Bound and Conjugated Pesticide Residues

29 Summary of Conjugate Papers HANS GEISSBUHLER

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Agrochemicals Division, CIBA-GEIGY Ltd., Basle, Switzerland

As a reviewer I might content myself with providing an abstract and mentioning some highlights of each of the papers presented. A more d i f f i c u l t but (maybe) more rewarding task would be to look at the presentations from a different angle. I shall attempt to follow the latter approach. The optics I have chosen are those of an industrial scientist whose daily managerial responsibility consists of providing answers to often reasonable, but sometimes not so reasonable biochemical and analytical questions raised by regulatory authorities all over the world. Before coming to this conference and without knowing the contents of the presentations, I had decided that I should try to review the various conjugate papers according to the following question scheme: (1) What are the type- or general conjugation reactions recognized with pesticides or pesticide moieties? How can they best be classified and in which organisms do they occur? (2) What is the present stage of methodology for synthesis, purification/separation and identification of conjugates? (3) What is the significance of conjugation reactions with regard to the behavior of pesticides in the environment, specifica l l y in terms of: - terminal residues - elimination and/or transport mechanisms - bioavailability. Thus, I purposely neglect the various aspects of enzymatic mechanisms involved (for which theme I do not feel competent) and I also omit to evaluate the significance of conjugation reactions in the chain of events leading to the biocidal/bioregulating effects or to the selective behavior of pesticides in target organisms. After having heard and read the papers I have to admit that the authors, who have approached their topics in quite different ways, have not exactly f a c i l i t a t e d my task. If, in spite of this diversity in presentation, I adhere to my original question 368

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

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Summary of Conjugate Papers

369

scheme, I f u l l y r e a l i z e that my review w i l l be no more than an incomplete and sketchy mixture o f f a c t s , o p i n i o n s , ideas and questions. I. Type, o r General Conjugation Reactions Recognized w i t h Pesticides With the i n f o r m a t i o n provided at t h i s meeting one would h o p e f u l l y l i k e t o be in a p o s i t i o n t o t a b u l a t e the major c l a s s e s o f p e s t i c i d e s and/or t h e i r primary metabolites according t o the conjugation r e a c t i o n s t o which they are subjected. However, I assume t h a t all o f us have r e a l i z e d that we still have a long way to go before we succeed in e s t a b l i s h i n g such a comprehensive l i s t i n g . Even if we attempt t o c l a s s i f y the chemical r e a c t i o n s which l e a d t o the d e s c r i b e d conjugates, we face what Dr. Hutson has c a l l e d the "xenobiocheraical dilemma". Both systems, c l a s s i f i c a t i o n by f u n c t i o n a l moieties conjugated, and by groups t r a n s f e r r e d have been used a t t h i s meeting and I am in no p o s i t i o n t o improve on t h i s by p r e s e n t i n g a u n i f i e d scheme. I f ,ins p i t e o f these l i m i t a t i o n s , I have t r i e d two k i n d s o f t a b u l a t i o n s , it is to demonstrate how the present o r accumulating i n f o r m a t i o n might u s e f u l l y be c o l l e c t e d and how the e x i s t i n g gaps might be visualized. Table I gives an overview o f the type r e a c t i o n s which appear to have been recognized w i t h p e s t i c i d e s . This l i s t is already impressive. However, if one compares it w i t h s i m i l a r t a b u l a t i o n s in recent textbooks o r reviews on drug metabolism Q > J0> it is to be expected t h a t a d d i t i o n a l type r e a c t i o n s w i l l be demonstrated with p e s t i c i d e s . Table I I attempts t o l i s t the main conjugation r e a c t i o n s d e s c r i b e d at t h i s meeting in terms o f groups o f biosystems in which they have been demonstrated o r in which they may be expected t o occur. The t a b l e r e v e a l s that there is reasonable documentat i o n on conjugate formation in mammals and p l a n t s , but a d e f i n i t e lack o f i n f o r m a t i o n on other v e r t e b r a t e s ( b i r d s , f i s h , etc.) and non-vertebrates ( p o s s i b l y except f o r i n s e c t s ) . Some o f these biosystems comprise species which may be important p a r t s o f the "food c h a i n " i n v o l v e d in the t r a n s p o r t and d i s t r i b u t i o n o f p e s t i c i d e m o i e t i e s . E s p e c i a l l y s u r p r i s i n g is the o b s e r v a t i o n that there is apparently no more than l i m i t e d demonstrated knowledge on conjugate formation in microorganisms such as a l g a e , f u n g i and b a c t e r i a , all o f which a r e prominent c o n s t i t u e n t s o f the soil/water ecosystem. However, conjugates produced by these organisms would be expected t o be r e l e a s e d i n t o the surroundings r a t h e r than being r e t a i n e d , accumulated o r t r a n s p o r t e d . I I . Stage o f Methodology In my personal judgement the i n f o r m a t i o n acquired under t h i s heading may have been the most rewarding o f the present



Table I

Amino A c i d Conjugates

Glycosides

Glucuronides

ο- ( E s t e r / E t h e r ) NS-glycine -glutamic -aspartic

έ

0- ( E s t e r / E t h e r ) Ν

Alkylated Metabolites

Acylated Metabolites

Sulfuric Acid Esters Glutathione Conjugates

AcetylFormylMalonic a c i d Methyl-

Alkyl (Aralkyl) A r y l (Epoxide) Alkene N-heterocycle

Aryl-S

Type o f Reactions Recognized in Conjugate Formation o f P e s t i c i d e s o r o f P e s t i c i d e M o i e t i e s ( l i s t compiled from r e a c t i o n s discussed in v a r i o u s conjugate papers).

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Table I I

371


P e s t i c i d e Conjugation Reactions Recognized o r Presumed t o Occur in D i f f e r e n t Groups o f Biosystems

Mammals

Glucuronides

Glycosides


Sulfuric Acid Esters

Glutathione Conjugates

Acylated Metabolites

Alkylated Metabolites

Legend:

Other NonPlants Vertebrates Vertebrates

•• ο • ο • ο • ο ο• • • ο • • •• • ο ο• ο

ο

MicroOrganisms

ο Q

Ο Ο

Ο Θ

ο ο

Conjugate formation demonstrated w i t h s e v e r a l p e s t i c i d e s or p e s t i c i d e moieties

Ο

Conjugate formation presumed t o occur from the presence o f enzymes and/or n a t u r a l conjugates

•OExceptional

conjugation r e a c t i o n


372

B O U N D AND

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

meeting. The authors have provided a number o f u s e f u l personal experiences, references and suggestions regarding methodology and i n s t r u m e n t a t i o n . This i n f o r m a t i o n ought t o a s s i s t us in coping w i t h the v a r i o u s s y n t h e t i c and a n a l y t i c a l problems o f conjugate chemistry, b i o c h e m i s t r y and p h y s i o l o g y . For t h i s type of work the p e s t i c i d e chemist needs the f o l l o w i n g : - s y n t h e s i s procedures f o r s m a l l - s c a l e p r e p a r a t i o n o f nonlabeled and l a b e l e d reference conjugates; - i n f o r m a t i o n on p h y s i c a l / c h e m i c a l p r o p e r t i e s o f conjugates t o a s s i s t in e s t a b l i s h i n g a n a l y t i c a l and p h y s i o l o g i c a l c r i t e r i a ; - procedures f o r p u r i f i c a t i o n and s e p a r a t i o n o f conjugates from extraneous m a t e r i a l s o f n a t u r a l o r i g i n ; and - methods f o r i d e n t i f i c a t i o n o f compounds at the submicrogram s c a l e . The p a r t on p u r i f i c a t i o n / s e p a r a t i o n and i d e n t i f i c a t i o n has found the most a t t e n t i o n at t h i s meeting. Dr. Frear has c l e a r l y p o i n t e d out the three c r i t e r i a which have t o be f u l f i l l e d f o r adequate i d e n t i f i c a t i o n o f conjugates: - p r o t e c t l a b i l e groupings; - i d e n t i f y the conjugate in i t s e n t i t y ; and - i d e n t i f y both the p e s t i c i d e moiety and the endogenous part a f t e r suitable hydrolysis. An attempt t o summarize the s a l i e n t i n f o r m a t i o n on methodol o g y in a short hand f a s h i o n is made in Table I I I . The most h e l p f u l and promising procedures as suggested by the authors themselves are l i s t e d t o a s s i s t in the s e l e c t i o n o f appropriate techniques from among the b e w i l d e r i n g v a r i e t y of p o s s i b i l i t i e s . Without going i n t o the d e t a i l s o f the t a b l e , it is evident t h a t column chromatography (ion exchange, a d s o r p t i o n , g e l permeation) and mass spectrometry are the most prominent t o o l s at t h i s time. However, new i n s t r u m e n t a t i o n in the yg-range, i . e . raicro-IR and F o u i e r - t r a n s f e r NMR appear to be very powerful a i d s f o r supplementing MS-data. High pressure l i q u i d chromatography (HPLC) has been mentioned as a p o s s i b i l i t y f o r speeding up and improving separations and according t o experience acquired in our own l a b o r a t o r i e s , ought to be u t i l i z e d without delay. To acquire more d e t a i l e d and d e f i n i t e q u a n t i t a t i v e data on conjugate formation under d i f f e r e n t environmental c o n d i t i o n s (see s e c t i o n I I I ) , s t a n d a r d i z e d and l e s s e l a b o r a t e procedures are r e q u i r e d which a l l o w clean-up and determination o f major conjugates on a r o u t i n e - s c a l e . I I I . S i g n i f i c a n c e o f Conjugation

Reactions

To evaluate the s i g n i f i c a n c e o f conjugation r e a c t i o n s w i t h regard t o the behavior o f p e s t i c i d e s in the environment, I wish t o demonstrate w i t h two s i m p l i f i e d models the v a r i o u s p o i n t s which have to be considered:



PC, TLC, LC ( i o n exchange) MS ( d e r i v a t i z a t i o n ) , B i o s y n t h e s i s

Legend :

Identification Mass Spectrometry I n f r a r e d Spectrometry Nuclear Magnetic Resonance Spectrometry

I: MS: IR: NMR:

Purification/Separation

L i q u i d Chromatography Gas L i q u i d Chromatography Thin Layer Chromatography Paper Chromatography

P/S:

LC: GLC: TLC: PC:

Glutathione Conjugates

P/S: I:

PC, TLC, LC ( i o n exchange), E l e c t r o p h o r e s i s MS

P/S: I:


LC ( i o n exchange, g e l f i l t r a t i o n ) IR

TLC ( p r e p a r a t i v e ) , LC ( i o n exchange, a d s o r p t i o n , g e l f i l t r a t i o n ) MS ( G L C / d e r i v a t i z a t i o n ) , NMR (as s u p p o r t i n g evidence)

P/S: I:

Glycosides

S u l f u r i c A c i d P/S: I: Esters

LC ( i o n exchange, a d s o r p t i o n ) , GLC ( d e r i v a t i z a t i o n ) , PC MS ( G L C / d e r i v a t i z a t i o n ) ; f o r aglycone: NMR, IR

P/S: I:

Methods and Instrumentation Recommended f o r P u r i f i c a t i o n , S e p a r a t i o n and I d e n t i f i c a t i o n o f P e s t i c i d e Conjugates

Glucuronides

Table I I I

374

BOUND AND

CONJUGATED PESTICIDE

RESIDUES

- a model c o n s i s t i n g o f crop p l a n t s and mammals ( F i g . 1 ) ; and - a model comprising soil and an aqueous biotope ( F i g . 2 ) . Provided the parent compound o r any o f i t s primary metab o l i t e s are s i g n i f i c a n t t e r m i n a l r e s i d u e s on a p l a n t crop, the study o f t h e i r forming conjugates becomes p a r t o f the r o u t i n e mammalian metabolism i n v e s t i g a t i o n ( F i g . 1, step A ) . I f conj u g a t i o n r e a c t i o n s do occur in t r e a t e d p l a n t s , it has t o be e s t a b l i s h e d in q u a n t i t a t i v e terms if they represent a s i g n i f i cant f r a c t i o n o f the t e r m i n a l r e s i d u e in the raw a g r i c u l t u r a l commodity (B). Determination o f t h e i r r o l e in the formation o f non-extractable o r "bound" r e s i d u e s (C) is not a requirement but may p r o v i d e some u s e f u l i n f o r m a t i o n on the s t r u c t u r a l i d e n t i t y o f the l a t t e r f r a c t i o n . Provided conjugates are s i g n i f i cant t e r m i n a l r e s i d u e s , t h e i r b i o a v a i l a b i l i t y has t o be examined in an a p p r o p r i a t e mammalian organism (D). The s i g n i f i c a n c e o f conjugate formation in a soil/water biotope can be v i s u a l i z e d by an analogous sequence o f p o t e n t i a l p h y s i o l o g i c a l and biochemical events ( F i g . 2 ) . However, p o l a r conjugates formed by microorganisms (B) in such a system would normally appear t o be t o o u n s t a b l e t o become p a r t o f the termina l r e s i d u e f r a c t i o n in soil o r water. I f they do, t h e i r b i o a v a i l a b i l i t y (D) t o both p l a n t s and exposed soil o r aqueous fauna ( f i s h , e t c . ) would need t o be examined as d e s c r i b e d above f o r t e r m i n a l crop r e s i d u e s . A l a r g e p o r t i o n o f the i n f o r m a t i o n p r o v i d e d a t t h i s meeting has d e a l t w i t h s t e p A ( F i g . 1 ) , i . e . the d i f f e r e n t conjugates formed from parent p e s t i c i d e s in mammalian systems. However, t h i s i n f o r m a t i o n has mainly been q u a l i t a t i v e , and, w i t h a few e x c e p t i o n s , little has been s a i d about the q u a n t i t a t i v e aspects o f conjugate f o r m a t i o n . We need t o d i s t i n g u i s h between major and minor pathways as t h i s p r o v i d e s some e s s e n t i a l i n d i c a t i o n s r e g a r d i n g the behavior o f the examined compound in the mammal. Thus, simple appearance o f a p a r t i c u l a r conjugate in very s m a l l q u a n t i t i e s has t o be p l a c e d in proper p e r s p e c t i v e . As regards conjugate formation in p l a n t s (step B, F i g . 1) we had an overview o f the d i f f e r e n t type r e a c t i o n s which do o r can occur in young v e g e t a t i v e organs and t i s s u e s . Again, q u a n t i t a t i v e i n f o r m a t i o n appears t o be minimal a t t h i s time. As w i t h mammals, we have t o d i s t i n g u i s h between major and minor pathways. In a d d i t i o n , we do have t o c o n s i d e r the p h y s i o l o g i c a l age o f the system. The d i s c u s s e d conjugates may indeed r e f l e c t important metabolic routes in the e a r l y stages o f p l a n t growth. However, what is f i n a l l y s i g n i f i c a n t is the presence o f these conjugates as measurable t e r m i n a l r e s i d u e s in the raw a g r i c u l t u r a l commodity. As regards steps C and D ( F i g . 1 ) , which r e f e r t o the imp l i c a t i o n o f conjugates in the formation o f n o n - e x t r a c t a b l e p l a n t r e s i d u e s and t o t h e i r b i o a v a i l a b i l i t y in the mammalian organisms, it is obvious from t h i s meeting t h a t we are a t present in the i n i t i a l stages o f experimentation. These experiments


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Figure 1. Scheme of simplified model consisting of crop plant and mammal(s) for demonstrating the significance of con jugate formation from pesticides applied to plants. Steps A to D describedintext.

ι

Parent Compound

®

®

#

^

©

Terminal ^ Residues

"Bound Residues"

Figure 2. Scheme of simplified model comprisingsoiland an aqueous biotope for demonstrating the potential signifi cance of conjugate formation from pesti cides applied to or collected bysoil.Steps A to Ό analogous to those of Figure 1 and describedintext.


376

B O U N D AND

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

ought t o l e a d us to an appropriate design of more d e f i n i t e s t u d i e s . The same remarks apply t o the s i g n i f i c a n c e o f conjugate formation in a soil/water biotope ( F i g . 2 ) . An adequate design of experimentation is needed to p r o p e r l y evaluate the r o l e o f conjugates in such a system. At t h i s p o i n t , and s i n c e it r e f e r s in p a r t t o the model s i t u a t i o n s j u s t d e s c r i b e d , I wish t o make some comments on Dr. Dorough's i n t r o d u c t o r y paper which has d e a l t w i t h c e r t a i n aspects o f b i o a v a i l a b i l i t y and t o x i c i t y t e s t i n g o f p e s t i c i d e conjugates. According t o my personal o p i n i o n it has s e t the stage f o r some d i s c u s s i o n s which were beyond the purpose and s c i e n t i f i c competence of t h i s meeting: - a p e s t i c i d e conjugate formed in p l a n t leaves o r stems, even if present in l a r g e q u a n t i t i e s is not a s i g n i f i c a n t residue unless it has been e s t a b l i s h e d t h a t it represents a major p o r t i o n o f the t e r m i n a l residue in the raw a g r i c u l t u r a l commodity as observed under p r a c t i c a l a p p l i c a t i o n conditions. - even if the b i o a v a i l a b i l i t y to mammals o f such an i d e n t i f i e d o r n o n - i d e n t i f i e d conjugate f r a c t i o n has been demonstrated, t h e r e is no v a l i d s c i e n t i f i c reason t o subject it as an e n t i t y t o r o u t i n e t o x i c i t y t e s t i n g . Such t e s t i n g may be i n d i c a t e d if the p e s t i c i d e moiety or aglycone r e leased from the conjugate has been i d e n t i f i e d and has s t r u c t u r a l features which set it d e f i n i t e l y apart from the major primary mamalian metabolites o f the parent compound. - a l l u s i o n t o drug metabolism in t h i s connection has to be made w i t h care. Drugs are chemicals designed to have s p e c i f i c b i o l o g i c a l e f f e c t s in mammalian systems and t h i s in concentrations s e v e r a l orders of magnitude h i g h e r than those o f p e s t i c i d e t e r m i n a l r e s i d u e s . Biochemists f a m i l i a r w i t h the mammalian metabolism o f f o r e i g n compounds know t h a t the dose a p p l i e d has a d e f i n i t e e f f e c t in q u a n t i t a t i v e terms on the operation of d i f f e r e n t pathways which represent the t r a n s f o r m a t i o n p a t t e r n o f a compound. - as regards the proposed m u t a g e n i c i t y / c a r c i n o g e n i c i t y t e s t i n g of conjugates w i t h microorganisms and the 90-days t o x i c i t y experiments w i t h l a b e l e d conjugates, I wish to v o i c e some r e s e r v a t i o n s which, I b e l e i v e , would be shared by t o x i c o l o g i s t s much more competent than myself. I may r e f e r t o a r e c e n t l y p u b l i s h e d r e p o r t by a group o f experts convened by WHO who have expressed t h e i r o p i n i o n on some o f these s u b j e c t s (3). IV. Concluding Remark I do hope t h a t the present meeting has demonstrated the need f o r a major s c i e n t i f i c e f f o r t by u n i v e r s i t i e s , government i n s t i t u t i o n s and i n d u s t r i a l l a b o r a t o r i e s . Once t h i s e f f o r t is accom-


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p u s h e d then the proper s i g n i f i c a n c e o f conjugate formation in d i f f e r e n t biosystems can r e a l l y be evaluated. Acknowledgments I g r a t e f u l l y acknowledge the a s s i s t a n c e o f Gino J. Marco in p r e p a r i n g t h i s review and the h e l p p r o v i d e d by the authors o f conjugate papers in c o l l e c t i n g the data f o r Tables I t o I I I . References 1.

2.

3.

Foreign Compound Metabolism in Mammals (D.E. Hathway, senior rep.). Vols. 1 to 3. Specialist Periodical Reports, The Chemi c a l Society, Burlington House, London, WIV OBN (1970, 1972, 1975). Metabolic Conjugation and Metabolic Hydrolysis (W.H. Fishman, ed.). Vols. 1 to 3. Academic Press, New York and London (1970, 1973). Assessment of the Carcinogenicity and Mutagenicity of Chemi c a l s . Report of a WHO Scientific Group, Technical Report Series 546, World Health Organisation, Geneva (1974).


Bound and Conjugated Pesticide Residues

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