Effects of the herbicide EPTC and the protectant DDCA on incorporation and distribution of [2-14C]acetate into major lipid fractions of maize cell suspension cultures

The rapid effects of the herbicide EPTC (S-ethyl dipropylthiocarbamate) and the protectant DDCA (N,N-diallyl-2,2-dichloroacetamide) on [2-¹⁴C]acetate incorporation into lipids of maize cell cultures were studied in order to determine whether they act at similar sites of lipid synthesis. DDCA, at 0.05 mM and 0.1 mM, increased the incorporation of [2-¹⁴C]acetate into neutral lipids of a total lipid extract within 2 h. It had very little effect on the major polar lipid constituents. DDCA altered neither the distribution of label within the major lipid classes, nor turnover of the major lipids within 2 h. EPTC (0.1 mM) inhibited overall uptake of [2-¹⁴C]acetate into both neutral and polar lipids by about 30% after a 2-h incubation. The major polar lipid affected was an unidentified glycolipid. In addition to reducing the quantity of lipids synthesized, EPTC changed the lipid profile, altering the distribution of label, mainly within the neutral lipid fraction. A crude membrane fraction from maize cells contained both polar lipids and some neutral lipids. DDCA stimulated [2-¹⁴C]acetate incorporation into different lipid species. EPTC inhibited incorporation of [2-¹⁴C]acetate into both neutral and polar membrane lipids but altered significantly only its distribution into neutral lipids. DDCA (0.1 mM) given together with EPTC (0.2 mM) partially counteracted the effect of EPTC within the neutral lipid fraction. It is suggested that DDCA has a rapid effect on lipid synthesis, but it is probably not sufficient to account for the entire mode of action of the protectant.     

Competition between a thiocarbamate herbicide and herbicide protectants at the level of uptake into maize cells in culture

The rapid interactions between the herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the structurally similar herbicide protectant N,N-diallyl 2,2-dichloroacetamide (DDCA) at the level of herbicide uptake were examined in maize cell cultures. When the two compounds were given simultaneously, DDCA inhibited uptake of [¹⁴C]EPTC into maize cells measured for 30 min. A Lineweaver-Burk plot indicated this inhibition to be competitive. N,N-Diallyl 2-chloroacetamide (CDAA), a compound similar in structure to DDCA, inhibited uptake to a lesser extent. Other protectants having no similarity in structure to either DDCA or EPTC had no inhibitory effect on the uptake of EPTC. The data suggest that competition between DDCA and EPTC for a site of uptake may be related to their similarity in chemical structure. Experiments with metabolic inhibitors suggested that uptake of EPTC is not via an active transport mechanism. We suggest that competition for uptake between EPTC and DDCA may represent the first step in a complex series of interactions between the herbicide and its protectant that contributes to the protection of maize from herbicide injury.     

Sulfoxidation of thiocarbamate herbicides and metabolism of thiocarbamate sulfoxides in living mice and liver enzyme systems

The microsome-NADPH system of mouse liver oxidizes each of benthiocarb, butylate, cycloate, EPTC, molinate, pebulate, and vernolate herbicide chemicals to the corresponding thiocarbamate sulfoxide which is then cleaved by the liver soluble-glutathione system. These sulfoxides are also detected as transient metabolites in the liver of mice injected with EPTC, molinate, pebulate, and vernolate but not with the other three thiocarbamates. Thiocarbamate sulfones are not detected as metabolites of the thiocarbamates. Studies in vivo and in vitro with [¹⁴C]EPTC and -pebulate or their corresponding sulfoxides and/or sulfones further indicate that sulfoxidation is the initial metabolic step in cleavage of the thiocarbamate ester group. Sulfoxidation appears to be a detoxification mechanism for thiocarbamate herbicides in mammals.     

Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione S-transferase activity

Glutathione (GSH) content and GSH S-transferase activity are consistently increased in corn roots on 24-hr exposure of corn seedlings to part per million levels of N,N-diallyl-2,2-dichloroacetamide (R-25788) and related antidotes for thiocarbamate herbicide injury in susceptible corn varieties. This combined enhancement of enzyme activity and cofactor level leads to rapid detoxification of thiocarbamate sulfoxides, which are proposed to be the active herbicidal compounds formed on metabolic sulfoxidation. S-(N,N-Dipropylcarbamyl)-GSH is formed by this enzyme-catalyzed detoxification of EPTC sulfoxide. This hypothesis on antidote mode of action is supported by studies on 32 dichloroacetamides and related compounds and on the concentration- and time-dependent relationships of R-25788 action. The liver GSH content is normal in mice injected with high doses of R-25788, but the content is reduced when EPTC or EPTC sulfoxide is administered. EPTC sulfoxide also carbamoylates the thiol group of coenzyme A in neutral aqueous medium.     

Gibberellin influence on membrane disruption by thiocarbamate herbicides

Vernolate (0, 8, 16, 31, 62, 125.0, or 250.0 ppbw) incorporated into sand inhibited the growth of wheat (Triticum aestivum L. cv Holley) at 125.0 ppbw. These growth inhibition and morphological responses were virtually identical to wheat response to EPTC at 125 ppbw. ¹⁴C from vernolate (carbonyl labeled) (125.0 ppbw) was absorbed into wheat seedlings at approximately 1.8 μM on the presumption that the ¹⁴C present was [¹⁴C]vernolate. Since the response of wheat to the thiocarbamate herbicides resembles a gibberellic acid (GA) deficiency and cell enlargement requires the presence of functional plasmalemmas and tonoplasts, the question of membrane disruption by excessive concentrations of thiocarbamate herbicides and potential reversal thereof by GA₃ was studied by measuring the efflux of K⁺, Na⁺, and Mg²⁺. GA₃ (0.003 μM) stimulated lettuce leaf disc growth in diameter and fresh weight. This GA-stimulated increase in size and weight was reversed by 1 mM EPTC. Betacyanin efflux from beet leaf tonoplasts was increased by 1 mM EPTC and this efflux was not reversed by exogenous GA₃ (0.3 μM). This influence by supraoptimal EPTC concentrations was shown to be via membrane disruption, which obviated any possible GA influence by eliminating the functionality of the membranes requisite to the development of a GA response. It is concluded that viable mode-of-action studies must measure physiological responses consistent with the symptomology of herbicide responses normally observed with each herbicide at field concentrations.     

Metabolism of EPTC by a pure bacterial culture isolated from thiocarbamate-treated soil

A bacterial strain has been isolated from an enhanced thiocarbamate degradation soil and identified as Corynebacterium sp. The strain was capable of rapidly metabolizing EPTC in a liquid culture where the herbicide was the sole source of carbon. Evolution of high quantities of [¹⁴C]carbon dioxide was coupled with a rapid decline of [¹⁴C]EPTC in the medium; after 12 h incubation these accounted for, respectively, 60% and 0% of the recoverable radioactivity. Radioactivity in the polar extract increased gradually up to 20% after 6 h of incubation and then declined slowly. TLC analysis and identification based on comparison to reference compounds showed that the polar extract consisted of EPTC sulfoxide and two conjugates, EPTC-GSH and EPTC-cysteine (1·8%, 3·4%, and 16%, respectively). Piperonyl butoxide and tetcyclasis, but not tridiphane, were found to be effective inhibitors of EPTC metabolism in the bacterial culture, suggesting that the breakdown of EPTC might be carried out by a cytochrome P-450 monooxygenase-type activity. The thiocarbamate extender, dietholate, also strongly inhibited the metabolism of EPTC in bacterial culture. Based on these results it was postulated that the bacteria metabolize EPTC mainly by hydroxylation of the α-propyl carbon finally to release [¹⁴C]carbon dioxide, while EPTC sulfoxidation appears to be a minor route.     

Comparative metabolism of atrazine and EPTC in proso millet (Panicum miliaceum L.) and corn

The purpose of this study was to examine the differential activities of proso millet (Panicum miliaceum L.) and corn (Zea mays L.) with respect to atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine] and EPTC (S-ethyldipropyl thiocarbamate) metabolism. GSH-S-transferase was isolated from proso millet shoots and roots. When assayed spectrophotometrically using CDNB (1-chloro 2,4-dinitrobenzene) as a substrate, the shoot enzyme had only 10% of the activity of corn shoot enzyme while the root enzyme had 33% the activity of corn root enzyme. However, when proso millet shoot GSH-S-transferase was assayed in vitro using ¹⁴C-ring-labeled atrazine, it degraded the atrazine to water-soluble products at the same rate as the corn shoot enzyme. Incubation of excised proso millet and corn roots with [¹⁴C]EPTC indicated that uptake of EPTC was similar in both plants. However, proso millet metabolized the EPTC to water-soluble products at only half the rate of corn. Glutathione levels of proso millet roots were 35.9 μg GSH/g fresh wt, compared with 65.4 μg GSH/g fresh wt for corn. However, a 2.5-day pretreatment with R-25788 (N,N-diallyl-2-2-dichloroacetamide) elevated proso millet GSH levels to 62.7 μg GSH/g fresh wt. R-25788 did not elevate the activity of proso millet GSH-S-transferase, in contrast to its effects on corn. We conclude that differences in response to atrazine and EPTC in proso millet and corn are a result of their differential metabolism.     

Inhibition of conversion of geranylgeranyl-chlorophyll to phytol-chlorophyll by S-ethyl dipropylthiocarbamate (EPTC)

The influence of EPTC (S-ethyl dipropylthiocarbamate) on the hydrogenation of geranylgeranylchlorophyll (GG-Chl) to phytol-Chl was studied during the greening (6-, 12-, 18-, 24-, and 48-hr incandescent light exposure) of etiolated wheat [Triticum aestivum (L.) cv “Stacy”] and sorghum [Sorghum bicolor (L.) Moench cv “G 522DR”] seedlings grown in nutrient solution containing ¹⁴C-labeled sodium acetate. Chloroplast pigment synthesis occurred and small quantities of GG-Chl were found in both Chl^?a and Chl^?b. When wheat seedlings were greened for 48 hr in an EPTC concentration series (1 nM to 100 μM), geranylgeraniol (GG) content increased from 11% (control) to 60% (100 μM EPTC) of the isoprenoid alcohol esterified to chlorophyllide a, but Chl-b contained ≤1% GG-Chl at all concentrations of EPTC. Sorghum seedlings greened for 48 hr in the same EPTC concentration series contained about 3% GG (control) while 100 and 40% GG esterified to chlorophyllide a and chlorophyllide b, respectively, after 48 hr exposure to 100 μM EPTC. Thus, EPTC prevented hydrogenation of GG-Chl to phytol-Chl on the Chl molecule more in sorghum than in wheat.     

Differential effect of diclofop-methyl on fatty acid biosynthesis in leaves of sensitive and tolerant plant species

The herbicide diclofop-methyl caused an early and pronounced inhibition of the incorporation of [¹⁴C]acetate into leaf lipids of the sensitive plant species maize (Zea may L.), wild oat (Avena fatua L.), and barnyardgrass (Echinochloa crus-galli L.). With an EC₅₀ value of approximately 10^?7M inhibition was already apparent 0.5–4 hr after herbicide application. The fatty acid biosynthesis of tolerant bean (Phaseolus vulgaris L.), sugar beet (Beta vulgaris L.), and soybean (Glycine max L.) was not affected, with one exception [wheat (Triticum aestivum L.) belongs to the more tolerant species]; the inhibition of fatty acid biosynthesis, however, was in the same order of magnitude as in sensitive plants. More detailed studies showed that in wheat a recovery from inhibition of fatty acid biosynthesis occurred. Four days after herbicide application (0.18 kg diclofop-methyl/ha) in wheat normal fatty acid biosynthesis was restored, whereas in sensitive maize a 60% inhibition was maintained over the whole experimental period (8 days). The results support the view that tolerance of wheat to diclofop-methyl is based on its inactivation in leaves, whereas the tolerance of dicotyledonous species may probably lie at the level of the site of action of diclofop-methyl. In experiments with intact leaves, the inhibition of fatty acid biosynthesis resulted in an enhanced flow of [¹⁴C]acetate into organic acids and amino acids. This effect, however, was not always reproducible in experiments with leaf pieces or isolated root tips.     

Effect of pyridazinone herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of five substituted pyridazinones (pyrazon, San 133-410H, San 9774, norflurazon, and San 6706) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1-¹⁴C]acetate, [³²P]orthophosphate, and [³⁵S]sulfate was significantly inhibited by these herbicides and the magnitude of inhibition varied, depending on the substituents. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, no effect was observed in the case of [1-¹⁴C]acetate but there was significant inhibition in the incorporation of the other two precursors, suggesting that pyridazinones interfere with the metabolism of the phospholipids and the sulfolipid. None of these compounds affected the uptake of [methyl-¹⁴C]choline but all inhibited its incorporation into phosphatidylcholine indicating that phosphatidylcholine metabolism is vulnerable to pyridazinones. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the pyridazinones at 0.1–0.5 mM concentrations. San 9774 showed the most potent inhibition. In addition, the pyridazinone herbicides significantly inhibited sn-glycerol-3-phosphate acyltransferase(s) in both chloroplast and microsomal fractions but showed no effect on phosphatidic acid phosphatase. The magnitude of inhibition of fatty acid synthetase and acyltransferase(s) is related to the nature of the substituent groups on the herbicide. Trifluorophenyl substitution at position 2 or amino substitution at position 5 of the pyridazinone molecule caused the maximum inhibitory effect.     

Gibberellin precursor biosynthesis inhibition by EPTC and reversal by R-25788

S-ethyl dipropylthiocarbamate (EPTC) inhibited gibberellic acid (GA) precursor biosynthesis in a cell-free enzyme preparation from unruptured, etiolated sorghum (Sorghum bicolor L. cv. G522 DR) coleoptiles. EPTC, 1 μM, inhibited incorporation of [¹⁴C]mevalonic acid into kaurene 60%, while 10 μM EPTC inhibited ¹⁴C incorporation into kaurene 90%. The precursor of kaurene cyclization (GGPP) increased in ¹⁴C content at both EPTC concentrations. R-25788 reversed the EPTC inhibition of kaurene synthesis. Kaurene oxidation was modified by both EPTC and R-25788. Hypothesized modes of action for EPTC and R-25788 are (a) inhibition of GA synthesis, (b) increased peroxidase activity resulting in increased lignification, (c) increased detoxification by sulfoxidation and carbamoylation, and (d) inhibition of fatty acid synthesis and/or desaturation. These hypotheses are discussed with three of them being incorporated into one working unit which correlates with EPTC and R-25788 symptom phenology. The fourth hypothesis could also fit into this general pattern.     

Biochemical response of inbred and hybrid corn (Zea mays L.) to R-25788 and its distribution with EPTC in corn seedlings

The average endogenous GSH content of eight lines of inbred corn was almost twofold greater than ten varieties of hybrid corn. When inbred and hybrid corn lines were treated with R-25788, the average GSH content increased by 56 and 95%, respectively. R-25788 protected two special inbred corn lines, GT 112 (atrazine susceptible) and GT 112 RfRf (atrazine resistant) from EPTC injury by increasing the GSH content and GSH S-transferase activity in roots. Most of the radiolabel from [¹⁴C]R-25788-treated plants remained in the root tissues whereas the radiolabel in [¹⁴C]EPTC-treated plants was evenly distributed between foliar and root tissues. From radiolabel experiments, hybrid corn seedlings were found to absorb more R-25788 from soil than EPTC. There was no difference between inbred and hybrid corn in the amounts of R-25788 or EPTC taken up or in the enhancement of GSH S-transferase activity caused by R-25788.     

Influence of the herbicides hexazinone and chlorsulfuron on the metabolism of isolated soybean leaf cells

The effects of the herbicides hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione] and chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide) on the metabolism of enzymatically isolated leaf cells from soybean [Glycine max (L.) Merr., cv. ‘Essex’] were examined. Photosynthesis, protein, ribonucleic acid (RNA), and lipid syntheses were assayed by the incorporation of specific radioactive substrates into the isolated soybean leaf cells. These specific substrates were NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetate, respectively. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 μM of both herbicides. Photosynthesis was the most sensitive and first metabolic process inhibited by hexazinone. RNA and lipid syntheses were also inhibited significantly by hexazinone whereas the effect of this herbicide on protein synthesis was less. The most sensitive and first metabolic process inhibited by chlorsulfuron was lipid synthesis. Photosynthesis, RNA, and protein syntheses were affected significantly only by the highest concentration of this herbicide and longest exposure. Although these two herbicides may exert their herbicidal action by affecting other plant metabolic processes not examined in this study, hexazinone appears to be a strong photosynthetic inhibitor, while the herbicidal action of chlorsulfuron appeared to be related to its effects on lipid synthesis.     

Antifungal mode of action of imazalil

Imazalil had no effect on the initial growth of mycelia of Penicillium italicum (for 10 hr) or Aspergillus nidulans (for 2 hr). In P. italicum during this period neither respiration nor cell permeability was affected, but uptake of [³²P]phosphate, [¹⁴C]leucine, or [¹⁴C]uridine was partially inhibited. The initial (5 hr) inhibition of substrate uptake coincided with a 50% reduction in ergosterol content. Within 0.5 hr, incorporation of [¹⁴C]acetate into C-4-desmethyl sterols was strongly inhibited in mycelia of A. nidulans treated with 0.5 μg/ml of imazalil. However, radioactivity in C-4-methyl and dimethyl sterols exceeded that of control cultures. Concentrations of imazalil as low as 0.005 μg/ml caused short-term (1 hr) declines of incorporation into desmethyl sterols and increases into the C-4-methyl and dimethyl sterols. Incorporation into phospholipids, triglycerides, and free fatty acids was not affected. These data suggest that the primary antifungal action of imazalil is inhibition of demethylation in the biosynthesis of ergosterol.     

Working Group on Genetical Methods of Pest Control,Bucharest, September, 1974

Abstract

Treating maize seed and cowpea seed with activated carbon or naphthalic anhydride permitted highly selective and economic early control of grasses and some broadleaved weeds with respectively EPTC and linuron and EPTC and chloramben. Without protectant every herbicide treatment produced less crop yield than with protectant. Early season control of grasses with herbicides followed by one row-cultivation controlled weeds throughout the crop.     

The role of waxes in the uptake of metolachlor into sorghum in relation to the protectant CGA 43089

The grass weed herbicide metolachlor (2-chloro-N-[2-ethyl-6-methylphenyl]-N-[2-methoxy-1-methylethyl]acetamide) which is especially effective against wild millets, inhibits the formation of epicuticular waxes on sorghum leaves. The metolachlor protectant CGA 43089 [α - (cyanomethoximino) - benzacetonitrile] prevents the depletion of the waxes on the leaves of metolachlor-treated sorghum plants, as demonstrated by scanning electron microscopy. This alteration of the plant surface polymers also changes their permeability to the herbicide. ¹⁴C-metolachlor uptake into isolated coleoptiles and first leaves of sorghum which had been pretreated with the herbicide was increased. Incubation with added protectant reduced the uptake of ¹⁴C-metolachlor. It is postulated that the modifications caused by metolachlor and its protectant to sorghum surface structures influence the action of the herbicide in two ways:

• 1 The selectivity observed against sorghum and millet grasses could occur because of an increased uptake of metolachlor through cuticles which are particularly sensitive to the structural changes caused by the herbicide, since the composition of the plant waxes is very species-specific.
• 2 The loss of cuticular integrity is prevented by the protectant CGA 43089, which greatly reduces penetration of metolachlor.

     

Physiological responses of corn (Zea mays) to AC 243,997 in combination with valine,leucine, and isoleucine

Various physiological processes were measured in corn after treatment with AC 243,997. Neutral sugar levels in leaves increased 39% over the control 24 hr after application of AC 243,997. Protein synthesis, measured by [¹⁴C]leucine and [¹⁴C]cystine incorporation, and lipid synthesis were not inhibited 24 hr after application of 150 μM of AC 243,997, while respiration and RNA synthesis were inhibited 32 and 15%, respectively. DNA synthesis was severely inhibited (70–90%) by 150 μM of the herbicide 24 hr after application. The inhibition of DNA synthesis by AC 243,997 did not begin until 5 to 7 hr after application. Although protein synthesis rates were apparently unaffected by AC 243,997, the level of the soluble proteins decreased 40% while free amino acid levels increased 32% 24 hr after application of the herbicide. An exogenous supply of valine, leucine, and isoleucine to corn prevented the inhibition of growth and reversed the inhibition of DNA synthesis caused by AC 243,997. All three amino acids at a concentration of 1 mM were needed to provide maximum protection. The results support the hypothesis that AC 243,997 kills plants by interfering with the biosynthesis of valine, leucine, and isoleucine.     

The effect of α-hexachlorocyclohexane on liver growth in intact and adrenalectomized rats

α-Hexachlorocyclohexane was administered as a single oral dose (100 mg/kg body wt) to both intact and adrenalectomized male Sprague-Dawley rats. In the intact rats there was a peak in [³H]thymidine incorporation into DNA at 30 hr and an increase in relative liver weight (liver weight/body wt × 100) which peaked at 60 hr post α-HCH dose. However, in α-HCH-dosed adrenalectomized rats [³H]thymidine incorporation into DNA remained elevated throughout the study period and relative liver weight was not significantly increased. In corticosterone-supplemented α-HCH-dosed Adx rats, [³H]thymidine incorporation into DNA was significantly reduced and RLW was increased comparable to that seen in intact animals. These results suggest that α-HCH-induced liver growth is mediated by corticosterone.     

Metabolism of EPTC in germinating corn: Sulfone as the true carbamoylating agent

Corn (Zea mays L. single cross hybrid Mv 620) was germinated in a petri dish with addition of carbonyl[¹⁴C]EPTC (S-ethyl-N,N-dipropylthiocarbamate). The shoots and roots of 4-day-old seedlings were crushed and extracted in 80% methanol. On the chromatogram of the extract three radioactive peaks were found. The main peak was identified as S-(N,N-dipropylcarbamoyl)-glutathione. For the comparison of carbamoylating ability [¹⁴C]EPTC, [¹⁴C]EPTC-sulfoxide, and [¹⁴C]EPTC-sulfone were incubated with glutathione. Only EPTC-sulfone reacted in the 10-day incubation time. In aquatic solutions EPTC and EPTC-sulfoxide proved to be stable during the 10 days compared to EPTC-sulfone which quickly degraded, S-(N,N-Dipropylcarbamoyl)-glutathione was converted to S-(N,N-dipropylcarbamoyl)-cysteine in corn shoot homogenate. [¹⁴C]EPTC, [¹⁴C]EPTC-sulfoxide and [¹⁴C]EPTC-sulfone were added to corn shoot homogeneates and each of the three mixtures were analyzed by chromatography after 1 day incubation. EPTC was partly oxidized to EPTC-sulfoxide. EPTC-sulfoxide did not change and EPTC-sulfone produced similar metabolites as had been found in the germination experiment.     

Physiological interactions between the herbicide EPTC and selected analogues of the antidote naphthalic anhydride on two hybrids of maize

The efficacies of nine structural analogues of the herbicide antidote naphthalene-1,8-dicarboxylic acid anhydride (naphthalic anhydride, NA) for the protection of maize (Zea mays L. cv. DeKalb XL72AA and DeKalb XL67) against injury by the herbicide S-ethyl dipropyl(thiocarbamate) (EPTC) were elevated under greenhouse conditions. The chemical analogues of NA tested were: acenaphthenequinone (ACQ); 4-aminonaphthalene-1,8-dicarboxylic acid anhydride (NH₂NA); 1,8:4,5-naphthalenetetracarboxylic acid dianhydride (NDiA); naphthalene- 1,8-carboximide (NHNA); 4-chloronaphthalene-1,8-dicarboxylic acid anhydride (C1NA); biphenyl-2,2′-dicarboxylic acid anhydride (diphenic anhydride; DA); 2-phenylglutaric anhydride (PGA); phthalic anhydride (PHA); phenalen-1-one (PA). Pre-plant incorporated applications of EPTC at 2.2, 4.5, 6.7, and 9.0 kg ha^?1 were highly toxic to XL67 maize. Appreciable injury to XL72AA maize by EPTC was observed only with the high rates of EPTC (6.7 and 9.0 kg ha^?1). Of the analogues tested PGA and PA were very toxic and inhibited germination of both maize hybrids. NA, ACQ, NH₂NA, NDiA, NHNA, C1NA, DA, and PHA applied as seed dressings at 5.0 and 10 g per kg of seed offered satisfactory protection to XL72AA maize against EPTC rates higher than 6.7 kg ha^?1. The same antidotes significantly antagonised the EPTC activity against XL67 maize but the overall protection obtained was partial and not agronomically important. The presence of the dicarboxylic anhydride group and of at least one aromatic ring attached directly to the anhydride appeared to be essential for the exhibition of protective activity by the structural analogues of NA. NA was slightly toxic to both hybrids of maize and chlorination of NA increased the phytotoxicity of this molecule. A genetic component that is present in the thiocarbamate-tolerant XL72AA hybrid but absent from the thiocarbamate-susceptible XL67 hybrid of maize appeared to be important for the phytotoxic activity of EPTC and may be involved in the protective activity of NA and its structural analogues.