%«‘<,M..nflv1fl.h'h \szm‘lJ 1--. Id! ‘IIJ‘INm. ' _.’.am . w : g — . u" 3‘ ‘IYIV * ABSTRACT LIPID AND DDT DYNAMICS IN DEVELOPING ‘BROOK TROUT EGGS AND FRY BY Gary James Atchison The movement and metabolism of lipids and DDT in develop- ing brook trout eggs and fry were investigated. The relation- ship of DDT—3H and lipid-14C movement within the embryonic system was correlated with DDT-caused fry mortalitieies reported in the literature. Several changes in lipid composition accompanied develop- ment. Free fatty acids increased significantly after hatch- ing, denoting lipid metabolism. An increase in the percentage of saturated fatty acid methyl esters occurred in the embryo but not in the yolk. Increases in 16:0 and 22:6 fatty acids and a decrease in 1821 were the most apparent changes in fatty acid composition. About 50-35% of the DDT-3H was converted to DDE-EH and DDD-aH by the time of yolk depletion. DDT metabolism was believed to be due to microsomal enzymes rather than intes- tinal microflora° A major reduction in lipid weight and a decrease in oil droplet radioactivity occurred during the 10-20 days following Gary James Atchison hatching. The greatest increase in rate of 3H uptake by the embryo occurred at 65-70 days and was attributed to phospho- lipid mobilization and release of DDT. This period corres— ponded to the final stages of yolk sac absorption and the period of fry mortalities reported in the literature. The release of DDT by metabolized phospholipids was felt to be of more importance in previously described fry mortalities than the utilization of the triglyceride oil droplet. LIPID AND DDT DYNAMICS IN DEVELOPING BROOK TROUT EGGS AND FRY BY Gary James Atchison A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Fisheries and Wildlife 1970 ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. Howard Johnson for his advice and guidance during this study. I am grateful to Drs. Eugene Roelofs, Ronald Monroe and Paul Fromm for serving as members of my guidance committee. I wish to thank Dr. Jerry Hamelink and Dr. Norman Leeling for their encouragement and advice. I wish to thank my wife, Gayle, and son, Bradely, for making the last three years worthwhile. This study was financially supported by a Traineeship (No. 5-T14WP-109-O4) and Predoctoral Research Fellowship (No. 1-F14WP-26, 559-01) Sponsored by the Federal Water Quality Administration of the United States Department of the Interior and by funds from Grant ROi-FD-OOZZS-OS provided by the Food and Drug Administration of the United States Department of Health, Education and Welfare. ii TABLE OF CONTENTS PART I CHANGES IN THE COMPOSITION OF LIPIDS DURING THE DEVELOPMENT OF BROOK TROUT EGGS AND FRY INTRODUCTION. . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . ARESULTS . . . . . . . . . . . . . . . . . . . . . . . DISCUSSION. . . . . . . . . . . . . . . . . . . . . . BIBLIOGMPHY O O O O C O O O I O O O O O O O O O 0 O 0 PART II METABOLISM.OF DDT BY BROOK TROUT EGGS AND FRY INTRODUCTION. . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . RESULTS . . . . . . . . . J . . . . . . . . . . . . . DISCUSSION. . . . . . . . . . . . . . . . . . . . . . BIBLIOGMPIIIY O O O O O O O O O O O O 0 O O O O O O O 0 PART III DDT AND LIPID DYNAMICS AND RELATIONSHIPS IN .DEVELOPING BROOK TROUT EGGS AND FRY IMRODUCTION . O O O . O O 9 O O O O o . O O O O O O 0 MATERIALS AND METHODS . . . . . . . . . . . . . . . . iii Page 20 25 28 29 52 37 59 42 45 ,\-i.-Ar-- 190‘ . «‘Aj“; 1-“: $1154. .‘ '5’ n 5.. o “I. GM TABLE OF CONTENTS--continued Page ”SULTS O O O O O O O O O O O O O O O O O O O O O O O 49 DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O O 65 BIBLIWRAPI‘IY. O O O O O O O O O O O O O O O O O O O 0 70 iv ‘W'amm *“r ' LIST OF TABLES TABLE PART I Changes in several average weight parameters of develOping brook trout eggs and fry. . . . . . . Average percentage radioactivity based on total radioactivity present on a silica gel G thin layer plate. Sample is of brook trout total lipid extract. . . . . . . . . . . . . . . . . . Calculated fatty acid composition of the total lipid extracted from developing brook trout eggs and fry 0 O O O O O O O O O O O O O O O O O O O 0 Fatty acid percentages of the lipids extracted from develOping brook trout eggs and fry based on the degree of unsaturation. . . . . . . . . . PART II Average concentrations and percentage composi- tion of DDT, DDD and DDE in developing brook trout eggs and fry (concentrations based on dry weight). . . . . . . . . . . . . . . . . . . . . PART III rAverage weight changes based on duplicate samples of the various components of deve10ping brook trout eggs and fry. .A denotes linolenic acid treated and B linolenic acid and DDT treated. . . . . . . . . . . . . . . . . . . . . Page 10 13 18 19 55 55 LIST OF FIGURES FIGURE PART I Changes in several average weight parameters of develOping brook trout eggs and fry. . . . . . . Percentage composition of certain components of brook trout eggs and fry. Percent residue and percent lipid were based on dry weight . . . . . PART II The approximate percentage composition of total DDT-3H as related to time in development . . . . PART III Specific activity of DDT -3H in the components of developing brook trout eggs and fry with a high concentration of DDT- H. . . . . . . . . . . . . Specific activity of DDT ~3H in the components of develOping brook trout eggs and fry with a low concentration of DDT- 3H. . . . . . . . . . . . . Distribution of 14C among the components of developing brook trout eggs and fry treated with linolenic-1-14C only 0 o o o o o o o o o o o o 0 Distribution of 14C and 3H among the components of developing brook trout eggs and fry treated With linOIeniC-1-14C and DDT- 3H. o o o o o o o 0 Amounts of DDT-3H in developing brook trout eggs and fry from high and low 3H concentration groups 0 O O O O O O O O O O O O O O O O O I O 0 Specific activity of 14C in deve10ping brook trput eggs and fry treated with linolenic acid- 1- C. o o o o o o o c o o o o o o o o o o o o 0 vi Page 12 15 55 51 53 58 60 62 64 PART I CHANGES IN THE COMPOSITION OF LIPIDS DURING THE DEVELOPMENT OF BROOK TROUT EGGS AND FRY INTRODUCTION In recent years several instances of excessive mortality in salmonid fry have been attributed to DDT which had accumu- 1ated in the eggs of the parent fish (Burdick g; al., 1964; Johnson and Pecor, 1969; and Macek, 1968). In most instances the mortalities have occurred during a "critical period" of embryo development just prior to initial feeding. It has been suggested that the mortality resulted when a lethal concentra- tion of DDT was released from egg lipids during the last stages of yolk absorption. Smith (1957) reported that the unpublished histochemical data of Mathur showed that phospholipids and lipoproteins were metabolized early in development of fish embryos and the tri- glyceride oil droplets were not metabolized until a late phase of the yolk sac period, shortly before initial feeding and complete yolk absorption. Burdick §£_§l, (1964) utilized this information to describe a relationship between lipid metabolism and DDT toxicity that could account for the mortality of lake trout fry in New York hatcheries. They be- lieved that DDT was stored in the triglyceride oil droplets of the eggs and as these droplets were utilized by the de- veloping fry, a lethal dose of DDT was released. They suggested that the DDT associated with the phospho- lipids of the egg yolk was less important in the mortalities because the phospholipids were utilized at a slow rate which would not usually result in a lethal concentration of DDT in the embryo. Smith (1958) demonstrated that phospholipids were an important energy source for rainbow trout embryos. Phopho- lipids played a minor role only during hatching and during a 5-10 day period shortly before complete yolk absorption. During hatching, protein was the main energy source and just prior to feeding triglyceride was of greatest importance. Blaxter (1969) pointed out that there is some ambiguity about the role of lipids in energy production. The study described here was part of an investigation of the dynamics of lipids and DDT in fish embryos. This study was undertaken because of the lack of knowledge about the metabolism of lipids in developing fish eggs and fry and because changes in lipid composition were felt to be important in the DDT-caused fry mortalities. The objectives were to describe the changes in lipid composition during the develop- ment of fish embryo, to relate these changes to lipid utili— zation by the embryo and to assess the importances of lipid utilization to the "critical period" in DDT-caused fry mortalities. MATERIALS AND METHODS Mature brook trout (Salvelinug fontinalis) with a mean weight of 2269 were obtained from the Grayling Research Station, Michigan Department of Natural Resources. A ration of dry pelleted food was fed at a rate approximating 2% body weight per day. Daily lighting was gradually reduced from 15.5 hrs. at the onset of the experiment to 11.1 hrs. at the time of spawning. The water temperature ranged from 12-130C. Utilizing a procedure similar to that described by Nakatani (1962), each fish was force fed five weekly doses of 0.5 u Ci (2.65 ug) linolenic acid—1—14C contained in a gelatin capsule. Feeding was begun 6 weeks and ended 2 weeks before spawning. After 8 weeks of holding the adults, eggs were stripped from the females and fertilized by milt from one or two males. Fifteen fish were used, six of which were females, but eggs were obtained from only four of these. Approxi— mately 550-400 eggs from each of these females were kept in separate 1000 ml beakers in a flow through system and incu- bated in the dark in water ranging from 11—150C.. One week after hatching the fry were removed to baskets in troughs where the temperature ranged from 10.5-120C. The eggs hatched abOUt 40 days after fertilization and the yolk sac was com- pletely absorbed by 72 days after fertilization. The fry were not fed and starvation began during the last 5 days of the experimental period after complete yolk absorption. .Characteristics of the water throughout the eXperiment were: Dissolved oxygen, 5.7-9.8 ppm; pH, 7.7-8.0; free carbon dioxide, 5.7-15.0 ppm, and total alkalinity as CaCOs, 291-520 ppm. Samples of 20 eggs from two of the females were collected every 10 days and stored under nitrogen at -15°C until analyzed. All values presented in this study are mean values for the duplicate sample. The eggs and fry from the remaining two females were utilized in the study of DDT and lipid dynamics (Part III). Twenty eggs were blotted dry, weighed and comminuted several minutes in a Sorval Omni—Mixer. .Fry samples received the same treatment except the yolk sac and embryo body were analyzed separately. ,The extraction procedure was similar to that of Bligh and Dyer (1959) and utilized a mixture of chloroform-methanol (2:1). The lipid extracted material was dried and weighed. The extracted lipids were diluted to 10 ml with solvent and divided into three portions. One aliquot of 1 ml was used to determine total lipid weight. A 4 m1 aliquot was stored under nitrogen for later examination of lipid class composition and the remaining 5 ml was used for fatty acid analysis. .‘V-wzz;8'-a-'« ' w ~. ' ‘ I‘ZJ ' To determine the relative distribution of the lipids in the yolk sac and embryo during development, thin layer chrom- atography (TLC) utilizing silica gel G plates was used to separate specific lipid classes and the relative distribution of radioactivity was determined on a liquid scintillation spectometer. The solvent system for TLC was petroleum ether- ethyl ether-acetic acid (85:15:1.5). Standards of phospho- lipid, cholesterol, free fatty acid, triglyceride, fatty acid methyl ester and cholesteryl ester were used to identify the lipid classes. Detection of bands was accomplished using 2,7-dichlorofluorescein as a spray and then viewing the plate under ultraviolet light. The bands were placed into liquid scintillation vials with 15 ml scintillation—fluor mix (toluene—ethylene glycol monomethyl ether (2:1) with 5g/l PPO and 0.19/1 POPOP) and the sample counted in a Nuclear Chicago Mark I liquid scintillation spectrometer. Counting periods were of sufficient time (10—20 min.) to give statis- tically valid data (cpm value corrected for background 1 10% at 95% confidence level). Counting efficiency was determined by use of quenched standards of carbon14 and an external standard source. The data were recorded as decompo- sitions per minute (dpm) and reported as the percentage of radioactivity in each lipid class based upon the total radioactivity on the TLC plate. A 5 ml portion of the lipid extract was utilized for the formation of fatty acid methyl esters by the method of -“ iMfitcalfe §£_a;, (1966). The sample was saponified with methanolic sodium hydroxide, the fatty acids esterified with BFa-methanol and the methyl esters extracted with hexane— ethyl ether (1:1) and dried over anhydrous sodium sulfate. The sample was protected from air by a nitrogen atmosphere whenever possible. .Methyl esters were stored under nitrogen in a refrigerator until analyzed by gas-liquid chromatography (GLC). The time period between preparation and analysis never exceeded 5 days. Fatty acid methyl esters were analyzed with a Hewlett Packard F&M 700 gas chromatograph equipped with a hydrogen flame detector. .The column used was 1/8 inch by 6 feet stainless steel and packed with 12% diethylene glycol succinate (HI—EFF~1B;Applied Science Laboratories, Inc.) on Chromsorb P 80-100 mesh. .Relative concentrations of fatty acids were determined by multiplying the peak height by one-half base width of a given fatty acid methyl ester. Comparison of the retention times of the fatty acid methyl esters obtained from the eggs and fry with fatty acid methyl ester standards obtained from the Serdary Research Laboratories and Applied Science Laboratories, Inc. aided in the direct identification of peaks on the chromatogram. Standards used were: 8:0, 10:0, 12:0, 14:0, 15:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 18:5, 20:0, 20:1, 20:4, 20:5, 20:6, and 22:1. .The number prior to the colon designates ‘the number of carbon atoms and the number following the colon refers to the number of double bonds. Thin layer plates (silver nitrate-silica gel G) were used to separate the component methyl esters in bands according to the number of double bOnds present in the fatty acids (Morris, 1962). The bands were detected by spraying 2,7-di— chlorofluorescein on one edge of the plate and viewing under ultraviolet light. The bands were then scraped from the plate, extracted with hexane and injected into the gas chrom- atograph: Complete separation was not accomplished but the procedure proved helpful in identifying many of the fatty acid methyl esters. RESULTS The materials comprising the residue (lipid extracted dry weight) decreased from 11.1 mg/egg at fertilization to 9.6 mg/fry at hatching and to 6.0 mg/fry on the 80th day (Table 1, Figure 1). The values for residue weight and lipid weight for the 10th day are probably invalid. All lipid values and thus residue values for the 10th day through- out the study show large differences from surrounding values. The specific activity value for the 10th day is another example (Table 2). Lipid values for the 10th day will not be considered valid in the remainder of the discussion. .The lipid weight did not decline to a significant degree until between 50 and 60 days at which time a drop of from 3.7 mg to 2.2 mg/fry occurred. The decrease in wet weight at hatch- ing was due to chorion loss. The dry weights were calculated without the chorion so no decrease in dry weight attributable to chorion loss was seen. The percentage lipid weight increased between fertiliza- tion and 50 days and then decreased to a level below that at the beginning of development (Figure 2). Since the residue and lipid weights make up 100%;of the dry weight, the rela- tive change in residue had a pattern opposite to that of the 10 mm.om oo.mm o.m o.o o.m o.mm Issue om ma.oo mm.mm m.m e.o o.m o.mo issue oe om.am oo.mm m.m o.e o.oa «.mm Aamuouv oo he.em mm.a~ e.a o.m e.o «.me Aosunsme oo mm.mo mm.mm m.o m.m o.m m.aa Axaose oo «a.me ao.mm e.m e.o ¢.Na o.e¢ Ismuouv om ma.am ma.an e.a e.m e.m m.mm Iowansoe om a~.ao em.mm o.m o.m o.» m.ma Axaoso om mm.oo om.mm m.m o.m «.ma m.mm Aamuoue ow mm.me em.mm o.o o.m m.m o.aa Aomnname oe mm.mo om.om o.m o.» o.oa m.om ixsome oe mm.oo oe.em o.a o.oa o.ea o.me on om.oo am.o~ m.m o.oa m.ea «.ma om mm.mo am.ma o.m m.ma m.ea «.me oa mm.ao eo.om a.e a.aa m.ma o.mm o ARV at 3.5 35 3.5 3.5 Agog Hmumk flamed cannfim Mom omunfim Mom omuflfim mom owunfim Mom new» unmams pom .us wsoammm unmams sun unmams um: nmuaaauuom EOHM GEAR .wnm pom ammo usonu xooun mnemoam>mu mo mnoumfimumm uanmB mmmnm>m Hmuw>mm cw momsmnu_ .d magma Figure 1. 11 Changes in several average weight parameters of developing brook trout eggs and fry. mg/EMBRYO 12 60- 55- 50- 45 4O 35- 30- 25'- 20- I5 IO 0 O Wet Wei ht (mg) Dry Weight (mg) Lipid Weight (mg) hatching I I L lb» l I 1 I0 20 30 4O 50 60 7O 80 DAYS AFTER FERTILIZATION Figure 1 ooo.m oo.e.a m.~ m.oa o.ma ~.mo issue om mom.ea em.o.a m.m e.ae e.oa m.mm issue ob mom.om mm.o.a o.m o.mm m.ma o.om Iowansme oo aaa.am oe.o.a e.m o.me m.aa o.em ixaoso oo www.mm mo.o.a o.e o.ee o.oa «.mm iosuosme om www.mm oo.o.a m.aA m.mm e.m e.mm Assess om ome.am aua m.e «.me a.m «.me AosuoEme oe ”w oea.em me.a.a o.m m.oe o a.em Axaose oa mom.mm ea.a.a e.o m.ae o m.me om ano.am mm.o.a e.m m.me o m.a¢ om oea.ma mm.o.a a.o a.om o o.«m oa woa.a~ m>.o.a o.¢ o.mm o o.ae o unmflm3 mun pwmwaosmwonm Amwmpv mfi\zmn ou mpwumu Hmumm mpaumo mpwud moaned soap >uw>wu04 Imamflua Hmnmumm thamwna muumm mmnm Ionmmonm Imuwawuumm oamaomom mo oaumm -Hono scum mafia .uomuuxm panda Hobo» usouu xooun mo ma mamsmm .mumHm mommH saga 0 How mowawm m so ucmmmum muw>wuom0womu Hmuou so Comma >uw>auomoaomu mmmusmoumm mmmnm>m .N magma ‘ _--v * *- q-rr" “Frugal-gm. ' 14 Figure 2. Percentage composition of certain components of brook trout eggs and fry. Percent residue and percent lipid were based on dry weight. PERCENT 15 IOO 90 - Percent Res/due 4 80 --¢ 70 60 ' Percent Water 50 _. ---- Embryo 30 P ”I Percent Li id ‘4 20 p '0 (- hatching O 4 l l T I I l 0 IO 20 30 40 50 60 70 80 DAYS FROM FERTILIZATION Figure 2 16 lipid weight. The water in the yolk showed a less marked change with development than that of the embryo. The water in the embryo increased from 66.52% in the egg at 50 days to 86.58% in the 80 day fry. The water in the yolk de- creased from 66.52% on day 50 to 61.71% on the 50th day and then increased to 68.58% on the 60th day. {5’ Analysis of lipid class behavior demonstrated a very pronounced increase in free fatty acids following hatching (Table 2). The increase was greater in the embryo (2.4% at 40 days to 19.2% at 60 days) than the yolk (0.0% at 40 days to 11.9% at 60 days). The cholesteryl esters increased from 4.6% on the day of fertilization to 9.4% at 50 days but after hatching the values remained quite constant at a per- centage slightly below that at the beginning of development. Triglycerides were the most abundant lipid class until the 50th day. Phospholipids then became more abundant until the 50th day at which time triglycerides again were more plenti— ful. On the 80th day, the phospholipids clearly predominated, comprising 65.2% of the radioactive lipids. Cholesterol was present but its radioactivity was extremely low and its separation from the phospholipids incomplete at times. For this reason, the percentage of radioactivity contained in cholesterol was not recorded and this radioactivity appeared as a slight addition to the percentage of phospholipid. The specific activity of “PC based on dry weight in- creased between the 20th day and the 40th day, returned to 17 near the level present at fertilization by the 50th day and decreased rapidly after 60 days (Table 2). As previously discussed, the specific activity value for the 10th day is (erroneous. .The greatest increase in relative radioactivity that corresponded to the increased specific activity at hatching was in the yolk phOSpholipids at 40 days. The fatty acid composition of the brook trout eggs and fry underwent some very noticeable changes during develop- ment (Table 5). The saturated fatty acids (14:0, 16.0 and 18:0) increased in the embryo after hatching. The only other notable increase was in the 22:6 content of the embryo. Oleic (18:1) and linoleic (18:2) acids showed pronounced decreases and the remaining fatty acids showed no gross. changes. The relative composition of the fatty acids in the yolk remained approximately the same or changed only slightly in the opposite direction of the embryo as compared to the percentages at the beginning of development. Saturated fatty acids and fatty acids with six double bonds increased at the expense primarily of those with 1, 2 and 5 places of unsaturation (Table 4). .The ratio of satur— ated to unsaturated fatty acids remained rather consistent until 50 days after which the ratio decreased. This ratio changed very little in the yolk lipids. 18 .oumd .ouOd .oum "Rm.d smnu mama mo mucsoEm CH .mumm pom «HNN .mumm .duom .ouwfi .aued usmmmum muw3 mpwom muumm mcw3oaaom one .x. m.am m.s ~.m o.m o.m m.e m.om e.o o.m e.ma e.e om m.ma m.o a.m m.a m.m e.oa o.em a.e m.o o.oa m.m op m.me .e.o o.m o.m o.m m.m m.em m.o m.o m.oa o.m Aomnnsmv oo m.ma m.m o.m o.m e.e a.ma N.om e.o e.o a.ea m.m ixsose om o.ma e.o e.~ w.m m.m m.oa w.¢~ e.o o.m m.ea o.m Iowansme om m.ma o.o o.m o.m ~.e m.aa e.om m.o e.o m.ma m.m Axaome om m.ea a.o o.m m.m o.m m.aa m.om m.m o.o o.ma m.m homunsme o4 e.oa e.o o.m m.m m.m m.aa o.em o.m o.m m.ea e.m ixaome oa o.ma e.o o.m o.m o.e e.aa o.om m.m o.o o.ea m.m om e.ma o.o o.m a.m m.m o.aa m.em m.m e.o m.ma o.m om «.ma o.e a.m ~.m m.m o.ma o.om o.m o.e m.¢a o.m oa o.ea m.o o.m m.m o.e m.ea ¢.em o.m m.o m.ma e.m o o.mm euom muom m.om m.ma mums sums ouma a.oa o.oa o.ea cos» i Imuaaauumm *Aucmoumm%cowuwmomfioo wand %upmm Hmuw4.m%mn .hnm cam mmmm usonu xooun mcamoHo>mp Eoum Umuomuuxm pamwa Hmuou on» no GOAUHmomfioo pwum muumm owumHDUHmo .m manna 19 m.m.a m.am woman m.e m.m m.m e.om m.om om m.m.a m.ma moms» m.o e.m m.ma m.om o.om ob m.m.a m.ma momma e.o m.o m.ma e.om e.mm iosunsmo oo «.mua m.ma moms“ m.m ¢.e «.ma o.mm o.em ixsose oo m.m.a o.ma moans e.o e.m o.ma o.om o.o~ iosnn2me om m.m.a m.ma momma o.o m.e e.ea m.em o.mm Asaose om m.m.a m.ea moans e.o o.o o.ea «.mm «.mm iosunsmv oe e.m.a e.oa momnu a.o m.o m.ea e.mm m.mm ixaosv ow m.m.a o.ma momma e.o o.e a.ea. e.mm «.mm on m.m.a e.ma moms» o.o m.o m.ea m.em e.mm om n.m.a a.na momma o.e o.o m.ma o.em a.mm oa o.m.a o.ea moans m.o m.o m.ea. m.mm m.am o oopmuspmm o m e m m a o coast IGD ou . Imuwawbumm pmumusumm GOAUMHSDMmcD mo common >9 wmmusmoumm Ufiom muumm Eonm whom no oaumm .coflumusummss mo common may no comma mum cam ammo usouu xoonn mnemon>mU Scum Umuumuuxo mpwmfia may mo mommucmunwm pwom hunch. .¢ magma l‘ DISCUSSION The increase of lipids on a percentage dry weight basis has been reported for the eggs of several salmonids (Smith, 1952 and Phillips and Dumas, 1959). Phillips and Dumas (1959) believed this increase was due to conversion of pro- tein material to lipids during the last few weeks before hatching. Lafon (1947, reported by Smith, 1952) showed that 55% of rainbow trout egg lipids were bound to protein, 50% were phospholipid and the remainder were free fat. Mathur (unpublished, reported by Smith, 1957) demonstrated that most non-triglyceride fat formed a phOSpholipid-protein complex. Lipoprotein material is not as extractable as unbound tri- glycerides and phospholipids. Concurrent to protein metabol- ism, some protein bound lipids may be separated from the complex and remain in the yolk sac. These would be extracted and could be an explanation for the increased Specific activ— ity of 14C on a dry weight basis at hatching (Table 2). The phospholipid content increased in the yolk from 41.2% on day 20 when most of the egg was yolk and the embryo body was small to 57.1% on the 40th day when hatching occurred. A sig- nificant decrease in the percentage of phospholipid occurred in the yolk during the 10 days after hatching. The Specific 20 21 activity also showed a large decrease in this same time period. An increase in phOSpholipid radioactivity in the yolk accompanied by an increase in Specific activity prior to hatching would lend evidence to the idea that the phos- pholipids become more available to extraction during the period of peak protein utilization at hatching as suggested by Smith (1957). Sndth (1958) reported the contribution of protein and lipids to the daily heat production of rainbow trout eggs and fry. ,PhOSpholipids and protein contributed most of the energy until 60 days after fertilization (hatching occurred at 56 days). Between 29-40 days, metabolism‘was confined mainly to protein. PhOSpholipids accounted for 60-70% of the daily energy production between 40-60 days after which 75%iof the energy was produced by phOSpholipids. Terner _§_§l, (1968) stated that triglycerides are converted to phOSpholipids. The reduction in fat weight after 50 days (Table 1) could be attributed to triglyceride metabolism. Phospholipids are a more stable component of the embryo and thus make up about 65% of the lipid material at 80 days. Terner (1968) demonstrated that the radioactivity taken up by trout embryos incubated with acetate-1-14C was released slowly as 1‘."C02. “Terner _£_al, (1968) stated that free fatty acids and glycerides probably serve as substrates for endo- geneous metabolism. This decrease in radioactivity plus the dilution of the lipid pool by protein-derived-non-labeled 22 lipids just prior to hatching would account for the progres- sive decline in Specific activity during the last 50 days of development (Table 2). Lovern (1956), Kyte (1956) Ando (1962) and Gruger EE.§£3 (1964) reported values of about 85% unsaturation in salmonid egg fatty acids. Ando (1962) indicated that the percent saturation of acetone soluble lipids decreased with develop- ment of rainbow trout eggs and fry. This conflicts with data presented in Table 4. Acetone probably does not give as com- plete extraction as chloroform-methanol. Ando's data Show an increase in C18 unsaturated fatty acid from 55% at ferti- lization to 58% at hatching and 55% at swim-up. The present study indicated 45% unsaturated C13 at fertilization to 50% at the end of the yolk sac stage. Palmitic (16:0) and docosahexaenoic (22:6) acids showed the greatest increase. The composition of yolk fatty acids did not change appreciably with development but that of the embryo was altered considerably from that of the yolk. Brenner gg_§l, (1965) stated that palmitic acid was synthe- sized in_§9£9_by Pimelodus maculatus (a fresh water fish). On a fat free diet the palmitic acid content increased. Linolenate (18:2) not only reduced the level of palmitic acid to normal but also increased the level of arachidonate (20:4). Ackman (1967) believed that C18 polyunsaturated fatty acids were stored when food was abundant and was only converted to more highly unsaturated fatty acids of longer chain length 25 when necessary as water temperature fell or food became scarce. .In non-fed fry, of course, food does become scarce after complete yolk absorption. Terner §£_gl, (1968) demonstrated that trout embryo lipids were quite dynamic. They believed that the preformed long-chain fatty acids of the neutral lipids of the yolk sac were not utilized as such but broken down and embryo fatty acids synthesized d§_ngyg_from the acetyl coenzyme A units derived from their breakdown. They stated that the phOSpho- lipids were derived from triglycerides and that there was a smaller reserve of phospholipids in the yolk. The increase in free fatty acids of the brook trout in this study indi- cated that either the phOSpholipidS or the triglycerides were being metabolized. Ando (1962) also noted a rapid increase in free fatty acids during the development of rainbow trout just after hatching. From the above discussion, the following sequence of events can be outlined. As the protein becomes scarce and phOSpholipid stores are utilized the triglycerides begin to be converted to free fatty acids. Free fatty acids are ab- sorbed by the embryo and either broken down to acetyl co- enzyme A, then resynthesized (eSpecially to palmitate and to a lesser extent myristate); undergo chain extension to form long chain polyunsaturated fatty acids (eSpecially doco- sahexaenoic acid): or are utilized for energy. The synthe- sized or lengthened fatty acids are restructured to form 24 triglycerides or phospholipids. The triglycerides are even- tually utilized as fuel if starvation sets in after yolk sac absorption and the phoSpholipids are left to predominate the lipid composition of the fry. .A reduction in linoleic and linolenic (18:5) acids is due to their use as precursors of longer chained acids. For most fry mortalities reported in the literature and attributed to DDT toxicity, the “critical period" of embry- onic development was juSt prior to initial feeding. There was a large decrease in triglycerides in the brook trout fry on the 80th day of development but this was about 10 days after the yolk sac was completely absorbed. Whether these triglycerides were directly from the yolk lipids or from lipids Synthesized by the embryo could not be ascertained from the present experiment. The major decrease in yolk lipids occurred between the 50th and 60th days which was before the time of the "critical period". These changes cannot be ruled out as possible contributing factors in fry mortalities but they would cast doubt on the occurrence of a major utiliza- tion of triglycerides and release of DDT at the end of the yolk sac period (about 70 days). The significance of changes in lipid composition to DDT-caused fry mortalities will be discussed more fully in Part III. a v—-- -——_—. rm BIBLIOGRAPHY Ackman, R. G. 1967. Characteristics of the fatty acid composition and biochemistry of some freshwater fish oils and lipids in comparison with marine oils and lipids. Comp. Biochem. Physiol., 22; 907—922. Ando, K. 1962. Change of the lipid during development of rainbow trout eggs. Bull. Jap. Soc. Sci. Fisheries, 28: 75—76. Blaxter, J. H. S. 1969. Development: Eggs and larvae, p. 177 -252. In W. S° Hoar and D. J. Randall [ed. ]. Fish physiology, Vol. III. Academic Press, Inc., New York. Bligh, E. G. and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Canadian J. Biochem. Physiol., 51; 911—917. Brenner, R. R., D. V. Vazza and M. E. DeTomas. 1965. Effect of a fat-free diet and of different dietary fatty acids (palmitate, oleate, and linoleate) on the fatty acid composition of fresh-water fish lipids. J. Lipid Res., 4; 541—545. Burdick, G. E., E. J. Harris, H. J. Dean, T. M. Walker, J. Shea, and D. Colby. 1964. The accumulation of DDT in lake trout and the effect on reproduction. Trans. Amer. Fish. Soc., 95: 127—156. Gruger, E. H., Jr., R. W. Nelson and M. E. Stansby. 1964. Fatty acid composition of oils from 21 species of marine fish, freshwater fish and Shellfish. J. Amer. Oil Chem. Soc., 415 662-667. ’ Kyte, R. M. 1956. Constituent fatty acids of salmon egg fat. J. Amer. Oil Chem. Soc., 553 146-149. Lafon, M. 1947. Recherches sur l‘utilisation des réserves vitellines au cours de l'embryogénese. (1) Données sur l'embryon de truite (Salmo fairio et Salmo irideus). Arch. Intern. Physiol., gig 125-152. 25 26 Lovern, J. A. 1956. Fat metabolism in fishes. VIII. Changes in fat of ripening salmon eggs. Biochem. J., §_O_; 20-240 Metcalfe, L. D., A. A. Schmitz and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem., 58: 514-515. Mbrris, L. J. 1962. Separation of higher fatty acid iso- mers and vinylogues by thin-layer chromatography. Chem. Ind. 21: 1258-1240. Nakatani, R. E. 1962. A method for force-feeding radio- isotopes to yearling trout. Prog. Fish-Cult., 24: 56-59. Phillips, A. M., Jr., and R. F. Dumas. 1959. The chemistry of develOping brown trout eyed eggs and sac fry. Prog. Fish-Cult., 21: 161-164. Smith, S. 1952. Studies in the development of the rainbow trout (Salmo irideus). II. The metabolism of carbo- hydrates and fats. J. Exp. Biol., 29; 650-666. Smith, S. 1957. Early development and hatching, p. 525-559. In_M. E. Brown [ed.]. The physiology of fishes, Vol. I. Academic Press, Inc., New York. Smith, S. 1958. Yolk utilization in fishes, p. 55-55. In_D. Rudnick [ed.]. Embryonic nutrition. Univ. Chicago Press, Chicago, Illinois. Terner, C. 1968. Studies of metabolism in embryonic develop- ment--I. The oxidative metabolism of unfertilized and embryonated eggs of the rainbow trout. Comp. Biochem. Physiol., 24: 955-940. Terner, C., L. A. Kumar and T. S. Choe. 1968. Studies of metabolism in embryonic development--II. Biosynthesis of lipids in embryonated trout ova. Comp. Biochem. Physiol., 24: 941-950. PART II METABOLISM OF DDT BY BROOK TROUT EGGS AND FRY 27 INTRODUCTION There are five principle routes of DDT metabolism in various organisms: Oxidation to DDA, Kelthane or dichloro— benZOphenone; dehydrochlorination to DDE; or reductive dechlorination to DDD (O'Brien, 1967). Menzie (1969) re- viewed the literature on this subject. Routine analyses of fish generally reveal DDE and DDD when DDT is present (Henderson §£_§l,, 1969). These two metabolites are the only compounds with substantial evidence linking them.with ‘DDT breakdown in fish. Degradation of DDT in fish has been demonstrated by Allison §£_§l, (1965,1964), Buhler gg.al. (1969), Greer and Paim (1968), Grezenda §t__l, (1970), Prather and Ferguson (1966), Premdas and Anderson (1965) and Wedemeyer (1968). All of these studies utilized juvenile or adult fish. Wedemeyer (1968) and Cherrington §£_al, (1969) indicated that part of this degradation, eSpecially to DDD was due to microbial action in the gut. Wedemeyer (1968) demonstrated that liver enzymes converted DDT to DDE in rain— bow trout. The metabolism of DDT by fish eggs and fry has not been previously reported. A knowledge of the degree of degrada- tion and the developmental stages involved is important in understanding the effects of DDT on egg and fry development and survival. 28 MATERIALS AND) METHODS General rearing conditions, feeding rates, methods of isotope introduction and egg collection, fertilization and rearing were previously described (Part I). Brook trout received five weekly force feedings of 0.5 uCi (2.65 ug) of linolenic acid—1-14C and 1.0 uCi (114 ug) DDT-3H (ring labeled)contained in gelatin capsules. The linolenic acid was utilized in lipid studies run concurrently and it was assumed to have no affect on DDT metabolism. Eggs were stripped from two females, fertilized, and incubated for 80 days in 1000 ml beakers. Samples of eggs or fry were col- lected every 10 days and stored at -15°C until analyzed. Egg and fry samples of approximately 1g were ground with anhydrous sodium sulfate, extracted three times with ethyl ether—petroleum ether (6:94) and cleaned up on activated florisil (Mills g£_§l,, 1965). Solvent from the eluted frac- tion was evaporated to near dryness on a rotary evaporator and quantitatively transferred to glass stoppered sample tubes. Gas chromatographic (GC) analysis for DDT and metabolites was carried out on a MicroTek 220 instrument equipped with a electron capture detector and a 1/4 inch by 6 foot glass column packed with 5% SE—50 on 60—80 mesh Gas Chrom—Q. 29 50 The column temperature was 175°C with a nitrogen carrier gas flow rate of 70 ml/min. Confirmation of sample identity was accomplished by thin layer chromatographic analysis and by injection on a second column packed with 5% DC 200 and 11% QF-1 on 60—80 mesh Gas Chrom-Q. The entire remaining portion of the sample was spotted across a silica gel G thin layer chromatographic plate and developed in ethyl ether—hexane (2:98). Standards were placed on one side of the plate and after development were Sprayed with rhodamine B and viewed under ultraviolet light. The individual components of the sample were scraped from the plate into liquid scintillation vials. Gas chromatography checks on the procedure demonstrated that the separation was complete. Fifteen ml of scintillation-fluor mix (toluene- ethylene glycol monomethyl ether (2:1) with Sg/l PPO and 0.19/1 POPOP) was added to each vial. ‘ .Radioanalyses were made with a Nuclear Chicago Mark I liquid scintillation Spectrometer. Counting periods were of sufficient time (20-40 min.) to give statistically valid data (1.10% at 95% confidence level). Counting efficiency was determined by quenched standards of tritium and carbon14 and an external standard source. Only trace quantities of 14C from the linolenic acid-1-14C remained in the sample after cleanup and did not interfere with tritium determinations. Dry weight and water content were determined on samples run concurrently with the pesticide samples. These dry 51 weight data were utilized in conversion of pesticide concen- tration data to parts per million (ppm) dry weight. This procedure corrected for any loss in water during storage. RESULTS DDT was not noticeably metabolized by brook trout eggs prior to hatching (40 days) but metabolism did occur after hatching, particularly during the final 20 days of develop- ment (Table 1, Figure 1). The eggs contained some non- labeled DDT which was probably derived from the commercial pelleted dry food fed to the adult brook trout. An analysis of the food Showed an average concentration of 16 ppb (parts per billion) DDD, 56 ppb DDT and traces of DDE. At fertili— zation, 62.77% of the non-labeled DDT complex (DDT, DDD, and DDE) was p,p'-DDT and 92.40% of the DDT—5H complex was p,p'-DDT—3H. Thus the p,p'—DDT—3H was diluted by the non- labeled p,p'-DDT. Some of the DDT ingested in the food was probably metabolized to DDE and DDD by the adult and deposited in the eggs in approximately the concentrations indicated by the 0 day values (Table 1). The changes demonstrated by the isotope method of analysis were masked in the GC analysis due to the dilution of isotope with non-labeled DDT, DDD and DDE. Thus an increase in DDD was not demonstrated by the GC method but was by the isotope method. Both methods of analy- sis did demonstrate metabolism of DDT to DDE. Of the two procedures utilized in this study, the isotope method 52 0>.Ne d>.ee mm.Ne hoo.b oomh. ooem. mooe. Noon. Om pm.om wo.> oe.em mom.o Noeo.e econ. whoo. omem. OP Ne.mm o>.> oe.mm om>.o ommw. mNoN. Ommo. Heme. oo oe.oN eo.o om.eo >oe.o oooo. oeoe. emeo. onoe. om eo.oN ee.> N>.mo mmo.o mmno. ease. omeo. whee. oe % oo.mN oo.m oo.oh e>o.m oeoo. emme. Como. epoe. om mm.mm >e.> om.mo omm.o neon. whoa. domo. eomm. om oe.em oe.> ee.eo hoe.> oeom. ooee. mmwo. mNNN. oe em.om mo.> >>.eo on.e boom. mooe. eomo. oomm. o >HM\HO AEQQV Afimmv AEmmv Afimmv mace snow“ some“ mmm\mc Hmmme was con eon mama .Aunmflm3 wnp co woman mcoflumnucmocoov mum pom wmmo usouu xooun mcflmoao>wp SA man pom can .HQQ wo cofluflmomfioo ommpcoouwm pom mcoHumuuswusou omnuw>< .H wanna .usmamoaw>mo ca. mfiau Ou. ooumamn mm menace Hmuou mo seauamomfiou wmmucmonwm wumfiexoummm was in musmfim H OHDmHh mhzw2d04w>mc z. m>.o 0o oe.no oo.mo No.ee oe.ne N>.ee Ne.ee ho.o oo.o oN.N NN.N ow nm.o> N>.o> oo.ee oe.ee Nn.o mm.> oo.o ne.o oe.o Nn.o oo mo.e> me.e> Nn.ee ee.ne oo.o om.o oe.o «5.0 oo.> mm.o on oe.>o oo.o> we.ne mn.ee on.m ee.m on.o en.o on.NH em.ee oe ne.oo hm.mo oo.ee eo.ee He.e No.0 >e.o ee.o 0N.ne o>.Ne on >m.oo No.no om.ne on.ne II In oo.o on.o on.ne em.ee om oe.eo on.no oo.ne mn.ne II I: o>.o oo.o oe.ne on.ee oe eo.eo mn.>n od.nd n>.me II II eo.o on.o nn.ee oe.me o m < m aw m d m 4 .m d Houmz AONHAEOVme NONHmEonEw Noxumaommfiw AONHQEm\mmm Ammmpv unwouom uzmfloz hum Chunfim Hwo MACH wEwB .poumwnu Baa pom oaom oasoaosfla m pom pmummuu Uflom GasmHOSHH mouocop d .mum can name usouu xooun mnemoao>mo mo mucocomfioo msoaum> may mo nmamfimm muMUHHmSo so woman momcmso usmflo3 ommuw>¢ .e wanna lilll’l‘ll 56 development (Figures 5 and 4). The relative distribution of lipid-14C in treated and nontreated samples was essentially the same throughout development. The percent 14C in the yolk and oil began to decrease before hatching and continued at an increased rate following hatching. At fertilization, about 80%Iof the 14C and 70%»of the 3H was present in the yolk (Figure 4). The 14C increased to about 85% by the 80th day in the embryo. DDT-3H radioactivity showed a parallel decrease in the oil droplet as compared to the 14C and a parallel increase in the embryo. Hewever, loss of DDT-3H activity from the yolk lapsed behind the decrease in lipid radioactivity indicating perhaps a different relationship between DDT and lipid utilization in the yolk and DDT and utilization of the oil droplet. Uptake of DDT-3H by the embryo increased in rate between the 48th and 54th days and again between the 66th-and 70th days. As mentioned above, no change in Specific activity of DDT-3H occurred between the just fertilized egg and the late fry stage, but a definite decrease in the amount of DDT-3H did occur after about 60 days (Figure 5). The high concen- tration sample lost 52.8% of its 3H and the low concentration sample 12.4%. Specific activities of lipids decreased by-more than 50% from fertilization to the end of the yolk sac stage (Figure 6). The patterns of change were similar between treated and nontreated samples and there was no significant difference in Specific activities between the two groups. Figure 5. Distribution of 14'C among the components of developing brook trout eggs and fry treated with linolenic-l-l‘c only. ‘a—~—.__— PERCf/VT fiAD/OACT/V/TY IOO 58 9O 80- 70- 60- 50- 4o- 50* 20- I08 O X Yolk O 0 Oil x O . x . IEmbryo H/mfmgouu..." 0 0 IO 20 50 4O 50 60 7O 80 DAYS AFTER FERTILIZATION Figure 5 59 .moiaan pom Ovaleloacmaocaa nuH3 ooummuu may now ammo usouu xooun mnemoam>oo mo mucocomsoo ecu mcoEm mm pom 0e.fl mo SOAUSQHHumHQ .e ousmam e musmflm ZO_._.wp SA Uea mo mua>wuom oamaummm .o musmflm 65 64 Co o musmflm ZO_._.