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1 Glen c. Sanderson, Head Section of Wildlife Research Illinois Natural History Survey Urbana, Illinois GLEN C. SANOERSOf'o. I 6 ~J:J 19ft~ ~~J;~~ f.t(,w ~-~,_) <-<? S01"1E EFFECTS OF INGESTED COPPER SHOT ON 'vj I LD-TRAPPED /-\ALE hal LARDS Glen c. Sanderson, Illinois Natural History Survey, Urbana, IL Paul D. Beamer, College of Veterinary Medicine, University of 11 linois, Urbana, IL Abstract.--Eighty wild male mallards (Anas platyrhynchos) v1ere divided into 8 groups of 10. Each duck in an experimental group was given either 1 No. 4 lead shot or 1, 2, 4, 8, 16, or 32 No. 4 copper shot. The lead shot in the gizzards eroded much faster than the copper shot. The rate of erosion for each copper shot in the gizzard did not decrease with time up to 160 days, the length of the experiment. Although the data are inconclusive, ingested copper shot may cause the death of some captive wild mallards. In ducks that survive, copper shot are retained in the gizzards longer than lead shot and subject the ducks to relatively long periods of exposure to copper. This study did not demonstrate a conclusive effect of ingested copper on body weights of ducks on a diet of corn, and the possible effect of copper on the body weights of ducks on a diet of duck pellets needs additional study. Both morta 1 i ty and v1e i ght losses of cant ro 1 s and of ducks dosed vji th copper shot indicate that corn is an inadequate diet for wild male mallards in captivity for a period as long as 100 days. Ingested lead shot probably results in a decrease in the weights of livers and spleens, and dosing with copper may cause an increase in the weights of these same organs. Numerous cellular changes indicated that copper was toxic to captive wild male mallards on a diet of corn. Several possible effects of copper in ducks on a diet of duck pellets were also indicated by cellular changes. The most pronounced effects I l ~~-4 ~ ~r) c~ ~, a~tr r~-jj,,, ( < )L'-1 ~~-'- I\_ l '.,. J 1("'!. ' ':! )' :", ((...:.,_/,_ -- ; ~/1Cif r: ~ 1_,_, )~-, a~

2 Sanderson et aj. 2 of copper appeared to be on the liver and possibly on spermatogenesis. Results of this study are not conclusive because of the adverse effects of a diet of corn and because of deficiencies in the study; however, the study suggests that copper shot is not a satisfactory substitute for lead shot for hunting waterfowl. Before copper receives further consideration for this purpose, additional studies will need to be made. INTRODUCTION The purpose of this study was to determine the effects of ingested copper shot on wild-trapped male mallards. Because ingested lead shot causes substantial mortality of wild waterfowl each year (Bellrose 1959), there has long been an interest in finding a suitable substitute for lead shot for hunting waterfowl. As the waterfowl resource continues to decline because of losses in habitat, increasing demands are placed on the remaining resource. Thus, the need for a suitable substitute for lead shot for waterfowl hunting has intensified in recent years. Copper is ballistically feasible as a substitute for lead in the manufacture of shot. However, it appeared desirable to conduct additional tests on the effects of ingested copper shot on ducks. Dr. Edward L. Kozicky, Director of Conservation, Winchester-Western Division, Olin, suggested that this study be made. Olin provided funds for duck food, for some equipment, and for analyzing copper residues in the livers of 7 ducks and provided the copper shot. Stewart Laboratories, Inc., Knoxville, Tennessee, analyzed 7 duck livers for copper. Laurie J. Sanderson assisted with the autopsies. The following personnel of the Illinois Natural History Survey assisted with the study: Frank c. Bellrose planned the experiment and provided assistance and advice; Robert Crompton live-trapped the ducks and assisted in many other ways; Dr. Kenneth E. Smith conducted all but 7 of the analyses for copper in the organs of the ducks and analyzed the duck pellets

3 Sanderson et al. 3 for zinc and iron; Stephen P. Havera ran the statistical analyses; and William L. Anderson, James Seets, Ronald L. Duzan, and Ann Cummings assisted with the autopsies and helped in other ways. William L. Anderson and Frank c. Bellrose made suggestions regarding the manuscript and Helen c. Schultz edited the manuscript. METHODS Previous work indicated that females were more susceptible to lead poisoning than males (Jordan and Bellrose 1951:21). Thus, we chose to work with males on the assumption that if copper shot were found to have an effect on males, the effects would probably be even greater on females. Earlier work also indicated that diet had a substantial effect on the toxicity of ingested lead shot in ducks. Ducks on a diet of corn alone were most sensitive to the toxic effects of lead shot (Jordan and Bellrose 1951: 18-20). Thus, we decided to use a diet of corn for the present experiment. Wild mallards were captured in live traps on the Chautauqua Refuge near Havana, Illinois, during the fall of The male mallards captured were examined by fluoroscopy and all those with lead shot in the gizzards or vital organs were released. Eighty apparently healthy male mallards were held in 3 outdoor pens at the Illinois Natural History Survey Laboratory, Chautauqua National Wildlife Refuge, Havana, Illinois. The pens were 9.1 X 3.6 X 0.6 m (30' X 12' X 2 1 ) (Jordan and Bellrose 1950:157). Each pen was provided with an ample supply of running water. The ducks were fed daily all the whole and cracked corn they would eat. The pens were constructed of wire mesh on a wooden frame and had wire bottoms that were approximately.9 m (36 Inches) off the ground. Each bird was banded with a leg band for individual recognition. All birds were weighed and examined by fluoroscopy at the beginning of the experiment (22 November 1972), at various intervals during the experiment, and at

4 Sanderson et al. 4 autopsy. In this report, ''at death" indicates that the ducks were found dead in the pens, either from the dosing with shot or from other causes. Ducks that were sacrificed are indicated to distinguish them from the birds found dead in the pens. The ducks were randomly divided into 8 groups of 10 ducks each. The controls were handled as were the ducks in the other groups except that they received no shot. A second group of ducks each received 1 No. 4 lead shot. The remaining groups of ducks were given, respectively, 1 1 2, 4, 8, 16 1 and 32 No. 4 copper shot each. The copper shot were made from ETP copper, which is essentially 99.9% copper (E. L. Kozicky, Personal communication). The shot were placed In the gullets by menas of a plastic tube passed through the esophagus. Ducks found dead in the pens were tagged with the date of death, weighed, and placed in a freezer (-15 C) for autopsy at a later date. At 100 days after the beginning of the experiment (2 March 1973), 1 duck was selected at random from the control group and from each of the groups dosed with copper shot and was killed by decapitation. All ducks dosed with lead shot were dead before this time. fluoroscopy at this time. Surviving ducks were weighed and examined by The brain, liver, spleen, heart, both kidneys, and both testes from each sacrificed duck were weighed to the nearest 0.1 g. A piece of the brain, liver, spleen, lungs, kidneys, and a piece of one femur were preserved in 10% buffered neutral formalin for histological study (luna 1968). A piece of the brain, breast muscle, liver, and kidneys was frozen for determination of residues of copper. All ducks surviving at 160 days after dosing {I May 1973) were handled in a similar manner except that the testes also were preserved for histological study and the pancreases were weighed and preserved for histological study. Because all ducks were in poor physical condition 100 days after dosing, and some controls as well as dosed ducks had died, the diet of corn was

5 Sanderson et al. 5 changed to a diet of duck pellets. On 30 April 1973 a number of dosed ducks and all remaining controls were stolen from the pens. After these ducks disappeared, the only remaining survivors were 4 ducks that had received copper shot each, 3 ducks that had each received 2 copper shot, 3 ducks that had each received 8 copper shot, and 2 ducks that had each received 16 copper shot. RESULTS Rate of Erosion of Lead and Copper from Shot in the Gizzard In the 6 ducks with 1 lead shot in each gizzard at death, from 77.7 to mg (mean, mg) of lead had been eroded from each shot at the time of death (Table 1). The rate of loss of lead was 7.55 mg per shot per day. Because these ducks had been given only I shot each, the rate of loss of lead was also 7.55 mg per duck per day. The ducks dosed with lead survived for 10 to 25 days (mean, 17.2 days) after dosing (Table 1). In the 6 ducks killed 100 days after dosing, there was an average erosion of mg of copper per shot per day or an average erosion of mg of copper from all of the shot per duck per day (Table 2). In the 12 ducks sacrificed 160 days after dosing, there was an average loss of mg of copper per shot per day and an average loss of mg from all of the copper shot per duck per day (Table 2). Thus, the rate of erosion per shot increased slightly after the diet of corn was replaced by duck pellets. The rate of erosion per shot might be expected to decrease with time as there would be progressively less surface on each shot; however, Irwin et al. (1974:5) have found that lead shot erode more rapidly when ducks are fed a pellet diet than when fed a diet of corn. In the 21 ducks that died after dosing with copper shot (26 to 122 days after dosing, mean, 60.7 days), there was a loss of mg of copper per shot per day and mg from all of the copper shot per duck per day (Table 3).

6 Sanderson et al. 6 There were no significant differences ~?0.05) in the mean rates of copper eroded per shot per day or per bird per day among the ducks autopsied at 100 or 160 days and ducks that died in the pens. Also, there were no significant differences {P70.05) in the rates of copper erosion per shot per day between the mean for each of the 3 groups and the mean rate for all ducks. The differences were not significant between the mean rate of copper eroded per duck per day for all ducks and for ducks autopsied 100 days after dosing and for ducks that died in the pens. The only significant difference (f<0.05) in the rate of copper eroded per bird per day was the lower rate for ducks autopsied 160 days after dosing compared with the mean rate for all ducks. Effects of Copper Mortality.--The average survival time of 21 ducks that died after being dosed with copper shot was 60.7 days, with a range from 26 to 122 days (Table 3) and after losing an average of 30.8% of their body weight. For comparison, all 10 ducks that were dosed with 1 lead shot each died between 10 and 25 days (mean, 17.2 days) after dosing (Table I) and after losing an average of 33.6% of their body weight. Six of 10 controls died from 10 to 116 days (mean, 58.2 days) after dosing and after losing an average of 25.7% of their body weight (Table 3). Three controls were stolen and I was sacrificed 100 days after dosing. These data appear to indicate no increased mortality caused by dosing with copper shot. In the 17 ducks that retained all the copper shot in their gizzards at death, there was an average loss of 5.6 mg from the copper per bird per day and mg of copper eroded from all of the shot per duck by the time they died at an average of 62.0 days after dosing (Table 4). The correlation coefficients between the number of days to death and the amount of copper eroded from all of the shot per bird per day (!, ) and the total amount of copper eroded per bird (~, ) were not significant.

7 Sanderson et al. 7 The 10 ducks dosed with I lead shot each died from 10 to 25 days after dosing (mean, 17.2 days). From 77 7 to mg (mean, mg) of lead had been eroded from the shot in each of 6 of these ducks. The rates of erosion of lead are based on only 6 ducks because the lead shot were passed from the gizzards in 4 ducks prior to death (Table 1). Based on the calculated rate of erosion of copper in the gizzards, in only 4 ducks dosed with copper was as much as mg of copper eroded in 17.2 days (Table 4), the average amount of lead eroded from the shot in each duck by the mean time of death for the ducks dosed with lead. Thus, copper might be more nearly as toxic as lead to ducks if the actual rates of physiological exposure were the same for both. In the 17 ducks that expelled no copper shot prior to death, an average of only 95.1 mg of copper (range, mg) was eroded from all of the shot in 17.2 days. Two (No. 175 and No. 186) of the 4 ducks in which more than mg of copper was eroded from all of the shot in 17.2 days were the first birds to die among ducks that retained all the copper shot administered (Table 4), and (No. 187) was the fourth duck in this group to die. However, in duck No. 161 that died 39 days after dosing, only 30.9 mg of copper was eroded from all of the shot in its gizzard in 17.2 days, and duck No. 191 that recieved mg of copper from all of the shot in its gizzard in 17.2 days did not die until 104 days after dosing. Of the 17 ducks that expelled no copper shot from their gizzards prior to death, 13 survived for 46 days or longer. In these 13 ducks, erosion of copper from the shot in their gizzards was at a slower than average rate in all but 1 (No. 191, Table 4). Thus, the toxicity of copper shot to ducks may be partially determined by the rate at which the copper shot is eroded in the gizzard. The conditions in a duck that cause the copper shot to erode at a faster rate may also increase the susceptibility of that duck to copper toxicity. These data

8 Sanderson et al. 8 suggest that ingested copper may cause the death of some ducks even though the average survival times of controls and ducks dosed with copper shot were not significantly different. It is not unusual for ducks dosed with lead shot to pass the shot before complete erosion occurs (Jordan and Bellrose 1951:22). In the present experiment, 4 of 10 ducks (40.0%) dosed with 1 lead shot each had passed the lead shot prior to death, 14 to 25 days after dosing (Table 1). Of 39 ducks dosed with copper shot and subsequently examined, only 8 (20.5%) had passed 1 or more shot each, and these 8 ducks passed only 58 of a possible 410 shot {14.1%, Table 2, 3). The lead shot were absent from the gizzards in an average of 17.2 days after dosing (Table 1) and the copper shot in an average of days. Size of the pellet may be an important factor determining when a shot is passed or the physiological effects of lead intoxication may be involved. In any case, it is apparent that in the absence of death, copper shot will be retained in the gizzard;for a longer period than lead shot and will subject the duck to a relatively long period of exposure to copper. Body Weight of Surviving Ducks.--After 28 and 100 days on a diet of corn, there were no significant (E~0.05) differences in body weights of surviving ducks among the control group and the 6 groups of ducks dosed with copper shot (Table 5). As indicated above, at 100 days after dosing the diet was changed from corn to duck pellets because all surviving ducks, including nondosed controls, were in poor physical condition. By 160 days after dosing, when all surviving ducks were sacrificed, the weights of the ducks dosed with I copper shot each were significantly ~'0.05) heavier than the weights of birds dosed with 2, 8, and 16 shot each. There were no significant differences among the mean weights of the latter 3 groups, and no controls or ducks dosed with 4 shot each were available for comparison. The 1 surviving duck dosed with 32

9 Sanderson et al. 9 copper shot had only 1 shot in its gizzard 160 days after dosing and its body weight was similar to the weights of ducks dosed with I shot (Table 5). Thus, dosing with more than I copper shot may have inhibited weight gains of ducks on a diet of duck pellets in comparison with the weight gains of birds dosed with only I copper shot each. Body Weights of Ducks That Died~ the Pens.--There were no significant differences (f~o. 10) 1 at the start of the experiment, among the mean weights of the 8 groups for the ducks that died in the pens. There were no significant differences (P~0.25) among the mean weights of the ducks in the 8 groups at death (Table 6). Differences among weights of ducks dosed with 2 1 8, 16, and 32 copper shot all approached significance; however, average weights of ducks that died in the pens after dosing with 16 or 32 copper shot were higher than the average weights of the other ducks. The small sample sizes probably contributed to the lack of significance in the body weights of ducks that died in the pens. we conclude that this study has demonstrated no clear effect of dosing with copper shot on the body weights of male mallards on a diet of corn, and that the possible effect of copper on the body weights of male mallards on a diet of duck pellets needs further study. Effects of Diet ~ Body Weight.--Diet apparently had a greater effect on body weights than dosing with copper shot. All surviving ducks, including controls, lost weight from 0 to 28 to 100 days after dosing on a diet of corn and then gained weight between 100 and 160 days after the diet had been changed to duck pellets {Table 5). The mean weight of all 80 ducks at the start of the experiment was g, by 100 days after dosing the mean weight of 46 surviving ducks was g {the low point in body weight), and the mean weight of 12 ducks surviving at 160 days (all dosed with copper shot) was 1,286 g.

10 Sanderson et al. 10 The mean body weight (910 g) of the 7 ducks sacrificed 100 days after dosing (Table 7) was significantly Jess (f~o.ool) than the mean body weight (1,286 g) of the 12 ducks sacrificed 160 days after dosing (Table 8). We conclude that corn alone is an inadequate diet for wild male mallards in captivity. Organ Weights.--For the 6 ducks sacrificed 100 days after dosing (Table 7), the correlation coefficients between the number of copper shot dosed and weights of the brain, liver, spleen, heart, kidneys, and testes and the ppm of copper in the liver were not significant (f>o.os). The ppm of copper in the liver was positively correlated with the number of shot given whereas all organ weights were negatively correlated with the number of shot. These ducks were on a diet of corn. Although the small sample size apparently precluded statistical significance, the weights of testes and spleens appeared to be adversely affected by dosing with copper shot. The effects of copper shot on weights of the kidneys, heart, and brain were less evident. There was some indication that dosing with copper shot caused an increase in the weight of the liver (Table 7). As noted below, there were marked cellular changes in the livers, spleens, and kidneys of copper-dosed ducks after 100 days on a diet of corn. For the ducks dosed with copper shot, placed on a diet of duck pellets at 100 days after dosing, and sacrificed 160 days after dosing (Table 8), none of the correlation coefficients between the number of copper shot given and the organ weights was significant (f~o.lo). The correlation signs of the weights of the spleen, heart, and kidneys were positive whereas at 100 days they were all negatively correlated with the number of shot. The change in diet at 100 days may have been responsible for the change in the signs of the correlation coefficients.

11 Sanderson et a The average weights of the brain (f.70.20), spleen (f.::>-0.20), heart (f..c: 0.02), and kidneys (f.::>-0.10) all increased from 100 to 160 days with the improved diet, and the livers decreased (f...::o.oi). Copper residues in the livers of the 12 ducks sacrificed 160 days after dosing with copper shot averaged 62.0 ppm wet weight (range, ) whereas copper residues in 6 ducks sacrificed 100 days after dosing with copper shot averaged 4.98 ppm (range, ). Thus, any changes in organ weights between 100 and 160 days after dosing can probably be ascribed to the improved diet. Apparently the more than 10-fold increase in copper residues in the liver from 100 to 160 days after dosing were a result in the change of diet from shelled corn to duck pellets. The most dramatic change was In the weights of testes of ducks sacrificed 100 and 160 days after dosing. On day 100 (2 March) the control bird's testes weighed 2.0 g, 5 times as much as the testes of No. 178 (0.4 g), the dosed bird with the heaviest testes {Table 7) The mean weight (0.22 g) of the testes from the 6 copper dosed ducks on day 100 was significantly (f.c:::o.ool) less than the mean weight (10.28 g) of the testes from the 12 ducks (all dosed with copper) sacrificed 160 days (1 May) after dosing (Table 8). The mean weight of 2 testes of 2 mallards collected in 1 of the Royal Parks in London during February and March was g {Hohn 1947:288). No control birds were available 160 days after dosing (I May) for our study; however, the smallest testes weighed 5. I g, and the mean weight for all birds dosed with copper was (Table 8). In spite of the dramatic changes in weights of testes with the improved diet and possibly as a result of seasonal changes, the lack of control birds precludes the conclusion that dosing with 1 Qr more copper shot did not affect weights of the testes. The heaviest testes among the dosed ducks weighed 19.1 g. The smallest testes {5.1 g) were in the I surviving duck of the group that was dosed with 32 copper shot, but this duck retained only I copper shot in its gizzard at autopsy. The mean weight of 2 testes of 3 mallards

12 Sanderson et al. 12 collected in one of the Royal Parks in London during May was g (Hohn 1947:288). The effects of a corn diet on weights of testes in captive wild mallards seems to be more important than the effects of copper in the diet. Sanderson et al. (In Press) reported the weights of wild male mallards live trapped in Illinois on 20 March 1974 and placed in captivity at Havana, Illinois (in the same pens used for the present study). These ducks were given a diet of duck pellets and shelled corn until their study began on 15 May 1974, when the diet consisted of shelled corn. At the end of the experiment on 12 June 1974, the average weight of two testes from 9 control ducks (not dosed) was 1.4 g and for 9 ducks each dosed with 5 No. 4 steel shot the average weight was 1.3 g. On the basis of the data cited by Hohn (1947:288) and the weights, after a diet of duck pellets for 60 days, of testes of ducks dosed with copper in the present study, it seems that a combination of captivity and a diet of corn suppresses growth of the testes in captive wild mallards. There were no significant differences ~~0.25) among the weights of testes of dosed and undosed ducks that died (Table 9) The mean weights of the testes of these ducks were 0.18 g for controls and 0.20 g for ducks dosed with lead or with copper. All these weights are in the same range as the weights of the testes of the copper-dosed ducks sacrificed 100 days after dosing (Table 7). Thus, it appears that the physiological parameters associated with the deaths of the control birds may have prevented the testes of these birds from growing normally. Two of the control birds died 116 days after dosing. Their testes weighed 0.3 and 0.2 g, respectively, much less than the 2.0 g that the testes of 1 control duck weighed 100 days after dosing (Table 7). This control duck may have had enlarged testes because of some factor not related to our tests. Before we can make definitive conclusions regarding the effects of copper on the testes of mallards, it will be necessary to conduct additional tests.

13 Sanderson et a Histological studies were not made of the testes of the ducks sacrificed 100 days after dosing, but such studies would have been desirable. Mean weights of brains (f.>0.25) and hearts (f.~o.lo) were also not significantly different at death among controls, ducks dosed with lead, and ducks dosed with copper {Table 9). The weights of livers were not significantly different (f.~0.05) in all groups; however, livers of ducks dosed with lead weighed the least and livers of copper-dosed ducks weighed the most (Table 9). Although weights of spleens were not significantly different (P~0.25) among the 3 groups, spleens showed the same kinds of weight changes as livers. The weights of livers and spleens from the ducks dosed with lead shot were significantly lower ~~ 0.001) than the mean weights of these organs for all ducks not dosed with lead that died after dosing (Table 9). The weights of kidneys were not significantly different (f.::::-0.25) among the 3 groups, but kidneys from ducks dosed with copper weighed more than the kidneys from the other 2 groups. There were no significant differences (P>O.lO) in weights of pancreases among the 3 groups, but the weights of pancreases from ducks dosed with lead were less than those from the other 2 groups. We conclude that dosing with lead shot probably results in a decrease in the weights of the livers and spleens in wild male mallards in captivity and that dosing with copper shot may cause an increase in the weights of these two organs. Cellular Changes.--A lack of change in the gross weight of an organ does not indicate that changes have not occurred. Thus, we attempted to correlate cellular changes in various organs with dosing with copper. As with the weight data, our understanding is hampered by the toss of all remaining control birds as well as several of the birds dosed with copper shot prior to the end of the

14 Sanderson et al. 14 experiment. Also, ducks that died in the pens were frozen until they were autopsied at a later date. No attempt was made to do histological studies of the organs of these birds. One duck from each surviving group was sacrificed 100 days after dosing (all ducks dosed with lead were dead prior to this time), and various organs were examined for possible effects of dosing with copper. All, or selected representatives of these tissues, were stained with hematoxylin and eosin, Prussian blue, ammoniacal silver, periodic acid Schiff, and Sudan black. The most prominent changes were found in the livers, kidneys, and bone marrow. The alterations found in the liver and kidney were indicative of intoxication. The bone marrow hypoplasia and hemosiderin deposits are consistent with excessive antemortem blood destruction. Copper poisoning in mammals is said to be accompanied by destruction of brain tissue. Some investigators have described brain cavitation. Such changes were not present in these duck brains. Since the changes were qualitatively similar and varied quantitatively in relation to the numbers of administered copper shot, we have described the tissues as a group rather than as individual ducks. LIVER- The liver cells were diffusely swollen and vacuolated. Karyolysis was well marked. Karyorrhexis was less prominent. There was well-marked deposition of hemosiderin in all ducks dosed with copper shot, suggesting excessive antemortem blood destruction. Similar pigment was present in the control bird (No. 133), but in limited quantity. The hepatocytes probably are vacuolated by reason of water inhibition. The fat stains and glycogen stains were negative. Liver damage in mammals that is related to copper poisoning is said to be centrilobular fatty metamorphosis. SPLEEN- There was well-marked hemosiderin deposition in the splenic white pulp of the birds dosed with copper shot. Only trace hemosiderin was found in the spleen of the control duck.

15 Sanderson et aj. 15 KIDNEY- Well-marked tubular degenerative changes and necrosis were present in the birds dosed with copper shot. The glomerular tufts often had a smudged appearance. Additionally, in the glomeruli of some of the birds that received larger numbers of shot, there were spicules of eosinophilic material about I or 2 microns by 6 or 8 microns. These occurred in clumps with the spicules arranged parallel to each other. The physical and chemical character of these spicules was not determined but their appearance suggested that they were crystalline structures. LUNGS - Moderate to large amounts of dark brown to black pigment was present around many bronchioles. This pigment was both intracellular and extracellular. It reduced ammoniacal silver, suggesting that it was melanin. Traces of hemosiderin were present adjacent to bronchioles of birds No. 155 and No BONE MARROW- There was well-marked reduction in numbers of hematopoietic elements in the bone marrow of those birds receiving copper shot as compared with the control bird. The cellular hypoplasia appeared to be roughly proportionate to the numbers of shot administered. BRAIN- Any alterations present in the brain were subtle and limited. There appeared to be some widening of the perivascular spaces, suggesting edema. Two arteries with microhemorrhage in the adventitia and adjacent perivascular space were seen in the brain of bird No There was occasional satellitosis, neuronophagia, and focal gliosis indicating neuronal necrosis. We do not know, however, that there changes suggest neuronal loss beyond normal neuronal loss in ducks. Only 12 ducks, all dosed with copper shot, remained 160 days after dosing. The other birds died, were sacrificed 100 days after dosing, or were stolen. The 12 remaining ducks were sacrificed 160 days after dosing (Table 2, 8) and histological examinations were made of the testes, spleens, kidneys, brains, lungs, livers, and pancreases.

16 Sanderson et al. 16 TESTES - There was active spermatogenesis in the testes of 7 ducks. Spermatogenesis was less active in No. 153 (2 Cu shot, testes 7.7 g) and No. 174 (8 Cu shot, testes II. 1 g). Spermatogenesis was moderate in No. 183 (16 Cu shot, testes 13.2 g) and low in No. 179 (16 Cu shot, testes 7. I g). Spermatogenesis was poorly to moderately active in No. 189, the bird that was dosed with 32 copper shot and whose testes weighed only 5 1 g at autopsy. apparent morphologic cause for the reduced spermatogenesis. There was no The mean weight of 2 testes in the 7 ducks with active spermatogenesis (11.30 g) was not significantly different (P~ 0.20) from the mean weight (8.84 g) of 2 testes in the 5 ducks with less active spermatogenesis. LIVER - Four (Nos. 150, 168, 174, and 179) of the 12 ducks dosed with copper shot and sacrificed 160 days after dosing had relatively large numbers of lymphocytes at the hepatic portal area; however, we could not say for certain that they were beyond the normal limits for ducks. The liver of No. 139 showed moderate fatty changes, and the liver of No. 174 had moderate hemosiderosis. The mean weight (13.30 g) of these 5 livers showing some possible abnormalities was significantly (f~o.oi) lower than the mean weight ( g) of the livers from the remaining 7 ducks in the group. The mean weight of livers from the 12 ducks was g (Table 8). All these weights are significantly (P~O.OI) lower than the mean weight of g (Table 7) for the livers of 6 ducks dosed with copper and sacrificed 100 days after dosing, while on a diet of corn. It appears that copper had an effect {both on weight and on cellular changes) on the livers of ducks on a diet of corn and on the livers of at least some of the ducks after they had spent 60 days on a diet of duck pellets after 100 days on the diet of corn. SPLEEN- The spleen of one duck (No. 139) showed well-marked hemosiderin deposition, and there was moderate hemosiderosis in 4 spleens (No.s 140, 174, 183, and 189) and limited hemosiderosis in one spleen (No. 179). The spleen of

17 Sanderson et al. 17 No. 183 also showed moderate to marked amyloidosis. The mean weight (0.78 g) of the spleens of these 6 ducks was not significantly heavier {f>0.05) than the mean weight (0.47 g) of the spleens of the remaining 6 ducks in the group. The mean weight (0.62 g) of spleens from the 12 ducks sacrificed 160 days after dosing (Table 8) was not significantly heavier (f?o. 10) than the mean weight {0.38 g) for the spleens from the 6 copper-dosed ducks sacrificed 100 days after dosing {Table 7). It appears probable that copper had an effect (both on weights and on cellular changes) on the spleens of the ducks dosed with copper shot while the birds were on a diet of corn, but that copper had less effect on the spleens of ducks dosed with copper shot after the birds were placed on a diet of duck pellets for 60 days. BRAIN - In 1 duck {No. 139) dosed with 1 copper shot, the brain showed many neuron cell bodies encircled by glial elements, suggesting that the neurons were undergoing toxic degeneration. Because the brain of this duck was the only that showed these symptoms, we cannot say that they were caused by dosing with copper. LUNGS, KIDNEYS AND PANCREASES - No significant histological changes were noted in any of the lungs, kidneys, or pancreases of the 12 ducks sacrificed 160 days after dosing. Pancreases from the birds sacrificed 100 days after dosing were not examined, and the effects noted in the lungs and kidneys of the copper-dosed ducks 100 days after dosing, while on a diet of corn, had disappeared after 60 days on a diet of duck pellets. We observed numerous cellular changes indicative of copper toxicity in wild male mallards in captivity on a diet of corn and dosed with copper shot. The data suggest that inhibition of normal growth of the testes may be one effect of copper toxicity in ducks on a diet of corn. Several possible effects of copper

18 Sanderson et al. 18 toxicity in ducks on a diet of duck pellets were indicated by cellular changes. Possible effects of copper toxicity on spermatogenesis and on the livers were noted in ducks on a diet of duck pellets. DISCUSSION Suttle and Mills (1966) in a study of copper poisoning in pigs showed that high intakes of copper raised the requirements for zinc and iron. Slight increases in zinc and iron in the ration prevented adverse effects from copper at the rate of 250 ppm or higher. Underwood {1971:103) stated that 11 it is already evident that the toxicity of a particular dietary intake of copper is determined not only by the level of copper, but by the ratio of this level to the dietary levels of zinc, iron, calcium, molybdenum, inorganic sulfate, and of other dietary components, some of which have yet to be identified. Copper intakes alone can be as misleading in determining potential copper toxicity as they are in determining potential copper deficiency. 11 Anderson and Stewart {1969:260) showed that samples of corn from 3 counties in Illinois contained from 19 to 28 ppm zinc and from 27 to 50 ppm iron. The duck pellets fed to ducks in our experiments contained 61 ppm zinc and 105 ppm iron. Thus, some or most of the effects of copper toxicity in the ducks in the present study might be related to an added requirement for zinc and iron, which were low in the corn diet, in the presence of a high level of copper. Lindow et al. (1929) reported that individual tissues and species differ in their ability to vary their copper content with differences in intake of copper. The copper content of the liver, kidney, blood, spleen, and lungs changes most readily and of the muscles, heart, and endocrine glands least readily. Underwood {1971:62) reported that the ppm of copper on the dry basis in the liver of a mature domestic fowl on a normal diet was 14.8 ppm and in

19 Sanderson et al. 19 mature domestic ducks on a normal diet was 153 ppm (about 36 to 48 ppm wet weight). He also reported (p. 63) that ducks tend to carry higher levels of copper in their livers than chickens and turkeys on simi Jar diets. This level of about 36 to 48 ppm wet weight of copper in the livers of ducks on a normal diet is compared with the mean of 4.98 ppm (wet weight) of copper found in the livers of the 6 ducks dosed with copper shot and 0.94 ppm in the 1 iver of the control duck (Table 5) after 100 days on a diet of corn, perhaps giving further evidence that the ducks in the present study were affected more by an imbalance of minerals {relative deficiency of zinc and iron) in the diet of corn rather than by an excess of copper. Twelve ducks dosed with copper had a mean of 62.0 ppm copper in their livers (Table 6) after 100 days on a diet of corn and 60 days on a diet of duck pellets. Our results are not conclusive, largely, we believe, because of the adverse effects of a diet of corn and because several ducks were stolen prior to the end of the study. However, possible adverse effects of copper on spermatogenesis and cellular changes indicating copper toxicity, especially coupled with a diet of corn, are enough to suggest that copper shot is probably not a satisfactory substitute for lead shot for hunting waterfowl. As a minimum, additional studies should be made with copper before it is given further consideration as a substitute for lead in shot for waterfowl hunting. One factor to consider is that copper shot appears to remain in the gizzard for a longer period than lead shot. Thus, copper shot would expose ducks to a relatively longer period of toxicity than lead shot. LITERATURE CITED Anderson, W.L., and Peggy L. Stewart Relationships between inorganic ions and the distribution of pheasants in Illinois. J. Wildt. Manage. 33(2): 254-2]0.

20 Sanderson et al. 20 Bellrose, F.C Lead poisoning as a mortality factor in waterfowl populations. Illinois Nat. Hist. Surv. Bull. 27(Article 3): Hohn, E.O Sexual behaviour and seasonal changes in the gonads and adrenals of the mallard. Proc. Zool. Soc. London 117 (Parts I I and I II): Plates 1 and 2. Irwin, J.c., L.N. Karstad, and R.G. Thomson Progress report on lead poisoning in waterfowl March 1973 to March Univ. Guelph, Guelph, Ont. 6pp. (Mimeo). Jordan, J.S., and F.C. Bellrose Shot alloys and lead poisoning in waterfowl. Trans. North Am. Wild). Conf. 15: ' and Lead poisoning in wild waterfowl. Illinois Nat. Hist. Surv. Bioi. Notes No pp. Lindow, c.w., w.h. Peterson, and H. Steenbock The copper metabolism of the rat. J. Bioi. Chern. 84(1): Luna, L.G., ed Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. The Blakiston Division, McGraw Hill Book Company, New York. 258pp. Sanderson, Glen c., Horace w. Norton, and Sarah S. Hurley. ingested lead-iron shot on wild-trapped male mallards. Some effects of 111. Nat. Hist. Survey Bioi. Notes (In Press). Suttle, N.F., and c.f. Mills Studies of the toxicity of copper to pigs. Brit. J. Nutr. 20(2): and Underwood, E.J Trace elements in human and animal nutrition. 3rd ed. Academic Press, New York and London. 543pp.

21 1 Table 1. Wild-trapped Mallard Drakes Dosed with 1 Pb Shot and Autopsied at Death. Number of Pb Shot Days Percent Weight Total Mg Total Mg Pb Eroded Percent of Total ~ At to Body of Pb Shot of Pbb Percent Weight per Shot Weight of Pb 1/1 a 0 Number Given Autopsy- Death Wt. Lost at Autopsy E rodec:fj of Pb Eroded per Day Eroded per Day Ql I I I I l to n l Average ! In gizzard. Average weight of one Pb shot at time of dosing = mg. 1/ Ql ::;, Cl, ~ r+

22 1 Table 2. Wild-trapped Mallard Drakes Dosed with Various Numbers of Copper Shot and Autopsied 100 Days (2 March 1973) or 160 Days After Dosing (1 May 1973). - No. Days Autopsied After Tota J mg Vl Ql Dosing Number of Cu Shot Percent Av. Weight of Cu Eroded Total Mg Cu Eroded eer Day Percent of :I 0.. and Band At Body 1 Cu Shot atb Per Per Percent Weight Per (I) a Per Total Weight of., Number Given Autopsy- Wt. Lost Autopsy (mg)- Shot Bird of Cu Eroded Shot Bird Cu Eroded per Day ~ I 00 Days Ql I o o o J , o. 24 Average days I # 4.662~ Days I o I I I o J a. 281 o I J o J7.67 o o. II o I , I ). 5 1, Table 2 - continued. :I (I) rt

23 l Table 2. Continued. Average days + ].2 Average to 160 days &d o.4o~ 0.442~ 2. 49aJ' ~ ~ In gizzard v ~ ~ ~ ~ ~ ~ ~ ~ ~Average weight of one copper shot at time of dosing = mg. ~ Not including the contro~oucks that passed 1 or more shot were not used in tabulating this figure. d ~Wei~hted average.

24 raore J wrra-trappea Mar rara urakes uosea wttn varrous Numbers of Copper Shot and Autopsied at Death. A 11 These Ducks Were Found Dead in the Pens. Total mg Mg Cu Eroded Number of Cu Shot Percent Av. Weight of Cu ErodecF Total E!er Da~ Percent of Total Band At Days to Body 1 Cu Shot atb Per Per Percent Weight a Per Per Weight of Cu Number Given Autopsy- Death Wt. Lost Autopsy (mg)- Shot Bird of Cu Eroded Shot Bird Eroded per Day - v :s R , VI :s 25.8 (D " o I ] o o o o , o.4oo , Average rfi o.45jf. 4.63oft o. 25 Table 3 - continued.

25 l Table 3 Continued.!!. In gizzard. ~Average weight of one copper shot at time of dosing = mg. ~We assumed that all shot weighed mg when dosed~average date to death of ducks that retained all shot was 62.1 days ~--- ~ t ~ Ducks that passed one or more shot were not used in tabulating this figure. f - Weighted average. v Cll j Q B.., "' 0 j ~,.. Cll

26 Sanderson et a 1. Table 4. Rate of Erosion from Copper Pellets in Gizzards and Survival Time for Male Mallards That Died While Retaining All Copper Shot Placed in Their Gizzards. Amount of Co~~er Eroded ~er Bird (mg} Duck Days to a b At Death Number Death 17.2 Days- - Per Day Total ' so so.o I o , ]2.0 Average s a -Average survival time for 10 male mallards each dosed with 1 lead shot. b A. - ssum1ng a uniform rate of erosion from dosing to death.

27 Sanderson et al. Table 5. Mean Body Weight of Surviving Ducks at Dosing, 28, 100, and 160 Days After Dosing. Number Mean Body Weight of Surviving Ducks {g} Shot 28 Days 100 Days 160 Days Day of Dosed After Dosing After Dosing After Dosing Dosing 0 a 1 ' a:=- 1, , Pb 1 ' Cu 1, ' , , Cu 1 ' , b 1, , Cu 1, , , Cu 1, , c 1, ' Cu 1, , , , Cu 1, , a - Number of ducks. b - One duck had only copper shot at autopsy. - c One duck had only 5 copper shot at autopsy. d -Only 1 copper shot in gizzard at autopsy. d 1 ' ,

28 Sanderson et a 1. Table 6. Mean Body Weight of Ducks That Died in the Pens. Ducks Were Dosed on 22 November Number Shot Mean Weight of Ducks Mean Number Number Dosed That Died in Pens (g) Days to Death Ducks Pb Cu Cu Cu Cu Cu Cu

29 Sanderson et a 1. Table 7 Body Weights and Organ Weights of Six Wild-trapped Shot and Sacrificed 100 Days After Dosing, 2 March 1973 Male Mallards Dosed with Copper Duck Number Number of Cu Shot At At a Dosing Autopsy- Cu in Li verb (ppm)- We i9ht {9} Two Two Body Brain Liver Spleen Heart Kidneys Testes , , o o. I I c Mean ! In gizzard Wet Weight. ~ Mean of copper-dosed ducks.

30 l Table 8. Body Weights and Organ Weights of 12 Wild-trapped Male Mallards Dosed with Copper Shot and Sacrificed on I May 1973, 160 Days After Dosing. Number of Cu Shot Duck At At a Number Dosing Autopsy- 137 I I I I Cu in Li verb (ppm)- Body 89 1, , I,364 weight (g} Bra In Liver Spleen Heart Two Kidneys Two Testes ] 11.9 ] I 5 I (/) Ql :::J 0. () Pancreas.., Ill 0 :::J 2. I () rt 1.5 I. 9 Ql I 121 1, I , ].6 I , II I, I I, I , I I 1 I ' II I I I,24o I 85 I, ] II Mean 62.0 I, ! In gizzard. E. wet weight.

31 Sanderson et al. Table 9. A Comparison of Body Weights and Organ Weights of Wild-trapped Male Mallards That Died After Dosing on 22 November Number Days Ducks to Mean Weight (g} in Death Two Two Dose Group (mean) Body Brain Liver Spleen Heart Kidneys Testes Pancreas 0 Shot o Pb Shot oo Cu Shot