Aid-S Ito . v (fr-3;. {It (D'thSV'; 0.52.5. {.5 1-.(1. p 1.! it... {0.5}.- .- (very-92...: c.1395!!! 4‘15 (11.0... z.) [7»... r4 . 5r:- fo. {(4.}! (to? 5‘. ritacabali 15.... .élotf...it 5; I... f #2.? 1.4; .Iiftivila ‘ If... elvittfr e .5 .(l. ‘02 .fg3... (AI .14“... .t‘ rib-l». .. 5543.11.13: 5 Vt}; .f... 1 . .. «v .. T.) . . .J.l..( .. . ..f:}.lvoivll£l .v. (I: yifrli ,4 lay-.539!!! I IV: I)! a la .' VIII}: [I ’51:)?171r’lf fr.l.oa\lrl.!rf . .rllalf‘v’)?! I! ¢ .rpll.illt’)l it"s/VD!!! , 3?.) t): refirllllifrflfng (Iir;£f}l11.3: ., Ifllfr ‘r: A . . . ‘ 1...... . 3. . _ ‘ 573:... . :. ) LC, .. . . lllllllllllllllllllllllllllllllllllllllllllllllllfl||||||||llll| 3 1293 00791 45 i LIBRARY A ‘ 1 Michigan State 1‘ University This is to certify that the thesis entitled THE EFFECTS OF HEPTACHLOR ON REPRODUCTION IN MINK AND EVALUATION OF A TECHNIQUE TO ENHANCE THE ELIMINATION OF HEPTACHLOR EPOXIDE presented by JEFFREY A. CRUM has been accepted towards fulfillment of the requirements for ".3. degree in Ali'imaI SCience MWofessor Date November 20, 1992 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE sspzbim A ." ‘ltq/ FEE; d: L" 3,19% MSU Is An Affirmative Action/Equal Opportunity Institution c:VcIrc\datedue.pm&p.' THE EFFECTS OF HEPTACHLOR ON REPRODUCTION IN MINK AND EVALUATION OF A TECHNIOUE TO ENHANCE THE ELIMINATION OF HEPTACHLOR EPOXIDE By Jeffrey A. Crum A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1992 ABSTRACT THE EFFECTS OF HEPTACHLOR ON REPRODUCTION IN MINK AND EVALUATION OF A TECHNIOUE TO ENHANCE THE ELIMINATION OF HEPTACHLOR EPOXIDE By Jeffrey A. Crum Adult female mink were fed diets containing 0, 6.25, 12.5 and 25 ppm technical grade heptachlor prior to and during the reproductive period to evaluate the effects of heptachlor consumption on reproduction and offspring viability and to assess the extent of placental and mammary transfer of heptachlor epoxide (HE) to mink offspring. Whelping success of the 6.25 ppm females was not affected, while early adult mortalities in the 12.5 and 25 ppm groups resulted in decreased reproduction. Postnatal exposure of kit mink from the 6.25 and 12.5 ppm dams to HE through lactation and/or dietary heptachlor resulted in reduced growth and survival. Consumption of heptachlor-free diets provided ad Iibitum or as a diet containing 10% mineral oil and restricted by 45% of ad libitum intake for 21 days caused a significant reduction in total HE body burdens and whole-body HE concentrations in adult female and kit mink previously exposed to heptachlor. To my family with love. ACKNOWLEDGEMENTS I am sincerely grateful to my major professor, Dr. Steven Bursian, for his enduring assistance, support, guidance and encouragement throughout the preparation of this document. Most of all i am indebted to Dr. Bursian for his patience as I attempted to attain a balance between developing this thesis and advancing in my professional career. I also wish to thank the other members of my graduate committee: Drs. Richard Auierich and Darrell King. Appreciation is extended to the Hawaii Heptachlor Research and Education Foundation who provided the funding for this research. I would like to thank Dr. John Gill, Dr. Ellen Lehning, and Bob Day for their guidance in performing the statistical analyses. Special gratitude is expressed to Phil Summer, Angelo Napolitano and Chris Bush for their help in breeding and taking care of the animals. My colleagues Dennis Bush and Natalie Biondo are appreciated for their assistance in the various facets of this work. Sincere gratitude is expressed to Ms. Carol Daniel for her assistance in wordprocessing. i am also indebted to my occupational supervisor, Chris Flaga, for her patience and understanding. Lastly, I would like to thank the most important people in my life. A heartfelt thank you is expressed to Cheryl Egres, who provided relentless encouragement and inspiration when I needed it most. And finally, I thank the members of my family who patiently stood by me throughout my academic career. TABLE OF CONTENTS LISTOFTABLES ............................. vi LIST OF FIGURES ...................................... vii INTRODUCTION ....................................... 1 LITERATURE REVIEW ................................... 4 Development and Uses of Heptachlor .................... 4 Chemical and Physical Properties of Heptachlor ............. 7 Pharmacokinetics of Heptachlor ........................ 8 Biological Effects of Heptachlor ........................ 13 Heptachlor Contamination Incidents ..................... 26 Decontamination Methods ........................... 30 CHAPTER I. THE REPRODUCTIVE EFFECTS OF DIETARY HEPTACHLOR IN MINK (MUSTELA VISOM ..................... 34 Abstract ........................................ 34 Introduction ..................................... 35 Materials and Methods .............................. 36 Results ......................................... 42 Discussion ...................................... 53 CHAPTER II. THE EFFICACY OF MINERAL OIL COMBINED WITH FEED RESTRICTION IN ENHANCING THE ELIMINATION OF HEPTACHLOR EPOXIDE FROM MINK (MUSTELA VISONI ........................... 62 Abstract ........................................ 62 Introduction ..................................... 63 Materials and Methods .............................. 66 Resuns ......................................... 69 Discussion ...................................... 79 SUMMARY .......................................... 85 REFERENCES ......................................... 87 LIST OF TABLES CHAPTER I. Page 1. Mortality and survival time of mink fed control and heptachlor- contaminated diets ................................ 47 The effect of dietary heptachlor on reproductive performance in adult female mink ................................. 49 The effect of heptachlor on body weight and survival of kit mink .......................................... 50 The effect of dietary heptachlor on total body burden and whole- body concentration of heptachlor epoxide (HE) and percent fat in mink kits at birth and three and six weeks of age ............ 52 The effect of dietary heptachlor on total body burden and whole- body concentration of heptachlor epoxide (HE) and percent fat in adult female mink after 181 days ...................... 54 The effect of dietary heptachlor on total body burden and whole- body concentration of heptachlor epoxide (HE) and percent fat in two to three-month-old mink kits euthanized at the end of the 181-day study period ............................... 55 CHAPTER II. Study design for the 21-day withdrawal period ............. 67 Body weights of adult female and kit mink at the beginning (day 0) and end (day 21) of the withdrawal period ........... 73 Body burdens and whole-body concentrations of heptachlor epoxide (HE) and percent fat in adult female mink at the beginning (day 0) and end (day 21) of the withdrawal period ........... 75 Body burdens and whole-body concentrations of heptachlor epoxide (HE) and percent fat in three to four-month-old mink kits at the beginning (day 0) and end (day 21) of the withdrawal period ......................................... 78 vi LIST OF FIGURES LITERATURE REVIEW 1. 2. Chemical structure of heptachlor ...................... Metabolism of heptachlor in mammals (Tashiro and Matsumura 1978) ........................................ CHAPTER I. 1. The effect of dietary heptachlor on feed consumed by adult female mink during the first 12 weeks of the study. Feed consumption by females in the 6.25 ppm group was not significantly different from controls throughout the 12 week period; feed consumption in the 12.5 ppm group was significantly less than controls at weeks 3, 4, 10 and 12; feed consumption in the 25 ppm group was significantly less than controls from weeks 2 through 8, and week 11. Significance (p < 0.05) ..................... The effect of dietary heptachlor on adult female mink body weights during the first 12 weeks of the study. Body weights of females in the 6.25 ppm group were not significantly different from controls throughout the 12 week period; body weights in the 12.5 ppm group were significantly less than controls from weeks 7 through 12; body weights in the 25 ppm group were significantly less than controls from weeks 4 through 12. Significance (p < 0.05) ............................ The effect of dietary heptachlor on adult female mink body weights at whelping and at three and six weeks post-whelping. No females fed 25 ppm heptachlor whelped. Asterisk indicates significant difference (p < 0.05) from control ............. CHAPTER II. 1. Consumption of the ad libitum (AL) diet during the 21-day withdrawal period by adult female mink previously fed control (—) and 6.25 ppm ( - - ) heptachlor for 181 days ............. Consumption of the ad Iibitum (AL) diet during the withdrawal period by kit mink (3 to 4 months of age) previously weaned by dams fed control (—) and 6.25 ppm ( - - ) heptachlor. Kits were also fed diets identical to their dams for approximately one month prior to the withdrawal period. ....................... Page 5 43 45 46 7O INTRODUCTION In recent years, there has been a growing awareness and concern regarding the environmental implications of pesticide use. Of all the chemicals produced, pesticides may have the greatest potential for causing adverse effects in the environment due to their broad application. In addition, many of the compounds manufactured were not highly selective and consequently threatened the livelihood of several environmentally important species. More importantly, the extensive use of pesticides has inadvertently resulted in exposure of the human population (Murphy 1986). Of particular Concern are the organochlorine insecticides, including DDT (dichlorodiphenyltrichloroethane) and the cyclodienes chlordane, heptachlor, aldrin, endrin and toxaphene. These chemicals, due to their physicochemical properties, are among the most persistent insecticides. The breakdown products of organochlorine compounds may be even more resistant to environmental degradation. Furthermore, these chemicals are lipophilic molecules which can accumulate in biological systems and thus have the potential to biomagnify through the food chain. Collectively, these characteristics permit translocation of the chemical from the site of application, thereby enhancing their potential to affect nontarget organisms (Murphy 1986). Indeed, the prolonged historical use of organochlorine insecticides on agricultural crops along with their environmental stability has resulted in detection of insecticide residues in numerous plant and animal species. Several studies have associated the presence of these residues with the development 1 2 of various adverse effects in animals including death. Studies in animals have also demonstrated that many of these chemicals are potentially carcinogenic. As a result, several organochlorine insecticides were banned by the US Environmental Protection Agency (EPA) in the 19703 (Murphy 1986). However, because alternative methods for insect control had not been developed to protect certain agricultural crops, some insecticides were cancelled subject to a "phased out" schedule which permitted or limited their use for a specified period of time on economically important crops. The chlorinated cyclodiene insecticide heptachlor was one such example (US EPA 1976, 1978). In Hawaii, heptachlor was the only remaining insecticide effective in controlling a commensal relationship between ants and mealybugs which resulted in damage to pineapple plants, the state's major source of agricultural revenue (Smith 1982). Therefore, following the cancellation proceedings of heptachlor in 1978, EPA authorized the continued application of heptachlor to pineapples through 1982, since there was no adequate alternative (US EPA 1978). However, in January 1982, routine testing of milk samples for pesticides revealed that violative concentrations (0.3 ug/g fat or 0.3 ppm) of heptachlor residues were present in milk from dairy farms on the island of Oahu, Hawaii (Smith et al. 1984; Le Marchand et al. 1986). In addition, testing of stored samples showed that milk containing heptachlor residues in excess of the current EPA action level of 0.1 ppm (fat basis) had been sold for a 27-29 month period (Le Marchand et al. 1986). Hence, there was concern regarding the potential for the development of adverse effects in the Oahu population, 3 particularly among pregnant mothers and nursing infants. Because heptachlor and its rapidly formed metabolite heptachlor epoxide (HE) are lipophilic chemicals, continued exposure can result in accumulation and bioconcentration of HE in adipose tissue (Bruce et al. 1965; Radomski and Davidow 1953). HE has been shown to cross the human placenta (Curley et al. 1969) and has been detected in the breast milk of nursing mothers (Curley and Kimbrough 1969; Takahashi et al. 1981 ; Takei et al. 1983). Reproductive and developmental toxicity in laboratory animals have also been demonstrated as a result of heptachlor exposure (Mestitzova 1967; Green 1970; Akay and Alp 1981). Since the mink has been shown to be sensitive to low concentrations of chlorinated compounds, particularly in terms of reproductive effects and offspring viability (Auierich and Ringer 1977; Bleavins et al. 1984a; Hochstein et al. 1988), it was considered the most appropriate test species for evaluating the reproductive effects of heptachlor. Therefore, the objectives of this study were to determine the subchronic toxicity of heptachlor to mink, the effect of dietary heptachlor on the reproductive performance of mink and on offspring viability, the placental and mammary transfer of HE to mink kits and whole-body HE concentrations in adult female mink fed heptachlor. This information is presented in Chapter I. Immediately following this experiment a second phase of the study was designed to determine whether consumption of a diet containing 10% mineral oil and restricted by 45% of ad libitum intake would enhance the elimination of HE from adult female and kit (young) mink previously exposed to heptachlor (Chapter II). LITERATURE REVIEW A. Development and Uses of Heptachlor Heptachlor is a chlorinated cyclodiene insecticide. Its chlorinated endomethylene bridge structure, common to all cyclodienes, distinguishes it from other organochlorine insecticides (Figure 1). Additional cyclodiene compounds include chlordane, which is the parent compound of heptachlor, as well as aldrin, dieldrin, endrin, endosulfan and toxaphene (Matsumura 1985; Murphy 1986). The basis for the development of insecticides was primarily economical in nature. The annual worldwide damage to agricultural crops from pests has been estimated at approximately 80 billion dollars. In the early 19705, insects caused agricultural losses in the United States of approximately 3.5 billion dollars annually (Sittig 1971, as cited in Matsumura 1985). Therefore, it was evident that a means for controlling insects was necessary to ensure an adequate production of food for human consumption (Brooks 1979). The first major insecticide to gain acceptance and eventual world-wide recognition was dichlorodiphenyltrichloroethane, or DDT. The effectiveness of DDT for insect control was demonstrated in 1939, which led to the insecticide’s patenting in 1942. Within 10 years after the introduction of DDT, a number of other organochlorine compounds had been synthesized for use as insecticides (Brooks 1979; Murphy 1986). Among them was heptachlor, which was first introduced for agricultural use by the Velsicol Chemical Corporation Cl H H CI I CI-C-CI I H \l/ CI y CI CI * H Figure 1. Chemical structure of heptachlor. 6 in 1948 under the designations "E3314" and ”Velsicol 104" (Brooks 1979). Other tradenames included Agroceres, Drinox, H-34, Heptagran, Heptamul and Rhodiachlor (IARC 1979). Heptachlor was officially registered in the United States in 1952 as a commercial insecticide for foliar, soil and structural applications. Throughout the 19603 and early 19703, heptachlor was used extensively by farmers to kill insects in seed grains and on agricultural crops. It was also used for subterranean termite control (Dynamac 1989). Other registered uses of heptachlor included treatment of seeds, application to Hawaiian pineapple plants and dipping of roots and tops of nonfood plants for insect control (Stehr- Green et al. 1986; Dynamac 1989). . Heptachlor achieved wide acceptance as an insecticide. In 1971 , it was registered for use in the United States on 22 agricultural crops (US EPA 1971). In 1974, an estimated 930,000 kg were used in the United States. However, in 1978, positive results in carcinogenicity assays conducted by EPA led to the suspension of most uses of compounds containing heptachlor. In addition, evidence indicated that heptachlor persisted in the environment and therefore was capable of considerable movement from the site of application. In March 1978, EPA's heptachlor/chlordane cancellation proceeding was completed and an agreement was reached over its contested uses (US EPA 1978). The settlement permitted the following uses: (1) treatment of seeds, (2) control of corn cutworms, (3) insect control on citrus crops in Florida, (4) control of ants on Hawaiian pineapples, and (5) control of termites and the narcissus bulb fly. The sole producer of heptachlor, the Velsicol Chemical Corporation, responded 7 by decreasing production to 1.3, 0.4 and 0.1 million lbs in 1978, 1980 and 1982, respectively. The sale of heptachlor was voluntarily discontinued by Velsicol in 1987 (Dynamac 1989). B. Chemical and Physical Properties of Heptachlor The synthesis of heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a- tetrahydro-4,7-methanoindene) involves free-radical chlorination of chlordene in benzene containing 0.5 to 5.0 percent catalyst, such as Fuller’s earth. Chlordene used in this reaction is produced by a Dials-Alder condensation of hexachlorcyclopentadienewith cyclopentadiene (Brooks 1979; Dynamac 1989). Its molecular formula is CstCl7 and its molecular weight is 373.5 (IARC 1979). Heptachlor is formulated in two grades. The analytical grade is 99% heptachlor and the technical grade consists of approximately 73% heptachlor, 22% transchlordane and 5% nonachlor. The analytical compound is a white crystalline solid which has a characteristic camphor-like odor. Its solubility in water (0.056 ppm @ 25-29° C) is considerably lower than its solubility in organic solvents. Solubilities at 27° C, expressed in g/100 ml of solvent, are 4.5 for ethanol, 75 for acetone, 102 for xylene, 106 for benzene and 112 for carbon tetrachloride (Brooks 1979; IARC 1979). The analytical grade has the following properties (Brooks 1979; IARC 1979): 95-96° C 135-145° C @ 1.5 Torr 3 x10“ mmHg @ 25° C Melting Point Boiling Point Vapor Pressure The technical product is a soft, waxy solid that is tallow in appearance. The 8 technical grade has the following properties (Brooks 1979). Melting Point = 46-74° C Density = 1.65-1.67 g/ml @ 25° C Viscosity 46-66 centistokes @ 71° C Vapor Pressure 4 x 10“ mmHg @ 25° C These physicochemical characteristics of heptachlor contribute to its effectiveness as an insecticide. However, emphasis placed on the development of desirable insecticidal properties has resulted in neglectful consideration of potential environmental effects and effects on non-target species (Murphy 1986). Due to its resistance to environmental degradation and its lipophilic nature, it accumulates in biological and non-biological media, Several human contamination incidents have been reported. These characteristics, along with its carcinogenic potential, have resulted in the elimination of heptachlor as a useful insecticide. C. Pharmacokinetics of Heptachlor An important metabolic function in animals is the detoxification of potentially harmful exogenous compounds. Animals have a number of biochemical processes that ordinarily transform lipophilic substances to more water-soluble forms, thereby promoting their elimination from the body. However, some chemicals are converted into metabolites which may be as lipophilic as the parent compound, and thus will be reabsorbed and redistributed to body tissues (Sipes and Gandolfi 1986). Although heptachlor can be absorbed through the skin, it is more readily absorbed through the gastrointestinal tract. Gaines (1969) demonstrated the variation in heptachlor absorption by showing that the dermal LD50 was nearly 9 twice the oral LDso in rats. Since exposure to heptachlor is most likely to occur through ingestion of contaminated food, gastrointestinal absorption of heptachlor has been the most commonly studied exposure route for evaluating its subsequent distribution and fate within the body. An intravenously administered dose of heptachlor to rats resulted in systemic distribution of the compound, with elevated levels found in areas heavily perfused, particularly the liver, kidney and to some degree, skin (Fendick et al. 1990). Mizyukova and Kurchatov (1970) showed that a single dose (120 mg/kg) injected directly into the stomach of female rats reached all tissues and organs within one-half to one hour. Rats fed heptachlor in the diet daily for two months had markedly higher concentrations of heptachlor (primarily as heptachlor epoxide) in adipose tissue compared to much lower concentrations in kidney, liver and muscle, and none in the brain (Radomski and Davidow 1953). Increasing concentrations of heptachlor epoxide in adipose tissue was also reported by Mizyukova and Kurchatov (1970) after a single administration of heptachlor. Moreover, they found only traces of heptachlor in adipose tissue only a month after dosing, demonstrating rapid metabolic conversion of heptachlor to its epoxide. Heptachlor epoxide has a high propensity to bioconcentrate in adipose tissue. Bruce et al. (1965) demonstrated that dairy cows fed heptachlor epoxide in the diet for 12 weeks had heptachlor epoxide concentrations in butterfat ranging from nine to 22 times the amount present in the diet, and from five to 14 times the concentration in body fat. Radomski and Davidow (1953) showed that rats fed 30 ppm heptachlor attained maximum concentrations of heptachlor epoxide in fat within two to 12 weeks, indicating 10 that accumulation of the epoxide may plateau. In addition, they showed that females accumulated approximately six times more heptachlor epoxide in fat than males. The authors speculated that the difference in storage was due to variations in metabolism which may have a hormonal basis. Placental transfer of chlorinated hydrocarbon compounds to the developing fetus is well documented in several animal species. Several studies on mink (Mustela vison) and the European ferret (Mustela putorius furo) have demonstrated placental and mammary transfer of polychlorinated biphenyls, polybrominated biphenyls and hexachlorobenzene (Bleavins et al. 1981, 1982, 1984b). Curley et al. (1969) detected heptachlor epoxide in cord blood of newborn babies and in the liver, kidney, heart, adrenal glands and adipose tissue of stillborn infants. Moreover, Polishuk et al. (1977) reported higher concentrations of heptachlor epoxide in extracted lipids of fetal blood and placenta than in maternal blood and uterine muscle of humans. Selby et al. (1969) determined the heptachlor epoxide distribution between the placenta and maternal blood to be 5.8:1. These data are significant, considering that the fetus is extremely vulnerable at specific developmental stages. The metabolic fate of heptachlor in mammalian species is dependent on the hepatic microsomal mixed-function oxidase (MFO) system. Generally, this system catalyzes epoxidation, N- and 8- oxidation, aromatic and aliphatic hydroxylation, O-, N- and S- dealkylation, oxidative deamination, and oxidative desulfuration reactions. Products from these reactions are conjugated with endogenous moieties which normally cause the final product to become more polar and less lipid-soluble, thus enhancing its excretion (Sipes and Gandolfi 11 1986). In vivo metabolism of heptachlor can be complex in aquatic species, but is fairly simple in mammals. The predominant pathway for the metabolic degradation of heptachlor in mammals is epoxidation to heptachlor epoxide (Radomski and Davidow 1953; Figure 2). This reaction, mediated by the hepatic MFO system, requires the presence of NADPH and 02 (Wong and Terriere 1965; Greene 1972). Heptachlor epoxide is a lipid-soluble substance that is extremely resistant to further metabolic action (Lu et al. 1975) and is therefore retained in adipose tissue (Radomski and Davidow 1953). The stability of the epoxide is due to the absence of the reactive allylic structure (CH=CHCHCI) which would otherwise promote hydrolysis to 1- hydroxychlordene 2,3-epoxide (Lu et al. 1975). Heptachlor can also be directly hydrolyzed to form 1 -hydroxychlordene, a compound that is considerably less toxic and more water-soluble than heptachlor (Brooks and Harrison 1965). However, this reaction occurs to a limited extent in mammalian species because of the resistance of the C1-Cl bond to microsomal oxidation (Lu et al. 1975). 1-Hydroxychlordene may then be oxidized to form 1-hydroxychlordene 2,3-epoxide. Tashiro and Matsumura (1978) were able to quantify the formation of these metabolites by examining feces from rats administered a 1"'C-Iabeled dose of heptachlor. Constituent analysis of the feces at 10 days postdosing revealed 26.2% unaltered heptachlor,19.5%1-hydroxychlordene,17.5%1-hydroxy-2,3-epoxychlordene, 13.1% heptachlor epoxide, 3.5% 1,2-dihydroxy-chlordene and 19% unidentified metabolite. 12 dehydrogenated derivative 1-hydroxy—2.3—exo-epoxychlordane of 1-hydroxy~2.3-axo- apoxychlordene 1. 2-dihydroxydihydrochlordana Figure 2. Metabolism of heptachlor in mammals (Tashiro and Matsumura 1978) 13 Heptachlor is eliminated from rats primarily via the feces (90%), with urinary excretion (10%) playing a more limited role (Scheufler and Rozman 1984). Tashiro and Matsumura (1978) recovered 62% of a 1“C-labeled dose of heptachlor in the feces as opposed to 6% in the urine 10 days after treatment. In contrast, Smith et al. (1987a) reported that sheep eliminated 67% of administered heptachlor in the urine as opposed to 33% in feces. Perhaps the most significant and critical pathway of heptachlor excretion is through the milk of nursing females. Heptachlor elimination from lactating cows occurs predominantly through milk lipids (Huber and Bishop 1962; King et al. 1967). Conversely, excretion of heptachlor through milk was not shown to be a major pathway in lactating sheep (Smith et al. 1987b). Mammary transfer of heptachlor through the milk of lactating humans has also been reported (Curley et al. 1969). The importance of this route of elimination is exemplified when considering the 1982 Hawaiian milk contamination episode and evidence which indicated that exposure to heptachlor residues may have occurred over a 27-29 month period (Le Marchand et al. 1986). D. Biological Effects of Heptachlor The acute toxicity of heptachlor has been evaluated in a number of mammalian species. Doses lethal to 50% of the test population (LDso) vary considerably depending on species, sex, age and route of exposure. Gaines (1969) reported oral LD.50 values for technical—grade heptachlor of 100 and 162 mg/kg'body weight (bw) for male and female Sherman rats, respectively. The dermal LD50 for technical heptachlor in male and female Sherman rats was 14 195 and 250 mg/kg bw, respectively. Of the 19 chlorinated hydrocarbon insecticides tested, heptachlor was the seventh most potent. Podowski et al. (1979) examined the acute toxic effects of analytical- grade heptachlor in male Charles River rats and determined an oral L050 of 71 mg/kg bw. Harbison (1975) also reported an LDso of 71 mg/kg bw for heptachlor administered intraperitoneally to adult Sprague-Dawley rats, but found decreased toxicity in newborn rats (LD50 = 531 mg/kg bw). Oral LD50 estimates for the mouse, rabbit and hamster were 70, 80-90 and 160 mg/kg bw, respectively (Gak et al. 1976, Gleason et al. 1969; Cabral et al. 1979). Although no toxicity studies could be found regarding inhalation exposure to heptachlor, this route may be important due to its extensive use in many homes as a termiticide (Fendick et al. 1990). Radomski and Davidow (1953) were the first to demonstrate that heptachlor epoxide was more toxic than heptachlor. Sperling and Ewenike (1972) reported oral LDso, of 62 mg/kg bw for heptachlor epoxide and 112 mg/kg bw for heptachlor in male Charles River rats. Podowski et al. (1979) found only negligible differences in the lethality of these two chemicals, oral L050, of 60 mg/kg bw for heptachlor epoxide and 71 mg/kg bw for heptachlor in male Charles River rats. lntraperitoneal injection of heptachlor epoxide in male Swiss-Webster mice yielded an LD$0 of 18 mg/kg bw (lvie et al. 1972). Administration of heptachlor intraperitoneally to rats resulted in an LD.50 of 71 mg/kg bw (Harbison 1975). Oral toxicity studies on one- to two- week-old dairy calves indicated that heptachlor epoxide was nearly 10 times more toxic than heptachlor (Buck et al. 1959). 15 Subchronic studies (3 to 6 months) examining the lethality of heptachlor and heptachlor epoxide are lacking. The National Cancer Institute (NCI) (1977) conducted a six-week study to identify the maximum tolerated doses of technical-grade heptachlor in rats and mice for application in a chronic study. Increased mortality in rats and mice was observed with increasing concentrations of heptachlor administered orally. Male rats were slightly more tolerant of heptachlorthan females, with a lowest-observed-adverse-effect level (LOAEL) of 320 ppm (16 mg/kg bw/day) in males and 160 ppm (8 mg/kg bw/day) in females. The no-observed-adverse-effect level (NOAEL) was 80 ppm (4 mg/kg bw/day) for both male and female rats. A NOAEL of 40 ppm (5.2 mgikg bw/day) was reported for male and female mice (NC) 1977). Chronic oral intake of heptachlor, heptachlor epoxide, or in combination at concentrations lower than their respective LDSO, can also be lethal. Female B6C3F1 mice fed technical-grade heptachlor at a time-weighted average (TWA) concentration of 18 ppm for 80 weeks had significantly (p < 0.02) increased mortality over controls (NCI 1977). Based on the mortality data for females, the NOAEL was determined to be 9 ppm (1.2 mg/kg bw/day). No significant effects on mortality were observed in male mice fed TWA concentrations of 6.1 and 13.8 ppm when compared with controls. A dose-dependent increase in mortality was observed in female CD rats fed diets containing 5, 7.5, 10 or 12.5 ppm heptachlor/heptachlor epoxide (75%:25%) for two years. Mortality in the high dose group was 50% as opposed to 21% in the control group (Jolley et al. 1966, as cited in Epstein 1976). Heptachlor epoxide administered to C3H male and female mice was found to be considerably more toxic than 16 heptachlor over a 104 week period. Mortality for males and females combined was 91.5% for those fed 10 ppm heptachlor epoxide as opposed to 60% for those consuming 10 ppm heptachlor (Davis 1965, as cited in Epstein 1976). The most characteristically observed effect resulting from exposure to heptachlor is abnormal stimulation of the central nervous system. In fact, all chlorinated cyclodiene insecticides (CCl) such as chlordane, aldrin, dieldrin, isodrin, endrin and endosulfan are classified as neurotoxicants. The acute neurological effects arising from heptachlor intoxication are hyperexcitability, tremors, and convulsions which routinely result in the death of the animal (Gosselin et al. 1976). Exaggerated cortical and motor responses to peripheral nerve stimulation have been reported following acute exposure to heptachlor (Joy 1976; St. Omer and Ecobichon 1971). Additional signs reported include hypertension, arrythmia (Joy 1976), dyspnea (St. Omer and Ecobichon 1971) and hypothermia (Hrdina et al. 1974). Intravenous administration of single doses of heptachlor (2-10 mg/kg bw) in cats resulted in tonic-clonic type seizures (Joy 1976). Of the CCls tested, heptachlor and aldrin produced convulsions after 20 to 30 minutes, whereas dieldrin, endrin and lindane were more rapid acting convulsants. The authors suspected that the delay in onset of activity may have been due to time needed to metabolically convert heptachlor and aldrin to their more potent epoxides, heptachlor epoxide and dieldrin, respectively. A latency period between application and appearance of signs (convulsions) was also reported by Albrecht (1987) in mice and Hrdina et al. (1974) in rats. A number of studies have examined the mode and site of action of 17 heptachlor, heptachlor epoxide and several other cyclodiene insecticides in animals. Although the specific mode of action of heptachlor has not been elucidated, two mechanisms have been postulated. The first involves the inhibition of Ca2+, Mg2+ -ATPase. Ca2+, Mg2+ - ATPase regulates the concentration of intra- and extracellular Ca“ in the presynaptic axon terminal. Heptachlor epoxide inhibits this enzyme causing increased concentrations of intracellular Ca2+ (Yamaguchi et al. 1979, 1980). The increase in Ca2+ stimulates the release of excitatory neurotransmitters such as acetylcholine (ACh) and glutamate from nerve terminals by a process known as exocytosis. Rat brain synaptosomes treated with heptachlor epoxide sequestered more Ca2+ and released less Ca2+ than untreated synaptosomes. The increased release of excitatory neurotransmitters results in convulsive seizure-type behavior usually culminating in death due to respiratory failure (Brooks 1979). The more recently proposed hypothesis concerning the CNS effects of heptachlor epoxide involve gamma-aminobutyric acid (GABA). GABA is one of the principle inhibitory neurotransmitters in the CNS (Vander et al. 1985). The putative target site of heptachlor epoxide is the GABA receptor complex which consists of three distinct binding sites: (1) a site for binding of GABA, (2) a site that binds benzodiazepines and (3) a site considered to be the chloride ionophore channel that binds known convulsants such as picrotoxin and t- butylbicyclophosphorothionate (TBPS) (Abalis et al. 1986). Normally, the binding of GABA causes an increase in postsynaptic membrane permeability to chloride (Cl') ions. CI' ion influx functions to prevent excitatory impulses, such 18 as those carried by ACh and glutamate, from initiating postsynaptic action potentials (Vander et al. 1985). Studies using rat brain microsacs (in vitro) have shown Cl' ion influx to be inhibited to a greater degree when GABA is administered in combination with picrotoxinin or TBPS, than if given alone (Abalis et al. 1985; Gant et al. 1987). TBPS has a higher affinity for the chloride channel of the GABA receptor complex than does picrotoxinin (Squires et al. 1983). However, TBPS was displaced by heptachlor and heptachlor epoxide in the rat brain, with ICSO, (i.’e. the concentration that inhibited 50% of TBPS binding) of 400nM and 70nM, respectively (Abalis et al. 1985). The epoxides of all cyclodiene insecticides tested were determined to be more effective at inhibiting Cl' ion influx than their parent compounds (Abalis et al. 1985, 1986; Gant et al. 1987). The greater affinity of heptachlor and heptachlor epoxide for the GABA receptor complex decreases the efficiency of GABA, which in turn decreases Cl' ion influx. This ultimately results in increased nerve stimulation. It is apparent that the neurological effects of heptachlor and heptachlor epoxide may be the consequence of several biochemical and physiological events occurring in the body. Endpoints such as feed consumption and body weight can be useful indicators of more subtle toxic effects occurring in animals exposed to heptachlor/heptachlor epoxide. In several studies reviewed by Epstein (1976), there was conflicting results of decreased food consumption in rats and mice chronically fed diets containing heptachlor/heptachlor epoxide. However, a dose-related decrease in feed consumption was observed for standard dark 19 mink consuming technical grade heptachlor in the diet for 28 days (Auierich et al. 1990). Body weight losses were also directly related to the concentration of heptachlor in the diet. After the fourth week of exposure, mink consuming 25, 50 and 100 ppm heptachlor lost an average of 12.6, 26.5 and 38.2 percent of their initial body weights, respectively. A number of other studies have also reported reduced body weight gain in mice and rats exposed to heptachlor (Shain et al. 1977; IRDC 1973 as cited in Epstein 1976; NC) 1977). The liver is the primary organ impacted following exposure to heptachlor/heptachlor epoxide (Enan et al. 1982, as cited in Dynamac 1989). This is true for nearly all the chlorinated hydrocarbon insecticides, as the major function of the liver is to detoxify potentially harmful foreign compounds. Several morphological, histological and biochemical alterations have been reported subsequent to heptachlor intake. Liver weights were significantly increased in male and female mice fed a diet consisting of 75% heptachlor epoxide/25% heptachlor for 18 months at concentrations of 5 and 10 ppm (IRDC 1973, as cited in Epstein 1976). Female mice fed 1 ppm also had significantly increased liver weights relative to controls. A marked dose-related increase in liver weights was observed in female CFN rats consuming heptachlor epoxide at dietary concentrations ranging from 0.5 ppm (0.05 mg/kg/d) to 10 ppm (1 mg/kg/day) for 108 weeks (Witherup 1959, as cited in Epstein 1976). In contrast, intramuscular injection of heptachlor (3 and 15 ppm) or heptachlor epoxide (1 and 5 ppm) for 45 days significantly decreased (p < 0.05) liver weights in male Wistar rats (Kacew et al. 1973). Male standard dark mink fed technical-grade heptachlor in the diet 20 for 28 days at concentrations up to 100 ppm did not have significantly increased liver weights compared to controls (Auierich et al. 1990). Other morphological alterations in mink fed 100 ppm heptachlor included significantly decreased kidney and spleen weights, and significantly increased adrenal gland weights (Auierich et al. 1990). Intramuscular injection of 15 and 5 ppm heptachlor and heptachlor epoxide, respectively, caused no change in adrenal, thymus, heart, kidney or testicular weights of male Wistar rats (Kacew et al. 1973). Microscopically, heptachlor intake has caused liver necrosis, hepatic cell vacuolization (Krampl 1970) and fatty liver degeneration (Dynamac 1989). In addition to hepatocytomegaly reported by IRDC (1973, as cited in Epstein 1976), hypertrophic-hyperplastic lesions classified as nodular hyperplasia were observed in a number of mice fed 5 and 10 ppm heptachlor epoxide (75%)/ heptachlor (25%) for 18 months. Various biochemical alterations in the livers of heptachlor/heptachlor epoxide-exposed animals have been reported. Changes resulting from acute exposure to heptachlor included increased serum concentrations of aldolase, glutamic-pyruvic transaminase (GPT), alkaline phosphatase, bilirubin and cholesterol, all of which are indicative of hepatic injury (Krampl 1970; Enan et al. 1982, as cited in Dynamac 1989). Gluconeogenesis in the liver and kidney cortex of male Wistar rats was enhanced following a single dose of heptachlor (200 mg/kg) (Kacew and Singhal 1973). Heptachlor increased the activities of four gluconeogenic enzymes resulting in significantly elevated serum glucose and urea, and a concomitant decrease in liver glycogen. Repeated 21 intramuscular injections of heptachlor (3 and 15 ppm) and heptachlor epoxide (1 and 5 ppm) in male Wistar rats produced a dose-related increase in glucose synthesis (Kacew et al. 1973). The authors hypothesized that the increased formation of glucose was due to an initial stimulation of the cyclic AMP- adenylate cyclase system in the liver and kidney cortex. All chlorinated cyclodiene insecticides are inducers of the hepatic MFO system, also known as the cytochrome P-450 system (Hodgson et al. 1980). The MFO system has a number of enzymes that generally convert xenobiotics to more water soluble and excretable forms, which are often less toxic by adding or exposing a functional group for subsequent conjugation (Sipes and Gandolfi 1986). Heptachlor and heptachlor epoxide were shown to induce three hepatic MFOs (phosphorothioate detoxification, O-demethlyase and N- demethylase) in a dose-related manner (Kinoshita and Kempf 1970). Relative to other cyclodiene insecticides, heptachlor and heptachlor epoxide were the more potent and persistent inducers tested (Gillet and Chan 1968; Kinoshita and Kempf 1970; Den Tonkelaar and Van Esch 1974). Den Tonkelaar and Van Esch (1974) compared no-effect levels for enzyme induction with no-effect levels based on morphological changes in the liver in male Wistar rats administered heptachlor (99%) for two weeks. The lowest dose which produced enzyme induction was 2 ppm, as compared to 5 ppm which caused no liver abnormalities. The lowest no-observed-effect-level (NOEL) for induction of hepatic microsomal enzymes by heptachlor is approximately 1 ppm (Gillet and Chan 1968; Kinoshita and Kempf 1970; Den Tonkelaar and Van Esch 1974). Heptachlor induced aniline hydroxylase, 22 aminopyrine demethylase and hexobarbital oxidase at concentrations of 2 to 50 ppm, 2 to 50 ppm and 5 to 50 ppm, respectively (Den Tonkelaar and Van Esch 1974). The ability of heptachlor and heptachlor epoxide to cause gene mutations has been evaluated in several microbial systems. Nearly all mutation assays were negative with and without metabolic activation. Heptachlor and heptachlor epoxide tested with a number of Salmonella typhimurium strains in the Ames assay were not mutagenic in the absence (Marshall at al. 1976) or the presence of rat liver enzymes (Moriya et al. 1983). Heptachlor was not mutagenic in assays with strains of Escherichia co/i (Moriya et al. 1983), Saccharomyces cerevisiae (Gentile et al. 1982) and Bacillus subtilis (Shirasu et al. 1976). Heptachlor epoxide was also not mutagenic in bacteria (Moriya et al. 1983). Gentile et al. (1982) reported a 1.5 to 2-fold increase in reversions of two strains of Salmonella (TA98 and TA100) for technical grade heptachlor, this occurring in the presence of rat liver microsomes. The results, however, were shown to be inconclusive due to questionable control values and administration of only a single dose. The carcinogenic effects of heptachlor and heptachlor epoxide are well established in mice. Virtually all carcinomas reported in mice were of the liver. Heptachlor and heptachlor epoxide induced a significant increase in liver carcinomas in C3H mice fed 10 ppm in the diet for two years (Davis 1965, as cited in Epstein 1976). A 3:1 mixture of heptachlor epoxidezheptachlor fed to Charles CD-1 mice for 18 months resulted in a dose-dependent induction of hepatocellular carcinomas with significant increases at 5 and 10 ppm in males 23 and at 10 ppm in females (IRDC 1973, as cited in Epstein 1976; Reuber 1987). The NCI (1977) reported a dose-related increase in hepatocellular carcinomas in male and female B6C3F1 mice fed diets containing technical-grade heptachlor for 80 weeks at time-weighted average concentrations of 6.1 and 13.8 ppm, and 9 and 18 ppm, respectively. The carcinogenic effects of heptachlor and heptachlor epoxide in the rat are less conclusive. Heptachlor (unknown purity) fed to Carworth Farm rats at dietary concentrations ranging from 1.5 to 10 ppm for 110 weeks caused a significant increase in the incidence of multiple site and other tumors in females receiving 7 and 10 ppm (Witherup et al. 1955, as cited in Epstein 1976). In contrast to the common liver tumors in mice, many of the rats had tumors of the endocrine organs. In a similar test with heptachlor epoxide, an increase in hepatomas and multiple site malignant tumors were discovered in treated male and female rats (Witherup et al. 1959, as cited in Epstein 1976). lnadequacies in the methodology and documentation of these studies precluded definitive conclusions. The NCI (1977) reported no hepatocellular carcinomas in 187 rats fed heptachlor-contaminated diets for 80 weeks. The development and high incidence of liver tumors in mice is an attribute of carcinogens which lack the ability to interact with DNA (Williams and Weisburger 1986). Indeed, heptachlor and several other organochlorine compounds have been shown to be non-genotoxic in a number of mutation assays, including systems using cultured liver cells. Heptachlor was not mutagenic in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in an adult rat liver epithelial cell line (Telang et al. 1981), nor did 24 heptachlor induce unscheduled DNA synthesis (UDS) in primary cultures of rat, mouse and hamster hepatocytes (Williams 1980; Probst et al. 1981). Ahmed et al. ( 1977) demonstrated qualitative evidence of UDS in SV-40 transformed human fibroblasts by heptachlor and heptachlor epoxide in the presence of rat liver enzymes. Subsequently, heptachlor was investigated for its ability to act as an epigenetic liver tumor promotor. Telang et al. (1982) demonstrated that heptachlor and a closely related compound, chlordane, significantly inhibited intercellular communication between cultured liver cells, a typical feature exemplified by many tumor promoting Chemicals. Conversely, benzo[a]pyrene, a known genotoxicant, did not produce this effect. Male 86C3F1 mice given diethylnitrosamine (DEN), a known tumor initiating agent, for 14 weeks followed by 25 weeks of chlordane or heptachlor had twice as many liver tumors as those on control diets (Williams and Numoto 1984). Heptachlor did not promote the occurrence of lung and stomach tumors that had developed in mice solely administered DEN. Moreover, increased production of neoplasms did not occur in mice exposed to heptachlor 25 weeks before initiation with DEN or 25 weeks without previous initiation. Evidence from this study and supporting studies (Williams 1980; Williams and Weisburger 1986; Chuang et al. 1991) shows that heptachlor and several structurally related organochlorine insecticides are epigenetic carcinogens of the promotor class. Currently, both heptachlor and heptachlor epoxide are regulated as probable human carcinogens based on studies which demonstrated benign and malignant tumor induction in three strains of mice of both sexes (US EPA 25 1992). Production of liver tumors in mice by four structurally related compounds (chlordane, aldrin, dieldrin and chloendic acid) further supports the present carcinogenic classification of heptachlor and its epoxide (US EPA 1992). These findings become increasingly important in light of the bioaccumulative and persistent nature of this insecticide. The reproductive effects of heptachlor and its epoxide in animals are well documented. Heptachlor and its epoxide fed to male mice which were subsequently mated with untreated females did not cause any adverse reproductive effects (Epstein et al. 1972). However, when male and female mice were given 50, 100 or 200 ppm (6.5, 13, or 26 mg/kg bw/day, respectively) heptachlor in the diet for 10 weeks, they failed to produce a new generation (Akay and Alp 1981). Feeding 5 ppm (0.25 mg/kg bw/day) heptachlor to male and female Sprague-Dawley rats for two generations resulted in decreased pregnancy rates in the second generation only. The first generation females achieved a 72% pregnancy rate as opposed to a 0% pregnancy rate for the second generation (Green 1970). In utero and postnatal exposure to heptachlor and heptachlor epoxide may have significant effects on offspring viability. Placental and mammary transfer of heptachlor and heptachlor epoxide has been confirmed by several researchers in humans (Curley and Kimbrough 1969; Curley et al. 1969; Polishuk et al. 1977; Takei et al. 1983), but no adverse effects on neonates have been reported (Burch 1983, as cited in Le Marchand et al. 1986). Mestitzova (1967) reported a marked decrease in litter size of the first and succeeding generations of rats fed 6 ppm heptachlor. Survivability of the 26 newborn rats was significantly reduced, with the highest mortality occurring between 24 to 48 hours postpartum. Rats fed diets containing heptachlor and/or heptachlor epoxide at levels up to 10 ppm for three consecutive generations had a small increase in newborn mortality, but no teratogenic effects were reported (Witherup et al. 1976a, as cited in WHO 1984; Witherup et al. 1976b, as cited in WHO 1984). High pup mortality occurred in beagle dogs administered 10 ppm heptachlor epoxide in the diet for two generations, but no congenital malformations were observed (WHO 1984). A 46% reduction in survival was observed for newborn rats from mothers fed 5 ppm (0.25 mg/kg/day) heptachlor for 60 days prior to mating and during gestation (Green 1970). Conversely, heptachlor epoxide fed to pregnant rabbits at 5 mg/kg bw/day during gestation days 6-1 1 did not cause adverse effects in the young (WHO 1984). Eisler (1968) reported that a mixture of heptachlor and heptachlor epoxide (ratio not specified) administered to male and female rats at a dose of 7 ppm for three generations did not retard postnatal development or cause teratogenic effects. E. Heptachlor Contamination Incidents Due to the extensive past use of organochlorine insecticides on agricultural crops and because of their environmental persistence, there has been periodic detection of insecticide residues in human food products. Invariably, a number of food contamination incidents have been associated with the livestock industry. In 1968, a considerable part of Montana’s milk supply 27 was contaminated with heptachlor after chlordane, the parent chemical, was applied to alfalfa subsequently fed to dairy cows (Smith et al. 1984). Grain and mash sold for cattle feed by a gasohol plant in Arkansas were found to contain concentrations of heptachlor residues far above the United States Food and Drug Administration action level of 0.1 ppm (fat basis) (Stehr-Green et al. 1986). The more toxic and rapidly-formed metabolite of heptachlor, heptachlor epoxide, was found at concentrations of 89.2 ppm (fat basis) in raw milk samples collected from dairy cattle consuming this feed. This incident occurred in 1986 and resulted in the quarantine of 33 farms in Arkansas and five others in Oklahoma and Missouri. From an economic standpoint, the most devastating incident took place in January, 1982 when nearly the entire milk supply in Oahu, Hawaii was contaminated with heptachlor. The source of the contamination was traced to heptachlor-treated pineapple plant foliage that was used as a supplement to dairy cattle feed (Le Marchand et al. 1986). This incident resulted in a recall of approximately nine million pounds of milk from market shelves and an estimated loss of nine million dollars (Smith et al. 1984). Of greater importance was concern that adverse human health effects might result, particularly among pregnant mothers and nursing infants. Younger children who consume large amounts of milk and have poorly developed immune systems were also considered to be potentially at risk. The problem was further confounded by reports indicating that violative levels were present in milk bottled nearly two years earlier (Smith et al. 1984; Le Marchand et al. 1986). This generated a great deal of public skepticism regarding the duration of exposure. 28 The Hawaiian milk contamination event was contingent upon the economical importance of the pineapple industry to Hawaii, as it was the state’s fourth largest revenue source. Figures from 1974 placed Hawaii as the world’s largest producer of pineapple, with the gross income reported at 124.3 million dollars. Unfortunately, pineapple production is endangered by mealybugs which suck sap from the plants, thereby creating a condition known as "mealybug wilt.” If left alone, this disease can spread rapidly, resulting in fields of plants that are incapable of producing marketable pineapple (US EPA 1976). In a typical infestation, mealybugs produce a secretion called "honeydew" which they would normally succumb to were it not for the assistance of the ant, since the honeydew provides a substrate for fungi that would naturally cover and kill the mealybugs. The ant, being attracted to the sweet substance, consumes it and in return also protects the mealybug from its predators as well as transporting them to other plants (US EPA 1976; Smith 1982). In the 19403, the pineapple growers decided that eliminating the ant was the most effective means for controlling the problem. This disrupted the commensal relationship between the two insects, hence the mealybugs’ self-destruction. The widely acclaimed organochlorine insecticide DDT was the first chemical used against the ant, followed by mirex and finally heptachlor. All three insecticides were banned by EPA during the 19703 because of their environmental persistence and potential carcinogenicity. However, an exemption was granted for heptachlor to sustain Hawaii’s economically important pineapple industry. Hawaii was granted the exemption, with the 29 stipulation that pineapple growers wait one year after the last application of heptachlor before harvesting the plants for cattle feed (US EPA 1976; Smith 1982). The harvesting of "green chop" was considered a cheap alternative for imported dairy feed, and had been a routine practice since 1958. Unfortunately, heptachlor’s breakdown product, heptachlorepoxide, was found to contaminate the plant. Furthermore, development of a more efficient harvesting machine in the 19703 resulted in even higher concentrations in the forage. Dairy cattle consuming the contaminated feed concentrated and excreted heptachlor epoxide into the milk. This resulted in violative levels of heptachlor residues in milk destined for market shelves (Smith et al. 1984). The Hawaiian milk contamination incident provided a unique opportunity to evaluate the human reproductive effects of heptachlor consumption. Because of the reported reproductive effects in animals and additional documentation demonstrating the propensity of heptachlor epoxide to bioconcentrate in the milk of dairy cows, exposed human populations were suspected to be potentially at risk. Concentrations of 2.8 ppm (fat basis) were detected in milk available to consumers (Le Marchand et al. 1986). An investigation by Le Marchand et al. (1986) failed to show any increase in congenital malformations during the 27-29 month exposure period. Burch (1983 as cited in Le Marchand et al. 1986) also reported no adverse effects on human reproduction during this time period. Additionally, there was no evidence of hepatic effects in individuals consuming heptachlor-contaminated milk (Stehr-Green er al. 1988). Although no adverse effects have been 30 documented in humans, evidence that heptachlor epoxide is concentrated in human milk four to five times higher than in cows’ milk warrants concern (Luquet et al. 1974, as cited in Mussalo-Rauhamaa et al. 1988). F. Decontamination Methods The use of organochlorine compounds such as heptachlor on agricultural crops has resulted in contamination of livestock consuming treated forages. This has often led to large economic losses to farmers, and incidental exposure of humans through consumption of animal products. Consequently, methods to enhance the removal of contaminants from animals have received considerable attention, particularly for those animals of economic importance. Three decontamination strategies include (1) increasing the activity of hepatic enzymes that metabolize organochlorine compounds, (2) enhancing fecal excretion of xenobiotics through adsorption to administered agents and (3) mobilizing body fat. Since heptachlor routinely undergoes a bioactivation (toxication) reaction to form heptachlor epoxide in vivo, there would seemingly be little justification for induction of phase I enzymes to facilitate the elimination of heptachlor and its epoxide. However, Street et al. (1966) found that feeding DDT along with heptachlor resulted in decreased accumulation of heptachlor epoxide in the adipose tissue of rats, sheep and swine. In contrast, phenobarbital, a phase I enzyme inducer, failed to expedite the clearance of heptachlor epoxide from dairy cattle (Stanley et al. 1984). Interestingly, phenobarbital has been effective in eliminating other organochlorines such as DDT and dieldrin (Cook 31 and Wilson 1971). Efforts to stimulate induction of phase II enzymes has also yielded mixed results. Trans-stilbeneoxide (TSO), a powerful inducer of glutathione S-transferase which reacts with epoxides, decreased the half-life of radioactive 1“C-heptachlor by a factor of three in male Sprague-Dawley rats. This reduction was verified by measurable decreases in adipose tissue and blood concentrations of heptachlor (Rozman 1984). Oddly, intraperitoneally administered TSO was not effective in reducing the body burden of heptachlor epoxide in cattle (Kendall et al. 1988). T80 was also unable to enhance the rate of heptachlor clearance from ovines (Smith et al. 1989) A related Phase Il-inducer, butylated hydroxyanisole (BHA), was also unsuccessful in promoting heptachlor epoxide excretion from pigs and cattle (Raisbeck et al. 1986). The use of aliphatic hydrocarbons, such as mineral oil, liquid paraffin and hexadecane, in feed to decrease body burdens of organochlorine compounds in livestock has been very effective. These therapeutic agents, in addition to activated charcoal and cholestyramine, function by adsorbing to the contaminant in the intestinal tract thereby preventing reabsorption and allowing fecal excretion. Experiments with rats, rhesus monkeys and livestock have demonstrated a 3 to 13-fold increase in fecal elimination of several lipophilic toxicants resulting from orally administered mineral oil (Richter et al. 1977; Rozman et al. 1981a,b; Rozman et al. 1982a,b). Mineral oil was also effective in enhancing the removal of polychlorinated biphenyl (PCB) residues from goats (Polin et al. 1987). In contrast, hexadecane was unsuccessful in promoting heptachlor 32 clearance from rats (Rozman 1984) and mineral oil failed to increase heptachlor elimination from ovines (Smith et al. 1989). In addition, the adsorbent cholestyramine was not effective in reducing heptachlor epoxide in milkfat of dairy cattle (Stanley et al. 1984). Raisbeck et al. (1989) also reported that mineral oil administered to swine and dairy cattle at 5% in the diet was not effective in significantly reducing heptachlor epoxide body burdens when compared to those fed control diets. The third decontamination method (mobilizing body fat) would appear to be the most practical, as this would allow for the excretion of lipophilic compounds. However, depleting fat reserves in dieldrin- and heptachlor- contaminated cattle did not reduce the body burdens of these contaminants (Hironaka 1968; Raisbeck 1988 as cited in Raisbeck et al. 1989). Two disadvantages to this approach are redistribution of the contaminants to other lipid-containing tissues, and increased plasma concentrations of the toxicants during animal weight loss (Raisbeck er al. 1989). Wyss et al. (1982) reported that nearly half of hexachlorobiphenyl (6-CB) was translocated from adipose tissue into the skin of food-restricted rats. Polin et al. (1985, 1986a,b) were able to overcome redistribution and bioconcentration of contaminants by combining the last two decontamination methods. They reported successful elimination of PCB, polybrominated biphenyl (PBB), hexachlorobenzene (HCB) and pentachlorophenol (PCP) residues from chickens by combining feed restriction, thereby forcing utilization of adipose tissue, with feeding mineral oil which served to prevent reabsorption of the mobilized xenobiotic. Polin et al. (1991) later showed this to be effective 33 in rats treated with PBBs, with the highest degree of elimination occurring in rats administered 10% mineral oil combined with 45% feed restriction. Mutter et al. (1988) also reported large increases in fecal excretion of dichlorodiphenyldichloroethene (ODE) from gerbils relative to controls when food restriction was combined with sucrose polyester (SPE), a nonabsorbable lipophilic binding agent. Other combinations such as activated carbon and phenobarbital, have been equally effective in removing pesticides from dairy cattle (Cook and Wilson 1971). CHAPTER I. THE REPRODUCTIVE EFFECTS OF DIETARY HEPTACHLOR IN MINK (MUS TELA VISON) Abstract Adult female mink were fed diets containing 0 (control), 6.25, 12.5 and 25 ppm technical grade heptachlor prior to and throughout the reproductive period (181 days) to evaluate the effects of heptachlor consumption on reproduction and offspring viability and to assess the extent of placental and mammary transfer of heptachlor epoxide to mink offspring. Feeding 12.5 and 25 ppm resulted in significant reductions in feed consumption and body weights of female mink. Mortality was 0, 8, 67 and 100% for the control, 6.25, 12.5 and 25 ppm groups, respectively. All females in the 25 ppm group died within 88 days. Mink fed the two higher heptachlor diets displayed clinical signs indicative of central nervous system involvement just prior to death. Females were mated with males on the same dietary treatments. Whelping success rates were 67, 83, 27 and 0% for the control, 6.25, 12.5 and 25 ppm groups, respectively. High mortality in the 12.5 and 25 ppm groups accounted for the lack of reproductive success. Gestation length, litter size and birth weight of kits were not significantly affected by adult female consumption of 6.25 ppm heptachlor while kits whelped by females on the 12.5 ppm diet weighed significantly less than control kits at birth. Survival of kits in the 12.5 ppm group from birth to three weeks of age was also adversely affected. At three and six weeks of age, kit body weights in both the 6.25 and 12.5 ppm groups were significantly less than body weights in control kits. Examination 34 35 of heptachlor epoxide concentrations in newborn and developing kits indicated both placental and mammary transfer of the chemical from the dams to the kits. The LC50 for the 181-day exposure period for female mink was 10.5 ppm heptachlor and the LOAEL, based on reduced kit growth, was 6.25 ppm. Introduction Heptachlor (1,4,5,6,7,8,8-heptachlor-3a,4,7,7a-tetrahydro-4,7- methanoindene) is a chlorinated cyclodiene insecticide that was used extensively on a wide variety of agricultural crops in the 19605 and early 19703 (Dynamac Corp 1989). As with other chlorinated hydrocarbon compounds, heptachlor is persistent in the environment, existing primarily in its oxidized form as heptachlor epoxide (HE) (Nash and Harris 1973). Heptachlor and HE have been detected in several plant and animal species (Fox et al. 1964; Boddicker et al. 1971; Clark et al. 1980, 1983; Chawla et al. 1981; DeWeese et al. 1986) and in some instances have been associated with wildlife mortalities (Henny et al. 1983; Blus et al. 1984). Despite restrictions placed on the use of heptachlor by the US Environmental Protection Agency (EPA) in 1978 (US EPA 1978), contamination of human food products has occurred in Arkansas, Missouri, Oklahoma (Stehr-Green et al. 1986; Flora 1989) and Hawaii (Smith et al. 1984; Le Marchand et al. 1986). In 1982, the milk supply on the island of Oahu, Hawaii was found to be contaminated with heptachlor as a result of dairy cattle consuming heptachlor- treated pineapple plants being used as a feed supplement (Le Marchand et al. 1986). Because animal studies have shown adverse reproductive effects from 36 heptachlor exposure (Mestitzova 1967; Green 1970; Akay and Alp 1981) and because placental and mammary transfer of HE and other chlorinated hydrocarbons have been documented in human studies (Curley et al. 1969; Curley and Kimbrough 1969; Takahashi et al. 1981; Takei et al. 1983; Mussan-Rauhamma et al. 1988) there has been particular concern for pregnant women and nursing infants exposed to these chemicals (Le Marchand et al. 1986). Since the mink has been shown to be very sensitive to low concentrations of chlorinated hydrocarbons, particularly in terms of reproductive effects and offspring viability (Aulerich and Ringer 1977; Bleavins et al. 1980, 1984a; Aulerich et al. 1985; Hochstein et al. 1988), it was considered to be an appropriate species for evaluating the subchronic toxicity of heptachlor. In addition, short-term studies conducted in our laboratory have shown mink to be sensitive to dietary heptachlor (Aulerich et al. 1990). The objectives of the present study were to determine the subchronic toxicity of heptachlor to mink, the effect of dietary heptachlor on the reproductive performance of mink and on offspring viability, the placental and mammary transfer of HE to mink kits and whole-body HE concentrations in adult mink fed heptachlor. Materials and Methods Sixty adult standard dark mink (Mustela vison) from stock raised at the Michigan State University Experimental Fur Farm were randomly divided into three treatment groups and a control group. Each ‘group consisted of 37 12 females and three males. Mink were fed ad lib/tum a basal diet1 supplemented with either 0 (control), 6.25, 12.5 or 25 ppm technical-grade heptachlor2 for 181 days. The dietary concentrations selected were based on the results of a 28—day study in mink (Aulerich et al. 1990). The heptachlor- contaminated diets were formulated by dissolving the desired quantity of heptachlor (corrected for purity) in acetone and adding the solution to finely ground mink cereal to make a premix. After the acetone was evaporated, the heptachlor-cereal premix was combined with the basal diet to yield the nominal concentrations of heptachlor. Feed concentrations of heptachlor as determined by gas chromatographic analysis (see below) varied little from nominal concentrations. Samples from the control, 6.25, 12.5 and 25 ppm diets yielded concentrations of 0.01 i 0.01, 5.15 _+_ 0.28, 11.98 _+_ 1.54 and 21.24 i 4.15 ppm heptachlor, respectively. Mink were housed individually in wire cages (76 cm long x 61 cm wide x 46 cm high) suspended above the floor in an animal room. Routine mink farm procedures were followed in feeding, care and breeding of the mink. Water was provided ad libitum throughout the study. The photoperiod was regulated by timeclock to simulate natural light conditions. Temperature within the ‘ The basal diet consisted of 25% mink cereal, 20% chicken by-products, 20% ocean fish trimmings, 5% liver and 30% water. As fed, the diet contained 13.9% protein, 6.8% fat, 4.1% ash and 62.6% moisture (National Environmental Testing, Inc., Chicago, IL). Velsicol Chemical Corp., Memphis, TN; Technical grade heptachlor; purity 72%, Lot No. 53714421. 38 animal room approximated ambient temperature, except during cold periods when it was maintained above 0°C by thermostatically controlled electric heaters to prevent freezing of drinking water. Ventilation was provided by an exhaust fan and ceiling vents. The mink were acclimated to the test facilities for seven days prior to receiving the test diets. The trial was initiated on January 2, 1989 and continued to July 2, 1989 (181 days). Body weights were measured weekly. Feed consumption was determined over a two day period each week, except during the reproductive period. Mink were observed daily for overt signs of toxicity including abnormal behavior and mortality. Gross necropsies were performed on all mortalities and body and organ (liver, kidneys, adrenal glands, spleen, heart, lungs and brain) weights were recorded. After 42 days on treatment (February 13), females were mated to males within their respective dietary groups. Due to early mortality of males in the 25 ppm group, a small number of females in this treatment group were mated to males in the 12.5 and 6.25 ppm groups. Each female was given additional opportunities to mate following routine commercial mink farm breeding procedures. Successful matings were verified by the presence of motile spermatozoa in a vaginal aspiration taken after copulation. Females were checked daily for newborn young (kits) during the whelping period. During the later stages of pregnancy and during lactation, body weights of the females were not recorded, except at whelping and at three and six weeks post- whelping. Kit body weights were also determined at whelping and at three and six weeks (weaning) of age in the control, 6.25 and 12.5 ppm groups. No 39 females in the 25 ppm group whelped and none of the mink in this group survived beyond 88 days on treatment. Five kits were randomly chosen from the control and 6.25 ppm groups and euthanized (C02) at birth and at three and six weeks of age to determine whole-body HE concentrations for evaluation of placental, lactational and dietary transfer of HE. Only three, two and two kits could bersampled in the 12.5 ppm group at birth and at three and six weeks of age, respectively, due to the low number of females whelping in this group. Kits euthanized at three and six weeks of age were taken from the same litters as those euthanized at birth. Maternal milk samples were collected from six randomly selected females from the control as well as the 6.25 ppm group between 30 and 35 days post- whelping (Jones et al. 1980) to determine HE concentrations. At the end of the 181-day study, four, three and four adult female mink were randomly selected from the control, 6.25 and 12.5 ppm groups, respectively, and euthanized (C0,) to determine whole-body concentrations of HE. Similarly, nine and eight kits were euthanized on the 181st day from the control and 6.25 ppm groups, respectively, to evaluate the same parameter. All adults and kits were frozen after euthanization. While frozen, the mink were ground in a Hobart meat grinder to a hamburger-like consistency and refrozen for subsequent chemical analysis. Whole-body and milk HE concentrations were determined according to modifications of the procedure of Price et al. (1986). A 50 g aliquot of whole ground mink carcass was frozen in liquid nitrogen and blended to a fine powder in a Waring blender. A 20 9 portion was 40 mixed thoroughly with 100 g anhydrous NaZSO4, packed into a 2.5 x 30 cm column between two layers (approximately 1 cm each) of Na2804, and the fat (containing the chlorinated pesticides) extracted by percolation with 300 ml of diethyl ether/petroleum ether (1:1). The solvent was collected and evaporated in a tared 400 ml beaker, and the total fat content determined. A 200 mg aliquot of fat was suspended in 1 ml hexane and chromatographed on a column of Silica Gel 60 (Alltech Associates, Inc., Deerfield, IL) according to the procedure of Price et al. (1986). Fractions 86-1, 86-2, and 36-3 were collected following elution with 15 ml hexane, 20 ml hexane, and 20 ml benzene, respectively, beginning the collection of 36-3 when the translucent layer reached within 1.5 cm of the glass wool support. Heptachlor, if present, eluted in 56-2, HE eluted in 86-3. For this study, in which residue free animals were exposed to heptachlor only, both 86-2 and 86-3 were combined for glc analysis. Mink fetuses, which were too small to blend in a Waring blender, were weighed, minced with scissors, and mixed with 3 times their weight of anhydrous NaZSO4. They were then ground to a fine powder with a mortar and pestle, adding additional NaZSO4 if necessary to maintain a dry consistency. The mixture was quantitatively transferred to a 400 ml beaker and extracted 3 times with 100 ml of diethyl ether/petroleum ether (1:1) with gentle heating in a 60° water bath. The organic extracts were combined in a 400 ml tared beaker and the fat recovered and fractionated on a silica gel column as above. Milk was extracted by lightly vortexing a 5 9 sample with 15 ml acetone/hexane (1:1), the layers separated by centrifugation (2000 g), and the 41 organic fraction dried through anhydrous Nazso4 and transferred to a tared 50 ml conical tube. The milk was re-extracted consecutively with 15 ml hexane and 15 ml CHZCIZ. All three organic phases were combined and evaporated to dryness to determine the total fat content. The fat was taken up in hexane, quantitatively transferred to a silica gel column, and fractionated as above. The combined chlorinated pesticide fractions (36-2 and SG-3) recovered from silica gel chromatography were evaporated to dryness under a stream of N2 and resuspended in an appropriate volume of ethanol/iso-octane (20:80). Heptachlor and heptachlor epoxide were determined by gas chromatography on a Shimadzu GC-4BM chromatograph with 63Ni electron-capture detector. The compounds were chromatographed on a 3 mm x 1.8 m column packed with 1.5% SP2200/1.95% SP2401 (Supelco, lnc., Bellefonte, PA) at 200°C, with a N2 carrier gas flow rate of 50 ml/minute. Peak areas were integrated on a Spectra Physics SP2470 integrator and quantified by extrapolation from a 5 point standard curve (3.12, 6.25, 12.5, 25, and 50 pg). Differences in adult female mink body weights and feed consumption between treatment groups and the control group were determined by one-way analysis of variance (ANOVA) followed by nonorthogonal designed contrasts using Bonferroni’s 1‘ statistic (SAS Institute 1985). These parameters were not statistically analyzed for male mink due to the small number in each treatment group. The median lethal concentration (LC50) at 181 days for female mink was determined by the probit method using the computer software program Toxstat (Gulley at al. 1989). Adult female body weights and kit body weights at birth and at three and six weeks post—whelping, length of gestation and litter 42 size were also evaluated using Toxstat (Gulley et al. 1989). These data were analyzed for normality and homogeneous variance with the Chi-square test and Bartlett's test, respectively, before comparison of means. ANOVA, followed by Bonferroni's t statistic, were conducted when the data were normally distributed and the variance of the control and treatment groups was homogeneous. If the data failed to fulfill the assumptions required for ANOVA, the Kruskal-Wallis nonparametric test was performed to compare group means. Relationships between whole-body HE concentrations (pg/g body weight (bw)) and whole-animal HE body burdens (pg/animal) in adults and kits with dietary heptachlor concentrations were determined by linear regression analysis using a Macintosh SE computer with the software program Stat-View 512+ (Brain Power Inc., Calabasas, CA). Statements of significance are based on p < 0.05. Results Feed consumption by female mink was similar for all groups during the acclimation period, designated as week 0 (Figure 1). During the subsequent 12 weeks, 6.25 ppm heptachlor had no significant effect on feed intake while 12.5 ppm heptachlor caused significant decreases at weeks three, four, 10 and 12 when compared to controls. Feed consumption by female mink fed 25 ppm heptachlor was significantly depressed from weeks two through eight and at week 11. Heptachlor intake was 1.0, 1.7 and 3.1 mg heptachlor per kg body weight (bw) per day over the 12 week period for adult female mink fed diets containing 6.25, 12.5 and 25 ppm heptachlor, respectively. 43 .606 v 3 850555 .3 x83 05 .m 50:85 N 8603 th 22.50 :ms. 30. 3:35:03 am; 9.80 Ea: mm 9.: :_ :ofiEsmcoo 98 ”we U5 0.. .v .m 9.83 “a m_ob:oo :ms. 80. 350558 mm; 965 End mdw o£ :_ :OnQEamcoo poo. ”boron x83 we 05 50:99.5 m_ob:oo E9..— EoLoEo 3:35:06 “0: mm; 965 End mwd o5 :_ mofiEs .3 :oaaEamcoo boom .52.» 05 :0 $325 9. 5...... 05 0:50 x:_E oREo: :33 >9 UoEzmcoo Doc: :0 5.503ro >520 :0 Soto och .r 230E 2mm; 3 3 or m m H o m v o u r o b _ _ _ _ F r _ _ _ H _ b Eda 9mm I I I / \\ / EQQ m.NF ........... / xx x / Eda mm o ..... \ / \ / \ / \ / 6E8 \ / x / \ / x x \ r \All/ \ .VHJ: ......... II\.a. /// om 00.. cm? OON 0mm Mammal/6) dawnSNoo oaad 44 Adult female mink body weights during the same 12 week period decreased in a dose-related manner (Figure 2). Although females fed 6.25 ppm heptachlor weighed less than controls, the difference was not significant. Females fed 12.5 ppm heptachlor weighed significantly less than control mink from weeks seven through 12 while body weights of the 25 ppm group were significantly below those of controls from weeks four through 12. The body weights of females that whelped in the 12.5 ppm group were significantly reduced at whelping and three weeks post-whelping when compared to control dams, but not at six weeks post-whelping (Figure 3). None of the 25 ppm female mink survived to the whelping period. Adult female mink mortality for the 181-day exposure period was 0, 8, 67 and 100% for the control, 6.25, 12.5 and 25 ppm groups, respectively (Table 1). The median lethal concentration (LC50) for the 181-day period was 10.5 ppm heptachlor for female mink. All females in the 25 ppm group died within 88 days, with an average consumption of 177 mg heptachlor prior to death. Female mortalities in the 12.5 ppm group occurred through day 107 and the single mortality in the 6.25 ppm group occurred on day 178. All male mink in the control and 6.25 ppm groups survived the 181-day trial, whereas one male in the 12.5 ppm group died on day 90 and all males in the 25 ppm group died within 51 days. Mink fed 12.5 and 25 ppm heptachlor generally displayed clinical signs indicative of central nervous system involvement prior to death, such as hyper- excitability and seizures. Hindlimb ataxia was observed in several animals prior to more pronounced neurological effects. Bloody stools were observed 45 $0.0 v 3 8:85:90 .0. cmzofiv 0x00; E0: 0.09:8 :05 000. 3:005:90 203 030.6. End mm 05 :_ 3:90; >00: ”N F :m:o:£ K 0x003 Eot m_ob:00 car: 80. 2:005:90 22> 0305 End mar 05 :_ 3:903 >000 ”noted x003 N? 9.: 50:03.5 0.0bcoo E0: E2350 3:005:90 Co: 9.03 0306 Eng mm.o o£ :_ mofiEe 00 3:90? 50m .2030 05 :0 0x003 we 0.0.5 0£ 0:50 2:903 >000 x:_E 20E2 =39... :0 :oEowfio: 3320 :0 “00:0 2F .N 059: v_mm>> N? _.r o_. m m h w m v o N r o E _ _ _ _ _ _ _ L _ _ _ _ EQQ 9mm . | | | San mdr ............. EQQ mmd ....... \ / _oh:oo OON 00v com com 000. w CON; 00v... (6) LHeIaM A008 46 )1. j ._ob:00 E9: $0.0 v 8 00:20E0 E00550 020205 22:20.: 0020:; 02:02.00: Eda mm 00: 020E9— oz .m:_Q_o:>>-..00Q 9.003 50 0:0 02$ «0 0:0 00:60:; “a $5902, >00: x:_E 20:3: 2300 :0 250030: >820 00 0.00:0 or; .o 050E 0522308: 050.2338: 9.83 0 9.025 a 052055 - l L V o 1 8N T T 84 1 l 08 I . I cow * I l 80; En: mN— E 1 E00 AND E 1 com; .2080 I 8v; (6) LHeIaM A008 47 00:0: 05 00 28:20:00 :_ 0:00Enz :25 0:00:05 I+I :00E 00 0000055 00.00 _. :mém. mflmm cop QM mm 00 mm . Q P m.~P -l O m\0 mmd 0 ma 0 00.0.2 50.03 0 H um 00? N (up mm K0702 m H mm no N20 m.~P wt 0 N P: mwd 0 N :0 0 wu_mEun_ .2902 >:_0toE .x. :05: 6:020 .05 .803 020 005 25E 3:00:05. 520030: 00 0E0. _0>_>5m >020 .320 030:_E0E00 «5200000: 0:0 65:00 000 xEE .6 0E: 0223 0:0 3:00:05. .F 0.00:. 48 frequently in mink fed 25 ppm heptachlor. Nearly all mink stopped eating for one to two weeks before death. Ultimately, the animals became listless and then convulsed just prior to dying. Fatty livers, ulcerations and bloody mucus in the stomach were observed at necropsy in mink fed 12.5 and 25 ppm heptachlor. The effects of dietary heptachlor consumption on reproduction are presented in Table 2. All female mink fed 25 ppm heptachlor died prior to the whelping period (mid-April to early May). At necropsy, no fetuses were observed in the uteri of the five bred females in the 25 ppm group. Seven of 11 bred females fed 12.5 ppm heptachlor also died prior to whelping. Of these, two females contained fetuses in their uteri. Three of the four remaining mink successfully whelped. Feeding 6.25 ppm heptachlor had no adverse effect on female reproductive performance. Gestation length and litter size were not significantly affected by heptachlor consumption (Table 2), however, the percentage of stillborn kits was increased in the 12.5 ppm group relative to the control and 6.25 ppm groups (Table 2). A single morphologic anomaly was observed in a kit from the 12.5 ppm group. The front legs and tail were considerably shortened and the kit survived for only one day. Feeding male mink 6.25 and 12.5 ppm heptachlor had no effect on sperm motility or morphology. Male mink in the 25 ppm group died before they could be mated. Consumption of 6.25 ppm heptachlor by female mink prior to breeding and during gestation had no significant effect on kit birth weights while kits whelped by dams in the 12.5 ppm group weighed significantly less than kits from control mothers at whelping (Table 3). At three and six weeks of age, kit 49 00:90 0005 :_ 0.:_E :050 :0 05000:: 00E0>0:0 >~..0:.:0E >_:0m a 000.05.. 505 00.0E0: 00:05 :0 0:00:00 2 00005500 :. :00E02 o .:0::0 0:00:05 I...I :00E 00 00000000 0500 a 050:: 50000000 .05 05 :0 0000 05 :0 00000 . l - l I- l- 10. m5 mN K m NF mdflfim TNHoém LBN. :5 mdp mm m mm 0.0 H. Nd 9N H. 9mm 82 N {or mmd mm m 0.0 Nd I+I N6 mé H. mdm Lab. «(m o .20. 0000 02.4. ..020 4.600. 00:.0E 00.0E0: .E00. . 5:... 0.0 .0. .02 :05... 50:0. :00E0:\000_0:>> 5.5000: 02.0 05. :0505000 00.0E0: :00E02 2050.0 0.5:: 0.0E0: 0.000 :. 00:0E:0t00 05000900: :0 8200500: 2050.0 :0 00050 0:... .N 0.00... 50 .0:_0> .0:::00 E0:: .000 v 0. ::0:0::_0 >_::00.:.:0.0 .. ..00.:00 0E.: :00: :0 020 0:0. :0 :0060: .0... 000 0.0800 0: :0:0: 0000:5000 :. 0:00:52 .:0::0 0:00:0:0 H 000:: 00 00000508 0:00 . 00: 00 0.0. 0. :0 H 0.00: 0.0. 0.0 H 0.00 n.0:. 0.0 H 0.0 0.0: 00 00 :00. 0.0: H 0.000 3.00. 0.0 H 0.00 .00. 0.0 H 0.0 00.0 00: 00 .:0. 0.0 H 0.::0 .00. 0.0 H 0.00: .:0. 0.0 H 0. :: 0 8.003 0 8.00.5 0 9.002: 0 8.002. 0 :::.0 .E00. 0: 8.00:5 0 0: :::.0 3.000500 20:05 .0. .0225 :0. .a. £0.02, 28.. :0. .v.:.E :0. :0 .0>.>:00 0:0 50.0.5 >000 :0 5.00080: :0 :00::0 0:... .0 0.00 .0 51 body weights in both the 6.25 and 12.5 ppm groups were significantly below those of control kits. Kit survival from birth to three weeks of age was reduced in the 12.5 ppm group compared to control and 6.25 ppm kits, whereas survival from three to six weeks of age was comparable for all groups. In utero exposure to 6.25 and 12.5 ppm heptachlor resulted in HE body burdens of 8.9 and 24.8 ug/animal and whole-body concentrations of 0.86 and 3.08 ug/g bw (ppm), respectively, in kits at birth (Table 4). The relationships between heptachlor concentration in feed consumed by dams prior to breeding and during gestation and HE body burdens and whole-body HE concentrations in newborn kits were linear, with coefficients of determination (R2) of 0.70 and 0.77, respectively. HE body burdens continued to increase in the 6.25 and 12.5 ppm kits through lactation (birth to three weeks of age), but when expressed as a function of body weight, whole-body HE concentrations declined during this period when compared to concentrations at birth (Table 4). However, a significant positive relationship between whole-body HE concentrations in kits and the dams' dietary treatment level continued to three weeks of kit age (R2 = 0.87). After three weeks of age, kits were provided with the same diet as their respective dams in addition to heptachlor exposure throughnursing. Body burden levels and whole-body concentrations of HE at six weeks of age were considerably elevated over those at three weeks for kits in the 6.25 and 12.5 ppm groups (Table 4). Whole-body HE concentration in kits at six weeks of age was directly related to the dams treatment concentration (R2 = 0.76). Milk samples obtained between 30 to 35 days postpartum from dams fed 6.25 ppm heptachlor had a mean HE concentration 52 .900 :00 v. 000:0. .v.:.E :0. 00~.:0:::0 28 3:000:00: £0.02. >000 :005. .. :0::0 0:00:0:0 H 000:: 00 0000298 0:00 . 0.0 H 0.0 00.0 H 00.0 0.00 H 0.00: 0. : :0 H 0.000: 0 0.0: 0.: H 0.0 00.0 H 00.0 0.00 H 0.000 0.00: H 0.000 0 00.0 0.: H 0. :: 00 0.00 H 0.0:0 00 0 0 gala 0.: H 0.0 00.0 H 00.0 0.0: H :00 :.0: H 0.00 0 0.0: 0.: H 0.0: 00.0 H 00.0 0.0: H 0.0:: :. : H 0.00 0 00.0 0.0 H 0.0: 00 0.0 H 0.00: :.0 H :.0 0 0 003240 :.0 H 0.: 00.0 H 00.0 0.0 H 0.0 0.0 H 0.00 0 0.0: 0.0 H 0.: 0 :.0 H 00.0 0.: H 0.0: 0.0 H 0.0 0 00.0 0.0 H 0.: 00 0.0 H 0.0: no 0 0 00.4. .00. :0: .35 0.0:. 4.3. .._0::.:m.0:. 2.0 Ea... >000 :0.:0:::00:00 £0.03 c0050 >000 0: 0.0E00 5.00050: 0... >000-0.0:>> >000 >:0:0.0 IIIIIII|IIIII|IIIIIIIIIIIIIL .000 :0 0x003 :00 0:0 00:0: 0:0 :::.0 :0 0:0. x:.E :. :0: ::00:00 0:0 .01. 000800 :0.:00:00: :0 :0.:0:::0o:00 >000-0_0:>> 0:0 :00:00 >000 .0:0: :0 050090 5.00060: >:0:0.0 :0 :o0::0 0:... .0 0.00... 53 of 4.6 ppm as compared to trace amounts in the milk of control dams. The amount of fat in milk collected from control (15.8%) and 6.25 ppm (16.8%) dams was not significantly different. Adult female mink in the 6.25 and 12.5 ppm groups euthanized at the end of the 181-day feeding periOd had whole-body HE concentrations of 8.87 and 13.83 ug/g bw, respectively (Table 5). HE concentrations in the body were directly related to the concentration of heptachlor in the diet (R2 = 0.77). The percent body fat of the euthanized adult females decreased with increasing amounts of heptachlor in the diet (Table 5). Kits between two and three months of age from the 6.25 ppm group (no kits remained in the 12.5 ppm group) showed continued accumulation of HE when euthanized at the end of the 181-day trial (Table 6) compared to concentrations observed at six weeks of age. There were no mortalities in kits from the control and 6.25 ppm groups during the period from 6 weeks of age to the end of the trial. Discussion Diets containing 25 ppm heptachlor (dose equivalent to 3.1 mg/kg bw/day) fed to female mink caused a reduction in feed intake and a concomitant decrease in body weight within 28 days on treatment. In a 28-day feeding trial with male mink, Aulerich et al. (1990) also reported decreased feed consumption at dietary concentrations of 25 ppm (3.11 mg/kg bw/day) heptachlor, but no significant body weight loss. They observed no effects on these parameters in male mink fed 12.5 ppm (1.79 mg/kg bw/day) heptachlor. Female mink fed 12.5 ppm (1.7 mg/kg bw/day) heptachlor in the present study 54 .v.:.E 0.0E0: :.:00 00~.:0::=0 >.:0 0::000:00: £0.02. >000 :00—2 0 .:0::0 0:00:0:0 H :00E 00 0000000 0:00 . :.0 H 0.0: 00.0 H 00.0: 00: H :00 0.0000 H 0.0000: 0 0.0: 0.0 H 0.0: 00.0 H 00.0 :0 H 000 0.000 H 00:00 0 00.0 0.: H 0.00 0.0 H :06 :0 H 000: 0.: H 0.0 0 0 .3... :0: .32: 0.0:. 4...... 20.03 0.060003 2.0 .500. >000 . :0.:0:::00:00 >000 :0050 >000 0: 0.0E00 5200:00: 01 38-29:; >:0:0.0 ||||||I|||Il .0>00 :0: :0::0 v.:.:: 0.0E0: :.:00 :. :0: >000 ::0o:00 0:0 .01. 000800 5.00000: :0 :0.:0:::00:00 >000-0_00>> 0:0 :00:00 >000 .0:0: :0 :0.:00:00: >:0:0.0 :0 :00::0 0:... .0 0.00.0 55 .20: :00 v. 89:. .:0::0 0:00:0:0 H :00E 00 0000000 0:00 a .v.:.E 03: 00:.0::00 :0.:>> 030:0 .0:::00 0:: :. 0.0E00 0:0 :0: :000X0 0.0E00 :000 000.:0E00 0:0. 00:: .0 . 0.: H 0. :: 00.0 H 00.0 :0: H 000 0.000 H 0.0000 0 00.0 0.:H0.0: oo 00 H000 0.: H0.: 0 0 0.0. :0: .35 0.03 .3. £0.02, 00500.00. .000 .60... >000 :0_:0:::00:00 >000 :00::0 >000 01 0.0E00 520000: 01 >000-0.0:>> . >:0:0.0 .I|IIIIIIIIII|III|| .00.:00 >030 >00-:0: 0:: :0 0:0 0:: :0 00~.:0:::0 0:0. :20: 0.0.5020 -00::: 0: 02.: c. :0: >000 ::00:00 0:0 .01. 000800 :0.:00:00: :0 :0.:0:::00:00 >000-0.0:>> 0:0 :00::0 >000 .0:0: :0 :0.:00:00: >:0:0.0 :0 :00::0 0:... .0 0.00... 56 had decreased feed intake after 21 days on treatment, but experienced no reduction in body weight until day 49. Feeding 6.25 ppm (1 mg/kg bw/day) heptachlor to female mink had no adverse effect on feed consumption or body weight. Similarly, rats fed 0.1 to 10 ppm heptachlor for 21 days displayed no decrease in feed intake or body weights (Polin, personal communication). Consumption of 25 ppm (3.1 mg/kg bw/day) heptachlor was highly toxic to male and female mink causing 100% mortality within 88 days. The earliest deaths occurred in two male mink after only 31 and 32 days on treatment, and 87% of all mink in this group died by day 57. Aulerich et al. (1990) observed no mortality in male mink fed 25 ppm (3.11 mg/kg bw/day) heptachlor for 28 days. They also reported no deaths in males fed 12.5 ppm (1.79 mg/kg bw/day) heptachlor, whereas the same dietary concentration caused one female mink mortality after 34 days in the present study. However, most of the mortalities for mink fed 12.5 ppm (1.7 mg/kg bw/day) heptachlor came later in the trial, with seven of eight female deaths occurring between 86 to 107 days and one male death on day 90. Consumption of 6.25 ppm (1 mg/kg bw/day) heptachlor caused a single death in a female on the 178th day of the trial. Assuming the death of this female was due to heptachlor toxicity, the lowest-observed-adverse-effect level (LOAEL) in female mink for the 181-day study period (26 weeks) was 6.25 ppm (1 mg/kg bw/day). In comparison, the LOAEL for female mice fed heptachlor for 80 weeks was 18 ppm (2.3 mg/kg bw/day) and the no-observed-adverse-effect level (NOAEL) was 9 ppm (1.2 mg/kg bw/day) (NCI 1977). Heptachlor, like other chlorinated cyclodiene insecticides, is a central 57 nervous system stimulant (Murphy 1986). Consumption of 12.5 (1.7 mg/kg bw/day) and 25 ppm (3.1 mg/kg bw/day) heptachlor by adult mink generally caused hyperexcitability, loss of hindlimb coordination, and tonic-clonic seizures prior to death. Similar signs were reported in rats following acute exposure to much higher doses of heptachlor (200 mg/kg bw) and HE (100 mg/kg bw) (Hrdina et al. 1974). Cats exhibited seizure-type behavior within 20 to 40 minutes after receiving an intravenous dose of 2 to 10 mg heptachlor/kg bw (Joy 1976). Aulerich et al. (1990) observed no neurotoxic effects in male mink fed 12.5 (1 .79 mg/kg bw/day) and 25 ppm (3.11 mg/kg bw/day) heptachlor for 28 days. However, several mink fed 100 ppm (6.19 mg/kg bw/day) exhibited neurological aberrations consistent with those observed at 12.5 (1.7 mg/kg bw/day) and 25 ppm (3.1 mg/kg bw/day) dietary heptachlor in this study. Fatty infiltration of the liver was observed at necropsy in nearly all mink fed 12.5 (1.7 mg/kg bw/day) and 25 ppm (3.1 mg/kg bw/day) heptachlor. Aulerich et al. (1990) also reported liver steatosis in male mink fed diets containing 100 ppm (6.19 mg/kg bw/day) heptachlor. Fatty livers have been noted in rats following acute (Pelikan 1971, as cited by Dynamac 1989) and chronic (Witherup et al. 1955) oral exposure to heptachlor. The effects of heptachlor consumption prior to breeding and during gestation on reproductive performance were difficult to assess due to high mortality in the 12.5 and 25 ppm groups before the whelping period. No fetuses were observed in the uteri of the five bred females fed 25 ppm heptachlor, whereas two of seven females in the 12.5 ppm group contained fetuses at time of death. Reproductive performance was not adversely affected 58 in mink fed 6.25 ppm (1 mg/kg bw/day) heptachlor. in contrast, a dietary level of 5 ppm heptachlor (0.25 mg/kg bw/day) fed to male and female rats for two generations caused a decrease in pregnancy rates. The first generation pregnancy rate was 72% versus 94% in the controls, while none of the second generation heptachlor-treated females became pregnant (Green 1970). Male and female mice consuming 50 ppm (7.5 mg/kg bw/day) heptachlor in the diet for 10 weeks also failed to reproduce (Akay and Alp 1981). In utero exposure of kits to heptachlor from dams fed 6.25 ppm had no adverse effect on kit body weights or survival rates at birth. However, feeding 12.5 ppm (1.7 mg/kg bw/day) heptachlor to adult female mink did cause a decrease in kit birth weights and survival rates compared to controls. Gestation length was also slightly reduced relative to control females. However, this value was well within the reported range for mink (Sundqvist et al. 1989). No difference was observed in litter size of females fed 6.25 (1 mg/kg bw/day) and 12.5 ppm (1.7 mg/kg bw/day) heptachlor. However, marked decreases in litter size have been reported by Mestitzova (1967) in rats fed considerably higher concentrations (6 mg/kg bw/day) of heptachlor. Although concentrations of 6.25 ppm heptachlor in the present study did not affect the reproductive capacity of female mink, it did have an adverse effect on postnatal survival of kits within individual litters. Kit survival was only 25% in one litter from the 6.25 ppm group during the period from birth to three weeks of age, while survival in another litter was 29% from three to six weeks of age. However, overall kit survival to three weeks of age was 83% for the 6.25 ppm (1 mg/kg bw/day) group. In a similar study conducted by 59 Green (1970), diets containing 5 ppm (0.25 mg/kg bw/day) heptachlor fed to rats prior to mating and during gestation resulted in 16% survival of newborns to three weeks of age. In the present study, 45% of the kits from dams fed 12.5 ppm (1.7 mg/kg bw/day) group survived to three weeks of age, indicating that rat pups are more sensitive to heptachlor than mink kits. In contrast, Witherup et al. (1976, as cited in WHO 1984) reported only slightly decreased pup survival in rats at two and three weeks of age in second generation litters exposed to 10 ppm heptachlor. Postnatal growth, as assessed by body weight gain, was decreased in kits from the 6.25 and 12.5 ppm groups. In contrast, there was no growth retardation in suckling rats from dams fed 10 ppm heptachlor over three generations (Eisler 1968). The presence of HE in kits whelped by dams fed 6.25 and 12.5 ppm heptachlor confirms the work of previous researchers concerning its ability to cross the human placenta (Curley et al. 1969; Polishuk et al. 1977). Transplacental transfer of other chlorinated hydrocarbons has also been demonstrated in mink (Bleavins et al. 1981, 1982, 1984b). The concentration of HE in kits at birth increased with respect to the dams’ treatment level, with kits whelped by dams fed 12.5 ppm heptachlor having 3.6 times greater HE concentrations than kits in the 6.25 ppm group. Although total body burdens of HE increased in kits from the heptachlor-treated groups during the lactational period (birth to three weeks of age), whole-body HE concentrations declined. This suggests that the growth rate of kits exceeded the accumulation of HE residues derived from nursing, as kits are entirely reliant upon the dam’s milk during this period. A comparison of HE body burdens at birth with those at 60 three weeks of age for kits in the 6.25 ppm group indicates that transfer of HE via lactation is greater than through the placenta. HE body burden results for kits in the 12.5 ppm group were less convincing. However, this may be due to lower production of milk by dams in this group as they were considerably underweight. Milk samples collected from dams in the 6.25, ppm group four to five weeks after whelping, contained a mean HE concentration of 4.6 ppm (27.6 ppm, fat basis). Although this result was obtained after three weeks of kit age, it does reflect the potential for considerable uptake of HE via lactation. Heptachlor residues up to 5.0 ppm (fat basis) have been reported in milk for human consumption from Oahu, Hawaii (Le Marchand et al. 1986). Kits were provided the same diets as their dams after reaching three weeks of age. The exponential increase of total body burdens and whole—body HE concentrations in the 6.25 and 12.5 ppm kits from three to six weeks of age is due to the increased intake of heptachlor from this dietary supplementation. Total body burdens and whole-body concentrations in kits (two to three months of age) euthanized at the end of the 181-day study demonstrate a continued accumulation of HE. Body burdens of HE increased nearly five-fold and whole-body concentrations doubled compared to values at six weeks of kit age. Whole-body concentrations of HE in adult female mink from the 6.25 and 12.5 ppm groups euthanized at the end of the 181-day study period were 8.87 and 13.83 pg/g bw, respectively. Rats fed 1 ppm heptachlor for the same duration had a whole-body HE concentration of 0.24 pg/g bw (Polin, personal communication). Using the regression of whole-body HE concentrations in 61 adult female mink on dietary heptachlor concentrations of O, 6.25 and 12.5 ppm, a concentration of 1 ppm fed to female mink would predict a whole- body HE concentration of 1.64 ug/g bw. This indicates that mink accumulate HE residues to a greater extent than rats. The results of this study indicate that consumption of 6.25 ppm dietary heptachlor for approximately 120 days prior to whelping did not affect the ability of female mink to produce viable offspring. However, postnatal exposure of young mink in this group to low levels of HE through lactation and/or dietary heptachlor resulted in reduced growth and survival compared to control kits. These findings are supported by large increases in total body burdens and whole-body concentrations of HE in kits during this period. Diets supplemented with 25 ppm heptachlor were highly toxic, causing 100% mortality within 88 days on treatment. Feeding 12.5 ppm heptachlor caused early mortality (67%) in this group, thereby precluding an accurate assessment of reproductive performance. However, the reproducing females in the 12.5 ppm group produced an increased number of stillborn kits and there was a reduction in postnatal survival and growth of the kits. Using postnatal body weight gain and survival as endpoints, the LOAEL is 6.25 ppm (1 mg/kg bw/day) heptachlor. Depending on body weight, 25 pg of HE in mink kits at whelping may be lethal and continued exposure to HE from dams fed 12.5 ppm heptachlor during the lactational period is also detrimental to survival. CHAPTER II. THE EFFICACY OF MINERAL OIL COMBINED WITH FEED RESTRICTION IN ENHANCING THE ELIMINATION OF HEPTACHLOR EPOXIDE FROM MINK (MUSTELA VISONI Abstract Adult female mink previously fed 0 (control) and 6.25 ppm dietary heptachlor for 181 days were administered heptachlor-free diets ad libitum {AL} or the same diet containing 10% mineral oil and restricted by 45% ofAL intake (MO/R) for 21 days to evaluate the efficacy of these diets in enhancing the withdrawal of heptachlor epoxide (HE) from mink. Kit mink whelped by dams of the control and 6.25 ppm groups, were also fed the withdrawal diets for 21 days. Daily consumption of the AL diet by kit mink was significantly greater than consumption of the same diet by the adult females. Body weights of the control adult females and control and 6.25 ppm kits were significantly reduced by feeding the MO/R diet. Two adult females from the control group and one adult female from the 6.25 ppm group fed the MO/R diet died during the withdrawal period. No mortalities occurred in kit mink fed the MO/R diet or in any mink fed the AL diet. Administration of the MO/R diet caused a considerable reduction in body fat of the control and 6.25 ppm adult female and kit mink. However, lower body fat percentages were not associated with greater elimination of HE. Evaluation of total HE body burdens and whole-body HE concentrations in adult female and kit mink from both prior treatment groups at day 21 indicated that consumption of the MO/R diet resulted in no greater effect on withdrawal of HE, and that both regimens were equally effective in terms of elimination of HE from mink. Elimination of HE was more extensive in 62 63 kit mink, particularly in the 6.25 ppm kits which experienced an approximate 15% greater reduction of HE compared to adult females. These results indicate that HE exists as a labile pool in adult female and kit mink. Introduction The extensive and widespread past use of the insecticide heptachlor (1,4,5,6,7,8,8-heptachlor—3a,4,7,7a-tetrahydro-4,7-methanoindene) together with its recalcitrant properties have inadvertently resulted in exposure of non- target organisms. The most common and economically significant exposures have occurred in the livestock industry as a result of heptachlor’s use on agricultural crops. Despite restrictions placed on the use of heptachlor by the US Environmental Protection Agency (EPA) in 1978 (US EPA 1978), secondary contamination of human food products has occurred in Missouri, Oklahoma (Raisbeck et al. 1986; Stehr-Green et al. 1986), Hawaii (Smith et al. 1984; Le Marchand et al. 1986), and most recently in Arkansas (Flora 1989). From a human health and economic perspective, the most significant of these contamination incidences occurred in 1982 on the island of Oahu, Hawaii where milk was found to contain violative concentrations of heptachlor (Le (Marchand et al. 1986). The milk supply was contaminated as a result of dairy cattle consuming feed supplemented with heptachlor-treated pineapple plant foliage. Approximately nine million pounds of milk were condemned resulting in an estimated economic loss of nine million dollars (Smith et al. 1984). Of greater importance was concern that adverse health effects could develop in the Oahu population, since testing of stored milk samples indicated that milk 64 containing heptachlor residues in excess of the current EPA action level of 0.1 ppm (fat basis) had been sold on the island for a 27 to 29 month period before the first recall (Le Marchand et al. 1986; Smith et al. 1984). Because heptachlor and its primary metabolite, heptachlor epoxide (HE), are lipophilic molecules that accumulate in adipose tissue (Radomski and Davidow 1953), methods to remove these chemicals from tissue stores are of considerable interest to the livestock industry and are ultimately important for the protection of humans from potential long-term health effects. A review of various HE decontamination strategies indicates that the efficacy of the method is dependant on the animal species tested. In addition, the majority of techniques have focused on enhancing intestinal elimination since most residual chlorinated hydrocarbons are excreted primarily in the feces (Rozman 1985). Metabolic inducers of biotransformational enzymes in the liver enhanced the clearance of 1“C-heptachlor from rats (Rozman 1984), but were not effective in increasing the elimination of 1“C-heptachlor from sheep (Smith et al. 1989) or in reducing HE body burdens in cattle (Raisbeck et al. 1989). Intestinal adsorbents (lipotropic binding agents) such as mineral oil, liquid paraffin and hexadecane, were highly effective in enhancing fecal elimination of several polyhalogenated hydrocarbons from rats, rhesus monkeys and goats (Richter et al. 1977; Rozman et al. 1981a,b; Rozman et al. 1982a,b; Polin er al. 1987), but failed to increase elimination of heptachlor residues from rats, sheep, pigs and cattle (Rozman 1984; Smith et al. 1989; Raisbeck er al. 1989). Since adipose tissue is the principal reservoir of most lipophilic toxicants, techniques to mobilize and reduce body fat stores have been tested. However, 65 this method was not effective in reducing body burdens of HE-contaminated cattle (Raisbeck et al. 1989), while experiments on other halogenated compounds in rats resulted in redistribution of the contaminant to other lipid storage sites (Wyss et al. 1982). Various combinations of the above methods have consistently resulted in successful elimination of many xenobiotics from several animal species. Cook and Wilson (1971) demonstrated that a combination of activated carbon and phenobarbital was more effective than either separately in removing dieldrin from cattle. Mutter et al. (1988) reported increases in fecal excretion of DDE from gerbils when feed restriction was combined with administration of sucrose polyester, a lipophilic binding agent. Feed restricted diets containing mineral oil were also successful in hastening the withdrawal of PCBs, PBBs, hexachlorobenzene and pentachlorophenol from chickens (Polin et al. 1985, 1986a,b). Subsequent experiments using this method in rats exposed to PBBs indicated that the greatest reductions in body burdens occurred in rats fed diets containing 10% mineral oil and restricted by 45% of ad libitum consumption (Polin et al. 1991). This treatment was recently shown to cause the greatest reduction in HE body burdens in rats exposed to heptachlor (Polin, personal communication). The purpose of the present study was to determine the efficacy of a diet containing 10% mineral oil and restricted by 45% of ad Iibitum intake in enhancing the withdrawal of HE from adult female and kit (young) mink previously exposed to heptachlor in a 181 day feeding trial (Crum er al. 1993). Materials and Methods Adult female standard dark mink (Mustela vison) from stock raised at the Michigan State University Experimental Fur Farm that were previously fed 0 (control) and 6.25 ppm technical—grade heptachlor1 for 181 days (Crum et al. 1993) were randomly divided into two dietary treatment groups consisting of a basal mink diet2 fed ad Iibitum (AL) or a basal diet containing 10% mineral oil and restricted to 45% of the ad Iibitum intake of an adult female mink (MO/R). Young mink (kits ranging from two to three months of age) whelped by dams fed control or 6.25 ppm heptachlor in the previous trial were also randomly and equally divided into the two treatment groups described above (Table 1). Animals were maintained on the two diets for 21 days (withdrawal period), beginning on July 2 and ending on July 22, 1989. To assure that animals fed the MO/R diet were truly receiving 45% less feed than the AL animals, a three- day moving average of feed consumed per day by adult female and kit mink fed the AL diet was calculated. Therefore, adjustments to the MO/R diet were made every fourth day with a separate corresponding average calculated for ‘ Velsicol Chemical Corp., Memphis, TN; Technical grade heptachlor; purity 72%, Lot No. 53714421. 2 The basal diet consisted of 25% mink cereal, 20% chicken by-products, 20% ocean fish trimmings, 5% liver and 30% water. As fed, the diet contained 13.9% protein, 6.8% fat, 4.1% ash and 62.6% moisture (National Environmental Testing, Inc., Chicago, IL). 66 67 I Table 1. Study design for the 21 —day withdrawal period. I Withdrawal treatment ll MO/Rc Prior dietary heptachlor No. No. No. (ppm)" adults kits adults kits 0 4 9 4 6.25 4 9 4 9 " Dietary treatments fed to mink during the 181-day feeding trial (Crum et al. 1993). b Basal diet fed ad Iibitum. ° Basal diet containing 10% mineral oil fed at 45% of ad Iibitum consumption. 68 adult female and kit mink. Mink were housed individually in wire cages (76 cm long x 61 cm wide x 46 cm high) suspended above the floor in an animal room. Animals were observed daily for any adverse response to the treatments, including mortality. Water was provided ad libitum throughout the withdrawal period to all mink. Temperature within the animal room approximated ambient temperature, which averaged 72 degrees farenheit during the 21-day period (NOAA 1989). Ventilation was provided by an exhaust fan and ceiling vents. Prior to the beginning of the present study, four control and three 6.25 ppm adult mink from the previous investigation were euthanized (C02) to determine whole-body HE concentrations. Similarly, eight and nine kits from the control and 6.25 ppm groups, respectively, of the previous study were euthanized for HE analysis. At the completion of the withdrawal period, all mink were euthanized (C02) to determine whole-body HE concentrations. Methods for preparation and analysis of mink carcasses were previously described (Crum et al. 1993). Statistical analyses to determine the effects of the withdrawal treatments on feed consumption, body weight, body fat content, total HE body burdens and whole-body HE concentrations in adult female and kit mink from the prior heptachlor treatment groups were conducted using the computer software program Toxstat (Gulley et al. 1989). The Student t-test was used to evaluate feed consumption and body weight differences within and among prior heptachlor treatment groups for adult females and kits separately, while feed consumption differences between adults and kits fed the AL diet were 69 compared using Tukey’s method of multiple comparisons of means. Tukey's method was also used to examine differences in body fat content, total HE body burden and whole-body HE concentrations within prior heptachlor treatment groups for adult female and kit mink separately, except where data were found to be heterogeneous. If the data were determined to be heterogeneous, via Bartlett’s test, they were then transformed by taking the square root of the values. If the transformed data passed Bartlett’s test for homogeneous variance they were evaluated by Tukey’s method. If the transformed data failed this test, the KruskaI-Wallis nonparametric test was performed to compare group means. Statements of significance are based on p < 0.05. Results Feed consumption by adult female mink fed the AL diet is presented in Figure 1. The quantity of feed consumed per day by females previously fed the control diet (0 ppm heptachlor) for 181 days varied considerably during the withdrawal period compared to female mink formerly fed the 6.25 ppm heptachlor diet, and was generally less than the 6.25 ppm group. The average daily intake of the control (131 g/day) and 6.25 ppm (141 g/day) groups over the withdrawal period was not significantly different. However, when expressed on a body weight (bw) basis, feed consumption by the 6.25 ppm females was significantly greater than that of the control females (157 versus 110 g/kg bw/day, respectively) during the withdrawal period. Feed consumption by kit mink (approximately three to four months of 70 .9000 :0: 612000500 A. -. E00 mud 000 T... 60000 00: 2030380 0.0.0: 0.0E0: 0:00 >0 02:8. 03805.; .60. :0 on. 00.50 :20 as 83.0.: 00 on. .o 8:08:28 .: 0:30.“. w><0 rNONGPthrmrmewmrNrerrmONomVONr b _ _ _ P _ _ _ _ _ F _ _ _ _ _ _ g _ _ _ O 1 cm I 00.. . . . . ~III ‘3. X x .0 a s a sIlI ss 0; s . H x a s I I s o Its . X I; I \III ‘ 1 ss I. . .I o. . t . .x . . I 00: \ o as i . CON (AepMUIw/B) dawnSNoo aaaa 71 age) from both the control and 6.25 ppm groups fed the AL diet was markedly similar over the withdrawal period, with the greatest daily fluctuations occurring during the first eight days (Figure 2). Control kits consumed an average of 175 g/day, while kits from the 6.25 ppm group ingested an average of 169 g/day. Daily feed intake for both kit groups was significantly greater than intake by adult female mink in the same groups. Based on the mean body weight of these two groups at the end of the withdrawal period, feed consumption was estimated at 171 and 185 g/kg bw/day for the control and 6.25 ppm kits, respectively. However, these values are likely underestimates as kits were rapidly growing during this period of their lives. Feed consumed by adult female mink on the MO/R diet ranged from 65 to 100 g/day, while kits on this diet consumed 85 to 105 g/day. These quantities equaled the allotted amount of feed supplied to these mink based on mean consumption of the AL animals. ‘Both adults and kits consumed the entire allotted quantity of food each day, with the exception of a small number of mink that either rejected feed or consumed only a small portion of the diet the first two days. One control adult female consumed a very small portion of the MO/R diet the first two days and then totally rejected the feed for the next 15 days before dying. Body weights of the control and 6.25 ppm adult female mink assigned to the AL or MO/R diet were not significantly different on day 0 of the withdrawal period (Table 2). However, after 21 days of withdrawal, the surviving adult females from the control group fed the MO/R diet weighed significantly less than control females fed AL. Although the percent body 72 00.000 03005.3 00: 0: 5.0 0:008 000 200E08000 :0: 0E00 :00: o: 02:000. 000 00: 00.0 0:03 05. 00.00.2000 A- -. 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NO: .I.I..I. 000 0.0.2 .0. 00 H 0:0 02 .0. mm H ~00 .0. mm + 000 .:x 00.0 ..0. 00 H 000 02 ..N. 00 H 000 .0. 00: H 000 0.0.2 .0. 00 H 000: 02 .0. 0:: H :00: .0. 00: H 00:: L.\ 09:000. 0 :0. >00 0 >00 :0 >00 .0 >00 20500:: 3.600. .0>>0:00:.>> :0_000:000 >:0:0.0 :000 0:.v. 0:.30< .3. £0.02, >000 .00.:00 .030:00:.>> 00: :0 .:N >00. 000 000 .0 >00. 00.05000 00: :0 0.0.0: :0. 000 0.0E0: :_300 :0 0:00.03 >000 .N 0.00... 74 weight loss of surviving female mink from the 6.25 ppm group (22%) fed the MO/R diet was similar to control females (25 %l on the same diet, there was no significant difference in body weights between 6.25 ppm females fed AL or a MO/R diet at day 21 of withdrawal. Although body weights for kit mink were not recorded on day 0 of the withdrawal period, there was no significant difference in body weights of eight and nine kit mink from the former control and 6.25 ppm groups, respectively, which were euthanized at the end of the 181-day feeding trial (immediately prior to the beginning of this study). The body weights of control and 6.25 ppm kit mink fed the MOIR diet were significantly below the body weights of kits from the same groups fed AL at day 21 (Table 2). Final body weights for the control and 6.25 ppm kits were 41 and 37% less, respectively, than body weights of kits from the same groups fed AL. Moreover, kits consuming the MO/R diet for 21 days had body weights that were below body weights of their corresponding litter mates that were euthanized three weeks earlier. Adult female and kit mink fed the MO/R diet were extremely hyperactive throughout the withdrawal period. Two adult female mink from the control group fed the MO/R diet died on days 18 and 21 of the trial. The female which died on day 18 had consumed only a minute amount of feed up to its death, while the other female consumed the allotted amount of feed provided in the MO/R diet up to its death. Interestingly, the female dying on day 18 had a body fat content of 35.1%, which was greater than the mean of the control ' females on the AL diet (28.2%; Table 3). The other control female that consumed the MOIR diet up to its death on day 21 was nearly devoid of fat 75 .00.:0050000 0:000... 00 :0 $00 :0 00: ..0 .0:00.0: $0: 00.0.0:000 :0.0 .0000 . .883... 00 00: :0.0 .0000 u .000 v 0. :00:0::.0 >.:000.:.00.0 0:0 0:0.:00:00:0 :00:0::.0 0:.3 :00E:00:: >:0:0.0 500 0000 :0: 005.00 00:00 0.0:.>> 00005. c .0 >00 :0 00.0> 05:: 00.:0000: :000:00 0. 000050200 0. :00052 .:0::0 0:00:0:0 H 0000: 00 00:0000:0 0:00 a .000: .:0 :0 05:0. 0>00 :0: :0: 0.0.0: 0.050: 0000 0: 00: :0.000:000 :0 000.:0::000000 >:0:0.0 . .00. .02 .:.0 H 0. :: .0000 H 000.0 .0000 H 0.000: 0 0.0.2 .00. .00. 0.0 H 0.0: .:0:.0 H :0>.: 3.000 H 0.000: 0 .:0. :0 >00 .00 H 0.0: .0050 H 000.0 .0000 H 0.0 :00 0 0 >00 00.0 .00. .00. .0: H 0.:. .0 H 000.0 .:.0 H 0.0 N .055. .00. .:0. .00 H 0.00 .0000 H 000.0 .00 H 0.0 0 L0 3 >00 .>.: H 0.00 .:000 H 000.0 .0: H 0.0 0 0 >00 .6008. 0 00.0... :0: 3.050 003 "3.0.0.0003 0~.0 :00E:00:: .030:00:.>> ..0:00. >000 00.:0::000000 00050 >000 0... 0.0E00 .030:00:.>> :0 >00 :0.000:000 mI >000-0_00>> >:0:0.0 :0.:n. .00.:00 .0>>0:00:.>> 00: :0 .:0 >00. 000 000 .0 >00. 00.05000 00: :0 0.0.0: 0.00:0: :.:00 0. :0: :000:00 000 .01. 000800 :0.000:000 :0 000.:0::000000 >000-0_00>> 000 000050 >000 .0 0.00... 76 with a body fat content of 1.5%. Analysis of the control females dying on days 18 and 21 revealed total HE body burdens of 6.3 and 1.5 ug/animal, respectively. One mortality occurred in an adult female from the 6.25 ppm heptachlor group fed the MO/R diet. This mink died on the 8th day and consumed the daily allocated amount of feed up to its death. Analysis of this mink showed a body fat content of 1.2% and a total HE body burden of 1,548.8 ug, which was equivalent to a whole-body HE concentration of 3.3 ug/g bw. There were no mortalities of kits fed the MOIR diets, nor were there any deaths among the adults and kits fed the AL diet. The efficacy of the AL and MO/R diets in enhancing the elimination of HE from adult female mink is presented in Table 3. Neither withdrawal treatment resulted in a significant decline of total HE body burdens or whole-body HE concentrations in control adult females when compared with values at day 0 of withdrawal. However, there were numerical reductions in HE body burdens of 41 and 66% for control females fed the AL or MOIR diet, respectively. The two withdrawal diets were equally effective in reducing whole-body HE concentrations and total HE body burdens in adult female mink previously fed 6.25 ppm heptachlor (Table 3). Total body burdens of HE decreased 80 and 78% for mink fed AL and the MO/R diet, respectively. Body. fat was significantly reduced among control adult female mink fed the MOIR diet, whereas control females fed AL had a nonsignificant increase in body fat content relative to body fat percentages of females at day 0 of withdrawal. Neither withdrawal diet consumed during the 21-day period had a significant 77 effect on body fat content in the 6.25 ppm females, although mink fed the MO/R diet had reduced percentages, with values comparable to those reported for control mink fed the same diet. Similar to adult female mink from the prior control group, kits (three to four months of age) whelped by dams in this group contained only trace amounts of heptachlor prior to the beginning of the withdrawal trial (Table 4). Consumption of either withdrawal diet for 21 days resulted in no significant change in whole-body HE concentrations and HE body burdens. Nonetheless, total HE body burdens decreased 75 and 70% in the control kits fed the AL and MOIR diet, respectively. Both withdrawal treatments were significantly effective in reducing whole-body HE concentrations and total HE body burdens in the 6.25 ppm kits (Table 4). The percent reduction in total HE body burden for kits fed AL or MO/R was 93 and 96%, respectively, with similar decreases in whole-body HE concentrations (95 and 96%, respectively). Therefore, the withdrawal treatments administered to the 6.25 ppm kits for 21 days resulted in an approximate 15% greater increase in HE removal compared to percent reduction values in the 6.25 ppm adult females fed these diets. Body fat content increased significantly in the developing control kits fed AL, but was significantly reduced for kits on the MOIR diet when compared to control kit percentages at day 0 of withdrawal (Table 4). Body fat was nearly 2-fold greater in the 6.25 ppm kits fed AL compared to the percent body fat in kits euthanized at the termination of the 181-day heptachlor feeding trial. Feeding the MOIR diet to the 6.25 ppm kits caused a significant decrease in body fat compared to percentages in the 6.25 ppm kits fed the AL diet. 78 .00.:0050000 05:3... 00 :0 $0.0 :0 00: ..0 .0:00.0: $0: 00.0.0:000 :0.0 .0000 . .5300... 00 8: :0.0 0.00 . .000 v 0. :00:0::.0 20:80.09. 0:0 0:0.:00:00:0 :00:0::.0 0:.>> :00E:00:: >:0:0.0 500 0000 :0: 005.00 00:00 0.002: 0000.). . 0:... 0.0.0: 00:0: :0 00.0E00 00.000 00:0: 0. 00.0 0.0E00 .0 >00 :0 0:_0> 05:: 00.8000: E0800 0. 00000::0:00 0. :00052 .:0::0 0:00:0:0 H 0000: 00 0800005 0:00 . .000: .:0 :0 05:0. 02,905.; 28... 0500008 0000080. 000 000:: 0.: 0: 00.0000 0. 00.002: 00.0 0: 0>.: E0:: 0:0.0 0000: 00050000 0:.v. . .00. .00. .:.0 H :0 .0000 H 000.0 .00 H 0.00: 0.0.2 .00. .00. .:.: H «.00 .0000 H 00.0 .00.. H 0.30 .:x :0 >00 .00 H 0. :: .0000 H 0 :0.0 .0400 H 0.000.. 0 >00 00.0 .00 +. .00. .0 : H 0 0 .0 H 000.0 .00 H 0.: 000.2 .00. .00. .0: H 0.00 .:00.0 H :000 .0: H 0.: .0... :0 >00 .00 H 0.0: .:00.0 H 000.0 .00 H :0 0 >00 22:08. 0 .00.. :0: 2.25 0.0... ....._:_E.03 506:8: 02,905.; 00:00. >000 00.:0::000000 00050 >000 w... .030:00:.>> :0 >00 :0.000:000 0: 38-29.; >:0:0.0 :000 .00.:00 .030:00:.>> 00: :0 .:0 >00. 000 000 .0 >00. 00.00.000 00: :0 0:0. x0.E 0.0-5000: 50: 0: 00:0: 0. :0: :000:00 000 .01. 000800 :0.000:000 :0 000.:0::000000 >000-0_00>> 000 000050 >000 .0 0.00... 79 However, feeding the MO/R diet to kits in the 6.25 ppm group did not significantly reduce the body fat content below that of kits from the same group euthanized after the 181-day trial. Discussion Mean daily feed consumption during the withdrawal period by adult control and 6.25 ppm female mink fed AL was approximately 30 to 50 g/day less than was measured over a 12-week period for mink in these groups during the prior 181-day heptachlor feeding trial (Crum et al. 1993). This was probably due to the seasonal increase in ambient temperature between January through March (heptachlor feeding trial) to July (heptachlor withdrawal trial). Mink are generally less active during warm temperatures, thus their feed intake decreases in response to lower energy requirements. However, when daily feed intake was computed on a body weight basis, the feed intake of the 6.25 ppm females (157 g/kg bw/day) compares very closely with intake values reported for female mink by Bleavins and Aulerich (1981). Data compiled from Crum et al. (1993) also yielded similar values; 148 and 154 g/kg bw/day for female mink fed control and 6.25 ppm heptachlor diets, respectively. The mean daily intake of 110 g/kg bw/day by the control females during the withdrawal period was still considerably below these values. The consistency in daily feed intake by the 6.25 ppm animals may reflect an attempt to regain weight that was lost from the heptachlor feeding trial (Crum et al. 1993). Unlike the pattern of daily food consumption by adult female mink from the control and 6.25 ppm groups, daily food intake by kit mink fed AL was 80 nearly identical for the two groups throughout the withdrawal period. Kit mink also consumed greater quantities of feed and more feed per unit body weight than adult female mink fed the same diet. However, this is not unusual for kit mink three to four months of age, which are still growing and developing. Restricting feed intake by 45% of the AL intake and adding 10% mineral oil to the diet caused considerable weight loss in adult female and kit mink from both the control and 6.25 ppm groups. The lack of a significant difference in body weight between the 6.25 ppm adult females fed Al. or a MO/R diet at day 21 of withdrawal was probably due to a lower body weight at day 0 for the Al. animals compared to the weight of the MO/R animals in this group. Since dietary mineral oil alone has not been shown to affect the body weight of sheep and rats (Rozman et al. 1982a; Polin et al. 1991), and feed restriction alone caused reductions in body weight of rats (Wyss et al. 1982; Polin et al. 1991; Polin, personal communication), it is believed that body weight loss of mink in this study was due to restricting dietary intake. However, it is important to note that a diet containing 10% mineral oil and restricted by 45% resulted in the greatest body weight reduction when fed to rats previously exposed to control and heptachlor diets (Polin, personal communication). Prior exposure to 6.25 ppm heptachlor did not result in an increased effect on- body weight loss in mink when administered the MOIR diet. The percent weight loss of the surviving 6.25 ppm females fed the MO/R diet was similar to that of the control females on the same diet (22 vs 25%). Although day 0 body weights were not recorded for kit mink, the day 21 body weights for control and 6.25 ppm kits administered the MOIR treatment were very 81 similar, further indicating that prior exposure to heptachlor did not cause an increased effect on body weight loss in adult female or kit mink during the withdrawal period. Experiments conducted on rats given a similar withdrawal regimen also showed no differences in body weight loss between control rats and rats previously exposed to heptachlor (Polin, personal communication) and polybrominated biphenyls (PBBs) (Polin et al. 1991). Restricting feed intake by 45% was noticeably stressful on all adult and kit mink fed the MO/R diet. As stated above, these mink consumed the entire quantity of allotted feed each day and were also hyperactive at feeding time. However, the MO/R diet which was supplemented with extra vitamins, was enough to sustain all control kits and kits previously exposed to heptachlor despite considerable reductions in body weight gain. In comparison, a basal rodent diet administered in the same manner and for the same duration as the MOIR treatment used in this study caused increased mortality in rats previously exposed to 10 and 100 ppm PBBs (Polin et al. 1991 ). In contrast, the rodent MO/R diet fed for 21 days caused no mortalities among rats previously fed 1 or 10 ppm dietary heptachlor (Polin, personal communication). Of the three mortalities that occurred among adult female mink fed the MOIR diet, two (one control and one 6.25 ppm) had the lowest body weights at day 0 of withdrawal. The body weight of the 6.25 ppm female (646 g) on day 0 of withdrawal was nearly half of its weight 26 weeks earlier at the start of the heptachlor feeding trial, while the day 0 body weight of the control female (724 g) was only slightly greater. It is evident that these mink were not in a relatively healthy condition to survive on a diet restricted by 45%. The 82 other control female that died had the highest body weight (1293 g) of the four administered the MO/R diet, but since it did not eat the MO/R diet for 17 straight days, it apparently died of innatiation. Evaluation of total HE body burdens and whole-body HE concentrations in adult female and kit mink from both prior treatment groups at day 21 of withdrawal indicated that both withdrawal regimens were equally effective in enhancing the removal of HE from mink (i.e. consumption of the MO/R diet was no more effective in eliminating HE from mink than was feeding mink AL). This contradicts the findings of Polin (personal communication) who fed heptachlor to rats for 181 days and then administered the same withdrawal treatments for 21 days. Rats fed the equivalent of an MO/R treatment had a 71 and 73% greater reduction of whole-body HE concentrations and total HE body burdens, respectively, than rats fed AL. However, experiments using younger rats with shorter prior exposures to heptachlor showed that the MOIR treatment was only slightly more effective in reducing HE body burdens and whole-body HE concentrations than was the AL diet (Polin, personal communication). The MOIR treatment administered to 6.25 ppm kit mink, also resulted in a slightly greater decrease in total HE body burdens and whole-body HE concentrations compared to the kits in the same group on the AL treatment. However, the significance of these comparisons are limited by the small number of samples analyzed in the present study. Overall, removal of HE was more successful in kit mink than in adults, particularly in the 6.25 ppm kits which experienced a near 100% reduction in total HE body burden and whole-body concentration of HE. This may have 83 been due to the shorter exposure duration of the kits to heptachlor prior to the withdrawal period, whereas the adult females had been consuming 6.25 ppm heptachlor for 181 days before withdrawal. Polin (personal communication) showed that rats exposed to heptachlor for shorter durations prior to withdrawal also had greater reductions in HE compared to rats exposed for longer periods before withdrawal. However, the short-term experiment used younger rats not fully developed, while the long-term study was conducted with mature rats. Therefore, it is also conceivable that natural growth and developmental changes occurring in kits during the withdrawal period of the present study could have resulted in larger HE reductions compared to adults. Consumption of either withdrawal diet by the 6.25 ppm kits decreased whole-body HE concentrations below concentrations determined at birth in kits whelped from dams fed 6.25 ppm heptachlor prior to and during the reproductive period (Crum et al. 1993). However, total HE body burdens were still much greater than the body burdens of newborn kits, but were comparable to amounts in kits from this group at three weeks of age. This suggests that HE exists as a labile pool within the bodies of kit mink, and that the magnitude of adverse effects, such as decreased postnatal growth (Crum et al. 1993). could be reduced in a relatively short time span simply through consumption of a heptachlor-free diet. Although consumption of the MOIR diet caused considerable reductions in body fat content over the 21-day withdrawal period in adult female and kit mink from both prior treatment groups, lower body fat percentages could not be associated with increased reductions in HE body burdens or whole-body HE 84 concentrations. In evaluating all withdrawal treatment combinations of O, 5 and 10% mineral oil (MD) with O, 15, 30 and 45% feed restriction (FR), Polin (personal communication) also found no consistent relationship between body fat content and HE body burdens or whole-body HE concentrations in rats fed heptachlor prior to withdrawal. However, it was demonstrated that the largest elimination of HE and P833 (Polin et al. 1991) occurred in rats fed 10% MO combined with 45% FR, which also had the lowest body fat percentages among all the groups tested. Since mink displayed no substantial differences in HE body burdens or whole-body HE concentrations whether fed the AL or MO/R diet, but had considerably different body fat percentages, these data suggest that mink respond much differently to the MO/R diet than rats after prior exposure to heptachlor. Therefore, restricting dietary intake to mobilize fat-laden HE residues does not appear necessary to achieve substantial removal of the HE from mink. SUMMARY The subchronic and reproductive effects of heptachlor were investigated during a six-month feeding period in adult female mink. Immediately following this study, a 21-day experiment was conducted to determine if a heptachlor- free diet containing 10% mineral oil and fed at 45% of ad Iibitum intake would enhance the elimination of heptachlor epoxide from adult female and kit mink exposed to heptachlor in the first study. Consumption of 25 ppm heptachlor was highly toxic to adult female mink causing 100% mortality within 88 days. Substantial mortalities also occurred among adult females fed 12.5 ppm heptachlor, with the majority of deaths transpiring during the reproductive period. As a result, an (assessment of the reproductive effects of feeding these two dietary treatments could not be determined. Of the kits that were whelped by dams in the 12.5 ppm group, nearly half were stillborn. Feeding adult female mink 6.25 ppm heptachlor prior to breeding and during the reproductive period caused no adverse effects on whelping success and survival of newborn kits, and consumption of this dietary concentration was generally non-toxic. Although the results are somewhat conflicting in the literature, rats generally appear more sensitive than mink to heptachlor in terms of reproductive effects since similar concentrations fed to rats have caused decreased survival among newborns. Although consumption of 6.25 ppm heptachlor did not affect the ability of adult female mink to produce viable offspring, postnatal exposure of kits to HE through lactation resulted in decreased kit survival and growth. Postnatal 85 86 growth and survival were also reduced in the 12.5 ppm kits. These results are supported by the fact that total HE body burdens at three weeks of kit age were considerably greater than at birth, indicating that lactational transfer of HE to kits is more significant than placental transfer to the fetus. This has also been demonstrated with other chlorinated hydrocarbons in mink. Administering a heptachlor-free diet containing 10% mineral oil and restricted by 45% of ad libitum intake for 21 days to adult female and kit mink previously fed 6.25 ppm heptachlor, resulted in a significant reduction in total HE body burdens and Whole-body HE concentrations. 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