LOCATEON OF LENDANE; MELDREM AND DDT IN EGGS. Thesis for the Degree of Ph. Dc MICHIGAN STATE UNIVERSITY MARY ELLEN ZABIK 1970 IIIIIIIIIIIIIII III III IIIIIIIII'I/ 31293 01066 3486 \fiES‘S This is to certify that the thesis entitled LOCATION OF LINDANE, DIELDRIN, AND DDT IN EGGS presented by MARY ELLEN ZABIK has been accepted towards fulfillment of the requirements for _Eh_._D_._degree in FOOD SCIENCE W@m9¢ Maj?! professv Date MAY 12, 1970 0-169 LIBRARY 1 Michigan State University ABSTRACT LOCATION OF LINDANE, DIELDRIN, AND DDT IN EGGS BY Mary Ellen Zabik In order to study the effect of the amount and type of lipid present on pesticide residue accumulation, three groups of eggs were collected from hens fed feed contaminated with 25 ppm lindane, dieldrin, and DDT, separated, and the yolk fractionated using salt solution separation techniques in combination with ultracentrifugation into lipovitellin, lipo- vitellenin, livetin and phosvitin fractions. Electron cap- ture GLC was used to determine the pesticide residue levels; p,p'-DDE and o,p'-DDT-DDD were also found in the eggs. The albumen, vitelline membrane and yolk fractions were also ana— lyzed for moisture, protein, phosphorus, total, neutral, and phospholipids. The ppm of lindane and dieldrin associated with the albumen was within the limits of water solubility but the DDT compounds were present in amounts exceeding this value. The lipid-free phosvitin also contained amounts of dieldrin and DDT compounds in excess of their respective water Mary Ellen Zabik solubilities. Protein pesticide complexes are probably responsible for these associations. Although all three pesticides were fed at the same level, wide variation occurred in the amount transferred to the yolk and these amounts reached a plateau with the second group. The amount of lipid within the egg yolk fraction exerted the greatest influence on the residue accumulation. Lipovitel- lenin contained 80 to 85% of the total residues with lipovi- tellin containing approximately 10% and livetin 5%. The proportion of pesticide residues in the lipovitellenin frac- tion decreased with increasing concentrations of pesticide residues in the eggs while the proportion in lipovitellin correspondingly increased. When the amount of lipid in each fraction was compen- sated for, no significant differences occurred among the egg yolk fractions even though lipovitellin possessed higher residue levels. Correlating lipid composition to pesticide residue accumulation in lipovitellin, lipovitellenin and livetin fractions indicated dieldrin, p,p'-DDT and p,p'-DDE related to a slightly greater extent to the weight of neutral lipid present in the fraction than to the amount of total lipid. Lindane deposition, however, appeared to be related to the proportion of phospholipid present in the fat. Mary Ellen Zabik A corollary study into the potential of freeze-drying for residue removal from whole eggs and egg yolks was con- ducted. Significant pesticide reductions of 79% for lindane, 57% for dieldrin, 57% for p,p'-DDT, and 51% for o,p'-DDT-DDD occurred in freeze-dried whole eggs. Half the lindane was removed by freeze—drying egg yolk but only minimal removal occurred for the other pesticides. Approximately a 20% increase in p,p'-DDE occurred in both the freeze-dried whole eggs and yolks. The success of freeze-drying for pesticide removal from eggs appeared related to both the vapor pres- sure of the pesticide and the amount of contamination in the whole egg. LOCATION OF LINDANE, DIELDRIN, AND DDT IN EGGS BY Mary Ellen Zabik A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1970 ACKNOWLEDGMENTS The author expresses her gratitude to Dr. LeRoy Dugan Jr. for his kindness, helpfulness, and guidance during her graduate program. His encouragement toward independent re- search and his continuing interest in the project are deeply appreciated. Sincere thanks are expressed to Dr. Pearl Aldrich, Dr. Lawrence Dawson, Dr. Charles Stine, and Dr. Duane Ullrey for the advice and encouragement given as members of the guidance committee. Sincere appreciation is also expressed to Dr. Robert Ringer and the Poultry Science Department for providing the hens and the facilities to keep the hens during the collection of pesticide contaminated eggs. Gratitude is also expressed to the Pesticide Analytical Laboratory of the Pesticide Research Center for assistance in developing gas chroma- tographic procedures for residue analyses and to Dr. Robert Brunner for aid in protein separations. Sincere thanks are expressed to Dr. Dena Cederquist for her encouragement to initiate a graduate program and her sup- port during periods of combining graduate study with a teaching-research position in the Department of Foods and Nutrition. Appreciation is also expressed to the General Foods Corporation for supplying fellowships to encourage graduate study. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . Egg Yolk Composition. . . . . . . . . . Granules . . . . . . . . . . . . . Lipovitellin. . . . . . . . . Phosvitin . . . . . . . . . . Lipovitellenin . . . . . . . . . . Livetins . . . . . . . . . . . . . Macromolecular Composition During Oogenesis Yolk Components During Embryogenesis . Chlorinated Hydrocarbon Pesticides. . . Pesticide Residues in Poultry and Eggs Residue Removal by Volatilization and/or Heat 0 O O O O I O O O I O O 0 EXPERIMENTAL PROCEDURE . . . . . . . . . . . Source of Eggs. . . . . . . . . . . . . Separation of Eggs. . . . . . . . . . . Freeze-Drying . . . . . . . . . . . . Chemical and Pesticide Residue Analyses Lipid Analyses . . . . . . . . . Pesticide Residue Analyses . . . . Eggs. . . . . . . . . . . . . Feed. . . . . . . . . . . . . GLC Analyses. . . . . . . . . Analyses of the Data. . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . Lipid Composition . . . . . . . . . . Pesticide Analyses. . . . . . . . . . . Lindane. . . . . . . . . . . . . . Dieldrin . . . . . . . . . . . DDT and Related Compounds. . . . Relation of Pesticide Content to Lipid Sition. O O O O O O O O O O 0 iii 58 58 4O 45 44 45 46 46 47 48 48 50 53 57 57 62 66 7S TABLE OF CONTENTS--Continued Potential of Freeze-Drying for Reducing Pesti- cide Levels in Eggs. . . . . . . . . . . . SUMMARY AND CONCLUSIONS. . . . . . . . . . . . PROPOSALS FOR FUTURE RESEARCH. . . . . . . . . . . . REFERENCES CITED . . . . . . . . . . . . . . . . . . APPENDICES . . . . . . . . . . . . . . . . . . . . . I. Feed Composition . . . . . . . . . . . . . . II. Average Moisture Contents of the Albumen, Vitelline Membrane, Egg Yolk and Egg Yolk Fractions. . . . . . . . . . . . . . . . . . III. Percentages of Protein, Lipid and Phosphorus as well as Lipid and Protein Phosphorus of Egg Yolk Fraction (Based on Solids). . . . IV. Summary of Analyses of Variance. . . . . . . V. Distribution of Pesticide Expressed as a Function of the Amount of Phospholipid and Neutral Lipid in the Lipovitellin, Lipovitel- lenin, and Livetin Fractions . . . . . . . . iv Page 80 85 90 92 101 101 103 105 104 105 LIST OF TABLES TABLE 1. Lipid composition of lipovitellins and lipovitel— lenin (Martin g£_§l,, 1965). . . . . . . . . . . 2. Fatty acid distribution in lipovitellin and lipo- vitellenins (Martin §t_al,, 1965). . . . . . . . 5. Total DDT residues in egg yolk, abdominal fat and other tissue of broilers and hens (Liska §£_gl,. 1964). . . . . . . . . . . . . . . . . . 4. Diminution of pesticide residues (Stadelman §£_§l,. 1965). . . . . . . . . . . . . . . . . . 5. Pesticide residues found in fortified cottonseed oils at various stages of processing (Smith §£_§l,. 1968). . . . . . . . . . . . . . . . . . 6. Yields, percentage recovery, and percentage dis- tributions of yolk fractions for three egg col- lection groups . . . . . . . . . . . . . . . . . 7. Percentage total lipids, neutral lipids, and phospholipids,determined both by adsorption on activated silicic acid and from phosphorus con- tent, in the egg yolk fractions. . . . . . . . . 8. Total lindane in yolk and its distribution and recovery in the egg yolk fractions . . . . . . . 9. Lindane residues in albumen, vitelline membrane, whole yolk and yolk fractions (ppm based on ‘ total solids). . . . . . . . . . . . . . . . . . 10. Lindane residues in the vitelline membrane, whole yolk, and yolk fractions (ppm based on lipid content) . . . . . . . . . . . . . . . . . 11. Total dieldrin in yolk and its distribution and recovery in the egg yolk fractions . . . . . Page 10 12 52 55 57 52 55 58 59 61 65 LIST OF TABLES--continued TABLE 12. 15. 14. 15. 16. 17. 18- 19. Dieldrin residues in albumen, vitelline membrane, whole yolk and yolk fractions (ppm based on total solids). . . . . . . . . . . . . . . . . . -Dieldrin residue in the vitelline membrane, yolk and yolk fractions (ppm based on total lipid). . DDT compounds in yolk and its distribution and recovery in the egg yolk fractions . . . . . . DDT compounds and its isomers in albumen, vitel- line membrane, whole yolk and yolk fractions (ppm based on total solids). . . . . . . . . ADDT compounds in the vitelline membrane, yolk and yolk fractions expressed as a ppm of the total lipid concentration. . . . . . . . . . . Correlation of the mg of each pesticide with the weight of total, neutral, or phospholipid present in each egg yolk fraction. . . . . . Correlation of ppm (based on lipid) of each pesticide with percentage neutral or phospho— lipid. . . . . . . . . . . . . . . . . . . . . Summary of the effect of freeze-drying on pesti- cide residue contents of whole egg and yolk (ppm based on solids) . . . . . . . . . . . . . . . vi Page 64 67 69 71 74 78 78 85 FIGURE 1. LIST OF FIGURES Summary of pesticide distribution in egg yolk fractions based on their respective solids content. . . . . . . . . . . . . . . . . . . . .Summary of pesticide distribution in egg yolk fractions based on their respective lipid con- tent . . . . . . . . . . . . . . . . . . . . . Distribution of pesticides in liquid and three samples each of freeze-dried whole egg and egg yolk . . . . . . . . . . . . . . . . . . . . . vii Page , 76 . 77 . 82 INTRODUCTION When DDT was introduced for the control of malaria, it was lauded as one of the great contributions to the welfare of mankind, leading to the awarding of the Noble prize to its discoverer. This universal acceptance of DDT has now been reevaluated so that in 1969 the use of DDT was banned first in Michigan and then in many other states. Although no one can dispute the great contribution pesticides have made to the control of insect borne diseases, to increased agricultural production, and to the control of household pests thus improving the quality of living the world over, the almost universal occurrence of pesticide residues in soil, water, air, and living organisms has lead to pesti- cides now being looked upon as environmental contaminants or more spectacularly in some of the popular press as insidi- ous, uncontrollable poisons (Mitchell, 1966). The chlori— nated hydrocarbon insecticides have attracted particular attention because of their "persistence" and adverse affects to some birds and to aquatic eco-systems. Due to their lipid solubility, the chlorinated hydro- carbon pesticides are readily partitioned into the fatty material of living organisms. Numerous studies have shown variation in pesticide content among selected tissues and species (Casarett t 21,, 1968; Durham, 1967; Matthysse t $1., 1966), gt 31,, 1968: Rumsey t 31,, 1967; Wesley but information concerning the distribution of pesticides within lipid-containing material is almost non-existent. A better understanding of the mode of pesticide association within the lipid-containing material of the organism may lead to theories of how pesticides are stored and then mobilized possibly leading to a better understanding of their action within the organism. Egg yolk lipoproteins differ considerably both in amount of lipid material and in the relative proportion of neutral to phospholipids. The low density lipoprotein, lipovitel- lenin, contains 80-90% lipid of which approximately 26% is phospholipid; whereas the high density lipoprotein, lipovi- tellin, contains 18-25% fat which is made up of about 50% phospholipid (Cook, 1968). Several studies have shown that feeding pesticide contaminated feed to hens results in the deposition of the pesticide in the eggs (Cummings g; $1., 1966; Herrick t 31,, 1969; Stadelman gt al,, 1965: Wesley §£_§l,, 1966, 1969). Thus, in order to investigate the effect of the amount and type of lipid present on the amount of selected pesti- cides associated with each fraction, hens were fed contami- nated feed, and pesticide residues were determined in the whole yolk, in the lipovitellenin and lipovitellin fractions as well as in the protein fractions, phosvitin and livetin. Residue analyses were also conducted on the albumen and the vitelline membrane. In order to further investigate the relation of lipid solubility of the chlorinated hydrocarbon pesticides to their location of incorporation, the following pesticides were used: Lindane (gamma isomer of 1,2,5,4,5,6-hexachlorocyclo- hexane), dieldrin (1,2,5,4,10,10-hexachloro-6,7-epoxy- 1,4,4a,5,6,7,8,8a-octahydro-1,4-gggngxgr5,8-dimethano- naphthalene), and DDT (1,1,1-trichloro—2,2-bis(pfchloro- phenyl)ethane) which are 6.80, 0.195, and 0.025 ppm water soluble, respectively at 25°C (Gunther t 33., 1968). Even though the mere presence of pesticide residues in living organisms does not necessarily jeopardize public health, it seems prudent to minimize the pesticide residues in our food supply until more definitive studies elucidate the long range effects of accumulating pesticide residues in human and animal tissues. Chlorinated hydrocarbon pesticides have been removed from milk by molecular distil- lation (Bills and Sloan, 1967), from oil during commercial processing, particularly deodorization (Gooding, 1966; Smith 33 333, 1968) and from water by codistillation (Mitchell, 1966). In addition, Ruzicka _3.3;. (1967) reported a 15-25% loss in selected chlorinated hydrocarbon insecticides during the roller-drying of whole milk. Thus, it seemed feasible that drying of eggs might also be useful in reducing the residue level. Therefore, a corollary study into the pos- sible removal of lindane, dieldrin, and/or DDT and its isomers from whole eggs and egg yolks by freeze—drying was carried out. REVIEW OF LITERATURE Since the primary emphasis of this investigation was to study the possible effects of the amount and type of lipid present in egg, particularly in the egg yolk lipoproteins, upon the accumulation of chlorinated hydrocarbon pesticides, studies concerning egg yolk composition are reviewed in the first portion. The second section of this review summarizes information concerning the chlorinated hydrocarbon pesticides particularly studies relating to their accumulation in eggs and poultry as well as studies concerned with methods of removal by volatilization and/or heat. Egg Yolk Composition According to Cook (1968) egg yolk contains protein, lipid, and water in the proportion of 1:2:5. Approximately 2% of the solids are lost on dialysis; the rest of the com- ponents are present as macromolecules. Using centrifugation techniques, the egg yolk macromolecules can be readily sub- divided into three fractions: the sedimenting granules (primarily lipovitellin and phosvitin) representing 25%:3f the yolk solids; the low density fraction (lipovitellenin accounting for 66%tof the yolk solids; and the dissolved protein (livetin). All these proteins have been further fractionated. Lipovitellin and lipovitellenin both contain lipid whereas phosvitin and the livetins are lipid-free. These yolk contents are surrounded by the vitelline membrane, separating them from the egg albumen. This vitel- line membrane is composed primarily of mucin and keratins (Parkinson,.1966). Doran and Mueller (1961) stated that these occurred in two layers with the protein being the mem- brane proper and the second mucin layer corresponding to the chalaziferous membrane. Granules When yolk is diluted with an equal volume of water or 0.16 M NaCl and centrifuged at 44,000 G for 1 hour, a bright yellow pellet comprising 25% of the total yolk solids is obtained (Cook, 1968). The granules can be further subdi- vided into lipovitellin, including a small portion of con- taminating low density fraction, and phosvitin. Burley and Cook (1961) reported that ionic or secondary forces are involved in the binding of the phosvitin to the lipovitellin in the sedimenting granule. Lipovitelllg, As early as 1900, Osborne and Campbell used brine precipitation to separate a yolk protein which they named nucleovitellin (now lipovitellin) and whose composition they reported to consist of protein, lecithin, and phosphoric acid. Little further work had been reported on the separation of egg yolk proteins until Chargaff (1942) isolated the lipovitellin complex by diluting the yolk with sodium chloride, extracting with ether, and precipitating the protein by dilution. He determined its lipid, nitrogen, phosphorus, and sulfur contents to be 18%, 15%, 1.5%, and 0.9%, respectively. Both lecithin and cephalin were found to be components of the phospholipids. Alderton and Fevold (1945) obtained a lipovitellin preparation with a slightly lower lipid content by first obtaining the granule. Both preparative procedures failed to remove all the phosvitin (Cook, 1968) so that the protein phosphorus of these preparations was over 1%. Using differential precipitation from magnesium sulfate solutions to remove the phosvitin, Joubert.and Cook (1958a) reduced the protein phosphorus content of their lipovitellin preparation to 0.50%. Wallace (1965) found phosvitin to be soluble in 100% saturated ammonium sulfate and collected the precipitated lipovitellin by high speed centrifugation. From electorphoretic and ultracentrifugal investiga- tions, lipovitellin was found to be heterogeneous (Sugano, 1959; Sugano and Watanabe, 1961). Bernardi and Cook (1960a) resolved the two lipovitellins electrophoretically and termed them d- and B-lipovitellin. Both the d- and B-lipovitellin sediment as a single boundary in 1 M NaCl but at higher pH values they both dissociate to yield a slower sedimenting component, with B—lipovitellin dissociating at lower pH values than d-lipovitellin. These researchers concluded that both lipovitellins appear to behave as reversible association- dissociation systems. Using chromatography on hydroxyapatite columns, Bernardi and Cook (1960b) separated the d- and B-lipovitellins. B-lipovitellin was eluted with a 0.6 M phosphate buffer and 01,-» with a.2M buffer. Phosvitin remained on the column. Burley and Cook (1961) first removed the phosvitin using a Dowex-l column but reported that recovery of d- and B-lipo— vitellin from the hydroxyapatite column was incomplete. Radomski and Cook (1964) modified the procedure and used a triethylaminoethyl-cellulose (TEAE) column with gradient elution, obtaining essentially complete recoveries. Wallace (1965) also used TEAE cellulose to separate proteins of avian and amphibian yolk granules. Bernardi and Cook (1960b) stated that both a- and B- lipovitellin have similar molecular weights of 4.0 x 105. These workers (1960c) further reported both lipovitellins to dissociate in 4 M urea to give a molecular weight of half that observed in 1 M NaCl. Moreover, B-lipovitellin will dissociate at a lower urea concentration than will the a- fraction. The lipovitellin dissociation in urea is partial- ly reversible upon reducing the concentration of urea. More recently, sedimentation analyses and equilibrium molecular weight determinations of a- and B-vitellins in 6 M guanidinium chloride provided evidence for a light molecular weight frac- tion in the range of 50,000 and a heavy fraction of molecular weight 110,000-140,000 in both proteins. Both lipovitellins had also been shown to dissociate in acid solvents at pH 2.0 (Kratohvil _£_3;,, 1962) but the irreversible change which occurred precluded quantitative studies. Bernardi and Cook (1960b) thought a- and B-lipovitellin to have similar amino acid compositions; but a later study indicated B-vitellin had a lower proportion of histidine (Cook 33 333, 1962). The protein phosphorus has been re- ported as 0.50 and 0.27% for d- and B-lipovitellin, respec- tively, but lipid content was more variable, ranging from 14.6 to 22% (Burley and Cook, 1961; Radomski and Cook, 1964; Wallace, 1965). Using solubility in petroleum ether and ethanol, Evans and Bandemer (1961) determined the amount of tightly and loosely bound lipid in lipovitellin. Their preparation con- tained 21.7% lipid, most of which was firmly bound. The loosely bound lipid was nearly all neutral fat whereas phos- phatidyl choline was the major constituent of the ethanol- extractable lipid. The neutral fat consisted primarily of palmitic and oleic acids while the phosphatidyl choline was very high in palmitic acid with smaller amounts of stearic and oleic acids. Martin 33 33, (1964) also studied the lipid distribution in egg yolk lipoproteins. The composition of the egg yolk lipoproteins is presented in Table 1. Their B-lipovitellin preparation, prepared using a hydroxyapatite column, was O 1 .mxcmHQ How omuomuuou .oascmum CH cowuomuw mufimcmo 30H u 0mQQ¢ .cofluomum muwmcmo 30H m mane .oflmwa Hmuusoc ca ooosauch .mcoflumcflauoumo mumoflamwuu Mom mmmum>m we musmwm gonna mm.o mm.m «m ea.o m.¢d whoa mm m¢.o m.ma ewmnq m.ma e.¢e o.m ma.o mm.m s.mm m.ms om .eo.o o.¢e mag w.ma o.H> m.m Nm.o mo.¢ m.mm m.¢> mm Ha.o o.¢a man m.ma m.m> m.m mm.o mo.¢ hm da.o o.¢a when a.>a m.m> m.m ¢H.O oa.¢ m.mm m.>m mm #m.o m.ma cflaamuw>0dwaa m.sfi «.me o.e Hm.o ma.¢ m.mm a.oe mm $0.0 m.ma cussmuu>omuuta madam GHQ» mcwamcmmuomma umumm moum onHH oamfla & ,Rm R2 wamfimm IHocmsuo leooq .cwsuflomaomwa NHoumummHoco Iosmmonm Hmuusmz .oflmfla. camuoum 1H>owu .cflamonmcflcmm R .wmmHH mo coHuHmeroo Hmuos Imnmmosm &.ofimaHoanosm moacoflwwmomaoo .Ammma .aflm um cwuumzv newsmaamufl>omfia ocm mceaaoufi>omaa mo cofluflmomaou named .H OHQMB 11 slightly higher in lipid than the a—component. It also con- tained a higher proportion of phospholipid. Their neutral lipid component was composed of 40 to 50% oleic acid with another 25 to 55%Iof palmitic acid as shown in Table 2. The authors reported that the slight differences in fatty acid distribution between the two lipovitellins were of minor importance. In contrast to the fatty acid composition of phosphatidyl choline reported by Evans and Bandemer (1961), these workers found about equal proportions of oleic and palmitic acids, with the former being slightly higher. In a general discussion of lipoprotein structure, Cook and Martin (1962) related the protein, phospholipid and neutral lipid ratio to their structure. These authors sug- gest that lipoproteins occur in two classes which are dis- tinguished by a transition in their composition in the region in which the previously mentioned constituents are present in 1:1:1 proportions. The higher protein lipoproteins of which the lipovitellins are examples, were considered to have more ordered structures and to be molecular in contrast to the micelle or microemulsion description for low protein lipoproteins. tMore recently, Evans 3£_3l, (1968), presented data re- flecting the influence of various dissociating treatments and of enzyme digestion upon the ether-extractable lipid of lipovitellin. Their data indicated that most of the lipid is held within the protein network with about 57% or less of 12 .mcoflumummmum o3u mo msam> mmmum>¢a o.e s.afi s.mm m.ma e.mm s.fi mmcuaoeo Iamoeumcmmosm «.03 o.sm m.mm fi.m e.mfi m.a mmcusmaocmnum uasenumsmmoem o.av >.m m.mm m.m N.mm m.m ofimfla Hmuusmz acuaamuu>omuqum m.a m.ae m.mm m.ma «.mm o.m mmcuaosu namwflpmnmmosm fi.m m.mm a.¢m m.oe 3.0m m.a menusmaocmsum nameuumnmmogm o.fiv m.ma o.me m.ma m.om H.m asses Hmuusmz acuaamuu>omuuna o.ev fi.m m.om m.m m.mm «.m was o.ev e.m e.me m.m m.em ¢.m mag e.m m.me e.oe o.oe 0.0m o.mv m.e ¢.mfi m.mm s.ma a.mm m.mv auasmuu>omuuuu a.m a.ma H.me m.oa s.em o.mv m.a m.ma m.mm s.ae m.am «.mv auaamuu>omuqta «now ouma sums mama oumfi sums mamsmm .Ammma .mm cuuumzv mcflcmauwun>omua new auaamuu>omus as couusnuuumue chum spams .m magma 15 the lipid bound on or near the surface of the lipovitellin. This surface lipid consisted of 17 molecules of triglycer- ide, 4 molecules of sterol, 4 molecules of lecithin and 1 molecule each of cephalin and.mono-cn:diglyceride. Other experiments showed this amount of surface lipid to vary with the method of preparation, solvent composition and other un- known variables. Treatment of lipovitellin with 8 M urea or 6 M guanidine hydrochloride not only did not increase the ether-extractable lipid, indicating hydrogen bonding was not primarily responsible for the binding of the lipid to the protein, but actually substantially reduced the lipid extract- ability. Nonionic detergents, such as Brij-55, Tween-80, etc., also appeared to stabilize the lipid binding. Evans _3_3l, (1968) also stated that hydrophobic bonding appears to be involved in binding part of the lipids to the protein or in holding the protein molecule in shape so that the lipids are unextractable since sodium dodecyl sulfate treatment increased the ether-extractable lipid by 54%. Enzymatic digestion also substantially increased lipid extract- ability. These authors concluded that lipovitellin appears to consist of a protein chain folded in a globular configur- ation with lipid, equal to one-fourth of the weight of the protein held in pockets throughout the molecule. Phosvitin. Mecham and Olcott (1949) first isolated phosvitin by diluting yolk with magnesium sulfate rather than sodium chloride which had been used in all previous separations. 14 In the former system, phosvitin is precipitated at ionic strengths which will retain lipovitellin in solution. Joubert anxi Cook (1958b) precipitated phosvitin from an egg yolk solution in 0.4M magnesium sulfate by adding equal volumes of water. The purified phosvitin was lipid free and contained 9.6% phosphorus. Phosvitin has also been isolated by butanol extraction (Sundararajan, Sampath-Kumar, and Sarma, 1960); ammonium sulfate precipitation of lipovitellin followed by diethylaminoethyl cellulose chromatography puri- fication of the remaining phosvitin (Wallace 33_3l,,1966)7 and chromatographic separation on TEAE cellulose (Radomski and Cook, 1964). Ultracentrifugation studies show phosvitin to be homo- geneous with molecular weights ranging from 5.6 x 104 (Cook, 1961) to 4.0 x 104 (Mok _£__l,, 1961; Allerton and Perlmann, 1965). Nevertheless, Mecham and Olcott (1949) had shown phosvitin to be heterogeneous using electrophoretic studies and this was substantiated by the work of Bernardi and Cook (1960a). The heterogeneity appeared to be dependent upon the solution characteristics including ionic strength and the concentration of bivalent cations. More recently, Mok 33_3l, (1966) fractionated phosvitin into two components by countercurrent distribution. Both phosvitin components had similar nitrogen and phosphorus compositions. Allerton and Perlmann (1965) reported the amino acid composition of phosvitin. Serine residues comprise approxi- mately 60% of the entire molecule with minor amounts of 15 various basic amino acids making up the rest. Sulfur and proline are absent; in addition, its very low tyrosine and tryptophane contents distinguish it from the other egg yolk proteins. Most of the high phosphorus content is present as phosphoserine residues; Williams and Sangar (1959) estab- lished that at least six phosphoserine residues are grouped together in the polypeptide chain, while Belitz (1966b), using trypsin digestion of the partially dephosphorylated phosvitin, obtained peptides with 5,4, or 6 serine residues separated by 1 or 2 basic amino acids. Belitz (1966a) also supported the heterogeneity of phosvitin by reporting 1 mole of phosvitin contained 2 moles of alanine and approximately 1 mole of serine as N-terminal amino acids. Previously only alanine had been found (Mok 3£_3;., 1961; Neelin and Cook, 1961). Lipovitellenin Fevold and Lausten (1946) first isolated a second lipo- protein from diluted egg yolk by repeated centrifugation followed by ether extraction of the supernatant. This preparation which they termed lipovitellenin could be dis- tinguished from lipovitellin since it contained approximately 40% lipid and only 9-10% nitrogen. Once ultracentrifugal techniques were established, numerous separations of a low density lipoprotein from egg yolk were reported. In 1954, Nichols and associates isolated a low density lipoprotein from egg yolk by flotation and 16 reported this preparation was 89% lipid. .Sugano (1958) re- ported the preparation of a low density lipoprotein from eggs which he termed B-lipovitellin and described his prep- aration as the major component of Fevold and Lausten's lipovitellenin. Vandegaer 33_3l, (1956) reported a study of whole yolk using the analytical centrifuge. They observed the floating low density fraction gave a pattern suggesting two components were present. ~Electrophoretic and ultracentrifugal investi- gations by Sugano (1959) collaborated the heterogeneity of lipovitellenin. Sugano and Watanabe (1961) separated lipo- vitellenin into two components which gave distinctly differ- ent ultracentrifugal patterns but had similar lipid content of approximately 84%. They calculated the molecular weights of these fractions to be 9.0 x 106 and 4.8 x 106. This com- pares to an unfractionated molecular weight of 5.7-4.5 x 105 (Vandegaer 33.33., 1956; Joubert and Cook, 1958a). Martin and coeworkers (1964) reported that about one- fifth of the low density fraction was LDFl with a mean mole- cular weight of 10.5 x 106 and the remaining was LDFg with a molecular weight of 5.5 x 106. Both fractions were poly- disperse. LDFl had slightly higher total lipid and phos- pholipid contents with 86.8% and 27.5% as compared to 85.2% and 22.4%, respectively, for LDFg. Saari and associates (1964) reported similar differences in total lipid content between the two components of lipovitellenin and further 17 established that the two components reacted differently to heat and papain digestion. They theorized that the component with the lower protein content may have more protein—phos— pholipid interactions thus rendering the polypeptides more resistant to proteolysis. Both vitellenin fractions are glycoproteins (Augustyniak and Martin, 1968). Neelin and Cook (1961) found both arginine and lysine were N-terminal amino acids of vitellenin while Cook 3£_3l, (1962) reported the amino acid composition as well as phosphorus and sulfur contents. Vitellenin can be distinguished from vitellin by its lower sulfur and phosphorus content as well as differences in the amount of 10 amino acids, thus establishing basic differences in these egg yolk lipo- proteins beyond the lipid composition. aEvans and Bandemer (1961) reported most of the 82% lipid in their lipovitellenin preparation to be loosely bound. Phosphatidyl ethanolamine was the principle constituent of the tightly bound lipid and palmitic, stearic, and oleic com- prised the major portion of its fatty acids. Martin 33.33, (1965) reported that the low density fraction of egg yolk contained approximately 89% lipid having 27% phospholipid, 69% triglyceride, and 4%3cholesterol and choleSterol eSters (see Table 1, page 10). The fatty acid composition of lipo- vitellenin was also given in Table 2, page 12. Oleic acid laccounts for 50% of the fatty acids, palmitic for 28%, and linoleic and linolenic each for 12%. In contrast to the 18 results of Evans and Bandemer, phosphatidyl choline was reported to be the major phospholipid as it had been for lipovitellin and was earlier reported to be for whole yolk by Rhodes and Lea (1957). From the partiCle size and lipid content of the low density lipoproteins, a protein film of 8 A could cover two- thirds of the surface area of each of the two lipovitellenins (Martin 3£_3;,,1964). Earlier, Cook and Martin (1962) had theorized that the low density lipoprotein belonged to the "lipid-core" type of lipoproteins with a hydrophilic surface of protein and phospholipid. More recently, Steer and co- workers (1968) demonstrated that 20% of the protein of native low density lipoprotein was not accessible to Pronase attack. They envisioned three types of protection that could account for this unavailability: the backbone of the resistant pep- tide is interdigitated between surface lipid; the peptide is located at a contact area between associated lipoprotein molecules; or the peptide is buried within the neutral lipid core. For the latter to occur, the peptide would also have to be folded to accommodate the relatively high proportion of polar amino acids in the residual peptide. Studying the type of lipid released from the low density lipoprotein following detergent, urea and similar compounds and enzyme treatments, Evans 3£__;, (1969) reported that trypsin digest released 84%Iof the protein as soluble pep- tides. Treatment of the residue with pepsin or Pronase 19 resulted in further release of both protein and lipid. Neither nonionic detergents, urea, nor guanidine hydrochloride increased the ether-extractable lipid but sodium dodecyl- sulfate and sodium deoxycholate increased the ether lipid extractability by 50%. These authors concluded their data supports the view that the low density lipoproteins are a sphere of lipid surrounded by a layer of protein and phos- pholipid with hydrophobic groups on the inside and hydro— philic groups on the outside resulting in water solubility and insolubility in organic solvents. Evans and associates also stated that variation in size of the low density lipoproteins is related to lipid content. Citing other data from the same laboratory, they reported the molecular weight of the protein portion remained constant but the amount of triglycerides increased in the larger molecular weight low density lipoproteins as did the amount of phospholipid but to a lesser extent. Apparently size in- creases results from increases in neutral lipids in the core of lipovitellenin and by increased phospholipid in between the protein strands on the lipovitellenin's surface shell. Livetins Plimmer (1908) named the water soluble proteins of egg yolk, livetin. In 1949, Shepard and Hottle reported the livetin fraction could be separated electrophoretically into three or more components. The heterogeneity of livetin was confirmed by Martin and co-workers (1957) who termed their 20 three electrophoretically separable components a, 6—, and y-livetin with molecular weights of 8.0 x 104 and 4.5 x 104 for d- and B-livetin, respectively. Martin and Cook (1958) obtained y-livetin by precipitation with ammonium sulfate and their preparation had a molecular weight of 1.5 x 105. Williams (1962) and Mok and Common (1964a,b) used im- munoelectrophoresis to establish that the antigens for the egg yolk livetins were present in hen serum thus indicating the yolk livetins to be serum proteins. Hui and Common (1966), using starch gel electrophoresis, resolved sixteen zones, of which seven were major, six minor, and the remain- ing three faint and diffuse. Two of the major zones were identified as transferrin. Williams (1962) had previously identified d-livetin as chick serum albumin, B-livetin as ag—glycoprotein, and y-livetin as y-globulin of blood serum. The starch gel electrophoresis showed 8- and y-livetin also contained other proteins but in lesser amounts. Macromolecular Composition During Oogenesis MacKenzie and Martin (1967) determined the relative proportion of granules (lipovitellin-phosvitin), low density fraction (lipovitellenin), and water soluble fraction (livetins) during yolk maturation. Although total yolk solids and the amount of each fraction increased exponentially dur- ing yolk formation, differential rates of formation were established. 21 The proportion of the low density fraction increased rapidly to a maximum of 76%‘When the yolk weighed 2-5 g and then decreases to approximately 65%; thus, it is always the predominant fraction. In contrast, the proportion of gran- ules decrease until the egg yolk weighs 2-5 9 and then raises slightly. The proportion of phosvitin in the granule in- creased slightly as the yolk went from 2-5 9 to greater than 15 g. The proportion of the water soluble fraction also de- creases slightly until the yolk'weighs 2-5 9 and then remains steady. Sedimentation and electrophOretic analyses of this fraction indicated the proportion of a- and B-livetin in- creased while the y-livetin decreased as the egg yolk matures. Yolk Components During Embryogenesis Saito _3.3l. (1965) reported that although only small quantitative and qualitative changes occurred in whole yolk and its fractions from fertile eggs during the first 12 days of incubation, marked changes took place during the latter portion of incubation. The proportion of granules and low density fractions decreased while the water soluble portion increased. Qualitatively, the lipid rich low density frac— tion remained constant but the granules lost phorphorus and both ultracentrifugal and electrophoretic patterns indi- cated at least one new component was formed. Martin and Saito (1967) further studied.the lipovitellin- phosvitin complex of the granules during incubation. 22 The relative proportion of a—lipovitellin decreased but its protein phosphorus content was stable whereas the relative proportion of B-lipovitellin remained stable but it exhibited a reduced protein phosphorus. Two new granule components appeared after 15 days of incubation but were not resolved. Earlier Saito and Martin (1966) investigated further the in- crease in the water soluble fraction. Ovalbumen, evidently entering from the egg white, represented a large proportion of the increase in the water soluble fraction and its presence made detection of changes in the livetins more difficult but d-livetin (serum albumin) apparently disappeared most rapidly and most completely. Without identifying the specific proteins, Mulkern and Clegg (1968) reported that the fast moving phosphoproteins disappeared during the 14th to 19th days of incubation. The P/N ratios indicated nitrogen was used more rapidly be- tween the 7th and 14th days and the reverse was true between the 14th and 19th days. Noble and Moore (1965,1967) studied changes-in yolk lipids during embryogenesis. A marked increase in the phos- phatidyl choline to phosphatidyl ethanolamine ratio occurred during incubation. Moreover, phosphatidyl ethanolamine was the only phospholipid fraction which exhibited any consistent change in fatty acid composition, with the concentration of docosahexenoic acid decreasing from 8% at the start of incu- bation to 1.6% after 21 days. 25 The transport of yolk lipid to membrane was extremely active during the 15th to 17th day of incubation so that by the 17th day, the yolk membrane contained as much lipid as the yolk itself. There was an increase in the esterified to free cholesterol ratio in both yolk and yolk-sac membranes giving evidence that the transport of lipid is associated with extensive esterification of yolk cholesterol, mainly with oleic acid. By comparing the fatty acid compositions and positional distributions, the authors concluded the major proportion of yolk triglycerides were absorbed intact by the yolk-sac membrane. Chlorinated Hydrocarbon Pesticides The chlorinated hydrocarbon pesticides include a wide number of compounds which have been grouped as DDT and its derivatives including DDE(1,1-dichloro-2,2-bis(p-chlorophenyl) ethene) and DDD(1,1-dichloro-2,2-bis(p-chlorophenyl)ethane), and related compounds including Kelthane and methoxychlor; cyclodiene compounds including aldrin, dieldrin, endrin, and heptachlor; and miscellaneous residual compounds such as benzenehexachloride (BHC) and lindane. All of these com— pounds possess very low water solubility and are relatively persistent. Dieldrin is not less than 85% of 1,2,5,4,10,10-hexa- chloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4-33397333r 5,8-dimethanonaphthalene (HEOD). It is a broad spectrum 24 insecticide with high contact and stomach toxicity. Feeding dieldrin to rats established its acute oral LDso to be 100 mg/kg but no changes were observable in the rats after con- tinuous feeding of 5 ppm of dieldrin for up to two years. Tinsley (1966) reported that 20 ppm of dieldrin accentuated essential fatty acid stress in that it stimulated the trans— formation of linoleate to arachidonate as well as inducing other mixed function oxidases. DDT is 1,1,1-trichloro-2,2-bis(pfchlorophenyl)ethane. Its action spectrum is by ingestion and contact. DDT posses- ses an acute oral LDSO in male rats of 115 mg/kg; continuous feeding of dietary levels of 5 and 400 ppm to rats and dogs, respectively, produced minimal or no effect (Mitchell, 1966). Lindane is the gamma isomer of not less than 99% purity of 1,2,5,4,5,6-hexachlorocyclohexane. Its action spectrum is that of a stomach poison with persistent contact toxicity and fumigant action against a wide variety of insects. Continuous feeding for up to two years of 50 ppm to rats and greater than 15 ppm to dogs produced minimal or no effect. Various factors influence the storage and excretion of the chlorinated hydrocarbon pesticides. Besides inherent differences for the compounds themselves, storage is affected by mode and efficiency of absorption, intensity and duration of dosage, sex, age, species, and tissue type (Hayes, 1965). Although the physical characteristics of DDT and dieldrin are quite similar, different modes of absorption have been 25 cited. Schafer (1968), reporting the data of Rothe and co- workers, stated that when radioactive DDT was administered orally to rats with cannulated thoracic lymph ducts, approxi- mately half the absorbed radioactivity appeared in the chyle. Absorption reached a peak 1.5 to 2.5 hours after administra- tion; one-half of the total DDT administered was absorbed within seven hours and 95% by 18 hours. Their data did not rule out the possibility of some absorption from the in- testine via the hepatic portal system, but it appeared un- likely. In contrast, Heath and Vandekar (1964) observed that most C136-labeled dieldrin was absorbed via the portal vein whereas only 1/7 of the radioactivity was recovered from the lymph. Schafer (1968) stated that 61% of the total dieldrin in whole blood was present in serum. Previously, Moss and Hathaway (1964) presented data concerning the distribution of c14-1abe1ed dieldrin and Telodrin. Most of the radioac- tivity occurred in the plasma and erythrocyte contents with very little occurring in leucocytes, platelets, or stroma. These researchers reported similar g; M and i_3 yi_1_:_rp_ distribution among soluble proteins in the blood and cited a 1:2 ratio between cells and plasma. Erythrocyte stroma is freely permeable to dieldrin and Telodrin. Hemoglobin was largely responsible for binding the pesticide in the erythrocyte contents, but an unidentified component bound a small portion. 26 Moss and Hathaway (1964) further stated that binding in the plasma involves interactions with serum albumin and a constituent of the globulin fraction which may be lipo- protein in nature. The binding to the serum proteins is not decreased with increasing concentration of insecticide nor is increasing concentration accompanied by a spill over to other proteins. Although the binding of these insecticides closely resembles the complex binding of corticosterols and thyroid hormones, the actual pattern for distribution of the insecticides appears specific for these substances. Following absorption, the initial distribution of pesti- cides is quite universal, but 33_yiyg_redistribution soon causes the majority to be found in the fat (Schafer, 1968; Heath and Vandekar, 1964). This redistribution occurs to a greater extent after repeated doses than after a large single dose. Rumsey 33 33, (1967) reported total DDT isomers appeared to be evenly distributed throughout beef tissues when calculated on the fat basis although their values ranged from 285 ppm in the diaphram to 1422 ppm in the psoas major. These researchers, however, cited work of Harrison and Shanks which showed differences in the distribution of pesticide in sheep fat. There is also some indication that the occurrence of DDT and dieldrin in tissues other than adipose tissue is deter- mined by their lipid content and especially by their neutral fat content (Hayes, 1965). Nevertheless, Hayes went on to 27 say that the distribution of DDT in the visera before and after starvation suggests factors other than fat content _p§£_33 are involved. Recently, Hugunin and Bradley (1969) fractionated, by ultracentrifugation, buttermilk prepared from washed cream naturally contaminated with dieldrin and Kelthane and re- ported a significant decreasing order of residue per gram fat for high density pellet, serum phase, and free fat. These authors indicated their data suggested organochlorine pesticides are associated with the phospholipid in milk fat. The relative dosage of pesticide influences the amount of tissue storage. DDT, aldrin, endrin, and isodrin are less efficiently stored at higher dosage levels but lindane is stored with the same efficiency (Hayes, 1965). At a constant daily dosage, the storage of DDT increases steadily but eventually reaches a maximum or plateau in man and other species reflecting the level of intake. DDE and DDD also reach a plateau storage level but at a later time than is required for DDT. Studies with rats indicated a reduction of storage of dieldrin following peak absorption but this could be related to a decreased food intake as the rats reached maturity (Hayes, 1965). Gillett (1968) related the level of DDT in the diet to microsomal epoxidation of aldrin to dieldrin. Less than 2.0 ppm had no effect but greater than 2.5 ppm produced sig- nificant increases in 6 weeks of feeding female rats and in 28 2 weeks for males. He went on to state that the extent of induction appears directly related to the concentration of DDT in the diet. Thus, there is an interaction among pesti- cides. Feeding DDT and dieldrin simultaneously results in a decrease in tissue accumulation of each. True excretion or elimination of previously absorbed chlorinated hydrocarbon pesticides or their metabolites may occur by way of expired air, urine, feces, milk, dermal secretion, and even the fetus. Braund and co-workers (1967, 1968) reported placental transfer of dieldrin to dairy calves born to dams contaminated during gestation. Approximately 0.9%Iof the ingested dieldrin (5.4 mg/kg diet) was recovered in the calves; the residue then decreased with age. These authors also stated that neither stage of gestation nor dieldrin concentration in body fat of contaminated dams ap- peared to affect residue levels found in the calves at birth. The existence of an enterohepatic circulation of pesti- cides appears to be certain (Hayes, 1965). Cannulation of the bile duct of rats increased the excretion of unchanged dieldrin from 5 to 10% (Heath and Vandekar, 1964). The bile appears to be the principal source of DDT metabolites in the feces. Other pesticides, such as lindane, are excreted in a much higher proportion in the urine. Excretion must account for the fact that equilibrium storage is reached and stores are reduced when dosage is discontinued. The rate of storage loss, however, is not 29 constant after dosage is discontinued. Gannon and Decker (1960) reported that the concentration of dieldrin in milk fell more rapidly at first following removal of contaminated feed, but then became more and more gradual. Wesley 3£_3l, (1969) established the persistence of some DDT in eggs and chicken tissues even when the hens were starved or given the fat mobilizing hormone, androgen, so that 18 to 20 weeks were required to reduce DDT to fairly low levels. Since the concentration of these chlorinated hydrocarbon pesticides can be eventually reduced, they must be able to be mobilized from the storage sites. The mode and factors influencing this mobilization are unknown at the present time. However, the metabolites and/or degradation products of these pesticides have been very recently extensively re- viewed (Menzie, 1969). Pesticide Residues in Poultry and Eggs In a study on the effect of commercial benzene hexa- chloride dust (12.5% gamma isomer) and a deodorized form (99% gamma isomer) on egg and meat quality, taint was de- tected after 4 weeks of feeding 25 ppm and after 6 weeks at 5 ppm of the former whereas eggs were tainted 16 and 18 weeks, respectively, after feeding the later (Milne, 1955 reported by Lindgren 3£_3;,, 1968). YOung chicks fed for 27 days on a mash containing 4, 16, or 64 ppm lindane showed no clinical signs of toxicity but accumulated an average of 19.1, 56.4, 50 and 156.0 ppm, respectively, of lindane in the body fat (Lindgren 33_3;,, 1968). Naber and Ware (1961) reported detectable lindane residues in eggs and tissues of chickens consuming less than 1 ppm of lindane in the diet. Using lindane vapor, Whitacre and‘Ware (1967) reported a significant storage of lindane in egg yolks of hens exposed for 47 days. Although the Hens exhibited considerable variation in the amount transferred to the egg, there was a steady increase with time until the 27th day when the values fluctuated but did not increase over previous values. Liver, fat, and kidney residue levels in- creased throughout the period but the amount in the fat increased to a much greater extent reaching levels of approxi- mately twenty times that in the liver and kidney at the 47th day. Thompson 33_3l, (1967) reported much lower residue con- tamination in egg yolks from hens fed diet contaminated with up to 100 ppm methoxychlor. After 28 days of eating feed containing 100 ppm methoxychlor, these authors reported levels of 0.51 ppm in the yolk. Methoxychlor residues of 0.91 ppm were obtained in the feces of these hens. No residue was found in the liver, brain, or kidney of any of the hens; hOWever, one hen fed a diet with 100 ppm methoxychlor had 1.4 ppm in the visceral fat. This hen also exhibited higher levels of methoxychlor in both the eggs and feces thus vari_ ation may be due either to the amount of food consumed or to the rate of methoxychlor excretion. 51 Oral consumption of 250 or 500 ppm of the organophos- phate malathion did not result in any detectable residues in the egg or in chicken tissues (Marion 33_ 1., 1968). Direct application of a 1% malathion spray (1.5 gal/100 ft2) to nests, lower side walls, floors, and hens themselves re- sulted in significant residues on the skin and feathers and in some instances on the egg shell but only trace amounts occurred in the egg contents. Herrick 33_3l, (1969) studied the effect of chlorinated hydrocarbon contamination of "safe" insecticide dusts (Co-Ral, malathion, and Sevin) on the residue level found in the eggs. No methoxychlor was found in the eggs even when 5% Sevin dust was contaminated with 2000 ppm. In contrast, 200 ppm levels of BHC and DDT resulted in levels of 0.55 and 0.55 ppm, respectively, in the whole eggs. After 50 days of feeding hens a ration contaminated with 0.1 and 0.5 ppm DDT, Liska 33,33, (1964) reported DDT accumu— lation in eggs and tissues of hens and broilers as shown in Table 5. Low levels of DDT in the ration resulted in higher tissue levels in the broilers than in mature laying hens. This may be explained by the much higher rates of tissue formation in broilers or by the hens having the possible mechanism of excreting~fat soluble contaminants via the egg. Further studies (Stadelman 3£__l,, 1965) indicated that supplying the equivalent of 0.1 to 0.15 ppm of lindane, dieldrin, heptachlor, and DDT in the feed to laying hens in 52 Table 5. Total DDT residues in egg yolk, abdominal fat and other tissue of broilers and hens (Liska 33_3;, 1964). Hens Broilers 0.1 ppm DDT 0.5 ppm DDT 0.1 ppm DDT 1.0 ppm.DDT Sample ration ration ration ration Egg yolk <0.1 1.5 Body fat 0.5 2.6 0.90 2.55 Skin 0.2 1. 0.80 1.25 Thigh <0.1 0.5 0.75 1.25 Breast <0.1 0.2 0.60 0.85 a capsule form for 14 days resulted in 0.2 to 0.5 ppm accumu- lation of lindane and DDE in the abdominal fat but this was no longer detectable 4 weeks after pesticide feeding was discontinued. Residues could not be detected in the eggs from these hens. However, feeding the same pesticides at a level equivalent to 10 to 15 ppm for 5 days resulted in sig— nificant residues which were extremely persistent (Table 4) in eggs and depot fat. t 3;. (1966) studied the effect of using semi- Wesley starvation followed by either a high or low protein diet on the removal of DDT from egg yolk and abdominal fat of laying hens. .Diet did not affect the initial deposition of DDT in either the abdominal fat or the egg yolk. Eight weeks after DDT was injected, DDT removal from both the abdominal fat and egg yolk was most efficient using the reduced feeding 55 N.0 0.0 N.0 0.0 m.0 0.0 mm m.0 N.0 H.0 0.0 m.0 0.0 ha «.0 0.0 m.0 H.0 0.d a.0 0a xaom 00m 0.0 m.a N.d m.0 N.d a.0 m m.0 0.0 m.0 0.0 0.0 0.0 a 0.0 0.0 m.0 0.0 0.0 0.0 mm 0.0 0.0 0.0 d.0 0.H 0.0 00 0.0 o.a 0.e e.0 0.m «.0 0e new Loewe N.a m.m N.0 0.0 m.¢ m.O m 0.0 0.0 0.0a. N.0 0.m v.0 H man Ban mofixomm HOHSU cwuoamfln unspeaq musmomxm monsom Heanumumwm Imummm Hmuwm mxmmz Emu .Ammma ..Hm MM.cmEHmoMumv mwsoflmmu mowofiumwm mo cofluscflawn .0 manna 54 with a high protein diet, followed by the reduced feeding with a low protein diet. These differences continued to be significant until 16 and 22 weeks for egg yolks and fat, respectively, at which time very low residues were found in all instances. The chickens fed a reduced high protein ration lost the most weight and were so depleted in abdominal fat that these researchers found it necessary to scrape the intestinal organs and abdominal cavity to obtain one—gram fat samples. Wesley 3£_3l. (1969) further studied the effect of three different periods of starvation, incorporation of the fat mobilizing hormone, androgen, and three levels of protein on DDT residue removal from hen abdominal fat and from egg yolk. Only the inclusion of androgen had any significant effect on the level of DDT in the abdominal fat with the DDT residue being slightly higher in the fat of hens fed the hormone. This was also true for egg yolk levels. The length of star- vation and the amount of protein in the diet also signifi- cantly affected the amount of DDT in the yolk. Reduction of DDT levels was slightly greater in hens starved for 48 hours in comparison with 0 or 96 hr. Diets with 45% protein resulted in slightly greater DDT depletion in egg yolks after 8 weeks than did diets with 20 or 75% protein. Following the combined feeding of 0.05 to 0.45 ppm of lindane, heptachlor epoxide, dieldrin, endrin, and DDT, Cummings 3$.33, (1966, 1967) determined the pesticide residues 55 in the hen's abdominal fat, breast muscle, liver, and whole eggs. All pesticide residues reached a plateau whose level reflected the amount of pesticide in the diet. The time for the residue to reach this plateau varied with the type and level of pesticide, being shorter for lindane and longer for DDT and its isomers. The greatest storage was in the fatty tissue where dieldrin and'heptachlor epoxide attained plateau levels approximately 10 times the respective levels in the feed while endrin, p,p'-DDT, and lindane were stored to a lesser degree. Dieldrin and heptachlor epoxide were also stored to the greatest extent in the eggs reaching levels approximately equal to that in the feed. The respec- tive storage tendencies of the pesticides were also similar for the breast and liver but the levels of storage were much less. Upon return to the basal diet for one month, only lindane values dropped to the background level in the eggs and breast tissue and were approaching this level in the abdominal fat. Residue Removal by Volatilization and/or Heat In a research note in 1966, Gooding reported commercial processing resulted in an oil free of chlorinated organic pesticide residues. Commercial grade aldrin, BHC, chlordane, DDT, dieldrin, heptachlor, heptachlor epoxide, Kelthane, lindane, methoxychlor, sesone, strobane, TDE, and Toxaphene in amount of three to ten times the highest established FDA tolerance for oil bearing seeds was used to spike cottonseed oil prior to processing. 56 Smith 33,33, (1968) verified that normal commercial processing of crude vegetable oils for human consumption effectively removes any chlorinated hydrocarbon pesticides present in the crude oil. Using fortification levels of 1.0 ppm of endrin, DDE, aldrin, dieldrin, and heptachlor -epoxide and 21.0 ppm of DDT to crude cottonseed oil, the authors reported residue levels after refining, bleaching, and deodorization (Table 5). Neither alkali-refining nor subsequent bleaching reduced the pesticide content. Hydro- genation prior to deodorization reduced endrin contamination, but the greatest loss in pesticide contamination resulted from the deodorization step in processing. -Deodorization, with or without prior hydrogenation, eliminated the chlori- nated hydrocarbon pesticides. Mitchell (1966) cited data concerning the loss of in- secticides from water by codistillation for 20 hrs at 26.50C. Over 90% of aldrin and heptachlor codistilled whereas lindane and p,p'-DDT exhibited only 50% codistillation. Dieldrin was intermediary with 55% codistillation. Mitchell, however, further reported nearly quantitative removal of aldrin and dieldrin from water in one to two hours by aeration. .Bills and Sloan (1967) reported 95 to 99% of added lindane, heptachlor, heptachlor epoxide, aldrin, DDT, DDE, and TDE were successfully removed from milk fat by a laboratory scale molecular distillation apparatus. Langlois 33H3L. (1964) processed milk contaminated with lindane by feeding the cows 57 Table 5. Pesticide residues1 found in fortified cottonseed oils at various stages of processing (Smith 33.33,, 1968). Refined Bleached Deodorized oil oil oil PPm .Endrin 0.42 0.65 BDL2-0.08 DDT 11.1 12.5 BDL -0.08 DDE O . 92 O . 85 BDL Aldrin 0.55 0.65 BDL Dieldrin 0.72 > 0.64 BDL Heptachlor 0.58 0.69 BDL Heptachlor epoxide 0.77 0.86 BDL lAverage pesticide values of two oils with three independent laboratory analytical values per oil. 2Below detectable limit of analysis. and with DDT by spiking. Although condensing had no effect on the pesticide residues, spray—drying reduced the DDT from 26.00 ppm (based on fat content) to 9.85 ppm and the lindane from 25.00 ppm to 4.58 ppm. Drum drying also reduced the lindane content but to a lesser extent (9.27 ppm). These authors, however, reported drum drying resulted in an almost two-fold increase in DDT in the dried milk but gave no explana- tion for this phenomenon. Roller drying resulted in a 15% loss of dieldrin and p,p'-DDE, a 20% loss in B—BHC, p,p'-TDE (DDD) and p,p'-DDT, and a 25% loss in a—BHC, y-BHC and hepta- chlor epoxide from whole milk (Ruzicka 33.3;,, 1967). EXPERIMENTAL PROCEDURE In order to investigate the relationship of the type and amount of lipid present to the storage of selected chlorinated hydrocarbon insecticides, eggs from hens fed a contaminated ration were subdivided and analyzed. In addi- tion, whole eggs and yolks were freeze-dried to determine the potential of this process for residue removal. Source of Eggs Ten single comb White Leghorn hens which were approxi- mately 10 months of age at the time of the experiment were fed a standard laying ration which was contaminated with 25 ppm each of Lindane (99+%, Applied Science Labs, Inc., State College, Pa.), Dieldrin (recrystallized, 99+%, Shell Chemical Company, New York), and p,p'-DDT (ESA pesticide reference standard, 991%, City Chemical Corp., New York) . From the start of laying at approximately.22 wk, these hens had been maintained in individual cages, lOXlBXlBjJL” in a room main- tained at 25120C and these conditions were continued through- out the experimental egg collection. One week prior to the feeding of the pesticide contami- nated food, the hens were switched to the laying ration 58 59 whose composition is cited in the Appendices. All birds were in egg production at the time they were switched to this ration. One hundred lbs of the laying ration were contaminated with 25 ppm each of the previously mentioned pesticides as follows: Four hundred g of the non-contaminated laying ration was removed for pesticide residue analyses, and re- placed by 400 g Wesson oil (mixture of cottonseed and soy- bean oils) in which 1.154 g each of Lindane, Dieldrin, and p,p'-DDT had been dissolved by stirring at room temperature 2 hr on a magnetic stirrer set on low. The pesticide con- taminated oil was mixed for 25 min with 4140 9 feed in a Liquid-Solids Blender, model LB-4598. This mixture was divided into ten 450 9 portions and each portion was added to 4100 g of the original feed and mixed for 5 min. To insure even pesticide distribution, half the contents of two por- tions were remixed together for an additional 5 min. The contaminated feed was then placed in polyethylene bags and after four 100 9 samples were removed at random from four different bags for residue analyses, stored at refrigeration temperature until needed for feeding the chickens. During the experimental feeding period, 10 lb of feed was kept in the cage room until entirely used. The hens were fed the contaminated feed and water 33 libitum. After the hens had been on the contaminated feed for 7 days, the eggs were collected daily Monday through 40 Friday and once on the weekends, dated, and stored at 40C prior to analyses. Egg collection was continued for 25 days and the eggs from the first two 7-day periods designated as Groups I and II, respectively, and the eggs from the remain- ing time as Group III. Separation of Eggs Each group of eggs were broken; the albumen was sepa- rated and pooled. Each yolk was rolled on absorbent towel- ing to remove any adhering albumen and then placed on a double layer of cheese cloth secured over a beaker. The vitelline membrane was punctured allowing the liquid yolk to drain overnight at 4°C. All the yolk from each group of eggs were pooled and 0.02% butylated hydroxy toluene added to minimize oxidation. The vitelline membranes were recovered by alternately soaking the cheese cloth coated with the membranes in de- ionized water and rinsing the ydlk particles away under running deionized water. Final removal of yolk was accomplished by gently swirlingfiimzseparated membranes in three rinses of deionized water using a magnetic stirrer operating at a low speed. Prior to analyses, the final rinse water was removed with a Buchner funnel. The yolk was further subdivided into the following four fractions: lipovitellin, lipovitellenin, phosvitin, and livetin. Approximately 540 g of yolk was diluted 1:1 with 41 deionized water and centrifuged at 40C for lé-hr using a 856 rotor operating at 15,000 rpm in an International High Speed Refrigerated Centrifuge, model HR-1, to sediment the yolk granules. The supernatant, containing lipovitellenin and livetin fractions, was carefully decanted from the granules. After the yield of granules had been determined, the granules were transferred to a 400 ml beaker using 1 M NaCl to wash the centrifuge tubes. One hundred ml of 1 M NaCl was then added to the granules, the beaker securely covered and held at 4°C overnight. By this procedure the granules softened sufficiently so that they could be com- pletely dissolved in the 1 M NaCl by stirring for 1 hr with a magnetic stirrer operating at a medium speed. To further subdivide the granules into lipovitellin and phosvitin fractions, the dissolved granules were diluted with 2 volumes of 100% saturated ammonium sulfate as outlined by Wallace 3£_3;, (1966). The precipitated lipovitellin was recovered by centrifuging at 15,000 rpm for 1é-hr at 40C as described previously. The floating solid lipovitellin was freed of residual phosvitin by three reprecipitations from .ammonium sulfate (Wallace, 1965). After each centrifugation, saturated ammonium sulfate was used to remove any lipovitel- lin particles adhering to the centrifuge tubes after the clear water subnatant had been drained from the tubes. The purified lipovitellin was dialyzed extensively against deion- ized water until free of both chloride, using the silver 42 nitrate test, and sulfate, using the barium chloride test. The clear subnatant fraction from the lipovitellin preparation and purifications contained the protein phos- vitin. To recover this protein, the material was dialyzed extensively against deionized water until it was free of chloride and sulfate. The dialyzed liquid was centrifuged to remove any contaminating pieces from the lipovitellin pel- let and this supernatant adjusted to pH 1.5 with 1 N HCl to precipitate the phosvitin. In order to obtain quantitative yields, the filtrate at pH 1.5 was held overnight at 40C according to the procedure of Wallace 3£_3l, (1966) and the precipitated phosvitin obtained by centrifuging 1 hr at 40C in the 856 rotor of the International centrifuge operating at 12,000 rpm. The supernatant from the original granule precipitation contained the lipovitellenin and livetin fractions. Suffi- cient sodium chloride was added to make this supernatant 1 M NaCl and this was centrifuged for 18 hr at 40C in a No. 50 rotor operating at 50,000 rpm in a Beckman Ultracentrifuge, model L-2. After centrifugation the floating yellow gela— tinous material and bright yellow liquid was removed. The latter was accomplished with the aid of a 50 ml syringe equipped with a No. 8 needle which had been cut at a 900 angle. ‘All the centrifuge tubes and their fittings were rinsed with 1 M NaCl to minimize loss during initial prepara- tion and purifications. Deionized water was used after the 45 final purification. To purify the lipovitellenin, the crude preparation was redissolved in 1 M NaCl and recentrifuged and separated twice (Evans, 1969). The purified lipovitel— lenin was dialyzed against deionized water until free of chloride. The subnatant from the lipovitellenin preparation and purifications contained the livetin fraction. These com- bined subnatants were also dialyzed against deionized water until free of chloride. After total yields were obtained, all protein fractions were stored in tightly sealed glass containers at 40C until further analyses. Freeze-Drying Three 50-g samples each of whole egg and yolk from the third group of eggs were freeze-dried. Yolk was prepared as previously described. Whole eggs were blended for three 15-sec periods with an Osterizer Blender set on low to insure homogeneity. The aliquots were placed in 400 ml beakers, giving a depth of approximately 5/4 in., covered securely with aluminum foil, and frozen at -250C overnight. The frozen samples were transferred to a Stokes Freeze-Dryer, model 2005F-2, and freeze-dried for 24 hr with a system pressure of 100 u. The freeze-dried samples were removed from the beakers, ground with a mortar and pestle, and the powder stored in tightly sealed glass vials at 40C for chemi- cal and pesticide residue analyses. 44 Chemical and Pesticide Residue Analyses All chemical and pesticide residue analyses were carried out in duplicate except for the vitelline membrane whose small quantity did not allow for duplication. The chemicals used were ACS reagent grade and the solvents were redistilled from glass. In addition, all glassware was acetone-rinsed after thorough washing for the pesticide analyses. Hexane, nanograde from Mallinckrodt Chemical Works, was used for all pesticide determinations and preparation of pesticide stand- ards for gas chromographic analyses. In addition to the lindane, dieldrin, and p,p'-DDT listed for the feed contami- nation, standard pesticide solutions in hexane were also pre- pared using o,p'—DDT (98+%, Analytical Standard, Pesticide Repository, Pesticide Research Laboratory, Perrine, Fla.) and p,p'-DDE (98+%, Analytical Standard, Pesticide Repository, Pesticide Research Laboratory, Perrine, Fla.). Percentage solids were determined by drying samples to a constant weight in a Precision Thelco Vacuum Oven, model 29, at 60°C and a vacuum of 28-29 in. of Hg_as outlined by Steer 33_3l,, 1968. ‘Excess moisture was first removed by drying in an air oven at 110°C for 15 min before vacuum dry+ ing. Protein was determined using the microkjeldahl method (AOAC, 1960). Percentage phosphorus of both the egg yolk fractions and the total lipids of each fraction was obtained using the procedures described by Morrison (1964). 45 Lipid Analyses Total lipids of the albumen and whole yolk as well as the lipovitellin, lipovitellenin, and livetin fractions were measured using the chloroform-methanol extraction technique of Bligh and Dyer (1959) with the following modi- fications. The total blending time was increased to 10 min to obtain consistent results and acid-washed Hyflo Super Cel (John Manville Products) was used to facilitate filtration. The filter taid and.precipitated protein were then washed with two 25 ml portions of chloroform-methanol (1:1) followed by two washings with chloroform. Aliquots of the solvent were evaporated under a stream of N2. Differences in per- centage moisture were compensated for in order to maintain the ratio of 2:2:1.8 for chloroform, methanol, and water. The total lipids of the phosvitin, vitelline membrane, and freeze-dried samples were estimated by drying, under a stream of N2, aliquots of hexane following the hexane-methanol ex- traction for pesticide determinations. The proportion of neutral to phospholipids was obtained in two ways. The amount of phospholipids was estimated by multiplying the percentage phosphorus of the total lipids by 25 as outlined in several previous studies (Augustyniak 33.33,, 1964; Martin 3£_3l,, 1965; Nobles and Moore, 1967; Wittcoff, 1951). The second method utilized differential solubility of the lipid components in chloroform and methanol after the 46' total lipid had been adsorbed on activated silicic acid, a modification of the procedure described by Zook, 1968. The silicic acid was activated by heating at 1100C for 6 hr. The total lipid, weighing approximately 0.05-0.10 g, was dissolved in chloroform and added to 5.0+.1 g activated silicic acid and allowed to stand for 50 min at room tempera- ture. The neutral lipids were then removed with 500-400 ml of chloroform during filtration in a 150 ml-60M Buchner funnel, equipped with a Kimflow fritted disc. Following removal of the neutral lipids, the phospholipids were removed from the silicic acid with 200-500 ml methanol. One hundred ml washes of each solvent was continued until no additional organic material was obtained, 3,3,, no charring with con- centrated sulfuric acid. Solvents were evaporated under a stream of N2 and percentages of neutral and phospholipids were calculated from the weights obtained. Pesticide Residue Analyses 3393, Sample sizes for pesticide residue analyses were as follows: 50 g for albumen, lipovitellin, lipovitellenin, and livetin; 25 g for whole yolk, 7-10 9 for phosvitin, 0.8- 1.0 g for vitelline membrane; 50 g for whole egg: and 5 g for freeze-dried samples. The samples were extracted with 200 ml of 1:1 hexane-methanol for 10 min in a Waring Blender. The mixture was filtered through a Buchner funnel fitted with Whatman No. 1 filter paper and 15:0.1 g HYflo Super Cel. The Super Cel and precipitated protein were washed with 100 47 m1 of 1:1 hexane-methanol followed by four 50-ml hexane washes to insure complete removal of pesticides. Soxhlet extractiOn of the Super Cel and filter paper with hexane for 48 hr (20 min cycle) yielded no additional pesticides verify- ing the adequacy of the washing procedure. Because of the small quantity, the membranes were ex- tracted using a Virtis '45‘ homogenizer. The weighed membrane was transferred to a 250 ml Pyrex homogenizer flask and ex- tracted for three 10 min periods with 50 ml of 1:1 hexane- methanol. The solvent was carefully decanted from the mem- brane material between extractions. All solvent was filtered through a 150 ml-60M Buchner funnel, equipped with a Kimflow fritted disc... An additional 25 ml of hexane was used to rinse the homogenizer flask and the precipitated protein. The hexane-methanol solutions were transferred to 500- ml separatory funnels and the methanol washed out with four 100-ml portions of 10% sodium chloride solution after which the hexane was dried over anhydrous sodium sulfate for 50 min. No further cleanup or concentration was required for quantitative analyses except for the membrane samples. The latter had to be concentrated to volumes of approximately 10 ml. £333, The composite samples of feed before and after pesticide contamination were blended individually in a 1 gal Waring Blender for 50 sec to obtain a homogeneous mixture. Fifty-gram samples were weighed into 500 ml stainless steel 48 containers for the Omni—Mix Blender and extracted three times with 200 ml portions of acetone-hexane (1:1) during a 5 min blending period. The solvents were combined, acetone washed out with 10% sodium chloride solution, dried over anhydrous sodium sulfate for 50 min, and then concentrated to 10 ml using a Kuderna Danish evaporator. The concentrate was cleaned up on a Florisil/Celite (5:1) column and eluted with 600 ml of hexane. This hexane was then conCentrated to approximately 10 ml as previously described. GLC Analyses. Gas chromatographic analyses were carried out using a Beckman GC-4 chromatograph equipped with a dis- charge electron capture detector. It was fitted with a 6 ft (1:85 m) x fi-in. (5.5 mm) I.D. Pyrex column packed with 11% QF-1 plus 5% DC—200 on 60/80 mesh Gas Chrom Q and was operated at column, inlet and detector temperatures of 210, 260 and 280°C, respectively. Helium flow rates used were 40 ml/min for the column and 120 ml/min for the discharge side of the detector. Standards were injected at the beginning of each run, after every 10 to 15 samples, and at the end of the run. Quantitations were based on the peak height of the standards and concentrations were expressed on the wet weight, dry weight, weight of the fat components as well as the total pesticide in each fraction. Analyses of the Data The pesticide concentrations based on solids and on fat were analyzed for variance. Duncan's Multiple range test 49 (1957) was then used to sort out differences revealed by the analyses of variance. Simple correlation coefficients were calculated between the pesticide concentration (based on the amount of fat in the sample) and the percentage of phospho- lipid or neutral lipid in the sample. The 2 statistic was used to establish significant differences between mean pesti- cide concentrations in the liquid and freeze-dried whole eggs and egg yolk (Dixon and Massey, 1957). RESULTS AND DISCUSSION Eggs were obtained from hens on contaminated diets in order to establish whether the type of lipid present influ- enced the chlorinated hydrocarbon pesticide distribution. Pesticide analyses of the feed before and after contamina- tion showed the feed contained less than 0.01 ppm each of lindane, dieldrin, and p,p'-DDT before contamination and the specified 25:0.1 ppm each after contamination. One of the hens started molting immediately after the ten hens were switched to the LB-68 feed one week prior to pesticide contamination. Egg production for the remaining nine hens averaged 64.1%‘which was well within the amount anticipated from their previous laying history and substan- t 3;, (1966) that pesti- tiates the conclusion of Cummings cide contamination does not affect egg production. The albumen was separated and the yolk fractionated into lipovitellin, lipovitellenin, livetins and phosvitin. The percentages of moisture were determined for all the egg com- ponents and the egg yolk fractions and are summarized in the Appendices. Since the exhaustive dialysis required to remove salt solutions used in egg yolk fraction separation resulted in great variation in moisture in the samples, all yields 50 51 were calculated on the solids contents. The yields, per- centage recovery and percentage distribution for the three time groups are presented in Table 6. The percentage re- coveries varied from 85.7 to 90.5%. The percentage distri- butions among the egg yolk fractions were fairly consistent except that the percentage of livetin decreased slightly and phosvitin increased slightly, for the third group. These percentage distributions closely agree with the average percentage distribution of egg yolk fractions cited by Cook (1968L He indicated that the low density fraction comprised 66.0%, the water soluble fraction (livetin) 10.6%, phosvitin 5.7%, lipovitellin 16.5% and the low density frac- tion in the granules 5.0%. Since the latter two were not separated in this study, these values should be considered together. The protein, lipid, and phosphorus values tabled in the Appendices were used to verify the fraction identification. ~Even though the percentage of the phosvitin fraction was lower than previously reported, the low protein phosphorus values of 0.57 to 0.55% for lipovitellin closely compare with 0.49% reported by Joubert and Cook (1958a) . Therefore, it seems more likely that phosvitin was lost in fractionation than inadequately separated from the lipovitellin. Although increased recovery might have been possible using DEAE or TEAE cellulose columns (Wallace 33H3£,, 1966; Radomski and Cook, 1964), column techniques were avoided to eliminate 52 How mummcmmeoo ou Ammmav Hawuumz ocm upm3 .mflmwamwo smsounu umoH mmuwaouuomaw cw moanmu Eoum ooumHsono mosam> nmfia R0¢.Om fihm.mm Rmm.mm >Hm>oumm e.ema m.mea «.mme encasemem Hence 0.0 4.0 0.0 Heme em.m e.m mm.a 0.a em.a 0.a caee>moem He.0 0.0a 0s.aa 0.0a am.ma 0.0a meeem>aq 00.00 0.00s 00.e0 0.m0 00.e0 H.00 enemaemua>oaeq 00.0m e.mm mm.mm 0.Hm mm.em 0.mm eaHHmen>omaq m.eea e.eee e.0ea xHo» ememeoaeumhecs 0 0 0 e meadow e meaaom e meanom newcomeoo HHH HH Hi mmm QDOHO .mmsoum Goduumaaoo mom mmnnu msu Mom mcofluomum xaow mo mcoHusQHHumHo monucmuumm ocm .muw>oomu ommucmoumm .moamww .0 manna 55 possible loss of pesticides. Cook 33_3l, (1962) reported vitellenin to be 0.15%;phosphorus which along with the lipid composition substantiates the separation of lipovitellenin. Lipid Composition Only traces of lipid material were found to be present in the albumen (0.195, 0.256 and 0.101% based on solids for Groups I, II and III, respectively). Romanoff (1967) indi- cated that albumen contains 0.12 mg lipid material per 100 mg dry material of which one-half is phospholipid and the remaining is carotenoid. Phosphorus determinations of the albumen lipid material showed much lower phospholipid con- tents of 15.9, 12.5 and 5.6% for Groups I, II and III, respectively. However, Smith (1959) reported that diffusing yolk lipids are primarily triglyceride in nature, thus some of the very small quantity of lipid present in the albumen could have resulted from diffusion from the yolk. Due to the small quantity of membrane material present, lipid was estimated by evaporating an aliquot of the hexane from the pesticide extraction. Based on dry weight, vitel- line membranes from Groups I, II and III contained 17.95, 14.92 and 15.47% lipid, respectively. Phosphorus analyses of these materials indicated only traces of phospholipid were present (142%). These dried membranes were slightly cream in color so carotenoids contributed to this lipid material but due to the quantity available no attempt was made to 54 quantify the contents. The lipid material could have orig- inated in the yolk as only water was used to remove con- taminating yolk material; more efficient lipid solvents could not be used since these also extract the chlorinated hydrocarbon pesticides. Neither Parkinson (1966) nor Doran and Mueller (1961) reported any lipid component for the vitellineimembrane. Chloroform-methanol extraction of the unfractionated yolk indicated total lipid contents of 52.58, 55.49 and 52.15% for Groups I, II and III, respectively. Lipid composition of the egg yolk fractions was investigated following separa- tion of the yolk. The results of these total lipid determin- ations, neutral and phospholipids as established by adsorp— tion on activated silicic acid and elution with chloroform followed by methanol, and phospholipid estimated from the percentage lipid phosphorus are summarized in Table 7. The total lipid content of the phosvitin fraction was estimated by evaporating an aliquot of hexane from the pesticide frac— tion. Since the phosvitin preparations were found to be lipid-free as previously reported (JOUbert and Cook, 1958b), this fraction is not included in the data presented in Table 7. The total lipid composition of lipovitellenin varied from 81 to 89% but is well within the range expected from previous studies (Martin 3£_3;., 1965; Sugano and Watanabe, 1961; Evans 33_3l,, 1969). Agreeing closely with the data 55 .mCOHumcHEHmumo mumowamso mo wmmnm>mm .000000 :0 pmmmmN .mmHMIUHQHH ma 0» 02500 003 C0u0>mosma 00.00 00.0 00.00 00.00 00.00 0000>00 00.00 00.00 00.00 00.00 00.00 000....000000000.H 000 00.00 00.00 00.00 00.00 00.00 0000000>0000 00.00 00.00 00.00 00.00 00.00 0000000 00.00 00.00 00.00 00.00 00.00 00000000000000 00 00.00 00.00 00.00 00.00 00.00 000000000000 00.00 00.00 00.00 00.00 00.00 0000>00 00.00 00.00 00.00 00.00 00.00 000000000>0000 0 00.00 00.00 00.00 00.00 000.00 0000000>0000 e e e e R 00000 00000 00000 00000 N0000.0 00000000 00000 Iozmmosm Iocmmonm losmmosm 0000502 00008 00000>< 000002 000002 0000 0000000 00 .cowuumum xaom mom 0:» c0 .ucmucoo msuonmmonm Eoum new 0000 0000000 pmum>fluum co nodumnomom mg 0000 0000000000 .0000000000000 000 .0000070000000 .000000 00000 0000000000 .0 00000 56 of Martin §£_3l, (1965, phospholipid accounted for approxi- mately one-quarter of the lipovitellenin's total lipid for Groups I and II and approximately one-fifth for Group III. The latter Group also possessed the lowest total lipid. This fraction had been frozen prior to lipid analyses thus freezing may have increased the strength or number of lipid- protein interactions on the surface of the lipovitellenin shell making total lipid extraction more difficult. Increasing the time of chloroform-methanol extraction did not improve the lipid extractability. Evans E; El, (1969) had reported freezing did not affect the ether extractable lipid of lipovitellenin. Lipovitellin consisted of approximately 26% lipid of which about 40%‘was phospholipid. Previous reports had shown purified lipovitellin to have as high as 60% phospholipid (Martin gt al,, 1965), however the small amount of low density fraction which is associated with lipovitellin in the granules, and is called LDFG, was not separated by the fractionation procedures used in this study. Since the LDFG has a lipid composition similar to other low density lipoproteins (Cook, 1968), its lower phospholipid content would result in a lowering of the proportion of phospholipid in the lipovitel- lin fraction. .Nevertheless, the proportion of phospholipid in these lipovitellin preparations was still lé-to 2 times that of lipovitellenin. The relatively high proportion of lipid in the livetin fraction resulted from free lipid released from the 57 lipovitellenin during the extensive centrifugations. The high proportion of neutral lipid supports its origin in lipovitellenin. Evans §£_§l, (1969) reported the easily extractable lipid of lipovitellenin to be higher in neutral lipid than that of the total lipid. Probably some of this lipid material also resulted from contamination with lipo- vitellenin pg; gg since precise separation of the two materials during the fractionation was extremely difficult. Pesticide Analyses The hens were fed lindane, dieldrin, and p,p'-DDT. Gas chromatographic analyses showed that the DDT isomers and degradation products, o,p'—DDT or DDD and p,p'-DDE were also present. Lindane Recovery and distribution of lindane in the egg yolk fractions are presented in Table 8. The recovery ranged from 85 to 98% and is comparable to the recovery of the fractions pg£_§§, Lipovitellenin contains the major portion of the lindane residues, however this percentage decreased slightly from Group I to Group III whereas the proportion present in the lipovitellin correspondingly increased. Table 9 summarizes the lindane concentrations expressed as ppm based on solids for the albumen and the vitelline membrane as well as yolk and its fractions. The distribution of the lindane in the albumen as compared to the yolk is in .0 00009 00 c3000 00 U0c000no 00000000 £000 00 009050 00000 000 8000 000 000000000 000000 00 0009 x0om no 005050 00000 00m me 00 00000500000 58 Rmm.¢m Rma.mm Rmm.>m >H0>000m mm.¢ mfi.m mm.m 0:0000000 :0 000GB om.o 00.0 m0.o 00.0 0N.o 00.0 C000>monm mm.m mm.o 0o.m mm.o no.0 NN.O C000>00 00.00 00.0 00.00 00.0 00.00 00.0 00000000000000 00.00 00.0 00.00 00.0 00.0 00.0 0000000>o000 om.m m>.m ¢>.m x00» U000c00000umcb R we . R me . R _ _ ma 0c0comEOU 000 Hm l0 00.0 900.00 0.0:0000000 000% mm0 0:0 :0 >00>oo00 Uc0 c000sn00000p 000 0:0 000cm :0.0c0©:w0 000GB .m 00909 59 Table 9. Lindane residues in albumen, vitelline membrane, whole yolk and yolk fractions (ppm based on total solids). Egg Group, Component Component I II III Averagel Albumen 0.92 1.0 1.2 1.0 Vitelline Membrane 5.1 5.6 1.5 2.7 Whole Yolk: 20.9 55.1 52.7 28.9 Lipovitellin 9.4 17.9 19.1 15.5a Lipovitellenin '51.5 47.0 59.0 59.2 Livetin 11.1 15.6 18.7 15.2a Phosvitin ‘2.6 2.8. 2.2 2.5 Group Average1 15.1 25.5' 22.4 lWhole yolk and yolk fraction averages superscripted b same letter are not significantly different at the 1%? of probability (Duncan 1957). 2Average of two determinations except for one analysis of vitelline membrane. 60 the ratio of 1 to 50 (approximately 1 to 150 on the liquid basis). Cummings _£_§l, (1966) had previously reported a ratio of 10/90 when feeding levels of 0.15 to 0.45 ppm. These authors indicated this relationship was in the order of the solubility of lindane in water but use of the ultra- centrifuge to determine water solubility more precisely has shown the water solubility of lindane to be in the order of t al,, 1968). The data from the current 6.8 ppm (Gunther study showed that the lindane associated with the albumen is far below that amount which is soluble in water portion of the albumen. Analysis of variance, summarized in the Appendices, established very highly significant differences among the lindane residues in the yolk and its fractions. Use of Duncan's Multiple range test showed that lipovitellenin con- tained more lindane than the unfractionated yolk whereas lipovitellin and livetin contained less (Table 9). Lindane concentrations increased from Group I to Group II (p fi_0.05) but remained fairly constant between Groups II and III. When the lindane content was based on the total lipid in each component (Table 10), the order of magnitude was reversed in that the lindane in lipovitellin was slightly higher than that in lipovitellenin and both were slightly higher than the lindane content of the unfractionated yolk whereas livetin's lindane content was slightly lower. Statistical analysis of these values revealed no differences 61 Table 10. Lindane residues in the vitelline membrane, whole yolk, and yolk fractions (ppm based on lipid content). Egg Group Component Component I II III Average Vitelline Membrane 17.51 19.8 10.8 16.0 Yolk: 55.8 50.6 55.1 45.8 Lipovitellin 56.9 67.8 75.1 59.9 Lipovitellenin 55.4 56.2 48.1 46.6 Livetin 29.4 40.2 46.5 58.7 Group Average 50.6 46.9 46.7 1Based on two determinations except for one for vitelline membrane. 62 among these yolk and egg yolk fraction lindane values although the vitelline membrane had significantly'less lindane (p §_0.05) than either the yolk or its fractions. Dieldrin Total recovery of dieldrin from the egg yolk fractions was from 85 to 91% of that in the unfractionated yolk (Table 11). Lipovitellenin contained approximately 85% of the di- eldrin whereas lipovitellin possessed about 10% although the relative proportion of dieldrin decreased slightly in the lipovitellenin fraction from Group I to Group III. Simul- taneously, the proportion of dieldrin residue in the lipo- vitellin fraction increased. Dieldrin contents, expressed as a function of the solids present, of the albumen, vitelline membrane, yolk and yolk fractions are listed in Table 12. As would be expected from dieldrin's extremely low solubility in water (0.195 ppm at 25°C, Gunther g; al., 1968), a much lower proportion of dieldrin was found in the albumen as compared to that in the yolk than was previously cited for lindane. .The dieldrin concentration in the albumen (0.05 ppm on a liquid basis) is still below dieldrin's solubility in water portion of egg albumen. Cummings g£_gl, (1966) had reported a slightly higher ratio of 1 to 99 for dieldrin occurrence in albumen versus yolk. The phosvitin fraction was also found to be lipid free. This fraction, however, contained approximately 2 ppm 63 .m magma ca czozm mm vwcwMqu GOAHUMHM zoom mo uCDOEm Hauou mnu Eoum pom mcofluomumncfimuno on com: xaoa mo ucsoem Hmuou mom me mm Umumasuamoa momom eomnm Rana 338mm em.¢d m¢.HH mm.m mcouuomuucu Hmuoa as.o oa.o am.o no.0 mm.o ao.o chun>mosm mm.m s>.o mm.m ¢¢.o >¢.¢ mm.o chum>uu m¢.am em.da mH.mm ma.m «m.mm mm.« cuamaamuu>omuq mm.ma am.a. mm.oa ma.a. mm.m am.o cuaamuu>omuq mm.ma en.ma mm.m xflom cmumcoauomumcs R mE R we & ma ucwcomeoo wnH +mm H mom msouw xao> mmw msu cw >Hm>oumu pom Godusnwuumwp muw pom axHo> cw canvamwp Hmuoa H.:owuomnm .dd magma 64 Table 12. Dieldrin residues in albumen, vitelline membrane, whole yolk and yolk fractions (ppm based on total solids). Egg Group Component Component I II III Average1 Albumen 0.52 0.4 0.7 0.5 Vitelline Membrane 12.8 11.4 4.6 9.6 Whole Yolk 37.1 76.2 90.2 67.8a Lipovitellin 15.6 57.2 51.4 54.7b Lipovitellenin 49.3 105.7 116.4 90.5a Livetins 12.8 26.2 58.9 52.6 Phosvitin 7.4 27.6 28.1 21.9 Group Average; 24.4 54.6C 69.0C 1Groupvaverages as well as whole yolk and yolk fraction averages superscripted by the same letter are not signifi- cently different at the 1% level (Duncan, 1957). 2Average of two determinations except for one for vitelline membrane. 65 dieldrin on the wet basis greatly exceeding the water solu- bility of dieldrin. Since phosvitin is closely associated with lipovitellin in the granule of the intact yolk, this pesticide residue could have resulted from contamination from the lipovitellin fraction but lipid contamination did not occur simultaneously. .Moss and Hathaway (1964) have previously reported protein-dieldrin complexes to be im- portant in the transport of dieldrin in plasma. These types of interactions may also contribute to dieldrin's occurrence in the phosvitin fraction. The van der Waals' radii of chlorine are very similar to those of methyl groups so that this type of interaction could bind dieldrin to the phos- vitin molecule. An analysis of variance among the dieldrin concentrations of the yolk and its fractions revealed very highly significant differences among fractions as well as groups. This analysis as well as that for dieldrin expressed as a function of lipid is summarized in the Appendices. Use of Duncan's Multiple range test established that the dieldrin concentrations of lipovitellin, livetin, and phosvitin fractions were less (p £_0.01) than that in the yolk or lipovitellenin fraction (Table 12). The latter two were significantly different from teach other at the 5% level of probability. Dieldrin concen- tration in the yolk and fractions increased significantly (p £_0.01) from Group I to Group II and III, so for a time hens can eliminate increasing amounts of contamination via the egg but this amount then levels off. 66 When dieldrin concentrations were based on the lipid content as illustrated in Table 15, lipovitellin contained the largest proportion of dieldrin, lipovitellenin contained a residue level similar to that of the unfractionated yolk whereas the dieldrin in the livetin fraction was slightly lower. However, as had been true for the lindane residues, none of these differences were significant. DDT and Related Compounds Gas chromatographic analyses revealed that a portion of the p,p'-DDT fed the hens was dechlorinated to form DDE (1,1-dichloro-2,2—bis (prchlorophenyl)ethene). This conver- sion of DDT to DDE by fowl has been reported in numerous other investigations (Cummings §£_§l,, 1966, 1967; Ritchey _£Mgl,, 1967, 1969). A third peak was observed in the DDT series which was considered to be o,p'—DDT since the standard -DDT reference used to contaminate the feed contained a minimal contamination of o,p'-DDT (approximately 0.05 ppm o,p'-DDT in a 1.0 ppm solution of p,p'-DDT). The proportion of o,p'-DDT to p,p'—DDT was slightly higher than this very low contamination in the standard so this peak may also contain minimal quantities of the DDT degradation product DDD (1,1- dichloro-Z,2-bis(2-chlorophenyl)ethane), which has been previously reported in chickens (Wesley gt al,, 1969). The column used to obtain the gas chromatographic analyses reported did not separate these two compounds. The very low values for this pesticide did not warrant further subdivision. 67 Table 15. Dieldrin residue in the vitelline membrane, yolk and yolk fractions (ppm based on total lipid). Egg Group Component Component I II III Average Vitelline Membrane 71.02 62.6 54.4 56.0 Yolk: 60.1 116.6 146.4 107.7 Lipovitellin 61.0 140.8 200.9 154.2 Lipovitellenin 55.5 126.4 145.4 108.4 Livetin 54.0 67.5 142.5 81.5 Group Average1 56.5 102.6a 155.5a 1Group averages superscripted by the same letter are not significantly different at the 5% level of probability (Duncan. 1957). 2Average of two determinations except for one for the vitelline membrane. 68 Total DDT compounds were also calculated for the data ex- pressed as ppm based on both solids and lipids. The recoveries of DDT compounds in the yolk fraction were comparable to those for lindane and dieldrin (Table 14). The distribution among the fractions of the egg yolk showed p,p'-DDT to be present in the lipovitellenin fraction in the highest proportion of any of the pesticides obtained whereas o,p'-DDT-DDD combination was present in lipovitellenin in the lowest proportion. Since lipovitellenin contained the largest amount of lipid and particularly of neutral lipid, the former could be related to its extremely low polarity. As had been true for both lindane and dieldrin, the percentage distribution of DDT compounds in the lipovitellenin fraction decreased from Group I to Group III with a corres— ponding increase of the percentage distribution occurring in the lipovitellin fraction. For p,p'-DDT, the lipovitel- lenin fraction contained 88.5% of the total in Group I and 84.5% in Group III while lipovitellin contained 6.2% in Group I and 10.1% in Group III. Lipovitellenin also con- tained 86.2% of the p,p'-DDE and 85.5%lof the o,p'-DDT-DDD combination for Group I and 82.0% and 76.7%, respectively, for Group III while the lipovitellin had 7.9% of the total DDE and 10.6%'o,p'-DDT-DDD for Group I with 12.5% and 15.5%, respectively, for Group III. Although lipovitellenin is always the predominant lipo— protein in the yolk even during oogenesis, its proportion 69 .w wanma a“ c30£m mm meflMDQO cofluomum 30mm mo UCSOEM Hmuou Eoum Ucm mcofluumum cflmuno Cu @009 xaow mo DCDOEm Houou umm m8 mm UmumHsoamoe R0e.00 R00.00 wa.e0 mum>oumm «00.0 000.0 000.0 00000000000 H6009 00.0 ee0.0 00.e 000.0 ee.0 a00.0 neue>monm 00.0 000.0 00.0 e00.0 00.0 000.0 chum>0q 000.900-40.o 00.0e 000.0, 00.0e 000.0 00.00 000.0 seameemue>omeq 00.0e 000.0 mm.ee 000.0 e0.0e 000.0 eeeemuaeomflq 000.0 000.0 e0m.0 x00» 00000000000000 R00.e0 R00 00 Roe.me wuw>oomm ~0.e 00.e 000.0 00000000000 Hmuoe 00.0 00.0 00.0 «0.0 e0.0 000.0 0000>0000 00.0 00.0 00.0 00.0 00.0 00.0 eeum>0q moou.m.m 00.e0 me.e 00.00 ee.a ee.00 0e.0 chameemue>omfiq 00.0e 00.0 00.0e ee.0 00.e 00.0 neeamue>omeq mm.a Nw.a oa.d xaom omum:00uomumcs R00.em R00 00 Rem.00 mum>oowm om.m Hm.> Hm.¢ mGOwuomumcH Hmuos 00.0 00.0 00.0 00.0 0e.0 e0.0 0000>mosm 00.0 00.0 0e.e 00.0 0e.0 00.0 chum>00 aoo-.m.m 0e.¢0 00.e ee.00 00.0 ee.00 00.0 eecmaemue>ooeq ee.0e 00.0 0e.0 me.0 0e.0 00.0 cheamue>omeq Ne.0 00.0 00.0 sec» 00000000000000 R il DE R m8 R m8 ucmcomeoo mpflowumwm H H H 000 nacho a.:owuomum xaow mmw may :0 >Hw>oomu 6cm COwusflfluumwp mu“ 0cm axao> cw mpcsomeoo EGG .fifi magma 70 is relatively higher in the early stages of yolk formation (MacKenzie and Martin, 1967). Thus with ever increasing amounts of pesticide contamination in the hen, it can be speculated that a higher concentration in lipovitellin toward the end of the feeding of pesticide-contaminated feed could have resulted simply from the hen's eliminating greater quantities of the pesticide in the portion of the yolk being formed in the largest proportion. It is also possible that the level of pesticide to be eliminated through the egg affects its location, thus at the lower levels more is in- corporated into the major lipid component of the egg but with ever increasing amounts of pesticide, a greater proportion is selectively incorporated or perhaps just spills over into the relatively low fat, but high phospholipid lipovitellin. A slightly longer holding period occurred between the collect- ing and breaking of the eggs for Group III, thus transloca- tion of the pesticide within the yolk could have occurred during storage but it does not seem feasible that the pesti— cide residues would move, even if translocation is possible, from a very high concentration of neutral lipid to a lower ‘ one considering their very high degree of non-polarity. The concentration of DDT compounds calculated on a solids basis are presented in Table 15. The amount of p,p'-DDT in the albumen was approximately three times the concentration of dieldrin and slightly higher than the concentration of lindane. The concentrations of DDT compounds with the .020H3808 msflaamufl> How mco ammoxw maceumcwEHmumv o3u mo wmmuw>¢m .A0mma .cmucsav H0>0H Ra msu um ucwummmwp wauCMUflmwcmwm uoc mum Hmpuma mean 0:» >9 vmumwuumummsm mmmmum>m coHuumum Mach Cam xaow waon3 mm HH03 mm mmmmum>m msoum xaowa 7 m.am «.00 0.mN m.m m.N H.H 0.0 m.0 m.m Qm.0¢£m.mm 0.06m0>¢ msouw xaow 0.0a 0.mm H.mfi 0.0 mm.m 0.m m.m 0.0 Q0.N 0.0 a.m 0.H 00.0 0.0a 0.0« w.m :Huw>monm 00.0N 0.00 0.0m 0.mamm.a m.H m.fi 0.0 QN.¢ 0.0 $.mu.m.m Q0.HN m.mm 0.0a 0.Nd cflum>wq .1 0.00 N.¢m 0.00 m.m¢~0.m 0.n N.m H.N mm.ad 0.0H ¢.NH 0.0 m.N0 0.00 m.a0 0.00cacmaamuw>oqu .mm.¢m 0.mm $.0m m.aamm.a 0.0 0.0 0.0 AN.¢ m.0 0.0 0.Nn.0d.mfi 0.0m 0.NN 0.0 cwaamufl>omflq 0.0m 0.007%.00 0.0mmm.m m.m 0.N m.d 00.0 0.0a m.m d.0 N.0w 0.00 0.00 «.mm “xaow maonz N.0 0.0 6.0d 0.0 d.0 0.0 m.0 0.0 m.a m.fi m.d 0.0 m.0 $.m m.0. m.0 mcmunawz" mcwaamufl> 0.N m.m 0.0. 0.H N.0 N.0 N.0 N.0 m.0 0.0 «.0 m.0 m.d 0.d_ 0.H NN.a amasnad a.m>¢ HHH HH H H.m>¢ HHH HH H a.m>¢ HHH HH H H.m>< HHH HH H ucwcomeoo .QEOU .mEoo .mEoo .mEoo« _ mom QMOHG QSOHG asouo nwouw wmsoo son Hmuoe oootaoor.m_o moou.0.m sootwmsmu , 00HUHpm0m h .A00000 00000 no 00000 2000 mCOwuomuw xHo> 0cm xaow 0HO£3 .mcmunfiwa mcwaamufi> «cwfisnam aw mpcsomfioo 899 .00 manna 72 fat-free phosvitin were also considerably higher than those for lindane but less than the amount of dieldrin associated with the phosvitin fraction. Specific protein-DDT interactions and/or surface adsorp- tion contribute to these values of DDT's occurrence with albumen and phosvitin. Gunther g; 1;. (1954) first presented data to show that DDT is bound to protein by van der Waals' forces. More recently, Matsumura and O'Brien (1966a,b) com- bined DDT with components of the central nervous system using Sephadex techniques to remove non-reacted pesticide and re- ported binding of DDT to the proteins occurred. However, a third study (Hatanaka §£_§l,,.1967) indicated that electro- phoretic separation of the protein after "binding" of DDT failed to substantiate their previous work. These workers did report that a bathochromic shift in the ultraviolet spectra of serum bovine albumin occurred after the protein was interacted with DDT thus establishing complex formation in this system. Very recently, Shin (1970) reported adsorption isotherms showing adsorption of DDT on alfalfa proteins to be very similar to adsorption to colloidal particles of various types of soil. Therefore, the colloidal nature of the proteins as well as specific complexing of DDT to the proteins through van der Waals' attractions and possibly hydrophobic bonding in certain interior pockets of the protein molecules account for the association of DDT with egg albumen and phosvitin. 75 The distribution of DDT compounds between the albumen and yolk resulted in.a ratio of 1 to 600 for p,p'-DDT and p,p'-DDE and of 1 to 200 for o,p'-DDT-DDD component. After feeding much lower concentrations of pesticides, Cummings _£_gl, (1966) reported ratios of 7 to 95 for p,p'-DDT and 5 to 95 for p,p'-DDE. However it is feasible that whenever the level of pesticide residues are extremely low, a much greater portion could be adsorbed and/or complexed with lipid-free proteins. The concentrations of DDT compounds based on the solids content in the yolk and yolk fractions were analyzed for variance. As is shown in Table 15, lipovitellenin contained more p,p'-DDT and total DDT compounds whereas lipovitellin and livetin contained significantly less p,p'—DDT, p,p'-DDE and total DDT than did the yolk or lipovitellenin fraction (p 5 0.01). There were no significant differences among the egg yolk or egg yolk fractions for the concentration of o,p'-DDT-DDD component. Again, as had been the case for lindane and dieldrin, there were no significant differences among the DDT compounds of the yolk and yolk fractions when calculated on the basis of the lipid content. ‘These values are given in Table 16. These data are similar to the earlier findings of Rumsey §£_§;, (1967) who reported total DDT isomers were evenly dis- tributed throughout beef tissues when expressed as a function of the lipid content. .0CMHQEmE mcHHku0> mzu Mom mco ammoxm maoHumcfleumumU o3u mo mmmum>0H Rm 0:0 pm pawnmmmHU >Hucmowm0cmwm uoc mum umuumH.mEmm 0:» >9 Umumwnomummsm mmmmum>m msouwa 00.0001m.00 00.00 00.0 00.0 00.0 00.000m.0000.0 00.0000000 0.00 0.0>0 00000 0.00 0.00 0.00 0.00 0.0 0.0 0.0 0.0 0.00 0.00 0.0 0.0 0.00 0.00 0.00 0.00 0000>00 Mno.mm 0.000 m.m00 N.0m m.m 0.m m.m m.m 0.n0 0.00 0.00 0.0 N.m0 $.00 m.mm m.m¢ cwcwHHmu0>Om0H 0.0m 0.0m0 m.000 0.00 0.0 w.m N.m N.m n.00 0.0N 0.00 0.0 0.M0 mtfifinm.mw m.mm GHHHmuH>Om00 0.00 0.000 00000 0.00-0.0 .0.¢. N.¢ 0.N 0.00 0.00 «.00 0.0 m.m0 0.00 m.m0 070w uxaow 00033 0.00 0.0m 0.00. m.mm ¢.N m.N 0.0 0.N 0.0 m.m 0.0 «.00 0.0m 0.0m w.m¢m¢.0¢ msmunfimz 00000000> .m>< HHH HH H .m>< HHH HH H .m><_HHH HH H .950 HHH HH H #:0089000 .0800 ,msouo .0800 anouw .mEOU msouo .0800 @5000 mom 000000500 ago 00000 000189.06 0.de Human.m.mw 000009000 .soHumuucmocoo 00000 00000 map Ho Ema mm vwmmmumxm mCOHuomum x00» 0cm .xHo> .m:muQEmE 0GHHHmuH> may :0 mvcsomaou 990 .00 00308 75 Relation of Pesticide Content to Lipid Composition Chlorinated hydrocarbon pesticides have been variously reported to be primarily associated with neutral lipids (Hayes, 1965) and with phospholipids (Hugunin and Bradley, 1969). Since the proportion of neutral to phospholipid varies considerably in the egg yolk lipoproteins, it was hoped that the data reported from this study would contribute informa- tion to resolve these discrepancies. Figures 1 and 2 illustrate the average distribution of lin— dane, dieldrin, and total DDT compounds eXpressed as functions of solids and lipid content, respectively. Lipovitellenin con- tained significantly more and lipovitellin contained signifi- cantly less pesticide residues when expressed on a solids basis, but no significant differences occurred among the egg yolk and the egg yolk fractions when the pesticide content was calculated as a function of the lipid content. Neverthe- less, lipovitellin always contained higher proportions of pesticides expressed on a lipid basis. To further investigate whether any relationship existed between the amount of pesticide and the lipid composition.two sets of simple correlation coefficients were calculated. The first set of correlation coefficients related the mg of each pesticide in the total lipovitellin, lipovitellenin-and livetin fraction for each group to the corresponding weight of total lipid, neutral lipid, or phospholipid. These corre— lation coefficients are summarized in Table 17. As antici- pated, very highly significant correlation coefficients were 76 nlL Elm Illilohwfln [UIIlimflth00 00.0 000 00 00000000000 * .00000000000 00 00>00 00 000 00 00000000000 * ** .2. .00000000000 00 00>00 00 000 00 00000000000 .x. >N>.0 0.000.00 0.000.00 909 00009 000.0 0.000.m 0.000.m 000I900I.m.o *N00.m 0.000.m0 0.on.00 monI.0.0 000.0 0.000.00 0.000.00 900I.0.0 $00.0 0.000.00 0.NHN.00 00000009 ***omm.0 0.000.00 0.000.0N 0000000 x000 ***mm0.00 0.000.00 N.NH>.0> 900 00009 ***000.0 0.000.m 0.000.m 000I900I.0.o **0>N.m 0.000.00 0.000.00 manl.m.m ***>m¢.¢m O.an.hN m.HHN.¢® BQQI.Q~Q ***mo0.0 0.00N.mm 0.000.000 00000000 ***000.0m 0.000.0 0w000.om 0000000 00003 000000000; 000000>00_0000cmum 000000>0Q 00000000 000000000 000 N + + 00 0009 :00: 000H0I0N00um 0002.000000 .0000000 00 00000 8000 M00» 000 000 00003 00 00000000 0000000 000000000 :0 000>H0I0N0000 00 000000 0:» 00 0000500 .00 00009 84 Langlois gt 2;. (1964) had reported a 60%lloss in DDT and a 80% loss in lindane by spray-drying milk contaminated with approximately 25 ppm of each pesticide residue on a fat basis. Ruzicka g£_gl, (1967) reported the same rank order of loss of pesticides from roller drying of milk but showing lower losses of 15% for dieldrin, 20% for p,p'-DDT, and a 25% loss for y-BHC. These workers also reported a 15% loss of p,p'-DDE. The greater ease in the removal of pesticides from whole egg could be due to the lower concentrations of pesticide residues present on.a liquid basis or to greater ease of volatilization from a less dense or less viscous substance. Dilution of the egg yolk material with the egg white proteins may result in a different crystal structure or a greater sur- face area being formed during freeze-drying which could : facilitate vaporization 0f.the pesticides; SUMMARY AND CONCLUSIONS This research project investigated the effect of the amount and type of lipid present on the lindane, dieldrin, and DDT residue accumulation in egg albumen, vitelline mem- brane, whole egg yolk and the egg yolk fractions: lipovitel- lin, lipovitellenin, livetin and phosvitin. .Hens were fed diets contaminated with 25 ppm each of these pesticides and eggs were collected from three time periods. Pesticide feeding did not affect egg production. The eggs were separated and the yolks fractionated using salt separation techniques in combination with ultracentrifu- gation. Moisture, protein, phosphorus, total lipid, neutral lipid, and phOSpholipid analyses as well as pesticide residue determinations using electron capture GLC were conducted for the albumen, vitelline membrane, yolk and yolk fractions. The recovery of total yolk material in the fractions varied from 84 to 91%, but the percentage distributions among egg yolk fractions were comparable for all three groups. Protein and phOSphorus compositions of the egg yolk proteins were used to verify their identification. The egg albumen contained only traces of lipid of which only a small portion was found to be phOSpholipid. The ligUid albumen contained an average of 0.1 ppm lindane, 0.05 ppm 85 86 dieldrin, and 0.2 ppm DDT compounds. The concentrations of lindane and dieldrin were below the water solubilities re- ported for these compounds using ultracentrifugation tech- niques while the level of DDT greatly exceeded that antici- pated from the water solubilities alone. The lipovitellenin preparations contained 81 to 89% lipid of which approximately 25% was phospholipid. The lipo- vitellin preparation contained approximately 25% lipid with 40% of this being phospholipid. Although the livetin pro- teins of the egg yolk are themselves lipid free, this prepara- tion contained considerable lipid resulting from free lipid released from the lipovitellenin during extensive centrifuga- tions. This lipid material was extremely high in neutral lipids with only 15 to 18% phospholipid. The recovery of pesticide residues in the egg yolk frac- tions was in the same order or slightly higher than the recovery of the solids gel ge. The percentage distributions of the pesticides among the egg yolk fractions showed the high lipid containing lipovitellenin contained the majority of the pesticide residues with lipovitellin containing approximately 10%, livetins 5%, and phosvitin less than 1%. Although all three pesticides were fed at the same level, wide variation occurred in the amount transferred to the egg, reaching plateau levels at the second group. In addition to the three pesticides fed, p,p'-DDE and o,p'-DDT were found in small quantities in the eggs. The proportion of these pesticide residues decreased in the lipovitellenin fraction 87 with increasing concentration of pesticide in the eggs while the proportion in the lipovitellin fraction correspondingly increased. Analyses of the pesticide residue concentrations based on solids verified that lipovitellenin contained the major portion of pesticide residues being significantly higher in lindane and p,p'-DDT than the unfractionated yolk. Lipo- vitellin contained significantly less of the pesticide residues than the yolk itself. However when the amount of lipid present was compensated for by expressing the pesticide concentrations as a function of lipid content, no significant differences were found although the values for the pesticide residues in the lipovitellin fraction were higher than those for lipovitellenin. Correlating lipid composition to pesticide residue ac- cumulation in lipovitellenin, lipovitellin and livetin frac- tions showed dieldrin, p,p'—DDT, and p,p'-DDE related to a slightly greater extent to the weight of neutral lipid pres- ent while lindane deposition appeared to be better related to the amount of phospholipid in the lipid material. Although representing a small proportion of the total pesticides present in the yolk, the lipid free egg yolk pro- tein phosvitin had average solids residue levels of 2.5, 21.0 and 14.6 ppm for lindane, dieldrin and DDT compounds, respectively. Since the phosvitin fraction was 7 to 10% solids, the values for dieldrin and DDT greatly exceed their 88 relative water solubilities. Phosvitin is bound by ionic or secondary forces to lipovitellin in the egg yolk granule. Thus, it is feasible that the residues could have resulted from contamination from the lipoprotein rather than selective incorporation in the phosvitin pg; gs but lipid contamination did not occur simultaneously. .A corollary study investigated the feasibility of pesti- cide removal by freeze-drying. ‘Significant reduction in residue levels of freeze-dried whole eggs were obtained for all pesticides except p,p'-DDE. In whole eggs, freeze-drying reduced lindane by 79%, dieldrin by 37%, p,p'-DDT by 57% and o,p'-DDT by 51%. Freeze-drying was much less successful in pesticide residue removal from egg yolk giving only 4minimal reduction of dieldrin, p,p'LDDT and o,p'-DDT. Freeze- dried yolks did contain significantly less lindane but the reduction of 49%*was considerably less than for the whole eggs. From an evaluation of the data obtained in this investi- gation, it can be concluded that: 1. The amount and type of lipid present influences the accumulation of lindane, dieldrin, and DDT com- pounds in egg yolk and its fractions. 2. The amount of lipid p§;_§g_is the single most important factor in determining the amount of residue accumulation. 3. The location of dieldrin, p,p'-DDT and p,p'-DDE! relates to a slightly greater degree of the amount of 89 neutral lipid present than it did to the amount of total lipid alone, whereas lindane's distribution correlated positively with the proportion of phospholipid. 4. The total amount of pesticide present in the egg was also shown to affect the relative proportions present in the egg yolk fractions. 5. The amount of dieldrin and DDT in the albumen and phosvitin suggests some protein pesticide complexes are formed in the egg system. 6. Freeze-drying has potential in reducing the levels of lindane, dieldrin and p,p'-DDT in whole egg and lindane in egg yolk; the amount of residue reduction appears to be related both to the vapor pressure of the pesticide and the degree of contamination. PROPOSALS FOR FUTURE RESEARCH As with any investigation, as many questions can be raised on which further investigations can be based as conr clusions can be drawn so that many more facets of the mode of association of pesticides with lipid materials should be explored as well as studies to elaborate on the data reported in this thesis. The following areas of research are pro- posed: 1. The trend toward different proportional distri- bution of chlorinated hydrocarbon pesticides with increasing concentrations should be explored further starting with lower levels of pesticide to delay the plateau level occurring in the egg. 2. Correlating increasing amounts of pesticide residues with lipid composition which remains essen- tially constant is difficult. The results of this study should be verified using batteries of hens so that multi- ple lots of eggs at one time interval are available. 5. The feasibility of pesticide removal by freeze- drying warrants further investigation using different levels of contamination and other pesticides as well as extending the study to other methods of dehydration. 90 91 4. Further information concerning the distribution of pesticide residues in the egg and mode of mobiliza- tion could be obtained by following pesticide location throughout oogenesis and embryogenesis. 5. To further study the mode of pesticide associ- ation in fatty tissue and its turnovér, fat pads of varying metabolic activity could be followed under dif- ferent dietary conditions, i323, normal versus semi- starvation, and at various growth periods using radioactive labeled pesticide in conjunction with histological as well as chemical studies of pesticide location. REFERENCES CITED REFERENCES CITED Alderton, G. and H. L. Fevold, 1945. Preparation of the egg yolk lipoprotein, lipovitellin. Arch Biochem. 8: 415- 4 O ~Allerton, S..E. and; G. E. Perlmann, 1965. Chemical charac- terization of the phosphoprotein phosvitin. J. Biol. Chem. 240: 5892-5898. Association of Official Agricultural Chemists, 1960. , "Official Methods of Analysis" 9th ed., pp. 643-644. Association of Official Agricultural Chemists, Washington, D. C. Augustyniak, J. 2. and W. G. Martin, 1968. Characterization of glycopeptides derived from the low-density lipopro- tein of hen's egg yolk. Can. J. Biochem. 46: 985;988. Augustyniak, J. 2., W. G. Martin, and W. H. Cook, 1964. Characterization of lipovitellenin components and their relation to low-density lipoprotein structure. Biochem. Biophys. Acta 84: 721-728. Belitz, H. D., 1966a. Egg yolk proteins and their frac- tionated products. V. The N-terminal amino acid residues of phosvitin. Z. Lebensm. Untersuch. Forsch. 150(1): 12—16 CA 65: 5710h (1966). Belitz, H. D., 1966b. Egg yolk proteins and their frac- tionated products. VI. The typical cleavage products from enzymatically partially dephosphorylated phosvitin. .Z. Lebensm. Untersuch. Forsch. 130 (5):,152-157 CA 65: 10856h (1966). Bernardi, G. and W. H. Cook, 1960a. An electrophoretic and ultracentrifugal study on the proteins of the high density fraction of egg yolk. Biochim. Biophys. Acta 44:i86;96. Bernardi, G. and W. H. Cook, 1960b. Separation and charac— terization of the two high density lipoproteins of egg yolk, d- and B-lipovitellin. Biochim. Biophys. Acta 44: 96-105. 92 93 Bernardi, G. and W. H. Cook, 1960c. Molecular weight and behavior of lipovitellin in urea solution. Biochim. Biophys. Acta 44::105-109. Bills, D. D. and J. L. Sloan, 1967. Removal of chlorinated insecticide residues from milk fat by molecular distil- lation. J. Agr. Food Sci. 15: 676-678. Bligh, E. G. and W. J. Dyer, 1959. A rapid method of total lipid extraction and purification. Can J. Biochem. Biophys- 57: 911-917. Braund, D. G., L. D. Brown, J. T. Huber, N. C. Leeling, and M. J. Zabik, 1968. Placental transfer of dieldrin in dairy heifers contaminated during three stages of ges- tation. J. Dairy Sci. 51:.116-119. Braund, D. G., L. D. Brown, N. G. Leeling, M. J. Zabik, and J. T. Huber, 1967. Storage, excretion, and placental transfer of dieldrin by dairy heifers contaminated dur— ing three stages of gestation. J. Dairy Sci. 50: 991- 994. Burley, R. W. and W. H. Cook, 1961. Isolation and composi- tion of avian egg yolk granules and their constituent a- and B-lipovitellin. Can. J. Biochem. Physiol. 59: 1295-1507. Casarett, L. J., G. C. Fryer, W. L. Yanger Jr.,,and H..W-. Klimmer, 1968. Organochlorine pesticide residues in human tissue. Hawaii Arch.-Environ. Health 17: 506-511. Chargaff, E., 1942. A study of lipoproteins. J. Biol. Chem. 142: 491-504. Cook, W. H., 1961 Proteins of hen's egg yolk. Nature 190: 1175-1175. Cook, W. H., 1968. Macromolecular components of egg yolk. in "Egg Quality: A Study of the Hen's Egg," T. C. Carter, ed. Oliver and Boyd, Limited, Edinburgh. Cook, W. H., R. W. Burley, W. G. Martin, and J. W. Hopkins, 1962. Amino acid compositions of the egg yolk lipo- proteins and a statistical comparison of their amino acid ratios. Biochim. Biophys. Acta 60: 98-105. Cook, W. H. and W. G..Martin, 1962. Composition and proper- ties of soluble lipoproteins in relation to structure. Can. J. Biochem. Physiol. 40: 1273-1285. 94 Cummings, J. G., K. T. Zee, V. Turner, F. Quinn, and R. E. Cook, 1966. Residues in eggs from low level feeding of five chlorinated hydrocarbon insecticides to hens. J. Assoc. Offic. Anal. Chem. 49: 554-564. Cummings, J. G., M. Eidelman, V. Turner, D. Reed, and K. T. Zee,.1967. Residues in poultry tissues from low level feeding of five chlorinated hydrocarbon insecticides to hens. J. Assoc. Offic. Anal. Chem. 50: 418-425. Dixon, W. J., and F. O. Massey Jr., 1957. "Introduction to Statistical Analyses" 2nd ed. pp. 119-124. McGranHill Book Co., Inc., New York. Doran, B. M. and W. J. Mueller,.1961. The development and structure of vitelline membrane and their relation to yolk mottling. Poultry Sci. 40: 474-478. Durham, W. F., 1967. The interaction of pesticides with other factors. Residue Rev. 18: 21-105. Evans, R. J. and S. L. Bandemer,.1961. Lipide distribution and egg yolk lipoprotein complexes. Poultry Sci. 40: 597-605. Evans, R. J., S. L. Bandemer, K. Heinlein, and J. A. Davidson, 1968. Binding of lipid to protein in lipovitellin from the hens egg. Biochem. 7: 5095-5102. Evans, R. J., S. L. Bandemer, J. A. Davidson, K. Heinlein, and S. S. Vaghefi, 1969. Binding of lipid to protein in the low-density lipoprotein from the hen's egg.- Biochim. Biophys. Acta 164: 566-574. ' Duncan, D. B., 1957. Multiple range test for correlated and heteroscidastic means. Biometrics-15: 164-176. Franzen, J. S., C. M. Bobik, R. N. Stuchell, and L. D. Lee, .1968. Polypeptide subunits of lipovitellin. Arch. Biochem. Biophys. 125:.127-152. Gannon, N. and G. C. Decker, 1960. The excretion of dieldrin, DDT, and heptachlor epoxide in milk of dairy cows fed on pastures treated with dieldrin, DDT and heptachlor. J. Econ. Entomol. 55: 411-415. Gillett, J. W., 1968. "No effect" level of DDT induction of microsomal epoxidation. J. Agr. Food Chem. 16: 295-297. Gooding, C. M. B., 1966. Fate of chlorinated organic pesti- cide residues in the production of edible vegetable oils. Chem. and Ind. 1966: 544. 95 Gunther, F. A., R. C. Blinn, G. E. Carman, and R. L. Metcalf, 1954. Mechanisms of insecticidal action. The struc- tural topography theory and DDT-type compounds. Arch. Biochem. Biophys. 50: 504-505. Gunther, F. A., W. E. Westlake, and P. S. Jaglan, 1968. Pesticide solubilities in water. Residue Rev. 20: 142. Hatanaka, A., B. D. Hilton, and R. D. O'Brien, 1967. The ap— parent binding of DDT to tissue components. J. Agr. Food Chem. 15: 854-857. Hayes, W. J., Jr., 1965. Review of the metabolism of chlori— nated hydrocarbon insecticides especially in mammals. Ann. Rev. Pharm. 5: 27-52. Heath, D. F. and M. Vandekar, 1964. Toxicity and metabolism of dieldrin in rats. Brit. J. Ind. Med. 21: 269-279. Herrick, G. M., J. L. Fry, W. G. Fond, and D. C. Golden, 1969. Pesticide residues in eggs resulting from the dusting and short-term feeding of low levels of chlori— nated hydrocarbon insecticides to hens. J. Agr. Food Chem. 17: 291-295. Hugunin, A. G. and R. L. Bradley Jr., 1969. Location of organochlorine pesticides in milk. Presented at the -64th Annual Meeting of American Dairy Science Associa- tion, June 22-25, Minneapolis, Minnesota. Hui, S. F. and R. H. Common, 1966. Studies on the livetins of egg yolk. III. Heterogeneity and immunoelectro- phorectic behavior of B-livetin. Can. J. Biochem. 44: 1557-1564. Joubert, F. J. and W. H. Cook, 1958a. Separation and char- acterization of lipovitellin from hen egg yolk. Can. J. Biochem Physiol. 56: 589-598. Joubert, F. J. and W. H. Cook, 1958b. Preparation and char- acterization of phosvitin from hen egg yolk. wCan. J. Biochem. Physiol. 56: 599-408. Kratqhvil,JZ P., W. G» Martin, and W. H. Cook, 1962. Mole- cular weight and dissociation behavior of d- and 8- lipovitellin in acid solvents. Can. J. Biochem. Physiol. 40: 877-885. Langlois, B. E., B. J. Liska, and D. L. Hill, 1964. The effects of processing and storage of dairy products on chlorinated insecticide residue. I. DDT and lindane. J. Milk Food Tech. 27: 264-267. 96 Lindgren, D. L., W. B. Sinclair, and L. E. Vincent, 1968. Residues in raw and processed food resulting from post-harvest insecticidal treatment. Residue Rev. 21: 1-125. Liska, B. J., B. E. Langlois, G. C. Mostert, and W. J. Stadelman. 1964. .Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci 45: 982-984. MacKenzie, S. L. and W. G. Martin. 1967. The macromolecu— lar composition of hen's egg yolk at successive stages of maturation. Can. J. Biochem. 45: 591-601. Marion, W. W., J. M. Ning, and S. M. Ning, 1968. Application of malathion to the laying hen. Poultry Sci. 47: 1956- 1961. Martin, W. G., J. Augustyniak, and W. H. Cook, 1964. Frac- tionation and characterization of the low-density lipoproteins of hen's egg yolk. Biochim. Biophys. Acta 84: 714-720. Martin, W. G. and W. H. Cook, 1958. Preparation and molecu- lar weight of v-livetin from egg yolk. Can. J. Biochem. Physiol. 56: 155-160. Martin, W. G. and Z. Saito, 1967. Lipovitellin, phosvitin, and other granule components in avian yolk during embryogenesis. Can. J. Biochem. 45: 495-501. Martin, W. G., N. H. Tattrie, and W. H. Cook, 1965. Lipid extraction and distribution studies of egg yolk lipo- proteins. Can. J. Biochem. Physiol. 41: 657-666. Martin, W. G., J. E. Vandegaer, and W. H. Cook, 1957. “Fractionation 6f livetin and the molecular weights of the d- and B-components. Can. J. Biochem. Physiol. 55: 241-250. Matsumura, F., and R. O'Brien, 1966. Insecticide reaction with nerve. Interactions of DDT with components of American cockroach. J. Agr. Food Chem. 14: 59-45. Matsumura, F., and R. O'Brien, 1966 Insect mode of action. Absorption and binding of DDT by central nervous system of American cockroach. J. Agr. Food Chem. .14: 56-59. Matthysse, J. G., W. H. Gutenmann, and R. Gigger, 1968. Sheep ectoparasite control. II. Toxicity to sheep and residues of diazinon and lindane. J. Econ. Entomol. 61: 207-209. 97 Mecham, D. K. and H. S. Olcott, 1949. Phosvitin, the prin- cipal phosphoprotein of egg yolk. J. Am. Chem. Soc. 71: 5670-5679. Menzie, C. M., 1969. Metabolism of pesticides. Bureau of Sport Fisheries and Wildlife Special Scientific Report-- Wildlife No. 127. Washington, D. C. Mitchell, L. E. .1966. Pesticides: properties and prognosis. Adv. in Chem. 60: 1-22. Mok, C.-C., C. T. Grant, and G. Taborsky, 1966. Counter- current distribution of phosvitin. Biochem. 5: 2517— 2525. Mok, C.-C., and R. H. Common, 1964a. Studies on the livetins of hen's egg yolk. I. Identification of paper-electro- phoretic and immunoelectrophoretic livetin fractions with serum protein antigens by immunoelectrophoretic analysis. Can. J. Biochem. 42: 871-881. Mok, C.-C. and R. H. Common, 1964b. Studies on the livetins of hen's egg yolk. II. Immunoelectrophoretic identi- fication of livetins with serum proteins. Can. J. Bio- chem. 42: 1119-1151. Mok, C.-C., W. G. Martin, and R. H. Common, 1961. A compari- son of phosvitin prepared from hen's serum and from hen's egg yolk. Can. J. Biochem. Physiol. 59: 109-117. Morrison, W. R., 1964. A fast, simple and reliable method for the microdetermination of phosphorus in biological materials. Anal. Biochem. 7: 218-224. Moss, J. A. and D. E. Hathaway, 1964. Transport of organic compounds in the mammal. Partition of dieldrin and Telodrin between the cellular components and soluble proteins of the blood. Biochem. J. 91:75842595. Mulkern, J. J. and R. E. Clegg, 1968. Changes in the phos- phoproteins of the yolk during the development of the chick embryo. Poultry Sci. 47: 644-648. Naber, E. C. and G. W. Ware, 1961. Effect of BHC or lindane ingestion on its occurrence in eggs and body tissues. Poultry Sci. 40: 1455. Neelin, J. M. and W. H. Cook, 1961. Terminal amino acids of egg yolk lipoproteins. Can. J. Biochem. Physiol. 59: 1075-1084. 98 Noble, R. C. and J. H. Moore, 1965. Metabolism of the yolk phospholipids by the developing chick embryo. Can. J. Biochem. 45: 1677-1686. Noble, R. C. and J. H. Moore,.1967. -The partition of lipids between the yolk and yolk sac membrane during the de- velopment of the chick embryo. Can. J. Biochem. 45: 949-959. Osborne, T. B. and T. L. Campbell, 1906. The proteins of egg yolk. J. Am. Chem. Soc. 22: 415. Parkinson, T. L., 1966. The chemical composition of eggs. J. Sci. Fd. Agr. 17: 101-111. Plimmer, R. H. A., 1908. The proteins of egg yolk. J. Chem. Soc. 95: 1500-1506. Radomski, M. W. and W. H. Cook, 1964. Chromatographic separation of phosvitin, d- and 8-lipovitellin of egg yolk granules on TEAE cellulose. Can. J. Biochem. 42: 1205-1215. Rhodes, D. N. and C. H. Lea, 1957. Phospholipids 4. On the composition of hen's egg phospholipids. Biochem. J. 65: 526-555. 1 Ritchey, S. J., R. W. Young, and E. O. Essary, 1969. Cooking methods and heating effects on DDT in chicken tissues. J. Food Sci. 54: 569-571. Ritchey, S. J., R. W. Young, and E. O. Essary, 1967. The effects of cooking on chlorinated hydrocarbon pesticide residues in chicken tissues. J. Food Sci. 52: 258-240. Romanoff, A. L., 1967. "Biochemistry of the Avian.Embryo." p. 221 Interscience Publishers, New York. Rumsey, T. S., P. A. Putman, R. C. Davies, and C. Corley, 1967. Distribution of p,p'-DDT residues in adipose and muscle tissue of beef cattle. J. Agr. Food Chem. 15: 898-901. Ruzicka, J. H. A., J. H. Simmons and J. O'G. Tatton, 1967. Pesticide residues in food stuffs in Great Britian. IV. Organochlorine pesticide residues in welfare foods. J. Sci. Fd. Agr. 18: 579-582. Saito, Z. and W. G. Martin, 1966. Ovalbumin and other water- soluble proteins in avian yolk during embryogenesis. -Can. J. Biochem. 44: 295-501. 99 Saito, Z., W. G. Martin, and W. H. Cook, 1965. Changes in the major macromolecular fractions of egg yolk during embryogenesis. Can. J. Biochem. 45: 1755-1770. Saari, Agt W. D. Powrie, and O. Fennema, 1964. Isolation and characterization of low-density lipoproteins in native egg yolk plasma. J. Food Sci. 29: 507-515. Schafer, M. L., 1968. Pesticides in blood. Residue Rev. 24: 19-59. Shin, Y.-O., 1970. Adsorption of DDT by soils and biological materials. PhD Thesis, Michigan State University Library, East Lansing, Michigan. Smith, C. F., 1959. Shell egg deterioration: diffusion of yolk lipids into albumen as a natural cause of failure in performance. Poultry Sci. 58: 181-192. Smith, K. J., P. C. Polan, D. M. DeVries, and F. B. Coon, 1968. Removal of chlorinated pesticides from crude vegetable oils by simulated commercial processing procedures. J. Am. Oil Chem. Soc. 45: 866-869. Stadelman, W. J., B. J. Liska, B. E. Langlois, G. C. Mosterti, and A. R. Stemp, 1965. Persistence of chlorinated hydrocarbon insecticide residues in chicken tissues and eggs. Poultry Sci. 44: 455-457. Steer, D..C., W. G. Martin, and W. H. Cook, 1968. Structural investigation of the low-density lipoprotein of hen's egg yolk using proteolysis. Biochem. 7: 5509-5515. Sugano, H., 1958. Studies on egg yolk proteins. III. Preparation and properties of two major lipoproteins of the egg yolk, d- and B-lipovitellin. J. Biochem. 45: 595-401. Sugano, H., 1959. Studies on egg yolk proteins. V. Electro- phoretic and ultracentrifugal investigations on the homogeneities and some properties of d- and B-lipo- vitellin. J. Biochem. 46: 417-425. Sugano, H. and I. Watanabe,.1961. Isolation and some proper- ties of native lipoproteins from egg yolk. J. Biochem. 50: 475-479. Sundararjan, T. A., K. S. V. Sampath Kumar and P..S. Sarma, _1960. A simplified procedure for the preparation of phosvitin and vitellin. Biochem. Biophys. Acta 58: 560-561. _ 100 Thompson, E. M., G. J..Mountney, and G. W. Ware, 1967. .Methoxychlor residues in chicken eggs. J. Econ. Entomol. 60: 255-257. Tinsley, I. J., 1966. ,Nutritional interactions in dieldrin toxicity. J. Agr. Food Chem. 14: 565-565. Vandegaer, J. E., M.-E. Reichmann, and W. H. Cook, 1956. Preparation and molecular weight of lipovitellin from egg yolk. .Arch. Biochem. BiOphys. 62: 528-557. Wallace, R. A., 1965. Resolution and isolation of avian and amphibian yolk-granule proteins using TEAE-cellulose. ~Anal. Biochem. 11: 297-511. Wallace, R. A., 1965. Studies on amphibian yolk. III. A resolution of yolk platelet components. Biochem. Biophys. Acta 74: 495-504. Wallace, R. A., D. W. Jared, and A. Z. Eisen, 1966. ‘A general method for the isolation and purification of phosvitin from.vertebrate eggs. Can. J. Biochem. 44: 1647-1655. Watt, B. K. and A. L. Merrill, 1965. vComposition of foods. Agricultural Handbook No. 8. U. S. D. A. Washington, D. C. ’ Wesley, R. L., A. R. Stemp, B. J. Liska, and W. J. Stadelman, 1966. vDepletion of‘DDT from commercial layers. Poultry Sci. 45: 521-524. Wesley, R. L., A. R. Stemp, W. J. Stadelman, B. J. Liska, R. L. Adams, and R. B. Harrington, 1969. Further studies on the depletion of DDT residues from commercial layers. Poultry Sci. 49: 1269-1275. .Whitacre, D. M. and G. C. ware, 1967. Retention of vaporized lindane by plants and animals. J.-Agr. Food Chem. 15: 492-496. Williams, J.,.1962. Serum proteins and the livetins of hen's egg yolk. Biochem. J. 85: 546-555. Williams, J. and F. Sangar, 1959. The grouping of serine phOSphate residues in phosvitin and casein. Biochim. BiOphys. Acta 55: 294-296. Wittcoff, H., 1951. "The PhOSphatides." p. 162. Reinhold Publishing Corp., New York. Zook, B. J., 1968. Some reaction conditions for methylation of triglyceride and phOSpholipids. MS thesis, Michigan State University Library, East Lansing, Michigan. APPENDICES APPENDIX I FEED COMPOSITION LB-68 RATION (FED'WITH PESTICIDE) Corn Soybean Meal, 49% Alfalfa Meal,17% Meat & Bone Meal, 50% Vitaproil, 55% Whey, dried Limestone Dicalcium Phosphate 24 Ca. 18.5 Phos. Salt Premix. R-5 Choline Chloride, 25% Zinc Oxide Fat, Stab. animal veg. TOTAL CalculatedzAnalysis Crude Protein, Fat, Fiber, % Calcium, % Phosphorus, % Total Available Productive Energy Cal/lb. Metabolizable Energy 101 Pounds 1500 510 60 50 6O 40 120 22 2000.25 17.18 5.84 5.21 5.04 0.70 0.50 965 1261 APPENDIX II AVERAGE MOISTURE CONTENTS; OF THE ALBUMEN, VITELLINE MEMBRANE, EGG YOLK AND EGG YOLK FRACTIONS Egg Group Component I II III .76 % 95 Albumen 87.49 89.01 87.79 Vitelline Membrane 80.66 91.82 84.61 Yolk: 47.56 48.74 47.85 Lipovitellin 95.69 95.29 96.18 Lipovitellenin 82.99 86.40 88.04 Livetin 97.77 98.28 98.56 Phosvitin 95.06 95.10 90.11 1Based on duplicate determinations except one for vitelline membrane. 102 Hmuou CH msuonmmonm 6cm pHmHH a“ monogamonm mnu CH .mCOADMCHEHmumU 03u mo wmmHm>monm No.0 hm.o m«.o mm.>m mm.aw cuum>uq HHH ma.o am.o mm.o mm.om mm.9a adamaamua>oqu m9.o sm.a om.o mm.m~ ad.ma cuaamufl>omuq Hp.m . u . «0.0m cauu>moam am.o m9.o om.o 9m.mm mm.mm cuum>aq HH «9.0 90.9 mm.o om.mm mo.ma aflcmaamuu>omuq mm.o «5.9 mm.o mm.m~ Ho.ms cuaamuu>omuq om.m . I . mm.mm cauu>mosm 99.0 mm.o sm.o mm.m9 Hp.mm cuum>an. H aa.o mm.o mm.o am.mw m¢.aa cflcmaamua>omaq sm.o mm.a m>.o 9>.mm mma.m> cHHHmuu>oqu a be m be he acwmuoum pwmflq cofluumum flamed cflmuoum ucwcomaoo msonw monommmonm AmQHAOm ZO Qmm¢mv ZOHBUfiMh MAOM Dwfi m0 mDMDmmmomm.. ZHNBOflQ QZ¢7QHQHA mfl.qflfi3 m4 Mbmommmomm 92¢ QHmHA .ZHmaomm ho mm0<fizm0mmm HHH NHDZMWQG .muwaanmnoum mo Hm>ma fin may um ucmoflmwcmflm * .wuflawnmnonm mo Hm>wa Rd on“ um DCMUHMHcmflm * .muuaflnmnoum mo Hm>ma as.o may um pcmoauacmam .x. * ** m.¢ww 0.0 o.aa N.mnm o.¢mmd >.mm m Hounm *H.«Nmm m.m *m.mm *m.m>ma *s.mmma m.omm m museum swoBumm pagan w.mmm N.N m.am m.omm m.mmmm *d.mam e mcofluomum cmwBumm we Hmuoe m.m¢ m.o N.H «.pm m.¢ma >.Na m Houum ***>.NmOH **b.N ***m.>N **m.0m® ***N.¢mmm *m.OOH N mQDOHm cmwzumm pwaom ***m.N¢ON H.H ***m.mm ***fi.0m¢H ***m.¢m¢N ***m.mmm fl mQOHuUMHM cmwBumm «a Hmuoa Ban Hmuos ano|.m.o mann.m.m anal.m.m campamflo mcmpcwq .m.p mucmaum> co Umwmn . . .7 mo Aammv ucmusou mumsvm :mmz mousom mQHUHummm @024Hm¢> m0 meNAH NHDZflmmd 104 .mCOHuMQHEHmumU oBu mo mmmum>¢9 0.999 0.00 9.99 9.099 0.999 0.99 0.000 9.009 0.09 900 90009 9.0 0.0 0.0 0.9 9.0 9.0 9.09 9.0 0.0 900..0.o 9.09 0.09 0.9 0.00 9.90 0.09 0.99 0.00 9.09 000-.0.0 09099 . Hmuusmz 0.00 9.00 9.00 0.099 9.909 0.00 0.999 0.099 0.90 900-.0 0 0.009 9.00 0.09 9.999 0.099 0.09 9.990 0.090 0.90 09909090 0.90 0.09 9.00 0.00 0.00 0.99 0.909 0.099 0.00 0000099 0.000 0.900 9.000 0.000 9.000 0.000 0.090 0.000 0.999 900 90009 0.00 9.00 0.99 9.09 9.99 0.0 9.99 0.09 9.0 900-.0.o 0.009 0.99 0.00 0.00 9.00 9.00 0.00 0.00 9.00 000-.0.0 0m099 9.000 0.000 0.009 0.009 0.900 0.909 0.090 9.009 9.00 900-.0.0 -00 0000 0.000 0.000 9.000 0.999 9.909 0.000 9.009 0.000 0.009 09009090 0.000 9.990 09099 0.000 0.990.0.999 09099 9.00990.00 0:00:99 999 99 9 999; 99 9 999 99 9 009099000 00 00009 .mDOHU - tr, mmOHw @5096 8mm cflum>wq - CHGmHHmuH>OQHA : C9HHGHH>OQWJ s09u009m x90» omm 020990009 2999>99 020 .292099999>0099 .2999me9>0099 909 2H QHmHA Q¢MBDWZ QZ¢ QHmHflommmomm ho BZDOZ¢ $38 no ZOHBUZDm d mfi Qfimmmmmxm mQHUHBmmm m0 ZOHEDMHMBmHQ > NHQZNQQG 105