The decline of the California Condor during the last century has been attributed to high mortalities from hunting and poisoning. More recently, several deaths among the remaining wild Condors have been correlated to the ingestion of lead bullet fragments embedded in abandoned carcasses. Lead toxicity is therefore considered to be a major hazard to the California Condor. The purpose of this study is to determine the relative toxicities of bullets composed of copper and an alloy of tin-bismuth-tungsten to Condors by orally administering metal fragments to a surrogate species, the turkey vulture (Cathartes aura).

Thirty turkey vultures were trapped and housed individually in 36"x36" cages. The average weight of the birds was 1.68 Kg at capture. Birds were randomly divided in 3 groups of 10 birds. Two of the groups were fed bovine hearts containing either Cu or TTB pellets (2.3g/Kg body weight). The third group was not fed any pellets. Retention and movement of the pellets within the bird was monitored radiometrically over an eight week period. Retention of the pellets was highly variable and birds were individually re-dosed weekly to maintain a constant dosage throughout the study. Blood samples were collected from the brachial vein prior to and following metal exposure on a weekly basis for a period of 8 weeks. Liver wedge biopsy samples were also collected after eight weeks of metal exposure. The birds showed no visible or behavioral signs of toxicological stress and were released one week after surgery.

Statistical comparisons between the CBC and chemistry panel between the treatment and control groups showed no significant differences. Similarly, ICPMS analyses showed no change in copper concentration in the sera. An increased tin concentration was found in liver and sera in the TTB treatment. No evidence of cellular toxicity was observed by transmission electron microscopy. Evidence of metal deposition is currently being studied by energy dispersive X-ray microanalysis.

Copper or TTB based munitions could provide a non-toxic alternative to lead based munitions and this conversion could help the re-establishment of the California Condor population in the wild. However, further studies need to be performed on the blood and liver samples before a concrete conclusion and recommendation can be made regarding the relative toxicities of copper or TTB.

Key Words: Turkey Vulture, California Condor, Electron Microscopy, Copper, TTB, Tungsten, Tin, Bismuth, Avian Hepatic Tissue, Wildlife, Endangered Species, Restoration Programs, US Fish and Wildlife Service.

INTRODUCTION

The California Condor Recovery Program is a Federally funded project which aims to reintroduce the California condor into its native habitat. The program which was established in 1975 has followed a three phase approach:

1. Capture of all of the 23 remaining native wild birds. 2. Breeding the captive birds while maintaining the genetic diversity of the offspring. 3. Reintroduction of the captive reared offsprings into the wild.

Capture, breeding, and maintenance of genetic diversity of the offsprings have been successful. There has been a consistent increase in the population of the captive birds at the Los Angeles Zoo and the San Diego Wild Animal park.

The initial reintroduction of the captive reared condors to the wild, however, has met numerous setbacks and there has been high mortality among the initial group of reintroduced birds. Attempts to rationalize the mortalities have indicated that the deaths result largely from of a lack of necessary survival skills in the wild, i.e., perching on power poles, poor foraging, and geniality to humans gained while in captivity. As a result of such setbacks, some of the released birds have been recaptured to ensure their survival. However, there is considerable concern also about the potential for lead poisoning together with shooting as a causal factor in the decline of the wild California condor population (Weimeyer et al. 1984). Poisoning from lead intoxication appears to occur from ingestion of lead bullet fragments embedded in abandoned carcasses. Research conducted in the 1980's documented four California condor deaths, three of which were attributed to lead poisoning (Janssen at al. 1986, Weimeyer et al. 1988). In addition, five of the fourteen wild condors captured during 1981-1986 had high levels of lead in their blood (average of 0.69 ppm). Lead poisoning accounts for 20-25% (3 of 12) of known losses (Snyder 1986) and 60% (3 of 5) of the confirmed mortalities (Weimeyer et al. 1988). It can, therefore, be inferred that a significant number of earlier, undocumented condor deaths can be attributed to lead intoxication.

The purpose of this study is to evaluate the relative toxicities of bullets composed of copper and/or TTB to avian scavengers by orally administering metal fragments to a surrogate species, the Turkey Vulture (Cathartes aura). These metals may provide non-toxic alternatives to lead based munitions which are known to be highly toxic to non-target avian species when ingested.

Since the California condor is an endangered species, it cannot be used in this study. For this reason, the turkey vulture was chosen as a model to study the effects of two possible, non-lead based alternative munitions. Another major reason for choosing the turkey vulture as a surrogate is that it is a member of the same family as the California Condor and is common in Southern California.

An alloy of Tungsten-Tin-Bismuth (TTB ) and copper based bullets are being considered as alternative bullets to lead based munitions that are currently in the market. Both copper and TTB bullets are claimed to be ballistically superior to steel shots by their respective manufacturers. Toxicological studies using Mallards, Anas platyrhynchos have shown TTB to be less toxic than lead (Sanderson et al. 1992, Ringleman et al. 1993).

In contrast, copper has been found to be potentially toxic to Mallards, Canada Geese, Branta canadensis, Mute Swans, Cygnus olor, and broiler chicks (Sanderson et al. 1974, Henderson and Winterfield 1975, Kobayashi et al. 1991, Jensen et al. 1991). At the present time, the of these materials to raptors relative to lead is not fully know. This study provides vital information on the relative toxicities of ingested copper and TTB pellets on California condors and other avian scavengers.

Toxicity testing is generally conducted using LD50 assays which evaluate the concentration of toxicant required to induce death in 50% of the individuals in a 96 hour period. However, the dosage used in the present study was based on an estimated maximal amount of metal that a wild California condor could consume upon feeding on carrion from an abandoned carcass. Environmentally relevant dosages were estimated to be equivalent to two bullets.

MATERIALS AND METHODS

Thirty turkey vultures, 11 from Rancho Mission Viejo in Orange County and 19 from San Luis Obispo, were trapped and housed individually in 36"x36" cages. The average weight of the birds was 1.68 Kg at capture. Birds were randomly divided in 3 groups of 10 birds. The birds were kept in a ranch at the Starr Ranch Sanctuary of the National Audubon Society in Rancho Santa Margarita, California. The birds were initially fed rats then bovine heart. Two of the groups were fed bovine hearts containing either Cu or TTB (55%W, 44%Sn, 1%Bi) pellets (2.3g/Kg body weight). The third group was not fed any pellets. Retention and movement of the pellets within the bird was monitored radiometrically over an eight week period.

Blood samples were collected from the brachial vein weekly for metal quantification by Inductively Coupled Plasma Mass Spectroscopy (ICPMS), a complete blood count (CBC), chemistry panel, and LDH assay. Liver wedge biopsy samples were also collected, by veterinarians at the San Diego Wild Animal Park, Escondido, Ca. and Serrano Animal and Bird Hospital, Lake Forest, Ca., after eight weeks of metal exposure for metal quantification by ICPMS, histopathology, transmission electron microscopy (TEM), energy dispersive X-ray microanalysis (EDX), and enzyme cytochemistry.

Hepatic tissue sample preparation for TEM.

Liver samples were fixed with 1% formaldehyde and 2% glutaraldehyde in 0.05 M Millonigs phosphate buffer pH 7.4 at room temperature. Followed by secondary fixation with 2% osmium tetroxide in Millonigs-buffer and dehydration with graded ethanol series and propylene oxide. The dehydrated samples were embedded in spurr epoxy resin, sectioned using a Sorvall MT-2 Utra Microtome, and viewed in a JEOL 1200 EXII TEM. TEM micrographs were taken using the same instrument.

Scanning electron micrographs were taken, using a JOEL T200 SEM, of pellets collected from the feces and unused pellets to determine whether there was leeching out of metal particles on the surface of the pellets.

RESULTS

Transmission electron microscopy of liver biopsy specimens revealed no observable ultrastructural evidence of cellular toxicity between the control and treatment groups (see Fig. 1-12). Figures 1, 2, and 3 are micrographs of liver wedge biopsy samples from control birds, figures 4-8 are from TTB treated birds, and figures 9-12 are from copper treated birds. Energy dispersive X-ray microanalysis is currently being performed to determine evidence of metal deposition in the hepatic cells.

SEM of the pellets showed evidence of leeching out of surface metal particles from copper and TTB pellets that were found in the feces. There was a higher degree of leeching of TTB pellets compared to copper pellets ( Fig. 13-17).

Retention of the pellets was highly variable and birds were individually re-dosed weekly to maintain a constant dosage throughout the study. The birds showed no visible or behavioral signs of toxicological stress and were released one week after surgery. CBC and chemistry panel data showed no changes in whole blood composition. ICPMS data showed that copper concentrations in the blood and liver did not change during 0, 6, and 8 weeks post ingestion. However, there was an increase in tin concentration in the liver after 8 weeks of TTB exposure. Elevated levels of tin in the blood was also found after 6 weeks of TTB exposure.

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  Figure 1. Shows the nuclei (N), nucleoli (No), mitochondria (M), and glycogen particles (G) from a control bird. x 10,000.

  

  Figure 2. Shows two control hepatocytes. x 5,000.

  

  Figure 3. Is a high power TEM shot of a mitochondrion (M) and a lysosome (Ly) from a control hepatocyte. x 50,000.

  

  Figure 4. Hepatocytes from a TTB treated bird. x 5,000.

  

  Figure 5. Hepatocyte containing peroxisomes (P) from a TTB treated bird. x 12,000.

  

  Figure 6. High power TEM micrograph of a peroxisome (P) from a TTB treated bird. x 20,000.

  

  Figure 7. High power TEM shot showing a lysosome (Ly), myeloid body (MB), glycogen particles (G), and mitochondria (M) from a TTB treated bird. x 25,000.

  

  Figure 8. Hepatocyte from a TTB treated bird containing a relatively high amount of lysosomes (Ly) and lipid droplets (L) than neighboring cells. It also contains a unique vesicle that contains an electron dense material. x 8,000.

  

  Figure 9. Hepatocyte from a copper treated bird showing a nuclei (N),mitochondria (M), lipid droplets (L), and glycogen particles (G). x 8,000.

  

  Figure 10. Hepatocytes adjacent to a bile canaliculi (C) from a copper treated bird. Lipid droplets (L) and collagen fibers (Co) appear to be abundant in this area. x 5,000.

  

  Figure 11. Vesicles (V) and peroxisomes (P) present in a hepatocyte of a copper treated bird. The vesicles contain a small amount of electron dense that was also found in TTB treated birds. x 15,000.

  

  Figure 12. High power TEM micrograph of collagen fibers (Co). x 10,000.

  

  Figure 13. Low power scanning electron micrograph of an unused copper pellet. x 30.

  

  Figure 14. Low power scanning electron micrograph of a copper pellet collected from the feces. Leeching of the surface of the metal is evident. x 30.

  

  Figure 15. Low power scanning electron micrograph of an unused TTB pellet. x 30.

  

  Figure 16. Low power scanning electron micrograph of a TTB pellet collected from the feces. A higher degree of leeching (arrows) compared to the copper pellet shown in figure 14 is apparent. x 30.

  

  Figure 17. High power scanning electron micrograph of the lesion from the TTB pellet from figure 16. x 25,000.

DISCUSSION

Vesicles which appear to contain electron dense materials, possibly metals, have been observed in sections from TTB and copper treated birds (Fig. 8 and Fig. 11 respectively). Vesicles from copper treatment appeared to contain less electron dense material than vesicles from the TTB treatment. These vesicles containing electron dense material observed from TTB and copper treatments could potentially serve as storage compartments of excess metals present in the sera. Excess tin that was present in the sera (week 6) of TTB treated birds could have been sequestered in the liver during the 8th week of exposure since tin sera levels returned to baseline levels in week 8 of the experiment. Energy dispersive X-ray microanalysis is currently being performed on the sections containing the said vesicles to determine the elemental composition of the observed electron dense bodies. If the said vesicles are found to contain tin, in TTB treated animals, this data could support the increased levels of hepatic tin found upon ICPMS metal quantification analysis of the liver samples. Furthermore, the increase in hepatic and sera levels of tin could be a result of a higher degree of leeching of TTB particles than copper (Fig. 14, 16, and 17).

Although initial results gathered from the TEM examination of hepatic tissues, CBC analysis, and chemistry panel showed no evidence of toxicological effects among the treatments, it is currently premature to recommend the use of copper or TTB based munitions as an alternative to lead bullets. Further analysis of the blood and liver samples need to be performed before a concrete conclusion and recommendation can be drawn from the study.

CITATION

Beck, A. B. 1955. The copper content of the liver and blood of some vertebrates. Aus. J. of Zool. 1-18.

Henderson, B. M., and Winterfield*, R. W., 1975. Acute copper toxitosis in the Canada Goose. Avian Dis. 19:385-387.

Janssen, D. L., Oosterhuis, J. E., Allen, J. L., Anderson, M. P., Kelts D. G., and Weimeyer, S. N. Lead poisoning in free ranging California condors. JAVMA. 189(9): 1115-1117.

Jensen, L. S., Dunn, P. A., Dobson, K. N. 1991. Induction of oral leasions in broiler chicks by supplementing diet with copper. Avian Dis. 35:969-973.

Kobayashi, Y., Shimada, A., Umemura, T., Nagai, T. 1992. an outbreak of copper poisoning in Mute swans (Cygnus olor). J. Vet. Med. Sci. 54(2): 229-233.

Ringleman, J. K., Miller, M. W., and Andelt. 1993. Effects of ingested tungsten-bismuth-tin shot on captive Mallards. J. Wildl. Manage. 57(4):725-732.

Sanderson, G. C., Beamer, P. D., and Walker, K. 1974. Some effects of ingested copper shot on wild trapped male mallards. Illinois Natural History Survey. Unpublished Report.

Sanderson, G. C., Wood, S. G., Foley, G. L., Brawn, J. D. 1992. Toxicity of bismuth shot compared with lead and steel shot in Game-farm Mallards. Trans. 57th N. A. Wildl. & Nat. Res. Conf. 526-540.

Snyder, N. F. R. 1986. California condor recovery program, p. 56-71. In S. E. Senner, C. M. White, and J. P. Parish [eds.], Raptor conservation in the next 50 years. Raptor Res. Rep. No.5 Raptor Research Foundation, Hastings, MN.

Weimeyer, S. N., Jurek, R. M., and Moore, J. F. 1984. Environmental contaminants in surrogates, foods, and feathers of California Condors. Environmental Monitoring and Assesment. 6:91-111.

Weimeyer, S. N., Scott, J. M., Anderson, M. P., Bloom, P. H., and Stafford, C. J. 1988. Environmental contaminants in California condors. J. Wildl. Manage. 52(2):238-247.

ACKNOWLEDGEMENTS

I would like to thank Dr. Thomas G. Douglas for his expertise and technical assistance in the TEM and SEM. I would also like to thank David and Sheryl Clendenen, Dr. Robert Risebrough, Edward and Judy Henckle, Pete Bloom, Pete DeSimone, Scott Weldy, Dr. Jeffrey Zuba, Dr. Meg-Sutherland Smith, Danny Tang, Mory Guadalupe, Tara Wood, Paula Spencer, Rick Desai, Lan Troung, Alyson Silverman, Samantha Hua for their support and assistance. Finally, I would like to extend my sincere gratitude to Dr. Andrew Zed Mason for his advice and support. This project is funded by the United States Fish and Wildlife Service, California Condor Recovery Program, Ventura, Ca.

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zedmason@csulb.edu