'WWWI“Wall‘fll l 100 026 THS_ gsow I bl LIERI’ERY Michigan State University UN IVERSITY LIBRARIES IIIIIIIIIIIIIIIIIIIIIIII II III II I II 3 1293 006299 I L This is to certify that the dissertation entitled THE EFFECT OF AMMONIA ON OREOCHROMIS NILOTICUS (NILE TILAPIA) AND ITS DYNAMICS IN FERTILIZED TROPICAL FISH PONDS presented by Abdelmoez A.F . Abdalla has been accepted towards fulfillment of the requirements for Fisheries& Wildlife MM. MM: Major professdl' Date (9/\©/Kq MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove thIs checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE m 2 I.“ Time a {312 ll IJ’) I‘WVMWJ ' ll ‘LIIIIE I? n 7""7’ MSU Is An Afflrmdive ActiorVEquel Opportunlty Institution THE EFFECT OF AMMONIA ON OR EOCHROMIS NILOTICUS (NILE TILAPIA) AND ITS DYNAMICS IN FERTILIZED TROPICAL FISH PONDS By Abdelmoez A.F. Abdalla A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY Department of Fisheries and Wildlife 1989 N") I I r- r II‘JVJ -.._~) ABSTRACT THE EFFECT OF AMMONIA ON OREOCHROMIS NILOTICUS (NILE TILAPIA) AND ITS DYNAMICS IN FERTILIZED TROPICAL FISH PONDS By Abdelmoez A.F. Abdalla This study was designed to investigate: (l) acute effects of unionized ammonia on 10.7 g f ingerlings of Oreochromis niloticus at two temperatures (23°C and 33°C), and at 28°C with two sizes of fish (3.4 g and 45.2 g), (2) chronic effects of unionized ammonia on growth of 0. niloticus at two temperatures typical of tropical ponds (28°C and 33°C), and (3) loss of ammonia to the atmosphere and uptake by algae as mechanisms that reduce ammonia concentrations and toxic effects in fertilized ponds stocked with 0. m'loticus. In acute toxicity tests at 23°C and 33°C, 96 hr LCSOs were 2.2 and 2.3 mg/l unionized ammonia. The 96 hr LCSO at 28°C was 1.4 mg/l unionized ammonia for small fish (3.4 g) and 2.8 mg/l for large fish (45.2 g). There was not a significant effect of temperature on acute toxicity of unionized ammonia with 10.7 g fish, while there was a significant effect due to fish size at 28°C. In chronic tests, there was a linear decrease in fish weight gain with increasing unionized ammonia concentrations at both 28°C and 33°C. The level of no growth effect was 0.06 mg/ I unionized ammonia at both temperatures. Bf f ective concentrations for 50% growth reduction were 0.77 and 0.87 mg/l at 28°C and 33°C respectively. No-growth concentrations were 1.48 and 1.67 mg/l unionized ammonia at these temperatures. The relative growth rate of fish in controls was significantly higher at 28°C than at 33°C. Three fertilizer treatments were used in ponds in a field experiment. Substantial quantities of ammonia were lost from ponds in each treatment during daylight hours. Increasing initial total ammonia present at dawn increased net primary productivity. Losses to the atmosphere were relatively small, varying from 1-9% of total diurnal ammonia losses. Uptake of ammonia by algae was a more important mechanism for removal of ammonia from ponds. Algae uptake accounted for 37% to greater than 100% of the ammonia lost during daylight hours. Fish mortalities occurred in ponds with the highest fertilizer treatment (3150 g N/mz/wk). The average diel unionized ammonia measured in these ponds was 0.7 mg/l. Interaction between unionized ammonia, low dissolved oxygen and high pond temperatures appeared implicated in mortalities. To my beautiful kids, Sarah and Zakaryia, who always make me smile and to my family iv ACKNOWLEDGEMENTS First, I would like to thank Almighty ALLAH for everything HE gave me. Great appreciation goes to Dr. Clarence McNabb my major advisor and director of my research especially for his suggestions and intense review of this manuscript despite of his busy schedule, and for supplying me with a fellowship without which this research would have been impossible. Thanks go to Dr. D. Garling my committee member for his help with the experimental design, his bright ideas throughout this study, and for facilitating the use of his fisheries laboratory for me to use. I would also like to thank my other committee members, Dr. J. Tiedje and Dr. D. Ullery, for their significant advice during the course of this study. Warmest thanks are expressed to the rest of the Limnological Research Laboratory family, Dr. Ted Batterson, Ralph Beebe, Tim Fiest, for their microcomputer assistance and the good time. Thanks go also to Dr. J. Giesy for supplying the micro-computer program used in toxicity studies. Finally, I can't forget the financial help, confidence and encouragement by my wife, Demoree, especially during the last period of finishing this research. This research was supported by the American Agency for International Development and Michigan State University. TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................ vii LIST OF FIGURES ........................................... - ........................... viii INTRODUCTION ......................................................................................................... 1 MATERIALS AND METHODS ................................................................................... 4 RESULTS ...................................................................................................................... 13 DISCUSSION ................................................................................................................. 35 SUMMARY ................................................................................................................... 44 LIST OF REFERENCES ............................................................................................... 46 APPENDIX .................................................................................................................... 52 vi LIST OF TABLES Table Page 1. Average concentrations (mg/l) of elements in water used in laboratory experiments. NDA is non detectable amount ................................................ 5 2. Environmental conditions for acute tests. Values are means with ranges in parentheses -- ------ ................................ l4 3. Results of acute toxicity tests ........................................................................... 15 4. Environmental conditions for chronic tests. Values are means with ranges in parentheses .................................................................................................... l7 5. Mean concentrations of inorganic nitrogen and dissolved oxygen for the period of the experiment. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea .................................................................................... 22 6. Average ammonia loss to the atmosphere (flux) from treatment ponds. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea .................................................................................................................... 25 7. The relationship between ammonia concentrations in ponds and loss to the atmosphere (flux) in treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ......................................................... 26 8. The relationship between algae uptake of ammonia and net carbon fixation in treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea .................................................................................... 31 9. The lethal level of unionized ammonia with varying concentrations of dissolved oxygen ............................................................................................... 42 vii Figure 10. A-2. LIST OF FIGURES The system used for acute and chronic experiments ....................................... Toxicity curves for unionized ammonia in acute tests .................................... The chronic effect of unionized ammonia on average weight gain of fish in trials at 28°C and 33°C ...... - - - - ...................... The chronic effect of unionized ammonia on percent decrease in fish weight gain in trials at 28°C and 33°C ............................................................. Diel total ammonia variations in treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ........................................................ Diel rates of dissolved oxygen in treatment A with 500 kg/ha/wk chicken manure ............................................................................................................... Diel rates of dissolved oxygen change in treatment B with 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea .......................................................... Diel rates of dissolved oxygen change in treatment C with 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ......................................................... The relationship between cumulative net carbon fixation and cumulative total ammonia loss at 1 hr intervals starting with the 7-8 am interval and proceeding through the day to the 4-5 pm interval for 3 treatments tested. Total ammonia loss is corrected for loss to the atmosphere. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ................. Diel unionized ammonia in ponds of three treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ................................ Diel total ammonia cycle in three layers of the water column in treatment A with 500 kg/ha/wk chicken manure ............................................................ Diel total ammonia cycle in three layers of the water column in treatment B with 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea ........................ viii Page 6 l 6 19 20 23 28 29 30 32 34 52 53 Figure Page A-3. Diel total ammonia cycle in three layers of the water column in treatment C with 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea .................... 54 A-4. Mean pH values in ponds of Treatments A, B and C. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ....................................... 55 A-S. Mean temperatures in ponds of treatments A, B and C. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ............................... 56 A-6. Diurnal loss of nitrogen to the atmosphere from ponds in treatment A with 500 kg/ha/wk chicken manure ......................................................................... 57 A-7. Diurnal loss of nitrogen to the atmosphere from ponds in treatment B with 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea .................................... 58 A-8. Diurnal loss of nitrogen to the atmosphere from ponds in treatment C with 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea ................................ 59 A-9. Diel unionized ammonia cycle in three layers of the water column in treatment A with 500 kg/ha/wk chicken manure .......................................... 60 A—10. Diel unionized ammonia cycle in three layers of the water column in treatment B with 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea ....... 61 A-ll. Diel unionized ammonia cycle in three layers of the water column in treatment C with 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea .. 62 ix INTRODUCTION Accumulation of ammonia is a major constraint on fish production in intensively managed aquaculture systems (Smith 1972; Harader and Allen 1983). It can originate from either allochthonous or autochthonous sources. While source water used to fill ponds and raceways may contain ammonia from nitrogenous materials in sewage treatment plant effluents, industrial effluents, and runoff of fertilized agricultural land and animal f eedlots, principal allochthonous sources in intensive aquaculture are fertilizers and feeds applied directly to ponds. Autochthonous sources of ammonia include excretions of end products of protein catabolism in aquatic fauna (e.g. fish and zooplankton), and bacterial mineralization of the organic nitrogeneous matter. Two chemical species of ammonia occur in aqueous solutions: unionized (NH,) and ionized (NH,*) forms. Fractions of these species in the total ammonia present depend on pH and temperature (Emerson et a1. 1975), and to a lesser degree on ionic strength of the solution. Increasing the pH and temperature in aquaculture systems tends to increase the unionized form, while increasing ionic strength tends to decrease the unionized form of ammonia. Unionized ammonia is by far the more toxic of the two forms of ammonia (Thurston et a1. 1981). It moves with relative ease across cell membranes due to lipid solubility and lack of charge (Coombe et a1. 1960). Fish exposed to acute concentrations suffer disturbances of the nervuos system. They exhibit hyperexitability, an increase in gill ventillation, a loss of equilibrium, convulsions, coma, and finally death (Russo 1985, WHO 1986). In general, reported median lethal concentrations of unionized ammonia in 4 day bioassay tests ranged from 0.083 to 1.1 mg/l for salmonids, and from 0.14 to 4.6 mg/l for non-salmonids (Thurston et a1. 1984). However, much lower concentrations were observed to cause the sublethal effects. Fish exposed to sublethal concentrations of unionized ammonia suffer a histological changes in tissues of the gills, liver, kidneys, eyes, brain, fins and blood (Thurston et a1. 2 1986), with a summation result of decreased weight relative to unexposed cohorts (Colt and Tchobanoglous 1978). Many factors can modify the toxicity of ammonia on aquatic organisms. These include pH (Tabata 1962; Thurston et al. 1981; Broderius 1985), temperature (Brown 1968; Colt and Tchobanoglous 1976), dissolved oxygen (Downing and Merkens 1955; Selesi and Varmos 1967), previous acclimitization to ammonia (Vamos 1963; Redner and Stickney 1979), carbon dioxide concentration (Allan et a1. 1958), salinity (Harder and Allan 1983), calcium content of the water (Tomasso et a1. 1980),and presence of other toxicants (WHO 1986). Concentrations that cause acute or chronic effects will also vary with the size and species of fish (Sprague 1985). Research reported in this dissertation was part of overseas experiments aimed at evaluating water quality criteria for high fish production in intensively managed tropical ponds. High rates of fertilizer application were under study. The experimental fish was Nile tilapia, Oreochromis niloticus. It was chosen for these experiments because of its ability to efficiently convert food web products growing in response to manure fertilizers into high quality protein while resisting relatively poor water quality and diseases (Balarin and Haller 1982). While earlier work (Boyd 1983) suggested that ammonia would accumulate in experimental ponds, no data were available in the literature for acute or chronic effects of ammonia on this species. Under conditions of the field experiments, ammonia losses from water columns of ponds was expected to occur in several ways, thus decreasing its toxicity to fish. Uptake as a nutrient by microflora, absorption on sediments, volatization to the atmosphere, and nitrification and denitrification were viewed as protective processes. Field data relating to these processes were poorly developed in the aquaculture literature. On the other hand, the work of Gallaway (1980) suggested that volatization of ammonia from eutrophic ponds could serve to protect fish from toxic effects. Bouldin et al. (1974) reported that 2- 38% of the NH,+ concentrations present was volatized per day from ponds. Also, in laboratory studies, Harrison (1978) reported a strong positive correlation between NH} assimilation and photosynthetic carbon assimilation. Lara and Romero (1986) found that the utilization of 3 nitrate or ammonia by cyanobacteria was strictly dependent on light and the availability of carbon dioxide. They concluded that the light requirement was derived from the requirement for photosynthetically generated reductant and ATP needed by the corresponding assimilatory processes, whereas the CO, requirement arose from different interactions between carbon and nitrogen metabolism. From field studies, Murphy and Brownlee (1982) reported higher ammonia concentrations in the morning than in the afternoon. Healy and Hendzel (1976) noted nitrogen deficiency caused by algae blooms in hypertrOphic lakes. Shilo and Rimon (1982) noted ammonia depletion by cyanobacteria blooms prior to their collapse in intensively managed fish ponds. This work predicted a coupling between the diel rate of photosynthetic carbon fixation, algae uptake, and the loss of ammonia that would moderate ammonia toxicity in heavily fertilized ponds. In this background of information, the following objectives were developed for this work: 1) to determine the lethal concentrations of unionized ammonia on Oreochromis niloticus at two different temperatures (23°C and 33°C), and with two different sizes of fish (3.4 and 45.2 g) at 28°C. 2) to determine the sublethal growth effects of unionized ammonia on Oreochromis niloticus at two different temperatures typical of tropical ponds (28°C and 33°C). 3) to relate the diel ammonia losses in ponds to atmospheric flux and uptake by algae as mechanisms that protect against fish toxicity. The first two objectives were achieved by running a series of toxicity tests in the Limnological Laboratory at Michigan State University. The last objective was achieved by a field experiment conducted in Thailand. Sampling for the experimental design was carried out by MSU and Thai scientists under the Pond Dynamics/Aquaculture Collaborative Research Support Program (CRSP) sponsored by the United States Agency for International Development. MATERIALS AND METHODS LABQBAIQRX W Laboratory experiments were divided into two parts; tests for acute (lethal) and chronic (sublethal) toxicity of unionized ammonia. Methods for acute tests followed standard procedures recommended by the American Society for Testing and Materials (1980) and APHA (1985). Criteria for the end point of acute tests was death, recognized by cessation of movement and no response after stimulation with a glass rod. In chronic tests, growth of fish held at different ambient concentrations of unionized ammonia was measured and results were compared to controls. The source water used in experiments was wellwater used for drinking in Lansing area. Elemental analysis of the water is shown in Table 1. It entered a fiberglass head tank that held approximately 635 liters. Water in the head tank was aerated to oxidize souble ferrous iron and precipitate it as ferric iron. This prevented the accumulation of iron compounds in tubing and tanks of the test system. Chlorine concentrations were measured in head tank water to assess its potential for adverse effects on growth. Eren and Langer (1973) found that small tilapia (3-5 g) were sensitive to chlorine concentrations above 0.5 mg/l, and that large fish could withstand concentrations up to 1 mg/l. Free and total chlorine concentrations were measured daily during experiments using a HACH test kit. Total chlorine never exceeded 0.05 mg/l in the head tank. A preliminary test of fish survival in source water was run for 40 days using 10 aquaria with 10 fish each. No deaths or signs of disease were observed. The toxicant delivery system consisted of a reservoir of concentrated ammonia (reagent grade ammonium chloride dissolved in distilled water), a variable speed ten channel Autoclude pump, mixing chambers and an agitator (Figure l). The ammonia reservoir was a 23 liter nalgene carboy. The concentrated ammonia solution dripped from it into a bucket with a 7 liter capacity. The bucket was connected to the pump by using appropriate sizes of Table 1. Average concentrations (mg/l) of elements in water used in laboratory experiments. NDA is non detectable amount. Element Average Detection Limit Aluminum NDA 0.030 Arsenic NDA 0.050 Barium 0.1 0.005 Boron 0.02 0.010 Calcium 93 0.100 Cadmium NDA 0.010 Cobalt NDA 0.010 Copper 0.02 0.005 Iron 0.1 0.050 Mercury NDA 0.050 Potassium 1.8 0.100 Magnesium 25 0.050 Manganese 0.02 0.010 Molybdenum N DA 0.020 Sodium 8 0.010 Phosphorous 0.5 0.100 Lead NDA 0.050 Zinc 0. I 0.005 £53109”. 015qu ES 8:3 8* v8: 88»? 2:. A cam—h 7 plastic tubing. Different sized tubes came out from the pump and were connected to the mixing chambers. The mixing chambers were one liter vacuum flasks painted black to prevent algal growth. Different amounts of ammonia coming from the pump were mixed with dilution water coming from the head tank to get the concentrations used in each treatment. Water inflow to each mixing chamber was controlled by a stopcock. Vacuum flasks were put on a shaker to ensure good mixing prior to delivery to aquaria. Plastic tubes were used to carry ammoniated water with different concentrations from the sidearm of each mixing chamber to a corresponding aquarium. A water flow of about 150 ml/min was delivered to each aquarium. The water retention time in each aquarium was about 4.2 hour. A timer was used to obtain a 14 hr light period and a 12 hr dark period during experiments. The work of Palachek and Tomasso (1984) pointed out the toxic effect that nitrites could have in aquaria of the test system. They found the 96hr LC50 to be 16.2 mg/l. Nitrite was measured each week during chronic tests, and once on the last day of acute tests. It was measured using the sulphanilamide method of APHA (1985). The nitrite concentration was never above 0.85 mg/l in individual measurements. The mean concentration in aquaria for acute toxicity tests was 0.55 mg/l, and 0.25 mg/l in those for chronic tests. Acute toxicity tests for ammonia were run at two different temperatures: 23°C, and 33°C. Small fish were tested at each temperature. Fish used at 23°C had a mean weight of 10.6 g, while those used at 33°C had mean weight of 10.8 g. Large and small size fish, with a mean weight of 45.2 g and 3.4 g respectively, were also tested at 28°C to determine the effect of size on toxicity. Chronic tests were run at 28°C and 33°C. One aquarium was used at each concentration of unionized ammonia at acute or chronic tests. Three control aquaria were used in 28°C and four control aquaria were used in 33°C chronic tests. Two control aquaria were during acute tests. Temperatures in test aquaria were controlled by regulating temperature in the head tank relative to heat gain or loss in portions of the delivery system below the head tank. Temperature in the head tank was regulated by a constant flow through of source water and mercury thermoregulators that controlled immersion heaters. Additional small heaters were required in each aquarium to maintain temperature during acute and chronic tests at 33°C. 8 The test fish used was Oreochromis niloticus. Adults were obtained from Auburn University and spawned in the Fisheries Research Laboratory at Michigan State University. Spawning procedures involved stocking one ready male and three ripe females in an aerated rectangular tank (635 liter). The tank was well aerated and heated to about 28°C. Fish were fed twice daily to satiation and the tank was cleaned twice daily. Young were collected after released from their mothers' mouths and transferred to a circular tank (742 liter) where they were fed daily about 3% of their body weight using small size purina trout chow. These fish were used in the acute and chronic tests at 28°C. However, water flow stopped by accident and this stock was lost. A second batch of fish was brought from Auburn University and divided into size classes for use in other experiments. During holding time, these fish were fed trout chow to maintenance level: about 1% of body weight daily as suggested by Brett et al. (1969). Temperature was held at about 22°C. The mortality rate of fish was less than 1% during holding time. Ages of fish used in experiments ranged from 2-6 months. In all cases, fish were acclimatized for three weeks at the desired temperature before beginning either the acute or chronic tests. Fish were fed to satiation twice daily during the acclimatization period. The fish grew during this time. Any weak or diseased fish were eliminated from the random choice of the fish used for specific test. Acriflavine was used to prevent tail and fin rot disease which was observed in a few cases during both holding and acclimatization period. Two ml/liter were added weekly to each aquarium from a stock solution containing 10 mg/l as recommended by Duf in (1973). Fish were exposed to ammonia in 38 liter aquaria. Aquaria were covered with a screen to prevent fish from jumping. Ten fish were used per aquarium, except in the case of testing large fish (45.2 g), when six were used per aquarium. Fish were not fed one day before exposure to ammonia, and for acute tests, they were not fed during exposure. Fish in chronic tests were fed to satiation twice a day. Debris was cleaned from aquaria daily. Total ammonia was measured daily in each aquarium during the acute tests, and three times a week in the chronic tests. An ammonia electrode was connected to a Corning pH - ion meter No.135 and used according to the approved method of APHA (1985). Unionized 9 ammonia in test aquaria was determined from measurements of total ammonia, pH, and temperature by the formula of Emerson et al. (1975), based on the ionization constants of Bates and Pinching (1949). Temperature, pH and dissolved oxygen were measured at the same time daily during the acute tests, and three times a week in the chronic tests. Temperatures were measured by a thermometer to the nearest 0.5°C. Measurements of pH were made using a model 221, digital pH/temp meter with Ross combination pH electrode (ORION). Dissolved oxygen concentration was measured with a YSl model 54 A no meter. Alkalinity and hardness were measured each week in the chronic experiment, and once during acute tests. Total alkalinity was measured by titration with sulfuric acid (APHA 1985; Wetzel and Likens 1979). Hardness was calculated using the procedure of Wetzel and Likens (1979). In acute toxicity tests, the number of fish that died were recorded each 24 hours, up to 96 hours. Chronic tests lasted for 5 weeks. Length (cm) and weight (gm) of fish were measured in the begining and at the end of each chronic test. The probit analysis (Stephan's 1984 probit program ) was applied to calculate the median lethal concentration (LC50) at 24, 48, 72, and 96 hours. A test of parallelism (Litchfield and Wilcoxn 1949, APHA 1985) was used to test for significant differences between LC50 values. Linear regression analyses were performed between the weight gain of fish (Y) and unionized ammonia (X) in the chronic experiment to determine the effect of different concentrations of ammonia on fish growth. One way analysis of variance was used to determine the effect of temperature alone on the relative growth rate of fish. Test of independence or contingency tables (Gill 1978) were developed to test the effect of temperature and unionized ammonia concentration on the mortality rate of fish in the chronic experiments. HELD W Ammonia dynamics in fertilized fish ponds was studied in Thailand. The experiment was run at the Bang Sai Station of the Royal Thai Department of Fisheries near Ayutthaya at 14.2°N. and 100.5°E. The experiment had three treatments with four ponds in each 10 treatment. Ponds had a surface area of 0.022 ha. They were filled with water from the Chao Phraya River and maintained at a depth of 0.9 m. Ponds were stocked with Nile tilapia, at a density of two fish per square meter. Fingerlings had a mean weight of 20.4 g at planting. The experiment lasted for four months. Three fertilizer treatments were used in the experiment: (A) chicken manure at a rate of 500 kg/ha/wk, (B) chicken manure at 44 kg/ha/wk plus 24 kg/ha/wk of urea, and (C) chicken manure at 500 kg/ha/wk plus 280 kg/ha/wk of urea. Treatment A had a chicken manure loading rate commonly recommended to fish farmers in the region to promote growth of algae and general productivity in pond food webs. However, earlier experiments on the site had shown that algae in ponds fertilized with 500 kg/ha/wk had inadequate nitrogen for algae growth (Diana et al. 1985). It was also found that chicken manure was deficient in nitrogen relative to phosphorus and the approximate 7:1 ratio of N:P by weight that algae have for normal growth (Batterson et al. 1988). The chicken manure used in treatment A had a mean N:P ratio of 0.6. Treatment A was expected to be nitrogen limited. The rate of total nitrogen loading in the fertilizer for treatment A was 271 g/mz/wk. In treatment B, the amount of nitrogen added to ponds weekly was the same as for treatment A. The chicken manure contained enough phosphorus to make a 7:1 ratio of N:P in the fertilizers. Algae productivity in treatment B was expected to be nearly the same as in treatment A. Fertilizers in treatment C also had an N:P ratio of 7. The nitrogen loading rate was much higher than for other treatments: 3150 g/mz/wk. High algae productivity was predicted for ponds in this treatment compared to other treatments, and high nitrogen loading was expected to result in ammonia concentrations that might effect the growth of fish. In this study, fertilizers were broadcast over ponds on day-0 of each week, and measurements of pond parameters were made on day-3 of the fertilizing cycle. Thus, data given here represent conditions in ponds at mid-points between weekly applications. Measurements of total ammonia were made on samples collected every two weeks at 0600 hr, 1000 hr, 1200 hr, 1400 hr, I600 hr, 1800 hr, 2300 hr and 0600 hr the next day. Water samples were taken from top (25 cm below the surface), middle (50 cm below the 11 surface) and bottom (25 cm above the sediment) layers of the water column. Temperature and pH were measured at the time of sampling at each of the three depths. A thermistor was used to measure temperature, and a standardized meter with combination electrode to measure pH. Total ammonia was measured by the nesslerization method (APHA 1985). Unionized ammonia concentrations were calculated with procedures used in the laboratory study. The cadmium reduction method (APHA 1985) was used to determine NO,-N + NO,-N in composite samples of water columns collected near mid-morning at intervals of two weeks. Volatization rate, or flux of ammonia to the atmosphere, was determined from field data using a model developed originally by Galloway (1980). It predicts ammonia flux from total ammonia concentration, pH, temperature, pond depth, and wind speed. The model assumes that flux depends on both unionized and ionized ammonia. Mean daily wind speed was measured with an anemometer, comparable with a Weathertronics Model 5210, set at 1 m above pond surfaces. Pond depth was measured daily from staff gauges in each pond. The model was put in a spreadsheet for micro-computer use at MSU’s Limnological Research Laboratory. Dissolved oxygen concentrations were measured every two weeks at 0600 hr, 1000 hr, 1200 hr, 1400 hr, 1600 hr, 1800 hr, 2300 hr and 0600 hr the next day. Measurements were made in top, middle and bottom layers of water columns with a YSI model 57 meter. These data were used to obtain estimates of primary productivity in ponds. The single curve method described by Odum (1956, 1957) and Hall and Russel (1975) was used to calculate primary productivity. Averages of biweekly measurements of dissolved oxygen were used to calculate mean oxygen concentrations in water columns in g/m3 for different times of the day. Oxygen rates of change between time intervals of measurement were determined in g/m’/hr. Mean temperatures of water columns were obtained in degrees Celsius. Dissolved oxygen concentrations in water at atmospheric equilibrium with specified temperatures were obtained from APHA (1985). Percent O, saturation for water in ponds was then calculated. Rates of oxygen exchange with the atmosphere were obtained from Boyd (1983), based on data of Schroder et a1. (1975), in g/mz/hr. Oxygen rates of change between time intervals of measurement were corrected for atmospheric exchange. Graphs were then drawn for each 12 treatment with with time of day on the X-axis and oxygen rate of change in g/m3/hr on the Y—axis. Areas under curves where the oxygen rate of change was positive were then integrated to obtain net photosynthesis for light periods of days. Calculations of net photosynthesis based on oxygen were converted to units of carbon using a photosynthetic quotent of 1.2 (Strickland 1960). RESULTS W EXPERIMENTS Acute and chronic toxicity tests were run under conditions shown in Tables 2 and 3. The pH of test aquaria ranged from 7.8 to 8.2. Dissolved oxygen concentrations were relatively high, and varied from 92% to 115% of saturation. Total alkalinity and hardness were also high relative to surface waters that are available for aquaculture in various parts of the world (Wetzel 1983). The mean coefficient of variation for total ammonia concentrations, a measure of precision and reproducibility of the toxicant delivery system used in tests, varied from 15 to 18. This range of variation was low relative to range of 13 to 156 obtained by Redner and Stickney (1979) for their variable dosing apparatus in their work with ammonia effects of Tilapia aurea. No mortalities were observed in controls of any of the acute experiments. Toxicity curves for acute tests are shown in Figure 2. The 95% confidence limits of LC50 values are shown in Table 3 with slopes of lines obtained by regressing the percent mortality at different times during tests against unionized ammonia concentrations. At 23°C, the median lethal concentrations of unionized ammonia (LC50) at 24, 48, 72 and 96 hr were 2.9, 2.6, 2.4 and 2.2 mg/l respectively. Slopes of regressions ranged from 15 to 13. At 28°C with small fish (3.4 g), the median lethal concentrations were somewhat smaller: 1.9, 1.7, 1.6 and 1.4 mg/l at 24, 48, 72 and 96 hr respectively. Slopes of regressions ranged from 10 to 5 at different times during this test. At 33°C, median lethal concentrations of unionized ammonia at 24, 48, 72 and 96 hr were 3.1, 2.9, 2.6 and 2.3 mg/l respectively. Slopes ranged from 8 to 7 at the different time intervals. At 28°C with large fish (45.2 g), the median lethal concentrations of unionized ammonia were the highest observed in tests, namely, 4.0, 3.3, 2.8 and 2.8 at 24, 48, 72 and 96 hr respectively. Slopes of regressions were 5 at each interval of time during the test. Statistical analyses showed no significant differences (p < 0.05) between the LCSOS at any time during tests at 23°C and 33°C when 13 Table 2. 14 Conditions for acute toxicity tests. Values are means with ranges in parentheses. Test Number 1 2 3 4 Fish Size (3) 10.6 10.8 3.4 45.2 (8.5-12.6) (9.2-13.4) (2.2-5.1) (38.3-48.7) Temperature 23 33 28 28 (°C) (22.5-23.5) (32-33.5) (27.5-28.5) (27-28.5) pH 8.2 8.2 8.1 8.0 (7.9-8.4) (7.9-8.5) (7.8-8.4) (7.8-8.3) D.O (mg/L) 7.9 8.3 8.0 8.0 (7.7-8.5) (7.9-8.7) (7.8-8.4) (7.6-8.3) Alkalinity 341 326 334 332 (mg/L) (329-352) (317-337) (313-350) (316-343) H a r d n e s s 354 335 342 346 (mg/L) (343-359) (326-342) (324-353) (326-362) CV1 15 17 18 17 (1 1-23) (10-30) (1 1-29) (10-37) 1. CV. is the coefficient of variation for the distribution of total ammonia concentrations. 15 Table 3. Conditions for chronic toxicity tests. Values are means with ranges in parentheses. Test 1 Test 2 Fish Size (g) 6.1 5.8 (5.8-6.7) (5.4-6.9) Temperature (°C) 28 33 (27-28.5) (32-34) pH 7.8 8.0 (7.6-8.1) (7.7-8.2) D.O (mg/l) 8.3 8.2 (7.9-8.8) (7.9-8.7) Alkalinity (mg/l) 334 331 (325-347) (322-343) Hardness (mg/l) 353 346 (337-365) (331-358) CV.1 18 17 (12-27) (1 1-28) 1. CV. is the coefficient of variation for the distribution of total ammonia concentrations. 16 .38“ 2:3 3 «158.5 63301:. Ba 0050 335,—. a Semi 33.5 985.5 “causes: .0 825.880 353 58.2 or Emu 80$ 0.3 If Em: 9.9 0.8 II Em: and: 0.8 1.... ES mod: 0.8 II 4 l 1 l I l or 8.. (smou) emu l7 Table 4. Results of acute toxicity tests. Time LC50 95% C.I.‘ Slope (hr) (mg NHJL) (mg NHJL) of Regression2 Test 1 - 23 °C (10.6 g fish) 24 2.9 2.7-3.1 15 48 2.6 2.4-2.8 15 72 2.4 2.2-2.6 15 96 2.2 2.0-2.4 13 Test 2 33 °C (10 8 g fish) 24 3.1 2. 8- 3. 4 8 48 2.9 2. 6- 3. 2 8 72 2.6 2. 3- 2 .9 8 96 2.3 2. 0- 2. 6 7 Test 3 - 28 °C (3. 4 3 fish) 24 1.9 1 .8- 2 .0 10 48 1.7 1.5-1 .9 7 72 1.6 1 .3-1 .8 6 96 1.4 1.1- 1. 7 5 Test 4 - 28 °C (45.2 g fish) 24 4.0 3. 1- 5.2 5 48 3.3 2. 4- 4. 5 5 72 2.8 l .9 4. l 5 96 2.8 1.9-4.1 5 l. C.I. is confidence interval. 2. Percent mortality at different times regressed on unionized ammonia concentrations. 18 average fish weights were 10.6 g and 10.8 g respectively. In tests conducted at 28°C using both small fish and large fish, the LC50 at any time interval was always significantly smaller (P < 0.05) with small fish. Fish of approximately 6 g size were used in tests of sublethal concentrations of unionized ammonia on growth. Temperatures of 28°C and 33°C were used in these chronic tests. Results are shown in Figure 3. Regression analysis of fish weight gains (Y) and unionized ammonia concentrations (X) revealed a high and significant negative correlation at both temperatures. At 28°C, the equation of the regression analysis was: Y - 30.2 - 20.3(X), r’ - 0.91 and p < 0.001. Extrapolating to zero unionized ammonia concentration, the average fish weight gain was 30.2 g in the absence of unionized ammonia. At 33°C, the equation of the regression analysis was: Y - 25.0 - 15.0 (X), r’ - 0.95 and p <0.001. The average fish weight gain was 25 g extrapolating to zero unionized ammonia concentration. Fish weight gain was 10 g/fish/35 days when the unionized ammonia concentration was 1 mg/l at both of the temperatures tested. Fish weight gain was higher at any concentration of unionized ammonia below 1 mg/l at 28°C than at 33°C. However, the reverse was observed at concentrations of unionized ammonia higher than 1 mg/l. Slopes of the regression lines were 20.3 and 15.0 at 28°C and 33°C respectively. There was a significant difference between slopes (p <0.05). The relationship in chronic tests between percent decreases in fish weight gain relative to controls (Y), and unionized ammonia concentrations (X) was developed for tests run at 28°C and 33°C. Results are shown in Figure 4. At 28°C, the equation for the regression analysis was : Y - - 4.1 + 70.4 (X), r2 = 0.89 and p <0.001 At 33°C, the regression equation was: Y - - 3.7 + 62.0 (X), I’ - 0.94 and p < 0.001 The no effect for unionized ammonia (ECO) was extapolated from the equation for the concentration when the percent decrease in fish weight gain was equal to zero. The median 19 0.9 as 0.8 a as: a in Co ea :33 amass 8 “aces“ ease? co some 385 2F .m 2:5 see. 523.8..8 $85.5 258.5 m; we we F md 0.0 To NO 0 _ I q A _ _ _ _ O or 1 m * * W [or .m. N a D mw * 008 1 WP w. I. ‘ low w. I D a M» I so a w a M * ( 0.. * 0.00 1m: > on s Ohm 1*! 00mm mm 20 .Uomm can Uowm «a £05 3 Emu 2303 am E 030800 2.8qu no Soon—Ea Enema: we vote 02020 2:. 33.5 congcgcoo mEoEE< 35:25 v. —. N. _. o. .. $6 0.6 V6 Nd * U D * or N AW 8 * D a a ’ D I. D a 00mm * * 0 mm D O * * comm * .v 0.:qu ulee IuBIeM usL-j ul eseeloea 96 cow 21 effective concentration (EC50) and the no weight gain effect of unionized ammonia (EC100) were taken from the equation for conditions when the percent decreases in fish weight gain were 50% and 100% respectively. ECO, ECSO and EC100 were 0.06, 0.77 and 1.48 mg/l unionized ammonia at 28°C, and 0.06, 0.87 and 1.67 mg/l unionized ammonia at 33°C. No mortalities were observed at any of the unionized ammonia concentrations used for 28°C chronic tests. However, 21 of 140 fish used in treatments at 33°C died. Contigency tables were constructed to evaluate the combined effect of temperature and ammonia concentrations on the mortality of these fish. There was a deleterious combined effect of ammonia concentrations and higher temperature (33°C)on mortalities observed at this higher temperature. However, data from controls showed that temperature alone did not have a significant effect on mortality (p < 0.001). Temperature did, however, have a significant effect on growth in controls. The relative growth rates of fish (RGR), that is, RGR - [total weight gain per f ish/initial weight per fish x days of testinglx 100, was higher at 28°C than at 33°C (p < 0.05). Relative growth rates were 13.8 %/day at 28°C, and 11.8 %/day at 33°C. HELD W Concentrations of inorganic nitrogen and dissolved oxygen were measured at regular intervals during the four month field experiment conducted in Thailand. Average concentrations for these parameters are given in Table 5. Nitrite + nitrate nitrogen was found in increasing concentrations in ponds with fertilizer treatments A through C. Ammonia nitrogen, the difference between total inorganic nitrogen and nitrite + nitrate nitrogen, also increased in treatments A through C. Mean diel dissolved oxygen minima and maxima were nearly the same for treatments A and B. Ponds in treatment C had lower oxygen diel minima than other treatments, 2.8 mg/l on average, and higher maxima. Diel variation in total ammonia concentrations was measured in thiee layers of water in experimental ponds. Mean diel concentrations from samples collected in each layer over four months of the experiment are shown in Figures A-l through A-3 of the Appendix. Figure 5 shows mean diel variation for whole water columns in ponds receiving one of three fertilizer treatments. The total ammonia diel cycle exhibited two phases in each of the 22 Table 5. Mean concentrations of inorganic nitrogen and dissolved oxygen for the period of the experiment. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea. Treatment NO,+NO,-N Total Dissolved Oxygen (mg/L)l (mg/L) Inorganic-N (ms/L) Diel Minimum Diel Maximum A 0.04 0.15 4.2 8.4 0.24 0.56 4.3 8.4 C 1.51 4.56 2.8 10.5 1. Diel minimum is average of measurements made at 0600 hr and diel maximum is average of measurements made at 1600 hr. 3.00 2.75 2.50 ' 2.25 ' 2.00 ’ Total Ammonia (g Nlmz) I; O 0.15 0.10 0.05 0.00 Treatment C l l l L l l l l i l l U I I -- Treatment A -- Treatment B 6:00am 2:00pm 10:00pm 6:00am Figure 5. Tlme of Day Diel total ammonia variations in treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea. 24 treatments. Concentrations decreased during daylight hours from near 6:00 am to 6:00 pm, then increased during nighttime hours. Substantial amounts of ammonia were lost from ponds between successive dawns in the diel cycle. These losses were compensated for by weekly additions of fertilizers on day-0 of the fertilizer schedule. Average amounts of total ammonia nitrogen present in ponds at 6:00 am were 1 1 1, 298 and 2907 mg N/m’ for treatments A, B and C respectively. Losses during 12 hr daylight periods averaged 78, 183 and 1040 mg N/m’/ 12 hr. Percentages of ammonia nitrogen present at dawn that were lost from ponds during 12 hr daylight periods were 71, 62 and 36% for treatments A, B and C respectively. Measurements were made in ponds for the purpose of examining two mechanisms involved in these daytime losses of ammonia: volatization to the atmosphere and uptake by algae. Ammonia flux to the atmosphere from ponds depended primarily on ammonia concentrations in ponds (Figure 5), pH, temperature and wind speed. pH in ponds increased from sunrise until 3:00 or 4:00 pm, after which time it decline until the following dawn (Figure A-4). Pond temperatures behaved much as pH (Figure A-5). On average, wind speed over ponds increased during days from 0.97 m/sec at 6:00 am to about 2.1 m/sec at 4:00 pm. Table 6 shows calculated flux losses under average conditions that prevailed at the site. Losses early in daylight periods were less than daily maxima in 4:00 to 6:00 pm periods, and were low at night relative to daytime maxima. Curves drawn for flux at intervals during days are shown in Figures A-6 through A-8. Areas under these curves were integerated to determine the amount of ammonia nitrogen volatized from treatment ponds during 12 hr light periods. Results are given in Table 7. Amounts of ammonia nitrogen volatized were 0.7, 3.7, and 93.7 mg N/m2/12 hr light period for treatments A, B, and C respectively. By methods used in this work, percentages of daytime losses of ammonia nitrogen due to flux from ponds were relatively small : 0.9, 2.0, and 9.0 % for treatments A, B, and C respectively. Daytime losses of ammonia due to algae uptake from water in ponds was evaluated using estimates of daily net primary productivity and the assumption that algae in ponds used nitrogen in a ratio of 7:40 by weight with carbon that was fixed daily in net photosynthesis (Round 1973, Wetzel 1983). Daily net primary productivity was determined by the single 25 Table 6. Average ammonia loss rate to the atmosphere (flux) in treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea. Time Flux in Treatment Flux in Treatment Flux in Treatment (ms NIm’lhr) (ms Nl/Bm’mr) (ms NIthr) 6:00 am 0.07 0.27 4.43 10:00 am 0.03 0.26 5.62 2:00 pm 0.04 0.28 7.21 4:00 pm 0.12 0.45 10.32 6:00 pm 0.09 0.39 7.24 11:00 pm 0.05 0.21 4.00 6:00 am 0.02 0.10 1.74 26 000003052 033033 .0 .80 86 00 80008 08085 A ca 0.8 82 8% 0.03 0 0.0 mm m: 08 0.00 0 .3 8 we 5 08 < 3 «Quiz may p32 0&0 930030 5E 2 Ease? may ~03 .8an 38. 8003 0=Q 003 Q6 “BE 08085 30H 83.: 0&0me 808000; .008 0300\3 RN 030 08.808 09.030 :3\0:\wx 8n 02,0000 0 808.005 “80 008 «BEARD— vN 029 08008 000.020 {(053 3. 003000. m 8080005 .08808 004030 0.30am: 8m 00330. < 808000.: 8 008m .988 002300800 08 o. 0.0. 000— 000 00000 8 80:05:02.8 080880 000.33 038050—00 05. H 030,—. 27 curve oxygen method for 12 ponds used in three treatments of this experiment. Average rates of oxygen change in ponds are shown in Figures 6 through 8. Areas under curves above 0.0 rate of change were integrated to obtain net photosynthesis for days. For each treatment, net primary productivity was low in the hours after dawn, and increased until about 12:00 noon. Thereafter, net photosynthesis decreased until it reached zero at about 5:00 pm. Converting net mg of oxygen evolved daily to unit weights of carbon, mean net primary productivities were 0.86, 1.1 and 1.99 g C/m’/day for treatments A, B and C respectively. Results of one way analysis of variance revealed there was not a significant difference in net primary productivity between treatment A and B, while there was a significant difference between treatment B and C (p < 0.05). Table 8 shows the relationship between ammonia loss during 12 hr daylight periods, net carbon fixation per day, daily nitrogen requirements of algae, and percentage of daily ammonia loss from ponds due to algae uptake. The percent loss due to algae uptake of ammonia was estimated at 194, 107 and 37% of flux-corrected total ammonia daytime loss from ponds for treatments A, B and C respectively. Figure 9 shows the relationship between flux-corrected cumulative losses of ammonia from treatment ponds and cumulative net carbon fixed, on average, during days of the experiment. Points on the figure are for daylight time intervals when net photosynthesis occurred (Figures 6 through 8). Points near the origin of each curve are for the 7 to 8 am interval, and points proceed away from the origin at one hour intervals through the day to the 4-5 pm interval. Analysis of these data showed a strong positive correlation between cumulative ammonia loss and cumulative net primary productivity for all the values in the three experimental treatments (r - 0.86 and p < 0.001), as well as between cumulative ammonia loss and cumulative net primary productivity at the different time intervals (r > 0.98). Diel variation in unionized ammonia concentration was calculated in three layers of water in experimental ponds. Mean diel concentrations from samples collected in each layer over four months of the experiment are shown in figures A-9 through A-ll of the Appendix. Average unionized ammonia concentrations were always higher in surface layers in any 28 .2808 000—030 0.30:}: 8m a? < 808.00.: 8 09:30 .0bi 00280:. Lo 0200 35.0 03mm E086? >00 8 08F 5080 5080 E0005. 8080 q d A q u u u 6.01 . to. \. 0.? \ o.o . 0.0+ - 0.0+ 0.0+ (Iuéw/B) eBueuo 10 area uefillxo 29 580qu .83 {<33 3 33 0558 50.030 {(3}.— 3 £5 M 30.500: E 05 combs 32% no 00.8 35 >00 .0 08:. 5890 .880 5892 .h 0gb Emomnw \ n \ 0.0. ‘1: 9 N 9 O. C: «6+ v.o+ 0.0+ (llléul/fi) afiueuo 10 9193 uefiAxo 30 .005 {00 M6 2:: £086. 2080 5800 5805. 5080 l \ 0.0. 0.0. 0.0 0.0+ 0.0+ (Jq/aw/fi) efiueuo go 9193 uefifixo 31 Amwafi 40303 $83 .000on :30? >2 90> 00 0000 0 £ 03000 000 003.90 000.0 0030 .05 0000000000 00 02030—00 .N 000008000 0.0 8 0.8. 08 03000.80 :30 8 0300 08¢ >00 000 0.8— 005880 38,—. A hm men 83 03 Gm: U 80 A «3 8: 2.0 find 0 03 A $4 cow R find < 23% 300 2 E 3052 may 3 0588 0.5 0.. 3032 may 23330 00D 3Q.— >=0Q 0000000050m 000005 003 503 00 § 0300000m «swabs/H 03000 002 00009002. 300. 00wohmz 000000000. .35 0.3300 80 83 0338 5030 0333 Sw 00000000 0 00000005 000 0000 x3\00\wx «a 030 0000000 000030 x3\0a\wx 3. 0030000 m 0000000: 000000. 000—030 {(00}.— oow 0030000 < 00000000 5 0.0000 £008.00: 5 00080.0 03000 000 000 00090000 00 00.0000 00% 000500 038000—00 05. .w 030,—. 32 .8... 03300.. 80 3.0 8:53 88.0 0330.. Sn 3:82 o .858: 08 3... 0350.. a 2.... 035a 80% 0333 3. 3082 0 .858: case 8020 .323 can 3082 < 0008000.: 8 0.000.— 000.3850 0.0 3 0.3— .5.. 000000.80 mm 0.06- 080880 180. .0038 8008000.: m a. 0:23 .8 m... 2: 2 5 2: 0085 8088a 08 0:23 5 0.0. 20 a? 0530 £223 0— a .0 08— 008880 ~59 900—0800 000 000000 03000 000 3003800 0003.00 038000—00 0.3. 00500 500:... 59.00 .02 $00555 8.0 m: on; 00.— 8., who 8.0 00.0 o q q u - _ u u o o .. 0 E0503. I? o 0 80500:. 10: 1 < E00000; 1T d 0.0—Eh o O n to m M m. a 0.0 m W O .u.. a to m {x mm no W (z 0.0 33 treatment. Average daily maximum concentrations of unionized ammonia , at 4:00 pm, in the surface layers were 0.01, 0.03 and 0.6 mg/l for treatments A, B and C respectively. Figure 10 shows mean diel variation for unionized ammonia in whole water columns in ponds receiving fertilizer treatments. Concentrations increased from near 10:00 am to around 4:00 pm in treatments A and B. However, in treatment C it increased gradually from sunrise till it reached its maxima by 4:00 pm. Average daily maximum concentrations of unionized ammonia, at 4:00 pm, were 0.004, 0.017 and 0.37 mg/l for treatments A, B and C respectively. Unionized Ammonia Concentration (mg/L) 0.4 0.3 - 0.2 0.1 r- Treatment C 0.020 0.015 0.01 O 0.005 0.000 -°- Treatment A -'- Treatment 8 6:00am Figure 10. 2:00pm 10:00pm 6:00am Time of Day Diel unionized ammonia in ponds of three treatments. Ponds in treatment A received 500 kg/ha/wk chicken manure, treatment B received 44 kg/ha/wk chicken manure plus 24 kg/ha/wk urea and treatment C received 500 kg/ha/wk chicken manure plus 280 kg/ha/wk urea. DISCUSSION AT B R M Toxicity tests were used to detect and evaluate effects of ammonia on 0. niloticus. This work was centered on the unionized species of ammonia because it is 300 - 400 times more toxic than the ionized form (Thurston et al. 1981). Acute tests were run under two sets of conditions: temperature was varied using fish of the same size, and size of fish was varied using the same temperature. In the first case, where fish of 10.7 g were exposed to ammonia at 23°C and 33°C, no significant difference (p < 0.05) was observed between LCSOS at any of four 24 hr intervals of measurement during tests. In the only report of acute ammonia toxicity for species of tilapia found in the literature, Redner and Stickney (1979) obtained a 48 hr median lethal concentration (LC50) for Tilapia aurea of 2.4 mg/l unionized ammonia at 27°C. Their test fish had lengths in the range of 7-9 cm. The 48 hr LC50 for 0. niloticus in this study, using 10.7 g fish with lengths in the same range as Redner and Stickney, was somewhat higher at 2.6-2.9 mg/l. One of the main features of the probit analysis, used with acute toxicity data in this study, is the slope obtained with points from successive time intervals in each treatment. The slope is an index of sensitivity to chemicals within test samples of fish. Sprague ( I969) reported that the flatter the slope, the more heterogenous the population, or the less toxic the compound. Rand and Petrocelli (1985) stated that a flat slope may be indicative of slow absorption, rapid excretion or detoxification, or delayed toxification. A steep slope usually indicates rapid absorption and rapid onset of effects. For fish 10.7 g in size, values of slopes during tests were higher from 48 to 96 hr at 23°C than at 33°C. For 96 hr LC50s, slopes were 13 and 7 for tests at 23°C and 33°C respectively. This suggests that toxicity of unionized ammonia was greater at 23°C than at 33°C. The temperature effect of toxicants on aquatic organism differs depending on toxicant. The response also differs between cold and warm water species (Sprague I985). 35 36 Most information in the literature for warmwater fish suggests a decrease in toxicity of ammonia with increasing temperature in a range from 20°C—30°C. Reinbold and Pescitelli (1982) found that the toxicity of ammonia decreased with increasing temperature from 43°C to 24.5°C for bluegill and fathead minnow. The same trend was reported for fathead minnows over the temperature range from 12°C to 22°C (Thurston et al. 1983), and for bluegill, channel catfish, and largemouth has over the temperature range from 22°C to 30°C (Roseboom and Richey 1977). The 96 hr LCSOS for channel catfish in a static toxicity testing system were 2.4, 2.9, and 3.8 mg/l of ammonia at 22°C, 26°C, and 30°C respectively (Colt and Tchobanoglous 1976). Power (I920) reported an increase in toxicity of ammonium chloride for blunt nose minnows between 142°C and 24.9°C, and for straw-colored minnows at between 15.6°C and l9.8°C. A second set of acute tests was run in this study to measure the effect of fish size on toxicity of ammonia. Two lots of fish, one with mean individual weight at 45.2 g, and one with individuals at 3.4 g, were exposed to ammonia at a temperature of 28°C. This temperature was within the Optimum range of 28°C to 33°C reported for growth for 0. niloticus by Beamish (1972) and Chervinski (1982). Significant differences (p < 0.05) were observed between LC508 at all of four 24 hr intervals when measurements were made during tests. The 96 hr LC50 for 3.4 g fish was 1.4 mg/l unionized ammonia. For 45.2 g fish, the 96 hr LC50 was 2.8 mg/l. Results in this study show that, in the optimum range of temperature for growth, small f ingerlings of the test species are more susceptible to acute ammonia toxicity than larger f ingerlings. Reports dealing with species of tilapia and effects of size on ammonia toxicity were not found in the literature. However, the work of Anderson and Spear (1980) supports the generality that larger fish were expected to be more tolerant of the toxicant. They used pumpkinseed sunfish (Lepomis gibbosus) in tests with copper. They found the following useful relationship: log LC50 - log a + b log W where log a is the y intercept, W is weight of fish and b is slope. It shows that a positive change in b from zero indicates that smaller fish accumulate ammonia at a greater rate, 37 receive a larger dosage per unit body weight than large fish, and thus are more susceptible to the toxicant. It should also be noted that, in addition to results obtained in this study in tests with small fish and large fish at 28°C, LCSOs at all of four 24 hr intervals when measurements were made were always higher for large fish than for small fish, regardless of temperatures used in tests. Sublethal concentrations of unionized ammonia caused growth reductions during tests with 0. niloticus. Growth related effects of ammonia at the level of tissues and organs are known to impair growth of fish. Gill hyperplasia and prof ileration were observed by Smith and Piper (1975) in fish exposed to ammonia. Brockway (1950) reported that fish lost the ability to use oxygen as the ammonia concentration in the water increased, since blood carbon dioxide increased about 15% causing hemoglobin to take up considerably less oxygen. Maetz (1972) reported inhibition of sodium uptake. Livers of exposed fish showed reduced glycogen storage and scattered areas of dead cells which became more extensive with increasing exposure time (Smith et al. 1975). Thurston et al. (1978) reported that kidneys suffered a hydropic degeneration due to elevated ammonia. Fromm and Gillett (1968) found that an increase of ammonia in the blood during exposure to ammonia inhibited ammonia excretion. Loyd and Orr (1969) found an increase in urine flow which increased loss of ions and increased cell permeability to water inflow. High ammonia concentrations at the cellular level have also been associated with depletion of alpha-keto-glutarate resulting in impairment of krebs cycle ( Waren and Schenker I964), NADH depletion slowing down the generation of ATP (Worcel and Erecinska 1962), depletion of ATP due to glutamine formation by the glutamine synthetase system ( Forster and Goldstein 1969), depletion of ATP causing a decrease in cerebral acetyl-choline (Braganca et al. 1953), and stimulation of membrane ATPase producing increased nerve cell excitability and activity (Hawkins et al. 1973). Tissue changes associated with ammonia exposure were observed in eyes, brains, fins, and blood of fish by Thurston et al. (1986). Detoxification of ammonia also occurs in tissues. It costs the organisms energy that might otherwise be used for growth. Two biochemical reactions which fix ammonia into less toxic organic compounds during conditions of hyperammonia (Berl et al. 1962, Cooper et al. 38 1979) are: Alpha Ketoglutarate + NADH + NH,“ --> Glutamate 4» NAD“ + H,O Glutamate 4» ATP + NH,“ :2 Glutamine + ADP + Pi The first reaction is catalyized by glutamine dehydrogenase (GDH), and the second by glutamine synthetase (GS). Both detoxification reactions have energy costs in the form of NADH or ATP. Chronic effects on growth of 0. niloticus were measured in this study over a range from 0.02 to 1.4 mg/l unionized ammonia. Increasing unionized ammonia concentrations caused linear decreases in fish weight gain at both 28°C and 33°C. There was a significant difference between slopes of lines regressing fish weight gain and unionized ammonia concentration (p < 0.05) at the two temperatures tested. There was a decrease of 20.3 g and 15.0 g in fish weight gain with each increase of 1 mg/l of unionized ammonia at 28°C and 33°C respectively. Because of steeper slope, the chronic effect of unionized ammonia on growth was greater at 28°C than at 33°C. No fish mortalities occurred in chronic tests at 28°C, while partial mortality occurred at 33°C. Statistical analysis showed there was a significant (p < 0.05) combined effect of ammonia concentrations and temperature on the mortality of these fish. Overall, because of mortality the effects ammonia were expected to be greater in intensely managed aquaculture ponds that have temperatures in the neighborhood of 33°C than in cooler ponds. The safe level of unionized ammonia (ECO) in chronic experiments was 0.06 mg/l at both temperatures tested. Median effective concentrations that reduced growth by 50% relative to controls (EC50) were 0.77 and 0.87 mg/l unionized ammonia at 28°C and 33°C respectively. No weight gain concentrations (EC100) were 1.48 and 1.67 mg/l unionized ammonia at 28°C and 33°C respectively. Colt and Tchobanoglous (1978) found the EC50 for channel catfish at 0.52 mg/l unionized ammonia, and the EC100 at 0.97 mg/l unionized ammonia. They worked at at 28°C. 0. niloticus was more tolerant of sublethal concentrations of unionized ammonia than channel catfish at this temperature. Laboratory studies of both acute and chronic effects of unionized ammonia demonstrated the importance of keeping concentrations low enough to avoid either mortality 39 or reduced growth rate of fish in order to promote an economic return from aquaculture. W Nitrogen was loaded into fish ponds in Thailand at rates of 271, 271 and 3150g/m3/wk in treatments A, B, and C respectively. Chicken manure was used alone in treatment A, while combinations of chicken manure and urea were used in treatments B and C. Concentrations of ammonia decreased in ponds during days between weekly applications of fertilizers. Diel losses of ammonia were expected to occur as a result of uptake by pond microflora, volatization to the atmosphere, absorption on sediments, and nitrification and denitrification processes. The first two of these were examined in this study as potential mechanisms to protect fish against ammonia toxicity. From measurements made in ponds during the experiment, it appeared that the environment was not suitable for rapid rates of denitrification, particularly in regard to oxygen concentrations. Garcia and Tiedje (1982) reported that major enviromental factors that control denitrification are oxygen, availability of organic matter, supply of nitrate, and to a lesser degree temperature. Woldendorp (1968) found that dissolved oxygen concentration needed to be low, less than 0.2 ppm, before denitrification began. However, dissolved oxygen concentrations never fell below 0.4 ppm in any of the measurements made. In the typical diel cycle of total ammonia observed in ponds of each treatment, concentrations were highest at sunrise, decreased with daylight to sunset, and increased during the dark period at night. The same trend was observed under intensive fish culture conditions by Shilo and Rimon (1982), and in a hypertrophic prairie lake (Murphy and Brownlee 1982). In flooded rice fields, often used for fish culture in the tropics, Filery et al. (1984) observed that total ammonia concentrations also reached maxima at 0600 to 0700 hr and were lowest in the afternoon. Average amounts of total ammonia nitrogen present at dawn in ponds in Thailand were 111, 298 and 2907 mg N/m2 for treatments A, B and C respectively. Percentages of these concentrations lost during days were 71, 62 and 36% for treatments A, B and C respectively. While Boyd (1983) stated that considerable amounts of ammonia may be lost from fish ponds to the atmosphere by volatization, few authors have made estimates of this loss. 40 Bouldin et a1. (1974) did report that losses from total ammonia concentrations due to volatization ranged from 2-38% per 24 hr of total ammonia concentrations in their ponds. Diel flux to the atmosphere in ponds of each treatment in Thailand was low at dawn, increased with daylight up to 4:00 pm, and then declined. Pond temperatures and pHs, as well as wind speed over ponds, all important variables in the volatization model used to calculate flux (Galloway 1980), had the same diel pattern. Estimated percentages of daytime losses of total ammonia nitrogen from ponds due to flux were 0.9, 2.0 and 9.0% for treatments A, B and C respectively. Daily losses relative to concentrations present in ponds at dawn were 0.6, 1.2 and 3.2%. Flux losses from ponds in Thailand were low compared to losses reported by Bouldin et al. (1974). Daytime losses of ammonia due to algae uptake from water in ponds was evaluated using estimates of net primary productivity, and the assumptions that algae in ponds used nitrogen in a ratio of 7:40 by weight with carbon that was fixed in net photosynthesis (Round 1973, Wetzel 1983) and ammonia was a preferred nitrogen source relative to other soluble forms of nitrogen (Syrett 1962, Liao and Lean 1978, Harrison 1978). A pattern of changes in net primary productivity was noted in ponds of each treatment. Net productivity increased from dawn, reached maxima about noon, and decreased thereafter until it reached zero about 1700 hr. A similar pattern was noted by other authors (Odum 1956; Dotty and Oguri 1957; Capeland et al. 1962; Kevern and Ball 1965). Results from the experiment in Thailand showed that flux-corrected cumulative total ammonia loss at each interval of the daylight period was significantly associated (interdependent) with cumulative net primary productivity. The flux-corrected percentages of total daily ammonia losses assignable to algae uptake were estimated at 196, 108 and 37% for ponds in treatments A, B and C respectively. Results of this work indicate that algae uptake was an important mechanism for ammonia removal, more important than flux to the atmosphere. Algae uptake and flux accounted for more than 100% of losses that occurred in treatments A and B, and for an estimated 43% of the loss from ponds in treatment C. Nitrification and sediment adsorption were likely processes for removal of the remainder of daily ammonia lost in treatment C. 41 The surplus of ammonia in treatment C appeared to cause a deleterious effect on fish. Seventy-eight percent of stocked fish were harvested from ponds in treatments A and B at the end of four months. There was no significant difference in growth between fish in these treatments (p < 0.05). However in treatment C, there was 100% mortality in week 10 of the experiment in two of four ponds used in the treatment. An average of only 59% of stocked fish were retrieved at harvest from the other two ponds in treatment C. The average unionized ammonia concentration measured in ponds with 100% mortality in week 10 was 0.7 mg/l. Mean diel maximum concentrations, at 4:00 pm, of unionized ammonia in surface layers during the experiment were 0.01, 0.03 and 0.60 mg/l in ponds of treatments A, B and C (Appendix Figures A-9 through A-l 1). Laboratory tests of acute toxicity would not have clearly predicted lethal effects from unionized ammonia concentrations found in ponds of treatment C. Fish that died were relatively large (76 g). A median lethal concentration of unionized ammonia greater than 2.0 mg/l was required for mortality of large fish (45 g) at 28°C in acute tests. Mean diel water temperatures in ponds of treatment C ranged from 285°C to 308°C (Appendix Figure A-5). Mean temperatures were above 30°C from 1200 to 2200 hr. While no mortalities occurred in chronic toxicity tests at 28°C, mortalities did occur at 33°C with unionized ammonia concentrations in a range from 0.1-1.1 mg/l. An interaction between unionized ammonia and high temperature was shown to be involved in these mortalities. Table 9 shows results from studies of Thurston et al. (1981) and Selesi and Vamos (1976) on the effect of dissolved oxygen on the concentration of unionized ammonia required for lethal effects. Data in the table show that the lethal concentration of unionized ammonia decreased in their studies as dissolved oxygen concentration decreased. In related studies, Redner and Stickney (1979) reported no mortalities to blue tilapia during 35 days of exposure to 0.43-0.53 mg/l unionized ammonia under the laboratory conditions with an average dissolved oxygen concentration of 6.5 mg/l. However, Stickney et al. (1977) and McGeachin and Stickney (1982) obtained total mortality of blue tilapia at nearly the same concentrations of unionized ammonia (0.52-0.63 mg/l) when dissolved oxygen was low at 2.6-2.7 mg/l. Dissolved oxygen concentrations were seldom less than 8 mg/l in aerated aquaria used for 42 Table 9. The lethal level of unionized ammonia with varying concentrations of dissolved oxygen. Species Dissolved Oxygen Unionized Ammonia Reference (ms/L) (ms/L) Rainbow trout 3 0.38 Thurston et al. (1981) 6 0.58 9 0.75 Carp 5 0.2 Selesi and Vamos (1976) 10 1.2 43 toxicity tests in this study. It fluctuate from highs at the end of daylight periods to predawn lows in ponds in Thailand. Mean minimum diel concentrations measured at 0600 hr were 4.2, 4.3 and 2.8 mg/l for ponds in treatments A, B and C respectively. It appears that periodic low concentrations of dissolved oxygen in ponds of treatment C, combined with conditions of high temperature, may have been implicated in mortalities that occurred in ponds of treatment C at moderate concentrations of unionized ammonia. Finally, it should be noted that pond parameters were measured in this study at the mid point of weekly fertilization cycles. Fish were likely exposed to concentrations of unionized ammonia that were higher in ponds on days immediately following fertilization than at mid cycle. Taken as a whole, laboratory and field work done for this dissertation provides broad guidelines for the design of fertilizer regimes for warm tropical fish ponds that protect against toxic effects of unionized ammonia on 0. niloticus. SUMMARY WW 1. Temperature effect on acute toxicity of unionized ammonia to 0. niloticus was not significant (p < 0.05). However, LC50's were always lower at 23°C than at 33°C at any time period tested. Toxicity of unionized ammonia decreased significantly with increasing fish size. There was a linear decrease in fish weight gain with increasing unionized ammonia concentrations in chronic experiments at 28°C and 33°C. The safe level of unionized ammonia (ECO) was 0.06 mg/l in chronic tests at 28°C and 33°C. The median effective concentrations (ECSO's) were 0.77 and 0.87 mg/l unionized ammonia at 28°C and 33°C respectively. No weight gain concentrations (EC100's) were 1.48 and 1.67 mg/l unionized ammonia for 28°C and 33°C chronic tests respectively. The slopes of the lines regressing unionized ammonia concentrations and fish weight gain at both chronic experiments at 28°C and 33°C were significantly different. Therefore, the chronic toxicity effect of unionized ammonia was higher at 28°C than at 33°C. Relative growth rate of 0. niloticus in control tanks was significantly higher at 28°C than at 33°C. There was a synergistic effect between unionized ammonia concentrations and temperature on partial mortality of fish at 33°C in chronic tests. 52151811111125 1. Fertilized ponds lost ammonia during daylight periods. In this experiment, daily loss from ponds ranged from 71% to 36% of the maximum diel concentrations observed at dawn. Increasing initial total ammonia present in early morning increased net primary productivity. 44 45 Ammonia loss to the atmosphere was relatively small. It ranged from 1 to 9% of total ammonia loss during the 12 hour day light period. A coupling between decreasing total ammonia concentrations and increasing net primary productivity was observed in all treatments tested. Percent of flux-corrected total daily ammonia loss due to algae uptake was estimated to ranged between 37% and 100%. An average fish mortality of 70% was observed in four ponds with the highest fertilizer loading (3150 g N /m’/wk) and the highest unionized ammonia concentrations (0.6 mg/l mean diel maximum). High pond temperature (> 30°C) and low dissolved oxygen (2.8 mg/l at dawn) appeared to interact with concentrations of unionized ammonia that were moderate, relative to laboratory results, to cause mortality in ponds. LIST OF REFERENCES Allan, I.R.H., D.W.M. Herbert, and LS. Alabaster. 1958. A field and laboratory investigation of fish in a sewage effluent. Fish. lnves. 6(2): 1-76. American Society for Testing and Materials. 1960. standard practice for conducting toxicity tests with fishes, macroinverteberate, and Amphians. ASTM E 729- 80. Philadelphia. Anderson, J.W., and J. Done. 1977. Polarographic study of ammonia assimilation by isolated chloroplasts. Plant Physiol. 60: 504-508. Anderson, P.D., and RA. Spear. 1980. Copper pharmacokinetics in fish gills. 11. Body size relationships for accumulation and tolerance. Water Res. 14: 1107-1111. APHA. 1985. Standard methods for the examination of water and wastewater, 16th ed. Washington, D.C. Balarin, J.D., and RD. Haller. 1982. The intensive culture of Tilapia in tanks, raceways and cages. Pages 265—355 In J.F. Muir and R.J. Roberts, editors. Recent advances in aquaculture. Westview Press, Colorado. Bates, R.G., and GD. Pinching. 1949. Dissociation constant of aqueous ammonia at 0 to 50° from e.m.f. studies of the ammonium salt of a weak acid. J. Amer. Chem. Soc. 72: 1393-1396. ’ Batterson, T.R., C.D. McNabb, C.F. Knud-Hansen, H.M. Eidman, and K. Sumatadinata. 1988. Effects of chicken manure additions on fish production in ponds in West Jafa, Indonesia. CRSP research reports 88-8, Pond Dynamics/Aquaculture Collaborative Research Support Program, Office of International Research and Development, Oregon State University, Corvallis. Beamish, F.W.H. 1970. Influence of temperature and salinity acclimation on temperature pref erenda of the eurhaline fish T. niloticus. J. Fish. Res. Bd. Can. 27(7): 1209-1214. Berl.S., G. Takagaki, D.D. Clarke, and H. Waelsch. 1962. Metabolic compartments in vivo. Ammonia and glutamic acid metabolism in brain and liver. J. Biol. Chem. 237: 2562-2569. Bouldin, D.R., R.L. Jonson, C.Burda, and C. Kao. 1974. Losses of inorganic nitrogen from aquatic systems. Journal of Environ. Qual. 3: 107-114. Boyd, CE. 1982. Water quality management for pond fish culture. Elsevier scientific publishing company, New York. Braganga, B.M., P.Faulkner, and J.M. Quastel. 1953. Effects of inhibitors of glutamine synthetase in brain slices by ammonium ions. Biochim. Biophys. Acta. 10: 83-88. 46 47 Brett, J .R., J.E. Shelbourn, and C.T Shoop. 1969. Growth rate and body composition of f ingerling sockeye salmon Oncorhynchus nerka, in relation to temperature and ration size. J. Fish. Res. Bd. Can. 26: 2363-2394. Brockway, DR. 1950. Metabolic products and their effects. Prog. Fish-Cult. 12(3): 127-129. Broderius, S., R. Drummand, J. Fiandt, and M.C. Russo. 1985. Toxicity of ammonia to early life stages of the small mouth bass at four pH values. Environ. Toxicol. Chem. 4: 87-96. Brown, V.M. 1968. The calculation of the acute toxicity of mixtures of poisons to rainbow trout. Water Res. 2 (10): 723-733. Chervinski. J. 1982. Environmental physiology of Tilapias. Pages 119-140 In R.S.V. Pullin and R.H. Lowe-McConnel, editors. The biology and culture of Tilapias. ICLARM, International Center for living Aquatic Resources Management. Manila, Philippines. Colt, J.E., and G. Tchobanglous. 1976. Evaluation of the short term toxicity of nitrogenous compounds to channel catfish, Ictalurus punctatus. Aquaculture 8(3): 209-224. Colt, J .E., and G. Tchobanglous. 1978. Chronic exposture of channel catfish, Ictalurus Punctatus to ammonia: effects on growth and survival. Aquaculture 15(4): 353- 372. Coombe, J .B., D.E. Tribe, and J .w. Morrison. 1960. Some experimental observations on the toxicity of urea to sheep. Aust. J. Agric. Res. 11: 247-256. Cooper, A.J.L., J.M. McDonald, A.S. Gelbard, R.F. Gledhil, and TE. Duffy. 1979. The metabolic fate of 13N labelled ammonia in rat brain. J. Biol. Chem. 254: 4892-4992. Copeland, B.J., J.L. Butler, and W.L. Shelton. 1961. Photosynthetic productivity in a small pond. Proc.Oklahoma Acad. Sci. 42: 22-26. Diana, J.S., C.K. Lin, P. schneeberger, T. Bhukaswan, and V. Sirsuwanatach. 1985. Progress report -CRSP Pond Dynamics Thialand, Pond Dynamics/Aquaculture Collaborative Research Support Program, The University of Michigan, Great Lakes and Marine Water Center, International Programs, Ann Arbor. Dotty, MS, and M.Oguri. 1957. Evidence for a photosynthetic daily periodicity. Limnol. Oceanogr. 2: 37-40. Downing, K.M., and LC. Merkens. 1955. The influence of dissolved oxygen concentration on the toxicity of unionized ammonia to rainbow trout (Salmo gairdneri Richardson). Ann. Appl. Biol. 43(2): 243-246. Duif in, C.V.Jr. 1973. Diseases of fishes. Cox and Wyman Ltd., London. Emerson, K., R.C. Russo, R.E Lund, and R.V. Thurston. 1975. Aqueous ammonia equilibrium calculations: effect of PH and temperature. J. Fish. Res. Board. Can. 32: 2379-2383. Eren, Y., and Y. Langer. 1973. The effect of chlorination on Tilapia fish. Bamidgeh 25(2): 56-60. 48 Filery, I.R.P., J .R. Simpson, and SK. DeDatta. 1984. Influence of field environment and fertilizer management on ammonia loss from flooded rice. Soil Sci. Soc. Am. J. 48: 914-920. Forster, R.P., and L. Goldstein. 1969. Formation of excretory products. Pages 313- 350 In W.S. Hoar, and DJ. Randall, editors. Fish Physiology, Vol. 1. Academic Press, New York. Fromm, P.O., and J.R. Gillette. 1968. Effects of ambient ammonia on blood ammonia and nitrogen excretion of rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 26: 887-896. Galloway, J .E. 1980. Aquatic nitrogen cycling: The processing of nitrate by Chlorella vulgaris and the possibility of ammonia loss to the atmosphere. Doctoral dissertation, Michigan State University, East lansing. Garcia, J.L., and J.M. Tied je. 1982. Denitrification in rice soils. Pages 187-208 In Y.R. Dommergues and HG. Diem, editors. Microbiology of tropical soils and plant productivity. Junk Publishers. Gill, J .L. 1978. Design and analysis of experiments in the animal and medical sciences. Vol 2. Iowa State University Press, Ames. Hall, C.A.S., and R. M011. 1975. Methods of assesing aquatic primary productivity. Pages 19-53 In H. Leath and R.H. Whittaker, editors. Primary productivity of the biosphere. Springer-Verlag, Inc., New york. Harader,R.R., and G.H. Allen. 1983. Ammonia toxicity to chinook salmon parr: reduction in saline water. Trans. Am. Fish. Soc. 112: 834-837. Harrison, W.G. 1978. Experimental measurements of nitrogen reminerlization in coastal waters. Limnol. Oceangr. 23(4): 684-694. Hawkins, R.A., A.L. Miller, R.C. Nielsen, and R.L. Veech. 1973. The acute action of ammonia on rat brain metabolism in vivo. Biochem. J. 134: 1001-1008. Healey, F.P., and LL. Handzel. 1976. Physiological changes during the course of blooms of Aphanizomenon flos-aqua. J. Fish. Res. Bd. Can. 33: 36-41. Kevern, N. R., and R. C. Ball. 1965. Primary productivity and energy relationships in artificial streams. Limnol. Oceanogr. 10: 74- 87. Lara, C., and J .M. Romero. 1986. Distinctive light and CO, fixation requirements of nitrate and ammonium utilization by the cyanobacterim Anacystis nidulans. Plant Physiol. 81: 686-688. Liao, C.F-H., and D.R.S. Lean. 1978. Seasonal changes in nitrogen compartments of lakes under different loading conditions. J. Fish. Res. Bd. Can. 35: 1095-1101. Litchfield, J.T.Jr., and F. Wilcoxon. 1949. A simplified method of evaluating dose- effect experiments. J. Pharmacol. Exp. Ther. 96: 99-113. Lloyd, R., and L.D. Orr. 1969. The diuretic response by rainbow trout to sublethal concentrations of ammonia. Water Res. 3: 335-344. Maetz,J. 1972. Branchial sodium exchange and ammonia excretion in the gold fish Carassius auratus. Effects of ammonia loading and temperature changes. J. Exp. Biol. 56:601-620. 49 McGeachin, R.B., and R.R. Stickney. 1982. Manuring rates for production of blue tilapia in simulated sewage lagoons receiving layinghin waste. Prog. Fish - Cult. 44(1): 25-28. Murphy, T.P., and B.G. Brownlee. 1982. Blue green algae ammonia uptake in hypertrophic prairie lake. Can. J. Fish. Aquat. Sci. 38: 1040-1044. Odum, H.T. 1956. Primary production of flowing waters. Limnol. Oceanogr. 1: 102- 117. Odum, H.T. 1957. Trophic structure and productivity of silver springs, Florida. Ecol. Monogr. 27: 55-112. Palachek, R.M., and J.R. Tomasso. 1984. Toxicity of nitrite to channel catfish (Ictalurus punctatus), tilapia (Tilapia aurea), and largemouth bass (Micmpterus salmoides): Evidence for a nitrite exclusion mechanism. Can. J. Fish. Aquat. Sci. 41: 1739-1744. Powers, EB. 1920. Influence of temperature and concentration on the toxicity of salts to fishes. Ecology 1:95-112. Rand, G.M., and SR. Petrocelli. 1985. Introduction. Pages 1-28 In G.M. Rand and SR. Petrocelli, editors. Fundamental of aquatic toxicology, Methods and applications. Hemisphere Publishing Corporation, Washington. Redner, B.D., and R.R. Stickney. 1979. Acclimatization to ammonia by Tilapia aurea. Trans. Am. Fish. Soc. 108(4): 383-388. Reinbold, K.A., and S.M. Pescitelli. 1982. Acute toxicity of ammonia to channel catfish, Illinois Natural History Survey Champaign, Illinois. Roseboom, D.P., and D.L. Richey. 1977. Acute toxicity of residual chlorine and ammonia to some native Illonis fishes, Illonis State Water Survey. Perport of investigation 85. Urbana, Illonis. Round, F .E. 1973. The biology of the algae. St.Martin Press, New York. Russo, R.C. 1985. Ammonia, Nitrite, and Nitrate. Pages 455-471 In G.M. Rand and SR. Petrocelli, editors. Fundamental of aquatic toxicology, Methods and applications. Hemisphere Publishing Corporation, Washington. Schroder, G.L. 1975. Nighttime material balance for oxygen in fish ponds receiving organic wastes. Bamidgeh 27: 65-74. Selesi, D., and R. Vamos. 1976. Factors affecting the lethal concentration of ammonia in fish ponds. Ichthyologia 8(1): 115-121 (with English Summary). Shilo, M., and A. Rimon. 1982. Factors which affect the intensification of fish breeding in Israel. Part 2. Ammonia transformation in intensive fish ponds. Bamidgeh 34(3): 101 -1 14. Smith, CE. 1972. Effects of metabolic products on the quality of rainbow trout. Am. Fishes U.S. Trout News. 17(3): 7-8. Smith, CE, and R.G. Piper. 1975. Lesions associated with chronic exposture to ammonia. Pages 497-514 In W.E. Ribelin and W.E. Migaki, editors. the pathology of fishes. University of Wisconsin press, Madison. 50 Sprague, J.B. 1969. Measurements of pollutant toxicity to fish. I. Bioassay methods for acute tests. Water Res. 3: 793-821. Sprague, J.B. 1985. Factors that modify toxicity. Pages 124-163 In G.M. Rand and SR. Petrocelli, editors. Fundamental of aquatic toxicology, Methods and applications. Hemisphere Publishing Corporation, Washington. Stickney, R.R., L.O. Rowland, and J.H. Hesby. 1977. Water quality-Tilapia aurea interactions in ponds receiving swine and poultry wastes. Proc. World Maricult. Soc. 8: 55-71. Strickland, J.D.H. 1960. Measuring the production of marine phytoplankton. Bull. Fish. Res. Bd. Can. No. 122. Syrett, P.J. 1962. Nitrogen assimilation. Pages 171-188 In R.A. Lewin, editor. Physiology and biochemistry of algae. Academic Press, New York. Tabata, K. 1962. Toxicity of ammonia to aquatic animals with reference to the effect of pH and carbon dioxide. Hokoku 34: 67-74 (English translation). Thurston, R.V., and R.C. Russo. 1983. Acute toxicity of ammonia to rainbow trout. Trans. Am. Fish. Soc. 112: 696-704. Thurston, R.V., R.C. Russo, and C.E.Smith. 1978. Acute toxicity of ammonia and nitrite to cutthroat trout fry. Trans. Am. Fish. Soc. 107(2): 361-368. Thurston, R.V., G.R. Philips, R.C. Russo, and S.M. Hinkins. 1981. Increased toxicity of ammonia to rainbow trout (Salmo gairdneri) resulting from reduced concentrations of dissolved oxygen. Can. J. Fish. Aquat. Sci. 38(8): 983- 988. Thurston, R.V., R.C. Russo, and G.R. Philips. 1983. Acute toxicity of ammonia to fathead minnows. Trans. Am. Fish. Soc. 112: 705-711. Thurston, R.V., R.C. Russo, R.J. Luedtke, CE. Smith, E.L. Meyn, C. Chakoumakos, K.C. Wang, and C.J.D Brown. 1984. Chronic toxicity of ammonia to rainbow trout. Trans. Am. fish. Soc. 113(1): 56-73. Thurston, R.V, R.C. Russo, and R.K. Zajdel. 1986. Chronic toxicity of ammonia to fathead minnows. Trans. Am. Fish. Soc. 115: 196-207. Tomasso, J.R., C.A. Goudie, B.A. Simco, and K.B. Davis. 1980. Effects of environmental pH and calcium on ammonia toxicity in channel catfish. Trans. Am. Fish. Soc. 109(2): 229-234. Vamos, R. 1963. Ammonia poisoning in carp. Acta. Biol. Szeged. 9 (1-4): 291-297. Warren, K.S., and S. Schenker. 1964. Effect of an inhibitor of glutamine synthesis (methionine sulfoximine) on ammonia toxicity and metabolism. J. Lab. Clin. Med. 64: 442-449. Wetzel, R.G., and GE. Liken. 1979. Limnological analyses. W.B. Saunders Company, Philadelphia. Wetzel, R.G. 1983. Limnology. Saunders College Publishing, New York. 51 WHO. 1986. Ammonia. WHO Environmental Health Criteria 54. World Health Organization, Geneva. Woldendorp, J.W. 1968. Losses of soil nitrogen. Strikstof (no. 12). central Nitrogen Sales Organization, Ltd., The Hague, Netherland. Worcel, A. and M. Erecinska. 1962. Mechanism of inhibitory action of ammonia on the respiration of rat liver mitochondoria. Biochem. Biophys. Acta. 65: 27- 33. APPENDIX 52 gas sauna “.333 8m 55 < .8585 3 nae—8 ~29.» 05 we 39?— oocfi 3 296 3.88:3 :33 35 .14. Paw—h >8 no 05.... 88on £892 583 .585 — q u u q o 7 - 8.0 0. .- r V0.0 7.. c 1 00.0 r 00.0 1.0 ..N_..0 VP.0 2051—00111 w._00_21ml aOhlol . 9.0 ('I/Bw) BIUOWUJV lam 54 .3... is} as a... .aaa 5%. 3%.. 8m .33 U 3253b 3 55:8 333 2: we 39?— ooufl 3 396 £888.. .38 .05 .m-< chew—a— >mo Lo 9a.... 5286 5:899 zdoouw 5286 (1/6w) aluowwv 18101 2052—.001111 mJOnZIImI EOPIOI m...” 55 .003 .323 8a a... 05...... 8.3.... .323 8a 828.... o .853: a... 3... .333. 3 a... 055... an...» .323 3 8382 m .853: .035... 8.1% 333 com 3330.. < 3253: E mun—om .0 can m :4. 33.535 no mecca am 82.3 In :32 >50 Co 95... 58on £892 £83 ....< 03m... .586 - d d u q 0 “cm—Swot. Imr. m #:95wa 1.1 < “cognac... I? v.5 Ym 56 .503 .339. an a... 2:8... 8.3... .339. Sn .8382 o .858: a... 8... .339. a a... .58... 8.2.... .33.... a. .382 m .858: .223... 8.3.... .33.... 8m 3232 < 3053.: am venom .0 .05 m .< 35.535 me «doom 3 853.368 :32 >8 .0 oEF Emoono :58qu anofi .m-< Paw—rm Emoonm d u _ — a 0 50:500.... Iml m «cw—509:. 1...! < “cw—tune... lml 9mm *0 0. 81 81 ( 0.) elnteredwel é». a 8 0. PM 57 .035... ............o .323. 8m 5.3 < 3058.. ... 68.. as. 23.85.. 2.. o. 8.8:... .o .8. .853 .3. 2......— >mo .o 8...... Eu 85 En 8N Eu 8.? Eu 86 \\ o .. 3.0 H m m 5 m «to 49 McGeachin, R.B., and R.R. Stickney. 1982. Manuring rates for production of blue tilapia in simulated sewage lagoons receiving layinghin waste. Prog. Fish - Cult. 44(1): 25-28. Murphy, T.P., and B.G. Brownlee. 1982. Blue green algae ammonia uptake in hypertrophic prairie lake. Can. J. Fish. Aquat. Sci. 38: 1040-1044. Odum, H.T. 1956. Primary production of flowing waters. Limnol. Oceanogr. l: 102- 117. Odum, H.T. 1957. Trophic structure and productivity of silver springs, Florida. Ecol. Monogr. 27: 55-112. Polachek, R.M., and J.R. Tomasso. 1984. Toxicity of nitrite to channel catfish (Ictalurus punctatus), tilapia (Tilapia aurea), and largemouth bass (Micropterus salmoides): Evidence for a nitrite exclusion mechanism. Can. J. Fish. Aquat. Sci. 41: 1739-1744. Powers, EB. 1920. Influence of temperature and concentration on the toxicity of salts to fishes. Ecology 1:95-112. Rand, G.M., and SR. Petrocelli. 1985. Introduction. Pages l-28 In G.M. Rand and SR. Petrocelli, editors. Fundamental of aquatic toxicology, Methods and applications. Hemisphere Publishing Corporation, Washington. Redner, B.D., and R.R. Stickney. 1979. Acclimatization to ammonia by Tilapia aurea. Trans. Am. Fish. Soc. 108(4): 383—388. Reinbold, K.A., and S.M. Pescitelli. 1982. Acute toxicity of ammonia to channel catfish, Illinois Natural History Survey Champaign, Illinois. Roseboom, D.P., and D.L. Richey. 1977. Acute toxicity of residual chlorine and ammonia to some native Illonis fishes, Illonis State Water Survey. Perport of investigation 85. Urbana, Illonis. Round, F .E. 1973. The biology of the algae. St.Martin Press, New York. Russo, R.C. 1985. Ammonia, Nitrite, and Nitrate. Pages 455-471 In G.M. Rand and SR. Petrocelli, editors. Fundamental of aquatic toxicology, Methods and applications. Hemisphere Publishing Corporation, Washington. Schroder, G.L. 1975. Nighttime material balance for oxygen in fish ponds receiving organic wastes. Bamidgeh 27: 65-74. Selesi, D., and R. Vamos. 1976. Factors affecting the lethal concentration of ammonia in fish ponds. Ichthyologia 8(1): llS-lZl (with English Summary). Shilo, M., and A. Rimon. 1982. Factors which affect the intensification of fish breeding in Israel. Part 2. Ammonia transformation in intensive fish ponds. Bamidgeh 34(3): 101 -l 14. Smith, CE. 1972. Effects of metabolic products on the quality of rainbow trout. Am. Fishes U.S. Trout News. 17(3): 7-8. Smith, C.E., and R.G. Piper. 1975. Lesions associated with chronic exposture to ammonia. Pages 497-514 In W.E. Ribelin and W.E. Migaki, editors. the pathology of fishes. University of Wisconsin press, Madison. 50 Sprague, J.B. 1969. Measurements of pollutant toxicity to fish. I. Bioassay methods for acute tests. Water Res. 3: 793-821. Sprague, J .B. 1985. Factors that modify toxicity. Pages 124-163 In G.M. Rand and SR. Petrocelli, editors. Fundamental of aquatic toxicology, Methods and applications. Hemisphere Publishing Corporation, Washington. Stickney, R.R., L.O. Rowland, and J.H. Hesby. 1977. Water quality-Tilapia aurea interactions in ponds receiving swine and poultry wastes. Proc. World Maricult. Soc. 8: 55-71. Strickland, J.D.H. 1960. Measuring the production of marine phytoplankton. Bull. Fish. Res. Bd. Can. No. 122. Syrett, P.J. 1962. Nitrogen assimilation. Pages 171-188 In R.A. Lewin, editor. Physiology and biochemistry of algae. Academic Press, New York. Tabata, K. 1962. Toxicity of ammonia to aquatic animals with reference to the effect of pH and carbon dioxide. Hokoku 34: 67-74 (English translation). Thurston, R.V., and R.C. Russo. 1983. Acute toxicity of ammonia to rainbow trout. Trans. Am. Fish. Soc. 112: 696-704. Thurston, R.V., R.C. Russo, and C.E.Smith. 1978. Acute toxicity of ammonia and nitrite to cutthroat trout fry. Trans. Am. Fish. Soc. 107(2): 361-368. Thurston, R.V., G.R. Philips, R.C. Russo, and S.M. Hinkins. 1981. Increased toxicity of ammonia to rainbow trout (Salmo gairdneri) resulting from reduced concentrations of dissolved oxygen. Can. J. Fish. Aquat. Sci. 38(8): 983- 988. Thurston, R.V., R.C. Russo, and G.R. Philips. 1983. Acute toxicity of ammonia to fathead minnows. Trans. Am. Fish. Soc. 112: 705-711. Thurston, R.V., R.C. Russo, R.J. Luedtke, C.E. Smith, E.L. Meyn, C. Chakoumakos, K.C. Wang, and C.J.D Brown. 1984. Chronic toxicity of ammonia to rainbow trout. Trans. Am. fish. Soc. 113(1): 56-73. Thurston, R.V, R.C. Russo, and R.K. Zajdel. 1986. Chronic toxicity of ammonia to fathead minnows. Trans. Am. Fish. Soc. 115: 196-207. Tomasso, J.R., C.A. Goudie, R.A. Simco, and KB. Davis. 1980. Effects of environmental pH and calcium on ammonia toxicity in channel catfish. Trans. Am. Fish. Soc. 109(2): 229-234. Vamos, R. 1963. Ammonia poisoning in carp. Acta. Biol. Szeged. 9 (l-4): 291-297. Warren, K.S., and S. Schenker. 1964. Effect of an inhibitor of glutamine synthesis (methionine sulfoximine) on ammonia toxicity and metabolism. J. Lab. Clin. Med. 64:442-449. Wetzel, R.G., and GE. Liken. 1979. Limnological analyses. W.B. Saunders Company, Philadelphia. Wetzel, R.G. 1983. Limnology. Saunders College Publishing, New York. 51 WHO. 1986. Ammonia. WHO Environmental Health Criteria 54. World Health Organization, Geneva. Woldendorp, J.W. 1968. Losses of soil nitrogen. Strikstof (no. 12). central Nitrogen Sales Organization, Ltd., The Hague, Netherland. Worcel, A. and M. Erecinska. 1962. Mechanism of inhibitory action of ammonia on the respiration of rat liver mitochondoria. Biochem. Biophys. Acta. 65: 27- 33. APPENDIX 52 .93qu 3.320 «BEAR.— Sn 5? < 3253: am :838 .833 05 «o 9.9?— 025 am 396 «Eon—Ea 39 ~05 .~-< Pawwm >3 .0 oEF 28on .585. £83 .585 q u a q u o 7 - 8... ‘ J1 . .- 1 V0.0 o . .. 00.0 .. 00.0 $0 ._NF.0 .130 IOthmlwl m._00_21ml aOhlol . 0P0 (“I/6w) eluowwv new. 53 .33 x3\§\w._ g 33 0.558 54030 {(3}.— 3. 5.3 m 3253: am 5:38 .895 2: 00 En?— ooh: E 0.90 «Eon—Ea 3o. .30 .N-< oSwE >8 .o 95... Eocene E893 anonw Emoonm _.¢.0 IOPFOmlil WJOO.2lml QOPIOI 0.0 (1/Bw) aluowwv 13101 54 8:. :12? 8a 83 case :32“. .323 8m 55 U 3253.: um 5:38 333 05 yo E9?— ooufi 3 0?? «Eon—8a 32 .05 >8 .0 9E... .25on .m-< ouawE 2.865? Emoonw $286 IOPFOQ I...l MJOD .2 lml mOF IOI (1mm) aluowwv Moi m6 55 dob.— .333 8a 83 eaaa genie 333 Sn 3‘82 0 songs: ea 85 ~33? tn «.23 0558 98.039 {5?wa 3 3332 m .853: .9558 uoquu {influx 8n 8:82 < .353 a 38.. o as m .< 3558: a. 88.. a 82: E. :32 >3 3 mEF .585 5899 anoN .v.< 2..me Emoono — u d d q 0 «ngwmhh Iml m 305$th I...I < «cmgmofi. I¢I vN To 56 .505 {<23 8m 8:. Baas 8.35 as}? can 3‘82 o .553: as 83 ”EBB. tn 33 0.55:. 5:01? {(33 3. 3282 m 3253: .9558 acacia {<59— ocn 3332 < 3253: um wax—om .0 23 m .< 8553: Ho 8.59 3 3.32382 :32 >8 ,6 mEF E306 E899 anonm .n-< paw—h— o $.58: :T m E0559... 1.1 < 50539... I®I o. 5 ( o ) exmmadwal 57 case :23”. “323 8m 55 < 2.253: a 8.8a 89a 2238.5“ 2: 2 comeba— uc 82 1335 .o.< Paw...”— >8 8 mEF Eu 8qu Eu 85 q - a o 1 vod H m w 6 N we . 3... w «to 58 as 323 a an 2:3 53. 323 3. 55 m 3253; 3 £309 Bah 80.—among 05 3 coach? «a 33 .5ng .b-< 0.59."— >8 B 95... En 85 En 8mm Ea 85? En 86 o R . \ u \ m w 5 \\ m . «.0 W3 \ -2 a... 59 do... {<23 8a 3.. case 849% “32E. 8m 5m? 0 3253; am «.989 Boa guinea—Ha 05 8 couch? mo 82 3955 28 3 «E.L. Ea 86 En 8mm . Ea 86F .w.mo .0 9E... .o-< usur— Ew 86 Eu 8qu En 8N Em 86 c E038 lil 1 0522 Iml no... I¢I Good .86 $86 wood $86 o 56 (1/Bw) uomanuaouoo aluouuuv pezluolun 61 .3... “333. 3 Ba 22a... 86% «is? 3. a? m 30535 E 5:38 333 05 no E9?— 35 3 29>". «Eon—Ea conica— EQ .8 -< 253m >8 .o 95... Em 86 En 86F Ea 8mm Em 86 \\ - 5o Eaton I...l 0.022 Iml n no... I¢I (1/6u1) uonanueouoo Bluowwv pez1uolun 62 35 “353 8m 83 case :23... “32R“. 8“ a? D 32535 um 5:28 .533 05 «a E??— ooufi E 296 £5.55 35?: 35 .:-< 23E $0 .0 «E.L. Em 86 En 85F Ea 8mm Em 86 (1/Bw) uonanueouog aluowwv pazlumun "‘7ll'i'7fliflfljfliflifllifllifllfliflr