Enantioselective separation and phytotoxicity on rice seedlings of paclobutrazol

The environmental significance of enantioselectivity in chiral insecticides and herbicides has been widely studied. However, little information is currently available on the enantioselective behavior of chiral plant growth regulators. In this study, paclobutrazol enantiomers were resolved and prepared by chiral high-performance liquid chromatography with a Sino-chiral OJ column. The relationship among absolute configuration, optical activity and circular dichroism of paclobutrazol enantiomers was established. The enantioselective behavior of paclobutrazol, including enantioselective effect of paclobutrazol on the growth of rice seedlings and cyanobacteria and enantioselective loss of paclobutrazol in rice seedling growth media, in rice culture system was studied. The (2S,3S)-(-)-enantiomer was almost 3.1 times more active than the (2R,3R)-(+)-enantiomer toward shoot growth as measured by 7 day EC50 values. Enantioselectivity could not be determined with respect to root growth of rice seedlings because a typical dosage response was not observed in the range of the concentrations studied. The dissipation of paclobutrazol in rice growth medium is not enantioselective. Enantiomers and diastereoisomer of paclobutrazol all facilitated the growth of cyanobacteria, which increase the effectiveness of rice biofertilizers. The (2S,3S)-(-)-enantiomer showed stronger stimulatory activity on Microcystis aeruginosa cyanobacteria than the (2R,3R)-(+)-enantiomer, whereas the latter was a more potent stimulator of Anabaena sp. growth. These observations indicate that application of the (2S,3S)-(-)-enantiomer of paclobutrazol and Microcystis aeruginosa in rice cultivation is a good strategy for improving rice seedling performance.     

Enantioselective environmental behavior of the chiral herbicide fenoxaprop-ethyl and its chiral metabolite fenoxaprop in soil

The enantioselective degradation behavior of fenoxaprop-ethyl (FE) and its chiral metabolite fenoxaprop (FA) in three soils under native conditions was investigated. Two pairs of enantiomers were analyzed by high-performance liquid chromatography (HPLC) with an amylose tri-(3,5-dimethylphenylcarbamate) (ADMPC) chiral column. The degradation of racemic FE in three soils showed the herbicidally inactive S-(-)-enantiomer degraded faster than the active R-(+)-enantiomer. FE was configurationally stable in soils because no interconversion to the respective antipodes was observed during incubation of the enantiopure S-(-)- or R-(+)-FE. The main metabolites of FE were confirmed as FA and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB), and the formation of the chiral metabolite FA showed enantioselectivity in soils. The degradation of rac-FA was also enantioselective with the S-(-)-FA preferentially degraded: the half-life (t(1/2)) of the S-form in the three soils ranged from 2.03 to 5.17 days, and that of R-form ranged from 2.42 to 20.39 days. The inversion of the S-(-)-enantiomer into the R-(+)-enantiomer occurred in two of the three soils when the enantiopure S-(-)- and R-(+)-FA were incubated. The data from sterilized control experiments indicated that the enantioselectivity of FE and FA was attributed to microbially mediated processes.     

Enantiomeric resolution and biotoxicity of methamidophos

Despite the fact that the biological processes of chiral pesticides are enantioselective, the biotoxicity of methamidophos with respect to enantioselectivity has so far received limited research. In this study, the enantiomeric separation and biotoxicity of the two enantiomers of methamidophos were investigated. Successful enantioseparation of methamidophos using high-performance liquid chromatography on a Chiralcel OD column was achieved. As indicated by the optical rotation and circular dichroism detection, (+)-methamidophos was eluted prior to (-)-methamidophos. The t test at the 95% level of confidence indicated significant differences between the enantiomers in their in vitro inhibition toward acetylcholinesterases of bovine erythrocytes and Electrophorus electricus and in vivo acute aquatic toxicity to Daphnia magna. The in vitro assays showed that (-)-methamidophos was about 8.0-12.4 times more potent to the enzymes than its (+)-form. In contrast, the (+)-enantiomer was 7.0 times more toxic to D. magna in 48 h tests. The toxicity of racemic methamidophos was intermediate in both in vitro and in vivo bioassays. These results suggest that the biotoxicity of chiral OPs to nontargeted organisms is enantioselective and therefore should be reevaluated with their pure enantiomers.     

Enantioselective degradation and chiral stability of malathion in environmental samples

The dissipation behaviors of the two enantiomers of the organophosphorus pesticide malathion (MA) in environment samples were elucidated using a normal-phase high-performance liquid chromatography with a cellulose-tris(3,5-dimethylphenylcarbamate) (CDMPC) chiral column. A validated chiral residue analysis method in soil and water was established; the average recoveries for the two enantiomers were 88-102% in soil and 81-99% in water. Racemic and enantiopure R-(+)- and S-(-)-MA were incubated in five soil and water systems. The results of the degradation of racemate in all of the environment samples showed the inactive S-(-)-enantiomer degraded more rapidly than the active R-(+)-enantiomer, resulting in a relative enrichment of the R-form. Moreover, when the enantiopure S-(-)- and R-(+)-MA were incubated in three well-chosen soil and water samples, respectively, inversion from one enantiomer to another was found, indicating that using the optically pure enantiomer will not help to increase the bioactivity and reduce the environmental pollution.     

Application of capillary electrophoresis to study the enantioselective transformation of five chiral pesticides in aerobic soil slurries

The enantiomers of five chiral pesticides of environmental interest, metalaxyl, imazaquin, fonofos (dyfonate), ruelene (cruformate), and dichlorprop, were separated analytically using capillary electrophoresis (CE) with cyclodextrin chiral selectors. For metalaxyl, imazaquin, and fonofos, aqueous slurries of soil samples from two sites in Georgia and one in Ohio were spiked with the racemate of each pesticide at 50-60 mg/L of aqueous phase of the slurry, and CE analyses were performed at various time intervals to determine enantiomer fractions (EF). Metalaxyl underwent enantioselective transformation; in one soil, the half-life of the target active R-(+)-enantiomer was 17 days while that for the S-(-)-enantiomer was 69 days. Transformation occurred more slowly in the other two soils but was still selective for the R-(+)-enantiomer. Imazaquin and fonofos exhibited nonselective enantiomer loss over their 3 months of incubation time; this could have been due to abiotic or nonselective microbial reactions. Ruelene and dichlorprop were transformed selectively in a variety of soils in a previously reported study (7) that showed the influence of environmental changes on the transformation of chiral pollutants in soils; analytical methods used in that study are reported here to further illustrate the application of CE. CE is shown to be a simple, efficient, and inexpensive way to follow the transformation of chiral pesticides in laboratory microcosms where concentrations can be made high enough (25-50 mg/L initial racemate concentration) for detection of residual parent enantiomers during most of the process.     

Enantioselective residue dissipation of hexaconazole in cucumber (Cucumis sativus L.), head cabbage (Brassica oleracea L. var. caulorapa DC.), and soils

In this study, the enantioselective dissipation behavior of hexaconazole was investigated in cucumber fruit, head cabbage, and two different types of agricultural soils. The dissipation kinetics was determined by reverse-phase liquid chromatography-tandem mass spectrometry on a cellulose tris (3-chloro-4-methylphenylcarbamate) chiral column. Dissipation rates of hexaconazole enantiomers followed first-order kinetics; the residues of (+)-enantiomer decreased more rapidly than (-)-enantiomer in cucumber and head cabbage, resulting in relative enrichment of the (-)-form, while the two enantiomers showed similar degradation rates in the tested soils. These results indicate substantial enantioselectivity in the residue dissipation of hexaconazole enantiomers in cucumber and head cabbage; however, nonenantioselective dissipation was observed in the tested soils.     

Enantioselective degradation and ecotoxicity of the chiral herbicide diclofop in three freshwater alga cultures

Aryloxyphenoxypropanoates are a class of chiral herbicides. They have a pair of enantiomers, only the R(+) form of which is herbicidally active. Diclofop, the model compound of these herbicides, is commercialized as the racemate of the ester form, diclofop-methyl, consisting of a 1:1 mixture of the enantiomers. This study evaluated the enantioselectivity in aquatic toxicity and biodegradation of diclofop and diclofop-methyl. The herbicidally inactive S(-) enantiomers of both diclofop-methyl and diclofop were similar to or higher than the corresponding R(+) forms in toxicity to algae, depending on specific species. Although no enantiomeric conversion occurred for diclofop-methyl and diclofop, the difference in the enantioselective degradation of these herbicides observed in algae cultures suggested that their application forms were an important factor determining their enantioselective environmental behavior. The cell permeability and heat treatment of algae revealed that the enantioselective degradation of diclofop in algae cultures was governed primarily by the facilitated uptake by algae, whereas the enantioselective toxicity was primarily governed by the passive uptake. These results suggested that the acute toxicity test such as the 96 h EC 50 was insufficient to assess the ecological risk of chiral pesticides because of the differential degradation as well as possibly differential action sites of enantiomers. From this study, it was concluded that the enantioselective degradation and toxicity of chiral herbicides may result in their ecotoxicological effects being difficult to predict and that specific attention should thus be paid to currently used racemic pesticides as less active or inactive enantiomers may pose higher ecological risks.     

Determination of gossypol enantiomer ratio in cotton plants by chiral higher-performance liquid chromatography

A celulose tris(3,5-dimethylphenylcarbamate) coated onto APS silica (Nucleosil, particle size, 7 microm; pore size, 500 A) was used under a reversed-phase condition to measure the enantiomeric ratios of gossypol enantiomers in cottonseeds, flowers, and roots in a number of cultivars samples of different Gossipium species. While unidimensional chromatography was used for measuring the enantiomeric ratio of all the samples of G. hirsutum, G. mustelinum, and in the seeds of G. barbadense, multidimensional chromatography was necessary for the analysis of samples of roots and flowers of G. barbadense. In the latter case, an ODS Hypersil column was used in the first dimension for sample clean up, and the enantiomers were resolved on the second dimension by the chiral column. As expected, all the seed samples of G. hirsutum and G. mustelinum showed the (P)-(+)-enantiomer in excess, whereas the seeds of G. barbadense showed the (M)-(-)-enantiomer. However, (P)-(+)-gossypol was found in enantiomeric excess in three samples examined of roots and in one of flower of G. barbadense. These results are discussed in this paper.     

Single and joint acute toxicity of isocarbophos enantiomers to Daphnia magna

Although enantioselectivity in the toxicity of chiral pesticides has received considerable attention over recent years, how coexisting enantiomers interact with each other during their toxic action remains unknown. In this study, we attempted to resolve the enantiomers of a chiral organophosphate insecticide, isocarbophos, and investigated the acute toxicity of individual enantiomers and various enantiomer mixtures. Baseline enantiomeric separation of isocarbophos was achieved on a Chiralcel OD column with the mobile phase of n-hexane/isopropanol (90/10, v/v) at a flow rate of 0.8 mL/min. The resolved enantiomers were differentiated by their responses on a circular dichroism detector. The median lethal concentrations (LC 50) of racemate, (+)-enantiomer, and (-)-enantiomer of isocarbophos toward Daphnia magna were 13.9, 7.08, and 353 microg/L, respectively, after 48 h of static exposure, displaying a 50-fold difference between the enantiomers. Toxic unit (TU) analysis was employed to evaluate the joint toxicity of isocarbophos enantiomer mixtures. The calculated TU mix for the acute toxicity (48 h test) of various binary mixtures ranged from 0.83 to 1.04, suggesting a mode of additive effect. Further evaluation of available literature data for chiral organophosphorus insecticides showed that the joint toxicity of enantiomers may be additive, synergistic, and antagonistic. Therefore, when significant enantioselectivity exists for a chiral pesticide, it is important to also evaluate the interaction of enantiomers in the joint toxicity effect when enantiomers are present in a mixture.     

Different enantioselective degradation of pyraclofos in soils

This study investigated the enantioselective degradation behavior of pyraclofos in three soils (NC, HZ, and ZZ) under native and sterilized conditions. The absolute configuration of pyraclofos enantiomers has been determined by the combination of experimental and calculated electronic circular dichroism spectra. S-(+)- and R-(-)-Pyraclofos were separated and determined on a cellulose tri-(4-chloro-3-methylphenylcarbamate) (Lux Cellulose-4) chiral column by reversed-phase high-performance liquid chromatography-tandem mass spectrometry. Pyraclofos enantiomers were configurationally stable in three soils and no interconversion was observed during the incubation of enantiopure S-(+)- or R-(-)-pyraclofos under native conditions. The enantioselective degradation behavior of chiral pyraclofos was dramatically different in three soils under native conditions, with half-lives (t(1/2)) of pyraclofos in NC, HZ, and ZZ soils of 2.6, 13.4, and 7.8 days for S-(+)-pyraclofos and 9.2, 9.3, and 8.2 days for R-(-)-pyraclofos. Compared to the half-lives (t(1/2)) of rac-pyraclofos of 21.5, 55.9, and 14.4 days in sterilized NC, HZ and ZZ soils, the degradation velocity was greatly improved in native soils, indicating that degradation was greatly attributed to microbially mediated processes in agricultural cultivating soils.     

Biogenetic studies in Mentha x piperita. 2. Stereoselectivity in the bioconversion of pulegone into menthone and isomenthone.

Mentha x piperita shoot tips and first leaf pairs were fed with aqueous solutions of different deuterium-labeled pulegone and various enantiomeric distributions. The essential oil was extracted by solid-phase microextraction and analyzed using enantioselective multidimensional gas chromatography/mass spectrometry. The genuine p-menthan-3-ones (-)-menthone and (+)-isomenthone as well as their labeled analogues were analyzed simultaneously. Both enantiomers of labeled pulegone were converted into the corresponding labeled p-menthan-3-ones by Mentha x piperita, indicating an unspecific reduction process. The generation of 4S- and 4R-configured p-menthan-3-ones differed in their stereoselectivities. Labeled (S)-pulegone was reduced by Mentha x piperita more rapidly rather than (R)-pulegone. From a comparison of labeled pulegone enantiomers the bioconversion preferrably led to 4S-configured diastereomers.     

Toxicity of (+)- and (-)-gossypol to the plant pathogen,Rhizoctonia solani

The dimeric sesquiterpene gossypol occurs naturally in cottonseed and other parts of the cotton plant. Gossypol exists as enantiomers because of the restricted rotation around the central binaphthyl bond. The (-)-enantiomer is toxic to nonruminant animals while the (+)-enantiomer exhibits little, if any, toxicity to these animals. Developing cotton plants with low levels of the (-)-gossypol could expand the use of cottonseed as a feed source. Gossypol also may play a role in protecting the plant from pathogens. The relative toxicity of (+)- and (-)-gossypol to plant pathogens has not been reported. We measured the concentration of (+)- and (-)-gossypol in roots from cotton seedlings that were treated with the biocontrol agent Trichoderma virens that induces biosynthesis of gossypol and related terpenoids in cotton roots. (-)-Gossypol was the minor enantiomer in control and treated roots, but levels were slightly higher in roots from T. virens-treated seed. We also determined the toxicity of the gossypol enantiomers and the racemate to the seedling disease pathogen Rhizoctonia solani. The (+)- and (-)-enantiomers of gossypol and the racemate are equally effective in inhibiting growth of this pathogen. The lethal doses of the gossypols required to kill the pathogen appeared to be similar, but their toxicities are significantly less than those of related cotton and kenaf sesquiterpenes. The results indicate that altering the enantiomeric ratio in cotton roots will not adversely affect the resistance of seedlings to the seedling pathogen R. solani.     

基质类型及烯效唑对不同秧龄晚稻机插质量和产量的影响

为明确基质类型及烯效唑对不同秧龄晚稻秧苗生理特征及本田期生长发育特性的影响,确定适宜晚稻机插育秧的基质类型、烯效唑浸种措施及秧龄,该研究以嘉58为供试晚稻品种,以2种代表性育秧介质(营养土和轻型无土育秧基质)为研究对象,采用2种(清水浸种和25 mg/L烯效唑浸种)外源生长调控措施,阐明了不同育秧介质和烯效唑浸种对不同秧龄晚稻秧苗生理特征、机插质量的影响及调控技术。结果表明,随晚稻秧龄延长,秧苗地上部分干物质量显著增加,叶片SPAD值和根系活力先增加后降低;水稻全生育期延长,大田生育期缩短。采用烯效唑浸种可提高秧苗素质,浸种处理秧苗茎基宽约增加0.1~0.2 mm,株高显著降低,叶片SPAD值和根系活力显著提高。短秧龄条件下烯效唑浸种对秧块质量和机插质量影响较小,长秧龄条件下烯效唑浸种可有效提高成苗率和大田基本苗数,降低漏插率和伤秧率。采用营养土育秧时最适机插秧龄为18 d,轻型无土基质育秧适宜机插秧龄为18~24 d。秧龄大于24 d时,烯效唑浸种可提高营养土育秧晚稻有效穗数和每穗粒数,增加晚稻产量,且秧龄越长,烯效唑浸种的增产效果越显著。烯效唑浸种对轻型无土基质育秧的产量影响较小。采用轻型无土育秧基质培育机插晚稻秧苗,产量高于常规营养土处理,适宜晚稻机插育秧。研究结果可为提升晚稻种植机械化水平提供重要参考。     

Determination of enantiomers of synthetic pyrethroids in water by solid phase microextraction - enantioselective gas chromatography

Solid phase microextraction (SPME) is an ideal sample preparation technique because of its speed and solvent-free features. Sampling by SPME is selective and only the dissolved concentration is measured, which allows measurement of the bioavailable fraction of a contaminant in aqueous media. One potential application of SPME is for analysis of enantiomers of chiral contaminants in environmental samples. In this study, a method was developed for determining enantiomers of (Z)-cis-bifenthrin and cis-permethrin in water using coupled SPME and enantioselective gas chromatography (GC). Following SPME sampling, enantiomers of (Z)-cis-bifenthrin and cis-permethrin were separated at the baseline on a beta-cyclodextrin-based enantioselective column, and analyte enrichment onto the SPME fiber was not enantioselective. The GC response increased as sampling time was increased from 0 to 240 min, and as sampling temperature was increased from 20 to 40 degrees C. Organic solvents such as methanol, acetone, and acetonitrile enhanced, while soil extracts slightly decreased, the GC response. The integrated SPME-enantioselective GC method was used to analyze surface runoff samples. The analysis showed preferential degradation of the 1S-3S enantiomer over the 1R-3R enantiomer for both (Z)-cis-bifenthrin and cis-permethrin. The concentrations detected by SPME-GC were substantially smaller than those determined following solvent extraction, suggesting that SPME-enantioselective GC analysis selectively measured the dissolved fraction.     

Enantioselective analysis of triazole fungicide myclobutanil in cucumber and soil under different application modes by chiral liquid chromatography/tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of myclobutanil enantiomers by chiral liquid chromatography coupled with tandem mass spectrometry. The separation and determination were performed using reversed-phase chromatography on a Chiralcel OD-RH column, with ACN-water (70/30, v/v) as the mobile phase under isocratic conditions at 0.5 mL/min flow rate. The matrix effect, linearity, precision, accuracy, and stability were evaluated. The proposed method then was successfully applied to the study of enantioselective degradation of rac-myclobutanil in cucumber and soil under different application modes. The results showed that the preferential degradation of (+)-myclobutanil resulted in an enrichment of the (-)-myclobutanil residue in plant and soil. Moreover, in cucumber, the stereoselective intensity of myclobutanil under root douche treatment was stronger than that under foliar spraying treatment, whereas in soil, the intensity was exactly opposite. The probable reasons underlying these enantioselective effects were also discussed. This study highlighted the importance of examining the fate of both enantiomers in the greenhouse system for the correct use of chiral pesticides.     

Enantiomeric separation and toxicity of an organophosporus insecticide, pyraclofos

Despite the fact that the biological processes of chiral pesticides are enantioselective, knowledge of the toxicities of pyraclofos due to enantiospecificity is scarce. In this study, the optical isomers of pyraclofos were separated and their toxicities to butyrylcholinesterase (BChE) and Daphnia magna were assessed. Baseline resolution of the enantiomers was obtained on both Chiralcel OD and Chiralpak AD columns. The effect of the mobile phase composition and column temperature were then discussed. The resolved enantiomers were characterized by their optical rotation and circular dichroism signs. The anti-BChE tests demonstrated that (-)-pyraclofos was about 15 times more potent than its (+)-form. However, acute aquatic assays suggested that (+)-pyraclofos was about 6 times more toxic than its antipode. Moreover, the joint toxicity of pyraclofos enantiomers to D. magna was found to be an additive effect. These results demonstrated that the overall toxicity of pyraclofos should be assessed using the individual enantiomers.     

香石竹GA20-oxidase基因的克隆及RNA干扰载体的构建

根据已发表的菠菜、烟草等植物GA 20-oxidase基因序列在保守区设计简并引物,通过RT-PCR和RACE的方法克隆了Marster香石竹（Dianthus caryophyllus L. cv. Marster）GA 20-oxidase基因的全长cDNA（1 179 bp）,命名为Dc20ox。同源性分析表明该基因与其它作物上发表的GA 20-oxidase基因的氨基酸序列同源性为66%~75%。在此基础上选用同源性相对较高的400 bp DNA片段,构建了RNA干扰（RNAi）载体pART400。     

Enantiomeric resolution of chiral pesticides by high-performance liquid chromatography

Successful enantiomeric separation of 10 chiral pesticides by high-performance liquid chromatography (HPLC) using cellulose-tris(3,5-dimethylphenylcarbamate) (CDMPC) chiral stationary phase (CSP) was performed. The mobile phase was n-hexane modified by ethanol, propanol, 2-propanol (IPA), butanol, or isobutanol. The effects of mobile phase composition and column temperature on the separation were investigated. Baseline separation was obtained with ethofumesate, fluroxypyr-meptyl, malathion, benalaxyl, diclofop-methyl, methamidophos, vinclozolin, and lactofen, whereas near baseline separation was obtained with profenofos and acetochlor. Butanol was the best modifier for benalaxyl; isobutanol was the best modifier for lactofen, malathion, diclofop-methyl, and ethofumesate; and IPA was the best modifier for the other five. Better separations were not always at low temperature. The elution orders of the eluting enantiomers were determined by a circular dichroism (CD) detector. The quantitative analysis methods for the enantiomers of ethofumesate, benalaxyl, and diclofop-methyl were established. Validation parameters include linearity, precision, and limit of detection (LOD). The enantiomeric residual analysis procedures in soil and water samples were also developed using acetone extraction and C(18) solid phase extraction. The methods were reliable for residual analysis of the enantiomers.     

Structure elucidation, enantioselective analysis, and biogenesis of nerol oxide in Pelargonium species.

A novel enantioselective synthesis of nerol oxide (3, 6-dihydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran) was used for the determination of the absolute configuration at C-2. The order of elution of the enantiomers on octakis-(2, 3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-gamma-cyclodextrin in OV 1701-vi as the chiral stationary phase in enantioselective GC was determined as (2R) before (2S). Enantioselective multidimensional GC/MS (enantio-MDGC/MS) was used for the determination of the enantiomeric ratios of nerol oxide in different geranium oils. As a result, in all investigated oils nerol oxide occurs as a racemate. The biogenesis of nerol oxide in Pelargonium species was investigated by feeding experiments using deuterium-labeled neryl glucoside as the precursor. The Pelargonium plants were able to convert the fed precursor into racemic nerol oxide, which has to be considered as a "natural racemate".     

Distribution and organoleptic impact of sotolon enantiomers in dry white wines

The enantiomers of sotolon, a flavor compound typical of oxidized white wines, were separated by preparative HPLC to determine their perception thresholds and distribution in wines. The enantiomeric ratios of chiral sotolon were evaluated in several dry white wines using gas chromatography and a chiral column (beta-cyclodextrin) connected to a 2 m precolumn (BP20). The perception threshold of (S)-sotolon (0.8 microg/L) in model wine solution was 100 times lower than that of the (R) form (89 microg/L), indicating that (S)-sotolon contributes to the characteristic aroma of prematurely aged dry white wines. Both enantiomers are detected in white wines. Analysis of commercial dry white wines from various vintages and origins revealed three types of distribution patterns: the racemic form, an excess of R, and an excess of S. The proportions found in these wines may be partially explained by the slow racemization kinetics (20 months) of optically active sotolon.