Acute toxicity of ingested bismuth alloy shot in game-farm mallards. Toxicity of ingested bismuth alloy shot in game-farm mallards : chronic health effects and effects on reproduction Digitized by the Internet Archive in 2011 with funding from University of Illinois Urbana-Champaign http://www.archive.org/details/acutetoxicityofi35sand I I J 55:3-4 3 £ ILLINOIS ^^^J NATURAL HISTORY SURVEY Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards Glen C. Sanderson, William L. Anderson, George L. Foley, Loretta M. Skowron, Jeffrey D. Brawn, James W. Seets, and Karen L. Duncan Illinois Natural History Survey Bulletin Volume 35, Articles 3 and 4 April 1997 Natural History .Survey . Library Illinois Natural History Survey, Edward J, Arrhbrust, Acting;Chief A Division of the Illinois Department of Natural Resources A catalog of the publications of the Illinois Natural .History Survey is available without charge from the address below. A price list and an order blank are included with the catalog. Illinois Natural History Survey Distribution Center Natural Resources Building 607 East Peabody Drive _..'." Champaign, Illinois 61820 Citations: Sanderson, G.C, W.L. Anderson, GJL. Foley, L.M. Skowron, J.D. Brawn, and J.W. Seets. 1997. Acute toxicity of ingested bismuth alloy shot in game-farm mallards. Illinois Natural History Survey Bulletin 35(3): 185-2 16. Sanderson, G.C, W.L. Anderson, G.L. Foley, K.L.. Duncan, L.M. Skowron, J.D. Brawn, and J.W. Seets. 1997. Toxicity of ingested bismuth alloy shot in game-farm mallards: chronic health effects and effects on reproduction. Illinois Natural History Survey Bulletin 35(4):217-252. Editors: Thomas- E. Rice and Charles Warwick US ISSN 0073-4918 Printed by Authority of the State of Illinois (MJ412759-1M-4-97) Printed with soy ink on recycled and recyclable paper. ILUNOIS LIBRARY Contents Article 3: Acute Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards Acknowledgments ii Abstract 185 Introduction 185 Literature Review 185 Methods 187 Toxicity Study 187 Chemical Analyses 189 Storage of Samples 189 Digestions of Samples 189 Digestions for ICP Analysis 1 89 Digestions for GFAA Analysis 189 Analytical Methods 189 ICP 189 GFAA 190 Quality Control 190 Calculations 190 Statistical Analyses 190 Results 191 Survival 191 Retention and Dissolution of Shot 191 Body Weight 192 Organ Weights 192 Gizzard 192 Liver 192 Kidneys 192 Gonads 192 Hematocrit (Hct) 192 Heavy Metals and Essential Elements in Organs and Blood 192 Kidneys 195 Liver 195 Gonads 202 Plasma and Blood Cells 204 Feces 208 HlSTOPATHOLOGY 208 Gonadal Lesions 208 Female 208 Male 208 Liver 211 Kidneys 211 Gizzard 211 Discussion 212 Copper 212 Phosphorous 212 Iron 212 Calcium 212 Feces 212 Conclusions 213 Literature Cited 214 Continued on next page Article 4: Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards: Chronic Health Effects and Effects on Reproduction Acknowledgments ii Abstract 217 Introduction 217 Methods 217 Toxicity Sudy 217 Chemical Analyses 220 Storage of Samples 220 Digestions of Samples 220 Digestions for ICP Analysis 220 Digestions for GFAA Analysis 221 Analytical Methods 221 ICP 221 GFAA 221 Quality Control 221 Calculations 221 Statistical Analyses 222 Results 222 Chronic Toxicity Test 222 Survival 222 Hematocrit 222 Body Weight 222 Dissolution of Shot 222 Shot Retention 226 Organ Weights 228 Analyses of Tissues and Other Materials 231 Kidneys 231 Liver 231 Gonads 234 Blood 236 Reproduction 236 Eggs 236 Ducklings 240 Egg Weights 244 Egg Shell Thickness 244 Fertility Rates 244 Hatchability Rates 244 Egg Shell Analysis 244 Egg Content Analysis 244 Age of Embryo at Time of Death 247 HlSTOPATHOLOGY 247 Adults 247 Kidneys 247 Liver 247 Gonads 248 Heart 248 Lungs 248 Ducklings 248 Liver 248 Kidneys 248 Heart 248 Discussion 248 Conclusions 250 Literature Cited 251 ILLINOIS NATURAL HISTORY SURVEY Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards: Chronic Health Effects and Effects on Reproduction Glen C. Sanderson Illinois Natural History Survey William L. Anderson Illinois Department of Natural Resources and Illinois Natural History Survey George L. Foley University of Illinois and Illinois Natural History Survey Karen L. Duncan University of Illinois Loretta M. Skowron Illinois State Water Survey Jeffrey D. Brawn Illinois Natural History Survey James W. Seets Illinois Natural History Survey Illinois Natural History Survey Bulletin Volume 35, Article 4 April 1997 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Acknowledgments Bradley W. Zercher and James W. Sergent, Illinois Natural History Survey, fed the ducks, cleaned the pens, collected and weighed the eggs, and assisted with other phases of the study. Stephen P. Havera, Michelle M. Georgi, Aaron P. Yetter, and Christopher S. Hine, all with the Waterfowl Research Laboratory, Forbes Biologi- cal Station, Illinois Natural History Survey, Havana, Illinois, assisted with weighing, dosing, and collecting blood from the ducks. Patrick W. Brown, Brian W. Wilm, Trina H. Simpson, Angela M. Young, Anne E. Zielske, and Linda K, Campbell, all with the Illinois Natural History Survey, and Beverley C. Sanderson and J. William Sanderson, volunteers, assisted with weighing, dosing the ducks, and collecting and processing blood. Beverley C. Sanderson also assisted with many tasks in the preparation of this report. William R. Manuel, retired, College of Veterinary Medicine, University of Illinois, provided his expertise in the collection of blood. Judy K. Holding, Jenny Huffington, and Karen Bischoff, DVM, of the College of Veterinary Medicine, University of Illinois, assisted with the collection of blood. Helen M. Parker, College of Veterinary Medicine, University of Illinois, assisted with the necropsies. Veronica Lasovsky, Illinois State Water Survey, prepared the samples for analysis by ICP and by graphite furnace AA. Saada E. Hamdy, Illinois State Water Survey, conducted the analyses by graphite furnace AA. Gale D. Taylor, Head, Program of Laboratory Animal Medicine, University of Illinois, in- spected Pb-dosed sick ducks when we called him and authorized euthanasia when it was apparent that ducks would not survive. Without the enthusiastic assistance and support of all these individuals, the study would have been much more difficult. Their assistance is gratefully acknowledged. We thank Jerry L. Longcore, Leader, Patuxent Wildlife Research Center, Orono, Maine; Lawrence J. Blus, Wildlife Research Biologist, Biological Resources Division, U.S. Geological Survey; and Louis N. Locke, Wildlife Pathologist and Milton Smith, Chemist, National Wildlife Health Center, for their reviews of the manuscript. Petersen Publishing Com- pany, Los Angeles, California, provided financial support for the research and costs of this publica- tion. Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 217 Abstract In a 150-day study, we tested for chronic toxicity and effects on reproduction of bismuth/tin (Bi/Sn) alloy shot dosed in game-farm mallards (Anas platyrhynchos). Histopathology of livers, kidneys, gonads, hearts, and lungs showed no significant group-related differences among 0-dosed (controls), iron (Fe)-dosed (8, No. 4, steel shot), and Bi-dosed (8, No. 4, Bi/Sn alloy shot) adult ducks or among ducklings from pairs of these dosed groups. Bi shot, under our test conditions, did not elicit toxicity in mallard ducks or affect their reproduction or offspring. Introduction The present study is sequential to the investiga- tion by Sanderson et al. (1997a). In the present study, our first objective was to determine if Bi/ Sn alloy shot (i.e., "Bi shot") is chronically toxic to game-farm mallards. Our second objective was to determine if ingested Bi shot affected the ability of game-farm mallards to reproduce under a test protocol as specified by the Canadian Wildlife Service (CWS) (Environment Canada 1992) and modified by the U.S. Fish and Wildlife Service (USFWS), January 1995. We attempted to associ- ate toxic effects, if they occurred, with concentra- tions of elements in tissues. EnvironmentCanada (1 992) provided guide- lines for chronic toxicity and reproductive tests that were necessary to approve a candidate shot as nontoxic for waterfowl hunting in Canada. The original protocol for the present study was de- signed to comply with these guidelines. Dr. Simon Nadeau, CWS, and Dr. Keith A. Morehouse, USFWS, reviewed the protocol prior to initiation of the study. The reader is referred to Sanderson et al. (1997a) for additional introductory material and to Sanderson et al. (1992, 1997a) for reviews of Bi literature. Methods We randomly assigned ducks, doses, pens, pair- ings of ducks, and ducklings in our tests. One hundred twenty individual pens were numbered sequentially. The leg band number became the duck number and determined the duck's random assignment to a pen. Four slips of paper, labeled 0, 8 No. 4 Fe, 8 No. 4 Bi, or 8 No. 4 Pb, were placed individually in gelatin capsules, which were placed in a container. The first capsule removed (8, No. 4, Fe) determined that the first 18 male and female bands drawn were assigned to the Fe- dosed group. The procedure was repeated for the remaining three dose assignments. All female bands were placed in one con- tainer and all male bands in a second container. The bands were removed one at a time to deter- mine dose assignments. Subsequently, all bands for female ducks in each dosing group (e.g., 8, No. 4, Bi shot) were placed in one container and all bands for male ducks for the same dosed group were placed in a second container. One band at a time was selected from each container to deter- mine the female:male pairs. This procedure was repeated to determine which five females and which five males from each dosed group were selected for analyses of elements in blood, liver, kidney, and gonads and for necropsy and histo- logical study. However, female and male bands were selected independently and only five ducks were selected for each sex and dose. Ten ducklings from each dosed group were randomly selected for necropsy and analysis of blood, liver, and kidneys for nine elements. To insure randomization, the numbers of adult fe- males (those that produced live ducklings) in each dosed group were placed in separate gelatin capsules, which were placed in a container. The first 10 numbers selected from each dosed group of females determined the ducklings chosen for tissue analysis, necropsy, and histopathologic study. Because of their small size, samples from the first two ducklings produced by each pair were combined. For this report, 0- (sham-) dosed ducks are controls. Fe-dosed ducks are those that were dosed with eight, No. 4, Fe shot on Day and (for the survivors) again on Days 30, 60, and 90. Bi- dosed ducks are those that were similarly dosed with Bi shot. Pb-dosed ducks are those that were dosed with eight, No. 4, Pb shot on Day 0. For ducklings, 0-dosed, Fe-dosed, and Bi-dosed indi- cate that the ducklings were hatched from eggs laid by a female of 0-, Fe-, or Bi-dosed pairs. Toxicity Study Sixty-five male and 65 female wild-type game- farm mallards, 6 to 8 months old, were purchased from Whistling Wings, Hanover, Illinois, and transported to Champaign, Illinois, in crates in an enclosed van on 4 January 1995. The ducks were reared on a 60-acre lake. 218 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Ducks were weighed and randomly assigned, one to a pen, on 4January 1995 . Males and females were randomly assigned to one of the four groups (18 males and 18 females to each of three groups or 6 males and 6 females to one group). Pens were consecutively-numbered, elevated, 1-m2 , and constructed of vinyl-coated, 25.4-mm mesh, 14-gauge wire (Sanderson et al. 1992). A 9.1- x 36.6-m pole barn (metal roof, sides, and ends covered with heavy-duty polyethylene tarpaulin [black outside, silver inside, brass grommets 0.6 m apart, McMaster-Carr, Chicago]), housed the pens and excluded light. These facilities were inspected by several members of the Laboratory Animal Care Committee, University of Illinois, before ducks were placed in pens. The committee also inspected facilities twice during the study. Beginning 4 January 1995, the ducks were offered commercial duck pellets (Heinhold™ 1 4% Duck Developer pellets, Heinhold Feeds, Inc., Kouts, Indiana) and water ad libitum and exposed to ambient light. After allowing 3 weeks for ducks to acclimatize, males were moved on 26 January 1995 (Day 0) into pens with previously assigned females. At that time, each duck was given one of the following doses: eight, No. 4, (3.30mm diam- eter), Fe shot (18 females and 18 males); eight, No. 4, Bi shot (18 males and 18 females); eight, No. 4, Pb shot (6 females and 6 males); or no shot (con- trols, 18 males and 18 females). All surviving ducks were redosed on Days 30, 60, and 90 with the original dosing regime. Each dose of eight shot was weighed to the nearest 0.1 mg and stored in a numbered vial before it was placed in the duck. The Bi shot were provided by William S. Montgomery, Jr., Bismuth Cartridge Co., Dallas, Texas. Seven shot were analyzed in the labora- tory of the Illinois State Water Survey,Champaign, Illinois, before dosing the ducks. Concentrations of Bi in the shot ranged from 97.27% to 100.05% (x = 98.35%, SD = 0.86%) and Sn ranged from 1.69% to 1.98% (x = 1.90%, SD = 0.10%). Other elements averaged <0.1% each; Pb ranged from 0.0040 to 0.0186% (x = 0.0094%,SD= 0.0054%). Fe and Pb shot were obtained from commercial 12- gauge shotgun shells and were not analyzed. Seventeen dosed ducks (four of each sex of Fe-dosed and Bi-dosed, plus the one surviving Pb-dosed duck [a male]) were radiographed on 6 February 1995 (Day 11) and (except for the Pb- dosed duck), again on 6 March (Day 39) and on 6 April (Day 70) to determine the number of shot retained in the gizzards. We made a dorsal- ventral and a right-left view radiograph for each duck. Each duck was placed in a square 1.9-L cardboard milk carton with its top open and a hole cut in the bottom to reduce struggling in order to obtain a dorsal-ventral and a right-left side view. For each duck, the dorsal-ventral and right-left views were recorded on opposite halves of a single sheet of 35.6- x 43.2-cm X-ray film. When the ducks were initially dosed (Day 0, 26 January 1995), light was restricted to 8 hr per day (0800-1600 hr, CST) for 90 days. Beginning on the 91 st day (27 April 1 995), the daily illumination was gradually increased over 2 weeks to 18 hr per day. Half the daily increase was added in the a.m. and half in the p.m. (approximately 20 minutes each). An Indoor/Outdoor Digital 7-day Timer (Double Pole Single Throw, Model EZ-701-2, EZ Controls Co.—McMaster-Carr, Chicago) was pro- grammed one week at a time to increase the daily light by the proper amount each morning and evening. When 18 hr of light per day were at- tained (10 May 1995), the light regime was held constant (0500-2300 hr, CST) for the remainder of the study. On Day (26 January 1995), we weighed ducks and collected blood samples. On this same date, we removed commercial duck pellets and provided shelled corn ad libitum for 60 days, at which timewe switched the diet to Mazuri Water- fowl Breeder pellets (PMI™ Feeds, Inc., St. Louis) for the duration of the study. We used a small plastic funnel fitted with a plastic tube (9.5 mm outside diameter, 22.9 cm long) that was inserted through the pharynx to place the shot in the proventriculus. To reduce friction, we kept the tube in a pail of water when not in use. We poured shot into the funnel and flushed them into the proventriculus with 5mL of water. Controls were treated in the same manner except that no shot was placed in the proventricu- lus. At dosing, each shot dose was matched with its randomly selected duck. On Days 30, 60, and 90, the 8-shot doses for each shot type were ran- domly selected and placed in the same numbered vials that were used on Day 0. We collected blood from the wing vein of all ducks in heparinized microhematocrit capillary tubes to determine hematocrits (Hcts). In addi- tion, we collected 4 mL of whole blood with 5.0- mL syringes (20-gauge, 25.4-mm needles) from the wing vein of each of five 0-dosed, five Fe- dosed, five Bi-dosed females, and five ducks of each dose/ sex group to determine major elements (>1% by wt in shot—Bi, Sn, Pb, and Fe) and major nutritionally essential elements (Ca, P, Mg, Zn, April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 219 and Cu). We selected these ducks at random. Because we expected high mortality of the Pb- dosed ducks, we collected blood from all 12 Pb- dosed ducks. Although Fe and Pb were not present in the candidate shot, we analyzed for these metals because the USFWS (1986:42102) procedures for the approval of nontoxic shot re- quire that "...physiological parameters caused by the candidate shot must be significantly less than those caused by lead shot and must not be signifi- cantly greater than those caused by steel shot." Whole blood was injected into 10-mL lithium heparinized Vacutainer tubes and frozen until analyzed. We weighed ducks and collected blood from all survivors on Days 0, 30, 60, 90, 120, and 150. Afterwe had collected 24 hematocrit samples, we centrifuged the hematocrit tubes and read them on site in a mobile field laboratory/office. We spun the tubes for 5 minutes at 1 1 ,500 RPM at 13,000-g force. As we collected each sample of whole blood for analysis, we placed tubes in metal racks and put them on ice in a styrofoam cooler. After all samples were collected, we stored them in a freezer (-10°C) until thawed for analyses. After we killed adult ducks, livers, kidneys, gonads, hearts, and lungs from 20 females and 20 males (those chosen for collection of blood for analysis—5 ducks of each sex from each dosed group) were examined by the pathologist for gross and microscopic lesions. Livers, kidneys, and gonads of these 40 adult ducks were analyzed for major elements in candidate shot and essential major and trace elements. We excised gizzards from all ducks, removed the contents, and weighed the gizzards. The contents of gizzards of all dosed ducks were washed through a series of fine screens to recover shot, which were sorted by size (to identify the date dosed), counted, and weighed. We deter- mined the percent of shot retained at death and the percent of the weight of metal dissolved from each dosing. When necropsying the 40 randomly selected ducks, the pathologist examined and weighed the kidneys, livers, and gonads; a representative sample of each organ was fixed in 10% formalin for histopathology. Hearts and lungs also were examined and samples preserved for histopathol- ogy. The residual tissues of these organs were placed in separate, numbered, plastic bags and stored in a freezer until thawed for analysis. For the remaining 60 ducks, the same organs were removed and weighed, placed in individual, num- bered, plastic bags and stored in the freezer to serve as backup samples. When ducks began laying eggs, pens were visited at least twice daily but usually more often. Eggs were removed, weighed, numbered with a felt-tipped marking pen, held overnight at room temperature, and stored for 1 week at 12.8° to 15.6°C and a relative humidity of 75%. The num- bering system included the hen's ID number and the sequential order in which the egg was laid, e.g., the 12th egg laid by hen number 205 was numbered 205-12. For each female, we collected eggs until 21 uncracked eggs were obtained or until Day 150, whichever occurred first. When the 21st egg was collected, the female and her mate were weighed, bled for a blood sample, and killed. We removed organs and weighed them. As pre- viously indicated, organs were excised from 40 ducks and stored for chemical analysis; these ducks were necropsied and tissues were saved for histopathology. All uncracked eggs collected during each 7 days (except the 11th egg for each pair) were placed in an incubator. The temperature in the incubator was maintained at 37.5°C and the rela- tive humidity was 84-87% . After 6 days of incuba- tion, eggs were candled to determine fertility; we removed infertile eggs. Eggs were transferred to a hatcher 4 days before their expected hatching date. The temperature in the hatcher was main- tained at 37.2°C and the relative humidity was 87- 93%. Fertile eggs that failed to hatch were opened to determine age of embryos at death. Each of six trays in the hatcher was separated into nine compartments by thin pieces of Masonite™, each compartment was 18.42 x 18.42 cm. Eggs from each female were placed in sepa- rate compartments and each tray was fitted with a 0.6-cm mesh hardware cloth cover to prevent the ducklings from moving among compartments. Thus, individual ducklings were associated with their parents. We removed ducklings from the hatcher ap- proximately 1 8 hr after they hatched, then weighed and banded them with Size 8 sequentially num- bered, aluminum leg bands (National Band and Tag Co., Newport, Kentucky). (Note: these bands are too small to remain on mallard ducklings past 7 days of age.) We maintained the temperature in the brooders at 37.8°C with thermostat-controlled heat lamps. The brooders were constructed of vinyl-covered, 1.3-cm mesh welded wire. Each brooder compartment was 82.6 x 88.9 cm, provid- ing 245 cm2 of floor space for each of 30 ducklings. The minimum requirement for each duckling <7 220 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 days of age is 239 cm2 (personal communication, Laboratory Animal Care Committee, University of Illinois). Thus, the ducklings were free to move about and choose a preferred temperature. Water was provided ad libitum via waterers equipped with standard 1.9-L jars, which were refilled at least twice daily. Starter mash (Purina™ Duck Grower, 16% protein, Purina Mills, Inc., St. Louis) was provided ad libitum in metal feeders. When ducklings were 7 days old, we sexed and weighed them, collected blood to determine hematocrits, and killed themby decapitation. Ten ducklings, each of different parentage, were se- lected atrandom from each dosing group; samples of blood, liver, and kidneys were collected from each bird. These samples were analyzed for the same elements as the tissues from adults. These ducklings also were necropsied; liver and kidney samples (and several hearts) were preserved for histopathology. Because the amounts of kidney and blood from a single duckling were often inadequate for the required analyses, we aug- mented our samples by adding kidneys and blood from the next clutch mate of the selected duck- lings. Because of their small size, gonads were not collected for analysis. Thickness of shells of the 11th egg laid by each female was measured with a Digimatic Out- side Micrometer™ accurate to 0.001 mm (Metutayo, Japan). Measurements were taken at three locations (two each at the apex, cap, and equator) of each egg and averaged. The shell and contents of the 11th egg from each female were saved and analyzed separately for nine elements. The shells were stored at room temperature, and the egg contents were frozen until analyzed. Pb-dosed ducks were examined periodically by the institutional veterinarian in the Office of Laboratory Animal Resources, University of Illi- nois, who at various times reported that four ducks were moribund. These four ducks were euthanized. Five of the remaining eight Pb-dosed ducks died during a night when the temperature inside the test facility fell to -20.6°C. The last Pb-dosed ducks died 9 February 1995 (14 days after dosing), which was before most gonads began to respond to the approaching breeding season. Because most gonads were too small to analyze for all elements, we analyzed them by GFAA for Pb and Bi. Chemical Analyses Storage of Samples We inventoried samples (labeled by number and type of tissue) and stored them at -10°C in a freezer, which was monitored daily. Some Vacutainer tubes containing blood broke during freezing. Ifnoticed while still frozen, some samples were transferred to polypropylene test tubes and not lost. Digestions of Samples We allowed samples to thaw, then used acid to digest samples of blood, liver, kidney, gonad, egg contents, and eggshell for metal analyses. The analyses were performed with either inductively- coupled, argon plasma emission spectroscopy (ICP) or graphite furnace atomic absorption spec- troscopy (GFAA) or both. Because we wanted concentrations expressed on a wet-weight basis forblood and organs,we did not dry these samples before they were digested. Metals we sought were either present in the test shot (Bi, Sn, Fe, and Pb) or were essential elemental nutrients (Ca, Mg, P, Zn, and Cu). We used ICP to measure for these metals, and we analyzed for beryllium (Be) as an internal standard. GFAA was used to measure Pb and Bi when concentrations were low. Digestions for ICP Analysis A mixed portion of the sample (0.5 to 1.0 g) was placed in a tared 50-mL conically tipped polypro- pylene centrifuge tube and weighed to 0.1 mg with an electronic top-loading balance. Centri- fuge tubes were precleaned by soaking for 24 hr in a 10% nitric acid (HN03 ) bath and rinsing with deionized water. Samples and tubes were tared, then we added 1 to 2 mL of hydrogen peroxide (H2 2 ) and reweighed. We then added 30 to 50 mL of 2% HNO3, and 10% hydrochloric acid (HC1) and the internal standard solution of Be (2 mg/L). We homogenized samples into a slurry with a sawtoothed generator manufactured with tita- nium and TFE-fluorocarbon (Pro Scientific, Mon- roe, Connecticut). The internal standard solution was used to rinse excess materials from the gen- erator, with theamount ofrinse solution accounted for in the total weight. Sample preparations were completed using a SpectrPrep™ System automated microwave di- gestion system (CEM Corporation, Matthews, North Carolina). We used a 15-mL sample loop. After heating, cooling, and filtering, about 12.5 mL of the sample were collected and deposited by autosampler into a 15-mL polypropylene test tube. This digestate was then used for ICP analysis. Eggshells tended to clog the small-diameter tub- ing of the microwave system, but homogenation of the sample mixture, followed by a few hours in April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 221 a warm ultrasonic bath, effectively reduced par- ticle size. Digestions for GFAA Analysis A mixed portion of the sample (0.5 to 1.0 g) was placed in a tared TFE-fluorocarbon beaker and weighed to 0.1 mg on an electronic top loading balance. We added 20 mL of deionized water (DI H20), 0.250 mL concentrated HN03 , and 1 mL of hydrogen peroxide (H2 2 ). We heated the mix- ture on a hot plate at 95°C until the solution started to clear (about 0.5 hr). Approximately 20 mL DI H2 and 2 mL H2 2 were added. Upon further heating the mixture cleared and "foamed up." We rinsed down contents from the beaker walls with DI H20. Beakers were then covered with TFE-fluorocarbon watch glasses and allowed to reflux for approximately 1 hr. The resulting solutions were usually clear to yellow. The samples were brought to 50 mL with a volumetric flask, filtered through a 0.45-mm nitrocellulose filter, and stored in acid-washed linear polyethyl- ene bottles. The final acid concentration used was 0.5% HN03 . High purity acids and hydrogen peroxide (Baker Ultrex™ and Fisher Optima™) were used for all digestions. Analytical Methods Tissues were analyzed "blind" by the chemists — that is, they did not know either the gender of duck or which test shot it had received. ICP We used aThermo Jarrell Ash (TJA) AtomComp™, Model 61, vacuum spectrometer, with the polychromator configured with 44 fixed chan- nels, including analytical lines for variable con- centrations of Ca and Mg. Although we reported results for only a few elements, we measured for 30 elements to monitor for spectral interferences, which we did not detect. Blank subtraction and background correction were used. We used USEPA Method 200.7, (Office of Research and Development 1994). We used a different digestion process and we measured for Bi, which was not a listed analyte. We chose Be as an internal standard because it was not in the samples, it caused no spectral or background interference, and it was precisely detectable. Because samples of eggshells were mostly calcium carbonate, the amounts of Ca were be- yond the analytical range of the system. To cope with this situation, we analyzed eggshells by ICP to quantify all the elements except Ca, then we diluted samples with an acid blank solution (10% HC1, 2% HNO3) and reanalyzed for Ca. We could reconstruct the actual Ca values by making com- parisons with the internal standard. GFAA We used a Thermo Jarrell Ash Model 957 Atomic Absorption Spectrophotometer coupled with a Model 188 Furnace Atomizer and FASTAC autosampler. Samples were introduced as a spray and deposited directly into a carbon cuvette at 1 00°C to obtain drying on contact. Method 31 1 3 of Greenberg et al. (1992) was used. We analyzed samples in triplicate and reported the means. Quality Control We calibrated instruments daily with the stan- dard curve being verified with traceable, quality- control samples (QCS) from the National Institute of Standards and Technology (NIST). Samples (usually 10) were bracketed by calibration blanks, laboratory fortified blanks, and instrument per- formance check solutions during analysis, and we performed periodic checks on the internal stan- dard solution. The ICP instrument was pro- grammed to compensate for drift. The calibration was accomplished by recalculating the slopes of the calibration curves when any analyte was more than +5% of the true value while determining the ICP check standard. When an analyte was > +10% of the true value for a sample, the instrument was recalibrated and the affected sample reanalyzed. The ICP check standard was formulated to equal a concentration at the midpoint of the calibration curve and was traceable to NIST Standard Refer- ence Materials (SRMs). The QCS for the GFAA initially had to be within 10% of the true value. Subsequent measurement of the bracketed internals had to be +15%; if these limits were exceeded, we recalibrated the instrument and reanalyzed affected samples. We digested and analyzed in duplicate 10% of the samples, half of them spiked. Also, we prepared digestion blanks and spiked digestion blanks at a frequency of 10%. They underwent the same digestion and analytical process as did the samples. Calculations Data produced by ICP analysis were transferred to database files with ThermoSpec (TJA) Enable OA software. We then imported these into Enable spreadsheets for tabulations and calculations. We saved the Enable spreadsheets in a Lotus 1-2-3 format on diskette. For the GFAA instrument, results were recorded and data printed on an 222 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 instrument printer as concentrations (|ig / L) based on measurement of peak area. Data were then manually entered into spreadsheets to tabulate and perform calculations. Statistical Analysis Statistical comparisons among doses for variables measured only once (usually after necropsies) were made with one-way analyses of variance (ANOVA), except two-way ANOVAs were used when there were sex differences. Equality of variances among groups was evaluated with Levene's test (BMDP 1992). In instances where heteroscedasticity (P < 0.05) was detected, Brown- Forsyte statistics and approximate degrees of free- dom were used. Pairwise differences among groups were evaluated with Bonferroni compari- sons. In instances where variables were measured for two or more periods, dose groups were com- pared and tested for variation over time with a repeated-measures ANOVA. When necessary, significance levels based on the Huynh-Feldt (BMDP 1992) adjustment were used. Because of unbalanced data sets (caused by animals dying during the experiment), we used Wald statistics in a restricted maximum-likelihood model to es- timate parameters to test for differences among doses. We performed all tests with the BMDP statis- tical software package, version 7.0 (BMDP 1992). When we report two values as "different" or that they "differ," we mean that they were statistically different at the 95% level of confidence (P <0.05). Results Chronic Toxicity Test Survival All 12 Pb-dosed ducks died within 14 days after dosing; mean survival was 9.9 days, and no differ- ence in survival existed between sexes. All 0- dosed and Fe-dosed ducks survived until sacri- ficed; time from Day to sacrifice averaged 115.6 days for 0-dosed ducks and 121.1 days for Fe- dosed ducks. Only one Bi-dosed duck died (on Day 1 31 , after laying 1 6 eggs). Survival time for Bi- dosed ducks (including the one that died) aver- aged 120.5 days; mean survival times were not different among the three dosage groups. Both ducks of each pair were sacrificed when the fe- male had laid 21 uncracked eggs. Thus, these survival times only indicate that most ducks sur- vived until sacrificed and that no differences ex- isted among 0-dosed, Fe-dosed, and Bi-dosed ducks in the mean time required to lay 21 uncracked eggs. Hematocrit Mean Hcts for Pb-dosed ducks declined from 44.6 to 25.2 (Table 1, Figure 1) during their 9.9-day mean survival. However, we obtained Hcts at necropsy for only 4 of the 12 Pb-dosed ducks. For the other dosage groups, mean Hcts of males did not decline through Day 120 and at necropsy; however, mean Hcts of females de- clined in all three groups of surviving ducks by Day 90 and at necropsy were lower than mean Hcts of males (Figure 2). Mean Hcts in 0-dosed females declined from 46.3 on Day to 38.2 at necropsy, in Fe-dosed females from 46.2 on Day to 37.9 at necropsy, and in Bi-dosed females from 46.0 on Day to 36.2 at necropsy (Table 1 ) . Except for Pb-dosed ducks, no difference existed among doses in the mean Hcts in the present study (Table 1). Body Weight All males weighed more than all females from Day through Day 60. By Day 90, the mean weights of males and females did not differ, and on Day 120 and at necropsy females were heavier than males (Figure 3). Changes in weight were caused primarily by gains in females rather than losses in males (Table 2). The gain by females was accompanied by a decline in average Hct (Table 1). Weights of females (and of males) among the three dosed groups were not different at necropsy. Pb-dosed males weighed more than Pb-dosed females on Day 0. At necropsy, Pb-dosed males and Pb-dosed females weighed about one-third of their mean body weights on Day 0, but mean weights of the Pb-dosed ducks were not different between sexes (Table 2). The only dose-related difference in mean bodyweightswas associated with Pb-dosed ducks, which weighed less at necropsy than 0-dosed, Fe- dosed, or Bi-dosed ducks (Table 2, Figure 4). Dissolution of Shot Lead—In our study, Pb-dosed females dissolved an average of 31.5% of the weight of eight, No. 4, shot in an average of 9.3 days—3.4% per day. Males dissolved 31.2% of the weight of dosed Pb shot in an average of 10.5 days—3.0% per day. At death, females retained in their gizzards 81.2% and males 87.5% of the number of dosed Pb shot (Table 3). Four of 15 shot not recovered from the Continued on page 226 April 1997 50 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 223 M E A 40 N H E M A T C ? 30 T S iDayO I Necropsy20 Females Males Figure 1 . Mean Hcts of game-farm mallard ducks dosed with 8, No. 4, Pb shot on Day 0. n = 12 for Day O and 4 for necropsy. Table 1. Mean Hcts of adult male and female game-farm mallard ducks dosed with shot (controls); eight, No. 4, Fe shot; eight, No. 4, Bi shot; or eight, No. 4, Pb shot. Dose Sex Days after first dosing 30 60 90 120 Neca 46.3 49.5 50.2 43.5 38.8C 38.2 0.63b 0.72 0.55 1.52 1.12 1.08 46.4 48.4 48.2 45.0 45.4C 45.3 0.44 0.47 0.50 0.63 0.65 1.19 46.2 50.3 50.6 46.2 40.2d 37.9 0.61 0.68 0.66 1.29 0.82 1.02 46.7 48.4 49.5 46.9 48.4d 44.6 0.40 0.54 0.49 0.56 0.61 0.84 46.0 47.9 47.9 43.9 37. 1' 36.2' 0.72 0.84 1.05 1.31 1.11 1.03 47.3 49.1 48.8 46.0 48.0e 47.4 f 0.62 0.63 0.50 0.46 0.89 0.62 44.6d 25.2s 0.61 1.65 Fe Bi Pb F M F M F M F&M Ducks were necropsied when one member of a pair died, when 21 uncracked eggs were collected from the pair, or at 150 days post dosing, whichever occurred first. Mean survival was 115.6 days for 0-dosed ducks, 121.6 days for Fe-dosed ducks, and 120.5 days for Bi-dosed ducks. All Pb-dosed ducks died <14 days post dosing and only 2 samples from each sex were collected at necropsy. Difference in Hcts: ' SE. e n = 8. Between sexes: « n = 7 ' n = 17. d n = 12. Bn = 4. n = 18 for all other samples. = 15.31; P = 0.0003. Among doses: F = 1.41; P = 0.2533. Over time: F = 101.80; P< 0.00001. 224 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 52 50 48 46 44 42 40 38 36 34 £a Necropsy Females Males Figure 2. Mean Hcts of game-farm mallards dosed with shot (controls); 8, No. 4, Fe shot; or 8, No. 4, Bi shot on Days 0, 30, 60, and 90. The three groups of ducks were combined for this graph, n = 54 females and 54 males for Days 0, 30, 60, and 90; n = 27 females and 27 males for Day 120; and n = 53 for females and 54 for males at necropsy. Table 2. Mean body weight (kg) of adult male and female game-farm mallard ducks dosed with (controls) shot; eight, No. 4, Fe shot; eight, No. 4, Bi shot; or eight, No. 4, Pb shot. Sex Days after first dosing Dose 30 60 90 120 Nee1 F 1.05 1.00 0.99 1.13 1.28h 1.25 0.04c 0.02 0.02 0.03 0.03 0.03 M 1.15 1.14 1.10 1.19 1.16b 1.20 0.02 0.03 0.02 0.03 0.04 0.04 Fe F 1.02 1.01 0.97 1.13 1.24d 1.25 0.04 0.03 0.03 0.04 0.04 0.04 M 1.20 1.14 1.11 1.20 1.20d 1.17 0.02 0.02 0.02 0.02 0.02 0.02 Bi F 1.05 1.02 0.99 1.14 1.19- 1.22 0.03 0.03 0.03 0.03 0.06 0.04 M 1.18 1.17 1.12 1.20 1.16e 1.18 0.02 0.03 0.03 0.02 0.04 0.03 Pb F M 1.03' 0.03 1.22' . 0.04 0.68 f 0.03 0.79» 0.05 " Ducks were necropsied when one member of a pair died, when 21 uncracked eggs were collected from the pair, or at 150 days post dosing, whichever occurred first. Mean survival was 115.6 days for 0-dosed ducks, 121.6 days for Fe-dosed ducks, and 120.5 days for Bi-dosed ducks. All Pb-dosed ducks died <14 days post-dosing. h n = 7. Difference in body weight: 1 SE. Between sexes: F = 7.05; P = 0.0107. d n = 12. e n = 8. Among doses: F = 1.28; P = 0.2870. n = 18 for all others. Over time: = 54.88; P< 0.00001. April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 225 13n 1.2 1.1- 1.0 DayO Day 30 Day 60 Day 90 Day 120 Necropsy Females Males Figure 3. Mean body weight (kg) of game-farm mallards dosed with shot (controls); 8, No. 4, Fe; or 8, No. 4, Bi shot on Days 0, 30, 60, and 90. The doses were combined for this graph, n = 34 for each sex for Days 0, 30, 60, and 90; n = 27 for each sex for Day 120; n = 53 for each sex for necropsy. 1.3 I Females i Males Controls Fe Bi Pb Figure 4. Mean body weight (kg) at necropsy of game-farm mallards dosed with shot (controls); 8, No. 4, Fe shot; or 8, No., 4 Bi shot on Days 0, 30, 60, and 90, or 8 No. 4 Pb shot on Day 0. n = 18 for female 226 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 continued from page 222 gizzards were found in the feces of one duck. Because the time between dosing and recovery of the Pb shotwas short (Table 4), the 1 1 missing shot were probably voided, but were not found in the feces. Eight ducks that each retained all eight dosed Pb shot at death dissolved an average of 330.5 mg of Pb from the shot in their gizzards. To compare the rates at which Pb, Fe, and Bi shot were dissolved in the gizzard, we measured the mean daily rate of dissolution of Pb shot in 9.9 days—themean survival time for Pb-dosed ducks. We then used multiple regression and estimated that the Fe-dosed ducks dissolved eight, No. 4, Fe shot at a rate of 1 .4% per day in 9.9 days, and eight Bi-dosed ducks dissolved Bi shot at a rate of 2.5% per day in 9.9 days. In contrast, 12 ducks dosed with Pb shot on Day dissolved eight, No. 4, Pb shot at a rate of 3.3% per day in an average of 9.9 days (Table 4). Iron—Fe-dosed females dissolved an average of 99.9% of the weight of the eight, No. 4, Fe shot dosed on Day in a mean of 121 .2 days—0.8% per day. They dissolved 48.6% of the weight of Fe shot dosed on Day 90 in a mean of 31.2 days — 1.6% per day (Table 3). Many of the Fe shot dosed on Day were probably completely dissolved in less than 121.2 days as only 1.4% of the number of Fe shot dosed in females on Day were recovered from giz- zards. Males dissolved an average of 96.6% of the weight of Fe shot dosed on Day in a mean of 121 .2 days—0.8% per day. Males dissolved 27.5% of the weight of Fe shot dosed on Day 90 in 31.2 days—0.9% per day. Each female dissolved an average of 3.9 g of Fe from all Fe shot dosed and each male 3.1 g over a mean period of 121.2 days after the first shot were dosed (Table 3). In our previous toxicity study, the weight of six, No. 4, Fe shot dosed was 69.2% dissolved in 30 days (Sanderson et al. 1997a). This rate com- pares with 48.6% of the weight of Fe shot dis- solved from eight, No. 4, Fe shot dosed on Day 90 in females in a mean of 31.2 days in the present study. On Day 90, the ducks in the present study retained most or all of the Fe shot dosed on Days 0, 30, and 60. These results suggest that the higher the number of shot in the gizzard, the slower the rate that individual pellets dissolve. Bismuth—Bi-dosed females dissolved a mean of 98.9% of the weight of eight, No. 4, Bi shot dosed on Day in an average of 120.5 days—0.8% per day. They dissolved 52.5% of the weight of Bi- shot dosed on Day 90 in an average of 30.5 days — 1 .7% per day. Bi-dosed males dissolved a mean of 99.2% of the weight of Bi shot dosed on Day in an average of 120.5 days—0.8% per day. Bi-dosed males dissolved an average of 55.5% of Bi shot dosed on Day 90 in an average of 30.5 days—1 .8% per day. Females dissolved a mean of 5.4 g of metal and males a mean of 5.2 g, from all dosed Bi shot over a mean of 120.5 days (Table 3). Fe-dosed females dissolved 7.5 times asmuch metal from shot as did Pb-dosed females, which all died. Fe-dosed males dissolved 5.9 times as much metal from shot as did Pb-dosed males—all also died. Bi-dosed females dissolved 10.4 times as much metal, and males 10.7 times as much metal, as their counterparts dosed with Pb. Simi- larly, Bi-dosed females dissolved 1.4 times as much metal in their gizzards as did Fe-dosed females. Bi-dosed males dissolved 1.8 times as much metal as was dissolved by Fe-dosed males (Table 3). All Fe-dosed ducks and all but one Bi- dosed duck survived until euthanized at the end of the study, whereas all Pb-dosed ducks died within 14 days after they were dosed. Shot Retention From the radiographs made on 6 February 1995 (Day 11), the eight pellets that were dosed on Day were identified in the gizzard of each of the 17 ducks selected for examination by radiographs. Usually eight pellets showed in both views (dor- sal-ventral and right-left), but sometimes the count was questionable in one view. From radiographs made on 6 March 1995 (Day 39), the eight pellets dosed on 24 February 1995 in each of the four male and four female Fe- dosed and Bi-dosed ducks were clearly identi- fied. In addition, for the eight Fe-dosed ducks, 16 shot were counted in each of five gizzards, a minimum of 10 shot in one gizzard, and 15 shot in each of two gizzards. In gizzards of the eight Bi- dosed ducks, 16 shot were identified in each of six gizzards and a minimum of 15 shot in each of two gizzards. Although all shot dosed on 26 January 1995 probably were retained by all ducks on 6 March, this presumption could not be verified by radio- graphs. With 16 shot compressed in the gizzard, some shot obscured the view of others. Radio- graphs obtained on 6 April 1995 showed the eight shot dosed on 27 March 1 995 in each gizzard of the eight Fe-dosed and eight Bi-dosed ducks. Twenty- four shot were identified in each of two gizzards of Fe-dosed ducks and two Bi-dosed ducks. A mean of 1 7.8 shot was identified in the gizzards of Continued on page 228 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 227 r/able 3. Mean weight of eight, No. 4, Fe, Bi, and Pb shot dosed in game-farm mallard ducks, mean weight of shot recovered from :he ducks, number and percent of dosed shot recovered, and percent and weight of shot dissolved in the gizzard. DavO Dav30 Dav60 Dav90 Sex Sex Sex Sex Dose F M F M F M F M 1.197 1.200 0.003a 0.002 1.649 1.663 0.005 0.003 1.658 1.666 0.008 0.009 0.001 0.025 0.001 0.008 0.018 0.024 0.008 0.012 1.136 1.147 0.165 0.160 99.9 96.6 0.07 1.43 98.9 99.2 0.51 0.39 31.5 31.2 9.87 9.62 1.196 1.174 0.003 0.009 1.631 1.639 0.011 0.012 0.522 0.519 0.163 0.160 0.111 2.78 0.111 0.62 1.94 2.61 0.70 0.78 6.50 7.00 0.96 1.00 1.4 34.7 1.39 7.80 24.3 32.6 8.78 9.70 81.2 87.5 11.97 12.50 121.2 121.2 2.92 2.92 120.5 120.5 4.32 4.32 9.3 10.5 0.760 0.922 Mean weight (g) of 8 shot dosed 1.202 1.202 1.198 0.001 0.002 0.002 1.654 1.661 1.644 0.004 0.005 0.006 Mean weight (g) of shot recovered 0.003 0.232 0.273 0.001 0.029 0.036 0.089 0.070 0.272 0.023 0.024 0.041 Mean % of weight dissolved from shot dosed 99.8 80.7 76.8 0.12 2.41 2.96 93.7 95.8 83.4 1.94 1.43 2.49 Mean weight (g) dissolved from shot dosed 1.199 0.975 0.924 0.002 0.031 0.036 1.566 1.591 1.372 0.024 0.024 0.042 Mean number of shot recovered from shot dosed 0.889 7.00 6.56 7.67 0.403 0.40 0.59 0.20 4.67 4.17 6.06 6.06 0.642 0.64 0.63 0.70 Mean % of the number of shot recovered from shot dosed 11.1 87.5 81.2 95.8 5.04 4.95 7.44 2.48 58.3 52.1 75.7 75.0 8.02 7.97 7.92 8.69 Mean No. of days that shot dosed were in the gizzard 91.2 91.2 61.2 61.2 1.201 1.199 1.200 0.002 0.003 0.003 1.654 1.646 1.653 0.004 0.010 0.005 0.628 0.616 0.878 0.032 0.046 0.033 0.252 0.787 0.735 0.054 0.098 0.099 47.8 48.6 27.5 2.78 3.84 2.78 82.8 52.5 55.5 3.68 5.88 5.99 0.573 0.582 0.324 0.033 0.046 0.035 1.402 0.860 0.918 0.053 0.093 0.099 90.5 90.5 60.5 60.5 7.83 7.89 0.121 0.111 7.28 7.72 0.463 0.177 97.2 98.6 2.16 1.39 91.0 96.5 5.79 2.22 31.2 30.5 31.2 30.5 228 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Table 4. Rates at which eight, No. 4, Fe, Bi, and Pb shot dissolved after 10 to 120 days in the gizzards of game-farm mallards (2nd, 3rd, and 4th doses of 8 Fe or 8 Bi shot were dosed on Days 30, 60, and 90). Dose Day Dosed Mean No. Days Shot in Gizzard Mean % Wt of Shot Dissolved per Day Fe Bi Pb 30 60 90 30 60 9.9 121.1 2.03 91.1 2.03 61.1 2.03 31.1 2.03 9.9 120.5 3.01 90.5 3.01 60.5 3.01 30.5 3.01 9.9 0.60 1.4a 0.62b 0.8 0.02 0.9 0.06 1.0 0.05 1.3 0.08 2.5 a 0.26 0.8 0.02 1.0 0.04 1.4 0.06 2.2 0.17 3.3 0.75 Estimated by multiple regression. SE. Difference in rate shot were dissolved: Between doses: Dosed Day 60; P < 0.0001 . Dosed Day 90; P< 0.0001. continued from page 226 the remaining six Fe-dosed ducks and a mean of 19.7 shot in the remaining six Bi-dosed ducks. All shot dosed on 27 March (Day 60) were readily identified, but it was not possible to distinguish all shot dosed on Day from shot dosed on Day 30. Each of the ducks was dosed with 8 shot on 26 January 1995 (Day 0), 24 February 1995 (Day 30), and 27 March 1995 (Day 60); most of the 24 shot were retained on 6 April 1995 (Day 90), the last date that ducks were dosed. At necropsy, rem- nants of all 32 shot were found in one gizzard of a Fe-dosed duck on Day 99, and all shot were present in each gizzard of three Bi-dosed ducks on Days 109 (2) and 118. In addition, one gizzard con- tained 30 Bi shot on Day 118, one gizzard con- tained 31 Bi shot on Day 95, and 30 shot were retained in each gizzard of three Fe-dosed ducks on Days 109, 112, and 132. In our present study, six females retained an average of 81.2% of the number of dosed Pb shot to an average of 9.3 days. Six males retained an average of 87.5% of the number of the dosed Pb shot to an average of 10.5 days (Table 3). These results show that ducks void ingested Pb shot at a faster rate than they do Fe or Bi shot. Organ Weights Gizzard—The mean weights of gizzards ranged from 19.2 g for Bi-dosed females to 26.5 g for Pb- dosed males (Table 5). No sex differences existed in any of the four dosed groups. Gizzards of Pb- dosed ducks were heavier than gizzards of 0-, Fe- and Bi-dosed ducks, but no difference was de- tected among gizzard weights of 0-, Fe-, and Bi- dosed ducks (Table 5). In our study, ducks were on a diet of com- mercial duck pellets from Day 61 to necropsy—an average of58 days. Furthermore, Pb-dosed ducks, all of which died in February 1995, had heavier gizzards than the 0-, Fe-, and Bi-dosed ducks, which were euthanized in April, May, or June 1995. The lower average gizzard weights in our study also may be related to the extended repro- ductive period of the 0-, Fe-, and Bi-dosed ducks, whichwas not experienced by the Pb-dosed ducks. Liver—Mean weights of livers ranged from 1 7.7 g for Pb-dosed females to 46.6 g for Fe-dosed fe- males (Table 5). Livers of 0-dosed, Fe-dosed, and Bi-dosed females weighed more than twice as much as livers of Pb-dosed females and of males in each dosed group (Figure 5). No difference was Continued on page 230 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 229 Table 5. Mean weights (g) of gizzard, liver, kidneys, and gonads of adult male and female game-farm mallard ducks with shot (controls); eight, no. 4 Fe shot; eight, no. 4 Bi shot; and eight, no. 4 Pb shot, n = 18 for each sex for O-, Fe-, and Bi-dosed ducks, n = 6 for each sex for Pb- dosed ducks. Dose Sex Gizzard Liver Kidneys F 21.0 0.83' M 21.4 0.81 F&M 21.2 0.57 Fe F 19.7 0.93 Fe M 20.1 0.52 Fe F&M 19.9 0.53 Bi F 19.2 0.77 Bi M 21.2 0.76 Bi F&M 20.2 0.56 Pb F 23.2 1.20 Pb M 26.5 2.28 Pb M&F 24.8 1.33 42.2 2.02 18.5 0.91 30.4 2.27 46.6 1.97 20.0 0.64 33.2 2.47 43.9 2.36 18.0 0.94 30.9 2.53 17.7 1.81 18.6 2.57 18.1 1.51 9.3 0.28 6.2 0.16 7.8 0.31 9.1 0.30 6.3 0.23 in 0.30 9.0 0.36 6.2 0.19 7.6 0.31 8.6 0.29 8.7 0.95 8.7 0.48 43.5 2.53 33.1 2.46 38.3 1.95 38.9 3.08 36.8 2.32 37.9 1.91 45.2 2.37 36.1 4.26 40.6 2.52 0.6 0.10 1.2 0.20 0.8 0.14 " SE. Differences between sexes in organ weights. Only significant differences are shown. Liver: 0-dosed F = 115.45; P < 0.00001. 1,24 Fe-dosed F = 164.68; P < 0.00001 . 1,21 Bi-dosed F = 104.82; P < 0.00001 . 1,22 Kidneys: 0-dosed F = 95.44; P < 0.00001. Fe-dosed F = 53.48; P < 0.00001. 1,34 Bi-dosed F = 50.49; P < 0.00001. 1,34 Gonads: 0-dosed F = 8.65; P = 0.0059. 1,34 Pb-dosed F = 7.29; P = 0.0223. 1,10 Difference among doses in organ weights: Gizzard: F = 6.72; P = 0.0003. Liver: F = 15.43; P< 0.00001. 3,112 Kidneys: F = 2.70; P = 0.0492. 3,112 Gonads: F = 113.91; P< 0.00001. 3,79 230 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Females I Males Controls Fe Bi Pb Figure 5. Mean weight (g) of livers of game-farm mallards dosed with shot (controls); 8, No. 4 Fe, shot; or 8, No. 4, Bi shot on Days 0, 30, 60, and 90, or 8, No. 4, Pb shot on Day 0. n = 18 for each sex for 0-, Fe, and Bi-dosed ducks, and n = 6 for each sex for Pb-dosed ducks. continued from page 228 detected in the mean weights of livers of males among the four dosed groups. Mean weights of livers of females in our present study were much higher than the mean weights of livers of females in our previous toxic- ity study (Sanderson et al. 1997a). These differ- ences were manifestations of long-term egg lay- ing. Ducks in the previous study were killed on 12 May 1994, at the start of the reproductive season, whereas ducks in the present study were killed after each female had laid 21 uncracked eggs (most were killed in late May or June 1995). Kidneys—The mean weights of kidneys ranged from 6.2 g for 0-dosed and Bi-dosed males to 9.3 for 0-dosed females (Table 5). No difference was detected among doses in the mean weights of kidneys of females, or of 0-dosed, Fe-dosed, and Bi-dosed males. The kidneys of male Pb-dosed ducks weighed more than the kidneys in the other dosed groups. Kidneys of female 0-dosed, Fe- dosed, and Bi-dosed ducks weighed more than kidneys ofmales in the respectively dosed groups. The mean weights of kidneys of female and male Pb-dosed ducks did not differ (Table 5). As with livers, mean weights of the kidneys of males in the present study (Table 5) were simi- lar to the mean weights of kidneys of males in the earlier study (Sanderson et al. 1997a). Mean weights of kidneys of females in the present study were higher than the mean weights of kidneys of females in the earlier study. Gonads—No differences were detected among mean weights of gonads for 0-, Fe-, and Bi-dosed ducks. Gonads of 0-dosed females were heavier than gonads of 0-dosed males, and gonads of Pb- dosed males were heavier than gonads of Pb- dosed females. The mean weights of female go- nads ranged from 0.6 g for Pb-dosed birds to 45.2 g for Bi-dosed ducks (Table 5). The mean weights of gonads did not differ between sexes for Fe- dosed and Bi-dosed ducks. The mean weights of gonads of both female and male Pb-dosed ducks were lower than the mean weights of gonads of April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 231 the respective sexes of 0-dosed, Fe-dosed, and Bi- dosed ducks. These weight differences are, no doubt, the result of the terminal condition of the Pb-dosed ducks; they died on an average date of 5 February, before the gonads had begun their seasonal growth. Thus, effects on weight of go- nads from dosing with Pb were not measured in our study. Analyses of Tissues and Other Materials We used the Method Detection Limit (MDL) (Glaser et al. 1981 ) to establish the detection limits for levels of elements in tissues and other materi- als. The MDL procedure must produce a value that averages >two times larger than the MDL value to be considered meaningful (Glaser et al. 1981; see Sanderson et al. 1997a for a definition of MDL). Because results of ICP analyses for Bi and Pb were usually lower than the MDLs, we usually analyzed the kidneys, livers, gonads, and blood by GFAA for these two elements. Kidneys and livers of all Pb-dosed ducks contained Pb levels several times higher than the MDLs as analyzed by ICP. Concentrations of Pb in the kidneys and livers of Pb-dosed ducks were determined by ICP. Kidneys At necropsy, with doses combined, females had higher mean concentrations of Ca than males (138.7 vs 109.7 Ug/g). Compared with females, males had higher mean concentrations of P (3706 vs 3542 Ug/g),Mg (234.5 vs 221 .4 Ug/g), Zn (34.02 vs 30.51 ug/g), and Cu (9.62 vs 7.07 ug/g). No other sex differences were detected in the concen- tration of the nine elements of interest in the current study. With sexes combined, a higher mean concen- tration of Pb was detected in the kidneys of Pb- dosed ducks (213 Ug/g) compared with the kid- neys of 0- (0.448 Ug/g), Fe- (0.198 Ug/g), and Bi- dosed (0.574 Ug/g) ducks. A higher mean concen- tration of Pb was detected in the kidneys of 0- dosed ducks versus Fe-dosed ducks, but no dif- ferences existed in the mean concentrations of Pb in the kidneys of 0- and Fe- versus Bi-dosed ducks (Table 6). In spite of the high mean concentrations of Pb in the kidneys of Pb-dosed ducks, we de- tected no dose-related histopathologic differences in the kidneys. In our study, Bi-dosed ducks had higher mean concentrations of Bi in their kidneys (1.54 Ug/g) than in their livers (0.637 Ug/g). Our Bi- dosed ducks were exposed to Bi dissolved from Bi shot in the gizzard from Day to necropsy—an average of 120.5 days. A higher mean concentration of Bi (1.54 ug/ g) was detected in the kidneys of Bi-dosed ducks than in the kidneys of 0-, Fe-, or Pb-dosed ducks; all three of the latter dosed groups had 2xMDL. The apparent higher mean concentra- tion of Bi in the gonads of the Pb-dosed females probably resulted from the low gonad weights of Pb-dosed females, which died before the seasonal increase in gonad size. The high dilution ratio associated with the small samples probably caused the apparent higher levels of Bi. Thus, we con- cluded that no differences existed among doses in the mean concentration of Bi in the gonads. All mean concentrations of Sn were below MDL for Sn in gonads. The mean concentrations of Ca and P were lower in gonads of Pb-dosed females than in gonads of 0-, Fe-, and Bi-dosed females, but no difference was found among the latter three dosed groups. Although Pb-dosed males had higher mean concentrations of Ca and P in their gonads than 0-, Fe-, and Bi-dosed males, these differences were not significant. Pb-dosed females had a higher mean concen- tration of Mg in their gonads than 0-, Fe-, or Bi- dosed females. No differences were detected in the mean concentrations of Mg in the female gonads of 0-, Fe-, and Bi-dosed ducks, or among doses in the male ducks (Table 8). Pb-dosed females had a lower mean concen- tration of Zn in their gonads than 0-, Fe-, or Bi- dosed females. Fe-dosed females had a higher mean concentration of Zn in their gonads than 0- dosed females. No differences were found in the mean concentrations of Zn in the gonads of Fe- dosed and Bi-dosed females, or between Bi and 0- dosed females. Lead-dosed males had a higher mean concentration of Zn in their gonads than 0-, Fe-, or Bi-dosed males (Table 8). Blood The mean values for Pb, Bi, and Sn were all <2xMDL and all but 6 of the 57 means for these three elements were 90 to 120 days. ' n = 5. 1 Blood samples taken from >120 to 150 days. k n = 4. 1 n = 2. MDL: Bi - 0.081 ug/g. Sn-2.14 ug/g. Pb- 0.132 M.g/g. Cu- 0.180 ug/g. Difference over time in females: Ca: DF5, Chi-square 313.8533; P< 0.00001. Zn: DF5, Chi-square 183.6148; P< 0.00001. Cu: DF5, Chi-square 20.4004; P = 0.0011. April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 239 Table 10. Mean number of days required for 0-dosed (controls), Fe-dosed, and Bi-dosed game-farm mallard female ducks to lay 21 eggs and mean number of days after Day the first egg was laid. Sample sizes are in parentheses. Dose Mean days to lay 21 eggs Mean Days after Day first egg was laid 27.4(17) 2.04" 83.8(17) 4.30 Fe 25.7(18) 1.56 94.0(18) 4.12 Br 25.9(15) 1.26 91.6(17) 3.66 -1 One 0-dosed and one Bi-dosed female laid no eggs; they suffered from egg yolk peritonitis. h SE. c One Bi-dosed hen died 24 days (and 16 eggs) after laying her first egg and one Bi-dosed female was sacrificed on Day 150 when she had laid 17 eggs in 35 days after laying her first egg. These two females are not included. 64 n G R A M S Controls Figure 8. Mean weight (g) of hatched and not hatched fertile eggs from 0-dosed (controls), Fe-dosed, and Bi-dosed game-farm mallard pairs. See Table 11 for sample sizes. 240 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 continued from page 236 92 (27 April) for Bi-dosed females (Table 10). These dates do not differ statistically. Two 0-dosed females each laid 21 eggs in 21 days, and one 0-dosed female required 54 days to lay 21 eggs. Three Fe-dosed females each laid 21 eggs in 21 days, and one Fe-dosed female re- quired 46 days to lay 21 eggs. Three Bi-dosed females each laid 21 eggs in 21 days, and one Bi- dosed female required 45 days to lay 21 eggs. One 0-dosed and one Bi-dosed female laid no eggs by Day 150, and both hens had egg yolk peritonitis. Probably activities associated with catching, weighing, bleeding, and dosing these ducks when egg yolks were about to be released into the infundibula resulted in the yolks being discharged into the body cavity, causing peritoni- tis. One Bi-dosed female died ofunknown causes 24 days after laying her first egg on Day 113 and after she had laid 16 eggs. One Bi-dosed female laid 17 eggs in 35 days after laying her first egg and by the time she was sacrificed on Day 150. We detected no differences among doses in 0-dosed, Fe-dosed, and Bi-dosed females in the mean date laying was initiated or the mean number of days required to lay 21 eggs. Differences were found between the weights of fertile eggs that hatched and those that did not hatch in all dosed classes. For 0-dosed and Fe- dosed pairs, fertile eggs that hatched were heavier than eggs that did not hatch. For Bi-dosed pairs, fertile eggs that did not hatch weighed more than eggs that hatched (Figure 8). Differences existed among doses in the weights of both hatched and unhatched fertile eggs. Hatched eggs from Fe- dosed pairs were heaviest followed by hatched eggs from 0-dosed pairs and Bi-dosed pairs. Fer- tile eggs that did not hatch from Bi-dosed pairs weighed more than nonhatched fertile eggs from 0-dosed and Fe-dosed pairs, in that order. With doses combined, fertile eggs that hatched weighed more (61.9 g) than fertile eggs that did not hatch (60.9 g) (Table 11). Ducklings Body Weight—All ducklings were weighed at the time of hatching, and a difference existed in mean body weights among dose groups. Ducklings from Bi-dosed pairs weighed approximately 2 grams less, on the average, than either the 0-dosed or the Fe-dosed ducklings. However, by day 7, we found no difference in body weights of ducklings among the dosed groups. Body weights did not differ between sexes at hatching or at Day 7 (Table 12). Survivability—All but two ducklings survived the first 7 days after hatching. These deaths resulted from the ducklings entangling their legs in the wire floor of the brooder. One of the ducklings experienced neurologic deficits in the affected leg and the other duckling suffered a fractured leg. Both ducklings, offspring of Fe- dosed pairs, stopped eating and were emaciated at death. Hematocrit—Mean Hcts for ducklings at 7 days of age ranged from 35.0 for Fe-dosed ducklings to 35.8 for Bi-dosed ducklings (Table 13). Hcts were not different among doses. Mean Hcts for adult (parent) ducks ranged from 44.6 to 47.3 prior to dosing (Table 1). Sex Ratios—Of 399 ducklings hatched, 382 were identified as to sex: 189 females and 193 males. We found no differences among doses in the sex ratios of ducklings (Table 14). Organ Weights—The mean weights of kidneys of ducklings were: 0-dosed ducklings—1.76 g, Fe- dosed ducklings 1 .64 g, and Bi-dosed ducklings — 1.56 g (Table 13). The mean weights of livers of ducklings were: 0-dosed—5.70 g, Bi-dosed—5.15 g, and Fe-dosed—5.20 g. Neither mean kidney weights nor mean liver weights differed among doses. Because of their small sizes, gonads of ducklings were not weighed. Elements in Kidneys—No differences were de- tected among doses in the mean concentrations of the elements studied in the kidneys of 7-day-old ducklings. The mean concentrations of Bi in the kidneys were 0.05. Hatchability rates: F = 1.07; P > 0.05. 244 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 continued from page 240 Pb, Bi, and Sn in the blood were all 85%), and the few pairs of ducks with low fertility rates had pathology of the male reproductive tracts. The mean fertility rates of the 0-dosed and Bi-dosed pairs were equal and the mean fertility rates of the Fe-dosed pairs were higher (Table 19). However, we found no statis- tical difference in the fertility rates among the dosed groups. Hatchability Rates The normal incubation period for mallard duck eggs is reported to be 28 days, but a majority of eggs that hatched during our study did so in 25 or 26 days. Most eggs that had not hatched by the 27th day were found to contain dead embryos. Hatchability rates were measured as a ratio of number of hatched eggs to the total number of fertile eggs. The hatchability rates varied widely forunknown reasons and were low for each dosed group. The hatchability rates for the Fe-dosed and Bi-dosed groups exceeded the hatchability rate for the 0-dosed group (Table 19), but we detected no difference in the hatchability rates among the dosed groups. Egg Shell Analysis The only differences among doses for the nine elements studied were higher mean concentra- tions of Pb in shells of eggs from 0-dosed ducks (x= 0.300 ng/g) and Bi-dosed ducks (x= 0.261 \ig/ g) than in shells of eggs from Fe-dosed ducks (x= 0.145 |J.g/g). No difference existed in the mean concentrations of Pb in the egg shells from eggs of 0-dosed and Bi-dosed ducks (Table 20). As with other organs and tissues in this study, high con- centrations of Fe in the diet resulted in lower concentrations of Pb in egg shells. Egg Content Analysis The contents of the 11th egg from each female were saved and analyzed for the nine elements. No differences were detected in the mean concen- trations of seven elements—Bi, Sn, Ca, P, Mg, Zn, and Cu—among the three dosed groups of ducks (Table 21). Mean concentrations of Pb were higher in contents ofeggs from 0-dosed and Bi-dosed ducks than in contents of eggs from Fe-dosed ducks. These differences were manifested by a reduction in the concentration of Pb in the Fe-dosed eggs because no difference was found between 0-dosed and Bi-dosed ducks. Yip et al. (1981) found in- creased mean concentrations of Pb in children as Fe deficiency increased. The contents of eggs from Fe-dosed ducks contained higher mean concentrations of Fe (x = 40.3 |ig/g) than contents of eggs from Bi-dosed ducks (x= 33.0 |ig/g), but no other differences were found among the dosed groups. However, we found suggested differences (P <0.10) between contents of eggs from the Fe-dosed group and contents of eggs from the 0-dosed group (x = 34.4 |ig/ g) . Underwood (1971) reported that, in ducks, iron in serum was elevated by a factor of almost five during the laying season. Also, suggested differences (P <0.10) were found between Cu in contents of eggs from Fe-dosed ducks (x= 1.30 |ig/g) and contents of eggs from 0-dosed ducks (x = 1.37 ug/g), and between Cu in contents of eggs from Fe-dosed ducks and contents of eggs from Bi-dosed ducks (x= 1.43 |ig/g). Continued on page 247 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 245 Table 20. Mean concentrations (j^g/g) of nine elements in egg shells from 0-dosed (controls), Fe-dosed, and Bi-dosed game-farm mallards, n = 17 each for all except n = 18 each for Pb and Bi from Fe-dosed ducks. Dose Pb Fe Bi Sn Ca P Mg Zn Cu 0.300 5.32 0.232 1.40 377106 1732 1364 0.936 29.2 0.042'1 1.31 0.079 0.184 11342 56 39 0.137 0.856 Fe 0.145 6.29 0.305 1.17 392059 1695 1397 0.960 29.8 0.020 2.70 0.079 0.146 3877 47 28 0.181 1.771 Bi 0.261 7.87 0.353 1.45 381559 1658 1381 0.871 29.4 0.027 3.60 0.092 0.222 12960 57 32 0.142 1.166 a SE. MDL: Pb - 0.072 ug/g. Bi- 0.050 ug/g. Sn-1-93 Ug/g. Differences among doses: Pb in egg shells: F = 7.0404; P = 0.002. Table 21. Mean concentrations (ug/g) of nine elements in the contents of eggs from 0-dosed (controls), Fe-dosed, and Bi-dosed game-farm mallards, n = 52 for all samples. Dose Pb Fe Bi Sn Ca P MS Zn Cu 0.170 34.4 0.037 1.28 1186 2696 124.7 18.3 1.37 0.024a 1.79 0.007 0.21 48 98 4.40 1.04 0.04 Fe 0.092 40.3 0.035 1.05 1113 2498 122.8 16.4 1.30 0.013 2.16 0.006 0.14 45 109 2.54 0.84 0.05 Bi 0.185 33.0 0.024 0.91 1161 2488 120.5 16.6 1.43 0.027 1.94 0.002 0.00 31 101 2.44 0.74 0.10 SE. MDL: Pb - 0.064 ug/g. Bi - 0.045 ug/g. Sn-1.82 ug/g. Differences among doses: Pb: F - 5.26; P = 0.0086. 2,49 Fe: F - 3.96; P = 0.0255. Cu: F = 2.47; P = 0.0950. 2,47 246 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Table 22. Numbers of embryo deaths per day (expressed in percentages of the embryos available to die on a specific day) for embryos from 0-dosed (controls), Fe-dosed, and Bi-dosed pairs of game-farm mallards. Day of Dose Incubation Fe Bi10 2 3 3.2 1.5a 4 0.3 0.7 0.3 0.5 5 6 7 0.6 0.4 8 9 10 0.2 0.2 11 0.6 0.6 0.4 0.4 12 0.3 0.3 0.9 0.3 0.3 0.7 13 0.6 0.4 14 0.9 0.3 0.5 0.3 15 0.6 1.0 0.3 0.4 0.5 0.3 16 0.6 1.4 0.4 0.4 0.9 0.3 17 1.6 0.6 0.7 0.6 0.4 0.5 18 2.3 1.1 1.6 1.2 0.5 1.0 19 . 1.7 2.5 4.3 0.8 1.1 1.3 20 5.3 4.6 4.1 2.2 1.7 1.1 21 5.1 9.2 3.7 1.6 2.2 0.8 22 13.2 8.8 8.5 2.9 2.3 2.2 23 16.9 23.6 11.8 4.2 4.0 3.6 24 16.3 21.7 16.2 3.5 6.2 3.7 25 9.5 3.7 7.4 4.0 1.8 2.4 26 3.4 4.5 2.4 1.6 1.6 1.6 27 4.5 6.0 3.5 2.3 3.0 1.8 28 4.3 0.4 5.0 2.3 0.4 2.2 a=SE. April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 247 Table 23. Mean age at death of embryos in fertile, but unhatched, eggs from 0-dosed (controls), Fe-dosed, and Bi-dosed game-farm mallard pairs. Sample sizes are in parentheses. Dose Age at Death (days) Fe Bi All doses 21.6(189) 0.31" 20.9(206) 0.38 22.2(147) 0.27 21.5(542) SE. Difference among doses: Age at death of embryos: F = 4.43; P = 0.0125. continued from page 244 Age of Embryo at Time of Death A written protocol for determining the age of mallard duck embryos at the time of death was not found. Thus, determining the ages of em- bryos in the study was accomplished by combin- ing published criteria for wood duck and turkey embryos and by comparing mallard duck em- bryos extracted from eggs opened at various stages of incubation. The criteria used for aging the embryos relied primarily on overall body length, extent of feathering, and size of the yolk. As with wood duck and turkey embryos, the criteria of eye closure and bill length were inconsistent among the mallard duck embryos, and therefore were not used. The highest rate of embryo deaths (63.2% of the embryos at risk died) occurred from Day 20 through Day 25 of incubation (Table 22). The embryos from the Fe-dosed pairs experi- enced low peaks of embryonic death at Days 3 and 4. Embryos from neither of the other two dosed groups experienced similar peaks early in incuba- tion. Differences were found among the dosed groups in the ages at which embryos died, par- ticularly embryos from Fe-dosed ducks and Bi- dosed ducks (Table 23). Embryos from Bi-dosed ducks died at a later age, on the average, than embryos from the other two dosed groups. Histopathology Thomas et al. (1988:120) reported, "One of the commonest toxic effects [of Bi] recorded is that of renal tubular damage, extending to acute tubular necrosis with some renal failure. Nephrotic syn- drome as a result of glomerular damage has also been described. The liver can be affected with jaundice, various bleeding disorders, and multi- focal hepatic necrosis being described." None of these effects was observed in our Bi-dosed ducks or their offspring. Adults Kidneys All but seven ducks had slight inflammatory changes in the ureters of the kidneys. This change was noted regardless of the dosed group and is considered normal for this group of ducks, based on results from this and previous histologic ex- aminations. Two ducks (one Bi-dosed and one Fe- dosed) had focal granulomas in the kidney paren- chyma. These small granulomas were not related to dose. Six of the seven ducks with no significant lesions (NSL) were in the Pb-dosed group. This pattern indicates that the mild inflammatory changes become more common with age (also evident by the lack of inflammatory kidney changes in ducklings) as the Pb-dosed ducks died at an earlier age than the other dosed groups. Liver Four histologic changes (inflammation, fatty change, hepatocellular swelling, and hemosid- erosis) were noted in all dosed groups. Inflamma- tory changes were mild to moderate in severity and lesions and numbers affected were similar in all groups. Fatty change was most frequently associated with egg production and should be considered within a normal range. Hepatocellu- lar swelling occurred in equal proportion in the Bi-, Fe-, and 0-dosed ducks. Hemosiderosis was most pronounced in the Pb-dosed group (9 of 12 ducks) and had a slightly higher incidence in the Fe-dosed group. No hemosiderosis was detected in the 0-dosed ducks. Pb-dosed ducks did not have any degree of fatty change nor hepatocellu- lar swelling. The lack of these histologic changes, 248 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 which reflect fat mobilization and glycogen stor- age/mobilization, is consistent with the emacia- tion associated with Pb toxicity. Gonads Ovaries were morphologically normal in all groups/ducks examined. Three ducks had vary- ing degrees of egg yolk peritonitis that could negatively impact fertility. Of the three ducks, two came from the Bi group and one came from the control group. Testes from the Bi-, Fe-, and 0-groups were normal. One Fe-dosed duck and one Bi-dosed duck had small areas of inflammation but normal spermatogenesis. One Fe-dosed duck had nor- mal spermatogenesis and mild vacuolization of the seminiferous epithelium. The minimal degree of vacuolization is not judged to be significant to fertility. Of the Pb-dosed ducks, five of the six males did not have spermatogenesis; however, Pb-dosed ducks died shortly after the start of the experiment—before the breeding season. Heart All hearts examined were normal. Three ducks in the Pb-dosed group had varying degrees of in- flammation, most likely related to Pb toxicity and secondary systemic illnesses. Lungs All lung parenchyma was normal in the ducks examined. All groups had mild degrees of peribronchiolar inflammation and lymphoid hy- perplasia. The Pb-dosed ducks had the lowest incidence of inflammatory lesions around bron- chi, which is most likely related to their early demise in the experiment. The remaining dosed groups had varied incidence of this mild inflam- matory lesion: 12 of 12 in Bi-dosed ducks, 5 of 9 in Fe-dosed ducks, and 7 of 10 in 0-dosed ducks. The inflammatory change is not judged to be signifi- cant to the health of the animals and probably represents a range of normal for these ducks. Ducklings Liver The most common finding was a minimal to mild hepatocellular swelling. Based on the ducklings' young age, this condition is considered normal and due primarily to glycogen storage of the ducklings. Kidneys The kidneys were free of any histologic lesions with the exception of two Fe-dosed ducklings and one 0-dosed duckling. Both Fe-dosed ducklings had minimal lesions. The 0-dosed duckling had an inflammatory lesion that probably represented a systemic illness as supported by a small granu- loma in the heart. Heart Several hearts of ducklings were examined and no significant lesions were found. Discussion Only one duck (a Bi-dosed female) died of "natu- ral" causes during our study. She died on Day 131, after laying 16 eggs. She weighed 0.97 kg when initially dosed compared with the mean weight of 1.04 kg for all females on Day 0. Al- though her body weight was lower than the mean weight of all females, she maintained her weight throughout the study and weighed the same (0.97 kg) at the time of death (10 June 1995) as on Day 0. The pathologist necropsied the duck, but post- mortem changes prevented histopathological study. He identified no cause of death. This duck was not selected for collection of blood. Thus, no blood samples were available for analysis. Sanderson et al. (1992) reported a mean Hct of 25.5 in six game-farm mallards 30 days after they were dosed with eight No. 2 Pb shot. In our present study, we found a mean Hct of 25.2 in four Pb-dosed ducks after a mean survival of 9.9 days. In our acute toxicity study (Sanderson et al. 1 997a), on Day 30 the mean Hcts were 49.6 for 0-dosed, 50.8 for Fe-dosed, and 49.6 for Bi-dosed ducks, sexes combined. In our present study, mean Hcts for 0-, Fe-, and Bi-dosed males did not decline through Day 120. We did not expect an effect on Hcts by dosing with Bi shot as Slikkerveer and deWolf (1989) stated that anemia had never been associated with ingestion of Bi. Hcts of 0-, Fe-, and Bi-dosed females all declined about 9% from Day to Day 120, perhaps as a result of stresses asso- ciated with egg laying. We found no effect of dosing with eight, No. 4, Bi or Fe shot on body weight compared with 0- dosed ducks. Puis (1988) found that 1,000 ppm of Bi in the diet had no effect on body weight in chickens. Kimball and Munir (1971 :364) ".. .believe that the effect of the grinding action of the gizzard is to prevent the accumulation of the corrosion prod- ucts on the surface of the pellet." In our study, females dissolved Fe shot that were in the gizzard for a mean of 31.2 days at a faster rate than they dissolved Fe shot that were in the gizzard for a April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 249 mean of 121.2 days. The higher dissolution rate for the former probably resulted from more sur- face area exposed to dissolution per day, on aver- age, for the shot dosed on Day 90 than for the shot dosed on Day 0. From radiographs made on Days 1 1 and 39, we clearly identified all eight, No. 4, Fe or Bi pellets dosed in each of eight female and eight maleducks. Sanderson etal. (1997a) radiographed 20 ducks on Day 23 of their study and identified all shot in the gizzards of five female and five male ducks each dosed with six, No. 4, Bi shot or six, No. 4, Fe shot. The mean weights of gizzards in our present study ranged from 19.2 g for Bi-dosed females to 26.5 g for Pb-dosed males. Sanderson et al. (1997a) reported gizzard weights ranging from 29.3 g to 32.2 g for 0-, Fe-, and Bi-dosed ducks on 12 May 1994, Day 30 of the acute toxicity study. These latter relatively heavy gizzards may be a seasonal phenomenon or they may be related to diet. In Sanderson et al. (1997a), ducks were on a diet of shelled corn for the 30 days before necropsy, whereas in our current study, ducks were on a diet of breeder pellets before necropsy. Mean weights of livers ofmales in the present study (Table 5) were similar to the mean weights of livers of males in the acute toxicity study (Sanderson et al. 1 997a), but mean weights ofboth livers and kidneys of females were higher than mean weights of these organs in the earlier study. These differences may be related to long-term egg laying by females in our present study. Because of season-related increases, gonads were heavier in both sexes in the present study compared with weights reported by Sanderson et al. (1997a). We found that Bi-dosed ducks had higher mean concentrations of Bi in their kidneys than in their livers, but Gregus and Klaasen (1986) re- ported that feces and urine were equally impor- tant in the excretion of Bi. Krigmanetal. (1985:65) estimated a half-time of about 5 days for elimina- tion of Bi from the whole body of humans. Our Bi-dosed ducks had a mean concentra- tion of 1 .54 Ug/g of Bi in their kidneys. Hamilton et al. (1972/1973) reported that humans with no known exposure to Bi had the following concen- trations of Bi at autopsy (ug/g wet wt): kidney - 0.4, muscle - 0.007, and liver - 0.004. Our 0-, Pb-, and Fe-dosed ducks had <0.054 ug/g of Bi in their kidneys. We found a higher mean concentration of Fe in the kidneys of Fe-dosed ducks than in the kidneys of 0-, Bi-, and Pb-dosed ducks. Forth and Rummel (1971) and Skoryna and Waldron-Ed- ward (1971) reported that absorbed Fe differs from other metals by its slow rate of excretion. Sanderson et al. (1997a) found that mean concen- trations of Fe were more than double in the liver and feces of Fe-dosed ducks, but not in the kid- neys, gonads, plasma, and blood cells, as com- pared with 0- and Bi-dosed ducks. The high mean concentration of Fe in the livers of Fe-dosed ducks, as compared with 0- and Bi-dosed ducks in our present study, probably is a result of the low excretion rate of Fe once it is absorbed (Forth and Rummel 1971; Skoryna and Waldron-Edward 1971). Also, Gregus and Klaassen (1986) found that the percentage of Fe in the liver increased as the dose increased, and corresponded to a reduced percentage of the Fe in bone, blood, plasma, heart, lung, and brain. We found a much higher concentration ofCu in the liver than in the kidneys, blood, and go- nads. Copper is reported to concentrate in the livers of domestic ducks (37-555 Ug/g) (Underwood 1971:62). Underwood (1971) re- ported that Cu concentrations in the liver are affected by the levels of Fe and Zn in the diet in rats (an Fe-deficient diet results in high concentra- tions of Cu in the liver). In our present study, we found no difference among doses in the mean amounts ofCu in the liver. Sanderson et al. (1997a) reported means of 3,081 Ug/g P in livers of 0- dosed, 3,108 Ug/g in Fe-dosed, and 3,026 Ug/g in Bi-dosed game-farm mallards on Day 30 after dosing with 0, six, No. 4 Fe, or six, No. 4, Bi shot. No differences existed in the mean concentrations of P in the livers of ducks on Sanderson et al's (1997a) study. Our current study found higher concentrations of P in the livers of 0- and Pb- dosed ducks versus Fe- and Bi-dosed ducks. There seems to be little agreement as to the concentrations of Bi in the blood that are diagnos- tic for intoxication. Krigman et al. (1985) reported that blood Bi concentrations in humans adminis- tered oral therapeutics differ between those who exhibit side effects from chronic use and those who do not. Those with no symptoms usually have Bi concentrations <0.05 Ug/g in blood and those with neurological symptoms have concen- trations >0.05 Ug/g. Hillemond et al. (1977) and Serfontein and Mekel (1979) concluded that 0.05 Ug/g Bi in blood is an index of potential neurotox- icity in humans. Dipalma (1988) said that Bi should not ex- ceed 0.02 Ug/g in blood of humans, and Locke et al. (1987) reported neurotoxic effects at Bi concen- trations of <0.1 ug/g in blood. Ross et al. (1988) suggested that 6 Ug/g of Bi in the brain of labora- 250 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 tory mice showed neurologic symptoms and that a concentration of>0.5-2.0(ig/gof Bi in blood had to be maintained for several weeks to accumulate enough Bi in the brain to cause neurotoxicity. Thomas et al. (1988:124) reported that concentra- tions of Bi in blood of more than 0.1 |4g/g were potentially dangerous in humans and indicated that treatment with Bi should be stopped. Con- centrations between 0.05 and 0.1 Ug/g indicate that patients should be carefully monitored, and concentrations of less than 0.05 M-g/g are consid- ered safe. In our present study, we found no effect of dosing ducks with Bi shot on egg laying com- pared with 0- and Fe-dosed ducks. Hermayer et al. (1977) added 1, 10, 100, and 1,000 ppm Bi trioxide to the diet of female chickens and found no effect on feed intake, number of eggs laid, or changes in body weight. Puis (1988) found that 1,000 ppm Bi in the diet had no effect on egg production in chickens. Conclusions We conclude that under the conditions of this study, eight No. 4, Bi shot, repeatedly dosed in game-farm mallards, resulted inno demonstrable toxic effects on adult ducks or the eggs and duck- lings they produced. Survival of game-farm mallards was not af- fected during a 150-day test in which groups of ducks were dosed with eight, No. 4, Bi shot and compared with survival of 0-dosed and Fe-dosed ducks. All ducks dosed with eight, No. 4, Pb shot died within 2 weeks. No adverse effects on tissues were detected and concentrations of residues of elements in tissues were not different for 0-, Fe-, and Bi-dosed ducks. No adverse effects were manifest for egg fertility, egg weight, eggshell thickness, egg hatch- ability, duckling weight at Day 7, and survival of ducklings to Day 7, for ducks dosed with eight, No. 4, Bi shot. Values for these variables were not different from those of 0- and Fe-dosed ducks. The only clear difference between Bi-dosed ducks and 0- and Fe-dosed ducks was in the timing of embryonic mortality, which was later for Bi-dosed ducks than for 0- and Fe-dosed ducks. 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Trace elements in human and animal nutrition. 3rd Ed. Academic Press, New York and London. 543 pp. U.S. Fish and Wildlife Service. 1986. Migratory bird hunting: nontoxic shot approval procedures. Federal Register 51(225):42098-42102. UNIVER9ITY OF ILLIN0I9-URBANA 3 0112 044858188 Illinois Natural History Survey Natural Resources Building 607 East Peabody Drive Champaign, Illinois 61820 217,333-6880 A Division of the Illinois Department"of Natural Resources ILLINOIS NATURAL RESOURCES