine brine uptake. Muscle pH of fresh and brined fillet will be measured at the cranial, middle and caudal third of each fish using a surface probe. Prior to thermal processing, a sample will be removed from one fish for protein solubility determination and myosin and actin quantification using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The remaining fish will be weighed to determine cook yield. Fish fillets will be placed on smoke racks and thermally processed in a microprocessor-controlled smokeoven (Model CVU- 490; Enviro-Pak, Inc. (Clackamas, OR). Following thermal processing to an internal temperature of 65�C, fish fillets will be weighed and vacuum packed for texture analyses.

Proximate analyses will be conducted for fresh and smoked trout using standard AOAC (I 990) procedures. The pH measurements will be carried out using a pH/Ion analyzer (Coming, Inc.; Coming, NY). Proteins will be extracted using 2% NaCl, and extract pH will be subsequently measured using the procedure described by Nayak et al. (I 996). Characterization of soluble proteins will be carried out by SDS-PAGE, and actin and myosin will be quantified as described in an earlier report (Nayak et al., 1996) using video-image analysis of gels. Expressible moisture will be measured according to Jauregui et al. (1981). Percent NaCl will be determined using QUANTAB Chloride Titrators (Environment Test Systems, Inc., Elkhart, IN). Cook yields will be determined based on weights prior to and following thermal processing. Brine uptake will be evaluated based on weights of fresh fish and brined fish prior to thermal processing.

Model System Evaluations: A trout mince will be prepared from five fish per treatment combination. Either 2.0% NaCl or 2.0% NaCl and 0.4% sodium tripolyphosphate (STPP) will be added to 300 g of ground trout muscle. A water addition of 10% will be used in each evaluation. Water, ingredients, and ground trout will be chopped in a Culsinart food processor (Model DLC-8M, Cuisinart Corp., NJ) for a total of 45 seconds. Chopping will be interrupted at 15 sec intervals to scrape the sides of the bowl. Following chopping, pre-blends will be vacuum packaged and held for 12 h at 4� C. After 12 h, each batch will be stuffed into three, 2.8 cm dia. polypropylene centrifuge tubes and heated to an internal temperature of 65�C. Internal temperature will be recorded at one-minute intervals on a temperature recorder (Model 205 Datalogger, Beckman Industrial, Carico Inc.; Overland Park, KS). Gels will be cooled to room temperature, and three 12.7 mm thick X 19.1 mm diameter cores will be removed from each cooked trout gel. Using a Texture Analyzer (Model TA-HDI; Texture Technologies; Scarsdale, NY) each core will be axially compressed at 127 mm/min to 50% of its original height for two cycles. Gel hardness (Boume, 1978; peak force of the first compression) and cohesiveness (energy of second compression as a percent of energy of the first compression) will be determined. A portion of the raw batters will be extracted with either a 2.0% NaCl or 2.0% NaCl and 0.4% STPP solution that mimics the concentration of these ingredients in the aqueous phase of the raw batter. Total soluble protein and myosin will be determined (Nayak et al., 1996). Myosin will be separated from total soluble protein using SDS-PAGE. Gels will be stained with Coomassie blue, and following destaining, the gel image will be captured for evaluation by the Optimas image analysis system (Optimas Inc., Edmonds, WA). The gels will be scanned and individual bands of myosin quantified against a standard amount of myosin loaded in a reference lane. In addition to myosin quantification and to provide a more thorough characterization of protein changes, differential-scanning calorimetry will be used to develop endothermic peaks for soluble proteins. Because protein-water and protein-protein interactions are significant to the functionality of muscle foods, any differences in the utility of the raw material from different operations will be rationalized on the basis of differences in these functional traits assessed by SDS-PAGE and differential scanning calorimetry.

OBJECTIVE: Determine and implement appropriate technology transfer activities to disseminate information generated by this project to the aquaculture industry in Appalachia, to state agencies with aquaculture-related responsibilities, and to the general public.

Personnel: The investigators in the other objectives will participate in this objective. Specific initiative within this objective will be coordinated by Kenneth J. Semmens and Richard Zimmerman.

Expected Outcome: An appropriate technology transfer component will disseminate the results of this applied research and will result in improved opportunities for development of aquaculture enterprises in rural areas of Appalachia.

Procedures: The approach of this objective is to develop appropriate procedures, including research to determine more effective methods, and to disseminate information to producers, processors, related businesses, public agencies, and the general public. Consequently, as the research results become available, programs will be designed, tested, and initiated to improve communications with the aquaculture industry, other public agencies and the general public. The investigators will continue to team up with the Freshwater Institute's Aquaculture Program, the West Virginia Department of Agriculture, the West Virginia Aquaculture Association, and other organizations to deliver research findings in the most "user friendly" manner possible. Evaluations of alternative delivery mechanisms will be carried out to assure that the more effective methods are determined and utilized.

Work with the WV Department of Tourism to identify methods and materials required to make fee fishing more visible to tourists in West Virginia and incorporate these materials into their promotional activities.

OBJECTIVE: Characterization of effluents from West Virginia trout production facilities, optimize a working system, and evaluate of impaired water from mine sites.

Expected Outcome: The expected research outcomes are the establishment of baseline conditions unique to West Virginia trout production operations using flowing water systems. An additional outcome is the determination of the technical feasibility of developing impaired waters or production of rainbow trout through development of a facility near the WVU campus.

Personnel: Roger C. Viadero, Jr., Assistant Professor of Civil and Environmental Engineering, and Kenneth J. Semmens, WVU State Extension Specialist for Aquaculture.

Background: Commercial trout production enterprises in West Virginia must ultimately satisfy National Pollution Discharge Elimination System (NPDES) requirements; however, most facilities have not yet applied for permits. While conventional wisdom has been that the WV Department of Environmental Protection (DEP) will not require permits for facilities that apply <20,000 lb. of feed on an annual basis, it is expected that regulatory pressures will extend to those facilities that are currently believed to be exempt. The objective established for this research is to develop baseline water quality data for a variety of trout production facilities in West Virginia. To meet the emerging needs of the aquaculture industry, a water quality assessment of West Virginia trout producers will be performed to develop baseline water quality data for the industry. The knowledge gained from this study will provide relevant and timely information necessary for industry, State, and Federal stakeholders to make informed decisions regarding effluent management and the efficient future use of water resources.

Task 5. 1: Baseline Water Quality Data and Characterization of Effluents from West Virginia Trout Production Facilities

Water quality assessments in the aquaculture industry have been performed by other investigators, with notable research conducted in North Carolina (North Carolina Department of Environment, Health, and Natural Resources, 1994) and Virginia (Seelong and Helfrich, 1998). However, the primary focus of such studies has been on downstream impacts of aquaculture operations on receiving bodies in which increased nutrient loading, algae blooms, etc. have been reported.

To ensure the anonymity of producers and their corresponding water quality data, all facilities selected for inclusion in this study will immediately be assigned a code number for all future reference, For reporting purposes, the results would be pooled without identifying reference to a particular facility. Specific water quality data regarding individual production facilities will be shared only with individual operators.

Site Selection: Seven (7) West Virginia trout production enterprises will be selected for participation in the baseline water quality assessment. To enable inter-facility comparisons, each of the selected production enterprises will raise rainbow trout in a flowing water system. Site selection will be based upon the following criteria:

Water source. To account for the diverse nature of water resources in West Virginia, trout production enterprises utilizing waters from natural springs, ground(/surface waters, and mine waters will be considered for inclusion in the study. As an aid in developing data on the flow and initial characteristics of each water source, particular attention will be paid to the depth and breadth of data available on each water source from the US Geologic Survey and/or the West Virginia Department of Natural Resources (US Geologic Survey, 1984).

Facility size. Since NPDES permits have traditionally been issued based on the annual rate of feed applied, a range of facility "sizes" will be investigated. Three size ranges have been preliminarily identified:

Dr. Kenneth J. Semmens, West Virginia University Extension Service Aquaculture Specialist will assist with the identification of suitable sites, as he has an in-depth knowledge of the West Virginia aquaculture industry.

Effluent treatment processes: Due to the wide variety of water resources and hatchery sizes, many different treatment processes are used by trout producers in West Virginia. Treatment processes range from direct discharge into receiving bodies from culture raceways to more sophisticated designs involving sedimentation and bio-filtration. Through the inclusion of facilities with a range of treatment operations, the benefits of additional treatment processes on raceway trout production facilities can be preliminarily ascertained.

Operator feeding practices. Each facility will be characterized according to the type and quantity of feed used. Attention will be paid to the inclusion of producers who use "high yield" fish feeds. The aim is to ascertain whether such feeds reduce waste loading for the particular case of raceway trout production in West Virginia. To arrive at a comparative conclusion regarding the effect(s) of high yield feed, at least one facility that uses a conventional feed will be included in the water quality study. Further, the water quality effects of altered feeding practices such as decreased feeding during times of high environmental stress (high ambient temperature and low water flows typically experienced during summer months) can be ascertained through effluent monitoring.

Analytic Parameters The following are required water quality parameters which must be monitored and reported for current NDPES regulated fish production enterprises in West Virginia: waste flow rate; five-day biochemical oxygen demand (BOD5); total suspended solids (TSS); ammonia nitrogen (NH₄⁺-N); settable solids; dissolved oxygen (DO); and pH. A summary of typical water quality parameters and regulatory limits required by the West Virginia DEP are presented in Table I (Personal communication, Michael V. Shingleton, WV Department of Natural Resources).

Water Quality Parameter	Regulatory Limit
BOD5 Total suspended solids Total settable solids NH₄⁺-N pH	60 mg/L 60 mg/L 0.2 mL/L 10.44 mg/L 6 - 9

Once facilities have been selected for inclusion in the study, an analytic sampling plan will be devised through consultation between the investigators and hatchery operator(s). Water quality parameters to be measured include influent and effluent (field measurements will be made for parameters marked with an asterisk[*]):

Further, it is proposed that influent, process water, and effluent turbidity be added to the list of analytical procedures conducted during field water sampling. A correlation between turbidity and analytic laboratory data (BOD5, TSS, TDS, etc.) will be developed with the goal of enabling the use of turbidity as an indicator of real-time water quality, process performance, and pollutant loading. Consequently, a simple field assay such as turbidity may then be used as a predictive tool to make informed decision regarding changes in water quality.

Additionally, particle size analysis (PSA) will be conducted on water quality samples to study the dissolution of solids in process waters. Particle size analysis will provide insight into the transfer of nutrients from the solid to the aqueous phase which will then be used to quantify the kinetics of dissolution and consequently, nutrient loading.

Influent and effluent water quality parameters will be measured in order to perform a "before" and "after" comparison to ascertain the net effects of the hatchery on water quality. Each parameter will be measured according to the applicable "Standard Method" (APHA 1998) or US EPA (I 997) approved analytic method as outlined in subsequent sections. Special sampling events will be arranged with hatchery operators to ascertain the water quality effects of applicable operational events such as raceway cleaning, bio-filter back-flushing, etc. Such sampling events will be made on an as needed basis, as determined by the operational routine of the individual facilities selected for inclusion in the study.

Concentration Measurements: Sample analyses will be conducted according to the applicable APHA Method, as presented in Table 2.

Table 2. Water Quality Permit Parameters, Analytic Methods, & Typical Analytic Values

Water quality parameter	APHA Method¹	Typical Values²
BOD5 Total suspended solids Total settable solids NH₄⁺-N pH	5210 B. 2540 B. 2540 F. 4500 C. NH4+-N 4500 B. H+	30 mg/L 30 mg/L -- -- 7.26

Routine Sampling: To ensure sufficient baseline data, routine water quality sampling will be performed twice per month for the first six months of the study. Routine water quality sampling will consist of sample collection at the following points in the process:

After the first six months, routine sampling will then be conducted at a rate of one event every month for the balance of the study, except under special circumstances.

Special Sampling: Periodic operational events such as bio-filter back flushing will necessitate special sampling to ascertain the effect(s) of such an operation on effluent water quality and process performance. The frequency of such activities is seasonal in nature; thus, water quality impacts must be ascertained in special sampling events. Prior to bio-filter back flushing or solids removal from the sedimentation basin, water quality samples will be taken at each of the routine sampling points to establish baseline water quality conditions in the system. Attention will be paid to ensure routine sampling and special sampling occur at the same time, when operationally possible. (Routine samples will correspond to pre-flushing or pre-solids removal samples.) Consequently, it is estimated that at least one special sampling event will occur in each quarter resulting in additional analytic samples (sludge solids and/or flushing solids concentration).

Technical and Analyses: Water flow and quality data will be used to determine the technical effectiveness and feasibility of applying each treatment to aquaculture processing and waste streams. The technical effectiveness of each process will be based on the reduction in solids, ammonia nitrogen, BOD5, etc. achieved by each unit operation.

This quality assurance narrative (PQAN) is not intended to take the place of a more rigorous Quality Assurance Plan (QAP) which will be formulated and submitted for approval prior to initiating laboratory studies. Details on experimental conduct and sampling locations, frequency, etc. have been provided previously in the Experimental Approach and Rationale section. Thus, quality practices related to sample analysis and data reduction are the primary focus of this proposal quality assurance narrative.

The objective of the proposed research is to develop an engineering-based rationale for characterizing and consequently improving on a solids and nutrient removal system currently used in an operating trout culture facility. As a result, much of the data collected will be based on field sampling and analysis.

Analytic samples collected in the course of the study will be labeled and a chain of custody will be maintained throughout the life cycle of each sample, as per ANSI/ASQC E4, "Specifications and Guidelines for Quality Systems for Environmental Data Collection and Environmental Technology Programs." Further, all samples will be collected and preserved according to the applicable Standard Method for the Analysis of Water and Wastewater (APHA 1998). Additional information on the methods used in sample analysis was specified in a previous subsection of the proposal (see Table 2). An audit of sample collection and custody practices in addition to instrument calibration and maintenance procedures will be performed quarterly and reported in the QA/QC section of each technical progress report.

Particle size distribution and number count will be ascertained. Particle size analysis will be conducted using a Coulter LS 230 particle size analyzer capable of measuring particles in a range of 0.25 to 250 �m (typical size of solids in aquaculture systems are approximately < 200 [tm (Summerfelt and Wade, 1997). The minimum sample volume necessary for particle size analysis is 2 mL.

In the event analytically determined concentrations are below the detection limits, statistical data reduction methods outlined in Section 4.7 of EPA QA/G-9 will be followed. The following four-tiered quality assurance/quality control procedure will be adhered to in the processing of all analytical samples:

All statistical analysis and data reduction will be performed in accordance with guidelines established in EPA QA/G-9, Guidance for Data Quality Assessment, Practical Methods for Data Analysis. As experimental data are analyzed, a formal structure to identify and account for "outliers" will be established and data will be further reduced according to Section 4.4 in EPA QA/G-9. When triplicate experiments are performed, statistical comparison mechanisms prescribed in EPA QA/G-9 will be followed.

Task 5.2: Technical Assessment of Impaired Water Resources Suitable for Production of Trout in West Virginia

The goal of Task 5.2 research is to ascertain the technical feasibility of utilizing impaired mine waters for future production of rainbow trout in West Virginia. This task will indicate how impaired waters may be used to grow trout from an engineering perspective, whereas the mine water assessment in objective two will determine where sites are located and the suitability for aquacultural development.

Background: Much of the mine water in northern West Virginia is associated with coal seams high in sulfur and pyrite (FeS). Oxidation of pyrite causes a decrease in pH and a corresponding increase in dissolved metal concentrations. In the mine water inventory study conducted by Jenkins et al. (1 995), sixteen sites with flow rates of 2,000 to 5,000 gpm were reported; however, only 31% of the sixteen sites were deemed useable without treatment for acid mine drainage (AMD). Of the top 20 sites listed in the inventory, only four were from southern West Virginia. Mining companies spend considerable sums to treat water discharged under the NPDES program. The reliability of treatment methods and the associated risks posed by spiking of critical water quality parameters tend to discourage investment in production facilities associated with these water sources, regardless of the actual risk and associated costs.

West Virginia University is favorably positioned to study this problem, as there are a number of mine sites in proximity to the campus which have impaired water flow rates in excess of 1,000 gpm. Further, expertise in mining and mine-related issues is available throughout the campus and across the State. The Department of Civil and Environmental Engineering will prove integral to this line of research. The investigators will also collaborate with the Freshwater Institute as this project develops. This line of research has potential usefulness to engineers, mining companies, environmental groups, resource managers, trout producers, and other diverse disciplines.

Experimental Approach: A successful aquaculture system utilizing impaired waters consists of three basic components: (1) pretreatment, (2) production, and (3) effluent treatment. Further details of each stage are provided below:

Pretreatment: Systems to safely deliver the quantity and quality of water necessary for a traditional flowing water system will be assessed. Methods currently employed for mine water treatment as well as traditional water treatment technologies used in other industries will be surveyed and evaluated as potential means to remediate the impaired water sources with minimal effects on water flow rate. Attention will be paid to system reliability, the potential effects of treatment on the fish culture system, and pretreatment cost.

Production: Based on the physical characteristics of sites identified earlier, preliminary layouts for actual production units of sufficient size necessary to conduct production-scale research will be made. A primary focus in the layout of such facilities will be on siting production units to make maximum use of West Virginia's unique topography as a means to defray costs associated with water pumping.

Effluent Treatment: Systems that can recover and remove bio-solids from the effluent of the production cells will be assessed. Specific items to be considered in the assessment of effluent treatment processes will be similar to those detailed above in the pretreatment section. Care will be taken to coordinate the work conducted in this task with work conducted by investigators who will be evaluating effluent characteristics in studies conducted in Task 1 research.

The mine water survey presented by Jenkins, et al. (1995) contained primarily economic data based on water flow rates reported as part of NPDES permit requirements. Only a limited summary of water quality data was also provided. Consequently, where reliable water quality data are not available for selected sites, samples will be collected and tested for the presence of metals (especially iron, aluminum, and manganese); pH; temperature; and alkalinity. Additionally, mine water flow rates will be measured in the field when water quality samples are collected.

The findings of the above research will be summarized in a formal engineering-based technical feasibility report. Specific features of impaired West Virginia mine waters that make the development of the resources advantageous to the aquaculture industry will be identified. The investigators will present a detailed identification of the technical and economic hurdles that must be overcome to realize the efficient use of impaired waters for aquaculture. Engineering- based solutions to each technical hurdle will be forwarded, when appropriate.

OBJECTIVE: Conduct research at the farm level focusing on production efficiency of facilities growing food size rainbow trout and fish health.

Expected Outcomes: Development and implementation of a protocol synthesizing best management practices for production of food size rainbow trout on two West Virginia trout farms. Verification of yield will validate research recommendations and serve as a conduit of information exchange between fan-n application and current or proposed research projects. This project will also provide outstanding training to participating county agents.

Personnel: Scientists responsible for accomplishments in this task are Kenneth J. Semmens (W-VU), Jonathan Eya (West Virginia State College) and Julia Detabbio (Bluefield State College).

Task 6.1: Develop and Implement a Pilot Yield Verification Program for Food Size Rainbow Trout in Flowing Water Systems at Two Commercial Facilities

Background: Yield verification programs have proven successful with a variety of crops, including cotton and soybeans. They also have been used for channel catfish. A program brings together people working in research, extension, and on the farm in a framework that focuses on verification of production methods and results. The heart of a yield verification program is a protocol forged with a consensus by all parties regarding how the product (in this case rainbow trout) is to be stocked, tended, and evaluated. Participating farmers agree to devote part of their facilities to the program. Extension personnel assist with collection of data on the farm and researchers analyze the data. Actual production results are then examined by all parties and the protocol is reevaluated. With a consensus, the protocol is then modified and implemented in another production cycle. Thus, through a series of production cycles there is incremental improvement in a variety of production parameters. This program provides a mechanism for all participants to contribute within their discipline to improve farm level performance.

The objectives of the yield verification program are to conduct on-farm trials and verify the utility of research-based extension recommendations. The expected results include:

Research Plan: This will be a cooperative effort with aquaculture extension and research personnel from across the state and the two largest private producers of food size trout in West Virginia. High Appalachian, LLC operates two large Debased systems with coal mine water sources. They also operate a processing facility for their product near Beckley. Trout Lodge and Anglers Resort near Lindside, West Virginia is a raceway system with a spring water source. Most fish grown at Trout Lodge and Angler's Resort are marketed to recreational outlets.

A Yield Verification Technical Committee will be created to develop a protocol for growing food size rainbow trout and to implement the project. The Committee will be composed of Jonathan Eya (West Virginia State College), Julie Delabbio (Bluefield State College) and. Kenneth Semmens (West Virginia University). They will discuss and agree on recommended technology, data collection and production procedures appropriate to each facility and will consult with additional research personnel when appropriate. Jonathan Eya will coordinate yield verification of the High Appalachian facility and Julie Delabbio will coordinate yield verification of the Trout Lodge and Angler's Resort. This arrangement takes advantage of additional aquaculture expertise in West Virginia academic institutions, and the relative proximity of these institutions to participating trout producers. Coordinators will be responsible for collection of required data on a weekly basis and during stocking and harvest activities. Coordinators will also be responsible for organizing and summarizing data into products useful to other project participants. Water quality, flow rates, production inputs, stocking and harvest data will be collected from two production units at each facility. Kenneth Semmens, a research assistant, and county extension staff will assist with data collection as needed.

A production cycle lasting approximately six months will permit two to three cycles through the two-year project. Cooperating farms will be provided with the feed required to grow fish in the two production units not to exceed one ton per production cycle. This will permit the flexibility to select a feed different from the diet normally fed by the producer. The first cycle will serve as a baseline and will reflect production efficiency of each farm as it operates prior to adoption of a production protocol. This provides opportunity to consider the standard operating procedures utilized by each farm which will serve as a basis for development of production protocol version one. In the second production cycle the chosen protocol will be implemented. In subsequent cycles the protocol will be examined and modified as deemed appropriate.

This program provides a framework within which work from other objectives can be applied and validated. The production information derived from this program permit modification to enterprise budgets based on West Virginia data. Simulation models (Objective 2) will also benefit from this hard data from West Virginia farms. The farms cooperating in this study will also participate in the Health Survey (Task 6.2 ) and perhaps the Waste Characterization study (Objective 5). The health information may lead to strategies for minimizing mortality and maximizing fish condition.

Results from this project will be summarized on the WVU Cooperative Extension Service aquaculture web site. They will also be summarized and presented at a joint meeting of WVU Cooperative Extension Service and the West Virginia Aquaculture Association. Further, results will be distributed by direct mail to individuals and businesses on the "Fish List" maintained by WVU Cooperative Extension Service and on request, through Bluefield State College and West Virginia State College.

Expected Outcome: Managers of trout production facilities will determine if fish grown in their production facilities satisfy health requirements for transportation of live fish in Maryland and Pennsylvania. This will permit certain facilities to become certified and enhance their opportunity for interstate trade of live trout. This survey will also determine the presence or absence of restricted pathogens in trout production facilities throughout West Virginia and may assist with preventing the transport of pathogens currently restricted by law.

Personnel: Scientists responsible for accomplishments in this task are Julie Bebak, DVM, Ph. D., The Freshwater Institute; Ana Baya, Ph.D., Maryland Department of Agriculture; and Kenneth J. Semmens, Ph.D., West Virginia University.

Background: From the perspective of the West Virginia trout producer, aquatic animal health policies in adjacent states have become an effective barrier to marketing live fish transported across state lines. West Virginia and Pennsylvania require testing for viral pathogens (IHNV and IPNV or VHSV). Maryland recently implemented a policy covering all species of aquatic animals and eighteen pathogens listed in four categories. West Virginia vendors wishing to sell live trout into Maryland's recreational markets face the prospect of paying for at least seven tests to satisfy the new Aquatic Animal Health Policy. Presently the cost for these tests is $550, plus 60 fish and transportation of the sample to the diagnostic laboratory. Most private production enterprises in West Virginia rely on spring water and purchase eggs certified to be free of specific pathogens. Thus, they may be producing trout free from the pathogens of concern, but cannot demonstrate this without testing for their presence. Virtually none of the trout producers in West Virginia have conducted testing to certify their facilities under the new policy and may consider the health policies to be a bureaucratic barrier to trade. While wild and cultured fish populations must be protected from the introduction of fish pathogens, there also is a need for the producer to conduct business in compliance with developing aquatic animal health policies. Whether real or perceived, many of the barriers can be removed through testing and education. Obtaining consistent results for detection of fish pathogens provides a basis for encouraging the trout grower's trust in the soundness of public aquatic animal health policy.

Research Plan: This activity provides a mechanism by which trout farmers may become certified for interstate transport of fish through participation in a state-wide health survey. The task requires participation by an aquaculture veterinarian, who will oversee sample collection and shipping, maintain records, report results