除草剂氟磺胺草醚对土壤酶、微生物与蚯蚓的生态毒理研究
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摘要
氟磺胺草醚是一种二苯醚类除草剂,广泛应用于大豆、花生等作物田用来防除阔叶性杂草。该除草剂是长残留除草剂,使用不当或长期大量重复使用可以对后茬作物产生药害,并且可能会对土壤生态环境造成不利影响。因此本文利用室内培养实验,采用常规生理生化方法结合变性梯度凝胶电泳(DGGE)和限制性片段长度多态性(T-RFLP)等分子生物学技术评价了氟磺胺草醚对土壤酶和土壤微生物的生态效应。同时还研究了氟磺胺草醚对土壤中生物标志物蚯蚓的氧化胁迫和DNA损伤,为合理评价氟磺胺草醚的环境生态效应提供了科学依据。主要研究结果如下：
1.利用HPLC法测定了氟磺胺草醚在土壤中的残留动态,结果表明氟磺胺草醚在受试土壤中的消解动态符合一级动力学方程,降解半衰期为38.9d。采用平板计数法测定了不同浓度氟磺胺草醚对土壤可培养微生物数量的影响,结果表明培养40d内氟磺胺草醚在土壤中浓度为10、100和500μg/kg时对土壤中细菌和放线菌表现出明显的刺激作用,对真菌表现出轻微的抑制作用,培养60d后,细菌、放线菌和真菌数量均回到对照水平,整体来说实验浓度下的氟磺胺草醚对土壤微生物毒性较小。
2.本文测定了低剂量氟磺胺草醚对土壤磷酸酶、脲酶、脱氢酶和p-葡糖苷酶的影响。结果发现氟磺胺草醚对土壤中酸性磷酸酶、碱性磷酸酶和β-葡糖苷酶在染毒前30d有刺激作用,对土壤脲酶呈现出先抑制再刺激逐渐恢复至对照水平的作用,而土壤脱氢酶在60d的实验周期内均呈现出刺激作用,且呈现剂量效应关系。实验结果表明土壤脲酶和脱氢酶对低剂量氟磺胺草醚较为敏感,可以作为评价氟磺胺草醚对土壤生态效应影响的指标之一
3.采用PCR-DGGE分子指纹技术研究了低剂量(≤500μg/kg)氟磺胺草醚在不同培养时间对土壤中细菌和真菌群落结构的影响。结果发现在整个试验周期(60d)氟磺胺草醚对土壤中细菌生长起到一定的刺激作用,特别是对Emticicia、Luteibacter、 Bacillus和Pontibacter菌属代表的细菌生长刺激作用较强,且在某个实验阶段呈现出一定的剂量效应关系,说明低剂量(≤500μg/kg)氟磺胺草醚不会对土壤中细菌有毒性作用,但从相似度指数上可以看到随土壤染毒浓度的提高相似度指数与对照相比相似性逐渐变小,这说明氟磺胺草醚可以在一定程度上改变土壤中细菌的群落结构；氟磺胺草醚对真菌群落结构也有一定程度的影响,在氟磺胺草醚处理20d以内,10μg/kg的氟磺胺草醚对土壤中真菌生长有刺激作用,而100和500μg/kg氟磺胺草醚对土壤真菌有轻微的抑制作用,表现出轻微的毒性效应,当培养30d以后,各浓度氟磺胺草醚和对照相比均对土壤中Trichosporon、Ascobolus和一些未培养的真菌生长起到刺激作用,且呈现一定的剂量效应关系。
4.采用PCR-RFLP技术研究了低剂量(≤500μg/kg)氟磺胺草醚对土壤中细菌群落结构的影响。结果发现氟磺胺草醚施用后10d对土壤细菌影响较大,对土壤中不同细菌种群既有刺激作用也有抑制作用,但是从整体上刺激作用大于抑制作用；10d后对土壤细菌抑制作用基本消失,但在培养30天以内对土壤细菌的刺激作用仍然较为明显,该研究结果与平板计数和PCR-DGGE实验研究结果基本一致。在细菌种群多样性方面,氟磺胺草醚培养30d以内可以使土壤细菌多样性降低,但是可以使某些细菌种群的优势度增加,30d以后氟磺胺草醚对土壤细菌种群多样性的影响基本消失。在整个实验周期内TRF138菌群相对含量和对照相比均较高,这可能说明TRF138片段代表的细菌种群能够充分利用低剂量氟磺胺草醚作为能源,为筛选氟磺胺草醚降解菌株奠定了基础。
5.利用OECD人工土壤研究了低剂量(≤500μg/kg)氟磺胺草醚对蚯蚓的氧化胁迫和DNA损伤,发现蚯蚓在氟磺胺草醚暴露前14d,体内抗氧化酶(SOD、CAT和POD)活性、ROS水平变化较为明显,除第14d在100和500、μg/kg氟磺胺草醚影响下蚯蚓体内SOD活性下降外,其余时间SOD、CAT和POD活性以及ROS水平均比对照水平高；低剂量氟磺胺草醚不能引起蚯蚓体内明显的脂质过氧化；彗星实验表明低剂量氟磺胺草醚施用前期(14d)可以引起蚯蚓体腔细胞的DNA损伤,但是DNA损伤程度多为轻微损伤和中度损伤。随着暴露时间的延长,在蚯蚓机体自我防御功能的保护下,氟磺胺草醚对蚯蚓的生态毒性逐渐消失。
Fomesafen is a diphenyl ether herbicide that is mainly used in soybean and peanut fields to control the broad-leaved weeds. The improper use, long-term use and large amounts use of fomesafen, long residual herbicide, caused not only carryover injury to sensitive crops but also potential damaged the soil ecosystem. To evaluate the safety of fomesafen on the soil environment, studies were conducted concerning its influences on soil enzymes and soil microorganisms by conventional physiological and biochemical methods and combining with the denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) technologies. In addition, the oxidative stress and DNA damage in the earthworm Eisenia fetida induced by low doses of fomesafen were also investigated. The main results were summarized as follows:
1. The fomesafen was determined in culture soil by high-performance liquid chromatography (HPLC), which showed that the dissipation kinetics of fomesafen in soil was described using a first order kinetics model, the half-time was38.9d. Followed that, the soil microbe quantity in the soil samples treated with different doses of fomesafen were measured by the plate counting method. Results showed that the number of bacterial and actinomyces in the10,100and500μg/kg fomesafen treated soil were higher than those in the control groups, but the fungi was slightly inhibited in the40days of incubation. With the incubation time prolonged, the number of bacteria, actinomyces and fungi were gradually returned the control levels at60d. In the whole, low doses (≤500μg/kg) of fomesafen has less effect on the soil microorganism quantity
2. Effect of low doses of fomesafen on soil enzyme activities under the laboratory simulation conditions was investigated. Five enzymes, i.e., Acid phosphatase, alkaline phosphatase, urease, dehydrogenase and β-glucosidase, were selected. The results found that the activities of acid phosphatase, alkaline phosphatase and P-glucosidase were significantly stimulated by the fomesafen in the30days of incubation. The activity of soil urease presents the effect of inhibit-stimulate-return to a control level. In the60days of incubation, the dehydrogenase activities of soils treated with fomesafen were higher that those of the controls, and showed a statistical dose-dependent relationship. Because of the sensitive to fomesafen, the soil urease and dehydrogenase can be used as one of the indicator to evaluate its soil ecological effect.
3. The potential influences of fomesafen on soil bacterial and fungal community in brown soil were assessed by16S rDNA-PCR-DGGE. During the60days of incubation, fomesafen could stimulate the bacteria populations, especially for Emticicia, Luteibacter, Bacillus and Pontibacter. These mean that low doses (≤500μg/kg) of fomesafen had no toxicity on soil bacteria. But the Jacarrd indices calculated from DGGE indicated that the similarity gradually reduced with the increased of fomesafen concentration in soil compared to the controls. These showed that fomesafen could change the soil bacteria community structure in some extent. Soil fungi community was also affected by low doses of fomesafen. In the20days of incubation, soil fungi were stimulated by minimum concentration (10μg/kg) of fomesafen, but the inhibited effect was found at the higher concentrations (100and500μg/kg). After30days of incubation, DGGE profiles showed that Trichosporon、Ascobolus and uncultured fungi were simulated under the stresses of fomesafen.
4. T-RFLP was used to assess the impact on soil bacterial community by low doses of fomesafen in this study. The soil bacterial was significantly affected by fomesafen during10days of incubation. Some soil bacterial floras were inhibited, but this inhibition effect gradually disappeared after days of10. Another some soil bacterial floras were always stimulated by fomesafen during the whole cultural period (60d). It was found that these findings were similar to results obtained through DGGE and plate counting method. The biodiversity of soil bacterial community (Shannon index, H') reduced during the30days of cultural period and then recovered to that in the control. The Simpson index was increased under the stress of fomesafen, which showed that fomesafen could stimulate the growth of some bacteria. The relative content of TRF138flora was always higher than that in the control. This showed that some strains could utilize fomesafen as the energy sources for growth. And this may be also providing the foundation for isolating fomesafen degrading bacterium.
5. Oxidative stress and DNA damage in the earthworm Eisenia fetida induced by low doses (≤500μg/kg) of fomesafen were investigated in the OECD soil. Compared to the control, the SOD activity increased on the3rd and7th days but decreased on the14th day due to treatment with100and500μg/kg of fomesafen. The activities of CAT and POD increased significantly on the3rd,7th and14th days of exposure. In addition, the ROS level was significantly enhanced throughout the entire experimental period. When exposure period was prolonged21and28days, the activities of SOD, CAT, and POD returned to the control levels. Slight increase of ROS could not effect the changes of three enzymes activity on the exposure days of21and28. Compared to the controls, MDA contents had no obvious changes, in addition to the seventh day had significance increase. Based on the results of the present study, low doses of fomesafen (≤500μg/kg) could not lead to oxidative stress and peroxidation in E. fetida after exposure of21days. The slightly DNA damage in earthworm coelomocytes was observed after treatment with low doses fomesafen in14days of exposure. With the extension of exposure time, the DNA damage in earthworm coelomocytes gradually disappeared under the defense mechanism of earthworm.
1.利用HPLC法测定了氟磺胺草醚在土壤中的残留动态,结果表明氟磺胺草醚在受试土壤中的消解动态符合一级动力学方程,降解半衰期为38.9d。采用平板计数法测定了不同浓度氟磺胺草醚对土壤可培养微生物数量的影响,结果表明培养40d内氟磺胺草醚在土壤中浓度为10、100和500μg/kg时对土壤中细菌和放线菌表现出明显的刺激作用,对真菌表现出轻微的抑制作用,培养60d后,细菌、放线菌和真菌数量均回到对照水平,整体来说实验浓度下的氟磺胺草醚对土壤微生物毒性较小。
2.本文测定了低剂量氟磺胺草醚对土壤磷酸酶、脲酶、脱氢酶和p-葡糖苷酶的影响。结果发现氟磺胺草醚对土壤中酸性磷酸酶、碱性磷酸酶和β-葡糖苷酶在染毒前30d有刺激作用,对土壤脲酶呈现出先抑制再刺激逐渐恢复至对照水平的作用,而土壤脱氢酶在60d的实验周期内均呈现出刺激作用,且呈现剂量效应关系。实验结果表明土壤脲酶和脱氢酶对低剂量氟磺胺草醚较为敏感,可以作为评价氟磺胺草醚对土壤生态效应影响的指标之一
3.采用PCR-DGGE分子指纹技术研究了低剂量(≤500μg/kg)氟磺胺草醚在不同培养时间对土壤中细菌和真菌群落结构的影响。结果发现在整个试验周期(60d)氟磺胺草醚对土壤中细菌生长起到一定的刺激作用,特别是对Emticicia、Luteibacter、 Bacillus和Pontibacter菌属代表的细菌生长刺激作用较强,且在某个实验阶段呈现出一定的剂量效应关系,说明低剂量(≤500μg/kg)氟磺胺草醚不会对土壤中细菌有毒性作用,但从相似度指数上可以看到随土壤染毒浓度的提高相似度指数与对照相比相似性逐渐变小,这说明氟磺胺草醚可以在一定程度上改变土壤中细菌的群落结构；氟磺胺草醚对真菌群落结构也有一定程度的影响,在氟磺胺草醚处理20d以内,10μg/kg的氟磺胺草醚对土壤中真菌生长有刺激作用,而100和500μg/kg氟磺胺草醚对土壤真菌有轻微的抑制作用,表现出轻微的毒性效应,当培养30d以后,各浓度氟磺胺草醚和对照相比均对土壤中Trichosporon、Ascobolus和一些未培养的真菌生长起到刺激作用,且呈现一定的剂量效应关系。
4.采用PCR-RFLP技术研究了低剂量(≤500μg/kg)氟磺胺草醚对土壤中细菌群落结构的影响。结果发现氟磺胺草醚施用后10d对土壤细菌影响较大,对土壤中不同细菌种群既有刺激作用也有抑制作用,但是从整体上刺激作用大于抑制作用；10d后对土壤细菌抑制作用基本消失,但在培养30天以内对土壤细菌的刺激作用仍然较为明显,该研究结果与平板计数和PCR-DGGE实验研究结果基本一致。在细菌种群多样性方面,氟磺胺草醚培养30d以内可以使土壤细菌多样性降低,但是可以使某些细菌种群的优势度增加,30d以后氟磺胺草醚对土壤细菌种群多样性的影响基本消失。在整个实验周期内TRF138菌群相对含量和对照相比均较高,这可能说明TRF138片段代表的细菌种群能够充分利用低剂量氟磺胺草醚作为能源,为筛选氟磺胺草醚降解菌株奠定了基础。
5.利用OECD人工土壤研究了低剂量(≤500μg/kg)氟磺胺草醚对蚯蚓的氧化胁迫和DNA损伤,发现蚯蚓在氟磺胺草醚暴露前14d,体内抗氧化酶(SOD、CAT和POD)活性、ROS水平变化较为明显,除第14d在100和500、μg/kg氟磺胺草醚影响下蚯蚓体内SOD活性下降外,其余时间SOD、CAT和POD活性以及ROS水平均比对照水平高；低剂量氟磺胺草醚不能引起蚯蚓体内明显的脂质过氧化；彗星实验表明低剂量氟磺胺草醚施用前期(14d)可以引起蚯蚓体腔细胞的DNA损伤,但是DNA损伤程度多为轻微损伤和中度损伤。随着暴露时间的延长,在蚯蚓机体自我防御功能的保护下,氟磺胺草醚对蚯蚓的生态毒性逐渐消失。
Fomesafen is a diphenyl ether herbicide that is mainly used in soybean and peanut fields to control the broad-leaved weeds. The improper use, long-term use and large amounts use of fomesafen, long residual herbicide, caused not only carryover injury to sensitive crops but also potential damaged the soil ecosystem. To evaluate the safety of fomesafen on the soil environment, studies were conducted concerning its influences on soil enzymes and soil microorganisms by conventional physiological and biochemical methods and combining with the denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) technologies. In addition, the oxidative stress and DNA damage in the earthworm Eisenia fetida induced by low doses of fomesafen were also investigated. The main results were summarized as follows:
1. The fomesafen was determined in culture soil by high-performance liquid chromatography (HPLC), which showed that the dissipation kinetics of fomesafen in soil was described using a first order kinetics model, the half-time was38.9d. Followed that, the soil microbe quantity in the soil samples treated with different doses of fomesafen were measured by the plate counting method. Results showed that the number of bacterial and actinomyces in the10,100and500μg/kg fomesafen treated soil were higher than those in the control groups, but the fungi was slightly inhibited in the40days of incubation. With the incubation time prolonged, the number of bacteria, actinomyces and fungi were gradually returned the control levels at60d. In the whole, low doses (≤500μg/kg) of fomesafen has less effect on the soil microorganism quantity
2. Effect of low doses of fomesafen on soil enzyme activities under the laboratory simulation conditions was investigated. Five enzymes, i.e., Acid phosphatase, alkaline phosphatase, urease, dehydrogenase and β-glucosidase, were selected. The results found that the activities of acid phosphatase, alkaline phosphatase and P-glucosidase were significantly stimulated by the fomesafen in the30days of incubation. The activity of soil urease presents the effect of inhibit-stimulate-return to a control level. In the60days of incubation, the dehydrogenase activities of soils treated with fomesafen were higher that those of the controls, and showed a statistical dose-dependent relationship. Because of the sensitive to fomesafen, the soil urease and dehydrogenase can be used as one of the indicator to evaluate its soil ecological effect.
3. The potential influences of fomesafen on soil bacterial and fungal community in brown soil were assessed by16S rDNA-PCR-DGGE. During the60days of incubation, fomesafen could stimulate the bacteria populations, especially for Emticicia, Luteibacter, Bacillus and Pontibacter. These mean that low doses (≤500μg/kg) of fomesafen had no toxicity on soil bacteria. But the Jacarrd indices calculated from DGGE indicated that the similarity gradually reduced with the increased of fomesafen concentration in soil compared to the controls. These showed that fomesafen could change the soil bacteria community structure in some extent. Soil fungi community was also affected by low doses of fomesafen. In the20days of incubation, soil fungi were stimulated by minimum concentration (10μg/kg) of fomesafen, but the inhibited effect was found at the higher concentrations (100and500μg/kg). After30days of incubation, DGGE profiles showed that Trichosporon、Ascobolus and uncultured fungi were simulated under the stresses of fomesafen.
4. T-RFLP was used to assess the impact on soil bacterial community by low doses of fomesafen in this study. The soil bacterial was significantly affected by fomesafen during10days of incubation. Some soil bacterial floras were inhibited, but this inhibition effect gradually disappeared after days of10. Another some soil bacterial floras were always stimulated by fomesafen during the whole cultural period (60d). It was found that these findings were similar to results obtained through DGGE and plate counting method. The biodiversity of soil bacterial community (Shannon index, H') reduced during the30days of cultural period and then recovered to that in the control. The Simpson index was increased under the stress of fomesafen, which showed that fomesafen could stimulate the growth of some bacteria. The relative content of TRF138flora was always higher than that in the control. This showed that some strains could utilize fomesafen as the energy sources for growth. And this may be also providing the foundation for isolating fomesafen degrading bacterium.
5. Oxidative stress and DNA damage in the earthworm Eisenia fetida induced by low doses (≤500μg/kg) of fomesafen were investigated in the OECD soil. Compared to the control, the SOD activity increased on the3rd and7th days but decreased on the14th day due to treatment with100and500μg/kg of fomesafen. The activities of CAT and POD increased significantly on the3rd,7th and14th days of exposure. In addition, the ROS level was significantly enhanced throughout the entire experimental period. When exposure period was prolonged21and28days, the activities of SOD, CAT, and POD returned to the control levels. Slight increase of ROS could not effect the changes of three enzymes activity on the exposure days of21and28. Compared to the controls, MDA contents had no obvious changes, in addition to the seventh day had significance increase. Based on the results of the present study, low doses of fomesafen (≤500μg/kg) could not lead to oxidative stress and peroxidation in E. fetida after exposure of21days. The slightly DNA damage in earthworm coelomocytes was observed after treatment with low doses fomesafen in14days of exposure. With the extension of exposure time, the DNA damage in earthworm coelomocytes gradually disappeared under the defense mechanism of earthworm.
引文
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