«: AGROCHEMICALS: FATE IN FOOD AND THE ENVIRONMENT PROCEEDINGS OF A SYMPOSIUM, ROME, 7 - 1 1 JUNE 1982 JOINTLY ORGANIZED BY IAEA AND FAO l^J I N T E R ...»
However, the main objective here is to consider the application o f nuclear techniques to studies o f the metabolism o f pesticides in plants, but similar methods are also used to study enzyme activities and normal metabolic pathways. Since living organisms are so closely interdependent, having similar basic metabolites and pathways in c o m m o n and because it is sometimes difficult to distinguish chemical from biological degradation, it is perhaps not surprising that the nuclear techniques used to study metabolism in plants are much the same as those used to study degradation o f chemicals on surfaces or in soil, water or organic solvents or in the vapour phase, and by other organisms from microorganisms to mammals.
Techniques must obviously be adapted to the morphology and chemistry o f the system being investigated and to the ultimate objectives o f the studies.
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Pesticides are here defined as chemicals used to control pests, and in broad terms pests may be defined as any organism (insects, fungi, plants, microorganisms) that causes crop losses or deterioration o f stored products. T o do this safely the pesticides must remain active long enough to control pests but without leaving harmful residues.
Simply to measure persistence is not enough. If the chemical can no longer be detected we need to know why not; has it moved on or has it been modified?
We need to know the fate o f the modified chemical to ensure efficient and safe use. Thus, we are led to study pathways o f degradation and metabolism.
Isotopic labelling is not essential for all studies, especially if products are formed that can be recognized and assayed by other methods. However, this is not always the case and isotopes have obvious advantages in that they facilitate detection and identification o f degradation products by providing a unique nuclear property to distinguish them from normal materials.
A detailed and comprehensive review is neither appropriate nor possible here and would merely supplement existing compilations [ 2 - 6 ]. The aim, therefore, is to present an overall view o f the subject illustrated by reference to selected publications.
Historically, the introduction and rapid spread o f the use o f organophosphorous insecticides caused much anxiety because most o f these compounds were toxic to mammals; anxiety was not allayed when some, which are taken up into and move in plants, began to be used. At about the same time radioisotopes became freely available and labelled pesticides were synthesized and used to study the behaviour o f these pesticides.
In 1951 Hartley and Heath [ 1 ] published work using 32 P-labelled octamethylpyrophosphamide and they concluded that this systemic insecticide was metábolized by plants. The decomposition products formed in plants differed from those formed by chemical hydrolysis, which was also much slower. The parent compound was separated from decomposition products by partition between water and chloroform. Differential solubility in the presence o f sodium salts and solvent partition was used to confirm that products o f chemical decomposition differed from those in the plant.
This early demonstration o f the metabolism o f pesticides in plants emphasizes both the importance o f establishing the identity, if not the structure, o f radioactive materials and the need for methods to do this. The radioactive isotope 32 P
extracts cause quenching and have to be removed by some means; in this instance column chromatography was used. Many alternative methods have been devised to avoid quenching, each tailored to suit particular problems; methods include solvent extraction [ 1 3 ] separation by thin-layer chromatography [14| and even by total combustion to destroy the interfering compound, the 14C02 being absorbed in an alkaline medium for counting. Combustion is also valuable to assay labelled metabolites associated with other insoluble material from which it cannot be separated, as was found with some metabolites of Etrimfos .
The increased range o f nuclear techniques now available is complemented by the development o f analytical techniques, and in the study o f Etrimfos leaves were rinsed with water then macerated and extracted with chloroform, followed by acetone and then water. Solvent extracts and leaf residues were examined separately, each fraction providing a partial separation o f materials. Solutions were chromatographed on thin layers o f silica, using seven different solvents in all, to separate or purify materials that were located by radioautography. Separated materials were assayed by scintillation counting or subjected to gas chromatography and mass spectrometry for identification. Oxidation and hydrolysis products were identified, as well as conjugated metabolic products, although neither recovery o f 14C nor identification o f metabolic products was complete.
Metabolism o f the oxide carbamate aldicarb in plants [15, 16] was examined using similar methods: extraction, separation by solvents, thin-layer and ionexchange chromatography for isolation and identification o f metabolites. Liquid scintillation counting was used for quantitive measurements o f activity. Radioautography was used to locate material separated on thin-layer plates and authentic samples o f unlabelled materials were used for tentative identifications, which were confirmed by mass spectrometry and NMR. It was shown that the major pathway o f degradation was by oxidation o f the thioether group to the sulphoxide and by hydrolysis to liberate the corresponding oximes. Recovery of radioactivity declined with time, and only 50% or less was accounted for after one month. No attempt was made to determine if the loss resulted from incorporation into insoluble materials, root or leaf exudation, or by complete metabolism to C 0 2.
Similar methods were used by Belgian workers [17, 18] to investigate the metabolism o f 14 C-aldicarb in sugar-beet plants, but the work was extended to investigate conjugated products o f metabolism that were not soluble in the solvent (50% aqueous ethanol) used for extraction.
Results from localized applications o f aldicarb to sugar-beet plants growing in the field showed that at harvest only 13% o f the radioactivity in the leaves was IAEA-SM-263/38 217 present as aldicarb or its sulphoxide or sulphone; about 15% appeared to be incorporated into plant polymers. Much o f the remaining 14C was probably conjugated to plant materials, as shown by liberation by hydrolysis with strong acids or an enzyme (-glucuronidase). When roots were examined for compounds labelled with 14C the proportions o f aldicarb metabolites in the form o f conjugates differed from those in leaves and as much as a fifth o f the 14C found in roots occurred as sugar, indicating incorporation o f label into the normal metabolic pools o f sugar-beet. On reflection it is not surprising that no 14 C-sugar was found in leaves, since the root is the normal sink for sugar in sugar-beet.
FURTHER EXAMPLES OF PESTICIDE METABOLITES ENTERING
METABOLIC POOLS A F T E R BREAKDOWN OF PESTICIDESIncorporation o f 14 C from aldicarb into sugar is not a unique example o f metabolic products from pesticides entering normal metabolic pathways. Such incorporation is to be expected when small molecules containing carbon result from degradation, as in the case o f the organophosphorous systemic insecticide mephospholan . Applied to rice, about half the residual 14 C from mephospholan could not be extracted with methanol for a period o f up to 18 weeks after treatment, and 14C was shown to be incorporated into the glucose or starch, cellulose and lignin o f the plants. Labelled material extracted by methanol was almost all the unchanged parent compound, indicating rapid breakdown o f hydrolysis products containing 14 C.
Cellulose, lignin and many other normal metabolites were shown to be derived from the 14 C-labelled fungicide cymoxanil . After foliar application to grapes, tomatoes and potatoes the fungicide was rapidly metabolized and much (30—55%) o f the 14C was incorporated into glycine and other amino acids, while another considerable proportion (7—15%) o f the 14C was reported as sugar or starch, the proportions depending on the crop and its physiology or biochemistry.
Among other compounds identified as containing 14C were polycarboxylic acids, such as citric acid and fatty acids.
Residues o f the fungicide and its hydrolytic degradation products were sought using thin layer chromatography, the radioactive bands being located using a TLC radio scanner (not radioautography ), and assay was by scintillation counting o f bands scraped from TLC plates. Various analytical methods were used to separate and identify other radioactive products, including derivatization, assay by radio gas chromatography or by gas chromatography trapping column affluents for assay o f radioactivity by scintillation counting. Chemical structures were confirmed by GC/MS.
MORE PERSISTENT STRUCTURES A N D CONJUGATES
Fragments o f pesticides once incorporated into normal metabolic pools behave indistinguishably from material derived from natural sources and so have no toxicological interest and cannot be recognized as pesticide residues except in so far as their origin may be determined by the presence o f unusual isotopes.
However, many substances are less completely metabolized and parts, if not all, o f the pesticide structure may remain chemically recognizable, if not intact, so that the residues may have significant biological activities.
Recent investigations o f such pesticides provide examples of the use o f all types o f techniques and instrumentation already mentioned for identification, location and assay both o f radioisotopes and chemicals.
Development o f this methodology makes possible identification and measurement o f the multiplicity o f metabolites that may be derived from compounds such as pentachloronitrobenzene [21—23], and some of the products may represent less than 1% o f the original pesticide [20, 22].
Metabolism o f pentachloronitrobenzene in onions and peanuts provided evidence o f a whole range o f mechanisms, including reduction o f elimination o f the N 0 2 group, formation o f phenols, their methylation and acetylation, introduction, methylation and oxidation o f a thiol group into the aromatic ring, and dechlorination. Such reactions occur separately or in sequence to produce a variety o f products which may in turn form conjugates. A variety o f conjugates containing cysteine or glutathione were identified, but some insoluble radiolabeled products remain unidentified [22—24].
Complex patterns o f metabolism occur with many pesticides that are susceptible to degradation at several sites. Typically, pathways o f metabolism o f pesticides with aromatic groups are related and a range o f pesticides may have some metabolites in c o m m o n so that subsequent pathways o f metabolism are also the same. Phenols and carboxylic acids, whether pesticides or degradation products, are likely to be conjugated with a variety o f substances, such as amino acids and sugars. Examples include herbicides, such as phenoxyacetic acids [5, 25] and synthetic pyrethroid insecticides, reported to be degraded in plants by hydrolysis at the ester group, followed by oxidation and conjugation with sugars o f both the cyclopropyl and aromatic moieties o f the molecule [6,26,27].
SIGNIFICANCE OF PESTICIDE METABOLISM IN PLANTS
METABOLISM, ISOTOPES AND THE FUTUREThere seems little doubt that while chemicals continue to be developed and applied to crops there will remain a need for continued studies of metabolic pathways and metabolites. Doubtless, studies o f metabolism in plants and other organisms will continue to be made in parallel, using radioisotopes to label either all or parts o f the pesticide molecule. There is evidence o f the continuing development o f techniques and instrumentation for both isotopic and chemical assay, which will further increase power to gather information.
Resolution o f the problem o f unidentified residues has begun with demonstration o f the incorporation o f 14C from pesticides into normal plant constituents.
Increasing information on conjugates o f pesticides and their metabolites suggests that understanding o f their toxicological importance may emerge.
Work on the identification o f a conjugate o f a pyrethroid metabolite [ 2 7 ] presents an interesting application o f 14C glucose, which may find wider applications o f labelled normal metabolites to investigate conjugates.
Radioimmunoassay techniques have been applied to determination o f pesticides (benzimidazoles [ 3 2 ] and parathion ). It is an extremely sensitive technique and if it can be adapted to the assay o f metabolites closely related t o pesticides it may advance metabolic studies, but the method has yet to be fully assessed for specificity.
More studies are needed to assess the biological significance, if ány, o f metabolites and also to develop a more systematic approach to the metabolism o f groups o f related compounds, such as substituted phenols, which may be derived from pesticides containing similarly substituted aromatic groups but with widely differing biological activities. Such systematic investigations would provide information to allay anxieties about effects on non-target organisms more economically than detailed investigations o f individual compounds.
[ 1 ] HARTLEY, G.S., HEATH, D.F., Decomposition of radioactive octamethylpyrophosphoramide in living plants, Nature (London) 167 (1951) 816.
 LYKKEN, L., CASIDA, J.E., Metabolism of organic insecticide chemicals, Can. Med.
Assoc. J. 1 0 0 ( 1 9 6 9 ) 145.
 PAULSON, G.D., Conjugation of foreign chemicals by animals, Residue Rev. 76 (1980) 31.
 MULLA, M.S., MIAN, L.S., KAWECKI, J.A., Distribution, transport and fate of the insecticides malathion and parathion in the environment, Residue Rev. 81 (1981) 1.