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Metabolic Pathways of Agrochemicals Part 1: Herbicides and Plant Growth Regulators
By Terry R. Roberts
The Royal Society of ChemistryCopyright © 1998 The Royal Society of Chemistry
All rights reserved.
Isoxaben, propyzamide and tebutam form a small group of herbicides classified as amides. Two are mono-N-substituted arylamides and tebutam is a di-N-substituted amide of pivalic acid. They have no common mode of action and thus, from biological and chemical viewpoints, they form a somewhat heterogeneous group. A common feature, however, is their phytotoxicity to germinating grasses. The amide herbicides are neutral molecules of intermediate lipophilicity and low vapour pressure. They have the stability associated with the amide bond, being stable to hydrolysis and relatively insensitive to photodegradation. Isoxaben is exceptional in being somewhat base-labile because of the nature of the N-substituent.
These herbicides are stable enough to be applied to the soil and because of this and their activity profile, they are mostly used pre-emergence. There are few common features in their metabolism and environmental degradation because of the disparate nature of their structures. They are, however, all biodegradable in soils, plants and animals.
Uses Isoxaben is a selective pre-emergence herbicide used widely for the season-long control of dicotyledonous weeds in winter cereals. It is also used for control in turf, fruit, vines and ornamental trees and shrubs.
Mode of action Isoxaben is mainly absorbed by the roots. It acts via the inhibition of protein synthesis.
The photodegradation and the fate of isoxaben in soils and plants has been reported; metabolism in the rat has been studied but not reported in the literature. The expected amide bond cleavage is a relatively minor reaction in plants and soils. The most important cleavage reaction occurs at the amide N-substituent bond (see below). Hydroxylation in the aliphatic side chain of the intact molecule is the major route of metabolism in plants.
Isoxaben is stable to hydrolysis in the pH range 5-9. It is hydrolysed outside of this range but in aqueous solution the usual amide CO-N bond cleavage (to 13 and 14) was not observed (see Scheme 2 for structures). Instead isoxaben was cleaved to 2,6-dimethoxybenzamide (6) and the 5-isoxazolinone [3-(1-ethyl-1-methylpropyl)isoxazolinone, 10] (Rouchaud et al., 1993a). This unexpected reaction was probably due to the positive charge on C5 of the isoxazole ring. The chemistry is relevant to the metabolism of isoxaben in soil, where the same reaction was observed as a major pathway.
The photolysis of isoxaben in dilute aqueous solution and adsorbed on silica gel plates has been studied under natural and simulated sunlight (Mamouni et al., 1992). Its half-life in sunlight in aqueous solution was 14 days, but more rapid with simulated sunlight, in which case 8 metabolites were identified. At about 65% conversion of isoxaben, photodecomposition in water afforded 2 (25%), 3 (12%), 4 (4%), 5 (10%) and 6 (15%) as major products and 7, 8 and 9 as minor products. Only 2, 3, 5 and 6 were observed in the adsorbed phase. The photolysis rate decreased during the experiment and the kinetics were initially first order but changed after a few hours. This was due to the reversal of the reaction, in particular 2 to 1, resulting in formation of the original herbicide. The primary reaction was thought to be N-O bond cleavage, followed by ring-closure to the azirine 2. Cleavage of the C-C bond of the azirine ring in 2 lead to the oxazole 3. Photodegradation of 3 and 4 (structure unknown) also gave the azirine. Other products were formed by further chemical reactions: dimethoxybenzamide (6) was a major product formed by the cleavage of the benzamide-isoxazole linkage. The benzonitrile 5 and the methyl benzoate 7 were derived from 6. The formation of the methyl ester 7 was due to the presence of methanol in the system; the free acid was not observed. 8 and 9 were formed as minor products by elimination and rearrangement. These reactions are shown in Scheme 1. The rate of photodegradation was slower in the presence of humic acids (which usually act as photosensitisers).
Degradation in soils
Non-radiolabelled isoxaben was applied to a manured sandy loam and winter wheat was later over-planted. At various times up to harvest (281-282 days after application) soil was analysed for known and putative metabolites of isoxaben (6 and 10-14, Scheme 2) (Rouchaud et al., 1993b). All of these were observed in the 0-10 cm layer; no leaching occurred to lower levels. Though radioactive isoxaben was not used, recoveries indicated that most of the applied herbicide was accounted for as parent compound and known metabolites. The half-lives were in the range 75-199 days. Taking one green-manured plot (half-life of isoxaben 106 days) as an example, metabolite yields at 118 days (% total) were: 6 (14%), 10 (12%), 11 (11%), 12 (3%), 13 (3%) and 14 (12%). Isoxaben accounted for 45%.
Thus the observed reactions were: demethylation (to 11), 5-isoxazolinone formation (giving 10, see Rouchaud et al. (1993a) above) and amide bond cleavage (to 13 and 14). The latter appears to be of relatively minor importance. The major terminal metabolites (282 days) were 2,6-dimethoxybenzamide (6), the 5-isoxazolinone (10) and demethoxyisoxaben (11). The pathways are shown in Scheme 2 in comparison with those in plants.
Metabolism in plants
Seedlings of winter wheat (tolerant) and oilseed rape (sensitive) were treated with [14C-5-isoxazole]isoxaben by root uptake. Rape plants absorbed more radioactivity (by a factor of 2-3) than did wheat plants but translocated less radioactivity to their shoots. Metabolism occurred only in the shoots and proceeded at a similar rate in both species to give the same metabolites (Cabanne et al., 1987). The major component after 2 days metabolism (70% conversion) was the secondary-hydroxy-isoxaben (15); both enantiomers were assumed. The primary-hydroxy-isoxaben (16) and an unknown metabolite (17) were also found. Ratios of 15 to 16 were about 2:1 in wheat and 11:1 in rape. These metabolites were also present as enzyme-hydrolysable conjugates. The conjugate of 15 accounted for up to 30% of the radioactivity after 4 days. Isoxaben is rapidly metabolised by wheat and barley (Cabanne et al., 1987) affording at maturity 65% and 41% extractable radioactivity, respectively. The remainder was lignin-bound. Metabolites 15 and 16 and their conjugates were detected in the straw of both species. Hydrolysis to the benzamide 6 occurred as a minor route. These pathways are shown in Scheme 2 in comparison with those in soils.
Metabolism in animals
Isoxaben was rapidly eliminated following oral administration to rats, with 90% of the dose appearing in the faeces within 48 hours. Approximately 10% of the absorbed herbicide was converted to 15 or more metabolites which were excreted in the urine (PM).
Uses Propyzamide is used for the control of annual and perennial grasses and some broad-leaved weeds in fruit, vines, lettuce, chicory, brassicas and other vegetables, oilseed rape, forage crops (alfalfa, clover and trefoil) and ornamentals, and on fallow land and forestry. It is used either pre-emergence or early post-emergence.
Mode of action Propyzamide is a systemic herbicide, absorbed by the roots and translocated. It inhibits the growth of sensitive species by disrupting the mitotic sequence in dividing cells.
Photodegradation in solution and degradation in soil have been studied, as have metabolism in plants, the rat and a cow. Cyclisation to an oxazoline is a common metabolic feature which plays a part in both abiotic degradation and biotransformation. Metabolic pathways in soils, plants and animals provide very similar pathways, although plant metabolism studies have not distinguished between metabolites formed in plants and those absorbed from the treated soil. The comparative metabolism in the three groups is discussed below in the section on animals.
Propyzamide is stable in water at pH 5-9, showing <10% degradation over 28 days at 20 °C. However, it is degraded at pH values outside of this range: in 1N nitric acid the half-life is about 70 days but in 1N NaOH it is 2-3 hours (25 °C). The rate of degradation is unaffected by iron and copper salts but silver ions very effectively catalyse the cyclisation via complexing with the propynyl group (Bastide and Coste, 1978). This route of degradation is relevant to the environmental fate studies because the transformation (Scheme 1) is seen in light and in soil (Yih et al., 1970) and also plays a role in biodegradation, with the oxazoline 2 and the ketoamide 3 featuring in most pathways.
The DT50 of propyzamide on soil thin-layers in artificial sunlight was 13-57 days. The sequence 1 to 2 to 3 was seen when propyzamide was subjected to photolysis in organic solvent (Méallier et al., 1980). Also seen was aryl dechlorination (to dechloropropyzamide, 16), tertiary carbon-nitrogen bond fission (to the benzamides 17 and 18) and further degradation of the ketoamide 3 to 5 and 19. Product 19 appears to be unique to photodegradation. Other reported products found in this experiment were: ethylbenzamide, acetaldehyde, acetone, butan-2-one and 3-methyl-butan-2-one (isopropyl methyl ketone). The products are shown in Scheme 1 (the numbering used in Scheme 1 is designed to retain the authors' original sequence in the main comparative metabolic pathways shown in Scheme 2).
Degradation in soils
The DT50 values for propyzamide in a variety of soils (organic matter 1.38-7.3% and pH values 4.8-7.2) fell in the range 23-42 days, degradation being faster at higher moisture contents (Bastide and Coste, 1978) and slower at lower temperatures (60-120 days at 15 °C; Rouchaud et al., 1987). Degradation was faster in biologically active soil but some degradation was also observed in sterile soils (Cantier et al., 1986) and thus abiotic degradation is a significant element in the fate of propyzamide.
[14C-carbonyl]Propyzamide in a clay-loam soil under laboratory conditions was 13% mineralised to 14CO2; under the same conditions carbonyl- and ring-labelled 3,5-dichlorobenzoic acid (20) were 81% and 51% mineralised, respectively (Fisher, 1974). This study indicates that the benzoic acid, if formed in soil, may not be detected. However, the substantial amount of soil-bound residue in an outdoor treatment over-planted with lettuce was released under alkaline conditions as 20 (Rouchaud et al., 1987). A full analysis of the metabolites of [14C-carbonyl]propyzamide applied to a range of soil types under laboratory conditions and in the field over-planted with alfalfa has been presented by Yih and Swithenbank (1971a). After 90 days, the parent herbicide (1) and 9 metabolites were found. Most of the radioactivity extracted was associated with propyzamide (10%), the initial cyclisation product 2-(3,5-dichlorophenyl)-4,4-dimethyl-5-methyleneoxazoline (2, 9%) and its ketoamide hydrolysis product N-(1,1-dimethylacetonyl)-3,5-dichlorobenzamide (3, 77%). The chemical transformations illustrated in Scheme 1 emphasise the abiotic contribution to this process. The other products 4-10 were each present in 0.1-1.6% amounts and were mostly products of the biotransformation of the (originally) acetylenic carbon atoms after the hydrolysis of cyclisation product 2. The pathways are shown in Scheme 2 in comparison with plant and animal results which are essentially similar. Comparative aspects are discussed below in the section on animals.
Metabolism in plants
Alfalfa was treated with [14C-carbonyl]propyzamide and sampled at 17,50 and 112 days (Yih and Swithenbank, 1971a). The residues of unchanged propyzamide at these times were 21.9, 1.0 and 0.075 mg kg-1, respectively. Taking 50 days as an example, the distribution of extracted radioactivity (95%) was mainly between parent (61%), 7 (3.4%), 8 (16.4%), 10 (4.1%) and 12 (6.8%). The structure of 12 was unknown but it was methanolysed to methyl 3,5-dichlorobenzoic acid and was therefore regarded as a conjugate of 20. Metabolite 8 and parent were the major products found at 112 days and together accounted for 52% of the extracted radioactivity. At this time 46% of the radioactivity was not extractable (with methanol). Compared with the soil metabolites, only the unidentified 12 is unique to alfalfa. It is not known if metabolites 2-10 were formed by the plant or absorbed via the roots as soil metabolites. Lettuce planted in soil treated with propyzamide was found to contain propyzamide, the ketone 3 and benzoic acid 20 at harvest (Rouchaud et al., 1987).
A comparative study of Witloof chicory (resistant) and the common amaranth (sensitive weed) revealed no clear differences in absorption, translocation or metabolism. Thus it would appear that the selectivity of propyzamide is not metabolically-mediated (Mersie, 1995). The pathways are shown in Scheme 2 with those of soil and animals; comparative aspects are discussed in the section on animals below.
Metabolism in animals
[14C-carbonyl]Propyzamide is extensively metabolised in the rat; 12 metabolites were identified (Yih and Swithenbank, 1971b). The metabolites were eliminated in the urine and faeces, with unidentified conjugates of 3,5-dichlorobenzoic acid (13 and 15) predominating in the urine. Major unconjugated urinary metabolites were 9 (22%) and the α-hydroxyacid 11 (19%) which was found only in rat urine and cow urine (cf. soil and plants). Unchanged propyzamide (54%) and 2-(3,5-dichlorophenyl)-4,4-dimethyl-5-hydroxymethyloxazoline (4, 15%) predominated in rat faeces. Only four metabolites were found in the urine of treated cow: 8, 9, 11 and 14. The α-hydroxyacid 11 was the major of these, accounting for 71% of the urinary radioactivity. Metabolite 14 (19%) was a conjugate of 3,5-dichlorobenzoic acid.
The metabolic pathways in animals are shown in Scheme 2 together with those in soils and plants. Several features common to soils, plants and animals are noteworthy. Two series of metabolites are formed: (i) N-(1,1-dimethylacetonyl)-3,5-dichlorobenzamide (3) and other metabolites oxygenated at the penultimate carbon atom of the side-chain (5,7,10 and 11) and (ii) the terminally oxygenated 6 and 8. The former arise via the cyclisation product 2. The latter are postulated to be formed via hydroxylation of the acetylenic carbons. Most of these metabolites are common to soils, plants and the rat. The presumed conjugates (12, 13, 14 and 15) are not found in soil; 12 is unique to the plant (alfalfa); 13 and 15 are unique to rat; 14 is common to rat and cow. Based on this species distribution, it is likely that 12 is a glucoside and that 13, 14 and 15 are glucuronic acid and amino acid conjugates of 3,5-dichlorobenzoic acid. Metabolite 11, the α-hydroxyacid, is unique to animals but it may well have been formed in soils and plants and oxidised to the keto acid 10.
Excerpted from Metabolic Pathways of Agrochemicals Part 1: Herbicides and Plant Growth Regulators by Terry R. Roberts. Copyright © 1998 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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