四种除草剂对蚯蚓生理生态的影响及其降解特征
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摘要
除草剂使用范围非常广泛,然而过量和不合理使用,引起了一系列的生态和环境问题。上世纪80年代后世界主要发展高效、低毒、广谱、低用量的除草剂,一般认为比较低毒的阿特拉津、绿麦隆、乙草胺和丁草胺近年来被广泛使用的除草剂,其产量和用量非常大,但是它们在环境中的行为以及对土壤生态系统的影响尚缺乏比较系统的研究。
本文在调查昆明市农业生产中除草剂使用状况的基础上,以赤子爱胜蚓为材料,模拟常规除草剂使用剂量,在室外模拟培养环境下研究了蚯蚓SOD、CAT等氧化酶及热激蛋白HSP27和HSP40对各类除草剂的时间一效应和剂量—效应关系,分析了它们对土壤脲酶活性的影响,探讨了除草剂在土壤中的降解动态,为这些除草剂的合理使用和生态安全性评估提供数据支持。主要结果如下,
(1)除草剂对蚯蚓生物量的影响的结果表明,除草剂浓度对蚯蚓鲜重的影响总体表现为暴：露剂量越小,体重降低程度越小,暴露剂量越高,体重降低越大。各种浓度除草剂处理的蚯蚓鲜重呈降低趋势。随着处理时间的延长,蚯蚓鲜重随着处理浓度的提高而降低,不同处理浓度引起蚯蚓鲜重降低开始的时间不同,不同剂量最后引起的降低程度不同。
(2)各种处理条件下除草剂对蚯蚓SOD活性的影响均表现为先升高后降低,只是SOD最高活性出现的时间早晚有差异。小于126mg/kg低浓度阿特拉津处理中蚯蚓SOD活性在第7d最高,大于316mg/kg的高浓度阿特拉津处理在第10d蚯蚓SOD活性最高；绿麦隆在低于1OOmg/kg时显著促进SOD活性,高于158mg/kg时SOD活性显著受到抑制；乙草胺在小于33mg/kg范围内促进SOD活性,SOD活性在第7d最高,之后SOD活性降低,低浓度13 g/kg处理被诱导程度最大,而大于83mg/kg时对SOD活性有极显著抑制作用；丁草胺浓度在小于50mg/kg内促进SOD活性,SOD活性在第7d最高,之后SOD活性降低,低浓度32mg/kg处理被诱导程度最大,而大于126mg/kg时对SOD活性有极显著抑制作用。根据双因素方差分析结果表明,阿特拉津、乙草胺和丁草胺处理浓度与时间及浓度与时间的交互作用均对土壤中蚯蚓体组织SOD活性具有极显著的影响(P<0.001)。绿麦隆处理时间、时间与浓度的交互作用均对土壤中蚯蚓体组织SOD活性具有极显著的影响(P<0.001)。
(3)随着除草剂暴露时间的延长,CAT活性对乙草胺处理变化范围为284.8-335.5U/g蛋白,平均为309.68 U/g蛋白,而各处理蚯蚓CAT活性表现为先升高后降低。阿特拉津在低于126mg/kg时显著促进CAT活性,达到316mg/kg以上时对CAT活性有显著抑制作用；绿麦隆在低于1OOmg/kg时,显著促进CAT活性；高于158mg/kg对CAT活性有极显著抑制作用。双因素方差分析结果表明,阿特拉津、乙草胺和丁草胺处理浓度与时间及浓度与时间的交互作用均对蚯蚓体CAT活性具有极显著的影响(P<0.001)；绿麦隆处理浓度对蚯蚓CAT活性具有极显著的影响(P<0.001),而与时间、浓度与时间的交互作用对土壤中蚯蚓体组织CAT活性的影响不显著(P=0.78,>0.05)。
(4)暴露于四种除草剂的处理条件下,蚯蚓体内的热激蛋白(HSP)有新的表达,经Western Blotting免疫印迹检测,确定27kDa为HSP27,40kDa为HSP40。相对于对照而言,随着处理时间的延长,HSP27和HSP40的表达强度先升高后降低。HSP27和HSP40对阿特拉津暴露最为敏感,然后依次为乙草胺、丁草胺和绿麦隆。蚯蚓体组织HSP27和HSP40对除草剂的表达曲线都呈现出倒U型剂量—效应特征。双因素方差分析结果表明,阿特拉津、乙草胺及丁草胺处理浓度与时间及浓度与时间的交互作用均对蚯蚓HSP27和HSP40具有极显著的影响(P<0.001)；绿麦隆处理浓度、时间均对蚯蚓HSP27具有极显著的影响(P<0.001)。
(5)对同一处理浓度而言,随着处理时间的延长,除草剂对土壤脲酶活性的影响表现是先呈升高趋势,随着作用时间的延长脲酶活性下降。除草剂对土壤脲酶活性的影响呈现出抛物线型。双因素方差分析结果表明,阿特拉津、乙草胺和丁草胺处理浓度、时间及浓度与时间的交互作用均对土壤中蚯蚓脲酶活性具有极显著的影响(P<0.001)。而绿麦隆处理浓度、时间均对土壤中蚯蚓脲酶活性具有极显著的影响(P<0.001)。
(6)四种除草剂在土壤中的降解动力学过程符合一级动力学方程,低浓度降解曲线呈平滑下降,而高浓度快速下降；在供试浓度范围内,随着除草剂处理浓度的增加,降解半衰期缩短,且存在一定线性关系；未接种赤子爱胜蚓时,阿特拉津、绿麦隆、乙草胺和丁草胺的半衰期变化范围分别为26.4～28.3d、51.36～42.03d、11.2～6.50d和14.7～8.60d；接种蚯蚓后,阿特拉津、绿麦隆、乙草胺和丁草胺的半衰期分别为24.2～25.6d、41.24～48.56 d、5.2～10.1d和6.30～12.50d之间。方差分析表明,处理浓度与处理时间显著影响四种除草剂在土壤中的降解率。虽然接种蚯蚓能降低土壤中除草剂的半衰期,加速其降解,但蚯蚓在其中的贡献作用达不到显著水平。
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.
本文在调查昆明市农业生产中除草剂使用状况的基础上,以赤子爱胜蚓为材料,模拟常规除草剂使用剂量,在室外模拟培养环境下研究了蚯蚓SOD、CAT等氧化酶及热激蛋白HSP27和HSP40对各类除草剂的时间一效应和剂量—效应关系,分析了它们对土壤脲酶活性的影响,探讨了除草剂在土壤中的降解动态,为这些除草剂的合理使用和生态安全性评估提供数据支持。主要结果如下,
(1)除草剂对蚯蚓生物量的影响的结果表明,除草剂浓度对蚯蚓鲜重的影响总体表现为暴：露剂量越小,体重降低程度越小,暴露剂量越高,体重降低越大。各种浓度除草剂处理的蚯蚓鲜重呈降低趋势。随着处理时间的延长,蚯蚓鲜重随着处理浓度的提高而降低,不同处理浓度引起蚯蚓鲜重降低开始的时间不同,不同剂量最后引起的降低程度不同。
(2)各种处理条件下除草剂对蚯蚓SOD活性的影响均表现为先升高后降低,只是SOD最高活性出现的时间早晚有差异。小于126mg/kg低浓度阿特拉津处理中蚯蚓SOD活性在第7d最高,大于316mg/kg的高浓度阿特拉津处理在第10d蚯蚓SOD活性最高；绿麦隆在低于1OOmg/kg时显著促进SOD活性,高于158mg/kg时SOD活性显著受到抑制；乙草胺在小于33mg/kg范围内促进SOD活性,SOD活性在第7d最高,之后SOD活性降低,低浓度13 g/kg处理被诱导程度最大,而大于83mg/kg时对SOD活性有极显著抑制作用；丁草胺浓度在小于50mg/kg内促进SOD活性,SOD活性在第7d最高,之后SOD活性降低,低浓度32mg/kg处理被诱导程度最大,而大于126mg/kg时对SOD活性有极显著抑制作用。根据双因素方差分析结果表明,阿特拉津、乙草胺和丁草胺处理浓度与时间及浓度与时间的交互作用均对土壤中蚯蚓体组织SOD活性具有极显著的影响(P<0.001)。绿麦隆处理时间、时间与浓度的交互作用均对土壤中蚯蚓体组织SOD活性具有极显著的影响(P<0.001)。
(3)随着除草剂暴露时间的延长,CAT活性对乙草胺处理变化范围为284.8-335.5U/g蛋白,平均为309.68 U/g蛋白,而各处理蚯蚓CAT活性表现为先升高后降低。阿特拉津在低于126mg/kg时显著促进CAT活性,达到316mg/kg以上时对CAT活性有显著抑制作用；绿麦隆在低于1OOmg/kg时,显著促进CAT活性；高于158mg/kg对CAT活性有极显著抑制作用。双因素方差分析结果表明,阿特拉津、乙草胺和丁草胺处理浓度与时间及浓度与时间的交互作用均对蚯蚓体CAT活性具有极显著的影响(P<0.001)；绿麦隆处理浓度对蚯蚓CAT活性具有极显著的影响(P<0.001),而与时间、浓度与时间的交互作用对土壤中蚯蚓体组织CAT活性的影响不显著(P=0.78,>0.05)。
(4)暴露于四种除草剂的处理条件下,蚯蚓体内的热激蛋白(HSP)有新的表达,经Western Blotting免疫印迹检测,确定27kDa为HSP27,40kDa为HSP40。相对于对照而言,随着处理时间的延长,HSP27和HSP40的表达强度先升高后降低。HSP27和HSP40对阿特拉津暴露最为敏感,然后依次为乙草胺、丁草胺和绿麦隆。蚯蚓体组织HSP27和HSP40对除草剂的表达曲线都呈现出倒U型剂量—效应特征。双因素方差分析结果表明,阿特拉津、乙草胺及丁草胺处理浓度与时间及浓度与时间的交互作用均对蚯蚓HSP27和HSP40具有极显著的影响(P<0.001)；绿麦隆处理浓度、时间均对蚯蚓HSP27具有极显著的影响(P<0.001)。
(5)对同一处理浓度而言,随着处理时间的延长,除草剂对土壤脲酶活性的影响表现是先呈升高趋势,随着作用时间的延长脲酶活性下降。除草剂对土壤脲酶活性的影响呈现出抛物线型。双因素方差分析结果表明,阿特拉津、乙草胺和丁草胺处理浓度、时间及浓度与时间的交互作用均对土壤中蚯蚓脲酶活性具有极显著的影响(P<0.001)。而绿麦隆处理浓度、时间均对土壤中蚯蚓脲酶活性具有极显著的影响(P<0.001)。
(6)四种除草剂在土壤中的降解动力学过程符合一级动力学方程,低浓度降解曲线呈平滑下降,而高浓度快速下降；在供试浓度范围内,随着除草剂处理浓度的增加,降解半衰期缩短,且存在一定线性关系；未接种赤子爱胜蚓时,阿特拉津、绿麦隆、乙草胺和丁草胺的半衰期变化范围分别为26.4～28.3d、51.36～42.03d、11.2～6.50d和14.7～8.60d；接种蚯蚓后,阿特拉津、绿麦隆、乙草胺和丁草胺的半衰期分别为24.2～25.6d、41.24～48.56 d、5.2～10.1d和6.30～12.50d之间。方差分析表明,处理浓度与处理时间显著影响四种除草剂在土壤中的降解率。虽然接种蚯蚓能降低土壤中除草剂的半衰期,加速其降解,但蚯蚓在其中的贡献作用达不到显著水平。
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.
引文
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