THE USE OF SELECTED BIOLOGICAL MATERIALS PRODUCED BY TERTIARY WASTEWATER' TREATMENT PONDS IN THE DIET OF TWO SPECIES OF FISH Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY CURTIS L. KERNS 1976 ABSTRACT THE USE OF SELECTED BIOLOGICAL MATERIALS PRODUCED BY TERTIARY NASTENATER TREATMENT PONDS IN THE DIET OF TWO SPECIES OF FISH By Curtis L. Kerns Fragments of aquatic plants are routinely found in the stom- achs of fish not normally considered herbivores. Consequently, a series of laboratory feeding trials were conducted at Michigan State University using a filamentous algae, Oedogonium, in the diet of steelhead trout (Salmo gairdneri), and varying levels of a 50:50 mix- ture of fresh poultry waste and Elodea canadensis in the rations of Israeli carp (Cyprinus carpio). A preliminary feeding trial demon- strated growth of steelhead fed a diet containing 34.5% Oedogonium was comparable to the growth of control fish on a nutritionally com- plete commercial trout ration. Oedogonium, when coarsely chopped and combined with other ingredients, formed a floating pellet readily consumed. Poultry waste and g, canadensis were fed to Israeli carp in order to evaluate the inclusion of non-protein-nitrogen (NPN) in the diet of a stomachless fish. Growth rate and feed conversion were found to be inversely related to the amount of poultry waste in the regimen. 6/0203? THE USE OF SELECTED BIOLOGICAL MATERIALS PRODUCED BY TERTIARY WASTENATER TREATMENT PONDS IN THE DIET OF TWO SPECIES OF FISH By {‘6‘ Curtis L. Kerns A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1976 This thesis is dedicated to all the people who have had faith in me: my Mother and Father, my senior advisor, and above all, my lady. ii ACKNOWLEDGMENTS This work was supported by the U. S. Department of the Interior with Allotment Grant A-082-Michigan administered through the Michigan State University Institute of Water Research, by the Michi- gan State University Agricultural Experiment Station, and by the Environmental Protection Agency Training Grant No. T90033l. We also thank the Michigan Department of Natural Resources and the Department of Fisheries and Allied Aquacultures at Auburn University for pro- viding the test eggs and fish. Thanks especially to Dr. C. J. Flegal of the Department of Poultry Science of Michigan State University for kindly providing excellent counsel and many of the diet ingredients. TABLE OF CONTENTS LIST OF TABLES INTRODUCTION . Cultural Eutrophication Resource Loss . . Michigan State University Water Quality Management Project. . . . . . . . . . . PART I FILAMENTOUS ALGAE (Oedogonium) IN THE DIET OF A SALMONID METHODS Diet Formulation Diet Production . Feeding Experimental Animals Environmental Conditions . Data Collection and Analysis RESULTS AND DISCUSSION Floating Feed . . Submerged Aquatic Plants . Pigments . . PART II POULTRY WASTES IN THE DIET OF ISRAELI CARP . MATERIALS AND METHODS . Diet Formulation Diet Production . Feeding . Experimental Animals Environmental Conditions . Data Collection and Anaysis . iv Page vi u—l IO 12 12 I3 IS 17 I8 20 22 22 24 29 29 3O 31 Page RESULTS AND DISCUSSION . . . . . . . . . . . . 32 CONCLUSION . . . . . . . . . . . . . . . . 38 APPENDIX . . . . . . . . . . . . . . . . . 39 BIBLIOGRAPHY . . . . . . . . . . . . . . . . 41 Table @01th \I 10. II. LIST OF TABLES Proximate Analyses of Daphnia magna in % Dry Weight, Average of Two Values . . . . . . . Amino Acid Content of Oedogonium as % Dry Weight Formulas of Test Diets . Summary of Results of Steelhead Feeding Trial #2 Composition of Carp Diets as % Dry Weight Composition of Poultry Waste Expressed as % Dry Weight . . . . . . . . Amino Acid Content of Dehydrated Poultry Wastes Chemical Composition of Elodea canadensis, as % Dry Weight, Average of Two Tests . . . . Summary of Results of Israeli Carp Fed Varying Amounts of Poultry Waste and Elodea canadensis Summary of Statistical Analyses . Final Proximal Analysis of Israeli Carp as % Dry Weight (n- 9); Vicerated, Nubbed . . . . vi Page II 16 23 25 26 27 33 34 35 INTRODUCTION Before a large quantity of domestic wastewater is discharged into a receiving water that is meso- or eutrophic, tertiary wastewater treatment is desirable. Nutrient removal in the main reverses cul- tural eutrophication and lessens the loss of an agriculturally quite Significant non-renewal natural resource, phosphorus. Cultural Eutrophication Relatively few lakes in North America have been studied in great detail before and after modification by artificial enrichment (20, p. l29). None the less, it may be said that during the first centenary of the United States most surface waters acted as nutrient sinks, that is, were able to ecologically process any increases in nutrient loads without significant changes in Species diversity. The lakes were in trophic equilibrium, and most of their hypolimnions remained aerobic. With the concomitant demographic transition and rapid human and domestic animal population growth of the last of the 18th and early l9th centuries, certain lacustrine systems became culturally eutrophic. Their finite capacity to sequester and cycle nutrients had been exceeded; they became transport systems instead of sinks. Anoxic conditions oftentimes develop in the hypolimnion of a eutrophic lake; all economically significant fish native to North America require relatively high dissolved oxygen levels. The fish must move to the shallower, often warmer, water. Consequently, there can occur a shift in species composition to the warm water tolerant organisms; commercially important fish species decline. Nuisance growths of aquatic plants occur; rapid geological aging occurs (58, pp. 330-332). During the second centenary domestic water use increased at an exponential rate (r2 = 0.991,calculated from data from Metcalf & Eddy). By the end of the second centenary, practically every major water body, including most estuaries, in the United States had shown signs of nutrient overloading. The oceans have even been adversely effected. The area off New York City, the "Dead Sea" is quite defin- itely no longer a sink. That Southern California coastal waters have lost much of their bull kelp (Laminaria) beds and that Florida waters have periodic blooms of often toxic dinoflagellates (Gonyaulax polyedra) should not go unnoticed. While precise data on the amount of phosphorus discharged are not available for all cities, an estimate may be made (Appendix). If all the phosphorus in domestic wastewater were to be absorbed by 6 aquatic plants assuming a density of 500 g/m2, some l9.65 x lo ha 6 or 98.26 x l0 mt (dry weight) of aquatic plants would be produced yearly (Appendix). Resource Loss Cultunaleutrophicationis a rather salient effect of dis- charging too much phosphorus; however, the most significant aspect of the practice may well be the loss, for the geological present, of this most important resource. Phosphorus and potassium unlike the other plant macro-nutrients, carbon, nitrogen, and water, do not recycle back into the atmosphere. Cathcart and Gulbrandsen (18) say of United States phosphate deposits: These reserves can supply domestic demands for scores or hundreds of years, but political, economic, and ecologi- cal demands may radically alter the traditional supply patterns in the U.S., and at sometime in the future the supply of phosphate rock to the eastern seaboard may possibly come from foreign sources. A rather innocuous statement until one considers that 83% of the United States production comes from Florida and North Carolina and so would have to be imported, and that the United States has the world's largest supply of identified reserves (l8). The U.S.S.R. and North Africa have most of the remaining world's mineable reserves. Michigan State University Water Quality Management Project The Michigan State University Water Quality Management Proj- ect is a research facility that is designed to receive two million gallons per day of secondarily treated domestic wastewater into a series of four ponds. The ponds have an aggregate area of 40 acres and are located on a 320-acre site on the Michigan State University campus. The project's main goal is to evaluate and demonstrate the potential for tertiary treatment of domestic wastewater by biologi- cally concentrating the nutrients into useful products within aquatic and terrestrial ecosystems. It is felt that the use of biological materials produced by tertiary wastewater treatment ponds in animal rations can reduce cultural eutrophication of aquatic environments, increase efficiency of resource utilization, and augment protein supplies. Three items were used in two separate laboratory-scale bio- logical feeding trials: the zooplankter, Daphnia magna harvested from pond l; a common filimentous green algae, Oedogonium from pond 3; and a submerged aquatic vascular plant, Elodea canadensis, drawn from pond 4. Section I of this work presents the results of the first feeding trial performed, a l2-week test with steelhead trout (Salmo gairdneri) fed a mixture of Oedogonium, Daphnia, soybean meal, and fish meal. Section II describes a feed assessment trial using Israeli carp (Cyprinus carpio) fed varying levels of a 50:50 mixture of poultry waste and Elodea. PART I FILAMENTOUS ALGAE (Oedogonium) IN THE DIET OF A SALMONID The use of aquatic plants for feedstuffs has lagged behind that of terrestrial plants. Tracts of aquatic vascular plants and filamentous algae can grow so dense that other beneficial uses of the water body are interfered with (8). Supply is not the problem. Aquatic plants sequester and biologically complex large amounts of nutrients. Culley and Epps (l9) estimate that a duckweed, Spirodela oligorrhiza, can uptake l85 kg of nitrogen/month/ha (T65 lb/ month/acre) and 60 kg of phosphorus/month/ha (53 lbs/month/acre). Boyd (lO) calculates that water hyacinth (Eichhornia crassipes) can remove 1980 kg of nitrogen/year/ha and 322 kg of phosphorus/ year/ha. Several researchers have been actively engaged in seeking uses for aquatic plants (8,9,l9,30,3l,38,4l,56). There is a limited use of water weeds in animal rations (ll,3l,38). Many species of fish consume aquatic plants; fragments of aquatic plants are found in the gut of fish normally not considered herbivorous, such as trout (Salmo gairdneri), bluegills (Lepomis macrochirus), and channel catfish (Ictaluris punctatus) (28,35). Other fish, especially as adults, reported to be almost exclusively plant feeders, e.g., grass carp (Ctenopharyngodon idella), can control a variety of aquatic weeds (2). Many fish species includ- ing Cyprinus carpio, Tilapia heudeloti, I, mossambica, I, nilotica, and I, gillj_consume specific plant species (2,33). A few feeding trials have been conducted using aquatic plants in the diet of fish. Liang and Lovell (30) using channel catfish conclude: The most feasible nutritional contribution of water hyacinth appears to be when the plant meal is fed as a low percentage in vitamin-poor diets as a source of growth factors. When fed to fingerlings, water willow (Justicia americana) was found to have good palatability, water hyacinth (Eichlornia crassipes) fair to good, and alligatorweed (Alternanthera philoxeroides) poor acceptability. KitcheIl and Windell (28) found that bluegills do gain some nutritional value from Charg_but cannot obtain sufficient dietary calories from the algae alone. The purpose of this section of the thesis is to present the results of a preliminary laboratory biological feeding trial con- ducted at the Fisheries Research facility of Michigan State University from August, l975, through February, 1976. Two sequential experi- ments were conducted in order to evaluate the inclusion of high per- centages of Oedogonium in the diet of steelhead trout (Salmo gairdneri). METHODS Diet Formulation Two diets meeting the essential amino acid and protein C requirements (40) of Chinook salmon (Oncorhynchus tschawytscha) were separately computer formulated. Formulation was performed using Michigan State University's CDC 6500 computer; the software employed was the Agriculture Economics Linear Program Package, Version 2 (27). The control diet was Purina Trout Chow,an extruded, nutritionally complete ration containing 40% crude protein (cp). Ringrose (48) working with brook trout (Salvelinus fontinaIis) has fOund that a calorie/protein ratio of 75 gives approximately optimum growth and feed conversion efficiency. Therefore, meta- bolizable energy was forced into the formulation at 2976 kcal/kg (l350 kcal/lb). The essential amino acid and metabolizable energy content of the diet ingredients were calculated using National Acad- emy of Science (NAS) values (39,40). A proximate analysis was run on the Daphnia (Table l); an amino acid analysis was performed on the Oedogonium (Table 2). The metabolizable energy values were estimated using 8.0 kcal/g of lipid, 3.9 kcal/g of protein, and l.6 kcal/g of starch again as per NAS (40); a value of 888 kcal/kg was used for Oedogonium and l024 kcal/kg for Daphnia. Amino acid values for Daphnia were taken from the literature (44). 9 TABLE l.--Proximate Analyses of Daphnia magna in % Dry Weight, Average of Two Values. Sample Number 1 2 3 Crude protein 42.8 53.7 53.7 Ether Extract 3.46 6.00 6.29 Ash 40.82 23.00 23.01 Cell Walls 15.84 N.A. N.A. TABLE 2.--Amino Acid Content of Oedogonium as % Dry Weight. Amino Acid Value Thr 1.582 Ser 1.538 Glu 3.786 Gly 1.703 00a 1.895 Va1 1.103 Cys --- Met 1.892 150 0.975 Leu 2.173 Tyr 0.668 Phe 1.386 Lys 2.218 His 0.478 Arg 1.381 Sum of Amino Acids 22.778 IO The possible ingredients were Oedogonium, Daphnia magna, blood meal, fish meal, soybean meal, dried whey, brewer's yeast, fish and corn oil, and methionine; kelp meal, salt w/trace minerals, and vitamins were forced in (Table 3). Diet Production The Oedogonium was harvested during August from pond 3 of the Michigan State University Water Quality Management Project (W.Q.M.P.). a wastewater recycling research facility. The plants were hand-wrung and forced-air dried at 51 C (124 F) for 12 hours. A W-W 5-hp compost grinder was used to coarsely chop the algae before freezer storage. Before inclusion into diet 1, the plants were finely ground using a Clifford laboratory cutting mill with a screen equivalent to a 20-mesh Tyler (0.833 mm, 0.0328 inch). The Oedogonium used in diet 2 was not finely ground. The Daphnia were seined from pond l of the W.Q.M.P. during July and August, 1976. The zooplankton used in diet 1 were air dried to a moisture content of approximately 50% and then frozen; the Q. magna_used in diet 2 were quick frozen on dry ice in l-liter plastic bags. The dry dietary components were mixed in a large bag by hand tumbling beginning with the ingredient smallest in mass and proceed- ing sequentially. After mixing they were finely ground in the lab- oratory mill. The oil was added just before pelletizing. Final mixing and pelleting was accomplished by using a 1.5-hp Hobart food grinder. Sufficient water was added to bring the mixture 11 TABLE 3.--Formulas of Test Diets. Ingredient % Dry Weight Diet 1, 10/11/75-11/7/75 Oedogonium 29.2 Daphnia 44.2 Corn Oil 10.7 Blood Meal 1.0 Soy Bean Meal (49% cp) 11.4 Salt w/trace Mineralsa 1.5 Kelp Meal 0.5 Vitaminsb .__lg§ 100.0 Diet 2. 11/25/75-2/17/76 Oedogonium 34.50 Daphnia 8.97 Fish Meal (Menhaden) 46.78 Fish Oil (Cod Liver) 5.00 Kelp Meal 2.00 Salt w/trace Mineralsa 1.25 Vitaminsb 1.50 100.00 aTrace minerals supplied per kg of diet: Manganese Sulfate- 55.0 mg; Magnesium Oxide-500.0 mg; Iron-80.0 mg; Copper-4.0 mg; Zinc-80.0 mg; Selenium-0.1 mg; MSU Poultry Mineral Mix #2. bVitamin content supplied per kg of diet: A-33,33 IU; 0 -3.333 IU; E-33 IU; MSBC-6.6 mg; Thiamine-10.0 mg; Riboflavin- 3§.0 mg; Pantothenic acid-50.0 mg; niacin-333 mg; Pyridxin-20 mg; Biotin-500 mcg; Folacin 10 mg; 812=33 mcg; MSU chick starter #2. 12 to a moisture content of 45%; the feed was passed through the grinder four times. Pellet diameter was 8 mm (3/16"); all longer-than- average pellets were hand broken. Diet 1 was forced air dried at 51 C (124 F) for 12 hours to a moisture content of approximately 7%; diet 2, which was prepared at a later date was air dried for 10 hours to a moisture level of 33.63%. The pellets were stored frozen under a nitrogen atmosphere in glass containers. 3229109 Brett (13) has found with sockeye salmon (Oncorhynchus nerka) the greatest increase in appetite occurs between 7 and 11 hours of fasting. Therefore, the fish were fed all they would consume once every 8 hours. The fish were fed on a 7-day per week basis. A modified version of the electric clock-based automatic feeder described by McCauley (34) was used; control was by a 2E026 Dayton timer. Experimental Animals The test fish were se1ected for uniform size from fish hatched on the premises. The eggs were supplied by the Michigan Department of Natural Resources' Platte River Hatchery, Honor, Michi- gan. The steelhead were assigned growth tanks using a random number generator. Three replications of 15 to 17 fish were used for the two treatments. At the conclusion of the first test all fish were ran- domly reassigned. The main phase of the two tests were of 4 and 10 weeks duration. respectively. 13 During the 2-week acclimation period preceeding the tests, the fish were fed at 1.8% of body weight. At the beginning of the second acclimation period, the second Oedogonium diet was mixed with the control diet in 1:7, 1:3; and 1:1 ratios; each mixture, start- ing with the former, was fed the test fish for three days. There- after, the Oedogonium-based test diet was fed. Environmental Conditions The fish were kept in 150-1iter oval, fiberglass tanks covered with screens. The center stand pipe was adjusted to allow a volume of 120 liters. The fish density was very low, 7.413 kg/m3 maximum. At the start of the first test, cleaning was performed daily, but was found to unduly stress the fish. So the frequency was reduced to biweekly. During the second test, weekly cleaning was deemed sufficient. All tanks were, however, scrubbed during the biweekly weighing. The filtered well water had a temperature of 12.6 :_0.5 C (54.5 :_0.9 F). In earlier tests, the water hardness as CaCO3 was found to be 329 mg/liter, alkalinity as CaC03 - 332 mg/liter, and the pH ranged from 7.0 to 7.6. The water flow rate was 2 liters/min. Dissolved oxygen ranged from 6.1 to 6.7 mg/liter. The fish were kept under constant artificial light. Data Collection and Analysis At the beginning and end of the experiments the fish were anesthetized with a 25-ppm MS-222 solution and individually 14 displacement weighed to the nearest one-tenth of a gram on a 1200-g Mettler balance. During the experiments the fish were displacement weighed biweekly to the nearest gram by lot on a 10-kg Mettler balance. The plant materials were washed with distilled water before analysis; an endeavor was made to remove all animal biomass present, although some of the smaller organisms undoubtedly did remain. A Virtis freeze dryer was used to desiccate materials to be analyzed. Proximate and amino acid analyses were conducted using A.0.A.C. standard procedures by the Botany and Aminal Science departments of Michigan State University. Statistical analysis of the test results consisted of one- way analysis of variance. RESULTS AND DISCUSSION The first ration tested was very poorly accepted by the steelhead. The pellets were dense and rapidly sank; the fish con- sumed very little food. The pellets that were eaten appeared to pass through the fish's digestive tract almost intact. Trout typically , do not masticate their prey before swallowing (51) although some trituration does occur in the gut after exposure to strong gastric fluids. Little growth resulted from the test diet, while the control fish grew satisfactorily. Consequently, after 4 weeks the trial was ceased. An excessively fine grind of the Oedogonium was thought to be the principal cause of the problem. A test for trimethylamine performed by the Food Science Department revealed little oxidative rancidity of the lipid fraction of the ration. Coarsely chopped Oedogonium was used in the second diet. The pellets floated well and were readily consumed by the fish. The growth rate of the test fish slightly exceeded that of the control fish (Table 4), but the difference was not statistically significant, P < 0.05 level. The test diet was a preliminary formulation; the . development of a practical, that is, low in fish meal, production ration was beyond the scope of this experiment. 15 16 TABLE 4.--Summary of Results of Steelhead Feeding Trial #2. Item Units Test Diet Control Diet Average Initial Weight grams 20.4 19.2 Standard Deviation 8.42 9.37 Skew 0.76 0.91 Average Final Weight grams 55.6 51.6 Standard Deviation 25.28 22.92 Skew 0.75 0.60 Total Food Fed grams 3,055.4 3,050.4 Total Weight Gain grams 1,387.3 1,281.9 Feed Conversion 2.20 2.38 Weight Gain % 273 269 Weight Gain as a % of Control % 101.5 100.0 Daily Growth Increment % 1.20 1.18 Mortalities 0 0 Cost of Feed per kg $ 0.241 (Estimated) 0.430a per (lb) $ (0.109) (0.195) Cost of Feed per kg of Weight Gain $ (0.443) (0.809) per (lb) $ (0.201) (0.367) aJune, 1976 prices. 17 Floating Feed The use of a floating pellet in a salmonid regimen is highly desirable. Artificial foods are not readily consumed off the bottom; trout feed almost exclusively in the water column and at the air- water interface. A 30-minute test of a grab sample of 45 pellets was conducted. After 1 minute, 39 (87%) remained afloat; at the end of the test, 21 (47%) were still buoyant. Extrusion cooking is the main process employed to produce commercial floating pellets. Unfortunately, the extrusion process is very capital and energy intensive (50); floating rations conse- quentl y cost more. Furthermore, extruded foods must include a high starch base; starch, even after being subjected to high temperatures, has been demonstrated to be incompletely utilized by salmonids (40), at 1.4 kcal/g vice 4.0 kcal/g for mammals. The most highly biologically ordered, and expensive, component of a trout ration is protein. High temperatures, > 80 C,can result in significant protein loss because of the Maillard reaction (non- enzymatic browning), unless the feedstuff has a moisture content of less than 10% and is rapidly cooled (26). Extrusion cooking may involve temperatures of "150-175°C (300-350°F)" according to Sanderude (50). Submerged Aquatic Plants Dense and diverse animal communities are often found in and among submerged aquatic plants (16,28,43,45). Protozoa, rotifers, 18 annelids, crustaceans, insects, and molluscs have been reported to make up 17.5% of the dry weight of submerged aquatic plants (45). The production lot of Oedogonium used contained large numbers of Cladocera, Coleoptera and Hyalella; the sample tested which was well-rinsed had a sum of amino acids of 22.78% (Table 2). Boyd and Scarsbrook (12) report crude protein values of from 4.5% to 31.3% and phosphorus concentrations of 0.05% to 0.56% on a dry weight basis in filamentous algae. The fauna associated with certain aquatic plants may make a further contribution to the plant's amino acid pro- file. Pigments Aquatic plants contain relatively large amounts of photo- reactive carotenoid pigments. Nelson and Palmer (41) reported that Vallisneria spiralis had a carotenoid content of 100mg/lOOg. The red crab (Pleuronodes planipes), a Species noted for its carotenoid content, has from 56.9% ug/lOOg to 85.7 Ug/lDOg (53). Visual examina- tion of the flesh of fish sacrificed at the termination of the experiment showed no difference between dietary treatments; the muscle tissue of either lot of fish was not highly pigmented. The largest fish weighed about 1259; a larger size or greater age may have to be reached before flesh pigmentation occurs. If the carotenoids in aquatic plants are biochemically available to fish, a desirable flesh pigmentation may occur. PART II 19 POULTRY WASTES IN THE DIET 0F ISRAELI CARP Calvert (17) reports that in the United States, 28.8 million kg (63.5 million lbs.) of nitrogen is excreted by animals as waste each day. Substantial interest has been shown in converting the presently underutilized non-protein-nitrogen (NPN) in animal wastes into animal biomass (5,6,17,22,23,25,32,42,47,52,55). NPN in the form of animal wastes has been demonstrated to be sparing of protein-nitrogen in the diet of ruminant animals (5,42,55). The mineralized nitrogen is incorporated biologically into microbial biomass in the rumen. However, with a non-ruminant animal, broiler- chicks, Flegal and Zindel (23) report "feed efficiency was inversely related to the level of DPW (Dried Poultry Wastes) in the diet." The inclusion of poultry wastes and other forms of NPN into the diet of fish has produced ambiguous results. Shiloh et a1. (52) found the inclusion of DPW at a 10% and 20% level was deleterious to the growth of carp (Cyprinus carpio); but the addition of 4% and 5% soybean oil resulted in an improvement in growth of 3.2% and 3.5% respectively over that of the control diet. Leray (29) states some- what cryptically that with mullet (Mugilidae), "one-half of the protein-nitrogen could be replaced by urea-nitrogen without modifying the growth curve." Lu and Kevern (32) found the growth of goldfish (Carassius auratus) fed a 30% DPW ration was superior to that of the 20 21 control group fed Ewos F49, a salmon feed. However, with channel catfish (Ictalurus punctatus) growth of the control fish, again Ewos- fed, was significantly better than fish fed 30:70, 70:30, and 100:0 ratios of DPW:Ewos (P < 0.05). Fish fed 100% DPW showed little or no growth. Fowler reports that channel catfish fed a ration of 25% DPW grew better than the control group (25). The purpose of this section of the thesis is to present the results of a laboratory-scale biological feeding trial conducted at the Fisheries Research facility of Michigan State University from January through April, 1976. The experiment was conducted to evalu- ate the inclusion of NPN in the form of poultry waste in the diet of Israeli carp. MATERIALS AND METHODS Diet Formulation Four diets equal in metabolizable energy and essential amino acids were formulated on the MSU CDC 6500 computer. The software employed was the Agriculture Economics Linear Program Package, Version 2 (27). The control diet selected was a production ration extensively used in Israel (52,57). Three diets utilizing varying levels of a 50:50 mixture of fresh poultry waste and §19g§g_ canadensis, a source of fiber, along with other ingredients were tested (Table 5). The essential amino acid and metabolizable energy content of the control diet was calculated using National Academy of Science values (39,40). Proximate and amino acid analyses were run on the Elodea. Metabolizable energy value for glggga_was estimated using 8.0 kcal/g of lipid and 3.9 kcal/g of protein (40). A value of 4.0 kcal/g of carbohydrate, the physiolgical fuel value for mammals, was used instead of the value commonly used for salmonids and ictalurids, l.6 kcal/g (40). This substitution was felt justified because of the carp's high polysaccharide digestion coefficient (7). Poultry waste amino acid and energy values came from the litera- ture (22,47). When formulating diets using ingredients of low nutrient concentration, it is often difficult to get enough of the ingredients into the ration. Poultry waste and the particular batch of Elodea 22 23 TABLE 5.--Composition of Carp Diets as % Dry Weight. Diet Number Item 1 2 3 (ConErol) FORMULA Elodea canadensis 12.50 20.00 27.23 Poultry Wastes 12.50 20.00 27.23 Wheat 42.28 20.77 70.00 Soybean meal (49% cp) 38.47 41.06 43.55 15.00 Fish meal (menhaden) 15.00 Fish oil (cod 1iver)a 3.18 7.15 10.98 Vit.b'C 1.0 1.0 1.0 Methionine 0.07 0.03 Feeding rate as % of control 109.00 110.01 109.99 100.00 PROXIMATE ANALYSIS Crude protein 30.80 33.75 35.18 28.04 Cell walls 27.52 26.52 24.06 27.12 Ether extract 1.85 5.78 9.13 2.42 Ash 9.88 13.44 16.98 5.94 aStabi1ized with 200 ppm Lenox 26 (F.D.A. Standard for human consumption). bMSU chick starter vit. mix #2. CVitamin Content: A—33,333 IU; D3-3,333 ICU; E-33 IU; MSBC-6.6 mg; Thiamine-10.0 mg; Riboflavin 33.0 mg; Pantothenic acid- 50.0 mg; naicin-333 mg; Pyridxin-20 mg; Biotin-500 mcg; Folacin 10 mg; 312-33 mcg per kg of diet; MSU chick starter vitamin mix #2. 24 used have a rather low food value (Tables 6, 7, and 8). Consequently, test diets l, 2, and 3 were allowed to fluctuate in quantity up to 110% of the control (Table 5). When the quantity of the test diets to be fed was held to be equal to that of the control, the computer formulation always included fish meal. But at the 110% quantity level, no fish meal was needed. The latter option was chosen because it was felt that the inclusion of fish meal would obscure the experimental objective. Diet 3 was computer formulated on a least-cost basis to be equal to the control diet in the ten essential amino acids and meta- bolizable energy. The possible ingredients were: a 50:50 mixture of poultry waste and Elodea, soybean meal, fish meal, wheat, fish oil, methionine, and vitamins. Vitamins were forced in at a 1% level, metabolizable energy was forced in at 2965 kcal/kg (1345 kcal/lb). Diets l and 2 were formulated as per diet 3 except lower levels of poultry waste and Elodea were forced in (Table 5). Diet Production The Elggga_was harvested in July from pond 4 of the Michigan State University Water Quality Management Project, a wastewater recy- cling research facility. The plants were air dried for four hours to a moisture content of about 50% and then coarsely chopped using a W-W 5-hp compost grinder. Forced air drying at a temperature of 51 C (124 F) for 12 hours reduced the moisture content to 6.03%. The E19dea_was stored in doubled 150-liter (40 gal) plastic trash bags. Before inclusion into the diets the plants were finely ground with a 25 TABLE 6.--Composition of Poultry Waste Expressed as % Dry Weight. Item Value Remarks Source Crude 33.92 Fresh Benne (4) Protein n = 23 Moisture 75.00 Fresh ' Benne (4) n = 29 Total 6.73 CP = 42.06% White et a1. Nitrogen I59) % of 70.2 urinary 61.2% uric acid Total N 9.0% NH4 Salts 29.8 Fecal Unutilized Pro- Flegal (24) tein from food Sloughed off gut Microorganisms Feathers P205 4.54 White et a1. (59). K20 2.03 Ash 20.41 Organic 66.29 Matter 26 TABLE 7.--Amino Acid Content of Dehydrated Poultry Wastes.a Essential Amino Acids gm/100 gm Crude Protein % dry wt as Tested Arg. 1.93 0.65 HIS 0.82 0.28 ISO 2.05 0.70 LEU 3.32 1.13 LYS 2.01 0.68 MET + cvs 4.88 1.65 PHE + TYR 2.91 0.99 THR 2.05 0.70 TRY --- --- VAL _g;§§_ _5ng3 22.55 7.67 aF1ega1 & Zindell (22). 27 TABLE 8.--Chemica1 Composition of Elodea canadensis, as % Dry Weight,Average of Two Tests. Item Amount Proximate analysis Crude protein 9.9 Crude fiber 14.74 Ether extract 0.84 N-free Extract 48.66 Essential Amino Acids ARG 0.533 HIS 0.204 ISO 0.254 LEU 0.574 LYS 0.454 MET + CYS 0.081 PHE + TYR 0.718 THR 0.373 TRY -- VAL 0.393 28 Clifford laboratory cutting mill with a screen equivalent to a 20- mesh Tyler (0.833 mm, 0.0328 in). Benne (4) has demonstrated a marked nitrogen loss in poultry waste during the drying process. Therefore, fresh poultry waste.: 24 hours old,was collected from the Michigan State University Poultry Barns from a production lot of Leghorn-type layers and was immedi- ately frozen. Just before using, the frozen poultry waste was ground with a 6 mm (1/4") screen on the compost grinder. The Elggg§_and poultry waste were combined and thorough mixing was insured by pass- ing the material through the compost grinder three times. All other dry ingredients were mixed by tumbling the combined ingredients in a plastic container for 1 min. The two ingredients smallest in mass were mixed first; the next larger in mass was then added and tumbled. After mixing, all dry ingredients except the wheat were finely ground in the laboratory mill. The wheat could not be passed through the fine screen in the Clifford mill as the screen would quickly plug. Instead, the wheat was repeatedly passed through the next coarser screen until a fairly fine grind resulted. Final mixing and pelleting was accomplished using a 1.5-hp Hobart food grinder. Sufficient distilled water was added to each diet to bring the moisture content to 45%. All diets were passed through the grinder three times. Shaking the fresh pellets in a plastic bag served to break most of the pellets. The diets were air dried at 18 C (64.6 F) for 12 hours to a moisture content of 9%. 29 E91119 Cyprinids have a stomachless digestive system and therefore feed at shorter intervals than do many other fish. Rozin and Mayer (49) found that goldfish, when allowed free access to a self-feeder distributed their feeding responses fairly evenly over time. There- fore, the test fish were fed once every 2 hours on a 24-hour basis. A modified verion of the electric clock-based automatic feeder described by McCauley (34) was used; control was by a 2E026 Dayton timer. The feeding rate was 4% of body weight per day for the control fish (7,52,57); diets 1, 2, and 3 were fed at 4.36%, 4.4%, and 4.4% body weight, respectively, which provided equal metabolizabel energy and essential amino acids. The fish were fed on a 7-day per week basis with pellets 5 mm (1/8") in diameter for the first two months and 8 mm (3/16") in diameter thereafter. Any longer than average pellets were hand broken before feeding. Experimental Animals The test fish were selected from a group of 350 full sibs that originated from Auburn University, Auburn, Alabama. The fish were maintained without mortality in 12.5 C filtered wellwater in three 150-liter fiberglass tanks until ready for use. A 45% crude protein (Purina No. 4) trout ration was fed at 1% of body weight. The fish increased from 119 to 189 mean weight during the 9 months they were held prior to the experiment. For the test, the 240 closest to median sized fish were selected; all malformed individuals were excluded. The fish were 30 assigned to growth tanks using a random number generator. Three replications of 20 fish each were used for each of the four diet treatments. Two acclimations periods were allowed. The first period was just after tank assignment and lasted four weeks during which water temperature was increased in daily increments of l C until the test temperature was reached. The fish were fed on the control ration at 2% of body weight daily. A further two-week acclimation period was allowed on the respective test diets again at 2% of body weight. The main phase of the test ran for ten weeks. Environmental Conditions The fish were kept in twelve 80-liter plastic barrels, round in cross section. The water volume was held at 201iters (5.3 gal) by using a center stand pipe. The resulting fish density was con- sidered medium by comparison with Israeli cage culture(52) at 75 kg/ma. In earlier tests, the water hardness as CaCO3 was found to be 329 mg/liter, alkalinity as CaCo3, 332 mg/liter, and the pH ranged from 7.0 to 7.6. Water temperature was controlled by a Powers 11A regulator @ 22.5 :_0.5 C (72.5 :_0.9 F). The water flow was 1 liter/ min. The influent water was directed at a slight angle from the vertical in order to power a slow, radial current. The tanks were self-cleaning. Because of the water current, physical action of the fish, and high water flows little aufwuchs developed, consequently no additional cleaning was performed. Dissolved oxygen (0.0.) fluctuated over the course of the experiment. Winkler determinations taken during the first four weeks 31 averaged 5.1 mg 02/liter. At the beginning of the sixth week 0.0. fell to less than 2 mg/liter in one of the control tanks; the aver- age of the other four was 4.4 mg/liter. Air stones delivering an estimated 60 incheS3/min were immediately added to each tank and an aspirator was added to the facility's main water supply increasing 0.0. from 64% saturation to 86% saturation. For the rest of the experiment, 0.0. averaged 7 mg/liter. The fish were kept under natural photoperiod dUring a time of increasing day length. Data Collection and Analysis At the beginning and end of the experiment the fish were anesthetized with a 25-ppm MS-222 solution and individually displace- ment weighed to the nearest one-hundredth of a gram on a 12009 Mettler balance. During the experiment the fish were displacement weighed biweekly to the nearest gram by lot on a lO-kg Mettler. Proximate analysis of the fish at the conclusion of the experiment was obtained from a grab sample of three individuals netted from each tank. The trunk region, vicera removed, from each of the 36 fish was used in the analysis. Proximate and amino acid analyses were conducted using A.0.A.C. standard procedures by the Biochemistry, Botany, and Animal Science departments on the Michigan State University campus. Statistical analysis consisted of one-way analysis of vari- ance, Duncan's new multiple range, and linear regression tests (54). RESULTS AND DISCUSSION The growth rate of the test fish was inversely related to the 2 = 0 975). The amount of poultry waste in the diet (Table 9, r growth rate of the control fish was significantly different from the growth rate of the test fish (P > 0.005, Tables 9 and 10)- Feed conversion efficiency was also inversely related to the level of poultry waste in the diet; the most expensive diet, the control, pro- duced the most economical weight gain and protein production. The reduced weight gain of the fish fed the test diets indicates poor utilization of poultry waste; the amino acids found in the poultry waste were not efficiently incorporated into fish biomass. Despite the diets being isocalorically formulated, the test fish had lower tissue lipid levels (Table 11). It would appear that the inclusion of NPN in the diet of Israeli carp has a high metabolic cost for the fish. The tissue ash content of the test fish varied directly with the level of poultry waste in the diet (r2 = 0.962). The amount of dietary mineral retained has been found to be proprotional to the quantity ingested (40). Dry matter varied inversely with level of poultry wastes fed (r2 = 0.947). Poultry waste contains high levels of calcium, phosphorus, potassium, and other minerals (Table 6); however, direct ingestion of 32 .APNV 5666 666666 66666666666 33 666662 6666 66.6 66.6 66.6 66.6 6666666 66 66.6 66.6 66.6 66.6 6666666 66666666 6666666 66 666 6666 6666.6 6666.6 6666.6 6666.6 6666666 66 6666.6 6666.6 6666.6 6666.6 6666666 6666 666663 66 666 6666 666. 666. 666. 666. 6666666 66 666.6 666.6 666.6 666.6 6666666 666 666 6666 66 6666 6 6 6 6 66666666662 66.6 66.6 66.6 66.6 63 6666666 6 666666666 663666 66666 66.666 66 66 66.66 66.66 6666666 66 6 6666 666663 66.666 66.666 66.666 66.666 6 6666 666663 66.6 66.6 66.6 66.6 6666 63 666666 6 6666663666 6666 6.666.6 6.666.. 6.666.. 6.666._ 66666 666666 666663 66666 666.6 666.6 666.6 666.6 66666 666 6666 66666 66.6 66.6 66.6 66.6 3666 66 66 66.66 66.66 66.66 666666366 66666666 66.66 66.66 66.66 66.66 66666 .63 66666 6666636 66.6 66.6 66.6 66.6 3666 66.6 66.6 66.6 .66.6 666666366 66666666 66.66 66.66 66.66 66.66 66666 .63 6666666 6666636 6666666 . 6 6 6 6 66666 6666 . m wmcwbmcmo 666066 666 66663 xgupzoaht666czoe< mcwxgm> 666 agmu 6666666 mo mupzmmm mo 666556m11.m mAm 0.005. Duncan's New Multiple Range Test Diet Number 3 2 l 4 (Control) Final Mean Weight 45.25 54.20 56.26 75.09 P > 0.01 P > 0.05 Any two means underscored by the same line are not significantly different. Growth Equationsa Diet 1 Y = 21.41 + 3.36X Diet 2 Y = 21.51 + 3.23X Diet 3 Y = 21.43 + 2.45X Diet 4 Y = 17.35 + 5.61X aWhere Y = weight in grams X week of feeding trial 35 TABLE ll.--Final Proximal Analysis of Israeli Carp as % Dry Weight (n=9); Vicerated, Nubbed. Diet Number Item 1 2 3 4 Ash 8.59 9.69 11.86 5.47 Ether extract 30.44 18.86 16.43 38.64 Crude protein 71.56 73.13 75.69 56.56 Nitrogen-free extract 3.30 0.28 --- 0.51 Dry matter (wet weight) 26.89 24.29 23.25 29.48 36 Ca,P, Co, and Cl is not necessary for fish. Fish absorb minerals directly from their environment (40) across their gill membranes. Flegal and Zindel (22) found dehydrated poultry waste to contain an average of 26.1% crude protein. About 44.7% of the crude protein is protein-nitrogen consisting of unutilized ingested feed, microorganisms, Sloughed-off gut, and feathers (24) as indicated in Table 6. The remaining nitrogen fraction is non-proteinaceous and consists of uric acid, urea, and ammonia salts. White et a1. (59) found 70.2% of the nitrogen in poultry wastes to be urinary in origin. In a ruminant, NPN is absorbed by mircoorganisms in the rumen and biologically complexed along with fiber, as an energy source, to form the essential amino acids required by the host animal. Diges- tion and absorption of the microbial protein occurs in the abomasum and intestine. In order for fish to use NPN and fiber an analogous microbial process would have to occur. Salmonids and ictalurids do, at times, support high populations of intestinal microflora. However, bacteria are not usually found in the intestines of fasting fish (36). The presence of microorganisms depends upon the ingestion of microbially contaminated food and the presence of food as a substrate. The bacteria present are not regarded as commensal (36), although the presence of bacteria may have some beneficial side effects. It is, however, doubtful that bacteria in the intestine of fish with stomachs contribute significant amounts of protein to the fish. 37 Based on his own work and that of others, Beauvalet (3) as reported by Al-Hussaini (1) states that "when fishes have a stomach the secre- tions of their intestines do not effect proteins to any appreciable degree." Carp along with other cyprinids do not have stomachs. The pH in their digestive systems is neutral to alkaline. Protein diges- tion occurs mainly in the 3rd (final) limb of the intestine and in the rectum. Carp have strong carbohydrate digesting enzymes but rather weak proteinases (l). Carp were selected to be the test fish as they are widely cultured, have a high fiber diet, and it was felt had the best chance of profitably using NPN in their diet. ,However, they do not appear to do so economically. All fish fed actively during the experiment; little uneaten food was noted. The daily growth increment of the control fish was 1.90%, higher albeit not strictly comparable to that reported by other researchers with larger carp fed the same diet, 1.09% (52). Boyd (11) has found that aquatic plants in differing states of maturity show a marked variation in crude protein content. E12922. has been demonstrated to contain 26.81% crude protein by Nelson and Palmer (41). The Elggga_used in this experiment had a crude protein content of 9.9% (Table 8). Therefore, this experiment could not evaluate the plant nutritionally; it was included mainly as a source of fiber. CONCLUSION NPN in the form of poultry waste does not appear to be suit- able for inclusion into the diet of small Israeli carp. Israeli carp do not appear to be able to satisfy dietary protein requirements via the digestion of microbes produced in their gut. Growth of non- ruminants such as fish on diets containing NPN is probably not because of, but rather in spite of, the dietary inclusion of items such as poultry manure. 38 APPENDIX 39 APPENDIX POTENTIAL OF PHOSPHORUS IN WASTEWATER FOR AQUATIC PLANT PRODUCTION Assumptions: U. S. Population 1976 - 223 x 106 personsa Phosphorus in Wastewater - 10 mg/lb Wastewater production - 378.541 liter/day/capita Aquatic plant standing crop - 500 g/m2 (5 mt/ha) Percent P in aquatic plants - 0.3138%C 223 x 106 x 378.541 liter/day/capita x 0.01 g P/liter = 844 mt P/day x 365.25 days/year = 308,324 mt P/year 6 308,324 mt 3 0.003138 = 98.255 x 10 mt aquatic plants/year e 5 mt/ha = 19.651 x 106 ha/year aTrend analysis using Bureau of the Census data (15). bMetcalf & Eddy (37), p. 231. cBoyd (12) average of 79 species of aquatic plants. 40 BIBLIOGRAPHY 41 BIBLIOGRAPHY Al-Hussaini, A. H. 1949. On the functional morphology of the alimentary tract of some fish in relation to differences in their feeding habits; Cytology and Physiology. Quarterly Journal of Microscopial Science, vol. 90, pt. 4, p. 323-353. Avault, J. W., R. 0. Smitherman, and E. W. Shell. 1968. Evaluation of eight species of fish for aquatic weed control. Proc. World Symp. on Warm-water Fish Culture, Rome, Italy. May 18-25, 1966. FAO Fisheries Report, 44, vol. 5: VII/E-3, p. 109-122. Beauvalet, H. 1933. C. R. Soc. Biol. Paris, vol. 112, p. 640. Quoted by Al-Hussaini (l). Benne, E. J. 1971. A compilation of some samples of DPW. In G. C. Sheppard (Ed.). Poultry Pollution: Research Results. Michigan State Agricultural Experimental Station Research Report No. 152. Bhattacharya, A. N. and J. P. Fontenot. 1965. Utilization of different levels of poultry litter nitrogen by sheep. Journal of Animal Science, vol. 24, p. 1174-1178. Blair, R. 1974. Evaluation of dehydrated poultry wastes as a feed ingredient for poultry. Federation Proceedings, vol. 33, no. 8, p. 1934-1935. Bondi, A., A. Spandorf, and R. Calmi. 1957. The nutritive value of various feeds for carp. Bamidgeh, vol. 9, no. 1, p. 13-18. Boyd, C. E. 1968. Fresh-water plants: a potential source of protein. Economic Botany, vol. 22, no. 4, p. 359-368. Boyd, C. E. 1968. Evaluation of some common aquatic weeds as possible feedstuffs. Hyacinth Control Journal, vol. 7, p. 26-27. 42 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 43 Boyd, C. E. 1970. Vascular aquatic plants for mineral nutrient removal from polluted waters. Economic Botany, vol. 24, no. 1, p. 95-103. Boyd, C. E., and R. 0. Blackburn. 1970. Seasonal changes in the proximate composition of some common aquatic weeds. Hyacinth Control Journal, vol. 8, p. 42-44. Boyd, C. E., and E. Scarsbrook. 1975. Chemical composition of aquatic weeds. Agricultural Experimental Station, Auburn University, Auburn, Alabama (Unpublished). Brett, J. R. 1971. Saturation time, appetite, and maximum food intake of sockeye salmon (Oncorhynchus nerka). Journal of Fisheries Research Board of Canada, vol. 28, p. 409-415. Buhler, D. R., and J. E. Halver. 1961. Nutrition of salmonid fishes--carbohydrate requirements of Chinook salmon. Journal of Nutrition, vol. 74, p. 307-318. Bureau of the Census. 1975. Statistical Abstract of the U. S. 1975 (96th Edition, Washington, D.C.), 1050 p. Burton, G. J. 1973. Feeding of Simulium hargreavesi (Gibbons) larvae on Oedogonium algae filaments in Ghana. Journal of Medical Entomology, vol. 10, no. 1, p. 101-106. Calvert, C. C. 1974. Animal wastes as substrates for protein production. Federation Proceedings, vol. 33, no. 8, p. 1938-1939. Cathcart, J. B., and R..A. Gulbrandsen. 1973. Phosphate Deposits. In Brobst, D. A., and W. P. Pratt (Eds.) U. S. Mineral Resources, U. S. Geological Survey Professional paper 820, 722 p. Culley, D. 0., Jr., and E. A. Epps. 1973. Use of duckweed for waste treatment and animal feed. Journal Water Pollution Control Federation, vol. 45, no. 2, p. 337-347. Edmondson, W. T. 1969. Eutrophication in North America. In Eutrophication: Cause, Consequences, Correctives. National Academy of Sciences, Washington, D.C., 661 p. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 44 Feedstuffs. 1975. The Ingredient Market, vol. 47, no. 50, Dec. 12. Flegal, C. J., and H. C. Zindel. 1970. The utilization of poultry waste as a feedstuff for growing chicks, p. 21-28. In C. C. Sheppard (ed.) Poultry Pollution: Problems and Solutions. Michigan State Univers- ity Aggiculture Experimental Station Research Report no. . Flegal, C. J., and H. C. Zindel. 1971. Dehydrated poultry-waste (DPW) as a feedstuff in poul- try rations. Proceedings of the International Symposium on Livestock Wastes, p. 305-307. Flegal, C. J. 1976. Personal Communication. Fowler, C. J. 1973. Poultry pointers. Southwestern Poultry Times, April 14. Goering, H. K., and P. J. VanSoest. 1973. Relative susceptibility of forages to heat damage as affected by moisture, temperature, and pH. Journal of Dairy Science, vol. 56, no. 1, p. 137-142. Harsh, S. B., and J. R. Black. 1975. Agricultural Economics Staff Paper No. 75-10. Michigan State University. Kitchell, J. F., and J. T. Windell. 1970. Nutritional value of algae to bluegill sunfish Lepomis macrochirus. Copeia, vol. 1, p. 186-190. Leray, C. 1970. Experimental approaches to artificial feeding of some sea fish. In J. Gaudet (ed.) Report of the 1970 Workshop on Fish Feed Technology and Nutrition. Resource publica- tion No. 102. Bureau of Sport Fisheries and Wildlife, p. 169-171. Liang, J. K., and R. T. Lovell. 1971. Nutritional value of water hyacinth in channel catfish feeds. Hyacinth Control Journal, vol. 9, no. 1, p. 40-44. Little, E. C. S. (ed.). 1968. Handbook of utilization of aquatic plants. FAD. Rome, Italy, 121 p. Lu, J. D., and N. R. Keveren. 1975. The feasibility of using waste materials as supple- mental fish feed. Progressive Fish-Culturist, vol. 37, no. 4, p. 241-244. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 45 McBay, L. G. 1961. The biology of Tilapia nilotica Linnaeus. Proceedings Southeastern Association of Game and Fish Commissioners, vol. 15, p. 208-18. McCauley, R. W. 1970. Automatic food pellet dispenser for walleyes. Progres- sive Fish-Culturist, vol. 32, no. 1, p. 42. Mathur, D. 1970. Food habits and feeding chronology of channel catfish in Conowingo Reservoir, Proceedings Southeastern Association of Game and Fish Commissioners, vol. 24, p. 377-386. Margolis, L. 1953. The effect of fasting on the bacterial flora of the intestine of fish. Journal Fisheries Research Board of Canada, vol. 10, no. p. 62-63. Metcalf and Eddy, Inc. 1972. Wastewater Engineering. McGraw-Hill Book Co., New York, 782 p. Misric, V. 1936. Lake vegetation as a possible source of forage. Science, vol. 83, p. 391-392. National Academy of Sciences. 1971. Atlas of Nutritional Data on U. S. and Canadian Feeds. Subcommittee on Feed Composition. Washington, D.C. 782 p. ‘National Research Council, National Academy of Sciences. 1973. Nutrient Requirements of Trout, Salmon, and Catfish. Subcommittee on Fish Nutrition. Washington, D.C. 57 p. Nelson, J. W., and L. S. Palmer. 1938. Nutritive value and chemical composition of certain fresh water plants of Minnesota. Bulletin Minnesota Agri- cultural Extention Station Technical Bulletin, vol. 136, p. 4-31. Noland, P. R., B. F. Ford, and M. L. Ray. 1955. The use of ground chicken litter as a source of nitro- gen for gestating lactating ewes and fattening steers. Journal of Animal Science, vol. 14, p. 860-865. Odum, H. T. 1957. Trophic Structure and Productivity of Silver Springs, Florida. Ecological Monographs, vol. 27, p. 55-112. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 46 Ogino, C. ' 1963. Studies on the chemical composition of some natural foods of aquatic organisms. Bulletin of Japanese Society of Scientific Fisheries, vol. 29, p. 459-462. Petr, T. 1968. Population changes in aquatic invertebrates living on two water plants in a tropical man-made lake. Hydrobiologia, vol. 32, p. 449-485. Phillips, A. M., A. V. Tunison, and R. D. Brockway. 1948. The utilization of carbohydrates by trout. New York Conservation Department Fisheries Research Bulletin, vol. 11, p. 44. Polin, D., S. Varghese, M. Neff, M. Gomez, C. J. Flegal, and H. Zindel. 1971. The metabolizable energy value of dried poultry waste, p. 33-44. In C. C. Sheppard (ed.), Poultry Pollution: Research Results. Michigan State University Agricultural Experimental Station Research Report, no. 152. Ringrose, R. C. 1971. Calorie-to-protein ratio for brook trout. Journal Fisheries Research Board of Canada, vol. 28, p. 113-117. Rogin, P., and J. Mayer. 1961. Regulation of food intake in the goldfish. American Journal of Physiology, vol. 291, no. 5, pp. 968-974. Sanderude, K. G. 1970. Excerpts from extrusion cooking principles and applica- tions for snack foods, meats, and other products. In Report of the 1970 workshop on fish feed technology and nutrition. Bureau of Sport Fisheries and Wildlife, Research Publication no. 102, p. 68-70. Shapavalori, L. and A. C. Taft. 1954. The life histories of the steelhead rainbow trout (Salmo gairdneri gairdneri) and silver salmon (Qngorhynchus kisutch) with Special reference to Waddell Creek, Ca., and recommendations regarding their management. California Department of Fish and Game Bulletin, vol. 98, p. 1113-1117. Shiloh, S., E. Hamifray, and S. Viola. 1973. Experiments in the nutrition of carp growing in cages. Bamidgeh, vol. 25, no. 1, p. 17-31. Spinelli, J., L. Lehman, and D. Wieg. 1974. Composition, processing and utilization of red crab (Pleuroncodes planipes), as an aquacultural feed ingredient. Journal Fisheries Research Board of Canada, vol. 31, p. 1025- 1029. 54. 55. 56. 57. 58. 59. 47 Steel, R. G. 0., and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, 481 p. Tinnimit, P., Y. Yu, K. McGuffey, and J. W. Thomas. 1972. Dried animal waste as a protein supplement for sheep. Journal of Animal Science, vol. 35, p. 431-435. Traux, R. 1972. Duckweed for chick feed. Louisiana Agriculture, vol. 1, no. 8. Viola, S. 1975. Experiments on nutrition of carp growing in cages. Bamidgeh, vol. 27, no. 2, p. 40-48. Warren, E. E., and P. Doudoroff. 1971. Biology and Water Pollution Control. W. B. Saunders Co., Philadelphia, Pennsylvania. 434 p. White, J. W., F. K. Holben, and A. C. Richer. 1944. Production, composition and value of poultry manure. Pennsylvania Agricultural Experiment Station Bulletin, no. 469. HICHIGRN STATE UNIV. LIBRQRIES 11111 111 1 312931022 4498