....... ...... .......... DISTRIBUTION AND ELIMINATIONOF pIsLDIIINBveIIIsIaII mnsn (LEPIIM-Is CYANIELLUS,'RAFINESQUE). c > FOLLOWING ADMIN'ISTRATIQN.VOF A SINGLE ORAL DOSE, . Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY WILLARD L. GROSS 19,69 ill “ I, \IIIIIIIIIIIIII \IW ‘ TO“ IJBRf ”.Y Michigan .‘ rate Universal}! This is to certify that the thesis entitled Distribution and elimination of Dieldrin by Green Sunfish (Legomis cyanellus, Rafinesque) following administration of a single dose . presented by Willard L. Gross has been accepted towards fulfillment of the requirements for Ph.D degree in Fisheries & Wildlife {/J/m, Major professfl Date November 21,1969 0-169 , _____-.. Wfd ABSTRACT DISTTIBUTION AND ELIMINATION OF DIELDRIN BY GREEN SUNFISH (LEPOMIS CYANELLUS, RAFINESQUE) FOLLOWING ADMINISTRATION OF A SINGLE ORAL DOSE By Willard Louis Gross Green sunfish were administered a single oral dose of approximately 95 pg of dieldrin and placed in individual flow-through chambers (flow rate approximately 80 ml/min) for 3—day intervals over a 15-day period. Dosed fish, awaiting placement in chambers, were held in flow-through aquaria (flow rate ADO ml/min). Two fish in the chambers were cannulated to collect urine; feces were collected from the four remaining fish in the chambers; and the dieldrin content of water passing over these fish was monitored. At the end of a three—day interval, these fish were sacrificed and selected tissues and organs analyzed for dieldrin residue levels. Green sunfish demonstrated an 86.6% efficiency in absorbing dieldrin from the digestive tract. The distribution of dieldrin in tissues and organs analyzed remained constant with only the liver and gonads exhibiting considerable variability. A difference was noted in dieldrin residue levels in the liver of males Willard Louis Gross and females which was probably associated with ovarian development. Dieldrin levels generally declined in tissues and organs. However, the combined intestine and pyloric caeca samples demonstrated a slight increase in residue levels. Dieldrin was lost from the fish at an exponential rate; the biological half-life for total dieldrin was 25.8 days. Rates of dieldrin loss from individual tissues and organs varied considerably, with storage tissues such as adipose tissue and ovary demonstrating long dieldrin half-lives. Dieldrin was not detected in mucus and only small quantities were detected in urine late in the experiment suggesting that very little, if any, dieldrin was eliminated via these pathways. Essentially all of the insecticide was eliminated via the intestine (in feces) and from the gill. The gill was the most important pathway with a minimum of 55% of the dieldrin lost via this route. DISTRIBUTION AND ELIMINATION OF DIELDRIN BY GREEN SUNFISH (LEPOMIS CYANELLUS, RAFINESQUE) FOLLOWING ADMINISTRATION OF A SINGLE ORAL DOSE By Willard L. Gross 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 1969 Copyright by WILLARD LOUIS GROSS 1970 ACKNOWLEDGMENTS Many people have been helpful during_this endeavor all of whom cannot be acknowledged, therefore I would like to express my gratitude to all who have been involved. I would like to express a special thanks to the members of my committee: Dr. Paul O. Fromm, Dr. Peter I. Tack, Dr. Robert C. Ball, Dr. Howard Johnson and above all to my major professor Dr. Eugene W. Roelofs. I am especially indebted to both Dr. Roelofs and Dr. Ball who have been extremely helpful and understanding throughout my graduate training. I would like to express a debt of gratitude to Dr. Jerry Hamelink for introducing me to gas chromatography and for his many helpful suggestions. For her excellent editing of the manuscript I am indebted to Miss Norma Dombrock. In addition, a special thanks to my wife, Lorraine, for her persistence, patience and devotion throughout this period. Lastly, I would like to thank the taxpayer and the F.W.P.C.A. for their financial support of this research under the Training Grant 5T1-WP-lO9. ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . LIST OF TABLES . . . LIST OF PIG URES . . . . . . . . LIST OF APPENDICES INTRODUCTION . . . . . . . . . Uptake of Insecticides by Fish Insecticide Distribution in Fish Tissues Insecticide Elimination by Fish METHODOLOGY . . . . . . . . . Experimental Animals and Apparatus Collection Procedures . . . Analytical Procedures RESULTS AND DISCUSSION . . . . SU LI Dieldrin Uptake . . . . . . Dieldrin Distribution and Translocation Distribution in Tissues . Translocation in Tissues . . Dieldrin Losses from Individual Tissues Dieldrin Elimination . . . . in Tissues Dieldrin Elimination Via the Integument . . . Dieldrin Elimination Via the Kidney Dieldrin Elimination Via the Intestine Dieldrin Elimination Via the Gill Comparison and Discussion of the Various Elimination Routes MMARY . . . . . . . . . TERATURE CITED . . . . . APPENDICES . . . . . . . . . . iii Page ii iv vi 13 13 2O 22 26 26 31 31 142 A7 55 57 58 61 66 72 77 82 87 Table \I‘I L ST OF TABLES Page Dieldrin distribution in fish tissues ranked ordinally according to their average dieldrin concentrations within each sample period . . 32 Dieldrin distribution in selected fish tissues at various periods of the experiment . . . 3A Blood dieldrin concentrations in male and female fish during the first nine days of the experiment . . . . . . . . . . . . A2 Average dieldrin concentrations in fish tissues at various sample periods . . . . A3 Summary of pertinent statistics for dieldrin losses from selected tissues whose slopes demonstrated a significant difference from Zepo 0 O O O O O 0 O O O O O O O “9 Summary of the conditions and results of the water-feces interaction experiment . . . . 62 Average dieldrin quantities and concentrations in feces at each sample period . . . . . 62 Average dieldrin quantities and concentrations in water at each sample period . . . . . 68 The average and total quantity of excreted dieldrin recovered in waste products in each sample period and the percentage of total dieldrin recovered in water . . . . . . 73 iv Figure LIST OF FIGURES Diagram depicting the general design of the experiment . . . . . . . . . Diagram of the experimental apparatus Total dieldrin recovered from fish at various times during the experiment . . . . . Semi-log plot of calcualted least-squares regression lines for changes in dieldrin con— centrations in liver and gonad of a fish after administration of a single oral dose (9A.93 Hg) . . . . . . . . . . . Semi-log plot of calculated least—squares regression lines for changes in dieldrin con- centrations in intestine + caeca, stomach, gill and kidney of a fish after oral administration of a single oral dose (9A.93 Hg) . . Semi—log plot of calculated least—squares regression lines for changes in dieldrin con- centrations in blood and muscle of a fish after administration of a single oral dose (94.93 ug) . . . . . . . . . . Semi-log plot of calculated least—squares regression lines for changes in dieldrin con— centrations in adipose tissue and gall bladder + bile of a fish after administration of a single oral dose (94.93 Hg) . . . . . . The average quantity of dieldrin eliminated into water from the branchial region of a green sunfish after administration of a single oral dose of 9A.93 ug of dieldrin . . . Page 15 18 27 51 52 53 5A 67 LIST OF APPENDICES Appendix Page A. Procedures for extraction and cleanup of samples 88 B. Dieldrin amounts and concentrations found in tissues and waste products of each fish for each sample period . . . . . . . . . 9“ vi INTRODUCTION The history of synthetic organic insecticides, par— ticularly the chlorinated hydrocarbon group (organochlorine insecticides), spans a relatively short period of time (approximately 30 years). Yet, in this short period, their impact upon man and the environment has been of such magni- tude that it has resulted in intensive research on these compounds. Volumes of literature exist regarding how these substances affect man and his environment. Chlorinated hydrocarbon insecticides are noted for three properties: a broad spectrum of high toxicity, a long residual life, and an ability to penetrate biological tissue on contact. These prOperties are highly desirable in an insecticide; and these compounds, particularly DDT, appeared to be a panacea for insect control. Consequently, they were widely used. However, it was not long before scientists recognized that their widespread, indiscriminate use had resulted in a wide variety of ecological problems. One of the problems attributable to these prOperties is the indiscriminate destruction of beneficial insects, frequently weakening this link of the food chain. Often, other desirable components of the ecosystem have been seriously depleted or obliterated. A second problem is the l accumulation of several of these compounds in the natural environment. Most of our soils contain detectable amounts of DDT and its metabolites; in agricultural areas, dieldrin is also found. The soils and sediments appear to act as storage reservoirs for these pesticides. The presence of pesticides in water is also widespread. Although chlorinated hydrocarbon insecticides are not very water soluble, DDT and its analogs are found in water samples from all major river basins in the United States. Dieldrin was also detected from many of these basins (Weaver, et al., 1965). In addition to contamination of the soil and water substrata, their long residual life and high solu— bility in fats, oils, and waxes has resulted in the incorpora— tion of several chlorinated hydrocarbons, notably DDT and dieldrin, in practically all living biological organisms including man. This accumulation in biological organisms has produced a magnification of insecticide concentrations through the food chain posing a threat to the survival of animals occupying the higher trophic levels. Rachel Carson's book, Silent Spring, focused public attention on the perils of insecticide use and initiated intensive research on the effects of pesticides in ecologi— cal systems. In addition, interest was aroused in the long-term effects of these compounds at sublethal concen— trations. A mass of scientific data, plus more recent economic losses from the confiscation of insecticide- contaminated foodstuffs, has awakened the public to the hazards of these compounds and resulted in the banning of DDT in several countries, as well as in several states within the United States. The banning of other highly toxic, persistent insecticides, including dieldrin is being attempted. Even if the decision to ban the use of persistent chlorinated hydrocarbon insecticides is accepted, we are still faced with the problem of accrued concentrations of pesticides--notably DDT, its metabolites, and dieldrin—-in the environment. A recent article in Time Magazine (Anonymous, 1969) stated, "Some scientists estimate as much as two-thirds of the 1.5 million tons of DDT [and its metabolites] produced by man may still be adrift [in the environment]." Therefore, we should not abandon our research endeavors, but continue to probe the effects these compounds have on the natural environment, particularly the biota. An important part of gaining a thorough understanding of the effects of insecticides involves a knowledge of their dynamics in living organisms. This includes knowl- edge of amounts and rates of insecticide uptake, distribu— tion and translocation to tissues, metabolism, and elimina- tion. The uptake of various insecticides from water by fish has been studied, and knowledge of the distribution of various insecticides in fish tissues also exists. However, relatively few studies have been concerned with the process of insecticide elimination in fish. The knowledge that most chlorinated hydrocarbon insecticides accumulate and are stored in biological tissue for varying periods of time appears to occlude the concept of actual excretion of these compounds. A review of our present knowledge regarding the uptake, tissue distribution, and elimination of organochlorine compounds by fish follows. Uptake of Insecticides by Fish Insecticides may be taken up by fish directly from the water via the gills, by absorption across the skin, or across the gut from contaminated food. Large differ- ences in DDT concentrations in the skin and mucus of brown trout as compared to water, led Holden (1962) to conclude that DDT may be absorbed across the integument. Similar conclusions were reached by Premas and Anderson (1963) in studies of DDT uptake by Atlantic salmon and also by Ferguson and his associates (1966) in studies of endrin uptake by mosquitofish. However, all three studies con- cluded that the primary route of uptake is via the gill rather than the integument. In fact, Premas and Anderson (1963) found appreciable quantities in salmon tissues after only five minutes of exposure which could only be accounted for by absorption across the gills. Recently, Fromm and Hunter (1969) demonstrated that dieldrin can be transferred from environmental water to the vascular system of isolated perfused gills of rainbow trout. Relatively little information is available regarding uptake across the fish gut. However, Mount (1962) did observe high endrin levels in the digestive tract and low levels in the gills of bluntnose minnows. On the basis of these data, he concluded that endrin enters the body through the intestine as a result of the fish's drinking water. His work, however, has been largely disputed and the gill is now accepted as the primary route of uptake. Yet, the digestive tract should not be discounted as a site of absorption as evidenced by the food—chain concen- tration studies observed in aquatic systems (Hunt and Bischoff, 1960; Hickey, et al., 1966). Both routes are undoubtedly important, with the gut increasing in impor— tance with the presence of contaminated food (Johnson, 1968). A few studies have compared uptake via the gill and the intestine; Lenon (1968) compared whole body concentra— tions in bluntnose minnows exposed to dieldrin in water with groups fed dieldrin-contaminated daphnia, and noted higher body concentrations in fish exposed to dieldrin in water. Reinert (personal communication, 1968) reached a similar conclusion working with a daphnia-guppy system. Allison, g£_§1. (1963) conducted chronic toxicity studies of DDT in cutthroat trout by exposing some lots of fish to DDT in water and periodically feeding DDT contaminated food to different lots. They drew no conclusions regarding differences in body concentrations except to note that body concentrations increased more rapidly in fish fed DDT in the diet. Insecticide Distribution in Fish Tissues Insecticide residues in fish tissue vary widely from organ to organ and even within the same organ or tissue. This variability persists even when tissues from different studies are simply ranked on an ordinal scale and compared in this manner. A few generalizations, however, may be noted: adipose tissue invariably contains the highest insecticide concentrations in studies which examined this tissue, while muscle and/or blood usually contains the lowest concentrations. Holden (1962) observed inter— mediate concentrations in the muscle of brown trout indi- cating that dieldrin concentrations in this tissue may vary with its fat content. Fish liver was found to contain considerable concen- trations after exposure to several different insecticides (Premas and Anderson, 1963; Bridges, etugl., 1963; Holden, 1962; Allison, et al., 1963; and Gakstatter, 1966). Only Cope (1960) failed to observe DDT or any of its metabolites in the liver of fish from the Yellowstone River after con- tamination from a spraying project. Gakstatter (1966) included analysis of the gall bladder and observed high concentrations in this tissue as well. His results were highly variable, but demonstrated a gradual increase in concentration during a three-day recovery from exposure to a sublethal concentration of dieldrin. The digestive tract also accumulates relatively large amounts of chlorinated hydrocarbon insecticides, although studies of various portions of the gut provide conflicting results. Holden (1962) observed higher concentrations in the stomach than either the pyloric caecae or the intestine of brown trout exposed to lethal concentrations of DDT in water. Gakstatter (1966), working with DDT and dieldrin, found the reverse situation--low concentrations in the stomach and higher in the other two regions. Most other body tissues contain intermediate, but variable concentrations of insecticides. Holden (1962) indicates that residues in such tissues as spleen, kidney, heart and, to some extent, gill, can be attributed to the blood volume of these organs. Tissues such as the brain and gonad appear to have intermediate to high insecticide concentrations with results reported in the literature being quite variable. Brain dieldrin concentrations appear highly dependent upon degree of exposure and the amount of time elapsed after exposure. Gakstatter (1966) observed rapid declines in dieldren and DDT concentrations in brain tissue after exposure to a sublethal level. Concentrations of insecti- cide in the gonads appear dependent upon the maturity of the fish and the seasonal condition of the gonads. In most studies, the ovaries of mature females acculumate large amounts of insecticide. Variable levels of dieldrin have been reported in testes; some studies report low con— centrations, whereas others report very high concentrations. The reason for these conflicting results for the testes is apparently unknown. Many factors influence pesticide concentrations in fish tissue. Some of these factors are size, sex, age and condition of the fish. Also, the concentration to which fish are exposed, the length of time that they are exposed, and the time lapse between exposure and sampling appear to influence concentrations in tissues. Consequently, only general statements as cited above are possible. Insecticide Elimination by Fish Although excretion of pesticides has been noted in mammals for some time, very little research has been con— ducted on elimination of these compounds by fish. Several references to possible elimination of insecticides by fish can be found in the literature and were the primary motiva- tion for initiation of this project. Recently, however, Ferguson, et al., (1966) provided definitive proof of insecticide excretion by fish in his studies on the resistance mechanisms of mosquitofish to endrin. One of their experiments involved placing a resistant female mosquitofish (previously exposed to 1,000 ppb endrin solution for 36 hours and rinsed four times) in 10 liters of tap water with five mosquitofish of a sus— ceptible strain. Within 38.5 hours all susceptible fish had died. They did not measure the concentration of the water, but based upon bioassay tests of the susceptible strain, the endrin concentration in the water was approxi- mately£2to 3 ppb. This means that the resistant female had to excrete 20 to 30 micrograms of endrin in the 38.5 hours. Additional studies by this group demonstrated that both live and dead fish, exposed to varying concentrations of endrin for 7.5 hours, eliminated sufficient quantities of endrin in two liters of tap water in 8.25 hours to kill susceptible fish when they were placed in the jars of con- taminated water after the exposed fish had been removed. After initiation of the present research project, a study of the elimination of several chlorinated hydrocarbon insecticides by fish was reported by Gakstatter and Weiss (1967). Their studies with labeled DDT, dieldrin, and lindane showed that lindane was rapidly eliminated from goldfish and bluegills within two days and that 90% of the initial concentration of dieldrin was eliminated in the first two weeks of recovery. However, less than 50% of the labeled DDT was eliminated during the 32—day recovery period. The authors observed that both uptake and eliminae tion of these insecticides were related to their water solubility, i.e., the least soluble compound (DDT) being lO eliminated at the slowest rate. Recently Gakstatter (1968) obtained similar evidence on elimination of the aldrin—dieldrin complex from goldfish. . Although the above references afford evidence that chlorinated hydrocarbon insecticides are eliminated by fish, no one has determined the pathway or route of elimina— tion. Most of the published information regarding excre- tion routes of various insecticides and their metabolites deals with mammals. This literature has been reviewed by O'Brien (1967) and Menzie (1967). In mammals, chlorinated hydrocarbon insecticides are primarily eliminated in feces with lesser amounts lost in urine. The findings are quite variable and, in a few instances contradictory. Whether different routes exist for different insecticides could not be ascertained from the literature. One proposed pathway for dieldrin elimination in rats (Heath and Vandekar, 196A) involves removal from blood by the liver and transfer to the gall bladder, from there it is transported to the intestine in the bile and eventually eliminated in the feces. Gakstatter (1966) proposed a similar mechanism for elimination of DDT, dieldrin, and lindane in goldfish and bluegills based upon insecticide concentrations observed in the stomach, pyloric caeca, and intestine. Other investigators have observed comparable concentrations of different insecticides in the fish gut and support this proposed route of elimination by fish. 11 Other elimination pathways in fish include the kidney, the skin, and lastly, the branchial region. Mount (1962) observed high endrin concentrations in the kidney of blunt- nose minnows and suggested that endrin might be eliminated via this organ. However, Weiss (1967) has also observed high levels of dieldrin in the kidney and believed this might indicate an inability to excrete dieldrin. Up to this time, no one has analyzed fish urine to resolve this matter. No individuals have considered the integument or gill as possible elimination routes. However insecticides could feasibly be eliminated from the skin with the mucus from mucus-secreting glands as the mucus is sloughed from the fish. The gill is also a plausible excretory route since it is known to function as a secondary excretory organ in the elimination of various ionic compounds and nitrogenous wastes (Prosser and Brown, 1961). As mentioned previously, the gill is the primary route of insecticide uptake when fish are exposed to insecticide-contaminated water. Although the chlorinated hydrocarbon insecticides are relatively insoluble in water, the large surface area of the gill plus the close proximity of the blood to the external medium could permit a passive transport across the gill down a concentration gradient. The continuous exchange of water at the gill surface would aid in maintaining such a concen- tration gradient. 12 No studies of insecticide elimination by fish have included all possible elimination pathways, or the analysis of excretion products. Except for Gakstatter's estimates of the time necessary for elimination of DDT, dieldrin, and lindane, no information is available on the dynamics of insecticide elimination in fish. The present research was undertaken to conduct a detailed study of the elimination of dieldrin in green sunfish. Dieldrin elimination was observed from several viewpoints: analysis of excretory products or the receiving medium (in the case of gill), analysis of organs and tissues associated with possible elimination pathways, and lastly, an analysis based on actual body losses of administered dieldrin. Oral administration of dieldrin provided information on uptake efficiency of dieldrin from the fish gut. METHODOLOGY Experimental Animals and Apparatus The green sunfish (Lepomis cyanellus, Rafinesque) utilized for this study were obtained from the Michigan State University Lake City Experiment Station. Their size range was 10.3 to 17.2 cm in total length (average 14.7 cm) and 20 to 106 g in weight (average 65.9 g). Fish used for different phases of the research varied in average size. Smaller fish (average length 10.0 cm and average weight 38.78 g) were utilized as unexposed aquarium controls, and the larger fish (average length 16.2 cm and average weight 83.A g) were selected for cannulation to maximize urine collection. The treated fish and their controls which were employed for the main part of the study averaged 1A.6 cm in total length and 6A.57 g in weight. Analyses of the fish prior to use revealed only small amounts of DDT and its metabolites, particularly DDE. Dieldrin was not detected in any fish. In pilot studies, dieldrin was administered by dis- solving it in corn oil and placing 0.05 or 0.1 milliliters in a gelatin capsule which was then force fed to the fish. This procedure was found to be unsatisfactory because the fish could not assimilate this volume of oil and some of it 13 14 passed through the digestive tract to contaminate the water. Therefore, subsequent dieldrin administration involved force feeding the fish a dieldrin—treated dry food pellet. The treated pellets were prepared by applying five micro-liters of a dieldrin/acetone solution to the pellet surface with a 10 microliter syringe. The dieldrin (Shell Chemical Corporation—-99% pure) was obtained from Dr. R. E. Monroe of the Entomology Department, Michigan State University. The intended dosage was 100 micrograms per pellet, but analysis of the treated pellets indicated an average dose per pellet of 94.93 t 1.37 micrograms of dieldrin. Therefore, the average dose received by a fish was 1.A6 mg/kg (based on the average weight of the fish). This value is well within dosages reported as sublethal for fish in other studies (Macek, 1968). The experimental design (Figure l) was to dose all fish at one time, and then divide them into groups which were placed in the experimental chambers for successive three-day periods. Each three—day group of fish consisted of two treated fish which were cannulated for urine col— lection, another three treated fish for studying other routes of elimination, and one treated fish fed a pellet dosed with acetone to serve as a control (referred to as chamber control). Thus, data regarding elimination was obtained for a 15-day experimental period from five dif-i ferent groups of fish placed in the experimental chambers l5 5 fish fed an 25 fish fed a acetone-treated dieldrin-treated pellet pellet Ha r A V 1 I I n I I AA e.c LIB L'L/ b.c 0.x CIC cc : I I : I 2 I l ' 2 1 : 2 Holding '"""“ "'"'" "1"" "T" -_.‘_- ”'1‘“ “+“ 7..-...“ Aquaria I I AIA DIX XIC a:x x:s c,:cl cl:x x:x . L. #I l 1 1 L 1 I I—'—JI I I Four Chamber Three treated fish plus an barre treated fish to be used Control Fin. aquarium control fish (X) in for cannulation plus an each aquarium aquarium control fish in each aquarium Aquarium control fish sacrificed at time a group of fish were placed in the experimental chambers. Tissues taken for analysis included mucus, blood, fat, and remaining carcass. I p Experimental A B C D it, C2 Chambers Chamber Treated Treated Treated Cannulated Treated fish L4control fish fish fish 114 l I l Fish utilized for evaluatinr dieldrin elimination Fish utilized only for from the gill, analyzed for dieldrin residues stomach, pyloric caeca, gonad, blood, kidney, muscle, and mucus. liver, integument and intestine. included intestine, gall bladder, "w ,. . linuucu L‘Tiil, visceral adipose tissue, evaluatlnr from the kidney. analyzed for dieldrin residues included intestine, stomach, dieldrin elimination Tissues pyloric caeca, gill, visceral adipose tissue, blood, mucus, and occasional samples of other tissued. Figure l.--Diagram depicting the general design of the experiment. 16 for successive three-day periods. A total of thirty experimental fish was employed. After dosing the fish, they were assigned to indi- vidual compartments of 20-gallon aquaria having a flow rate of approximately #00 ml/min. The fish to be cannulated and the acetone-treated controls for the experimental chambers were placed in pre—selected aquaria. The other dieldrin-treated fish were randomly assigned to any of three compartments in each of the remaining aquaria. In each of these latter aquaria, a fourth compartment contained a small untreated fish (referred to as aquarium control fish) to serve as a monitor of resorption of excreted dieldrin while the exposed fish were held in the aquaria. Whenever a particular aquarium of treated fish was selected for place- ment in the experimental chambers, this untreated fish was sacrificed and tissues preserved for residue analysis. Not all treated fish of a three-day group were cannu- lated for urine collection since previous cannulation attempts always imposed a stress condition which impaired the fish's ability to survive in the chambers. Therefore, data on possible dieldrin excretion via the kidney was obtained from two previously selected, large sunfish placed in experimental chambers on the same three-day schedule as other fish. The only mortality experienced during the experiment was among the cannulated fish. A fish from the third sample 17 period (7-9 days) died of anoxia on the eighth day when the water siphon to the experimental chamber air—locked and stopped the flow. A fish in the fifth sample period was in very poor condition at the time it was sacrificed. The experimental apparatus is shown in Figure 2. A lSO—gallon continuous flow reservoir served as a common water source for both the experimental chambers and the holding aquaria. Water from the reservoir was siphoned to the holding aquaria and the flow regulated by a nylon valve. Water to the experimental chambers was pumped from the reservoir to a constant head tank from which it was siphoned to the chambers. The water supply was university tap water which was passed through a glass wool filter to trap iron oxide and other particulate matter. In preliminary studies, a charcoal filter was employed but was found to be unsatis— factory because during its use, unknown compounds were added to the water which caused interference on the chromatographic traces for water samples. The water temperature was maintained at 20° i O.5°CL with use of a refrigeration and mixing unit. The experimental chambers were glass cylinders 20.3 cm long and 5.7 cm in diameter, closed at each end with rubber stoppers fitted with glass ports. In the posterior half of the chamber, glass tubing was glued to the inside to maintain the fish in an upright position. The experimental chamber emptied into the bottom of a “-02. food jar employed 18 Lepmaswmm 30am pmapso .o Loocfiamo mQMSUMLw Loumaswom 30am pchH .2 dose moomm Esfipmsc< wcHUHom .2 nonsmno Hmpcmefipmaxm Gamma poem: .q some boom undamcoo xfislwmz .x dESm manfimhoensm m . Suspmddw Hmucoeflpmdxo one o #41 J‘s: h-------- E .s sac: coapmtmmfipomm .H poapso pflo>powmm .m poHcH Lao>pommm .o tmpafim .m pfio>pommm mo Empwmfialu.m msswfim <£IDOQLIJ 19 as a feces trap. The outlet from the trap was a U-shaped glass tube which conducted the surface water to the water extraction apparatus, but allowed the feces to remain in the bottom of the trap. The flow rate through the chamber and trap was regulated by the height and degree of bend in this outlet tube. The flow rate varied slightly between chambers, but was never less than 77 ml/min nor more than 85 ml/min. Flow rates were measured daily and adjusted as close to 80 ml/min as possible. Daily differences in rate never exceeded 5 ml/min and the maximum differences between chambers was only 3 ml/min over the three-day period any one group was in the chambers. Each experimental chamber had a volume of 355 ml without a fish; so that, at an average flow rate of 80 ml/min, a chamber had a maximum turnover time of u.u minutes or turned over a minimum of 328 times each 2“ hours. The experimental chambers utilized for cannulation were modified by placing a 22 gauge syringe needle through the rubber stOpper so that the cannula could be conducted to the exterior and the urine collected in graduated centrifuge tubes. Except for the rubber stoppers, the entire system, from chamber through the water extraction apparatus was con- structed of glass. 20 Collection Procedures Dieldrin in the water passing over the fish was measured daily. Urine samples were also collected daily unless the volume was insufficient, in which case, two—day samples were collected. Fecal material was allowed to accumulate in the trap until the fish were removed. At the end of a three—day period, the fish were anesthetized, removed from the chambers, measured and weighed, and various tissues and organs removed for residue analysis. All visceral fat and connective tissue around the gastro-intestinal tract was removed. The fat about the intestine, pyloric caeca and in the omentum was saved for residue analysis. In addition, the stomach and intestine were opened and the contents (mainly a yellow viscous fluid) discarded. Gill samples consisted of the entire branchial apparatus (including the gill arches) from one side of the fish. Muscle samples were taken from the expaxial or hypaxial masses on the right side of the fish, and blood samples were obtained from the caudal artery by severing the caudal peduncle. The gall bladder was removed intact so that analysis of this organ included the bladder plus bile. All samples taken were placed in tared screw- capped vials, reweighed to obtain tissue weights, sealed, and stored at -lO° C. The remaining fish carcasses were individually weighed, placed in plastic bags which were sealed to exclude air, and then stored at -lOO C. Later 21 the carcasses were homogenized with water and aliquots taken for residue analysis. Cannulated fish were also sacrificed at the end of a three-day period and the various tissues and organs removed for analysis. Feces and water samples were not collected for these fish. The cannula consisted of No. 20 polyethylene tubing heat-sealed at one end to form a smooth rounded surface, with numerous small openings made about the end with a heated needle. About 0.6 cm from the sealed end, below the openings, a small flange was formed around the tubing with a medical adhesive. The cannula was inserted into the urogenital opening, and sutured to the abdominal wall with -surgical thread. The adhesive flange formed an excellent area for attaching the thread to the cannula, permitted a tight fit about the urogenital opening, and lastly, pre- vented further insertion of the cannula during movements of the fish in the chamber, thereby avoiding internal injuries. This type of cannula appeared to have several advantages over the flared-end type: easier insertion, less apparent irritation, no tendency to become blocked, and lastly, facilitated collection of increased urine volumes. A continuous extraction apparatus was designed for monitoring the dieldrin content of the water passing over the fish in the experimental chamber. Water from the trap outlet (Figure 2) was conducted to the bottom of a 500-ml 22 graduated cylinder containing 150 ml of carbon tetrachloride. The graduate was positioned on a Magmix and thus, the water was continually mixed with the carbon tetrachloride. Since carbon tetrachloride is denser than water, it remained in the bottom of the cylinder while the water moved to the top and spilled over into a drain. Extraction efficiency was not especially high. Six A—hour and two 2A-hour tests, utilizing two different dieldrin concentrations (0.1 and 0.5 micrograms/liter), had an average extraction efficiency of ”6.82 t 5.27%. Differ- ences in extraction efficiency between length of tests or between dieldrin concentrations were not significantly different. In calculating the extraction efficiency, the data had to be corrected for carbon tetrachloride losses due to its slight solubility in water (average loss per 2A hours was 25 i “.2 ml). Although the efficiency was low and quite variable, the system appeared the best method in view of the volume of water utilized over a 2U—hour period (average llU.6 liters/day; range 110—112 liters). Analytical Procedures Different methods of extraction and cleanup for recovery of dieldrin had to be employed for the different types of samples; details of the specific procedures used are given in Appendix A. All dieldrin concentrations reported are corrected for extraction efficiencies. All reagents were redistilled before used and some were refined, 23 if necessary, according to methods described by Hamelink (1969). Provided all reagents were clean, good chromatographic traces for quantitating the dieldrin in water were obtained by evaporation of the carbon tetrachloride and transfer of the dieldrin to petroleum ether. (Overall recovery efficiency for the continuous water extraction procedure was A6.82 : 5.27%.) General methods described for extraction and cleanup of mammalian excretory products were not applicable to the small samples obtained from fish. A suitable extraction procedure was designed for urine which involved a 1:1 dilution with distilled water and extraction with petroleum ether. Chromatographic traces of samples extracted in this manner showed few extraneous peaks and no interference, so that additional cleanup steps were unnecessary. Efficiency tests, employing addition of dieldrin in acetone to the samples and evaporation of the acetone under partial vacuum, demonstrated an average recovery of 82.18 t 7.95%. Extraction and cleanup of fecal material was par- ticularly difficult. The technique developed involved extraction of dried material with acetonitrile, partition- ing the acetonitrile with a small amount of petroleum ether as a cleanup step, and finally, partitioning diluted acetonitrile with petroleum ether. The method was not entirely satisfactory as chromatographic traces frequently 24 contained artifacts and at times, the dieldrin in a sample was eluted above a broad sloping shoulder of some unknown compound. Additional cleanup steps such as saponification, adsorption with Nu—Char Attaclay, or elution from a micro-Florisil column did not improve the traces and always resulted in decreased recovery efficiencies. Although the chromatographic traces were not "perfect,' sufficient resolution existed for quantifying the samples. Recovery efficiency averaged A5.82 i 2.A8%. Dieldrin was extracted from fish tissue by a modifi- cation of the alcoholic—KOH procedure described by Schafer, et_gl. (1963). Twelve efficiency tests, involving addition of dieldrin in acetone to alcoholic-KOH solutions containing at least one of each of the tissues analyzed, gave an average recovery efficiency of 86.17 i 2.18%. Dieldrin levels in all samples were determined on Wilkens-Aerograph gas chromatographs (Models 600—0, 665, and 550—B) equipped with a tritium foil (activity 250 me) electron-capture detector. The basic operating conditions of the chromatographs were as follows: Pyrex column 5' x 1/8" packed with approximately 3% QF-l Gas Chrom Q; oven temperature 1800 0.; detector temperature 2000 C.; nitrogen carrier gas having a flow rate of “0 ml/min. Actual Operating conditions varied slightly with different columns and samples to facilitate resolution of dieldrin 25 on the traces. A Micro-Tek (Model MT—220) gas chromato— graph with electron capture or microcoulometric detector was employed on occasion to check for metabolites of dieldrin. Samples were quantified by comparing the peak height of samples with heights obtained for known standards. Each sample was replicated three times on the gas chromatograph with three dieldrin—in-benzene standards (either 0.1 or 0.05 ppm) interspersed between sample injections. The average response of the chromatograph to the standards was employed to quantify each sample repli— cate and the average of the three replicates considered as the concentration in the sample. Preliminary studies of quantitative methods indicated that peak height was the least variable, hence, the most reliable of the methods. To avoid errors due to photo—oxidation or evaporation in the dieldrin standards, these were made up in benzene and stored frozen at —100 C. when not in use. New standards were prepared every three or four weeks from a 1.0 ppm dieldrin/benzene stock solution. FacUlfC AND “1 CUSSlf I-e...,.1 - I» U lle.nrin ijtake Although the primary intent of this project was to evaluate the elimination of dieldrin by green sunfish, some inform tion regarding uptake and absorption effi- ciency across the gut was obtained. The total amount of dieldrin F9’«V”FJ1 from the tissues and carcass of each treated fish was calculated and the results graphed as n Figure 3. (he total dieldrin content decreased during F). the experimental period at an exponential rate. The equation for the line has the following form: Where YO amount of dieldrin present at time zero b = slope of the line t = time The slope multiplied by 2.303 is a constant usually designated as "k" and represents the fractional rate of change in dieldrin. 1e calculated regression equation for the amount of dieldrin lost was -0.0.269t I‘U O\ Micrograms of Dieldrin Recovered 27 1001 MO - 30 - 20 Regression Equation: log Y = 1.91“? + (-0.0ll7)t T%: 25.8 days k: 0.0269 10 - .! 7 I j 3 6 9 12 15 Days Post Administration Figure 3.-—Total dieldrin recovered from fish at various times during the experiment. Each point represents one fish. 28 A statistical test to determine if a true regression did exist--i.e., test whether the slope (b) is significantly different from zero-—indicated that the regression was *1 highly significant F = 22.2 > F 9.07). Conse- .01 quently, an estimate of the amount of dieldrin initially taken up by the fish is given by the point where the regression line crosses the ordinate (YO), which in this instance equaled 82.23 micrograms. The rate of dieldrin uptake across the fish gut is rapid. The rate at which the food pellet passed through the digestive tract was not determined in this experiment. However, estimates of the rate of food passage through the gastrointestinal tract of green sunfish, at comparable temperatures, were obtained in preliminary studies where dieldrin was administered with oil in gelatin capsules. Since the dye in the capsule tainted the fecal material a pinkish color, the time at which the capsule was eliminated from the body could be easily ascertained. Nine observa- tions showed that the average time for passage of the capsule through the digestive tract was 3A.7 hours, with a range of 26 to 52 hours. The passage rate of the food pellet was probably comparable. The data also permitted determination of the efficiency of dieldrin absorption from the gastrointestinal tract. The average amount of dieldrin taken up by a fish was 82.23 micrograms. As previously mentioned, the average 29 amount of dieldrin per food pellet was 9A.93 micrograms. Therefore, tne green sunfish gut demonstrated an efficiency of 86.62% for absorbing dieldrin. This is the first known measurement of the efficiency of insecticide uptake from the digestive tract. Other fish studies in which insecti- cides were administered orally (Allison, et al., 1963; Buhler, et al., 1969) involved feeding over a long time period rather than administering a single dose and thus could not readily be used to determine absorption effi— ciency. Observations of the dieldrin concentrations in various portions of the digestive tract (stomach, pyloric caeca, and intestine) indicate that the intestine and pyloric caeca are primary areas of absorption. This agrees with areas of primary fat absorption (anterior intestine) observed in Tilapia (Sivadas, 1965). The fact that green sunfish are capable of absorbing approximately 86% of an orally ingested dose of dieldrin has important implications in aquatic ecosystems. Even in the absence of dieldrin in the water (so that the gills are not a route of uptake) this degree of efficiency would easily permit a rapid accumulation by fish through feeding upon contaminated organisms. This high efficiency value certainly affords evidence of the importance of food- chain concentration in aquatic systems. Generally, insecticides do not persist in the water for long periods 30 of time. In streams and other lotic environments, con— taminated water passes through the area and continues downstream. Under lentic conditions, photo-oxidation, microbial decomposition, absorption, and adsorption by the living and non-living organic components result in a relatively rapid decline of insecticides to non-detectable levels in the water. However, because of the high affinity of many of the chlorinated hydrocarbon insecticides for organic matter, large amounts of these insecticides remain tied up in the ecosystem. Further movement and concentra- tion then occurs via the food chain which become important in understanding the fate and effect of pesticides in the environment. Whether the efficiency of dieldrin absorption across the fish gut is as high under natural conditions as it was in this study (86%) is not definitely known. I believe the value observed in this experiment would represent a maximal value under field conditions. Much of the dieldrin was probably adsorbed onto the pellet rather than absorbed into the actual components. Consequently, the dieldrin was probably more readily available to the fish than dieldrin would be in natural food (insects, fish, etc.). 31 Dieldrin Distribution and Translocation in Tissues In selecting tissues and organs for residue analysis, emphasis was placed on those associated with possible insecticide elimination routes. Tissues such as blood (transport vehicle for insecticides), visceral fat (chief insecticide—storage site), and muscle (most prevalent tissue) were analyzed with an interest in detecting trans- location of dieldrin within the body. The dieldrin con- centrations observed in the various tissues and organs of each fish are given in Appendix B. Aquarium control fish indicated that little, if any, resorption of eliminated dieldrin occurred while the fish were in the holding aquaria. Analysis of mucus samples and the carcass of these fish showed no dieldrin, while the visceral adipose tissue from three of four control fish showed only trace amounts. The continuous water flow through the aquarium was apparently effective in diluting and removing the dieldrin. Therefore, it can be assumed that dieldrin concentrations, translocation, and rates of elimination were not affected by resorption from the water. Distribution in Tissues In order to evaluate dieldrin distribution in the various tissues, they were ranked (highest to lowest) according to the amount of dieldrin present per gram of tissue (Table 1). This ordinal ranking permitted comparing 32 .mmzmmflp pmzpo mo mcomflpmoEoo mumuflafiomm 0p omcfimpmp momqm .UOflhoo mcflomompo CH xcmp mflnp pm mammflp pom maomHfim>m poc opmom maomsg mHomzz maomsz maomsz maomzz meEILo>HA oooam LomEOpm oooam mHmEILm>fiq mopmo9 gomEOpm oooam howsopm cocam nooam mmomo9 IIIIIIIIII mmpmm9 moumm9 nomEOpm xmcoflx llllllllll mamélho>flq comEOpm mmcowm mHmEILo>flq mmcoflx hmcoflx hocoflx maflo + maflo + Looomam dado LooomHm flame mmmopmo mmmopmo mmmopmo maflb + mmmopmo masosmo LocomHm Hamo mpm>o zpm>o llllllllllllllllllll th>o .Emmnso>flq HHHU Haflm Haflm HHflo Hafiu oaflp + mafia + pmoowam Hams IIIIIIIIII mlnlunlnnln .Emmnpo>flq Looomam flame .EmMIpo>aq moomo .m mommu .m momma .m .ome .m mommo .m + mewpmoch + mcflomoch + meaowmch + mcfipmmch + mcflpmmch .9 omoofiod .9 mmooflo< .9 mmrdoow .9 mmooflo< .9 mmoqfio< m:molma mmmolma mxmonm mzmolw mmmolm .wofipoo maoemm comm Canvas mcoflpmspcmocoo Cannamflo mmmpm>m Laws» on mcfiohooom maamcfiopo omxcmp mmSmmflp swam CH coapzoflppmfio CHLUHmHQII.H mqm<9 33 the tissues without becoming confused by variations in actual dieldrin concentrations in the tissues at different sampling periods. Visceral adipose tissue, combined intestine and pyloric caeca, and muscle maintained the same ranks throughout the experiment. The dieldrin dis- tribution ranks for the remaining tissues varied consider— ably. The two most variable tissues were liver and gonad. As indicated in Table 1, large differences in residue levels were noted in male and female liver. The carcass samples were not homogeneous, in some instances, not all the visceral fat was taken for separate analysis and in other cases, not enough fat was available for a sample and therefore not removed. If the carcass, liver, and gonads are omitted from the table, the remaining tissues maintain a relatively constant rank throughout the experiment Initially, the gall bladder + bile and gill ranked third and fourth in residue levels, with the two organs reversing this order between the sixth and ninth days. However, the dieldrin concentrations in both organs were very similar for the first six days (Table A, p. U6) so that assignment of a definite rank was somewhat arbitrary. Due to a more rapid decline in dieldrin levels in the gall bladder than in the gill after the sixth day, the residue levels were markedly different and thus the organs could be properly ranked. In general, stomach ranked sixth 3A .H mahm9 ca mm Umxcmp zaamcflopo osm mmSmmH9x maomsz maomsz maomsz maomsz maomsa soofim sooam gassesm sooam soon zomEOpm somEOHm mooam nomEOQm gomEOpm assess smcsfle smcsflx smcsax smcsfix sass + mfius + mass + ssesmfim Hams caveman Hflms tossaam Hams HHHU HHHs mass + mass + HHHU HHHU HHHQ pmowmfim Hamo pmoowam Hamo moomu .d domwo .m ommo .m momma .m momwo .m + mcflpmmch + mchmoch + mcfipmmch + mcfipmmch + mcflpmmch .9 awesome .9 mmoassa .9 mmosnsa .w smoouma .e mmoafls< mhmonma mmmonmfi mmmonm mAMwlw mmwolm III'IIIIII'IIIII‘II ecu mo mooflhmd mzoflhm> pm mommmfiu smfim Umpomamm CH *.pcmEHbmoxm coijpwhpmau CHLUHmHQII.m mqm<9 35 and blood ranxed seventh in dieldrin residues; although in one instance (at nine days), blood showed a higher dieldrin concentration than did the stomach. This may have resulted from sample variation, since both tissues had similar concentrations, never differing by more than 0.1 ppm at any sample period. The tissues and organs analyzed in the experiment can be grouped according to dieldrin residue levels with visceral adipose tissue in a group by itself--containing dieldrin levels approximately ten—fold higher than any other tissues or organs. A second group of tissues possessing l-A ppm of dieldrin includes intestine and caeca, gill, ovary, gall bladder + bile, and female liver. A third group showing low dieldrin concentrations (O.U-O.8 ppm) includes kidney, male liver, testes, blood, and stomach. Muscle tissue could be included in this group or actually represent a fourth group containing 0.1 ppm dieldrin or less. It was not possible to determine the sex of green sunfish except by dissection and as a result the two sexes were not equally represented in the experiment. Of the 15 fish for which data are reported, only five were females. They were represented in only the first three sample periods (through nine days), while males were present in all sample periods except at nine days. This disparity prevented comparing the two sexes throughout the experiment. f 30 In general, however, dieldrin levels in the ovaries (1.55 ppm) were approximately three—fold higher than in the testes (0.55 ppm). All fish examined were mature and the gonads appeared to be at the stage of rapid development which precedes spawning in the spring. When comparing dieldrin residues in the gonads with residue levels in other tissues (Table l), ovaries would rank high (comparable to gills), whereas dieldrin levels in the testes would rank low (comparable to concentrations in the stomach and blood). Male and female fish also demonstrated substantial differences in dieldrin residues in the liver. Although individual dieldrin measurements within each sex were quite variable; the average dieldrin levels in the female liver exceeded levels in the male liver several fold and the ranges for the two never overlapped. Comparing dieldrin levels in the liver of each sex with levels in other tissues, the females would again contain relatively high dieldrin concentrations with the male liver exhibiting much lower levels, comparable to the kidney (Table 1). In other insecticide studies (Gakstatter, 1966; Holden, 1962; Premas and Anderson, 1963), actual dieldrin concentrations in tissues and organs vary considerably and the assignment of ranks becomes quite arbitrary, often influenced by the type and number of tissues analyzed. Consequently, comparing the results of this experiment 37 with others reported in the literature could be misleading. Despite these difficulties, the rank of the tissues and organs according to residue levels in this study are comparable with other investigations——where exposure was to dieldrin in water (Gakstatter, 1966; Holden, 1962; Mount, 1962). These authors noted that visceral fat accumulated dieldrin in much higher concentrations than observed in any other tissue or organs; the gill and digestive tract also ranked high in dieldrin residues, while muscle and blood were generally much lower in rank. Consequently, the method of administering the insecticide does not appear to affect the pattern of insecticide accu- mulation in tissues. However, absolute dieldrin levels in this study were lower than levels observed in the above studies. Generally, those organs associated with possible elimination routes possessed higher dieldrin levels than other tissues. The high residue levels in the combined intestine and pyloric caeca sample were expected initially since this was the route that the dieldrin was administered. However, oral administration would not necessarily account for the continuously high levels throughout the experiment. The proposed pathway of elimination via the intestine (Gakstatter, 1966) appears plausible based upon dieldrin concentrations observed in these organs. The initial high concentrations of dieldrin in the gall bladder + bile 38 provide additional evidence of possible elimination Via this route. The fish gill also contained considerable amounts of dieldrin when compared with other tissues, ranking third among the tissues analyzed. Both Gakstatter (1966) and Holden (1962) also noted high concentrations in this organ. However, both were short-term studies in which fish were exposed to a lethal level of DDT (Holden) or a chronic level of DDT or dieldrin in water (Gakstatter). In View of this method of exposure, one would expect reasonably high dieldrin concentrations in the gill. Gakstatter demonstrated that uptake from water was correlated with the solubility of the insecticide in water. The less soluble the insecticide, the more it will adsorb to the mucus surfaces of the fish--particularly the gills. Holden did not measure blood dieldrin levels, but he believed that DDT concentrations observed in the gill were present largely in the blood rather than in the gill tissue itself. In this study, fish gills received little, if any, eXposure to dieldrin in the water. The most probably instance of exposure was while the fish were in the holding aquaria, prior to placement in the experimental chambers. However, analyses of aquarium control fish carcass, adipose tissue, and mucus revealed no dieldrin accumulation in these fish. The water supply showed no traces of dieldrin, and gill samples from chamber control fish also contained no 39 dieldrin. The possibility exists that some contamination of the gills may have taken place while fish were in the experimental chamber, but this exposure was certainly minimal since the chamber water turned over rapidly and the water flow minimized mixing. Therefore, in this study, dieldrin concentrations in the gill cannot be attributed to adsorption of dieldrin from water into the mucus or gills. The dieldrin residues in the gill do not appear to be the result of the blood present (as postulated by Holden) because the dieldrin levels in the gill were always five to ten-fold higher than in blood samples. If dieldrin in the gill were due solely to the blood volume, the additional weight of gill tissue would depress the concentrations below that observed in the blood and this was not the case. Consequently, dieldrin accumulation in the gill appears to represent insecticide actually absorbed by the tissue and not attributable to the tissue's vascularity. Therefore, the data suggest that the gill serves as either a storage site or a site for dieldrin elimination. The fish kidney exhibited low dieldrin levels relative to other organs possibly associated with dieldrin elimina- tion (gills - intestine), but high levels relative to other tissues such as stomach, muscle, and testes. The size of the blood component of the sunfish kidney compared to the fluid component of the forementioned tissues is not MO definitely known. Hoffert (1966) observed a higher blood volume in the kidney than in the liver, muscle, spleen and swimbladder of lake trout. Consequently, it appears plausible that the high insecticide levels observed in the kidney in this as well as other studies (Mount, 1962; Cope, l960; Weiss, 1967) may be partially due to the size of the fluid (blood) component. However, some dieldrin must be stored in kidney tissue since dieldrin levels there were approximately two-fold higher than observed in blood. The difference noted in dieldrin levels for male and female liver were unexpected. To my knowledge, no such difference has been previously reported. A comparison of dieldrin levels in the male liver for the first six days with levels in the female liver for nine days indicated that dieldrin levels in the liver of the two sexes were significantly different (t = 3.6“ > = 2.365). Only 13.95 the first six-day data were included for the male liver because no data were available at nine days and inclusion of any later sample period data would have induced a bias caused by dieldrin elimination from male liver in the later periods. The high dieldrin levels observed in the female liver may be associated with ovarian deveIOpment. The ovary is noted for the accumulation of fats and oils which are deposited in the eggs to serve as nourishment for the Al embryo in the event of fertilization. The liver is acknowledged as the site of lipid synthesis and conse- quently would be expected to contain high fat concentra- tions as a result of ovarian activity. Since dieldrin is primarily deposited in lipid, one would expect higher residue levels in the female liver than in the liver of males. In conjunction with this hypothesis, one might expect elevated dieldrin levels in the blood of females resulting from a rapid turnover of body lipids to provide energy for increased metabolism and also to provide fatty acids for the synthesis of egg lipids. For the period for which females were included in the experiment, their blood possessed higher residue levels than the blood of male fish (Table 3). However, the dieldrin levels between the two sexes were not significantly different. Presumably, if data were available for males at the ninth-day sample period, their average blood concentration would have been lower because of dieldrin excretion and differences in the blood residues levels would have been more apparent. This statement is substantiated by the average dieldrin concen— tration for three male fish after 12 days (0.27 ppm). Since differences in dieldrin residues in the liver appear to be associated with ovarian development, this phenomena is probably a seasonal event. To what extent this sex—linked factor applies to residue levels in other A2 TABLE 3.-—Blood dieldrin concentrations in male and female fish during the first nine days of the experiment. Days after Dieldrin Blood Dieldrin Concentrations (ppm) Administration Males Females 0.A8 o.u1 3 0.40 0.33 0.55 6 0.U9 0.33 9 0.30 0.75 Average 0.A25 Average 0.468 tissues and organs is unknown. However, it appeared that differences in residue levels may also exist in the blood. Translocation in Tissues Except for the fact that blood serves as the transport medium for insecticides in the body, little is known about translocation of dieldrin or other insecticides within the body, such as movement from the vascularized tissue to principle storage sites or to organs associated with elimination. In the present study, movement within the fish was evaluated by observing changes in dieldrin residues in tissues at successive sample periods (Table A). “3 .mmpzp coho mmma mfi deEmm CH swam mo LooEsc mucmmmpdop mammcucoLMQ CH hopedz .U®CHDEOO wommo Ucm mCflpmmpCH Lorw MDMQM mom.o mam.o 900.0 mmm.H omm.a .mmmosmo 999.0 Amv mmm.o msH.H ssm.m Amv Hms.m baseman Hams .......... ssm.m AHV mao.m AHV osm.m mfimsmousmsfia ssfi.o ssm.o ..... Amv ems.o Amv mas.o mamsusmswa sfis.o mmm.o sam.o 9mm.o sas.o smcswm soa.o smo.o mmH.o msa.o sms.o magma; .......... osm.a AHV smm.a AHV mom.a ssm>o ssfi.o msm.s uuuuu Amv som.o Amv som.o mousse 3mm.H Hmo.m smm.a smm.m mms.m Haws Am mmw.sa mas.sm msm.m osm.mm Amv ma:.mm mammwp mmoswsa om9.m Hom.m mam.m msm.m mom.m moses + ocflpmopcfl oocHoEoo mom.a mos.fi Hom.o .......... mamas ossoasm msa.fl mmm.m mfis.a .......... mcwpmmscH msm.o mmm.o m m.o oms.o mmm.s consosm smm.o Hem.o mos.o sma.s oms.o soon mmwolma somlm nmmmnm mxmonw wmonm mommfl9 cflppamflu mo omoa Hoso mo ecspassmACwssa amuse mama .ooooc Coos poooxm swam bongo go ommpm>m who mucomohdmp moam> zoom pm moSmme cmfiu CH Agog mm ommmmhdxov mcofiomhpcmocoo Cahoamfio ommho> AU Individual tissues and organs displayed increases in dieldrin concentrations at two different times--between the 3-day and 6—day sample periods and again between the 9—day and l2—day sample periods. The change in residue levels between the latter sample periods reflects a recovery from the low tissue dieldrin levels in the three female fish sampled at nine days rather than evidence of dieldrin translocation. Increased dieldrin levels in tissues between the 3—day and 6—day sample periods may reflect one or more of the following conditions: 1. redistribution of dieldrin among the various organs and tissues. 2. continued dieldrin absorption from the intestine, despite previous voidance of the food pellet. 3. random sample variation. The increased dieldrin levels in blood, adipose tissue, muscle, kidney, and male liver suggest further absorption from the intestine. However, the changes in dieldrin concentrations observed were never statistically signifi— cant, so that in terms of probability, these changes represent sample variation. As a consequence, relatively few inferences regarding translocation of dieldrin in fish are possible. “5 Except for the slow increase in dieldrin levels in the combined intestine and pyloric caeca sample, the tissues and organs showed a general decline with time suggesting that translocation was mainly from sites of storage to sites of elimination. This general decline in dieldrin residue levels also suggests that elimination rates were probably equal to rates of mobilization from fish tissues. In fact, the rate of elimination may be limited by the rate at which dieldrin was transported to elimination sites. Heath and Vandekar (196A) noted that disposal of dieldrin in rats was not controlled by the capacity of the rat to metabolize dieldrin, but rather by the flow of dieldrin to the place where it was metabolized (liver). The differences in dieldrin levels observed in por— tions of the digestive tract were also of interest. The combined intestine and pyloric caeca was the only tissue sample which demonstrated a slight total increase in dieldrin levels during the experiment. When the two organs were analyzed separately, later in the experiment, the intestine appeared to maintain a relatively constant dieldrin level whereas the pyloric caeca increased in residue levels. In contrast to these two organs, dieldrin levels in the stomach declined continuously throughout the experiment. Holden (1962) considered the high DDT residues in brown trout caeca and intestine as an indication that these organs served as storage ites, while Gakstatter (1966) ([1 associated the DDT and dieldrin levels in these two organs with dieldrin elimination via the intestine (in the bile). Neither statement appears entirely satisfactory for explaining the residue levels in intestine and caeca observed in this study. Since dieldrin declined in all other tissues, including visceral adipose tissue, one would expect a similar decrease in dieldrin stored in these organs. In addition, the removal of the digestive tract contents (except in pyloric caeca) at the time of sampling, should have removed any dieldrin being eliminated. An alternate explanation is that some of the dieldrin transported to the lower digestive tract with bile was resorbed by the intestine and pyloric caeca. The addition of resorbed dieldrin to amounts already stored in these organs would account for the constant dieldrin concentra— tions observed during the experiment. If resorption exceeded losses of stored dieldrin, the levels could increase as observed in the caeca. Exactly where the bile duct enters the intestine in green sunfish is unknown, but Lagler, et_al,, (1962) indi— cates that the bile duct enters at the beginning of the intestine in the pyloric region. Other evidence suggesting that dieldrin may be reabsorbed in the intestine include knowledge that bile salts function as fat emulsifiers which facilitate the hydrolysis of fats, and also that fat 47 absorption is intensified in the pyloric caeca of some fish such as the genus Salmo (Op. cit.). Additional data are needed to verify this hypothesis; however, it seems probable that dieldrin may be recycled internally between the digestive tract and other parts of the fish body. Dieldrin Losses from Individual “is VJ (1 ‘SL‘LE’S In the previous section, the distribution and trans- location of dieldrin among tissues were considered. The application of regression analyses to these data provided information on dieldrin losses from individual tissues. Tests of the linearity of the calculated least—square regression lines indicate that dieldrin was lost from fish tissues and organs in an exponential manner (Figures U, 5, 6 and 7). However, all of the regression lines may not represent accurate descriptions of the dynamics of dieldrin in the tissues. For several of the tissues, the slopes of the regression lines were not significantly different from random sample variation. For these particular tissues——liver, visceral adipose tissue, ovary, and the combined intestine + pyloric caeca sample--the regression line could have been drawn as a horizontal line indicating no change in dieldrin concentrations throughout the experi- ment (slope equal to zero). Consequently, any inferences regarding changes in dieldrin concentrations in these tissues must be interpreted with caution. A8 The slopes of the regression lines for the remaining tissues were statistically significant from zero (Table 5) and reflect dieldrin losses from these tissues under the conditions of this experiment. Dieldrin appeared to be eliminated from this second group of tissues at varying rates. However, an analysis of covariance demonstrated that the slopes of the regression lines for these tissues were not significantly different, i.e., that there was no significant departure from parallelism. Consequently, definite statements regarding differences in rates of dieldrin loss from individual fish tissues must await further study. The rates of dieldrin loss from various tissues can provide additional information on the dynamics of dieldrin in fish. Both visceral adipose tissue and the ovary-—which are known sites of dieldrin storage—~exhibited very slow rates of loss. In fact, the biological half-life for dieldrin loss from adipose tissue Ta = 2A.3 days) was . nearly the same as the half—life observed for the entire fish (Ta = 25.8 days). This similarity reflects the impor— tance of lipid deposits as storage sites and further demonstrates that elimination from fish may be strongly influenced by the rate of mobilization from sites of Storage. In addition, fish blood had an intermediate rate Of loss (Ta = 12.1 days), attesting to its role as a tPansport medium for dieldrin in the fish body. It was “9 O sens s u us "compmsoo Hmflpcmcooxo on» he coapmbpcmoooo cflboamflo CH omcmho mo mums Hocoflpombm who mpcmmmpoms mflc9 .mom.m moEHp odoam Apcmpmcoov :xzo ooflhmd oEHp zoo u mafia one mo mooam homebopcfi z map mo woa Apv meflp mcm no composucoocoo mo woa u x o m m ”whom: ND+®H% ocfla pcmflmsom m how compmsdm no mo Show CH oopComobq compmsomm sash.ou m.s snosso.o-v + ommm.o u s mos smsemas Hams ammo.o: m.s sflmmmo.onv + mmsm.9 n s mos messes mmso.sn 3.0fl sAmmmo.ouv + same.9 u s mod eQmEOpm msmo.ou H.mH sgssmo.ouv + msms.9 n s mos soofim osso.s! m.sH sAmomo.s-V + mmmm.9 u s moH shows: maso.sn m.efi sflmmao.onv + Hass.o u s mow Haws sasso.ou m.sH asAmsHo.ouv + msom.9 u s was secede mama :x: omflfilmamz coapmsom cofimmohwmm mommfi9 pcmomcoo amoflwofioflm .opom Eonm mocohmMMHp quOflmficwfim m ompmhpmcoEoU modoam omens mosmmflu oopooaom Eonm mommoa Caspaoflo Lou moapmflpmpm psoCaphmo wo zomEESmII.m mqm<9 50 also interesting to note that despite the great differences in dieldrin residue levels in the liver of male and female fish, the rate of dieldrin loss was similar (Ta = 8.A and 8.1 days for male and female, respectively). Attempts to resolve the issue of whether fish tissues eliminate dieldrin at different rates by comparing the present data with previous fish studies was not possible because, to my knowledge, calculation of rates of dieldrin loss from individual fish tissues has not been previously attempted. However, one study (Robinson, eg_§l., 1967) determined the rates of dieldrin loss from muscle, fat, brain and liver of pigeons. They observed that the rate of loss for these tissues was approximately the same (Ta = A0 to 57 days). Since statistical significance was lacking in the present experiment, it cannot be definitely stated that the situation is different in fish. However, the fish tissues demonstrated much greater differences in rates of dieldrin loss (Table 5) than was observed in the pigeon. Therefore, although the results of this study were sta- tistically inconclusive, it appears that fish tissues may exhibit different rates of dieldrin loss and that further research is warranted. Additional research in this area could be particularly beneficial in evaluating the dynamics, especially translocation, of dieldrin within the fish body. Dieldrin Concentration (ug/g) 51 10.0 q .7 3 male liver . mmwm female liver 4 --"- testis ovary I I I l l 3 6 9 12 15 Days Post Administration Figure A.--Semi-log plot of calculated least-squares regression lines for changes in dieldrin concentrations in the liver and gonad of a fish after administration of a single oral dose (9A.93 Hg). Data for females is based upon five fish present during the first nine days. Data for males is based upon nine fish. Dieldrin Concentrations (Hg/g) 52 8.01 -——- intestine + caeca .1 q -—~— gill . .... kidney 4 stomach q 1.0-I dp . . . PM... ........ ‘ ““3...“ .......... ‘ MM ........... d n.” q d 0.1 I I l I I 3 6 9 12 15 Days Post Administration Figure 5.—-Semi-log plot of calculated least— squares regression lines for changes in dieldrin concen— trations in the intestine + caeca, stomach, gill and kidney of a fish after oral administration of a single oral dose (9A.93 pg). Data are based upon 15 fish for each tissue. Dieldrin Concentration (pg/g) 53 1.0. d d . -—- Muscle d ‘A‘“ 0.1- “~~.. A q .1 d d '01 r I I I i 3 6 9 12 15 Days Post Administration Figure 6.--Semi—log plot of calculated least-squares regression lines for changes in dieldrin concentrations in the blood and muscle of fish after oral administration of a single oral dose (9A.93 ug). Data are based upon 15 fish for each tissue. Dieldrin Concentration (pg/g) s 100.0 5 LLlII . -—- Adipose Tissue d -—-- Gall Bladder 10.0: 111 l 1111: / / l l 6 9 l2 15 Days Post Administration LU-I Figure 7.--Semi—log plot of calculated least-squares regression lines for changes in dieldrin concentrations in the adipose tissue and gall bladder + bile of a fish after oral administration of a single oral dose (9A.93 Hg). Data for adipose tissue based upon 13 fish. Data for gall bladder + bile based upon 12 fish. 55 Dieldrin Elimination As previously indicated, dieldrin elimination from fish can be examined in two ways: dieldrin losses from the body (either on the basis of absolute quantity or body con- centration) and secondly, by measurement of dieldrin in waste products. In the present study, dieldrin elimination was observed from both viewpoints thereby providing two independent estimates of dieldrin elimination. In addition, the analysis of waste products for dieldrin provided a means for evaluating the importance of the various possible routes as insecticide elimination pathways in fish. Fish excretory organs are the liver, kidney, integu— ment, and gill. The manner in which these various organs may function in insecticide elimination was discussed in the introduction. To evaluate dieldrin excretion via these organs, analyses were performed on feces, urine, mucus, and water passing over the gill respectively. The quantities and concentrations of dieldrin in the waste products as well as excretory organs are given in Appendix B. The results obtained on dieldrin elimination from green sunfish are not conclusive because the two methods for determining dieldrin elimination were not in agreement. Only 63.3% of the total quantity of dieldrin lost from a fish during the experiment (27.38 ug) was recovered in the waste products (17.33 ug). This discrepancy prohibited definitive statements regarding the rate of dieldrin loss and routes of dieldrin elimination. 56 Since dieldrin is known to be metabolized in mammals, a possible explanation for the low recovery would be degradation of the dieldrin to undetected metabolites. Korte and Arnt (1965) found six dieldrin metabolites in the excretia of rabbits after oral administration of labelled dieldrin; but only one of these metabolites (6,7, trans- dihydroxy—dihydroaldrin) was of major importance, con— stituting 86% of the total metabolites found. Other investigators (Heath and Vandekar, 196A; Morsdorf, e£_al., 1963; Datta, e£_§1,, 1965) have observed a variable number of dieldrin metabolites in rats. All the studies agreed that the metabolites were more polar (hydrophilic) than dieldrin and hence, more soluble in water. Heath and Vandekar observed one metabolite, representing 78% of the total radioactivity in the bile, that they considered a probable glucuronide of a dieldrin derivative. Although unknown peaks were obtained on chromatograms of most extracted materials in this study, no dieldrin metabolites were suspected. To verify the presence or absence of metabolites, all samples of a particular tissue or waste product were combined, concentrated, and then analyzed on a Micro-Tek gas chromatograph equipped with a microcoulemetric detector. The amount of dieldrin in the combined samples was small in relation to the sensitivity of the detector so that the sample produced only a small response (5-20% of the chart paper). However, in all cases, only one peak, whose retention time matched that of the dieldrin standard was observed on the trace, demonstrating that only one halogenated compound——name1y dieldrin--was present. Grzenda (personal communication, 1968) also found that neither gas—liquid chromatography nor radio—thin-layer chromatography indicated metabolism of dieldrin in goldfish. Consequently, metabolism of the dieldrin did not appear responsible for the low recovery of dieldrin in the waste products. An explanation of the low dieldrin recovery from several waste products will be discussed later in conjunction with elimination from the intestine (p. 62). Dieldrin Elimination Via the Integument As indicated previously, no one has considered the integument a possible pathway for insecticide elimination. However, Ferguson, gg_gl. (1966) noted that dead mosquito fish exposed to 1.0 ppm of endrin for 11.5 hours, washed four times in tap water and once in acetone, and transferred to clean water, released small amounts of endrin into the water. The mechanism of this release is unknown, but since the fish were dead, one might suspect diffusion across the integument as a possible explanation. Since the sunfish in this study received little, if any, exposure to dieldrin in the external medium, its presence in mucus ‘would indicate elimination across the integument. 58 Mucus samples were collected by scraping the sides of a fish with a spatula immediately after it was removed from the experimental chambers. The mucus was placed in vials and stored in the same manner as the tissue samples. Mucus samples from treated fish in the three-day and six—day sample periods contained no detectable dieldrin (limit of detectability was 0.1 ppm). Consequently, only two mucus samples from the three fish in each succeeding sample period were randomly selected for analysis. Again, dieldrin was not detected in these samples. Hence, on the basis of these findings, one can conclude that dieldrin was not eliminated across the integument in the green sun— fish. In addition, the mucus samples from chamber control fish and aquarium control fish did not contain detectable dieldrin. The absence of dieldrin in mucus from these fish was of interest because it afforded evidence of the minimal exposure of the experimental fish to dieldrin in the external medium. Absorption or adsorption of dieldrin to the gill from the water was definitely minimal. Dieldrin Elimination Via the Kidney A total of 21 urine samples were collected from the ten cannulated fish in the experiment (two fish for each of the five three-day sample periods). Eighteen samples were daily samples and three consisted of combined two—day samples. Daily urine volumes ranged from 1.0 ml to 9.3 ml with an average volume of 3.7 ml/day. The average urine flow for green sunfish was 57.5 ml/kg/day. Despite the significant amounts of dieldrin observed in the fish kidney, no dieldrin was detected in fish urine during the first nine days of the experiment. A tenth- day urine sample of one fish contained a small quantity of dieldrin (12 ng) for a concentration of 2.26 ppb. Urine from the same fish contained 79 ng in a combined 11-12 day sample of 1.3 ml of urine giving a dieldrin concentration of “5 ppb in the urine. Urine from the second cannulated fish con— tained no dieldrin at 10 or 11 days and no urine was collected on the last day. Both cannulated fish in the last sample period (13— 15 days) contained small quantities of dieldrin in the urine, although one fish was negative on the thirteenth day. But, again, the dieldrin amounts recovered were low and the maximum concentration was only 5.8 ppb. Most of the dieldrin levels observed in urine were close to the minimum detectable limits of the gas chromatograph (1-2 ppb). The data indicate that some dieldrin may be eliminated from fish via the kidney after 10 or 12 days, but the quantities observed in urine were small compared with levels observed in feces and water as will be shown later. The rather short duration of the present experiment may have failed to reveal the nature of dieldrin elimination from the kidney. herte and Arnt (1965) and Heath and Vandekar (196A) ,n observed a slow increase in radioactive sub- (.7 O .—+ stances in urine after cessation of feeding labelled ' dieldrin or intravenous perfusion of labelled dieldrin in rats over a period of A to 26 weeks. Fish may exhibit a similar action and levels in urine may have increased with time. The urine flow for sunfish observed in this study 22 (57.5 ml/kg/day) may have been lo er than normal, thereby -1 N O ‘ 0 _ ‘ V' _. ,.. V depressing dieldrin 1cJ‘ (*1 F—J 01 oh (' (9 served in urine. U"ine output for green sunfish or other centrarchids was not available from the literature. Hut, "ata on urine flow in rainbow trout snow volumes of 101 1 8 ml/kg/day (Fromm, 1963) and a maximal flow of 91.9 ml/kg/day at 2A hours post— catherization (Hahn, 1569). Therefore, sunfish urine volume was approximately one-half that recorded for rainbow trout. The data demonstrate that dieldrin levels in the kidney are not indicative of dieldrin excretion by this organ. Dieldrin was not observed in the urine until levels had decreased in the i'idney. Even if the urine output for green sunfish was low, one can infer that very little, if any, dieldrin is eliminated via the kidney. However, additional studies over a longer time period may produce slightly different results. Apparently, the kidney serves more as a low level storage site than as an excretory site. OK 1'4 (-+ r (L r4 '1) s c. '- (T) ’ L d V >1 (I This pathway involves removal of insecticides from the blood by the liver followed by tranSport to the intestine in the bile and elimination in the feces. Some dieldrin may be transported to the intestine by alternate pathways; heath and Vandekar (196A) observed dieldrin in fecal material of rats when the bile duct was cannulated. 1h=y believed some dieldrin was eliminated across the intestinal mucosa. ff1\ .ne water passing over a fish was in contact with feces which accumulated in the trap over a three-day period. Little exchange of dieldrin was expected because of the high affinity of chlorinated hydrocarbon insecticides for organic material. However, to verify whether an inter— action between water and feces existed, a short three-day experiment was conducted. Two samples of feces with known amounts of dieldrin were placed in the chambers and both feces and water monitored for their dieldrin content. An average of 3A.2L of the total dieldrin recovered was found in the water (Table 6). This amount of dieldrin lost to the water from feces was probably a maximal value since the dieldrin concentra- tion employed for the test was higher than that normally encountered in fish f ces and also because most of the (I) dieldrin applied prob bly remained on the surface rather than being incorporated into fecal material. it was 62 TABLE 6.-—Summary of the conditions and results of the water—feces interaction experiment. Sample 1 Sample 2 Weight of feces 11.2 mg 55.U mg Dieldrin concentration U28 pg/g 86 pg/g Dieldrin recovered in feces at 72 hours 2.366 pg 2.592 pg Dieldrin recovered in water 1.362 pg 1.188 pg Per cent dieldrin recovered in water 36.5“% 31.84% assumed that a similar dieldrin loss from feces existed during the experiment and all feces values were corrected for this loss as well as recovery efficiency. Dieldrin levels in the first three-day fecal sample were much higher than those observed in subsequent sample periods (Table 7) because the sample included dieldrin voided with the food pellet residue and not absorbed by TABLE 7.—-Average dieldrin quantities and concentrations in feces at each sample period. Days After Administering Dieldrin 3—days 6-days 9-days l2-days lS—days Total dieldrin recovered (pg) 1.166 0.083 0.038 0.07u 0.0u9 Dieldrin con- centration (us/s dry wt. of feces) lU2.609 0.905 0.672 0.899 0.503 63 the fish. The pattern of dieldrin elimination in feces in the subsequent sample periods was difficult to determine. Excluding the first three-day period, the differences noted in feces dieldrin concentrations were not significantly different from one another and could, therefore, be attributed to sample variation. This would infer that the dieldrin in feces remained constant during the latter part of the experiment rather than declining. Consequently, dieldrin concentrations in feces was con- sidered as constant with an average of 0.7uu pg/g dry weight feces/3 days or 0.2U8 pg/g dry weight feces/day. The amount of dieldrin a fish would excrete in the feces per day was estimated from the average dry weight of feces obtained from all fish in the study. The average weight amounted to 60.03 : 12.01 mg feces/fish/3 days. Using this value in conjunction with the average dieldrin concentration in feces, the average quantity of dieldrin excreted by a fish was calculated as 0.0AU7 pg dieldrin/ 3 days or 0.015 pg dieldrin per day. Assuming that all dieldrin in fish was eliminated by this route, it would require 5,U82 days or 15 years to eliminate the average quantity of dieldrin taken up by a green sunfish (82.23 pg). This time period is incon— sistent with losses observed from fish. The total quantity of dieldrin lost during the experiment was 27.33 pg and based upon the exponential decline observed 64 (Figure 3), the biological half—life was only 25.8 days. Consequently, other routes for dieldrin elimination must exist. The dieldrin quantities eliminated in fish feces was much lower than is the case for mammals which excrete approximately 90% of incorporated dieldrin or its meta— bolites via this route (Heath and Vandekar, 196M; Ludwig, gt_al., 1964). In view of the importance of this route in mammals and the high dieldrin concentrations in the intestine, pyloric caeca, and the combined gall bladder- bile samples, one might question whether the dieldrin levels observed in feces accurately represent elimination via this route in fish. This is particularly true when one recalls the disagreement between dieldrin quantities lost from the fish (27.38 pg) and the amounts recovered in all waste products (17.33 pg). Of the four possible elimination pathways, it appears most plausible that the missing dieldrin was eliminated via the intestine. The low recovery of dieldrin from feces may have resulted from not extracting and detecting dieldrin bound to proteinacious material in the feces. It has been shown that dieldrin and other organochlorine insecticides may be bound to proteinacious material (probably lipoproteins) and not completely extracted with standard solvent extraction methods. Witt, et_al, (1966) demonstrated that only two—thirds of the DDT in cow's 65 blood was extracted by a simple ethyl-ether extraction. Grzenda (personal communication, 1968) encountered diffi- culty in extracting dieldrin from fish testes and had to resort to a formic acid digestion procedure to obtain adequate recoveries. Some evidence exists that a similar situation may have existed in this study. A large quantity of the unabsorbed dieldrin which initially passed through the fish digestive tract with the treated food pellet, was not recovered in the fecal material during the first sample period. Only 1.166 pg of an estimated 12.7 0% of dieldrin not absorbed was actually recovered. Since dieldrin was not detected at this time, it appears plaus— bile that additional dieldrin may have escaped detection during the rest of the experiment. No data on dieldrin quantities in waste products of fish were available in the literature. However, Grzenda (personal communication, 1968) recovered substantial quantities of labelled dieldrin in the feces of goldfish. Consequently, although I cannot definitely state that the missing dieldrin lost from the fish was eliminated in the feces, the above discussion indicates that this was the ‘ most probable route. In summary, it appears that the quantity of dieldrin observed in fish feces was possibly much less than is actually eliminated via this route. More adequate 66 extraction procedures such as alkaline or acid hydrolysis are probably necessary to accurately measure dieldrin quantities in feces. Dieldrin Elimination via the Gill As indicated previously, dieldrin elimination from the fish branchial region was determined from the corrected dieldrin content of water passing over the fish. The water could not be isolated from dieldrin contamination from other excretory routes, but dieldrin levels were corrected for contamination from urine and also amounts leached from feces as a result of the water-feces interaction in the feces trap. The quantity of dieldrin present in water rather than concentration was emphasized in this case since it better lreflects the dieldrin losses from the gill. Because of the large volume of water passing through the experimental apparatus each day, dieldrin concentrations were very small, rarely exceeding 10 pptr (parts per trillion). Water showed a pattern of dieldrin content similar to that found for feces except that the total amounts of (dieldrin were much greater in water. More dieldrin was present in the first three-day sample periods than in subsequent sample periods (Figure 8) with the highest ciieldrin quantity observed on the second day (2.305 pg). Tiuereafter, the daily measurements demonstrated a rapid Dieldrin Quantity—-micrograms 67 2.5 ' R /\ / \ “" Daily Average // \ ____. 3-day Average 2.0 1 / ‘ J 1.5 1 i 1.0 ' 0.5 ‘ T V I T I I I I I ' U 1 I f a 3 6 9 12 15 Days Post Administration Figure 8.-—The average quantity of dieldrin eliminated into water from the branchial region of a green sunfish after administration of a single oral dose of 9U.93 micro- grams of dieldrin. 68 decline to about the fourth day. Dieldrin in the water further declined slowly to the seventh day after which it slowly increased to a second peak at thirteen days and then began to decline once more at termination of the experi— ment. These fluctuations in daily measurements apparently were random sample variations since the average quantity of dieldrin during the last four sample periods were not significantly different in an analysis of variance test of the mean dieldrin levels in each period (Table 8). TABLE 8.--Average dieldrin quantities and concentrations in water at each sample period. 3—days 6—days 9-days l2-days l5—days Total dieldrin recovered (pg) 1.879 0.92u 0.722 0.868 0.853 Dieldrin concen- tration (ng/liter = pptr) 16.8 7.9 6.1 7.U 7.6 The high dieldrin levels in the first three-day _period suggested contamination of water by dieldrin which passed through the digestive tract and was not absorbed by the fish. However, an average quantity of 1.912 pg of (iieldrin was recovered from water on the first day--before true food pellet was voided from the digestive tract. Cknqsequently, the high dieldrin levels were apparently not true result of fecal contamination. 69 Presumably, the dieldrin concentration in blood was much higher during the first two days than that observed on the third day, which thus resulted in increased elimina— tion from the gill during this period. Using the blood and water data from subsequent sample periods, I calculated the probable levels in blood which would account for the dieldrin quantities observed in the water. The calculations showed that the dieldrin concentrations in blood necessary to account for the quantities observed in the water during the first and second days were 0.968 ppm and 1.170 ppm, respectively. These values are comparable to blood dieldrin levels observed in catfish (Gakstatter, 1966) and endrin levels in the blood of both catfish (Mount, 1966) and golden shiners (Ludke, gt;al., 1968) shortly after exposure to these insecticides in water. Therefore, the dieldrin quantities observed in water the first three days could be attributed to dieldrin being eliminated from the gill during this period. The above explanation infers that the rate of dieldrin elimination from the gill is proportional to the dieldrin concentrations in the blood when the gill region is bathed in insecticide-free water. However, this was not the case throughout the experiment. The average quantity of dieldrin eliminated into the water appeared to level off after the fourth day to a constant rate of 0.852 pg/ fish/day, despite continued decreases in blood dieldrin 70 levels. These results suggest that a threshold exists for blood dieldrin levels below which the relationship is no longer proportional. This value for green sunfish blood is near 0.A3 ppm (the dieldrin concentration in blood at the third day). Fromm and Hunter (1969) demonstrated that dieldrin uptake from water into blood plasma across isolated perfused trout gills could take place by passive transport. Dieldrin and other insecticides are probably lost in a similar manner when insecticide levels in the water bathing the gills are sufficiently below their solubility limit and below existing insecticide concentrations in the blood. In this study, the average dieldrin concentration in water after the fourth day was 0.0075 ppb which was far below the solubility limit of 1U0-180 ppb reported by Robeck, et_al, (1965) and was at least 5000 fold less than con— centrations observed in fish blood. In addition, the continuous flow of insecticide-free water across the gills would facilitate maintenance of a concentration gradient between insecticide levels in blood and the water. The actual process of passive transport across the gill is unclear. Dieldrin was lost from the blood, gill, and other tissues at an exponential rate and, as a con- sequence, one would have expected the rate of excretion to also decline at an exponential rate. However, the dieldrin 71 recovered from water was constant during the last 12 days. During this same period, blood dieldrin levels declined “7% and dieldrin levels in the gill declined 38%. The reason for this inconsistency in the manner in which dieldrin was lost from the fish and recovered in water is not definitely known. Possibly, the rate of a dieldrin transport into the gill epithelial cells may ’ differ from the rate of transport of dieldrin from these cells to the environment. If so, transport across the ’3 gill epithelium could occur at a constant rate with the exponential rate of loss from the gill representing an interaction between dieldrin in the gill and blood com- partments. Unfortunately, analysis of the sunfish gill was not limited to gill tissue exclusively, but included dieldrin present in the vascular compartment (blood and lymph). In addition, other factors such as variations in blood flow patterns under different physiological condi— tions as shown by Richards and Fromm (1969) may play a role in dieldrin elimination from the gill. These results and speculations demonstrate that the dynamics of dieldrin elimination from the fish gill merits further study. The presence of dieldrin in water passing over the fish and the high residues in the gill afford evidence of the gill's function as an insecticide excretory organ. .After the initial dieldrin loss during the first three days, ‘the average amount of dieldrin eliminated from the gill 72 was 0.852 pg/fish/day which represented the greatest recovery of dieldrin from any of the waste products. Assuming that the gill served as the only route of dieldrin elimination in fish, approximately 90 days would be required to completely eliminate the average amount of dieldrin taken up by a fish (82.23 pg). The 90 days com— pares favorably with the 25.8 day half-life calculated for loss of dieldrin from the entire fish. Comparison and Discussion of the Various Elimination Routes Of the four possible insecticide excretion systems in fish, dieldrin was detected in the waste products of three systems (Table 9). Only the integument (mucus) failed to show any evidence of dieldrin excretion. The presence of dieldrin in urine, late in the experiment, suggested that some dieldrin may be eliminated via the kidney, but the quantities were very small and it is doubt— ful that a significant amount (less than 1%) is actually eliminated via this route in fish. Dieldrin quantities eliminated by way of the intestine and feces were several fold higher than those observed in urine, but much lower than quantities detected in the water (Table 9). Only 2 or 3% of the absorbed (iieldrin recovered in waste products was collected from feces with 95—98% recovered in the water. 73 TABLE 9.--The average and total quantity of excreted dieldrin recovered in waste products in each sample period and the percentage of total dieldrin recovered in water. Sample Water Feces Urine Total Per Cent Period (Hg) (Hg) (Hg) (Hg) in Water 3-days 5.6349 1.1665 0.0000 6.8014 82.8 6—days 2.7708 0.0826 0.0000 2.8534 97.0 9—days 2.16u9 0.0377 0.0000 2.2026 98.3 12-days 2.6053 0.07A2 0.0859 2.725“ 95.6 l5-days 2.6812 0.0u9u 0.0177 2.7u83 97.6 Based on the actual quantities of dieldrin which were observed in waste products, the most important path— way of dieldrin elimination in green sunfish was across the gill——the same structure which serves as the primary route of insecticide uptake. Excluding the first sample period, at least 95% of all dieldrin recovered was eliminated via this route. However, if the assumption that the dieldrin lost from the fish but not recovered in waste products was actually eliminated with the feces, about two micrograms of dieldrin could be added to the amount actually observed in feces in each sample period. The intestinal pathway would then be much more important than the data actually indicated. If this assumption is correct, approximately 55% of the dieldrin excreted by a fish was eliminated via the gill and the remainder (85%) 7“ via the feces. Regardless of the actual amounts of dieldrin detected in feces, the data demonstrates that the gill serves as an important route for dieldrin elimi- nation. A comparison of these results with those of others shows that green sunfish exhibited a slower rate of dieldrin loss than other species studied. Gakstatter and Weiss (1967) found that 90% of the absorbed dieldrin was eliminated from goldfish and bluegills in 1“ days. This is about six times faster than the 85 days determined for the green sunfish. Grzenda (personal communication, 1968) found that seven weeks (M9 days) was required for complete turnover of dieldrin in goldfish, which was also sub- stantially faster than the rate of loss from sunfish. Attempts to explain these large differences in rates of dieldrin elimination from fish are difficult because the techniques and experimental conditions vary consider- ably. However, it appears that the rate of dieldrin elimination may be related to the levels of dieldrin stored in the fish. Gakstatter and Weiss (1967) exposed their fish to a toxic concentration of dieldrin in water until the fish began to exhibit signs of insecticide poisoning. Under these conditions, the fish probably received a large dose. In the studies by Grzenda, the goldfish were fed a small dose daily until dieldrin reached an equilibrium level in the fish tissues. The 75 fish were then placed on an insecticide-free diet and the rate of dieldrin loss determined. The total body accumu- lation was probably less than in the fish used by Gakstatter and Weiss but this cannot be definitely verified since Grzenda's work has not been published. In the present study with green sunfish, the fish received a single sublethal dose and would have taken up less dieldrin than fish in either of the other two studies. If one can assume that dieldrin is accumulated in fish as postulated, then the rate of dieldrin elimination followed an inverse order. Additional evidence that the rate of elimination may be inversely related to the levels stored in fish was demonstrated by the increased dieldrin levels observed in water the first two days of the experiment when blood levels were presumably higher. The fact that dieldrin was eliminated from fish in the feces and particularly from the gill has important implications with reference to the cycling of dieldrin and other insecticides in aquatic ecosystems. Hamelink (1969) proposed that DDT in a free or unbound state was a primary factor controlling equilibrium relationships between the ruitural environment and animals of the various trophic levels. The dieldrin eliminated via the gill would probably exist in such a form and thus perpetuate the presence of unbound dieldrin in water. In this form, the eliminated dieldrin would contribute to the continued 76 cycling of dieldrin among the various trophic levels of the ecosystem. Gakstatter and Weiss (1967) observed that both dieldrin and DDT were readily transferred from insecticide— exposed fish to control fish maintained in the same aquarium during recovery periods. Knowledge of the rates of insecticide elimination from fish may have some practical implications. If one were permitted a choice of insecticides for use in a. particular situation, it would appear plausible to select an insecticide which was rapidly excreted, providing other factors were the same for the choices available. Another possible application of knowing elimination rates would be in situations in which repeated doses of insecticide were necessary. Calculating the rate at which the insecticide was lost from a natural population of fish would provide meaningful data as to when it would be per- missible to make a second application to avoid excessive losses of wildlife. Although many other factors which exist in such situations would also have to be taken into consideration, elimination rates would definitely be one useful criterion. SUMMARY The uptake, distribution, and elimination of a single oral dose of approximately 95 micrograms of dieldrin administered to green sunfish was determined. All fish were dosed at the same time and separate lots placed in specially constructed flow-through chambers at three-day intervals over a 15 day experimental period. Dieldrin losses via various excretory routes were monitored while fish were held in these chambers. Dieldrin distribution was evaluated from measurements of residue levels in tissues and organs removed from fish when they were recovered from the chambers at the end of a three—day interval. Application of regression analyses to changes in residue levels of various tissues, organs, and remaining carcass permitted estimates of the rate of dieldrin loss from the entire fish and also from individual tissues and organs. Procedural problems and the lack of statistical significance in the data occasionally precluded making definite statements regarding dieldrin elimination from fish. However, I believe the data adequately demonstrated the following findings: 1. Green sunfish absorbed 82.23 pg of an average 9u.93 pg of dieldrin administered demonstrating an 86.6% 77 j!!! i 78 efficiency for absorbing dieldrin across the intestinal tract. 2. Ranking the tissues and organs according to their dieldrin concentrations in each sample period demonstrated that dieldrin distribution remained relatively constant throughout the experiment, with the exception of the liver and gonad. 3. The tissues and organs analyzed could be grouped together according to their similarities in dieldrin resi- due levels. Visceral adipose tissue represented a level by itself containing dieldrin levels at least six—fold higher than in any other tissue. A group, possessing l to 4 ppm dieldrin, included gill, ovary, female liver, combined intestine and pyloric caeca sample, and lastly the gall bladder plus bile sample. Generally, those organs associated with insecticide elimination routes were in this latter group. A second group, having low dieldrin concentrations (less than 1.0 ppm), included kidney, male liver, testes, blood, stomach and muscle tissue. u. In addition to noting previously observed dif- ferences in dieldrin residue levels in ovary and testis, differences in dieldrin concentrations in the liver of male and female fish were also noted. The higher dieldrin residue level observed in female livers was associated with ovarian development and probably represents a seasonal giliiiflli 79 phenomena since such a difference has not be previously observed. 5. Very little translocation among tissues was actually observed during the experiment. However, dieldrin levels declined in all tissues except the combined intes- tine and pyloric caeca sample which suggests that any translocation was mainly from sites of storage to sites of elimination. This general decline in residue levels in tissues plus the similarity of the biological half-lives for dieldrin loss from the fish body and from adipose tissue suggests that mobilization rates from dieldrin storage sites, particularly adipose tissue, may be the chief factor controlling dieldrin elimination rates. 6. The combined intestine and pyloric caeca sample demonstrated a slight increase in dieldrin levels during the experiment. Separate analyses of these two organs later in the experiment indicated that the residue levels in the intestine remained essentially constant, but that levels in the pyloric caeca increased with time. It is hypothesized that dieldrin transported to the intestine for elimination in the feces is resorbed from the (digestive tract, particularly in the pyloric caeca. 7. Dieldrin was lost from fish at an exponential :rate. The biological half-life for dieldrin in the entiie'fish was 25.8 days. The data suggest that dieldrin 80 was lost from individual tissues at different rates, but the results were not statistically significant. 8. Dieldrin was not detected in mucus and there- fore, is probably not eliminated across the integument. Some dieldrin was eliminated in urine after the tenth day, but the quantities were small and it is doubtful that significant amounts of dieldrin are lost via the fish kidney. 9. The importance of the intestine and feces as an excretory pathway for insecticides was not definitely demonstrated. More dieldrin was lost from the fish than was actually recovered in waste products and it is thought that the missing dieldrin may have been eliminated, undetected, in the feces. Based upon the dieldrin amounts actually recovered in feces, less than 5% of the absorbed dieldrin was eliminated via this pathway. If, however, the missing dieldrin was lost via this route, the intes- tine may account for as much as “5% of the dieldrin elinflnated from the fish. 10. The gill represented the major route for dieldrin eliflunation from fish. At least 95% of the dieldrin :recovered was from the water passing over the fish. Even if“the actual amount of dieldrin eliminated in feces was grweater than that observed, the gill still accounted for slgightly more than half (55%) of the dieldrin lost during the experiment . 81 A paradoxical condition existed in that, except for the first sample period, dieldrin levels in the water remained constant (average was 0.852 pg dieldrin/fish/day) whereas dieldrin levels in the gill and remaining fish declined at exponential rates. “I .4; L LITERATURE CITED 82 LITER TURE CITED Allison, D., B. J. Kallman, O. B. Cope and C. C. VanValin. 1963. Insecticides: effects on cutthroat trout of repeated exposure to DDT. Science, 142: 958-961. Anonymous. 1969. Pesticide into pest. Time Magazine. July 11: 9“, 56-57. Bridges, W. R., B. J. Kallman and A. K. Andrews. 1963. Persistence of DDT and its metabolites in a farm pond. Trans. Am. Fish. Soc., 92: “21-427. Buhler, D. R., M. E. Rasmusson and W. E. Shanks. 1969. Chronic oral DDT toxicity in Juvenile coho and Chinook salmon. Toxic. App. Pharmacol., 14: 535-555. Carson, R. 1962. Silent Spring. Houghton-Mifflin Co., Boston. 368 pp. Cope, O. B. 1960. The retention of DDT by trout and Whitefish. Trans. of the 1959 Seminar on Biological Problems in Water Pollution, 72-75. Datta, P. R., E. P. Laug, J. 0. Watts, A. K. Klein and M. J. Nelson. 1965. Metabolites in urine of rats on diets containing aldrin or dieldrin. Nature, 208: 289—290. Ferguson, D. E., J. L. Ludke and G. G. Murphy. 1966. Dynamics of endrin uptake and release by resistant and susceptible strains of mosquitofish. Trans. Am. Fish. Soc., 95: 335-344. Fromm, P. O. 1963. Studies on renal and extra-renal excretion in a freshwater teleost, Salmo gairdneri. Comp. Biochem. Physiol., 10: 121-128. Fromm, P.<3.and R. C. Hunter. 1969. Uptake of dieldrin by isolated perfused gills of rainbow trout. J. Fish. Res. Ed. Canada, 26: 1939-1942. 83 84 Gakstatter, J. H. 1966. The uptake from water by several species of freshwater fish of p,p'-DDT, dieldrin, and lindane; their tissue distribution and elimination rate. Ph.D. Dissertation. U. of North Carolina, Chapel Hill, N.C. 149 pp. Gakstatter, J. H. 1968. Rates of accumulation of 14C_ dieldrin residues in tissues of goldfish exposed to a single sublethal dose of lL‘C-aldrin. J. Fish. Res. Ed. Canada, 20: 1797-1801. Gakstatter, J. H. and C. M. W 183. 1967. The elimination of DDT-cl“, dieldrin-Cl , and lindane-C11“ from fish following a single sublethal exposure in aquaria. Trans. Am. Fish. Soc., 96: 301-307. Hamelink, J. L. 1969. The dynamics of DDT in the lentic environment. Ph.D. Dissertation, Michigan State University, East Lansing, Mich. 173 pp. Heath, D. F. and M. Vandekar. 196“. Toxicity and metabolism of dieldrin in rats. Brit. J. Industr. Med., 21: 269-279. Hickey, J. J., J. A. Keith and F. B. Coon. 1966. An exploration of pesticides in Lake Michigan ecosystem. J. Appl. Ecology, 3 (Suppl. ): 141- 154. Hoffert, J. R. 1966. Observation on ocular fluid dynamics and carbonic anhydrase in tissues of lake trout (Salvelinus namaycush). Comp. Biochem. Physiol. 17: 107-11“. Holden, A. V. 1962. A study of the absorption of Cl“- labelled DDT from water by fish. Ann. Appl. 8101., 50: 467-477. Hunn, J. B. 1969. Chemical composition of rainbow trout urine following acute hypoxic stress. Trans. Am. Fish. Soc., 98: 20-22. Hunt, E. G. and A. I. Bischoff. 1960. Inimical effects on wildlife of periodic DDD applications to Clear Lake. Calif. Fish and Game, “6: 91-106. Johnson, D. W. 1968. Pesticides and fishes--a review of selected literature. Trans. Am. Fish. Soc., 97: 398-42“. 85 Korte, F. and H. Arent. 1965. Metabolism of Insecti- cides. IX. Isolation and identification of dieldrin metabolites from urine of rabbits after oral administration of dieldrin-Cl“. Life Sciences, 4: 2017-2026. Lagler, K. F., J. E. Bardach and R. R. Miller. 1962. Ichthyology. John Wiley and Sons, Inc., New York. 535 pp. Lenon, H. L. 1968. Translocations and storage equilibria involving sublethal levels of dieldrin in aquatic ecosystems. Ph.D. Dissertation, Michigan State University, East Lansing, Mich. 85 pp. Ludke, J. L., D. E. Ferguson and W. D. Burke. 1968. Some endrin relationships in resistent and susceptible populations of golden shiners, Notemigonus crysoleucas. Trans. Am. Fish. Soc., 97: 260-263. Ludwig, G., J. Weis and F. Korte. 196“. Excretion and distribution of aldrin-Clu and its metabolites after oral administration for a long period of time. Life Sciences, 3: 123-130. Macek, K. J. 1968. Reproduction in brook trout (Salvelinus fontinalis) fed sublethal concentrations of DDT. J. Fish. Res. Ed. Canada, 25: 1787-1796. Menzie, C. M. 1966.' Metabolism of pesticides. U.S. Bur. Sport Fish. Wildl., spec. Sci. Rept., Wildl. No. 96. 274 pp- Morsdorf, K., G. Ludwig, J. Vogel an F. Korte. 1963 Die ausscheidung von aldrin-Cl und dieldrin-Ola sowie ihrer metaboliten durch die Galle. Med. Expt1., 8: 363-370. Mount, D. I. 1962. Chronic effects of endrin on blunt- nost minnows and guppies._ U.S. Bur. Sport Fish. and Wildl., Res. Rept. No. 58. 38 pp. Mount, D. I., L. W. Vigor and M. L. Schafer. 1966. Endrin: use of concentration in blood to diagnose acute toxicity to fish. Science, 152: 1388-1390. (D'Brien, R. D. 1967. Insecticides: Action and Metabolism. Academic Press, New York. 332 pp. Premdas, F. H. and J. M. A derson. 1963. The uptake and detoxification of cl -labelled DDT in Atlantic salmon, Salmo salar. J. Fish. Res. Ed. Canada, 20: 827-837. 86 Presser, C. L. and F. A. Brown, Jr. (1961. Comparative Animal Physiology. 2nd ed. W. B. Saunders Co., Philadelphia. 688 pp. Richards, B. D. and P. O. Fromm. 1969. Patterns of blood flow through filaments and lamellae of isolated-perfused rainbow trout (Salmo gairdneri) gills. Comp. Biochem. Physiol., 29: 1063-1070. Robeck, G. G., K. A. Dostal, J. M. Cohen and J. F. Kreissel. 1965. Effectiveness of waste treatment processes in pesticide removal. J. Amer. Water Works Assoc., 57: 181-199. Robinson, J., A. Richardson and V. K. H. Brown. 1967. Pharmacodynamics of dieldrin in pigeons. Nature, 213: 734-736. Schafer, M. L., K. A. Busch and J. E. Campell. 1963. Rapid screening method for DDT in milk with gas chromatography. J. Dairy Sci., XLVI: 1025-1032. Sivadas, P. 1965. Absorption of fat in the alimentary canal of Tilapia mossambica (Peters) (Teleostei). J. Cell. Comp. Physiol., 65: 249-251. Weaver, L., C. G. Gunnerson, A. W. Breidenbach and J. J. Lichtenberg. 1965. Chlorinated hydrocarbon pesticides in major U.S. river basins. Pub. Health Rept. 80: “81-493. Weiss, C. M. 196“. Use of fish to detect insecticides ' in water. A review and assessment of current knowledge. Proceedings 19th Annual Industrial Waste Conference. Purdue University, Lafayette, Indiana. 22 pp. Witt, J. M., W. H. Brown, G. 1. Shaw, L. S. Maynard, L. M. Sullivan, F. M. Whiting and J. W. Stull. 1966. Rate of transfer of DDT from the blood compartment. Bull. Environ. Contam. Toxicol. 1: 187-197. APPENDICES 87 APPENDIX A PROCEDURES FOR EXTRACTION AND CLEANUP OF SAMPLES 88 APPENDIX A 8222. The carbon tetrachloride was separated from the water by pouring the contents of the graduate into a separatory funnel and draining off the carbon tetrachloride into a 250 ml erlenmeyer flask. Approximately two grams of sodium sulfate (Na2804) was added to the flask and the carbon tetrachloride evaporated down until the Naesou was just moist, by drawing an air current across the surface with an aspirator while the erlenmeyer was immersed in a warm-water bath. Benzene (approximately 10 ml) was added, swirled with the Na2SO“ and evaporated down. This step was repeated two or three times with petroleum ether to remove traces of carbon tetrachloride. The dieldrin was eluted off the Nazsou with more petroleum ether, decanted into glass-stoppered centri- fuge tubes with several petroleum ether rinses and finally, brought to a constant volume (10 or 15 ml). 89 90 929.2 Urine was poured from a storage vial into a 125 m1 separatory funnel, rinsing the vial once with distilled water and twice with petroleum ether. The urine was then diluted with an equal volume of distilled water. Petroleum ether was added (10 m1) and the mixture shaken vigorously for two minutes after which the two immiscible phases were allowed to separate. The mixture was then agitated a second time. After partitioning, the aqueous phase was discarded. The petroleum ether was dried with Na2SOu and then decanted into glass-stoppered centrifuge tubes. The separatory funnel and Na2SOu were rinsed with aliquots of ether and added to the centrifuge tube. The sample was then evaporated down to a constant volume (usually 4 ml). Ease. Fecal material was separated from water in the feces trap by filtration (under vacuum) through a tared piece of glass wool. The feces and glass wool were dried for at least 2“ hours in an oven at “0° C. After removal from the oven, the material was cooled and weighed to obtain the dry weight of the feces. 91 The feces and glass wool were transferred to a mortar and ground with 4 or 5 m1 of acetonitrile (CH3CN). This step was repeated three times, transferring the acetonitrile (with filtration) to a 250 ml separatory funnel. Finally, the mortar, pestle, and filter were rinsed with CH3CN. Approximately 8 m1 of petroleum ether was added and the mixture shaken for one minute (Clean-up step). The acetonitrile was drawn off into a clean l-liter 'm‘ separatory funnel and the ether shaken with another 5 ml of CH CN which was added to the original CH CN. 3 3 (Total volume of CH CN was 30 m1.) 3 The acetonitrile was diluted 20 fold with 600 m1 of a 1.0% sodium sulfate distilled water solution. Sixty milliliters of petroleum ether were added and the mixture shaken for two minutes. The phases were allowed to separate and the aqueous portion was discarded. The ether was dried with NaZSO“ and decanted into a 250 m1 erlenmeyer flask, adding the ether from three rinses of the separatory funnel. The petroleum ether was evaporated down in a current of air drawn across the solution surface with vacuum pressure, transferred to graduated centrifuge tubes and brought to a constant volume (usually 5 m1). 92 Tissues Each sample was placed in a tared 100 m1 beaker and weighed. Twenty milliliters of a 20% (w/v) KOH- methanol solution (prepared fresh daily) was then added. The samples were heated on a hot plate with occasional stirring until all the tissue had digested (approxi- mately 15 minutes). With blood, the alcoholic-KOH was added directly to the storage vial and heated until completely digested. The sample was allowed to cool and then transferred to a 125 ml separatory funnel, rinsing the beaker once with a small amount of distilled water and twice with petroleum ether (total volume of ether was 20 ml). The mixture was shaken vigorously for four minutes, the two phases allowed to separate, and the aqueous portion discarded. The ether was shaken once more with 10 ml of distilled water to remove traces of the alcoholic-KOH. After separation, the water was discarded. The petroleum ether was then decanted into an erlen- meyer, adding the ether from three separate rinses of the separatory funnel. The ether was evaporated down to a few milliliters with a current of air while the erlenmeyer was immersed 93 in a warm-water bath. Using a disposable pipette, the ether was transferred to a graduated centrifuge tube, together with three ether rinses of the erlenmeyer, and brought to a constant volume (from 3 ml to 25 ml, depending upon the tissue). APPENDIX B DIELDRIN AMOUNTS AND CONCENTRATIONS FOUND IN TISSUES AND WASTE PRODUCTS OF EACH FISH FOR EACH SAMPLE PERIOD 95 0000032200 pmuMHsccmo 0000.0 0000.0 0000000000 coaomtpcmoeoo 0000.0 0000.0 0000 seaoee , ocflpa 0000.0 0000.0 000.0 000000\000 coaompoceocoo 0000.0 0000.0 0000.0 0000 000000 00003 000.00 000.000 000.000 00\000 coaomapcmocoo 0000.0 0000.0 0000.0 0000 000050 mmomm 00000000 00003 0000.0 0000.0 0000.0 0000000 0cac00E00 I- 0000.0 0000.0 0000000 0000 0000.0 0000.0 0000.0 00>aq 0000.0 0000.0 0000.0 mmcoflx 0000.0 0000.0 0000.0 000002 0000.0 .. .. 000>0 .. 0000.0 0000.0 000000 0000.0 0000.0 0000.0 0000 0000.00 0000.00 .. 000000 emodaoa 0000.0 0000.0 0000.0 00000 .0 ece 000000000 0000.0 0000.0 0000.0 0000000 0000.0 0000.0 0000.0 ooofim AEQQ N w\WJV 0:00pmppcmocoo 0000000 0 0000 0 0000 m 0000 00000 0:00 000000 000200 000-0 96 0000.0 0000.0 Apmpfia\w:v coapmpucmocoo 0000.0 0000.0 0000 pesoea mafia: 0000.0 0000.0 0000.0 0000000000 co000000000o0 0000.0 0000.0 0000.0 0000 oesos< 00003 it In 0000.0 Aw\wnv coapmpucmocoo an I: 0000.0 0000 peaoea mmomm whooaomm m9m¢3 0000.0 0000.0 0000.0 0000000 000000200 0000.0 0000.0 0000.0 0000000 0000 0000.0 0000.0 0000.0 00>00 0000.0 0000.0 0000.0 000000 0000.0 0000.0 0000.0 00000: 0000.0 I: .. 000>o .. 0000.0 0000.0 000000 0000.0 0000.0 0000.0 0000 0000.00 0000.00 0000.00 000000 00o0000 0000.0 0000.0 0000.0 00000 .0 0:0 0000000c0 0000.0 0000.0 0000.0 000eo00 0000.0 0000.0 0000.0 oooam AEQQ fl W\w1v 0coapmppcmocoo 00000He 00 0000 H0 0000 0000000000 0000000000 0 e000 0 0000 0 e000 00000 0:00 000000 000200 000-0 97 0000.0 0000.0 A00000\wnv 2000000200000 0000.0 0000.0 Away 0csoE< 0000: 0000.0 0000.0 0000.0 000000\000 0000000000000 0000.0 MOHm.0 0000.0 Away 003054 00003 0000.0 0000.0 0000.0 Amxmnv 0000000000000 0000.0 0000.0 0000.0 0000 000000 mmomm 00000000 0000: 0000.0 0000.0 0000.0 0000000 000000000 1. 0000.0 0000.0 0000000 0000 0000.0 ozmm.m mmm0.m 00>0q 00m0.0 mm0:.0 000000 00:00x 0000.0 0000.0 0000.0 00000: m00m.0 m0mm.0 000m.0 000>o u- u: us 000000 0000.0 0000.0 0000.0 0000 0000.00 0000.00 0000.00 000000 0000000 0000.0 0000.0 0000.0 00000 000 000000000 0000.0 0000.0 0000.0 00000 0000000 0000.0 0000.0 0000.0 000000000 0000.0 0000.0 0000.0 0000000 0000.0 0000.0 0000.0 00000 0809 I w\wnv C00 awhucwocoo 000000 00 0000 00 0000 0000000000 0000000000 0 0000 0 0000 0 0000 Amwfiq :m::.o moo:.o :mop.o amcofix mmmo.o mmmo.o woma.o maomsz II II I: >Lw>o mm:m.o mmoa.o mmo:.o mfipmme mmmw.a mmON.H mwmm.m Haas mamm.mfi omoo.mm wwmq.am mammaa mmoafic< mpma.m ommm.m mm:m.m mommo new mcfipmmch oomo.a omom.o mma:.m wommo OHLOHmm ma:a.z ommm.a mmmm.H mcfipmmch mm:m.o :o:m.0 HHNm.o somEOpm mmom.o mmmm.o omzm.o cooam mcoapMchmocoo mmammHe m* swam H§ gmfim umpmasccmo nmpmasccmo a swam o cmfim m cmfim Amw.o Away pesos< prm3 Hmm:.o mmmm.o :mpm.o Aw\wnv soapmppcmocoo afimo.o mfimo.o mmmo.o Away pcsoe< mmomm meoaaomm mem<3 Naom.o mmm~.o mmmm.o ammopmo mcficfimsmm ommm.o mmmm.o wmoo.a gmcumam flame Hmmm.o Homo.o Fzma.o pm>fia momm.o mmo:.o mmmm.o zmcofix mmma.o omoa.o mmmo.o maowsz II In :1 Ahm>o mmma.o mmma.o oamfi.o mfiumme mowm.fi Fuzm.fi :mmm.a Haas o~mm.:fl .. wawm.mfl mammfie mmoafic< momm.m mmmm.m o~m~.m mommo cam mcfipmmch Hmmm.H wowm.m :mm:.a mommo oapoaam mamw.a mmmm.m mmmm.a mcfipmmch :mom.o mmmm.o Hmmm.o consoum momm.o momm.o mm:m.o oooam Asaa u wanv COHDMhquoCOO mmammHa m* swam H§ swam cmpmfizccmo omumasccmo a swam o cmfim m swam Amw