foes ane tte eee Baer LOE ee we Cade VS tos tov recto, Fh, fie Ra aad So Sa Ahn Bae Nehin Dating Bi BE ge BEN aM he ba be AVA AEE L ie tin dgoR ee Ws A sitelex Touts Sod Behe lg Restle lone GAS ANE Ai wow yg fa Neate oT, Behe et hot Hebe te Me dae bet oot, at aie Ay ict oP tte ele 5 By ages BAe eA oe ey FB Lot iy Nabe hiltatieliony Beton, Sede Sasaiea net De, Fate ee that 5 Ma Hobe Mee ae CLs Rohit Ny Se reg ey Meet nt Fue ds ay 2 a5 PeEe fon Hara Fit ae Mathie aoe hig Fife Ay hoe Da lie te Fa Se He teks Faby Mohyfincncn Od athe Ey Sy PPAF I BAbe bgt vats Z ior, Fasttore Far koh Peta ity te fed VIDEO OLR ave. ke tg ayiei Sa ee een Lda aden PPI OATS ait Bui pore meee ee : eer IND Ah al "% des ao hee ae fay ts Sactacer ‘ Fave B46 akan feces ae Ee pecan eta Warner Bute ESR aN ist Beha Am DWH « eotes ee erase ares 5 ASS WEAR LAW oh stam otcn annus Scie: PAG HSRaeot Osi Boe oo 20 aT ea ear se SEI SS ot ote OPW ASE Aone Seven SBS an SN NET A whats TAP iba hit Sr ee BNF oagsenk, Se Seen reine art OMT Fae TR pet Rieti Bema vA SOA V Ln, ve R ER may SUA AA sew eg Satepa cy tan Protea wa carts sheik SiN tae me 1 te. Sarbanes Reig eis gh SOs, bohny. Pe eens Picker Neen weno Woe hae Aemwten ye nay vb WIN ech cM en cael etna Met é We RS] Shee RANA (Rata omen be oe eeateenet o WSN Rthawctialt tne Made e ea oaths eect Tea Ratan eee dR Mi anak EWN oe SP AU AS alt Ee Son SF REE AAR olathe AMT UNIVERSITY oF ILLINOIS LIBRARY NATURAL HIST. SURVEY Digitized by the Internet Archive in 2011 with funding from University of Illinois Urbana-Champaign http://www.archive.org/details/acutetoxicityofig5sand Va) Peer” ey tke vt. | eae ; 1 »® j i ro q i Ae =~ = od 1 a | rT, 7 5 ‘ 1 - / nel uf ; 4 = gig : ‘ 6s,’ ‘ ’ ; re ‘ A f y i 4 i @ if 4: 3 | | 6 yee Ee EENODG he.) * ~ NATURAL _ HISTORY SURVEY Toxicity of Ingested Bismuth ££ —* Shot i eee iam ue | GlenC. c. Eades. Wilkiam be 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 3and4_. April 1997 = = | Natural History Survey Library Illinois Natural History Survey, Edward J. Armbrust, Acting: Chief A Division of ins Illinois rae of Natural Resources OF 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 pees are included with the ee Illinois Natural History Survey Distribution Center — ‘Natural Resources Building. 607 East Peabody Drive. Champaign, Illinois 61820 °° Citeidns: Sanderson, G. en W. Li Anderson; .G.L. Foley, L.M. Skowron, J, D. Brawn, and _J.W. Seets. 1997. Acute toxicity of ingested bismuth alloy shot in game- -farm mallards. eiots Natural History ey, Bulletin 35(3):185- 216. 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 shotin . - _ game-farm mallards: chronic health effects and effects on. reproduction. Illinois Natural History eae Bulletin 35(4): 217- ae - Editors: Thomas ie. Rice and Charles Warrick ; 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. WNIVERSILY: 9) ILEINOES LIBRARY 3 SHAME ay ~ sare SURVEY Contents Article 3: Acute Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards Acknowledgments _ ii ABSTRACT 185 INTRODUCTION 185 LITERATURE REviEW 185 MeETHOoDs 187 Toxicity Study 187 Chemical Analyses 189 Storage of Samples 189 Digestions of Samples 189 Digestions for ICP Analysis 189 Digestions for GFAA Analysis 189 Analytical Methods 189 ICP 189 GFAA 190 Quality Control 190 es Calculations 190 a Statistical Analyses 190 Resutts 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 HIsTOPATHOLOGY 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 MetHops 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 NR 221 GFAA 221 Quality Control 221 Calculations 221 Statistical Analyses 222 RESsuLTs 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 HISTOPATHOLOGY 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 Acute Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 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 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 3 April 1997 Illinois Natural History Survey Bulletin Acknowledgments James W. Sergent and Wendy L. Grethen, Illinois Natural History Survey, fed the ducks, cleaned the pens, examined the feces for voided shot, saved fecal samples for chemical analysis, and assisted with other phases of the study. The following people assisted with weighing and dosing ducks and collecting and processing blood: Stephen P. Havera, Michelle M. Georgi, Aaron P. Yetter, and Christopher S. Hine, all with the Waterfowl! Re- search Laboratory, Forbes Biological Station, IIli- nois Natural History Survey, Havana, Illinois; Ed- ward J. Heske, Linda K. Campbell, and Anne E. Zielske, all with the Illinois Natural History Sur- vey; and volunteers Beverley C. Sanderson and J. William Sanderson. Beverley C. Sanderson also assisted with many tasks in the preparation of this manuscript. William R. Manuel, retired, College of Veterinary Medicine, University of Illinois, pro- vided his expertise in the collection of blood. 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 Company, Los Angeles, California, provided fi- nancial support for the research and costs of this publication. Vol. 35 Art. 3 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 185 Abstract Ina30-day study involving penned game-farm mallards (Anas platyrhynchos), no harmful health effects were detected from dosing with either six, No. 4, bismuth/tin (Bi/Sn) alloy shot or six, No. 4, steel (Fe) shot, as compared with sham (0 shot) dosing. Survival, hematocrit (Hct) values, body weight, and mean weights of kidneys, livers, gonads, and gizzards were not affected. Mean concentrations of nutritionally essential elements (calcium [Ca], phosphorous [P], magnesium [Mg], zinc [Zn], copper [Cu], Fe, and Sn) were different among doses and between sexes in kidneys, livers, and gonads. However, concentrations of these elements in these organs and tissues in Bi-dosed ducks were not different from both 0- and Fe-dosed ducks. Bi/Sn alloy shot, as tested in this study, elicited no indications of toxicity in game-farm mallard ducks. Introduction To protect waterfowl from poisoning caused by ingested lead (Pb) shot, nontoxic shot regulations were implemented for waterfowl hunting on areas with severe problems with Pb poisoning (“hotspots”) in the United States beginning in the early 1970s (Anderson 1992). Federal regulations became nationwide in 1991. Several European countries have converted or are planning to convert to nontoxic shot for waterfowl hunting (Moser 1992) and Canada will implement a nationwide ban on lead shot for all migratory bird hunting in 1997 (Canadian Wildlife Service [CWS] 1995). From the 1970s to the early 1990s, Fe was the only shot material approved as nontoxic by the U.S. Fish and Wildlife Service (USFWS) (Longcore et al. 1974). Although hunters have generally adapted to using Fe shot, some have urged that a search for alternative shot with greater ballistic capability be continued. Specifically, they wanted a shot that is non- toxic, inexpensive, and ballistically similar to lead shot. There have been numerous evalua- tions of potential substitute shot. Irby et al. (1967) evaluated three types of plastic-coated Pb, two Pb/Mg alloys, Fe, Cu, Zn-coated Fe, and molybdenum-coated Fe. Longcore et al. (1974) evaluated Pb shot with nickel coatings, Pb/phosphor Sn alloy shot, Pb shot with Sn/ nickel alloy coatings, steel shot with Pb coatings, Pb/Sn alloys, two types of disintegrable Pb shot, and Pb shot with biochemical additives. Haseltine and Sileo (1983) evaluated uranium. No satisfactory alternative was found until 1990, when John E. Brown, St. Catherines, Ontario, was awarded a U.S. patent for Bi shot. Our primary objective was to determine if Bi/Sn alloy shot caused toxic effects in captive game-farm mallards. Secondly, if toxic effects were manifest, we wanted to associate toxic ef- fects with amounts of Biand other elements in the tissues. Our study complied with the “Acute Toxicity Test” guidelines of the USFWS and the CWS. Dr. Simon Nadeau, CWS, and Dr. Keith A. Morehouse, USFWS, reviewed our protocol be- fore we initiated our study. Literature Review The first known report of metallic Bi dosed in birds was by Hanzlik and Presho (1923), who administered metallic Bi, Pb, and other heavy metals to pigeons. The fatal dose of metallic Pb in their studies ranged from 0.6 to 2.28 g/kg. By contrast, none of the four Bi-dosed pigeons died after receiving doses that averaged 1.39 g/kg, and the researchers concluded that Pb is more toxic— and mortality is higher with smaller doses—than other heavy metals, including Bi. Sanderson etal. (1992) conducted the first comprehensive study to determine the toxicity of ingested Bishot (100% Bi) in birds (mallards). They followed with tests on ingested Bi/Sn alloy shot (Sanderson et al. 1997b) and the present study. Sanderson et al. (1997b) reported that reproduction of game-farm mallards was not affected after chronic dosing with Bi/Sn alloy shot. Sanderson et al. (1997a) reported no toxic effects of Bi shot embedded in the breast muscles of game-farm mallards. The International Commission on Radiologi- cal Protection (ICRP) (1960) reported that Bi is rapidly excreted by the kidneys except for small amounts retained in these organs. Some Bi is lost in bile. The estimated half-time for elimination in humans was about 5 days. Hamilton et al. (1972/ 1973) reported 0.4 ug/g Bi (wet weight) in kid- neys and 0.004 g/g Bi in livers of autopsy cases (humans) with no known exposure to Bi. Kidneys of 22 individuals who had been given Bi salicylate had 33 ug/g Bi and livers had 6.8 ug/g Bi. 186 Lee (1981) reported that after treatment was terminated, Bi declined about 2.6% in the urine daily, with half purged from the body in about 20 days. Fowler and Vouk (1979:348) stated, “In- gested bismuth is largely eliminated unabsorbed in feces. Model values for the daily balance of bismuth in reference man are: dietary intake 20 ug/g, fecal elimination 18 ug/g, urinary excretion 1.6 ug/g. ... Absorbed bismuth is mainly ex- creted in the urine.” Oehme (1979) reported that soluble and in- soluble Bi salts, suspended in oil to maintain levels in blood, were injected to treat syphilis. Other Bi compounds were used to treat malaria and amebiasis. Medicinal use of Bi decreased with the advent of newer treatments. Most hu- man exposure to Bi is in compounds that are insoluble and not readily absorbed whether in- gested or applied to the skin. Apparently tissue binding is slight, even when Bi is absorbed. An equilibrium is established among tissues, blood, and urine. Kidneys have the highest amounts of Bi, with the liver generally a poor second. With few exceptions, Bi compounds present no prob- lems whether by ingestion, inhalation, or dermal application. Poisoning from industrial exposure is rare (Oehme 1979). There are no federal standards for Bi or its compounds and no evidence linking Bi or Bi compounds with industrial poisoning. Also, all episodes of Bi poisoning were from soluble com- pounds used in medicine, and fatalities and near fatalities were mainly from intravenous or intra- muscular injection of soluble salts (Key et al. 1977). Venugopal and Lukey (1978) reported that low solubility limits the toxicity of Bicompounds, which are highly toxic. Locke et al. (1987) re- ported neurotoxic effects at Bi concentrations of < 0.1 ug/g in blood. Thomas et al. (1988:124) reported, “Since tox- icity resulting from environmental or industrial exposure to bismuth or any of its compounds is not a problem, levels of tolerance have not been identified ....” Bi telluride, which is used as a semiconductor in the electronics industry (Oehme 1979), is an exception. Thomas et al. (1988) re- ported, however, that the French Ministry of Health banned the sale and use of all Bi com- pounds, and that Australia restricted the use of Bi subgallate. These authors suggested that in hu- mans amounts of Bi in blood > 0.48 umol/L (0.1 ug/mL) are potentially dangerous, that amounts > 0.05 and <0.1 ug/mL call for careful monitoring of patients, and that amounts < 0.05 ug/mL are considered safe. Illinois Natural History Survey Bulletin Vol. 35 Art. 3 Krigman etal. (1985) reported that levels of Bi in blood of humans differ between individuals who show side effects from chronic use of Bi and those who do not. Those who show no effects usually have <0.05 ug/g Biin their blood whereas those who show symptoms have > 0.05 ug/g Biin their blood. These authors conclude that levels of Bi in blood > 0.05 ug/g indicate a high risk and amounts < 0.05 ug/g indicate a low risk. Other investigators (Hillemond et al. 1977; Serfontein and Mekel 1979) concluded that 0.05 ug/g Bi in blood is potentially neurotoxic. Dipalma (1988) stated that levels of Bi in blood should not be > 0.02 ug/g. Dipalma (1988) reported that exposure of humans to Bi is not considered a serious indus- trial hazard. According to him, there are few data on Bi concentrations in blood from either oral or topical applications because of the assumption that absorption of Bi is low. Dipalma (1988) indicated that bacteria in the intestine might me- thylate Bi to form a soluble compound. He re- ported (p. 244), “In animals, trimethyl bismuth is highly toxic and causes an encephalopathic syn- drome similar to that seen in man. Blood levels of bismuth should not exceed 20 ug per L (20 ppb).” In their review, Slikkerveer and de Wolff (1989) summarized the effects of Bi in mammals and reported a peak of Biin blood 45 minutes after oral dosing with colloidal Bi in humans. Others had reported peaks between 4.7-21 ug/g 15-60 minutes after dosing. With continued dosing, 3- 4 weeks were necessary to reach a steady-state of Bi in plasma. Persons who had not received Bi therapy had between 1 and 15 ug/L of Bi in their blood. Although the site of Bi absorption in the gastrointestinal tract is unknown, Slikkerveer and de Wolff believed that absorption after oral dos- ing is dependent on solubility and that cysteine, sorbitol, and lactic acid may promote absorption of Bi. They suggested that colloidal Biis absorbed in the small bowel and stomach. Meaningful reference values for Bi levels in tissues are not available because of large varia- tions in experimental and analytical techniques, and the chemical form of Bi in blood is unknown. The highest concentrations of Bi were always in the kidney. After 14 months of dosing with colloi- dal Bi subcitrate in rats, Bi concentrations ranked from high to low in kidney, lung, spleen, liver, brain, and muscle tissues. When bone concentra- tions were measured, they were usually 10-20 times lower than in the kidney. Slikkerveer and de Wolff (1989) reported that Biis found in both urine and feces. The Biin feces April 1997 comes from Bi excreted in bile, which concen- trates plasma Bi by a factor of 10, and from intes- tinal secretion. In humans showing symptoms of Bi toxicity after exposure, concentrations in bone were 1.5-6.7 ug/g wet weight compared with < 1 ug/g wet weight in nonexposed individuals. Bi encephalopathy is mainly supported by elevated blood Bi. A steady-state Bi concentration of > 100 ug/L (ppb) of blood in humans was arbitrarily suggested as an “alarm” level and 50 ug/L was considered a “safety” level, but no proof supports these choices. Concentrations of blood Bi from 10 to 4,600 ug/L were found in 618 Bi encephalopa- thy patients. Abbracchio et al. (1985) administered tri-po- tassium-dicitrato bismuthate intraperitoneally and by gavage in laboratory rats. After intraperi- toneal injection, Bi reached peak concentrations in blood within 30 minutes and declined rapidly. When a dose 10 times higher was given by gastric intubation, much lower blood concentrations were detected. They found no Bi in the brain after oral administration and concluded that there was ap- parently little risk of neurotoxicity after dosing with this derivative. They stated (p. 143), “This could also be the reason why no appreciable side effects have ever been described after use of this drug in humans.” Gregus and Klaassen (1986) reported that feces and urine were equally important for the excretion of injected Bicompounds in rats. Biliary excretion apparently determined the fecal excre- tion of Bi, but the percentage of dosed Bi excreted in the bile was independent of the amount dosed. Woods and Fowler (1987) reported that little information was available on the effects of Bi in mammals in general, but noted that toxic effects in the liver, kidneys, and blood have been found in humans and laboratory animals after exposure to Bi compounds. In their studies with rats (P. 276), they found that “...bismuth significantly im- pairs the activities of both hepatic ALA synthetase and heme synthetase at all dose levels.” Ross et al. (1988) injected 2,500 ug/g of Bi subnitrate intraperitoneally in laboratory mice. Although Bi concentrations in blood and brain tissues of mice that showed signs of neurotoxicity were significantly higher than in dosed mice that showed no signs, they concluded that the concen- tration of Bi in blood did not predict neurologic signs. They suggested that 6 ug/g of Bi in the brain show neurologic symptoms and that a con- centration of > 0.5-2.0 ug/g of Bi in blood had to be maintained for several weeks to accumulate enough Bi in the brain to cause neurotoxicity. Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 187 They also concluded that 5-10 ug/g Biin the brain was associated with motor dysfunction in hu- mans and mice and that concentrations above 50 ug/L (ppb) are necessary to produce frank en- cephalopathy in humans. Slikkerveer and de Wolff (1989) reported, however, that following oral dosing of trimethyl Bi to dogs, the level of Bi was higher in the liver than in the kidney, probably because of the or- ganic character of the molecule. They reported that early toxic effects of Bi may be related to effects on enzymes of the haem synthesis but that anemia has never been associated with ingestion of Bi. Methods The Bi/Sn shot used in this study contained 0.0040% to 0.0186% Pb K=0.0094%,SD =0.0054%). Because Pb made up < 0.1% of the test shot, Environment Canada (1992) guidelines did not require that tissues be analyzed for Pb. We recog- nized, however, that researchers are interested in Pb, so we included this metal, albeit at a some- what high detection limit, in the analyses for residues. Seventy-five female and 75 male wild-type game-farm mallards 6 to 8 months of age were purchased from Whistling Wings, Hanover, IIli- nois. The ducks, reared on a 60-acre lake, were transported from Hanover to Champaign, Illi- nois, by truck in crates on 22 March 1994. Toxicity Study The ducks were weighed and one duck was ran- domly assigned to each pen. Forty ducks (20 females and 20 males) were randomly assigned to one of the three treatments—dosed with Bi shot, dosed with Fe shot, or sham dosed with 0 shot (controls). Five male and five female ducks were randomly selected from each dosing group for collection of feces to be analyzed for excreted Bi, Fe, and Sn. Sanderson et al. (1997b) describe the methods we used to randomize the doses, ducks, and pen assignments for the present study. The pens were consecutively numbered, el- evated, outdoor, 1-m2 structures. They were cov- ered with vinyl-coated, 25.4-mm mesh, 14-gauge wire. A 9.1-m x 36.6-m pavilion (roof but no sides) covered the pens (see Sanderson et al. 1992 for more details). Facilities for holding the ducks were inspected and approved by several members of the Labora- tory Animal Care Committee, University of IIli- nois, after the ducks were placed in the pens. The 188 Illinois Natural History Survey Bulletin committee also inspected the facilities once dur- ing the study. Commercial duck pellets (Heinhold 17% Duck Finisher Pellet™, Heinhold Feeds, Inc., Kouts, Indiana) were provided ad libitum during the 3-week acclimatization period. The duck pellets contained a minimum of 17.0% protein. On the date of dosing, the pellets were replaced with whole shelled cornad libitum for the duration of the study. Protocols of the CWS and the USFWS specified these diets (Environment Canada 1992). The three groups of ducks were each dosed as follows: sham dosed (controls); six, No. 4 (3.30 mm diameter), Fe shot; or six, No. 4, Bi shot. Ducks in each group are hereafter referred to as 0- dosed (controls), Fe-dosed, and Bi-dosed. We began the study on 12 April 1994 (Day 0) when we weighed, collected blood samples, and dosed the ducks. A small plastic funnel fitted witha plastic tube (9.5 mm outside diameter, 22.9 cm long) was inserted through the pharynx into the proventriculus. To reduce friction, the tube was kept ina pail of water between dosings. Each dose of shot was poured into the funnel and flushed into the proventriculus with approxi- mately 5 mL of water. Controls were treated the same except that no shot were included. Before dosing, the shot were counted, weighed, and placed in individual vials in the laboratory. The type, number, and weight of each dose of shot were recorded on the top of each vial and on a computer printout for each duck. At dosing, the shot dose was matched with the corresponding duck. Blood was collected from the wing vein in heparinized microhematocrit capillary tubes for hematocrit determination and in 2.5-mL syringes for separation into cells and plasma. The plasma samples were analyzed for major elements (> 1% by wtin shot) and for major nutritionally essential elements (Ca, P,Mg, Zn, and Cu). Twenty-gauge, 25.4-mm needles were used (Baxter Healthcare Corporation, Scientific Products Division, McGaw Park, Illinois). The whole blood was injected into 10-mL lithium heparinized Vacutainer™ tubes and centrifuged to separate cells and plasma. Body weights were recorded and blood samples collected on Days 0, 15, and 30. As each group of 24 hematocrit samples was collected, the samples were centrifuged at the site in a mobile laboratory (house trailer). The tubes were spun for 5 minutes at 11,500 RPM at 13,000- g force, after which the values were read and recorded. The whole blood samples also were centri- fuged at the site, in groups of 12 samples (capacity Vol. 35 Art. 3 of the centrifuge) at a time. The tubes were spun for 5 minutes at 3,000 RPM. The plasma was removed with micropipettes and placed in 5-mL nonheparinized Vacutainer tubes. The cells were retained in the 10-mL lithium heparinized tubes. As the plasma and cells were separated, the tubes were placed in racks and put on ice ina styrofoam cooler. All samples were stored in a freezer (-10°C) until analyzed. The Bi shot were provided by William S. Montgomery, Jr., Bismuth Cartridge Co., Dallas, Texas. Seven shot were chemically analyzed in the laboratory of the Illinois State Water Survey, Champaign, Illinois, before the ducks were dosed. Mean (+SD) percentages of elements in these shot were as follows: Bi = 98.35%, + 0.86%; and Sn = 1.90%, + 0.10%. Other elements averaged < 0.1% each; Pb ranged from 0.0040% to 0.0186% (x=0.0094%, + 0.0054%). Fe shot were removed from commercial 12-gauge shotgun shells and were not analyzed. The 120 ducks were weighed and blood was collected from the wing veins, as scheduled, on Day 30 (12 May 1994). Following these proce- dures, the ducks were killed by decapitation and necropsied on the same day (with the exception noted below). The gizzard, liver, kidneys, and gonads were excised from each duck. Two changes were made in the methods as originally approved by the CWS. First, because voided shot were not found in the feces, 20 dosed ducks were radiographed to obtain a positive record of shot retention in the gizzards. The 20 ducks for which daily fecal samples were being collected were chosen so that fecal material could be re-examined if the radiographs indicated dosed shot were missing from the gizzards. A dorsal- ventral and a right or left lateral view radiograph were made for each duck on Day 23 (5 May 1994) by the College of Veterinary Medicine, University of Illinois. The other change in the methods involved killing the ducks and performing the necropsies over 2 days (instead of 1) to ensure that tissue samples were obtained from freshly killed birds. The pathologist necropsied 30 ducks on 12 May and the other 30 ducks on 13 May 1994. After the pathologist had examined, weighed, and fixed representative samples of kidneys, liver, and gonads in 10% formalin for histopathology, the remaining residual tissue from these organs was placed in separate, numbered, plastic bags and stored ina freezer as backup samples. Organs from the remaining 60 ducks, which were not necropsied, were removed and weighed, placed April 1997 in individual, numbered, plastic bags, and stored in the freezer as additional backup samples. We took representative samples, after fixing in 10% formalin, from each of the 60 necropsied ducks and examined the samples histopathologi- cally. Sections of gonad (testis or ovary), liver, kidney, and gizzard were embedded, trimmed, and sectioned at 4 microns. Tissues on glass slides were stained with hematoxylin and eosin by stan- dard methods. All ducks were examined by a veterinary pathologist, who did not know the dose history of the ducks. Later, we associated group assignment and weight data with histo- logic findings to aid in interpretation. Chemical Analyses Storage of Samples Samples were inventoried when received, stored at -10°C, and monitored daily. Samples were allowed to thaw to room temperature, then pre- pared for metal analysis by labelling by tissue type and a number for identification. The sex of the duck and the shot dose it received were not disclosed to the individuals who analyzed the samples. Digestions of Samples Blood cells, blood plasma, livers, kidneys, go- nads, and feces were acid digested before analysis for metals withinductively coupled, argon-plasma emission spectroscopy (ICP) and graphite-fur- nace, atomic-absorption spectroscopy (GFAA). Because wet weight concentrations of the blood and organs were desired, these samples were not dried before digestion. Feces were dried at 104°C to determine percent moisture. The concentra- tions of metals measured in fecal samples are on a dry-weight basis. We analyzed for Bi, Sn, Fe, Pb, Ca, Mg, P, Zn, and Cu. ICP was used to measure these metals; beryllium (Be) was used as an inter- nal standard. GFAA was used to measure Pb and Bi when they were at low concentrations. Digestions for ICP Analysis We used samples of 0.5 to 1.0 g. A mixed portion of the sample was weighed to 1.0 mg with an electronic, top-loading balance and placed into a tared 50-mL conically tipped polypropylene, cen- trifuge tube. The tubes were precleaned for 24-hr with a 10% nitric acid (HNO3) soak then rinsed in deionized water. Samples of feces were weighed to 0.1 mg. Approximately 30 to 50 mL of an acid and internal standard solution were added to the sample after taring. The final acid concentrations were 2% HNO; and 10% hydrochloric acid (HC). Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 189 The Be concentration was targeted at 2.00 mg/L. The samples were then homogenized into a slurry using a saw-toothed generator made of titanium and TFE-fluorocarbon (Pro Scientific, Monroe, Connecticut). The internal standard solution was used to rinse excess materials from the generator and the amount was accounted for in the total weight. Samples were prepared with the SpectrPrep System™, an automated microwave-digestion- system (CEM Corporation, Matthews, North Caro- lina). A 15-mL sample loop was used. After heating, cooling, and filtering, about 12.5 mL of the sample were collected and deposited by autosampler into 15 mL polypropylene test tubes. This digestate without further treatment was then used for ICP analysis. The automated microwave digestion system was a relatively new technique to prepare samples. A few problems arose in adjusting to the system; most were associated with clogging of the small-diameter tubing. A thorough homogenation followed by a few hours in a warm, ultrasonic bath usually improved the operation. Digestions for GFAA Analysis We used samples of 0.5 to 1.0 g. A mixed portion of the sample was weighed to 1.0 mg with an electronic, top-loading balance and placed into a tared TFE-fluorocarbon beaker. Approximately 20 mL of deionized water (DI HO), 0.250 mL concentrated HNO3, and 1 mL of hydrogen per- oxide (H,O,) wereadded. The mixture was heated at approximately 95°C until the solution started to clear (about 0.5 hr). Approximately 20 mL of DI H,0 and 2 mL H,O, were added. Upon further heating the mixture cleared and “foamed up.” DI H,0 was used to rinse contents from the sides of the beaker. The beakers were then covered with TFE-fluorocarbon watch glasses and allowed to reflux for approximately | hr. The resulting solu- tions were usually clear to yellow. The samples were increased to 50 mL in a volumetric flask, filtered through 0.45-~m nitrocellulose filters, and stored in acid-washed, linear, polyethylene bottles. The ultimate acid concentration was 0.5% HNO. High-purity acids and hydrogen peroxide (Baker Ultrex™ and Fisher Optima™) were used for all digestions. Analytical Methods @e Weuseda ThermofJarrell Ash (TJA) AtomComp™, Model 61, vacuum spectrometer. The instrument has a polychromator configured with 44 fixed 190 channels, including analytical lines for high and low concentrations of Ca and Mg. Although we reported results for only a few elements, we mea- sured 30 analytes to monitor for spectral interfer- ences, which we did not detect, with blank sub- traction and background correction. We used USEPA Method 200.7, Revision 4.4, Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma- Atomic Emission Spectroscopy for our work. We modified the method and used a different diges- tion process, and we measured Bi, not a listed analyte. We chose Be as an internal standard because it was not present in the samples, it does not cause spectral or background interferences, and it is precisely detectable. GFAA Weused 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 100°C so that samples dried on contact. We used method 3113 of Greenberg et al. (1992). We ana- lyzed samples in triplicate and reported the mean. Quality Control We calibrated instruments daily and we verified the standard curve using National Institute of Standards and Technology (NIST) traceable, qual- ity control samples (QCS). Samples (usually 10) were bracketed by calibration blanks, laboratory fortified blanks, and instrument-performance, check solutions during analysis as well as peri- odic checks on the internal-standard solution. The ICP instrument was programmed to compen- sate for drift by recalculating the slopes of the calibration curves if any analyte was more than +5% of the true value while measuring the ICP check standard. If an analyte measured greater than +10% of the true value for this sample, the instrument was recalibrated and the affected samples reanalyzed. The ICP check standard was formulated for a concentration at the midpoint of the calibration curve. It was traceable to NIST Standard Reference Materials (SRMs). The GFAA QCS initially were required to be within 10% of the true value. Subsequent measurement of the bracketed internals was required to be +15%. If these limits were exceeded, the instrument was recalibrated and the affected samples reanalyzed. Ten percent of the samples were digested and analyzed in duplicate, half of them spiked. Additional liver samples were treated as dupli- Illinois Natural History Survey Bulletin Vol. 35 Art. 3 cates as part of the process of evaluating the automated, microwave-digestion equipment. Digestion blanks and spiked digestion blanks were prepared at a frequency of 10%. They were processed through the complete digestion and analytical system in the same manner as the samples. Calculations We saved the ICP data during analysis in data- base files with ThermoSpec (TJA) software utiliz- ing Enable OA. Data were then imported into Enable spreadsheets for tabulations and calcula- tions. The Enable spreadsheets were saved in a Lotus 1-2-3 format on diskette. The GFAA results were recorded on an instrument printer as con- centrations in ug/L based upon peak-area mea- surements. These data were manually entered into spreadsheets for tabulations and calculations. The Method Detection Limit (MDL) (Glaser et al. 1981) was used to establish the detection limits for concentrations of elements in tissues and other materials. Glaser et al. (1981:1426) describe the MDL as “a new performance crite- rion for chemical analysis . . . defined as that concentration of the analyte that can be detected at a specific confidence level.” Also, “The detec- tion limit should be related to the standard devia- tion of the measured value at or near zero concen- tration of the analyte... .” They further report (1427), “MDL is considered operationally mean- ingful only when the method is truly in the detec- tion mode, i.e., analyte must be present. The method detection limit is defined as the minimum concentration ofa substance that can be identified.” To be considered a meaningful difference, the MDL procedure is required to provide a value that averages > two times the MDL (Glaser et al. 1981). For statistical analysis, values < MDL were en- tered as one-half the MDL value. Most values for elements in the tissues were determined by ICP. Results of ICP analyses for Bi, Pb, and Sn were usually lower than the MDLs. Thus, selected samples of kidneys, livers, and gonads were analyzed for Bi and Pb by GFAA. The remaining amounts of plasma and blood cells after analysis by ICP were inadequate for further analysis by GFAA. Graphite-furnace atomic ab- sorption is not a satisfactory method to analyze for Sn. Statistical Analyses In this report, when two values are reported as “different” or that they “differ,” it means that they differ in a statistical sense at an alpha of (P < 0.05). April 1997 Differences in concentrations of various ele- ments in livers, kidneys, and gonads; weights of organs (post-mortem); numbers of shot recov- ered; and dissolution rates of shot were tested by one- or two-way ANOVA using sex and dose (shot type) as grouping factors. Homogeneity of variances among groups was assessed with Levene’s test. Brown-Forsythe or Welch statistics were used in instances where variances could not be assumed equal. In instances where the overall test of differences among groups was significant, pairwise comparisons were performed and sig- nificance evaluated based on the Bonferroni cor- rection. In instances where comparisons were made with controls, Dunnett’s procedure was used. Variation in body weights, hematocrit counts, and concentrations of elements in plasma and red blood cells (all measured at Days 0, 15, and 30) were evaluated using repeated-measures ANOVA. As above, sex or dose or both, were used as between-subject factors. Within-subject tests for variation over time were also performed as were tests for interactions between dose and time. When assumptions of compound symme- try were violated, Huynh-Feldt-adjusted signifi- cance probabilities were used. Results Survival All 120 ducks (controls, Bi-dosed, and Fe-dosed) survived to the end of the 30-day test period. Retention and Dissolution of Shot No voided shot were found in the feces from the 20 dosed ducks (5 female and 5 male Bi-dosed and 5 female and 5 male Fe-dosed ducks) for which feces were saved for chemical analysis. Radio- graphs on Day 23 readily identified all six shot in the gizzard of each of these ducks. Six pellets, which were sometimes dissolved tosmall disks, were recovered from 38 of the 40 Bi- dosed ducks. One male contained only five Bi disks in his gizzard. Because most of the shot were highly dissolved, it is probable that the sixth pellet had dissolved. A second male contained four tiny Bi particles in his gizzard. The combined particles of Bi weighed only 42.1 mg, and the fifth and sixth pellets probably had dissolved. Six pellets were recovered from 35 of the 40 Fe-dosed ducks. One female had five tiny pellets in her gizzard that weighed 77.7 mg. This duck had the second highest (2,339 ug/g) concentra- Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 19] tion of Fe in the liver. The mean concentration of Fe in livers of Fe-dosed ducks was 1086 ug/g. Thus, the sixth pellet undoubtedly had dissolved. One female contained no shot in her gizzard, but she had 1,782 g/g Fe in the liver. This duck probably also had dissolved the shot. The re- maining three ducks, one male and two females, each contained five pellets in their gizzards. All of these pellets were small, collectively weighing from 147.4 to 282.9 mg for each duck. One of these females had the highest concentration (2,412 tg / g) of Fe in her liver of any duck. The other two ducks contained 645 ug/g and 1,043 ug/g Fe respectively, in their livers. The sixth pellet in each of these three ducks may have been voided, but they probably were dissolved. None of the dosed ducks with missing shot in their gizzards was among the ducks that were radiographed and for which feces were saved for analysis. The retained Bi and Fe shot differed in ap- pearance. The Fe shot were usually round, al- though many were pitted or had empty spaces on their surfaces, whereas the Bishot were generally disk-shaped or flattened. In several instances, five Bi disks plus two, three, or four tiny pieces (not flakes) of Bi were recovered from the gizzard. Obviously, when a Bi disk became thin enough, it disintegrated into several pieces. A small number of flakes of Bi were found ina few gizzards. This finding for Bi/Sn alloy shot is in contrast to the abundance of tiny flakes of Bi found in the dosing study that used 100% Bi shot (Sanderson et al. 1992). The dissolution rates were variable in both Fe-dosed and Bi-dosed ducks. Based on the shot recovered from the gizzards on Day 30, females dosed with Bi shot dissolved a mean of 69.5% and males 72.5% (Table 1) of the metal’s original weight in 30 days (dissolution in individual ducks ranged from 38.2% to 96.4%). No difference between the sexes was detected for Bi-dosed ducks. Fe-dosed females dissolved an average of 69.2% and Fe- dosed males 55.6% of the metal’s original weight in 30 days (range for individual doses was from 38.0% to 89.6%). The different dissolution rates between sexes for Fe-dosed ducks was expected (Table 1). Females approaching the breeding season in spring eat more food than males and thus produce more acid in their gizzards. Asa result, Fe shot, which dissolve readily in the acid (HCI) environment of the gizzard, dissolve more rapidly in females than in males during this sea- son. Males dissolved more of the weight (72.5%) of the Bishot than of the Fe shot (55.6%) in 30 days. 192 Illinois Natural History Survey Bulletin Females dissolved no more (69.5%) of the weight of the Bishot than of the Fe shot (69.2%) in 30 days. An interaction, which was caused by the lower rate of dissolution of Fe shot by males as com- pared with the dissolution rate of Fe shot by females and no difference in the dissolution rates of Fe shot and Bishot by females, existed between sex and dose (Table 1). Body Weight All groups of dosed ducks, except Bi-dosed males, lost from 1.8 to 5.0% of their body weight during the 30-day study. All groups lost from 4.5 to 9.6% of their body weight from Day 0 to Day 15, prob- ably because of the switch from duck pellets to a whole corn diet. By Day 30, most of the birds had regained weight lost after the change in diet. Bi- dosed males gained only 1.6% in body weight from Day 0 to Day 30 (Table 2). Males weighed more than females, and an interaction in weight between sex and time ex- isted, with females losing a larger percentage of their weight from Day 0 to Day 30 than males. Although ducks lost weight over time, the aver- age weight losses for females from Day 0 to Day 30 were only -3.8% for 0-dosed, -3.8% for Fe-dosed, and -5.0% for Bi-dosed females. The average weight changes for males from Day 0 to Day 30 were -1.8% for 0-dosed, -3.2% for Fe-dosed, and +1.6% for Bi-dosed ducks. No difference existed in body weights among doses (Table 2). Organ Weights Gizzard Mean gizzard weights ranged from 29.3 g for Fe- dosed females to 32.2 g for Bi-dosed males (Table 3). No difference was detected in the weight of gizzards between sexes or among doses. As a percentage of total body weight, mean gizzard weights ranged from 2.5% for each of Fe- dosed and Bi-dosed males to 3.0% for 0-dosed females (Table 3). Gizzards of females contrib- uted a higher percentage of the total body weight than males. No difference was recorded among doses in the percentage that gizzards contributed to total body weight. Liver Mean weights of livers ranged from 19.3 g for Bi- dosed females to 21.7 g for Fe-dosed females. No differences existed between sexes or among doses (Table 4). When considered as a percentage of total body weight, mean values for livers ranged from 1.6% for Bi-dosed and 0-dosed males to 2.0% for Vol. 35 Art. 3 Fe-dosed and 0-dosed females. Livers of females comprised a higher percentage of the total body weight than the livers of males. No difference was detected among doses in the mean percentage that livers contributed to the total body weight. Kidneys Weights of kidneys, the organ most involved in excretion of Bi, differed least between sexes and varied least among doses of the organs weighed. Mean weights of kidneys ranged from 6.4 g for Bi- dosed females, Bi-dosed males, and Fe-dosed fe- males to 6.6 g for 0-dosed males (Table 5). No differences were found between sexes or among doses in the weights of kidneys. As with weights of livers, when kidney weights were expressed as a percentage of total body weight, sex differences were detected. Mean percentages ranged from 0.5% for each group of males to 0.6% for each group of females. Kidneys of females comprised a larger percentage of the total body weight than males, but no differences existed among doses. Gonads No differences among doses in the mean weights of gonads were found (Table 6). As was expected, mean weights of gonads differed between the sexes: 6.4 g for 0-dosed females, versus 26.4 g for 0-dosed males; 10.1 g for Fe-dosed females, ver- sus 28.0 g for Fe-dosed males; and 4.3 g for Bi- dosed females versus 22.5 g for Bi-dosed males. These sex differences also were evident in gonad weights when expressed as a percentage of total body weight; the means ranged from 0.4% for Bi- dosed females to 2.4% for Fe-dosed males (Table 6). No differences appeared among doses, but male gonads contributed a larger percentage of the total body weight than did the female gonads. Hematocrit (Hct) Mean hematocrits were not different among doses for the three sample times: Days 0, 15, and 30 (Table 7). The mean percentage changes in Hct values from Day 0 to Day 30 did not differ be- tween the sexes. With sexes combined the mean percentage change in Hct from Day 0 to Day 30 increased (P< 0.00001) by 6.6% for controls, 11.8% for Bi-dosed ducks, and 12.8% for Fe-dosed ducks. Heavy Metals and Essential Elements in Organs and Blood For consistency in presentation of the data, usu- ally the mean concentrations of elements in each organ or tissue for each sex and for sexes com- Continued on page 195 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 193 Table 1. Percent of the dosed shot accounted for and mean percent of weight of dosed shot dissolved in 30 days in the gizzard——-six, No. 4, Fe shot or six, No. 4, Bi shot——in female and male game-farm mallards (n = 20 females and 20 males in each dosed group). % of Dosed Shot Mean % Wt of Sex Dose Accounted for* Shot Dissolved? F Fe 93.3 69.2 5.04 2.84 M Fe 99.2 55.6 0.84 22? F Bi 100.0 69.5 0.00 4.01 M Bi 97.5 72.5 1.82 3.59 * Based on the shot recovered from the gizzards on Day 30, when the ducks were killed. BYE. Interaction between sex and dose: F a 4.53; P = 0.0374. Difference between sexes for percent of Fe shot dissolved in 30 days: F = 14.89; P = 0.0014. 1,37 Difference between doses for males: F =17.42: P=0.0002. 131 Table 2. Mean body weight on Days 0°, 15, and 30° of female and male game-farm mallards each dosed with 0 shot; six, No. 4, Fe shot; or six, No. 4, Bi shot, and mean percentage change in body weight from Day 0 to Day 30 (n = 20 females and 20 males in each group). Mean Body Weight (Kg) iste tn ee a Mean % change in body Sex Dose Day 0 Day 15 Day 30 wt-Day 0 to Day 30° F 0 1.11 1.06 1.06 -3.8 0.024¢ 0.027 0.030 1.12 M 0 2.24 1.19 1.22 -1.8 0.020 0.025 0.022 1.38 F Fe 1.10 1.04 1:05 - 3.8 0.020 0.018 0.021 0.97 M Fe 1.24 1.18 1.20 = 3.2 0.023 0.027 0.025 1.12 F Bi 1.08 1.02 1.03 - 5.0 0.026 0.025 0.026 114 M Bi 1.23 124 1.24 + 1.6 0.019 0.025 0.039 231 * Ducks were dosed on Day 0. ® Ducks were killed on Day 30. © Because of rounding error, these means are sometimes slightly different than if calculated by differences in the Day 0 and Day 30 columns. SE. Mean body weight: Difference between sexes: F =66.42; P< 0.00001. 1,114 Interaction between sex and time: Ea oe OOLZA 2,228 Difference among doses: E =011- P= 0:8995: 2,114 Change over time: F =33.17; P < 0.00001. 8 2,2 194 Illinois Natural History Survey Bulletin Vol. 35 Art. 3 Table 3. Mean weight of gizzard and mean percentage it contributed to total body weight in game-farm mallards 30 days after dosing with 0 shot; six, No. 4, Fe shot; or six, No. 4, Bi shot (n = 20 females and 20 males in each group). Mean Mean % of Sex Dose Weight(g) body wt F 0 31.8 3.0 1.04? 0.07 M 0 31.6 2.6 0.75 0.08 F Fe 293 2.8 1.07 0.08 M Fe 30.7 25) 0.98 0.08 F Bi 30.2 2.8 1.00 0.13 M Bi 622. 25 1.08 0.13 2OH. Difference among doses in weight of gizzard: E1250 72 102265: 4 2,1 Difference between sexes in percentage gizzard contributed to total body weight: F i 34.43; P < 0.00001. Difference among doses in percentage gizzard contributed to total body weight: Es = 1.99; P = 0.1422. 2,10 Table 4. Mean weight of liver and mean percentage it contributed to total body weight in game-farm mallards 30 days after dosing with 0 shot; six, No. 4, Fe shot; or six, No. 4, Bi shot (n = 20 females and 20 males in each group). Mean Mean % of Sex Dose Weight(g) body wt F (0) 21.1 2.0 1.498 0.10 M 0 20.0 1.6 0.94 0.06 F Fe 21.7 2.0 1.54 0.12 M Fe 20.5 ley 0.94 0.08 F Bi 19.3 1.9 0.92 0.08 M Bi 19.5 1.6 0.97 0.04 Stoke Difference among doses in weight of liver: F = 1.10; P= 0.3370. 2,14 Difference between sexes in percentage liver contributed to total body weight: F =22.21; P< 0.00001. 1,84 Difference among doses in percentage liver contributed to total body weight: F = 1.80; P= 0.1706. 2,114 April 1997 continued from page 192 bined are listed in the tables. When no statisti- cally significant differences existed between sexes, usually only P values for the combined sexes are provided. Kidneys The MDL for analysis by ICP for Bi in kidneys was 17.8 ug/g (wet wt). The mean concentra- tions were < MDL in kidneys of all but 2 of 120 ducks. Because the MDL for Bi by ICP in the kid- neys was unacceptably high, kidneys of 10 0- dosed and 11 Bi-dosed ducks were selected for analysis by GFAA. No sex differences were detected in the mean concentration of Bi in the kidneys of Bi-dosed ducks (Table 8). The mean concentration (6.86 ug/g) of Bi in the kidneys of Bi-dosed ducks, with sexes combined, was higher than the mean concentration of Bi in 0-dosed ducks (0.334 ug/g). The mean concentration of Bi in the kidneys of Bi-dosed ducks was much higher than the mean concentration (2.23 ug/g) of Bi in the livers of Bi-dosed ducks (Table 10). The MDL for Pb in the kidneys was 6.54 ug / g (wet wt) by ICP. All mean values for Pb in the kidneys were < MDL by this method. By GFAA, no sex differences existed in the concentration of Pb in the kidneys of 0-dosed or Bi-dosed ducks. No difference was found in the concentration of Pb in the kidneys of 10 (sexes combined) 0-dosed ducks (0.440 ug/g) compared with 11 Bi-dosed ducks (0.313 ug/g) (Table 8). The MDL for Sn in the kidneys was 9.47 ug/ g (wet wt). Only six ducks had concentrations of Sn >MDL. The mean concentration of Sn in the kidneys of these six ducks was 14.3 ug/g and ranged from 10.5 to 19.7 ug/g. Three of these ducks were Bi-dosed (x = 6.7 ug/g), two were 0- dosed (x = 1.0 ug/g), and one was Fe-dosed (13.7 g/g). No difference existed between sexes in the mean concentrations of Cu in the kidneys (Table 9). With sexes combined, no differences were detected among doses in the mean concentration of Cu in the kidneys: 6.31 ug/g in 0-dosed, 7.31 ug/g in Fe-dosed, and 6.14 ug/g in Bi-dosed ducks. No sex differences existed in the mean con- centrations of P in the kidneys (Table 9) and no differences existed among doses in the mean concentrations of P in the kidneys. However, an interaction was found between sex and dose. No difference existed between sexes in the mean concentration of Fe in the kidneys (Table Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 195 9), but mean concentrations of Fe in the kidneys differed among doses. Fe-dosed ducks, sexes combined, had higher mean concentrations (145 ug/g) of Fe in their kidneys than 0-dosed ducks (123 ug/g) or Bi-dosed ducks (123 g/g). No difference was detected in the mean concentra- tions of Fe in the kidneys of 0-dosed and Bi-dosed females (Table 9). Females, with doses combined, had higher mean concentrations of Ca in the kidneys than males, but no difference was found in the mean concentrations of Ca in the kidneys among doses. Mean concentrations of Mg in the kidneys did not differ between sexes, and with sexes combined, no difference existed among doses (Table 9). Mean concentrations of Zn in the kidneys of 0-dosed, Fe-dosed, and Bi-dosed ducks did not differ between sexes within each dose, but with doses combined, males had higher mean concen- trations of Zn in the kidneys than females (Table 9). With sexes combined, mean concentrations of Zn in the kidneys varied among doses. Bi-dosed ducks had higher mean concentrations of Zn in their kidneys (28.2 ug/g) than Fe-dosed ducks (25.2 ug/g), but not higher mean concentrations than 0-dosed ducks (26.6 ug/g) (Table 9). No difference was detected in mean concentrations of Zn in the kidneys of 0-dosed and Fe-dosed ducks. Liver The MDL (by ICP) for Bi in livers was 18.45 ug/g (wet wt). No concentration of Bi exceeded the MDL in the liver of any duck. Analysis by GFAA produced values of Bi in the livers of 11 Bi-dosed ducks that averaged 2.23 ug/g (0.63 to 5.63 ug/g). Mean amounts of Bi in the liver (Table 10) were not different between sexes. With sexes com- bined, Bi-dosed ducks contained a higher (2.23 ug/g) mean amount of Bi in the liver than did 0- dosed ducks (0.193 ug/g ). The MDL (by ICP) for Pb in the liver was 7.51 ug/g. The concentrations were all below the MDL. The concentration of Pb in the livers, as determined by GFAA, were from Ducks were killed on Day 30. 2 SEL Difference among doses: Pe —D7i;P= 04961: 2,117 Change over time: je = 50.10; P < 0.00001. 2,234 Mean Het Day 15 48.2 0.41 49.7 0.58 48.7 0.47 Mean % change in Hct—Day 0 Day 30 to Day 30 49.6 + 6.6 0.48 125 50.8 + 12.8 0.55 2.49 49.7 + 11.8 0.52 a02. Table 8. Mean concentrations (ug/g wet wt) of Bi and Pb in kidneys of game-farm mallards 30 days after dosing with 0 shot (controls) compared with ducks dosed with six, No. 4, Bi shot, as measured by GFAA. Dose Bib Element Sex 02 Bi F 0.140 0.000° M 0.528 0.058 F&M 0.334 0.070 Pb F 0.138 0.070 M 0.742 0.640 F&M 0.440 0.320 8.05 1.14 4.77 1.45 6.86 0.99 0.427 0.179 0.112 0.033 0.313 0.121 MDL = Method Detection Limit (ug/g wet wt) by GFAA for Bi in kidneys = 0.10 ug/g for 10 ducks and 0.27 ug/g for 11 ducks and Pb = 0.27 ug/g for 10 ducks and 0.15 ug/g for 11 ducks. 2 N=10. Sa 1h OE: Bi Difference between sexes in Bi-dosed ducks: F = 3.09; P=0.1127. 19 Difference between doses: F =43.32; P=0.0001. 1,10 Pb Difference between sexes in 0-dosed ducks: F = 0.88; P=0.4011. 14 Difference between sexes in Bi-dosed ducks: F = 1.68; P=0.2274. 19 Difference between doses: EOS. —0)7055: 1,19 198 Illinois Natural History Survey Bulletin Vol. 35 Art. 3 Table 9. Mean concentrations (ug/g wet wt) of Cu, P, Fe, Ca, Mg, and Zn (by ICP) in kidneys of game farm- mallards 30 days after dosing with 0 shot (controls) compa-red with ducks dosed with six, No. 4, Fe shot or six, No. 4, Bi shot (n = 20 females and 20 males in each group). Element Sex 0 Cu F 5.50 0.38? M 7.13 0.45 F&M 6.31 0.34 P F 2758 53 M 3167 203 F&M 2962 112 Fe F 110 6.0 M 136 7.9 F&M 123 5.6 Ca F 84.0 Holl M 79.9 Wes) F&M 81.9 5.0 Mg F 196 Bao) M 216 14.8 F&M 206 Well Zn F 25.3 0.8 M 27.9 1.5 F&M 26.6 0.9 a SE. Cu Difference among doses: F = 0:32; P = 0.7280. |? Difference among doses: F = 0.63; P = 0.5384. 2,55 Interaction between sex and dose: F =3.48; P = 0.0540. 2,17 Fe Difference among doses: F =4.98; P = 0.0103. Interaction between sex and dose: F _ =3.01; ao P = 0.0574. Difference between 0-dosed and Fe-dosed ducks: P <0.05. Difference between 0-dosed and Bi-dosed ducks: P>0.10. Difference between Fe-dosed and Bi-dosed ducks: P < 0.05. Dose Fe Bi 5.34 5.74 0.26 0.49 9.08 6.60 Bro, 0.65 7.31 6.14 1.79 0.40 2937 3050 80 67 2903 3006 30 48 2919 3030 40 42 152 118 8.5 8.4 139 129 8.2 8.1 145 123 6.0 5.8 84.4 87.2 5.3 4.5 70.2 TRAP 1.8 1.6 76.9 80.4 3.1 3.0 199 206 4.2 3.8 197 204 2.4 3.7 198 205 2.3 2.6 24.7 27.4 0.6 1.2 25.6 29.3 0.8 1.1 25.2 28.2 0.5 0.8 Ca Difference between sexes:F = 6.99; P =0.0107. 1,55 Difference among doses: F = 0.41; P = 0.6680. 2,33 Mg Difference among doses: F _ = 0.80; P = 0.4536 2,55 Zn Difference between sexes:F = 4.45; P = 0.0395. 1,55 Difference among doses: F = 4.56; P = 0.0147. Difference between Fe-dosed and Bi-dosed ducks: P <0.05. April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 199 Table 10. Mean concentrations (ug/g wet wt) of Bi and Pb in livers of game-farm mallards 30 days after dosing with 0 shot (controls) compared with ducks dosed with six, No. 4 Bi, shot (analyses by GFAA). Element Sex 0 Bi F 0.140? 0.000° M 0.2462 0.033 F&M 0.193 0.024 Pb F 0.068 0.018 M 0.552 0.376 F&M 0.310 0.195 Dose Bi 2.79° 0.675 E255 0.347 2228, 0.492 0.184 0.053 0.110 0.030 0.157 0.036 MDL = Method Detection Limit (ug/g wet wt) by GFAA for Bi = 0.27 for 10 ducks and 0.10 for 11 ducks and for Pb = 0.10 for 10 ducks and 0.15 for 11 ducks. Difference between doses: F = 17.14; P = 0.0020. 1,10 Pb Difference between doses: F = 0.65; P = 0.4294. 1,19 continued from page 195 The mean concentration of Cu in the livers of 0-dosed females was 85.5 ug/g versus 191ug/gin males, 56.3 ug/g in the livers of Fe-dosed females versus 172 ug/g in males, and 78.3 ug/g in the livers of Bi-dosed females versus 149 ug/g in males (Table 11, Figure 1). Males had higher mean concentrations of Cu in the liver than fe- males, but we found no differences among doses in the mean concentration of Cu in the livers. Females consistently had more P in their livers than males (Table 11): 0-dosed, 3,164 ug/g in females versus 2,998 P ug/g in males; Fe-dosed, 3,258 ug/g in females versus 2,958 ug/g in males; and Bi-dosed, 3,154 ug/g in females versus 2,897 ug/g in males. We found no differences among doses in the mean concentrations of P in the livers (Table 11). We detected no difference between sexes in the mean concentrations of Fe in the livers, but the mean concentrations of Fe differed among doses: 411 ug/g in 0-dosed ducks versus 1086 ug/g in Fe-dosed ducks versus 399 ug/g in Bi-dosed ducks, sexes combined. Differences were detected in the mean concentrations of Fe in 0-dosed versus Fe- dosed ducks and in Fe-dosed versus Bi-dosed, but not in 0-dosed versus Bi-dosed ducks (Table 11). The mean concentrations of Ca in livers did not differ between sexes, but with sexes com- bined, the mean concentrations of Ca in the liver were different among doses. The mean concen- tration of Ca in the livers was higher in 0-dosed ducks (62.8 ug/g) than in Fe-dosed ducks (50.4 ug/g), but was not higher in 0-dosed than in Bi- dosed ducks (51.4 ug/g) (Table 11). We detected no difference in the mean concentrations of Ca in the livers of Fe-dosed and Bi-dosed ducks. The mean concentration of Mg in the livers ranged from 211 ug/gin0-dosed males to 224 Ug / g in Fe-dosed females, but no difference existed between sexes. With sexes combined, no differ- ences were detected among doses in the mean concentrations of Mg in the livers: 0-dosed ducks, 213 ug/g; Fe-dosed ducks, 219 mg/g; and Bi- dosed ducks, 214 ug/g (Table 11). Continued on page 202 200 Illinois Natural History Survey Bulletin Vol. 35 Art. 3 Table 11. Mean concentrations (ug/g wet wt) of Cu, P, Fe, Ca, Mg, and Zn (by ICP) in livers of game-farm mallards 30 days after dosing with 0 shot (controls) compared with ducks dosed with six, No. 4, Fe shot or six, No. 4, Bi shot (n = 20 for each sex). Dose Element Sex 0 Fe Bi Cu F ~ Seles: 56.3 78.3 16.4" pel 15.6 M 191 172 149 37.6 31.8 32.6 F&M 138 114 114 21.9 19.2 18.7 Re F 3164 3258 3154 126 ih 93 M 2998 2958 2897 TA 88 63 F&M 3081 3108 3026 72 62 59 Fe F 416 1158 435 37.8 9] 43.6 M 406 1015 362 58.8 10 24.9 F&M 411 1086 399 34.1 iD 161.0 Ca F 66.4 54.0 52.9 7.0 Dae) 3.6 M 5OW. 46.8 49.8 7.9 3.8 4.6 F&M 62.8 50.4 51.4 53 ZS) 2.9 Mg F 2N5 224 216 8.4 4.6 4.5 M 211 214 DAP: 49 6.5 5.0 F&M PANE: 219 214 4.8 4.0 ee: Zn F 535 48.4 50.8 on 2.6 Or. M 48.9 48.1 45.4 2.8 2.8 19 F&M Silail 48.2 48.1 Dep 1.9 1.9 a SE. Ca difference between sexes: es = 20.64; P< 0.00001. Difference among doses: Ee a= 3.43; P=0.0356. difference among doses: F Miss 0.56; P=0.5721. Difference between 0-dosed and Fe-dosed; P< 0.05. ag Difference between 0-dosed and Bi dosed; P>0.05. ifference between Fe-dosed females and Fe-dosed Difference between Fe-dosed and Bidosed; P>0.10. males: P <0.05. Meg difference between 0-dosed females and 0-dosed Difference among doses: F = 0.66; P=0.5182. males: P <0.05. 2s difference between Bi-dosed females and 0-dosed Zn males: P< 0.05. Difference among doses: F = 0.70; P= 0.5005. Sn oes difference between sexes: Bye = 11.07; P =0.0012. The mean level was < MDL: = 12.8 ppm. difference among doses: Ie sie 0.45; P =0.6380. difference among doses: Fs v= 67.53; P < 0.00001. Jifference between 0-dosed and Fe-dosed; P < 0.01. difference between 0-dosed and Bi-dosed; P > 0.10. Jifference between Fe-dosed and Bi-dosed;P < 0.01. April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 201 200 RAXKXXKXRRK He Serpette os RRXX RRXKX at * RAKKKRKARKKKH RRKKKKRKEKR RKRARKRKRKRKXRKK Pitetatetetitatetatatatetes pppott tee Ph eb t eh RARRRKKKX 100 xx RARKKRKKARKRR A SPran AMV HSEYADODODO-=E Controls RARAKKRKARKKKK? RKRKKKKRK KD: x x RRRX x x Z MRXNN Figure 1. Mean concentrations of copper (ug/g wet weight) in liver of game-farm mallards 30 days after dosing with 0; 6, No. 4, steel (iron); or 6, No. 4, bismuth shot. 202 Illinois Natural History Survey Bulletin Vol. 35 Art. 3 Table 12. Mean concentrations (u/g wet wt) of Bi and Pb in gonads of game-farm mallards 30 days after dosing with 0 shot (controls) compared with ducks dosed with six, No. 4, Bi shot (by GFAA Furnace). Dose Element Sex Bi? Bi F 0.050 0.677 0.000 0.455 M 0.050 0.155 0.000 0.048 F&M 0.050 0.468 0.000 0.277 Pb F 0.080 0.093 0.000 0.013 M 0.080 0.100 0.000 0.020 F&M 0.080 0.096 0.000 0.011 MDL = Method Detection Limit for gonads (ug/g wet wt) by GFAA = 0.15 for Pb and 0.10 for Bi. 2 N= 20: PSE: Bi Difference between 0-dosed males and Bi-dosed females: F =6.2821; P = 0.0406. i continued from page 199 The mean concentration of Zn in the livers ranged from 45.4 ug/g for Bi-dosed males to 53.3 ug/g for 0-dosed females, and values were not different between sexes. Withsexes combined, no differences were found among the mean concen- trations of Zn in livers of 0-dosed ducks, 51.1 ug/ g; Fe-dosed ducks, 48.2 ug/g; and Bi-dosed ducks, 48.1 ug/g (Table 11). Gonads The MDL for Bi in gonads by ICP was 12.0 ug/g for 33 ducks and 13.2 ug/g for 28 ducks. All but five values for Bi in gonads were MDL ranged from 15.2 to 27.8 ug/g and averaged 19.9 ug/g. As determined by GFAA Furnace, the con- centrations of Bi in gonads of 0-dosed ducks were all MDL. The mean concentrations of Pb, as determined by GFAA furnace, in gonads of 0-dosed and Bi-dosed ducks were all MDL (17.8 ug/g) of Snin the liver. Mean concentrations of Cu in the gonads differed by sex in 0-dosed ducks, but not in Fe-dosed and Bi-dosed ducks. With doses combined, mean concentrations of Cu differed by sex with males having lower concen- trations than females (Table 13). No differences were found for the mean concentrations of Cu in the gonads of mallards among doses. Mean concentrations of P in the gonads were not different by sex within doses. With sexes combined, no difference was detected in the con- centrations of Pin the gonads among doses (Table 13). Differences in the mean concentrations of Fe in the gonads of males and females were substan- tial (Figure 2) in all dosed groups: 0-dosed, 56.4 ug/g in females versus 12.5 ug/g in males; Fe- dosed, 53.5 ug/g in females versus 10.7 ug/g in males; and Bi-dosed, 40.3 ug/g in females versus 16.8 ug/g in males. Mean concentrations of Fe in the gonads of ducks within doses were not differ- ent (Table 13). Females contained up to 17 times more Ca in their gonads than males (Figure 3): 0-dosed Continued on page 204 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 203 Table 13. Mean concentrations (g/g wet wt) of Cu, P, Fe, Ca, Mg, and Zn in gonads of game-farm mallards 30 days after dosing with 0 shot (controls) compared with ducks dosed with six, No. 4, Fe shot or six, No. 4, Bi shot (N = 10, 11, or 12 for each sex). Dose Element Sex 0 Fe Bi Cu F 1.76 1.70 1.48 0.215? 0.152 0.183 M 0.985 1.12 1.34 0.149 0.113 0.174 F&M 1.37 1.41 1.41 0.155 0.114 0.125 P F 3132 3102 2566 388 395 342 M 2662 2717 2917 41 91 128 F&M 2897 2910 2726 197 202 195 Fe F 56.4 53.5 40.3 We 8.0 5.4 M 1225 10.7 16.8 2.8 i? 5.4 F&M 34.4 32.1 29.1 6.4 6.3 4.6 Ca F 540.0 590.1 334.4 165.4 179.2 102.5 M 34.5 34.4 139.6 1.7 1.6 104.5 F&M 287.2 312.2 241.7 QP 108.0 74.6 Mg F 113 27 126 15.9 14.6 . 16.2 M 203 206 201 25 6.3 11.8 F&M 158 166 162 13.0 11.9 13.0 Zn F 23.7 24.6 19.3 4.0 3.6 2:9 M 13.9 14.3 16.4 0.4 0.6 2.0 F&M 18.8 19.4 18.0 DED 2.1 1.8 aaSE: Cu Ca Difference between sexes: ae = 13.20; P = 0.0006. Difference between sexes: ae = 19.50; P < 0.00001. Difference between sexes in 0-dosed ducks: P < 0.05. Difference among doses: ie oa 0.22; P = 0.8021. Difference among doses: F = 0.03; P = 0.9664. Mg > Difference between sexes: F = 65.70; P < 0.00001. ie 3 Difference among doses: B ys = 0.23; P =0.7965. Difference among doses: F _ = 0.22; P =0.8051. Fe Zn Difference between sexes: Bes = 64.51; P = 0.00001. Difference between sexes: be = 12.66; P = 0.0012. Difference between sexes in 0-dosed ducks: P< 0.01. Difference among doses: F = 0.17; P=0.8402. Difference between sexes in Fe-dosed ducks: P < 0.01. Difference between sexes in Bi-dosed ducks: P < 0.05. Difference among doses: F = 0.57; P=0.5704. 204 continued from page 202 females (540.0 mg/g) versus 0-dosed males (34.5 mg/g); Fe-dosed females (590.1 mg/g) versus Fe- dosed males (34.4 mg/g); and Bi-dosed females (334.4 mg/g) versus Bi-dosed males (139.6 mg/ g). With doses combined, females also contained higher mean concentrations of Ca in their gonads than males. No differences existed, however, in the mean concentrations of Ca in the gonads among doses within each sex (Table 13). Males had higher mean concentrations of Mg in their gonads than females (Table 13), but no differences were detected among doses within each sex. With doses combined, females had higher mean concentrations of Zn in their gonads than males (Table 13). With sexes combined, Zn values in gonads varied little among doses. Plasma and Blood Cells The MDLs (by ICP) for Biin plasma were 7.38 ug / g (wet wt) for Day 0,21.8 ug/g for Day 15, and 11.8 ug/g for Day 30. The MDLs for Bi in blood cells were 8.72 ug/g for Day 0, 9.35 ug/g for Day 15, and 16.3 ug/g for Day 30. All mean levels were MDL only for Day 30, the only data included in this report. Females had higher mean concentra- tions of Zn in their plasma than males (Table 14). Continued on page 208 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 205 60 50 M | C R oO 40 =: R M S30 P S R 20 G R A 10 - iB Controls 0 Females Males Figure 2. Mean concentrations of iron (ug/g wet weight) in gonads of game-farm mallards 30 days after dosing with 0; 6, No. 4, steel (iron); or 6, No. 4, bismuth shot. 700 600 500 400 300 RRRAKKX: 200 Z=rymon Wmv n=f=rADoDOWO-=E 100 Females Males Figure 3. Mean concentrations of calcium (ug/g wet weight) in gonads of game-farm mallards 30 days after docino with 0: & No 4 cteel (iron): or 6 No 4 hiemiith chot 206 Illinois Natural History Survey Bulletin Vol. 35 Art. 3 Table 14. Mean concentrations (ug/g wet wt) of Cu, P, Fe, Ca, Mg, and Zn in plasma of game-farm mallards dosed with 0 shot (controls) compared with ducks dosed with six, No. 4, Fe shot or six, No. 4, Bi shot (N = 18, 19, or 20 for each sex). Elements Dose Sex Day Detected 0 Fe Bi F&M 0 Cu 0.334 0.304 0.370 0.0362 0.031 0.048 F 0 P 179 204 196 18.0 20.9 13.9 15 245 268 225 14.1 12.5 8.2 30 291 303 270 20.4 14.3 13.3 M 0 220 202 215 19.4 11.8 17.0 15 257 262 282 15 6.3 ODES 30 259 252 251 9.6 7.3 14.0 F&M 0 199 203 206 13.5 11.8 11.0 15 251 265 252 10.2 6.9 12.4 30 275 Day 260 11.5 9.0 9.8 F 0 Fe 8.40 14.5 10.7 ile. 3.5 A 15 6.31 7.55 5.08 0.8 0.6 0.8 30 7.47 9.07 6.85 0.9 1.0 0.7 M 0 15.4 7.62 13.8 4.5 1.0 3.4 15 5.94 5.67 8.19 0.8 0.5 2 30 7.71 6.08 5.17 1.1 0.6 0.5 F&M 0 11.8 11.1 Dy 2.3 1.9 1.9 15 6.14 6.61 6.59 0.5 0.4 0.7 30 7.48 7.58 6.03 0.7 0.7 0.4 F 0 Ca 88.3 87.9 87.0 6.1 5.7 5.8 15 144 140 120 12.2 7.8 3.8 30 176 169 168 17.3 12.6 12.4 M 0 83.0 80.1 80.4 6.3 5.8 6.2 15 110 117 115 5.4 1.7 1S 30 107 111 109 NA 1.3 0.5 F&M 0 85.7 84.0 84.1 3.9 4.0 4.2 15 127 129 118 ee 4.3 2.1 30 141 140 139 10.2 7.8 8.2 F 0 Mg 17.4 19.2 17.8 ily 1.4 1.2 15 23.3 24.5 Diler 1.0 0.8 0.5 30 26.6 27.0 26.2 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 207 Table 14 continued Elements Dose Sex Day Detected 0 Fe Bi M 0 18.0 16.3 17.1 15 EL il) 15 22.5 22.7 23.8 2 0.5 1.0 30 24.4 24.3 24.4 0.4 0.4 0.6 F&M 0 WZ 17.8 U5 0.8 0.9 0.9 15 22.8 23.6 22.6 0.8 0.5 0.6 30 25.0 2a 233 0.6 0.6 0.5 F 30 Zn 4.56 4.27 4.63 0.39 0.34 0.30 M 30 2.83 DITITL De 0.13 0.08 0.17 F&M 30 3.69 hay 3.64 0.25 0.21 0.23 @ SE. Cu Difference among doses; Day 0: FE Was 0.74: P = 0.4789. Mean concentrations for Days 15 and 30 were N=3. © N=10. °N=4. EOE. 8’ N=9 MDL = Method Detection Limit by ICP: MDL for Bi = 103 ug/g (dry wt) for Days 0, 1, 2, and 1-10 and 58.9 ug/g for Days 11-30. MDL for Sn = 19.5 ng/g (dry wt) for Days 0, 1, and 2 and 14.9 ug/g for Days 11-30. MDL for Fe = 31.8 ug/g (dry wt) for Days 0, 1, and 2 and 19.2 ug/g for Days 11-30. continued from page 208 and one Bi-dosed duck. In the Fe-dosed duck, a small-sperm granuloma was found, but the re- sidual parenchyma was normal. The Fe-dosed duck had evidence of a locally extensive tubular atrophy consisting of a decreased height in the seminiferous epithelium in one zone of the exam- ined testis. The adjacent tubules were within normal limits with no evidence of inflammation. The testis of the Bi-dosed duck contained scattered aggregates of lymphocytes and plasma cells, and multinucleated giant cells (presumably sloughed spermatocytes) were observed within scattered tubules. Evidence of normal produc- tion of spermatozoa was present on the slide. Asmall percentage of ducks inall three groups had mild vacuolar changes in the seminiferous epithelium. These ducks had spermatozoa within the genital ducts and in seminiferous epithelium. Liver Nearly all ducks had a variable number of lym- phocytes and plasma cells within the liver. The most common pattern was around the portal tri- ads. Occasionally, the inflammatory cells formed small nodules scattered in the parenchyma. One 0-dosed male had abscesses within the liver, which probably represented an acute bacterial infection. The hepatic lipidosis seen histologically seemed to correlate with livers that were heavier. Kidneys Nearly all ducks had a variable number of lym- phocytes and plasma cells in the wall of the ureter. Gizzard On Day 30, the contents of the gizzards from all 120 ducks were removed and saved, and those from Fe-dosed and Bi-dosed ducks were exam- DAZ ined for retained shot. The linings of all gizzards appeared to be unaffected, and there was no pattern of variation among doses. No lesions were detected in any of the gizzard sections exam- ined. Discussion Copper Approximately twice the concentration of Cu was detected in the livers of male ducks as in the livers of female ducks in all dosed groups (Table 11). Hanson and Jones (1974) found significantly higher concentrations of Cu in the feathers of female Ross’ geese (Anser rossii) than in males. They presumed estrogen was responsible for the difference. Underwood (1971:61, 63), discussing Cu in the liver, stated, “There is no effect of sex, except in the Australian salmon (Arripis trutta) in which the female carried higher concentrations than the male.” Van Campen (1971:214) reported that “Ad- ministration of estrogens induces large increases in serum copper in humans, rats, and swine.” He also reported that androgens increased serum Cu concentrations in humans. Hill and Matrone (1961) found that when both Cu and Fe were low in the diet, an increase in one partly compensated for the deficiency of the other. Matrone (1960) concluded that Cu absorption is not directly af- fected by Fe. Thus, the Fe:Cu interaction is af- fected by something other than absorption. In the present study, the diet (corn) of the ducks (both females and males) was low in Fe, but dosing with Fe shot did not have a significant effect on the level of Cu in the livers. Van Campen (1971:221-222) stated, “The fac- tors that are most influential in determining the tissue levels of copper are age, hormones, disease and diet. . . . calcium apparently can either in- crease or decrease copper absorption, depending on the composition of the diet to which they are added.” In our study, females had higher mean con- centrations of Cu in their gonads than males, which is in contrast to kidneys and livers where males always had higher mean concentrations of Ca Phosphorous The lower concentrations of P in livers of Fe- dosed males, as compared with females, resulted from decreases of P in the livers of Fe-dosed males. Concentrations of P were only slightly higher in the livers of Fe-dosed females than in 0- Illinois Natural History Survey Bulletin Vol. 35 Art. 3 dosed females. Dosing with Bi shot also caused a decrease in the concentration of P in livers of males, which resulted in a significant difference between the sexes. Concentrations of P were essentially the same in 0-dosed females and Bi- dosed females (Table 11). Iron Dosing with Fe shot resulted in large concentra- tions of Fe deposits in the livers, but dosing with Bi shot did not significantly affect the concentra- tions of Fe in the liver (Table 11). Although females dissolved a higher percentage of the dosed Fe shot than males (Table 1), the Fe-dosed females did not have significantly higher concentrations of Fe in their livers than the Fe-dosed males. Females had significantly higher concentra- tions of Fe in their gonads than males. These sex differences in the concentrations of Fe in the go- nads may be related to the preparation of the ovaries for egg laying. No differences were found in the mean concentrations of Fe in the gonads attributed to dosing with either Fe shot or Bi shot. Calcium It appears that dosing with Fe or Bi shot is associ- ated with lower concentrations of Ca in the livers and kidneys of ducks as compared with controls (Table 9). Forth and Rummel (1971:182) reported that increases in Fe or Ca mutually inhibited each other in their transfer through the small intestine of the rat. They concluded that it was possible there is “. ..a common transport mechanism for iron and calcium... .” Perhaps Bi induces a similar reduction in the transfer of Ca, although Bi has apparently not been studied in this context. Ca increased substantially in the plasma of both males and females for all dosed groups from Day 0 to Day 15 to Day 30 (Table 14). The increase cannot be related to increase in Ca in the diet because after dosing all ducks were ona corn diet, and corn is low in Ca. Ca in the plasma among doses did not vary statistically. Feces Both Biand Sn greatly increased in the feces of Bi- dosed ducks the day after dosing. Birds excreted Bi in the feces at high concentrations to the end of the 30-day study. Mean concentrations of Bi were not substantially different between 0-dosed and Fe-dosed ducks on Days 0,1, and 2. Bi was much higher in feces of Bi-dosed ducks than in either 0- dosed or Fe-dosed ducks on Days 1 and 2. It appears that almost all of the Bi dissolved from Bi April 1997 shot in the gizzards is excreted in the feces of ducks. The mean concentration of Sn in the feces of Bi-dosed ducks was higher at the end of the 30- day study than the background level found the day prior to dosing. However, the mean concen- tration of Sn in the feces of Bi-dosed ducks de- clined substantially after Day 10. These findings for Sn in the feces seem to support Underwood (1971), who reported that the available evidence for humans shows that Sn is poorly absorbed, poorly retained, and excreted primarily in the feces. In humans, the amount of Sn ingested with food was approximately the same as the amount excreted in the feces. Underwood’s conclusion was that Sn shows little toxicity, probably because it is absorbed slowly and is excreted rapidly in feces. The mean concentration of Fe in feces de- clined sharply for both 0-dosed and Bi-dosed ducks starting on Day 1. The decline continued to the end of the study in Bi-dosed ducks. Feces of 0- dosed and Fe-dosed ducks were not analyzed for the entire study. The decline of Fe in feces of 0- dosed and Bi-dosed ducks was probably a result of switching on Day 0 from a diet of commercial duck food to corn, which is low in Fe. Conclusions We detected no toxic effects in game-farm mal- lards dosed with six Bi/Sn alloy shot and ob- served for 30 days. Survival, body weight, Hct, and weights of organs were not affected. Gross and microscopic examination of the kidneys, liver, and gonads of the ducks also revealed only slight tissue changes. Our data support the conclusions of Sanderson et al. 1992, who reported no toxic effects in game-farm mallards dosed with 100% Bi shot. A number of differences in weights of organs and in mean concentrations of individual ele- ments were detected between females and males. These differences appear related to physiological changes associated with the onset of breeding, especially in egg-laying females. Although a few “anomalies” were linked to dosing with Fe shot or with Bishot, no toxic effects were detected with either. For example, livers and kidneys of both Bi-dosed and Fe-dosed ducks had lower mean concentrations of Ca than livers and kidneys of 0-dosed ducks. The difference in the mean concentrations of Ca in the livers of Fe- dosed ducks versus Bi-dosed ducks was not sig- nificant (Table 11). Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards ZAS With two exceptions (kidneys of males, mean = 0.528 ug/g [range 0.36 to 0.72 ug/g], and livers of males, mean = 0.246 g/g [range 0.18 to 0.37 ug/g]), Bi was not found in the livers, kidneys, or gonads of 0-dosed ducks. The mean concentra- tions of Biin kidneys of Bi-dosed ducks were 8.05 ug/g for females (range 4.69 to 12.6 ug/g) and 4.77 ug/g (range 2.02 to 8.82 ug/g) for males. The mean concentration of Bi in livers of Bi-dosed females was 2.79 ug/g (range 1.19 to 5.63 ug/g). The differences for Bi-dosed versus 0-dosed ducks were significant for both kidneys and livers. Both macro and micro histological observations de- tected no toxic effects of Bion the kidneys, liver, or gonads. 214 Literature Cited Abbracchio, M.P., W. Balduini, A. Cavallaro, P. Adamoli, M. Fittipaldi, F. Muzio, S. Malandrino, and F. Cattabeni 1985. Brain and blood levels of bismuth after oral or parenteral administration of tripotassium-dicitrato bismuthate to rats. NeuroToxicology 6(3):139-144. Anderson, William L. 1992. Legislation and law- suits in the United States and their effects on nontoxic shot regulations. Pages 56-60 in D.J. Pain, ed. Proceedings of an international waterfowl and wetlands research bureau workshop, Brus- sels, Belgium. 1991. IWRB Special Publication 16, Slimbridge, UK. Canadian Wildlife Service. 1995. Minister Copps acts on two toxic substances. News release dated 11 July 1995. 6 pp. Dipalma, J.R. 1988. Bismuth toxicity. American Family Physician 78(5):244-246. Environment Canada. 1992. Guidelines regard- ing the toxicity tests required for the approval of candidate non-toxic shot (to be submitted to the meeting of the executive in January 1993). Envi- ronment Canada. 9 pp. Forth, W., and W. Rummel. 1971. Absorption of iron and chemically related metals in vitro and in vivo: specificity of the iron binding system in the mucosa of the jejunum. Pages 173-191 in S.C. Skorya and D. Waldron-Edward, eds. Intestinal absorption of metal ions, trace elements and ra- dionuclides. Pergamon Press, Oxford, New York, Toronto, Sydney, Braunschweig. 431 pp. Fowler, B.A.,and V. Vouk. 1979. Bismuth. Pages 345-353 (Chapt. 20) in L. Friberg, G.F. Nordberg, and V. Vouk, eds. Handbook on the toxicity of metals. Elsevier/North-Holland Biomedical Medical Press, Amsterdam, New York. Glaser, J.A., D.L. Foerst,G.D. McKee, S.A. Quave, and W.L. Budde. 1981. Trace analyses for waste- waters. Environmental and Science Technology 15(12):1426-1435. Greenberg, A.E., L.S. Clesceri, and A.D. Eaton, eds. 1992. Standard method for the examination of water and wastewater. American Public Health Association, American Waste Water Works Asso- ciation, and Water Environment Federation. 18th Illinois Natural History Survey Bulletin Vol. 35 Art. 3 ed. Section 3113 Metals by electrothermal atomic absorption spectrometry: 3-20 through 3-28. Gregus, Z.,and C.D. Klaassen. 1986. Disposition of metals in rats: a comparative study of fecal, urinary, and biliary excretion and tissue distribu- tion of eighteen metals. Toxicology and Applied Pharmacology 85:24-38. Hamilton, E.J., M.J. Minski, and J.J. Cleary. 1972- 1973. The concentration and distribution of some stable elements in healthy human tissues from the United Kingdom. Science of the Total Environ- ment 1:341-374. Hanson, H.C., and R.L. Jones. 1974. An inferred sex differential in copper metabolism in Ross’ geese (Anser rossii): biogeochemical and _physi- ological considerations. Arctic 27(2):111-120. Hanzlik, P.J.,and E. Presho 1923. Comparative toxicity of metallic lead and other heavy metals for pigeons. Journal of Pharmacology and Experi- mental Therapeutics 21(2):145-150. Haseltine, S.D., and L. Sileo. 1983. Response of American black ducks to dietary uranium: a pro- posed substitute for lead shot. Journal of Wildlife Management 47:1124-1129. Hill, C.H., and G. Matrone 1961. Studies on copper and iron deficiencies in growing chickens. Journal of Nutrition 73:425. Hillemond, P., M. Palliere, B. Laquais, and P. Bauvet. 1977. Traitment bismuthique et bismuthemie. Semaine des Hopitaux de Paris 53:1663-1669. International Commission on Radiological Pro- tection. 1960. Report of Committee II on permis- sible dose for internal radiation. International Commission on Radiological Protection, Publica- tion No. 2. Pergamon Press, Oxford 218-219. Irby, H.D., L.N. Locke, and G.E. Bagley. 1967. Relative toxicity of lead and selected substitute shot types to game farm mallards. Journal of Wildlife Management 31:253-257. Key, M.M.,A.F. Henschel, J. Butler, R.N. Ligo,and I.R. Tabershha, eds. Lorice Ede, manuscript ed. 1977. Occupational diseases. A guide to their rec- ognition. Bismuth and compounds. Page 338. United States Department of Health, Education, April 1997 and Welfare, Public Health Service, Center for Disease Control, National Institutes of Occupa- tional Safety and Health. Revised Edition. Krigman, M.R., T.W. Bouldin, and P. Mushak. 1985. Metal toxicity in the nervous system. Mono- graphs in Pathology 58-100. Lee, S.P. 1981. Studies on the absorption and excretion of tripotassium dicitrate bismuthate in man. Research Communications in Chemical Pa- thology and Pharmacology 34:359-364. Locke, M., H. Nichol, and C. Kotola-Pirie. 1987. Binding of bismuth to cell components: clue to mode of action and side effects. Canadian Medi- cal Association Journal 137:991-992. Longcore, J.R., R. Andrews, L.N. Locke, G.E. Bagley, and L.T. Young. 1974. Toxicity of lead and proposed substitute shot to mallards. U.S. Department of the Interior, Fish and Wildlife Service, Special Scientific Report—Wildlife No. 183. 23 pp. Matrone, G. 1960. Studies on copper and iron deficiencies in growing chickens. Journal of Nu- trition 93:425. Moser, M. 1992. International Lead Poisoning Newsletter. International Waterfowl and Wet- lands Research Bureau, September. 1992. 17 pp + appendices. Oehme, F.W., ed. 1979. Pages 603-605 in Toxicity of heavy metals in the environment. Part 2. Bis- muth. Marcel Dekker, Inc., New York and Basel. Ross, J.F., Z. Sahenk, C. Hyser, J.P. Mendell, and C.L. Alden 1988. Characterization of a murine model for human bismuth encephalopathy. NeuroToxicology 9(4):581-586. Sanderson, G.C.,S.G. Wood, G.L. Foley, and J.D. Brawn 1992. Toxicity of bismuth shot compared with lead and steel shot in game-farm mallards. Transactions of the North American Wildlife and Natural Resources Conference 57:526-540. Sanderson, G.C., W.L. Anderson, G.,L. Foley,S.P. Havera, L.M. Skowron, J.W. Brawn, G.D. Taylor, and J.W,. Seets. 1997a. Effects of lead, iron, and bismuth alloy shot embedded in the breast muscles of game-farm mallards. Journal of Wildlife Dis- eases. In press. Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 21S Sanderson, G.C., W.L. Anderson, G.L. Foley, K.L. Duncan, L.M. Skowron, J.D. Brawn, and J.W., Seets 1997b. Toxicity of ingested bismuth/tin alloy shot in game-farm mallards: chronic health effects and effects on reproduction. Illinois Natu- ral History Survey Bulletn 35(4):217-252. Serfontein, W.J., and R. Mekel. 1979. Review of bismuth blood and urine levels in patients after administration of therapeutic bismuth formula- tions in relation to the problem of bismuth toxicity in man. Research Communications in Chemical Pathology and Pharmacology 26:391-411. Slikkerveer, A., and F.A. de Wolff 1989. Pharma- cokinetics and toxicity of bismuth compounds. Medical Toxicology and Adverse Drug Experi- ence 4:303-323. Thomas, D.W., T.F. Hartley, P,. Coyle, and S. Soecki. 1988. Bismuth. Chapter 11, pages 115-127 in H.G. Seiler and H. Segil, eds. Handbook on toxicology of inorganic compounds. Marcel Dekker, Inc., New York and Basel. Underwood, E.J. 1971. Trace elements in human and animal nutrition. 3rd Ed. Academic Press, New York and London. 543 pp. Van Campen, D.R. 1971. Absorption of copper from the gastrointestinal tract. Pages 211-227 in S.C. Skoryon and D. Waldron-Edward, eds. In- testinal absorption of metal ions, trace elements and radionuclides. Pergamon Press, Oxford, New York, Toronto, Sydney, Braunschweig. 431 pp. Venugopal, B., and T.D. Luckey. 1978. Chemical toxicity of metals and metalloids. Pages 215-219 and 354-401 in Metal toxicity in mammals. 2. Plenum Press, New York and London. Woods, J.S., and B.A. Fowler 1987. Alteration of mitochondrial structure and heme biosynthetic parameters in liver and kidney cells by bismuth. Toxicology and Applied Pharmacology 90:274- 283. 216 Illinois Natural History Survey Bulletin Vol. 35 Art. 3 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 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. Vol. 35 Art. 4 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards Abstract 217 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. Environment Canada (1992) 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 etal. (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 0 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 Ducks were weighed and randomly assigned, one toa pen,on4 January 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™ 14% Duck Developer pellets, Heinhold Feeds, Inc., Kouts, Indiana) and water ad libitum and exposed toambientlight. 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, (3.30 mm 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 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 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 91st day (27 April 1995), 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 0 (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 time we 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 5 mL 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 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. After we 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 11,500 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 ina 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. Weexcised 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- Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 219 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 at37.2°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 18 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 cm? of floor space for each of 30 ducklings. The minimum requirement for each duckling <7 220 days of age is 239 cm? (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 them by 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 ege contents were frozen until analyzed. Pb-dosed ducks were examined periodically by the institutional veterinarian in the Office of Laboratory Animal Resources, University of IIli- 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 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 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 for blood 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 ina 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 hrin a 10% nitric acid (HNO3) bath and rinsing with deionized water. Samples and tubes were tared, then we added 1 to 2 mL of hydrogen peroxide (H,O,) and reweighed. We then added 30 to 50 mL of 2% HNO3, and 10% hydrochloric acid (HCl) 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 the amount of rinse 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 intoa 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 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 HO), 0.250 mL concentrated HNO3, and 1 mL of hydrogen peroxide (H,O>). 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 H,O and 2 mL H,O, were added. Upon further heating the mixture cleared and “foamed up.” We rinsed down contents from the beaker walls with DI H,O. 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 witha 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% HNO3. 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 Weused a Thermo 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% Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 221 HCl, 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 asa spray and deposited directly into a carbon cuvette at 100°C to obtain drying on contact. Method 3113 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 (GRMs). The OCS 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 ata 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 instrument printer as concentrations (ug/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 0 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 131, after laying 16 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- Illinois Natural History Survey Bulletin Vol. 35 Art. 4 isted among 0-dosed, Fe-dosed, and Bi-dosed ducks in the mean time required to lay 21 uncracked eggs. Hematocrit Mean Hets 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 0 to 38.2 at necropsy, in Fe-dosed females from 46.2 on Day 0 to 37.9 at necropsy, and in Bi-dosed females from 46.0 on Day 0 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 0 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 body weights wasassociated 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 N Nm WwW April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 50 M E A 40 N H E M A if O - = HRKKRE ses cieb ieieniet R | 30 T S Day 0 20 SESE S SSSR ESS Necropsy Females 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 Hets of adult male and female game-farm mallard ducks dosed with 0 shot (controls); eight, No. 4, Fe shot; eight, No. 4, Bi shot; or eight, No. 4, Pb shot. Days after first dosing Dose Sex 0 30 60 90 120 Nec? 0 F 46.3 49.5 50.2 43.5 38.8° 38.2 0.63" 0.72 0.55 R52) Ten) 1.08 M 46.4 48.4 48.2 45.0 45.4° 45.3 0.44 0.47 0.50 0.63 0.65 1.19 Fe F 46.2 50.3 50.6 46.2 40.24 37.9 0.61 0.68 0.66 1.29 0.82 1.02 M 46.7 48.4 49.5 46.9 48.49 44.6 0.40 0.54 0.49 0.56 0.61 0.84 Bi F 46.0 47.9 47.9 43.9 BYLilE 36:2" 0.72 0.84 1.05 iil 1.11 1.03 M 47.3 49.1 48.8 46.0 48.0° 47.4! 0.62 0.63 0.50 0.46 0.89 0.62 Pb F&M 44.64 25.28 0.61 1.65 * 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 Hets: See Sete) Between sexes: Cn=7 f n=17. Ebola — 00003: 4 n=12. §n=4. ao n = 18 for all other samples. Among doses: a F i =a — i) 25oo: Over time: F =101.80; P < 0.00001. 30 224 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 52 50 48 46 RARKXARKARARRRXKXKX RRXXXXXRAXXXXXXXXRRX RRXXRXXKXKRXAXXXKXXK bbs ichcboetbeb cpa cht M E A N H Ea M A T 42 O C R | ee Day 0 il; UPL YSU Ss Day 30 38 Bee Day 60 Day 90 36 Day 120 34 Necropsy Females Males Figure 2. Mean Hcts of game-farm mallards dosed with 0 shot (controls); 8, No. 4, Fe shot; or 8, No. 4, Bishot 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 0 (controls) shot; eight, No. 4, Fe shot; eight, No. 4, Bi shot; or eight, No. 4, Pb shot. Days after first dosing Dose Sex 0 30 60 90 120 Nec? 0 F 1.05 1.00 0.99 1.13 1.28° 1225 0.04° 0.02 0.02 0.03 0.03 0.03 M NMS 1.14 1.10 1.19 1.162 1.20 0.02 0.03 0.02 0.03 0.04 0.04 Fe F 1.02 1.01 0.97 iL) 1.244 1.25 0.04 0.03 0.03 0.04 0.04 0.04 M 1.20 1.14 el 1.20 1.20¢ iW 0.02 0.02 0.02 0.02 0.02 0.02 Bi F 1.05 1.02 0.99 1.14 1.198 1.22 0.03 0.03 0.03 0.03 0.06 0.04 M 1.18 il tl7/ 12: 1.20 1.16° 1.18 0.02 0.03 0.03 0.02 0.04 0.03 Pb 1 1.03! 0.68' 0.03 0.03 M 1.22! 0.79! 0.04 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. Deri Difference in body weight: SE. Between sexes: F = 7.05; P = 0.0107. tne 12. He “n=8. Among doses: F = 1.28; P = 0.2870. aii 6! ae) n = 18 for all others. Over time: F =54.88; P< 0.00001. Nw Nw Nn April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards ON=PADOOr--A Day 90 ie Day 120 Eg Necropsy Females Males Figure 3. Mean body weight (kg) of game-farm mallards dosed with 0 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. ee KRARARKAXAKKKXRARX KKARKKXKXKRXKXKARX: x x. x x x x x x x x x x x x x x x x rt x xx x x xx x x xx xx x x * x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x Uv 1.1 1.0 eH RXXXXXX?> XKXXXX x xx RRKREX KRARKARXXd fH H x x x NS PADOOC-—-A Females Males Controls Fe Bi Pb Figure 4. Mean body weight (kg) at necropsy of game-farm mallards dosed with 0 shot (controls); 8, No. 4, Fe shot; or 8, No., 4 Bishot on Days 0, 30, 60, and 90, or 8 No. 4 Pb shot on Day 0. n= 18 for female Tian f 7rme ohO6Uf 226 Illinois Natural History Survey Bulletin continued from page 222 gizzards were found in the feces of one duck. Because the time between dosing and recovery of the Pb shot was short (Table 4), the 11 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—the mean survival time for Pb-dosed ducks. We then used multiple regression and estimated that the Fe-dosed ducks dissolved eight, No. 4, Fe shot ata 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 0 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 0 ina 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 0 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 0 were recovered from giz- zards. Males dissolved an average of 96.6% of the weight of Fe shot dosed on Day 0 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 0 in an average of 120.5 days—0.8% per day. They dissolved 52.5% of the weight of Bi- Vol. 35 Art. 4 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 Bishot dosed on Day 0inan 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 as much 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 0 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 1995 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 17.8 shot was identified in the gizzards of Continued on page 228 | April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 22 Table 3. Mean weight of eight, No. 4, Fe, Bi, and Pb shot dosed in game-farm mallard ducks, mean weight of shot recovered from the ducks, number and percent of dosed shot recovered, and percent and weight of shot dissolved in the gizzard. Day 0 Day 30 Day 60 Day 90 Sex Sex Sex Sex Dose F M E M F M F M Mean weight (g) of 8 shot dosed Fe 1.197 1.200 1.202 1.202 1.198 1.201 1199 1.200 0.003* 0.002 0.001 0.002 0.002 0.002 0.003 0.003 31 1.649 1.663 1.654 1.661 1.644 1.654 1.646 1.653 0.005 0.003 0.004 0.005 0.006 0.004 0.010 0.005 >b 1.658 1.666 0.008 0.009 Mean weight (g) of shot recovered Be 0.001 0.025 0.003 0.232 0.273 0.628 0.616 0.878 0.001 0.008 0.001 0.029 0.036 0.032 0.046 0.033 3i 0.018 0.024 0.089 0.070 0.272 0.252 0.787 0.735 0.008 0.012 0.023 0.024 0.041 0.054 0.098 0.099 >b 1.136 1.147 0.165 0.160 Mean % of weight dissolved from shot dosed “e 99.9 96.6 99.8 80.7 76.8 47.8 48.6 27.5 0.07 1.43 0.12 2.41 2.96 2.78 3.84 2.78 3i 98.9 99.2 93:7) 95.8 83.4 82.8 5250 55:5 0.51 0.39 1.94 1.43 2.49 3.68 5.88 See) -b ep les) S12 9.87 9.62 Mean weight (g) dissolved from shot dosed “e 1.196 1.174 1.199 0.975 0.924 0.573 0.582 0.324 0.003 0.009 0.002 0.031 0.036 0.033 0.046 0.035 3i 1.631 1.639 1.566 591 L372 1.402 0.860 0.918 0.011 0.012 0.024 0.024 0.042 0.053 0.093 0.099 -b 0.522 0.519 0.163 0.160 Mean number of shot recovered from shot dosed fe 0.111 2.78 0.889 7.00 6.56 7.67 7.83 7.89 0.111 0.62 0.403 0.40 0.59 0.20 O24 0.111 3i 1.94 2.61 4.67 4.17 6.06 6.06 7.28 VAP 0.70 0.78 0.642 0.64 0.63 0.70 0.463 0.177 >b 6.50 7.00 0.96 1.00 Mean % of the number of shot recovered from shot dosed 7e 1.4 34.7 ie 87.5 81.2 95.8 97.2 98.6 1.39 7.80 5.04 4.95 7.44 2.48 2.16 1,39 3 24.3 32.6 58.3 52.1 737 75.0 91.0 96.5 , 8.78 9.70 8.02 Te Tee 8.69 572 Papa) 81.2 87.5 11.97 12.50 Mean No. of days that shot dosed were in the gizzard fe 1212 121-2 91.2 Bi? 61.2 61.2 ely oie. i 120.5 120.5 90.5 90.5 60.5 60.5 30.5 30.5 228 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 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 mallards (2nd, 3rd, and 4th doses of 8 Fe or 8 Bi shot were dosed on Days 30, 60, and 90). Mean No. Days Mean % Wt of Shot Dose Day Dosed Shot in Gizzard Dissolved per Day Fe 0 9.9 Ae 0.62° 0 121.1 0.8 2.03 0.02 30 91.1 0.9 2.03 0.06 60 61.1 1.0 2.03 0.05 90 31.1 1.3 2.03 0.08 Bi 0 9.9 DSF 0.26 0 120.5 0.8 3.01 0.02 30 90.5 1.0 3.01 0.04 60 60.5 1.4 3.01 0.06 90 30.5 Dee 3.01 0.17 Pb 0 9.9 3.3 0.60 0.75 * Estimated by multiple regression. Bobs 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 0 from shot dosed on Day 310) 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 ata faster rate than they do Fe or Bi shot. Difference in rate shot were dissolved: Between doses: Dosed Day 60; P < 0.0001. Dosed Day 90; P < 0.0001. 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 of 58 days. Furthermore, Pb-dosed ducks, all of which died in February 1995, had heavier gizzards than the O-, 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, which was not experienced by the Pb-dosed ducks. Liver—Mean weights of livers ranged from 17.7 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 0 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 Gonads 0 F 21.0 42.2 9.3 43.5 0.832 2.02 0.28 2.53 0 M 21.4 18.5 O2 33.1 0.81 0.91 0.16 2.46 0 F&M DileD 30.4 7.8 38.3 0.57 DDT. 0.31 1.95 Fe F 19.7 46.6 9.1 38.9 0.93 1.97 0.30 3.08 Fe M 20.1 20.0 6.3 36.8 0.52 0.64 0.23 2.32 Fe F&M 19.9 33.2 Wodl 37.9 0.53 2.47 0.30 1.91 Bi F 19.2 43.9 9.0 45.2 0.77 2.36 0.36 Doi Bi M Pile? 18.0 6.2 36.1 0.76 0.94 0.19 4.26 Bi F&M 20.2 30.9 7.6 40.6 0.56 2.53 0.31 2.52 Pb F DBD 17.7 8.6 0.6 1.20 1.81 0.29 0.10 Pb M 26.5 18.6 8.7 1e2. 2.28 Dui 0.95 0.20 Pb M&F 24.8 18.1 8.7 0.8 1.33 1.51 0.48 0.14 2 SIE. Differences between sexes in organ weights. Only significant differences are shown. Liver: O-dosed F =115.45; P <0.00001. 1,24 Fe-dosed F =164.68; P < 0.00001. 21 Bi-dosed F =104.82; P < 0.00001. 1,22 95.44; P < 0.00001. Kidneys: 0-dosed F 1,27 Fe-dosed lg 1,34 Bi-dosed E 1,34 Gonads: 0-dosed F 13. 53.48; P < 0.00001. 50.49; P < 0.00001. 8.65; P = 0.0059. 4 Pb-dosed F = 7.29;P=0.0223. 1,10 Difference among doses in organ weights: Gizzard: [EO = (6.72312 = (010/008). 3,116 Liver: F = 15.43; P< 0.00001. 3,112 Kidneys: 2 3,112 Gonads: F =113.91; P < 0.00001. 3,79 2.70; P = 0.0492. ON=S=P>ADOH Controls Fe Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Females Males Bi Pb Figure 5. Mean weight (g) of livers of game-farm mallards dosed with 0 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 of males 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 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). Inspite 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 0 to necropsy—an average of 120.5 days. Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 231 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 Pin 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= : eh samples taken from >120 to 150 days. n=4. tm =2, MDL: Bi - 0.081 ug/g. Sn - 2.14 we/g. Pb - 0.132 ug/g. Ca 61.0 1.03 68.8 6.60 62.8 1.60 65.3 1.47 63.4 5.55 64.7 6.81 60.2 2.25 71.2 8.61 57.2 1.74 62.1 2.98 125.7 32.1 92.4 18.49 104.4 22.4 173.4 37.4 164.8 37.3 149.4 30.2 207.0 89.1 154.2 38.6 278.5 Use) Mg 78.2 1.79 83.2 4.04 78.4 2.00 78.8 1.05 78.8 13S 83.6 1.87 80.2 2.34 83.9 1.00 84.8 1.59 79.2 2.63 80.1 1.57 84.8 1.14 81.1 1.44 80.5 1.25 82.0 2.78 79.9 1.77 75.6 6.75 82.2 1.39 81.8 12.65 Zn 5.67 0.10 5.96 0.27 6.02 0.14 6.02 0.24 5.91 0.20 5.95 0.22 6.15 0.13 6.25 0.30 5.93 0.14 6.01 0.16 6.78 0.60 6.32 0.28 6.49 0.35 7.55 0.70 7.54 0.84 7.78 0.80 7.70 1.65 75u 1.08 9.21 1.29 Cu 0.385 0.077 0.420 0.055 0.478 0.058 0.566 0.061 0.460 0.083 0.525 0.045 0.418 0.053 0.550 0.030 0.477 0.047 0.508 0.063 0.518 0.041 0.509 0.036 0.425 0.037 0.493 0.087 0.487 0.074 0.490 0.059 0.602 0.201 0.645 0.095 0.632 0.032 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 229 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 0 the first egg was laid. Sample sizes are in parentheses. Mean days to Mean Days after Day 0 Dose lay 21 eggs first egg was laid 0° 27.4(17 83.817) 2.04° 4.30 Fe 25.7(18) 94.0(18) 1.56 4.12 Bi 25.9(15) 91.6(17) 1.26 3.66 * One 0-dosed and one Bi-dosed female laid no eggs; they suffered from egg yolk peritonitis. BASE: * 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. N= PDA Hatched Not Hatched . Controls Fe Bi 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 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 of unknown 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); Illinois Natural History Survey Bulletin Vol. 35 Art. 4 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. 2A9 Hatchability rates: F = 1.07; P > 0.05. 2,48 244 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 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 fertile eggs. The hatchability rates varied widely for unknown 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 ug/g) and Bi-dosed ducks (x= 0.261 ng / g) than in shells of eggs from Fe-dosed ducks (x= 0.145 ug/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 of eggs 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 ug/g) than contents of eggs from Bi-dosed ducks (x= 33.0 ug/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 ug/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 ug/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 g/g). Continued on page 247 April 1997 Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 245 Table 20. Mean concentrations (ug/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 lt Mg Zn Cu 0 0.300 5132 0.232 1.40 377106) 1732 1364 0.936 A) 0.042° ol 0.079 0.184 11342 56 oo 0.137 0.856 Fe 0.145 6.29 0.305 ile S22059 2m 1695 1397 0.960 29.8 0.020 2.70 0.079 0.146 3877 47 28 0.181 Le7aL 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 maSE. MDL: Pb - 0.072 ug/g. Bi- 0.050 ug/g. Sn-1.93 yg/g. Differences among doses: Pb in egg shells: F = 7.0404; P = 0.002. 2,49 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 Mg Zn Cu 0 0.170 34.4 0.037 1.28 1186 2696 124.7 18.3 1.37 0.024? 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 a 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: 2,49 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 0 Fe Bi il 0 0 0 yp 0 0 0 3 0 BP 0 1253 4 0.3 0.7 0 0.3 0.5 5 0 0 0 6 0 0 0 7 0.6 0 0 0.4 8 0 0 0 9 0 0 0 10 0.2 0 0 0.2 11 0 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 0 0.4 14 0.9 0.3 0 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 Deo 1.1 1.6 17 0.5 1.0 19 17, 2.5 4.3 0.8 1.1 1.3 20 5.3 4.6 4.1 Dp 1.7 1.1 21 5.1 9.2 3.7 1.6 DD, 0.8 22 13.2 8.8 8.5 2.9 8) Dp 23 16.9 23.6 11.8 4.2 4.0 3.6 24 16.3 21.7 16.2 35 6.2 By 25 B5 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 DB 3.0 1.8 28 4.3 0.4 5.0 2.3 0.4 Dp 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 0 Fe Bi All doses eeSE: Difference among doses: Age at death of embryos: F = = 4.43; P = 0.0125. 2,539 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- Age at Death (days) 21.6(189) Ot" 20.9(206) 0.38 22.2(147) 0.27 21.5(542) 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 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 Allhearts 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 10in0-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 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 one 0-dosed duckling. Both Fe-dosed ducklings had minimal lesions. The 0-dosed duckling had aninflammatory 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 Het of 25.2 in four Pb-dosed ducks after a mean survival of 9.9 days. Inouracute toxicity study (Sanderson etal. 1997a), 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 0 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. Puls (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 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 11 and 39, we clearly identified all eight, No. 4, Fe or Bi pellets dosed in each of eight female and eight male ducks. Sanderson etal. (1997a) radiographed 20 ducks on Day 23 of their study and identified allshotin 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 of males in the present study (Table 5) were similar to the mean weights of livers of males in the acute toxicity study (Sanderson et al. 1997a), but mean weights of both 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. Krigman et al. (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 Biin 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 Biin 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- Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 249 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 of Cu 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 of Cu 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 Biin 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 Biin 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 Biin the brain of labora- 250 Illinois Natural History Survey Bulletin Vol. 35 Art. 4 tory mice showed neurologic symptoms and that aconcentration of >0.5-2.0 ug/g of Biin 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 ug/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 ng/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. Puls (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, Bishot. 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. We believe that the overall low hatchability of eggs, regard- less of dose, might be related to repeatedly dis- turbing the ducks on Days 0, 30, 60, and 90 to weigh, dose, and bleed them and to collect eggs twice daily. April 1997 Literature Cited BMDP 1992. Statistical software manual, 7.0 soft- ware release. University of California Press, Ber- keley. Dipalma, J.R. 1988. Bismuth toxicity. American Family Physician 78(5):244-246. Environment Canada. 1992. Guidelines regard- ing the toxicity tests required for the approval of candidate non-toxic shot (to be submitted to the meeting of the executive in January 1993). Envi- ronment Canada. 9 pp. Forth, W., and W. Rummel. 1971. Absorption of iron and chemically related metals in vitro and in vivo: specificity of the iron binding system in the mucosa of the jejunum. Pages 173-191 in S.C. Skorya and D. Waldron-Edward, eds. Intestinal absorption of metal ions, trace elements and ra- dionuclides. Pergamon Press, Oxford, New York, Toronto, Sydney, Braunschweig. Glaser, J.A., D.L. Foerst,G.D. McKee, S.A. Quave, and W.L. Budde. 1981. Trace analyses for waste- waters. Environmental and Science Technology 15:1426-1435. Greenberg, A.E., L.S. Clesceri, and A.D. Eaton. 1992. Standard methods for the examination of water and wastewater. Section 3113 Metals by electrothermal atomic absorption spectrometry. American Public Health Association, Washing- ton, D.C. 18th Ed:3-20—3-28. Gregus, Z., and C.D. Klaassen. 1986. Disposition of metals in rats: a comparative study of fecal, urinary, and biliary excretion and tissue distribu- tion of eighteen metals. Toxicology and Applied Pharmacology 85:24-38. Hamilton, E.J., M.J. Minski, and J.J. Cleary. 1972/ 1973. The concentration and distribution of some stable elements in healthy human tissues from the United Kingdom. Science of the Total Environ- ment 1:341-374. Hermayer, K.L., P.E. Stake, and R.L.Shippe. 1977. Evaluation of dietary zinc, cadmium, tin, lead, bismuth and arsenic toxicity in hens. Poultry Science 56:1721-1722. Hillemond, P., M. Palliere, B. Laquais, and P. Bauvet. 1977. Traitment bismuthique et Toxicity of Ingested Bismuth Alloy Shot in Game-farm Mallards 251 bismuthemie. Semaine des Hopitaux de Paris. 53:1663-1669. Irving, J.T. 1973. Calcium and phosphorous metabolism. Academic Press, New York and London. 246 pp. Kimball, W.H., and A.A. Munir. 1971. The corro- sion of lead shot in a simulated waterfowl giz- zard. Journal of Wildlife Management 35:360- 365. Krigman, M.R., T.W. Bouldin, and P. Mushak. 1985. Metal toxicity in the nervous system. Mono- graphs in Pathology 58-100. Locke, M., H. Nichol, and C. Ketola-Pirie. 1987. Binding of bismuth to cell components: clue to mode of action and side effects. Canadian Medi- cal Association Journal 137:991-992. Office of Research and Development. 1994. Meth- ods for the determination of metals in environ- mental samples—Supplement I. Revision 4.4. U.S. Environmental Protection Agency. EPA/600/R- O41 EE -7-E— 57: Puls, R. 1988. Minerals in animal health. Diag- nostic data. Sherpa International, Clearbrook, British Columbia. Ross, J.F., Z. Sahenk, C. Hyser, J.P. Mendell, and C.L. Alden. 1988. Characterization of a murine model for human bismuth encephalopathy. NeuroToxicology 9:581-586. Sanderson, G.C.,S.G. Wood, G.L. Foley, and J.D. Brawn. 1992. Toxicity of bismuth shot compared with lead and steel shot in game-farm mallards. Transactions of the 57th North American Wildlife and Natural Resources Conference 526-540. Sanderson, G.C.,W.L. Anderson, G.L. Foley, L.M. Skowron, J.D. Brawn, and J.W.Seets. 1997a. Acute toxicity of ingested bismuth alloy shot in game- farm mallards. Illinois Natural History Survey Bulletin 35(3):185-216. Sanderson, G.C., W.L. Anderson, G.L. Foley, S.P. Havera, L.M. Skowron, J.D. Brawn, G.D. Taylor, and J.W. Seets. 1997b. Effects of lead, iron, and bismuth alloy shot in breast muscles of game- farm mallards. Journal of Wildlife Diseases. In press. US) Illinois Natural History Survey Bulletin Vol. 35 Art. 4 Serfontein, W.J., and R. Mekel. 1979. Review of bismuth blood and urine levels in patients after administration of therapeutic bismuth formula- tions in relation to the problems of bismuth toxic- ity in man. Research Communications in Chemi- cal Pathology and Pharmacology 26:391-411. Skoryna, 5.C., and D. Waldron-Edward. 1971. Intestinal absorption of metalions, trace elements, and radionuclides. Pergamon Press, Oxford, New York, Toronto, Sydney, and Branuschweig. 431 jee) Slikkerveer, A.,and F.A. de Wolff. 1989. Pharma- cokinetics and toxicity of bismuth compounds. Medical Toxicology and Adverse Drug Experi- ence 4:503-323. Thomas, D.W.,T.F. Hartley, P. Coyle, and 5. Soecki. 1988. Bismuth. Chapter 11, pages 115-127 in H.G. Seiler and H. Segil, eds. Handbook on toxicology of inorganic compounds. Marcel Dekker, Inc., New York and Basel. Underwood, E.J. 1971. 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. wii Illinois Natural History Survey Natural-Resources Building . - 607 East Peabody Drive. ent z Champaign, Illinois 61820. | cat i ear "217-333-6880, ~ A Division of the Illinois Department of Natural Resources ILLINOIS: DEPARTMENT OF , a i Sone NATURAL eS a a 2 RESOURCES ; r ¥ M i, L' ; rt a ee Ce OF tae Lory Hh : ae Te meh id My He ‘y iy v oe i We ' | URBANA | i | | ca) o = = ot uw oO -—_—_—— = wn c wi = z = —— Sa ——— seyiSitodiejmbcjausraceiemn DEEPER WN tees Sree weteaats Scary A ced PAPA OP eat ce RSS WATS aye ye . : i encore een ie sesh AA TN aqnye oes) Pratt = F anton ge User pees Os ce 2M WON pee Oey Cierre arse dene Sete sys roar yes arise Beye ita ge we . SS ust ge te. reas ota SURG es SiMe ton pins ‘ me : ’ TP ante : % ; 1S wey te ’ 5 : aba ace : Or try aba e Bs SS TR UMEAIS a ey yi ae raphe ee oy WU yrange areca e a2) ‘ ™ . - Say riractyas WSLS cours > S _ eit Gi va care near €or ” i