Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.     

Enantioselective effects of optically active alpha-methylbenzyl-s-triazine on the root growth of rice and Echinochloa plants and their herbicidal activity

The chiral requirement on the alpha-methylbenzyl moiety of 2,4-diamino-6-chloro-s-triazine for sufficient inhibition of root growth was similar towards both rice and barnyard millet. With the monoalkylamino series, the most suitable configuration was markedly changed by the substituent on the other amino moiety. However, for the dialkylamino series, the (S)-enantiomer was an active inhibitor. Clear species selectivity between rice and barnyard millet was observed in the series for the (R)-enantiomers, providing high herbicidal activity toward Echinochloa plants and safety toward rice. The enantioselectivity against barnyard millet increased with increasing inhibitory activity of the active enantiomers, following Pfeiffer's rule. R-EtNH (3) controlled the growth of barnyardgrass with leaf-burning (LB) under paddy conditions, and S-EtNH (4) and S-Et2N (20) controlled the growth without LB. The RS-EtNH derivative is an interesting inhibitor controlling the growth of barnyardgrass from the just-germinated stage (by the (R)-enantiomer) to early-middle growth stage (by the (S)-enantiomer).     

Japanese barnyard millet (Echinochloa utilis,poaceae) in Japan

Two species are included in barnyard millet:Echinochloa utilis andE. frumentacea. These differ from each other in their genomic constitution and phylogeny. The former species originated fromE. crus-galli probably in eastern Asia, and is grown in Japan, Korea, and the northeastern part of China; the latter originated fromE. colona probably in tropical Asia, and is grown in Pakistan, India, and Nepal. “Japanese barnyard millet” is suggested as a suitable English common name forE. utilis; “Indian barnyard millet,” forE. frumentacea. In the past, Japanese barnyard millet was important in Japan as the staple food crop in districts where soil, weather conditions and irrigation systems were not suitable for paddy rice cultivation. When the rice crop suffered serious cool weather damage, the millet relieved people from starvation, especially in northeastern Japan. But the acreage devoted to the millet gradually decreased during and after the 1880s. Only the northern part of Iwate Prefecture is an exclusive Japanese barnyard millet cropping region at present. The breeding of cool- weather- resistant rice varieties and improvements in rice- growing techniques are mainly responsible for the decrease in acreage of the millet.     

Antimicrobial Activity of 3-Phenylimidazolidine-2,4-diones and Related Compounds

The structure-activity relationships of 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were investigated by the agar dilution method using Sclerotinia sclerotiorum as a test microbe. Several compounds were tested for antimicrobial spectrum in vitro with other pathogenic microbes and for foliage protective activity in green house tests with rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. It was found that the antimicrobial activity was enhanced when the 1-position of imidazolidine ring was substituted by an alkyl group but was reduced when the 5-position was substituted by alkyl groups. Generally, 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were active against Scierotiniaceae, Corticiaceae, Dematiaceae, Polystigmataceae or Pleosporaceae. In green house tests, some of these compounds showed high protective activity against rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. Results of the green house tests on the above mentioned diseases correlate well with those of in vitro tests except in the case of kidney bean stem rot.     

Induced accumulation of tyramine,serotonin, and related amines in response to Bipolaris sorokiniana infection in barley

The inducible metabolites were analyzed in barley leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of barley. HPLC analysis revealed that B. sorokiniana-infected leaves accumulated 4 hydrophilic compounds. They were purified by ODS column chromatography and preparative HPLC. Spectroscopic analyses revealed that they were tyramine (1), 3-(2-aminoethyl)-3-hydroxyindolin-2-one (2), serotonin (3), and 5,5′-dihydroxy-2,4′-bitryptamine (4). Among these, 2 and 4 have not been reported as natural products. They showed antifungal activity in an assay of inhibition of B. sorokiniana conidia germination, suggesting that they play a role in the chemical defense of barley as phytoalexins. The accumulation of 1–4 was examined also in the leaves of rice and foxtail millet. Rice leaves accumulated 2, 3, and 4, whereas foxtail millet leaves accumulated 3 and 4 in response to pathogen attack, suggesting the generality of accumulation of 3 and 4 in the Poaceae species.     

Plantlet regeneration from somatic hybrids of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola Vasing)

Summary Somatic hybridization of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola), a close relative of barnyard millet, was attempted using electrofusion and a new culture method developed for rice protoplasts (Kyozuka et al. 1987) to incorporate some of the agronomically important characters of the latter species into rice. Selection of hybrids was based on inactivation of rice protoplasts by iodoacetamide and the inability of barnyard grass protoplasts to divide. A total of 166 calli were identified as hybrids by isozyme and chromosome analyses. Hybrid calli were highly morphogenic, and 44 shoots were obtained. Most of them, however, were abnormal, and nine grew to plantlets whose morphology was distinct from that of either parent. Our study clearly demonstrates the totipotency of protoplasts in graminaceous monocots.     

Maternal and developmental toxicity of halogenated 4'-nitrodiphenyl ethers in mice

In an ongoing effort to delineate structure-activity relationships in the developmental toxicity of diphenyl ethers, we evaluated the maternal and developmental toxicity of 10 diphenyl ethers related to the herbicide nitrofen. All possible trichlorophenyl 4'-nitrophenyl ethers were evaluated, as were the 2,4-difluorophenyl and 2,4-dibromophenyl 4'-nitrophenyl ethers. We also evaluated bifenox and chlomethoxyfen, which are 2,4-dichlorophenyl congeners with meta-substituents on the 4'-nitrophenyl ring. Nitrofen (2,4-dichlorophenyl 4'-nitrophenyl ether) was included for comparison. Identity of the halogen affected the postnatal (but not prenatal) mortality induced by 2,4-dihalogenated 4'-nitrophenyl ethers. The presence of 3'-substituents on the 4'-nitrophenyl ring reduced both pre- and postnatal toxicity of 2,4-dichlorinated congeners. Among chlorinated 4'-nitrophenyl congeners without meta-substituents on the nitrophenyl ring, the position of chlorine substituents strongly affected the congener's potential for inducing prenatal vs. postnatal syndromes. All congeners increased liver to body weight ratios in unmated females, but such increases were not well-correlated with either prenatal or postnatal embryotoxicity.     

Degradation of 3-(3′,5′ -Dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione by Plants,Soil and Light

Studies on uptake, tissue distribution, and metabolsim of 3-(3′,5′ -dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione (DDOD) by bean and grape plants, and degradation by soil and light were carried out using ¹⁴C- or ³H-DDOD. DDOD injected at stem or absorbed through roots of bean plants was transported mainly to leaf tissues. No downward movement of the label was observed. DDOD was decomposed in the nutrient solution to N-3,5-dichlorophenyl-N-α-hydroxyisobutyryl carbamic acid (DHCA) and α-hydroxyisobutyryl-3,5-dichloroanilide (HDA). Metabolism of DDOD in bean plants or on grape berries themselves occurred to only a small extent if at all. When injected into grape trees, DDOD underwent some degree of metabolization to HDA and, probably, 3-(3′,5′ -dichloro-4′ -hydroxyphenyl)-5,5-dimethyloxazolidine-2,4-dione. In soil, DDOD broke down to DHCA, HDA, and 3,5-dichloroaniline, but formation of tetrachloroazobenzene was not observed under the present experimental conditions. DDOD decomposed to some degree when irradiated with a xenon lamp.     

Biotransformation of 3-(3′,5′ -Dichlorophenyl)-5,5-dimethyl oxazolidine-2,4-dione

Using 3-(3′,5′-dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione labeled with ¹⁴C or ³H, absorption, excretion, and tissue distribution in male Wistar rats were studied, and metabolites excreted were identified. At the dosage rates of 100, 300, 1000 and 3000 mg/kg, the maximum excretion of orally administered radioactivity occurred within 24 hr. Increase in the dosage rate was paralleled by decrease in the proportion of urinary elimination. Essentially all the radioactivity was excreted in 2 weeks. DDOD level was generally low in most tissues. Adipose tissue contained higher radioactivity compared with others. Most of the urinary metabolites identified were characterized by hydroxylation at the 4′ position of the benzene ring moiety, and hydrolytic or oxidative modification of the oxazolidine ring portion.     

Comparative performance of pearl millet- and sorghum- based diets vs. wheat- and rice-based diets for trace metal bioavailability.

Pearl millet and sorghum offer a cheap source of energy compared to wheat and rice and are widely consumed by rural communities in many parts of the world. Due to the low consumption of vegetables and animal foods, millets also are the major suppliers of micronutrients especially for low-income groups. It is of prime importance to study how millets perform in terms of bioavailable contents of trace metals. Investigations were carried out using weanling mice which offer a model for the initial testing of bioavailability of trace metals before human trials. Four isocaloric diets differing only in the type of cereal, i.e. pearl millet, sorghum, wheat and rice, were prepared representing habitual dietary patterns observed by National Nutrition Monitoring Bureau (NNMB) of India. Mice were allocated randomly to 4 groups of 8 mice each, and housed individually in metal free metabolic cages. A fifth group of 8 mice fed a balanced synthetic diet served as control. All the groups were fed ad libitum. The absorption of zinc and iron averaged for 3 periods of 5 days each was significantly higher for the wheat and pearl millet group than for the other 2 experimental groups (p < 0.05), as were also the levels of liver zinc and iron. The weight gain was also highest (6.9 +/- 1.2 g) in the pearl millet group as compared to sorghum (1.58 +/- 0.59 g), wheat (1.66 +/- 1.27 g) and rice (-0.72 +/- 0.62 g) groups. The levels of liver copper were comparable in all the 5 groups. These results further confirm our earlier in vitro results indicating the superiority of pearl millet but not sorghum in bioavailability of zinc and iron.     

Interaction between C4 barnyard grass and C3 upland rice under elevated CO2: Impact of mycorrhizae

Atmospheric CO₂ enrichment may impact arbuscular mycorrhizae (AM) development and function, which could have subsequent effects on host plant species interactions by differentially affecting plant nutrient acquisition. However, direct evidence illustrating this scenario is limited. We examined how elevated CO₂ affects plant growth and whether mycorrhizae mediate interactions between C₄ barnyard grass (Echinochloa crusgalli (L.) Beauv.) and C₃ upland rice (Oryza sativa L.) in a low nutrient soil. The monocultures and combinations with or without mycorrhizal inoculation were grown at ambient (400 ± 20 μmol mol^?1) and elevated CO₂ (700 ± 20 μmol mol^?1) levels. The ¹⁵N isotope tracer was introduced to quantify the mycorrhizally mediated N acquisition of plants. Elevated CO₂ stimulated the growth of C₃ upland rice but not that of C₄ barnyard grass under monoculture. Elevated CO₂ also increased mycorrhizal colonization of C₄ barnyard grass but did not affect mycorrhizal colonization of C₃ upland rice. Mycorrhizal inoculation increased the shoot biomass ratio of C₄ barnyard grass to C₃ upland rice under both CO₂ concentrations but had a greater impact under the elevated than ambient CO₂ level. Mycorrhizae decreased relative interaction index (RII) of C₃ plants under both ambient and elevated CO₂, but mycorrhizae increased RII of C₄ plants only under elevated CO₂. Elevated CO₂ and mycorrhizal inoculation enhanced ¹⁵N and total N and P uptake of C₄ barnyard grass in mixture but had no effects on N and P acquisition of C₃ upland rice, thus altering the distribution of N and P between the species in mixture. These results implied that CO₂ stimulation of mycorrhizae and their nutrient acquisition may impact competitive interaction of C₄ barnyard grass and C₃ upland rice under future CO₂ scenarios.     

Environmental fate of the triazole fungicide propiconazole in a rice-paddy-soil lysimeter

Environmental fate of the triazole fungicide propiconazole, 1-[[2(2,4-dichlorophenyl)-4-propyl-1,3-diox olane-2-yl]methyl]1H-1,2,4-triazole, in soil was investigated using lysimeters simulating a rice-paddy-soil conditions. Two lysimeters composed of different soil types, a sandy loam (lysimeter A) and silty clay (lysimeter B), were used. Propiconazole (Tilt 250^R EC) plus [U-¹⁴C]-propiconazole was applied over a two-year period to the soil surface of the lysimeters. Propiconazole fate in the lysimeters was assessed by measuring total radioactivity in the leachate, evolved ¹⁴CO₂, and ¹⁴C-residues in the soil and rice plants. The amounts of applied ¹⁴C in the leachate from lysimeter A were 4.4 and 5.2% in the first and second year, respectively. A background level of (0.00005% of applied) ¹⁴C in the leachate from lysimeter B was detected, suggesting negligible movement of the fungicide to groundwater in the silty clay soil. The amount of ¹⁴CO₂ evolved from lysimeter A accounted for 7.8 and 12.2% of applied ¹⁴C in the first and second year, respectively, whereas those from lysimeter B were 5.7 and 7.1%. Total ¹⁴C detected in the rice plants grown in lysimeter A were 7.3 and 9.8% of applied ¹⁴C in the first and second year, respectively, which compared to 3.0 and 7.6% in lysimeter B. Most of the applied ¹⁴C was detected in the top 10 cm soil layer, suggesting that propiconazole remains close to the soil surface after application in soil. Degradation products of propiconazole identified in the lysimeter soils were 1-[[2(2,4-dichlorophenyl)-2-(1,2,4-triazole -1-yl) ketone (DP-1), 1-(2,4-dichlorophenyl)-2-(1,2,4-triazole-1- yl) ethanol (DP-2) and 1-[[2(2,4-dichlorophenyl)-4-hydroxypropyl-1,3-dioxolane-2-yl]methyl]1H-1,2,4-triazole (DP-3 and DP-4).     

Synthesis and antimicrobial activity of some new substituted pyrido[3′,2′:4,5]thieno[3,2-d]-pyrimidinone derivatives

A series of new 3-substituted-7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one derivatives were synthesized as antimicrobial agents using 7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methyl-4H-pyrido[3′,2′:4,5]thieno[3,2-d]-[1,3]oxazin-4-one as a starting compound. Its condensation with substituted aniline derivatives or phenyl hydrazine gave the corresponding N-substituted derivatives. Treatment of the starting compound with hydrazine hydrate afforded the corresponding N-amino derivative, which was reacted with substituted phenylisocyanate and phenylisothiocyanate derivatives to give the corresponding semicarbazides and thiosemicarbazide derivatives. All the newly synthesized compounds were evaluated for their antimicrobial activities in comparison to streptomycin and fusidic acid as positive controls. The structure assignments of the new compounds are based on chemical and spectroscopic evidence.     

Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of 3-phenyloxazolidine-2, 4-diones, 3-phenyl-4-imino-oxazolidine-2-ones and n-phenylcarbamates were investigated on Sclerotinia sclerotiorum by the agar medium dilution method. In addition, antimicrobial spectra of several compounds against other 15 pathogenic microbes were investigated by the same method. In each series, 3, 5-dihalo-substituents on benzene ring are essential to high antifungal activity against Sclerotinia sclerotiorum and in the case of n-phenylcarbamates, it is necessary that the α-position of alcohol moiety is substituted by such a group as cyano group, ethoxy-carbonyl group or carbamoyl group. α-Cyanoisopropyl N-(3, 5-dichlorophenyl) carbamate, 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyl-4-iminooxazolidine-2-one and 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyloxazolidine-2,4-dione were the most effective and completely inhibited the mycelial growth of Sclerotinia sclerotiorum at 3.2 γm (about l.0 ppm). In general, 3-(3′, 5′-dichlorophenyl) oxazolidine-2, 4-diones and related compounds are highly active against Sclerotinia sclerotiorum and Botrytis cinerea, and fairly active against Cochliobolus miyabeanus, Pellicularia filamentosa, Pellicularia sasakii and Alternaria kikuchiana.     

The chemical-mediated allelopathic interaction between rice and barnyard grass

Aims

The possible involvement of the chemical-mediated interaction in allelopathy between rice and barnyard grass was investigated.

Methods

Effcts of rice seedlings and rice root exudate on the alleloapthic activity of barnyard grass were determined and a key compound invovled in the allelopathic interaction between rice and barnyard grass was isolated.

Results

Allelopathic activity of barnyard grass was increased by the presence of rice seedlings. Rice root exudates also elevated the allelopahtic activity of barnyard grass. A key compound, which increased the allelopathic activity of barnyard grass, in the rice root exudates was isolated and determined as momilactone B. Momilactone B increased the allelopathic activity of barnyard grass at concentrations greater than 3 μM, and increasing the momilactone B concentration increased the activity.

Conclusions

Momilactone B is known to act as a potent rice allelochemical and to possess strong growth inhibitory activity against barnyard grass. The present research suggests that barnyard grass may response to the presence of neighboring rice by sensing momilactone B in rice root exudates and increase allelopathic activity. Thus, momilactone B may not only act as a rice allelochemical but also play an important role in rice-induced allelopathy of barnyard grass. The induced-allelopathy may provide a competitive advantage for barnyard grass through the growth inhibition of competing plant species including rice. Barnyard grass allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyard grass. Rice allelopathy was also reported to be increased by the presence of barnyard grass through increasing production and secretion of momilactone B into surrounding environments. During the evolutional process, rice and barnyard grass may have developed the chemical cross talk to activate the defense mechanisms against some biotic stress conditions by detection of certain key compounds.     

The Effect of Metabolic Poisons on ATP Level and on Active Phosphate Uptake in Chlorella pyrenoidosa

Influence of light and dark aerobic conditions and of various metabolic poisons [3-(3,4-dichlorophenyl)1,1-dimethylurea (DCMU), 2,4-dinitrophenol (2,4-DNP), carbonyl cyanide-m-chlorophenylhydrazone (m-CCCP), α,α′bis(hexafluoroacetonyl) acetone (1799), N,N′-dicyclohexylcarbodiimide (DCCD), sodium azide, fatty acids] on the active phosphate uptake and on the ATP level has been investigated. Cells in the dark have the same level of ATP as in the light though the phosphate transport is decreased. Similar findings are observed with DCMU. Other poisons used inhibit phosphate uptake and decrease ATP level. However their effect is more drastic on phosphate transport than on the ATP level. There is no good correlation between the ATP level and phosphate transport.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Xanthones from the bark of Garcinia pedunculata

Three new xanthones, pedunxanthones A–C (1–3), together with five known compounds, 1,5-dihydroxy-3-methoxy-6′,6′-dimethyl-2H-pyrano(2′,3′:6,7)-4-(3-methylbut-2-enyl)xanthone, 1,5-dihydroxy-3-methoxy-4-(3-methylbut-2-enyl)xanthone, dulxanthone A, garbogiol and oleanolic acid, were obtained from a petroleum ether extract of the bark of Garcinia pedunculata. The new structures were elucidated using spectroscopic methods, mainly 1-D and 2-D NMR.     

Metabolism of 2,4,4′-Trichlorobiphenyl by Acinetobacter sp. P6

Metabolism of 2,4,4′-trichlorobiphenyl by Acinetobacter sp. strain P6 has been studied. When the incubation was carried out without shaking at 15°C, two isomeric monohydroxy compounds, a dihydrodiol compound, a dihydroxy compound, a meta-cleaved yellow compound and a dichlorobenzoic acid were detected by combined gas liquid chromatograph-mass spectrometry. As an additional metabolite, dichlorodihydroxy biphenyl, a dechlorinationhydroxylation product, was also detected. When the incubation mixture was shaken at 30°C, a meta-cleaved yellow compound was readily produced and predominantly accumulated in the reaction mixture upon further incubation. The major pathway of 2,4,4′-trichlorobiphenyl by Acinetobacter sp. P6 was considered to proceed oxidatively via 2.′3′-dihydro-2′,3′-diol compound, concomitant dehydrogenated 2′,3′-dihydroxy compound and then the 1′,2′-meta-cleaved yellow compound, i.e., 3-chloro-2-hydroxy-6-oxo-6(2,4-dichlorophenyl)hexa-2,4-dienoic acid.