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" MO 51113 1 1 11": 1331""..1 1"".[311' 3“ 1,1 1 11': , , 3,1,1, . 133,311. 7:313:3313“: 3, ', I I 1'1: 1 333.3%" " l1‘1 MH,’ 9“ ”23‘5“ “,‘;;’\‘,1‘-"“"““‘ ”1‘.‘ 1311,:1‘11 "'11‘4'1111“ £1.33” 313333 335113).“ I ‘ ‘3. :31 '1 . ': 3|“|' ’ 3.51121 1:: :‘g‘n‘1 033313? I“ i ,, .... ,, 111,1-.v ~. ..3"! I3-333'1'31n‘1‘“‘”‘"w‘1" 1’13: ' “ 13‘1‘33' 3' 1.3“?)3“‘ I 3" 1.1.": :1 ’3' 1 ‘1 .33” 1 H 3111' I LIBRARY 'tfnfi‘»: MlChig‘an S" University This is to certify that the thesis entitled POLYBROMINATED BIPHENYL REDUCTION DURING PRESSURE COOKING OF CHICKEN presented by Susan Katherine Smith has been accepted towards fulfillment of the requirements for M . S . d . Foods egree 1n “2 z‘ . //%M 64: 72% Manf/Ellen Zahj/A/ Major professor Date 11/17/77 0-7 639 POLYBROMINATED BIPHENYL REDUCTION DURING PRESSURE COOKING OF CHICKEN by Susan Katherine Smith A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1977 ABSTRACT POLYBROMINATED BIPHENYL REDUCTION DURING PRESSURE COOKING OF CHICKEN by Susan Katherine Smith Effects of cooking on polybrominated biphenyls (PBB) were studied. Thigh meat, thigh skin, drumstick and breast (with skin) from half of each chicken fed 0, 30, HS, 60 or 90 ppm of PBB were analyzed raw. Pieces from paired halves were analyzed following pressure cooking separately in deion- ized water. Cooking yields were obtained and the cooked meat and broth were analyzed separately for PBB. PBB residues were found in all tissues analyzed with considerable variation in quantity found. Residue levels in tissue increased with increased levels of PBB in feed, and were related to fat level in tissue (wet basis). Levels of P883 expressed as ppm (solids basis) were lower in cooked samples than in corresponding raw pieces. Part of the PBB lost being found in the drip. PBB recoveries in cooked tis— sue and broth ranged from 68.1% (thigh skin) to 84.6% (drum- stick) with approximately two-thirds found in the cooked meat. vlu «\U u A Q» ACKNOWLEDGEMENTS The author expresses her gratitude to Dr. Mary Zabik for her guidance and helpful support throughout the graduate program. Her encouragement and continuing interest in this project are most deeply appreciated. Sincere thanks to Dr. Lawrence Dawson for his interest and advice throughout the graduate program as a guidance com- mittee member. A special thanks to Dr. Dawson for his assis- tance in the slaughter and dressing ofhens for this study. Sincere appreciation is expressed to Dr. Robert Ringer for the advice and encouragement given as a member of the guidance committee. Also, to Dr. Ringer and to the Poultry Science Department, a genuine thanks for the provision Of the hens used in this study. Gratitude is also expressed to the Pesticide Analytical Laboratory, Pesticide Research Center, for the assistance and use of instrumentation in sample analyses and quantitation. A sincere thanks is extended to Mrs. Melissa Shafer for the elaboration of the computer program for data analyses. Genuine thanks to Mrs. Marce Weaver for her assistance in refinement of the laboratory procedures, and for her en- couragement throughout the study. Appreciation is also expressed to my family for their ever present support. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES LIST OF APPENDICES. INTRODUCTION. REVIEW OF LITERATURE. Polybrominated Biphenyls. . . Chemical and Physical Properties - Uses Residues in the Environment . . . Residues in Biological Systems. Toxicity of Polybrominated Biphenyls. Mammalian Toxicity. . . . . . Avian Toxicity. . . . . . Low Level Contamination Human Exposure to P88 Contamination in Michigan. . . . . . . . . . . . . . . Polychlorinated Biphenyls - Chlorinated Hydrocar- bons. . . . . . Chemistry - Use Occurance Animal Toxicity Human Toxicity. Halogenated Hydrocarbon and Pesticide Residue Removal from Food EXPERIMENTAL PROCEDURE. Feeding PBBs. Slaughter and Preparation Cooking . . . . . iii Page vi vii viii 33 33 35 37 39 A0 A6 A6 “7 TABLE OF CONTENTS (cont.) Chemical and Residue Analyses Moisture Analyses . . . Lipid Analyses. . Residue Extraction and Cleanup. GLC Analyses. Confirmation of PBBs. Data Analyses RESULTS AND DISCUSSION. Chicken Meat and Broth Yields Percentage Fat and Solids Residue Analyses. . . . . PBB in Wet Tissue . PBB Content on a Fat and Solids Basis Recovery and Distribution of P888 After Cooking . SUMMARY AND CONCLUSIONS SUGGESTIONS FOR FUTURE RESEARCH . BIBLIOGRAPHY. . . . . . . . . . . . APPENDICES. iv Page 148 ‘49 A9 50 50 53 53 55 55 62 7O 72 76 80 8A 86 101 12 13 lil Table 10 ll 12 13 1“ LIST OF TABLES Specific industrial uses and approximate alloca- tions of FiremasteHE>BP- 6 produced. . . . Livestock known to be contaminated. Livestock and products destroyed because of PBB contamination . . LD-SO experimental test results for selected sub- stances when fed orally to rats . . . . . . . Comparison of PCB and PBB (ppm in feed) minimum effective level - as seen in chicks . . . . . Comparison of PCB and P88 (ppm in feed) minimum effective level - as seen in adult hens Analyses of variance for percentage of meat and broth yields. . . . . . . . . . . . . . . . . Means and standard deviations of the means for percentage chicken meat yield Means and standard deviations of the means for percentage chicken broth yield. Analyses of variance of percentage fat and solid in raw and cooked chicken pieces and chicken broth 0 0 0 O O O O O O O O 0 O O O O O O 0 Percent fat content of raw and cooked chicken and chicken broth . . . . . . . . . . . Percent solids content of raw and cooked chicken and chicken broth PBB levels expressed as ppm of wet tissue of raw and cooked chicken and chicken broth. PBB levels expressed as ppm in fat of raw and cooked chicken and chicken broth. Page 14 15 22 29 3O 56 57 59 6O 61 63 6A 65 LIST OF TABLES (cont.) Table Page 15 FEB levels expressed as ppm in solids of raw and cooked chicken and chicken broth . . . . . . 66 16 Analyses of variance of P88 levels in hens . . . 67 vi LIST OF FIGURES Figure Page 1 GLC tracing of P88 in standard and poultry tissue sample . . . . . . . . . . . . . . . . . . 52 2 PBBs in raw hen tissue, ppm wet weight basis. . . 69 3 PBBs in wet tissue of raw and cooked hen pieces . 71 A PBBs in the fat of hen pieces; raw, cooked and broth state . . . . . . . . . . . . . . . 73 5 Distribution of recovered PBBs between cooked tissue and broth. . . . . . . . . . . . . . . . . 78 vii LIST OF APPENDICES Appendix Page A Layer Ration for PBB Experiments . . . . . . . . 101 B Smith, S. K, M. E. Zabik and L. E. Dawson. 1977. Polybrominated biphenyl levels in raw cooked chicken and chicken broth. Poultry Science 56:1289-1296 102 viii 196' 196 and INTRODUCTION The tremendous influx of synthetic compounds and their accompanying waste materials has led to increasing concern over environmental pollutants by people world wide. Of these compounds much attention has been directed toward halogenated hydrocarbons. The nearly ubiquitous presence of chlorinated hydrocarbon residues in many levels of the biosphere is well documented (Risebrough and de Lappe, 1972; Zitko et al., 1972; Martel et al., 1975; Risebrough et al., 1968) and their pres- ence is expected to remain in the ecosystem for an extended period of time. Organochlorine pesticides and polychlorin- ated biphenyls (PCBs) with their lipophylic properties have been found in the fat of virtually all animals (Holmes et al., 1967; Holden and Marsden, 1967; Reynolds, 1969; Jensen et al., 1969; Koeman et al., 1969; Anderson et al., 1969; Longcore and Mulhern, 1973), and have been detected in human tissues as well (Biros et al., 1970; Yobs, 1972; Hammer et al., 1972; Price and Welch, 1972; Musial et al., 197A; Dyment et al., 1971; Savage et al., 1973; Solly and Shank, 197A; Doguchi and Fukano, 1975; Akiyama et al., 1975). Because of the high degree of biochemical stability (Fries et al., 1973) and lipid solubility these compounds have accumulated through food chains and in the human diet (Fishbein, 197A; Kolbye, l K1 8.8 th to. lmc COT ‘V men 001'] 1972; Fujiwara, 1975; Berglund, 1972; Fries, 1972; FAQ/WHO, 197A; Manske and Corneliussen, 197A). Concomitantly, several reports provide evidence that would indicate a substantial proportion of the population of the United States has been exposed to PCBs (Jenlinek and Corneliussen, 1975; Kurtz and Yang, 1975; Dennis, 1975; Humphrey et al., 1976; Hesse, 1975a; Kurtz and Strassman, 1975). Occurance of contamination of human tissue are generally associated with ingestion Of food containing low levels of these compounds. Although precise information concerning toxicity of halogenated compounds at very low levels is not known, the concern is for possible chronic effects of their continual assimulation and accumulation in body fat. Polybrominated biphenyls (PBBs) are members of the halo— genated hydrocarbon class of compounds with structure, reac- tivity, use and toxicity similar to those attributed to PCBs of higher chlorination. They are not present in the environ- ment in as large amounts as PCBs, and would not be of great concern had not an incredible error occured in Michigan dur- ing the manufacture of feed in May of 1973. Michigan Chemical Corporation manufactured the PBB com- pounds, Firemaster‘EFf-l.l These compounds were mistaken for magnesium oxide (trade name Nutrimaster also produced by Michigan Chemical Corp.) and mixed into high protein dairy l Firemastefifi>BP-6 mixed with the anticaking agent "Flo—Gard" which is manufactured by PPG Industries and which contains 83% silicone dioxide and a maximum of 7% calcium oxide. pellets (Jackson and Halbort, 197M; Dunckcl, 1975). Through this contamination and consequent cross contamination of other feeds, dairy animals, poultry, swine, and concomitantly animal products were affected. This mistake has cost Michi- gan farmers untold dollars in loss of animals, income from those animals and the cost of clean up procedures to rid their farms of this contaminant. In the poultry industry alone, approximately two million birds and nearly five mil- lion eggs were destroyed. Since this large scale exposure, the circumstances surrounding the accident and the herculian effort to rectify the problem has been published in several reports (Dunckel, 1975; Ball, 1975; Hoeting, 1975; Welborn, 1975; Isleib and Whitehead, 1975). Toxicity studies have shown PBBs to have similar mode of action to other halogenated compounds, namely PCBs, in that they produced porphyria, liver injury, tremor and loss of weight (Strik, 1973a, b; Jackson and Halbert, 19?”; Pre- witt et al., 1975; Lee et al., 1975; Aftosmis et al., 1972; Kimbrough et al., 1975), dysfunction of the thyroid (Bastom- sky, 197A) effect on various enzymes (Pardini, 1971; Babish et al., 1975), changes in the liver to body weight ratios (Gartnoff et al., 1975), and to alter the level of utiliza- tion of corticosteriods (Wasserman et al., 1973), and vita- min A, D and E (Cecil et al., 1973; Wong et al., 197U; Combs et al., 1975). Feeding studies have shown that bromine levels in bio- logical systems elevate rapidly and then plateau but when the 1|. animals are fed "clean feed" again the levels decrease. This decrease occurs more rapidly in milk, eggs, liver and muscle than in fatty tissue which loses its PBB store much more slowly. These data raise concern about the possible impact on fish and wildlife in the environment and the health of human beings. Studies concerning the possibility of removing halo- genated compounds from food stuffs upon processing thus of great interest. Several studies have been conducted concern- ing the removal of lipophilic compounds from such products as poultry (Zabik, 1974; Morgan et al., 1972; Ritchey et al., 1967, 1969, 1972; Liska et al., 1967; McCaskey et al., 1968), eggs (Zabik and Dugan, 1971), sausage patties (Funk et al., 1971), bacon (Yadrick et al., 1971), pork loins (Maul et al., 1971), pork muscles (Yadrick et al., 1972), beef loaves with texturized soy (Shafer and Zabik, 1975), dairy products (Murata, 1976), and salmon (Smith et al., 1973). Most of the losses seen were attributed to fat rendering or leaching out during cooking - the more severe the rendering the greater the loss. Different levels of success in elimination of contamin- ants were seen depending upon the compound worked with, the levels of contamination and the tissue from which the extrac- tion was accomplished. The purpose of this study was to investigate the effect of cooking on the PBB levels in chicken tissue and chicken broth. The ease of residue removal from skin in contrast to tissue was evaluated by cooking thigh meat and skin separately. REVIEW OF LITERATURE Polybrominated biphenyls (PBBs) are fairly new compounds, and are mainly incorporated into plastics as fire retardants for the manufacture of safe end-use products. They are the bromine analogs of the halogenated biphenyl compounds, and share many chemical properties with polychlorinated biphenyls (PCBs) and organochlorine pesticides. PCBs have been reported to affect a large number of bio— logical systems. To the extent that it has been reported, PBBs affect most of the same systems in a similar fashion. PCBs are a class of large volume and multi-use industrial chemicals which are known to be widely distributed in the global ecosystem. Many of the toxic effects of these chemi— cals have been extensively studied in both animals and humans.’ PBBs, on the other hand, have received little atten- tion because their industrial use is limited and they are not recognized as significant environmental contaminants. As a consequence, the toxicological research conducted with these chemicals has been limited in scope. This review presents the uses, characteristics and toxi- cities of PBBs, and attempts to summarize the similarities of properties of PBBs and P088. Due to the durth of studies concerning the effect of processing on PBB levels in foods, evaluation of the effect of processing on PCB and chlorinated hydrocarbon pesticide levels in foods are also reviewed. 5 CE bi ch. Fl: de] Polybrominated Biphenyls ‘Chemical and Physical Properties - Uses Due to the chemical and physical properties of PBBs (particularly that of thermal stability) they provide effec- tive and economically feasible plastisizers and fire retard- ants for incorporation into flame resistant polymers. Michigan Chemical Corporation manufactured a product called FiremasteH®)BP-6. BP—6 is a mixture of brominated biphenyl with an average of approximately 63% hexabromobi- phenyl, qu heptabromobiphenyl, 10.5% pentabromobiphenyl, 2% tetrabromobiphenyl and others unidentified (Kerst, 197“). Recent analysis with nuclear magnetic resonance (NMR) and chemical studies have identified the principle component of FiremastegaBP-6 as 2,2',A,A',5,5' - hexabromobiphenyl as depicted below (Sudstrom et al., 1976). General properties of BP-6 are: solid at room temperature, with a softening point of 72°C; decomposing at 300-AOO°C; very low solubility in water (11 ppb at 25°C); soluble in most organic solvents; 8 and a vapor pressure of 5.2 x 10- mmHg at 25°C (Kerst, l97h). The production, distribution and usage of PBBs has not been as widespread as PCBs; and unlike PCBs, may not be physically located in a position of chemical reactivity. BP-6 has been used extensively in thermoplastics. Some examples of major uses are in business machines, electrical products and fabricated products (Table l). PBBs are not used in food or feed, nor are they used in products that come in contact with human skin as in flame retarding fabrics (Kerst, 1974). Of the possible degradation routes of halogenated aroma— tic compounds in the environment, photolytic degradation is especially important since these materials do not appear to be readily metabolized by microorganisms (Norris et al., 1975). Studies indicate that BP-6 undergoes reductive de-halogenation when exposed to UV light under certain conditions (Ruzo and Zabik, 1975). This reaction has also been shown to occur in mono- and di—brominated biphenyls (Bunce et al., 1975) and octabromo-biphenyls (Norris et al., 197“). This photoreac- tivity seems to take place about 7 times more rapidly in bro- minated compounds than the corresponding chlorinated biphenyls (Ruzo and Zabik, 1975). This may be due to steric interfer- ence of the bromines and the lower C - Br bond energy (Ruzo and Zabik, 1975; Bunce et al., 1975), which is 71.0 k cal/mole compared to the C - Cl bond energy of 85.6 k cal/mole (Kerst, 197A). Presumably ortho halogens preferentially cleave from PBBs upon photoexcitation. Photodegradation of halogenated aromatics in water with UV light seems to occur through ’_._l Table 1. Specific industrialfififes and approximate alloca— tions of Fire Maste BP-6 produced. Approx. alloca- Industrial Use tion of tot 1 Examples Fire Maste BP-6 produced % Business machines and Typewriter, calcula- industrial equipment “8 tor and microfilm reader housings; bus- iness machine hous- ings Electrical 35 Radio and TV parts, thermostats, shaver and hand test hous- ings Fabricated products 12 Projector housings, movie equipment cases Transportation 1 Miscellaneous small automotive parts, i.e. electrical wire con- nectors, switch con- nectors, speaker grills Miscellaneous A Small parts for elec- trical applications, motor housings, com- ponents for industri— al equipment 1 Kerst, 197a. hydroxylation leading to the formation of phenolic compounds as seen below (Joschek and Miller, 1966). Once photodegrada- Photoreduction XI-'<::> -+lfii =9 <::> + RX Photohydroxyla- X - O — 0H + H2O—*HO-O -OH + HBr tion tion of a polyhalogenated aromatic molecule in water is ini- tiated, it should accelerate as electron with drawing halo— gens are replaced by electron releasing hydroxyls. Also as hydroxyl replacement of halogen proceeds, the resulting species would be expected to absorb more strongly in the long- er wave lengths and this ultimately could result in the rup- ture of the aromatic ring (Norris et al., 197A). Michigan Chemical Corporation contends that BP—6, after initial degra- dation has proceeded, would degrade relatively rapidly then to carbon dioxide, water and bromine ion (Kerst, 197U). Residues in the Environment The chemical characteristics of PBBs are similar to those of PCBs, of higher chlorination, and the chlorinated hydrocarbon pesticides such as DDT. Thus, one can make strong inferences about the behavior of PBB in the environ- ment based on the past experience with PCB and DDT (Fries, 1975a). Important characteristics are high solubility in fat, low solubility in water, high resistance to breakdown by 10 living organisms or chemical processes in the environment. Therefore, after PBB is introduced into the environment it will persist. Initially most of it will remain in the gen- eral area in which it was introduced. However, as time pass— es, there are a number of processes by which PBBs can be redistributed to other areas. After the initial disaster in Michigan of the introduction of BP-6 into animal feed, this feed was distributed to a number of farms through normal mar- keting Channels. PBB was redistributed further by the move- ment of animals, animal food products and byproducts. Also, PBB attached to soil particles can move both by water and wind erosion to distant areas. Some small fraction of PBB can volatilze and can subsequently wash out in rain or other precipitate (Fries, 1975a). To illucidate information concerning the movement of PBB, Hesse (1975b) studied the extent of contamination pro- duced by what was the major Michigan Chemical Corporation PBB production site located on the Pine River at St. Louis, Michigan. Water sampling revealed concentrations which ranged from 3.2 ug/l in samples taken 75 yards downstream from the plant to 0.01 ug/l in samples from 8 miles below the site. Concentrations at sample sites 12 and 20 miles downstream were below the sensitivity limit of 0.01 ug/l. PBB concen- trations in near shore stream sediments in the area of plant out falls were as high as 77,000 ug/kg. Downstream from the St. Louis Reservoir the sediment concentrations showed a gradual decline from 6,200 ug/kg l/A mile downstream, to 100 ll ug/kg 2A miles downstream. Elevated PBB levels were found in Pine River fish ranging up to a maximum Of 1.33 mg/kg/ in carp captured in the reservoir in the vicinity of Michigan Chemical Corporation. Sampling 3 miles downstream showed con- tinued high concentrations of 1.25 mg/kg and measurable con— centrations (0.09 mg/kg) in carp captured 8 miles downstream. These levels in local fish populations were sufficiently high to issue health warnings against their consumption. A study undertaken at Michigan State University by Jacobs et a1. (1976) evluated the persistance in the soil and plant uptake from the soil of PBB. It was shown that PBBs were extremely persistent with only one pentabromobiphenyl isomer showing any significant disappearance after 2“ weeks of incu- bation in selected soils. Consistent with this finding, PBBs were detected in soils from a field which had received manure from a PBB contaminated dairy herd 10 months earlier (Jacobs et al., 1976). Orchard grass and carrots grown in soil con- taminated with PBBs showed none or only very minor uptake of PBBs, respectively. Jacobs et a1. (1976) concluded that the potential hazards from PBB contaminated soils are low since not only were the PBBs not taken up by the plants, they were also not leached below the depth of their incorporation. Filonow et a1. (1976) and Islieb et al. (197“) concurred that PBBs do not leach down into the soil. Michigan Chemical Corporation (1970) has predicted that under normal use of PBBs, its migration in the soil would be minimal; based on the fact that BP-6 is known to be tightly er: the gal am l2 bound into the plastic into which it is incorporated. BP-6 is expected to react in a manner which is similar to the more highly chlorinated PCBs. These exhibit an extremely small migration tendency. Residues in Biological Systems Accidental substitution of FiremasteflB’BP-6 in place of magnesium oxide in animal feed resulted in widespread con- tamination of cattle, poultry and swine during the fall of 1973 and the winter of 197“ (Jackson and Halbert, 197A; Gut- enmann et al., 1975; Detering et al., 1975b; Prewitt et al., 1975; Islieb and Whitehead, 1975; Fries et al., 1975). The first herd reported to be affected has been des- cribed by Jackson and Halbert (197a). In September, 1973, the “00 head Halbert herd, located near Battle Creek, Michi- gan, experienced a problem with milk cows refusing to eat and a dramatic deterioration of animal health. Extensive testing was carried out by the Michigan Department of Agri- culture (MDA). Finally, in late April, 197“, George Fries in the USDA laboratory at Beltsville, Maryland identified the feed contaminating compound as PBB, and the problem was traced back to its origin. Upon identification of PBB as the unkown contaminant, an intensive effort was made (by the Food and Drug Admini— stration (FDA), Detroit District; MDA; Michigan Department Of Public Health; persons at Michigan State University and others) to exclude contaminated food and feed from further 13 use. The second objective was to learn as much as possible of the human, animal health and environmental consequences of PBB contamination. Tolerances were established on May 10, 1974 by the US Food and Drug Administration. They were: meat and milk, 1.0 ppm; eggs, 0.1 ppm; and feed 0.3 ppm. These toler- ances were revised downward on November A, 197“ to: milk and meat, 0.3 ppm on fat basis; and eggs and finished feed, 0.05 ppm. Recently (August, 1977) a newly revised tolerance (pf 0.02 ppm in meat has been established. These action gguidelines allowed the MDA to determine how many herds and :flocks in the state were contaminated above the guideline Ilevels. This was necessary so these units could be quaran- txined and provision could be made for disposal of animals, txirds and products rendered useless by contamination. Tables 22 and 3 show portions of the May 10, 1976 PBB contamination Status Report prepared by the MDA. The efforts of monitoring for PBB by the USDA, MDA, FDA, Enid public health authorities of the food supply have been <30ntinuous since PBB contamination was discovered in 197”. DR) foods are being offered for sale which are above the guidelines set by the FDA. For example, no trace of PBB has kneen found in processed milk in Michigan since early June 197“ (Cooperative Extension Service Bulletin, April, 1976; Islieb, 1976). Concern has been directed toward levels of residues in, and.excretion of, PBBs from affected animals - from the 14 «OLSBHSOfiLw< mo pCoEupmme :wwHQOHz .owma .OH mm: .ppoams mdpmpm coapmcfiempcoo mmm Eopm H m mmmmo cam mxoso m mmmpom mm moanpmm ems aaeoe m mpmou ooo.mm mmfi sppfizom ooo.omm.a seaflzom 0mm m gmmcm msz.H ammcm co: so mcfizm mma.m mcfizm ooo.mH we: mfipsmo ooo.mm masses meEHc< wmmHEmpm Hmefice amnesz HmEHC< mo honesz mo pmnesz coapmcHEMBCOQ Hm>mq 304 pm mmmHEmpm czocx zsmfl .m as: mocfim BO>HO>CH xooumm>fiq H .UmpBCHEmpcoo on Op czocx xOOpmo>Hq .m magma .msmH .OH Am: .mezp nHBOHpm< mo pcoEppmdmo cmeQOHz .upoamp mzpmpm :oHpmcHEMpcoo mmm Eopm H m mmmpom mwmm Rmm.ssm.: mmwm mm mpHnnmm mu mnH 3mm.m swapsm m mpmoo mnH ooo.:m wsosuosa xHHE sea Ham.H nmmcm mpH szm.RH mmmmzo mmo.: mcHzm cos mom atom :Hm.mm mHspmo pcsoe< EOOH unpasz HmEHc< Umzoppmmo muozpopm OpHm mxmmmex pm Umesm xOOpmo>HH H.20HpmcHEmpsoo mmm mo wmsmOmn cmzopummp mposcopq cam xooumm>HH .m mHnt 16 standpoint of safety of human consumption of such animals and their products. Several studies concerning PBB residues have shown that PBB is excreted into the milk of cows and eggs of chickens (Gutenmann et al., 1975; Fries et al., 1975; Willet and Irving, 1975). Willet and Irving (1975) found PBB appeared in the plasma, milk and feces of cows after experimental intraruminal doses of the product were given. Gutenmann and Liska (1975) reported the distribution of PBB residues in the tissues of cows with the highest concentra- tion found in fatty tissues. A two year rat study conducted by Norris et a1. (1974) providing 0.1 mg decabromodiphenyl oxide (DBDPO)/kg/day in the diet, revealed the bromine concentration reached a plateau in the liver within thirty days while the concentration in adipose tissue slowly increased. A comparable octabromobi- phenyl (OBBP) study (Norris et al., 197“) revealed liver and adipose tissue bromine levels increased rapidly with no pla— teau reached in the 180 day study. Neither compound produced an accumulation in other tissues. Fries et a1. (1975) con- curred with this data and found a higher percentage PBB in milk fat, due to the increased percentage of fat content. Several other studies also observed this same concen— tration of PBB in fat. Fries et al. (1973b) observed that PBB was accumulated in body fat by hens and cows at levels above those of the diet. PBB levels in eggs declined rapidly when hens were switched to clean feed, although body fat lev- els remained high. PBB in milk fat also exceeded the concentra- tion in the diet, and diminished upon removal of PBB from the l7 cow's feed. Detering et al. (1975c) found the content of PBBs in blood, milk and body fat of cows contaminated 7-9 months previously showed that body fat and milk fat con- tained 600 and 300 times, respectively, of that which the blood contained. Fries et al. (1973a) demonstrated that resi- ,dues of PBBs in animal systems behave similarly to the resi- dues of PCBs with a comparable degree of halogenation. In examining the excretion rate of FiremasteHE>BP—6, PCB and DDE into the milk of cows, Fries and Marrow (1975) found that the half life of PBB in milk was 58 days. The decay rate and steady state excretion levels were similar to those for PCBs and chlorinated hydrocarbons. Studies done by Ringer and Polin (1975) showed that the accumulation of PBB in the egg of the hen is in direct rela- tion to the level in the diet, and reaches a constant concen- tration in the egg as soon as the yolk is fully formed in the ovary (taking 7-10 days). The concentration is 1.5 times that in the diet. The biological half-life of PBB in the egg, once the drug has been removed from the diet, is about 17 days. Nearly 60% of the daily intake of PBB in feed goes directly into an egg (yolk portion). That which remains goes into fat depots, liver, muscle, or is excreted in the droppings (about 10-11% of daily intake is excreted). The -results of these feeding studies are similar to those found with chlorinated hydrocarbon insecticides (Cummings et al., 1965). Stadelman et a1. (1965) studied the persistance of 18 selected hydrocarbons after a measured exposure and found up to twenty-six weeks were required to completely remove resi- dues from hen abdominal fat and egg yolk. Liska et al. (196“) looked at DDT residues in eggs and tissues of chickens on low DDT level rations. The residue levels in both eggs and fat increased sharply, thus the fatty tissue concentrated the contaminant. Feeding studies show bromine levels in biological sys- tems build up, seem to level Off and remain stable, and then when the animals are again on "clean feed" the levels decrease. More rapid decreases occur in milk, eggs, liver and muscle than in adipose tissue which loses its PBB store much more slowly. Toxicity of Polybrominated Biphenyls The primary concern with the toxicity of PBBs revolved around its structural similarity to PCBS. Since both human and animal toxicological data are available for PCBs, compari- sons are made between the two groups of compounds. For brominated biphenyls limited toxicity data are only available on mixtures containing predominantly hexa- and octa-bromobiphenyl. Both mixtures differ sufficiently in isomeric composition so that their toxic effects may quanti- tatively be quite different, and also different from PCBS. The problem of toxic contaminants such as dibenzofurans and naphthalenes, has not yet been resolved for both PBBs and PCBS. 19 Aftosmis et al. (1972a, b) described toxicity of the more highly brominated PBB mixture. This mixture was prin- Cipally octabromobiphenyl (OBB). Liver wenfln;increase was dose-related, and bromine accumulated in liver, fat and muscle tissue. Bromine levels remained elevated after 18 weeks on "clean feed" with no decrease in level in fat tis- sue; however, levels in liver and muscle decreased. Histo- pathological changes in the liver of rats receiving 100 ppm and 1,000 ppm OBB were observed. Abnormal fetuses were observed in litters from mothers on 1,000 ppm and 10,000 ppm OBB diets, but no conclusion as to compound relationships of these abnormalities was reached by the authors. Bromine was diluted in fetuses of mothers receiving OBB in their diets. Hexabromobiphenyl (HBB) was shown to be more toxic than OBB by acute skin absorption when applied at 35 percent in corn oil paste. Also, HBB, OBB and decabromobiphenyl (DBB) were compared by direct interperitoneal injection (in corn oil) in rats. After one week, HBB (especially a preparation con- taining lower bromologs) accumulated in body fat far more than OBB or DBB. These observations caused PBBs to be rejected as candidates for flame retardants in polyester fibers (Aftosmis et al., 1972a, b). The work of Norris et al. (197“) demonstrated hematolo- gical changes, liver enlargement, and liver and kidney lesions at all levels of OBB (1.0, 01, 0.01%) in the diet. Decabromobiphenyl oxide was less toxic. Lee et a1. (1975) also induced enlargement of the liver due to hypertrophy of 20 liver cells in rats fed 100 and 1,000 ppm OBB for 2 and A weeks, respectively. Microscopic lesions and pathological changes also occured in the liver. Analysis of the tissues revealed a dose related build up of bromine predominantly in the fat as well as the liver. The available data suggests PCBs and PBBs containing six or fewer halogen atoms are readily absorbed from the gut of higher animals. The available data also implies that PCBs are not excreted to an appreciable extent prior to metabolism to more polar compounds, and that long term PCB storage is in the skin and adipose tissue (Matthews and Anderson, 1975a). Laboratory studies have demonstrated that the rate of PCB metabolism and thus excretion is approximately inversely proportional to the degree of PCB chlorination so long as there are two adjacent unsubstituted carbon atoms on the biphenyl ring. When two adjacent unsubstituted carbon atoms are not present the biological half-life of the given PCB may be a matter of years, and accumulation of high tissue con- centrations with continued exposure is inevitable (Matthews and Anderson, 1975b). The major constituent of FiremasteHE> BP-6 does not have two adjacent unsubstituted carbon atoms, and the corresponding PCB has been shown to have an extremely long half-life in the laboratory rat and probably the human population as well. Poor metabolism and excretion of P885 may lead to long retentions, predominantly in adipose tissue, with accumulation to very high levels on continued exposure. Whether this would 21 lead to sufficient recirculation of the chemicals to cause toxic effects on target organs is presently not known. Toxicological evaluation by way of the oral lethal dose (LDSO) comparison of PBB with other halogenated compounds shows that it has low acute toxicity. The data with regard to acute toxicity in rats given in Table A suggests PBBs are "practically non-toxic." The varying degree of toxicity of P888 in animals is most often correlated to the concentration of these chemicals at which the animals are exposed. The LD50 dose for Japanese quail exceeds 1.0 g/kg (Strik, 1973), for rats the value found is in the range of 21.5 g/kg, and the acute derimal LD5O for rabbits is between 2.15-10.0 g/kg (Michigan Chemical Corporation, 1970). Patch application produced slight erythema and edema. No irritative effects were produced in the eyes of albino rabbits by application of the test material, and no mortalities resulted from inhala- tion of a PBB dust concentration of 71.1 mg/liter (Michigan Chemical Corporation, 1970). Based on these results, PBB was described by Michigan Chemical Corporation as non-toxic by ingestion or dermal application, not a primary skin irritant or a corrosive material, not an eye irritant, and not highly toxic by inhalation exposure. However, PBBs are fat soluble and, as already stated, accumulate in the body. Field experience with highly contam- inated feed (2,000-A,000 ppm) indicates there is toxicity especially in younger animals and those not lactating or pro- ducing eggs (Hoeting, 1975). 22 Table A. LD50 experimental test results for selected sub- stances when fed orally to rats.1 Substance Mg/Kg Body Weight Dieldrin A0 DDT 285 PCB 10,000 Fire retardant - PBB 21,500 Heptachlor 90 Lindane (benzene hexachloride delta isomer 1,000 Metathione 1,156 Methoxychlor 5,000 Sodium chloride (table salt) 3,000 l Cooperative Extension Service, East Lansing, MI. April, 1976. 23 The clinical symptoms of animals involved in the Michi- gan PBB contamination incident are complex and confusing because there seems to be little consistency between the levels at which the animals were exposed and the symptoms they exhibit. Most dairy animals exposed to toxic levels of PBB showed symptoms of malnutrition and starvation which couldn't be readily distinguished from poor rations. Chemi- cal analysis is the only positive method of determining PBB exposure. However, as the following studies demonstrate, PBBs have a similar mode of action as halogenated compounds in that they produce porphyria, liver injury, tremor, loss of weight and retarded growth. Mammalian Toxicity Preliminary results of studies (Gartoff et al., 1975) with rats to determine biochemical toxicologic characteris- tics of PBB were reported by Kolbye (1975). These studies were initiated in October, 197A by scientists from the Bureau of Foods, with rats being fed diets containing various con- centrations (5, 50 and 500 ppm) of Aroclon3’125A (PCB) or BP-6 (PBB). The results obtained indicate that both PCB and PBB cause dramatic alternations in normal biochemical and physiological processes. These effects occurred over many levels of biological organization, from macroscopic retarda— tion of growth to submicroscopic changes in the relationships between cellular molecules. Many of these specific biochemi- cal effects have been reported in previous work on the toxi- cology of PCB (Mehlmann et al., 197Aa, b; Mackerer et al., 2A 1973). It was found that the most obvious effect of PCB and PBB on these rats was a retardation of growth seen at the high level of exposure (500 ppm fed). Growth depression is a constant finding in studies where high levels of PCB were fed to lab animals (Mehlmann et al., 197Aa, b; Allen and Abrahamson, 1973; Buckner et al., 197A; Koller and Zinkl, 1973; Kimbrough et al., 1972), and was a symptom reported in the farm animals ingesting PBB contaminated feed. The depressed growth of rats found by Gartoff et a1. (1975) study was due primarily to decreased feed efficiency and secondar- ily to decreased feed consumption and feed efficiency. In these rats disruptions in the energy metabolism of ‘the cell were produced by even three weeks of exposure to PCB zand PBB. Greater disruption of mitochondria energy produc- ‘tion was caused by PBB than PCB. Further analysis of the intermediary metabolites in the liver of rats fed PBB and PCB indicated a perturbation of the redox state of the cell. The :redox state is intimately related to cellular energy produc- tion. This study also observed (at 50 and 500 ppm level) that increased cell size and resultant liver enlargement were more marked with PBB than PCB. The changes in the liver composi- tion which were associated with liver enlargement were also greater in animals fed PBB. These changes included an increased percentage dry weight and increased percentage liver lipids consisting of increased cholesterol, phospho- lipid and neutral lipid. The Changes seen in the liver 25 composition with PBB fed are an established part of the now classical response to PCB (Kolbye, 1975). This response includes proliferation and concentric array formation of the smooth endoplasmic reticulum, fatty liver degeneration and induction of microsomal mixed function oxidase (MFO) activity (Allen and Abrahamson, 1973; Bruckner et al., 197A). Farber and Baker (197A) found that, on a molar basis, hexabromobiphenyl was approximately 5 times more potent than PCB ArocloHB>125A as an inducer of mixed MFO enzymes at 5 Ppm level in rats. A three fold difference was observed by Gartoff et a1. (1975) between FiremastefiB>BP-6 and Arochlona 12AA in enzyme induction. Among PCBs it appears as if their potency increases with increasing chlorination and chlorine substitution in the para>ortho>meta positions, respectively (Echobichon and Comeau, 1975). Echobichon and Comeau (1975) suggest that since induction of MFO enzymes may result in increased hormone metabolism of carcinogen activation, expo- sure to the PCBs and PBBs should be limited on that basis alone. Plasma and serum components were also analyzed in these PCB and PBB fed rats (Gartoff et al., 1975). The only sig- nificant effects were a change in blood glucose and choles- terol levels. PCB caused a decreased blood glucose level in animals fed 50 and 500 ppm while PBB caused a slight decrease only at 50 ppm. On the other hand, cholesterol was increased by a diet with 5 ppm PBB, but only by a diet with 500 ppm PCB (Kolbye, 1975). 26 In a study carried out by Corbett et al. (1975) BP-6 was shown to be weakly teratogenic causing exencephalia in the offspring of the mice that received both 100 and 1,000 ppm dosages. Cleft palate and defective kidneys were noted in offspring at the 1,000 ppm level. BP-6 fed to pregnant rats and mice (100 and 1,000 ppm) resulted in dose dependent decrease in mean fetal weight. Liver abscesses, enlargement and hepatic pathological changes were the most common symptoms of acute and chronic toxic effects seen in the Michigan cattle (Jackson and Hal- bert, 197A). These were seen when the level of PBBs in the body fat were approximately 200 ppm. Hematomas and abscesses in the peritoneal and thoracic cavities of these cows were also observed. From field observations of PBB contaminated cattle, Prewitt et a1. (1975) reported that animals which had over 20 ppm in their milk fat at parturition, had unrelaxed pelvic ligaments resulting in difficult labor, and the calves were stillborn or died shortly after birth. Metritis (inflamma- tion of the uterus) and retained placentas, as well as liver and kidney adhesions, were also common. Examinations of dead calves showed PBBs were transferred through the placenta to the fetus and were embryo-toxic (Detering et al., 1975b). PBB concentration in the body fat of these calves was in the range of 50 to A00 ppm. 27 Avian Toxicity Strik (1973b) reported hexabromobiphenyl (100 and 250 mg/kg body weight) fed to Japanese quail and chickens induced porphyria, especially in the liver. Hepatic porphyria is characterized by an accumulation of porphyrins in the liver, kidneys, intestine and other organs. Strik (1973b) saw species differences in porphyria and attributed these dif- ferences to those within and between species, the physiolo- gical state of the animal, dosage, route of administration, age and feeding of the animal. Adequate protein diet seems to provide some protection against porphyria (Strik, 1973b). Hepatic microsomal oxidase enzymes of Japanese quail were induced by dietary PBB at 10, 20, and 100 ppm (Babish et al., 1975). Egg production was reduced with none of the eggs hatching from quail hens fed 100 ppm PBBs. Forty percent of the embryos died in the first day or two of development. How- ever, when the feed contained 10 and 20 ppm of PBBs no effect was observed. Also, there was no egg shell thinning in any of the treated groups. Cecil et a1. (1975) Studied the response time to phenobarbitol as indication of microsomal enzyme activity in Japanese quail. Hexabromobiphenyl behaved similarly to the higher molecular weight polychlorinated terephenyls in that it did induce enzyme activity and was less active than the PCBs studied. Lillie et al. (197A) fed poultry various PCBs and PBBs at 2 and 20 ppm levels. Egg production, feed consumption and 28 body weight gain of the progeny were reduced by hexabromo- biphenyl at the 20 ppm feed level. Toxic symptoms in poul- try as enumerated by Ringer and Polin (1977) seem to be liver and thyroid enlargement, decrease in spleen size, a state of anemia (hematocrit and hemoglobin content decreased), edema (manifested as hydropericardium interfering with normal heart function), and depression of egg production, hatchabil- ity, progeny survival and progeny growth. Consumption of PBB did not influence egg shell thickness, egg weight or ability of egg to be fertilized by sperm at levels studied. A com— parison of PBB and PCB and minimum contaminant levels at which there is an observable toxic effect in chicks and in adult hens is shown in Tables 5 and 6, respectively. Gener- ally, the contamination level at which changes are seen is higher in PBB than in the PCBs tested. Low Level Contamination In order to evaluate the effects of low level contam- ination (levels above the previous FDA guideline of 0.3 ppm PBB in fatty tissue), Deming (1975) visited 72 quarantined low level herds of dairy cattle. Sixty four percent of the herds visited had experience 20-50% milk production drops with a substantial proportion of the drop attributed to high incidence of sterility problems in the herd. Retarded growth of young stock was also significant which Deming believed to be due to some form of hormone interference in these low I“) Table 5. Comparison of PCB and PBB (ppm in feed) minimum effective level - as seen in chicks.1 Affected Parameter PCB PBB Body weight 125A + 50 + 75 Liver weight 12A2 t 25 t > 25 > 50 Thyroid weight no effect t 100 Comb weight 12A2 l < 100 + 50 Testes weight 12A2 + 100 l 200 Spleen weight 12A2 i 100 l > 50 > 100 Hematocrit 125A + 50 + 75 Hemoglobin 12A2 + 25 + 75 Hydropericardium 12A2 t 50 t 75 Ringer and Polin, 1977. Legend: Aroclor8>l25A - biphenyl of 5A% average chlor- ination. ArocloHE)l2A2 - biphenyl of A2% average chlor— ination. + - decreased effect due to contaminant. t — increased effect due to contaminant. Number following arrow indicates ppm of contam- inant in feed. 30 Table 6. Comparison of PCB and PBB (ppm in feed) minimum effective level - as seen in adult hens. Affected Parameter PCB PBB Feed consumption + ? + 125 Egg production l2A8 + 10 + A5 Eggshell thickness no effect no effect Egg weight no effect no effect Fertility no effect no effect Hatchability 12A8 + 10 + A5 Progeny survival 12A8 + 20 + 30 Progeny growth 12A8 l 20 l > 25 < A5 Liver microsomal enzyme induction + + Pentobarbital sleeping time t + 1 Ringer and Potin, 1977. Legend: ArocloHE>l25A - biphenyl of 5A% average chlor- ination. ArocloHE>l2A2 - biphenyl of A2% average chlor- ination. i - decreased effect due to contaminant. i - increased effect due to contaminant. Number following arrow indicates ppm of contam- inant in feed. 31 levels. Body functions suspected to be involved were, meta— bolism, hormone, and blood cell producing mechanism. Fries (1975b) reported on a long term observation on the effect of PBB on health and production in dairy cows. Four cows were fed 10 mg PBB/day for 60 days. These cattle were evaluated two years later and it was found that there were no important effects on them as compared to the control group. A dairy herd health survey was conducted by Meroser et a1. (1975), again to assess those herds with low level (those with trace to 1 ppm PBB in body fat or milk fat) contamina- tion; to see if they showed greater health problems than did non-contaminated herds. Based on these data, it was concluded that there were no herd health problems observed that could be attributed to the presence of low levels of PBB. Human Exposure to PBB Contamination In Michigan It has been estimated that between the onset of BP-6 contamination in the fall of 1973 and the establishment of the quarantine of affected herds and flocks in the spring of 197A, 8,000-10,000 Michigan residents have been exposed to PBB through the consumption of contaminated eggs, meat, milk and other dairy products (Kolbye, 1975; Schmidt, 1976). A considerable amount of variation in both length and level of exposure has probably occured. As a group, the farm family members have been at greatest risk followed by those indivi- duals who purchased products from contaminated farms on a ’) C. regular basis. In order to determine whether or not persons exposed to PBB contaminated products had suffered any acute adverse health effects, the Michigan Department of Public Health undertook a series of studies in the summer and fall of 197A. Study participants for the exposed group were dairy farm residents from farms which had been quarantined by the MDA. Non-exposed subjects were randomly selected from a list of dairy producers in the same geographical areas where farms had been quarantined. A total of 298 persons were interviewed for exposure data, illness histories and physical examination and/or blood samples were obtained from 110 persons in the exposed group and 10A persons from the control group. This study revealed no disease, symptoms, or laboratory findings that occured consistently in the exposed group, or that was significantly more frequent among the exposed individuals as compared with the controls. There was no positive relationship detected between PBB blood levels and the occurance of any symptom in the individuals. Physical examination of adults and children showed no unusual abnormalities of the heart, liver, spleen or nervous system. Urinalysis and complete blood count did not reveal a significant excess of unusual abnormalaties related to exposure or PBB levels. Several exposed females delivered normal babies without complication. Tests showed concentrations of PBB in breast milk to be considerably high- er than found in paired blood plasma, as high as 175:1, 33 respectively (Michigan Department of Public Health, 1975b; Humphrey and Hayner, 1975; Fine, 1976). The concern still remained, however, that there might be long-term effects of the BP-6 contamination. The clini- cal observations in Japan relating to PCB ingestion suggested skin, liver, nutritional and neurologic changes, seen in individuals ingesting PCBs, might be signs of PBB toxicity. Therefore, a long term epidemiologic evaluation sponsored by the Michigan Department of Public Health, the US Food and Drug Administration and the Center for Disease Control has begun (Michigan Department of Public Health, 1975a). Four thousand persons are taking part in this study and are being followed closely for the development of skin disease, liver disease, metabolic and neurologic changes. Polychlorinated Biphenyls - Chlorinated Hydrocarbons Chemistry - Use Polychlorinated biphenyls are quite stable and are con— sidered "industrial chemicals" with a variety of uses. They had important applications as plasticizers in plastics, and modifiers in many paints and other products, as lubricants, electrical insulators and fire retardants. They were also used in printing inks, textile oils and heat transfer fluids and many other products (US Dept. of HEW, 1969). PCBs have 3A also been used for mixing with chlorinated insecticides to suppress vaporization and extend their "kill life" (Sullivan et al., 1953; Lichtenstein, et al., 1969). Now the use of PCBs is limited to closed systems for which there is no sub- stitute. In the US PCBs were manufactured by Monsanto Chemical Company with the trade name ArocloflBA this year their produc- tion of PCBs has stopped. Aroclors were mixtures of com- pounds with the content of individual Aroclors varying from approximately 10-70% chlorine by weight, and the physical properties varying with chlorine content. Those with low percentage chlorine are very fluid liquids; as percentage chlorine increases the products become more viscous or become solids. The series of numbers designating individual Aro- clors are as follows: the 1200 series indicates a biphenyl and the last two numbers in the four number series indicates percentage chlorine content. For example ArocloHB>l26l is a biphenyl with 61% chlorine. PCBs are considered chemically inert, are not hydrolyzed in water, and are resistant to alkalies, acids and corrosive chemicals. They have low volatility and their boiling points range from 278°C for ArocloHE>122l to A15°C for ArocloH®>l268. All are stable to prolonged heating at 150°C. PCBs are in- soluble in water and very soluble in hydrocarbon solvents (Peakall and Lincer, 1970). 35 Occurance The nearly ubiquitous presence of PCBs is fully docu- mented. Studies indicate that sources of these chemicals are generally associated with waste disposal materials (Carnes et al., 1972; Schmidt et al., 1971; Fujiwara, 1975; Martell, 1975; Hammer et al., 1972; Nisbet and Sarofim, 1972; Trout, 1972). With sludge disposal taking place by incinera- tion, land fill, and crop or pasture application, the contam- ination of PCBs in the environment seems obvious. PCBs and organochlorine insecticides are persistant and accumulate in oil and fat so that the highest levels are found in fatty tissues Of birds and in the blubber of certain marine animals. One difference between the two groups of compounds is that, whereas insecticides are found in signifi- cant quantities in nearly all species of animal and plant life, significant amounts of PCBs are usually associated with the marine or aquatic environments. Thus, the higest levels of PCBs have been found in fish-eating birds and in marine mammals (Jenson et al., 1969; Anderson et al., 1969; Holden and Marsden, 1967; Peakall et al., 1972; Dennis, 1975). In the US, minimal human PCB exposure is due to food, air and water; while significant human exposure appears to be limited to sports fishermen consuming fresh water fish from contaminated streams and lakes, and to occupational exposure in industrial workers. A number of studies show that human tissue contains low levels of halogenated com- pounds. Yobs (1972) reported PCBs, and Biros et a1. (1970) 36 found PCBs and DDE in adipose tissue. Fujiwara (1975) found PCBs, DDT, DDE and several BHC isomers, and Solly and Shanks (197A) reported on PCBs and organochlorine pesticides in human adipose tissue. Also,analysis of human blood showed varying concentration of PCBs, BHC, DDT, and DDE residues (Doguchi et al., 1975; Fujiwara, 1975), depending on the area studied, those with greater environmental contamination had increased levels in the blood analyzed. A range of levels of PCBs in human milk has been detect- ed. In Colorado (Savage et al., 1973) levels were not appreciable, while in Japan, the PCB concentrations found were relatively high and it was estimated that babies would ingest between A.8-5.3 mg/kg body weight of PCBs/day (Fuji- wara, 1975). Musial et a1. (197A) found that milk from people of provinces of Canada contained PCBs as well as DDT and DDE. Dyment et a1. (1971) found PCBs in human milk and serum from Texas and human milk from New Guinea. Other works that have reported the occurance of organochlorine pesticides in human tissue (Price and Welch, 1972; Brown, 1967; Abbot et al., 1968; Dyment et al., 1971) contribute to the evidence of the ultimate disposition of these compounds in man. The major source of human contamination has generally been associated with ingestion of food containing these com— pounds (Kolbye, 1972; Fujiwara, 1975; FAO/WHO, 197A). Jelinek and Corneliussen (1975) reviewed the data from the FDA Total Diet Study (1971-1975). They listed three sources of PCB food contamination: 1) environmental contamination - 37 levels in fish from lakes and streams, 2) industrial acci- dents - isolated incidents involving leakage and spillage of PCB fluids and other PCB materials on animal feeds, feed ingredients or food, 3) food packaging materials - PCB migra- tion to foods packaged in PCB contaminated paper products (Trout, 1972). Jelinek and Corneliussen (1975) noted that all food classes of the total diet have declined to no occur- ance and no calculated daily intake of PCBs except in the meat—fish-poultry composites. About A0% of these composites contain detectable PCBs although only traces (below 0.05 ppm) in some have been detected in later years with tighter con— trols. It is estimated that PCB in the total diet for the general public is 5—10 ug/day (Jelinek and Corneliussen, 1975). Low level findings are primarily due to the fish in these composites. Animal Toxicity The range of sensitivity to PCB is enormous. Inverte- brates show effects at levels of a few ppb (Duke et al., 1970), while at the other extreme Escherichia coli appear to thrive at thousands of ppm (Keil, 1972). Fish, birds, and mammals fall between these extremes. The different commercial mixtures of PCBs elicit differ- ent toxic responses in animals, and different animal species seem to vary in their susceptibility to the toxic effects of PCBS. Reproduction is severely affected in milk at a 38 dietary level of 5 ppm Aroclong)l25A and a slight effect is still noted at a dietary level of 1 ppm (Ringer et al., 1972). In the Rhesus monkey, reproduction was reduced at a dietary level of 2.5 ppm Arocloéfi)l2A8 (Allen, 1975). In rats, a dietary level of 20 ppm ArocloHE)125A depressed reproduction and in the same laboratory with the same rat strain a level of 500 ppm Arocloég)l260 was necessary to reduce reproduction (Linder et al., 197A). Hepatic porphyria has been reproduced in a number of species including the chicken (Vos and Koeman, 1970), rabbit (Vos and Beems, 1971), and the rat (Iverson et al., 1975). Hepatic porphyria occurs along with an increase in aminolevu- 1inic acid synthetase (ALA) in the liver. Mixed function oxidases (MFO) are also induced in the liver, and comparative studies with PCB isomens have suggested that when the A,A' positions on the biphenyl ring are occupied by chlorine atoms the effect is more pronounced (Ecobichon and Comeau, 1975). MFO enzymes are induced in the rat with as little as 10 ppm in the diet if the animal is exposed for a long enough period of time (Turner and Green, 197A). Liver enlargement is shown in the rat (Kimbrough, 1975) concomitant with lipid accumulation and increase in cell breakdown with either higher doses or longer exposure. In the primate, in addition to the effect on the liver, the gastric mucosa is affected (Allen and Norback, 1973), the skin is affected and bone marrow is depressed (Allen, 1975). In the rabbit, atrophy of the thymus is seen in addition to 39 liver pathology (Vos and Beems, 1971). Fluid accumulation occurs in chickens (Vos, 1972; Cecil, 1973) and the lymphatic system is affected in mink (Ringer et al., 1972). Decreased hatchability and teratogenic effects in chick embryos were recorded by Cecil et a1. (197A). Human Toxicity Adverse human health effects resulting from PCB expo- sure have come primarily from occupational exposure and from exposure through the ingestion of contaminated rice oil by people in Japan. Schwartz (1936) provided some of the ear- liest reports of adverse effects due to occupational exposure in the US, in which he described skin lesions and symptoms of systemic poisoning among workers who were reported to have inhaled chlorodiphenyls. There have been numerous reports over the years describing such skin eruptions and of syste- matic manifestations as well, among marine electricians, machinists, capacitor and transformer manufacturing workers and others occupationally exposed to PCBS. The skin lesions described by Schwartz in 1936 have come to be designated as "chloracne." Part of the chloracne lesion resembles adoles- cent acne, but is generally more severe and the lesion dis- tribution is inconsistent with adolescent acne. Typical clinical findings in the human exposure to PCBs which occured in Japan in 1968 and which resulted from ingestion of rice oil (contamination as high as 2,500 ppm in the canned oil) A0 included chloracne and increased pigmentation of the skin, increased eye discharge, transient visual disturbances, feel— ing of weakness, numbness in limbs and some disturbance in liver function (Kuratsune et al., 1972). Halogenated Hydrocarbon and Pesticide Residue Removal from Foods Surveys of food in the market place indicate varying amounts of pesticide residues in many commercial foods (FAO/ WHO, 197A; Jelinek and Corneliussen, 1975; Duggan and Weather— wax, 1967; Duggan and Lipscomb, 1969). The total dietary exposure varies from one part of the country to the other and depends upon the dietary habits of the individual or family. Few, if any, foods are completely free from some degree of pesticide residue. As stated previously PCBs and chlorinated hydrocarbon pesticides have been detected in human tissues and their occurance is generally associated with the ingestion of food containing low levels of these compounds. Although precise information concerning the toxicity of these compounds at very low levels is not known, the interest in removing resi- due compounds from food stuffs with processing comes from the possible chronic effects of continued assimilation and accumulation of these compounds in body lipids. Numerous studies have been conducted concerning the amount and procedures most effective in the removal of Al pesticides and chlorinated hydrocarbons from our food supply. The effectiveness of molecular distillation in removal of pesticides from milk fat has been evaluated by Bills and Sloan (1967). Heat, deodorization, steam deodorization and freeze drying have been used to study their effect on insec— ticide residues in milk fat (Kroger, 1967). The effect of processing and storage of dairy products made from milk con- taminated with DDT and lindane, were studied by Langlois et a1. (196A), and from milk contaminated with organochlorine pesticide residues were evaluated by Li et a1. (1970). A study completed by Murata et a1. (1976) concluded that spray drying showed promise for the removal of P885 from milk. Zabik et a1. (1971) studied the potential of freeze drying for removal of chlorinated hydrocarbon insecticides from eggs. The success of freeze drying appeared related to both the vapor pressure of the pesticide and the amount of contamina- tion in the whole egg. Studies of particular interest to this work are of those which showed effectiveness of cooking procedures in removal of chlorinated hydrocarbons from meat. Evidence indicates residues of chlorinated hydrocarbon pesticides are concen— trated in fats and fatty tissues of animals; hence, pesti- cide residue should be reduced when fat is removed during cooking or other preparational procedures. The effect of cooking on removal of PCB and DDT from Chinook and Coho salmon from Lake Michigan were studied by Smith et a1. (1973). Reduction of these compounds from the A2 salmon steaks was slight; poaching and baking reduced 2—8% of the residues, while baking in nylon bags caused a reduc- tion of 11-16%. No difference was found between Chinook steaks cooked with and without skin. There was no apparent relation between the level of PCB or pesticide residues and concentration of fat in these spawning low fat fish. The effectiveness of curing, heat processing and cook- ing to reduce dieldrin residue levels in pork bellies was investigated by Yadrick et a1. (1971). Dieldrin was reduced A7-80% in the cooked bacon as compared to what was originally present in the uncured samples. Most of the losses were attri- buted to fat rendering during cooking, although heat destruc- tion, co-distillation, and/or aeration may have occured. No consistent pattern was shown for animal, cooking method, or in curing in reducing dieldrin residue levels. Funk et a1. (1971) determined the effect of three cook- ing methods (pan frying, baking and microwave enePEY) on dieldrin residue levels in pork sausage. Sausage patties cooked by the three methods showed no difference in dieldrin residue level, however, differences were noted among animals from which the sausages were made. Analysis of the drip from samples showed residues can be reduced by cooking. Maul et a1. (1971) found that cooked pork loin samples showed lower levels of dieldrin residues than uncooked sam— ples, and residues were present in cooking drip. Ranked in order of increasing percentage of total cooking losses were samples cooked by braising, microwave, roasting and broiling. 43 In a study completed by Yadrick et a1. (1972) dieldrin levels, total lipid composition, and the relative proportion of neutral lipids to phospholipids were determined in selected raw and cooked pork muscles. A consistent reduction in resi- due levels based on fat occured with roasting. Most of the loss of residues apparently accompanied volatile losses in the meat during cooking, since drip losses were minimal. The dark adductor muscle had a significantly higher (P<0.001) level of phospholipid and a lower (P<0.001) level of neutral lipid than all other muscles. The dark adductor tended to have the highest dieldrin levels, where the light femoris muscle had the lowest. The data may indicate some preferen- tial deposition of the dieldrin into the phospholipid compon- ent of the fat. Liska et a1. (1967) first reported the feasibility of removal of selected hydrocarbon pesticides by cooking. The cooking methods used were more severe than normal procedures might be - simmering at 88-93°C for 2-3 hours and autoclaving at 15 psi for 3 hours. It was found the amount of insecti- cide residues in raw chicken tissues was related to fat con- tent, and with autoclaving at 15 psi for 3 hours there was a reduction of 10-90% in the amount of residues detected in cooked tissues. This indicated a rendering out of fat and insecticides, however, in the case of DDT the cooking opera- ‘tion caused a change in chemical structure to DDE and DDD. Ritchey et a1. (1967) found DDT and lindane, which had beeui incorporated into chicken tissues via feed, were reduced AA considerably when the birds were cooked by baking and frying. In a later study Ritchey et a1. (1969) found DDT incorporated into chicken tissues during the growing period was also reduced during cooking. Total losses of residue were greater when tissues were fried or steamed than when samples were either baked or heated in a closed container. Losses of resi— dues from chicken primarily occured through leaching out of fat during the cooking process. Lindane, endrin, heptachlor, dieldrin and aldrin were fed at 10 ppm to broilers for the 8 week growing period (Ritchey, 1972) and tissue was then cooked by baking, frying or steaming. Losses of these residues occured primarily by leaching with fat and water, although there was some destruction of lindane and heptachlor epoxide by heating. Morgan et a1. (1972) evaluated the levels of lindane, dieldrin, DDT, DDE and DDD in meat and broth from selected parts Of hens cooked by simmering and pressure cooking and compared these values to levels in the raw meat. Both cook- ing methods resulted in similar levels of pesticide reduc- tion. Sixty six percent of the lindane and seventy five per- cent of the dieldrin was recovered in the broth, therefore, discarding the broth and drippings after cooking could greatly reduce pesticide consumption. The effect of cooking on PCB residue levels in chicken studied by Zabik (197A) showed cooking by pressure or stew- ing were similar in effective reduction of PCB levels. Ren- dering of the PCBs with the fat appears to be the major mode A5 of removal. Except for adipose tissues, which lost most of its PCB content to the broth, recovered PCBs were about equally divided between cooked samples (drumsticks, breast pieces, thigh meat and thigh skin) and the broth. on tr be we Ap pp: gr. 12 Na: SUI age A5: Dla 31a EXPERIMENTAL PROCEDURES To facilitate the investigation of the effect of cooking on PBB levels in chicken tissue and chicken broth, the Poul- try Science Department of Michigan State University donated hens which had been fed PBBs. Feeding PBBs Fifteen white Leghorn hens approximately 9 months of age were fed for five weeks a standard cage layer ration (see Appendix) which was contaminated with 0, 30, A5, 60 and 90 ppm of Firemasteé3)FF-l. Three hens were in each feeding group; each group was housed in colony cages designed to hold 12 birds in a Poultry Science Research and Teaching Center. Water and feed were available ad libitum. Feed was dispensed by a mechanical feeder controlled by a time clock. Feed con- sumption was not affected by the level of PBB fed and aver- aged 82, 77, 79, 79, and 81 gm/wk/bird for feed with 0, 30, A5, 60 and 90 ppm PBB, respectively. At the end of five weeks, the first three groups of hens (those fed feed contaminated with 0, 30, A5 ppm levels of PBBs) were slaughtered. The remaining two groups were placed on clean feed for an additional 8 weeks and then slaughtered. A6 A7 Slaughter and Preparation The hens were slaughtered by external severing of the jugular vein, hung in killing funnels, bled, scalded in water at 56°C, hand picked, eviscerated and labeled before being chilled overnight under water. The next morning they were carefully dissected to provide breast pieces, drumsticks, thigh meat, and thigh skin from the right side for raw analy- ses and those from the left side for cooking and subsequent analyses. The breasts were split so the keelbone remained with the half to be cooked. All samples were wrapped in aluminum foil, labeled as to bird identification, ppm in feed, piece and state in which they were to be analyzed. The sam- ples were then frozen and held at -20°C pending analysis. Samples, as they were analyzed, were removed from the freezer and thawed at A-5°C for approximately 2A hours. The raw samples were deboned, the meat and skin of the breast and drumstick were combined, while the thigh and thigh skin were kept separate. Samples were homogenized using a Waring blender; the amount necessary for analysis was removed, and the remainder frozen in glass jars with foil-lined lids. Cooking Each chicken piece to be cooked was processed under 15 psi pressure for 15 minutes in 500 ml deionized water, in a 3.8 1 aluminum pressure sauce pan. Heating time to reach the A8 designated pressure was approximately 8 minutes and did not vary significantly from one piece to another. After the pan had cooled 5 minutes the pressure was released. Percentage yields of cooked chicken pieces were calculated as the weight of cooked chicken pieces minus bone divided by the raw piece weight times 100. Broth percentage yields were calculated as the cooked broth volume divided by 500 ml times 100. Cooked samples were homogenized, placed in glass jars, with foil-lined lids and then frozen and held at -20°C pending analysis. The broth was not frozen to eliminate the possible problem of bottle breakage upon freezing; but rather, held at A-5°C in glass bottles and analyzed with one month. Chemical and Residue Analyses All chemical and residue analyses were carried out in duplicate. The chemicals used were ACS reagent grade and the solvents were redistilled from glass. All glassware was acetone-rinsed following by petroleum ether rinsing before being used. Standard solutions were prepared in petroleum ether using the commercial FiremastenB>BP-6, hexabromobiphenyl (Lot No. 51A3, Michigan Chemical Corporation, Chicago, IL). A9 Moisture Analyses Moisture analyses were carried out in a Hotpack vacuum oven, model 633, at 100°C and a vacuum of 660-711 mm (26-28 in) Hg to a constant weight. Sample size weighed to the near- est 0.1 mg were as follows: A-6 g for raw and cooked breast and drumstick (with skin) and thigh (without skin) and 2-A g samples of raw and cooked thigh skin. Aluminum drying cups were loosely covered with perforated aluminum foil caps to prevent loss of fat due to spattering. The percentage of total solids expressed as the dry weight of the samples was used in residue calculations. Lipid Analyses Lipid in the raw and cooked chicken tissues was deter- mined using soxhlet extraction (AOAC Method 11.23). The dried samples were placed in the soxhlet thimble and extracted for 5-6 hrs with petroleum ether. Two ten ml aliquots of this extract were transfered separately to predried 50 ml Erlen- meyer flasks. The petroleum ether was removed over a steam bath followed by drying under a vacuum of 711 mm at 70°C. Percentage lipid was calculated as follows: g lipid X total m1 petroleum ether wet sample weight X 100 For the broth, duplicate lA-l5 g samples were weighed after first homogenizing the entire broth sample in a Waring 50 blender. The broth was stirred for 30 min with 150 ml petroleum ether using a 2.5 cm magnetic stirrer at medium speed. The broth extracts were then washed with 1% sodium sulfate solution to remove any polar compounds remaining and dried over anhydrous sodium sulfate for 30 minutes. Ali- quots of lipid in petroleum ether were taken and treated as previously described. Residue Extraction and Cleanup Petroleum ether soxhlet extraction (AOAC Method 11.23) was used for removal of PBBs from tissue samples. The broth was stirred with 150 m1 petroleum ether for 30 minutes using a 2.5 cm magnetic stirrer at medium speed. The broth extrac- tions were then washed with 1% NaZSOu solution to remove any polar compounds remaining and dried over anhydrous sodium sulfate for 30 minutes. Both broth and tissue sample extracts were treated with acetonitrile partitioning (AOAC Method 29.001), Kuderna Danish concentration and Florisil cleanup (AOAC Method 29.01A). The Florisil column used was 50 cm long x 20 mm id. The packing consisted of 12 mm anhydrous sodium sulfate, 11 cm florisil and again 12 mm sodium sulfate on top. The sample was carefully applied to the top of the packing and eluted with a 6% solution of ethyl ether in petroleum ether collecting 200 ml of eluate. For the 0 ppm in feed sample the eluate was evaporated to dryness with Kuderna Danish and then the sample was brought 51 up to 1/2 ml for GLC analyses. The 30, A5, 60 and 90 ppm in feed samples were taken from the 200 ml eluate without evaporation for GLC analyses. Blanks were carried through the complete extraction and cleanup procedure to insure that no contamination of either solvents or glassware was occuring. GLC Analyses PBBs were quantitated by comparing the area of the 6- isomer peak produced by the sample and the area of the 6- isomer produced by the standard (FiremasteHE)BP-6, Lot No. 51A3, Michigan Chemical Corporation, Chicago, IL). The sam- ples and standards in petroleum ether solution and nanograde hexane, respectively, were run on a Tracor 560 GLC, equipped with a 63N1 electron capture detector and interfaced with a Digital PDP-8e-Pamila GC Data System. The column for the GLC was a pyrex column, 1.83 m long x A.0 mm id, packed with 3% OV-l (Methyl Silicone) on Chromosorb W 60/80 mesh. The carrier gas was nitrogen with a flow rate of A0 ml/min. Temperatures were 270, 2A0, and 300°C, at the injection port, column and EC detector, respectively. Standards were injected at the beginning of each run, after every 6-7 samples and at the end of the run. Quantitations were based on the peak area of the standards (hexabromobiphenyl peak). Figure 1 shows a GLC tracing of a PBB standard and sample containing PBB. 52 A LIA Figure l. GLC tracing of PBB in standard and poultry tissue sample. a) A g of 1 ppm PBB b) Sample thigh skin raw standard. from bird fed 60 ppm PBB in diet (162 ppm fat weight basis). PBB levels were expressed as ppm (wet weight), ppm of solids and ppm of fat. Total micrograms of PBBs in raw and cooked chicken pieces as well as in broth were used to calcu- late percentage recovery and percentage distribution of PBBs recovered in the cooked meat and broth. Percentage recoveries of the PBB residues by the util— ized method were determined, in spiked samples at levels of 0.02, 50, and 100 ppm, to be 55, 66 and 97%, respectively. Confirmation of PBBs The presence of the PBB residues was confirmed by ultra violet irradiation and mass spectrometric analysis. Ultra violet test was carried out in a Rayonet photochemical reac- tor, model 1162, equipped with short wave UV lamps. Mass spectrometric analysis was run on a pool of all of the extracted samples. A GC-65 gas chromatograph interfaced to a DuPont 2l-A90 mass spectrometer which was in turn inter- faced to a Digital PDP-l2 computer was used. The mass spec- tra was obtained at an ionizing voltage of 70 eV with a source temperature of 210°C. Data Analyses PBB levels expressed as ppm wet tissue, ppm in solids, and ppm in the fat as well as percentage fat and percentage solid were analyzed for variance due to state, i.e. raw, cooked, or broth, chicken piece, and level of PBB fed using 5A the MSU Stat System Version A.2 for the Michigan State Uni- versity CDC 6500 computer. Percentage meat and broth yield as well as percentage of total recovered PBBs and recovered PBBs in the meat were analyzed for variance due to chicken piece and level of PBB fed. The studentized range test (Duncan, 1957) was used to sort out any significant differences established by these analyses. RESULTS AND DISCUSSION To investigate the effectiveness of cooking in reducing brominated biphenyl levels in chicken tissue, hens that had been fed five levels of PBB were analyzed. Thigh meat, thigh skin, drumstick with skin and breast with skin from the right half of each bird were analyzed raw; whereas, pieces from the left half, and their resultant broth were analyzed following pressure cooking. Chicken Meat and Broth Yields Analyses of variance of percentage meat and broth yield (Table 7) indicate the level of PBBs fed did not signifi- cantly affect these parameters. The type of piece cooked, however, did significantly affect both average meat and broth yield. The cooked meat yield (Table 8) for breast (61.6%) and thigh skin (59.2%) pieces was significantly higher (P<0.01) than the drumstick (53.3%) or thigh meat (50.2%) pieces. The average weight in grams of the pieces were 216.1 g, 7A.6 g, 85.7g and 13.5 g for breast, drumstick, thigh and thigh skin, respectively. 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Remov- al of skin may have contributed to greater loss from the thigh meat since Funk et a1. (1968) reported ground beef cylinders wrapped in fat exhibited significantly lower cook- ing losses than did unwrapped cylinders. The broth yield (Table 9) of the breast piece (89.6%) was significantly higher (P<0.01) than that of the thigh skin (77.7%). The breast pieces were comparatively so large that the moisture lost from them contributed more to the drip and therefore increased the broth yield more than did the smaller pieces. Percentage Fat and Solids Analyses of variance of percentage fat and solids in raw and cooked chicken pieces and broth (Table 10) indicate a highly significant difference (P<0.001) in percentage fat and percentage solids among states analyzed (raw, cooked and broth) and among the four pieces. The average percentage of fat in the cooked chicken pieces (1A.0%) were significantly lower (P<0.001) than in the raw pieces (21.1%) showing that rendering had occured during cooking (Table 11). In addition, the average fat con- tent of the drumstick (A.6%) was less (P<0.05) than that of the breast piece (7.9%) or thigh meat (7.5%); both which had less fat (P<0.01) than the thigh skin (27.7%). 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Greater rendering of fat from thigh skin during cooking probably contributed to the significant interactions shown. Neither the average lipid nor solids content were affected by the level of PBB fed to the hens. High standard deviations of the means of especially A5 and 90 ppm PBB fed birds, seen in percentage meat and broth yields (Tables 8 and 9) and percentage fat and solids (Tables 11 and 12) point out the great variability among birds. Residue Analyses Data concerning PBB levels in the raw and cooked chick- en as well as in the chicken broth, expressed as ppm in wet tissue, ppm in fat and ppm in solids for each of the five levels fed are summarized in Tables 13, 1A, and 15. Analyses of variance established a highly significant (P<0.001) inter- action between state, piece and level PBB fed expressed as ppm wet weight (Table 16). On a ppm solid and ppm fat basis a highly significant difference between levels of PBB fed is seen. The major factor in this interaction was that a marked increase in ppm PBB residue was seen in each tissue with increase in level fed. 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This unexpected presence of a low level of contamination may have resulted from PBB particles being airborne on dust. Although all groups of birds were housed in separate cages, they were all in the same cage room and were fed their respective rations at the same time. The blanks that were constantly run showed no PBB contamination and hence the birds themselves must have been contaminated and not reagents or glassware. The levels of PBBs in the tissues increased progressively with increasing levels in the feed and were highest in the high fat thigh skin (Figure 2). This phenomenon was also shown by Ritchey et al. (1967) where raw tissues were found to contain residue levels reflecting DDT and lindane intake. It was expected that the PBB levels in the tissues of hens fed feed containing 60 and 90 ppm PBBs and which were subsequently fed "clean food" for eight additional weeks, would be lower than that in tissue from birds fed 30 and A5 ppm PBB in the feed since the half life of eggs, liver and muscle had been found to be approximately 3 weeks (Polin and Ringer, 1975). The samples in this study, however, either contained skin or were higher in fat and thus these factors may have affected our reduced rate of elimination. This could be in agreement with studies that have shown PBB accu- mulates to a greater degree and is metabolically eliminated more slowly from fatty tissue than from liver and muscle (Gutenman and Lisk, 1975: Fries et al., 1973a). In addition, Stadelman et a1. (1965) reported that feeding high levels of p.p.m. 69 6O Breast 55 Thigh 50 A5 Thigh skin A0 Drumstick 35 30 25 20 15 10 3O p.p.m. A5 p.p.m. 6O p.p.m.* 90 p.p.m.* p.p.m. PBB in feed 5 weeks Figure 2. PBBs in raw hen tissue, ppm wet weight basis. * Birds were fed clean feed for 8 additional weeks. 70 several pesticides resulted in greater residue persistance in eggs and abdominal fat of hens than was found when low levels were fed. As can be seen from the high standard deviations of the means in Tables 13, 1A and 15 wide variation in the PBB lev- els and fat content occured in the birds within each group. However, duplicate values (within 1-10% difference except samples from 0 ppm PBB fed birds where differences varied as much as 50-100% due to limits of sensitivity) on any one tissue gave reasonable agreement. We When the chicken pieces were cooked, the level of PBBs in the wet tissue decreased slightly (Table 13). Part of the PBB lost was recovered in the broth. The average level of 0.7A ppm P885 in the broth of the breast pieces from birds fed 30, A5, 60 and 90 ppm PBB was higher than the average level in the broth of the other pieces. The drumstick, thigh, and thigh skin contained a mean ppm PBB in their broth of 0.19, 0.35 and 0.28, respectively. Since the amount of cook- ing water was constant, greater amounts of P883 in the breast broth resulted from the larger amount of fat rendered from the larger piece. Figure 3 depicts the reduction Of ppm PBB in cooked pieces expressed on a wet weight basis. The greatest reduc- tion is seen in the higher fat thigh skin and large breast pieces, and the least in the low fat drumstick. These data 71 30" 25_‘ Raw l;;]] Cooked 20- 15 a p.p.m. 10 ~ 8 ~ ~ “pm 6 - r A-H 1]. H Breast Thigh Thigh Drumstick skin .Figure 3. PBBs in wet tissue of raw and cooked hen pieces (data shown are composite of all 15 birds analyzed). 72 reflect the preferential distribution of PBBs in the lipid phase and rendering of P883 with the fat appears to be a mode of removal. Several previous studies (Ritchey et al., 1967, 1969, 1972; Liska et al., 1967; Morgan et al., 1972; Zabik, 197A) have indicated that rendering during cooking signifi— cantly reduced levels of chlorinated hydrocarbon pesticides in chicken. WW5 When the PBB content was expressed on a fat basis there was no significant difference among chicken pieces (Table 1A). On a solids basis the thigh skin had a higher (P<0.05) level of PBBs than the other pieces (Table 15). Cooking did not significantly affect the level of PBBs expressed on a solids basis, however, the average P883 in the fat of the broth (52.6 ppm) was less (P<0.05) than that in the fat of the raw (86.1 ppm) or cooked pieces (92.2 ppm). Thus, the average values in the fat of cooked pieces were slightly higher than those in the fat of raw pieces (Figure A). While rendering of fat is undoubtedly an important mode of PBB reduction, the amount of PBB reduced is not directly proportional to fat removal. Supporting this finding Lee et a1. (1970) also Observed this nonproportionality to fat removal in work with several chlorinated hydrocarbon pesticides. Murata et a1. (1976) found PBB levels were higher in butter, cheese and freeze— dried cream than levels in buttermilk, cheese whey and cream, 73 Raw E Cooked [ND 120— Broth [::] m 110— All IOOH 901 E 80- W W E E E E E = :2 :2 6o— : :: — E E . 50— E 71 E E E E E E E 7 E '1 aho- E E E E 30‘ E E E E 20- E E : : :.—. :: l: : _- .__7 l--— r—— 10- : :2 : L: 0 E 2 E Breast Thigh Thigh Drumstick Figure A. PBBs in the fat of hen pieces; raw, cooked and broth state (data shown are a composite of all 15 birds analyzed). 7“ respectively, reflecting the preferential distribution of P883 in the lipid phase of the products. However, the researchers also found PBB levels in skim milk, buttermilk, and cheese whey were slightly higher than PBB levels in pasteurized whole milk, per fat unit, suggesting some PBB may be associated with lipoprotein and or may be soluble in the "serum" of milk. Data from this study suggest that PBBs may associate to a greater extent with lipoproteins present in the tissue than some other lipophilic compounds and therefore not be as easily removed with fat in cooking. Whether this is due to chemical properties, such as greater n interaction of the parent PBB biphenyl rings with phospholipids (lipid moiety of some lipoproteins) or the size of the bromine atoms attached which contribute a greater electron cloud (there- fore decreased solubility) than chlorine atoms, is of inter— est. Besides variable amounts of neutral lipids, muscle is found to contain small amounts of cholesterol and larger quantities of phospholipid. Those muscles requiring the production of greater amounts of energy, the cardiac muscles and the muscles of locomotion, contain the greater amount of phospholipid, and tend to be darker in color (Orten and Nei- haus, 1975). Lawrie (1966) concurs with this and states; dark (red) muscles are characterized by being high in respir— atory activity and constantly in action, light(white) muscles generally operate in short bursts of activity with little 75 capacity for respiratory activity. In a work by Peng (1965) it was found the phospholipid content of total lipids in chicken leg muscle (dark meat) tend to be higher than in lipids of the breast muscle (white meat). Katz et al. (1966) found the opposite to be the case with the phospholipid content of poultry white meat being higher than the phospholipid content of dark meat. However, the poultry tissue contained by far the greater percentage of phospholipids than the skin and depot fat. Beecher et al. (1968) in studying pork muscle, stated total lipid content tends to be higher in light (white) muscle than in dark (red) muscle and muscles with lower total lipids possess a greater proportion of phospholipids. In agreement with this, Luddy et al. (1970) found the dark por— tion of the semitendinosis muscle of pork contained a greater proportion of phospholipids than did the white; that is the light muscle contained 20% more lipid, 20% more glycerides, and u0% less phospholipids than the the dark muscle. In addition, Yadrick et al. (1971) reported that dark pork muscles with a lower percentage neutral lipid content possessed the greater percentage of phospholipids compared to the light pork muscle studied. The authors also found increased dieldrin levels in dark muscle and lower percentage reduction of dieldrin the cooking process. This may mean dieldrin follows phospholipid disproportionately. Looking at the fatty acid content of phospholipids pre- sent in muscle it was shown by Luddy et al. (1970) that the 76 fatty acids of light pork muscles are predominately monoenes while the dark muscle phospholipid fatty acids were higher in polyunsaturates. Kutz et al. (1966) found poultry tissue, both white and dark meat, to be higher in the fatty acid arachidonic acid than was skin or depot fat. In addition, Peng (1965) noted fatty acid composition of the phospholipid sphingomyelins was such that the dark meat contained a num- ber of the longer chain fatty acids from C21-C25 whereas the white muscle had none. Polyenoic acids may have greater n interactions with the parent rings of PBB and hence hold them tighter than monoenoic acids. The conclusion could be drawn that structure may contribute to increased bonding with halo- genated hydrocarbons and perhaps more so with brominated hydrocarbons and decreases their liklihood of following the fat proportionately in rendering out of a meat during cook— ing. In this study there was less reduction of P885 in cook- ing of the drumsticks which consist of dark muscle with increased levels of phospholipids. Recovery and Distribution of PBBs After Cooking Total micrograms of P888 in the cooked chicken and broth were compared to the level in the respective raw chicken piece to calculate the percentage PBB recovery. No signifi- cant differences occurred among the percentage of PBB recov- ered in any of the four pieces; total percentage recoveries from raw were 68.1% in thigh skin, 75.8% in the breast piece, 77 83.9% in the thigh meat, and 8u.6% in drumsticks. PBB recov- eries, however, did tend to be higher in the chicken pieces which contained less fat (i.e. drumstick and thigh meat) even though these pieces had lower percentage meat yields (greater cooking losses) (Table 8) than did the higher fat breast piece or thigh skin. This may again be pointing to greater association of PBBs with phospholipids in tissue. The distribution of the recovered PBBs between the cooked meat and broth is illustrated in Figure 5. The percentage of recovered PBBs in the meat did not differ significantly among the four pieces evaluated and ranged from 65.5% in the cooked thigh skin, 67.1% in cooked thigh, 71.2% in cooked breast to 72.9% in the cooked drumstick. The proportion of the recov- ered PBB in the cooked meat is considerably higher than that found in previous studies. Recovered PCBs were about equally distributed between the cooked meats and broth (Zabik, 197“) while only l/H to 1/3 of recovered lindane, dieldrin and DDT compounds occurred in the cooked hen pieces (Morgan et al., 1972). The bulkier size and higher molecular weight of the PBB molecules in comparison to the organochlorine pesticides studied, may contribute to a smaller proportion of the recov— ered PBB material being found in the broth. Another factor in the recovery of greater amounts of PBBs in the meat as compared to other studies, might be the level of contamination of the tissue from which the PBBs are being extracted. Murata et al. (1976) observed losses of PBB from spray dried skim milk (61—69%) were greater than from 78 90-q 30 1 7o .. I1 60 ‘ so a no — 30 — 20 - 10'- Breast Thigh Thigh Drumstick skin Figure 5. Distribution of recovered PBBs between cooked tissue and broth (data shown is a composite of all 15 birds analyzed). 79 spray—dried whole milk (30-36%). These results suggested to the authors that PBBs may be more easily removed from low fat products and/or when PBB levels in the original product were low. In this study, the levels of contaminants fed were quite high (up to 90 ppm) compared to the 25 ppm fed by Zabik (197“). Thus, these high levels of PBB contamination may have influenced the distribution recovered. Also of inter- est, for pieces from control hens which showed low level con- tamination( 0.02-0.07 ppm wet, 0.1u-0.26 ppm fat basis in raw tissue, Figures 14 and 15) the proportion of recovered PBBs in the meat was slightly less, ranging from 39.8% in the drumstick to 58.7% in the thigh meat. Therefore, it is possi- bhecooking has an even greater potential for reducing low levels of P883 such as might have reached the market during 1973-197“. SUMMARY AND CONCLUSIONS Thigh meat, thigh skin, drumsticks with skin, and breast with skin were analyzed to investigate the effectiveness of cooking in reducing PBB residue levels in contaminated chic- ken tissue. Pieces from the right half were analyzed raw whereas pieces from the left half, and their resultant broth, were analyzed following pressure cooking (15 psi for 15 min). These hens had been fed either 0, 30, N5, 60, and 90 ppm PBBs. Analyses of variance of percentage meat and broth yield indicate the level of P885 fed did not significantly affect these parameters. However, the type of piece cooked did sig- nificantly affect both average meat and broth yields, with the breast and thigh skin (the higher percentage fat pieces, 7.9 and 27.7%, respectively) producing the greatest yields of cooked meat (61.6 and 59.2%, respectively). Also the broth yield of the breast pieces was significantly higher than the broth yield of the thigh skin pieces. Analyses of variance of percentage fat and solids in raw and cooked chicken pieces and broth indicated a highly sig- nificant difference in percentage fat and percentage solid between states analyzed (raw, cooked and broth) and among four pieces. The percentage fat of raw and cooked and broth was 21.1, 1A.O, and 0.7%, respectively; and of the four 80 81 pieces, breast with skin was 7.9%, drumstick with skin was A.6%, thigh meat was 7.5%, and thigh skin was 27.u% fat. These data indicate a rendering out of fat in the cooking process and show that breast meat and thigh skin had the highest percentage fat. The breast was considered higher in fat than thigh meat even though it was very close in percen- tage fat mainly because of its large size. The breast pieces averaged 2 1/2 times the size of the next smallest piece which was the thigh piece. PBB residue analysis on a wet basis revealed that PBB levels increased with increasing levels fed, were the high- est in high fat pieces and were reduced in the cooked sample from the paired raw sample. These data reflect the prefer- ential distribution of PBBs in the lipid phase and rendering of PBBs with the fat appears to be a mode of removal. Residue analyses on a fat basis revealed a highly sig- nificant difference between levels of PBB fed - as level fed increased, ppm PBB residue increased. The birds fed 60 and 90 ppm PBB in their ration for five weeks and then on "clean feed" for 8 additional weeks before slaughter were expected to show levels similar to the 30 and H5 ppm level fed birds since the half life in eggs, liver and muscle had been found to be approximately 3 weeks. However, the samples either contained skin or were higher in fat content and the slower rate of elimination of residue shown here would be in agree- ment with studies showing that PBB accumulates to a greater degree and is metabolically eliminated more slowly from fatty 82 tissue than from liver and muscle. On a fat basis there was no significant difference among pieces in PBB residue content. In cooking the average PBB residue content of the fat in the broth was less than in the fat of the raw or cooked pieces, i.e. average values in the fat of cooked pieces were slightly higher than those in the fat of the raw pieces. Therefore, while rendering of fat is an important mode of PBB reduction, the amount reduced is not directly proportional to the amount of fat removed as has been shown in some work with PCBs. The reasons for nonpro- portionality of fat and PBB removal may be due to increased n bonding of parent PBB biphenyl rings with phospholipids in the tissue, or the larger size of the bromine atom which would contribute a larger electron cloud with increased association and possibly reduced fat solubility. Total micrograms of PBBs in the cooked chicken and broth were compared to the level in the respective raw chicken piece to calculate the percentage recovery. Percentage recoveries were 68.1, 75.8, 83.9 and 84.6% for thigh skin, breast, thigh meat and drumstick, respectively. PBB recov- eries tended to be higher in the chicken pieces with less fat (i.e. drumstick and thigh meat) even though these pieces had greater cooking losses. The percentage of recovered P883 in the cooked meat did not differ significantly among the four pieces analyzed, the range was from 65.5% in thigh skin to 72.9% in drumstick pieces. The proportions of recovered PBBs in cooked meat 83 was considerably higher than that recovered in previous studies. The high levels of contamination may have contri- buted to the lower amounts found in the broth since several studies have indicated halogenated hydrocarbons may be more easily removed from low fat products and/or when PBB levels in the original product were low. Thus, the percentage of recovered PBBs in the tissue of control birds (low level contamination samples) was slightly less ranging from only 39.8% in drumsticks to 58.7% in thigh meat. It appears cook- ing may have an even greater potential for reducing low levels of PBB such as might have reached the market during 1973-197“. SUGGESTIONS FOR FUTURE RESEARCH In this study the lipophilic compound PBB did not com— pletely follow the fat in the process of rendering out of fat in cooking. The reasons for this and under what condi— tions more PBBs could be removed in processing should be investigated. Studies with light and dark meat which con- tain different lipoproteins and different levels of lipopro- teins might be used in such a study. The correlated higher reduction of PBBs in lower fat content tissue should be elaborated further with a larger number of samples with varying P883 and lipid concentrations. Greater percentage reduction of PBBs from low PBB resi- due level tissue was observed. This was shown in 0 ppm fed birds which were contaminated possibly due to airborne dust carrying PBB. An evaluation of this result could be done with the two above studies. Studies done with DDT elimination from poultry tissue have found some retention of DDD (the converted DDT isomer) in the meat. Some unknown peaks appeared in this study, particularly at low levels where the sample was highly con- centrated. This may possibly be due to partial debromina- tion of the PBBs in the processing or perhaps through meta- bolism. These peaks should be further investigated. This 8“ 85 should then be extended to include a toxicological evalua- tion of lower brominated biphenyl compounds. This study as observed differences in PBB and PCB in ease of removal from tissue, and the maintenance of high tissue levels following a clean diet. This may be due to chemical differences between chlorine and bromine atoms or it may be due to solubility differences between chlorine and bromine atoms. Some of the answers to these differences could be elucidated in the above suggested studies, but fur- ther work looking at these specific questions should be pur- sued. BIBLIOGRAPHY BIBLIOGRAPHY Abbott, D. C., R. B. Harrison, J. O' G. Tatton and J. Thom- son. 1968. Organochlorine pesticides in the atmos- phere. Nature 211:259. Aftosmis, J. G., R. Culik, K. P. Lee, H. Sherman and R. S. Waritz. 1972a. The toxicology of brominated biphen- yls. I. Oral toxicity and embryo toxicity. Presen— ted at the Society of Toxicology, Williamsburg, VA. March 8, 1972. Aftosmis, J. G., O. C. 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B. and H. A. Irving. 1975. Distribution and clearance of polybrominated biphenyls by cows. J. Dairy Sci. 58:764. 100 Wong, R. G., H. G. Nowicki and A. W. Norman. 1974. Effects of polychlorinated biphenyls on calciferol (Vitamin D) mediated calcium metabolism. Pestic. Biochem. Physiol. 4:170. Yadrick, M. K., K. Funk and M. E. Zabik. 1971. Dieldrin residues in bacon cooked by two methods. J. Agri. Food Chem. 14:491. Yadrick, M. K., M. E. Zabik and K. Funk. 1972. Dieldrin levels in relation to total neutral and phospholipid composition in selected pork muscles. Bull. Environ. Contam. Toxicol. 8:284. Yobs, A. R. 1972. Levels of polychlorinated biphenyls in adipose tissue of the general population of the nation. Environ. Health Perspectives 1:79. Yoshimura, H. and H. Yamamoto. 1975. A novel route of excretion of 2,4,3344 tetrachlorobiphenyl in rats. Bull. Environ. Contam. Toxicol. 14:681. Zabik, M. E. 1974. Polychlorinated biphenyl levels in raw and cooked chicken and chicken broth. Poultry Sci. 53:1785. Zabik, M. E. and L. R. Dugan, Jr. 1971. Potential of freeze drying for removal of chlorinated hydrocarbon insec- ticides from eggs. J. Food Sci. 36:87. Zitko, V., O. Hutzinger and P. M. K. Choi. 1972. Contam- ination of the Bay of Fundy - Gulf of Maine area with PCBs, PCTs, chlorinated dibenzo dioxins and dibenzo furans. Environ. Health Perspectives 1:47. APPENDIX A Layer Ration for PBB Experiments 1 Ingredient Parts/1000 Corn, #2 yellow 572.2 Soybean meal, 50% 200 Meat and bone meal 30 Alfalfa, 17% 40 Tallow 55 Methionine hydroxy analogue 0.8 Dicalcium phosphate 18 Limestone 69 Salt, iodized 3 Choline chlgride, 50% 2 Vitamin mix 5 Mineral mix3 5 1 Calculated analysis: protein - 17.5%; ME - 3.02 kcal/g; Ca - 3.5%; P (available) - 0.6%; fat - 8.3%; fiber - 2.8%; methionine - 2.0% of protein. Vitamin mix supplies per kg: IU; vitamin D3 - 1,000 ICU; vitamin A - 10,000 dl-x-tocopherol - 10 IU; menadione sodium bisulfite - 4.0 mg; riboflavin - 4.0 mg; niacin - 10.0 mg; biotin - 100 mcg; vitamin B12 - 8 mcg; mg; ethoxyquin - 125 mg. 3 Mineral mix supplies per kg: pyridoxine - 4.0 magnesium - 500 mg; manganese - 50 mg; zinc - 50 mg; copper - 5 mg; iodine - 1.0 mg; cobalt — 3 mg. 101 102 Reprinted from Poultry Science. Vol. 56. July. 1977. Pages 1289-1296. Polybrominated Biphenyl Levels in Raw and Cooked Chicken and Chicken Broth' S. K. SMITH, M. E. ZAHIK and 1.. F.. DAWSON Department a] Food Science and Human Nutrition. Michigan State University, East Lansing. Michigan 48824 (Received for publication December 9. 1976) ABSTRACT S.C.W.L. laying hens were fed either 0, 30. 45. 60 or 90 p.p.m. PBBs for five weeks. Hens fed 0. 30 and 45 p.p.m. of PBBs were sacrificed at the end of this five week period whereas those hens fed 60 and 90 p.p.m. of PBBs were fed a clean standard laying ration for an additional eight weeks at which time they were sacrificed. Thigh meat. thigh skin. drumstick and breast (with skin) from half of each bird were analyzed raw whereas pieces from the other half were analyzed following pressure cooking separately in 500 ml. of deionized water at 15 p.s.i. for l 5 min. Cooking yields were obtained. and the cooked meat and broth were analyzed separately for PBBs. PBB residues were found in all tissues analyzed from birds receiving each level of PBB treatment with considerable bird variation in quantity found. Residue levels in tissue increased with increased levels of P88 in feed. and were related to fat level in tissue (wet basis). The level of PBBs expressed as p.p.m. on a solids basis were lower in the cooked sample than in corresponding raw piece with part of the PBBs lost being found in the drip. Recoveries of PBBs in cooked tissue and broth ranged from 68.1% in the thigh skin to 84.6% in drumstick with approximately two-thirds of the recovered PBBs found in the cooked meat itself. INTRODUCTION The growing concern over environmental contamination most likely stems from the rapid influx of synthetic compounds and their ac- companying waste materials. Of these com- pounds, much attention has been focused on halogenated hydrocarbons. Organochlorine pesticides and polychlorinated biphenyl (PCBs) with their lipophylic properties have been found in the fat of virtually all animals, and have been detected in human tissues as well (Biros at al., 1970: Fujiwaru. 1975: Musial er al., 1974). These compounds resist biological degradation (Fries et al., 197 3) and consequent- ly are expected to be present in the ecosystem for an extended period of time. Occurrences of contamination in human tissue are generally associated with ingestion of food containing low levels of these compounds. Although pre- cise information concerning toxicity of halo- genated compounds at very low levels is not known, the concern is for possible chronic ' Michigan Agricultural Experiment Station Journal Article No. 7 895. 'FiremaaterO BM mixed with the anticaking agent “Flo-Gard" which is manufactured by PPG Industries and which contains 8396 silicone dioxide and a maximum of 7% calcium oxide. Poultry Science 56:1289-1296. 1977 effects of their continual assimilation and ac- cumulation in body fat. Polybrominated biphenyls (PBBs) are mem- bers of the halogenated hydrocarbon class of compounds with structure, reactivity, use and toxicity similar to those attributed to PCBs of higher chlorination. Michigan Chemical Corpo- ration manufactured a product called Fire- master® BP-6. 8P6 has an average of 6 bromine atoms attached to the biphenyl rings and to meet specifications is brought up to 75% bromine by weight. General properties include: solid at room temperature with a softening point of 72°C.; decomposes at 300-400°C.: very low solubility in water, 11 p.p.b. at 25°C.; soluble in most organic solvents; and a vapor pressure of 5.2 x 10" mm. Hg at 25°C. (Kerst, 1974). PBBs are industrial compounds used mostly as fire retardants and plasticers. Some examples of major uses are business machines, electrical uses. and fabricated products. PBBs are not used in food or feed. nor are they used in products that come in contact with human skin as in flame retarding fabrics (Kerst, 1974). However. in May, 1973. an incredible error was made during feed manufacture in Michigan: the PBB compounds. Firemaster® FF-l2 was mistaken for magnesium oxide (trade name Nutrimaster) and mixed into high protein dairy pellets (Jackson and Halbert. 1974; Dunckel, 1289 103 1290 1975). Through this contamination and conse- quent cross contamination of other feeds. dairy animals, poultry and swine were affected. This mistake has cost Michigan farmers untold dol— lars in loss of animals, income from those animals. and the cost of clean-up procedures to rid their farms of this contaminant. In the poultry industry alone. approximately two mil- lion birds and nearly five million eggs were destroyed. Toxicity studies have shown PBBs to have a similar mode of action to other halogenated compounds in that they produced porphyria, liver injury, tremor and loss of weight (Strik. I973 ; jackson and Halbert. I974; Prewitt et al., 1975; Babish et al., 1975: Lee et al., 1975; Aftosmis et al., 1972). Feeding studies have shown that bromine levels in biological syStems elevate rapidly and then plateau but when the animals are fed on “clean feed" again the levels decrease. This decrease occurs more rapidly in milk, eggs, liver and muscle than in fatty tissue which loses its PBB store much more slowly. Studies concerning the possibility of remov- ing halogenated compounds from food stuffs upon processing are of great interest. Several studies have been conducted concerning the removal of lipophylic compounds from such products as poultry (Zabik, 1974: Morgan er al., 1972; Ritchey et al., 1967. 1969. 1972); eggs (Zabik and Dugan, I971): sausage patties (Funk er al., 1971): bacon (Yadrick er al., 1971); pork loins (Maul et al., 1971): pork muscles (Yadrick et al., 1972): and beef loaves with texturized soy (Shafer and Zabik, 1975). Most of the losses seen were attributed to fat rendering or leaching out during cooking—the more severe the rendering the greater the loss. Different levels of success in elimination of the contaminants were seen depending upon the compound worked with, the levels of contami- nation and the tissue from which the extraction was accomplished. The purpose of this study was to investigate the effect of cooking on the PBB levels in chicken tissue and chicken broth. The ease of residue removal from skin in contrast to tissue was evaluated by cooking thigh meat and skin separately. METHODS AND MATERIALS Sample: Fifteen White Leghorn hens approx- imately 9 months of age were fed a standard cage layer ration for 5 weeks which was SMITH, ZABIK AND DAWSON contaminated with O, 30, 45, 60, and 90 p.p.m. Firemastcré" I’F-l (3 hens/feeding level). At the end of five weeks the first three groups (0, 30 and 4S p.p.m. levels) were slaughtered. The remaining two groups were placed on clean feed for an additional 8 weeks and then slaughtered. At the time of slaughter the hens were picked. eviscerated and chilled overnight in ice water before being carefully dissected to provide breast pieces. drumsticks, thigh meat. and thigh skin from the right side for raw analyses and those from the left side for cooking and subsequent analyses. The breasts were split so the keelbone remained with the half to be cooked. All samples were wrapped in aluminum foil, labeled as to bird identification. p.p.m. in feed. piece and state in which they were to be analyzed. The samples were then frozen and held at —20°C. until analyzed. Samples. as they were analyzed. were re- moved from the freezer and thawed at 4—S°C. for approximately 24 hours. The raw samples were deboned, the mat and skin of the breast and drumstick were combined. while the thigh and thigh skin were kept separate. Samples were homogenized using a Waring blender; the amount necessary for analysis was removed. and the remainder frozen in glass jars with foil-lined lids. Cooking: Each chicken piece to be cooked was processed under 15 p.s.i. pressure for 15 minutes in 500 ml. deionized water. in a 3.8 l. aluminum pressure sauce pan. After the pan had cooled 5 minutes the pressure was released. Percentage yields of cooked chicken pieces were calculated as the weight of cooked chick- en pieces minus bone divided by the raw piece weight times 100. Broth percentage yields were calculated as the cooked broth volume divided by 500 ml. times 100. Cooked samples were homogenized. placed in glass jars. with foil-lined lids and then frozen and held at -20°C. pending analysis. The broth was not frozen to eliminate the possible problem of bottle breakage upon freez- ing; but rather held 4—S°C. in glass bottles and analyzed within one month. Analyses: Moisture and lipid determinations were carried out in duplicate by drying at 100°C. and 70°C.. respectively, under vacuum of 711 mm. (28 in.) Hg. Lipid samples dried ‘ were aliquots of the petroleum ether extracts used for PBB analyses. Duplicate 4-6 g. samples of raw and cooked breast and drumstick (with skin) and thigh PBBS IN COOKED CHICKEN 1291 TABLE 1 . - Meansa and standard deviations of the mean jar the percentage chicken meat and broth yield Level I" of PBB "“ Measure fed Breast Drumstick Thigh meat Thigh skin Meat yield% 0 60.3 t 3.5 54.0 11.1 51.2 2 1.2 57.2 t 4.3 30 63.9 1 2.8 56.1 t 0.4 51.0 t 1.2 55.5 1 8.9 45 63.6 t 2.2 53.4 t 1.9 51.5 t 0.8 66.7 2 8.7 60b 58.9 2 2.9 50.9 2 2.9 47.2 2 2.2 56.6 2 2.5 90b 61.2 2 1.3 51.8 21.2 50.2 2 0.9 59.9 213.2 Broth yield 96 0 87.3 3 10.6 83.3 2 6.1 76.9 a 17.3 74.0 a 8.0 30 85.7 t 7.9 79.7 2 5.1 82.5 1 3.3 76.1 t 6.4 45 91.7 2 8.0 80.1 2 8.0 84.4 t 5.0 77.6 2 4.1 60b 87.9 2 10.4 83.1 2 7.2 81.3 2 1.8 82.7 2 7.0 90b 95.4 2 8.5 85.3 2 4.0 91.0 2 2.2 78.1 2 6.7 'Based on 3 hens. Significant studentized range values for comparing two consecutive means (P<0.05) are 7.5 and 12.6 for percentage meat and broth yield. respectively (Duncan. 1957). b TABLE 2. — Fat and solids content‘ of raw and cooked chicken as well as chicken brath Fed clean feed 8 additional weeks before slaughter. Level Piece of PBB Measure State fed Breast Drumtick Thigh meat Thigh skin Fat as Raw 0 11.4 2 5.2 8.4 2 5.4 14.9 2 3.9 54.1 2 1.6 30 14.4 2 7.2 9.2 2 4.9 15.9 2 7.1 55.0 a 2.1 45 10.3 2 3.2 6.0 21.2 9.6 2 2.7 53.3 2 13.3 60b 14.4 2 3.4 7.5 2 1.7 10.1 21.5 48.2 2 4.5 909 12.3 2 8.3 7.3 2 2.6 14.2 26.5 56.5 2 18.5 Cooked 0 8.4 2 2.6 6.7 2 22 8.8 2 2.9 27.3 2 3.8 30 13.4 2 6.7 6.4 t 2.3 11.8 2 6.0 40.6 2 4.5 45 8.9 2 0.5 5.4 2 2.4 9.1 2 2.0 30.6 2 14.0 60b 7.8 2 3.2 5.0 2 2.0 6.4 2 0.5 23.4 2 4.6 90b 11.4 2 4.3 5.9 2 2.4 8.9 2 3.0 33.7 2 18.3 Broth 0 1.3 2 0.6 0.3 2 0.0 0.6 2 0.2 0.4 2 0.0 30 0.8 2 0.3 0.3 2 0.2 0.7 2 0.1 0.6 2 0.0 45 1.3 2 0.7 0.2 2 0.1 0.7 2 0.3 0.8 2 0.4 60b 1.3 2 0.6 0.4 2 0.1 0.5 2 0.4 0.4 2 0.1 90b 1.9 2 1.3 0.4 2 0.1 0.7 2 0.4 0.7 2 0.9 Solids as Raw 0 31.5 2 2.6 29.3 2 3.3 37.0 2 3.1 65.3 2 2.6 30 33.8 2 2.8 31.0 2 3.4 33.4 2 3.7 64.9 2 3.6 45 31.7 2 2.9 2725 2 1.6 30.2 2 2.5 70.0 2 4.9 60b 34.7 2 1.8 28.4 2 2.0 30.7 2 1.5 59.6 2 3.3 90b 35.4 2 7.7 28.7 2 0.8 30.9 2 9.1 58.7 2 17.7 Cooked 0 40.0 2 2.8 37.4 2 1.2 40.4 2 0.3 43.1 2 2.9 30 49.3 2 3.9 38.2 2 1.3 42.2 2 3.6 54.2 2 5.1 45 45.2 2 7.2 37.7 2 2.2 40.5 21.6 45.9 2 9.7 60b 41.8 2 4.4 37.0 2 1.2 39.3 2 1.5 40.2 2 4.4 90b 52.1 2 12.6 39.1 2 1.3 40.8 2 2.1 52.0 2 16.1 'Mean and standard deviation of the mean for 3 hens. Significant studentized range values for comparing two consecutive means (P<0.05) are 7.1 and 5.3 for percentage fat and moisture. respectively (Duncan. 1957). b Fed clean feed 8 additional weeks before slaughter. 105 SMI'I‘II, ZAISIK ANI) DAWSON £853.. 833 8.83 a 3:022». ea .8 to“... 50.9 no". a ...... .5855. 58.88.! .... .52.... .5. 22.8 .52.... .83 5...... 5. .... .85 ..o. ...... o: .....ov... 2.35 02.3828 03. 8.5958 8. 8...! one: 88:83. Snowman... .80.. . 8. 50:. o... .6 8.8.28.8 885.. .8... 50:. ......8 2:37 «.6. 8 3.2... can. u 3.. 8 .3. 8 ...... 8 ...... 8.... 8.... 2...... 8.. .... .... .9. o... ...o. 8 ...... 8 .... 8 ...... 8 8.. 8 .... 8 .8. 8 .... 8 8.... 8.... ...? ....8 v... 8.. .... 3.. ..oo .... 8 .... 8 .3. 8 ...o. . a... 8 .... 8 .8. 8 8.. 8 2.... 8.... ...... ...... .... .e.. ..8. ..¢. 3 ...o. 8 .... 8 ...o. 8 ....o. 8 ..o. 8 28. 8 .... 8 v... 8 ...... .3. ...... ...... 8o. .... .2. .... o. ....o . mud u 5.6 a «No 8 8°. « N8. 8 80. a .8. u ...o . ... a ..o ...... .8. 8o. .8. .8. o ...o... 8...... 3.8.8 8...... 8.28 .3... 8 ......8 .....8 .....8 ...... 8 8.. 8 .... 8 .....8 ...... 3...... .9... ....o. .....o. 3.... ...... 8.8 o... ...: .8. o... 88 ......o 8 ...... 8 8.3 8 3.... 8 .oo. 8 a... 8 ...... 8 8.. 8 .....8 .... 8 ...o 8 ...o 8 ...o.. 8.... 8...: ...... 3... ...... .... ...... ...... .... .... 8... 88 ...... n 2.... 8 o..... 8 8... 8 8o. 8 8.. 8 ...o 8 8.. 8 8.6.8 8.. 8 .... 8 .... 8 8.... ...... ...... 2.8 3... ....o. .... .oo. .... .... o... 8.. .. .... N 3.... N 8... 8 ...... 8 .... 8 .... 8 .2... 8 8.. 8 x... 8 .... 8 ...... 8 .... 8 .... ...... .8... 8.... ...... .o... ...... .... 8.... 3.... 8.. 2... o. .3. .. ....o 8 ...o 8 8... 8 ...... 8 2... 8 .8 8 8... 8 .6... 8 8... 8 8... 8 8... 8 o... ....o ...o 8... 6.... ...o .6... 8... 8... 8.6 8... .o... o .8838 So... .... 8 2.8.8 2.3.8 ...o. 8 3.28 ....N8 .....8 ......8 ...... 8 .... 8 8.28 ...... ...... ...... 2.8. ...... .... .v... .....o ...8 ...... ...: .... so. ...... 8 ......o 8 8.6. 8 ...... 8 ...... 8 ......8 .... 8 .....8 $.38 ...... 8 8.. 8 .928 8. ... 8. .o. ...... .. 8.. .. a ..8 ...... 8... ...... .o... 8.. . 8.. ...... 88 .... 8 .... 8 3...: 8 8...: 8... 8 .... 8 ...... 8 ......8 84.8 .... 8 :8 8 .9. 8 .o. .. 8.8 8.8 8.... .. ...... 8.... 8... 8.... ...... .9. c... 8... ... ...... 8 ...... 8 ....o. 8 ...... 8 .... 8 .....8 o... 8 o... 8 .... . .9. 8 o... 8 ...o 8 ...8 ...... .... ...... ...? B... 2.... 8... ....8. ...+ o... 8.. o. 8... 8 ...o 8 .oo 8 ...o 8 ...... 8 8... 8 8... 8 .o... 8 ...... 8 8... 8 ...... 8 No.6 8 I... o... ...... ....o ...o ...o 8... 8... .o... 8.6 So 3... o 2:. Ex. .38 so... .80.... :3. you... :93. .82: :2. =95 gut. 83.5 «.0. 88m ...... 5...... .58.. ...... 8.... .53.. 8...... 2...... .52.. ...: .o .262— ..o .... 5 .Ed... 8 68.9.9.0 no... .0 8:0. .3 ...—...... 8 «.0829.» mm.— .e 2.8.» .25 em ...—2.... 8 .3889... mm.— a.o.£ 530...... ... =03 .... 540.5... ‘32.... he: 3.... \o .83.». mam I .n waned. 106 1294 (without skin) and 2*4 g. samples of raw and cooked thigh skin were weighed to the nearest 0.1 mg. and analyzed for PBB content. Dupli- cate 14—15 g. samples of broth were taken after first homogenizing the total broth recov- ered. Petroleum ether Soxhlet extraction (A.O.A.C. Method 11.23) was used for removal of PBBs from tissue samples. The broth was stirred with 150 ml. petroleum ether for 30 minutes using a 2.5 cm. magnetic stirrer at medium speed. The broth extractions were then washed with 1% NaSO. solution to remove water, and dried over anhydrous sodium sulfate for 30 min. Both broth and tissue sample extracts were treated with acetonitrile partion- ing (A.O.A.C. Method 29.001 ), Kuderna Danish Concentration and Florisil cleanup (A.O.A.C. Method 29.014) with 6% solution of ethyl and petroleum ethers collecting 200 ml. of eluate. PBBs were quantitated by comparing the area of the 6-isomer peak produced by the sam- ple and the area of the 6-isomer produced by the standard (FiremasterO BP-6. lot #5143, Michigan Chemical Corporation, Chicago, Illi- nob) The samples and standards in petroleum ether solution and nanograde hexane, respec- tively were run on a Tracor 560 GLC. equip- ped with a ‘3 Ni electron capture detector and interfaced with a Digital PDP-Be-Pamila GC Data System. The column for the GLC was a pyrex column, 1.83 m. long x 4.0 mm. i.d., packed with 3% 0V on Chromosorb W 60/80 mesh. The carrier gas was nitrogen with a flow rate of 40 mI./min. Temperatures were 270°C. 240°C, and 300°C. at the injection port. column and EC detector. respectively. Stan- dards were injected at the beginning of each run. after every 6-7 samples and at the end of the run. Quantitations were based on the peak area of the standards (hexabromobiphenyl peak). The presense of the P88 residues was confirmed by ultraviolet irradiation and mass spectrometric analysis. Ultraviolet test was car- ried out in a Rayonet photochemical reactor, model 1162. equipped with short wave UV lamps. Mass spectrometric analysis was run on a pool of all of the extracted samples. A 6065 gas chromatograph interfaced to a DuPont 21-490 mass spectrometer which was in turn interfaced to a Digital PDP-IZ computer was used. The mass spectra was obtained at an ionizing voltage of 70 eV. with a source temper- ature of 210°C. SMITH. ZABIK AND DAWSON Percentage recoveries of the P83 residues by the utilized method were determined. in spiked samples at levels of 0.02, 50. and 100 p.p.m.. to be 55, 66 and 97%. respectively. PBB levels were expressed as p.p.m. (wet weight), p.p.m. of solids and p.p.m. of fat. Total micrograms of P885 in raw and cooked chicken pieces as well as in broth were used to calculate percentage recovery and percentage distribution of PBBs recovered in the cooked meat and broth. The data were analyzed for variance and the studentized range test (Dun- can, 1957) was used to sort out any significant differences established by these analyses. RESULTS AND DISCUSSION Analyses of variance of percentage meat and broth yield indicate the level of P885 fed did not significantly affect these parameters. The type of piece cooked, however, did significantly affect both average meat and broth yield (Table l). The cooked meat yield for breast (61.6%) and thigh skin (59.2%) pieces was significantly higher (P<0.01) than that of drumstick (53.3%) or thigh meat (50.2%) pieces. In addition, the broth yield of the breast piece (89.6%) was significantly higher (P<0.01) than that of the thigh skin (77.7%). The breast piece weighed 2.5 times more than the next heaviest piece and thus had less surface area per unit of weight which would contribute to the reduced cooking losses. The average percentage of fat in the cooked chicken pieces (14.0%) were significantly lower (P<0.001) than in the raw pieces (21.1%) showing that rendering had occurred during cooking (Table 2). In addition, the average fat content of the drumstick (4.6%) was less (P<0.05) than that of the breast piece (7.9%) or thigh meat (7.5%); both of which had less fat (P<0.01) than the thigh skin (27.7%). Because of its higher fat content. the thigh skin also had significantly average higher (P<0.01) solids (37.1%) content than did the other chicken pieces used which were 26.7, 24.6. and 22.4% for breast. thigh meat and drumstick, respec- tively (Table 2). Neither the average lipid nor solids content were affected by the level of PBB fed to the hens. PBB levels in the raw and cooked chicken as well as in the chicken brath for each of the five levels fed are summarized in Table 3. The control birds had low levels of 9885 in their tissues; the highest level occurred in the high fat containing thigh skin. This unexpected low 107 level of contamination may have resulted from PBB particles being airborne on dust. Although all groups of birds were housed in separate cages, they were all in the same cage room and were all fed their respective rations at the same time. The levels of P885 in the tissues increased progressively with increasing levels in the feed and were highest in the high fat thigh skin. It was expected that the PBB levels in the tissues of hens fed feed containing 60 and 90 p.p.m. P885 and which were subsequently fed clean food for eight additional weeks, would be lower than that in tissues from birds fed 30 and 4S p.p.m. PBB in the feed since the half life in eggs, liver and muscle has been found to be approximately 3 weeks (Polin and Ringer, 1975). These samples, however, either con- tained skin or were higher in fat and thus these factors may have affected the rate of elimina- tion. As can be seen from the high standard deviations of the mean in Tables 2 and 3. wide variation in the P88 levels and fat content occurred in the birds of each group. Duplicate values on any one tissue, however, gave reason- able agreement. When the chicken pieces were cooked, the level of “His in the wet tissue decreased slightly (Table 3). Part of the PBBs lost was recovered in the broth. The level of PBBs in the broth of breast pieces was higher than that in the broth of the other pieces. Since the amount of cooking water was constant, greater amounts of 983s in the breast broth resulted from a larger amount of fat rendered from the larger piece. When the PBB content was expressed on a solids or fat basis, there was no significant difference among chicken pieces. Cooking did not significantly affect the level of PBBs ex- pressed on a solids basis. however, the average 933s in the fat of the broth (52.6 p.p.m.) was less (P<0.05) than that in the fat of the raw (86.1 p.p.m.) or cooked pieces (96.2 p.p.m.). Average values in the fat of cooked pieces were slightly higher than those in the fat of the raw pieces. Thus, while rendering of fat is undoubto edly an important mode of P83 reduction. the amount of PBB reduced is not directly propor- tional to fat removal. Total micrograms of PBBs in the cooked chicken and broth were compared to the level in the respective raw chicken piece to calculate the percentage recovery. No significant differ- ences occurred among the percentage of PBB recovered in any of the four pieces, percentage recoveries were 68.1% in thigh skin, 75.8% in the breast piece, 83.9% in the thigh meat, and 84.6% in drumsticks. Recoveries, however, did tend to be higher in the chicken pieces which contained less fat (i.e. drumstick and thigh meat) even though these pieces had lower percentage meat yields (Table 1) than did the higher fat breast piece or thigh skin. The distribution of the recovered PBBs between the cooked meat and broth is illus- trated in Figure l. The percentage of recovered P885 in the meat did not differ significantly among the four pieces evaluated and ranged from 65.5% in the cooked thigh skin to 72.9% in the cooked drumstick. The proportion of the recovered P88 in the cooked meat is consider- ably higher than that found in previous studies. Recovered PCBs were about equally distributed between the cooked meats and broth (Zabik, 1974) while only 1/4 to 1/3 of recovered lindane, dieldrin, and DDT compounds oc- curred in the cooked hen pieces (Morgan er al., 1972). The bulkier size and higher molecular weight of the P88 molecules may contribute to a smaller proportion of the recovered material being found in the broth. Moreover, Stadleman er al. (1965) reported that feeding high levels of several pesticides resulted in greater residue persistance in eggs and abominal fat of hens than was found when low levels were fed. Thus, the high levels of contamination may have influence this distribution. For pieces from control hens, the proportion of recovered PBBs '00? {5] near [:1 acorn 80 ’ 60 " as 335. so F 35}. {5}. 5351 20 » 355; 5:5: :35; Breast Drumsiiek Thigh Moot Thigh Skin FIG. 1. Distribution of recovered PBBs in cooked chicken and chicken broth. 108 in the meat was slightly less, ranging from 39.8% in the drumstick to 52.7% in the thigh meat. ACKNOW LEDG EMENT The authors express their appreciation to Drs. R. K. Ringer and D. Polinand the Poultry Science Department of Michigan State Univer- sity for supplying the hens. REFERENCES Aftosmis, J. G., O. C. Dashiell, F. D. Griffith, C. S. Hornberger, M. E. McDonnell, H. Sherman, F. O. Tayfun and R. S. Waritz. 1972. Toxicology of brominated biphenyls. ll. Skin, eye and inhalation toxicity and an acute test method for evaluating hepatotoxicity and accumulation in body fat. Toxicol. Appl. Pharmacol. 22:316-317. Association of Official Analytical Chemists, 1970. Official Methods of Analysis of the Association of Official Analytical Chemists. 11th Ed. Association of Official Analytical Chemists, Washington, DC. Babish, J. G., W. H. Gutenmann and G. S. Stoewsand. 1975. 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