Bulletin ILLIMTOIS ^a.tii.ra.1 History BULLETIN Comparative Uptake and Biodegradability of DDT and Methoxychlor by Aquatic Organisms iturah A. Reinbold der P. Kapoor illiam F. Childers illis N. Bruce )bert L. Metcaif NATURAL r.iST3R7Sliae NOV ? 1971 LIBRARY ATE OF ILLINOIS EPARTMENT OF REGISTRATION AND EDUCATION ATURAL HISTORY SURVEY DIVISION RBANA, ILLINOIS THE LIBRARY OF THE OCT 14 m\ UNIVERSITY OF 1LUN0I3. AXURBANA-CHA.V.r'«lGH VOLUME 30, ARTIC JUNE, 1971 1 ILLINOIS aturail Histoz^y Surv-ey BULLETIN Comparative Uptake and Biodegradability of DDT and Methoxychlor by Aquatic Organisms ah A. Reinbold P. Kapoor in F. Childers N. Bruce t L. Metcalf )F ILLINOIS iTMENT OF REGISTRATION AND EDUCATION IRAL HISTORY SURVEY DIVISION iNA, ILLINOIS VOLUME 30, ARTICLE 6 JUNE, 1971 STATE OF ILLINOIS DEPARTMENT OF REGISTRATION AND EDUCATION BOARD OF NATURAL RESOURCES AND CONSERVATION WILLIAM H. ROBINSON, Chainnan; Thomas Park, Ph.D., Biology; L. L. Sloss, Ph.D.. Geology; Roger Adams, Ph.D.,D.Sc., Chemistry; Robert H. Anderson, B.S.C.E., Engineering; Charles E. Olmsted, Ph.D., Forestry; W. L. Everitt. E.E., Ph.D., Representing the President of the Uni- versity of Illinois; Roger E. Beyler, Ph.D., Representing the President of Southern Illinois University. NATURAL HISTORY SURVEY DIVISION, Urbana, Illinois SCIENTIFIC AND TECHNICAL STAFF George Sprugel, Jr., Ph.D., Chief Alice P. Campbell, B.A., Secretary to the Chief Section of Economic Entomology VViLLiA.M H. Luckmann, Ph.D., Entomologist and Head Willis N. Bruce, Ph.D., Wayne L. Howe, Ph.D., Stevenson Moore, III, Extension Howard B. Petty, Ph.D., Entomologist Entomologist Ph.D., Entomologist, on Entomologist, Exten- Ph.D., Associate Bntomol-James E. Appleby, ogist Edward J. Armbrust^ Ph.D., Associate Ento- mologist Marcos Kogan, Ph.D., Associate Entomologist Joseph V. Maddox, Ph.D., Associate Entomol- ogist Ronald H. Meyer, Ph.D., Associate Entomol- ogist ROBERT D. Pausch, Ph.D., Associate Entomol- ogist Ralph E. Sechriest, Ph.D., Associate Entomol- ogist George L. Godfrey, Ph.D., Assistant Entomolo- gist Clarence E. White, B.S., Assistant Entomolo- gist Keun S. Park, M.S., Assistant Chemist SUE E. Watkins, Supervisory Assistant Roscoe Randell, Ph.D., Assistant Professor, Extension Donald E. Kuhlman, Ph.D., Instructor, Exten- sion Tim CooleYj M.A., Assistant Specialist, Exten- sion Jean G. Wilson, B.A., Supervisory Assista^it Martha P. Nichols, M.S., Research Assistant Keturah Reinbold, M.S., Research Assistant LOWELL Davis, Technical Assistant Nancy D. DeWitt, B.S., Technical Assistant Marcia Janes, B.S., Technical Assistant Lu-PiNG Kan, M.S., Technical Assistant Marie Monkman, M.S., Technical Assistant Stephen Roberts, B.S., Technical Assistant Douglas K. Sell, B.S., Technical Assistant JOHN T. Shaw, B.S., Technical Assistant Section of Botany and Plant Pathology J. CEDRic Carter, Ph.D., Plant Pathologist and Head Robert A. Evers, Ph.D., Botanist Junius L. Forsberg, Ph.D., Plant Pathologist Eugene B. Himelick, Ph.D., Plant Pathologist R. Dan Neely, Ph.D., Plant Pathologist D. F. Schoeneweiss, Ph.D., Associate Plant Pathologist J. Leland Crane, Ph.D., Assistant Mycologist Walter Hartstirn, Ph.D., Assistant Plant Pathologist Betty S. Nelson, Technical Assistant Gene E. Reid, Technical Assistant Section of Aquatic Biology George W. Bennett, Ph.D., Aquatic Biologist and Head D. Homer Buck, Ph.D., Aquatic Biologist R. Weldon Larimore, Ph.D., Aquatic Biologist William C. Starrett, Ph.D., Aquatic Biologist Robert C. Hiltibran, Ph.D., Biochemist William F. Childers, Ph.D., Associate Aquatic Biologist Donald F. Hansen, Ph.D., Associate Aquatic Biologist Richard J. Baur, M.S., Research Assistant Dennis L. Dooley, Technical Assistant Mary Frances Martin, Technical Assistant Robert F. Randall, Ph.D., Technical Assistant Kenneth R. Walker, Technical Assistant C. Russell rose. Field Assistant Warren U. Brigham, M.S., Junior Technical Assistant Section of Founistic Surveys and Insect Identification Philip W. Smith, Ph.D., Taxonomist and Head Wallace E. LaBerge, Ph.D., Taxonomist Milton W. Sanderson, Ph.D., Taxonomist Lewis J. Stannard, Jr., Ph.D., Taxonomist ROBERT W. POOLE, Ph.D., Assistant Taxonomist JOHN D. UnziCKEr, Ph.D., Assistant Taxono- mist Donald W. Webb, M.S.. Assistant Taxonomist RODERICK R. Irwin, Research Affiliate Bernice p. Sweeney, Junior Professional Scientist Section of Wildlife Research Glen C. Sanderson, Ph.D., Wildlife Specialist and Head Frank C. Bellrose, B.S., Wildlife Specialist RICHARD R. GRABER, Ph.D., Wildlife Specialist Harold C. Hanson, Ph.D., Wildlife Specialist William L. Anderson, M.A., Associate Wildlife Specialist W. W. Cochran, jr., B.S., Associate Wildlife Specialist William R. Edwards, M.S., Associate Wildlife Specialist Jack A. Ellis, M.S., Associate Wildlife Special- ist Ronald F. Labisky, Ph.D., Associate Wildlife Specialist Charles M. Nixon, M.S., Associate Wildlife Specialist Stanley L. Etter, M.S., Assistant Wildlife Spe- cialist ROBERT E. Greenberg, M.S., Assistant Wildlife Specialist G. Blair Joselyn, M.S., Assistant Wildlife Spe- cialist George B. Rose, Ph.D., Assistant Wildlife Spe- cialist David R. Vance, M.S., Assistant Wildlife Spe- cialist RONALD L. Westemeier, B.S., Assistant Wildlife Specialist RONALD E. DUZAN, Technical Assistant Norma J. Hubbard, Technical Assistant Mary Ann Kjos, Technical Assistant Helen C. Schultz, M.S., Technical Assistant Hilda Wiesenmeyer, Technical Assistant Eleanore Wilson, Technical Assistant Robert D. Crompton, Field Assistant James W. Seets, Laboratory Assistant Section of Administrative Services Robert O. Watson, B.S., Administrator and Head Supporting Services Wilma G. Dillman, Property Control and Trust Accounts ROBERT O. Ellis, Assistant for Operations Lloyd E. Huffman, Stockroom Manager J. William Lusk, Mailing and Distribution Melvin E. Schwartz, Financial Records James E. Sergent, Greenhouse Superintendent Publications and Public Relations Owen F. Glissendorf, M.S., Technical Editor ROBERT M. Zewadski, M.S., Associate Technical Editor Shirley MCClellan, Assistant Technical Edi- tor Richard M. Sheets, Technical Illustrator Wilmer D. Zehr, Technical Photographer Technical Library Doris F. Dodds, B.A., M.S.L.S., Technical Li- brarian Jean ICKES, B.A.. M.S.L.S., Assistant Technical Librar-ian CONSULTANTS: PARASITOLOGY, NoRMAN D. Levine, Ph.D., Professor of Veterinary Parasitology and Veterinary Research, University of Illinois; Wildlife Research, Willard D. Klimstra, Ph.D., Professor of Zoology and Director of Cooperative Wildlife Research, Southern Illinois Univer- sity; Statistics, Horace W. Norton, Ph.D., Professor of Statistical Design and Analysis, Univer- sity of Illinois; Entomology, gilbert p. Waldbauer, Ph.D., Associate Professor of Entomology, University of Illinois. CONTENTS Acknowledgements 405 Materials and Methods 406 Radiolabeled Compounds 406 Radioassay Methods 406 Chromatographic Techniques 406 Bioassay Methods 407 Results and Discussion 408 Uptake and Metabolism by Fishes 408 Uptake and Metabolism by Daphnia and Snails 411 Uptake and Metabolism by Guppies in a Daphnia-to-Fish Food Chain 413 Summary 414 Literature Cited 415 Index 416 This paper is published by authority of the State of Illinois, IRS Ch. 127, Par. 58.12. Keturah A. Reinbold is a Research Assistant, Section of Economic Entomology, Illinois Natural History Survey; Dr. Inder P. Kapoor is a Research Assistant, Department of Entomology, Uni- versity of Illinois; Dr. William F. Childers is an Associate Aquatic Biologist, Section of Aquatic Biology, Illinois Natural History Survey; Dr. Willis N. Bruce is an Entomologist, Section of Economic Entomology, Illinois Natural History Survey; and Dr. Robert L. Metcalf is Professor of Zoology, Entomology, and Agricultural Entomology and Head of the Department of Zoology, University of Illinois. (30448—5M—6-71) Frontispiece. —Organisms and compounds used in this investigation. On a background of dophnia swimming (top to bottom) the green sunflsh, guppy, tilapio, and snails. Comparative Uptake and Biodegradability of DDT and Methoxychlor by Aquatic Organisms DDT IS SOLUBLE in water to about 0.002 ppm, but it is soluble in animal fats to about 100,000 ppm. Be- cause DDT possesses such a high lipid- to-water partition value and resists attack by multifunction oxidase detoxi- fying enzymes, this insecticide has be- come a ubifjuitous environmental pollu- tant and is found in animal tissues everywhere. For example, DDT is pres- ent in Lake Michigan bottom muds at about 0.014 ppm. It has been found in concentrations of 3-6 ppm in fishes, such as coho salmon and lake trout, taken from Lake Michigan and in even higher concentrations in the tissues of fish-eat- ing birds living near the lake ( Harrison et al. 1970:505). DDT used for spruce budworm control in the Yellowstone River system was found to persist in the aquatic environment for more than 2 years (Cope 1961:242-244). Bridges et al. ( 1963 ) found concentrations of DDT in a number of organisms in a farm pond treated with 0.02 ppm of DDT. Fish in aquaria have been shown to eliminate DDT very slowly after a single sublethal exposure (Gakstatter & Weiss 1967:305). Because animals concentrate DDT in their tissues and eliminate it very slowly, aquatic pollu- tion by DDT, whether it results directly from blackfly and mosquito control programs or indirectly from urban treatments for elm bark beetle control or agricultural applications for fruit pests, is particularly deleterious to en- vironmental quality. The need for a persistent but bio- Kelurah A. Reinbold Inder P. Kapoor William F. Childers Willis N. Bruce Robert L. Metcalf degradable substitute for DDT is urgent. Methoxychlor is being widely considered as such a substitute, espe- cially for control of blackllies, bark beetles, and fruit and garden pests. It has been reported that, after 28 days of exposure to 0.04 ppm of methoxychlor, bluegills had metabolized or excreted most of the compound which they had taken up. After 56 days no methoxy- chlor was found in the fish ( Kennedy et al. 1970:12). Preliminary studies in a laboratory model ecosystem (Kapoor et al. 1970:1151) indicate that me- thoxychlor does not accumulate in fishes, as does DDT, but reaches a dy- namic ecjuilibrium. The studies reported in this paper were designed to further test the bio- degradability of methoxychlor and to compare in a laboratory aquatic en- vironment (i) the uptake of DDT and methoxychlor directly from water by fishes, from water by crustaceans of the genus Daphnia and by snails of the genus Phijsa, and from daphnia by guppies in a daphnia-to-fish food chain and (ii) the subsequent elimination of the insecticides from the organisms. ACKNOWLEDGEMENTS We are indebted to several members of the Illinois Natural History Survey staff for their contributions to this in- vestigation. Dr. William H. Luckmann, Head of the Section of Economic Entomology, provided encouragement throughout the study and reviewed the 405 406 Ilunois Natxjral History Survey Bulletin Vol. 30, Art. 6 manuscript, which was edited by Rob- ert M. Zewadski, Associate Technical Editor. Richard M. Sheets, Technical Illustrator, prepared the graphs and designed the frontispiece, and VVilmer D. Zehr, Technical Photographer, pro- vided most of the photographs. The photograph of the tilapia appearing in the frontispiece was loaned by the Sec- tion of Aquatic Biology. We wish to thank the Natural His- tory Survey and the University of Illi- nois for the laboratory space and equip- ment provided for this investigation. This study was supported in part by funds supplied by U. S. Department of Agriculture Regional Projects NC-85, Reduction of Hazards Associated with the Presence of Insecticidal Chemicals in the Environment, and NC-96, En- vironmental Implications of Pesticide Usage, both Hatch projects. These funds were administered by the Uni- versity of Illinois College of Agriculture. The study was also supported in part by U.S. PubHc Health Service grants FD-00271 and FR-07030. and FWQA 16050 EHH. MATERIALS AND METHODS Radiolabeled Compounds The investigations were conducted with 3H-ring substituted methoxychlor obtained by the procedure of Kapoor et al. (1970:1146). The compound was 99.9 -f percent pure according to our evaluation of it by thin-layer chroma- tography, and it had a specific activity of 4.81 mC/mM. Ring-labeled "C- DDT was obtained from the World Health Organization of the United Nations, Geneva, Switzerland. The DDT had a specific activity of 5.48 mC, mM and a purity of 99.9+ percent. Radloassay Methods The radioactivity in 1-ml water samples containing radiolabeled com- pounds was counted in 10 ml of ^H scintillation fluid (200 grams of naph- thalene, 10 grams of PPO, 0.25 gram of POPOP in dioxane to make 1 liter) in a Beckman S-250 scintillation coun- ter. All organisms used in our investi- gations were freeze-dried and ground to a powder with a mortar and pestle. Each of three 10-mg portions of each sample of the powdered material was placed in 15 ml of scintillation fluid. The samples were kept in the dark overnight before counting to quench phosphorescence. The radioactivity in each sample was determined by aver- aging the results of the three counts. Quenching, if any occurred, was corrected for, using the appropriate quench curves. All insecticide concen- trations in the organisms were calcu- lated on a dry-weight basis. Chromatographic Techniques Thin-layer chromatography ( TLC ) \\'as performed in the usual manner using glass plates coated with 0.25 mm of silica gel. To determine the relative proportions of parent compounds and their metabolites in tilapia {Tilapia mossambica) and green sunfish {Le- pomis cyanellus), portions of the pow- dered organisms were extracted with acetonitrile (2 ml for tilapia, 4 ml for green sunfish). About 0.05 ml of the extract from each sample was spotted on a TLC plate. Non-radiolabeled parent compounds and model metab- olites were incorporated as internal standards for cochromatography by spotting them on the plate over the acetonitrile extracts of the fishes. Me- thoxychlor chromatograms were devel- oped in a solvent system consisting of 3 parts diethyl ether and 1 part petro- leum ether (b. p. 60° to 68° C), and those of DDT in petroleum ether (b. p. 60° to 68° C) alone. Radioautographs of the '^C-labeled DDT metabolites were made by ex- posing Eastman Kodak No Screen Medical X-ray film to the developed chromatograms. Black spots which had developed on the film were matched with the incorporated internal stand- ards to determine the identity of the metabolites. The corresponding areas of silica gel were scraped from the June, 1971 Reinbold et al. : Biodegradability of DDT and Methoxychlor 407 chromatograms and counted by scintil- lation counting; from these counts the relative percentages of each parent compound and its metabolites were then calculated. The relative propor- tions of methoxychlor and its metabo- lites were determined by scraping ap- propriate spots or strips of silica gel from the chromatograms and radio- assaying them. Bioassay Methods ^Ve used cylindrical Pyrex jars 8 inches deep and 8' 4 inches in diameter in all of the experiments reported on here. Each jar contained 5 liters of synthetic hard water (Cairns 1969:8) and was covered by a plate glass lid. The water was constantly aerated by compressed air bubbling through a glass tube extending down the inside wall of the jar. Both DDT and methoxy- chlor were added to the water in these jars from standard 0.0025-percent solutions in acetone. The water-insec- ticide solutions were allowed to equil- ibrate for 24 hours before any aquatic organisms were placed in the jars. The animals used were daphnia {Daphnia magna) (hereafter referred to as "daphnia"), snails {Phijsa sp.) (hereafter referred to as "snails"), tila- pia {Tilapia mossambica) (hereafter referred to as "tilapia"), green sunfish (Lepomis ctjanellus), and guppies (Lebistes reticitlatiis). At 2-day, 3-day, or 10-day intervals, the organisms were moved to fresh water or were surface rinsed with clean water and then frozen. Subsequently, the organisms were freeze-dried, powdered, and radio- assayed. In all experiments organisms held under the same conditions as were the test animals but not exposed to in- secticides were prepared and assayed in the same manner as were the test samples. Comparative uptake of DDT and methoxychlor from water by tvvo fish species, tilapia and green sunfish, was studied at insecticide levels of 0.001, 0.003, and 0.01 ppm. The tilapia experi- ment was duplicated using three fish (0.6-1.8 grams per fish) in each jar. Two jars of tilapia were used at each of the three levels of each insecticide ( 12 jars ) . Only one green sunfish ( 5-9 grams) was placed in each of three jars at each concentration of each com- pound ( 18 jars ) . One fish was removed from each jar (two tilapia from each insecticide concentration and one green sunfish from each concentration) on the 3rd, 10th, and 31st days, after which the experiment was terminated. On the 10th and 20th days those fishes which remained were transferred to jars containing insecticide concentra- tions equivalent to those in which the fishes had been placed at the beginning of the experiment. The uptake of insecticides from water by daphnia (1 gram per jar) was studied at the insecticide levels used in the fish experiments. The daphnia from half of the jars at each concentration of each insecticide were removed on the 3rd day and from the remaining jars on the 6th day. All samples were then radioassayed. In another experiment tilapia weigh- ing 0.2-1.4 grams each were exposed, five fish per jar in each of five jars for each insecticide, to 0.003 ppm of DDT or methoxychlor for 12 days. At 3-day intervals one fish from each jar was removed for radioassay, and the rest were transferred to water freshly treated with 0.003 ppm of insecticide. After 12 days the remaining fish were moved to water containing no insec- ticide. Samples of one fish each were then taken after additional periods of 2, 5, 9, 11, 12, and 15 days and assayed for radioactivity to determine the com- parative insecticide excretion rates. Daphnia ( 2 grams ) and snails ( 1 gram) were exposed together in jars to a level of 0.003 ppm of DDT or methoxychlor for 2, 4, or 6 days. In one series of jars the daphnia and snails were exposed for 2 days, the animals in one jar were removed for radioassay, and the remaining organisms were moved to water without insecticide. They were transferred to fresh water 408 Ilunois Natubal History Survey Bulletin Vol. 30, Art. 6 without insecticide every 2 days, and a one-jar sample was removed after 1, 2, 4, or 6 days. The organisms in another series of jars were transferred to water containing the original level of insecticide after 2 and 4 days. They were also sampled for radioassay after 4 days. After 6 days a sample was re- moved for radioassay, and the remain- ing organisms were moved to water without insecticide. Again, they were transferred to fresh water every 2 days and were sampled after 1, 3, or 5 days. Insecticide uptake in a daphnia-to- guppy food chain was studied by hold- ing 2-5 grams of daphnia in water con- taining 0.003 ppm of DDT or methoxy- chlor for 48 hours and then feed- ing them to guppies held in water without any insecticide. Three jars of daphnia exposed to methoxychlor were combined. One-third of these daphnia were analyzed for insecticide content by scintillation counting and the re- maining animals were fed to guppies held in two jars, each containing 10 fish. The same procedure was followed with daphnia exposed to DDT and fed to two additional jars of fish. By ex- posing additional jars of daphnia to the insecticides at 2-day intervals, we repeated the process and fed the fish every other day. Fish were removed from each jar on the 6th, 8th, and 20th days and assayed for radioactivity. RESULTS AND DISCUSSION Uptake and Metabolism by Fishes The results of the investigation of the uptake from water of DDT and methoxychlor by tilapia and green sun- fish are presented in Table 1. At the 0.001-ppm level the concentration of methoxychlor in tilapia dropped from 0.8 ppm after 3 days of exposure to 0.2 ppm after 10 days, remaining at 0.2 ppm at the end of 31 days. The DDT concentration decreased from 1.3 ppm after 3 days to 0.7 after 10 days; it then increased markedly to 6.8 ppm at 31 days. The same pattern occurred at the higher insecticide levels. The pattern was repeated in the green sunfish, which differed from the tilapia, however, in having lower DDT-to-methoxychlor ratios. These lower ratios may have been the result of the green sunfish's lower proportion of body fat and its consequent smaller capacity for con- centrating DDT in fat tissues. The results show far greater con- trasts when we examine the concen- tration factor (Fig. 1 and 2). While Toble 1. — Comparative uptake from water of **C-DDT and ^H-methoxychlor by tilapia and green sunfish. Insec- ticide Concen- tration in Water June, 1971 Reinbold et al. : Biodegradability of DDT and Methoxychlor 409 T 10 15 20 EXPOSURE TIME (DAYS) T 30 Fig. 1, — Comparative uptake from water of "C-DDT and 'H-methoxychlor by tilapio. Tfie vertical arrows indicate the days when fish were transferred to jars containing insecticide levels equivolent to those which the fish had been placed in at the beginning of the experiment. the insecticide concentration in me- thoxychlor-exposed fishes reached an equihbrium, which indicated that the compound was being metaboUzed and excreted, DDT was increasingly con- centrated in the fishes. In tilapia ex- posed to the 0.01-ppm insecticide level at the end of 31 days, even after the DDT had been concentrated 10,600 times it had not reached a steady state. Furthermore, when tilapia were held in water containing 0.003 ppm of insec- ticide for 12 days and subsequendy were left for 15 days in jars containing water with no insecticide, the radio- active compounds in methoxychlor-ex- posed fish were rapidly excreted (Fig. 4). At the end of 15 days in uncon- taminated water there was a 10,000- fold difference between the concentra- tions in tilapia of methoxychlor and of DDT (Table 4). The metabolic pattern (Table 2) in- dicates that both fishes metabolized methoxychlor to a greater extent than they did DDT. Tilapia metabolized DDT to a greater extent than green sunfish metabolized it. Tilapia contain- ed DDD as a major metabolite, but the major metabolite of the green sun- fish was DDE. Methoxychlor was rapidly metabolized by tilapia, which contained considerably higher propor- tions of the mono-O-demethylated prod- uct [2- ( p-methoxyphenyl ) - 2 - (p-hy- droxyphenyl ) - 1, 1,1- trichloroethane] 410 Ilunois NATxmAL History Survey Bulletin Vol. 30, Art. 6 but only slightly greater proportions of trichloroethane] than did the green the bis-O-demethylated metabolite [2, sunfish. 2-bis- (;3-hydroxyphenyl) -1, 1, 1- The data from these experiments in- -- 9 -I Fig. 2. — Compan arrows indicate the d vhich the fish had been placed uptoke fi when fish T 1 1 T 10 15 20 25 EXPOSURE TIME (DAYS) rom woler of '*C-DDT ond ^H-methoxychlo were transferred to jars containing insect t the beginning of the experiment. by gr. :ide le' sunfish. The vertical equivalent to those Table 2. — Distribution of "C-DDT and Sh- owing exposure to water containing these ins ethoxychio :ticides. etobolites in tilapia and green sunfish Insecticide June, 1971 Reinbold et al. : Biodegradability of DDT and Methoxychlor 411 dicate that methoxychlor is readily bio- degradable in tilapia and green sunfish. It seems likely that this insecticide is also biodegradable in other fish species. Uptake and Metabolism by Daphnia and Snails Daphnia also concentrated both me- thoxychlor and DDT taken up from water. The DDT-to-methoxychlor ratio (Table 3) in daphnia after 3 days of exposure at the three insecticide levels used in these experiments ranged from 1.8 to 2.5, a pattern similar to that dis- played by the fishes we studied. When daphnia and snails were held together in jars containing 0.003 ppm of insecticide, daphnia concentrated the two insecticides at about the same rate, but the snails concentrated me- Table 3. — Comparotive uptake fr< 3ter of "C-DDT and ^H-methoxychlor by daphni( Insecticide Concen- tration 412 Ilunois Natubal History Sxjrvey Bulletin Vol. 30, Art. 6 a.SQ •S-fi 2 o2Ci Sa 0-2 S r; V. bO I O rH Cq TlH (D O 00,000 ^ CO CO , CD *' * -H o o o o * cq o 00 N oq c4 ' o o o c^ \o a> y-i a to \ June, 1971 Reintjold et al. : Biodegradability of DDT and Methoxychlor 413 thoxychlor much more than they con- centrated DDT- 18 ppm of DDT: 38 ppm of methoxychlor after 6 days of ex- posure to the insecticides (Table 4 and Fig. 3). When daphnia and snails were held in water free of insecticide after exposure to DDT or methoxychlor, both organisms excreted radioactive com- pounds. Daphnia excreted methoxy- chlor more rapidly than DDT, while the opposite was true of the snails (Table 4 and Fig. 4). The snails ap- 41 -I parently cannot readily metabolize methoxychlor and, as a result, retain relatively persistent, high concentra- tions of that insecticide once they have taken it up. This observation agrees with that of Kapoor et al. (1970:1151). The snails, like the other organisms studied, also tend to retain DDT. Uptake and Metabolism by Guppies in a Daphnia-to-Fish Food Chain The results of the daphnia-to-guppy food chain study are shown in Table 5 DDT METHOXYCHLOR - DAPHNIA — FISH -- SNAILS .0001 DAYS IN WATER WITHOUT INSECTICIDES Fig. 4. — Loss of radiooctivity from tilapio, snails, and daphnia in water without insecticides following exposure to 0.003 ppm of DDT or methoxychlor in water. Periods of exposure to insecticides: fish, 12 doys; snails, 6 doys; daphnia, 2 doys. 414 Illinois Natural History Survey Bulletin Vol. 30, Art. 6 Toble 5. — Uptake of '*C-DDT and ^H-methoxychlor in a daphnia-to-guppy food chain. Insecticide Concentration^ in Daphnia in ppm Days Fish Fed on Daphnia Exposed to Insecticide Insecticide Concentration" in Fish in ppm Rate of Insecticide Uptake From Food By Fish ppm in Fish ppm in Daphnia Ratio of Rates of Uptake by Fish, DDT to Methoxychlor Methoxychlor 22.6 21.3 21.7 DDT 25.1 25.9 26.6 20 20 0.14 0.07 0.17 3.10 3.69 7.80 0.006 0.003 0.008 0.124 0.143 0.293 21.0 48.0 38.0 "All such concentrations were calculated on a dry-weight basis. and Fig. 5. There was little difference in the uptake of the two insecticides from water by the daphnia. However, when the daphnia entered the food chain, the methoxychlor content in the 10 -> ? 8- 6- 4- 2- DDT • METHOXYCHLOR 10 20 EXPOSURE TIME (DAYS) Fig. 5. — Uptake of "C-DDT and ^H-methoxy- chlor by guppies in a daphnia-to-guppy food choin. The dophnio were held in wafer containing 0,003 ppm of DDT or methoxychlor for 48 hours before being fed to the fish. fish rapidly reached a steady state, while the concentration of DDT con- tinued to increase. From the 6th to the 20th day the methoxychlor level in the guppies increased only from 0.14 to 0.17 ppm, but the DDT level increased from 3.10 to 7.80 ppm. The differences in concentration of the two compounds are shown by the relative values of the rates of uptake by the fish from the daphnia (column 4 of Table 5) and by the ratios of the rates of uptake (col- umn 5). The difference in accumula- tion is due to the presence of alkoxy groups on the aryl rings in methoxy- chlor, which cause it, unlike DDT, to be subject to attack by multifunction oxidases of the fish and, therefore, to be rapidly metabolized and excreted. In general, our investigations show that methoxychlor appears to be read- ily biodegradable in fishes and in some, though not all, other aquatic organisms. Thus, it seems to be a safer insecticide than DDT to use in or near aquatic environments. SUMMARY Comparative studies were made of the uptake and metabolism by three species of fishes, by daphnia, and by a snail of radiolabeled methoxychlor and DDT. Tilapia and green sunfish ex- posed over a 31-day period to the June, 1971 Reinbold et al. : Biodegradability of DDT and Methoxychlor 415 radiolabeled insecticides at levels of 0.001, 0.003, and 0.01 ppm in water concentrated DDT as much as 10,600- fold and methaxychlor about 200-fold. However, when tilapia were transferred to water with no insecticide following 12 days of exposure to 0.003 ppm of insecticide in water, the residues of methoxychlor decreased within 15 days from 8 ppm to 0.0001 ppm and DDT from 13 to 1 ppm, a 10,000-fold differ- ence between the concentrations of DDT and methoxychlor. Significant differences were found between the rates at which tilapia and green sunfish metabolized DDT and methoxychlor. Tilapia metabolized DDT to a greater extent and contained DDD as a major metabolite, while green sunfish contained only small amounts of DDD. Tilapia also metab- olized methoxychlor more rapidly than green sunfish metabolized it and con- tained higher amounts of mono- and bis-phenols produced by 0-demethyla- tion. Daphnia in water containing DDT or methoxychlor concentrated DDT at nearly the same rate at which they con- centrated methoxychlor to about twice that rate. When daphnia containing either radiolabeled DDT or methoxy- chlor were fed to guppies to complete a food chain, DDT was rapidly con- centrated in the fish, reaching levels of about 8 ppm in 20 days, while me- thoxychlor concentrations never rose beyond 0.17 ppm. Thus, methoxy- chlor appears readily biodegradable in fishes. However, the snail used in this investigation could not rapidly metabolize either DDT or methoxy- chlor and accumulated both to high levels. LITERATURE CITED Bridges, W. R., B. J. Kallman, and A. K. Andrews. 1963. Persistence of DDT and its metabolites in a farm pond. American Fisheries Society Transactions 92(4): 421-427. Cairns, John, Jr. 1969. Fish bioassays—re- producibility and rating. Revista de Bio- logia 7(l-2):7-12. Cope, Oliver B. 1961. Effects of DDT spraying for spruce budworm on fish in the Yellowstone River system. American Fish- eries Society Transactions 90(3) :239-251. Gakstatter, Jack H., and Charles M. Weiss. 1967. The elimination of DDT-C", dieldrin-Cl+, and lindane-Ci* from fish following a single sublethal e.xposure in aquaria. American Fisheries Society Trans- actions 96(3):301-307. Harrison, H. L., O. L. Loucks, J. W. Mitch- ell, D. F. Parkhurst, C. R. Tracy, D. G. Watts, and V. J. Yannacone, Jr. 1970. Systems studies of DDT transport. Science 170(3957):503-508. Kapoor, Inder p., Robert L. Metcalf, Robert F. Nystrom, and Gurchahan K. Sangha. 1970. Comparative metabolism of methoxychlor, methiochlor, and DDT in mouse, insects, and in a model ecosystem. Agricultural and Food Chemistry Journal 18(6):1145-1152. Kennedy, Harry D., Lafayette L. Eller, and David F. Walsh. 1970. Chronic effects of methoxychlor on bluegills and aquatic invertebrates. U.S. Department of the In- terior, Bureau of Sport Fisheries and Wild- life Technical Paper 53. 18 p. INDEX Acetonitrile, 406 Food chain, 405, 408, 413-414, 415 Freeze-dried organisms, 406, 407 Bioassay metliods, 407-408 Bluegill, 405 Green sunfish (see Lepomis cyanellus) Guppy (see Lebistes reticulatus) Carboni* (iiC),406 Chromatography cochromatography, 406 diin-layer chromatography, 406 Coho salmon, 405 :iH, 406 Insecticide ( sec DDT and methoxychlor ) Internal standard, 406 Daphnia magna, 405, 407^08, 411-415 DDT elimination or excretion of, 405, 412, 413, 415 environmental pollution by, 405 Lake Michigan pollution by, 405 metabolism of by organisms, 409, 410, 415 metabolites of DDD, 409, 410, 415 DDE, 409, 410 radiolabeled, 406, 414 solubility in water and fats of, 405 specific activity of radiolabeled, 406 uptake and/or accumulation of by organisms birds, 405 coho salmon, 405 daphnia, 411, 412,414, 415 green sunfish, 408, 410, 414-415 guppy, 414, 415 lake trout, 405 snail, 411, 412, 413, 415 tilapia, 408, 409, 412, 415 use of in control programs blackfly, 405- elm bark beetle, 405 mosquito, 405 spruce budwomi, 405 Yellowstone River system pollution by, 405 Dioxane, 406 Ecosystem, laboratory model, 405 Ether diethyl, 406 petroleum, 406 Fislics (see under individual species) Lake Michigan, 405 Lake trout, 405 Lebistes reticulatus, 405, 407, 408, 413-414, 415 Lepomis eijanellus, 406, 407, 408-411, 414-415 M Metabolism {see DDT and methoxychlor) Metabolites (see also DDT and metlioxychlor ) model metabolites, 406 Methoxychlor biodegradability of, 405, 411, 414, 415 elimination or excretion of, 405, 409, 412, 413 dynamic equilibrium ( steady state ) of, 405, 409, 414 metabolites of 2- ( p-methoxyphenyl ) -2- ( p-hydroxyphenyl ) -1,1,1- trichloroethane, 409, 410, 415 2,2-bis- ( p-hydroxyphenyl ) - 1,1,1-trichloroetlrane, 410, 415 metabolism of by organisms, 405, 409-410, 413, 415 radiolabeled, 406, 414 sjiecific activity of radiolabeled, 406 substitute for DDT, 405, 414 uptake and/or accumulation of by organisms daphnia, 411, 412, 414, 415 green sunfish, 408, 410, 415 guppy, 414, 415 snail, 411,412,413,415 tilapia, 408, 412, 415 Multifunction oxidase enzymes, 405, 414 N Naphtlialcne, 406 416 June, 1971 Reinbold et al. : Biodegradability of DDT and Methoxychlor 417 P Phy-sci, 405, 407, 411, 412, 413, 414, 415 Phosphorescence, 406 POPOP, 406 PPO, 406 Q Quench curves, 406 Raclioassay, 406, 407, 408 Radioautograph, 406 Radiolabeled insecticide ( sec under DDT and metlioxychlor ) ScintillaHon countins, 406, 407, 408 Scintillation fluid, 406 Silica gel, 406, 407 Snail ( sec Tlujsa ) Specific activity ( see tmder DDT and methoxychlor ) Snvthetic hard water, 407 Tilapia mossambica, 406, 407, 408-411, 412, 414, 415 Uptake of insecticides ( see under DDT and metlioxychlor ) Scintillation counter, 406 Yellowstone River system, 405 Some Publications of the ILLINOIS NATURAL HISTORY SURVEY BULLETIN Volume 29, Article 3—Hybridization of Four Species of Sunfishes ( Centrarchidae ) . By William F. Childers. September, 1967. 55 p., frontis., 2 fig., color plate, bibliogr., in- dex. Volmiie 29, Article 4.—The Thrips, or Thysa- noptera, of IllinoLS. By Lewis J. Stannard. May, 1968. 338 p., frontis., 310 fig., bib- lioRr., inde.v. \'oliune 30, Article 1 .—Largemoutli Bass and Other Fishes in Ridge Lake, Illinois, 1941- 1963. By CeorRe W. Bennett, H. Wick- lifte Adkiiis, and William F. Childers. Sep- tember, 1969. 67 p., 10 fig., bibliogr., in- dex. Volmne 30, Article 2.—D>iiamics of One- Species Populations of Fishes in Ponds Subjected to Cropping and Additional Stocking. By D. Homer Buck and Charles F. Thoits III. March, 1970. 97 p., 10 fig., bibliogr., index. Voliune 30, Article 3.—Migrational Behavior of Mallards and Black Ducks as Deter- mined from Bajiding. By Frank C. Bell- rose and Robert D. Crompton. September, 1970. 68 p., frontis., 25 fig., bibliogr., index. Volume 30, Article 4.—Fertilization of Es- tablished Trees: A Report of Field Stud- ies. By Dan Necly, E. B. Himelick, and ^^'ebster R. Crowley, Jr. September, 1970. 32 p., frontis., 8 fig., bibliogr., index. Vokmie 30, .Article 5.—A Survey of the Mus- sels (Unionacea) of tlie Illinois River: a Polluted Stream. By William C. Staixett. February, 1971. 137 p., 17 fig., bibliogr., index. BIOLOGICAL NOTES 62.— Nighthghting: Its Use in Capturing Pheasants, Prairie Chickens, Bobwhites, and Cottontails. By Ronald F. Labisky. Octo- ber, 1968. 12 p., 8 fig., bibliogr. 63.—Selected Minerals in Soils, Plants, and Pheasants: An Ecosystem Approach to Un- derstanding Pheasant Distribution in Illinois. By Robert L. Jones, Ronald F. Labisky, and William L. Anderson. December, 1968. 8 p., 1 fig., bibliogr. 64.—The Value of In Vitro Fungicide Tests. By Dan Neely. January, 1969. 8 p., bibliogr. 65.—Trends in Pheasant Abundance in Illi- nois: 1958 to 1968. By Ronald F. Labiskv. May, 1969. 8 p., 4 fig., bibliogr. 66.—Tree and Shrub Hosts of Verticillium albo-atnmi. By E. B. Himelick. July, 1969. 8 p., bibliogr. 67.—Concentrations of Chemical Elements in Phcasiuit Tissues. By William L. Anderson and Peggy L. Stewart. April, 1970. 15 p., bibliogr. 68.-Illinois Birds: Mimidae. By Richard R. Graber, Jean W. Graber, and Etlielyn L. Kirk. September, 1970. 38 p., 32 fig., bibliogr. 69.—The Life History of the Dusky Darter, Percina sclera, in tlie Embarras River, Illi- nois. By Lawrence M. Page and Philip W. Smith. September, 1970. 15 p., U fig., bibliogr. 70.—An Ecological Study of Four Darters of tlie Genus Percina (Percidae) in tlie Kas- kaskia River, Illinois. By David L. Thomas. December, 1970. 18 p., 11 fig., bibliogr. 71.—A Svnopsis of Common and Economic Illinois Ants, with Keys to the Genera ( H.Miienoptera, Fonnicidae ) . By Herbert H. Ross, George L. Rotramel, and Wallace E. LaBerge. Januaiy, 1971. 22 p., 27 fig., bibliogr. 72.—The Lfse of Factor Analjsis in Modeling Natural Comnimiities of Plants and Animals. By Robert \\^ Poole. February, 1971. 14 p., 14 fig., bibliogr. CIRCULAR 46.—Illinois Trees: Their Diseases. By J. Cedric Carter. June, 1964. (Third printing, witli alterations.) 96 p., frontis., 89 fig. 49.—The Dunesland Heritage of Illinois. By Herbert H. Ross (in cooperation witli Illi- nois Department of Conservation). August, 1963. 28 p., frontis., 16 fig., bibliogr. 51.—Illinois Trees: Selection, Planting, and Care. By J. Cedric Carter. August, 1966. 123 p., frontis., 108 fig. 52.—Fertihzing and Watering Trees. By Dan Neely and E. B. Himehck. December, 1968. (Second printing.) 20 p., 9 fig., bibliogr. 53.—Dutch EDn Disease in Illinois. By J. Cedric Carter. October, 1967. 19 p., fron- tis., 17 fig. List of available publications mailed on request No charge is made for publications of the Illinois Natural History Survey. A single copy of most publications will be sent free to anyone requesting it until the supply becomes low. Costly publications, more than one copy of a publication, and pubUcations in short supply are subjects for special correspondence. Such correspondence should identify the wTiter and explain the use to be made of the publication or publications. Address orders and correspondence to the Chief, Illinois Natural History Survey Natural Resources Building, Urbana, Illinois 61801