Phytotoxicity and metabolism of chlortoluron in two wheat varieties

Varietal susceptibility of winter wheat to chlortoluron, 1-(3-chloro-4-methylphenyl)-3,3 dimethylurea, has been studied in two varieties, Corin (susceptible) and Clement (tolerant). After a 24-hr root absorption of the herbicide, phytotoxicity was estimated from growth measurements. When administered at 12 to 96 μM concentrations, the herbicide reduced the growth of both varieties. A significant selective effect was found at 96 μM. Measurements of chlorophyll fluorescence-induction kinetics allowed to discriminate between the two varieties treated with 12 to 48 μM chlortoluron. The metabolism of chlortoluron was studied following absorption of 24 μM solutions. Both varieties produced the same pattern of metabolites but the tolerant variety degraded the herbicide and the phytotoxic mono-N-demethylated metabolite at a slightly higher rate. An unexpected result was that the more susceptible variety possessed a very significant ability to metabolize chlortoluron. In conclusion, it appears that further studies are necessary before deciding whether the differences in susceptibility of the two varieties can be explained by the only metabolic factor.     

The metabolism of chlorotoluron in the rat

The metabolic fate of ¹⁴C-labeled chlorotoluron, i.e., 1-(3-chloro-4-methyl[⁴C]-phenyl)-3,3-dimethyl urea, was followed in rats. After a single oral dose the radioactivity was preferably excreted with the urine. Nine of the eleven urinary metabolites isolated, were identified by spectroscopic and derivatization techniques, whereas the structure of the remaining two metabolites was only partially elucidated. N-Demethylation and stepwise oxidation of the ring methyl group to hydroxymethyl and carboxyl derivatives were found as the major metabolic mechanisms. Both mechanisms proceeded simultaneously so that the isolated metabolites showed all combinations of N-demethylation and ring methyl group oxidation in their structures. One of these metabolites was an N-formyl derivative, being probably an intermediate product of demethylation. In the urine of rats fed doses of [¹⁴C]chlorotoluron higher than 50 mg/kg three additional metabolites with different degrees of N-dealkylation were found, the ring methyl group of which was transformed to a methylthio methyl group. The metabolites identified in the faeces were of the same type as those found in the urine. Based on the structures of the metabolites elucidated, a metabolic pathway of chlorotoluron in the rat is presented.     

Oxidative degradation of chlortoluron,propiconazole, and metalaxyl in suspension cultures of various crop plants

The metabolism of chlortoluron, (N′-(3-chloro-4-methylphenyl)-N,N-dimethylurea), propiconazole (1-[2-(2′,4′-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl-methyl]-1H-1,2,4-triazole), and metalaxyl (dl-N-(2,6-dimethylphenyl)-N-(2′-methoxyacetyl) alanine methyl ester) was investigated in suspension cultures of crop species for which differences in metabolism had been demonstrated at the intact plant level. Uptake and metabolism of chlortoluron by cultures of Italian rye-grass (Lolium multiflorum) was slow, the metabolites detected (42% of applied radioactivity after 13 days) being products of ring methyl oxidation and N-monodealkylation reactions. In sharp contrast, metabolism in a cotton (Gossypium hirsutum) suspension culture was extremely fast (72% after 4 hr) and was attributed to extensive N-didealkylation in addition to rapid ring methyl oxidation. This rate of metabolism also implied a very rapid uptake of chlortoluron by the cotton culture. ¹⁴C-labelled propiconazole became rapidly associated with cells of wheat (Triticum aestivum) and rice (Oryza sativa) following treatment of suspension cultures. Uptake was initially more rapid in the rice culture (36% of applied radioactivity after 8 h compared to 19% for wheat) which, together with a slower rate of metabolism, resulted in more unchanged propiconazole being associated with rice cells. On a parts-per-million basis these results indicated an apparent 10-fold accumulation of propiconazole in rice cells compared to 5-fold in the wheat culture. Propiconazole metabolite patterns were similar in cultures of both species and indicated side-chain hydroxylation as the principal pathway. Uptake of metalaxyl was slow in suspension cultures of both lettuce and potato (≅ 20% after 17 days). Subsequent metabolism was also slow but appeared to be limited by the poor rate of uptake. Both cultures were found to be similarly versatile with respect to metabolic attack on metalaxyl, which included ring methyl hydroxylation, aryl hydroxylation, ester cleavage, ether cleavage (O-dealkylation), and N-dealkylation (side-chain cleavage), the hydroxylation reactions being quatitatively the more important. The results for all three pesticides are compared to those obtained previously from studies with intact plants of the same species.     

Biotransformation of dimethoate by cell culture systems

Dimethoate [O,O-dimethyl S-(N-methylcarbamoylmethyl) phosphorodithioate] was oxidatively metabolized by primary human embryonic lung cells in culture. Over 95% of the recovered radioactivity after incubation with ¹⁴C-labeled dimethoate resulted from oxidative metabolites, with the remainder being water soluble. Thus, oxidative metabolism of dimethoate predominated over hydrolytic metabolism in the cell culture system, in contrast to the whole rat where the opposite is true. The sequence of reactions was similar to that found in rats. Dimethoate was desulfurated to yield dimethoxon and both compounds were N-demethylated. Metabolism of dimethoate in mouse fibroblast L-929 cell cultures revealed up to 35% of dimethoate carboxylic acid as the only compound other than dimethoate present.     

Enzyme activities and cytochrome P-450 levels of some Japanese quail tissues with respect to their metabolism of several pesticides

In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl-N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)-N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)-N′-methylurea.     

Behavior of 14C oryzalin in soil and plants

Radiochemical studies of field soil treated with ¹⁴C oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) indicated that the compound was readily degradable. One year after soil treatment with oryzalin, 45% of the original radioactivity had dissipated, 25% was extractable, and 30% was “soil bound”. The extractable fraction contained oryzalin and several degradation products, some of which were isolated and identified. No single degradation product accounted for more than 3% of the applied oryzalin. The “soil-bound” radioactivity was extractable with hot alkali. No significant radioactive residues were detectable in either seed or forage of soybean and wheat plants. No specific metabolites of oryzalin were identified in soybean plants. Trace amounts of radioactivity found in plant tissue appeared to be associated with the various plant constituents.     

Chlorotoluron Metabolism in Leaves of Resistant and Susceptible Biotypes of the Grass Weed Alopecurus myosuroides

The metabolism of the herbicide chlorotoluron by susceptible and resistant biotypes of the grass weed, Alopecurus myosuroides, was examined. After administration of radiolabelled herbicide to leaves, metabolites were extracted and analysed. The metabolites identified consisted of mono-demethylated-, di-demethylated- and ring methyl-hydroxylated chlorotoluron. Metabolism was more extensive in the resistant biotype, yielding principally the non-phytotoxic ring methyl-hydroxylated metabolite. The metabolites observed are characteristic of the activity of cytochrome P450 mixed-function oxygenase action. The specific cytochrome P450 inhibitor, 1-aminobenzotriazole, reduced accumulation of the ring methyl-hydroxylated metabolite in the resistant biotype.     

Metabolism of N-hydroxymethyl dimethoate and N-desmethyl dimethoate in bean plants

N-Hydroxymethyl [carbonyl-¹⁴C] dimethoate (0.43 ppm) and N-desmethyl [carbonyl-¹⁴C] dimethoate (0.50 ppm) were stem-injected into bean plants (Phaseolus vulgaris) in two separate experiments. Plants were harvested periodically, extracted, fractionated, and analyzed for metabolites. The resulting pattern of metabolites formed from the administration of these two compounds was different. Radioactivity was not detected in the organic fraction 2 days after N-desmethyl dimethoate administration. N-Desmethyl dimethoate was rapidly broken down to dimethoate carboxylic acid and other polar metabolites, then further degraded into materials which became part of the plant constituents. N-Hydroxymethyl dimethoate was quite stable in the plant. Most of the material not remaining as parent became rapidly conjugated and constant levels of conjugate were maintained. Very little radioactivity was bound in the plant marc. The metabolic pathway of these compounds is as follows: N-hydroxymethyl to the glucoside or N-desmethyl derivative; the N-desmethyl metabolite degrades primarily to the carboxylic acid but also to N-desmethyl dimethoxon, either of which in turn may be degraded to dimethoxon carboxylic acid. The conversion of -NHCH₂OH to -NH₂ is a slow reaction so that conjugation becomes the route of choice when the plant is treated with N-hydroxymethyl dimethoate.     

The metabolism of the herbicide flamprop-isopropyl in barley

The metabolism of the wild oat herbicide, flamprop-isopropyl, [Barnon, isopropyl (±) N-benzoyl-N-(3-chloro-4-fluorophenyl)-2-aminopropionate] in barley grown to maturity has been examined under glass-house and outdoor conditions. [¹⁴C]Flamprop-isopropyl labeled separately in two positions was used. The major metabolic route of the herbicide was by hydrolysis to the corresponding carboxylic acid, II, which occurred in free and conjugated forms. Flamprop-isopropyl also underwent hydroxylation in the 3 and 4 positions of the benzoyl group, and the 3-hydroxybenzoyl analogue of II was detected. The hydroxylated metabolites were also present in the plants as conjugates. Additional minor metabolites detected only in glass-house samples were N-benzoyl-3-chloro-4-fluoroaniline, 2-[3-chloro-4-fluorophenylamino]-propionic acid, and benzoic acid. The soil in which the plants were grown received part of the spray application of the herbicide. Residues in the 0–10-cm layer at barley harvest comprised the unchanged herbicide, the carboxylic acid II, and unidentified polar material.     

The interaction between planting depth of four winter wheat cultivars, Alopecurus myosuroides Huds. and Bromus sterilis L. and their susceptibility to post-emergence applications of isoproturon and chlorotoluron

Seeds of four winter wheat cultivars, Slejpner, Galahad, Avalon and Penman, were sown at depths ranging from 6–75 mm in soil in pots, and isoproturon or chlorotoluron was then applied to the soil surface. For chlorotoluron-treated plants (both pre- and post-emergence) the dose required to produce a 50% effect (ED₅₀) was unaffected by depth of planting. In contrast, for isoproturon applied pre-emergence, the ED₅₀ for both Avalon and Slejpner was strongly affected by sowing depth. Although chlorotoluron was much more active in a second experiment when applied post-emergence to Slejpner wheat, the ED₅₀ for both herbicides increased with greater depth of sowing. Protection of wheat from isoproturon damage by deeper planting was enhanced if the adsorption capacity of the soil was raised from K_d 0.5 to 2.0 by incorporation of activated charcoal in the soil. Isoproturon entry into plants (as measured by the effect on rate of photosynthesis) was slower in those that had been sown deeper and were growing in more adsorptive soils, but there was no obvious relationship between these observations and isoproturon distribution in the soil profile. In nutrient culture the four wheat cultivars responded similarly to a range of doses of isoproturon. The chlorotoluron-sensitive cultivars, Slejpner and Galahad, were damaged by much lower doses of chlorotoluron than were Avalon and Penman. Bromus sterilis L. responded similarly to wheat with regard to its interaction with isoproturon and planting depth. Alopecurus myosuroides Huds., however, was less damaged by isoproturon when the zone above the seed was protected from the herbicide by growing the shoot through a plastic straw.     

Inhibitory effect of isoprothiolane on metabolism of a phosphoramidate by isolates of Pyricularia oryzae cav. in relation to fungicide sensitivity

The inhibitory effect of isoprothiolane(diisopropyl 1,3-dithiolan-2-ylidenemalonate), a fungicide for rice blast control, on the metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by 20 isolates of Pyricularia oryzae was examined in relation to sensitivity of the isolates to the reference fungicide IBP(S-benzyl diisopropylphosphorothiolate). The isolates were divided into five groups based on the modes of BPA metabolism and the inhibition of BPA metabolism by isoprothiolane. Every isolate in groups I and II, which was either a field isolate or a stock culture, decomposed BPA rapidly and produced both hydroxylated and N-demethylated BPA as metabolites. BPA decomposition by these isolates was strongly inhibited by isoprothiolane, resulting in the decreased production of both metabolites in group I and of the hydroxylated metabolite in group II. These isolates were almost equally sensitive to isoprothiolane. Isolates in groups III, IV, and V were all obtained from selection of the fungus mutants found growing on media containing isoprothiolane. Isolates in group III, derived by plating large numbers of conidia, did not decompose BPA to any extent. Mutants of groups IV and V were obtained from fast-growing sectors on agar containing isoprothiolane. Both these groups decomposed BPA, but isolates belonging to group IV produced copious amount of N-demethylated BPA whereas isolates in group V did not. BPA metabolism by these in vitro mutants in groups III, IV, and V was not inhibited by isoprothiolane. Thus, the inhibitory effect of isoprothiolane on BPA metabolism was correlated with sensitivity of an isolate to isoprothiolane. The inhibitory effect of IBP on BPA metabolism was not always correlated with the sensitivity of an isolate to IBP.     

Metabolism of bromoxynil octanoate in growing wheat

[¹⁴C]ring-Bromoxynil octanoate was applied to the leaves of wheat seedlings, which were cultivated in a growth cabinet under controlled conditions for 14 days. Fractionation of the metabolites present in the treated leaves, which accounted for about 63% of the radioactivity applied, indicated a complex metabolic pathway resulting from initial hydrolysis to free bromoxynil, followed by three consecutive or concurrent steps (a) hydrolysis of the cyano group to the amide and carboxylic acid, followed by decarboxylation to 2,6-dibromophenol (0.5% of the ¹⁴C applied), (b) replacement of one or both bromine atoms by hydroxy groups to 3-bromo-4,5-dihydroxybenzonitrile (1.3 %) and 3,4,5-trihydroxybenzo-nitrile (0.6 %) or their hydrolysis products, (c) replacement of one or both bromine atoms by hydrogen, giving 3-bromo-4-hydroxybenzonitrile (1.9 %) and 4-hydroxy-benzonitrile (0.6%) or their hydrolysis products. Some of the phenolic acids or phenols formed are natural plant constituents. The metabolites identified represented in all about 11 % of the herbicide applied, but no individual metabolite accounted for more than a small proportion of it.     

Mercapturic acid formation in the metabolism of propachlor,CDAA, and fluorodifen in the rat

When [¹⁴C]F₃-fluorodifen (2,4′-dinitro-4-trifluoromethyl diphenylether), carbonyl-[¹⁴C]CDAA (N,N-diallyl-2-chloroacetamide), and carbonyl-¹⁴C-propachlor (2-chloro-N-isopropylacetanilide) were fed to rats, 57 to 86% of the ¹⁴C was excreted via the urine within 48 hr. Although very little radioactivity was excreted in the feces of CDAA-treated rats, 15–22% of the ¹⁴C was excreted in the feces of propachlor- of fluorodifentreated rats and an average of 8% of the ¹⁴C remained in these rats 48 hr after treatment. Oxidation of the ¹⁴C label to [¹⁴C]O₂ was not a major process in the metabolism of these herbicides. The only major radioactive metabolite present in the 24-h urine of fluorodifen-treated rats, 2-nitro-4-trifluoromethylphenyl mercapturic acid, accounted for 41% of the administered dose of ¹⁴C. In the metabolism of CDAA, the corresponding mercapturic acid accounted for 76% of the dose; it was the only major metabolite present in the 24-h urine. In contrast, three major metabolites were detected in the 24-h urine of propachlortreated rats, and the mercapturic acid accounted for only 20% of the dose. The mercapturic acid of each herbicide was identified by mass spectrometry.     

Metolachlor (2-chloro-N-[2-ethyl-6-methylphenyl]-N-[2-methoxy-l-methylethyl] acetamide) and the metolachlor safener CGA 43089 [α-(cyanomethoximino)-benzacetonitrile] in sorghum seedlings: Correlations between morphological effects and ethylene formation

Treatment of germinating sorghum [Sorghum bicolor (L.) Moench] seeds with the grass herbicide, metolachlor (2-chloro-N-[2-ethyl-6-methylphenyl]-N-[2-methoxy-1-methylethyl] acetamide), causes growth retardation, promoted by thickening of the first leaf and thus inhibition of unfolding of secondary leaves, and increased ethylene production. Sorghum seeds pretreated with the safener CGA 43089 [α-(cyanomethoximino)-benzacetonitrile] exhibit neither morphological deformations nor ethylene production upon metolachlor treatment. Aminoethoxyvinylglycine [l-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid], a specific inhibitor of ethylene formation in higher plants, decreases ethylene formation by metolachlor-treated sorghum seedlings; the observed deformations, however, remain unchanged. Sorghum control seedlings which grow against a covering plate build up ethylene concentrations as after herbicide treatment, but without induction of the morphological symptoms. These observations suggest that the plant hormone ethylene is a symptom and not the inducer of the morphological effects visible after metolachlor treatment of sorghum seedlings.     

Conversion of [14C]buturon in soil and leaching water under outdoor conditions

[¹⁴C]Buturon, a urea herbicide, was sprayed on soil and winter wheat as an aqueous formulation (2.98 kg/ha) under outdoor conditions. Three months after application, a total of 49.2% of the applied radiocarbon was recovered: 46.9% in the soil, 0.3% in the leaching water (depth > 50 cm), and 2.0% in the plants. Radioactive residues in the soil were distributed to a depth of 50 cm and decreased with increasing depth of the soil. An average of 47% of the radioactivity present in the soil could be extracted with cold chloroform; by this extraction method, the formation of artefacts was avoided. Between one and two thirds of the extracted radioactivity was unchanged buturon. In the soil extracts, the following eight conversion products were isolated and identified by combined gas chromatography/mass spectrometry: N-(p-chlorophenyl)-N-methyl-O-methyl carbamate; N-(p-chlorophenyl)-O-methyl carbamate; N-(p-chlorophenyl)-N′-methyl-N′-isobutenyl-urea; N-(p-chlorophenyl)-N′-methyl-urea, N-(p-chlorophenyl)-N′-methyl-N′-isobutenylol-urea; p-chloroaniline in “biologically bound” form; N-(p-chlorophenyl)-N′-methyl-N′-methoxyisobutenyl-urea; and N-(p-chlorophenyl)-N′-methyl-N′-ethoxyisobutenyl-urea. In the leaching water, which contained only 0.005–0.006 mg/liter of radioactive substances, the following three conversion products were isolated and identified by gas chromatography/mass spectrometry: p-chloroformanilide; N-(p-chlorophenyl)-N-methyl-O-methyl carbamate; and an N-hydroxyphenyl-N′-methyl-N′-isobutinyl-urea. The results are discussed in relation to the factors responsible for the formation of these products.     

The degradation of the herbicide benzoylprop ethyl on the foliage of cereal seedlings

The breakdown of the herbicide benzoylprop ethyl [SUFFIX, ethyl N-benzoyl-N-(3,4-dichlorophenyl)-2-aminopropionate] has been examined in wheat, oat, and barley seedlings after application of ¹⁴C-labeled herbicide to the foliage.Within 15 days of the application the route and rate of the breakdown were similar in the plants of all three species. Some of the herbicide was present in the plants in a complexed form which could be extracted from the plant with organic solvents and converted back into the herbicide on treatment with hot acid. Evidence was obtained for hydrolysis of the herbicide in the plant to give its des-ethyl analog which conjugated with plant sugars. There was some evidence for a small degree of degradation of benzoylprop ethyl by debenzoylation to give products which also conjugated or complexed.There was no evidence for the formation of 3,4-dichloroaniline in the plants.     

Metabolism of isopropyl carbanilate (propham) in alfalfa grown in nutrient solution

Alfalfa plants, Moapa variety, were grown in nutrient solution containing isopropylring-[¹⁴C] carbanilate (43.8 μCi/liter propham). After 8 days, 41.2% of the radioactivity initially added to the nutrient culture was recovered; 10.9% of this was from shoots, 3.4% from roots and 26.9% from nutrient medium. Nonextracted residues accounted for 23% of the radioactivity in shoots and 62% of that in roots. The parent herbicide constituted 53 and 38% of the radioactivity extracted from shoots and roots, respectively. The balance of extracted ¹⁴C was polar metabolites which were purified and subjected to enzymatic and acid hydrolysis. Four aglycones were isolated, three of which were purified by thin-layer chromatography and characterized by mass spectrometry. The principal aglycones were: isopropyl-2-hydroxycarbanilate, isopropyl-4-hydroxycarbanilate, and 1-hydroxy-2-propylcarbanilate. The fourth aglycone was not identified.     

Behaviour of the herbicide EL-107 in wheat and rape grown under controlled conditions

The behaviour of ¹⁴C-EL-107 has been evaluated in winter wheat and rape, which are tolerant and susceptible, respectively, under field conditions. After 10- to 13-days’growth under controlled conditions, seedlings were allowed to absorb the herbicide through the roots. Two experiments were conducted to study the absorption and the metabolism of EL-107. Absorption was estimated during a 5-day treatment at the rate of 1–47 μM, and metabolism was studied after a 1-day treatment at 14.7 μM. The results showed that (i) rape plants absorbed more herbicide than wheat, and translocated less radioactivity into their shoots, and (ii) the metabolism of EL-107 proceeded actively only in the shoots, where EL-107 disappeared at similar rates in the two species, giving rise to the same metabolites. In conclusion, the respective degrees of susceptibility of the two species could be partly related to differences in the concentration of the herbicide in the roots, where it can exert its phytotoxic effect.     

DETOXIFICATION OF FLUOMETURON BY CITRUSTISSUES*

Summary. The metabolism of [¹⁴C]fluometuron in Citrus was studied by feeding the herbicide to either young seedlings or to excised organs. Most of the uptake of fluometuron occurred via the roots during the first 24 h and radioactivity was found after 16 days to be in the rootlets (36·5%), mainroot (34·5%), stem (13·7)% and leaves (15·2%). By feeding [¹⁴C]fluometuron to excised organs it was established that although most of the fluometuron breakdown occurred in the rootlets, other plant parts were also capable of metabolizing the herbicide. Therefore, the presence of metabolites in the upper plant organs was not entirely due to translocation from the rootlets. These results suggest that the resistance of Citrus to fluometuron is due to its breakdown in the tissues, probably induced by an N-demethylase enzyme system, similar to that reported for cotton (Frear, Swanson & Tanaka, 1969), in which harmless metabolites arc formed. Détoxification du fluométuron par les tissus de Citrus     

Metabolism of chlorsulfuron by plants: Biological basis for selectivity of a new herbicide for cereals

A major factor responsible for the selectivity of chlorsulfuron [2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide] (formerly DPX-4189), as a postemergence herbicide for small grains is the ability of the crop plants to metabolize the herbicide. Chlorsulfuron is the active ingredient in Du Pont “Glean” weed killer. Tolerant plants such as wheat, oats, and barley rapidly metabolize chlorsulfuron to a polar, inactive product. This metabolite has been characterized as the O-glycoside of chlorsulfuron in which the phenyl ring has undergone hydroxylation followed by conjugation with a carbohydrate moiety. Sensitive broadleaf plants show little or no metabolism of chlorsulfuron.