SURVIVAL, REPRODUC'HON, AND MERCURY LEVI-13 ' i IN THE USSUEs or RING-NECKEDPHEASANTS ’ f CONSUMING Iwo MERCURIAL FUNGICIDES Thesis for the Degree. of :M. S. MICHIGAN STATE UNIVERSITY WILLIAM I. ADAMS 1971. . .. ' . ‘ V " I .I ‘Ewilgig...’ _v.-r ~ . . , n‘.'--.rn.‘- THES g, LIBRARY Michigan State University ABSTRACT SURVIVAL, REPRODUCTION, AND MERCURY LEVELS IN THE TISSUES OF RING-NECKED PHEASANTS CONSUMING TWO MERCURIAL FUNGICIDES BY William J. Adams Female pheasants (Phasianus colchicus) were fed methyl mercury dicyandiamide and phenyl mercuric acetate for 74 days during the reproductive period. Twelve con— centrations of each fungicide were fed daily and five con- centrations were fed every third day. A cumulative consumption of more than 4 mg of methyl mercury dicyandi- amide significantly reduced hatchability of eggs at a rate of 6.8 t 0.5 percent per mg increase in methyl mercury dicyandiamide. Egg production and food con- sumption declined and death occurred after consumption of 15.8 mg i 0.9 mg, 17.0 mg i 3.0 mg, and 24.7 mg i 0.8 mg, respectively. Phenyl mercuric acetate had no effects on egg production, food consumption, or survival. Hatchability was reduced after consumption of 20 mg at a rate of 0.20 i 0.06 percent per mg increase in phenyl mercuric acetate. William J. Adams Based on recommended dosages for seed treatments a wild pheasant would consume 4 mg of methyl mercury dicyandiamide from treated wheat and corn in approximately 6.5 and 0.8 days, respectively. Quantities consumed in excess of 4 mg would be capable of reducing pheasant numbers. The concentration of total mercury in the tissues was related to the relative toxicity of the two compounds. The highest mercury levels were in the tissues of birds that died from methyl mercury dicyandiamide. Greater quantities of phenyl mercuric acetate were consumed, but relatively low concentrations of total mercury were pre- sent in all tissues with brain and breast tissues showing the smallest increase over levels in control birds. The relative distribution of total mercury was similar for both compounds with the greatest concentrations in the kidney and liver followed by breast muscle, and brain and gonad. The proportional increase of total mercury in the tissues as the total quantity of mercury consumed in- creased was more rapid for methyl mercury dicyandiamide than for phenyl mercuric acetate. This was reflected by smaller concentrations of mercury in the feces of the birds fed methyl mercury dicyandiamide. These results indicate that the use of methyl mercury dicyandiamide for seed treatment presents a much greater threat to pheasant pOpulations than phenyl mercuric acetate. SURVIVAL, REPRODUCTION, AND MERCURY LEVELS IN THE TISSUES OF RING-NECKED PHEASANTS CONSUMING TWO MERCURIAL FUNGICIDES BY we ' William J. Adams A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1971 ACKNOWLEDGMENTS My appreciation is given to my graduate committee, especially to Dr. Harold Prince, chairman, and members, Dr. Frank D'Itri, Dr. Howard Johnson, and Dr. Matthew Zabik. I wish to thank Dr. William Youatt, Gordon Zorb, and Martin Pollack, of the Michigan Department of Natural Resources, for providing research facilities and assistance throughout this experiment, and Dr. Dean Haynes for pro- viding statistical advice. Thanks are due to the Michigan Agricultural Experi- ment Station for providing financial support, to the Wisconsin Department of Conservation for supplying the birds for this experiment, and NOR-AM Agricultural Pro- ducts, Inc., Woodstock, Illinois, for providing the mercury fungicides. ii TABLE OF CONTENTS Section INTRODUCTION 0 O O O O O O O O I O O EXPERIMENT I: SURVIVAL AND REPRODUCTION OF RING-NECKED PHEASANTS CONSUMING TWO MERCURIAL FUNGICIDES O O O O I O O O O O I O C Materials and Methods . . . . . . . . Treatments . . . . . . . . . . . Management . . . . . . . . . . . Statistical Analysis . . . . . . . . Results 0 O O I O O O O O O O O I Survival of Adult Females . . . . . . Reproduction. . . . . . . . . . . Discussion . . . . . . . . . . . . EXPERIMENT II: LEVELS OF MERCURY IN THE TISSUES OF PHEASANTS CONSUMING TWO MERCURIAL FUNGICIDES Materials and Methods . . . . . . . . Results 0 O O O O O O O O O O O 0 Distribution of Mercury in the Tissues . . Mercury Content in the Feces . . . . . Discussion . . . . . . . . . . . . LITERATURE CITED. . . . . . . . . . . iii Page mm 16 21 21 22 22 32 34 38 Table 1. LIST OF TABLES The Experimental Design . . . The Wet Acid Digestion Procedure Used to Prepare Tissues for Analysis of Total Mercury. 0 O O O O O 0 Mercury Concentrations in the Tissues of Male and Female Pheasants Fed Methyl Mercury Dicyandiamide and Phenyl Mercury Acetate. . . . . . . . Relationship of the Total Mercury (mg/kg) in the Feces from Hens Fed Methyl Mercury Dicyandiamide and Phenyl Mercuric Acetate with Mercury Concen- trations on the Food (mg/kg). iv Page 23 24 33 Figure 1. LIST OF FIGURES Page The Effects of Varied Concentrations of Methyl Mercury Dicyandiamide Fed Daily on Survival Time of Nine Pheasant Hens . . 7 The Total Weight of Methyl Mercury Dicyandi- amide Consumed by Eleven Pheasant Hens Given Treated Food Each Day. . . . . . 8 Average Daily Food Consumption of Hen Pheasants Influenced by the Cumulative Amount of Methyl Mercury Dicyandiamide Eaten . . . . . . . . . . . . . 10 The Relationship of the Number of Days in Production to the Concentration of Methyl Mercury Dicyandiamide on the Food Consumed Daily and Every Third Day . . . . . . 11 The Relationship of the Number of Eggs Pro- duced to the Concentration of Methyl Mercury Dicyandiamide on the Food Consumed Each Day . . . . . . . . . . . . 12 Hatchability of Eggs Related to Total Amount of Methyl and Phenyl Mercury Consumed by Hen Pheasants . . . . . . . . . . 14 Hatchability of Eggs Related to Total Amount of Methyl Mercury Consumed by Hen Pheasants. . . . . . . . . . . . 15 Distribution of Embryonic Mortality of Eggs from Hens in the Control, Methyl, and Phenyl Mercury Treatment Groups . . . . 17 Distribution of Mercury in the Tissues of 11 Control Birds . . . . . . . . . 26 Figure Page 10. Distribution of Mercury in the Tissues of 7 Hens That Died from Consumption of Methyl Mercury Dicyandiamide . . . . . 28 11. Distribution of Mercury in the Tissues of Male Pheasants as Related to the Total Consumption of Methyl Mercury Dicyandi- amide. . . . . . . . . . . . . 30 12. Distribution of Mercury in the Tissues of Female Pheasants as Related to the Total Consumption of Phenyl Mercuric Acetate. . 31 vi INTRODUCTION The widespread use of mercurial fungicides on cereal grains and other agricultural crops has been a common practice since the 1940's. The use of alkyl- mercury fungicides has been recently restricted and the use of aryl-mercury compounds has been encouraged. The effects of mercury compounds on pheasants (Petoskey 1948, Leedy and Cole 1950, and Carnaghan and Blaxland 1957) and chickens (Heuser 1956, and Smart and Lloyd 1963) have not been considered serious until recently when some of these compounds were linked to cases of mercury poisoning in Swedish wildlife, including pheasants (Borg et a1. 1969, and Tejning 1967a). There is a paucity of data on relative distri- bution, excretion, and lethal mercury concentrations in tissues of pheasants. Borg et al. (1969) studied the effects of methyl mercury dicyandiamide on the survival of male and female pheasants and the distribution and retention of mercury in the tissues. Other members of the order Galliformes have been studied more intensely than pheasants. The distribution and excretion of mercury compounds in chickens have been studied by Kiwimae et a1. (1960), Miller et a1. (1960), Miller et a1. (1961), Swensson and Ulfvarson (1968), Swensson and Ulfvarson (1969), Smart and Lloyd (1963), and Tejning and Vester- berg (1964), and in quail by Backstrom (1969). The purpose of this study is to determine the effects of two mercurial fungicides, methyl mercury dicyandiamide, and a common subsitute, phenyl mercuric acetate, on survival and reproduction of hen pheasants and to determine the relative distribution, excretion, and mercury concentrations in body tissues resulting from the consumption of the two compounds. EXPERIMENT I: SURVIVAL AND REPRODUCTION OF RING-NECKED PHEASANTS CONSUMING TWO MERCURIAL FUNGICIDES Materials and Methods Treatments Two mercurial fungicides were used, Panogen 15, containing 2.2 percent methyl mercury dicandiamide, and Panomatic, containing 3.4 percent phenyl mercuric acetate (Table 1). Adult pheasant hens were fed Purina game breeder pellets ad libitum treated with varied levels of the two mercurial fungicides. The birds were divided into two groups, those fed treated food every day and those fed treated food every third day. There was an array of twelve concentrations of each compound for the daily treatment and five concentrations of each compound for the hens fed every third. There was one hen per con- centration in all treatments. The mercury compounds were sprayed with an aspirator on food pellets in a rotating jar. The food was treated in one kilogram quantities under a forced air hood. Table l. The experimental design.a Mercury Compounds (mg/kg) Treatment Methyl Mercury Phenyl Mercury Dicyandiamide Acetate Daily 0.0 0.0 2.6 3.6 5.1 7.1 7.7 10.7 10.2 14.3 12.8 17.9 15.4 21.5 17.9 25.1 20.5 28.7 23.0 32.2 25.6 35.8 30.7 43.0 Every 3rd Day 0.0 0.0 5.1 7.1 10.2 14.3 20.5 28.7 30.7 43.0 a 0 One hen per concentration. Management The birds were placed in outdoor pens and given three weeks to acclimate prior to receiving mercury treated food. Each female occupied a 3 x 4 meter pen containing natural vegetation. A male was placed with each hen for a 24-hour period every fourth or fifth day for insemi- nation. New food was given each day with the previous day's food being removed and weighed to obtain a measure of consumption. Eggs were collected daily and stored at 10° C for no longer than seven days. They were incubated at 37.5° C, 67 percent humidity, and rotated eight times daily. The eggs were candled and transferred to individual hatching trays on the let day of incubation. All chicks were weighed, banded, and placed in brooder houses. The con- tents of all eggs that were candled and removed or did not hatch were classified by the scheme developed by Hamburger and Hamilton (1951) and Labisky and Opsahl (1958) . Statistical Analysis Standard statistical procedures (chi square and regression analysis) were used (steel and Torie 1958) and all values were tested at 0.05 level. Variation about the mean is denoted by the standard error. Results Survival of Adult Females A11 birds given untreated food survived the 74-day trial. One of 11 hens fed phenyl mercury daily died. Hens given methyl mercury began to die on the 25th day with 9 of 11 fed daily and 1 of 4 fed every third day dying. Two of 4 males died that consumed methyl mercury treated food each day. Using a logarithmic transformation of the data, there was a linear decrease of survival time as the con- centration of methyl mercury on the food increased (Fig. l). The cumulative amount of methyl mercury consumed prior to death was 24.7 mg i 0.8 mg (Fig. 2). The total amount of methyl mercury consumed by the two hens surviving the daily treatment was 11.2 mg and 22.5 mg suggesting they had not yet consumed a lethal quantity. Although the hen that consumed 22.5 mg of methyl mercury had not died, symptoms of acute mercury poisoning were visible when the experi- ment terminated. The first visible indications of poisoning occurred after the hens had consumed a total of 13 to 17 mg of methyl mercury. This was usually about 2 weeks prior to death and there was also cessation in egg pro- duction at this time. Obvious changes in behavior of the hens typically included ruffed head feather, tameness, refusal to attempt flight, and slight incoordination in walking. This progressed into acute ataxia until the .mcmc ucmmmm£d mafia mo mEHu Hm>fl>nsm co haflmp pom mGHEmHUcmmoHU musouma ahsumfi mo mcoflumuucmocoo Umfinm> mo muommmm was .H .mflm 3&2: 89. z. 585: .5152 .._o .028 ad» on own mm 93 ON 2.. a. ram. 0. mg. o6 nu - H q 1 q . q . 4 . q . a O. o. 1 on «m x 066-62....» 8.. .. o~ l on 1 O? J on 1 ow L O p. HIVBO OJ. SAVCI it S.E. @ Died during Exp. Living at termination 24.66! 0.77 of Exp. CUMULATIVE AMOUNT OF METHYL MERCURY CONSUMED (M9) 0 2.5 50 25 IO l2.5 l5 l7.5 20 22.5 25 225 30 325 CONC. OF METHYL MERCURY IN FOOD (Mg/Kg) Fig. 2. The total weight of methyl mercury dicyandi- amide consumed by eleven pheasant hens given treated food each day. birds were unable to fly and walking became difficult. Food consumption diminished from about 58 g per day prior to a 15 mg culminative total consumption of methyl mercury until nothing was eaten during the last two or three days prior to death (Fig. 3). In the most advanced stages of poisoning the birds were unable to walk, muscle twitching of wings and legs occurred, and the birds were often found lying on their chests in a comatose state. Reproduction The hens given untreated food produced eggs during the entire 74-day trial. Fertility of the treatment and control groups was constant throughout the experiment and averaged 96.1 and 90.4 percent, respectively. Egg pro- duction of the hens given methyl mercury declined during the second week of the experiment with hens on the higher concentrations going out of production first. There was a linear decrease in the number of production days after logrithmic transformation as the concentration of methyl mercury on the food increased on the daily and three-day treatments (Fig. 4). No decline in the number of egg production days was observed for either of the phenyl mercury treatments. The total number of eggs laid during the period of treatment by hens fed methyl mercury daily followed the same pattern of decline as the number of days in production (Fig. 5). This is not surprising since AVERAGE DAILY FOOD CONSUMPTION (o) (x: s. E.) Fig. 10 60— 50 4o- 30- H 20 — IO - O 5 IO IS 20 25 225 CUMMULATIVE AMMOUNT OF METHYL MERCURY CONSUMMED (mg) 3. Average daily food consumption of hen pheasants influenced by the cumulative amount of methyl mercury dicyandiamide eaten. DAYS IN PRODUCTION 11 30 I' Treatment Every 3rd.Day 70 - ..‘ ‘ Log is 2.06 - 0.02! x 60 C \ 2 p- \ \ R 3 .974 \\x 40 .- \ \ 30A ‘\\ \Q x 20 . Treatment Daily '0 - Log is I.78l-0.03l x 9 l- 2 a b R-.83 7 _ 6 L l J J P 1 L 1 I J J_ l J 2.5 5.0 7.5 l0.0 l2.5 l5.0 IT.5 20.0 22.5 25.0 27.5 30.0 32.5 CONC. OF METHYL MERCURY IN FOOD (Mo/Kg) Fig. 4. The relationship of the number of days in production to the concentration of methyl mercury dicyandiamide on the food consumed daily and every third day. 12 .hmu some pmfismcoo noom may no OOHEOHOGO>OH© wusoumfi Hmsumfi mo GOHDMHDCOOCOU map on pmonconm homo mo Hmnfisc on» mo mfinmcoflumamn was 3:2: 88 2. $852 $1sz to .028 mean on 0.3 on menu .8 3.. 0.9 nd. ed. as 0.» «MN .m omflm 1 q d u u - q u u q d u u 6.4. . n . N I. Q x -o.o-~._e._.+o3 . N .. o A m I o. I cm .0 Lon 3993 so aaawnN 'IVIOJ. 13 pheasants will lay an egg per 1.25 days (Labisky and Jackson 1969). Hens fed higher concentrations of methyl mercury laid fewer eggs and went out of production sooner than those on lower concentrations. There was a decrease from a high of 40 eggs to a low of 10 eggs for the hens fed methyl mercury every third day. The total amount of methyl mercury consumed prior to the last egg laid by those hens on a daily treatment was 15.8 mg i 0.9 mg and 15.1 mg i 1.3 mg by the hens fed treated food every third day. Egg production stopped after the consumption of approximately 16 mg of methyl mercury, even though this occurred over a period of 10-58 days. There seems to be no reduction in egg production due to any of the phenyl mercury treatments within the limits of this experiment. Both methyl and phenyl mercury compounds affected hatchability (Fig. 6). Hatchability of the eggs laid by hens fed methyl mercury daily increased significantly above the control value of 74 percent to 93 percent until about 3.5 mg of methyl mercury had been eaten. Then hatchability decreased at a rate of 6.8 t 0.5 percent for each additional mg of methyl mercury eaten (Fig. 7). Hatchability decreased at a rate of 0.20 t 0.06 percent for each mg of phenyl mercury consumed on a daily basis. Embryonic mortality in the control and treatment groups was greatest during the first and last parts of PERCENT HATCHABILITY IOO so so 70 so so 40 30 20 IO Fig. 14 is .780— .002 x 2 e e 3.,499 e \ I- \ Phenyl Mercury e P \ i‘ . ‘Methyl Mercury \ .‘s t . I.Os4 -.0 as x 2 R-.962 l l l l l I l J l J l L l IO 20 30 4O 50 60 TO 80 90 IOO llO IZO ISO TOTAL Mq. MERCURY EATEN BY ADULT HENS 6. Hatchability of eggs related to total amount of methyl and phenyl mercury consumed by hen pheasants. 15 0| 0: st 0 O O I l T .h C) 1 (M C) I is l.054 - 0.068 x 2 R8362 e PERCENT HATCHABILITY RD (3 3 T 1, J 1 I 41. 1 1 L l I 14 0 L5 3.0 4:: 6.0 7.5 9.0 l0.5 l2 l3.5 l5 l6.5 Mg. METHYL MERCURY EATEN BYADULT HENS Fig. 7. Hatchability of eggs related to total amount of methyl mercury consumed by hen pheasants. 16 the incubation period (Fig. 8). There was a shift in the distribution of embryonic mortality from hens fed methyl and phenyl mercury compounds. A significant decrease of mortality in the 20-24 day developmental period occurred for both treatment groups with consistently higher per- centages of mortality in the first three developmental periods. The amount of embryonic mortality occurring during the first 14 days of the developmental period was 70, 62, and 48 percent for the methyl mercury, phenyl mercury, and control groups, respectively. Discussion Methyl mercury dicyandiamide affected survival and reproduction in pheasants. There was a range of 31 days between the time the first and last hen went out of egg production and a range of 34 days between the first and last death on high and low concentrations, respec- tively. The average amount of methyl mercury consumed at termination of egg production and at death was 15.8 mg i 0.9 mg and 24.7 mg i 0.8 mg, respectively. The low variation about the mean over the range of concentrations suggests that methyl mercury accumulates in the body in an additive manner which is a function of concentration and time . The hatchability of eggs from hens fed methyl mercury dicyandiamide declined at a rate of 6.8 percent per mg consumed. Borg et a1. (1969) reported a decline 17 I Control Methyl Mercury PII AV .0 E Phenyl Mercury nv Ru o““.... CG1NN‘.‘.. OWOMOOOOOOOODO OHONONOWOWOWOWO’ODODODO. 2 . VII/llllllllz m as ...A mh€v_mw unlu . Embroyanlc Age (5-day range) None.“ N 7”. . .106:...4« we. tone???“ 9 VII/1 .... .vO- . 14000- . 114.0400- a {1100-.- c c. .ODONQNO.0..O..DON.WOWOW..Q...’...D.HOWO.....OW....D.NOWO ?/////////////////////////Z a. nu l b m m o 30L .m . .8. a: 232 methyl, and phenyl mercury treatment Distribution of embryonic mortality groups. of eggs from hens in the control, 8. Fig. 18 in hatchability to 55 percent after feeding pheasant hens 15-20 mg/kg methyl mercury dicyandiamide for 9 days. Based on an average daily consumption of 54 grams of food, their birds would have consumed 8.5 mg which according to our regression equation would give 51 percent hatchability. Similar results have been reported for domestic fowl (Gallus gallus) by Tejning (1967b) who found that after 28 days of continuous feeding methyl mercury dicyandiamide at rates of 18.4 mg/kg and 9.2 mg/kg hatchability was reduced to 10 percent and 17 percent, respectively, as compared to a control value of 61 percent. Successful hatching is related to total accumu- lation of mercury in the bird and in the egg. Eggs which were laid after a small amount of methyl mercury had been consumed (0-4 mg) had much better success in hatching than those laid after consumption of larger amounts (5-12 mg). This suggests that a certain concentration of methyl mercury, and phenyl mercury to a smaller extent, must be accumulated in the hen's body and incorporated into the eggs before hatchability will be reduced. Tejning (1967b) reported similar results with domestic chickens showing that hatchability depends on the concentration of methyl mercury dicyandiamide in the food, number of days the food has been consumed, and the concentration of the mercury in the egg. 19 Phenyl mercuric acetate, in comparison with methyl mercury dicyandiamide, is much less toxic. There were no effects on survival, egg production, or food consumption attributed to phenyl mercuric acetate. Hatchability was affected declining at a rate of 0.20 t 0.06 percent for each mg of phenyl mercuric acetate consumed and did not decline below the control value until 20 mg had been con- sumed. Calculations of L.D.100 by Grolleau and Giban (1966) also show phenyl mercuric acetate to be much less toxic than methyl mercury dicyandiamide. Although no data are available on the amount of mercury consumed by wild pheasants, Tejning (1967a) has estimated the average spillage of seed grain in Sweden to be 1 percent of the total amount planted with an average mercury concentration of 16 mg/kg. Tejning (1967a) and Ulfvarsson (1965) have shown that the high levels of mer- cury found in Swedish pheasants can be attributed to the amount of grain left on the ground after spring and fall planting. In the past, recommended dosages for the use of methyl mercury dicyandiamide in the United States have ranged from 10 mg/kg for cereal grains to 82 mg/kg for corn, peas, soybeans, and navybeans. Based on recom- mended dosages for wheat and corn and an average daily consumption of 60 grams it would take a wild pheasant hen 6.5 days and 0.8 days, respectively, to consume 4 mg of methyl mercury dicyandiamide. The 4 mg level is the 20 point at which hatchability declines below the control value. Consumption of more than 4 mg would seriously decrease hatchability and would result in a cessation of egg production if approximately 16 mg were consumed. We believe that methyl mercury dicyandiamide could limit pheasant numbers if treated seed is available. EXPERIMENT II: LEVELS OF MERCURY IN THE TISSUES OF PHEASANTS CONSUMING TWO MERCURIAL FUNGICIDES Materials and Methods Two mercurial fungicides (NOR-AM Agricultural Products, Inc.) were used, Panogen 15, containing 2.2 per- cent methyl mercury dicyandiamide, and Panomatic, contain- ing 3.4 percent phenyl mercuric acetate (Table 1). Adult hen pheasants were fed Purina game breeder pellets ad libitum treated with varied levels of the two mercurial fungicides. The birds were divided into two groups, those fed treated food every day and those fed treated food every third day. There was an array of 12 concentrations of each compound for the daily treatment and 5 concen- trations of each compound for the birds fed every third day. There was one hen per concentration in all treat- ments except the three-day treatments which had one cock with each hen. The experiment was terminated on day 74 and all birds living at this time were sacrificed for mercury analysis. At least 10 grams of breast muscle and liver and the entire kidney, gonad, and brain were collected 21 22 and frozen in plastic containers for subsequent analysis. Similar samples were collected from all birds that died during the experiment as soon as possible after death. Fecal material was collected and frozen for subsequent analysis on day 10, 18, and 24 of the experiment from the hens fed methyl and phenyl mercury daily. All tissues and fecal samples were prepared for analysis by wet acid digestion according to Zabik (unpub- lished), Dept. Ent. M.S.U., E. Lansing, Mich. (Table 2). Samples were analyzed with a flameless atomic absorption spectrophotometer (Jerrell Ash model 800) for total mer- cury content with the method provided by the Jerrell Ash Company. All values are reported as mg/kg (ppm). Results Distribution of Mercury in the Tissues Samples were analyzed for total mercury content from 11 control hens, ll hens and 6 cocks fed methyl mercury dicyandiamide, and 14 hens and 7 cocks fed phenyl mercuric acetate (Table 3). The mean concentration of mercury in the tissues of the control birds ranged from 0.027 t 0.006 mg/kg in the brain tissue to 0.090 f 0.042 mg/kg in the breast muscle (Fig. 9). There were no sig- nificant changes in the mercury concentrations of 4 hens sacrificed at the start and 7 hens at the conclusion of the experiment. The greatest concentration of mercury 23 .coflumHOmbm OHEODO mmmaoamam an mflmmawcm Ono musouoe HmucoEOHm on COHDOSOOH How womou Bo: mw OHQEmm use .COADmomHU Hmuou mnu ou Ammumz spas as com on amusafia mefiuoflao saflwom I>\3V wom No as om can mummasm maflsaax Iona»; A>\3V wmm No as omv mummasm mafiamaxonasaumufluoaso asHOOM Co as om 00¢ .oumcomcmfiuwm EsflmmmDOQ mo cowusaom concusumm m SDHB HOHOO xcflm unmfla 0 Op oumuufls .coflumomflp vHom onu ou v doum Eoum ucmumcuomsm osu com .mnson Hsom How Ucmum “0H paw OOHMOHOQ somoupws mo HE m HOGOADHUOO cm ppm umoao no: MH coflusHOM 0:» MH .coHDDHOM 30HH0> uzmwa m m>Hm vasonm many “Amuson may unmwcuo>o ocmum OOH mam Ho>oo .ummmflc Ufiom canssmasm map on moflxoumm comoupms wom mo HE m ppfi .cumn 00H map :a H503 0:0 Mom wcmum DOH ocm .:0HumomH© mcu CH desH how an xmmum .Ufiom vansnmasw HE m sufl3 moo manuauhcmo on» omcflm .Anumn 00H :0 Ga 0 cm 0p OOHOOOV Owom OHHDSQHSM pmumuucoocoo mo HE om on manuauucoo 0gp Eoum oucuflmfioonm on“ GUM .m>mm cam Hm>oo .xmmam m oucfl ucmumcuomsm Hoom .mmuschs ma Ooh o x ooom um mmsmfluucwo .Uflom oaumomoHoHsowuu woa mo HE m nuHB Mano HOUQOHQ 05p mmcflm .afis 0H Mom aflom OaumomonoaaONNO woa no as ow guflz manna» m 0H camam D HNMV‘LO mo mamMHMGM How nonmmwu mummmum 0p poms ouswoooum cofluwomww Ufiom #03 one .mmsouofi Hmuog .N mqmdB 4 2 00.0H «H.0H wo.Hm mo.m0 mn.mH n.0m = n.0m : mH mm.0 0m.~H 00.0m n~.Hm mn.mm 0.0m = 0.mm = «H vm.m vm.mH nm.nv mm.mm on.om 0.0m = m.0m = NH mm.mm II mm.mm II 0H.mm n.m~ = o.nH = 0H 00.0H II 0H.0m II 00.0H m.HN = v.mH = m 00.MH II mv.nm II wm.mH m.0m = m.mH = m 00.0H 00.0 0H.0v mv.w0 mn.mH v.mm OOHQ N.0H = n n0.m mm.n Hm.mH om.mH 0m.Hm m.mm : H.m = w OUHEO 0m.0 00.m H0.NH mm.mH mm.n m.vH = m.oH e: mm IHOCmmOHU an.a ma.m mm.HH om.oa mm.m N.CH = 0.N = m succuma vm.o 0m.m mm.n Hm.m 00.0 0.n OOOHmHuomm H.m e: mm Hmnpoz No.0 No.0 no.0 no.0 nH.o 0 = 0 = nH = No.0 00.0 m0.o NH.0 00.0 o = o = m = No.0 no.0 no.0 mo.0 H0.o o = o = 00m = mo.o mo.o 00.0 no.0 H0.o 0 = 0 = mom = 00.0 00.0 mm.0 HH.0 no.0 0 = 0 = mom = no.0 00.0 HH.0 00.0 mH.0 0 = o = Hom = no.0 No.0 No.0 m0.o m0.o 0 = 0 = nmm = .H0.0 Ho.o H0.o H0.0 Ho.0 0 = 0 : 0mm : H0.o H0.0 H0.o Ho.o No.0 0 = o = mmm = H0.0 H0.0 No.0 H0.o Ho.o o = 0 = Hmm = Ho.o Ho.o Ho.0 Ho.0 Ho.o 0 UOOHMHHowm 0 .6 0mm HouuCou UCCOO CHmum >0C©Hx H0>Hq oaomsz umwmum A080 on no Amx\mEv COMM .oz CoHumfidmCOO oumm 0:» mo m xmm 0Com uCoaummue Amx\mEv 05mmHe OCH CH >CCOC02 mo OCOU .mpmuoom musonmfi HmCOCm 0C0 OCHEMHUC0>OHO >usou0§ Hmsuofi 00m mqummozm 0H0E0m 0C0 OHCE mo mmsmmHu OCH CH mCoHumuuCOOCoo >C50H02 .m mamas 25 n I .000 OHHCu mu0>0 0000 wwummup 000 mwuHm* ma.o H~.o Cm.o mo.o C~.o mm.o Co.o m~.0 nm.m 00.m 4H.o 00.H 00.0 00.0 00.0 00.H 00.0 00.0 H0.0 00.0 .HH.aH 00.H n0.0 v0.H 00.H 00.0 n0.0H 00.0 00.0 00.0 00.0 00.H no.0 00.H0 0H.0 00.0H n0.0H 00.nH 00.0H 00.0 00.0 vo.n 00.0 0H.0 0n.0 0H.H nw.H 00.00 0v.HH 00.0H 0n.v 00.0 00.0 0n.0 0H.H H0.0 HH.0 HH.0 00.0 00.0 00.0 00.0 00.0 00.0 00.H 00.0 0v.H 00.H 00.0 00.0 0H.0 00.0H 00.0H 00.0 00.0 0H.0 n0.0 0H.H n0.0 0n.0 HH.0 H0.H 00.H 00.0 0H.0 0H.0 00.0 0H.0 n0.H 0H.0 00.0 H0.0 00.0 0H.0 0H.0 00.n0 nn.HH 00.0 0H.0 00.0 n0.0 wmonHnomm GOHQ umOHCHComm 0.00In.00 H.00I0.vH n.0HI0 00.00 «0.0H «0.0 0 0.00 0.00 n.00 H.00 0.H0 0.nH 0.0a 0.00 n.0H n.00I0.00 n.nl0 00.00 «0.0H 0H.0 0 00 H0 0H mva 0HOH nHmH 000a 00 00 n0 00 00 00 00 00 00 00 0H 00 0H n0 HH H 000a 000H 000H 000a oumumom OHHCUHOE Hmcmsm 0000000 OHCsoumfi Hmcmcm mesm IHchHOHo wusoumfi H0000: 26 .I4 - .l3- 'T" 3 .|2.— XI 18E. x \ — E, .II ~—' .IO r w -- i: 139 - -'_H' .S’.’ *- 00+- 4) 5 .07 — -0. .E .06- -- Z‘ 3 .05 - 4b _ .I- B .04- F" E _ .03 - "' 2 0 ' . 2 I- .2 .0I P 0 Breast Liver Kidney Brain Ovary Tissue Fig. 9. Distribution of mercury in the tissues of 11 control birds. 27 occurred in the breast muscle and the kidney with signifi- cantly lower quantities in the ovary and brain. The liver values were between the two groups. The concentration of mercury in the tissues of 7 of 9 hens which had consumed 24.7 i 0.8 mg of methyl mercury dicyandiamide prior to death showed a significant increase from the control hens and ranged from 12.33 t 1.97 mg/kg in the gonad to 53.62 i 6.39 mg/kg in the kidney (Fig. 10). The breast muscle contained signifi- cantly less mercury than the kidney and liver, but more than either the ovary or brain. Similar results have been reported by Borg et al. (1969) who fed methyl mer- cury dicyandiamide to male and female pheasants until death at a rate of 21 mg/kg food and found concentrations of mercury in mixed liver and kidney to range from 30 to 130 mg/kg and breast muscle from 20 to 45 mg/kg. Tejning (1967b) fed methyl mercury dicyandiamide to chickens for an average of 85.5 days at a mean concentration of 18 mg/kg food and found the mean concentration of mercury in the liver, kidney, breast muscle, and brain to be 55.2, 47.8, 15.8, and 13.3 mg/kg, respectively. The amount of methyl mercury dicyandiamide con- sumed by 5 cocks ranged from 3.42 to 45.3 mg. The cock which consumed the largest amount died on day 41 of the experiment. There are positive correlations between in- creasing consumption of methyl mercury dicyandiamide and levels of mercury in the kidney, breast muscle, and gonad Total mercury in the tissue (mg/kg) 28 GOP - 32:1 3.5. 01' 50- 1 J_ -0 4O " J... CH C) I Hi if a; C) l Breast Liver Kidney Brain Ovary Tissue Fig. 10. Distribution of mercury in the tissues of 7 hens that died from consumption of methyl mercury dicyandiamide. 29 (Fig. 11). The highest rate of increase was in the kidney followed by the breast muscle and gonad. Consumption of 10 mg of phenyl mercuric acetate resulted in significant increases in mercury concen- trations of all tissues analyzed except brain when com- pared to control birds. Consumption of approximately 100 mg of phenyl mercuric acetate resulted in mercury levels in the brain that were higher than control birds. The concentration of mercury in hens continued to increase as additional phenyl mercuric acetate was consumed in all tissues except the breast muscle (Fig. 12). The corre- lation coefficient between the concentration of phenyl mercuric acetate on the food and the concentration of mercury in the breast muscle was not significant (r = 0.49). The highest concentration in the breast muscle was 1.61 mg/kg after 191.2 mg of phenyl mercuric acetate had been consumed. All but three of the values were 0.50 mg/kg or smaller. The greatest accumulation of mercury occurred in the kidney followed by the liver and gonad, breast muscle, and brain. Similar distributions of phenyl mercury com- pounds have been reported for chickens and quail (Swensson and Ulfvarson 1969; Backstrom 1969). Miller et a1. (1960) reported concentrations of ppm phenyl mercuric acetate in the liver and kidney to be about the same, but total mercury concentrations were higher in the kidney than in the liver, similar to this study. 30 .00HEMH0C000H0 0000008 Hhsumfi mo CoHumECmCoo Hmuou may 00 wouMHmu m0 muCMmmmcm onfi 00 mosmmHu 0CD CH 0050005 00 CoHuanuumHo .HH .mHm 3:: 028200220 >582: 3508 00 00:08:28 .20... ac 00 00 on 00 00 0. o. 0 d u H q _ u 1 O 00.0 um x x n~.~ + .m.~_ no :50. 00. 00. 0¢ 9v on on 00 ON 0. q q d O. n .40. . x... med + N0..- .0 00:00 nc O¢ on on 00 00 0. o. 0 ‘ 1 4 u d d $0 1.. x x 8.0 + mo.» .0 0.035 8020 O .O ; 0. ha 10. I w I». 0 Nu ION m [A L00 m. we. a o It o 00 m D «U o. W n. / W 00 J l\ JON 31 .0000000 00050005 H>c0£m mo 00000850000 H0000 0:0 00 0000000 00 000000050 0H0E0m no 0050000 0:» 00 m05000E mo cowu5nfl0umwa 3:: 22000 0.580.: Sauna 00 8:05:28 .20... OON 00. OO. O! ON. 00. On Ow O¢ ON O 1 q _ _ m 0 O $6 4.0 10.0 O . nNoum loo xxnood+-oduuo no. c_o.m OON Om. Om. OS ON. 00. ON 00 O.v ON O fi 4 q 0 fl . 1 04 o o o .mdu~_- 0.. «3.0 +9.06- " 0 v2.00 1 O O. ON On O¢ On 00 Oh Om. OO. O! ON. 00. ON GO O¢ ON O -I J u 5.0 X x bN_.O + 3.6- amen. m a I < x 1 .m0 .000 0N On Om. 00. OS ON. OO. 00 OO 0..» ON O — a # q H q q T q q ofioum .02 08.0 + 08.0 a o bo>fid 1 O O O. ON On 0.» On Om Oh (bx/6w) anssu am ug Amman: IMO]. 32 The hens fed phenyl mercuric acetate every third day tended to show higher concentrations of mercury in the breast muscle and liver and lower concentrations in the kidney than the hens fed similar amounts of phenyl mercury acetate daily (Table 3). The concentrations in the brain and gonad showed no differences for the two treatments. The reason for this difference is not clear and should be investigated further. The amount of phenyl mercuric acetate consumed by 6 cocks, all living at the termination of the experiment, ranged from 4.4 to 152.7 mg. There is a positive corre- lation (r = 0.95) between increasing consumption of phenyl mercuric acetate and levels of mercury in the kidney tissue. This correlation was not significant for any other tissues. The greatest concentration of mercury occurred in the kidney followed by the liver, breast muscle, and brain and gonad. Mercury_pontent in the Feces Mercury concentrations in the feces of birds fed methyl mercury dicyandiamide were lower than the levels in the feces of the birds fed phenyl mercuric acetate and ranged from 0.15 to 3.15 mg/kg and 1.24 to 29.76 mg/kg, respectively, with corresponding concentrations of mercury on the food ranging from 2.6 to 30.7 mg/kg and 3.6 to 43.0 mg/kg, respectively (Table 4). The average 33 om.m0 .mv.¢0 00.0 o.m¢ . om mm.m 00.00 00.m0 ~.~m . mm u- mn.mm u- h.m~ . km 00.0 «v.m m0.0 0.m~ = mm 00.0 .u- m~.m m.0m . vm 00.0 mo.m0 .. m.n0 = mm ll mmoH II MoVH : NN mo.m 00.0 mm.m 0.00 . om mm.~ I- m0.0 m.» . m0 0000000 0m.~ 0m.0 00.0 0.m . m0 00050000 mo.o m0.o mo.o o . 00 00cmgm om.0 mm.0 .. 0.0m . m0 GVoN II VMoO WoMN : @H om.o mm.o 00.o o.mm = m0 .. mo.0 00.0 m.om = N0 00.0 0m.o mm.o m.00 = 00 mmoo II HMoO @omH : m m0.m no.~ mm.o m.m0 .. m ll $0.0 Hmoo N.OH : P m~.o mm.o m0.o 0.0 . m 00000 mm.c .. m0.o 0.0 = 0 -00cmmo00 m0.o mm.o 0m.o 0.m . m 0050000 ~o.o mo.o 0o.o o .o N 000002 «N 000 m0 000 o0 000 .mxme. 00cm .02 000 00 .00 x00 000800009 00000 000 00 Amx\m8v mm 00009 00 .0000 0000 ..m0\mev 0o00 0:0 co 00000000000000 0050008 0003 0000000 00050008 000000 000 0008000000000 0050008 000008 000 0000 8000 00000 000 00 Amx\m8v 0050008 00000 000 00 000000000000 .v 00009 34 concentration of mercury in the feces of the control birds was 0.07 i 0.02 mg/kg. Although mercury concentrations from one time period to the next were not significantly different from one another, the mercury levels in feces of hens fed methyl mercury dicyandiamide tended to increase with time. The correlation coefficients for mercury concen— trations in the feces and mercury concentrations on the food for hens fed methyl and phenyl mercury were, r = 0.77, 0.57, 0.49, and 0.08, 0.67, and 0.98 for day 10, 18, and 24, respectively. The day 10 methyl mercury and day 18 and 24 phenyl mercury r values are significant. The correlation between the concentrations of methyl mercury dicyandiamide fed and excreted decreased with time while the correlation between the concentrations of phenyl mercuric acetate became more apparent with time and subsequent consumption of additional quantities of the compound. Discussion A comparison of methyl mercury dicyandiamide and phenyl mercuric acetate on survival and reproduction of pheasants during a 74-day experiment indicates there are major differences between the effects of these two com- pounds on the birds. None of the birds consuming phenyl mercuric acetate died during the experiment while an average consumption of 24.9 t 0.8 mg of methyl mercury 35 dicyandiamide was lethal to 9 hens. Two cocks that con- sumed 37.0 and 45.3 mg also died. The concentration of total mercury in the tissue was related to the relative toxicity of the two compounds. The highest mercury levels were in the tissues of birds that died from methyl mer- cury dicyandiamide. Although the birds consumed greater quantities of phenyl mercuric acetate, up to 191.2 mg, relatively low concentrations of total mercury were pre- sent in all tissues with brain and breast tissues showing the smallest increase over levels in the control birds. Investigations on chickens and quail by Swensson and Ulfvarson (1969) and Backstrom (1969) have shown that alkyl mercury compounds do accumulate in greater quanti- ties than aryl mercury compounds. Other studies (Swensson and Ulfvarson 1968, Miller et al. 1960, and Miller et a1. 1961) have shown that some phenyl mercury compounds may accumulate in greater amounts than methyl mercury com- pounds. There is a great deal of variation in the rela- tive distribution of mercury when different compounds and different methods (intravenously and orally) are used to get the compound into the bird. Giban (1953) and Miller et a1. (1960) have shown the lethal single oral dose of methyl mercury dicyandiamide and phenyl mercuric acetate in chickens to be 12 to 18 and 60 mg/kg body weight, respectively, indicating that methyl mercury dicyandiamide is more toxic than phenyl mercuric acetate. The proportional increase of total 36 mercury in the tissues as the total quantity of mercury consumed increased was more rapid for methyl mercury dicyandiamide. This was also reflected by smaller con- centrations of total mercury in the feces of birds fed methyl mercury dicyandiamide than in the feces of birds fed phenyl mercuric acetate. The relative distribution of total mercury was similar for both compounds. Similar results have been reported for pheasants by Borg et a1. (1969) and by Tejning and Vesterberg (1964) with chickens using methyl mercury dicyandiamide and by Miller et a1. (1960) and Swensson and Ulfvarson (1969) with chickens using phenyl mercuric acetate. The two cocks that died from methyl mercury dicyandiamide consumed an average of 41.2 mg prior to death as compared to the 24.7 mg consumed by the hens. The mercury concentrations in the tissues of the one cock analyzed were higher than the hens. Borg et a1. (1969) has reported that male pheasants fed methyl mercury dicyandiamide have a longer survival time than females. Backstrom (1969) has also shown for quail that males tend to concentrate greater amounts of methyl mercuric nitrate than females in all tissues tested. He also found that males concentrate less phenyl mercuric nitrate than females in all tissues but liver. The use of methyl mercury dicyandiamide for seed treatment presents a much greater threat to pheasant 37 populations than phenyl mercuric acetate and it appears that females are more vulnerable to alkyl mercury com- pounds than males. LITERATURE CITED Backstrom, J. 1969. Distribution studies of mercuric pesticides in quail and some fresh water fishes. Acta pharmacol. et. toxicol. 27, suppl., 3:1-103. Borg, K., H. Wanntorp, K. Erne, E. Hanko. 1969. Alkyl mercury poisoning in terrestrial Swedish wildlife. Viltrevy 6(4):301-379. Carnaghan, R. B. A., and J. D. Blaxland. 1957. The toxic effects of certain seed-dressings on wild and game birds. Vet. Rec. 69(2):324-325. Giban, J. 1953. Toxicity of ingesting mercuric chloride and fungicide products with a methoxyethyl mercury base. Ann. Epiphyt. 4(l)201-220. Grolleau, G., and J. Giban. 1966. Toxicity of seed dressings to game birds and theoretical risks of poisoning. J. App. Ecol. Suppl. 3:199-212. Hamburger, V., and H. L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49-92. Heuser, G. F. 1956. Feeding chemically treated seed grains to hens. Poultry Sci. 35(1):161-162. Kiwimae, A., A. Swensson, U. Ulfvarson and G. Westoo. 1969. Methyl mercury compounds in eggs from hens after oral administration of mercury com- pounds. Agr. and Food Chem. l7(5):1014-1016. Labisky, R. F., and J. F. Opsahl. 1958. A guide to aging of pheasant embryos. Ill. Nat. Hist. Sur., Bio. Botes No. 39. 4 p. Labisky, R. F., and G. L. Jackson. 1969. Production and weights of eggs laid by yearling, 2-, and 3- year-old pheasants. J. Wildl. Mgmt. 33(3):?18- 721. 38 39 Leedy, D., and C. Cole. 1950. The effects on pheasants of corn treated with various fungicides. J. Wildl. Mgmt. l4(2):218-255. Miller, V. L., P. A. Klavano and E. Csonka. 1960. Absorption, distribution and excretion of phenyl mercuric acetate. Toxicol. and App. Pharmacol. 2(1):344-351. Miller, V. L., P. A. Klavano, A. C. Jestad and E. Csonka. 1961. Absorption distribution and excretion of ethyl mercuric chloride. Toxicol. and Appl. Pharmacol. 3(2):459-468. Petosky, M. 1948. Effects of mercury treated grains on pheasants. Paper presented at Mich. Acad. Sci. Arts and Letters, Ann Arbor, Mich. Smart, N. A., and M. K. Lloyd. 1963. Mercury residues in eggs, flesh, livers of hens fed on wheat treated with methyl mercury dicyandiamide. J. Sci. Food Agr. 14:734-740. Swensson, A., and U. Ulfvarson. 1968. Distribution and excretion of various mercury compounds after single injections in poultry. Acta Pharmacol. et toxicol. 26(1):259-272. Swensson, A., and U. Ulfvarson. 1969. Investigations on the toxic effects of different mercury com- pounds on young, white leghorn cocks. Poultry Sci. 48(5):1567-1574. Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Co., Inc., New York. 481 p. Tejning, S. 1967a. Mercury in pheasants (Phasianus colchicus) deriving from seed grain dressed with methyl and ethyl mercury compounds. Oikos 18(2): 334-344. Tejning, S. 1967b. Biological effects of methyl mercury diacyandiamide-treated grain in the domestic fowl Gallus gallus L. Oikos Suppl. 8:1-116. Tejning, S., and R. Vesterberg. 1964. Alkyl mercury- treated seed in food grain. Poultry Sci. 43(1): 6-11. Ulfvarson, U. 1965. Mercury, aldrin and dieldrin in pheasants. Srensk Kemisk Tidskrift. 77(2): 235-246. "TIT 11111111131) filfliflfliflflifllfilfll’hfi