Bound and Conjugated Pesticide Residues 9780841203341, 9780841202948

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Bound and Conjugated Pesticide Residues

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Bound and Conjugated Pesticide Residues Donald D. Kaufman, EDITOR Gerald G. Still, EDITOR Gaylord D. Paulson, EDITOR U.S Department f Agricultur Suresh K. Bandal, EDITOR 3M Company

A symposium sponsored by the Division of Pesticide Chemistry at Division Workshop, Vail, Colo., June 22-26, 1975

ACS SYMPOSIUM SERIES 29

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1976

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Library of Congress CIP Data B o u n d and c o n j u g a t e dp e s t i c i d er e s i d u e s . (ACS s y m p o s i u ms e r i e s ; 29 I S S N 0 0 9 7 6 1 5 6 ) I n c l u d e sb i b l i o g r a p h i c a l r e f e r e n c e s and i n d e x . 1. P e s t i c i d e s — E n v i r o n m e n t a la s p e c t s — C o n g r e s s e s . I . K a u f m a n , D o n a l d D e V e r e , 1933. II. A m e r i c a n C h e m i c a l S o c i e t y . D i v i s i o n of P e s t i c i d e C h e m i s t r y . III. S e r i e s : A m e r i c a n C h e m i c a l S o c i e t y . ACS s y m p o s i u m s e r i e s ; 29. Q H 5 4 5 . P 4 B 6 3 6 3 2 ' . 9 5 ' 0 4 2 7 6 1 3 0 1 1 I S B N 0 8 4 1 2 0 3 3 4 2 A C S M C 8 29 1-396

Copyright © 1976 American Chemical Society All Rights Reserved. No part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. P R I N T E D IN THE

U N I T E DS T A T E S OF A M E R I C A

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

ACS Symposium Series Robert F. G o u l d , Editor

Advisory Board Kenneth B. Bischoff Jeremiah P. Freeman E. Desmond Goddard Jesse C. H. Hwa Philip C. Kearney Nina I. McClelland John B. Pfeiffer Joseph V . Rodricks Aaron Wold

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The 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. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PREFACE roduction of adequate supplies of food andfiberpresently requires the use of pesticides. Pesticides are used deliberately to alter the ecology, that is, to eliminate or restrict undesirable species in favor of species considered necessary for mans continued existence. The ubiquitous nature of many biological and biochemical processes makes it likely that even highly specific pesticides will affect some nontarget organisms. It is therefore imperative to determine what ecological changes pesticides may produce, which changes are permanent or temporary, and to decide which are acceptable or unacceptable. In the past, the inability to reisolate a pesticide or its degradation products enabled us to conclude glibly that it was detoxified, degraded, metabolized, or eliminate for further concern. Radiolabeled pesticides and more sophisticated analytical technology, however, have brought a halt to such practices. We now recognize that our inability to isolate a chemical does not constitute metabolism or complete detoxication to innocuous products, but rather it constitutes a complex environmental research problem requiring the most sophisticated inputs of a multitude of scientific disciplines. This workshop was organized because of current interest and concern for bound and conjugated pesticide residues in animals, plants, and soils. The objective of the conference was to bring together scientists with biological, chemical, and physical expertise in environmental fate of pesticides so that they could examine in some detail the formation and fate, synthesis, extraction, and methods of characterization of such pesticide residues, and if possible, provide scientific insights to future considerations regarding their overall significance. In the absence of definitive conclusions it was thought that the information discussed would provide the foundation for further research toward such a goal.

P

U.S. Department of Agriculture Beltsville, Md.

DONALD D. K A U F M A N

U.S. Department of Agriculture Fargo, N. Dak.

GERALD G. STILL GAYLORD D. PAULSON

3M Company St. Paul, Minn. January 20, 1976

SURESH K. BANDAL

xi

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1 Bound and Conjugated Pesticide Residues DONALD D. KAUFMAN Pesticide Degradation Laboratory, Agricultural Environmental Quality Institute, ARS, U. S. Department of Agriculture, Beltsville, Md. 20705

Man has developed the capacity to manufacture and use on a vast scale, organic compound our own body and to our environment. Such synthetic organic compounds are used as drugs for sickness, pesticides of various kinds for agriculture and health purposes, coloring matters, emulsifiers and stabilizing agents for food and drink, dyes for clothes, plasticizers, lubricants, coolants, and cleansing agents for a l l sorts of purposes, flame retardants, beauty preparations, spermicides and ovicides for population control, explosives and poison gases for military use and so on. Past research experience has clearly demonstrated that certain of these compounds, or their degradation products can enter into almost every phase of our environment and civilized existence. It is therefore essential that we know what happens to these compounds i f and when they enter our bodies, our foodstuffs, and our environment so as to avoid any damaging effects or, if they cause damage, their use can be avoided in favor of less harmful compounds, or the risks associated with their continued use can be adequately evaluated. Almost as rapidly as man has learned to generate complex new organic chemicals our environment has adjusted to cope with not a l l , but the majority of these chemicals. Investigations of the environmental fate, behavior, and metabolism of synthetic organic chemicals have revealed many new and fascinating environmental processes, metabolic pathways, and chemical and physical reactions which heretofore were either not recognized, or their significance not fully understood or appreciated. We a l l quickly recognize where man would be without the development of synthetic and natural organic chemicals in medical science. Similarly, where would environmental science and food production technology be today i f it were not for the advent of agricultural chemicals. We know that agricultural chemicals are dissipated or utilized by many mechanisms and biochemical processes including oxidation, reduction, hydrolysis, dehalogenation, dehydrohalogen1

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BOUND AND CONJUGATED PESTICIDE RESIDUES

a t i o n , r i n g cleavage, e t c . In the past metabolic changes o f f o r e i g n o r g a n i c compounds were r e f e r r e d t o as " d e t o x i c a t i o n . " I n the past the i n a b i l i t y to i s o l a t e a chemical enabled us t o g l i b l y conclude t h a t i t was degraded, metabolized, o r by some unknown mechanism e l i m i n ated from any need f o r f u r t h e r concern. The advent o f r a d i o l a b e l e d p e s t i c i d e s and more s o p h i s t i c a t e d a n a l y t i c a l t e c h n o l ogy, however, g r a d u a l l y c a l l e d a h a l t t o what many have r e f e r red t o as "bathtub c h e m i s t r y . " We now recognize t h a t our i n a b i l i t y t o i s o l a t e a chemical does not c o n s i t u t e metabolism or complete d e t o x i c a t i o n t o inocuous p r o d u c t s , but r a t h e r , i t c o n s t i t u t e s a complex environmental research problem r e q u i r i n g the most s o p h i s t i c a t e d i n p u t s o f not o n l y a m u l t i t u d e o f s c i e n t i f i c d i s c i p l i n e s , but economic, resource management, and development techniques We know now t h a t a l occur w i t h a p e s t i c i d e do not n e c e s s a r i l y l e a d t o complete degradation o r m i n e r a l i z a t i o n o f the p e s t i c i d e molecule. In some i n s t a n c e s a parent p e s t i c i d e molecule may be converted t o a more t o x i c substance. Indeed, a few p e s t i c i d e s a c t u a l l y r e q u i r e molecular changes f o r a c t i v a t i o n . Other p e s t i c i d e s or t h e i r m e t a b o l i t e s e n t e r i n t o s y n t h e t i c r e a c t i o n s which f r e q u e n t l y r e s u l t i n the formation o f molecules f a r more complex than the parent p e s t i c i d e molecule. It i s this latter phenomenon which b r i n g s us t o our present conference r e g a r d i n g bound and conjugated p e s t i c i d e r e s i d u e s . A review o f r a d i o l a b e l e d p e s t i c i d e degradation o r d i s s i p a t i o n s t u d i e s r e v e a l s numerous b a s i c s i m i l a r i t i e s . Briefly, these s i m i l a r i t i e s can be c h a r a c t e r i z e d as three experimental fractions: A. V o l a t i l i z e d o r e l i m i n a t e d products. B. E x t r a c t a b l e products. C. Unextractable (or r e s i d u a l ) products. V o l a t i l i z e d m a t e r i a l s would i n c l u d e r e s p i r e d m a t e r i a l s , i . e . , parent o r i n t e r m e d i a t e p r o d u c t s , and CO^, as w e l l as m a t e r i a l s l o s t by the p h y s i c a l processes o f v o l a t i l i z a t i o n . Other products may be e l i m i n a t e d i n wastes o r e x c r e t a . G e n e r a l l y speaking, the products l o s t by these mechanisms are r e a d i l y trapped and c h a r a c t e r i z e d once the process and the e n v i r o n mental f a c t o r s a f f e c t i n g the process are recognized. For purposes o f t h i s p r e s e n t a t i o n e x t r a c t a b l e products are considered as those products r e a d i l y removed from the t r e a t e d m a t e r i a l by any one o r more o f a v a r i e t y o f s o l v e n t s and e x t r a c t i o n techniques. Although c h a r a c t e r i z a t i o n o f e x t r a c t a b l e products has c h a l l e n g e d our very best minds and technology, they too represent at present a more e a s i l y worka b l e and i d e n t i f i a b l e f r a c t i o n . Included i n t h i s f r a c t i o n are some o f the parent compound and i t s many metabolic products r e s u l t i n g from both s y n t h e t i c and degradative r e a c t i o n s . G e n e r a l l y , but not always, degradative r e a c t i o n s lead toward

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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more p o l a r products, hence the tendency to use s o l v e n t e x t r a c t i o n techniques designed f o r maximum p o l a r product removal. Included i n t h i s p o l a r s o l v e n t e x t r a c t are products r e s u l t i n g from synt h e t i c r e a c t i o n s o c c u r r i n g i n the t r e a t e d m a t e r i a l . Experience has demonstrated t h a t these s y n t h e t i c products are f r e q u e n t l y the r e s u l t o f v a r i o u s conjugative processes which occur w i t h i n the t r e a t e d m a t e r i a l . Part o f the purpose o f t h i s conference i s t o examine i n some d e t a i l the s y n t h e s i s , e x t r a c t i o n , and methods o f c h a r a c t e r i z a t i o n of these products, i . e . , conjugates, and i f p o s s i b l e provide s c i e n t i f i c i n s i g h t s t o f u t u r e c o n s i d e r a t i o n s regarding t h e i r o v e r a l l s i g n i f i c a n c e . I s o l a t i o n and removal o f the e x t r a c t a b l e products from the o r i g i n a l pesticide treated material invariably involves s a c r i f i c ing the n a t u r a l p h y s i c a l s t a t e o f the t r e a t e d m a t e r i a l . While the r e s u l t i n g mass o f u n e x t r a c t a b l and d e s c r i b a b l e i n p l a n t carbohydrates; i n animals as: l i p i d s , f a t s , p r o t e i n s , and s k e l e t a l m a t e r i a l s ; and i n s o i l as: sand, s i l t , c l a y , and organic matter; i t s p r e c i s e chemical nature has g e n e r a l l y d e f i e d complet e l y meaningful and accurate d e s c r i p t i o n . I t i s f r e q u e n t l y w i t h i n t h i s p o r t i o n o f the p e s t i c i d e t r e a t e d m a t e r i a l t h a t anywhere from a few percent t o n e a r l y 100% o f some p e s t i c i d e r e s i dues w i l l remain i n an e x t r a c t a b l e form. I t i s t h i s u n e x t r a c t able and h e r e t o f o r e l a r g e l y undescribable p e s t i c i d e residue which has been l o o s e l y c h a r a c t e r i z e d by many s c i e n t i s t s as the "bound residue. As w i t h many such general q u a s i - s c i e n t i f i c terms, however, t h i s terminology i s not f u l l y understood or appreciated by a l l p e s t i c i d e s c i e n t i s t s , r e g u l a t o r y agencies, or t h e i r administ r a t o r s . Thus, a second p a r t o f our conference i s t o examine what i s known about the "bound r e s i d u e " : What i s i t ? How can i t be c h a r a c t e r i z e d , d e f i n e d , o r i d e n t i f i e d ? and i f not, What i s i t s s i g n i f i c a n c e ? How f a r must we go i n c h a r a c t e r i z i n g i t ? I f i t i s t r u l y "bound", and not r e a d i l y a v a i l a b l e t o s i g n i f i c a n t b i o l o g i c a l systems, must i t be f u l l y c h a r a c t e r i z e d ? What does the q u a l i f i c a t i o n o f " r e a d i l y a v a i l a b l e " mean? These are but a few o f the questions we hope t o e i t h e r answer o r l a y the foundat i o n f o r f u r t h e r research t o f i n d the answer f o r d u r i n g t h i s conference. 11

Conjugates There are very few chemicals which enter i n t o b i o l o g i c a l systems t h a t are not subject t o chemical changes. A few " b i o c h e m i c a l l y i n e r t " compounds remain unchanged, although they may be t o x i c o l o g i c a l l y a c t i v e . The type o f change which occurs depends p r i m a r i l y upon the s t r u c t u r e of the compound, but other f a c t o r s such as s p e c i e s , route of entrance, and n u t r i t i o n a l b a l ance may a l s o be important. There are s e v e r a l types o f s y n t h e t i c r e a c t i o n s common t o p e s t i c i d e s . Conjugate type r e a c t i o n s which have been observed i n c l u d e (jL): A. Reactions w i t h carbohydrates ( g l y c o s i d e formation):

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1. Glucuronic a c i d c o n j u g a t i o n . 2. Glucoside formation. 3. Other ( r i b o s i d e , g e n t i o b i o s i d e ) . B. Reactions w i t h aminoacids: 1. Simple amino a c i d s ( g l y c i n e , a l a n i n e , e t c . ) 2. Complex amino a c i d s ( g l u t a t h i o n e , c y s t e i n e , etc.) C. Reactions i n v o l v i n g s u l f u r ( s u l f a t e c o n j u g a t i o n ) : and D. Reactions i n v o l v i n g a l k y l a t i o n and a c y l a t i o n . Reactions o f p e s t i c i d e s and p e s t i c i d e metabolites w i t h carbohydrates and amino a c i d s are common i n p l a n t and animal metabolism. Few, i f any, c a r b o h y d r a t e - p e s t i c i d e conjugates have been i s o l a t e d from s o i l . This i s somewhat s u r p r i s i n g i n view o f the r e l a t i v e ease w i t h which simple sugars can be reacted w i t h aromati other hand such conjugate microorganisms and t h e r e f o r e seldom i s o l a t e d . S u l f a t e conjugation i s a common r e a c t i o n i n animal metabol i s m . Reports have a l s o i n d i c a t e d the occurrence o f s u l f a t e conjugation i n p l a n t s and microorganisms. A l k y l a t i o n and a c y l a t i o n r e a c t i o n s are common t o p l a n t s , animals, and s o i l microorganisms. Other types o f s y n t h e t i c r e a c t i o n s i n v o l v i n g p e s t i c i d e residues and metabolites i n c l u d e condensation type r e a c t i o n s y i e l d i n g dimeric and polymeric compounds such as have been observed i n s o i l s . These w i l l be d i s c u s s e d f u r t h e r under s o i l bound r e s i d u e s . Whether o r not a given compound w i l l undergo any o f the above syntheses depends upon i t s possessing p a r t i c u l a r chemical groups or r e a c t i v e s i t e s . I f the compound does not i n i t i a l l y c o n t a i n such a group, i t may aquire one by o x i d a t i o n o r reduct i o n o r some other process. Perhaps the s i m p l e s t example o f such a r e a c t i o n i s the h y d r o x y l a t i o n o f benzene t o phenol which i s subsequently conjugated through the h y d r o x y l group. Several hypotheses have been put forward r e g a r d i n g the purpose o r s i g n i f i c a n c e o f conjugative r e a c t i o n s i n b i o l o g i c a l systems. These hypotheses i n c l u d e : A. Chemical defense ( 2 ) . B. Surface t e n s i o n ( 3 ) . C. Increased a c i d i t y ( 4 ) . B r i e f l y , the chemical defense hypothesis i s based on the assumption t h a t metabolic products o f f o r e i g n compounds are l e s s t o x i c and more s o l u b l e than t h e i r p r e c u r s o r s . Such i s not always the case, however. The surface t e n s i o n hypothe s i s observes t h a t compounds which lower the s u r f a c e t e n s i o n o f water tend t o accumulate at c e l l surfaces and thereby a t t a i n t o x i c c o n c e n t r a t i o n s . Conjugated products do not lower s u r f a c e tensions a p p r e c i a b l y , and thus do not accumulate t o t o x i c concentrations at s u r f a c e s . The hypothesis o f increased a c i d i t y notes that conjugation g e n e r a l l y changes a weak a c i d

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which the body can not e l i m i n a t e t o a s t r o n g a c i d which i t can e l i m i n a t e . While a l l three hypotheses seem t o account f o r c e r t a i n aspects o f the problem, none o f them p r o v i d e s a general e x p l a n a t i o n o f how and why such processes occur. I t i s not the primary o b j e c t i v e o f t h i s conference t o determine why such conjugates form but r a t h e r , how are they formed, how are they i s o l a t e d and c h a r a c t e r i z e d , and what i s t h e i r p o s s i b l e r o l e i n the formation o f the u n e x t r a c t a b l e o r bound residues observed i n v a r i o u s b i o l o g i c a l systems. A l s o , what i s t h e i r p o s s i b l e s i g n i f i c a n c e i n terms o f b i o l o g i c a l a v a i l a b i l i t y and t o x i c i t y i n subsequent food chain organisms. In general conjugates are c o n s i d e r a b l y more p o l a r and l e s s l i p o p h l i c than the parent p e s t i c i d e molecule, and as such are t h e r e f o r e more r e a d i l y e l i m i n a t e d from animals. P l a n t s , however, do not have e f f i c i e n while conjugation may i t does not n e c e s s a r i l y l e a d t o e l i m i n a t i o n . The r o l e o f such conjugates i n c a t a b o l i s m and u l t i m a t e b i n d i n g o f the p e s t i c i d e molecule i s not c l e a r . An a l t e r n a t i v e mechanism t o b i n d i n g , however, may be the d i r e c t i n t e r a c t i o n o f the p e s t i c i d e w i t h f u n c t i o n a l groups on p r o t e i n o r complex carbohydrate molecules. Bound Residues Perhaps the f i r s t o b j e c t i v e o f t h i s s e c t i o n should be t o d e f i n e the term "bound r e s i d u e . " L i k e many new terms, the d e f i n i t i o n o r i n t e r p r e t a t i o n o f what a bound r e s i d u e i s , has v a r i e d w i t h every i n d i v i d u a l s c i e n t i s t , and i s t o a very l a r g e degree dependent upon the e x t r a c t i o n techniques used. To a c e r t a i n e x t e n t , i t has been an e l u s i v e concept not only t o the p e s t i c i d e s c i e n t i s t , but t o our a d m i n i s t r a t o r s and r e g u l a t o r y agencies as w e l l . Concern f o r bound r e s i d u e s has v a r i e d a l l the way from t o t a l preoccupation w i t h c h a r a c t e r i z a t i o n o f t h a t e l u s i v e few percent o f u n e x t r a c t a b l e r a d i o l a b e l e d chemical, t o i n c r e d u l o u s d i s b e l i e f t h a t an u n e x t r a c t a b l e product should be o f any concern whatsoever. Responsible s c i e n c e , however, d i c t a t e s t h a t we know at l e a s t something about t h a t u n e x t r a c t a b l e e n t i t y . I f we can not d e s c r i b e i t p r e c i s e l y as (e.g.) a r a d i o l a b e l e d a s p a r t i c a c i d molecule l i n k i n g together other components o f a p r o t e i n , then we should at l e a s t be able t o i n d i c a t e t h a t when passed through another l i v i n g organism which i s most l i k e l y t o encounter t h a t t r e a t e d product, i t i s o r i s not b i o l o g i c a l l y a v a i l a b l e t o t h a t organism, and i f i t i s a v a i l a b l e i t i s e v e n t u a l l y e l i m i n a t e d o r has no s i g n i f i c a n t e f f e c t on t h a t organism. I f i t i s an unknown s o i l r e s i d u e i n c o r p o r a t e d i n t o s o i l o r g a n i c matter and i s s l o w l y r e l e a s e d at a r a t e r e p r e s e n t i n g o n l y a s m a l l percent o f what was o r i g i n a l l y a p p l i e d , can i t be considered safe and acceptable? These are o n l y a few o f the many questions

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which we hope t o answer i n the course o f t h i s conference. I am not aware o f any a l r e a d y e x i s t i n g d e f i n i t i o n of a bound r e s i d u e i n p l a n t s o r animals. While i t i s evident t h a t i n d i v i d u a l s c i e n t i s t s have developed t h e i r own concepts o f bound r e s i d u e s i n p l a n t s o r animals, there does not appear to have been any coordinated e f f o r t t o provide a d e f i n i t i o n . Last F a l l (1974) an American I n s t i t u t e o f B i o l o g i c a l SciencesEnvironmental Chemistry Task Group attempted to provide such a d e f i n i t i o n f o r s o i l bound r e s i d u e s . As some o f you are aware, t h i s was a committee organized by AIBS f o r The E n v i r o mental P r o t e c t i o n Agency t o develop a s e r i e s o f p r o t o c o l s designed t o provide the i n f o r m a t i o n necessary t o meet the Pesticide Registration Guidelines. The d e f i n i t i o n which we cast at t h a t time was considered o n l y an " i n t e r i m d e f i n i t i o n a more p r e c i s e or meaningfu t h i s very conference. The d e f i n i t i o n : A s o i l bound r e s i d u e i s "that u n e x t r a c t a b l e and c h e m i c a l l y u n i d e n t i f i a b l e p e s t i c i d e r e s i d u e remaining i n f u l v i c a c i d , humic a c i d , and humin f r a c t i o n s a f t e r exhaustive s e q u e n t i a l e x t r a c t i o n w i t h nonpolar organic and p o l a r s o l v e n t s . " In r e t r o s p e c t , perhaps a b e t t e r d e f i n i t i o n would have i n c l u d e d reference to p l a n t r o o t s , o r to decomposition by s o i l microorganisms, o r r e s i s t a n c e t o r e l e a s e by s p e c i f i c c e l l f r e e enzymes. I t seems e n t i r e l y reasonable, however, t h a t analogous d e f i n i t i o n s could be cast f o r u n e x t r a c t a b l e p e s t i c i d e r e s i d u e s i n p l a n t s and animals. Such a d e f i n i t i o n based on a more n e a r l y u n i v e r s a l methodology has the advantage o f p r o v i d i n g a standard p o i n t o f reference w i t h which t o more o b j e c t i v e l y evaluate i n d i v i d u a l chemicals and groups of chemicals. The great d i f f i c u l t y w i t h a l l f r a c t i o n a t i o n procedures, however, i s that the methods employed e i t h e r separate out products which are not d e f i n i t e chemical e n t i t i e s , o r form a r t i f a c t s which do not have the p r o p e r t i e s o f the o r i g i n a l m a t e r i a l . Nevertheless, the v a r i o u s f r a c t i o n a t i o n procedures can prove u s e f u l f o r i n v e s t i g a t i o n and charact e r i z a t i o n o f bound p e s t i c i d e r e s i d u e s . There are many c r i t i c a l questions t o be asked concerning bound r e s i d u e s . Perhaps the three most c r i t i c a l questions are: 1. What i s t h e i r nature and/or i d e n t i t y ; 2. What i s t h e i r s i g n i f i c a n c e ( t o x i c i t y , a v a i l a b i l i t y , accumulative n a t u r e , e t c . ) ; and 3. What i s t h e i r source? Within c e r t a i n l i m i t s answering e i t h e r one o f the f i r s t two questions can o b v i a t e the need f o r answers t o the other two. For example, i f the r a d i o l a b e l e d bound r e s i d u e i s i d e n t i f i e d as a n a t u r a l product i t s s i g n i f i c a n c e can p o s s i b l y be assumed on the b a s i s o f previous knowledge. On the other hand i f i t s i d e n t i t y i s not determined, but i t s a v a i l a b i l i t y and/or t o x i c i t y i s determined t o be o f no s i g n i f i c a n c e , then

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i t s i d e n t i t y becomes academic. Question three can be important i f i t i s known through hydroponic o r s t e r i l e r o o t i n g medium s t u d i e s t h a t absorbed r a d i o l a b e l e d p e s t i c i d e o r i t s p l a n t metabolites are not t r a n s l o c a t e d i n t o e d i b l e p o r t i o n s o f the p l a n t . Radiolabeled products e n t e r i n g i n t o e d i b l e p o r t i o n s o f p l a n t s grown i n p e s t i c i d e t r e a t e d n o n s t e r i l e s o i l , t h e r e f o r e must come from s o i l degradation products o f the p e s t i c i d e . Knowledge o f the source and the p o s s i b l e s o i l degradation products a v a i l a b l e t o the p l a n t can provide a c l u e as t o what r a d i o l a b e l e d product might be present i n the p l a n t and what e x t r a c t i o n procedures are necessary t o i s o l a t e and c h a r a c t e r i z e them. Concern f o r the i d e n t i t y o r nature o f the bound residue centers around the question o f whether o r not the bound residue consists o f intact pesticid t i o n products which ar i n the p l a n t , animal, o r s o i l m a t r i x and may be r e l e a s e d a t some f u t u r e date, o r whether i t i s a common o r d i n a r y metabolite which has reached a metabolic s t a t e where i t can be r e i n c o r p o r ated i n t o normal organic b u i l d i n g m a t e r i a l s . In s o i l , concern has a l s o been expressed f o r the long range e f f e c t s o f " p o l y c h l o i n a t e d o r p o l y t r i f l u o r o m e t h y l a t e d " s o i l organic matter on v i t a l s o i l processes and c o n d i t i o n s . Considerable i n f o r m a t i o n e x i s t s regarding adsorption mechanisms and s i t e s f o r p e s t i c i d e s i n p l a n t s , animals, and s o i l c l a y and organic matter. D i s c u s s i o n o f t h i s i n f o r m a t i o n and i t s s i g n i f i c a n c e w i l l be presented by s e v e r a l o f the speakers. There i s a l s o a growing body o f i n f o r m a t i o n which i n d i c a t e s t h a t a number o f a l i p h a t i c and simple aromatic p e s t i c i d e s are e x t e n s i v e l y metabolized i n one o r more systems and subsequently r e i n c o r p o r a t e d as n a t u r a l products. In the past we have been o p t i m i s t i c i n b e l i e v i n g that a l l p e s t i c i d e s would o r should be completely m i n e r a l i z e d t o CO2, H2O, e t c . , a f t e r t h e i r f u n c t i o n has been f u l f i l l e d . In these times o f energy c r i s e s and shortages we should take comfort i n knowing t h a t nature does not wantonly d i s c r i m i n a t e i n the u t i l i z a t i o n o f a simple organic a c i d , aminoacid, o r sugar molecule o r g i n a l l y d e r i v e d from a p e s t i c i d e molecule over one d e r i v e d from i t s own s y n t h e t i c e f f o r t s . There are a number of p e s t i c i d e s f o r which complete metabolic pathways are known from the parent p e s t i c i d e molecule t o simple o r g a n i c a c i d s . For example, the h e r b i c i d e dalapon i s metabolized t o pyruvate and a l a n i n e ( 5 , 6 ) ; TCA i s metabolized t o s e r i n e ( 7 ) ; the 2 , 4 - d i c h l o r o p h e n o l i c p o r t i o n o f 2,4-D goes througH a long series o f reactions ultimately y i e l d i n g succinic acid ( 8 ) . While i t i s t r u e t h a t most o f these m e t a b o l i c pathways have been worked out i n i s o l a t e d systems f r e e o f many competitive adsorptive o r metabolic r e a c t i o n s , i t i s reasonable t o expect t h a t a t l e a s t a s m a l l percent o f the parent p e s t i c i d e molecule w i l l be metabolized through t o such n a t u r a l l y o c c u r r i n g product

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i n the environment. We are always r e l i e v e d i n s o i l metabolism i n v e s t i g a t i o n s t o see l a r g e q u a n t i t i e s o f evolved from p e s t i c i d e t r e a t ments. T h i s may be an unreasonable e x p e c t a t i o n , however, when f o l l o w i n g metabolism o f ^ - a r o m a t i c m o i e t i e s i n s o i l . The formation o f humic substances i n s o i l i s a dynamic process i n v o l v i n g the a c t i o n o f s o i l microorganisms on p l a n t m a t e r i a l s and other organic r e s i d u e s . Macromolecules are formed at the expense o f carbohydrates o f p l a n t o r i g i n . These i n c l u d e b a c t e r i a l gums, a l g i n i c and p e s t i c a c i d s , and o t h e r l e s s w e l l - d e f i n e d polymeric c a r b o x y l i c a c i d s . Aromatic polyphenols formed by way o f quinone o x i d a t i o n can condense w i t h amino acids to u l t i m a t e l y give h u m i c - l i k e substances. Basidiomycetes as w e l l as o t h e r microscopic f u n g i have been found to degrade l i g n i n (polyphenol) an l i k e polymers. Phenoli pounds have been i n c o r p o r a t e d i n t o fungus s y n t h e s i z e d polymers. I t i s g e n e r a l l y b e l i e v e d t h a t there i s a g e n e r i c r e l a t i o n between the v a r i o u s humic substances. F u l v i c a c i d i s b e l i e v e d to c o n s i s t of polycondensation m a t e r i a l from s i m p l e r molecules. C o n t i n u a t i o n o f the p o l y m e r i z a t i o n process and chemical m o d i f i c a t i o n leads to the l e s s s o l u b l e humic a c i d and e v e n t u a l l y to i n s o l u b l e humin which has the h i g h e s t molecular weight and s t r u c t u r e most r e s i s t a n t t o degradation. When r a d i o l a b e l e d p e s t i c i d e degradation occurs i n s o i l we become concerned when l a r g e amounts o f the r a d i o l a b e l remain adsorbed or bound i n the s o i l f o r long p e r i o d s o f time. The concern f o r the r e l e a s e o f i n t a c t p e s t i c i d e o r p e s t i c i d e metabolites and contamination o f subsequent crops i s r e a l and j u s t i f i a b l e . I t i s j u s t as r e a l and j u s t i f i a b l e , however, to expect t h a t those o r other residues w i l l remain i n s o i l f o r long periods o f time i n an inocuous manner, o r be r e l e a s e d so s l o w l y as t o be i n s i g n i f i c a n t i n comparison to o t h e r products produced i n the s o i l system. Sorenson (9) s t u d i e d the degradation o f ^^C-labeled glucose and c e l l u l o s e i n three s o i l s . A f t e r a r a p i d i n i t i a l breakdown, h a l f - l i v e s o f 5 to 9 years were reported f o r the remaining C to be degraded. Other i n v e s t i g a t o r s have demons t r a t e d the formation o f humin from r e a d i l y decomposable organic compounds (10,11). These data imply t h a t even r e a d i l y metabolized compounds are i n c o r p o r a t e d i n t o humic substances and the extent t o which degradation occurs i s l i m i t e d . Although Fuhr (12) has shown t h a t 1 4 c - l i g n i n and -humic a c i d are not taken up by p l a n t s , the s i g n i f i c a n c e o f p l a n t uptake o f p e s t i c i d e s or p e s t i c i d e products i n c o r p o r a t e d i n t o s o i l o r g a n i c matter i s not f u l l y understood. In our own research w i t h c h l o r o a n i l i n e residues we have i s o l a t e d and t e n t a t i v e l y i d e n t i f i e d l a r g e polymeric type s t r u c t u r e s from both s o i l and i s o l a t e d m i c r o b i a l c u l t u r e s . These m a t e r i a l s appear analogous i n s t r u c t u r e t o those products o f 4 - c h l o r o a n i l i n e i d e n t i f i e d 14

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by Holland and Saunders (13). The r a t e o f decomposition and r e l e a s e o f a n i l i n e s from such products i s not known. The question now a r i s e s , given the s t r u c t u r e o f such a molecule and r e c o g n i z i n g t h a t i t i s made completely o f p e s t i c i d e d e r i v e d aromatic m o i e t i e s , "What s i g n i f i c a n c e must be attached to t h i s r e s i d u e ? " I s there a general " r u l e o f thumb" which could be put f o r t h i n d i c a t i n g t h a t molecules over a given s i z e need not be considered s i g n i f i c a n t i n terms o f p l a n t uptake? Summary Bound and conjugated p e s t i c i d e r e s i d u e s occur i n v i r t u a l l y a l l b i o l o g i c a l systems. As i n d i c a t e d at i n t e r v a l s throughout t h i s d i s c u s s i o n there are many questions t o be asked and perhaps d e f i n i t i o n s t o be c a s t . Are there a l s o some general p r o t o c o l s which we can recommend r e g a r d i n g i s o l a t i o n charact e r i z a t i o n , and e v a l u a t i n Our AIBS-ECTG put f o r t r e s i d u e s . C e r t a i n l y , the recommendations and p r o t o c o l s may change w i t h time as new i n f o r m a t i o n and concepts are developed. Can analogous type recommendations be made f o r p l a n t and animal bound r e s i d u e s ? What c o n s i d e r a t i o n s should be given t o conjugates o f p e s t i c i d e s and p e s t i c i d e m e t a b o l i t e s ? How f a r must we go i n c h a r a c t e r i z i n g these v a r i o u s types o f r e s i d u e s ? Are there e x t r a c t i o n schemes which we can o u t l i n e t h a t w i l l s u i t a b l y c h a r a c t e r i z e a bound r e s i d u e from p l a n t s , animals or s o i l s and know t h a t i n a l l good conscience t h i s i s a reasonable end t o t h a t p a r t i c u l a r requirement, beyond which a more d e f i n i t i v e answer i s p u r e l y academic? I s i t reasonable t o assume t h a t i f a bound r e s i d u e i n p l a n t s r e a d i l y passes through a mono- or p o l y g a s t r i c animal, and enters the s o i l where i t i s o n l y degraded at an i n f i t e s i m a l r a t e per year, t h a t t h i s residue can be considered i n s i g n i f i c a n t . For example, has our knowledge o f bound r e s i d u e s progressed t o the p o i n t t h a t we can recommend a general procedure (or procedures) f o r the e x t r a c t i o n o f bound r e s i d u e s from p l a n t s , animals and s o i l s (presumably a d i f f e r e n t procedure i s needed f o r each o f the three systems)? The f u r t h e r charact e r i z a t i o n o f the e x t r a c t s produced by such procedures would be dependent upon the percent o f the parent product l a b e l present i n any s p e c i f i c f r a c t i o n . In the absence o f p r e c i s e chemical i d e n t i f i c a t i o n , t h e i r p r o c e s s i n g through o t h e r b i o l o g i c a l systems, i . e . , s o i l , animals, e t c . would p r o v i d e an i n d i c a t i o n o f the b i o l o g i c a l a v a i l a b i l i t y o f such m a t e r i a l . S i g n i f i c a n c e determinations o f the a v a i l a b i l i t y o f the bound r e s i d u e s could be based on s e v e r a l f a c t o r s : 1. A c t u a l percent o f bound m a t e r i a l r e l e a s e d ; 2. Side e f f e c t s upon the consuming system; 3. T o x i c o l o g i c a l p r o p e r t i e s o f parent p e s t i c i d e o r most t o x i c m e t a b o l i t e s ; 4. E t c .

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The advantage o f such a system i s t h a t i t provides everyone w i t h a s i m i l a r format w i t h which t o assess the environmental f a t e and behavior o f t h e i r compound and the s i g n i f i c a n c e o f i t s bound r e s i d u e s . The disadvantage o f such a system i s that once e s t a b l i s h e d i t can become an endpoint beyond which no one, o r at l e a s t very few people are w i l l i n g t o progress. The format o f t h i s conference was designed w i t h the i n t e n t t h a t each o f these questions could be faced, d i s c u s s e d , and i f adequate i n f o r m a t i o n appears t o be a v a i l a b l e , then t o r e s o l v e the l e v e l o f need f o r any f u r t h e r c o n s i d e r a t i o n o f these q u e s t i o n s . In the absence o f s a t i s f a c t o r y answers i t i s intended t h a t new and more p r o d u c t i v e avenues o f research w i l l be e n v i s i o n e d which w i l l u l t i m a t e l y enable s u i t a b l e r e s o l u t i o n o f t h i s p e r p l e x i n g problem o f bound and conjugated pesticide residues. Literature Cited 1.

Williams, R. T., "Detoxication Mechanisms", (1959) 2nd ed., John Wiley & Sons, Inc., New York. 2. Sherwin, C. P., Physiol. Reviews (1922), 2, 264. 3. Berczeller, L., Biochem. Z. (1917), 84, 75. 4. Quick, A. J . , J . Biol. Chem. (1932), 97, 403. 5. Kearney, P. C., Kaufman, D. D., and Beall, M. L., Biochem. Biophys. Res. Comm. (1964), 14, 29. 6. Beall, M. L., M. Sc. Thesis. (1964). Univ. Maryland, College Park, Md. 7. Kearney, P. C., Kaufman, D. D., Von Endt, D. W., and Guardia F. S. J . Agr. Food Chem. (1969), 17, 581-584. 8. Kaufman, D. D., In W. D. Guenzi (ed.) "Pesticides i n Soil and Water", (1974), Soil S c i . Soc. Am., Madison, Wis. p.164. 9. Sorensen, L. H., Soil Biol. Biochem. (1972), 4, 245. 10. Chekalar, K. I., and Illyuvieva, V. P., Pochvovedenie (1962), 5, 40. 11. Sinha, M. K., Plant and Soil (1972), 36, 283. 12. Fuhr, F., Zeits. Pflanzenernachr. Bodenk. (1969), 121, 43. 13. Holland, V. R., and Saunders, B.C., Tetrahedron (1968), 24, 585.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2 Biological Activity of Pesticide Conjugates H. W. DOROUGH Department of Entomology, University of Kentucky, Lexington, Ky. 40506

Conjugation, by evolutionary design, is a metabolic process whereby endogenous as wel to polar components for the purpose of facilitating their removal from the site(s) of continuing metabolic processes. In animals, this is accomplished by the excretion of the more polar metabolites from the body. Elimination may be via the urine and/ or the bile. For example, glucuronide conjugates are excreted mainly in the urine i f their molecular weight is approximately 300 or less ( 1 ) . Those having a molecular weight of 700 or over are excreted largely in the bile, while those of intermediate size are eliminated by both routes. In plants, some elimination of the conjugates may occur but most are simply stored as terminal metabolites in the tissue. Most pesticides, like other exogenous compounds, are subject to a variety of conjugative reaction by living organisms. It is the intent of the current discussion to consider conjugates of these toxicants from the standpoint of their biological activity and potential significance to man and other life. Conjugated metabolites of pesticides are but one of the many types of residues which may result from those chemicals used to control various pests. True, they are not among those metabolites such as DDE, paraoxon, etc. which are immediately recognized by all pesticide chemists and most laymen, but, nevertheless, they are pesticide residues and must be treated as such. This is stated simply to infer that the significance of pesticide conjugates should not be over estimated, under estimated, or ignored. But, rather, they must be evaluated in much the same fashion as any other pesticide residues and judgements of their significance based on sound scientific data. While this view appears highly virtuous and is congruent in respect to one's scientific inclinations, pesticide conjugates possess some rather unique characteristics which severely challenge its practicality. Of uppermost importance is the fact that conjugation increases the polarity of the pesticide or its metabolite. Consequently, the resulting metabolites become very 11

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much akin to each other and to a multitude of endogenous chemicals found in all biological systems. Thus, the problems involved in isolation, purification and identification of conjugates are magnified tremendously as compared to apolar metabolites, and indeed have proven to be insurmountable in far too many cases. Much could be said to document the adverse effects increasing polarity has on attempts to identify and subsequently determine the biological activity of pesticide metabolites, especially those formed by conjugation. However, it is necessary only to peruse past progress in this regard to see the real importance of metabolite polarity. With few exceptions, one will find that the ease and rapidity with which metabolites of a particular pesticide have been identified and toxicologically evaluated is inversely proportional to the polarity of the individual metabolites under consideration. Concomitant with th jugates is a general lack of knowledge relative to the synthesis of these compounds. Even in those instances where the pesticidecontributing moiety has been identified and the endogenous chemical fairly well defined, there has been l i t t l e success in chemically synthesizing the suspect intact conjugate. As a result, sufficient quantities of the metabolites are not available for determining their biological activity as is routinely done with non-conjugate metabolites of pesticides. While there is no doubt that the chemical synthesis of many pesticide conjugates is extremely difficult, the primary reason that synthetic conjugates are not commonly available is that few concentrated efforts have been placed on their preparation. This will likely remain the case until such time biological activity data relative to pesticide conjugates are included in the numerous requirements for commercial utilization of pesticidal chemicals. There is ample evidence to suggest that pesticide conjugate synthesis is feasible (2-4)and that pesticide conjugates can, and will, be prepared i f ever deemed essential for evaluating the safety of pesticides to man and the environment. Having attempted to place pesticide conjugates in their proper prospective as pesticide residues with certain properties quite unlike the non-conjugate materials, i t is now desirous to address generally the significance of pesticide metabolites whether they be free or conjugated. Metabolite Significance. The term "metabolite" is used here to denote any derivative of the parent pesticide molecule formed subsequent to its preparation. This includes products formed spontaneously during storage, and those formed chemically and biochemically after their application. It would exclude impurities remaining in the product following synthesis and normal purification procedures. Generally, a metabolite should be judged significant or

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potentially significant unless adequate information is available which reasonably assures that estimated maximum levels of exposure (a) to man will in no way adversely effect his wellbeing or that of future generations, and (b) to other animal species will not endanger the survival or integrity of the exposed species. The reason for considering man separate from other animal species is simple. With man, there can be no compromise, but with other animal species some harm may have to be tolerated for the benefit of man. However, the degree of harm must be minimal and of short duration. For a pesticide metabolite to become significant in animals, i t must be available. While the manner of exposure to animals may have a bearing on the ultimate significance of the metabolite, the most important thing is exposure, per se. The basic sources of exposure are shown in TABLE I Common Sources of Pesticide Metabolites. External

Internal

1. Food

1. Enzymatic Formation

2. Environmental Contaminates

2. Non-Enzymatic Formation

Of the external sources, the food must be considered as a major means by which man is exposed to pesticide metabolites, especially the conjugates. Successful production of most crops require pesticide treatments of some type, and i t is generally in this way that metabolites find their way into the diet. "Environmental contaminates" is a catch-all phrase to cover all external sources of pesticide metabolites other than food. Pesticide metabolites in the air, on dust particles, and on various surfaces are examples of this type of exposure. This usually is not a major source of conjugate residues. Internal sources of metabolites refer to those generated in the animal body. They may be formed in a number of ways, chemically and biochemically, in both man and in animals which constitute a portion of his diet. In the broadest sense, there are 3 different categories of pesticide metabolites, free, conjugated and bound. The free metabolites are those which are derived from the parent molecule and have not reacted further with natural components of a biological system. With apolar pesticides, the free metabolites are usually considered as those extractable from the substrate and which partitioned from water into an organic solvent such as chloroform or ether. Conjugated metabolites are derivatives of the pesticide which have reacted chemically with a natural component of the organism

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to form a new material. Generally, this involves a free metabol i t e , usually hydroxylated, conjugated as a glucuronide, glucoside, sulfate, etc. These metabolites are usually extractable from the substrate with polar solvents but do not partition from water into solvents such as chloroform or ether. Bound or unextracted metabolites also are conjugates but are derivatives of the pesticide which can not be removed from the substrate by thorough extraction. Little is known of their chemical nature, but i t is suspected that they represent derivatives of the pesticide which have reacted with components of the organism such as proteins, cell membrane and/or various other cellular inclusions. Assuming that the metabolic pathway of a pesticide has been completely defined, how does one go about determining the significance of these metabolites? Ask a toxicologist and he will stress the need for acut ecologist will emphasize the need for determining their impact on the environment, while an enzymologist would question their effect on animal enzyme systems. This type of response can go on indefinitely. Actually, i t is very difficult to determine just what information should be obtained for evaluating the significance of a metabolite. Asking the right question is just part of the problem. There remains the task of obtaining appropriate information to answer the question. All experimental approaches and procedures are not the same and, thus, some must be better than others. The best approach should be determined to the best of our ability before engaging in research designed to evaluate metabolite significance. To gather data is one thing; to properly utilize it is another. Data collected in studies designed to determine metabolite significance will be meaningful only i f correctly interpreted. It is imperative that we know before conducting the experiments that the results are subject to interpretation and that the qualifications of the interpretors are as good as technology will allow. In summary, then, there are 3 basic questions to be asked, and answered, before initiating research in the area of metabolite significance: 1. What do we need to know? 2. How do we go about obtaining this information? 3. How can this information best be utilized? It is readily apparent that an interdisciplinary approach is essential i f these questions are to be answered satisfactorily. Some insight into the scientific diversity required in determining metabolite significance may be gained by considering just a few areas of concern (Table II) which must be considered with every metabolite which is encountered. Potential adverse effects of pesticide metabolites are not confined to this list. Nor should any list made at any time, even by the most qualified scientists, be considered as final.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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15

Like the requirements for determining metabolite significance, the factors to be considered will continue to change as our knowledge increases. TABLE II Some Possible Detrimental Expressions of Biologically Active Pesticide Metabolites Acute Toxicity

Teratogenesis

Carcinogenesis

Reduced Fecundity

Mutagenesis

Altered Behavior

Regardless of the area of concern selected for evaluation, there is certain informatio aid in that evaluation. Among the more basic requirements are: 1. Levels attainable in the body. 2. Fate in the animal. 3. Site(s) of concentration and/or storage of the metabolites and its derivatives. By knowing the maximum levels likely to be encountered by an animal, the duration of these levels, and the sites of concentrations of the metabolite or its derivatives, an expert in any area of concern could better determine the potential significance of the chemical. More important, this information would aid in designing research to more clearly define the significance of a metabolite. Those who have worked in the area of pesticide metabolism readily acknowledge the difficult task involved in evaluating the significance of pesticide metabolites. It is known, for example, that the majority of the terminal residues of many pesticides exist as conjugates and bound metabolites. Almost no direct information exists which might be used to determine their significance in animals. Therefore, their potential significance must be estimated using indirect evidence until pesticide conjugates are identified and evaluated individually. While not all pesticide metabolites which possess biological activity are necessarily significant, none is significant without possessing some type of biological activity. As a starting point, then, the potential significance of pesticide conjugates can be estimated to some degree by gaining an insight into the biological activity of any conjugate, pesticide or non-pesticide. The critical point initially is not the type of activity, but only i f conjugates might be expected to be biologically active. Naturally, activity falling within those areas of concern mentioned earlier (toxicity, carcinogenesis, etc.) would be of special interest, particularly i f associated with pesticide conjugates. If biological activity of conjugates is indicated, the next step would be to consider the aforementioned information basic

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

16

to evaluating the significance of metabolites, i.e., levels attainable in the body, fate in animals, and storage in the body. Currently, the only information available on pesticide conjugates, and this is meager, relates to the fate of the metabolites in the animal. The potential for accumulation and storage, however, may be estimated from the metabolism data. Finally, one must consider the possible use of simple screening techniques for determining the chronic effects of pesticide conjugates on biological systems. Such assays are used extensively in estimating the potential carcinogenic, mutagenic and teratogenic characteristics of drugs, chemicals, and various environmental pollutants, and may hold promise for similar evaluations of the pesticide conjugates. The following discussion is predicated on the approach outlined above. No attempt was made to cover all known conjugates, or even all pesticide conjugates that demonstrated the points being made and which would serve as a nucleus for continued discussions of the biological activity and significance of pesticide conjugates. Conjugate Nomenclature. A discussion of pesticide conjugates is made exceedingly awkward because of the absence of a simple, consistent system of nomenclature. The terms aglycone and glycone apply only to the glycoside conjugates where the former denotes the non-sugar moiety and the latter the sugar moiety of the conjugate. Pesticides are conjugated with a number of different endogenous materials other than sugar, and often times their identity is not known. In these cases, there is no simple terminology which readily differentiates the exogenous moiety from the endogenous portions of the conjugate. In this paper, a simple self-explanatory system of nomenclature applicable to all conjugates formed from the reaction of an exogenous compound with an endogenous compound is used. Definitions: Exocon - That portion of a conjugate derived from an exogenous compound. Used to denote this portion when existing as a precusor to conjugation, a part of the conjugate complex, or after cleavage of the conjugate linkage. Endocon - That portion of a conjugate derived from an endogenous compound. This system is particularly useful when one of the components of the conjugate is unknown. From this standpoint, it is very appropriate that the terms exocon and endocon be used in a paper dealing with pesticide conjugates.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Biological Activity of Pesticide Conjugates

Another area which needs clarification terminology is the differentiation of unbound and bound conjugates. Usually, the term conjugate is used to define those exocon-endocon complexes which can be extracted from the biological substrates. This, then, indicates that those not extracted, the bound residues, are not conjugates. Of course, these residues are conjugates and should be designated as such. From the biological activity standpoint, the important thing is the availability of conjugates to living organisms, particularly to animals and those plants consumed in the animal diet. The extraction characteristics of the residues really have l i t t l e meaning unless related to their bioavailability. With this in mind, pesticide conjugates should be categorized as follows: Bioavailable Conjugates - If from animals and plants, those pesticide conjugate to animals, are absorbed from the gastrointestinal tract. If from soils, those conjugates which are taken up by plants and/or soil-inhabiting animals. Bound, or Bio-unavailable, Conjugates - If from animals and plants, those pesticide conjugates which, when administered orally to animals, are not absorbed from the gastrointestinal tract and are excreted in the feces. If from soils, those conjugates which are not taken up by plants and/or soil-inhabiting animals. With the animal- and plant-derived pesticide conjugates, it is rather simple to determine bioavailability as defined above. For example, combined extractable and unextractable C-conjugates could be given orally to rats and the urine and feces radioassayed. That material excreted via the urine would be bioavailable; usually, that eliminated in the feces would be unavailable and classified as bound. That the fecal l*C-residues indeed were not absorbed from the gut could be confirmed by canulating the bile duct and radioassaying the bile. The availability of pesticide conjugates in soils could be determined by growing various plants in soils that had the free metabolites removed. Availability to soil-inhabiting animals could be evaluated by assaying earthworms, insects, etc. at designated times after being placed in the conjugate-containing soil. Certain criteria, such as test species, exposure times, etc., would have to be established and standardized, but these should be worked out easily by pesticide chemists and biologists who have experience in pesticide uptake studies. It is important to note that the bioavailability studies do not necessarily have to be qualitative in nature. Once conjugation has been established by conventional means, i t is essential only to quantitate the fate of the residues in plants and animals. Naturally, the bioavailable conjugates should be identified and/or their toxicological significance determined. 14

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

18

BOUND AND CONJUGATED PESTICIDE RESIDUES

Conjugation and Biological Activity. It is quite likely that the vast majority of conjugate reactions taking place in living organisms has l i t t l e direct influence on the biological activity of the exocon involved. The reason for this is two-fold. First, the precusors themselves may be void of any biological activity and the process of conjugation serves only to enhance elimination. Secondly, an exogenous compound, even i f biologically active in its initial form, is subject to a number of biochemical reactions (hydrolysis, oxidation, etc.) which may yield an inactive exocon prior to conjugation. Again, the role of conjugation would be related to elimination rather than directly to deactivation. This type of an effect on the biological activity of toxic exogenous compounds may be referred to as "indirect deactivation". "Direct deactivation direct reaction of an active exocon with an endocon to yield an inactive derivative. With pesticides, this usually involves conjugation of a toxic free metabolite formed by hydroxy1 ation of the parent molecule. Certain pesticides, drugs, etc. containing nitrogen may react directly with endogenous chemicals to form Nconjugates. In any event, the immediate exogenous precursor, the exocon, must be active and the resulting conjugate inactive i f the reaction is to be considered a direct deactivation. If the indirect deactivation reactions were combined with those involving direct deactivation, then there is no question but that conjugation may be classified generally as a detoxication mechanism. This is an important concept in considering the biological activity of pesticide conjugates. Since so l i t t l e is actually known concerning the latter, i t is somewhat comforting to know that the possibility of their being biologically active, or toxicologically significant, is not very great. This line of thinking is probably the major factor contributing to our current lack of knowledge about the pesticide conjugates. However, it is not without substantial validity and should be kept in mind in making any predictions relative to the potential significance of conjugate metabolites. Contrary to the situation with pesticides, conjugative deactivation of drugs has been demonstrated for a number of compounds. As early as the 1880*5, i t was reported that the hypnotic activity of chloral hydrate was lost when converted to trichloroethyl glucuronide (5,6). Only when the glucuronide was administered at very high doses was there any sign of activity and this probably resulted from the free alcohol formed upon hydrolysis of the compound in the gut. Over the years, many other conjugate metabolites of drugs have been shown to be void of therapeutic activity. These include drugs exhibiting antibacterial, hypnotic, and various other types of biological activity (7-11). Recently, i t has been suggested that the antihypotensive agent dopamine (3,4-dihydroxyphenethylamine)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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generated from L-dopa [3-(3,4-dihydroxyphenyl)-L-alanine] in dogs was inactivated by conjugation (12). While the endocon was not characterized, strong acid hydrolysis of the plasma released high levels of dopamine. Dopamine infusions confirmed that such levels would produce excessive cardiac stimulation or hypertension i f present in the free form. Some of the conjugates just discussed represent indirect deactivation while others are clearly direct deactivations. Unless the exocon is isolated, identified and assayed, i t is impossible to differentiate between indirect and direct deactivation reactions. To establish that a conjugate is inactive is very meaningful, but this information does not indicate whether cleavage of the material would yield an active or inactive exocon. The importance of knowing the type of conjugative deactivation should become obvious in the following two sections where the toxicities o are considered separately. Indirect Deactivation. Examples where conjugation has no direct influence on the biological activity of exogenous compounds are shown in Fig. 1. Meprobamate is a tranquilizer with an LD50 to mice of 800 mg/kg. In animals, the drug is metabolized to hydroxymeprobamate (Fig. l),a product virtually non-toxic to mice (13). Once this action has occurred, the toxicological consequences of glucuronide conjugation are n i l . The meprobamate glucuronide is non-toxic and is devoid of pharmacological activity (14J.

9

SF*

(HgN-C-O-CH^g-C^CHg^CHa M e p r o b a m a t e ^ D ^ 800 mg/kg) R-CHo-CHOH *

1

CH OH-Meprobamate LD 7000+ mg/kg

Carbary^LDgQ 430mg/kg)

3

5 0

>L R—CH2"" CHOC^HgOg CH

3

R-OH l-Naphthol(LD5o 2590mg/kg) R-OCeHgOfc

Figure 1. Indirect conjugative deactivation of the drug meprobamate (13) and the insecticide carbaryl (15) by glucuronidation

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20

BOUND AND CONJUGATED PESTICIDE RESIDUES

Carbaryl, an insecticide, has an LD50 to rats of 430 mg/kg. One of the major metabolites of carbaryl formed in most animal systems is 1-naphthol Q5). This compound is only one-sixth as toxic to rats as carbaryl and further detoxication resulting from conjugation would not be of great importance. Even though conjugation is not the initial step in the deactivation of some exogenous compounds, its role in the overall detoxication process of such compounds should not be minimized. Compounds like hydroxymeprobamate and 1-naphthol may be relatively non-toxic, but without efficient conjugation mechanisms the excretion rates would not be sufficient to prevent their accumulation to toxic levels. A lack of an efficient conjugative system may also result in the accumulation of toxic exogenous materials which otherwise would be rapidly degraded. Carbaryl, for example, is metabolized extremely efficiently b micromoles of NADPH and 5 micromoles of UDPGA (16). Under these conditions almost 60% of the carbamate was converted to glucuronide conjugates (Table III). As glucuronidation was supressed by limiting the quantity of UDPGA, the conjugates were reduced as expected. The unexpected results occurred with the parent compound where its total metabolism was reduced in proportion to the reduction in conjugation. TABLE III Effect of Reduced Glucuronide Conjugation on Total Carbaryl Metabbolism by Rat Liver Microsomes + NADPH (16). Umoles

% Distribution of Metabolites

UDPGA added

Carbaryl

Free

Glucuronides

5

26

3

32

17 20

48

0.3

55

20

25

0

71

19

10

Carbaryl-naphthyl-C used. addition of NADPH.

57

No metabolism occurred without

Since the NADPH concentration was the same in all incubations it was anticipated that oxidative metabolism to yield the free metabolites would continue, and that they would accumulate. However, the effect of reduced conjugation was reflected solely in the increased metabolic stability of carbaryl, per se. The possible consequences of decreased degradation of pesticides in vivo are obvious, and could occur with carbaryl and possibly other toxicants i f conjugation was reduced by the monoamine oxidase inhibitors or other drugs (V7).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Biological Activity of Pesticide Conjugates

Direct Deactivation and Reactivation. Although direct deactivation by conjugation occurs less frequently than indirect, its toxicological significance is much greater. As mentioned earlier, the exocon is an active product and, i f not conjugated, may not be deactivated and/or eliminated. Moreover, the exocon may be released i f the conjugate is consumed by animals and thereby be in a form to express its biological activity. From this standpoint, direct deactivation of toxicants by conjugation, particularly in plants, could provide a source of biologically active materials, the exocons, which otherwise would not be available. There is also a very real possibility that the conjugate would provide protection of the toxic exocon against metabolic degradation and/or facilitate transportation of the exocon to its site of action. While there are numerous examples of direct deactivation by conjugation, the data in potential toxicological significance of this phenomenon. Cyclohexylamine(l) and the 4- and 5-hydroxy derivatives of carbaryl (4) are toxic exocons of glycoside conjugation (Fig. 2). TABLE IV Toxicity of Exocon and Conjugate to Mice When Administered IP 24 Hr LD , mg/kg

Compounds Cyclohexyl ami ne

50

100

a

600

Glucuronide form

55

4-Hydroxycarbaryl

b

1550

Glucoside form 5-Hydroxy carbaryl

50

b

950

Glucoside form a ( 1 ) , b (4)

The cyclohexylamine has an LD of 600 mg/kg when conjugated as a glucuronide. A more pronounced deactivation is noted with the carbaryl metabolites. In the free form the LD50 to mice is approximately 50 mg/kg; their glucoside conjugates have LD50 values of 1550 and 950 mg/kg for the 4- and 5hydroxy carbaryl derivatives, respectively. Obviously, the conjugation of cyclohexylamine and the hydroxy carbaryl compounds is an effective detoxication mechanism. With these particular toxicants, there are no indications that the conjugate forms protected the exocons from metabolic degradation or served as a "carrier" of the exocons 50

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22

BOUND AND CONJUGATED PESTICIDE RESIDUES

o II

Cyclohexylamine Glucuronide

OC^H-pOs 4 - a n d 5-Hydroxy c a r b a r y l Glucoside

Figure 2. Direct detoxication of exocons by glycoside conjugation (see Table TV)

to the site of action. Results of similar studies with various chemotherapeutic agents do suggest that active exocons are released when the conjugated drugs are administered (18-20). Generally, the conjugates are less active than the free compounds although the estrogen, estriol, was no more active than its glucuronide when both forms were administered orally (21). When injected, the estriol retained its activity while the glucuronide was only weakly active. These data suggest that the primary site of conjugate cleavage to yield the exocon was in the gut. They also demonstrate that the route of administration can affect the biological activity of conjugates to a greater degree than that of nonconjugated compounds. Activation. Although there are many conjugates of drugs, and of some pesticides, which exhibit varying degrees of biological activity, there is little evidence suggesting that the desired effects, therapeutic or pesticidal, are dependent upon conjugative activation. In fact, there is no indication of such an occurrence relative to pesticides and their toxic action. The activity reported for the conjugates usually is not as great as for the parent compound, or liberated exocon, and the observed activity is probably due, at least partially, to reactivation. This is not too surprising with most pesticides since a relatively high lipid solubility is required for them to penetrate to the site of action. It is difficult to imagine an inactive, apolar chemical being converted to a potent nerve poison by a process such as conjugation which increases its polarity. Carcinogenicity. Acute toxicity is not always the most obvious or most significant type of detrimental biological activity exhibited by conjugates or other chemicals. Card no-

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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genesis, mutagenesis and teratogenesis may be induced by certain chemicals and these dastardly consequences must be accepted as a possibility from exposure to pesticide conjugates. Thus far, pesticide conjugates have not been strongly implicated as causative agents of the above abnormalities. However, a brief discussion of the carcinogenic activity of certain nonpesticidal conjugates justifiably increases one's concern about the potential significance of pesticide conjugates. Perhaps the most striking form of biological activity attributed to conjugate compounds is the ability of certain ones to induce cancer. Most of the studies relative to carcinogenic conjugates have centered around the aromatic amines, especially 2-acetylaminofluorene (AAF). While the parent compound is an active carcinogen, i t has been established beyond doubt that metabolic activation is required for its carcinogenic properties to be expressed (22-24) The first step in the biochemical activation of AAF (Fig. 3) is the formation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Many studies have demonstrated that this metabolite is a greater carcinogen than AAF and that i t , or a subsequent metabolite, binds more efficiently with hepatic protein and nucleic acids (22,23,25-27).

AAF

N-OH-AAF

Sulfate

Figure 3. Metabolic activation of the carcinogen 2-acetylaminofluorene. The sulfate form has been identified as the active carcinogen.

More recently, several investigators have shown that the real active carcinogen involved is the sulfate ester of N-OH-AAF (28-31). Glucuronide and phosphate esters are also formed from the N-OH-AAF (30,32), but the evidence overwhelmingly supports the sulfate ester as the active carcinogen. There is evidence, however, that conjugates other than sulfates are potent carcinogens. 2-Naphthylamine, for example, induces bladder cancer when implanted therein (33). A more active carcinogen is produced when the compound is conjugated as an 0-glucuronide. There are many other examples where conjugates are implicated as active carcinogens and this type of biological activity cannot be ignored when considering pesticide conjugates.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

Chemotherapeutic Conjugates. It is quite natural for one to think in negative terms when considering the possible biological activity of pesticide conjugates. Nevertheless, there is a substantial number of conjugates which exhibit very desirable pharmacologic action, and further demonstrate that certain conjugates are definitely biologically acti ve. Glycosides constitute the bulk of the chemotherapeutic conjugates. Most are naturally occurring and are derived from plants even for commercial purposes. Their source, chemistry and therapeutic uses have been previously covered in detail (34), and only a few examples will be presented here. The cardioactive glycosides are commonly used drugs which upon hydrolysis yield one or more sugars and a host of rather complex alcohols. Digitoxin from Digitalis sp. and is the 3-0-glycoside of digitoxigenin (Fig. 4j. Other glycosides occur in the plant but digitoxin is the most active. The many other cardioactive glycosides (ouabain, lantoside C, etc.) are similar in structure to digitoxin but vary markedly in biological activity.

In addition to the cardioactive glucosides, many other plant conjugates have therapeutic value. The plant source and chemical structures of the aglycones vary widely, as do the prescribed uses of these drugs. Their biological activity is such that they are used as cathartics, emetics, diuretics, vasoconstrictors, and as antirheumatic agents. Although not of current commercial use, certain plasma protein-nitrogen mustard conjugates show promise as anti-tumor agents (35-36). As with most other conjugation, the toxicity of mustards to animals was reduced by protein conjugation but the

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Biological Activity of Pesticide Conjugates

inhibitory potency of the chemical was not drastically altered. This is demonstrated in Table V by the data obtained with aniline mustard and its protein conjugates. It was hypothesized that the proteins enveloped the cytotoxin groups and protected them from hydrolytic degradation before they reached their active site. TABLE V Toxicity and Tumor Inhibitory Potency of Aniline Mustard and its Protein Conjugates in Mice (36). Compound Aniline Mustard

LD 0

IDgO

LD50/ LD

112

1.4

80

450

2.3

193

5

90

Globulin Conjugat Fibrinogen Conjugate ID

90

- 90% inhibitory dose.

Protein conjugation of the carcinogen, 2-anthrylamine, served as an immunizing agent when administered to rats prior to a single oral dose of the carcinogen (37). Up to 50% tumor inhibition was achieved, leading the authors to conclude that they had produced animals resistant to the action of 2anthrylamine in producing neoplasia. The in vivo conjugation of some drugs may yield compounds which contribute significantly to the intended therapeutic action of the non-conjugated material administered. One such case was noted in a stucty designed to explain why patients with renal failure showed an increased sensitivity to the hypotensive effect of methyldopa (38,39). The evidence obtained showed that methyldopa-0-sulfate,formed metabolically, and normally eliminated rapidly, accumulated in the plasma of patients with impaired elimination. It was this conjugate, the author believed, that was acting in the same manner as methyldopa and, consequently, gave an additive effect when the patients were again dosed with the antihypertensive drug. Possibly, conjugates of other drugs contribute to the biological activity of the free therapeutic agent in the same way. However, it is not likely to be recognized unless some unique physiological responses occur which alter the expected action of the drug. Effects on Plants. It is unlikely that pesticide conjugates other than those formed from herbicides would have any appreciable effect on plant growth. With the herbicides, however, conjugates could be formed which retained herbicidal activity directly or which

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

could be cleaved to yield an active exocon. Evidence that such conjugates are formed from 2,4-D has recently been reported (40). Using cultured callus tissues of soybean cotyledon, 2,4-D was shown to be rapdily conjugated with a variety of amino acids which, in turn, were synthesized and their biological properties evaluated. Of the 20 amino acid conjugates of 2,4-D tested for their ability to stimulate cell division and elongation, all were active to some degree. Selected data from these studies are shown in Table VI. In many cases, the growth stimulation of the conjugates exceeded that produced by 2,4-D. TABLE VI Biologically Active 2,4-D Amino Acid Conjugates (40). Tissue Response Conjugates 2,4-D, Free

39

224

Glutamic Acid

64

288

Phenylalanine

49

433

Arginine

71

274

a

Elongation of Avena coleoptile sections at 10 M concentration. Mg soybean callus tissue at 10 M concentration.

It was pointed out that 2,4-D could be formed metabolically from the conjugates and, thus, might be the active component. The point i s , however, that the conjugates did elicit a growth response and were active either directly or served as a mechanism for obtaining greater concentrations of 2,4-D at the site of action. The authors concluded that amino acid conjugation of 2,4-D could not always be considered as a detoxication mechanism. Fate of Pesticide Conjugates in Animals. Only rarely is it possible to obtain pure pesticide conjugates in quantities suitable for thorough acute and chronic toxicological evaluations. Moreover, the data currently available do not demonstrate that such evaluations are essential for establishing the safety of pesticidal chemicals. The fact remains, however, that "proof of safety" of the pesticide conjugates has yet to be documented. This situation requires that we continue to evaluate the significance of these metabolites using whatever approaches likely to yield useful information. In our laboratory, we have taken an indirect approach in attempting to establish parameters for estimating conjugate significance. The studies have centered around the carbamate

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Biological Activity of Pesticide Conjugates

27

insecticides and are designed to determine the fate of certain conjugate metabolites in mammals. Just as fate studies are useful in predicting the safety of parent pesticides, they should be of value in estimating the potential significance of their conjugates. Since plants would probably provide the major source of the conjugates to man, we have chosen to use plants to generate conjugate and bound residues from radioactive carbamates, and to use these in our fate studies with rats. Also, a limited number of radioactive conjugates of the carbamate insecticide carbaryl (1-naphthyl N-methylcarbamate) has been chemically synthesized and similarly studied in the rat. Some of the data obtained in these studies are presented below. Carbaryl, when injected into bean plants, is metabolized sequentially into water soluble compounds, or conjugates, and then to unextractable residues or bound metabolites (41_). After 20 days, there are sufficien bound materials resulting from a carbaryl-naphthyl- C treatment to administer to rats (Fig. 5) (42). Most of these metabolites contain the intact carbamate ester linkage (41,43). 14

O II

O-C-NHCH3

Rats C o n j ugates—30 * 'o • 24 Hrs

20 Days

Urine

88 %

Feces

5°

Rats

Urine

2^

24 Hrs

Feces

90°*

Free —20%

Bound—48%

Figure 5. Nature of carbarvl naphthyl- C in bean plants 20 days after injection an^ fate of conjugated and bound materials when administered orally to rats (42) 14

When the conjugate metabolites were given orally to rats, over 90% of the radiocarbon was excreted within 24 hours (Fig.5), mostly via the urine. Although the bound residues were excreted equally as efficiently, almost all of the elimination occurred in the feces. These data prove that the conjugate metabolites were absorbed from the gut and were available to the animal. The bound residues did not appear to be available to the animal since elimination was rapid and via the feces. Using these data alone, one would have to predict that the conjugate metabolites were potentially more significant than the bound ones. The animal data obtained with the carbaryl plant metabolites are similar to those using several other carbamate insecticides, except ethiofencarb. The latter compound [(2-ethylthiomethyl)phenyl N-methylcarbamate] is a Bayer product under development in the United States by Chemagro. Unlike most carbamates, this

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

28

material is hydrolyzed within the plants and, even with the Bering-labeled compound, there is no appreciable buildup of the bound residues (Fig. 6). Hydrolysis of the ester to yield B(X)2 was indicated by the fact that only 18% of the injected radiocarbon remained in plants 20 days after injection with ethiofencarb-carbonyl- C After the same period, 98% of the ring- c material could be recovered from the plants. 14

14

24%

co2 —NHCH3

38%

Rat

* Conjugate— 3% • Urine 24 Hrs 13% Feces a,

2%

C H ^ C ^ j S Rat

Beans 20 D a y s

Figure 6.

Urine

85%

24Hrs Feces

2%

Nature of ethiofencarb C-metabolites in bean plants 20 days after injection and fate of conjugated materials when administered orally to rats (42) 14

The C-carbonyl conjugates were metabolized by rats to respiratory i**C02 (24%) and to products eliminated in the urine (38%). Only 2% of the dose was eliminated in the feces. Fecal elimination of the B e r i n g J 9 approximately the same, but no c02 was produced and most of the dose (85%) was voided in the urine. The bound ^-materials were not administered to rats because of the excessive bulk needed to obtain the required radiocarbon. As for the usefulness of these data in estimating metabolite significance, two things are obvious. First, the terminal residues of ethiofencarb in bean plants are predominately non-carbamate in nature which should lessen their potential significance. Second, the conjugative metabolism does not produce the bound metabolites usually encountered in plants and, thus, need no evaluation. Add to this the fact that the conjugates are almost all ethiofencarb hydrolysis products which are rapidly eliminated from the body, and the significance of the conjugates become even less apparent. In addition to working with the total 0conjugate bound carbaryl metabolites formed in plants, the glucosides of certain free metabolites have been synthesized and fed to rats. The primary purpose of these studies was to test the metabolic stability of the exocon-O-glucriside linkage and to compare the c o n

u

a t e

w

a

s

14

14

a

n

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

d

2.

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29

Biological Activity of Pesticide Conjugates

fate of conjugates 1n animals with that of the free metabolite. 1-Naphthyl- c glucoside was excreted intact, about 20% of the dose, in the urine of rats treated orally (Table VII) (43). The major metabolite was the glucuronide form (24%) while naphthyl sulfate and 1-naphthol in the urine each constituted 10% of the dose. Identical studies with the ' c-gi ose-labeled conjugate confirmed that the naphthyl glucoside in the urine contained the same sugar moiety as administered. It also showed that the glucuronide was not formed by the oxidation of 1-naphthyl glucoside but resulted from the cleavage of the glucoside linkage to form 1-naphthol, which was then conjugated with endogenous glucuronic acid. The free hydrolytic metabolite of carbaryl, 1-naphthol, was excreted more rapidly than its glucoside, apparently because the free hydroxy1 group allowed direct glucuronidation. 14

4

UC

Fate of 1-Naphthol-Bc and its Glycoside in Rats (44). Percent of Dose in 0-24 Hour Urine as Compound 1-Naphthol- C Naphthyl-14c Glucoside 14

Naphthyl Glucoside- C 14

1Naphthol 1

Naphthyl Glucoside

Naphthyl Glucuronide

Naphthyl Sulfate

0

73

15

10

19

24

10

0

16

.1

0

Unlike 1-naphthyl glucoside, the glucoside linkage of 4- and 5-hydroxycarbaryl glucoside was almost completely cleaved by the rats (45). While 30% of the dose was excreted, only a small amount, 2 to 3% of the dose, contained the intact glucoside bond; over 90% of this was the parent conjugate while the remainder was the 4- or 5-glucoside of 1-naphthol. Other radiocarbon eliminated by the rats appeared to lack the 1-naphthyl moiety. The naphthyl- c-labeled forms of the 4- and 5-hydroxycarbaryl glucosides have not been synthesized and, consequently, the fate of the free and conjugated metabolites have not been compared. 14

Screening for Carcinogenic/Mutagenic Potential. That certain conjugate compounds, formed in vivo, can induce cancer in animals is an established fact. This does not mean that the pesticide conjugates will exhibit carcinogenic and/or mutagenic properties, but i t does add considerably to one's concern about just what type of biological activity they do, or do not possess. Because of the problems relating to the isolation,

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

30

identification, synthesis, etc., of pesticide conjugates, i t is currently impossible to fully evaluate the carcinogenic and mutagenic potential of these metabolites. However, there are techniques available which have proven useful in estimating this potential for a variety of chemicals, including pesticides, that may be applicable to pesticide conjugates (46-49). The most promising method for screening pesticide conjugates for mutagenic and carcinogenic activity appears to be one u t i l i zing bacterial strains designed specifically for this purpose (48,49). Four strains of Samonella typhimurim were developed which could not synthesize histidine but were reverted back to the wild type by particular mutagens and carcinogenes. While the genetics and biochemistry of the system are quite complex, the assays are very simple. Basically, the procedure calls for counting the number of colonies which develop from the mutant strains in the presence histidine for a few cell divisions. If no mutation occurs, there is no colony formation because the mutant strains cannot biosynthesize the compound. However, i f mutation occurs, the colonies thrive and their growth rate can be correlated to the potency of the mutagen. Preliminary tests have been conducted in our laboratory using the screening technique with several pesticides (42). The experiments showed that the system was very sensitive to nitrosocarbaryl and captan (Table VIII). Both of these materials previously have been shown to be potent mutagens (47,50). No effect was observed with the other insecticides at the concentrations tested. Certain of these are shown in Table VIII. TABLE VIII Bacterial Mutagenesis of Pesticides and Related Compounds (42). Minimum concentration, uq/plate, for Mutagenesis

Growth Retardation

Heptachlor epoxide

100 +

2500 +

Oiazinon Carbaryl

100 +

1000 +

100 +

2500 +

Compound

Nitrosocarbaryl Captan

1

200

2.5

50

Salmonella typhimurium strain TA 1535; method of Ames et a l . , 1973 (48). + indicates that these concentrations were inactive. Some of those pesticides which did not show any activity are among those pesticides banned because of their carcinogenic properties. Thus, this test, like all others, will not provide all the answers. However, all means available must be used when

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

DOROUGH

31

Biological Activity of Pesticide Conjugates

dealing with such a vital topic and future studies using the bacterial carcinogenic/mutagenic screening test will include isolated pesticide conjugates. Significance of Pesticide Conjugates. Sufficient numbers of conjugates have demonstrated varying forms of biological activity to establish that the pesticide conjugates are potentially biologically active. It seems necessary, therefore, to determine i f , and which, conjugates are active and the significance of this activity. Taking into account current technical problems discussed earlier, one possible approach to accomplishing this is outlined in Table IX. TABLE IX Sequential Approac Significance of Pesticide Conjugates Factors to be Determined

Results & Priority a. Available

1.

- high

b. Unavailable - low

2. Degradation/excretion rates — a. b. a. 3. Carcinogeni c/mutageni c potential b. 4. Ninty-day feeding studies

5. Tests 1-4

6. Identity and synthesis of active component(s) 7. Full toxicological significance

—»

2

high

— » 3,4

Rapid Positive

low - high

—• —•

3 4

Negative

-

—»

5

—•

6

- high — •

7

Slow

-

low

a. Effect

-

high

b. No effect

-

low

a. Positive

-

high

b. Negative a. Successful

b. Unsuccessful - higher (halt production) a. Hazardous (halt production) b. Safe

Those conjugates found to be bioavailable should be isolated, their fate in animals determined, and assayed for carcinogenic/ mutagenic potential. A concentrated effort is needed immediately to select the best screening method for the latter. If the bioavailable conjugates accumulate in the animal and/or show

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

32

BOUND AND CONJUGATED PESTICIDE RESIDUES

carcinoaenic/mutagenic potential, a 90-day feeding stucty of the crude l^C-conjugate preparation should be performed. Levels fed should be the maximum which could occur in a normal diet, and the criteria used for determining detrimental effects the same as those used in similar studies of major free pesticide metabolites. In addition, total accumulation of C-residues in the animals over the 90-day period should be determined. After the feeding study has been completed, all data collected to that point should be reviewed. Those conjugates which accumulated extensively, showed strong carcinogenic potential and/or produced i l l effects in the 90-day feeding study should be further evaluated. Components of the crude conjugate preparation should be isolated and identified. If this proved unsuccessful, the discontinued use, or development, of the parent pesticide would have to be seriously considered. The synthetic conjugates should be re-evaluate potentially harmful subjected to a full-scale toxicological study Results of these studies would determine the commercial fate of the parent compound. It is not important whether one accepts or rejects the approach suggested above. The important thing is that this approach, and others, be considered, and that a well designed and well organized program be set in motion to determine the significance of pesticide conjugates. 14

Literature Cited. 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Smith, R.L. and R.T. Williams, "Implication of the conjugation of drugs and other exogenous compounds" in Glucuronic Acid: Free and Combined, pp 457-491, G.J. Dutton (ed). Academic Press, New York (1966). Steller, W.A. and W.W. Brand, J. Agr. Food Chem. (1974) 22, 445. Ecke, G., J. Agr. Food Chem. (1973) 21, 792. Cardona, R.A. and H.W. Dorough, J. Agr. Food Chem. (1973) 21, 1065. Kulz, E., Arch. Ges. Physiol. (1882) 28, 506. Kulz, E., Z. Biol. (1884) 20, 157. McChesney, E.W., J. Pharmacol. Exptl. Therap. (1947) 98, 368. Woods, L.A., J. Pharmacol. Exptl. Therap. (1954) 112, 158. Koechlin, B.A., W. Kernand R. Engleberg, Antibiot. Med. Clin. Therapy (1959) 6, Suppl. 1, 22. Keberle, H., K. Hoffmann and K. Bernhardt, Experientia (1962) 18, 105. Glazko, A.J., L.M. Wolf, W.A. Dill and A.C. Bratton, J. Pharmacol. Exptl. Therap. (1949) 96, 445. Tjandramaga, T.B., L.I. Goldberg and A.H. Anton, Proc. Soc. Exp. Biol. Med. (1973) 142, 424. Ludwig, B.J., J.F. Douglas, L.S. Powell, M. Meyer and F.M. Berger, J. Med. Pharm. Chem. (1961) 3, 53. Tsukamoto, H., H. Yoshimura and K. Tatsumi, Chem. Pharm.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2. DOROUGH

Biological Activity of Pesticide Conjugates

Bull. (1963) 11, 1134. 15. Dorough, H.W., J. Agr. Food Chem. (1970) 18, 1015. 16. Mehendale, H.N. and H.W. Dorough, Pest.Phy. (1971) 1, 307. 17. Culver, D.J., T. Lin and H.W. Dorough, J. Econ. Entomol. (1970) 63, 1369. 18. Schuller, J . , Z. Biol. (1911) 56, 274. 19. Dodgson, K.S., G.A. Garton, A.L. Stubbs and R.T. Williams, Biochem. J. (1948) 42, 357. 20. Wilder Smith, A.E. and P.C. Williams, Biochem. J. (1948) 42, 253. 21. Odell, A.D., D.I. Skill and F.G. Marrian, J. Pharmacol. Exptl. Therap. (1937) 60, 420. 22. Miller, E.C., J.A. Miller and H.A. Hartmann, Cancer Res. (1961) 21, 815. 23. Miller, E.C., J.A. Mille (1964) 24, 2018. 24. Irving, C.C., Conjugates of N-hydroxy Compounds In: W.H. Fishman (ed.). Metabolic Conjugation and Metabolic Hydrolysis, pp 53-119. New York: Academic Press, Inc. (1970). 25. Miller, J.A. and E.C. Miller, Progr. Exptl. Tumor Res., (1969) 11, 273. 26. Marroquin, F. and E. Farber, Cancer Res. (1965) 25, 1262. 27. Lotlikar, R.D., J.D. Scribner, J.A. Miller and E.C. Miller, Life Sci. (1966) 5, 1263. 28. DeBaun, J.R., J.Y. Rowley, E.C. Miller and J.A. Miller, Proc. Soc. Exp. Biol. Med. (1966) 129, 268. 29. DeBaun, J.R., E.C. Miller and J.A. M i l l e r , Cancer Res. (1970) 30, 577. 30. Irving. C.C., Xenobiotica (1971) 1, 387. 31. Weisburger, J.H., R.S. Yamamoto, G.M. Williams, P.H. Matsushima and E.K. Weisburger, Cancer Res. (1972) 32, 491. 32. Lotlikar, P.D. and M.B. Wasserman, Biochem. J. (1970) 120, 661. 33. Allen, M.J., E. Boyland, C.E. Dukes, E.S. Horning and J.G. Watson, Brit. J. Cancer (1957) 11, 212. 34. Claus, E., V. Tyler and L. Brady, "Pharmacognosy" 6th ed., pp 79-130, Lea and Febiger, Philadelphia (1970). 35. Szekerke, M., R. Wade and M.E. Whisson, Neoplasma (1972) 19, 199. 36. Szekerke, M., R. Wade and M.E. Whisson, Neoplasma (1972) 19, 211. 37. Peek, R.M. and E.B. Peck, Cancer Res. (1971) 31, 1550. 38. Myhre, E., O. Stenback, E.K. Brodwall and T. Hansen, Scand. J. Clin. Lab. Invest. (1972) 29, 195. 39. Myhre, E., E.K. Brodwall, O. Stenback and T. Hansen, Scand. J. Clin. Lab. Invest. (1972) 29, 195. 40. Feung, C., R.O. Mumma and R.H. Hamilton, J. Agr. Food Chem. (1974) 22, 307. 41. Dorough, H.W. and O.G. Wiggins, J. Econ. Entomol. (1969) 62, 49.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

42. Marshall, T.C. and H.W. Dorough, Unpublished data (1975). 43. Kuhr, R.J. and J.E. Casida, J. Agr. Food Chem. (1967) 15, 814. 44. Dorough, H.W., J.P. McManus, S.S. Kumar and R.A. Cardona, J. Agr. Food Chem. (1974) 22, 642. 45. Cardona, R.A. and H.W. Dorough, Unpublished data (1973). 46. Epstein, S.S. and H. Shafner, Nature (1968) 219, 385. 47. Legator, M.S., F.J. Kelly, S. Green and E.J. Oswald, Ann. N.Y. Acad. Sci. (1968) 160, 344. 48. Ames, B.N., F.D. Lee and W.E. Durston, Proc. Nat. Acad. Sci. (1973) 70, 782. 49. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Proc. Nat. Acad. Sci. (1973) 70, 2281. 50. Elespuru, R.K. and W. Lijinsky, Fd. Cosmet. Toxicol. (1973) 11, 807.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

3 Pesticide Conjugates—Glycosides D. S. FREAR Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Fargo, N. Dak. 58102

Abstract Selected examples O-glucoside, N-glucoside, S-glucoside, glucose ester, acylated glucoside and gentiobioside metabolites of pesticides and plant growth regulators are discussed from the standpoint of the isolation, identification, metabolism and significance in plants and insects. Introduction In the past, pesticide metabolism studies have emphasized the isolation, identification, and toxicity of primary reaction products. Recently, however, increased interest has been focused on the nature and significance of a variety of conjugated pesticide metabolites, including a number of glycosides. Increasing evidence suggests that the rate and extent of glycoside formation is a significant factor in regulating the biological activity and the selectivity of pesticides and their toxic metabolites (Figure 1). However, relatively few studies have reported the isolation and identification of pesticide metabolites as glycosides. Many workers simply note that unknown polar metabolites are present. Other reports only show that unknown polar metabolites are hydrolyzed by acids, bases or various glycosidic enzymes. Also, l i t t l e information is available concerning the distribution, specificity, activity and regulation of the enzyme systems responsible for the biosynthesis, hydrolysis and further metabolism of glycoside metabolites. The continuing search for more selective and less persistent pesticides, together with increased concern about the nature of "terminal" pesticide residues, suggest that Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 35

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

36

BOUND AND CONJUGATED PESTICTOE RESIDUES

a d d i t i o n a l b a s i c s t u d i e s of g l y c o s i d e metabolism should provide v a l u a b l e and needed i n s i g h t s i n t o the behavior and f a t e of pesticides. An examination of the n a t u r a l products l i t e r a t u r e r e v e a l s that a vast number and v a r i e t y of g l y c o s i d e s are present i n nature. Comparative metabolism s t u d i e s , however, have shown t h a t p e s t i c i d e s and t h e i r metabolites are normally conjugated as 3-glucuronides i n v e r t e b r a t e s and as 3-glucosides i n p l a n t s , I n s e c t s and other i n v e r t e b r a t e s (1). Since the f o l l o w i n g paper w i l l discuss g l u c u r o n l d e s , the present d i s c u s s i o n w i l l be l i m i t e d to glucosides and t h e i r metabolites i n p l a n t s and insects• S e v e r a l d i f f e r e n t types of simple and complex glucosides have been i s o l a t e d and i d e n t i f i e d i n c l u d i n g ; a v a r i e t y of O-glucosides, s e v e r a l N-glucoside glucose e s t e r s , a c y l a t e no attempt w i l l be made to c a t a l o g or discuss a l l of the p e r t i n e n t references i n the l i t e r a t u r e . Instead, s e l e c t e d examples of each type of g l u c o s i d e w i l l be used t o i l l u s t r a t e some of the methodology that has been used and the problems t h a t have been encountered i n the i s o l a t i o n , i d e n t i f i c a t i o n and metabolism of t h i s Important c l a s s of conjugated p e s t i c i d e metabolites. O-Glucosides Many i n v e s t i g a t o r s have suggested that O-glucosides represent a major c l a s s of conjugated p e s t i c i d e metabolites i n p l a n t s and i n s e c t s . A number of p e s t i c i d e s are s u b s t i t u t e d phenols, and many p e s t i c i d e s are metabolized t o phenols or a l c o h o l s by o x i d a t i o n or h y d r o l y s i s . I t i s g e n e r a l l y assumed t h a t these phenols or a l c o h o l s are conjugated as O-glucosides i n p l a n t s and i n s e c t s . However, i n very few s t u d i e s has a m e t a b o l i t e a c t u a l l y been i s o l a t e d and i d e n t i f i e d as a g l u c o s i d e . T e n t a t i v e i d e n t i f i c a t i o n i s g e n e r a l l y provided by enzymic h y d r o l y s i s and/or a n a l y s i s of the aglycones a f t e r h y d r o l y s i s . Frequently, the i d e n t i f i c a t i o n and a n a l y s i s of the carbohydrate moiety i s omitted because of d i f f i c u l t i e s i n removing n a t u r a l l y o c c u r r i n g carbohydrate i m p u r i t i e s . N a t u r a l l y o c c u r r i n g p h e n o l i c compounds e x h i b i t a v a r i e t y of metabolic a c t i v i t i e s i n p l a n t s and animals, and t h e i r r o l e as mediators of metabolism i s w e l l e s t a b l i s h e d (2-5). Phenolic p e s t i c i d e s o r p e s t i c i d e metabolites a l s o a f f e c t a number of metabolic processes and b i o l o g i c a l f u n c t i o n s . A p o s s i b l e means f o r r e g u l a t i n g c e l l u l a r concentrations of b i o l o g i c a l l y a c t i v e p h e n o l i c Intermediates i n p e s t i c i d e metabolism i s o u t l i n e d i n F i g u r e 2. Support f o r such a hypothesis has been provided by a number of s t u d i e s . Examples of such studies w i l l be mentioned b r i e f l y . A review of carbamate i n s e c t i c i d e metabolism i n p l a n t s and i n s e c t 8 by Kuhr (6) suggests t h a t d i f f e r e n c e s i n the t o x i c i t y

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

FREAR

Pesticide Conjugates—Glycosides

SECONDARY METABOLITES More active

1

PESTICIDE

'TERMINAL* RESIDUES OR EXCRETION

PRIMARY METABOLITES

7

Lets active

Figure 1. Role of glycosides in pesticide metabolism and the bioregulation of pesticide toxicity

Insoluble Residue

^

Secondary Oxidation Product(s)

0

"Oxidase" Pesticide

Biologically Active ^~ Phenol Intermedia

Target Site or System

\/

GlucosylII Glucosidase Transferase 11 (

O-Glucoside

Insoluble Residue

Excretion (Insects)

Figure 2. Proposed scheme for the bioregulation of phenol intermediates in the metabolism of pesticides

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

38

BOUND AND CONJUGATED PESTICIDE RESIDUES

of these chemicals to v a r i o u s i n s e c t s may be due, i n p a r t , t o d i f f e r e n c e s i n the r a t e of conjugation of primary metabolites as glucosides and other w a t e r - s o l u b l e conjugates. Studies by B u l l and Whitten (_7) i n d i c a t e t h a t enzymic O - g l u c o s y l a t i o n of £m e t h y l s u l f o n y l phenol, a m e t a b o l i t e o f 0,0-dimethyl 0-(4-methylthiophenyl)phosphate, i s more a c t i v e i n r e s i s t a n t than i n s u s c e p t i b l e tobacco budworms. A recent review of s e v e r a l s t u d i e s by S t i l l , Rusness and Mansager (8) suggests that O - g l u c o s y l a t i o n of 2-hydroxychlorpropham (isopropyl-5-chloro-2-hydroxycarbanilate) may provide an e f f e c t i v e means f o r r e g u l a t i n g the p h y t o t o x i c i t y of t h i s p h e n o l i c intermediate of chlorpropham ( i s o p r o p y l - 3 c h l o r o c a r b a n i l a t e ) metabolism i n p l a n t s . A l s o , the d i r e c t involvement of p h e n o l i c intermediates i n the formation of " t e r m i n a l " p e s t i c i d e r e s i d u e s has been suggested i n recent p l a n t metabolism s t u d i e s w i t methyl-l-pyrrolidinecarboxanilide t h a t a r e a c t i v e p h e n o l i c m e t a b o l i t e of c i s a n i l i d e i s o x i d i z e d f u r t h e r and serves as a precursor o f an i n s o l u b l e residue f r a c t i o n . Both p l a n t s and Insects have a c t i v e phenol oxidase systems. The s i g n i f i c a n c e o f these enzymes i n the formation of " t e r m i n a l " p e s t i c i d e residues should be determined. The primary mechanism of O - g l u c o s y l a t i o n i n p l a n t s (JL, 10-11) and i n s e c t s (1, 11-13) appears to i n v o l v e UDPG as the most e f f e c t i v e g l u c o s y l donor, a UDP-glucosyltransferase, and v a r i o u s phenol and a l c o h o l acceptor groups. Examples of s e v e r a l O-glucoside metabolites i s o l a t e d from p e s t i c i d e t r e a t e d p l a n t s are shown i n F i g u r e 3. M e t c a l f et a l . (14) e x t r a c t e d the O-glucoside of 2,3-dihydro-2,2-dimethyl-3keto-7-hydroxybenzofuran from c o t t o n leaves w i t h aqueous e t h a n o l . I s o l a t i o n of the m e t a b o l i t e was achieved by chromatography on s i l i c i c a c i d columns and TLC. The s t r u c t u r e of the O-glucoside was determined u n e q u i v o c a l l y by IR s p e c t r a and mass s p e c t r a of the TMS d e r i v a t i v e . A molecular i o n at m/e 628 was reported together w i t h expected fragment i o n s f o r both the glucose and the phenol m o i e t i e s . S t i l l and Mansager (15) e x t r a c t e d the O-glucoside of 2-hydroxychlorpropham from soybean r o o t s by a m o d i f i e d Bligh-Dyer procedure (16). The g l u c o s i d e was p u r i f i e d by n-BuOH e x t r a c t i o n of the water s o l u b l e m e t a b o l i t e s , a d s o r p t i o n of I m p u r i t i e s on b a s i c aluminum oxide and c e l l u l o s e i o n exchange chromatography. I d e n t i f i c a t i o n of the O-glucoside was obtained by GLC-MS a n a l y s i s of the a c e t y l a t e d d e r i v a t i v e , 3-glucosidase h y d r o l y s i s , and mass s p e c t r a l a n a l y s i s of the phenol aglycone and i t s methylated d e r i v a t i v e . In metabolism s t u d i e s w i t h the h e r b i c i d e , c i s a n i l i d e , two O-glucoside metabolites were not completely separated by a v a r i e t y of chromatographic procedures i n c l u d i n g TLC, a d s o r p t i o n on A m b e r l i t e XAD-2, c e l l u l o s e i o n exchange w i t h DE-52 and g e l f i l t r a t i o n on B i o g e l P-2 (9). The a n a l y t i c a l problems a s s o c i a t e d w i t h a mixture of O-glucoside metabolites and a

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

3.

FREAR

Pesticide Conjugates-Glycosides

39

probable degradation of TMS o r a c e t y l a t e d d e r i v a t i v e s d u r i n g attempted GLC s e p a r a t i o n were circumvented by enzyme h y d r o l y s i s of the i s o l a t e d g l u c o s i d e mixture, TLC s e p a r a t i o n o f the aglycones, IR, MS and FT-PMR a n a l y s i s of aglycone s t r u c t u r e s , and q u a n t i t a t i v e a n a l y s i s o f glucose w i t h glucose oxidase. The s e p a r a t i o n o f the mixed O-glucoside m e t a b o l i t e s by a more e f f e c t i v e and l e s s d e s t r u c t i v e chromatographic technique, such as HPLC, would permit a d i r e c t a n a l y s i s of each g l u c o s i d e , N-Glucosides Numerous p e s t i c i d e s are s u b s t i t u t e d a n i l i n e s . Others may be metabolized t o a n i l i n e s by o x i d a t i o n , r e d u c t i o n or h y d r o l y s i s . In p l a n t s , s e v e r a l h e r b i c i d e s are e i t h e r d i r e c t l y or i n d i r e c t l y metabolized to N-glucoside formation of chlorambe dinoben ( 3 - n i t r o - 2 , 5 - d i c h l o r o b e n z o i c a c i d ) , p r o p a n i l (3',4 d i c h l o r o p r o p i o n a n i l i d e ) and pyrazon (5-amino-4-chloro-2-phenyl3(2H)-pyridazinone) N-glucoside m e t a b o l i t e s are shown i n Figure 4. Studies w i t h chloramben (17, JL9, 21, 25, 26) and pyrazon (23, 24, 22, 28, 29) have shown t h a t the r a t e and extent o f N-glucoside formation i s an important f a c t o r i n the movement, p h y t o t o x i c i t y and s e l e c t i v i t y of these h e r b i c i d e s i n p l a n t s . The p h y t o t o x i c i t y of s u b s t i t u t e d a n i l i n e m e t a b o l i t e s , such as 3 , 4 - d i c h l o r o a n i l i n e , formed by the h y d r o l y s i s of p r o p a n i l (22) may a l s o be a f f e c t e d by N-glucoside formation. The i n v i t r o b i o s y n t h e s i s of N - g l u c o s y l chloramben and other N - g l u c o s y l arylamlnes has been reported i n s t u d i e s w i t h p l a n t s (30, 31). A UDP-glucosyl t r a n s f e r a s e from soybean was s p e c i f i c f o r the n u c l e o t i d e g l u c o s y l donors UDPG and TDPG, but e x h i b i t e d a r e l a t i v e l y broad s p e c i f i c i t y toward acceptor arylamlnes (Figure 5). The N-glucosides of chloramben and pyrazon are s t a b l e i n v i v o and appear to p e r s i s t i n soybean (26) and sugarbeet (24) as " t e r m i n a l " m e t a b o l i t e s . P r o p a n i l metabolism s t u d i e s i n r i c e (22, 32), however, suggest t h a t the N-glucoside of 3,4-dichloroa n i l i n e may undergo f u r t h e r metabolic r e a c t i o n s t o y i e l d other g l y c o s i d e s and a methanol-insoluble " l i g n i n " complex. The N-glucosides o f arylamlnes are hydrolyzed by d i l u t e a c i d s , but do not appear t o be hydrolyzed by 3-D-glucosidase. At the present time, the c o n f i g u r a t i o n o f the N - g l u c o s i d i c l i n k a g e has not been e s t a b l i s h e d . I t i s p o s t u l a t e d , however, that i n v e r s i o n of c o n f i g u r a t i o n occurs during the UDP-glucosylt r a n s f e r a s e c a t a l y z e d r e a c t i o n and t h a t the 8-D-glucose anomer i s formed. H e t e r o c y c l i c N-glucosides have a l s o been r e p o r t e d . Kamimura et a l . (33) i d e n t i f i e d the N-glucoside and the 0g l u c o s i d e of 3-hydroxy-5-methylisoxazole, a s o i l f u n g i c i d e , as major m e t a b o l i t e s i n cucumber, tomato and r i c e p l a n t s , and i n f

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Figure 3.

Pesticide metabolites characterized as O-glucosides

CI COOH

CHfOH

CI COOH

Chlorotnbtn

CI COOH

CI Dlnob«n

CHfOH

CI Chlorombtn CI

Figure 4.

CI COOH

CI COOH

OH

CI

CI

CHfOH

CI

Pesticide metabolites characterized as N-glucosides

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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tobacco c a l l u s . The N-glucoside was q u i t e s t a b l e t o a c i d h y d r o l y s i s and was not hydrolyzed by 3-glucosidase. The s t r u c t u r e o f the i s o l a t e d N-glucoside m e t a b o l i t e was determined by a n a l y s i s of MS, PMR, I R and UV s p e c t r a and by i d e n t i f i c a t i o n of the a c i d h y d r o l y s i s products. Linkage o f the aglycone t o the C - l o f glucose was e s t a b l i s h e d by a f a i l u r e t o detect a f r e e reducing group and by the i s o l a t i o n of methyl penta-O-me thy1-3glucopyranoside a f t e r permethylation and methanolysis o f the g l u c o s i d e . O p t i c a l r o t a r y d i s p e r s i o n s t u d i e s suggested t h a t the glucose l i n k a g e was the 3 - c o n f i g u r a t i o n . Studies on the f a t e of both the 0- and the N-glucoside metabolites i n cucumber s e e d l i n g s showed that the O-glucoside was hydrolyzed and converted t o the N-glucoside w h i l e the Ng l u c o s i d e remained unchanged (33). Recent i n v i t r o enzyme s t u d i e s (34) support thes UDPG as the g l u c o s y l dono Zeatin[6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine], a p l a n t hormone w i t h c y t o k i n i n a c t i v i t y , and 6-benzylaminopurine, a r e l a t e d s y n t h e t i c c y t o k i n i n , are a l s o metabolized t o N-glucos i d e s i n p l a n t s (35). I n s t u d i e s w i t h 6-benzylaminopurine and derooted r a d i s h s e e d l i n g s , N-glucosides accounted f o r 90% of the e x t r a c t a b l e metabolites a f t e r 24 hours (35, 36). N - g l u c o s y l a t i o n occurs a t e i t h e r the 7- o r 9 - p o s i t i o n of the purine r i n g as shown i n Figure 7. Both glucosides appear to be s t a b l e m e t a b o l i t e s . The 7-glucosldes of c y t o k i n i n s are q u i t e s t a b l e and appear t o accumulate, p o s s i b l y as storage forms (35, 37^, ^38). I n r a d i s h s e e d l i n g s , 7 - g l u c o s y l z e a t i n was not t r a n s l o c a t e d (39). Guern e t a l . (40), however, reported that s y n t h e t i c 6-benzylamino-9-3-Dg l u c o s y l p u r i n e was r e a d i l y t r a n s l o c a t e d without a p p r e c i a b l e enzymic m o d i f i c a t i o n i n c h i c k pea. The p h y s i o l o g i c a l s i g n i f i c a n c e of c y t o k i n i n N-glucosides i s obscure. In s t u d i e s by Parker e t a l . (35), N-glucoside m e t a b o l i t e s o f c y t o k i n i n s were i s o l a t e d by c e l l u l o s e i o n exchange chromatography, TLC and paper chromatography. S t r u c t u r e s were determined by u l t r a v i o l e t and mass spectroscopy. Glucose was determined w i t h glucose oxidase a f t e r a c i d h y d r o l y s i s w i t h a polystyrene sulphonic a c i d r e s i n (H+ form) at 120° f o r 1 hour. Glucose E s t e r s S e v e r a l p e s t i c i d e and p l a n t growth r e g u l a t o r s are a c i d s o r r e a d i l y h y d r o l y z a b l e e s t e r s . Studies w i t h a number o f these p e s t i c i d e s have shown t h a t they are r a p i d l y complexed as waters o l u b l e metabolites and e a s i l y hydrolyzed t o the f r e e a c i d by treatment w i t h a d i l u t e base or an a c i d . I n some of these s t u d i e s , i t has been speculated t h a t glucose e s t e r s were formed. U n f o r t u n a t e l y , very few of these w a t e r - s o l u b l e complexes have been i s o l a t e d o r i d e n t i f i e d . However, a number o f r e p o r t s (4149) have shown t h a t auxins and p l a n t growth r e g u l a t o r s are metabolized t o glucose e s t e r s i n h i g h e r p l a n t s (Figure 8 ) .

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

CI COOH

—I

CI

CH 0H 2

glucosyl transferase

i OH

CI COOH

I CI

Figure 5. Biosynthesis of N-glucosyl chloramben

CH

3

Figure 6. Proposed scheme for the metabolism of 3-hydroxy-5methylisoxazole in plants

glucose 6-benzylamino-9-glucosyl purine Figure 7. Cytokinin metabolism in plants—N-glucoside formation

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Enzymatic s y n t h e s i s o f the 2-0, 4-0 and 6-0 e s t e r s o f IAA ( i n d o l e - 3 - a c e t i c acid ) and glucose have been reported by Kopcewicz e t a l . (47). A crude enzyme from mature sweet corn k e r n e l s r e q u i r e d ATP and CoA as c o f a c t o r s and suggested that IAA-CoA t h i o l e s t e r formation was r e q u i r e d f o r a c y l a t i o n o f glucose. A proposed r e a c t i o n sequence i s shown i n F i g u r e 9. The presence of three i s o m e r i c forms was a t t r i b u t e d to a c y l m i g r a t i o n . Zenk (45) has suggested that the b i o s y n t h e s i s o f the 1-0-ester o f IAA and glucose proceeds by a d i f f e r e n t mechanism and i s c a t a l y z e d by a UDP-glucosyItransferase, as shown i n Equation 1. A d d i t i o n a l support f o r a UDP-glucosyltransferase mechanism of glucose e s t e r b i o s y n t h e s i s has been provided by J a c o b e l l i et a l . (50) and Corner and Swain (51). I n s t u d i e s w i t h enzymes i s o l a t e d from germinating l e n t i l s and geranium l e a v e s , the formation o f s e v e r a a c i d glucose e s t e r s r e q u i r e v i t r o s t u d i e s are needed to understand the mechanism(s) o f glucose e s t e r b i o s y n t h e s i s . I n a d d i t i o n t o glucose e s t e r s , the b i o s y n t h e s i s and i s o l a t i o n of s e v e r a l IAA-m^o-inositol e s t e r s have a l s o been reported (47, 48). Glucose e s t e r s are s e n s i t i v e to m i l d a l k a l i n e h y d r o l y s i s , and are a l s o hydrolyzed by a c i d s . H y d r o l y s i s w i t h 3-glucosidase has been reported f o r glucose e s t e r s of IAA, NAA (1-naphthalenea c e t i c a c i d) and 2,4-D (2,4-dichlorophenoxy a c e t i c a c i d ) . However, the glucose e s t e r o f a b s c i s i c a c i d was not hydrolyzed by 3-glucosidase (42). I t has been suggested by s e v e r a l authors that glucose e s t e r b i o s y n t h e s i s and h y d r o l y s i s may be an important f a c t o r i n the b i o r e g u l a t i o n of p l a n t hormone l e v e l s (47, 52). I t i s i n t e r e s t i n g t o speculate t h a t the s e l e c t i v e p h y t o t o x i c i t y o f a number of h e r b i c i d e s may a l s o be a f f e c t e d by d i f f e r e n t i a l r a t e s o f glucose e s t e r formation and h y d r o l y s i s . S-Glucosides Various g l u c o s i n o l a t e s have been i s o l a t e d as n a t u r a l products from s e v e r a l p l a n t s p e c i e s . A general s t r u c t u r e f o r these S-glucosides i s shown i n Figure 10. The R group may be a l i p h a t i c o r p a r t l y aromatic. G l u c o s i n o l a t e s do not appear t o be hydrolyzed by emulsin, but are hydrolyzed i n c e r t a i n p l a n t species by a mixture of enzymes c a l l e d myrosinase. Recent i n v e s t i g a t i o n s (53-55) of b e n z y l g l u c o s i n o l a t e b i o synthesis i n p l a n t s have shown that an i n t e r m e d i a t e , phenylacetothiohydroximate, i s g l u c o s y l a t e d by a UDP-glucosyltransf e r a s e (Figure 11a). Examples of p e s t i c i d e S-glucosides are l i m i t e d . However, s t u d i e s by Kaslander et a l . (56) have shown that the f u n g i c i d e , dimethyldithiocarbamate, i s metabolized i n potato s l i c e s and cucumber s e e d l i n g s to the S-glucoside (Figure l i b ) . I n i n s e c t s , Gessner and Acara (57) have shown

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Figure 8.

Figure 9.

Glucose esters of auxins and plant growth regulators

Proposed biosynthesis of 2-0, 4-0, and 6-0 glucose esters of IAA

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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45

Pesticide Conjugates—Glycosides CH.OH

H

0

OH

H

Equation 1.

CHfOH

/ ~ ys-c0

HC^lf

N-O-SOi OH

Figure 10. General structure of glucosinolates

CH OH 2

(a)

HS

-C-CH Yo) NOH

2

>

'

UDP-glucosyl transferase

-C-CH N ' "NOH 2

HO^l/

8

OH CH 0H 2

/CH (b)

S

HS-C-N S \ H *

UDP-glucosyl transferase

/

A—Q _

/CH

5

VS-C-N CH

9

OH

CH^H UDP-glucosyl transferase OH

(c)

CH OH 2

N—'

UDP-glucotyl transferase OH

Figure 11. Biosynthesis of S-glucosides in plants and insects

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

f

46

BOUND AND CONJUGATED PESTICIDE RESIDUES

t h a t thiophenol and 5-mercaptouracil a r e metabolized t o S-glucosides (Figure 11c). The S-glucosides were i s o l a t e d from e x c r e t a and t r e a t e d t i s s u e . I n v i t r o s t u d i e s w i t h f a t body t i s s u e s e s t a b l i s h e d t h a t S-glucoside b i o s y n t h e s i s was c a t a l y z e d by a UDP-glucosyltransferase. Complex Glycosides Recently, a few s t u d i e s have demonstrated t h a t glucosides of p e s t i c i d e metabolites may be subject t o f u r t h e r metabolism in plants. E x t e n s i v e e a r l y s t u d i e s by M i l l e r (58) showed that s e v e r a l x e n o b i o t i c a l c o h o l s and phenols were conjugated as g e n t i o b i o s i d e s by higher p l a n t s . Apparent d i f f e r e n c e s i n enzyme s p e c i f i c i t y toward aglycone and sharp c o n t r a s t s i d i f f e r e n t species t o form e i t h e r glucosides o r g e n t l o b i o s i d e s were reported. These g l u c o s i d e s and g e n t i o b i o s i d e s were not t r a n s l o c a t e d from t i s s u e s i n which they were formed. Studies w i t h the h e r b i c i d e , diphenamid (N,N-dimethyl-2,2diphenylacetamide), have shown that a primary o x i d a t i o n product, N-hydroxymethyl-N-methyl-2,2-diphenylacetamlde, i s conjugated as a 3-glucoside and a 3-gentiobioside (59). S t r u c t u r e s were determined by MS o f a c e t y l a t e d d e r i v a t i v e s and a n a l y s i s o f h y d r o l y s i s products. Time course s t u d i e s (60) suggest t h a t t h e g l u c o s i d e i s a precursor of the g e n t i o b i o s i d e as shown i n F i g u r e 12. Support f o r such a hypothesis has been provided by Yamaha and C a r d i n i (61). They i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d a UDP-glucosyltransferase from wheat germ that c a t a l y z e s the b i o s y n t h e s i s of g e n t i o b i o s i d e s from phenolic-mono-3-Dglucosides as shown i n Equation 2. UDPG + phenol g l u c o s i d e

p

UDP +• phenol g e n t i o b i o s i d e

I t has been suggested (62) t h a t diphenamid s e l e c t i v i t y may be determined, t o some e x t e n t , by inherent d i f f e r e n c e s i n UDPg l u c o s y l t r a n s f e r a s e and/or g l y c o s i d a s e a c t i v i t i e s between t o l e r a n t and s u s c e p t i b l e p l a n t s p e c i e s . I t i s a l s o i n t e r e s t i n g t o note that t h e a c t i v i t i e s o f these enzymes are a f f e c t e d by environmental f a c t o r s such as ozone l e v e l s (59, 60) o r l i g h t i n t e n s i t y and humidity (63). The i s o l a t i o n and i d e n t i f i c a t i o n o f the g l u c o s i d e and g e n t i o b i o s i d e o f N-hydroxymethyl-N-methyl-2,2-diphenylacetamide presented some i n t e r e s t i n g and c h a l l e n g i n g problems (59). The most d i f f i c u l t problem was t h e s e p a r a t i o n of the h i g h l y p o l a r g e n t i o b i o s i d e from n a t u r a l l y o c c u r r i n g g l y c o l i p i d s . A l l of t h e procedures that were t r i e d i n c l u d i n g p r e p a r a t i v e TLC, anion exchange chromatography (DEAE c e l l u l o s e ) and g e l permeation chromatography ( B i o g e l P-2) f a i l e d t o achieve the p u r i t y needed f o r s t r u c t u r e determination. A s o l u t i o n t o t h i s problem was

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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achieved by r e a c t i o n o f the p a r t i a l l y p u r i f i e d g e n t i o b i o s i d e w i t h T r i - S i l Z, TLC s e p a r a t i o n o f the TMS d e r i v a t i v e , h y d r o l y s i s of the i s o l a t e d TMS d e r i v a t i v e (90% MeOH r e f l u x at 70°C f o r 2 hours) and f i n a l TLC o f the o r i g i n a l g e n t i o b i o s i d e . I n our experience, TMS d e r i v a t i v e s o f glucosides are u s u a l l y s t a b l e enough t o be handled i n t h i s manner and provide a simple method f o r changing the p o l a r i t y o f a g l y c o s i d e m e t a b o l i t e f o r s e p a r a t i o n purposes. The m i l d c o n d i t i o n s needed f o r the recovery of the unchanged g l y c o s i d e are a l s o h e l p f u l i n s i t u a t i o n s where the g l y c o s i d e i s e a s i l y hydrolyzed. Another i n t e r e s t i n g l e s s o n learned i n diphenamid metabolism s t u d i e s (59) was that f a i l u r e t o demonstrate metabolite h y d r o l y s i s w i t h emulsin does not preclude the presence o f a 3-D-glucoside. The g l u c o s i d e metabolite of N-hydroxymethyl-Nmethyl-2, 2-diphenylacetamid the g e n t i o b i o s i d e was hydrolyze t o y i e l d the g l u c o s i d e . O p t i c a l r o t a t i o n s t u d i e s w i t h the g l u c o s i d e and i t s t e t r a a c e t a t e c l e a r l y i n d i c a t e d , however, that the metabolite was a 3-glucoside. I n r e t r o s p e c t , PMR a n a l y s i s may have provided an unequivocal assignment o f anomeric c o n f i g u r a t i o n . Even though emulsin i s the enzyme g e n e r a l l y used t o hydrolyze 3-D-glucosides, other 3-glucosidases w i t h d i f f e r e n t substrate s p e c i f i c i t i e s are a v a i l a b l e from a v a r i e t y of sources. One of these enzymes, a crude hesperidlnase from A s p e r g i l l u s n i g e r , contained a 3-glucosidase that c a t a l y z e d the complete h y d r o l y s i s of both the g l u c o s i d e and the g e n t i o b i o s i d e m e t a b o l i t e s . Enzyme h y d r o l y s i s with hesperidlnase at pH 5.25 provided an e x c e l l e n t means o f i s o l a t i n g and i d e n t i f y i n g the a c i d l a b i l e N-hydroxymethyl aglycone. Even m i l d a c i d h y d r o l y s i s of the g l u c o s i d e r e s u l t e d i n the l o s s of formaldehyde and the formation of N-methyl-2,2-diphenylacetamide. S e v e r a l p l a n t pigments have been i d e n t i f i e d as malonate hemi-ester d e r i v a t i v e s of 3-glucosides (64-66). S p e c t r a l and chemical s t u d i e s w i t h a c y l a t e d betacyanins (64) and i s o f l a v o n e s (65) have e s t a b l i s h e d that the 0-malonyl group i s l o c a t e d at the C-6 o f the glucose moiety. Hahlbrock (67) i s o l a t e d a malonyl CoA t r a n s f e r a s e from p a r s l e y c e l l c u l t u r e s that c a t a l y z e d the t r a n s f e r of malonate from malonyl CoA t o flavone g l y c o s i d e s . Moore and Wilson (68) reported the enzymatic h y d r o l y s i s o f a c y l a t e d flavone g l y c o s i d e s and showed t h a t p a r t i a l l y p u r i f i e d enzymes from p a r s l e y and c h i c k pea leaves c a t a l y z e d the h y d r o l y s i s o f the malonate e s t e r l i n k a g e . Recently, Shimabukuro et a l . (69) reported the i s o l a t i o n and t e n t a t i v e i d e n t i f i c a t i o n of 6-0-malonyl-3-D-glucoside of JJn i t r o p h e n o l as a major m e t a b o l i t e of f l u o r o d i f e n (p-nitrophenylo t , c x , a - t r i f l u o r o - 2 - n i t r o - p - t o l y l ether) i n peanut. I n these s t u d i e s , glucoside metabolites of f l u o r o d i f e n were e x t r a c t e d w i t h 80% methanol and the concentrated aqueous e x t r a c t was p a r t i t i o n e d w i t h hexane and then w i t h i s o p r o p y l ether t o remove unreacted f l u o r o d i f e n and f r e e p - n i t r o p h e n o l . Two major

American Chemical Society Library 1155

16th St. N. W.

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48

BOUND AND CONJUGATED PESTICEDE RESIDUES

g l u c o s i d e m e t a b o l i t e s , a n e u t r a l g l u c o s i d e and an a n i o n i c malonyl g l u c o s i d e of p - n i t r o p h e n o l were separated by g e l f i l t r a t i o n on Sephadex G-10 and c e l l u l o s e i o n exchange chromatography on DE-52. The I s o l a t e d malonyl glucoside was s t a b l e t o weak a c i d s . Only l i m i t e d h y d r o l y s i s to the g l u c o s i d e was reported during i t s p u r i f i c a t i o n . M i l d a l k a l i n e h y d r o l y s i s , however, r a p i d l y l i b e r a t e d the glucoside . Enzymatic h y d r o l y s i s was achieved w i t h h e s p e r i d l n a s e , but not w i t h emulsin. S i m i l a r r e s i s t a n c e to emulsin h y d r o l y s i s has been reported by Minale et a l . (64) f o r the 6-0-malonyl-3-D-glucoside of b e t a n i d i n . The s t r u c t u r e of the I s o l a t e d O-malonyl-3-D-glucoside of p - n i t r o phenol was e s t a b l i s h e d by MS a f t e r methylatlon w i t h diazomethane and a c e t y l a t i o n w i t h a c e t i c a c i d anhydride and Zn&2« Deuterium exchange of one or both of the methylene protons of the malonyl group was demonstrated l o c a t e d at the C-6 of th make an unequlvocable s t r u c t u r e assignment based on PMR and p o s s i b l y 13cMR s t u d i e s . A proposed pathway f o r the b i o s y n t h e s i s of t h i s a c y l a t e d glucoside i s shown i n Figure 13. Studies on the i s o l a t i o n and i d e n t i f i c a t i o n of complex g l u c o s i d e m e t a b o l i t e s have j u s t begun. The extent, v a r i e t y and s i g n i f i c a n c e of these metabolites remain l a r g e l y unknown. I n p l a n t s , complex g l y c o s i d e metabolites may be intermediates i n the formation o f " t e r m i n a l " p e s t i c i d e r e s i d u e s . Recent s t u d i e s by S t i l l and Mansager (70) I n d i c a t e that p h e n o l i c Intermediates i n the metabolism of chlorpropham by a l f a l f a may be g l y c o s y l a t e d as a homologous s e r i e s of methanol-water s o l u b l e o l i g o s a c c h a r i d e d e r i v a t i v e s . P a r t i a l h y d r o l y s i s of these h i g h l y p o l a r complex g l y c o s i d e metabolites was achieved by repeated treatment w i t h c e l l u l a s e . A d d i t i o n a l s t u d i e s on the i s o l a t i o n , i d e n t i f i c a t i o n , and b i o s y n t h e s i s of complex g l y c o s i d e metabolites are needed. Methodology A v a r i e t y of techniques and procedures are a v a i l a b l e f o r the i s o l a t i o n and i d e n t i f i c a t i o n of g l y c o s i d e m e t a b o l i t e s . U n f o r t u n a t e l y , an adequate d i s c u s s i o n or c o n s i d e r a t i o n of the many methods that have been used i s not p o s s i b l e i n the time a v a i l a b l e t h i s morning. Besides, each g l y c o s i d e m e t a b o l i t e must be considered, t o a l a r g e extent, as a unique i s o l a t i o n and i d e n t i f i c a t i o n problem. What works i n one s i t u a t i o n may not work i n another, depending on the nature of the p a r t i c u l a r g l y c o s i d e , the aglycone and the endogenous m a t e r i a l s i n the t i s s u e e x t r a c t . I t i s f o r t u n a t e , t h e r e f o r e , that a v a r i e t y of techniques and approaches are a v a i l a b l e t o the i n v e s t i g a t o r . S e v e r a l p o i n t s should be s t r e s s e d , however, i n the i s o l a t i o n and i d e n t i f i c a t i o n of g l y c o s i d e m e t a b o l i t e s : (a) the importance of p r o t e c t i n g l a b i l e g l y c o s i d i c linkages from h y d r o l y s i s during e x t r a c t i o n and chromatographic s e p a r a t i o n ; (b) the n e c e s s i t y f o r f r e e i n g the i s o l a t e d g l y c o s i d e from

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

3.

49

Pesticide Conjugates—Glycosides

FREAR

0 i

\

M

i? 8 , N-C-CH

CH/

HOCHfy NAOPH O N-C-CHi microtomol CH,' dtmtthylatt" t

UOPG>lM p. uD

U p V glucotyllrantftratV 0

CHfOH

*UDPglucotyltrontftrotV

Figure 12. Diphenamid metabolism in tomato—proposed biosynthesis of gentiobioside and metabolite

NO*

CHt-0-&-CHt-COOH

OH

Figure 13. Proposed biosynthesis of pnitrophenyl-6-O-malonyl-p-D-glucoside in peanut

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

contamination by n a t u r a l products; and (c) the requirement t h a t the s t r u c t u r e of the g l y c o s i d e be determined e i t h e r as the i n t a c t molecule or as a d e r i v a t i v e and supported by an a n a l y s i s of the h y d r o l y s i s products. Enzymatic h y d r o l y s i s by a g l y c o s i d e s e or q u a l i t a t i v e a n a l y s i s of h y d r o l y s a t e s are o b v i o u s l y not adequate f o r s t r u c t u r a l determination. In a number of s i t u a t i o n s , c u r r e n t methodology has not been adequate i n s o l v i n g d i f f i c u l t problems of g l y c o s i d e i s o l a t i o n and s t r u c t u r e d e t e r m i n a t i o n . F o r t u n a t e l y , s e v e r a l new and improved techniques are on the h o r i z o n , and may be very h e l p f u l . In some cases, n o n - v o l a t i l e , h i g h molecular weight g l y c o s i d e s and thermally unstable g l y c o s i d e s have l i m i t e d the usefulness of GLC as an e f f e c t i v e s e p a r a t i o n method. H o p e f u l l y , p r e p a r a t i v e HPLC w i l l s o l v e some of these problems, and provide more e f f i c i e n t s e p a r a t i o f glycoside tha d adequately by the chromatographi E l e c t r o n Impact mass spectroscopy i s a very s e n s i t i v e and powerful t o o l i n the s t r u c t u r a l a n a l y s i s of g l y c o s i d e s (71-74). Ion fragmentation i s e x t e n s i v e , however, and primary molecular i o n s needed f o r a determination of molecular weight and elemental composition are o f t e n absent. Recent s t u d i e s have shown t h a t c h e m i c a l - i o n i z a t i o n (CI) and f i e l d d e s o r p t i o n (FD) mass s p e c t r a of g l y c o s i d e s e x h i b i t s t r o n g q u a s i molecular i o n peaks and l i m i t e d i o n fragmentation (74-76). These techniques should provide a s e n s i t i v e and more d i r e c t means f o r determining the s t r u c t u r e of i n t a c t g l y c o s i d e s . Nuclear magnetic resonance (NMR) spectroscopy i s another important method f o r s t r u c t u r e e l u c i d a t i o n i n s t u d i e s w i t h carbohydrates and t h e i r d e r i v a t i v e s (77, 78). In the p a s t , the use of NMR spectroscopy i n p e s t i c i d e metabolism s t u d i e s has been l i m i t e d . Recently, however, the a v a i l a b i l i t y of new instrumenta t i o n , p a r t i c u l a r l y F o u r i e r transform NMR, has made NMR s p e c t r o scopy a p r a c t i c a l and v e r y u s e f u l technique f o r g l y c o s i d e s t r u c t u r a l a n a l y s i s . Thus f a r , most a p p l i c a t i o n s have i n v o l v e d PMR spectroscopy. However, recent reviews (77, 79) suggest that n u c l e i other than protons may a l s o be u s e f u l i n s t r u c t u r a l s t u d i e s of g l y c o s i d e m e t a b o l i t e s . Summary Glucosides o f t e n account f o r a major p o r t i o n o f the p e s t i c i d e metabolites i n p l a n t s and i n v e r t e b r a t e s . T h e i r importance and s i g n i f i c a n c e should not be overlooked. Even though the i s o l a t i o n and i d e n t i f i c a t i o n of p e s t i c i d e s and t h e i r m e t a b o l i t e s as g l y c o s i d e s has been l i m i t e d , the d i v e r s i t y of i s o l a t e d g l u c o s i d e s i s already apparent and includes'; O-gluc o s i d e s , N-glucosides, glucose e s t e r s , S-glucosides, a c y l a t e d g l u c o s i d e s and g e n t i o b i o s i d e s . Undoubtedly, many a d d i t i o n a l g l u c o s i d e s and other g l y c o s i d e s w i l l be i s o l a t e d i n the f u t u r e . I n f o r m a t i o n about the nature and extent of g l y c o s i d e formation

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Pesticide Conjugates-Glycosides

51

i s needed before t h e i r r o l e i n p e s t i c i d e metabolism can be determined. At t h e present time, glucosides have been i m p l i c a t e d as s i g n i f i c a n t f a c t o r s i n the b i o r e g u l a t i o n o f p e s t i c i d e t o x i c i t y and s e l e c t i v i t y i n p l a n t s and i n s e c t s . U n f o r t u n a t e l y , the enzyme systems r e p o n s i b l e f o r g l u c o s i d e formation and h y d r o l y s i s have not been s t u d i e d t o any great extent. A l s o , t h e f u r t h e r metabolism of g l u c o s i d e m e t a b o l i t e s has r e c e i v e d l i t t l e a t t e n t i o n . I n p l a n t s , the p o s s i b l e r o l e and s i g n i f i c a n c e of these complex g l y c o s i d e metabolites i n the formation o f " t e r m i n a l " p e s t i c i d e r e s i d u e s has been suggested.

Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Smith, J. N., Advan 3, 173. Finkle, B. J., In: "Phenolic Compounds and Metabolic Regulation," pps. 3-24 (1967), Eds. Finkle, B. J. and Runeckles, V. C., Appleton-Century-Crofts, N.Y. Hanson, K. R., Zucker, M., and Sondheimer, E., In: "Phenolic Compounds and Metabolic Regulation," pp. 69-93 (1967), Eds. Finkle, B. J., and Runeckles, V. C., AppletonCentury-Crofts, N.Y. Ramwell, P. W., and Sherratt, H. S. A., In: "Biochemistry of Phenolic Compounds," pp. 457-510 (1964), Ed. Harborne, J. B., Academic Press, N.Y. Daly, J. W., In: "Phenolic Compounds and Metabolic Regulation," pp. 27-66 (1967), Eds. Finkle, B. J., and Runeckles, V. C., Appleton-Century-Crofts, N.Y. Kuhr, R. J., J. Agr. Food Chem. (1970), 18, 1023. Bull, D. L., and Whitten, C. J., J. Agr. Food Chem. (1972), 20, 561. S t i l l , G. G., Rusness, D. G., and Mansager, E. R., ACS Symposium Series No. 2 (1974), "Mechanism of Pesticide Action," pp. 117-129. Frear, D. S., and Swanson, H. R., Pest. Biochem. Physiol. (1975), 5, 73. Pridham, J. B., Phytochemistry (1964), 3, 493. Towers, G. H. N., In: "Biochemistry of Phenolic Compounds," pp. 249-294 (1964), Ed. Harborne, J. B., Academic Press, N.Y. Trivelloni, J. C., Enzymologia (1964), 26, 6. Mehendale, H. M., and Dorough, H. W., J. Insect Physiol. (1972), 18, 981. Metcalf, R. L., Fukuto, T. R., Collins, C., Borck, K., Abd El-Aziz, S., Munoz, R., and Cassil, C. C., J. Agr. Food Chem. (1968), 16, 300. S t i l l , G. G., and Mansager, E. R., Phytochemistry (1972), 11, 515.

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41. 42. 43.

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Bligh, E. G., and Dyer, W. J., Can. J. Biochem. Physiol. (1959), 37, 911. Colby, S. R., Science (1965), 150, 619. Swanson, C. R., Kadunce, R. E., Hodgson, R. H., and Frear, D. S., Weeds (1966), 14, 319. Colby, S. R., (1966), Weeds 14, 197. Colby, S. R., 152nd Amer. Chem. Soc. Meeting, (1966), Abstr. No. A-33, New York. Frear, D. S., In: "Degradation of Herbicides," 2nd Edition, Eds. Kearney, P. C., and Kaufman, D. D., Marcel Dekker, Inc., N.Y. (In press). S t i l l , G. G., Science (1968), 159, 992. Ries, S. K., Zabnik, P. J., Stephenson, G. R., and Chen, T. M., Weed Sci. (1968), 16, 40. Stephenson, G. R. d Ries S d Sci (1969) 17 327. Stoller, E. W., Plant Physiol. (1966), 44, 854. Swanson, C. R., Hodgson, R. H., Kadunce, R. E., and Swanson, H. R., Weeds (1966), 14, 323. Frank, R., and Switzer, C. M., Weed Sci. (1969), 17, 365. Stephenson, G. R., and Ries, S. K., Weed Res. (1967),7,51. Frear, D. S., Hodgson, R. H., Shimabukuro, R. H., and S t i l l , G. G., Advan. Agron. (1972), 24, 327. Frear, D. S., Swanson, C. R., and Kadunce, R. E., Weeds (1967), 15, 101. Frear, D. S., Phytochemistry (1968),7,381. Yih, R. Y., McRae, D. H., and Wilson, H. F., Science (1968), 161, 376. Kamimura, S., Hishikawa, M., Saeki, H., and Takahi, Y., Phytopathology (1974), 64, 1273. Murakoshi, I., Ikegama, F., Kato, F., Tomita, K., Kamimura, S., and Haginiwa, J., Chem. Pharm. Bull. (1974), 22, 2048. Parker, C. W., Wilson, M. M., Letham, D. S., Cowley, D. E., and MacLeod, J. K., Biochem. Biophys. Res. Commun. (1973), 55, 1370. Wilson, M. M., Gordon, M. E., Letham, D. S., and Parker, C. W., Jour. Exptl. Bot. (1974), 25, 725. Deleuze, G. G., McChesney, J. D., and Fox, J. E., Biochem. Biophys. Res. Commun. (1972), 48, 1426. Parker, C. W., and Letham, D. S., Planta (1973),114,199. Gordon, M. E., Letham, D. S., and Parker, C. W., Annals of Bot. (1974), 38, 809. Guern, J., Doree, M., and Sadorge, P., In: "Biochemistry and Physiology of Plant Growth Substances," pp. 1155-1167 (1968), Eds. Wightman, F., and Setterfield, G., Runge Press, Ottawa. Koshimizer, H. Fukui, and Mitsui, T., Agr. Biol. Chem. (1968), 32, 789. Milborrow, B. V., J. Exptl. Bot. (1970), 21, 17. Hiraga, K., Yokota, T., Murofushi, N., and Takahashi, N., Agr. Biol. Chem. (1972), 36, 345.

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53

Shindy, W. W., Jordon, L. S., Jolliffe, V. A., Coggins, C. W., and Kumamoto, J., J. Agr. Food Chem. (1973), 21, 629. Zenk, M. H., Nature (1961), 191, 493. Ehmann, A., Carbohydrate Res. (1974), 34, 99. Ropcewicz, J., Ehmann, A., and Bandurski, R. A., Plant Physiol. (1974), 54, 846. Ehmann, A., and Bandurski, R. S., Carbohydrate Res. (1974), 36, 1. Thomas, E. W., Loughman, B. C., and Powell, R. G., Nature (1964), 204, 286. Jacobelli, G., Tabone, M. J., and Tabone, D., Bull. soc. chim. biol. (1958), 40, 955. Corner, J. J., and Swain, T., Nature (1965), 207, 634. Hiraga, K., Yamane, H., and Takahaski, N., Phytochemistry (1974), 13, 2371 Matsuo, M., and Underhill Commun. (1969), 36, 18. Matsuo, M., and Underhill, E. W., Phytochemistry (1971), 10, 2279. Matsuo, M., Kirkland, D. F., and Underhill, E. W., Phytochemistry (1972), 11, 697. Kaslander, J., Sijpesteinj, A. K., and VanderKerk, G. J. M., Biochem. Biophys. Acta (1961), 52, 396. Geasner, T., and Acara, M., Jour. Biol. Chem. (1968), 243, 3142. Miller, L. P., In: "Phytochemistry," p. 311, Vol. I (1973), Ed. Miller, L. P., Van Nostrand Reinhold Co., N.Y. Hodgson, R. H., Frear, D. S., Swanson, H. R., and Regan, L. A., Weed Sci. (1973), 21, 542. Hodgson, R. H., Dusbabek, R. E., and Hoffer, B. L., Weed Sci. (1974), 22, 205. Yamaha, T., and Cardino, C. E., Arch. Biochem. Biophys. (1960), 86, 133. Schultz, D. P., and Tweedy, B. G., J. Agr. Food Chem. (1971) , 19, 36. Schultz, D. P., and Tweedy, B. G., J. Agr. Food Chem. (1972), 20, 10. Minale, L., Piatelli, M., DeStefano, S., and Nicolaus, R. A., Phytochemistry (1966), 5, 1037. Beck, A. B., and Knox, J. R., Aust. J. Chem. (1971), 24, 1509. Kruezaler, F., and Hahlbrock, K., Phytochemistry (1973), 12, 1149. Hahlbrock, K., FEBS Letters (1972), 28, 65. Moore, A. L., and Wilson, S. B., Biochem. J. (1972), 129, 18P. Shimabukuro, R. H., Walsh, W. C., Stolzenberg, G. E., and Olson, P. A., Abs. Weed Sci. Meetings (1975), p. 65, No. 171 (Washington, D. C.).

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A N D C O N J U G A T E D PESTICIDE

RESIDUES

S t i l l , G. G., and Mansager, E. R., Chromatographia (1975), 8, 129. Kochetkov, N. K., and Chizhov, O. S., Advan. Carbohydrate Chem. (1966), 21, 39. Pearl, I. A., and Darling, S. F., Phytochemistry (1968), 7, 831. Kochetkov, N. N., and Chizhov, O. S., Methods Carbohyd. Chem. (1972), 6, 540. Lönngren, J., and Svensson, S., Advan. Carbohydrate Chem. Biochem. (1974), 29, 41. Lehman, W. D., Schulten, H. R., and Beckey, H. D., Org. Mass Spectrom. (1973), 7, 1103. Foltz, R. L., Chemtech (1975), January, 39. Angyal, S. J., In: "The Carbohydrates" IA 2nd Ed., (1972), pp. 195-215, Eds Press, N.Y. Coxon, B., Methods Carbohydrate Chem. (1972), 6, 513. Wilson, N. K., and Stothers, J. B., In: "Topics in Stereochemistry," (1974), 8, Eds. Eliel, E. L., and Allinger, N. L., 1, John Wiley and Sons, N.Y.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

4 Recent Advances in the Isolation and Identification of Glucuronide Conjugates JEROME Ε. BAKKE Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Fargo, N. Dak. 58102

The chemistry and biochemistr of glucuronic acid and glucur onide conjugates has bee G. J. Dutton (1). The chapte y Jayl Pasqualin methods for extraction, fractionation, and identification of steroid glucuronides. The present discussion will cover methods that have recently been applied to the extraction, separation, and identification of glucuronide conjugates from mammalian plasma, urine, and bile. These methods will be discussed in the sequence in which they are usually applied in practice and with bias toward the methods that the author has used. Extraction of Glucuronides Four materials have recently been successfully applied to the extraction or concentration of glucuronides present in aqueous solutions. These are: Amberlite XAD-2 (Rohm & Haas Co.), a synthetic polystyrene polymer, Porapak Q (Waters Assoc.), a gas chromatography column packing, liquid anion exchangers (tetraheptylammonium chloride, Eastman Organic Chemicals; methyl t r i caprylyl ammonium chloride, General Mills), and Sephadex LH-20 (Pharmica Fine Chemicals). ( R )

(R)

(R)

Amerlite XAD-2 . This bead form polymer has been used for the extraction of glucuronide conjugates from aqueous solutions (urine and salt solutions; 2,3). The XAD-2 column is first washed with methanol to remove contaminants and then washed with water prior to adding the aqueous solution containing the glucuronides. Materials in the sample that do not bind to the polymer are washed through the column with water and the glucuronides and other bound materials are recovered from the column by elution with organic solvents (usually methanol or acetone). (R)

Porapak Q . Columns of Porapak Q have been used in our laboratory for the extraction of glucuronides and other metabo­ lites from urine, plasma, and various aqueous solutions or 55

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AND CONJUGATED

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RESIDUES

e x t r a c t s . Porapak Q i s used i n the same manner as XAD-2 and i s assumed to f u n c t i o n i n the same manner, i . e . , by e s s e n t i a l l y a reversed phase chromatographic process. It i s p o s s i b l e to e f f e c t some s e p a r a t i o n of the m a t e r i a l s bound to Porapak Q by e l u t i o n with d i f f e r e n t organic solvents or stepwise gradients of methanol or acetone i n water. Aschbacher (4) u t i l i z e d a stepwise gradient of methanol i n water to separate u r i n a r y d i e t h y l s t i l b e s t e r o l (DES) from DES-glucuronide. The glucuronide e l u t e d from the Porapak Q with 80% methanol. The DES was e l u t e d from the column with methanol. In another case, the separation of the u r i n a r y metabolites from crufomate (5), using a stepwise e l u t i o n s e r i e s of hexane, d i e t h y l ether, methanol, i t was p o s s i b l e to e f f e c t a separation of u r i n a r y metaboli t e s i n t o two s o l u b i l i t y c l a s s e s . Hexane d i s p l a c e d the water from the Porapak Q column metabolites, and methano o l i t e s which included seven glucuronides. The plasma metabolites from crufomate, which contained the same seven glucuronides, a l s o bound to Porapak Q when the plasma was a p p l i e d d i r e c t l y to the column. Again, the metabolites were q u a n t i t a t i v e l y recovered by subsequent e l u t i o n of the column with methanol. Some compounds that do not absorb to Porapak Q from aqueous s o l u t i o n can be retarded on the column by an apparent s a l t i n g out reversed phase process. For example, the major u r i n a r y metaboli t e from cyclophosphamide (6) was not bound to Porapak Q from a simple aqueous s o l u t i o n ; however, i n urine i t remained on the column u n t i l the bulk of the u r i n a r y s o l i d s had been e l u t e d . L i q u i d Anion Exchangers. Matt ox e_t a l . (7) have demonstrated the a p p l i c a b i l i t y of l i q u i d anion exchangers d i s s o l v e d i n organic solvents f o r the e x t r a c t i o n of glucuronides from aqueous s o l u t i o n s . This e x t r a c t i o n i s assumed to i n v o l v e mainly an ion exchange process. The more p o l a r s t e r o i d glucuronides were ext r a c t e d l e s s e f f i c i e n t l y ; however, the completeness of the ext r a c t i o n could be increased i f the aqueous phase was made 4M with ammonium s u l f a t e . The glucuronides were recovered from the organi c phase by e x t r a c t i o n with ammonium hydroxide. A l l three of the above procedures have a p p l i c a b i l i t y to the e x t r a c t i o n of glucuronides from aqueous media; however, none of these processes i s s p e c i f i c f o r glucuronides. The XAD-2 and Porapak Q w i l l e x t r a c t many nonpolar m a t e r i a l s and the l i q u i d anion exchangers w i l l e x t r a c t any anion that can compete with the counter ion that i s present. Sephadex LH-20 (H 0). A 120 X 2 cm column of w a t e r - e q u i l i brated LH-20 has been used i n our l a b o r a t o r y f o r the p r e l i m i n a r y f r a c t i o n a t i o n of u r i n a r y and plasma metabolites from x e n o b i o t i c s (6). Using water as the eluent, t h i s column u s u a l l y separates these metabolites by a t y p i c a l reversed phase chromatographic 2

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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process Into polarity or s o l u b i l i t y classes* In three separate studies, a l l the glucuronide conjugates of xenobiotic metabolites appeared i n one fraction that had an elution volume range of 0.4 to 0*9 of the t o t a l column volume* The xenobiotics studied were crufomate (seven glucuronides), 0,0-dimethy1-0-(3,5,6-trichlorpyridyl)phosphorothionate (one glucuronide) and propachlor (sheep 2 and rat 5 glucuronides)• Since many urinary solids elute from this column with this glucuronide containing-fraction, i t was advantageous to use the Porapak Q column before or after use of the IH-20 column to remove many very polar materials and inorganic salts* The general applicability of this procedure for the concentration of. glucuronides w i l l have to await i t s application i n many more cases* Counter Current Distribution has been applied to th ides and sulfate esters (8). The application of this technique to the separation of these two groups of conjugates from b i o l o g i cal fluids was not reported; however, the authors relate that such application would require a prepuriflcation to remove materi a l s that would Interfere with the partition systems used (salts)• This technique may have application to the fractionation or concentration of glucuronides i f the salts can be removed using XAD-2 or Porapak Q* Fractionation of Glucuronides Several methods have been applied to the separation of glucuronide conjugates* These have been anion ion exchange c e l lulose columns, counter current distribution, the amino acid analyzer colums, paper chromatography, LH-20 columns, and gas l i q u i d chromatography of glucuronides rendered v o l a t i l e by derivatization. A number of these methods have been well studied with test mixtures but have not, as yet, been applied i n actual practice* Anion Exchange Cellulose* Knaak et a l . (9) separated four urinary glucuronide conjugates of carbaryl metabolites using a column of DEAE cellulose* The metabolites were eluted from the column using a tris-HCl buffer gradient* Knaak et a l . (10) later pointed out with other compounds, that this technique did not adequately separate closely related glucuronides and demonstrated that acetyl and trlmethylsilyl derivatives of methyl esters of a test mixture of glucuronides could be separated by gas l i q u i d chromatography* This procedure was suggested as a method for Identifying glucuronides i f standards were available* Van Der Wal and Huber (11) have studied the separation of a test mixture of steroid glucuronides by high-pressure l i q u i d chromatography (HPLC) using anion exchange celluloses* ECTEOLAcelluloses were found to be best suited for HPLC using acetate

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

buffers. The application of this procedure to glucuronides from biological fluids has not been reported. R

DEAE-Sephadex( ). Paulson and Jacobsen (12) used a DEAE Sephadex column eluted with a gradient of water to M KBr for the p a r t i a l separation of glucuronides from sulfate esters. KBr was used for the gradient to f a c i l i t a t e the preparation of micro pellets for Infrared analysis. This DEAE Sephadex-KBr gradient technique was applied to the separation of the glucuronides from crufomate (5) • The metabol i t e s i n the urinary glucuronide fraction from the LH-20 column [see Extraction of Glucuronides: LH-20 (H 0)] separated into five fractions on the DEAE column. The last four fractions to elute from the column contained three, one, two, and one glucuronides, respectively. This wa interpretation of the mas s u f f i c i e n t l y free from other urinary constituents after elution from the column and removal of the KBr using Porapak Q for derivat i z a t i o n and glc. 2

Sephadex LH-20 (HCOQ. A 115 X 0.9-cm column of LH-20 equilibrated and eluted with 0.065 M ammonium bicarbonate was used to p a r t i a l l y fractionate the glucuronides from crufomate metabolism (5). The metabolites from the glucuronide fraction from the LH-20 (H 0) column [see Extraction of Glucuronides: LH-20(H2O)] separated into three glucuronlde-contalning fractions. These fractions contained 4, 2, and 1 glucuronides, respectively, as determined by derivatization, glc, and mass spectral interpretation. The fraction containing four glucuronides separated into two glucuronide containing fractions on glc. One contained three glucuronides that have not been separated. These were the same glucuronides that were inseparable using the DEAE column. The LH-20 (HC03~) and DEAE-KBr columns have not given ideal separations of glucuronide mixtures. Closely related glucuronides do not separate. I f the glucuronide derivatives are stable to glc, some separations are possible. Mass spectrometry has been the only method used i n this laboratory for the detection of mixtures of glucuronide derivatives i n such samples. 2

Cation-Exchange Resin Chromatography. Urinary glucuronides of metabolites from terbutryne (2-methylthio-4-tert-butylamino-6ethylamino-sj-triazine) were separated using the amino acid analysis column eluted with a citrate buffer gradient. The column technique has been reported (13) and a preliminary report on the structures of the glucuronides has been presented (14)• This column separated the five glucuronides into four fractions. The two glucuronides that did not separate were separated by glc of the perTMS derivatives. Three of the glucuronides were characterized as S-glucuronides and two as alkyl-O-glucuronides (mass spectrometry).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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The general methods used to isolate these glucuronides consisted of the following steps. The metabolites from terbutryne were extracted from the urine on either an XAD-2 or Porapak Q column and eluted from the column with methanol. The residue i n the methanol eluate was dissolved i n water, adjusted to pH 3 and chromatographed on the amino acid analyzer. The buffer salts were removed from the separated fractions using the Porapak Q column technique. The fractions were further separated from contaminating materials by paper and thin-layer chromatography. For f i n a l purification prior to mass spectral analysis, the metabolites were silylated with excess N,N-bis(trimethylsilyl)trifluroacetamide containing 1Z t rime thy lchlorosilane and gas chromatographed (SE-30). Glucuronidase and hydrolysis studies were also used to characterize the aglycones. The cation exchang cases where the aglycon give the glucuronide the zwitterionic character of amino acids. It i s of interest that these glucuronides eluted from the resin without the use of organic solvents when they would bind to XAD-2 or Porapak Q from aqueous solution. Liquid Anion-Exchange Paper Chromatography. Hattox et a l . (15) have described a paper chromatographic system for the separation of steroid glucuronides using the liquid anion exchanger, tetraheptylammonlum chloride (THAC), i n the mobile phase. A l though this system has not been applied (to the author's knowledge) i n xenobiotic metabolism studies, i t would appear to be of value i f the glucuronides can be recovered from the chromatograms for structural characterization. The technique has only been applied to a test mixture of steroid glucuronides and i t i s unknown how much preliminary cleanup of biological fluids would be required before the system would become functional. Gas-Liquid Chromatography. Utilization of gas-liquid chromatography (glc) for the isolation of glucuronides requires the conversion of these conjugates to v o l a t i l e derivatives. The v o l a t i l i z a t i o n of glucuronides by derivatization not only makes It possible, In many cases, to purify and possibly separate glucuronides by glc, but also makes i t possible to obtain mass spectral data for structural determination. The derivatives that have been used for the v o l a t i l i z a t i o n of glucuronides are the methyl (aglycony1-2,3,4-tri-O-acety1glucopyranosid)uronate8 (acetyl-methy1; 10, 16); the totally methylated glucuronides (permethyl; 16, 17, 18, 19, 20, 21); the methyl (aglyconyl-2,3,4-tri-0-trimethylsilyl-glucopyranosid)uronates (TMS-methyl; 10, 16, 22, 23); and the totally trimethyl silylated glucuronides (perTMS; 23, 24) • In a l l cases, the aglycone w i l l also be derivatized i f functional groups are present that react with the reagent used.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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In general, the permethyl glucuronides have shorter retention times on the glc column than the other derivatives (16). They also exhibit good thermal s t a b i l i t y ; however, the permethyl derivative of chloramphenicol glucuronide was not stable to glc (21). The acetyl-methyl glucuronides exhibit much less s t a b i l i t y during glc separation (16, 24, 25); however, they are stable to mass spectral analysis (24, 25). The perTMS and TMS-methyl derivatives of glucuronides exhibit good s t a b i l i t y to glc and their retention times tend to be between those of the permethyl and acetyl-methyl glucuronides (16)• The perTMS derivatives offer an added advantage i n that the glucuronide can be trapped from the glc and the TMS groups easily removed with aqueous methanol to recover the intact glucuronide. The glucuronide can then be subjected to other derivatization techniques and/or glucuronidas Further investigatio separation and purification of glucuronide derivatives w i l l be needed before any general rules as to the expected s t a b i l i t y of various glucuronides and glucuronide-derivative types can be made. However, the perTMS and TMS-methyl derivatives of approximately twenty glucuronide conjugates of xenobiotic metabolites have been subjected to glc at this laboratory and a l l exhibited good thermal s t a b i l i t y . These derivatives ranged i n mass from 600 to 860 and included glucuronide conjugates of metabolites from diethyl s t l l besterol (one glucuronide), terbutryne (five), crufomate (seven), propachlor (five), and 3,5,6-trichloropyridin-2-ol (one). These were a l l chromatographed using temperature programming (10° per min from 100°C) on 6 X 1/8" 3% SE-30 columns. Of these twenty perTMS glucuronides (characterized by mass spectrometry), five eluted from the glc between 225 and 234°C, seven eluted between 240 and 250°C, and eight between 270 and 280°C. It i s , therefore, apparent that, under the conditions used, glc cannot be relied upon to separate closely related TMS-methyl or perTMS glucuronides. However, i f mass spectrometry i s used, especially high resolution, for the identification (characterization) , structures can be assigned to the glucuronides i n a mixture. ?

Identification Methods—Mass Spectrometry The classical methods involving glucuronidase hydrolysis and identification of the aglycone w i l l not be covered in this discussion. The properties of glucuronidase have been covered (1), and methodology for Identification of aglycones involves the s k i l l s required to identify any metabolite. This discussion w i l l Involve mainly electron impact mass spectrometry as a tool to characterize the structure of derivatized glucuronide conjugates. Mass spectrometry i s rapidly becoming the method of choice for the i n i t i a l characterization of glucuronide conjugates. In conjunction with hydrolysis (enzymatic or chemical) and identification of the aglycone using infrared, proton magnetic resonance, and mass

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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spectra, most glucuronides can be identified. I f the glucuronide can be rendered v o l a t i l e by derivatization, i t s mass spectrum can be obtained by conventional means ( i . e . , electron impact). I f the derlvatized glucuronide i s unstable or does not give sufficient aglycone containing fragment ions by electron impact, chemical ionization can be attempted (24) or possibly f i e l d desorption (27, 28). These latter two methods require special ion sources not generally available on existing spectrometers. If the derlvatized glucuronide i s stable to glc, the gas chromatograph can be used as a means of Introduction of the derivative into the mass spectrometer (21, 23)» The presence of a derlvatized glucuronide i n the sample introduced into the mass spectrometer i s readily determined by fragment ions i n the mass spectrum that are characteristic for the derlvatized glucuronic acid moiety determined for the permethy methyl derivatives (29), and the TMS-methyl and perTMS derivatives (23). These fragment ions are l i s t e d i n Table I.

Table I.

Fragment Ions Diagnostic for the Derlvatized Glucuronic Acid Moiety.

Permethyl (m/e)

233 or 2321/ 201*1/ 141 116 101 88 75

1/ 2/ 3/

Acetyl-Methyl (m/e)

317 257 215 197 173 155* 127 43

TMS-Methyl (m/e)

PerTMS (m/e)

407 406 317* 217 204 4231/

465 464 375* 217 204 , 4811/

m/e 232, when present, indicates a phenolic glucuronide; m/e 233, when present, Indicates an aliphatic glucuronide. An asterisk Indicates an Intense ion. These ions, when present, Indicate an aliphatic O-glucuronide.

Some glucuronides w i l l form the 4-5 dehydro analogue of the derlvatized glucuronic acid moiety during either derivatization, glc, or thermally i n the mass spectrometer (16, 21, 30). This chemical or thermal degradation process cannot be distinguished from the electron impact fragmentation mode which gives glucuronic acid moiety fragment ions of the same mass. This degradation

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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product, though undesirable, s t i l l gives interpretable mass spect r a l data since usually only the highest mass ion for each derivative l i s t e d i n Table I i s missing. This degradation reaction (elimination of methanol, acetic acid or t rime thy Is Hanoi) w i l l also be apparent i n the molecular ion region. In Interpretation of electron impact mass spectra for the presence of acetyl-methyl glucuronides, one oust be aware that the mass spectrum from methyl (1,2,3,4-tetra-O-acetyl-glucopyranosid)uronate contains a l l the fragment ions l i s t e d i n Table I and no molecular ion. Also the mass spectrum from permethyl glucuronic acid contains a l l ions i n Table I except for the m/e 232 and/or 233 ions and also no molecular ion (21). The ions at m/e 232 and/or 233 are not always present or can be of very weak intensity i n the spectra from permethyl glucuronides. Therefore, unless the techniques which would i s not present i n the sample, other ions i n the mass spectra must be present to confirm the presence of a glucuronide. These are ions that contain both the glucuronic acid and aglycone moieties (Table I I ) • These ions are Important for determining the mass of the glucuronide ( i f the molecular ion i s not present) and the mass of the aglycone moiety which w i l l be discussed later. Of the fragment ions l i s t e d i n Table I for the TMS-methyl and perTMS derivatives of glucuronides, only the Ions at m/e 217 and 204 are present In mass spectra from me thy 1(1,2,3,4-tetra-0trimethylsilyl-glucopyranosid) uronate and perTMS-glucuronic acid (23) • The remaining fragment ions l i s t e d for each derivative are confirmation that a glucuronide i s present, i . e . , something other than TMS i s the aglycone. Once the presence of a derlvatized glucuronide i s established, the mass of the aglycone (which w i l l be appropriately derlvatized) can easily be established i f the molecular ion i s present. The number of methyl, acetyl, or TMS moieties added to the aglycone during derivatization can be determined using the appropriate deuterium labeled reagents since the number of derivatizable functional groups on the glucuronic acid moiety remains constant. If the molecular ion i s not present, i t s mass can usually be deduced from the fragment ions l i s t e d i n Table I I . The ions l i s t ed result from obvious fragmentations and eliminations from the glucuronic acid moiety. The deduced molecular Ion can usually be confirmed by the presence i n the mass spectra of the aglycone containing fragment ions or rearrangement ions l i s t e d i n Table I I I . I t i s interesting to note a difference between these aglycone ions from the various derivatives of aromatic glucuronides. The TMS derivatives give aglycone containing ions that result from the elimination of the glucuronic acid moiety with rearrangement of a TMS to the aglycone. The permethyl and acetyl-methyl derivatives give aglycone-containing ions that result from the same elimination except that a proton rearranges to the aglycone.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Table I I . Fragment Ions used t o P r e d i c t the Mass o f the M o l e c u l a r Ion.

Permethyl (m/e)

Acetyl-Methyl (m/e)

TMS-methyl & PerTMS (m/e)

M -30 (CH 0)

M -59 (CH^COO)

M -31 (CH 0H)

M -60 (CH^COOH)

3

3

M -15 (CH ) 3

M -90 [ ( C H ^ S i O H ]

Table I I I . Aglycone C o n t a i n i n g Fragment Ions from V a r i o u s A l i p h a t i c and Aromatic Glucuronides^/

Fragment ion

Glycosidic linkage

m/e

Permethyl

Gl-0(aro)-Agl Gl-0(allph)Agl

(M -232) (M -249)

[H0-A l]t [AgllT

Acetyl-methyl

Gl-0(aro)-Agl

(M -316)

[H0-Agl]t

TMS-methyl

Gl-0(aro)-Agl Gl-S(aro)-Agl Gl-0(allph)-Agl

(M -334) (M -334) (M -423)

[TMS-OAgl]t [TMS-S-Agl]t [Agl]+

PerTMS

Gl-0(aro)-Agl Gl-S(aro)-Agl Gl-0(allph)-Agl

(M -392) (M -392) (M -481)

[TMS-OAgl]t [TMS-S-Agl]T [Agl]

Derivative

1/

Gl Agl = aro • aliph =

?

+

The d e r l v a t i z e d g l u c u r o n i c a c i d moiety. The d e r l v a t i z e d aglycone moiety, Aromatic a c e t a l l i n k a g e , Aliphatic acetal linkage.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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The fragment ions i n Tables I and I I I can a l s o be used to p r e d i c t the presence of a d e r l v a t i z e d glucuronide and i t s molecul a r i o n i f i t can be e s t a b l i s h e d that a glucuronide was present and not a mixture of g l u c u r o n i c a c i d and some m e t a b o l i t e . These ions are e s p e c i a l l y important i f the molecular i o n and the ions i n Table I I are not present o r are of very weak i n t e n s i t y . This method c o u l d be a p p l i e d to the TMS d e r i v a t i v e s w i t h more confidence s i n c e the ions i n Table I would r u l e out the presence of g l u c u r o n i c a c i d . The method could be a p p l i e d to permethyl d e r i v a t i v e s w i t h equal confidence i f e i t h e r the m/e 232 or 233 ion were present. In summary, mass spectrometry of glucuronide d e r i v a t i v e s can y i e l d the f o l l o w i n g i n f o r m a t i o n : a) The presence of a glucuronide from the fragment ions l i s t e d i n Table I . b) The molecular weight of the d e r l v a t i z e d aglycone ) Th numbe f d e r i v a t i z a b l func t i o n a l groups on the aglycon reagents, d) The elementa compositio aglycon from h i g h r e s o l u t i o n data, e) The type of g l y c o s l d i c l i n k a g e , whether a l i p h a t i c or aromatic from the designated ions l i s t e d i n Tables I and I I I . This i n f o r m a t i o n , along w i t h data from the f r e e aglycone, u s u a l l y y i e l d s enough i n f o r m a t i o n to d i r e c t s y n t h e s i s e f f o r t s toward the r i g h t compound f o r i d e n t i f i c a t i o n of the a g l y cone. The author p r e f e r s to use the perTMS d e r i v a t i v e s f o r the f o l lowing reasons: a) A one-step d e r i v a t i z a t i o n . b) R e l a t i v e l y good s t a b i l i t y of these d e r i v a t i v e s to g l c . c) The presence of a perTMS glucuronide can be determined w i t h confidence, d) Most glucuronides have given molecular ions and a l l have given M -15 fragment i o n s . 4) The TMS moieties can be e a s i l y removed a f t e r g l c p u r i f i c a t i o n f o r subsequent glucuronidase s t u d i e s . 5) The presence of f u n c t i o n a l groups on the aglycone that react w i t h diazomethane can be determined by comparing the mass spectrum from the perTMS d e r i v a t i v e w i t h that from the methyl TMS d e r i v a t i v e . The major disadvantage to the TMS d e r i v a t i v e s i s t h e i r l a b i l i t y to h y d r o l y s i s . The mass spectrum should be obtained as q u i c k l y as p o s s i b l e a f t e r p r e p a r a t i o n or t r a p p i n g from the g l c . A l s o , l i q u i d chromatographic techniques are not a p p l i c a b l e to these d e r i v a t i v e s due to t h e i r ease o f h y d r o l y s i s . Two examples w i l l p o i n t out the a p p l i c a b i l i t y of mass spectrome t r y and exact mass determinations to the i d e n t i f i c a t i o n and d i f f e r e n t i a t i o n of i s o m e r i c glucuronides. Two of the seven perTMS glucuronides of crufomate metabolites gave the same molecular i o n and fragment ions i n Table I I . Both mass s p e c t r a contained the fragment ions d i a g n o s t i c f o r perTMS glucuronides (Table I) except that one contained the m/e 481 i o n . The elemental composition of the m/e 481 i o n was determined by p r e c i s e mass measurement. I t s composition was c o n s i s t e n t w i t h the ion r e s u l t i n g from the homolytic cleavage of the bond between the e x o c y c l i c a c e t y l oxygen and the aglycone w i t h the charge remaining on the g l u c u r o n i c a c i d moiety. The mass spectrum from the other

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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perTMS glucuronide contained an i n t e n s e c h l o r i n e c o n t a i n i n g M -392 (Table I I I ) fragment i o n (m/e 344) which i n d i c a t e d a p h e n o l i c glucuronide• The two perTMS glucuronides were trapped from the gas chromatography the TMS groups removed w i t h aqueous methanol, and the f r e e glucuronides s u b j e c t e d t o glucuronidase h y d r o l y s i s . The same aglycone was obtained from both g l u c u r o n i d e s . The s t r u c t u r e o f t h e aglycone was confirmed by s y n t h e s i s to be 4-(l ,1'-dimethy1-2'hydroxyethy1)-2-chlorophenol. The TMS-methyl d e r i v a t i v e s o f both glucuronides were prepared. The mass spectrum from the TMS-methyl d e r i v a t i v e o f the perTMS glucuronide t h a t gave the m/e 481 i o n showed t h a t i t had r e a c t e d w i t h two moles o f diazome thane and the m/e 481 i o n now appeared at m/e 423 (Table I ) . Therefore, the g l y c o s i d e l i n k a g e i n t h i s glucuronide was through th The mass spectrum fro glucuronide showed t h a t i t had r e a c t e d w i t h one mole o f diazome thane and s t i l l contained the i n t e n s e c h l o r i n e - c o n t a i n i n g i o n a t m/e 344 (M -334, Table I I I ) . The above mass s p e c t r a l data from the two d e r i v a t i v e s along w i t h i d e n t i f i c a t i o n o f the aglycone e s t a b l i s h e d the s t r u c t u r e s o f these two i s o m e r i c g l u c u r o n i d e s . I n t h i s example, i t was e s s e n t i a l that the two glucuronides be separated from one another before mass s p e c t r a l a n a l y s i s , f o r glucuronidase s t u d i e s would have given the same aglycone. This s e p a r a t i o n was e f f e c t e d u s i n g the LH-20(HCO^"") column. Two i s o m e r i c glucuronides were i s o l a t e d (as perTMS d e r i v a t i v e s separated by g l c ) from the u r i n e o f sheep dosed w i t h propachlor ( N - i 8 o p r o p y l - a - c h l o r o a c e t a n i l i d e ) . The perTMS d e r i v a t i v e s o f both isomers gave molecular i o n s o f weak i n t e n s i t y and the fragment ions l i s t e d i n Table I I . Fragment ions were a l s o present which were d i a g n o s t i c f o r the presence o f perTMS glucuronides (Table I ) . One perTMS d e r i v a t i v e gave a weak i n t e n s i t y m/e 481 i o n (Table I) and an i n t e n s e M-481 i o n (Table I I I ) . The other gave an i n t e n s e M -392 i o n (Table I I I ) . From these d a t a , i t was assumed t h a t the former was an a l i p h a t i c glucuronide and the l a t t e r an aromatic g l u c u r o n i d e . The aglycones were obtained by glucuronidase h y d r o l y s i s . Mass s p e c t r a were obtained from the TMS d e r i v a t i v e s o f the aglycones and exact masses o f the molecular i o n s and major fragment ions were obtained. The molecular ions from both TMS-aglycones had the same e l e mental composition. Both mass s p e c t r a contained i n t e n s e fragment ions a t M -79. The 79 amu l e a v i n g group was c a l c u l a t e d t o have a mass o f 78.98389, i . e . , a n e g a t i v e mass d e f e c t , which i n d i c a t e d the presence o f oxygen, s u l f u r , o r s i l i c o n o r a combination o f any two or a l l o f these elements. A computer search f o r elemental composi t i o n s f o r t h i s mass gave SO2CH3 as the best f i t . This SO2CH3 group was assumed t o have r e p l a c e d the c h l o r i n e i n the o r i g i n a l p r o p a c h l o r molecule. f

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BOUND AND CONJUGATED PESTICIDE RESD3UES

The most Informative d i f f e r e n c e s i n the mass s p e c t r a from the two TMS-aglycones were t h a t the a l i p h a t i c TMS-aglycone gave an i n t e n s e i o n at M -103 [103 - ( O ^ ^ S i O O ^ ] u s u a l l y i n d i c a t i n g the TMS e t h e r of a primary a l c o h o l , and the aromatic TMS-aglycone gave an i n t e n s e i o n at M -42 which r e s u l t e d from the e l i m i n a t i o n of propene from the molecular i o n . Both o f these were confirmed by exact mass d e t e r m i n a t i o n s . The e l i m i n a t i o n of propene from the aromatic TMS-aglycone supported the presence of the N - i s o p r o p y l group and, t h e r e f o r e , the placement of the SO2CH3 moiety on the a c e t y l group. The s t r u c t u r e of the aromatic aglycone was c o n f i r m ed by s y n t h e s i s and was shown to be r i n g - h y d r o x y l a t e d i n the para position. The a l i p h a t i c aglycone has not been s y n t h e s i z e d however; the absence of the M -propene i o n and the presence of the M - ( 0 1 3 ) 3 S10CH2 i o n p l a c e the h y d r o x y i s o p r o p y l moiety w i t h th acetate. From these d a t a , the s t r u c t u r e s were assigned t o the two i s o meric g l u c u r o n i d e s . This example demonstrates the value of h i g h r e s o l u t i o n mass spectrometry f o r determining the elemental composit i o n s of fragment ions i n the assignment of s t r u c t u r e s because of the unexpected metabolic t r a n s f o r m a t i o n s of the x e n o b l o t i c which l e d t o glucuronide formation. I n t h i s case, s e p a r a t i o n of the two glucuronides would not have been e s s e n t i a l , f o r glucuronidase s t u d i e s would have demonstrated the presence of two aglycones. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Dutton, G. J., "Glucuronic Acid," Academic Press, New York, N.Y., 1966. Mattox, Vernon R., Goodrich, June E. Vrieze, Wiley D., Biochemistry (1969) 8, 188. Chang, T.T.L., Kuhlman, Ch. F., Schillings, R. T., Sisenwine, S.F., Tio, C. O., Ruelius, H. W., Experientia (1973) 29, 653. Aschbacher, P. W. (personal communication). Bakke, J. E. (unpublished data). Bakke, J. E., Feil, V. J. Fjelstul, C. E., Thacker, E. J., J. Agr. Food Chem. (1972) 20, 384. Mattox, Vernon R., Litwiller, Robert D., Goodrich, June E., Biochem. J. (1972) 126, 533. Assandri, Alessandro, Perazzi, Antonio, J. Chromatog. (1974) 95, 213. Knaak, J. B., Tallant, N. J., Bartley, W. J., Sullivan, L. J., J. Agr. Food Chem. (1965) 13, 537. Knaak, J. B., Eldridge, J. M., Sullivan, L. J., J. Agr. Food Chem. (1967) 15 605. Van Der Wal, Sj., Huber, J.F.K., J. Chromatog. (1974) 102, 353. Paulson, Gaylord D., Jacobsen, Angela M., J. Agr. Food Chem. (1974) 22, 629.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BAKKE

Glucuronide Conjugates

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13. Bakke, J. E., Robbina, J. D., Fell, V. J., J. Agr. Food Chem., (1967)15,628. 14. Larsen, G. L., Bakke, J. E. Abstr. 168th ACS National Meeting, Atlantic City (1974) Division of Pesticide Chemistry Abstr. #4. 15. Mattox, Vernon R., Goodrich, June E., Litwiller, Robert D., J. Chromatogr. (1972) 66, 337. 16. Imanari, Toshio, and Tamura, Zenzo. Chem. Pharm. Bull. (1967) 15, 1677. 17. Thompson, R. M., Desiderio, D. M., Biochem. Biophys. Res. Comm. (1972) 48, 1303. 18. Thompson, R. M., Gerber, N., Seibert, R. A., Desiderio, D. M., Res. Commun. Chem. Pathol. Pharmacol. (1972) 4, 543. 19. Gerber, N., Seibert, R. A., Thompson, R. M., Res. Commun. Chem. Pathol. Pharmacol. (1973) 6 499 20. Gerber, N., Olsen, M. L., Pharmacologist (1974) 16, 225. 21. Thompson, R. M., Gerber, N., Seibert, R. A., Desiderio, D. M., Drug Metabolism and Disposition (1973) 1, 489. 22. Horning, E. C., Horning, M. G., Ikekawa, N., Chambaz, E. M., Jaakonmaki, P. I., Brooks, C.J.W., J. Gas Chromatog. (1967) 5, 283. 23. Billets, Stephen, Lietman, Paul S., Fenselau, Catherine, J. Med. Chem. (1973 16, 30. 24. Chang, T.T.L., Kuhlman, Ch. F., Schillings, R. T., Sisenwine, S. F., Tio, C. O., Ruelius, H. W., Experientia (1973) 29, 653. 25. Paulson, G. D. (private communication). 26. Games, D. E., Games, M. P., Jackson, A. H., Olavesen, A. H., Rossiter, M., Winterburn, P. J., Tetrahedron Letters (1974) 2377. 27. Schulten, H. R., Games, D. E., Biomed. Mass Spec., in press. 28. Paulson, G. D., Zaylskie, R. G., Dockter, M. M., Anal. Chem. (1973) 45, 21. 29. Björndahl, H., Hellerqvist, C. G., Lindberg, B., Svensson, S., Angew. Chem. Int. Ed. Engl. (1970)9,610.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5 Amino A c i d Conjugates RALPH O. MUMMA and ROBERT H. HAMILTON Departments of Entomology and Biology, Pesticide Research Laboratory and Graduate Study Center, The Pennsylvania State University, University Park, Pa. 16802

Amino acid conjugates of pesticides have been reported in both plants and animals. Perhaps the most well known conjugates are the glycine conjugate found in animals (1-3) hippuric acid, was first isolated by Liebig in horse urine in 1829 (4). Knoop found in 1904 that long chain phenylalkyl acids fed to dogs were excreted as glycine conjugates of either phenylacetic acid or benzoic acid suggesting the existence of the β-oxidation pathway (5). Nicotinic acid also is excreted in urine of man as the glycine conjugate (nicotinuric) while phenylacetic acid is excreted as the glutamine conjugate (5). Birds excrete both these substances as the diornithine conjugates (5). Most of the pesticides that are recognized to form amino acid conjugates in plants are acidic insecticides, fungicides and herbicides but primarily the latter. The aspartic acid conjugate of indole-3-acetic acid, phenoxy herbicides and auxin-like plant growth regulators has been reported in plants by numerous laboratories (6-25). To further complicate the picture Feung et al. (26, 27) identified six additional amino acid conjugates (glutamic acid, alanine, valine, leucine, phenylalanine and tryptophan) of 2,4-D in soybean callus tissue. Figure 1 shows some examples of simple amino acid conjugates a l l involving con­ jugation through anα-amidebond. More complex amino acid conju­ gates have been reported such as the glutathione conjugate of triazines and diphenylether herbicides (28-33). However, in this case the glutathione is conjugated by means of a sulfur-carbon bond and its biochemical origin is different from amide linked amino acid conjugates. The glutathione conjugates are covered in more detail in another chapter (see Chapter by D. H. Hutson). Some other amino acid conjugates not linked through amide linkages with theα-aminogroup have been reported. For example 3-amino-1,2,3-triazole has been reported conjugated with alanine, glycine or serine (34) and alanine conjugates of N,N-dialkyldithio carbamates have also been reported (35).

68

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Amino Acid Conjugates

Although amino a c i d conjugates o f a u x i n type h e r b i c i d e s would now appear t o be commonly found I n p l a n t s ; t h e i r l e v e l s , mechanism of formation and b i o l o g i c a l s i g n i f i c a n c e remain t o be evaluated. With regard t o l e v e l s o f s p e c i f i c conjugates, Feung e t a l . (36) have shown that w h i l e 2,4-D amino a c i d conjugation was common t o f i v e p l a n t c a l l u s t i s s u e s , the k i n d and percentage o f each conjugate was species s p e c i f i c . I n a d d i t i o n the l e v e l s and amounts of conjugates a l s o v a r i e d w i t h time, thus when soybean c a l l u s was incubated w i t h 2,4-D the e t h e r - s o l u b l e metabolites (amino a c i d conjugates) a r e very r a p i d l y formed (Figure 2) but degrade w i t h time (8). This was p a r t i c u l a r l y s i g n i f i c a n t i n the case o f the glutamic and a s p a r t i c a c i d conjugates (Figure 3 ) . Other metabol i t e s accumulated i n longer exposure times a t the expense o f t h e glutamic and a s p a r t i c conjugates. Although no enzymatic conjug a t i n g system has y e t bee the a s p a r t i c conjugatio s i g n i f i c a n c e of these conjugates a l s o demands greater a t t e n t i o n s i n c e the 2,4-D amino a c i d conjugates a r e b i o l o g i c a l l y a c t i v e , they s t i m u l a t e p l a n t c e l l d i v i s i o n and c e l l e l o n g a t i o n a t concent r a t i o n s t y p i c a l of auxins (38). The chemical, p h y s i c a l and b i o l o g i c a l p r o p e r t i e s , the i s o l a t i o n , i d e n t i f i c a t i o n and a n a l y t i c a l methods f o r amino a c i d conjugates w i l l be d i s c u s s e d . Since t h i s r e p o r t i s t o r e f l e c t the s t a t e of the a r t of work w i t h amino a c i d conjugates, most o f the examples w i l l be taken from our own i n v e s t i g a t i o n s of amino a c i d conjugates o f 2,4-D. These data represent the most extensive i n v e s t i g a t i o n o f amino a c i d conjugates. Chemical and P h y s i c a l P r o p e r t i e s Most amino a c i d conjugates behave as weak a c i d s . They a r e s o l u b l e i n water under b a s i c c o n d i t i o n s and i n s o l u b l e under a c i d i c c o n d i t i o n s . A t pH 7 they a r e u s u a l l y s o l u b l e i n p o l a r organic s o l vents such as methanol, e t h a n o l , 1-butanol and acetone. At pH 3 or lower most amino a c i d conjugates a r e nonionized and e x t r a c t a b l e i n t o e t h y l ether. A l s o 1-butanol e x t r a c t s the conjugates out of water a t a l l pH's. Some amino a c i d conjugates complicate the e x t r a c t i o n procedure because o f being d i f u n c t i o n a l such as t h e d i c a r b o x y l i c amino a c i d s , glutamic and a s p a r t i c a c i d s , and t h e b a s i c amino a c i d s such as a r g i n i n e , l y s i n e , and h i s t i d i n e . The l a t t e r three are i o n i z e d o r z w i t t e r ions a t a l l pH s and do not e x t r a c t w i t h e t h y l ether. A t pH 3 four e x t r a c t i o n s a r e u s u a l l y necessary t o e f f e c t i v e l y e x t r a c t the amino a c i d conjugates out o f water w i t h e t h y l ether. The amino a c i d conjugates o f 2,4-D can be e a s i l y c r y s t a l l i z e d from aqueous-alcoholic s o l v e n t s under a c i d i c c o n d i t i o n s . A l l possess low v o l a t i l i t y and high m e l t i n g p o i n t s . Most o f these conjugates a r e r e l a t i v e l y s t a b l e i n a c i d and b a s i c s o l u t i o n s a t room temperatures. The amino a c i d conjugates a r e r e a d i l y hydrolyzed i n f

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

70

BOUND AND CONJUGATED PESTICIDE RESIDUES

« Cl-V

COOH

j~X.O C H . C - N H CIH

-

00H

V0CH C-NHCH CH

Cl-V

2

V

CH_

2

2,4-0-Asp

2,4-0-Gly

2,4-D-Phe

OOH

COOH Hippufic Acid

Figure 1.

IAA-ASP

Typical amino acid conjugates

O-

-i 5

1 10

1 15

INCUBATION

ETHER SOLUBLE FRACTION

1 20

1 25

1 30

I 35

TIME (DAYS)

Figure 2. Distribution of the radioactivity taken up by the soybean callus in water-soluble, ether-soluble (amino acid conjugates), and residue fraction

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

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AND HAMILTON

Amino Acid Conjugates

71

6 N HC1 a t 70°C i n 24 hours t o f r e e amino a c i d s and the parent acidic pesticide. Amino a c i d conjugates r e a d i l y form e s t e r s w i t h a l c o h o l s and t h i s sometimes causes problems i n the p u r i f i c a t i o n o f the i s o l a t e d m e t a b o l i t e s . Since methanol, e t h a n o l , and 1-butanol are used i n our i s o l a t i o n procedure and i n the chromatographic s o l v e n t s , s i g n i f i c a n t amounts o f these e s t e r s were i s o l a t e d and r e a d i l y detected by mass spectroscopy. A l l the amino a c i d conjugates o f 2,4-D that were t e s t e d were p a r t i a l l y hydrolyzed by Emulsin (27, 39) which i s a crude enzyme p r e p a r a t i o n used t o hydrolyze 3-glucosides. With these r e s u l t s one could assume the i s o l a t e d amino a c i d conjugates a c t u a l l y were B-glucosides, however, Emulsin hydrolyzed the s y n t h e t i c amino a c i d conjugates a l s o . E v i d e n t l y the enzymatic p r e p a r a t i o n contains s u f f i c i e n t peptidase t Nineteen amino a c i r e a c t i o n o f 2 , 4 - d i c h l o r o p h e n o x y a c e t y l c h l o r i d e w i t h the c o r r e s ponding L-amino a c i d i n aqueous sodium hydroxide (40) as i s shown i n F i g u r e 4. The N - l y s i n e conjugate was prepared i n a s l i g h t l y d i f f e r e n t manner. The b a s i c e-amino group o f l y s i n e was d e r l v a t i z e d t o a carbobenzoxy group which was e v e n t u a l l y removed by hydrogenation (42). A s l i g h t l y modified r e a c t i o n has been used t o prepare the amino a c i d conjugates of i n d o l e - 3 - a c e t i c a c i d (43). Since both paper and t h i n - l a y e r chromatography are so important i n the i s o l a t i o n , p u r i f i c a t i o n , and i d e n t i f i c a t i o n of amino a c i d conjugates, s p e c i a l emphasis must be placed on the proper s e l e c t i o n of good s o l v e n t systems. Since t h e o r e t i c a l l y n e a r l y twenty amino a c i d conjugates are p o s s i b l e , the chromatographic s o l v e n t system must be a b l e t o separate most o f the conjugates. Table I shows the m o b i l i t y of t e n s e l e c t e d amino a c i d conjugates of 2,4-D i n t h i n - l a y e r (TLC) and paper chromatographic (PC) s o l vent systems (42). V a l i n e , l e u c i n e and i s o l e u c i n e conjugates have s i m i l a r chromatographic p r o p e r t i e s and are d i f f i c u l t t o separate. Chromatographic techniques must be used f o r i d e n t i f i c a t i o n purposes when only t r a c e amounts (0.01 yg) mass s p e c t r o m e t r i c a n a l y s i s can be extremely u s e f u l . For example, n e a r l y a l l of the amino a c i d conjugates of 2,4-D o r of i n d o l e - 3 - a c e t i c a c i d gave molecular ions and c h a r a c t e r i s t i c fragmentation p a t t e r n s t y p i c a l o f both the amino a c i d and of the parent p e s t i c i d e (42, 43). The upper r e g i o n of the s p e c t r a (>m/e 219) i s c h a r a c t e r i s t i c of the s p e c i f i c conjugate and p a r t i c u l a r l y u s e f u l f o r i d e n t i f i c a t i o n purposes. F i g u r e 5 i l l u s t r a t e s the 01

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

72

BOUND

A N D CONJUGATED

PESTICIDE

RESIDUES

8 16 INCUBATION TIME (DAYS) Figure 3. Relative amounts of the ether-solubles isolated from soybean callus tissues grown for different times in 2,4-D-l- C. E * =» glutamic acid conjugate, E =- aspartic acid conjugate. 14

CI

Figure 4.

t

t

7

CI

General scheme for the synthesis of amino acid conjugates of 2,4-D

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

MUMMA

Table I .

AND HAMILTON

R

Amino Acid Conjugates

73

Values of Amino A c i d Conjugates of 2,4-D. Solvent System

Compound

I

II

Gly

0.00

0.27

Ala

0.35

Ser

5

TLC IV

V

VI

PC VII

0.00

0.17

0.00

0.80

0.68

0.33

0.48

0.20

0.54

0.77

0.74

0.14

0.17

0.12

0.11

0.35

0.73

0.65

Val

0.36

0.39

Leu

0.40

0.42

He

0.42

0.43

0.56

0.27

0.58

0.74

0.82

Asp

III

0.17

0.01

0.26

0.03

0.31

0.71

0.36

Glu

0.13

0.02

0.21

0.03

0.30

0.71

0.43

Phe

0.33

0.37

0.49

0.25

0.49

0.74

0.80

Trp

0.27

0.28

0.32

0.18

0.42

0.80

0.80

I, benzene-dioxane-formic a c i d (90:25:2, v / v / v ) ; I I , c h l o r o form-methanol-concentrated ammonium hydroxide (70:35:2, v / v / v ) ; I I I , d i e t h y l ether-petroleum ether (60-70°)-formic a c i d (70:30:2, v / v / v ) ; IV, benzene-triethylamine-methanol-concentrated ammonium hydroxide (85:15:20:2, by v o l ) ; V, benzene-methanol-cyclohexaneformic a c i d (80:10:20:2, by v o l ) ; V I , 1-butanol-acetic a c i d water (90:20:10, v / v / v ) ; and V I I , l-butanol-95% ethanol-3 N ammonium hydroxide (4:1:5, v / v / v ) .

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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AND CONJUGATED

PESTICIDE

RESIDUES

mass fragmentation (>m/e 219) of 2,4-D-Ile. I t gives a strong molecular i o n (53%, m/e 333) and fragments t y p i c a l of the amino a c i d . The main fragments >m/e 219 can be grouped i n t o four types as f o l l o w s : (a) p a r e n t - C l (P-35); (b) P-COOH (P-45); (c) P-H 0 (P-18); and (d) P-side chain fragmentation. The s i d e chain fragmentation i s s i m i l a r to the s i d e chain fragmentation p r e v i ously reported f o r peptides and d e r i v a t i v e s and are c h a r a c t e r i s t i c of the amino a c i d (44). 2

Figure 6 shows the prominent mass s p e c t r a l ions a r i s i n g from the fragmentation of the 2,4-D p o r t i o n of the molecule and are t y p i c a l f o r a l l the amino a c i d conjugates as w e l l as 2,4-D (42). The presence of the c h a r a c t e r i s t i c c h l o r i n e isotope peaks permits i d e n t i f i c a t i o n of metabolites even when s i g n i f i c a n t i m p u r i t i e s are present. I s o l a t i o n , P u r i f i c a t i o n and I d e n t i f i c a t i o n In our hands the procedure of the e x t r a c t i o n of the p l a n t t i s s u e and the time involved i n t h i s procedure depends upon the t i s s u e being examined. P e s t i c i d e metabolism s t u d i e s with p l a n t c a l l u s t i s s u e s o f f e r s many advantages over using the whole p l a n t . C a l l u s t i s s u e does not r e q u i r e a l i g h t e d growth chamber. I t i s s t e r i l e , uses inexpensive equipment, r e q u i r e s l i t t l e space, u s u a l l y does not c o n t a i n many i n t e r f e r i n g substances and o f f e r s v e r s a t i l i t y i n comparing metabolism i n d i f f e r e n t p l a n t s a t the same time by using d i f f e r e n t p l a n t c a l l u s t i s s u e . Whole p l a n t s , on the other hand, do r e q u i r e c o n t r o l l e d environmental growth chambers or greenhouse space and c o n t a i n s i g n i f i c a n t i n t e r f e r i n g phenolic substances and pigments. Obtaining s t e r i l e i n t a c t p l a n t s i s a l s o not u s u a l l y f e a s i b l e and r e s t r i c t s the method of treatment i f m i c r o b i a l metabolism i s to be avoided. U s u a l l y we can i s o l a t e and i d e n t i f y a metabolite from p l a n t c a l l u s t i s s u e i n 1/5 to 1/15 the time i t takes to work with the whole p l a n t . The l a r g e amount of p l a n t pigments and sugars o f t e n causes s t r e a k i n g of chromatograms and thus does not give good separations as i s t y p i c a l of c a l l u s t i s s u e . Therefore, most of our i d e n t i f i c a t i o n of metabolites have been performed with p l a n t c a l l u s t i s s u e s . However, once the metabolites have been i d e n t i f i e d the r e l a t i v e amount and types of metabolites must be

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

MUMMA

AND

0 Cl-^

HAMILTON

Amino

Acid

75

Conjugates

COOH

VoCH,C-NHCH

m/e 298 100 %(base)

/=\

HCH Cl-(^J>-OCH C-NHCf CH, m/e 252 14% CH.

-CI

Cl-f'

° NHCH 2

VOCH^C-NHCH

m/e 3 3 3 53% -C H 4

0

C l

CI I—

8

COOH

OCHXNHCH l H m/e 2 7 7 14%

Figure 5.

Figure 6.

2

Prominent mass spectral ions arising from fragmentation of 2,4-D-Ile >219)

(m/e

Prominent mass spectral ions arising from the fragmentation of the 2,4-D portion of the molecule of amino acid conjugates of 2,4-D

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

76

BOUND

A N D CONJUGATED

PESTICIDE RESIDUES

determined i n the whole p l a n t i n order to determine the s i g n i f i cance of the metabolites. Figure 7 shows the i s o l a t i o n scheme of amino a c i d conjugates of 2,4-D from p l a n t c a l l u s t i s s u e . The f r o z e n t i s s u e was homogenized i n 95% ethanol i n a Waring Blendor. The homogenate was f i l t e r e d with s u c t i o n , and the r e s i d u e r i n s e d thoroughly with 80% ethanol. The pooled f i l t r a t e was evaporated and the aqueous concentrate (adjusted to pH 3.0) was e x t r a c t e d four times with e t h y l ether. The aqueous l a y e r contains the g l u c o s i d i c conjugates i n c l u d i n g the hydroxylated m e t a b o l i t e s . The water f r a c t i o n was then u s u a l l y extracted four times with equal volumes of 1-butanol (water s a t u r a t e d ) . The e t h y l ether f r a c t i o n contains the amino a c i d conjugates of 2,4-D as w e l l as any f r e e 2,4-D. The e t h y l ether e x t r a c t was concentrated and the components separated on paper chromatography. Th the paper chromatogram The solvent systems that we found best s u i t e d f o r our work are given i n Table I . The t h i n - l a y e r tanks always contained a paper l i n e r which does a f f e c t the m o b i l i t y of the s o l v e n t system and the s i l i c a g e l l a y e r was a c t i v a t e d (135°C f o r 4-8 h r ) . Figure 8 shows a t y p i c a l s e p a r a t i o n on paper chromatograms of whole p l a n t and c a l l u s t i s s u e e x t r a c t s . As i n d i c a t e d the c a l l u s t i s s u e e x t r a c t s give much b e t t e r r e s o l u t i o n . U s u a l l y only one a d d i t i o n a l t h i n l a y e r chromatographic separation of each eluted band i s necessary to o b t a i n s u f f i c i e n t p u r i t y f o r mass s p e c t r a l a n a l y s i s . A l l compounds e l u t e d from r a d i o a c t i v e bands are subjected to a c i d hyd r o l y s i s (6 N HC1, 70°C, 24 h r ) , enzymatic h y d r o l y s i s (Emulsin, N u t r i t i o n a l Biochemical Company) and chromatographic c h a r a c t e r i z a t i o n i n a l l the l i s t e d t h i n - l a y e r and paper chromatographic s o l v e n t s , i n c l u d i n g comparison with standard s y n t h e t i c compounds. Following t h i s procedure the sample i s analyzed i n a mass s p e c t r o meter v i a a s o l i d probe i n l e t . Unfortunately mass spectrometric a n a l y s i s destroys the sample and r e q u i r e s a r e l a t i v e l y pure f i n i t e (>0.01 yg) amount of compound. T h i s amount of m a t e r i a l i s sometimes hard to o b t a i n when metabolites are present i n small q u a n t i t i e s . When i n s u f f i c i e n t amounts of sample are a v a i l a b l e f o r mass spectrometric a n a l y s i s , i d e n t i f i c a t i o n must be based p u r e l y upon chromatographic data. Since the c o n c e n t r a t i o n of amino a c i d conjugates i n the c a l l u s t i s s u e v a r i e s g r e a t l y with time of exposure (8) i t i s des i r a b l e to determine the c o n c e n t r a t i o n of metabolites a f t e r s e v e r a l time i n t e r v a l s as i s i l l u s t r a t e d i n Figure 2 f o r soybean c a l l u s t r e a t e d with 2,4-D. The e t h e r - s o l u b l e f r a c t i o n (conjugates) decreases with time while the water-soluble metabolites i n c r e a s e . In f a c t the major conjugates (glutamate and aspartate) both i n crease and l a t e r decrease over d e f i n i t e time i n t e r v a l s , f i r s t the glutamic and then the a s p a r t i c (Figure 3). A comparison of the metabolism of 2,4-D (Table II) shows the r e l a t i v e amount of 2,4-D-Asp and 2,4-D-Glu i n the e t h y l ether e x t r a c t of s i x d i f f e r e n t p l a n t c a l l u s t i s s u e s ( c a r r o t , jackbean,

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

MUMMA

AND

HAMILTON

77

Amino Acid Conjugates

C a l l u s Tissue (2,4-D) 1.

Ethanol homogenization

RESIDUE

ETHANOL EXTRACT 1. 2.

Concentrate E t h y l ether, pH 3

ETHER EXTRACT (2,4-D and amino a c i d conjugates)

WATER SOLUBLE

l-Butanol Extract (mostly glycosides)

water saturated 1-butanol

Water soluble

Figure 7. Scheme of isolation of amino acid conjugates of 2,4-D from plant callus tissue

Figure 8. Radioautographu of decending paper chromatograms of ether-soluble (pH 3.0) metabolites of 2,4-D-l* C isolated from soybean plant (A) and soybean callus tissue (B). Solvent system: 1-butanol-ethanol (95% )-ammonium hydroxide (3N) (4:1.25:1, v/v/v); Whatman No. 1 paper. 4

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

78

BOUND AND CONJUGATED PESTICIDE RESIDUES

soybean, sunflower, tobacco and corn) (36). Once the m e t a b o l i t e s have been thoroughly c h a r a c t e r i z e d i n the c a l l u s t i s s u e , i t i s e a s i e r t o recognize and q u a n t i f y them i n the whole p l a n t e x t r a c t s . A l l whole p l a n t s and p l a n t c a l l u s t i s s u e examined i n our l a b o r a t o r i e s to date, contained some amino a c i d conjugates but i n v a r y i n g amounts.

Table I I . R e l a t i v e Percentage of 2,4.-D-Asp and 2,4-D-Glu i n S i x C a l l u s Tissues Incubated w i t h 2,4-D f o r 8 Days Callus Tissue

2,4-D-Asp

2 4-D-Glu f

0

23.8

Soybean

3.7

12.9

Sunflower

0

5.0

Tobacco

0.8

6.7

Corn

1.6

1.3

Carrot Jackbean

Biological

P r o p e r t i e s and Metabolism

U n f o r t u n a t e l y the l i t e r a t u r e does not c o n t a i n many examples where the b i o l o g i c a l a c t i v i t y of amino a c i d conjugates has been determined. Twenty amino a c i d conjugates of 2,4-D and s e v e r a l amino a c i d conjugates of i n d o l e - 3 - a c e t i c a c i d have been r e p o r t e d to possess b i o l o g i c a l a c t i v i t y (38, 39). They s t i m u l a t e p l a n t c e l l d i v i s i o n and c e l l e l o n g a t i o n (38, 39). Table I I I i n d i c a t e s the e l o n g a t i o n of Avena c o l e o p t i l e s e c t i o n s and Table IV the s t i m u l a t i o n of soybean cotyledon c a l l u s t i s s u e induced by s e l e c t e d amino a c i d conjugates of 2,4-D. As i n d i c a t e d i n these Tables the amino a c i d conjugates of 2,4-D are b i o l o g i c a l l y a c t i v e a t p h y s i o l o g i c a l c o n c e n t r a t i o n s ( 1 0 ~ - 1 0 ~ M) and i n some cases considerably more a c t i v e than 2,4-D. Their p h y s i o l o g i c a l e f f e c t i s theref o r e t y p i c a l of the e f f e c t of the parent h e r b i c i d e . At higher than p h y s i o l o g i c a l c o n c e n t r a t i o n these amino a c i d conjugates possess h e r b i c i d a l p r o p e r t i e s and the D-amino a c i d conjugates of 2,4-D have been observed t o s t i m u l a t e f r u i t growth (41). I n a d d i t i o n we have determined the t o x i c o l o g y of a number of L-amino a c i d conjugates of 2,4-D i n r a t s and showed t h e i r L D 5 0 t o be s i m i l a r to that of 2,4-D. 6

7

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

MUMMA

AND HAMILTON

Table I I I .

79

Amino Acid Conjugates

Growth of Soybean Cotyledon C a l l u s T i s s u e Induced by Amino A c i d Conjugates of 2,4-D R e l a t i v e Percent Greater or• Less than

2,4-D or Conjugate

5

10" M

7

10" M

6

10" M

C o n t r o l (no a d d i t i v e )

2,4-D 8

10- M

A l l Died

2,4-D-Gly

+13

- 4

-14

- 35

2,4-D-Glu

+56

+29

+53

+ 85

2,4-D-Leu

+26

2,4-D-Phe

+92

+93

+ 9

+110

Table IV.

E l o n g a t i o n of Avena c o l e o p t i l e Sections Induced by Amino A c i d Conjugates of 2,4-D

2,4-D or Conjugate

% Elongation 8

10" M

10- M

10- M

10" M

2,4-D

39

74

45

39

2,4-D-Asp

57

35

26

22

2,4-D-Ile

45

55

47

24

2,4-D-Phe

49

59

32

26

2,4-D-Try

66

41

24

22

5

6

7

The amino a c i d conjugates are capable of being metabolized to other b i o l o g i c a l l y a c t i v e compounds (8). 2,4-D-Glu i s metabolized by soybean c a l l u s t i s s u e to 2,4-D, 2,4-D-Asp and to the hydroxy1ated metabolites; 4-hydroxy-2,5-dichlorophenoxyacetic a c i d and 4-hydroxy-2,3-dichlorophenoxyacetic a c i d . I n t e r e s t i n g l y 2,4-D-Glu i s more r a p i d l y metabolized by soybean c a l l u s t i s s u e than i s 2,4-D, (Table V) e s p e c i a l l y to the hydroxylated m e t a b o l i t e s . Of s p e c i a l note i s that 2,4-D-Glu i s metabolized to other amino a c i d conjugates, p a r t i c u l a r l y the a s p a r t i c conjugate.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

80

BOUND

Table V.

A N D CONJUGATED

R e l a t i v e Percentage Metabolites of 2,4-D Incubated with Soybean C a l l u s Tissue

Ether Soluble Metabolite

RESIDUES

and 2,4-D-Glu

Water Soluble

% In T i s s u e * 2,4-D

Metabolite

2,4-D-Glu

2,4-D-Asp

3.7

11.7

2,4-D-Glu

12.9

6.7

2,4-D

33.7

6.0

Others

11.9

1.9

Total

PESTICIDE

% In T i s s u e * 2,4-D

(4-OH-2,5-D, 4-0H-2,3-D)

2,4-D-Glu

26.3

54.9

2,4-D

0.8

4.2

Others

6.7

11.7

62.2

*2,4-D Incubated 12 days, 2,4-D-Glu Incubated 8 days. A n a l y t i c a l Methods Although amino a c i d conjugates of p e s t i c i d e s have been i s o l a t e d f o r many years no comprehensive i n v e s t i g a t i o n has been reported concerning the development of a n a l y t i c a l methods f o r these compounds. Recently, i n t h i s l a b o r a t o r y Arjmand (45) developed an a n a l y t i c a l method f o r the a n a l y s i s of nineteen metabolites of 2,4-D i n c l u d i n g the amino a c i d conjugates. T h i s technique i n volved the gas chromatographic a n a l y s i s of the t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s . He showed that s i x t e e n amino a c i d conjugates could be separated and q u a n t i f i e d when analyses were performed i n two separate columns (OV-1 and OV-17 s t a t i o n a r y phases) with temperature programming c o n d i t i o n s . A t y p i c a l separation i s shown i n Figure 9. The proper d e r i v a t i z a t i o n reagent and c o n d i t i o n s were found to be important. Hexamethyldisilazane gave monosilyl a t e d amino a c i d conjugates while more stronger s i l y l a t i n g r e a c t i o n c o n d i t i o n s always r e s u l t e d i n a mixture of mono- and d i s i l y l a t e d products. A l l the TMS d e r i v a t i v e s of the amino a c i d conjugates of 2,4-D were s t a b l e and gave a l i n e a r response with a flame i o n i z a t i o n detector i n the range of 1-10 yg. Unfortunately e l e c t r o n capture detectors were not a p p l i c a b l e s i n c e temperature program c o n d i t i o n s were employed and the TMS d e r i v a t i v e s do not work w e l l with t h i s d e t e c t o r . Figure 10 shows a t y p i c a l GLC s e p a r a t i o n of the ether e x t r a c t of soybean c a l l u s t i s s u e f o r t i f i e d with 30 ppm amino a c i d conjugates and hydroxylated 2,4-D metabolites. Unfortunately the percentage recovery of the amino a c i d conjugates from the f o r t i f i e d c a l l u s t i s s u e v a r i e d g r e a t l y as evidenced i n Table VI.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

MUMMA

i 0

•

1 2

1

1 4

1

81

Amino Acid Conjugates

AND HAMILTON

1 6

1

1

•

1

8

1

1

10

1

1

12

' 14

r 16

T I M E (min) Figure 9. GLC separation of TMS derivatives of 2,4-D metabolites and amino acid conjugates. Column: 1% OV-17 on 80/100 mesh Supelcoport, 6' X 4 mm i.d. glass. Temperature programmed at 5°/min up to 280°C, initial temperature 180°C.

1 0

1

1 2

1

1 4

'

1 6

1

1 8

'

1 10

1

1

' 12

1 14

1

1

'

1

16

r

18

TIME (min) Figure 10. GLC of ether extract of soybean callus tissue fortified with 2,4-D metabolites and amino acid conjugates. Lower tracing is control tissue extract without fortification. Column: 2% OV-1 on 100/120 mesh Supelcoport, 6' X 4 mm i.d. glass. Temperature programmed at 5°/min up to 280°C, initial temperature 180°C.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

82

Recovery ranged from 18.5 t o 91.4%. No i n v e s t i g a t i o n was conducted on ways t o improve the recovery which o b v i o u s l y needs f u r t h e r study.

Table VI.

Percentage Recovery o f 2,4-D-Conjugates from Soybean C a l l u s Tissue F o r t i f i e d w i t h 30 ppm each

Compound

ppm Recovered

% Recovery

2,4-D

25.6

85.49

2,4-D-Ala

21.7

72.33

2,4-D-Val

27.4

91.40

2,4-D-Leu 2,4-D-Asp 2,4-D-Phe

5.6 21.6

18.53 71.88

Discussion Amino a c i d conjugates a r e o b v i o u s l y more wide spread i n p l a n t t i s s u e than once e n v i s i o n e d . A s p a r t i c a c i d conjugates have been found t o be most abundant, however, conjugates w i t h glutamic a c i d , a l a n i n e , v a l i n e , l e u c i n e , phenylalanine and tryptophan have been i d e n t i f i e d . Probably as more t i s s u e s and p l a n t s a r e examined conjugates w i t h a d d i t i o n a l amino a c i d s w i l l be found. Since d i f f e r e n t p l a n t t i s s u e s c o n t a i n d i f f e r e n t concentrations of amino a c i d conjugates, perhaps the c o n c e n t r a t i o n o f the conjugate i n the t i s s u e r e f l e c t s a f r e e amino a c i d p o o l s i z e and should be examined f u r t h e r . Since i t i s now c l e a r t h a t the glutamic a c i d conjugate i s a major m e t a b o l i t e , a number of r e p o r t s of the a s p a r t i c a c i d conjugates must be examined c r i t i c a l l y e s p e c i a l l y s i n c e i t i s d i f f i c u l t t o separate the glutamic and a s p a r t i c conjugates by chromatography. Although i t seems c l e a r that amino a c i d conjugates a r e important i n p l a n t c a l l u s t i s s u e , j u s t how s i g n i f i c a n t these compounds are i n the whole p l a n t remains t o be proved. U n f o r t u n a t e l y , almost a l l of the 2,4-D metabolism s t u d i e s w i t h c a l l u s t i s s u e has been performed a t p h y s i o l o g i c a l concentrations and metabolism of 2,4-D a t h e r b i c i d a l concentrations might be s i g n i f i c a n t l y d i f f e r e n t . The c o n c e n t r a t i o n o f amino a c i d conjugates found i n soybean c a l l u s t i s s u e e x h i b i t e d a temporal r e l a t i o n s h i p . The highest c o n c e n t r a t i o n was found the f i r s t day of exposure of the p l a n t t o 2,4-D and the amino a c i d conjugates s t e a d i l y decreased w i t h a concomltment i n c r e a s e i n the c o n c e n t r a t i o n of the n o n b i o l o g i c a l l y

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

MUMMA

AND HAMILTON

Amino Acid Conjugates

83

a c t i v e hydroxylated m e t a b o l i t e s . Whether a s i m i l a r temporal r e l a t i o n s h i p e x i s t s i n the whole p l a n t remains t o be determined. Although a l l the amino a c i d conjugates of 2,4-D possess a u x i n - l i k e p r o p e r t i e s , whether they express these p r o p e r t i e s as the amino a c i d conjugate o r some d e r i v a t i v e o r hydrolyzed product i s not known. The evidence would suggest that perhaps the conjugates may have i n v i v o b i o l o g i c a l a c t i v i t y . The f a c t that they are so r a p i d l y formed and s t i m u l a t e p l a n t c e l l d i v i s i o n (at p h y s i o l o g i c a l c o n c e n t r a t i o n s ) i n excess o f that of 2,4-D i s very suggestive. Venis (37) has shown the amino a c i d c o n j u g a t i o n o f i n d o l e - 3 - a c e t i c a c i d and other aromatic a c i d s i s c a t a l y z e d by an auxin (2,4-D, naphthyl a c e t i c a c i d , i n d o l e - 3 - a c e t i c a c i d ) i n d u c i b l e enzyme. I t i s i n t e r e s t i n g that the s t r u c t u r a l r e q u i r e ments f o r i n d u c t i o n are more s p e c i f i c than the s u b s t r a t e r e q u i r e ments. Since 2,4-D-Gl c o n c e n t r a t i o n s than 2,4may possess an enzyme capable of c a t a l y z i n g the d i r e c t conversion of 2,4-D-Glu t o 2,4-D-Asp. The hydroxylated m e t a b o l i t e s are more r a p i d l y formed from 2,4-D-Glu than from 2,4-D thus r a i s i n g the q u e s t i o n , can the amino a c i d conjugates be d i r e c t l y hydroxylated and i f so are they r e q u i r e d f o r h y d r o x y l a t i o n ? To our knowledge the i n v i t r o h y d r o x y l a t i o n o f 2,4-D has not been demonstrated i n a c e l l f r e e system and warrants f u r t h e r i n v e s t i g a t i o n s employing amino a c i d conjugates as s u b s t r a t e s . U s u a l l y the hydroxylated 2,4-D m e t a b o l i t e s are present as the g l u c o s i d e s , however, s m a l l amounts of the f r e e aglycone were reported i n bean p l a n t s ( 8 ) . Amino a c i d conjugates of hydroxylated 2,4-D m e t a b o l i t e s have not been r e p o r t e d , however, we do have p r e l i m i n a r y evidence f o r t h e i r occurrence. A s i g n i f i c a n t q u a n t i t y o f 2,4-D i s e v i d e n t l y present as a glucose e s t e r ( 7 ) . The glucose e s t e r s of the amino a c i d conjugates have not y e t been r e p o r t e d , however, i t i s q u i t e p o s s i b l e that they may e x i s t . Small amounts o f the amino a c i d conjugates are o f t e n found i n the water s o l u b l e f r a c t i o n a f t e r Emulsin treatment. The amino a c i d conjugates would undoubtedly possess d i f f e r e n t p e r m e a b i l i t i e s than the parent p e s t i c i d e t o cytoplasmic and subc e l l u l a r membranes. Thus, the b i o l o g i c a l a c t i v i t y and r a p i d metabolism of the amino a c i d conjugates might be owing t o t h e i r more r a p i d p e n e t r a t i o n o f the c e l l than the parent p e s t i c i d e , which r e s u l t s i n an accumulation a t the t a r g e t s i t e s o f b i o l o g i c a l a c t i v i t y and metabolism. Although o n l y the amino a c i d conjugates o f 2,4-D and i n d o l e 3 - a c e t i c a c i d have so f a r been s t u d i e d i n depth, probably other amino a c i d conjugates are e q u a l l y important. P o s s i b l y a l l the a c i d i c a u x i n - l i k e h e r b i c i d e s form amino a c i d conjugates and need to be reexamined i n l i g h t of c u r r e n t t h i n k i n g . A d d i t i o n a l s t u d i e s a r e needed t o determine i f the d i f f e r e n t h e r b i c i d a l d e r i v a t i v e s o f 2,4-D, such as the amine s a l t s , the b u t y l e s t e r and the butoxyethanol e s t e r , a l s o g i v e r i s e t o the

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amino a c i d conjugates when a p p l i e d to p l a n t s . The metabolism of 2,4-D has been examined i n only a few p l a n t s and a d d i t i o n a l p l a n t s should be i n v e s t i g a t e d . These data c o l l e c t i v e l y demonstrate the wide d i s t r i b u t i o n of amino a c i d conjugates, t h e i r b i o l o g i c a l s i g n i f i c a n c e and p l a n t species s p e c i f i c i t y . Hopefully these r e s u l t s w i l l stimulate other i n v e s t i g a t o r s to be more aware of amino a c i d conjugates. A n a l y t i c a l methods should be modified so that amino a c i d and e s t e r conjugates can be determined i n residue work. F i n a l l y the use of s t e r i l e p l a n t t i s s u e c u l t u r e s o f f e r s many advantages f o r metabolism s t u d i e s .

Literature Cited 1. Knaak, J. B., Sullivan 16, 454. 2. Wit, J. G., Van Genderen, H., Biochem. J. (1966) 101, 698. 3. Lethco, E. J., Brouwer, E. A., J. Agr. Food Chem. (1966) 14, 532. 4. Fieser, L. F., Fieser, M., "Organic Chemistry", p. 2, Reinhold Publishing Corp., New York, 1956. 5. Fruton, J. S., Simmonds, S., "General Biochemistry", John Wiley & Sons, New York, 1953. 6. Andrea, W. A., Good, N. E., Plant Physiol. (1961) 32, 566. 7. Klämbt, H. D., Planta (1961) 57, 339. 8. Feung, C. S., Hamilton, R. H., Witham, F. H., Mumma, R. O., Plant Physiol. (1972) 50, 80. 9. Andrea, W. A., Good, N. E., Plant Physiol. (1955) 30, 380. 10. Andrea, W. A., van Ysselstein, M. W. H., Plant Physiol. (1956) 31, 235. 11. Andrea, W. A., van Ysselstein, N. W. H., Plant Physiol. (1960) 35, 225. 12. Good, N. E., Andrea, W. A., van Ysselstein, N. W. H., Plant Physiol. (1956) 31, 321. 13. Fang, S. C., Theisen, P., Butts, J. S., Plant Physiol. (1959) 34, 26. 14. Bennet-Clark, T. A., Wheeler, A. W., J. Exp. Botany (1959) 10, 468. 15. Thurman, D. A., Street, H. E., J. Exp. Botany (1962) 13, 369. 16. Wightman, F., Can. J. Botany (1962) 40, 689. 17. Zenk, M. H., Collow. Intern. Centre, Nat. Recherche Sci., Paris (1963) 123, 241. 18. Sudi, J., Nature (1964) 201, 1009. 19. Sudi, J., N. Phytotologist (1966) 65, 9. 20. Winter, A., Thimann, K. V., Plant Physiol. (1966) 41, 335. 21. Olney, H. O., Plant Physiol. (1968) 43, 293. 22. Robinson, B. J., Forman, M., Addicott, F. T., Plant Physiol. (1968) 43, 1321. 23. Morris, D. A., Briant, R. E., Thomson, P. G., Planta (1969) 89, 178.

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24. Beyer, E. M., Morgan, P. W., Plant Physiol. (1970) 46, 157. 25. Lau, O. L., Murr, D. P., Yang, S. F., Plant Physiol. (1974) 54, 182. 26. Feung, C. S., Hamilton, R. H., Witham, F. H., J. Agr. Food Chem. (1971) 19, 475. 27. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1973) 21, 637. 28. Frear, D. S., Swanson, H. R., Pesticide Biochem. and Physiol. (1973) 3, 473. 29. Shimabukuro, R. H., Lamoureux, G. L., Swanson, H. R., Walsh, W. C., Stafford, L. E., Frear, D. S., Pesticide Biochem. and Physiol. (1973) 3, 483. 30. Shimabukuro, R. H., Swanson, H. R., Walsh, W. C., Plant Physiol. (1970) 46, 103. 31. Shimabukuro, R. H. W. C., Plant Physiol 32. Lamoureux, G. L., Stafford, L. E., Shimbukuro, R. H., Zaylskie, R. G., J. Agr. Food Chem. (1973) 21, 1020. 33. Shimabukuro, R. H., Walsh, W. C., Lamoureux, G. L., Stafford, L. E., J. Agr. Food Chem. (1973) 21, 1031. 34. Carter, M. C., Physiol. Plant (1965) 18, 1054. 35. Kaslander, J., Sijpesteijn, A. K., Van Der Kerk, G. J. M., Biochim. Biophys. Acta (1962) 60, 417. 36. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1975) 23, 373. 37. Venis, M. A., Plant Physiol. (1972) 49, 24. 38. Feung, C. S., Mumma, R. O., Hamilton, R. H., J. Agr. Food Chem. (1974) 22, 307. 39. Hamilton, R. H., Feung, C. S., Myer, H. E., Mumma, R. O., Fifth Annual Meeting American Society Plant Physiologists, June 20, Cornell University, Ithaca, New York, 1974. 40. Wood, J. W., Fontaine, T. D., J. Org. Chem. (1952) 17, 891. 41. Wood, J. W., Fontaine, T. D., Mitchell, J. W., U. S. Patent No. 2,734,816 (1956). 42. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1973) 21, 632. 43. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1975) in press 44. Biemann, K., Cone, C., Webster, B. R., J. Amer. Chem. Soc. (1966) 88, 2597. 45. Arjmand, M., "Quantitative Gas-Liquid Chromatographic Analysis of 2,4-D Metabolites", Masters Thesis, 1975, The Pennsylvania State University, University Park, Pa.

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6 Sulfate Ester Conjugates—Their Synthesis, Purification, Hydrolysis, and Chemical Spectral Properties G. D. PAULSON Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Fargo, N. Dak. 58102

Since the early repor phenol to phenyl sulfat developed about sulfate ester conjugation. Sulfoconjugates are a very diverse and widespread group of compounds that are found in microorganisms, plants (2), Insect (3-5), mammals, birds, reptiles, amphibia, arthropods, and mollusks (6). Dodgson and Rose (7) have classified these compounds in the following way: 1) compounds with P-O-SO- linkages; 2) compounds with C-O-SO3" linkages; 3) compounds with N-SO3- linkages; 4) compounds with N-O-SO- linkages; and 5) compounds with S-SO3- linkages. This review is restricted primarily to compounds with the C-O-SO3linkage, and particularly to aryl sulfates because these are most commonly encountered by the pesticide chemist and have been most extensively investigated. Some aspects of compounds with the P-O-SO- and N-O-SO- linkages will be discussed as they apply to xenobiotic metabolism. 3

3

3

3

Biosynthesis and Metabolic Fate The early studies by DeMeio (8), DeMeio and Tkacz (9), DeMeio et al. (10), DeMeio et al. (11), Bernstein and McGilvery (12, 13), Segal (14), and others demonstrated that phenols were converted to aryl sulfates by the soluble fraction of rat liver homogenates when incubated with sulfate ions, ATP, and Mg ions. Bernstein and McGilvery (12, 13) and Segal (14) discovered that an active sulfate was formed from ATP and sulfate ion and that the active sulfate reacted with a phenol to give phenyl sulfate. Robbins and Lipmann (15-17) showed that the active sulfate was adenosine-3'-phosphate-S'-phosphosulfate (PAPS), and that two enzymes were involved in the formation of PAPS (15, 16). The first enzyme, ATP-sulfurylase (ATP:sulfate adenyItransferase, 2.7.7.4) catalyzes the reaction of ATP andSO42-ion to give adenosine-5'-phosphosulfate (APS) and pyrophosphate, and the second enzyme APS-kinase (ATP:adenyl sulfate 3'-phosphotransferase, 2.7.1.25) catalyzes the phosphorylation of APS to give PAPS. Baddiley et al. (18, 19) 2+

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confirmed t h e s t r u c t u r e o f both APS and PAPS by s y n t h e s i s . More d e t a i l e d d i s c u s s i o n s o f these and r e l a t e d s t u d i e s are a v a i l a b l e (7, 20-24). The thermodynamically unfavorable formation o f APS (AF • +11 kCal) from ATP and s u l f a t e i s d r i v e n by the h y d r o l y s i s o f pyrophosphate and by t h e r a p i d u t i l i z a t i o n o f APS by APS-kinase (7, 2£, 22). A T P - s u l f u r y l a s e p u r i f i e d from yeast r e q u i r e s M g i o n and i t s pH optimum i s 7.5-9.0. However, Dodgson and Rose (7) d i s cussed species d i f f e r e n c e s and s t r e s s e d the importance o f s e v e r a l v a r i a b l e s when assaying f o r the s u l f a t e a c t i v a t i n g systems i n other p r e p a r a t i o n s . Roy (20, 21) reviewed the methods a v a i l a b l e f o r assay o f A T P - s u l f u r y l a s e a c t i v i t y , i n h i b i t o r s o f t h i s enzyme, and s t u d i e s on p u r i f i c a t i o n o f t h i s enzyme. The conversion o f APS t o PAPS by the APS kinase i s o l a t e d from yeast i s e s s e n t i a l l y i r r e v e r s i b l i o n ; t h e optimum pH f o apparently no d e t a i l e d i n f o r m a t i o n on t h i s enzyme i n animal s y s tems although i t i s presumed t o be present i n a l l t i s s u e s t h a t form PAPS (21). Assay techniques and other s t u d i e s on t h i s enzyme, as w e l l as t h e p r o p e r t i e s o f APS and PAPS, have been reviewed by Roy (20, 21) and Dodgson and Rose (7). Recently, Wong (25) r e ported a new method f o r measuring the a c t i v i t y o f the enzymes that generate PAPS and o f the t r a n s f e r a s e enzymes; i t was p o s t u l a t e d that ATP acts as an a l i o s t e r i c m o d i f i e r o f one o f the enzymes r e s p o n s i b l e f o r t h e s y n t h e s i s o f PAPS. The a b i l i t y t o s y n t h e s i z e PAPS (an e n e r g e t i c a l l y expensive process f o r t h e organism) i s common t o a wide v a r i e t y o f p l a n t s , animals, and microorganisms. The involvement o f PAPS i n s u l f a t e r e d u c t i o n , s u l f a t e t r a n s p o r t , s u l f o c o n j u g a t l o n o f carbohydrates, and g l y c o l i p i d s and i n many other d i v e r s e metabolic r e a c t i o n s ( 7 , 20, 21) i s beyond t h e scope o f t h i s review. Rather, t h i s d i s cussion w i l l be r e s t r i c t e d p r i m a r i l y t o t h e involvement o f PAPS and s u l f o t r a n s f e r a s e s i n the b i o s y n t h e s i s o f a r y l s u l f a t e e s t e r s and r e l a t e d compounds formed i n the metabolism o f x e n o b i o t i c s . The b i o s y n t h e s i s o f a r y l s u l f a t e e s t e r s i s accomplished by t r a n s f e r r i n g t h e s u l f a t e group i n PAPS t o a receptor (ROH) t o form ROSO3". Evidence f o r t h e formation o f d i s u l f a t e conjugates o f d i - and t r i - h y d r i c phenols has been reported (26). I n some cases, amines but not t h i o l s can s u b s t i t u t e f o r ROH as acceptors (21). Apparently there i s no c o n c l u s i v e evidence f o r the b i o s y n t h e s i s o f s u l f a t e e s t e r s o f hydroxylamines i n v i v o (which may be due t o the inherent i n s t a b i l i t y o f these compounds); however, the evidence f o r t h e formation and t r a n s i e n t e x i s t e n c e of such compounds i n v i t r o i s c o n v i n c i n g (27-31). Apparently, no one has i s o l a t e d a s u l f o t r a n s f e r a s e i n pure form (21), but i t i s w e l l e s t a b l i s h e d t h a t there are many d i f f e r ent s u l f o t r a n s f erase enzymes 07, 20^, 2JL, 32-39) . Some o f the s u l f o t r a n s f e r a s e s apparently have a high degree o f s u b s t r a t e s p e c i f i c i t y but t h i s c o n c l u s i o n must be v e r i f i e d w i t h p u r i f i e d enzymes• ?

2 +

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The i n t r a c e l l u l a r l o c a t i o n o f the s u l f a t e a c t i v a t i n g and s u l f o t r a n s f erase enzymes has not been s t u d i e d f o r many t i s s u e s , but i n t h e l i v e r they are present i n the s o l u b l e f r a c t i o n o f the c e l l . Although s u l f a t e e s t e r formation has been most e x t e n s i v e l y s t u d i e d i n mammalian l i v e r , many t i s s u e s , i n c l u d i n g t h e kidney, i n t e s t i n e , b r a i n , a d r e n a l , mast c e l l s , ovary, and t e s t i s , a l s o have the a b i l i t y t o synthesize PAPS (21) . Powell e_t a l . (40) demonstrated that the gut o f t h e r a t r a p i d l y converted phenol t o phenyl s u l f a t e and have questioned t h e b e l i e f o f others t h a t the l i v e r i s the major organ i n v o l v e d i n t h e metabolism o f compounds o f t h i s nature. T h e i r r e s u l t s support the c o n c l u s i o n t h a t phenols per se are not transported from t h e gut but are conjugated before e n t e r i n g the c i r c u l a t o r y system. The q u a n t i t a t i v e importance o f s u l f a t e e s t e r conjugation v a r i e s w i t h many f a c t o r s whic animal (35, 4 1 ) ; specie mal ( 4 3 ) ; s i z e o f dose (44, 4 5 ) ; s u l f u r n u t r i t i o n a l s t a t e o f a n i mal (46, 4 7 ) ; time a f t e r dosing (47); disease s t a t e (48); s u b s t i t uent e f f e c t s (49); and i n h i b i t o r s (43, 5 0 ) . Roy (20, 2 1 ) , Dodgson (51), Gregory and Robbins (22) , Dodgson and Rose ( 7 ) , and Young and Maw (52) have reviewed the evidence f o r t h e s y n t h e s i s o f s u l f a t e e s t e r s by metabolic routes other than those u t i l i z i n g PAPS. Perhaps the best evidence f o r a l t e r n a t e pathways has been obtained w i t h lower animals such as mollusks, but there are suggestions t h a t other routes may occur i n higher animals as w e l l . A s c o r b i c a c i d 3 - s u l f a t e and unknown s u l f a t e donors have been i m p l i c a t e d . Some proposed mechanisms have been discounted (21); but t h e p o s s i b i l i t y o f a l t e r n a t e routes o f s u l f a t e e s t e r b i o s y n t h e s i s has not been completely i n v e s t i g a t e d . The metabolic f a t e o f some s u l f a t e e s t e r s i n animals has been i n v e s t i g a t e d . S t u d i e s , such as those reported by Flynn e t a l . (53) and Hawkins and Young (54) demonstrated that many s u l f a t e e s t e r s are q u i c k l y e l i m i n a t e d i n the u r i n e w i t h l i t t l e or no metabo l i s m . Park (6) reported t h a t a r y l s u l f a t e s were e l i m i n a t e d i n the u r i n e by a c t i v e t r a n s p o r t . C u r t i s e t a l . (55) compared the r e n a l clearance o f i n u l i n and a s e r i e s o f a r y l s u l f a t e s at d i f f e r ent plasma concentrations and found t h a t s u l f a t e e s t e r s were s e c r e t e d by the r e n a l c e l l s . The c o n t r i b u t i o n o f the r e n a l s e c r e t o r y process t o o v e r a l l u r i n a r y e x c r e t i o n ranged from 22 t o 87%. They concluded that the r a p i d e l i m i n a t i o n of the a r y l e s t e r s s t u d i e d was due t o t h e r a p i d s e c r e t i o n of these compounds r a t h e r than prevention o f t u b u l a r r e s o r p t i o n . However, some s u l f a t e e s t e r s are e x t e n s i v e l y metabolized t o a v a r i e t y of products which i n c l u d e mercapturic a c i d d e r i v a t i v e s (56), doubly conjugated d e r i v a t i v e s (53, 5 7 ) , other compounds formed without removal o f the s u l f a t e group ( 5 8 ) , and other u n i d e n t i f i e d metabolites (59-61). The degree o f metabolism o f s u l f a t e e s t e r s may vary w i t h t h e sex o f the animal (62). Studies have shown that p e r i t o n e a l b a r r i e r s were permeable t o some a r y l s u l f a t e e s t e r s but other b a r r i e r s were n o t ; f o r i n s t a n c e , r a d i o a c t i v i t y

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d i d not pass i n t o the c e n t r a l nervous system when [35s]aryl s u l f a t e e s t e r s were given t o r a t s (63). C u r t i s e t a l . (55) reported t h a t some a r y l s u l f a t e e s t e r s were bound to plasma p r o t e i n s i n v i v o . B i l i a r y s e c r e t i o n , which i s o f q u a n t i t a t i v e importance w i t h some s u l f a t e e s t e r s , can be i n f l u e n c e d by e x t e r n a l f a c t o r s . For i n s t a n c e , Powell et a l . (64) reported t h a t the b i l i a r y e x c r e t i o n of phenolphthalein d i s u l f a t e by the r a t was decreased by admini s t r a t i o n of d i e t h y l s t i l b e s t r o l s u l f a t e and d i e t h y l s t i l b e s t r o l monoglucuronide• S u l f a t a s e Enzymes Because the p e s t i c i d zymes t o cleave s u l f a t the k i n e t i c c h a r a c t e r i s t i c s and p r o p e r t i e s o f the v a r i o u s members of t h i s d i v e r s e group o f enzymes i s e s s e n t i a l . A t l e a s t s i x gene r a l groups o f enzymes are r e s p o n s i b l e f o r the h y d r o l y s i s o f s u l f a t e e s t e r s which can be c l a s s i f i e d on the b a s i s o f t h e i r subs t r a t e s as: a r y l s u l f a t a s e s , s t e r o i d s u l f a t a s e s , mucopolysaccha r i d e s u l f a t a s e s (chondrosulfatase and heparin s u l f a t a s e s ) , g l y c o s u l f a t a s e s , myrosulfatases, and a l k y l s u l f a t a s e s (22). Roy (65) and Dodgson and Rose (7) have used s i m i l a r c l a s s i f i c a t i o n schemes. This d i s c u s s i o n i s r e s t r i c t e d p r i m a r i l y to the a r y l s u l f a t a s e s because they have been s t u d i e d i n g r e a t e s t d e t a i l and are of most I n t e r e s t to the p e s t i c i d e chemist. The s t u d i e s t h a t l e d t o the d i s c o v e r y of a r y l s u l f a t a s e enzymes have been reviewed ( 7 ) . The general p r o p e r t i e s and c l a s s i f i c a t i o n of these enzymes, as w e l l as k i n e t i c and i n h i b i t o r s t u d i e s , have been summarized (7, 20_ ,22, £5, 66) . Enzymes f o r the h y d r o l y s i s o f a r y l s u l f a t e e s t e r s are widespread i n nature (22, 65), but the most d e t a i l e d s t u d i e s have been conducted w i t h enzymes from mammals, mollusks, and microorganisms (22). The s u b s t r a t e s p e c i f i c i t y and p r o p e r t i e s o f a r y l s u l f a t a s e s from d i f f e r e n t sources vary and f a i l u r e t o recognize t h i s f a c t l e d to confusing and appare n t l y c o n t r a d i c t o r y r e s u l t s i n the e a r l y s t u d i e s ( 7 ) . The more d e t a i l e d s t u d i e s (67-72) which c l a r i f i e d these p o i n t s have been summarized ( 7 ) • I t i s now known t h a t the mammalian l i v e r contains two a r y l s u l f a t a s e s (designated A and B) i n the lysosomes and a t h i r d form (designated C) i n the microsome f r a c t i o n . A r y l s u l f a t a s e A and B from mammalian lysosomes are i n h i b i t e d by S O 4 " , HPO^"", and F~, are not i n h i b i t e d by QT*, have a low pH optimum, and are most a c t i v e i n the h y d r o l y s i s o f substrates such as n i t r o c a t e c h o l s u l f a t e . These mammalian enzymes and a r y l s u l f a t a s e enzymes from other sources t h a t have s i m i l a r s u b s t r a t e s p e c i f i c i t i e s and behavi o r toward i n h i b i t o r s have been c l a s s i f i e d as "Type I I enzymes." I n c o n t r a s t , a r y l s u l f a t a s e C i n the microsome f r a c t i o n of mammali a n l i v e r has a pH optimum of 8 , i s most a c t i v e on simple subs t r a t e s such as £-nitrophenyl s u l f a t e , and i s i n h i b i t e d by CN~ 9

2

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but not by HPO4 . This mammalian enzyme and a r y l s u l f a t a s e s from other sources w i t h s i m i l a r p r o p e r t i e s have been c l a s s i f i e d as "Type I Enzymes." The v a l i d i t y of the s u b d i v i s i o n of the a r y l s u l f a t a s e s i n t o Type I and Type I I enzymes and i n c o n s i s t e n c i e s that sometimes a r i s e when the m u l t i c r i t e r i a c l a s s i f i c a t i o n system i s used have been discussed (66). This c l a s s i f i c a t i o n system has shortcomings, but i t i s f u n c t i o n a l and should remind the p e s t i c i d e chemist that the p r o p e r t i e s of a r y l s u l f a t a s e s from d i f f e r e n t sources may be q u i t e d i s s i m i l a r . The a r y l s u l f a t a s e s c a t a l y z e the h y d r o l y s i s of the 0-S bond, and t h e only known s u l f a t e acceptor i s water. There i s no e v i dence t h a t a metal i s i n v o l v e d , and the r e a c t i o n i s apparently i r r e v e r s i b l e . N i c h o l l s and Roy (66) suggested t h a t the a c t i v a t i o n energy i s probably about 12-14 Kcal/mole; however, the thermodynamics of the a r y l s u l f a t a s s t u d i e d i n d e t a i l . The s t r a t e s and i n h i b i t o r s of the a r y l s u l f a t a s e s , the a c t i v e s i t e s on these enzymes, and the k i n e t i c and p h y s i c a l p r o p e r t i e s of these enzymes have been summarized (7^, 66) . The procedures t h a t have been used t o assay a r y l s u l f a t a s e a c t i v i t y have u s u a l l y i n v o l v e d measuring the l i b e r a t e d phenol c o l o r i m e t r i c a l l y o r determining the a n i o n i c form o f the phenol i n the v i s i b l e o r u l t r a v i o l e t regions o f the spectrum. Dodgson and Spencer (73) reviewed the methods, l i m i t a t i o n s , and problems that have been encountered w i t h these procedures. More recent s t u d i e s have been reported on d i r e c t cytochemical assay of a r y l s u l f a t a s e s (74) , assays f o r s u l f a t a s e s A and B (75, 76) , the i n fluence o f the s t a t e o f molecular aggregation on the enzymic h y d r o l y s i s of a r y l s u l f a t e s (77), the t i s s u e d i s t r i b u t i o n of a r y l s u l f a t ases A and B (76), k i n e t i c c h a r a c t e r i s t i c s and i n h i b i t o r s of a r y l s u l f a t a s e A (78), e l e c t r o p h o r e t i c s e p a r a t i o n and c h a r a c t e r i z a t i o n of a r y l s u l f a t a s e A and B ( 7 9 ) , and evidence that cerebroside s u l f a t e s and a r y l s u l f a t e s are degraded by the same enzyme (80). The l a t t e r r e p o r t i s of i n t e r e s t s i n c e humans w i t h metachromatic leukodystrophy, a human s p h i n g o - l i p i d storage d i s e a s e , are d e f i c i ent i n a r y l s u l f a t a s e A (81). I n d u c t i o n of a l k y l s u l f a t a s e s i n microorganisms has been r e ported (82, 83). Whether i n d u c t i o n a l s o occurs i n higher animals and w i t h other c l a s s e s of compounds, such as a r y l s u l f a t e s , has not been reported but may be worthy of f u r t h e r study. Laboratory

Synthesis

The most w i d e l y used methods f o r the synthesis of a r y l s u l f a t e s employ s u l f u r t r i o x i d e o r S03-amine adducts; the chemistry of s u l f u r t r i o x i d e , and i t s d e r i v a t i v e s has been reviewed i n d e t a i l (84). Many reagents have been used i n t h i s type o f s u l f a t i o n react i o n i n c l u d i n g c h l o r o s u l f o n i c a c i d (45, 77, 85-89), t r i e t h y l a m i n e s u l f u r t r i o x i d e (90, 9 1 ) , and p y r i d i n e s u l f u r t r i o x i d e (92, 93)• Other reagents t h a t have been used i n the s y n t h e s i s of s u l f a t e

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e s t e r s i n c l u d e p y r o s u l f a t e (94). fuming s u l f u r i c a c i d , and s u l f a m i c a c i d (84) . The use o f c h l o r - t ^ S ] s u l f o n i c a c i d i n the p r e p a r a t i o n of [ S ] - l a b e l e d a r y l s u l f a t e e s t e r s has been d e s c r i b e d (S3, 54, 95). Although not widely used by p e s t i c i d e chemists, the r e a c t i o n of H2SO4 w i t h a v a r i e t y of compounds i n the presence of d i c y c l o hexylcarbodiimide and a p o l a r s o l v e n t (96-98) warrants c a r e f u l c o n s i d e r a t i o n . This method gives s u l f a t e e s t e r s i n good y i e l d and i s e s p e c i a l l y u s e f u l i n the p r e p a r a t i o n of s u l f a t e e s t e r s o f compounds t h a t are unstable to reagents such as c h l o r o s u l f o n i c a c i d and p y r i d i n e s u l f u r t r i o x i d e . Moreover, i f the c o n d i t i o n s are j u d i c i o u s l y a d j u s t e d , t h i s procedure can be used f o r the s e l e c t i v e s u l f a t i o n of p o l y f u n c t i o n a l molecules (98). This method i s p a r t i c u l a r l y good f o r the s y n t h e s i s of T ^ S ] - s u l f a t e e s t e r s because [35so^] i s r e a d i l y a v a i l a b l e d i r e c t l y without conversio sulfur trioxide. Mumma (99) reported t h a t a s c o r b i c a c i d 2 - s u l f a t e and isopropopylidene a s c o r b i c a c i d s u l f a t e acted as an i n v i t r o s u l f a t i n g agent at e l e v a t e d temperatures and/or i n the presence of o x i d i z i n g agents. For example, a l c o h o l s such as 1-octanol and 33c h o l e s t a n o l were r e a d i l y s u l f a t e d when incubated w i t h i s o p r o p y l i dene a s c o r b i c a c i d s u l f a t e i n the presence of bromine or when incubated at 100°C. Quadri et a l . (100) and Mumma et a l . (101) reported on the s y n t h e s i s and c h a r a c t e r i z a t i o n of L - a s c o r b i c a c i d 2 - s u l f a t e . The p o s s i b l e use of a s c o r b i c a c i d s u l f a t e and/or i t s d e r i v a t i v e s as a p r e p a r a t i v e method f o r s u l f a t i n g phenols appare n t l y has not been reported but may be worthy o f f u r t h e r evaluation. Recently, Nagasawa and Yoshidome (89) reported on the C u ( I I ) c a t a l y z e d r e a c t i o n of 8 - q u i n o l y l s u l f a t e i n the s y n t h e s i s o f D-galactose 6 - s u l f a t e , adenosine 5 ' - s u l f a t e and dextran s u l f a t e . Whether t h i s procedure can be used to prepare s u l f a t e e s t e r s o f phenols, a l c o h o l s , and s t e r o i d s w a i t s f u r t h e r i n v e s t i g a t i o n . Boyland and Nery (102) reported on the s u l f a t i o n of phenylhydroxy lamine and r e l a t e d compounds w i t h p y r i d i n e s u l f u r t r i o x i d e and other reagents t o form N - s u l f o n i c and O - s u l f o n i c a c i d d e r i v a t i v e s (the product formed depended on the r e a c t i o n c o n d i t i o n s and b l o c k i n g groups used). These compounds were i s o l a t e d as t h e i r ammonium and potassium s a l t s . Boyland and Nery (102) a l s o made the important observation t h a t phenyl hydroxylamine-O-sulfonic a c i d rearranged t o 2-amino-phenyl s u l f a t e . The b i o s y n t h e s i s of s u l f a t e e s t e r s w i t h i n v i t r o t i s s u e prepa r a t i o n s , f o r t i f i e d w i t h PAPS or PAPS generating systems, has been used by many workers (9», 12, 13, 32, 36, 37^, 103-109). This technique lends i t s e l f w e l l t o the s y n t h e s i s o f [ ^ S ] - l a b e l e d s u l fate esters. The p h e n o l s u l f o t r a n s f e r a s e r e a c t i o n i s r e a d i l y r e v e r s i b l e when the a r y l s u l f a t e e s t e r i n v o l v e d i s r e a c t i v e (34, 110, 111). For i n s t a n c e , Brunngraber (110) demonstrated the t r a n s f e r o f

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s u l f a t e from £-nitrophenyl s u l f a t e t o m-aminophenol i n the presence of phenol s u l f o t r a n s f e r a s e and PAP. This f a c t has been e x p l o i t e d as a convenient assay procedure. However, the p o s s i b l e use o f t h i s technique f o r t h e s y n t h e s i s o f s u l f a t e e s t e r s should be cons i d e r e d . This approach could be e s p e c i a l l y u s e f u l when the acceptor molecule has one o r more l a b i l e l i n k a g e s . Properties A r y l s u l f a t e e s t e r s are u s u a l l y s t a b l e as t h e i r a l k a l i s a l t s , e s p e c i a l l y when s t o r e d i n t h e dark at low temperatures. Havinga et a l . (112) described t h e photochemical a c c e l e r a t e d h y d r o l y s i s of n i t r o p h e n y l s u l f a t e s . A r y l s u l f a t e e s t e r s are h i g h l y s o l u b l e i n water and appreciably s o l u b l e i n a l c o h o l s . An e s p e c i a l l y usef u l s o l v e n t i s N-butano a r y l s u l f a t e e s t e r s fro e s t e r s form s a l t s w i t h organic bases such as j j - t o l u i d e n e (113) , £-bromoaniline (114), methylene blue (115, 116), and the amlnoa c r i d i n e s (72) i s u s e f u l f o r t h e i s o l a t i o n of a r y l s u l f a t e s because most o f them can be e x t r a c t e d from aqueous s o l u t i o n s w i t h organic s o l v e n t s . Dodgson e t a l . (72) used 5-aminoacridine t o i s o l a t e the a r y l s u l f a t e s excreted i n t h e u r i n e o f r a b b i t s fed £-chlorophenol and r e l a t e d compounds. Roy and Trudinger (117) and Young and Maw (52) discussed t h e a p p l i c a t i o n o f t h i s p r i n c i p l e t o the i s o l a t i o n and i d e n t i f i c a t i o n of a r y l s u l f a t e s . The e a r l y work o f Burkhardt (118, 119) and others e s t a b l i s h e d that a r y l s u l f a t e e s t e r s are r e a d i l y hydrolyzed by a c i d s ; the r a t e of a c i d h y d r o l y s i s i s increased when t h e s u l f a t e moiety i s a t t a c h ed t o a p o s i t i o n o f low e l e c t r o n a v a i l a b i l i t y . For example, £n i t r o p h e n y l s u l f a t e i s e a s i l y hydrolyzed w i t h a c i d . The mechanism of a c i d h y d r o l y s i s o f a r y l s u l f a t e s has been s t u d i e d (120-122). Roy and Trudinger (117) discussed the problems w i t h a r t i f a c t f o r mation when some s u l f a t e e s t e r s are a c i d hydrolyzed. Batts (123) reported t h a t t h e r a t e o f h y d r o l y s i s o f s u l f a t e e s t e r s was i n creased by a f a c t o r of 10^ when the s o l v e n t was changed from pure water t o moist dioxane. L a t e r , Goren and Kochansky (124) extended these s t u d i e s and found t h a t t h e s o l v o l y s i s r e q u i r e d i n i t i a t i o n by t r a c e s o f i m p u r i t i e s , presumably a c t i n g as an e l e c t r o p h i l e . For i n s t a n c e , 2-octanol s u l f a t e i n clean t e f l o n v e s s e l s was s t a b l e t o hot, moist dioxane. In c o n t r a s t t o t h e i r l a b i l i t y under a c i d c o n d i t i o n s , most a r y l s u l f a t e s a r e q u i t e s t a b l e under b a s i c c o n d i t i o n s 07 , S7_ 117119). For example, Burkhardt and Lapworth (87) heated a r y l s u l f a t e s t o 150°C f o r 4 hours i n strong a l k a l i o r h a l f - c o n c e n t r a t e d ammonia t o b r i n g about h y d r o l y s i s . 9

Separation

and P u r i f i c a t i o n Techniques

A s s a n d r i and P e r a z z i (125) reported on t h e s e p a r a t i o n o f phenolic O-glucuronides and p h e n o l i c s u l f a t e e s t e r s by m u l t i p l e

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l i q u i d - l i q u i d p a r t i t i o n . T h e i r methods i n v o l v e d a counter current technique w i t h continuous flow of the s o l v e n t s . However, the I s o l a t i o n o f s u l f a t e e s t e r s from b i o l o g i c a l f l u i d s , such as u r i n e , by t h i s technique r e q u i r e d p r e - p u r i f i c a t i o n o f the crude m a t e r i a l before the counter current f r a c t i o n a t i o n procedure; i m p u r i t i e s , such as s a l t s , i n t e r f e r e d w i t h the p a r t i t i o n systems. Because of the p o l a r nature o f a r y l s u l f a t e e s t e r s , i t i s not s u r p r i s i n g t h a t i o n exchange chromatography has been used extens i v e l y i n the p u r i f i c a t i o n o f these compounds (12, 13, 45, 88, 126-131. Sephadex G-10 columns e l u t e d w i t h water (45, 88, 103, 130, 131), Sephadex G-15 columns e l u t e d w i t h water (132), and Sephadex LH-20 columns e l u t e d w i t h e i t h e r CH3OH or H 0 (45, 103, 129-131) have been used f o r the p u r i f i c a t i o n o f a r y l s u l f a t e s . I t should be noted t h a t mixed s a l t forms o f s u l f a t e e s t e r s are e x c r e t ed by animals and t h a t thes on Sephadex LH-20 column atographic procedures t h a t have been used t o separate and p u r i f y s u l f a t e e s t e r conjugates o f p e s t i c i d a l compounds i n c l u d e : B i o g e l P-2 columns (131) ; XAD-2 columns (94^, 116) ; Porapak Q columns (88, 129); and paper chromatography (88, 133, 134). FaakonmaTci (135) reported on a d i r e c t gas chromatographic a n a l y s i s o f s t e r o i d s u l f a t e s and glucuronides. Mass spectroscopy showed t h a t the s t e r o i d s u l f a t e s l o s t H2SO4 and a double bond was formed g i v i n g a m o l e c u l a r i o n 18 mass u n i t s lower than t h a t o f the f r e e s t e r o l . I n c o n t r a s t , the g l u c u r o n i c a c i d conjugates gave the parent s t e r o l . The a p p l i c a b i l i t y o f t h i s procedure, i f any, t o a r y l s u l f a t e s and glucuronides apparently has not been reported. P r e l i m i n a r y s t u d i e s w i t h e l e c t r o n impact mass spectrometry a t t h i s l a b o r a t o r y i n d i c a t e d that a r y l s u l f a t e e s t e r s (K s a l t s ) thermally degrade t o give a fragment corresponding to the phenol ( u s u a l l y base peak) and fragments a t lower masses. 2

D e r i v a t i z a t i o n Procedures There apparently i s l i t t l e or no i n f o r m a t i o n i n the l i t e r ature concerning attempts t o d e r i v a t i z e the s u l f a t e group i n a r y l s u l f a t e s . McKenna and Norymberskl (136), P a s q u a l i n i et a l . (137) , and E m i l i o z z i (138) reported on the formation of methylated der i v a t i v e s when s t e r o i d s u l f a t e s were t r e a t e d w i t h diazomethane• Studies at t h i s l a b o r a t o r y i n d i c a t e d t h a t a r y l s u l f a t e s were not methylated by diazomethane o r , i f they were, the products were not s t a b l e ; the l a t t e r e x p l a n a t i o n seems most l i k e l y . However, f u r t h e r study i s needed t o c l a r i f y t h i s p o i n t . In some cases, i t i s p o s s i b l e t o d e r i v a t i z e other f u n c t i o n a l groups i n a molecule without c l e a v i n g or d e r i v a t i z i n g the s u l f a t e group. For i n s t a n c e , Dodgson e t a l . (70) methylated the f r e e h y d r o x y l o f a monosulfate e s t e r of 4-chlorocatechol i s o l a t e d from r a b b i t u r i n e . The m e t h y l a t i o n of the monosulfate e s t e r s o f i s o p r o p y l 3,4-dihydroxycarbanilate w i t h diazomethane ( l e a v i n g the s u l f a t e e s t e r group i n t a c t ) f o l l o w e d by replacement o f the s u l f a t e

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e s t e r w i t h an acetoxy group made p o s s i b l e the c h a r a c t e r i z a t i o n and subsequent s y n t h e s i s and i d e n t i f i c a t i o n o f propham metabolites (140). A one-step method f o r r e p l a c i n g t h e s u l f a t e group i n a r y l and s t e r o i d s u l f a t e e s t e r s w i t h an acetoxy group (139) i s u s e f u l f o r c h a r a c t e r i z a t i o n o f compounds that are unstable t o conventional h y d r o l y s i s c o n d i t i o n s . For example, u t i l i z a t i o n o f t h i s technique made i t p o s s i b l e t o i d e n t i f y c a r b a r y l metabolites i n chicken u r i n e as conjugated forms o f 1,5-dehydroxynaphthalene and 1 , 5 , 6 - t r i hydroxynaphthalene, compounds that are unstable t o normal h y d r o l y s i s c o n d i t i o n s (103). This technique was a l s o used i n the chara c t e r i z a t i o n o f s u l f a t e e s t e r - c o n t a i n i n g metabolites of propham (129, 140) and £-chlorophenyl N-methylcarbamate (45, 131) . Spectral Analysis Hearse e t a l . (95) reporte ary e s t e r s e x h i b i t e d s t r o n g absorption from 240-280 my (maxima 250-275 my); whereas t h e parent phenols absorbed s t r o n g l y from 270-310 my (maxima 280-295 my). Moreover, the e x t i n c t i o n c o e f f i c i e n t of the phenols was much g r e a t e r than t h a t of the s u l f a t e e s t e r s . The marked s h i f t i n t h e Amax and t h e i n c r e a s e i n the e x t i n c t i o n coe f f i c i e n t ' a s s o c i a t e d w i t h t h e conversion of the p h e n o l i c hydroxy1 to i t s i o n i z e d form were not shown by t h e corresponding a r y l s u l f a t e e s t e r . This behavior has been e x p l o i t e d t o develop assays f o r t h e h y d r o l y s i s o f a r y l s u l f a t e s (73, 141). Nuclear magnetic resonance s t u d i e s (NMR) a t t h i s l a b o r a t o r y have demonstrated t h a t a r y l s u l f a t e e s t e r s s h i f t the absorption of r i n g protons downfield r e l a t i v e t o t h e i r p o s i t i o n i n the spectrum o f t h e parent phenol. As expected, the s h i f t was g r e a t e s t f o r t h e proton ortho t o t h e s u l f a t e group. For i n s t a n c e , absorpt i o n s o f t h e protons ortho and meta t o t h e hydroxy i n £-nitrophenol were a t 6.93 and 8.10 ppm, r e s p e c t i v e l y , (solvent-d£-DMSO) and these absorptions were s h i f t e d t o 7.4 and 8.18 ppm, respect i v e l y , i n t h e spectrum o f £-nitrophenyl s u l f a t e . NMR s p e c t r o s copy has been used i n a s s i g n i n g s t r u c t u r e s t o the mono and d i s u l f a t e e s t e r s o f 4-chlorocatechol (131). Further s t u d i e s are underway t o more thoroughly i n v e s t i g a t e t h e e f f e c t of s u l f a t e e s t e r s on NMR absorption o f aromatic compounds. Since a l k a l i s a l t s of s u l f a t e e s t e r s are s o l i d s and are only s l i g h t l y s o l u b l e i n most organic s o l v e n t s , the i n f r a r e d s p e c t r a of these compounds are u s u a l l y measured i n a KBr p e l l e t o r i n a N u j o l m u l l . Chihara (142) reported t h a t the s p e c t r a of s u l f a t e e s t e r s obtained from KBr p e l l e t s and mulls were not appreciably d i f f e r e n t . However, i t should be noted that the i n f r a r e d s p e c t r a of d i f f e r e n t s a l t forms o f a r y l s u l f a t e e s t e r s are d i s t i n c t l y d i f f e r e n t (103). Chihara (142) s y s t e m a t i c a l l y s t u d i e d t h e i n f r a red s p e c t r a o f a s e r i e s o f a l k y l and a r y l s u l f a t e e s t e r s . He assigned t h e two bands at 1210-1220 and 1240-1260 cm" t o the S 0 asymmetric s t r e t c h i n g v i b r a t i o n ; these absorptions were very 1

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s t r o n g and not g r e a t l y s h i f t e d by a v a r i e t y o f s u b s t i t u e n t s . He assigned t h e s t r o n g a b s o r p t i o n a t 1040-1081 cm" t o the SO3 symm e t r i c v i b r a t i o n and the l e s s i n t e n s e bands a t 550-590 cm-i (sometimes s p l i t ) and 617-650 c n r t o SO3 bending v i b r a t i o n s . The abs o r p t i o n from 757 t o 838 c n r was assigned t o the S-O-C s t r e t c h . L l o y d e t a l . (143) s t u d i e d the i n f r a r e d s p e c t r a of the s u l f a t e e s t e r s o f a l c o h o l s , amino a l c o h o l s , and hydroxylated amino a c i d s and reported s i m i l a r conclusions ( a b s o r p t i o n bands a t 1210-1260 cm" , 1030-1050 cm" , and 770-810 cm" , assigned t o the s u l f a t e e s t e r ) . Related s t u d i e s on t h e i n f r a r e d s p e c t r a o f p o l y s a c c h a r i d e s u l f a t e s (144) and monosaccharide s u l f a t e s (145) have been r e p o r t ed. Hummel (146) discussed the i n f r a r e d s p e c t r a of primary (abs o r p t i o n bands a t 1220-1267 cm" , 1075-1100 cm" , and 834-840 cm" ) and secondary ( a b s o r p t i o n bands 1228-1250 cm" , 1063-1075 cm" , and 926-945 cm"*Colthup eit a l . (147) presente which showed a b s o r p t i o n bands a t approximately 820-840 cm" , 1 2 0 0 1280 cm" , and 1060-1080 cm" which were assigned t o the s u l f a t e moiety. I n f r a r e d spectroscopy has been used t o c h a r a c t e r i z e s u l f a t e e s t e r s o f drugs (94, 148), s t e r o i d s (96, 138), and s u l f a t e e s t e r c o n t a i n i n g metabolites o f p e s t i c i d e s which i n c l u d e mobam (88) , £-chlorophenylmethylcarbamate (45, 131), propham (129, 140), c a r b a r y l (103), chlorpropham (134), and barban (134). The technique o f l a s e r i o n i z a t i o n mass spectrometry (149) has been used by Mumma and V a s t o l a (150) t o o b t a i n the mass s p e c t r a of t h e sodium and potassium s a l t s o f 1 - h e x y l , 1 - d e c y l , and 1-octadecyl s u l f a t e . The molecular species p l u s a c a t i o n ([M + N a ] o r [M + K ] ) was one o f the more i n t e n s e peaks i n the s p e c t r a but no other "organic i o n s " were observed. "Inorganic i o n s " that were abundant i n the s p e c t r a i n c l u d e d [NaS04]+, [ ^ 3 8 0 4 ] * , and t h e corresponding potassium-containing fragments. Approximatel y 1 mg o f sample was used f o r these assays; but the authors r e ported "good s p e c t r a can be obtained on l e s s sample." This procedure has been used t o c h a r a c t e r i z e a number o f s t e r o i d , a l k y l , and a r y l s u l f a t e e s t e r s (97, 9 8 ) . R e c e n t l y , Games e t a l . (151) p u b l i s h e d a b r i e f report d e a l i n g w i t h t h e u t i l i t y o f f i e l d d e s o r p t i o n mass spectrometry i n the a n a l y s i s o f s u l f a t e e s t e r s and r e l a t e d compounds. They found t h a t n-hexyl, n - d e c y l , and n-undecyl s u l f a t e s gave quasi molecular ions at m/e 259, 315, and 329, r e s p e c t i v e l y , but no fragment ions were observed. C y c l o h e x y l p h e n y l - 4 - s u l f a t e gave a quasi-molecular i o n ([M + K ] ) at m/e 333 but a l s o gave an i o n a t m/e 176, presumably r e s u l t i n g from cleavage and hydrogen t r a n s f e r t o form the parent phenol. Thus, there i s reason f o r optimism t h a t f i e l d - d e s o r p t i o n mass spectrometry may be a u s e f u l t o o l . However, much a d d i t i o n a l work needs t o be done t o determine i f t h i s approach w i l l be o f p r a c t i c a l importance i n t h e i d e n t i f i c a t i o n o f s u l f a t e e s t e r s — p a r t i c u l a r l y s u l f a t e e s t e r s from b i o l o g i c a l preparations where c o m p l i c a t i n g f a c t o r s , such as mixed s a l t forms, may be a problem. H o p e f u l l y , i n f o r m a t i o n t o answer t h i s and other questions about 1

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f i e l d - d e s o r p t i o n mass spectrometry of s u l f a t e e s t e r s w i l l soon be available. General D i s c u s s i o n There i s a l a r g e and growing body of i n f o r m a t i o n concerning the b i o s y n t h e s i s , chemical s y n t h e s i s , i s o l a t i o n , c h a r a c t e r i z a t i o n , and enzymology of the s u l f a t e e s t e r s . However, a review of the published l i t e r a t u r e d e a l i n g w i t h p e s t i c i d e metabolism reveals that many p e s t i c i d e chemists are not f u l l y u s i n g the i n f o r m a t i o n and technology t h a t i s a v a i l a b l e . For i n s t a n c e , i n most of the r e ported s t u d i e s , the s u l f a t e e s t e r s were hydrolyzed e i t h e r chemica l l y or e n z y m a t i c a l l y and then only the "nonpolar" h y d r o l y s i s product was i d e n t i f i e d . Because some of these compounds, such as the N-hydroxy s u l f a t e s , are the i s o l a t i o n and i d e n t i f i c a t i o t a n t . The observation by Boyland and Nery (102) that the s u l f a t e e s t e r of phenyl hydroxylamine rearranged to 2-amino phenyl s u l f a t e suggests t h a t a r t i f a c t s may be produced by c o n d i t i o n s such as those used t o hydrolyze conjugated m e t a b o l i t e s . In many i n s t a n c e s , s t r u c t u r e s of s u l f a t e e s t e r s have been assigned on the b a s i s of enzyme h y d r o l y s i s s t u d i e s and c h a r a c t e r i z a t i o n of only the h y d r o l y s i s product. I n c o r r e c t assignment of s t r u c t u r e because of enzyme preparations that are contaminated w i t h other h y d r o l y t i c enzymes, as w e l l as contaminates from nonezymatic h y d r o l y s i s , are always p o s s i b i l i t i e s that must be considered. Often the markedly d i f f e r e n t p r o p e r t i e s of Type I and Type I I s u l fatases (see previous d i s c u s s i o n ) have been ignored i n s e l e c t i n g assay c o n d i t i o n s and/or i n s e l e c t i n g the type of enzyme to be used f o r the h y d r o l y s i s of d i f f e r e n t c l a s s e s of compounds. Some workers have synthesized the suspected s u l f a t e e s t e r and then c h a r a c t e r i z e d t h e i r unknown metabolites by co-chromatography s t u d i e s only. This i s unfortunate because a d d i t i o n a l and u s u a l l y d e f i n i t i v e d a t a can be obtained by comparative UV, NMR, and IR spectroscopy. These i n s t r u m e n t a l procedures are not d e s t r u c t i v e and r e q u i r e only s m a l l samples ( f o r UV and IR, a sample of 10 ug or l e s s i s u s u a l l y s u f f i c i e n t ) . We have found IR spectroscopy to be e s p e c i a l l y u s e f u l i n c h a r a c t e r i z i n g s u l f a t e e s t e r conjugated p e s t i c i d e m e t a b o l i t e s ; a l l of the s u l f a t e e s t e r s that we have examined have shown the c h a r a c t e r i s t i c absorptions p r e v i o u s l y discussed and the f i n g e r p r i n t region almost i n v a r i a b l y showed sharp, intense bands that are i d e a l f o r comparative IR spectroscopy s t u d i e s . Most p e s t i c i d e chemists have not confirmed the s t r u c t u r e of suspected s u l f a t e e s t e r conjugated metabolites by s y n t h e s i s . This i s s u r p r i s i n g i n l i g h t of the f a c t t h a t there are s e v e r a l methods i n the l i t e r a t u r e f o r the synthesis of s u l f a t e e s t e r s i n c l u d i n g methods that are s u i t a b l e f o r the s u l f a t i o n of compounds w i t h r e l atively l a b i l e linkages. Information concerning the b i o l o g i c a l a c t i v i t y of s u l f a t e e s t e r conjugates of p e s t i c i d e s and/or t h e i r primary metabolites i s

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very l i m i t e d . Studied t o determine the e f f e c t o f d i e t a r y f a c t o r s , drugs, hormones, disease s t a t e s , and r e l a t e d f a c t o r s on the s u l f a t e e s t e r conjugation o f p e s t i c i d e s and t h e i r primary m e t a b o l i t e s by animals would be o f v a l u e . The f a t e o f s u l f a t e e s t e r s i n s o i l and p l a n t systems would a l s o be o f i n t e r e s t . F i n a l l y , there i s a need f o r b e t t e r and f a s t e r methods o f i s o l a t i n g s u l f a t e e s t e r s from b i o l o g i c a l p r e p a r a t i o n s . The e x i s t ing techniques f o r c h a r a c t e r i z a t i o n o f s u l f a t e e s t e r s need t o be improved. A d d i t i o n a l s t u d i e s on NMR and f i e l d d e s o r p t i o n mass spectrometry o f t h i s c l a s s o f compounds may be e s p e c i a l l y f r u i t f u l .

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77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105.

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Baxter, T. H., Kostenbauder, H. B., J. Pharm. Sci., 58: 33 (1969). Stinshoff, K., Biochim. Biophys. Acta, 276: 475 (1972). Dubois, G., Baumann, N., Biochem. Biophys. Res. Comm., 50: 1129 (1973). Stinshoff, K., Jatzkewitz, H., Biochim. Biophys. Acta, 377: 126 (1975). Austin, J., Bolasubramanian, A., Pattabiraman, T., Saraswathi, S., Basu, D., Bachhawat, B., J. Neurochem., 10: 805 (1963). Fitzgerald, J. W., Dodgson, K. S., Payne, W. J., Biochem. J., 138: 63 (1974). Dodgson, K. S., Fitzgerald, J. W., Payne, W. J., Biochem. J., 138: 53 (1974). Gilbert, E. E., "Sulfonatio science Publishers Feigenbaum, J., Neuberg, C. A., J. Amer. Chem. Soc., 63: 3529 (1941). Burkhardt, G. N., Wood, H., J. Chem. Soc., 141 (1929). Burkhardt, G. N., Lapworth, A., J. Chem. Soc., p. 684 (1926). Robbins, J. D., Bakke, J. E., Fell, V. J., J. Agr. Food Chem., 18: 130 (1970). Nagasawa, K., Yoshidome, H., J. Org. Chem., 39: 1681 (1974). Dusza, J. P., Joseph, J. P., Bernstein, S., Steroids, 12: 49 (1968). Hardy, W. B., Scalera, M., J. Amer. Chem. Soc, 74: 5212 (1952). Peterson, J.Y.F., Klyne, W., Biochem. J., 43: 614 (1948). Sobel, A. E., Spoerri, P. E., J. Amer. Chem. Soc., 63: 1259 (1941). Fujimoto, J. M., Haarstad, V. B., J. Pharmacol. Exp. Therap., 165: 45 (1969). Hearse, D. J., Olavesen, A. H., Powell, G. M., Biochem. Pharmacol., 18: 173 (1969). Mumma, R. O., Lipids, 1: 221 (1966). Hoiberg, C. P., Mumma, R. O., Biochim. Biophys. Acta, 177: 149 (1969). Hoiberg, C. P., Mumma, R. O., J. Amer. Chem. Soc., 91: 4273 (1969). Mumma, R. O., Biochim. Biophys. Acta, 165: 571 (1968). Quadri, S. F., Seib, P. W., Deyoe, C. W., Carbohydrate Res., 29: 259 (1973). Mumma, R. O., Verlangieri, A. J. Weber, W. W., Carbohydrate Res., 19: 127 (1971). Boyland, E., Nery, R., J. Chem. Soc., 5217 (1962). Paulson, G. D., Zaylskie, R. G., Zehr, M. V., Portnoy, C. E., Fell, V. J., J. Agr. Food Chem., 18: 110 (1970). Goldberg, I. H., Delbrück, A., Fed. Proc., 18: 235 (1959). Layton, L. L., Frankel, D. R., Arch. Biochem., Biophys., 31: 161 (1951).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133.

Sulfate Ester Conjugates

101

Raub, H. R., Hobkirk, R., Can. J. Biochem., 44: 657 (1966). Raub, H. R., Hobkirk, R., Can. J. Biochem., 46: 749 (1968). Raub, H. R., Hobkirk, R., Can. J. Biochem., 46: 759 (1968). Torday, J., Hall, G., Schweitzer, M., Giroud, C.J.P., Can. J. Biochem., 48: 148 (1970). Brunngraber, E. G., J. Biol. Chem., 233: 472 (1958). Fendler, E. J., Fendler, J. H., J. Org. Chem., 33: 3852 (1968). Havinga, E., DeJongh, R. O., Dorst, W., Recueil, 75: 378 (1956). Barton, A. D., Young, L., J. Amer. Chem. Soc, 65: 294 (1943). Laughland, D. H. 657 (1944). Goertz, G. R., Crepy, O. C., Judas, O. E., Longchampt, J. E., Jayle, M. F., Clin. Chim. Acta, 51: 277 (1974). Miyabo, S., Kornel, L., J. Steroid Biochem., 5: 233 (1974). Roy, A. B., Trudinger, P. A., "The Biochemistry of Inorganic Compounds of Sulphur," University Press, Cambridge (1970). Burkhardt, G. N., Evans, A. G., Warhurst, E., J. Chem. Soc., p. 25 (1936). Burkhardt, G. N., Ford, W.G.K., Singleton, E., J. Chem. Soc., p. 17 (1936). Kice, J. L, Anderson, J. M., J. Amer. Chem. Soc., 88: 5242 (1966). Batts, B. D., J. Chem. Soc. (B), p. 551 (1966). Benkovic, S. J., Dunikoski, L. K., Biochemistry, 9: 1390 (1970). Batts, B. D., J. Chem. Soc. (B), p. 547 (1966). Goren, M. B., Kochansky, M. E., J. Org. Chem., 38: 3510 (1973). Assandri, A., Perazzi, A., J. Chromatog., 95: 213 (1974). Jenner, W. N., Rose, F. A., Nature, 252: 237 (1974). Anders, M. W., Latoree, J. P., J. Chromatog., 55: 409 (1971). Hobkirk, R., Davidson, S., J. Chromatog., 54: 431 (1971). Paulson, G. D., Jacobsen, A. M., Zaylskie, R. G., Feil, V. J., J. Agr. Food Chem., 21: 804 (1973). Paulson, G. D., Jacobsen, A. M., J. Agr. Food Chem., 22.: 629 (1974). Paulson, G. D., Zehr, M. V., Docktor, M. M., Zaylskie, R. G., J. Agr. Food Chem., 20: 33 (1972). Slotkin, T. A., Distefano, V., Au, W.Y.W., J. Pharmacol. Exp. Ther., 173: 26 (1970). Tocco, D. J., Buns, R. P., Brown, H. D., Matzuk, A. R., Mertel, H. E., Harman, R. E., Trenner, N. R., J. Med. Chem., 7: 399 (1964).

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102

134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Grunow, W., Böhme, C., Budczies, B., Fd. Cosmet. Toxicol., 8: 277 (1970). Faakonmäki, P. I., Acta Endocr. (Kbh), Suppl. 119, p. 118 (1967). McKenna, J., Norymberski, J. K., J. Chem., Soc., p. 3893 (1957). Pasqualini, J. R., Zelnik, R., Jayle, M. F., Bull. Soc. Chim. Fr., p. 1171 (1962). Emiliozzi, R., Societe Chimique De France, p. 911 (1960). Paulson, G. D., Portnoy, C. E., J. Agr. Food Chem., 18: 180 (1970). Paulson, G. D., Docktor, M. M., Jacobsen, A. M., Zaylskie, R. G., J. Agr. Food Chem., 20: 867 (1972). Dodgson, K. S., Spencer, B., Biochem. J., 53: 444 (1953). Chihara, G., Chem 8 988 (1960) Lloyd, A. G., Tudball Acta, 52: 413 (1961). Lloyd, A. G., Dodgson, K. S., Price, R. G., Rose, F. A., Biochim. Biophys. Acta, 46: 108 (1961). Lloyd, A. G., Dodgson, K. S., Biochim. Biophys. Acta, 46: 116 (1961). Hummel, D., "Identification and Analysis of Surface Active Agents," Interscience Publishers, New York, London, Sydney (1962). Colthup, N. B., Daly, L. H., Wiberly, S. E., "Introduction to Infrared and Raman Spectroscopy," Academic Press, New York and London (1964). Yeh, S. Y., Woods, L. A., J. Pharm. Sci., 60: 148 (1971). Vastola, F. J., Mumma, R. O., Pirane, A. J., Organic Mass Spectrometry, 3: 101 (1970). Mumma, R. O., Vastola, F. J., Organic Mass Spectrometry, 6: 1373 (1972). Games, O. E., Games, M. P., Jackson, A. H., Olavesen, A. H., Rossieter, M., Tet. Letters, p. 2377 (1974). Lotikar, P. D., Scribner, J. D., Miller, J. A., Miller, E. C., Life Sci. 5: 1263 (1966). DeBaun, J. R., Rawley, J. Y., Miller, E. C., Miller, J. A., Proc. Soc. Expt. Biol. Med., 129: 268 (1966). Lin, M. S., Walden, D. B., Exp. Cell Res., 86: 47 (1974).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7 Glutathione Conjugates D. H . HUTS0N Shell Research Ltd., Tunstall Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent, England

Almost 100 years ago, tw the fate of halobenzene , unwittingly the study of glutathione (GSH) conjugation. Baumann and Preusse (1) isolated a cysteine derivative from the acidifed urine of mammals treated with bromobenzene. Jaffé (2) isolated a similar metabolite of chlorobenzene. Their results were published in the same volume of Chemisches Berichte in 1879. The derivatives were called mereapturic acids and were shown later to be S-aryl-N-acetyl-L-cysteines (Figure 1). The mechanism of formation of this type of mercapturic acid is not immediately obvious from a consideration of the structures of precursor and product and almost 100 years elapsed before the reaction pathway was fully elucidated. Waelsch (3), and Brand and Harris (4) suggested in the 1930's that glutathione (GSH) was the source of the cysteine for the biosynthesis of mercapturic acids, but as late as the 1940's and 1950's, dietary protein and tissue cysteine (5)(6)(7) were s t i l l being considered as sources. Glutathione was shown to be the source in 1959 by James and her co-workers (8)(9) in England. As far as the mammal is concerned, mercapturic acid formation and GSH conjugation are inextricably linked and the former must be included in this discussion. In most cases the initial reaction in mercapturic acid biosynthesis is the enzyme-catalysed reaction between GSH and either the foreign compound itself or a metabolite of that foreign compound. The metabolic activation of a compound to the ultimate reactant with GSH is an important facet of this type of conjugation and its significance will be discussed later. It is now clear that mercapturic acid biosynthesis from a precursor R-X (for example benzyl chloride) proceeds in four stages which are shown in Figure 2 . The final product, benzyl mercapturic acid, is excreted in the urine but i t is the first step, conjugation with GSH, which is crucial in the destruction 103

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND

A N D CONJUGATED

PESTICIDE

Figure 1. Mercapturic acid derived from bromobenzene

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RESIDUES

7.

HUTSON

105

Glutathione Conjugates

of the benzyl chloride. Most of the background information available up to 1973 on GSH conjugation and mercapturic acid formation i s obtainable from reviews by Boyland and Chasseaud (10), Wood (11) and Hutson (12). In addition Chasseaud has published two reviews (13)(14) dealing s p e c i f i c a l l y with the enzymes which catalyse the i n i t i a l transfer - the glutathione transferases. 1. The Chemical Requirements For Glutathione Conjugation Much of the structure-activity data on this mode of conjugation has been acquired v i a observations of mercapturic acid excretion i n the urine of animals treated with the precursor. A disadvantage of this approach has been that the yields of mercapturic acids i n th of GSH conjugation. Thes and they may also be extensively metabolised i n the l i v e r , and intestinal tract, or during enterohepatic circulation. An advantage of the approach however, i s that i t reveals mercapturic acid formation from substrates which do not themselves react with GSH and transferase. Undoubtedly the best way to investigate GSH conjugation by the mammal i n vivo i s to analyse metabolites excreted i n the b i l e . This can be done relatively simply by cannulating the b i l e duct under general anaesthesia prior to dosing the animal. Such experiments may be short-term, e.g. 3 hour8, without recovery from the anaesthetic, or longterm, e.g. several days, during which time the b i l e can be c o l lected i n a surgically implanted vessel and the animal can be allowed to live normally in a metabolism cage f i t t e d for the collection of urine and faeces. The use of these various techniques over the last 20 years or so has demonstrated that several general types of compounds are substrates for GSH conjugation. We face the usual xenobiochemical dilemma of how to make a useful chemical c l a s s i f i c a t i o n of these substrates. Classification by function of compound is not very enlightening. Classification on the basis of the group transferred (e.g. alkyl, aryl, etc) f a i l s to acknowledge the importance of the activation effected by the leaving group. Thus i n the generalised reaction shown i n Figure 3 the nature of X i s as important as that of R in determining the rate of reaction. However, the notion of group transfer i s well entrenched and w i l l be used below, but the reader i s cautioned (i) that the s p e c i f i c i t y does not l i e solely with the group which i s transferred and ( i i ) certain classifications of substrates are only important special cases of group transfer (e.g. aromatic 'epoxide transfer i s only a special case of aryl transfer). 9

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AND

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RESIDUES

A l k y l Transfer A l k y l H a l i d e s . The simple fumigants such as methyl bromide, ethylene dibromide and many other halogenated p a r a f f i n s form GSH conjugates (15)(16). The sequence of r e a c t i v i t y l i e s i n the p r e d i c t a b l e order I > Br > CI > F. The e f f i c i e n c y of d e c h l o r i n a t i o n depends on a c t i v a t i o n of the carbon-chlorine bond by e l e c t r o n withdrawing groups e.g. carbonyl and n i t r i l e . The r e a c t i o n r a t e s of some h a l i d e s are shown i n Figure 4. A l k y l A l k y l s u l p h o n a t e s . The a l k y l a t i n g agents methyl methanesulphonate (17) and e t h y l me thanesulphonate (18) (Figure 5) are metabolised v i a GSH conjugation. Both the a l k y l h a l i d e s and the sulphonates react spontaneously w i t h GSH i n v i t r o but the r e a c t i o n r a t e i s considerabl enhanced b th f h e p a t i c GSH a l k y l t r a n s f e r a s e Organophosphate I n s e c t i c i d e s . Methyl p a r a t h i o n (19), methyl paraoxon (20), t e t r a c h l o r v i n p h o s and s e v e r a l other dimethyl phosphoric a c i d t r i e s t e r s (21) (Figure 6) are demethylated by the a c t i o n of GSH t r a n s f e r a s e . The r e a c t i o n i s very important i n l i m i t i n g the acute t o x i c i t y of these compounds i n c e r t a i n s p e c i e s . The pathway i s u s u a l l y o n l y e f f e c t i v e f o r methyl groups. I n a recent study u s i n g a mixed methyl e t h y l alkenylphosphate (temivinphos, F i g u r e 6) we have found that demethylation was the only observable r e a c t i o n i n v i t r o . However, the O-desethylation of the nematacide Mocap (Figure 7) by r a b b i t l i v e r c y t o s o l has been detected (22), the e t h y l group being t r a n s f e r r e d t o GSH. Methyl g l u t a t h i o n e and methyl merc a p t u r i c a c i d ( i n b i l e and u r i n e , r e s p e c t i v e l y ) are b a r e l y detectable i n the metabolism of the organophosphate i n s e c t i c i d e s because the former i s r a p i d l y f u r t h e r c a t a b o l i s e d , the methyl group being e l i m i n a t e d as CO2 (23). However we have detected s m a l l q u a n t i t i e s of methyl mercapturic a c i d , j>-methyl c y s t e i n e and S-methyl c y s t e i n e oxide i n the u r i n e of r a t s dosed w i t h [14c-methyl]dichlorvos (24). Dimethyl organophosphate t r i e s t e r s are weak methylating agents; they undoubtedly methylate GSH but our s t u d i e s i n v i t r o (21) revealed only very low r a t e s of r e a c t i o n w i t h GSH i n the absence of enzyme. A r a l k y l Transfer Although a r a l k y l t r a n s f e r may j u s t be a s p e c i a l case of a l k y l t r a n s f e r , there i s a tendency f o r s t a b i l i s a t i o n of the carbonium i o n d e r i v e d from these s u b s t r a t e s , and there may t h e r e f o r e be an element of SNi r e a c t i o n p o s s i b l e w i t h some s u b s t r a t e s , f o r example benzyl c h l o r i d e . A r a l k y l H a l i d e s . Benzyl c h l o r i d e i s a mercapturic a c i d precursor (Figure 2; F i g u r e 8) and Boyland and Chasseaud (25)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

HUTSON

Glutathione Conjugates

In the reaction : R-X+GSH—•RSG

+ HX

the nature of X is as important as that of R in determining the possibility of reaction

Figure 3.

CH I

100

* C CH-CI H

55

N=C CH CI

20

3

0


C H S 3

7

7

O » P-OH + GSCH CH / 2

3

C H S

7

3

7

Mocap

S-ethyl glutathione Biochemical Pharmacology

Figure 7.

(22)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

HUTSON

Glutathione Conjugates

109

have carried out some studies on the enzyme which catalyses the formation of jS-benzyl glutathione. It has been distinguished from the other GSH transferases by heat inactivation, precipitation, and species distribution. Aralkyl Sulphates. Benzyl, 1-menaphthyl (Figure 8) and phenanthr-9-ylmethyl sulphates are substrates for the transferase. The corresponding aralkyl alcohols (which are not substrates) are mercapturic acid precursors i n vivo and i t would seem likely that £-sulphate conjugation i s an obligatory intermediate step i n this process (26). This i s an unusual role for O-sulphates; they are usually terminal metabolites. Aryl Transfer Substrates for ary nitrobenzene, l,2,4,5-tetrachloro-3-nitrobenzene, 4-nitropyridine N-oxide and sulphobromophthaleine (a dye used to test liver function). The structures, and points of attack of GSH, are shown in Figure 9. Paraoxon and methyl paraoxon are deary late d by a GSH transferase (20)• We see i n this situation (Figure 10), GSH acting at alternative points on the same molecule. If R • methyl, demethylation predominates, i f R ethyl, de-arylation predominates* The GSH-dependent cleavage of a diphenyl ether has recently been reported. Fluorodifen (2,4 -dinitro-4-trifluoromethyl diphenyl ether), one of a new class of herbicides used for the control of broad-leaved weeds in soya beans, peanuts, cotton and rice, i s de-arylated by a GSH aryl transferase. The enzyme ha8 been isolated from the epicotyl tissue of pea seedlings (27) and i s one of the few examples of plant GSH transferases reported to date. The products of reaction have been identified as S(2-nitro-4-trifluoromethyl phenyl) glutathione and £nitrophenol (Figure 11). Limited substrate s p e c i f i c i t y studies have shown that substitutents causing a large decrease i n electron density at C - 1 are necessary for enzyme activity. This is commensurate with nucleophilic attack of glutathione sulphur at this carbon atom. Bromobenzene, the precursor of the f i r s t known mercapturic acid, i s not a substrate for glutathione aryl transferase. This indicates that the reaction i n vivo i s not a hydride ion d i s placement but that another reaction precedes GSH conjugation. ,

Epoxide Transferases When rats and rabbits are dosed with bromobenzene, careful extraction of the urine affords N-acetyl-j>-(4-bromo-l,2-dihydro2-hydroxyphenyl)-Jj-cysteine (Figure 12). This was isolated as i t s cyclohexylamine salt (28) which could be decomposed i n acid solution to the mercapturic acid shown i n Figure 1. This

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND

A N D CONJUGATED

PESTICIDE

benzyl chloride

^ M

0

II

CH-OS-0"

Figure 8. Substrates for GSH aralkyl transferase

NO,

a

CI

CI

ci

I o

Br

HONaO^S

A Na0 S 3

Figure 9. Substrates for GSH aryl transferase

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RESIDUES

IU.TSON

Glutathione

Conjugates

GSH

Life Sciences Figure 10. Alternative GSIIdependent pathways for the

Pesticide Biochemistry and Physiology Figure 11.

GSH-dependent

cleavage of a diphenyl ether (27)

— SCH^CH COOH I NHCOCH3

-SCH CH COOH I NHCOCH., 7

Biochemical Journal Figure

12.

Mercapturic acid formation from hydrocarbons (28)

aromatic

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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observation, together with the now c l a s s i c work on arene oxides at N.I.H. (29), and the discovery of GSH epxoide t r a n s f e r a s e by Boyland and coworkers ( 3 0 ) has led to the r e c o g n i t i o n that aromatic hydrocarbons form GSH conjugates and mercapturic acids only a f t e r oxygenation to epoxides. A t y p i c a l r e a c t i o n pathway i s shown i n F i g u r e 12. A l i p h a t i c epoxides, e.g. styrene oxide (29) and 1-phenoxyprop-2-enyl oxide (31) (Figure 13), are also substrates f o r a GSH t r a n s f e r a s e and form mercapturic a c i d s . f

Alkene T r a n s f e r Compounds containing a c t i v a t e d carbon-carbon double bonds, with the general formula shown i n Figure 14 are conjugated with GSH by the enzyme GSH alken the l i v e r s of various mammalia of the r a t (32). Chasseaud (33) has studied the enzyme(s) i n some depth. E f f i c i e n c y of the r e a c t i o n of the substrate depends on the electron-withdrawing power of the group X and on e l e c t r o n r e p u l s i o n or a t t r a c t i o n exerted by groups R, R' and R . Reaction occurs at the 6 carbon atom of the substrate to y i e l d the product shown i n Figure 14. Some e f f e c t i v e substrates of the enzyme are i l l u s t r a t e d i n Figure 15 which shows that group X may be carbonyl, n i t r i l e , sulphone or n i t r o . ,f

A l k y l Mercapto Transfer The f i r s t example of a t o x i c a t i n g ( b i o a c t i v a t i o n ) r e a c t i o n mediated by a GSH transferase was discovered by Casida and coworkers (34). I t i s catalysed by a s o l u b l e l i v e r enzyme and involves the attack of GSH at the thiocyanate sulphur of an a l k y l thiocyanate r e s u l t i n g i n the l i b e r a t i o n of HCN (Figure 16). The existence of t h i s r e a c t i o n leads to an unusual p r o t e c t i v e e f f e c t which we can now e x p l a i n . The i n s e c t i c i d a l dimethyl phosphate t r i e s t e r , f e n i t r o t h i o n , reduces the t o x i c i t y of s e v e r a l organic thiocyanates to mice, e i t h e r by lowering hepatic GSH or by competitively i n h i b i t i n g the glutathione t r a n s f e r a s e . Transfer of Nitrogen

Heterocycles

A paper published i n 1970 reported the conjugation of a t r a z i n e (Figure 17) with GSH i n sorghum l e a f s e c t i o n s (35). T h i s paper was important because not only d i d i t describe the f i r s t example of a conjugate of a sym-triazine with GSH, but i t demonstrated f o r the f i r s t time that p l a n t s possessed the c a p a b i l i t y f o r GSH conjugation. At t h i s time, during a study of the metabolism of another sym-triazine h e r b i c i d e , cyanazine (Figure 17), i n the r a t , we i s o l a t e d the f i r s t example of a s y m - t r i a z i n y l mercapturic acid (36) (of N-desethyl cyanazine) and subsequently i d e n t i f i e d the p r e d i c t a b l e GSH conjugates i n

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

HUTSON

Glutathione

i

Conjugates

y— CH

V=/

CH,

\ „ /

^

OCH CH 2

\

/

CH

2

\ / ^o/

R

Figure 13. Substrates for GSH epoxide transferase

R

\ R

113

/

/ C=C

, GS

\

•

v X

R

R

1

'

S G

X

Figure 14. The action of GSH alkene transferase

c =cr

(

\

/

\

C=C ' \

/

H

frans-benzylidene acetone

r

\

CN

CS riot control agent

CH =CH SChk II

3

O methyl vinyl sulphone

cyclohex—2-ene-J-one

(3—0—nitrovinyDindole

Biochemical Journal Figure 15. Substrates for GSH alkene transferase

(33)

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PESTICIDE

RESIDUES

b i l e and the " t r i a z i n y l t r a n s f e r a s e " i n l i v e r c y t o s o l (37). A t r a z i n e and i t s metabolites which r e t a i n the 2-chloro group have been shown r e c e n t l y to conjugate s i m i l a r l y i n v i t r o (38). I t was p u z z l i n g to f i n d that Simazine (Figure 17) i n our hands, though y i e l d i n g GSH conjugates i n v i t r o , did not a f f o r d mercapturic acids i n the urine when dosed to r a t s . The conjugation i n plants has been found to occur i n sugar cane leaves, com leaves, sorghum leaves, b a r l e y shoots, Johnson grass and Sudan grass and to be a general r e a c t i o n of 2-chlorosym-triazines (39)(40). Further s t u d i e s (41) have shown that the " a t r a z i n y l g l u t a t h i o n e " i n i t i a l l y formed i s c a t a b o l i s e d in p l a n t s f i r s t by loss of g l y c i n e , then glutamic a c i d (cf the reverse order f o r mammals shown i n Figure 2) to y i e l d the Sis u b s t i t u t e d c y s t e i n e . T h i s undergoes an JS transt r i a z i n y l a t i o n reactio t N-substituted cystein which the forms a l a n t h i o n i n e conjugat r e a c t i o n s are summarise A p o s s i b l y analogous r e a c t i o n of d i a z i n o n (Figure 19) has been reported (42), however, t h i s may be an analogue of aryl transfer. Miscellaneous Casida and co-workers (43) have r e c e n t l y reported that thiocarbamate h e r b i c i d e s are a l s o a c t i v a t e d f o r GSH conjugation by ^-oxygenation i n mouse l i v e r . The sulphoxide can be i s o l a t e d but the conjugates are unstable (Figure 20). General Comment T h i s b r i e f review has demonstrated the wide v a r i e t y of s t r u c t u r e s which may be i n v o l v e d i n GSH conjugation. There are undoubtedly other r e a c t i o n s to be discovered. There i s however, an important u n i f y i n g feature about a l l of the substrates of the GSH t r a n s f e r a s e s . They a l l possess, to a greater or l e s s e r extent, an e l e c t r o p h i l i c carbon atom which has been marked i n many of the f i g u r e s so f a r . The r e a c t i o n s a l l possess a marked s i m i l a r i t y to an SN2 r e a c t i o n of a sulphur n u c l e o p h i l e with e l e c t r o p h i l i c carbon. T h i s i s c l e a r l y the main d r i v i n g f o r c e f o r the r e a c t i o n . In t h i s property, GSH conjugation d i f f e r s from the other two general types of conjugation, i n that the a c t i v a t i o n f o r the r e a c t i o n d e r i v e s mainly from the i n t r i n s i c r e a c t i v i t y of the x e n o b i o t i c . Thus we can recognise the three major types of conjugation (Figure 21) as (a) i n which the f o r e i g n acceptor substrate reacts enzymatically with an endogenous r e a c t i v e donor, as with glucuronide formation from UDPGA (b) i n which the f o r e i g n substrate becomes a c t i v a t e d to a donor v i a an endogenous mechanism and then r e a c t s with an acceptor, as with the forma t i o n of hippurates from benzoic acids and (c) i n which the

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

HUTSON

Glutathione

G S H

Conjugates

115

l > [ RCH SSG ] + HCN 9

I ^SH • RCH SH 2

Pesticide Biochemistry and Physiology Figure 16. Bioactivation of alkyl thiocyanates by GSH alkyl transferase (34)

+ GSSG

Cl

CI

CI

CH, Et N "

-CH

H

H

EtN'

CH

atrazine

H 3

N H

CN

CH

EtN"

N Et

H

3

cyanazine

Figure 17.

Substrates of GSH triazinyl transferase

Glut-Cyst

Lanthionine conjugate

Journal of Agricultural and Food Chemistry Figure 18. Catabolism of a sym-triazine conjugate in plants (41)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

B O U N D A N D C O N J U G A T E D PESTICIDE

RESIDUES

OEt

Pesticide Biochemistry and Physiology Figure 19.

Detoxication of diazinon

(42)

GSH

R-S-C-N II

O

/ \ .

[01 R

1>

2

\

* O

i

"

S-C-N II O

/ \

• products R

2

Pesticide Biochemistry and Physiology Figure 20.

PHENOL

Sulfoxidation as an activation step for GSH conjugation (43)

active d o n o r

BENZOIC ACID

UDPGA

•

+• e n z y m e

activation^ via C o A

PHENYL

BENZOYL CoA

—

no further activation needed

Figure 21.

Glycine ^

(active d o n o r )

ALKYL HALIDE

GLUCURONIDE

ggpjZOYL

+ enzyme

•

ALKYL

GLUTATHIONE

+ enzyme

Classes of conjugation reaction

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

GLYCINE

7.

HUTSON

Glutathione

Conjugates

f o r e i g n substrate i t s e l f possesses

117 the required r e a c t i v i t y .

2. Some Techniques Used To I n v e s t i g a t e Cysteine and Conjugates

GSH

Mercapturic Acids These present no s p e c i a l problems as they can u s u a l l y be extracted from u r i n e at pH 2-3 with ether, or from f r e e z e - d r i e d urine by e x t r a c t i o n with methanol. They can then be methylated and subjected to the usual p h y s i c a l methods such as nmr s p e c t r o metry and mass spectrometry. I f the carbon - sulphur bond i s s t a b l e to a c i d , i t i s u s e f u l to remember that a mercapturic a c i d of a n e u t r a l compound i s only a n i o n i c before h y d r o l y s i s , but amphoteric a f t e r h y d r o l y t i These changes can be monitore and pH 2. A s e n s i t i v e microtest f o r a carbon - sulphur bond (sodium formate f u s i o n t e s t and d e t e c t i o n of H2S with lead acetate paper) i s a l s o an a i d to i d e n t i f i c a t i o n . T h i s t e s t works at the 5 ug l e v e l . T h i s combination of t e s t s was used to i d e n t i f y the mercapturic a c i d derived from cyanazine (36), with the exception that a c i d h y d r o l y s i s s p l i t the C-S bond. A general t e s t f o r mercapturic a c i d formation i s to l a b e l the sulphur pool of r a t s by treatment with [35s]cysteine or by feeding on a d i e t c o n t a i n i n g [35s]yeast, and then c h a l l e n g i n g some of the animals with the t e s t compound. Mercapturic acids i n the u r i n e can be d i s t i n g u i s h e d r e a d i l y from the background of sulphate e t c . Synthesis. These compounds may be r e a d i l y synthesised by r e f l u x i n g the sodio d e r i v a t i v e of N - a c e t y l - L - c y s t e i n e with the precursor. I f the true precursor i s an a c t i v e metabolite, t h i s method w i l l not be a p p l i c a b l e i f the metabolite i s not to hand. However, i n t h i s s i t u a t i o n i t i s often p o s s i b l e to devise other routes u t i l i s i n g chemically synthesised intermediates. B i o s y n t h e s i s . T h i s i s not u s u a l l y p r a c t i c a l i n a c o n t r o l l e d manner i n v i t r o because there are too many stages i n the o v e r a l l reaction. C e r t a i n of the techniques discussed below f o r GSH conjugates are, however, a p p l i c a b l e to the mercapturic a c i d s . Glutathione Conjugates These conjugates present more problems than do the merc a p t u r i c acids because of t h e i r high p o l a r i t y and amphoteric nature• I s o l a t i o n . The conjugates w i l l u s u a l l y have to be i s o l a t e d from aqueous media composed of preparations from i n s e c t s , p l a n t s , mammalian l i v e r or mammalian b i l e . They are best i s o l a t e d from

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

118

BOUND

A N D C O N J U G A T E D PESTICIDE RESIDUES

freeze-dried preparations (from which lipophilic materials have been extracted) by extraction with methanol containing 5 to 25% of water. Purification. The concentrated methanol extract may be purified by thin-layer chromatography, column chromatography using s i l i c i c acid and very polar solvents (37)(41), e.g. butanol:formic acid:water (70:7:7 v/v), or by paper chromatography using butanol:acetic acid:water mixtures* Methods of detection w i l l depend on the compound but radiochemical methods and UV absorbance w i l l be common. The GSH conjugates are ninhydrin-positive and this provides a sensitive and very helpf u l indicator, particularly i f another method (e.g. radiochemistry) i s simultaneously available for the xenobiotic portion of the conjugate. Ion-exchang by Lamoureux et a l (35 require much investment of time to optimize. As a glutathione conjugate i s progressively purified, preparative paper electrophoresis u t i l i s i n g the amphoteric nature of the molecules may be useful as a penultimate purification step. The buffer ions w i l l then have to be removed by a further chromatographic step. Structural Identification. One of the most complete identifications of a glutathione conjugate published i n recent years i s that of the plant atrazine conjugate by Lamoureux et a l (35). This may be a reflection of the surprise at d i s covering the mechanism in plants. Figure 22, adapted from the publication of these workers, illustrates the steps taken to effect the identification. (a) Chemical Hydrolysis. Acid hydrolysis (6N HC1, 100°, 20 h) followed by routine application of an amino acid analyzer serves to identify glycine and glutamic acid. Cysteine w i l l be identified (as cystine) i f the C-S bond i s acid-labile, but normally i t w i l l appear as an j>-substituted cysteine. This can be derivatised as the N-T,4-dinitrophenyl derivative and methylated to afford a derivative which may well be suitable for mass spectrometry. S-Aryl glutathiones are labile to base (e.g. N NaOH, 40°, 2~h) yielding the thiophenol (as dimer i f oxidised). During this process the cysteinyl moiety undergoes 3-elimination to yield the tripeptide, y-glutamyldehydroalanylglycine, which can be hydrolysed in acid to glutamic acid, glycine and pyruvic acid (44). (b) Hydrogenolysis. Refluxing with Raney nickel i n aqueous ethanol can be an excellent and simple way of demonstrating the site of sulphur substitution on the xenobiotic portion of the molecule. The latter should then be amenable to mass spectrometry.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HUTSON

Glutathione Conjugates

OH

Journal of Agricultural and Food Chemistry

Figure 22. Identification of a glutathione conjugate (35)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

120

B O U N D A N D C O N J U G A T E D PESTICIDE

RESIDUES

(c) Terminal Analyses. C l a s s i c C and N-terminal amino a c i d a n a l y s i s are c l e a r l y necessary f o r unequivocal i d e n t i f i c a t i o n when t h i s i s r e q u i r e d . (d) Spectroscopy. The change i n UV spectrum of the x e n o b i o t i c ( p r e f e r a b l y w i t h a m e t h y l t h i o analogue i n comparison) may be suggestive of c o n j u g a t i o n . IR spectroscopy may be o f l i m i t e d v a l u e , but the c h a r a c t e r i s t i c s of the amide bonds may be recognisable. (e) NMR Spectroscopy. Proton NMR spectroscopy of the GSH conjugates i s very complex and not normally used f o r i d e n t i f i c a t i o n . The C NMR s p e c t r a of the GSH conjugate of cyanazine has been measured (45) as an a i d t o the i d e n t i f i c a t i o n o f s y n t h e t i c compound. F i g u r e 23 i l l u s t r a t e th s p e c t r f GSH th cyanazine conjugate an cyanazine. A t present applicatio l i t e s i s l i m i t e d by i t s s e n s i t i v i t y (about 1-5 mg). 1 3

( f ) Mass Spectrometry. W h i l s t the fragments of the GSH conjugates can normally be s u i t a b l y d e r i v a t i s e d and presented t o a mass spectrometer f o r e l e c t r o n impact and chemical i o n i z a t i o n s t u d i e s , I am not aware of a GSH conjugate which has been succ e s s f u l l y d e r i v a t i s e d as a whole and so analysed. The new technique o f f i e l d - d e s o r p t i o n mass spectrometry (FDMS) would seem t o be i d e a l f o r the c h a r a c t e r i s a t i o n of these conjugates. I t s major s t r e n g t h i s i t s a p p l i c a b i l i t y t o p o l a r m a t e r i a l s (46). Chemical Synthesis of GSH Conjugates. Chemical s y n t h e s i s should provide no r e a l problems because i t i s e s s e n t i a l l y a question of f i n d i n g the c o r r e c t e l e c t r o p h i l i c s p e c i e s t o present to the e x c e l l e n t sulphur mucleophile i n the c o r r e c t medium. However, the problem can sometimes be tedious even when the parent x e n o b i o t i c i s r e a c t i v e . A major d i f f i c u l t y i s that g l u t a thione i s i n s o l u b l e i n o r g a n i c s o l v e n t s and the x e n o b i o t i c precursors are u s u a l l y i n s o l u b l e i n water. Each problem has t o be faced i n d i v i d u a l l y , the i d e a l s o l u t i o n being t o c h e m i c a l l y a c t i v a t e the p r e c u r s o r i n such a way that i t can be presented to GSH i n aqueous medium ( t h i s i s probably what the t r a n s f e r a s e a c h i e v e s ) . T h i s approach can be e x e m p l i f i e d by c o n s i d e r i n g cyanazine again. Cyanazine was incubated f o r about 24 h at 40° w i t h a s a t u r a t e d s o l u t i o n o f GSH i n DMSO i n the presence of s o l i d sodium b i c a r b o n a t e . A poor y i e l d of the conjugate was l a b o r i o u s l y p u r i f i e d from the r e a c t i o n mixture. However, when cyanazine i s reacted w i t h trimethy1amine i n acetone, a 2trimethylammonium c h l o r i d e analogue i s formed i n e x c e l l e n t y i e l d (37). The product i s s t a b l e , f r e e l y s o l u b l e i n water, and r e a c t s very r a p i d l y w i t h GSH i n the presence o f a mole of sodium b i c a r b o n a t e . The r e a c t i o n i s e q u a l l y s u i t a b l e f o r the s y n t h e s i s of gram q u a n t i t i e s and s u b - m i l l i g r a m q u a n t i t i e s ( e . g .

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Figure 23.

•kwkm^^

S3

CM

C

13

2

2H

2

0

2

C

7

H

2

1

7

2

2

2

2

2

2

3

2

2

2

v

N

1

6

3

9

14.8 CH, CH, NH 163.2 35.7 or

13.04 ^ C H

2 1 3

27.7 CHa 46.9

3

NH-C-CN 163.2 U 1 or 27.7 163.9

N

S 1 179.9 179

2—Methylmercapto analogue of cyanazine

2

f " O 45.1 O 47.4 H N CH CH CH -C-NH CH-C-NH-CH C0 H 54.0 31.2/31.9 I CH 26.8 S ^j^178.1 N 26.9 CH 14.3 35.9 || J | 123.1 NH-C-CN CH CH NH I 163.2 CH, 26.9

5

?! ° R71 0 il 43.0 II H N CH-CH CH -C-NH-CH-C-NH-CH C0 H 55.2 27.5 32.7 26.9 I SH

Nuclear magnetic resonance spectra of glutathione and a conjugate (45)

TMS

(1: 3

* wcvjP«4 CM

Glutathione

122

BOUND

A N D CONJUGATED

PESTICIDE

RESIDUES

radiosynthesis)• The reactions are shown in Figure 24* 3. Enzymic Synthesis and Degradation of Glutathione Conjugates The biosynthesis and biodegradation of these conjugates are helpful i n their characterisation and to an understanding of the mechanisms of their formation* Biosynthesis Crude transferase may be prepared from rat or rabbit l i v e r cytosol (100,000 g supernatant) and stored frozen for long periods. If i t is dialysed before storage (to remove GSH) and partially purified by ammonium sulphate precipitation (21)(37), the requirement for GSH b demonstrated b includin control reaction containin GSH should be added a pH 7.5. The GSH conjugate of cyanazine can be synthesised directly by this method (37). Some conjugates cannot be so prepared however because bioactivation steps (e.g. epoxidation) occur i n vivo. These reactions must be initiated i n v i t r o by incubating with the transferase i n the presence of liver microsomes and cofactors. The post-mitochondrial supernatant w i l l often substitute for the latter mixture, but i t i s preferable to carry out the various stages separately to understand what events are occurring. The f i r s t studies of GSH conjugation i n plants were effectively in v i t r o studies, u t i l i s i n g the action of excised sorghum leaf sections on atrazine (35). The enzyme has been purified 8-fold from com leaves by extraction, centrifugation, ammonium sulphate fractionation and gel f i l t r a t i o n , (40). Biodegradation Y-Glutamyltranspeptidase should remove the glutamic acid from a GSH conjugate and carboxypeptidase should remove glycine. Few controlled studies have been carried out however. An exception i s the hydrolysis of the j>-atrazine derivative of glutamyl-cysteine isolated from sorghum (41). The metabolite was incubated with hog kidney y-glutamyltranspeptidase and the product was identified as the S-atrazine conjugate of cysteine. We have also observed the hydrolysis of the GSH conjugate of cyanazine under similar conditions, but using alanine as the glutamate acceptor. The use of C - S lyase i n the characterisation of the lanthionine conjugate of atrazine (41) i s also of great value in the specialised area of sym-triazine biochemistry.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

HUTSON

123

Glutathione Conjugates

Some Properties of the Glutathione

S-transferases

Virtually a l l of the mammalian transferases reported are located in the cytosol of the l i v e r c e l l . The main impression gained from working in this area i s that there i s an enzyme for every substrate. The careful work of Chasseaud in d i f f e r entiating between several alkene transferases (33) has been followed by the purification of three other transferases from rat liver cytosol to homogeneity. Six reactions were used to monitor the purifications (31). The results were not encouraging in terms of substrate s p e c i f i c i t y . The three proteins possessed a considerable overlap in s p e c i f i c i t y and they were specific neither for the leaving group nor for the carbon skeleton of the transferred group. However, the protein Menton kinetics, they ar for GSH. Glutathione methyl transferase (46-fold purified) i s stereospecific i n i t s demethylation of dimethyl 1-naphthyl phosphorothionate (47). A very thorough kinetic study has been carried out on 76-fold pure GSH aralkyltransferase (menaphthyl sulphate) (48). The reaction product activates the enzyme when GSH i s saturating and the concentration of substrate i s low. The enzyme may exist in two sub-units separable by isoelectric focussing. The thiol group of GSH does not possess any special nucleophilic properties (towards benzene oxide) which would not be predicted from i t s pKa (49), therefore the s p e c i f i c i t y of GSH relative to other endogenous thiols must be conferred by the enzyme(s). It has been suggested (50) that ligandin, a soluble protein in liver c e l l s which binds several organic ions, may be identical with GSH aryltransferase. This protein comprises about 4Z of that in the liver c e l l . The multiplicity and overlapping substrate specificity of these transferases is d i f f i c u l t to acknowledge, and i t may yet prove possible to rationalise the a c t i v i t i e s as being due to isoenzymes. The s i t uation i s somewhat analogous to that of cytochrome P450, the terminal mono—oxygenase of l i v e r microsomes. This material i s present in relatively massive amounts and i t s identity as one enzyme, or as a plethora of related enzymes, i s s t i l l the subject of much discussion. The mechanism of action of the enzyme(s) i s not yet known. It i s thought that both substrates are bound to protein. It i s l i k e l y that this interaction allows the highly polar, solvated GSH molecule into a reasonably dipolar aprotic environment in which an efficient SN2 reaction with the l i p o p h i l i c foreign compound can take place. Spectacular increases in the rates of SN2 reactions have been observed when reaction conditions are changed from protic solvents to dipolar aprotic solvents (e.g. acetone, DMSO) (51).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

124 5.

BOUND AND

The Occurrence of GSH

CONJUGATED PESTICIDE

RESIDUES

Transferases

The transferases appear to be vide spread in nature. Mammals Investigations of inter-species variations are patchy, and have not routinely been carried out, but what information exists suggests that the cytosol of a l l mammalian liver contain the enzyme(s). Chasseaud (33) made a six-substrate comparison of GSH alkene transferases"^ livers of rat, mouse, ferret, rat, dog, rabbit, guinea-pig, hamster, human adult and human foetus* Level8 were generally of the same order, but those for humans were low on average* The methyl transferase (organophosphate insecticides rabbit, dog and pig (21) i s present in the livers and kidneys of rat, mouse, guinea-pig, rabbit, lamb (very high), ox, monkey, pig and cat(16). The aryl transferase i s widely distributed in mammals, including sheep (52)• Organ variation of the enzyme i s now being studied increasingly. A ten-substrate study of alkene transferase i n rat liver and kidney showed that the activity in liver exceeded that in kidney by factors of about one and a half to seven fold depending on the substrate used for assay. Methyl transferase (methyl iodide) has been found i n the rat l i v e r , kidney and adrenal but not in the heart, lung, spleen, blood or brain (16)• However, brain, spleen, lung, heart, kidney and muscle are reported to be active in the GSH-dependent demethylation of methyl parathioa (19). Epoxide transferase i s also present in extra-hepatic tissues including those of the foetus (53). A recent study of GSHepoxide transferase using naphthalene-1,2-oxide and styrene oxide as substrates and [35s]GSH for quantitation has revealed that this enzyme i s widely distributed in rat tissues, though liver and kidney contain about 10 and 7 times the mean value found i n the other tissues (54). The same technique was used to show that the transferase to naphthalene-1,2-oxide was present in livers from a variety of species as follows: (activity as nmoles conjugate formed/g wet wt/min. in parentheses) sheep (463); horse (300); cattle (330); pig (86); monkey (96); rabbit (152); guinea-pig (448); male rat (228); female rat (187); male mouse (665) and female mouse (327) (54). Aryl transferase has been found in the lung of rabbit at about one-fifth of the specific activity found in l i v e r . Birds Alkene transferase i s present in pigeon liver (33)* Aryl transferase has been found in the livers of several wild birds in the order pheasant > gull > coot > duck > eider > grebe >

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

HUTSON

Glutathione Conjugates

125

goosander (55)* Chicken l i v e r and kidney contain low alkyltran8ferase activity (16)* Insects Alkyltransferase occurs in the midgut region of the horn beetle, in s i l k worm larvae (19), and i n houseflies (56)* Aryl transferase occurs i n grass grubs (52)• Aralkyl transferase occurs i n the locust (57), housefly, flourbeetle, cockroaches, cattle tick, cotton staTner and turnip beetle (58)• The insect enzyme can often be distinguished from the mammalian enzyme by d i f f e r e n t i a l inhibition (52)(58)• Plants We do not yet kno plants but aryl transfer (27)(44) and several triazinyltransfers (39) have now been characterised at the USDA Laboratory at Fargo* Some organophosphate insecticides are O-dealkylated in plants (59) and this reaction may well be medTated by such a transferase. The Normal Role of the Enzymes In mammals GSH transferases play a role in the metabolism of steroidal estrogens i n vivo (13)* Two conjugates of 17-3estradiol have been biosynthesised (60)* The 2,3-unsaturated acylcoenzyme A thiol esters are also substrates for a transferase (61). Their reaction with GSH could be the i n i t i a t i n g mechanism for the excretion of the normal j>-(carboxyalkyl) cysteines found i n animal and human urine. 6. The Significance of GSH Conjugation General The formation of a GSH conjugate effects a dramatic change in the physical properties of a molecule. Thus a small, lipop h i l i c , neutral molecule may be altered in one or two biosynthetic steps into a molecule which i s about twice as large, very lipophobic and possessing both anionic and cationic properties. I t i s therefore highly l i k e l y that, regardless of the chemical changes that have taken place, the physical changes w i l l destroy the biocidal properties of the parent molecule. The formation of the conjugates however, may have different consequences i n plants, insects and animals.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND

126

A N D CONJUGATED

PESTICIDE

Pesticide Biochemistry and Physiology

Figure 24.

Alternative syntheses of GSH conjugate (37)

SG Figure 25.

Glutathione conjugates of 17 fi-estradiol

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RESIDUES

7.

HUTSON

Glutathione Conjugates

127

Plants Plants do not have efficient excretory mechanisms for GSH conjugates, however, the reaction normally leads to destruction of biological activity of a compound. Therefore the possession of an appropriate transferase by a particular species may be expected to confer a degree of resistance to a foreign compound* This has been shown to be the case with atrazine in susceptible and resistant plant species (62)(63)* However, i t seems that plants, like animals, possess a range of enzymes with various s p e c i f i c i t i e s . The transferase that catalyses the cleavage of fluorodifen i s different from the enzyme that catalyses the metabolism of atrazine* Therefore, the finding that fluorodifen- resistant plants like cotton, soyabean and peanut are susceptible to atrazin of certain herbicides The cataboli8m of GSH conjugates and subsequent biochemical incorporation of a substituted cysteine may be a mechanism of such binding. Alternatively the incorporation may be due to the direct interaction of the precursor with t h i o l groups of proteins. In view of the specificity of the proteinsynthesis ing systems, the former mechanism would seem unlikely. Insect8 Certain of the transferases effect the detoxication of organophosphate insecticides in insects and there i s some evidence that resistance to these insecticides i n the tobacco budworm i s associated with higher levels of alkyl transferase than are present in susceptible strains(64)* The excretion processes for GSH conjugates in insects have not been studied in d e t a i l . Mammals There are three major consequences of GSH conjugation i n the mammalian l i v e r : (i) specific bioactive properties are lost; ( i i ) the conjugate i s ideally structured for b i l i a r y secretion and the compound i s therefore e f f i c i e n t l y removed from the organ; ( i i i ) electrophilic compounds/metabolites are scavenged. The last of these consequences i s probably the most important one. It i s now established that many foreign compounds exert their long-term toxic effects by reaction of electrophilic centres in the parent molecules (or of centres generated by metabolism) with DNA, RNA, proteins and other c r i t i c a l sites in the c e l l . Glutathione, being present i n a l l c e l l s i n relatively high concentrations and containing the nucleophilic

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thiol group, is a protective agent par excellence. G i l l e t t e and coworkers have clearly demonstrated this protective effect against the hepatic necrosis induced by massive doses of the drug, acetaminophen. The degree of necrosis correlates with the extent of oxidative metabolism of acetaminophen (65). The severity of the necrosis i s proportional to the amount of covalent binding of a metabolite to liver protein (66). The binding i s mediated by cytochrome P450 (67). Acetaminophen causes a dose-related decrease in hepatic GSH. Experimental depletion of GSH potentiates the necrosis and increases the covalent binding. Administration of cysteine reverses these effects (68). A similar situation exists with bromobenzene. which is oxidatively metabolised to i t s 3,4-oxide (Figure 12) which i s then detoxified by the action of GSH and GSH epoxide transferase. However, GSH depletion, the centrilobula subject to necrosis (69). These regions are close to the portal blood supply and therefore subjected to the highest concentration of compound. Glutathione can be progressively depleted from these cells as i t i s u t i l i s e d i n reaction with the bioactived bromobenzene (the epoxide). When GSH levels are severely depleted (>90%), the epoxide reacts with other components of these c e l l s . The apparently wide occurrence of the GSH transferases in other mammalian organs i s also an important aspect of the protection afforded by GSH. Generally speaking, the enzyme which plays the largest part i n the metabolic generation of electrophiles (the microsomal mono-oxygenase), i s less active in extra-hepatic tissues, i n comparison with the l i v e r . Nevertheless, GSH and the transferase(s) are present i n these tissues which may, therefore, be better protected from covalent interactions than the l i v e r . It would be surprising, however, i f there were not many exceptions to this generalisation because the balance between oxygenase and the transferase w i l l vary with substrate and tissue.

Literature Cited 1. 2. 3. 4. 5. 6.

Baumann, E. and Preusse, C. Chem. Ber. (1879), 12, 806. Jaffé, M. Chem. Ber. (1879), 12, 1092. Waelsch, H. Arch, exp. Pathol. Pharmakol. (1930), 156, 356. Brand, E. and Harris, M. M. Science, (1933), 77, 589. Stekol, J. A. J. biol. Chem. (1939), 128, 199. Smith, J. N., Spencer, B. and Williams, R. T. Biochem. J . (1950), 47, 284. 7. Mills, G. C. and Wood, J. L. J. biol. Chem. (1956), 219, 1. 8. Barnes, M. M., James, S. P. and Wood, P. B. Biochem. J. (1959), 71, 680.

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9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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Bray, H. G., Franklin, T. J. and James, S. P. Biochem. J. (1959), 71, 690. Boyland, E. and Chasseaud, L. F. Adv. Enzymol. (1969), 32, 173. Wood, J. L., in "Metabolic Conjugation and Metabolic Hydrolysis", p. 261, Ed. W. H. Fishman, Academic Press, New York, Vol. 2, 1970. Hutson, D. H., in "Foreign Compound Metabolism in Mammals", The Chemical Society, London; Vol. 3, 1975, p.537; Vol. 2, 1972, p.385; Vol. 1, 1970. p.364. Chasseaud, L. F., in "Glutathione; Proceedings of the 16th Conference of the German Society of Biological Chemistry, Tübingen, 1973", Eds. L. Flohé, H. Ch. Benöhr, H. Sies, H. D. Waller and A. Wendel. G. Thieme, Stuttgart, 1974, p.90. Chasseaud, L. F. Johnson, M. K. Biochem. J. (1966), 98, 38. Johnson, M. K. Biochem. J. (1966), 98, 44. Pillinger, D. J., Fox, B. W. and Jackson, H., in "Isotopes in Experimental Pharmacology", p.415, Ed. L. J. Roth, University of Chicago Press, 1965. Booth, J., Boyland, E. and Sims, P. Biochem. J. (1961), 79, 516. Fukami, J. and Shishido, J. J. econ. Entomol. (1966), 59, 1338. Hollingworth, R. M., Alstott, R. L. and Litzenberg, R. D. Life Sci. (1973), 13, 191. Hutson, D. H., Pickering, B. A. and Donninger, C. Biochem. J. (1972), 127, 285. Iqbal, Z. M. and Menzer, R. E. Biochem. Pharmacol. (1972), 21, 1569. Hollingworth, R. M., in "Biochemical Toxicology of Insecticides", p.75, Ed. R. D. O'Brien and I. Yamamoto, Academic Press, New York, 1970. Hutson, D. H. and Hoadley, E. C. Xenobiotica, (1972), 2, 107. Boyland, E. and Chasseaud, L. F. Biochem. J. (1969), 115, 985. Gillham, B. Biochem. J. (1971), 121, 667. Frear, D. S. and Swanson, H. R. Pest. Biochem. Physiol. (1973), 3, 473. Gillham, B. and Young, L. Biochem. J. (1968), 109, 143. Daly, J. W., Jerina, D. M. and Witkop, B. Experientia, (1972), 28, 1129. Boyland, E. and Williams, K. Biochem. J. (1965), 94, 190. Pabst, M. J., Habig, W. H. and Jakoby, W. B. Biochem. Biophys. Res. Commun. (1973), 52, 1123. Boyland, E. and Chasseaud, L. F. Biochem. J. (1967), 104, 95.

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33. Chasseaud, L. F. Biochem. J. (1973), 131, 765. 34. Ohkava, H., Ohkava, R., Yamamoto, I. and Casida, J. E. Pest. Biochem. Physiol. (1972), 2, 95. 35. Lamoureux, G. L., Shimabukuro, R. H., Swanson, H. R. and Frear, D. S. J. Agric. Fd. Chem. (1970), 18, 81. 36. Hutson, D. H., Hoadley, E. C., Griffiths, M. H. and Donninger, C. J. Agric. Fd. Chem. (1970),18,507. 37. Crayford, J. V. and Hutson, D. H. Pest. Biochem. Physiol. (1972), 2, 295. 38. Dauterman, W. C. and Muecke, W. Pest. Biochem. Physiol. (1974), 4, 212. 39. Lamoureux, G. L., Stafford, L. E. and Shimabukuro, R. H. J. Agric. Fd. Chem..(1972), 20, 1004. 40. Frear, D. S. and Swanson, H. R. Phytochemistry,(1970), 9, 2123. 41. Lamoureux, G. L., Zaylskie, R. G. J. Agric. Fd. Chem. (1973), 21, 1020. 42. Shishido, T., Usui, K., Sato, M. and Fukami, J. Pest. Biochem. Physiol. (1972), 2, 51. 43. Casida, J. E., Kimmel, E. C. Ohkawa, H. and Ohkawa, R. Pest. Biochem. Physiol. (1975), 5, 1. 44. Shimabukuro, R. H., Lamoureux, G. L., Swanson, H. R., Walsh, W. C., Stafford, L. E. and Frear, D. S. Pest. Biochem. Physiol. (1973), 3, 483. 45. Leworthy, D. P. (1975). Sittingbourne Research Centre, unpublished work. 46. Schulten, H. R., Prinz, H., Beckey, H. D., Tomberg, T. W., Klein, W. and Korte, F. Chemosphere, (1973), 2, 23. 47. Hutson, D. H. Med. Fac. Landbouwwet. Gent, (1973), 38, 741. 48. Gillham, B. Biochem. J. (1973), 135, 797. 49. Reubens, D. M. R. and Bruice, T. C. J.C.S. Chem. Comm. 1974, 113. 50. Kaplowitz, N., Percy-Robb, I. W. and Javitt, N. B. J. Exp. Med. (1973), 138, 483. 51. Miller, J. and Parker, A. J. J. Am. Chem. Soc. (1961), 83, 117. 52. Clark, A. G., Darby, F. J. and Smith, J. N. Biochem. J . (1967), 103, 49. 53. Juchau, M. R. and Namkung, M. J. Drug Me tab. Disposit. (1974), 2, 380. 54. Hayakawa, T., Lemahieu, R. A. and Udenfriend, S. Arch. Biochem. Biophys. (1974), 162, 223. 55. Wit, J. G. Europ. J. Pharmacol. (1968), 5, 100. 56. Ishida, M. and Dahm, P. A. J. econ. Entomol. (1965), 58, 602. 57. Cohen, A. J. and Smith, J. N. Biochem. J. (1964), 90, 449. 58. Cohen, A. J., Smith, J. N. and Turbert, H. Biochem. J. (1964), 90, 457. 59. Beynon, K. I., Hutson, D. H. and Wright, A. N. Residue Rev. (1973), 47, 55.

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60. Kuss, E. Z. physiol. Chem. (1969), 350, 95. 61. Speir, T. W. and Barnsley. E. A. Biochem. J. (1971), 125, 267. 62. Shimabukuro, R. H., Swanson, H. R. and Walsh, W. C. Plant Physiol. (1970), 46, 103. 63. Shimabukuro, R. H., Lamoureux, G. L., Frear, D. S. and Bakke, J. E. IUPAC Internat. Symp. on Terminal Residues, Tel Aviv, Ed. A. S. Tahori, Butterworths, London, 1971, p.323. 64. Bull, D. L. and Whitten, C. J. J. Agric. Fd. Chem. (1972), 20, 561. 65. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R. and Brodie, B. B. J. Pharmacol. Exptl. Therap. (1973), 187, 185. 66. Jollow, D. J., Mitchell Gillette, J. R. an Therap. (1973), 187, 195. 67. Potter, W. Z., Davies, D. C., Mitchell, J. R., Jollow, D. J., Gillette, J. R. and Brodie, B. B. J. Pharmacol. Exptl. Therap. (1973), 187, 203. 68. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R. and Brodie, B. B. J. Pharmacol. Exptl. Therap. (1973), 187, 211. 69. Reid, W. D. and Krishna, G. Exp. Mol. Pathol. (1973), 18, 80.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8 Miscellaneous Conjugates—Acylation and Alkylation of Xenobiotics in Physiologically Active Systems JORG IWAN Schering AG, Berlin/Bergkamen, Plant Protection Division, D-1 Berlin 65, Postfach 650311, West Germany

"Conjugation", as used by the p e s t i c i d e chemist, represents a c o l l e c t i v e term f o r reactions u s u a l l y catalyzed by enzymes an are l i n k e d to xenobiotics compounds takes place at f u n c t i o n a l groups that may already be present in the parent molecules or added in the course of the metabolic process. Formation of conjugates can a f f e c t the b i o l o g i c a l a c t i v i t y of a p e s t i c i d e or drug in two ways: Through decrease of lipid s o l u b i l i t y and through the a l t e r a t i o n of molecu l a r structures e s s e n t i a l f o r the exertion of physiol o g i c a l e f f e c t s . Therefore, conjugation normally r e s u l t s in d e t o x i f i c a t i o n but, as f a r as a c y l a t i o n s and a l k y l a t i o n s are concerned, t h i s is not always the case. 1. ACYLATION 1.1. A c e t y l a t i o n . Transfer of acetate from acetyl-coenzyme A (whose biosynthesis, as a reminder, is shown in fig. 1)to an amino group of a xenobiotic, is c e r t a i n l y the best understood a c y l a t i o n r e a c t i o n observed in biotransformations of f o r e i g n compounds. This r e a c t i o n , which is catalyzed by arylamine N — a c e t y l t r a n s f e r a s e s , represents a general metabolic pathway of aromatic amines, sulfonamides and hydrazino compounds as well as nonaromatic amines in mammals (1 - 4). There are strong i n d i c a t i o n s that generation of the N-acetyl d e r i v a t i v e s proceeds v i a a simple "ping-pong" mechanism i n v o l v i n g two consecutive steps: Formation of a c e t y l a c e t y l t r a n s f e r a s e through a r e a c t i o n between acetyl-CoA and a c e t y l t r a n s f e r a s e and, secondly, r e a c t i o n of the enzyme complex with a s u i t a ble substrate to produce the N-acetate and a c e t y l transferase (5 - 7). 132

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Studies on d e t o x i f i c a t i o n of k,6-dinitro-oc r e s o l i n the r a b b i t , conducted by Smith, Smithies and Williams in 1952 (8), belong to the early investigat i o n r e v e a l i n g N - a c e t y l a t i o n of a p e s t i c i d e or or one of i t s degradation products ( f i g » 2). Among the DNOC metabolites extracted from u r i n e these same workers found 6 - a m i n o - 4 - n i t r o - o - c r e s o l and 6 - a c e t a m i d o - 4 - n i t r o - o - c r e s o l , the N - a c e t y l compound (as an O-glucuronide) being the most abundant convers i o n product. Detection of these substances, whose i d e n t i f i c a t i o n was based on paper chromatography as w e l l as on UV spectrophotometry, and comparison with authentic standards demonstrated that i n the r a b b i t , r e d u c t i o n and subsequent a c e t y l a t i o n of d i n i t r o - o c r e s o l c o n s t i t u t e d the predominant mechanism of inactivation. Intensive research on m i c r o b i a l decomposition of a n i l i n e d e r i v a t i v e s o c c u r r i n g as degradation products from a v a r i e t y of p e s t i c i d e s i n the s o i l environment s t a r t e d approximately i n the middle s i x t i e s . In the course of these studies which r e c e i v e d a s p e c i a l momentum by the discovery of m i c r o b i o l o g i c a l azobenzene formation, N - a c e t y l a t i o n of a n i l i n e s was recognized as a metabolic conversion common i n a v a r i e t y of f u n g i , b a c t e r i a and algae (9 - 15)* Some of the compounds studied are shown i n f i g . 3» During t h e i r i n v e s t i g a t i o n s on the metabolism of metobromuron by s e l e c t e d s o i l microorganisms Tweedy and coworkers detected the r a p i d and q u a n t i t a t i v e a c e t y l a t i o n of p-bromoaniline (9) {fig* *0 • These authors suggested that conversion of a n i l i n e s to t h e i r r e s p e c t i v e N - a c e t y l d e r i v a t i v e s may be competitive with the concentration dependent o x i d a t i v e coupling to azobenzenes i n s o i l . I d e n t i f i c a t i o n of the a c e t a n i l i d e s was accomp l i s h e d through cochromatography of s o i l and c u l t u r e extracts with known standard compounds or t h i n l a y e r and gas chromatography combined with mass spectrometry, A c h a r a c t e r i s t i c of the mass spectra obtained using e l e c t r o n impact i o n i z a t i o n was the loss of ketene from the molecular ions thus g i v i n g r i s e to the appearance of the corresponding a n i l i n e r a d i c a l ions i n high r e l a t i v e abundance. Needless to say these modern studies were c a r r i e d out employing carbon 1^ l a b e l e d parent compounds. Highly i n t e r e s t i n g as to the way of i t s format i o n , i t s mass spectrometric fragmentation pattern and i t s ultimate metabolic f a t e was the d e t e c t i o n of 4 - c h l o r o - 2 - h y d r o x y - a c e t a n i l i d e i n c u l t u r e s of Fusarium oxysporum fed with p - c h l o r o a n i l i n e (13) ( f i g . 5)•

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Figure 1.

O

OH

°

2

N

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C

2

H

—

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OH

it

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2

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2

Figure 2.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Acylation and Alkylation of Xenobiotics

Figure 5.

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136

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Upon e l e c t r o n impact, generation of a benzoxazole r a d i c a l i o n by o r t h o - e l i m i n a t i o n of water was observed ( f i g . 6). Discovery of such a fragment may be a u s e f u l diagnostic t o o l f o r the i n t e r p r e t a t i o n of respective mass spectra; however, we must bear i n mind that ohydroxy-acetanilides w i l l undergo the same c y c l i z a t i o n on heating thus forming benzoxazoles as a r t e f a c t s . This behaviour could lead to misinterpretations i f a glc-ms combination i s used f o r i d e n t i f i c a t i o n of metabolic products. As f a r as f u r t h e r degradation i s concerned, ohydroxylation may be one of the f i r s t steps toward m i c r o b i a l r i n g f i s s i o n of a n i l i n e s . Conversion of m-arainophenol to m-hydroxy-aceta n i l i d e on sugar bee desmedipham provides an example of the p a r t i c i p a t i o n of higher plants i n the a c e t y l a t i o n of a f o r e i g n compound (~16) (fig. 7). However, the N - a c e t y l d e r i v a t i v e was detected among the chloroform soluble materials recovered from the l e a f r i n s e of treated beets. M i c r o b i a l i n t e r a c t i o n at the plant surface thus e f f e c t i n g N - a c e t y l a t i o n , t h e r e f o r e , cannot be excluded. A c e t y l t r a n s f e r from ^-(N-hydroxyacetaraido)biphenyl to 4-aminoazobenzene, a r e a c t i o n catalyzed by a s p e c i a l acetyltransferase occurring i n rat l i v e r , may i l l u s t r a t e that transacylations do not n e c e s s a r i l y b r i n g about d e t o x i f i c a t i o n (17). In t h i s case a c e t y l a t i o n and deacetylation apparently play an important r o l e i n the carcinogenic act i v i t y of arylamlnes. By way of a c e t y l t r a n s f e r from the hydroxamic a c i d to another arylamine - the metabo l i c step shown i n f i g . 8 - an arylhydroxylamine i s released that can be o x i d i z e d to the corresponding n i t r o s o d e r i v a t i v e . T h i s , i n t u r n , i s more carcinogeni c than any of i t s precursors. 1.2. Formylation. Besides a c e t y l a t i o n , conjugat i o n with other carboxylic acids i s also p o s s i b l e . The simplest representative of the f a t t y a c i d homologues, formic a c i d , has been found i n a v a r i e t y of a c t i v a t e d forms i n l i v i n g c e l l s . Formyltetrahydrofolic a c i d occuring as N-10-formyl and N-5-N-1O-methenyl-tetrahydrofolate ( f i g . 9) i s c e r t a i n l y the most important formate c a r r i e r . F s s e n t i a l metabolic steps i n the biosynthesis of purines ( f i g . 10) depend on the a v a i l a b i l i t y of t h i s formyl source. Other b i o l o g i c a l l y a c t i v e compounds may also be considered as formate donors. Formyl-CoA, f o r i n -

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Acylation and Alkylation of Xenobiotics H c

,A^ogC.

-CH -C« O / / 2

C H 3

\ -H20 \

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CI

3

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m/e 143 r.a. 100

Figure 6.

I

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OH O

OH

N

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N

acetyl

N

transferase

^

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Figure 8.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

H

H

N -Formyl-tetrahydrofolate 10

N5-Nio-Methenyl-tetrahydrofolate

„THF"

Fumarate Figure 10.

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Acylation and Alkylation of Xenobiotics

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139

stance has been demonstrated i n microorganisms and higher plants as shown i n f i g . 11 which gives a part i a l view of the glyoxylate c y c l e . N-Formyl-L-kynurenine an intermediate i n t r y p t o phane metabolism, was found to be capable of transf e r r i n g formate to a n i l i n e , naphthylamine and ant h r a n i l i c a c i d i n v i t r o (18) ( f i g . 12). The transformylation was catalyzed by kynurenine formamidase from guinea-pig l i v e r . 2-Formamido-1naphtyl hydrogen sulphate, detected as a metabolite of 2-naphthylamine i n the urine of dogs and r a t s , may have received i t s formyl group by r e a c t i o n with t h i s enzyme system (19)• M i c r o b i a l transformation of a n i l i n e s to formanilides i n s o i l and pure fungal cultures was observed by Kaufman 20) ( f i g . 13). 3,^-Dichloroformanilide, as a metabolite of 3t^d i c h l o r o a n i l i n e i n s o i l , could be i d e n t i f i e d a f t e r p u r i f i c a t i o n by column and preparative t h i n l a y e r chromatography using i n f r a r e d and mass spectrometry. Loss of 28 mass u n i t s from the parent i o n was i n d i c a t i v e of the e l i m i n a t i o n of a formyl group as carbon monoxide, a fragmentation analogous to the loss of ketene from a c e t a n i l i d e s • I n t e r p r e t a t i o n of the spect r a was confirmed by synthesis of authentic 3t^dichloroforraanilide obtained from 3,4-dichloroaniline through r e a c t i o n with a c e t i c formic anhydride or r e f l u x i n g i n e t h y l formate under atmospheric pressure. 9

f

1.3* Malonic a c i d conjugation. Conjugation with malonic a c i d has been observed as a mechanism f o r d e t o x i f i c a t i o n of D-amino acids i n higher plants (21, 22). Malonyl-CoA which plays a c e n t r a l r o l e i n the biosynthesis of f a t t y acids ( f i g * 1*0 presumably mediates t h i s r e a c t i o n . Malonyl t r a n s f e r to a f o r e i g n compound by microorganisms was reported by Ross and Tweedy (2^)• Studi e s on the fate of chlordimeform i n mixed m i c r o b i a l cultures revealed that ^ - c h l o r o - o - t o l u i d i n e , occurring through stepwise h y d r o l y t i c a l cleavage of the parent compound, was transformed to the respective malonanilic a c i d d e r i v a t i v e ( f i g . 15)• In some experiments t h i s metabolite amounted to as much as k6 $ of the t o t a l administered r a d i o a c t i v i t y thus suggesting that malonic a c i d conjugation may be an important mechanism f o r d e t o x i f i c a t i o n of aromatic amines. I d e n t i f i c a t i o n of the conjugate was accomplished using t h i n l a y e r chromatographic p u r i f i c a t i o n and mass

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

I I

Formyl CoA

I i

! I

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if CO.

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o

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/

\ \ NADP ^| \\CoA-SH4l

^A-FAD • E V - f H 0 2

Glyoxylate

V

H2^2 0 NH,j o

k

A"

4 COOH

1h II II II

2

3

I'

!!H OI !'|2 'I 5

—I

I

Pyr P

L-Glutamate

Figure 11.

Figure 12.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8.

141

Acylation and Alkylation of Xenobiotics

IWAN

HS R

Cx

e

ooc

c

o HaC

CH

s

2

Bk*ln~COO

e

HS

e

R A c CH

O il OOC sC CH SCoA N

X

S

2

x

2

Makmyl-CoA NADP®

0

HC

II /Cv

3

O

-

11

SCoA

°OOC\ / \ CH S C

0

2

o CH H3C

M CH S

- - 6 C0

o

O

II

11

HsC H 0* 2

CH

2

H3C

S * ^ NADPH NADP* + H «

2

CH S 2

HS

Figure 14.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

v

BOUND AND CONJUGATED PESTICIDE RESIDUES

142

spectrometry. C h a r a c t e r i s t i c of the mass spectrum was a loss of kk mass u n i t s from the parent i o n to 183/ 185 which i n d i c a t e d the e l i m i n a t i o n of carbon d i o x i d e . From t h i s point downward i n the d i r e c t i o n of lower mass numbers the spectrum became i d e n t i c a l to that of 4-chloro-o-acetotoluidide the base peak representing the t o l u i d i n e r a d i c a l i o n at § 1^1/143. Synthesis of authentic k -chloro-2 -methylmalonanilic a c i d confirmed these r e s u l t s . Evidence f o r the capacity of higher plants to conjugate a n i l i n e derivates with malonic a c i d was r e cently obtained i n studies on the metabolism of 2,6d i c h l o r o - 4 - n i t r o a n i l i n e i n soybeans (2k) ( f i g » 16). The metabolic pathway involved reduction of DCNA to the corresponding p-phenylenediamine and subsequent malonyl t r a n s f e r to phenyl)-malonamic a c i d as the major conversion produ c t . Spectrometric i d e n t i f i c a t i o n could be confirmed by synthesis from 2,6-dichloro-p-phenylenediamine through r e a c t i o n with e t h y l chloroformylacetate f o l lowed by mild h y d r o l y s i s . 1

1

*\,k. Miscellaneous a c y l a t i o n s . Various other carboxylic acids are known to e x i s t as a c t i v a t e d subs t r a t e s i n l i v i n g c e l l s . Assuming the a v a i l a b i l i t y of a s u i t a b l e t r a n s f e r a s e , a l l of them might be regarded as p o t e n t i a l a c y l a t i o n reagents f o r xenobiotics i f these f o r e i g n compounds can penetrate to the s i t e of enzymatic a c t i o n . The studies of Smith showing that Streptomyces venezuelae can produce a c e t y l , propionyl and b u t y r y l as w e l l as probably pentanoyl and hexanoyl analogues of chloramphenicol, i f the c h l o r i d e i o n conc e n t r a t i o n i n the growth medium i s l i m i t e d , may i l l u s t r a t e t h i s point (25). 2. ALKYLATION 2.1. Methylation. As early as i n 189^ i t was suggested by Hofmeister that methylation of organic and inorganic compounds i n animal t i s s u e s may occur v i a t r a n s f e r of an i n t a c t methyl group (26). A particu l a r compound a c t i n g as a p o s s i b l e methyl source, however, was not mentioned. In 1933t 39 years l a t e r , Challenger, Higginbottom and E l l i s succeeded i n e l u c i dating the nature of the s o - c a l l e d Gosio-gas, a v o l a t i l e arsenic compound produced by several fungi growi n g on arsenic containing media (27)• These authors demonstrated that Gosio-gas e n t i r e l y consisted of t r i m e t h y l a r s i n e , and they proposed a mechanism i n v o l v i n g stepwise a d d i t i o n of formaldehyde to arsenious

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8.

IWAN

143

Acylation and Alkylation of Xenobiotics

CI

Figure 15.

OH

9

o N0

NH

2

2

NH

2

HN-C-CH -COOH 2

HN-C-CH

3

-C^OH NH NH v>H 0 2 NHo ? ? HN-C-CH -C-OC2H NM CI-C-CH -C-OC2H ?

2

2

,,
90* >90 >90' #

i l l i t e > kaolinite. Comparative studies between known clay minerals and organic s o i l s suggest that most, but not all, pesticides have a greater a f f i n i t y for organic surfaces than for mineral surfaces. Scott and Weber (15) found that the phytotoxicities of 2,4-D, prometone, and CIPC to the test plant were reduced to a much greater extent by addition of an organic s o i l to the growth media than by addition of montmorillonite or kaolinite. Donerty and Warren's (16) results show that both fibrous peat and a well-decomposed muck were more adsorptive than bentonite for pyrazone, linuron, prometone, and simazine. Hance (17) concluded that diuron was a more effective competitor for water at organic matter surfaces than at mineral surfaces, and Deli and Warren (18) found that organic

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15.

STEVENSON

185

Reactions Involving Pesticides in Soil

l

NH

I

Figure 3. Clay-metal-organic matter complex

40 -

0

8

16

24

32

Kd Figure 4. Relationship between organic matter content and amount of atrazine adsorbed by 36 soils. Kd *= fimoles adsorbed per g/fimoles per ml equilibrium solution. From Ref. 3 as adapted from Ref. 13.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

186

BOUND AND CONJUGATED PESTICIDE RESIDUES

Table 1.

Chemical designations o f organics mentioned i n t e x t .

Common name

Chemical

formula

s-Triazines Atrazine Simazine At ratone Ametryn Prometon Prometryn Propazine

2-chloro-4-ethylamino-6-isopropylamino-striazine 2-chloro-4,6-bis(ethylamino)-s-triazine 2-methoxy-4-ethylamino-6-isopropylamino-striazlne 2-methylthio-4-ethylamino-6-isopropylamino-s 2-methoxy-4,6-bis(isopropylamino)-s-triazine 2-methylthio-4,6-bis(isopropylamino)-striazine 2-chloro-4,6-bis(isopropylamino)-s-triazine

S u b s t i t u t e d ureas Diuron Monuron Fenuron Linuron Neburon

3-(3,4-dichlorophenyl)-1,1-dimethylurea 3-(p-chlorophenyl)-1,1-dimethylurea 3-phenyl-l,1-dimethylurea 3-(3,4-dichlorophenyl)-1-methoxy-l-methylurea l-butyl-3-(3,4-dichloropheny)-1-methylurea

Phenylcarbamate CIPC

isopropyl

m-chlorocarbanilate

Bipyridylium quaternary s a l t s 1

1

6,7-dihydrodipyrido(1,2-a:2 ,1 -c)pyrazidinium s a l t 1,1'-dimethyl-4,4 dipyridinium s a l t

Diquat

1

Paraquat

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15.

STEVENSON

Reactions

Involving

Pesticides

in Soil

187

Table 1 (Cont'd) Common name

Chemical formula

Others Amiben 2,4-D Picloram Dalapon Diphenamid Trifluralin

3-amino-2,5-dichlorobenzoic a c i d 2,4-dichlorophenoxyacetic a c i d 4-amino-3,5,6-trichloropicolinic acid 2,2-dichloropropionic a c i d N,N-dimethyl-2,2-diphenylacetamide ot

DCPA DNPB Amitrole Pyrazone Lindane DDT

dimethyl-2, β, 5 , 6 - t e t r a c h l o r o t e r e p h t h a l a t e 4,6-dinitro-o-sec-butylphenol 3-araino-l,2,4-triazol 5-amino-4-chloro-2-phenyl-3-(2H)-pyridazone 1,2,3,4,5,6-hexachlorocyclohexane 1,1,l-trichloro-2,2-bis(p-chlorophenyl) ethane

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

188

BOUND AND

CONJUGATED PESTICIDE

RESIDUES

matter was more e f f e c t i v e i n adsorbing diphenamid than c l a y ( b e n t o n i t e ) . In other s t u d i e s , Weber, P e r r y , and I b a r a k i (19) found t h a t , on a weight b a s i s , an organic s o i l was more e f f e c t i v e than m o n t m o r i l l o n i t e i n reducing the p h y t o t o x i c i t y of prometone to wheat. For the s - t r i a z i n e s , prometone and prometryne may p r e ­ f e r m i n e r a l surfaces (20). Laboratory s t u d i e s have, i n g e n e r a l , corroborated f i e l d observations i n d i c a t i n g t h a t organic matter p l a y s a major r o l e i n the performance of s o i l - a p p l i e d p e s t i c i d e s . This work has gen­ e r a l l y i n v o l v e d m u l t i p l e c o r r e l a t i o n a n a l y s i s f o r p e s t i c i d e ad­ s o r p t i o n by a s e r i e s of s o i l s w i t h w i d e l y d i f f e r e n t p r o p e r t i e s , the u s u a l s o i l parameters being organic matter content, t e x t u r e ( c l a y c o n t e n t ) , c l a y m i n e r a l type, pH, and c a t i o n exchange capa­ c i t y . I n a t y p i c a l study, a given q u a n t i t y o f s o i l is added to a p e s t i c i d e s o l u t i o n of allowed to e q u i l i b r a t e , the s o l u t i o n phase is estimated. The amount of p e s t i c i d e adsorb ed is subsequently c a l c u l a t e d from the change i n c o n c e n t r a t i o n and is u s u a l l y expressed by such u n i t s as μ moles adsorbed per Kg of s o i l (x/m). By r e p e a t i n g the measurements a t s e v e r a l p e s t i ­ c i d e c o n c e n t r a t i o n s , an adsorption isotherm can be obtained by p l o t t i n g the q u a n t i t y adsorbed (x/m) v s . the e q u i l i b r i u m concen­ t r a t i o n (C). In most i n s t a n c e s , a s t r a i g h t l i n e is obtained when the data are p l o t t e d as l o g x/m v s . l o g C, according to the F r e u n d l i c h a d s o r p t i o n equation, x/m

- KC

1 / n

where Κ and η are c o n s t a n t s . The constant Κ provides a measure of the extent of a d s o r p t i o n and has been used i n c o r r e l a t i o n s t u d i e s aimed a t determining the r e l a t i v e importance of the v a r i o u s s o i l parameters on a d s o r p t i o n . A l t e r n a t e l y , a d i s t r i b u t i o n c o e f f i c i e n t , Kd, can be obtained f o r a given s o l u t i o n c o n c e n t r a t i o n as the r a t i o of the amount of p e s t i c i d e adsorbed to the amount remaining i n s o l u t i o n K
t r i f l u r a l i n > 2,4-D > diphenamid > DCPA > DNPB > amiben > paraquat (no adsorption)· The most readily desorbed herbicide was 2,4-D; CIPC and DNPB showed little or no desorption. The p o s s i b i l i t y of using activated charcoal to detoxicate herbicide treated s o i l has been discussed by Ahrens (39) and Coffee and Warren (38). Special Role of Fulvic Acids Because of their low molecular weights and high a c i d i t i e s , fulvic acids are more soluble than humic acids, and they may have special functions with regard to herbicide transformations. F i r s t they may act as transporting agents for pesticides i n s o i l s and natural waters· Ogner and Schnitzer (40), and Schnitzer and Ogner (41), suggested that fulvic acids act as carriers of alkanes and other normally water-insoluble organic substances i n aquatic environments, and it is possible that these constituents also function as vehicles for the transport of pesticides. According to Ballard (42), the downward movement of the insecticide DDT i n the organic layers of forest soils is due to water-soluble, humicl i k e substances. Second, fulvic acids by virtue of their high a c i d i t i e s , may

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

192

BOUND AND CONJUGATED PESTICIDE RESIDUES

c a t a l y z e the chemical decomposition o f c e r t a i n p e s t i c i d e s . The suggestion has been made, f o r example, t h a t these c o n s t i t u e n t s might c a t a l y z e the h y d r o x y l a t i o n o f the c h l o r o - s - t r i a z i n e s (30). For a d d i t i o n a l i n f o r m a t i o n regardin g f u l v i c a c i d s and t h e i r r e ­ a c t i o n s , the reader is r e f e r r e d t o the recent book o f S c h n i t z e r and Khan (43). P o t e n t i a l Chemical Reactions I n v o l v i n g P e s t i c i d e s and Organic Substances i n S o i l There seems little doubt but t h a t the organic f r a c t i o n o f the s o i l has the p o t e n t i a l f o r promoting the n o n b i o l o g l c a l degradation of many p e s t i c i d e s . Organic compounds c o n t a i n i n g n u c l e o p h i l i c r e a c t i v e groups o f the types b e l i e v e d t o occur i n humic and f u l v i c a c i d s (e.g., COOH, p h e n o l i c phatic-OH, amino, h e t e r o c y c l i o t h e r s ) are known t o produce chemical changes i n a wide v a r i e t y of p e s t i c i d e s (7, 44). Of a d d i t i o n a l i n t e r e s t is that humic sub­ stances are r a t h e r s t r o n g reducing agents and have the c a p a b i l i t y of b r i n g i n g about a v a r i e t y o f r e d u c t i o n s and a s s o c i a t e d r e a c t i o n s , as discussed by Crosby ( 7 ) . The occurrence o f s t a b l e f r e e r a d i ­ c a l s i n humic and f u l v i c a c i d s f u r t h e r i m p l i c a t e s organic matter i n chemical transformations o f p e s t i c i d e s . F o r example, t h e h e t e r o c y c l i c r i n g o f amitrοle is known t o be h i g h l y s u s c e p t i b l e to a t t a c k by f r e e r a d i c a l s (45, 46). B a s i c amino a c i d s and s i m i l a r compounds have the p o t e n t i a l f o r c a t a l y z i n g the h y d r o l y s i s o f organophosphorus e s t e r s (47), as w e l l as the d e h y d r o c h l o r i n a t l o n o f DDT and l i n d a n e (48)· Miskus et a l . (49) demonstrated that c e r t a i n c h l o r o p h y l l degradation products (reduced porphyrins) can convert DDT t o DM). Substances i n s o i l organic matter which c o n t a i n hydroxy1 and amino groups, such as humic and f u l v i c a c i d s , are p o t e n t i a l l y capable o f being a l k y l a t e d by the a c t i o n o f c h l o r i n a t e d a l i p h a t i c a c i d s (e.g., c h l o r o a c e t i c , d i c h l o r o p r o p i o n i c ) , as shown below (50). R-NH R-OH

2

+ +

Cl-CH -(CH2) -C00H -> R - N H - ( C H ) 2

n

2

n + 1

Cl-CH -(CH ) -C00H -> R - 0 - ( C H ) i " 2

2

n

2

-

C 0 0 H

C O O H

n +

S p e c i f i c examples o f n o n b i o l o g l c a l transformations brought about by the o r g a n i c f r a c t i o n of the s o i l i n c l u d e s h y d r o x y l a t i o n of t h e c h l o r o - s - t r i a z i n e s (51-56) and decomposition o f a m i t r o l e (45>46). w i t h regard t o the former, Armstrong and Chesters (51) concluded t h a t h y d r o l y s i s o f a t r a z l n e r e s u l t e d from the sequence of events shown i n F i g u r e 5. Adsorption was b e l i e v e d t o take p l a c e between a r i n g n i t r o g e n atom and a protonated COOH group o f t h e organic matter. Hydrogen bonding o f the r i n g n i t r o g e n was b e l i e v ­ ed t o cause the withdrawal o f e l e c t r o n s from the e l e c t r o n d e f i ­ c i e n t carbon atom bonded t o the c h l o r i d e ; thereby enabling water to r e p l a c e the c h l o r i d e atom. Nearpass (55)found t h a t propazine h y d r o l y s i s was enhanced i n the presence o f organic matter i r r e s p e c ­ t i v e o f the pH o f the system and was r e l a t e d i n some way t o

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15.

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Reactions Involving Pesticides in Soil

a d s o r p t i o n . I n other work, Hance (53) was unable to e s t a b l i s h a r e l a t i o n s h i p between r a t e o f a t r a z l n e decomposition and extent o f adsorption. The review of Crosby (7) should be consulted f o r other examples o f n o n b i o l o g l c a l degradation of p e s t i c i d e s by r e a c t i o n w i t h o r g a n i c substances. Chemical B i n d i n g o f P e s t i c i d e s and T h e i r Decomposition Products S u b s t a n t i a l evidence e x i s t s t o i n d i c a t e t h a t p e s t i c i d e d e r i v e d r e s i d u e s can form s t a b l e chemical l i n k a g e s w i t h o r g a n i c substances and t h a t such b i n d i n g g r e a t l y i n c r e a s e s the p e r s i s t ence o f the p e s t i c i d e r e s i d u e i n the s o i l (57-61). Two main mechanisms can be e n v i s i o n e d : ( i ) d i r e c t chemical attachment o f the r e s i d u e s t o r e a c t i v ( i i ) incorporation into f u l v i c a c i d s d u r i n g the h u m i f i c a t i o n process ( 3 ) . A key to the f a t e o f p e s t i c i d e s and t h e i r i n t e r m e d i a t e decomposition products may be provided by c o n s i d e r a t i o n o f the p r o cess whereby humic and f u l v i c a c i d s are formed. The l i g n i n - p r o t e i n theory i n i t s o r i g i n a l form is now b e l i e v e d by many i n v e s t i gators to be o b s o l e t e , and the modern view is t h a t humic substance are formed by a m u l t i p l e stage process which i n c l u d e s : (1) decomp o s i t i o n o f all p l a n t components, i n c l u d i n g l i g n i n , i n t o s i m p l e r monomers, (11) metabolism of the monomers w i t h an accompanying i n c r e a s e i n the s o i l biomass, ( i l l ) repeated c y c l i n g of the b i o mass carbon w i t h s y n t h e s i s of new c e l l s , and (jLv) concurrent p o l y m e r i z a t i o n o f r e a c t i v e monomers i n t o high-molecular-weight polymers (8-11). The g e n e r a l consensus is t h a t polyphenols (quinones) s y n t h e s i z e d by microorganisms, together w i t h those l i b e r a t e d from l i g n i n , polymerize alone o r i n the presence o f amino compounds (amino a c i d s , e t c . ) t o form brown c o l o r e d polymers. An a l t e r n a t e pathway is by condensation of amino a c i d s and r e l a t e d substances w i t h reducing sugars, a c c o r d i n g t o the M a i l l a r d r e a c t i o n s . The r e a c t i o n between polyphenols and amino compounds i n v o l v e s simultaneous o x i d a t i o n o f the polyphenol t o the quinone form, such as by polyphenol oxidase enzymes. The a d d i t i o n product r e a d i l y polymerizes t o form brown nitrogenous polymers according to the general sequence shown i n F i g u r e 6. I n the case o f the M a i l l a r d r e a c t i o n , the i n i t i a l step i n v o l v e s a d d i t i o n o f the amine to the C«0 group o f the sugar, w i t h the formation o f an aldosylamine ( F i g u r e 7 ) . This is f o l l o w e d by the Amador1 rearrangement t o form the N - s u b s t i t u t e d keto d e r i v a t i v e , which subsequently undergoes dehydration and fragmentation to y i e l d a v a r i e t y o f unsaturated intermediates (62, 63). I n the f i n a l stages o f browning, the intermediates polymerize i n t o brown polymers and copolymers. The r a t e o f the r e a c t i o n i n c r e a s e s w i t h temperature, pH, and the b a s i c i t y o f the amine. Under l a b o r a t o r y c o n d i t i o n s , brown polymers can r e a d i l y be s y n t h e s i z e d from amino acid-sugar mixtures w i t h i n hours i n aqueous s o l u t i o n a t 50 C. c

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

194

BOUND

A N DCONJUGATED

PESTICIDE

RESIDUES

8Φ Η. δθ CI

I CI

N^S SORPTION N^N5-HO-?-SOM Hx L ï M +SOM-COOH-^zz=^ „ f V R ^ ' N T ^ R DESORPTON R . ^ % M

N

CHLORO - 1 - TRIAZINE

A

CHLORO - £ - TRIAZINE (SORBED)

HYDROLYSIS OH

OH

N ^ N

DESORPTIO + HCI

HYDROXY-S-TRIAZINE

HYDROXY - £ - T R I A Z I N E ( SORBED)

+ SOM-COOH SOM-COOH = CARBOXYL

FUNCTIONAL

GROUP ON SOIL ORGANIC MATTER. Pesticides in Soil and Water

Figure 5. Proposed model for the sorption-catalyzed hydrolysis of chloro-striazines by soil organic matter (6)

Q

NH -CH -COOH 2

H

2

ρ

NH -CH -COOH 2

2

/OH

N-CH -COOF 2

4H +4e +

Condensation of intermediates

NH CH COOH 2

OH

OH NH

B r o w n nitrogenous

CH-COOH

II 2

polymers CHO-COOH NH

NH

I

I CH -COOH 2

CH -COOH 2

Figure 6. General scheme for the formation of brown nitrogenous polymers by con­ densation of polyphenols and amino acids as exemplified by the reaction between catechol and glycine

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15.

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195

Condensation r e a c t i o n s between p e s t i c i d e s and t h e i r degradat i o n products with organic substances i n s o i l would be enhanced by such processes as f r e e z i n g and thawing, wetting and d r y i n g , and the i n t e r m i x i n g of r e a c t a n t s w i t h m i n e r a l matter having c a t a l y t i c properties. I t is r a t h e r evident that r e a c t i o n s s i m i l a r to these shown i n F i g u r e s 6 and 7 could be i n v o l v e d i n p e s t i c i d e transformations i n s o i l . Many weakly b a s i c compounds, i n c l u d i n g amino a c i d s , p y r r o l s , amides, amines, and imines, are known to have the a b i l i t y to combine c h e m i c a l l y with an array of c a r b o n y l - c o n t a i n i n g substances, i n c l u d i n g reducing sugars, reductones, the common aldehydes and ketones, and f u r f u r a l . Many of the common p e s t i c i d e s f a l l i n t o one of these c a t e g o r i e s . Those p e s t i c i d e s which are b a s i c i n character, have the p o t e n t i a l f o r forming a chemical l i n k a g e with C*0 c o n s t i t u e n t s of s o i group are t h e o r e t i c a l l y uents. Condensation and conjugate r e a c t i o n s of p e s t i c i d e s with metabolic products have been p o s t u l a t e d to c o n s t i t u t e a form of p e s t i c i d e transformations by microorganisms and high p l a n t s . Another f a c t o r to consider is that the p a r t i a l degradation of many p e s t i c i d e s by microorganisms leads to the formation of c h e m i c a l l y r e a c t i v e intermediates which can combine with aminoor C=0 c o n t a i n i n g compounds, as i l l u s t r a t e d i n Figure 8. Thus, l o s s of the s i d e chain from the phenoxyalkanoic a c i d s by enzymatic a c t i o n leads to the formation of p h e n o l i c c o n s t i t u e n t s which can e i t h e r be o x i d i z e d f u r t h e r v i a the enzymatic route or undergo condensation (probably as quinones) w i t h amino compounds to form "humic-like" substances. On the other hand, amines (or c h l o r o amines) produced by b i o l o g i c a l decomposition of such h e r b i c i d e s as the a c y l a n i l i d e s , phenylcarbamates and phenylureas may r e a c t with C=0 c o n s t i t u e n t s o c c u r r i n g n a t u r a l l y i n s o i l . Entry i n t o the carbon c y c l e by t h i s mechanism may c o n s t i t u t e a form of n a t u r a l detoxification. Thus, it must be concluded that some p e s t i c i d e s or t h e i r decomposition products can become p a r t of the p o o l of precursor molecules f o r humus s y n t h e s i s , and, i n so doing, l o s e t h e i r identity. Bartha (57), Bartha and Pramer (58), and Chisaka and Kearney (59) concluded that the bulk of c h l o r o a n i l i n e s l i b e r a t e d by part i a l degradation of the phenylamide h e r b i c i d e s ( a c y l a n i l i d e s , phenylcarbamates, and phenylureas) becomes immobilized i n s o i l by chemical bonding to organic matter. The chemically bound residues could not be recovered by e x t r a c t i o n with organic s o l vents or i n o r g a n i c s a l t s ; p a r t i a l r e l e a s e was p o s s i b l e by a c i d or base h y d r o l y s i s (60). According to Hsu and Bartha (60), b i n d i n g occurs when the amino group of the a n i l i n e s react with C=0 and COOH groups a p p r o p r i a t e l y p o s i t i o n e d on the humic a c i d core with formation of a h e t e r o c y c l i c r i n g . The soil-bound c h l o r o a n i l i n e r e s i d u e s r e s i s t a t t a c k by microorganisms (57, 61).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

196

BOUND AND CONJUGATED PESTICIDE RESIDUES

ALDOSE SUGAR

AMINO COMPOUND

N-SUBSTITUTED GLYCOSYLAMINE

AMADORI REARRANGEMENT

1-AMINO-1 - DEOXY - 2 - KETOSE (1,2-ENOL FORM)

DEHYDRATION • oc-AMINO ACID

11

STRECKER DEGRADATION

FISSION PRODUCTS

ALDEHYDE

DEHYDROREDUCTONE FURFURALS

AMINO COMP'D

AMINO COMP'D

J

FRAGMENTATION

ETC.)

AMINO COMP'D

ι

|BROWh^JJTROGENOU^

Figure 7. Formation of brown nitrogenous polymers according to the MaiUard reaction

PHENYLCARBAMATES PHENYL UREAS

PHENOXYALKANOIC ACIDS

SIDE CHAIN

SIDE CHAIN

PHENOLIC

AMINES

CONSTITUENTS

ENZYMATIC

ENZYMATIC *OH OH AMINES FROM O.M.

CHEMICAL

•C=0's FROM O.M.

HUMIC-LIKE SUBSTANCES Environmental Quality

Figure 8.

Chemical reactions involving intermediate products of herbicide decompo­ sition and constituents of soil organic matter (3)

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197

Fate of Organics In Sediments The r o l e o f organic matter i n chemical transformations deserves s e r i o u s a t t e n t i o n i n determining the long-time f a t e o f p e r s i s t e n t p e s t i c i d e s i n the environment. I n d i r e c t i n f o r m a t i o n on t h i s s u b j e c t is provided by the many biogeochemical s t u d i e s d e a l i n g w i t h the f a t e of n a t u r a l l y o c c u r r i n g organics i n sediments and sedimentary rocks (64). Since t h i s subject is beyond the scope o f the present review, the d i s c u s s i o n which f o l l o w s w i l l be confined to a c o n s i d e r a t i o n o f the diagenesis o f amino a c i d s i n sediments, as o u t l i n e d elsewhere (65)· The net e f f e c t of n o n b i o l o g l c a l r e a c t i o n s i n v o l v i n g amino a c i d s (see Figures 6 and 7) is i n c o r p o r a t i o n of n i t r o g e n i n t o the s t r u c t u r e s of humic and f u l v i c a c i d s . Thus, whereas amino a c i d s c o n s t i t u t e 80% or more o sue (biomass), they accoun n i t r o g e n i n s o i l and marine humus. F o l l o w i n g b u r i a l i n sediments, f u r t h e r changes occur, w i t h t r a n s f e r o f amino acid-N i n t o the humified remains. The s i g n i f i c a n c e of chemical transformations o f amino a c i d s f o l l o w i n g b u r i a l can be i l l u s t r a t e d by c o n s i d e r a t i o n o f data obtained f o r the forms of n i t r o g e n i n sediments o f the Argentine B a s i n (66) and the Experimental Mohole (67). I n the case o f the Argentine Basin sediments (estimated maximum age o f 125,000 y e a r s ) , data were reported (65) f o r t o t a l amino a c i d s and organic carbon i n 72 i n d i v i d u a l samples from two cores (V-15-141 and V-15-142). As i n d i c a t e d i n F i g u r e 9, there was a p r o g r e s s i v e decrease i n the percentage o f the organic carbon as amino a c i d s w i t h i n c r e a s i n g age. A s i m i l a r r e s u l t was obtained f o r the somewhat o l d e r Experimental Mohole sediments, f o r which q u a n t i t a t i v e data were a v a i l able f o r e i g h t samples ranging i n age from 3 t o 14 m i l l i o n y e a r s . These data are shown i n F i g u r e 10. For both b a s i n sediments, the disappearance o f amino a c i d s from the sedimentary organic matter, as estimated from composit i o n a l s t u d i e s , was c o n s i d e r a b l y greater than would be a n t i c i p a t e d from k i n e t i c s t u d i e s o f amino a c i d s i n aqueous s o l u t i o n . This o b s e r v a t i o n lends support t o the c o n c l u s i o n t h a t , during d i a g e n e s i s , amino a c i d s are transformed to other products as a consequence o f chemical r e a c t i o n s w i t h other o r g a n i c s , presumably by r e a c t i o n s of the type shown i n Figures 6 and 7. D i a g e n e t i c changes i n the amino a c i d s o f sediments have a l s o been observed f o r Saanich I n l e t , B r i t i s h Columbia, where it was found t h a t l o s s e s o f amino a c i d s w i t h depth exceeded that f o r organic carbon (68), Abelson (69) e a r l i e r p o s t u l a t e d t h a t nonb i o l o g l c a l processes i n v o l v i n g complex heteropolymers were r e s p o n s i b l e f o r the disappearance o f amino a c i d s from sediments. Coupling of amino a c i d s w i t h porphyrins may be o f geochemical s i g n i f i c a n c e (70). The h i g h s t a b i l i t y o f amino a c i d s i n petroleum b r i n e s has been a t t r i b u t e d t o t h e i r l i n k a g e w i t h phenol- and quinone-containing substances (71).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESEDUES

YEARS BEFORE PRESENT Advances in Organic Geochemistry

Figure 9. Relationship between estimated age of Argentine Basin sediments and the percentage of organic carbon which occurred as amino acids. Regression equation: Y =- 22.92 - 0.05 x(r = 0.47, significant at ρ — 0.01) (65).

30h

z ό Ο • EM 8-11 EM 8-9 \

S»' Figure 10. Relationship between age of sedimentary material in Experimental Mohole sediments and the percentage of organic nitrogen which occurred as amino acids. Value shown in brackets in the upper left hand corner represents an average for the younger Argentine Basin sediments. Regression equation: Τ «= 32.03 — 0.001 χ (τ = 0.95, significant at ρ — 0.01 ).

EM 8-13 E M 8-141 • \ EM 8-12 V

10 4

8

MILLIONS OF YEARS

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

12

15.

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U s i n g the analogy given above, one would expect t h a t n a t u r a l organics would e x e r t an a p p r e c i a b l e k i n e t i c i n f l u e n c e on the disappearance o f c e r t a i n p e r s i s t a n t p e s t i c i d e s (or t h e i r i n t e r mediate decomposition p r o d u c t s ) . Rate o f l o s s would, o f course, depend upon the nature o f the compound and environmental c o n d i t i o n s e x i s t i n g i n the sediment, such as pH and temperature. A d s o r p t i o n Mechanisms Bonding mechanisms f o r the r e t e n t i o n o f p e s t i c i d e s by o r g a n i c substances i n s o i l i n c l u d e Ion exchange, p r o t o n a t i o n , H-bonding, van der Waal's f o r c e s , and c o o r d i n a t i o n through an attached metal i o n ( l i g a n d exchange). In a d d i t i o n , nonpolar molecules may be p a r t i t i o n e d onto hydrophobic s u r f a c e s through "hydrophobic bondi n g ." For some p e s t i c i d e s r e v e r s i b l e (18, 20, 32, importance i n determining the environmental impact o f p e s t i c i d e s i n s o i l and water. Ion Exchange and P r o t o n a t i o n . A d s o r p t i o n through i o n exchange is r e s t r i c t e d t o those p e s t i c i d e s which e i t h e r e x i s t as c a t i o n s (diquat and paraquat) o r which become p o s i t i v e l y charged through p r o t o n a t i o n ( s - t r i a z i n e s ; amitrole). Diquat and paraquat, b e i n g d i v a l e n t c a t i o n s , have the potent i a l f o r r e a c t i n g w i t h more than one n e g a t i v e l y charged s i t e on s o i l humic c o l l o i d s , such as through two COO i o n s ( i l l u s t r a t e d below f o r d i q u a t ) , a COO" i o n p l u s a phenolate i o n combination, o r a COO" i o n (or phenolate i o n ) p l u s a f r e e r a d i c a l s i t e .

Diquat

On the b a s i s o f i n f r a r e d s t u d i e s , Khan (T3 74) suggested t h a t b i p y r i d y l i u m h e r b i c i d e s form c h a r g e - t r a n s f e r complexes w i t h humic substances. T h i s could not be confirmed by Burns e t a l , (75) who s u b j e c t e d some paraquat-humic a c i d complexes t o u l t r a violet analysis. Paraquat has been found to be complexed i n g r e a t e r amounts by humic and f u l v i c a c i d s (73), and by an organo-clay complex ( 7 4 ) , than d i q u a t . A h i s t o s o l and i t s humic and f u l v i c f r a c t i o n s has 9

f

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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200

a l s o been observed t o show s e l e c t i v e preference f o r paraquat (76). F a c t o r s which i n f l u e n c e the a v a i l a b i l i t y o f exchange s i t e s f o r a d s o r p t i o n i n c l u d e the presence of competing metal c a t i o n s and pH. S o i l pH has a d i r e c t b e a r i n g on the r e l a t i v e importance of o r g a n i c matter and c l a y i n r e t a i n i n g o r g a n i c c a t i o n s . U n l i k e c l a y , o r g a n i c c o l l o i d s have a s t r o n g l y pH-dependent charge. Therefore, the r e l a t i v e c o n t r i b u t i o n o f o r g a n i c matter t o c a t i o n exchange c a p a c i t y , and subsequently r e t e n t i o n o f c a t i o n s , w i l l be h i g h e r i n n e u t r a l and s l i g h t l y a l k a l i n e s o i l s than i n a c i d i c ones. For each u n i t change i n pH, the change i n c a t i o n exchange c a p a c i t y f o r o r g a n i c matter is s e v e r a l times greater than f o r c l a y . Less b a s i c compounds, such as the s - t r i a z i n e s , may become c a t i o n i c through p r o t o n a t i o n . Whether o r not p r o t o n a t i o n occurs w i l l depend upon: (1) the nature o f the compound i n q u e s t i o n , as r e f l e c t e d by i t s p l ^ , an humic c o l l o i d s . Reaction as p o s t u l a t e d by Weber e t a l . ( 7 7 ) , are shown by the f o l l o w i n g equations : Τ + H 0 2

^

RCOOH + H 0

RCOOH + Τ

+

+

+

OH"

R-COO" +

2

RC00~ + HT

HT

^

[1]

H 0

+

3

[2]

R-COO-HT

[3]

= ^ R-COO-HT

[4] +

where R is the o r g a n i c c o l l o i d , Τ the s - t r i a z i n e molecule, HT the protonated molecule, and the hydronium i o n . Equation [1] represents pH-dependent a d s o r p t i o n through p r o ­ t o n a t i o n i n the s o i l s o l u t i o n w h i l e equation [2] represents i o n i ­ z a t i o n o f the c o l l o i d COOH group. I o n i c a d s o r p t i o n o f t h e c a t i o n i c s - t r i a z i n e molecule, formed by r e a c t i o n [ 1 ] , is shown by equation [ 3 ] . A d s o r p t i o n through d i r e c t p r o t o n a t i o n on the s u r ­ face o f the o r g a n i c c o l l o i d is shown by r e a c t i o n [ 4 ] . A m i t r o l e is another example o f a weak base that can be adsorbed t o s o i l o r g a n i c matter through i o n exchange (78). S o i l pH has a profound e f f e c t on a d s o r p t i o n o f s - t r i a z i n e s and other weakly b a s i c p e s t i c i d e s by o r g a n i c matter. S o i l reac­ t i o n governs not o n l y the i o n i z a t i o n o f a c i d i c groups on the o r g a n i c c o l l o i d s but the r e l a t i v e q u a n t i t y o f the p e s t i c i d e which occurs i n c a t i o n i c form, i n accordance w i t h equation [ 1 ] . The p K o f a c i d i c groups i n humic a c i d s (COOH p l u s p h e n o l i c - and/or enolic-OH) is o f the order o f 4.8 t o 5.2. Thus, it would appear t h a t i o n exchange would not be an important mechanism f o r adsorp­ t i o n o f weakly b a s i c p e s t i c i d e s w i t h p K s much lower than 3.0. I t should be p o i n t e d out however, t h a t the pH a t the s u r f a c e o f s o i l organic c o l l o i d s may be as much as two pH u n i t s lower than that o f the l i q u i d environment. The a d s o r p t i o n c a p a c i t i e s o f s o i l o r g a n i c matter p r e p a r a t i o n s f o r the s - t r i a z i n e s has been a

f

a

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found t o f o l l o w t h e order expected on the b a s i s o f p K values f o r the h e r b i c i d e s , w i t h maximum a d s o r p t i o n o c c u r r i n g a t pH values near t h e p K o f t h e r e s p e c t i v e compound (78). Ion exchange is but one o f s e v e r a l mechanisms f o r a d s o r p t i o n of t h e s - t r i a z i n e s t o organic c o l l o i d s , as i l l u s t r a t e d below: a

a

s> s-Triazine

On the b a s i s o f an i n f r a r e d study o f some s - t r i a z i n e - h u m i c a c i d complexes, S u l l i v a n and Felbeck (79) concluded t h a t one secondary amino group was bound t o e i t h e r a C«0 o r quinone group of the humic a c i d through a hydrogen bond whereas the other secondary amino group became protonated and was bound by i o n exchange t o a COO" group. I t should be noted t h a t the mechanism described above is somewhat d i f f e r e n t from that shown e a r l i e r i n Figure 5. Walker and Crawford (13) suggested that a d s o r p t i o n o f the s - t r i a z i n e s by organic matter could best be regarded as p a r t i t i o n i n g out o f s o l u ­ t i o n onto hydrophobic surfaces (discussed l a t e r ) . For a n i o n i c p e s t i c i d e s , such as the phenoxyalkanoic a c i d s , r e p u l s i o n by the predominantly n e g a t i v e l y charged s u r f a c e o f organic matter may occur. P o s i t i v e a d s o r p t i o n o f a n i o n i c h e r b i ­ c i d e s a t pH values below t h e i r pKa can be a t t r i b u t e d t o a d s o r p t i o n of t h e unionized form o f t h e h e r b i c i d e t o organic s u r f a c e s , such as by Η-bonding between t h e COOH group and C«0 o r NH2 groups o f organic matter. Η-Bonding, van der Waals Forces, and C o o r d i n a t i o n . Adsorption mechanisms f o r r e t e n t i o n o f n o n i o n i c p o l a r p e s t i ­ c i d e s , such as the phenylcarbamates and s u b s t i t u t e d ureas, a r e i l l u s t r a t e d i n F i g u r e 11. The great importance o f Η-bonding i n r e t e n t i o n is suggested, w i t h m u l t i p l e s i t e s being a v a i l a b l e on both p e s t i c i d e and organic matter s u r f a c e . Other a d s o r p t i o n mechanisms i n c l u d e van der Waals f o r c e s ( p h y s i c a l a d s o r p t i o n ) , l i g a n d exchange ( - M 0»C), and, f o r p e s t i c i d e s c o n t a i n i n g an i o n i z a b l e COOH group, a s a l t l i n k a g e through a d i v a l e n t c a t i o n on the organic exchange s i t e . For c h l o r i n a t e d phenoxyalkanoic a c i d s , such as 2,4-D, p h y s i c a l adsorption t o aromatic c o n s t i t u e n t s

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of o r g a n i c matter may be i n v o l v e d ; Η-bonding w i l l be l i m i t e d t o a c i d c o n d i t i o n s where COOH groups are u n i o n i z e d . Considerable v a r i a t i o n can be expected i n the a d s o r p t i o n c a p a c i t y o f o r g a n i c matter f o r n o n i o n l c p o l a r h e r b i c i d e s , depend­ i n g upon s t e r i c e f f e c t s and the number and kinds of e l e c t r o ­ n e g a t i v e atoms i n the molecule. Hydrophobic Bonding P a r t i t i o n i n g on hydrophobic s u r f a c e s has been proposed as a mechanism f o r r e t e n t i o n of nonpolar o r g a n i c p e s t i c i d e s by s o i l o r g a n i c matter. A c t i v e s u r f a c e s i n c l u d e the f a t s , waxes, and r e s i n s , as w e l l as p o s s i b l e a l i p h a t i c s i d e chains on humic and f u l v i c a c i d s . Weber and Weed (80) p o i n t e d out that "humus," by v i r t u e o f i t s aromatic may c o n t a i n both hydrophobi P i e r c e e t a l . (81, 82) suggested that c h l o r i n a t e d hydrocar bons (such as DDT) would have g r e a t e r a f f i n i t y f o r hydrophobic s i t e s on o r g a n i c substances than f o r c l a y and that scavenging by o r g a n i c p a r t i c u l a t e s provided a means whereby these p e r s i s t e n t p o l l u t a n t s are t r a n s p o r t e d through the water column and concen­ t r a t e d i n bottom sediments. For DDT, it would be noted that c h l o r i n e atoms on the e t h y l group may impart a s l i g h t n e g a t i v e charge to the molecule (83); consequently, p a r t o f the a d s o r p t i o n a t t r i b u t e d to hydrophobic bonding may be due to a t t r a c t i o n to p o s i t i v e l y charged s i t e s such as t o an amino group. Considerable emphasis has been given to hydrophobic bonding as a mechanism f o r a d s o r p t i o n o f the s - t r i a z i n e s (13) and the phenylureas (17) by s o i l o r g a n i c matter. These claims r e q u i r e c o n f i r m a t i o n ; f o r these p e s t i c i d e s , the b u l k of the evidence favors the i d e a that s p e c i f i c a d s o r p t i o n s i t e s ( f u n c t i o n a l groups) are i n v o l v e d (see e a r l i e r d i s c u s s i o n ) . R e l a t i v e A f f i n i t i e s o f P e s t i c i d e s f o r Organic Matter The d e l i b e r a t i o n s o f the previous s e c t i o n serve to empha­ s i z e t h a t the v a r i o u s p e s t i c i d e s d i f f e r g r e a t l y i n t h e i r r e l a t i v e a f f i n i t i e s f o r s o i l o r g a n i c c o l l o i d s . The approximate order f o r some common h e r b i c i d e s are given i n F i g u r e 12. Thus, the c a t i o n i c h e r b i c i d e s (diquat and paraquat) would be expected to be the most s t r o n g l y bound, followed by those weakly b a s i c types capable of b e i n g protonated under moderately a c i d i c c o n d i t i o n s . For the s - t r i a z l n e s , d i f f e r e n c e s i n a d s o r b a b i l i t y can be account­ ed f o r by v a r i a t i o n s i n pKg, w i t h the more b a s i c compounds (high pKa) b e i n g adsorbed the s t r o n g e s t . H e r b i c i d e s i n the next order of a d s o r p t i o n are those having v e r y low pK values but which con­ t a i n one or more p o l a r groups s u i t a b l e f o r H-bonding. A n i o n i c p e s t i c i d e s may o r may not be adsorbed, depending upon s o i l pH. a

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15.

203

Reactions Involving Pesticides in Soil

STEVENSON

PHENYLCARBAMATES SUBST UREAS 0

/

, ,

R,

0

VN-C-O-CH \=J Η V R

f

R,

2

+

VAN DER WAALS

R, II 1 R - N - C ,*C-N-R Η Ν Η

2

PHENOXYALKANOIC ACIDS

s-TRIAZINES

CI ^ ~ ^ - Q - C H - C O O H 2

3

CI

+

+

+

+

-

+ (R,'OH)

-

-

-

-

+

+

-

+

+

+

-

-

—

—

—

+ (pH>7.0)

H-BONDING N

/

H

^ 8 "

- O H — · ••••0= R -C-O — · \ '

-

HAJ

HOHN = HA

0

£ C = 0- ••·

H

HN-

(pH< K ) P

0

LIGAND EXCHANGE Z

M *-(HÂ]

^c=o

SALT LINKAGE -C-0-M-O-C-fHÂ] λ

ο

d

Environmental Quality

Figure 11.

Typical bonding mechanisms for adsorption of some common herbicides by soil organic matter (3)

ION

DIQUAT

EXCHANGE

A N D PARAQUAT PROMETONE ATR ATONE PROMETRYNE AMETRYNE

ATRAZINE SIMAZINE

PHENYLCARBAMATES SUBSTITUTED UREAS OTHERS

Environmental Quality

Figure 12. Relative affinities of herbicides for soil organic matter surfaces (3)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

Literature Cited 1. 2. 3. 4.

5.

6.

7.

8. 9. 10. 11.

12.

13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Bailey, G. W., and White, J . L., J . Agr. Food Chem. (1964) 12, 324-333. Hayes, M. H. B., Residue Rev. (1970) 32, 131-174. Stevenson, F. J., J . Environ. Quality (1972) 1, 333-343. Weed, S. B., and Weber, J . B., In Guenzi, W. D. (ed.), "Pesticides i n Soil and Water," pp. 39-66. American Society of Agronomy, Madison, Wisc. (1974). Wolcott, A. R., In "Proceedings Int. Symposium on Pesticides i n S o i l . " pp. 128-138. Michigan State University, East Lansing. (1970). Armstrong, D. Ε., and Konrad, J . G., In Guenzi, W. D. (ed.), "Pesticides i n S o i l and Water," pp. 123-131. American Society of Agronomy Crosby, D. G., In i n S o i l , " pp. 86-94. Michigan State University, East Lansing. (1970). Dubach, P., and Mehta, N. C., Soils Fert. (1963) 26, 293-300. Kononova, Μ. Μ., "Soil Organic Matter," pp. 544. Pergamon Press, Inc., New York. (1966). Scheffer, F., and Ulrich, Β., "Humus and Humusdüngung. Bd I," pp. 266. Ferdinand Enke, Stuttgart, Germany. (1960). Stevenson, F. J . , and Butler, J . Η. Α., In Englinton, G., and Murphy, M. T. J . , (eds.), "Organic Geochemistry," pp. 534-557. Springer-Verlag, Berlin. (1966). Steelink, C., and T o l l i n , G., In McLaren, A. D., and Peterson, G. H. (eds.). "Soil Biochemistry," pp. 147-169. Marcel Dekker, Inc., New York. (1967). Walker, Α., and Crawford, D. V., In "Isotopes and Radiation in S o i l Organic Matter Studies," pp. 91-108. Atomic Energy Agency, Vienna, Austria. (1968). Bailey, G. W., and White, J . L., Residue Rev. (1970) 32, 29-92. Scott, D. C., and Weber, J . B., S o i l Sci. (1967) 104, 151-158. Doherty, P. J., and Warren, G. F., Weed Res. (1969) 9, 20-26. Hance, R. J.,Weed Res. (1965) 5, 108-114. Deli, J., and Warren, G. F., Weed Sci. (1971) 19, 67-69. Weber, J . B., Perry, P. W., and Ibarki, K., Weed Sci. (1968) 16, 134-136. Talbert, R. E., and Fletchall, O. H., Weeds (1965) 13, 47-52. Day, B. F., Jordan, L. S., and J o l l i f f e , V. Α., Weed Sci. (1968) 16, 209-213. Dubey, H. D., Sigafus, R. Ε., and Freeman, J . F., Agron. J . (1966) 58, 228-231. Grover, R., Weed Res. (1968) 8, 226-232. Harris, C. I., and Sheets, T. J., Weeds (1965) 13, 215-219. Liu, L. C., Cibes-Viade, H., and Koo, F. K. S., Weed Sci. 18, 470-474. Nearpass, D. C., Weeds (1965) 13, 341-346.

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15. 27. 28. 29. 30.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

STEVENSON

Reactions Involving Pesticides in Soil

205

Obien, S. R., Suehisa, R. H., and Younge, O. R., Weeds (1966) 14, 105-109. Stevenson, F.J.,J.Amer. O i l Chem. Soc. (1966) 43, 203-210. Swincer, G. D., Oades, J. Μ., and Greenland, D. J., Adv. Agron. (1969) 21, 195-325. Hayes, M. H. B., Stacey, Μ., and Thompson, J. M. In "Isotopes and Radiation i n S o i l Organic Matter Studies," pp. 75-90. Atomic Energy Agency, Vienna, Austria (1968). Dunigan, E. P., and McIntosh, Τ. Η., Weed Sci. (1971) 19, 279-282. Tompkins, G. Α., McIntosh, Τ. Η., and Dunigan, E. P., S o i l Sci. Soc. Amer. Proc. (1968) 32, 373-377. Sherburne, H. R., and Freed, V. H., J. Agr. Food Chem. (1954) 2, 937-939. Lichtenstein, E. P. Agr. Food Chem. (1968 Hilton, H. W., and Yuen, Q. H., J. Agr. Food Chem. (1963) 11, 230-234. Yuen, Q. H., and Hilton, H. W., J. Agr. Food Chem. (1962) 10, 386-392. Weber, J. B., Perry, P. W., and Upchurch, R. P., Soil Sci. Soc. Amer. Proc. (1965) 29, 678-688. Coffee, D. L., and Warren, G. F.,Weed Sci. (1969) 17, 16-19. Ahrens, J. F.,Proc. Ν. E. Weed Control Conf. (1965) 19, 364. Ogner, G., and Schnitzer, M.,Geochim et Cosmochim. Acta (1970) 34, 921-928. Schnitzer, M., and Ogner, G., Israel J. Chem. (1970) 8, 505512. Ballard, T. M., Soil Sci. Soc. Amer. Proc. (1971) 35, 145-147. Schnitzer, M., and Khan, S. U., "Humic Substances i n the En­ vironment," pp. 327. Marcel Dekker Inc., New York. (1972). Burchfield, H. P., and Schechtman, J. , Contrib. Boyce Thompson Inst. (1958) 19, 411-416. Kaufman, D. D., Plimmer, J. R., Kearney, P.C., Blake, J., and Guardia, F. S.,Weed Sci. (1968) 16, 266-272. Plimmer, J. R., Kearney, P. C., Kaufman, D. D., and Guardia, F. S. ,J. Agr. Food Chem. (1967) 15, 996-999. Gatterdam, P. E., Casida, J. Ε., and Stoutamire, D. W., J. Econ. Entomol. (1959) 52, 270-276. Lord, Κ. Α., J. Chem. Soc. (London) (1948), 1657-1661. Miskus, R. P., B l a i r , D. P., and Casida, J. E., J. Agr. Food Chem. (1965) 13, 481-483. Kearney, P. C., Harris, C. I., Kaufman, D. D., and Sheets, T. J., Adv. Pest Control Res. (1965) 6, 1-30. Armstrong, D. E., and Chesters, G., Environ. Sci. Technol. (1968) 2, 683-689. Armstrong, D. Ε., Chesters, G., and Harris, R. F., Soil Sci. Soc. Amer. Proc. (1967) 31, 61-66. Hance, R. J., Soil B i o l . Biochem. (1974) 6, 39-42.

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54. Harris, C. I., J. Agr. Food Chem. (1967) 15, 157-162. 55. Nearpass, D. C., Soil Sci. Soc. Amer. Proc. (1972) 36, 606-610. 56. L i , G. C., and Felbeck, G. T., Jr., Soil Sci. (1972) 114, 201-209. 57. Bartha, R., J. Agr. Food Chem. (1971) 19, 385-387. 58. Bartha, R., and Pramer, D., Adv. Appl. Microbiol. (1970) 13, 317-341. 59. Chiska, Η., and Kearney, P. C., J. Agr. Food Chem. (1970) 18, 854-858. 60. Hsu, T-S., and Bartha, R., S o i l Sci. (1974) 116, 444-452. 61. Hsu, T-S., and Bartha, R., Soil Sci. (1974) 118, 213-220. 62. E l l i s , G. P., Adv. Carbohydrate Chem. (1959) 14, 63-134. 63. Hodge, J. E., J. Agr. Food Chem. (1953) 1, 928-943. 64. Englinton, G., an pp. 828. Springer-Verlag 65. Stevenson, F. J., In Tissot, B., and Bienner, F. (eds.). "Advances i n Organic Geochemistry," pp. 701-714. Editions Technip, Paris. (1974). 66. Stevenson, F. J., and Cheng, C-N., Geochim. et Cosmochim. Acta (1972) 36, 653-671. 67. Stevenson, F. J., and T i l o , S. N., In Hobson, G. D., and Speers, G. C. (eds.) "Advancesin Organic Geochemistry" pp. 237-263. Pergamon Press, London. (1970). 68. Brown, F. S., Baedecker, M. J., Nissenbaum, Α., and Kaplan, I. R., Geochim et Cosmochim. Acta (1972) 36, 1185-1203. 69. Abelson, P. H., Carnegie Inst. of Washington Yearbook (1958) 58, 181-185. 70. Hodgson, G. W., Holmes, Μ. Α., and Halpern, B., Geochim. et Cosmochim. Acta (1970) 34, 1107-1119. 71. Degens, Ε. T., Hunt, J. M., Reuter, J. Η., and Reed, W. E., Sedimentology (1964) 3, 199-225. 72. Grover, R., Weed Sci. (1974) 22, 405-408. 73. Khan, S. U., Can. J. Soil Sci. (1973) 53, 199-204. 74. Khan, S. U., J. Soil Sci. (1973) 24, 244-248. 75. Burns, E. G., Hayes, Μ. Η. Β., and Stacey, M., Pesticide Sci. (1973) 4, 201-209. 76. Best, J. Α., Weber, J. B., and Weed, S. B., Soil Sci. (1972) 114, 444-450. 77. Weber, J. B., Weed, S. B., and Ward, T. M., Weed Sci. (1969) 17, 417-421. 78. Nearpass, D. C., Soil Sci. Soc. Amer. Proc. (1969) 33, 524-528. 79. Sullivan, J. D., J r . , and Felbeck, G. T.,Jr., S o i l S c i . (1968) 106, 42-52. 80. Weber, J. B., and Weed, S. B., In "Guenzi, W. D. (ed.) "Pesticides i n S o i l and Water," pp. 223-256. American Society of Agronomy, Madison, Wisc. (1974). 81. Pierce, R. H., Jr., Olney, C. E., and Felbeck, G. T., Jr., Environ. Lett. (1971) 1, 157-172.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15.

STEVENSON

82. 83.

Reactions Involving Pesticides in Soil

207

Pierce, R. H., J r . , Olney, C. E., and Felbeck, G. T.,Jr., Geochim. et Cosmochim. Acta (1974) 38, 1061-1073. Champion, D. F., and Olsen, S. R., Soil Sci. Soc. Amer. Proc. (1971) 35. 887-891.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

16 Clay—Pesticide Interactions JOE L. WHITE Department of Agronomy, Purdue University, West Lafayette, Ind. 47907

This paper is concerned with an evaluation of the current status of information on clay-pesticide interactions as related to the problem of "bound w i l l be made to point ou soil-pesticide interactions which need to be overcome i n order to provide a better basis for prediction and understanding of the behavior of pesticides i n soils on a long-term basis. In addition, some examples of experimental approaches useful i n such studies w i l l be given. Whether or not a pesticide may pose a problem as a "bound" or unavailable residue depends largely on the nature and extent of the interactions of the pesticide with the s o i l constituents having high surface areas. Since high specific surface is us­ ually associated with small particle size, the colloidal frac­ tion of the s o i l w i l l be the controlling factor i n interactions between pesticide molecules and the s o i l . Several comprehensive reviews of the factors which i n f l u ­ ence adsorption, desorption and movement of pesticides i n soils have been made recently (1, 2, 3, 4, 5, 6). Mortland (7) and Theng (8) have provided detailed treatments of clay-organic complexes and interactions. S t r u c t u r e and P r o p e r t i e s of Clay M i n e r a l s T h i s c o n t r i b u t i o n is concerned w i t h the nature of the i n ­ t e r a c t i o n s between the c o l l o i d a l m i n e r a l f r a c t i o n of s o i l s and p e s t i c i d e s . The minerals i n the c o l l o i d a l f r a c t i o n form very s t a b l e complexes w i t h components of the s o i l o r g a n i c matter. The behavior of t h i s n a t u r a l c l a y - o r g a n i c complex w i t h respect t o the a d s o r p t i o n of p e s t i c i d e s has r e c e i v e d little a t t e n t i o n . The r o l e of o r g a n i c matter i n i n t e r a c t i o n s between p e s t i c i d e s and s o i l s has been discussed by Stevenson ( 9 ) . The c l a y f r a c t i o n (JLl)* T n e ESR-signal is one o f the arguments in favor f o r the presence o f q u i n o i d a l s t r u c t u r e s in humic s u b s t r a n c e s . For a f u l v i c a c i d a s p i n count o fO.58X 10 per g was measured, and from t h i s value it was estimated that one semiquinone r a d i c a l

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

BARTHA

AND

Hsu

Soil

Organic Matter

263

is present f o r every 44,000 carbon atoms (28). The o r i g i n of the free r a d i c a l s is as i n t r i g u i n g as t h e i r p e r s i s t e n c e , and the most p l a u s i b l e theory is that they were formed during and preserved s i n c e the o r i g i n a l b i o l o g i c a l o x i dation that lead to the formation of the humic compound. The b i o l o g i c a l e f f e c t o f these s t a b i l i z e d free r a d i c a l s and t h e i r p o t e n t i a l to i n i t i a t e polymerization reactions or the binding of p e s t i c i d e residues to humic m a t e r i a l s are v i r t u a l l y v i r g i n areas awaiting experimental work. Nuclear Magnetic Resonance (NMR) S p e c t r a . The nuclei of c e r t a i n atoms e x h i b i t a magnetic s p i n momentum. The most important o f these atoms are 7-H,13-C.,19-F and 31-P. When placed in a homogeneous magnetic f i e l d and e x c i t e d with radiowaves, energy t r a n s i t i o n s , as evidenced by radiowave a b s o r p t i o n , take place. A g a i n , the molecula the resonance energy r e s u l t i n g in a resonance s h i f t r e l a t i v e to the a r b i t r a r y reference point o f the t r i m e t h y l s i l a n e (TMS) s i g n a l (19). NMR spectrometry is a powerful tool f o r the exp l o r a t i o n o f the immediate chemical environment of a proton (32) that has been d e f i n i t e l y u n d e r u t i l i z e d in the study of soil organic matter. The l i m i t e d s o l u b i l i t y of the humic compounds in the s u i t a b l e deuterated solvents presents a problem, but the r a p i d l y i n c r e a s i n g s e n s i t i v i t y o f the instruments is l i k e l y to improve t h i s s i t u a t i o n . To date the NMR-technique was a p p l i e d only to hydrogen protons in humic a c i d . Barton and S c h n i t z e r (33) investigating a low molecular weight methylated f u l v i c a c i d noted the absence of aromatic and o l e f i n i c protons. This s u r p r i s i n g r e s u l t seems to i n d i c a t e that p r a c t i c a l l y all hydrogens are replaced by s u b s t i t u e n t s on the aromatic core of humic substances. Felbeck (34) a p p l i e d NMR spectrometry to products of hydrogenolysis of soil organic matter and noted the lack of deuterium-exchangeable protons on nitrogen atoms, and a l s o noted a prevalence of methylene peaks over methyl and methine. The l a t t e r f i n d i n g was i n d i c a t i v e o f a low degree of branching o f the carbon c h a i n s , at l e a s t in the material modified by h y d r o g e n o l y s i s . General L i m i t a t i o n s o f Spectroscopic C h a r a c t e r i z a t i o n o f Humic Substances. Spectroscopic methods work best in the c h a r a c t e r i z a t i o n of homogeneous substances of small or i n t e r mediate molecular s i z e . Under such circumstances spectra are sharp, reasonably s i m p l e , and can be i n t e r p r e t e d with r e l a t i v e ease. In case of humic substances the i n d i v i d u a l molecules are large and complex and d i s s i m i l a r to each o t h e r . The overlap of a multitude of absorption bands r e s u l t s in broad areas o f absorption rather than in d i s t i n c t absorption maxima. For t h i s reason, the s p e c t r a contain a s e v e r e l y reduced amount of useful information and, in a d d i t i o n , the i n t e r p r e t a t i o n o f the r e s i d u a l information is fraught with complexity. S o l u b i l i t y of humic

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND

264

A N D CONJUGATED

PESTICIDE

RESIDUES

compounds in s u i t a b l e solvents at high enough concentrations may present t e c h n i c a l problems. Molecular s i z e and a consequent lack of v o l a t i l i t y l a r g e l y prevents the a p p l i c a t i o n of mass s p e c t r o ­ metry to i n t a c t humic compounds and, t h e r e f o r e , d i s c u s s i o n of mass spectra was omitted here. However, e s p e c i a l l y in combina­ t i o n with gas chromatography, t h i s technique can be very useful in c h a r a c t e r i z a t i o n of degradation or p y r o l y s i s products. Spectrometric Studies

on P e s t i c i d e Residue - Humus i n t e r a c t i o n s .

The reduced a c t i v i t y o f many preemergence h e r b i c i d e s in high humus s o i l s (35.36) is a general i n d i c a t i o n of the a b i l i t y of humic compounds to bind man-made chemicals by various mechanisms. Such b i n d i n g t y p i c a l l y leads to increased p e r s i s t e n c e conbined with immobilization and are marked exceptions t and DDT (39) were reported to be m o b i l i z e d by absorption to or complexing with water s o l u b l e f u l v i c a c i d s . The absorption mechanisms of h e r b i c i d e s to humic compounds was r e c e n t l y subject to a l u c i d review by Stevenson {j). He l i s t s ion exchange, Η - b o n d i n g , van der Vaals forces and c o o r d i n a t i o n through a metal ion as the prevalent modes o f attachment. The molecular s t r u c ­ ture of h e r b i c i d e s determines the predominant mechanism, but more than one absorption mechanism may act on the same h e r b i c i d e .

humic acid

diquat

Figure 2. Charge transfer complex of diquat with humic acid. After Ref.40 B i p y r i d y l i u m H e r b i c i d e s . Herbicides of cat i o n i c n a t u r e , such as the two b i p y r i d y l i u m h e r b i c i d e s paraquat and diquat are bound by ion exchange r e a c t i o n s . This type o f binding was s u c c e s s f u l l y i n v e s t i g a t e d by Khan (40,41) u s i n g IR spectrometry. Based on the s h i f t o f C-H o u t - o f - p l a n e bending v i b r a t i o n s (from 815 cm" 1 to 825 cm" 1 f o r paraquat, from 792 cm"*1 to 765 cm*"1 for d i q u a t , r e s p e c t i v e l y ) he deduced the formation of a charge t r a n s f e r complex (Figure 2). Less b a s i c h e r b i c i d e s may undergo s i m i l a r reactions due to protonation (40).

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

BARTHA

AND

Hsu

Soil

humic acid

-o-c^o ο 4

R

'~

N H

265

Organic Matter

M

H

2 ~ j £ Jj" " 2 N

R

s-tnazine

CI Figure 3. Attachment of s-triazines to humic acid by charge transfer and hydrogen bonding mechanisms. After Ref. 42

5-Trîazînes. H - b o n d î n g may take place between C=0 groups of the humic compounds and the secondary amino groups o f s - t r i a z i n e s (Figure 3 ) . Evidence f o r t h i s type of bonding was obtained from IR-spectra by S u l l i v a n and Felbeck (42). These workers reacted e t h a n o l i c s o l u t i o n s of humic acids with various t r i a z i n e h e r b i cides. Carbonyl absorption (1720 c m " 1 ) was reduced in every case and in a d d i t i o n , the bands at 2,900 c m " 1 (C-H) and 3,300 cm""1 (-0H) were reduced to varying degrees. New absorption bands appeared at 1,625 c n T 1 (C00~ of humic a c i d and/or C=N of the s - t r i a z i n e s ) and at 1,390 cm'" 1 ( i n d i c a t i v e of a s a l t of a carboxylic acid). From these data it was concluded that the primary binding s i t e s of the humic a c i d are carboxyl groups with a c o n t r i b u t i o n from phenolic h y d r o x y l s . Since in some cases the reduction in carbonyl groups (1,720 c m " 1 ) was not accompanied by an increase o f the C00" (1,625 cm" 1 ) and carboxyl s a l t (1,390 c m " 1 ) bands, it was suggested that C « 0 from quinones may have a l s o served as a binding s i t e . As there was no evidence for involvement of any other p o r t i o n o f the h e r b i c i d e molecule, the amino group was proposed as the a c t i v e binding s i t e of the s-triazines. A d d i t i o n a l work on the binding of s - t r i a z i n e s by other techniques (14,43) tended to support the above c o n c l u s i o n s . P o t e n t i a l A p p l i c a t i o n s . Aside of the reviewed IR work, we are not aware of studies on h e r b i c i d e absorption by humic material which used spectrometric techniques as t h e i r primary research tool. In the l i g h t of widespread speculations that free r a d i c a l s may play a r o l e in h e r b i c i d e residue b i n d i n g , the lack of studies on quenching o f humus ESR s i g n a l s by p e s t i c i d e residues is rather surprising. Another spectrometric technique that has great p o t e n t i a l in bound p e s t i c i d e residue research is the 13~C NMR (44),

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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13-C emits a r e l a t i v e l y weak NMR s i g n a l , but modern instrumenta t i o n using the F o u r i e r transformation p r i n c i p l e and computerprocessed m u l t i p l e scannings to f i l t e r out random n o i s e , have enormously increased the s e n s i t i v i t y and t i m e - e f f i c i e n c y o f the NMR instruments, and made t h e i r a p p l i c a t i o n f o r routine s t r u c t u r al i n v e s t i g a t i o n s p o s s i b l e . The natural abundance of 13"C is low (1.1%) but a r t i f i c i a l l y enriched 13"C compounds are becoming a v a i l a b l e for research. NMR-spectrometry of humic compounds with 13-C enriched bound p e s t i c i d e residues would give extremely useful information not otherwise o b t a i n a b l e , s i n c e it would e l u c i d a t e the actual b i n d i n g environment. In case of 14-C l a b e l i n g , t h i s is to be deduced i n d i r e c t l y from degradation s t u d i e s , g r e a t l y i n c r e a s i n g the danger o f a r t i f a c t s , s i d e r e actions and, consequently, erroneous or ambiguous r e s u l t s . The cost and l i m i t e d a v a i l a b i l i t s t i l l an o b s t a c l e ; one Çh11 ο roan i 1 i ne Res i dues. Covalent bond formation between p e s t i c i d e residues and humic compounds was not covered in Stevenson's review (7) and we have some ongoing work to report in t h i s area although, to d a t e , spectrometric techniques played only a minor r o l e in t h i s i n v e s t i g a t i o n . The b i o d é g r a d a t i o n of phenylami de h e r b i c i d e s r e s u l t s in release of a n i l i n e moieties (45,46,47). Studies with 14-C labeled 3,4-dichloroani1ine (DCA) and î^chToroani1ine, compounds representative o f the a n i l i n e moieties of several phenylamide h e r b i c i d e s , showed that these c h l o r o a n i 1 i n e s are subject to absorption as well as to covalent binding in soil ( 4 8 ) . The mineral part of soil plays only a minor r o l e in a b s o r p t i o n , the greater p o r t i o n o f the a n i l i n e s becomes attached to the soil organic matter. The nature of the non-covalent b i n d i n g was not i n v e s t i g a t e d in d e t a i l , but the b a s i c character of the a n i l i n e s suggests ion exchange and hydrogen bonding mechanisms. This r e v e r s i b l e a b s o r p t i o n , by b r i n g i n g the a n i l i n e molecules in intimate contact with the humic a c i d molecules is probably of importance a l s o for the subsequent covalent b i n d i n g . At 5 ppm a p p l i c a t i o n r a t e , 85-90% of the a p p l i e d a n i l i n e is c o v a l e n t l y bound w i t h i n 5 days. About 50% o f the bound DCA is released in unchanged form a f t e r a c i d or a l k a l i n e h y d r o l y s i s ; 50% of the r a d i o a c t i v i t y remains attached to the humic compounds. We have no d i r e c t evidence to prove that t h i s attached r a d i o a c t i v i t y s t i l l represents i n t a c t c h l o r o ani l i n e , but we b e l i e v e t h i s to be a reasonable assumption. To e l u c i d a t e the nature of both the hydrolyzable and the non-hydrolyzable a n i l i n e b i n d i n g , we compared IR-spectra of DCA, of humic a c i d and o f the DCA-humic a c i d complex (Figure 4 ) . The complex contained less than O.25% DCA by weight, and no obvious changes in spectrum, as compared to the untreated humic a c i d , could be d i s c e r n e d . Being unsuccessful in t h i s p r e l i m i n a r y spectrometric approach, we turned to model reactions in order to gain i n s i g h t i n t o the p o s s i b l e mechanisms o f the b i n d i n g .

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

Soil

BARTHA AND H s u

4000

2500

267

Organic Matter

1800

1400 1200

800 750

KKX) 900

FREQUENCY, CM*

1

Figure 4. IR spectra of 3,4-dichloroaniline (DCA), humic acid (H), and their complex (H-DCA) (recordedinKBr)

Under ambient conditions we obtained hydrolyzable binding o f chloroani1ines to aldehydes and to qui nones in form o f a n i l s and ani1inoquinones, r e s p e c t i v e l y (Figure 5 ) . For the nonhydrolyzable binding o f a n i l i n e s , on t h e o r e t i c a l b a s i s , a number o f reactions (Figure 6) can be suggested from the

R-C=O

•

Η Ν-0?α 2

* aldehyde

5• (Mjuinone DCA

\&

.

f00m

in

water

DCA

R-C=N-^CI w

3.4-dicMoroonil

sœ- Çr^^A^' 2-iOCA).

2,5-di(DCA)-

quinone

quinone

Figure 5. Reactions of 3,4-dichloroaniline (DCA) with aldehydes and p-quinone

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND

A N D CONJUGATED

PESTICIDE

RESIDUES

a v a i l a b l e chemical l i t e r a t u r e (kS). We are now in the process of t e s t i n g some o f the more relevant reactions as models f o r the nonhydrolyzable attachment o f DCA to humic a c i d . Our p r e l i m i n a r y r e s u l t s are encouraging. Spectrometric methods w i l l undoubtedly be o f great use in the c h a r a c t e r i z a t i o n of these model r e a c t i o n products, and armed with s p e c i f i c i n f o r -

Heterocyclic Chemistry

Figure 6. Chemical reactions that lead to non-hydrolyzable attachment of antlines (49)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

BARTHA AND HSU

Soil Organic Matter

269

mation we may be more successful in the spectrometric i n v e s t i gation of DCA-humic a c i d complexes in the f u t u r e . While much work remains to be done, we b e l i e v e that the phenomenon of the c o v a l e n t l y bound a n i l i n e residues may resemble a very f a m i l i a r but in i t s d e t a i l s s t i l l obscure natural process of ammonia and amino a c i d attachment to humus that occurs both in hydrolyzable and in non-hydrolyzable forms (50), The mechanisms by which phenoxazines are formed from 4-methylcatechol and ammonia (Figure 7,A), and phenazines are formed from quinones and ammonia (Figure 7,B) r e s u l t i n g in h e t e r o c y c l i c nonhydrolyzable nitrogen (50 have very obvious analogies to the

Figure 7. Reactions of 4-methykatechol (A) and of p-quinone (B) with ammonia, leading to non-hydrolyzable incorporation of nitrogen into phenoxazine (A) and phenazine (B) type compounds. After Ref. 51

type o f reactions we propose for ani1ine b i n d i n g . If we look at DCA as ammonia tagged by a s t a b l e and e a s i l y recognizable chlorophenyl r i n g , we can imagine that our a p p l i e d research on DCA binding to humus may eventually c o n t r i b u t e to the understanding of much more fundamental aspects of soil chemistry. LITERATURE CITED.

1.

2.

Swain, F. M. "Geochemistry of Humus". In: Organic Geochemistry. (I.A. Berger, ed.) pp. 81-147, Pergamon Press, New York, 1963. Kononova, M. M. "Soil Organic Matter." Pergamon Press, New York, 1966.

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3. 4. 5.

6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29. 30. 31. 32.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Haider, K. and Martin, J. P. Soil. Biol. Biochem. (1970) 2:145-156. Martin, J. P. and Haider, K. Soil Sci. (1971) 111:54-63. Stevenson, F. J. In: C. A. Black (ed.) "Methods of Soil Analysis" pp. 1409-1421, American Society of Agronomy, Madison, 1965. Stevenson, F. J. and Butler, J. H. In: Organic Geochemistry - Methods and Results. (G. Eglington and M. T. J. Murphy, eds.) pp. 534-557, Springer, New York 1969. Stevenson, F. J. Bioscience (1972) 22:643-650. Tan, Κ. H. Soil Sci. Soc. Amer. Proc. (1975) 39:70-73. Mathur, S. P. Soil Sci. (1972) 113:136-139. Felbeck, G. T. J r . Adv. Agron. (1965) 17:327-368. Felbeck, G. T. J r . Soil Sci. (1971) 111:42-48. Flaig, W. Soil Sci. Haworth, R. D. Soil Hayes, M.H.B. Res. Rev. (1970) 32:131-174. McLaren, A. D., Pukite, A. H., and Barshad, I. Soil Sci. (1975) 119:178-180. Haider, Κ., Frederick, L. R. and Flaig, W. Plant and Soil (1965) 22:49-64. Ladd, J. N. and Butler, J. H. A. Austral. J. Soil Res. (1966) 4:41-54. Schnitzer, M. In:"Soil Biochemistry" Vol. 2 (A. D. McLaren and J. Skujins, eds. pp. 60-95, Dekker, New York, 1971. Williams, D. H. and Fleming, I. "Spectroscopic Methods in Organic Chemistry" McGraw H i l l , London 1973. Nakanishi, K. "Infarared Absorption Spectroscopy Practical." Holden-Day, San Francisco, 1962. Stevenson, F. J. and Goh, Κ. M. Soil Sci.(1972) 113:334-345. Schnitzer, M. and Skinner, I. M. Soil. Sci. (1965) 99:278-284. Stevenson, F. J. and Goh, Κ. M. Soil Sci. (1974) 117: 34-41. Schnitzer, M. Soil Sci. (1974) 117:94-102. Schnitzer, M. and Skinner, S. I. M. Soil Sci. (1963) 96:86-93. Schnitzer, M. and Hoffman, I. Soil Sci. Soc. Amer. Proc. (1964) 28:520-525. Steel ink, C. and T o l l i n , G. In: Soil Biochemistry (A. D. McLaren and G. Peterson, eds.) pp. 147-169, Marcel Dekker, New York, 1967. Schnitzer, M. and Skinner, S. I. M. Soil Sci. (1969) 108:383-390. Atherton, Ν. M., Cranwell, P. Α., Floyd, A. J., and Haworth, R. D. Tetrahedron (1967) 23:1653-1667. Cheshire, M.V. and Cranwell, P.A. J. Soil Sci. (1972)23:424-430. Haworth, R. D. Soil Sci. (1971) 111:71-79. Bible, R. H. J r . "Interpretation of NMR Spectra" Plenum Press. New York, 1965.

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33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

45.

46.

47. 48. 49. 50. 51.

BARTHA

AND

Hsu

Soil

Organic Matter

271

Barton, D. H. R. and Schnitzer, M. Nature (1963) 198:217218. Felbeck, G. T. J r . Soil Sci. Soc. Amer. Proc. (1965) 29:48-55. Bailey, G. W. and White, J. L. J. Agr. Food Chem. (1964) 12:324-333. Upchurch, R. P. Res. Rev. (1966) 16:46-85. Matsuda, K. and Schnitzer, M. Bull. Env. Contam. Toxicol. (1971) 6:200-204. Ogner, G. and Schnitzer, M. Science (1970) 170:317-318. Ballard, T. M. Soil Sci. Soc. Amer. Proc. (1971) 35:145-147. Khan, S. U. Can. J. Soil Sci. (1973) 53:199-204. Khan, S. U. J. Env. Qual. (1974) 3:202-206. Sullivan, J. D. Jr (1968) 106:42-52. L i , G.-C. and Felbeck, G. T. J r . Soil Sci. (1972) 113:140-148. Levy, G. C. and Nelson, G. L. "Carbon 13 Nuclear Magnetic Resonance for Organic Chemists" Wiley-Interscience, New York, 1972. Geissbühler, H. In: Degradation of Herbicides. (P. C. Kearney and D. D. Kaufman, eds.) pp. 79-111, Dekker, New York, 1969. Herrett, R. A. In: Degradation of Herbicides. (P. C. Kearney and D. D. Kaufman, eds.) pp. 113-145, Dekker, New York, 1969. Bartha, R. and Pramer, D. Adv. Appl. Microbiol. (1970) 13:317-341. Hsu, T.-S. and R. Bartha. Soil Sci. (1974) 116:444-452. Joule, J. A. and Smith, G. F. "Heterocyclic Chemistry" van Nostrand Reinhold, London, 1972. Nömmik, H. Plant and Soil (1970) 33:581-595. Lindbeck, M. R. and Young, J. L. Anal. Chim. Acta (1965) 32:73-80.

ACKNOWLEDGEMENT: This paper of the Journal S e r i e s , New Jersey A g r i c u l t u r a l Experiment S t a t i o n , New Brunswick, N.J., was supported by RR,NE-63 and Hatch funds.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20 Classification of Bound Residues Soil Organic Matter: Polymeric Nature of Residues in Humic Substance R. W. MEIKLE, A. J. REGOLI, and N. H . KURIHARA Dow Chemical U.S.A., Ag-Organics Research, 2800 Mitchell Drive, Walnut Creek, Calif. 94598 D. A. LASKOWSKI Dow Chemical U.S.A., Ag-Organics Department, Midland, Mich. 48640

In the course of studyin foreign organic compound associated with the soil organic matter. This associated radioactivity has generally not been identified because of the d i f f i c u l t y of working with the material. As the incubation time for the organic compound in soil increases, the amount of radioactivity in the soil organic matter also generally increases. Consequently, it has become increasingly important to have some notion of how this radioactivity is combined structurally with the soil organic matter. The organic matter of soils consists of a mixture of plant and animal products in various stages of decomposition, of substances synthesized biologically and/or chemically from the breakdown products, and of microorganisms and small animals and their decomposing remains. To simplify this very complex system, organic matter is usually divided into two groups: (a) nonhumic substances and (b) humic substances. Nonhumic substances include compounds of known chemical characteristics. To this class of compounds belong carbohydrates, proteins, peptides, amino acids, fats, waxes, resins, pigments and other low-molecular-weight organic substances. In general, these compounds are relatively easily attacked by microorganismsinthe soil and have a relatively short survival rate. The bulk of the organic matter in most soils consists of humic substances. These are amorphous, brown or black, hydrophilic, acidic, polydisperse substances of molecular weights ranging from several hundreds to tens of thousands. Based on their solubility in a l k a l i and acid, humic substances are usually divided into three main fractions: (a) humic acid (HA), which is soluble in dilute alkaline solution but is precipitated by acidification of the alkaline extract; (b) f u l v i c acid (FA), which is that humic fraction which remains in the aqueous acidified solution, i.e., it is s o l uble in both acid and base; and (c) the humic fraction that cannot be extracted by dilute base and acid, which is referred to as humin. 272

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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There is i n c r e a s i n g evidence t h a t the chemical s t r u c t u r e and prope r t i e s o f the humin f r a c t i o n a r e s i m i l a r t o those o f HA, and t h a t i t s i n s o l u b i l i t y a r i s e s from the firmness w i t h which it combines w i t h i n o r g a n i c soil and water c o n s t i t u e n t s . Data a v a i l a b l e a t t h i s time suggest t h a t s t r u c t u r a l l y the three humic f r a c t i o n s are s i m i l a r to each o t h e r , but t h a t they d i f f e r in molecular weight, u l t i m a t e a n a l y s i s , and f u n c t i o n a l group content. The FA f r a c t i o n has a lower molecular weight but a higher content o f oxygen-containing f u n c t i o n a l groups per u n i t weight than do HA and the humin f r a c t i o n . While the f r a c t i o n a t i o n scheme is a r b i t r a r y — the f r a c t i o n s a r e s t i l l m o l e c u l a r l y heterogeneous — it has nonetheless been w i d e l y accepted. The a b i l i t y o f s y n t h e t i c c r o s s - l i n k e d polydextran g e l s t o separate molecules by t h e i r molecular s i z e has become i n c r e a s i n g l y important in the study o d e s c r i b e s the use o f Sephadex humic substances e x t r a c t e d from soil a f t e r the soil has been incubated w i t h ^ j r a d i o a c t i v e d i t a l i m f o s f u n g i c i d e , 0 , 0 - d i e t h y l phthalimido-1C-phosphonothioate. Experimental Three soil samples were used in t h i s study and t h e i r p h y s i c a l p r o p e r t i e s are d e s c r i b e d in Table I . Mechanical analyses were c a r r i e d out u s i n g the hydrometer method ( 2 ) . Soil pH was measured in water a t a 1:1 soil:solution r a t i o w i t h a g l a s s e l e c t r o d e assembly ( 3 ) . Organic matter content of soil was determined u s i n g a wet combustion method ( 4 ) . The moisture content of the s o i l s a t 1/3 bar t e n s i o n was a l s o determined ( 5 ) . TABLE I Some p r o p e r t i e s o f the s o i l s used in the study o f d i t a l i m f o s decomposition. Sand, %

Silt, %

Clay, %

Organic carbon, %

Soil moisture content at 1/3 bar tension

pH

Loam, D a v i s , California

46

35

18

O.86

21.75

6.4

Sandy Loam, No. Dakota

66

22

12

2.2

22.52

7.3

S i l t y Clay Loam, Geneseo, Illinois

14

54

32

4.2

26.31

5.8

Soil t e x t u r a l classification and source

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These s o i l s were incubated w i t h r a d i o a c t i v e d i t a l i m f o s and then analyzed f o r t h i s compound and i t s decomposition products a t a p p r o p r i a t e times. The r e s u l t s of t h i s study w i l l be r e p o r t e d elsewhere. The s o i l s were first e x t r a c t e d w i t h a c i d i f i e d ether to remove e x t r a c t a b l e r a d i o a c t i v e compounds, r i n s e d w i t h water, and d r i e d at ambient temperature. Humic substances were obtained from the e x t r a c t e d s o i l s by shaking 2-g. soil samples f o r 18 hours a t ambient temperature w i t h 3g. of DOWEX® A - l c h e l a t i n g r e s i n (sodium form) of 50 t o 100 mesh and 25 ml. of water. The t o t a l nominal c a p a c i t y of the r e s i n was 2.6 meq./3g. The soil suspensions were c e n t r i f u g e d a t 12,000 χ G. A l i q u o t s of these r a d i o a c t i v e s o l u t i o n s were examined by g e l chromatography, s e p a r a t i o n i n t o humin-humic a c i d - f u l v i c a c i d f r a c t i o n s , and d i a l y s i s . G e l chromatography and G-100) were prepared as recommended by the manufacturer ( 6 ) . The column used was 2.6 χ 70 cm. The v o i d volume (V ) was determined e m p i r i c a l l y by u s i n g Blue Dextran 2000 (Pharmacia). V as shown on the f i g u r e s i n d i c a t e s the first excluded Blue Dextran f r a c t i o n . The t o t a l bed volume (V ) was obtained by water c a l i b r a t i o n of the column b e f o r e packing trie bed. V f o r the G-50 columns was 248 ml and f o r the G-100 column, 266 ml. The g e l d i d not compact d u r i n g e l u t i o n . F i v e - m l . f r a c t i o n s of column e f f l u e n t were c o l l e c t e d . The f l o w r a t e was maintained a t O.5 ml/ min. The b u f f e r systems used as e l u a n t were O.025M sodium borate (pH 9.1) f o r the G-50 g e l and O.1M sodium hydroxide f o r the G-100 gel. The f r a c t i o n a t i o n ranges of the g e l s are r e p o r t e d (6) to be as f o l l o w s : G-50, s o l u t e s w i t h molecular weights from 500 to 10,000; c o r r e s p o n d i n g l y f o r G-100, s o l u t e s w i t h molecular weights from 1,000 to 100,000. These v a l u e s are based on c a l i b r a t e d dextrans (Pharmacia). Over a c o n s i d e r a b l e range, the e l u t i o n volume (V ) of a p o l y ­ mer from a dextran g e l column is approximately a l i n e a r f u n c t i o n of the l o g a r i t h m of the molecular weight ( 2 j j B , 2 , 1 0 ) . The g e l columns were c a l i b r a t e d f o r molecular weight u s i n g samples of c a l i b r a t e d dextrans (Dextran T®, Pharmacia). Dextran in the e l u t e d f r a c t i o n s was determined by the method of Dubois, et a l (11). A l i n e a r r e g r e s s i o n of I n molecular weight on the " g e l a f f i n i t y c o n s t a n t " (K ) allowed the c a l c u l a t i o n of apparent molecular weights of eYuted r a d i o a c t i v i t y from the e l u t i o n volume (V ). The constant, Κ , is d e f i n e d by Laurent and K i l l a n d e r ( 9 ) and it is r e l a t e S to V as f o l l o w s : Κ — e av (V - V ) / ( V -V ). Κ is independent of column geometry and packing d e n s i t y . TrîYs constant we d e f i n e as the " g e l a f f i n i t y constant" where i t s magnitude bears a d i r e c t r e l a t i o n s h i p to the a f f i n i t y of the e l u t e d molecule f o r the g e l . e

Q

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F r a c t i o n a t i o n of soil e x t r a c t s . A t r a d i t i o n a l f r a c t i o n a t i o n of the soil r e s i n e x t r a c t s w i t h a l k a l i i n t o humic a c i d ( p r e c i p i t a t e d from a l k a l i n e s o l u t i o n by a c i d ) , f u l v i c a c i d (that p a r t o f the a l k a l i n e s o l u t i o n not p r e c i p i t a t e d by a c i d ) , and humin (organic m a t e r i a l not s o l u b l e in a l k a l i ) was performed as described by S c h n i t z e r and Kahn (12). D i a l y s i s o f soil e x t r a c t s . The soil r e s i n e x t r a c t s were a l s o submitted t o d i a l y s i s a g a i n s t running tap water in c e l l u l o s e acetate a t ambient temperature. R e s u l t s and D i s c u s s i o n Of the l a r g e number o f e x t r a c t a n t s that have been t e s t e d , d i l u t e aqueous sodium hydroxid and q u a n t i t a t i v e l y the mos humic substances from s o i l s . When the incubated s o i l s l i s t e d in Table I were e x t r a c t e d w i t h hot IN sodium hydroxide s o l u t i o n or w i t h DOWEX A - l c h e l a t i n g r e s i n and water, a f t e r first being e x t r a c t e d w i t h a c i d i f i e d ether t o remove e x t r a c t a b l e r a d i o a c t i v e compounds, we found that the two e x t r a c t i o n methods were e q u a l l y e f f i c i e n t a t removing r a d i o a c t i v e humic substances. These r e s u l t s are shown in Table I I . TABLE I I E x t r a c t i o n of r a d i o a c t i v i t y from s o i l s u s i n g DOWEX A - l Resin and IN sodium h y d r o x i d e -

Chelating

Soil t e x t u r a l classification and source Loam, D a v i s ,

Incubation c o n d i t i o n s time,days temp,°C

R a d i o a c t i v i t y , % in..— resin NaOH extract extract

California

56

15

95

82

40

35

76

80

33

25

82

88

Loam, D a v i s , California Sandy Loam, No. Dakota S i l t y Clay Loam, Geneseo, I l l i n o i s

175

15 91 85 Ave. 86 84 a/ S o i l s had been incubated w i tSt'd. h d i t ea rl ri om rf o s - 4 C (5 ppm) and sub2 sequently e x t r a c t e d w i t h ether/O.IN HC1 (1.5/1.0 v / v ) . b/ These values a r e % o f that present in soil a f t e r a c i d i f i e d ether e x t r a c t i o n . The i n i t i a l v a l u e s were 37%, 31%, 35% and 30% o f the a p p l i e d r a d i o a c t i v i t y in the s o i l s as l i s t e d in the table.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

Resin e x t r a c t i o n of soil r e s u l t s in e f f i c i e n t removal of p o l y v a l e n t c a t i o n s t h a t b i n d o r g a n i c substances in soil. T h i s i n c r e a ses the d l s p e r s i t y of humic substances and a l s o i n c r e a s e s t h e i r s o l u b i l i t y by d i s r u p t i n g the hydrogen bonds of the f i x e d m e t a l l i c c a t i o n s . Sodium hydroxide accomplishes much the same t h i n g but is a more severe reagent. Thus, e x t r a c t i o n of s o i l s w i t h a c h e l a t i n g r e s i n w i l l u s u a l l y r e s u l t in l e s s degradation t o soil organic matter (13). When a l i q u o t s of the r e s i n soil e x t r a c t s were submitted to g e l chromatography the r e s u l t s shown in F i g u r e s 1 to 5 were o b t a i n ed. In each case, a p o r t i o n of the r a d i o a c t i v e m a t e r i a l placed on the column was e l u t e d in two main f r a c t i o n s . The apparent molec u l a r weights and percent recovery based on a p p l i e d r a d i o a c t i v i t y are i n d i c a t e d on the f i g u r e s . I t is recognized t h a f i g u r e s are o n l y approximate c a l i b r a t e t h e i r p o l y d e x t r a n c r o s s - l i n k e d g e l s . I f the humic substance molecules are more asymmetric than the dextrans used f o r c a l i b r a t i o n , as seems l i k e l y , then any p a r t i c u l a r grade of g e l w i l l exclude lower molecular weight humic substances than the nominal v a l u e would i n d i c a t e . Put another way: For equal molecular weight substances, a h i g h e r degree of molecular asymmetry is e q u i v a l e n t to a l a r g e r s i z e . Thus, the apparent molecular weight v a l u e s in these f i g u r e s are probably h i g h . The evidence c l e a r l y i n d i c a t e s there are a t l e a s t two r a d i o a c t i v e polymer f r a c t i o n s in each of the soil samples. The North Dakota soil (Figure 3) appears to have f i v e a d d i t i o n a l r a d i o a c t i v e f r a c t i o n s but t h i s degree of s e p a r a t i o n would need c o n f i r m i n g . The range of apparent molecular weights f o r these polymer f r a c t i o n s is 2100 t o >10,000. However, when the North Dakota soil sample e x t r a c t was submitted to g e l chromatography u s i n g a g e l w i t h l e s s c r o s s - l i n k i n g , thus extending the e x c l u s i o n l i m i t of the g e l , it was found t h a t the h i g h molecular weight f r a c t i o n in the e x t r a c t could be assigned an apparent molecular weight >100,000 (Figure 5 ) . The apparent molecular weight range of humus is reported to be 600 to 300,000 (14). To f u r t h e r c h a r a c t e r i z e the r a d i o a c t i v e polymeric substances in the DOWEX A - l r e s i n e x t r a c t s of soil, a sample of the North Dakota soil e x t r a c t was separated i n t o f u l v i c a c i d , humic a c i d and humin u s i n g the t r a d i t i o n f r a c t i o n a t i o n scheme d e s c r i b e d by S c h n i t z e r and Kahn (12). The p r o p o r t i o n of r a d i o a c t i v i t y in humic a c i d to that in f u l v i c a c i d was 1.8:1.O. A hot, IN sodium hydroxide e x t r a c t i o n of t h i s same soil, f o l l o w e d by s e p a r a t i o n i n t o humic a c i d and f u l v i c a c i d , r e s u l t e d in a r a d i o a c t i v e humic a c i d - f u l v i c a c i d p r o p o r t i o n of O.6:1.O. In the one case, where soil was e x t r a c t e d w i t h a c h e l a t i n g r e s i n , the r a d i o a c t i v e humic a c i d f r a c t i o n was h i g h r e l a t i v e to the r a d i o a c t i v e f u l v i c a c i d ; in the other — e x t r a c t i o n w i t h hot sodium hydroxide — the r a d i o a c t i v e humic a c i d f r a c t i o n was low. The reason f o r t h i s r e v e r s a l is that hot sodium hydroxide causes g r e a t e r degradation of the humic a c i d polymers (high molecular weight) than does the c h e l a t i n g r e s i n . The r e s u l t i n g

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Bound Residues in Soil Organic Matter

MEIKLE ET AL.

60

1

40

I DU"

15% = 0 IC av mw , >10,000 / 2% g Κ = O.65 ^ av . ^mw = 3200

U uU

j

y * |] IP

y «•

U

UL'

20 +

ν

ι

ν

t

E l u t i o1n volume 1-(ml)

Figure 1. Elution diagram for radioactive polymersinchelating resin extract of Davis, Calif,soil(15°C): Sephadex G-50, O.025M sodium borate, pH 9.1

16% Κ =0 av mw = >10,000

t

70

140 210 280 E l u t i o n volume (ml)

Figure 2. Elution diagram for radioactive polymersinchelating resin extract of Davis, Calif,soil(35°C): Sephadex G-50, O.025M sodium borate, pH 9.1

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

278

BOUND AND CONJUGATED PESTICIDE RESIDUES

12%

•9

6%

138 207 276 E l u t i o n volume (ml)

Figure S. Elution diagram for radioactive polymersinchelating resin extract of North Dakotasoil(25°C): Sephadex G-50, O.25M sodium borate, pH 9.1

80

160 240 320 E l u t i o n volume (ml)

Figure 4. Elution diagram for radioactive polymersinchelating resin extract of Illinoissoil(15°C): Sephadex G-50, O.025M sodium borate, pH 9.1

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

MEIKLE

ET AL.

Bound Residues in Soil Organic Matter

279

decomposition products have a lower molecular weight and tend to f r a c t i o n a t e as f u l v i c a c i d s . Dormar (15) has shown that e x t r a c t i o n of organic matter w i t h c h e l a t i n g r e s i n provides humic substances w i t h minimum a l t e r a t i o n . The humic and f u l v i c a c i d f r a c t i o n s separated from the DOWEX A - l r e s i n e x t r a c t o f the North Dakota soil were each submitted t o g e l chromatography and the r e s u l t s appear in Figures 6 and 7. We see h i g h molecular weight r a d i o a c t i v e m a t e r i a l in the humic a c i d f r a c t i o n and it comprises the major p a r t of the moveable r a d i o ­ a c t i v i t y in t h i s f r a c t i o n . The lower molecular weight r a d i o ­ a c t i v e m a t e r i a l appears in the moveable p o r t i o n of the f u l v i c a c i d f r a c t i o n w i t h some overlap o f 2300 Dalton polymers i n t o the humic a c i d f r a c t i o n . Thus the molecular weight d i s t r i b u t i o n of r a d i o a c t i v e f r a c t i o n s in the soil e x t r a c t s f o l l o w s the p a t t e r n expected f o r f r a c t i o n a t i o When a l i q u o t s o f th soil were d i a l y z e d through cellophane ( c e l l u l o s e acetate) an average 53% o f the r a d i o a c t i v e m a t e r i a l was r e t a i n e d by the membrane. That p o r t i o n of the n o r t h Dakota soil e x t r a c t r e t a i n e d by the cellophane membrane was submitted t o g e l chromatography using g e l G-50. The r e s u l t s are shown in Figure 8. We see that the r a d i o a c t i v e polymers w i t h Κ >0, apparent molecular weight, 10,000 were r e t a i n e d and appear in Figure 8 (see Figure 3 f o r comparison). This is another demons­ t r a t i o n that a p o r t i o n of the r a d i o a c t i v i t y in the r e s i n e x t r a c t s of soil is a s s o c i a t e d w i t h n o n - d i a l y z a b l e , h i g h molecular weight humic substances. The recovery o f r a d i o a c t i v e m a t e r i a l from the Sephadex g e l columns v a r i e d from 17% to 31% of that put on the column. I n the case o f the d i a l y s i s experiment, only 17% of the r a d i o a c t i v i t y a p p l i e d t o the column appear in the e l u a t e as a s i n g l e peak in F i g u r e 8. Apparently a l a r g e p a r t (83%) o f the high molecular weight (>10,000) r a d i o a c t i v e m a t e r i a l is in some way s t r o n g l y adsorbed by the g e l . When t h i s g e l was removed from the column and segments were assayed f o r r a d i o a c t i v i t y , 93% of the r e t a i n e d a c t i v i t y was found in the first i n c h and 100% in the first 5 inches. Sephadex gels are known to adsorb some p r o t e i n s (16), aromatic and h e t e r o c y c l i c compounds (17), and humum molecules (14). This phenomenon probably accounts f o r the low recovery o f r a d i o a c t i v e m a t e r i a l from the g e l columns used in our work. ^ I t has been shown in our work w i t h d i t a l i m f o s - C / s o i l that the s p e c i f i c r a d i o a c t i v i t y of the humus f r a c t i o n s , as dpm/mg. o f carbon, bears an i n v e r s e r e l a t i o n s h i p to molecular weight. The data showing the r e l a t i o n s h i p are reproduced as Table I I I . These changes in the s p e c i f i c a c t i v i t y of the soil organic carbon f r a c t i o n s are c o n s i s t e n t w i t h the concept that the more s o l u b l e f r a c t i o n s have a more r a p i d turnover. Thus, if humin represents organic carbon that is formed and broken down more s l o w l y than f u l v i c a c i d , f o r example, then a s m a l l e r p r o p o r t i o n of the t o t a l carbon of the humin w i l l be "new" carbon c o n t a i n i n g

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

16%

11%

a

E l u t i o n volume (ml) Figure 5. Elution diagram for radioactive polymersinchelating resin extract of North Dakotasoil:Sephadex G-100,O.1Msodium hydroxide

K = O.85 av m = 2300

60 +

40 4-

49

98 147 196 E l u t i o n volume (ml)

Figure 6. Elution diagram for radioactive humic acid from chelating resin extract of North Dakota soil: Sephadex G-50,O.025Usodium borate, pH 9.1

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Bound Residues in Soil Organic Matter

60

g. ο

4.

9% 8% Κ O.88 Κ = 1.03 av av mw = 2100 raw = 1700 χ

40

,

/

Figure 7. Elution diagram for radioactive fulvic acid from chelat­ ing resin extract of North Dakota soil: Sephadex G-50, O.025M sodium borate, pH 9.1

50

100 150 E l u t i o n volume (ml)

200

Figure 8. Elution diagram for radioactive polymers retained by membrane after dialysis of chefoting resin extract of North Dakota soil: Sephadex G-50,O.025M sodium borate, pH 9.1

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

282

BOUND

AND

C O N J U G A T E D PESTICIDE

RESIDUES

TABLE I I I S p e c i f i c a c t i v i t i e s , dpm/mgC, f o r f r a c t i o n a t e d soil organic matter a f t e r i n c u b a t i o n of s o i l s w i t h d i t a l i m f o s C.

FRACTION I n c r e a s i n g molecular weight ( f u l v i c a c i d ) humic a c i d )

Soil Sample Davis 15°

soil,

Davis 35°

soil,

2080

> (humin)

2074

467

216

No. Dakota soil, 25°

641

582

281

Illinois soil, 15°

638

281

46

Formation of humic substances in soil is a dynamic process o c c u r r i n g through the a c t i o n of microbes on p l a n t m a t e r i a l (18). Macromolecules are formed at the expense of carbohydrates of p l a n t o r i g i n . These macromolecules i n c l u d e b a c t e r i a l gums, a l g i n i c a c i d , p e c t i c a c i d , and other l e s s w e l l - d e f i n e d polymeric c a r b o x y l i c a c i d s . Aromatic polyphenols formed by way of o x i d a t i o n of quinones can condense w i t h amino a c i d s to u l t i m a t e l y g i v e h u m i c - l i k e substances. Basidiomicetes as w e l l as other microscopic f u n g i have been found to degrade l i g n i n to form appreciable^ amounts of humic a c i d - l i k e polymers (19). P h e n o l i c u n i t s from C-labeled phenolase l i g n i n have been shown to be i n c o r p o r a t e d i n t o f u n g i - s y n t h e s i z e d polymers (20). The general consensus appears to be t h a t there is a genetic r e l a t i o n between the v a r i o u s humic substances. F u l v i c a c i d is considered to represent poly-condensation m a t e r i a l formed from s i m p l e r molecules. C o n t i n u a t i o n of p o l y m e r i z a t i o n and chemical m o d i f i c a t i o n leads to the l e s s s o l u b l e humic a c i d and e v e n t u a l l y to i n s o l u b l e humin, thought to have the h i g h e s t molecular weight and most r e s i s t a n t s t r u c t u r e . The e a r l i e r , and probably more r a p i ^ y formed, f u l v i c a c i d s w i l l be c l o s e r to e q u i l i b r i u m w i t h the C p o o l of simpler and s m a l l e r molecules than w i l l m a t e r i a l s f a r t h e r down the sequence and would, t h e r e f o r e , have a h i g h e r s p e c i f i c acj£vity. During t h i s sequence of r e a c t i o n s the incorporated C becomes an i n t e g r a l p a r t of the molecular s t r u c t u r e without r e c o g n i z a b l e r e l a t i o n s h i p to the parent molecule from which it is d e r i v e d . The r a t e of humin degradation is very slow (21)• Sorenson (22) s t u d i e d the degradation of l a b e l e d glucose and c e l l u l o s e in

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

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three s o i l s . A f t e r a r a p i d i n i t i a l b r e a ^ o w n , h a l f - l i v e s of 5 to 9 years were r e p o r t e d f o r the remaining C to be degraded. These data imply t h a t , even w i t h r e a d i l y metabolized compounds, i n c o r ­ p o r a t i o n i n t o humic substances occurs and l i m i t s the extent to which complete degradation to C0~ proceeds. L i k e w i s e , p e s t i c i d e molecules degrade and the products u l t i m a t e l y become i n c o r p o r a t e d in humic m a t e r i a l s . These macromolecules so formed are i n d i s t i n ­ guishable from those d e r i v e d from carbon compounds n a t u r a l to soil. Other authors have demonstrated the formation of humin from r e a d i l y decomposable o r g a n i c compounds (23,24). In summary, we have shown that when an o r g a n i c compound i n c o r p o r a t e d in soil is decomposed, a p a r t of the decomposition products u l t i m a t e l y become a s s o c i a t e d w i t h the soil organic m a t e r i a l . These products are sometimes r e f e r r e d to as "bound m a t e r i a l " . In r e a l i t y a can be s o l u b i l i z e d w i t h hydroxide or DOWEX A - l c h e l a t i n g r e s i n and water. The l a t e r is p r e f e r r e d because it is much l e s s d e s t r u c t i v e to the organic matter. F u r t h e r , we have shown t h a t a p a r t of the decomposition products are combined w i t h the e x t r a c t e d o r g a n i c m a t e r i a l in such a way t h a t the products are an i n t e g r a l p a r t of the p o l y ­ molecular s t r u c t u r e of the o r g a n i c m a t e r i a l . F i n a l l y , we have shown t h a t the f r a c t i o n s of soil organic m a t e r i a l , commonly known as f u l v i c a c i d , humic a c i d and humin, c o n t a i n i n c o r p o r a t e d decomposition products. These macromolecules can be separated i n t o r a d i o a c t i v e f r a c t i o n s having apparent molecular weights ranging from 2100 to >100,000. LITERATURE CITED 1. 2.

3.

4.

5.

6.

7. 8.

Altgelt, Κ. H., and Segal, L., "Gel Permeation Chromatography", Dekker, New York (1971). Day, P. R., Particle fractionation and particle-size analysis, pages 545-566 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron., Inc., Madison, Wisc. (1965). Peech, Μ., Hydrogen-ion activity, pages 914-925 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron., Inc., Madison, Wisc. (1965). Allison, L. E., Organic Carbon, pages 1367-1378 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron. Inc., Madison, Wisc. (1965). Richards, L. Α., Physical condition of water in soil, pages 131-137 in C. A. Black (ed), "Methods of Soil Analysis", Amer. Soc. of Agron., Inc., Madison, Wisc. (1965). Anon., "Sephadex-gel f i l t r a t i o n in theory and practice", Pharmacia Fine Chemicals, Inc., 800 Centennial Ave., Piscataway, N. J. 08854. (1966). Carnegie, P. R., Nature (1965) 206, 1128. Andrews, P., Biochem. J. (1965), 96, 595.

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9. 10. 11. 12. 13.

14. 15. 16 17. 18. 19.

20. 21

22. 23. 24.

BOUND AND CONJUGATED PESTICIDE RESIDUES

Laurent, T. C., and Killander, J., J. Chromatog. (1964), 14, 317. Carnegie, P. R., Biochem. J. (1965) 95, 9P. Dubios, M., G i l l e s , Κ. Α., Hamilton, J. Κ., Rebers, P. Α., and Smith, F., Anal. Chem. (1956), 28, 350. Schnitzer, Μ., and Kahn, S. U., "Humic Substances in the Environment", p. 17, Dekker, New York, (1972). Bremner, J. Μ., Organic nitrogen in s o i l s , pages 93-149 in W. V. Bartholomew and F. E. Clark (eds), "Soil Nitrogen", Amer. Soc. Agron., Madison, Wisc. (1965). Gjessing, E. T., Nature (1965), 197, 1091. Dormaar, J. F., Bull. Ass. Fr. Etude Sol (1973), (2), 71-9. Chem. Abstr. (1974), 80, 107113t. Glazer, Α. Ν., and Wellner, D., Nature (1962), 194, 862. Gelotte, B., J. Chromatograph McLaren, A. D., Scienc Hurst, Η. M. and Burger, W. Α., Lignin and Humic Acids, pages 260-286 in "Soil Biochemistry", McLaren, A. D., and Peterson, G. H. (eds), Dekker, New York (1967). Martin, J. P., and Haider, Κ., Soil Sci. (1971), 111, 54. Stevenson, I. L., Biochemistry of Soil, Page 242, in "Chemistry of the Soil No. 160", Bear, F. E. (ed), Reinhold Publ. Corp., New York (1964). Sorenson, L. H., Soil Biol. Biochem. (1972), 4, 245. Chekalar, Κ. I., and Illyuvieva, V. P., Pochvovedenie No. 5, pp. 40-5 (1962). Sinha, Μ. Κ., Plant and Soil (1972), 36, 283.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21 Chemical Extraction of Certain Trifluoromethanesulfonanilide Pesticides and Related Compounds from the Soil SURESH K. BANDAL, HENRY B. CLARK, and JAY T. HEWITT 3M Co., 3M Center, St. Paul, Minn. 55101

The 3M Company, S a i n t P a u l , Minnesota, has d i s c o v e r e d t h r e e p r o m i s i n g new a g r i c h e m i c a l s of the N-aryl 1,1,1-trifluoromethanesulfonamid compounds:

®

Perfluidone (DESTUN Herbicide)

®

Fluoridamid (SUSTAR Plant Growth R e g u l a t o r )

MBR 12325 ( E x p e r i m e n t a l Herbicide/Plant Growth Regulator)

285

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

286

BOUND AND CONJUGATED PESTICIDE RESIDUES

Perfluidone shows a dramatic control of nutsedge (Cyperus sp.) and is also h e r b i c i d a l to a variety of important grassy and broadleaf weeds. Perfluidone has been granted a temporary permit for use on cotton by the EPA, and p e t i t i o n s for full r e g i s t r a t i o n for use on cotton and for establishment of n e g l i g i b l e residue tolerance in cottonseed have been submitted. Fluoridamid is f u l l y registered by the EPA for certain a p p l i ­ cations as a turf growth retardant. MBR 12325 is un­ dergoing development as a grass and ornamental plant growth retardant and as an agent for enhancing sugar content in sugarcane. Because these compounds are either applied dir­ ectly to soil or are applied f o l i a r l y , in which case an appreciable amoun eventually lodge on study the degradation of each compound in soil. In addition, it was of i n t e r e s t to study the behavior in soil of several known or p o t e n t i a l soil metabolites of these three compounds in order to elucidate the react­ i v i t y of the various functional groups present. The ten compounds which were studied are shown in Figures 1 to 4; for convenience, they are divided into four groups based on s i m i l a r i t i e s of chemical structure. The known soil metabolites are Compound II (Figure 1), which is the major soil metabolite of perfluidone (1) and Compound VII (Figure 3) which has been shown to be a major soil metabolite of MBR 12325 (2). Materials A l l c h e m i c a l s were of g r e a t e r than 99% p u r i t y . A p p r o p r i a t e amounts of n o n r a d i o l a b e l e d c h e m i c a l s were mixed w i t h the c o r r e s p o n d i n g carbon-14 l a b e l e d compounds to y i e l d the d e s i r e d s p e c i f i c a c t i v i t i e s . P e r f l u i d o n e and m e t a b o l i t e α were u n i f o r m l y l a b e l e d on the t r i s u b s t i t u t e d r i n g ; all o t h e r s were u n i f o r m l y l a b e l e d on the benzene r i n g . A l l r a d i o l a b e l e d com­ pounds were of g r e a t e r than 99% r a d i o c h e m i c a l p u r i t y . The s p e c i f i c a c t i v i t i e s of the v a r i o u s compounds a r e l i s t e d in T a b l e I . The soil used was a sandy loam o b t a i n e d from B r a i n e r d , M i n n e s o t a , and c o n t a i n e d 57% sand, 32% s i l t , 11% c l a y , and 2.0% o r g a n i c m a t t e r . The pH of the soil was 6.5. Methods T h i n - l a y e r Chromatography ( t i c ) . S i l i c a g e l F254 p r e c o a t e d c h r o m a t o p l a t e s (20 χ 20 cm, MN brand,

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

BANDAL

287

Trifluoromethanesulfonanilide Pesticides

ET AL.

Perfluidone

Metabolite

ΒΑ

α

7209

Figure 1. Group 1 compounds: lJ,l-trifluoromethanesuîfonanilides containing no other —NHR groups

NHS0 CF 2

3

(IV)

NHS0 CF 9

Fluoridamid

Q

3

O] 2

(V)

MBR

12325

NHCCH, C H

3

Figure 2. Group 2 compounds: 1,1,1-trifluoromethanesulfonanilides containing a 3-acetamido group

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

288

BOUND AND CONJUGATED PESTICIDE RESIDUES

N

CH , HS0 CF 3

CH

2

N H 3

S0 CF 2

3

(VI)

BA

5974

(VII)

BA

15753

3

CH,

(VIII)

BA

8315

NH„ CH (IX)

BA

15733

NHCCH. CH. Figure 3.

Group 3 compounds: 1,1,1-tHfluorornethanesulfonanilides or acetanilides containing one free —NH group t

Figure 4.

Group 4 compound: compound containing two free —NH groups

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9

21.

Table

I.

289

Trifluoromethanesulfonanilide Pesticides

BANDAL ET AL.

S p e c i f i c A c t i v i t i e s o f t h e Compounds U s e d a n d TLC S o l v e n t S y s t e m s U s e d f o r C l e a n u p and T w o - D i m e n s i o n a l TLC A n a l y s i s , TLC

Specific DesignatActivity, Compound i n g Number dpm/yg

Solvent

System Second Cleanup and D e v e l o p F i r s t D e v e l - ment in 2opment in 2- D i m e n s i o n Dimensional a l Analysis Analysis

Perfluidone

I

Metabolite α

II

972

D

Ε

ΒΑ 7209

III

958

A

C

Fluoridamid

IV

199

Β

C

MBR 12325

V

830

Β

C

BA 5974

VI

1355

Β

C

BA 15753

VII

1975

Β

C

BA 8315

VIII

1957

Β

C

BA 15733

IX

2006

Β

C

TDA

X

2248

Β

c

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

290

BOUND

AND

CONJUGATED PESTICIDE

RESIDUES

o b t a i n e d from Brinkmann I n s t r u m e n t s , Incorporated, W e s t b u r y , New Y o r k ) w e r e u s e d a t O.25 and O.50 mm g e l t h i c k n e s s f o r a n a l y s i s and a t 1.0 and 2,0 mm gel t h i c k n e s s f o r p r e l i m i n a r y cleanup of soil extracts. The s o l v e n t s y s t e m s u s e d and t h e i r a l p h a b e t i c a l d e s i g n a t i o n s a r e as f o l l o w s : A: Ethyl acetate-toluene-chloroform-formic acid (1-1-1-O.06), B: Ethyl acetate-acetic acid (49-1), C: Chloroform-methanol-acetic a c i d (45-5-1), D: Chloroform-methanol-acetic acid-water ( 2 5 - 1 5 - 4 - 2 ) , and E: η-Butanol-water-acetic a c i d (3-1-1). Two-dimensional cochromatography of a r a d i o ­ l a b e l e d component, d e t e c t e (Kodak N o - S c r e e n X - r a u n l a b e l e d compound in two d i f f e r e n t s o l v e n t s y s t e m s , was c o n s i d e r e d s u f f i c i e n t to c o n s t i t u t e t e n t a t i v e i d e n t i f i c a t i o n of the component. The t i c s o l v e n t s y s t e m s u s e d f o r e a c h compound a r e a l s o l i s t e d in Table I. Q u a n t i t a t i v e d a t a w e r e o b t a i n e d by s c r a p i n g r a d i o a c t i v e g e l r e g i o n s , a s d e t e c t e d by radioauto­ graphy, i n t o s c i n t i l l a t i o n v i a l s f o r r a d i o c a r b o n c o n t e n t d e t e r m i n a t i o n by d i r e c t l i q u i d scintillation counting ( l s c ) . E a c h v i a l was recounted after addition o f a known amount o f t o l u e n e - l ^ C t o d e t e r m i n e c o u n t i n g efficiency. P r e p a r a t i o n of Soil Samples. F o r e a c h compound, f o r e a c h t i m e p e r i o d s t u d i e d , 100 gram p o r t i o n s o f soil (dry w e i g h t b a s i s ) were g e n t l y p a c k e d i n t o g l a s s j a r s (7 cm h i g h X 7 cm in d i a m e t e r ) . E a c h compound was a p p l i e d to the soil s u r f a c e as a s o l u t i o n o f 1 mg of the d i e t h a n o l a m i n e o r p o t a s s i u m s a l t o f t h e compound in 5 ml o f d i s t i l l e d w a t e r (pH ^8) so t h a t t h e c o n c e n t r a t i o n o f t h e compound in soil was 10 ppm. The treated soil samples were w a t e r e d p e r i o d i c a l l y but a l l o w e d t o d r y o u t b e t w e e n w a t e r i n g s so t h a t simulated f i e l d c o n d i t i o n s o f a l t e r n a t e w e t t i n g and d r y i n g w e r e achieved. The soil s a m p l e s w e r e h e l d in t h e g r e e n h o u s e and G R 0 L U X ( M o d e l FR 96T12-GRO-VHO-WS, S y l v a n i a c o r p o r a t i o n , Salem, M a s s a c h u s e t t s ) r e f l e c t o r i z e d wide s p e c t r u m lamps were s u s p e n d e d at a d i s t a n c e o f a p p r o x ­ i m a t e l y 30 i n c h e s f r o m t h e t o p o f t h e soil with a d a y - n i g h t c y c l e o f 18:6 hours. R

Soil A n a l y s is. F o r e a c h c o m p o u n d , a t d e s i r e d t i m e i n t e r v a l s r a n g i n g up t o two m o n t h s a f t e r soil treatment duplicate soil samples were s e p a r a t e l y soaked w i t h 15% (V/W) o f d i s t i l l e d w a t e r f o r 12 h o u r s and t h e n s o x h l e t -

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

BANDAL ET AL.

Trifluoromethanesulfonanilide Pesticides

291

e x t r a c t e d f o r 16 h o u r s w i t h an a c e t o n i t r i l e - w a t e r azeotrope (5-1); a t s a m p l e was s i m i l a r l y e x t r a c t e d but w i t h o u t s o a k i n g . The r a d i o c a r b o n c o n t e n t o f e a c h e x t r a c t was d e t e r m i n e d by s u b m i t t i n g an a l i q u o t t o l s c . The e x t r a c t s w e r e e v a p o r a t e d t o d r y n e s s in vacuuo u s i n g a r o t a r y e v a p o r a t o r w i t h a water bath m a i n t a i n e d a t 4 5 ° C o r l e s s t o m i n i m i z e t h e r m a l decomposition. The c o n c e n t r a t e d e x t r a c t s w e r e t h e n a n a l y z e d by t i c and r a d i o a u t o g r a p h y . The s o l v e n t e x t r a c t e d soil samples were a i r - d r i e d and p u t i n t o o n e - p i n t j a r s , m e c h a n i c a l l y r o t a t e d f o r a t l e a s t 24 h o u r s t o i n s u r e t h o r o u g h m i x i n g , and a s a m p l e ( a p p r o x i m a t e l y 1-1.5 g r a m s ) a n a l y z e d by comb u s t i o n - l s c to determine r e s i d u a l u n e x t r a c t e d r a d i o carbon content. The radiocarbon applied u n e x t r a c t a b l e r a d i o c a r b o n was c a l c u l a t e d , w h i c h r e p r e s e n t e d t h e l o s s o f r a d i o c a r b o n by v o l a t i l i z a t i o n o f p a r e n t compound o r d e r i v e d p r o d u c t s , s u c h as 1 ^ C 0 2 . The soil was t h e n f r a c t i o n a t e d i n t o h u m i c a c i d , f u l v i c a c i d , and h u m i n f r a c t i o n s a c c o r d i n g t o t h e method o f S t e v e n s o n (3). The r a d i o c a r b o n c o n t e n t o f e a c h o f t h e s e f r a c t i o n s was d e t e r m i n e d by l s c o r combustion-lsc. The h u m i c a c i d f r a c t i o n was dried o v e r P 2 ° 5 and w e i g h e d ; t h e f u l v i c a c i d f r a c t i o n was l y o p h i l i z e d , d i a l y z e d a g a i n s t d i s t i l l e d water to remove s o d i u m c h l o r i d e , l y o p h i l i z e d a g a i n , and w e i g h e d . The p e r c e n t a g e o f r a d i o c a r b o n in e a c h f r a c t i o n was then c a l c u l a t e d . Q

Results Acetonitrile-Water Extraction Analysis. Figures 5 t o 14 show, f o r t h e v a r i o u s compounds a t v a r i o u s t i m e p e r i o d s , t h e r a d i o c a r b o n a c c o u n t e d f o r in t h e s o x h l e t e x t r a c t s , t h e amounts o f e x t r a c t e d r a d i o c a r b o n a c c o u n t e d f o r as p a r e n t compound and as p o l a r comp o n e n t s ( i . e . , t h o s e t h a t d i d n o t move f r o m t h e o r i g i n in t i c a n a l y s i s u s i n g n o n - p o l a r s o l v e n t s y s t e m s ) t h e amount o f u n e x t r a c t a b l e r a d i o c a r b o n , and t h e amount o f r a d i o c a r b o n l o s t as v o l a t i l e c o m p o n e n t s . The two G r o u p 1 compounds c o n t a i n i n g an - S O 2 R g r o u p , Compounds I and I I , showed t h e l e a s t reactivity o f t h e compounds s t u d i e d , b o t h compounds e x h i b i t i n g nearly i d e n t i c a l behavior in soil. B o t h showed h i g h e x t r a c t a b i l i t i e s of r a d i o c a r b o n from soil (about 90% a f t e r two m o n t h s ) w i t h n e a r l y all o f t h e r a d i o c a r b o n r e p r e s e n t e d by p a r e n t compound. Low l e v e l s o f p o l a r e x t r a c t a b l e c o m p o n e n t s and u n e x t r a c t a b l e r a d i o c a r b o n w e r e f o u n d , a n d r a d i o c a r b o n l o s s as v o l a t i l e c o m p o n e n t s

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

292

BOUND AND CONJUGATED PESTICIDE RESIDUES

0

2

4

6

8

10

Time AfterSoil-Treatment,Weeks

Figure 5. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled perfluidone. . , radiocarbon extracted; A> varent- C extracted; |, loss as volatiles; •, unextractable ("bound*) radiocarbon; X, extractable C-polar substances. u

14

Time AfterSoil-Treatment,Weeks

Figure 6. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled metabolite «. . , radiocarbon extracted; A> parent- C extracted; I , loss as volatiles; •, unextractable ("bouncT) radio­ carbon; χ, extractable C-polar substances. u

u

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

BANDAL

293

Trifluowmethanesulfonanilide Pesticides

E T AL.

100

Ι­

Ο

2

4

β

8

10

Time AfterSoil-Treatment,Weeks

Figure 7. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled Β A 7209. . , radiocarbon extracted; A> varent- C extracted; |, loss as volatiles; Π> unextractable ("bound*) radiocarbon; χ, extractable C-polar substances. 14

14

loo

L-

I

Time AfterSoil-Treatment,Weeks

Figure 8. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled fluoridamid. . , radiocarbon extracted; A , parent- C extracted; |, l° as volatiles; Q unextractable ("bound) radiocar­ bon; χ, extractable C-polar substances. 14

ss

14

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

294

BOUND AND CONJUGATED PESTICIDE RESIDUES 100

t

Time AfterSoil-Treatment,Weeks

Figure 9. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled MBR12325. . , radiocarbon extracted; A> parent- C extracted; |, loss as vohtiles; •, unextractable ("bound') radiocarbon; X, extractable C-polar substances. 14

14

100

Ι­

Ο

2

4

6

8

10

Time AfterSoil-Treatment,Weeks

Figure 10. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled BA 5974. . , radiocarbon extracted; A , parent- C extracted; |, loss as volatiles; Π , unextractable ("bound*) radiocarbon; X, extractable C-pofor substances. 14

14

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

BANDAL

ET AL.

Trifluoromethanesulfonanilide Pesticides

100 h

Time AfterSoil-Treatment.Weeks

Figure 11. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled Β A 15753. . , radiocarbon extracted; A , varent- C extracted; |, loss as volatiles; •, unextractable ("bound*) radiocarbon; X, extractable C-polar substances. 14

14

Time AfterSoil-Treatment,Weeks

Figure 12. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled BA 8315. . , radiocarbon extracted; A , parent- C extracted; A loss as vohtiles; •, unextractable ("bound*) radiocarbon; χ, extractable C-polar substances. 14

14

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

295

296

BOUND AND CONJUGATED PESTICIDE RESIDUES 100

r-

Time AfterSoil-Treatment,Weeks

Figure 13. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled BA 15733. . , radiocarbon extracted; A , varent- C extracted; loss as vofotiles; •, unextractable ("bound') radiocarbon; X, extractable C-polar substances. u

14

100

Ι­

Ο

4

2

6

8

10

Time AfterSoil-Treatment,Weeks

Figure 14. Radiocarbon accountability at various time periods aftersoil-treatmentwith radiolabeled TDA. . , radiocarbon extracted; A , parent- C extracted; •, loss as vofotiles; •, unextractable ("bound*) radiocarbon; χ, extractable C-polar substances. 14

14

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

BANDAL ET AL.

Trifluoromethanesulfonanilide Pesticides

was

297

q u i t e l o w ( 5 % o r l e s s a f t e r two m o n t h s ) . The t h i r d G r o u p 1 c o m p o u n d , Compound I I I , e x h i b i t e d f a r d i f f e r e n t b e h a v i o r , w i t h the major route o f d i s s i m i l a t i o n b e i n g l o s s by v o l a t i l i z a t i o n (^70% a f t e r two m o n t h s ) . I t s h o u l d be p o i n t e d o u t t h a t in a separate experiment ( 2 ) u s i n g biométrie f l a s k s a c c o r d i n g t o a m o d i f i c a t i o n o f t h e method o f B a r t h a and P r a m e r ( 4 ) , o n l y a r e l a t i v e l y m i n o r amount o f 14c02 was c o l l e c t e d t h r o u g h two w e e k s ; t h e r e f o r e , it a p p e a r s t h a t u n d e r f i e l d c o n d i t i o n s a v e r y l a r g e amount o f Compound I I I may v o l a t i l i z e . Because o f t h i s , r a d i o c a r b o n e x t r a c t a b i l i t y was f a r l e s s t h a n w i t h Compounds I a n d I I , w i t h a b o u t o n e - h a l f o f t h e e x t r a c t e d r a d i o c a r b o n b e i n g accounted f o r as p a r e n t compound. R a d i o c a r b o w i t h about 20% o f r a d i o c a r b o u n e x t r a c t a b l e f r a c t i o n a f t e r two m o n t h s . The two G r o u p 2 compounds s h o w e d p a r a l l e l behavior. Radiocarbon e x t r a c t a b i l i t y dropped s h a r p l y f o r a b o u t one w e e k , t h e n l e v e l e d o f f ; p a r e n t compound accounted f o r about o n e - f o u r t h t o o n e - t h i r d o f extractable radiocarbon. The amount o f " b i n d i n g " c o n c o m i t a n t l y rose s h a r p l y , then l e v e l e d o f f . Radioc a r b o n l o s s was a b o u t 2 5 % a f t e r two m o n t h s . Compound V, w h i c h c o n t a i n s a 4 - m e t h y l g r o u p , showed l e s s b i n d i n g t h a n Compound I V . The f o u r G r o u p 3 c o m p o u n d s , e a c h o f w h i c h h a s one f r e e a m i n o g r o u p , all b e h a v e d s i m i l a r l y in soil. E x t r a c t a b i l i t i e s o f r a d i o c a r b o n and r a d i o c a r b o n " b i n d i n g " w e r e s i m i l a r t o t h e two G r o u p 2 c o m p o u n d s , t h e m a j o r d i f f e r e n c e b e i n g in t h e v e r y r a p i d d i s a p p e a r a n c e o f p a r e n t compound w i t h t h e G r o u p 3 compounds. E v e n a t t ( a c t u a l l y a b o u t 15 m i n u t e s ) t h e r e c o v e r i e s o f p a r e n t compounds w e r e c o n s i d e r a b l y l e s s t h a n t h e e x p e c t e d 1 0 0 % , r a n g i n g f r o m 20 t o 6 0 % , and r e c o v e r i e s r a p i d l y d r o p p e d t h e r e a f t e r . ( I n c o n t r a s t , w i t h all o f t h e G r o u p 1 a n d 2 compounds all o f e x t r a c t e d r a d i o c a r b o n a t was d u e t o p a r e n t compound). W i t h t h e two G r o u p 3 compounds n o t h a v i n g a 4 - m e t h y l g r o u p , d i s a p p e a r a n c e o f p a r e n t compound was c o m p l e t e in l e s s t h a n a week. W i t h a 4 - m e t h y l g r o u p p r e s e n t , d i s a p p e a r a n c e o f p a r e n t compound was d e l a y e d somewhat b u t was s t i l l c o m p l e t e a f t e r e i g h t weeks. The G r o u p 4 compound was t h e most r e a c t i v e ; o n l y a b o u t 1 0 % o f t h e r a d i o c a r b o n was e x t r a c t a b l e , and t h i s v a l u e r e m a i n e d a b o u t c o n s t a n t t h r o u g h two months. No p a r e n t compound was d e t e c t e d a t any t i m e p e r i o d , i n c l u d i n g t . R a d i o c a r b o n l o s s , h o w e v e r , was c o m p a r a b l e t o t h a t o f t h e Group 2 and 3 compounds. 0

0

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND

298

A N D CONJUGATED

PESTICIDE

RESIDUES

F r a c t i o n a t i o n o f Soil O r g a n i c M a t t e r . Table I I shows t h e r e c o v e r y o f soil r a d i o c a r b o n a s h u m i c a c i d , f u l v i c a c i d , a n d h u m i n f r a c t i o n s two weeks and two months a f t e r soil application of various radiolabeled compounds. The v a l u e s r e p r e s e n t p e r c e n t o f t h e t o t a l r a d i o a c t i v i t y p r e s e n t in t h e s o x h l e t - e x t r a c t e d soil, i . e . the unextractable f r a c t i o n . I t is a p p a r e n t t h a t w i t h t i m e t h e amount o f r a d i o c a r b o n in t h e h u m i c a c i d d e c r e a s e d , a n d t h a t , c o n c o m i t a n t l y , t h e r e was an i n c r e a s e in t h e amount o f r a d i o c a r b o n a s s o c i a t e d with the f u l v i c acid f r a c t i o n . The o n l y e x c e p t i o n t o t h i s o b s e r v a t i o n was MBR 1 2 3 2 5 . Discussion The r e s u l t s o f t h a t t h e -NHSO2CF3 g r o u p , u n d e r t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s , does n o t b i n d a p p r e c i a b l y t o soil; Compounds I a n d I I a r e r e a d i l y e x t r a c t e d f r o m soil e v e n two m o n t h s a f t e r soil-treatment, a n d Compound I I I r e a d i l y v o l a t i l i z e s from t h e soil. Compound I I I undergoes a h i g h e r degree of c o n v e r s i o n to other soil p r o d u c t s t h a n Compounds I a n d I I , s u g g e s t i n g t h a t t h e -SO2R g r o u p , l a c k i n g in I I I , c o n f e r s s t a b i l i t y t o w a r d s soil d e g r a d a t i o n under these c o n d i t i o n s . I n t h e two m - p h e n y l e n e d i a m i n e compounds w i t h b o t h a m i n o g r o u p s b l o c k e d , f l u o r i d a m i d a n d MBR 1 2 3 2 5 , r a d i o c a r b o n e x t r a c t i o n is l o w e r a n d soil-binding o f r a d i o c a r b o n is h i g h e r t h a n w i t h t h e G r o u p 1 comp o u n d s , s u g g e s t i n g t h a t t h e a c e t a m i d o g r o u p may b e i m p o r t a n t f o r " b i n d i n g " t o soil p a r t i c l e s . In the f o u r m - p h e n y l e n e d i a m i n e compounds w i t h o n e f r e e a n d one b l o c k e d a m i n o g r o u p , t h e s i m i l a r b i n d i n g b e h a v i o r t o t h e G r o u p 2 compounds p l u s t h e r a p i d disappearance o f p a r e n t compound s u g g e s t t h a t b i n d i n g f o ralls i x compounds o c c u r s v i a a f r e e -NH2 g r o u p ; in t h e c a s e o f t h e G r o u p 2 compounds t h i s is o b t a i n e d b y c l e a v a g e o f t h e N-C b o n d o f t h e a c e t a m i d o g r o u p . The f a c t t h a t t h e G r o u p 1 compounds do n o t b i n d i n d i c a t e s t h a t t h e N-S b o n d o f t h e -NHSO2CF3 g r o u p is n o t r e a d i l y cleaved. The b e h a v i o r o f TDA r e i n f o r c e s t h e t h e o r y t h a t f r e e -NH g r o u p s a r e r e s p o n s i b l e f o r t h e soil " b i n d i n g " b e h a v i o r o f t h e compounds s t u d i e d . W i t h two f r e e -NH2 g r o u p s , b i n d i n g was a l m o s t i n s t a n t a n e o u s a n d no p a r e n t compound was d e t e c t e d a t any t i m e . The r e l a t i o n s h i p b e t w e e n t h e a m o u n t s o f r a d i o - , c a r b o n a s s o c i a t e d w i t h humic and f u l v i c a c i d s a t t h e two t i m e p e r i o d s s t u d i e d s u p p o r t s t h e f i n d i n g s o f S c h n i t z e r ( 5 ) in t h a t t h e a m o u n t s o f e x t r a c t a b l e 2

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

Table

II.

299

F r a c t i o n a t i o n of the U n e x t r a c t a b l e Soil R a d i o c a r b o n i n t o Humic A c i d , F u l v i c A c i d and Humin Two Weeks and Two M o n t h s A f t e r Soil-Application of I n d i c a t e d Radiolabeled Compound.

Radiocarbon Recovery, % of T o t a l Present in S o l v e n t - E x t r a c t e d Soil Humin Humic A c i d Fulvic Acid 2 Wk

Compound

No

Trifluoromethanesulfonanilide Pesticides

BANDAL ET AL.

Free

-NH

2

Group a

„ >

Perfluidone

a

22.7

α „ >

a

47.0

„ > a

30 .3

17.5

„ >

38.4

>

44 .1

a

Metabolite

„ > a

7209

38.9

23.6

29 .9

46 .2

31.2

30 .2

Fluoridamid

42.8

41 .6

23.4

37.0

33.8

21 .4

MBR

33.7

42.8

44 .7

34 .9

21.7

22 .3

ΒΑ

12325

Free

-NH

0

G r o u p ( s ) Près ent

BA

5974

57.1

38.2

36.9

53.2

6.0

8 .6

BA

15753

56.2

33.3

40.0

54.2

3.8

12 .5

BA

8315

48.2

29 .2

28.1

67.8

23.7

3 .0

BA

15733

46.6

25.9

34 .1

58 .2

19 .3

15 .9

38.7

40 .4

36 .4

41.0

24 .9

18 .6

TDA

Fractionation

not done.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

300

BOUND AND CONJUGATED PESTICIDE RESIDUES

humic a c i d s were n e g a t i v e l y c o r r e l a t e d w i t h t h e p r o ­ c e s s o f h u m i f i c a t i o n , whereas t h e amounts o f e x t r a c t a b l e f u l v i c a c i d s were p o s i t i v e l y c o r r e l a t e d . I t is p o s s i b l e that w i t h time the degradation products of m e t a b o l i s m o f t h e s e compounds p a r a l l e l t h e p r o c e s s o f h u m i f i c a t i o n and a r e i n c o r p o r a t e d m a i n l y in t h e fulvic acid fraction. E x c e p t f o r MBR 1 2 3 2 5 , t h e f i n d ­ i n g s on t h e r a d i o c a r b o n d i s t r i b u t i o n b e t w e e n h u m i c and f u l v i c a c i d s o b s e r v e d in t h e p r e s e n t s t u d y a r e in a c c o r d a n c e w i t h A l e x a n d r o v a ( 6 ) who s t a t e s t h a t h u m i c a c i d b r e a k s down i n t o f u l v i c a c i d , a l t h o u g h f u l v i c a c i d does n o t p o l y m e r i z e i n t o humic a c i d . The g e n e r a l l y h e l d b e l i e f t h a t t h e g e n e r i c relation­ s h i p between v a r i o u s f r a c t i o n s of the soil organic m a t t e r , where the p r o c e s from f u l v i c a c i d s t s u b s t a n c e s ( h u m i n ) , was n o t c l e a r l y e v i d e n t in t h e present i n v e s t i g a t i o n . Literature

Cited

(1) Bandal, S. Κ., Η. Β. C l a r k , and S. C. Anderson. 1973. Paper p r e s e n t e d at the 167th N a t i o n a l Meeting of the American Chemical S o c i e t y , Los A n g e l e s , California (2) Bandal, S. Κ., Η. Β. C l a r k and J. T. H e w i t t . 1975. Unpublished results. (3) Stevenson, F. J. 1965. Methods of Soil A n a l y s i s , e d i t e d by B. A. B l a c k , Am. Soc. Agron., Volume 2, pages 1409-1421. (4) B a r t h a , R. And D. Pramer. 1965. Soil Sci., 100, 68. (5) S c h n i t z e r , M. 1967. Can. J. Soil Sci., 47, 245. (6) A l e x a n d r o v a , L. N. 1966. I n t e r n . Soc. Soil Sci. T r a n s . I I , IV, Comm., Aberdeen, p. 73.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22 Biological Unavailability of Bound Paraquat Residues in Soil D. RILEY and W. WILKINSON ICI Plant Protection Division, Jealott's Hill Research Station, Bracknell Berkshire, U. K. Β. V. TUCKER Chevron Chemical Co., Ortho Division, Richmond, Calif. 94804

1.

INTRODUCTION

The herbicidal propertie bipyridylium ion] were discovere Research Station, England, in 1955. Paraquat is now used commercially worldwide. It is normally manufactured as the dichloride salt.

Paraquat is a broad spectrum, rapidly acting contact herbi­ cide which is highly effective against grasses and most broad leaved species. A unique property of paraquat is its rapid and complete adsorption onto soil. Paraquat that reaches the soil is rendered unavailable to plant roots. It is used to k i l l emerged weeds anytime before planting a crop or before the crop emerges. Consequently, paraquat is widely used in agriculture for preplant and preemergence weed control and it has an im­ portant use in minimum tillage farming systems. It is also used for weed control between trees and as a directed spray in row crops. Due to its desiccating properties, it finds wide application as a harvest aid. The properties of paraquat have been reviewed by Calderbank (1) and Akhavein and Linscott (2). This paper summarizes studies on the biological unavailability of 'bound' paraquat r e s i ­ dues in soil.

301

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

302 2.

N A T U R E AND

A M O U N T S OF P A R A Q U A T SOIL RESIDUES

P a r a q u a t is n o r m a l l y a p p l i e d as a s p r a y at r a t e s of O.1 to 2 k g / h a . * S o m e of the p a r a q u a t r e a c h e s the soil d i r e c t l y and is a d s o r b e d by c l a y m i n e r a l s o r o r g a n i c m a t t e r . The r e m a i n d e r is i n t e r c e p t e d by the t a r g e t w e e d s . P a r a q u a t a d s o r b e d on p l a n t s u r f a c e s is s u b j e c t to p h o t o c h e m i c a l d e c o m p o s i t i o n by s u n l i g h t . The m a i n p h o t o c h e m i c a l d e g r a d a t i o n p r o d u c t s a r e 4 - c a r b o x y - l m e t h y l p y r i d i n i u m c h l o r i d e and m e t h y l a m i n e h y d r o c h l o r i d e (3). The f o r m e r has a l o w t o x i c i t y and is r a p i d l y d e g r a d e d in soil and c u l t u r e s o l u t i o n s (1, 4, 5). M e t h y l a m i n e o c c u r s n a t u r a l l y and is r e a d i l y d e g r a d e d (5). Paraquat adsorbed o glas slide thin-layer f soil is photoc h e m i c a l l y d e g r a d e H o w e v e r , p h o t o c h e m i c a l d e g r a d a t i o n of s i g n i f i c a n t a m o u n t s on soil s u r f a c e s in the f i e l d has not b e e n c l e a r l y d e m o n s t r a t e d . The a m o u n t of p a r a q u a t e v e n t u a l l y r e a c h i n g the soil o b v i o u s l y depends on f a c t o r s s u c h as d e n s i t y of w e e d c o v e r and s u n l i g h t i n t e n s i t y to c a u s e p a r a q u a t p h o t o d e g r a d a t i o n on plant s u r f a c e s . A n a l y s i s of s o i l s f r o m o v e r 50 s i t e s has s h o w n that the a m o u n t of p a r a q u a t w h i c h r e a c h e s the soil c a n r a n g e f r o m 10 to a l m o s t 100% of that a p p l i e d . If 5 0 % of a 1 k g / h a a p p l i c a t i o n r e a c h e d the soil, t h i s w o u l d r e s u l t in O. 5 jug p a r a q u a t / g soil if the r e s i d u e s w e r e u n i f o r m l y i n c o r p o r a t e d into the top 15 c m . In f i e l d e x p e r i m e n t s , l a b e l l e d p a r a q u a t was s p r a y e d onto a g r a s s s w a r d , onto b a r e soil, o r i n c o r p o r a t e d into the soil. A f t e r 1 y e a r t h e r e was no s i g n i f i c a n t d e g r a d a t i o n of p a r a q u a t in the soil; at l e a s t 9 0 % of the labelled residues w e r e paraquat ( B . C . B a l d w i n - u n p u b l i s h e d data). B e c a u s e p a r a q u a t is f i r m l y bound to soil (see below) it is i m m o b i l e u n l e s s the a d s o r b e n t itself moves. P a r a q u a t i n i t i a l l y a d s o r b e d onto p l a n t d e b r i s i n c o r p o r a t e d into soil o r onto soil o r g a n i c m a t t e r t r a n s f e r s to c l a y m i n e r a l s w h i c h a d s o r b p a r a q u a t m u c h m o r e s t r o n g l y (1). In d i l u t e s u s p e n s i o n s the t r a n s f e r f r o m soil o r g a n i c m a t t e r to c l a y is r a p i d (6). A l s o w h e n m o n t m o r i l l o n i t e c l a y was m i x e d w i t h a m o i s t peat soil c o n t a i n i n g a v a i l a b l e p a r a q u a t r e s i d u e s the p a r a q u a t was r a p i d l y d e a c t i v a t e d (7). T h i s shows that in m o i s t s o i l s , as w e l l as s l u r r i e s , p a r a q u a t r a p i d l y t r a n s f e r s f r o m w e a k a d s o r p t i o n s i t e s on the c l a y m i n e r a l s . T h i s is not s u r p r i s i n g s i n c e m u c h of the soil o r g a n i c m a t t e r is c l o s e l y a s s o c i a t e d w i t h the c l a y s u r * Note:

T h r o u g h o u t t h i s p a p e r r a t e s and c o n c e n t r a t i o n s r e f e r to p a r a q u a t c a t i o n .

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22.

RILEY

ET AL.

Bound Paraquat Residues

303

f a c e s . O n peat s o i l s c o n t a i n i n g o n l y s m a l l a m o u n t s of c l a y s e v e r a l w e e k s m a y be r e q u i r e d f o r the t r a n s f e r of p a r a q u a t f r o m o r g a n i c m a t t e r to the c l a y (8). The a d s o r p t i o n of p a r a q u a t o n s o i l s , c l a y s and o r g a n i c m a t t e r has b e e n e x t e n s i v e l y i n v e s t i g a t e d (j6, ], 9. to 27). C a l d e r b a n k (1) r e v i e w e d the n a t u r e of p a r a q u a t a d s o r p t i o n in soil. H a y e s et a l (26) and K h a n (27) r e v i e w e d the m e c h a n i s m of p a r a q u a t a d s o r p t i o n on c l a y s and o r g a n i c m a t t e r , r e s p e c t i v e l y . The q u a n t i t y of p a r a q u a t a d s o r b e d b y s o i l s is a l w a y s l e s s t h a n the b a s e exchange c a p a c i t y f o r i n o r g a n i c c a t i o n s , s u c h as a m m o n i u m i o n s . F u r t h e r m o r e , a l t h o u g h s o m e p a r a q u a t c a n be d i s p l a c e d by h i g h c o n c e n t r a t i o n s of a m m o n i u m ions w h e n the a d s o r p t i o n s i t e s of the soil a r e s a t u r a t e d w i t h p a r a q u a t d i s p l a c e m e n t is complet and a p o r t i o n of the p a r a q u a quantity of a d s o r b e d p a r a q u a t d e c r e a s e s , d i s p l a c e m e n t b e c o m e s p r o g r e s s i v e l y m o r e d i f f i c u l t and m o r e c o n c e n t r a t e d s a l t s o l u t i o n s a r e r e q u i r e d . M a l q u o r i and R a d a e l l i (17) c o m p a r e d the r e l a t i v e e f f e c t i v e n e s s of N H , K + , C a , Mg^+ and Na+ f o r r e l e a s i n g p a r a q u a t p r e v i o u s l y a d s o r b e d on f i v e d i f f e r e n t c l a y m i n e r a l s at varying concentrations. K and N H w e r e , in g e n e r a l , m o r e e f f e c t i v e t h a n the o t h e r c a t i o n s s t u d i e d , but no p a r a q u a t was r e l e a s e d w h e n i t s c o n c e n t r a t i o n on the c l a y was b e l o w a c e r t a i n l i m i t , w h i c h v a r i e d c o n s i d e r a b l y w i t h the type of m i n e r a l . The o n l y e f f e c t i v e m e a n s of d i s p l a c i n g p a r a q u a t f r o m soil w h e n it is p r e s e n t in r e l a t i v e l y l o w c o n c e n t r a t i o n s , e v e n f r o m v e r y sandy s o i l s , is to r e f l u x the s a m p l e w i t h s t r o n g a c i d s , e.g. 18 N, s u l f u r i c a c i d . B o i l i n g w i t h s t r o n g s u l f u r i c a c i d r e p r e s e n t s m o r e t h a n e l u t i o n . T h e s t r u c t u r e of the c l a y is p a r t i a l l y d e s t r o y e d and the b i n d i n g s i t e s a r e thus e l i m i n a t e d . E v e n v e r y s m a l l a m o u n t s , b e l o w 1.0 /ug/g p a r a q u a t , c a n be q u a n t i t a t i v e l y r e c o v e r e d f r o m s o i l s w h e n t r e a t e d in t h i s way. O n a m o l e c u l a r s c a l e , the p h e n o m e n o n of f i r m a d s o r p t i o n is a s s o c i a t e d w i t h the shape and c h a r g e d i s t r i b u t i o n of the p a r a q u a t i o n . The two p y r i d i n e r i n g s of p a r a q u a t c a n r o t a t e about the int e r r i n g bond and r e a d i l y a s s u m e a c o p l a n a r s t r u c t u r e - a p r e r e q u i s i t e of h e r b i c i d a l a c t i v i t y . T h i s f l a t c o n f i g u r a t i o n undoubte d l y f a c i l i t a t e s t h e i r i n t e r a c t i o n w i t h the c l a y m i n e r a l s u r f a c e s . P a r a q u a t is h i g h l y p o l a r i z a b l e and i t s n o r m a l c h a r g e d i s t r i b u t i o n is d i s t o r t e d in the v i c i n i t y of the n e g a t i v e l y c h a r g e d c l a y s u r f a c e s thus c h a r g e t r a n s f e r c o m p l e x e s a r e f o r m e d (12), r e i n f o r c i n g the n o r m a l c o u l o m b i c a t t r a c t i o n f o r c e s . P a r a q u a t is r e a d i l y d i s p l a c e d q u a n t i t a t i v e l y f r o m c a t i o n exchange r e s i n s (28) as d i s t i n c t f r o m c l a y m i n e r a l s w h i c h f u r t h e r s u g g e s t s that o t h e r a d s o r p t i o n f o r c e s , in a d d i t i o n to c o u l o m b i c f o r c e s , a r e i n v o l v e d in the p a r a +

2 +

4

+

+

4

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

304

quat-clay system. P u b l i s h e d data and a l a r g e n u m b e r of u n p u b l i s h e d e x p e r i m e n t s c o n d u c t e d by I C I P l a n t P r o t e c t i o n D i v i s i o n in the U n i t e d K i n g d o m and the C h e v r o n C h e m i c a l C o m p a n y in the U.S.A. have s h o w n that the p a r a q u a t soil r e s i d u e s r e s u l t i n g f r o m n o r m a l a p p l i c a t i o n s of p a r a q u a t a r e f i r m l y bound. C l a y s c a n f i r m l y b i n d up to about 5 0 , 0 0 0 ^ i g p a r a q u a t / g c l a y (50 meq/100 g). The a m o u n t and s t r e n g t h of p a r a q u a t a d s o r p t i o n by soil depends on the a m o u n t and type of c l a y m i n e r a l s p r e s e n t . H o w e v e r , a l m o s t all a g r i c u l t u r a l s o i l s c o n t a i n s u f f i c i e n t c l a y to f i r m l y b i n d 50 to 5000 jug p a r a q u a t / g soil. The o n l y e x c e p t i o n s a r e v e r y sandy and peat s o i l s c o n t a i n i n g l e s s t h a n 5 % c l a y ; h o w e v e r , e v e n m a n y of t h e s e s o i l s w i l l f i r m l y b i n d t l e a s t 50/ug/g Peat adsorb larg a m o u n t s of p a r a q u a t bu c l a y s and m o s t of the p a r a q u a t is d e s o r b e d w i t h 5 M N H 4 C I (24). 3.

U N A V A I L A B I L I T Y OF B O U N D P A R A Q U A T S O I L R E S I D U E S TO P L A N T S

P a r a q u a t is an e x t r e m e l y a c t i v e h e r b i c i d e w h e n a p p l i e d to p l a n t r o o t s ( g r o w n in n u t r i e n t s o l u t i o n ) as w e l l as t h e i r l e a v e s . T h i s is i l l u s t r a t e d in F i g u r e 1. P u r e s o l u t i o n s of p a r a q u a t d i c h l o r i d e , w i t h and without H o a g l a n d and S n y d e r n u t r i e n t s o l u t i o n s (29), w e r e b i o a s s a y e d w i t h p r e g e r m i n a t e d w h e a t s e e d l i n g s ( T r i t i c u m a e s t i v i u m c u l t i v a r K o l i b r i ) . The r e s u l t s g i v e n in F i g u r e 1 a r e the m e a n of r e s u l t s f r o m t h r e e b i o a s s a y e x p e r i m e n t s c o n d u c t e d at d i f f e r e n t t i m e s ; at e a c h t i m e e a c h t r e a t m e n t was r e p l i c a t e d t h r e e t i m e s . The s e e d l i n g s f a i l e d to g r o w in s o l u t i o n s c o n t a i n i n g m o r e t h a n 1 jag p a r a q u a t / m l . In s o l u t i o n s b e l o w 1 jug/ml i n c r e a s i n g the p a r a q u a t c o n c e n t r a t i o n gave c o r r e s ponding r e d u c t i o n in the e l o n g a t i o n of both r o o t s and shoots. The e f f e c t was e s p e c i a l l y m a r k e d on the r o o t s , the a c t i v i t y - c o n c e n t r a t i o n c u r v e h a v i n g a m u c h s h a r p e r c u t - o f f at a s l i g h t l y l o w e r c o n c e n t r a t i o n t h a n w i t h the shoots. The s m a l l e r e f f e c t on shoot e l o n g a t i o n was p r o b a b l y due to p o o r t r a n s l o c a t i o n of p a r a q u a t ( B . C . B a l d w i n , u n p u b l i s h e d data). T h e r e was no c h l o r o s i s o r n e c r o s i s of the l e a v e s of t r e a t e d p l a n t s e x c e p t that w h e n p l a n t s w e r e e x t r e m e l y stunted (above O.1jug/ml) they t h e n b e c a m e c h l o r o t i c and d i e d . The e f f e c t as m e a s u r e d by r o o t and shoot e l o n g a t i o n was g r e a t e r t h a n the e f f e c t as m e a s u r e d by the d r y w e i g h t of the s e e d l i n g s . S u c c e s s i v e b i o a s s a y s on the s a m e p a r a q u a t s o l u t i o n s showed that the p a r a q u a t c o n c e n t r a t i o n in the s o l u t i o n s dec r e a s e d 2-3 f o l d d u r i n g e a c h b i o a s s a y . The l o w e s t c o n c e n t r a t i o n of p a r a q u a t w h i c h had a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t (p = O. 05)

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22.

RILEY

ET AL.

Bound Paraquat Residues

305

on r o o t length r a n g e d f r o m O. 005 t oO.02 jug/ml a n d f o r shoots it r a n g e d f r o m O. 01 toO.1u g / m l . T h e c o n c e n t r a t i o n o f p a r a q u a t w h i c h r e s u l t e d in a 5 0 % r e d u c t i o n in the lengths o f r o o t s r a n g e d f r o m O. 005 t oO.04 μg/ml and f o r shoots r a n g e d f r o m O. 03 t o O.10 μg/ml. T h e v a l u e s depended o n g r o w t h c o n d i t i o n s a n d w h e t h e r o r not n u t r i e n t s w e r e added d u r i n g the b i o a s s a y . P l a n t r o o t s a r e in i n t i m a t e c o n t a c t w i t h soil p a r t i c l e s a s w e l l as w i t h the soil s o l u t i o n . T h e r e is e v i d e n c e f o r a b r i d g e o f " m u c i g e l " b e t w e e n the r o o t s a n d soil p a r t i c l e s (30), w h i c h m i g h t p r o v i d e a r o u t e b y w h i c h c h e m i c a l s a d s o r b e d o n soil s u r f a c e s c a n m o v e t o r o o t s w i t h o u t e n t e r i n g the soil s o l u t i o n . It has b e e n s u g g e s t e d that n u t r i e n t s s u c h a s p o t a s s i u m c a n be a d s o r b e d b y r o o t s d i r e c t l y f r o m a d s o r p t i o n s i t e s o n soil p a r t i c l e s (30 31) a n d itisc o n c e i v a b l e that a d s o r b e d i r e c t c o n t a c t . H o w e v e r , it h a s b e e n s h o w n that s o m e h e r b i c i d e s , s u c h as a t r a z i n e , a r e s u p p l i e d to r o o t s m a i n l y v i a the soil s o l u ­ t i o n (32). D u r i n g the p a s t 15 y e a r s p a r a q u a t has b e e n a p p l i e d to m i l l i o n s of h e c t a r e s throughout the w o r l d . T h e r e have b e e n a n i n s i g n i f i ­ cant n u m b e r of r e p o r t s of p a r a q u a t h a v i n g a n y r e s i d u a l h e r b i c i d a l a c t i v i t y in the soil. T h e r e f o r e ,itm u s t be c o n c l u d e d that the p a r a q u a t soil r e s i d u e s a r e u n a v a i l a b l e t o p l a n t s . It h a s a l s o b e e n s h o w n in g l a s s h o u s e e x p e r i m e n t s that 'bound' p a r a q u a tisu n ­ a v a i l a b l e t o p l a n t s (25). In m o s t s o i l s , p a r a q u a t has t o be a p ­ p l i e d at s e v e r a l h u n d r e d t o s e v e r a l t h o u s a n d t i m e s the n o r m a l r a t e of a p p l i c a t i o n b e f o r e the r e s i d u e s have any e f f e c t on p l a n t s . A f t e r s u c h e x t r e m e l y h i g h r a t e s of a p p l i c a t i o n p a r a q u a t c a n be d e t e c t e d in the e q u i l i b r i u m soil s o l u t i o n (16). A l s o s o m e of the a d s o r b e d p a r a q u a t r e s i d u e s c a n be d e s o r b e d w i t h h i g h c o n c e n t r a ­ t i o n s of i n o r g a n i c s a l t s o l u t i o n s , s u c h as 5 M a m m o n i u m c h l o r i d e (24). We have u s e d two d i f f e r e n t a p p r o a c h e s to c h a r a c t e r i z e the a v a i l a b i l i t y of p a r a q u a t r e s i d u e s t o p l a n t s :

A.

A

D e t e r m i n a t i o n of the c a p a c i t y of s o i l s to r e d u c e the c o n ­ c e n t r a t i o n of p a r a q u a t in the e q u i l i b r i u m s o l u t i o n b e l o w phytotoxic levels.

Β

D e t e r m i n a t i o n of the c a p a c i t y of s o i l s t o b i n d p a r a q u a t s o t i g h t l y thatitisnot d e s o r b e d w i t h s a t u r a t e d (5M) a m m o ­ nium chloride.

C a p a c i t y of S o i l s t o R e d u c e P a r a q u a t in the E q u i l i b r i u m Solution Below Phytotoxic Levels T h e s e e x p e r i m e n t s showed that any r e s i d u a l a c t i v i t y o f

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

306

BOUND AND CONJUGATED PESTICIDE RESIDUES

p a r a q u a t , r e s u l t i n g f r o m e x t r e m e l y h i g h r a t e s of a p p l i c a t i o n to soil, a r e due to uptake of p a r a q u a t f r o m the soil s o l u t i o n ; p a r a q u a t a d s o r b e d on soil p a r t i c l e s is not t a k e n u p by p l a n t s . D e t a i l s of 4 E n g l i s h s o i l s s t u d i e d a r e g i v e n in T a b l e I. P a r a q u a t a d s o r p t i o n i s o t h e r m s w e r e d e t e r m i n e d by s h a k i n g 10 g of soil w i t h 200 m l p a r a q u a t d i c h l o r i d e s o l u t i o n s in O. 01M C a C l 2 . T h e O.01M C a C l 2 s o l u t i o n was u s e d to s i m u l a t e the s a l t c o n c e n t r a t i o n o f t e n found in the soil s o l u t i o n (33). A f t e r s h a k i n g f o r 16 h o u r s , the s u s p e n s i o n s w e r e c e n t r i f u g e d and the s u p e r n a tant s o l u t i o n s a n a l y z e d c o l o r i m e t r i c a l l y a f t e r r e d u c t i o n w i t h a l k a l i n e d i t h i o n i t e s o l u t i o n (1). C o n c e n t r a t i o n s in the r a n g e O.01 to O.1 /ug/ml w e r e d e t e r m i n e d a f t e th t had b e e t r a t e d 10 f o l d u s i n g Z e r o l i b r i u m soil s o l u t i o n s w e r e a l s o b i o a s s a y e d w i t h wheat s e e d l i n g s as d e s c r i b e d above, w i t h o u t any a d d i t i o n of n u t r i e n t s , o t h e r t h a n those e x t r a c t e d f r o m the soil.

Glasshouse Studies Soil b i o a s s a y s w e r e a l s o p e r f o r m e d on the 4 s o i l s g i v e n in T a b l e I. P a r a q u a t d i c h l o r i d e s o l u t i o n s (200 m l ) w e r e t h o r o u g h l y m i x e d w i t h 2 k g s a m p l e s of e a c h of the 4 s o i l s . E a c h t r e a t m e n t was r e p l i c a t e d 3 t i m e s . The s o i l s w e r e p l a c e d in p l a s t i c pots and a r r a n g e d in a r a n d o m i z e d b l o c k d e s i g n in the g l a s s h o u s e . A f t e r l e a c h i n g out e x c e s s c h l o r i d e s a l t s (there was n e g l i g i b l e l e a c h i n g of p a r a q u a t ) and the a d d i t i o n of n i t r o g e n , p h o s p h o r o u s and p o t a s s i u m n u t r i e n t s , the pots w e r e b i o a s s a y e d w i t h a s e r i e s of c r o p s . B e t w e e n e a c h c r o p the soil was a i r d r i e d and m i x e d . E a c h c r o p was g r o w n f o r about 6 w e e k s b e f o r e h a r v e s t . D r y w e i g h t s of shoots and in s o m e c a s e s l e n g t h and d r y w e i g h t of r o o t s (after w a s h i n g ) w e r e m e a s u r e d . The c r o p s g r o w n w e r e w h e a t ( T r i t i c u m a e s t i v u m , c u l t i v a r K o l i b r i ) , r a d i s h (Raphanus s a t i v a 8 . c u l t i v a r S u t t o n s c a r l e t G l o b e ) , peas ( P i s u m s a t i v u m c u l t i v a r M e t e o r ) and l e t t u c e ( L a c t u c a s a t i v a , c u l t i v a r Suttons Unrivalled). A f t e r t h e s e b i o a s s a y s had b e e n c o m p l e t e d s a m p l e s of e a c h t r e a t e d soil w e r e b i o a s s a y e d w i t h the a q u a t i c w e e d L e m n a p o l y r h i z a w h i c h has b e e n r e p o r t e d to be v e r y s e n s i t i v e to p a r a quat (34J, Soil s a m p l e s (10 g) w e r e m i x e d w i t h 100 m l d i s t i l l e d w a t e r p l u s 1 m l H o a g l a n d and S n y d e r n u t r i e n t s o l u t i o n (29). S a m p l e s of L e m n a w e r e f l o a t e d on the s u r f a c e of the m i x t u r e . The i n c r e a s e in the d r y o r f r e s h w e i g h t of the L e m n a was d e t e r m i n e d a f t e r 7 d a y s g r o w t h . S a m p l e s o f the t r e a t e d S a n d y H i l l s

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

-

65.0

-

-

42.3

7.3

Fen peat (Muck)

Methwold

27.8

31 48

21

4.8

7.8

Calcareous

Tarlton

10.0 65

4.5 18

m

Sand

17

loam

Sandy Loam

Broadricks

1.9

6.7

Sandy Loam 5.8

m

79

Silt

S2à-

Clay

Cation Exchange Capacity (meq/100g a t D H 7.0)

10

m

Organic M a t t e r

11

pH 1.7

Type

Sandy H i l l s

Name

AND DEACTIVATION STUDIES

PROPERTIES OF SOILS USED IN PARAQUAT ADSORPTION

TABLE I

308

BOUND AND CONJUGATED PESTICIDE RESIDUES

soil w e r e a l s o b i o a s s a y e d w i t h L e m n a in the p r e s e n c e of O. 05 and O.1 M C a C l 2 ; the L e m n a d i d not g r o w in the 0. LM C a C ^ . Pure s o l u t i o n s of p a r a q u a t d i c h l o r i d e p l u s n u t r i e n t s o l u t i o n w e r e s i m i l a r l y bioassayed. S a m p l e s (400 g) of the t r e a t e d Sandy H i l l s and M e t h w o l d s o i l s w e r e b i o a s s a y e d w i t h S23 P e r e n n i a l R y e g r a s s ( L o l i u m perenne) in the p r e s e n c e of 0, O.5 and O.25 M C a C l 2 . The C a C l 2 c o n c e n t r a t i o n in the soil s o l u t i o n was m o n i t o r e d and kept constant by p e r i o d i c a d d i t i o n s of C a C l 2 s o l u t i o n , to r e p l a c e C a C l 2 l e a c h e d f r o m the pots d u r i n g w a t e r i n g . F i g u r e 2 shows the a m o u n t of p a r a q u a t a d s o r b e d by the 4 s o i l s w h e n the c o n c e n t r a t i o n of p a r a q u a t in the e q u i l i b r i u m soil s o l u t i o n is in the r a n g e 1-20 u g / m l F i g u r e 3 shows the a m o u n t of p a r a q u a t a d s o r b e d w i t s o l u t i o n . C l e a r l y the s o i l s have v e r y d i f f e r e n t a d s o r p t i o n i s o t h e r m s . F o r e x a m p l e , the peat (Methwold) a d s o r b s l a r g e r a m o u n t s of p a r a q u a t t h a n the B r o a d r i c k s and T a r l t o n s o i l s w h e n the c o n c e n t r a t i o n of p a r a q u a t in the e q u i l i b r i u m s o l u t i o n is 5/ug/ m l , but the peat (Methwold) a d s o r b s l o w e r a m o u n t s of p a r a q u a t t h a n the B r o a d r i c k s and T a r l t o n s o i l s w h e n the e q u i l i b r i u m s o l u t i o n c o n t a i n s l e s s t h a n O.1 u g / m l . R e s u l t s of the b i o a s s a y of the e q u i l i b r i u m s o l u t i o n s a r e g i v e n in F i g u r e 4. O n l y r e s u l t s f o r the r o o t g r o w t h a r e g i v e n b e c a u s e they w e r e a f f e c t e d m o r e t h a n shoots; as they w e r e in p u r e p a r a quat d i c h l o r i d e s o l u t i o n s . T y p i c a l r e s u l t s f o r the soil b i o a s s a y s a r e g i v e n in F i g u r e 5 ( f i r s t wheat c r o p on all s o i l s ) and F i g u r e 6 (wheat, pea, and l e t t u c e c r o p s on B r o a d r i c k s soil); all the soil b i o a s s a y r e s u l t s a r e s u m m a r i z e d in T a b l e II. The r e s i d u a l a c t i v i t y of the p a r a q u a t d e c r e a s e d s l i g h t l y d u r i n g the first few b i o a s s a y s . T h i s was p r o b a b l y due to a s l o w e q u i l i b r a t i o n of the p a r a q u a t w i t h the a d s o r p t i o n s i t e s . A n a l y s i s of the s o i l s showed it was not due to d e g r a d a t i o n of the p a r a q u a t . The r e s i d u a l a c t i v i t y of p a r a q u a t was v e r y s i m i l a r on all the plant s p e c i e s tested. The d i f f e r e n c e between the s e n s i t i v i t i e s of r o o t s and shoots to p a r a q u a t was not as g r e a t in the soil b i o a s s a y as in the s o l u t i o n b i o a s s a y . T h i s was p r o b a b l y b e c a u s e the e m e r g i n g shoots as w e l l as r o o t s w e r e in contact w i t h the r e s i d u a l p a r a q u a t in the soil b i o a s s a y , but not in the s o l u t i o n b i o a s s a y . A l s o , in s o l u t i o n c u l t u r e s s e v e r e l y stunted r o o t s c a n m a i n t a i n n o r m a l shoot g r o w t h , w h i l e in the m o r e a u s t e r e soil e n v i r o n m e n t the stunted r o o t s m a y not be able to s u p p l y the shoots w i t h s u f f i c i e n t w a t e r and n u t r i e n t s .

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22.

RILEY E T A L .

Bound Paraquat Residues

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

310

BOUND AND CONJUGATED PESTICIDE RESIDUES

0

0O5 0-1 015 PARAQUAT IN SOLUTION ( j i ^ m l )

Figure 3.

Ο

200

400

0*2

Paraquatsoiladsorption isotherms

BOO

Θ

SANDY HILLS

+

BROADRICKS

800

1000

120O

PARAQUAT CONCENTRATIONS IN EXTRACTED SON. */Q>

Figure 4. Wheat bioassay of equilibrium solutions from soils treated with paraquat

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RILEY

ET

Bound Paraquat Residues

AL.

311 Θ SANDY HILLS

Ο

400

800

BROADRICKS

À

TARLTON

120O

PARAQUAT IN SOIL

Figure 5.

+

1600

2000

iy^g)

Wheat bioassay of soih treated with paraquat (first crop)

Δ WHEAT 3rd crop

Ο PEA •

Ο

Figure 6.

200

400 600

8 0 0 100O 1400 PARAQUAT IN SOtLQi^g)

LETTUCE

1800

Bioassay of Broadrickssoiltreated with paraquat

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

312

BOUND

A N D C O N J U G A T E D PESTICDDE

RESIDUES

T A B L E II CAPACITIES OF SOILS TO DEACTIVATE PARAQUAT

Date of Bioassay

Soil

Bioassay

Sandy

Soil treated Wheat 1st crop

March 1971

Wheat 2nd crop

June 1971

Wheat 3rd crop

July 1971

Wheat 4th crop Wheat 5th crop

Sept.1971 Jan. 1972

Radish 1st crop Radish 2nd crop

Mar.1972 May 1972

Lemna Lemna (+O.05MCaClo) Ryegrass

July 1972 July 1972 June 1973

Ryegrass (-K).05MCaCl ) June 1973 2

Ryegrass (+O.25MCaCl2)June 1973

Methwold

Soil treated Wheat 1st crop Wheat 2nd crop Radish

Dec. 1971 Feb. 1972 A p r i l 1972 June 1972

Lemna Pea

Aug.1972 Oct. 1972

Wheat 3rd crop

Feb. 1973

Lettuce

June 1973

Soil treated Wheat 1st crop

May 1972 June 1972

Observed effect of paraquat

Lowest concentration of paraquat which s i g n i ficantly (P=O.05) affected crop ()ug paraquat/g soili

Increased Decreased Increased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased

300 dry wt.shoots > 450 dry wt.shoots 300 dry wt.roots > 450 dry wt.roots 300 length roots 500 dry wt.shoots 450 length roots 500 dry wt.shoots 550 dry wt.shoots .550 dry wt.roots 500 length roots > 700 dry wt.whole plant 600 fresh wt.whole plant 600 dry wt.whole plant 550 dry wt. 500 dry wt. 600 dry wt.shoots 550 length roots 600 dry wt.shoots 550 length roots 600 dry wt.shoobs length roots 450

Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased

dry wt.shoots dry wt.shoots fresh wt.whole plant dry wt.whole plant fresh wt. dry wt.shoots length roots dry wt.shoots length roots dry wt.shoots length roots

Decreased dry wt.shoots Decreased length roots

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1400 1200 1600 1600 400 1800 1400 1400 1000 1400 1200 75 50

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T A B L E II. CAPACITIES OF SOILS TO DEACTIVATE PARAQUAT (CONT.)

Soil

Bioassay

Meth- Wheat 2nd crop wold (Cont.) Lemna Soil treated Wheat 3rd crop

Date o f Bioassay

Observed e f f e c t of paraquat

Aug

Decreased dry wt.shoots

1972

Sept.197 May 1972 Sept. 1972

Wheat 4th crop

Oct. 1972

Wheat 5th crop

Jan. 1973

Wheat 6th crop

Mar. 1973

Ryegrass

June 1973

Ryegrass(+O.05MCaCl )June 1973 2

Ryegrass(+O.25MCaCl2)June 1973 Tarlton

Soil treated Wheat 1st crop Wheat 2nd crop Radish

Aug. 1971 Mar. 1972 A p r i l 1972 Aug. 1972

Wheat 3rd crop

Oct. 1972

Wheat 4th crop

Dec. 1972

Wheat 5th crop

Jan. 1973

Lemna

Sept.1973

Lowest concentration of paraquat which s i g n i ficantly (P-O.05) affected crop (pg paraquat/g soil)

Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased

dry wt .shoots length roots dry wt .shoots length root 8 dry wt .shoots length roots dry wt shoots length roots dry wt shoots length roots dry wt shoots length roots dry wt, shoots length roots

Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased

dry wt.shoots dry wt.shoots fresh wt.whole plant dry wt.whole plant > dry wt.shoots length roots dry wt.shoots length roots dry wt.shoots length roots fresh wt. >

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75

75 50 200 50 100 75 200 75 200 200 200 100 100 25 300 450 1350 1500 1050 450 1350 450 600 600 1500

314

BOUND AND CONJUGATED PESTICIDE RESIDUES

C o m p a r i s o n of F i g u r e s 4 a n d 5 shows that the e f f e c t s of p a r a ­ quat o n g r o w t h w e r e s i m i l a r in s e p a r a t e d e q u i l i b r i u m soil s o l u ­ t i o n s a n d in the t o t a l s y s t e m of soil p l u s e q u i l i b r i u m s o l u t i o n . T h e r e f o r e , the p a r a q u a t bound to the soil w a s not a v a i l a b l e to the p l a n t s a n d the p h y t o t o x i c i t y o b s e r v e d w a s due t o f r e e p a r a q u a t in the soil s o l u t i o n . T h e c o n c e n t r a t i o n of bound p a r a q u a t in the soil w a s a p p r o x i m a t e l y 10,000 t i m e s g r e a t e r than the p a r a q u a t c o n ­ c e n t r a t i o n in the soil s o l u t i o n w h e n p h y t o t o x i c i t y w a s first ob­ served. U n d e r o u r c o n d i t i o n s , the L e m n a b i o a s s a y h a d a s i m i l a r s e n s i t i v i t y t o that of the w h e a t b i o a s s a y ; O.02 μg/ml p a r a q u a t s e v e r e l y r e d u c e d g r o w t h . In the L e m n a b i o a s s a y of the s o i l s , r o o t s w e r e s u s p e n d e d in th e q u i l i b r i u s o l u t i o abov th soil but c a m e into c o n t a c t w i t g r o w t h p e r i o d . R e s u l t s f o r the L e m n a b i o a s s a y w e r e s i m i l a r to t h o s e of the wheat b i o a s s a y s of b o t h s o i l s a n d e q u i l i b r i u m s o l u ­ t i o n s ( T a b l e II). W h e n the c o n c e n t r a t i o n of p a r a q u a t in soil is v e r y h i g h it is p o s s i b l e to d i s p l a c e s o m e of the a d s o r b e d p a r a q u a t w i t h h i g h c o n c e n t r a t i o n s of i n o r g a n i c c a t i o n s (1). N e v e r t h e l e s s , the e f f e c t of soil r e s i d u e s o n the g r o w t h of L e m n a w a s not i n c r e a s e d in the p r e s e n c e of O.05 M C a C l 2 . L e m n a w o u l d not g r o w in the p r e s ­ ence of h i g h e r c o n c e n t r a t i o n s of C a C ^ . O.05 a n d O.25 M C a C l 2 o n l y s l i g h t l y r e d u c e d the c a p a c i t i e s of Sandy H i l l s and M e t h w o l d (peat) s o i l s t o d e a c t i v a t e p a r a q u a t w i t h the e x c e p t i o n of O. 25 M C a C l 2 o n the M e t h w o l d peat; O.25M C a C l 2 i t s e l f r e d u c e d the g r o w t h of r y e g r a s s b y about h a l f . T h e O.25M c o n c e n t r a t i o n o f C a C l 2 is h i g h e r than the s a l t c o n c e n t r a t i o n found in m o s t soil s o l u t i o n s (33) a n d s u r r o u n d i n g f e r t i l i z e r bands (35). T h e r e f o r e , t h e r e is no d a n g e r of i n o r g a n i c c a t i o n s in s o i l s d i s p l a c i n g bound paraquat residues. T h e d e t e r m i n a t i o n of the c a p a c i t y of s o i l s to d e a c t i v a t e p a r a ­ quat by b i o a s s a y i n g s o i l s t r e a t e d w i t h h i g h r a t e s of p a r a q u a t in l o w v o l u m e s of t r e a t m e n t s o l u t i o n is not p r a c t i c a l b e c a u s e it t a k e s at l e a s t s e v e r a l m o n t h s f o r the p a r a q u a t t o e q u i l i b r a t e in the soil. H o w e v e r , in d i l u t e s l u r r i e s e q u i l i b r a t i o n is c o m p l e t e a f t e r o v e r n i g h t s h a k i n g a n d the e q u i l i b r i u m s o l u t i o n c a n t h e n be a n a l y z e d p h o t o m e t r i c a l l y o r b i o a s s a y e d a s d e s c r i b e d above. The p a r a q u a t 'Strong A d s o r p t i o n ' c a p a c i t y of a soil d e t e r m i n e d by b i o a s s a y i n g e q u i l i b r i u m s o l u t i o n s w i t h wheat is d e f i n e d a s the c o n c e n t r a t i o n of soil-adsorbed p a r a q u a t w h e n the c o n c e n t r a t i o n of p a r a q u a t in the e q u i l i b r i u m s o l u t i o n is s u f f i c i e n t to r e d u c e the l e n g t h of 14-day o l d w h e a t r o o t s by 5 0 % ; t h i s s o l u t i o n c o n c e n t r a ­ t i o n is about O.Oljug p a r a q u a t / m l ( T a b l e III). The c o n c e n t r a t i o n

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Bound Paraquat Residues

TABLE

III

'STRONG ADSORPTION' CAPACITIES OF SOILS

Paraquat 'Strong Adsorption C a p a c i t y (1) frig p a r a q u a t / g soil)

Soil Sandy H i l l s Tarlton Broadricks Methwold Pure paraquat dichloride solution

concentra-

y (jug paraquat /ml) O.05 O.01 O.02 O.004

400 300 800 40

O.005

1.

Mean o f 2 determinations

2.

Assuming a bulk d e n s i t y o f 1.2 g / c c

3.

Assuming a bulk d e n s i t y o f O.5 g / c c

kg paraquat/ha r e q u i r e d to

15 cm 720 540 1440 30

y soil (2) (2) (2) (3)

- O.04

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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B O U N D A N D C O N J U G A T E D PESTICIDE

RESIDUES

of p a r a q u a t w h i c h r e d u c e d r o o t l e n g t h b y 5 0 % is o n l y s l i g h t l y h i g h e r than that w h i c h first s i g n i f i c a n t l y a f f e c t s r o o t l e n g t h (see above), 5 0 % r e d u c t i o n of r o o t g r o w t h is u s e d t o m e a s u r e the S t r o n g A d s o r p t i o n c a p a c i t i e s of s o i l s b e c a u s e it c a n be e a s i l y d e t e r m i n e d by v i s u a l e x a m i n a t i o n of the data, w h e r e a s the first e f f e c t c o n c e n t r a t i o n depends v e r y m u c h o n the v a r i a b i l i t y of the e x p e r i m e n t and the data have t o be s t a t i s t i c a l l y a n a l y z e d . B i o a s s a y of the e q u i l i b r i u m s o l u t i o n s and thus 'Strong A d ­ s o r p t i o n ' c a p a c i t y v a l u e s , g i v e s a good e s t i m a t e of the c a p a c i t y of s o i l s t o d e a c t i v a t e p a r a q u a t ( c o m p a r e T a b l e s II and III). If a n y t h i n g , t h e 'Strong A d s o r p t i o n ' c a p a c i t y v a l u e s tend t o u n d e r e s t i m a t e the c a p a c i t y of s o i l s t o d e a c t i v a t e paraquat. This s o l u t i o n b i o a s s a y technique has b e e n w i d e l y u s e d b y I C I P l a n t P r o t e c t i o n D i v i s i o n to d e t e r m i n vate paraquat. f

1

Field Trials The r e l a t i o n s h i p b e t w e e n 'Strong A d s o r p t i o n ' c a p a c i t y v a l u e s and the c a p a c i t y of s o i l s to d e a c t i v a t e p a r a q u a t in the f i e l d has b e e n s t u d i e d . P a r a q u a t w a s a p p l i e d at r a t e s of 1/2, 1 a n d 4 t i m e s the 'Strong A d s o r p t i o n ' c a p a c i t y found by the e q u i l i b r i u m soil s o l u t i o n b i o a s s a y t e c h n i q u e . T h e effect of p a r a q u a t soil residues on crop growth and crop residues were a c c u r a t e l y pre­ d i c t e d ; r e s i d u e s b e l o w the 'Strong A d s o r p t i o n ' c a p a c i t y b e i n g un­ available to plants. One e x p e r i m e n t w a s c o n d u c t e d at F r e n s h a m in E n g l a n d . T h e l o a m y sand soil c o n t a i n s 9% c l a y , 8 % s i l t and 8 3 % sand and 2 . 0 % o r g a n i c m a t t e r ; it h a s a p H of 6. 6 and c a t i o n exchange c a p a c i t y of 5 meq/100 g at p H 7.O. T h e 'Strong A d s o r p t i o n ' c a p a c i t y of the soil is 120 u g p a r a q u a t / g soil. P a r a q u a t a d s o r p t i o n i s o ­ t h e r m s , d e t e r m i n e d in d i l u t e s l u r r i e s as d e s c r i b e d above, a r e g i v e n in F i g u r e 7. The t r i a l w a s l a i d out in 3 b l o c k s . E a c h b l o c k w a s s p l i t into two h a l v e s a n d e a c h h a l f w a s s p l i t into 4 p l o t s , 40 m χ 5 m. F o u r p l o t s o f o n e - h a l f o f e a c h b l o c k w e r e t r e a t e d w i t h 0, 15, 33 and 120 k g / h a of p a r a q u a t ; t h i s w a s then l i g h t l y i n c o r p o r a t e d into the t o p 2 c m soil u s i n g a hand r a k e . T h e 4 p l o t s o n the o t h e r h a l f o f e a c h b l o c k w e r e t r e a t e d w i t h 0, 90, 198 a n d 720 k g / h a o f paraquat; t h i s w a s t h o r o u g h l y r o t o v a t e d into the top 15 c m . T h e p a r a q u a t w a s a p p l i e d in N o v e m b e r , 1971, a n d the p l o t s w e r e left u n d i s t u r b e d t h r o u g h the w i n t e r . In the s p r i n g , w e e d s w e r e c o n ­ t r o l l e d by a n o v e r a l l s p r a y o fO.5 k g / h a paraquat. In M a r c h , 1972, the t r i a l a r e a w a s s e e d e d w i t h b a r l e y ( H o r d e u m v u l g a r e -

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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PARAQUAT IN SOLUTION (HQ/ml)

Figure 7.

Paraquat adsorption isotherms on Frensham soil

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

317

BOUND AND CONJUGATED PESTICIDE RESIDUES

318

c u l t i v a r J u l i a ) by d i r e c t d r i l l i n g w i t h a t r i p l e d i s c d r i l l , i . e . , by z e r o t i l l a g e . In A u g u s t , w h e n the c r o p had r e a c h e d m a t u r i t y , s a m p l e s of g r a i n and s t r a w w e r e a n a l y z e d f o r p a r a q u a t r e s i d u e s . A s e c o n d c r o p of b a r l e y was s i m i l a r l y g r o w n in 1973. A l s o in 1973, l a r g e soil s a m p l e s w e r e t a k e n f r o m the top 15 c m of the deep i n c o r p o r a t e d t r e a t m e n t s . T h e s e soil s a m p l e s w e r e u s e d to g r o w c a r r o t s (Daucus c a r o t a - c u l t i v a r New M o d e l R e d C o r e ) o u t d o o r s in 20 c m d i a m e t e r , 20 c m deep pots. The c a r r o t s w e r e h a r v e s t e d 119 d a y s a f t e r s o w i n g , t h o r o u g h l y w a s h e d and a n a l y z e d for p a r a q u a t r e s i d u e s . In 1972, the 90 k g / h a i n c o r p o r a t e d and 15 and 33 k g / h a s u r ­ f a c e t r e a t m e n t s o n l y s l i g h t l y a f f e c t e d e a r l y g r o w t h of b a r l e y and the c r o p q u i c k l y and c o m p l e t e l d ( T a b l IV) Th 20 kg/ha surface treatmen had a s i g n i f i c a n t e f f e c t on b a r l e y g r o w t h and r e d u c e d the n u m b e r of p l a n t s . H o w e v e r , the r e m a i n i n g p l a n t s g r e w n o r m a l l y and a l t h o u g h g r a i n y i e l d s w e r e s l i g h t l y l e s s t h a n on the c o n t r o l p l o t s t h e r e was no s t a t i s t i c a l d i f f e r e n c e in the y i e l d at the 9 5 % c o n f i ­ dence l e v e l . T h e r e was an a l m o s t c o m p l e t e c r o p f a i l u r e on the 720 k g / h a i n c o r p o r a t e d t r e a t m e n t . H o w e v e r , a few p l a n t s s u r ­ v i v e d and they g r e w n o r m a l l y ; they w e r e h a r v e s t e d f o r r e s i d u e a n a l y s i s . The r e s i d u a l a c t i v i t y of the p a r a q u a t was l e s s in 1973 t h a n 1972. T h i s was due to a s l o w e q u i l i b r i u m of the l a r g e a m o u n t s of p a r a q u a t w i t h a d s o r p t i o n s i t e s ; t h e r e was no s i g n i f i ­ cant d e g r a d a t i o n of p a r a q u a t d u r i n g the e x p e r i m e n t . In 1973, the 15 and 33 k g / h a s u r f a c e t r e a t m e n t s and 90 and 198 k g / h a i n c o r p o ­ r a t e d t r e a t m e n t s had no s i g n i f i c a n t e f f e c t s on b a r l e y g r o w t h . The 120 k g / h a s u r f a c e t r e a t m e n t s l i g h t l y a f f e c t e d b a r l e y g r o w t h but d i d not s i g n i f i c a n t l y a f f e c t g r a i n y i e l d ( T a b l e IV). The 720 k g / ha i n c o r p o r a t e d t r e a t m e n t was a g a i n s e v e r e l y p h y t o t o x i c but m a n y m o r e p l a n t s s u r v i v e d and g r e w n o r m a l l y t h a n in 1972. R e s i d u e s in b a r l e y g r a i n and s t r a w w e r e n e g l i g i b l e , e x c e p t f o r the 720 k g / h a i n c o r p o r a t e d t r e a t m e n t (Table I V ) . T h i s c o u l d have b e e n due to c o n t a m i n a t i o n w i t h t r a c e s of soil c o n t a i n i n g l a r g e p a r a q u a t r e s i d u e s as w e l l as a d s o r p t i o n by the c r o p . R e s i ­ dues in the c a r r o t s w e r e v e r y low, i . e . , O.04 (ug/g o r l e s s (Table IV) A f u r t h e r t r i a l was c o n d u c t e d on B r o a d r i c k s f i e l d at J e a l o t t ' s H i l l R e s e a r c h S t a t i o n . D e t a i l s of the soil a r e g i v e n in T a b l e I. It has a 'Strong A d s o r p t i o n c a p a c i t y of 800 jug p a r a q u a t / g soil. Two 6 χ 6 m p l o t s w e r e t r e a t e d w i t h 5 a n n u a l a p p l i c a t i o n s of 112 k g / h a p a r a q u a t ; t h i s r e s u l t e d in r e s i d u e s of 1000 μg/g in the top 2. 5 c m soil. S23 p e r e n n i a l r y e g r a s s s o w n in the top 2. 5 c m of soil f a i l e d to g e r m i n a t e o r was s e v e r e l y stunted. H o w e v e r , a f t e r f

1

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. .

1973 Paraquat r e s i d u e s in c a r r o t r o o t s Qug/g f r e s h weight)