nu HI v 'I- v ’ it ‘~ u" ‘ and" «91.! 'w ' a F L fig: In .~ hx‘ YI I . .1 .,~".‘“-tf'1 - -‘T‘v ‘ "7711 V ”5‘- n; ' I ‘ .: '1“.U..|'l::"‘lj" ,. ‘ ' "lot-:l‘; ‘ ;; . 'r‘ 'I I” «1:: " ‘ "1"”?4‘“ "HILUI 'l I'l. ’,.""'f':: 4» ' " .nl‘ H v I 'IGHI: .LWK_'.‘:‘HIM\. al'u‘n-HI u!) .MJ I 0 n" . ' ‘ .. 92 6902 " LIE-5MB? [THESIS flsfdgam State L..- U vanity J This is to certify that the dissertation entitled DEVELOPMENT OF DIETARY L050 AND REPRODUCTION TEST PROTOCOLS USING MINK AND FERRETS AS REPRESENTATIVE MAMMALIAN CARNIVORES presented by Thomas C. Hornshaw has been accepted towards fulfillment of the requirements for Ph 0 D . degree in Animal SC’i ence ajor professor Date NQMBHM MSU is an Affirmatiw Action /Equal Opportunity Institution 0-12771 MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this Checkout from Ailing—Ian. your record. FINES will be charged if book is returned after the date stamped below. 5950 m. JAN"); 1 2000 V APR? 1'33 50032 DEVELOPMENT OF DIETARY LCSO AND REPRODUCTION TEST PROTOCOLS USING MINK AND FERRETS AS REPRESENTATIVE MAMMALIAN CARNIVORES by Thomas C. Hornshaw A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science and Center for Environmental Toxicology 1984 5 3 $414.) '/ ACKNOWLEDGEMENTS I wish to thank my wife, Elena, for the many years of support, help, reassurance, and love she has provided during my academic endeavors. I also extend these thanks to both of our families, especially to my parents, in memoriam. I wish you were here to see this folks. I owe a deep debt of gratitude to Dr. Richard J. Aulerich. His help, insight, encouragement, and guidance throughout my graduate education have been a tremendous aid to my develop- ment as a student and as a person. He has given me the free- dom to pursue my interests and has stood by me in good times as well as bad. I deeply appreciate him as a major professor and as a friend. I am similarly indebted to Dr. Robert K. Ringer, who has also had a profound effect on my academic development. His expertise in many areas has been invaluable in providing insight and guidance for me and he has also given me the freedom to pursue research where it leads. I must also thank him for making me even more aware of the importance of the correct use of language. Dr. Steven J. Bursian, another member of my academic committee, has also been a good friend and a good listener when I start rambling on axnnza research project. I appreciate his help with some of my studies and with providing insight into the meanings of the results. I also would like to thank Dr. Matthew J. Zabik for his assistance and input in my research and for being a member of ii my academic committee.. While I didn't become as deeply involved in the analytical details of this experiment as I did in my Master's project, his help with the problems I encountered was deeply appreciated. Finally, I would like to express my gratitude to Mr. Angelo Napolitano, Ms. Darlene Krause, and Ms. Carol Streigler for their assistance with the "drudge" work involved with these experiments, and to Ms. Carol Daniel for typing the manuscript. This research was supported by U.S. EPA/NCAA Interagency Agreement #AD13F28800. iii ABSTRACT DEVELOPMENT OF DIETARY LC50 AND REPRODUCTION TEST PROTOCOLS USING MINK AND FERRETS AS REPRESENTATIVE MAMMALIAN CARNIVORES by Thomas C. Hornshaw Representative mammalian wildlife species have not been designated as toxicological models for testing substances of environmental concern. The mink (Mustela vison) and the European ferret (M; putorius furo) have been suggested as representative mammalian species since they are among the most sensitive mammalian species to the toxic and reproductive effects of several substances. Also, as carnivores, these species are subject to the effects of bioaccumulation of lipo- philic compounds. Therefore, dietary LC50 and reproduction tests were initiated with these species, using sodium mono- fluoroacetate (Compound 1080), o-cresol, tetramethylthiuram disulfide (thiram), and a polychlorinated biphenyl (Aroclor 1254) as test substances to develOp protocols for these tests. The results of the various tests demonstrate that mink and ferrets may be used in subacute and reproduction tests with a wide range of test substances, since the test substances used representated a wide range of solubilities, volatilities, acute toxicities, modes of action, and chemical classes. Since the tests were conducted indoors under controlled conditions, it is expected that the results should be reproducible in similarly equipped laboratories. Factors demonstrated in these tests that may affect the determination of toxicity of a substance include the age of the animal at the beginning of a test and the carrier used to introduce the substance into the diet, while the diet's composition (as long as it meets the nutrient requirements of the test species) and the season of the year may have little or no effect on the results of the test. Diet- ary LC50 tests may be conducted with as few as 32 animals (3 test concentrations plus control, 8 animals per concentration) if an accurate acute oral LDsd is available, while reproduction tests may require 64 animals (3 test concentrations plus con- trol, 16 animals per concentration) if unproven breeders are used. TABLE OF CONTENTS Page Acknowledgements ....................................... ii List of Tables ......................................... iv List of Figures ... ..................................... vi Introduction ........ ................................... 1 Review of Literature . .................................. 4 Legal Framework .................................... 4 Test Protocols ..................................... 8 Test Substances .................................... 12 Sodium monofluoroacetate (Compound 1080) ............................. 12 o-cresol .. .................................... 17 Thiram ........................................ 21 Aroclor 1254 .................................. 26 Materials and Methods . ................................. 30 Test Animals .......... ..................... ........ 30 Housing ............................................ 31 Diet ..... . ..... .... ................................ 34 Reproduction ....................................... 36 Weights and Measurements ........................... 37 Pre-test Procedures .................................... 38 Acclimation .......... .............................. 38 Dietary Concentrations ............................. 39 Definitive Test .... ...... . ............................. 42 LC50 Tests ......................................... 42 Reproduction Tests ...... . .......................... 44 Results and Discussion ................................. 43 Experiment I-Compound 1080 .............................. 49 Results ........ . ................................... 49 Mink LC50 Test ........ .. ...................... 49 Ferret LC50 Test ....... ................ . ..... . 56 Mink Reproduction Test ........................ 65 Discussion ..... .................................... 66 Conclusions ..... ............................ . ...... 78 Experiment II-O-Cresol .................................. 79 Results .... ........................................ 79 Mink LC50 Test ................................ 79 Ferret LC50 Test .............................. 86 Mink Reproduction Test ........................ 91 Discussion ........ ................................. 98 Conclusions ........................................ 100 iv Experiment III-Thiram ................................... 100 Results . ............................................ 100 Mink LC50 Test ... .............................. 100 Ferret LC50 Test ............................... 109 Mink Reproduction Test ......................... 115 Ferret Reproduction Test ....................... 123 Discussion ...... .................................... 130 Conclusions ..... ....................... _ ............. 135 Experiment IV;Aroc10r 1254 .............................. 135 Results ............................................. 136 Discussion .......................................... 147 Conclusions ......................................... 151 Experimental Protocols .................................. 151 Results ....... . ..................................... 151 Discussion .......................................... 152 Conclusions ......................................... 158 References .............................................. 160 Appendices ............................................ 170 Appendix A - Mammalian Wildlife (Mink and Ferret) Dietary LC50 Test ..................... 170 Appendix B - Mammalian Wildlife (Mink and Ferret) Reproduction Test ..................... 189 Table Table Table Table Table Table Table Table Table Table Table 10. 11. LIST OF TABLES Composition and nutrient analysis of basal diet .................................... 35 Reproductive parameters of control groups from reproductive trials involving mink fed several compounds .......... 47 Results of range-finding study with mink exposed to sodium monofluoro- acetate (Compound 1080) by gavage ............. 50 Average body weight changes and feed and compound consumed by mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days ................................... 51 Initial and final body weights of male (M) and female (F) mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days ................................... 52 Mortality pattern of mink fed sodium monofluoroacetate (Compound 1080) during a 28-day LC50 test ..................... 53 Blood parameters of mink fed various concentrations of sodium monofluoro- acetate (Compound 1080) for 28 days ........... 54 Body and organ weights of male (M) and female (F) mink fed various concentrations of sodium monofluoro- acetate (Compound 1080) for 28 days ........... 55 Average body weight changes and feed and compound consumed by young or old ferrets fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days ............................. 58 Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days ................................... 60 Mortality pattern of ferrets fed sodium monofluoroacetate (Compound 1080) during a 28-day LC50 test ............... 61 Table Table Table Table Table Table Table Table Table Table 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. Page Blood parameters of growing (young) and fully grown (old) ferrets fed various concentrations of sodium monofluoro- acetate (Compound 1080) for 28 days ........... 62 Body and organ weights of growing male (M) and female (F) ferrets fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days ............................. 63 Body and organ weights of fully grown male (M) and female (F) ferrets fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days ............................. 64 Average body weight changes and feed and compound consumed by mink fed various concentrations of sodium monofluoroacetate (Compound 1080) during a reproduction test .................... 67 Initial and final body weights of male (M) and female (F) mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 8 weeks prior to breeding ................. 68 Reproductive performance of female mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 23 weeks ............................ 69 Average kit body and litter weights and kit survival for dams fed various concentrations of sodium monofluoro- acetate (Compound 1080) for 23 weeks .......... 70 Blood parameters of mink fed various concentrations of sodium monofluoro- acetate (Compound 1080) for 6 months .......... 71 Body and organ weights of male (M) and female (F) mink fed various con- centrations of sodium monofluoro- acetate (Compound 1080) for 6 months .......... 72 Results of range-finding studies with mink and ferrets exposed to o-cresol by gavage ............................ 80 vii Table Table Table Table Table Table Table Table Table Table Table 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Page Average body weight changes and feed and compound consumed by mink fed various concentrations of o-cresol for 28 days Initial and final body weights of male (M) and female (F) mink fed various concentrations of o-cresol for 28 days ................................... 83 Blood parameters of mink fed various concentrations of o-creSol for 28 days Body and organ weights of male (M) and female (F) mink fed various concentrations of o-cresol for 28 days Average body weight changes and feed and compound consumed by ferrets fed various concentrations of o-cresol for 28 days Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of o-cresol for 28 days Blood parameters of ferrets fed various concentrations of o-cresol for 28 days Body and organ weights of male (M) and female (F) ferrets fed various concentrations of o-cresol for 28 days Average body weight changes and feed and compound consumed by mink fed various concentrations of o-cresol during a reproduction test Initial and final body weights of male (M) and female (F) mink fed various concentrations of o-cresol for 8 weeks prior to breeding ................. 93 Reproductive performance of female mink fed various concentrations of o-cresol for 23 weeks viii Table Table Table Table Table Table Table Table Table Table Table Table 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. Average kit body and litter weights and kit survival for dams fed various concentrations of o-cresol for 23 weeks .................................... Blood parameters of mink fed various concentrations of o-cresol for 6 months ................................... Body and organ weights of male (M) and female (F) mink fed various concentrations of o-cresol for 6 months ................................... Results of range-finding studies with mink and ferrets exposed to thiram by gavage ......................... Average body weight changes and feed and compound consumed by mink fed concentrations of thiram (added in water) for 28 days .................... Initial and final body weights of male (M) and female (F) mink fed various concentrations of thiram for 28 days .............................. Organ weights of mink fed various concentrations of thiram for 28 days ..................................... Average body weight changes and feed and compound consumed by ferrets fed various concentrations of thiram for 28 days .................... Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of thiram for 28 days .................................. Mortality pattern of ferrets fed thiram during a 28-day LC50 test ......... Blood parameters of ferrets fed various concentrations of thiram for 28 days .............................. Body and organ weights of male (M) and female (F) ferrets fed various concentrations of thiram for 28 days .............................. ix Page 95 96 97 .... 113 Table Table Table Table Table Table Table Table Table Table Table Table 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. Average body weight changes and feed and compound consumed by mink fed various concentrations of thiram during a reproduction test ............ Initial and final body weight of male (M) and female (F) mink fed various concentrations of thiram for 8 weeks prior to breeding ................ Reproductive performance of female mink fed various concentrations of thiram for 23 weeks .......................... Average kit body and litter weights and kit survival for dams fed various concentrations of thiram for 23 weeks ........ Blood parameters of mink fed various concentrations of thiram for 6 months ........ Body and organ weights of male (M) and female (F) mink fed various concentrations of thiram for 6 months ........ Average body weight changes and feed and compound consumed by ferrets fed various concentrations of thiram during a reproduction test ................... 124 Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of thiram for 8 weeks prior to breeding Reproductive performance of female ferrets fed various concentrations of thiram for 23 weeks Average kit body and litter weights and kit survival for dams fed various concentrations of thiram for 23 weeks ........ Blood parameters of ferrets fed various concentrations of thiram for 6 months ........ Body and organ weights of male (M) and female (F) ferrets fed various concentra- tions of thiram for 6 months Table Table Table Table Table Table Table Table 57. 58. 59. 60. 61. 62. 63. 64. Average body weight changes and feed and compound consumed by young mink fed various concentrations of Aroclor 1254 for 28 days .......................... Initial and final weights of young male (M) and female (F) mink fed various concentrations of Aroclor 1254 for 28 days and after a 7-day withdrawal period ......................... Average body weight changes and feed and compound consumed by older mink fed various concentrations of Aroclor 1254 for 28 days ............................... Initial and final weights of older male (M) and female (F) mink fed various concentrations of Aroclor 1254 for 28 days and after a 7-day withdrawal period ......................... Mortality pattern of young mink fed Aroclor 1254 during a 28-day test ...................................... Mortality pattern of older mink fed Aroclor 1254 during a 28-day test . ..... . ............................... Body and organ weights of growing male (M) and female (F) mink fed various concentrations of Aroclor 1254 for 28 days .......................... Body and organ weights of fully grown male (M) and female (F) mink fed various concentrations of Aroclor 1254 for 28 days .................. xi Page ... 139 ... 141 ... 143 ...144 ...145 Figure 1. Figure 2. LIST OF FIGURES Housing for test animals ..... Typical cage for test animals xii INTRODUCTION Since the late l960's-early 1970's, there has been an ever- increasing concern in this country about the effects of toxic compounds on man, his domestic animals, aquatic and terrestrial wildlife, and the environment. As this concern grew and as toxic compound-related incidents both large and small became more numerous, pressure mounted for a legal framework to better protect man and the ecosystem from the adverse effects of these compounds. As a result, several new laws were passed and some old laws were amended, and, in 1970, the Environmental Protec- tion Agency (EPA) was created. This agency was charged with, among other things, the regulation of most toxic or hazardous chemicals. Toward this end, EPA has promulgated many rules and regulations, so that, today, most toxic or hazardous chemicals are regulated "from cradle to grave". In the area of new compounds (or new uses of old compounds), the key to regulation of toxic or hazardous chemicals is toxi- cological testing of the compounds with an appropriate test species. Thus, on June 25, 1975, the Environmental Protection Agency published in the Federal Register (FR) proposed guide- lines for registering pesticides in the United States, enumera- ting test procedures and data reporting requirements for evaluating the toxicity of compounds to man, domestic animals, and fish and wildlife. After review and modification, these guidelines were reproposed and published in the Federal Register on July 10, 1978 and by the National Technical Information Service (EPA, 1983). The species of choice for testing the toxicity of a com- pound to man and domestic animals, as specified in these guidelines, are common laboratory animals such as rats, mice, rabbits, guinea pigs, and hamsters. Fathead minnows, bluegill sunfish, and rainbow trout were suggested as representative fish species for toxicological testing, while the mallard duck and bobwhite quail were chosen as representative avian wildlife species. However, no representative mammalian wildlife species has been designated either under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) or under the Toxic Substances Control Act (TSCA). In toxicological tests required on carnivorous species, the dog and cat have been the species of choice. However, paralleling the increasing concern about toxic compounds, there has been an increasing concern about using "pet" species in toxicological tests. And, for toxicological tests of all kinds, there has been an increasing awareness during this same time frame of the importance of using sensitive species as test animals. Since no wildlife mammalian species has been designated under FIFRA or TSCA for toxicological testing, since dogs and cats have fallen into disfavor as carnivorous animal models in toxicological testing, and since the importance of using sensi- tive test species has been stressed, the mink (Mustela vison) has been suggested as a representative mammalian carnivore for toxicological testing. In non-standard tests it has been shown to be among the most sensitive, if not the single most sensi- tive, mammalian species to polychlorinated biphenyl (PCB) toxicity. Research performed with mink was instrumental in setting U.S. water quality standards for PCBs (Aulerich and Bleavins, 1981). In subsequent studies, this species has been shown to be similarly sensitive to polybrominated biphenyls (PBBs) (Aulerich and Ringer, 1979), hexachlorobenzene (Bleavins gt 31., 1984), and sodium monofluoroacetate (Compound 1080) (present study), as well as to aflaxatoxins (Chou 35 31., 1976L Furthermore, since mink occupy a position high on the food chain, they may be subject to the effects of bioaccumulation of fat soluble compounds and thus, may be exposed to higher concentrations of a compound via the diet in the wild than species lower in the food chain. This research was initiated to develop standard dietary LC50 and reproduction tests using the mink as a representative mammalian carnivore. Four test substances were chosen for use in developing the test protocols, sodium monofluoroacetate (Compound 1080), o-cresol, Aroclor 12543(a PCB), and tetra- methylthiuram disulfide (thiram), ranging from water soluble, to slightly water soluble, to lipid soluble, to practically insoluble, respectively. The test substances represented a wide range of acute oral LDsos, ranging from highly toxic (Compound 1080) to practically non-toxic (Aroclor 1254), and also were representative of four different classes of chemical compounds. Where possible, the European ferret (Mustela putorius furo), a closely related carnivore exhibiting high sensitivity to the same compounds as the mink, although gener- ally not as sensitive as the mink, was tested and the results compared to those for mink using the same test procedures. REVIEW OF LITERATURE Legal Framework In addition to FIFRA and TSCA, toxic or hazardous sub- stances may be regulated under the Federal Food, Drug, and Cosmetic Act, the Federal Water Pollution Control Act, the Federal Hazardous Substances Act, the Occupational Safety and Health Act, and the Resource Conservation and Recovery Act of 1976. FIFRA provides for registration, re-registration, and classification of pesticides, while TSCA regulates most other toxic or hazardous substances_not specifically covered by one of the other federal acts. This review will concentrate on the regulation of pesticides under FIFRA. The Federal Insecticide, Fungicide, and Rodenticide Act (7U.S.C.136 et seq.) provides, under Section 3 of the Act, for the establishment of regulations for the registration, re- registration, and use classification of pesticides in the United States. The EPA is required to formulate Registration Guidelines for pesticides, which, after a suitable period of public comment, revision, further public comment, and final issuance, become a part of the Code of Federal Regulations (CFR). These Guidelines, which must specify the kinds of information to be required to support the registration of a pesticide, were published for public comment in the Federal Register on June 25, 1975 and republished after revision on July 10, 1978 as Guidelines for Registering Pesticides in the United States, 40 CFR Parts 162 and 163. The Act requires the EPA to register a pesticide if it can be determined that: 1. Its composition is such to warrant the proposed claims for it; 2. Its labelling complies with the requirements of the Act; 3. It will perform its intended function without unreasonable adverse effects on the environment; and 4. When used in accordance with widespread and commonly recognized practices, it will not cause unreasonable adverse effects on the environment. Toward these ends, various sections of the Guidelines deal with product performance, label deveIOpment, product chemistry, and hazard evaluation. Of particular interest is Section 162.8, dealing with hazard evaluation. Section 162.8 addresses the toxicological information necessary to assess the hazard of a pesticide to man and domestic animals, and to fish and wildlife. The data required by this Section enables the EPA: 1. To determine whether or not to approve a registration for a pesticide, and if approved, whether it should be classified for general or restricted use; 2. To determine whether the proposed labelling contains adequate warnings to protect pesticide users, and exposed humans, domestic animals, and fish and wild- life; 3. To determine residue tolerances for the protection of consumers of exposed foods; and 4. To determine conditions under which farm workers may safely re-enter treated fields. Required tests for all pesticide registrations include acute oral LD50, acute dermal LDso, primary dermal irritation, and primary eye irritation tests. For pesticides which may present a hazard to man and domestic animals, a series of conditional tests, depending on the physical and chemical nature of the pesticide, its proposed use, its expected residue on foods, and other "triggers", are enumerated. These tests range from acute inhalation LC50 to subacute oral and dermal to chronic feeding tests. For pesticides which may present a hazard to fish and wildlife (i.e., pesticides that will be used outdoors or that may contaminate water or other environmental resources), avian acute oral LDso and subacute dietary LC50, fish acute LC50, and aquatic invertebrate acute LC50 tests are also required. These tests constitute the first level, or tier, of tests for all pesticides. A second tier of longer term tests are required for certain pesticides intended for outdoor use, depending on the toxicity found in the first level tests, the probable environmental exposure calculated from the environmental chemistry data and the proposed use patterns, the species likely to be exposed to the pesticide, the probable routes of exposure of these species, and the persistence and expected degree Of bioaccu- mulation of the pesticide and/or its metabolites. These second tier tests include subacute, reproduction, and/or chronic feeding studies. Finally, if the second level tests arouse suspicions that unreasonable adverse effects on fish or wildlife may occur, a third tier of simulated or actual field tests may be required. 7 As mentioned previously, required tests for the registra- tion of a pesticide to be used outdoors include acute studies on rats, birds, fish, and aquatic invertebrates and a sub- acute dietary test on birds, while no tests so far are required on wildlife mammals. It was at first thought in the original issuance of the Registration Guidelines (FR, June 25, 1975) that the rat acute oral and, if performed, the subacute dietary and reproduction studies would be sufficient to assess the hazard of most pesticides to wildlife mammals, and that if unique conditions of preposed usage were identified as potential hazards to certain mammals, acute oral and subacute dietary studies could be required for these mammals. Among the approximately 80 sets of public comments received regarding this first issuance were several replies questioning the adequacy of protection provided to wildlife under the Guide- lines. Many of the questions raised were addressed in the reproposal of three sections of the Guidelines (FR, July 10, 1978), although this document still contains no mention of specific requirements for dietary LC50 or reproduction tests for wildlife mammals. Currently, if concerns exist about the effects of a proposed use of a pesticide on wildlife mam- ' mals, dietary LC50 and reproduction tests are required, following protocols established for these tests for man and domestic animals (i.e., using rats and one other non-rodent). Test Protocols Standard test protocols for rats have been in existenCe for two decades or more. Acceptable protocols for the study of acute oral (Hagan, 1965), subacute dietary (Fitzhugh and Schouboe, 1965), chronic (Barnes and Denz, 1954; Fitzhugh, 1965), and reproductive (Oser and Oser, 1956) toxicity are listed in the Registration Guidelines. The Guidelines also specify acceptable protocols for acute LC50 tests for fish and aquatic invertebrates (Stephan, 1975) and birds (Anony- mous, 1968), and subacute dietary LC50 and reproduction tests for birds (FR, July 10, 1978). The subacute dietary LC50 test for birds is designed to test the effects of a substance applied to plant matter in the field. It allows ingestion and degradation of the sub- stance, as well as absorption and metabolism over the five days of dietary exposure. The test also allows exposure to the compound via inhalation from the daily diet, and dermal exposure from contact of the feet and face with the daily diet. A 3-day withdrawal period, during which the birds are given untreated feed, allows for observation of delayed mortality or recovery. The test specifies the use of one aquatic species, preferably the mallard, and one upland species, preferably the bobwhite quail, and requires that hatchlings be used. Hatchlings are used to ensure that (l) the sub- stance is tested at the most sensitive period of the animal's life, and (2) the substance will be tested via the diet, since very young birds cannot survive five days without eating. In addition to an estimate of the dietary concentration of the substance lethal to young birds, this test also may provide (1) an estimate of the maximum concentration of the substance tolerated in the daily diet, (2) signs of intoxication not found in the acute LDso test, (3) indications of the target organ or organ system of the substance, and (4) indications of behavior changes, including rejection of the treated diet. The avian reproduction test should be used when the sub- stance or its metabolite(s) persists in the environment or when wildlife are subjected to repeated or continual exposure. Specifically, these studies are required when: l. The pesticide or major metabolites or degradation products is persistent in the environment; 2. The pesticide or major metabolites or degradation products is stored or accumulated in plant or animal tissues; 3. The pesticide is intended for use under conditions in which birds may be subjected to repeated or continued exposure to it; or 4. Other test information indicates reproduction may be affected adversely. The test also specifies the use of one aquatic and one upland species, as in the dietary LC50 test. It requires a period of exposure prior to egg-laying, and provides data on a longer term of exposure than the dietary LC50 as well as effects of the substance on the reproduction of adult birds and survival of the young. A short-term dietary LC50 test has been proposed for small mammals (McCann gt 31., 1981), based_on the avian dietary LC50 test. This test substitutes immature rats (90-120 9) for hatchling birds, using the same experimental protocol as that described for the avian test, with the exception that the withdrawal period for the rats was 9 days, and the housing was appropriate for rats. Dietary tests were conducted on 21 pesticides, and dietary LC503 were calculated for 17 of them. Where duplicate tests were run on a pesticide, good reprodu- cibility was usually obtained. A protocol is specified by the Canadian Environmental Protection Service (1975) for testing the effects of chemicals on mink reproduction. This protocol describes normal repro- duction in the mink, housing 'and lighting conditions adequate for conducting a reprOduction test with mink, two diets suit- able for mink and methods for incorporating a test chemical into the diet, and an experimental design utilizing 5 males and 15 females in each of 2 or 3 test concentrations plus a similarly-sized control group. Parameters measured in this test include adult mortality, weight of adults at the beginning and end of the test, feed consumption per week, number mated, number whelped, length of gestation, number of males with motile sperm, number of implantation sites (which persist in mink well past parturition, and can be counted at 3 weeks, the suggested time of termination), number of resorbed embryos, number and weight of newborn (kits) born alive and dead, deformed kits, number and weight of kits alive at l, 2, and 3 weeks post-partum, sex ratio of kits, organ weights of adults at 3 weeks post-partum, and pathology. The length of exposure to the test chemical is not specified in this protocol. It is important to note that, when conducting a toxico- logical test with a wildlife species instead of standard, often highly inbred laboratory test species, certain inherent factors will contribute to a greater degree of variability in the test. Age, sex, species and strain, and factors associated with unnatural conditions, such as diet, social (or anti- social) interactions, and stress are all known to influence toxicological test results (Hurni, 1970). The age of the animal at the beginning of a test can be very important. Hill and Camardese (1981) noted an increase in the subacute dietary LC50 with increasing age of the test birds in 60 subacute tests with four different classes of pesticides. It has also been shown that carriers can affect the dietary toxicity of a substance in the avian LC50 test. Gile gt 31. (1983) noted carrier-related differences in the toxicities of some pesti- cides tested with distilled water, corn oil, propylene glycol, or carboxymethylcellulose as the carrier. Carrier-related differences in feed consumption and body weight gain were also noted in this study. 11 Test Substances Sodium Monofluoroacetate (Compound 1080) Compound 1080 (analytical reagent; Pfaltz & Bauer, Stamford, CT) is a white, odorless, nearly tasteless hygroscopic powder, soluble in water and nearly insoluble in organic solvents. It has no vapor pressure. Ithas a molecular weight of 100.03. Its molecular formula and structure are: 4’0 C2H202FNa; FCH2C\\ 0‘Na+ Compound 1080 is a potent rodenticide and predacide approved for pest control in several countries and recently re-approved for coyote control in the United States. In the past in the U.S., it has been used for coyote, gOpher, ground squirrel, prairie dog, and field mouse control. However, it's extreme toxicity to non-target species and the possibi- lity of secondary poisoning led to its restriction to rodent control by licensed exterminators until its recent re-registra- tion for coyote control. It was first synthesized by Swarts (1896). Its synonyms and trade names include: SMFA; Gifblaar poison; ten-eighty; "1080"; sodium f1uoroacetate;and fratol. During World War II, when the major sources of red squill, thallium, and strychnine were cut off, intense research efforts on both sides of the Atlantic were directed at finding an effective rodenticide to replace those in short supply. SMFA was among ten compounds selected for study by the Fish and Wildlife Service, and was given the laboratory acquisition number 1080 (hence the name Compound 1080). The results with 12 laboratory rats were encouraging, and extensive field trials further showed SMFA's potential for pest animal control. Independently of this wartime research, Marais (1944) identi- fied monofluoroacetic acid (FCH2COOH) as the toxic agent of the Gifblaar plant, Dichapetalum cymosum (Hook), of South Africa, which had long been associated with livestock poison- ings in South Africa. Monofluoroacetic acid has since been identified as the toxic agent in certain plants of the genera Palicourea of South America and Gastrolobium, Oxylobium, and Acacia of Australia (Hall, 1972). Monofluoroacetate is itself an innocuous compound. Peters (1952) showed that SMFA must first be converted to fluoro- citrate (which he termed the "lethal synthesis") in mitochond- ria before a toxic response will be elicited. This conversion occurs in the Krebs cycle, similar to the synthesis of citrate from acetic acid. Monofluoroacetate is first converted to monofluoroacetic acid, which, in the presence of adenosine triphosphate (ATP), combines with coenzyme A (CoA) to form fluoroacetyl-CoA. Fluoroacetyl-CoA then combines with oxalo- acetate and water in the presence of "condensing enzyme" to form fluorocitrate. Whereas citrate continues through the Krebs cycle, fluorocitrate does not, constituting a block in the cycle which leads to a buildup of citrate (Buffa et 31., 1973). Fluorocitrate specifically inhibits aconitase (Morrison and Peters, 1954), which is responsible for cataly- zing the conversion of citrate to cis-aconitate, and succinate dehydrogenase (Fanshier gt g1., 1964), which is responsible 13 for catalyzing the conversion of succinate to fumarate. The resulting accumulation of citrate leads to interference with cellular respiration, permeability barriers, and energy production, and ultimately cellular function. Citrate tends to accumulate in cells of metabolically active tissues, such as kidneys, liver, and testes (Buffa and Peters, 1950). For example, Buffa and Peters (1949) found levels of citrate up to 70 times normal in kidneys from rats treated with SMFA. Eventually, the cellular disruptions caused by fluoro- citrate cause organ or organ system disruptions. There is a latent period of from k to 2% hours, during which vomiting usually occurs. This latent period is a product of the "lethal synthesis", since time is required for: (a) hydroly- sis of SMFA to monofluoroacetic acid; (b) synthesis of fluorocitrate from monofluoroacetic acid; and (c) fluorocitrate to disrupt cellular functions enough to produce gross clinical signs. When clinical signs begin, the onset is acute and the course rapid. The clinical signs of SMFA poisoning vary markedly among species. Man, monkeys, horses, and rabbits suffer cardiac arrhythmias and ventricular fibrilation, while canids suffer intermittent excitation and depression of the central nervous system (CNS), with death ultimately due to convulsions or subsequent respiratory paralysis. Animals that die from SMFA poisoning develop rigor mortis very rapidly, with the limbs usually rigidly extended (Edwards, 1977). In general, herbi- vores die as a result of cardiac disorders, carnivores as a 14 result of CNS disorders, and omnivores as a result of both types of disorders. Cold-blooded animals are less sensitive than warm-blooded ones to SMFA (Chenoweth, 1949). The LD50 for SMFA also varies markedly among species, ranging from 0.05 mg/kg for the dog (Buch gt g1., 1976) to over 500 mg/kg for the South African clawed toad (Chenoweth, 1949). The LD50 does not appear to be markedly affected by age in the cow (Robinson, 1970), but young mallard ducks appear to be approximately one-half as sensitive as older ducks (Hudson gt g1., 1972). The estimated LDso for man ranges from 0.7 mg/kg (Kaye, 1970) to 10 mg/kg (Harrison gt gt., 1952). The LD50 for mink has been reported to be approximately 1 mg/kg (Robinson, 1953), and 1.41 mg/kg for ferrets (Tucker and Crabtree, 1970). SMFA may be absorbed through the gastrointestinal tract, respiratory tract, mucous membranes, and abraded skin, but not through intact skin (Edwards, 1977). Its toxicity is approxi- mately the same regardless of the route of administration (Chenoweth and Gilman, 1946; Ward and Spencer, 1947), and the oral toxicity is also approximately the same whether the carrier is water, meat, grain, oil, gum acacia suspension, or gelatin capsule (Atzert, 1971). There are no pathognomonic lesions associated with SMFA poisoning. There is a generalized cyanosis of mucous membranes and other tissues, and the liver and kidneys usually exhibit marked congestion. The heart may be flaccid and may show subepicardial hemorrhages. Hyperglycemia and elevated citrate 15 levels, especially in the kidneys, will usually be present (Edwards, 1977). Several secondary biochemical effects have been reported with SMFA poisoning. The accumulation of citrate has been shown to inhibit phosphofructokinase (PFK) in the heart both it ztttg_(Bowman, 1964) and 12.21X2 (Williamson gt gt., 1964). The inhibition of PFK causes a decrease in glucose metabolism, resulting in a further decrease in energy production (Bowman, 1964), and a concurrent increase in free glucose, resulting in hyperglycemia (Elliott and Phillips, 1954). Ketonemia has also been reported (Engel gt g1., 1954). Roy (Shapira) gt g1. (1980) have found a significant decrease in ionized calcium in the blood of SMFA-poisoned cats (Citrate is a potent chela- tor of calcium ions), which contributes to the pathogenesis of SMFA poisoning. Repeated sublethal doses of monofluoroacetate can increase the tolerance to a subsequent challenging dose in some species. Golden eagles (Atzert, 1971), rats (Miller and Phillips, 1955), mice (Quin and Clark, 1947), and rhesus monkeys (Chenoweth, 1949) have been shown to acquire-a toler- ance for monofluoroacetate, although only for slightly increased challenging doses and for a limited period of time. Conver— sely, repeated sublethal doses of SMFA have accumulated in some species until they reached lethal levels. Dogs, guinea pigs (Foss, 1948), rabbits (Rowley, 1963), and mallards (Tucker and Crabtree, 1970) have exhibited this response. 16 SMFA has been shown to cause testicular atrophy, decreased testicular ATP concentration, and seminiferous tubular atrophy in rats at doses of 6.6 and 20 ppm in their drinking water for 7 days, and altered appearance and decreased numbers of spermatids even at the lowest dose of 2.2 ppm (Sullivan gt g1., 1979). Secondary poisoning of dogs eating the carcasses of ani- mals poisoned by SMFA has been reported (Meldrum gt gt., 1957L Such instances are quite rare, since the poison has been diluted. Of greater concern is the ingestion of the vomitus of an animal poisoned by SMFA by another animal, in which case a toxic dose may be consumed. O-cresol O-cresol (analytical reagent; Pfaltz & Bauer, Stamford, CT) is a light to dark brown volatile solid or liquid (mp = 30°C). It is slightly soluble in water (2.9 mg/l at 46°) and is miscible with alcohols, chloroform, and ether. Other 20 4 flash point = 81-83°; Vp = l at 36.6°. It has a molecular physical characteristics include: bp = l9l-l92°; d = 1.047; weight of 108.14. Its molecular formula and structure are: OH CH3 C7H80; O-cresol is used as a disinfectant, like phenol, often in a mixture with m-cresol and p-cresol known as cresyl, as a solvent, and as an intermediate in other chemical processes. A major use of o-cresol is in the manufacture of the herbicides 17 dinitro-o-cresol (DNOC) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) (Klapproth, 1976). The cresols are derived from petroleum or coal tar acids (McNeil, 1965), and they are also components of the phenolic wastes of certain manufacturing processes, and as such pose a threat to aquatic species both directly as a toxicant and indirectly as a behavior modifier (Buikema gt g1., 1979). Synonyms of o-cresol include: 2- methylphenol; o-cresylic acid; 2-cresol; 0-hydroxytoluene;and o-oxytoluene. One of the earliest uses of o-cresol was in the disinfec- tant Lysol. Lysol originally contained phenol as its active ingredient when it was introduced in 1860, but a new formula- tion, introduced in 1872, contained the cresols (6-50%) in glycerin or saponified linseed oil. Cresol was removed from Lysol in the U.S. in 1951 and replaced by o-phenylphenol (U.S. Dept. of Health, Education, and Welfare, 1978). Attempts at suicide involving ingestion of 4-120 ml of the Lysol prepara- tion with cresol have revealed the signs of acute intoxication in humans, which include abdominal pain and cramps, vomiting, burning sensation of the mouth, throat, esophagus, and epiga- strium, cyanosis, unconsciousness, and respiratory failure (Isaacs, 1922). In animals,clinical signs of o-cresol intoxication include twitching, pupil contraction followed by dilation, some sali- vation, and marked dyspnea upon original exposure in all species tested. In addition, rats showed continued twitching 18 and uncoordinated movements until death. Cats eventually became lethargic or comatose before death, while rabbits behaved similar to cats, but also exhibited asphyxial convul- sions just before death. Signs and symptoms of o-cresol intoxication, in general, resemble those for phenol, but the convulsions are less severe while the signs of weakness and/or depth of coma are more severe (Deichmann and Witherup, 1944). The main effect of o-cresol exposure is CNS depression, and death often results from respiratory failure. The cresols are also corrosive and can cause chemical burns and dermatitis. Absorption through the skin is rapid (Sax, 1963). Exposure to cresol vapors can lead to inflammation of mucous membranes, nervous irritation, and kidney damage (Deichmann and Witherup, 1944). The cresols are readily metabolized, being conjugated in the body to form glucuronides and sulfates (Bakke and Scheline, 1970). Rat oral LD50 estimates include values of 121 mg/kg for male white rats (Bio Fax Techniques, 1969) and 1350 mg/kg (Deichmann and Witherup, 1944) and 1470 mg/kg (Uzhdavini gt__1., 1974) for unspecified strains of rats. The mouse oral LD50 has been reported to be 344 mg/kg (UZhdavini et gt., 1974). 0-cresol is as toxic by the dermal route as by the oral. Uzhdavini E£.El- (1974) have reported the rat dermal LD50 to be 620 mg/kg, while LD50 estimates for rabbits include values of 890 mg/kg (Vernot gt gt., 1977) and 1380 mg/kg (Bio Fax Techniques, 1969). The clinical signs reported by Bio Fax Techniques included skin irritation, 'IO hyperemia, convulsions, and tremors. The inhalation LC50 for mice has been reported to be 0.179 mg/l (= 179 mg/m3) (Uzhdavini _t _t., 1974), but the conditions reported in this study included a degree of saturation of the air such that dermal contact was also likely. O-cresol is much more toxic to aquatic species. Pickering and Henderson (1966) reported 96—hr LC508 of 20.78, 12.55-13.42, 23.25, and 18.85 mg/l for bluegills, fathead minnows, goldfish, and guppies, respectively. Concentrations much lower than these values may lead to avoidance behavior by fish and/or tainting of edible flesh (Buikema gt gt., 1979). Very few longer-term studies have been conducted with o- cresol. Savolainen (1979) reported no effect on the body weights of rats receiving 0.3 g/l o-cresol in their drinking water for 20 weeks. The ingested cumulative dose exceeded the acute oral LD50 by the fourth week in this study. Biochemical effects of this exposure on the brain were inconspicuous. The cresols have been shown to be tumor promoters in mouse-skin assays, causing an increase in papillomas but not in carcinomas (Boutwell and Bosch, 1959). Reproduction tests with o-cresol are lacking in the literature (U.S. Dept. of Health, Education, and Welfare, 1978). Workplace exposure to cresol has been regulated since 1952. The recommended Threshold Limit Value (TLV) was set at 5 ppm (American Conference of Governmental Industrial Hygienists, 1952). This value was expressed as a Time Weighted Average (TWA), being 22 mg/m3 for an 8-hour workday and 40-hour work- week, in 1956 (American Conference of Governmental Industrial Hygienists, 1956). A "Skin" notation was added in 1961, acknowledging the importance of dermal exposure in toxicity and the rapid absorption of cresol through the skin (American Conference of Governmental Industrial Hygienists, 1961). The 0.5. Dept. of Health, Education, and Welfare (1978) has proposed lowering the TWA to 10 mg/m3 for a lO—hour workday and 40-hour week, and has proposed labeling and precautionary measures for workers exposed to cresol. The TSCA Interagency Testing Committee has recommended that cresol be a priority substance to be tested for carcino- genicity, mutagenicity, and teratogenicity (Federal Register, October 12, 1977). Thiram Thiram (analytical reagent; Pfaltz & Bauer, Stamford, CT) is a fine light green powder, insoluble in water, slightly soluble in alcohol (< 0.2%), ether (< 0.2%), acetone (1.2%), and benzene (2.5%), and more soluble in chloroform. It has a melting point of 70° and a density of 1.30. It has a molecular weight of 240.44. Its molecular formula and struc- ture are: S S H H C6H12st4; (CH3)2N-C-S-S-C-N(CH3)2 Thiram, a dithiocarbamate, is used as a rubber accelerator and vulcanizer, as a seed disinfectant, as a fungicide, and as a bacteriostat in soap (it is the main ingredient of the anti- septic spray Nobecutan). It is also used, when suspended in 21 water, as a paint or spray on ornamental plants to prevent gnawing. It is prepared by the oxidation of sodium dimethyl- dithiocarbamate by hydrogen peroxide or iodine. Its synonyms and trade names include: tetramethylthioperoxydicarbonic diamide; bis (dimethylthiocarbamoyl) disulfide; bis (dimethyl- thiocarbamyl) disulfide; tetramethylthiuram disulfide; TMTD; Thiurad; Thiuram; Thiosan; Thylate; Tiuramyl; Thiuramyl; Puralin; Fernasan; Nomersan; Rezifilm; Pomarsol; Tersan; Tuads; Tulisan; Arasan; TTD; and disulfuram. In acute human intoxication, thiram causes liver and kidney injury, and can cause brain damage (Sax, 1963). It is an irritant of the eye, nose, and throat, and can cause derma- titis. Sub-acute exposure can cause nausea, vomiting, diarrhea, hyperexcitability, weakness, or ataxia (Anon., 1975). The toxicity is greater in the presence of fats, oils, and fat solvents (Merck Index, 1976). The mammalian acute oral LD50 ranges from 350 mg/kg for rabbits (Matthiaschk, 1973) to 4000 mg/kg for male rats and mice (Lee gt g1., 1978). Sex differences have been reported for rats, with an LD50 of 4000 mg/kg for male CD rats and 1900 mg/kg for females (Lee gt g1., 1978). Strain differences are also apparent for rats, since Gaines (1969) has reported an oral LD50 of 620-640 mg/kg for male and female Sherman rats. Manifestations of acute exposure in animals include severe effects on the nervous system, with ataxia, incoordination (especially of the lower extremities), and clonic convulsions being reported (Fishbein, 1976). 22 In subacute exposure, whether by diet or by mouth, thiram has repeatedly been shown to significantly decrease feed con- sumption and body weight gain. Rats (Lee gt gt., 1978; Lowy gt g1., 1980), chickens (Waibel gt gt., 1957; Rasul and Howell, 1974), turkeys,and geese (Waibel gt g1., 1957) have been reported to reduce feed consumption upon exposure to 2 225 mg/kg (in diet), 178 mg/kg (po), 2 400 mg/kg (in diet), and 2 150 mg/kg (in diet), respectively. Further manifestations of toxicity in these studies included: tubular degeneration of the testes with atypical spermatids in the epididymis of rats fed 2500 ppm for 13 weeks (Lee gt g1., 1978); decreased weight of kidneys, epididymal and perirenal fat pads, testes, and seminal vesicles, and increased weight of livers of rats fed 2 225 ppm for 29 days (Lowy gt g1., 1979; 1980); abnormal gait in four of seven chicks due to enlarged epiphyses of the long bones, and degenerative changes in the epithelium of the seminiferous tubules, at a dose of 178 mg/kg po for 18 weeks, and mortality of five of five adults within 3 weeks exposure to the same dose (Rasul and Howell, 1974); and leg weakness and enlargement of the hock joint in chicks fed 2=150 ppm, poults fed 400 or 800 ppm, and goslings fed 400 or 800 ppm for 3 weeks (Waibel gt g1., 1957). Chronic and/or reproduction studies have revealed other adverse effects of exposure to thiram. Lee and Peters (1976) found further evidence of neurotoxicity in rats. They have reported demyelination and degeneration of peripheral nerves, with secondary degeneration of muscle, and behavioral deficiencies in gait and jump tests, and hyperactivity in an 80-week chronic study with rats fed 100, 400, or 1000 ppm thiram in the diet. They also found decreased weight gain in males at all doses and females at 400 and 1000 ppm, ataxia in 8 of 24 females fed 1000 ppm, alopecia in some animals of both sexes at 1000 ppm, and increased weights of thyroid and testes in high-dose males and liver, spleen, kidney, thyroid, ovary, and brain in high-dose females. The spleen weights of females fed the 400 ppm diet were also increased. No changes in blood chemistry were reported in this study. Adverse effects on several reproductive indices have been reported following exposure to thiram. Robens (1969) noted increased resorption of hamster fetuses at oral doses of 125 and 250 mg/kg, given from days 5-15 of gestation, and low birth weight and viability of surviving fetuses. Short gt gt. (1976) found, in addition to the tubular degeneration of testes of male rats already noted (Lee gt gt., 1978), that female rats fed 400 ppm thiram in the diet for 14 days prior to mating experienced a significant decrease in the number of implants and the number of pups per female. They also found that, at a dose of 2000 ppm, only 1 of 20 females mated and 5 of 20 had died by 4% weeks. The cause of the failure of these females to mate was determined to be a prolonging of the diestrus phase of the estrus cycle. Extension of the estrus cycle at the expense of the resting cycle has been reported for albino rats exposed to 3.8 mg thiram/m3 air for 5 hr/day, 5 days/wk, for 4.5 months (Davydova, 1973), and inhibition of ovulation has been reported in bobwhite quail at doses as low as 8.8 mg/kg/day (Wedig gt gt., 1968). In further studies, Short gt gt. (1976) reported only 33% survival of female rats given 200 mg/kg/day of thiram po during days 6-15 of gestation, decreased number of implants and 100% resorption of fetuses at 164 and 200 mg/kg, decreased number of pups/female at 136, 164, and 200 mg/kg, and decreased birth weight for pups in all treatment groups (40, 90, 136, 164, and 200 mg/kg). In a similar study with mice given 100 or 300 mg/kg/day po during days 6-14 of gestation, these authors reported 78% survival of females at the high dose, and no changes in weight gains of adults, litter sizes, incidence of resorption, or birth weight. In peri- and postnatal studies with rats fed 300 or 1000 ppm thiram in the diet from day 16 of gestation through day 21 of lactation, they found no effect at 300 ppm and decreases in Viability and growth at 1000 ppm. All offspring of dams fed 1000 ppm were dead by day 21 of lactation. A cross-fostering study conducted simultaneously determined that thiram is most toxic to the pups during the nursing period, suggesting that it can be transferred via the milk. Thiram has been shown to be teratogenic in hamsters (Robens, 1969), rats, and mice (Short gt gt., 1976). Major anomalies noted in hamsters were cranial abnormalities, fused ribs, and limb and tail abnormalities. Occasional heart anomalies and missing kidneys were also noted. The major anomaly found in rats and mice was hydrocephalus, and cardio- vascular defects were also noted in mice. The tumor incidence 25 in rats fed 100, 400, or 1000 ppm thiram in the diet for 80 weeks was found not to be different from controls (Lee gt gt., 1978). A dietary no-effect level of 48 ppm has been reported for thiram in the rat (WHO, 1965). This value has been confirmed by Lowy gt gt. (1979; 1980), who used a log-probit model to extrapolate a no-effect level of 38 ppm from a 29-day feeding study. They determined that the most sensitive parameters, in the rat, for measuring toxicitytnnxethe weights of the epididy- mal and perirenal fat pads, while the least sensitive parameter was kidney weight. Aroclor 1254 Aroclor 1254 (electrical grade, lot #KB-05-612; Monsanto Co., St. Louis, MO) is a clear to yellowish viscous, oily liquid, nearly insoluble in water and soluble in organic solvents. It is a mixture of over 100 individual chloro- biphenyls, made by reacting biphenyl with chlorine to produce a product that is 54% chlorine by weight. By virtue of its high chlorine content, Aroclor 1254 is virtually non-flammable. Aroclor mixtures have variable molecular weights, since they are mixtures of many congeners, and are usually designated by the percent chlorine in the mixture (i.e. Aroclor 1254 contains 54% chlorine). Its molecular formula and structure are: C12HlO"nCln7Cl <3) <‘> C1 26 Currently, Aroclor 1254 is restricted to use in existing electrical transformers as an insulating fluid, and is being phased out. Its manufacture is prohibited in the 0.8. Due to its excellent heat-resistance, it has been used widely in the past for many applications requiring non-flammable liquids, including electrical transformers and capacitors, as a heat exchange medium, as plasticizers in paints, as hydraulic fluids, and for many other uses. Due to its widespread use over a long period of time (over 40 years), the PCBs have become nearly ubiquitous in the environment (Peakall, 1975). Its synonyms and trade names include: PCBs, polychlorobi- phenyls; Kanechlor, Clophen, and Phenochlor. There are numerous theories of the mode of action of PCB toxicity. It has been shown to cause liver enlargement and induction of microsomal enzymes in almost all species tested. Decreased feed consumption and severe loss of body weight (sometimes called the "Wasting Syndrome") has also been associated with acute or subacute PCB exposure in most species. The nature of the toxicity of PCBs is still an area of intense investigation. Clinical signs of PCB intoxication are often species-specific, and can include inanition, weight loss, liver enlargement, edema, hydropericardium and/or ascites, chloracne, hyperkeratinization, gastric ulcers, and lethargy or unconsciousness. The PCBs have been the subject of numerous reviews, monographs, and symposia, and the reader is referred to Hutzinger gt gt. (1974) and to Kimbrough (1974; 1980). Nicholson and Moore (1979), and Poland and Knutson (1982) for comprehensive reviews of the chemistry and biology, respec- tively, of these compounds. This review will focus mainly on the effects of PCBs on mink and ferrets. Mink and ferrets appear to be among the most sensitive species to PCB toxicity. The first indication of this sensi- tivity to PCBs came in the mid-19605, when mink ranchers began to note reproductive problems with mink fed diets containing Great Lakes fish (Hartsough, 1965). Research by Ringer, Aulerich, and associates in the early 19705 ultimately isolated PCBS as the causative factor in these reproductive problems (Aulerich gt 1., 1970; Aulerich gt gt., 1971; Ringer gt gt., 1972; Aulerich gt_gt,, 1973). Further research by this laboratory showed that consumption of 2 or more ppm of Aroclor 1254 in the daily diet for 8 months (Aulerich and Ringer, 1977) or 5 or more ppm of Aroclor 1242 for 9 months (Bleavins gt gt., 1980) resulted in severe reproductive problems in mink. Recent research (Hornshaw gt_gt,, 1983) has shown that PCBS which have been "metabolized" by another animal before being fed to mink are even more detrimental to mink reproduction, a response also noted by Platonow and Karstad (1973). The PCBS are only slightly toxic to mink on an acute oral basis. The acute oral LDsos for Aroclors 1221, 1242, and 1254 are approximately 1, 3, and 4 g/kg (Aulerich and Ringer, 1977), and are slightly less than those reported for the rat (4-10 g/kg: Kimbrough gt gt., 1978). On a subacute or chro- nic basis, however, the PCBs are highly toxic to mink. Con- centrations of Aroclors 1242 and 1254 only slightly higher than those which caused reproductive problems resulted in outright mortality in mink during the 8-9 months of these studies,1eading to dietary LC50 estimates of 8.60 and 6.65 ppm for these two Aroclors (Ringer gt gt., 1981). Clinical signs of toxicity noted were: enlarged livers, as found in most species following exposure to PCBS, inanition, reduced growth rate, enlarged kidneys, and hemorrhagic gastric ulcers, which have also been observed in monkeys (Allen and Norback, 1973) and swine (Hansen gt gt., 1975). A further indication of the extreme sensitivity of mink to certain components of the Aroclor mixtures is the result of a recent study in which 50% of the female mink fed a diet containing 50 ppb of the PCB congener 3,4,5,3',4',5'-hexachlorobiphenyl died during 6 month's exposure (unpublished research, Michigan State Univer- sity). The ferret is also highly sensitive to PCB toxicity, although not as sensitive as the mink. Studies in which the two species have been tested at comparable concentrations of PCB mixtures have shown the ferret to be 2—4 times less sensitive than the mink. Ferrets fed up to 20 ppm of Aroclor 1242 in the diet survived 9 month's exposure, whereas 15 of 15 mink fed this same diet died during 9 month's exposure. Mink mortality'was noted at concentrations as low as 5 ppm in this study (Bleavins gt gt., 1980). Similarly, ferrets fed 30% Great Lakes fish in the diet experienced no reproductive problems, whereas mink suffered total reproductive failure at this level (Ringer, 1983). Several subacute or chronic dietary studies with PCBS with other species further illustrate the sensitivity of mink and ferrets to PCBS. 1000 ppm of Aroclor 1254 in the diet of 29 rats resulted in 50% mortality in an 8-month study (Kimbrough gt gt., 1972) and 100% mortality by 53 days in another study (Tucker and Crabtree, 1970). One of six rhesus monkeys fed 25 ppm of Aroclor 1248 for 2 months died after a 2-month withdrawal period, with a total PCB intake of approximately 400 mg (Allen, 1975). Five of five mice fed 10 ppm of 3,4,5,- 3',4',5'-hexachlorobiphenyl died after 36-47 days of exposure (Kimbrough gt gt., 1978), a dose 200 times greater than the concentration that caused 50% mortality in mink. MATERIALS AND METHODS At the beginning of each test a log book was kept, in which the name(s), reason(s) for entry, and time(s) of entry and departure of all personnel entering the test rooms were recorded. Also, the following were posted at the entrance to each test room: name(s), address(es), and telephone number(s) of person(s) to contact in case of emergency; compound(s) and concentrations being tested; and appropriate warning labels. The following materials and methods were used in four experiments (I. Compound 1080; II. O-cresol; III. Thiram; and IV. Aroclor 1254), unless specifically noted in the text. Test Animals The species used were the ranch mink (Mustela vison) and the European ferret (Mt putorius furo). The mink were the standard dark color variety, and the ferrets were the agouti variety. Both species were from stock raised at the Michigan 30 state University Experimental Fur Farm. All animals were immunized (at approximately 8 weeks of age) against botulism, virus enteritis, and canine distemper. Each animal was assigned a unique identification number. Only alert, active, injury-free animals were used in the tests. Housing Test animals were housed indoors (see Figure 1) in two adjoining 14.0 x 6.7 m rooms, with a maximum of 64 animals housed per room. Each animal was kept in a 61 (W) x 76 (L) x 46 (H) cm cage (see Figure 2) constructed of 2.54 x 2.54 cm galvanized steel welded wire on the front, back, and top, with the bottom wire being Vinyl coated. Solid fiberglass dividers separated adjacent cages, to prevent cross-contamina- tion and incidental contact between the animals. The cages were suspended 61 cm above the flOor. Each cage was provided with a 15.2 x 22.9 cm feed grid on the top of the cage and an aluminum water cup attached to the front. For reproduction tests, all cages of female animals were equipped with 1.27 x 1.27 cm mesh vinyl-coated false bottoms prior to whelping, to prevent the kits from falling through the bottom of the cages. The wire fabric used in the front and back of the cages contained a "Kit Guard", which consists of a 1.27 x 2.54 cm mesh on the lower 10.2 cm of the fabric, to prevent the newborn from falling out of the cage. All cages of female animals were also equipped prior to whelping with 38.1 (L) x 27.9 (W) x 26.7 (H) cm wooden nest boxes. The nest boxes were partially filled with a sugar cane product used as nesting 31 FIGURE 1. Housing for test animals. 32 FIGURE 2. Typical cage for test animals. 33 material. Wood shavings were spread below the cages before (the start of all trials and were changed monthly. The walls and ceilings of the test rooms were painted with an epoxy- based primer coat and an oil-based final coat, which provided an easily washable surface. The concrete floors were untreated. Each room was equipped with a thermostatically controlled 60 AMP, 250 VAC, 15 h.p. heater-blower system to maintain room temperature above freezing to facilitate estimation of feed consumption. During warm months, the temperature in the rooms was allowed to approximate ambient air temperature, and ventilation was provided by an exhaust fan and ceiling vents. Lighting was provided by four 200 watt incandescent bulbs per room, and was controlled by a timer to approximate ambient light/dark schedules. Fifteen minutes of light were added to the time of local sunrise and sunset to approximate twilight. The light/dark settings were changed weekly during reproduction tests and were held constant at the schedule in effect during the last week of the acclimation period for the LC50 test. All cages, nest boxes, walls, and floors were thoroughly cleaned with a high pressure sprayer after each test. m_et The basal diet (Table 1) was formulated to meet the nutrient requirements of the mink (National Research Council, 1982) and was comparable to diets used commercially. Studies have shown that this diet also supports adequate growth and repro- duction of the ferret (Bleavins gt gt., 1980). Feed ingredients were obtained locally, with the exception of the ocean fish 34 Table l. Composition and nutrient analysis of basal diet. Ingredient Percentage Commercial mink cereal1 l6.7 Whole chicken 20.0 Ocean fish scrap mix2 12.5 Beef tripe 6.7 Beef liver 3.3 Beef lungs 3.3 Beef trimmings 3.3 Cooked eggs 3.3 Added water 3 .9 l00.0 Nutrient Analysis3 Moisture 63.40 Protein 14.30 Fat 7.91 Ash 2.54 93.l5 ‘ xx-4o, xx Mink Foods, Thiensville, wt. 2 Consists of cod, haddock, and flounder; National Fur Foods, New Holstein, NI. 3 Analyzed by Rosner/Runyon Laboratories, Inc., Chicago, IL. 35 scraps and cereal. The feed ingredients were ground in a commercial meat grinder (except cereal), mixed in a commercial paddle-type mixer, and stored at -l8°C in metal containers. Feed and water were provided gg libitum. Any remaining feed was removed and discarded prior to feeding each day. Prior to the whelping period (April 15) and until the newborn were weaned (June 15), corn oil ("1% W:W) and supple- mental vitamin E (18.3 IU/kg) were added to the diets to pro- mote lactation and kit survival, as is standard practice in the mink industry. Reproduction Mating attempts were begun at the beginning of March for mink and the end of April for ferrets. In breeding the mink, each female was presented to a male and, if receptive, was allowed to mate. If not receptive, the female was removed and presented to a male 4 days later. Once a successful mating occurred (as verified by the presence of normal- appearing, motile spermatozoa in a vaginal smear taken just after copulation) the female was given the opportunity to mate a second time, either 8 days after the initial mating or the next day (if the first mating occurred late in the breeding season). In breeding the ferrets, females were presented to males when they were judged to be in estrus (determined by the extent of vulvar swelling) and left overnight. Vaginal smears were not taken from female ferrets, nor were they given the opportunity for an additional mating(s). 36 Mated females of both species were checked daily for newborn (kits) during the parturition (whelping) period (April 20-May 15 for mink, approximately 42 days after mating for ferrets). All kits (alive and dead) were counted and weighed on the day of birth and at 3 and 6 weeks of age. During the reproductive period undue handling and stress were avoided, including cessation of routine weighing and measurement of feed consumption, to maximize reproductive potential. Weights and Measurements During the definitive test, adult animals were weighed weekly (LC50 tests) or bi-weekly (every 2 weeks; reproduction tests) on a digital balance (Fisher Scientific Co., Model EWO-4010) to the nearest gram. Kits were weighed on a top- loading balance (Mettler Model P-1210) to the nearest tenth of a gram. In estimating feed consumption by mink or ferrets, several precautions were necessary. Since feed consumption can be adversely affected on a short-term basis by temperature, weather, and other factors, estimates were based on at least 2 consecutive days' consumption. These days were also days in which the animals were not handled (e.g. during weighing, moving, etc.), since handling can produce a temporary reduction in feed consumption. Since ranch-kept mink, and to a lesser extent ferrets, are known to attempt to store away or hide food, and to examine or "play" with objects (i.e. feed contai- ners) in their cages, certain measures were taken to ensure an accurate measurement of feed consumed. In these trials, 37 each cage was equipped with a "holder" for the cups in which the feed was presented. This holder consisted of a 7.5 cm by 33 cm length of cage material which was bent into a circle and fastened to the side and floor of the cage. Also, when feed consumption was measured, all cages (and nest boxes, during reproduction trials) and the wood shavings beneath them were examined for hidden, stored, or spilled feed. Where possible (i.e., if feed was stored inside a nest box or spilled in a discrete pile where it could be picked up without shavings) stored or spilled feed was returned to the feed cup. If this was not possible, the animal's consumption was not inclu- ded in the estimate for that group. To measure feed consumption, uniform feed cups were important. In these trials, #2 and #3 tuna cans (68 and 12 02.) were used for females and males, respectively. A cup was filled with a portion of feed in excess of the amount nor- mally consumed, weighed, and recorded to the nearest gram, and placed in the holder. At approximately the same time the next day, the cup was removed, weighed, and recorded to the nearest gram, emptied, and then the procedure was repeated. Average feed consumption was estimated on the basis of 2 con- secutive days' consumption. Feed consumption was measured weekly for LC50 tests and bi—weekly for reproduction tests. PRE-TEST PROCEDURE Acclimation Prior to the start of an LC50 test, the cages were divided randomly throughout the test room into groups of two or four cages, to hold the 10 animals (5 male, 5 female) per test group in sub-groups of 4, 4, and 2 animals (equal numbers of each sex). For the reproduction tests, the cages were divided into groups of 4, to accommodate the 16 animals (4 male, 12 female) per test group in sub-groups of 4 animals (1 male, 3 female). The animals used in a test were weighed, then randomly allocated to treatment groups (with the exception that litter- mates were not assigned to the same group), and were accli- mated to the test facilities for a minimum of 7 days, with most tests having an acclimation period of 14 days. Each animal was observed daily and weighed at weekly intervals until the beginning of the test. Feed consumption was measured at least once, and usually twice, during the acclimation per- iod. Any animal that died or exhibited sickness or reduced feed consumption was replaced. Dietary Concentrations For the LC50 tests, if a search of the literature failed to provide sufficient toxicological data to set dietary concentrations for the test substance, a range-finding trial was conducted in which the compound was administered by gavage to animals at several concentrations. The test compound was either dissolved or suspended in an innocuous vehicle (e.g. distilled water or corn oil) and serial dilutions of a stock ' solution were made so that the volume administered was equal for all animals. 39 The range-finding procedure varied slightly, depending on whether LD50 estimates were available from the literature. If LD50 estimates were found for other species, a dose (mg/kg of body weight) approximating the LD50 became the highest of a series of five concentrations, each one-half the previous concentration, administered to two animals (1 male, 1 female). If LD50 values were not found, the test substance was first 'administered to one animal at doses of l, 10, 100, or 1000 mg/kg of body weight to find a lethal concentration. If a lethal concentration was found, it became the median dose of a series of five concentrations, two concentrations being one-half and one-fourth of the median and two concentrations being two and four times greater than the median. These doses were admini- stered, as above, to two animals per dose. After dosing, all animals were observed for 2-3 hours for signs of intoxication and for vomiting. For either range-finding procedure, the approximate LD50 was the dose at which one or two animals died after an appropriate observation period (usually one week). Based on estimates of the L050 found in the literature or determined by range-finding techniques, the dietary concen— trations for the test substance were determined as follows: using the estimated LD50 and the estimate(s) of feed consump- tion during the acclimation period, the highest dietary concen- tration (in mg/kg of feed) was calculated such that an animal would consume a lethal dose in one day's feed, and four more concentrations were calculated, each being 1.8 times smaller than the previous concentration. If an estimate of 40 the LD50 was not determined, or if the animals vomited the oral dose during range-finding procedures, the highest dietary concentration was set at 5000 mg/kg (since concentrations higher than this are considered to be non—toxic), and four more concentrations were calculated as above. Finally, it was necessary to determine whether the animals would consume the feed dosed at the highest concentra- tion. One kg of the standard diet (enough to feed an animal for 3-5 days) was dosed with an appropriate weight of the compound or volume of the stock solution to yield a concentra- tion equal to the highest test concentration to be used. Two animals were then fed this diet to determine if they would reject or avoid the feed. (Due to time constraints, this procedure was not carried out in some of the trials, leading to problems in two of the trials). If it was determined that they would not eat the feed at this concentration, several lower concentrations were fed to different animals until a concentration was found which the animals would eat for 3-5 days. This then became the new highest concentration, and the four lower concentrations were calculated as before. The dietary concentrations of the test substance to be used in the reproduction tests were based on the results of the LC50 test performed previously with the test substance. This involved the determination of the maximum tolerated dose (MTD), which was the highest concentration of the substance found not to produce mortality or gross abnormalities in the LC50 trial. Since blood parameters and organ weights were 41 analyzed statistically, these factors were also examined to determine the MTD. The MTD then approximated the highest concentration for the reproduction test. Three concentrations plus a control were used, with each being one-fourth the previous concentration. After the dietary concentrations were set for the LC50 or reproductiOn test, enough feed to last each of the test groups approximately 30 days, based on feed consumption data from the acclimation period, was mixed. An appropriate amount of the test substance for the amount of feed mixed was either dissolved in an innocuous solvent (e.g. distilled water or corn oil), or dissolved in a volatile solvent (e.g. ace- tone, hexane), mixed with ground mink cereal, and evaporated to dryness. The resultant premix was then added to the basal diet and mixed thoroughly (the solvent alone was likewise added in the same manner as the compound to the control diet). A sample of each diet was frozen for subsequent residue analy- sis. The diets were placed in metal containers large enough to hold l-2 days feed and frozen at -18°C. In the case of volatile test substances, the containers were lined with sealable plastic bags. For reproduction tests, feed was mixed and frozen as needed throughout the trial. DEFINITIVE TEST LC50 Tests The prescribed length of the LC50 test was 28 days, with an optional withdrawal period (not more than 14 days) to be used if animals were still dying or exhibiting signs of 42 intoxication. For the LC50 test, the animals used were approxi- mately 6 months old (i 14 days) unless the test was designed specifically for younger animals. The use of 6-month old animals guarantees that the animals have attained the adult body size and that any body weight changes still occurring are due to fat deposition. Each group of 10 animals (5 males, 5 females) was randomly assigned to either the control group or one of the five dietary concentrations. Body weight and feed consumption were recorded, as described above. Each animal was observed at least once daily for signs of intoxication, abnormal behavior, or mortality. Mortalities were recorded, and gross examination of organs was performed as soon after death as possible. Weights of whole body, brain, heart, lungs (5 lobes only), liver, spleen, and kidneys (minus capsules) were recorded for all mortalities. Samples of these organs, as well as muscle and fat, were frozen for subsequent residue analysis. Upon termination of the test, a suitable volume of blood was taken from all survivors (2-3 ml, via toe clip) for analy- sis of blood parameters (HCT, RBC, WBC, Hb). The animals were then weighed and euthanized with C02 gas. C02 gas was used for terminal kills because, unlike decapitation or cervi- cal dislocation, bleeding does not occur, thus allowing blood to pool in organs. This allows comparison of organ weights of terminally-killed animals with mortalities during the test. Gross examination of organs was performed, and weights of brain, heart, lungs (5 lobes only), liver, spleen, and kidneys (minus capsules) were recorded. Samples of these organs, as well as muscle and fat, were frozen for residue analysis and preserved in 10% formalin for histopathological examination. The LC50 for the test substance, the slope of the dose- response curve, and the confidence limits for these values were determined according to the methods of Litchfield and Wilcoxon (1949). Blood parameters were analyzed via l-factor analysis of variance. Organ weights were transformed to per- centages of body and brain weights, and the body and transformed organ weights of male and female animals were then analyzed via 2-factor analysis of variance. Organ weights were trans- formed to percentage of brain weight to account for the possi- bility of a decrease in body weight not giving a corresponding decrease in organ weight. Since brain weight remains relatively constant regardless of changes in body weight, it was used as a benchmark against which to measure organ weight changes, relative to the organ weight: brain weight ratios of controls. Significant differences revealed by analysis of variance were then analyzed by Dunnett's method for comparison with a control (Gill, 1978). Reproduction Tests The prescribed length of the reproduction test was approxi- mately 23 weeks, divided into the following segments during which the animals were exposed to the test substance. 1. Pre-breeding period of 8 weeks, starting the beginning of January for mink and the middle of February for ferrets. 44 2. Breeding and whelping period of 9-12 weeks (depending on species), starting the beginning of March and lasting through mid-May for mink and starting the end of April and lasting through the end of June for ferrets. 3. Post-whelping period of 3 or 6 weeks. Animals used in the reproduction test were approximately 9 months old (:_14 days) at the beginning of the trial. Each group of 16 animals (4 males, 12 females) was randomly assigned to either the control group or one of the three dietary concen- trations. Body weight and feed consumption were recorded according to the methods described previously. All mating attempts were made within dietary groups, and no extraordinary methods (e.g. sedation, use of overaggressive males from ranch stock) were employed to breed uncooperative females. Use of first-year females may result in a limited number of females which will not breed under ordinary circumstances, or within the normal time range of the breeding season. If mortalities occurred, the animal was weighed and necropsy performed as soon as possible after death, with the necropsy report recorded. Upon termination of the test at 3 weeks post- partum or at weaning (6 weeks post-partum), the four males and four randomly chosen females were weighed, sampled for analysis of blood parameters, and euthanized with C02 gas. Gross examination of organs was performed and weights of brain, heart, lungs (S lobes only), liver, spleen, and kidneys (minus capsules) were recorded. Samples of organs, as well as muscle and fat, were collected logical analysis. Reproductive parameters were performance (see Table 2 for some values). The parameters measured dures used were: for residue and histopatho— calculated for the females' representative average and the statistical proce- 1. Percent whelped and not whelpeda 2. Live kits/female whelpedb 3. Total kits/female whelpedb 4. Length of gestationb 5. Average birth weightb 6. Average litter weightb 7. Percent kit survival to 3 weeksa 8. Average 3 week weightb a Analyzed by contingency table. then tested by Bonferroni's Chi- Significant differences were square test (Gill, 1978). Analyzed by l-factor analysis of variance. Significant differences were then tested by Dunnett's method for compari- son with a control (Gill, 1978). When subtle reproductive effectswwere observed (i.e., when statistical analysis yielded levels of significance of 0.1 < a < 0.2 for two or more reproductive parameters),it was sometimes advisable to combine these parameters according to the method of Brown (1975) to give a more accurate indication of the effect of a compound on reproduction. Any kit 46 mu_to_;ooa>; Eswuom 47 m muqu>th apm page _MH:me_tmaxm to; «En: month m xuaum mcwtmumowummotu .mcm~:maotopzumxmz q ausum cowuuzuotamg .m:m~=mnoto~;omxm: m mswt_=m_u E~t=_gu_s;smswtumh N mumpmomotozpwocoe Ezwuom _ mmm_ .guwtm.:< new ocmuw_oamz m.mo_ ~.mm «.mm om.m oo.o “.mm PF\F_ c.0omz Nwmp.uml.mm cuptmpz< o.mo_ 0.0“ m.m¢ oo.m __.m w._w _P\P_ mconmm mmm_..flu.mw ;u_twpz< N.mo_ o.¢~ o.m¢ ¢N.m om.m o.oo_ NF\NP mxmum>rms mmm_ .mcw>mm_m o.ma N.om m.em eo.m o_.o o.om om\cm emumo: mwmp .m:_>mm_m m.mm m._m o.¢v o_.m om.¢ o.oo_ o_\o_ mpumu: xvzpm mwnp m.om o.mm w.mm _m.m om.m m.mm N_\N_ Namtwcp muzum mwgp o._¢ o.mm m.me Fm.m om.v m.mw Np\m Fommto-o muzpm mwzp ©.wm m.¢m m.um oe.m mm.m o.m~ NF\N_ pomop mucmtmwmm Amw .x: m mxz m o» Amv .umz Amy .umz uma_m;3_ weapon: Pmuou ucaoasou .u 3 p_x _m>w>t:m tmuuwp notwn 0\mu_x N \umpoe mmmtm>< uwx & mmmtw>< mmmgm>< m>_4 mm_m2mm .mvcaanoo Pmtm>mm not xcwe mcw>Po>cw m_m_tu m>wuo:SOLamt sotw mazotm —otu:oo mo mLmHmEmtma m>_uu:u0tqmm .N m_nmp abnormalities were recorded. Blood parameters were analyzed via l-factor analysis of variance. Organ weights were trans- formed to percentages of body and brain weights, and the body and transformed organ weights of male and female animals were then analyzed via 2-factor analysis of variance. Significant differences revealed by analysis of variance were then analyzed by Dunnett's method for comparison with a control (Gill, 1978). RESULTS AND DISCUSS ION The following tests were conducted with the test substances: Experiment I. Compound 1080 - LC50 tests with mink and young and old ferrets; reproduction test with mink; Experiment II. O-cresol - LC50 tests with mink and ferrets; reproduction test with mink; Experiment III. Thiram - LC50 tests with mink and ferrets; reproduction tests with mink and ferrets; Experiment IV. Aroclor 1254 - LC50 tests with young and old mink. All tests followed the procedures outlined above, unless specifically noted. The results of each individual experiment will be discussed separately. A discussion of the protocols develOped from these experiments will follow. 48 Experiment I - Results Mink LC50 Test: Since a literature search yielded only an approximate LD50 for mink of around 1 mg/kg (Robinson, 1953), it was decided to perform a range-finding study. From the results, the LD50 was estimated to be 0.25 mg/kg of body weight (Table 3). Based on the results, dietary concentrations of 0, 0.50, 0.90, 1.62, 2.90, and 5.25 ppm were chosen. Distilled water was the carrier used. The nominal dietary concentrations and those determined analytically were: 0 ppm - 0 ppm 0.50 ppm - 0.51 ppm 0.90 ppm - 0.64 ppm 1.62 ppm - 1.31 ppm 2.90 ppm - 2.10 ppm 5.25 ppm - 3.93 ppm (Analysis performed by Howard H. Casper, Ph.D., Dept. of Veteri- nary Science, North Dakota State University, Fargo, ND 58105). It should be noted that the feed samples saved for analysis were thawed and refrozen several times during the development of the analytical procedure and this may have resulted in the loss of some of the 1080 residues in the feed. A 13-day acclimation period was begun on 27 October, 1981. The 28-day LC50 trial began on 9 November, 1981 and ended on 7 December, 1981. Signs of intoxication were first noted on day 5, when 2 animals fed the 5.25 ppm diet exhibited difficulty Tab1e 3. Resu1ts of range-finding study with mink exposed to sodium monof1uoroacetate (Compound 1080) by gavage. Dose Died/ (mg/kg) tota1 Comments 10.0 2/2 1.00 2/2 0.50 2/2 0.25 1/2 3 days to death 0.10 0/2 No effects observed 50 econ—zopau Jamezfita ..— ..zoz... 3.2.2... 2.: be 82...... c... .8 325 $5.... 232. 3.8 a 63.8.. 532533 a... so: $9.38... .... «9.29 232. 2.22.5.3 .2829: "waves“... .8: 3:32.. _ goo: u .5 x :o..:s=m=ou a..~c sot. co..a2=mcoo eczoaeou apxmmz .co..otu:ou=cu accum.= x =o..ae=m:cu some caugm>u sot» wage—=upmu =c.a=s=mcou vasoQEou a..oo a ...czaaamcou 3.3.. 2.3.53.8“. 25 1:. cafe: 2: .8 33.. 529523 3...“. a .m.o eo.— mm.~ Ne.— m~.— c Ax3\aevco_.as=m=ou eczoasou --- e~.o once .~.a m..o c Ae\aeV:o—.;e=mcou ceaanou --- 3 8 3 z ....N .23 .52....528 B: .3: 32:- .....- 93:- ~53- --- ...... .3 $55 232. a... $6 3.. ...w 3.. 5...: o 3395:..3..=5m=8 3:253 --- c~.o om.c n~.c mp.o c Av\oa~.::.=s=m:ou eczcaeou --- 8 z: ... 3 E 23 5:55.28 .5: . Us :52- 98- no..- .....- ...... .3 .326 23... 22 mm. . .3. N m: 2.. 2.. o 3:33 5:...528 .2895 --- 2... Na... .3... 2... a .253 8:55.28 2588 --.. 2... a. S. .... a... .23 539.528 2...; 68:23- n... - new- 2.... --- mg? . .3 2.56 232. 8.. nm.o .m._ mn._ mo.— e—.— c Ax:\9=w:o.aasam:ou ncaogsou 2... 2... 2... ...... c .29.. 52......28 232.8 --- 8. SN 2. .... EN .23 8.2.528 28 689.2 - NS- ..8. v.8- ...: .3 855 232. a... 3.. 8... a... 2... 3... a :iSw 53.2.5958 289:8 --- Np... 3.: S... 36 a 3B... 5.3.2.523 3.59:3 a... New 8. 2... :n .23 82.528 .5: 68:98? 3? ...... as? --- 3:. .3 .956 23.... ...... --- c c o c o 32E... 5.39.523 258:5... --- o o o o c Au\aa =c_uae=m=ou acaeeaou --- 8.. EN SN 3 as .33 529.528 8: 83:3... 9...... ...... .....- --- to? .3 as... 232. s m>..o.=e=u e n N. _ cc.uas_.uu< cutaway: “and. msomx e xmmz Jam: gum: gum: guamsmgum =o.uugucoucou .22. .... .3 .8... 2398: m.oouuooto=..o:os e:_uom be mac—aogaeouzou m:o.te> em. x=_s an unsamcau nucaoasou ecu sumo» can «coca;u “gm—o: swan wanto>¢ .v o_a~» Table 5. Initial and final body weights of male (M) and female (F) mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 28 days. Concentration Initial Final Change (ppm) Sex n wgt. (9) “qt. (9) (9) 0 M 5 1617.4 1899.8 +282.4 F 5 904.4 1108.4 +204.0 0.50 M 5 1540.4 1894.4 +354.0 F 5 882.2 935.8 + 53.6 0.90 M 5 1452.2 1487.8** + 35.6 F _5 1011.4 896.2 -115.2* 1.62 M 5 1490.4 1285.8** -204.6** F 5 956.6 840.8 -115.8* 2.90 M 5 1448.8 835.0** -613.8** F 5 1006.6 641.6** -365.0** 5 25 M 5 1474.4 929.6** -544.8** F 5 938.8 494.0** -444.8** Significantly different from control (P < .05). ** Significantly different from control (P S .01). 52 . . . . . . . . . . m~.m . N . om.~ ~m.. cm.o mo.o o 3113 £11 .wli ...N 1121131.? ...NlmCFILW1wfamv $1912-11F1c. a wk. a m e m ml. 2.5.: 98 age m:. a m.ce.ca do .oz :o.umc.:mu:ou ..mm. emu. smu-mw m o:.c=c .omo. neaoasoo. maauoumogoa..o=oe 5:.vom no. x=.a .o ccmuuma >u..u.toz .m m.nm. Table 7. Blood parameters of mink fed various concentrations of sodium mono- fluoroacetate (Compound 1080) for 28 days. Concentration RBC NBC Hb Hct (ppm) n (x105) (x103) (g/dl) W») 0 10 9.89a 19.9 23.2 53.0 :_0.189 :_ 5.56 :_0.90 :_1.94 0.50 10 9.55 23.3 22.3 54.3 :_0.554 :_11.21 :_0.97 :_2.99 0.90 10 9.61 13.4 22.7 53.7 :_0.633 :_10.51 :_1 12 :_2.72 1.62 10 9.63 22.3 21.8* 52.8 :_0.554 :_l4.90 :_l.l3 :_2.61 2.90 6 9.26 20.8 21.1** 51.0 :_0.737 :_10 73 + 1 04 :_3.18 5.25 l 9.62 6.6 21.3 50.5 i 0 :2 0 + 0 3: 0 a Mean :_S.D. * Significantly different from control (P g ** Significantly different from control (P s .01). 54 Table 8. Body and organ weights of male (M) and female (F) mink fed various concentrations of sodium mono- fluoroacetate (Compound 1080) for 28 days. Concentration Body Brain Liver Spleen Kidney Lung Heart Testes (me) wgt (g) (2 body) (3 body) (5 body) (5 body) (5 booy) (5 body) (2 body) 0 u 1899.8 0.51 4.28 0.24 0.53 0.57 0.54 0.04 Ea 1108.4 0.71 5.05 0.36 0.61 0.68 0.52 -- 0.50 M 1894.4 0.55 3.55 0.26 0.56 0.55 0.50 0 03 F 935.8 0.87 4.22 0.36 0.52 0.75 0.57 -- 0.90 M 1487.8** 0.68 3.99 0.28 0.60 0 72 0.55 0.04 E 896.2 0.90 “.99 0.30 0.62 0.82 0.57 -- 1.62 M 1285.8*‘ 0 83** 4.06 0.22 0.55 0.85 0.62 0.03 2 840.8 1 1* 4.02 0.33 0.66 0.75 0.66** -- 2.90 M 835.0** 1.21** 3.77 0.18 0.76** 1.01** 0.72** 0.05 E 641.6** 1.27** 4.58 0.32 0.76** 1.17** 0.78** -- 5.25 M 929.6** 1.17** 3.66 0.20 0.72** 1.74 0 70” 0 0‘ § 494.0** 1.64** 4.23 0.20** 0.87** 1.61 0.82** l-” (Organs expressed as a percent of brain weight) 0 M 839.8 48.0 104.4 112.4 106.7 9.0 F 714.7 50.1 86.0 96.5 74.4 -- c --- 49.0 -- -- -- -- 0.50 M 650.8* 47.9 103.2 101.1 91.8 4.9 F 486.9** 41.7 71.2 85.1 65.4 -- c --- 44.8 -- -- -- -- 0.90 M 602.8** 42.0 89.7 107.4 81.8** 6 7 s 448.1** 33.0 69.8* 91.9 63.6 -- C --- 37.5* -- -- -- -- 1.62 M 497.1** 27.2 67.5** 103.8 76.6** 3.3 F 399.2** 33.5 65.8** 74.0 65.5 ~- 0 --- 30.3** -- -- -- -- 2.90 M 321.3** 15.5 64.0** 84.0 59.9** 4.2 F 367.6** 26.8 60.6** 92.3 62.4 -- c --- 21.l** -- -- —- ~- 5.25 M 336.9** 19.3 63.9** 109.8 52.7** 3.6 F 263.1** 12.5 53.3** 92.9 50.4** -- C --- 15.9** -— -- -- -- a Sexes are combined where no significant difference (P < .05) was found between them. * Significantly different from control (P < .05). ii Significantly different from control (P s .01). 555 in moving their hind legs. (Feed consumption was affected earlier, since most of the animals fed the 5.25 ppm diet exhibited reduced feed consumption by day 3). Clinical signs included reduced feed consumption, incoordination or paralysis (especially of hindquarters), and unconsciousness. Body weight changes and feed and compound consumption are summarized in Tables 4 and 5. Initial body weights were not recorded, therefore, body weight changes have been calculated from the last week of the acclimation period. At the conclusion of the 28-day exposure period, 4 of 10 animals had died on the 2.90 ppm diet and 9 of 10 died on the 5.25 ppm diet. The mortality pattern is described in Table 6. The data yielded a LC50 of 3.2 ppm. ‘with a 95% confidence interval of 2.4 to 4.5 ppm, and a slope of 1.46, with a 95% confidence interval of 1.24 to 1.71. No gross lesions were noted at necropsy of mortalities during the test or at the terminal kill (8 December, 1981). The hematologic parameters, summarized in Table 7, showed signi- ficant decreases in hemoglobin content at 1.62 and 2.90 ppm. The transformed organ weights, summarized in Table 8, revealed significant effects on the liver, spleen, kidney, lungs, and heart. Ferret LC50 Tests: Based on a published LD50 value of 1.41 mg/kg of body weight for yearling domestic ferrets (Tucker and Crabtree, 1970), dietary concentrations of 0, 1.08, 1.94, and 3.50 mg/kg of feed were chosen for young, rapidly growing ferrets (based on their 56 average body weights during the last week of the acclimation period), and 0, 4.76, 8.56, and 15.40 mg/kg of feed for adult ferrets. Distilled water was the carrier used.- Actual con- centrations of Compound 1080 in the test diets have not been determined. A 7-day acclimation period was begun on.26 July, 1983. The 28-day LC50 trial began on 2 August, 1983 and ended on 30 August, 1983. The animals used were approximately 2% months old and > 1 year old for the tests with growing and mature animals, respectively, since one of the objectives of the trial was to compare growing and mature animals' responses in an LC50 test. One adult ferret on the 4.76 ppm diet died the fourth day of the test from enteritis (not related to effects of the compound) and was not included among the results of the trial. Also, one adult ferret in the control group showed signs of the same illness, losing over 100 g over the last week of the trial and was thus excluded from the results. Body weight changes and feed and compound consumption are summarized in Tables 9 and 10. Signs of intoxication were first noted on day 2 among the adult animals, when 2 animals fed the 15.40 ppm diet were observed to have lost coordination of their hindquarters. Reduced feed consumption was noted for some animals on all 3 treatment groups by day 2, also. Among the rapidly growing animals sign of intoxication were first noted on day 5, when a slight loss of coordination of the hindquarters was noted in 2 animals fed the 3.50 ppm diet. This group also showed a reduction in feed consumption A.a.=oov o~.o_ oe.m c—.v nc.v —~.~ --- sz\aev :o_.;§:m=ou cznoasou --- c~.c om.o no.c mm.c --- A=\aav :o_ua§:m:ou v::ccsou --- m- so— .m— ——_ ms Au\o. co_u:s:a:ou can; uxmvm.m-. a..~. c.c~ . o._m+ ~.cm . o.c~_+ Au. «mango usa.~= an.” —o.__ ~—.n ~o.~ ~_.n m—.~ --- Agzxoav co.uaszm=ou uconEcu --- mc.c sn.c mo.c pn.c --- .v\oev cowuqeamzou uczoqeou --- cMN ma. cmm am. am. .a\a. co.zas=.=ou some uxm.o.co~. ~.eq+ o._o + a.mm. m._~ + m.mn_+ Am. uacagu “£5.82 4a.. ~—.~ Nm.— _m.— cc.— n~._ --- Axsxaav =c_uaa:m:ou azaocsou --- m~.o o~.c e~.o m~.o --- A=\a5. =o_~aszm:3u acsoaeou --- .0N amw CNN an ax. .e\a. =o_zas=m=au sum. u.m.~.e_n+ s.mm. o.aa . ...k. m.ma + ..on.. Am. macagu ug=_m: so.— na- o o c o c Auz\asv =c_aa§:m:oo azaoasou --- c c c o c Acxasv :cmuzszmzou uczoae:u --- ~¢~ .xmw NNN c¢~ an. Ac\m. cozuaa=m=cu cool uxa.m.m_e. a.cm. c.om_. ...m. ..k... ~._~_. .9. oazozu ug¢.m= o mama» o>_uoe:—=u v n ~ _ covaoe_—uu< cutaway: A§;a. m4uoz v sum: xmaz “on: gum: guuosutum :o_aa;¢:uu:oo s=.uom uo «co..otd:oucou «DDT—Q) an: .mxac am to» gens. cczaasouv odabmuooto=_.o=ca about». u—o to m:=ox so Quezmcou ascsocsou new goose can muscu:u ago—u: xeoa omotu>< .a mpaop SE3 nouapau—cu :o_bae=mcou acaoaeou >_4ouz «co..atu=uuccu xtaao_u x :o.ucs:m=ou some oa~gu>a eat. soda—:u_ou co.bnsamcou uczoaeou >__aa .m_o.ou :. coucaau as: .m.u.tu.=a 3“.) umbu._uua .aewcu 9:: a .mummgacogea :. exczm m—oa_=a a>.— go Logan: may co canon caeagu u=a_u3 o>_aa_:s=o u .s x co.uae=m=ou >._oc set. a .co..;a:mcou «.mos o>.a:umm:ou are ac maa5m>a as» co sumo: ca.aae:mcou saw; a -.- «m.~ mm.m .e.m no.v --- “Jaxas. :c_.aszm=cu u::o:§oo --- mo._ cm.c -.c om.c --- Aa\mav :o.u:5:mcau sczoasoo --- so wm cm Km —e A=\a. co_uce:acou and o; u... can- c.4m- ~.cm - c.~n_- m.co.- ..mN + As. accagu z;._a= a..m. :3 ~m.m~ oc.c— mm.m eo.m mo.~ --- Aa:\o§~ :o_dqe:m:ou uczoasou --- nv.— mm.c -.o am.o --- nu\as. =o_u;a=m=ou vzzoasoo --- so— ac «a cc n__ Av\mv co.»aszmcou veal all...~c~- M.“ . m.o - o.na- o.o._- ..m. + As. macszu .==_o= em.s 3.2 3.“ -.m m5... Ed --.. 339.; ._o_ua..5m:cu 3:35;... --- ~o.. m~.o em.o m~.c --- Acxasv =o_u;aam:ou cczoaeoU --- QNN. an. m_. as _~_ .2\@. co.zas=m=ou cool a.u.o.~.m__- ~.m_+ a.~ . k.cc- m..o - a... +. Am. ascogu zg¢.a= o~.. --- c a c c --- Ax:\aa. :=_ua5:m:oo nanoasou --- c c o o --- Au\as. zo.uas=m=ou assessou --- me. No. as. «a Na .u\ov =a_zas=m=ou was. e.uas.a..o . n.-+ m.~_ + a.” . m..~ . a.m_ - Am. macagu .gm.u= o . wmm o>.ua.:s:u e n N — cc.u5.._uu< autzmous Raga. mama: v sum: sou: 3mm: gum: touaeataa co_uatu=uu=ou ...Llllll A...cou..m o_sap Table l0. Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of sodium monofluoroacetate (Compound l080) for 28 days. . Concentration Initial Final Change (ppm) Sex n wgt. (9) wqt- (9) (9) Young 0 M 4 692.2 l288.2 +596.0 F 4 5ll.5 746.5 +235.0 l.08 M 4 729.5 ll75.0** +445.5** F 4 550.0 737.0 +l87.0 1.94 M 4 756.0 ll27.2** +37l.2** F 4 545.2 695.2 +150.0** 3.50 M 4 730.8 lO72.5** +34l.7** F 4 561.5 679.2 +ll7.7** 01_d O M 4 l300.2 1387.2 + 87.0 F 3a 634.5 677.0 + 42.5 4.76 M 4 l390.5 l280.0 -llO.5* F 3a 670.0 597.0 - 73.0 8.56 M 4 1299.5 l080.0 -2l9.5** F 4 7ll.5 53l.5** -l80.0** l5.40 M 4 1353.5 890.8** -462.7** F 4 680.5 428.2** -252.3** a One animal died from causes not related to effects of the excluded. * Significantly different from control (P s .05). ** Significantly different from control (P < .01). 60 compound and was __ ... ‘N 2...... p mm.m p . cu.v om.m em.— mo.p c mzzo> as 33: 2 was. sszsrfifzfi :a_amhomem~_ 2...: “may eclch\nmmav m—ms.:a to .62 . =o_ueco:oo=ou .amob smog >a2-m~ a a:.c=u Remap ceaozsoov oomuoucoco=_cocoa 22,16» so» nausea; co sewage: mu.paugoz ... m_amp Table 12. Blood parameters of growing (young) and fully grown (old) ferrets fed various concentrations of sodium monofluoro- acetate (Compound 1080) for 28 days. Concentration RBC NBC Hb Hct (ppm) n (x106) (x103) (g/di) (%) Young 0 8 11.26a 12.0 20.1 46.7 :_1.020 :_5.18 :_l.82 :_1.94 1.08 8 9.43** 6.9** 20.0 45.4 10.719 10.84 i 1.53 i 2.12 1.94 8 9.06** 6.9** 18.3 45.1 :_0.320 :_2.70 :_1.81 :_1.64 3.50 8 9.36** 5.0** l7.0** 43.0** :_l.170 :_1.18 :_2.54 :_1.69 91d. 0 8 11.16 6.4 19.8 51.7 :_2.029 :_4.63 :_2.43 :_5.54 4.76 6 10.15 4.3 20.3 47.7 :_l.971 : 0.98 :_3.08 i 7.36 8.56 7 10.71 4.4 19 1 41.4* :_2.017 i 2.54 i 4.19 :_8.96 15.40 1 11.15 3.0 21.6 54.8 :0 :0 :0 :0 a Mean :_S.D. * Significantly different from control (P p S ** Significantly different from control ( < .01). 62 .:9. v .3 99.59.99 9.999 999.9999... 92:99.9.995 . .999. v 99 9999999 5999 9999999.: 9.999999999_m 9 .59.: 999599 9:599 9...: 39. v .3 9999999999 9:99-99:39 9: 9.99;: 999.4999 9.99 99999 9 II III II III II II III 0 -- 9.99 9.99 9.~9 9.99 ..99 ~.99~ 9 99.9 99.99 ~.~m 9.99. ..~.~9 9.99 «9.999 x 99.9 -- --- -- --- .. -- - --. D -- 9.99 9.99 9.99 9.99 ..99 9.999 9 99.9 9.99 9.99 9.99. m.~9 9.99 9.999 x 99.— I' III -I III -I -I 3.. U -- 9.99 9.99 9.99 9.99 9.99 9.999 9 9.9 9.99 9.99 9.99. 99.99 9.99 9.999 x 99.— II III II I.II II II III U .. ~.n9 9.99 9.99 . 9.99 . 9.nm 9.999 9 ~.9. ~.9__ 9.99 9.9.. 9.99 9.99 9.999 x 9 . 99999: 99999 99 9999999 9 99 9999999x9 999999 .. 99.9 99.9 .. 99.9 -- ~9.n .. --- 9 -- 99.9 99.9 «No.9 99.9 99.9 99.9 n... ~.999 9 99.9 99.9 99.9 999.9 99.9 99.9 ~9.n 9.99.9 c.9.~99_ x 99.9 -- .9.9 999.9 -- 99.9 .. 99.» .. .-- 9 -- 99.9 99.9 99.9 99.9 99.9 99.9 99.9 ~.999 9 99.9 99.9 99.9 999.9 99.9 99.9 No.9 «No.9 a.~.9~__ : 99.— -- 99.9 99.9 -- ~9.9 .. . n~.n .- -.. 9 -- 99.9 99.9 99.9 99.9 99.9 99.n 99.. 9.999 9 99.9 99.9 99.9 .9.9 99.9 99.9 99.9 999.9 999.99.. x 99.— . -- 99.9 99.9 -- ~9.9 .. «9.9 u. --. 99 -- 99.9 99.9 ~9.9 99.9 99.9 99.9 99.. 9.999 9 99.9 99.9 99.9 99.9 99.9 99.9 99.» 99.9 ~.99~. : 9 35.. s 35.. 99 3.3 99 35.. 99 35.. 9. 33.. .9 3.2. 99 :93 99 .9» .99.. 2...... 999999 999999 9999: 99:9 99:99x 999.99 99999 99999 999 9999999999999. .9999 9w 999 «999. 99999299. 99.99999999999995 29.999 99 99999999999999 9999999 999 9999999 999 9.9299 999 9:. 9.92 99.2999 99 9999993 99999 999 9999 .n— 9.99— ...9. v 9. .99.:99 .999 99999.9.9 9.9999...=9.9 . ..99. v 9. .99.:99 9999 99999.9.9 9.99.9...99.9 . .59... 99939.. 99999 99: 39. v .... 999999.. 9.9 9999.....939 99 999.... 999389 999 9999... 9 II II III III III III 9 -- 9.99 9.9. 9.99 9.99 ~..99 9 ..99 ...9 9.... ...9 ..9.99 .9.999 x 99.9. II II III III III III U -- 9.99 9... 9.99 9.99 ..999 9 9.99 ..99 9.99. 9.99. 9.99 9.999 9 99.9 II II III III III III 9 -- . ..99 9.9. 9.99 9.99 9.9.9 9 9.99 9.99 9.99. 9.99. ..99. 9.9.9 9 9... II II III III III III U . -- 9.99 9.9. 9.99 9.9. 9..~9 9 9.99 9.99 9.99. ..9.. 9..9. 9.9.. a 9 999.9: 9.999 99 9999999 9 99 9999999x9 999999 -- -- ..N... ...9.9 -- -- -- --- 9 -- ...9.9 9... 99.9 99.9 .....9 ...... ..~.9~9 9 .9.9 .9..9 9... 99.9 99.9 9..9 ..99.9 ..9.999 x 99.9. -- -- 9..9 .9..9 -- -- -- --- 9 -- ...9 99.9 9..9 99.9 .9.9 9... ..9..99 9 99.9 99.9 9..9 9..9 ...9 .9.9 ...9 9.999. x 99.9 -- -- ...9 99.9 -- -- -. --- . 9 -- 99.9 .9.9 .9.9 99.9 99.9 99.. 9..99 9 99.9 99.9 99.9 99.9 9..9 99.9 99.9 9.99.. 9 9... -- -- 99.9 .9.9 -- -- -- ..- .9 -- 99.9 99.9 99.9 .9.9 9..9 99.9 9...9 9 99.9 .9.9 99.9 .9.9 ...: 99.9 99.9 ...99. x 9 ..999 9. .999. 9. ..999 9. .999. 9. .9999 9. .999. 9. .9999 9. .9. .99: .999. . 99.99. .999: 99:. 9999.3 999.9m 999.. 9.999 9999 99.9999999999 ---III“ ' III-.-II‘II I .9999 9N 999 .999. 99999599. 9.999999999.99:9§ 29.999 99 9:9..99.:99=9u 999.99> 999 9.9999. ... 9.959. 9:9 .z. 9.95 :3999 >..:9 99 9.99.9: 99999 999 9999 .9; 9.99. by day 2. No signs of intoxication were noted in the groups fed the 1.08 or 1.94 ppm diets. Clinical signs among the adult animals included reduced feed consumption, incoordination or paralysis (especially of hindquarters), and unconsciousness, while among the younger animals the only signs noted were reduced feed consumption and occasional loss of coordination of the hindquarters. At the conclusion of the 28-day exposure period, 1 of 7 animals had died on the 4.76 ppm diet, 1 of 8 had died on the 8.56 ppm diet, and 7 of 8 had died on the 15.40 ppm diet among the adult ferrets. No animals died among the young ferrets. The mortality pattern of the adult ferrets is described in Table 11. The data yielded an LC50 of 9.4 ppm, with a 95% confidence interval of 6.1 to 14.5 ppm, and a slope of 1.56, with a 95% confidence interval of 1.01 to 2.42. No gross lesions were noted at necropsy of mortalities during the test or at the terminal kill (30 August, 1983). The blood parameters, summarized in Table 12, show significant effects on all measurements for younger animals and a decrease in hematocrit for the older animals. The transformed organ weights, summarized in Tables 13 and 14 for young and old ferrets, respectively, reveal effects on the liver, kidneys, thymus, and testes of the young animals and liver and spleen of the older animals. Mink Reproduction Test: Based on the results of the LC50 trial, where adverse effects were noted at concentrations 2 0.90 ppm, dietary concentrations of 0, 0.05, 0.20, and 0.80 ppm were chosen. Distilled water was the carrier used. Actual concentrations of Compound 1080 in the test diets have not been determined. The reproduction test was initiated on 7 January, 1982 and terminated on 29 June, 1982. Weight changes were recorded bi-weekly until 4 April, 1982, and feed consumption was measured only for weeks 6 and 8. These data are summarized in Tables 15 and 16. No signs of intoxication or mortalities were noted, nor were there any obvious birth defects found. The reproduc- tive indices are presented in Tables 17 and 18. No gross lesions were noted at necropsy at the terminal kill (29 June, 1982). The hematologic parameters, summarized in Table 19, reveal a decreased WBC count for the 0.80 ppm group. The transformed organ weights, listed in Table 20, show effects on the heart weights of males fed the 0.20 and 0.80 ppm diets. DISCUSSION Several noteworthy results can be obtained from an inspec- tion of the LC50. feed consumption, and body weight data. First, it is apparent from the cumulative weight changes that a dose- dependent response was elicited upon exposure to Compound 1080 in both mink and ferrets. Second, from the cumulative compound consumption data, it would appear that an upper limit may exist for the ingestion of Compound 1080 over 28 days, being approximately 7.5 mg for mink and 23.5 mg for ferrets. Simi- larly, from the daily compound consumption data, it would :o—uasam=ou uczoasoo gum: ~ m:o_uotu=mocou agoum.u mue.a :o.aa53m:ou some mmaco>c sage undo-zopou covuae:m=ou acaoasou »—.oa .m-mm¥uo= co masto>o 20 canon .couue_umm mun—o> v .Amopanu w. .mo—ae «v «pas_cu a. so. moa_a> :a woman umcozu u:a_wz xcaa u .9. «ma_d :a_ua5=m=ou >__n= soak code—:o_au a .co.uas=mcoo m.>no u>_a:uom:oo or“ “a ouugm>u and co woman :o_aae:m:oo cool a sv._— ~_.~ oo.~ --- --- --- Ax: N\mav ca.s;e:m:ou e==aaecu --- _m_.o m~_.c --- --- --- Asxmsv =o.das:m:oo czaoaacu --- o.mm_ o.o- --- --- --- A=\s. ¢c_saa=m=ou was. u~.~a- o.a~. ..ee. ..mm- m.co_- ~.~n As. cacogu uga.~=. os.a um.n mo.o c~.o --- --- --- Ax: «\as. =o_gas:m=ou u==aaeou --- mcc.o cmo.o --- --- --- Auxas. co.u;s=m=oo scaoaeoo --- m.o- ..cmN --- --- --- .u\a. co.nas=m:ou coal uo.c_- a.-- c.3m. a.n_- o.om- ¢.¢m As. mecogu.u;a_o: o~.c vo.c a—.o ~—.c --- --- --- Ax: ~\aav =o..a§:m:ou azaoasou --- n_c.o ~_o.c --- --- --- As\¢ev :o_uas=m:oo uczoaeou --- «.mcw m.oo~ --- an: .-- Ao\mv co.d:5:m:cu one; Um.v_. ..m.+ ”.mk. m.mm- ...o- e.a.. Am. eacogu sga.u= mo.c o o a . --- --- --- Ax3.~\ae =o_ga5:m:oo uzzoasou --- c c --- --- --- A=\osw co.~as=m:ou ucaoaaou --- o.o- o.mm. --- --- --- As\a. co.uas=m=ou cyan um.~ + ..k.. c.0e. m.~ - n.nm- ~.ee+ Ase mocogu aza.oz o o>.uo_=sau a-~ o-m o-n ~-_ co.u~s.puu< swtzmoge Asagv mama: a axon: mama: axon: «Jam: cadaEatam =o_untu=ao:=o «unauuoOtoapco=aa s:_uom 5o .umo. copauzootaoa a 3:.gaz Acao— cananou. mco.aatacuo=ou m:o_ta> so. x=_e a: sasamcou nuczoqsou use swoon use maa:u;u agape: >coa oungo>< .mp opasp 6'7 Table 16. Initial and final body weights of male (M) and female (F) mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 8 weeks prior to breeding. Concentration Initial Final Change (ppm) Sex n wot. (9) wet. (9) (9) 0 M 4 1786.8 1844.0 +57.2 F 12 981.8 973.2 - 8.7 0.05 M 4 1788.2 1792.5 + 4.2 F 12 956.9 974.8 +17.9 0.20 M 4 1616.2 1645.2 +29.2 F 12 924.8 900.9 -23.9 0.80 M 4 1899.5 1822.8 -76.8 F 12 955.2 871.2* -90.2** * Significantly different from control (P s .05). ** Significantly different from control (P S .01). 68 .mcaosm _a=_ma> a? cm>cwmno mcmz aoNoumEcoam m>w_ o: as; mm—ma cmuamoom mmpmame tango m a .a.m H 58: ... o H e ow. m on 2 N : 3% 85 an H or. .2 H A: a; H ...? 2: NS: 85 3.. H ...... 3.. .... as N: u N: :3 wt: mod 3.. ..u E. 8.. H as woos ... 2; N2... 25 o cog—oz: o wag—m5; o Amamcv not; m.o _mboa Asaav \mu_x .auoh \mqu m>_4 =o_umummo \cmapmzz m.o \umca m.o :owumcuemocou .mxmmz mm to; AcmOP assessouv monsoon -oto=_uo:oe s=_uOm co mzopumcucmucou m:o_cm> no» gees «peace to mo:cscomcoa m>wauzvocamz .~_ «paws 69 .A_o. w av _ocu=ou sort semtacc_a s_o=ao_c_=a_m.. .Amo. w av .ocucoo acne acmcmem_e x~ucao_hvcm_m « .saa so sm__1x ms_x ma=_ue_ do: mace a .a.m.H cam: a o .H _o._.H o o --- --- e.me --- --- m.m oe.o ¢.m_e.u mm.ea_.w mm.om.H NR.¢N.H mo._N.H _m._.u a~.mo am.ma ch_ o.mem N.mm ..~.Na~ ..a.mc_ m.a o~.o m.ooe.u mo.p_~.fl mk.__.fl aa.m¢.H . N_.m_.fl as...“ se.m~ ~.mm .oN_ ¢.mke _.mm m.~e~ e.ma a.m mc.o N.¢Nm.w em.ke_.fl _m.N_.H em.ee.H c_.o~.H new...“ m.em m.ea mmm. o.~m¢ e.~m m.mm~ e.mm e.a o xmmz c xmmz m sum: 0 goo: m zucwm xomz c goo: m zwwwmli Agaev po>_>c:m a flaw u;m_m3 cmuu_— mmmcm>< flaw u;m_oz moon u_x mmccm>< copumcucmucou .mxmmz mm to» onop czzoaaouv muoaaoaocozpmaeos E:_1om mo meo_uccu:mocoo m=o_cm> emu mama toe —m>_>c:m u_x use mu;m_oz imbue. use awe: avg ommcm>< .m. w—nmp 70 Table 19. Blood parameters of mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 6 months. Concentration RBC WBC Hb Hct (ppm) n (x105) (x103) (9/d1) (%) 0 8 9.27a 28.0 22.4 53.4 _+_0.739 : 13.77 10.85 i 3.06 0.05 8 8.50 19.3 22.1 53.1 10.756 : 6.97 311.76 _+_ 3.07 0.20 8 9.44 45.3 22.7 54.6 i 0.579 _+_ 24.94 i 1.17 1 2.66 0.80 8 9.10 56.0* 23.3 55.5 i 0.560 3: 28.34 _+_ 0.79 i 2.57 a Mean 1 5.0. * Significantly different from control (P < .05). 7]. Table 20. Body and organ weights of male (M) and female (F) mink fed various concentrations of sodium monofluoroacetate (Compound 1080) for 6 months. Concentra— Body Brain Liver Spleen Kidney Lung Heart tion wgt. (% (% (% (% (% (% (ppm) (9) body) body) body) body) body) body) 0 M 1456.0 0.62 3.99 0.29 0.59 0.93 0.78 F 899.0 0.84 4.28 0.45 0.65 .0.86 0.64 Ca ---- -- 4.14 -- -- 0.90 0.71 0.05 M 1570.2 0.59 4.25 0.26 0.49 0.84 0.62 F 871.2 0.91 4.45 0.42 0.64 0.95 0.71 C ---- -- 4.35 -- -- 0.89 0.66 0.20 M 1437.0 0.66 4.11 0.31 0.58 0.95 0.65 F 881.8 0.87 4.58 0.48 0.63 0.96 0.66 C ---- -- 4.35 -- -- 0.95 0.66 0.80 M 1447.0 0.66 4.22 0.25 0.61 0.81 0.62 F 843.8 0.95 4.50 0.41 0.66 0.95 0.64 C ---- -- 4.36 -- -- 0.88 0.63 (Organs expressed as a percent of brain weight) 0 M 659.9 50.0 97.0 150.2 128.0 F 518.0 53.4 79.5 103.5 76.9 C --- 51.7 -- --- --- 0.05 M 608.2 38.8 83.8 120.3 106.6 F 494.3 47.5 70.8 104.9 78.3 C --- 43.1 -- --- --- 0.20 M 620.1 46.6 87.2 142.8 98.5* F 527.2 56.3 72.9 109.7 76.0 C ' --- 51.5 -- --- --- 0.80 M 642.1 38.5 92.2 121.4 92.7** F 480.8 43.9 71.6 100.5 68.1 C --- 41.2 -- --- --- Sexes are combined where no significant difference (P < .05) was found between them. .05). Significantly different from control (P s Significantly different from control (P < .01). *‘k 72 appear that the upper limit for consuming 1080 in one day would be approximately 0.35 mg for mink and 1.4 mg for ferrets (approximately equal to the oral LDsos). Finally, from the feed consumption and weight change data, it would appear that tolerance to the compound has occurred by the third week in the mink, since feed consumptionimas considerably above the first 2 week's levels (except in the 5.25 ppm diet), and after 2-3 weeks exposure in the older ferrets. The same phenomenon may have occurred with the younger ferrets, especially those fed the 3.50 ppm diet. Tolerance to Compound 1080 has been reported by several authors (Quin and Clark, 1947; Chenoweth, 1949; Miller and Phillips, 1955; Atzert, 1971), although the protection provided was only slight. Inspection of the data for the 1.08 ppm diet for the young ferrets reveals another interesting possibility. Since the feed consumption for all four weekswas nearly equal, yet the cumulative weight gain showed a difference of nearly 100 g and a highly significant difference for males, it may be possible that Compound 1080 had an effect on weight gain by ‘growing animals unrelated to feed consumption (perhaps related to nutrient uptake, feed conversion, etc.). Examination of the blood parameters from the two LC50 tests (Tables 7 and 12) revealed a significant decrease in the hemoglobin content for mink fed the three highest concentra- tions of 1080 (including the 5.25 ppm group, even though a statistical analysis was not performed since there is only one value), indicating a possible anemic condition in the mink. The older ferrets fed corresponding dietary concentrations, however, did not show this decreased hemoglobin content, suggesting that the mink may be more sensitive to Compound 1080 than the ferret. For the younger ferrets, significant decreases were noted in all hematologic parameters at 3.50 ppm 1080 and in RBC and WBC counts at the other two dietary concentrations. This may be an indication of an effect on the hematopoietic system in the young ferrets, although further tests would be necessary to confirm this result. The organ weight data from the mink and ferret LC50 tests (Tables 8 and 13) also revealed interesting information about the two species' responses to 1080 exposure. The most notable observations from Table 8 were: (1) a significant decrease in the liver weight when expressed as a percentage of brain weight for all treatment groups (although these groups do not show a decrease when the liver weight was expressed as a percentage of body weight, since the body weights declined with increasing concentration of 1080); (2) significant decrea- ses of spleen, kidney, and heart weights (as a percentage of brain weight) for all dietary concentrations except 0.50 ppm for at least one sex (although the kidneys were significantly increased as a percentage of body weight at 2.90 and 5.25 ppm and the heart was increased at 1.62, 2.90, and 5.25 ppm); (3) a significant increase in the lung weight expressed as a percentage of body weight for at least one sex at 1.62, 2.90, and 5.25 ppm coupled with no significant difference in lung weight as a percentage of brain weight, which may be an 74 indication that the lungs (and heart) are the last organs to decline in size as the body loses weight; and (4) no signifi- cant decrease in testicular weight. Previous research with rats exposed to Compound 1080 has shown significant decreases in testicular weight during subacute exposure (Sullivan 33 31., 1979). However, it must be noted that the animals used in this test were not in active spermatogenesis at the time of necropsy. In contrast with the mink, the adult ferrets did not experience widespread decreases in organ weights upon dietary exposure to Compound 1080. The only significant findings were decreases in liver and spleen weights as percentages of brain weight for males fed 15.40 ppm. This may be a further indica- tion of the greater sensitivity of mink to 1080. In the LC50 test with young ferrets, significant decreases were noted for liver, kidney, thymus, and testes of males fed 3.50 ppm and for testes at 1.94 ppm as well. The decrease in thymus weight may be correlated with the reduction in WBCs noted above. The decrease in testis weight of the young ferrets fed 1.94 and 3.50 ppm of 1080 may be indicative of a preferential effect on this organ, although it must be noted that the testis is relatively undeveloped in ferrets at this age. Taken i£_t2tg, the results from these LC50 tests indicate a systemic deleterious effect on adult mink and ferrets upon exposure to dietary concentrations of Compound 1080 of 2 0.90 ppm and 2 8.56 ppm, respectively. This is consistent 75 with the mode of action of Compound 1080, in which fluoro— acetate, when converted to fluorocitrate, constitutes a block in the Krebs cycle, and produces an accumulation of citrate. This citrate accumulation leads to interference with energy production and cellular function and is likely involved in the decrease in body and organ weights noted above, and prob- ably also in the anemia seen at the higher dietary concentra- tions in mink. In comparing the responses of the young and old ferrets, Compound 1080 at first glance appears to affect the mature animals more severely, since all three dietary groups exhibited reduced feed consumption almost immediately, whereas only the 3.50 ppm group showed this response among the young, rapidly growing animals,and mortalities were only recorded among the adult ferrets. However, due to this rapid growth the young ferrets were able to "outgrow" the dose (which was calculated on the basis of an approximate body weight of 500 grams), so that at the end of the 28 days of the trial the animals on the 3.50 ppm diet were actually receiving a dose of approxi- mately 0.9 mg/kg of body weight instead of 1.4 mg/kg. Since young, rapidly growing ferrets appear capable of "outgrowing" a lethal dose within a 28-day LC50 trial, it would appear that this age group is not suitable for use in a trial of this length. If it is desirable to conduct LC50 trials using young, rapidly growing animals, either the length of the trial should be shortened or the dietary concentration of the com- pound should be changed weekly to account for changes in body weight. 76 The younger ferrets do appear to be more sensitive to hematological derangements upon exposure to Compound 1080. There were significant decreases noted for all parameters at the highest concentration fed to the young ferrets and decreased RBC and WBC counts even at the lowest dose, whereas the only decrease noted among the older ferrets was the hematocrits of the 8.56 ppm group. Comparison of the LC50 values obtained from the two tests (3.2 ppm for mink, 9.4 ppm for ferrets) indicates that the mink is approximately 3 times more sensitive to Compound 1080 than the ferret. This is also reflected in the apparent upper limits for daily 1080 consumption (0.35 mg/d vs. 1.4 mg/d), and the apparently greater effect on the blood parameters and organ weights noted above. In the reproduction test with Compound 1080, adverse effects on body weight were found for female mink fed 0.80 ppm, although there was only a slight decrease in feed consump- tion noted. This dietary concentration proved to have an adverse effect on the reproductive performance of the mink, although this effect was only seen in males in this test. The impaired reproduction in this group is presumed to be due to oligo- or aspermia, since motile spermatozoa were only seen twice in vaginal aspirations taken after c0pulation, even though the males were repeatedly accepted by the females in this group. Testes degeneration and altered spermatogenesis have been reported in rats exposed to sodium monofluoroacetate (Sullivan gt 31., 1979). Further tests are necessary to 77 evaluate the reproductive effects of 0.80 ppm of Compound 1080 in the diet on females. No significant differences were noted in reproductive per- formance between controls and dietary concentrations of 0.05 and 0.20 ppm, although the decrease in the number of live kits per female and average litter weight for the 0.20 ppm group indicate that there may be subtle effects on reproduction at this dietary concentration. (The significant increase in average kit body weight at 3 and 6 weeks was a product of decreased litter size, which allowed individual kits to attain relatively larger sizes. Note the decrease in the corresponding litter weights at 3 and 6 weeks). Examination of Tables 19 and 20 reveals that the maximum tolerated dose estimated by the LC50 test was quite accurate, since the only parameters significantly different from the controls were the WBC counts of the 0.80 ppm group and the heart weights as a percentage of brain weight of the males of the 0.20 and 0.80 ppm groups. CONCLUSIONS Based on the results of tests conducted with mink and ferrets, which were fed sodium monofluoroacetate (Compound 1080) in the diet, the following conclusions may be drawn: 1) The dietary LC50 for mink and ferrets is 3.2 ppm and 9.4 ppm, respectively. 2) Dose-dependent decreases in body weight and feed con- sumption are elicited in both species upon dietary exposure to Compound 1080. 78 3) Mink are more severely affected by dietary exposure to Compound 1080 than ferrets. 4) Young, rapidly growing animals may be unsuitable test animals for LC50 tests, since they may be able to "outgrow" a lethal concentration. 5) Red and white blood cell counts are negatively affected by dietary concentrations of Compound 1080 as low as 1.08 ppm fed to young, rapidly growing ferrets. 6) 0.80 ppm of Compound 1080 in the diet causes reproduc- tive failure in mink. The presumed cause of this failure is oligo- or aspermia. Experiment II - Results Mink LC50 Test: Since a literature search yielded acute oral LD50 values ranging from 121 mg/kg (Bio Fax Techniques, 1969) to 1470 mg/kg (Uzhdavini §t_31., 1974) for rats, range-finding tests were performed for mink and ferrets. From the results (Table 21), LDsos were estimated to be between 100 and 500 mg/kg for mink and between 300 and 500 mg/kg for ferrets. Based on these results, dietary concentrations of 0, 240, 432, 778, 1400, and 2520 ppm were chosen for mink. Corn oil was the carrier used. Actual dietary concentrations of o-cresol, analyzed by a modification of an analytical technique published by Supelco Co. (19751 were: Table 21. Results of range-finding studies with mink and ferrets exposed to o-cresol by gavage. Dose (mg/kg) Died/total Comments Mink ————1000 1/1 500 1/1 300 0/2 Both unconscious after dosing 200 1/2 4 days to death 100 0/2 Both lost coordination after dosing 50 0/1 No effects observed Ferrets 500 1/1 400 1/1 1 day to death 300 0/2 1 unconscious, 1 lost coordination after dosing 200 0/1 Lost coordination after dosing 80 0 ppm - ‘ 0 ppm 240 ppm - 213.5 ppm 432 ppm - 473.1 ppm 778 ppm - 862.3 ppm 1400 ppm - 1533.7 ppm 2520 ppm - 3680.3 ppm A l3-day acclimation period was begun on 27 October, 1981. The 28-day trial began on 9 November, 1981 and ended on 7 December, 1981. No overt signs of toxicity were observed during the test. No animals died during the test and there were no gross lesions noted atthe terminal kill (8 December, 1981). Body weight changes and feed and o-cresol consumption are summarized in Tables 22 and 23. Initial body weights were not recorded for the mink, therefore, body weight changes have been calculated from the last week of the acclimation period. Feed consumption was negatively affected at first at 2520 ppm, but returned to normal levels shortly afterward. Weight gain was affected at this concentration also, with levels of significance of a = .05 for the males and a = .10 for the females. The hematologic parameters, summarized in Table 24,showed significant decreases in RBC counts at 1400 and 2520 ppm and a decrease in hemoglobin at 2520 ppm. The organ weight data, summarized in Table 25,revea1ed an increase in liver weight (as a percentage of body weight) at all concentrations except 240 ppm and an increase in heart weight at 2520 ppm. However, no significant changes were seen in the organ weights when expressed as percentages of brain weight. 81 .oo_c;; :o_.ee__ooc 6;“ sec» catamas= m. woeczo age—oz m>_be_:e:o “coccoooc do: nuza_:z _ xeoz . a momegdzmcea :. exocm «_oa.:c o>__ Co 59:53: oz“ co some; occrzo u;a_u: set; .~ mw5_d =c_S;s:.cco >_—es soc. co_uae:m:oo w::o:5¢o »_susz .:c_bacb:ec:co xcc 6.: m9=¢u =a_u;52m:oo comb acacm>a Eon. swam—:o—eo :c_g;e=mcco c::c;5co >_.e: a .:o_aes= coo wxcu o>.u=uom=ou ox“ be enclose oz“ =o comma :o..as:m::o stale ¢.Nommp o.em~n n.4fiem omen e.-_~ a Arz\asw =6.zas=n=ou seaoesec ivo m.nmm 54mm c.m¢m m.mcm G 29:3. :O—un—Ezmzcu fizzoaaou --- ~_N sew \_N _N. _NN Ae\e. e6_zes=n=66 eat. 6.6Ao_ve.e~ 5.4N. N.“ - o.em. -- a.___+ . .mv 65:6;6 see.6: swam m.o¢mm N.~e- ..Noo_ ~.n~:N o.e~_~ o Arz\msw =6.zaa=neou ueaoaaao --- n.9Nm c.-~ n.6eN m.mam o Au\ae eo.zae=n=ou sesoaaou --- saw me. N_~ ~_~ new .a\a. =6_zas=n=au seal a.olapck.co_. e.c - e.~_- a.” - -- n._c. . As. aceceu 553.63 oee_ ~.nmmm e._Q¢— n.s_e_ a.ee_P ~...~_ o sz\ms. =6..as=m=oo ceasesco --- m.maw ~.~:N m.m¢_ o.m~_ c. Au\mev cc.dae:m:ou s:=o:&ou --- new cam __~ NNN ecu .u\a. =a_zas=m=66 some v.ufio_vm.oMp+ c.~e+ ~.on+ ...—i _ .. e._c+ “av macezo u;c*m3 ckk Néomn aim: wécm Nd: $.52“ o :335W co......==m:ou 959.599 --- n.c__ c.mmp —.c:_ e.mm— c Ao\oE cc.a:s:m:ou nanosecu --- SN :6. SN :8 can :{3 835.356 2.: s.oxo_.,.n¢~. a.Cm n..m. o.~_- -- m.e__4 As. ameneu .;a_6: NM. 0.6—m— —.:mn m.a~m. 9. con KKK? O ad3\m=; €039=3m=ou 9.302500 --- c.co ~.en e. _m v._o c Atxcev :o..ae:m:ou cezcesou --- sew aim e_~ emN sNN .6\a. =6.zea=.=66 one. v.ofio_V—.~m_+ a.“ + o.~m+ e.n + -- ~._e+ Am. mmeonu aza.m2 ccm o o c a o c Axx\aa co.daszm=ou uzzoascu --- c c c o c At\ae co.u:5=.:ou ===oa§cu --. nmw new :mm e_~ _am .au\o. =o.a;s:mccu atom e.6.o_.a.¢=m. ¢.ms. N._N. m.=.. -- a.ec_. . as. massed .;=.a= c m>_bc_:s:u e n ~ _ cc.ume__uo< euczmmee Asaav also: e see: 3663 3262 x622 LudoEncoa co_uoc.cmuccu III" I‘ll!!! .‘lill'tu-"!-'-l"l'll". In-.. 310!‘ {1'1""lli In .....It '1‘ .wxo: m to. _Omoco-o _o m:a_dacd:ao:ou w=o_cs> and ¥:_2 x; euaam:ou gscaozsco =:c mama. 3:6 mw==6;o u;a_a; sac; eaocm>< .Nm e_;a~ a). Table 23. Initial and final body weights of male (M) and female (F) mink fed various concentrations of o-cresol for 28 days. Concentration Initial Final Change (ppm) Sex n wgt. (9) wgt. (9) (9 0 M 5 1537.0 1836.2 +299.2 F 5 969.8 1080.4 +110.6 240 M 5 1515.2 1763.4 +248.2 F 5 980.6 1006.6 + 26.0 432 M 5 1544.4 1966.8 +422.4 F 5 941.8 1005.6 + 63.8 778 M 5 1528.2 1767.0 +238.8 F 5 1030.4 1053.0 + 22.6 1400 M 5 1592.2 1806.8 +214.6 F 5 1013.2 1012.0 - 1.2 2520 M 5 1582.6 1592.6 + 10.0* F 5 938.2 887.4 - 50.8 * Significantly different from control (P s .05). 83 Table 24. Blood parameters of mink fed various concentrations of o-cresol for 28 days. Concentration RBC WBC Hb Hct (ppm) n (x105) (x103) (g/du 1%) 0 10 11.51a -b 24.4 53.4 :_0.700 :_1.43 :_3.01 240 10 11.21 - 24.5 54.8 :_0.449 :_1.97 :_1.95 432 10 1 .14 - 25.9 55.7 i 0.544 :_l.38 1.2.19 778 10 10.92 - 25.4 53.7 _+_0.926 : 1.72 i 2.63 1400 10 9.53** - 23.1 54.9 :_0.398 :_1.12 :_2.72 2520 10 9.62** - 22.5* 54.3 :_0.707 :_0.80 :,2.04 a Mean :_S.D. b Not analyzed due to problems with lysing agent. s .05). ** Significantly different from control (P < .01). * Significantly different from control (P 84 Table 25. Body and organ weights of male (M) and female (F) mink fed various concentrations of r-cresol for 28 days. Concentration Body Brain Liver Spleen Kidney Lung Heart Testes (ppm) wgt (g) (5 body) (5 body) (5 body) (5 body) (i body) (2 body) (1 oooy) 0 M 1836.2 0.54 3.50 0.25 .49 3.55 0.45 6.04 F 1080.4 0.81 3 83 0.32 0.56 3.70 0.49 ~- Ca ---- -- -- 0.29 -- -- -- -~ 240 M 1763.4 0.56 3.98 0.25 3.54 0.63 0.50 0.06 F 1006.6 0.82 4.79 0.32 0.60 0.56 0.55 -~ C ---- -- -- 0.28 -- -- -- -- 432 M 1966.8 0.53 4.48 0.28 0 53 0.67 0.46 0.05 F 1005.6 0.85 5.03* 0.28 0.59 0.75 0.52 -- c ---- -- -- 0.23 -- —- -- -- 778 M 1757.0 0.59 4.32 0.28 0.53 0.55 0.50 0.04 F 1053.0 0.79 5.03* 0.32 0.60 0.77 0.55 ~- C ---- -- -- 0.30 - -- -- ~- 1400 M 1806.8 0.59 4.55 0.32 0.53 0. 5 0. 0.06 F 1012.0 0.81 5.19* 0.26 0.61 0.79 0 S -- C ---- -- -- 0.29 -- -- -- ~- 2520 M 1592.6 0.64 S.31** 0.29 0.53 0.66 0.54* 0.36 - F 887.4 0.94* 5.95** 0.38 0.67 0.75 0.61** -- C ~--- -- -- 0.33 -- -- -- -- (Organs expressed as a percent of brain weight) 0 M 671.4 47.2 91 8 102.0 84 3 8.2 F 475.9 39.9 69.9 66.6 61.9 -- f‘ --- -_ __ ___ -_ __ 240 M 722.2 44 7 97.2 115.1 59.7 10.4 F 594.3 38.7 73.3 81.1 67.5 -- c --- -- -- --- -- -- 432 M 849.5 52.3 101.7 127.9 87 2 9.5 F 592.5 32.6 69.7 88.3 61.6 -- c --- -- -- --- -- -- 778 M 741.2 47 9 93 6 101.4 86 9 6.2 F 648.5 39.9 76.6 99.0 69.3 -- c --- -- -- --- -- -- 1400 M 768.9 44.7 87.1 109.7 8 8 5.8 F 650.1 39.5 76.0 93.3 6’.8 -- C --- -- -- --- -- -- 2520 M 845.0 44.9 84.5 104.4 86.7 3.3 F 639.3 40.3 71.5 80.2 65.9 -- c --- -- -- .-- -- -- a Sexes are combined where no significant difference (P s .05) was found between them. * Significantly different from control (P < .05). it Significantly different from control (P < .01). £35 Ferret LCSQ Tests: Based on the estimated LD50 (Table 21), dietary concentrations of 0, 432, 778, 1400, 2520, and 4536 ppm were chosen, providing overlap of four dietary concentrations with the mink LC50 test. The overlapping diets were taken from the same batch of feed prepared for the mink. Analytically determined dietary concentra- tions were: 0 ppm - 0 ppm 432 ppm - 473.1 ppm 778 ppm - 862.3 ppm 1400 ppm - 1533.7 ppm 2520 ppm - 3680.3 ppm 4536 ppm - 5188.7 ppm A 13-day acclimation period was begun on 27 October, 1981. The 28-day trial began on 9 November, 1981 and ended on 7 December, 1981. No overt signs of toxicity were observed during the test. No animals died during the test and there were no gross lesions noted at the terminal kill (9 December, 1981). Body weight changes and o-cresol consumption are summarized in Tables 26 and 27, which show a slight reduction in feed consumption at 4536 ppm but no effects on body weight. The hematologic parameters, summarized in Table 28, show a reduced RBC count at 4536 ppm, while the organ weight data (Table 29) reveals increases in liver and kidney weights and a decrease in the lung weight (as a percentage of body weight) at some dietary concentrations. When the organs were expressed as a percentage of brain weight, however, the only changes were increases in livers at concentration 2 1400 ppm, and an increase in the females' kidney weight at 4536 ppm. .momo;.:cgua :. czcnm m—cs.=a u>.— Co cocszc as“ so come: mocczo .:s.oz stczo .~ was.» :3..:E:¢:oo x..eu soc. co.uaa=mcou u::aa5cu >..eaz .ea..ee.:o.:oo xtnua.v was.“ :o_aea=m:ou new. mango): Eon; to...=u_eo :c_u:e=mc=u u::ceEco >._e: .co.u:a=mcou axe: o>.a=uomccu oz. .6 maeec>c mg. co women :o..:s:¢=co cem.e L 5.5.m5. o.===. ....ee ...... ..eemm a ..x\ae. ee..ee=3=eo eeseeEeu --- n.=ce m.ano c.amc n.ea~ o Auxaev =c..:5:m=co =:=c;acu --- ... ... 5.. .5. so. ..e\a. ee..ee=meee see. 6.5.....@ n.e + 5.5.. ..m.. ..a . ..Nm. .5. eeeeeo .ee.e: 2... ...oo.. n.oamm ..am.m ..ee.n ..ewam o ..z\aaw ee..ea=m=eu eeeoaeeo . 1-1 o.ch ~.nvm c.5ev n.o¢m o At\mE :o_aaE:mcou c=zcaaou --- as. ~.~ =.. ..N .:N .e\m. ee..es=meee sea. e....m.~..+ m.... ..c.+ a.c¢+ ¢.m~+ c.¢e4 . .a. seemed 3:5.62 cam“ o.~ees a.aec. «.5... ..C55. 6.5... o .x;\se =6..;e=m=ed eesoeeeo --- e.c«~ m.cum c.mm~ ..cem c Auxoe ce_.;s:meoo ===oa2¢u --- ac. mm. _c. NR. n~_ A=\ov =o..ea=m=oo new. u.o..v.oe + ~.~.. «.... m.n.+ ¢.~.+ ~.mo+ .5. seemed .gc.e: ass. ..omCe ~.omo. e..=o n.a.a ~.m__. o .¥:\asv =o.uee=mcou eczoesou --- o.ce. ~.c:.. n._m. c.am— o Anxaev eo.~:s=m:co vcacescu --- cm. ca. as. sea .5. ..e\s. ee..e55neea see. u.c..¢.o. e.~ . o.a.. 5.... 5.... a..e. .5. 655556 .;e.ez 5.. e.eee~ m.o:e n.~.m. ....m m.eem o ..:\as =e..ae=n;eu ecsoeeeo --- o.cc ~.n. o.m~ m.na c Ac\oe =o.a;e:m:cu ac:ocsou --- as. as. ... no. ... .6\m. 5.2.526U e55. u.o..e.me + ...... 5.. + m.em. ”.... ..oe. .5. eaeeeu .ga.e= N.5 c o o o o o Ax3\ae =o_aas:n:ou czao;sou --- o c c c o Ac\csw =a_.:a:m:ou szzoascu --- cc. 2;. ac. as. mow Aexm. :o..ae:m=ou new. u.c....~. . a.c - 5.5.. ~.e~. q.¢m. ...o.. .5. 6555.6 .35.53 a o>..~.=e:u q n ~ . ee.uea..uu< catameos Assn. mgmmz v x023 xooz goo: x003 Luuoamgca :o_umga:oo:c. -..-II t .mxsc nu t2. _onu.3-5 .5 n:5_.=.4:uo:33 n:5..1> so. n.5..:. x: svzznzou gazaoasou ::o case. u:m amazezo .:m_az x23; easem>< .om v.25. -~ Table 27. Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of o-cresol for 28 days. Concentration Initial Final Change (ppm) Sex n wgt. (9) 1491:. (9) (9) 0 M 5 1780.6 1913.2 +132.5 F 5 950.6 962.2 + 11.6 432 M 5 1637.0 1781.0 +144.0 F 5 952.4 945.2 - 7.2 778 M 5 1708.4 1816.8 +103.4 F 5 972.0 1004.4 + 32.4 1400 M 5 1709.6 1795.2 + 85.6 F 5 960.2 933.4 - 25.8 2520 M 5 1799.4 199°.4 +199.0 F 5 1000.4 1026.0 + 25.6 4536 M 5 1689.8 1803.2 +113.4 F 5 905.4 914.8 + 9.4 88 Table 28. Blood parameters of ferrets fed various concentrations of o-cresol for 28 days. Concentration RBC NBC Hb Hct (ppm) n (x105) (x103) (9/dl) (%) 0 10 10.46a 27.5 20.9 50.4 :_0.671 :_ 9.03 :_1.96 :_2.95 432 10 10.64 25.2 22.2 51.8 :_0.975 :_ 8.22 :_1.76 i 2.59 778 10 10.66 24.9 21.2 50.8 :_0.762 :_ 7.95 :_1.90 :_2.77 1400 10 10.52 30.2 21.5 49.9 :_0.510 :_10.34 :_1.29 :_2.04 2520 10 10.59 36.2 21.4 52.4 i 0.806 :_10.60 :_1.31 :_3.51 4536 10 9.52* 28.3 19.7 49.1 :_0.380 + 15.09 :_1.04 :_2.83 a Mean i 5.0. * Significantly different from control (P < .05). 89 Table 29. Body and organ weights of male (M) and female (F) ferrets fed various concentrations of a—cresol for 28 days. Concentration Sody Brain Liver Spleen kidney Lung Heart Testes (ppm) wgt. (g) (2 body) (5 body) (5 body) (L bedy) (1 bOOy) (2 booy) (3 body; 0 M 1913.2 0.42 3.74 0.65 0.49 0.54 0.33 0.16 F 962.2 0.64 4.85 0.61 0.54 0.70 0.42 -~ Ca ---- -- -- 0.63 -- -- -- -- 432 M 1781 0 0.41 4.00 0.67 0.58 0.5 0.36 0.14 F 945.2 0.66 5.29 0.60 0.60 0.6 0 49** -- C ---- -- -- 0.64 -- -- -- ~- 778 M 1816.8 0.42 4.12 0.59 0.55 0.54 0.37 0.14 F 1004.4 0.64 5.72 0.65 0.63 0.64 0.46 -- C ---- - -- 0.62 -- -- -- -- 1400 M 1795.2 0.43 4.93* 0.69 0.53 0.5 0.39* 0 15 F 933.4 0.65 6.12* 0.69 0.66* 0.6 0.45 -- C --—- -- -- 0.69 -- -- -- -- 2520 M 1993.4 0.40 4.51 0.56 0.55 0. 0.35 0 19 F 1026.0 0.59 6.26* 0.73 0.59 0.56' 0.43 -- C ~--- -- -- 0.64 -- -- -- -- 4536 M 1803.2 0.45 5.19** 0.75 0.58 0.4 0.38 0.19 F 914.8 0.69 7.41** 0.68 0.77** 0.6. 0.50** -- C ---- —- -- 0.71 -- -- -- -- (Organs expressed as a percent of brain weight) 0 M 904.5 1.6.5 119.9 130.8 81.2 33.3 F 760.5 91.3 84.8 110.2 66.2 -- -c ---- --- --- --- -- -- 432 M 956.7 165.0 141.5 129.5 39.5 33.5 F 814.4 92.7 91.3 102.5 73.8 -- c ---- --- --- --- o- -- 778 M 991.9 141.7 135.6 129.6 39.3 33.8 F 895.4 102.8 93.7 99.6 72.: -- c ---- --- --- --- -- -- 1400 M 1156.7* 163.8 137.0 126.1 90.3 35.8 F 936.3 104.8 100 7 92.9 68 7 -- c ---- --- --- --- -- -- 2520 M 1135.5 142.2 140.5 111.3 89.1 47.8 F 1089.7*' 128.3 103.6 96.9 74.8 -- c ---- --- --- --- -- -- 4536 M 1168.7* 168.4 131.7 110.3 55.4 43.1 F 1077.8'* 93.3 112.4* 98.9 72.4 -- c ---- --- --- --- -- -- a . . .. Sexes are combined where no Significant difference (P < .05) was found between them. * Significantly different from control (P < .05). 1* Significantly different from control (P < .01). 9() Mink Reproduction Test: Based on the results of the LC50 trial, where adverse effects were noted at 2520 ppm, dietary concentrations of O, 100, 400, and 1600 ppm were chosen. Corn oil was the carrier used. Actual dietary concentrations of o-cresol have not been analyzed. A l4-day acclimation period was begun on 23 December, 1981. The reproduction test was initiated on 7 January, 1982 and terminated on 29 June, 1982. Weight changes were recorded bi-weekly until 4 April, 1982, and feed consumption was measured only for weeks 6 and 8. One female assigned to the 1600 ppm diet was later discovered to be a male during weighing for week 2 and was replaced from farm stock, and one female on this diet died from virus enteritis, not related to effects of o-cresol exposure. Weight changes and feed and o—cresol consumption are summarized in Tables 30 and 31. No effect on feed consumption was observed, although the males fed 1600 ppm differed in weight gain from control at the .10 level of significance. No signs of intoxication were observed during the test. The reproductive indices, summarized in Tables 32 and 33, show the average birth weight of kits from the 100 ppm diet to be increased over control kits' birth weights. No obvious birth defects were noted in this study. The hematologic parameters, summarized in Table 34, reveal a significant increase in RBC count for the 1600 ppm group. The organ weight data, presented in Table 35, show'an increase in females' liver weight at 1600 ppm. 91 ..mo.aso. N. .uu.as c. m.us.sa o. co. mas—o) so uomes masseso usa.az sues .v— «mama ..3.—a9=3m:00 a..au ace. =o..ae=m=ou ueaaseoo x..eez-.a .so..ec.:oo=ou scene.u was.. so.uassnsou uee. uaaLo>o soc. uo.~.=u.au =o..ae=mcoo assessed ...ea .so..ss:msou mxau o>.»:uomsoo or. so uaoco>o as. so usage so..as:msou use. ..ma.oso. o. .ma.as v. m.ae.sa s. so uomas omsoso uso.uz muss .o.ee a as o. uasmsoom.u was 6.556. use use mosobmaam and. us. he muuu.uo ca uoue.ms ass msmaou soc. uo.u c.655. use a .m1m mama: so emacu>a so ummcs .uu.ae.omm mw=.o> 82mm 38.. 3...; -1 -1 o ...: 29... 8.2.5528 55.2.58 11 «.mmm o.oom 11 11 o .uxae. so.uss:msou usaosssu 11 m- smm 11 11 11 .uxa. so..ss:msou use. 8.21 .... 1 ”...? ...... 1 4.2- ..m .. .2 69.20 225.. OS. umomm ~.mmo. c.mmo. 11 11 o .8; Nxme. so.uss:m=ou usaossou 11 o.m~ a... 11 11 c .uxaE. so.ase:msou uszossou 11 mm. em. 11 11 11 .uxm. so..se:msou uom. do... 1 5.2- 4.3. ......N- 9:- 5...? .3 69.2.6 226.. 8.. uwmm. ~.m- o.~m~ 11 11 o .32 mxmev so.uae:msou ussosaou 11 ..a. «.m. 11 11 o .u\oe. so..assmsou usaossou 11 .m. cm. 11 11 11 .u\m. so.uss:nsou ups. 6.3... ...NN- 9...... ...21 .....- 5...: .3 69.9.6 25...... 9... o o o o o c .33 Nxmev so..as:msou ussoasou o c a o o c .uxms. so..as:mssu usaosEou 11 mm. -~ -1 11 11 .u\m. so..ae=nsou uom. one? .....- 5.8. 8.5.. 92- n8 1 .3 69.2.0 22...: o u>..a.:e=u a1. e1m e1m N1. so.uos..uu< umssmees .sas. axon; a mxuoz «goo: memo: mxuoz souo5asoa so..os.soosou 1uauossos a ms.s=u .omuso1o .o mso..ssusaosoo ass.sa> um. 3:.5 xn .umm. so.. uoE:msou aussoseou use sumo. use moasesu .sm.u: xuoa omos~>< .om 5.2n. 92 Table 31. Initial and final body weights of male (M) and female (F) mink fed various concentrations of o-cresol for 8 weeks prior to breeding. Concentration Initial Final Change (ppm) Sex 0 wgt- (9) wgt- (9) (9) 0 M 4 1748.5 1822.5 + 74.0 F 12 958.2 1009.5 + 42.8 100 M 4 1713.5 1796.0 + 82.5 F 12 997.2 986.8 - 10.4 400 M 4 1697.5 1648.0 - 49.5 F 12 955.5 971.2 + 15.8 1600 M 4 1752.8 1606.8 -14 .0 F 10a 946.5 982.6 + 36.1 a One female died from causes not related to effects of the test substance, one female found to be male, both not included in totals. 93 .a.m.H cum: 8 om._ +~_.m mm.~ + mN.e NV.N + 8.48 a \m __\m coo. NP.~.H NN.m om...“ am.¢ ...o.fl 8.44 ~_\m N_\N_ ace oo.N.u mm.m Na...“ “5.: ~_.8.H 8.44 __\m N.\.. co. o~.~.u ~_.m mm.~.H ow.¢ 6_N.~.H m.o¢ ¢\m ~_\m o cog—mg: o cog—mzz.o Amxmcv omen m.o page“ Aagqv \mo_¥ .8064 \mo_x a>_4 =o_uaumao \6aa_a=3 m.8 \6626 6.6 =o_uacb=aacou .mzomz mm com _ommcuuo mo m=o_aaca=oo:ou m:o_cc> cab x:_a upmamu mo mocuagomgma o>_uo:wogquz .Nm u_ac~ 94 .Amo. w my _ocucoo seem ucmgmmm_u a_u:mu_m_:o_m 11 k. .o.m + com: a :3 H 358 + 3. a + 8.3 H 2.: H m: H m.mm ~.¢m mme. o.m~e m.mm c.~e~ m.oo o.m oom— oéc H 3.88 H 2.2 H 8% H 3.8 H 8; H N.mo ..vw _mo_ ~.mmm ¢.N¢ a.e- N.~m N.m cow 99.3 H 3.28 H 2.8 H 3.2 H 8.3 H 2: H «.mm ~.~m ¢v__ m._ce ~.o¢ m.oem . o.om av.m so. :2 H 22% H 2.3 H 3.: H 8.2 H K: H ~.N~ o.m~ omop o._me m.m¢ m.Om~ o._m mum o xmmz c xmmz m xmmz a 3mm: m zucmm xmmz m xmoz m cucmm Azagv _c>_>s:m & lflmv .umr gmuu_p .mw< Aav .umz zoos a_x .m>< :o_uacuzmo:ou .mxmoz mm com Pemmco1o mo m:o_uecu:ou:ou m:o_cc> way ”so: cog _c>_>c=m a_¥ ace muzm_m3 cmuu_— u:m hues “_x macgo>< .mm 9.46» 95 Table 34. Blood parameters of mink fed various concentrations of o-cresol for 6 months. Concentration RBC WBC Hb Hct (ppm) n (x105) (x103) (9/d1) (%) 0 8 9.37a -b 23.0 54.1 i 0.525 _+_ 1.47 i 2.43 100 8 9.13 - 22.8 53.0 _+_0.612 : 1.32 _+_ 3.07 400 8 9.52 - 24.5 55.8 10.532 1 1.73 i 2.41 1600 8 10.42** - 23.6 55.0 :O.642 11.27 i 2.87 a Mean :_S.D. b Not analyzed due to problems with lysing agent. * Significantly different from control (P < .01). 'k 96 Table 35. Body and organ weights of male (M) and female (F) mink fed various concentrations of o-cresol for 6 months. Concentra- Body Brain Liver Spleen Kidney Lung Heart tion wgt. (% (% (% (% (% (% (ppm) (9) body) body) body) body) body) body) 0 M 1822.0 0.56 3.59 0.29 0.55 0.66 0.52 F 1009.5 0.93 4.01 0.32 0.61 0.73 0.58 Ca ---- -- -- -- 0.58 -- 0.55 100 M 1796.0 0.60 3.97 0.28 0.60 0.75 0.66 F 968.8 0.88 4.43 0.30 0.64 0.77 0.66 C ---- -- -- -- 0.62 -- 0.66* 400 M 1648.0 0.61 4.33 0.32 0.59 0.67 0.56 F 971.2 0.84 4.81 0.48* 0.65 0.79 0.60 C ---- -- -- -- 0.62 -- 0.58 1600 M 1606.8 0.64 4.52 0.31 0.59 0.76 0.59 F 982.6 0.96 5.62** 0.44 0.73 0.88 0.66 C ---- -- -- -- 0.66 -- 0.63 (Organs expressed as a percent of brain weight) 0 M 641.2 50.8 99.4 118.9 92.7 F 445.6 35.7 67.0 80.0 63.3 C --- 43.2 --- --- --- 100 M 670.6 47.3 102.6 125.3 109.9 F 508.5 33.9 74.0 88.4 75.5 C --- 40.6 --- --- --- 400 M 729.6 53.4 98.9 109.8 94.1 F 579.3 57.1 77.7 94.7 72.7 C --- 55.3 --- --- --- 1600 M 712.9 48.7 94.7 120.7 94.3 F 596.4 46.1 78.0 92.3 69.6 C --- 47.4 --- --- --- a Sexes are combined where no significant difference (P < .05) was found between them. * Significantly different from control (P < .05). ** Significantly different from control (P < .01). 97 DISCUSSION Examination of the body weight and feed consumption data revealed that, with the exception of weight change for male mink fed 2520 ppm, no significant results were observed in the mink and ferret LC50 tests. Since the cresols are known to be easily excreted as conjugated glucuronides and sulfates (Bakke and Scheline, 1970), it is assumed that the animals were able to excrete enough ingested o-cresol from any one feeding during the day that a toxic dose was not reached, even though the mink consumed in excess of the estimated LD50 in an average day's feed consumption on the 2520 ppm diet, and possibly on the 778 and 1400 ppm diets. Similarly the ferrets consumed in excess of the estimated LD50 on the 2520 and 4536 ppm diets. Similar results have been reported for rats exposed to 0.3 g/l of 0-cresol in their drinking water. Savolainen (19791 found no effect on body weight at this concentration, even though the rats had consumed a cumulative dose in excess of the acute oral LD50 by the fourth week of this study. In comparing results from diets that overlapped on the two tests, it appears that mink are more sensitive than ferrets to o-cresol. At a dietary concentration of 2520 ppm, both male and female ferret weight changes were in excess of control values, while the male mink weight change was significantly less than controls at the .05 level of significance, and females' weight change was also well below that of controls. Feed consumption was negatively affected in the first week for mink fed 2520 ppm, while it was slightly above control levels for ferrets in the first week at this dietary concentration. This sensitivity is also reflected in the hematologic para- meters. Significant decreases in RBC count were found in the mink fed 1400 and 2520 ppm and in the hemoglobin concentration of mink fed 2520 ppm, while no significant changes were noted in ferrets fed the same concentrations. It must be noted, however, that the RBC counts of the mink fed 1400 and 2520 ppm were well within the normal range for mink at this time of year. In fact, the control and 240 ppm groups' RBC counts were actually quite high for this time of year (Fletch and Karstad, 1972). Another puzzling result from the blood data was the decreased RBC count with increasing dietary concentration of o-cresol in the mink LC50 test, whereas the RBC count increased with concentration in the reproduction test. In regard to the reproduction test, no biologically signi- ficant results were obtained. No significant negative results were observed in the weight change data or in the reproductive indices, although male weight change on the 1600 ppm diet was significantly different from control at the .10 level of signi- ficance. Average birth weight of kits born on the 100 ppm diet was significantly greater than control, although the average of 9.4 g is within the range of normal birth weights. In fact, the 8.0 9 average birth weight for kits on the 1600 ppm diet is near the range of abnormally low birth weights, but is not significantly different from the 8.3 9 average of the control group (see Table 2). 99 CONCLUSIONS Based on the results of tests conducted with mink and ferrets, which were fed o-cresol in the diet, the following conclusions may be drawn: 1) The dietary LC50 for mink is > 2520 ppm, and for' ferrets is > 4536 ppm. 2) Since o-cresol is easily metabolized and conjugated, mink and ferrets appear able to excrete enough of the ‘compound ingested during a meal to avoid consuming a lethal dose during a whole day's feed consumption. 3) Mink are more sensitive to o-cresol than ferrets, based on results from diets of equal concentration fed to both species. 4) Dietary concentrations of o-cresol up to 1600 ppm have no significant effect on reproduction in mink. Experiment III - Results Mink LC50 Test: A literature search yielded acute oral LD50 values ranging from 350 ppm for rabbits (Matthiaschk, 1973) to 4000 ppm for male rats and mice (Lee at al., 1978), so a range-finding study was performed with mink and ferrets (Table 36). In the mink range-finding study, thiram was administered first at concen- trations of 10, 100, and 500 mg/kg suspended in water (since this was the intended carrier for mixing thiram in the test diets), with no observable effect. Since the toxicity of thiram has been reported to be greater in the presence of fats and oils (Merck Index, 1976), it was then decided to 100 Table 36. Results of range-finding studies with mink and ferrets exposed to thiram by gavage. Dose Died/ (mg/kg) total Comments Mink 2000 0/2 Corn oil carrier; both vomited dose within 20 min. 1000 0/2 Corn oil carrier; both vomited dose within 20 min. 1000 0/2 Water carrier; no effects observed 500 0/2 Corn oil carrier; both vomited dose within 20 min. 500 0/2 Water carrier; no effects observed 100 0/2 Water carrier; no effects observed 10 0/2 Water carrier; no effects observed Ferrets 200 0/2 Water carrier; both vomited dose within 20 min. 100 0/2 Water carrier; both vomited dose within 20 min. 50 0/2 Water carrier; 1 animal vomited dose after 14 min. lOl administer the compound suspended in corn oil. Doses of 500, 1000, and 2000 Emmi were administered, and were vomited by all animals. Since a lethal dose could not be found, 5000 ppm was selected as the highest concentration to be fed to the mink in the LC50 test. It was decided to perform a palatability test, since one of the uses of thiram is as a protective coating for ornamentals to prevent gnawing. A small portion of the basal diet was supplemented with enoughvaa_:azo o m e n N . co.“ vuu:mevs Aeaav. ace: 9 sea: gas: are: 32:3 sea: son: nas._uu< guacaeaoa cowucsacoucou .m»:: an to. Agode: :_ ll. .Ilu‘ll-.. cztccv Eag.za C: n::..=;s:95::u m::.;1> =2. . 'l I'l1- -.IIIIIIII'II 4:0 00. .‘III 1 n I... 9 0.! .l --I’O‘!’-.l'l|-ll ...:s 3 31.5259 3.5.53.3”. ....n 159.. 2:. 95:2? .........3 >25 in-.- -... . cull: I 0.! . II -.I 25......2 . R 5.....— io‘... ......I. III ‘0. l 11' .. 1‘ .0 10...... . x__cv sat. :o_u;E:wcoo c::ocscu z_xuc3 ..e.cu Co co.uat=v gab ——o econ :. suave Eas.gu ca.) “0.: to. u.es.:n a:.=.o§mz .~ any: a=.s=u 09.: .omaa no. m.ae.:o o=.=.aEmz o 42.5 5: 50.... page"... 3...: can £25235: Baum—.3 9...... 9.qu 25m .33 a2~ ..o 25:95:92.8 b t .mmmuzacocma :. cream n—os_=u o>._ be 5032:: co tomes mua=ozu “game: seen u m=o_secucmu=ou acouo.v was.“ eo.uae=m=ou emu» monco>n sac. ceso.:u.eu co.aas:m=oo sc=aasco x_.~= .s 3...... 3392.259 a .cc.aas:mcou whee u>.u:uumcou oz» Co emacc>n as“ no eons; :o.sas=m:eu ago. a c.eee~p cam Vmu nem+ IIIIO‘I'O'!’ Ill!- la.‘ . u>.ue_:s:u goo: e =.cN_m ..meen ~.~eon. m.o=m~ an- oc.me~ e szxae. :o.aaE:»:ou aczozscu n=.mee am.mac om.mme aa.=cv -11 on.mm o Ap\aav =o..:a:m=0u acacaaou can _Nm .mw cow .1. mw amn Ac\mv co.ucs=u:cu puma =.c~- w.no_. m.~m_+ Cc.m~¢ em.n + _.cmn- o.eov Am. mezegu aza.m: enem~ -.~o~ eo.ma— a szxaa :owuaesm:ou czzozsou we.~n ~m.o~ a Acxasw =o.d:6=m=ou azzoascu cw _n mom .:\a. =c.uas:m:oo sou. A_~_ts its. --- a.asm- o.oe+ .a. seemed 8:3.62 _ as: m=.cn— co.ne~ e szxae. =o_aaa=m=ou e::o;scu me.:_ -.en a Av\osv :o_.;s:m=ou veaoasou an nN sen Auxav :o.uas:u:ou come A_e.ca buoy P.nm~- e.nonu c.mn¢ “my eacozu aza.e: eke e m e n ~ _ so.“ outsmaos Azaa. x95: gas: gun: goo: goo: nas._uu< gouoaocea co_snc.=oo=au £55: .‘cllu ‘.Ill. 11!-" Ill'.lI| .I ‘1 n 0“] 'I.I 06! 0" "l‘ Onion! ’1 ... I '0'!". o. IOD'. I'llnllll '-I.- I. .I‘ 0'- .I o.l.ll‘|ll»6"ll.n.llcc. ....zas. .Nn 6.5a. .... I. .111- 0- -‘IIII.O'O|III.C’ Table 38. Initial and final body weights of male (M) and female (F) mink fed various concentrations of thiram for 28 days. Initial Final Concentration weight weight Change (ppm) Sex n (9) (9 (9) o M 5 1752.8 1867.0 +114.2 F 5 962.0 973.6 + 11.6 45 M 2 1453.0 1626.0 +173.o F 2 1200.0 1248.5 + 48.5 82 M 2 1677.0 1468.5 -208.5 F 2 1053.0 934.5 -118.5 147 M 5 1536.0 1213.0 -323.o F 5 1135.2 907.2 -228.0 265 M 4 ' 1576.5 1071.5 -505.o F 4 920.0 636.8 -283.2 1543 (611) ‘ M 4 1231.5 1628.2 +396.7 F 4 713.2 1003.8 +290.6 Table 39. Organ weights of mink fed various concentratios of thiram for 28 days. Concentration Brain Liver Spleen Kidney Lung Heart (ppm) n (% body) (2 body) (74 body) (7. body) (% body) (% body) 0 10 0.66 4.72 0.27 0.53 0.71 0.59 45 4 0.68 4.36 0.42 0.59 0.60 0.52 82 4 0.77 4.59 0.42 0.55 0.76 0.66 1543 (oil vehicle) 8 0.72 4.54 0.45* 0.58 0.70 0.61 (Organs expressed as a percent of brain weight) 0 10 739.5 40.9 83.6 110.7 92.4 45 4 645.9 59.6 86.4 88:2 77.2 82 4 . _ 604.9 55.3 72.7 99.5 88.1 1543 (oil vehic1e) 8 645.1 62.2** 82.1 98.5 87.1 * . Significantly different from control (P < .05). g ** Significantly different from control (P .01). 108 not every episode resulted in death. Curiously,two mink in groups that were removed from the trial were observed to have these convulsions after several days on clean feed. No gross lesions were noted at necropsy of mortalities during the trial or at the terminal kill (7 December, 1982). The only hematologic parameter analyzed in the 28-day test was hematocrit. Averages obtained for the 0, 45, and 82 ppm groups were 57.6, 51.2, and 48.5, respectively, with the two treated groups being significantly different from control (P < .01). The hematocrit for the 1543 ppm group. 51.9, was also significantly different from control (P < .01). The organ weight data, summarized in Table 39, was not analyzed by sex as in other tests, due to the small numbers involved. Significant increases were found in the spleen weight of the 1543 ppm group, however. Ferret LC50 Test: Since the ferret LC50 test was conducted after the mink test, the previously mentioned problems were avoided. Doses of 50, 100, and 200 ppm, suspended in water, were administered, and 5 of 6 animals likewise vomited the dose.‘ Again, no lethal concentration was found in the range-finding test, so a palat- ability test was conducted. Concentrations of 100, 500, and 1000 ppm of thiram were added to the basal diet in water and fed to previously-starved ferrets, and only the 100 ppm diet was consumed in normal amounts. Therefore, dietary concentrations of 0, 8, 20, 50, 125, and 312 ppm were chosen (each concentra- tion being 2-5 times the previous concentration, since it was decided that a wider range of dietary concentrations would be necessary to ensure finding a concentration that would be eaten for 28 days. A 21-day acclimation period was begun on 16 December, 1982 for the ferrets. The 28-day test began on 6 January, 1983 and ended on 2 February, 1983. Signs of intoxication were first noted on day 4, when two animals fed the 312 ppm diet were found to have tarry stools. Reduced feed consumption was noted in this group, and a transient reduction in feed consumption was also noted for some animals in the 50 and 125 ppm groups in the first week. Body weight changes and feed and compound consumption are summarized in Tables 40 and 41. Clinical signs were only noted in the 312 ppm group, and included inanition,tarry stools, listlessness, uncoordination, and the same type of convulsions described for the mink trial. All animals on the 312 ppm diet had died by day 16, while no deaths were noted on any other dietary concentration. The mortality pattern is described in Table 42. No gross lesions were noted at necropsy of mortalities during the trial or at the terminal kill (3 February, 1983). The data obtained from the LC50 test was unsuitable for analysis. The LC50 for thiram for ferrets was between 125 and 312 ppm. The hematologic parameters, summarized in Table 43, showed decreases in RBC count and hemoglobin concentration at 50 and 125 ppm. A significant decrease in hematocrit was seen in the 50 ppm group, but not the 125 ppm group. The organ weight data, summarized in Table 44, revealed significant increases in 110 3.8.. so... ..o.........8_.8u 2.2.8858 8888588.»... ... 5.9.8 8.8.8.... 8..: .8.. .8885: 8.: .... 888.... 89.888 2.38.. ...8.... 8 2.88... 2.8.88.5...8883 8.38:. 88...: 882958.88 :88; 09:8... ...8.... 8323—88 ....Zaeacou 9.588.888 8:3 68.885883 .8828 838888883 ...: .8 888.888 8.: cc 8888.. 88.88.888.88 88... 8 .8 3...: 88.8.8533 p ...888 --- --- 88.88. 8..8.8 8 ..8.88. 88.8888m888 88888888 --- --- --- 8..88 .8.88 8 .8.88. 88.8888.8oo 88888888 --- --- --- 8.. .8. 88. .8.8. 88.88888888 888. --- -- -- 8.88.- 8.8..- 8.8.. .8. 888888 888.8: 8.. 88.888 88.88. 88.8.. 88.... 88.8.. 8 ..8.88 88.88888888 88888888 . --- 88... 88.8. 8..88 88... 8 .8.88 88..8888888 88888888 --- .8. 888 .8. 88. 888 .8.8. 88.88888888 888. 8.8.88.88.- 8.... 8.8 . 8.8. - 8... - 8.88. .8. 888888 888.88 8.. 8.. 8. 88.88 8...8 88... .8.88 8 ..:.88. 88.8888m888 88888888 --- .8.8 88.8 88.8. .8.8 . 8 .8.88. 88.88888888 88888888 --- ... 88. 888 88. .88 .8.8. 88.88888888 888. 8.8....8. . 8...- ..8.. ... 8 8.. - ~..8+ .8. 888888 888.88 88 88.8.. ...8. 8.... 88.88 .8... 8 ..:.88w 88.88888888 88888888 --- 88.8 8..8 88.8 .8.. 8 .8.88 88.88888888 88888888 --- 8.8 .8. 88. .8. ..~ .8.8. 88.88888888 888. 8.8..8.8 4 8...- 8.8.. 8.8. - 8.8. 8 8.88. .8. 888888 888.88 8. 88..8 .8... 88... 8..8. ...8. 8 ..8.88. 88.88888888 88888888 -- 3. _ .3. _ N... ~ an; a A29... :c_u.......m..cu 9:68—85”. -- .8. ... .88 .8. .88 .8.8. 88.88888888 888. 8.8..8..8 . 8...- 8.8.. 8.8. + 8.88 + 8.88. .8. 888888 888.88 8 o a a o o o :33... :o_a.....:8..ou 9.59.85... -- c a = c o 283.... cc.a.......8....u ......c.....cu --- 8.. 88. 88. 8.. 88. .8.8. 88..8888888 888. 8.8....88 . 8.. - 8.88. 8.8 8 ..88 8 8.8.. .8. 888888 888.83 8 8.. . 8 8 . .885 8 8 ~ — .... . 8 88.58.85 A583 8.8.8: ...8.... ...8.... 8.88: 18.... 38¢ ...... 988...... .3. ...-5:3..3 "Oill all- . I I - 8...... ..m .5. 8.8.2: .o 8.538.258.58 8.5.2.... ...... 8.8-...... I'll..- '1’. '11 ‘llo. '0! I --.!7III‘ 'I dulll il1 l 3.- . II’I'.I|" ‘0 'I- '9'-a--1'i‘-‘Oi7"l""| Ill 1 .. I1 80"!.'-n. 3 89.58.58 .....5.......ou ....o 8.888... ...... 8..... III. '5'17' ...-'0' :23 8:38: 3:... 8......82 .3 ...—...8. Table 41. Initial and final body weights of male (M) and female (F) ferrets fed various concentrations of thiram for 28 days. Concentration Initial Final Change (me) Sex n wgt. (g) wgt. (g) (g) 0 M 5 l923.6 208l.8 +l53 2 F 5 933.4 950.d + l7 0 8 M S l399.6 2074.4 +l74.3 F 5 795.6 802.8 + 7.2 20 M 5 2033.2 2097.4 + 5-,2* F 5 906.8 86l.8 - 45.0 53 H 5 l906.6 l957.2 + 60.5* F 5 860.8 834.4 - 26.; l25 H 5 l993.2 l875.3* -l22.;** F 5 90l.6 337.4 - 4,28. 3l2 M 5 l95l.6 l623.0** -323 ~** F 5 949.2 763.0 -l86 2** * ** Significantly different from control (P < .0l). Significantly different from control (P S .05). 112 _ — N N m N . mwm em cm o ’l S. a- a Q. -N _N i 2 ......2.-.£ m. .3 2 N. E S a a N ... m Q n N _ :53 ......I. ......-.!....I.-‘.!. 32?: .2 $39...-»WWQEE t .2. ....Eszszs O '0'" i'il.‘ I... l‘-.vn" -Oq‘- .IOZ-“ 'II‘I'.I.AI1'-'1-I. .‘III‘ .I 1“. Ila."|l.\-lll0- I I.» "l': .gmoa emu; >c=-mm c azmgza Eag.;u aw» mumggmu be :gmuum: xu__cagc= .we m_;ep Tab1e 43. B1ood parameters of ferrets fed various concentrations of thiram for 28 days. Concentration RBC NBC Hb Hct (ppm) n (x105) (x103) (g/dl) (%) o 10 11.29a 10.0 20.9 48.8 :_0.682 :_3 55 :_1.31 :_3.37 8 10 10.57 8.6 20.1 47.6 :_0.875 :_2 14 :_1.75 :_5.37 20 10 10.24 18.5 18.7 48.4 :OJm :nzz ‘izm :231 50 10 8.19** 22.4 16.6** 42.3* :_1.549 :84.08 :_3.02 :_6.92 125 10 8.57** 16.8 17.1** 46.0 :_1.151 191.57 :_2.17 :_4.24 312 o --- --- --- --- a Mean :_S.D. * Significant1y different from contro1 (P < .05). ** Significant1y different from contro1 (P < 114 Tab1e 44. Body and organ weights of ma1e (M) and fema1e (F) ferrets fed various concentrations of thiram for 28 days. Concentration Body Brain Liver Sp1een Kidney Lung Heart (ppm) wgt. (g) (% body) (% body) (% body) (% body) (% body) (% body) 0 M 2081.8 .386 3.359 .561 .480 .432 .348 F 950.5 .657 3.846 .393 .484 .574 .435 Ca --- -- -4- .477 -- -- -- 8 M 2074.4 .375 3.524 .572 .475 .413 .314 F 802.8 .773* 3.859 .481 .506 .621 .489 C --- -- --- .527 -- -- -- 20 M 2097.4 .405 3.960 .682 .497 . .434 .389 F 861.8 .764* 3.913 .551 .539 .592 .519* C --- -- --- .617 -- -- -- 50 M 1957.2 .411 3.744 .704 .520 .481 .406 F 834.4 .716 4.160 .911 .536 .612 .517* C --- -- --- .807* -- -- -- 125 M 1875.8* .436 3.928 .796 .516 .495 .418 F 807.4 .813** 4.168 1.003 .571* .678 .526** C --- -- --- .900** -- -- -- 312 M 1628.0** .501* 4.091* .668 .525 .655** .460** F 763.0 .892** 4.455 .825 .642** .877** .596** C --- -- --- .746* -- -- -- Organs expressed as a percent of brain weight 0 M 869.3 145.4 124.7 112.0 90.1 F 592.6 60.4 74.2 87.4 66.5 c --- --- --- --- -- 8 M 949.4 153.4 127.5 110.8 85.1 F 500.5 62.0 65.5 80.3 63.4 c --- --- --- --- -- 20 M 985.1 170.3 123.0 107.0 96.7 F 520.4 73.7 71.1 77.7 68.4 C ..-- --- --.. ..-- -- 50 M 913.6 169.9 126.7 117.1 99.3 F 577.7 122.0 74.4 84.9 71.8 C --- --- --- --- -- 125 M 901.9 184.0 118.5 113.9 96.2 F 517.7 125.3* 70.4 83.0 65.1 C --- ..-- ..-- -..- .. 312 M 824.1 133.5 106.0 131.3* 92.4 F 501.3 92.5 72.1 98.8 67.0 C --- --- --- --- -- a Sexes are combined where no significant difference (P < .05) was found between them. * Significantly different from contro1 (P < .05). ** Significant1y different from contro1 (P < .01). all organs as a percent of body weight at 312 ppm, but only in the lungs of males when expressed as a percent of brain weight. An increase in spleen weight was also seen at 125 ppm, both as a percentage of body and of brain weight, and the kidneys and hearts of females, expressed as a percentage of body weight, were increased at 125 ppm. Mink Reproduction Test: Based on the results of the LC50 trial, where adverse effects were noted in mink at 82 ppm, dietary concentrations of 0, 2.5, 10.0, and 40.0 ppm were selected. Distilled water was the carrier used. A Zl-day acclimation period was begun on 16 December, 1982. The reproduction test was initiated on 6 January, 1983 and terminated on 22 June, 1983. Weight changes were recorded bi-weekly until 3 March, 1983, and feed consumption was measured during weeks 1, 3, 5, and 7. Body weight changes and feed and compound consumption are summarized in Tables 45 and 46. No signs of intoxication or mortalities due to effects of the compound were noted (one female mink died of virus enteritis), but male mink fed 40 ppm gained significantly less weight than did controls over the 8 weeks of the pre-breeding period. No obvious birth defects were found, and no gross lesions were noted at necropsy at the terminal kill (22 June, 1983). The reproductive indices for mink (Tables 47 and 48) revealed no significant effects of thiram on the reproductive performance of the females except decreased birth weight at 116 .Amo_asoc __ .mopns 9v m—ns_=o m. co some; moccagu u:m_o3 xaoa “museumaam awe“ and mo muumuuu cu noun—ac do: mumano eon. co_v «.cég. 9:0 3 .Amo_nSmu w. .mo—ae «v aposvca o— ecu uo=_n> :6 come: maacezo “za_u; seam .v— was.» :c.daa:n:ou >—.au sot. :o_uas=mcco ccaoasou a—xmux _a .co.unc~=aucoo munuumv mos.“ co—aasamcou some umuuu>a sore noun—:o_uo co.uas:mcou czaoasoo >__e: .:o_uas:m:ou. «has o>.u:uomcou ex» ea maoto>a age so come: :a..;s:n:ao any.« a on.o~n nn._a. No.nm o~.n__ n=.no_ a Ax; ~\ms en_nae=n=oo neznasno --- o~.~ ma.m cm.a mm.~ . o Au\a5 =o.uasam:ou scaoasau --- Ne. nn. now an. no. .n\s. =o_nae=neo« n««u n.~n- n.on- n.n_- e.n + ..nn- o.nn+ .a. «mango ngs.«z o.=e n«.,~_ Nn.cm en._n n~.on am.n~ Q As; ~\ms eo_naa=n=o« senoaenu --- a_.~ c~.~ o—.~ so.~ o Avxms :o_uasam:ou uzzoqsou -- s_~ nNN n_~ new an. An\a. nn_nas=neo« n««a n«.n~- m.mn- n.-+ ..n + n.~ - n.o~+ any «mango n=s_«3 o.o. oa.m~ c~.u -.u as.“ em.~ o Ax: ~\me :o_uaszm=ou u::anou --- mm.c we.c mm.c mm.c o Aexas :o_aas:mcou assessou --- cNN No. awn -~ on. An\s. eo_nas=n=o« n««u on.“ - ~.nn. n.n_+ o.n_. ~.n + o.nn+ . An. «mango n=a_«= n.~ o c o c o o as: «\as co.u;samcou venoasou c o o o o o auxas :o_uasam:au uczoaaou --- «a. n_~ .nn own n_~ , .n\a. no_naa=nen« n««a «o.n + ..nn- ..n + m.n n n.n~+ c.nm. .9. «nennu n;a_«: o o>.un_:s:u m-n u-m o-m ~u~ so.“ uncanny: Azaav _ «any: a «gem: «sum: axooz axon: uns.—uu< tuaoaaunm =o_dotucou=ou :o_ao:uouquu a m:_t:u Eag.za no m:o_~oua=oucoo m:o_te> - It’ll {4! I‘ll .umuu an. x:.& as uosaucou aucaanOu age «can. ecu maacnzu n=m_ox sue: «antu>< .me «_anp l 1.7 Tab1e 46. Initia1 and fina1 body weights of ma1e (M) and fema1e(F) mink fed various concentrations of thiram for 8 weeks prior to breeding. . Concentration Initia1 Fina1 Change (ppm) Sex n wgt. (9) wgt. (9) (9) 0 M 4 2095.2 2222.0 +126.8 F 12 1127.8 1089.6 ~ 38.2 2.5 M 4 1790.2 1849.2 + 59.0 F 12 1060.8 1033.5 - 27.3 10.0 M 4 1831.5 1840.0 + 8.5 F 11a 1142.3 1101.2 - 41.1 40.0 M 4 2079.5 1940.2 -139.2 F 12 1108.6 1044.7 - 63.9 a One fema1e died from causes not re1ated * Significant1y different from contro1 (P < .05). to effects of the test substance. 118 .:o_u_;=uenn ;u_z umum_oommn msm_aoca soc» uw_v opnseu ace 1. n .o.m + mcamz n on._ + n.n .n.~ + n.n kn.~ + ..ne N_\n ~_\N_ c.on snunn znuné Sewage ::_ .2: 92 no.__H.n.m ne.~.fl o.m n_.n.u m.nn m \nn ~_\m m.~ an...“ _.n mo.~.u o.m nnn.m.u a.mn N.\op N_\~_ o uma_m;3 0 weapon: a Amznuv toga m.o _nuou AEQQV \nn_x _nnnn \nn_x «>25 eo_nnnn«u \n«a_«nz n.« \n«nn n.« nonnnnnenneou .mxmoz mm to» anc_;a no m:o_uncu=mo:oo m:o_c«> no» x:_a «pose» no moanELomcma m>_au:nocamz .me m_nnp 119 .A_o. w av _onneou sort n=«n«cc_n s_nen«_c_=m_m n; .mocmumnzm “may no muomccm ca noun—occz mmmzno ou man Emu no gamma seem umo— twuu__ muzpo:_ no: moo: u .ann an n«___x n«3n__ «n=_«e_ no: n«na n .a.m.fl manna n :8 H 22.: H 8. a H can H 38 H in. .... w «N.Nn «n.nn n___ m.mnn n.m~ ..nmm n.nn ..m.n o.on 1. n.m.n.fl an.nnm.fl nn._~.fl no.nn.H mn.o_.H as...“ n.nn n.nn new. ~.mnn ..nq ..c.n_n ..~.n__ o.n o.o_ m._nn.H mm.nnm.fl nn.N~.H ~_.~n.fl _m.N~.fl an._.w o.on o.nn _no_ n.nam «.nn N.on~ n.nm m.m m.~ n._nn.fl an._m_.H _n.oN.H on.nn.u n_.n_.u nan._.u no.nn o.mm nn__ ..o_e m.nn N.nnn n.na n.m o mxmoz c mxmoz m mxooz c mxmm: m :ugwm mxmmz c mxmmz m zucmm Asaav _n>_>c:m a AmMu:m_mz cmuamp wmncm>< va u;a_m3 moon u_x omncm>< conumcucoozou .mxmmz mm to» Scene“ to m:o_uncu:mu:oo m:o_cn> no» msnu to» _n>_>e:m u_x czn mu=a_mz Lmuu__ new xuoa “_x mmncm>< .we m_anh Tab1e 49. B1ood parameters of mink fed various concentrations of thiram for 6 months. NBC (x103) Hct (%) Concentration RBC (ppm) n (X105) 0 8 11.05a :_0.606 2.5 8 10.38 1.1.073 10.0 8 10.05 1.1.172 40.0 7 8.79** + 0.761 7 68 0 16 .8 .70 .8 .69 I + |+ l+ Q’s: |+ 09.4 .2 .74 .2 .86 .8 .18 .2* .11 a Mean :_S.D. * Significant1y different from contro1 ( ** Significant1y different from contro1 ( .A_c. w as _nnnnon sort n=«««cc_n »_nen«,c.=anm . .1 m.oo ~.mw —.m~ «em.wm «.mem u c.8— ~.:; —.:: 25.3 ~.:w z 9.3 m.mw ~.mw p.o~ ~.mm c.e~m . m m.oo ~.wm m.mm ~.- e.oco . z c.o_ ..mo N.om m.wc m._m _.mmc u o.oo m.xpp m.mop m.em o.woo : m.~ 3.0N N.ow ~.o~ ¢.me c.c~e u o.~m e.m_p m.mm w.mm m.omm z o u;mmm3 specs to acmorma a ma commmtaxm mzcmco o_m. mum. mam. cam. ~_~.e cum. o.cmm u m_c. new. cam. 2mm. emo.e mam. ~.e_cp z o.oe moo. mac. _oo. woe. om_.¢ was. m.~oo_ u Nmm. mam. com. nme. m—~.m «pm. m.~mm— z o.o_ own. mmw. Mmm. oom. mae.m omm. m.o~m u new. _mo. coo. N—m. aee.n mum. m.emm— z m.~ ear. mmu. :30. wow. _Nm.m mew. m.~_a u vac. moo. com. wmm. “mm.m msm. o.mmmp z o Aston xv . Aston a Axvc: xv Axcoa NV Axaoa xv Assoc av «my .ua: Aaaav beam: azab xmcvmx com—am Lo>_4 cpucm atom copuntucoozou | ...- .2322. m .8.. scc_;n do mzawuetazoozoo mzowcn> sob xzwa Adv wpmsw» 5:8 sz opus no muzmmoz cauto use macs ..xw m_anp 40 ppm. In fact, significant increases in kit body weight at 3 and 6 weeks were seen for the 10 ppm group. The hematologic parameters (Table 49)showed decreases in RBC count, hemoglobin concentration, and hematocrit at 40 ppm. The organ weight data (Table 50) revealed a significant increase in the spleen (as a percentage of brain weight) for both sexes at 40 ppm. Ferret Reproduction Test: Based on the results of the LC50 test, where adverse effects were noted in ferrets at 20 ppm, dietary concentrations of 0, 4, l6, and 64 ppm were selected. Distilled water was the carrier used. A 9-day acclimation period was begun on 7 February, 1983. The reproduction test was initiated on 17 February, 1983 and terminated on 7 July, 1983. Weight changes were recorded bi- weekly until 14 April, 1983, and feed consumption was measured during weeks 1, 3, 5, and 7. Body weight changes and feed and thiram consumption are summarized in Tables 51 and 52. Female ferrets fed 64 ppm lost less weight than did controls during the 8 weeks of the pre- breeding period. No signs of intoxication or mortalities due to effects of the compound were noted during the test, but necrOpsy at the terminal kill revealed white nodules ranging in size from 0.5-3 mm on the lungs of some ferrets on all dietary concentrations, including controls, and may be indica- tive of pneumonia in the ferret strain. 123 .Amo_n59» ~— .mopes av m_aswcu e. to. mo=_a> :o conga mucaazo o;a_:; \232 a >._nu sate :o_oas:m:co uzzoaaoo x_gac:-_a aco_~nc~:aoaoo acasa_u mas_u co_uae:m:oo come omnum>u soc. uu.a_=o_au =a_oae:n:co eaaeasco x__na .e— m25_. ::__;§:n::o a .co.u;e:m:ou.wxnu o>_u:uum:ou ozu no oanga>c as“ a: noun: :a.s;szm=oo can. . a c Ax: ~\osv cowsnsamzoo cazoascu ca.n_m 4a.... a~.a__ Nm.cm_ an.nn_ --- mm.“ m .c w:.a em.o_ a Auxmsv co_u;E:u:0o szzoaaou --- nm. Mn. nm. No. mo~ A=\a. ao_oas=naao noon «can - ...8- man- an - 3.... 9... n .3 «aanao 2.32. in $6: 8.2 8.: 2.: 8.3 a o... 295 539.528 958.3 --- o~.~ me.~ .e.~ -.~ o ,vxos~ =o_uas:m:ou aczoeaou --- nn_ mm. _n_ on. o_~ a\av aa_oasanaoo a««a oo.nm - n.c=- n.~m- ..._- m.am+ a.m + an. «mango naa_«= c.a_ «a.nn na.n on.n ~m.a an.o_ a Ax: ~\aa. aa.naaa«aao naaaaaao --- Km.o mm.o mm.o m~.o c Auxos. :o_uas:m:ou nanoQEou --- N". am. an. an. _o~ .a\a. aa_nasanaao n««u o_.n~_- ..ca- n.nm- m.o - o.nn. a.c_+ .m. «mango oaa_«z o.n o o o c o c as: ~\oev cowuaszm:oo uzzoaaou --- o o o o o avxmev :o_uaaamcoo scacasou --- an. as. on. as. m_~ Acxa. ao_oae:naoo noou on.a~.- n.aa- «.mm- ..m + o.~n. n.nm+ Am. «canao oaa.«: a o>.uu—:e=u v n m _ co.“ nocznsga Asaav 3903 36:3 awe: . 3093 loam—uu< gvaofinsog co_aasu:cu:cu -uzaoaauu a m:_c:= Eacpza no m:o_uucu=oo:oo m:a_;n> as» .Il‘l ousll'i .dnou :9.“ “button as ensemcou aucacasou can some» uco mamznzo g:m_uz xco: mancu><. _n u_an_ 1224 Tab1e 52. Initia1 and fina1 body weights of ma1e (M) and fema1e (F) ferrets fed various concentrations of thiram for 8 weeks prior to breeding. Concentration Initia1 Fina1 Change (ppm) Sex n wgt. (9) wgt. (g) (g) 0 M 4 1980.2 1831.8 - 98 5 F 12 925.0 787.2 -137 8 4.0 M 4 1819.2 1709.8 -109.5 F 12 877.8 743.2 -134.5 16.0 H 4 1728.0 1658.0 - 70.0 F 12 869.8 777.1 - 92.7 64.0 M 4 1779.8 1689.2 -127.8 F 12 909.7 833.1 - 76.5* ‘r p \ Significant1y different from controi (P < .85). 125 A; H 2.82 a .:o_awt=ucaa :u_3 uoan_oommo msmpaogc soc; uo_u o_nsou mac n a a ..-- to NE 93 n: H E z; .... Na and .+. 9:. SB 25 an: 8n H n: 3.. H W: :5 ..n n: N2... NS. 3. 88 H ~.: a: .+. 9.: name ..... W; . N: nm. 23. o van—mg; o cog—«=3 o Amxncv coca m.o _nbou Asaav ma_x _nuoh \muw_ m>p4 :o_uoummu \uma_ozz m.o \voca m.o :o_uncu=mo:ou ..Il .mxmmz MN com snt_;a to meowuncucmozoo m=o_cn> cm; mumccmm m_nswm to mucoscomcmq o>_no:cocamm .muw m_an» 126 .A_o. w av _aanaao scan oa«a«aa_a s_nano_a_aa_m a: .Ana. w an _onoaao eon. oa«a«ac_a s_oano_cnaa_m I. an .a.m + mane: n -- -- --- --- --- --- --- --- o.em a.nam.fl an.a_~.u an.n~.fl n~._n.w nm.n~.fl an...“ m..@ m.om «nomm_ «a~.eoo «~.mo e.oom «am.o~ ~.w c.o_ N.nnm.w n~.an_.w mn.n_.m no.nm.H on.n_.H ma...“ ~.mm _.mm oamm ~.Nom ~.mm «am.~¢~ NCNQ o.m o.¢ n.-n.H om.ma .H _n.n_.H a_.mm.w na.n_.m nnm._.H P.em m.mm mmmm ¢.omm ~.ea m.o~m m.em m.w o xmmz o gum: m xmmz o 3863 m sauna gum; o goo: m zua_m Asaav _n>_>c=m N Amwu;m_mz Lmuum_ mmnam>< Amy uzmpmz zoos up; mmncm>< =o_unao=mo=ou .mxmoz m~ to» Enc_:u mo m:o_uneu:oo:oo m:o_am> to» want to» _n>_>c:m u_x new mu=m_03 gmuu_— use xuon u_x mmncm>< .41“ c.2np 127 Tab1e 55. B1ood parameters of ferrets fed various concentrations of thiram for 6 months. Concentration RBC WBC Hb Hct (ppm) 0 (x105) (x103) (9/d1) (%) 0 8 12.08a 11.5 22.4 57.3 i 1.069 _+_ 3.65 i 1.71 i 2.54 4 8 11.00 12.3 21.4 56.2 i 0.457 1 2.47 i 1.43 _+_ 3.98 16 8 10.91 13.1 21.5 55.8 i 0.826 3*. 4.47 _+_ 2.46 16.37 64 8 8.92** 14.8 18.8** 49.2** _+_ 1.587 16.84 +1.66 2‘. 5.59 a Mean : S.D. * 128 * Significant1y different from contro1 (P < .01). .APO. w av _ocucoo soc» uzwcmue_v x—ucmo¢u_=mwm ¥ * .Amo. 0 av _aanaao sane oaoaoccna s_oano_caaa_m i .565“ :mozbwa scaoc mm: Ame. w av mocmtmcu_u accopmmcmwm o: mews: cmcpaaoo one mmxom a ill _ III III III III Q m.oo m.mm m.mm ~.em m.mmm u m.mo_ m.om_ w.m_~ na_.mm~ N.e- 2 co III III III III Ill 0 N.mm o.om m.m~ m.mm ~._em u m.om p.np_ ~.Nm_ 4_.m_N m.o- z @— III ill III III III U _.NN _.oa m.mw o._m ~.m_m u m.mm o.wp_ c.oN_ o.¢~_ .w.~_~ z e III III III III III U ..ek m.mo w.mw v._m o.m~m u m.¢o~ o.w~_ m.wmp w.w- o.ocm z o u;o_mz :_mca mo bemoama a an ummmmtaxm manage - - _No. «nwmm.— 1- -1 -- 0 man. 4a~_w. moo. ~N~.p mum.e mmm. N.wm u mam. cox. mew. mew.. mkw.m _em. w.o~v_ 2 no - - omo. ««_om. 1-1 . - -- 9 com. NmN. mmo. Nam. owm.e o_w. o.~m u __m. wpo. new. om_._ “no.8 Kmm. o.vme_ z o— - - «No. axe. 111 - -- 0 _mm. max. mac. one. ~_N.e «mm. o.~a~ u me. ewe. com. www. o_a.m mmm. w.mmc_ z a - - owe. vwm. -- - 11- no Nmm. ace. moo. mom. mmp.¢ pmk. o.m_m u _mm. mmo. axe. moo. moo.e mmm. m.mem_ z c Anna; av Anson no Anson NV Anson NV lanes nv “anon RV Aav .oaa Aaaa. “Lao: can; Aw:v_x compam Lm>p4 c_mcm Atom :omumcucoucoo .mgucos o to» smcwzb do mzomuacucoozoo maowcc> won mbmccmw Adv m—msm» vzn sz o_as no mbzm_oz =cmco can atom . om m_amp No obvious birth defects were observed. Reproduction was prevented in the 64 ppm diet, and the reproductive indices (Tables 53 and 54) revealed effects on the growth of the kits at 4 and 16 ppm as well. The hematologic parameters (Table 55) showed decreases in RBC count, hemoglobin concentration, and hematocrit at the highest dietary concentration, as in the mink reproduction test. Likewise, the organ weight data (Table 56) revealed increased spleen weight at the highest concentration, as in the mink reproduction test, as well as at the middle level. An increase in the lung weight (as a percentage of body weight) of females fed 64 ppm was also seen. DISCUSSION Thiram affected feed consumption and body weight gain in mink at dietary concentrations of 82 ppm and greater and in ferrets at 50 ppm and greater. Feed consumption and body weight have been reported to be negatively affected in rats (Lee 33 31., 1978; Lowy gt_al,, 1980), chickens (Waibel 33 31., 1957; Rasul and Howell, 1974), turkeys, and geese (Waibel 35 51., 1957) during subacute exposure to 150 to 400 ppm of thiram. It is interesting to note that at_20 ppm male ferret body weight was significantly affected, even though feed consumption during the test was approximately equal to controls. This illustrates: 1) the greater sensitivity of ferrets to thiram than the rat, chicken, turkey, and goose, and 2) the possibility that thiram affects body weight in a manner not entirely due to feed intake. The effect of 20 ppm dietary thiram was also more severe in the males, illustrating the fact that, since the average male body weight is approximately twice the average female body weight (in both ferrets and mink), the male has more weight (as body fat) to lose during a subacute poisoning. It is difficult to draw strong conclusions from the mink LC50 data, or to make comparisons to the ferret data based on such fragmentary results. It is apparent that 82 ppm thiram added in water was detrimental to mink, while the results from the 45 ppm group indicate that this concentration may be safe for mink (it must be remembered that only four animals were exposed in both of these groups). In stark contrast to these results, the eight mink fed 1543 ppm thiram added in oil con- sumed a normal amount of feed over 25 days (with a cumulative intake of > 12000 mg of thiram) and regained all the weight lost during the week of exposure to 1543 ppm thiram added in water. (During this week, the mink's feed consumption averaged only 25 g/d, and two animals died). From these results, it is apparent that the claim that the toxicity of thiram is enhanced in the presence of fats and oils (Merck Index, 1976) does not hold for mink. In fact, the opposite was true in this test. With regard to the reproduction tests, an inspection of the body weight data reveals a dose-dependent decrease in weight change for male mink, with a significant decrease at 40 ppm (P < .05), although the average weight of the males in this group was within the range of normal body weights for male mink. No such pattern was evident for the ferrets. In fact, 131 the weight loss for female ferrets fed 64 ppm was significantly less than the loss by controls (P < .05). This may be indica- tive of a disruption in the normal pre-estrus pattern of female ferrets, which is marked by reduced feed consumption and weight loss. Further evidence of a disruption of the normal reproductive cycle of female ferrets was evident from the number of females bred. In the 64 ppm group, only 7 of 12 females were judged to be in estrus (as determined by the extent of vulvar swelling) at the termination of the 8-week exposure period, and none of the 12 whelped. Evidence of disruption of the normal repro- ductive cycle of female mink is more difficult to quantify, since female mink do not normally exhibit decreased feed consumption and weight loss of the same magnitude as female ferrets, nor do they exhibit noticeable vulvar swelling during estrus. Thiram has been associated with disruptions of the estrus cycle in previous experiments. Davydova (1973) found an extension of the estrus cycle at the expense of the resting cycle in rats exposed to 3.8 mg thiram/m3 of air for 5 hr/day, 5 days/wk for 4.5 months. A prolonging of the diestrus phase has been reported in rats fed 400 ppm thiram in the diet for 14 days prior to mating. Wedig et 31. (1968) reported inhibi- tion of ovulation in bobwhite quail at oral doses as low as 18.8 mg/kg/day. I The only reproductive effect of thiram on female mink was a significant decrease in the average kit birth weight at 40 ppm (P < .05). All other reproductive indices were within the range of normal values (although the number of females whelped and the number of live kits per female at 40 ppm was quite low). Actually, the 3 week and 6 week body weights of kits on the 10 ppm diet were significantly greater than controls, and were somewhat above the normal range. Since the litter size was not decreased at 10 ppm this high body weight was not due to fewer but larger kits. The reason for this increase is unknown. The reproductive effect of thiram on female ferrets was more severe. Reproduction was prevented at 64 ppm, and kit body weight at 3 weeks and litter weight at birth, 3, and 6 weeks are all significantly less than controls (P S .05) at 16 ppm. Six week survival was also less than control (P < .10). (The same trend of decreasedéiweek survival was apparent in the mink reproduction test, although the decrease at 40 ppm was not significant). From the results of these two reproduction tests, it would appear that female ferrets are more sensitive to thiram than female mink. Decreases in the number of offspring per female, birth weight, and/or offspring viability have been reported in several studies with thiram. Robens (1969) has reported increased resorption of fetuses, low birth weight, and decreased viabi- lity of offspring in hamsters given 125 or 250 mg/kg thiram orally from days 5-15 of gestation. Short 33 El. (1976) found a significant decrease in the number of implants and number of pups/female in rats fed 400 ppm in the diet for 14 days prior to mating. These authors also reported no reproduction due to 100% resorption at oral doses of 164 and 200 mg/kg, decreased number of pups/fema1e at 136, 164, and 200 mg/kg, and decreased live birth weight at all doses (40, 90, and 136 mg/kg) for rats given thiram po during days 6-15 of gestation. In similar studies with mice, these authors noted no changes in litter sizes, incidence of resorption, or birth weight at oral doses of 100 or 300 mg/kg administered during days 6-14 of gestation. Finally, these authors found no effect on viability or growth with rats fed 300 ppm of thiram in the diet from day 16 of gestation through day 21 post partum, but 100% mortality of pups by day 21 post partum at a dietary concentration of 1000 ppm. Examination of the blood and organ weight data of the four tests revealed a consistent pattern of response in mink and ferrets upon dietary exposure to thiram. The hematologic parameters showed an anemic condition at the highest dietary concentrations for all tests, while the spleen weights (as a percentage of brain weight) at these same levels were increased over controls. Whether these results are related would require further tests. Increased spleen weight has been reported by Lee and Peters (1976) in rats fed 400 and 1000 ppm thiram in the diet for 80 weeks, but not in rats fed 100 ppm for this length of time. These authors also reported increased weights of liver, kidney, and brain at 1000 ppm, results seen only in the ferret LC50 test among these experiments. They also reported no changes in blood chemistry in this study, while decreases in RBC count, hemoglobin concentration, and hematocrit occurred at the highest dietary concentration in 134 all tests in which these parameters were determined. Increased lung weight was occasionally seen in these experiments, although the cause and significance of these findings are unclear. The dietary no effect level of thiram for rats has been reported to be 38 ppm (Lowy at 31., 1979; 1980) or 48 ppm (WHO, 1965). In the LC50 tests of this experiment, a dietary no effect level was not found for mink (since a decrease in hematocrit was seen at 45 ppm), while the no effect level for ferrets may have been 8 ppm (if the increase in brain weight of females at this dietary concentration is regarded as not biologically significant). These results again illustrate the relative sensitivity of mink and ferrets to toxic compounds. In fact, the results of an 80 week chronic feeding study with rats (Lee and Peters, 1976) show just how sensitive mink and ferrets are to thiram in relation to rats. In this study, some rats survived the highest dietary concentration, 1000 ppm, for the 80 weeks of the study, while in the 28-day LC50 tests, mink were removed from the test at concentrations of 2 147 ppm (to prevent the animals from starving) and all ferrets fed 312 ppm had died by the 16th day of the test. CONCLUSIONS The following conclusions may be drawn from tests per- formed with thiram on mink and ferrets: l) Mink reject feed containing 2 82 ppm thiram. 2) The dietary LC50 for ferrets lies between 125 and 312 ppm. 135 3) Thiram added to the diet with water as the carrier affects mink much more severely than thiram added in corn oil. 4) Signs of thiram intoxication in mink and ferrets include reduced feed consumption or feed rejection, weight loss, tarry stools, listlessness, and occasional convulsions accompanied by high-pitched vocalizations. 5) Reproduction is prevented in ferrets at 64 ppm. Inter- ference with the estrus cycle is the probable cause of the reproductive problem. 16 ppm of thiram in the diet of ferrets causes low birth weight, as does 40 ppm in the diet of mink. 6) Ferrets appear to be more sensitive to the reproduc- tive effects of thiram than mink. 7) Mink and ferrets appear to be more sensitive to the effects of thiram in the diet than rats. Experiment IV - Results Two dietary LC50 tests were conducted with Aroclor 1254. One of the objectives of these tests was to determine differ- ences in the effects of PCB intoxication on young, rapidly growing animals and older animals whose body weight changes are primarily due to fat deposition. The ages of animals used in these tests were approximately 13 to 17 weeks old for the younger animals and > 1 year old for the older animals. Based on results of previous experiments conducted in this laboratory, in which an approximate LC50 for Aroclor 1254 over a 9 month study was calculated to be 6.65 mg/kg 136 (Ringer gt $1., 1981), dietary concentrations of 0, 10.0, 18.0, 32.4, 58.3, and 105.0 ppm were chosen for the 28-day studies. The Aroclor was dissolved in acetone, mixed with a small amount of ground mink cereal, and evaporated to dryness. The pre-mix was then added to the basal diet. Actual dietary concentrations of Aroclor 1254 have not been determined. A 21-day acclimation period was begun on 29 July, 1982 for young animals. The 28-day LC50 trial began on 19 August, 1982, and ended on 16 September, 1982, followed by a 7-day withdrawal period. The test was terminated on 23 September, 1982. A 21-day acclimation period was begun on 7 October, 1982 for older animals. The 28—day LC50 trial began on 28 October, 1982, and ended on 25 November, 1982, followed by a 7-day withdrawal period. The test was terminated on 2 December, 1982. Signs of intoxication were first noted on day 12 for the young animals, when 2 animals fed the 105.0 ppm diet were noted to be listless, and also on day 12 for the older animals, when several animals on the two highest diets were observed to have tarry stools. Feed consumption was affected earlier in both groups, since some animals in both tests exhibited reduced feed consumption by day 4. Reduced feed consumption appeared to be more pronounced among the older animals, being noted earlier and at lower dietary concentrations than for the young animals. Body weight changes and feed and com- pound consumption are summarized in Tables 57 and 58 for young animals and Tables 59 and 60 for older animals. 137 .mommzacotoa :. czasm m_ae_=n o>_— .5 Logan: «2“ co some: uaango u:a_az seen u .5 uya_o :o..ae:m=:o >__nu soc. co_oae:mcoo scaoaeoo x—xoaz m:o_uacu:ooaou xcouo_u was.» conuasamcou use» umncm>a sate saga—:u_no =a_uaazmcou a::oa&co >._n: a .:o_uaa:m:ou who: o>.u:umncou oza no manao>n 82s as sauna :o_s;e:m:ao any; n _o.nn~ _o.aa~ -- an.nn na.an n~.mn ~n.a__ a Aaz\aa. ao_oaaanaao naaaasao -1 111 -- om.m mm.m am.m oc.s— O Aume. :o.u26:m=ou acasasou -- -- 1- mm _a cm we. ooN Auxav copuasancoo use; «la . --- oam in.nnn- -- ~.n~_- n.nm_- o.an.- n._n- ~.ea. lav «aanao oaan«= a.ma_ mn.na~ ~n.no~ o no.nn an.nn an.nq Nm.oa a Ax:\aaw aa_oaaanaoo naaaaaao -- -- o cm.m e~.c on.o cm..— o Av\ms co.aas:ucou uczoaeou --- --- mm. na no. a__ na_ Pan Aa\a. aa_oaaanaao noon «Am Co.a~n- «An V~.na~- n.an- a.aa . ~.n._- n.n__- n.nn- n.~n. .av «aanao oaa_«= n.nn no.9mp no.om_ o m~.~m —~.em am.~m ~m..m c szxmsv copuaeamaou uaaoasou 1- -1- o ~n.m mm.o _~.e nn.~ c Au\as~ :o_uae:naoo vazoasou --- --- an. an. _m_ ma. . «Nu mmN Au\mv aa_oaaanaao aoaa o.a ...nan- oao_...nn_- a._n- ~._n - a.an - ..mn - ~.m + n.nn. As. «aanao naa_«= n.~n -.c__ -.o__ o om.n~ o~.o~ m~.o~ um.mm o sz\ms. co_o;s=n:oo a:=oa50u 1- 11- o —v.m nm.m ~m.m mo.m c Auxoav covuaszmcou usaoasou --- --- oo~ aa— na_ ~_~ _n~ can A=\a. aa_naaanaao anon o1a_v_.~o_- «Ao_.~.a - a.n~- ~.n_ - ~.nn - a.m~ - ..on+ n.n=. .a. «mango oaa_«= 5.x. na.nn no.nn a Na N. ~n.o_ na.n_ n_.a_ a Aaz\maw aa_oaaanaao naaaaaao 1- 1-1 o em.. mm.~ on.~ q~.~ c Auxme :owae53mcou scaoaeou --- --- naN nn. nnn on~ nn~ ca~ An\mv aa_naEanaao a««l «10....nn - «Ac..n.mn 1 n.nn- a.~ - n.nn - a.n_ 1 ..na. m.nn+ .a. «aanao n;a_«= a.a_ c c o o o o o c szxosv cowoasamzou acaoasou -- 111 c c o a o o Auxae. :o_uas:m:ou ccaoasou --- --- can n- nan . mnn awn can Aa\a. aa_.aaanaao n««a uAc_.o.cc_+ u.c_vm.eo_+ m.— + o.o_ + ..m_ - e.am + e.pe+. m.em+ Amy «mango u:m_uz o u>_ua_=e:u o>.on_:e:u pnzncu v n N — co.“ vacancy: Aaaav “my: m goo; v -za_z gum: ago: xoa: “no: -ne__uo< assesses; ao_~ncoaoo:ou ow to. «mm. ao_ooa< no m:o_onCoaoiaco n:c_an> vac za_s maaom xa sesamaoo guzzoaeoo can neao. can noaaa;o o;a_az sea; «an.«>< .mm a~aa_ -1 .mxsv. 1.3 8 Tabie 58. Initia1 and fina1 weights of young ma1e (M) and fema1e (F) mink fed various concentrations of Aroc1or 1254 for 28 days and after a 7- day withdrawa1 period. Change Concentration Initia1 Fina1 Change after with- (ppm) Sex n wgt. (g) wgt. (g). (g drawa1 (g) 0 M 5 1232.6 1376.7 +144.0 -158.0 F 5 860.8 925.8 + 65.0 + 63.6 10.0‘ M 5 1310.0 1381.0 + 71.0 + 23.6 F 5 908.8 889.0 - 19.8 - 99.8 18.0 M 5 1296.6 1231.4 - 65.2** ' - 85.8** F 5 912.4 821.2 - 91.2 -118.4* 32.4 M 5 1279.4 1075.4* -204.0** -295.6** F 5 826.8 676.6 -150.2** -182.4** 58.3 M 5 1330.6 848.8** -463.4** -507.6** F 5 910.2 643.6* -266.6** -272.2** 105.0 M 5 1324.8 741.2** -583.6** -383.6** -F 5 915.0 536.6** -378.4** -378.4** Significant1y different from contro1 (P < .05). X. * Significant1y different from contro1 (P < .01). 139 .manususosns s“ szosm «_ns_sa o>._ no census use so uowas mosaso usm_oz xuom u .~ w9=_. so.ussansoo >—_nu soc. so.s;s:msou usaosEoo x—sumz "so..ncusmosou scans.u was.» so_uss:msou use» amaso>o sous uasn_:o_no sc_ussamsoo ussossoo >__cc s .so_ase:msoo.m>eu u>.uauamsou use us conso>o as“ so uomas so.usesnsoo use; n n~.__~ n~.__~ a nn._m _n.~e o_.nn na.an a .a:\aaw aasnaaanaao naaaaaao -- 1-1 o av.“ no.9 _o.—— _~.m o Auxms sonuassmsou ussoaaou --- --- an. an an me. an .a~ .n\as ansoaaanaao none or. . n_n- «An sm.n~n- an- m.na_- a.na_- a.nn - ~.nn.- a.m_+ .a. «aanao oaa.«: a.ma_ am.cn_ nm.on_ s nn.~n _n.nn ns.an an.an s Anz\aaw aa.oaaamaoo aaaoaaao --- --- a ...n nm.n on.n as.m a Aa\aa aa.oaaanaao naaaaaao --- --- on. me. no ~n. so. anw .nxm. aanoaaanaao soon osn .~.nan- oln sn.n~m- n.an- n.~n_- a.nn_- c.aa_- a.n~_- n.1n. Am. «aanao oaa_«= n.nn n~.mn_ n~.nn_ a ~_._n na.nn no.mn mn.nn a .xx\aa. aa_oaaanaoo aaanaaao --- --- a nn.n ma.n ~_.m an.n a au\aav aa_oaaanaoo naaaaaao --- --- an. kn. an. an. on. Nnn .n\a. aa.oaa=«aao noon osa sn.an~- ola_.n.non- a.n . n.nn - n.nn - n.nn - n.nn - n.~n+ Ans «aanao oam.«= n.~n nn.an "n.0n o na.n~ nn.- nn.- an.a_ a .xz\aas aasoaaanaao naaaaan .1..- 1-1 6 mm." m—.n mN.n NQ.N 0 2:95 covuaszmzou «Essa—sou --- --- no. as. nn. _a_ km. ann. .n\a. aasnaaanaao n««s ole—sn.nn~- o.a_.a..nn- n.a_. n.nn - n.an - a.na_- n... - n._n. Ra. «aanao oaa_«= a.a_ nn.sn nn.On a nn.s_ Nn.~_ an.n. ma.n_ a As:\aa aa_oaaanaao naaaaaao --- --- a an._ on.. as.— aa._ a .n\aa annoaaanaoo naaoaaao --- --- non an. on. as. am. Nan .nxm. aa_oaaanaoo nnou «so sn.oa_- osa_sn.nn_-. n.nn. n.nn - n..m - a.an - n..~ - 8.8.. .a. «aanao oaa_«= a.a_ c c o o o c c c :3\msv :o—uassmzou venous—cu -- 1- o c c c c o Auxaav so_usssmsso ussoQEou --- --- aa_ _nN m_n own nNn nnn An\a. aa_oaaanaao n««s oaa_.n.nn + o.a_.n.no_. n.a~- n.n + n._n . n.n n n.nn . n.~n+ .a. «aanao oaa_«z a o>_Sa—=e=u. a>_sa_:e=u _nzasu e n w . _ so.u uncanny: AESQV goo: m xuoz e -s«.: x093 Joe: Joe: gum: 1as__ou< consensus so_umsusuosou .mxnu mm to» emw. so_oos< Co mso.uosusuusou m:o_sa> use xs_s tau—o so uoaamsau susaoQEOo use queue use muusaso osm_sz xuos mansu>< .am a_sn_ 1.41) Tab1e 60. Initia1 and fina1 weights of o1der ma1e (M) and fema1e (F) mink fed various concentrations of Aroc1or 1254 for 28 days, and a ter a 7- day withdrawa1 period. Change Concentration Initia1 Fina1 Change after with- (ppm) Sex 0 wgt. (9) wgt. (9) (9) drawal (9) 0 M 5 1753.6 2000.2 +246.6 +210.4 F 5 1055.0 1017.2 - 37.8 - 60.8 10.0 M 5 1711.0 1557.0** -150.8** - 98.6** F 5 1080.4 939.2 -141.2 -130.6 18.0 M 5 1703.8 1370.2** -333.6** -290.0** F 5 1103.0 893.0 -210.0 -215.0 32.4 M 5 1592.6 1215.2** -377.4** -392.0** F 5 1076.0 842.2 -235.8* -211.2 58.3 M 5 1570.4 978.0** -594.4** -605.2** F 5 954.2 575.2** -379.0** -402.2** 105.0 .M 5 1710.6 983.8** -719.6** -731.6** F 5 953.0 494.8** -458.2** -458.2** Significant1y different from contro1 (P < .05). Significant1y different from contro1 (P S .01). ** 141 Clinical signs included reduced feed consumption, weight loss, listlessness, and unconsciousness prior to death. Some animals also had bloody or tarry stools near death. At the end of the 28-day exposure period for young animals, 2 of 10 animals had died on the 58.3 ppm diet and 7 of 10 had died on the 105.0 ppm diet. After the withdrawal period, 1 of 10 animals had died on the 32.5 ppm diet, 5 of 10 died on the 58.3 ppm diet, and 10 of 10 died on the 105.0 ppm diet. The mortality pattern is described in Table 61. For the older animals, 5 of 10 animals on the 58.3 ppm diet and 8 of 10 animals on the 105.0 ppm diet had died by the end of the 28—day exposure period. After the withdrawal period, 1 of 10 animals had died on the 10.0 and 32.5 ppm diets, 6 of 10 died on the 58.3 ppm diet, and 9 of 10 died on the 105.0 ppm diet. The mortality. pattern is described in Table 62. Blood-filled intestines were usually found at necropsy of mortalities, and ascites and/or hydropericardium was occasionally noted. Occasional gastric ulcers were also noted at necropsy of mortalities and at the terminal kill. The data yielded LC50's of 105.0 ppm, with a 95% confidence interval of 79.4 to 139.0 ppm, and a slope of 1.57, with a 95% confidence interval of 1.45 to 1.70, for the 28-day exposure, and 58.3 ppm, with a 95% confidence interval of 39.1 to 87.0 ppm, and a slope of 1.58, with a 95% confidence interval of 1.16 to 2.15, for the 28-day exposure period plus the with- drawal period for the young mink. For the older animals, the data yielded Lcso's of 84.0 ppm, with a 95% confidence interval 142 — — ~ — — p — — o.mo_ _ p p — n.mm an on ma Nn pm on mu mm sw cw mm ow nN NN —~ cu m. a. m- o. m— s— n— ~p _— up m11mllh1lm11m11h11hllm11ur11 Amx\aev one» Co aou\ms,au mpns.sa no oz so_oasasmosou .umou emu; sou-o~ « as_s:u emw. sopooc< use xs_s usaox so scaoona sun—sous: .po u_sap 143 o.map 1 4.4 v.~m 9.x— c.9— oN a. m- s. u. m. e. mp ~91 __ op- a m h. m m c mI-w — Aau\usv mm“ o amu\ms.xw1mpoe_so be u: so_ansusmosou .umou smog sou-mm o mspssu camp so—uos< use 1s_s sou—o Co stooges xu_.nusoz .Nu «_snp ‘ Tab1e (13. Body and organ weights of growing ma1e (M) and fema1e (F) mink fed various concentrations of Aroc1or 1254 for 28 days. Concentration Body Brain Liver Sp1een Kidney Lung Heart (ppm) wgt. (g) (% body) (% body) (% body) (% body) (% body) (5 body) 0 M 1390.6 0.77 4.03 0.29 0.62 0.67 0.74 F 924.5 0.93 4.13 0.29 0.59 0.68 0.69 Ca --- -- 4.08 0.29 -- 0.68 0.72 10.0 M 1333.6 0.79 5.34 0.26 0.56 0.73 0.63 F 809.0 1.17 5.17 0.32 0.67 0.84 0.67 C --- -- 5.25** 0.29 -- 0.79 0.65 18.0 M 1210.8 0.86 5.75 0.37 0.65 0.87 0.70 F 794.0 1.12 5.89 0.36 0.69 0.98 0.68 C --- -- 5.82** 0.36 -- 0.92* 0.69 32.4 M 983.8** 1.12 5.45 0.33 0.76* 1.18 0.75 F 644.4* 1.34* 6.09 0.43 0.87** 1.00 0.83 C --- -- 5.77** 0.38 -- 1.09** 0.79 58.3 M 804.6** 1.43** 5.57 0.34 0.91** 1.11 0.72 F 625.6* 1.45** 6.46 0.36 0.95** 1.19 0.73 C --- -- 6.01** 0.35 -- 1.15** 0.73 105.0 M 739.4** 1.51** 5.47 0.31 0.93** 1.25 0.76 F 491.0** 1.77** 5.25 0.35 1.11** 1.22 0.78 C 5.36** 0.33 -- 1.24** 0.77 Organs expressed as a percent of brain weight 0 M 520.9 37.2 80.4 86.6 96.3 F 441.2 31.1 64.0 73.9 73.0 C --- 34.1 -- -- -- 10.0 M 678.9 33.6 71.8 93.4 80.2 L 466.2 28.9 58.3 73.4 59.1 b --- 31.2 -- -- -- 18-0 M 676.8 44.2 76.7 100.6 81.8 F 525.5 31.8 61.7 87.5 60.7 C --- 38.0 -- -- -- 32.4 M 496.7 31.4 69 1 105.2 67.6** F 455.5 32.6 65.4 75.7 61.9 C --- 32.0 -- -- -— 58.3 M 443.2 29.1 68.4 84.4 56.9** F 470.0 27.1 67.7 85.2 52.5* C --- 28.1 -- - -- 105.0 M 366.0 20.3 62 8* 84 9 51.4** t 296.7 19.6 62.5 68.4 43.8** C --- 19.9* -- -- -- a . . . . . Sexes are combined where no Sign1f1cant difference (P < .05) was found between them. * Significant1y different from contro1 (P ** Significant1y different from contro1 (P / § < \ .05). .01) 145 Tab1e (#1. Body and organ weights of fu11y grown ma1e (M) and fema1e (F) mink fed various concentrations of Aroc1or 1254 for 28 days. Concentration Body Brain Liver Sp1een Kidney Lung‘ Heart (ppm) wgt. (g) (% body) (% body) (% body) (% body) (% body) (% body) 0 M 1964.0 0.52 4.16 0.20 0.48 0.61 0.52 F 994.2 0.79 5.29 0.34 0.59 0.81 0.66 C6 --- -- -- -- 0.53 -- 0.59 10.0 M 1610.4* 0.61 5.73 0.31 0.60 0.92 0.65 F 1038.8 0.80 5.95 0.35 0.60 0.91 0.70 C --e -- -- -- 0.60 -- 0.68 18.0 M 1413.8** 0.72 6.41** 0.28 0.69 1.04* 0.91 F 888.0 0.96 7.64** 0.50 0.85 1.01 0.83 C --- -- -- -- 0.77** -- 0.87** 32.4 M 1200.6** 0.83* 6.87** 0.32 0.79 1.13** 0.77 F 864.8 1.01 6.96* 0.38 0.76 1.07 0.77 C --- -- -- -- 0.78** -- 0.77** 58.3 M 965.2** 1.02** 6.02* 0.28 0.87 0.98* 0.68 F 552.0* 1.45** 6.00 0.34 0.95 1.32** 0.67 C --- -- -- -- 0.91** -- 0.67 105.0 M 979.0** 1.15** 5.14 0.25 0.89 0.99* 0.69 F 494.8** 1.72** 5.53 0.29 0.96 1.43** 0.78 C --- -- -- -- O.92** -- O.73* Organs expressed as a percent of brain weight 0 M 816.9 39.6 92.6 119.3 101.7 F 662.1 42.3 74.6 102.1 83.0 C --- 40.9 -- --- --- 10.0 M 930.6 50.4 98.7 149.3 106.9 F 744.8 43.7 75.0 113.5 88.4 C --- 47.4 -- --- --- 18.0 M 890.4 38.5 95.4 145.2 127.0 F 809.8 50.5 87.6 106.5 87.6 C --- 44.5 -- --- --- 32.4 M 845.1 38.9 97.2 139.6 95.8 F 716.9 38.0 76.6 112.1 79.6 C --- 38.4 -- --- --- 58.3 M 630.4 28.8 89.2 97.4 68.0** F 413.8* 22.7 65.2 91.3 45.9** C --- 25.7* -- --- --- 105.0 M 480.7** 24.5 80.6 89.2 63.8** F 325.0** 16.5 56.1 84.7 45.4** C --- 20.5** —- --- --- a Sexes are combined where no significant difference (P S .05) was found between ** them. * Significant1y different from contro1 (P < P S Significant1y different from contro1 ( 1.46 .05). .01). of 53.5 to 131.9 ppm, and a slope of 2.44, with a 95% confidence interval of 1.43 to 4.16, for the 28-day exposure, and 47.0 ppm, with a 95% confidence interval of 29.6 to 74.6 ppm, and a lepe of 2.11, with a 95% confidence interval of 1.34 to 3.33, for the 28-day exposure period plus the withdrawal period. Hematologic parameters were not determined for these tests. The organ weight data, summarized in Table 63 for young mink and Table 64 in older mink, revealed increases in liver weight (as a percentage of body weight) for all dietary concentrations for the young mink and all but the 10 and 105 ppm diets for older mink. Increases in brain, kidney, and lung (as percentages of body weight) were seen in the young mink at several concentrations, while these same organs plus the heart were increased in older mink. When the organs were expressed as percentages of brain weight, only the spleen, kidney, and heart were affected (reduced) in the younger animals, while the liver, spleen, and heart were affected (again reduced) in the older animals. DISCUSSION Several factors lead to the conclusion that the young, rapidly growing mink were less sensitive to the effects of Aroclor 1254. Obviously, the LC50 values for the.older animals were lower at the end of the 28-day exposure and after the withdrawal period, although not dramatically different. It must be noted, however, that the initial body weights of the younger animals were considerably smaller than the initial 147 body weights of the older animals, especially for the males, and therefore the dose, on a mg/kg of body weight basis, for the younger animals was relatively higher. Thus, the difference between the corresponding LC50's was actually somewhat greater than it would appear. Other factors that point to an increased sensitivity for the older animals are: (1) a more pronounced decrease in feed consumption, both in the magnitude of the differences between controls and exposure groups (e.g. for week 1, young controls consumed 288 g/d while young 105.0 ppm-fed animals consumed 162 g/d, and for week 1, older controls consumed 323 g/d while older 105.0-fed animals consumed 54 g/d), and in the number of dietary groups affected (e.g. for week 1, feed consumption was noticeably reduced from controls at 32.4, 58.3, and 105.0 ppm among the young animals, while feed consumption is notice- ably reduced at all exposure groups among the older animals); (2) a more pronounded effect On cumulative weight change, again both in the magnitude of the differences and in the extent of the effect among the various exposure groups (although this effect was mediated somewhat by the fact that the older animals had more weight to lose); (3) a faster time to death for older mink (e.g. 26.5 days for young mink and 25.6 days for older mink fed 105.0 ppm, and 27.8 days for young and 25.5 days for older mink fed 58.3 ppm); and (4) a larger cumulative intake of Aroclor 1254 over the 28-day exposure period for all dietary concentrations of young mink versus older mink, which did not produce correspondingly larger weight losses or mortality. Several other noteworthy results were obtained from these tests. It was apparent from the cumulative weight changes that a dose-dependent response was elicited upon exposure to Aroclor 1254. It also appears that males were more severely affected, although this was probably due to the fact that, in mink, the average body weight of the male is approximately double that of the female, and therefore, the male has more weight (as body fat) to lose. From the data for the withdrawal period, it would appear that a limited recovery was occurring in those animals still able to eat at the end of the 28-day exposure period, since feed consumption increased in nearly all treat- ment groups over week 4 levels, although in most instances the animals continued to lose weight. It was also clear from the data of these tests that a withdrawal period is necessary in LC50 tests with certain substances to obtain an accurate picture of the toxicity of the test substance. The organ weight data revealed an increase in liver weight (as a percentage of body weight) in both age groups, as has been reported previously for mink (and most other animals) exposed to PCBs (Ringer 35 31., 1981). However, the weight of the organ (as a percentage of brain weight) increased up to 18 ppm in the younger animals and 32.4 ppm in the older animals, then decreased at higher concentrations. This res- ponse was probably a reflection of the fact that the animals fed the higher concentrations were either dead or dying at necropsy, and had probably used most or all of the glycogen store in the liver, leading to the lower liver weights. Other effects noted in these studies are consistent with previous research with mink fed PCBs. Reduced feed consumption, reduced growth rate, enlarged kidneys, hemorrhaigc gastric ulcers, tarry stools, and hydropericardium/ascites have been reported by Ringer gt 31. (1981), and these results were also seen in these tests. It is interesting to note that, had a reproduction test been performed based on the results of the LC50 test with the older mink, dietary concentrations of approximately 0.5, 2.0, and 8.0 ppm would have been chosen, based on the adverse effects noted at 10 ppm on body and liver weight. Since reproduction tests very similar to those conducted in these experiments have been conducted by this laboratory with Aroclor 1254, it is useful to review the findings of those experiments (Aulerich and Ringer, 1977). These authors tested the reproductive effects of 0, l, 2, 5, 10, and 15 ppm of Aroclor 1254 in the diet, and found severe effects on reproduction at 2 2 ppm and outright mortality at 15 ppm. No reproductive effect was noted at 1 ppm. The dietary concentrations that would have been chosen based on the LC50 test results would have found both a dietary no effect level and an adverse dietary concentration in the reproduction test conducted. according to the protocol presented here, based on the results of Aulerich and Ringer. CONCLUSIONS The following conclusions may be drawn from the tests performed with Aroclor 1254 on mink: l) The 28-day dietary LC50 for young mink is 105 ppm and 2) 3) 84 ppm for older mink. Following a 7-day withdrawal period, these values are 58.3 and 47.0 ppm, respec- tively. A dose-dependent weight loss occurs upon dietary exposure to Aroclor 1254. Clinical signs of exposure include inanition, lethargy, and occasional unconsciousness and tarry stools. Enlarged liver and kidneys, gastric ulcers, ascites and/or hydropericardium, and blood filled intestines are often seen at necropsy. Young mink appear to be less sensitive to the effects of Aroclor 1254 in the diet than older mink. 5) A withdrawal period is important in assessing the toxicity of a test substance if mortality or other signs of intoxication are still occurring at the end of the 28-day exposure. Experimental Protocols - Results The protocols developed for 28-day dietary LC50 and reproduction tests for wildlife mammalian carnivores as a result of theses tests have been submitted to the Environmental Protec— tion Agency, and appear in Appendices A and B, respectively. 1R1 DISCUSSION Various sections of the protocols are discussed within the final drafts submitted to EPA. This discussion will focus on an overview of the results of the experiments as they pertain to the development of the protocols. The protocols developed for dietary LC50 and reproduction tests in these experiments allow the testing of a wide range of test substances (theoretically, any substances that can be mixed uniformly in the diet). The test substances used in these experiments covered a wide range of physico-chemical and biological properties, including: solid and liquid sub- stances; water soluble, lipid soluble, and relatively insoluble compounds; volatile and non-volatile substances; and extremely toxic to relatively non-toxic test materials in acute oral exposure. The mode of action of the test substances ranged from metabolic poisons to neurotoxins to compounds of unknown biological mode of toxicity. The protocols as submitted thus appear to be capable of assessing the subacute or reproductive toxicity of most test substances (if they are not too unstable in air), providing an estimate of either a toxic level or a no effect level of the test substance. The experiments have been conducted indoors under control- led conditions, suggesting that dietary LC50 and reproduction tests may be conducted in a reproducible fashion by properly equipped laboratories. It is interesting to note that, in 152 experiments initiated in this laboratory to assess the secon- dary toxicity of certain test substances, Aroclor 1254 was used as the test substance in 28-day dietary LC50 tests. In the primary toxicity test conducted in conjunction with these tests, using the protocols developed here, a dietary LC50 of > 74.8 ppm was found after 28 days' exposure, and an LC50 of 48.5 ppm, with a confidence interval of 36.5 to 64.5 ppm and slope of 1.92, was calculated after a lO-day with- drawal period (unpublished research, Michigan State University). These results are in very close agreement with the values calculated from the LC50 test with older mink in this study, even though the tests were conducted during different seasons (summer vs. late fall), different years, and had different protein sources in the diet (20% chicken vs. 40% rabbit). The value of an accurate estimate of the acute oral LD50 of a test substance, obtained from either the literature or range-finding techniques, cannot be overstated. As evidenced by the ferret LC50 test with Compound 1080, a statistically valid dietary LC50 can be calculated with as few as 32 animals, using 3 dietary concentrations plus control and 8 animals per concentration, if an accurate LD50 is available from which to specify the test concentrations. Similarly, a good estimate of the dietary LC50 (or more specifically, a dietary no effect level in an LC50 test) is very helpful in conducting a repro- duction test. A good estimate of the no effect level can provide valuable information in establishing dietary concentra- tions which will maintain the animals in a physical condition 153 such that they will be able to mate. This permits testing the reproductive effects of a test substance rather than other physiological effects. An excellent example of the consequences of choosing dietary concentrations for a reproduction test not based on the no effect level of an LC50 test is seen in the ferret reproduction test with thiram. In this test, dietary concen- trations of O, 4, 16, and 64 ppm were chosen in spite of adverse effects at 20 ppm in the ferret LC50 test. These concentrations were chosen in part because dietary concentra- tions of O, 2.5, 10, and 40 ppm had been selected for the concurrent mink reproduction test, based on a no effect level of 45 ppm in the mink LC50 test. The dietary concentrations for the ferret reproduction test thus reflected the repeated experience of this laboratory that ferrets are less sensitive than mink to toxic substances. As the results showed, 64 ppm thiram in the diet proved to be too high a dietary concentra- tion, resulting in no reproduction and only 7 of 12 females coming into estrus. If the LC50 test data had been employed, dietary concentrations of approximately 0, l, 4, and 16 ppm would have been tested (4 and 16 ppm actually were tested), and statistically valid differences would have been found at 16 ppm. Since the lowest concentration actually tested, 4 ppm, resulted in decreased kit body weight at 6 weeks post- partum, as well as a reduction in the whelping percentage, a no effect level may not have been found in this reproduction test. 154 Several other problems occurred during these tests. The importance of a palatability test was underscored by the results of the mink and ferret LC50 tests with thiram. Several animals were "wasted" in the mink test when they had to be removed from the test due to feed rejection. This could have been avoided if a palatability test was conducted properly, as in the ferret test where no such problems were encountered. This test also points out, in a rather drastic manner, the importance of the carrier in a test, since vastly different results were obtained with distilled water or corn oil as the carrier. Similar results have been reported by Gile et al. (1983), who noted carrier-related differences in the toxicities of some pesticides in avian LC50 tests. The age of the test animals at the start of an LC50 test can also have a dramatic effect, as witnessed by the ferret LC50 tests with Compound 1080. In this test, young, rapidly growing ferrets were capable of "outgrowing" a lethal dietary concentration during the 28 days of the test, since the body weight gain during the test far outdistanced the increase in feed consumption, thus "diluting" the dose on a mg/kg of body weight basis. On the other hand, the LC50 tests with Aroclor 1254, using young and old mink, showed only slight differences in the toxicity of the PCB to the two age groups, with the younger group being less sensitive. It may be that young, rapidly growing animals can be successfully used with lipid soluble compounds that accumulate in the body, while this age group is unsuitable for testing water soluble substances. 155 Hill and Camardese (1981) have found an increase in the sub- acute dietary LC50 with increasing age of test birds for four different classes of pesticides, starting the tests with birds with ages ranging from one day to 21 days old. The length of the LC50 test was set at 28 days for several reasons. A 28-day test permits absorption, distribution, metabolism, enzyme induction, redistribution, bioconcentration, and elimination to occur in the body, similar to that which might occur in animals subacutely exposed to a substance in the environment via the daily diet. A 28-day test allows the testing of slow-acting or bioaccumulating substances, especi- ally if a withdrawal period is employed. This is exemplified by the LC50 tests with Compound 1080 and Aroclor 1254, in which prolonged and delayed mortality were seen (Tables 6, ll, 61, and 62). These results may not have been recorded in tests of shorter duration. In some instances, it may be possible to achieve satisfactory results with a shorter test using higher dietary concentrations, although the possibility of feed avoidance or rejection, as occurred in the 1080 and thiram tests, becomes greater with increased dietary concentrations. Also, certain substances cause delayed effects and/or mortality whether administered as a single acute dose or as multiple subacute doses. Increasing the concentration of the substance does not necessarily shorten the time to death. Examples of this phenomenon include the PCBS (as seen in the Aroclor 1254 LC50 tests) and some delayed neurotoxins. The standard avian dietary LC50 test length is 5 days' exposure in birds of a very young age. This test is designed to test the substance with birds at their most susceptible age. It also guarantees that the compound will be tested via the diet, since very young birds are not able to survive 5 days without eating. Such a protocol would be impractical if applied to mink and ferrets, however, since weanling mink and ferrets can vary widely in body weight (see, for example, the standard deviations of 6—week body weights in the repro— duction tests of this study), and can probably survive 5 days without eating. As previously mentioned, young mink can be less sensitive to some substances than older mink (e.g. Aroclor 1254). In regard to the reproduction tests, especially with mink, it is important to note some reproductive anomalies which may occur in a test, especially if first-year animals are used. A small percentage of first-year females will be found in a cohort which will not accept males during the normal repro- ductive season, are barren, or which do not have normal maternal instincts, while a small percentage of males will not attempt to mate or will produce no viable spermatozoa. Each of these anomalies was seen at least once during the course of these experiments, and points out the importance of assign- ing at least 12 females and 4 males per dietary concentration if inexperienced breeders are used. If proven breeders are used, it may be possible to conduct a statistically valid reproduction test with as few as 8 females and 2 males per concentration. Several advantages can be envisioned in the use of mink and/or ferrets as the species of choice for toxicological 157 tests with carnivores. First, these two species have been shown to be among the most sensitive of the wildlife species to com- pounds of environmental interest, as stated previously. Mink, and to a lesser extent, ferrets may dispel part of the growing opposition to the use of "pet" species as test subjects in scien- tific investigations. Mink and ferrets can be purchased, gener- ally, for approximately one-half the cost of standard laboratory beagles, and in the case of mink may permit recovery of part of the cost in the pelt. Also, mink and ferrets generally require less cost for maintainence than the dog. Negative factors that may arise in the use of mink and/or ferrets as toxicological models include: handling problems, especially with mink; a limited reproduction season, especially with mink; the natural range of these species, which may preclude their use in certain hot climates; and, of course, the limited background data for these species in the literature. CONCLUSIONS The following conclusions may be drawn from tests per- formed with several test substances on mink and ferrets: l) Statistically valid dietary LC50 tests may be per- formed with mink and ferrets, using 32-60 animals assigned to 3-5 dietary concentrations plus a control, with 8-10 animals per concentration. 2) Statistically valid reproduction tests may be per- formed with mink and ferrets, using 64 animals assigned 3) 4) 5) to 3 dietary concentrations plus a control, with 4 males and 12 females per concentration. Factors which may affect the results of these tests include the age of the animals at the beginning of the test, the carrier used to mix the test substance into the diet, the accuracy of the estimate of the toxicity of the test substance, and the palatability of the test substance. A wide range of test substances can be assessed using these protocols. The tests can be conducted indoors, allowing repro- ducible results in properly equipped laboratories. 159 REFERENCES Allen, J.R., 1975. Response of the nonhuman primate to poly- chlorinated biphenyl eXposure. Fed. Proc. 34:1675-1679. Allen, J.R. and D.H. Norback, 1973. Polychlorinated biphenyl- and triphenyl-induced gastric mucosal hyperplasia in primates. Science 179:498-499. American Conference of Governmental Industrial Hygienists, 1952. Threshold Limit Values for 1952. AMA Arch. Ind. Hyg. Occup. Med. 6:178-180. American Concerence of Governmental Industrial Hygienists, 1956. Threshold Limit Values for 1956. AMA Arch. Ind. Health 14:186-189. American Conference of Governmental Industrial Hygienists, 1961. Threshold Limit Values for 1961. Cincinnati, ACGIH, p. 4. Anonymous, 1968. Pesticide Regulation Division, U.S. Dept. of Agriculture. "Bobwhite quail laboratory acute oral toxi- city test method (test A-2)". Federal Register, Vol. 40, No. 123 (June 25, 1975), p. 26914. Anonymous, 1975. Toxic and Hazardous Industrial Chemicals Safety Manual. (International Technical Information Institute, Tokyo). 591 pp. Anonymous, 1978. Guide for the Care and Use of Laboratory Animals. DHEW Publication No. (NIH) 78-23, U.S. Dept. Health, Education, and Welfare. Anonymous, 1979. Dept. of Health, Education, and Welfare, Food and Drug Administration. "Nonclinical Laboratory Studies, Good Laboratory Practice Regulations", Federal Register, Vol. 43, No. 247, pp. 59986-60025. Anonymous, 1980. Environmental Protection Agency. "Proposed Environmental Standards; and Proposed Good Laboratory Practice Standards for Physical, Chemical, Persistence, and Ecological Effects Testing", Federal Register, Vol. 45, No. 227, pp. 77332—77365. Atzert, S.P., 1971. A review of sodium monofluoroacetate (Compound 1080) its properties, toxicology, and use in predator and rodent control. U.S. Dept. of the Interior Fish and Wildlife Service, Special Scientific Report - Wildlife No. 146, 1-34. 160 Aulerich, R.J. and M.R. Bleavins, 1981. Potential of mink as an animal model in testing in area of toxicology. The l982 Blue Book of Fur Farming (Communications Marketing, Inc., Eden Prairie, MN):30-32. Aulerich, R.J., S.J. Bursian, A.C. Napolitano, and M.R. Bleavins, 1982. Evaluation of oral larvicide with mink. Fur Rancher 62(5):4,8. Aulerich, R.J., A.C. Napolitano, and R.H. Ross, Jr., 1983. Larvadex insect growth regulator for controlling flies on mink farms. The Z984 Blue Book of Fur Farming (Communications Marketing, Inc., Eden Prarie, MN):48-51. Aulerich, R.J. and R.K. Ringer, 1977. Current status of PCB toxicity to mink and effect on their reproduction. Arch. Environ. Contam. Toxicol. 6:279-292. Aulerich, R.J. and R.K. Ringer, 1979. Toxic effects of dietary polybrominated biphenyls on mink. Arch. Environ. Contam. Toxicol. 8:487-498. Aulerich, R.J., R.K. Ringer, and S. Iwamoto, 1973. Reproduc- tive failure and mortality in mink fed on Great Lakes fish. J. Reprod. Fert. Suppl. 19:365-376. Aulerich, R.J., R.K. Ringer, P.J. Schaible, and H.L. Seagran, 1970. An evaluation of processed Great Lakes fishery products for feeding mink. Feedstuffs 42(42):48-49. Aulerich, R.J., R.K. Ringer, H.L. Seagran, and W.G. Youatt, 1971. Effects of feeding Coho salmon and other Great Lakes fish on mink reproduction. Can. J. Zool. 49:611—616. Bakke, O.M. and R.R. Scheline, 1970. Hydroxylation of aromatic hydrocarbons in the rat. Toxicol. Appl. Pharmacol. 16: 691—700. Barnes, J.M. and F.A. Denz, 1954. Experimental methods used in determining chronic toxicity. Pharmacol. Rev. 6:191- 242. Bio Fax Techniques, 1969. Methods Used in Determining Biolo- gical Activity of Chemicals. Industrial Bio-Test Labora- tories, Inc., Northbrook, IL. Vols. 3-5, 4-5, 5-5. Bleavins, M.R., 1983. Toxicological manifestations of hexa- chlorobenzene exposure in the mink (Mustela vison) and the European ferret (Mustela putorius sofas. Ph.D. Thesis, Michigan State Univ., E. Lansing, MI. 153 pp. Bleavins, M.R., R.J. Aulerich, and R.K. Ringer, 1980. Poly- chlorinated biphenyls (Aroclors 1016 and 1242): Effect on survival and reproduction in mink and ferrets. Arch. Environ. Contam. Toxicol. 9:627-635. 161 Bleavins, M.R., R.J. Aulerich, and R.K. Ringer, 1984. Effects of chronic dietary hexachlorobenzene on the reproductive performance and survivability of mink and European ferrets. Arch. Environ. Contam. Toxicol. 13:357-365. Boutwell, R.K. and D.K. Bosch, 1959. The tumour-promoting action of phenol and related compounds for mouse skin. Cancer Res. 19:413—424. Bowman, R.H., 1964. Inhibition of citrate metabolism by sodium fluoroacetate in the perfused rat heart and the effect on phosphofructokinase activity and glucose utilization. Biochem. J. 93:13C-15C. Brown, M.B., 1975. A method for combining non-independent one-sided tests of significance. Biometrics 31:987-992. Buch, W.B., G.D. Osweiler, and G.A. VenGelder, 1976. Fluoro- acetate and fluoroacetamide. Clinical and Diagnostic Veterinary Toxicology, G.A. VanGelder, ed. Kendall/Hunt (Dubuque, IA). pp. 233-237. Buffa, P., V. Guarriero-Babyleva, and R. Costa-Tiozzo, 1973. Metabolic effects of fluoroacetate poisoning in animals. Fluoride 6:224—247. Buffa, P. and R.A. Peters, 1949. Formation of citrate in vivo induced by fluoroacetate poisoning. Nature 163:914. Buffa, P. and R.A. Peters, 1950. The in vivo formation of citrate induced by fluoroacetate poisoning and its signi- ficance. J. Physiol. (London) 110:488-500. Buikema, A.L. Jr., M.J. McGinnis, and J. Cairns, Jr., 1979. Phenolics in aquatic ecosystems: A selected review of recent literature. Mar. Environ. Res. 2(2):87—182. Carpenter, J.W. and C.N. Hillman, 1978. Husbandry, reproduc- tion, and veterinary care of captive ferrets. Am. Assoc. Zoo Vet. Ann. Proc., Knoxville, TN. pp. 36-47. Chenoweth, M.B., 1949. Monofluoroacetic acid and related compounds. J. Pharmacol. & Exptl. Therap. 97:383-424. Chenoweth, M.B. and A. Gilman, 1946. Studies on the pharma- cology of fluoroacetate. I. Species response to fluoro- acetate. J. Pharmacol. & Exptl. Therap. 87:90-103. Chou, C.C., E.H. Marth, and R.M. Shackelford, 1976. Experi- mental acute aflatoxicosis in mink (Mustela vison). Am. J. Vet. Res. 37(10):1227—1231. 162 Davydova, T.B., 1973. [The effect of tetramethyl thiuram disulfide (thiram) inhaled on the estrous cycle and the reproductive function of animals]. Gig. Sanit. 38:108- 110 (Rus.). Deichmann, W.B. and S. Witherup, 1944. Phenol studies. VI. The acute and comparative toxicity of phenol and o-, m-, and p-cresols for experimental animals. J. Pharmacol. Exptl. Ther. 80:233-240. Dunnett, C.W., 1964. New tables for multiple comparisons with a control. Biometrics 20:482-491. Edwards, W.G., 1977. Sodium‘fluoroacetate (Compound 1080) poisoning. Curr. Vet. Ther. 6:119—120. Elliott, W.B. and A.H. Phillips, 1954. Effect of fluoroacetate on glucose metabolism in_vivo. Arch. Biochem. 49:389-395. Engel, F.L., K. Hewson, and B.T. Cole, 1954. Carbohydrate and ketone body metabolism in the sodium fluoroacetate poisoned rat: "SFA diabetes". Am. J. Physiol. 179:325-332. Environmental Protection Agency, 1983. Pesticide Assessment Guidelines, Subdivision E: Wildlife and Aquatic Organisms. NTIS, No. T883-153908. Environmental Protection Service (Canada), 1975. Protocol for Testing Effects of Chemicals on Mink Production. Regula- tions, Codes, and Protocols Report EPS 1-EC-75-2. 10 pp. Fanshier, D.W., L.K. Gottwald, and E. Kun, 1964. Studies on specific enzyme inhibitors. VI. Characterization and mechanism of action of the enzyme inhibitory isomer of monofluorocitrate. J. Biol. Chem. 239:425-434. Federal Register, June 25, 1975. U.S. Environmental Protection Agency, Vol. 40, No. 123, pp. 26912-26928. Federal Register, October 12, 1977. U.S. Environmental Protec- tion Agency, Vol. 42, No. 197, pp. 55056-55057. Federal Register, July 10, 1978. U.S. Environmental Protection Agency, Vol. 43, No. 132, pp. 29728-29729. Fishbein, L., 1976. Environmental health aspects of fungicides. I. Dithiocarbamates. J. Toxicol. Environ. Health 1:713- 735. Fitzhugh, O.G., 1965. Appraisal of the safety of chemicals in foods, drugs, and cosmetics - chronic oral toxicity. Assoc. of Food and Drug Officials of the U.S., Topeka, KS, pp. 36-45. 163 Fitzhugh, O.G. and P.J. Schouboe, 1965. Appraisal of the safety of chemicals in foods, drugs, and cosmetics - subacute toxicity. Assoc. of Food and Drug Officials of the U.S., Topeka, KS, pp. 26-35. Fletch, S.M. and L.H. Karstad, 1972. Blood parameters of healthy mink. Can. J. Comp. Med. 36:275-281. Foss, G.L., 1948. The toxicology and pharmacology of methyl fluoroacetate (MFA) in animals with some notes on experi- mental therapy. Brit. J. Pharmacol. 3:118-127. Fur Rancher Blue Book of Fur Farming (published annually). Communications Marketing, Inc. (Eden Prairie, MN). Gaines, T.B., 1969. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14:515-534. Gile, J.D., J.B. Beaver, and R. Fink, 1983. The effect of chemical carriers on avian LC50 toxicity tests. Bull. Environ. Contam. Toxicol. 31:195-202. Gill, J.L., 1978. Design and Analysis of Experiments in the Animal and Medical Sciences, Vol. 1 (The Iowa State Univ. Press, Ames, IA). 409 pp. Hagan, E.C., 1965. Appraisal of the safety of chemicals in foods, drugs, and cosmetics - acute toxicity. Assoc. of Food and Drug Officials of the U.S., Topeka, KS, pp. 17-25. Hall, R.J., 1972. The distribution of organic fluoride in some tropical plants. New Phytol. 71:855-871. Hansen, L.G., C.S. Byerly, R.L. Metcalf, and R.F. Bevill, 1975. Effect of a polychlorinated biphenyl mixture on swine reproduction and tissue residue. Am. J. Vet. Res. 36:23-26. Harrison, J.W.E., J.L. Ambrus, and C.M. Ambrus, 1952. Fluoro- cetate (1080) poisoning. Ind. Med. Surg. 21:440-442. Hartsough, G.R., 1965. Great Lakes fish now suspect as mink food. Am. Fur Breeder 38(10):25-27. Hill, E.F. and M.B. Camardese, 1981. Subacute toxicity testing with young birds - response in relation to age and intertest variability of LC50 estimates. Avian and Mammalian Wildlife Toxicology:Second Conference. ASTM STP 757; D.W. Lamb and E.E. Kenaga, eds., Am. Soc. for Testing and Materials. pp. 41-65. Hornshaw, T.C., R.J. Aulerich, and H.E. Johnson, 1983. Feeding Great Lakes fish to mink: effects on mink and accumula- tion and elimination of PCBs by mink. J. Toxicol. Environ. Health 11:933-946. 164 Hudson, R.H., R.K. Tucker, and M.A. Haegele, 1972. Effect of age on sensitivity. Acute oral toxicity of 14 pesticides to mallard ducks of several ages. Toxicol. Appl. Pharmacol. 22:556-561. Hurni, H., 1970. The provision of laboratory animals. IN: Methods in Toxicology. F.A. Davis Co. (Philadelphia, PA). pp. 11-48. Hutzinger, O., 8. Safe, and V. Zitko (eds.), 1974. The Chemi- stry of PCBS. CRC Press (Boca Raton, FL). 269 pp. Isaacs, R., 1922. Phenol and cresol poisoning. Ohio State Med. J. 18:558-561. Kaye, S., 1970. Handbook of Emergency Toxicology. 3rd edition. C.C. Thomas, Publ. (Springfield, IL). 514 pp. Kimbrough, R.D., 1974. The toxicity of polychlorinated poly- cyclic compounds and related chemicals. CRC Crit. Rev. Toxicol. 2:445-489. Kimbrough, R.D. (ed.), 1980. Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins, and Related Products. Elsevier/North Holland Biomedical Press (Amsterdam, NY). 406 pp. Kimbrough, R., J. Buckley, L. Fishbein, G. Flamm, L. Kasza, W. Marcus, S. Shibko, and R. Teske, 1978. Animal Toxi- cology. Environ. Health Perspect. 24:173-185. Kimbrough, R.D., R.E. Linder, and T.B. Gaines, 1972. Morpho- logical changes in livers of rats fed polychlorinated biphenyls. Arch. Environ. Health 25:354-364. Klapproth, E.M., 1976. Cresols and cresylic acids. IN: Chemical Economics Handbook. Stanford Research Insti- tute (Menlo Park, CA). pp. 637.5030A-637.5530E. Lee. C.C. and P.J. Peters, 1976. Neurotoxicity and behavioral effects of thiram in rats. Environ. Health Perspect. 17:35-43. Lee, C.C., J.Q. Russell, and J.L. Minor, 1978. Oral toxicity of ferric dimethyldithiocarbamate (ferbam) and tetra- methylthiuram disulfide (thiram) in rodents. J. Toxicol. Environ. Health 4:93-106. Leonard, A., 1966. Modern Mink Management. Ralston Purina Co. (St. Louis, MO). 206 pp. Litchfield, J.T. and F. Wilcoxon, 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. & Exptl. Therap. 96:99-113. 165 Lowy, R., G. Griffaton, L. Brigant, B. Ardouin, and F. Dupuy, 1979. The dietary no-effect level of a dithiocarbamate fungicide, thiram, as evaluated from measurement data on rats. II. The various sensitivities of the various parameters. Toxicol. 14:39-53. Lowy, R., G. Griffaton, F. Dupuy, B. Ardouin, and P. Manchon, 1980. Dietary no-effect level of a dithiocarbamate fungicide, thiram, evaluated from measurement data on rats. I. Choice of the model of the dose-response relationship. J. Toxicol. Environ. Health 6:403-419. Marais, J.S., 1944. Monofluoroacetic acid, the toxic principle of "gifblaar" Dichapetalum cymosum (Hook) Engl. Onder- spoort J. Vet. Sci. 20:67-73. Matthiaschk, G., 1973. Uber den einfluss von L-cystein auf der teratogenese durch thiram (TMTD), bei NMRI-mausen. Arch. Toxikol. 30:251-262 (Ger.). McCann, J.A., W. Teeters, D.J. Urban, and N. Cook, 1981. A short-term dietary toxicity test on small mammals. Avian and Mammalian Wildlife Toxicology: Second Conference, ASTM STP 757, D.W. Lamb and E.E. Kenaga, eds., Am. Soc. for Testing and Materials. pp. 132-142. McNeil, D., 1965. Cresols. IN: Kirk—0thmer Encyclopedia of Chemical Technology, 2nd edition, Vol. 6, John Wiley and Sons (NY). pp. 434-444. Meldrum, G.K., J.T. Bignell, and I. Rowley, 1957. The use of sodium fluoroacetate (Compound 1080) for the control of the rabbit in Tasmania. Aust. Vet. J. 33:186-196. Merck Index, 9th Edition, 1976. M. Windholz, ed. Merck and Co. (Rahway, NJ). 1313 pp. Miller, R.F. and P.H. Phillips, 1955. Effects of feeding fluoroacetate to the rat. Proc. Soc. Exptl. Biol. Med. 89:411-413. Morrison, J.F. and R.A. Peters, 1954. The biochemistry of fluoroacetate poisoning; the effect of fluorocitrate on purified aconitase. Biochem. J. 58:473-479. Napolitano, A.C. and R.J. Aulerich, 1982. Sodium hypochlorite usage with farm-raised mink. The l983 Blue Book of Fur Farming, Communications Marketing, Inc. (Eden Prairie, MN):51-52. National Research Council (NRC), 1982. Nutrient Requirements of Domestic Animals. No. 7. Nutrient Requirements of Mink and Foxes. National Academy of Sciences (Washington, D.C.). 72 pp. 166 Nicholson, W.J. and J.A. Moore, 1979. Health effects of halo- genated aromatic hydrocarbons. Ann. NY Acad. Sci. 320: 1-730. Oser, B.L. and M. Oser, 1956. Nutritional studies on rats on diets containing high levels of partial ester emulsi- fiers. II. Reproduction and Lactation. J. Nutr. 60: 489-505. Peakall, D.B., 1975. PCBs and their environmental effects. CRC Crit. Rev. Environ. Control 5:469-508. Peters, R.A., 1952. Lethal synthesis (Croonian Lecture). Proc. Royal Soc. Lond. (Biol.) 139:143-170. Pickering, Q.H. and C. Henderson, 1966. Acute toxicity of some important petrochemicals to fish. J. Water Pollut. Control Fed. 38:1419-1429. Platonow, N.S. and L.H. Karstad, 1973. Dietary effects of polychlorinated biphenyls on mink. Can. J. Comp. Med. 37:391-400. Poland, A. and J.C. Knutson, 1982. 2,3,7,B-tetrachlorodibenzo- p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Ann. Rev. Pharmacol. Toxicol. 22:517-554. Quin, J.I. and R. Clark, 1947. Studies on the action of potassium monofluoroacetate (CHzFCOOK), (Dichapetalum cymosum (Hook) Engl.) toxin on animals. Onderspoort J. Vet. Sci. 22:77-90. Rasul, A.R. and J. McC. Howell, 1974. The toxicity of some dithiocarbamate compounds in young and adult domestic fowl. Toxicol. Appl. Pharmacol. 30:63-78. Ringer, R.K., 1983. Toxicology of PCBs in mink and ferrets. IN: PCBs: Human and Environmental Hazards. F.M.D'Itri and M.A. Kamrin, eds. Butterworth Publishers (Woburn, MA). pp. 227-240. Ringer, R.K., R.J. Aulerich, and M.R. Bleavins, 1981. Biolo- gical effects of PCBS and PBBs on mink and ferrets - A review. IN: Halogenated Hydrocarbons: Health and Ecological Effects. M.A.Q. Khan, ed. Pergamon Press, Inc. (Elmsford, NY). pp. 329-343. Ringer, R.K., R.J. Aulerich, and M. Zabik, 1972. Effect of dietary polychlorinated biphenyls on growth and reproduc- tion of mink. 164th National Meeting, American Chemical Society 12(2):l49-154. 167 Robens, J.F., 1969. Teratologic studies of carbaryl, diazinon, norea, disulfiram, and thiram in small laboratory animals. Toxicol. Appl. Pharmacol. 15:152-163. Robinson, W.B., 1953. Coyote control with Compound 1080 stations in national forests. J. Forestry 51:880-905. Robinson, W.H., 1970. Acute toxicity of sodium monofluoro- acetate to cattle. J. Wildl. Manage. 34:647-648. Rowley, I., 1963. Effect on rabbits of repeated sublethal doses of sodium fluoroacetate. CSIRO Wildl. Res. 8:52-55. Roy (Shapira), A., U. Taitelman, and S. Bursztein, 1980. Evaluation of the role of ionized calcium in sodium fluoroacetate ("1080") poisoning. Toxicol. Appl. Pharma- col. 56:216-220. Savolainen, H., 1979. TOxic effects of peroral o-cresol intake on rat brain. Res. Commun. Chem. Pathol. Pharmacol. 25(2):357-364. Sax, N.I., 1963. Dangerous Properties of Industrial Chemicals, 2nd Edition (Reinhold, NY). 1343 pp. Short, R.D., J.Q. Russel, J.L. Minor, and C.C. Lee, 1976. Developmental toxicity of ferric dimethyldithiocarbamate and bis(dimethylthiocarbamoyl) disulfide in rats and mice. Toxicol. Appl. Pharmacol. 35:83-94. Shump, A.U. and K.A. Shump, Jr., 1978. Growth and development of the European ferret (Mustela putorius). Lab. An. Sci. 28(1):89-91. Sokal, R.R. and F.J. Rohlf, 1969. Biometry. W.H. Freeman and Co. (San Francisco, CA). Stephan, C.E., Chairman, 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. Committee on Methods for Toxcity Tests with Aquatic Organisms. U.S. Environmental Protection Agency, Ecol. Res. Series, EPA 660/3-75-009. 61 pp. Sullivan, J.L., F.A. Smith, and R.H. Garman, 1979. Effects of fluoroacetate on the testis of the rat. J. Reprod. Fert. 56:201-207. Supelco, 1975. Supelco Bulletin #742D-Tar Acids (phenolic compounds). Supelco, Inc., (Bellefonte, PA). 7 pp. Swarts, F., 1896. Bull. Acad. Belg. Clas. Sci. IIIes 31:675. Travis, H.E. and P.J. Schaible, 1960. Fundamentals of Mink Ranching. Cir. Bul. 229, Mich. State Univ. 101 pp. 168 Tucker, R.K. and D.G. Crabtree, 1970. Handbook of toxicity of pesticides to wildlife. Bureau of Sport Fisheries and Wildlife. Resource Publ. 84, U.S. Dept. of Interior (Washington, D.C.). 131 pp. U.S. Dept. of Health, Education, and Welfare, 1978. Criteria for a recommended standard --- occupational exposure to cresol. DHEW (NIOSH) Publ. No. 78-133. 117 pp. Uzhdavini, E.R., N.K. Astafyeva, and A.A. Mamayeva, 1974. [The acute toxicity of lower phenols]. Gig. Tr. Prof. Zabol. 18:58-59 (Rus.). Vernot, E.H., J.D. MacEwen, C.C. Haun, and E.R. Kinkead, 1977. Acute toxicity and skin corrosion data for some organic and inorganic ocmpounds and aqueous solutions. Toxicol. Appl. Pharmacol. 42(2):417-424. Waibel, P.E., B.L. Johnson, B.S. Pomeroy, and L.B. Howard, 1957. Toxicity of tetramethylthiuram disulfide for chicks, poults, and goslings. Poultry Sci. 36:697-703. Ward, J.C. and D.A. Spencer, 1947. Notes on the pharmacology of sodium fluoroacetate-Compound 1080. J. Am. Pharmaceu. Assoc. 36:59-62. Wedig, J., A. Cowen, and R. Hartung, 1968. Some of the effects of tetramethyl thiuram disulfide (TMTD) on reproduction of the bobwhite quail. Toxicol. Appl. Pharmacol. 12:293-297. WHO, 1965. Evaluation of the toxicity of pesticide residues in food. WHO/Food Addit. Ser. 27.65:181-184. Williamson, J.R., E.A. Jones, and G.F. Azzone, 1964. Metabolic control in perfused rat heart during fluoroacetate poisoning. Biochem. Biophys. Res. Commun. 17:696-702. Willis, L.S. and M.V. Barrow, 1971. The ferret (Mustela utorius furo LL) as a laboratory animal. Lab. An. Sci. 21(5):712-716. Zar, J.H., 1974. Biostatistical Analysis. Prentice-Hall Inc. (Englewood Cliffs, NJ). 169 APPENDIX A MAMMALIAN WILDLIFE (MINK AND FERRET) DIETARY LC50 TEST R.K. Ringer, T.C. Hornshaw, and R.J. Au1erich Department of Anima1 Science Michigan State University East Lansing, MI 48824-1225 December, 1983 170 1. 2. MAMMALIAN WILDLIFE (MINK AND FERRET) DIETARY LC50 TEST 8221: 1.1. This protoco1 describes a method for determining the subacute dietary toxicity of a test substance (that can be mixed uniform1y into the diet) administered to anima1s in their dai1y diet. Toxicity is expressed as the median 1etha1 concentration of the test substance (LC50) and the s10pe of the dose-response curve. 1.2. This protoco1 is intended for use with carnivorous species, such as the mink (Muste1a vison) and European ferret (Muste1a putorius furo). Other carnivorous species may be used with apprOpriate modifications. Summary 2.1. Groups of anima1s of the same Species and age (both sexes) are fed diets containing a test substance in a geometric series of concentrations for 28 days to measure 1etha1ity. This exposure period may be fo11owed by a withdrawa1 period during which 1etha1ity is 6150 measured. 2.2. Dai1y observations for signs of toxicity and morta1ity are reported. 2.3. Data derived from treatment and contro1 groups are compared statisti- ca11y to detect changes in body weight and feed consumption. Significance 3.1. This protoco1 provides a means of measuring the toxicity of a test substance in the dai1y diet of a carnivore under contro11ed conditions. The use of a 28—day dietary exposure period a11ows metabo1ic transformations of the test substance to occur. 3.2. This protoco1 provides data for assessing the potentia1 adverse effects of chemica1s to mamma1ian carnivores exposed through dietary intake, the norma1 exposure route in the environment. The mamma1ian carnivore occupies a position high on the food chain, thus it may be subject to the effects of bioaccumu1ation of chemica1s. 171 3.3. This protoco1 permits co11ection of data on signs of toxicity in addi- tion to morta1ity. 3.4. The dose-response curve provides additiona1 information about the susceptibi1ity of carnivores to a test substance. 3.5. This test provides a basis for deciding whether additiona1 toxicity testing shou1d be conducted. Definitions 4.1. LC50: The ca1cu1ated concentration of a test substance which causes 50 percent 1etha1ity of a test anima1 p0pu1ation under the conditions of the test. 4.2. Test substance: The e1ement, chemica1 compound, formu1ation, known mixture, or materia1 mixed in diets and fed for the purpose of determining an LC50. 4.3. Concentration: The weight of the test substance per unit weight of the diet (expressed as mg/kg of diet). 4.4. Acc1imation period: A period of at 1east 7 days immediate1y preceeding the exposure period during which the test anima1s are housed in the test faci1ities under test conditions and fed the untreated diet and drinking water to be used during the exposure period. 4.5. Exposuregperiod: The 28-day period during which the test animals are fed diets containing the test substance. 4.6. Withdrawa1 period: The period fo11owing an exposure period during which 611 anima1s are fed an untreated diet to a11ow for observation of de1ayed morta1ity. 4.7. Conventiona1 diet: Feed consisting of both fresh and dried ingredients with water added to provide a semi-so1id consistency. 4.8. Dry diet: Feed consisting of on1y dried ingredients fed in pe11eted form. 172 Precautions 5.1. Contact with a11 test substances, so1utions, and mixed diets shou1d be minimized with appr0priate protective c1othing, g1oves, eye protection, etc. The use of fume hoods and increased venti1ation in test rooms is necessary when hand1ing vo1ati1e substances. Information on other mamma1ian toxicity and specia1 hand1ing procedures shou1d be known before this pro- toco1 is used. 5.2. Disposa1 of excess test substances, so1utions, mixed diets, excreta, and treated anima1s shou1d be done with consideration for hea1th and environ— menta1 safety, and in accordance with 611 federa1, state, and 10ca1 regu1a- tions. 5.3. C1eaning and rinsing of g1assware, feeders, and other equipment with vo1ati1eso1vents shou1d be performed on1y in we11-venti1ated areas. 5.4. Since this protoco1 addresses the use of carnivores, appropriate precautions shou1d be used in the hand1ing of test anima1s. Test Anima1s 6.1. This protoco1 is intended for use with carnivorous species, such as the mink (Muste1a vison) and European ferret (Muste1a pgtorius furo). Other carnivorous species may be used with appropriate modifications. 6.2. A11 anima1s for a given test must come from one source and strain and be of approximate1y the same age to minimize variabi1ity. Test anima1s may be obtained from commercia1 sources or may be reared in 1aboratory co1onies, but they must not have been used in a previous test. It is recommended that test anima1s be immunized against diseases common1y aff1icting the test species (for mink and ferrets, immunization shou1d inc1ude: canine dis- temper, virus enteritis, and botu1ism). Anima1s that are deformed, injured, emaciated, or phenotypica11y different from norma1 anima1s must not be used 173 as test subjects. The p0pu1ation of anima1s from which the test subjects (treated and contro1) are se1ected sha11 be considered unsuitab1e for testing if morta1ity exceeds 5% during the acc1imation period. 6.3. It is preferab1e to use anima1s that have approached their mature body size (for mink and ferrets this is about 18-20 weeks of age). 01der anima1s can a1so be used to determine the LC50. The use of younger anima1s may yie1d a distorted LC50 va1ue because the change in body weight far exceeds the change in feed consumption resu1ting in a decreased amount of test sub- stance consumed per unit of body weight over the 28-day period. Faci1ities 7.1. Space requirements for most carnivores have not been standardized. However, adherence to genera] guide1ines and princip1es of good 1aboratory anima1 care (1) in addition to 1iterature pub1ished on individua1 species (see Tab1e 1) shou1d provide a basis for adequate space requirements. Cages must be constructed so as to prevent cross-contamination and contact between anima1s. Species not conducive to co1ony rearing, such as mink, must be caged individua11y. 7.2. Construction materia1s in contact with anima1s shou1d not be toxic, nor be capab1e of excessive1y adsorbing or absorbing test substances. Stain1ess stee1, ga1vanized stee1, or materia1s coated with perf1uorocarbon p1astics are acceptab1e materia1s, but other construction materia1s may 6150 be usefu1. » 7.3. Adequate venti1ation shou1d be provided at a11 times (1). 7.4. If test is conducted indoors, the phot0period shou1d simu1ate ambient day1ight conditions at the date of initiation of the definitive test. m 8.1. Diets must be formu1ated in accordance with the nutrient requirements of the test species (2). Any unmedicated commercia1 diet that meets the 174 10. minimum nutritiona1 standards of the test species is acceptab1e. 8.2. Fresh diets and water must be provided dai1y and fed gg_1ibitum. Diet Preparation 9.1. Know1edge of the physica1, chemica1, and bio1ogica1 properties of the test substance is important in test diet preparation. 9.2. Test diets can be prepared by mixing the test substance direct1y into the feed or by disso1ving or suspending the test substance in a so1vent or carrier prior to mixing with the feed. The use of so1vents or carriers may be necessary to achieve uniformity. If so1vents or carriers are used, they must 6150 be added to the contro1 diet. A 9.3. When conventiona1 diets are used, sufficient diet shou1d be mixed to provide adequate feed for the 28-day exposure period. The amount of feed to be mixed may be based upon the feed consumption data obtained during the acc1imation period. When mixed, the diet shou1d be stored frozen in con- tainers 1arge enough to ho1d 1-2 day's feed. In testing volati1e substances, sea1ab1e containers must be used. When dry diets are used, they shou1d be stored so as to maintain the stabi1ity of the test substance in the diet. The frequency with which the feed is mixed is dependent upon the physica1/chemica1 pr0perties of the test substance. 9.4. It is recommended that a11 diets be ana1yzed for the concentration of the test substance in the diet. Procedure 10.1. Range finding_test: In order to determine the test concentrations to be used in the definitive test, a range finding test may be conducted using severa1 wide1y spaced concentrations. 10.2. Acc1imation,period: A11 anima1s sha11 be conditioned to the test faci1ities, inc1uding: photoperiod, temperature, and caging for a minimum 175 of 7 days. During this period 611 anima1s sha11 be given the untreated (control) diet and drinking water, as used during the definitive test. Test anima1s shou1d be weighed at the start of the acc1imation period. It is recommended that feed consumption be measured during the 1atter part of the acc1imation period. 10.3. Definitive test: 10.3.1. Each test anima1 sha11 be random1y assigned to a specific test diet concentration and be unique1y identified. 10.3.2. The test diets must be fed for 28 days. For some test sub- substances, it may be necessary to inc1ude a withdrawa1 period, during which the test diets are rep1aced with untreated feed, in order to observe pro1onged or de1ayed toxicity. 10.3.3. Individua1 body weights must be recorded at the initiation of the definitive test and at week1y interva1s thereafter, and on the day of death. Feed consumption must be measured week1y for the exposure and withdrawa1 periods, and shou1d be based on a minimum of two consecutive day's feed consumption. 10.3.4. Morta1ity, behaviora1 abnorma1ities, and other signs of toxicity sha11 be recorded dai1y during the test. 10.3.5. For tests conducted indoors, the photOperiod sha11 be main- tained at the same schedu1e in effect at the conc1usion of the acc1ima— tion period. 10.3.6. A minimum of eight anima1s for each test concentration sha11 be used. The test concentrations shou1d be geometrica11y spaced so as to resu1t in at 1east 2 dietary concentrations yie1ding 10-90% morta1ity. These resu1ts usua11y can be obtained with 4-6 dietary concentrations, inc1uding a contro1. 10.3.7. A test sha11 be considered inva1id if more than 12.5% of the contro1 anima1s die during the definitive test. 176 11. 12. 10.3.8. It is strong1y recommended that a dietary concentration group shou1d be removed from testing when food consumption measurements indicate that 1 % or 1ess feed, compared to contro1s and/or acc1ima- tion period va1ues, is consumed dai1y for the first two week's feed consumption measurements. 10.3.9. Necropsies sha11 be performed on a11 morta1ities. At the termination of the test a11 surviving test anima1s sha11 be ki11ed by accepted humane methods and necr0psies performed. Qua1ity Assurance 11.1. In order to assure the qua1ity and re1iabi1ity of data deve1oped using this protoco1, good 1aboratory practices shou1d be fo11owed (3,4). Reporting Requirements 12.1. Name of the investigator(s), 1aboratory, 1aboratory address, 1ocation of raw data, and date of initiation and termination of test. 12.2. Name of species tested, inc1uding scientific name, source, and age of the anima1s at the beginning of the test. 12.3. A detaiIed description of the test substance inc1uding its chemica1 name, synonyms, structure, formu1ations, purity, source, batch, 1ot number, and physica1/chemica1 properties and name of so1vent or carrier, if used. 12.4. Description of the test faci1ities and housing conditions, inc1uding test cages, temperature, and photoperiod. 12.5. Name and source of feed, inc1uding description and proximate ana1ysis of diet. 12.6. The dietary concentrations; number of anima1$ per concentration; body weights; feed consumption; signs of toxicity; behaviora1 changes; % mortaIity for each concentration; significant necropsy findings; ca1cu1ated LC50 va1ues and 95% confidence 1imits, s10pe of the dose-response curve and 95% confidence 1imits, and the name and reference of the statistica1 method used; highest 177 dietary concentration at which no signs of toxicity were observed; anything unusual about the test; any deviations from the protocol; and other relevant information. 13. Discussion of Protocol by Section 3.1. It is recommended that, if possible, tests be conducted indoors. Indoor tests a1low greater control of test conditions, and therefore, greater reproducibility. Indoor faci1ities, if heated, allow conducting tests at any time of the year. Indoor faci1ities also make the possibility of escape of test anima1s less likely. 3.5. The 28-day dietary LC50 test can be used as a basis for further tests. Results from the 28-day test may indicate the need for subsequent reproduction or chronic tests with the test species. These results might also indicate the need for other types of tests, such as aquatic, inhalation, secondary toxicity, etc., or tests designed for a target organ or organ system. 4.4;10.2. An acc1imation period is required to condition the animals to the test faci1ities, diet, water, temperature, and lighting system to be used in a test. A minimum of 7 days is required, but a longer period may be necessary, eSpecially if the animals to be used in a test are changed from outdoor to indoor housing (or vice-versa) or if the diet or water to be used in a test is different from that which the animals are accustomed to consuming. It is important to measure feed consumption during this period as these measurements can provide an indication of how much feed the animals should consume once the test starts, how much diet to mix for the test, and can also serve as a "control" value for each group. 4.5;10.3.7. The prescribed length of the mammalian dietary LC50 test is 28 days for several reasons. A 28-day test allows time for absorption, distri- bution, metabolism, enzyme induction, redistribution, bioconcentration, and elimination to occur, similar to that which might occur in animals subacutely 178 __ ... . . _. ._ _ an p N p . cm.N 83 an 8 en 8 NN _N on are. S 2an Bnrza_aanemnm~_ 35:8. saw» us Neu\ms,»u m_sewsn es .uz sonussosuosou .umus smog xau sN s asmtsu Aesop ussssesuv muuuoosssosptssos Esmusm um» xsns mu stooges sun—sass: .N m_ss» — — — _ — N . 3...: p. 35 11“ I! p 26 o en. R an a an 2. ...a _N an. arc: erefmeqmlmr-wtu S a m u. e m n m w _ Bias. : 38 .3 u 2955. Cu .3. 1 suturessossu .38 can: an... E a 9.2.6 82.: ussssaauv 338833589: 5.5.3. um» Soto“ be seeing 5:33: .m 323 1Qn exposed to a substance via diet in the environment. A 28-day test also allows testing of slow-acting or bioaccumulating substances. Such tests could prove negative or misleading in a test of shorter duration. For example, prolonged mortality patterns were observed in 28-day tests with mink (Table 2) and ferrets (Table 3) fed Compound 1080, in which morta1ites were observed up to the end of the test. Delayed mortalities were observed in a 28-day test with mink fed Aroclor 1254 (Table 4), in which mortalities were observed during a 7-day with- drawal period as well as during the exposure period. In some instances, it may be possible to achieve satisfactory results with a test of shorter duration using higher concentrations of the test substance, but the possibility of feed rejec- toin or avoidance becomes greater with increasing concentrations. For example, in the 1080 tests already noted, signs of feed avoidance appeared in the first week of both tests in a dose-related manner. Increasing the concentration in these tests would have resulted in nearly complete avoidance of the feed and subsequent removal of the highest dietary concentrations from the test for humane reasons. Also, certain substances cause delayed morta1ity, whether administered as a single dose or multiple dosages. Increasing the concentration of the substance does not necessarily shorten the time to death. An example of this phenomenon is seen in the Aroclor 1254 test. 4.6;10.3.2. A withdrawa1 period is recommended when animals are still exhibiting signs of toxicity at the end of the exposure period. This period provides a more accurate estimation of the true toxicity of a test substance, especially if the substance causes delayed or cumulative injury. By observing the animals and measuring feed consumption during this peirod, the permanence of the injury can also be estimated. It is recommended that a withdrawal period not exceed 14 days. 5.4. Several precautions must be mentioned if the researcher is to use the mink as a test species. Mink are by nature extremely aggressive and may attack 181 a handler if given the opportunity. Appropriate cautions, such as leather gloves and arm-coverings, should be used. Mink can also transmit certain diseases, most importantly tetanus, and also tuberculosis and rabies if contracted from outside sources, so researchers should take precautions for these diseases. The possibi- lity of TB infection can be reduced to nearly zero by avoiding the use of pork products in the diet, and pr0per caging and isolation of test facilities from wild animals can similarly reduce the possibility of rabies infections to nearly zero. 7.1. While space requirements for mink and ferrets have not been determined, individual cages measuring 61 (L) x 76 (W) x 46 (H) cm (24 x 30 x 18 in) have proven adequate for tests performed in conjunction with the development of this protocol, and are within the range of cage dimensions widely used in the fur industry. In designing a caging system for carnivores, and especially for mink, it is important to prevent both cross-contamination of treatment groups and contact between individual animals. As stated previously, mink are extremely aggressive and may attack neighboring animals, if contact can occur. This can be prevented by providing adequate space between adjoining cages if wire mesh cage material is used throughout the cage, or by use of solid dividers between adjoining cages. 7.1;10.2;10.3.5. Phot0period is maintained at the schedule in effect near the conclusion of the acclimation period because a changing photoperiod subjects mink and ferrets to slight adjustments in normal body rhythms with respect to feeding, sleeping, fat mobilization or deposition, coat development, and hormonal activity. It should be noted that the effects of the length of the light/dark cycle on toxicity have not been investigated in these species. 9.2. It is very important to assure uniform distribution of a test sub- stance in the diet. In many instances, this will be more easily accomplished using the conventional diet, since many substances can be mixed into a diet more 182 uniform1y if the diet is semisolid and capable of being machine-mixed. For some test substances, especially water soluble ones, this may be the only method of assuring uniform distribution, since pe11eted diets are not conducive to being coated with aqueous solutions, but rather tend to become a mash. No matterwhich type of diet is used it is recommended that innocuous solvents or carriers, such as distilled water or corn oil, be used if possible. (It is recomnended that, unless the amount of test substance to be added is large, solvents or carriers be used to introduce the substance to the diet to ensure uniform distribution). If an innocuous solvent or carrier cannot be found, it is recommended that a volatile solvent, such as acetone or hexane, be used to dissolve the test substance, if possible, and the resultant solution be added to a small amount of either the dry diet or a dry ingredient (e.g. cereal) of the conventional diet. After the solvent is evaporated the pre-mix can then be mixed with the rest of the diet uniform1y. (If this procedure is used, it must likewise be used on the control diet). 9.3. If the researcher chooses to use the conventional diet, it is impor- tant not to freeze the diets in containers too large, since the diets will not remain fresh under refrigeration for more than 2-3 days. 10.1. In most cases, L050 estimates for mink or ferrets will not be avail- able to aid in setting dietary concentrations for the L050 test. Therefore, range-finding procedures can be employed to save both time and animals by reducing errors or miscalculations in setting these concentrations. LC50 estimates for other species may be helpful in setting dietary concentrations, although in general mink and ferrets are more sensitive to toxic compounds than laboratory animals. For this reason, if L050 estimates are available for other species, these values can be used as the upper limit of doses in the range- finding procedure. This procedure can be a geometrica11y spaced series of doses (e.g. in multiples of 2 or 8) administered by gavage to 2 animals per dose, in 183 which case the approximate L050 is the dose at which 1 or 2 animals die after an appropriate period of observation (often one week). (It is suggested that, when administering an oral dose to mink or ferrets by gavage, a piece of plastic large enough to force the animal's mouth Open, with a small hole in the center, be used. The tube can then be inserted through the opening without the animal biting it - see Figure 1). If L050 estimates are not available for other species, wide1y-spaced doses (e.g. l, 10, 100, and 1000 mg/kg) can be administered to one animal to find a lethal dose. The range-finding procedure described above can then be employed, centering on the lethal dose. If range-finding procedures yield an approximate L050 value, the highest dietary concentration should then be set to ensure that an animal will consume the equivalent of an L050 dose in one day's feed. If a lethal dose is not found, the highest dietary concentration should then be set at 5000 mg/kg, since concentrations above this value are assumed to be non-toxic. Palatability tests may also be employed to save time and animals. It is recommended that the highest preposed dietary concentration be fed for several days to 2-4 animals to determine if they will eat the diet at this concentration of the test substance. If not, it may be necessary to reduce the highest con- centration to a level at which the diet will be eaten. 10.3.3. In estimating feed consumption by mink or ferrets, several precau- tions are necessary. Since feed consumption can be adversely affected on a short-term basis by temperature, weather, and other factors, estimates should be based on at least two consecutive day's consumption. These days should also be days in which the animals are not handled (e.g. during weighing, moving, etc.), since hand1ing can produce a temporary reduction in feed consumption. 10.3.6. It is possible to conduct an L050 test with as few as 3 dietary concentrations and 8 animals per concentration if a good estimate of the LD50 for the test species is available. In many instances, however, accurate results 184 can be achieved with 5 dietary concentrations and 10 animals per concentration if a good estimate of the L050 is available from range-finding procedures. 10.3.9. It is suggested that necropsies be performed on all test animals, either on the day of death or at the termination of the test. Valuable informa- tion on the mode of action and target organs or organ systems of the test sub- stance can sometimes be gained from gross observation of the test animals at necropsy, and histopathological examination can sometimes provide more informa- tion. Weights of internal organs of control and treated animals can be compared statistically to determine effects of the substance, although the effects of starvation can sometimes be confounded with effects of the substance. 185 321 O d ; 9 = 10cm Figure 1. Device for gavage. 186 Table l. Cage dimensions as reported in the literature for mink and ferrets. Species Cage dimensions (L X W X H in cm) Reference Mink 76.2 45.7 38.1 5' to X to X to 121.9 61.0 61.0 91.4 X 45.7 X 38.1 6 76.2 38.1 22.9 7 to X to X to 91.4 61.0 45.7 Ferret 76 X 76 X 46 8 187 REFERENCES Anonymous, Guide for the Care and Use of‘Laboratory Animals. DHEW Publica- tion No. (NIH) 78-23, U.S. Department of Health, Education, and Welfare, 1978. National Research Council (NRC). Nutrient Requirements of Mink and Foxes. Nutrient Requirements of Domestic Animals series. Washington, 0.C.: National Academy of Sciences, 1982. Anonymous, Department of Health, Education, and Welfare, Food and Drug Administration. "Nonclinical Laboratory Studies, Good Laboratory Practice Regulations", Federal Register, Volume 43, No. 247, pp. 59986-60025, 1979. Anonymous, Environmental Protection Agency. "Preposed Environmental Standards; and Proposed Good Laboratory Practice Standards for Physical, Chemical, Persistence, and Ecological Effects Testing“, Federal Register, Volume 45, No. 227, pp. 77332-77365, 1980. Travis, H.E. and P.J. Schaible, Fundamentals of'Mink Ranching, Cir. Bul. 229, Michigan State University, 1960. Leonard, A., Modern Mink Management, Ralston Purina Co., St. Louis, MO, 206 pp., 1966. ‘ Fur Rancher Blue Book of'Fur Farming (published annually). Communications Marketing Inc., Eden Prairie, MN. Shump, A.U. and K.A. Shump, Jr., Growth and development of the Eur0pean ferret (Mustela putorius). Lab. Anim. Sci. 28(1):89-91, 1978. l 88 APPENDIX B MAMMALIAN WILDLIFE (MINK AND FERRET) REPRODUCTION TEST R.K. Ringer, T.C. Hornshaw, and R.J. Au1erich Department of Animal Science Michigan State University East Lansing, MI 48824-1225 December, 1983 189 MAMMALIAN WILDLIFE (MINK AND FERRET) REPRODUCTION TEST 1-_S_c_929_ 1.1. This protocol describes a method for determining the reproductive toxicity of a test substance (that can be mixed uniform1y into the diet) administered to animals in their daily diet. Reproductive toxicity may be expressed as an adverse effect on: a) adu1t survival; b) oogenesis and/or spermatogenesis; c) embryo or fetus development; d) reproductive indices; or e) offspring growth and survival. 1.2. This protocol is intended for use with carnivorous species, such as the mink (Mustela viSon) and European ferret (Muste1a putorius furo). Other carnivorous species may be used with appr0priate modifications. 2. Summary 2.1. Groups of animals of the same species and age (both sexes) are fed diets containing a test substance in a series of concentrations, plus a control, for 8 weeks prior to breeding, during breeding, gestation, and parturition, and for 3 weeks of lactation (approximately 23 weeks) to measure reproductive toxicity. 2.2. Animals are observed daily and mortalities are reported. 2.3. Data derived from treatment and control groups are compared statisti- cally to detect changes in body weight; feed consumption; length of gestation; percent of females bearing offspring; total offspring born per female (live and dead); average birth weight of offspring; average live litter weight; average weight of offspring at 3 weeks; and percent offspring survival to 3 weeks. 3. Significance 3.1. This protocol provides a means of measuring the reproductive toxicity of a test substance in the daily diet of a carnivore under controlled conditions. 190 3.2. This protocol provides data for assessing the potential adverse effects 3.3. 3.4. of chemicals to mammalian carnivores exposed through dietary intake, the normal exposure route in the environment. The mammalian carnivore occupies a position high on the food chain, thus it may be subject to the effects of bioaccumulation of chemicals. This protocol permits collection of data on signs of toxicity and mortality over an extended period of dietary exposure, such as may occur in nature. This test provides a basis for deciding whether additiona1 toxicity testing should be conducted; Definitions 4.1. 4.2. 4.3. 4.4. 4.5. Test substance: The element, chemical compound, formu1ation, known mixture, or material mixed in diets and fed for the purpose of deter- mining reproductive toxicity. Concentration: The weight of the test substance per unit weight of the diet (expressed as mg/kg of diet). Acclimation period: A period of at least 7 days immediately preceding the exposure period during which the test animals are housed in the test facilities under test conditions and fed the untreated diet and drinking water to be used during the exposure period. Conventiona1 diet: Feed consisting of both fresh and dried ingredients with water added to provide a semi-solid consistency. Dry diet: Feed consisting of only dried ingredients fed in pe11eted form. Precautions 5.1. Contact with all test substances, solutions, and mixed diets should be minimized with appropriate protective clothing, gloves, eye protec- tion, etc. The use of fume hoods and increased ventilation in test 191 5.2. 5.3. 5.4. 5.5. rooms is necessary when handling volatile substances. Information on other mamma1ian toxicity and special handling procedures should be known before this protocol is used. Disposal of excess test substances, solutions, mixed diets, excreta, and treated animals should be done with consideration for health and environmental safety, and in accordance with all federal, state, and local regulations. Cleaning and rinsing of glassware, feeders, and other equipment with volatile solvents should be performed only in well-ventilated areas. Since this protocol addresses the use of carnivores, appr0priate precautions should be used in the handling of test animals. Since mink and ferrets are known to be sensitive to handling and other disturbances during the first 2 weeks post-partum, contact and outside disturbances should be minimized during this period. Test Animals 6.1. 6.2. This protocol is intended for use with carnivorous species, such as the mink (Muste1a vison) and European ferret (Mustela putorius furo). Other carnivorous species may be used with appropriate modifications. All animals for a given test must come from one source and strain and be of approximately the same age to minimize variabi1ity. Test animals may be obtained from commercial sources or may be reared in laboratory colonies, but they must not have been used in a previous test. It is recommended that test animals be immunized against diseases commonly afflicting the test species (for mink and ferrets, immunization should include: canine distemper, virus enteritis, and botu1ism). Animals that are deformed, injured, emaciated, or phenotypica11y different from normal animals must not be used as test subjects. The population of 192 6.3. animals from which the test subjects (treated and control) are selected shall be considered unsuitable for testing if morta1ity exceeds 5% during the acclimation period. It is preferable to use animals that are proven breeders. However, availability and cost may dictate that animals in their first breeding season be used. 7. Facilities 7.1. 7.2. 7.3. Space requirements for most carnivores have not been standardized. However, adherence to general guidelines and principles of good labora- tory animal care (1) in addition to literature published on individual species (see Table 1) should provide a basis for adequate space require- ments. Cages must be constructed so as to prevent cross-contamination and contact between animals. Species not conducive to Colony rearing, such as mink, must be caged individua11y. Construction materials in contact with animals sh0uld not be toxic, nor be capable of excessive1y adsorbing or absorbing test substances. Stain- 1ess stee1, galvanized steel, or materials coated with perfluorocarbon plastics are acceptable materials, but other construction materials may also be useful. A nest area and nesting material must be provided for all females prior to the parturition period. All materials must be free of contaminants. 193 8. 7.4. 7.5. Adequate ventilation should be provided at all times (1). If test is conducted indoors, the photoperiod shou1d simulate ambient daylight conditions throughout the acclimation period and definitive test. This protocol addresses the use of mink and ferrets during their natural breeding seasons. Photoperiodic manipulation may permit the use of this protocol at other seasons. Since very low intensities of light may alter the reproductive cycle, care must be taken to ensure that total darkness is maintained during the appropriate periods. If test is conducted outdoors, care must be taken to ensure that the photoperiod is not altered by extraneous light sources. Diets 8.1. Diets must be formulated in accordance with the nutrient requirements of the test species (2). Any unmedicated commercia1 diet that meets the minimum nutritional standards of the test species is acceptable. 8.2. Fresh diets and water must be provided daily and fed 2g libitum. Diet Preparation 9.1. Knowledge of the physical, chemical, and biological properties of the 9.2. 9.3. test substance is important in test diet preparation. Test diets can be prepared by mixing the test substance directly into the feed or by dissolving or suspending the test substance in a solvent or carrier prior to mixing with the feed. The use of solvents or carriers may be necessary to achieve uniformity. If solvents or carriers are used, they must also be added to the control diet. When conventiona1 diets are used, sufficient diet shou1d be mixed to provide adequate feed for approximately 4 weeks. The amount of feed to be mixed may be based upon the feed consumption data obtained during the acclimation period. When mixed, the diet should be stored frozen in containers large enough to hold 1-2 day's feed. In testing volatile 194.. substances sealable containers must be used. Feed should be similarly mixed and frozen as needed for the duration of the test. When dry diets are used they should be stored so as to maintain the stability of the test substance in the diet. The frequency with which the feed is mixed is dependent upon the physical/chemica1 properties of the test substance. 9.4. It is recommended that all diets be analyzed for the concentration of the test substance in the diet. 10. Procedure 10.1. Dietary concentrations of test substance. 10.1.1. Establishing the dietary concentrations of a test substance for a reproductive study is a difficult but essential first step in determining an environmental effect of a chemical substance upon reproduction. A number of procedures exist for establishing the dietary concentrations to be used. Three are presented in this protocol. 10.1.1.1. If a mammalian wildlife dietary LC50 test (see pp - of this volume) has been conducted with the species under con- sideration, the highest dietary concentration at which no signs of toxicity were observed shou1d approximate the highest of a series of geometrica11y spaced dietary concentrations, plus a control. 10.1.1.2. Another method of establishing dietary concentrations utilizes known or expected environmental concentrations of the test substance. Two or more dietary concentrations, plus a control, should be used. Examples of series of concentrations that may be used include 1X, 3X, and 5X or 1X, 3X, and 10X, where X equals the environmental concentration. 195 10.1.1.3. If LC50 data are lacking, it is useful to conduct a pre- liminary study with several widely spaced dietary concentrations of the test substance. The dietary concentrations may be established from these preliminary studies. It is recommended that 3 or more dietary concentrations plus a control be tested in the definitive test if this procedure is followed. 10.2. Experimental Design 10.2.1. This protocol is intended for use with individually caged animals only. Males and females will be paired only during breeding attempts, and one male will be assigned to a treatment group for each 3 or 4 females. Thus, this protocol is primarily designed to test female reproductive effects, and provides only limited data on male repro- ductive effects. If data on male reproductive effects are desired, a different experimental design Will be necessary. 10.2.2. A minimum of 12 females is recommended per dietary concentration and an equal number for the control group. In addition, a minimum of 3 males should be housed per treatment group. It is recommended that breeding attempts be made only between males and females within the same treatment group. Use of proven breeders may reduce the number of animals per treatment group, but not to less than 8 females and 2 males per group. 1 10.2.3. If the experimental design is selected as per Section 10.2.2., one of the following criteria must be met: 1) One dietary concentration must produce an effect. 2) The highest dietary concentration must contain at least 1000 mg/kg. 3) The highest dietary concentration must be at least 100 times the highest known or expected environmental concentration. 196 If the researcher selects an experimental design based on con- siderations of Type I and Type II error, the number of females per treatment group may be specified by the researcher's levels of power, significance, and difference between means to be detected. 10.2.4. Each test animal shall be randomly assigned to a specific test diet concentration and be uniquely identified. 10.3. Acclimation Period 10.3.1. All animals shall be conditioned to the test facilities, including: photoperiod, temperature, and caging for a minimum of 7 days. During this period all animals shall be given the untreated (control) diet and drinking water, as used during the definitive test. Test animals should be weighed at the start of the acclimation period. It is recommended that feed consumption be measured during the latter part of the acclimation period. 10.4. Definitive Test 10.4.1. The test diets must be fed daily throughout the pre-breeding, breeding, gestation, parturition, and lactation periods, a duration of approximately 23 weeks. 10.4.1.1. Pre-breeding period: Individua1 body weights must be recorded at the initiation of the definitive test and bi-weekly (once every other week) thereafter for the 8 weeks of the pre- breeding period. Feed consumption must also be measured bi- weekly during the pre-breeding period, and should be based on a minimum of two consecutive day's feed consumption. 10.4.1.2. Breeding period: This period lasts approximate1y 3-4 weeks, during which the females are presented to the males 1197 within treatment groups for breeding. Matings are confirmed by microscopic examination of vaginal aspirations for viable sperma- tozoa, and recorded by date. Females with confirmed matings are given the opportunity for a second mating either 1 or 8 days following the initial confirmed mating. Generally, body weight changes and feed consumption measure- ments are not recorded during this period. These measurements may be performed if animals are not excessively disturbed. 10.4.1.3. Gestation period: This period lasts approximately 6 weeks for ferrets and 7-8 weeks for mink. During this period animals should not be weighed, handled, or unduly disturbed. 10.4.1.4. Parturition period: This period lasts up to 3 weeks, depending on species. During this period females are checked daily for newborn. All newborn are counted, weighed, and recorded within 24 hours post-partum. 10.4.1.5. Lactation period: Individua1 body weights of a11 survi- ving newborn are recorded at the end of this 3 or more week period. This period should not extend beyond 6 weeks, the normal weaning time for mink and ferret offspring. During this period, offspring may come in contact with or eat (after 3 weeks) the maternal diet. 10.4.1.6. Termination: At the termination of the test all males and an equal number of females chosen at random from each dietary group should be killed by accepted humane procedures and necropsies performed. Tissue residue analyses and histo- pathological examination may be helpful. Measuring organ weights may also provide useful information about the test substance. 198 10.5. 10.4.2. General considerations 10.4.2.1. All animals must be observed dai1y. All overt clinical signs and any abnormal behavior must be recorded when observed. If mortality occurs, the date and body weight must be recorded and necr0psy performed. 10.4.2.2. A test may be considered invalid if more than 20% of the control animals die during the definitive test. Reproductive indices 10.5.1. The following reproductive indices must be calculated: 1) Length of gestation: The time, in days from the last con- firmed mating until parturition. 2) Number whelped, not whelped: The number of females giving birth and not giving birth in a treatment group. Number whelped includes females that die during the process of whelping from problems associated with parturition. This value is expressed as the number of females whelped or not whelped per the number of females with confirmed matings in a treatment group. 3) Live newborn/fema1e whelped: The average number of live newborn produced by all females that give birth in a treatment group. This value does not include females that die during the process of whelping from problems associated with parturi- tion. 4) Average birth weight: The average weight of all live newborn born in a treatment group, weighed to the nearest tenth of a gram within 24 hours post-partum. 5) Average litter weight: The average weight of a11 litters (live newborn on1y) born in a treatment group, weighed to the 199 7) nearest tenth of a gram within 24 hours post-partum. Percent newborn survival to 3 weeks: The number of live newborn in a treatment group surviving to 21 days of age, expressed as a percentage of a11 live newborn born in a treatment group. Average 3 week body weight: The average weight of all live newborn in a treatment group, weighed to the nearest gram on the let day after birth. 10.5.2. The following reproductive indices may also be useful: 1) 2) 3) 10.6. Statistical Total newborn/fema1e whelped: The average number of all newborn (alive and dead) produced by all females that give birth in a treatment group. This value includes females that die during the process of whelping from problems associated with parturition. Percent newborn survival to 6 weeks: Identical to 21 day survival, but extended to 42 days. Average 6 week body weight: Identical to 21 day weights, but measured at 42 days of age. ana1ysis 10.6.1. The following variables may be analyzed by analysis of variance (3) and significant differences may be tested by Dunnett's method for comparison with control (4): 1) 2) 3) 4) 5) 6) 7) Body weight changes Feed consumption Length of gestation Live offspring/female whelped Total offspring/fema1e whelped Average birth weight Average litter weight 2()O 8) Average 3 week body weight 9) Average 6 week body weight 10.6.2. The following variables may be analyzed by contingency tables (5) and significant differences may be tested by Bonferroni's Chi- square test (6): 1) Number whelped, not whelped 2) Percent newborn survival to 3 weeks 3) Percent newborn survival to 6 weeks 10.6.3. Other valid statistica1 procedures may be used to analyze data. 11. Quality Assurance 12. 11.1. In order to assure the quality and reliability of data developed using this protocol, good laboratory practices should be followed (7,8). ReportingiRequirements 12.1. 12.2. 12.3. 12.4. 12.5. Name of the investigator(s), 1aboratory, 1aboratory address, location of raw data, and date of initiation and termination of test. Name of species tested, including scientific name, source, and age of the animals at the beginning of the test. A detailed description of the test substance including its chemical name, synonyms, structure, formu1ations, purity, source, batch, lot number, and physical/chemica1 pr0perties and name of solvent or carrier, if used. ' Description of the test facilities and housing conditions, including test cages, temperature, and photoperiod. If conducted outdoors, adverse weather conditions may alter test resu1ts, especially during the parturition period, and should be reported. Name and source of feed, including description and proximate analysis of diet. 201 12.6. The dietary concentration; number of males and females per concentra- tion; body weights; feed consumption; signs of toxicity; abnormal behavior; mortality; reproductive indices; statistical methods employed; significant necropsy findings (including organ weights, if recorded); anything unusual about the test; any deviations from the protocol; and other relevant information. 13. Discussion of Protocol by,Section .gyl; It is recommended that, if possible, tests be conducted indoors. Indoor tests allow greater control of test conditions, and therefore, greater reproduc- ibility. Indoor faci1ities, if heated, allow more accurate measurements of feed consumption than outdoors, especially during sub-freezing conditions. Indoor facilities also make the possibility of escape of test animals less likely. 3444_ The reproduction test can be used as a basis for further tests. Results from a reproduction test may indicate the need for subsequent chronic tests with the test species. These results might also indicate the need for other types of tests, such as aquatic or inhalation, or tests designed for a target organ or organ system. This protocol can provide limited data on the effects of a substance on male reproductive performance. However, if such effects are noted, it would be necessary to conduct further tests employing a different experimental design than the one described in the protocol to quantify ma1e effects. §ygy_ Several precautions must be mentioned if the researcher is to use the mink as a test species. Mink are by nature extremely aggressive and may attack a handler if given the opportunity. Appropriate cautions, such as leather gloves and arm-coverings, should be used. Mink can also transmit certain diseases, most importantly tetanus, and also tuberculosis and rabies if contracted from outside sources, so researchers should take precautions for these diseases. The possibi- lity of TB infection can be reduced to nearly zero by avoiding the use of pork 2132 products in the diet, and proper caging and isolation of test facilities from wild animals can similarly reduce the possibility of rabies infections to nearly zero. 241; While Space requirements for mink and ferrets have not been determined, individual cages measuring 61 x 76 x 46 cm (24 x 30 x 18 in) and nest boxes measuring 38.1 x 27.9 x 26.7 cm (15 x 11 x 10.5 in) have proven adequate for tests performed in conjunction with the develOpment of this protocol, and are within the range of cage and nest box dimensions widely used in the fur industry. In designing a caging system for carnivores, and especially for mink, it is important to prevent both cross-contamination of treatment groups and contact between individual animals. As stated previously, mink are extremely aggressive and may attack neighboring animals, if contact can occur. This can be prevented by providing adequate space between adjoining cages if wire mesh cage material is used throughout the cage, or by use of solid dividers between adjoining cages. Z;§;_ It is very important to ensure that newborn are protected from toxic compounds. Of special importance is the nest material provided for the females. The researcher should be sure to use nest materials which are free of toxicants. A particular area of concern is wood by-products which may be contaminated with compounds to which mink are suspected to be sensitive. .112; In order to bring mink and ferrets into breeding condition indoors, it is necessary to gradually increase the length of daylight during the test. If the animals are held indoors for an extended period of time prior to the test, it is also necessary to gradually decrease daylight prior to the acclimation period to provide a necessary quiescent period of sexual development for the animals. Igygy It is very important to assure uniform distribution of a test sub- stance in the diet. In many instances, this will be more easily accomplished using the conventional diet, since many substances can be mixed into a diet more uniformly if the diet is semisolid and capable of being machine-mixed. For 203 some test substances, especially water soluble ones, this may be the only method of assuring uniform distribution, since pe11eted diets are not conducive to being coated with aqueous solutions, but rather tend to become a mash. No matter which type of diet is used it is recommended that innocuous solvents or carriers, such as distilled water or corn oil, be used if possible. (It is recommended that, unless the amount of test substance to be added is large, solvents or carriers be used to introduce the substance to the diet to ensure uniform distribution). If an innocuous solvent or carrier cannot be found, it is recommended that a volatile solvent, such as acetone or hexane, be used to dissolve the test substance, if possible, and the resultant solution be added to a small amount of either the dry diet or a dry ingredient (e.g. cereal) of the conventional diet. After the solvent is evaporated the pre-mix can then be mixed with the rest of the diet uniform1y. (If this procedure is used, it must likewise be used on the control diet). 9;§;_ If the researcher chooses to use the conventional diet, it is impor- tant not to freeze the diets in containers too large, since the diets will not remain fresh under refrigeration for more than 2-3 days. 10.1.1.3. If an estimate of a dietary concentration at which signs of toxicity are not observed is lacking, it is recommended that a pre1iminary study be con- ducted to aid in establishing dietary concentrations for the definitive test. This study may be patterned after the protocol for mammalian dietary LC50 tests, using several widely spaced concentrations over a short period (e.g. 7-14 days) to determine an approximate no effect concentration. Since the data which will be generated from a study such as this would be expected to be fragmentary, it is suggested that at least 3 dietary concentration's be tested in the definitive test in order to maximize the possibility of meeting the criteria for an acceptable test while minimizing the possibility of wasting time, money, and animals. 204 10.2.2. Since relatively few reproduction tests have been conducted with mink and ferrets, and experimental procedures have varied in those tests, few back- ground data are available to aid in determining the pr0per number of females to use to detect a significant difference for a given reproductive index. Thus, the minimum number of females per dietary concentration specified in this protocol was based on reproduction tests performed in conjunction with the development of this protocol and on other reproduction tests with mink and ferrets. Due to considerations of cost and availability of proven breeders, it generally will be necessary to use animals which have not had breeding experience. If this is the case, it is recommended that a minimum of 12 fema1es per treatment group be used, to provide a margin of safety against fema1es which will not accept males, are barren, or which do not have proper maternal instincts (each of these reproductive anomalies will be exhibited by a small percentage of first year fema1es within a cohort). Since the male's only function in reproduction is the mating act, it is not necessary to house equal numbers of males and females, unless male reproductive effects are expected. Thus, it is only necessary to house one male for every 3 or 4 females per dietary concentration. Again, if first year animals are used, it is suggested that the malezfemale ratio be 1:3, to provide a margin of safety against males which will not attempt to mate or which produce no viable spermatozoa. If proven breeders are used, it may be possible to meet the criteria for an acceptable test with as few as 8 females and 2 males per dietary concen- tration. .1g,;,_ An acclimation period is required to condition the animals to the test facilities, diet, water, temperature, and lighting system to be used in a test. A minimum of 7 days is required, but a longer period may be necessary, especially if the animals to be used in a test are changed from outdoor to indoor housing (or vice-versa) or if the diet or water to be used in a test is different 205 from that which the animals are accustomed to consuming. It is important to measure feed consumption during this period as these measurements can provide an indication of how much feed the animals should consume once the test starts, how much diet to mix for the test, and can also serve as a "control" value for each group. 10.4.1. The suggested length of the mammalian reproduction test of approxi- mately 20-23 weeks is designed to conform to the normal reproductive seasons of mink (March through June) and European ferrets (April through July), with an 8 week exposure period prior to the reproductive season. The total length of the mink exposure period can be expected to be somewhat longer than the ferret exposure period because mink exhibit a variable delay in implantation of ferti- lized ova, while ferrets do not. Thus, the gestation period for mink can range naturally from approximately 40 to 60 days, whereas for ferrets the gestation period will normally be approximately 42 days. The length of this test allows ample time for absorption, distribution, metabolism, enzyme induction, re-distribution, bioconcentration, and elimination to occur, and for tolerance to be acquired, similar to that which might occur to animals chronically exposed to a substance in the environment. 10.4.1.1. In estimating feed consumption by mink or ferrets, severa1 precau- tions are necessary. Since feed consumption can be adversely affected on a short-term basis by temperature, weather, and other factors, estimates should be based on at least two consecutive day's consumption. These days should also be days in which the animals are not handled (e.g. during weighing, moving, etc.), since handling can produce a temporary reduction in feed consumption. 10.4.1.2. Under natural conditions, mating attempts are begun at the beginning of March for mink and the end of April for ferrets. In breeding mink, a female is presented to a male and, if receptive, is allowed to mate. If not receptive, the female is removed and presented to a male approximately 4 days later. Once a successful mating occurs (as verified by the presence of viable 206 spermatozoa in a vaginal aspiration taken just after copulation), the female is given the opportunity to mate a second time, either 8 days after the initial mating or the next day (if the first mating occurs late in the breeding season). In breeding ferrets, females are presented to males when they are judged to be in estrus (determined by the extent of vulvar swelling) and left overnight. Vaginal aspirations are not normally taken from female ferrets, and they are not given the opportunity for additional matings. If the researcher has reason to su5pect male reproductive effects, vaginal aspirations may be taken for examina- tion of spermatozoa. Generally, it is advisable to discontinue recording body weights and measuring feed consumption once breeding attempts are begun. The increased hand- ling of the animals during the breeding period causes perturbations in the animals' daily routines, resulting in decreased feed consumption by some animals. In addition, some animals respond to increased handling by becoming excitable. Repeated breeding attempts, coupled with routine weighings,can produce some females that are so excitable that breeding them becomes extremely difficult. Once the breeding period is over, it is best that the animals are left undisturbed as much as possible, especially during the first 2 weeks post-partum. l0.4.l.4. It is suggested that in checking for newborn, care is taken not to excessively disturb the females. If a nest box is not employed, visual inspection often is sufficient to determine whether a litter has been born. If a nest box is employed, it may be necessary to exclude the female from the nest box while checking the nest for newborn. If the female refuses to leave the nest box, this is often an indication that parturition has occurred. l0.4.l.6. It is suggested that necropsies be performed on selected test animals at the termination of the test. Valuable information on the mode of action and target organs or organ systems of the test substance can sometimes be gained from gross observation of the test animals at necrOpsy, and histopathological 207 examination of tissues can sometimes provide more information. Heights of internal organs and blood parameters of controls and treated animals can also be compared statistically to determine effects of the substance. l0.4.2.2. It is highly unlikely, based on the results of tests conducted in conjunction with the development of this protocol and on general mortality patterns derived from the fur industry, that more than 20% of a population of healthy mink or ferrets would die over the course of a 23 week reproduction test. If a researcher suffers the loss of greater than 20% of control animals in a test, it is possible that problems may exist in his diet or husbandry practices, or that disease has affected his stock. 19;§;_ The reproductive indices required in this protocol are selected based on features of the reproductive performance of mink and ferrets. Heights of all offspring (live and dead) are not required to be tested and reported because mink and ferrets are known to consume dead or stillborn young, thus, testing this reproductive index may produce incorrect or misleading results. Percent survival and weights of offspring are required at 3 weeks to allow minimal disturbance of dams and offspring during the critical period after birth and to ensure that nourishment received by offspring is almost totally of maternal origin. Percent survival and weights of offSpring at 6 weeks is not required because the young usually begin consuming at least some solid feed by 4 weeks of age. As mentioned previously, mink are known to exhibit a variable delay in implantation of fertilized ova, thus the length of gestation may not be useful in assessing effects of a substance on gestation in mink. It may, however, be very useful in assessing these effects in ferrets. lgLQL- The statistical procedures suggested are only a few of the valid statistical methods which may be used. Use of more advanced methods may prove more powerful in detecting significant differences. Certain procedures may per- mit testing two or more combined reproductive indices to assess the true effect 208 of a substance on reproductive performance, even though none of the indices by themselves are statistically significant (l5). 209 x w.mm x m.m¢ A¢_v o.om ------------ Am_v N.m_ x N.m_ x m.N_ ---- ....... - AN_V mm x mm x mm me x cm x we “magma N.m¢ o._o ¢._m ------------ OH x a» x ca A__v a.NN _.mm ~.©N m.om Ao_v as x m.om x m.om ..mm x N.m¢ x ¢._m ¢.mN ©.mm N.m¢ o._© o.Po m._~_ ca x a.m~ x as OH x op x ca Am v m.om m.om _.mm N.me N.@~ xcwz mucmcmmmm xoa ummz ammo mmwumgm Aau :_ I x 2 x 4v mco_m:mswa .mumceww new x:_e com mcaumgmuw_ mzu cw omucoamc mm meowmcmsmu xon umm: new mono ._ mpnmh 210 10. ll. REFERENCES Anonymous Guide fbr the Care and Use of'Laboratory Animals. DHEW Publica- tion No. (NIH) 78-23, U.S. Department of Health, Education, and Welfare. l978. National Research Council (NRC). Nutrient Requirements of Mink and Foxes. Nutrient Requirements of Domestic Animals series. Washington, D.C.: National Academy of Sciences. l982. Sokal, R.R. and F.J. Rohlf. Biometry. W.H. Freeman and Co., San Francisco. l969. Dunnett, C.N. New tables for multiple comparisons with a control. Biometrics 20:482-49l. l964. Zar, J,H, Biostatislieal Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ. 1974. Gill, J.L. Design and Analysis oj'Experimenzs in the Animal anj medical Sciences, V01- 1 (The Iowa State University Press, Ames, IA).409 pp., l978. Anonymous. Department of Health, Education and Welfare, Food and Drug Administration, "Nonclinical Laboratory Studies, Good Laboratory Practice Regulations", Federal Register, Vol. 43, No. 247, pp. 59986-60025. l979. Anonymous. Environmental Protection Agency, "Proposed Environmental Standards; and Proposed Good Laboratory Practice Standards for Physical, Chemical, Persistence, and Ecological Effects Testing", Federal Register, Vol. 45, No. 227, pp. 77332-77365. l980. Fur Rancher Blue Book of’Fur Farming (published annually). Communications Marketing, Inc., Eden Prairie, MN. Travis, H.E. and P.J. Schaible. Fundamentals of'Mink Ranching. Cir. Bul. 229, Michigan State University. 1960. Leonard, A. Mbdern Mink Mbnagement. (Ralston Purina Co., St. Louis, MO). 206 pp. l966. 211. 12. l3. l4. 15. Shump, A.U. and K.A. Shump, Jr. Growth and develOpment of the European ferret (Mustela putorius). Lab. Anim. Sci. 28(1):89-9l. l978. Willis, L.S. and M.V. Barrow. The ferret (Mustela putorius fare L.) as a laboratory animal. Lab. Anim. Sci. 2l(5):7l2-7l6. l97l. Carpenter, J.W. and C.N. Hillman. Husbandry, reproduction, and veterinary care of captive ferrets. Amer. Assoc. Zoo Vet. Ann. Proc., Knoxville, TN, pp. 36-47. 1978. Brown, M.B. A method for combining non-independent one-sided tests of signi- ficance. Biometrics 3l:987-992. l975. 212