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If... .L 3%.”: HZ). — "a! p.» ‘MV‘IIJVI 1-0.0.L L..I.P~.d. .... . _..........o.....:. .. .. i. f...1o.;1€.-I I. Q’. .1. I .. ..0o:0;.........“4JoUo"..hMW.& (”1.5V .. _ tun . .. L. . T ... _. a..... L ..8.8:, cm..‘nv..yo§§.aé.. I. . c o . I u . .. o I. . oroo .. . J Illllllllllllllllllllllllll‘llllll h. 1/ ‘ 3 1293 01089 0949 This is to certify that the thesis entitled Biological Effects of Methyl Mercury in Aquatic Systems on Mallards and Scaup presented by James Russell Ford has been accepted towards fulfillment of the requirements for Ph.D. degreein Fisheries 5 Wildlife. llama $4 92,“; Major professor .....J 2. 5...... gfi‘ ABSTRACT BIOLOGICAL EFFECTS OF METHYL MERCURY IN AQUATIC SYSTEMS 0N MALLARDS AND SCAUP By James Russell Ford The effects of mercury contamination in an aquatic system on the survival and reproduction of two waterfowl species were determined by estimating mercury consumption rates and relating them to experi- mental data. The rate of mercury consumption was determined by formu- lating a diet based on esophagus. crop, and proventriculus contents of field collected birds and analyzing these components for mercury content. Mallards (Anas platyrhyncos) and scaup (Aythya spp.), during October and November, were estimated to have daily total mercury con- sumption rates of 0.008 and 0.681 mg, respectively. Scaup, during March, were estimated to consume 0.175 mg total mercury daily. Feed- ing experiments with mallards indicate that consumption of methyl mercury can lower both survival and reproduction. Lethal effects were observed after both male and female mallards consumed 60.3 i 13.4 mg of methyl mercuric chloride at varied doses above 0.5 mg per day over an 120 day period. Reduction in hatthability of eggs and survival of ducklings were observed at doses less than 0.5 mg per day. Although food consumption and egg production did not decline when females were fed 0.8 mg of the compound on alternate days for a 60 day period, James Russell Ford hatchability of eggs decreased by l9 percent after 1.6 mg was consumed and then stablized for the remainder of the experimental period. Duck- ling survival from 0 to 21 days decreased at a rate of l.3 percent per mg methyl mercuric chloride consumed by the female. Time, sex and reproductive status affected the decline of total mercury levels in breast, brain, liver and kidney tissues of mallards after a single 8 mg dose. When significantly different the levels of mercury in males exceeded females, non-reproductive exceeded reproductive birds and were highest in liver tissue followed by kidney, breast and brain. It took from 30 to more than 90 days for mercury levels in tissues to decline below 1 ppm after a 8 mg dose. BIOLOGICAL EFFECTS OF METHYL MERCURY IN AQUATIC SYSTEMS ON MALLARDS AND SCAUP By James Russell Ford 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 l975 ACKNOWLEDGMENTS I wish to express my gratitude to Dr. H. H. Prince for his guidance and advice. My thanks also goes to Dr. F. M. D'Itri for his advice and the use of equipment in his laboratory. I would also like to express appreciation to Dr. R. F. Ringer, Dr. H. E. Johnson and Dr. M. J. Zabik for-theiradvise and aid in preparing this manu- script. Financial support for this project came from the Office of Water Resources Research Grant AOSZ—Mich. The Institute of Water Research, Biomedical Support Grant, and The Michigan State Agricul- tural Experiment Station. Special appreciation is to be expressed to my wife for her endurance and help. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES . INTRODUCTION . LITERATURE REVIEW MATERIALS AND METHODS Study Area . . . . . . . . . Field Sample Collection and Analysis . Management of Experimental Stocks . Experiment 1: Lethal Effects . Experiment II: Reproductive Effects . Experiment III: Distribution and Excretion. Mercury Analysis RESULTS Field Investigation . . Experiment I: Lethal Effects . . Experiment 11: Reproductive Effects . Experiment III: Distribution and Excretion. DISCUSSION CONCLUSIONS BIBLIOGRAPHY . Page iv vi Table LIST OF TABLES The percentage weight and occurrence and mercury concen- tration of food items in crop and proventriculus of mallards collected during October and November of 1970 and 1971 at three locations in southeast Michigan The percentage weight and occurrence and mercury concen- trations of food items in crop and proventriculus of scaup collected during October and November of 1970 and 1971 and March 1972 in the Lake Erie area of Michigan . . . . . . The total mercury level (ppm) in the liver of mallards collected in October and November of 1970 and 1971 at three locations in southeast Michigan by age and sex The total mercury level (ppm) in the liver of scaup collected in October and November of 1970 and 1971 at two locations in southeast Michigan by age and sex The total mercury level (ppm) in the liver of scaup collected in the Lake Erie area of Michigan during three periods of the year . . . . . . The initial body weights, days to death, and quantities of methyl mercuric chloride consumed by 8 male and 18 female mallards given 7 different daily doses for 120 days . . . . . . . . . . . Mean level of mercury (ppm) in the tissues of mallards that died from consumption of 60. 3 + 13. 4 mg methyl mercuric chloride . . heights (9) of eggs and ducklings from female mallards receiving no treatment and those receiving 0.8 mg of methyl mercuric chloride on alternate days for 60 days . Mercury levels (ppm) in eggs of female mallards receiv- ing 0.8 mg of methyl mercuric chloride on alternate days in relationship to number of ovulations after treatment . . iv Page 22 24 26 28 29 30 33 34 38 Table Page 10. The mortality of mallard embryos and ducklings from untreated females and treated females fed 24 mg of methyl mercuric chloride at a rate of 0.8 mg on alternate days for 60 days . . . . . . . . . . 44 ll. Decline in mercury levels (ppm) in the tissues of reproductive (R) and non-reproductive (N), male (M) and female (F) mallards after an 8.0 mg oral dose of methyl mercuric chloride . . . . . . . . . . . 50 12. Mercury levels (ppm) in eggs from female mallards after a single oral 8.0 mg dose of methyl mercuric chloride . 51 13. Results of statistical comparisons of mercury concentra- tion in tissues of reproductive and non-reproductive female and male mallards that received a single 8.0 mg oral dose of methyl mercuric chloride . . . . . . 52 14. Mercury levels (ppm dry weight) in excreta after an 8.0 mg oral dose of methyl mercuric chloride . . . . . 58 15. Calculated excretion of mercury via eggs and feces of laying females and feces of non-laying females after a single 8000 pg oral dose of methyl mercuric chloride . 59 16. Comparison of mercury levels (ppm) in tissues of mal- lards and pheasants that died from the consumption of methyl mercury . . . . . . . . . . . . . . 64 LIST OF FIGURES Figure Page 1. Study area map showing location of collection areas and known sources of mercury pollution: "A" is Lake St. Clair collection area; "B" is Lake Erie collection area; and "C" is Maumee Bay collection area . . . . . . ll 2. The relationship between the daily dose of methyl mercuric chloride given to male and female mallards and the number of days to death . . . . . . . . . . 31 3. Mercury levels in eggs from female mallards receiving 0.8 mg of methyl mercuric chloride on alternate days for 60 days . . . . . . . . . . . . . . . 36 4. Hatchability of eggs from 5 female mallards receiving no treatment and 23 female mallards given 0.8 mg of methyl mercuric chloride on alternate days for 60 days . 39 5. Survival of mallard ducklings from hatching to three weeks of age that hatched from eggs laid by females given 0.8 mg of methyl mercuric chloride on alternate days for 60 days. The total effect for the period from fertilization of the egg until three weeks of age is represented by the equation below the figure . . . . 42 6. Distribution of post-hatch duckling mortality from female mallards ingesting 0.8 mg of methyl mercuric chloride on alternate days for 60 days . . . . . . 45 7. The percentage of embryonic and duckling mortality of progeny from individual female mallards that consumed 24 mg of methyl mercuric chloride at a rate of 0.8 mg on alternate days for 60 days . . . . . . . . . 47 8. Total mercury levels in breast muscle, brain, liver and kidney tissues in reproductive and non-reproductive mallards of both sexes after an 8.0 mg oral does of methyl mercuric chloride . . . . . . . . . . . 54 9. Mercury levels in eggs (ppm wet weight) and excreta (ppm dry weight) of laying and non-laying female mal- lards after an 8.0 mg oral dose of methyl mercuric chloride . . . . . . . . . . . . . . . . 56 vi INTRODUCTION The presence of mercury contamination in the Great Lakes was first reported by the Ontario Water Resources Commission in Feburary, 1970. Since then it has been established that the areas of greatest contamination are in the continuous area formed by the St. Clair River, Lake St. Clair, Detroit River and Lake Erie (Bails 1972). Both fish (Bails 1972) and aquatic birds (Dustman et a1. 1972) associated with these waters have been reported to contain excessive quantities of mercury. Waterfowl frequent these waters in large numbers during certain periods of the year and the presence of mercury may be a danger to their reproduction and survival. The primary concern of this study was to determine the poten- tial effect of mercury contamination in an aquatic system on water- fowl survival and reproduction. Mallards and scaup were selected for study because of their different dietary habits and widespread dis- tribution within the area of mercury contamination in Southeastern Michigan. The first objective was to estimate the rate of mercury uptake by these species. Since food is probably the major source of mercury to ducks, the rate of mercury intake can be estimated by analysis of the dietary items for mercury content. It is not suffi- cient to know the rate of mercury consumption unless there is infor- mation about toxicity. Therefore the other objective of this study was to determine the toxic effects of methyl mercury comsumption on waterfowl survival and reproduction. Mallards were selected for these experiments because of their ubiquitus nature and adaptability to con- fined conditions. LITERATURE REVIEW Excessive levels of mercury were first noted during the mid 1950's in Swedish terrestrial wildlife (Borg et a1. 1969). These high mercury levels were believed to be associated with the decline of some avian populations. The source of contamination was linked to the use of mercurial fungicide seed dressings. Banning further use of these products resulted in an immediate downward trend of mercury levels in the affected species (Wanntorp et al. 1967, Berg et a1. 1966). Mercury contamination of aquatic organisms was brought to public attention during this same period in Minamata, Japan, where consumption of mercury contaminated shell fish resulted in at least 150 afflictions and over 50 deaths among humans (Takeuchi 1972). In an aquatic system the reduction of mercury input may not necessarily lead to an immediate reduction of contamination of organisms in that system. Some investigations indicate mercury is dispersed through- out the biotic elements and sediment deposits may serve as a continu- ing source of contamination (Johnels et a1. 1967, Johnels et a1. 1968, Hasselrot 1968, Hannerz 1968). The problem in aquatic systems is further complicated by biological methylation of inorganic mercury to monomethyl and dimethyl mercury (Jensen and Jernelov 1968, Wood et a1. 1968). Methyl mercury because of its chemical and toxicological prop- erties is the most important of the five mercurial compounds associated with aquatic systems. These mercurials are (1) methyl mercury; (2) inorganic mercury; (3) phenyl mercury; (4) alkoxyalkyl mercury; and (5) metalic mercury (D'Itri 1972). Swensson and Ulfvarson (1969) tested the relative toxicity of compounds from the first four classes by administering them to chickens (Gallus gallus) by injection and by continuous feeding of dressed wheat. Inorganic mercury was the most toxic of these mercurials following intravenous injection. Phenyl mercury was the second most toxic and the methyl mercury and alkoxyalkyl mercury compounds were the least toxic. But when consumed on dressed wheat, the methyl mercury compound exhibited the greatest toxicity. This difference was related to the slow excretion rate and the tendency of methyl mercury to accumulate in tissues (Swensson and Ulfvarson 1968a, Swensson and Ulfvarson 1968b, Swensson and Ulfvarson 1969). The biological methylation of inorganic mercury to methyl mercury by certain bacteria in the sediments of aquatic systems has been well established by Jensen and Jernelov (1969) and Wood et a1. (1968). In addition there is evidence to indicate that mercurials, other than methyl mercury, may be converted to methyl mercury after being ingested. Kiwinae et al. (1969) reported that the mercury in the breast muscle and eggs of chickens following an oral dose of mercury (II) nitrate consisted of 85 to 100 percent methyl mercury. Phenyl mercury given in the same manner underwent a similar conversion. Wood (1972) suggested that microflora, such as those in the digestive tracts of herbivores, may be capable of methylating inorganic mercury. The biological methylation of other mercurials to methyl mercury, along with its toxicity makes methyl mercury a greater environmental danger than the other mercurials. The analysis of tissues from animals collected in an area contaminated with mercury indicates that a large percentage of the mercury is methyl mercury. Westoo (1969) reported that muscle tissue samples of reindeer (Rangifer tarrandus), beef (Bos taurus) and pigs (Sus scrofa) collected in Sweden contained 86, 92, and 97 percent methyl mercury, respectively. In this same study albumen of chicken and merganser eggs (Mergus spp.) contained 91 and 99 percent methyl mecury, respectively. Wahlberg et al. (1971) reported that the albumen of common merganser (Mergus merganser) eggs, also collected in Sweden, contained 78 percent methyl mercury. Whole contents of common tern (Sterna hirundo) eggs collected in the vicinity of the mercury contaminated waters of Ball and Wabigoon Lakes in Ontario, Canada, were reported to have methyl mercury percentages of 65 and 82, respectively (Fimreite 1974). Whole egg methyl mercury concentra- tions would be expected to be lower because the yolk of eggs gener— ally contain less methyl mercury than the albumen (Backstrom 1969). Fimreite (1974) and Vermeer et al. (1973) analyzed breast muscle of three species of ducks from this same region of Ontario and reported that the percentage of methyl mercury ranged from 65 to 99. Fimreite (1974) analyzed the stomach contents from one mallard and seven common mergansers collected in this area. He found 71 percent of the total mercury was methyl mercury in the stomach contents of the mallard while the percent methyl mercury in the common merganser stomach con- tents ranged from 41 to 88. These data are further indications that methyl mercury is the dominant mercurial in animals environmentally contaminated with mercury. Hannerz (1968) studied the accumulation and retention of methyl mercury of various aquatic organisms in experimentally contaminated concrete ponds. Fish were found to accumulate methyl mercury directly from the water and food. The uptake of mercury from water exceeded that derived from food. Jernelov and Lann (1971) have reported similar findings. The concentration factor in pike (Esox lucius) has been placed at about 3,000 times the mercury level in the water (Johnels et a1. 1967). Hannerz (1968) reported that invertebrates in methyl mercury treated ponds had mercury concentrations which were 110 to 8,470 times greater than the water. The highest concentration occurred in ggrjxa_sp. and the lowest values were found in the annelids. Snails concentrated mercury by a factor of 3,500. Hannerz (1968) found that methyl mercury was not taken up by aquatic plants in appreciable amounts, but surface adsorption did cause high concentrations to occur on the submerged portions of plants. Methyl mercury concentration factors for submerged vegetative parts of Scirpus spp., Polygonum amphibium, Carex spp. and Lemna minor ranged from 1,000 to 3,000. The concentration factors for the emergent portions of these plants ranged from 4 to 25. (These low concentration factors in the emergent portions of the plants indicate a low rate of mercury accumulation by plants from soils and water. Yamada (1968) also reported similar findings for rice plants grown in mercury contaminated soils. These data indicate the rate of mercury consumption by animals consuming - aquatic organisms would depend on which organisms or plant parts they consumed. The mallard and scaup represent two Species of waterfowl which have widely differing food habits. The mallard diet has been reported to consist of 82 to 98 percent plant material during the fall, winter and spring months (Stoudt 1944, Martin et al. 1951, Anderson 1959, Dillon 1959, Junca et al. 1962). The utilization of seeds from cul- tivated crops and other terrestrial plants indicates that mallards are not dependent on aquatic food sources during these months. Juven- ile mallards between 0 and 4 weeks of age and laying females have a higher percentage of animal material in their diets and have closer ties to aquatic food sources (Chura 1961, Bartonek 1972). Scaup in contrast make more extensive use of animal foods. Examinations of esophagus, cr0p, proventriculus and gizzard contents indicate the quantity of animal material in the diet ranges from 64 to 99 percent of an annual basis (Cronan 1957, Anderson 1959, Rodgers and Korschgen 1966, Harmon 1962, Dirschl 1969, Bartonek and Hickey 1969, Bartonek and Murdy 1970, Thompson 1973). Cottam (1939) reported lower per- centages of animal materials in the diets of scaup, but his compila- tion was based on gizzard contents and was probably biased toward seeds which are more resistant to grinding than soft bodied animals. The actual diet may be influenced by the availability of foods. Dirschl (1969) studying lesser scaup (Aythya affinis) diets in Sas- katchewan reported that the seeds of Nuphar varigatum accounted for 85.6 percent of the diet during one period of his study. This change in diet corresponded to the release of seeds from this plant into the water. Cronan (1957), Harmon (1962) and Thompson (1973) all indicate the percent composition of items in the diet were a good reflection of their availability. It is apparent from these studies that, although the dietary makeup may shift with location or season, scaup are totally dependent on aquatic systems for food. Investigations of the effects of methyl mercury compounds on reproduction and survival of avian species have been limited to domestic chickens, ring—necked pheasant (Phasianus colchicus) and mallards. Tejning (1967) reported a significant reduction in hatch- ability when chickens were fed grain treated with methyl mercury dicyandiamide. The treatment did not affect egg production, but the number of floor eggs and shell-less eggs were found to increase with continued treatment. Consumption of methyl mercury dicyandamide treated grain decreased hatchability in ring-necked pheasant eggs (Borg et al 1969, Fimreite l97l, Tejning 1971, Adams and Prince 1972). Adams and Prince (1972) found, that after cumulative consump- tion of 4 mg of methyl mercury dicyandiamide, hatchability in ring- necked pheasants declined 6.8 i 0.5 percent for each additional mg consumed. Borg et a1. (1969), Tejning (1971) and Adams and Prince (1972) did not report declines in egg production by ring—necked pheasants that consumed methyl mercury. But Fimreite (1971) did report a moderate decline in egg production in methyl mercury treated ring-necked pheasants. Adams and Prince (1972), however, did report a cessation in egg production after an average of 15.8 i 0.9 mg of methyl mercury dicyandiamide had been consumed by ring-necked pheas- ants. Lethal effects were also reported for both chickens and ring- necked pheasants (Tejning 1967, Adams and Prince 1972). Tejning (1967) reported the death of one cock after it had consumed 44.07 mg of methyl mercury dicyandiamide. The consumption of this same com- pound by ring-necked pheasants resulted in death after an average of 24.7 i 0.8 mg had been consumed (Adams and Prince 1972). Heinz (1974) reported preliminary findings which indicate that 3 ppm methyl mercury dicyandimide (as methyl mercury) in the diet will reduce the reproductive success of mallards. He found the duration of egg produc- tion for treated mallards was reduced to 21 weeks as compared to 43 weeks for controls. The treated birds had a higher frequency of cracked eggs and a lower hatchability as compared with the controls. The most notable effect was the reduced survival of ducklings from the treated group. Heinz (1974) also reported 3 ppm methyl mercury dicyandiamide fed for a 12 month period was not lethal to adult mal- lards. It is apparent from these data that reproductive effects will occur at a low methyl mercury consumption rate without causing adult mortality. MATERIALS AND METHODS Study Area The study area included portions of the aquatic system formed by the St. Clair River, Lake St. Clair, Detroit River and western Lake Erie (Figure 1). It has been well established that fish (Bails 1972) and wildlife (Dustman et al. 1972) associated with this system have excessive tissue levels of mercury. The excessive mercury levels in fish has been related to industrial activities in this area, espe- cially, two chloralkali plants which had direct discharges of both metalic and mercuric mercury into the St. Clair River at Sarnia, Ontario, and the Detroit River at Wyandotte, Michigan. Two sites for the collection of samples were selected because of their relative position to these industrial plants. A third area, the Maumee Bay area, was chosen because it is adjacent to but separate from the waters of Lake Erie. All collection areas are used extensively by migrant and resident waterfowl and have a long tradition as hunting areas. The Lake St. Clair collection area extends along the northern shore of Lake St. Clair from Harsen Island to the western shore of Anchor Bay. Anchor Bay is open with most of the area having water depths of 3 meters or less. A series of marshy islands extends east- ward from Anchor Bay across the mouth of the St. Clair River. Most of these islands are included in the St. Clair Flats State Game Area. Water from the river flows through the marsh areas allowing possible 10 11 Figure l.--Study area map showing location of collection areas and known sources of mercury pollutions: "A" is Lake St. Clair collection area; "B" is Lake Erie collection area; and "C" is Maumee Bay collection area. 12 Scale in miles 253:}; Collection Area >1! Mercury Pollution Site DETROIT L—l WINDSOR WYANDOTTE ' Detroit River State Game Area Lake Erie ‘a‘a".'.‘.‘.' ...... .......... // Maumee Bay / SARNIA Harsens Island Lake St. Clair l3 deposition of mercury from the Sarnia discharge. The area is used by mallards and scaup during fall and spring migratory periods for feeding and resting. Mallards also breed in this area with an esti- mated production of one duckling per acre of broad habitat (E. Mikula, personal communication). The Lake Erie collection site extends southward from the middle of the Trenton Channel at the lower end of the Detroit River to the southern end of Point Mouillee State Game Area. The eastern half of this area consisted of the Open waters of Lake Erie which were gener- ally less than 4 meters deep. The Point Mouilee State Game Area is located at the mouth of the Huron River and consists largely of cat- tail islands. Fifty hectares of this area is diked and managed for production of natural waterfowl foods. The marsh area is Open to free movement of lake water and is frequently flushed by seiches. Both mallards and scaup use this area for feeding and resting during migratory periods. Also a significant number of scaup remain in this area during the winter. Breeding activity by mallards is low. The Maumee Bay area is located along the western shore of Lake Erie just north of the Michigan-Ohio border. The collection site consisted of marsh lands managed by the Erie Shooting Club which are separated from Maumee Bay by dikes. The main source of water in the marsh comes from a large spring within the dike system. This area is used by mallards and other puddle ducks throughout the year. Use by scaup is limited. 14 Field Sample Collection and Analysis The objectives of the field study were to determine the mer- cury concentration in the mallards and scaup frequenting the study area and estimate the rate of mercury consumption while in the area. The total mercury concentration in the liver was used as an index for body levels. Food contents of the esophagus, crap and proventriculus served as a basis for estimating the rate of mercury consumption. Collections of mallards and scaup were made during October and Novem- ber of 1970 and 1971. Additional collections of scaup were made in January and March of 1972. The October-November collections were made from ducks killed by hunters. The January and March specimens were collected by the investigator and the Michigan Department of Natural Resources personnel. During the two year period samples were collected from 448 mallards with 188 from the Lake Erie area, 136 from the Maumee Bay area, and 124 from the Lake St. Clair area. A total of 431 samples were collected from scaup in this same period with 383 from Lake Erie and 48 from Lake St. Clair. In addition 90 scaup were collected in January and March from the Lake Erie area. The esophagus, crop, proventriculus, and liver were removed from all birds. The entire viscera was removed intact from hunter killed birds by making an incision along the ventral surface of the neck and another in the abdomen. The esophagus and crop was sepa- rated from the trachea and thoracic connective tissue. The intact viscera was then removed through the abdominal incision, placed in a plastic bay, sealed and frozen. 15 The contents of the esophagus, crop and proventriculus were removed, segregated, and identified. Identification of plant material was based on the Seed Identification Manual (Martin and Barkley, 1961) and A Mannual of Aquatic Plants (Fassett, 1957). Animal material was identified with the aid of Freshwater Invertebrates of the United States (Pennak, 1953). The weight of the segregated items was deter- mined after blotting dry on paper toweling. In samples containing large numbers of items from two or more sources the total weight was determined and five portions containing at least 50 of each item were selected and weighted. The items in each portion were segregated, counted and weighed. Total quantity by species was estimated from the proportion in the subsample applied to the total weight. The sepa- rated specimens were placed in an individual glass vial, labeled and refrozen for analysis of mercury content. The percent weight, percent occurrence and mercury concentra- tion was determined for the food items. The analyses were separated on a generic basis by collection sites. It was necessary to form three general groups, gastropods, vegetation and miscellaneous seeds. All of the seeds in the miscellaneous group occurred in incidental amounts or only once. Management of Experimental Stocks The mallards used in this investigation were progeny from a parent flock of 20 female and 20 male "game farm" mallards. The parent flock was obtained from the Max McGraw Wildlife Foundation, Dundee, Illinois. During experiments the ducks were housed in l6 Petersime finishing batteries with 30 individual compartments, each measuring 68 x 70 x 28 cm. The temperature of the room containing the finishing batteries was maintained at about 22°C. A photoperiod of 14 hours of light was used in experiments involving reproductive birds. Nine hours of light was used in experiments involving non- reproductive birds. The birds were allowed a minimum of two weeks to acclimate before treatment. Experiment 1: Lethal Effects The methyl mercuric chloride was dissolved in acetone and sprayed on food pellets in eight evenly spaced treatment levels rang- ing from 0.00 to 2.00 mg per 15 g of pellets. During preparation of the food pellets precautions were taken to minimize personal contact with the vapor by spraying in a negative pressure hood and by wearing protective clothing and an air respirator. Fifteen g of food pellets from the designated treatment level were fed each bird daily. To ensure that the birds consumed the treated pellets, food was with- held each night and treated food was given each morning. After the treated food was consumed untreated food was given ad_libitum until evening. The water trays were removed during treatment to reduce losses due to dabbling. A group of 18 female and 8 male one year old mallards in reproductive condition were fed mercury treated food over a 120 day period. One male and two females were treated at the 0.00, 0.28, 0.57, 0.87, 1.14, 1.71 mg levels while one male and three females were treated at the 1.43 and 2.00 mg levels. The liver, kid- ney, breast muscle and brain of birds that died were removed and analyzed for total mercury concentration. l7 Experiment II: Reproductive Effects This experiment was designed to evaluate the effects of methyl mercury consumption over a 60 day period on hatchability of eggs and survival of progeny. Thirty 20 month old female mallards were paired with 20 males of the same age and each pair was housed in individual finishing battery compartments. Five females were designated as controls (untreated food) with individual compartments selected to represent all possible positions in the finishing cages. The remaining 25 hens received methyl mercuric chloride at a rate of 0.8 mg every second day. Treatment was presented on 10 g of layer-ration pellets prepared and given to the birds as previously described. The males were removed from the compartments each morning during treatment periods. The treatment was initiated after all but one of the females had produced four or more fertile eggs. Eggs were collected daily, washed and stored at 11°C and either analyzed for total mercury or incubated. All of the eggs from two of the treated females were held for mercury analysis. Eggs from the other females (treated and untreated) were incubated with one egg being held for analysis every 6 days from each female. If no egg was laid by a female on that particular day, the egg laid on the next nearest day was used. Egg collection for mercury analysis from the 23 treated females was staggered so that analysis was being made for half of the group every three days. Shell thickness of all unincu- bated eggs was determined as the mean of four measurements around the transverse midline of each shell. Eggs were set every 7 days 18 and incubated in a Petersime Model 4 incubator at 37.500 and 70-80 percent humidity for 23 days and then transferred to individual com- partments in a David Bradley incubator maintained at 37°C and 95-100 percent humidity for hatching. Ducklings were wing tagged and weighed at approximately 18 hours post-hatch. They were reared in a Petersime brooder unit with food and water gg_libitum and sacrificed at 3 weeks of age and weighed and sexed. The ducklings were checked daily throughout the rearing period for mortality. Experiment III: Distribution and Excretion This experimental set was designed to compare the distribution and excretion of methyl mercury in 27 female and 12 male non-repro- ductive and 30 female and 12 male reproductive mallards that were 20 and 24 months of age, respectively. A single oral dose of 8 mg (i 2.5 percent) of methyl mercuric chloride in a number 3 gelatin capsule was given to each bird. Food and water was given gg_libitum. The non-reproductive mallards were fed a maintenance diet while those in the reproductive state were given a layer ration. Food consumption was monitored and eggs were collected daily from the reproductive females. The eggs from 6 hens laid on day l, 2, 3, 4, 6, 8, 10, 12, 16, 20, 45, and 60 were analyzed for total mercury. Three females were sacrificed on 4, 8, 12, 16, 20, 30, 45, 60, and 90 days after treatment with an additional group of reproductive hens being sacri- ficed at 75 days. Three non-reproductive males were sacrificed on 4, 12, 30, and 60 days after treatment while three reproductive males were sacrificed on days 4, 12, 30, and 90. The liver, kidneys, breast 19 muscle and brain were removed and stored by freezing for mercury analysis after death by electrocution. The excreta from each bird was collected during the 24 hour period prior to death and analyzed for mercury. Mercury Analysis Mercury concentrations were determined by wet digestion and flameless atomic absorption spectorphotometry using a modification (Annett et al. 1972) of a procedure reported by Uthe et a1. (1970). The entire sample was homogenized. A Sunbeam blender was used for samples with a volume greater than 100 cc and a Polytron homogenizer was used on samples less than 100 cc. Subsamples of 0.4 to 0.6 g were placed into 100 m1 volumetric flasks and 5 ml of concentrated sulfuric acid was added to each flask which was then loosely covered with parafilm and placed in a 60-7OOC water bath. After the particu- late material dissolved, the flask was cooled in a 4-6OC water bath and 20 ml of a 6 percent w/v potassium permanganate solution was slowly added. After the reaction subsided at room temperature, the flask was placed into a 60-700C water bath until the reaction sub- sided. Five ml more of the potassium permanganate solution was added and the contents were heated to the boiling point. The solution was allowed to cool to room temperature and cleared with a 10 percent vw/v hydorxylamine hydrochloride solution and brought up to volume 1vi th distilled water. An apprOpriate aliquot of the solution was trréinsferred to the reaction vessel and reduced with a stannous ch 1 oride-hydorchloric acid solution. The liberated mercury vapor 20 was washed through the detector cell with nitrogen gas attached to a Jerrell Ash model 800 atomic absorption spectrophotometer. A stand- ard curve was prepared for each group of samples processed. The mercury analysis results for all, except fecal material, are reported on a wet weight basis. RESULTS Field Investigation Sixty-eight percent or 303 of the mallards collected con- tained an average of 10.40 i 24.98 g of food in their esophagus, crop and proventriculus. Seeds from cultivated and endemic plant species composed an average 99.5 percent of the food on a wet weight basis. Seeds from cultivated plants usually accounted for the greatest per- centage of the weight (Table 1). In the Lake Erie area 98.7 percent of the food weight was composed of seeds. The seeds from corn (gee_ mays) and wheat (Triticum avestivum) accounted for 83.5 percent of the total weight. Two endemic plant species, Polygonum spp. and Cerex_spp., contributed 5.7 and 6.1 percent of the total weight respectively. The food of mallards collected in the Maumee Bay area was composed of 99.8 percent seeds. Two plant species, corn and Polygonum spp. accounted for 85.1 percent of the total weight. Although seeds of cultivated and endemic plants accounted for 99.9 percent of the mallard diet in the Lake St. Clair area, cultivated plants, corn and buckweat (Faygopyrum spp.)., accounted for a smaller percentage of the diet. Seeds of Echinochola spp. and Amaranthus spp. accounted for 19.8 and 18.9 percent of the weight respectively. The miscellaneous group, which consisted of 17 plant species, composed 8.6 percent of the total weight. 21 22 .vmecoecma mm: mwmxpmcm xcsoeme o: page mmumowncw :mmc < N mpnmuompmo uoz u ozF m., Paewca Peace muoa_;ae< avamcc< mnoaocummw ~ N F auommcfi _aswca PPdOC) I mmmm F060 oHHH . m ~.wo mcmmm acm_a Peace o.o .qm Ezcxmomxmu o o ~N.o m.m OKN En>mumwm Eauwuwmfiu m m m.mm mNmE.MmN I ,— 1... I [\OO r—OO m meaam p=a_a emoa>_a_=u N m N._ maom:m__momwz H .aam mmmmmmumam e .qam mzocwom .o .aam m_o;oocw;ow F .aam mcmuwm .o .aam xmcau m .aam Escomxmmm, mummm ucmpa owewvcm m a m o N: m e m—.o _.P w m m o — N P I oomnomoooo VMNQ’MMON N'— N GHHOHHH oz oz P mo.o 1.0 m a: Egg mucocgzooo unmwmz m: Egg mocmccaooo uzmwmz 0: Egg wocmccaooo unmwmz Eaaa coca Amoucv Lea—u .om axes Aeo_n=v saw amaze: Amm_n:v mac“ axed .cmmwzowz pmmmcpsom cw meowumooF woes» on .mmP use onp we Lmn5m>oz ecu Lmnouoo m:_c:u nmuomFFOU mucePFQE mo m:_:owcp ico>oca ecu aoco cw memo? woo» co :owpmeucmocoo xcsocme new mocmcczooo new agave: mmmpcmoema m:~--.~ mom<~ 23 Although seeds from cultivated plants formed the bulk of the mallards diet on a weight basis, the seeds from several species of endemic plants occurred with greater frequency. The frequency of occurrence of corn in the Lake Erie area samples was exceeded by seeds from five species of endemic plants (Table 1). Over 50 percent of the mallards from this area contained seeds of Polygonum Spp. and Echinochola spp. The other species which occurred more frequently than corn were Carex spp., Bidens spp. and Scirpus spp. In the Mau- mee Bay area seeds of Polygonum spp. and Echinochola spp. occurred more frequently than corn. A similar pattern occurred for the mallards collected in the Lake St. Clair area. Seeds of Polygonum spp. had the highest rate of occurrence and the seeds of Echinochola spp., Scirpus spp. and Amaranthus spp. occurred more frequently than corn. Analysis for total mercury content of some food items collected from the eSOphagus, crop and proventriculus of mallards suggests there was not difference between areas (Table 1). The mean level of mercury was 0.06 i 0.06 ppm with values for individual species ranging from not detectable to 0.22 ppm. The wheat samples collected from the Lake Erie area had the highest mercury levels. It was believed that the birds obtained the wheat from an illegal baiting operation to attract ducks for shooting. Food items were found in 13 percent of the scaup collected in October and November. The average weight of the crop and proventriculus contents was 1.23 i 3.39 9. Animal material accounted for 83.7 per- cent of the total weight (Table 2). Gastropods formed 59.3 percent 24 TABLE 2.--The percentage weight and occurrence and mercury concentra- tions of food items in crap and proventriculus of scaup collected during October and November of 1970 and 1971 and March 1972 in the Lake Erie area of Michigan. October-November (N-53) March (N=30) Food Item Weight Occurrence Hg++ppm Weight Occurrence1 Animal Gastropoda 59.3 45.3 0.46 64.1 26.7 Pelecypoda 20.5 9.4 4.76 0.2 10.0 Pisces 3.2 1.9 -2 0.3 3.3 Amphipoda 0.6 3.9 - 0.0 0.0 Insecta 0.1 1.9 - 3.8 3.3 Total 83.7 - 68.4 Plant Seeds Potamogeton spp. 0.3 3.8 - 0.0 0.0 Polygonum spp. 0.9 5.7 — 8.8 20.0 Scirpus spp. T 7.6 - 0.0 0.0 Echinochola Spp. T 1.9 - 0.4 10.0 Bidens spp. 0.0 0.0 - T 3.3 Miscellaneous 0.3 15.1 - T 3.3 Total 1.5 9.4 Vegetative Materials 14.8 45.3 0.24 22.2 53.3 1No mercury analysis of March foods. 2A dash indicates thatrwimercury analysis was performed. 25 of the food material weight. Valvata spp., Physa spp. and Amnicola spp. were the most common gastropods. Pelecypoda, Sphaerium spp., made up 20.5 percent of the weight while vegetative materials such as the leaves and shoots of Vallisneria spp. and the leaves and stems of Ceratophyllum Spp. and Chara Spp. accounted for 14.8 percent of the total weight. Gastropods and vegetative material occurred most fre- quently in the scaup collected in October and November. Forty-one percent of the scaup collected in March had food in their crop and proventriculus. The average weight of these contents was 0.26 i 0.91 9. Animal material accounted for 68.4 percent of the total weight with gastropods being the most important item (Table 2). Although importance of the pelecypods on a weight basis decreased, the frequency of occurrance was similar between periods. Vegetative material and seeds of Polygonum spp. accounted for 22.2 and 8.8 per- cent of the total weight respectively. Vegetative material occurred most frequently. Four other items occurred with frequencies of 10 percent or greater; gastropods, pelecypods and seeds of Polygonum Spp. and Echinochola spp. The tissues of pelecypods consumed by scaup contained 4.76 ppm mercury (Table 2). Gastropod tissue mercury concentrations were one tenth of those in pelecypod tissues. The vegetative material con- tained 0.24 ppm of mercury. The mercury levels in all of these food items exceeded those in mallard foods. Twenty-five percent of the mallard livers collected in October and November were analyzed for total mercury concentration (Table 3). 26 Pm.o «m.o NNF m©.o m¢.o mm No.0 mm.o mm w¢.o Pm.o mm om.o mN.o ow Pouch mw.o cm.o mm o_.~ Fm.o m mm.o mo.o m mw.o mm.o m mm.o Pm.o op mepu .pm oxm; Nm.o -.o Ne Rm.o _e.o m ee.o om.o w em.o _m.o o. .N.o m_.o e_ sam amaze: mN.o em.o me em.o mN.o F, N_.o m_.o m ¢_.o e_.o _P Pe.o mm.o e_ awed axed om .m g cm .m g cm .m c om .m : om .m c _aaoe mesaaseu SF=e< meaaaeea “Faea meEmu ope: .xmm ucm mom ma cmmwzowz ummmcuzom cw mcowmeOP mecca um FumF new oump Co cmaew>oz new canouoo cw cmuomFFoo mccmppme mo cm>w_ on“ cw Agony _m>m_ xcsoeme Fancy w:#--.m momoz ecu cmnouoo cw umpomFFoo azmom Co Lm>wp on“ cw AEQQV Pw>wp mczoeme papa» ochlu.¢ momwm moco__oe oposoo mF oco oFoE m »o oosomcoo mowcopco owcoocoe pxgpos Co mowuwucoso oco .soooo op mxoo .mpcmwoz aooo Fo_pwcw o;»-.o momqp 31 Figure 2.--The relationship between the daily dose of methyl mercuric chloride given to male and female mallards and the number of days to death. Days to Death 120 100 80 60 40 20 32 x = males 0 = females 1?: 98.8-35.5X R= 0.77 x N= 19 l l 0.5 l 1.5 mg CH3 Hg 01 / day 33 TABLE 7.--Mean level of mercury (ppm) in the tissues of mallards that died from consumption of 60.3 i 13.4 mg methyl mercuric chloride. Females Males Total (10) (6) (16) 1’ so I so Y so Liver 32.3 15.5 37.5 12.3 35.6 14.0 Kidney 27.8 6.1 33.5 10.8 30.6 22.7 Breast 14.8 6.2 14.6 5.2 14.7 5.7 Brain 12.0 4.1 12.7 5.0 12.6 4.3 Experiment II: Reproductive Effects Twenty-five of a group of 30 female mallards in reproductive condition were given 0.8 mg of methyl mercuric chloride every other day for 60 days amounting to a total dose of 24 mg. 0f the 1,474 eggs produced, 961 were incubated and 542 were either used for shell thick- ness measurement and/or analyzed for total mercury. Consumption of methyl mercuric chloride did not significantly affect egg production. The five untreated birds produced a mean of 53.0 i 4.8 eggs compared to a mean of 48.0 i 7.5 eggs for the treated birds. Food consumption by the pair of birds was relatively constant throughout the experiment and not significantly different between the control and treated groups. The estimated average daily food intake of females paired with males was 129.30 i 18.23 g. The equivalent concentraiton of methyl mercury in the food, based on a daily consump- tion of 0.4 mg of methyl mercuric chloride, would have been 2.6 ppm. 34 The weights of the eggs prior to incubation and ducklings at 18 hours and 3 weeks post-hatch from the treated and untreated groups were not significantly different (Table 8). Within the treated group TABLE 8.--Weights (g) of eggs and ducklings from female mallards receiving no treatment and those receiving 0.8 mg of methyl mercuric chloride on alternate days for 60 days. Eggs Ducklings 18 Hours of Age 3 Weeks of Age n i' SD n i" 50 n I' SO Treated Survived 414 57.4 4.21‘1l 408 35.9 2.92al 337 380.3 67.39a Died 124 55.6 5.94b 118 34.7 2.62b --- Untreated 163 56.0 4.56b 158 35.1 2.91b 158 377.1 57.07631 Note: Similar superscripts indicate means which are not significantly different. the ducklings that died originated from lower weight eggs and weighed less at 18 hours when compared to those that survived (p < 0.05). The sex ratio of the two groups of ducklings did not deviate signifi- cantly from the expected 1:1 ratio (x2 = 0.28 with 1 d.f.). The shell thickness of 238 unincubated eggs from treated females and 49 unincubated eggs from untreated females averaged 0.342 t 0.033 and 0.348 t 0.023 mm, respectively and were not signifi- cantly different. 35 The total mercury level in the eggs laid after the initial 0.8 mg dose ranged between 0.0 to 1.0 ppm (Figure 3). The mercury level in the eggs continued to increase until about 16 mg had been consumed, which was on the 40th day of the experiment. Although the increase was curvilinear, it was variable with the equation accounting for 28 percent (r2) of the variability. Part of the unaccounted variability was due to the treatment being given every other day and whether or not an egg had been laid on the previous day (Table 9). The mercury level was significantly higher in eggs formed by the first ovulation compared to eggs formed by the second ovulation after treat- ment (p < 0.05). Although the previous days laying record was import- ant, it was apparent only in mercury level of eggs from the second ovulation after treatment. Individual variation was another source of variability. The concentration of mercury in the eggs among individual birds averaged 3.54 t 0.97 with a 27 percent coefficient of variation. The consumption of methyl mercuric chloride caused a signifi- cant reduction in hatchability of eggs from treated females (p < 0.05). The hatchability of the untreated group remained relatively constant throughout the experiment at 88.7 i 7.9 percent compared to a hatch- ability of 69.4 i 11.3 percent for the treated group (Figure 4). This decrease occurred after 1.6 mg had been consumed on the 4th day of the experiment and remained relatively constant. No significant corre- lation (r = —0.39) existed between the hatchability of eggs for the treated group and the estimated mercury concentration in the eggs by female and time period. 36 .mxoo om Loo when opoccoppo co oomeopco owcooeos Fxspos wo ms m.o mcw>wooog moLoP—os opoEoe Eoce mmmo cw m_o>op »c:ocoz-.m ocomwm 37 = l. 189 +0.3160X " 0.0084 X2 1' 0.0526 1 0.0020 III.Lllllllllljllllllllllllll 5 IO 15 20 25 mg CH3 HgCl Consumed 38 TABLE 9.--Mercury levels (ppm) in eggs of female mallards receiving 0.8 mg of methyl mercuric chloride on alternate days in relationship to number of ovulations after treatment. First Ovulation Second Ovulation Egg laid on prev1ous day n i- SD n ;- SD Yes 86 3.91 1.50 120 2.83 1.29 No 31 4.04 1.64 34 3.64 2.00 39 Figure 4.--Hatchability of eggs from 5 female mallards receiving no treatment and 23 female mallards given 0.8 mg of methyl mercuric chloride on alternate days for 60 days. % Hatchability 100 (D O O) O .b O 20 40 Cl Control ZiSD=88.717.9 I Treated XtSD=69.41-ll.3 F 5 IO 15 20 25 mg CH3 Hg Cl Consumed 41 There was a significant linear decrease of 1.3 percent per mg of mercury consumed by the females and survival of ducklings from hatching until 3 weeks of age (Figure 5). There was also a signifi- cant correlation (r = -0.58) between duckling survival and mercury concentration in the egg. The duckling survival was reduced over the 60 day period to an estimated 62 percent compared to a mean survival of 96.7 i 4.8 percent for ducklings from untreated females. The total mortality of mallard embryos and ducklings after the females had consumed 24 mg of methyl mercuric chloride over 60 days was 46.0 percent compared to 16.1 percent mortality for the untreated group (Table 10). Although the embryonic mortality of the treated group was higher, the distribution of the mortality throughout the period of embryonic development remained similar for both treated and untreated. There was a large increase in post-hatch mortality in ducklings from the treated group. Ninety-nine percent of the duckling mortality from the treated females occurred within 12 days after hatching with the greatest percentage dying on day 4 (Figure 6). Thirty-six of these ducklings were submitted to Dr. A. L. Trapp of the Michigan State University Diagnostic Clinic for pathological examina- tion. All of these ducklings were reported as having acute encephalo- malacia; a disruptive swelling of the brain tissue. The mortality of embryos and ducklings was analyzed on the basis of the 60 day record of each individual female (Figure 7). The total mortality of progeny for untreated females varied from 5 to 21 percent while total mortality varied from 15 to 87 percent for individual 42 Figure 5.--Survival of mallard ducklings from hatching to three weeks of age that hatched from eggs laid by females given 0.8 mg of methyl mercuric chloride on alternate days for 60 days. The total effect for the period from fertilization of the egg until three weeks of age is represented by the equation below the figure. 43 100 ~.\——‘l + __ ‘ ’ e 30. l .\\\ ___J . \\\' .. 60» . N a: 40 .. 1:] Control x150 -- 96.7 14.8 — Treated ?=93.5- l.3X r = 0.67 20- 5 IO 15 2O 25 mg CH3HgCI Consumed Total effect it: 67.2 -I.| x r = 0.66 44 TABLE 10.--The mortality of mallard embryos and ducklings from untreated females and treated females fed 24 mg of methyl mercuric chloride at a rate of 0.8 mg on alternate days from 60 days. Treated Untreated Number Percent Number Percent Fertile Eggs Set 769 192 Embryonic 229 29.8 29 15.1 0- 6 days 32 13.9 5 17.2 7-12 days 0 0.0 2 0.0 13-18 days 5 2.2 0 6.9 19 days--Hatch 192 83.9 22 75.9 Posthatch 124 16.2 2 1.0 Total 353 46.0 31 16.1 45 Figure 6.--Distribution of post-hatch duckling mortality from female mallards ingesting 0.8 mg of methyl mercuric chloride on alternate days for 60 days. °/o Mortality 20 5 46 Duckfings Sacrificed lIlIlIJ nIrI 6 B IO 12 14 Days After Hatching 16 18 20 47 .mzoo co co» memo opocgop_o no as m.o mo ope; o um oowcono oweoocoe Fxnooe Co as om oosomcoo pogo mocoppoe opoeoe Fooow>wocw Eoge xoomoco mo zu_9opcos m:__xo:o oco owcoxgoeo mo omopcoocoo ozh-.N moaned 48 mm 8.95... .828 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ $533.. .3222: 5:262 3225 E 5:28: oEoroEm B 36:5... 382... - Om II...|I ‘ 49 females consuming the mercury compound. The coefficient of variation for embryonic mortality was 8 percent among untreated females and 24 percent among treated females. The coefficient of variation for duck- ling mortality was greater with the coefficients for untreated and treated females being 8 and 42 percent, respectively. The response by individual females ranged from no apparent treatment affects to almost complete mortality of progeny. There was no significant correlation (r = -0.03) between embryonic and duckling mortality. The relation- ships between embryonic and duckling mortality and body weight of the female, numbers of eggs laid by a female, weight of eggs and mercury concentration in the eggs were examined by multiple regression and no significant relationships were observed. Experiment III: Distribution and Excretion Thirty female and 12 male mallards in a reproductive status and 27 female and 12 male mallards in a non-reproductive status were treated with a single 8.0 mg dose of methyl mercuric chloride. Indi- viduals from these groups were sacrificed in groups of three over a 90 day period. The mercury levels in the liver, kidney, breast muscle, brain tissues, and excreta were determined for each bird (Table 11). Eggs from six females were also analyzed for total mercury (Table 12). None of the birds died during the experiment and there was no apparent reduction of food consumption in any of the groups. An analysis of variance of the realtionships between tissues, time, sex and reproductive status using the data for 4, 12, and 30 days 50 TABLE ll.-—Dec1ine in mercury levels (ppm) in the tissues of reproductive (R) and non-reproductive (N), male (M) and female (F) mallards after an 8.0 mg oral dose of mercuric chloride. Sex Number of days after 8.0 mg dose Tissue and status 41 8 12 16 20 3o 45 60 75 90 Kidney F R i" 7.93 5.87 3.69 3.45 4.73 1.13 0.56 0.31 0.20 0.11 SD 0.86 2.39 0.39 1.31 4.45 0.75 0.25 0.06 0.07 0.05 F N 2 10.85 9.70 3.25 1.82 1.44 1.54 0.71 0.40 0.46 - SD 3.65 0.99 1.34 0.25 0.59 0.59 0.24 0.05 0.16 M R R' 8.98 8.09 8.47 1.45 SD 2.45 1.36 _1.82 0.14 M N ‘R 15.21 10.06 9.43 7.38 SD 2.58 2.27 0.31 1.57 Liver F R 2' 9.53 5.42 3.38 2.00 5.34 0.92 0.92 0.31 0.29 0.18 SD 2.88 2.72 0.63 0.58 6.91 0.36 0.36 0.12 0.19 0.05 F N 'I 9.34 9.86 11.62 7.25 6.79 7.84 4.70 2.66 3.48 SD 2.01 3.07 4.18 1.86 0.69 3.95 1.40 0.12 1.30 M R i' 7.08 8.14 5.31 2.40 SD 2.62 0.82 3.80 0.14 M N I 13.64 12.72 9.76 8.01 SD 4.22 3.77 1.79 0.53 Breast F R I' 3.89 3.40 3.58 2.09 2.33 1.66 1.21 0.11 0.15 0.00 muscle 50 0.33 0.57 0.41 1.01 1.49 0.15 0.40 0.65 0.08 0.00 F N E’ 5.40 4.91 5.64 4.08 3.27 4.19 1.58 1.14 ' 1.40 SD 1.63 0.12 0.01 0.44 0.56 1.90 0.39 0.70 0.31 M R 27 4.79 3.73 3.19 0.30 SD 0.27 0.33 1.88 0.05 M N ET 4.10 3.26 3.85 2.88 SD 1.44 0.63 0.86 0.36 Brain F R 7' 2.29 1.94 1.90 1.78 1.41 0.55 0.33 0.16 0.11 0.11 SD 0.58 0.32 0.25 0.48 1.16 0.24 0.13 0.04 0.08 0.04 F N 2' 0.55 2.68 2.16 1.71 1.66 1.85 0.92 0.53 0.69 SD 0.16 0.22 0.53 0.35 0.12 0.64 0.47 0.07 0.21 M R "I 2.13 3.25 2.55 0.61 SD 0.18 0.71 0.66 0.06 M N I’ 3.16 3.49 2.83 2.10 SD 0.79 0.72 0.28 0.20 1Three samples per day. 51 TABLE 12.--Mercury levels (ppm) in eggs from six female mallards after a single oral 8.0 mg dose of methyl mercuric chloride. Days after Treatment n x SD 2 6 13.58 2.59 3 6 10.60 2.25 4 6 7.76 1.65 6 6 6.72 1.10 8 6 4.82 0.60 10 6 3.72 0.51 12 6 4.58 1.52 16 6 2.57 1.02 20 6 1.52 0.55 30 6 0.89 0.33 45 6 0.36 0.23 60 6 0.10 ‘ 0.10 indicated a significant 3-way interaction between tissues, time and reproductive status (Table 13). This led to further partioning of the data. Blocking out tissues within the time periods of 4, 12, 30, 60, and 90 days indicated significant differences between tissues within all time periods (p < 0.05) (Table 13). Comparison of the tissue means within periods by orthogonal contrasts showed that liver and kidney levels always exceeded those of the breast muscle and brain tissues. There was also a tendency for the liver levels to exceed those in the kidney (p < 0.05). The data were further analyzed on the basis of sex and reproductive status within tissues and time periods. Where significant sex or reproductive status differences were found, 52 .cozoEBE “an: cc... .8... 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