Developing the Mine Water Resource
Use of Farm Raised Fish in Recreation

Commercial Production of Trout and Developing the Mine Water Resource
Use of Farm Raised Fish in Recreation
Technology Transfer

Project Description

Economic development in West Virginia is critical, particularly in rural communities where traditional economic activities (principally coal and timber) have declined.  Rural economic development has been a principal objective of the Aquaculture Food and Marketing Development Project (AFMDP).   Strategies where aquaculture can impact economic development in West Virginia and adjacent states are:   (1)  development of mine water resources for commercial production of foodfish, and  (2)  use of farm raised fish in recreation.   Conducting work in both of these areas will encourage development of a dual market for growers in the region strengthening both components of the industry.

Six years of funding have been approved for this project and the work is in varying stages of completion.   Work presented in this proposal is designed to complement the work which has been completed and presently underway.  

Objectives (FY 2003)

Production Evaluation of Two Commercial Diets for Rainbow Trout in Treated Mine Water.   One diet treatment contains fish meal and the other does not.  The evaluation will include estimates of feed conversion ratio, growth rate, cost, quantity and characteristics of solid and dissolved waste production, and the concentration of organochlorine compounds (e.g., polychlorinated biphenyls, dioxins, etc.) in fish flesh grown in WVU’s mine water raceway. 

 Protein and Lipid Recovery from Fish Processing By-products:  Process Scale-up from Laboratory-batch to Continuous Semi-industrial Application.  The objective is to scale up the protein and lipid recovery batch process to continuous semi-industrial application.

 Assessment and Development of a Legal/ Institutional Framework for the Development of the Aquaculture Industry in West Virginia.   Legal, regulatory and institutional issues affecting the aquaculture industry in West Virginia will be defined, current laws relating to aquaculture issues in West Virginia, other states and the federal level will be examined, alternative prescriptive strategies will be developed, and educational and technical materials will be developed for the dissemination of information to individual policy makers and the aquaculture industry.

 Developing and Marketing Fishing Based Travel Packages.  Study sites which show the greatest potential for forming recreational travel packages consistent with the user segment needs and wants will be identified and recommendations will be developed for recreational travel packages targeting selected markets.

 Nutrient Removal from Trout Raceway Effluent Utilizing Watercress. Watercress will be evaluated as a tool to remove to remove nutrients from aquaculture effluent.  Operational conditions which affect nutrient removal and watercress growth will be determined.

  Technology Transfer.  Information developed from this research and previous research will be integrated into an effort to educate people regarding how aquaculture is conducted and its role in economic development.

Progress Report

Developing the Mine Water Resource

 Much of the research supported by the Aquaculture Food and Marketing Development Project has involved production and processing of cool water food fish.  A long term objective is to develop a modular raceway system and work to improve raceway system design for commercial production of trout as a foodfish.

 Honeycomb Fiber Reinforced Polymer (HFRP) Materials The initial design of the HFRP raceway system was based on the requirements of the users, who defined the length, width and height of the individual units. A detachable quiescent zone was also designed to allow for future modifications while at the same time performing research on various waste removal techniques.

 Beam type samples were produced and tested to evaluate the bending stiffness. Two different types of computer models were developed to verify the experimental results. Two different designs for the side-to-bottom panel connections were tested in the linear range to obtain connection stiffness. Based on these results, modifications to the preliminary design were suggested before the tanks were installed at Dogwood lakes. Beam samples were subsequently tested to failure to evaluate bending strength properties and failure modes. These results showed consistency with the finite element model results. Based on the above tests, a computer model to predict the raceway behavior in the field was developed.

Torsion tests on the sandwich beams with different fiber lay-ups was has been completed. Research on the delamination of facesheet from the core and studies on the facesheet-core interface stresses are underway. A comprehensive article with a detailed discussion on the work done so far will be submitted to the Journal of Composites for Construction.

 Production of trout in treated mine water   In previous work, it was demonstrated that the use of treated coal mine water for rainbow trout (Oncorhynchus mykiss) culture in a cage was technically feasible (based on water quality studies and a 50 fish bioassay).  However, no work on production related issues was conducted.  To further advance the use of treated mine water, work was conducted to assess the effects of using treated coal mine water for the intensive production of rainbow trout in a flow through system.  During this study, comprehensive water quality data were collected to supplement fish weight and length data collected during routine monthly sampling events.  The 8,000 fish grew well in the raceway system over the nine months of production, where a feed conversion ratio of 1.4 and a condition factor of 0.0005 were measured at a stocking density of 52.6 kg/m³ (3.3 lb/ft³).  Further, total net production was 3,657 kg (8,045 lb) with only 1.4% mortalities.  Throughout the study, dissolved ion concentrations (Fe, Al, Mg, Ca, and SO₄) often exceeded recommended limits.  Further, elevated ammonia nitrogen concentrations generated from a component of the mine water treatment process were identified as a potential limiting factor for aquaculture development.  However, when the non-ideal effects of high ionic strength and the speciation of dissolved metal–ligand complexes were taken into account, the concentrations of free metal ions were within recommended limits.

 The modular raceway system, constructed previously in Grant 3, was stocked with a total of 10,000 rainbow trout in October 2003.  Goals of this study are to further study production aspects related to rearing fish in treated mine water, compare the production performance of two trout diets, and to assess solids removal in the quiescent zone and develop methods to enhance the performance of effluent management techniques. Further, data collected in this and subsequent work will be used to develop and calibrate a model for trout production using treated mine waters in a flow through raceway system.  A monthly sampling regime was implemented consisting of assays made on water collected from the head box and each quiescent zone for: nutrients (NH₃, NO₃^-, NO₃^-, total phosphorous), metals (Al, Ca, Fe, Mg, Mn, Ni, Zn, and Cu), alkalinity, acidity, pH, water temperature, specific conductance, salinity, dissolved oxygen (DO), sulfates, 5 day biochemical oxygen demand (BOD₅), and total suspended solids.  Routine fish counts and measurement of mass and length in each raceway segment are carried out.  Yellow Spring Instruments (YSI) data sondes (instruments capable of measuring water quality parameters on a continual basis, without operator intervention) were employed at the inlet and outlet waters of the facility to develop a continuous stream of water quality data.  Further, a mechanism for sampling solids from the quiescent zone of each raceway segment was designed.

 Physiology and Contaminants  Three strains of juvenile rainbow trout were grown in a flow-through raceway supplied with reclaimed mine-water (treatment fish) from October 2002 to May 2003.  Sibling counterparts (control fish) for each of the strains were grown in flow-through, circular fiberglass tanks at the USDA Center for Cool and Cold Water Research during the same time period.  Throughout the study, all fish were fed daily with commercial trout chow.  Growth, physiological status, heavy metal content, and water quality parameters were measured at least monthly throughout the study using standard methods and materials.  Results for the physiological and heavy metal assessments are presented here.  Blood samples were collected using heparinized syringes for assessment of plasma chloride, glucose, and lactate concentrations, and whole fish were collected for assessment of selenium, magnesium, iron, manganese, and aluminum concentrations.

 Results of the study show mean plasma glucose and lactate concentrations (indicators of energy-balance and anaerobic activity, respectively) were within the normal physiological ranges of rainbow trout.  No differences were noted among the three strains of treatment fish, or between fish grown in reclaimed mine-water and the control fish grown at the USDA facility.  Plasma chloride differed significantly between the mine-water fish and the control fish, averaging 118.2 (+ 1.3 SEM) mEq/L and 104.7 (+ 0.3) mEq/L, respectively.  However, both means were within the normal range reported for rainbow trout (approximately 100 to 135 mEq/L); the dissimilarity likely resulted from general water quality differences between the two sites, especially pH and the carbonate constituents which have been shown to directly influence plasma chloride steady-state concentrations through the carbonate equilibrium system and concomitant ion balance. 

 Whole body heavy metal concentrations were highly variable among individual fish from all strains and locations for the study dates.  The following table shows the high and low means for whole organism heavy metal concentrations among all study groups, as well as the overall mean and standard deviation for each of the heavy metals assessed in fish between October 2002 and January 2003.  Heavy metal analysis is still continuing. 

* The unit of measurement for all heavy metals was mg/kg, except mercury.  Mercury was measured in ug/kg.

 Results did not indicate notable differences among study groups or among strains for any of the metals assessed to date.  However, because the responses among individual fish within treatment and control groups were highly variable, such results should be interpreted with caution.  Individual fish may show increased or decreased susceptibility to bioaccumulation of heavy metals, which in turn may affect whether individual fish are safe for human consumption.

 Economic analysis of impaired water sources/mine water aquaculture  With the aquaculture industry growing rapidly, the feasibility of alternative water sources for raising fish is being explored. Accordingly, we undertook a comprehensive economic analysis of mine water aquaculture.  We found that mine water aquaculture is profitable under the conditions investigated and can be a potent economic development strategy; however, like other types of aquaculture, it is risky. Theoretically, a combination of species and sales outlets is a desirable diversification (or risk reduction) strategy; once more data become available, we will investigate the profit-risk relationships of different combinations.  In addition, studies are needed to document consumer acceptance of fish produced in mine water environments. We demonstrated that mine water aquaculture can be beneficial from the viewpoint of individual aquaculture entrepreneur and statewide economic development.  Economic benefits to the mining company can also result and need to be quantified in subsequent research. 

 Effluents  Due to the impending promulgation of new federal effluent discharge regulations in the aquaculture industry, it is necessary to formulate methods of efficiently separating solid wastes from discharge waters.  However, before alternate designs can be considered, the hydrodynamic properties of traditional systems must be studied to adequately establish baseline characteristics of typical designs.  An Acoustic Doppler Velocimeter (ADV) was used to characterize the water velocity at specific locations in typical raceway systems and thus, establish baseline hydrodynamics.  The ADV is used to measure water velocity in three dimensions, making it possible to understand flow patterns at any point within a raceway.  Velocity measurements were made in flow-through systems raceway systems at multiple depths and distances from the raceway walls along the entire length of the raceway, with greatest emphasis placed on the characterization of quiescent zone hydraulics.

 Health Survey.  In 2001 and 2002, West Virginia salmonid farmers participated in a Fish Health Survey.  Six private farms and nine state facilities were sampled in 2001.  Eight private farms and seven state facilities were sampled in 2002.  This study could enable a farm to obtain Fish Health Certification (FHC).  

 Number of farms positive for certifiable pathogens

 Cost for FHC was $3,000.00 to $5,000.00 per farm.  Sampling for Myxobolus cerebralis (whirling disease) represented about 20 to 30% of the cost.

 Survey of private producers – experience and attitudes

• Producers sold live fish to WV, VA and MD.   Even for states where certification was required, no farmers were asked for proof of certification.  One producer commented that in more than 10 years of selling fish in WV, MD and VA, he was never asked for FHC paperwork.
• Number that agree that the following is true of fish health certification (out of 10 facilities)
  • It is an important part of a biosecurity program – 9/10
  • It has no value except as an annoying regulatory requirement – 0/10
  • It helps protect my farm from introduction of unwanted disease organisms – 10/10
  • It gives me confidence that I am shipping healthy fish to my buyers – 10/10
  • I can charge more for certified fish – 6/10
  • The regulations need to be changed so they are the same from state to state – 5/10
  • The regulations need to be more logical, that is, based on risk – 3/10
• Eight of ten responders replied they could not afford the full cost of certification ($3,000-$5,000).  However, eight of ten indicated they would pay for part of the service, ranging from 10% to 100% of the cost.
• If a program of on-call fish health services were available, eight of ten farms indicated they would definitely use the service, one would probably use it and one would not use it. 

 Certifiable pathogens (e.g., IPNV, Aeromonas salmonicida and Myxobolus cerebralis) that are endemic in the northeastern U.S. were detectable, but at low prevalence, in the WV salmonid industry.  The non-endemic pathogens IHNV and VHSV were not detected. These results present an opportunity to the industry to quickly benefit from the implementation of a state-wide biosecurity and fish health management program.  Enforcement of FHC requirements by importing states needs improvement. Fish health certification costs are too high for most producers to pay the full cost.  However, the majority are willing to pay some portion of the costs.The majority of the participants understand the value of FHC as a biosecurity and product quality tool.  No participants viewed it as simply an annoying regulatory requirement.

 Feeding Rainbow trout a finishing diet supplemented with Vitamin E   Supplementation of fish feed with antioxidants increases tissue levels of antioxidants and may reduce lipid oxidation depending on postmortem storage and handling practices.  Vitamin E supplementation increased fillet α-tocopherol content (p<0.05) from 56.9 mg/kg at week 2 to 156.5 mg/kg at week 9.  Storage through 14 days at 0 °C did not affect fillet vitamin E content (p>0.05).  Fillet vitamin E content did not affect lipid oxidation during refrigerated storage; thiobarbituric reactive substances ranged from approximately 0.5 to 2 mg/kg.

 Transport and Stunning of Arctic Char  Arctic char were transported in AQUI-S™, CO₂, ice-slurry, or water for 5.5 h followed by CO₂ or percussion stunning.  Transportation in an ice slurry followed by percussion stunning resulted in the highest pH, and CO₂ stunning reduced muscle pH, regardless of transport treatment.  Fillets from control fish that were not transported were firmer than all transport treatments.  Stunning with CO₂ resulted in firmer cooked fillets than percussion stunning, and this effect was evident through 4 days postmortem.  Transport and stunning did not affect muscle pH after 4 days of  refrigerated storage.

 Results also indicated that the anesthetic treatments investigated in this project (AQUI-S™, ice-slurry, and carbon dioxide) were not effective in consistently reducing the physiological responses of Arctic Char during simulated transport.  Treatments did not provide a beneficial effect over the controls.

 Effects of nitrite and carbon dioxide on Rainbow trout (Oncorhynchus mykiss) 

Objectives were to determine the effects of nitrite and carbon dioxide on the survival and physiological responses of rainbow trout.  Results from this project indicate that nitrite toxicity is affected by elevations in environmental carbon dioxide, and conversely, carbon dioxide tolerance is affected by environmental nitrite concentrations.  We feel that additional experiments examining the interacting effects of nitrite and carbon dioxide on fish are needed to identify the physiological mechanisms or effects that lead to increased nitrite and carbon dioxide toxicities in fish.

 Cryprotection and Restructured Trout   Vacuum tumbling was evaluated as a means to enhance sucrose penetration into trout fillets and thereby protect against protein denaturation during frozen storage.  Vacuum tumbling for extended time increased (p<0.05) sucrose content of the muscle; all tumbling treatments reduced (p<0.05) thaw loss, and gels produced from these treated muscles were firmer and more cohesive (p<0.05).  Subsequently, various cryprotectant treatments ( 8% sucrose/sorbitol, 8% trehalose, 8% trehalose/sorbitol, and 2% sodium lactate) were evaluated in restructured trout following 1, 3, 5, 10 and 15 freeze-thaw cycles (-20 °C; 8 h and 3 °C; 16 h).  Activa™, a commercial restructuring technology using transglutaminase, was employed to produce these products.  Sucrose/sorbitol, trehalose, and trehalose/sorbitol reduced thaw loss and product toughening caused by freeze/thaw cycling.  Sodium lactate reduced lipid oxidation and bacterial growth in these products without adversely affecting color.

 Proteins and Lipids Recovered From Trout Processing By-products   Trout mechanical filleting yields 40% of fillets from raw fish.  The remaining 60% of by-products contain valuable muscle proteins; primarily myofibrillar and sarcoplasmic.  These proteins, if recovered, could be used to develop value-added foods.  Fish oil, rich in omega-3 fatty acids is also lost in the 60% of by-products.  Precipitation of muscle proteins at their isoelectric point can be used to recover these proteins from the by-product stream.  Separation by centrifugation during isoelectric precipitation of muscle proteins can facilitate isolation of fish oil from the by-products.

Solubility studies showed best solubility of trout myofibrillar and sarcoplasmic proteins in two pH ranges; acidic pH = 2.5 and basic pH = 12.5.  Precipitation of myofibrillar and sarcoplasmic proteins was highest at pH = 5.5.  Sarcoplasmic proteins unlike myofibrillar proteins are water soluble.  Precipitation of sarcoplasmic proteins was enhanced by higher ionic strength.  Higher ionic strength shifted peak precipitation of myofibrillar proteins to pH = 4.5.

 Based on trout muscle protein solubility characteristics, a laboratory scale protein recovery batch process was designed and tested for recovery yields at various pH conditions.  A trout sample was (1) homogenized, (2) solubilized at acidic or basic pH, (3) protein-rich solution was separated from oil and insoluble fraction by centrifugation, (4) the protein-rich solution was precipitated at isoelectric point, and (5) water was separated from muscle protein pellet by centrifugation.  Protein recovery yields were slightly higher at acidic pH range, approaching 90%.  Protein recovery at basic pH was generally above 80%. 

 The laboratory process is also capable of recovering trout lipids (fish oil).  Fatty acid profile (FAP) of trout muscle and trout lipids recovered at pH = 2.0, 2.5, 3.0, 12.0, 12.5, and 13.0 was analyzed to determine quality of FA.  The concentration of the omega-3 and omega-6 FA in the recovered lipids was generally 3 times higher than in trout muscle.  The concentration of linolenic (18:3n3), EPA (20:5n3), DHA (22:6n3), linoleic (18:2n6), and arachidonic (20:4n6) in the recovered lipids was generally 5, 4, 3, 3, and 3 times higher than in trout muscle. 

Trout muscle proteins recovered in the laboratory process (solubilization at pH = 2.0 and precipitation at pH = 5.5) were used to develop protein gels.  The recovered proteins were chopped, mixed with ice and salt, and then cooked at 90°C for 15 minutes.  Developed gels were used to determine texture and color properties of trout muscle proteins in comparison to highest grade commercial Alaska Pollack surimi.  The gels obtained from trout exhibited superior gel strength, however, they were more yellow (higher b*) than gels obtained from Alaska Pollack surimi. 

 Currently tests are being conducted to determine quality of recovered muscle proteins, protein isoelectric point, protein gelation properties, and texture and color properties at various pH.  These tests will enhance our understanding of isoelectric precipitation of trout muscle proteins and are necessary to implement this technology to recover trout muscle proteins from filleting by-products.  In addition, a continuous system with a processing volume of 120 liters/hr is being designed to scale-up current laboratory scale batch process.

Omega-3 Fortified Trout as Functional Food  In 2002, the Food and Drug Administration (FDA) approved health claim that w-3 PUFA (omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA)) reduce risk of cardiovascular disease, giving a marketing leverage for functional foods fortified with the w-3 PUFA.  Unlike terrestrial animals that must be provided with the w-3 PUFA in the diet, trout synthesize w-3 PUFA from shorter-chain essential fatty acids (EFA), linoleic and linolenic by their elongation and desaturation.  Therefore, trout food products could be marketed as functional foods if w-3 PUFA  were enhanced through dietary modification.  Flax oil contains highest concentrations of EFA from plant sources, linoleic at 12.7% and linolenic at 53.3% that fish might use to synthesize  w-3 PUFA.

 A 4-month feeding experiment with nine dietary treatments was designed as a randomized complete block.  Ziegler Bros. Inc. (Gardners, PA) manufactured the diets with various concentrations of flaxseed oil and vitamin E supplementation.  A raceway system was constructed from honey comb reinforced fiber in Rayman Memorial farm in Wardensville.  A system of screens was placed in raceways to partition experimental units in order to comply with the statistical design (randomized complete block).  There are four levels in the raceway system, each level has two adjacent raceways.  There are 10 experimental units per level, resulting in 40 experimental units in the entire raceway system.  Trout approximately 10 inch long were stocked in mid November 2003 at a density of 75 fish per experimental unit.  The feeding experiment started on November 21, 2003.  Trout and feed samples are being taken in one-month intervals.  The feeding experiment will be completed in March 2004.

 The trout and feed samples are being analyzed for total fat, fatty acid profile (FAP), lipid oxidation, and concentration of vitamin E (a-tocopherol).  Following completion of the feeding experiment, the trout will be filleted and storage stability will be determined at various temperatures.  The same analyses will be performed as in the feeding experiments.  In addition, trout fed diets containing the highest concentration of flaxseed oil and vitamin E will be used in clinical trials in collaboration with Dr. D. Krummel from WVU Health Sciences.             

 Recalibrate trout budgets based on experimental results       Farm level trout budgets were updated  based on yield and feed conversion data obtained from WVU research.  These budgets are available on-line through the WVU Extension web site [http://www.wvu.edu/%7Eagexten/aquaculture/bdgt1-01.pdf] and are set up to allow for periodic updating based on changes in expected yields and prices.  In addition to benefiting producers, accurate budget information is important because it is also utilized in other research activities such as estimating economic models undertaken as part of other tasks.  

Use of Farm Raised Fish in Recreation

We are analyzing the demand and assessing the opportunity for marketing recreational fee fishing in West Virginia to travelers and tourists who do not have a West Virginia fishing license and would view fishing in their overall travel and tourism experience as a complementary/secondary recreational activity rather than as a primary recreational activity.  Since the last report, the questionnaire was mailed to a sample of 5000 individuals  who have in the past two years expressed an interest in visiting the State of West Virginia.  This sample was taken from a list provided by the West Virginia Division of Travel and Tourism.  From that survey 372 questionnaires were returned due to bad addresses.  Of the 4628 individuals who received the questionnaire 691 or 14.9 percent responded to the questionnaire.  Those responding represented 47 states and the District of Columbia.  The majority of the respondents were from states located within one day’s driving distance from the West Virginia.   Presently the data from the survey are being analyzed.  Preliminary results appear to support the notion that fee fishing would be attractive to potential visitors to West Virginia.  The results also appear to favor the fee fishing activity as being part of a package that could include lodging, meals, and other recreational activities.

 Hybrid Bluegill Production   Hybrid Bluegill were stocked in earthen ponds at five (low), ten (medium), and twenty thousand (high) fingerlings/acre (35 g each)  and fed a high protein (42% protein, 16% fat) diet for approximately 400 days.  Fingerling largemouth bass (100/acre) and grass carp (200/acre) were stocked to control bluegill reproduction and growth of unwanted vegetation.  Net production of hybrid bluegill was 672, 1488, and 2151 lb/acre, respectively for low, medium and high stocking rates after about 400 days.  Gross production of hybrid bluegill was 3560, 2130, and 1011 lb/acre and bluegill reproduction was 57, 85, and 12 lb/acre for treatments of 5000, 10,000, and 20,000 fingerlings/acre, respectively. Proportion by weight of market size (>7 inches TL) to submarket decreased with increasing stocking density.   Survival of stocked fingerling hybrid bluegill was 64%, 49%, and 55%, respectively for stocking rates of twenty, ten, and five thousand fingerlings/acre.  This disappointing survival was due in part to chronic Aeromonas  infection in the fall and spring seasons.

 Diet and strain affects growth in hybrid bluegill.       A study was designed to determine the effects of strain and diet on growth and efficiency of nutrient retention in hybrid bluegill.  A diet containing 42% crude protein and 16% fat was determined to be the best of the five diets tested for both strains used in the study.  Moreover, it was determined that the strain of bluegill known as the Georgia Giant grew faster than a commercial strain of hybrid bluegill. 

 Cumulative weight gain of a commercial strain of hybrid bluegill and the strain known as the Georgia Giant.

Feed efficiency of two strains of hybrid bluegill fed five different diets for 12 weeks.

With respect to feed efficiency, the commercial hybrid bluegill and the Georgia Giant were not different (see figure 2 below).  However, there were differences in the efficiency of retention for some amino acids.  This suggests that different strains of fish use different amino acids with different efficiencies and that amino acid requirements probably differ within a species depending on the strain.  That the 42/16 diet was the optimal diet for growth of bluegill is very important because this is not the diet the feed manufacturer typically suggests for bluegill production.  This work has been presented at the Experimental Biology ’03 meetings in San Diego, Ca. and is currently under review at the North American Journal of Aquaculture.  This body of work also represents part of one M.S. student’s thesis.

 Customer Satisfaction and Appropriate Fee Structure for Fee Fishing Enterprises. 

 A two-phase study was conducted to determine angler characteristics as well as effective pricing structure and program format for hybrid bluegill in order to better understand how West Virginia pay fishing businesses and anglers can best utilize farm-raised hybrid bluegill.  Phase One was conducted prior to the hybrid bluegill stocking project to determine anglers willingness to pay for this new fishing opportunity.  Phase Two was conducted following the stocking project to better understand this new market.

 Phase One of this study suggests that hybrid bluegill could be a profitable alternative species for pay fishing businesses in West Virginia.  The production cost for 3.3 pounds of hybrid bluegill is much lower than the $30 that 43 percent of anglers were willing to pay under a catch-and-keep format.

 Phase Two findings indicate that the primary market segment for hybrid bluegill at West Virginia pay fishing businesses is families with children whose goal is to enjoy the fishing experience together.  Businesses can enhance the family experience by maintaining clean and accessible facilities, and offering the convenience of bait and tackle sales.  Fishing contests can add to the fishing excitement.  Unlike fishing public waters, these additional pay pond services make it easier for families to enjoy fishing.

 Technology Transfer

Output from the Aquaculture Food and Marketing Development project has resulted in numerous  presentations and publications ranging from extension bulletins to refereed manuscripts. Approximately seventy scientific publications or presentations at scientific meetings have been created to date.  Each January we host a state wide aquaculture meeting featuring latest information from this research project and speakers from the commercial aquaculture industry. Demonstrations and workshops include bioassays of Arctic Char and rainbow trout in mine water sources around the state, development of an abandoned Acid Mine Drainage treatment plant as a fee fishing venue, marketing arctic char, and an aquaculture session at the Joint Conference of the American Society for Mining and Reclamation Meeting West Virginia Surface Mine Drainage Task Force Symposium. A project web site has been created at http://www.caf.wvu.edu/afmdp/project_info.shtml.    Numerous presentations on aquaculture were conducted in response to requests from county agents and other groups.

Procedures

Commercial Production and Development of the Mine Water Resource

Production Evaluation of Two Commercial Diets for Rainbow Trout in Treated Mine Water.

A schematic of the raceway system is presented in Figure 1.  The system consists of two parallel channels on four levels, for a total of eight discrete raceway segments.  Each of the eight segments has the following dimensions: 9.1 m (l) x 0.9 m (w) x 1.1 m (d) (30 ft x 3.0 ft x 3.5 ft).  The head loss from the top of the dam boards to the surface of the water in the next segment is 1.1 m (42 inches).

Figure 1.  Schematic of the raceway system (not to scale). 

The “paired raceway system” at Dogwood Lakes will be stocked with large rainbow trout fingerlings.  Each raceway segment in the first experiment will be stocked with approximately 1000 fish in the fall and harvested in the spring.   Fish on one side of the system will be fed a commercially available fish meal based diet (e.g. Ziegler Trout Grower 42/16).   Fish on the remaining side will be fed a comparable commercial fish meal free diet (e.g. VegiPRO Trout 42-12).  Feeding will be conducted using demand feeders and mass of feed applied will be tracked throughout the study. Comparisons based on water quality, solids production, economics, and total organochlorine body burden will be made.

 The following water quality characteristics will be monitored: water and air temperature; water flow rate; five-day biochemical oxygen demand (BOD₅); total suspended solids (TSS); ammonia nitrogen (NH₄⁺-N and NH₃-N); settlable solids; dissolved oxygen (DO); and pH.  These parameters are current NPDES regulated water constituents for fish hatcheries in West Virginia.  Influent, process water, and effluent turbidity measurements will also be taken during field water sampling events.  Additionally, alkalinity, acidity, dissolved metals and sulfates will be measured, as each is an important constituent of water chemistry, which can affect fish growth and production.  It is important to note that these analyses will also support continuity in water quality and fish production data developed over the past three years at the mine water treatment site.  Data collected will contribute to the modeling objective funded in a previous proposal.  Additionally, CONSOL Energy, Inc. is constructing a new feed line to increase the flow rate of water going to the mine water treatment facility.  It is quite likely that such alteration to the treatment process will result in changes in water quality.  In order to obtain a complete characterization of water quality in the pilot-scale aquaculture system under production conditions, the headbox and final quiescent zones will be outfitted with an in situ water quality monitor capable of measuring pH, temperature, turbidity, conductivity, DO, and depth.

 Water samples will be collected at the inlet to the raceway system, the effluent from each quiescent zone and the outlet of each raceway in an effort to enable comparisons between fish feeds.  Particular emphasis will be placed on the sampling of solids from the quiescent zone of the raceways to benchmark differences in solid waste production.  Each parameter will be measured according to the applicable “Standard Method” (APHA 1998) or US EPA (1998) approved analytic method.  Production parameters such as condition factor, loading densities, feed conversion rate, average growth rate, etc. will be determined and compared for both diets and compared with previous data.

 Fish samples will be taken before stocking and at harvest to assess the potential for bioaccumulation of organochlorine compounds and metals in fish fillets.  These assays will be performed according to USEPA guidelines (USEPA 1993).  Additionally, samples of both fish feeds will be analyzed to determine concentrations of organochlorines.  It should be noted that the concentrations of organochlorine compounds in raw feeds may be below detection limits.  However, measurable concentrations conceivably may be found in fish, due to bioaccumulation and biomagnification.

  Protein and Lipid Recovery from Fish Processing By-products:  Process Scale-up from Laboratory-batch to Continuous Semi-industrial Application.

A two-dimensional process scale-up is proposed, from laboratory-scale to semi-industrial application.  The scale-up will involve simultaneous increase of capacity, from 1 lb/day to 26 lbs/hr and change of the operation mode, from batch operation to continuous operation. 

 The continuous system will be based on by-product emulsifier (step 1), two bio-reactors (steps 2 and 4), and two continuous centrifuges (steps 3 and 5).  The fish processing by-products will be obtained from High Appalachian, LLC. (Sophia, WV).

 Specific objectives are: (1) equipment purchasing, installation, training, and set-up; (2) optimization of by-product/water ratio; (3) optimization of pH, agitation, homogenization and viscosity; (4) optimization of reaction time and flow rate (dwell time); (5) determination of protein solubility profile, and protein and lipid recovery yields; (6) optimization of continuous separation of proteins, lipids, water, and insolubles; (7) quality evaluation of recovered proteins and lipids; (8) texture and color evaluation of gels developed from recovered proteins; (9) development of value-added food products (in collaboration with Gourmet Central, Inc., Romney, WV).

 The following methods are proposed:

 (1)           Equipment will be purchased, installed and set up in the investigator’s laboratory in the Division of Animal and 
           Veterinary Sciences.  Investigator has sufficient laboratory space to accommodate the equipment and has the 
           necessary instruments to test the recovered proteins and lipids as well as the proposed system.

(2)              In our experiments we have learned that during the pH shifts (steps 2 and 4) the viscosity increases significantly at two pH ranges 3.5-4.5 and 9.5-10.5.  Viscosity is crucial for efficient separation by centrifugation (steps 3 and 5).  Therefore, it is necessary to add water to the by-products during homogenization (step 1).  We determined that for batch operation at the lab scale best by-product/water ratio was 1/9.  The by-product/water ratio will be tested for the continuous system in order to optimize this factor for best protein and lipid recovery.  The water added to the proposed system will be recovered in second centrifugation (step 5).  It was determined in our experiments that the recovered water is protein-free and clear, and therefore can be recycled in the system.

(3)              The pH is critical for protein solubility and precipitation (Figure 4), and thus directly relates to the recovery yield (Figure 3).  Therefore, various pH’s will be tested.  Homogenization reduces particle size, and therefore facilitates reaction between proteins and lipids with water.  In the continuous system, the homogenized slurry (water + by-products; step 1) will have to be pumped to bio-reactors.  Therefore, homogenization will be critical for efficient protein solubility and lipid separation as well as prevention of tube clogging during pumping.  Bioreactors are equipped with agitation, which facilitates reaction between proteins and lipids with water.  At the same time, agitation is affected by the slurry viscosity.  Therefore, the agitation and viscosity will be optimized for efficient reaction between proteins and lipids with water, but without promoting excessive foaming.

(4)              Objectives 1-3 are related to the flow rate, which is defined as the volume that will flow through the recovery system (two bio-reactors + two centrifuges) per unit time.  The shorter the dwell time, the more efficient the continuous system becomes Following completion of objectives 1-3, the flow rate will be optimized for the shortest dwell time (highest flow rate) that will result in highest protein and lipid recovery.  From our experiments with the lab-scale batch operation, we have determined that 5 minutes is sufficient for the reaction between proteins and lipids with water to take place.  Therefore, the recovery of 26 lbs/hr mentioned above is based on the 5-min reaction time.

(5)              Protein solubility and recovery yields will be determined as in previous experiments with the lab-scale batch process (Figures 3 and 4).

(6)              The centrifugation time and g force will be tested for efficient continuous separation of proteins, lipids, insolubles and water.  This objective is related to the objective (3), in which viscosity of the slurry will be optimized.

(7)              Protein gelation is the most critical quality parameter for development of restructured value-added food products.  Protein denaturation destroys gelation ability.  Proteins undergo irreversible denaturation when subjected to heat or ionizing radiation, and reversible denaturation when subjected to extreme pH.  Therefore, gelation properties of recovered muscle proteins will be evaluated with a dynamic rheometer available in the investigator’s laboratory.  Our technology also recovers fish lipids.  Fish lipids are rich in polyunsaturated fatty acids (PUFA) including omega-3 FA (EPA, DHA, and ALA) that are used in nutraceuticals, fortified foods, dietary supplements, and food formulations.  The PUFA are susceptible to lipid oxidation, which results in development of rancidity.  Extreme pH, similar to heat and ionizing radiation, induces lipid oxidation.  Therefore, it is necessary to determine quality of lipids recovered using the proposed continuous system.  The quality of lipids will be evaluated by determining fatty acid profile (FAP) using a gas chromatograph (GC).  We routinely determine FAP in our laboratory.

(8)              Texture and color properties of the food products developed from recovered proteins will be evaluated with Hamann gelometer and L*a*b* Chroma meter, respectively.  Both instruments are available in our laboratory and are routinely used.

The investigator will work together with a Postdoc, lab technician, and graduate student to accomplish the proposed objectives in a timely manner.  One year of support for a Research Associate (Postdoc) and two months for a Research Assistant I (laboratory technician) will be provided through existing projects.  The investigator recruited a graduate student who will start his research program in Fall semester, 2004.  This person has extensive experience in  fish protein and fish food product development throughout his 27-year long career in Alaskan seafood business.  This person will bring a comprehensive practical knowledge of product development and the seafood market along with his extensive industry network to the investigator’s research team. 

Assessment and Development of a Legal/ Institutional Framework for the Development of the Aquaculture Industry in West Virginia.

In order to gain information dealing with current laws and regulations, research will be conducted in several areas.  A comprehensive set of specific issues facing aquaculture in West Virginia will be identified and defined.  A panel of aquaculture experts will be utilized in this process.  This panel will identify a complete and thorough list of issues that are currently affecting aquaculture producers in West Virginia, or are hindering individuals seeking to become involved in the aquaculture industry.  The panel will be convened monthly throughout the duration of the project to both identify issues and review project results as they are developed.  The West Virginia legal code will then be applied to each of these issues to determine the efficacy of current laws and regulations.  An overlay of federal guidelines, which relate to aquaculture will be developed for application to the issues.

 Once important issues affecting the West Virginia aquaculture industry have been identified and state and federal laws have been applied to these issues, the laws relating to aquaculture in other states will be reviewed.    Laws of other state, which have made legal provisions to accommodate the aquaculture industry will be summarized, along with the strategies these states have implemented.  The resulting product will be an input for the aquaculture policy making process in West Virginia.

 After a through review of the issues and laws that deal with the West Virginia aquaculture industry, project investigators will develop a set of instructional and educational materials for dissemination of this information.  Extension publications, information for workshops and consulting, and informational materials for distribution to policy makers will result from this study.

 The legal research will be conducted by a WV attorney who currently teaches two applied courses in agricultural and natural resources law at WVU. The attorney will be assisted by a student with training in legal issues and a technical writer.  This team will develop a set of comprehensive legal and regulatory guidelines to guide aquaculture producers in West Virginia under the supervision of the project PI. 

Use of Farm Raised fish in Recreation

Developing and Marketing Fishing Based Travel Packages

The data collected by the College of Business and Economics faculty focused on two segments of the recreational user market:  1. West Virginia fishing license holders (both in-state and out-of-state) where fishing (fee and non-fee fishing) was a primary activity and 2. potential travelers to West Virginia where fishing (fee fishing and non-fee fishing) would be viewed as an alternative or secondary recreational activity.  For both user segments the research addressed the level of interest in participating in a recreational travel package.  The research did not address the providers of recreational travel packages (fee fishing services, other recreational activities, lodging facilities, and restaurants) and their willingness to provide and participate in recreational travel packages which include fee fishing. Developing recreational travel and tourism packages with fee fishing as a component will have a positive impact on the travel and tourism industry within West Virginia.  West Virginia should experience an increase in the demand for aquaculture output.  By expanding available recreational activities West Virginia will offer potential travelers additional incentives to select West Virginia as a travel destination.  Finally, the availability of additional recreational activities for visitors to participate in could lead to increasing the length of stay of those visitors.

 Faculty from the College of Business and Economics will work with faculty from the Recreation, Parks, and Tourism Resources Program to complete Phase II of the analysis.  The research effort will be a two-part project.

 Part 1

Data collected from the various tourism need assessments of the user market segments (phase I) have provided the foundation for developing fishing based, and targeted, recreational travel packages.  Proposed for Part 1 is an assessment of the provider market that are of interest to the user segments.  This assessment will include the following:  

• Identifying existing providers such as lodging facilities, fee fishing services, and other recreational activities that will go into forming recreational travel packages of interest to various user segments that have been identified.  A library that describes the providers/production process for fishing experiences, including recreational activities, associated costs, and liabilities will be completed.  These library materials will compliment the products being produced by other project investigators (e.g., materials that describe the production strategies, methods, costs, and case studies associated with potential fish species).  The library materials will be shared with tourism providers (e.g., trout unlimited, economic development agencies, Travel and Tourism, fish producers, and fee fishing businesses) to develop packages that target specific markets such as social-relaxation, trophy fishing, escape, family oriented recreation, or food and fun opportunities.

• Survey selected providers to assess their willingness to participate in recreational travel packages for the user market segments. For example, interviews will be conducted with selected lodging facility managers (e.g., resorts, bed and breakfast, and cabin rentals) throughout West Virginia.  Study sites will be selected based on their proximity to a fee fishing business and their ability to provide meals and other services and recreational activities. 

 Part 2

Data collected from Part1 of Phase II will be used to better understand existing and potential fishing experiences available to visitors to West Virginia.  For selected regions of the state, the supply-side potential (providers) of the tourism market will be compared with tourism packages demanded by tourists (users).  From these data recreational travel packages will be proposed.  Part 2 will focus on the following: 

• Identify study sites which show the greatest potential for forming recreational travel packages consistent with the user segment needs and wants.
• Develop recommendations for recreational travel packages for selected target markets.

 The results of this study will be shared with study participants and other referral and support sources (e.g., WV Travel and Tourism) to help develop and market desired travel packages that include fee fishing as an activity. The proposed study has the potential to expand the network of anglers for fee fishing as a recreational industry in West Virginia as well as enhance the overall economic initiatives of the travel and tourism industry within the state.

 Nutrient Removal from Trout Raceway Effluent Utilizing Watercress

Experimental variables were selected to provide the necessary initial cultural information to optimize nutrient removal by watercress in an aquaponic system.  The following variables will be successively manipulated along with water velocity in a 2 factorial experiment: watercress variety, plant material source, plant density, growing medium and harvest schedule. 

• Watercress variety (3 different varieties including wild watercress variety from the Wardensville site).  Different varieties of the same genus and species can have different growth rates and nutrient uptake levels.  By comparing 3 different varieties, including the wild watercress variety from the Wardensville site, it can be determined if there is a difference in varieties which will maximize production and nutrient uptake and which variety is the best of the varieties tested in this aquaponics system.
• Seedlings vs. vegetatively propagated stem cuttings.  Typically most plants are started from seeds and then transplanted to the location of final use.  Most growers grow directly from seed, which is less labor intensive but the advantage of using propagation methods other than seed such as stem cuttings, is that you have the plant sooner than you do with seeds (Hartmann et al., 2001).  Stem cuttings are initially larger physically with preformed axillary meristems present than seedlings of the same age.  Watercress naturally forms roots at each node making it ideally suited for vegetative propagation via stem cuttings.  This variable will determine which plant source (seed or cutting) will maximize production and nutrient uptake.
• Plant density (0.04, 0.09 and 0.16 plants·cm^-2).  Plants compete for the necessary components such as light and nutrients for growth and development.  Higher plant densities allow for more plants to be present in a given area but limit the amount of growth and development that each individual plant is capable of.  This variable will determine which density will maximize production and nutrient uptake.
• Growing medium.  Typically, leafy-type plants that are produced in a hydroponic system are produced using a floating system with the plant placed on a raft or similar structure as the growing medium (Acquaah, 2002).  Other growing media such as rock wool and sand will be compared with the floating planters and evaluated as to the ease of installation and harvestability; the effects on growth and development and nutrient uptake capabilities.
• Harvest schedule.  Typically the harvest schedule of watercress that is grown in hydroponics production is carried out every 7-10 days.  If the plants are harvested sooner then new growth will be initiated and if harvest is longer than 7-10 days then the plants will be larger.  The amount of active growth and size of each individual plant may have an effect on nutrient uptake but marketable harvest size of the watercress must be maintained.  By evaluating different harvest schedules, this variable will determine which schedule will maximize production and nutrient uptake.

 Each experiment will require 27 channels allowing 3 replicates of each treatment combination.  In addition there will be a control channel where no plants will be grown.  Experiments will be conducted over the course of a year allowing natural variation in available light, air and water temperature.  Controlled experiments will be performed to evaluate nutrient removal and growth rates of watercress under varied conditions with the aim of maximizing both nutrient removal and plant growth.

 Experimental channels will be constructed of plywood covered by a rubberized plastic sheet making the system waterproof.  The channels will be constructed so that they receive floating planters commonly used in hydroponics.  Each channel will be 7’ long allowing 3 planters that will hold from 18 to over 300 plants to be in each individual channel.  Thirty channels, in groups of five, will be constructed (Figure 5).

 Figure 5.  Diagram of off-line channels to be used for nutrient removal experiments.

 Effluent from the raceways will be pumped to the experimental channels and distributed via a manifold with flow rates to each trough individually controlled by a valve.  Water velocities of 0.1, 0.05, and 0.01 ft·s^-1, spanning the range found in aquaculture and hydroponics, will be used (Soderberg 1995). 

 The following water quality characteristics will be monitored: water temperature; water flow rate; ammonia; nitrate, nitrite, total phosphate, dissolved oxygen (DO); and pH.  Samples will be collected at the end of the raceway system and then at the bottom of each of the experimental channels.  Water samples will be taken at roughly 3 week intervals.  Biomass and nutrient concentrations (total C, total N and total P) of the plant material will be determined at the beginning and end of each experiment to determine growth and nutrient sequestration.  Each parameter will be measured according to the applicable “Standard Method” (APHA 1998) or US EPA (1998) approved analytic method.  In addition, air temperature and available light will be monitored.

Technology Transfer

Investigators in the Aquaculture Food and Marketing Development Project (AFMDP) have selected two areas of emphasis regarding role of aquaculture in the region’s economic development:

 1.)                Utilization of groundwater discharged from coal mining sites to grow food size coldwater fish.
 2.)                Recreational uses of farm raised fish.

After five years of research conducted in the Aquaculture Food and Marketing Development Project, the need for technology transfer is expanding.  Requests for literature and information have doubled since the inception of the project.  There is greater demand for site visits to assess opportunities, and assist individuals growing or utilizing farm raised fish.   Administrative duties increasingly require the attention of the Extension Specialist in his role as Principal Investigator making it increasingly difficult to meet all requests and reach out to new stakeholder groups. Consistent with this demand and the selected areas of emphasis, two technology transfer positions have been created. Each position assists and complements the efforts of the Extension Specialist – Aquaculture.  

Development of the Mine Water Resource.  Mine water has emerged as the most important source of water for commercial production of salmonids in West Virginia.  Each site must be evaluated on its unique topography, water quality, water volume, etc.  We continue to engage two large coal companies and numerous mine sites.  We will continue to work with coal companies and economic development agencies in the step by step process of site assessment and education required for development of the mine water resource.  We will also coordinate with processing plants and others to assist with determination production capacity and appropriate facility design once a site has been chosen for development.  The individual responsible for this area of work will coordinate with various investigators to develop workshops, a newsletter, and publications useful for development of the mine water resource.

 Aquaculture and Recreation.  Most fish farmers in West Virginia sell their fish in the recreational market. Approximately 300,000 lb is sold to fee fishing businesses around the state.  In addition, live fish are sold to fishing clubs, large companies, housing associations, and private individuals for recreational use in both private and public waters. Building upon the data developed in previous research, it is timely that this information be combined with the experiences of fish farmers serving the recreational industry and extend it to a wide variety of recreational stakeholders.  Such stakeholders would include resorts, attractions, fishing clubs, communities developing strategies to enhance tourism, and the general public. 

 There is a need to develop materials which describe how farm raised fish can be utilized in recreational activities.   Our long term goal is to develop a set of resources to assist with development of fishing based recreational travel packages.  This will include describing potential of fishing experiences and how to generate them as well as a set of extension publications describing of production methods for each potential fish species.  Specific efforts will be made to assist with development of recreational travel packages which require farm raised fish.   Site visits and assistance to farmers targeting the recreational market will also be an important component of this work.  There is opportunity to develop partnerships between segments of the tourism industry, fish farmers, fisheries managers, and the resource base to respond to the recreational opportunities described by marketing research.   Rodney Kiser  will be assigned to this segment of the Technology Transfer work and will tend to focus first on the eastern part of the state and work with the facility at Wardensville.

 Product Development.   Food Science research has assessed new technologies, but has not applied the information in product development.   Activa was favorably evaluated as a binder for trout flesh.  Protein and lipids have been extracted from processing by-products.  In this grant cycle the extraction process will be enhanced and is expected to yield adequate volume for initial product development.  A separate, but related development has been the creation of a “Fish Wagon” for preparing and serving farmed fish products at WV fairs and festivals.  The fish sandwich currently available from the Fish Wagon would benefit from further product development and offers an opportunity to directly apply these new technologies.  A subcontract with Gourmet Central is proposed as a vehicle to accomplish this task.  Rodney Kiser will facilitate activities between WVU Food Science investigators, and Gourmet Central.

 Implementation of technology transfer activities is to disseminate information generated by this project to the aquaculture industry in Appalachia, to government agencies with aquaculture-related responsibilities, and to the general public.  The investigators will continue to collaborate 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.   In January we will host the Aquaculture Forum – an annual meeting designed to reach aquaculture stakeholders and present a program of research results and practical commercial perspectives. 

 Justification

Production Evaluation of Two Commercial Diets for Rainbow Trout in Treated Mine Water.

In a recent study by Hites  et al. (2004), it was demonstrated that farm raised salmon are found to contain organochlorine compounds such as polychlorinated biphenyls and dioxins far in excess of wild fish.  The source of PCBs in farm raised salmon was traced to fish meal used in many fish feeds.  It was reported that bioaccumulation and subsequent magnification resulted in increased organochlorine body burdens in fish products which are rendered and subsequently used in the manufacture of fish meal.  Due to the environmental persistence of such compounds, there is little degradation of the organochlorines and further uptake is facilitated.  Further, the investigators concluded through risk analysis that the high levels of PCBs and related compounds in farm raised Atlantic salmon (Salmo salar) detracted from the positive health benefits of consuming fish.  At this point, trout growers have not identified PCBs as a potential contaminant in their fish, but they may not be looking for it either.

 Recently, an Ohio feed manufacturer began marketing a trout diet that is “fish meal free” called VegiPRO.  Presently, there are no other manufacturers with this type of diet.  As part of its marketing strategy, the company reminds producers that PCBs may be associated with fish meal and that an all vegetable diet is not expected to have this risk. The company claims that VegiPRO “reduces excess products that pass through the fish and that the diet reduces the amount of fecal phosphorous”. In addition, they contend that the fillets are whiter and “lighter” and “juicer”.    Price complicates the picture as the new diet can be obtained for a lower price than a control fish meal based diet.  West Virginia growers need data to verify or refute these claims.

 Due to the great potential impacts these findings may have on the aquaculture industry in WV, it is important to determine whether a difference in organochlorine body burdens can be detected between fish raised on a fish meal based diet and those grown using an all vegetable feed.  In addition to fish production related benefits, vegetable-based feeds are reported to decrease solids and dissolved waste production, as a greater fraction of the total feed is utilized efficiently by the fish.  Impending federal regulation of aquaculture operations has made effluent minimization a key area of concern to fish growers in West Virginia and the United States at large.  Consequently, the assessment of the use of vegetable-based feeds is one opportunity to have positive effects on effluent water quality. 

 Protein and Lipid Recovery from Fish Processing By-products:  Process Scale-up from Laboratory-batch to Continuous Semi-industrial Application

Filleting trout requires removal of bones, skin, head, and viscera (by-products).  Mechanical filleting of 100 lbs of trout yields approximately 40 lbs of fillets and 60 lbs of by-products.  The 60 lbs of by-products contains approximately 20 lbs of meat and 5 lbs of fish oils (lipids).  The by-products are reduced to animal feed or are land-filled.  Trout processors incur expenditures to remove processing by-products from their plants.  The by-products are also an environmental bio-burden. 

 To sustain the WV aquaculture industry, increased profitability is necessary.  Therefore, development of technologies that recover proteins and lipids from by-products will increase profitability of fish processors and broaden their product offerings as well as may create new jobs in West Virginia.  The use of recovered proteins and lipids for human food instead of for animal feed or land-filling will increase the value of these materials.  Therefore, human food products developed using proteins and lipids recovered from processing by-products are typical examples of value-added food products.

 This research will allow implementation of the protein and lipid recovery technology developed in our lab to semi-industrial application.  If this research proves that this technology can be implemented at the semi-industrial scale, then the next step would be full-scale industrial production.  Therefore, the proposed research will fill the gap between technology development at the university laboratory bench-top scale that our laboratory has done so far and full industrial production that is still a far perspective. 

 The proposed research will also allow protein and lipid recovery in sufficient quantities for development of marketable value-added food products.  Therefore, the proposed research will bridge university development of scientific food samples (i.e., protein gels) developed in our lab with the development of marketable value-added food products (i.e., Gourmet Central).

 The proposed research will enable the investigator to establish a protein and lipid recovery system that to the investigator’s knowledge has not been previously constructed.  Therefore, our laboratory would have a privilege of being at the forefront in protein and lipid recovery research.  This would result in increased feasibility for attracting external research funds from federal sources such as USDA-NRI (program 71.1 – value-added product research: food characterization/process/product), Northeastern Regional Aquaculture Center (NRAC) and National Science Foundation (NSF).  The USDA-NRI and NSF promote young investigators.  Therefore, the investigator would qualify for the New Investigator Award from both agencies due to Assistant Professor status.  The proposed recovery system will be universal, meaning that the proposed system will be as applicable to any other food animal species as to trout, as long as the isolectric point and solubility of the other species’ proteins are determined.  Therefore, this will open additional external funding opportunities to agencies such as U.S. Egg and Poultry, Fats and Proteins, and National Fisheries Institute.  Since the proposed system will be at the semi-industrial scale, the investigator anticipates increased interest and funding from private food animal processing businesses.  Since the proposed system will utilize food animal processing by-products that otherwise would be disposed of being an environmental bio-burden, funding from the Environmental Protection Agency may be feasible. 

 Assessment and Development of a Legal/ Institutional Framework for the Development of the Aquaculture Industry in West Virginia.

West Virginia Laws relating to fish and their commerce originated in a time when “wild” fish were harvested from the natural, usually public, environment.  Currently, though many fish are still considered wild and are harvested through recreational or commercial fishing, there is a steady increase in the number of fish produced, harvested, and marketed through commercial aquaculture.  For the past few decades, the aquaculture industry has moved fish and their commerce into the realm of agriculture, where fish are farmed by private individuals and business entities in private water.  These farm raised fish are sold both live and processed in local, national, and international markets.  Although thousands of pounds of fish are produced and harvested annually in West Virginia through fish farming, the aquaculture industry is largely regulated by the laws that apply to “wild” fish.  The lack of applicable, comprehensive regulations contributes to risk and cost, either directly or indirectly, for producers and potential producers.  Because the laws have not adequately adjusted to the change in fish farming methods, development of an aquaculture industry in WV and throughout the United States is impacted.

 Legal and regulatory constraints represent a “first hurdle” in the course to developing a successful aquaculture business.  They can effectively exclude individuals or business entities from competing.  Although they are not necessarily the limiting constraint for development of aquaculture, they do represent a real or perceived barrier increasing cost and/or risk to the prospective entrepreneur.

 Some of the issues affecting aquaculture producers in West Virginia currently are water availability and usage, effluent discharge (waste water discharge), transportation of live fish across state lines, exotic species, restriction on threatened or endangered species, handling and processing, taxation, insurance, biosecurity, and the confusing permitting process.  The industry is in need of a viable regulatory structure which address these issues in a way that protects our natural resources as well as allows the development of an aquaculture industry in West Virginia.  In order to enact policy and regulatory changes to better accommodate the aquaculture industry, research is needed to examine current laws in West Virginia, other states and at the federal level as they relate to regulation of fish farming.  Presently, aquaculture is not viewed as a form of agriculture in West Virginia, therefore the farming, marketing and transportation of the product is subject to a different set of regulations than other agricultural commodities.  There also is a general lack of knowledge among existing and potential producers as to the legal/regulatory framework for aquaculture production and marketing.  This knowledge gap leads to difficulties for those individuals who are currently or would like to contribute to the development of the aquaculture industry.

 Developing and Marketing Fishing Based Travel Packages

Fee fishing is a recreational activity that represents only one component of a much larger sector within West Virginia.  That sector which contributes significantly to the economic development of the state is the travel and tourism industry.  The recreational experience is similar to other market goods; the productivity of the industry is likewise influenced by the perceived quality and quantity of the products produced.  In recreation, perceptual events are the production units and are packaged as a set of activities and experiences (Pierskalla and Lee, 1998; Pierskalla et al., in press).  Therefore, West Virginia fee fishing opportunities can contribute to the productivity of the tourism industry by providing tourists with more to see and do (i.e., providing a more eventful visit). 

 Nutrient Removal from Trout Raceway Effluent Utilizing Watercress

There is increasing concern regarding nutrient loading from various sources including  aquaculture operations on receiving streams.  Both Selong and Helfrich (1998) and Loch et al. (1996) demonstrated deleterious effects of flow-through aquaculture systems on downstream benthic communities.  In addition to local impacts, nutrient inputs from upstream sources have negative effects on downstream estuaries.  In the Chesapeake Bay, as well as other estuaries, there are strong linkages between increases in nutrient loading and large algal blooms that lead to anoxia and toxic or harmful impacts on fisheries, human health and recreation (Anderson et al. 2002).  As a result, the states with major rivers flowing into the Chesapeake Bay have pledged to reduce nutrient loading to the bay.  Aquaponics, the simultaneous culture of fish and plants, has been proposed as a nutrient removal system for aquaculture systems that has the benefit of producing a value-added byproduct. 

 Aquaponics has been developed in conjunction with recirculating aquaculture systems (RAS).  Rakocy et al. (2000) developed a system that produced 3.1 mt of tilapia and 1,248 cases of lettuce over a 2.5 year period.  Adler et al. (1996, 2000) developed an off-line system that uses trout effluent to grow lettuce and basil.  In this system NO₃ concentrations were reduced from 25 to 3 mg·l^-1 and PO₄ concentrations were reduced from 0.7 to <0.001 mg·l^-1.  RAS are characterized by high nutrient concentrations and low volumes when compared with flow-through aquaculture systems. 

 In contrast, aquaponics has not been tried in flow-through systems, the more common aquaculture system in West Virginia, which typically generate greater volumes of more dilute effluent than RASs.  In addition, many of the aquaponics systems developed in conjunction with RASs operate off-line, removing the nutrients so that the conditioned water can be returned to the fish production unit.  Flow-through systems offer the possibility of simultaneous culture of plants and fish within the raceways rather than requiring a separate facility to grow the plants.  However, this will only be feasible if the plants are capable of removing nutrients from a relatively high flow, low concentration water source.  Controlled experiments, examining the interrelationships between effluent flow rates and various culture practices, will demonstrate whether aquaponics is a feasible nutrient removal technology for flow-through aquaculture systems.

 Literature Review

Production Evaluation of Two Commercial Diets for Rainbow Trout in Treated Mine Water.

 As a result of coal mining in West Virginia, millions of gallons of groundwater, which require perpetual treatment, have collected in abandoned underground mines.  According to one estimate, 8.78 x 10⁵ m³ (232 million gallons) of water are discharged from active and abandoned mines each day in West Virginia, of which it was further estimated that ~6.44 x 10⁵ m³/d (170 million gallons/day [MGD]) may be of suitable quality for additional use either directly or with treatment (Jenkins et al. 1995).  Due to the sulfur content of geologic strata disturbed during mining operations, acid mine waters containing dissolved metals are produced (Barlow 1974).  Generally, iron, aluminum, magnesium, and manganese are the most abundant dissolved metals encountered in mine waters in West Virginia, due to their relative natural abundance (Skousen and Ziemkiewicz 1996).  When compared with the water quality criteria required for salmonids (Meade 1989), presented in Table 1, raw mine waters may not be suitable for direct use in fish culture.  However, the perpetual treatment of such waters is required under NPDES regulations.

 In previous proof-of-concept work conducted by Viadero and Tierney (2003), it was shown that the use of treated coal mine water for rainbow trout (Oncorhynchus mykiss) culture in a cage was technically feasible, though only a fifty fish bioassay was grown and no work on production related issues was conducted.  To further advance the use of treated mine water, an under-utilized water resource throughout Mid-Appalachia, work was conducted to assess the effects of using treated coal mine water for the intensive production of rainbow trout in a flow through system (Viadero and Tierney, 2004).  During this study, comprehensive water quality data were collected to supplement fish weight and length data taken during routine monthly sampling events.  The eight thousand fish grew well in the raceway system over the nine months of production, where a feed conversion ratio of 1.4 and a condition factor of 5.1x10^-4 were measured with stocking and harvest densities of 26.4 and 50.2 kg/m³, respectively.  Further, total net production was 3,275 kg (7,220 lb) with 98.6% survival.  Throughout the study, dissolved ion concentrations (Fe, Al, Mg, Ca, and SO₄) often exceeded recommended limits.  Further, elevated ammonia nitrogen concentrations generated from a component of the mine water treatment process were identified as a potential limiting factor for aquaculture development.  However, when the non-ideal effects of high ionic strength and the speciation of dissolved metal–ligand complexes were taken into account, the concentrations of free metal ions were within recommended limits.

 Protein and Lipid Recovery from Fish Processing By-products:  Process Scale-up from Laboratory-batch to Continuous Semi-industrial Application

 The pH-driven protein recovery. Protein solubility is lowest at a protein’s isoelectric point (pI) (Srinivasan 1996).  The pI is the pH at which the protein molecule’s net electric charge is zero.  As pH diverges from the pI, the increased protein-protein electrostatic repulsion facilitates protein-water interaction, resulting in protein solubilization (Srinivasan 1996).  Conversely, as pH approaches pI, the decreased protein-protein electrostatic repulsion decreases protein-water interaction, allowing for protein-protein hydrophobic interactions, thereby leading to protein precipitation (Srinivasan 1996).  Precipitated proteins can be separated from lipids, debris (bones, skin, etc.), and water by centrifugation.  This isoelectric solubilization-precipitation cycle can be used to isolate proteins and lipids from the trout processing by-products. 

 Gel-forming ability.  To be useful in making surimi seafood products, muscle proteins recovered from the by-products must retain their gel-forming ability (Lanier 2000).  Heat-induced gelation is a result of protein denaturation, leading to inter- and intramolecular covalent and non-covalent interactions (Lee and Lanier 1995).  Solubilized proteins undergo denaturation followed by an ordered aggregation to form a gel network (Srinivasan 1996).  Therefore, solubility and denaturation are the critical prerequisites for heat-induced protein gelation. 

 Fish oil and omega-3 fatty acids.  Fish oil contains high concentrations of omega-3 polyunsaturated fatty acids (w-3 PUFA).  In 2002, the Food and Drug Administration (FDA) approved a health claim for the w-3 PUFA.  According to the FDA, “consumption of w-3 PUFA reduces the risk of coronary heart disease”.  This approval created a niche market in the functional foods and dietary supplements arenas for fish oils.  The concentrations of the w-3 PUFA in a fatty acid profile (FAP) of fish oil vary depending on species, diet and processing.  Therefore, determination of the FAP is critical.   

 Current research in the field.  Hultin and Kelleher (1999) applied the isoelectric solubilization/precipitation to recover muscle proteins from mackerel and demonstrated that solubilization of mackerel muscle proteins at pH 2-3, followed by precipitation at pH 5.5 and centrifugation, resulted in high recovery of muscle proteins and lipids.  Choi and Park (2000) applied the isoelectric solubilization/precipitation to recover muscle proteins from Pacific whiting (similar pH as Hultin and Kelleher), which increased muscle protein recovery by 80% as compared to conventional surimi production (sequential washings with water).  However, the poor texture of the surimi seafood product prepared from Pacific whiting proteins recovered at acidic pH was attributed to activation of proteases at acidic pH.  Yongsawatdigul and Park (2001) further investigated protein recovery of fish muscle using an alkaline pH, which resulted in improved texture of the surimi seafood product. 

 There are no reports on application of the isoelectric solubiliation/precipitation to recover muscle proteins from food animals processing by-products, including trout.  To the investigator’s knowledge, a construction of the continuous system at the semi-industrial scale using isoelectric solubilization/precipitation of muscle proteins has not occurred.   

 Assessment and Development of a Legal/ Institutional Framework for the Development of the Aquaculture Industry in West Virginia.

A review of the literature available focusing on the aquaculture industry in West Virginia reveals a lack of easily accessible current information dealing with what potential producers need to do with regard to permits and regulations to begin an aquaculture enterprise.  This lack of a reliable, single source of information affects current producers in a similar manner.  Some other states have more concise regulations that specifically apply to aquaculture, and many have none.  In several states, such as Iowa, Michigan, Indiana, and Illinois, aquaculture is regulated mainly by the department of natural resources.  Most states require specialized fees and permits to enter the aquaculture industry, especially in terms of the importation of fish.

 Wirth and Luzar (2000)  report that state level variations in legal, regulatory, and development programs targeted toward the aquaculture industry can affect aquaculture firm decision making, including site location and species selection decisions.  West Virginia ranked in the upper one-third of the study relative to regulatory stringency.  Wirth and Luzar (2001)  examine the stringency of the regulatory climate regarding finfish on a national survey.  Results of the study indicate that establishing a formal state aquaculture development plan and transferring regulatory enforcement authority to state departments of agriculture from state fish and wildlife agencies will significantly reduce regulatory stringency and the negative impacts of state institutional constraints on aquaculture. An example of such a plan was developed for Georgia in 1996 (Lewis, 1996).

 In 1990, legal issues associated with the recreational use of natural resource4s in West Virginia were addressed in nine papers presented at a WVU Conference (Grafton et al, 1990).  The issues investigated included premises liability, easements, land use and environmental regulations, nuisances, risk management, leases, and recreational use statuates.  In 1979, an extension bulletin outlining land owners rights and responsibilities in West Virginia was developed to aid property owners in production decisions and to avoid disputes (Doyle, Ferrise, and Smith, 1979).  The bulletin addresses issues such as trespass, water rights, fences, wildlife, mineral and timber rights, and animals.  Avault (2004) addressed aquaculture issues such as permits, species, ownership, certification, and chemical use for Louisiana, other states, and the US.

 Developing and Marketing Fishing Based Travel Packages

An understanding of how fee-fishing opportunities can be included in packages that better meet various tourist needs can enhance the quality-side of the productivity equation as well as serve as an incentive for travelers when deciding a destination location (Aguilo, Alegre, and Riera, 2001; Pizam and Mansfeld, 1999; Eagles, 1995).  To contribute to this understanding, the first phase of a two phased gap analysis has been completed by the investigators in the Aquaculture Food and Marketing Development Project (AFMDP).  Phase I of the gap analysis included an assessment of angler needs and travel market characteristics.  Where Phase I assessed the user of the recreational offerings, this proposed phase (Phase II) of the gap analysis would be an assessment of the providers of the recreational offerings.  Phase II would be an assessment of recreational providers and their willingness to participate in and be a part of recreational travel packages.  Lodging facilities play a central role in this packaging process and will be the focus of the proposed second phase of the analysis.  The first phase of the study focused on customers and is summarized in this report as Current Work. 

Nutrient Removal from Trout Raceway Effluent Utilizing Watercress

Watercress (Nasturtium officinale R.Br.) is an aquatic perennial plant found naturally in still or slowly flowing shallow water systems such as springs, spring-fed stream, and ditches.  It is a marketable horticultural crop, considered a culinary herb, grown for its leaves and young stems which are used as a garnish, salad or soup in upscale restaurants around the U.S.  Culinary herbs are well suited to small-scale production because of unique growing conditions and intensive labor needs. Production can be on small acreage, marginal land, and without heavy machinery or with modified equipment. Potential markets are in selling fresh-cut herbs to restaurants, at local farmers’ markets, and through some specialty grocers.  Watercress is exceptionally rich in vitamins and minerals (calcium, iron, potassium, Vitamin K, Vitamin A (beta carotene), Vitamin B-1 (thiamin), Vitamin B-3 (niacin), and Vitamin C) and has long been valued as a food and medicinal plant (USDA, 2004).  Rose et al. (2000) reported that a group of compounds found in relatively low levels in watercress have a strong potency in activating a protective reaction to cancer when watercress is eaten.

 Watercress was selected for initial trials in the aquaponic system as it prefers cool water temperatures, approximately 12 – 18°C, such as those found in spring-fed systems.  It has been used in pilot-scale aquaponic systems in North Carolina, and there is an available high-end market.  Additionally, it grows naturally in the spring-fed streams at Wardensville, providing an initial locally adapted plant source.

Current Work

Protein and Lipid Recovery from Fish Processing By-products:  Process Scale-up from Laboratory-batch to Continuous Semi-industrial Application

We have developed a batch operation for protein and lipid recovery at the laboratory scale that allows us efficient recovery of functional muscle proteins and lipids from trout.  The batch operation unlike continuous operation mode is defined as a cyclic operation that requires repetitive cycles of (1) loading substrate (fish processing by-products), (2) processing (isoelectric solubilization and precipitation of fish muscle proteins), and (3) unloading the products (recovered fish muscle proteins and lipids).  In contrast to batch operation, continuous operation mode allows continuous feeding of substrate, continuous processing, and continuous harvest of the products.  Therefore, the continuous operation mode is a preferred operation type for the protein and lipid recovery from fish processing by-products.  The protein recovery yield with our technology is approximately 90% on weight basis (Figure 1).  The recovery technology is based on isoelectric solubilization and precipitation of trout muscle proteins (Figure 2).  The recovered trout muscle proteins retain their functionality – gelation, which is critical in development of restructured value-added food products.  We have also developed protein gels from recovered proteins.  The laboratory-developed gels mimic restructured value-added foods and allow scientific determination of texture and color properties, which are the two most important quality attributes for these foods.  The texture of our gels developed from trout proteins exhibited superior properties in comparison to gels developed from highest grade commercial Alaska Pollack surimi.  Color properties of both gels were comparable.

We have developed five steps (Figure 2) necessary to recover muscle protein and lipids: (1) homogenization that simplifies sample handling and increases surface area of proteins and lipids, and therefore facilitates interaction between proteins and lipids with water; (2) first pH shift that results in protein solubilization due to increased electrostatic interaction between proteins and water, and facilitates separation of lipids from water due to increased hydrophobic interaction between lipids; (3) separation by first centrifugation that results in bottom fraction (“real by-products” – bones, skin, and insoluble proteins), middle fraction (muscle proteins solubilized in water), and top fraction (fish lipids rich in omega-3 fatty acids as confirmed by our experiments), (4) the middle fraction is recovered is subjected to the second pH shift that results in isoelectric precipitation of muscle proteins due to increased repulsion between protein and water and increased hydrophobic interaction between proteins, and (5) separation by second centrifugation that results in separation of precipitated functional muscle proteins from water.  The water separated in this step is protein-free and clear, and therefore can be recycled in the process.

 Developing and Marketing Fishing Based Travel Packages

Researchers have completed several projects to better understand West Virginia’s fee fishing market.  The College of Business and Economics faculty completed a study of West Virginia fishing license holders (both in-state and out-of-state) who view fishing as a primary recreational activity.  Faculty have also surveyed potential travelers to West Virginia where fee fishing would be viewed as an alternative or secondary recreational activity.  The focus of this research was on the following:

• An assessment of the demographic characteristics of individuals who have expressed an interest in visiting West Virginia.
• An assessment of the length of stay, lodging accommodations, and activities participated in while visiting West Virginia.
• An assessment of the visitor’s level of interest in participating in fee fishing activities during future visits to West Virginia.
• An assessment of the viability of putting together a recreational travel package for visitors to West Virginia that would include fee fishing as a secondary activity.

 The Recreation, Parks and Tourism Resources Program completed a study of West Virginia fee fishing anglers (on-site) to better understand their recreational needs and preferences in the following areas:

• An assessment of setting characteristic preferences.
• An assessment of desired experiences or motivations.
• An assessment of catch related standards of quality including preferred fish species, sizes, and catch numbers.
• An assessment of willingness to pay for various fee fishing experiences.

These combined market data provided the foundation to begin the development of recreational travel packages for West Virginia residents with fishing licenses, non-residents with West Virginia fishing licenses whose primary recreational activity is fishing; and visitors to West Virginia who do not have a West Virginia fishing license and would view fishing in their overall travel and tourism experience as a secondary recreational activity rather than a primary recreational activity.  A range of packages were identified and include experiences such as social-relaxation, trophy fishing, escape, family outings, and food and fun.  These recreational travel packages would draw primarily from existing resources and include but not be limited to lodging, meals, supplies, and various recreational activities.

Facilities and Equipment

The physical facilities of West Virginia University will be utilized for developing, analyzing and reporting the study.  Facilities of the Davis College of Agriculture, Forestry and Consumer Sciences will draw upon resources from the three divisions of Animal and Veterinary Science, Resource Management (Resource Economics), and Forestry (Recreation and Parks).  The Department of Civil and Environmental  Engineering,  in the College of Engineering, the Marketing Department in the School of Business and Economics, and the West Virginia Extension Service will provide additional resources.  Personal computers and software provided by the institution will be used in implementing this project.

 Proposed work will take place at two pilot scale raceway systems.  Each system is composed of HFRP material with four levels of paired units, each 30 feet long capable of maintaining approximately 1000 lb of fish.  Combined, the systems are supplied with about 1000 gallons/minute, and have a total of 16 experimental units.  The facility at Dogwood Lake, approximately 15 miles west of WVU main campus is supplied with treated mine water.  The facility at Reymann Memorial Farm is part of the WV Agricultural Experiment Station near Wardensville, WV and is fed by spring water.

 The raceway system at the Reymann Memorial Farm in Wardensville, WV, was selected to study nutrient recovery with watercress.  The spring feeding the facility naturally supports watercress and is representative of small springs found throughout the region.  Minimizing nitrogen in the Potomac River headwaters is an important consideration for growers in this area.   A temporary building has been installed enclosing both the raceways and additional space where controlled experiments may be performed. 

 The protein recovery experiments will be performed in the Division of Animal and Veterinary Sciences (A&VS).  Dr. Jaczynski has a sufficient laboratory space in the A&VS, which houses the following equipment spectrophotometer, SDS-PAGE and isoelectric focusing cell, homogenizer, ultra-speed refrigerated centrifuge, pH meter, universal food processor, water bath, colorimeter, torsion gelometer, dynamic rheometer, viscometer, texture analyzer, gas chromatograph, and other analytical equipment.  Dr. Jaczynski has a sufficient laboratory space where the new equipment (i.e., bio-reactors with chillers, continuous centrifuges, and homogenizer) for which funds are requested in the proposal budget will be set-up.  Dr. Jaczynski’s existing equipment will be used for laboratory analyses necessary to conduct the proposed research. 

 Facilities of the College of Engineering and Mineral Resources contains in excess of 5000 ft² of laboratory space.  Analytical equipment includes: one atomic absorption spectrophotometer; three gas chromatographs with multiple detector arrays (PID and FID) and a purge and trap apparatus; two total organic carbon analyzers; three automated pH titrators; one scanning electron microscope; and one UV-visual light absorbance spectrophotometer.  The National Research Center for Coal and Energy (NRCCE) located adjacent to the engineering facilities will conduct water quality analyses.  The laboratory is EPA certified for performing analysis under the National Pollutant Discharge Elimination System of the Clean Water Act. All analytical methods are  EPA approved and have a standard Quality Assurance/Quality Control protocol.

Project Timetable

Most components of this proposal will take a year to complete.  Not all components will begin at the same time, however and may depend on work presently underway to be completed. Analysis and technology transfer efforts will require additional time such that projects are expected to be complete in two years.  The diet evaluation and protein recovery objectives will require 12 months each.  The travel package objective will require two years.  The legal assessment will require about 12 months, and we are requesting permission to incur pre-award costs for this objective. Other components of the project will begin after August, 2004 and be completed by September, 2006.

REFERENCES

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Adler, P.R., J. K. Harper, F. Takeda, E.M. Wade and S.T. Summerfelt. 2000. Economic evaluation of hydroponics and other treatment options for phosphorous removal in aquaculture effluent. HortScience 35:993-999. 

Adler, P.R., F. Takeda , D.M. Glenn., E.M. Wade, S.T. Summerfelt and J.K. Harper. 1996. Nutrient removal: ecological process sows a cost-saving idea for enhancing water quality. Water Environment and Technology 8:23-24. 

Aguilo, P.M., Alegre, J. and Riera, A. 2001. Determinants of the price of German tourist packages on the island of Mallorca. Tourism Economics. 7:59-74. 

American Public Health Association, 1998. Standard Methods for the Analysis of Water and Wastewater, 20^th edition. American Public Health Association, Washington, DC. 

Anderson, D.M., P.M. Gilbert and J.M. Burkholder. 2002. Harmful algal blooms and eutrophication: nutrient sources, composition and consequences. Estuaries 25: 704-726. 

Avault, James W.  2004.  “Legal Considerations in Commercial Aquaculture: Parts I and II.  Aquaculture Magazine. Volume 30, Number 1 &2. 

Barlow, J., 1974.  Coal and Coal Mining In West Virginia, Coal-Geology Bulletin No.2, West Virginia Geological and Economic Survey, Morgantown, WV. 

Choi YJ, Park JW. 2000. Feasibility study of new acid-aided surimi processing method for enzyme-laden Pacific whiting. Abstract # 51A-4. Presented at the Institute of Food Technologists Annual Meeting (Dallas, TX). June 10-13. 

Doyle, D., A. Ferrise, and D.K. Smith. 1979.  Property: Land Owners Rights and Responsibilities in West Virginia.  WVU Extension Service, Morgantown, WV. 

Eagles, P.F. 1995. Understanding the market for sustainable tourism. In: McCool, S. and Watson, A.E. (Comps.). Linking tourism, the environment, and sustainability—topical volume of compiled papers from a special session of the annual meeting of the National Recreation and Park Association: 1994 October 12-14; Minneapolis, MN. Gen. Tech. Rep. INT-GTR-323. Ogden, UT: U.S. Department of Agriculture, Forest Service, Intermountain Research Station. 

Grafton, W.L., A Ferrise, D. Colyer, D.K. Smith, and J.E. Miller, 1990.  Conference Proceedings: Income opportunities for the private landowner through management of natural resources and recreational access.  WVU Extension Service, Morgantown, WV.   

Hartmann, H.T., D.E. Kester, F.T. Davies, Jr., R.L. Geneve. 2002. Plant propagation: principles and practices (7th ed.).  Upper Saddle River, NJ. 

Hites, R., Foran, J., Carpenter, D., Hamilton, M., Knuth, B., and S. Schwager, 2004.  “Global Assessment of Organic Contaminants in Farmed Salmon,” Science, 303, 226 – 229. 

Hultin HO, Kelleher SD. 1999. Process for isolating a protein composition from a muscle source and protein composition. US Patent No. 6,005,073. Issues Dec. 21.  

Jenkins, M., Wade, E., Fletcher, J., and Hankins, J., 1995. Economic Analysis of Non-Traditional Water Resources for Aquaculture in West Virginia. The Conservation Fund’s Freshwater Institute, Shepherdstown, WV. 

Lanier TC. 2000. Surimi gelation chemistry. In: Park JW, editor. Surimi and surimi seafood. New York: Marcel Dekker. p 237.  

Lee H, Lanier TC. 1995. The role of covalent crosslinking in the texturizing of the muscle proteins sols. J Muscle Foods 6:125-138. 

Lewis, George W.  1996.  Aquaculture Development Plan.  University of Georgia, Athens, GA.  

Loch, D. D., J. L. West, and D. G. Perlmutter. 1996. The effect of trout farm effluent on the taxa richness of benthic macroinvertebrates. Aquaculture 147:37-55. 

Pierskalla, C.D. and Lee, M.E. 1998.  An ecological perception model of leisure affordances. Leisure Sciences. 20:67-79. 

Pierskalla, C.D., Lee, M.E., Stein, T.V., Anderson, D.H., and Nickerson, R. 2004.  Understanding relationships among recreation opportunities: A meta-analysis of nine studies. Leisure Sciences. 26:1-18. 

Pizam, A. and Mansfeld, Y. (Eds.) 1999.. New York, NY: The Haworth Hospitality Press. Consumer behavior in travel and tourism 

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Rose P., K. Faulkner, G. Williamson, and R. Mithen.  2000.  7-Methylsulphinylheptyl and 8-methylsulphinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes. Carcinogenesis 21: 1983-1988. 

Skousen, J. and Ziemkiewicz, P., 1996. Acid Mine Drainage Control and Treatment, 2^nd Edition, West Virginia University, Morgantown, WV. 

Selong, J. H. and L. A. Helfrich. 1998. Impacts of trout culture effluent on water quality and biotic communities in Virginia headwater streams. Progressive Fish-Culturist 60:247-262. 

Soderberg, R., 1995.  Flowing Water Fish Culture.  Lewis Publishers, Boca Raton, Florida.

Srinivasan D. 1996. Amino acids, peptides, and proteins. In: Fennema OR, editor. Food Chemistry 3^rd ed. New York: Marcel Dekker. p 322.

Stein, T.V., Clark, J.K., and Rickards, J.L. 2004. Assessing nature’s role in ecotourism development in Florida: Perspectives of tourism professionals and government decision-makers. Journal of Ecotourism. 2:155-172.

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 Viadero, R. and Tierney A., 2003. Use of Treated Mine Water for Rainbow Trout (Oncorhynchus mykiss) Culture – A Preliminary Assessment. Journal of Aquacultural Engineering, 29, 43-56.

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 Wirth, FF, Luzar, EJ,  2001.   Regulatory climate toward finfish aquaculture: The impacts of state institutional structure.  Aquaculture Economics & Management, Vol. 5, no. 1-2, pp. 99-114.

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Key Personnel

Kenneth J. Semmens. Dr. Semmens, is State Extension Specialist for Aquaculture and specializes in aquaculture with twenty years of experience producing and marketing a wide variety of warm, cool and coldwater fish species.  He holds a joint appointment with the Cooperative Extension Service and the West Virginia Agricultural and Forestry Experiment Station.  He will oversee the execution of the project’s activities, assist the co-investigators with problems and issues that arise, facilitate resource allocation and integrate project activities in support of the aquaculture industry in West Virginia.

Karen M. Buzby is a postdoctoral fellow in the Department of Civil and Environmental Engineering at West Virginia University.  She will collaborate with Dr. Todd West in the evaluation of the efficacy of watercress in the removal of nutrients from trout raceway effluent.  She will focus on water chemistry.

 Jacek Jaczynski is an assistant professor of food science in the Division of Animal and Veterinary Sciences at West Virginia University.  His interests are aquatic foods and food safety.  He will lead the two tasks, Omega-3 fortified Arctic Charr as functional food, and development of value-added food based on  proteins and lipids recovered from trout processing by-products.

 Cyril M. Logar, Professor of Marketing, will collaborate with Dr. Pierskalla on the objective to develop and market fishing based travel packages. Responsibilities include survey design, administration and analysis and development of marketing plans based on survey results.

 Daniel Miller, M.S., is Research Associate in the Agricultural and Resource Economics Program.  Mr. Miller’s work experience emphasizes the biological and production aspects of aquaculture and fisheries.  He has served as a manager of and consultant on several aquaculture‑related projects in various parts of the U.S. and overseas.   His work on the project will focus on technology transfer particularly development of the mine water resource for commercial production of salmonids.

Chad D. Pierskalla, Ph.D., Assistant Professor in Recreation, Parks and Tourism

Resources, will collaborate with Dr. Logar on the objective to develop and market fishing based travel packages.  Dr. Pierskalla has research experience in the human dimensions of natural resources management.

 Dennis K. Smith, Professor of Agricultural and Resource Economics, and Associate Dean of Academic Affairs will coordinate the assessment and development of a legal framework for the aquaculture industry in West Virginia. Dr. Smith has extensive research and teaching experience in agri-business management, rural development and small business .

 Aislinn E. Tierney, is an associate engineering scientist in the department of Civil and Environmental Engineering. She coordinates water quality and effluent analysis at the WVU raceway facility utilizing treated mine water and assists Dr. Viadero with research on the engineering aspects at two mine water aquaculture facilities.

 Roger C. Viadero, Jr., Assistant Professor of Civil and Environmental Engineering will conduct task development and verification of a model to describe production of tainbow trout in raceway systems.  Dr. Viadero has led research on engineering aspects of water treatment in recirculating aquaculture systems used to raise yellow perch. 

 Todd West, Assistant Professor of Horticulture, Nutrient Recovery from Trout Raceways Utilizing Watercress.  He will collaborate with Dr. Viadero and Dr. Buzby to evaluate if watercress, a marketable horticultural crop can be effectively grown in a trout raceway system for nutrient recovery.