THE TOXICITY AND RESIDUE DYNAMICS OF SELENIUM I IN FISH AND AQUATIC INVERTEBRATES Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY WILLIAM JAMES ADAMS 1976 TwFQI“: ' IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I This is to certify that the thesis entitled THE TOXICITY AND RESIDUE DYNAMICS OF SELENIUM IN FISH AND AQUATIC INVERTEBRATES presented by William James Adams has been accepted towards fulfillment of the requirements for Ph.D. Fisheries & Wildlife degree in déMé/w Major fessor 0-7639 0N ABSTRACT THE TOXICITY AND RESIDUE DYNAMICS OF SELENIUM IN FISH AND AQUATIC INVERTEBRATES By William James Adams Selenium concentrations in water, sediment, zooplankton and fish from the western basin of Lake Erie were determined by spectrophotometric procedures. The average concentration of selenium (dry weight) in ten fish species ranged from 8.12 i 1.02 ppm for sheepshead to 1.80 i 0.12 ppm for common shiners. The selenium concentration in yellow perch increased in proportion to the length and weight of the fish. Higher concentrations in unfiltered water (0.023 i 0.00M ppm) than in filtered water (0.003 i 0.001 ppm) were attributed to selenium adsorbed on suspended solids and contained in the plankton. The average concentration of selenium in sediment and zooplankton samples (dry weight) was 0.36 i 0.07.ppm and 2.5h i 0.1h ppm, respectively. ‘The concentrations of selenium in the fish from western Lake Erie are higher'than reported for other areas of the Great Lakes and this may be attributed to the fact that western Lake Erie is heavily industrialized and a recipient of multiple waste discharges. The acute toxicity of sodium selenate and sodium selenite to several species of fish and invertebrates was determined by static and continuous flow tests. Sodium selenite was found to be more toxic than sodium selenate and both compounds were more toxic in the continuous flow tests than in the static tests. A slow accumulative William James Adams mortality occurred in all continuous flow tests and it was found that an exposure period of at least 96 days is needed to determine the asmyptotic L050 for inorganic selenium compounds. The NB day L050 values for sodium selenate and sodium selenite were 2.0 mg/l and 1.1 mg/l, respectively, with fathead minnows. The comparative A8 day L050 values for sodium selenite with bluegills and rainbow trout were 0.h0 mg/l and 0.50 mg/l. The 96 day L050 value for rainbow trout exposed to sodium selenite was 0.28 mg/l. Coho salmon larvae appeared to be more sensitive to sodium selenite than other species of fish as indicated by the A8 day L050 value of 0.16 mg/l. The 96 hour L050 for sodium selenate with HyaZZeZa azeteca was 0.76 mg/l. The hatchability of fathead minnow eggs exposed to sodium selenite at concentrations of l—hO mg/l was unaffected, however, the eggs hatched prematurely at concentrations greater than 15 mg/l and the median survival time of the resulting fry was reduced at all concentrations. The uptake of selenium by fathead minnows exposed to 10, 25 and 50 ug/l Se occurred in a curvilinear manner with a rapid period of accumulation during the first 8 days followed by a slower rate of accumulation over the next 88 days. The average concentration of selenium (wet weight), after 96 days exposure to 50 ug/l Se, in the viscera, gill, head—tail, and muscle was 2.hh, 0.58, 0.5h and 0.hh mg/Kg. The data suggests that the accumulation of selenium in the various tissues is directly related to the exposure concentration. The elimination of selenium from fathead minnows occurred in a curvilinear manner and was asmyptotic with the time axis after 96 days. Elimination from the viscera was most rapid (half—life 5.1 days); the half-life of selenium in other tissues exceeded 50 days. William James Adams Rainbow trout exposed to sodium selenite (0.22 mg/l) for A8 days had average concentrations of 81.7, 61.9, 29.b, 8.5, 7.0, h.6, h.5, 3.5, 1.9 and 0.h5 mg/Kg in the spleen, liver, heart, pyloric caeca, kidney, intestine, blood, brain, gill and muscle, respectively. Selenium concentration in the muscle was consistently lower than in other tissues with only 20 percent of the total body residue in the muscle of rainbow trout and 27 percent in the muscle of fathead minnows.~ Trout which died after exposure to sodium selenite contained 1.61 i 0.18 mg/Kg as compared to 0.90 i 0.11 mg/Kg in the muscle of the trout which survived a 96 day period of exposure. Bioconcentration factors for both fathead minnows and rainbow trout were inversely related to the exposure concentration. THE TOXICITY AND RESIDUE DYNAMICS OF SELENIUM IN FISH AND AQUATIC INVERTEBRATES By William James Adams A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Fisheries and Wildlife 1976 ACKNOWLEDGMENTS My appreciation is given to my graduate committee, Dr. Thomas G. Bahr, Dr. Frank M. D'ltri, Dr. Niles R. Kevern and especially to Dr. Howard E. Johnson, chairman, for the assistance and guidance he provided throughout my doctoral program. I wish to thank Mark T. Halter for his advice and assistance in setting up many of my experiments and for the many other times he helped me during the course of this study. 1 wish to thank Dr. Duane Ullrey for his advice concerning analytical techniques and for independently analyzing several fish samples for selenium content. I want to thank my parents who made all of this possible. Their financial assistance and encouragement throughout my college career is greatly appreciated. Most of all I want to thank my wife, Jeanne, for the support, encouragement and understanding she always provided and for the many things she did that helped me to obtain this degree. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION SECTION I: A SURVEY OF THE SELENIUM CONTENT IN THE AQUATIC BIOTA OF WESTERN LAKE ERIE METHODS AND MATERIALS RESULTS AND DISCUSSION SECTION II. TOXICITY AND SELENIUM RESIDUE DYNAMICS IN FISH AND AQUATIC INVERTEBRATES .1. METHODS AND MATERIALS Acute Toxicity Tests Uptake, Distribution and Elimination Experiments Fish Source and Maintenance Water Characteristics and Source Exposure Systems . Static Toxicity Tests with Fathead Minnows and Rainbow Trout . Fathead Minnow Egg Exposure Exposure System for Continuous Flow Toxicity Tests Egg—Alevin Exposure Amphipod Exposure . Fathead Minnows — Static ExpOSure to Selenite— 75 . . Fathead Minnow and Rainbow Trout Exposure — Uptake, Distribution and Elimination Experiments Preparation of Stock Solutions Sampling and Analytical Procedures Water Samples Fish Samples . Analytical Procedure 10 22 TABLE OF CONTENTS (cont‘d) Page RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . ho Static Toxicity Tests . . . . . . . . . NO Continuous Flow Toxicity Tests with FiSh . . . . . . Ah- Invertebrate Exposure . . . . . . . . . . 5h Uptake of Selenium — Static Exposure . . . 55 Uptake, Distribution and Elimination of Selenium — Continuous Flow Exposure System . . . . . . . 60 Accumulation and Distribution of Selenium in Rainbow Trout . . . . . . . . . . . . . . . . . . 78 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 83 Toxicity Tests . . . . . . 83 Uptake, Distribution and EliminatiOn Studies . . . . 85 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . 103 iv Table 10 LIST OF TABLES Comparison of selenium concentrations in fish samples analyzed by both spectrophotometric and fluorometric methods of analysis. Concentration of selenium in four sets of water samples collected at six locations in western Lake Erie. Selenium concentration in two sets of sediment samples collected at six locations in western Lake Erie. Sample one was collected on 6/2h/73 and sample two was collected on 8/28/7h. Selenium concentration in three sets of zooplankton samples collected at six locations in western Lake Erie. Samples one, two and three were collected on 6/2u/73, 7/26/73 and 8/2h/7u, respectively. Concentration of selenium in fish collected at six locations in western Lake Erie. Chemical and physical characteristics of the test water. Description of the fish and chemical and physical characteristics (mg/1) of the test water used for static toxicity tests. Description of the fish and chemical and physical characteristics (mg/1) of the test water used for continuous flow toxicity tests. Description of the fish and chemical and physical characteristics (mg/l) of the test water used for measuring uptake and elimination of selenium in fathead minnows. The mean concentration of selenium (i 1 S.E.) in the Water used for continuous flow toxicity tests. -Nomina1 concentrations are in parentheses. A single water sample was analyzed from each test chamber once a week. ll 13 1h 16 27 29 31 36 LIST OF TABLES (cont'd) Table 11 12 13 1h 15 16 17 A2 A3 AA The 96 hour L050 values, confidence intervals and slope values for sodium selenate and sodium selenite determined with fathead minnows and rainbow trout by static toxicity tests. Percentage hatch of fathead minnow eggs exposed to sodium selenite.. Initial exposure began at 2 days of age (50 eggs per concentration). Percent mortality and median survival time (MST) of groups of 25 fathead minnow fry exposed to sodium selenite (50 fry per concentration). 'The L050 values, confidence intervals and slope values of two selenium compounds with three species of fish and one invertebrate determined by continuous flow toxicity tests. Accumulation of selenium in fathead minnows exposed to three concentrations of selenium for 96 days. Regression values were obtained by plotting log concentration (ug/Kg) against log days. Half—life and rate of elimination of selenium from fathead minnows exposed to three concentrations of selenium for 96 days. Regression values were obtained by plotting log tissue concentration (pg/Kg) against time (days). Average concentration of selenium in the tissues of rainbow trout exposed to four concentrations of selenium. Standard errors are in parentheses. Determination of the loss of selenium, due to volitilization during digestion, from the muscle of fish which had been exposed to selenite—75. Percent recovery of selenium from fish muscle. Determination of the loss of selenium, due to volitilization, from Daphnia magna which had been exposed to selenite—75 and dried at 60 C for 30 hours. Determination of the loss of selenium, due to volitilization, from the muscle of fish which had been exposed to selenite—75 and then dried at 60 0 for 30 hours. vi AS M6 71 79 92 93 9h 95 LIST OF TABLES (cont'd) Table A5 Comparison of the selenium content in water samples (2 ml) using radioisotope and stable analysis. A6 Concentration of selenium in fish collected from six locations in western Lake Erie. vii Figure 10 11 12 LIST OF FIGURES A map of the study area located along the near shore area of western Lake Erie at Monroe, Michigan. A correlation of the concentration of selenium in fish muscle with the length and weight of the fish. A comparison of the concentration of selenium in water, sediment, zooplankton and fish samples collected from western Lake Erie. Relationship between temperature and the static 96 hour L050 for juvenile fathead minnows exposed to sodium selenite. Median survival time of fathead minnow larvae exposed to sodium selenite. The line was fitted by eye. The effect of time on the toxicity of sodium selenate to juvenile fathead minnows. The line was fitted by eye. The effect of time on the toxicity of sodium selenite to juvenile fathead minnows. The line was fitted by eye. The effect of time on the toxicity of sodium selenite to fingerling rainbow trout. The line was fitted by eye. The accumulation of selenium by juvenile fathead minnows during static exposure to selenite—75. The elimination of selenium by juvenile fathead minnows after static exposure to selenite—75. The accumulation of Selenium in the viscera of adult fathead minnows. The accumulation of selenium in the gills of adult fathead minnows. viii 2O 21 AB II8 119 59 61 62 LIST OF FIGURES (cont'd) Figure ‘ Page 13 The accumulation of selenium in the head and tail 63 of adult fathead minnows. 1A The accumulation of selenium in the muscle of adult 6A fathead minnows. 15 Whole—body accumulation of selenium by adult fathead 65 minnows. 16 The average concentration of selenium (i l S.E.) in 69 the tissues and whole—body of adult fathead minnows after 96 days of exposure to sodium selenite at concentrations of 10, 25 and 50 ug/l. 17 A comparison of the bioconcentration factors observed 72 at various intervals for fathead minnows exposed to four concentrations of selenium. 18 The elimination of selenium by the viscera of adult 73 fathead minnows. 19 The elimination of selenium by the gills of adult 7A fathead minnows. 20 The elimination of selenium by the head and tail of 75 adult fathead minnows. 21 The elimination of selenium by the muscle of adult 76 fathead minnows. 22 Whole—body elimination of selenium by adult fathead 77 minnows. 23 The average concentration of selenium (i 1 S.E.) in 81 the tissues and whole—body of rainbow trout after A8 days of exposure to sodium selenite at a concentra— tion of 0.22 mg/l. ix INTRODUCTION The element selenium can be traced in an orderly sequence from its origin in the earth's»crust to specific geologic formations, to distribution of specific genera and groups of plants which require the element for grOwth, to the accumulation in vegetation and to its subsequent toxicity to birds or mammals that consume the seleniferous feeds (Allaway et a1., 1966; Hoffman et a1., 1973). The disease syndrome produced by selenium is a disease of antiquity and has been described in widely separated areas of the world. It has been reported as early as 1295 by Marco Polo (1926) when he wrote of his travels in China and by Stein (1912) when his horses became poisoned in 1906 while traveling in Turkestan and Western China. The earliest account of the form of selenium poisoning known as alkali disease was reported in the United States in 1860 by T. 0. Madison (1860). Since this time numerous other incidences of selenium poisoning have been reported (Anderson et a1., 1961; Franke and Moxon, 1936; Franke and Painter, 1938; Moxon and Rhian, 19A3; Muth and Binns, 196A; and Rosenfeld and Death, 196A). More recently, Hosseinian et a1. (1972) have reported selenium poisoning in a mixed flock of sheep and goats in Iran. The symptoms of selenium poisoning are typical of heavy metal poisoning, including nervous disorders, loss of hair and nails and malformation and abortion of embryos. Until 1957 the main concern about selenium has been due to its toxicity. In the last 19 years several compelling reasons for the reexamination of the role of selenium in biology have arisen. Selenium was discovered by Schwarz and Foltz (1957) to be an essential component. of a factor, called factor three, that prevented liver degeneration in rats maintained in diets low in vitamin E (tocopherol). This was immediately followed by the finding that selenium is effective in the prevention of a number of economically important diseases of farm animals (Erwin et a1., 1961; Nesheim and Scott, 1958; Patterson et a1., 1957; Rahman et a1., 1960; Schwarz et a1. , 1957; and Scott et a1., 1967). Selenium has recently been shown by Thompson and Scott (1969) to be an essential nutrient in its own right, independent of a complimentary effect of vitamin E. The amount of selenium required by livestock varies greatly depending on the amount of vitamin E present although levels of 0.01—0.l mg/kg in the diet are known to protect cattle and poultry from white muscle disease and exudative diathesis, respectively (Schubert et a1., 1961; Thompson and Scott, 1969). The role of selenium as an essential nutrient became more clear when Rotruck et a1. (1972) discovered that selenium is an integral part of glutathione peroxidase, an enzyme responsible for destroying lipid peroxides, preventing erythrocyte hemolysis and oxidative destruction of cell membrane lipids. Selenium is also being reexamined in view of the fact that it may be of some benefit in treating certain forms of cancer (Mickelsen, 1970; Shamberger and Frost, 1969) and because it has been shown to be of benefit in detoxifying other metals including silver, mercury, cadmium, lead and some forms of arsenic (Burch et a1., 1973; Ganther et a1., 1972a; Ganther et a1., 197A; Groth et a1., 1972; Hill, 1972, 197A; Levander and Argrett, 1969; and Parizek and Ostadalova, 1967). At present, nearly all cases of selenium poisoning have been in domestic livestock and not in humans, nevertheless, environmental contamination of selenium is of special importance because of the possibility of human injury resulting from the consumption of meat, vegetables, dairy products and fish from affected areas. Extensive investigations of the poisoning of animals have already been made (Cerwenka and Cooper, 1961; Cousins and Cairney, 1961; Daize and Beath, 1935; Kubota et a1., 1967; Moxon and Rhian, 19A3; Rosenfeld and Beath, 196A; and Schroeder, 1967) and limited studies on consumption by humans have been reported (Hamilton and Hardy, 19A9; Smith et a1., 1936; Smith and Lillie, 19A0; Schroeder et a1., 1970; and Thompson et a1., 1975), but research on the concentration of selenium in aquatic organisms and the toxicity of selenium compounds to fish and other aquatic organisms is lacking. Several sources of selenium pollution do exist. Selenium is mainly produced as a by—product of copper refining and is used extensively as a decolorizer for glass and ceramics. It is also used for photo cells, xerography, rectifiers, solar batteries, television cameras, traffic lights, enamels, brighteners for copper plating, vulcanizing, antioxidant in oils, insecticides and fungicides, paint, varnish, glue remover and various other uses. Environmental contamination from the misuse of these materials could result in a possible threat to aquatic life. A major source of selenium in the aquatic environment has been attributed to fallout from stack emissions of fossil fuel power plants and industries (Pakkala et a1., 1972). Kesseler et al. (1971) and Shah et a1. (1970) have reported the concentration of selenium in coal and oil to be as high as 5.0 ppm and 1.A ppm, respectively. Other potential sources of selenium pollution have been demonstrated by Hashimoto et a1. (1970), Johnson (1970) and Pillay et a1. (197A) who have found concentrations of selenium in excess of 1.0 ppm in coal, petroleum, and rubber products, up to 8.0 ppm in solid wastes and as high as 1A.O ppm in particulate stack emissions. The proximity of fossil fuel power plants, steel mills and refining and metal plating factories along the shores of the Great Lakes increases the chance of contaminating the aquatic biota with selenium. Agricultural runoff may also cause some contamination because selenium is used in certain pesticides (Moxon and Rhain, 19A3) and is reported to be present as an impurity in fertilizers (Wells, 1966). A survey conducted by Copeland (1971) in Lake Michigan showed an average of 0.5 ppm selenium in bottom sediments and as high as 7.0 ppm in zooplankton. Ayers (1970) has reported levels of selenium, in Lake Michigan for phytoplankton, zooplankton and benthos to be as high as 1.05 ppm, 3.90 ppm and 3.10 ppm, respectively. Copeland (1970) originally suggested that selenium may accumulate in the aquatic ecosystem through the food chain with successive trophic levels accumulating greater amounts of selenium. However, more recent data (Copeland et a1., 1973) have not Substantiated this theory. A recent survey of fish from New York waters, Lake Erie and Lake Ontario indicated that the level of selenium in fish from these areas is usually between 0.1 and 1.0 ppm (wet weight). Similar values have been reported for Lake Michigan by Copeland et a1. (1973). Sandholm et a1. (1973) have found similar levels of selenium in fish taken from Finland, Baltic Sea and the Atlantic Ocean with the concentrations ranging between 0.9 ppm and 2.3‘ppm (dry weight). Schroeder et a1. (1970) have reported somewhat higher concentrations ranging between 1.0 ppm to 2.2 ppm (wet weight) in certain sea foods including smelt, lobster, shrimp, herring, and fish flour. Similar findings have been reported by Kifer et a1. (1969), Lindberg (1968), Lunde (1968, 1970), and Soares and Miller (1970). While the above types of data provide information concerning the availability of selenium in aquatic environments they do not clearly indicate whether selenium is accumulated through the food chain, nor do they suggest what influence selenium may have on fish populations. Only a minimal amount of research has been undertaken to interpret these environmental levels in terms of the effects on fish populations and studies have not been conducted to determine the distribution and rates of uptake and elimination of selenium in fish. In addition, the toxicity of various selenium compounds to fish has been just recently investigated (Huckabee and Griffith, 197A; Niimi and LaHam, 1975; and Weir and Hine, 1970). Although a paucity of data exists on the effects of selenium on aquatic organisms, the death of stocked game fish in a Colorado reservoir has been associated with high levels of selenium in the bottom sediments (Barnhart, 1958). In view of the scarcity of information on the presence of selenium in the aquatic environment and its affect on aquatic organisms the following three areas of investigation were chosen: (1) to determine the selenium content in selected components of the aquatic biota of western Lake Erie; (2) to measure the acute toxicity of selenium to several species of fish; and (3) to determine the distribution and rates of uptake and elimination of selenium in selected species of fish. SECTION I: A SURVEY OF THE SELENIUM CONTENT IN THE AQUATIC BIOTA OF WESTERN LAKE ERIE METHODS AND MATERIALS The method selected for selenium analysis was a spectrophotometric procedure described by Cummins et a1. (1965). This procedure is based on the method of complexing selenium with diaminobenzidine as described by Hoste and Gillis (1955) and refined by Cheng (1956). Some modifica— tions of the method were made including the use of a Beckman DK—2A spectrophotometer instead of a spectronic 20, a pH meter was used for all pH adjustments and the digestion solution was changed to a 1:1 mixture of sulfuric and perchloric acids. Twenty grams of sodium molybdate dissolved in 100 milliliters of water were added to each liter of digestion solution. A certified standard stock solution of selenium dioxide (Fisher Scientific Co.) was used to establish standard curves. The digestion procedure Was tested by analyzing fish which had been exposed to radioactive Selenite—75. The average loss of selenium during the digestion was 2.78 percent (Table A1). The minimum detectable concentration by this method is 0.1 mg/kg. The percent recovery of selenium added to fish muscle was found to be 103.0 1 1.0 percent (Table A2). In order to ascertain the accuracy of this method fish samples were independently analyzed for selenium by fluorometric analysis and compared with analysis of the same tissues using the described spectrophotometric procedure (Table 1). Table 1. Comparison of selenium concentrations in fish samples analyzed by both spectrophotometric and fluorometric methods of analysis. Concentration of Selenium (ppm) Sample No. Species Spectrophotometricl Fluorometric2 183 Fathead minnow 0.36:0.02 0.A0 153 Yellow perch 0.A2i0.03 0.Al 157 Yellow perch O.A7i0.06 0.A6 lThe spectrophotometric values are given with their respective standard errors. Each sample was analyzed four times. 2The fluorometric analysis was conducted independently in the Dept. of Animal Science, Mich. State Univ., E. Lansing, Mich. Each sample was analyzed twice. Fish, zooplankton, sediment and water samples were collected during the summers of 1973 and 197A from six different stations in western Lake Erie (Figure 1). The fish were collected with an otter trawl and gill nets, placed in plastic bags and frozen until each fish could be fileted, skinned, and dry homogenized with a Polytron Blender. The homogenate was placed in glass vials and kept frozen until analyzed. Water samples were collected in glass bottles and analyzed the same day to minimize the loss of selenium due to adsorption. Plastic bottles were found to adsorb greater amounts of selenium than glass bottles and therefore they were not used. Samples of filtered (0.A5 u Millipore filter) and non—filtered water were analyzed using 100 milliliters of water and following the same procedure as before except that the samples were not heated. The method of analyzing for selenium in water was checked by employing Selenite—75 and comparing stable and radioactive determinations on the same samples. The average deviation of the stable analysis from the radioactive analysis was 1.85 i 0.99 percent (Table 3A). Zooplankton samples were collected with a 500 micron plankton tow net and kept frozen until analyzed. An Ekman dredge was used to collect sediment samples. A subsample of about 100 grams was taken for analysis from the Ekman hauls at each station. Both the zooplankton and sediment samples were first air dried in a drying oven at 6000 for 2A hours. Experiments with radioactive Selenite—75 indicated that no significant loss due to volatilization of selenium from the samples should occur at this temperature for up to 30 hours of heating (Tables AA and 5A). One gram of fish and sediment, and 0.5 grams of zooplankton were normally used for analysis. Sediment and zooplankton values were Figure 1. A map of the study area located along the near shore area of western Lake Erie at Monroe, Michigan. Lake Erie 1km 1 PrevaHing Current 0 CoHecHng Station 10 calculated on a dry weight basis whereas the fish values were calculated on a wet weight basis and converted to dry weight using the average percent moisture for each species. Standard statistical procedures (variance, correlation, t—tests, and Duncan's multiple range test) were used to analyze the data (Steel and Torrie, 1955). All values Were tested at the 0.05 probability level and the term significance is used to indicate this throughout this section. Variation about the mean is denoted by the standard error. Logarithims to the base 10 were used for all data transformations. The fish collected and analyzed for selenium content were yellow perch (Perca fZavescens), common shiners (Notropis cornutus), spottail shiners (Notropis hudsonius), sheepshead (Aplodinotus grunniens), carp (Cyprinus carpio), white bass (Roccus chrysops), goldfish (Carassius auratus), gizzard shad (Dorosoma cepedianum), walleye (Stizostedion vitreum), and white sucker (Catastomus commersoni). RESULTS AND DISCUSSION The average concentration of the unfiltered water samples for each collection date was found to be significantly higher than in the filtered samples (Table 2). This difference was attributed to adsorption of selenium on plankton and suspended solids in the unfiltered samples. No relationship was found between the concentration of selenium in the water and the site of sample collection. The concentra— tion of selenium in the filtered samples is in agreement with the values reported by Bowen (1966) (0.02 ppm or less) as commonly occurring in freshwater lakes, however, these values are significantly higher 11 Table 2. Concentration of selenium in four sets of water samples collected at six locations in western Lake Erie. Concentration of Selenium (ppm) Station Date Filtered Unfiltered 1 5/11/73 0.005 0.012 2 " 0.006 0.026 3 " 0 006 0.015 A " 0&005 0.012 S ” 0.005 0.019 6 " 0.005 0.020 MeaniS.E. 0.005i0.001 0.017i0.001 1 5/30/73 0.001 0.036 2 " 0.001 0.036 3 " 0.002 0.017 A " 0.001 0.025 S " 0.005 0.032 6 " 0.003 0.071 MeaniS.E. 0.002:0.001 0.036:0.008 1 7/18/73 0.001 0.028 2 " 0.002 0.031 3 " 0.005 0.0A3 A " 0.005 0.02A 5 " 0.003 0.020 6 " 0.003 0.02A MeaniS.E. 0.003:0.001 0.028i0.003 l 8/28/7A 0.001 0.011 2 " 0.001 0.012 3 " 0.002 0.010 A " 0.002 0.009 5 " 0.001 0.010 6 " 0 001 0.015 MeaniS.E. 0.001i0.001 0.011i0.001 12 than those reported by Copeland and Ayers (1972) (0.083 ppb) for Lake Michigan. Two sets of sediment samples collected on 6/2A/73 and 8/28/7A had average concentrations of 0.56 i 0.06 and 0.16 i 0.0A ppm dry weight, respectively (Table 3). In the first set of samples, stations five and six were found to be significantly higher than station two and station six was also significantly higher than station three. There were no significant differences between stations in the second set of samples. The selenium concentration was not correlated with the organic content of the sediments at any of the stations. Sediment selenium values obtained in the present study are lower than the values reported by Copeland and Ayers (1972) for Lake Michigan. They found the average concentration of selenium in bulk sediments to be 0.60 ppm wet weight. Wiersma and Lee (1971) surveyed the selenium content in the sediment from several lakes in Wisconsin and found the concentrations to range from 1.0—3.0 ppm dry weight. The data obtained in this study indicates that the selenium content in the sediments of western Lake Erie appears to be less than in other areas of the Great Lakes region, however, the data does fall within the range of values (0.05—0.60 ppm wet weight) reported by Bowen (1966) for sediments on a world wide basis. Three sets of zooplankton samples were collected with the average concentration of all the samples being 2.5A : 0.1A ppm dry weight (Table A). There were no significant differences between the sets of samples nor did any of the stations tend to show consistently higher concentrations of selenium in the zooplankton. The data obtained in this study agrees with the concentrations of selenium found in zooplankton in Lake Michigan by Ayers (1970) and Copeland and Ayers (1972). Table 3. Selenium concentration in two sets of sediment samples collected at six locations in western Lake Erie. Sample one was collected on 6/2A/73 and sample two was collected on 8/28/7A. Selenium (ppm dry wt.) 1 1 Std. Error Station Sample #1 Sample #2 1 0.52:0.06 0.10:0.01 2 0.35i0.07 0 l7i0 03 3 0.A7:0.12 0.3710.07 A 0.6010.0A 0.10t0.01 5 O.6510.12 0.10t0.01 6 0.75:0.05 0.10:0.01 Mean 0.56i0.06 0.16:0.0A Table A. Selenium concentration of three sets in zooplankton samples collected at six locations in western Lake Erie. Samples one, two and three were collected on 6/2A/73, 7/26/73 and 8/2A/7A, respectively. Selenium (ppm dry wt.) 1 1 Std. Error Station Sample #1 Sample #2 Sample #3 1 2.71:0.23 2.27:0.12 2.17:0.22 2 2.21:0.16 2.0710.07 ——— 3 ——— 1.70a 0 78a A 2.12i0.1A 3.51a ___ 5 2.7Ai0.27 3.13i0.12 3.88:0.19 6 2.11:0.17 ——— 2.70i0.03 Mean 2.38:0.1A 2.5A:0.3A 2.3810.6h aInadequate amount of zooplankton was collected to allow for more than one analysis. .IllllllIIII . 15 The average dry weight concentration for all of the fish samples combined was 3.59 i 0.17 ppm (0.7A 1 0.03 ppm wet weight). The average dry weight concentrations ranged from 1.80 t 0.12 ppm for common shiners to 8.12 i 1.02 ppm for sheepshead (Table 5). Both dry and wet weight values for all the fish analyzed are presented in Table 6A. Yellow perch were examined most intensively because they are one of the most abundant species in western Lake Erie and are often taken by sports and commercial fishermen. The average concentration of selenium in 79 yellow perch was found to be 3.32 i 0.22 ppm dry weight. Although no definite relationships between selenium content in fish samples and collection site were established, yellow perch were found to have significantly higher concentrations at station one than at any other station. Pakkala et a1. (1972) surveyed the selenium content in yellow perch from eastern Lake Erie and reported the average concentration to be 0.32 i 0.01 ppm (wet weight) as compared to 0.7A 1 0.05 ppm (wet weight) in the present study. The mean concentration of selenium in yellow perch taken from Lake Michigan was 0.57 i 0.03 ppm wet weight (Copeland et a1., 1973). The average concentration (wet weight) of selenium in sheepshead, white bass, and walleye from eastern Lake Erie was found to be 0.A3 : 0.02 ppm, 0.A2 i 0.02 ppm, and 0.29 i 0.02 ppm, respec— tively (Pakkala et a1., 1972). These values are significantly lower than the values obtained for the same species in the present study (Table 5). The range of values reported by Copeland et a1. (1973) for Lake Michigan fish, are only slightly less than the values obtained in this study. They reported the average concentration of 16 Table 5. Concentration of selenium in fish collected at six locations in western Lake Erie. Average Concentration (ppm) i l S.E. Number Species Station of Fish Wet Weight Dry Weight Yellow perch l 20 0.89 i 0.07 A.00 : 0.31 " 2 25 0.7A r 0.11 3.33 i 0.51 " 5 15 0.57 r 0.09 2.5A t 0.A6 " 6 19 0.65 r 0.09 2.90 i 0.39 Average _ 79 0.7A 1 0.05 3.32 i 0.22 Common shiner 2 11 0.AA i 0.05 1.80 i 0.19 " 5 10 0.A3 : 0.0A 1.76 r 0.17 Average 21 0.AA i 0.03 1.80 i 0.12 Spottail shiner ‘ 1 10 0.52 r 0.05 2.12 r 0.21 " 6 6 0.96 i 0.18 3.9A : 0.73 Average 16 0.69 t 0.09 2.82 r 0.37 Sheepshead 1 3 0.97 r 0.17 5.23 r 0.92 " 3 1 1.A8 7.96 " A 2 1.13 6.08 " 6 7 1.85 r 0.23 9.95 i 1.25 Average 13 1.51 r 0.19 8.12 r 1.02 Carp 5 3 1.02 i 0.19 A.A8 i 0.83 " 6 3 0.61 r 0.01 2.67 i 0.38 Average 6 0.82 i 0.13 3.57 i 0.55 White bass 2 1 0.80 A.17 " 5 3 0.82 r 0.19 A.29 i 0.98 Average A 0.82 i 0.13 A.26 i 0.69 Goldfish A 1 1.60 6.96 " 5 1 1.11 A.83 Average 2 1.36 5.89 Table 5 (cont'd) Average Concentration (PPm) i l S. Number Species Station of Fish Wet Weight Dry Weight Gizzard shad 5 A 0.73 i 0.07 3.69 _ 0.3A Walleye A 3 0.32 i 0.07 1.52 0.35 " 5 A 0.67 : 0.1A 3.13 0.65 Average 7 0.52 i 0.11 2.AA _ 0.52 White sucker 2 2 0.59 3.01 18 selenium in all species of fish combined to be 0.5A 1 0.01 ppm (wet weight) as compared to 0.7A 1 0.0A ppm (wet weight) in this study. Selenium content was not significantly different between the sexes for any of the species collected. A significant correlation between selenium concentration and both length and weight was found for yellow perch at station one (Figure 2). Collections at other stations did not include sufficient size range to allow this type of comparison for yellow perch or for other species. In order to compare the concentrations of selenium in the fish from western Lake Erie with fish from other areas which are subject to less municipal and industrial wastes, seven yellow perch from the northern tip of Lake Huron were analyzed and found to contain significantly less selenium than the yellow perch from western Lake Erie. The average values for the Lake Huron and Lake Erie yellow perch were 0.60 i 0.0A ppm (wet weight) and 0.7A : 0.05 ppm, respec— tively. Although the difference between these two means is statistically significant, there is insufficient data to determine whether or not man's activities have influenced the concentration of selenium in western Lake Erie. Beal (197A), however, has indicated that selenium levels in Canadian fish generally declined from areas of high population to areas of low population density. He reported that fish from the Great Lakes had an average concentration of 0.5 ppm (wet weight) whereas fish from Northwestern Ontario, Manitoba, Saskatchewan, Alberta and Northwest Territories had a combined average concentration of 0.2 ppm. A comparison of the concentration of selenium in the various trophic levels indicated that selenium progressively increased from water to sediment to zooplankton and to fish (Figure 3). Similar results 19 reported by Copeland et a1. (1973) showed the concentration of Selenium to increase from water to sediment and from water to phytoplankton to zooplankton, but the levels in the fish did not exceed those found in the zooplankton.. Insufficient data has been collected to determine whether this increase in selenium concentration through the trophic levels is the result of biological magnification or simply a reflection of the relative rates of accumulation of the organisms at each trophic level. Figure 2. A correlation of the concentration of selenium in fish muscle with the length and weight of the fish. Length [cm] lgml Weight 15 - 10 4 . 5 Y: 4.7 + 10.6 x r: 0.74 U l I 5 1.0 l 5 904 O 70" .7 O 50. 304 Y=-27.4 + 77.2 x l'= 0.77 10‘ . 9 30.. .'5 1.0 1.5 PPM Selenium [wet wt.) 21 Figure 3. A comparison of the concentration of selenium in water, sediment, zooplankton and fish samples collected from western Lake Erie. All values are reported as ppm dry weight except water. Concentration of Selenium (ppm) 3.0. 2.0- .OOI- water sediment zooplankton fish SECTION II: TOXICITY AND SELENIUM RESIDUE DYNAMICS IN FISH AND AQUATIC INVERTEBRATES METHODS AND MATERIALS Acute Toxicity Tests Static and continuous flow toxicity tests were used to measure the toxicity of sodium selenate and sodium selenite to fish and aquatic invertebrates. The acute toxicity of sodium selenite to fathead minnows (Pimephales promelas) (at several temperatures), fathead) ‘minnow eggs and rainbow trout (SaZmO gairdneri) was determined by static toxicity tests. Static tests were also conducted with sodium selenate and fathead minnows. Continuous flow toxicity tests were used to measure the toxicity of sodium selenate to fathead minnows and amphipods (HyaZZeZa azeteca). The toxicity of sodium selenite to fathead minnows, bluegills (Lepomis macrochirus), rainbow trout fingerlings and alevin, and coho salmon (Oncorhynchus kisutéh) alevins was also determined by continuous flow toxicity tests. All tests were conducted according to the general methods outlined in Standard Methods for the Examination of Water and Wastewater (American Public Health Association, 1971) and the recommendations of the Committee on Methods for Toxicity Tests with Aquatic Organisms (Stephan, 1975). The term "asmyptotic L0 50", as described by Brown (1973), is used in this paper to refer to the concentration of selenium at which 50 percent of the test organisms can survive for an indefinite period of time. 22 Uptake, Distribution and Elimination Experiments As an initial attempt to measure the uptake and elimination of selenium in fish, juvenile fathead minnows were exposed, under static conditions, to radioactive selenite-75 (as H2Se03). Uptake was measured over a 28 day period after which the remaining fish were placed in a continuous supply of water without selenium for 96 days and the elimination of selenium was measured. Adult fathead minnows were exposed for 96 days to a mixture of radioactive selenite—75 and stable sodium selenite by means of a continuous flow delivery system. The uptake and distribution of selenium in the fish was measured during this time. At the termination of the exposure period the fish were placed in a continuous supply of water without selenium for 96 days and the elimination of selenium from the fish was measured. Fingerling rainbow trout were also exposed to a mixture of radio- active selenite—75 and stable sodium selenite by means of a continuous flow delivery system. The exposure concentrations were selected so that fish would be expected to die at the higher concentrations but not at the lower concentrations. Ten larger fish (12.0 i 0.5 cm) , were placed in the tank with the lowest exposure concentration (0.22 mg/l) so that tissues not easily dissected from the smaller fish (6.5 i 0.1 cm) could be analyzed for selenium content. The percentage of dead fish at each concentration was used to calculate a 96 day LC 50 value for the smaller trout. The experiment was terminated after A8 and 96 days for the larger and smaller trout, respectively, and all fish remaining alive were sacrificed and analyzed for selenium content. 2A Fish Source and Maintenance Fathead minnows and bluegills were obtained from populations maintained in ponds at the Michigan State University, Department of Fisheries and Wildlife Research Facility. Fish of a known age were obtained by collecting young of the year and maintaining them in the laboratory in 70 gallon fiberglass tanks supplied with a continuous flow of well water. All fish were maintained indoors for at least one month prior to being used. The fish were acclimated to test temperatures for one week before initiating the toxicity tests. Rainbow trout and coho salmon were reared in the laboratory from eggs obtained from the Michigan Department of Natural Resources' Platte River Fish Hatchery. Alevin trout and salmon were initially fed Ewos salmon starter diet (Aktiebolaget Ewos 00., Sodertalje, Sweden) several times each day and were later fed twice each day with a 1:1 mixture of #A Ewos pellets and Oregon Moist diet. The fish were not fed 2A hours prior to or during the static toxicity tests. In all other experiments the fish were fed #A Ewos pellets once each day with the amount of food corresponding to 2 percent of their body weight. Debris was siphoned from the tanks every other day, all tanks were checked in the morning and evening, and dead fish were removed and recorded. Water Characteristics and Source Well water, which was passed through an aeration tank and sand filter, was the water source for all experiments. The chemical characteristics of the well water are summarized in Table 6. Water chemistry, including dissolved oxygen, pH, temperature, total 25 Table 6. Chemical and physical characteristics of the test water. Filtered Characteristic Well Water Alkalinity (mg/l Ca003) 331.9 Ammonia (mg/l—N) 0.A2 Carbon, total (mg/l—C) 79 Chloride (mg/l—Cl) 0.6 Specific conductance (umoho/cm3 at 25 0) 610 Copper (mg/l—Cu) <0.05 Dissolved oxygen (mg/1) 8.2 Hardness (mg/l—CaCOB) 329.1 Iron (mg/l—Fe) 1.0 Lead (mg/l—Pb) <0.3 Nitrate (mg/l—N) 0.03 Nitrite (ug/1~N) <5 Nitrogen, total kjeldahl (mg/l—N) 1.11 Phosphorus, total (mg/l—P) 0.01 Selenium (ug/l—Se) <1 Solids, total (mg/1) 317 Sulfate (mg/l—SOh) 5.0 Temperature (C) 12 26 alkalinity and hardness, was meaSured at the beginning and end Of each static toxicity test (Table 7). Total alkalinity, hardness and pH were measured weekly and dissolved oxygen three times a week for all continuous flow toxicity tests and uptake, distribution and elimination experiments (Tables 8 and 9). All measurements were made in the control tanks except for temperature which was measured in a different exposure tank each day. A Beckman Chem—mate pH meter was used to measure pH and all other measurements were done according to Standard Methods for the Examination of Water and Wastewater (American Public Health Association, 1971). Exposure Systems Static Toxicity Tests with Fathead Minnows and Rainbow Trout The test containers consisted of 15 liter glass aquaria placed in a temperature controlled water bath. The aquaria were filled with water one day prior to the beginning of a test and aerated for 12 hours. The test tanks were not aerated during the tests. ~The toxicant was added to the aquaria by thoroughly mixing an appropriate amount of concentrated stock solution with the water in the test tanks. Duplicate test tanks were used for each test concentration and 10 fish were placed in each tank according to a random sorting scheme. At the termination of each test the 20 control fish were weighed and measured (Table 7). Fathead Minnow Egg_Exposure Fathead minnow eggs were collected from breeding populations maintained in the laboratory. Eggs 1 to 2 days old were placed in hr-.. , o.H»m o o.owm om.w s.: w.:m IIII IIII HssHm IIII IIII IIII IIII IIII mo.onH.mm wo.onmm.o MH.0H>m.z .m.m H H new: 0.00m o.OH 0.30m om.w m.w o.mm IIII IIII HmeHsH AmsoanE omoflpomv opHcmHom Edeom o.omm o o.mmm oo.w m.w m.mH IIII IIII HosHm IIII IIII IIII IIII IIII Ho.ono.om o>.ono>.o HH.OHom.: .m.m H H use: o.me o.OH o.OHm om.w w.w 0.0m IIII IIII HmeHqH Amzoans omompmmv OPHQOHom EdHeom o.mom 0 0.00m mm.~ H.s w.MH IIII IIII anHh IIII IIII IIII IIII IIII wo.onm.mH wo.oawm.o MH.OHHm.: .m.m H H new: o.o:m o 0.2mm mm.s m.m m.MH IIII IIII HanHsH szoodHS downpmmv ouHGOHom adHeom 0.00m o o.mHm om.w m.m o.mH IIII IIII quHm IIII IIII IIII IIII IIII mo.onw.:H Ho.onwo.o mo.onm:.m .m.m H H new: o.wmm 0.0: 0.2mm o:.w o.OH 0.:H IIII IIII HmeHQH AmzoceHs enonpnwv cpmnOHOm adHuom Amoonov mm Amoomov mm Amoooov mm mm sowhxo oMSPmMOQSoB AEmv AEOV mOHoomm one .323on 323812 moosonsm eoiomma Emacs season pqsoasoo anoe Hmesnm ONHm anm .mpmop thOonp OHpcpw pom pom: hopes pmop one mo AH\wEv mOHpmHhopoosnno HnOHmhnm can HSOHSOQO can smHm one mo GOHpmHnomoQ .N OHQwB 28 o.wmm o.NH 0.0mm om.m m.m o.mH IIII IIII HmnHm IIII IIII IIII IIII IIII mm.OHm.:H mo.onmm.o mo.ono:.: .m.m H H use: o.wmm o.wH 0.0mm o:.w w.m m.MH IIII IIII HmeHqH Aesonp SoanosV oquoHom EdHeom Amoooov mm Amoooov mm Amoonov mm mm comhxo manpmaomsoe AEmV ASOV wOHoomm use 323ng 32332 m 88.2% 83038 Emacs summon essoosoo Hcpoe Hchsmm ONHm anm Ae.ssoov s oases Acmmemmv AwmmInmmv Am.siz.sv Am.wim.ev Ao.mHIm.:HV Aon.OIwH.ov Ae.miz.mv Sam m.m m.H IIII wane mo.o mo.o mo.o sosaon o.m:m m.mmm ms.» oo.® 0.:H Hm.o 0H.m onoo : AmHmImmmv AosmIommv As.eIm.sv Ao.mIs.mv Am.omIm.sHv Amm.oIso.ov Ao.mI:.mv m.m s.a IIII Hm.o s.o Ho.o no.0 ssh recap m.mHm 0.1mm om.s mm.s :.sH mH.o we.m SopsHom : Amnmuosmv AwmmIommv Am.sIm.sv As.eIm.mv Am.mHIm.MHv Awo.mHIem.Hv AH.OHIH.mV mcflasomcfim m.o v.0 IIII NH.o H.o MH.o OH.o Pdopp m.m:m ®.:mm o:.~ mm.m w.:H ms.m em.m BOQsHmm : .698di Smmdamv Splat Ademov 8.88.2; 23.358 3611.1: s.m :.H IIII wH.o m.o :H.o :H.o anmsdm m.omm o.wam we.» we.» e.ma me.a mm.m Hammosam : Q/ . 2 AasmIonv AmmmIommv Aa.wIm.sV As.mIH.mV Ad.mHIs.sav Ao.mIm.ov AH.mIm.sv m.m s.m IIII sH.o , H.o mo.o wo.o SOGQHS SPHQOHmm s.Hmm m.emm ow.s om.w m.mH ss.o ss.s escapee ssHeom AommIsomv Amsmemmv AH.wIm.sv Am.wIm.ev Am.wHIw.sHv Aos.Hst.ov vo.mIm.sv H.H m.H IIII oa.o m.o no.0 pma.o Eocene opssoaon m.MHm m.smm oo:.> m®.s m.mH sw.o mwo.m omonpmm szHeom Amoooov no Amoooov mm mm somhxo omzpnsomsoe Aswv Aaov mOHoomm oqdomsoo thsHHoMH< mmoaosmm ©o>HommHQ panoz npmsoq anoe ONHm anm .mpmop thOonp BOHm msossHpsoo pom pom: nouns pmop one mo AH\msv mOHpmHn0poonmno HoOHmhnm can HwOHsmno can anm 039 mo COHpQHsomom .w oHnoB 30 .pmop hoe snow amp mo one one musquop one we comma monanm onp mo came one .OSHN> mm anpoZo . e mode> mo omsmmo o .nonso enmeampm moo msoHa so dem 65Hm> smozp. demands opacoHom . o O . . . IIII IIII o mHm w mmm : N mm S cm mH SHNNNBmm ssHUom Amoomov mm AMOONOV mm mm cowhxo onfipnsomEoE Asmv AEOV mOHoomm Undomsoo seasnasga< noosenom , rename season Hence ©o>H0mmHQ onam gong he.psoov m oases 31 .oHO.H.H® UHGUCNPm 0G0 mSGHE .HO WSHQ 05Hfl> Qdmz .oSHm> mm cmeozp .mode> mo owommo p.d AwHMIozmv AQNMIommV A:.>Im.sv A:.wI®.sv A0.mHI0.mHv Am.mim.HV Am.wlm.wv w.0 0.0 IIII m0.0 H.0 wH.0 0H.0 AsoHpmcHaHHov w.m:m 4.3mm 0m.s 20.0 m.MH wm.m 00.0 SOHh moochpsoo Azzmlmmmv AQNMImev Aw.SI:.>V AN.PI0.:V A0.0HI0.mHV Am.:I0.HV A0.®Io.mv N.H N.0 IIII MH.0 H.o 00.0 00.0 wampmdv m.H:m ®.mmm 0m.N :H.m ©.mH H®.m m0.» BOHb mSOSCHPGOO Sme pHSU< AmMMImmmv AomMImmmv Am.~IH.~V Am.wIm.mv A®.0mls.va Amm.0Imm.ov Am.mls.mv :.N m.m IIII mm.0 m.0 m0.0 No.0 AGOHPSGHSHHMV 3.0mm w.m:m 0m.» Hm.~ 0.0H m:.0 0H.: BOHm msodsHpcoo Ammmlommv AmNMImmmv Aw.SIm.sv A0.sim.mv Am.>mIH.Hmv Asm.HImm.ov oAm.me.mV o.m ®.w IIII 00.0 m.o no.0 sso.o Aoedeasv 0.0mm s.mmm 000.» m0.: H.mm mm.0 mmH.: OHpmpm . snag cameosse Amoomov mm moooov mm mm somhxo chapmsomsoe . Aamv Aaov pmog mo came spasaadxa< noosonem oo>aonnaa pawns: season Hobos oNHm anm . .mSOQQHs omonpmm GH EdHcoHOm mo QOHPmnHEHHo 0cm oxopmd wsHadmooE sow wows nope: pmbp 03p mo AH\wEv mOHpmHhopowsw£o HSOHmSQQ one HmOHSono 0cm anm 0:9 mo COHpmHsomom .0 OHQmB 3? egg cups made of nitex screening and 1 inch polyvinyl chloride pipe. The egg cups were suspended in 1 liter beakers which contained eight concentrations of sodium selenite ranging from 1 to A0 ppm. Each test concentration was duplicated once and 25 eggs were used in each test container. The water and toxicant were changed every other day and the temperature was maintained at 25 C by means of a water bath. An air stone was placed under each egg cup and allowed to bubble slowly to provide some agitation of the water around the eggs. The air was turned off after hatching was complete. The larval fish were kept in the same beakers after hatching to compare percent mortality with the controls. No attempt was made to feed the larval fish. Exposure System for Continuous Flow Toxicity Tests A proportional diluter (Mount and Brungs, 1967) was used to deliver 500 ml of test water per cycle to each exposure tank. Water for the diluter system was fed by gravity flow from a 70 gallon fiber- glass head tank where it was heated by 2 lOO—watt aquaria heaters to maintain a temperature near 15 C in all test tanks (Table 8). Test water flow rates averaged 125 ml/min for all toxicity tests except for rainbow trout fingerlings which averaged 150 ml/min. The replacement times (90%) were 7 and 9 hours, respectively (Sprague, 1969). These values agree with the replacement times suggested by Alabaster and Abram (1965) and Sprague (1969). The test tanks were screen—covered glass aquaria with water volumes of 30 liters. A11 aquaria were covered with black plastic and the entire diluter system was surrounded by plastic curtains to 33 protect the fish from any laboratory disturbances. Overhead fluorescent lamps provided light on a 13 hour/day photoperiod. Five concentrations of sodium selenate or sodium selenite and a control were used for each test, except when testing fingerling rainbow trout in which case only four concentrations and a control were used. Thirty fish were randomly assigned to each tank. At the beginning of each test an additional 25 fish of the same size and species as being tested were removed from the holding tanks and weighed and measured (Table 8). Egg—Alevin Exposure The effects of sodium selenite on rainbow trout eggs—alevin and coho alevin were tested for 31 days and A3 days, respectively. One hundred rainbow trout eggs, in the eyed stage, and 75 2—day old coho alevin were placed in plastic frame boxes (19 cm x 9 cm x 9 cm) with nitex bottoms and sides. The boxes were placed so that the water delivered to the exposure tanks flowed through the boxes and over the eggs and alevin. The rainbow trout eggs were in the boxes 8-10 days before hatching was complete. On the 11th day the number of rainbow trout alevin was reduced to 50 per box. The rainbow trout and coho alevin were held in these boxes for 20 days and then released into the test tanks. The eggs and alevin were protected from direct light by covering the tops of the test tanks with black plastic sheets. At the conclusion of the experiment, a subsample of 25 surviving rainbow trout fry from each test tank were weighed and measured. Mean weights and lengths were compared by Duncan’s multiple range test (Steel and Torrie, 1960). This was not possible with the coho fry because of the high mortality rate during the experiment. AmphipOd Exposure Immature HyaZZeZa azeteca were exposed to sodium selenite for 96 hours by means of a continuous flow delivery system. Thirty amphipods were placed in each of six plastic—frame, nitex—screen boxes of the same type as previously described. The amphipods were originally collected from the Red Cedar River at Michigan State University and cultured indoors for several months prior to this experiment. Fathead Minnows - Static Exposure §g_Se1enite—75_ Twenty juvenile fathead minnows were randomly assigned to each (3f eight 30 liter glass aquariums. The fish in four tanks were used ‘to measure uptake of selenite from water (Table 9) and those in the :remaining four tanks were used to measure elimination rates. One tank :in each set of four contained the control fish. The exposure concentration in all tanks was 0.083 ng/l (13.53 IlCi/l). During the 28 day period of exposure one half of the water iri each aquarium was replaced every other day. A sufficient amount cxf radioactiVe selenite was added to each exposure tank with the ITEplacement water to maintain a concentration of 0.083 mg/l. The radio— a£Itive water which was removed from the exposure tanks during this eJQperiment and was filtered on succeeding experiments through several la-Yers of charcoal and polyurethane foam before it was discharged into tkle sanitary sewer. At the end of the exposure period the fish saved for measurement (Df‘ elimination were placed in clean aquaria containing 30 liters of W"Euler and no selenite. Water was supplied at a rate of 125 ml/min and thus replacement time (90%) was 9 hours. Elimination Experiments Proportional diluters were uSed to deliver a mixture of radioactive selenite—75 and stable selenite to adult fathead minnows and fingerling rainbow trout. Flow rates, during the period of uptake and elimination for the fathead minnows, were maintained at 100 ml/min and 150 ml/min, respectively, and the replacement times (90%) were 12 and 9 hours, respectively. The flow rate and replacement time (90%) for the trout during the period of uptake was 150 ml/min and 9 hours, respectively. The diluter system was modified to deliver four duplicate concentrations, 0, 10, 25, and 50 ug/l for the fathead minnows. Five concentrations ranging from 0.22 mg/l to 0.95 mg/l, were tested with rainbow trout (Table 10). Sixty fathead minnows were placed in each of eight 30 liter tanks and 30 rainbow trout were placed in each of four 15 liter tanks. Ten ‘trOut were also placed in a 15 liter tank and used to measure the distribution of selenium in various fish tissues. Preparation of Stock Solutions Stock solutions were made with demineralized water and reagent gruade sodium selenate (NaQSeOh) and sodium selenite (Na2SeO3) (A. P. lViackay Inc. and Alfa Products, respectively). Stock solutions for all eJCperiments using a continuous flow delivery system were placed in a 7 liter mariotte bottle and connected to the diluter system as dfiscribedby Mount and Warner (1965). Two mCi of radioactive selenite—75 (H2Se03) (New England Nuclear) Were used to prepare the stock solutions for all uptake, distribution 811d elimination experiments. The half—life was 120.A days and the 36 A0.0A0 A0.mv Aom.mv Amm.AV . Amo.ov ersvon 00.AA 0e.m A0.m mm.A 0m.o : sovone eNoNNomm ssAoom Amm.A0 A00.00 nom.ov Aom.0v Aom.00 AAAAV mo.0 A 00.A 00.0 A mm.0 m0.0 A 0m.0 A0.o A sm.o A0.o A 0A.0 : sosAon 0000 : n00.AV Aom.0v Amm.ov AmmA.00 A00.00 AAAAV 0A.0 A 00.0 A0.0 A An.0 A0.0 A 00.0 Ao.0 A mA.0 no.0 A 00.0 : AsoAA eoserm s Amm.AV A00.00 nom.00 Aom.00 nom.00 AmsAAAomsAev A0.0 A no.0 A0.o A sm.0 no.0 A An.0 A0.o A Am.o A0.0 A 00.0 : AsoAA soosAem : Amm.A0 A00.ov nom.00 nom.00 nom.00 00.0 A 00.0 no.0 A nm.o no.0 A nm.0 00.0 A mm.o Ao.0 A 0A.o : AmAAssm AAAmosAm : A0.0HV A0.mv A0m.mv Amm.Hv Amm.ov opHGOHom no.0 A 0m.0 0m.0 A 0m.n mm.0 A 00.A 00.0 A m0.A no.0 A Am.o : : ssAeom no.0A0 A0.m0 nom.mv Amm.A0 n00.00 oAssvoo 0m.0 A 00.0A 0A.0 A mm.m SA.0 A 0e.m no.0 A mm.A no.0 A mm.0 A00.0 v goesAs oeoeAes ssAoom m n m m H Hoppsoo mmHoomm endomsoo msoflamno Pmoe me CH AH\wEV COHpmspsoocoo ESHGOHom .M003 0 coco Awesome @009 £000 scam oonhHmoo mo: OHQEom nope: onsHm < .momonpeosom cw one mmowpmhpmmocoo HmmHsoz .mpmop SHHOHEOP SOHm msodsHpqoo sow 00m: 90003 map QH A.m.m H HV ssflsmHmm mo moprapcmoqoo cums mae .QH magma 37 specific activity was 163 mCi/mg. Stock solutions were made by placing a known amount of stable sodium selenite in a 7 liter mariotte bottle and adding to this 7 ml of radioactive selenite—75, taken from a stock solution consisting of 2 mCi/l. The total volume was brought to 7 liters by adding deionized water. The selenite—75 contributed less than 0.001 percent of the total amount of stable selenium present and was considered insignificant. Sampling and Analytical Procedures Water Samples Water samples (200 ml) were collected weekly from each exposure tank, for all experiments except the static toxicity tests. Samples were placed in glass bottles and analyzed within 2—3 hours to minimize the loss of selenium due to adsorption (TablelO). Stable isotope analysis was conducted according to the colorimetric procedure (Cummins et a1., 1965) described earlier (Section I). Radioactive water samples were analyzed by counting 10 ml of water in a gamma spectrometer. Fish Samples Eighteen sets of fish samples were collected during the 28 day static exposure of fathead minnows to selenite—75. Each sample set consisted of A fish which were collected on days 0.5, 1.5, 2.5, 3, A.5, 5.5, 6.5, 7, 7.5, 9, 10, 11, 13, 1A.5, 18, 23, 25, 28. During the period of elimination 8 sets of samples with 8 fish per sample were collected on days 1, 2, A, 9, 16, 32, 67 and 9A. 38 The fish samples were rinsed with tap water and the whole fish placed in counting vials. Standards which occupied approximately the same space and provided a constant geometry were used to determine the concentration of selenium in the fish. After the fish were counted they were scraped with a knife to remove the slime and then recounted to determine the concentration of selenium in the slime by difference. During the period fathead minnows were expOSed to stable and radioactive selenite, 10 sets of samples were collected on days 1, 2, A, 8, 16, 2A, 32, 6A and 96. Each sample consisted of 3 fish from each of the duplicate concentrations. Eight sets of samples were collected during the period of elimination on days 2, A, 8, l2, 16, 32, 6A and 96. Rainbow trout which died were rinsed with tap water, wrapped in aluminum foil and frozen until the completion of the experiment at which time they were analyzed for selenium content together with those fish which survived the exposure period. The 10 larger trout were dissected into the following tissues: brain, gill, head—tail, heart, entire intestine, kidney, liver, muscle, pyloric caeca and Spleen. Blood samples were also collected from each fish prior to dissection by severing the caudal peduncle and quickly collecting three or four drops of blood in a counting vial. The smaller rainbow trout and fathead minnows were dissected to provide samples of muscle, gill, entire viscera, and remaining head and tail. Fish tissues were weighed and placed in counting vials containing 5 ml of a 2:1 mixture of nitric and perchloric acid.' The vials were capped, placed in a water bath at 60 C for 12 hours and then allowed to 6 39 cool to room temperature. The volumes were brought up to 10 ml with the acid mixture. Samples which were incompletely digested were reheated for an additional 2—3 hours. The samples were then counted and compared against a 10 ml standard.‘ Analytical Procedure Radioisotope analysis was performed with a Nuclear-Chicago 512 channel gamma Spectrometer equipped with an automatic sample changer and a sodium iodide, thallium activated detector. All samples were counted for selenium 75 activity (at the maximum energy peak of 0.A65 MeV) with constant geometry, compared with calibrated standards and corrected for physical decay and instrument efficiency. Counting times sufficient to give 95 Percent statistical reliability were used for all samples (Seelye, 197A). The results of all selenium analysis are reported as the concentration of the element selenium rather than the respective compounds. RESULTS Static Toxicity Tests The toxicity of sodium selenite to fathead minnows was directly related to water temperature (Figure A). The average 96 hour L050 values for sodium selenite with fathead minnows at 13 C, 20 C and 25 C were 10.9, 6.7 and 2.8 mg/l, respectively (Table 11). These data are not conclusive, however, because the period of exposure was too short and the exposure concentrations were greater than the asmyptotic L050 for sodium selenite. Fathead minnows were also exposed to sodium selenate (15 C) and the average 96 hour L050 was 11.8 mg/l. A comparison of the L050 values for the two selenium compounds suggests that sodium selenite is more toxic than sodium selenate. The results of static toxicity tests conducted with rainbow trout suggest that 96 hours is not a sufficient period of time to adequately determine the asmyptotic L050 for sodium selenite. The average 96 hour and 120 hour L050 values were A.35 mg/l and 2.72 mg/l, respectively (Table 11). A further decrease in the L050 value would be expected if the tests were conducted for a longer period of time. The above data also suggest that rainbow trout are more sensitive to sodium selenite than fathead minnows. Sodium selenite, at concentrations ranging from 1 to A0 mg/l, had no effect on the hatchability of fathead minnow eggs with an average of 99.1 percent of the eggs hatching (Huckabee and Griffith, 197A). A0 men w an 00.1736 own m e _. mm.H wo.mI®0.m 0m.: < mH mpsosp sononm : 0®.m .m 0H.w 0m.mI~:.H 0m.m m : : mm.H H0.:I®w.m 0:.m < mm SossHs emonpmm : 00.0 .m omH wwééfim 0nd. m : : Pm.H 00.0I0H.m 00.0 4 0m BOQQHE emonpom : 00.0H.w wn.H . mm.mHImn.m 0m.HH m : : 0m.H ww.mHIsm.w 0m.0H d MH BoscHs ooonpnb OPHQOHom ssHoom m>.HH.W nn.H me.nHImm.oH 0m.mH o .. : wN.H m:.mHIms.m 00.HH m : : 0m.H n0.:HImm.m 00.HH < mH SocsHs owonpmm oposoHom EdHeom Amv Hm>sopsH AH\mev opmoHHmom A00 mOHOOQm ecsomsoo oQOHm oocopHmsoo OmDH manpmnogsme Rmm an 00 pmoe .mpmop SAHOHxOP OHpmpm an psosp sochmn 0cm meochs ewonpmm ans oocHEAopoo opHcOHom ESHUOm .HH cHnt 0cm opmsoHom ESHUOm mom modHo> oQOHm can mHm>n0psH mosopHmcoo .mode> omoq Ado: 00 one A2 .msSon 0mH one 00 pm Pmop thOonp oemm onp Eosm popmHSOHmo moSHm> QmQH Dam mew w NH.H J©.N|Pm.m WLI.N m : : mm.H HH.MImm.m 00.m < mH deosp SopsHmm opHsoHow ESHUom Amv Hm>smpsH AH\msv omeHHmom A00 mOHoomm ossomsoo OQOHm oosoonsoo OmQH oedemaomsoB end . AsIod Anon SS A0.Asoov HA onsee. Figure A. Relationship between temperature and the static 96 hour L050 for juvenile fathead minnows exposed to sodium selenite. 96 Hour LC 50 [mg/l] 124 10- ° y: 2049-070 r2: 0.96 10 15 20 25 Temperature AA and Griffith (197A) and Niimi and LaHam (1975) have also reported that selenium has no effect on the hatchability of carp and zebrafish eggs at concentrations up to 5 mg/l and 10 mg/l, respectively. The concentra- tion of selenium in this experiment did, however, significantly reduce the incubation time of the eggs at concentrations of 15 mg/l and higher (Table 12) and caused a significant reduction in the post-hatch median survival time at 1 mg/l and higher (Table 13). Niimi and LaHam (1975) did not find an increase in mortality until zebrafish larvae were exposed to at least 3 mg/l of selenium dioxide (SeO ). This 2 compound in water would also form selenite (H Se03) suggesting that 2 zebrafish are not as sensitive to selenite as are fathead minnow larvae. The toxicity curve, presented in Figure 5, is not asmyptotic with the time axis suggesting that additional mortality of the fathead minnow' larvae would occur if concentrations less than 1 mg/l had been used. Continuous Flow Toxicity Tests With Fish Fathead minnows were exposed to sodium selenate and sodium selenite for A8 days and the LC50 values were found to be 2.A8 mg/l and 1.08 mg/l, reSpectively (Table 1A). Both values are considerably lower than the comparative static L050 values and they agree with the initial finding that sodium selenite is more toxic than sodium selenate. This has also been demonstrated by Franke and Moxon (1936) using rats and by Kumar and Prakash (1971) with blue-green algae. The extended period of time during which mortality occurred (Figures 5 and 6) indicates that selenium is accumulative as has been suggested by Gortiner and Lewis (1939) and Niimi and LaHam (1975). This same phenomenon has also been described by Pickering (1972) with cadmium and bluegills. A5 Table 12. Percentage hatch of fathead minnow eggs exposed to sodium selenite. Initial exposure began at 2 days age (50 eggs per concentration). Selenium % Hatch, Hours After Exposure Median Concentration Incubation (mg/l) ' A8 72, 8A 96 120 Time (hrs) Control 0 0 A 50 100 96 1‘ O O Q 28 100 99 5 0 0 0 A8 100 98 10 0 0 0 A0 96 97 15 0 A8 100 73 20 0 8A 100 57 25 0 8A 96 57 30 0 AA 100 7A A0 56 100 A7 A6 NA 00A II on on nA 00A 00 0N om AA 00A II no Nm mN mN 00A N0 II on NA 0N mm 00A 00 II nn 0N mA N0 00A N0 N0 0m 0N 0N II n 0A m0 00A 00 on NA NA NA II 0 m 0NA 0m on Nm nN 0A NA NA II 0 A 00A 00A 0n 0m 0A 0A NA NA NA NA NA II 0 AoAAsoo Anwev em: noN ANA 00A NmA 00A N0 no 00 on em nN NA AA\ws0 nopmnipmom soHpoppsoouoo noose AoAA< 0A000 ssAevom .AsoHpmspcoosoo pom ham OmV opHGOHom esHoom Op oomomxo ham SoosHs escapee mo Aemzv oer Ho>H>H5m soHuoE one thHmpnos psooaom .mH OHQNB A7 Table 1A. The LC50 values, confidence intervals and slope values of two selenium compounds with three species of fish and one invertebrate determined by continuous flow toxicity tests. Number of L050 95% Confidence Slope Compound Species Days (mg/l) Interval (mg/l) (8) Sodium Fathead A8 2.00 1.56—2.56 1.66 selenate minnow " HyaZZeZa A 0.76 0.A9—1.l9 2.11 azeteca Sodium Fathead A8 1.08 0.99-1.18 1.20 selenite minnow " Bluegill A8 0.A0 0.35—0.A6 1.16 " Rainbow A8: 0.50 0.A7-0.53 1.10 trout 96 0.28 0.26—0.30 1.07 " Rainbow 21 0.A6 0.A3—0.A9 1.19 trout (fry) " Coho A3 0.16 0.15—0.17 1.15 salmon (fry) a’bBoth values were calculated from the same test after A8 and 96 days of exposure. A8 oeAA one .98 SD 00013.“ was .opHnoHom ESHHVOm O0. pomomxo ON>HH BossHs 000030.“ .8 003.9 HN>H>HSm 9300: .m oAsmAs l O 1- N ll I—o—I -0 1' S l- / l-O-l -$2 I-O-I Ho 'Q’ -<") ~01 l A I I 1 I l I | O O O O O O CO I!) N O) (D (‘0 1'- 1- 1- snII MHBHOW lUSOJad 09 019W”. Concentration of Selenium [ppm] 0S9 .0h0 he 00PPH0 003 0eHH .kaCQHe 000:900 0HH20>sw Op 0pms0H0m adHeom mo thOHAOP 039 so oer mo #00000 one .0 0HSmHm 30 40 50 I _O N .O ‘— ”l0 '1’ '(O F—.——' "N l l I T I I O O 10 1' C’) N N ‘- u/BIuI sameA 0931 -as to uoneuuaouog Time Idaysl 50 To determine the toxicity of selenium to another species of fish, bluegills were exposed to sodium selenite for A8 days and the L050 value was found to be 0.A0 mg/l. This value is lower than the A8 day LC50 value obtained for fathead minnows suggesting that of the two species bluegills are the most sensitive. The above tests were terminated when it appeared that no additional mortality would occur, however, the toxicity curves (Figures 6 and 7) are not asmyptotic with the time axis suggesting that A8 days is not a sufficient length of time to determine the asmyptotic LC50 of selenium. To further investigate this and to determine the toxicity of sodium selenite to an additional species of fish, rainbow trout Were exposed to sodium selenite for 96 days. The LC50 values after A8 and 96 days were 0.50 mg/l and 0.29 mg/l, respectively (Table 1A). The L050 was decreased by almost one half by extending the period of exposure an additional A8 days. These data suggest that the length of time required to determine the aSmyptotic L050 should definitely be longer than A8 days and probably longer than 96 days (Figure 8). For the three species tested, the data indicates that fathead minnows are the least sensitive and rainbow trout are the most sensitive to sodium selenite. During the course of conducting acute toxicity tests with both selenium compounds a series of symptoms were observed in the fish which lasted for a period of one to two weeks prior to death. The first symptom observed was a pronounced swelling of the abdomen followed by an obvious swelling of the entire mid—region of the fish. This condition was followed by exopthalmia and the distension of scales along the lateral line. Slight to severe hemmorrhage along the ventral midline and in the branchiostegal regions was frequently observed 51 Figure 7. The effect of time on the toxicity of sodium selenite to juvenile fathead minnows. The line was fitted by eye. III. I . A _ A J _ 10 1 1 :\mE_ mo:_m> onOnnow no :ozmtcoocoo __ 987654 3 2 1 10 Time Idaysl Figure 8. The effect of time on the toxicity of sodium selenite to fingerling rainbow trout. The line was fitted by eye. Concentration of Se-LC5O Values [mg/ll 1.0~ 0.8- 0.6 - 0.4 - I—I\I\§ \EI‘I-I l | I I I I l l 20 40 60 80100 Time [days] prior to death. Post mortem examination revealed additional signs of hemmorrhage in the lining of the peritoneum and accumulation of excess fluids in the peritoneal cavity. The liver and spleen appeared pale and the intestines and stomach appeared in a degenerative state. The appearance of these symptoms was directly related to the concentration of selenium in the water and the length of the exposure period with the fish at the highest concentrations being the first to show signs of selenium poisoning. These symptoms were most pronounced in the fathead minnows with approximately 90 percent showing at least the initial symptom of abdomenal swelling. This was true for both sodium selenate and sodium selenite. Bluegills generally showed abdomenal swelling, but less than 50 percent showed any signs of hemmorrhage. Rainbow trout fingerlings and fry and coho fry showed the same symptoms, but to a lesser degree with only approximately 30 percent of the fish showing signs of hemmorrhage. Rainbow trout eggs, in the eyed—up stage, were exposed to sodium selenite. After 10 days of exposure hatching was complete and ranged from 92—97 percent with no significant differences between any of the concentrations. The period of exposure was terminated 21 days after hatching was complete because of excessive mortality of the control fish. This mortality was presumably due to the failure of the fry to begin feeding. The L050 based on the 21 days of exposure was found to be 0.A6 mg/l. Because of the short period of exposure this value underestimates the toxicity of sodium selenite to rainbow trout fry. The growth of the rainbow alevin—fry was adversely affected during the 21 day exposure period. A significant reduction in both the length and weight of the fish occurred at concentrations of 0.25 mg/l Se and greater. 5A Seventy—five 2—day old coho salmon alevin were placed in each of six tanks and exposed to sodium selenite for A3 days. The test was terminated at this time because 50 percent mortality had been exceeded at all exposure concentrations, except the control which had 17 percent mortality. The estimated A3 day LC50 value is 0.16 mg/l. While this is lower than any of the previously determined L050 values, it does not properly reflect the toxicity of sodium selenite to coho salmon. Had the coho larvae been exposed to lower concentrations over an extended period of time the aSmyptotic LC50 would have been lower. Fathead minnow larvae were also exposed to sodium selenite, however, the data are inclusive because of the high mortality in the control fish, but it does suggest that the LC50 for fathead minnow larvae is less than 0.1 mg/l. Invertebrate Exposure Immature amphipods were expOsed to sodium selenate in a continuous flow system for 96 hours with the resulting L050 value of 0.76 mg/l. The A and 1A day LC50 values for the same species using sodium selenite was determined in an earlier study (unpublished) and was found to be 0.3A mg/l and 0.07 mg/l. These values once again reflect the greater toxicity of sodium selenite and they Suggest that amphipods may be somewhat more sensitive to selenium than fish. However, this may not be true because 50 percent mortality was exceeded at all exposure concentrations with the coho larvae. It is quite probable that the asmyptotic LC50 for coho larvae is smaller than the observed LC50 value (0.16 mg/l) and it may be as small as the 1A day LC50 for amphipods. 55 A chronic bioassay conducted with Daphnia magna in an earlier set of experiments (unpublished) revealed the maximum acceptable toxicant concentration for sodium selenite to be 0.28 mg/l. The lA day LCSO was determined to be 0.A3 mg/l. These values are in general agreement with the LC50 values determined for bluegills and rainbow trout in this experiment (Table 1A) and suggest that daphnia are no more sensitive to selenium than fish. Uptake of Selenium — Static Exposure The uptake of selenium occurred in a linear manner throughout the entire 28 day period suggesting that a longer period of exposure is needed for equilibrium to be reached in the fish. Equilibrium, according to Macek (1975), may be defined as that time during the period of exposure where means obtained at three successive sampling periods do not statistically differ from each other and therefore indicate that. the rate of elimination equals the rate of accumulation. IThe uptake data are presented using a log transformation of the tissue concentra— tions to demonstrate the initial rapid period of accumulation (Figure 9). A log transformation of both the tissue concentration (ng/kg) and the number of days (x103) was used to facillate the calculation of an equation for the rate of accumulation. The mathematical expression employed to characterize the rates of uptake and elimination for this and all subsequent experiments was a simple linear regression of selenium concentration on a wet weight basis against the exposure time in days. The uptake and elimination data are described by the general regression equation of the form: Y = a + b (X) 56 Figure 9. The accumulation of selenium by juvenile fathead minnows during static exposure to selenite—75. Concentration of Selenium (ng/kg) 500 100 50 log y (ng/kg): —2.793 +1.928 r2=0.93 log XX‘IO3 I l l I I I .5 1 5 10 30 Time (days) 57 where: Y = the concentration of total selenium residue in the fish tissue a = the y—intercept of the regression line b = the rate of uptake or elimination (slope) x = the exposure time in days. The y—intercept, slope and coefficient of determination (r2) for the rate of accumulation using the described log—log conversion are —2.793, 1.298 and 0.931, respectively. The rate of accumulation of selenium may be expressed using the slope value, as 1.928 ng/Kg of body weight per (log) day (x103). Bioconcentration factors were determined by dividing the maximum residue concentration (mg/Kg) by the mean concentration (mg/l) of selenium in the water during the total period of exposure. The maximum bio— concentration factor determined during this experiment was A,AA3. This occurred on day 28 when the average whole body selenium residue in the fish was 368.7 ng/Kg. This large bioconcentration factor suggests that selenium is readily taken up by fish when present in the water in extremely small amounts. Of the total amount of selenium in the fish only 6.36 i 0.70 percent occurred in the slime. This low percentage of selenium in the slime plus the large bioconcentration factor suggests that selenium is not merely adsorbed on the fish but is absorbed across the gill membranes, assuming that uptake via the gastrointestinal tract is minimal. Fish which Were used to measure uptake and elimination were kept in separate tanks and on day 28 when the last set of samples for uptake and the first set of samples for eliminatiOn were analyzed the fish collected for measurement of elimination were found to contain significantly 58 less selenium than the fish sampled for uptake (Figures 9 and 10). This difference is due to the fact the average concentration of selenium in the water was 0.083 i 0.001 ng/l for the tanks designated for the uptake study and only 0.075 i 0.005 ng/l in the tanks designated for measurement of elimination rates. The different selenium concentrations in the two sets of tanks resulted from the fact that during the period of uptake 18 sets of fish samples were collected from the tanks designated for measurement of accumulation, but no fish were collected from the tanks designated for measurement of elimination rates. Elimination was measured over a 96 day period and occurred in a curvilinear manner. In order to describe the rate of elimination a log transformation of the time axis (days) was used so that a straight line could be fitted to the data by linear regression analysis (Figure 10). The y—intercept, slope and coefficient of determination for this regression is 119.89, —51.09 and 0.95, respectively. Calculation of the biological half—life, the time for 50 percent of the selenium residue to be eliminated, was done graphically and by substituting 50 percent of the calculated initial body burden as ng/Kg into the regression equation. The half—life was found to be 10.3 days. Using the calculated negative slope for the regression equation, the rate of elimination may be expressed as 59.09 ng/Kg of body weight per (log) day. The amount of selenium remaining in the fish after 96 days was 13.05 ng/Kg, 10.3 percent of the initial amount. At this time the elimination curve was asmyptotic with the time axis and no further significant elimination of selenium was expected. 59 Figure 10. The elimination of selenium by juvenile fathead minnows after static exposure to selenite—75. Concentration of Selenium (ng/kg) 140‘ 120 ~ 100 - 80- 60‘ 40- 20- y=119.89-59.09|og x+1 T r2: 0.95 T1/2 : 11 Days I' l I i I it 2 ('3 4 5 10 2'0 3'0 4'0 5'0 9'0 — Time (days) 60 Uptake, Distribution and Elimination 9f_Selenium — Continuous Flow Exposure System The mean concentrations of selenium in the duplicate tanks during « the periods of exposure were compared by Students t test (P = 0.01) and no significant difference was found. The average concentrations of selenium in the water during the period of exposure were 11.57 i 0.A2 ug/l, 2A.A2 i 0.8A ug/l and 50.57 i 1.A9 ug/l. These concentrations were selected because it was thought that information on the exposure of fish at natural levels would be of aid in evaluating the concentra- tions found in the muscle tissue of fish collected from Lake Erie (Table 5). The concentrations of selenium used in this experiment were not lethal to the fathead minnows although there was some initial mortality, less than 5 percent in all treatments and controls. No significant difference was found between replicate fish samples collected during the periods of accumulation and elimination. The data was combined and the results are presented as the mean of six samples for each fish at each sampling date. The accumulation of selenium occurred in a curvilinear manner in the whole fish and all tissues with a rapid period of accumulation occurring in approximately the first 8 days and a slower rate of accumulation occurring during the remaining 88 days. The data for the whole fish and individual tissues are presented using a log trans- formation of the tissue concentrations in order to provide an accurate description of the initial rapid period of accumulation (Figures 11—15). In each tissue it appears that the equilibrium concentration was being approached after 96 days of exposure. However, the data does not adequately meet the previously stated definition of equilibrium 61 .msogfls emwspmm Panes mo mnmomfir map Ga gasoaom .Ho soapmadfisoom meg .HH madman _m>oo_ oEC. mm two 0.0 om ow om _ p _ _ \\ .553 .. Imam“ o :maom o mcoszcoocoo oczmoaxm ON or F 0.0 -O.m [Bx/6w] wngue|es 10 uonenuaouoo 62 .msossfls emonpwe vases mo maaam map sfi saflsoaom mo noHpeadsdooo one .ma undone .232 9:: 0m two 00 o_m OW o.m CW 0% I . Pod uNod :msow I [no.0 {mama o . Canon 0 .bywoo acetateoocoo [mod ohsmonxm O -—.o O 0 1N6 [Md 0 IV.O O -md -50 [fix/6w] LunguaIas JO uonenueouog 63 .msosnfls pmogpdw vases wo Hams. was com: 9:. CH Eflsoamm mo soapoadsdoom one .ma mesmna .32: we: mm: mo 00 om ov on CW or mt .: r _ _ . mood _ — 600.0 .3: or I a .3: mm o . #06 In: an o .. 2029. 2.00.30 oasmoaxm rNOd .mod [moo Tho -Nd -od 10.0 [Bx/6w] wngua|es J0 uonenuaouoo 6h .msossas . eamepan panes mo maomds may 2H Edam . . maom mo so Hemassdoom 0:9 .:H onsmam we we em on _\m: up u to: mm o _\m:om o _m>~u_me_p ow om w:0_umhucwocoo 0.500qu ON or p . wood -mood .wod -mod ero rfio -md .md [Bx/Bun wngua|as to uoneituaouoo 65 .msosnfls pomnpmw waned hp Edflsoamm mo soapmassdoom hooplmaonz .3 933a mm mo 00 on :91: .. :9. mm o :9. cm 0 mco_.m::oo:oo ocamonxm _m>~u_ws_p ow om ON mw or m o— _ _ mood -mood -Nood -FQo - MOO -mod +~Qo [MO -md .NO [Bx/6w] wngua|as 10 uonenuaouoo 66 concentration and therefore suggests that the time for true equilibrium to occur is longer than 96 days. It does appear, however, that if one additional set of samples had been collected at the sampling interval of 32 days, that equilibrium would have occurred at 128 days. A log transformation of both the tissue concentration and the sampling time was used to calculate a regression equation which accurately describes the rates of accumulation for each tissue. Based on this log—log transformation the rate of accumulation of selenium for the various tissues can be expressed by their respective slope values which are presented in Table 15 together with the confidence intervals on the slope values, y—intercepts and coefficients of determination. The viscera consistently accumulated a greater amount of selenium than the other tissues regardless of exposure concentration. The largest concentration of selenium that occurred was 2.AA mg/Kg in the viscera of fish exposed to 50 ug/l for 96 days. The viscera also showed the largest amount of variability. Part of this variability may be due to the amount of selenium adsorbed on the food and would reflect the individual feeding habits of the fish. Sandholm et al. (1973) have demonstrated that a commercial fish food (Tetra Min) is capable of adsorbing selenite—75 from water in significant amounts over a 2A hour period. To reduce this variability and to minimize the uptake of selenium via the gastrointestinal tract the fish were fed only once a day. The food was usually consumed within.a few minutes after feeding. The possibility that some unknown amount of selenium may have been adsorbed through the gastrointestinal tract, however, cannot be entirely dismissed. 67 .mhop mm mo owopmcfl enamomxm mo mace mw poems popoadoamo .x no a mo GOflmmoamop nomqfla one Mom cowpwswammpmo mo pcofloflwmooo Q m o.NH :mm.o mwm.on zom.o H:N.H om : m.ma Nem.o Nam.on maw.o :No.a mm : N.mm mmm.o m:m.on mom.o smsyo OH Sway oaonz m.oa mwm.o mmm.on mmm.o osm.o om : w.:a wwm.o mam.on wmm.o mew.o mm : o.>m :mm.o N:H.OH me.o :H:.O OH Hamplowom e.m: mmw.o mmm.on was.o see.a cm : Q.o.mm wa.o wmm.on mw~.o mos.H mm : 0.0:H w:w.o em:.on www.o :Hm.H OH whoomfl> w.aa mom.o mm:.on mmm.o mmq.a cm : m.sa mmm.o wmm.on mmm.o :mm.a mm : 0.2m mam.o mwm.on mew.o ONH.H OH HHHO w.w Hmm.o wma.on HNQ.H m:m.o 0m : w.m mwa.o mmo.on Hmm.o H:m.© mm : o.wH Nem.o Hmo.on emo.a mwa.o OH oaomsz movemm ANMV omoam so Ammoamv Amx\w1 woav Aa\wnv odmmfle coapmnpqoosoooflm coaponflsnopom Hm>mmpsH coaumassdood pmmohopsH w coapmspooosoo omo pcoflofimmooo mosoeflwcoo mo 09mm madmomxm . paoopmm mm . .mhmw woa pmofimwm Amx\m1v qoflpmnpqoocoo moa wcfippoam an pocflopno one? mosam> sowmmopmom .mhop om pow afifleoamm mo mcoflpmppcoonoo oopnp Op pomomxo msossfle poonpmm CH Edflsoaom mo coHPmHSESoo< .ma mapoe 68 The maximum concentration of selenium in the gill, head—tail, muscle and whole fish was 0.58, 0.5A, 0.AA and 0.60 mg/Kg, respectively, and occurred after 96 days of exposure to 50 ng/l of selenite (Figure 16). The largest concentrations of selenium were consistently found in the tissues of the fish exposed to 50 ug/l Se and they were signifi- cantly greater than the residue levels in the tissues of the fish exposed to 10 ug/l Se. These data suggest that tissue residue levels are directly related to the exposure concentration. However, by using the previously described log—log transformation for the data in Figures ll—l5, it was found that the three regression lines for each tissue were not significantly different. Therefore, there is not i enough evidence to definitely conclude that exposure concentration influences the tissue concentration, but the data do suggest this. This view is further supported by comparing the whole—body accumulation of selenium by the fish in both this and the previous experiment (Figures 9 and 15). The concentration of selenium in the muscle of fish exposed to 10, 25 and 50 ug/l for 96 days was 0.18, 0.25 and 0.AA mg/Kg, respectively. These values are smaller than the residue levels found in most of the fish (0.7A mg/Kg) analyzed from Lake Erie even though the exposure concentrations were somewhat larger than the reported levels of selenium in Lake Erie waters (Tables 2 and 5). 0n the basis of this experiment it does not appear that the concentration of selenium (as selenite) in the water can entirely account for the residue levels in the Lake Erie fish. Data supporting this view has also been reported by Sandholm et a1. (1973). 69 .H\ma Om one mm «OH mo msoflpdnpcoosoo pm opflnmaom Edflpom Op ondwomxm MO made mm nwpmm mSOGsflE pomnpmm paged mo hoonloaozs and moSmmflp emu ca A.m.m a HV Edfismaom mo soapmhpcoosoo owmpo>m 0mg .ma madmaa (fix/6w) Lungua|as so uoulenuaouoé VISCERA WHOLE FISH GILL HEAD-TAIL MUSCLE 4000 o 0' 0/0‘ 0 O O 1000 0 ° 0 0.083 ng/I A 10.0 ug/l 1 . 25.0 ug/l . 50.0 ug/l Factor \0 100— Bioconcentration l I I IJ¥OI 20 30 40 50 60 65” 96 Days m- H- O 73 Figure 18. The elimination of selenium by the viscera of adult fathead minnows. Concentration of Selenium (mg/kg) 2.. 2.2 2.0 - 1.8 - 1.6»- 1.4- 1.2 — 1.0- 0.8- 0.6 - 0.4 T 0.2- _Exposure Concentrations O 50 ug/l O 25 ug/l I 10 ug/l \. 10 20 I 30 I I T 40 go 60 70 Tim e (days) I 80 T I 90 96 7h Figure 19. The elimination of selenium by the gills of adult fathead minnows. 0.8m Exposure ‘ Concentrations 0.71 o 50 ugll 0 25 ugll I 10 ugll .0 or I .0 on 1 0.31 Concentration of Selenium (mg/kg) 0 h l .0 N l 0.1- I I I I I I 1 I I If 10 20 30 40 5O 6O 7O 80 90 96 Time(days) 75 Figure 20. The elimination of selenium by the head and tail of adult fathead minnows. Concentration of Selenium (mg/kg) 0.7 l- Exposure ConcentraHons 0'6.“ o 50 Ug/I | _ _ O 25 ugll I I 10 ugll 05- 0.4. - 0.3- 0.2- 0.1- 1 I I I I I I ' I T I— 10 20 30 40 5O 60 70 80 90 96 Time (days) 76 Figure 21. The elimination of selenium by the muscle of adult ' fathead minnows. Concentration of Selenium (mg/kg) T 0.5- Exposure Concentrations 04 N O 50ug/l ' ‘ l 0 25ug/l I 10ug/l 0.34 “ 0.2- 0.1T Time (days) '77 ' Whole—body elimination of selenium by adult fathead minnows. Figure 22. Concentration of Selenium (mg/kg) 07‘ 06 Exposure Concentrations o 50 ug/l 0 25 ug/I - 10 ug/l l I I l' I I I I l I 10 20 3O 40 50 60 70 80 90 96 Time (days) 78 The rate of elimination of selenium from the viscera was found to be significantly faster than the other tissues. The viscera had an average half-life of 5.1 days as compared to half—lives generally in excess of 50 days for the other tissues (Table 16). This suggests that the rate of exchange of selenium between the internal organs, including the liver, spleen and kidney, with the plaSma is more rapid and complete than in the muscle and gill. The elimination of selenium from the muscle and whole fish, as depicted by their half—lives, appears to be inversely related with the exposure concentration. This relationship is not apparent in the other tissues, however, a similar relationship has been reported by Lopez et a1. (1969) for sheep after administering selenite-75 both orally and intravenously at four different concentra— tions. Accumulation and Distribution of_Selenium ip_Rainbow Trout The largest concentration of selenium occurred in the viscera followed by the gill, head—tail and muscle. This same relative distribution was also found in fathead minnows in the previous experi— ment. The exposure concentrations of this experiment were approximately one order of magnitude larger than were used in the previous experiment and the residue levels in the trout remaining alive at the end of the exposure period were significantly higher than found in the fathead minnows (Table 17). This is consistent with the previous data which suggest that the accumulation of selenium is directly related to the exposure concentration. However, the average concentration of selenium in the tissues of the trout exposed to 0.Al mg/l and collected alive was .oHSmomxo mo made mo mmqomo AmN.ov Apo.ov ANH.OV Ao:.ov Awm.mv mm.m mm.o mm.H m:.: wN.mH P 0>HH< 0.00 Hm.o Awo.ov Amo.ov Aoa.ov flow.ov Aam.ov AmeOmv m~.m NN.H om.H mo.m wm.ma om econ o.os Hm.o Awa.ov Aaa.ov A:H.ov Amm.ov Aas.av m:.N Hw.o mm.a mm.N mm.ma NH obfla< o.mm H:.o $.98 8a.“: 8:: Schov ANTS Salt: mu m:.m mm.H mm.N mm.: m:.ma w coco m.m~ H:.o 3:: 21.8 STE 898 $98 87.3 om.m oo.m om.m m:.: o:.w NH econ m.wa Fm.o Asa.ov Ama.ov Amm.ov AHm.oV Amm.ov wflmalmav mw.m pm.a Hm.m om.m om.w PH comm 0.:H mo.o swam 3oz: 38:: 33.3% :8 808; sag Sea 323 SEE mo madmomxm soapospsoosoo AmM\wsv mmdmmflB one Ga Edflsmaom mo coapmnpsoosoo new: ponsdz mo oSHB ondwomxm omono>< .momoepnosom nfi 0am mnoaao panesspm .sdfisoaom mo mQOHpenpsmonoo snow on pomomxo eachp sonsfios mo mmdmmflp one Ca Edflsoaom go soaponpsoosoo owoho>< .NH canoe 8O ' lower than the concentration in the trout exposed to 0.31 mg/l, although the difference is not significant. The concentration of selenium in the tissues of the dead fish were generally higher than in the fish remaining alive at the end of the experiment, except for the fish exposed to 0.31 mg/l. At all concentra- tions the muscle contained significantly less selenium than any other tissue and the muscle of fish collected alive contained significantly less than the muscle of the fish collected dead. The average concen— trations of selenium in the muscle of the fish collected alive and dead were 1.61 i 0.18 mg/Kg and 0.90 i 0.11 mg/Kg, respectively. The bioconcentration factors in the trout exposed to 0.31 mg/l for 96 days were 62.1, 1A.3, 6.3, 3.2 and 10.5 for the viscera, gill, head—tail, muscle and whole fish, respectively. The maximum whole—body bioconcentration factors obtained by fathead minnows in the previous two experiments and the rainbow trout in this experiment were A,AA3, 29.2 and 10.5, respectively, and the exposure concentrations were 0.083 ng/l, 50.6 ug/l and 0.31 mg/l, respectively. These data clearly show the inverse relationship between bioconcentration factor and exposure concentration and they demonstrate that biocon— centration factors may vary by several orders of magnitude as a direct result of different exposure concentrations. The largest concentration of selenium in the tissues of six rainbow trout (12 cm) exposed to a mixture of radioactive and stable selenite (0.22 mg/l) for A8 days occurred in the spleen followed by the liver and heart (Figure 23). Because of the minute size of the spleen and heart these tissues were analyzed collectively and it was 81 . .Qms was go coHpohpnoosoo m we opHnoHom EdHeOm op chamomxm mo when me seems pSOMP SODsHmM mo hooploHonB one modmme one CH A.m.m H av ssHsoHom mo QOHpmhpooonoo owoho>o 0:9 .mm 0.83m BIO—ILL! u._rn:: L>-_IO¢—O O.N Hm.o N a m.NH m.OH OmH : : : ms.m :m.o m z m.o m.m osa : = : ®:.m . ®P.O N .m m.+~H m.OH QJH : : : :m.m as.o N 2 o.NH 0.0H PJH : : : :O.M ®©.O N b J.©H O.HH QJH : : : NN.: 20.0 N z 0.0H 0.0H mzH : : : ww.m ww.o N z :.m o.m :JH : : : es.m sw.o m a e.ma m.oa mes : e e @H.m mH.H N S ©.®m m.mH mPH : : : sa.s mo.o m 2 o.oo m.wH sea : e : mo.m om.a m a w.oo m.wa mes : e = m:.m HN.H N .m m.H~. m.®H NEH : : : mm.m mm.H N a m.ww 0.0N HSH : : : mm.e so.o m z m.me o.wa oea : = e NN.: :m.o N 2 N.w~ 0.0H me : : : mm.m me.o m z e.om m.sa moa = z : sm.o me.H m 2 H.ms 0.0H sea : e : m:.m HN.H N z m.ow 0.0H me m>\mN\m H gonna 3OHHow mA.p3 aeov A.p3 pozv mHmaHdn¢ xom Asmv ASUV .02 ode soHpopw moHoomm and see no .oz unmade summon was doapooaaoo monsooHHoo .oHsm oon nympho: CH mGOHpoQOH me 50mm eopomHHoo anm QH soHsoHow mo soHponpnooooo .w< oHpoB 98 00. H mm o O N mm m . 0m 0. HH OHH : : : mm.N Nm.0 N 2 2.20 0.0H 00H : : : 2s.N H0.0 N 2 0.20 0.0H 00H : = : 0N.0 02.0 N 2 2.00 0.0H 20H e e = 0~.H 02.0 N z H.N0 0.0H 00H : : : mH.N ®J.O N E 0.2.1. m.~..H mOH : : : 02.H 02.0 N 2 N.2H 0.0H 20H = e : mH.N 02.0 m 2 0.02 0.0H 00H m2\0H\2 : : mH.N @210 N E NHJW O.N.H Nm : : : 22.H Nm.0 N m H.0s 0.0H H0 : : : 20.H H2.0 H z 0.20 0.0H 00 : : : 0H.0 mH.H H z 2.20 0.2H 0m : : : 00.m 00.0 H 2 0.22 m.mH 00 = e = 00.2 00.H H a 0.H0 0.0H Nm N2\0M\0 : : N2.0 0H.0 N m 0.2NH m.HN 0 N2\0H\0 m : 20.0 HN.0 H z N.N0 0.0H m0 : : : 2H.© NM.H H E ®.®N. 0.0H Nw : : : ww.H Fm.O H rm H.@© 0.0N Hm : : : m2.0 0H.0 H a 2.00H m.mN 00 = e s 02.0 0H.0 H a H.00H 0.20 02 e = = 02.0 0H.0 H 2 0.Hs 0.0H 02 = e e 2H.0 sm.H H 2 2.00 m.0H 22 = e : 0H.2 N0.0 H z 0.00 0.0H 00 : : : 00.2 HH.H H 2 N.m~ 0.0H m» : : : m0.m 00.0 H z 2.00 0.0N 22 : : : 00.0 02.H H 2 N.0m 0.2H ms : = : H0.0 0H.0 H 2 H.m0 m.sH Ne : e = 02.0 0H.0 H 2 0.02 0.0H H2 : : : N0.0 00.H H 2 H.00 0.0H 02 : : : 00.0 0N.0 H z m.H0 0.0H 00 mw\0H\2 N 20200 30HH02 .93 hnmv A.p3 pozv mHthoa< 200 A800 . AEoV .oz @900 SOHpopm moHoomm Ema Ema 00 .02 pan03 newcoq woe sOHpooHHoo wchooHHoo Ae_oeoov 0< odee Hm.H NM.O N I 0.0H O.HH :m : : : Hm.H 2m.o N I 0.2H m.HH m0 .. : : 20.H H2.0 N I 0.0H 0.NH N0 m2\0H\2 N HosHsm soEEoo 00.0 02.0 m E 0.0HH 0.2N NOH : . : adonp 0202 H0.N 00.0 H a 0.0H m.HH HOH .. ._ ._ 00.N 02.0 N z 0.00 m.2H 00H : : : 00.0 00.0 H z 2.0m m.2H 02H : : : 00.0 20.0 m m N.00 0.0H 02H : : : 0N.N Hm.0 m a 2.00H n.0H 22H : = : 2N.N 00.0 N E 2.00N 0.2N 02H m2\00\0 soazm .H : H®.m m®.0 N l :.HH 0.0H JWH : : : 0H.0 mm.H N , l O.mH 0.HH me : : : 20.H 22.0 N I m.NH 0.0H NOH : : : 20.2 00.H N I N.HH 0.0H HOH : : : 20.H H2.0 m I 0.NH 0.0H 00H : : : m:.0 0H.0 N I N.0H 0.0H QmH : : : 9.70 0H.0 N I 2.0H 0.HH me : : : m0.H m2.0 m 2 m.N2 0.0H 20H : = = 0H.0 mH.H N 2 2.0m m.0H 0mH e e e 9 00.H 02.0 m .m 0.2% 0.mH mmH : : : o. 02.0 2H.0 N 2 2.02 0.0H 2mH = e : 0m.N 00.0 2 m 0.02 0.0H mmH m2\mN\0 : : QW.N 00.0 H l mw.N2. 0.0H .Jm : : : $0.: Hm.O H l @211 m.©H mm NP\©N\m : : $0.: 220.H H I N.Wm O.®H PM : : : 0N.N Hm.0 H l ®.®2. 0.0H mm : : : MH.: NQ.O H I 0.H0H 0.0H :m : : : 0N.H 0N.0 H I m.2HH 0.HN mm : : : 20.2 00.H H I 0.H2H 0.mm Nm N2\0m\0 0 26200 soHH02 0A.93 220v A.p3 #030 mHmann< Now Aswv A800 .02 open QOHpopm moHoomm Egg Ema mo .oz panoz 290202 009 SOHPooHHoo moHpooHHoo Ae.pcoov 0< oHoha 100 0min 0m.o N I 0.HH m.OH N2H : : : m0.H 02.0 N I 2.0H 0.0H H2H : = : OH.m 02.. O N I H.NH 0.NH 02H : : : OH.m ©2..O N l w.HH 0.HH OMH : : : 2.0.H HJ.O N l J.JH m.HH QMH : . . : : ®®.H mJ.O N I J.MH m.OH 2.MH : : : NN.H Om.O N I 0.NH m.HH WMH : : : 2.m.N m©.O N I m.2.H m.NH mMH : : : 00.N 02.0 N I O.JH m.HH . JMH : : : m2.N 00.0 m I 0.HH m.HH mmH m2\mN\0 H sosHem HHoopoam @m.H OM.O N l w.MH m.HH QNH : : : ®®.H $2.0 N l O.MH 0.HH wNH : : : OH.H 2.N.O N I 0.NH 0.HH 2.NH : : : Hm . H NM . O N I 0. HH m . OH QNH : : : 00.H 02.0 N I 2.mH 0.HH mNH : : : M2..N 2.0.0 N l H.©H m.NH JNH : : : mm.H mm.O N l J.MH 0.HH MNH : : : Hm.H 2.M.O N I N.JH m.HH NNH : : : P©.H HJ.O N l J.JH m.HH HNH : : : mm.N N0.0 N I w.mH m.HH ONH : m : Qm.H mm.O N I m.m2 O.JH NOH : : : m®.N mW.O N I M.HH m.OH HOH : : : N®.H 2.2.0 N I W.NH O.HH OOH : : : NO.M J2..O N I J.HH 0.HH QQ : : : OO.N ©J.O N | w.MH 0.HH mm : : : JH.H wN.O N I J.2.H 0.NH 2.0 : : : N0.0 0N.0 N I m.mH 0.HH 0o : e e 00.H 02.0 N I 0.NH 0.HH m0 m2\0H\2 N .0an0 002250 02.03 2200 2.03 0020 ufimsHoo< 200 2200 Asov .oz 0200 eodepm uoHooam sag Ema .Ho .02 020H03 20.0002 00.H. 00Hpo0HH00 mano0HH00 20.2ooov 0< oHoea 101 NA mm.o 0H.0 m z m.owa m.2m NmH : : : mm.H mm.0 N 2 m.mNN 0.NJ HMH : : : HH.N m:.o m m m.mmw 0.HJ oma m2\mw\m : wmmaamz mN.: :w.o m I 0.2m 0.NH mad : : : WN.J J®.0 N I N.2.m m.mH OHH : : : mm.m 2m.o m I N.Hm m.wH FHA : : : mm.m 8. o N I QR 0.3 w: mth: m 3% @323 MN.m No.0 N m m.HHm m.mN mad : : = MN.W ON.H H 2 ®.2.NN O.®N NHH : : : mm.m mw.o H z w.wwm m.wm HHH : m : 2a.: ow.o N m m.HwN m.mN 2w m2\wa\q N mmmp mpflgz :2.m mm.o m 2 m.::m o.mm om : : : mw.N HN.0 N z :.me 0.2N aw : = : Hm.m ow.o m E m.mmH m.mw mm : w : 2H.: mm.o N z N.:NH 0.:N mHH m2\wa\n = = NN.m :50 a I Himm o.wN S Ntomg .. .. :o.m mm.a a I m.o~a m.mm m m2\wa\m m mgmo mw.: HN.H H I m.NNH m.wH m: N2\om\m m : mm.m om.H m I H.0mH 0.0m ma N2\NH\© : gmflmwaoo IIII MJ.H H l J.NH 0.HH NON : : : IIII Jm.H N I W.NH m.HH HON : : : IIII Na. O N I O. OH m . HH OON : : : IIII HJ . O N I 2. . NH m. OH OOH : : : IIII 22.0 m I 0.HH m.oH wma : : : IIII m2.o m I :.ma 0.HH 20H m2\mw\m m pmcflgm HHNppomm .93 hhmv “.93 away mfimhawq< xmm Aawv A800 .oz mpmo COprpm mmflowmm Sam Ema mo .02 pnwfiwz meamq me soapomaaoo waflpomaaoo Ac.pcoov m< manwe .moflummm comm Mom mHSpmfloS pamopwm mmwpm>w amp hp mmdam> pnmflms pm: muflhamflpade ha Umnflapno mam? mmdad> pgwfimz hhow 02.N mm.o N 2 m.MMH m.mN mm : : : Nm.m mm.o N 2 N.mmg m.:m Hm m2\mH\: N pwxosm mpng 02.. OH 00. H N I H. HO m. 0H JmH : : : Nw.m Hm.H N I 0.1m m.wH mmH : = = mm.» N:.H N I m.om m.mH NmH : : : mN.m mm.H N I m.mm o.NN HmH = = : Hm.m wo.H N I m.ow m.mH omH m2\mN\m : : MN.MH wJ.N H I 0.2.W m.2.H MN : . : : NN.mH mw.N H I 0.NHH 0.HN NN : I w : Hm.H mN.o H I H.2m m.2H Hm : = : m 8.3 8H H I ob mNH Om Ntoma .. _. 1 mm.2 w:.H H 2 c.20H m.NN mw N2\NN\® m = mJ. w ON. H N I O.H© m.2.H OQH : : : Hm.m wo.H N I 2.©m m.mH NNH : = = ::.m No.0 N I m.mmH m.:N NNH mp\mN\m H cmogmgmmgm JOAN J®.O N E m.MOH O.MN JHH M2.\®H\J : : m®.N HW.O H E m.ON O.JH Pm : : : Nm.: No.0 N 2 :.mm o.wH mm = = : HN.H om.o H z H.mN m.:H mm m2\mN\m m mamHHmz NA.pB hymv A.p> pmzv mflmhawn< xmm Asmv Aaov .oz mpma QOprpm mmflommm 5mm 2mm No .02 panmz Spwcmq mwe QOHHUNHHOQ mchomHHoo “N.Hsoov NH meme LITERATURE C ITED LITERATURE CITED Alabaster, J. S. and F. S. H. Abram. 1965. Development and use of a direct method of evaluating toxicity to fish. Advances in Water Pollution Research Proc. 2nd Int. Conf., Tokyo 196A. Vol. 1, pp. h—SA. Pergamon Press, Oxford. Akiyama, Akio. 1970. Acute toxicity of two organic mercury compounds to the teleost, Oryzias Zatipes, in different stages of develop— ment. Bull. Jap. Soc. of Sci. Fish. 36(6):563—570. Allaway, w. H., D. P. More, J. E. Oldfield and o. H. Muth. 1966. -Movement of physiological levels of selenium from soils through plants and animals. J. Nutr. 88:h11. American Public Health Association. 1971. Standard methods for the examination of water and Wastewater. 13th ed. A.P.H.A., New York. 87h pp. Anderson, M. A., H. W. Lakin, K. C. Beeson, F. F. Smith and E. Thacker. 1961. Selenium in agriculture. Agr. Handbook No. 200, U. S. Dept. of Agric., Washington, D. C. Ayers, J. C. 1970. Lake Michigan environmental survey. Great Lakes Research Division, Ann Arbor, Michigan. Special Report No. A9. Barnhart, R. A. 1958. Chemical factors affecting the survival of game fish in a western Colorado reservoir. Colo. Coop. Fish. Res. Unit Quarterly Rep. hz25. Beal, Albert R. 197A. A study of selenium levels in fresh water fishes of Canada's Central Region. Fish Mar. Serv. Tech. Rep. Ser. No. CEN/T—7h—6. Benoit, D. A. 1975. Chronic effects of copper on the survival, growth and reproduction of the bluegill (Lepomis macrochirus). Trans. Am. Fish. Soc. loh(2):353—358. Blicoe, C. 1960. Whole-body turnover of selenium in the rat. Nature 186:398—h05. Bowen, H. 1966. Trace elements in biochemistry. London, New York, Academic Press. 261 pp. 103 10A Bransen, D. R., G. E. Blan, H. C. Alexander, D. R. Thielen and W. B. Neely. 197A. Bioconcentration of 2,2',h,h'—tetrachloro— biphenyl in trout as measured by an accellerated test. Trans. Am. Fish. Soc. 10u(h):785-792. Brown, V. M. 1973. Concepts and outlook in testing the toxicity of substances to fish. Pp. 73-95 in Bioassay Techniques and Environmental Chemistry. Ann Arbor Science Publishers, Inc., Ann Arbor, Michigan. Burch, R. E., R. V. Williams and J. F. Sullivan. 1973. Tissue trace metal content of rats fed selenium with and without cobalt. Trace Substances in Environmental Health — VII, p. 257. 'Univ. of Missouri, Columbia, Mo. Cerwemka, Edward A., Jr. and W. Charles Cooper. 1961. Toxicology of selenium and tellurium and their compounds. Arch. Environ. Health 3:71—82. Cheng, K. L._ 1956. Determination of traces of selenium 3,3'— diaminobenzidine as selenium (10) organic reagent. Anal. Chem. 28(11):1738-17A2. Copeland, Richard A. 1971. Selenium: Ithe unknown pollutant. Limnos 3(A):7-9. Copeland, R. A. and J. C. Ayers. 1972. Trace element distribution in water, sediment, phytoplankton, zooplankton and benthos of Lake Michigan: a baseline study with calculations of concentra— tion factors and build up of radioisotopes in the food web.' Environmental Research Group Special Report No. 1, Ann Arbor, Michigan. ’ Copeland, R. A., R. H. Beethe and W. W. Prater. 1973. Trace element distributions in Lake Michigan fish: a baseline study with calculations of concentration factors and equilibrium radioisotope distributions. Environmental Research Group Special Report No. 2, Ann Arbor, Michigan. Cousins, F. B. and I. M. Cairwey. 1961. Some aspects of selenium metabolism in sheep. Australian J. Agric. Res. 12:927—9A3. Cummins, Lawrence H., J. L. Martin and D. Maag. 1965. Improved method for determination of selenium in biolOgical material. Anal. Chem. 37(3):A30—h31. DaiZc, J. H. and O. A. Beath, 1935. Observations on pathology of blind staggers and alkali disease. J. Am. Vet. M.A. 86:753—760. Eaton, John. 197A. Chronic cadmium toxicity to the bluegill (Lepomis macrochirus Rafinesque). Trans. Am. Fish. Soc. lO3(A):739—7A5. Fleming, R. W. and M. Alexander. 1972. Dimethylselenide and dimethyl- telluride formation by a strain of penicillium. Appl. Microbiol. 2hzh2A—A29. 105 Franke, K. W. and A. L. Moxon. 1936. A comparison of the minimum fatal doses of selenium, tellurium, arsenic and vanadium. J. Pharm. and Exp. Therap. 58:h5h. Franke, K. W. and E. P. Painter. 1938. A study of the toxicity and selenium content of seleniferous diets: with statistical considera— tion. Cereal Chem. 15:1—2A. Ganther, H. E., P. A. Wagner, M. L. Sunde and W. G. Hoekstra. 1972. Protective effects of selenium against heavy metal toxicities. Trace Substances in Environmental Health — V1, p. 2A7. Univ. of Missouri, Columbia, Mo. ' Ganther, H. E. and M. L. Sunde. 197A. Effect of tuna fish and selenium on the toxicity of methylmercury: a progress report. J. Food Sci. 39(1):l—5. Gortiner, R. A., Jr. and H. B. Lewis. 1939. The retention and excretion of selenium after the administration of sodium selenite to white rats. J. Pharm. and Exp. Therap. 67:358—370. Groth, D. H., L. Vignati, J. Lowry, G. Mackay and H. E. Stokinger. 1972. Mutual antagonistic effects of inorganic selenium and mercury salts in chronic experiments. Trace Substances in Environmental Health — V1, p. 2A7. Univ. of Missouri, Columbia, Mo. Hamilton, A. and H. L. Hardy. 19A9. Industrial toxicology, Ed. 2, New York, Paul B. Hoeber, Inc. 239 pp. Hashimoto, Yoshikazu, Jaey Hwang and Saburo Yanagisawa. 19A0. Possible source of atmospheric pollution of selenium. Environ. Sci. and Tech. A(2):157—518. Hansen, D. J., P. R. Parrish, J. I. Lowe, A. S. Wilson, Jr. and P. D. Wilson. 1971. Chronic toxicity, uptake and retention of aroclor l25h in two estuarine fishes. Bull. of Environ. Contam. and Toxicol. 6(12):ll3—ll9. Hazel, G. R. and S. J. Meith. 1970. Bioassay of king salmon eggs and sac fry in copper solutions. Calif. Fish and Game 56:121— 12h. Hill, C. H. 1972. Interactions of mercury and selenium in chicks. Fed. Proc. 31(2):692. Hill, C. H. 197A. Reversal of selenium toxicity in chicks by mercury, copper and cadmium. J. Nutr. 10h(5):593—598. Hoffman, 1., K. J. Jenkins, J. C. Meranger and W. J. Pigden. 1973. Muscle and kidney selenium levels in calves and lambs raised in various parts of Canada: relationship to selenium concentrations in plants and possible human intake. Can. J. Anim. Sci. 53(1): 61—66. 106 Hopkins, L. L., Jr., A. L. Pope and C. A. Bauman. 1966. Distribution of microgram quantities of selenium in the tissues of rats and effects of previous selenium intake. J. Nutr. 88:61—66 Hosseinion, M., T. T. Bazargani, J. Nahani, H. Mohammadiha and A. Owlia. 1972. Selenium poisoning in a mixed flock of sheep and goats in Iran. Trop. Anim. Prod. h(3):l73—l7h. Hoste, J. and J. Gillis. 1955. Spectrophotometric determinations of traces of selenium with diaminobenzidine. Anal. Chim. Acta 12: 158—161. Huckabee, J. W. and N. A. Griffith. 197A. Toxicity of mercury and selenium to the eggs of carp (Cyprinus carpio). Trans. Am. Fish. Soc. 103(k): 822- 82h. Johnson, Henry. 1970. Determination of selenium in solid waste. Environ. Sci. and Tech. u(lo):850_853. Kessler, T., A. G. Sharkey, Jr. and R. A. Friedel. 1971. Spark source mass spectrometer investigation of coal particles and coal ash. Bur. Mines. Tech. Prog. Rep. A2, U. S. Dept. of the Int., Washington, D. C. p. 5. Koeman, J. H., W. S. M. van de Ven, J. J. M. de Goeij, P. S. Tjioe and J. L. van Haaften. 1975. Mercury and selenium in marine mammals and birds. The Sci. of the Total Environ. 3:279-287. Kubota, J., W. H. Allaway, D. L. Carter, E. E. Gary and V. A. Lazar. 1967. Selenium in crops in the United States in relation to selenium—responsive diseases of animals. J. Agr. Food Chem. 15(3):uu8—h53. Kumar, H. D. and G. Prakash. 1971. Toxicity of selenium to the blue green algae, Aaacystis nidulans and Anabaena variabilis. Am. Bot. 35(lhl):697—705. Levander, O. A. and L. C. Argrett. l969. Effects of arsenic, mercury, thallium and lead on selenium metabolism in rats. Toxicol. Appl. Pharmacol. lh:308. Lindberg, P. and M. Siren. 1963. Selenium concentration in kidneys of normal pigs and pigs affected with nutritional muScular dystrophy and liver dystrophy. Life Sci. 5:326 Litchfield, J. T. and F. W. Wilcox. 19h9. A simplified method for evaluating dose~effect experiments. J. Pharmac. Exper. Therapeutics 96:99—113. Lopez, P. L., R. L. Preston and w. H. Pfander. 1969. Whole body retention, tissue distribution and excretion of selenium—75 after oral and intravenous administration in lambs fed varying selenium intakes. J. Nutr. 972l23-l32. 107 Macek, K. J., c. R. Rodgers, D. L. Stalling and s. Korn. 1970. The uptake, distribution and elimination of dietary th—DDT and luc- dieldrin in rainbow trout. Trans. Am. Fish. Soc. 99(h):689—695. Macek, K. J., M. E. Barrows, R. F. Frasny and B. H. Sleight. 1975. Bioconcentration of th—pesticides by bluegill Sunfish during continuous aqueous exposure. Pp. 119-lh2 in Symposium on Structure—Activity Correlation in Studies of Toxicity and Bio- concentration with Aquafic Organisms. International Joint Commission, Windsor, Ontario. Madison, T. C. 1860. Sanitary report — Fort Randall. Written Sept., 1857. in Coolidge, R. H., Statistical Report on the Sickness and Mortality in the Army of the United States, Jan. 1855 to Jan. 1860. (U. S.) Congr. 36th, lst Session, Senate Exch. Doc. 52:37-h1. McKim, J. M. and D. A. Benoit. 1971. Effects of long term exposure to copper on survival, growth and reproduction of brook trout (Salvelinus fbntinalis). J. Fish. Res. Ed. Canada 28(5) 655—662. McKim, J. A. and D. A. Benoit. l97h. Duration of toxicity tests for establishing no effect concentrations for copper with brook trout. J. Fish. Res. Ed. Canada 31(h):hh9—h52. Mickelsen, O. 1970. Selenium and cancer. Nutr. Rev. 28:75—80. Mount, D. I. and R. E. Warner. 1965. A serial dilution apparatus for continuous delivery of various concentrations of materials in water. U. S. Public Health Serv. Publ. No. 999-WP—23, 16 pp. Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Research l(1):21—30. Mount, D. 1. and C. E. Stephan. 1967. A method for establishing acceptable toxicant limits for fish - malathion and the butoethanol ester of 2,h—D. Trans. Amer. Fish. Soc. 96:185—193. Mount, D. I. and C. E. Stephan. 1969. Chronic toxicity of copper sulfate to fathead minnows in soft water. J. Fish. Res. Ed. Canada 26(9):2h50—2h57. Moxon, A. L. and Morris Rhian. 19h3. Selenium poisoning. Physio— logical Review 23(h):305—3h0. Muth, 0. H. and W. Binns. 196A. Selenium toxicity in domestic animals. Ann. New York Acad. Sci. 1112583—590. Nelson, E. M., A. M. Hurd—Karrer and W. 0. Robinson. 1933. Selenium as an insecticide. Science 78:12h—l30. Niimi, A. J. and Q. N. Lattam. 1975. Selenium toxicity on the early life stages of zebra fish (Brachydanio rerio). J. Fish. Res. Ed. Canada 32 803—806. 108 Pakkala, I. S., W. H. Gutenmann, D. I. Lisk, G. E. Burdick and E. J. Harris. 1972. A survey of the selenium content of fish from A9 New York State waters. Pestic. Monit. J. 6(2):lO7—llh. Parizek, J. and I. Ostadaloua. 1967. The protective effect of small amounts of selenium in subimate intoxication. Experientia 23: lh2—lh3. Pickering, Q. H. and M. H. Gast. 1972. Acute and chronic toxicity of cadmium to the fathead minnow (Pimephales promelas). J. Fish. Res. Ed. Canada 29(8):1099—1106. Pillay, K. K. S., C. C. Thomas, Jr. and C. M. Hyclte. 197M. Neutron activation analysis of some of the biologically active trace elements in fish. J. Radioanal. Chem. 20(2):597—606. Polo, Marco. 1926. The travels of Marco Polo. Revised from Marsden's translation and edited with introduction by Manual Komroff. Liveright, New York. p. 81. Rosenfeld, I. 196A. Metabolic effects and metabolism of selenium in animals. IV. Excretion and retention of 758e in relation to ' modes of administration, toxicity and pregnancy in rats. Wyoming Agr. Exp. Sta. Bull. No. Alh, p. 35. Rosenfeld, I. and H. F. Fppsom. 196A. Metabolic effects and metabolism of selenium in animals. V. Metabolism of selenium in sheep. Wyoming Agr. Exp. Sta. Bull. No. hlh, p. 53. Rosenfeld, I. and 0. A. Beath. 196M. Selenium. Academic Press, New York. All pp. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman and W. G. Hoekstra. 1973. Selenium. Biochemical role as a component of glutathione peroxidase. Science 179:588—590. Sandholm, M., H. E. Oksanen and L. Personen. .1973. Uptake of selenium by aquatic organisms. Limnol. Oceanogr. 18(3):h96. Schroeder, H. A. 1967. Effects of selenate, selenite and tellurate on the growth and early survival of mice and rats. J. Nutr. 92(3):33u—338. Schroeder, H. A., D. V. Frost and J. J. Balassa. 1970. Essential trace elements in man: selenium. J. Chron. Dis. 23(h):227—2h3. Schwarz, K. and A. Fredga. 1969. Biological potentcy of organic selenium compounds. I. Aliphatic monoseleno and diseleno— dicarboxylic acids. J. Biol. Chem. 2hh221O3—2110. Shah, K. R., R. H. Filby and W. A. Haller. 1970. Determination of trace elements in petroleum by neutron activation analysis. J. Radio Anal. Chem. 6 ul3—h22. 109 Shamberger, R. J. and D. V. Frost. 1969. Possible protective effect of selenium against human cancer. Can. Med. Assoc. J. 100:682-690. Shrift, A. 196D. A selenium cycle in nature? Nature 20l(h926):130h— 1305. Skidmore, J. F. 1965. Resistance to zinc sulfate of the zebra fish (Brachydanio rerio Hamilton—Buchanan) at different phases of its life history. Ann. Appl. Biol. 56:h7—53. Smith, M. I., K. W. Franke and B. B. Westfall. 1936. The selenium problem in relation to public health. U. S. Treasury Pub. Health Reports 51(h0):lh96—1505. ' Smith, M. I. and R. D. Lillie. 19h0. Part 1. The chronic toxicity of naturally occurring food selenium. U. S. Public Health Serv., Nat. Inst. Health Bull. 17h, l—13. Sprague, J. B. 1969. Measurement of pollutant toxicity to fish. I. Bioassay methods for acute toxicity. Water Res. 3:793—831. Seelye, J. G. 197A. Counting times for low level radioactive samples. Michigan State University Extension Bulletin, Technical Report Series. Steel, R. G. D. and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw—Hill Book Co., Inc., New York. A81 pp. Stein, M. A. 1912. Through the richtohofen range of the nan—shah. Ruins of desert cathay. Macmillan, London, p. 202. Stephan, C. E. 1975. (Editor) Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. Ecological Research Series No. EPA—660/3—75—OO9. U. S. Environ. Protection Agency, Washington, D. C. 80 pp. Thompson, J. N., P. Erodody and D. C. Smith. 1975. Selenium content of food consumed by Canadians. J. Nutr. l05(3):27h—277. Wells, N. 1966. Selenium content of some minerals and fertilizers. New Zealand J. Sci. 9(2):h09—h15. Wier, P. A. and C. H. Hine. 1970. Effects of various metals on behavior of conditioned goldfish. Arch. Environ. Health 20:h5-51. Wiersma, J. H. and C. F. Lee. 1971. Selenium in lake sediments: analytical procedure and preliminary results. Environ. Sci. Technol. 5(12) 1203—1206. Wright, E. 1965. The distribution and excretion of radioselenium in sheep. New Zealand J. Agr. Res. 8:28h. HICHIGRN STQTE UN H lHE“1111111111161111111113 312 310159 28.