Pesticide and Xenobiotic Metabolism in Aquatic Organisms 9780841204898, 9780841206335, 0-8412-0489-6

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
Metabolism in vitro......Page 10
Metabolism and bioaccumulation in vivo......Page 12
Translocation and transformation in a freshwater model ecosystem......Page 17
Literature Cited......Page 26
2 Disposition of Polychlorinated Biphenyls in Fish......Page 28
Literature Cited......Page 42
3 Metabolism of Cyclodiene Insecticides by Fish......Page 44
(ii) Elimination......Page 46
(iii) Metabolism......Page 47
(i) Absorption......Page 48
(iii) Metabolism......Page 50
Literature Cited......Page 62
INCORPORATION OF HYDROCARBONS INTO TISSUES AND BODY FLUIDS......Page 64
BIOTRANSFORMATIONS......Page 71
CONCLUSIONS......Page 78
LITERATURE CITED......Page 80
Metabolism of Phthalate Esters by Microorganisms......Page 83
Metabolism of Phthalate Esters by Fishin vitro......Page 85
Other Studies of the Metabolism of Phthalate Esters by Aquatic Species......Page 95
Acknowledgements......Page 98
Literature Cited......Page 99
Material and Methods......Page 101
Results and Discussion......Page 104
Acknowledgement......Page 122
Literature Cited......Page 124
7 Biotransformation of Selected Chemicals by Fish......Page 126
Glucuronide Conjugation......Page 127
Acetylation......Page 129
N-Dealkylation......Page 130
S-Methylation......Page 131
Literature Cited......Page 132
Toxicity of PCP and Other Chlorophenols......Page 135
Biliary Excretion of PCP-glucuronide......Page 137
Renal Excretion of PCP-sulfate......Page 138
Whole View of Major Detoxification Pathways for PCP......Page 140
Effect of Pre-exposure to PCP on PCP-tolerance and Sulfate Conjugation Activity......Page 143
Literature Cited......Page 147
9 The Disposition and Biotransformation of Organochlorine Insecticides in Insecticide-Resistant and -Susceptible Mosquitofish......Page 148
Disposition......Page 149
Biotransformation......Page 153
Discussion......Page 159
Abstract......Page 160
Literature Cited......Page 161
Diflubenzuron......Page 163
Epifenonane......Page 167
Methoprene......Page 171
Literature Cited......Page 176
11 The Fate of Highly Brominated Aromatic Hydrocarbons in Fish......Page 179
Experimental......Page 180
Results and Discussion......Page 181
Literature Cited......Page 183
Materials and Methods......Page 185
Results and Discussion......Page 188
Literature Cited......Page 196
Chemicals and Experimental Chambers......Page 197
Results and Discussion......Page 202
Abstract......Page 217
Literature cited......Page 218
14 Investigation of Xenobiotic Metabolism in Intact Aquatic Animals......Page 219
Laboratory Metabolism Chambers......Page 220
Aquatic Metabolism Protocol......Page 223
p-Nitroanisole Pharmacodynamics in the Sea Urchin......Page 225
Conclusions......Page 229
Literature Cited......Page 232
15 Xenobiotic Transport Mechanisms and Pharmacokinetics in the Dogfish Shark......Page 234
Handling Advantages of the Dogfish Shark......Page 235
Initial Studies with the Model Compound Phenol Red......Page 241
Disposition of Phenol Red......Page 242
Effects of Interrupted Enterohepatic Circulation on Biliary and Urinary Handling of Phenol Red......Page 243
Concentrated Transfer of Phenol Red into Renal and Hepatic Compartments......Page 245
Saturation of Excretory Processes......Page 246
Inhibition of Phenol Red Execretion by Probenecid......Page 247
Plasma Binding of Phenol Red......Page 248
Distribution and Pharmacokinetics of Water Soluble Compounds......Page 251
Distribution and Pharmacokinetics of Lipophilic Pollutants......Page 252
Distribution and Pharmacokinetics of Metallic Compounds......Page 256
Literature Cited......Page 257
16 Disposition of Toxic Substances in Mussels (Mytiluscaliforianus): Preliminary Metabolic and Histologic Studies......Page 260
Methods and Materials......Page 261
Results......Page 263
Literature Cited......Page 277
Cytochrome P-450 mediated monooxygenase......Page 279
Active metabolites and carcinogenicity......Page 286
Conclusions......Page 290
Literature Cited......Page 291
18 Microsomal Mixed-Function Oxidation in Untreated and Polycyclic Aromatic Hydrocarbon-Treated Marine Fish......Page 297
Materials and Methods......Page 298
Results and Discussion......Page 301
Literature Cited......Page 315
19 Induction of Hepatic Microsomal Enzymes in Rainbow Trout......Page 319
Materials and Methods......Page 320
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)......Page 321
Results and Discussion......Page 322
Literature Cited......Page 333
20 Further Studies on the Effect of Petroleum Hydrocarbons on Mixed-Function Oxidases in Marine Organisms......Page 338
Attempts to Induce AHH in Invertebrates under Extreme Oil Exposure Conditions......Page 339
Induction of Bivalve MFO......Page 341
Induction of Fish AHH with Pure Hydrocarbons......Page 342
Induction of AHH in Fish with Oil and Oil Spill Dispersant Mixtures......Page 343
Conclusions......Page 344
Literature Cited......Page 345
Introduction......Page 347
Materials and Methods......Page 348
Results......Page 351
Discussion......Page 361
Acknowledgement......Page 366
Literature Cited......Page 367
22 Degradation of Pesticides by Algae and Aquatic Microorganisms......Page 369
Experimental......Page 370
Results......Page 371
Discussion......Page 382
Literature Cited.......Page 384
23 Dietary Case in Levels and Aflatoxin B1 Metabolism in Rainbow Trout (Salmo gairdneri)......Page 386
Experimental Procedures......Page 387
Results and Discussion......Page 389
Literature Cited......Page 395
24 Alterations in Rainbow Trout Liver Function and Body Fluids Following Treatment with Carbon Tetrachloride or Monochlorobenzene......Page 398
Materials and Methods......Page 399
Results......Page 400
Discussion......Page 405
Literature Cited......Page 409
A......Page 411
Β......Page 412
C......Page 413
D......Page 415
E......Page 416
G......Page 417
H......Page 418
I......Page 419
L......Page 420
M......Page 421
Ν......Page 423
P......Page 424
R......Page 426
S......Page 427
Τ......Page 428
W......Page 429
X......Page 430

Citation preview

Pesticide and Xenobiotic Metabolism in Aquatic Organisms

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Pesticide and Xenobiotic Metabolism in Aquatic Organisms M . A . Q . K h a n , EDITOR University of Illinois John J . Lech, EDITOR Medical College of Wisconsin Julius J . Menn, EDITOR Stauffer Chemical Company

Based on a symposium sponsored by the Division of Pesticide Chemistry at the 176th Meeting of the American Chemical Society, Miami Beach, Florida, September 11-17, 1978.

ACS

SYMPOSIUM

SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1979 In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

99

Library of Congress CIP Data

Pesticide and xenobiotic metabolism in aquatic organisms. (ACS symposium series; 9 Includes bibliographies and 1. Pesticides and wildlife—Congresses. 2. Xenobiotic metabolism—Congresses. 3. Aquatic animals, Effect of water pollution on—Congresses. I. Khan, Mohammed Abdul Quddus, 1935- . II. Lech, John J. III. Menn, Julius J. IV. American Chemical Society. Division of Pesticide Chemistry. V. Series: American Chemical Society. ACS symposium series; 99. [DNLM: 1. Pesticides—Metabolism—Congresses. 2. Biotransformation—Congresses. 3. Water pollution, Chemical—Congresses. 4. Marine biology—Congresses. 5. Water—Analysis—Congresses. WA240 P4735 1979] QH545.P4P478 574.2'4 79-4598 ISBN 0-8412-0489-6 ASCMC 8 99 1-436 1979

Copyright © 1979 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ACS Symposium Series Robert F. Gould, Editor

Advisory Board Kenneth B. Bischoff

James P. Lodge

Donald G. Crosby

John L. Margrave

Robert E. Feeney

Leon Petrakis

Jeremiah P. Freeman

F. Sherwood Rowland

E. Desmond Goddard

Alan C. Sartorelli

Jack Halpern

Raymond B. Seymour

Robert A. Hofstader

Aaron Wold

James D. Idol, Jr.

Gunter Zweig

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide a medium for publishin format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PREFACE

TPVuring the 1960's considerable research effort was directed towards elucidating the metabolism and fate of foreign compounds in mammals, notably therapeutic agents and pesticides. However, little attention was paid to the processes involved in the biotransformation and elimination of xenobiotic chemicals in aquatic species. During this period some studies in the literature indicate able to metabolize foreign compounds. Despite this finding, the consensus was thatfishdo not have nor do they need the ability to biotransform xenobiotic substances. Several pivotal studies in the late Sixties and early Seventies indicated conclusively that fish have the capacity to both oxidize and conjugate xenobiotic substances. These observations, along with a growth in concern for the aquatic environment from both the ecological and human health points of view, led to interest in the metabolism of xenobiotic chemicals in aquatic species. The expansion of research on biotransformation and disposition of xenobiotic chemicals in fish, along with studies of the effects of these chemicals on aquatic organisms, has led to the rapid development of aquatic toxicology research in the pastfiveyears. Monitoring for the presence of xenobiotic chemicals in aquatic organisms may pertain to both ecological and human health interests. Since many xenobiotic chemicals ultimately find their way into aquatic species, knowledge of the location and concentration of these chemicals is germane to both protection of these species and assurance of a safe food supply for humans. In addition, monitoring the state of chemical contamination of the aquatic environment itself is of great importance. Since it is now known that fish and other aquatic organisms can carry out a variety of biotransformation reactions, any surveillance program, regardless of its ultimate purpose, is incomplete unless procedures are designed to take into account the chemical in question and its metabolic products. This is important for two reasons :first,chemical residues may be present in the form of biotransformation products and the identification of these xenobiotic substances in a given aquatic species will not be valid unless the nature and quantity of metabolic product is known; secondly, because it is now known that several biotransformation processes lead to the

ix In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

creation of chemicals which may be more toxic than the parent com­ pounds, it is necessary to know the nature and number of metabolites which are formed when this situation arises. The formation of polar metabolites from nonpolar materials may actually facilitate monitoring programs—in many cases the polar chemi­ cals are highly concentrated in certain bodyfluidssuch as bile and urine. On the other hand, materials such as certain cyclodienes and polychlorinated biphenyls, which are very lipid soluble and resistant to metabolism, may accumulate and these chemicals may persist in the environment and may be transferred via the food chain to man. There is also interest in these biotransformation processes in lower organisms since the simplicity of these systems may lead to a better understanding of the phylogenetic development of xenobiotic metabolism. If in the future unforesee determining the precise toxicity of all the chemicals which may enter into the aquatic environment, as opposed to knowing which chemicals are formed and where and how long they persist, the latter may be more pertinent. While the former is important, a knowledge of precise toxicity of chemicals to many organisms in all situations may not be feasible, either economically or within a specified time frame. However, knowl­ edge of the toxicokinetic properties of chemicals and their biotransforma­ tion pathways in aquatic organisms will lead to surveillance programs reflecting the state of contamination of the aquatic environment. It is possible that this knowledge will aid in averting an ecological or human health crisis in the future. This international symposium presents research from laboratories concerned with the metabolism and disposition of pesticides and other xenobiotic substances in aquatic organisms. The studies were not re­ stricted to pesticides since current interest concerns a wide variety of chemicals and their disposition in aquatic organisms both in vivo and in vitro. This book is in two sections, one dealing with the in vivo metabo­ lism of chemicals in aquatic organisms and the other describing primarily mechanisms of metabolism and disposition of xenobiotic chemicals in these species. Some of the papers overlap these two sections but were placed in one or the other as a matter of convenience. The authors wish to acknowledge the cooperation and efforts of the contributors both in oral presentations at the 176th ACS Meeting in Miami, Florida in Sep­ tember 1978, and in the preparation of the manuscripts comprising this book. The authors wish to thank Ms. Cheryl Beyer, Ginger Marlow, and Pat Kelley for their typing and clerical assistance in planning for the symposium and book. Activities of the editors related to the symposium

χ In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

and book were supported by NIEHS Grants #ES 01080 and ES 01985 (J.J.L.)> EPA Grant #R803971-010 (J.J.L.)> NIEHS Grant #01479 (M.A.Q.K.), and by the Stauffer Chemical Company (J.J.M.). University of Illinois Chicago, IL 60680

M. A.

Medical College of Wisconsin Milwaukee, WI 53233

Q . KHAN

J. J.

Stauffer Chemical Company Mountain View, CA 94042

J. J.

LECH

MENN

January 16, 1979

xi In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1 Metabolism of Organophosphorus Insecticides in Aquatic Organisms, with Special Emphasis on Fenitrothion JUNSHI MIYAMOTO, YOSHIYUKI ΤΑΚΙΜΟΤΟ, and KAZUMASA MIHARA Research Department, Pesticides Division, Institute for Biological Science, Sumitomo Chemical Co., Ltd., 4-2-1 Takatsukasa, Takarazuka, Hyogo 665, Japan

The organophosphoru insecticides, and a certai the aquatic environment resulting either from the actual use on paddy fields or from unavoidable transmittance to waterways. However, possibly because of its relatively shorter persistence, the translocation and transformation of organophosphorus compounds in the aquatic environment has not been extensively investigated as compared with more persistent organοchlorine compounds. Fenitrothion, O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate, is widely used for the control of paddy field insects and forest protection in several countries, and since it is rather highly toxic to some aquatic organisms (LC 50 after 48hr exposure, 1.28 ppm for rainbow trout, 2.72 ppm for bluegill, 4.4 ppm for carp, LC 50 after 3 hr exposure, 0.0092 ppm for daphnia and no-effect dosage after 4 week exposure, 0.02 ppm for carp) (1, 2), the knowledge on degradation and metabolism of the compound in the aquatic environment is important for assessing short-term and long-term impacts on the non-target aquatic organisms. In this article metabolism and bioaccumulation of fenitro­ thion in several aquatic species are dealt with under laboratory conditions. Metabolism in vitro To acquire information on the intrinsic metabolic activity of aquatic organisms, liver of carp (Cyprinus carpio Linnaeus), rainbow trout (Salmo gairdneri) and freshwater snail (Cipangopaludina japonica Martens) was dissected out, homogenized in 0.1M phosphate buffer, pH 7.5, and centrifuged at 105,000 g for 60 min to obtain the microsome-equivalent (described as the microsomal fraction hereafter) fraction. The protein content of microsomal and sub microsomal (supernatant fractions by Lowry's method, micro­ somal P-450 content (3), activity of aniline hydroxylase (4) and aminopyrine N-demethylase (_5) were determined. Table I shows the results which reveal that the drug0-8412-0489-6/79/47-099-003$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Rat Rainbow trout Carp Snail

37.1 16.5 3.7 2.3 1.8

5.5 99%) was e x a m i n e d b y u s i n g t h e m i c r o s o m a l f r a c t i o n a n d determining o x i d a t i v e d e s u l f u r a t i o n t of e n i t r o o x o n , 0,0-dimethyl 0-(3-methyl-4-nitrophenyl) p h o s p h a t e , o x i d a t i o n o f t h e m-mehtyl g r o u p o f f e n i t r o o x o n i n t h e p r e s e n c e o f NADPH p l u s EDTA, a n d h y d r o l y t i c cleavage o f f e n i t r o o x o n t o 3-methyl-4-nitrophenol, with c a l c i u m i o n a s a c o f a c t o r (6_). A l s o i n t h e p r e s e n c e o f r e d u c e d g l u t a t h i o n e , O - d e m e t h y l a t i o n o f f e n i t r o t h i o n a n d f e n i t r o o x o n was t e s t e d i n the s u p e r n a t a n t f r a c t i o n . D u r i n g i n c u b a t i o n f o r 5-60 min a t 24°C ( f o r a q u a t i c a n i m a l s ) ( 7 ) o r 37°C ( f o r mammals), an a l i q u o t o f t h e i n c u b a t i o n m i x t u r e was s a m p l e d p e r i o d i c a l l y a n d analyzed f o r substrate disappearanc formed by two-dimensiona d e t e r m i n a t i o n o f r a d i o a c t i v i t y o f the s e p a r a t e d s p o t s . T a b l e I I summarizes the r e s u l t s t o g e t h e r w i t h the d e t a i l e d e x p e r i m e n t a l c o n d i t i o n s . As i s e v i d e n t , m e t a b o l i c a c t i v i t i e s w e r e d e t e c t a b l e i n t h e s e 3 a q u a t i c s p e c i e s , b u t t h e r a t e was f a r l o w e r as compared w i t h mammalian h e p a t i c enzume p r e p a r a t i o n s , a n d t h e o x i d a t i v e a c t i v i t i e s i n s n a i l were p a r t i c u l a r l y low a l t h o u g h t h e p o s s i b i l i t y was n o t r u l e d o u t o f t h e p r e s e n c e o f i n h i b i t o r s o f mixed-function oxidases i n the f r a c t i o n s . The O - d e m e t h y l a t i o n r e a c t i o n p r o c e e d s e x t r e m e l y s l o w l y i n t h e enzyme p r e p a r a t i o n o f a q u a t i c a n i m a l s , a t l e s s t h a n one h u n d r e d t h t h a t o f mammals. T h u s , a l t h o u g h o r g a n o p h o s p h o r u s compounds l i k e f e n i t r o t h i o n may b e m e t a b o l i z e d i n a q u a t i c o r g a n i s m s t h r o u g h o x i d a t i v e d e s u l f u ­ r a t i o n , s i d e chain o x i d a t i o n , h y d r o l y t i c cleavage of P - O - a r r y l l i n k a g e , as w e l l a s O - d e m e t h y l a t i o n , t h e t u r n - o v e r r a t e a p p a r e n t l y i s much l o w e r t h a n i n mammals. M e t a b o l i s m and b i o a c c u m u l a t i o n

i n vivo

In order t o obtain metabolic p r o f i l e s o f f e n i t r o t h i o n i n f i s h i n v i v o , 2 y e a r l i n g r a i n b o w t r o u t w e i g h i n g on an a v e r a g e 26.6 g w e r e m a i n t a i n e d i n 10 l i t e r s o f a e r a t e d w a t e r a t 18±0.5°C c o n ­ t a i n i n g 0.1 ppm o f r a d i o a c t i v e f e n i t r o t h i o n l a b e l e d a t t h e m-methyl p o s i t i o n (3.16 mCi/mmole, >99%) ( 8) . A t 6 and 2 4 h r , a n d a l s o a t 24 h r a f t e r t r a n s f e r t o f r e s h w a t e r o f f i s h exposed f o r t h e p r e c e d i n g 24 h r t o t h e fen i t r o t h i o n - c o n t a i n i n g w a t e r (24+24 i n F i g u r e 1 ) , t h e f i s h were s a m p l e d and s u b j e c t e d t o a u t o r a d i o g r a p h y . The p a t t e r n o f d i s t r i b u t i o n o f t h e a b s o r b e d r a d i o a c t i v i t y i s shown i n F i g u r e 1. A f t e r 6 h r s o f e x p o s u r e t h e c o n c e n t r a t i o n o f r a d i o a c t i v i t y was h i g h e s t i n g a l l b l a d d e r and i n t e s t i n e , and t h e r a d i o a c t i v i t y was d i s t r i b u t e d i n most t i s s u e s e x c e p t b r a i n a n d h e a r t a f t e r 24 h r . Twenty-four hrs a f t e r t r a n s f e r t o f r e s h water ( 2 4 + 2 4 ) , most o f t h e r a d i o a c t i v i t y i n t i s s u e s h a d d i s a p p e a r e d

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8.

KOBAYAsm

Pentachlorophenol

143

the assay o f s u l f a t e c o n j u g a t i o n a c t i v i t y f o r phenol. F i g u r e 3 (13) shows a p p r o x i m a t e l y 26 % a u g m e n t a t i o n i n t h e sulfate conjugation a c t i v i t y of l i v e r soluble fractions of the f i s h e x p o s e d t o PCP f o r 4 d a y s , when compared w i t h t h e c o n t r o l group. The p r e - e x p o s u r e t o PCP i n c r e a s e d b o t h t h e P C P - t o l e r a n c e and t h e s u l f a t e c o n j u g a t i o n a c t i v i t y i n g o l d f i s h . This suggests t h a t f i s h h a v e some a b i l i t y t o i n c r e a s e t h e i r P C P - t o l e r a n c e when the f i s h had been exposed t o s u b l e t h a l PCP-media, and a l s o t h a t the s u l f a t e c o n j u g a t i o n a c t i v i t y i s an important f a c t o r d e t e r ­ mining the PCP-tolerance o f f i s h .

Literature Cited 1. Cirelli, D.P., In "Pentachlorophenol", pp. 13-18(K.R. Rao, Ed.), Plenum Press New York Ν Y. 1978 2. Kobayashi, Κ., In Ed.), Plenum Press, , , 3. Kobayashi, Κ., H. Akitake, and K. Manabe, Bull. Japan. Soc. Sci. Fish., submitted. 4. Kobayashi, Κ., and H. Akitake, Bull. Japan. Soc. Sci. Fish., (1975), 41 (1), 87-92 5. Kobayashi, Κ., and H. Akitake, Bull. Japan. Soc. Sci. Fish., (1975), 41 (1), 93-99. 6. Kobayashi, K., S. Kimura, and E. Shimizu, Bull. Japan. Soc. Sci. Fish., (1977), 43 (5), 601-607. 7. Glickman, A.H., C.N. Statham, A. Wu, and J.J. Lech, Toxicol. Appl. Pharmacol., (1977), 41 (3), 649-658. 8. Akitake, H., and K. Kobayashi, Bull. Japan. Soc. Sci. Fish., (1975), 41 (3), 321-327. 9. Kobayashi, Κ., H. Akitake, and T. Tomiyama, Bull. Japan. Soc. Sci. Fish., (1970), 36 (1), 103-108. 10. Kobayashi, Κ., and N. Nakamura, Bull. Japan. Soc. Sci. Fish., submitted. 11. Tashiro, S., T. Sasamoto, T. Aikawa, S. Tokunaga, E. Taniguchi and M. Eto, J. Agr. Chem. Soc. Japan, (1970), 44 (3), 124-129. 12. Kobayashi, Κ., and Ν. Nakamura, Bull. Japan. Soc. Sci. Fish., submitted. 13. Kobayashi, Κ., and K. Higuma, Bull. Japan. Soc. Sci. Fish., submitted. 14. Kobayashi, K., S. Kimura, and H. Akitake, Bull. Japan. Soc. Sci. Fish., (1976), 42 (2), 171-177. 15. Kimura, S., and K. Kobayashi, Bull. Japan. Soc. Sci. Fish., submitted. RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9 The Disposition and Biotransformation of Organochlorine Insecticides in Insecticide-Resistant and -Susceptible Mosquitofish JAMES D. YARBROUGH and JANICE E. CHAMBERS Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762 S e l e c t i v e pressure r e s u l t e d i n the development o f a r e s i s t a n t (R) population of mosquitofish (Gambusia a f f i n i s ) which demonstrates up to a 500 f o l d greater tolerance of i n s e c t i c i d e s than does a corresponding s u s c e p t i b l e (S) population (Table I). This r e s i s t a n c e was caused by a g r i c u l t u r a l runoff o f p e s t i c i d e s used on cotton and soybean f i e l d s i n t o drainage ditches s u b j e c t i n g the f i s h to chronic exposures to i n s e c t i c i d e s . The r e s i s t a n c e i s g e n e t i c a l l y based, and i s not merely an expression of environmentally-induced tolerances. The major s e l e c t i v e pressure was organochlorine i n s e c t i c i d e s and the highest l e v e l s o f r e s i s t a n c e are to the c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e s (40 - 500 f o l d d i f f e r e n c e between the 48 hr LC50 values o f S and R p o p u l a t i o n s ) . The S population i s not abnormally s e n s i t i v e to organochlorine i n s e c t i c i d e s , as i n d i c a t e d by a l d r i n acute t o x i c i t i e s in other f i s h species {]_). Table I - Comparative 48-hr LC50 Values ( y g / l ) f o r I n s e c t i c i d e S u s c e p t i b l e (S) and R e s i s t a n t (R) Mosquitofisha

Insecticide

S

R

Fold

difference

DDT

18.9

96.2

5.0

Aldrin

36.2

2735.0

93.5

Dieldrin

8.0

433.6

54.0

Endrin

0.6

314.1

499.0

458.7

388.5

Toxaphene a

11.6 From C u l l e y and Ferguson

(2).

0-8412-0489-6/70/47-099-145$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

Work in our laboratory on various parameters i n R and S f i s h has i n v e s t i g a t e d the f a c t o r ( s ) responsible f o r r e s i s t a n c e . The r e s u l t s have i n d i c a t e d t h a t r e s i s t a n c e is m u l t i f a c t o r i a l , i n v o l v i n g a b a r r i e r to i n s e c t i c i d e p e n e t r a t i o n , i n s e c t i c i d e s t o r a g e , i n s e c t i c i d e metabolism, and an apparent " i n s e n s i t i v i t y " at the t a r g e t s i t e to the t o x i c e f f e c t s of the i n s e c t i c i d e . The present report concentrates on two of these f a c t o r s : insecticide d i s p o s i t i o n and metabolism. Disposition In g e n e r a l , aquatic vertebrates absorb i n s e c t i c i d e s through the g i l l s (3). As such, the i n s e c t i c i d e enters the e f f e r e n t limb of c i r c u l a t i o n and may p o t e n t i a l l y enter the brain d i r e c t l y as one of the f i r s t major organs r e c e i v i n g blood from the g i l l s . The s i t e of a c t i o n o f some organochlorine i n s e c t i c i d e s i s b e l i e v e d to be the centra i n s e c t i c i d e penetration organ. A comparison of the r a t i o of i n s e c t i c i d e accumulated in the l i v e r to that i n the brain (L/B) of R and S animals f o l l o w i n g in vivo exposure would be an i n d i c a t i o n of the e f f e c t i v e n e s s o f the brain b a r r i e r (Table II). In every c a s e , there i s c o n s i d e r ably more i n s e c t i c i d e in the l i v e r s than in the brains of resistant f i s h . In only one case is t h i s pattern seen in the S f i s h and in the case of endrin there i s 1.7 times as much in the brains as in the l i v e r s . Table II

- Ratios of I n s e c t i c i d e Accumulated in L i v e r s to That Brains in S u s c e p t i b l e (S) and Resistant (R) Fish Exposed to the ' ^ c - l a b e l l e d I n s e c t i c i d e f o r 6 hr

Exposure Concentrât!on,yg/l

in

L/B R

S

Reference

DDT

20

5.6

1.1

4

Aldrin

80

3.1

2.1

5

Dieldrin

30

2.9

1.2

5

Endrin

10

1.8

0.6

6

This b a r r i e r can be f u r t h e r i l l u s t r a t e d by comparing t i s s u e i n s e c t i c i d e r a t i o s between the S and R p o p u l a t i o n s . Radioactivity accumulated in major organs f o l l o w i n g exposure to 10 y g / l 14cendrin i s greater i n S f i s h than i n R f i s h f o r a l l organs studied except kidney (Table III). S i m i l a r l y , S f i s h accumulate more a l d r i n , d i e l d r i n and DDT i n t h e i r brains than do R f i s h .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

YARBROUGH

Organochlorine Insecticides

A N D CHAMBERS

147

With the exception o f DDT, there was a t l e a s t as much o r more r a d i o a c t i v i t y accumulated i n l i v e r s from S f i s h than i n l i v e r s from R f i s h (Table I V ) . Table III

- R a d i o a c t i v i t y Accumulated i n Tissues o f S u s c e p t i b l e (S) and Resistant (R) F i s h Exposed to 10 y g / l 1*Cendrin f o r 6 hr ng endrin equivalents/mg wet weight

Tissue

S/R

R

S

3

Brain

192.2

+ 19.2

6.9 + 0.5

27.8**

Liver

119.9

+ 23.6

12.5 + 0.6

9.6**

7.1

20.8 + 0.7

6.8**

+ 7.3

12.8 + 0.3

2.4**

+ 13.0

24.1

Gill Gall

78. Bladder

140.4+

Intestine

30.1

Spleen

288.1

Kidney

20.2 + 0.5

+ 0.8

12.0**

66.8 + 1.8

0.3**

a

Values are expressed as mean + SEM, 4-6 r e p l i c a t i o n s , 3 f i s h per r e p l i c a t i o n . From Hunsinger (6_). * * S i g n i f i c a n t d i f f e r e n c e s between the means o f the two populations (£_ < 0.01) as determined by the t - t e s t . Table IV - Ratios ( S u s c e p t i b l e / R e s i s t a n t ; S/R) o f I n s e c t i c i d e Accumulated i n Brains and Livers Following 6 hr o f R e l a b e l l e d I n s e c t i c i d e Exposure Exposure Concentration, y g / l

S/R Brain

Li ver

Reference

DDT

20

2.2

0.5

4

Aldrin

80

5.2

3.6

5

Dieldrin

30

2.1

0.9

5

American Chemical Society Library 1155 16th St. N. % Washington, D. C. 20035 In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

148

From the data presented, there i s o b v i o u s l y a more e f f e c t i v e b a r r i e r to i n s e c t i c i d e penetration i n R f i s h than i n S f i s h . F u r t h e r , t h i s b a r r i e r apparently operates over a wide range o f exposure l e v e l s . For example, when the t i s s u e concentration i n brains from R f i s h exposed to 10 y g / l are compared to t i s s u e concentrations i n brains o f R f i s h exposed to 314 y g / l e n d r i n , there i s a 10-fold increase i n endrin concentration i n brain t i s s u e (from 6.91 to 73.8 ng endrin equivalents/mg wet weight o f t i s s u e ) , although t h i s represents a 3 0 - f o l d increase i n the i n s e c t i c i d e exposure l e v e l . However, i n S f i s h , the l e v e l o f i n s e c t i c i d e i n brain t i s s u e increased 355-fold (from 0.5 to 192.2 ng endrin equivalents/mg wet weight o f t i s s u e ) when the i n s e c t i c i d e exposure l e v e l was r a i s e d 1 7 - f o l d (from 0.6 to 10 y g / l ) (submitted f o r p u b l i c a t i o n ) . It should be pointed out that the 314 y g / l exposure l e v e l s i n the R f i s h represents the 48-hr LC50 value while th 0 6 y g / LC50 value f o r S f i s h . Therefor sons o f the S and R populations at both e q u i t o x i c and equal exposure l e v e l s o f e n d r i n . In most s t u d i e s i n s e c t i c i d e uptake i s measured a t a s p e c i f i c time o f exposure and does not take i n t o account the d i f f e r e n c e s in t o l e r a n c e to the i n s e c t i c i d e w i t h i n a p o p u l a t i o n . In such s t u d i e s , the more t o l e r a n t i n d i v i d u a l s o f a population are a c t u a l l y s e l e c t e d . I f comparisons are made w i t h i n and between the R and S populations based on t o x i c e f f e c t s as expressed by the appearance o f symptoms o f p o i s o n i n g , a c l e a r e r understanding of the r e l a t i o n s h i p between u p t a k e / d i s p o s i t i o n and t o x i c i t y i s possible. The i n s e c t i c i d e concentration i n the brain expressed as a r a t i o o f symptomatic (s) to asymptomatic (a) would give a measure o f the e f f e c t i v e n e s s o f the membrane b a r r i e r and some i n d i c a t i o n o f other f a c t o r s which might be i m p l i c a t e d i n t o x i c i t y . A symptomatic/asymptomatic r a t i o greater than 1 would be expected i f i n s e c t i c i d e concentration i n the t a r g e t organ was the only determining f a c t o r i n t o x i c i t y . L i k e w i s e , i f varying t a r g e t s i t e s e n s i t i v i t y i s a f a c t o r , a r a t i o o f 1 o r l e s s would be expected. Such comparison o f endrin accumulation within the r e s i s t a n t population (R /Ra) demonstrated a r a t i o s i g n i f i c a n t l y greater than 1 f o r the various b r a i n segments (Table V) (7_)· This i s i n d i c a t i v e o f more e f f e c t i v e membrane b a r r i e r s t o i n s e c t i c i d e penetration i n the more t o l e r a n t R f i s h , which leads to varying degrees o f i n s e c t i c i d e t o l e r a n c e w i t h i n the R p o p u l a t i o n . When a s i m i l a r comparison i s made w i t h i n the S population between symptomatic and asymptomatic f i s h ( S / S ) the r a t i o s are l e s s than 1 (Table V ) . Since these comparisons are between l e s s t o l e r a n t i n d i v i d u a l s to more t o l e r a n t i n d i v i d u a l s w i t h i n the S p o p u l a t i o n , the S / S a r a t i o s i n d i c a t e a range i n t a r g e t s i t e sensitivity. Such a range shows a b a s i c f l e x i b i l i t y w i t h i n the unselected population upon which s e l e c t i v e pressures could have acted to produce the R p o p u l a t i o n . s

s

a

s

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

YARBROUGH AND CHAMBERS

Organochlorine

149

Insecticides

Table V - Ratios of R a d i o a c t i v i t y Accumulated in Brains of S u s c e p t i b l e (S) and Resistant (R) F i s h , Some of Which Were E x h i b i t i n g Symptoms (s) and Others not E x h i b i t i n g Symptoms (a) of I n s e c t i c i d e P o i s o n i n g . Treatment Consisted of a 6 hr Exposure to 1 4 c - e n d r i n . a

10 y g / l

1500 yg/1

Endrin

Endrin

Ss/Sa

Ss/Rab

Sa/Rab

Ss/Rsb

Rs/Rab

Ss/Rab

Forebrain

0.6

3.1**

5.1**

0.5**

3.7**

1.7*

Midbrain

0.7

2.9**

4.4**

0.5**

4.8**

2.2*

Hindbrain

0.6

a

From Scales {7). b A l l s t a t i s t i c a l comparisons are between components of each ratio. S i g n i f i c a n t d i f f e r e n c e s were determined by the J > t e s t , Ρ < 0.05 (*), Ρ < 0.01 (**). When comparisons are made between p o p u l a t i o n s , both the e f f e c t i v e n e s s of the membrane b a r r i e r in R f i s h and the s e n s i ­ t i v i t y of the t a r g e t s i t e can be demonstrated (Table V ) . When endrin S / R r a t i o s are compared, the r a t i o i s l e s s than 1; t h i s suggests that more i n s e c t i c i d e i s required to e l i c i t symptoms in the R than in the S f i s h . s

s

This implicated target s i t e i n s e n s i t i v i t y i s more e f f e c t i v e ­ l y demonstrated when actual amounts of endrin present in brain t i s s u e of Ss f i s h are compared to R f i s h (7). In S f i s h the endrin concentration in the f o r e b r a i n i s 24 ng endrin equiv­ alents/mg protein while in the same brain f r a c t i o n of R f i s h there was 1150 ng endrin equivalents/mg p r o t e i n . F u r t h e r , when the amount of endrin in various brain f r a c t i o n s from R f i s h exposed to 1500 y g / l ^ C - e n d r i n i s monitored with time, up to 4560 ng endrin equivalents/mg protein appears in the brain f r a c t i o n s of R f i s h a f t e r 24 hr endrin exposure (Table V I ) . a

s

a

a

This varying s e n s i t i v i t y of the t a r g e t s i t e might e x p l a i n why uptake data does not seem to r e l a t e d i r e c t l y to t o x i c i t y (LC50 v a l u e s ) . From our data i t i s p o s s i b l e to suggest that more t o l e r a n t i n d i v i d u a l s within the R population would probably possess a high i n s e n s i t i v i t y to i n s e c t i c i d e s at the target s i t e and an e f f e c t i v e b a r r i e r to i n s e c t i c i d e p e n e t r a t i o n . The l e s s t o l e r a n t might possess only one of these f a c t o r s , or varying degrees of f u n c t i o n a l e f f e c t i v e n e s s of one or both f a c t o r s .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC

ORGANISMS

I n d i v i d u a l s in the S population would probably not contain both f a c t o r s and the f a c t o r ( s ) , i f p r e s e n t , would not be as f u n c t i o n a l l y e f f e c t i v e as i n i n d i v i d u a l s in the R p o p u l a t i o n . Table VI - R a d i o a c t i v i t y in Brain F r a c t i o n s of Resistant F i s h Exposed to 1500 y g / l 14C-endrin not E x h i b i t i n g Symptoms of P o i s o n i n g 3

Brain F r a c t i o n ^ Exposure Time

Forebrain

Midbrain

Hindbrain

3

970 ± n o

640 +

40

950 +

6

1150 ± 140

940 +

80

1100 ± 130

9

128

90

12

2370 ± 370

1910 + 170

2240 ± 140

24

4220 ± 230

4350 + 210

4560 ± 270

a

From Scales (7_). b Each value represents a mean of 5 treatments of 3 f i s h each expressed as ng endrin equivalents/mg p r o t e i n ± SEM. Biotransformation The biotransformation systems involved in i n s e c t i c i d e metabolism have been studied in the R and S populations to determine any d i f f e r e n c e s which might be p o t e n t i a l c o n t r i b u t o r y f a c t o r s to or r e s u l t s of i n s e c t i c i d e r e s i s t a n c e . In a d d i t i o n , the p o s s i b i l i t y of mixed-function oxidase induction has been investigated. S p e c i f i c a l l y , the studies have encompassed a seasonal study of microsomal mixed-function oxidase (mfo) components, and studies of a l d r i n , d i e l d r i n and DDT metabolism. Seasonal Study of Mixed Function O x i d a s e s . — A seasonal study of hepatic microsomal mfo components has been conducted i n female R and S f i s h (submitted f o r p u b l i c a t i o n ) . Components studied were cytochromes P-450 and Jb5, NADPH-cytochrome c reductase, NADPH-dichlorophenolindophenol r e d u c t a s e , NADHcytochrome c^ reductase and NADH-cytochrome J55 reductase. All were monitored at 30°C by standard spectrophotometry methods f o l l o w i n g o p t i m i z a t i o n procedures (8, 9 1J0, 21, 12). Microsomal and t o t a l hepatic p r o t e i n (]3j and l i v e r weight to body weight r a t i o s were a l s o monitored. 9

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

YARBROUGH AND CHAMBERS

Organochlorine

Insecticides

151

The r e s u l t s i n d i c a t e d that microsomal mfo a c t i v i t i e s followed a d e f i n i t e seasonal p a t t e r n , with highest a c t i v i t i e s and l e v e l s o c c u r r i n g in the c o l d weather months. A l l parameters measured, except protein c o n c e n t r a t i o n , followed the same t r e n d s . Microsomal p r o t e i n concentration was r e l a t i v e l y constant throughout the study. Cytochrome P-450 and NADPH-cytochrome c reductase are presented as r e p r e s e n t a t i v e of the parameters i n v e s t i g a t e d ( F i g s . 1 and 2 ) . Both the seasonal patterns and the o v e r a l l range of a c t i v i t i e s were s i m i l a r in both p o p u l a t i o n s . The c y c l i c nature of the parameters i n v e s t i g a t e d may be the r e s u l t of the r e l a t i v e magnitude of microsomal hydroxylations during the year in r e l a t i o n s h i p to other microsomal processes such as b i o synthesis. Although the ranges of enzyme s p e c i f i c a c t i v i t i e s and cytochrome l e v e l s were about the same in both p o p u l a t i o n s , the c o n s i s t e n t l y greater r e l a t i v e l i v e r s i z e in the R than the S f i s h suggests a greater p o t e n t i a l f o r x e n o b i o t i c o x i d a t i o n ( F i g 3) Lower r e l a t i v e l i v e r s i z r e f l e c t s the greater proportio There has a l s o been a report of seasonal trends in mfo a c t i v i t i e s in the f e r a l roach (Leuciscus r u t i l u s ) with highest a c t i v i t i e s in the summer and of induction of mfo a c t i v i t y by environmental contaminants (14). A l d r i n and D i e l d r i n Metabolism.— The j_n vivo metabolism of the c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e s , a l d r i n and d i e l d r i n , has been measured. Fish were exposed to l ^ C - l a b e l l e d a l d r i n or d i e l d r i n f o r 6 hours. The metabolism of each compound was monitored by t h i n l a y e r chromatography of hexane and chloroformmethanol e x t r a c t s of l i v e r homogenates, followed by l i q u i d s c i n t i l l a t i o n counting of the spots ( 5 , 1 5 , 1 6 ) . When f i s h of both populations were exposed to 80 y g / l "I^Ca l d r i n , d i e l d r i n was the only detectable metabolite in the organic e x t r a c t s of the l i v e r . The percentage of r a d i o a c t i v i t y in the w a t e r - s o l u b l e f r a c t i o n in both populations was s m a l l . Although previous work had i n d i c a t e d a greater production of w a t e r - s o l u b l e m e t a b o l i t e ( s ) in the R population (1_5), more recent work i n d i c a t e d that the r e l a t i v e proportion of r a d i o a c t i v i t y in the w a t e r - s o l u b l e f r a c t i o n was s i m i l a r in both populations (Table VII). The r e l a t i v e conversion of a l d r i n to d i e l d r i n a l s o v a r i e d with the season, with the greatest conversion o c c u r r i n g in the winter (Table V I I I ) . This seasonal phenomenon c o r r e l a t e s with the above mentioned seasonal data i n d i c a t i n g greater mixedf u n c t i o n oxidase component a c t i v i t y in winter in both populations ( F i g . 1 and 2 ) .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

Figure

1.

ORGANISMS

Cytochrome P-450 contents in liver microsomes from insecticideresistant and -susceptible mosquitofish

Figure 2. Specific activity of ΝADPH-Cytochrome c reductase in liver micro­ somes from insecticide-resistant and -susceptible mosquitofish. A unit (U) of enzyme activity is defined as 1 μτηοΐ product formed/min.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

YARBROUGH AND CHAMBERS

Organochlorine

Insecticides

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

154

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

Table VII

- R a d i o a c t i v i t y i n Water-soluble F r a c t i o n s of L i v e r s of S u s c e p t i b l e (S) and Resistant (R) F i s h Exposed to 80 y g / l 1 4 C - a l d r i n f o r 6 h r a

Population

a

ng a l d r i n e q u i v a l e n t s / mg protein

Water s o l u b l e r a d i o a c t i v i t y χ 100/ t o t a l hepatic r a d i o a c t i v i t y

S

9 ± 3(11)

1.7 ± 0.3(11)

R

10 ± 2(24)

1.8 ± 0.2(20)

Values are expressed as mean ± SEM (N).

Table VIII -

Proportio hepatic r a d i o a c t i v i t y and Resistant (R) Fish Exposed to 80 y g / l aldrin for 6 hr

1

4

C-

a

Season

s

R

Dec-Feb

47.5 ± 7.2(4)

52.8 ± 2.6(10)

Mar-May

29.1 ± 4.3(2)

13.7 ± 0.0(2)

Sep-Nov

25.0 ± 3.9(4)

28.1 ± 8.7(4)

a

Values are expressed as mean ± SEM (N).

No metabolites o f d i e l d r i n were observed i n orqanic e x t r a c t s of l i v e r s of e i t h e r S or R f i s h exposed to 30 y g / l ' ^ C - d i e l d r i n f o r 6 hr (5) (Table IX). A small percentage o f the r a d i o a c t i v i t y was found i n the w a t e r - s o l u b l e f r a c t i o n . T h e r e f o r e , i t appears that l i t t l e metabolism o f d i e l d r i n occurs in mosquitofish l i v e r s in e i t h e r p o p u l a t i o n . In a d d i t i o n , the degree o f i n t o x i c a t i o n i n S f i s h d i d not appear to a f f e c t the metabolism e i t h e r q u a l i t a t i v e ­ l y or q u a n t i t a t i v e l y . DDT M e t a b o l i s m . - - The metabolism o f DDT has been studied i n R and S f i s h , f o l l o w i n g s i m i l a r protocols to c h l o r i n a t e d c y c l o ­ diene metabolism: organic e x t r a c t i o n ( a c e t o n i t r i l e ) , t h i n l a y e r chromatography o f organic e x t r a c t s , and l i q u i d s c i n t i l l a t i o n counting o f the r e s u l t a n t spots ( 4 · ) . When S and R f i s h were exposed to 60 y g / l o f 1 4 C - l a b e l l e d _p_,£'-DDT f o r 4 h r , r a d i o ­ a c t i v i t y was found in the spots which co-chromatographed with

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

YARBROUGH AND CHAMBERS

Organochlorine

155

Insecticides

DDT, DDD and DDE (Table X ) . There were no q u a l i t a t i v e q u a n t i t a t i v e d i f f e r e n c e s between the two p o p u l a t i o n s .

or

Table IX - R a d i o a c t i v i t y i n L i v e r s of S u s c e p t i b l e (S) F i s h E x h i b i t i n g Symptoms of Poisoning (s) and S and Resistant (R) F i s h not E x h i b i t i n g Symptoms of Poisoning ( a ) . Fish were Exposed to 30 y g / l 1 4 c - d i e l d r i n for 6 h r a

Dieldrin

Population

a

Water-soluble

ng d i e l d r i n e q u i v a l e n t s / ng d i e l d r i n mg protein % equivalents/mg protein

%

128.6

± 13.0(4

Ss

162.7

± 14.5(4)

95

7.8 ± 0.9(4)

5

Ra

136.3

±

98

3.2 ± 0.3(5)

2

8.8(5)

Values are expressed as mean ± SEM (N).

From Watkins

(j3).

Table X - DDT and Metabolite Concentrations i n L i v e r s of S u s c e p t i b l e (S) and Resistant (R) F i s h Exposed to 60 y g / l 14c-p,jj'-DDT f o r 4 h r a

S ng DDT e q u i v a l e n t s / mg protein 624 + 36(6)

DDT

R

% 67.0

ng DDT e q u i v a l e n t s / mg protein

%

483 + 36(7)

74.1

DDD

75 ±

5(5)

8.0

42 ±

4(5)

6.5

DDE

109 ±

7(6)

11.7

51 +

6(5)

7.9

7.3

48 ±

8(6)

7.4

Water Soluble a

68 ± 10(6)

Data are expressed as mean ± SEM (N).

From Hamilton

(4).

Mixed-function Oxidase I n d u c t i o n . - - I n d i r e c t evidence of environmental induction of d e t o x i f y i n g enzymes i n the R f i s h has been observed as an increase i n the acute t o x i c i t y of parathion and a simultaneous decrease i n NADPH-dependent parathion

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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d e a r y ! a t i o n in R f i s h with time held in the l a b o r a t o r y (17,18). However, d i r e c t attempts to induce mixed-function oxidase a c t i v i t y in mosquitofish have been d i f f i c u l t (19,20). Using DDT as a p o s s i b l e inducer r e s u l t e d in an i n c o n s i s t e n t degree of enzyme i n d u c t i o n , with some experiments y i e l d i n g no induction at all (19). Discussion The uptake and d i s t r i b u t i o n of organochlorine i n s e c t i c i d e s has been studied under a v a r i e t y of c o n d i t i o n s . Although the r e s u l t s i n d i c a t e that f u r t h e r study i s needed on a c h a r a c t e r i z a t i o n of extraneous f a c t o r s that a f f e c t d i s p o s i t i o n , the studies c l e a r l y demonstrate the presence of a membrane b a r r i e r to i n s e c t i c i d e penetration in the R p o p u l a t i o n . This membrane b a r r i e r would a i d in th from the i n s e c t i c i d e . Thi f a c t o r in r e s i s t a n c e to organochlorine i n s e c t i c i d e s in mosquitofish. F u r t h e r , by v i r t u e of t h e i r l a r g e r l i v e r s , the R f i s h have a greater xenobiotic biotransformation p o t e n t i a l . However, the in vivo s t u d i e s show few c o n s i s t e n t d i f f e r e n c e s in metabolism between the two p o p u l a t i o n s . Biotransformation may be a major c o n t r i b u t o r y f a c t o r in mosquitofish r e s i s t a n c e to other p e s t i c i d e s , f o r example, organophosphorus and botanical i n s e c t i c i d e s , s i n c e the l e v e l of r e s i s t a n c e to these chemicals i s very low (4 f o l d or l e s s ) (18,20,21). However, biotransformation does not appear to play a major r o l e i n organochlorine i n s e c t i c i d e r e s i s t a n c e . The l e v e l s of r e s i s t a n c e which the mosquitofish demonstrate toward the c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e s (40 - 500 f o l d ) are the most i n t r i g u i n g of those s t u d i e d , yet they are impossible to e x p l a i n i n terms of d i s p o s i t i o n and biotransformation a l o n e . Although i n s e c t i c i d e metabolism cannot be completely d i s c o u n t e d , i t c o n t r i b u t e s l i t t l e to c h l o r i n a t e d a l i c y c l i c r e s i s t a n c e . B a r r i e r s to i n s e c t i c i d e penetration undoubtedly c o n t r i b u t e to chlorinated a l i c y c l i c resistance. However, we are led to conclude that these extremely high l e v e l s of r e s i s t a n c e are the r e s u l t of a postulated i n s e n s i t i v i t y of the t a r g e t s i t e which allows these f i s h to t o l e r a t e elevated i n t e r n a l l e v e l s of these t o x i c a n t s . We have, t h e r e f o r e , been able to i n d i r e c t l y assess the importance o f three f a c t o r s involved in c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e r e s i s t a n c e in m o s q u i t o f i s h : d i s p o s i t i o n , metabolism and t a r g e t s i t e s e n s i t i v i t y . In a highly p o l l u t e d environment in which mosquitofish have been placed under severe s e l e c t i v e pressures by chronic exposure to i n s e c t i c i d e s , the system of metabolism appears to be of l i t t l e s i g n i f i c a n c e in r e s i s t a n c e ; the

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

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Organochlorine

Insecticides

157

system of d i s p o s i t i o n with the development o f i n t e r n a l and external b a r r i e r s to i n s e c t i c i d e penetration i s the next most important; and the i m p l i c a t e d t a r g e t s i t e i n s e n s i t i v i t y i s p o t e n t i a l l y the most s i g n i f i c a n t f a c t o r i n the s u r v i v a l o f the population.

Abstract An insecticide-resistant (R) population of Gambusia affinis demonstrates a 5 to 500 fold resistance to organochlorine insecticides when the 48-hr LC values between the R population and a corresponding susceptible (S) population are compared. Uptake and disposition studies indicate that there is greater insecticide accumulation in tissues of S fish than in those of R fish. However, the difference in uptake between the two populations is not proportional to the degre cides. Resistant fish ca insecticide without demonstrating symptoms of poisoning than can S fish. 50

Hepatic mixed-function oxidase activities demonstrated seasonal trends, with higher specific activities in the cold weather months in both populations with few differences in enzyme activities or cytochrome levels between the two populations. Metabolism of aldrin, dieldrin and DDT was similar between the two populations. R fish have larger relative liver size and, therefore, a greater potential for xenobiotic metabolism. However, biotransformation appears to be of minor importance in chlorinated alicyclic insecticide resistance in mosquitofish; barriers to penetration appear to be of greater importance; and an implied target site insensitivity appears to be the most important factor in resistance.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Acknowledgments This work was supported i n part by National I n s t i t u t e s of Health grants 5 ROI ES00412 and 5 ROI ES00840. Literature Cited

1. Rehwoldt, R.E., Kelley, E. and Mahoney, M. Investigations into acute toxicity and some chronic effects of selected herbicides and pesticides on several fresh water fish species. Bull. Environ. Contam. Toxicol. (1977) 18:361-365. 2. Culley, D.D. and Ferguson, D.E. Patterns of insecticide resistance in the mosquitofish. J. Fish Res. Bd. Can. (1969) 26:2395-2401. 3. Ferguson, D.E., Ludke, J.L. and Murphy, G.G. Dynamics of endrin uptake and release by resistant and susceptible strains of mosquitofish 95:335-344. 4. Hamilton, M.L. "Uptake and metabolism of DDT by insecticide-susceptibleand -resistant mosquitofish, Gambusia affinis." M.S. Thesis, Mississippi State University (1976) 48 p. 5. Watkins, J.H. "A comparative study of the uptake and metabolism of aldrin and dieldrin in insecticide-resistant and -susceptible mosquitofish (Gambusia affinis)." M.S. Thesis, Mississippi State University (1974) 39 p. 6. Hunsinger, R.N. "Uptake and disposition of endrin in insecticide-susceptible and -resistant mosquitofish (Gambusia affinis)." M.S. Thesis, Mississippi State University (1978) 26 p. 7. Scales, E.H. "Comparative studies of endrin uptake in insecticide-resistant and -susceptible mosquitofish (Gambusia affinis)." M.S. Thesis, Mississippi State University (1973) 32 p. 8. Omura, T. and Sato, R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. (1964) 239:2370-2378. 9. Strittmatter, P. NADH-cytochrome b5 reductase. "Methods of Enzymology, X." (Ed. by Estabrook, R.W. and Pullman, M.E.) p. 561-565. Academic Press. New York. 1967 10. Ernster, L., Siekevitz, P. and Palade, G.E. Enzyme-structure relationships in the endoplasmic reticulum of rat liver. J. Cell. Biol. (1962) 15:541-562. 11. Masters, B.S.S., Williams, C.H., Jr., and Kamin, H. The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. "Methods in Enzymology, X." (Ed. by Estabrook, R.W. and Pullman, M.E.) p. 565-573. Academic Press. New York. 1967.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

YARBROUGH

AND

CHAMBERS

Organochlorine

Insecticides

159

12. Mackler, B. Microsomal DPNH-cytochrome c reductase. "Methods in Enzymology, X." (Ed. by Estabrook, R.W. and Pullman, M.E.) p. 551-553. Academic Press. New York. 1967. 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. (1951) 193:265-275. 14. Dewaide, J.H. and Henderson, P. Th. Seasonal variation of hepatic drug metabolism in the roach, Leuciscus rutilus L. Comp. Biochem. Physiol. (1969) 32:489-497. 15. Wells, M.R., Ludke, J.L. and Yarbrough, J.D. Epoxidation and fate of [14C] aldrin in insecticide-resistant and -susceptible populations of mosquitofish (Gambusia affinis). J. Agric. Food Chem. (1973) 21:428-429. 16. Chambers, J.E. and Yarbrough, J.D. Aldrin metabolism in insecticide-resistant and susceptible mosquitofish. Federation Proc 17. _. Organophosphat -resistant and -susceptible populations of mosquitofish (Gambusia affinis). Pestic. Biochem. Physiol. (1973) 3:312-316. 18. _. Parathion and methyl parathion toxicity to insecticide-resistant and -susceptible mosquitofish (Gambusia affinis). Bull. Environ. Contam. Toxicol. (1974) 11:315-320. 19. Chambers, J.E. Induction of mixed-function oxidase activity by DDT in the mosquitofish. Federation Proc. (1976) 35:375. 20. Fabacher, D.L. and Chambers, H. Apparent resistance to pyrethroids in organochlorine-resistant mosquitofish. Proc. 26th Ann. Conf. Southeastern Game Fish Comm. (1972) p. 461-464. 21. _. Rotenone tolerance in mosquitofish. Environ. Pollut. 3:139-141. RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

10 Metabolism of Insect Growth Regulators in Aquatic Organisms DAVID A. SCHOOLEY and GARY R. QUISTAD Biochemistry Department, Zoecon Corporation, 975 California Avenue, Palo Alto, CA 94304 Insect growth regulator insecticides which act y y g develop mental processes, include compounds which mimic the juvenile hormone of insects as well as compounds which interfere with chitin biosynthesis. Chemicals with either mode of action have much greater specificity than earlier insecticides and have substantial advantages in certain applications. Since representatives of both classes of IGR are useful as mosquito larvicides, their fate in the aquatic environment has been investigated in some detail. This paper will review the degradation of several IGRs in water and aquatic organisms. Diflubenzuron Diflubenzuron (Dimilin®, TH-6040) is an IGR which inhibits the normal deposition of chitin. The metabolic fate of diflubenzuron has been studied in sheep (1, 2), cattle (1), rats (1, 3), house flies (4, 5), stable flies (5), chickens (6), swine (6), boll weevils (7), plants (8, 9), and soil (2, 8, 10). Since good reviews of diflubenzuron metabolism have been given by Ivie (11) and Verloop and Ferrell (9), we will present only a tabular summary of the degradation of diflubenzuron in nonaquatic systems for comparative purposes (Table I). The remaining discussion will focus on diflubenzuron degradation in the aquatic environment. H y d r o l y s i s . Schaefer and Dupras (12) i n v e s t i g a t e d the h y d r o l y t i c s t a b i l i t y o f d i f l u b e n z u r o n as a 0.1 ppm aqueous s o l u t i o n . At pH 7.7 d i f l u b e n z u r o n i s s t a b l e a t 10-24°, but g r a d u a l l y decomposes a t 38°. A t pH 10 i t i s s t a b l e a t 10°, but degrades slowly a t temperatures g r e a t e r than 24°. Photodegradation. The photochemical degradation products o f diflubenzuron i n s t r i c t l y aqueous s o l u t i o n are unreported, perhaps because o f the compound's r e f r a c t o r y s o l u b i l i t y . Exposure of t h i n f i l m s on g l a s s o r a 0.1 ppm aqueous s o l u t i o n t o s u n l i g h t 0-8412-0489-6/79/47-099-161$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

=P

=P

=

3

2

5

2

H

2

"3

*

*

*

***

Sheep

*

*

***

***

Cow

* * *

* * *

***

***

Rat

***

Soil

***

P l a n t s (5 species)

* = d e t e c t a b l e , but 10%

5

R =H

5

R =C0CH

3

o

R =C0NH

R

4

R =NH

4

R =NHCH C0 H

R =0H 4

I

2

2

=

R =OH

R

1

3

3

R = H ,

R

R =OH

2

»2

R =R =H,

R

=

=H

2 3

F

R^OH,

1

«1

R

F Q Q

Table I. Nonaquatic Degradation o f Diflubenzuron (5 species)

* * *

***

Insects

10.

S C H O O L E Y A N D QuisTAD

Insect Growth

163

Regulators

r e s u l t e d i n minimal photodecomposition ( 1 2 ) . M e t c a l f et al. (2) and Ruzo e t a l . (13) i r r a d i a t e d methanolic s o l u t i o n s o f d i f l u b e n zuron with a r t i f i c i a l l i g h t as p r e d i c t i v e models f o r environmental photodegradation (Table I I ) . Metcalf e t a l . (2) a l s o detected t r a c e amounts o f 4 - c h l o r o a n i l i n e and a n i l i n e from d i f l u b e n z u r o n a f t e r i r r a d i a t i o n f o r 4 h r (254 nm) i n aqueous dioxane while Ruzo et al. (13) found t r a c e s o f 4-chlorophenyl isocyanate i n methanol upon i l l u m i n a t i o n a t 300 nm. Table I I .

Photoproducts o f Diflubenzuron i n Methanolic S o l u t i o n % Yield Metcalf et al. (2)

^^-NC0 CH

18

2

CI-^-NC0 CH 2

Ruzo et al. (13)

45

trace

3

4

0 CNH

Table I I I .

68

2

49

M e t a b o l i t e s o f Diflubenzuron from B a c t e r i a and Bluegreen Algae, Booth and F e r r e l l ( 1 4 ) . 14 % Extractable C Bacteria Blue-green Algae

(Pseudomonas) (10 days) CI-Q-NCNH

2

C I - ^ - N H

5

2

5

unmetabolized diflubenzuron

(4 days) 53

u

2

C0 H

{Plectonema)

trace

36

5

Microorganisms. Diflubenzuron was s t a b l e t o degradation by u n c h a r a c t e r i z e d microorganisms from a sewage lagoon ( 1 2 ) . Pseudomonas putida ( s o i l microbe) a l s o was unable t o metabolize diflubenzuron upon i n c u b a t i o n o f pure c u l t u r e s (2). Using Pseudomonas sp. from aquatic Utah s o i l Booth and F e r r e l l (14) followed the metabolic f a t e o f d i f l u b e n z u r o n f o r twelve days

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PESTICIDE AND XENOBIOTIC

METABOLISM

I N AQUATIC

ORGANISMS

(Table I I I ) . Pseudomonas was capable o f using diflubenzuron as a sole carbon source, but b a c t e r i a l growth was g r e a t l y a c c e l e r a t e d when the medium was supplemented with acetate. Booth and F e r r e l l (14) found t h a t blue-green algae, Plectonema boryanum, were voracious degraders o f diflubenzuron (Table I I I ) . J u s t 5 mg o f algae c e l l s could metabolize almost 80% o f the a p p l i e d diflubenzuron i n j u s t 1 hr. C u r i o u s l y , t h i s pace was not sustained s i n c e 45 mg o f algae could degrade only 95% o f the a p p l i e d dose a f t e r four days. Aquatic Ecosystem and F i s h . Metcalf e t al. (2) studied the f a t e o f d i f l u b e n z u r o n ( r a d i o l a b e l e d s e p a r a t e l y i n three d i f f e r e n t p o s i t i o n s ) i n t h e i r model ecosystem. Diflubenzuron was dubbed "moderately p e r s i s t e n t " i n algae, s n a i l s , s a l t marsh c a t e r p i l l a r s , and mosquito l a r v a e as evidenced by l i m i t e d b i o d e g r a d a b i l i t y (Table IV). However, d i f l u b e n z u r o n and i t s nonpolar metabolites were not prone t o e c o l o g i c a lack o f bioaccumulation s u b s t a n t i a t e d by Booth and ' F e r r e l l (14) who used the channel c a t f i s h , Ictalurus, i n a simulated lake ecosystem. They t r e a t e d separate s o i l samples a t 0.007 and 0.55 ppm, r e s p e c t i v e l y . Table IV.

Degradation o f Diflubenzuron Model Ecosystem.

Water diflubenzuron

24-31

i n the Metcalf e t a l . (2)

% Extractable Radiolabel i n Mosquito Snail Alga Culex Oedogonium Physa 46-74

73-96

84-98

Fish

Gambusia 5-17

6-8

0-CO H

3-10

2

^ - C O N H

CI-^^-NH

2

11

2

0 CI-^^-NCCH

3

CI-^^-NCNH

2

CI-^-N(CH ) 3

10

2

5

12

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Regulators

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The s o i l was aged a e r o b i c a l l y f o r two weeks, then submerged under water f o r two weeks. F i n a l l y , a d d i t i o n a l water and c a t f i s h were added with monitoring o f residues f o r twenty-eight days. For s o i l t r e a t e d a t 0.55 ppm, 64% o f the i n i t i a l dose was r e l e a s e d from the s o i l , but only 2.3% o f the a p p l i e d r a d i o l a b e l was found i n the water upon termination o f the study. Water residues c o n s i s t e d mostly (>93%) o f 4-chlorophenylurea, accompanied by d i f l u o r o b e n z o i c a c i d (3-5%), 4 - c h l o r o a n i l i n e (0-1%), and diflubenzuron (0.4-1.5%). An average o f 66% o f the s o i l residues were e x t r a c t a b l e with methanol, c o n s i s t i n g predominantly o f unmetabolized d i f l u b e n z u r o n (74-84%) and 4 - c h l o r o a n i l i n e (11-17%). F o r s o i l t r e a t e d a t 0.55 ppm, f i s h residues q u i c k l y reached a p l a t e a u a f t e r three days a t about 4 and 10 ppb f o r muscle and v i s c e r a , r e s p e c t i v e l y . Hence, Booth and F e r r e l l (1£) concluded t h a t bioaccumulation o f d i f l u benzuron residues from marsh a p p l i c a t i o n s should be minimal. R-20458 An impressive l i s t o f degradative s t u d i e s has been performed with S t a u f f e r * s R-20458 i n c l u d i n g metabolism by e i g h t i n s e c t species (15_, 16) , r a t s (15, 17) , s t e e r s (18, 19) , mice (20) and mammalian enzymes (15, 20, 21). I t i s evident t h a t most p u b l i s h e d i n v e s t i g a t i o n s have concentrated on metabolism by mammals and insects. Insect metabolism o f R-20458 has been reviewed (22) and a summary o f nonaquatic metabolism i s given i n Table V. Several a d d i t i o n a l hydroxylated metabolites were i d e n t i f i e d by Hoffman e t a l . (17) from r a t s . 1

Photodegradation. C a s i d a s group (15, 20) has s t u d i e d the photodecomposition o f R-20458 on s i l i c a g e l and i n water. The major aqueous photoproducts are summarized i n F i g u r e 1. The predominant photoproduct i n aqueous s o l u t i o n r e s u l t e d from epoxide hydration t o the corresponding d i o l . The photoproducts on s i l i c a were q u i t e s i m i l a r t o aqueous products with an enhanced y i e l d o f diepoxide and diminished y i e l d o f d i o l . P h o t o s e n s i t i z e r dyes had l i t t l e e f f e c t on R-20458 photodegradation. Algae. G i l l et al. (20) studied the metabolic f a t e o f R-20458 i n the algae Chlorella and Chlamydomonas. Both algae e f f i c i e n t l y metabolized R-20458 with Chlorella demonstrating a higher metabolic c a p a c i t y . In Chlamydomonas, the main metabolite r e s u l t e d from hydration o f the parent epoxide t o the d i o l . After 48 h r , 78% o f the R-20458 was degraded by Chlorella, the metabol i t e s c o n s i s t i n g mainly o f d i o l s (Figure 2 ) . Epifenonane D e t a i l e d r e p o r t s o f the degradation o f t h i s Hoffman-LaRoche compound are l i m i t e d . Compared t o s e v e r a l other IGRs, epifenonane (Ro 10-3108) was r e l a t i v e l y s t a b l e a t pH 4 i n the dark (23).

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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I

ι

0.5 ppm recovered methoprene 6%

Cc]methoprene ^J!2!i_» ~ ° > ^ A A

60%

4

0

H

29% Figure 5.

Metabolism of methoprene by soil and aquatic microorganism (26, 27)

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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In summary, photodecomposition o f methoprene i s f a c i l e and leads t o a m u l t i p l i c i t y o f products. The lower p h o t o s t a b i l i t y i n s u n l i g h t o f methoprene compared t o epifenonane has been mentioned p r e v i o u s l y . Because o f i t s photochemical l a b i l i t y and i t s ready m i c r o b i a l degradation (see below), methoprene i s microencapsul a t e d f o r aquatic use as a mosquito l a r v i c i d e . Microorganisms. Outdoor incubation o f 0.42 ppm o f [10- H]methoprene with unknown aquatic microorganisms f o r three days l e d t o production o f the hydroxy e s t e r (7.0%), the methoxy a c i d (5.7%), and the hydroxy a c i d (2.6%). Recovered methoprene (60%) contained about 7% o f the analogous e t h y l e s t e r , while the hydroxy i s o p r o p y l e s t e r metabolite contained about 15% o f the analogous e t h y l e s t e r (Figure 5). The genesis o f these e t h y l e s t e r s i s u n c e r t a i n , but b e l i e v e d t o a r i s e from ethanol used t o dose the incubations ( f i n a l concentration only 0.08% ethanol i n water), with p o s s i b l e enzymi [5- C]methoprene a t 0.6 the same source, but c o l l e c t e d during August i n s t e a d o f February, l e d t o i s o l a t i o n o f a s i n g l e major metabolite i n 29% y i e l d . The metabolite, 7 - m e t h o x y c i t r o n e l l i c a c i d , i s a l s o known as a photoproduct. However, a simultaneous autoclaved c o n t r o l showed a lower y i e l d o f t h i s product. A l s o , 98% o f the r a d i o a c t i v i t y was recovered from the autoclaved c o n t r o l vs. 48% from the microb i a l l y - a c t i v e water. Since C - l o f 7 - m e t h o x y c i t r o n e l l i c a c i d i s the l a b e l e d p o s i t i o n , f u r t h e r m i c r o b i a l degradation o f 7-methoxyc i t r o n e l l i c a c i d t o [ C ] a c e t a t e and C02 can be i n f e r r e d . F a l l has studied the c a p a b i l i t y o f fourteen species o f microorganisms t o grow on methoprene as sole carbon source. One of these organisms, Cladosporium resinae, was able t o u t i l i z e methoprene as s o l e carbon source, while another, Pseudomonas citronellolis, was s i m i l a r l y able t o u t i l i z e 7 - m e t h o x y c i t r o n e l l i c a c i d . (41). S o i l microorganisms degrade methoprene r a p i d l y and extens i v e l y (27). The hydroxy e s t e r was i s o l a t e d as a minor metabolite; over 50% o f the a p p l i e d dose was evolved as C0z. R a d i o a c t i v i t y from [5- **c]methoprene incorporated i n t o the humic a c i d , f u l v i c a c i d , and humin f r a c t i o n s o f s o i l . 3

1

li+

1!|

lk

1

F i s h and Ecosystem Studies. When b l u e g i l l s u n f i s h are exposed t o a constant l e v e l o f methoprene i n a dynamic flowthrough system, they accumulate radiocarbon u n t i l a p l a t e a u i s reached a f t e r 7-14 days (33) . While l e v e l s o f methoprene i n f i s h were about lOOOx that i n water a t the p l a t e a u , t r e a t e d b l u e g i l l p l a c e d i n t o uncontaminated water showed a 93-95% radiocarbon r e d u c t i o n i n 14-21 days. A n a l y s i s o f f i s h t i s s u e s a t p l a t e a u l e v e l s revealed t h a t ^90% o f the radiocarbon was unmetabolized parent, 1% was the hydroxy e s t e r , while the remainder was p o l a r conjugates.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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The f a t e o f methoprene was a l s o s t u d i e d i n an aquatic ecosystem designed t o simulate environmental exposure o f f i s h t o a mosquito l a r v i c i d e . B l u e g i l l f i s h were maintained i n two meter diameter pools c o n t a i n i n g m i c r o b i a l l y - a c t i v e water, s o i l , and bear rush p l a n t s . The p o o l was t r e a t e d three times a t weekly i n t e r v a l s with [5- **C]methoprene s u f f i c i e n t t o give 0.011 ppm s o l u t i o n . Radioassay o f samples revealed a r a t h e r general d i s t r i b u t i o n o f r a d i o a c t i v i t y throughout the system. We analyzed f i s h t i s s u e s two and four weeks a f t e r the l a s t treatment. While radioassay revealed an apparent concentration o f 2-3 ppm equivalents o f methoprene i n b l u e g i l l , e x t r a c t i o n and d e t a i l e d a n a l y s i s revealed t h a t l e s s than 0.1% o f the radiocarbon was methoprene and i t s known primary metabolites. R a d i o a c t i v i t y i n nonpolar e x t r a c t a b l e f r a c t i o n s was c h a r a c t e r i z e d as t r i g l y c e r i d e s , d i g l y c e r i d e s , c h o l e s t e r o l , and f r e e f a t t y a c i d s (14, 0.2, 1, and 3% o f the t o t a l r e s i d u e r e s p e c t i v e l y ) a t two weeks posttreatment Over 50% o f the radiocarbo solubilized radioactivit on s o l u b i l i t y (33). T h i s study provides a t e l l i n g c r i t i c i s m o f ecosystem s t u d i e s , when performed on r e a d i l y degraded m a t e r i a l s , and/or when not accompanied by cautious a n a l y s i s o f samples. The f a t e o f methoprene was i n v e s t i g a t e d i n the Metcalf ecosystem (42) before the environmental l a b i l i t y o f [5- *C]methoprene was documented. I t seems l i k e l y that the l i m i t e d data presented i n t h a t work gave s p u r i o u s l y high residue l e v e l s due t o formation of nonmetabolite residues which i n t e r f e r e with the simple t i c analyses performed. 1

1

Summary Insect growth r e g u l a t o r s c o n s i s t o f d i v e r s e chemical s t r u c t u r a l types, but appear t o share a common f e a t u r e : quick degradation i n the aquatic environment.

Literature Cited 1 Ivie, G.W., J. Agric. Food Chem., (1978), 26, 81. 2 Metcalf, R.L., Lu, P.-Y., and Bowlus, S., J. Agric. Food Chem., (1975), 23, 359. 3 Post, L.C. and Willems, A.G.M., paper in preparation, cited in ref. 9 below. 4 Chang, S.C., J. Econ. Entomol., (1978), 71, 31. 5 Ivie, G.W. and Wright, J.E., J. Agric. Food Chem., (1978), 26, 90. 6 Opdycke, J.C., Miller, R.W., and Menzer, R.E., Toxicol. Applied Pharmacol., (1976), 37, 96. 7 Still, G.G. and Leopold, R.A., Abstr. Pap., 170th Meet., Amer. Chem. Soc., (1975), PEST 5. 8 Bull, D.L. and Ivie, G.W., J. Agric. Food Chem., (1978), 26, 515.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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9 Verloop, A. and Ferrell, C.D., in "Pesticide Chemistry in the 20th Century", (J.R. Plimmer, ed.), ACS Symposium Series, 37, (1977), pp 237-270. 10 Verloop, A. Nimmo, W.B., and DeWilde, P.C., submitted to Pestic. Sci. 11 Ivie, G.W., in "Fate of Pesticides in Large Animals", (G.W. Ivie and H.W. Dorough, eds.), Academic Press, New York, (1977), pp 111-125. 12 Schaefer, C.H. and Dupras, E.F., Jr., J. Agric. Food Chem., (1976), 24, 733. 13 Ruzo, L.O., Zabik, M.J. and Schuetz, R.D., J. Agric. Food Chem., (1974), 22, 1106. 14 Booth, G.M. and Ferrell, D., in "Pesticides in Aquatic Environments", (M.A.Q. Khan ed.), Plenum Press, New York, (1977), pp 221-243. 15 Gill, S.S., Hammock B.D. Yamamoto I. Casida J.E. in "Insect Juvenile Hormones and M. Beroza, eds.) pp 177-189. 16 Hammock, B.D., Gill, S.S., Casida, J.E., Pestic. Biochem. Physiol., (1974), 4, 393. 17 Hoffman, L.J., Ross, J.H., Menn, J.J., J. Agric. Food Chem., (1973), 21, 156. 18 Ivie, G.W., Wright, J.E., and Smalley, H.E., J. Agric. Food Chem., (1976), 24, 222. 19 Ivie, G.W., Science, (1976), 191, 959. 20 Gill, S.S., Hammock, B.D., and Casida, J.E., J. Agric. Food Chem., (1974), 22, 386. 21 Hammock, B.D., Gill, S.S., Stamoudis, V., and Gilbert, L.I., Comp. Biochem. Physiol., (1976), 53B, 263. 22 Hammock, B.D. andQuistad,G.B., in "The Juvenile Hormones", (L.I. Gilbert, ed.), Plenum Press, New York, (1976),p374. 23 Hangartner, W.W., Suchý, Μ., Wipf, H.-K., and Zurflüh, R.C., J. Agric. Food Chem., (1976), 24, 169. 24 Dorn, S., Oesterhelt, G., Suchý, M., Trautmann, K.H., and Wipf, H.-K., J. Agric. Food Chem., (1976), 24, 637. 25 Quistad, G.B., Staiger, L.E., and Schooley, D.A., J. Agric. Food Chem., (1974), 22, 582. 26 Schooley, D.A., Bergot, B.J., Dunham, L.L., and Siddall, J.B., J. Agric. Food Chem., (1975), 23, 293. 27 Schooley, D.A., Creswell, K.M., Staiger, L.E., and Quistad, G.B., J. Agric. Food Chem., (1975), 23, 369. 28 Quistad, G.B., Staiger, L.E., and Schooley, D.A., Pestic. Biochem. Physiol., (1975), 5, 233. 29 Hammock, B.D., Mumby, S.M., and Lee, P.W., Pestic. Biochem. Physiol., (1977),7,261. 30 Quistad, G.B., Staiger, L.E., Bergot, B.J., and Schooley, D.A., J. Agric. Food Chem., (1975), 23, 743. 31 Quistad, G.B., Staiger, L.E., and Schooley, D.A., J. Agric. Food Chem., (1975), 23, 750.

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32 Quistad, G.B., Staiger, L.E., and Schooley, D.A., J. Agric. Food Chem., (1976), 24, 644. 33 Quistad, G.B., Schooley, D.A., Staiger, L.E., Bergot, B.J., Sleight, B.H., and Macek, K.J., Pestic. Biochem. Physiol., (1976), 6, 523. 34 Chamberlain, W.F., Hunt, L.M., Hopkins, D.E., Gingrich, A.R., Miller, J.A., and Gilbert, B.N., J. Agric. Food Chem., (1975), 22, 736. 35 Davison, K.L., J. Agric. Food Chem., (1976), 24, 641. 36 Tokiwa, T., Uda, F., Uemura, J., and Nakazawa, Μ., Oyo Yakuri, (1975), 10, 471. 37 Hawkins, D.R., Weston, K.T., Chasseaud, L.F., and Franklin, E.R., J. Agric. Food Chem., (1977),25,398. 38 Quistad, G.B., Staiger, L.E., and Schooley, D.A., Life Sci., (1974), 15, 1797. 39 Schaefer, C.H. and Dupras, E.F., J. Econ. Entomol., (1973), 66, 923. 40 Quistad, G.B., Staiger Food Chem., (1975), 23, 299. 41 Fall, R.R., University of Colorado, personal communication, (1977). 42 Metcalf, R.L. and Sanborn, J.R., Ill. Nat. Hist. Surv. Bull., (1975), 31, 393. RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

11 The Fate of Highly Brominated Aromatic Hydrocarbons in Fish V. ZITKO Fisheries and Environmental Sciences, Fisheries and Oceans Canada, Biological Station, St. Andrews, N.B., Canada E0G 2X0 Previous studies o carbons by juvenile Atlanti that bromobiphenyls with up to 4 bromine atoms in their molecule accumulate similarly to the corresponding chlorobiphenyls (1). In contrast, penta- and higher bromobiphenyls accumulated in the fish to a lesser extent than similar chlorobiphenyls. The accumulation decreased with increasing bromine substitution and a hexabromobiphenyl was the last bromobiphenyl still accumulated from water (2). This apparent effect of molecular weight on the accumulation was also confirmed by the lack of accumulation of hexabromobenzene (1), and by a relatively low accumulation of tetrabromo-2-chlorotoluene and pentabromotoluene (3). The last two compounds were very slowly excreted by the fish. This indicated that their uptake may be very slow as well, which may result in an underestimation of the accumulation coefficient (4). Octa- and higher brominated biphenyls were accumulated by the fish to a small extent only when administered in food. The main compound found in this case was a hexabromobiphenyl, not present in the originally administered mixture of bromobiphenyls (2). These observations indicate three possible reasons for the low accumulation of highly brominated biphenyls and benzenes in juvenile Atlantic salmon: (i) The compounds are likely to have an extremely low solubility in water, preventing their transport to biological membranes. Accumulation generally increases with decreasing water solubility [b), but the relationship has been established only for compounds with water solubilities larger than approximately 5xl0" ymoles/L. Water solubility of highly brominated biphenyls and benzenes has not been measured. Calculations (6) indicate that it may be at least an order of magnitude below the above value. The accumulation/solubility relationship may not be valid in this range. (ii) The high molecular weight of these compounds may be 2

0-8412-0489-6/79/47-099-177$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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preventing or considerably d i m i n i s h i n g t h e i r permeation through b i o l o g i c a l membranes. The permeability of b i o l o g i c a l membranes is p r o p o r t i o n a l to Mp -j (Mrel = molecular weight of an none l e c t r o l y t e , r e l a t i v e to molecular weight of methanol, s = e m p i r i c a l exponent, ranging from - 2 . 9 to -6) (7). e

( i i i ) The compounds may form bound or polar m e t a b o l i t e s , not e x t r a c t a b l e under the c o n d i t i o n s used f o r the parent molecules. Covalent binding of bromobenzene to t i s s u e s has been observed pre­ v i o u s l y (8J, and the p a r t i a l debromination of h i g h l y brominated biphenyls i n d i c a t e s the p o s s i b i l i t y of the formation of r e a c t i v e metabolites. This paper describes experiments attempting to simulate the accumulation of halogenated aromatic compounds by measuring t h e i r t r a n s p o r t rate through a l a y e r of water i n t o hexane, and shows that hexabromobenzene i s not transported in t h i s system. In a d d i t i o n , data o th accumulatio f l brominated benzenes by j u v e n i l e A t l a n t i accumulation of brominate measured transport r a t e s . Experimental Measurement of Transport Rates. Halogenated compounds, d i s s o l v e d in hexane or hexane c o n t a i n i n g a small amount of toluene i n the case of hexabromobenzene (0.5-2 ml_), were applied to the bottom of a 25-mL REACTI-VIAL f l a s k (Pierce Chemical Co.) and the solvent was evaporated gently under a stream of n i t r o g e n . Tap water (25 mL) was added c a r e f u l l y , followed by p e s t i c i d e - g r a d e hexane (3 mL), and the f l a s k was closed by a T e f l o n - l i n e d septum. The f l a s k was kept in subdued l i g h t at room temperature ( 2 0 ± 1 ° C ) . Samples of hexane were withdrawn by a 1 0 - μ ί Hamilton syringe and i n j e c t e d i n t o a gas chromatograph. The compounds were used in mixtures, prepared in a manner convenient f o r gas chromatography. Each compound was tested at 2-4 f l a s k l o a d i n g s , ranging from 0.5-56 y g / f l a s k . At each l o a d i n g , transport rates (ug/h) were c a l c u l a t e d and p l o t t e d against the loading ( y g / f l a s k ) , y i e l d i n g a s t r a i g h t l i n e R(ug/h) = a C ( y g / f l a s k ) + b, where a , Jb = empirical c o e f f i c i e n t s . Experi­ ments were u s u a l l y c a r r i e d out over a period of about 60 h. R

Accumulation in F i s h . Experiments were c a r r i e d out as desc r i b e d p r e v i o u s l y (_1, 2 _3) f o r accumulation from water. Average weight of the f i s h was 2.1 g , and water temperature was 1 2 ° C . F i s h were exposed f o r 48 h and placed in clean running water f o r up to 384 h. Each sample c o n s i s t e d of 2 whole f i s h , analyzed individually. 9

Chemical A n a l y s e s . Water samples were analyzed as described p r e v i o u s l y (1). Whole f i s h were homogenized with tap water (20 mL) i n a S o r v a l l Omni-Mixer, using a 100-mL s t a i n l e s s steel

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container. The homogenate v/as t r a n s f e r r e d into a 500-mL roundbottom f l a s k , using an a d d i t i o n a l 250 mL of tap water, and d i s t i l l e d as described [9) f o r 1 h, using p e s t i c i d e - g r a d e 2 , 2 , 4 trimethylpentane as the e x t r a c t i o n s o l v e n t . For halogenated benzenes analyzed by t h i s procedure, the a n a l y s i s of spiked f i s h r e s u l t e d in >85% recovery of the compounds. Gas chromatography was performed as described p r e v i o u s l y (10), except that a column temperature of 170°C was used when a n a l y z i n g f o r di-tetrabromobenzene, dibromo-toluene and xylene. Results and Discussion Transport Rates. The amount of halogenated compounds in the hexane layer increased l i n e a r l y with time f o r up to 12 h f o r a l l compounds t r a n s p o r t e d . For many compounds the r e l a t i o n s h i p remained l i n e a r f o r at l e a s t 24 h and then i t s t a r t e d to level off. This i n c r e a s e , expresse l i n e a r f u n c t i o n of the amoun Table I Transport rate R (yg/h) as a f u n c t i o n of the amount in the f l a s k C (yg); R = aC + b Compound ρ,ρ'-DDT dieldrin 2,4',5-tribromobiphenyl 1,2,4,5-tetrabromobenzene -nonachlor hexachlorobenzene 1,3,5-tribromobenzene 1,2,42,5-dibromoxylene 2,5-dibromotoluene

a χ 10 -1.0 8.0 9.0 6.2 3.5 1.4 26.2 54.9 22.9 14.7

3

b χ 10

3

38 25 21 13 7 3 -10 -30 -32 -103

Hexabromobenzene at 14 and 56 yg per f l a s k was not detectable i n the hexane l a y e r . At 29 yg per f l a s k , 0.57 yg of hexabromo­ benzene was found at 2.5 h. This might have been caused by a p a r t i c l e of hexabromobenzene released a c c i d e n t a l l y from the bottom of the f l a s k . The amount of hexabromobenzene in the hexane layer was decreasing s t e a d i l y throughout t h i s experiment at a rate of about 5.6 n g / h . These data i n d i c a t e that hexabromobenzene is not transported as r e a d i l y as halogenated hydrocarbons l i s t e d in Table I. The c o e f f i c i e n t a is r e l a t e d to the water s o l u b i l i t y of the compound. A low transport rate can be expected when the f l a s k loading i s less than the amount of the compound that would d i s s o l v e in the volume of water in the f l a s k . The transport rate

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should increase once t h i s amount is exceeded. The data in Table 1 confirm t h i s assumption. The transport rate is p r a c t i c a l l y independent of the amount added to the f l a s k f o r compounds of low water s o l u b i l i t y , but increases considerably with the amount added i n the case of compounds appreciably s o l u b l e in water. The c o e f f i c i e n t Jb, an extrapolated transport r a t e , may be i n t e r p r e t e d as a r e l a t i v e " d r i v i n g f o r c e " of the transport and r e l a t e d to the accumulation of the compound in aquatic b i o t a . This i s i n d i c a t e d by the order of the compounds in Table 1, arranged according to decreasing b v a l u e s . The h i g h l y accumula­ t i v e ρ,ρ'-DDT leads the l i s t , and the b values of compounds, not expected to accumulate e x t e n s i v e l y , are much lower. Accumulation in F i s h . The accumulation c o e f f i c i e n t and e x c r e t i o n h a l f - l i f e of t r i - and tetrabromo-benzenes, dibromotoluene and x y l e n e , are within a range expected on the basis of s i m i l a r experiments (1_) Table II Accumulation and e x c r e t i o n in f i s h

Compound

1,2,4,5-tetrabromo­ benzene

Concn. at 48 h fish water Ug/g) (ng/L)*

15.5

21.6

Accumulation coefficient

Excretion half-life, h

1390

103

1,3,5-tribromobenzene

2.29

2.58

1130

95

1,2,4-tribromobenzene

4.75

5.20

1095

104

1430

86

470

90

2,5-dibromoxylene

30.3

2,5-dibromotoluene

14.0

43.3 6.60

*Mean c o n c e n t r a t i o n , 0-48 h P r e l i m i n a r y experiments i n d i c a t e that the accumulation c o e f f i c i e n t of_o- and m-dibromobenzene is about 200, and the e x c r e t i o n h a l f - l i f e i s approximately 18 h. Consequently, with the p o s s i b l e exception of pentabromobenzene, hexabromobenzene may be the only brominated benzene which does not accumulate in f i s h . C o r r e l a t i o n of Accumulation in F i s h with Transport Rates. Equation [1] expresses the accumulation c o e f f i c i e n t s (Table II)

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

as

11.

ZITKO

Brominated

a f u n c t i o n of b (Table

Aromatic

Hydrocarbons

181

I):

Accumulation c o e f f i c i e n t = 77362? + 1354 (n=5, r=0.875)

[1]

The amount of data i s very l i m i t e d at t h i s s t a g e , but i t appears that the measurement of transport r a t e s , as described above, has a potential to become a tool f o r p r e d i c t i o n s of accumulation of chemicals in f i s h . From equation [ 1 ] , the accumulation c o e f f i c i e n t s of 2 , 4 ' , 5 tribromobiphenyl and of hexachlorobenzene are 1512 and 1378, respectively. P r e v i o u s l y reported (4) accumulation c o e f f i c i e n t s f o r these compounds are 400-2000, and 753. In a d d i t i o n , the measurement of transport rates predicted c o r r e c t l y that hexabromobenzene w i l l not accumulate in f i s h . This p r e d i c t i o n would not be p o s s i b l e on the basis of the octanol/water p a r t i t i o n c o e f f i c i e n t (se Cone!usions Low water s o l u b i l i t y , p o s s i b l y a c t i n g in conjunction with low membrane p e r m e a b i l i t y , appears to be the main reason f o r the lack of accumulation of h i g h l y brominated biphenyls and hexabromobenzene from water by j u v e n i l e A t l a n t i c salmon. A method f o r the measurement of transport r a t e s , described in t h i s paper, i s p o t e n t i a l l y useful f o r p r e d i c t i n g the accumulation of chemicals in f i s h . Acknowledgments I thank Mr. W. G. Carson and Mrs. Sandra Cervantes f o r valuable t e c h n i c a l a s s i s t a n c e in a part of the study, Messrs. A. Sreedharan and G. Fawkes f o r a s s i s t a n c e with c a l c u l a t i o n s , and Mrs. Brenda McCullough f o r typing the manuscript.

Literature Cited 1. Zitko, V., Hutzinger, O., Bull. Environ. Contam. Toxicol. (1976), 16, 665. 2. Zitko, V., Bull. Environ. Contam. Toxicol. (1977),17,285. 3. Zitko, V., Carson, W. G., Chemosphere (1977), 6, 293. 4. Zitko, V., "Uptake and Excretion of Chlorinated and Brominated Hydrocarbons by Fish", Fisheries and Marine Service Technical Report No. 737, Biological Station, St. Andrews, N.B. E0G 2X0. 5. Chiou, C. T., Freed, V. H., Schmedding, D. W., Kohnert, R. L., Environ. Sci. Technol. (1977), 11, 475. 6. Moriguchi, I., Kanada, Y., Komatsu, K., Chem. Pharm. Bull. (1976), 24, 1799. 7. Lieb, W. R., Stein, W. D., Nature (1969), 224, 240.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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8. Gillette, J. R., "Reactive Metabolites of Organohalogen Compounds", in "Ecological Toxicology Research", McIntyre, A. D., and Mills, C. F., Editors, 107-122, Plenum Press, New York and London, 1975. 9. Veith, G. D., Kiwus, L. Μ., Bull. Environ. Contam. Toxicol. (1977), 17, 631. 10. Zitko, V., Choi, P. Μ. Κ., Wildish, D. J., Monaghan, C. F., Lister, Ν. Α., Pestic. Monit. J. (1974), 8, 105. 11. Neely, W. G., Branson, D. R., Blau, G. E., Environ. Sci. Technol. (1974), 8, 1113. RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

12 A Terrestrial-Aquatic Model Ecosystem for Evaluating the Environmental Fate of Drugs and Related Residues in Animal Excreta S. H. CABALLA, M. PATTERSON, and I. P. KAPOOR Agricultural Division, American Cyanamid Company, P. O. Box 400, Princeton, NJ 08540 When farm animals are treated with drugs both as a prophylactic or curative measure, majority of the drug or drug related residues are eliminated i n the excreta. Poultry as well as farm animal excreta is allowed to compost into manure and the manure is used on the farm land. The objective of the present study was to design a terrestrial-aquatic model ecosystem for evaluating the environmental fate of drugs and related residues i n the animal excreta used as manure. Metcalf et al. (1) developed a model ecosystem consisting of a terrestrial/aquatic interface and a seven-element food chain for obtaining valuable information on the biodegradability and ecological fate of numerous pesticides. This study, using a modified system, was initiated to determine the ecological fate of robenidine hydrochloride and related residues present i n turkey excreta. ROBENZ® Robenidine hydrochloride, (Figure 1), has been found to be an effective and safe feed additive product for the prevention of coccidiosis i n broiler chickens (2). For comparison, a parallel experiment using turkey excreta fortified with carbon-14 DDT as a positive control was conducted since the biodegradability and ecological fate of DDT and i t s analogs have already been extensively studied (1, 3). Materials and Methods Robenidine hydrochloride labeled with carbon-14 i n the amino guanidine carbon atom had a specific activity of 18.61 yCi/mg. Carbon-14 DDT, labeled i n the ring, had a specific activity of 83.4 uCi/mg.

®

R e g i s t e r e d Trademark o f A m e r i c a n Cyanamid Company 0-8412-0489-6/79/47-099-183$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

184

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

ROBENZ®

Cl-f

^ D E N O T E S

ORGANISMS

ROBENIDINE HYDROCHLORIDE

"\ CC HH == NN HH NN CC **NN HH NN == CC HH --/ \>-NH- HCI

\ - CI

CARBON-1

Figure 1

Figure 2. Schematic of the modified model ecosystem detailing a complete terrestrial/aquatic environment for the study of drug biodegradability and ecological magnification

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

12.

CABALLA ET AL.

Terrestrial-Aquatic

Model

Ecosystem

185

I n t h i s s t u d y , t h r e e e x p e r i m e n t a l t a n k s were e m p l o y e d : Tank A c o n t a i n i n g e x c r e t a from a t u r k e y t r e a t e d w i t h carbon-14 r o b e n i ­ d i n e ; Tank Β c o n t a i n i n g e x c r e t a f o r t i f i e d w i t h c a r b o n - 1 4 DDT w h i c h was u s e d a s a p o s i t i v e c o n t r o l a n d Tank C c o n t a i n i n g u n ­ t r e a t e d e x c r e t a w h i c h was u s e d a s a c o n t r o l . To o b t a i n e x c r e t a f o r t h i s s t u d y , a t u r k e y was f e d 250 grams o f a b a s a l d i e t c o n t a i n i n g 66 ppm r o b e n i d i n e h y d r o c h l o r i d e f o r t e n d a y s . On t h e e l e v e n t h d a y , t h e t u r k e y was d o s e d w i t h 17 mg o r 316.37 u C i o f c a r b o n - 1 4 r o b e n i d i n e i n a c a p s u l e . E x c r e t a was c o l l e c t e d f o r two d a y s a f t e r t r e a t m e n t . Excreta c o l l e c t e d p r i o r t o t h e t e n - d a y c o n d i t i o n i n g p e r i o d was a l s o c o l ­ l e c t e d a n d u s e d f o r t h e c o n t r o l a n d DDT e x p e r i m e n t a l t a n k s . The m o d e l e c o s y s t e m was e s s e n t i a l l y t h e same a s t h a t p r e v i ­ o u s l y d e s c r i b e d b y M e t c a l f et_ a l . (1) e x c e p t t h a t t h e t e r r e s t ­ r i a l p o r t i o n c o n s i s t e d o f u n s t e r i l i z e d sandy loam s o i l i n s t e a d o f w h i t e q u a r t z s a n d . I n p r a c t i c e 4.5 k g o f a q u a r i u m g r a v e l was washed t h o r o u g h l y t o remov m e a s u r i n g 2 χ 6 χ 12 i n c h e a q u a r i u m . A s o i l s h e l f was t h e n m o l d e d o n t o p o f t h e g r a v e l c o n s i s t i n g o f 7 k g o f P r i n c e t o n s a n d y loam s o i l . The t o p 2 i n c h e s o f t h e s o i l s h e l f was m o l d e d f r o m 2.5 k g o f s o i l m i x e d t h o r o u g h l y i n a b a l l m i l l w i t h t h e d r i e d t u r k e y e x c r e t a . The amount o f e x c r e t a added c o r r e s p o n d e d t o a n a p p l i c a t i o n r a t e o f 5 tons/acre. The t o t a l a r e a o f t h e p l a t e a u was 78 s q u a r e i n c h e s (6.5 χ 12 i n c h e s ) w i t h a t o t a l h e i g h t o f 6 i n c h e s ( F i g u r e 2 ) . The t o t a l r a d i o a c t i v i t y a p p l i e d t o t h e s o i l a s c a r b o n - 1 4 r o b e n i d i n e r e s i d u e s i n e x c r e t a was 167.54 m i c r o c u r i e s . F o r Tank B, 100 m i c r o c u r i e s o f c a r b o n - 1 4 DDT a n d 9 mg o f u n l a b e l e d DDT w e r e m i x e d i n a b a l l m i l l w i t h 2.5 k g o f s o i l a n d 52g o f c o n t r o l t u r k e y e x c r e t a . The amount o f DDT added t o t h e s o i l c o r r e s p o n d e d to a n a p p l i c a t i o n r a t e o f a p p r o x i m a t e l y 1.5 l b s / a c r e . F o r Tank C, c o n t r o l t u r k e y e x c r e t a was m i x e d w i t h t h e t o p two i n c h e s of s o i l as p r e v i o u s l y described. A f t e r t h e t e r r e s t r i a l p o r t i o n was f o r m e d , 8 l i t e r s o f r e f e ­ r e n c e w a t e r (4) w e r e added t o t h e s y s t e m . The e x c r e t a was a l l o w e d t o age f o r 4 weeks. D u r i n g t h e a g i n g p e r i o d , d i s t i l l e d w a t e r was added whenever needed t o keep t h e l e v e l o f t h e a q u a t i c portion constant. A t t h e e n d o f 4 w e e k s , f i f t y sorghum (sorghum h a l p e n s e ) s e e d s were sowed i n 5 rows a l o n g t h e f l a t t e n e d t e r ­ r e s t r i a l end. A f t e r 3 t o 4 d a y s when t h e s e e d s h a d g e r m i n a t e d , 3 l i t e r s more o f r e f e r e n c e w a t e r were added a n d t h e l e v e l o f w a t e r was k e p t c o n s t a n t t h r o u g h o u t t h e r e m a i n d e r o f t h e s t u d y . At this p o i n t , t h e f o l l o w i n g w e r e added t o t h e a q u a t i c p o r t i o n : 100 D a p h n i a magna, 10 G y r a u l i s s n a i l s , a s t r a n d o f a l g a e ( R h i z o ^ c I o n i u m and L y n g b i a ) a n d 10 m i l l i l i t e r s o f pond w a t e r w h i c h p r o v i d e d t h e p l a n k t o n c u l t u r e . When t h e s e e d l i n g s w e r e 3 weeks o l d , ten e a r l y f i f t h i n s t a r s a l t marsh c a t e r p i l l a r (Estigmene a c r e a ) l a r v a e w e r e p l a c e d o n t h e sorghum p l a n t s . Two t o t h r e e s e e d l i n g s were removed p r i o r t o a d d i t i o n o f t h e l a r v a e i n o r d e r to d e t e r m i n e t h e g r o s s u p t a k e o f r a d i o a c t i v i t y b y t h e p l a n t s .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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A f i n e mesh w i r e s c r e e n was t h e n f i t t e d o v e r t h e t a n k s t o c o n f i n e t h e l a r v a e on t h e p l a n t s . The c a t e r p i l l a r s consumed t h e p l a n t s w i t h i n 3 t o 4 days and c o n t a m i n a t e d t h e w a t e r w i t h t h e i r e x c r e t a and l e a f f r a s s . I n t h e i r s e a r c h f o r more f o o d , t h e c a t e r p i l l a r s a l s o ended up c o n t a m i n a t i n g t h e w a t e r t h e m s e l v e s . Approximately 300 m o s q u i t o l a r v a e ( A n o p h e l e s q u a d r i m a c u l a t u s ) were added t o t h e e c o s y s t e m a f t e r 26 days and 4 days l a t e r 50 were removed f o r d e t e r m i n a t i o n of gross r a d i o a c t i v i t y . At t h i s p o i n t , t h r e e m o s q u i t o f i s h (Gambusia a f f i n i s ) were i n t r o d u c e d and a l l o w e d t o e a t t h e r e m a i n i n g m o s q u i t o l a r v a e and t h e D a p h n i a . A f t e r t h r e e d a y s (Day 3 3 ) , t h e e x p e r i m e n t was t e r m i n a t e d and t h e d i f f e r e n t components o f t h e s y s t e m were a n a l y z e d f o r c a r b o n - 1 4 r e s i d u e s . R e s u l t s and

Discussion

A p p r o x i m a t e l y 60% o f t h e c a r b o n - 1 4 r o b e n i d i n e a d m i n i s t e r e d to t h e t u r k e y was r e c o v e r e About 84% o f t h e r a d i o a c t i v i t m e t h a n o l , a n o t h e r 9% was e x t r a c t e d w i t h a 1% h y d r o c h l o r i c a c i d / m e t h a n o l m i x t u r e l e a v i n g a b o u t 7% u n e x t r a c t e d . I t was f o u n d by TLC t h a t 75% o f t h e m e t h a n o l - s o l u b l e r a d i o a c t i v i t y was due t o t h e p r e s e n c e o f unchanged r o b e n i d i n e ( F i g u r e 3 ) . Metabolites 1, 2, and 3 a c c o u n t e d f o r 7, 2 and 0.7% o f t h e e x t r a c t a b l e radioactivity, respectively. Sorghum s e e d l i n g s r a d i o a s s a y e d f o r c a r b o n - 1 4 r e s i d u e s a t t h e t i m e o f l a r v a l f e e d i n g showed l o w l e v e l s o f 0.004 and 0.013 ppm f o r c a r b o n - 1 4 DDT and c a r b o n - 1 4 r o b e n i d i n e , r e s p e c t i v e l y . R e s i d u e l e v e l s o f c a r b o n - 1 4 i n w a t e r were l o w , e s p e c i a l l y i n t h e c a s e o f c a r b o n - 1 4 DDT, i n d i c a t i n g t h a t D D T - r e l a t e d r e s i d u e s r e m a i n bound t o t h e s o i l ( F i g u r e 4 ) . A f t e r an i n i t i a l c o n c e n t r a t i o n o f 0.009 ppb a t Day 1, t h e c o n c e n t r a t i o n o f c a r b o n - 1 4 DDT r e s i d u e s r e a c h e d an e q u i l i b r i u m o f a b o u t 0.02 ppb by t h e t h i r d day and t h e n d r o p p e d o f f s l i g h t l y t o 0.012 to 0.013 ppb a t t h e t i m e t h e m o s q u i t o l a r v a e and f i s h w e r e i n t r o d u c e d . Carbon-14 r e s i d u e s i n w a t e r d e r i v e d f r o m c a r b o n - 1 4 r o b e n i d i n e showed an i n i t i a l c o n c e n t r a t i o n o f 0.344 ppb and t h e n r e m a i n e d f a i r l y c o n s t a n t a t a b o u t 1 ppb t h r o u g h o u t t h e s t u d y , i n d i c a t i n g t h a t r o b e n i d i n e - r e l a t e d residues are p o l a r i n nature and r e a d i l y m i g r a t e i n t o t h e w a t e r p h a s e and r e a c h e q u i l i b r i u m very r a p i d l y . The n a t u r e o f t h e r a d i o a c t i v i t y i n t h e w a t e r , s o i l and f i s h f r o m t h e c a r b o n - 1 4 DDT e x p e r i m e n t was e x a m i n e d by t h i n - l a y e r c h r o m a t o g r a p h y as shown i n F i g u r e 5. The r a d i o a c t i v i t y i n t h e w a t e r was v e r y p o l a r i n n a t u r e and d i d n o t m i g r a t e a p p r e c i a b l y from the o r i g i n . About 78% o f t h e r a d i o a c t i v i t y i n t h e s o i l was e x t r a c t e d w i t h m e t h a n o l . The m a j o r m e t a b o l i t e i n t h e e x t r a c t a b l e f r a c t i o n was DDD w h i c h r e p r e s e n t e d 33% o f t h e t o t a l r a d i o activity. The r e d u c t i v e d e c h l o r i n a t i o n o f DDT t o DDD i s a known pathway u n d e r a n a e r o b i c c o n d i t i o n s and has been shown t o be due to m i c r o b i a l m e t a b o l i s m ( 5 ) . S i n c e c a r b o n - 1 4 DDT was i n c o r -

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CABALLA ET AL.

Figure 3.

Terrestrial-Aquatic

Model

Ecosystem

TLC of the extractable radioactivity from the excreta of turkeys

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CONCENTRATION IN THE

WATER

AT

SORQHUM

OF

CARBON-14

DIFFERENT PLANTS

BY

RESIDUES

DAYS

(CALCULATED

FOLLOWING

SALT

MARSH

LARVAL

A8

PARENT)

FEEDING

OF

CATERPILLAR

14C-ROBENIDINE

0.01 L _ B _

10

15

20

25

30

DAYS Figure 4

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

35

CABALLA ET AL.

T erre striai-Aquatic

Model

Ecosystem

189

Figure 5. TLC of the fish and soil extractable radioactivity and water from the C-DDT experiment. Solvent system used was petroleum etheridiethyl ether (9:1).

14

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PESTICIDE AND

XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

porated i n t o the s o i l w i t h the e x c r e t a i t i s l i k e l y t h a t a n a e r o b i c c o n d i t i o n s p r e v a i l e d . P a r e n t DDT a c c o u n t e d f o r 44% of the e x t r a c t a b l e r a d i o a c t i v i t y w i t h t r a c e s (1.1%) of DDE. A b o u t 13% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y was a component more p o l a r t h a n DDT and t h e r e m a i n i n g r a d i o a c t i v i t y s t a y e d a t o r near the o r i g i n . E x t r a c t i o n o f t h e f i s h showed t h a t 8 1 % o f t h e c a r b o n - 1 4 r e s i d u e s were o r g a n o s o l u b l e , 13% w e r e p o l a r w a t e r - s o l u b l e p r o d u c t s and 6% u n e x t r a c t a b l e . Chromatography of the organos o l u b l e r a d i o a c t i v i t y showed t h a t a l a r g e p r o p o r t i o n (87%) was s t i l l v e r y p o l a r i n n a t u r e w i t h DDT a c c o u n t i n g f o r 8%, DDE 3% and DDD 2%. T h i n - l a y e r chromatography of the water from the carbon-14 r o b e n i d i n e s t u d y showed a b o u t 12% (0.138 ppb) p a r e n t compound, 27% (0.319 ppb) o f M e t a b o l i t e No. 2 and 6 1 % (0.734 ppb) p o l a r r a d i o a c t i v i t y which d i d not m i g r a t e f a r from the o r i g i n (Figure 6). The r e s u l t s i n d i c a t p r e d o m i n a n t component o and e x t e n s i v e l y d e g r a d e d i n t o p o l a r compounds w h i c h end up i n the a q u a t i c phase. A b o u t 20% o f t h e r a d i o a c t i v i t y i n t h e s o i l f r o m t h e c a r b o n 14 r o b e n i d i n e t a n k was e x t r a c t a b l e a t t h e end o f t h e e x p e r i m e n t w i t h 80% r e m a i n i n g u n e x t r a c t a b l e . Chromatography of the e x t r a c t a b l e r a d i o a c t i v i t y showed e x t e n s i v e d e g r a d a t i o n o f t h e compound as shown i n F i g u r e 7. R o b e n i d i n e , w h i c h was t h e m a j o r component i n t h e e x c r e t a , r e p r e s e n t e d a b o u t 10% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y i n t h e soil. I n terms of t o t a l carbon-14 r e s i d u e s i n the s o i l , p a r e n t compound r e p r e s e n t e d 2.0%. M e t a b o l i t e 2, w h i c h was p r e s e n t o n l y i n t r a c e q u a n t i t i e s i n t h e e x c r e t a , a c c o u n t e d f o r 21% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y o r 4.2% o f t h e t o t a l c a r b o n - 1 4 r e s i d u e s i n the s o i l . T h i s m e t a b o l i t e was a l s o t h e o n l y s i g n i f i c a n t compound f o u n d i n t h e w a t e r . T h r e e o t h e r m e t a b o l i t e s a c c o u n t e d f o r a b o u t 18% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y i n t h e s o i l , n a m e l y , M e t a b o l i t e 3, 5.3%, M e t a b o l i t e 6, 7.6% and M e t a b o l i t e 10, 4.9%. P o l a r m a t e r i a l w h i c h was n o t r e s o l v e d f r o m t h e o r i g i n r e p r e s e n t e d 25% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y i n t h e s o i l . The r e m a i n i n g r a d i o a c t i v i t y was d i s t r i b u t e d among t e n m i n o r components. I n t h e f i s h , t h e l a s t e l e m e n t i n t h e f o o d c h a i n web, m e t h a n o l e x t r a c t e d a b o u t 58% o f t h e r a d i o a c t i v i t y r e s u l t i n g f r o m t h e c a r b o n - 1 4 r o b e n i d i n e t r e a t m e n t l e a v i n g 42% u n e x t r a c t e d , i n d i c a t i n g t h a t r o b e n i d i n e was b e i n g e x t e n s i v e l y d e g r a d e d by f i s h into very polar nonextractable products. T h i n - l a y e r chromatography of the e x t r a c t a b l e r a d i o a c t i v i t y d i d n o t show any p a r e n t compound e v e n t h o u g h r o b e n i d i n e was one o f t h e components i n t h e e n v i r o n m e n t ( w a t e r ) ( F i g u r e 8 ) . The c o n c e n t r a t i o n o f c a r b o n - 1 4 r e s i d u e s i n t h e d i f f e r e n t components o f t h e m o d e l e c o s y s t e m i s compared i n T a b l e 3,· The b i o c o n c e n t r a t i o n f a c t o r ( B C F ) , d e f i n e d as t h e r a t i o o f t h e

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CABALLA ET AL.

Figure 6.

Figure 7.

T err est rial-Aquatic

Model

Ecosystem

TLC of the water from the C-robenidine model ecosystem 14

TLC of the methanol-soluble radiactivity in the aged soil/excreta mixture from the C-robenidine model ecosystem 14

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

192

Figure 8.

ORGANISMS

TLC of the methanol-soluble radioactivity in the fish from the robenidine model ecosystem

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

lA

C-

12.

CABALLA ET AL.

Terrestrial-Aquatic

Model

193

Ecosystem

Table I . COMPARISON

OF

THE

CONCENTRATIONS*'

AND

BIOCONCENTRATION

AND

CARBON-14

FACTOR

ROBENIDINE

COMPONENT

PPM

(BCF>

MODEL

BCF

2 /

OF IN

CARBON-14 THE

RESIDUES

CARBON-14

EC08Y8TEM8

PPM

BOP

WATER

0.001

ALQAE

0.030

33

0.0022

138

0.0124

10

0.0034

214

M08QUITOLARVAE

0.181

178

0.0038

314

FISH

0.081

88

0.0180

1120

8NAIL8

1/ CALCULATED

2

'

B C F

AS

0.000014

PARENT

. CONCENTRATION CONCENTRATION

OF CARBON-14 RESIDUES IN THE OF CARBON-14 RESIDUES IN THE

ORGANISM WATER

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

DDT

194

PESTICIDE

A N D XENOBIOTIC

METABOLISM

IN AQUATIC

ORGANISMS

c o n c e n t r a t i o n o f carbon-14 r e s i d u e s i n t h e organism t o t h e c o n c e n t r a t i o n o f c a r b o n - 1 4 r e s i d u e s i n t h e w a t e r , i s a l s o shown. The d a t a c l e a r l y i n d i c a t e s t h a t t h e BCF v a l u e s o b t a i n e d i n a l l t h e e l e m e n t s o f t h e f o o d c h a i n were h i g h e r f o r t h e c a r b o n - 1 4 DDT e x p e r i m e n t compared w i t h t h o s e f o r t h e c a r b o n - 1 4 r o b e n i d i n e e x p e r i m e n t . T h i s d i f f e r e n c e i s more p r o m i n e n t i n t h e c a s e o f s n a i l s a n d f i s h , t h a t i s , 214 v s 10 and 1129 v s 6 8 , r e s p e c t i v e l y . A l l t h e s e r e s u l t s s u g g e s t t h a t t h e u s e o f e x c r e t a a s manure f r o m b i r d s k e p t on a d i e t c o n t a i n i n g r o b e n i d i n e w i l l n o t r e s u l t i n any b i o l o g i c a l m a g n i f i c a t i o n o f r o b e n i d i n e - r e l a t e d r e s i d u e s i n the elements o f t h e environment. Conclusion The m o d i f i e d t e r r e s t r i a l - a q u a t i c m o d e l e c o s y s t e m d e s c r i b e d h e r e h a s b e e n f o u n d t o be a u s e f u l t o o l i n s t u d y i n g t h e e n v i r o n ­ m e n t a l f a t e o f d r u g s an e x c r e t a u s e d as manure r e l a t i v e l y s i m p l e and y e t i t a l l o w s one t o s t u d y t h e complex metabolic transformations o f a drug o r r e l a t e d r e s i d u e s i n i t s v a r i o u s components. E s p e c i a l l y i n t e r e s t i n g i s t h e s t u d y o f t h e d e g r a d a t i o n o f a compound i n t h e s o i l i n t h e p r e s e n c e o f m i c r o ­ organisms found i n t h e animal e x c r e t a . This i n f o r m a t i o n i s i m p o r t a n t s i n c e i t e v e n t u a l l y d e t e r m i n e s w h e t h e r a compound a n d / or i t s m e t a b o l i t e s w i l l bioaccumulate i n t h e v a r i o u s elements o f the environment.

Literature Cited 1. Metcalf, R. L., Sangha, G. Κ., and Kapoor, I. P., Environ­ mental Science and Technology, (1971), 5, 709-713. 2. Kantor, S., Kenneth, R. L., Jr., Waletzky, E., Tomcufcik, A. S., Science, (1970), 168, 373. 3. Kapoor, I. P., Metcalf, R. L., Nystrom, R. F., and Sangha, G. K., J. Agr. Food Chem., (1970), 18, 1145. 4. Freeman, L., Sewage Ind. Wastes, (1953), 25, 845, 1331. 5. Burge, W. D., J. Agr. Food Chem., (1971), 19 375-378. RECEIVED

January 10, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

13 Modeling Aquatic Ecosystems for Metabolic Studies ALLAN R. ISENSEE, PHILIP C. KEARNEY, and GERALD E. JONES U.S. Department of Agriculture, SEA, AR, Agricultural Environmental Quality Institute, Pesticide Degradation Laboratory, Beltsville, MD 20705

M o d e l e c o s y s t e m s hav ure t h e d i s t r i b u t i o n an e n v i r o n m e n t . Over t h a t p e r i o d o f t i m e numerous d e s i g n c h a n g e s have e v o l v e d t h a t h a v e i n c r e a s e d t h e v e r s a t i l i t y o f t h e e c o s y s ­ tem a n d i m p r o v e d s i m u l a t i o n o f e n v i r o n m e n t a l c o n d i t i o n s . I n o u r l a b o r a t o r y , we have u s e d t h e s t a t i c model e c o s y s t e m p r i m a r i l y t o model t h e pond o r s m a l l l a k e e n v i r o n m e n t , a n d t o s i m u l a t e t h e l i k e l y r a t e s a n d modes o f p e s t i c i d e e n t r y ( 1 ) . More r e c e n t l y , we have d e v e l o p e d l a r g e r s y s t e m s c a p a b l e o f p r o v i d i n g s u f f i c i e n t biomass f o r a c c u m u l a t i o n and d i s s i p a t i o n r a t e d e t e r m i n a t i o n s (2) and f o r m e t a b o l i c s t u d i e s ( 3 ) . The p r i m a r y p u r p o s e o f t h i s p r o j e c t was t o d e m o n s t r a t e t h a t a q u a t i c model e c o s y s t e m s c o u l d b e f u r t h e r s c a l e d up i n s i z e t o p r o v i d e g r e a t e r amounts o f t h e components ( b i o m a s s , s o i l a n d water) t o s a t i s f a c t o r i l y study metabolism k i n e t i c s . We u s e d t r i f l u r a l i n , a d i n i t r o a n i l i n e herbicide, since i t s metabolic p a t h w a y s a r e w e l l known a n d t h e m e t a b o l i t e s were r e a d i l y a v a i l ­ able. Methods and M a t e r i a l s C h e m i c a l s a n d E x p e r i m e n t a l Chambers The c h e m i c a l name a n d s t r u c t u r e o f t r i f l u r a l i n a n d t h e e i g h t m e t a b o l i t e s used i n t h i s study are g i v e n i n T a b l e I . A l l n i n e compounds h a d a c h e m i c a l p u r i t y g r e a t e r t h a n 98.7% a n d t h e [ B e ­ r i n g ] t r i f l u r a l i n ( s p e c i f i c a c t i v i t y 45.25 yCi/mg) h a d a r a d i o p u r i t y g r e a t e r than 97%. F o u r t e e n k g o f M a t a p e a k e s i l t loam (pH 5.3, 1.5% O.M.; s a n d , s i l t , a n d c l a y c o n t e n t s o f 38.4, 49.4, a n d 12.7% r e s p e c t i v e l y ) were t r e a t e d w i t h [ - ^ c ] t r i f l u r a l i n a t t h e r a t e o f 10 ppm a n d i n t r o d u c e d i n t o t h e e c o s y s t e m t a n k s a s d e s c r i b e d b e l o w . The c o n t r o l tank contained f o u r t e e n kg o f u n t r e a t e d s o i l . G l a s s a q u a r i a , m e a s u r i n g 75 χ 29 χ 45 cm, were u s e d a s t h e e c o s y s t e m chambers ( F i g u r e s 1 a n d 2 ) . T w e n t y - f o u r s o i l s a m p l i n g

This chapter not subject to U.S. copyright. Published 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Compound No.

3

C

N0

NH

α,α,α-Trifluoro-2,6-dinitro-N(3h y d r o x y p r o p y l ) -p_- t o l u i d i n e

a,a,a-Trifluoro-N ,N -dipropyltoluene 3,4,5-triamine

4

N0

a,a,a-Trifluoro-2,6-dinitro-N(2h y d r o x y p r o p y 1 ) -p_- t o l u i d i n e

4

N0

2-Ethyl-7-nitro-l-peopyl-5(trifluoromethyl)-benzimidazole

2

2

2

2

2

7

:

2

NH 3

2

^-(C H )

2

2

N0

N0

3

HN-CH CH CH OH

HN-CH CHOHCH

and i t s M e t a b o l i t e s .

N0

Name

F

I. Trifluralin

2-Ethyl-4-nitro-6-(trifluoromethyl benzimidazole

Table

2

13.

isENSEE E T A L .

x

CM

ΓΜ

o

2

Modeling

Χ

2

(N

2

U

to

I

I

CM /—\

Χ to

Χ to U

ν—'

χ

CM ο

/—\

υ

2

Ecosystems

2

(Ν

Χ

Aquatic

2

(Ν

CM

S 2

(Ν

Q 2

ο 2

21

^

21

I

0

Ζ|·Η

ô

i

•Η G I LO I ο

I

% · to I ω u c ο ο

T3

c

•H

r-H ΜΗ •Η μ Ει

δ*

r—(

Ο

4-> ΓΗ

Χ

ΡΗ

ο

?Η

ο +->

•Η Ρ! •Η Τ3 I (D νΟ •Η (Ν Ό I •Η

Ο u ι-Η ο Ο +->

ι-Η ΜΗ a i •Η I ι-Η ι

ΡΗ

Ο

rH

ΡΗ

δ

ιι ° 2| I c •Η

lit g 2| ι ^3 ι •Η ο Π3 ΓΗ I 4-> ^ · •Η to ι I LQ ω I

Ο u

ο r—(

ι-Η ΜΗ •Η rH Ε­ ι

δ

Ο

-Ρ ι—ι >% ΡΗ

Ο

ΡΗ

·\ Ό•Η

2| I ι

Ο •Η •Η Τ3 I •Η \Ω Τ3 ·> •Η CM 3 I ι-Η ο Ο

u ο

ι-Η ΜΗ •Η rH Ε­ Ι

+->

pj ι rH Χ ΡΗ

Ο

rH

a ΡΗ •Η δ Ό ·\ δ

c

8

•8 E-

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

197

198

PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

Figure 1.

Side view of ecosystem chamber detailing rehtive proportion and distribution of ecosystem components

REFERENCE ,··* ELECTRODE

REDOX

ELECTRODES

END VIEW Figure 2.

End view of ecosystem chamber showing detail of daphnid chamber and positioning of redox electrodes

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

13.

ISENSEE E T A L .

Modeling

Aquatic

Ecosystems

199

t u b e s (2.5 cm. d i a χ 4 cm h i g h ; F i g u r e 1) w e r e r a n d o m l y l o c a t e d on t h e b o t t o m o f t h e t a n k s . A n aluminum w i r e hoop was cemented ( s i l i c o n s e a l a n t ) t o each t u b e t o a i d sample r e t r i e v a l . A t one end o f t h e c o n t r o l t a n k ( F i g u r e 2) t h r e e h o l e s were d r i l l e d , 1, 2, a n d 3 cm f r o m t h e b o t t o m a n d 2 cm a p a r t ( h o r i z o n t a l l y ) . Two p l a t i n u m d i s c ( t o p and bottom h o l e s ) and one c a l o m e l r e f e r e n c e ( c e n t e r h o l e ) e l e c t r o d e s were cemented i n p l a c e ( s i l i c o n r u b b e r sealant). T h e s e e l e c t r o d e s were u s e d t o measure t h e Eh o f t h e soil. The s o i l ( t r e a t e d o r u n t r e a t e d ) was u n i f o r m l y d i s t r i b u t e d over the bottom o f the tanks and i n the sampling tubes. Only t h e hoop o n t h e s a m p l i n g t u b e s p r o t r u d e d f r o m t h e d i s t r i b u t e d soil. The t h r e e t a n k s were t h e n f i l l e d w i t h 84 l i t e r s o f w a t e r . One day a f t e r f l o o d i n g , 75 b l u e g i l l f i s h (Lepomis m a c r o c h i n u s ) , 60 s n a i l s ( H e l i s o m a s p . ) , 2 grams a l g a e (Oedogonium c a r d i a cum) a n d s e v e r a l h u n d r e d d a p h n i d s ( D a p h n i a magna) w e r e added t o t h e chambers. The d a p h n i d (25 χ 20 χ 15 cm) s u s p e n d e t a n k ( F i g u r e 2 ) . An o p e n i n g i n t h e t a n k b o t t o m was c o v e r e d w i t h a s t a i n l e s s s t e e l s c r e e n w i t h a mesh s u f f i c i e n t l y s m a l l (0.38 mm) t o r e s t r i c t t h e p a s s a g e o f d a p h n i a . A p e r c o l a t o r w a t e r pump c o n t i n u o u s l y pumped w a t e r i n t o t h e d a p h n i d t a n k , e n s u r i n g u n i f o r m m i x i n g o f t h e w a t e r a n d t r a n s p o r t o f f o o d t o d a p h n i d s . The e x p e r ­ i m e n t was c o n d u c t e d i n t h e g r e e n h o u s e u s i n g n a t u r a l l i g h t a t a n a v e r a g e t e m p e r a t u r e o f 27 ί 3 C. S a m p l i n g and A n a l y s i s . W a t e r s a m p l e s ( t r i p l i c a t e 1 ml) w e r e t a k e n a t 2-day i n t e r v a l s a n d a n a l y z e d b y s t a n d a r d l i q u i d s c i n t i l ­ l a t i o n (LS) methods f o r t o t a l C ; 100 m l samples were t a k e n 2, 5, 9, 15, 2 2 , 3 0 , 4 3 , 4 8 , a n d 58 days a f t e r t h e s t a r t o f t h e e x p e r i m e n t , ; t h e s e were e x t r a c t e d t w i c e w i t h 50 m l o f e t h y l a c e t a t e : h e x a n e (7:3 v / v ) a n d t h e e x t r a c t s were r e d u c e d t o 20 m l and a n a l y z e d b y LS a n d T L C . Two s o i l s a m p l i n g t u b e s were removed f r o m e a c h t a n k 2, 5, 9, 15, 27, 3 0 , 4 3 , a n d 58, a n d 72 days a f t e r t h e s t a r t o f t h e e x p e r ­ iment, then f r o z e n and s t o r e d f o r l a t e r a n a l y s e s . For a n a l y s e s , t h e f r o z e n s o i l c o r e s were removed f r o m t h e g l a s s t u b e s (by b r i e f i m m e r s i o n i n h o t w a t e r ) , t h e n s e c t i o n e d i n t o f o u r 1-cm c y l i n d e r s r e p r e s e n t i n g 0-1, 1-2, 2-3, a n d 3-4 cm s o i l d e p t h s . Samples f r o m each d e p t h were s h a k e - e x t r a c t e d w i t h 100 ml e t h y l a c e t a t e : h e x a n e o v e r n i g h t , a n d a g a i n w i t h 100 m l m e t h a n o l o v e r n i g h t . Extracts w e r e f i l t e r e d , c o n c e n t r a t e d t o 20 m l a n d a n a l y z e d b y LS a n d TLC as d e s c r i b e d b e l o w . Samples o f o r g a n i s m s (7 f i s h , 6 s n a i l s , 0.5 g a l g a e , a n d 0.5 t o 1.5 g d a p h n i d s ) were t a k e n a f t e r 2, 5, 9, 1 5 , 2 2 , a n d 30 d a y s . A l l r e m a i n i n g o r g a n i s m s were removed a f t e r 42 d a y s a n d a d d i t i o n a l o r g a n i s m s were a d d e d (27 f i s h , 30 s n a i l s , 2 g a l g a e , a n d s e v e r a l h u n d r e d d a p h n i d s ) t h e same day. Samples o f o r g a n i s m s were a g a i n t a k e n o n Days 44, 4 8 , 58 a n d 72. A l l s a m p l e s w e r e w e i g h e d , t h e n f r o z e n f o r l a t e r p r o c e s s i n g . A l g a e s a m p l e s were o x i d i z e d t o de­ t e r m i n e t o t a l l ^ C . F i s h , s n a i l s , and daphnids were homogenized i n m e t h a n o l a n d a n a l y z e d b y LS a n d T L C . 1 4

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

200

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

The i d e n t i t y o f t r i f l u r a l i n and compounds 1-8 were d e t e r m i n e d by c o - c h r o m o t o g r a p h y as f o l l o w s . E x t r a c t s f r o m s o i l , w a t e r , and o r g a n i s m s were r e d u c e d ( u n d e r N ) t o 0.1 m l , c o m b i n e d w i t h 10 t o 20 u l e a c h o f compounds 1-9; t h e n s p o t t e d on s i l i c a g e l TLC p l a t e s (20 χ 20 cm FG-254, E. M e r c k , D a r m s t a d t ) . The p l a t e s were d e v e l ­ oped two d i m e n s i o n a l l y , f i r s t i n b e n z e n e f o r 15 cm, t h e n i n b e n ­ zene: e t h y l a c e t a t e : a c e t i c a c i d (60:40:1). Spots c o r r e s p o n d i n g t o t h e n i n e compounds were l o c a t e d v i s i b l y and by UV l i g h t , t h e n s c r a p e d and a n a l y z e d by LS. I n a d d i t i o n , t h e o r i g i n and a d i f f u s e zone b e t w e e n t h e o r i g i n and compound 1 were s c r a p e d and a r e t e r m e d " p o l a r " and " n o n p o l a r " m e t a b o l i t e s , r e s p e c t i v e l y ( F i g u r e 3 ) . 2

R e s u l t s and

Discussion

A l l o r g a n i s m s t h r i v e d i n t h e s y s t e m s w i t h no l o s s o f f i s h or s n a i l s . Numerous egg c l u s t e r s and s m a l l s n a i l s were e v i d e n t i n a l l t a n k s by Day 42 (whe ted). T h e s e egg c l u s t e r t h e s e c o n d s e t o f s n a i l s was s u f f i c i e n t l y l a r g e t h a t i d e n t i t y a t h a r v e s t was n o t a p r o b l e m . The a l g a e w e i g h t i n c r e a s e d f r o m 2 g ( i n i t i a l ) t o an a v e r a g e o f 9.4 g i n t h e t r e a t m e n t t a n k s and 26.3 g i n the c o n t r o l . However, f o r t h e s e c o n d s e t o f a l g a e , t h e r e was no g r o w t h d i f f e r e n c e d u r i n g t h e e x p o s u r e p e r i o d (Days 42 - 7 2 ) . The c o n c e n t r a t i o n o f t r i f l u r a l i n i n w a t e r d e c r e a s e d r a p i d l y w i t h t i m e ( w h i c h w i l l be d i s c u s s e d i n d e t a i l l a t e r i n t h i s p a p e r ) . Thus any i n i t i a l e f f e c t o f r e d u c i n g a l g a e g r o w t h was l o s t w i t h time. D a p h n i d s r e p r o d u c e d ( i n b o t h t r e a t m e n t and c o n t r o l t a n k s ) q u i c k l y i n c r e a s i n g t h e i r mass a t l e a s t 10 t i m e s i n 15 days and t h e n m a i n t a i n e d t h i s d e n s i t y f o r up t o 42 d a y s . The s e c o n d s e t of daphnids behaved s i m i l a r i l y . D e g r a d a t i o n o f T r i f l u r a l i n i n Submerged S o i l . The r e d o x p o t e n t i a l o f t h e s o i l became n e g a t i v e a f t e r o n l y 3 days and r e a c h e d a low o f -450 mv a f t e r 32 days ( T a b l e I I ) . T h e r e was l i t t l e d i f f e r e n c e i n t h e measurements between t h e 1 and 3 cm s o i l d e p t h s . We t h e r e f o r e assume t h a t a l l s o i l s a m p l e s ( e x c e p t p o s s i b l y a t Day 2) were a n a e r o b i c . O n l y a b o u t 6% o f t h e o r i g i n a l C a p p l i e d t o s o i l as Ct r i f l u r a l i n was l o s t a f t e r 72 d a y s ( F i g u r e 4 ) . A l s o , t h e a c e t a t e : hexane and m e t h a n o l e x t r a c t s ) s t e a d i l y d e c r e a s e d w i t h t i m e t o a low o f a b o u t 58% a f t e r 72 d a y s . ( V a l u e s shown i n F i g u r e 4 a r e t h e a v e r a g e o f t h e f o u r d e p t h s s i n c e t h e r e was no s i g n i f i c a n t d i f ­ f e r e n c e i n t h e C c o n t e n t b e t w e e n t h e m ) . T h e s e r e s u l t s show t h a t t h e d i f f u s i o n o f t r i f l u r a l i n a n d / o r i t s m e t a b o l i t e s f r o m a submer­ ged s o i l i s a s l o w p r o c e s s , w h i l e a t t h e same t i m e , c o n v e r s i o n t o t h e "bound" o r n o n e x t r a c t a b l e form (35 t o 40% a t 72 d a y s ) o c c u r s . P r o b s t e t a l . (4) f o u n d more r a p i d c o n v e r s i o n t o bound r e s i d u e s : n e a r l y 60% o f t h e t o t a l C f r o m a submerged s o i l was u n e x t r a c t ­ a b l e a f t e r o n l y 14 d a y s . The a n a l y s i s o f t h e s o i l e x t r a c t s by TLC i s shown i n T a b l e III. (The p e r c e n t a g e v a l u e s a r e b a s e d on t h e d i s t r i b u t i o n o f C 1 4

1 4

1 4

1 4

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

13.

ISENSEE E T A L .

Modeling

Aquatic

201

Ecosystems

15

t II Benzene

60

H O

h5 1 Non

Polar

Polar

jjjjjjjjj

Metabolites

iijjSj:

Metabolites

''l''

w

LQ

Benzene

-ι—ι 10

15

Figure 3.

ι 5

I 0

TLC system used to separate trifluralin and metabolites extracted from ecosystem components

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

202

T a b l e I I . Redox p o t e n t i a l s ( e x p r e s s e d i n m i l l i v o l t s ) a t two d e p t h s i n a f l o o d e d s o i l .

Soil

depth

a

Days

1 cm

3 cm

0

+300

+300

1

+205

+220

5

-210

-80

6

-260

-140

7

-290

-250

8

-320

-270

9

-340

-300

12

-360

-320

16

-380

-350

19

-405

-370

22

-430

-395

25

-450

-420

30

-460

-440

2

Measurements t a k e n 1 a n d 3 cm b e l o w s o i l

surface.

Days a f t e r s t a r t o f e x p e r i m e n t .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ISENSEE ET AL.

Modeling

Aquatic

203

Ecosystems

50"

4

0

10

20

30

40

50

60

70

DAYS Figure 4.

Residual and extractable C from C-trifluralin-treated 14

14

anaerobic soil

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

0

0.3 3.4 0.3 1.1 1.1 0.5 0.1 2.1 0.1 89.4

0.4

1.2

.9 5.0 0.5 0.6 3.6 0.6 0.2 1.8 2.8 78.9

3.0

2.1

i 4

2.1 2.5 0.9 0.9 2.2 2.0 0.2 7.4 13.3 47.9

15.0

5.6

4.2 1.6 1.0 1.0 2.0 4.6 0.3 2.7 13.0 44.8

18.0

6.8

4

Values represent percent o f t o t a l C recovered from s o i l a c e t a t e : h e x a n e a n d m e t h a n o l ) b y TLC m e t h o d s . 1 C r e c o v e r e d from t h e o r g i n . 14c r e c o v e r e d i n a zone b e t w e e n o r g i n a n d compound 1. 14c r e c o v e r e d b e t w e e n compounds 1 a n d 2.

0

P o l a r metabolites Nonpolar metabolites 1 Unknown^ 2 3 4 5 6 7 8 Trifluralin

extracts

16.4 1.6 1.0 1.2 2.2 6.2 0.4 3.0 7.9 40.4

11.7

8.0

(ethyl

14.3 1.2 1.0 1.3 2.5 5.9 0.3 4.5 1.3 39.3

10.6

17.8

Table I I I . Degradation o f [ C ] t r i f l u r a l i n i n a f l o o d e d s o i l (expressed as percent of extracted r a d i o a c t i v i t y ) . Days a f t e r s t a r t o f e x p e r i m e n t 9 22 30 42 71 Compounds 2

ISENSEE E T A L .

13.

Modeling

Aquatic

205

Ecosystems 4

r e c o v e r e d f r o m t h e i n d i v i d u a l TLC p l a t e s , r e p r e s e n t i n g t h e ^ C i n the e x t r a c t s , not thet o t a l s o i l ) . T r i f l u r a l i n appeared t o deg r a d e b o t h b y s e q u e n t i a l r e d u c t i o n o f t h e n i t r o g r o u p s (Compounds 8 a n d 5) a n d b y d e a l k y l a t i o n (Compound 7 ) , T a b l e I I I , F i g u r e 3. These a r e a l l r e c o g n i z e d d e c o m p o s i t i o n p r o d u c t s i n the a n a e r o b i c d e g r a d a t i o n p a t h w a y a s p r o p o s e d b y P r o b s t £t a l _ ( 4 ) . The i n t e r m e d i a t e s i n t h e f o r m a t i o n o f Compound 1 i n o u r s y s t e m a r e unknown, s i n c e i t c o u l d form e i t h e r from t r i f l u r a l i n d i r e c t l y ( v i a s e v e r a l i n t e r m e d i a t o r s ) o r f r o m Compound 7 ( F i g u r e 5 ) . However, o u r r e c o v e r y o f Compound 1 s u p p o r t s a p r e v i o u s o b s e r v a t i o n (5) t h a t i t forms under a n a e r o b i c c o n d i t i o n s , b u t p r o b a b l y i s a minor pathway. The a c c u m u l a t i o n o f p o l a r a n d n o n p o l a r m e t a b o l i t e s a l s o agreed w i t h p r e v i o u s s t u d i e s . Compounds 2, 3, 4, a n d 6 w e r e p r o b a b l y n o t p r e s e n t i n o u r system as i n d i c a t e d b y t h e low r e c o v e r y of C (1 t o 3 . 6 % ) . The l e v e l o f C was t o o l o w t o c o n f i r m t h e p r e s e n c e o f t h e s e compounds b y T L C In g e n e r a l , t h e d e g r a d a t i o submerged s o i l o f o u r e c o s y s t e ed b y p r e v i o u s i n v e s t i g a t o r s ( 4 , 6 ) . However, t h e r a t e o f t r i f l u r a l i n d e g r a d a t i o n was much s l o w e r . F o r e x a m p l e , a b o u t 8% o f t h e total C i n t h e s o i l was t r i f l u r a l i n a f t e r 58 d a y s ( c o n f i r m e d by e l e c t r o n c a p t u r e g a s c h r o m a t o g r a p h y ) a s compared t o a b o u t 6% a f t e r 14 d a y s b y P r o b s t et_ a l _ . (4) . S o i l o r g a n i c m a t t e r may h a v e been responsible f o r thedegradation rates. P a r r a n d S m i t h (6) m e a s u r ed t h e e x t e n t o f t r i f l u r a l i n d e g r a d a t i o n i n a s i l t loam, amended and unamended w i t h 1% a l f a l f a m e a l , u n d e r a n a e r o b i c c o n d i t i o n s . A f t e r 20 d a y s , t h e amount o f t r i f l u r a l i n r e c o v e r e d was 1% a n d 6 8 % i n t h e amended a n d unamended s o i l s , r e s p e c t i v e l y . The o r g a n i c m a t t e r i n o u r s o i l may n o t h a v e p r o m o t e d r a p i d m i c r o b i a l d e g r a d a t i o n and t h e r e f o r e r e s u l t e d i n a slower r a t e o f d e g r a d a t i o n . 1 4

1 4

%

1 4

1 4

D e g r a d a t i o n o f T r i f l u r a l i n i n W a t e r . The amount o f C r e c o v e r e d from w a t e r w i t h t i m e i s shown i n T a b l e I V . A b o u t h a l f of t h e t o t a l C ( d i r e c t c o u n t a n a l y s i s ) i n w a t e r was r e c o v e r e d by e x t r a c t i n g t w i c e w i t h e t h y l a c e t a t e : h e x a n e ( 7 : 3 ) , i n d i c a t i n g t h a t p o l a r m e t a b o l i t e s r a p i d l y formed. A l s o , t h e c o n c e n t r a t i o n of C i n w a t e r i n c r e a s e d most r a p i d l y d u r i n g t h e f i r s t 22 d a y s , a f t e r which t h e r a t e decreased. A n a l y s i s o f t h e water e x t r a c t s by TLC i s shown i n T a b l e V. T r i f l u r a l i n d i s a p p e a r e d v e r y r a p i d l y , d e c r e a s i n g t o n o n d e t e c t a b l e l e v e l s b e t w e e n 9 a n d 22 d a y s . Even as e a r l y as 2 d a y s o n l y 4 6 % o f t h e r e c o v e r e d C was t r i f l u r a l i n . A s e q u e n c i a l r e d u c t i o n o f t h e n i t r o group (decrease i n t h e concent r a t i o n o f Compound 8 w i t h t i m e f o l l o w e d by a n a c c u m u l a t i o n o f Compound 5) i s i n d i c a t e d b y t h e r e s u l t s ( F i g u r e 6 ) . Compound 1 a l s o i n c r e a s e d r a p i d l y i n c o n c e n t r a t i o n e a r l y i n t h e experiment t h e n m a i n t a i n e d a l o w e r b u t c o n s t a n t c o n c e n t r a t i o n b e t w e e n Days 22 and 5 8 . P o l a r m e t a b o l i t e s r a p i d l y i n c r e a s e d t o 5 2 % o f t h e t o t a l r e c o v e r e d C b y Day 9, t h e n g r a d u a l l y d e c r e a s e d w h i l e t h e c o n c e n t r a t i o n o f nonpolar metabolites continuously increased w i t h time. 1 4

i 4

1 4

1 4

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

206

N(C,H ) 7

2

° S N(C H ) 3

7

2

l^vjUCompound 8 CF

\

H-N-C3H7

\

LJJCompound 7 \

3

N(C,H ), 7

CF

3

« NO

2

Compound 1

^Compound 5 CF

1

3

I

Polar and Non Polar Metabolites Figure 5.

Postulated pathway of trifluralin degradation in an anaerobic soil from a model ecosystem

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

isENSEE E T A L .

Table IV.

Days

a

Modeling

Aquatic

207

Ecosystems

Concentration of C i n ecosystem water (expressed on ppb p a r e n t compound).

D i r e c t count ^

Extraction

4.7

2.8

9

11.0

5.4

15

19.1

11.0

22

27.5

9.9

30

29.9

13.6

43

34.8

15.4

48

36.2

14.7

58

38.1

-

72

37.3

-

2

c

5

Days a f t e r s t a r t o f e x p e r i m e n t l ^ c r e c o v e r e d f r o m 1 ml w a t e r 1 4

samples.

C r e c o v e r e d from water e x t r a c t e d 2 times w i t h acetate:hexane (7:3).

ethyl

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

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In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

13.

iSENSEE E T A L .

Modeling

Aquatic

N(CH ) 7

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Ecosystems

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In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

209

210

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

T r i f l u r a l i n i s known t o u n d e r g o r a p i d p h o t o d e c o m p o s i t i o n on p h y s i c a l s u r f a c e s (7) and d e g r a d e s r a p i d l y i n s h a l l o w w a t e r e x p o s ed t o s u n l i g h t ( 8 ) . The r e c o v e r y o f o n l y 46% o f t h e t o t a l C as t r i f l u r a l i n a f t e r 2 days s u g g e s t s t h a t p h o t o d e g r a d a t i o n s i g n i f i c a n t l y c o n t r i b u t e d t o t r i f l u r a l i n d e g r a d a t i o n i n our ecosystem. The d a t a s u p p o r t s t h i s c o n c l u s i o n s i n c e Compound 1, a known p h o t o p r o d u c t o f t r i f l u r a l i n ( 9 ) , r e p r e s e n t s a s u b s t a n t i a l amount o f t h e t o t a l ^ C d u r i n g the f i r s t 9 days o f the experiment ( c o r r e s p o n d i n g to the h i g h e s t c o n c e n t r a t i o n o f t r i f l u r a l i n ) . D e g r a d a t i o n o f T r i f l u r a l i n i n A q u a t i c O r g a n i s m s . The r a p i d degradation of t r i f l u r a l i n i n water s i g n i f i c a n t l y a f f e c t e d the t y p e and amount o f m e t a b o l i t e s r e c o v e r e d from f i s h , s n a i l s , daphn i d s and a l g a e ( T a b l e s V I - I X ) . I n g e n e r a l , t h e amount o f t r i f l u r a l i n r e c o v e r e d was h i g h e s t f o r t h e i n i t i a l s a m p l i n g p e r i o d s , t h e n d e c r e a s e d t o n o n d e t e c t a b l e l e v e l s between 22 and 42 d a y s . This trend c l o s e l y f o l l o w s the t r i f l u r a l i n c o n c e n t r a t i o n i n water where t r i f l u r a l i n a c c o u n t e ppb) on Day 2. T h i s ma counted f o r the h i g h e s t p r o p o r t i o n of the r e c o v e r e d , even f o r t h e f i r s t s a m p l i n g p e r i o d s . Compound 1 was t h e n e x t most p r e v a l e n t p r o d u c t r e c o v e r e d from organisms. The 14c d i s t r i b u t i o n was more c o m p l e x i n d a p h n i d s ( T a b l e V I I I ) t h a n f o r f i s h , s n a i l s , o r a l g a e , where compounds 8, 2 and n o n p o l a r m e t a b o l i t e s were a l s o detected. (No d a t a was shown i n T a b l e V I I I b e f o r e Day 15 s i n c e f o r TLC a n a l y s i s was i n s u f f i c i e n t ) . I n t e r p r e t a t i o n of the a q u a t i c o r g a n i s m d a t a i s d i f f i c u l t s i n c e we c o u l d n o t d e t e r m i n e w h e t h e r s p e c i f i c compounds ( p r i m a r i l y t h e p o l a r m e t a b o l i t e s ) w e r e f o r m e d i n t h e o r g a n i s m o r were a b s o r b e d f r o m w a t e r . However, w a t e r was p r o b a b l y t h e m a j o r s o u r c e o f t h e r e c o v e r e d compounds s i n c e l i t t l e d i f f e r e n c e was n o t e d b e t w e e n o r g a n i s m s a n a l y z e d on Day 42 and 44, r e p r e s e n t i n g e x p o s u r e t i m e s o f 42 v s . 2 d a y s , r e spectively. ( A l l o r g a n i s m s were removed from t h e t a n k s on Day 42 and r e p l a c e d w i t h a new s e t t h e same d a y ) . F a r more t r i f l u r a l i n was i n i t i a l l y r e c o v e r e d (Days 2 and 5) f r o m a l g a e t h a n any o f t h e o t h e r o r g a n i s m s ( T a b l e I X ) . However, a f t e r 30 days t h e p o l a r and n o n p o l a r m e t a b o l i t e s a c c o u n t e d f o r 75% o r more o f t h e r e c o v e r e d i n d i c a t i n g t h a t t h e a l g a e were a l s o r e s p o n d i n g t o t h e r a p i d l o s s o f t r i f l u r a l i n from w a t e r . The i n i t i a l r e l a t i v e l y h i g h c o n c e n t r a t i o n o f t r i f l u r a l i n may account f o r the lower accumulation o f a l g a e biomass i n the t r e a t e d tanks as compared t o t h e c o n t r o l . O n l y one o t h e r s t u d y has b e e n c o n d u c t e d t o e v a l u a t e t h e f a t e o f t r i f l u r a l i n i n an a q u a t i c model e c o s y s t e m ( 1 0 ) . I n t h e i r s y s tem, t r i f l u r a l i n p e r s i s t e d much l o n g e r i n w a t e r t h a n i n o u r s t u d y ( p r o b a b l y due t o l e s s p h o t o d e g r a d a t i o n t h r o u g h t h e use o f a r t i f i cial light). As a r e s u l t , t h e y r e p o r t e d much h i g h e r c o n c e n t r a t i o n s o f t r i f l u r a l i n i n s n a i l s and f i s h t h a n we f o u n d , b u t no residues i n daphinds. They a l s o r e p o r t e d t h e p r e s e n c e o f Compound 7 p l u s s e v e r a l o t h e r known and unknown m e t a b o l i t e s . 1 4

4

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

0

3.8 3.9 1.2 1.5 1.7 1.8 0 0 24.2

3.3

58.6

4.9 4.4 0.2 1.2 0.8 1.1 0 0 14.7

5.2

67.5

1 4

7.0 3.3 1.4 0.6 2.7 4.3 0 0 20.5

5.6

54.6

a

13.8 3.4 3.2 2.2 0.8 1.0 0 0 10.6

4.1

60.9

20.6 2.1 1.4 1.8 1.7 1.1 0 0 4.5

3.9

62.9

29.8 4.0 1.3 2.0 1.1 0.4 0 0 2.5

5.6

53.3

c

25.6 6.0 0.5 0.7 0.9 0.4 0 0 0

1.8

63.4

( m e t h a n o l ) by

28.8 4.6 0.9 1.2 0.4 0.6 0 0 1.8

1.5

60.2

Degradation o f [ C ] t r i f l u r a l i n i n b l u e g i l l f i s h (expressed as percent of e x t r a c t e d radioactivity) Days a f t e r s t a r t o f e x p e r i m e n t 2 5 9 22 15 30' 42 44

Values represent percent o f t o t a l C r e c o v e r e d from f i s h e x t r a c t s TLC methods, k r e c o v e r e d from t h e o r g i n . r e c o v e r e d i n a zone b e t w e e n t h e o r g i n a n d compound 1.

a

b

P o l a r metabolites Nonpolar metabolites 1 2 3 4 5 6 7 8 Trifluralin

Compound

Table V I .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

a

0

22.2 3.5 6.9 3.5 0 0 0 2.5 4.7

56.9

2

12.6 10.1 3.9 4.4 0 2.6 0 4.3 1.9

60.4

5

a

9

12. 0 7. 8 0 4. 9 0 2. 3 1. 8 3. 6 2. 6

C r e c o v e r e d from t h e o r g i n .

i n snails

1 4

C

15.5 6.6 3.5 6.7 0 0 0 3.3 6.0

57.5 8. 8 5. 6 2. 5 1. 3 10. 2 0 0 0 0

71. 6 10.9 5.3 2.4 3.0 1.1 0.8 0 0 0

76.5

44

21.9 2.1 0.6 0 2.0 0 0 0 0

73.6

48

16.6 2.7 1.4 2.0 1.6 0 0 0 0

75.4

58

10.3 2.1 4.6 2.7 0.9 0 0 0 0

79.4

72

( m e t h a n o l ) b y TLC methods.

18.7 4.6 0 1.4 0 0 0 0 0

75.8

r e c o v e r e d from s n a i l e x t r a c t s

7. 7 1. 0 1. 4 0 6. 2 0 1. 0 2. 8 1. 1

79. 0

experiment 42

( e x p r e s s e d as p e r c e n t o f e x t r a c t e d

Days a f t e r s t a r t o f 22 30 15

C]trifluralin

62. 7

Degradation of [ radioactivity)

Values represent percent o f t o t a l

Polar metabolites 1 2 3 4 5 6 7 8 Trifluralin

Compounds

Table V I I .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

0

14

3.9 12.1 0.3 1.5 0.9 0.8 0 7.5 9.7

3.6

1 4

9.8 15.7 0.3 2.8 1.7 0 0 2.3 4.7

8.1

54.6

7.0 6.7 0 1.4 1.0 0 0 0.7 1.5

7.8

73.9

16.0 5.5 0 0 6.0 0 0 0 0

7.9

64.6

9.4 15.8 0.4 1.6 1.2 1.0 0 0 0

8.3

62.3

0

1 4

Values represent percent of t o t a l C r e c o v e r e d from daphnid e x t r a c t s TLC m e t h o d s , b c r e c o v e r e d from t h e o r g i n . C r e c o v e r e d i n a zone b e t w e e n t h e o r g i n a n d Compound 1.

a

7.8 16.6 4.0 0 1.0 1.0 2.3 12.0 36.0

5.2

59.7

8.9 7.0 0.4 2.3 0 0 0 0 0

15.5

(methanol) by

11.2 11.0 0 1.2 0 0.7 0 0 0

10.0

65.9

65.9

1 3

14.1

P o l a r metabolites Nonpolar metabolites 1 2 3 4 5 6 7 8 Trifluralin

Compounds

D e g r a d a t i o n o f [ C ] t r i f l u r a l i n i n daphnids (expressed as p e r c e n t o f extracted radioactivity) . Days a f t e r s t a r t o f e x p e r i m e n t 72 58 48 42 44 30 22 15

Table V I I I .

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

C

5.4 1.8 0 0 0 0 11.0 5.4 74.5

0

0.6 1.7 0 0 1.7 1.2 7.6 1.2 82.0

0.6

3.4

5

14.3 0 8.6 0 5.7 0 0 0 28.6

8.6

34.2

15

i n algae

1 4

11.4 2.3 8.9 0 0 0 6.8 2.3 11.4

9.1

47.7

2.3 0 0 0 0 0 0 0 19.3

22.6

54.8

6.1 0 8.2 0 0 0 0 0 6.1

20.4

54.2

7.1 0 0 0 0 0 0 0 0

28.6

64.3

experiment 42 44

3.0 0 0 0 0 0 0 0 0

21.2

75.8

48

5.8 2.0 0 0 0 0 0 0 0

28.7

61.5

58

( e x p r e s s e d as p e r c e n t o f e x t r a c t e d

Days a f t e r s t a r t o f 22 30

C]trifluralin

13.1 2.0 0 0 0 0 0 0 0

27.7

57.2

72

b

Values represent percent o f t o t a l C r e c o v e r e d f r o m a l g a e e x t r a c t s ( m e t h a n o l ) b y TLC methods. 14c r e c o v e r e d f r o m t h e o r g i n . c 14 r e c o v e r e d i n a zone b e t w e e n t h e o r g i n a n d Compound 1.

a

0

1.8

P o l a r metabolites Nonpolar metabolites 1 2 3 4 5 6 7 8 Trifluralin

b

2

a

Degradation of [ radioactivity)

Compounds

Table IX.

13.

isENSEE E T A L .

Modeling

Aquatic

215

Ecosystems

The o b j e c t i v e o f t h i s i n v e s t i g a t i o n , t o d e m o n s t r a t e t h a t a q u a t i c model e c o s y s t e m s c a n b e s c a l e d up i n s i z e t o p e r f o r m t i m e r e l a t e d , m e t a b o l i c s t u d i e s , was o n l y p a r t l y a c h i e v e d . We s u c c e s s f u l l y demonstrated t h a t t h e d e g r a d a t i v e pathways a n d r a t e s o f t r i f l u r a l i n m e t a b o l i s m i n an a n a e r o b i c s o i l c a n b e d e t e r m i n e d i n an e c o s y s t e m j u s t as w e l l a s t h e y c a n i n l a b o r a t o r y s t u d i e s ( 4 ) . A l s o , t h e r e c o v e r y o f Compound 1 f r o m w a t e r s u b s t a n t i a t e d t h e s i g n i f i c a n c e o f p h o t o d e g r a d a t i o n , which had p r e v i o u s l y been measured u n d e r l a b o r a t o r y c o n d i t i o n s ( 9 ) . However, m e t a b o l i c s t u d i e s i n a q u a t i c o r g a n i s m s were n o t v e r y s u c c e s s f u l b e c a u s e ( i ) t h e t r i f l u r a l i n i n water decreased v e r y r a p i d l y , and ( i i ) t h e o r i g i n o f t h e few m e t a b o l i t e s t h a t were r e c o v e r e d was n o t c l e a r . A c c u m u l a t i o n o f t h e C - l a b e l e d compounds i n w a t e r b y t h e o r g a n i s m s was a p p a r e n t l y the predominant source. (The m a j o r p r o b l e m i n s t u d y i n g t h e m e t a b o l i s m o f p e s t i c i d e s i n a q u a t i c o r g a n i s m s r e t r i e v e d f r o m model ecosystems i s i d e n t i f y i n were t h e y f o r m e d i n t h o r were t h e y a b s o r b e d f r o m w a t e r ? ) . T h e s e r e s u l t s i n d i c a t e t h a t o u r s c a l e d - u p model e c o s y s t e m s a r e more u s e f u l f o r s t u d y i n g s y s t e m p r o c e s s e s t h a n p r o c e s s e s t h a t f u n c t i o n i n i n d i v i d u a l components o f t h e e n v i r o n m e n t . In this r e g a r d , a p r e l i m i n a r y l a r g e s c a l e ecosystem study c o u l d be v e r y u s e f u l t o i n d i c a t e parameter l i m i t s such as o v e r a l l degradation r a t e s a n d l i k e l y c o n c e n t r a t i o n s o f p a r e n t compounds p l u s metabol i t e s over time. Such i n f o r m a t i o n w o u l d be u s e f u l i n t h e d e s i g n o f m e t a b o l i c s t u d i e s i n v a r i o u s components o f t h e e c o s y s t e m . I n a d d i t i o n , t h e l a r g e s c a l e ecosystem study c o u l d a l s o be used t o d e t e r m i n e i f p r o c e s s e s d e r i v e d under l a b o r a t o r y c o n d i t i o n s cont i n u e t o f u n c t i o n a n d / o r p r e d o m i n a t e when combined i n a c o m p l e x system. 1 4

Abstract This project was designed to demonstrate that the static water model ecosystems can be scaled up in size to provide sufficient amounts of biomass, s o i l , and water to study metabolism kinetics of pesticides. Fourteen kg of s o i l , treated with [ 4C]trifluralin at 10 ppm, was flooded with 84 liters water. Bluegill fish, snails, daphnids, and algae were exposed to this system for 72 days. Samples of s o i l , water, and organisms were periodically analyzed for trifluralin and eight metabolites. The recovered metabolites and their rate of formation closely followed previously reported values for anaerobic soil and water, substantiating the u t i l i t y of the model ecosystem for metabolic studies. However, residues in the biomass were apparently derived primarily through absorption from water rather than metabolism within the organisms themselves. It was concluded that the scaled-up model ecosystem is more useful for studying system process than processes that function in individual components of the environment. 1

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

216

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

Literature cited 1. Isensee, A. R., Intern. J. Envirnomental Studies (1976) 10, 35. 2. Isensee, A. R., E. R. Holden, E. A. Woolson, G. E. Jones, J. Agric. Food Chem. (1976) 24, 1210. 3. Ambrosi, D., A. R. Isensee, J. A. Macchia, J. Agric. Food Chem. (1978) 26, 50. 4. Probst, G. W., T. Golab, R. J. Herberg, F. J. Holzer, S. J. Parka, C. V. D. Schans, J. B. Tepe, J. Agric. Food Chem. (1967) 15, 592. 5. Probst, G. W., T. Golab, W. L. Wright, "Herbicide: Chemistry, Degradation and Mode of Action" Vol. 1, p. 453 Marcel Dekker, Inc. New York, 1975. 6. Parr, J. F., S. Smith, Soi Sci. (1973) 115, 55. 7. Wright, W. L. G. F Warren Weed (1965) 13 329 8. Kearney, P. C., A Physiology (1977) 7, 242. 9. Leitis, E., D. G. Crosby, J. Agric. Food Chem. (1974) 22, 842. 10. Metcalf, R. L., J. R. Sandborn, Illinois Natural History Survey Bull. (1975) 31, 381. RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

14 Investigation of Xenobiotic Metabolism in Intact Aquatic Animals D. G. CROSBY, P. F. LANDRUM, and C. C. FISCHER Department of Environmental Toxicology, University of California, Davis, CA 95616

More than 130 year of hippuric acid (benzoylglycine fed pure benzoic acid and so ushered in our modern era of metabolism investigations on xenobiotics (foreign substances in the environment). In addition to the valuable basic knowledge of the biological processes of terrestrial animals provided by such studies, the advent of regulations controlling the use of pesticides stimulated research on the disposition of these chemicals by both mammals and insects (2). Among the requirements for registration of pesticides in the United States, the 1978 guidelines proposed by the U.S. Environmental Protection Agency (3) list general metabolism studies "in at least one mammalian species, preferably the laboratory rat . . . " Although similar tests have been conducted on other terrestrial species with increasing frequency, the small rodents have remained the principal source of metabolism data from intact animals. Standardized techniques and equipment for such investigations are in widespread use. Unfortunately, the same cannot be said for metabolism investigations in aquatic animals. Most of the world's animals exclusive of the insects --over 200,000 known species - - live at least a part of their lives in water; over 100 species have major economic importance; and they form the populations most often at risk of exposure to a growing number of chemical pollutants, but science remains largely ignorant of the disposition of xenobiotics by intact, living specimens of even the most common of the aquatic animals. In t h i s a r t i c l e , we propose to discuss some reasons f o r t h i s l a c k o f i n f o r m a t i o n , compare c h a r a c t e r i s t i c s o f the p r i n c i p a l experimental systems, and d e s c r i b e our current research on a s c i e n t i f i c protocol and technique which would provide the f u n c t i o n a l e q u i v a l e n t o f the standard t e r r e s t r i a l metabolism study but a p p l i e d to aquatic s p e c i e s .

0-8412-0489-6/79/47-099-217$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

218

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC

ORGANISMS

Laboratory Metabolism Chambers According to the EPA g u i d e l i n e s mentioned above, metabolism studies (in rodents) have the f o l l o w i n g purposes: (1) To i d e n t i f y a n d , to the extent p o s s i b l e , q u a n t i f y significant metabolites; (2) To determine any p o s s i b l e bioaccumulation and/or b i o r e t e n t i o n of the t e s t substance and/or m e t a b o l i t e s ; (3) To determine [ p e s t i c i d e ] absorption as a f u n c t i o n of d o s e ; (4) To c h a r a c t e r i z e routes and r a t e s of [ p e s t i c i d e ] excretion; ( 5 ) To r e l a t e [ p e s t i c i d e ] absorption to the duration of exposure of the animal ; and (6) To o b t a i n an estimate of binding of the t e s t substance and/or i t s metabolites by t a r g e t macromolecules in potential targe The r e q u i r e d data g e n e r a l l a measured dose o f the candidate compound - - often i s o t o p i c a l l y labelled to the r a t or mouse e i t h e r by i n j e c t i o n or per ojs_. The animal i s housed i n a glass metabolism "cage" wïïëre i t r e c e i v e s f o o d , w a t e r , and clean a i r , and i t s u r i n e , f e c e s , and r e s p i r e d gases are c o l l e c t e d and examined f o r the parent chemical and i t s m e t a b o l i t e s . Eventual postmortem t i s s u e a n a l y s i s and c a l c u l a t i o n of material balance complete the measurements necessary to s a t i s f y the above purposes of metabolism and pharmacokinetic experiments. While in v i t r o biochemical s t u d i e s are important a d j u n c t s , i t i s a l s o apparent that only experiments with i n t a c t , h e a l t h y , l i v i n g animals w i l l s u f f i c e to meet EPA c r i t e r i a . Why i s i t t h a t so l i t t l e of t h i s information e x i s t s f o r important aquatic species? The f o l l o w i n g represent some of the r e a s o n s : (1) Lack o f experimental animals. Highly standardized r a t s and mice are r e a d i l y a v a i l a b l e i n l a r g e numbers commercially, but few aquatics are s i m i l a r l y a v a i l a b l e a n d , when they a r e , suitable transportation i s d i f f i c u l t . (2) Maintenance and environmental c o n t r o l . While d i e t , b r e a t h i n g , temperature, and waste removal are v i r t u a l l y taken f o r granted i n most rodent work, they form s e r i o u s problems with aquatic a n i m a l s . Knowledge of d i e t a r y requirements and prepared d i e t s g e n e r a l l y are nonexistent f o r most s p e c i e s ; oxygen must be s u p p l i e d and t o x i c gases removed; temperature maintenance and water composition are very important; and the decay of food waste and excreta must be a v o i d e d . (3) Dosing. Rodents commonly are dosed by i n t u b a t i o n , i n t r a p e r i t o n e a l i n j e c t i o n , or i n measured amounts of d i e t . Each of these routes can become extremely d i f f i c u l t to use with most aquatic species - - e s p e c i a l l y very small forms - and absorption from the surrounding water becomes the primary

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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route of exposure ( l a r g e l y analogous to i n h a l a t i o n exposure i n mammals). (4) Control o f remetabolism. Contamination of bedding and coprophagy undoubtedly were drawbacks i n e a r l i e r rodent work, but modern metabolism chambers remove excretory products r a p i d l y . The excretory products of aquatics are released into the medium and so may be a v a i l a b l e f o r repeated reabsorption and remeta­ bol i s m . (5) Metabolite c o l l e c t i o n . Whereas the separation and c o l l e c t i o n of u r i n e , f e c e s , and r e s p i r e d gases i s a simple mat­ t e r with r o d e n t s , the q u a n t i t a t i v e i s o l a t i o n of microgram amounts of complex metabolites from l a r g e volumes of aqueous medium - - e s p e c i a l l y seawater - - has posed a major hurdle to s t u d i e s i n aquatic organisms. Experimental aquatic metabolism systems have taken one of four forms - - s t a t i c water i n ordinary a q u a r i a j a r s or beakers; s t a t i c "model ecosystems" and c o n t i n u o u s l y - f l o w i n comparison of t h e i r advantages i s shown i n Table II. For example, while the s t a t i c aquaria doubtless are by f a r the Table II.

Evaluation of aquatic metabolism chambers.

SA

Τ est s i m p l i c i t y Environment c o n t r o l Species f l e x i b i l i t y Dosing Remetabol ism control Metabolite c o l l e c t i o n a

a

+++ ++ ++ ++ + ++

ME

Ρ

F

+ ++ + + + +

++ + ++ + + +

++ +++ +++ +++ +++ +++

S A = s t a t i c aquarium, ME = model ecosystem Ρ = pond, and F = flow-through system

s i m p l e s t to s e t up and m a i n t a i n , they provide l e s s control of oxygenation, chemical l o s s , and other environmental f a c t o r s than do flow-through systems, as well as l e s s f l e x i b i l i t y i n the species used ( e . g . , some aquatic species are very s e n s i ­ t i v e to accumulated waste p r o d u c t s ) . S o - c a l l e d "model ecosystems" (12) o f f e r environmental c o n t r o l s i m i l a r to that i n s t a t i c aquaria but are much more complex and so f a r have been l i m i t e d to a few c a r e f u l l y s e l e c ­ ted and compatible species - - t y p i c a l l y , m i c r o c r u s t a c e a n s , small s n a i l s , and minnows - - which are hardy and t o l e r a t e each other i n reasonable b a l a n c e . Remetabolism and c o l l e c t i o n of

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5 Water Tissues Dealkylated and reduced analogs

Green S u n f i s h

Trifluralin

11 Bile Glucuronide

Sea lamprey Trout

3-Trifluoromethyl4-nitrophenol(TMF)

5 Water

Green S u n f i s h

Methoxychlor

Demethylated and dehydrochlori nated analogs

Bluegill

Methoprene

9

acid

Phthalic

Metabolite pool

phthalate

Four f r e s h water species

10

Dioctyl

Water Muscle

8 Water Tissues

3-Hydroxy analog and other phenols

Frog

4,4'-Di chlorobiphenyl

Tissue

7

Tissues

Chlorosalicyclic acid

Midge

2 ,5-Dichloro-4'nitrosalicylanilide

1

6

Water Tissues

2,4-D

Trout, bluegill , channel c a t f i s h

5

Water Tissues

D i e l d r i n and hydroxy analogs

Reference

Green S u n f i s h

in

4

Identified

Tissues

Metabolites

Dieldrin

Major

Formed by F r e e - L i v i n g Aquatic Animals

Fresh-water C r u s t a c e a n s , mussel , s n a i l

Animal

Examples of Metabolites

2,4-D Butoxyethyl E s t e r

Aldrin

Chemical

Table I.

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Animals

221

i d e n t i f i a b l e q u a n t i t i e s o f metabolites are s t i l l a s e r i o u s problem, and while the i n i t i a l input of chemical to the system may be known, the actual extent of exposure of each species p r e s e n t l y i s unmeasurable. Ponds can be f a i r l y simple and o f f e r the advantage of c o n s i d e r a b l e species f l e x i b i l i t y and a more natural environment - - there seems to be no reason why s i z e o f the t e s t animal should be l i m i t i n g i n such a system — although problems of dosing and metabolite c o l l e c t i o n can be s e v e r e . However, ponds add environmental degradation and evaporation processes which may obscure whether product formation and l o s s i s real or an a r t i f a c t . Closed flow-through l a b o r a t o r y - s c a l e systems appear to have the g r e a t e s t p o t e n t i a l f o r analogy to current t e r r e s t r i a l metabolism chambers. While obviously more complex than s t a t i c a q u a r i a , they allow c l o s e r control of environmental v a r i a b l e s such as oxygenation and wide range o f s p e c i e s , maximu and improved c o l l e c t i o n of waste p r o d u c t s . Dosing can be accomplished through immersion o r , beforehand, by i n j e c t i o n or i n t u b a t i o n where a p p r o p r i a t e . On the other hand, none of the four types of metabolism system i s ideal , and the most complete data probably w i l l be derived from a combination of methods. Aquatic Metabolism Protocol We have developed and tested a metabolism system and regimen which allows c o l l e c t i o n of data comparable to those from t e r r e s t r i a l a n i m a l s . The key to our experiments i s a metabolism chamber, d e s c r i b e d p r e v i o u s l y (13, 14) ( F i g . 1 ) , which can be operated i n e i t h e r the s t a t i c or flow-through mode. B r i e f l y , i n d i v i d u a l s or groups of animals are held at constant temperature i n the jacketed g l a s s chamber ( A ) , on a s t a i n l e s s s t e e l screen ( B ) , while pure water or t e s t s o l u t i o n i s passed over them (or held under s t a t i c c o n d i t i o n s ) . S o l i d wastes are separated i n a jacketed container (C) held near 0°C to minimize m i c r o b i a l a c t i o n , and the e f f l u e n t containing d i s s o l v e d metabolites i s passed onto a column of nonionic m a c r o r e t i c u l a r adsoprtion r e s i n where organic s o l u t e s are adsorbed from s o l u t i o n (D). The general experimental protocol i s summarized i n Table III.

Table III. Step Step Step Step

1. 2. 3. 4.

Proposed t e s t protocol f o r aquatic metabolism T o x i c i t y rangefinding ( s t a t i c ) Metabolism/pharmacodynamics rangefinding ( s t a t i c ) S t e a d y - s t a t e metabolism and d i s p o s i t i o n Metabolite i d e n t i f i c a t i o n and measurement

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC

ORGANISMS

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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223

A f t e r determining a concentration of t e s t compound which e l i c i t s no v i s u a l l y detectable response or e f f e c t i n the aquatic species over a period o f 48 hours (Step 1 ) , f r e s h animals are placed in the chamber, exposed to known concentrations of t e s t chemical ( u s u a l l y ' ^ C - l a b e l l e d ) , and the uptake r a t e and major metabolites determined (Step 2 ) . Depuration r a t e from the dosed animals a l s o can be estimated at t h i s point by t r a n s f e r to untreated water. Fresh animals a l s o can be exposed to a constant flow of t e s t s o l u t i o n u n t i l an a b s o r p t i o n - e x c r e t i o n e q u i l i b r i u m (steady s t a t e ) has been e s t a b l i s h e d , dosed b r i e f l y with l a b e l l e d compound, and r e l e a s e (turnover) r a t e determined (Step 3 ) . Excreted metabolites are c o l l e c t e d on the r e s i n column as a r e s u l t of both s t a t i c and s t e a d y - s t a t e exposures, and t h e i r separation i s accomplished by t h i n - l a y e r , g a s - l i q u i d , and/or high-pressure l i q u i d chromatography of the eluted residue (Step 4 ) . Separated metabolite by f i r s t scanning developed t h i n - l a y e chromatographic p l a t e s to l o c a t e r a d i o a c t i v e spots by means of a radiochromatogram scanner and then a c c u r a t e l y measuring r a d i o a c t i v i t y by s c i n t i l l a t i o n c o u n t i n g . Unknowns are i d e n t i f i e d by the usual chemical and spectrometric methods. Although previous a p p l i c a t i o n s of t h i s technique in our l a b o r a t o r y had been concerned with aquatic animal metabolism of p e s t i c i d e s such as DDT, p a r a t h i o n , c a r b a r y l , and t r i f l u r a l i n (14, 1 5 ) , we a l s o became i n t e r e s t e d i n comparing metabolic routesTby means o f a "metabolic p r o b e " . Such a compound i d e a l l y should be s t a b l e to n o n b i o l o g i c a l d e g r a d a t i o n , of low t o x i c i t y to maximize the d o s e , and subject to as many major routes of metabolism as p o s s i b l e without undue a n a l y t i c a l c o m p l e x i t y . p - N i t r o a n i s o l e , whose metabolism i n the mouse r e c e n t l y was i n v e s t i g a t e d by Trautman (16), provided a s a t i s f a c t o r y probe in which O-demethylation, r i n g - o x i d a t i o n , n i t r o r e d u c t i o n , and 0 - and N-conjugation might l o g i c a l l y be observed. Consequentl y , the metabolism of p - n i t r o a n i s o l e i n marine i n v e r t e b r a t e s was chosen f o r comparison with that of the mammal. p - N i t r o a n i s o l e Pharmacodynamics i n the Sea Urchin The aquatic species chosen was Strongylocentrotus p u r p u r a t u s , the purple sea u r c h i n . T h i s animal i s a common r e s i d e n t of the C a l i f o r n i a c o a s t , a frequent pest i n commercial kelp c u l t u r e , and a s p e c i a l t y food item o f growing i n t e r e s t . Phylogenetically, these echinoderms are considered to be in the i n v e r t e b r a t e c l a s s most d i r e c t l y l i n k e d to the v e r t e b r a t e s . -,, p - N i t r o a n i s o l e (PNA), uniformly r i n g - l a b e l l e d with C, was prepared from commercial T^C-p-nitrophenol by the method of Ross (17) and p u r i f i e d by t h i n - l a y e r chromatography i n benzene-methanol (9:1 v / v ) . Sea urchins were c o l l e c t e d at S a l t P o i n t , C a l i f o r n i a , and transported i n natural sea water

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

to our Davis laboratory where they were t r a n s f e r r e d to a r t i f i c i a l sea water (Instant Ocean ) at the o r i g i n a l ocean temperature of 1 0 ° C . The acute t o x i c i t y of PNA to the sea urchin was measured by immersion o f the animals at 12°C i n Instant Ocean containing known l e v e l s of t e s t compound (Table I V ) . A f t e r 24 h o u r s , the animals were observed and t r a n s f e r r e d to untreated w a t e r , R

R

Table IV.

Acute t o x i c i t y of p - n i t r o a n i s o l e i n S_. p u r p u r a t u s

(PNA)

a

Concentration (mg/1)

24 h r .

48 h r . b

10

No e f f e c t

No e f f e c t

50

2/3 Not a t t a c h e d

No e f f e c t

Spines i n d i s a r r a y

Spines in rows

None a t t a c h e d , Spines i n d i s a r r a y

A l l dead, Sloughing spines

100

C

500

C

a

Ν = 3

b

R 24 H r s . i n untreated Instant Ocean

c

S o l u b i l i t y of PNA i s 71 .8 mg/1 at 15°C

and observed again a f t e r 24 hours. PNA showed no e f f e c t on sea urchins at concentrations of 10 mg/1 (65 μ Μ ) , and our f u r t h e r experiments were conducted below t h i s l e v e l . For the metabolism rangefinding experiments, sea urchins were dosed by i n j e c t i o n at a l e v e l of 2.2 mg/kg of body weight with 14c-PNA i n t o the c e n t r a l c a v i t y , held i n untreated water at 1 2 ° C , and both the water and coelomic f l u i d sampled p e r i o d i c a l ­ l y over the course o f 8 h o u r s . Most of the PNA was eliminated r a p i d l y i n t o the water ( F i g . 2 ) , with a p s e u d o - f i r s t order h a l f - l i f e of appearance of 12 minutes; h a l f of the t e s t compound was l o s t from the coelomic f l u i d i n about 24 minutes. To determine absorption r a t e , another group of animals was immersed i n a 5 mg/1 s o l u t i o n of PNA in Instant 0cean (closed chamber) and the water sampled at i n t e r v a l s to measure the l o s s o f r a d i o a c t i v i t y compared to c o n t r o l s ( F i g . 3 ) . The data were normalized f o r t i s s u e weight, and e q u i l i b r i u m was reached i n about 8 hours at a body burden of 10 ug/g (10 ppm) e q u i v a l e n t to a bioconcentration f a c t o r o f 2.0 (winter animals). In another experiment, animals were c o l l e c t e d at i n t e r v a l s , s a c r i f i c e d , and t h e i r t i s s u e analyzed f o r 14c R

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Animals

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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

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HOURS Figure 3.

1

Absorption of PNA by S. purpuratus from a 5-mg/L solution.

1

0

Figure 4.

1

1

1

4 HOURS

1

1

1

Γ

1

Absorption rate of PNA from a 5-mg/L solution

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

14.

CROSBY E T AL.

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227

(Fig. 4). The i n i t i a l uptake r a t e was s i m i l a r f o r both types of samples, but the b i o c o n c e n t r a t i o n f a c t o r in the second instance was 4.0 (summer a n i m a l s ) . When placed i n pure w a t e r , the animals c o n t a i n i n g s t e a d y s t a t e concentrations of PNA l o s t i t at a rate of 9.4 u g / g / h r with a h a l f - l i f e (appearance i n water) of 22 minutes; when they were placed i n a 5 mg/1 s o l u t i o n of unlabel led PNA, the r a t e of l o s s (turnover) of C was s i m i l a r to the depuration rate ( F i g . 5). 1 4

p - N i t r o a n i s o l e Metabolism The s o l u b l e metabolites excreted from animals dosed by i n j e c t i o n were c o l l e c t e d on A m b e r l i t e XAD-4 r e s i n , the r e s i n eluted s e q u e n t i a l l y with d i e t h y l e t h e r , acetone, and methanol, and the s o l u t e s separate s i l i c a gel and quantitate The products were i d e n t i f i e d by t h e i r chromatographic c h a r a c t e r i s t i c s i n comparison with authentic standards ( F i g . 6) and represented 72% of the o r i g i n a l dose; a c i d d i q e s t i o n of the s e a - u r c h i n t i s s u e released another 21.5% of 1ÏC bound as unknown p r o d u c t s , f o r a t o t a l a c c o u n t a b i l i t y of 93.5%. According to Trautman (]J5), PNA was converted almost e x c l u s i v e l y to p-nitrophenol i n the mouse with 24 h o u r s , nearly a l l of which was excreted i n the urine as g l u c u r o n i d e , s u l f a t e , g l u c o s i d e , and unextractable p r o d u c t s ; the mouse t i s s u e r e t a i n e d only about 1% of the o r i g i n a l dose. By comparison, the sea urchin metabolized PNA s l o w l y ; p-nitrophenol and i t s conjugates accounted f o r o n l y about 6% o f the metabolites and most of the remainder (90%) was p - a n i s i d i n e (p-methoxyaniline) and i t s N-acetyl d e r i v a t i v e . R

Conclusions It i s apparent that the aquatic echinoderm and t e r r e s t r i a l mammal deal with a chemical probe by very d i f f e r e n t metabolic pathways. The e x c l u s i v e formation of o x i d i z e d (Oedèmethylated) product i n the mouse may p a r t l y r e f l e c t the animal 's h i g h l y o x i d i z i n g environment, while the r e l a t i v e l y anoxic marine environment i s represented i n the observed reduced metabolites of the sea u r c h i n . One purpose of metabolic transformations has been assumed to be conversion of a x e n o b i o t i c into more w a t e r - s o l u b l e form (18); as shown by p a r t i t i o n c o e f f i c i e n t s (19), the h y d r o p h i l i c i t y of p-nitrophenol (Kp 81) i s much greater than that o f PNA (Kp 107), and that of the corresponding nitrophenyl s u l f a t e and g l y c o s i d e s must be greater s t i l l . On the other hand, while p - a n i s i d i n e (Kp 9) i s r e l a t i v e l y s o l u b l e compared to PNA, the advantage would seem to be l o s t in conversion to " i n s o l u b l e p-methoxyacetanilide (Kp 1 4 ) . 11

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CROSBY

ET AL.

Intact Aquatic

Animais

UNI. >0.1%

Figure 6.

PNA metabolites excreted into water by S. purpuratus during 8 hr

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Our research shows t h a t the described system allows the measurement o f b a s i c pharmacodynamic p r o p e r t i e s (not e x p l i c i t l y included i n the EPA g u i d e l i n e s ) and i d e n t i f i c a t i o n of x e n o b i o t i c metabolites from excreta i n much the same way as they are obtained with t e r r e s t r i a l mammals. The o r i g i n a l purposes of general metabolism s t u d i e s , o u t l i n e d at the beginning of t h i s a r t i c l e , can be s a t i s f i e d metabolite i d e n t i f i c a t i o n and q u a n t i t a t i o n , b i o a c c u m u l a t i o n , absorption and e x c r e t i o n , and binding to t i s s u e as shown with a chemical probe such as p - n i t r o a n i s o l e . While i n v i t r o measurements a l s o give important i n s i g h t and have r e c e i v e d the major emphasis in aquatic metabolism s t u d i e s (20, 21_), i t i s obvious that t h e i r r e s u l t s p r e s e n t l y are not so~cTirectly a p p l i c a b l e . The system i s not without major needs and d i f f i c u l t i e s : (1) More uniform animals w i l l be r e q u i r e d . The w i l d animals (such as S_ purpuratus) are quite v a r i a b l e and the s t r e s s of c o l l e c t i o problem; (2) The system and protocol r e q u i r e extensive s t a n d a r d i z a t i o n and the s p e c i f i c a t i o n of optimum (or at l e a s t g e n e r a l l y s u i t a b l e ) c o n d i t i o n s of temperature, o x y g e n a t i o n , water c o m p o s i t i o n , flow r a t e s , e t c . ; (3) Comparison of r e s u l t s between these and the other major experimental chambers w i l l be important, both because the others are i n c u r r e n t use and because each can o f f e r unique b e n e f i t s ; (4) The model systems e v e n t u a l l y must be compared against natural h a b i t a t s . Although the r e s u l t s of the c o n t r o l l e d , standardized l a b o r a t o r y t e s t s give important b a s i c and p r a c t i c a l information (as do those from t e r r e s t r i a l metabolism chambers), there i s no reason to b e l i e v e t h a t they represent q u a n t i t a t i v e l y the behavior of the animals in Nature; (5) Model systems e v e n t u a l l y must be extended to accommodate and i n v e s t i g a t e populations of a species and communities composed of a number of s p e c i e s . Even more than with t e r r e s t r i a l a n i m a l s , the aquatic communities provide o p p o r t u n i t i e s f o r a wide range of modified exchange, uptake, e x c r e t i o n , and metabolism which represent more than the sum o f the a c t i v i t i e s o f the component species alone.

Literature Cited 1. Keller, W., Liebig's Ann., (1842), 43, 108. 2. Menzie, C.M., "Metabolism of Pesticides--an Update", Spec. Sci. Report Wildlife 184, USDI, Washington, D.C., 1974. 3. EPA, "Proposed Guidelines for Registering Pesticides in the United States, Part II", Federal Register, (1978), 43, (63), 37395.

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14.

4. 5.

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ET

AL.

Intact Aquatic Animals

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Khan, M.A.Q., A. Kamal, R.J. Wolin, and J. Runnels, Bull. Environ. Contam. Toxicol., (1972), 8, 219. Reinbold, K.A., and R.L. Metcalf, Pestic. Biochem. Physiol., (1976), 6, 401. Rogers, C.A., and D.L. Stalling, Weed Sci., (1972), 20,

6. 101. 7. Kawatski, J.A., and A.E. Zittel, in "Investigations in Fish Control", Fish and Wildlife Service, USDI, La Crosse, Wisc., No. 79 (1977). 8. Tulp, M.T.M., G. Sundstrom, andO.Hutzinger, Chemosphere, (1976), 6, 425. 9. Metcalf, R.L., G.M. Booth, C.K. Schuth, D.J. Hansen, and P.-Y. Lu, Environ. Health Perspect., (1973), 4, 27. 10. Quistad, G.B., D.A. Schooley, L.E. Staiger, B.J. Bergot, B.H. Sleight, and K.J. Macek, Pestic. Biochem. Physiol., (1976), 6, 523. 11. Lech, J.J., and C.N (1975), 31, 150. 12. Metcalf, R.L., in "Essays in Toxicology" (W.J. Hayes, Jr., ed.), (1974), 5, 17. 13. Crosby, D.G., Pure Appl. Chem., (1975), 42, 233. 14. Garnas, R.L., Ph.D. Thesis, University of California, Davis, CA., 1975. 15. Fischer, C.C., and D.G. Crosby, Abstr. N W Meeting, Amer. Chem. Soc., Honolulu, Hawaii, 1975. 16. Trautman, T.D., Ph.D. Thesis, University of California, Davis, CA. 1978. 17. Ross, J.H., University of California, Davis, CA., Personal communication, 1977. 18. Baldwin, E., "An Introduction to Comparative Biochemistry", Cambridge University Press, Cambridge, 1964. 19. Leo, Α., C.H. Hansch, and D. Elkins, Chem. Rev., (1971), 71, 555. 20. Chambers, J.E., and J.D. Yarbrough, Comp. Biochem. Physiol., (1976), 55, 77. 21. Adamson, R.H., and S.M. Sieber, in "Survival in Toxic Environments" (M.A.Q. Khan and J.P. Bederka, eds.), Academic Press, New York, 1974, p. 203. RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

15 Xenobiotic Transport Mechanisms and Pharmacokinetics in the Dogfish Shark A. M. GUARINO and S. T. ARNOLD Laboratory of Toxicology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014 and Mount Desert Island Biological Laboratory, Salsbury, ME 04672 Our interest in studyin aquatic species stems fro these substances might do to fish, but also because such studies might forecast problems which might arise in man. Of course, we would also like to know the ultimate sites and mechanisms of action of these compounds, but before such lofty goals are achieved, there is much painstaking work which must be done to study the absorption, distribution (including binding and storage), excretion and metabolism of compounds foreign to living organisms. Having such information enables one to answer a series of questions such as: 1) What kinds of chemicals might cause fish k i l l s ? 2) Where in the fish body do they accumulate and 3) Does the xenobiotic itself or some biotransformed product exert the major action? We were introduced to this fascinating area of research by Dr. David P. Rall, the current Director of National Institute of Environmental Health Sciences. We took what could be described as a pharmacokinetic approach to toxicology. In other words we considered that knowing where the body accumulates a xenobiotic and how long i t takes an animal to rid itself of a compound, are important biologic properties to identify. For example, just as one can never ignore a toxicologist's observation that a drug caused a lesion to a particular organ, similar evidence that a xenobiotic localizes in one kind of tissue or organ must be heeded. Thus, in introducing us to such concepts Dr. Rail had us investigate the fate and distribution of the two relevant pollutants, DDT and methyl mercury (MeHg) in a highly relevant species, the lobster. We reported (1, 2) that most of the DDT accumulated in the lobster hepatopancreas and MeHg localized in the tail muscle. These findings are of obvious and profound significance, especially since these animals have been exposed to such pollutants. Our research moved away from these crustaceans for two primary reasons. First, they are not typical of the more abundant aquatic species. Second, in one of the earliest xenobiotic This chapter not subject to U.S. copyright. Published 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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d i s p o s i t i o n s t u d i e s done i n f i s h , R a i l and Z u b r o d d i s c o v e r e d what a p p e a r e d t o be an a p p r o p r i a t e s p e c i e s s i n c e i t y i e l d e d d a t a s i m i l a r t o t h a t r e p o r t e d i n mammalian t e r r e s t r i a l s p e c i e s . The s p e c i e s u s e d was t h e d o g f i s h s h a r k ( s p i n y d o g f i s h , S q u a l u s a c a n t h i a s ^ ) and t h e x e n o b i o t i c was t h e a n t i m a l a r i a l q u i n i n e (3) . Thus t h e s e w o r k e r s f o u n d t h a t t h e m a j o r d i s t r i b u t i o n f e a t u r e s w h i c h a r e somewhat u n i q u e f o r t h i s d r u g , i . e . , h i g h e r c o n c e n t r a t i o n s o f t h e d r u g i n r e d b l o o d c e l l s , b r a i n and m u s c l e t h a n i n p l a s m a , w e r e t h e same i n t h e d o g f i s h and mammals. T h i s s h o u l d n o t be s u r p r i s i n g s i n c e t h e d o g f i s h s h a r k i s r e g a r d e d as anatomi c a l l y and p h y s i o l o g i c a l l y t y p i c a l enough t o be w i d e l y employed i n c o m p a r a t i v e anatomy and p h y s i o l o g y t e x t b o o k s and l a b o r a t o r y m a n u a l s ( 4 , 5 ) . More r e c e n t l y , t h e o r g a n d i s t r i b u t i o n o f m e t a l l i c i o n s i n f i s h was shown (6) t o f o l l o w t h a t s e e n g e n e r a l l y i n mammals. Handling Advantages of th F u l l e r e x p l o i t a t i o n o f the s h a r k as a model f o r x e n o b i o t i c d i s t r i b u t i o n s t u d i e s had t o a w a i t t h e d e v e l o p m e n t o f methods i n two a r e a s : 1) p r a c t i c a l t e c h n i q u e s f o r s a m p l i n g two i m p o r t a n t p h y s i o l o g i c c o m p a r t m e n t s , b i l i a r y and u r i n a r y ; a n d , 2) v a l i d a t i o n of pharmacokinetic parameters i n t h i s a q u a t i c s p e c i e s . The f o r m e r was r e c e n t l y a c c o m p l i s h e d Ç7, 8) w i t h two s u b s t a n c e s w i t h w e l l - k n o w n p r e d i l e c t i o n s f o r t h e h e p a t i c ( b r o m s u l p h a l e i n , BSP) and r e n a l ( p h e n o l r e d ) c o m p a r t m e n t s . T h u s , f o r t h e s e t y p e s o f s u b s t a n c e s , f l u i d c o l l e c t i n g t e c h n i q u e s as shown i n F i g s . 1 and 2 d e m o n s t r a t e d t h a t 80% o f t h e d o s e o f BSP was e x c r e t e d i n t o s h a r k b i l e i n 48 h r s . (_7) w h i l e i n t h i s same t i m e p e r i o d , 4 1 % o f t h e d o s e o f p h e n o l r e d a p p e a r e d i n u r i n e and 49% was e x c r e t e d v i a t h e b i l e i n t h e d o g f i s h ( 8 ) . I n o t h e r w o r d s most o f t h e s e d r u g s w e r e c l e a r e d f r o m t h e s h a r k v i a t h e b i l e and u r i n e . T h e r e f o r e , these s t u d i e s demonstrated t h a t the proposed technique g r e a t l y improved the "bookkeeping" f o r substances which would have o t h e r w i s e b e e n l o s t i f t h e s e e x c r e t i o n r o u t e s w e r e n o t a d e q u a t e l y sampled. F i n a l l y , t h i s f i s h m o d e l p r o v e d t o be h a r d y enough t o s u r v i v e t h e s i m p l e s t e x p e r i m e n t a l t e c h n i q u e s , s u c h as c o n f i n e m e n t i n an a q u a r i u m , m i n o r s u r g e r y , and e x p o s u r e t o t r a c e q u a n t i t i e s of p o l l u t a n t s . W i t h t h e s e g e n e r a l f e a t u r e s o f how t h e s h a r k h a n d l e s s u c h x e n o b i o t i c s Bungay e t . a l , (9) were a b l e t o b u i l d on t h e e a r l i e r r e s e a r c h o f o t h e r s ( 1 0 , 1 1 ) , t o e s t a b l i s h the a p p l i c a b i l i t y of s c a l i n g processes i n the d i r e c t i o n o p p o s i t e of t h a t u s u a l l y taken; i n t h i s case, p h a r m a c o k i n e t i c models d e v e l o p e d i n i n t e r m e d i a r y s p e c i e s s u c h as t h e mouse, were a d a p t e d t o t h e l o w e r f i s h s p e c i e s . Thus, t h e y showed ( F i g . 3) t h a t t h e p h a r m a c o k i n e t i c m o d e l gave p r e d i c t e d ( d o t t e d l i n e s ) v a l u e s f o r p h e n o l r e d i n k i d n e y , l i v e r , p l a s m a and ( F i g . 4 ) , b i l e and urine. I t c a n be r e a d i l y s e e n t h a t most o f t h e e x p e r i m e n t a l p o i n t s f e l l v e r y c l o s e t o t h e v a l u e s p r e d i c t e d by t h e pharmacok i n e t i c model.

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GUARiNo A N D A R N O L D

Pharmacokinetics A

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Technique

for collecting urine from the dogfish

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In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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TIME, HOURS Journal of Pharmacokinetics and Biopharmaceutics

Figure 3. Time coarse of tissue and plasma concentrations of phenol red: model predictions vs. experimental results. The lines are model predictions; the symbols are experimental data for iv injection of 10 mg/kg into the caudal vein of dogfish sharks. Each symbol represents the average of five to eight female sharks/time point with SD indicated by vertical bars. The limit of sensitivity of the assay was 25,15, and 5 μg/g or mL for (/\), kidney (K); Liver (L); and (O), plasma (P), respectively (9).

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TIME, HOURS Journal of Pharmacokinetics and Biopharmaceutics

Figure 4. Time course of accumulation of phenol red and glucuronide in the bile and urine. The lines are model predictions and the symbols are experimental data: (U), bile (B); (A), urine (U) (9).

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Our q u e s t i o n s b r o a d e n e d t o c o n s i d e r how t h e t r a n s p o r t a n d m e t a b o l i c c a p a b i l i t i e s o f t h e s e a q u a t i c s p e c i e s compare w i t h t h o s e o f mammalian s p e c i e s . One r e a s o n f o r a s k i n g s u c h a q u e s t i o n i s t o assess whether the presence o r absence o f these c a p a b i l i t i e s a l t e r s the a b i l i t y o f f i s h t o s u r v i v e i n t o x i c environments. S u r v i v a l mechanisms f a l l i n t o two c a t a g o r i e s - b e h a v i o r a l a n d physiologic. An example o f a b e h a v i o r a l mechanism c o u l d b e a s simple as a f i s h a v o i d i n g that area o f a stream which contains t o x i c q u a n t i t i t e s o f p h e n o l . When e x t e r n a l p e r t u r b a t i o n s c a u s e d by p o l l u t a n t s a r e s m a l l , h o m e o s t a t i c mechanisms s u c h a s t h o s e o f t h e l i v e r and k i d n e y , a l l o w f i s h t o a d a p t t o t h e body o f w a t e r i n which they e x i s t . The p r o b l e m t h e n i s r e l a t e d t o d e f i n i n g t h e l i m i t s t o w h i c h h o m e o s t a t i c phenomena c a n b e s t r e s s e d i n aquatic species. An i m p o r t a n t r e a s o n t o e s t a b l i s h such i n f o r m a t i o n i n f i s h i s that bodies o f water are the " u l t i m a t e s i n k " f o r a number o f p o l l u t a n t s ( 1 2 ) Thus w h i l behavioral such as removing i t s e l a v a i l a b l e t o a mammalia impossible f o r a f i s h i f a t o x i c x e n o b i o t i c occurs uniformly t h r o u g h o u t a n e n t i r e body o f w a t e r . What a r e some o f t h e g e n e r a l modes by w h i c h t e r r e s t r i a l a n i m a l s c a n d i m i n i s h t o x i c e f f e c t s o f x e n o b i o t i c s a n d how do t h e s e compare i n f i s h ? 1. R o u t e s o f e n t r y - f i s h and mammals s h a r e two p o t e n t i a l r o u t e s o f e n t r y , n a m e l y o r a l a n d p e r c u t a n e o u s . Mammals a l s o absorb x e n o b i o t i c s v i a the lungs w h i l e g i l l a b s o r p t i o n i s p o s s i b l e in fish. 2. Plasma b i n d i n g - both the q u a n t i t y and types o f p r o t e i n s i n plasma d i f f e r i n these c l a s s e s o f animals. Mammals t e n d t o h a v e l a r g e amounts o f p r o t e i n compared w i t h f i s h ( c a . 8 v s 3 g/100 ml) a n d a l b u m i n , t h e p r o t e i n w h i c h b i n d s most x e n o b i o t i c s , i s n e g l i g i b l e i n a number o f f i s h s p e c i e s . 3. Storage i n f a t depots - s i n c e the l i p i d s o l u b i l i t y o f a x e n o b i o t i c i s i m p o r t a n t i n i t s a b s o r p t i o n , o n c e i n t h e body i t i s n o t u n u s u a l t h a t t h e s e a g e n t s w o u l d a c c u m u l a t e i n body f a t . Mammals t e n d t o d i s t r i b u t e t h e i r f a t somewhat u n i f o r m l y i n t h e s u b c u t a n e o u s t i s s u e s . W h i l e some a q u a t i c s p e c i e s a l s o do t h i s , o t h e r s have d i s t r i b u t i o n p a t t e r n s w h e r e t h e m a j o r s i t e f o r s t o r i n g f a t i s i n one o r g a n . For example, the s h a r k and t h e l o b s t e r e x t e n s i v e l y u t i l i z e the l i v e r and hepatopancreas, r e s p e c t i v e l y , f o r f a t s t o r a g e ; furthermore the f a t content i n t h e s e o r g a n s c o n s t i t u t e s g r e a t e r t h a n 50% t h e t o t a l t i s s u e w e t w e i g h t . A f u r t h e r p o i n t o f c o m p a r i s o n i s t h a t when f i s h c a s t g g s , they o f t e n t r a n s f e r l a r g e q u a n t i t i e s o f f a t and t h e r e f o r e l i p i d s o l u b l e x e n o b i o t i c s , t o the y o l k (13). 4. Organ b l o o d f l o w and o r g a n c o n c e n t r a t i o n - o r g a n b l o o d f l o w i s an important f a c t o r which determines the d i s t r i b u t i o n o f a x e n o b i o t i c once i t h a s been absorbed. T h i s f l o w i s r e l a t e d t o b l o o d p r e s s u r e and w h i l e mammalian p r e s s u r e s a r e a r o u n d 100 mm Hg, f i s h s p e c i e s u s u a l l y are about o n e - f i f t h o r l e s s o f t h i s v a l u e . e

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O b v i o u s l y , t h e n , c a r d i a c o u t p u t and b l o o d f l o w t h r o u g h t h e l i v e r and k i d n e y d i f f e r c o n s i d e r a b l y b e t w e e n t h e s e two c l a s s e s o f animals. As has b e e n d i s c u s s e d (9) i f t h e d i s t r i b u t i o n o f a compound i s f l o w - l i m i t e d , t h i s f a c t o r c a n be r e a d i l y c o n s i d e r e d i n p h a r m a c o k i n e t i c m o d e l i n g . W h i l e t h e p e r f u s i o n o f an o r g a n may e x p l a i n t h e i n i t i a l l y h i g h l e v e l s o f a x e n o b i o t i c i n h i g h l y v a s c u l a r t i s s u e s , t h e mammalian l i v e r and k i d n e y h a v e an a d d i t i o n a l f a c t o r f a v o r i n g c o n c e n t r a t i o n o f compounds i n them, n a m e l y a h i g h c a p a c i t y t o b i n d c h e m i c a l s more e x t e n s i v e l y t h a n most o t h e r organs. Comparative b i n d i n g i n f o r m a t i o n i s d e s i r a b l e t h e r e f o r e , i n f i s h l i v e r , k i d n e y and o t h e r o r g a n s . 5. M e t a b o l i s m - a f i n a l f a c t o r i n need o f c o m p a r a t i v e s t u d i e s i s t h e m e t a b o l i s m o f x e n o b i o t i c s . One o b v i o u s d i f f e r e n c e b e t w e e n mammalian and f i s h s p e c i e s i s t h a t t h e i r b o d i e s u s u a l l y f u n c t i o n a t t e m p e r a t u r e s a t l e a s t 10°C d i f f e r e n t . This fact u n d o u b t e d l y e x p l a i n s som d i f f e r e n c e i m e t a b o l i r a t but when i n v i t r o i n c u b a t i o n i s a 10 - 100 f o l d h i g h e (14, 1 5 ) . In other words, the l e v e l of the x e n o b i o t i c - m e t a b o l i z i n g c a p a c i t y , e s p e c i a l l y f o r o x i d a t i v e pathways, of the p o i k i l o t h e r m i c animals i s c o n s i d e r a b l y lower than t h a t of the homeothermic species. E l s e w h e r e i n t h i s v o l u m e Dr. Bend has f o c u s e d on t h i s a s p e c t o f t h e h a n d l i n g o f x e n o b i o t i c s by f i s h ( 1 6 ) . I n summary t h e n , f i s h as w i t h most o r g a n i s m s , f u n c t i o n c o m p e t i t i v e l y i n t h e i r n a t u r a l ecosystem. With o n l y a m i l d a d d i t i o n a l s t r e s s , as m i g h t be imposed by an i n c r e a s e d p o l l u t a n t b u r d e n , c e r t a i n s p e c i e s c o u l d be p l a c e d a t a c o m p e t i t i v e d i s a d v a n t a g e due t o some d e g r e e o f s e l e c t i v i t y o f d i s p o s i t i o n a n d / o r m e t a b o l i s m . An e x c e l l e n t example o f m a k i n g a " p o l l u t a n t " w o r k i n s u c h manner t o a d v a n t a g e i s by t h e use o f 3 - t r i f l u o r o m e t h y l 4 - n i t r o p h e n o l i n t h e u p p e r G r e a t L a k e s as a s e a l a m p r e y l a r v i c i d e . T h i s compound has c o n s i d e r a b l y l e s s t o x i c i t y t o a f a v o r e d s p e c i e s , the t r o u t (17). A major reason f o r t h i s s e l e c t i v i t y i s m e t a b o l i c , i . e . , t h e t r o u t more r e a d i l y c o n v e r t s t h i s compound t o t h e e a s i l y excreted glucuronide. B u t compared w i t h t h e i n f o r m a t i o n a v a i l a b l e on t h i s compound, we a r e r e l a t i v e l y i g n o r a n t o f how f i s h handle thousands of o t h e r f o r e i g n c h e m i c a l s . I n i t i a l S t u d i e s w i t h t h e M o d e l Compound P h e n o l

Red

Our i n i t i a l i n t e r e s t was i n s t u d y i n g t h e d i s p o s i t i o n o f phenol red i n the shark. T h i s t u r n e d o u t t o be a good c h o i c e b e c a u s e n e a r l y e q u a l amounts o f t h e m o d e l compound a p p e a r e d i n t h e u r i n e and b i l e . A f t e r s o l v i n g t h e body f l u i d c o l l e c t i n g p r o b l e m s , we s t u d i e d i n g r e a t e r d e p t h , t h e t r a n s p o r t p r o p e r t i e s o f p h e n o l r e d i n b o t h t h e r e n a l and h e p a t i c s y s t e m s . The u r i n e c o l l e c t i n g t e c h n i q u e s ( F i g . 1 ) , w e r e r e v i s e d f r o m t h o s e u s e d by o t h e r i n v e s t i g a t o r s a t Mt. D e s e r t I s l a n d B i o l o g i c a l L a b o r a t o r y , S a l s b u r y Cove, ME. Female f i s h w e i g h i n g f r o m 3-7 kg were p r e p a r e d by l i g a t i n g one end o f a 10 cm l e n g t h o f P.E. 190

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

15.

GUARiNO AND ARNOLD

Pharmacokinetics

241

t u b i n g t o t h e u r i n a r y p a p i l l a and t h e f r e e end t o a s t u r d y rubber b a l l o o n . A l i g a t u r e was p a s s e d a r o u n d t h e n e c k o f t h e b a l l o o n s o t h a t t h e s y r i n g e p l u n g e r hub a n d b a l l o o n c o u l d be s u t u r e d t o t h e s k i n and m u s c l e . This procedure r e l i e v e d the t e n s i o n p l a c e d on t h e p a p i l l a w h i c h would o t h e r w i s e cause t e a r i n g when t h e f i s h swam. F o r c o l l e c t i o n p e r i o d s o f l e s s t h a n 4 h r s . , b a l l o o n s w i t h a 20-30 m l c a p a c i t y w e r e u s e d a n d f o r p e r i o d s o f 4-24 h r s . , 200-300 m l b a l l o o n s w e r e u s e d . F o r more t h a n 2 4 - h o u r c o l l e c t i o n s , b a l l o o n s w e r e changed d a i l y . The a v e r a g e u r i n a r y output o f these sharks i s about 1 ml/hr/kg. U s u a l l y , b i l e was c o l l e c t e d t e r m i n a l l y by p e r f o r a t i n g t h e g a l l b l a d d e r w i t h a 19 gauge n e e d l e a t t a c h e d t o a 6 m l s y r i n g e . To compare t h e e x c r e t i o n o f p h e n o l r e d i n t o h e p a t i c a n d g a l l b l a d d e r b i l e , u n a n e s t h e t i z e d f i s h were prepared s u r g i c a l l y w i t h b i l i a r y f i s t u l a e a s d e s c r i b e d i n F i g . 2 ( 7 ) . The c e p h a l a d portion of restrained fis w h i l e t h e common b i l e d u c incision. The g a l l b l a d d e r was d r a i n e d o f b i l e b y a s p i r a t i o n . T u b i n g (P.E. 2 0 0 ) , f i t t e d w i t h a b a l l o o n o f 5-10 m l c a p a c i t y , was i n s e r t e d i n t o t h e d i s t a l t i p o f t h e g a l l b l a d d e r s o t h a t t h e c a n n u l a o c c u p i e d most o f t h e lumen o f t h e c o l l a p s e d g a l l b l a d d e r . The c a n n u l a was e x t e r n a l i z e d , s u t u r e d i n p l a c e , a n d t h e wound c l o s e d by s u t u r i n g . The two b i l e c o l l e c t i n g t e c h n i q u e s w e r e r e l a t i v e l y comparable s i n c e t h e s h a r k s were n o t f e d ; thus t h e g a l l b l a d d e r was n o t e m p t i e d d u r i n g s t u d i e s c o n d u c t e d f o r up t o one week. I n j e c t i o n s o f d i f f e r e n t d o s e s o f p h e n o l r e d w e r e g i v e n v i a t h e c a u d a l v e i n a n d b l o o d s a m p l e s w e r e drawn f r o m t h i s same l o c a t i o n a t s p e c i f i e d t i m e i n t e r v a l s . A f t e r the animals were s a c r i f i c e d , u r i n e , b i l e , k i d n e y , l i v e r , m u s c l e and b l o o d ( h e p a r i n i z e d ) samples were c o l l e c t e d a n d , where a p p r o p r i a t e , t o t a l volumes and t o t a l w e i g h t s were r e c o r d e d . The c o l o r i m e t r i c p r o c e d u r e s have been d e s c r i b e d p r e v i o u s l y (8) and c o n s i s t e d o f h o m o g e n i z a t i o n o f p l a s m a o r t i s s u e s w i t h m e t h a n o l f o l l o w e d by measurement o f t h e o p t i c a l d e n s i t y o f a l k a l i n i z e d s o l u t i o n s . P h e n o l r e d g l u c u r o n i d e was d e t e r m i n e d a f t e r e i t h e r HC1 o r 3g l u c u r o n i d a s e h y d r o l y s i s . The p r e s e n c e o f t h e g l u c u r o n i d e was c o n f i r m e d by t h i n l a y e r c h r o m a t o g r a p h y i n t h r e e s y s t e m s ( 8 ) . D i s p o s i t i o n o f P h e n o l Red Phenol r e d i s r a p i d l y c l e a r e d b i p h a s i c a l l y from t h e plasma compartment w i t h a n i n i t i a l t o f a b o u t 46 m i n . ( F i g . 3) a n d a second phase w i t h a t o f 8.3 h r s . A s e a r l y a s 10 m i n . there a r e d e t e c t a b l e l e v e l s of p h e n o l r e d i n t h e k i d n e y . The c o n c e n t r a t i o n o f d r u g i n k i d n e y p e a k e d a t 30 m i n . a n d d e c a y e d w i t h a h a l f - t i m e o f about 9 h r s . There a l s o were d e t e c t a b l e l e v e l s o f p h e n o l r e d w i t h i n 10 min. i n l i v e r b u t t h e v a l u e s w e r e c o n s i d e r a b l y below those o f plasma and k i d n e y . Hepatic l e v e l s took l o n g e r (ca. 2 h r s . ) t o peak than d i d those i n k i d n e y , and then d e c a y e d w i t h a h a l f - t i m e o f a b o u t 10 h r s . The g l u c u r o n i d e

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC

242

ORGANISMS

c o n j u g a t e ( n o t shown i n F i g . 3 - o n l y t o t a l d r u g a p p e a r s h e r e ) was n o t d e t e c t e d i n p l a s m a , k i d n e y o r l i v e r . P h e n o l r e d and i t s g l u c u r o n i d e w e r e a p p a r e n t i n u r i n e w i t h i n 30 m i n . , i n c r e a s e d i n c o n c e n t r a t i o n f o r t h e f i r s t 2 h r s . a f t e r i n j e c t i o n , and t h e n declined. The amount o f g l u c u r o n i d e a p p e a r i n g i n u r i n e ( F i g . 4) was v a r i a b l e , r a n g i n g f r o m 7.5% t o 32.6% o f t o t a l m a t e r i a l , w i t h h i g h e r percentages appearing toward the l a t e r times. Unlike u r i n e , t h e b i l e h a d no d e t e c t a b l e l e v e l s u n t i l 2 h r s . and t h e r e was no p e a k i n t h e b i l i a r y c o n c e n t r a t i o n o f t h e p h e n o l r e d , s i n c e samples from the l o n g e s t o b s e r v a t i o n p e r i o d c o n t a i n e d the g r e a t e s t c o n c e n t r a t i o n o f b o t h f r e e and c o n j u g a t e d d r u g . The amount o f c o n j u g a t e d d r u g a p p e a r i n g i n t h e b i l e r a n g e d f r o m 15 t o 30% o f total material. C o n s i d e r a t i o n o f these d a t a i n terms o f m u l t i - c o m p a r t m e n t a l a n a l y s i s i s f o u n d i n F i g . 5. The r e n a l compartment n e v e r a c h i e v e d more t h a n 6.3% o f t h e a d m i n i s t e r e d d o s e and d e c l i n e d r a p i d l y a f t e r 1 h r . As e a r l y a about 18% o f t h e a d m i n i s t e r e a t 2 h r s . and c o n t i n u e d t o c o n t a i n l a r g e amounts o f p h e n o l r e d f o r up t o 12 h r s . T h e r e i s o n l y a s l i g h t d i f f e r e n c e b e t w e e n t h e amount o f p h e n o l r e d h a n d l e d by t h e u r i n a r y and b i l i a r y c o m p a r t ments i n 48 h r s . , 4 0 % and 4 8 % r e s p e c t i v e l y . I n e a c h compartment most o f t h e m a t e r i a l i s f r e e d r u g . E f f e c t s of Interrupted Enterohepatic U r i n a r y H a n d l i n g o f P h e n o l Red

C i r c u l a t i o n on B i l i a r y and

The v a l u e s i n T a b l e I compare b i l i a r y and u r i n a r y e x c r e t i o n o f p h e n o l r e d . The i n t a c t a n i m a l e x c r e t e s 4 9 % o f t h e a d m i n i s t e r e d d o s e i n 48 h r s . i n t o g a l l b l a d d e r b i l e and o f t h i s 20% i s e x c r e t e d Table I .

C o m p a r i s o n o f U r i n a r y and B i l i a r y E x c r e t i o n o f P h e n o l Red i n I n t a c t and i n F i s t u l i z e d D o g f i s h S h a r k a

P h e n o l Red % T o t a l Dose

% as

Glucuronide

Intact Urine Bile

41 49

33 20

23 47

30 44

Fistulized Urine Bile l

Data from ( 8 ) . of phenol r e d . treatment.

A n i m a l s w e r e t r e a t e d i n t r a v e n o u s l y w i t h 10 mg/kg V a l u e s a r e means f o r 4-6 a n i m a l s 48 h r . a f t e r

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

15.

GUARiNO A N D A R N O L D

Pharmacokinetics

243

DISTRIBUTION OF PHENOL RED IN DOGFISH SHARK 1001-

HRS Figure 5.

Time course of distribution of phenol red in terms of multicompartment analysis (S)

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

244

as t h e g l u c u r o n i d e c o n j u g a t e . Forty-one percent of the adminis­ t e r e d d o s e a p p e a r s i n 48 h r s . i n t h e u r i n a r y compartment o f i n t a c t a n i m a l s and o n e - t h i r d o f t h i s i s g l u c u r o n i d e . When b i l e i s c o l l e c t e d v i a f i s t u l a 47% o f t h e a d m i n i s t e r e d dose appears i n t h e b i l e b u t a b o u t t w i c e a s much o f t h i s t o t a l o c c u r s a s t h e g l u c u r o n i d e compared w i t h t h e i n t a c t a n i m a l ( 4 4 % v s 2 0 % ) . These d a t a suggest t h a t t h e l i v e r s y n t h e s i z e s t h e g l u c u r o n i d e and e x c r e t e s i t i n t o t h e b i l e , so t h a t i n t h e a n i m a l s w i t h f i s t u l a e a l a r g e p r o p o r t i o n o f t h e a d m i n i s t e r e d dose i s d i v e r t e d , p r e v e n t i n g the r e a b s o r p t i o n of t h e g l u c u r o n i d e (presumably a f t e r h y d r o l y s i s ) v i a the g a s t r o i n t e s t i n a l tract. The u r i n a r y e x c r e t i o n i n s u r g i ­ c a l l y t r e a t e d animals confirms t h i s l a t t e r p o i n t , s i n c e i n animals w i t h f i s t u l a e o n l y 23% o f t h e dose i s e x c r e t e d i n t h e u r i n e i n 48 h r s . compared w i t h 4 1 % i n t h e i n t a c t a n i m a l s . On t h e o t h e r hand, t h e range o f v a l u e s seen i n t h e s e w i l d a n i m a l s (8) o f t e n was 30-50% o f t h values d henc t h e r o f t e not s i g n i f i c a n t d i f f e r e n c e C o n c e n t r a t e d T r a n s f e r o f P h e n o l Red i n t o R e n a l and H e p a t i c Compartments By c o m p a r i n g t h e t i s s u e and f l u i d c o n c e n t r a t i o n s o f f r e e p h e n o l r e d , one c a n o b t a i n t h e d e g r e e o f c o n c e n t r a t i o n o c c u r r i n g i n a n i n d i c a t e d compartment ( T a b l e I I ) . R e l a t i v e t o p l a s m a , t h e Table

II.

C o n c e n t r a t i o n G r a d i e n t s f o r F r e e P h e n o l Red i n R e n a l and H e p a t i c Compartments i n D o g f i s h S h a r k a

Ratio o f :

Value

Kidney/ρlasma

6.4

Ur i n e / k i d n e y

4.7

Urine/plasma

30.4

Liver/plasma

3.9

Bile/liver

16.5

Bile/plasma

64.3

D a t a f r o m (8.). A n i m a l s w e r e t r e a t e d i n t r a v e n o u s l y w i t h 10 mg/kg phenol red. V a l u e s a r e mean o f 5-6 a n i m a l s 6 h r . a f t e r t r e a t m e n t . T h i s t i m e was c h o s e n b e c a u s e i t was t h e l a t e s t t i m e when p l a s m a l e v e l s were d e t e c t a b l e . c o n c e n t r a t i o n i n k i d n e y was t w i c e t h a t o f l i v e r . The g r a d i e n t b e t w e e n b i l e and l i v e r was a b o u t f o u r - f o l d l a r g e r t h a n was t h e

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

15.

GUARiNO A N D A R N O L D

245

Pharmacokinetics

u r i n e / k i d n e y r a t i o i n d i c a t i n g a dominance o f t h e b i l i a r y t r a n s p o r t mechanisms, w h i c h became more o b v i o u s a t l a t e r t i m e s ( F i g 5 ) . F i n a l l y , the o v e r a l l f l u i d / p l a s m a r a t i o s again favored the b i l i a r y r o u t e , 64.3 v s 30.4. Saturation o f Excretory Processes Attempts t o g a t h e r e v i d e n c e t h a t b o t h p h e n o l r e d and i t s g l u c u r o n i d e a r e e x c r e t e d by s a t u r a b l e t r a n s f e r p r o c e s s e s w e r e made by m e a s u r i n g d r u g d i s p o s i t i o n a t f o u r d i f f e r e n t d o s e s o f p h e n o l r e d . Of t h e f o u r d o s e s s t u d i e d (8) o n l y t h e two e x t r e m e d o s e s a r e shown i n T a b l e I I I . Over t h i s r a n g e o f d o s e s , t h e r e was no e v i d e n c e o f s a t u r a t i o n i n t e r m s o f c o n c e n t r a t i o n , o f e i t h e r t h e p l a s m a o r k i d n e y . T h e r e was no p r o p o r t i o n a l i t y b e t w e e n Table I I I .

E f f e c t s o f D i f f e r e n t Doses o f P h e n o l Red on i t s D i s t r i b u t i o n i n t h e D o g f i s h S h a r k

(form o f phenol

red)

% Dose

g/g o r m l

% Dose

Plasma

(free)

16

7.6

204

10.2

Kidney

(free)

83

1.6

948

2.2

Liver

(free)

38

41.5

155

14.3

Urine

(free)

711

13.3

1777

3.9

63

1.4

148

0.3

262

2.6

936

1.0

120

1.1

241

0.3

Urine (glucuronide) Bile

(free)

B i l e (glucuronide)

a

g/g o r m l

D a t a f r o m ( 8 ) . A n i m a l s were t r e a t e d i n t r a v e n o u s l y w i t h 10 o r 100 mg/kg p h e n o l r e d . V a l u e s a r e mean o f 5-6 a n i m a l s p e r d o s e 4 hrs. a f t e r treatment.

dose and the p e r c e n t a g e o f a d m i n i s t e r e d dose i n e i t h e r t h e r e n a l o r p l a s m a compartment. T h i s p r o v i d e s e v i d e n c e t h a t b o t h t h e s e compartments a r e n o t l i m i t e d i n t e r m s o f t h e p o r t i o n o f t h e a d m i n i s t e r e d dose which they can b i n d and/or c o n t a i n . In liver, d o s e s o f 100 mg/kg o f p h e n o l r e d w e r e s i g n i f i c a n t l y d i f f e r e n t f r o m t h e 10 mg/kg d o s e i n t e r m s o f c o n c e n t r a t i o n a n d p e r c e n t o f a d m i n i s t e r e d dose. The p e r c e n t a g e o f a d m i n i s t e r e d d o s e i n l i v e r demonstrates t h a t w i t h i n c r e a s i n g doses l i v e r i s l e s s a b l e t o s t o r e phenol red than i s kidney. Regarding u r i n a r y e x c r e t i o n ,

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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i n c r e a s e d doses o f phenol r e d l e a d t o l e s s than p r o p o r t i o n a l i n c r e a s e s i n f r e e d r u g a n d g l u c u r o n i d e c o n c e n t r a t i o n s and a decreased p e r c e n t o f t h e dose i n t h e u r i n e s u g g e s t i n g s a t u r a t i o n . S a t u r a t i o n o f b i l i a r y t r a n s p o r t f o r f r e e and c o n j u g a t e d p h e n o l r e d a p p a r e n t l y d i d o c c u r a t t h e 100 mg/kg d o s e . Glucuronide t r a n s p o r t p r o v e d t o be t h e most r e a d i l y s a t u r a t e d i n b o t h b i l e and u r i n e . I n h i b i t i o n o f P h e n o l Red E x e c r e t i o n by P r o b e n e c i d S i n c e p r o b e n e c i d i s used e x t e n s i v e l y as an i n h i b i t o r o f t h e u r i n a r y and b i l i a r y e x c r e t i o n o f c a r b o x y l i c , p h e n o l i c and s u l p h o n i c a c i d s i n many o t h e r a n i m a l s , i t was o f i n t e r e s t t o d e t e r m i n e i f p r o b e n e c i d would i n h i b i t t h e u r i n a r y and/or b i l i a r y t r a n s p o r t o f p h e n o l r e d i n t h e s h a r k ( T a b l e I V ) . The p l a s m a l e v e l s d e t e r m i n e d at 4 h r s . a f t e r a d m i n i s t r a t i o n of phenol r e d alone o r i n combination Table IV.

Effects of Probeneci o f P h e n o l Red i n t h e D o g f i s h S h a r k

Tissue or f l u i d (form o f phenol r e d )

P h e n o l Red A l o n e % Dose Mg/g

a

P h e n o l Red + P r o b e n e c i d % Dose yg/g 7.3

Plasma

(free)

16

7.6

14

Kidney

(free)

85

1.6

25

b

0.5

b

28.0

Liver

(free)

38

42.0

27

b

Urine

(free)

711

13.3

85

b

3.6

b

63

1.4

3

b

0.1

b

262

2.6

191

112

1.1

52

Urine (glucuronide) Bile

(free)

B i l e (glucuronide)

b

1.7 b

0.3

b

Data from ( 8 ) . A n i m a l s were t r e a t e d i n t r a v e n o u s l y w i t h p h e n o l r e d (10 mg/kg) a l o n e , o r w i t h p r o b e n e c i d (40 mg/kg) 30 m i n . p r i o r t o p h e n o l r e d (10 mg/kg) t r e a t m e n t . V a l u e s a r e mean o f 5-6 a n i m a l s 4 hrs. a f t e r r e c e i v i n g phenol r e d . b

Significantly different

(P