I'H .. -: ‘1 ‘34,» a?! .‘ A“ ‘ .,. V g . . A . ‘ 12%", v .huu. v- .u- u \.-~.,4~y -‘ um. .‘ :."x ‘.‘ ‘ , $ ‘3 . .. V l ' .1. . 36.1... .r. 4_._ .2: u u .I 1" L“ '.‘ . i 1:4“. A t. -..‘.v.. 'zqu-nww "M .. .‘1'7 ”w . fl, ‘30:.5'3‘ ~ ‘ . .Y' I ' ‘ ' . .‘l' ‘- ‘ an“: _. ‘ 5‘ .fl —» ’ ‘ "1 e ‘ $.13 .{fié‘fi 'V ,. ‘ '\ ( MI ' ~.::e$ ~. ‘9.).‘ '- "‘ . ..'. 112$; ‘ \. ‘ \ A ‘ H . . . . .. _ , k‘ w ‘.- ~ '- ~v‘a. ‘ . ~."«.th?€1,§'.‘.5. . .. -. 4. 'uv u , . . ‘4 “.33.“- vqt 1‘ sum; .m n. .m - n w ‘ m ~~ 3" v V - . (3:32»; :L ?‘ ’l V‘txfl 4 This is to certify that the thesis entitled Effects of Cooking on Distribution Pattern and Reduction of Congener Specific Polychlorinated Biphenyls (PCBs) in Carp presented by Jeong—Hee Song has been accepted towards fulfillment of the requirements for Master's degree in FOOd Science 77%? a 2 We Zglajor fifessor Datefirch 23, 1994 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution li‘lll‘lllllllll Y LIBRARY Mlchlgan State University PM? :ETEUR NBOX oremavo othbchockomfromVr..ourocord Summoner “Manda“. DATE DUE DATE DUE DATE DUE . E - -- f - g I l I l l l l - MSU hatAnAfflrm WWMMWMW EFFECTS OF COOKING ON DISTRIBUTION PATTERN AND REDUCTION OF CONGENER SPECIFIC POLYCHLORINATED BIPHENYLS(PCBB) IN CARP BY Jeong-Hee Song 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 1994 ABSTRACT EFFECTS OF COOKING ON DISTRIBUTION PATTERN AND REDUCTION OF CONGENER SPECIFIC POLYCHLORINATED BIPHENYLS(PCBS) IN CARP. BY JEONG-HEE SONG The retentional and distributional changes of PCBs by cooking were studied with carp (Cyprinus Carpio) harvested from Lake Erie and Lake Huron. Carp were pan fried or deep fat fried in order to compare PCBs in raw and cooked fish. In general, since fish contain great amount of PCBs in fatty areas under skin, carp were prepared with and without skin to study the differences in concentration of PCBs. PCBs were extracted with dichloromethane, cleaned up by GPC and florisil and silica gel chromatographic columns. The concentrations of 52 individual and total PCB congeners based on Aroclor®>1254 were analyzed by capillary column GC. The total PCBs were the summation of detectable congeners. In this study, cooked fish exhibited the lower levels of PCBs than uncooked fish. However, the statistical comparison (Tukey's Test) between two cooking methods showed no significant difference for the effectiveness in reducing PCB levels, even though deep fat frying was a little more effective than pan frying. The average percent reduction of total PCBs based on total micrograms per fillet was 30.2i 14.1% by pan frying, 38.1i15.6% by deep fat frying. The distribution of PCBs in carp and cooking effect on PCB homolog congeners were also determined. In both raw and cooked carp, the distribution of PCB homolog congeners was in order: penta > tetra— > hexa— > heptachlorobiphenyls. The total PCBs in skin-off carp were lower than skin-on carp. To my parents, Gi-Soon Choi, Jin-Gi Kim, Myung-Hee Cho, and Ki-Jeong Song iv ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Mary Ellen Zabik who guided and encouraged me to finish my graduate program and this thesis. I appreciate also the members of the guidance committee: Dr. Matthew Zabik and Dr. Alden M. Booren. I am also grateful to Sandy Wu Daubenmire for her helpfulness and kindness during my study. In addition to, thank to Jie Wang to help my research. I would also like to thank all my neighboring friends. Finally, I thank my husband, Kwang-Yong Song (SKY) for his love and support as well as my son, Jihoon for his cooperation. TABLE OF CONTENTS Page LIST OF TABLES ...................................... viii LIST OF FIGURES ..................................... x INTRODUCTION ........................................ 1 REVIEW OF LITERATURE ................................. 4 Polychlorinated Biphenyls (PCBs) ................ 4 Factors in Reducing Polychlorinated Hydrocarbon Chemicals during Cooking ............ 9 Cooking Temperature ........................ 9 Cooking Time ............................... 11 Fat Rendering and Cooking Surface Area ............................... 13 Skin and Adipose Tissue Removal ............ 17 Carp (cyprinus Carpio) .......................... 19 EXPERIMENTAL PROCEDURE ............................... 21 Materials ....................................... 21 Glassware Preparation ...................... 21 Solvents and Reagent Preparation ........... 21 Sample Collection and Processing ................ 22 Sample Preparation .............................. 26 Preparation of Raw Sample .................. 26 Cooking Methods for Cooked Sample .......... 26 Pan Frying ............................ 26 Deep Fat Frying ....................... 27 Polychlorinated Biphenyl analyses ............... 29 Lipid Extraction ........................... 29 Cleanup of Lipid Extract ................... 31 Gel Permeation Chromatography ......... 32 Florisil column chromatography ........ 33 Silica Gel Column Chromatography ...... 33 Qualification and Quantitation ............. 34 Statistical Analyses ............................ 38 RESULTS AND DISCUSSION ............................... 39 Cooking Losses .................................. 40 Cooking Effects on the Reduction of Total PCB Levels ................................ 42 vi TABLE OF CONTENTS--CONTINUED Cooking Effects on the Reduction of PCB Homolog Congeners and Specific Congeners ......... Skin Removal Effects on PCBs Reduction .......... CONCLUSION ........................................... PROPOSALS FOR FURTHER RESEARCH ....................... REFERENCES ........................................... APPENDICES ........................................... vii 55 77 81 85 87 94 LIST OF TABLES TABLE 1. Approximate Composition of Aroclor®>1254 ...... 2. Chlorinated Pesticide Residue per Gram of Fat in Chicken Meat at Different Temperatures ..... 3. Dieldrin Content in Sausage, ppm based on Fat ........................................... 4. The Amounts of DDT in Chicken Tissues Cooked for Varying Times ............................. 5. Effect of Heat Processing and Storage on Chlorinated Hydrocarbon Pesticides in Spinach and Apricots .................................. 6. Total Cooking Loss in Carp during Cooking ..... 7. Total Cooking Yield in Carp during Cooking 8. Total PCBs Expressed as ppm in Wet Tissue in Raw and Cooked Carp Fillets ................ 9. Total PCBs Expressed as ppm in Dry Tissue in Raw and Cooked Carp Fillets ................ 10. Total PCBs Expressed as Micrograms per Raw and Cooked Carp Fillet from Lake Erie and Percent Reductions of PCBs by Pan Frying and Deep Fat Frying ........................... 11. Total PCBs Expressed as Micrograms per Raw and Cooked Carp Fillet from Lake Huron and Percent Reductions of PCBs by Pan Frying and Deep Fat Frying ........................... 12-a. PCB Homolog Congeners Expressed as ppm Wet Tissue in Raw and Cooked Carp Fillets Harvested from Lake Erie ....................... 12-b. PCB Homolog Congeners Expressed as Micrograms per Fillet in Wet Tissue of Raw and Cooked Carp Harvested from Lake Erie ....................... viii 10 12 14 15 41 41 43 44 52 53 6O 61 LIST OF TABLE--CONTINUED l3—a. PCB Homolog Congeners Expressed as ppm Wet Tissue in Raw and Cooked Carp Fillets Harvested from Lake Huron ........................ 62 13-b. PCB Homolog Congeners Expressed as Micrograms per Fillet in Wet Tissue of Raw and Cooked Carp Harvested from Lake Huron ...................... 63 ix LIST OF FIGURES FIGURE Page 1. Biphenyl Structure and Numbering of Carbon Atoms in Each Ring ............................. 5 2. Portions of Fish steak by Cutting Vertically ..................................... 18 3. The Sample Collection: near Monroe in Lake Erie and Saginaw Bay in Lake Huron ............. 23 4. The Procedure of Fish Processing and Filleting ...................................... 25 5. Overall Analytical Procedures for Congener Specific PCBs .................................. 30 6. Cleanup and Separation on Procedure of PCBs and Pesticides ................................. 35 7. Examplified Chromatograms. Top, Packed Column; Bottom, Capillary Column ....................... 45 8. The Average total PCBs in Carp from Lake Erie and Lake Huron: Top, Capillary Column; Bottom, Packed Column .................................. 47 9-a. The Correlation between Length and Weight of Carp ......................................... 49 9-b. The Correlation between Weight and PCB Level.. 49 9-c. The Correlation between Length and PCB Level.. 50 10. The Percent reduction of Total PCB Congeners by Pan Frying and Deep Fat Frying ............. 54 lJn-a. Each Grouped PCB Congeners in Raw and Pan Fried Carp Harvested from Lake Erie .............. 56 llr-b. Each Grouped PCB Congeners in Raw and Deep Fat Fried Carp Harvested from Lake Erie ......... 57 LIST OF FIGURES--CONTINUED 12-a. Each Grouped PCB Congeners in Raw and Pan Fried Carp Harvested from Lake Huron ............ 58 12-b. Each Grouped PCB Congeners in Raw and Deep Fat Fried Carp Harvested from Lake Huron ......... 59 13-a. Percentage Reduction of Each Grouped PCB Congeners by Pan Frying Carp Harvested from Lake Erie ................................ 65 13-b. Percentage Reduction of Each Grouped PCB Congeners by Deep Fat Frying Carp Harvested from Lake Erie ................................ 66 l4-a. Percentage Reduction of Each Grouped PCB Congeners by Pan Frying Carp Harvested from Lake Huron ................................ 67 14-b. Percentage Reduction of Each Grouped PCB Congeners by Deep Fat Frying Carp Harvested from Lake Huron ................................ 68 15-a. Congener Specific PCBs in Raw and Pan Fried Carp from Lake Erie, Skin-on ............. 69 15-b. Congener Specific PCBs in Raw and Pan Fried Carp from Lake Erie, Skin-off ............ 7O 16-a. Congener Specific PCBs in Raw and Deep Fat Fried Carp from Lake Erie, Skin-on ............. 71 l6-b. Congener Specific PCBs in Raw and Deep Fat Fried Carp from Lake Erie, Skin-off ............ 72 l7—a. Congener Specific PCBs in Raw and Pan Fried Carp from Lake Huron, Skin-on ............. 73 l7-b. Congener Specific PCBs in Raw and Pan Fried Carp from Lake Huron, Skin-off ............ 74 18~a. Congener Specific PCBs in Raw and Deep Fat Fried Carp from Lake Huron, Skin-on ............. 75 18-h. Congener Specific PCBs in Raw and Deep Fat Fried Carp from Lake Huron, Skin-off ............ 76 xi LIST OF FIGURES--CONTINUED 19. The Average Total PCBs in Skin—on and Skin-off Carp from Lake Erie and Lake Huron ............... 78 xii INTRODUCTION It is well known that harmful chemicals are accumulated in food via concentration in the food chain from contaminated environments. Since foods are usually consumed after cooking, it is necessary to investigate the cooking effects on the concentration of harmful chemical residues in the finished product. For long time, a lot of efforts have been made to determine the level of pesticides and other environmental contaminant residues in food after processing and cooking (Liska et al., 1967; Smith et al., 1977; Trotteret al., 1989; Zabik et al., 1992). Polychlorinated biphenyls (PCBs) are one of the chemicals which have contaminated environments and accumulated in food chain. Since Jensen (1966) identified PCBs, the concerns for PCBs in food has increased rapidly. Until 1977, PCBs were commonly used in the electrical and industrial areas. PCBs have been frequently found in rivers, lakes, of course, in the fish. PCBs are highly toxic and can cause cancer (Pal et al., 1980; Das and Kulkarni,l981; Concon, 1988; McFarland and Clarke, 1989). Most of exposure of the general human population to 1 I?CBs appears to be by ingestion of fish in the diet (Lands, 3.986), although some PCBs ingestion has been associated with (eggs and milk. Significant levels of PCBs have been discovered in fish used for human consumption in many different countries (Nelson, 1972; Schwartz et al., 1983; Seiber, 1990). PCBs may pose a health threat to consumers of fish. The diet of fresh water fish may constitute a small percentage of the total diet of consumers in the United States, however, some individuals in selected communities may consume significant amount of fresh water fish. Thus, the investigation of these chemicals in fish as eaten is necessary to lower a health threat to consumers of fish. In the present research, the retentional and distributional changes of PCBs during cooking carp (Cyprinus carpio) which had been caught from Lake Erie and Lake Huron using sizes representative of those caught by sport fishermen were studied. Carp fillets were pan fried or deep fat fried in order to compare the levels of PCBs in cooked fish with those in raw fish. Also, since fish contain the chlorinated hydrocarbon chemicals in higher amounts in the fatty area under the skin (Reinert et al., 1972; Hora, 1981; Sanders and Haynes, 1988), fish samples were cooked with or without skin to investigate whether skinning reduced PCBs contents of the cooked fillets. The objectives of this study were to determine the eeffects of cooking carp (Cyprinus carpio) by pan frying and cieep fat frying on PCB levels, and to compare the effect of ‘two cooking methods on the reduction of congener specific PCBs commonly found in Aroclor 125m®. Also carp was cooked with and without skin to determine the effect of the skin removal from fish on reduction of those congener specific PCBS. REVIEW OF LITERATURE Polychlorinated Biphenyls (PCBs) PCBs are a group of chlorinated hydrocarbons synthesized by chlorination of the biphenyl molecule. PCBs are not naturally occurring compounds. Chlorobiphenyls may carry 1 to 10 chlorine atoms with varying degrees of chlorination. There are 209 possible PCB congeners (Appendix 1). The nomenclature of chlorinated biphenyls is based on the position and extent of substitution of chlorine atoms on the biphenyl ring structure as shown in Figure 1 (The United Nations Environment Programme and the World Health Organization, 1976; Pal et al., 1980). Usually commercial products are different mixtures of Chlorobiphenyls, rather than a single pure compound. There are some kinds of commercial mixtures: Aroclor (U.S.A.), Phenoclor (France), Kanechlor (Japan), and Fenclor (Italy). Individual manufacturers had their own system of identification for their products. In the AroclorC>series, a four digit code is used; biphenyls were generally indicated by 12 in the first two positions, while the last two numbers indicated the percentage by weight of chlorine 4 Figure 1. Biphenyl structure and numbering of carbon atoms in each ring: X represents either H or Cl depending on chlorination (Pal et al., 1980). :in the mixture. Thus, Aroclor 1254C’is a polychlorinated koiphenyl mixture containing 54% chlorine (The United Nations Ivaironment Programme and The World Health Organization, 1976). The approximate composition of Aroclor 1254‘:> is presented in Table 1. Typical properties of PCBs are high thermal and chemical stability, low vapor pressure, high dielectric constant, high electric resistivity, high density, substantially hydrophobic and high lipophilicity. Their chemical and physical stability has been responsible for the PCB environmental contamination problem. Their melting points range from 34°C to 198°C and boiling points are usually from 275”: to 420°C (Pal et al., 1980; MacKay et al., 1983; Concon, 1988). Historically, PCBs were first synthesized in the late 18008, but were not used for industrial purpose in the United States until 1929. Since that time, PCBs have been distributed worldwide and accumulated in food chain from contaminated environments (Henderson et al., 1971; Kolbye, 1972; Fujiwara, 1975). All manufacturing of PCBs was prohibited after 1977 (Ghirelli et al., 1983). PCBs are accumulated in living matter, particularly in lipid tissues and organs. Most bioaccumulating PCB congeners have five to seven chlorine atoms per module. They were synthesized in Table 1. Approximate composition of Aroclor 1254C) (Pal et al., 1980) Empirical # of isomers Composition(%) formula C12H9Cl 3 < 0.1 C12H8C12 12 0.5 C12H7Cl3 24 1.0 C12H6Cl4 42 21 C12H5Cl5 46 48 C12H4Cl6 42 23 C12H3Cl7 24 6 C12H2C18 12 - C12H1C19 3 - C120110 1 ' *' The composition of Aroclor mixture was ascertained by the gas chromatographic separation. high proportions in many Aroclor formulations and are likely to be prevalent in environment. The more highly chlorinated congeners are generally less available to organisms. Congeners with less chlorination are more readily metabolized and eliminated (Hutzinger et al., 1974; Metcalf et al., 1975; Varanasi et al., 1992). The toxicity of PCBs varies greatly from species to species, probably as a result of differences in metabolic rates and capabilities, physiological differences, etc. The tnoxicity of the PCB congeners varies widely. Of the possible 2209 PCB congeners, only a few — especially the planar cxongeners without chlorine substitution at the ortho pmositions of the biphenyl moiety - are demonstrably or pcrtentially toxic (Nelson, 1972; Hutzinger et al., 1974; Sarfe, 1987; McFarland and Clarke, 1989). PCB congeners that 6113 the most similar structurally to 2, 3, 7, 8 tEd:rachlorodibenzo-p-dioxin (TCDD) are most toxic. PCBs can caiise liver damage and immunosupression, reproduction Prcflolems and birth defect etc. (Vos and Koeman, 1970; V05, 19'72; The United Nations Environment Programme and the World HEEthh Organization, 1976; Giesy et al., 1986; Krahn et a]--»,1986; Kubiak et al., 1989; Park, 1991). The studies on thfi? toxicity of PCBs have generally been conducted as lahXDratory or occupational exposure at high levels. Thus the Ckflgree of toxicity and effects on man has been conversable. Factors in Reducing Polychlorinated Hydrocarbon Chemicals during Cooking There are several factors which affect the reduction of PCBs and other chlorinated hydrocarbon pesticides during cooking. Since PCBs are polychlorinated hydrocarbons such as the pesticides, dichloro-diphenyl-trichloromethane (DDT), dieldrin and heptachlor, the factors can be considered with general chlorinated hydrocarbon chemicals or pesticides, loased on the work of other researchers who have investigated tflie effects of cooking or processing on those harmful chemicals. Cooking Temperature The reduction of some chlorinated hydrocarbon péisticides by cooking was reported by Liska et a1. (1967). Thuey simmered at 88°C to 93°C for 2-3 hours or high—pressure CCKDked chicken at 15 psi for 3 hours. It was shown that the le‘flels of DDT, dieldrin, and heptachlor were reduced in white meat during simmering chicken. Also, they compared the effect of temperature on heptachlor residue reduction. High temperature treatment (pressure cooking) reduced greatly the le\nel of heptachlor in chicken meat (Table 2). Temperature effects on chlorinated pesticide residue reduction during cooking were also investigated by Maul et 10 Table 2. Chlorinated pesticide residue per gram of fat in chicken meat at different temperatures (Liska et al., 1967). Pesticide Content (ppm) Dark meat White meat Pesticide Raw Cooked Raw Cooked DDT 7.7 7.1 22.5 17.9 Dieldrin 12 6 16 6 26 0 23 3 Heptachlor(L) 6 1 6 3 9.1 6 7 Heptachlor(H) 3 8 0.7 8 4 1 6 DDT, Dieldrin Heptachlor(L) Heptachlor(H) simmering at 88°C to 93°C for 2—3 hours simmering at 88°C to 93°C for 2-3 hours high temperature treatment, pressure cooking at 15 psi for 3 hours 11 al. (1971), Funk et al. (1971), and Yadrick et al. (1971). In study by Funk et al. (1971), sausage was pan fried at 204 °C and baked 177i1°C. The higher temperature cooking method, pan frying was more effective in reduction of dieldrin residue. Some of the dieldrin removed from sausage was found in the drip. Sausage samples cooked by pan frying had, in general, lower levels of dieldrin than samples cooked by baking (Table 3). Also Hemphill et al. (1966) indicated that higher temperature cooking methods were more effective in the reduction of chlorinated hydrocarbon pesticides. The increased effectiveness of residue reduction at higher temperature might occur due to more fat loss and volatilization, in addition to heat destruction per se. Cooking Time Cooking time is also one of the factors affecting the reduction of chlorinated pesticides and PCBs in food. Longer cooking time might give the greater opportunity for removal of chlorinated hydrocarbon chemicals from food during cooking. DDT in chicken tissue was reduced during heating tissues in closed containers for varying lengths of time (Ritchey et al., 1969). Only the moisture and volatile components could escape. The longer heating time appeared 12 Table 3. Dieldrin content in sausage, ppm based on fat (Funk et al., 1971) Cooking Raw Sausage Drip Method Sample # (ppm) (ppm) (ppm) Pan-Frying l 23.99 11.82 16.66 2 20.57 15.19 2.93 Baking 1 23.99 16.51 11.15 2 20.57 18.21 3.67 Pan frying sausage at 204°C Baking sausage at 177i1%2 13 to be more effective in reduction of DDT residues (Table 4). The reduction of DDT increased as the heating time increased, even though some of DDT was converted to a metabolite, dichloro-diphenyl-dichloroethane (DDD), during the heating process. The losses of DDT residue during cooking can be also attributed to losses of residue in fat. DDT, dichloro-diphehnyl-ethane (DDE), and DDD were found to exist at relatively high levels in the fat portion of the drippings. Further study by Ritchey et al. (1972) indicated that heating alone also reduced lindane and heptachlor epoxide. Additionally, the effect of storage time on reduction of chlorinated hydrocarbons was studied with fruit and vegetable (Elkins et al., 1972). They analyzed chlorinated hydrocarbons before and after heat treatment, and also after storage 1 year at ambient and 100°F temperature (Table 5). Storage resulted in decreases of chlorinated hydrocarbons. Fat Rendering and Cooking Surface Area Since most of chlorinated hydrocarbon pesticides and PCBs are lipophilic, they tend to be deposited in fat tissues. Therefore, fat rendering is an important factor in reduction of chlorinated hydrocarbon chemicals in food. Fat rendering might be related to cooking temperature, cooking time, and the structure of meat tissue. 14 Thable 4. The amounts of DDT in chicken tissues cooked for varying times (Ritchey et al., 1969) ppm in dry matter Heating time DDT DDE DDD Total 0 (raw) 31.3 11.0 3.0 45.3 30 min 22.7 9.9 5.6 38.2 60 min 14.7 9.7 11.8 36.2 90 min 9.4 9.8 14.5 33.7 IHeating at 177°C for 30, 60, and 90 minutes. 15 Table 5. Effect of heat processing and storage on chlorinated hydrocarbon pesticides in spinach and apricots (Elkins et al., 1972) % Reduction in residue level Initial Heat Ambient 100°F Pesticide level (ppm) processed 1 yr 1 yr Spinach Captan 35.7 93 100 100 Lindane 10.1 33 49 99 Thiodan 1.84 19 19 85 Toxaphene 6.50 27 6O 95 Methoxychlor 12.6 21 65 100 Apricots Captan 88.5 97 99 100 Lindane 6.80 13 56 100 Thiodan 1.79 13 22 85 Toxaphene 6.80 7 35 92 Methoxychlor 12.5 0 82 100 Heat processing was 65/66/252 for spinach 65/50/217 for apricots, where the numbers are initial temperature (°F), length of process in minutes, and processing temperature (°F), respectively. 16 Some chlorinated pesticides and PCBs were rendered from meat into dripping. Yadrick et al. (1971), Maul et al. (1971), Morgan et al. (1971), Zabik (1974, 1979), and Shafer and Zabik (1975) compared the amount of those chemicals in broth and in meat. Most of the those chemical residue losses which occurred were attributed to fat rendering during cooking, even though significant differences of chemical losses and distribution between meat and broth occurred among meat pieces. A large portion of residue recovered was found in the broth. Great fat loss from adipose tissue contributed significantly to the loss of high proportion of chlorinated pesticides and PCBs into the broth. Therefore, fat rendering during cooking appears to be major mode of chlorinated pesticide and PCBs residues reduction in food, although there are some other differences. It is likely that the greater surface area allows fat rendering which then reduces more chlorinated pesticide and PCBs residues from food. Thus, reduction of chlorinated pesticides and PCBs may be increased by shaping cuts to have maximum surface area. Also, surface can be exposed to higher temperature than center of meat during cooking, so that higher heat may promote more volatile and fat loss (Zabik et al., 1980). Ritchey et al. (1969) suggested cooking procedures which leached fat from the foods were most effective in reducing chlorinated hydrocarbon residues, l7 alqthough long heating times were somewhat effective. Skin and Adipose Tissue Removal Most chlorinated pesticides and PCBs are lipid soluble. Yt>shida et al. (1973) observed the elevated levels of PCBs iri the skin and dark muscle of carp (Cyprinus carpio) due to a. higher lipid content than in white muscle. Concentrations (of? DDT residues were highest in parts with the highest fat <:cxntent in fish, such as dorsal, ventral and medial as shown .ir1 Figure 2 (Reinert et al., 1972). Also chlorinated riyndrocarbon pesticide residues were at a highest cxaxncentration in the abdominal fat, less in dark meat, and leeast in white meat of leghorn hens (McCaskey et al., 1968). The process which removes lipid from fish is likely to a1:ter the PCBs and chlorinated pesticide concentrations in tile fish. The effect of skin removal on the PCBs in fish Tfillets was determined to be used in fish advisories to help Iluman. Removal of skin from edible portions reduced the PCBs laced in glass containers, covered with aluminium foil, and (zapped. All samples were stored at -34°C. Cooking Methods for Cooked Sample For fish which would be in the cooked state, skin-on €3nd.skin—off fish were pan fried, according to the procedure (of Puffer and Gossett (1983) or deep fat fried, according to 'the procedure of Morehouse and Zabik (1989). All equipment \Nhich was in contact with fish was cleaned with a cotton iball dipped into acetone after each use. £31 Frxing A sample was removed from the freezer shortly before the pan temperature reached 175W3. Each side of fish fillet 27 was sprayed with no stick cooking spray (Pam®) for 3 seuzonds. A thermocouple was placed in the center of the trLickest portion of the fish sample and a second thuermocouple was placed on the surface of the frying pan to Hubriitor the internal temperature, and to keep the frying EDaII at 185i5W3, respectively. Sprayed fish fillet was placed on the frying pan, tlien the no stick cooking spray was sprayed lightly around tine fish. If the sample was a skin-on fish fillet, the skin Slide was placed on the frying pan surface first. Since the ffish skin acts as an insulator and keeps the temperature firom increasing, cooking the skin-absent side was the last Sitep. The fish fillet was cooked until the internal tnemperature reached 80°C, during which the fish piece was tnirned at each 20°C. After cooking, the sample was placed on wire cooling Irack for 1 minute. When a skin—on sample was used, the skin \Nas peeled off so only the muscle tissue was analyzed as the edible portion. Qgep Fat Frying The oil (1300i10g of Mikado, commercial soybean oil) was placed in a frying pan and heated to 180—185W3. The sample was remoVed from freezer just before the oil 28 temperature reached lSOWC..A thermocouple was placed in the center of the thickest portion of the fish sample. A second thermocouple was placed in the frying oil to monitor the frying temperature and to keep it at 180i5W3. The Sample was cooked until the internal temperature reached 80i3°C, and drained in a fryer basket for 15 seconds, then on a wire rack for an additional minute. Both pan fried and deep fat fried fish samples were homogenized or ground in an Omnimixer. Ground samples were placed in glass jars (prerinsed with acetone and hexane) and labeled. Samples were frozen and stored at —34°C. Total cooking losses were calculated for each cooking method (Appendix 3). Fish pan fried with skin had the muscle flaked away from the skin, and the cooked muscle was weighed as the edible portion. Since deep fat fried fish are usually coated with a batter or a breading, the consumers would generally eat the skin so that the skin was included in the cooked portion. Percentage of cooking yield was based on the relation of the cooked edible weight to the raw weight (Appendix 3). Cook yield % did not contain skin except deep fat frying samples but cooking loss % contained skin. Cooked Yield % = cooked edibleweight X 100 raw fish fillet weight 29 Cooking Loss % = raw fish fillet weight -cooked fish weight X 100 raw fish fillet weight Polychlorinated Biphenyl Analyses Extraction and cleanup of samples for PCB analyses were performed using the gel permeation chromatography (GPC), florisil and silica gel chromatography. Identification and quantification of PCBs were done by gas chromatography. Capillary column gas chromatographic procedure by Ribick et al. (1982) was applied in our laboratories. The analytical procedures are summarized in Figure 5. Lipid Extraction Since fish contain lipids and PCBs are deposited in lipids, their samples can be extracted by techniques similar to those used for adipose tissue. Lipid extract from fish sample contains organochlorine pesticides and PCBs. The organochlorine pesticides and PCBs are non-polar compounds. In most cases, they are co-extracted together. In column lipid extraction, fish samples were extracted With dichloromethane. The PCB congener 2,4,6 tri- chlorobiphenyl (IUPAC #30) was used as an internal standard for determination of concentrations and recoveries of PCBs. 30 SAMPLE(CARP) COLLECTION FROM LAKES ERIE & HURON U SAMPLE PREPARATION WITHIN 24HR AFTER HARVEST U COOKING PAN FRYING & DEEP FAT FRYING U LIPID EXTRACTION (CHLORINATED HYDROCARBON PESTICIDES) U SAMPLE CLEANUP 1. GPC 2. FLORISIL CHROMATOGRAPHY 3. SILICA GEL CHROMATOGRAPHY U IDENTIFICATION & QUANTIFICATION BY GAS CHROMATOGRAPHY EQUIPPED WITH 63Ni ECD Figure 5. Overall analytical procedures for congener specific PCBs 31 This internal standard has not been found in the environment. The column extraction has some advantages: less time consuming, less requirement of fragile equipment, no requirement of the multiple-solvent, and can handle numerous samples simultaneously. The lipid extraction was accomplished by following procedure. Sample of fish (10.0g) and 1 mL of internal standard PCB (IUPAC #30, 5 ppm) was homogenized with 40g of granular anhydrous NaZSO4 (activated and stored overnight at 130°C) and ground to a fine powder. The ground fish mixture was eluted in a 1 cm i.d. reservoir column with 200 mL of, dichloromethane at a flow rate of 3—5 mL/min, collected and reduced in the Turbo-Vap evaporator (Zymark) to approximately 0.5 mL volume at ambient temperature. Care was taken to avoid the dryness of extract. The concentrated lipid extract was then diluted to 5 mL with 1:1 (V/v) hexane and dichloromethane mixture and placed in a 5 mL volumetric flask. This mixture of hexane and dichloromethane was used to promote high recoveries and to facilitate compound separation during GPC. Cleanup of Lipid Extract The cleanup step in an analytical procedure removes 32 other compounds which interfere with the determination of PCBs, prior to GC analysis. Gel Permeation Chromatography (GPC) Cleanup by GPC results in removal of more than 98% lipid without alteration of residue composition or levels. GPC is a form of liquid chromatography in which solute molecules are separated selectively as they permeate the pores in the column packing. The larger lipid molecules are excluded from pores because of their size, and therefore they elute from the column before the smaller contaminants do. The automated GPC system provided for unattended operation with time control to remove lipid. Contaminant residues were collected and then a wash cycle flushed the system prior to the next sample. The following procedure of cleanup by GPC was: 4 mL concentrated extract was put in the vial. A 2 mL aliquot of this extract was then injected into GPC column attached to a Waters/590 Programmable HPLC pump and Waters fraction collector. The lipid part was dumped and PCB portion collected, then PCB portion was reduced in a Turbo-Vap evaporator to approximately 1 mL and diluted to 5 mL with hexane. The operating conditions are presented in Appendix 4. 33 Florisil Column Chromatographic Cleanup After removal of the lipids, the sample needed further cleanup to fractionate PCBs and other chlorinated hydrocarbon pesticides. Most chlorinated pesticides are eluted from a florisil column with a solvent 6% (by volume) of ethyl ether in petroleum ether. The procedure of florisil column chromatographic cleanup started column preparation. The column (1 cm i.d. x 51 cm) was prepared by placing 1 cm of granular anhydrous NaZSO4 on glass wool, followed by 5g of 60-80 mesh florisil (activated.at 130°C for 16 hours), then 1 cm of granular anhydrous NaZSO4, again. The prepared column was then washed (prewetted) with 20 mL of hexane. When the hexane reached the top of the upper layer of Na2804, the GPC concentrate was transferred to the column and allowed to drain onto a florisil bed and collected. Continuously, 40 mL of elution solvent (6% diethyl ether in petroleum ether) was added to the column. The collected eluent was reduced to approximately 5 mL volume in the Turvo—Vap evaporator (Zymark). Silica Gel Column Chromatographic Cleanup Silica gel column separates satisfactorily the PCBs from the majority of the remaining pesticides. The PCBs would be mainly eluted with 50 mL of 0.5% toluene in hexane. The column was prepared the same as described for the 34 florisil columns but with silica gel 60 (70-230 mesh) activated at 130°C for 16 hours. A 50 mL quantity of 0.5% toluene in hexane was used as elution solvent. The eluent was reduced to approximately 0.5 mL volume in the Turbo-Vap evaporator and transferred to the 1 mL volumetric flask, using hexane to rinse the Turbo-Vap tube. The concentrate was reduced to 0.5 mL volume under a stream of N2 gas and isooctane was added to make a final volume 1 mL. The procedures of cleanup and separation of PCBs and other chlorinated pesticides are summarized in Figure 6. Qualification and Quantification Concentration of individual PCB congeners and total concentration of PCBs were determined in the PCB extract by Gas Chromatography with 63Ni electron capture detector (Hewlett Packard 5890 series II), which was equipped with DB-5 capillary column (60 m x 0.25 mm i.d.). Electron capture detector is selective toward halogenated compounds. The separation from GC is effected by the interaction of the compounds with the gas (mobile phase) and liquid phase (stationary phase). The solubility in the liquid phase and Volatility affect the retention times of PCB congeners. PCBs would generally elute in order of chlorination:C12H9Cl first, C12Cllo last. The separated peaks from the GC column were detected at 35 5g Florisil (60~80 mesh, Fisher Scientific) activated at 130°C U 20 mL Hexane wash U Sample concentrate to Florisil U 40 mL 6% ethyl ether in petroleum ether Elution solvent U O,P'- & P,P'-DDT, DDD, DDE Toxaphene PCBs Mirex Heptachlor etc. U 5g Silica gel 60 (70~230 mesh, E.Merick) activated at 130°C U 20 mL Hexane wash U Sample concentrate Silica gel U 50 mL, 0.5% Toluene in Hexane U PCBs etc. Figure 6. Cleanup and separation on procedure of PCBs and pesticides (Ribick et al., 1981) 36 different times (retention times). The retention time of each PCB congener was compared to the retention time of the aauthentic standard 52 PCB congeners based on Arochlor 125m3 IHJD under the same conditions. The standard PCB congeners llSed are listed in Appendix 5. The internal standard PCB cxongener (IUPAC #30, 2,4,6—trichlorobiphenyl) was added to cxorrect the recovery during the entire analysis procedure. IAJnalysis for all 209 congeners is not necessary because some ENSB congeners have never been reported in environmental samples. All quantification was based on peak areas relative to t:11eearea of individual congener standards and congener #30. .3k standard curve was constructed for each PCB congener from cij_fferent concentrations of the standard mixture (:31:andard/50, standard/10, standard concentration). Detector ITéasnoonse factors were linear over a limited range. The total C3C>rucentration of PCBs was determined by summing the C3C>I‘.1<::entrations of all of the individual congeners listed in IXIDIDendix 5 which were detected from the GC in the extract. Final separation and quantification of PCBs were done VVTifth a Gas Chromatography, equipped with 63Ni electron (:Eipmure detector (ECD) under the following conditions: Detector: 300°C Inject: 220°C, 3 uL volume by auto sampler 37 Column: 60 m x 0.25 mm i.d. capillary DB-5 column Carrier gas: He, 1 mL/min. flow rate Oven temperature: programmed from 160°C to 280°C Detector make-up gas: N2 The samples were quantified by comparison of peak area Chetermined by a computerized integrator with appropriate :3t:andards of known concentration, using following eeczuations: Recovery % = 1000 GPC vol. X sample wt. (LS Of #30 X RA of #30) X Concentration = GLC vol. X 500 X LS of congener X RA of congener sample wt. X GPC vol. X Recovery % GPC vol.: Gas Permeation Chromatography volume (ml) GLC vol.: Gas Liquid Chromatography volume (ml) LS: Line Slope RA: Retention Area Sample wt.: Sample Weight (g) These analyses were completed by the Pesticide Research C3€3I1ter at Michigan State University. All cases were IrEEIblicated six times and 10% of samples were duplicated. 38 Statistical Analysis Procedure The data were analyzed by general linear models (GLM) using SAS statistical software(Cary, NC). procedure, ESignificant differences were determined at a 0.05 level of pyrobability using Tukey' Studentized Range Test. The main euffects of variable factors (lakes, cooking methods, raw or <:c>oked state, and skin-on or skin—off) as well as their I>c>ssible interactions were considered. Statistical analyses aizre summarized in Appendix 6. RESULTS AND DISCUSSION Carp obtain the bulk of their nourishment by sucking colrganic materials from bottom of lakes or stream. PCBs ee>chibit a high affinity for particulate, and sediments are <3c>nsidered to be a major environmental sink for these c:<>ntaminants. Young et al.(1976) and Seelye et al.(1982) ifcyund that PCBs were taken up to a great extent by mussels airici fish closer to the bottom. Therefore, it is likely that t:11€2 level of PCBs in carp would be relatively high. Carp from Lakes Erie and Huron in sizes representative (>15 those caught as sport fish, were pan fried or deep fat fTITiJed at 80°C with and without skin. After the fish fillets Were cooked, the extra oil and skin (if, skin-on fillets) VV€31362 discarded, except deep fat fried skin—on fish samples, F31?onr‘to PCB congener analyses. Since deep fat fried fish is frequently prepared with breading or a batter and the COnsumer would eat them with skin, the deep fat fried skin- (5:1 fish samples were analyzed with skin. Carp from Lake Erie were significantly larger than t1"lose from Lake Huron (Zabik et al., 1993). The average l‘EIIgth and weight were 51.8 cm, 1834 g in carp from Lake 39 40 Erie and 46.6 cm, 1573 g in carp from Lake Huron, respectively. Carcass % and as prepared yield % (AP Yield %) of the carp are listed in Appendix 3. As expected, the average AP yield % of skin-on carp (32.7%) was significantly greater than that of skin-off carp (25.6%), even though average carcass yield % of carp which would be skinned off was significantly greater than that of skin-on carp (Zabik et al., 1993, Appendix 6-2 and 6-3). Cooking Losses Total cooking losses and cooking yields by pan frying Eirlci deep fat frying are summarized in Tables 6 and 7. The presence or absence of skin did not affect the cooking losses, but the cooking methods did (significance, p < O - 05) . The total cooking losses in deep fat fried fish fillets (average 32.49%) was higher than those in pan fried fish fillets (average 19.86%) . Total cooking losses varied among the various cooking methods in a previous study (Zabik et al., 1982) which used Duncan's New Multiple Range test to Seek out significant differences among cooking methods. The highest cooking loss occurred in deep fat frying among the QOoking methods: poaching, roasting, deep fat frying, Q1'larbroiling, and microwave cooking. They reported that a 41 Table 6. Total cooking loss in carp during cooking Total Cooking Loss (%) Pan frying Deep fat frying PVith skin 21.4 31.5 Without skin 18.4 33.4 'T<>tal average 19.9 32.5 I1 = 12: 6 from Lake Erie, 6 from Lake Huron iPaak>le 7. Total cooking yield in carp during cooking Total Cooking Yield (%) Pan frying Deep fat frying With skin 70.7 68.4 Without skin 81.6 66.6 Total average 76.2 67.5 \ I1 2: 12: 6 from Lake Erie, 6 from Lake Huron 42 great decrease in moisture in deep fat fried fish fillets caused the great cooking loss, even though fat was absorbed chiring cooking. Cooking Effects on the Reduction of Total PCB Levels Means and standard deviations of total PCBs levels ciearived from the sum of the individual congeners were (Table 8), ppm dry tissue (Table ea>CZE3 congeners in this report will be reported as ppm on a unless specified otherwise, because most vveat: weight basis, C2<>risumers are familiar with the amount of PCBs in the edible vveat: fish fillet more easily. Since data expressed as ppm wet tissue will not consider weight loss during cooking, data ”Vi-1J1 also be presented as micrograms per fillet. The Food and Drug Administration (FDA) regulates the tZCDIJ—erance level of total PCBs for fish and shellfish as 2 IDFJDH in wet edible tissue based on packed column GC for all“Eilysis of sample. However in this study, the analysis was EDGEZli‘formed by capillary column GC. The chromatogram of (:EiIDillary column improves resolution and reveals many peaks CVE. hidden congeners as shown in Figure 7. Therefore, the Capillary column approach is likely to be more accurate than 43 Table 8. Total PCBs expressed as ppm wet tissue in raw and cooked carp fillets PCB Content (ppm) Imake Cooking Method Skin Raw Cooked Pan fried Skin-on *1.521i0.696 1.316i0.896 Exrie Skin-off 1.165i0.405 1.094i0.366 Deep Fat fried Skin-0n *3.547i2.109 3.413il.276 Skin-off 1.568i1.143 1.350i1.059 Pan fried skin-on 1.394i0.908 1.208i0.858 riLlron Skin-off 1.583i2.106 1.395i1.962 Deep Fat fried Skin-0n 1.598i0.949 1.188i0.721 Skin-off 0.897i0.345 0.903i0.399 ‘* I30th total PCB levels were resulted from different Enortions in the same fish, although each portion of fish 1'1 vvas randomly assigned to cooking methods. ~ _ 6: pan fried samples, deep fat fried samples included skin only muscle tissue analyzed 44 Table 9. Total PCBs expressed as ppm in dry tissues of raw and cooked carp fillets PCB Content (ppm) Lake Cooking Method Skin Raw Cooked Erie Pan fried Skin-on *5.905i3.068 4.147i2.664 Skin-off 5.468i1.829 3.626i1.012 Deep Fat fried Skin-0n *13.47i6.930 7.659i8.363 Skin-off 7.253i4.984 3.214i2.267 .Huron Pan fried Skin-0n 5.117:3.036 3.725:2.43o Skin-off 5.509i5.568 3.962i4.586 Deep Fat fried Skin-on 6.012i3.384 2.720i3.699 skin-off 3.637i1.337 2.122:o.733 *’ 130th total PCB levels were resulted from different Loortions in the same fish, although each portion of fish vvas randomly assigned to cooking methods. II == 6: pan fried samples, only muscle tissue analyzed. deep fat fried samples included skin. 45 °1 2:0 4:0 0T0 010 10.0 15.0 14.0 103 Tfllhht . IJ J) ("'1 W I V V I I U U V I I *w 20 a: 40 a» 00 :m 00 I) no Figure 7. Exemplified Chromatograms. Top, packed column; bottom, capillary column (Pellizzari et al., 1985). 46 the packed column approach (Pellizzari et al., 1985; Draper and Koszdin, 1991). It is likely the values by capillary column analyses would be relatively higher than those by packed column analyses (Figure 8). The average level of PCBs in skin-on fish from Lake Erie was 2.534 ppm in wet tissue. Four of the six fish from Lake Erie had higher than 2 ppm of total PCBs. Either a tail or head portion of same fish was randomly assigned to be pan fried or deep fat fried, however there were considerable variances among these portions in the same fish in Lake Erie samples. For instance, portions assigned to pan frying was 1.521 ppm of average total PCBs in wet tissue (5.905 ppm in dry tissue) but portions assigned to deep fat frying was 3.547 ppm in wet tissue (13.470 ppm in dry tissue). The high level of total PCBs and standard deviations in skin-off raw and pan fried fish from Lake Huron were due to only one fish which showed a very high PCBs level (5.872 ppm in raw fish, 5.387 ppm in pan fried fish). The other five fish contained lower than 1 ppm in wet tissue. The carp from Lake Erie had higher average levels of PCBs (skin-on; 2.534 ppm, skin-off; 1.367 ppm in wet tissue) than those from Lake Huron (skin-on; 1.496 ppm, skin-off; 1.240 ppm in wet tissue). The other previous studies (Thomas and Frank, 1983; Schmidt, 1989) reported that, in general, fishes from Lake Erie were more contaminated by PCBs than F“ 47 3 ,\25 ‘63 3 .s 2‘ g IISflHm JE15 E] :3 smnm 0 CL 1. 'B 7.5 05 __ 0‘ —-l Ede 3 A25 ‘33 3 .E 2‘ E ISM-0n 3151 :8 [Janna a .. IE 0 “'05< - 0‘ ._g Figure 8. The average total PCBs in same carp samples from Lake Erie and Lake Huron: top, capillary column; bottom, packed column at Michigan department of Public Health (Zabik et al., 1993) 48 those from Lake Huron. Generally the large fish showed the high level of total PCBs like the study by Hora (1981). The larger fish size might be partly responsible for higher level of PCBs in carp from Lake Erie. The correlation between length, weight and PCB level is presented in Figure 9-a, b, and c. Above all, the effects of cooking on total PCBs reduction of total PCBs and PCB homolog congeners (grouped PCB congeners by chlorination) were main concerns in this experiment. It is thought that the cooking effectiveness of reducing PCBs from fish depends on the species and their fat contents. Cooking by microwave, roasting, and broiling fat lake trout (ciscowet) from Lake Superior reduced PCBs by 26- 53% (Zabik et al., 1979). These fish averaged 25—29% fat. In contrast, the level of PCBs in carp harvested from Saginaw Bay, MI, with an average fat content of 7.7% was not affected by cooking (Zabik et al., 1982). The levels of PCBs in chinook and coho salmon, which were relatively low in fat, were reduced slightly by cooking (Smith et al., 1973). Fish higher in fat allowed for rendering of more fat. Thus cooking was effective in reduction of PCBs from fish. For the current study, the average total PCB levels after cooking carp are summarized in Tables 7 and 8. Samples cooked by the two methods had the lower levels of PCBs than uncooked samples, even though the average fat content in 49 23ml. I 2mm.» . ' 31500. ' " ' .— I I f; g; umo. ' 500‘) O c 4 : c e a 35 40 45 50 55 60 65 LunfihOmn) Figure 9-a. The correlation between length and weight (Correlation coefficient: 0.86) 0° .- 01-50! . t" . . ctunucnsaoum Total PCBs (ppm) N O 12M) lflm) 2mm ' 2R” 6' “hflnN(g) Figure 9-b. The correlation between weight and PCB level (Correlation coefficient: 0.84) 50 45v 4. fe35‘» g 3 I gg25<» 83 2.. j; L5» ,_ 1 4. 0.5 «I 0 : : . : . . 35 40 45 50 55 60 65 Lengflafinn) Figure 9-c. The correlation between length and PCB level (Correlation coefficient: 0.86) 51 these carp samples were relatively low. Lipophilic PCBs might be lost along with oil dripping and skin that was discarded after cooking. The statistical comparisons (Tukey test) between the two cooking methods revealed no significant difference for the effectiveness in reducing PCBs levels, even though deep fat frying was a little more effective. Skea et al. (1979) reported deep fat frying reduced PCBs more effectively than other cooking methods. However, some differences in the level of total PCB congeners were related to the lakes where the carp were caught. In order to determine the loss of PCBs during cooking, percentage reduction was determined based on the total micrograms of PCBs in raw and cooked fillets, because both fat and moisture are lost during cooking. The percentage reductions by total micrograms per fillet for all carp pieces with and without skin, are summarized in Table 10, and 11. Data expressed in Figure 10 shows the average effects of each cooking method. The percentage change of the total PCB congeners was calculated from the change in the sum of the individual congeners. Percentage change of total PCBs based on capillary column gas chromatography analyses ranged from 26% to 48% but that based on traditional packed column gas chromatography analyses by Michigan Department of Public Health (Zabik et al.,l993) ranged from 12% to 65%. 52 Table 10. Total PCBs expressed as micrograms per in raw and cooked carp fillets from Lake Erie and percentage reductions of PCBs by pan frying and deep fat frying. Total micrograms/fillet Cooking method Fish Raw Cooked % Change Pan frying Skin-on 1 114.59 62.35 45.59 2 318.31 145.31 54.35 3 122.85 90.70 26.17 4 466.12 400.34 14.11 5 204.46 94.89 53.59 6 316.82 243.97 22.99 average 36.13 Skin-off 1 151.95 93.84 38.24 2 145.50 104.57 28.13 3 223.23 150.60 32.54 4 113.01 89.45 20.85 5 80.05 68.49 14.44 6 65.81 52.24 20.62 average 25.80 Deep fat Skin-on frying 1 609.57 345.33 43.35 2 1452.52 615.79 57.61 3 358.84 266.85 24.59 4 585.45 520.24 11.14 5 179.99 159.69 11.28 6 387.52 287.25 25.88 average 28.98 Skin—off 1 182.31 113.97 44.80 2 137.00 77.57 50.52 3 650.00 366.11 48.89 4 116.23 81.12 38.90 5 76.13 36.39 57.78 6 61.09 39.04 44.27 average 47.53 n = 6 Pan fried samples: only muscle tissue analyzed. Deep fat fried samples: included skin. 53 Table 11. Total PCBs expressed as micrograms per in raw and cooked carp fillets from Lake Huron and percent reductions of PCBs by pan frying and deep fat frying. Total micrograms/fillet Cooking Method Fish Raw Cooked % Change Pan frying Skin-on 1 173.42 75.21 56.63 2 242.14 186.08 23.15 3 496.42 311.70 37.21 4 36.07 24.15 33.04 5 44.59 38.07 14.62 6 105.92 84.53 20.20 average 30.81 Skin-off 1 55.85 31.85 42.98 2 26.17 21.14 19.22 3 73.74 38.72 47.49 4 882.27 674.36 21.30 5 56.72 52.69 7.11 6 33.75 23.47 30.46 average 28.09 Deep fat Skin-on frying 1 148.44 77.69 47.67 2 381.77 145.65 61.85 3 439.34 262.22 40.31 4 93.54 37.70 59.70 5 58.94 42.53 27.84 6 94.20 52.02 44.77 average 47.02 Skin-off 1 61.76 29.81 51.73 2 31.42 26.74 14.88 3 47.00 36.35 22.66 4 218.76 175.54 19.76 5 64.13 46.26 27.87 6 59.13 37.01 37.40 average 29.05 n = 6 Pan fried samples: only muscle tissue analyzed. Deep fat fried samples: included skin. 54 SO-F Erie Huron 45-. 404» 0 In 0 a 3 c I MW 2 g Elmmnuqu ‘o O a 1* 1 I 4 a) m a) m '5. E a: E ? ? ? ? O O O O = a = (a Figure 10. The percent reduction of total PCB congeners by pan frying and deep fat frying 55 Cooking Effects on The Reduction of PCB Homolog Congeners and Specific Congeners The most potent PCB congeners are some of the congeners most resistant to degradation and metabolism and may be selectively enriched, relative to other PCB congeners (Tenabe et al., 1987). Since most of the threatening congeners are tetra-, penta-, hexa-, and hepta- congeners based on their abundance and potential toxicity, the concerning was focused on those congeners. The game fish and selected bottom feeders were analyzed at EPA (1992) to indicate the potential for risk to human health from fish consumption. In the EPA research entitled "National Study of Chemical Residues in Fish", concentration of hexachloro- biphenyls was highest, followed by pentachlorobiphenyls (hexa- > penta- > tetra- > heptachlorobiphenyls). The cooking effects on those homolog congeners and distribution pattern of this research are shown in Figures 11 and 12 and the actual means and standard deviations are presented in Tables 12 and 13. And since both fat and moisture are lost during cooking, expression based on micrograms per fillet which considered weight loss during cooking are also presented in Figures 11 and 12 which refract actual change during cooking. The levels of each PCB homolog congeners were reduced by both pan frying and deep fat frying. 56 PAN FRYING 0.9 0.8 Skin'On Skin-off 0.7 0.6 0.3 < 0.2 * 0.1 1 v0.5 1 So... O 1 IRow Clem _ II I l Tetra- Penta- Hexa- Hepta- Tetra- Penta- Hexa- Groupod PCB congeners 100 PAN FRYING ug lfillot (wot) 8 B 8 8 S 8 3 8 8 O -I Skin-on Skin-off .Row (Slocum .51.“...1 Grouped PCB congeners Figure ll-a. Each grouped PCB congeners in raw and pan fried carp harvested from Lake Erie. 57 DEEP FAT FRYING 12 1 Skin-on Skin-off ..06 g I Raw ”'06 5 [Bound °' 0.4 02. 0 I 6 6 6 6 b ‘E x '5 I93 g f g Grouped PCB congeners DEEP FAT FRYING 2d) 180 , . 160 Skin-on Skin-off §E140+ g 100« D t 80 .. cm s 60 I 401 20« I o. I I ' g a g .1 g a g a I- 8': I 3‘3 +- 8 I f Grouped PCB congeners Figure ll-b Each grouped PCB congeners in raw and deep fat . fried carp harvested from Lake Erie. 58 PAN FRYING 09 as Skin-on o7 =,oa~ § tetra— > hexa- > heptachlorobiphenyls), with only a little exception. Even though it was not consistent to EPA's report, the penta-PCB congeners might show a high level due to accumulation and abundance in environment. The distribution pattern may be affected by the difference in chlorine contents of various PCBs in an aquatic environment. However, it did not have a specific relationship between the reduction of PCBs during cooking and the number of chlorines in PCBs. The percentage reductions of each PCB homolog by cooking are shown in Figures 13 and 14. The pattern of specific PCB congener distribution in raw and pan fried and deep fat fried carp fillets from Lake Erie and Huron are shown in Figures 15 through 18, respectively. The values which are expressed as ppm wet tissue do not take into account the weight loss during the deep fat frying or pan frying. In contrast, the values expressed as micrograms per fillet do show loss during cooking. The specific PCB congener values in the cooked fillets were generally lower than in the raw. 65 PAN FRYING Skin-on Skin-off i‘ B 8 8 8 8 G 8 °/e PCB change Penta- Hexa- Hema- I 2 Q g. Grouped PCB congeners Figure l3-a. Percentage reduction of each grouped PCB congeners by pan frying carp harvested from Lake Erie (Skin-on pan fried sample had only muscle tissue) 66 DEEP FAT FRYING Skin-on Skin-off 8 8 8 8 8 a 8 % PCB change I I g g g “a g 32 g “a O O) O 0 '- n. I I *- o. I I Grouped PCB congeners Figure 13-b. Percentage reduction of each grouped PCB congeners by deep fat frying carp harvested from Lake Erie (Skin—on deep fat fried sample included skin) 67 PAAIFRYHVG ) 45 —— w in, 35 Jinan Skin-off % PCB change I I I I e a a a an is an a. on! a X h o- 2 5 E F I s a; a Grouped PCB congeners Figure l4-a. Percentage reduction of each grouped PCB congeners by pan frying carp harvested from Lake Huron (Skin—on pan fried sample had only muscle tissue) 68 DEEP FAT FRYING Skin-0" Skin-off 8 8 8 8 8 a 8 % PCB change Tetra- Penta- Hexa Hepta- Ttm Penta Hexa- Hema Grouped PCB congeners Figure 14-b. Percentage reduction of each grouped PCB congeners by deep fat frying carp harvested from Lake Huron (Skin—on deep fat fried sample included skin) 69 r .Raw [Janna 0.02 - 0 H, 11, sneezeogezazssgsasgaggg PCB Congener # B 23 8 23 8 [Deana uglflllet (wet) a h. d O — — — I I OI 0 " "" ageeseogezassessosgaggga § § PCB Congener it Figure lS-a. Congener specific PCBs in raw and pan fried carp from Lake Erie, skin-on. 7O 0.12 'l IRaw [lemma PCB Congener I 14 12 a O ugflillet (wet) _ 0 aaezsgogezasaogsosgs PCB Congener # Figure lS-b. Congener specific PCBs in raw and pan fried carp from Lake Erie, skin-off. 71 [jCaMM PCB Congener # 70 8 8 I Raw [Donna 8 uglfillet (wet) 10 0 P’Jl-l ass: eugezasasgsssgaggg I —J 8 PCB Congener # Figure 16-a. Congener specific PCBs in raw and deep fat fried carp from Lake Erie, skin-on. 72 025) 02 l “ofi g . Raw 5 Elana: a. 0.1 oos~ o PCB Congener # 35 so 25 =- g 20 I Raw 8 = l 1.. l PCB Congener it Figure 16-b. Congener specific PCBs in raw and deep fat fried carp from Lake Erie, skin-off. 73 ow 014 on F —.oA g .Raw 5 [lemma O. PCB Congener it z: 18 1o _ ‘.14 I a l :12 l .Raw w I I ll Dow = 37"" l l- I * 6 HIT. 1) l I 4.. 2 lllll|l|||1H I I 0 Illllllllflllllllnflli. M sac:sefigRansassissgsgggnggagsggagggsggagggg § § E E is PCBCongenerl Figure l7-a. Congener specific PCBs in raw and pan fried carp from Lake Huron, skin-on. 74 0.180 329::ocgezassagsasgsggg PCB Congener I Ihuw [Janna uglflllet (wet) a GI .A O 0 aasezccgaaasaagsaogaae P" PCB Congener # Ihuw [Janna carp from Lake Huron, skin-off. Figure l7-b. Congener specific PCBs in raw and pan fried 75 oo 05 04 g .Raw ~'oa E [floamn a o. 02 0 M 339:3””§2283395§9=§3§§§FEEEFSEEEEIE§QE§§§§Q g '3' § é s 55 PCBCongener# a) a) 70 :60 :50 .RIW 2 $340 [Joamul 3 a) 20 10 I la 0 Lil—Ma are:39e§2=====s§==enazgnszsasssaaaagaaggssa i § § 5 5'9. PCBCongener# Figure 18-a. Congener specific PCBs in raw and deep fat fried carp from Lake Huron, skin-on. 76 0.1 0.09 0.08 0.07 A 0.01 .Raw Doookod L—q-m- PCB Congener it uglfillet (wet) (a) PCB Congener # Figure l8-b. Congener specific PCBs in raw and deep fat fried carp from Lake Huron, skin-off. 77 Skin-on carp fillets which were deep fat fried were analyzed with skin since deep fat fried fish are usually ingested with skin by consumers. Only the muscle tissue of the pan fried skin-on carp fillets was used for the cooked carp analyses. Thus, it is difficult to compare the percent change of PCBs during cooking. It was likely that the percent reductions by deep fat frying skin-on carp would be lower than those by pan frying. However, it is suprising that deep fat fried skin—on carp harvested from Lake Huron (Figure l4-b) lost greater percent of PCBs than pan fried skin-on carp (Figure 14-a). Skin Removal Effects on PCBs Reduction Any food process which removes lipid from fish may alter the PCB levels in the fish. The removal of skin and its associated fat from fish reduced the specific congeners, homologs and total PCBs as expected. The removal of skin and its associated fat from fish per se reduced the total PCBs level. Skin—on carp fillets had significantly greater values of PCBs than skin-off carp as shown in Figure 19. The differences for skin-on and skin-off carp are illustrated by the following average group values expressed as ppm wet tissue: 78 E" E 2 3 .Sldn-on 3 [jsuum o a g .- Erie Huron Figure 19. The average total PCBs in skin-on and skin-off carp from Lake Erie and Lake Huron. 79 Group Skin—on Skin-off Tri— 0.037 > 0.026 Tetra- 0.469 > 0.349 Penta- 0.763 > 0.502 Hexa— 0.424 > 0.246 Hepta- 0.178 > 0.109 Octa- 0.028 > 0.013 Total PCBs 1.898 > 1.244 The Packed column GC analyses by Michigan Department of Public Health (Zabik et al., 1993) also determined the level of total PCBs in skin-on carp was higher than skin-off carp. The reduction of PCB levels in fish fillets through removal of skin was also determined by Zabik et al. (1979), Hora (1981) and Sanders and Haynes (1988). Since PCBs are lipid- soluble, some of the PCBs are dissolved in fat portion. Yoshida et al. (1973) observed the high levels of PCBs in the skin and dark muscle of carp. Therefore, it is recommended to remove the skin, fatty areas under skin, lateral line, dorsal fat, and belly flap before consuming, since all of these parts tend to have higher levels of PCBs and other chlorinated pesticides. The presence or absence of skin significantly affected the total PCBs and specific congener levels in fish fillets themselves (Appendix 6-6, 80 6-8). However skinning did not affect the effectiveness in reducing PCBs during cooking (Appendix 6-9). The PCBs, lipophilic contaminants may pose a threat to the health of consumers by contaminated fish from lakes. Even though State and Federal agencies have monitored environmental contaminants, consumers should try to reduce the contaminants in food. Therefore, they are encouraged to skin carp fillets before cooking. CONCLUSION One of the most frequently noted differences in health between fishing villagers and people in industrialized areas is the low incidence of heart attacks and other major diseases. The consumption of fish products can be related to such a low incidence (Lands, 1986). The Great Lakes are important in providing fresh water fish to people living inland. However the consumption of Great Lakes fish provides a PCB exposure to fish eaters. PCBs are no longer manufactured but they continue to be found in fish from the Great Lakes. PCBs may pose a health threat to fish consumers. These PCBs and other contaminants in fish from Great Lakes have resulted in a hesitation of fish, especially sport fish consumption. This study in which congener specific PCBs were analyzed by capillary column gas chromatography, has demonstrated that the average level of total PCBs based on summing individual congeners commonly found in Aroclor®> 1254 was 2.534 ppm in skin-on carp, 1.367 ppm in skin-off carp from Lake Erie and 1.496 ppm in skin-on carp, 1.240 ppm in skin-off carp from Lake Huron. The packed column analyses 81 82 at the Michigan Department of Public Health (Zabik et al., 1993) also showed the higher values in the carp harvested from Lake Erie. The average level of total PCBs in carp from Lake Erie was higher than those from Lake Huron, which might be partly due to larger size of carp from Lake Erie. Four of six carp from Lake Erie exceeded 2 ppm which is the FDA's tolerance level of PCBs for fish. The effects of pan frying and deep fat frying on reduction of total PCBs and PCB homolog congeners were also determined. Cooked samples showed the lower levels of PCBs than uncooked samples, in contrast to previous PCBs study in carp (Zabik et al., 1982), even though the average fat content in carp was relatively low. However the statistical comparison between two cooking methods showed no significant difference for the effectiveness in reducing PCB levels, even though deep fat frying was a little more effective than pan frying. The average percent reduction of total PCBs based on total micrograms per fillet was 30.2i14.1% by pan frying, 38.1i15.6% by deep fat frying. Since most of the threatening congeners are tetra-, penta-, hexa-, and heptachlorobiphenyls based on their abundance and potential toxicity, the distribution of PCBs in carp and cooking effect on those PCB congeners were determined in this study. In both raw and cooked carp, the distribution of each PCB homolog congeners was in order; 83 penta- > tetra- > hexa- > heptachlorobiphenyls. However the distribution of PCB homolog congeners in EPA study (1992) was in order: hexa- > penta— > tetra— > hepta homolog. Even though they were not consistent with each other, it is thought that the penta homolog showed a high level due to accumulation and abundance in environment, and low level of hepta homolog due to low abundance in environment. And it did not show the special relationship between the reduction of PCBs during cooking and the number of chlorines in PCBs. The average total PCB level in skin-off carp were lower than skin-on carp. Since PCBs are lipid-soluble and dissolved in fat portions of fish, it is possible to remove some of PCBs. Skin and fat removal did not significantly affect the cooking effectiveness in reducing PCBs in carp. The States and Federal agencies have monitored and regulated the level of PCBs in fish that commercially marketed. Therefore sports fishermen or subsistence fishermen need to be more careful and try to reduce the PCBs and other contaminants in fish. Cooking and appropriate processing can be an additional safety factor. Skin and fatty area under skin, the lateral line, belly flap should be removed before consuming fish. In addition to PCBs, many Great Lakes fish contain other toxic compounds, including pesticides such as DDT, dieldrin, chlordane, mirex, and toxaphene. They also contain 84 mercury. Therefore, fish consumption advisories and public education should provide information to reduce the health risk from contaminated fish. PROPOSALS FOR FUTURE RESEARCH This study had considered the level of total PCBs, the grouped homolog congeners, and some specific PCB congeners based on Aroclor®>1254. However the majority of PCBs apparently have no effect on mammalian system (McKinney and Singh, 1981). Therefore study of specific PCB congeners based on their structure will be needed, because the most toxicologically active PCB congeners are those having chlorine substitution at the para (4 and 4') position and no ortho (2,2',6 and 6') positions on the biphenyl moiety. For instance, coplanar Chlorobiphenyls (#77, 81, 126 and 169) might be highly toxic since they are most structurally simillar to 2,3,7,8—TCDD of all the congeners (Safe, 1987; Tenabe et al., 1987, 1989). The dioxin, 2,3,7,8-TCDD is generally considered the most potent synthetic environmental toxicant. Therefore reporting concentration data as total PCBs may not related well to the toxicity of PCBs and may be mislead. As microwave oven is getting a popular kitchen appliance, and there are a lot of varieties of microwavable food in the market, the study of effectiveness in reduction 85 86 of harmful chemicals by microwave cooking is also necessary.Zabik et al. (1979) cooked lake trout (ciscowet) by microwave and reduced the PCBs by an average of 26%, even though they did not show a significant differences in reduction of PCBs, comparing to other cooking methods; broiling, roasting. Cooking by microwave using different cooking times and more comparison of effectiveness of PCB reduction with other cooking methods are needed to be researched. E LIST OF REFERENCES Ackerman,D.G., Scinto,L.L., Bakshi,P.S., Delumyea,R.G., Johnson,R.J., Richard,G., Takata,A.M., and Sworzyn,E.M. 1983. Destruction and disposal of PCBs by thermal and non- thermal Methods. Noyes Data Corporation. Park Ridge: New Jersey. pp.1—6. Andrews,S.L. 1992. Selected readings in food science. Stipeds Publishing Com. ChampaignzIL. Brown,E.E., and Gratzek,J.B. 1980. Fish farming handbook. An Avi Book. Westportzconnecticut. p.44. Concon,J.M. Food toxicology: Part B. 1988. Marcel Dekker Inc.:NY. Das,K.G., and Kulkarni,P.S. 1981. Gas—Liquid chromatography: Pesticide Analysis. Marcel Dekker, Inc.:New York. Draper,W.M., and Koszdin,S. 1991. Speciation and quantitation of Arclors based on PCB congener data: Application to California mussels and white croaker. J. Agr. Food Chem. 39:1457-1467. Elkins,E.R., Farrow,R.P., and Kim,E.S. 1972. The effect of heat processing and storage on pesticide residues in spinach and apricots. J. Agr. Food Chem. 20:286-291. EPA. 1992. National study of chemical residues in fish. Volume I & II. Office of Science and Technology Standards and Applied Science Division U.S. Environmental Protection Agency. Washington, DC. Fujiwara,K. 1975. Environmental and food contamination with PCB's in Japan. Science of the Total Environnment. 4:219- 247. Funk,K., Zabik,M.E., and Smith,S.L. 1971. Dieldrin residues in sausage patties cooked by three methods. J. Food Science. 36:616-618. 87 88 Giesy,J.P., Newsted,J., and Garling,D.L. 1986. Relationships between chlorinated hydrocarbon concentrations and rearing mortality of chinook salmon (Oncorhynchus Tshawytscha) eggs from Lake Michigan. J. Great Lakes Res. 12(1):82-98. Hemphill,D.D., Baldwin,R.E., Deguzman,A., and Deloach,H.K. 1966. Effects of washing, trimming, and cooking on levels of DDT and Derivatives in green beans. J. Agr. Food Chem. 15:290-294. Henderson,C., Inglis,A., and Johnson,W.L. 1971. Residues in fish, wildlife, and estuaries. Pesticides Monitoring J. 5:1- 5. Hepher,B., and Pruginin,Y. 1981. Commercial fish farming. John Wiley & Sons Inc.:New York. Hora,M.E. 1981. Reduction of Polychlorinated Biphenyl (PCB) concentrations in carp (Cyprinus carpio) fillets through skin removal. Bull. Environ. Contam. Toxicol. 26:364-366. Hutzinger,O., Safe,S., and Zitko,V. 1974. The chemistry of PCB's. CRC Press, Inc.:Florida. Jensen,S. 1966. Report on a new chemical hazard. New Scientist. 32:612. Kolbye,A.C.Jr. 1972. Food exposures to Polychlorinated Biphenyls. Environ. Health Perspectives. 1:85-88. Krahn,M.M., Rhodes,L.D., Myers,M.S., Moore,L.K., MacLeod Jr.,W.D., and Malins,D.C. 1986. Associations between metabolites of aromatic compounds in bile and the occurrence of hepatic lesions in English sole from Puget sound. Arch. Environ. Contam. Toxicol. 15:61-67. Kubiak,T.J., Harris,H.J., Smith,L.K., Schwartz,T.R., Stalling,D.L., Trick,J.A., Sileo,L., Docherty,D., and Erdman,T.C. 1989. Microcontaminants and reproductive impairment of the Forster's tern on Green Bay, Lake Michigan. Arch. Environ. Contam. Toxicol. 18:706-727. Lands,W.E.M. 1986. Fish and human health. Academic Press Inc.:Orlando. Liska,B.J., Stemp,A.R., and Stadelman,W.J. 1967. Effect of method of cooking on chlorinated insecticide residue content in edible chicken tissue. Food Technology. 21:117-120. 89 MacKay,D., Shiu,W.Y., Billington,J., and Huang,G.L. 1983. Physical chemical properties of Polichlorinated Biphenyls in physical behavior of PCBs in the Great Lakes ed. by MacKay,D., Paterson,S., Einsenreich,S.J., and Simmons,M.S. Ann Arbor Science. Ann ArborzMI. Maul,R.E., Funk,K., Zabik,M.E., and Zabik,M.J. 1971. Dieldrin residues and cooking losses in pork loins. Journal of the American Dietetic Association. 59:481-484. McCaskey,T.A., Stemp,A.R., Liska,B.J., and Stadelman,W.J. 1968. Residues in egg yolks and raw and cooked tissues from laying hens administered selected chlorinated hydrocarbon insecticides. Poultry Science. 47:564-569. McFarland,V.A., and Clarke,J.U. 1989. Environmental occurrence, abundance, and potential toxicity of Polychlorinated Biphenyl congeners: Considerations for a congener—specific analysis. Environ. Health. Perspectives. 81:225-239. Metcalf,R.L., Sanborn,J.R., Lu,P., and Nye,D. 1975. Laboratory model ecosystem studies of the degradation and fate of radiolabeled Tri—, Tetra-, and Pentachlorobiphenyl compared with DDE. Arch. Environ. Contam. Toxicol. 3:151- 165. Morehouse,S.E., and Zabik,M.E. 1989. Evaluation of polydimethylsiloxane fluids as noncaloric frying media. J. Food Sci. 54:1061 Morgan,K.J., Zabik,M.E., and Funk,K. 1971. Lindane, dieldrin, DDT residues in raw and cooked chicken and chicken broth. Poultry Science. 51:470-475. Nelson,N. 1972. Polychlorinated Biphenyls—environmental impact. Environ. Res. vol.5. Pal,D., Weber,J.B., and Overcash,M.R. 1980. Fate of Polychlorinated Biphenyls(PCBs) in soil-plant systems. Residue Reviews, vol.74. Park,P. 1991. Great Lakes pollution linked to infertility. New Scientist. 28:18. Pellizzari,E.D., Moseley,M.A., and Cooper,S.D. 1985. Recent advances in the analysis of Polychlorinated Biphenyls in environmental and biological media. J. Chromatography. 334:277-314. 9O Puffer,H.A., and Gossett,R.W. 1983. PCB, DDT, and benzo(a)pyrene in raw and pan-fried white crocker (Genyonemus lineatus). Bull. Environ. Contam. Toxicol. 30:65 Reinert,R.E., Stewart,D., and Seagran,H.L. 1972. Effects of dressing and cooking on DDT concentrations in certain fish from Lake Michigan. J. Fisheries Research Board of Canada. 29:525-529. Ribick,M.A., Smith,L.M., Dubay,G.R., and Stalling,D.L. 1981. Analytical methods for monitoring. In aquatic toxicology and hazard assessment, Proceedings of the Fourth Annaual Symposium on Aquatic Toxicology ed. by Branson,D.R., Dickson,K.L. American Society for Testing and Materials, PhiladelphiazPa. pp.249-269. Ribick,M.A., Dubay,G.R., Petty,J.D., Stalling,D.C., and Schmitt,C.J. 1982. Toxaphene residues in fish: identification, quantification, and confirmation at part per billion levels. Environ. Sci. Technol. 16:310-317. Ritchey,S.J., Young,R.W., and Essary,E.O. 1969. The effects of cooking on chlorinated hydrocarbon pesticide residues in chicken tissues. J. Food Science. 32:238-240. Ritchey,S.J., Young,R.W., and Essary,E.O. 1972. Effects of heating and cooking method on chlorinated hydrocarbon residues in chicken tissues. J. Agr. Food Chem. 20:291-293. Safe,S. 1987. Determination of 2,3,7,8-TCDD toxic equivalent factors: support for the use of the in vitro AHH induction assay. Chemosphere. 16:791-802. Sanders,M., and Haynes,B.L. 1988. Distribution pattern and reduction of Polychlorinated Biphenyls (PCB) in bluefish pomatomus saltatrix (Linnaeus) fillets through adipose tissue removal. Bull. Environ. Contam. Toxicol. 41:670—677. Schmidt,W.A. 1989. Are Great Lakes fish safe to eat? National Wildlife. 27:17-19. Schwartz,P.M., Jacobson,S.W., Fein,G., Jacobson,J.L., and Price,H.A. 1983. Lake Michigan fish consumption as a source of Polychlorinated Biphenyls in human cord serum, material serum, and Milk. AJPH. 3:293—296. Seelye,J.C., Hesselberg,R.J., and Mac,M.J. 1982. Accumulation by fish of contaminants released from dredged sediments. Environ. Sci. Technol. 16:459. 91 Seiber,J.N. 1990. Industrial and environmental chemicals in the human food chain in Chemicals in The Human Food Chain. Van Nostrand Reinhold. pp.183-219. Shafer,M.A.M., and Zabik,M.E. 1975. Dieldrin, fat and moisture loss during the cooking of beef loaves containing texturized soy protein. J. Food Science. 40:1068-1071. Skea,J.C., Simonin,H.A., Harris,E.J., Jackling,S., Spangnoli,J.J., Symula,J., and Colguhoun,J.R. 1979. Reducing levles of mirex, aroclor 1254, and DDE by trimming and cooking Lake Ontario brown trout (Salmo Trutto Linnaeus) and sonallmouth bass (Micropterus Dolomieui Lacepede). J. Great Lakes Res. 5:153. Smith,S.K., Zabik,M.E., and Dawson,L.E. 1977. Polybrominated Biphenyl levels in raw and cooked chicken and chicken broth. Poultry Science. 56:1289-1296. Smith,W.E., Funk,K., and Zabik,M.E. 1973. Effects of cooking on concentrations of PCB and DDT compounds in Chinook (Oncorhynchus tshawytscha) and Coho (O. kisutch) salmon from Lake Michigan. J. Fisheries Research Board of Canada. 30:702—706. Tenabe,S., Kannan,N., Ono,M., and Tatsukawa,R. 1989. Toxic threat to marine mammals: Increasing toxic potential of non- ortho and mono-ortho coplanar PCBs from land to ocean. Chemosphere. 18:485-490. Tenabe,S., Kannan,N., Subramanian,A., Watanabe,S, and Tatsukawa,R. 1987. Highly toxic coplanar PCBs: occurrence, source, persistency and toxic implications to wildlife and humans. Environ. Pollut. 47:147-163. Thomas,R.L., and Frank,R. 1983. PCBs in sediment and fluvial suspended solids in the Great Lake in physical behavior of PCBs in the Great Lakes. Ann Arbor Science. Ann Arborle. Trotter,W.J., Corneliussen,P.E., Laski,R.R., and Vannelli,J.J. 1989. Levels of Polychlorinated Biphenyls and pesticides in bluefish before and after cooking. J. Assoc. off. Anal. Chem. 72:501-503. Tuinstra,L., Driessen,J., Keukens,H., Van Munsteren,T., Roos,A., and Traag,W. 1983. Quantitative determination of specified Chlorobiphenyls in fish with capillary gas chromatography and its use for monitoring and tolerance purposes. Intern. J. Environ. Chem. 14:147-157. 92 The United Nations Environment Programme and the World Health Organization. 1976. Polychlorinated Biphenyls and Terphenyls. World Health Organization. Geneva. Varanasi,U., Stein,J.E., Reichert,W.l., Tilbury,K.l., Krahn, M.M., and Chan,S.L. 1992. Chlorinated and aromatic hydrocarbons in bottom sediments, fish and marine mammals in US coastal waters: laboratory and field studies of metabolism and accumulation in Persistent pollutants in marine ecosystems. edited by Walker,C.H., and Livingstone,D.R. Pergamon Press. OxfordzNew York. Vos,J.G. 1972. Toxicology of PCBs for mammals and for birds. Environ. Health Perspectives. 1:105-117. Vos,J.G., and Koeman,J.H. 1970. Comparative toxicological study with Polychlorinated Bipheynils in chickens with special reference to porphyria, edema formation, and tissue residues. Toxicol. Appl. Pharmacol. 17:656-668. White,R.J., Kim,H.T., and Kim,J.S. 1985. PCBs in striped bass collected from the Hudson river, New York, during fall, 1981. Bull. Environ. Contam. Toxicol. 34:883-889. Yadrick,M.K., Funk,K., and Zabik,M.E. 1971. Dieldrin residues in bacon cooked by two methods. J. Agr. Food Chem. 19:491-494. Yadrick,M.K., Zabik,M.E., and Funk,K. 1972. Dieldrin levels in relation to total, neutral and phospholipid composition in selected pork muscles. Bull. Environ. Contam. Toxicol. 8:289-293. Yoshida,T., Takashima,F., and Watanabe,T. 1973. Distribution of [14C] PCBs in carp. Ambio. 2:111-113. Young,D.R., Heesen,T.C., and McDermott,D.J. 1976. An offshore biomonitoring system for chlorinated hydrocarbons. Pollut. Bull. 7:156. Zabik,M.E. 1974. Polychlorinated Biphenyl levels in raw and cooked chicken and chicken broth. Poultry Science. 53:1785- 1790. Zabik,M.E. 1979. Polychlorinated Biphenyls, dieldrin and DDT in lake trout cooked by broiling, roasting or microwave. Bull. Environ. Contam. Toxicol. 21:136-143. 93 Zabik,M.E., Defouw,C., and Weaver,C.H. 1980. Polybrominated Bipheynyl congener levels and distribution patterns in raw and cooked beef. Arch. Environ. Contam. Toxicol. 9:651—659. Zabik,M.E., Harte,J.B., Zabik,M.J., and Dickmann,G. 1992. Effect of preparation and cooking on contaminant distributions in crustaceans: PCBs in blue crab. J. Agric Food Chem. 40:1197-1203. Zabik,M.E., Hoojjat,P., and Weaver,C.H. 1979. Polychlorinated Biphenyls, dieldrin and DDT in lake trout cooked by boiling, roasting or microwave. Bull. Environ. Contam. Toxicol. 21:136—143. Zabik,M.E., Merrill,C., and Zabik,M.J. 1982. PCBs and other xenobiotics in raw and cooked carp. Bull. Environ. Contam. Toxicol. 28:710—715. Zabik,M.E., Zabik,M.J., and Humphrey,H. 1993. Assessment of contaminants in five species of Great Lakes fish at the dinner table. Final Report Part 1. Great Lakes Protection Fund. ChicagozlL. Zitko,V., Hutzinger,O., and Safe,S. 1971. Retention times and electron-capture detector responses of some individual Chlorobiphenyls. Bull. Environ. Contam. Toxicol. 6:160-163. APPEND ICE S 94 Appendix 1. Systematic Numbering of PCB Compounds Nb. Structure Nb. Structure Monochlorobiphenyls Tetrachlorobiphenyls 1 2 40 2,2',3,3' 2 3 41 2,2',3,4 3 4 42 2,2',3,4' 43 2,2',3,5 Dichlorobipheynls 44 2,2',3,5' 4 2,2 45 2,2',3,6 5 2,3 46 2,2',3,6' 6 2,3' 47 2,2',4,4' 7 2,4 48 2,2',4,5 8 2,4' 49 2,2',4,5' 9 2,5 50 2,2',4,6 10 2,6 51 2,2',4,6' 11 3,3' 52 2,2',5,5' 12 3,4 53 2,2',5,6' 13 3,4' 54 2,2',6,6' 14 3,5 55 2,3,3',4 15 4,4' 56 2,3,3',4' 57 2,3,3',5 Trichlorophenyls 58 2,3,3',5' 16 2,2',3 59 2,3,3',6 17 2,2',4 60 2,3,4,4' 18 2,2',5 61 2,3,4,5 19 2,2',6 62 2,3,4,6 20 2,3,3' 63 2,3,4',5 21 2,3,4 64 2,3,4',6 22 2,3,4' 65 2,3,5,6 23 2,3,5 66 2,3,4,4' 24 2,3,6 67 2,3,4,5 25 2,3',4 68 2,3,4,5' 26 2,3',5 69 2,3,4,6 27 2,3',6 70 2,3,4',5 28 2,4,4' 71 2,3,4',6 29 2,4,5 72 2,3,S,5' 30 2,4,6 73 2,3,5',6 31 2,4',5 74 2,4,4',5 32 2,4',6 75 2,4,4',6 33 2',3,4 76 2',3,4,5 34 2',3,5 77 3,3',4,4' 35 3,3',4 78 3,3',4,5 36 3,3',5 79 3,3',4,5' 37 3,4,4' 80 3,3',5,5' 38 3,4,5 81 3,4,4',5 39 3,4',5 95 Appendix 1-—-continued Nb. Structure No. Structure Pentachlorobiphenyls 82 2,2',3,3',4 121 2, 3' 4, 5' ,6 83 2, 2', 3, 3', 5 122 2' ,3 3' ,4, 5 84 2, 2' 3, 3' ,6 123 2' 3, 4, 4' ,5 85 2, 2' 3, 4, 4' 124 2' 3, 4, 5, 5' 86 2, 2' ,3 4, 5 125 2' ,3, 4, 5, 6' 87 2, 2' ,3,4,5' 126 3, 3' L 4' ,5 88 2, 2' ,3,4,6 127 3, 3' L 5, 5' 89 2, 2' ,3,4, 6 90 2, 2' ,3,4' ,5 Hexachlorobiphenyls 91 2, 2' ,3,4',6 128 2,2', 3, 3', 4, 4' 92 2, 2' ,3 5,5' 129 2 2' 3, 3' ,4 5 93 2, 2' ,3 5, 6 130 2 2' ,3 3' 4, 5' 94 2, 2' 3, 5, 6' 131 2 2' ,3 3' 4, 6 95 2, 2' ,3 5' ,6 132 2 2' ,3 3' ,4 6' 96 2, 2' ,3 6, 6' 133 2 2' 3, 3' L 5' 97 2, 2' ,3 L 5 134 2 2' ,3 3' ,5, 6 98 2, 2' ,3 L 6 135 2 2' 3, 3' L 6' 99 2, 2' L 4' ,5 136 2 2' 3, 3' ,6, 6' 100 2, 2' L 4' ,6 137 2 2' ,3 L 4' ,5 101 2, 2' 4, L 5' 138 2 2' 3, 4, 4' ,5 102 2, 2' 4, L 6' 139 2 2' ,3 4, 4' ,6 103 2, 2' L L ,6 140 2 2' L 4, 4' 6' 104 2, 2' ,4, 6, 6' 141 2 2' 3, 4, 5, 105 2, 3, 3' 4, 4' 142 2 2' 3, 4, L 106 2, 3, 3' 4, 5 143 2,2' 3, L 5, 107 2, 3, 3' L 5 144 2 2' ,3 4, 5' 6 108 2, 3, 3' 4, 5' 145 2 2' ,3 4, 6, ' 109 2, 3, 3' L 6 146 2 2' ,3 4' L 5 110 2,3, 3' ,4 6 147 2 2' 3, 4' 5, 6 111 2,3, 3' ,5, 5' 148 2 2' 3, 4' ,5 6 112 2,3,3, 5', 6 149 2 2' ,3 4' L , 113 2,3,3' ,4' ,6 150 2 2' ,3 4' ,6, 6 114 2,3,4,4',5 151 2 2' ,3 5, L ,6 115 2,3,4,5,6 152 2 2' ,3 L 6,6' 116 2,3,4,5,6 153 2 L L 4' 5, 5' 117 2,3,4',5,6 154 2 2' 4, 4' 5, 6 118 2,3',4,4',5 155 2 2' ,L 4' ,L 6' 119 2,3',4,4',6 156 2 3, 3', L 4' ,5 120 2,3' 4,5 5' 157 2 3, 3' L L ,5 Appendix 1.---continued 96 No. Structure No. Structure 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 Hexachlorobiphenyls 2,3, 3', L 4',6 4, 5,5' A 5, 6 L 5' 4' 4' I I 4 ,5 ' 5, 5' ,5 ,6 ,5 5' 5,6 ' ,6 , 6 4' I I 3' 3' 3' 3' 3' 3' 3 , , 4 ,6 ,5, 5' 5' I I I I 4 4' u'4’4u 4 4 , ,6 ,5, 5' I 2 3, 2 3, 2 3, 2 3, 2 3, 2,3, 2 3 2 3, 2 3' 2 3 3 3 I I I Heptachlorobiphenyls 2, 2', 3, 3', 4, 4', 5 L ,6 L 5 4, 6 ,4 6 ,4 ,6 4, 6' 4, 6 ,5, 6 5, 6 5 i 6 6 4' 5, 5, 5, 5' 6, 5, 5' 6, ,5, L ,5 5' 6 6, I 6' ,6 6! ,3 ,3 ,3 3, ,3 ,3 3, 3, ,3 ,3 ,3 3, 3, 3 , 3, 5' ,3 6, ,3, 5' ,6 3, , 6 6' I I I I I 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, L 2, 2 2 2 2 2 I I I ‘ I 2' 3' 2' 3' 2' 3' 2' 3' 2' 3' 2' 3' 2' 3' 2' 3' 2' 3' 2' 4, 2' 4, 2' 4, 2' 4, 2' 4, 2' 4, 2' 4, 2' 4, 2' 4 3 4 3 4 3 4 3 4 3 4 ‘ I 4' 4' 4' 4' 4' L L L 5 I 4' L 4', L 4 5' 5 8 5 I ,5 5 I I 5 6 ,6 ,6 6 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 Octachlorobiphenyls 2,2',3, 3', 4, 4',5,5' 22 33 44,5,6 2 2 3, 3' ,4 4 ,5', 6 2 2 3, 3' L 4' ,6, L 2 2 3, 3' 4, 5, 5' ,6 2 2 3, 3' L L 6,L 2,2',3, 3' L 5' L 6' 2 2 3, 3' 4' ,5, 5' ,6 2 2 3, 3' ,5, 5' ,L 6' 2 2 3, 4, 4' 5, L ,6 2 2 L 4,L ,5,6,6' 2 2 3' 4, 4' 5, 5' ,6 Nonachlorobiphenyls 2,2',3,3', 4, 4', L 5', 6 2,2',3,3', 4, 4' ,L 6, 6' 2,2',3,3', 4, L L ,6,6' Decachlorobiphenyls 2,2',3,3',4,4',5,5',6, 97 Appendix 2. Protocol for Fish Processing in Meat Laboratory All fish were received at the Michigan State University Meat Laboratory. The containers should be placed in a 0%: cooler. All fish should be processed within 24 hours of receipt. It was hoped that processing can occur as soon as possible after the fish have been received. Each fish were received head off with gills, kidney and viscera removed. Wherever practically possible, the fish flesh should not be exposed to plastic. The fish were identified as to right and left side. All fish should go through the following process protocol: 1. If dirt or contamination existed on the surface, the fish was washed or rinsed in cold water. 2 Appropriate random sample fish were selected. 3. All fish contributing to skin-on fillets were scaled and washed. 4. Belly flaps were removed up to the ribs. 5. All fish were filleted with rib bones removed last. In skin-on fillets, the fatty back strip from the appropriate area of fillet was removed carefully. 6. In skin-off fillets, the skin was removed and all dark tissue was trimmed away including the dark tissue from the lateral line. The fish pieces were appropriately 10. ll. 98 labeled with the random numbers. Left sides for cooking portion were wrapped in aluminum foil and vacuum packaged. Labels were placed both in the interior and on the outside of the package. The packages were frozen at -34°C. Right sides for raw samples (skin-on or skin-off) were ground to a uniform particle size. The method of particle reduction was grinding, chopping, or blending appropriately at a frozen state. . All raw ground fish were placed in glass containers, covered with aluminum foil, and appropriately capped. The random number was labeled on the inside of the cap and was placed on a tape label on the outside of the container. All samples were stored immediately at -34°C and held for transport to appropriate laboratories. 99 Appendix 3. Size, Processing and Cooking data for Carp from Lake Erie and Huron. Fish Cooked both with Skin- on and Skin-off. Cooking Methods Skin-on Skin-off Pan Fry Deep Fat Fry Pan fry Deep Fat Fry Lake Erie Weight (g) 1813i3371 1813i337 l855i404 1855i404 Length (cm) 52.3i4.5 52.3i4.5 51.2i3.6 51.2i3.6 Carcass Yield % 57.5i3.5 57.5i3.5 ------------ As Prepared Yield % 36.3i3.4 36.3i3.4 25.6i2.7 25.6i2.7 Total Cooking Loss % 22.6:4.8 32.9i5.9 21.6i5.3 36.5i4.9 Cooking Yield % 68.5i3.l2 67.1i5.9 78.4i5.3 63.5i4.9 Lake Huron Weight (9) 1567:398 l653i442 l482i587 l625i521 Length (cm) 46.7:4.0 48.0i4.6 45.5:5.4 46.1i4.9 Carcass Yield % 58.4i1.9 58.0i2.0 62.2i1.4 61.3i2.1 As Prepared Yield % 29.0i2.9 29.1i2.8 22.0il.6 21.5il.7 Total Cooking Loss % 20.1i5.0 30.2i5.4 15.1i3.1 30.4i3.8 Cooking Yield % 68.9i5.0 69.8i5.4 84.9:3.1 69.6i3.8 1Mean and standard deviation, n=6 2Skin included with fillet for deep fat fried fish only. 100 Appendix 4. HPLC Pump Operating Conditions -HPLC Pump- 1. 2. Turn on Waters/590 Programmable HPLC Pump. Set to manual mode by (page 3.2.2 of manual). A. Push blue "2nd func" button B. Push manual (next Param) button Set flow to 4.000 mL/min (page 3.2.2 of manual) A. Push "next Param" button until readout indicates "flow X.XXX mL" B. Push "4" button C. Push "Enter" button . Make sure methylen chloride, brown solvent reservoir is full. If not: A. Filter HPLC grade methylen chloride through HPLC filter flask. B. Fill solvent reservoir. . Make sure the air bubbles in plastic inlet line between methylen chloride reservoir and HPLC pump are out. If air in the line: (page 2-10 of the manual) A. Attach high pressure, 10 mL syringe to plastic tube at the draw off valve on pump with plunger in. B. Turn black plastic knob counterclockwise 3-4 turns. C. Pull out on syringe and empty. Set pressure limits (page 3—4 of manual). 101 A. Set low limit to 0. B. Set high limit to 600. 7. set compressibility compensation (page 2-5 of manual). Set to 0. 8. In case of problems (pressure overload, etc.) see manual. -Automatic Injector- 1. Turn on Waters 712 Wisp (bottom red button). 2. Set to auto mode (red LED on at auto). 3. Set sample number to 0 (page 4-5 of manual). A. Press down "sample No." key and release. B. Press down "0" key and release. C. Press down "Enter" key and release. 4. Set system message for auxiliary loop (page 4—7 of manual). A. Press "Sys Mes" key and release. B. Using numeric key enter 71. C. Press "Enter" key and release. D. Using numeric key enter 01. E. Press "Enter" key and release. F. system message display should read 7101. 5. Set injection volume (page 4—6 of manual). A. Press down "Inj vol" key and release. 10. 102 B. Using numeral keys enter 200 uL. C. Press down "Enter" key and release. Set run time (page 4-8 of manual). A. Press "Run Time" key and release. B. Using numeric keys enter 25 minutes. C. Press "Enter" key and release. Set Equilibration Delay (page 4-9 of manual). A. Press "Sys Mes" key and release. B. Using numeric keys enter 0000. C. Press "Enter" key and release. Set number of injections (page 4-10 of manual). A. Press "No of Inj" key and release. B. Using numeric key enter 1. C. Press "Enter" key and release. Set syringe speed (page 4-14 of manual). A. Press "Sys Mes" key and release. B. Using numeric keys enter 76. C. Press "Enter" key and release. D. Using numeric key enter 1 for event maker speed. E. Press "Enter" key and release. Set mark width (page 4-10 of manual). A. Press "Sys Mes" key and release. B. Using numeric keys enter 78. C. Press "Enter" key and release. D. Using numeric key enter O4. 103 E. Press "Enter" key and release. F. System message display should read 7804. -Fraction Collector- 1. Turn on Waters fraction collector (press green "Power" key. 2. Press red "End" key. 3. Press red/black "C/M" key. Approved by Dr. Matthew Zabik 104 Appendix 5. PCB Standard Congener preparation for Gas Chromatography Congener ! Structure Congener l Structure 30 2, 4, 6 114 2, 3, 4,4' ,5 31 2, 4' ,5 118 2, 3' 4, 4' ,5 42 2, 2' ,3 4 120 2, 3' 4, 5, 5' 44 2, 2' 3, 5' 121 2, 3' 4, 5' ,6 47 2, 2' 4, 4' 122 2' ,3, 3' ,4, 5 49 2, 2' 4, 5' 128 2 2' 3, 3' 4, 4' 52 2, 2' ,5, 5 132 2 2' ,3 3' 4, 6' 55 2, 3, 3' ,4 136 2 2' ,3 3' ,6, 6' 66 L 3' L 4 137 2 2' 3, 4, 4' ,5 7O 2, 3' ,4' ,5 138 2 2' 3, 4, 4' ,5 72 2, 3' ,5 5' 141 2 2' 3, L 5,5' 76 2' ,3, 4,5 149 2 2' 3, 4' ,5',6 79 3, 3' ,4 5' 153 2,2' ,4, 4' ,5, 5' 83 2, 2' ,3 3',5 157 2 3, 3' 4, 4' ,5 84 2, 2' ,3,3'6 158 2,3, 3' 4, 4' ,6 85 2, 2' ,3,4,4' 167 2 3' ,4 4' ,5 5' 87 2, 2' ,3,4,5 171 2 2' 3, 3' ,4 4' ,6 91 2, 2' ,3,4',6 179 2 2' ,3 3' ,5, 6, 6 92 2, 2' ,3,5,5' 180 2 2' ,3 4, 4' ,5, 5' 95 2, 2' 3, 5',6 181 2 2' 3, 4, 4' ,5 6 97 2, 2' ,3 ,4,5 183 2 2' ,3 4, 4' ,5' ,6 99 2, 2' L 4',5 185 2 2' ,3, 4, 5, 5' ,6 101 2, 2' 4, 5,5' 190 2 3, 3' ,4 4' ,5 6 103 2, 2' ,4, 5',6 198 2 2' 3, 3' 4, 5, 5' ,6 105 2, 3, 3' ,4,4' 200 2 2' 3, 3' 4, 5' ,6 6' 108 2, 3, 3' ,4,5' 110 2, 3, 3' ,4',6 105 Appendix 6—1. Analysis of General Linear Models - Carp Length Analysis Class Level Information Class Level Lake 2 Erie Huron Cook Method 2 Pan fry Deep fat fry Skin 2 Skin-on Skin-off Source DF Mean Square F value Pr > F lake 1 321.37 13.70 0.0006 cook method 1 2.71 0.12 0.7359 lake*cook method 1 2.71 0.12 0.7359 skin 1 20.02 0.85 0.3612 lake*skin l 0.61 0.03 0.8730 cook method*skin 1 0.30 0.01 0.9104 lake*cook method*skin 1 0.30 0.01 0.9104 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 51.750 24 Erie B 46.575 24 Huron 106 Appendix 6-2. Analysis of General Linear Models - Carp Carcass Yield Analysis Class Level Information Class Level Lake 2 Erie Huron Cook Method 2 Pan fry Deep fat fry Skin 2 Skin-on Skin-off Source DF Mean Square F value Pr > F lake 1 49.04 6.44 0.0166 cook method 1 1.64 0.22 0.6462 lake*cook method 1 0.82 0.11 0.7453 skin 1 77.54 10.18 0.0033 lake*skin 0 cook method*skin 1 0.42 0.06 0.8146 lake*cook method*skin 0 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 61.787 24 Skin-on B 57.853 12 Skin-off 107 Appendix 6-3. Analysis of General Linear Models - Carp As Prepared Yield Analysis Class Level Information Class Level Lake 2 Erie Huron Cook Method 2 Pan fry Deep fat fry Skin 2 Skin-on Skin-off Source DF Mean Square F value Pr > F lake 1 157.65 16.37 0.0002 cook method 1 40.09 4.16 0.0479 lake*cook method 1 40.09 4.06 0.0479 skin 1 599.75 62.29 0.0001 lake*skin 1 152.40 15.83 0.0003 cook method*skin 1 36.87 3.83 0.0574 lake*cook method*skin 1 36.87 3.83 0.0574 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 32.663 24 Skin-on B 25.593 24 Skin-off 108 Appendix 6-4. Analysis of General Linear Models - Carp Cook Loss Analysis Class Level Information Class Level Lake 2 Erie Huron Cook Method 2 Pan fry Deep fat fry Skin 2 Skin-on Skin-off Source DF Mean Square F value Pr > F lake 1 238.74 8.45 0.0059 cook method 1 1912.56 67.72 0.0001 lake*cook method 1 0.01 0.00 0.9838 skin 1 3.68 0.13 0.7200 lake*skin 1 40.06 1.42 0.2407 cook method*skin 1 70.35 2.49 0.1224 lake*cook method*skin 1 0.31 0.01 0.9177 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 28.404 24 Erie B 23.944 24 Huron A 32.486 24 Deep fat fry B 19.862 24 Pan fry Appendix 6-5. Analysis of General Linear Models - Carp Cook 109 Yield Analysis. Class Level Information Class Level Lake 2 Erie Huron Skin 2 Skin-on Skin-off Cook Method 2 Pan fry Deep fat fry Source DF Mean Square F value Pr > F cook method 1 703.11 26.84 0.0001 lake*cook method 1 2.95 0.11 0.7392 skin 1 366.14 13.97 0.0006 lake*skin l 66.48 2.54 0.1190 cook mrthod*skin 1 655.57 25.02 0.0001 lake*cook method*skin l 5.65 0.22 0.6449 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 75.168 24 Pan fry B 67.514 24 Deep fat fry 110 Appendix 6—6. Analysis of General Linear Models - Total PCB Congeners Analysis (ppm wet). Class Level Information Class Level Lake 2 Erie Huron Skin 2 Skin—on Skin-off Cook Method 2 Pan fry Deep fat fry Product 2 Raw Cooked Source DF Mean Square F value Pr > F lake 1 8.67 6.49 0.0126 skin 1 10.26 7.68 0.0068 cook method 1 5.37 4.02 0.0479 product 1 0.74 0.56 0.4582 lake*skin 1 6.04 4.52 0.0363 lake*cook method 1 12.51 9.37 0.0029 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 1.659 48 Raw A 1.483 48 Cooked A 1.872 48 Erie B 1.271 48 Huron Appendix 6-6 --- continued Tukey Grouping A B Mean 1.898 1.244 1.808 1.335 111 48 48 48 48 Skin-on Skin-off Deep fat fry Pan fry 112 Appendix 6-7. Analysis of General Linear Models - Total PCB Congeners Analysis (ppm dry). Class Level Information Class Level Lake 2 Erie Huron Skin 2 Skin—on Skin-off Cook Method 2 Pan fry Deep fat fry Product 2 Raw Cooked Source DF Mean Square F value Pr > F lake 1 121.43 9.87 0.0023 skin 1 72.55 5.90 0.0172 cook method 1 28.27 2.30 0.1330 product 1 167.61 13.63 0.0004 lake*skin 1 31.87 2.59 0.1110 lake*cook method 1 99.97 8.13 0.0054 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 6.546 48 Raw B 3.904 48 Cooked A 6.350 48 Erie B 4.100 48 Huron Appendix 6-7 --- continued Tukey Grouping A B Mean 6.094 4.356 5.768 4.682 113 48 48 48 48 Skin-on Skin-Off Pan fry Deep fat fry 114 Appendix 6-8. Analysis of General Linear Models - Total PCB Congeners Analysis (ug wet) Class Level Information Class Level Lake 2 Erie Huron Skin 2 Skin-on Skin-off Cook Method 2 Pan fry Deep fat fry Product 2 Raw Cooked Source DF Mean Square F value Pr > F lake 1 267456.32 7.22 0.0086 skin 1 371641.55 10.04 0.0021 cook method 1 68485.40 1.85 0.1773 product 1 171581.88 4.63 0.0341 lake*skin 1 194190.90 5.24 0.0244 lake*cook method 1 244653.74 6.61 0.0118 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 230.00 48 Raw B 145.44 48 Cooked A 240.50 48 Erie B 134.94 48 Huron Appendix 6-8 -—- continued Tukey Grouping A B Mean 249.94 125.50 214.43 161.01 115 48 48 48 48 Skin-on Skin-off Deep fat fry Pan fry 116 Appendix 6-9. Analysis of General Linear Models - Total PCB Congeners Analysis (% Change). Class Level Information Class Level Lake 2 Erie Huron Skin 2 Skin-on Skin—off Cook Method 2 Pan fry Deep fat fry Source jDF Mean Square F value Pr > F lake 1 8.98 0.05 0.8311 skin 1 116.50 0.60 0.4438 cook method 1 755.09 3.88 0.0559 lake*cook method 1 5.11 0.03 0.8722 lake*skin 1 626.84 3.22 0.0804 skin*cook method 1 139.26 0.71 0.4028 lake*skin*cook method 1 1461.25 7.50 0.0092 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 35.734 48 Skin-on A 32.618 48 Skin-off 38.142 48 Deep fat fry 30.210 48 Pan fry 117 Appendix 6—10. Analysis of General Linear Models - Total PCB Congeners Analysis (ug wet). Class Level Information Class Level Lake 2 Erie Huron Skin 2 Skin-on Skin-off Cook Method 2 Pan fry Deep fat fry Product 2 Cooked Raw Source DF Mean Square F value Pr > F lake 1 267456.32 7.22 0.0086 skin 1 371641.55 10.04 0.0021 cook method 1 68485.40 1.85 0.1773 product 1 171581.88 4.63 0.0341 lake*skin 1 194190.90 5.24 0.0244 lake*cook method 1 244653.74 6.61 0.0118 < Tukey's Studentized Range Test > Tukey Grouping Mean N A 230.00 48 Raw B 145.44 48 Cooked A 249.94 48 Skin-on B 125.50 48 Skin-off 118 Appendix 6-10 --- continued Tukey Grouping Mean N A 214.43 48 Deep fat fry A 161.01 48 Pan fry