LIBRARY MIchIgan State Universlty PLACE IN RETURN BOX to remove thII checkout from your rocord. TO AVOID FINES return on or before date duo. DATE DUE DATE DUE DATE DUE 1""; 1 . -._, .. A I (I. ‘ 1431 45;: §Z__JI__IOIE "“ m 2 7 135 3 If 9..— 0f“) 1 ‘3 ll: 5 3 r) .‘ c3 M4201— _—||—— MSU I. An Affirmative Mon/Equal OpporIunIIy Inflation W MI TITLE THE USE OF GREAT LAKES FISH SPECIES AS BIOINDICATORS OF ENVIRONMENTAL CONTAMINATION AND THE EFFECT OF FOOD PROCESSING ON THE REDUCTION OF POLYCHLORINATED BIPHENYL (PCB) CONGENERS, HOMOLOGS AND TOTAL PCBS BY Sandy Wu Daubenmire A DISSERTATION Sumitted to Michigan State University is partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1996 ABSTRACT THE USE OF GREAT LAKES FISE SPECIES AS BIOINDICATORS OF ENVIRONMENTAL CONTAMINATION AND THE EFFECT OF FOOD PROCESSING ON THE REDUCTION OF POLYCELORINATED BIPEENYL (PCB) CONGENERS, EONOLOGS AND TOTAL PCBS BY Sandy wu Daubenmire The purpose of this project was to determine the concentration and distribution pattern of PCB congeners and homologs in fish species: carp, Chinook salmon, lake trout (siscowets), walleye, and white bass. These were harvested from Lake Huron, Ontario, Michigan, Erie and Superior. The fish were processed into skin-on.and skin-off fillets and deep fat fried, pan fried, baked, charbroiled, salt boiled, smoked and canned. The effect of food processing —trimming, skin removal and cooking- on the reduction of total PCBS and their homologs was evaluated. Physical parameters of the fish - length, weight, age and sex - were measured. An average of 50% of lipid. content and 10% of total fish. weight ‘was eliminated from raw fish fillets through skin removal procedure. The results showed that skin removal, fat trimming, and the cooking process did not alter the distribution patterns of PCB congeners. Prominent congeners 87, 158, 66/95/121, 42, 84/101 and 118 were observed in both skin-on and skin-off carp fillets. PCB homologs grouped by chlorination for the Great Lakes fish had a distribution pattern similar to Aroclor@ 1254. Concentration of total PCBS for walleye gave a linear relation between GC-capillary column analysis and GC—packed column analysis without influence of fat content. Only six out of one hundred and twenty-seven fish fillets had total PCBS above FDA action level (2 ppm) based on GC-packed column analyses. Fish harvested at the same location had at least 20% less total PCBS when processed as skin-off fillets than those processed as skin-on fillets. Skin removal before cooking enhanced the reduction of PCB concentration, but the average reduction of PCBS through cooking and skin removal after cooking was 28% to 40%. The most effective cooking method in this study was smoking, which caused a 48% reduction of PCBS in lake trout, and the least effective in lake trout was salt boiling (21%). The relationship between size of the fish and levels of total PCBS is predominated more by length than by weight. 01996 Sandy Wu Daubenmire All rights reserved. No part of this work may be reproduced or transmitted in any form or by any means - graphic, electronic, or mechnical, including photocopying, recording, or information storage and retrieval systems — without written permission from the author. To Jesus Christ my Lord as the Life-Giving Spirit And His Body as the dee ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Mary E. Zabik, for her guidance, encouragement and support through my entire program at Michigan State University. She led me through this narrow and dark tunnel, and perfected me to discover and solve problems as a professional. Her distinguished scholarly manner continually encouraged me to complete my degree. Thanks also to my graduate committee, Al Booren, William Helferich and Matthew Zabik for their expertise, training and unlimited assistance throughout my doctoral studies. Thanks must also go to the College of Human Ecology and the Great Lakes Protection Fund in Chicago for financial support. Thank also for technical support to the Michigan Department of Natural Resources and the Michigan Department of .Public Health. I am also indebted to my former fellow food science grad- students Miriam Nettles, Jeoung-Hee Song, Melvin Pascal, and Peter Liu and the staff from Dr. Matthew Zabik's lab in the Pesticide Research Center. It was tough but we worked as a team to accomplish the mission. Without my husband, Joe, and three children - Paul, Margaret and John who forced me to complete my degree, I might have had my own way, with regrets. Thanks for all the vi pressure, help and understanding added in this process. Many thanks to my parents, brothers and in-laws for their support and.endurance and love which enabled me to finish the task and manage the pressure. vii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . xi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . xiv INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 6 Polychlorinated Biphenyls (PCBS). . . . . 6 Properties of Polychlorinated Biphenyls. . . 9 Toxicity of PCBs . . . 10 Mechanisms of PCBs in the Aquatic Environment 12 Great Lakes Monitoring Programs on PCBS . . . . . 14 International Joint Commission . . . . 14 Remedial Action Plan for Areas of Concern . 17 Great Lakes Protection Fund. . . . 18 National Wildlife Federation' s Great Lakes Natural Resources Center in Michigan. . 18 Biological Variability and Environmental Fluctuation . . . . . . . . . . . . . . 19 Fish Species . . . . . . . . . . 19 Carp (cyprinus carpio). . . 19 Chinook Salmon (Oncorhynchus tshawytscha)19 Lake Trout (salvelinus namaycush) . . . 20 Collection Sites . . . . . . . . . . . . 21 Age and Size of Samples. . . . 21 Collection Time and Environmental Fluctuation 23 Tissue Sampled . . . 23 Analytic Procedures and Methodology for. PCBs 25 Effects of Processing and Cooking on the Reduction of PCBs Levels in Raw and Cooked Fish Fillets . . . . . . . . . . 28 Effect of Trimming Procedures. . . . . . . . 28 Effect of Cooking Methods. . . 30 Effect of Trimming and the Selected Cooking Methods . . . . . . . . . . 32 MATERIAL AND METHODS . . . . . . . . . . . . . . . . . 34 Fish Procurement. . . . . . . . . . . . . . 34 Processing of Fish Fillets. . . . . . . . . . . . 36 Sample Preparation. . . . . . . . . . . . 37 Preparation of Raw Sample. . . . . . . . . . 37 viii Preparation of Cooked Sample . Cooking Methods for Fish Fillets Baking. . . . . . . . Charbroiling. Deep Fat Frying . Pan Frying Salt Boiling. Smoking . Pressure Canning. Analysis of Solids. Lipid Analysis. . . Congener Specific Polychlorinated Biphenyls Analyses. . . . . . . . . . Glassware Preparation. Solvents and Reagent Preparation Lipid Extraction . . . Cleanup of Lipid Extract . Gel Permeation Chromatography . Florisil Column Chromatography. Silica Gel Column Chromatography. Identification and Quantitation. . Statistical Analysis. RESULTS AND DISCUSSION . . Procurement Data for the Great Lakes Fish species. Source and Size of Fish Data . . Age and Sex of Fish Data Processing Data of the Great Lakes Fish Species Analyses of Solids and Lipids on Raw Fish Fillets Cooking Data of the Great Lakes Fish Species. Analyses of Solids and Lipids of Cooked Fish Fillets. Distribution Pattern of PCB Specific congeners. in Fish Fillets from the Great Lakes. Lake Effect Skin Removal Effect Cooking Effect. Species Effect. Distribution Pattern of PCB Homologs in Fish Fillets from the Great Lakes. . . Evaluation of Total PCBS by GC- -Capillary and GC- Packed Columns in Raw Fish Fillets from the Great Lakes. Remedial Action on the Reduction of Total PCBs and PCB Homolog Concentration in Fish Species Harvested from the Great Lakes Through Food Processing Methods I. Method of Skin Removal and Trimming Before Cooking. 37 39 39 39 39 41 41 41 42 42 42 43 43 43 45 47 47 47 48 49 51 53 53 53 55 58 68 74 76 77 79 82 85 89 98 112 114 II. Method of the Skin Removal After .Cooking. 119 ix III. Selective Cooking Method to Enhance the Safety. . . . . . . . . . . . . . . . . Relationship of Physical Parameters with Chemical Parameters of the Great Lakes Fish Species for the Consumption Advice Application of Measurement of Correlation. SUMMARY AND CONCLUSIONS RECOMMENDATIONS APPENDICES. LIST OF REFERENCES. 125 130 138 139 146 148 216 Ta Ta Ta Tat Table Table Table Table Table Table Table Table Table Table Table Table 1. 2. 9. 10. 11. 12. LIST OF TABLES Approximate composition of Aroclor0 1254. . . Characteristics of Aroclor0 1254. . . . . . . Morphometric characteristics of the Great Lakes and means of total PCBs concentration in waters, lake trout and coho salmon of the Great Lakes . . . . . . . . . . . . . . . Characteristics of carp, Chinook. salmon, lake trout walleye and white bass on length, weight, spawning period and water temperature for growth . . . . . . . . . . . . . . . . . . Summary of the information on fish species, source of lake (# Fish), location of catch and date of catch . . . . . . . . . . . . . . Summary of processing and cooking methods of fish fillets in five species harvested from Great Lakes. . . . . . . . . . . . . . . IUPAC numbers and structure of the PCB Congener 8 O O O I O O O O O O O I O O O O O O Source and size of the Great Lakes fish ' spec 1e8 . . . . . . . . . . . O . . O . . . . Sex and age of the Great Lakes fish species . Processing data as well as solid and lipid contents of raw fillets for carp from Lakes hie and Huron O O O O O O I O O O O O O O 0 Processing data as well as solid and lipid contents of raw fillets for chinook salmon from Lakes Huron and Michigan . . . . . . . . Processing data as well as solid and lipid contents of raw fillets for lake trout from Lakes Huron, Michigan, Ontario and Superior . xi 10 16 20 35 4O 44 56 57 61 62 63 Table Table Table Table Table Table Table Table Table Table Table Table 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Processing data as well as solid and lipid contents of raw fillets for walleye from Lakes Erie, Huron and Michigan. . . . . . . . . . . 64 Processing data as well as solid and lipid contents of raw fillets for white bass from Lakes Erie and Huron. . . . . . . . . . . . . 65 Cooking data and solid and lipid contents of cooked skin-on and skin-off fillets for carp from Lakes Erie and Huron . . . . . . . . . . 68 Cooking data and solid and lipid contents of cooked skin-on and skin-off fillets for chinook salmon from Lakes Huron and Michigan . 69 Cooking data and solid and lipid contents of cooked skin-on and skin-off fillets for lake trout from Lakes Huron, Michigan, Ontario and Superior. . . . . . . . . . . . . . . . . . . 70 Cooking data and solid and lipid contents of cooked skin-on fillets for walleye from Lakes Erie, Huron and Michigan. . . . . . . . . . . 71 Cooking data and solid and lipid contents of cooked pan fried skin-on fillets for white bass from lakes Erie and Huron. . . . . . . . 72 Mean concentration of PCB specific congeners and their percentage for raw skin-on and skin- off chinook salmon harvested from Great Lakes 78 Mean concentration of PCB specific congener and total PCBs from raw skin-on and skin-off fillets for carp and chinook salmon harvested from the Great Lakes. . . . . . . . . . . . . 80 Comparison of total PCBs determined by GC-packed column and GC-capillary column of the same raw fish fillets for carp, chinook salmon, lake trout, walleye and white bass from the Great Lakes . 99 Total PCB concentrations based on summing 53 congeners determined by GC-capillary column in all raw fish fillets for carp, chinook salmon, lake trout, walleye and white bass from the Great Lakes . . . . . . . . . . . . . . . . . 100 Correlation coefficient values of total PCBs determined between GC-capillary and GC-packed column among fish species from the Great Lakes . 107 xii Table Table Table Table Table Table Table Table Table 24a. 25. 26. 27. 28. 29. 30. 31. 32. Predicted equations for total PCBs from GC- capillary column analysis based upon total PCBs from GC-packed column analysis and fat content of fish sample. . . . . . . . . . . . . . . . 111 Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for raw carp fillet. O O O O I O O O O O O O I O O O O O I 116 Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for raw chinook salmon. O O O O O O O O O O O O I O O O O O O 117 Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for carp through cooking and skin removal after cooking process 0 O O O O O O O O O O O O O O O O O O C O O O 121 Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for chinook salmon through cooking and skin removal after cooking process . . . . . . . . . . . . 122 Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for walleye through cooking and skin removal after cooking process . . . . . . . . . . . . . . . . . . . 123 Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for white bass through cooking and skin removal after cooking process . . . . . . . . . . . . . . . 124 Effectiveness of cooking process on the reduction of total PCBs and PCB homologs for skin-off chinook salmon . . . . . . . . . . . 126 Percentage change of PCB homolog concentration and of total PCBs during cooking process. . . 128 xiii Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 4a. 9a. 9b. LIST OF FIGURES Basic structure of polychlorinated biphenyls and numbering of carbon atoms and position of chlorine atom in biphenyl ring system . . . . . . . . . . . . . . . 7 Simplified chemical analytical procedures for PCBs congener specific from fish tissue 46 Mean concentration of PCB specific congener for raw skin-on and raw skin-off chinook salmon fillets harvested from the Great Lakes . . . . . . . . . . . . . . . . . . 81 Mean concentration of PCB specific congener for raw and cooked skin-off chinook salmon harvested from Great Lakes. . . . . . . . 83 Percentage of individual PCB specific congener to total PCB concentration for raw and cooked skin-off chinook salmon from Lakes Huron and MiChigan O O O O O O O O O O O O O C C O O 8 4 Mean concentration of PCB specific congener for raw skin-off and cooked skin-off carp fillets harvested from the Great Lakes. . 86 Mean concentration of PCB specific congener for raw skin-on chinook salmon, walleye and white bass harvested from Great Lakes 87 Distribution pattern of PCB homologs for carp O O O O O Q 0 O O O O O O O O I O O O 9 0 Distribution pattern of PCB homologs for chinook salmon fillets. . . . . . . . . . 91 Distribution pattern of PCB homologs for lake trout fillet. . . . . . . . . . . . 92 Distribution pattern of PCB homologs for lake trout f 1 1 let 0 O O O O O I O O O O I 9 1 xiv Figure 10. Distribution pattern of PCB homologs for walleye fillet. e o e o o o o e o e e e e 94 Figure 11. Distribution pattern of PCB homologs for white bass fillet . . . . . . . . . . . . 95 Figure 12. Comparison of total PCB concentration in raw skin-on and skin-off carp fillets determined by GC-capillary column and GC-packed column in relation to their fat content. . . . . 101 Figure 13. Comparison of total PCB concentration in raw skin-on and skin-off chinook salmon determined by GC-capillary column and GC-packed column in relation to their fat content. . . . . 102 Figure 14. Comparison of total PCB concentration in raw skin-on and skin-off lake trout fillets determined by GC-capillary column and GC-packed column in relation to their fat content. . . . . 103 Figure 15. Comparison of total PCB concentration in raw skin-on walleye fillets determined by GC-capillary column and GC-packed column in relation to their fat content. . . . . 104 Figure 16. Comparison of total PCB concentration in raw skin-on white bass fillets determined by GC-capillary column and GC-packed column in relation to their fat content . . . . . 105 Figure 17. Levels of PCB homologs and total PCB concentration in raw skin-on and skin-off carp fillets harvested from Lakes Erie and Huron . . . . . . . . . . . . . . . . . . 114 Figure 18. Levels of PCB homologs and total PCB concentration in raw skin-on and skin-off chinook salmon fillets harvested from Lakes Huron and Michigan. . . . . . . . . 115 Figure 19. Concentration of total PCBs in relation to length and weight of carp harvested from Lakes Erie and Huron. . . . . . . . . . . 131 .Figure 20. Concentration of total PCBs in relation to length and weight of chinook salmon harvested from Lakes Huron and Michigan . 132 Figure 21. Concentration of total PCBs in relation to length and weight of lake trout harvested from Lakes Michigan and Superior. . . . . 133 XV Figure 22. Concentration of total PCBs in relation to length and weight of walleye harvested from Lakes Erie, Huron and Michigan . . . 134 Figure 23. Concentration of total PCBs in relation to length and weight of white bass harvested from Lakes Erie and Huron . . . . . . . . 135 xvi INTRODUCTION Polychlorinated biphenyls (PCBs) are the complex chemical mixtures of 209 possible PCB congeners which widely used in capacitors, transformers, lubricants and other industrial applications between 1929 and 1972. Their low aqueous solubility, hydrophobicity and resistance to degradation resulted in the aerial transport to atmosphere, to aquatic and sedimental environment (Eisenreich et al. , 1981; Hermanson et al., 1991) including fish (Mac and Schwartz, 1992), wildlife, even to plant (Shane and Bush, 1989), human adipose tissues (Schmid et al., 1992), serum (Schwartz et al., 1983,- Jacobson et al., 1989), and milk (Dewailly et al., 1989,- Hong et al., 1992a) . Currently PCBs are present throughout the global ecosystem (Ravid et al., 1985,- El Nabawi et al., 1987; Satsmadjis et al., 1988; Leonzio et al., 1992). Even after use of PCBs were commercially banned, PCB congeners continue to translocate in the environment by atmospheric transport and deposition, point source discharge, agriculture run-off, sewage waste, paper mill effluent into aquatic system and dredging with existing polluted sediment (Seelye et al, 1982) . Scientists (Thomann and Connolly, 1984,- Borlakoglu et a1. , 1988) have proved that the mechanism of PCB 2 congeners in aquatic ecosystem is modelled as the bioconcentration of PCB congeners in water, and biota (Wang et al., 1982), bioaccumulation of PCBs in plankton, and biomagnification of PCB congeners concentration in predators (Oost et al, 1988). Since PCBs can bioaccumulate in the aquatic ecosystem, species such as lake trout, chinook salmon, which are near the top of the food chain, can be useful as indicators of contaminant levels in the aquatic ecosystem. As the angler and sportsman seemingly consumed the highest level of PCBs in the food chain, the risk of consuming PCBs contaminated fish has been under investigation through various monitoring programs, such as the Remedial Action Plan (RAP) in Michigan (MDNR, 1992). The toxicity of PCB congeners has been studied in the past decade (Tanabe et al., 1987a) and the studies of biochemistry, toxicology and the mechanism of action of PCBs, and related toxic compounds have also been performed extensively (Safe, 1984 and 1990). They have focused on the structure-activity relationships (SARs) of PCBs with 2.3.7.8- tetrachlorodibenzo-p-dioxin (2 , 3 , 7 , 8-TCDD) for the development of toxic equivalency factors (TEFs) in order to predict the toxicity of PCBs from total PCBs concentration which are commonly used in the hazard and risk assessment of toxic halogenated aromatics in fish fillet (Williams and Giesy, 1992; Williams et al., 1992). The toxic effects of PCBs occurred in several accidental exposures which included the Yusho (Kashimoto et al. , 1981) and Yu Cheng poisoning (Chen et 3 al., 1980) have provided important knowledge on the toxic effects of PCB congeners on human health and also generated pressure to government and regulatory agencies to take action. on the assessment of PCBs. The initial step for hazard and risk assessment of any toxic compound is to determine and verify the qualitative and quantitative analyses. The determination of PCBs in the aquatic environment is possible through the analysis of water, sediments, biota and bioindicators such as birds and fish. The method of extraction of sample varies; up-to-date identification and quantitative analyses have mostly been measured by gas liquid chromatography and mass spectrometry (GC/MS) (Bush et al., 1989, Bush et al., 1990, Draper and Koszdin, 1991) . The progression of packed columns into capillary column enables the analysts to study the PCB congeners specific instead of total PCBs concentration (McFarlan and Clarke, 1989). Knowing the levels of PCB congeners in the environment samples and the effect of PCBs in animal experiments, government and regulatory agencies set upiaction.level - i.e., 2 ppm by FDA - for the protection of the public health in compliance with food safety requirements. Locally' the Michigan Department of Natural Resources and Public Health issues fish.guides to provide significant information for the sportsmenfs protection from.consuming contaminated.species of Great Lakes fish. Nationally the Environmental Protection Agency (EPA)(Clark et al., 1984; Capel and Eisenreich, 1985) 4 and Food. & Drug .Administration (FDA) have surveyed. the contaminants in the environment and in manufactured products. All the above agencies have impacted on the establishment of the fish consumption advisories (Clark et al., 1987). Globally USA, Canada and the Eurporean countries had various collaborated studies on PCBs in wildlife and fish (Falandysz, 1985; Marthinsen et al., 1991; Teschke et al., 1993). The utilization of food processing and cooking methods on the reduction of PCBs congeners levels in fish tissue or marine products seemed to become applicable in compliance with food safety issues (Smith et al., 1973; Zabik et al., 1979; Hora, 1981; Zabik et al., 1982; Armbruster et al., 1987; Sanders and Haynes, 1988; Stachiw et al., 1988; Armbruster et al., 1989; Trotter et al., 1989; Voiland et al., 1991, Zabik et al., 1992; Zabik et al., 1993). They all strongly agreed that effects of fat trimming procedures, skin removal and cooking are feasible to maximize the loss of PCBs and/or other organic toxicants in fish fillet. The purpose of the studwaas to examine the concentration of congener specific and distribution pattern of polychlorinated biphenyl homologs in five species of Great Lakes fish as bioindicators in order to provide data for public health and other agencies to quantitate the degree of exposure a human might receive from consumption of each of five commonly caught open water fish species prepared and cooked. by commonly 'used. methods or 'methods which. offer potential for significant contaminant reduction. The specific 5 objectives were the following: 1). To investigate the distribution pattern of congener specific and homologs in raw and cooked fish fillets; 2) . To determine the effect of trimming and cooking methods on reduction of total PCBs and PCB homologs in the Great Lakes fish species; 3). To correlate the ‘measurements of physical parameters - length and weight with chemical parameters — levels of PCB homologs and total PCBs of fish species. LITERATURE REVIEW Polychlorinated Biphenyls (PCBs) Polychlorinated biphenyls are a class of halogenated aromatic hydrocarbons that possess high thermal and chemical stability. They were formerly used in capacitors, transformers, hydraulics, carbonless copy paper and other industrial equipments as lubricants. The nature of PCBs is a mixture of PCB congeners. Congener is defined as a PCB compound with a specific chlorine substitution pattern. The possible numbers of congeners existing in PCBs are 209 based upon the locations and orders of chlorines. According to International Union of Pure and Applied Chemistry (IUPAC) definitive rules for nomenclature of organic chemistry, one ring system in the biphenyl ring assembly is assigned unprimed numbers and the other primed numbers (Figure 1). The groups with the same number of chlorines in PCB congeners are called homologs, such as 2,4,6-trichlorinated biphenyls (IUPAC # is 30) and 2,4’,5—trichlorinated biphenyls (IUPAC # is 31) are grouped as tri-chlorinated biphenyls (Tri-CBs) homolog. Prior to 1977, PCBs manufactured by Monsanta Chemical Co. in USA were sold under the tradename Aroclor" until the prohibition on PCBs manufacture and uses under Toxic X X [m eta-l [o rtho-l Figure 1. Basic structure of polychlorinated biphenyls and numbering of carbon atoms and position of chlorine atom in biphenyl ring system 8 Substances Control Act (TSCA) of 1976. Nevertheless, in the commercial preparation of Aroclor@, a four digit code was given to the product; the first two digits are the number of carbon atoms in the biphenyl group and the last two digits indicate the percentage by weight of chlorine in the mixture. Aroclor“D 1254 denotes a polychlorinated biphenyl mixture having 12 carbon atoms and containing 54% chlorine content by weight” Table 1 states the approximate composition of Aroclor® 1254 with the groups of PCB homolog and their numbers of isomers. Tetra-CBs, Penta-CBs and Hexa-CBs are considered to be the predominant homologs in Aroclor0 1254. Scientists (Maack and Sonzogni, 1988; Draper and Koszdin, 1991) 2,2’,3,4,5'- (87), 2,2’,4,5,5’- (101), 2,3,3’,4',6- (110), Table 1. Approximate composition of Aroclor® 1254 (Composed from Waid, 1986) Empirical Formula PCBs Homolog # of Composition(%) Isomers C,,H,Cl Mono-CBs 3 <0 . 1 C,,H,Cl, Di -CBs 12 0 . 5 C,,H,Cl, Tri-CBs 24 1 . 0 C,,H,Cl, Tetra- CBs 42 2 1 . 0 CMHsCl5 Penta-CBs 46 48 . 0 C,,H,Cl, Hexa-CBs 42 23 . 0 C12H3Cl, Hepta-CBs 24 6 . 0 C,,H,Cl, Octa-CBs 12 - C12HC1, Nona-CBs 3 - CuClm Deca-CBs 1 - 9 identified that 2,2'5,5’- (52) from tetra-CBs homolog, 2,3',4,4',5- (118) and. 2,3,3',4,4'- (105) frcmt penta-CBS homolog, and 2,2’,3,4,4’,5’- (138) from hexa-CBs homolog are the predominant congeners inAroclor® 1254 mixtures. Bush et al. (1989) stated that the quantities of 2,4,5,3',4'— (118) and 2,3,6,3’,4'- (110) which account for 5% and 2% of total PCBs weight, respectively, in an environmental residue were the indicator congeners for Aroclor® 1254 in a mixture of Aroclors 1221, 1016, 1242, 1254, and 1260. Properties of Polychlorinated Biphenyls Typical properties of PCBs are hydrophobic, low water solubility, high lipophilicity, high density, low vapor pressure, high dielectric constant and high octanol/water partition coefficient. The degree of lipophilicity is increased with increasing ring chlorination. The average boiling point of PCBs is about 360°C which makes the compound very stable and resistant to the breakdown by acids, bases, heat and hydrolysis. Viscosity of PCBs resembles a mobile oil or liquid oil at room temperature. Color of PCBs might be yellow or clear. vapor pressure of PCBs and solubility of PCBs in water decrease with the increased chlorination” With the combination of low water solubility and high octanol/water partition coefficients, PCBs have a high affinity for suspended solids, especially those high in organic carbon (Chou and Griffin, 1986). Their physical and chemical stability may be the contributing factors for the transport and fate of PCBs into environment, even for bioaccumulation in 10 the food chain. Table 2 lists some characteristics of Aroclor0 1254. Table 2. Characteristics of Aroclor“D 1254 (Composed from Waid, 1986) Characteristics Color Light yellow viscous oil Specific gravity 1.495-1.505 Molecular weight(average) 328.4 Boiling point (°C) 365 - 390 Density 1.53 Dielectric constant (at 25TH 5.0 Aqueous solubility (ppb) 42 n-Octanol/water partition coefficient (Km) 1,288,000 Soil sorption constant (KNJ 63,914 Toxicity of PCBs The toxicity of PCBs is a function of the structure of the individual congener which depends upon the numbers and position of the chlorine. The stereochemistry of the PCB molecules possess planar and non-planar conformations which relate to the positions of the chlorine substitution at the ortho, meta or para substitution. When the chlorine atom substituted in the either or both ortho positions causes the bond between two benzene rings to rotate and changes their configurations. Those congeners appear in their non-planar conformations. Studies (Safe, 1984 and 1990) proved that PCB 11 congeners elicit many of their toxicological effects through the same receptor-mediated mode of action as 2,3, 7, 8-TCDD (tetrachlorodibenzoap-dioxin). The toxicity of PCB congeners is the greatest for those congeners which are stereochemical most similar to TCDD based upon the structure-activity relationships (SARs) , and which can assume a planar structure. These planar congeners elicit adverse effects at concentrations many orders of magnitude less than non-planar congeners (Williams, 1993). Out of 209 possible PCB congeners, four co-planar PCB congeners have been recognized as the most toxic congeners which 3,3',4,4’-tetraCBs (77), 3,4,4’,5-tetraCBs (81), .3,3',4,4',5-pentaCBs (126) and 3,3',4,4',5,5’-hexaCBs (169) are approximate isostereomers of 2,3,7,8-TCDD in their coplanar conformation (Safe, 1990) . The planar PCB congeners appear to elicit their toxicity through the same *mode of action as 2,3,7,8-TCDD. Safe (1990) summarized the mechanism of action as 2,3, 7, 8-TCDD and planar PCBs bind to the Ab receptor induce the cytochrome P-4501A1 and cytochrome P-4501A2 hemoproteins and their associated hepatic microsomal monooxygenases, which include aryl hydrocarbon hydroxylase (AHH) and ethosyresoruf in O-deethylase (EROD) activities in laboratory animals and mammalian cells in culture. In the risk assessment of toxic substances, Toxic Equivalency Factors (TEF) has extensively been used which is defined as the potency of the each congener to elicit toxic effects relative to the potency of TCDD. The potency of a 12 mixture of the AHH-active compounds can be expressed as TCDD Equivalents (TEQs) which derives from. multiplying’ molar concentrations of PCBs congeners by the corresponding TEF for that congener (Williams, 1993). According to Safe (1990), a TEF value of 0.1 is recommended for 126, 0.05 is for 169 and 0.01 is for 77, 0.001 is for 114 and other mono-ortho coplanars, and 0.00002 is assigned to diortho-coplanar PCBs which are major components of the commercial products and extracts from environmental samples. The target organ in most mammalian species is the liver and the reproductive system in mammalian species is also affected (U.S. EPA, 1993). High concentrations of PCBs were also found in adipose tissue (Tanabe et al., 1987b; Williams and LeBel, 1991) and breast milk of humans (Hong et al., 1992a). Mechanisms of PCBs in The Aquatic Environment PCBs were discharged into the environment through leaking of capacitors, transformers or by product of paper mills (Rastogi, 1992). Often, PCBs can be detected in the lakes or atmosphere. When hydrophobic chemicals such as PCBs are found dispersed in lakes, the compounds partition.between water and various non—aqueous compartments such as biotic lipids, organic phases of sediments or suspended particles which. are not fully known.(Oost et al., 1988). Generally, it is assumed that these partitioning processes can be described by first order kinetics. For the partitioning of chemicals between water and lipid phases of organisms, the bioconcentration is defined as the 13 uptake of substance by an organism from the surrounding medium through gill membrane or other external body surfaces or from food consumed. Normally, PCB contaminated particles or sediments in the aquatic ecosystem can act as a source of this contaminant which then can be bioconcentrated in the organism. The bioaccumulation is defined as the uptake and retention of substances by an organism from its medium and from food. Bruggeman et al. (1984) stated that PCBs are released into the environment as mixtures, containing many congeners; therefore, the bioaccumulation capabilities of the individual congeners are determined by their physico-chemical properties and their molecular configuration. Bioaccumulation of the higher chlorinated PCBs increases with higher trophic level organisms. For the lower chlorinated compounds, both the elimination rate in organisms and the rate of desorption are relatively high; thus, equilibrium will be achieved relatively fast (Wang et al., 1982, Bush et al., 1989) . Bioaccumulation is predominantly controlled by equilibrium partitioning of the chemical between the internal lipids of the biota and ambient water (Shaw and Connell 1984). Maack and Sonzogni (1988) found that PCB levels in water are several orders of magnitude less than those found in fish. Meantime, Oost et al. (1988) reported that there were no detectable concentrations of PCBs congeners (IUPAC # 28, 52, 101, 138, 153 and 180) in the water samples in their investigation; however, PCBs congeners were found in most of the sediment and organisms samples in a freshwater near Amsterdam (i.e., plankton, molluscs, 14 crustaceans and eel). It is consistent with the fact that fish readily bioaccumulate PCBs. They concluded that the process by which the concentration of PCBs increasing in different organisms, occupying successive trophic levels refers to biomagnification process. The biomagnification factor denotes the ratio between the concentration of chemicals in organisms to the concentration of chemicals by fish from food in food chain” Biomagnification factor increases with increasing hydrophobicity of PCBs specific congeners which causes the decreasing elimination rate constant. Oliver and. Niimi (1988) also confirmed that biomagnification is generally the major mechanism of PCB deposition at higher trophic level. Great Lakes Monitoring Programs on PCBs International Joint Commission The Great Lakes contain 20% of the world’s surface fresh water, they are one of unique and rich natural resources of Michigan. In addition, these waters border with seven other states and also form a portion of the international boundary between the United States and Canada. Eight states and one province all have jurisdictions over the use of The Great Lakes. In order to protect the resource, International Joint Commission (IJC) was organized to surveillance the extent and cause of pollution in the Great Lakes. Out of IJC, Great Lakes Water Quality Agreement (GLWQA) was signed between two federal governments in order to coordinate and monitor the 15 restoration and enhancement of water quality in the Great Lakes system. A variety of federal and state agencies including the U.S. Environmental Protection Agency, the U.S. Fish and. Wildlife Service, state departments of natural resources and/or universities, and Michigan Department of Natural Resources are the representatives in IJC committees. International Joint Commission has classified nine organic compounds and two heavy metals of 362 toxic substances in the Great lakes ecosystem as Critical Pollutants because of their toxic effects to the human health, environmental persistence, and widespread occurrence throughout the Great Lakes ecosystem (MDNR, 1992). Besides total PCBs, there are dichlorodiphenyltrichloroethane (DDT) and metabolites, dieldrin, toxaphene, 2,3,7,8-TCDD, 2,3,7,8— tetrachlorodibenzofuran (2,3,7,8-TCDF), ‘mirex, 'mercury, alkylated lead, benzo(a)pyrene and hexachlorbenzene (HCB). Since most of the Critical Pollutants have the ability to be bioconcentrated by aquatic organisms and to bioaccumulate up the food chain, species such.as lake trout and chinook salmon, which are near the top of the food chain, are often used as indicators of contaminant levels in the aquatic ecosystem. According to the report from Michigan Department of Natural Resources (MDNR)(1992), levels of PCBs in waters and lake trout of the Great Lakes between 1980 to 1986 (Table 3) were decreased through the restriction and implementation of environmental protection programs. Table 3 also indicated that PCBs level in open water of the Great Lakes were not 16 Table 3. Morphometric characteristics of the Great Lakes and means of total PCBs concentration in waters, lake trout and coho salmon of the Great Lakes Total PCBs Water Average Total Surface Depth PCBs ix1’lake in coho Area (ft) in water trout(ug/g) salmon (ud3) (ng/l) 3(1980-.1984) s(ug/g) Superior 31,700 489 0.325 2.0 a 1.0 traces 1(1983) Michigan 22,300 279 1.200 10.0 a 4.5 1.93 1(1980) Huron 23,000 195 0.573 3.5 a 2.0 1.95 1(1984) Erie 9,910 62 1.159 ‘NA 1.07 1(1986) Ontario 7,340 283 1.201 ‘NA 2.90 1(1983) 1 indicates the surveyed year whole fish analysis monitering year; from DeVault et al. (1986) data not available from Clark et al., (1984) “OH” 17 below analytical detection limits, total PCBs levels in fish accumulated to high concentrations corresponding to the concentrations found in the environment. MDNR fish contaminant monitoring program for Lake Michigan from 1984 data revealed that contaminants in coho salmon and lake trout less than 20 inches in length had decreased to the point where 90% or more of the fish tested did not exceed FDA action levels (2ppm). But, contaminant levels in lake trout over 25 inches in length and in carp and brown trout, remained high (MDNR, 1992). Total PCBs levels in lake trout from Lake Michigan are higher than in lake trout from.either Lake Huron or Lake Superior. Lake trout from Lakes Superior, Michigan and Huron had higher total PCBs than coho salmon sampled from those Lakes (Table 3). Remedial Action Plan for Areas of Concern Michigan in conjunction with seven other states and Ontario, all of which have jurisdiction over a portion of the Great Lakes, developed and implemented a Remedial Action Plan (RAP) for specific Area of Concern (AOC) in 1985. The RAP monitors the change in the chemical, physical or biological integrity of the Great Lakes ecosystem sufficient to cause the damage to the water and environmental quality. A portion of fish harvested from the Great Lakes in this study was from the AOC, such as carp, walleye and white bass from both Saginaw Bay in Lake Huron and River Raisin into Lake Erie, and siscowet from Lake Superior near Marquette. 18 Great Lakes Protection Fund The: Great Lakes Protection Fund. (GLPF) was jointly established by the eight Great Lakes states in order to provide a sustained level of financial resources to address the issues identified in the Toxic Substances Control Agreement (TSCA) and Great Lakes Water Quality Agreement, it acts as a "shared resource pool" to fund activities which are not normally covered by state or federal appropriated funds (GLNPO, 1981). The present research project was endowed by GLPF in Chicago to enhance the food safety issues in consuming Great Lakes fish. National Wildlife Federation’s Great Lakes Natural Resources Center in Michigan The function of National Wildlife Federation (NWF) monitors the areas not covered by the U.S. Food and Drug Administration which mainly monitors and regulates levels of toxic chemicals in fish that are commercially marketed. The main objective of NWF is developing an.uniform.approach.based on scientific methods to evaluation the health risks of eating sport fish in order to protect sport anglers and their families who may be consuming contaminated fish (Schmidt, 1989). Locally the Federation's Great Lakes Natural Resource Center in.Michigan summarizes the research results and offers advice about ways to reduce exposure to the toxic substances contaminating Lake Michigan fish for Lake Michigan sport anglers (Anonymous, 1989). 19 Biological Variability and Environmental Fluctuation Biological variability* and environmental fluctuation should be considered in the assessment of the data generated from. monitoring programs on PCBs contamination in fish species. Comparing the research data from different laboratories may misinterpret the results which often vary by the fish. species, age and size of specimens, timing’ of collection, sex of fish, collection sites, tissues sampled, number of specimens and. pooling; procedures of chemical analyses and statistical analyses, etc.. The uniformity of the above parameters in the experimental design may enhance the valid comparisons between fish monitoring contaminant data sets from individual Great Lakes generated as part of independent agency programs (Clark et al., 1984) Fish Species Table 4 lists some characteristics of carp, chinook salmon, lake trout, walleye and white bass to illustrate the biological variabilities. Carp (cyprinus carpio) Carp accepts almost any type of food and obtains bulk of their nourishment by sucking organic material from bottom of lakes or rivers (Song, 1994). The spawning period for carp lasts from April to August, but generally spawning occurs in late May and June. 91W (Oncorhynchus tshawytscha) Chinook salmon belongs to the Oncorhynchus branch of the salmonidae family, commonly known as the king salmon. The flesh tissue is between.bright red to yellowish-red depending upon the specie 20 Table 4. Characteristics of carp, chinook salmon, lake trout, walleye and white bass on length, weight, spawning period and water temperature for growth (Rodger, 1991) Fish Length Weight Spawning water Species (cm) (kg) Period Temp.CTO Carp NA1 NA April to NA August Chinook 90 8.0 - 10.0 Spring to NA salmon early fall Lake trout 40-50 2.3 - 4.6 every second 40 - 50 Autumn Walleye 40-60 0.9 - 1.6 Spring to 45 - 48 ' early summer White bass .50-60 0.9 - 3.6 April to 63 - 70 June 1 not available and harvesting time. They often migrate great distances and return to spawn at varying ages, from 2 to 8 years. LBK§_LIQUL (Salvelinus namaycush) Lake trout belongs to the char (Salvelinus) branch of the Salmonidae family. The difference between lean lake trout (Salvelinus namaycush namaycush) and siscowet (Salvelinus namaycush siscowet) is lipid content. Miller et al. (1992) reported that total PCB accumulation rates were not significantly different between lean trout and siscowet collected from.Lake Superior, despite a two-fold. greater ‘mean concentration of lipids in the siscowet than in the lean trout. The higher contaminant burden in lake trout are partly related to their longevity 21 because it takes up 6 - 12 years for lake trout to reach sexual maturity. Miller and.Jude (1984) found that chinook salmon and carp contained in general higher concentration of PCBs than did whitefish fillet harvested from Lake Huron. Both salmon and lake trout are in the top predators which are often used as reflectors for the concentrations of contaminants in lower trophic levels. Collection Sites Scientists (Marcus and Mathews, 1987; Swackhamer and Armstrong, 1987; Bush.et al., 1989; Miller et al., 1992) found that there is a general trend in reduction of the amount of PCBs in water and in fisheries with increased distance from the point source discharge and/or with increased depth of water offshore. Miller et al. (1992) found that PCB concentrations in lake trout were higher in Lake Michigan, relative to similarly aged fish from.Lake Superior. Clark et al. (1984) found that skin-on fillets of coho salmon from Lake Ontario (2.90 ppm wet weight basis) are more heavily burdened with PCBs than samples from other lakes (<2.00 ppm) . However, Maack and Sonzogni (1988) reported that there was no correlation observed for total PCB concentrations and species or location of fish collected from various Wisconsin waters, including from Lake Michigan. Age and Size of Samples Fish consumption advisories are generally based on fish length.alone as a predictor of total concentrations of PCBs in 22 Michigan (Clark et al., 1987). However, scientists (Zabik et al., 1982; Miller and Jude, 1984; Kuwabara et al. 1986; Voiland et al., 1991; Williams et al., 1992) proposed that size (length and weight) could be good predictor of total concentrations of PCBs. The finding from the study of Zabik et al. (1982) using carp harvested from Saginaw Bay of Lake Huron indicated that length of fish correlated better with total PCBs for carp which ranged in size between <2 kg and >5.5 kg, while weight correlated better for carp which weighed between 4 to 5.5 kg. Miller and Jude (1984) concluded that based on analyses of whitefish fillets from Saginaw Bay that PCBs displayed a strong direct relationship with fish length, especially in males. Kuwabara et al. (1986) reported that total PCB levels in peeled shellfish was significantly correlated to shell length and weight (r= 0.78 and 0.77, respectively). Voiland et al. (1991) indicated that length, weight and age are highly correlated to the levels of PCBs in untrimmed and trimmed fish fillets of brown trout from Lake Ontario. ‘Williams et al. (1992) reported that fish length is aumorejpositive indicator of PCB concentration for the skin-on fillets of chinook salmon from Lake Michigan, while fish weight is a slightly better predictor than fish length in skin-off trimmed fillets of chinook salmon from Lake Michigan. Miller et al. (1992) used length of lean and siscowet lake trout from Lake Michigan and Lake Superior to estimate the age in lake trout and concluded.that the concentrations of PCBs in lake trout are significantly influenced by age (exposure time) ”I r' F? 6;) 1' r ). 23 of lake trout. Collection Time and Environmental Fluctuation Some research (Zabik et al., 1982; Greig and.Sennefelder, 1987) indicated that the total PCB concentrations were higher in.fish.before spawning time than.other seasons, especially in the reproductive tissues of the fisheries. Lake environmental fluctuation often results in resuspension of sediments from storms, currents, passing ships, dredge as well as direct inputs from populated areas which caused some significant increase of PCB levels in some nearshore areas (Capel and Eisenreich, 1985). They proposed that total PCB concentration in the water columm.in.Great Lakes increases in the winter due to the resuspension of contaminated sediments and the decline of volatilization from the surface water due to the cold water temperature and ice cover. The level of PCBs in the water column reaches the yearly maximum in spring then declines during the next few months. But many other factors, such as sunlight, snowmelt, growth of microorganisms, types of soils (Hankin and Sawhney, 1984) and rate of atmospheric deposition may also affect the photolysis and biodegradation of PCBs. Tissue Sampled Various studies (Zabik et al., 1982; Greig and Sennefelder, 1987; Sanders and Haynes, 1988; Gundersen and Pearson, 1992), found that total PCB concentration is the function of lipid content in tissue samples. Total levels of PCBs increased with the increase of the lipid values. Gundersen and Pearson (1992) stated that in sturgeon both the 24 mean percentage of lipid content and the PCB concentration in reproductive tissues were more than two times the values in muscle tissues. Greig and Sennefelder (1987) reported that levels of PCBs (0.30—2.70 ppm, wet weight basis) in livers of winter flounder were significantly higher than the levels of PCBs (0.03-0.43 ppm) in ovaries of winter flounder at the average fish length of 32 cm. Sanders and Haynes (1988) observed that red muscle tissue stores more lipid than white muscle tissue in bluefish fillets. They found that skin-on (untrimmed) fish fillets had.38% more lipid content than skin- off (trimmed) fish fillets according to the modified trimming fish method of Skea et al. (1979). Smith et al. (1973) stated that average fat content from anterior halves of raw (3.0%) and cooked (4.2%) fillets of chinook salmon were higher than posterior halves of raw (2.4%) and cooked (3.1%) fillets. 'The same trend was reported by Sanders and Haynes (1988) that the rib cage and the belly flap in the trunk of the fish had the highest (average 13.0%) lipid content in skin-off bluefish fillets compared to the lipid content of the caudal zone in the posterior of the fish (5.0%). Recent study by Zabik et al. (1992) found that total PCBs level in raw body muscle of blue crabs harvested from U.S. east coast with or without tissue of hepatopancreas was 0.31 or 0.27 ppm (wet wt. basis), respectivelyu The average total PCBs in raw claw'of blue crab was only 0.18 ppm. 25 Analytic Procedures and Methodology for PCBs The validity of analytical data strictly relies upon the chemical analytic procedures and methodology which are the crucial factors toward the determination and quantitation of congeners specific PCBs. Price et al. (1986) modified the methodology for cleanup of extracts contaminated by chlorinated. pesticides in fish adipose tissue from acetonitrile liquid—liquid partitioning to Florisil® chromatography and silica gel column chromatography in order to obtain additional recoveries of the 6% Florisil® fraction and separation of PCBs from toxaphene and chlordane which often coelute on most packed.GC columns. Ribick et al. (1982) studied the differences between packed column gas chromatography-mass spectrometry' (GC-MS) with. well—coated open-tubular (WCOT) capillary column GC. They found that capillary column GC yielded accurate and more precise values for the spiked catfish samples than packed-column GC techniques due to inadequate resolution to separate specific chemical compounds from interferences in the chromatogram. Electron capture detector is selective toward halogenated compounds according to the solubility of individual PCB congener in the liquid. phase and its volatility; The interaction of the compounds with the gas (mobile phase) and liquid phase (stationary phase) in the separation of individual specific congener of PCBs affects the retention time of the specific PCB congener. Rentention time determines individual congeners identification. Williams et al.(1992) 26 reported that total PCB values from packed column techniques is 16% less than capillary column values obtained in their study. Maack and Sonzogni (1988) found that a linearly correlation.coefficient.of 0.9854 between.the concentration.of total PCBs obtained from capillary column versus packed column analysis for fish from Lake Michigan and the Wisconsin.River. Capel et a1. (1985) investigated that the application of data generated from capillary GC of AroclorQD standards into a multiple-linear regression analysis to calculate the total PCB concentration in environmental samples. These authors found that calculated PCB values were in accordance with the sum of the individual congener concentrations. More recently, scientists (Hong' and. Bush, 1990; Hong' et al., 1992a,b; Williams, 1993) are able to determine and quantitate mono- and non-ortho coplanar PCBs in fish with the additional step of carbon chromatography on the basis of molecular planarity and degree of chlorination. besides the separation. on solute polarity through Florisilo and silica gel chromatography. In. spite of the complex: procedures, the analytical identification of congener specific PCBs is simply to match the relative retention time of each peak in the chromatogram of fish sample against peak in the authentic standard PCB congeners run under the same conditions. Recoveries for the specific PCB congener is determined.based on response factors and the peak area of the quantitation standard. In the past, the results of the total concentrations of PCBs in raw and cooked fish fillets (Smith et al., 1973; Zabik 27 et al., 1978 and 1982) were based upon ppm lipid content. In early studies, scientists (Skea et al., 1979; Zabik et al., 1982) stated, the extractable lipid content in cooked fish fillets is higher than extractable content in raw fish fillets, especially for deepfat fried fish fillets which have the higher fat adsorption on the tissue and greater moisture loss in the tissue. One mechanism proposed for this phenomena is cooking causes an increase in the amount of ether extractable material in the lean portion of meat over that found in raw meat, since sulfhydryl-disulfide interchange during heat aggregation of myofibrillar proteins allows for release of phospholipid bound to protein in the raw muscle thus increasing the proportion of phospholipid in lipid extracts from cooked meat (Zabik et al., 1982) which leads to the better fat extraction from cooked fish fillets. In recent publications ( Voiland, et al., 1991; Zabik et al., 1992; Zabik et al., 1993), total concentrations of PCBs or of PCB homologs in raw and cooked fish fillets were expressed as ppm in wet tissue, ppm in dry weight tissue and/or the total 'micrograms in the fish samples. The comparison between cooked fillet to paired raw fillet in total 'micrograms of PCBs determined percentage change in fillet due to the effect of trimming, skin removal or cooking (Zabik et al., 1993). 28 Effects of Processing and Cooking on the Reduction of PCBs Levels in Raw and Cooked Fish Fillets Many studies have proved the effect of processing and cooking on the reduction of PCBs congeners levels in raw and cooked fish fillets or marine products. The principle of the effect.of processing on the reduction.of PCBs concentration in fish fillets is to minimize the PCBs residues in the uncooked edible tissue through. trimming' processings *which. include removal of head and.tail portions, skin removal plus trimming off belly fat and all adipose tissues. Afterward the process of cooking which applies heat in various forms for selected times on trimmed fish fillet enhances the reduction of PCBs levels through fat leaching, protein denaturization, evaporation of moisture or PCBs (Armbruster, et al. , 1989) and volatization of volatile compounds, etc. So far, the removal of skin and associated fat combined with the selected cooking method prior to consumption of fish has been highly recommended in the fish consumption advisories to the sports fisherman.in.compliance with food.safety issues for the public health (Sherer and Price, 1993; Song, 1994; Zabik et al., 1995) in addition to avoiding consuming larger and older fish (Voiland et al., 1991). Effect of Trimming Procedures PCB mixture is non-polar compound, is mostly lipid- soluble. Concentrations of PCB residues were the highest in parts of fish with the highest lipid content, such as dorsal, ventral, medial and belly flap areas. To minimize ingestion 29 of fat-contaminants in high fat tissues, trimming is the most effective process to reduce the health risk of anglers and their families. In the early study done by Zabik et al., (1978) found that head sections of freshwater mullet from upper Great Lakes (Lakes Huron, Michigan.and.Superior) had the highest PCB levels as compared to other portions of the whole mullet. Hora (1981) concluded that the effectiveness of removal of the skin alone resulted in 26% to 30% of PCBs and lipids losses respectively in carp fillets from upper Mississippi river. Skea et al. (1979) stated that removal of the skin, dorsal and ventral fat, and the entire lateral line from.Lake Ontario smallmouth bass and brown trout resulted in 64% and 43% reduction of Aroclor" 1254 in fish fillets, respectively. A similar investigation by Sanders and Haynes (1988) showed.that total PCB level in bluefish fillets reduced 27%' after the removal of belly flap adipose tissues which was close to the 28 percent reduction.of lipid” .Armbruster et al. (1989) found that trimming bluefish fillets resulted in an average reduction of PCB residues of 59%; They also indicated that concentration of PCBs in skin contained about twice that found in the fillet muscle expressed on a ppm wet weight basis. Voiland et al. (1991) reported that percent loss of total PCBs and fat content in brown trout from Lake Ontario through skinning and fat trimming procedures was 46% and 62%, respectively. It may be said that more than 1/4 to 1/2 of total PCBs concentration found in the raw fish fillet is feasible to be eliminated through recommended trimming 30 procedures. Voiland et al. (1991) also confirmed that the effectiveness of the fat trimming procedure on the reduction of PCBs in fish fillet is consistent despite wide variation in the initial (untrimmed fillet) levels of contamination. Song (1993) reported that skin removal in carp fillets from the Great Lakes reduced 35% of total PCBs concentration which was from 1.90 ppm down to 1.24 ppm (wet wt.), the highest reduction.percent was for hexa-CBs homolog (42%), followed by hepta-CBs (38%), penta-CBs (34%), the least reduction was for octa-CBs. 'The presence or absence of skin and adipose tissues significantly affected the total PCBs and its homologs in the raw fish fillets. Effect of Cooking Methods The effectiveness of cooking on PCB reduction in fish has differed substantially in various reports. Smith et al. (1973) found that a small decrease in the PCB levels in Lake Michigan chinook and coho salmon occurred through baking and poaching. Statistical comparison showed no consistent pattern for PCBs residue removal due to cooking. Fish fillets of salmon baked in nylon.bags lost 11-16% of PCBs, while samples baking or poaching were reduced by only 2-8%. In was noted that posterior halves of fish fillets of chinook salmon lost more PCBs through cooking procedure than anterior halves, possibly due to the greater leaching of fat during cooking. Skea et al. (1979) tested smoking, broiling, and baking methods in relation to the reduction of total PCBs in Lake Ontario brown trout and smallmouth bass. They found that 31 there were zero reductions of PCBs in untrimmed brown trout and smallmouth bass during baking or broiling fish fillets, 12% reduction of PCBs during smoking untrimmed brown trout fillets. Zabik and coworkers (1979) found that broiling fat lake trout (siscowets) reduced PCBs by 53% while roasting or cooking by microwave resulted in losses ranging from 34 to 26%, respectivelyn However, there ‘were not significant differences in these values due to cooking method. In contrast, Cin and Kroger (1982) reported that baking, frying, poaching, and baking without skin in brown trout did not cause significant decreases of insecticide mirex. Similar study by Zabik et al. (1982) showed that various cooking methods (poaching, roasting, deepfat frying, charbroiling and microwaving) did not significantly affect the level of PCBs in carp. Song (1994) indicated that there were no significant differences between deepfat frying and pan frying carp fillets in relation to total PCBs reduction, but both methods reduced PCBs by an average of 34%. Armbruster et al. (1989) found that there was an average 7.5% reduction from. baking, broiling, frying or poaching on trimmed bluefish fillets. They suggested that vaporization of PCBs during the various cooking procedures contributes the major portion of the total loss from cooking. Stachiw et al. (1988) pointed that fat rendering and moisture evaporation.during cooking contributed to the majors factors in xenobiotic reduction in fish. They also found that increasing the end point cooking temperature and. surface area. of fillets statistically increased. the 32 percentage of TCDD loss in roasted and charbroiled restructured carp fillets” It seems that the effectiveness of reducing PCBs from fish during cooking depends largely on the species, its fat content, end point cooking temperature or surface area and depends less on the specific cooking method used. Effect of Trimming and the Selected Cooking Method Many studies have indicated the effects of trimming and the selected cooking method in the reduction of DDT and PCBs level (Reinert et al., 1971; Skea, et al., 1979; Zabik, et al., 1982; Armbruster et al., 1987 and 1989; Zabik, et al., 1992; Sherer and Price, 1993; Song, 1994). Zabik et al. (1979) reported that removal of skin from fat lake trout combined with roasting enhanced reduction by an additional 10% total PCBs loss. .Armbruster et al. (1989) found that the mean percentage reduction in PCB levels in both trimmed and cooked bluefish fillets was 66.9%. The orders of cooking methods in terms of PCBs reduction percent.were as follows: broiled, 71%; baked, 68%; fried, 68% and.poached, 60%. The combined effect of trimming and cooking methods resulted in 7.5% further reduction in bluefish fillets. Puffer and Gossett (1983) confirmed that the combined effect of skin removal and pan- fried in white croaker from southern California on PCBs reduction ranged from 28% to 65%. A current study (Zabik et al., 1995) showed.walleye and white bass from.the Great Lakes obtained further reduction of 1/4 to 1/3 of PCBs level during baking, charbroiling, and deepfat frying skin-on fillet after 33 trimming belly flap. Based upon the statement from.the Great Lakes sport fish consumption advisory (GLSFATF, 1993), a contaminant reduction factor of 50% due to trimming and cooking is a realistic expectation for all the lipophilic contaminants of concern in the Great Lakes. Skin removal prior to cooking’ appears preferable; however, the further reduction of the contaminant can also be compensated by simply discarding the skin after cooking (GLSFATF, 1993). MATERIAL AND METHODS Fish Procurement Carp (Cyprinus carpio) , chinook salmon (Oncorhyncush tshawytscha) , lake trout (lean) (Salvelinus namaycush namaycush) , siscowet (Salvelinus namaycush siscowet) , walleye (Stizotedium vi treum vi treum) and white bass (Morons chrysops) were chosen to be representative of the mean Creel census data from sports fisherman for 1990 for all fish except siscowets (Rakoczy, 1992) . Siscowet size was based on average catch data of Native American fisheries. A total of thirty carp, seventy-one chinook salmon, seventy-one lake trout, thirty- five siscowet, thirty-nine walleye and sixteen white bass were collected on designated days and locations by the Michigan Department of Natural Resources (MDNR) , New York Department of Natural Resources and private companies. Table 5 summarized the information of five fish species harvested from Great Lakes. All fish were assigned non-duplicated random numbers and weighed (grams), measured (centimeters) and sex identified after the catch. In order to maintain the fish in food grade condition, fish were deheaded and detutted (scrapin kindneys and viscera from the abdominal wall) within eighteen hours of collection. Flake ice was packed into the body cavity of fish 34 35 Table 5. Summary of the information on fish species, source of lake (# fish), location of catch and date of catch Species Lakes Locations Date (# fish) Carp IErie (15) 41°51.5N,83°20.1W 4-22-91 (near Monroe, MI) Huron(lS) Saginaw, MI 7-22-91 Chinook Huron(35) SwanRiverWeir, MI 9-12—91 salmon Michigan(ll) Manistee Weir, MI 9-12-91 Michigan(25) Consumers Power, 4-19-91 Ludington & to grid 1509 off 7-19-91 Pentwater, MI Lake trout Huron(8) South Point, MI 6-04-91 Ontario(8) Cape Vincent, NY 9-04-91 Michigan(20) Pentwater, MI 4-17/19-91 grid 1409,1509 & 5-14/15-91 Siscowet Superior (35) 46°41 . 6N, 87°19 . 2W 6-19—91 (near Marquette, MI) Walleye Erie(12) 41°51.5N,83°20.1W 4—22-91 (near Monroe, MI) Huron(ll) Saginaw Bay, MI 7-25-91 Michigan(16) north end of 4-12-91 Little Bay de Noc, MI White bass Erie(8) 41°51.5N,83°20.1W 4-22-91 (near Monroe, MI) Huron(8) Saginaw Bay, MI 7-22-91 or L- Side 36 and the fish were placed in ice in an Igloo cooler for transport to Michigan State University Meat Laboratory. Processing of Fish Fillets Fish were processed within 24 hours of receipt at the Meat Laboratory. According to experimental design, carp, chinook salmon, and.lake trout were processed into skin-on.and skinfoff fillets. Walleye and white bass were processed into skin-on fillets. Skin-on fillets had the belly flap trimmed off, while skin-off fillets had the belly flap as well as dark tissue from. the lateral line and. associated fat tissue removed” Using red. meat techniques, the fish. will be identified as to right and left side, i.e., a.person with left hand at the head and right hand at the tail of the fish (belly down) will reckon the left side as the side visible or facing the individual. Left side of fish would be cooked and right side would be used in a raw state to compare the PCBs level between the raw and the cooked fish” Each side of fish fillet of the species of carp, lake trout, chinook salmon, and walleye were processed into head and tail pieces. White bass and siscowet were used as whole fillet due to the small size of fish fillet. All processed fish fillets were wrapped in aluminum foil, labeled with assigned random numbers, and vacuum packaged. Labels were placed.both in the interior and on the outside of the package. The packages were blast frozen at -34PC for further use. The fish scales were also placed in prelabeled.plastic bags and.used for the determination of the 37 age of the fish. Processing data included sex, age, length, weight and percentage carcass yield and percentage AP (As Prepared) yield. Carcass yield was based on the deheaded and degutted weight of each fish species to the whole fish weight as well as AP yield was the ratio of total weight of both sides of trimmed fillet to the total fish weight. AP yield would be similar to an "As Purchased" yield for commercial fish. Sample Preparation Preparation of Raw Sample The frozen fillet of right side (skin-on or skin-off) was coarsely chopped by a hammer. The broken pieces of fish tissue were crushed with dry-ice in a high speed Tekmar analytical mill and pulverized for 2 minutes. The powdered sample was mixed thoroughly and placed in glass containers, covered with aluminum foil, labeled and capped. All samples were stored at —30°C. Preparation of Cooked Sample Commercial no stick cooking spray PamS was used on fish samples to reduce the surface loss and the variation. A thermocouple was inserted into the center of the thickest portion of the fish fillets for samples cooked by baking, charbroiling, pan frying, deepfat frying, and salt boiling was applied to ensure the internal temperature reached 80°C. A thermocouple was also used to monitor the temperature of the heating medium. All equipment which was in contact with fish 38 was washed with soap and water and then rinsed with acetone. Before cooking and after cooking, the weights of fish sample (skin-on and skin-off) were recorded to be able to calculate the total cooking loss and cooking yield. When a cooked skin-on sample was prepared, the skin was peeled off so only the cooked muscle tissue weight was recorded as the edible weight. This muscle tissue was used as the edible portion for chemical analyses for all the skin-on fillets for all cooking methods except deepfat frying to maximize residue reduction. The logic for treating skin-on deep fat fried fillet differently was that the public consumers would generally eat a deepfat fried fish fillet with a batter or breading coating and thus always consume the skin as well as muscle tissue. The calculation of percentage total cooking loss was the ratio of the difference between raw and cooked fillet weight to the raw fish fillet weight times 100. Cooking yield percent was based on the relation of the cooked edible weight to the raw fillet weight times 100. After cooked fish fillet cooled on the wire cooling rack, cooked fish fillet or edible portion of fillet was placed into an Omnimixer to be homogenized. The homogenization consisted of mixing on low speed initially and then gradually increasing the speed until the desired fineness of the sample had been reached. The ground sample which had a paste-like consistency was mixed thoroughly and then placed into 3-4 separate glass jars (prerinsed with acetone and hexane) which were covered with aluminum foil, labeled on the cap and sealed for moisture 39 determination, PCB analyses, fat analyses and the use of Department of Michigan Public Health, respectively. All samples were frozen and stored at -34°C. Cooking Methods for Fish Fillets Lake trout, chinook salmon, siscowet and walleye fillets were baked and charbroiled according to the procedure described by Stachiw et al (1988). Carp and white bass were pan_fried as outlined in Puffer and Gossett (1983). Carp and walleye fillets were deep fat fried following the procedure of Morehouse and Zabik (1989). Lake trout and siscowet fillets were smoked as outlined in the Michigan State University Cooperative Extension Bulletin E-1180, entitled "Processing Great Lakes Chub (Leucichtys hoyi)" by Bratzler and Robinson (1967) . Canning chinook salmon fillets following standard USDA procedures (1988) were used. Table 6 summarized the cooking methods of skin-on or skin-off fillets in five species harvested from Great Lakes. BABES Each fillet was removed from the freezer shortly before the oven was up to 177°C. Then, the sprayed fillet was placed on the broiler rack which was in the pan. Each fillet was cooked until the internal temperature of 80°C was reached. W Each fillet was prepared as above and was placed in the preheated charbroiler at 250°C. Each fillet was turned after the half-way temperature (40°C) was reached, cooked until the fish fillet reached 80°C. W The oil (Mikado, commercial soybean oil) was placed in the frier and heated to 180-195° C. The fillet 4O Table 6. Summary of processing and cooking methods of fish fillets in five species harvested from Great Lakes Species Lakes Process Cooking Number Method Carp Erie skin-on & -off deepfat frying 12 Huron skin-on & -off deepfat frying 12 Erie skin-on & -off pan frying 12 Huron skin—on & -off pan frying 12 Chinook salmon Huron skin-on & -off baking 12 Huron skin-on & -off charbroiling 12 Huron skin-on & -off charbroiling 12 (increased surface) Michigan skin-on & -off baking 12 Michigan skin-on & -off charbroiling 12 Michigan skin-on & -off charbroiling 12 (increased surface) Huron skin-off canning 6 Michigan skin-off canning 6 Lake trout Huron skin-off baking 6 Huron skin-off charbroiling 6 Michigan skin-off baking 6 Michigan skin-off charbroiling 6 Michigan skin-off salt boiling 6 Michigan skin-on smoking 6 Ontario skin-off baking 6 Ontario skin-off charbroiling 6 Siscowet Superior skin-off baking 6 Superior skin-off charbroiling 6 Superior skin-off salt boiling 6 Superior skin-on smoking 6 lulleye Erie skin-on baking 6 Erie skin-on charbroiling 6 Huron skin-on baking 6 Huron skin-on charbroiling 6 Michigan skin-on baking 6 Michigan skin-on charbroiling 6 Michigan skin—on deepfat frying 6 lhite base Erie skin-on pan frying 6 Huron skin-on pan frying 6 41 was placed in the preheated frier and frying temperature was kept at 18015°C. Each fillet was cooked until the internal temperature reached 80¢3°C; afterwhich it was drained and cooled in deepfat frying basket for five minutes. P n 'n Each fillet was placed in the pregreased frying pan with PAMG’ and preheated pan at 18515°C. Each fillet was turned when the internal temperature had increased 20°C and was cooked until the internal temperature reached 80°C. For the skin-on fillets, the skin side was cooked first for heat to penetrate the fillet because skin acts as an insulator to keep the temperature from increasing. W The frozen fish fillet was placed in basket and submerged into a 5% NaCl boiling liquid (1.5 inches above fish). The liquid was kept at a gentle boil (99°C) during cooking. Fish fillet reached an internal temperature of 80°C before removal. Smoking Skin-on fish fillets were thawed in a cooler(4-5°C) for 24-36 hours prior to brining. The fillets were brined in a 30° salimeter brine containing 7.89% salt, 92.11% water for 14 hours at 4°C (Cuppett et al., 1989). The ratio of fish fillet weight to brine volume was 1:2. Afterward, the fillet was rinsed in cold running water and placed on cooking racks coated with lecithin to minimize sticking. Using hickory sawdust for wood smoking, smoke-cooking was accomplished in a stainless steel smokehouse, until fish reached an internal temperature of 80°C for 30 minutes (Bratzler and Robinson, 1967) . 42 W The skin-on fillet was placed in pint size glass canning jar covered with distilled water leaving one inch head space. After the jars were sealed in an appropriate manner, they were placed into the pressure cooker and processed for 100 minutes at 11 lb psi. Analysis of Solids Solids were determined using AOAC method 24.002 (AOAC, 1984) oven drying method in order to express PCB congeners data on dry weight as well as wet weight basis. Lipid Analysis The determination of lipid content followed the procedures modified by Price et a1 (1986). Fish homogenate (209) was thoroughly mixed with 80 g anhydrous Na2S0, in a 250 mu beaker until the sample was dry, afterwhich the dry mixture was lightly packed into a 400 mm x 19 mm chromatography column. The beaker then was rinsed with 10 ml of 50% ethyl ether/petroleum ether(v/v) and the rinse quantitatively transferred to column, followed by remaining 190 ml of extracting solvent at an adjusted flow rate of 3-5 ud/min. The extract was collected in a tared 250 ml beaker and evaporated to dryness on top of a moderately heated water bath under a gentle stream of nitrogen to determine the lipid weight. 43 Congener Specific Polychlorinated Biphenyl Analyses Glassware Preparation All glassware used in the residue analyses (Erlenmeyer flasks, reservoir columns, Turbo-Vap evaporator tubes, chromatographic columns and 1 ml and 5 ml volumetric flasks, etc.) were washed with detergent, rinsed with hot tap water, and distilled water, then with acetone, followed by hexane. The cleaned glassware were dried in an oven at 110%: overnight. Solvents and Reagent Preparation Solvents: All solvents were pesticide quality. Acetone - 99.8%, Mallinckrodt Specialty Chemicals Co. (Paris, KY) Dichloromethane, Isooctane, and Toluene - 99.9%, Mallinckrodt Specialty Chemicals Co.(Paris, KY) Hexane - 85.0%, EM Science Diethyl ether - 99.9%, Baxter Burdick & Jackson Laboratories, Inc. (Muskegon, MI) Petroleum ether - 99.9% EM science Solvent mixture: Prepared by volume. 50% Hexane : 50% Dichloromethane 6% Diethyl ether in Petroleum ether 0.5% Toluene in Hexane Reference PCB internal standards (Table 7): 99% pure From.AccuStandard, New Haven, CT. 44 Table 7. IUPAC numbers and structure of the PCB congeners Sagas! :nnmc tuber Psgsuune tnses 31 245 1ene4es m2 223A’ 44 1235' 47 ZTAA' 49 zzAs' 52 2255' 55 113A 66 zaAA' 7o zsuns 72 2,3',S,5’ 76 zsus 79 asAs' FDNMMI: 83 ZTJJTS 84 223315 85 (96%) 2.23.4.4 87 zzsAs' 91 223A}; 92 2,2’.3.5,S' 95 221n45 97 2.233245 99 zzAAui 101 zzuss' 'ms zzAsus 105 2.3.3'.4.4' NB zasus' 110 2.3.3.426 114 2.3.4.435 118 2,334,435 'no er55' 121 ZFASWB 122 :nasus 123 2.3.4.435 Men-CI! 128 2.2'.3.3’.4.4' ‘32 zzssuur ‘86 zzssuur 137 2.2'.3.4,4',5 138 2,233,443? 141 2,2'.3,4.5.5' 149 2,2'.3,4'.5'.6 1 53 292.740a.0575. ‘66 ZLFAAHS ‘67 liyflflfi? Hm Zlyflflfli 167 zsuuuas We 171 2.2'.3,3',4.4'.6 179 (95%) 2.2'.3.3'.5.6.6' 180 2233.44.55 181 2.2'.3.4.4'.5.6 183 2.2'.3.4.4'.5'.6 185 2,2',3,4,5.5',6 190 2,3.3',4,d'.5,6 Cumin! 1MB zzumnmsss 200 2.2'.3.3'.4.5'.5.5' 45 Chemicals: All chemicals were pesticide grade. Sodium sulfate - granular anhydrous, activated and stored at 130°C (J.T. Baker Chemical Co..Phillipsburg, NJ). Florisil” - 60~80 mesh. activated at 130°C Fisher Scientific, Fair Lawn. NJ). Silica gel 60 - 70~230 mesh. activated at 130°C (Sigma Chemical Co.. St. Louis, MO). Extraction and cleanup of samples for PCB congener specific analyses were performed using the column extraction with Dichloromethane (MeClz) , Gel Permeation Chromatography (GPC). Florisil” and ‘ silica gel chromatography. Identification and quantification of specific PCB congeners were done by capillary column gas chromatography according to the modification of Ribick et a1 (1982)(Figure 2). An internal standard addition. 0.375 ug of congener 2.4.6 trichlorobiphenyl (IUPAC # 30) was added to be able to correct .for losses during the entire extraction procedure. This PCB congener was selected as an internal standard because it does not occur in commercial Aroclor" mixtures nor has it been detected in environmental samples (Williams, 1989) . The advantages of using column extraction are time efficiency. application of single solvent, equipment replaceable and multiple samples performance. Lipid Extraction Each sample of fish (10.0 g) and 1 ml of internal standard PCB congener #30 (concentration Sppm) was homogenized 46 Homogenated fish tissue sample (109) added internal standard (congener #30) u Spiked fish sample 3 Dry with Nazso. Extracted with MeCl2 fl Lipids and xenobiotics concentrate fl Gel permeation chromatography (GPC) fl PCBs and pesticides concentrate fl Florisil® chromatography u PCBs and some pesticides concentrate fl Silica gel chromatography u PCBs 3 Gas chromatography Equipped with “Ni BCD fl Identification & quantification of individual congener specific PCBs Figure 2. Simplified chemical analytical procedures for PCBs congener specific from fish tissue 47 in a mortar and ground to a fine powder with 40 g of granular anhydrous sodium sulfate (Na,SO,) which had been activated and stored overnight at 130°C in order to remove water from the sample. The ground dry fish mixture was eluted in a 1 cm i.d. reservoir column with 200 ml of MeCl2 mobile phase 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. Cleanup of Lipid Extract W The concentrated lipid extract was then diluted with 1:1 (v/v) hexane and MeCl2 mixture into 5 ml volumetric flask for the cleanup by GPC. Four ml concentrated extract was pipetted quantitatively into GPC vial. Afterwhich, 2 m1 aliquot was automatically injected into GPC column (19 mm i.d. x 300 mm Ultrastyragel 500 A resin) attached to a Waters/590 Programmable HPLC pump and Waters fraction collector (Millipore, Co.. Milford, MA). The mobil phase MeCl2 was pimped through the column at 3 ml/min. The automated GPC system provided for unattended operation with time control (35 min/sample) to separate lipids and PCBs fraction. The lipid was discarded and the fraction which contained congener specific PCB and pesticides was collected and reduced as above to 1 ml afterwhich the volume was adjusted to 5 ml with hexane. 21W The Florisil” glass chromatography column (1 cm i.d. x 51 cm) was packed as 1 g Na,SO,, 5 g of Florisil” (activated at 130°C for 16 hours), 1 48 g Na,SO, and solvent-extracted glass wool in reservoir colum. The prepared column was rinsed with 20 ml of hexane before the extract was applied. When the hexane reached the top of the upper layer of Na,SO,, the GPC concentrated extract was pipetted into the column and onto a Florisil‘!’ bed and was collected into a Turbo-Vap flask. Forty ml elution solvent of 6% (v/v) diethyl ether in petroleum ether was portioned into 5 and 35 ml. The first 5 mL of eluent was used to rinse the column walls before the remaining volume (35 ml) was added to the reservoir column. In this step. 6% (v/v) diethyl ether in petroleum ether was used as mobile phase and the Florisil‘” column as the stationary phase. Most of chlorinated pesticides and PCBs (nonpolar pesticides) were eluted. The collected fraction was reduced to 5 ml volume in the Turbo-Vap evaporator. W The preparation of silica gel column was the same as the Florisil" column only replacing Florisil" with silica gel 60 (70~230 mesh, activated at 130°C for 16 hours) . After the column was rinsed with 20 ml hexane, the collected concentrate was pipetted onto the column. Fifty ml of 0.5% toluene in hexane served as mobile phase with silica gel as the stationary phase to separate PCBs and other non-polar or less polar pesticides. The eluent was reduced to 0.5 ml volume in the Turbo-Vap evaporator and pipetted to the 1 ml volumetric flask, using hexane as a rinsing solvent. The concentrate was reduced to 0.5 ml volume, under a gentle stream of N, gas and isooctane was added to make a final 49 volume of 1 ml. Identification and Quantitation Individual PCB congeners in the PCB concentrate were separated and quantitated by gas chromatography (Hewlett Packard Model 5890 Series II) equipped with 63Ni electron capture detector (ECD), and a DB-5 capillary column (60 m x 0.25 mm i.d.). The detector was operated at 300°C and a split/splitless injector at ZZUTL. Aliquots of 3 ul volume of PCB extract were injected by an autosampler. Helium at 20.0 psi, flow rate of 1 ml/min was used as the carrier gas. The column temperature was temperature programmed from 160%: at 8°C/min until 200°C and then held for 5 min after which the temperature was programmed to 280°C at 2°C/min and then held a final 5 minutes. The complete run time was 55 min. The injection of standard was required prior to the analyses of every three pairs of samples (cooked and raw fish samples). Each standard congener was injected separately to determine retention times for peak identification, then all congeners were combined into one standard and injected at three levels of concentration (standard/50, standard/10, standard concentration) in. order t0> calculate the slope of each congener standard curve. These linear regressions have R’ values of .99 and higher. The integration was performed by the Hewlett Packard software. The stored data was transferred from.the Hewlett Packard software to an Excel spreadsheet (Microsoft windows 3.1) and corrected retention times were calculated. based on the so Wh. the C01} if: "ere 50 retention time of the internal standard, congener 30. All quantitation was based on peak areas relative to the individual congener standards and the area corrected to the internal standard congener 30. The coeluting'pnpy-DDE peak was omitted from all calculations. The individual congener was quantified by comparison of peak area with appropriate standards of known concentration with following equations. Recovery % = Qetegted gong, of 30 in ex; action 3 199 Conc. of internal std. 30 in fish sample Concentration (ppm in wet tissue) = W LS of 30 x RA of 30 LS : Line Slope RA : Retention Area The results were expressed as ppm in wet tissue, ppm solids as well as the micrograms in the raw and cooked samples which derived fromnweights of raw or cooked fish fillets times the ppm on a wet weight basis. The percentage change was calculated by the difference between the micrograms of each congener in the raw and cooked fish fillets. Positive values are percentage reductions. values for PCB congeners which were below the reported detection limit, i.e. non-detectable 51 (ND), are not included in the average or standard deviations. If the cooked sample had a ND level and the raw sample had a numerical value, the percent loss was arbitrarily set at 100%. In contrast, if the cooked sample had a higher numerical value than the raw sample, the normal equation was used to calculate the negative loss. ‘Thus, any negative loss was not limited to 100%. The homolog of PCBs was determined by summing the concentration of the same chlorination group of PCB isomers. The total concentration of PCBs was determined by summing the concentration of the homologs of PCB isomers. Statistical Analysis All statistical analyses were performed with the Statistical Analysis System, version 6.04 for Personal Computers (SAS Institute, 1987) or SYSTAT for Windows 5.03 (SYSTAT, 1993). The level of significance of main effects that was used in ANOVA was p < 0.2 - 0.05 for the specific fish specie harvested at the same location. T-test was selected for the comparison with fillets processed from the same fish. Information on correlations among variables, including Pearson product moments and p values, was obtained using the CORR procedure in SYSTAT. The REG procedure was used to determine the combination of physical measurements for predicting the concentration of total PCBs. Response surface graphic representatives was also carried out by SYSTAT to visually demonstrate relationship among variables. The project was designed to test the null hypotheses: 52 1. The lakes origin and fish biological variability do not influence the levels of total PCBs and its congeners. 2. The processing and cooking procedures do not reduce the levels of total PCBs and its congeners and homologs. 3. There is no difference among cooking methods in the reduction of the levels of total PCBs and its homologs. The expressions of PCB levels in wet tissue, ppm solids as well as the micrograms per fish fillets were applied.in.all the statistical analyses and modellings; however, only the optimum result from those three expressions was stated here. Some significant information will be listed in appendix. Sou I'EE dew. ha: vi: Wei 134 40. 89C RESULTS AND DISCUSSION Procurement Data for the Great Lakes Fish Species Source and Size of Fish Data The source and size of fish were chosen based on the Creel Census data from sports fisherman for 1990 so the fish would be representative of the most commonly caught by sport fishermen. Raw data of physical and chemical parameters of fish species were recorded in appendix 1 - 5. Some of the results quoted.here were also reported.in.detail with standard deviation by Zabik et al. (1993). The mean length of carp harvested from Lake Erie were measured as 51.8 centimeter(cm) with a range in length from 45.7 to 56.5 cm and the mean weight of carp was 1834.2 gram (9) with a range in weight from 1340.0 to 2520.0 9. Lake Huron carp ranged in length from 40.6 to 55.9 with.a mean length of 46.6 cm, and in weight from 890.0 to 2710.0 g with a mean weight of 1581.7 g. Chinook salmon harvested from Lake Huron were measured from 70.0 to 91.5 cm with a mean length of 80.8 cm, and from 3860.0 to 7700.0 g with a mean weight of 5689.7 9. Lake Michigan chinook salmon.were measured as 76.0 cmmwith a range in length from 67.0 to 91.5 cm, and weighed a mean of 4798.5 9 with a range in weight from 2720.0 to 8415.0 g. Lake Huron lake trout ranged in length from 59.5 to 66.5 53 54 cm with a mean length of 63.7 cm, and in weight from 2000.0 to 3150.0 g with a mean weight of 2666.7 gx Lake trout harvested from Lake Michigan were measured in length from 54.2 to 71.5 cm (mean 63.6 cm), and in weight from 1460.0 to 3620.0 g (mean 2677.1 9). Lake Ontario lake trout were measured with a mean length of 64.7 cm (ranging from 62.4 to 66.0 cm), and a mean weight of 2756.7 g (ranging from 2365.0 to 2985.0 9). Lake Superior lake trout (siscowet) ranged in length from 49.0 to 56.0 cm with a mean length of 52.8 cm, and ranged in weight from 1078.0 to 1492.0 g with a mean of 1271.6 9. Walleye from Lake Erie were measured in length from.41.9 to 48.3 cm with a mean length of 46.1 cm. and in weight from 750.0 to 1010.0 g with a mean weight of 896.7 g. Lake Huron walleye ranged in length from 48.3 to 50.8 cm with a mean of 48.6 mm, and in weight from 940.0 to 1230.0 g with a mean of 1064.2 g. Walleye from Lake Michigan had a mean length of 46.5 cm (ranging from 41.3 to 49.4 cm) and a mean weight of 775.0 g (ranging from 620.0 to 865.0 g). White bass from Lake Erie averaged 31.0 cm in length with a range from 26.7 to 34.3 cm, and averaged 676.7 g in weight with a range from.290 to 1140 g. Lake Huron.white bass ranged in length from 27.9 to 34.3 cm.with a mean of 31.5 cm, and in weight from 270.0 to 520.0 g with a mean of 381.7 g. Based upon the average and range data in length and in weight of the Great Lakes fish species, chinook salmon from Lake Huron and Lake Michigan.possessed the longest length and the heaviest weight, followed by lake trout, carp, walleye. 55 White bass was the smallest fish specie used in the project. Because the specified lengths and.weights of the Lake Superior siscowets were chosen to be representative of Native American fisheries, these were significantly smaller than those of the lake trout from Lakes Huron, Michigan and Ontario (Table 8). Age and Sex of Fish Data The effect of sex on contaminant level was also under examination.in.this experiment» The actual data on sex of the Great Lakes fish species was listed in Table 9. Half of the Lake Erie and Lake Huron carp were male. Sixty-seven percent and fifty-seven.percent of chinook salmon from.Lake Huron and Lake Michigan were male, respectively. Half of the lake trout from Lake Huron and Lake Superior were male. Sex was not recorded for the Lake Ontario lake trout. Eighty-three percent of Lake Michigan lake trout were female. All of the walleye from Lake Erie and Lake Michigan were male, and eighty-four percent of Lake Huron walleye were male. All of the Lake Erie white bass were male, while all of the Lake Huron white bass were female. Table 9 also presented the average age and the range of the ages for fish species from the Great Lakes used in this study. Lake Superior siscowets were the oldest fish with an average age of 9.2 years with a range of 8-11 years, compared to a mean age of lake trout harvested from Lake Huron (6.2 years), Lake Michigan (6.4 years) and Lake Ontario (5.3 years). The mean age of the Great Lakes fish species from greatest to smallest was in the 56 Table 8. Source and size of the Great Lakes fish species Size (Mean Values) Length Weight Fish Species Lakes (cm) (gm) (No.) Carp Erie 51.8 1834.2 24 Huron 46.6 1581.7 24 Chinook Huron 80.8 5689.7 42 salmon Michigan 76.0 4798.5 42 Lake trout Huron 63.7 2666.7 12 Michigan 63.6 2677.1 24 Ontario 64.7 2756.7 12 Siscowet1 Superior ' 52.8 1271.6 23 Walleye Erie 46.1 896.7 12 Huron 48.6 1064.2 12 Michigan 46.5 775.0 17 White bass Erie 31.0 676.7 6 Huron 31.5 381.7 6 1 Siscowet is fat lake trout. Size was based on catch data of Native American and other fishermen from the Upper Peninsula of Michigan. 57 Table 9. Sex and age of the Great Lakes fish species Species Lakes Sex Age(Range) Fish (%male : %female) (No.) Carp Erie 50:50 3.5(3-5) 24 Huron 54:46 3.2(2-7) 24 Chinook Huron 67:33 3.6(3—5) 41 salmon Michigan 57:43 2.6(2-4) 29 Lake trout Huron 50:50 6.2 (6-7) 12 Michigan 17:83 6.4(5-8) 24 Ontario NA1 5.3(5-6) 12 Siscowet2 Superior 54:46 9.2(8-11) 24 Walleye Erie 100:0 5.1(3-7) 12 Huron 84:16 4.2(3—6) 12 Michigan 100:0 4.1(3-5) 18 White bass Erie 100:0 2.8 (2-4) 6 Huron 0:100 2.7(2-4) 6 1 Not Available ’ Siscowet is fat lake trout. Size was based on catch data of Native American and other fishermen from the Upper Peninsula of Michigan. 58 following order: lake trout, walleye, carp and chinook salmon. white bass. There were some significant correlations observed among age, length and weight of fish species from the Great Lakes based uponthe Pearson correlation coefficient (P<0.001) (Appendix 6). All fish species had a positive correlation coefficient between the length and weight. White bass had no significant correlations among age, length and weight. The length of walleye‘was significantly correlated with the weight of walleye,- however, the age of walleye was not strongly correlated with either walleye length or weight. The explanation of these phenomena could have been the small sample size, i.e., white bass had only twelve fish. It was found for carp, chinook salmon and some of lake trout in this project that the older the fish, the greater in length and weight they were. Since Lake Superior siscowet had the older age (8 - 11 years) with a shorter length (49.0 - 56.0 cm) and lighter weight (1078.0 - 1492.0 9) range than all other lake trout, Pearson correlation coefficient had a negative correlation between age/length and age/weight for lake trout. Processing Data of the Great Lakes Fish Species According to the experimental design, carp, chinook salmon, and lake trout were processed into skin-on and skin- off fillets. Walleye and white bass were processed into skin- on fillets. Skin-on fillets had the belly flap trimmed off, while skin-off fillets had both the belly flap trimmed off and 59 dark tissue from the lateral line and associated fat tissue removed. The right side of a fillet was used in a raw state as a control sample and the left side as cooked samples. Carcass yield (%) was based on the deheaded and degutted weight of each fish species to the whole fish weight, while the As Prepared (AP) yield (%) was the total weight of both sides of trimmed fillets to the total fish weight (Zabik et al., 1993). Summaries of processing data along with the analyses of solid and lipid contents on raw fillets for five fish species from the Great Lakes are presented in Tables 10 - 14. Regardless of the size or species of the fish, carcass yield ranged in percentage from 57% to 71% which means at least one-third portion of fish discarded at the initial processing of the fish. The further belly flap trimming of fish fillets resulted in another one-fourth to one-half reduction on carcass yield which.varied according to the skin conditions. Skin-on fish fillets had.a range of 29% to 50% AP yield with an average AP yield of 40%, and skin-off fish fillets had a range of 22% to 33% AP yield with an average AP yield of 30%. Walleye skin-on fillets had the highest yield (50%) from Lake Michigan,- carp skin-off fillets from Lake Huron had the lowest AP yield (21.7%) because carp has very thick and coarse skin tissue. 'Therefore, skin removal processing had a significant effect on the fillet weights of carp, chinook salmon and lake trout fillets ,- another ten percent of total fish weight was trimmed off from the fish 60 tissue during skin removal procedures. Carp skin-on and skin- off fillets weighed about one-third and one-fourth of the total weight of fish, respectively. Chinook salmon and lake trout skin-on fillets in AP yields were equal to two-fifth of the total fish weight, and skin-off fillets in AP yields were equivalent to less than one-third of total fish weight. The origins of lakes also had some effects on carcass yield and AP yield (Table 10-14). There were no weight differences between left-side fillets and right-side fillets of the fish. which meant that the fish fillet samples were processed in.a uniform condition facilitating comparison between the control group and the treatment group. However, the weights of either skin-on or skin-off fish fillets for carp, chinook salmon, and lake trout differed among the lakes. Fillets processed from chinook salmon had the heaviest skin-on (over 1000 g) and skin-off fillets weights (approximate 760 g), and fillets processed from white bass had the lowest skin—on fillets weights (70 g) which were due mainly to the original small size of fish. Analyses of Solids and Lipids on Raw Fish Fillets All solids of raw fish fillets ranged in.percentage from 22 to 35 in skin-on fillets, and from 21 to 30 in skin-off. fillets, which indicated that skin-off fillets had a higher water content than the skin-on fillets. Skin removal and the lake from which the fish were harvested had significant - effects on the solid contents of skin-on and skin-off fish 61 Table 10. Processing data as well as solid and lipid contents of raw fillets for carp from Lakes Erie and Huron Lakes Skin Lake Removal Effect2 Carp Fillets Erie Huron Effect1 Carcass skin-on 57.51 58.19 Yes No Yield % skin-off NA3 61.79 As Prepared skin-on 36 . 26 29 . 07 Yes Yes Yield % skin-off 25.62 21.70 Right-Side skin-on 323 . 80 243 . 80 Yes Yes Fillet (g) skin-off 239.60 171.90 Left-Side skin-off 331.30 226.40 Yes Yes Fillet (g) skin-off 237.70 169.50 Solids % skin-on 26.21. 26.38 Yes Yes skin-off 21.27 24.57 Lipids % skin-on 7.75 6.44 Yes No skin-off 2.82 2.34 1 ANOVA indicated the effect of skin removal on skin-on fillets significant at the P 5 0.001 level 3 ANOVA indicated the effect of lakes significant at the ‘P 5 0.05 level 3 not available 62 Table 11. Processing data as well as solid and lipid contents of raw fillets for chinook salmon from Lakes Huron and Michigan Lakes Skin Lake Removal Effect2 Chinook Fillets Huron Michigan Effect1 salmon Carcass skin-on 62.75 66.43 No Yes Yield % skin-off 63.76 66.76 As Prepared skin-on 38 . 99 46 . 20 Yes Yes Yield % skin-off 28.22 33.60 - Right-Side skin-on 1105 . 70 1174 . 70 Yes Yes Fillet (g) skin-off 755.80 761.90 Left-Side» skin-on 1217.90 1183.90 Yes Yes Fillet (g) skin-off 791.50 750.40 Solids % skin-on 25.41 28.20 Yes Yes skin-off 23.21 25.74 Lipids % skin-on 4.17 11.63 Yes Yes skin-off 1.82 5.71 1 ANOVA indicated the effect of' skin-on or skin-off significant at the P < 0.001 level 2 ANOVA incidated the effect of lakes at the P < 0.001 level 63 Hw>ma mo.o v m on» um unmofiuflcmflm mmme mo homuum one UODMOflocfi <>oz<.« HO>OH mo.o v a who no unmofluflcmwm mumaaflu coucfixm so Hm>OEmH nflxm uo uomuum wnu UODMOfiUGfi <>Oz¢ 4 m¢.m mm.m Hr.m mm.m muoucfixm mm» mm» mm.mm 3.: 5.5% w 333 mm.m~ mH.mN or.om mm.wm muoncaxm mm» mm» mH.mm mm.am co-:fixm « moflaom 362 2.63 8.43 8.1.: 39:08 3. 6433 wow wow om.mmm oo.HNm Goucfixm mofimuuumq 31m: 8.2:. 8.84 843 39:83 :3 3:3 mm» mm; om . mmm om . «.3 5-303 33-932 m¢.bm pm.mm mm.mm mm.am muo-:fixm a name» mm» mm» Exmm ~N.~¢ couaflxm omnmnmum m< mm.mm mH.Hb mw.bm w¢.¢m muoucwxm * flame» mm» mow ha.bm mm.bm sousflxm mmmoumu flmuomuum uofiummsw ofiumuso someones. nouns mumaafim .usoue mxmq «homuum Hm>oEmm mxwa sflxm mmxma Hofiummdm pom OHHMDGO .cmmfinowz .cousm mwxmq Scum usoua mxmq MOM mumaafim 3mm mo mucousou pagan pom owaom mm Hams on some mnfimmwooum .ma wHDMB 64 Table 13. Processing data as well as solid and lipid contents of raw fillets for walleye from Lakes Erie, Huron and Michigan Lakes Lake Walleye Fillets Erie Huron Michigan Effect1 Carcass skin on 66.45 59.73 63.40 Yes Yield % As Prepared skin on 40 . 93 40 .89 50 . 08 Yes Yield % Right-Side skin on 185.20 221.90 191.90 Yes Fillet (g) Left-Side skin on 181.40 213.60 193 .10 Yes Fillet (g) Solids % skin on. 22.47 22.57 21.01 Yes Lipids % skin on 1.65 3.03 1.08 Yes 1 ANOVA indicated the effect of lakes significant at the P 5 0.001 level 65 Table 14. Processing data as well as solid and lipid contents of raw fillets for white bass from Lakes Erie and Huron Lakes Lake White bass Fillets Erie lHuron Effect1 Carcass skin on. 59.46 64.48 Yes Yield % As Prepared skin on 25 . 78 34 . 14 No Yield % Right-Side skin on 74.20 62.20 No Fillet (g) ‘ Left-Side skin on 74 . 80 64 . 20 No Fillet (g) Solids % skin on 23.64 23.63 No Lipids % skin on. (4.38 2.57 No 1 indicated the effect of lakes source significant at the P 5 0.001 level 66 fillets. Lake trout skin-on fillets had the highest solid contents (34%) when compared to carp (26%), chinook salmon (27%), walleye (22%) and white bass (24%) fillets. Skin-off Lake Erie carp fish fillets had the lowest solids content (21%) and the highest water content. Lipids of raw fish fillets had a wide range from 1% to 36.5% in skin-on fish fillets. Siscowets from Lake Superior skin-on fish fillets had the highest lipids content (36.5%), while Lake Erie walleye skin-on fillets had the lowest lipids content (1.08%). Trimming off belly flap and associated fat tissue showed significant reduction in lipid content. There was an almost 75% reduction of lipid content observed in siscowet skin-off fillets from.Lake Superior. An average of 50% of lipid reduction was achieved through skin removing processes for all fish fillets, which is an important value on the assessment of PCBs consumption. Because most of the PCBs were extracted from the fatty tissue of fish, skin removal procedures eliminated the majority of contaminated substances in fatty tissue up to 50%. This figure has been used by the Great Lakes Fish Advisories Committee (GLSFATF, 1993). Cooking Data of the Great Lakes Fish Species The weights of fish sample (skin-on and skin-off) were recorded before cooking and after cooking in order to calculate the total cooking loss (%) and cooking yield (%). When cooked skin-on fillet sample was prepared, the skin was peeled off so only the cooked muscle tissue weight was 67 recorded as the edible weight. The one exception was for deep fat fried skin-on fillets; skin was not removed since deep fat fried fish would normally be battered or breaded and thus eaten with the skin-on. The calculation of percentage total- cooking loss was the ratio of the difference between raw and cooked fillet weights to the raw fish fillet weight times 100. Cooking yield percent was derived from the relation of the cooked edible weight of fillet to the raw fillet weight times 100. Sum.of total cooking loss and cooking yield are 100% in all skin-off fish fillets. Totality of cooking loss and cooking yield is less than 100% in all skin-on fish fillets due to the fact that the weight of skin was excluded. Summaries of cooking data along with the analyses of solid and lipid contents on cooked.skin—on.and.skin-off fillets for five fish species from the Great Lakes are presented at Tables 15 - 19. Regardless of the fillet size or species of the fish, cooking loss of skin-on fillets and.skin-off fillets ranged in percentage from 17-36 and 12-36 with average of 26.5% and 24.0%, respectively, which means nearly one-fourth portion of fillet weight lost during cooking. The further skin removal after cooking of fish fillets resulted in lower cooking yield than cooked skin-off fillets in all fish species from the Great Lakes. Skin-on fish fillets had a range of 54% to 76% cooking yield with an average of 65% cooking yield and skin- off fish fillets had a range of 64% to 88% cooking yield with an average of 76% cooking yield. Approximately 10% of the total fillet weight was lost from cooked skin-on fillets 68 Table 15. Cooking data1 and solid1 and lipid2 contents of cooked skin-on and skin-off fillets for carp from Lakes Erie and Huron Cooking Methods Carp Fillets Panfry Deep—fat Fry Lake Erie Cooking Loss % skin-on 22.60 32.93 skin-off 21.61 36.47 Cooking Yield % skin-on 68.51 67.07 skin-off 78.39 63.53 Solids % skin-on 32.45 44.61 skin-off 29.96 41.31 Lipids % skin-on 7.65 17.27 skin-off 3.80 7.21 Lake Huron Cooking Loss % skin-on 20.10 30.17 skin-off 15.14 30.37 Cooking Yield % skin-on 68.91 69.83 skin-off 84.86 69.63 Solids % skin-on 31.66 42.94 skin—off 30.52 42.38 Lipids % skin-on 7.20 13.80 skin-off 3.72 10.36 :35 th 69 Table 16. Cooking1 data and solid1 and lipid2 contents of cooked skin-on and skin-off fillets for chinook salmon from Lakes Huron and Michigan Cooking Methods Chinook Fillets Bake Charbroil Can Salmon Regular Surface Increased Lake Huron Cooking skin-on 21.32 27.37 35.15 Loss % skin-off 23.58 27.96 28.82 26.84 Cooking skin-on 72.20 65.20 54.58 Yield % skin-off 76.42 72.04 71.18 73.16 Solids % skin-on 31.16 33.96 35.02 skin-off 32.15 32.54 33.96 27.42 Lipids % skin-on 5.10 5.70 5.23 skin-off 2.82 3.18 2.93 1.80 Lake Mi chigan Cooking skin-on 19.70 29.28 25.73 Loss % skin-off 21.10 24.24 25.28 25.96 Cooking skin-on 76.01 66.66 69.02 Yield % skin-off 78.90 75.76 74.72 74.04 Solids % skin-on 34.59 37.29 32.43 skin-off 32.07 32.86 35.85 31.59 Lipids % skin-on 11.87 9.63 9.10 skin-off 5.08 7.78 6.02 5.25 II II um 33:: 70 Table 17. Cooking1 data and solid1 and lipid2 contents of cooked skin-on and skin-off fillets for lake trout from Lakes Huron, Michigan, Ontario and Superior Cooking Methods Lake Fillets Bake Char- Salt Smoke Trout broil Boil Lake Huron Cooking skin-on Loss % skin-off 23.70 24.22 Cooking skin-on Yield % skin-off 76.30 75.78 Solids % skin-on skin-off 34.38 35.20 Lipids % skin-on skin-off 9.05 8.52 Lake liohigan Cooking skin-on 33.95 Loss 8 skin-off 17.62 23.88 12.25 Cooking skin-on 57.61 Yield 8 skin-Off 82.38 76.12 87.75 Solids % skin-on 38.65 skin-off 34.00 35.14 31.54 Lipids % skin-on 9.13 skin-off 7.41 9.78 8.68 Lake Ontario Cooking skin-on Loss % skin-off 17.26 19.92 Cooking skin-on Yield % skin-off 82.74 80.08 Solids % skin-on skin-off 30.83 31.28 Lipids 8 skin-on skin-off 8.80 6.83 Lake Superior Cooking skin-on 36.24 Loss 8 Chin-off 26.01 25.18 13.06 Cooking skin-on 53.61 Yield 8 lkin-off 73.99 74.82 86.94 Solids % skin-on 41.91 skin-off 35.03 32.42 32.38 Lipids % skin-on 22.36 skin-off 9.38 7.96 11.64 1 n-6 1 n-3 71 Table 18. Cooking1 data and solid1 and lipid2 contents of cooked skin-on fillets for walleye from Lakes Erie, Huron and Michigan Lakes Cooking Walleye Methods Erie Huron Michigan Cooking Loss % Bake 23.48 19.18 28.95 Charbroil 21.03 21.94 26.22 Deepfat fry 35.70 Cooking Yield % Bake 69.74 71.79 62.56 Charbroil 71.89 70.73 67.89 Deepfat fry 64.30 Solids % Bake 28.43 27.32 27.50 Charbroil 27.18 27.96 26.06 Deepfat fry 38.82 Lipids % Bake 2.20 3.03 1.53 Charbroil 2.45 2.08 1.70 Deepfat fry 8.89 Db nu was 72 1 Table 19. Cooking1 data and solid1 and lipid2 contents of cooked pan fried skin—on fillets for white bass from Lakes Erie and Huron Lakes White bass Fillets Erie Huron Cooking Loss % skin on 21.27 16.70 Cooking Yield % skin on 68.33 73.66 Solids % skin on 28.48 29.79 Lipids % skin on 5.14 3.18 73 during the combination of cooking and skin removal processes. Skin-on smoked lake trout fillets had the highest averaged cooking' loss (35%), and. lake trout skin-off salt-boiled fillets had the lowest averaged cooking loss (13%), all from Lakes Michigan and Superior. The differences of cooking loss between smoking and salt boiling were possibly related to the cooking time, cooking atmosphere and the presence of skin. According to the experimental design, all fillets either skin-on or skin-off were cooked to an internal temperature of 80°C, which resulting in the variations with the median cooking temperature and the appropriate cooking time. By monitoring the internal temperature, the variation of cooking loss between skin-on and skin-off fillets of the same cooking method was reduced. This phenomena was observed in cooked fillets for carp and chinook salmon. However, the effect of cooking methods on cooking loss of fillets was significantly less for pan-fried cooking method than deep-fat fried cooking method on skin-on and skin-off carp harvested from.Lakes Erie and Huron (P<0.05) (Table 15). Baked skin-on and skin-off chinook salmon fillets also had significantly less cooking loss than charbroiled (regular and surface increased) chinook salmon fillets from Lakes Huron and Michigan (P<0.05)(Table 16). Skin-off salt-boiled lake trout fillets had significantly less cooking loss than skin-off baked lake trout fillets, and skin-off charbroiled lake trout fillets (P<0.05)(Table 17). Smoked skin-on lake trout fillets, deep— fat fried skin-on walleye fillets and deep-fat fried skin-on 74 and skin-off carp fillets had similar values of the cooking loss. which were 35%, 36% and 33%, respectively. Lake effect on cooking loss and cooking yield of skin-on carp and skin-on chinook salmon fish fillets did not possess the same pattern as their skin-off fillets which were in the same trend within each species. Analyses of Solids and Lipids of Cooked Fish Fillets All solids of cooked fish fillets ranged in percentage from 26 to 45 in skin-on fillets, and from 30 to 42 in skin- off fillets with an average of 36% on both skin-on and skin- off fillets. As mentioned above, fillets cooked to the same internal temperature also reduced the variation of solid contents between skin-on and skin-off fillets. Deep—fat fried skin-on and skin-off carp fillets from Lakes Erie and Huron had significantly higher solid contents (43%) than lake trout skin-on and skin-off fillets (34%) , skin-on and skin-off chinook salmon (33%), walleye skin-on fillets (29%) and.white bass skin-on fillets (29%). Walleye charbroiled skin-on fillets from Lake Michigan had the lowest solid contents (26%) and the highest water content. Solid contents were significantly higher in cooked fillets than in raw fillets (Appendix 7). This mechanism is due mainly to the loss of water content (Zabik et al. , 1993) . Effects of cooking methods on solid contents of fillets were significantly higher for deep-fat fried carp (43%), smoked lake trout (40%), and deep-fat fried. walleye than baked (32%) or charbroiled 75 (regular (33%) and surface increased (34%)) chinook salmon; baked (34%), charbroiled (34%) or salt-boiled (32%) lake trout; and baked (28%) or charbroiled (27%) walleye (P<0.005). Smoked skin-on lake trout had the least water content among all the cooked fillets (Table 17). Lipid of cooked fish fillets ranged from 2% to 22% compared to the range of lipid content (1% to36%) of raw skin- on and skin-off fillets. Smoked skin-on siscowets from Lake Superior had the highest lipid content (22%), while baked skin-on walleye from Lake Michigan fillets had the lowest lipid content (2%). Lipid content of skin-on smoked lake trout fillets from Lake Superior (22%) was significantly higher than lipid content of panfried (6%) or deep-fat fried (12%) carp fillets; baked (7%), charbroiled (6%) or canned (4%) chinook salmon fillets; baked (9%), charbroiled (8%) or salt boiled (10%) lake trout fillets; baked (2%), charbroiled (2%) or deep-fat fried (9%) walleye fillets; and panfried (4%) white bass fillets. In addition, the lipid content of skin-on chinook salmon was significantly higher than that of skin-off chinook salmon in their specific cooking method (Table 16). Lipid content was significantly higher in cooked fillets than in raw fillets (Appendix 7). In this study, cooking media consisted of liquid cooking oil, corn oil spray, distilled water or a solution for brining containing lecithin. Cooking medium varied with the designed cooking method. The mechanisms of the adsorption of lipid. extraction of lipid, cooking time, and cooking temperature 76 caused some changes of the lipid contents in.a specific cooked skin-on.or skin-off fillets; deep-fat frying caused.four times the increase in the lipid content in cooked fillets compared to the raw fillets; however, smoking caused the 14% decrease of lipid content in cooked Lake Superior skin-on lake trout fillets significantly compared to the raw fillets. Distribution Pattern of PCB Specific Congeners in Fish Fillets from the Great Lakes In order to produce the accurate risk assessment of PCB toxicity, there is a need to quantitate individual PCB specific congeners. Based upon their chlorination and chlorine substituted position, the separation of individual PCB congeners became visible in.GC-capillary column analysis. In this study, there were 53 specific congeners selected which are existing in the commercial Aroclor0 1254. PCB congeners 66/95/121, 84/101, 79/99, 123/149, 105/132, 128/167, 156/171, and 157/200 were co-elutions based upon their retention time. All concentrations are reported on a wet weight basis. The proportion of individual congener was derived from each specific congener concentration to the total PCB concentration. Out of all specific congeners, 55, 76 and 120 were under the limit of quantitation in all samples; thus, the values for these congeners did.account as zero. 'This was also applied to any congener below the level of detection. The statistical analysis was done by.ANOVA, using Tukey test with significant level of p < 0.2 to find the effects of lake, skin 77 removal, cooking, and species. Lake_§ffiegt Table 20 represented the mean concentration of 53 secific PCB congeners for raw skin-on and skin—off chinook salmon harvested from Lakes Huron (42 fillets) and Michigan (42 fillets). Total PCBs for chinook salmon fillets were not affected by lakes. However, concentration of PCB 31, 52, 49, 47, 44, 42, 72, 70, 66/95/121, 91, 92, 84/101, 79/99, 87, 110, 153, 141, 137, 138. 183, 171/156, and 180 were significantly different between lakes for chinook salmon (ANOVA.p<0.2). Of the individual congeners and co-eluting congeners found, 87, 66/95/121, 118 and 138 were most prominent (each comprised > 5% of total PCBs), followed by, in decreasing order. 84/101, 153, 110, 79/99, 180, 105/132 (2.72%) in Lake Huron chinook salmon. Of Lake Michigan chinook salmon, the prominent congeners were 87, 66/95/121, and 84/101 (each comprised > 5% of total PCBs). followed by, in decreasing order, 118, 110, 79/99, 42, 83, 138 and 70 (2.93%). Maack and Sonzogni (1988) reported that the prominent congeners found in Wisconsin fish species were 153/132 (9—19% of total PCBs), followed by 138, 66/95, 110, 101, 180, 70/76, 146, 28/31, 149,118 and 105(1- 5%). Oliver and Niimi (1988) found that congeners 153, 101, 84, 110, 180, 87+97, 149, 187+192 and 105 constituted over half the total PCBs in salmonids from Lake Ontario. The above results indeed revealed that concentration of PCB specific congeners provided much more detailed information toward distribution pattern of PCBs in the aquatic species than just concentration of total PCB alone. However, chinook salmon 78 Table 20. Mean concentration of PCB specific congeners and their percentage for raw skin-on and skin-off chinook salmon harvested from Great Lakes PCB congeners Lake Huron Lake Michigan Lake Huron Lake Michigan (Mamet wt.) (ppmmet wt.) % % 31 0.021 ° 0.041 0.85 1.41 52 0.043 0.066 1.72 2.28 49 0.039 0.057 1.55 1.98 47 0.028 0.045 1.14 1.57 44 0.031 0.056 1.23 1.94 42 0.058 0.105 2.34 3.63 72 0.005 0.008 0.18 0.27 103 0.011 0.010 0.45 0.34 70‘ 0.063 0.085 2.52 2.93 76 n.d. n.d. n.d. n.d. 66/95/121 0.136 0.173 5.45 5.99 91 0.021 0.032 0.86 1.11 55 n.d. n.d. n.d. n.d. 92 0.065 0.088 2.60 3.06 84/101 0.122 0.151 4.87 5.22 79l99 0.081 0.101 3.26 3.49 83 0.072 0.103 2.36 3.37 97 0.040 0.049 1.60 1.68 87 0.605 0.748 24.25 25.86 120 n.d. n.d. n.d. n.d. 85 0.029 0.033 1.15 1.13 136 0.000 0.019 0.00 0.66 110 0.085 0.103 3.41 3.58 108 0.017 0.021 0.69 0.73 1491123 0.034 0.033 1.35 1.13 118 0.132 0.139 5.30 4.81 114 0.056 0.057 2.23 1.97 122 0.016 0.000 0.66 0.00 153 0.1 13 0.074 4.52 2.56 105/132 0.068 0.080 2.72 2.75 141 0.019 0.026 0.75 0.89 179 0.019 0.01 7 0.78 0.60 137 0.010 0.015 0.40 0.50 138 0.127 0.092 5.11 3.19 158 0.044 0.027 1.78 0.93 183 0.018 0.007 0.73 0.25 1671128 0.033 0.040 1.33 1.39 185 0.008 0.011 0.32 0.39 181 0.055 0.062 2.19 2.13 1 71 I1 56 0.027 0.011 1.07 0.38 157/200 0.007 0.005 0.27 0.18 180 0.073 0.027 2.92 0.92 190 10.031 0.035 1.25 1.22 198 0.046 0.048 1.83 1.64 TOTAL 2.495 2.893 100 100 '80Ided number means lake effect at significant level (p < 0.2) 79 harvested from Lake Michigan had significantly higher tri-, tetra- and penta-CBs, but significantly lower hexa-, hepta-, and octa-CBs than chinook salmon harvested from Lake Huron. Lower chlorinated biphenyls were significantly affected by the origin.of lakes; and.the persistence of PCB congeners remained in the environment and.detected in chinook salmon fillets more likely came from the higher chlorinated biphenyls, such as hexa-, hepta-, and octa-CBs. fikig_g§mggal_gffggt Skin-removal effect on PCB specific congeners from 36 raw skin-on and 48 raw skin-off chinook salmon fillets as well as 24 raw skin-on and 24 raw skin-off carp fillets was presented in Table 21. These skin-on and skin-off chinook salmon fillets were not processed from the same fish, but from the same location as well as carp skin-on and skin-off fillets. The result showed that the concentrations of over 30 PCB specific congeners and total PCBs were significantly reduced.by removing skin and trimming adipose tissue under skin for chinook salmon fillets; particularly on the prominent congeners, such as 66/95/121, 87, 118, 84/101 and 83. Figure 3 demonstrated that the reduction of concentration of PCB specific congeners was very effective through skin-removal and fat trimming, not only on the lower chlorinated biphenyls, such as 42 (35% reduction) but also on the higher chlorinated biphenyls, such as 181 (50% reduction) for chinook salmon fillets which normally have higher fat content than carp fillets (Tables 10 and 11). Levels of PCB specific congeners on 70, 92, 84/101, 99/79. 87 80 Table 21. Mean concentration of PCB specific congeners and total PCBs from raw skin-on and raw skin-off fillets for carp and chinook slamon harvested from the Great Lakes Total PCB concentration (ppm, wet wt.) 'Bolded number means skin removal effect at significant level (p < 0.2) PCB Chinook salmon Chinook salmon Carp ‘ _C_o_ngener raw skin-on raw skin-off raw skin-on raw skin-off 31 0.031 0.031 0.039 0.030 52 '0.070 0.046 0.091 0.074 49 0.060 0.040 0.086 0.067 47 0.044 0.031 0.054 0.040 44 0.046 0.042 0.067 0.051 42 0.105 0.068 0.143 0.100 72 0.008 0.005 0.021 0.005 103 0.013 0.010 0.018 0.044 70 0.088 0.066 0.047 0.030 76 n.d. n.d. n.d. n.d. 66I95/121 0.192 0.128 0.173 0.117 91 0.032 0.023 0.027 0.01 9 55 n.d. n.d. n.d. n.d. 92 0.094 0.065 0.074 0.048 841101 0.1 62 0.1 15 0.1 37 0.098 99179 0.1 1 0 0.076 0.084 0.055 83 0.1 07 0.056 0.032 0.022 97 0.053 0.039 0.041 0.028 87 0.830 0.556 0.227 0.137 120 n.d. n.d. n.d. n.d. 85 0.039 0.026 0.027 0.01 7 136 n.d. 0.100 n.d. 0.012 1 10 0.1 1 6 0.080 0.092 0.059 108 0.024 0.01 6 0.017 0.047 149I123 0.047 0.058 0.063 0.040 118 0.172 0.110 0.110 0.080 1 14 0.090 0.041 0.037 0.018 122 0.018 0.006 0.029 0.009 1 53 0.087 0.098 0.095 0.062 1 32I105 0.090 0.064 0.060 0.038 141 0.025 0.022 0.028 0.01 7 1 79 0.025 0.01 7 0.021 0.009 137 0.01 6 0.010 0.009 0.004 138 0.108 0.113 0.108 0.077 158 0.031 0.052 0.203 0.1 51 183 0.01 5 0.014 0.020 0.01 1 167/128 0.044 0.032 0.030 0.018 185 0.01 5 0.008 0.005 0.003 181 0.082 0.040 0.101 0.077 171/1 56 0.024 0.022 0.024 0.01 1 1 57I200 0.005 0.009 0.008 0.005 180 0.034 0.069 0.083 0.041 190 0.036 0.032 0.035 0.037 198 0.047 0.050 0.026 0.01 1 TOTAL 3.195 2.333 2.387 1 .561 81 86.3 «no.0 05 EB. 62822 92.: 55.3 880ch toéim 38 can 5.56....“ 26.. .6. Cocoa—.8 0:8on moo do 5:62.588 cows. .m 959... mhwcomcoo Stowam mom Nd 06 md ad ll m .8. w. an I. R 6 “mm mmmmxwmuwwwmawwwanmummwwwmwmawmmwwumwuawzmzi dine—:55..- . L- ed 1. .. 0.0 .. Nd :oEimtoéEmBED 88.851.24.38. - ad ('1M 16M ’uidd) uoglenuaouoo 83d 82 and 110 and total PCBs were also significantly reduced by removing skin and trimming fat for carp fillets (Table 21) . The distribution pattern of individual congeners was not affected by skin-removal process because the prominent congeners were 87, 158, 66/95/121, 42, 84/101, and 118 for skin-on carp fillets and 158, 87, 66/95/121, 42, 84/101, and 118 for skin—off fillets according to the decreasing order of their concentration; however. specific concentration of those prominent congeners was reduced through skin-removal and trimming fat process before cooking. Wt Figures 4 and 4a represented the mean concentration of PCB congeners for raw and cooked skin-off chinook salmon harvested from Lakes Huron and Michigan. Most congeners had higher concentrations in raw fillets than cooked fillets with the exceptions of 72, 83, 120, 137, 158 and 167/128 measured in ppm wet tissue (Figure 4). A comparison graph (Figure 4a) on percentage of individual congener to the total concentration of PCBs showed that more congeners in cooked chinook salmon were a higher % of the total PCBs (31, 52, 49, 44,42, 72, 66/95/121, 91, 92, 84/101, 99/79, 83, 87, 120, 85, 108, 118, 122, 132/105, 179, 137, 158, 183, and 167/128) than in raw salmon; despite this, the level of the specific congener had a higher ppm in raw wet tissue than in the cooked wet tissue, except for 72, 83, 120, 137 and 158. This occurrence was mainly because the total concentration of PCBs measured in ppm wet cooked tissue was reduced through the cooking process, including cooking losses and evaporation; the 83 — - _ l - — l - I E — — E g - - n _ _ I — — — - — - _ — — — _ _ - _ _ I s — _ - _ _ —_ .L .L I. l i i '9. ". Q 1‘! F. O O O O O 0.6 ('w 10M 'uidd) uoglenueouoo 86l- 06) 08) OOZILSL 95M“ l8l 98) 8leL9l 88) 891- 88L L8) 6“ Lil SOL/28L 89L ZZL 7H 8H €ZL/67l 80) Oil 98L 98 OZL L8 L6 88 6U66 £01778 Z6 99 i6 lZl/96I99 9!. 0!. 80 i Z]. Z? W L? 6? ZS L8 PCB specific congener number Figure 4. Mean concentration of PCB specific congeners for raw and cooked skin-off chinook salmon harvested from Great Lakes 25 I rawskin-off C] cooked skin-off 84 25 d— i F In 10 4 F 882):! “101 1° % 1— 86L 06L O8) OOZILSL 99H)“ i8i 98l 8ZLIL9) 88l 89L 881. LSL 6!.) W) SOL/ZS) 89) ZZL Vil- 8H. 8ZLI6VL 80) OH 98L :8 OZL 1.8 L6 88 61.166 I'm/78 Z6 99 L6 ' lZL/96I99 9!. 01 80 i Z4 Z? W 1.7 6? ZS L8 PCB specific congeners Figure 49. Percentage of individual PCB specific congener to total PCB concentration for raw and cooked skin-off chinook salmon from Lakes Huron and Michigan 85 proportion of individual congener to total PCBs in cooked chinook salmon appeared higher ‘than the proportion of individual congener to total PCBs in raw tissue. However, this phenomena occurred particularly in the lower chlorinated congeners instead of the higher chlorinated congeners, which might mean that the cooking process facilitates the reduction of higher chlorinated biphenyls instead of lower chlorinated biphenyls. Figure 5 also represented the cooking effect on the reduction of PCB congener levels during the cooking of carp fillets. Even though expressing the congeners on a wet weight.basis does not account for differences in fillet weight between raw and cooked samples, most of the congener concentrations were reduced through the panfrying or deepfat frying procedure. spggig§_gfifiggt Figure 6 presents the comparison of the effect of species on the distribution pattern of PCB specific congeners. In general, chinook salmon had a higher concentration of specific congener than walleye and white bass. White bass also had higher concentrations than walleye; this may have something to do with the fat content of a fish species because the lipid content of skin-on chinook salmon, white bass and walleye was 8%, 3.5% and 2%, respectively. There were significant differences between residues in the raw skin-on chinook salmon and the residues in the raw skin-on walleye for congeners 31, 52, 49, 47, 44, 42, 103, 70, 66/95/121, 91. 84/101, 99/79, 83, 97, 87, 85, 136. 110, 108, 118, 114, 132/105, 141, 179, 137, 167/128, 181, 198 and total D cooked skin-off carp Irawskin-offcarp 86 ‘11" 0.16 I _ _ - - — _ _= — _ 111111 3. 9‘. 3 8. 8. 3. 3. ° CO 0000 mm mm 'uidd) uogtenueauoo 80d 86) 06) 08) 00ZIL9) 99)/)L) )8) 98) 8ZLIL9) 88) 89) 88) £8) 6L) )7) 90)/Z€) 89) ZZ) 7)) 8)) SZIJBV) 80) 0)) 98) 98 OZ) L8 1.6 88 6U66 )0)IV8 Z6 99 )6 )Z)I96/99 9L OL 80) ZL Z? W L? 67 Z9 )8 PCB specific congeners Figure 5. Mean concentration of PCB specific congener for raw skin-off and cooked skin-off carp fillets harvested from the Great Lakes 87 0.9 v 3.3 §§e 53? IE]. 'é;.l.i..'..l.” 9°. 0 o o' o o' o' 0' run 18M 'uidd) uonenueouoa 86) 06) 08) OOZILS) 99)l)L) )8) 98) 8Z)IL9) 88) 89) 88) L8) 6L) )1?) 90)/Z8) 89) ZZ) 7)) 8)) 8Z)l6?) 80) 0)) 98) 98 OZ) L8 L6 88 6Ll66 )0)IV8 Z6 99 )6 )Z)I96IQQ 9L OL 80 ) ZL Z7 717 L7 6? Z9 )8 PCB specific congeners Figure 6. Mean concentration of PCB specific congeners for raw skin-on chinook salmon. walleye and white bass harvested from Great Lakes 88 PCBs with the exception of congeners 72, 123/149, 122. 153, 138, 158, 183, 185, 171/156, 157/200, 180, and 190. As compared between the residues in raw chinook salmon to the residue in raW' white bass fillets, there were over 20 congeners significantly different, such as 52, 49, 47, 44, 66/95/121, 91, 84, 99/79, 83, 97, 87, 85, 136, 110, 118, 114, 132/105, 179, 137, 167/128, 181, and 198 with exceptions of 31, 42, 72, 103, 108, 149/123, 122, 153, 141, 138, 158. 185, 171/156, 157/200, 180. and 190. Significant differences between congener specific PCB residues in walleye fillets and in white bass fillets occurred only for congeners 66/95/121, 110, 108, and 132/105. The prominent congeners for chinook salmon are 87, 65/95/121, 118, 84/101, 110, 181, 99/79. 83, and 138; for walleye are 87, 66/95/121, 149/123, 153, 138, 158, and 189; for white bass are 66/95/121. 87. 84/101, 110, 153, 138 and 189. The common prominent congeners are 87, 66/95/121 and 138. As mentioned before, the higher chlorinated biphenyls persisted in the environment longer than lower chlorinated biphenyls, particularly because fish species on the top of the food chain ‘may have the effect of biomagnification on concentrations of higher chlorinated biphenyls. Oliver and Niimi (1988) found that the chlorine content of the PCBs was observed to increase with trophic level in the Lake Ontario ecosystem. 89 Distribution Pattern of PCB Homologs in Fish Fillets from the Great Lakes The PCB patterns that are found in fish tend to have greater concentrations of the more chlorinated PCB homologs (congeners with five to seven chlorine substitutions), while water has been reported to have greater amounts of less chlorination biphenyls (Bush et al. 1989; Oliver and Niimi, 1988) . Grouping the congeners together makes the identification of the specific AroclorQ products visible. In this study, PCB homologs were grouped by the same chlorination for 53 individual congeners analyzed in fish tissue. For each species of fish, samples analyzed in the EPA "National Study of Chemical Residues in Fish" (1992) were from sites in Michigan, and are illustrated in the graphs 7, 9a, 10 and 11. Figures 7-11 demonstrated the distribution pattern of PCB homologs which was derived from the concentration of PCB homologs to the total PCB concentration (ppm, wet wt. basis) for five fish species harvested from the Great Lakes. Based upon the experimental design, data were generated from all the fillets both raw and cooked in these analyses. Ninety—six carp fillets, one hundred sixty-eight chinook salmon fillets, one hundred forty-four lake trout fillets, eighty-four walleye fillets and twenty-four white bass fillets were used. The homolog pattern of carp harvested from Lakes Erie and Huron possessed tri-, tetra-, penta-, hexa-, hepta-, and octa-CBs (2%, 30%, 35%, 23%, 9%, and 1%) and (2%, 27%, 48%, 17%, 5% and 1%), respectively. Penta-CBs homolog in carp 90 98 .8 329:9. mod Lo Eaten nonsense .N. 659“. 322.3... mon— 981 -85.. -255. secs: 8.... U arm 8.3 I Eu D it. (sissq '1M 18M 'urdd) saga mo; 10 9g, 91 26.... cores» 8.355 to. 329:9. mod Lo Eaton 5.59.85 .m e59”. .831 363:5: mod -93: .85.. SN. 5.33 U c.3235. 8.... I :23: 8.3 D TO— imp row imw tom 10¢ .13 to... (sissq '1M 19M 'uidd) 980d |8101 40 9g, % of Total PCBs (ppm, wet wt. basis) 92 so “ 1 1:] EPA (field) 45-1 40‘ I Lake Huron 354 E Lake Michigan 30 :1 1 1 ; Cl Lake Superior 25 ‘ 201 15; f ‘ .1. F 5" ;1 0" F T i Tri- Tetra- Penta- Hexa- Hepta— Octa- PCB Homologs Figure 9a. Distribution pattern of PCB homologs for lake trout fillet 93 40‘ ULakeOntario _ 35‘ IBreast milk 0 3 30- .3 , 25‘ “E 3... 20‘ ‘1 5 '3 . 15‘ .15 s ._ a 10-1 O- 32 El: Tri- Tetra- Penta- Hexa- Hepta- Octa- PCB Homologs Figure 9b. Distribution pattern of PCB homologs for lake trout fillet (Breast milk data quoted from Waid, 1986) 94 3:: 26:9; .2 329.3: mun. no Sousa 5.59me .0. 2:9”. 322.3... men -800 -88: -88: .528 -2qu _ _ _ :6 2.... U $.22: 2.... fl .35: on... D 3... (an I .33: (mm D L ,. (SEMI 'lM 18M) 880d IBIO‘L JO % 95 8:: was 222, .2 329:9. mom .o 5.53 9.259285 .— p 939“. 322.3: mon— nmx 0T. imp—50n— -m=O.—. :35. -800 ~~ 'i‘fi}. v ‘7‘ z! . l‘*:\ ~‘r .3 Lei-2,: .y in! ; f ”.e WW} afar :23... 8.... D 25 8.... I .22: (.3 D (mm um um saoa mm #0 as 96 fillets from Lake Huron had the same proportion as Aroclor09 1254 (48%) as well as hexa-CBs homolog for carp fillets from Lake Erie had the same composition as Aroclor® 1254 (23%)(Table 1). It was noted that carp, as a bottom feeder fish from.Lakes Erie and Huron, had higher tetra-CBS (29% and 27%, respectively) instead of hexa-CBs (23% and 17%, respectively). The distribution pattern of tetra-, penta-, hexa-, and hepta-CBs for carp collected from Escanaba River, Escanaba, Michigan (U.S. EPA, 1992) was 21%, 39%, 31% and 8%, respectively (Figure 7), which had the same tetra-CB proportion as AroclorGD 1254. Penta- and hexa-CBs were the dominant homologs (69%) in whole-body carp collected from Escanaba which was a historical PCB contaminated site. Chinook salmon fillets had a distribution pattern very similar to Aroclor" 1254 in the composition of tetra- and penta-CBs from Lake Michigan (21% and 49%) and penta- and hexa-CBs from Lake Huron (46% and 25%) (Figure 8). It was quite clear that chinook salmon fillet can be a good indicator for Aroclor" products in the aquatic environment. Both proportions of hepta- and octa-CBs were detected at 13% and 10% of total PCB concentration, respectively, fromfiLakes Huron and Michigan instead of trivial levels in commercialAroclorO 1254. The higher levels of hepta- and octa-CBs could.indicate that concentration of higher chlorination homologs may be biomagnified in predator fish tissue. The distribution.pattern of PCB homologs for lake trout caught from four large lakes was illustrated in Figures 9a and 97 9b. Lake trout (U.S. EPA, 1992) collected from Lake Michigan at Waukegan Harbor in Illinois, which was described as a Superfund site, had a high proportion of hexa-CBS (36%) . Lake Michigan lake trout fillets possessed some similar pattern as Aroclor0 1254 in proportions of penta- and hexa-CBs (47% and 25%) . Lake trout caught from Lake Huron and Superior had proportions of penta-CBs 44% and 42%, respectively. Lake Superior siscowet had a lower proportion of tri-CBs (4%) and a greater proportion of hepta-CBs (19%) than lake trout caught from other lakes. Lake trout caught from Lake Ontario had the highest octa-CBs (36%) compared to the other lake fish fillets (Figure 9b) . It might indicate that the persistent and less biodegradable congeners exist in Lake Superior and Lake Ontario regions. Lake trout is also a predator in the food chain which carries a similar pattern as chinook salmon from the same lake origin. Walleye harvested from Lakes Erie, Huron and Michigan all had penta—CBs as the dominant homolog, 43%, 41% and 42%, respectively (Figure 10). Particularly, walleye from Lake Huron demonstrated the distribution pattern of Aroclor" 1254 in fillets, tri-, tetra-, penta-, hexa-, hepta- and octa-CBs (1%, 24%, 41%, 22%, 9% and 2%, respectively). Walleye harvested from Lakes Erie and Michigan had higher total proportions of hexa-, hepta-, and octa-CBs (about 10%) than total proportions of hexa-, hepta-, and octa-CBs to Aroclor" 1254. EPA (1992) tested walleye fillets from Escanaba River, Escanaba, Michigan and reported that these walleye also 98 possessed a distribution pattern similar to Aroclor® 1254. White bass from Lakes Erie and Huron had different distribution patterns than those of white bass caught from Kalamazoo River, Saugatuck, Michigan (EPA, 1992)(Figure 11). Moreover, the order of proportion of PCB homologs for white bass from the current study was penta- > hexa- > tetra > hepta > octa-, then tri-CBs instead of tetra— > penta- > hexa- > tri- > hepta >, then octa-CBs for white bass caught from Kalamazoo, Michigan. Evaluation of Total PCBs by GC-Capillary and GC-Packed Column in Raw Fish Fillets From the Great Lakes According to the experimental design, Michigan Department of Public Health (MDPH) analyzed fifty percent of each specified fish groups from this project in order to determine the levels of the priority pesticides commonly found in the aquatic environment. GC-packed column was used by MDPH to quantitate the total PCBs. The average of total PCB concentration (ppm, wet basis) in raw skin-on and skin-off fish fillets which had PCBs determined both by GC-packed column and.GC-capillary column is shown.in.Table 22. Table 23 presents the total PCB.data.based.on summing the 53 individual congeners determined by capillary column GC for all raw fish from the study. Figures 12 - 16 represented the distributions of total PCB concentration for fish species determined by GC- packed column and GC-capillary column in relation to their fat contents which were determined by MDPH following the 99 Table 22. Comparison of total PCBs determined by GC-packed column and GC- capillary column of the same raw fish fillets for carp, chinook salmon, lake trout, walleye and white bass from the Great Lakes Fish Total PCBs No. of Fish to 1 Species Fillets (ppm, wet basis) (RangeHSDJ Total Fish No. GC-capillary column Carp skin-on 1.92 (0.853-3.071)(0.781) 6/12l50%) skin-off 1.117 (0.431-3.726)(0.856) 1/12( 8%) Chinook skin-on 2.28 (1.519-3.171)l0.497) 12/18l67%) salmon skin-off 1.547 (0.738-3.014)(0.536) 5/24(21 %) Lake skin-on 2.544 (1 .490-3.466)l0.775) 4/6 (67%) trout skin-off 1 .703 (0.824-3.084)(0.656) 8/27(30%) Walleye skin-on 0.673 (0.252-1 .604)(0.391) 0/20( 0%) White skin-on 1 .047 (0.547-1.924)(0.538) 0/6l 0%) bass GC-packed column Carp skin-on 1 .891 (0.283-6.620)( 1 .760) 4/12(33%) skin-off 0.611 (0.267-2.078)(0.484) 1/12( 8%) Chinook skin-on 1 .368 (0.593-2.173)(0.361 ) 1/1 8( 5%) salmon skin-off 0.813 (0.2874 .721 ”0.388) 0/24( 0%) Lake skin-on 0.928 (0.536-1.422)(0.322) 0/6( 0%) trout skin-off 0.76 (0.269-1 .940)(0.439) 0/28(0%) Walleye skin-on 0.277 l0.100-0.938)(0.1 96) 0/21 (0%) White skin-on 0.63 (0.242-1.379)(0.463) 0/6 (0%) bass 1 number of fish on total PCB concentration (ppm, wet weight) exceeding 2.0 ppm 100 Table 23. Total PCB concentrations based on summing 53 congeners determined by GC-capillary column in all raw fish fillets for carp, chinook salmon, lake trout, walleye and white bass from the Great Lakes # of Fish1 Fish Total PCBs (ppm,wet)(Range) to Total Species Fillets Fish # (Ratio) Carp skin-on 2.015 (0.618 - 7.556) 11/24(46%) skin-off 1.303 (0.419 - 5.872) 2/24 (8%) Chinook skin-on 2.161 (1.306 - 3.110) 23/36(64%) salmon skin-off 1.681 (0.738 - 3.014) 15/47(32%) Lake skin-on 2.398 (1.193 - 4.735) 5/L2(42%) trout skin-off 1.547 (0.583 - 4.324) 11/59 (19%) Walleye skin-on 0.850 (0.252 - 2.435) 2/41 (5%) White skin-on 1.279 (0.547 - 2.110) 1/12 (8%) bass INumber 6f_fish on tota1*PCB concentration (ppm,wet weight) exceeding 2.0 ppm 22:8 5. 55 s 8.3.2 s :58 86.8-00 Ea 5.3.8 52.38.00 3 85.52% as... 88 :32» 2a 85.» as. s 83588 mod .29 .6 c8858 .3 8:9". 7x; “€02.00 «6... I. up 0.. o o v N o k T k k k |+ w o D D D D nru D D m In.0 I I5 mrI I 1.. P I I U D I n I. D D -. m U i N W .o . d I D O a a I .i n m d l D I m m 11 V l M m Li W W 4r o 5:200 83.89000 a 55.8 5:38.00 . . l k 102 E0500 .0. :05 o. 0000.8 0. 050.00 09.03-00 0:0 5.5.00 t0.__000.00 .5 0058.200 ace—om 0.80.00 00.0.20 0:0 00.03038 0. 508E088 mom .30. 5 08.03050 .9 050K 33 E3000 «an. 8.9 8.9. 8.3. oo.Nw 8.2. 8.0 8.0 8.? SN 8.6 0 . . . . i T . i o B a mo 0 D D flu DU 1 n. n. D O 0 m Dan. 0 D I n. n. I I r F i I. D 0 00 I D n. I d D D U I I fir I I O C Lu I ~ I l m._. W m. m. I n. m. NIH I I I I D d I I N w l - . I. I DI I I M I . I . m I I I 1T m N W I I 1.. O I I L: m.” 55.8 8560-00 a 5.5.8 05.0800 .. 103 .858 a. 5.... o. 8.5.2 c. 55.8 888 -00 08 55.8 05.58.00 .5 85.52% 22.... Se. 9.0. 8.0.05 08 8.0....» 38 c. 8.8.08.8 mod .32 .0 080880 .3 050.“. mN Hr- ON 1*; uCOuCOU unn— '1’- 9 or m U D D 0m D flu .W D D D n. n. nuns I D I I In. 30 DIID c D I I I D II II I I I I I DD I I I I I I I I 85.00 80.80.00 6 85.8 8:80.00 _. l I l 1' md mé mN md run IeM'wdd) 880d “no; 104 .0250 .0. 0.05 o. 02.20.. 0. 0.00.00 09.00900 000 00.5.8 00.30000 >0 00505.00 0.0:: 05:03 00.00.» 2.0.. 0. 508.0038 mod .08. .0 000009000 .9 0.50.“. 7*. “cog—00 no". 0. 0 0 k 0 0 v 0 N . 0 . . J. l L. . . . . . 0 D DD 0 0 0 .0 00 i «.0 O a 9.0 D I ... .- ., 0.0 l D I O I 19 .. a... 0.0 m. .. l D I I N I . l 0.0 m. d U .0 LI _. W M l 0.. m i 0.. rt 005.00 09.08.00 0 . u 005.00 09.00000 0 .. . l 0.. 105 22:8 .0. 0.0... o. 02.0.2 c. 0528 00.08.00 0:0 5.5.00 b0__.000.ow .5 00580.00 0.0:... 0000 0:85 c220 30.. c. 5:00.588 00.. .05. .0 50:00:50 .0. 0.50.“. 1*; HCOHCOU «flu— ooN cm: 00.: CV... 0N._. 8.: . 00.0 8.0 ovd and cod _ _ _ a —v— —-1 —0-- — —1r— --0- dr— -00... :_ 5003:0050 mo: .08... 0:0 000.080... 000 .0 0.00.. .m— 050.“. 000.. .80» 0:. 000.020.. 00.. .03.: 0.00 0.00: 0.8... 0.:00 00.0... -0... 50-0.0.0. :oE.00 002:0..2 00.0.. - m.— .:o-:0.0. 006.00 c0020.... 00.0.8 (um‘mdd) uogsnuoouoo 80d 90-0.0.0. 58.00 :05... 00.0..- 1 N .5030. 58.00 :05... 00.0..D r md 116 Table 25. Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for raw carp fillet PCB Concentration (ppm, wet) PCB Homologs skin-on skin-off % difference £11101: £11101: (9‘) Tri-CBS 0.039 0.028 28.2 (0.230) Tetra-CBS 0.502 0.356 50.2 (0.090) Penta-CBs 0.806 0.530 34.2 (0.115) Hexa-CBs 0.444 0.262 41.0 (0.078) Hepta-CBs 0.195 0.114 41.5 (0.276) OCta-CBS 0.029 0.012 58.6 (0.039) Total PCBs 2.015 1.303 35.3 (0.075) 1 Probability; significant level at P < 0.2 117 Table 26. Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for raw chinook salmon fillet PCB Concentration (ppm, wet) PCB Homologs skin-on skin-off % difference (P1) Tri-CBS 0.032 0.025 21.9 (0.247) Tetra-CBs 0.410 0.273 33.4 (0.000) Penta-CBs 1.105 0.719 34.9 (0.000) Hexa-CBS 0.364 0.401 -10.1 (0.401) Hepta-CBs 0.203 0.164 19.2 (0.306) Octa-CBS 0.048 0.058 -20.8 (0.319) Total PCBs 2.161 1.641 24.0 (0.000) 1 Probability; significant level at P < 0.001 118 (Table 26) . The presence or absence of skin and adipose tissues significantly affected the total PCBs and its homologs in the raw chinook salmon and carp fish fillets based upon the above results. About 30% of tetra- and penta- homologs deposited on the skin tissue and associated fatty tissue of carp and chinook salmon fillets, respectively, can be reduced by removal of skin and associated fatty tissue before cooking, through skin removal and trimming procedures. An average of 30% (25 - 35%) of total PCBs can be eliminated through trimming procedures; these results are similar to the Hora (1981) study that the effectiveness of removal of the skin alone resulted in 26% to 30% of PCBs and lipids losses, respectively, in carp fillets from upper Mississippi river. Skea et al. (1979) found that removal of the skin, dorsal and ventral fat, and the entire lateral line from Lake Ontario smallmouth bass and brown trout resulted in 64% and 43% reduction of Aroclor" 1254 in fish fillets, respectively. Sanders and Haynes (1988) indicated that total PCB level in bluefish fillets reduced 27% after the removal of bellyflap adipose tissues which was close to the 28 percent reduction of lipid. Armbruster et al. (1989) stated that trimming bluefish fillets resulted in an average reduction of PCB residues of 59%. They also stated that concentration of PCBs in skin contained about twice that found in the fillet muscle expressed on a ppm wet weight basis. Voiland et al. (1991) reported that percent loss of total PCBs and fat content in brown trout from Lake Ontario through skinning and fat- 119 trimming procedures was 46% and 62%, respectively. It may be said that more than 1/4 to 1/2 of total PCBs concentration found in the raw fish fillet can be feasibly eliminated through recommended trimming procedures. Voiland et a1. (1991) also confirmed that the effectiveness of the fat- trimming procedure on the reduction of PCBs in fish fillet is consistent despite wide variation in the initial (untrimmed fillet) levels of contamination. Song (1994) reported that skin removal in carp fillets from the Great Lakes reduced 35% of total PCBs concentration, which was down to 1.24 ppm from 1.90 ppm (wet wt.) . that the highest reduction percent was for octa-CBs homolog (59%) , followed by hexa- (42%) , hepta- (38%) , penta-CBs (34%). and that the least reduction was for tri-CBs (28%). Zabik et al. (1995) found that average total DDT compounds in skin-on chinook salmon and carp were 0.79 ppm and 0.21 ppm, respectively, and the average level of the DDT complex for skin-off salmon fillets and carp were 0.38 ppm and 0.08 ppm, respectively. About 50% of the total DDT was eliminated through trimming and skin removal procedures. 11. Method of the Skin Removal After Cooking Skin removal after cooking also feasibly reduces the levels of contaminants in fish tissue. In processing, skin removal before cooking does require skillful hands and tools in order to reduce the loss of fish tissue and preserve the integrity of the fillet appearance. However, it is quite easy to peel off fish skin after cooking regardless of the cooking 120 methods. The effectiveness of Skin removal after cooking, including the effectiveness of cooking procedure, was obtained from skin-on carp (12 pairs) and chinook salmon (35 pairs), walleye (35 pairs) and white bass (11 pairs) based upon the levels of total PCBs and PCB homologs in dry fish weight by paired T-Test at level of 5%. Through the cooking process, cooked fillets normally had less.water content than uncooked fillets. Therefore, it would be inadequate to compare concentration of total PCBs and PCB homologs for raw and cooked fillets based upon their wet weight. The following tables (Tables 27-30) presented the effectiveness of skin removal after cooking on the reduction of total PCBs and PCB homologs for Great Lakes fish species based upon their concentration (ppm) in wet weight and in dry weight. The results (Tables 27-30) indicated that PCB concentration (ppm) expressed in dry weight is the more sensitive unit than ppm in wet weight for deriving the comparison between raw and cooked fish fillets from the same fish, unlike the previous comparison between raw skin-on fillets with raw skin-off fillets of which both had similar water content and solid contents, but were not processed from the same fish. It was also noted that concentration of tri- and octa-CBs homologs in dry weight for carp, chinook salmon, walleye, and white bass were significantly reduced by skin- removal after the cooking process, which was not seen as significant in the comparison of PCB concentration in wet weight. Effectiveness of skin removal after the cooking 121 Table 27. Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for carp through cooking and skin removal after cooking process1 PCB Homologs ,COnoentration ppm(wet wt) ppmtdry wt) ug (wet wt) Raw skin-on carp fillet Tri-CBs 0.026 0.105 3.180 Tetra-CBs - 0.373 1.464 49.622 Penta-CBs 0.627 2.359 84.199 Hexa-CBs 0.285 1.152 37.919 Hepta-CBS 0.087 0.349 12.102 Octa-CBs 0.018 0.070 2.551 Total PCBs 1.416 5.500 189.574 Carp fillet skin removal after cooking1 Tri-CBs 0.024 0.077' 2.313‘ Tetra-CBs 0.318 0.995‘ 32.721‘ Penta-CBs 0.558 1.691' 59.658‘ Hexa-CBs 0.254 0.793‘ 27.053' Hepta-CBs 0.081 0.250‘ 8.767‘ Octa-CBs 0.019 0.058' 1.935‘ Total PCBs 1.254 3.865‘ 132.447‘ :panfrying significant level at p<0.05 122 Table 28. Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for chinook salmon through cooking and skin removal after cooking process1 PCB Homologs Concentration ppm(wet wt) ppm(dry wt) ug (wet wt) Raw skin-on chinook salmon fillet Tri-CBB 0.032 0.118 18.310 Tetra-CBS 0.410 1.508 244.587 Penta-CBB 1.105 4.099 647.269 Hexa-CBS 0.364 1.374 211.570 Hepta-CBs 0.203 0.761 118.242 Octa-CBS 0.048 0.180 27.746 Total PCBs 2.161 8.041 1268.001 Chinook salmon fillet skin removal after cooking1 Tri-CBs 0.031 0.094‘ 13.234‘ Tetra-CBS 0.378 1.127‘ 165.079‘ Penta-CBs 0.971 2.916‘ 414.322‘ Hexa-CBs 0.312 0.963‘ 133.456‘ Hepta-CBs 0.162 0.493‘ 67.587‘ Octa-CBs 0.044 0.135‘ 18.826‘ Total PCBs 1.897 5.727‘ 812.504' :either baking or charbroiling significant level at p<0.05 123 Table 29. Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for walleye through cooking and skin removal after cooking process1 PCB Homologs Concentration ppm(wet wt) ppm(dry wt) ug (wet wt) Raw skin-on walleye fillet Tri-CBs 0.008 0.035 0.781 Tetra-CBs 0.158 0.716 15.821 Penta-CBs 0.368 1.670 35.497 Hexa-CBs 0.266 1.215 25.442 Hepta-CBs 0.094 0.429 9.139 Octa-CBs 0.015 0.066 1.460 Total PCBs 0.908 4.131 88.141 Walleye fillet skin removal after cooking1 Tri-CBs 0.006 0.022‘ 0.481' Tetra-CBs 0.134 0.493‘ 10.432‘ Penta-CBs 0.292 1.066' 21.709‘ Hexa-CBs 0.182 0.665‘ 13.394‘ Hepta-CBs 0.049 0.181‘ 3.704‘ Octa-CBs 0.012 0.045' 0.911‘ Total PCBs 0.675 2.471‘ 50.632‘ feither baking or charbroiling significant level at p<0.05 124 Table 30. Effectiveness of skin removal on the reduction of total PCBs and PCB homologs for white bass through cooking and skin removal after cooking process1 PCB Homologs Concentration ppm(wet wt) ppm(dry wt) ug (wet wt) Raw skin-on white base fillet Tri-CBs 0.009 0.040 0.728 Tetra-CBs 0.221 0.931 16.725 Penta-CBs 0.547 2.322 41.266 Hexa-CBs 0.357 1.519 26.610 Hepta-CBs 0.118 0.503 8.734 Octa-CBs 0.027 0.114 2.003 Total PCBs 1.279 5.428 96.066 White base fillet skin removal after cooking1 Tri-CBs 0.008 0.026‘ 0.469‘ Tetra-CBs ‘ 0.174 0.594' 10.525‘ Penta-CBs 0.451 1.551‘ 27.177‘ Hexa-CBs 0.326 1.115‘ 19.571‘ Hepta-CBs 0.112 0.381‘ 6.446‘ Octa-CBs 0.024 0.082‘ 1.478‘ Total PCBs 1.094 3.748' 65.666‘ :panfrying significant level at p<0.05 125 process on total PCBs and PCB homologs for carp, chinook salmon, walleye and white bass was significantly reduced when comparing the right side fillets with the left side fillets of the same fish based upon their dry weight measurement. An average of 29% reduction of total PCBs had occurred in carp and chinook salmon through the cooking and skin removal process based on dry weight. A 40% and 31% reduction of total PCBs were found in walleye and white bass, respectively, based on dry weight. Effectiveness of cooking process on the reduction of total PCBs and PCB homologs for skin-off chinook salmon was represented in Table 31. Compared to the cooked chinook salmon from Table 28 and 31, the total PCBs for cooked chinook salmon fillets was reduced from 5.730 to 4.424 ppm on dried basis There was 23% further reduction of total PCBs based on dry weight calculation. It may indicate that skin removal before cooking can further enhance the reduction of contaminants; perhaps through the increased cooking loss, fat dripping and other mechanisms. There is a need for more studies in the effectiveness of skin removal after the cooking process to ensure the safety aspect of consuming higher levels of contaminated fish species which should have had their skin removed before cooking . III. Selective Cooking lethod to Enhance the Safety It has been unpleasant for sportsman to catch fish and not consume them due to fear and doubt about their safety. In this section, seven different cooking methods on the reduction 126 Table 31. Effectiveness of cooking process} on the reduction of total PCBs and PCB homologs for skin-off chinook salmon PCB Homologs Concentration ppm(wet wt) ppm(dry wt) ug (wet wt) Raw skin-off chinook salmon fillet Tri-CBs 0.025 0.100 9.966 Tetra-CBs 0.273 1.083 101.029 Penta-CBs 0.719 2.883 260.902 Hexa-CBs 0.401 1.619 140.778 Hepta-CBs 0.164 0.764 58.586 Octa-CBs 0.058 0.239 20.153 Total PCBs 1.641 6.607 591.413 Cooked skin-off chinook salmon fillet Tri-CBs 0.023 0.065‘ 6.689‘ Tetra-CBs _ 0.248 0.732‘ 68.431‘ Penta-CBs 0.615 1.826‘ 167.607‘ Hexa-CBs 0.305 0.915‘ 80.318‘ Hepta-CBs 0.112 0.312‘ 27.179‘ Octa-CBs 0.042 0.125' 10.865‘ Total PCBs 1.337 3.974‘ 361.090‘ feither baking or charbroiling significant level at p<0.05 127 of total PCBs and PCB homologs will be discussed. Because fish species, origin of lakes, fillet size, cooking media, and cooking methods all influence the concentration of PCBs, percentage change (%) , derived from the difference between the micrograms of each congener in the raw and cooked fish fillets, was used to evaluate the effectiveness of the cooking method. Table 32 listed percentage change of total PCBs and PCB homolog concentration during the cooking process for the composite fish fillets. Positive 'values are jpercentage decreases and negative values are percentage increases. For skin-on fillets, smoked fillet (48%) had the highest total PCBs percentage change, followed by deep fat fried fillet (39%), charbroiled skin-on (38%), baked skin—on (33%) and panfried fillet (31%); among skin-off fillets, charbroiled fillets (36%) had the highest total PCBs percentage change, followed by baked fillet (32%), canned fillet (29%). deep fat fried fillet (28%), panfried fillet (27%) and salt-boiled fillet (21%). Charbroiling. baking, and deep fat frying are all convenient methods of cooking for reducing contaminants effectively. The effectiveness of cooking methods on the reduction of total PCBs and PCB homologs might be related to either long cooking time or large volume of cooking oil. Reinert et al. (1972) found that the broiling and frying method could reduce DDT 64-72% in Great Lakes lake trout. Zabik et al.(1982) also reported that PCB, dieldrin and DDT levels had been reduced 53, 48 and 39%, respectively, in lake trout by broiling; 34, 25 and 30%, respectively, by roasting 128 mém VOAN mmdm m.5v odm mmdm mo. pm mmém wmdm mvdm No.5m mmbm med .305 mmhm mwdm w P .vw —v.©m mmém mo.5p cm.w— mmd- mm.m~ 5mém N5.mm 0.0m mmuouoo “£0-88: N5.pm mmfip- Fmém 954.; mvév mfmm m5.—N pod— vvém mdm mmdv mm.5m mmém 9;: mmdm N054 w5.mm wodm PO.5N Nadm modm 5m.mm mvdv mm.mm mmuoxoz ovdm mm 5N.om Penn mN.5m mdv 5. pm PmdN mm. pm mfmw cm.wm MMNM 30-851 No.5 mud— mdm made m5.mm ohm moém NN.mN 50.2mm 5m.mw 05.; mmdm 6.8.25 68-3 modm 55.5 mm.NN vmmm- vm5m mvdm 5...©N vodm mod. vwd— emdm mdm oc_mm_E 29:3 95.. mm N N F to m P to N P co m P co N P to em co m P to 0m co mm to we so we to $383 Exw memo—2 .28. .. zonimm coo 96:5 31:50 Browne Brown. from.“ .oxmm exam €5.20 4:99:30 £5522 mmoooto ucfioou octet mood .39 so can cozobcoocoo 529:9. mod to mucosa 38:356. .Nm o3: 129 lake trout; and 26, 47 and 54%, respectively, by microwaving lake trout. A current study by Zabik et al. (1993) revealed that PCB levels based on packed-column GC—quantitation in lake trout can be reduced 10-17% through baking, 12-59% through charbroiling, and 10% through salt boiling. Ambruster et al. (1989) found that there was an average reduction of 7.5% from baking, broiling, frying or poaching on trimmed bluefish fillets. Song (1994) indicated that there were no significant differences between deepfat frying and pan frying carp fillets in relation to total PCBs reduction, but that both methods reduced PCBs by an average of 34%. Shearer and Price (1993) used a mass basis to summarize PCB loss from some earlier studies. They concluded that there was an average of 22% PCB loss by baking chinook salmon, lake trout, small mouth bass, and bluefish; 27% loss by broiling lake trout and brown trout; 56% loss by frying small mouth bass and white croaker; and 26% loss by microwaving lake trout. Zabik et a1 (1995) studied the reductions of PCBs quantitated by' packed-column GC analysis and pesticides in chinook salmon harvested from the Great Lakes; they found that average losses of pesticides, such as DDT complex, chlordane, oxychlordane, nonachlor, HCB, dieldrin, heptachlor expoxide, toxaphene and total PCBs from the chinook salmon ranged from 30 to 50%, and that those charbroiled with an increased surface area had a higher cooking loss than regular charbroiled fillets. Cooking will not alter the distribution pattern of PCB homologs; however, it facilitates the reduction of PCBs through cooking loss, 130 liquefying of fats, and volatilization. Relationship of Physical Parameters with Chemical Parameter of the Great Lakes Fish Species for the Consumption Advice With. all the collected. data (chemical and. physical parameters) of fish species from the Great Lakes, the relationship between the size of fish species and their concentration of total PCBs from specific raw skin-on fillets was determined. Linear or nonlinear regression models were used to examine the relationship between fish length, weight and total PCB concentration. The highest r’ generated by SYSTAT, with a minimum of 0.8, was used as the best fit regression equation. TWenty—four carp skin-on fillets from Lakes Erie and Huron; 36 chinook salmon skin-on fillets from Lakes Huron and Michigan; 12 lake trout skin-on fillets from Lakes Michigan and Superior; 41 walleye skin-on fillets from Lakes Erie, Huron, and Michigan; and 12 white bass skin-on fillets from Lakes Erie and Huron were used. The concentrations of total PCBs were derived from the addition of 53 individual PCB specific congeners measured.by GC-capillary column with the PCBs expressed as ppm wet basis. The results of the nonlinear relationship between the length.and the weight of fish.with total PCB concentration for each of the five species are presented in Figures 19 - 23. By selecting a quadratic relationship, it resulted in increasing 1? values from 0.1 to 0.8. Quadratic effects also influenced 131 Total PCBs - 58.458 + 1.331 * length - 8.846 * length3-r 0.001 * weight + (-8.846) * weight2 (R2 I 0.77) lawmakwnwefi u e o o 51 m N ‘ is Figure 19. Concentration of total PCBs in relation to length and weight of carp harvested from Lakes Erie and Huron 132 Total PCBs - -l.985 + (-0.07) * length + 1.086 * length2 (R2 I 0.954) (D N $7 § § 2 Figure 20. Concentration of total PCBs in relation to length and weight of chinook salmon harvested from Lakes Huron and Michigan 133 length’-+ 0.035 * weight + (-3.186) * weight2 (R2 I 0.94) Total PCBS :- (-294.196) + (-5.781) * length + 92.32 * 4° Figure 21. Concentration of total PCBs in relation to length and weight of lake trout harvested from Lakes Michigan and Superior 134 Total PCBS I (297.25) + 6.2 * length + (-85.584) * 1ength’-+ 0.004 * weight + (-0.l49) * weight2 (R2 - 0.826) 10m 9088 m we“ Figure 22. Concentration of total PCBs in relation to length and weight of walleye harvested from Lakes Erie, Huron and Michigan 135 Total PCBs . (137.434) + 4.751 * length + (-53.072) * length2 + (-0.019) * weight + 0.972 * weight’ (R2 :- 0.971) I'//////Il’;‘oo " fill/I49"! (Wm 440.8400.) ,- 7 7 ‘ °§°¢‘§\ 1.1/go”! ' O O O 7 2 r’¢¢§ 9‘s‘\~« . 4’17“”! O '0'0'...‘ 3 4 4 4 4‘8. , , ‘ 7...,424444 o o o o m g .:””:’§‘{"9235‘313249?t-£’4"’27"0':’:”’.%%. 7 O 7. 9‘ '42'.)"“?3‘3*?<35?“ deep fry with skin-on (39%) > Charbroil (37%) > bake (33%) > panfry (29%), can (29%) > deep fry with skin-off (28%) and salt-boil (21%) based on total micrograms per fillet. The differences of cooking methods on the reduction of total PCBs are related to fish species, lake effects, cooking media, cooking time and lipid adsorption on fish tissue. In order to protect the health of anglers and their family members, state and federal agencies have monitored Great Lakes fish continually, and fish consumption advisories implement guidance in order to reduce the risk of consuming contaminated fish” The most accessible method for the sports fishermen is to follow the fishing guide based upon the size of fish. The study validating the relationship between size of fish and levels of total PCBs is predominated by length more than by weight. Also, there are some other toxic chemicals existing in the Great Lakes ecosystem, including pesticides, dieldrin, transnonachlor, lindane, DDT and ‘mercury, which all can pose as hazards to the public and.Great Lakes fish eaters. States and local public health agencies will need to provide extended information and education to the public and anglers on the effects of belly flap trimming and skin removal before cooking or skin removal after cooking, as well as propagating the most effective cooking methods for maximum residue reduction. RECOMMENDATIONS Since Great Lakes fish species are very sensitive bio- indicators for aquatic contaminants, some future research should be implemented: 1. Determination of the effects of cooking time and volume of cooking oil used on the reduction of total PCBs. 2. Establishment of a standardized procedure for the determination of total PCBs and PCB congeners by federal regulatory agencies to be made available through the Internet for the public and private sectors. 3 . Improvement of measurement of fat content in fish fillets based on gel permeation chromatography and high.pressure liquid chromatography. 4. More studies on the effectiveness of skin-removal on the reduction of PCB specific congeners, after cooking by microwave. This is a simple and easy procedure for consumers. 5. More research on toxicity of individual congeners and combinations of specific congeners. 6. Future risk assessments should be established on congener specific environmental data and the health effects of exposure of individuals to specific congeners for more 146 147 accurate fish consumption advisory. Studies on the acceptance of fish advisory recommendations by sports fishermen and their families for the protection of their health; particularly among young children, females especially pregnant or lactating; Native American Indians, and other minority and ethnic population who consume whole fish instead of fish fillet. 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Eon 600.800: E. .0 600-200: 213 .5... 03.80. 0 2.0 x. ”Dunn—Ian: 0..—...: .3.... 08.000. .00..: 09.000. 38.: 08.000. .03 ~25 c. .25 c. is .03 c. a .3048... Eng 600.88 E .3388 214 Appendix 6. 'Pearson correlation matrix on age, length and weight of the great lakes fish procurement E C J I' 2 EE' . I Age/Wt. Length/Wt. Fish Species Age/Length Carp O.605*** Chinook salmon 0.862*** Lake trout -0.651*** Walleye 0.187 White bass 0.500 O.612*** O.855*** 0.840*** 0.909**. -O.665*** o.972*** 0.186 0.709*** 0.154 0.455 *** indicated the significant 0.001). Pearson correlation (P < Appendix 7. 215 Wilcoxon Signed Ranks Test on solids and lipid content of raw and cooked fish fillets from the Great Lakes Fish species 2 Test1 2 Test on Solids on Lipids Carp 6.031*** 3.429*** Chinook salmon 7.961*** 2.326** Lake trout 6.970*** 1.814* Walleye 5.646*** 3.076*** White bass 3.059*** 1.782* I 2: (sum of signed ranks) 7 square root (sum of squared ranks) * indicated the significant Wilcoxon Signed Ranks Test (P < 0.1). ** indicated the significant Wilcoxon Signed Ranks Test (P < 0.05). *** indicated the significant Wilcoxon Signed Ranks Test (P < 0.005). LIST OF REFERENCES LIST OF REFERENCES AOAC. 1984. "Official Methods of Analysis". 14th ed. Association of Official Agricultural Chemists , Washington, D.C. Anonymous. 1989. Sport Fish: What are the Risks? National Wildlife. 27:19. Armbruster, G., Gall, K.L., Gutenmann, W.H. and Lisk, D.J. 1989. 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