PLACE IN RETURN BOX to remove this checkout from you: record. TO AVOID FINES retum on or before dds duo. DATE DUE DATE DUE DATE DUE 21v V‘s p‘ :Ete 9,1); ll MSU I: An Aflimmlvo Adlai/Equal Opportunity Indltunon cmmG-pd EFFECTS ON REPRODUCTION OF RANCH MINK FED CARP FROM SAGINAW BAY, MICHIGAN BY Sylvia N. Heaton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Sciences 1992 1 t --J - 566 7” A / 0 ABSTRACT EFFECTS ON REPRODUCTION OF RANCH MINK FED CARP FROM SAGINAW BAY, MICHIGAN BY Sylvia N. Heaton Chemical contamination of fish food sources has been implicated as a cause of declining populations of wild mink and otter throughout the Great Lakes region. The objective of this study was to determine the effects of contaminated prey species on the survival and reproductive performance of ranch mink. Carp collected from Saginaw Bay, MI, containing 8.4 mg/kg total polychlorinated biphenyls (PCBs) , as well as other contaminants, were substituted for marine fish at levels of 0, 10, 20, and 40% of the diet. Experimental diets were fed to both male and female mink beginning seven weeks prior to breeding and continuing until the young were seven weeks old. The diets containing Saginaw Bay carp caused impaired reproduction and/or reduced surviVal of kits. Females fed 40% Sagniaw Bay carp whelped the fewest number of kits, all of which were stillborn or died within 2 4 hours . To Lyle, Zachariah and Emily, with love. ACKNOWLEDGEMENTS I extend my most sincere thanks and admiration to my major professor, Dr. Richard Aulerich, for his endless giving of help, encouragement, support, guidance, and above all, patience, throughout my graduate and professional career. I also extend my sincere thanks to the remaining members of my graduate committee: Drs. Steven J. Bursian, Harold H. Prince and W. Richard Dukelow. I am also deeply indebted to Dr. qohn Giesy for providing hours of guidance, support and encouragement throughout the preparation of this thesis. In addition, I would like to thank Dr. Don Tillitt and staff of the Presticide Research Center, as well as Timothy J. Kubiak and Robert Sills, for providing technical expertise. I extend a warm embrace and thanks to Phil Summer, Angelo Napolitano and Chris Bush for their help in caring for the animals. I wish to thank the Lansing and Clare District Fisheries staff for their assistance and cooperation in collecting the 3,000 pounds of carp! And finally, a sincere thanks to Ms. Carol Daniel for her friendship, help, and hospitality throughout my graduate career. ii TABLE OF CONTENTS LIST OF TABLES.............................................v LIST OF FIGURES............................. .......... ..viii LIST OF APPENDICES.........................................X INTRODUCTION....... ...... ..................................1 LITERATURE REVIEW..........................................4 Chemistry of PCBs.....................................4 Production and Use of PCBs............................9 Environmental Fate of PCBs...........................11 TCDD-Equivalents.....................................13 Human Exposure to PCBs...............................17 Laboratory Animal Exposure to PCBs...................18 Great Lakes PCB Contamination........................19 Wildlife Exposure to PCBs............................22 Effects of PCBs on Mink.......... ..... ...............23 MATERIALS ANDMETHODSOOOOOOOOOOOOOOOOOOOOO00.000.00.00000026 Fish Collection......................................26 Diet Preparation.....................................26 Chemical Analyses....................................32 Experimental Design and Animal Care..................36 Feeding Trial........................................36 Reproduction.........................................39 Blood Collection.....................................40 Necropsies...........................................41 Chemical Analyses of Liver Tissue....................42 Histopathology.......................................42 Data Analysis........................................43 RESULTSOOOO0.0.000000000000000000000000000000000000.000000044 Chemical Residues in Raw Carp........................44 Nutrient Analyses of Diets..... ..... .................44 Feed Consumption.....................................50 Chemical Residues in Diets...........................53 Adult Body Weights...................................61 Adult Mortality......................................61 Reproduction.........................................65 Kit Organ Weights....................................72 Adult Organ Weights..................................79 Histopathology.......................................79 Concentrations of PCBs and TCDD-EQs in Liver.........82 Hematologic Parameters...............................96 Thyroxine Profiles...................................96 NOAEL, LOAEL, and Reference Doses....................98 iii DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O O O O O O ..... O O O O O O O O O O O O O O O .102 SUMMARY AND CONCLUSIONS............. ..... ................131 APPENDICESOOCOOOOOOOOOOOOOOOOOOOO0.0...0.00.00.00.0000000132 Appendix A. Calculations of feed, PCB and TCDD-EQ consumption............................132 Appendix B. Total PCB concentrations in individual mink liver samples.....................133 Appendix C. Calculations for H4IIE-derived TCDD-EQs for individual female mink livers......135 BIBLIOGMPHYOOOOOOOOOOOOOOOOOOOOOO0.0.0.0....0.00.00.00.0137 iv Table 1. 8. 9. 10. 11. 12. 13. 14. LIST OF TABLES Page General physical properties of various Aroclors® ..... 10 Thiaminase activity of some common freshwater fish......................................... ........ 29 Composition of experimental diets............. ....... 31 Concentrations of PCBs in raw Saginaw Bay carp.......45 Average concentrations of individual congeners found in raw carp from Saginaw Bay.............. ..... 46 Average dietary concentrations of TCDD- EQs (pg/g) in raw carp from Saginaw Bay as calculated from TEF1 values derived using the H4IIE rat hepatoma bioassay...................... ..... ..................47 Average dietary concentrations of TCDD-EQs (pg/g) in raw carp from Saginaw Bay as calculated from TEF1 values derived using the H4IIE rat hepatoma bioassay.............................................48 Nutrient analysis of experimental diets..............49 Mean feed consumption of adult female mink fed various percentages of Saginaw Bay carp..............51 Mean feed consumption of adult male mink fed various percentages of Saginaw Bay carp..............52 Total PCB and H4IIE-derived TCDD-EQ consumption for female mink fed various percentages of saginaw Bay carPOOOOO0.0.00......OOOOOOOOOOOOOOOOO0.054 Total PCB and H4IIE-derived TCDD-EQ consumption for male mink fed various percentages of saginaw Bay carp...IOOOOOOOOOOIOOOOOOOOO...00.0.0000055 Average dietary concentration of TCDD-EQs (ng/kg diet) for each congener and as a total in diets of mink fed Saginaw Bay carp as estimated by congener specific analysis..............56 Average dietary concentrations of TCDD-EQs (ng/kg diet) for each congener and as a total in diets of mink fed Saginaw Bay carp as estimated by congener specific analysis..............57 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Average daily dietary dose1 of TCDD-EQs (ug/kg body weight/day) in diets of mink fed Saginaw Bay carp as estimated by congener specific analysis....................................59 Average daily dietary dose1 of TCDD-EQs (ug/kg body weight/day) in diets of mink fed Saginaw Bay carp as estimated by congener specific analysis....................................60 Mean body weight, body weight changes and percent body weight loss for female mink fed various percentages of Saginaw Bay carp........... ........... 62 Mean body weight, body weight changes and percent body weight loss for male mink fed various percentages of Saginaw Bay carp............... ..... ..63 The effect of feeding PCB-contaminated carp on breeding inmink.0.0...00....OOOOOOOOOOOOOOOOOOOO0.0.68 Reproductive performance of female mink fed various percentages of Saginaw Bay carp............. ......... 69 Average body weights of mink kits whelped and raised by dams fed various percentages of Saginaw Bay carPOOOOOOOCO0.0.0.0...OOOOIOOOOOOOOOOOOOOOOO00.070 Mean body and organ weights of six-week-old kits whelped and nursed by female mink fed diets containing Saginaw Bay carp..........................78 Mean organ weights expressed as percent brain weight of adult female mink fed diets containing various percentages of Saginaw Bay carp..............80 Incidence of histological changes in livers of mink fed diets containing varying percentages Of saginaw Bay carPOOOOOOOOOOOOOOOOOOOCOOOOOOO0.0.0.081 Treatment means for total PCB and H4IIE-derived TCDD-EQ (pg/g) concentrations in female mink livers...0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOO0.0.0.0...92 Concentrations of PCBs and TCDD-EQs and relative potencies of feed and liver tissue of female mink fed Saginaw Bay carp.................................95 Hematologic values of female mink fed diets containing various percentages of Saginaw Bay carp...97 vi 28. 29. 30. 31. 32. 33. 34. 35. Mean thyroxine (T4) and triiodothyronine (T ) concentrations in blood serum of female mink fed diets containing various percentages of Saginaw Bay carp.............................................99 NOAEL, LOAEL and reference concentrations of total PCBs determined as Aroclors® 1248, 1254 and 1260.00.00.00... .......... 000000000000 ...... 00000000100 NOAEL, LOAEL and reference concentrations of TCDD-EQs as determined by the H4IIE rat hepatoma cell bioassay in female mink................... ..... 101 Average residue concentrations of selected pesticides in carp collected from Saginaw Bay, MiChigan, 1990.0. 00000 0000.00.00.000000000000 00000000 103 Average PCB concentrations in tissues of wild mink reported in the literature. ("*" denotes mink body homogenates; "**" denotes liver tissue.).......120 A summary of PCB residues in liver and adipose tissue and corresponding reproduction data from mink fed various dietary PCB concentrations... ...... 122 Maximum Allowable Daily Intake (MADI) levels and Exceedance Values (EV) of various Great Lakes fish speCies000.0...0.0.0.000.0.0000.00.0.000000000000000126 Summary of the percentages of fish comprising the total diet of wild mink reported in the literature..129 LIST OF FIGURES Figure Page 1. Number of possible isomers of PCBs (taken from cairns et al.' 1986)0.0.0.00000000000.0.00....00..00.06 2. Chemical structures of PCB, DDT, dieldren and aldrin000.00.00.00.0000000000.00...00.0.000000000 00000 8 3. Chemical structure of 2,3,7,8-tetrachlorodibenzo- p-dioxin (2,3,7,8-TCDD)..............................15 4. The 42 Areas of Concern identified in the Great Lakes basin (modified from Government of Canada, 1991)0.000.000.0000000..0...0..00...00..0.00.0..0.00.21 5. Site of fish collections in Saginaw Bay, Michigan....28 6. Sunrise and sunset times at Lansing, Michigan, Eastern Standard Time (Nautical Almanac Office, United States Naval Observatory, Washington, D.C0)00.0.0.0000.0 ..... 0. 00000000 00000000.... 00000000 38 7. Ten "normal" fetuses from a female mink fed 10% Saginaw Bay carp.....................................67 8. Percent survival of mink kits at birth, three and six weeks of age. Numbers above the bars represent the average percent survival for the group...........73 9. Mink kit (right) whelped by a female fed 40% Saginaw Bay carp. Note edema in the head and neck region. Control kit (left).....................75 10. "Normal" control mink kit (above) and a stillborn kit whelped by a female fed 40% Saginaw Bay carp (below). Note the small size, deformed body and reduced mandible and maxilla.........................77 11. Mild periportal hepatocellular lipidosis 12. characterized by vacuolization of hepatocytes (A) observed in the liver of a female mink fed a 40% carp diet (100x, stained with hematoxylin and GOSin)0.0.0.000000000......00000000.0...000.0.0..084 Section of liver from a female mink showing no histological alterations. Hepatocytes are normal appearing with very little lipid accumulation (A). Sinusoids appear free of congestion (B). (100x, stained with hematoxylin and eosin)..................86 viii 13. 14. 15. 16. Moderate scattered portal lymphocytic infiltration of the liver (A) that was evident in several mink fed the 20% carp diet (400x, stained with hematoxylin and eosin)....................... ....... .............88 Marked congestion and mild, diffuse hepatocellular vacuolar lipidosis observed in the liver (A) of a female mink receving 40% carp in the diet (400x, stained with hematoxylin andeosin)............90 Relationship of total PCB concentrations to TCDD-EQ concentrations in livers of mink fed either a 0% (control), 10%, 20% or 40% Saginaw Bay carp diet..................... ................... 94 Michigan Department of Natural Resources native fish collection sites in the Great Lakes and connecting channels, 1989-1990 (taken from MDNR, 1991)................. ........ ...... ........ ........125 ix LIST OF APPENDICES Appendix Page A. Calculations of feed, PCB and TCDD-EQ consumption.................................. ...... .132 B. Total PCB concentrations in individual mink liver samples.00.000.00.00000000000000.0.000 00000000 133 C. Calculations for H4IIE-derived TCDD-EQs for individual female mink livers.............. ......... 135 INTRODUCTION Polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), collectively termed planar chlorinated hydrocarbons (PCHs), have sparked a myriad of controversies and concerns pertaining to their chronic effects in the environment. Increased concern of global contamination by chlorinated compounds has resulted in an extensive and comprehensive literature base on the physical and chemical properties of PCBs as well as their toxicity. Of particular concern are the polychlorinated biphenyls (PCBs), which, because of their environmental and thermal stability, resistance to oxidation, and tendency to bioaccumulate, have become widespread and persistent contaminants in terrestrial and aquatic ecosystems (Veith and Lee, 1970; Kalmaz and Kalmaz, 1979; Zimmerman, 1982). Environmental samples containing PCBs have been collected from England, Scandinavia, the Netherlands, Antarctica, the United States, Australia and Central America; thus making them truly ubiquitous pollutants (Koeman et al., 1969; Bagley et al., 1970; Gustafson 1970; Richardson and Waid, 1982). Significant concentrations of PCBs have been measured in nearly all abiotic and biotic compartments of the ecosystem including air (Delfino 1979; Eisler 1986), water (Nadeau and Davis, 1976; Rice et al., 1983; Swackhammer and Armstrong, 1987), sediments (Nadeau and Davis, 1976), fish 1 2 (Kleinhart et al., 1978; Copel and Eisenreich 1985), birds (Eisler 1986; Weseloh et al., 1989) freshwater and terrestrial mammals (Norheim, 1978; Bleavins et al., 1982b; Eisler, 1986; Foley et al., 1988), marine mammals (Gaskin et al., 1983; Martineau et al., 1987), plants (Klein and Weisberger, 1976; Anderson et al., 1982) and macroinvertebrates (Novak et al., 1988). Collectively, the Great Lakes basin is the largest body of freshwater in the world. In a publication reviewing the current Great Lakes contaminant issue, the author writes that the "production and release of chemicals associated with high densities of population and industry in the Great Lakes basin has led to widespread concern over the impact of human activity on the ecosystem and the implications of a contaminated ecosystem for health" (Government of Canada, 1991). These concerns have led to the nominations of several wildlife species or species groups as surrogates by both the scientific and regulatory communities (Kubiak and Best, 1991). These surrogates are considered to be indicator species for Great Lakes water quality. Among these species are the mink and otter. 3 The primary objective of the present study was to test the hypothesis that mink would be adversely affected by dietary exposure to contaminants in fish from Saginaw Bay. To fulfill this objective, the primary goal was to: 1) determine the PCH threshhold concentration where adverse reproductive effects would not be expected to occur by determining a dose-response relationship. The threshhold concentration could then be used to predict whether or not current concentrations of contaminants in either the food source or tissue of wild animals present a hazard to wild mink populations. LITERATURE REVIEW Chemistry—mg Polychlorinated biphenyls, systematically called 1,1'- biphenylchloro-derivatives (Ballschmiter et al., 1989), have the empirical formula C12H10-nCln with n ranging from 1-10. Theoretically, 209 different PCB congeners can be formed through partial or total chlorination of biphenyls (Figure 1). Approximately 25 of these account for 50-75% of the total mass of PCBs found in environmental samples (McFarland and Clarke, 1989). Chlorination of the biphenyl ring makes PCBs very similar in chemical structure to many of the chlorinated pesticides such as DDT, dieldrin and aldrin (Figure 2). Pure forms of PCBs are colorless crystals. However, commercial products may be represented as either liquids, if they contain a lower percentage of chlorination, or as viscous and even solid materials at higher degrees of chlorination (Burke and Fitzhugh, 1970; Ballschmiter et al., 1989). Electrophilic substitution in PCBs is favored by ortho and para positions, although commercial products as complex mixtures of isomers and congeners have no positional preference for halogen substitution (Hutzinger et al., 1974). Figure 1. Number of possible isomers of PCBs (taken from Cairns et al., 1986). 4 4' 5 6 6' 5' Chlorine Number of Substitution Possible Isomers Mono - 3 Di - . 12 Tri - 24 Tetra - 42 Penta - 46 Hexa - 42 Hepta - 24 Octa - 12 Nona- ' 3 Deca - 1 Figure 2. Chemical structures of PCB, DDT, dieldrin and aldrin. Cl ‘ 0Q?©q 0:4 ~01 ‘3' DOT CI CI CCRCHZO CI DIELDRIN Cl ALDRIN r ° U C s The first documented synthesis of PCBs was reported in 1881 by Schmidt and Shull as cited by Eisler (1986) and Tanabe (1988). Commercial production of PCBs began in 1929 by the Swann Corporation, which later became part of the Monsanto Company (Risebrough and Brodine, 1970). Production of commercial mixtures of PCBs peaked in 1970. In total, 1.5 million metric tons were produced between 1929 and 1979 (DeVoogt and Brinkman, 1989). In 1977, Monsanto ceased its production of PCBs. The Environmental Protection Agency, under the Toxic Substances Control Act, mandated that all 0.8. sales and distribution of PCBs end by July 1 of 1979 (Langford, 1979). Commercial mixtures of PCBs produced in the United States were given the trade name Aroclor® and designated by four digit numbers indicating the type of molecule and the chlorine content. For example, the first two digits of Aroclor0 1242 represent the molecular type - a chlorinated_ biphenyl, and the last two digits indicate 42% chlorine by weight. _However, not all Aroclors followed this convention (e.g., Aroclor0 1016). The chlorine content of the individual Aroclor mixtures varies from 10-70 percent by weight with physical properties varying with chlorine content (Table 1). PCBs exhibit a wide range of desirable chemical and physical properties that were quickly recognized as being particularly well suited for industrial use. Aroclors were 10 Table 1. General physical properties of various AroclorsO1 Vaporization Aroclor® Form Specific DR2 Rate No. Gravity (g/cmz/hr)3 1221 Clear, mobile Oil 1.182-1.192 275-320 0.00174 1232 Clear, mobile oil 1.270-1.280 290-325 0.000874 1242 Clear, mobile oil 1.381-1.392 325-366 0.000338 1248 Clear, mobile Oil 1.405-1.415 340-375 0.000152 1254 Light yellow viscous oil 1.495-1.505 365-390 0.000053 1260 Light yellow, . soft sticky resin 1.555-1.566 385-420 0.000009 1262 Light yellow, soft sticky resin 1.572-1.583 395-425 --- 1268 White to off- white powder 1.804-1.811 435-450 ---- 1 Source: Cairns et al., 1986. 2 DR = Distillation range. 3 Measured at 100°C. 11 first used in the industrial setting as dielectric fluids in transformers. After World War II, their use broadened to include capacitors, heat transfer systems and open systems including printer's ink, natural and synthetic rubber, fabric and paper coatings, brake linings, paints, varnishes, waxes, asphalt, adhesives and resins (Risebrough and Brodine, 1970). Environmental Fate of PCBs The identification of PCBs in environmental samples was first reported in 1966 by a Swedish scientist, Soren Jensen of the Institute for Analytical Chemistry at the University of Stockholm. Gas chromatographic (GC) analysis of eagle feathers dated as far back as 1944, for the insecticide DDT, showed the presence of several unknown peaks, determined to be PCBs, which interfered with the quantitative determination of the contaminated samples (Jensen, 1966). Since that time, a tremendous amount of research has been conducted on PCBs to document their molecular structures and intricately related biological activities (Bitman and Cicel, 1970; Cook, 1972; Tanabe et al., 1987; Safe, 1990). Unfortunately, the properties that made the chlorinated compounds industrially useful are the same properties that cause them to continually persist in the environment. Their resistance to oxidation and other types of chemical degradation have caused PCBs to persist in the environment 12 while solubility in non-polar solvents contributes to their absorption into fatty and liver tissues of exposed organisms (Gustafson, 1970). The PCB congeners with higher chlorination are more tightly bound in soils and sediments, making them generally less bioavailable. The penta-, hexa- and hepta- chlorobiphenyls are considered to be moderately chlorinated PCBs and tend to bioaccumulate to the greatest degree. The less chlorinated congeners are metabolized and eliminated more readily by organisms, thus, they bioaccumulate to a lesser degree (Safe et al., 1982; Bush et al., 1985). Because of the high lipophilic characteristic of PCBs in aquatic systems, their content in fish is an effective indicator of PCB contamination of aquatic ecosystems. This characteristic poses potential hazards.to humans and wildlife. However, evaluation of the potential impact of PCB mixtures on the environment is extremely complicated, since the individual congeners differ in physical, chemical and biological properties. Subtle differences between chlorobiphenyls are further amplified by differences among various animals in absorption, distribution, biotransformation and excretion of individual PCB isomers (Hansen et al., 1983). 13 - 'va e PCBs are also approximate isostereomers of PCDDs and PCDFs, which have no known industrial uses, but are present as contaminants in complex mixtures of chlorinated compounds in environmental samples (Birnbaum et al., 1985; Tillitt et al., 1991). These samples could theoretically contain up to 75 different PCDD congeners (Safe, 1987; McFarland and Clarke, 1989). This results in difficulty in evaluating whole complex PCH mixtures, since individual congeners have different toxic potencies and may elicit complex interactions of synergism, antagonism and additivity (Poland and Knutson, 1982; Tillitt et al., 1991). The most toxic PCB and PCDD congeners are those that are chlorinated in the lateral positions and can assume planar or nearly planar configurations (Greenlee and Neal, 1985; McFarland and Clarke, 1989) similar to that of the most potent PCDD congener, 2,3,7,8-tetrachlorodibenzo-p- dioxin (2,3,7,8-TCDD) (Poland et al., 1976; Niimi and .Oliver, 1989). The chemical structure of 2,3,7,8-TCDD is shown in Figure 3. The more toxic PCB, PCDD, and PCDF congeners have chlorine substitution in either three or four of the lateral benzene ring positions. These include the 2,3,7 and 8 positions for PCDDs and PCDFs, and the 3,3' (meta) 4,4' (para) and 5,5' (meta) positions for the PCBs (Poland and Knutson, 1982; Leece et al., 1985; Safe et al., 1985; McFarland and Clarke, 1989; Safe, 1990). These non- ortho, planar, chlorine-substituted congeners are the most 14 Figure 3. Chemical structure of 2,3,7,8-tetrachlorodibenzo- p-dioxin (2,3,7,8-TCDD). Cl 15 C1 C1 16 toxic isomers and elicit biological and toxicological responses similar to 2,3,7,8-TCDD, including immunotoxicity, teratogenicity and embryotoxicity (Safe, 1984; 1990). Due to the near mathematical and financial impossibility of experimentally determining the biological effects of these complex chemical mixtures, recent studies have concentrated efforts in evaluating the toxic potencies of planar hydrocarbons relative to the toxic response elicited by 2,3,7,8-TCDD (Kannan et al., 1988; Tillitt et al., 1991). Numerous studies have shown that the toxic properties of dioxin-like, planar, halogenated compounds are expressed by a common mode of action (Bradlaw and Casterline, 1979; Eadon et al., 1986; Safe, 1987; 1990). One of the most well characterized biochemical responses to PCHs is the induction of microsomal monooxygenase activity whereby a complex environmental mixture induces the activity of cytochrome P- 450-requiring enzymes, such as ethoxyresorufin-o-deethylase (EROD) (Bradlaw et al., 1980; Bandiera et al., 1984; Zacharewski et al., 1989; Tillitt et al., 1991). Recent studies have focused on the use of H4IIE rat hepatoma cells as an integrative predictor of the toxic potency of complex mixtures of PCBs (Sawyer and Safe, 1982; Mason et al., 1986; Zacharewski et al., 1989; Tillitt et al., 1991). The bioassay has been proposed for use as a screening technique to assess the relative in vitro toxic potencies of individual PCBs and complex environmental 17 samples relative Osimilar responses observed with TCDD (Bradlaw and Casterline, 1979; Mason et al., 1986; Tillitt et al., 1988). The H4IIE bioassay determines TCDD equivalents (TCDD-EQs) for Complex mixtures of PCBs by measuring EROD activity induced by PCHs in rat hepatoma H4IIE cells. The magnitude of the response is expressed relative to induction observed using TCDD (Casterline et al., 1983; Safe, 1990; Tillitt et al., 1991). Several studies have demonstrated the use of the H4IIE rat hepatoma cell bioassay for deriving TCDD-EQ concentrations using extracts from the rat, guinea pig, colonial water bird eggs and Great Lakes fish as models (Safe, 1987; Holcomb et al., 1988; Tillitt et al., 1988; Zacharewski et al., 1989). Human Exposure to PCBs . Consumption of PCB-contaminated fish in Michigan is cited as being the most important source of PCBs in human breast milk (Schwartz et al., 1983). Breast milk samples from 1,057 nursing Michigan mothers contained PCB residues of up to 5.1 ppm (Wickizer et al., 1981). Physical and behavioral effects have been reported in infants of mothers who have consumed contaminated fish from Lake Michigan. These mothers consumed from 1.2 to 41.7 kg of fish per year (Fein et al., 1984). Positive correlation exists between the number of fish meals consumed and concentrations of PCBs in sera of Wisconsin anglers (Fiore et al., 1989). In general, PCBs in the human population are probably a result 18 of life-long exposure (Wickizer et al., 1981; Mes, 1987), but consumption of fish is a major source of PCBs. o o A ' a u 0 CBS Exposure of laboratory animals to PCBs has been known to cause delayed reproduction, reproductive failure, birth defects, skin lesions, tumors, thymic atrophy, liver disorders, behavioral changes, histopathological lesions and alterations, body weight loss, decreased food consumption, and, in more sensitive species, death ( Aulerich et al., 1970; Barsotti et al., 1976; Eisler, 1986; Gillette et al., 1987a). However, a review of the extensive literature base pertaining to the effects of PCBs on animals shows that different commercial mixtures of PCBs elicit different toxic responses and that different animal species vary in their sensitivities to the toxic effects of PCBs. This is due to differences in absorption, distribution, biotransformation and excretion of individual PCB congeners among species (Hansen et al., 1983). For example, a dietary dosage of 25 mg/kg Aroclor0 1254 fed to nine-month-old mallards for approximately one month prior to egg-laying caused no detrimental effects on reproductive success or nest attentiveness (Custer and Heinz, 1980). However, Aroclor0 1242 at 10 mg/kg fed to chickens reduced egg production and hatchability, and caused eggshell thinning (Keplinger et al., 1971). Kimbrough et al. (1978) provides a comprehensive review of animal toxicity caused by PCB 19 exposure, which discusses among-species differences in response to PCBs. s m' o The late 1960's and early 1970's brought about increased environmental awareness and better laboratory analytical capabilities. These two changes, one societal and the other scientific, resulted in the discovery of widespread organochlorine contamination throughout the Great Lakes region (Delfino, 1979). It was for this reason that the United States and Canada signed the first Great Lakes Water Quality Agreement (GLWQA) which established objectives and criteria for the restoration and enhancement of water quality in the Great Lakes (Michigan Department of Natural Resources, 1988). Under the auspices of the International Joint Commission, an agency established to govern the rights and obligations of the U.S. and Canada regarding their common boundary waters, designated Areas of Concern (AOC) were established that defined problem areas where water pollution had caused degraded environmental and water quality conditions (Figure 4). According to the International Joint Commission, toxic chemicals have become the primary water quality-based issue in the Great Lakes basin (Delfino, 1979). Insecticides such as DDT, aldrin, dieldrin and endrin, and industrial chemicals such as PCBs, PCDDs and PCDFs are the most significant organic contaminants in biota of the Great Lakes. 20 Figure 4. The 42 Areas of Concern identified in the Great Lake basin (modified from Government of Canada, 1991). 21 --------- Canadaand USA Bou‘idily L'no ArcaolConccm Michigan Saginaw Bay . AOC 22 The most significant current versus historic source of PCB contamination in the Great Lakes is atmospheric deposition. However, once in the water column, partitioning of PCBs occurs into the more non-polar compartments of the ecosystem, due to the high lipophilic nature of this type of chlorinated organic compound (Eisler, 1986). The greater bioaccumulation of PCBs into some species of Great Lakes fish has led to fish consumption health advisories for many waters in the basin (Michigan Department of Natural Resources, 1991). These advisories have been issued in 36 of the 42 AOCs (Government of Canada, 1991). The current concern level for PCBs in Great Lakes fish in Michigan is 2 mg/kg as established by the Michigan Department of Public Health (Michigan Department of Natural Resources, 1991). A further discussion of PCB residues in Great Lakes fish and the risk posed to wildlife is presented in the Risk Assessment section of this thesis. An historical review of PCB toxicology in the Great Lakes region can be found in -.7 1‘". - - '1 he G_ea Lakes :12 - s- 7: ‘1 ‘ s, Volumes I and II (Government of Canada, 1991). Wildlife Exposere to BCBs Widespread environmental contamination in both the aquatic and terrestrial ecosystems has been implicated as a cause of reproductive dysfunction and mortality in certain. fish and wildlife species. Reproductive failure, and embryonic and fry mortality in Lake Michigan lake trout have 23 been associated with the exposure of chlorinated hydrocarbons, such as PCBs and DOE (Mac et al., 1985). Mortality, decreased reproductive success and several outbreaks of disease in colonial fish-eating birds in the Great Lakes basin have been thought to be caused by PCBs and TCDD. Observations have included embryonic mortality and congenital abnormalities in herring gulls from Lake Ontario (Gilbertson, 1983) and growth retardation, parental inattentiveness and nest abandonment in Forster's tern from Green Bay, Lake Michigan (Hoffman et al., 1987; Kubiak et al., 1989). Declining otter populations along the lower Columbia River in Oregon have been associated with elevated PCB concentrations in liver tissue of otter from this area (Henney et al., 1981). In addition, liver tissue of mink from the same area were at levels which have been associated with kit mortality in ranch-raised mink (Platonow and Karstad, 1973). Excellent reviews of effects on fish and .wildlife species exposed to chlorinated contaminants can be found in the review papers of Gilbertson (1989), Gilbertson et a1. (1991) and Wren (1991). Effeete_2f_£§§§_on_uink PCBs and other organochlorines have been implicated as causative agents in the decline of wild mink and otter populations throughout the world (Chanin and Jeffries, 1978; Kenny et al., 1981; Olsson et al., 1981; MacDonald 1983; 24 Mason et al., 1986). A review of mink feeding studies involving the dietary exposure of mink to commercial PCB mixtures has shown that these animals are highly sensitive to chlorinated compounds (Ringer et al., 1972; Platonow and Karstad 1973; Ringer et al., 1981; Hornshaw et a1, 1983; Aulerich et al., 1985). Reproductive complications and excessive kit mortality in ranch mink fed Great Lakes fish were first reported by Hartsough (1965). Feeding trials using coho salmon from Lake Michigan tributaries resulted in kit mortalities as great as 80% although the cause of the problem was undetermined (Aulerich et al., 1971 and 1973b). Aulerich and Ringer (1977) conducted feeding experiments using various species of Great Lakes and marine fish in order to investigate the cause of reproductive failure seen in previous studies. Results indicated that the presence of PCBs in the Great Lakes fish species and the environment from which they were obtained were directly related to the degree of reproductive impairment in the ranch mink. ‘ According to literature reports, reduced reproductive success in ranch mink fed diets containing various percentages of Great Lakes fish and fish by-products occurred at PCB concentrations below those found in diets containing commercial PCB mixtures (Hornshaw et a1, 1983). These results suggested that once PCBs underwent alteration 25 due to biological processes, their degree of toxicity may have been altered due to changes in the ratio of the congeners (Platonow and Karstad, 1973). Mink are useful indicator species for the effects of contaminants in the aquatic environment. Along with being semi-aquatic animals, mink are carnivores at the top of the food chain. This results in their accumulation of pesticides, PCBs, heavy metals and high molecular weight compounds found in prey of lower trophic status (Somers, 1985). The localized habits of wild mink, unlike those of avian species, could allow monitoring of PCB residues that would result in unique contaminant profiles of individual mink (Somers, 1985). However, the secretive nature and sparse distribution of mink, coupled with insufficient data on population trends and productivity, makes population monitoring difficult (Harris, 1988). Although mink are distributed widely throughout the Great Lakes region, only crude estimates exist pertaining to harvest location. The problem of population estimates is confounded by changes in the intensity of trapping and the value of pelts (Government of Canada, 1991). MATERIALS AND METHODS lle 'o Carp (Cyprinus carpio) were collected by electro-shock from the mouth of the Saginaw River near the Confined Disposal Facility of Saginaw Bay, Michigan, in December, 1988 (Figure 5). The fish were taken from depths ranging from 1-6 feet. Approximately 3,000 pounds of carp were collected and transported to the Michigan State University Experimental Fur Farm. The fish were ground and then blended for a minimum of 15 minutes in a paddle mixerl. The resulting homogenous mixture was used to obtain a single whole fish composite sample. Sub-samples of the homogenous ground carp were stored frozen in plastic bags at -5° Celsius for subsequent analysis of total PCB and TCDD-EQ concentrations. The remainder of the ground fish was stored frozen in sealed plastic containers at -5° Celsius until needed for diet preparation. Wm Before preparation of the experimental diets, the ground, raw carp was thawed and cooked in a large double boiler system to inactivate thiaminase activity, which is present in certain fish species such as carp (Table 2). Thiaminase cleaves the thiamine molecule and thereby induces 1 Weiler and Company, White Water, WI, Model 1170M. 26 27 Figure 5. Site of fish collections in Saginaw Bay, Michigan. 29 Table 2. Thiaminase activity of some common freshwater fish. Species Thiamine§e_agtixi§xz Bowfin 206 Amie calva Shad 112 Dorosoma cepedianum Smelt 47 Osmerus mordax Carp 2003 Cyprinus carpio Shiner 1418 notropis hudsonius Alewife 1533 Pomolobus pseudoharengus 1 Source: Graedinger and Krzeczkowski (1966). 2 Micrograms of thiamine hydrochloride destroyed in twenty minutes per gram of protein of the unheated raw fish. 3 Average of three values. 30 a thiamine deficiency, called Chastke's paralysis, in animals fed some species of raw fish. This disease causes severe damage to the central nervous system of ranch-raised mink and arctic fox, and can be fatal (Anglesea and Jackson, 1985; Gnaedinger, 1963). Therefore, to inactivate the thiaminase present in the carp, the ground fish was cooked for a minimum of 30 minutes, after the internal temperature reached 88° Celsius (Gnaedinger and Krzecskowski, 1966). Upon cooling, the fish was stored in the original plastic containers and refrozen until needed. A control and three treatment diets were mixed containing 0, 10, 20 and 40% Saginaw Bay carp. Each diet, consisted of a basal portion, containing water, XK-40 commercial mink cereal, raw poultry by-products and beef liver (Table 3). The fish portion of the control diet was composed of marine fish scraps. The relative proportions of marine fish and Saginaw Bay carp in the diets varied, in order to keep a constant 40% fish in the diet (Table 3). A total of eight batches of treatment diets was prepared for the 6-month exposure period. Rex® wheat germ oil2 was added to the diets (90 ml oil/100 lbs feed) during the breeding and whelping period from February 9, 1989 to May 11, 1989. After the diets were mixed, the feed was packed 2 Per 02., this wheat germ oil contained: 70 IU vitamin E, 25,000 USP Vitamin A, and 2500 IU Vitamin D (VioBin Corporation, Monticello, IL). 31 Table 3. Composition of experimental diets. DieEary greetment 0% carp 10% 20% 40% (Control) carp carp carp 0 io Saginaw Bay carp 0 10 20 40 Ocean fish scrap1 40 30 20 0 Water 25 25 25 25 Commercial mink cerealz 20 20 20 20 Raw Poultry by-products3 10 10 10 10 Beef Liver 5 5 5 5 1 Cod, haddock, pollack, flounder trimmings, GMF Brand Ocean Fresh Fish Product, C.B. Mercer Inc., Glouster, ME. 2 XK-40 mink food, XK Mink Foods, Inc., Plymouth, WI. 3 Tyson Foods, Inc., Ft. Smith, AR. 32 and stored frozen in one gallon tin containers. A single can was used as one day's ration per treatment group. 'ca ses Subsamples of the raw carp and triplicate samples of each of the diets were submitted to the Michigan State University Pesticide Research Center for total PCB residue analysis, congener specific analysis, and H4IIE bioassay derived TCDD-EQs. A 500 gram sample of each diet was also submitted to National Environmental Testing Inc., Chicago, IL, for analysis of nutritive content. The raw carp and diet samples were analyzed using standard operating procedures for residue extraction and analysis of total PCBs (Schwartz, 1982; Schmitt et al., 1985; Mora and Verbrugge, 1991). A minimum 10 gram subsample of either ground carp or prepared diet was supplemented with 2,4,6-trichlorobipheny1 (IUPAC #30; an internal standard for monitoring the condition of the chromatographic column) for sample preparation and homogenization. Lipid extraction was accomplished by placing a 1:4 mixture of NaSO4:sample (weight basis) in a 2x30 cm internal diameter chromatographic column and extracting with methylene chloride. Lipid content of the fish flesh was determined by allowing the solvent to evaporate from an extracted aliquot 33 for 12 hours and then weighing the remaining residue. The percentage of lipid in the sample was calculated as follows: % Lipid = Lieid_fleight x 100 Sample Weight PCBs were separated from lipids by gel permeation chromatography (GPC; ABC Laboratories, model 1001, Columbia, MO) with a 1:1 mixture of hexane and dichloromethane. The GPC column consisted of 6099 grams of resin (Biobeads SX-3) in a 2.5 cm x 48 cm internal diameter glass column. Pesticides and contaminants in the PCB sample extract were removed in a clean-up procedure described in detail in the standard operating procedures (Mora and Verbrugge, 1991). Volumes of the final extracts reached 1.0 ml and during gas chromatographic (GC) analysis, the samples were diluted to maintain a linear range of electron capture detection (ECD). Sample extracts were injected into a Perkin-Elmer (Model-8500) GC (Perkin-Elmer Corp., Norwalk, CT). A more detailed description of the extraction and analysis can be found in the standard operating procedures (Mora and Verbrugge, 1991). The chromatographed data sets were analyzed using Complex Mixture Statistical Reduction (COMSTAR, University of Wisconsin-Superior, Superior, WI). This PCB analytical. method is a multiple regression pattern analysis program used to quantify PCBs as a linear combination of Aroclors 34 (Burkhard and Weininger, 1987). This procedure makes use of a template that has been obtained from GC chromatograms of AroclorsO 1242, 1248, 1254 and 1260 that have each been supplemented with the internal extraction standard IUPAC #30. The resulting COMSTAR data is either rejected or accepted and reported as total PCB concentration in ug/kg, wet weight. If the composition of the sample could not be adequately represented by a linear combination of the Aroclor mixtures, then a total concentration of PCBs could not be determined (Burkhard and Weininger, 1987). Final determination of the total PCBs in the four experimental diets was calculated as time composite samples (i.e., adjustments in the final dietary concentrations of PCBs were made, depending on the number of days that the animals were fed a specific batch of feed). The diets were also analyzed for TCDD-EQs using the H4IIE rat hepatoma cell bioassay. The H4IIE rat hepatoma cells were obtained from the American Type Culture_ -Collection and cultured as described by Tillitt et a1. (1991). ~Ethoxyresorufin O-deethylase (EROD) activity of the closed cells was determined by a modification of the spectrofluorometric method of Pohl and Fouts (1980) by the staff of the Michigan State University Pesticide Research Center. EROD activity was calculated as pmol resorufin formed per mg protein per min (pmol/mg/min). The effective doses for half-maximal EROD induction (EDSO) were calculated by probit analysis (Finney 1978). A 35 TCDD standard curve was used to calibrate the environmental extracts in order to calculate the TCDD-EQ concentration. Calculations of extract potency for each dietary sample were made according to Sawyer et al. (1984) as follows: Extract potency = TCDD EDso/Extract E050 where; TCDD EDSO = pg/plate Extract EDSO = ul/plate Extract potency = pg TCDD-EQ/ul TCDD-EQs were calculated as: TCDD-EQ(pg/g) = a tenc CD -E t Vo u Sample weight(g) A more detailed description of the analytical procedure and theory for utilizing the H4IIE rat hepatoma bioassay can be found in Tillitt et a1. (1991). In addition to the bioassay results, the average total dietary concentration of TCDD-EQs in the raw carp and treatment diets were derived by congener-specific analysis using Toxic Equivalency Factors (TEFs) derived from both the relative induction of EROD in the H4IIE cells by the method of Tillitt et al. (1992) and TEF values as proposed by Safe (1990). ‘The TEFs proposed by Safe were derived from a number of biological and biochemical responses including acute lethality, body weight loss, thymic atrophy, teratogenicity, receptor binding affinity and hepatic microsomal AHH induction (Safe, 1990). The concentration of each congener was multiplied by its appropriate TEF to calculate the TCDD-EQ concentration for each congener. The 36 individual congener TCDD-EQ concentrations were then summed to obtain the total TCDD-EQs in the raw carp as estimated by congener specific analysis. Expenimentel Qesiqn and Animel Care On December 29, 1988, 60 standard dark mink were randomly assigned to four groups (each consisting of three males and 12 females) and placed on the four diets (Table 3). However, care was taken that littermates were not placed within the same treatment groups to compensate for any genetic predisposition to PCH toxicity. The mink were housed individually indoors in wire cages measuring 76 cm L x 61 cm W x 46 cm H and suspended from the ceiling. Food and water were provided ad 11bitum according to the mink farm guidelines (Fur Farm Animal Welfare Coalition, Ltd., 1988). The animals were provided artificial lighting and maintained on a diurnal cycle following sunrise and sunset times for Lansing, Michigan (Figure 6). Eeedins_Tria1 The feeding trial began on December 29, 1988 following a one week acclimation period whereby all animals were fed the control diet. Daily food consumption was determined once each week by averaging the amount of food consumed for two consecutive days. Females and males were fed in excess of what they would consume each day (approximately 350 and 450 grams of wet feed, respectively). All mink were weighed 37 Figure 6. Sunrise and sunset times at Lansing, Michigan, Eastern Standard Time. (Nautical Almanac Office, United States Naval Observatory, Washington, D.C..) 38 .8. a. 8i 3.- : I: I...» 2...»... .8 S... I. u! 23 83 3 23 23 83 83 83 83 83 83 83 33 33 an 23 83 83 83 3 83 83 83 23 83 83 83 83 83 83 3 B3 33 83 8" 83 33 8 23 83 83 83 83 83 83 23 23 33 83 83 83 83 83 83 83 83 83 8“ 83 23 83 83 8 =3 83 83 83 83 83 8" 33 83 83 83 83 83 83 83 83 83 B3 33 83 83 23 83 33 8 23 83 83 8" 83 83 83 83 83 33 3 83 83 83 83 83 23 83 83 83 83 23 83 83 8 23 83 83 83 23 83 83 83 83 83 83 83 83 83 83 83 83 83 3" 33 83 23 83 33 8 83 B3 f83 83 83 83 83 83 83 33 83 83 83 83 83 83 33 83 83 23 83 83 83 $3 8 3 3 83 83 83 83 «n3 3 83 3.3 83 83 83 83 83 3 83 n2 33 83 83 83 83 83 8 83 83 83 83 83 3 83 83 83 33 83 83 83 83 83 83 83 8" 33 B3 83 83 23 3» 8 3 83 23 83 83 33 83 83 83 33 83 23 83 83 83 3 83 83 33 83 23 83 83 8" 8 33 83 23 R3 83 83 83 83 33 8.3 23 23 83 83 3" 23 83 8" 33 83 23 83 B3 8” 8 33 83 23 83 3 33 23 8" 83 83 23 23 23 3 8“ 23 83 8" 83 83 23 83 83 83 8 83 83 23 83 33 83 :3 83 83 83 23 23 2H 33 $3 23 83 33 83 83 23 83 23 83 2 83 83 23 83 33 3.3 83 83 83 83 23 23 23 33 83 23 83 33 83 83 2" 33 23 8" 2 83 83 23 23 u 33 83 23 B3 83 23 23 23 33 33 23 83 3.3 83 83 2" 83 83 3 2 3 83 23 33 33 33 83 23 83 83 23 23 23 83 33 23 83 33 8" 83 23 83 83 83 2 83 83 23 83 83 33 8” 23 83 83 23 23 23 33 33 23 83 B3 83 33 3 83 83 83 2 83 83 23 83 8" 8.3 33 23 83 83 2" 23 23 33 33 23 23 83 8" 33 83 B3 83 83 2 83 3 23 83 33 3 33 23 83 83 23 23 23 33 33 23 23 83 8“ 33 8" 83 83 83 2 3 33 23 83 33 83 33 23 83 83 23 23 23 33 8.3 23 23 3 83 33 83 83 83 83 2 83 3" 23 83 83 83 33 23 83 83 23 23 2" 33 83 83 23 83 83 33 8" 83 83 8" 2 83 33 83 83 83 83 B3 23 83 83 23 83 2" 33 83 83 23 3 23 83 83 n 83 83 2 83 8“ 83 83 83 83 33 23 83 B3 23 83 23 83 83 83 23 83 R3 83 83 8“ 83 83 a 83 33 23 83 83 83 83 23 8.3 3 23 83 23 83 83 83 23 3 83 83 83 8" 83 83 I 83 33 83 23 83 83 83 83 33 83 23 83 23 83 83 83 23 2" 83 83 33 8» 3 83 u 83 33 83 23 23 83 83 83 33 83.23 83 23 83 83 8" 3 2" 23 3 33 3 23 83 o 83 33 83 23 .23 83 83 83 83 83 23 83 2" 83 83 83 3 2“ 33 83 83 u 23 83 a 83 33 83 23 23 83 83 83 83 23 23 83 2" 83 83 83 3 2" 83 83 33 8." 23 83 v 83 83 83 23 23 R3 83 83 83 33 23 83 23 83 83 83 3 2" 83 23 33 33 23 .83 n 83 83 83 23 1—3 83 23 83 33 8" 83 83 23 83 23 33 3 2" 83 23 33 33 23 83 u 83 83 33 23 83 83 23 33 33 83 83 83 3 83 23 83 83 83 83 23 33 33 23 83 _ ii .I.- .l‘ .I.- .Ii .I.o .03 .0.- .Ii .Ii .Ié .l.- 3.3 .I.- .03 .Ii .03 .3.- .lé .I.- 3.. 3.. .03 i.- 83 .3 33 a3 33 can 33 «3 on: «3 33 «3 03- 03 88 «3 on; com 33 03 8E 03 33 «3 8.- 58 a: 5338 sin-In «8 at... II... u! pg 19.! 2831 Hui 2: in“ g auto—I .83..- hC g 8 H28 39 on a weekly basis using an electronic balance in order to determine body weight gain or loss, and examined for general health throughout the initial 12-week exposure period. Body weights for the female mink were not determined after the first 12 weeks of the study to prevent any undue stress on the bred females. However, dietary exposure to PCBs continued for the duration of the six-month trial period. Reproduction Females were mated within their respective treatment groups between February 13 and March 13, 1989. Each female was given the opportunity for a second mating eight days after the first mating (a common mink ranch practice). All matings were verified with a microscope for the presence of motile spermatozoa in vaginal aspirations taken immediately after copulation. Nest boxes bedded with aspen shavings and wood wool (excelsior) were attached to the females' cages during the third week of gestation. False floors (one-half inch wire mesh) were also fitted into the cages to prevent the young mink kits from falling through the cage floors. All nest boxes were checked daily for kits during the whelping period from April 14 to May 11. All kits were sexed at birth and counted as either dead or alive. Kit body weights were‘ recorded at birth and at three and six weeks of age. Kits found dead were identified according to the female's identification number and frozen for subsequent 4O quantification of PCBs and TCDD-EQ analysis. At six weeks of age, a maximum of six kits from each dietary group were euthanized with carbon dioxide for three minutes and necropsied by a Michigan State University Board Certified Veterinary Pathologist. Organ weights (brain, liver, kidneys, heart, spleen, lungs, adrenals, thymus and thyroids) and incidental findings were recorded for all kits necropsied. Blood lelection On June 28, 1989, following weaning of the kits, the reproductive portion of the study was terminated. The 45 surviving females were anesthetized using 0.2 ml of ketamine hydrochloride (100 mg/ml; Veterinary Products, Bristol Laboratory, Syracuse, NY) and weighed, and a 5 ml blood sample collected via cardiac puncture. Blood samples were analyzed for hematocrit, hemoglobin, white (WBC) and red blood cell (RBC) counts and differential leukocyte counts. 'Three ml of each blood sample were used to determine thyroxine profiles. Of this blood, three pooled subsamples were collected from each treatment group. Serum was separated from whole blood by centrifuging the samples for 5 minutes at 650 rpm. Approximately 2 ml of the resulting supernatant were removed from each pooled sample and submitted to the MSU Animal Health Diagnostic Laboratory for analysis of both bound and free plasma thyroid hormones (T3 and T4). Duplicate hematocrit samples were collected in 41 mini capillary tubes and centrifuged (Micro-capillary Centrifuge, Model MB, International Equipment Co., Boston, MA). Hematocrit samples were read using a hematocrit reader (Mircro-capillary Reader, International Equipment Co., Boston, MA). Blood samples taken for determination of blood cell counts and hemoglobin concentration were collected in heparin-filled vacutainers to prevent clotting. Duplicate blood cell counts were made on a Coulter Counter0 (Coulter Electronics, Inc., Hialeah, FL) in 30 ml blood dilution vials. Operating conditions for the Coulter Counter were as follows: l/Amp = 2 Gain = 5 1/AC = 0.177 Lower Threshhold = 10-15 Matching = 20K Upper Threshhold = 100+ Background readings of the Coulter Counter were taken periodically as a reference standard. Prior to analyzing the blood samples, and periodically throughout the determinations, the instrument was flushed with an isotonic_ buffering solution. Hemoglobin counts were determined using a hemoglobinometer (Coulter Electronics, Inc., Hialeah, FL). 8 es After the final body weights were recorded and blood samples collected, the 45 remaining adult females were killed by cervical dislocation. Necropsies on the adult females were performed by a Michigan State University Board Certified Veterinary Pathologist. Organ weights (brain, 42 liver, kidneys, spleen, lungs, heart, adrenals and thyroids) and gross abnormalities were recorded. Throughout the study, all animals were observed daily for signs of intoxication, abnormal behavior, physiological stress and mortality. Those adult animals found dead were necropsied. Chemical Analyses of Liver Tissue Ten gram samples of liver were stored frozen for total PCB residue analysis. Approximately two grams of each liver were immediately placed in liquid nitrogen for determination of isomer specific PCB congener analysis by the Patuxent Wildlife Research Center in Laurel, MD. Total PCB and TCDD- EQ concentrations in individual liver samples were determined by the MSU Pesticide Research Center using procedures described above for dietary feed analysis. Histopatnglogy The remaining organs (brain, heart, spleen, lungs, adrenals and thyroids) and portions of the kidneys were fixed in 10% neutral buffered formalin for histopathological examination. Liver sections were fixed in 10% formalin, imbedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) according to routine histologic procedures for light microscopic examination by the Michigan State University Animal Health Diagnostic Laboratory. 43 W Data were analyzed using statistical software (SAS Institute Inc., 1987). Statistical treatment of the data to determine the effect of treatment was by one-way analysis of variance (ANOVA). Where the P value statistic was significant at p<.05, treatment means were tested by the Tukeys honestly significant difference (HSD) test statistic. RESULTS s’ u s 'n Raw Car Total PCB concentrations in the raw carp from Saginaw Bay ranged from 6.19 to 10.36 mg/kg, yielding an average total PCB concentration of 8.40:1.44 mg/kg measured as Aroclors® 1248, 1254 and 1260 (Table 4). The overall extraction efficiency was approximately equal to the ratio of areas for the standard peak IUPAC #30 (sample/standard) and was calculated as 44i5%. The percent recovery, accounting for the mechanical loss of 3/8 of the sample during the step of xenobiotic and lipid separation was reported as 70t8%. The results of the congener specific analysis of the raw carp samples are shown in Table 5. The predicted TCDD-EQ concentration in the,raw carp as calculated from congener specific analysis using the H4IIE bioassy-derived TEFs was 55.39 pg/g of fish, as shown in Table 6. The predicted TCDD-EQ concentration in the raw carp as calculated from congener specific analysis using TEF values proposed by Safe (1990) was 709.33 pg/g of fish (Table 7). H ! . ! E J E E' ! The nutrient analyses of the experimental diets are shown in Table 8. Fat content and caloric value increased. with increasing percent substitution of carp. The remaining 44 45 Table 4. Concentrations of PCBs in raw Saginaw Bay carp.1 or® ncentrat'ons Subsample Replicate _numb§r Number 1248 1254 1269 Total 1 1 5.56 3.67 1.10 10.34 2 5.68 3.60 1.08 10.36 3 5.29 3.35 1.14 9.78 2 l 4.94 3.74 1.11 9.80 2 4.35 3.13 1.04 8.52 3 4.91 3.13 1.01 9.05 3 1 3.67 3.05 0.989 7.71 2 3.54 2.89 0.894 7.32 3 2.72 2.69 0.774 6.19 4 1 3.80 3.03 0.924 7.75 3 3.33 2.58 0.779 6.69 Average Aroclor concentration (ppm) 4.27 3.15 0.98 Average Total PCB Concentration (ppm) i S.D. 8.40:1.44 1 PCB concentration measured as Aroclors® 1248, 1254, 1260. 2 Adjusted for sample weight and extraction efficiency. 46 dual congeners Congener Concentration found in raw carp from Saginaw Bay. Average concentrations of indivi Table 5. Congener "- """""”" 31422232222329.2222 8033018767 733 9 00000 0000000 31.231857560429100 no... 0...... ooooooooooooooooo 69651. 9629983 1.2444020820320162 2 1.2 31.4 1. 73 161. 2 63 31. 2 .. .5! . ... II5 . - - 5" — -' ' 8 8 .5 I55 6.445 ..I ...I ...II.III I6III 887 8887 55055l4|5.l I44! .III —-IIII .II4II4I4I5451I4 8776 887776 .II0 III I4 II IIIII I .IIII .IIIIII 54444444 I44I4|334 87464 8774664 IIIIIIII' II4 I4 III II!!! IIIIIII I444I4433'4 II '33. 73333 7343343 4 III3 IIII3 I434 II3 IIIII II3' III'II'IIIIII 32222 43 I33332333333223 1 Residue data courtesy of the USFWS. 47 Table 6. Average dietary concentrations of TCDD-EQs (pg/g) in raw carp from Saginaw Bay as calculated from TEF1 values derived using the H4IIE rat hepatoma bioassay. Congenerz IUPAC TEF x [Conc] 3 = TCDD-EQ # (pg/9) (pg/9) 2,3,7,8-TCDD - 1.0 26 26.0 2,3,7,8-TCDF - 6.44x10'3 39 0.25 3,4,4',5-TCB 81 1.91::10‘3 1,380 2.64 3,3',4,4'-TCB 77 1.81x10’5 2,100 0.04 2',3,4,4',5-PnCB 123 <1.0x10‘8 4,230 0.00004 2,3',4,4',5-PnCB 1184 <1.0x10'9 374,330 0.0004 2,3,3',4,4'-PnCB 105 5.49x10‘5 134,100 7.36 2,2',3,4,4'-PnCB 138 <1.0x10‘9 260,770 0.0003 3,3',4,4',5'-PnCB 126 2.24::10’2 810 18.14 2,3',4,4',5,5'-HxCB 167‘ <1.0x10"9 12,580 0.00001 2,3,3',4,4',5-cha 156 4.95::10'S 18,560 0.92 2,3,3',4,4',5'-chs 157 1.50x10'5 2,670 0.04 3,3',4,4',5,5'-cha 169 4.73x10‘4 0 0.0 Total 55.39 1 TEF values were derived from the relative induction of EROD by methods of Tillitt et al. are relative to 2,3,7,8-TCDD (Tillitt, personal communication). 2 TCDD (tetrachlorodibenzo-p-dioxin), TCDF (tetrachlorodibenzofuran), TCB (tetrachlorobiphenyl), PnCB (pentachlorobiphenyl), HxCB (hexachlorobiphenyl). 3 Average congener concentrations in raw carp courtesy of USFWS. (1991). All TEF values 48 Table 7. Average dietary concentrations of TCDD-EQs (pg/g) in raw carp from Saginaw Bay as calculated from TEF1 values derived using the H4IIE rat hepatoma bioassay. Congener2 IUPAC TEF x [Conc]3 == TCDD-EQ # (pg/9) (pg/9) 2,3,7,8-TCDD - 1.0 26 26.0 1,2,3,7,8-TCDD - 0.5 9 4.5 l,2,3,4,7,8-TCDD - 0.1 6 0.6 1,2,3,6,7,8-TCDD - 0.1 15 1.5 1,2,3,4,6,7,8-TCDD - 0.1 21 2.1 0000 - 0.001 17 0.03 2,3,7,8-TCDF - 0.1 39 3.9 1,2,3,7,8-TCDF - 0.1 16 1.6 2,3,4,7,8-TCDF - 0.5 42 21.0 1,2,3,4,7,8-TCDF - 0.1 9 0.9 1,2,3,6,7,8-TCDF - 0.1 9 0.9 2,3,4,6,7,8-TCDF - 0.1 8 0.8 1,2,3,4,6,7,8-TCDF - 0.1 13 1.3 3,3',4,4'-TCB 77 0.01 2,100 21.0 2',3,4,4',5-PnCB 123 0.001 4,230 4.2 2,3',4,4',5-PnCB 118 0.001 374,330 374.0 2,3,3',4,4'-PnCB 105 0.001 134,100 134.0 3,3',4,4',5'-PnCB 126 0.1 810 81.0 2,3',4,4',5,5'-chs 167 0.001 12,580 12.6 2,3,3',4,4',5-HxCB 156 0.001 18,560 18.6 2,3,3',4,4',5'-HxCB 157 0.001 2,670 2.7 3,3.',4,4',5,5'-cha 169 0.05 0 0.0 Total 709.33 1 TEF values as proposed by Safe (1990). 2 TCDD (tetrachlorodibenzo-p-dioxin), TCDF (tetrachlorodibenzofuran), TCB (tetrachlorobiphenyl), PnCB (pentachlorobiphenyl), HxCB (hexachlorobiphenyl). 3 Average congener concentrations in raw carp courtesy of USFWS. 49 Table 8. Nutrient analysis of experimental diets. Dimm 0% carp 10% 20% 40% (Control) carp carp carp W Moisture 68.10 65.20 63.10 61.90 Protein 15.10 13.80 13.80 14.60 Fat 5.35 6.70 8.10 9.95 Ash 5.52 3.51 4.23 4.80 Fiber, crude 1.40 1.27 1.34 1.19 Carbohydrates 4.53 9.52 9.43 7.56 Calories/100 gm diet 127 154 166 178 1 Analysis by National Environmental Testing,Inc., Chicago, IL. 50 constituents, moisture, protein, ash, crude fiber and carbohydrates were relatively constant in the four diets. Eeed Consumption The mean weekly and cummulative feed consumption based on the average of two consecutive days' consumption per week for adult female and male mink are shown in Tables 9 and 10, respectively. Daily feed consumption for male mink was statistically analyzed by four-week periods due to the large amount of data collected. Feed consumption during the acclimation period was not significantly different among treatment groups for either female or male mink. Average feed consumption in the control group for both sexes remained relatively constant throughout the exposure period, ranging from 248 to 280 g for the females and 264 to 369 g for the males (Table 9). Mean feed consumption for female mink receiving the treated diets decreased significantly from the controls in a dose-dependent manner “at the onset of the trial and continued to decrease for the remainder of the study. The average daily feed consumption by period for the males was signficantly different only in the 40% carp group. Female animals fed the 10% carp diet consumed less feed than controls except for weeks five through nine. Females consuming 20% carp in the diet had feed consumption that remained significantly less than controls for all weeks except for week nine. Animals in the 51 ..mo.0Anv uc0u0uuwc aaucmofiuwcmwm no: 0H0 umwuomu0oom 0300 0:» cufia 3ou 03.6% @Gu. 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H4IIE rat hepatoma bioassay-derived TCDD- EQ concentrations, evaluated on a time composite basis ranged from 1.03 ng TCDD-EQ/kg wet weight in control feed to 80.76 ng TCDD-EQ/kg wet weight in the 40% carp diet (Table 11). The cummulative dose (mg/mink) reported as total PCBs and TCDD-EQs differed significantly from controls (p<0.05) for both sexes in the treated groups and increased as expected with increasing percentages of dietary Saginaw Bay carp (Tables 11 and 12). .Determination of the amount of PCBs and TCDD-EQs ingested over the entire exposure period was determined by evaluating the dietary PCB and TCDD-EQ intake based on the number of days a specific batch of food lasted. The total PCBs and TCDD-EQs consumed were also reported as dose consumed/kg body weight/day to account for the variability in body weights of individual animals each ‘week. Calculations for'the parameters listed in Tables 9 to 12 are presented in Appendix A. The average total dietary concentration of TCDD-EQs (ng/kg) in each of the treated diets estimated from congener specific analyses is shown in Tables 13 and 14. In 54 .Amo.oA0v 000000000 0000000000000 000 000 00000000000 0500 0003 300 0500 000 00 0000: N :00: H 0000.00 0000.0 0000.0 0000.0 000\000003 0000 00 \000000>0000n0000 0: 0000.00 0000.0 0000.0 0000.0 000\000a\000000>0000u0009 m: 0000.0000 0000.000 0000.000 0000.00 0000a\000 0000 . . 0:000>0000u0000 0>000000000 000.00 000.00 000.00 000.0 00\000 0000000000000 0:000>0000u0000 0000000 0000.0 0000.0 0000.0 0000.0 000\000003 0000 00\000 05 0000.0 0000.0 0000.0 0000.0 000\000a\000 00 0000.00 0000.00 0000.00 00000.0 00000\050 0000 000 0>000005500 000.0 000.0 000.0 000.0 00\000 0000000000000 000 0000000 000.00 000.00 000.00 0000.00 00005\00 00000000000 0000 0>fiUOH5§O 3003 NH. 000000000 0000 no0 0000 now 0000 ao0 0000 «o 00000000010000000 000000> 000 .0000 >00 3000000 00 00000000000 £005 000500 000 00000500000 ominous 00>0000|mHH0m 000 000 00009 .00 0000B 55 Amo.OAOV 000000000 0005000005000 00: 000 00000000000 0500 £003 300 0500 0:0 :0 0500: N 500: 0 0000.0 0000.0 0000.0 0000.0 000\000003 0000 00 \000000>0000n0000 00 0000.00 0000.00 0000.0 0000.0 000\0000\000000>0000u0000 00 0000.0000 0000.0000 0000.0000 0000.00 00000\000 0000 0500000svmloooa 0>000095590 00.00 000.00 000.00 000.0 00\000 0000000000000 00000>0000u0000 0000000 0000.0 0000.0 0000.0 0000.0 000\000003 0000 00\000 00 0000.0 0000.0 0000.0 0000.0 000\x000\000 00 0000.00 0000.00 0000.00 00000.0 00000\000 0000 000 0>000000000 000.0 000.0 000.0 000.0 00\000 0000000000000 000 0000000 000.00 000.00 000.00 000.00 00000\0v 00000000000 0000 0>fiUMH5§O XOG3 NH 000000000 0000 000 0000 «on 0000 000 0000 we 0:00MMMHHINMMM000 .0000 000 3000000 00 00000000000 000000> 000 0000 0000 000 00000000000 00-0000 00>0000u00000 000 000 00000 .00 00000 Table 13. Average dietary concentration of TCDD-EQs (ng/kg diet) for each congener and as a total in diets of mink fed Saginaw Bay car congener specific analysis. p as estimated by Treatment; QIQQQ Congener2 IUPAC TEF3 10% 20% 40% No. carp carp carp 2,3,7,8- - 1.0 2.6 5.2 10.4 TCDD 2,3,7,8- - 6.44x10'3 2.5x10'2 5.0x10‘2 1.0x10'1 TCDF 3,4,4',5- 81 1.91x10'3 2.6x10'2 5.3x10'1 1.1 TCB 3,3',4,4'- 77 1..81x10'5 4.0x10'3 8.0x10‘3 1.6x10'2 TCD 2' ,3,4,4 ' ,5- 123 <1.0x10'3 4.2x10'6 1.0x10'5 2.0x10"5 PnCB 2,3 ' ,4,4 ' ,5- 1184 <1.0x10'9 4.0x10'5 1.0x10‘4 2.0x10'4 PnCB 2,3,3',4,4'- 105 5.49x10'5 7.4x10'1 1.5 2.9 PnCB 2,2',3,4,4'- 138 <120x1o‘9 3.0x10'5 1.0x10‘4 1.0x10'4 PnCB 3,3',4,4',5'- 126 2.24x10'2 1.81 3.6 7.3 PnCB 2, 3 ' ,4,4 ' ,5, 1674 <1.0x10‘9 1.0::10'6 2.0::10"6 4.0::10'6 5'-cha 2,3,3',4,4', 156 4.95x10'5 9.2x10‘2 1.8x10"1 3.7 s-cha 2,3,3',4,4', 157 1n50x10'5 4.0x10‘3 1.0::10‘2 1.6x10'2 S'-HxCB , , 3,3',4,4',5, 169 4.73x10“4 0.0 0.0 0.0 5'-cha Total 5.30 11.08 22.16 1 Dietary concentration calculated as the percentage of the TCDD-EQs in the raw carp depending on the percent substitution of carp in the diet. 2 TCDF (tetrachlorodibenzo), TCB (tetrachlorobiphenyl), PnCB (pentachlorobiphenyl), HxCB (hexachlorobiphenyl). 3 TEF values used in calculating the TCDD-EQs in raw carp as derived by the H4IIE rat hepatoma cell bioassay. 4 Some induction occurred, but an incomplete dose-response curve was developed when doses of up to 1 ng/plate were used. 57 Table 14. Average dietary concentration of TCDD-EQs (ng/kg diet) for each congener and as a total in diets of mink fed Saginaw Bay carp as estimated by congener specific analysis. Treatment_§2222____ Congener2 IUPAC TEF3 10% 20% 40% No. carp carp carp 2,3,7,8-TCDD - 1.0 2.6 5.2 10.4 1,2,3,7,8-TCDD - 0.5 0.45 0.9 1.8 1,2,3,4,7,8-TCDD - 0.1 0.06 0.12 0.24 1,2,3,6,7,8-TCDD - 0.1 0.15 0.3 0.6 1,2,3,4,6,7,8-TCDD - 0.1 0.21 0.42 0.84 OCDD - 0.001 0.003 0.003 0.007 2,3,7,8-TCDF - 0.1 0.39 0.78 1.56 1,2,3,7,8-TCDF - 0.1 0.16 0.32 0.64 2,3,4,7,8-TCDF - 0.5 2.1 4.2 8.4 l,2,3,4,7,8-TCDF - 0.1 0.09 0.18 0.36 1,2,3,6,7,8-TCDF - 0.1 0.09 0.18 0.36 2,3,4,6,7,8-TCDF - 0.1 0.08 0.16 0.32 1,2,3,4,6,7,8-TCDF - 0.1 0.13 0.26 0.52 3,3',4,4'-TCB 77 0.01 2.10 4.2 8.4 2',3,4,4',5-PnCB 123 0.001 0.423 0.85 1.7 2,3',4,4',5-PnCB 118 0.001 37.43 74.9 149.7 2,3,3',4,4'-PnCB 105 0.001 13.31 26.8 53.6 3,3',4,4',5'-PnCB 126 0.1 8.11 16.2 32.4 2,3',4,4',5,5'-cha 167 0.001 1.26 2.52 5.03 2,3,3',4,4',5-HXCB 156 0.001 1.86 3.71 7.42 2,3,3',4,4',5'-HXCB 157 0.001 0.267 0.53 1.07 3,3',4,4',5,5'-HXCB 169 0.05 0.0 0.0 0.0 Total 71.27 142.73 285.37 1 Dietary concentration calculated as the percentage of the TCDD-EQs in the raw carp depending on the percent substitution of carp in the diet. 2 TCDF (tetrachlorodibenzo), TCB (tetrachlorobiphenyl), PnCB (pentachlorobiphenyl), HxCB (hexachlorobiphenyl). 3 TEF values as proposed by Safe (1990). 58 comparison with the H4IIE bioassay results, TCDD-EQ concentrations estimated from congener specific data tended to be lower than those derived using the bioassay when using the H4IIE bioassy-derived TEFs, and greater when using TEFs as proposed by Safe (1990) (Table 11). In addition, the average daily dietary dose of TCDD-EQ (mg/kg body weight/day) as estimated by congener specific analysis when using H4IIE bioassy—derived TEFs was also less than the dosages determined by the H4IIE bioassay alone (Table 15). However, estimated, average daily dietary doses of TCDD-EQs from congener specific analysis when using Safe's TEFs are greater than the dosages determined by the H4IIE-rat hepatoma cell bioassy (Table 16). Calculation of total TCDD-EQ concentrations using TEFs proposed by Safe show that PCBs are of major importance in weathered chlorinated environmental mixtures. It should be noted that in estimating TCDD-EQ concentrations by summation of congener specific data, one assumes strict additivity, which does not take into consideration, the interactions among active congeners or actions of inactive congeners that modulate the toxicity of those that are active (Tillitt et al., 1991b) However, as stated above, the full range of dioxins and furans detected in the raw carp was not included in both sets of the congener calculations of TCDD-EQs. More accurate comparisons between the bioassay and congener results can be made when the full range of congener specific 59 Table 15. Average daily dietary dose1 of TCDD-EQs (ng/kg body weight/day) in diets of mink fed Saginaw Bay carp as estimated by congener specific analysis. Treatment group Congener2 IUPAC TEF3 10% 20% 40% No. carp carp carp 2,3,7,8- - 1.0 0.47 1.764 1.393 TCDD 2,3,7,8- - 6.44xclo‘3 0.004 0.010 0.025 TCDF 3,4,4',5- .81 1.91::10‘3 0.005 0.090 0.141 TCB 3,3',4,4'- 77 1.81x10'S 0.001 0.001 0.002 TCB 2',3,4,4',5- 123 <1.0x10'8 ---4 ---4 ---4 PnCB 2,3',4,4',5- 118 <1.0x10"9 —--4 ---4 ‘---4 PnCB 2,3,3',4,4'- 105 5.49x10‘5 0.132 0.250 0.393 PnCB 2,2',3,4,4'- 138 <1.0x10"9 ---4 ---‘ ---4 PnCB 3,3',4,4',5'- 126 2.24x10'? 0.328 0.616 0.977 PnCB 2,3',4,4',5,5'- 167 <1.0x10'9 ---4 ---4 ---4 HxCB 2,3,3',4,4',5- 156 4.95x10'5 0.017”2 0.031 0.049 HxCB _ 2,3,3',4,4',5'- 157 21.50x10’5 0.001 0.002 0.002 HxCB 3,3',4,4',5,5'- 169 4.73x10‘4 0.0 0.0 0.0 HxCB Total 0.96 2.76 2.98 1 Dietary dose calculated as the average daily feed consumed (grams) multiplied by the dietary concentration of the TCDD-EQs in each diet, divided by the average body weight of the mink. TCDD (tetrachlorodibenzo-p-dioxin), TCDF (tetrachlorodibenzofuran), TCB (tetrachlorobiphenyl), PnCB (pentachlorobiphenyl), HxCB (hexachlorobiphenyl). TEF values derived using the H4IIE rat hepatoma cell bioassay (Tillitt, personal communication). Congeners did not contribute a significant amount of dioxin-like activity based on TEF values derived using the H4IIE rat hepatoma cell bioassay. Some induction occurred but an incomplete dose-response curve was developed when doses of up to 1 ng/plate were used. 60 Table 16. Average daily dietary dose1 of TCDD-EQs (ng/kg body weight/day) in diets of mink fed Sagniaw Bay carp as estimated by congener specific analysis. Treatment group Congener2 IUPAC TEF3 10% 20% 40% No. carp carp carp 2,3,7,8-TCDD - 1.0 0.47 1.76 1.39 1,2,3,7,8-TCDD - 0.5 0.08 0.15 0.23 1,2,3,4,7,8-TCDD - 0.1 0.01 0.02 0.03 1,2,3,6,7,8-TCDD - 0.1 0.03 0.05 0.08 1,2,3,4,6,7,8-TCDD - 0.1 0.04 0.07 0.11 OCDD - 0.001 0.0003 0.001 0.001 2,3,7,8-TCDF - 0.1 0.07 0.13 0.20 1,2,3,7,8-TCDF - 0.1 0.03 0.05 0.08 2,3,4,7,8-TCDF - 0.5 0.39 0.7 1.06 l,2,3,4,7,8-TCDF - 0.1 0.02 0.3 0.05 1,2,3,6,7,8-TCDF - 0.1 0.02 0.3 0.05 2,3,4,6,7,8-TCDF - 0.1 0.02 0.03 0.04 1,2,3,4,6,7,8-TCDF - 0.1 0.02 0.04 0.06 3,3',4,4'-TCB 77 0.01 0.39 0.7 1.08 2',3,4,4',5-PnCB 123 0.001 0.08 0.14 0.22 2,3',4,4',5-PnCB 118 0.001 6.94 2.5 18.95 2,3,3',4,4'-PnCB 105 0.001 2.49 4.47 6.79 3,3',4,4',5'-PnCB 126 0.1 1.5 2.71 4.1 2,3',4,4',5,5'-HXCB 167 0.001 0.23 0.42 0.64 2,3,3',4,4',5-HXCB 156 0.001 0.34 0.62 0.94 2,3,3',4,4',5'-HXCB 157 0.001 0.05 0.09 0.14 3,3',4,4',5,5'-HXCB 169 0.05 0.0 0.0 0.0 Total 13.22 25.25 36.24 1 Dietary dose calculated as the average daily feed consumed (grams) multiplied by the dietary concentration of theTCDD-EQs in each diet, divided by the average body weight of the mink. 2 TCDD (tetrachlorodibenzo-p-dioxin), TCDF (tetrachlorodibenzofuran), TCB (tetrachlorobiphenyl), PnCB (pentachlorobiphenyl), HxCB (hexachlorobiphenyl). 3 TEF values as proposed by Safe (1990). ' 61 analysis for the treatment diets and raw carp samples is completed. MM Mean body weight, body weight changes and percent body weight loss for the female and male mink are summarized in Tables 17 and 18, respectively. No significant differences in initial or final body weights were observed in any of the treated groups as compared to controls. Mean body weights of females at the termination of the study increased proportionally to the percentage of carp in the diet. Percent body weight loss for females on the treated diets decreased concurrently with the addition of carp to the diet. The greatest loss in body weight occurred in the control females (10.55%) with the least observed in the 40% treatment group (4.37%). It is suspected that this may have been due to 1) the increased caloric value and fat content in the diets containing greater percentages of carp, and/or 2) the added stress and energy demand of reproduction. 1 However, this trend was not observed for males (Table 18). Adult gortality Clinical signs of PCB intoxication during the exposure period were first observed in mink fed the 40% carp diet (2.56 mg/kg PCB). These effects included nervousness, listlessness, and tarry stools. On week eight of the feeding trial, a solitary female in the 40% treatment group 62 .oofiumm vsHQHmn3 O» 050 «a £003 um unawumswauoumo unvfim3 upon mo :oHuosfiauoa e .Amo.oAqv usmumuuflo hausoofimeme #0: who umfiuomummsw meow spas 30H 050m 0:» ca mama: n .o u c muons .umwo auoo woe ummoxm ucosumwuu\x:wfi «H n H N .:003 on» no Hound unaccoum fl and: H 6.6 o.m 6.6 m.oa 000a panda: anon usuoumm mm: «on ¢h| omHI mmcosu «4.6nuomaa an.¢mwomsa «H.m~wmmaa <~.m~nmnoa «musmooxm no cam {Unnamoma «admfimwaa 4~.w~«~om.n m.m.a¢m.~nflmwaa HMMUficH A61 0:640: anon Aaouucoov undo woe ammo won sumo «oa numo «o usvwoz osouu >Hmmwwo .nuou ham zoswmmm mo momnucoouou msowum> com xswa macaw“ Mom mmoa Davao; upon ucooumm can mmwcoso unmwmz >603 .HBOwwa zoos :00: .ba manna 63 .Amo.oAmv usmummuflo aaucmowuwcuwm no: mum uafluomummsm 03mm saws 3ou 03mm on» ma msmw: m ..cmma may no Hound mumosmum H :mo: H o.sH v.m~ 6.6H m.mH anon unofims hmom unmoumm ppm: mflmn nnnu menu 06cmmo 4m.mmflmnma mo.oe Hamwa ¢¢.wmflmomd £9.5vafimbha munmomxm no Dam 40.4nHNHNN 4m.hnHHo>HN <¢.hmflmnNN N.d¢b.w¢HHNNH~ . HmfiuflcH Ame 0:640: soon Aaouucoo. mumo woe Qumo «om sumo «0H mumo wo usmfimz amouu >ummuwa .mumo amm 3msfimmm no mmomusmoumm msofinm> con xswa mama you mmoa unawma anon usuoumn mam mmvsmno unwed: >603 .usoam3 upon :mm: .ma magma 64 had tarry stools for several days, which were closely followed by paralysis of the rear limbs and severe epileptic-like seizures. Examination revealed the animal to be anorectic and unusually reactive to light and sound. Upon necropsy by a pathologist, all viscera were examined for gross lesions. The kidneys contained disseminated pinpoint white foci and the liver appeared diffusely yellow with a prominent lobular pattern due to congestion of the central veins. The urinary bladder contained two yellow irregular calculi and cervical muscles in the neck region contained a small amount of gelatinous fluid indicative of edema. Histopathological examination of tissues indicated a small amount of hemorrhage surrounding the spinal nerve roots of the thoracic region. The lungs were diffusely congested and the liver showed centrilobular congestion with moderate fatty infiltration. Hepatocytes were swollen and contained round non-staining, lipid-containing vacuoles of variable sizes. Laboratory findings concluded that the neurologic disease was of undetermined cause. Histopathologic examination of the liver did not suggest a cause for the clinical signs exhibited, however, all of these observations have been documented with PCB exposure in other animal species. During the trial, an additional female from the 40% dose group and one female from the 10% group died from undetermined causes. Gross necropsy of the female from the 10% group revealed no lesions or clinical signs. 65 Examination of the reproductive tract showed 10 normal- appearing fetuses (Figure 7). Gross necropsy of the other female (40% group) showed no signs indicative of PCB toxicity, although many of the animals exposed to 40% carp in the diet exhibited tarry stools. Reproduction Breeding results for the 48 females are shown in Table 19. 0f the 48 females bred, twenty-four whelped. The average number of confirmed matings/female across the treatment groups ranged from 2.3 to 2.8. The number of live kits whelped per female was normal for the control, 10% and 20% treatment groups (Table 20). However, reproductive effects in females fed the diet containing 40% carp were evident since an average of only 0.7 live kits were born per female that whelped. All kits whelped by females in this dose group were either stillborn or died within 24 hours. A dose-related decrease in kit birth weights compared to control body weight was seen in all three treatment groups (Table 21) and the differences were significant in the 20% and 40% groups. At birth, these kits were observed as being weak and having listless behavior, characterized by decreased responsiveness when touched. Females in the highest treatment group (40% carp) appeared to have a lack of interest in their kits. Mean kit body weights at three and six weeks of age were significantly less than the 66 Figure 7. Ten "normal" fetuses from a female mink fed 10% Saginaw Bay carp. 67 68 o m.~ 3 mm 3 3 sumo 2:. o n.~ Z on 3 NH ammo wow 6 m.~ ma mm ma 3 sumo «3 m m.~ 2 mm «H 3 30.353 ammo «o 60.3053 mam—mom mvswum: mmcfiumz 60pm: mmamamm mma mama \ 92.. um: 608.3 8.80 6mumamuu< mod mama . oz 95.5 .02 60.5.3300 uo umnasz mo non—.52 .oz Hmuoa acme—Hmong. ..x:fla cw osw6mmun so ammo 6mumswamus001mom msfi6mmu no uomuum one .ma magma 69 .Amo.oAmv accumuufl6 zausmofiuwcowm no: mum umwuomummsm 08mm spas sou 05mm as mammz .cmma on» no uouum 6um6smum H :mmz n .mcwmamn3 anammn 6mw6 mmamamu 6muma O39 N . .mcHaamna muowmn 6mw6 mHmEmu 6muma one a mn.m mm.m mn.m <>.m . Guam hopped momum>¢ mo.~ co.H 4m.a ¢>.o mama m>.o em.v am.m co.m 0>fiam "mswmamn3 mamamu .. \mmdamns mums .oz mm.H~«HmHH 2H.m~86oaa 6on mamamm mus.aub.mm 0>~.HH~.mm mme.aus.mm maes.awm.vm Ace unmama coflumummo no 36 . o o mudamna mmamsmm .oz Aaouucoov numb wow Qumo won Qumo woa ammo «o usMflMNmHHlxmmmmwa hmm amcwmmm mo .mumo mommusmoumn msoflum> 60“ xsfia mamamu no mommah0uumm m>wuos6oummm .oN magma 70 Table 21. Average body weights of mink kits whelped and raised by dams fed various percentages of Saginaw Bay carp. Saginaw Bay Kithodv weight(g) Carp(%) Birth 3 weeks 6 weeks 0 10.52A1 98.7A 247.9A (Control) 10.23 £1.93 £19.11 10 9.8AB 66.18 197.48 £0.34 £9.75 £16.24 20 8.7BC 65.88 100.58 £0.29 £4.80 £0.15 40 7.49C --- --- 10.48 --- --- 1 Mean standard error of the mean. Means in same column with same superscript are not significantly different (p>0.05). 71 controls in both the 10% and 20% treatment groups. No kits survived past 24 hours in the 40% carp group. Percent kit survival to six weeks of age (weaning) was 85% for the control animals and 28%, 11.5% and 0% for 10, 20 and 40% treatment levels, respectively (Figure 8). Gross examination of four kits stillborn to a female ingesting 40% carp in the diet revealed localized edemas in the head and tail region of three kits (Figure 9). Figure 10 shows the extremely small size and deformed body of one of these stillborn kits as compared to a control kit. A reduced mandible and maxilla size was also observed in the fourth kit. 8.1mm; Sixteen kits were euthanized at six weeks of age and submitted to the MSU Animal Health Diagnostic Lab for gross necropsy. No kits in the 40% carp dose group survived to six weeks of age; therefore, no kit organ weights were .available for comparison at this treatment level. kit organ weights are expressed on a percent brain weight and wet weight basis for comparison (Table 22). Brain, liver, spleen, kidney, heart and adrenal gland weights of kits whelped and nursed by females fed 10 and 20% carp in their diets were significantly less than the respective organ weights of control kits. Lung weights of kits in the 20% treatment level were also significantly less than control values. All organ weights, except those of thyroid, adrenal 72 Figure 8. Percent survival of mink kits at birth, three and six weeks of age. Numbers above the bars represent the average percent survival for the group. .65 s o. g .268 g 62 5. 65 s 8 I 65 .x. cm 5.5 9.33 o 73 3223 8 57*. 74 Figure 9. Mink kit (right) whelped by a female fed 40% Saginaw Bay carp. Note the edema in the head and neck region. Control kit (left). 75 76 Figure 10. "Normal" control mink kit (above) and a stillborn kit whelped by a female fed 40% Saginaw Bay carp (below). Note the small size, deformed body and reduced mandible and maxilla. 77 .Amo.oAsV unououuu6 sausmoumusmum no: mum usuuomuossm 03mm gnu: sou 05mm on» :u memo: .smoa on» uo uouuo 6um6cmum « smo: .Amomosusoums swv mamus nu usmuoa uo3 6cm unmwos cumun unmouos mm 6ommousxo munmwo3 cmvuo H 78 nun m.oou m.m>u m.sm~ unmuos soon nu: Auu.oum6.ov sou.oumn.ov Am~.owmm.ov an: mo.ouom.o em.ouon.m om.~umo.n mossna nun “so.oueu.ov «mo.oumo.ov «mo.oumu.ov nu: ¢~.ou~n.~ ue.o«>>.o em.ouso.u mumcoumm nun wno.ouom.ov gnu.oumu.ov «no.ouho.ov us: ou.ousm.n om.ouoo.m om.muom.n mouousae nun «mo.ouom.ov «mu.ounm.uv Mnn.ou~m.uv nun om.ou¢>.nu om.uusm.>u mm.nuo>.n~ puma: nun wms.oun.mv som.ouns.nv 4mm.ouoo.¢v unu 66.nuom.om um.m«nm.~m «mm.ouuoo.om moose nun dmu.oum6.uv «an.owmo.mv Ae<.oueu.nv nun ~m.owoo.m~ mo.~usm.nn «b.486m.mm mamcoux nun smu.ounm.ov deu.ouno.uv Amm.ouoo.mv nu: m~.o«ou.m mo.oumv.eu «Nu.oun~.m~ cmmusm nun wmo.unmn.sv ~6.~H-.~uv Amm.~umo.>uv nu: 06m.ouum.nou mm¢.o~u~m.omu coo.unuom.mo~ u0>uq nu: ouo.oumo.o mm~.ouno.> 23.386 Amy cumum o 6 o o z Auouuooov sumo wow sumo mom sumo «ca sumo wo msmouo usmawmwMHluummmus .sumo amm 3mcummm no mommusoouos msOuum> ocucumusoo wuou6 6mm xsua mamaou sn 6omuss 6cm 6osaonz muux 6H0Ixoo3nxwm no mucouo3 smvuo 6cm >6on :mo: .mm oanma 79 and thymus glands, decreased in a dose-dependent manner (Table 22). A§21£_QIQQD_H§iQh§§ Organ weights of adult female mink are summarized in Table 23. Expressed as percent of brain weight, all organ weights showed a general dose-dependent increase. Liver, spleen, lungs and adrenal gland weights for females in all three treatment groups were signicantly different from control values. Females in the 40% carp treatment group had kidney weights significantly greater than kidney weights for control mink. Heart weights were significantly greater for the 10% and 20% carp-fed animals and thyroid gland weights were significantly greater for the mink which consumed 20% or 40% carp in their diet. ho 0 Gross examination of the adult female mink at necropsy revealed enlarged livers that were diffusely yellow in many of the animals that were fed 40% carp in the diet. Histopathological examination of the livers indicated no lesions that were indicative of PCB toxicosis. Those lesions observed were considered incidental, even though numerous microscopic changes were observed in the livers of mink that have died from dietary exposure to PCBs (Table 24). Minimal to mild periportal hepatocellular lipidosis was seen in two mink from each treatment level, except those 80 .Amo.oAsV uc0u0uuu6 huucmowuusvwm uo: 0um usuuomuossm 03mm saw: 3ou 0Emm 0:» cu msm0z .Amamumv unwuos #03 mm 60mm0usx0 munow03 :mmuo H .sm0a 0:» mo uouu0 6um6cmum H smoz Anoo.ouom>o.ov Amo.oummmo.ov Auo.onm>oo.ov Anoo.oummmo.ov oou.ouu.u mso.oum.o mdmo.oum.o ¢m~.ous.o mumcmuom Amoo.oummmo.ov Auo.ou¢noo.ov Auo.oummeo.ov Auo.oumo¢o.ov oso.ous.o moo.oum.o aoo.ouo.o «mo.ouo.o mouousne A-.ouon.mv Asu.ou¢o.mv Amu.ou¢m.mv Asu.ouo~.mv amh.~«~.oo mos.uum.os mm~.~«¢.mo coo.~«o.mo uumom sum.ouum.wv Ann.ouem.sv Amu.o«¢~.pv uou.ou>m.ov omo.nuo.mou onu.muo.mm mem.~um.om cum.u«¢.mm moans Amn.oumn.mv Amu.oues.vv som.oun>.¢v Amu.o«mo.ee mms.muv.oo cem.uu~.mm com.~we.mm ano.uue.mm msmcmuu Am~.oumo.mv Ao~.ou~m.mv Am~.ouo>.mv uou.oumm.~v oe.~um.sn mom.uu¢.om mne.num.mm mo.uwu.m~ . cumusm w>¢.uumn.mme mm.oum~.mmv som.uum~.m~v Moe.o«~o.¢~v omu.muu~.>m¢ mom.ouus.mon mom.muu~.oom ano.uuuu.oom u0>uq ou.ouu.m ou.ouo.m ou.ono.m umu.o«u.m Ame cumum Auouucoov sumo woe sumo wow sumo wou sumo «o mcmouo ucwmwmwMHINMmmwua .sumo amm 3msummm mo m0mmus0ou0s msoMum> Dausumucoo mu0u6 60m xsus mamaou vas6m mo 9:6«03 swmun us0ou0s mm 60mm0usx0 munmwoa smouo smo: .mm 0Hnma 81 Table 24. Incidence of histological changes in livers of mink fed diets containing varying percentages of Saginaw Bay carp. Type of Tm Histopathological Liver Change 0% carp 10% 20% 40% (Control) carp carp carp Periportal hepatocellular lipidosis 2/121 2/11 0/12 2/11 Scattered portal lymphocytic infiltration 0/12 2/11 3/12 0/11 Diffuse hepatocellular vacuolar lipidosis 0/12 1/11 1/12 0/11 Diffuse pallor and swelling 0/12 0/11 1/12 0/11 1 Number of animals showing alteration per number examined. 82 animals fed the 20% carp diet (Figure 11). For comparison, Figure 12 shows a section of liver from a female mink having no histological alterations. The hepatocytes are normal- appearing with very little lipid accumulation. Minimal to moderate scattered portal lymphocytic infiltration had a higher occurrence in those animals that were fed the 20% carp diet (Figure 13), an indication that inflammation was present. Single animals in both the 10% and 20% carp groups had livers with marked congestion and diffuse hepatocellular vacuolar lipidosis (Figure 14). Sinusoids in the normal- appearing liver section were free of congestion (Figure 12). One animal in the 10% dose group had hemorrhages in the brain, edema in the neck region and lungs that were sharply demarcated with a linear band of pulmonary congestion. The spleens of six animals in the 20% treatment level were irregularly shaped and firm compared to spleens taken from control animals. Seven mink from each of the groups fed carp had spots on the uterine horns, however, -each of the affected animals whelped. Cogcegtrations of PCBs and TCDD-EQs in Liver Concentrations of total PCBs in livers of mink ranged from 0.066 ug PCB/g liver (wet weight) in controls to 10.62 ug PCB/g liver (wet weight) in the 40% carp-fed group (Appendix B). Concentrations of PCBs in livers among the three treated groups were significantly different from control values and increased in a dose-dependent manner 83 Figure 11. Mild periportal hepatocellular lipidosis characterized by vacuolization of hepatocytes (A) observed in the liver of a female mink fed a 40% carp diet (100x, stained with hematoxylin and eosin). 84 , (73.5 (id '\ ’ ‘ ‘ ' ‘ . . .3 .0 85 Figure 12. Section of liver from a female mink showing no histological alterations. Hepatocytes are normal appearing with very little lipid accumulation (A). Sinusoids appear free of congestion (B). (100x, stained with hematoxylin and eosin). 86 87 Figure 13. Moderate scattered portal lymphocytic infiltration of the liver (A) that was evident in several mink fed the 20% carp diet (400x, stained with hematoxylin and eosin). 88 89 Figure 14. Marked congestion and mild, diffuse hepatocellular vacuolar lipidosis observed in the liver (A) of a female mink receiving 40% carp in the diet (400x, stained with hematoxylin and eosin). 90 91 (Table 25). On a per gram lipid basis, PCB concentrations in liver tissue were comparable for the 10% and 20% treatment groups. There was a significant difference from controls for average TCDD-EQ concentrations found in liver samples from all three treated groups (Table 25). However, a direct dose-dependent increase in TCDD-EQs was not observed, as was seen for liver total PCB concentrations. Concentrations of total PCBs in the liver increased across treatment groups at a faster rate than did TCDD-EQ, as determined by the bioassay (Figure 15). Average concentrations of TCDD-EQ were below the limit of quantification (LOQ = 5 pg/g) in the control group, slightly greater in the 10% dose group than in the 20% treatment diet, and greatest in the livers of mink fed 40% carp. The bioassay-derived TCDD-EQs for the individual samples of mink liver ranged from less than the LOQ for control tissue to 950 pg/g for a liver in the 40% carp group (Appendix C). The relative potencies in food, defined as the ratio of extract-bioassay TCDD-EQs to total PCBs in the diet, were relatively constant across the treated groups (Table 26). However, the relative potency for the control diet was greater than those in the treated groups. Comparison of the relative potencies between diet and liver tissue indicated that toxicant enrichment occurred from food to animal in the treated groups, but did not occur in the control group. However, enrichment from food to animal unexpectedly 92 .w\vsm u couumouuuucmsv mo uuauq v .Amo.oAsv us0u0muu6 hausmOumuGOum uo: 0um usuuou0ssm 08mm cuwz 3ou 03mm 0:» Cu mcm0: n .COMUMwawo flHMGCflflm H Gmmz N .unouma 003 u omn.muom.mp muo.nums.m6 amo.nuuo.¢¢ cuu.oumo.~ “ousuu o\oov mom umuoa omm.o«>~.o oom.oum¢.m mu~.owmu.~ euo.owmo.o Ham\osv mom umuos .ououu o\oov auo.>mmum.momm omhuuum~.mmmo som.u~uumo.mu¢ou «mu.ouuoo.~uu ousug\omuoooa omu.emuho.omo ouv.moumo.omm mso.mn«mv.moe zoogv uuo\osv omuoooe ono.u«v~.m omos.oume.s mom.ou¢o.m «qo.owmo.m use ousuq mm.ouen.o m¢.oumm.w .n.~s¢.owu~.m nun Ame unouos ousamm uu mu uu mu umnsoz Auouucoov sumo woo sumo mom sumo wou sumo we unmammeH Numuufid .mu0>ua xsua 0Hma0u cu unauumuuc0ocoo Am\osv omnoooa 60>uu06lmHH¢= 6cm mos Hmuou uom mcm0a uc0aum0ue .mN magma 93 Figure 15. Relationship of total PCB concentrations to TCDD-EQ concentrations in livers of mink fed either a 0% (control), 10%, 20% or 40% Saginaw Bay carp diet. 94 _ o 93 *9. 4 960 $8 I 98 $2 0 .6560 663 55 E reumbcoocoo mod .83 o. m o e a o _ _ u _ _ 4 I I; 88.8 + an: n > 688 u a 98 nun. 00m 8s coop A903 .62.. S Owén—Oh 95 Table 26. Concentrations of PCBs and TCDD-EQs and relative potencies1 of feed and liver tissue of female mink fed various percentages of Saginaw Bay carp. Treerment Group 0% carp 10% carp 20% carp 40% carp (Control) Food Total PCB 0.015 0.72 1.53 2.56 019/9) TCDD-EQ 1.03 19.41 40.02 80.76 (Pg/9) Relative 68.67 26.96 26.16 31.55 Potency Liver Total PCB 0.09 2.19 3.45 6.27 (119/9) TCDD-EQ <5 495.48 380.03 656.07 (Pg/9) Relative 55.55 226.25 110.15 104.64 Potency Ratio of Potencies Food/Liver2 0.8 8.87 6.83 4.14 1 Defined as ratio of TCDD-EQs to PCBs. 2 Defined as ratio of relative potencies for food and liver. de I 21'! 96 decreased in a dose-dependent manner for the treated groups (Table 26). One possible explanation for this may be that higher dosages may cause a greater amount of antagonists such as 2,2',4,4',5,5'-HxCB (IUPAC #153) to accumulate in the liver at higher ratios (Safe, 1990). Hemetologic Parameters The dietary treatments caused significant effects on several hematologic parameters. RBC counts of female mink showed significant dose-dependent decreases (Table 27). WBC counts differed significantly from controls for the 10% and 20% carp groups. The relative numbers of neutrophils were significantly greater for all carp treatments relative to that of adult mink which were fed the control diet. The number of lymphocyte was significantly'greater in adult mink fed 20% and 40% carp. Numbers of monocytes were significantly less from the controls in all three carp treatment groups, while eosinophil counts were significantly greater from the controls in both the 10% and 20% treatment levels. Significant reductions from controls were also observed in hematocrit values of mink fed the two highest percentages of carp. However, no differences in hemoglobin values were observed for mink in any of the treated groups. Wes Concentrations of free and total thyroxine (T4) were greater in adult mink which consumed Saginaw Bay carp than 97 .m0m0nus0ums aw va m0sam> 0>uuma0m 0um usuuou0ssm 03mm 0:» nous 3ou 03mm 0:» cu msm0: .Amo.oAsv pc0u0muu6 sausmoumwsmum #0: .sm0fi 0:» mo uouu0 6um6smum H :m0: a Aum\aoe :uoouooam: Hm.OH6.mH mm.ou~.ma Hm.0Hm.wH Hm.OHN.mH mos.ouo.mm mmo.uum.¢m mes.ouu.nm mme.uu~.sm Awe uuuooumamm as.uv Am.uv .m.me .p.u. mmo.emuneu mu.mmueo~ mm.ovu¢mu mm.m~unnu maussocumom Am.ue Ame .o.~V Am.mv mn.m~unnu mm.meummu mm.~uunu~ ms.mmu-~ monsooco: 16.6mv some Am.¢nv some man.mmnumem~ mm.m~nwmmm~ m~.eunuumum m¢.u¢~u>6- mouaoomsssq some some Au.ome A¢.mmv mo.~woNHnHm¢ mo.amumom¢ mm.wn¢Hm¢N¢ <~.¢6nflhahm 60ucwfim0m "maflssouvsoz mucsoo Hmuus0u0uuuo m<>o.ouu.s mum.o8~.> mmm.ouu.h mom.o«q.m Anouxvnas ums om: omu.oum.m omu.ou~.m .mm~.ous.m umm~.ou~.h .mouxvnss ums omm ou mu uu mu scum mo .02 Aaouucoov mu0u0amums sumo wov sumo wow sumo wou sumo wo 6ooam unwflmmmMHINMmmwun .sumo >mm 3mswmmm mo movmucoouos msOHum> mswcwmusoo muow6 60m xswa 0ama0m no 005Hm> owooHouma0m .bm 0Hnm9 98 in controls (Table 28). Total and free concentrations of triiodothyronine (T3) were less in treated mink, in a dose- dependent manner, except for T3 concentrations in the 10% group, which were greater than in the controls. No statistical analyses of the T3 and T4 concentrations were made because the samples were pooled for analysis. NOAEL. LOAEL and Reference Dose Based on the results of the present study, the 10% carp treatment diet was the lowest level at which effects were observed (LOAEL) (Table 29). This equated to 0.134 mg PCBs/kg body weight/day or 3.60 ng TCDD-EQs/kg body weight/day (Table 30). The effects observed at these concentrations were depressed kit body weights and decreased kit survival at six weeks of age, and increased relative liver weights of adult females. Greater concentrations of PCBs in the diet resulted in smaller litter size, lesser birth weights of kits and early growth, and greater mortality of kits at birth, three and six weeks of age. 99 Table 28. Mean thyroxine (T4) and triiodothyronine (T ) concentrations in blood serum of female mink fed diets containing various percentages of Saginaw Bay carp. T:§§£E§DL.§£QBE§_______ Serum parameter 0% 10% 20% 40% carp carp carp carp (Control) Total thyroxine TT4 (nmol/L) 61.43 69.50 67.43 66.10 Free (unbound) T4 (pmol/L) 24.63 32.43 29.57 30.37 Total triiodothyronine TT3 (nmol/L) 1.17 1.60 1.07 0.93 Free (unbound) T3 (pmol/L) 3.0 2.83 2.87 2.73 1 Three pooled samples per treatment group. 100 Table 29. NOAEL, LOAEL and reference concentrations of total PCBs determined as AroclorsO 1248, 1254 and 1260. NOAELI Reference2 LOAEL3 Dietary 0.015 0.104 0.72 Concentration (m9/k9) Daily Dose 0.004 0.0251 0.157 (mg/mink/d) Cummulative 0.335 2.11 13.23 Dose (mg/mink) Daily Dose 0.004 0.023 0.134 (mg/kg body wtId) Liver 0.09 0.444 2.19 Concentration (“Q/9) Liver 2.03 9.52 44.61 Concentration ' (u9/9 lipid) 1 No Observable Adverse Effect Level (control group). 2 The geometric mean of the NOAEL and LOAEL. 3 Lowest Observable Adverse Effect Level (10% carp group). 101 Table 30. NOAEL, LOAEL and reference concentrations of- TCDD-EQs as determined by the H4IIE rat hepatoma cell bioassay in female mink. NOAELlQ Reference2 LOAEL3 Dietary 1.03 4.47 19.41 Concentration (fig/k9) Daily Dose 0.273 1.07 4.23 (fig/mink/d) Cummulative 22.99 90.41 355.6 Dose (mg/mink) Daily 0.251 0.951 3.60 Cummulative Dose (fig/k9 body wt/d) Liver <5 49.77 495.5 Concentration (PG/9) ‘ Liver <112.1 1081 10420 Concentration (pg/9 lipid) 1 No Observable Adverse Effect Level (control group). 2 The geometric mean of the LOAEL and NOAEL. 3 Lowest Observable Adverse Effect Level (10% carp group). DISCUSSION In addition to PCBs, the carp from Saginaw Bay contained numerous other organochlorine contaminants including PCDFs and PCDDs. Carp collected the next year from the same location in Saginaw Bay contained numerous other organochlorine compounds as shown in Table 31. These compounds have been reported to elicit many of the same reproductive effects on laboratory animals as PCBs, but at much lower concentrations (Poland and Knutson, 1982; Holcomb et al.,1988). Thus, it is possible that effects attributed to total PCBs in this study may be due in part to the presence of CDFs, CDDs and/or other organochlorines in the carp (Hornshaw, 1981). However, since the effects of the individual CDF or CDD compounds on mink have not been studied, or have been found to be considerably less toxic to mink than PCBs, as is the case with some organochlorine pesticides, it will be assumed that the effects noted are due primarily to dioxin-like PCBs, measured through bioassay-derived TCDD-EQs (Aulerich et al., 1973; Aulerich and Ringer, 1977; Bleavins et al., 1982; Aulerich et al., 1985, 1987; Hochstein et al., 1988; Brunstrom et al., 1991). The primary objective of this study was to determine the effects of PCH-contaminated prey species on the reproductive performance and survival of mink. Thus, an attempt was made to simulate the exposure of mink to a contaminated diet. 102 103 Table 31. Average residue concentrations of selected pesticides in carp collected from Saginaw Bay, Michigan, 1990. Pesticide Concentration1 (ppb) Lindane 1.13 Heptachlorepoxide 2.04 Oxychlordane 3.00 Gamma-chlordane 5.37 o,p'-DDE fiH MOM ”THROQTH mflflfiflmmh mum H Anomuv .um um 3mmmmuoz mmsumnz muux 0>uu oz m.¢ mm.e~ “humus .um um cmmcmn omoummz muux oz ~.< mo.om~ o.uu Annmuv .um um umocuz mmmumnz muux oz 6.: u~.6 Ncan Anomav 6mumumz 6cm sosoumus 60sH0n3 muux 0>ua oz m.n.¢ Ho.~u om.m mu: ¢~A usuusmmuo Anomav 6mumumz 6cm 3ocoumas mo Hm>u>usm oz m.¢ u~.u mo.o immunoae A»: 903. lease 00:0sm0m sauumuss mos .osoo mos 0os0u0m0m 0>wuo96ous0m 0usmosxm .osoo mos >umu0fio .msouumuucoocoo mos humuouo usoflum> 60m xCHE Scum mum6 COwuoo6ous0u onw620s00uuoo 6cm mommwu omoswom 6cm u0>wa cfi 00:6fl00u mos MO humfiadu 4 .nn 0Hnma 123 daily intake (MADI) levels of Great Lakes fish filets for mink were developed using various fish species with known PCB concentrations. The monitoring data used in the evaluation was obtained from the 1991 Fish Contaminant Monitoring Report published by the MDNR (1991). Locations of the fish collection sites are shown in Figure 16. The MADI levels were calculated by dividing each PCB concentration in the fish species in Table 34 by the reference dose generated from the mink feeding-reproduction study (Table 29). The reference dose is defined as the amount of PCBs consumed per mink per day that did not cause reproductive impairment. This was calculated as the geometric mean between the PCB level consumed that did not produce an adverse effect on reproduction and that level which did. These concentrations are respectively termed the No Observable Adverse Effect Level (NOAEL) and the Lowest Observable Adverse Effect Level (LOAEL) of the study. Based on these calculations, MADI levels ranged from 6 grams of carp per day from Lake Huron to 628 grams per day. for carp from Lake Michigan and yellow perch from Lakes Huron and Michigan. Daily intake levels by wild mink over the MADI could potentially have an adverse effect on mink reproduction and kit survival based on the results of the Present laboratory findings. In addition to the MADI, Exceedance values (EV) were also calculated and were defined as the ratio of the PCB concentration in Great Lakes fish in Inglkg to the reference dietary concentration in mg PCBs/kg 124 Figure 16. MDNR native fish collection sites in the Great Lakes and connecting channels, 1989-1990 (taken from MDNR, 1991). 14255 LAKE SUPERIOR Il-Z a .-1 5.25.26 38 I mm ‘ ~hnamw~.,h", 23’24 ' ' :’ Iliflo I: L}\r(EE tiLJFiCDPq . 14 4 15 “\1!\Z%E-22,26,28, 29 -17, 21 9b 7,’8 LAKE MICHIGAN go. . A 1990 Muuaguanwuv I 1989 1 MM 19- . 3. 1 K 13 “MM Lake L————— Erie 126 Table 34. Maximum Allowable Daily Intake (MADI)1 levels and Exceedance Values2 (EV) of various Great Lakes fish species. Site Waterbody Fish PCBs MADI EV No.4 Species (ppm) (g/d) 1 L. Superior Lk. Trout 0.41 61 3.9 2 L. Superior Lk. Trout 0.31 81 2.9 3 L. Superior Lk. Trout 1.34 19 12.9 4 L. Michigan Carp 2.42 10 23.3 5 L. Michigan Carp 0.04 628 0.4 6 L. Michigan Lk. Trout 2.63 10 25.0 7 L. Michigan Lk. Trout 0.91 28 8.8 8 L. Michigan Lk. Trout 0.76 33 7.3 9 L. Michigan Chinook 1.30 19 12.5 10 L. Michigan Chinook 1.15 22 11.1 11 L. Michigan Chinook 1.03 24 9.9 12 L. Michigan Br. Trout 1.35 19 13.5 13 L. Michigan Br. Trout 0.82 31 7.9 14 L. Michigan Br. Trout 0.98 26 9.4 15 L. Michigan Burbot 0.19 132 1.8 16 L. Michigan Lk. Whitefish 0.84 30 8.1 17 L. Michigan Lk. Whitefish 0.42 60 4.1 18 L. Michigan Lk. Whitefish 0.34 74 3.3 19 L. Michigan Lk. Whitefish 0.61 41 5.9 20 L. Michigan Y. Perch 0.04 628 0.4 21 L. Michigan Y. Perch 0.06 418 0.5 22 L. Michigan Y. Perch 0.05 502 0.5 23* L. Michigan Redhorse Sucker 0.48 52 0.2 24 L. Michigan Walleye 0.20 126 0.5 25 L. Michigan Walleye 0.06 418 0.6 26 L. Michigan White Sucker 0.07 359 1.5 Table 34 (cont'd.) Site Waterbody Fish PCBs MADI EV No.4 Species (Ppm) (g/d) 27 L. Huron Lk. Trout 0.69 36 6.6 28 L. Huron Lk. Trout 0.90 28 8.7 29 L. Huron Br. Trout 1.19 21 11.4 30 L. Huron Br. Trout 0.63 40 6.1 31 L. Huron Walleye 0.17 148 1.6 32 L. Huron Walleye 1.52 17 14.6 33 L. Huron - Ch. Catfish 1.40 18 13.5 34 L. Huron Carp 2.00 13 19.2 35 L. Huron Carp 4.10 6 39.4 36 L. Huron Chinook 1.13 22 10.9 37 L. Huron Burbot 0.16 156 1.6 38 L. Huron Y. Perch 0.04 628 0.4 1 Defined as the average daily reference dose of PCBs in mg/mink/day divided by the PCB concentration of Great Lakes fish in mg/kg. 2 Defined as the ratio of PCB concentration in Great Lakes fish (mg/kg) to the dietary reference PCB concentration (mg/kg). Values >1 indicate exceedance of no effect level determined in the mink reproduction study. 3 Fish contaminant monitoring data, courtesy of MDNR (1991). 4 Site number in table corresponds to location number on map in Figure 16. 127 food. Values greater than 1 indicated an exceedance of the NOAEL (control group) determined in the present mink reproduction study. The dietary reference concentration was calculated as the geometric mean of the NOAEL and LOAEL dietary concentrations from Table 29. Based on the exceedance values, yellow perch (from locations 20, 21, 22 and 38), redhorse sucker (from location 23), walleye (from locations 24 and 25) and carp (from location 5) fillets consumed by mink would not have PCB concentrations that could potentially cause adverse effects based upon the results of this mink reproduction study. However, lean fish species, such as the yellow perch and walleye, have greater concentrations of contaminants in the head and viscera. It is likely that a wild mink would consume the whole fish, rather than just the fillet. Therefore, wild mink that consumed these fish species could still be exposed to concentrations of PCBs that would impair reproduction. All other fish species from all other locations contained PCB concentrations that exceeded the no effect dietary reference concentration of 0.104 mg PCB/kg food. In addition, the higher the exceedance value, the greater the potential for adverse effects in reproduction and kit survival to occur. In order to determine whether or not wild mink could potentially consume the MADI levels of fish listed in Table 34, several references were used in calculating the 128 approximate percentage of fish that comprise the diet of mink in the wild (Table 35). Korschgen (1958), Sealander (1943), and Melquist et al. (1981) collected mink carcasses and identified the stomach contents to determine the percentage of fish in the diet. The remaining estimates were based on the identification of food types and frequency of occurrence in mink scats (Hamilton, 1940; Erlinge, 1967; Gerell, 1967; Gilbert and Nancekivell, 1982). Based on these studies, the average percentage of fish in the diet of wild mink was determined to be 29.7%. Female control mink in the present study consumed an average of 266 g of food per day. Therefore, it is estimated that a wild mink could consume up to 79 g of fish per day. In comparing the amount of fish consumed per day by a wild mink with the MADI levels, it can be concluded that a wild mink could potentially consume great enough quantities of the majority of fish species listed from locations shown in Table 34 to cause reproductive impairment based on our results with captive ranch mink. . It must be noted that, in conducting risk assessments for wild animals based upon laboratory data, certain assumptions must be made. The greatest of these assumptions in this case is that fish with these PCB concentrations are accessible and present as prey items. In addition, the availability and consumption of prey such as fish could fluctuate greatly with season and year. Melquist et al. 129 Table 35. Summary of the percentages of fish comprising the total diet of wild mink reported in the literature. Location Percent Reference Fish Missouri 19.91 Korschgen (1958) Southern 6.01 Sealander (1943) Michigan Idaho 59.01 Melquist et al. (1981) New York 27.22 Hamilton (1940) Northeastern 31.42 Gilbert and NanceKivell (1982) Alberta Northeastern 6.62 Gilbert and NanceKivell (1982) Alberta Sweden 25.02 Gerell (1967) Sweden 62.52 Erlinge (1967) Average 29.7 1 Estimates from mink stomachs. 2 Estimates from mink scats. 130 (1981) reported that larger fish eaten by mink are most likely those that are scavenged. The possibility exists that wild mink could obtain such PCB concentrations, since a mink's home range in Michigan may commonly be within 300 meters of a waterbody's shoreline (Marshall, 1936; Allen, 1986) and dead and dying fish washed ashore could be readily obtained. However, a tremendous amount of shoreline development has since occurred. Therefore, studies evaluating the exposure of a contaminated food source to wild populations of mink should include assessments of habitat quality and availability, as well as monitoring of wild mink population numbers. SUMMARY AN D CONCLUSIONS Chlorinated hydrocarbons such as the furans (PCDFs), dioxins (PCDDs) and polychlorinated biphenyls (PCBs) are both widespread and extremely persistent in the aquatic ecosystem. Research on the reproductive effects of these compounds in animals is quite extensive. The mink, used as a model in the present study, was shown to be very sensitive to the toxicological effects of these contaminants. Environmentally altered PCBs, as well as other organochlorine contaminants, in Great Lakes fish fed to mink six weeks prior to whelping significantly impaired reproduction. As little as 10% Saginaw Bay carp in the diet significantly decreased kit survival and growth rate to the age of weaning (six weeks). Ten percent dietary Saginaw Bay carp provided 0.72 mg/kg total PCBs in the diet and 19.41 ng/kg TCDD-EQs as determined by the H4IIE rat hepatoma cell bioassay. Adult female mink consuming 40% Saginaw Bay carp in the diet whelped kits that did not survive past 24 hours. Teratogenic effects were also observed at this level of carp in the diet. 131 APPENDIX A Appendix A Calculations of feed, PCB and TCDD-EQ consumption. A. Average Daily Feed Consumption (g/mink/day) [Day 1 Intake + Day 2 Intake] / 21 B. Cummulative Feed Consumption (g/mink) 21 (g/mink/d.) x (411111 C. Cummulative PCB Dose (mg/mink) c 1 B x dietary concentration D. Daily Average PCB Dose (mg PCB/mink/day) D = C / # days1 E. Adjusted Daily Average PCB Dose (mg/kg body weight/day) E = D / X weight1 1 Calculations for TCDD-EQ concentrations are the same. Units are nanograms TCDD-EQs (mg). 132 APPENDIX B APPENDIX B Total PCB concentrations in individual mink liver samples. Sample TCDD-EQ Total Total TCDD-EQ/ TCDD-EQ/ 10 No. pCB1 PCB pCB1 PCB (pg/9 lipid) (pg/9) (u9/9 lipid) (pg/9) (u9/9/9 lipid) Q§ earp ‘QQDEIQlI 01014 --2 0.084 1.75 --- --- 0 794 --- 0.074 2.18 --- --- 01102 --- 0.107 2.33 --- --- 0 984 --- 0.080 1.45 --- --- 01090 --- 0.084 2.80 --- --- 01652 --- 0.066 1.69 --- --— 0 800 --- 0.115 3.11 --- --- 0 600 --- 0.076 2.45 --- --- 01052 --- 0.109 0.96 --- --- 01040 --- 0.124 1.82 --- --- 0 614 --- 0.087 1.61 -—- --- 0 622 --- 0.121 2.24 --- --- 19$.25r2 0 618 12636.2 2.462 61.55 205.3 5132.5 0 544 10648.0 3.835 63.92 166.6 2776.5 0 602 11205.3 L.s.3 0 880 12865.6 1.710 50.29 255.8 7523.8 0 554 10069.3 2.809 39.01 258.1 3584.7 01654 8963.7 2.579 53.73 166.8 3475.6 0 796 11784.1 1.480 40.00 294.6 7962.2 01248 9066.2 2.451 31.42 288.5 3699.0 01158 14743.6 1.286 49.46 298.1 11464.7 01010 7477.5 2.048 39.38 189.9 3651.1 01194 9974.9 1.853 40.28 247.6 5383.1 0 530 5601.8 1.682 21.71 258.1 3440.9 221.9810 0 630 12782.7 2.103 38.94 328.2 6078.3 01100 14517.8 2.202 34.41 422.0 6593.0 0 550 3160.3 3.916 59.33 53.3 807.0 01150 2561.2 8.519 60.85 42.1 300.6 01660 12768.2 3.654 65.25 195.7 3494.3 0 532 3635.0 3.322 48.85 74.4 1094.2 0 566 6019.2 1.825 33.80 178.1 3298.2 0 684 5793.6 2.156 35.93 161.2 2687.2 01146 3919.8 5.428 55.39 70.8 722.2 0 462 8880.4 1.718 32.42 274.0 5169.0 0 594 3843.9 3.316 46.70 82.3 1159.2 01240 2899.9 5.585 50.77 57.1 519.2 133 APPENDIX B (cont'd.) Sample TCDD-EQ Total Total TCDD-EQ/ TCDD-EQ/ 10 No. pCB1 PCB pCB1 PCB (pg/9 lipid) (pg/9) (uglg lipid) (us/9) (pg/qlq lipid) 493.2312 01030 6460.4 6.070 75.88 85.1 1064.3 01092 11976.6 1.566 30.71 390.0 7647.9 01110 2960.5 5.063 31.25 94.7 584.7 01000 9500.7 9.245 92.45 102.8 1027.7 01198 6731.8 . 10.622 92.37 72.9 633.8 0 590 12801.9 4.433 98.51 130.0 2887.9 01134 7402.0 6.365 120.09 61.6 1162.9 01024 9050.5 8.956 89.56 101.1 1010.6 01050 10350.5 4.085 75.65 136.8 2533.8 01244 12450.1 L.S.3 1 Wet weight. 2 TCDD-EQ concentration was less than the level of detection (Spq/g). , 3 L.S. = lost sample for PCB analysis. 134 APPENDIX C 135 u.un u.o~e num.m ooou .¢.~ m~.ae 6.5 ms.su onm o m.mn m.mme ee~.> ooou hv.~ m~.me o.m ~m.eu emuuo m.mn «.mmn meo.m ooou he.~ m~.me m.m mm.mu ououo ~.en n.nmn mmo.m ooou he.~ m~.me 6.8 eu.eu emuuo m.ua ~.>os msu.» ooou m.u mo.um o.ou eo.~u memuo 6.3 6.62. 323 83 m.u 3.3 6.6 3.3 62. o s.me n.one m-.m ooou >.~ ma.oe o.u mm.uu «mouo u.meu o.m~e mmo.ou ooou s.~. mo.oe o.e~ ~n.m 6mm a «.me «.hme um>.n ooou .p.~ mm.oe o.du «o.~u one o m.~m m.eue oos.m ooou .n.~ ma.oe «.mu mu.ou mom a 6.3 668 6:6 ooou Em 3.3 0.3 3.3 38 o «.mm «.mom ome.m ooou e.m. mm.oe o.ou sm.~u mum a sumo wou 1 1 ooou 1 1 1 1 «mm o 1 1 ooou 1 1 1 1 «um o 1 1 ooou 1 1 1 1 oeouo 1 1 ooou 1 1 1 1 «mouo 1 1 ooou 1 1 1 1 com o 1 1 ooou 1 1 1 1 com o 1 1 ooou 1 1 1 1 «mouo 1 1 ooou 1 1 1 1 omouo 1 1 ooou 1 1 1 1 «mm o 1 1 ooou 1 1 1 1 mouuo 1 1 ooou 1 1 1 1 «on o .uouucoov 1 1 ooou 1 1 1 N1 «uouo sumo wo .ov .um. Ame. .ume umnaoz .o\mov umoumz mesuo> Awe omom Awe omou nu smouo .o.m mom1oooa musamm uomuuxu u>o coop u>o uomuuxu musamm ucmaummua U XHszmm< .mum>uu umua mumamu umsmu>umau uou mou1oooe mm>uumm1uuuez uou mcouumumoumo 136 .60u00u06 #02 N .so«um«um> mo usowowuu0oo 6 6.66 6.665 666.6 6666 6.6 66.66 6.66 66.66 66666 6.66 6.666 666.6 6666 6.6 56.66 6.6 66.66 66666 6.566 6.666 666.6 6666 6.6 56.66 6.66 65.6 66666 6.56 6.666 666.6 6666 6.6 56.66 6.6 .66.66 66666 6.66 6.656 666.6 6666 6.6 56.66 6.6 66.66 666 6 5.66 6.655 666.6 6666 6.6 56.66 6.66 66.6 66666 6.666 6.666 666.6 6666 6.6 56.66 6.66 66.6 66666 6.66 6.656 666.6 6666 6.6 66.66 5.6 66.56 66666 6.66 6.666 666.6 6666 6.6 66.66 6.5 66.66 66666 6.66 6.666 666.66 6666 6.6 66.66 6.66 66.66 66666 sumo 666 6.66 6.666 666.66 6666 6.6 66.66 6.6 66.66 66666 6.66 6.656 666.66 6666 6.6 66.66 6.6 66.66 666 6 6.66 5.656 666.66 6666 6.6 66.66 6.66 66.66 666 6 6.66 6.666 665.6 6666 5.6 66.66 5.5 66.66 66666 5.66 6.566 666.5 6666 5.6 66.66 6.5 65.66 666 6 6.66 6.666 666.5 6666 5.6 66.66 6.5 56.66 666 6 6.66 6.566 666.6 6666 6.6 66.66 6.6 66.66 666 6 6.666 6.665 666.5 6666 5.6 66.66 6.66 66.6 66666 6.66 6.666 666.6 6666 5.6 66.66 6.6 66.66 66666 6.66 6.666 666.66 6666 5.6 .66.66 6.6 66.66 666 6 6.666 6.666 666.66 6666 6.6 66.66 6.66 65.6 66666 6.66 6.666 666.5 6666 6.6 66.66 6.66 66.6 666 6 sumo 666 .66 .666 .osv .656 uonaoz .o\osv 666603 oam6o> Awe 6666 .66 6666 66 smouo .6.6 666166oe o6samm oomuoxu 6>o 6666 6>o oomuoxu o6samm usoaummua .66.»:ooe o xu6zuss6 BIBLIOGRAPHY BIBLIOGRAPHY Allen, A.W. 1986. 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Age and context affect the stereotypies of caged mink. Behaviora- 127:191- 229. SAS Institute Inc. 1990. WWW SAS Institute Inc., Cary, NC, pp. 1-1686. Tillitt, D. B., R. W. Gale, J. C. Meadows, J. L. Zajicek, P. H. Peterman, S. N. Heaton, P. D. Jones, S. J. Bursian, T. J. Kubiak, J. P. Giesy, and R. J. Aulerich. 1996. Dietary exposure of mink t0 carp from Saginaw Bay. 3. Characterization of dietary exposure to planar halogenated hydrocarbons, dioxin equivalents, and biomagnification. Environ. Science Tech 30):283-29l. .' 4 619376 £1362 (2755) QM g (.5va l GNP. ““003 "L 9 - A [973’ W443 Q 7311,] C6 Moshe-J e’f/th M’f of” 0 “1° l- I I'll fl {#4210 Of ”1,411.1: Mon 0.01/valid. J “23?. rs" ‘p‘llf" J gab-y chn Q/W( /i P 17 awful/14114 {’LoJ'm r1164 6n iwj‘Jfi/JL )n/// 44ij 30f Y C» )9: J F /’ V ,/ 5 1 (1;. ~‘ /~- ”’3 >/ 77 I \é/* fog/rd - PM-IL t if L w 7 2 J i i 6 3 (-1 8 6» 715.15 5 fin .. (7.11.626 / HP 370 5‘ D65 0071) 5‘1 ' ’V )4 4 I. KIA)? } C ’7 > {’5 # d4»?! 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