| Pesticides play an important role in the process of agriculture practice, and they are used to improve the production and quality of the agricultural products. Studies of the mode of action and characteristics of pesticides can provide support and guidance for developing effective and safe new agrochemicals. Consulting the current hot research issues in pesticides, enantioselective toxicity of chiral fungicide tebuconazole to non-target aquatic organism Daphnia manga and binding sites differences of two diamides on RyRs are studied in the present study and the main findings are listed below:1. Studies of toxicity differences of the tebuconazole and its enantiomers’to Daphnia magnaEnatiomers of tebuconazole, R-and5-teb were prepared using HPLC on CDMPC column and the48h acute immobilisation,21d reproduction and14d offspring recovery tests were performed to tell the toxicity differences of rac-, R-and5-teb. The48h EC50s of rac-,R-and5-teb to Daphnia were3.5(3.3-3.8) mg/L,3.3(3.0-3.8) mg/L and2.7(2.3-3.1) mg/L respectively. They are all medium toxic to Daphnia and have no significant toxicity differences. Based on the1/10of48h EC50s, neonates were exposed to0.01,0.05,0.10,0.20,0.30and0.40mg/L of rac-, R-and S-teb for21d reproduction test. During these exposures, rac-,R-and S-teb significantly reduce the total number of offspring [lowest-observed-effective-concentration (LOEC, mg/L) were0.05,0.05,0.10respectively), total number of broods (LOEC-0.30,0.05,0.10respectively), and size of the1st and3rd brood for per daphnia (LOEC-0.05,0.20,0.20and0.05,0.20,0.10respectively). Otherwise, the development of Daphnia was also impacted seriously as the molting retes (LOEC-0.20,0.30,0.10respectively) were reduced and the age at maturity (LOEC->0.40,0.20,>0.40respectively), days to the1st and3rt brood reproduction (LOEC-0.20,0.20,>0.40and0.10,0.10,0.10respectively) were all significantly delayed as well as the body legth (LOEC0.05,0.05,>0.40respectively). Under the same concentration, there are significant differences among the three tebs and the toxicity order is S>rac-b≥R-teb, which indicating the obvious stereoselective toxicity among tebs. The14d F1offspring-generation Daphnia recovery test showed that the impact of rac-teb on F1offsprings can generally recover when exposure concentrations to Fo Daphnia lower than0.20mg/L, but can’t be recoverd when concentrations higher than0.20mg/L. The toxic effects of S-tebs on F1offspring is>rac-≥R-teb. The lowest LOEC of S-teb to observed paramets is just0.01mg/L and no F2offsprings were found under0.30and0.40mg/L exposure. By now, China has no standards limiting the residue amount of teb in water but0.2-4.16μg/L of rac-teb was calculated in Brazil groundwater and as by USEPA records, teb in water generally should be lower than13μg/L. Comprehensively considering the significant unrecovery effects of rac-, R-and D-teb on the F1offspring-generation Daphnia at0.01mg/L, it is reasonable to speculate that rac-, R-and S-teb may impact the fecundity and development of daphnia in long-time exposure even the concentration of teb is lower than10μg/L. Thus, in general use of teb, it is not recommended to separate the enantiomers to improve the safety to non-target aquatic organisms and it is necessary to establish monitoring standards of teb in water.2Selective studies of binding sites of diamide insecticidesThis part of study was achieved by collaborations with Environmental Chemistry and Toxicology Laboratory at University of California at Berkeley. New radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole (Chlo) was synthesized at high specific activity and compared with phthalic diamide insecticide [3H]flubendiamide (Flu) and [3H]ryanodine (Ry) in radioligand binding studies with muscle membranes of different species to chosed to define species differences in the degree and mechanisms of diamide selective action at the binding sites.Lobster, rabbit and four insect species, house fly (Musca domestica), honey bee(Apis mellifera), tobacco budworm(Heliothis virescens) and black cutworm(Agrotis ipsilon) are used to prepare RyR membarnes, and the effect of ATP, Ca2+, Ry, Flu, Chlo and Cyan on three radioligands binding are calculated. Through the radioligand specific binding studies, the [3H]Ry site is observed in muscle of lobster, rabbit and four insect species whereas the [3H]Chlo site is evident in the four insects and the [3H]Flu site in only the two lepidoptera.[3H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit.[3H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory.[3H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis.[3H]Chlo and [3H]Flu binding are not dependent on added Ca2+or ATP whereas the other radioligand-receptor combinations are enhanced by Ca2+and ATP. A possible relationship is therefore evident between the Flu site but not the Ry and Chlo sites in the Ca2+and ATP modulation mechanisms. Using Heliothis with [3H]Chlo and [3H]Flu allows binding site structure-activity correlations for anthranilic and phthalic diamides. The anthranilic and phthalic diamide analogs stimulation of [3H]Ry binding greatly parallels their inhibition of [3H]Chlo and [3H]Flu binding. In Musca adults, anthranilic diamides stimulate [3H]Ry binding and inhibit [3H]Chlo binding. Phthalic diamide stimulate [3H]Chlo binding but has no significant effect on [3H]Ry binding. These results showed that two diamides have obvious species selectivity, Chlo and Flu have two different but coupled binding sites on Musca and Apis RyR, but they have same binding site on lepidoptera insect RyR. These observations establish species differences in diamide target site specificity and are important in considering diamide structure optimization and evaluation of resistance mechanisms. |
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