Bioactivity Difference Of Difenoconazole Stereoisomers And Their Stereoselective Environmental Behavior In Paddy Field

Difenoconazole, a broad spectrum systemic triazole fungicide, has been extensively used in paddy-growing system. It has two chiral element and four stereoisomers. The stereoisomers of difenoconazole may differ dramatically in bioactivity to pathogens, toxicity to non-target organisms, and degradation in plants and environment. In conventional risk evaluations, the chiral pesticide was often treated as one compound, and the data obtained were incomplete and nonspecific, bringing potential risks posed by the fungicide to environment and public health. However, little work was made to date about bioactivity and stereoselectivity degradation of difenoconazole stereoisomers in paddy-growing system.This paper reports the resolution and determination of the chiral triazole fungicide difenoconazole, in water, soil, paddy plant, rice and rice hull by UPC2–MS/MS. The separation was studied using three different polysaccharide-type chiral stationary phases inculding amylose tris-( 3, 5-dimethylphenylcarbamate), cellulose tris-(3, 5-dimethylphenylcarbamate) and cellulose tris-(3, 5-dichlophenylcarbamate) in combination with co-solvent of n-butanol/methanol. Compared with other CSPs, cellulose tris-(3, 5-dimethylphenylcarbamate)(Chiralpak? IB-3) exhibited higher resolving ability for chiral difenoconazole. Baseline resolution(Rs ≥ 1.56) of and high selectivity(LOQ ≤ 0.02 mg kg-1) for the four stereoisomers were achieved by postcolumn addition of 1.0 % formic acid–methanol to a Chiralpak IB-3 using CO2/n-butanol/methanol as the mobile phase at 25℃, 2200 psi, and for 8.5 min in electrospray ionization positive mode. The elution order of the stereoisomers was(2R, 4R)-stereoisomers,(2S, 4S)-stereoisomers,(2S, 4R)-stereoisomers and(2R, 4S)-stereoisomers with the retention time are 5.96, 6.38, 6.82 and 7.37 min, respectively. The interday mean recovery, intraday repeatability and interday reproducibility varied from 70.3 to 96.3 %, 0.7 to 9.7 % and from 1.4 to 10.2 %, respectively. The proposed method was used to study the pharmacokinetic dissipation of difenoconazole stereoisomers in paddy-growing system under open-field experiment.Next, the bioactivity of individual stereoisomers of difenoconazole against a number of fungi including Fusarium moniliforme Sheld., Pyricularia oryzae Cav., Rhizoctonia solani kühn and Ustilaginoidea oryzae(Patou.)Bref. were compared. Differences were observed in the bioactivity among the stereoisomers of difenoconazole on the test fungi. The bioactivity for the stereoisomers of difenoconazole have been found to be(2S, 4R)-stereoisomers >(2R, 4R)-stereoisomers >(2R, 4S)-stereoisomers >(2S, 4S)- stereoisomers. The ED50 ratio of enantiomers [(2R, 4R)-stereoisomers and(2S, 4S)-stereoisomers] for the fungi of Fusarium moniliforme Sheld., Pyricularia oryzae Cav., Rhizoctonia solani kühn, Ustilaginoidea oryzae(Patou.)Bref. were 0.28, 0.66, 0.29 and 0.62,respectively. The(2R, 4R)-stereoisomers was eudomer. The ED50 ratio of enantiomers [(2R, 4S)-stereoisomers and(2S, 4R)-stereoisomers] for this fungi were 2.57, 1.60, 1.38 and 1.44,respectivety. The(2S, 4R)-stereoisomers was eudomer.Then, the stereoselectivity of difenoconazole stereoisomers in paddy plant, soil and water was studies. The results indicated that the degradation of the four stereoisomers of difenoconazole followed first-order kinetics. The dissipation rates of the four stereoisomers followed the order of paddy plant > water > soil. In paddy plant, the enantiomers fraction of(2R, 4R)-stereoisomers and(2S, 4S)-stereoisomers(EFA) and the enantiomers fraction of(2R, 4S)-stereoisomers and(2S, 4R)-stereoisomers(EFB) are lower than 0.5, respectively. The(2R, 4R)-stereoisomers and(2R, 4S)-stereoisomers were preferentially degraded, resulting in relative enrichment of(2S, 4S)-stereoisomers and(2S, 4R)-stereoisomers. In soil, the EFA is equal to 0.5 as well as EFB is little higher than 0.5, respectively. The(2S, 4R)-stereoisomers were preferentially degraded, resulting in relative enrichment of(2R, 4S)-stereoisomers in soil. In water, the stereoselectivity was weakly because EFA and EFB are equal to 0.5.At last, difenoconazole was sprayed onto some plots four and other plots five times, with 7 days between applications. Representative samples were collected from each plot at pre-harvest intervals of 7, 14, and 21 days. The concentration of the four stereoisomers followed the order of paddy plant > rice hull > rice > soil. The stereoselective behavior was observed in paddy-growing system. In paddy plant and soil samples, the EFA is lower than 0.5 as well as the EFB is higher than 0.5,(2S, 4S)-stereoisomers and(2R, 4S)-stereoisomers ease to enrichment, respectively. In rice hull and rice samples, the EFA and EFB are higher than 0.5,(2R, 4R)-stereoisomers and(2R, 4S)-stereoisomers ease to enrichment, respectively.