Changes in composition of chlorophylls,carotenoids, and prenylquinones in green seedlings of Hordeum and Raphanus induced by the herbicide San 6706—an effect possibly antagonistic to phytochrome action

The substituted pyridazinone herbicide San 6706 (4-chloro-5-(dimethylamino)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone) inhibits accumulation of chlorophylls and carotenoids to about the same degree in Hordeum and Raphanus seedlings under continuous illumination. Stronger inhibition of pigment accumulation in general is correlated with a stronger inhibition of the prenylquinones plastoquinone-9,α-tocopherol, and α-tocoquinone; but the amounts of inhibition are much lower for the prenylquinones. Such an inhibition pattern, which is observed in the two plants of different ages and when different herbicide concentrations are applied, points to a site of action which regulates the biosynthesis of these prenyllipids together. There is a different degree in the change of the relative proportions (percentages of herbicide-treated plants as related to the respective control values) of the single carotenoids induced by the herbicide. In this sense there was the highest increase for zeaxanthin and lowest for β-carotene both in Hordeum and Raphanus. The order of relative change of the carotenoids analyzed is about the same as in etiolated barley seedlings of equal age illuminated with white light—but with an opposite sign. The relative proportions of the benzoquinones might be changed in an analogous way. It is suggested, that with respect to carotenoid synthesis and perhaps also benzoquinone synthesis San 6706 acts on the same reaction chain like phytochrome but in an antagonistic way, possibly at the cytoplasmic ribosomes.     

Photoreceptors and respiratory electron flow involvement in the activity of acifluorfen-methyl and LS 82-556 on nonchlorophyllous soybean cells

The diphenyl ether acifluorfen-methyl [AFM; methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate] and the pyridine derivative LS 82-556 [(S)-3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] induce light-dependent polyunsaturated fatty acid peroxidation, leading to general membrane disruption. Although devoid of functional chloroplasts, cultured soybean cells are sensitive to AFM and LS 82-556 only in the light. The possible involvement of carotenoids and respiratory electron flow was examined by monitoring ethane evolution, fluorescein release, and dry weight/fresh weight ratio alteration. Herbicide effects on cells exposed to white light or blue light (380–540 nm) remain unchanged when the cells are deprived of carotenoids by treatment with norflurazon [4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H)-pyridazinone]. Treatment with antimycin A reduced sensitivity to AFM and LS 82-556. In nonchlorophyllous cells, chromophores, other than carotenoids, absorbing between 380 and 540 nm are involved as photoreceptors. Furthermore, respiratory electron flow also participates in the toxic process.     

Mitochondrial involvement in the mode of action of acifluorfen

The herbicidal action of acifluorfen {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid} was studied with greened and expanded discs from cotyledons of cucumber (Cucumis sativus L.). Discs were floated on various treatment solutions for 20 hr in darkness before exposure to 400 μE m^?2 sec^?1 of white light. Herbicide damage, as measured by electrolyte leakage, began in the light after a 1- to 2-hr lag period. Cytochemical methods at the ultrastructural level indicated that acifluorfen caused marked increases in production of superoxide radical and hydrogen peroxide in the mitochondrion, but not in the plastid. The mitochondrial inhibitors antimycin A, rotenone, CCCP, and DNP antagonized the action of acifluorfen, lengthening the lag period and reducing the rate of electrolyte leakage. Ethanol, α-tocopherol, N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N′-phenylurea, and copper-penicillin also lengthened the lag phase and slowed the rate of damage, indicating that acifluorfen damage involves toxic oxygen species. PS II-inhibiting levels of DCMU, atrazine, or bentazon did not affect acifluorfen-induced ion leakage. Yellow tissue produced by treatment with tentoxin was supersensitive to acifluorfen, but white tissue produced by treatment with norflurazon was relatively insensitive. These data indicate that, after an initial carotenoid-acifluorfen interaction, the mitochondrion is involved in production of toxic oxygen species and that this process is closely tied to the mechanism of action of this herbicide.     

S-cysteinyl-hydroxychlorpropham transferase inhibition by chlorpropham analogs,sulfhydryl compounds,ortho- and/or para-substituted phenols,and firefly luciferin

Inhibition of S-cysteinyl-hydroxychlorpropham transferase from oat (Avena sativa L.) by various compounds was studied. The β-O-glucoside of the substrate, isopropyl-3′-chloro-4′-hydroxycarbanilate (4-hydroxychlorpropham), and isopropyl-3′-chlorocarbanilate (chlorpropham) did not inhibit the enzyme. Isopropyl-5′-chloro-2′-hydroxycarbanilate (2-hydroxy-5-chlorpropham), was a competitive inhibitor with respect to 4-hydroxychlorpropham, but 2-β-O-glucosyl-5-chlorpropham was not an inhibitor. The inhibition patterns exhibited by 2-hydroxy-5-chlorpropham and other aryl-hydroxylated analogs suggested that the site of aryl-cysteine thioether conjugation might be the ortho (2′) aromatic carbon. Inhibitions by 3-chloro-4-hydroxyaniline and ferulic acid suggest that related phenols and/or naturally occurring phenolic plant acids could serve as substrates for the enzyme system. Glutathione was a competitive inhibitor with respect to cysteine and could also form a conjugate with 4-hydroxychlorpropham. Atypical inhibitions of cysteine conjugation by cysteine ethyl ester or firefly d-luciferin were described. Similarities between S-cysteinyl-hydroxychlorpropham transferase and firefly luciferase were noted.     

Potential involvement of alkoxyl and hydroxyl radicals in the peroxidative action of oxyfluorfen

The potential involvement of hydroxyl and alkoxyl radicals in the peroxidative action of the p-nitro diphenyl ether herbicides acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxyl]-2-nitrobenzoic acid), acifluorfen-methyl (methyl ester of acifluorfen), nitrofen [2,4-dichloro-1-(4-nitrophenoxy)benzene], nitrofluorfen [2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene], and oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene] was evaluated under laboratory conditions. Methional was added to illuminated thylakoids from peas (Pisum sativum L., cv Little Marvel) and its oxidation to ethylene was used as an indicator of hydroxyl and alkoxyl radical production. Oxyfluorfen stimulated the rate of methional oxidation by 138% at 10 μM and 175% at 1 mM. This oxyfluorfen-induced stimulation of the rate of methional oxidation was dependent on light, photosynthetic electron transport, and hydrogen peroxide since it was not observed under dark conditions or in the presence of DCMU and catalase. Addition of Fe-EDTA, a catalyst of the Fenton reaction, stimulated the oxyfluorfen-induced enhancement of methional oxidation sixfold, suggesting that hydroxyl radicals are synthesized through a Fenton reaction. Acifluorfen, nitrofen, and nitrofluorfen inhibited the rate of methional oxidation whereas acifluorfen-methyl had no effect on the rate of methional oxidation, even at high concentrations (1 mM). Nitrofluorfen at 1 mM was the only p-nitro diphenyl ether herbicide tested to inhibit photosynthetic electron transport of pea thylakoids. In experiments with pea leaf disks, acifluorfen at low concentrations stimulated the rate of methional oxidation, whereas acifluorfen-methyl, nitrofen, and nitrofluorfen had no effect. These data indicate that hydroxyl and alkoxyl radicals could be involved in the mechanism of cellular damage caused by oxyfluorfen but they are not important for the activity of the diphenyl ether herbicides acifluorfen, acifluorfen-methyl, nitrofen, and nitrofluorfen.     

The inhibitory mode of action of the pyridazinone herbicide norflurazon on a cell-free carotenogenic enzyme system

The inhibition site of the phenylpyridazinone herbicide, norflurazon [SAN 9789, 4-chloro-5-(methylamino)-2-(3-trifluoromethylphenyl)-pyridazin-3(2H)one] was determined in a cell-free carotenogenic enzyme system from a mutant strain of Phycomyces blakesleeanus (Mucoraceae). The presence of norflurazon resulted in a reduced flow of radioactivity from [2-¹⁴C]mevalonic acid to phytoene (7,8,11,12,7′,8′,11′,12′-octahydro-ψ,ψ-carotene) and β-carotene (β,β-carotene), whereas an increased incorporation occurred in the C₃₀ terpenoids, squalene, and ergosterol. Furthermore, radioactivity accumulated in geranylgeranyl pyrophosphate. Since no radioactivity was found in prephytoene pyrophosphate and the radioactivity in phytoene decreased upon addition of norflurazon, this herbicide exerts its primary inhibitory action on the reaction catalyzed by phytoene synthetase. The nonbleaching phenylpyridazinone BAS 13761 [4-chloro-5-methoxy-2-phenyl-pyridazin-3(2H)-one] did not show this effect. Other inhibitory sites of norflurazon, either on prenyl pyrophosphate synthetase or on the desaturation of phytoene, were excluded.     

Degradation of acylanilide pesticides by Aspergillus niger

Aspergillus niger converts the herbicide 3′-chloro-2-methyl-p-valerotoluidide (solan) to 3′-chloro-4′-methylacetanilide and the fungicide 2,5-dimethylfuran-3-carboxanilide to acetanilide. The metabolites were formed by hydrolysis with an aryl acylamidase, followed by subsequent acetylation resulting in the corresponding acetanilides. Their structures were elucidated by mass spectrometric analysis and confirmed by comparison with synthetic compounds.     

S-cysteinyl-hydroxychlorpropham: Formation of the S-cysteinyl conjugate of isopropyl-3′-chloro-4′-hydroxycarbanilate in oat (Avena sativa L.)

Isopropyl-3′-chlorocarbanilate (chlorpropham) forms phenolic metabolites, isopropyl-3′-chloro-4′-hydroxycarbanilate (I), and isopropyl-5′-chloro-2′-hydroxycarbanilate (II), in several plant species. In oat, which is a chlorpropham-susceptible plant, I was converted to an S-cysteinyl-conjugate (III). The reaction in vitro was catalyzed by a partially purified, soluble enzyme. The formation of III by the enzyme preparation and by oat shoot sections was compared. Mass spectral data indicated the presence of an aryl-thioether bond, and chloro-, hydroxy-, and isopropylcarbanilate groups in III. The results of this investigation indicate that III was isopropyl-[(2-amino-2-carboxyethyl)thio]-chloro-hydroxycarbanilate (S-cysteinyl-hydroxychlorpropham).     

Partial purification and properties of S-cysteinyl-hydroxychlorpropham transferase from oat (Avena sativa L.)

S-Cysteinyl and glutathione conjugates of isopropyl-3′-chloro-4′-hydroxycarbanilate (4-hydroxychlorpropham) were synthesized directly in the presence of soluble enzyme systems isolated from etiolated shoots of oat seedlings. The enzyme systems responsible for these reactions were partially purified and charaterized. Enzyme A appeared to be a multicomponent system, equally reactive with either cysteine or glutathione. Enzyme B was twice as active as enzyme A in the formation of S-cysteinyl-hydroxychlorpropham. Affinity chromatography of enzyme A produced an enzyme fraction with properties similar to those of enzyme B. Both enzymes (A and B) were significantly inhibited by increased cysteine concentrations. The reaction of glutathione with enzyme B was limited. However, when low concentrations of a nonreacting effector, cysteine ethyl ether, were added, glutathione conjugation increased significantly. At higher concentrations, cysteine ethyl ester formed a conjugate with 4-hydroxychlorpropham. Isopropyl-5′-chloro-2′-hydroxycarbanilate (2-hydroxy-5-chlorpropham) did not conjugate with either cysteine or glutathione but did react with cysteine ethyl ester. Isopropyl-3′-chlorocarbanilate (chlorpropham) was not a substrate for thioether conjugation. These data suggest that para- and/or ortho-hydroxylated carbanilates and cysteine-related substrates may form thioether conjugates when incubated under appropriate conditions with these complex enzyme systems.     

Expression level of specific isozymes of maize catalase mutants influences other antioxidants on norflurazon-induced oxidative stress

The combined action of catalase (CAT) and superoxide dismutase (SOD) is critical in mitigating the effects of oxidative stress. The standard maize (Zea mays) CAT line (W64A), CAT-2/CAT-3 double null mutant (WDN), and high CAT-2 activity mutant (R6-67) were used to examine whether absent or high expression of specific CAT isozymes influences the levels of SOD isozymes or non-enzymic antioxidant carotenoids, in response to norflurazon (NF)-induced oxidative stress. In leaves and mesocotyls of NF-treated W64A, there was a significant reduction in carotenoid, chlorophyll, and total CAT activity, but an increase in total SOD activity in leaves and an increase in anthocyanin in mesocotyls. W64A and WDN showed a great decrease in carotenoids and chlorophylls, while R6-67 sustained carotenoid level high and remained dark green at low NF concentrations. WDN had a lower CAT activity than W64A in control tissues and its CAT activity decreased further in response to NF, whereas R6-67 showed a smaller decline in CAT activity. These results suggest the critical role of CAT-2 isozyme in NF-induced oxidative stress. Total SOD activity increased slightly in response to NF in leaves of W64A, but remained almost constant in leaves of other lines and in mesocotyls of all the maize lines examined. Chloroplastic SOD-1, a dominant pathway during NF-induced enzymatic scavenging in W64A, did not increase in leaves of NF-treated WDN and R6-67. These results demonstrate that absent or high CAT activity influences the levels of photoprotective carotenoids and SOD in response to NF-induced oxidative stress.     

Characterization of herbicidal injury by acifluorfen-methyl in excised cucumber (Cucumis sativus L.) cotyledons

Initial signs of herbicidal injury by several diphenyl ether herbicides were monitored by following the efflux of ⁸⁶Rb⁺ from treated cucumber (Cucumis sativis L.) cotyledons after exposure to light (600 μE m^?2 sec^?1; measured as PAR, i.e., photosynthetically active radiation between 400 and 700 nm). This very sensitive, rapid, and quantitative bioassay proved quite useful in (a) a structure-activity correlations study of the diphenyl ether compounds investigated and (b) an examination of herbicidal characteristics. The following diphenyl ether herbicides were analyzed: acifluorfen, sodium 5-[2-chloro-4-(trifluormethyl)phenoxy]-2-nitrobenzoate; acifluorfen-methyl (MC-10108), methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate; bifenox, methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate; nitrofen, 2,4-dichlorophenyl p-nitrophenyl ether; nitrofluorfen, 2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene; oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene; MC-7783, potassium 5-(2,4-dichlorophenoxy)-2-nitrobenzoate; and MC-10982, ethyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate. Of the compounds investigated, acifluorfen-methyl (AFM) had the greatest degree of herbicidal activity. Cucumber cotyledons placed in high light (600 μE m^?2 sec^?1; PAR) with 10 nM AFM showed a significant increase in the efflux of ⁸⁶Rb⁺ within 2 to 4 hr. Light was required for herbicidal activity by AFM, and when treated cotyledons were returned to darkness, no further damage to the tissue occurred. By decreasing the quantity of light, the effect of the compound was delayed, although the magnitudes of the responses at the different intensities (600, 300, 150, and 75 μE m^?2 sec^?1; PAR) were nearly equal. By increasing the length of time of dark pretreatment with 1 μM AFM, ⁸⁶Rb⁺ efflux could be detected as early as 10 to 15 min after exposure to light (600 μE m^?2 sec^?1; PAR). Following light activation of AFM there was a simultaneous efflux of ⁸⁶Rb⁺, ³⁶Cl^?, ⁴⁵Ca²⁺, 3-O-methyl-[¹⁴C]glucose, and [¹⁴C]methylamine⁺. These data suggest the initial response to the herbicidal activity of AFM is expressed as a general increase in membrane permeability.     

Diphenyl ether-chloroplast interactions

The diphenyl ethers acifluorfen (sodium-5-[2-chloro-4-(trifluoromethyl)-phenoxyl]-2-nitrobenzoate), acifluorfen-methyl (methyl-5-[2-chloro-4-(trifluoromethyl)-phenoxyl]-2-nitrobenzoate), and oxyfluorfen (2-chloro-1-[3-ethoxy-4-nitrophenoxy]-4-(trifluoromethyl)benzene have an absolute light requirement for herbicidal activity. CO₂-dependent O₂ evolution was inhibited in leaf disks obtained from 5-week-old spinach plants as a result of incubation in the light in the presence of each of the three diphenyl ethers. I₅₀'s were determined for inhibition by the diphenyl ethers of CO₂-dependent O₂ evolution in intact chloroplasts obtained from three species of varying susceptibilities (spinach, coffeeweed, and pea). Rankings obtained correlated well with relative susceptibilities and with relative effectiveness of the three compounds tested. Coupled and uncoupled photosynthetic electron transport in susceptible species were unaffected by the three compounds at concentrations in the I₅₀ range. Exposure to herbicidally inactive isomeric analogs of oxyfluorfen and acifluorfen did not affect photosynthesis in leaf disks but was effective in inhibiting photosynthesis in isolated chloroplasts. Photosynthetic abilities of intact tissue were not affected by herbicide treatment in red light. Red light was, however, as effective as white light in mediating the inhibition of photosynthesis in isolated intact chloroplasts by diphenyl ethers. The existence of two photoreceptors for diphenyl ether action, one located at the chloroplast envelope and a second outside of the chloroplast, is suggested as a possible basis for these findings.     

Correlation between structure and phytotoxic activities of nitrodiphenyl ethers

Various nitrodiphenyl ethers were assayed using a cell-free photosynthetic system and intact microalgae to approach a correlation between chemical structure and their effect upon three biochemical processes: (1) linear photosynthetic electron transport (Hill reaction); (2) photophosphorylation (energy-transfer inhibition by affecting the ATP synthetase); and (3) light-induced peroxidative degradation of thylakoid lipids (formation of short-chain hydrocarbons). Diphenyl ethers with a p-nitro group at one phenyl ring and one or two chloro substituents at the other exhibit energy-transfer inhibition. Analogs with different substituents are ineffective. A moderate electron-transport inhibition is seen with almost all derivatives. The most obvious phytotoxic effect is the occurrence of light-induced peroxidation (of polyunsaturated fatty acids) by p-nitro- and p-nitrosodiphenyl ethers. Substituents such as -OCH₃, -OC₂H₅, -NHC₂H₅, or -COOCH₃, -CONHCH₃ next to the p-nitro group enhance peroxidative activity and are apparently important for stabilization and/or reactivity of a diphenyl ether radical originating through photosynthetic electron flow. Activity is further enhanced by substituents with positive Hammett parameters in ortho or para positions at the phenyl ring not carrying the nitro group.     

The herbicidal mode of action of R-40244 and its absorption by plants

The herbicide R-40244, 1-(m-trifluoromethylphenyl)-3-chloro-4-chloromethyl-2-pyrrolidinone, was studied to elucidate its action and absorption by corn (Zea mays L. DeKalb XL-45A) and other plant species. R-40244 readily induced lead chlorosis in susceptible plants at relatively low rates of application. The leaf chlorosis was found to be related to a reduction in chlorophyll and β-carotene content and an accumulation of the β-carotene precursor, phytoene. The phytotoxic action of R-40244 occurred only under light conditions. R-40244 was readily absorbed by plant roots and translocated to foliar tissues. There were no discernible differences in R-40244 absorption between proadleaf and grassy species. However, uptake studies with eight plant species indicated that foliar accumulation tended to occur in susceptible species and root accumulation predominated in tolerant species.     

Action of R-40244 on chloroplast pigments and polar lipids

The herbicide R-40244 [1-(m-trifluoromethylphenyl)-3-chloro-4-chloromethyl-2-pyrrolidinone] blocked the accumulation of chloroplast pigments in cabbage (Brassica oleracea L.) and annual ryegrass (Lolium multiflorum L.). The total amount of polar (membrane) lipids synthesized and the proportion of unsaturated 18-carbon fatty acids were progressively reduced in shoots of annual ryegrass as the concentration of R-40244 increased from 1 to 100μM. Blocked chloroplast pigment accumulation and perturbations of membrane lipids were related to reduced growth of annual ryegrass. Growth and membrane lipids of cabbage were much less affected by R-40244. Light was not required for the action of R-40244 on membrane lipids. The combined data suggest that the accumulation of chloroplast pigments is blocked by R-40244 in all species sensitive to R-40244, but that species differ in sensitivity to action on membrane lipids. In sensitive species, where both pigments and membrane lipids are affected, the action on membrane lipids may limit growth independently of effects on pigments.     

Effects of various classes of herbicides on four species of algae

Chlorella pyrenoidosa, Chlorococcum sp., Lyngbya sp., and Anabaena variabilis were cultured in Bold's basal medium. They were treated with 0.1, 1.0, and 10 μM concentrations of 2-chloro-2′, 6′-diethyl-N-(methoxymethyl)acetanilide (alachlor), 2-chloro-4-(ethylamino)-6-(tert-butyl-amino)-s-triazine (terbuthylazine), 2-sec-butyl-4,6-dinitrophenol (dinoseb), 1,1-dimethyl-3-(α,α,α-trifluoro-2,6-dinitro-N-propyl-p-toluidine) (profluralin), 2, 4-bis(isopropylamino)-6-(methylthio)-s-triazine (prometryne), and (2,4-dichlorophenoxy)acetic acid (2,4-D). Growth of all algal species tested was markedly reduced by the triazines. Alachlor, dinoseb, and fluometuron inhibited growth of some algae at higher concentrations while 2,4-D and profluralin did not inhibit growth at the concentrations tested. Photosynthesis was greatly inhibited by the triazines, even at the 0.1 μM concentration. Fluometuron was very toxic to the blue-green algae but had less effect on the green algae tested. Lyngbya was most susceptible to photosynthesis reduction by the herbicides. The concentrations of herbicides tested had little effect on respiration of the algae species. It appears that effects on algal growth were due primarily to inhibition of photosynthesis rather than to other metabolic processes.     

Potassium uptake in atrazine-treated roots of sensitive and resistan plants

The action of atrazine and its biodegradation products on the membrane transport of potassium in roots was evaluated in both sensitive and resistant plants. Excised roots of maize and oat showed inhibition of potassium uptake efficiency in the presence of 1.4 × 10^?4M atrazine and 1.4 × 10^?4M deethylated atrazine. Other biodegradation products such as 2-chloro-4-amino-6-ethylamino-1,3,5-triazine,2-chloro-4,6-,bisamino-1,3,5-triazine, and 2-chloro-4-amino-1,3,5-triazine showed no inhibitory effect on the K⁺ uptake capacity. Two maize hybrids showing different uptake efficiency were inhibited differently by atrazine. We suggest that atrazine and deethylated atrazine inhibited the K⁺ transport interacting directly with the plant cell membranes without discerning between resistant and sensitive plants.     

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.     

Nitro free radical formation of diphenyl ether herbicides is not necessary for their toxic action

The diphenyl ether herbicides MC 15608 {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-chloromethylbenzoate} and MC 10878 {5-[2-chloro-4-(trifluoromethyl)phenoxy]methyl benzoate} are structurally similar to acifluorfen-methyl (methyl ester of 5-[2-chloro-4-(trifluoromethyl)phenoxy]-nitrobenzoic acid), except that the NO₂ is replaced by a Cl and H, respectively. These diphenyl ether herbicides required light for herbicide toxicity to the green unicellular alga Chlamydomonas eugametos and three major weeds (Xanthium pennsylvanicum, Abutilon theophrasti, and Ipomoea sp.). Acifluorfen-methyl and MC 15608 toxicity in Chlamydomonas decreased in an atmosphere of nitrogen, and in the presence of the free radical scavengers α-tocopherol and ethanol. Therefore, the mechanism of toxic action of these three different diphenyl ether herbicides is similar and appears to involve some type of free radical reaction. As confirmed by cyclic voltammetry studies, MC 15608 and MC 10878, unlike AFM, cannot readily accept electrons to become free radicals. Therefore, initiation of free radical reactions in polyunsaturated fatty acids of membranes does not necessarily involve direct reduction and reoxidation of the diphenyl ether molecule.     

Pyrazole phenyl ether herbicides inhibit protoporphyrinogen oxidase

Two isomeric pairs of pyrazole phenyl ether herbicides [AH 2.429, 4-chloro-1-methyl-5-(4-nitrophenoxy)-3-(trifluoromethyl)-1H-pyrazole; AH 2.430, 4-chloro-1-methyl-3-(4-nitrophenoxy)-5-(trifluoromethyl)-1H-pyrazole; AH 2.431, 5-((4-chloro-1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)oxy)-2-nitrobenzoic acid; and AH 2.432, 5-((4-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)oxy)-2-nitrobenzoic acid were evaluated for herbicidal activity in both intact plants and in tissue sections. Their capacity to induce accumulation of porphyrins in tissue sections and to inhibit protoporphyrinogen oxidase (Protox) in vitro were determined. In whole plant tests, the order of herbicidal activity was AH 2.430 AH 2.431 > AH 2.429 > AH 2.432. AH 2.430 consistently caused light-dependent membrane leakage in both green and far-red light grown cucumber cotyledon and barley primary leaf tissue sections after incubation for 20 hr in darkness in 0.1 mM solutions. The same treatment caused marked increases in protoporphyrin IX (PPIX) content during the 20-hr dark incubation. AH 2.429 and 2.431 were less effective and not effective in all tissues in causing herbicidal damage and PPIX accumulation. AH 2.432 was ineffective in tissue section assays. Mg-PPIX levels were not significantly affected by any of the compounds. Protochlorophyllide levels were decreased by AH 2.430 and 2.431 in barley and increased by AH 2.429, 2.431, and 2.432 in cucumber. A positive relationship was found between herbicidal activity and the amount of PPIX that was caused to accumulate by each compound. All of the compounds inhibited Protox activity. Positive correlations were found between herbicidal activity in planta over a 300-fold range and in vitro Protox inhibition and the amount of PPIX caused to accumulate in vivo. These data support the view that the pyrazole phenyl ethers exert their herbicidal activity entirely through inhibition of Protox.