Physiological Ecological Effects In Earthworms Treated With Four Herbicides And Their Degradation In Soils

Herbicides have a broad scope of application, but excessive and unreasonable use of herbicides has lead to many environmental and ecological issues. Athough the production of world's herbicide since 1980s has been mainly focused on producing those kinds of high efficiency, low toxicity, broad-spectrum and low dosage such as Atrazine, Chlorotoluron, Acetochlor, and Butachlor, the ecological effects of these four kinds of herbicides in soils have not been well understood. So far herbicides' behavior in the environment and its effect on soil ecological system need further investigation.On the basis of the survey of agricultural herbicide usage in Kunming,Yunnan, through the incubation of earthworm (Elsinia foetida) simulated as experimental material in field and lab pot by the regular treatment dosage of herbicides, we studied time-effect and dose-effect relationship of SOD,CAT,HSP27 and HSP 40 in the earthworms under treatment of the four herbicides, and the degradation of the herbicides in soil. In this paper, we reported the effect of the four herbicides on the activities of SOD and CAT, the expression of HSP 40, HSP27 in earthworms, and urease activity in soil, and discussed the degradation dynamics of herbicides. The goal of this work is to provide the theoretical basis for the reasonable usage and ecological safety evaluation of the four herbicides. The results and conclusions come as follows:(1) According to the result of herbicides'effect on the biomass of earthworm, the effect on the fresh weight of earthworm indicates that the lower concentration of herbicide exposure dose resulted in less weight loss in earthworm; With the prolongation of treatment time of the four herbicides, the less fresh weight of earthworm declined in accordance with the higher exposure concentration, but different exposure concentration of the four herbicides lead to different starting time of the decline of earthworm's fresh weight to different extents.(2) The SOD activity of earthworm increased and then decreased when treatment concentration was set a certain dosage, but it differed in expression time. For example, when the exposure concentration of Atrazine was lower than 126 mg/kg, the highest SOD activity appeared after 7 d treatment, and appeared after 10 d when treatment concentration was higher than 316 mg/kg. As to Chlorotoluron, there was a remarkable increase in SOD activity when the concentration was below 100mg/kg and significant decreased when it was above 158mg/kg. When the treatment concentration of Acetochlor was lower than 33mg/kg, the SOD activity was shown highest in 7 d, then it decreased. When treatment concentration was lower than 13mg/kg, the greatest SOD was induced; when it was above 83mg/kg the activity of SOD decreased dramatically. When the concentration of Butachlor was lower than 50mg/kg, the SOD activity is promoted to the highest in 7 d, then SOD activity decrease. According to two-factor analysis of variance showed that exposure concentration of Atrazine, Acetochlor and Butachlor and treatment time, relationship between concentration and time had significant effects on SOD activity in earthworm (P<0.001). while Chlorotoluron treatment time, relationship between time and concentration applied also had remarkable effects on the SOD activity in earthworm (P<0.001).(3) With the prolongation of Acetochlor treatment, CAT activity in earthworm ranged from 284.8U/g to 335.5U/g protein, averaged at 309.68U/g protein, while the CAT activity in earthworms increased at first and then declined. Atrazine below 126mg/kg could significantly increase the CAT activity, but it would be remarkablely inhibited when Atrazine treatment was above 316mg/kg; when Chlorotoluron was applied at the concentration less than 100mg/kg, it could significantly enhance the CAT activity; if treatment concentration was higher than 158mg/kg CAT activity was significantly restrained. Two-factor analysis of variance showed that exposure concentration of Atrazine, Acetochlor and Butachlor. and its treatment time, relationship between concentration and time had significant effects on CAT activity in earthworm (P<0.001), while Chlorotoluron treatment time, relationship between treatment time and concentration applied did not show significant effects on the CAT activity in earthworm (P=0.78,>0.05).(4) Being exposed with four herbicides, earthworm had new expression in heat shock protein (HSP). By Western Blotting Assay, it was been proved that the molecule with 27kDa was HSP27 and 40kDa was HSP40. Compared to the control test, with the extension of treatment time the expression level of HSP27 and HSP40 first rose and then decreased. The expression of HSP27 and HSP40 in earthworm was the most sensitive to Atrazine exposure, and then followed by Acetochlor, Butachlor and Chlorotoluron, which was consistent with the more lipophilic herbicides demonstrated greater toxicity. The expression curve of the HSP27 and HSP40 in earthworm tissues exposed to herbicide represented inverted U-shaped curves featuring dose-effect. Two-factor analysis of variance showed that exposure concentration of Atrazine, Acetochlor and Butachlor, and its treatment time, relationship between concentration and time had significant effects on the expression of HSP27 and HSP40 in earthworms (P<0.001), while Chlorotoluron treatment time, treatment concentration applied showed significant effects only on the HSP27 expression in earthworm (P<0.001).(5) With the extension of treated time, the effect of herbicides at the same treatment concentration on the activity of soil urease showed increase, then decreased. Herbicides'effects on the soil urease activity represented parabola-shaped curves. Two-factor analysis of variance showed that exposure concentration of Atrazine, Acetochlor and Butachlor, and its treatment time, relationship between concentration and time had significant effects on soil urease activity of earthworm (P<0.001), while only Chlorotoluron treatment time, treatment concentration applied showed significant effects on the urease activity of earthworm (P<0.001).(6) The kinetics of the degradation of these four herbicides showed linear kinetic equation, the degradation curve at lower treatment concentrations smoothly went down, while it revealed a rapid decline at higher concentrations. Ranged within the tested concentration, with the increasing concentrations of herbicide treatment, the half-life of degradation became shorter. When earthworm Elsinia foetida was not vaccinated into soils, the half-life of Atrazine, Chlorotoluron, Acetochlor and Butachlor differed in the range of 26.4-28.3d,51.36-42.03d,11.2-6.50d and 14.7-8.60d. After inoculation of earthworms, the half-life of Atrazine, Chlorotoluron, Acetochlor and Butachlor decreased as 24.2-25.6d,41.24-48.56 d.5.2-10.1d and 6.30-12.50d.Analysis of variance showed that treatment concentration and treatment time significantly affected the degradation rate of the four kinds of herbicides in the soil. Although the inoculation of earthworms could reduce the half-life of herbicide degradation in soil or might accelerate degradation, but the role that earthworms played in this process needed further studied.