土壤中甲磺隆结合残留的风险评价及水稻对其响应基因型差异的机理研究
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摘要
随着人口的迅速增长、工农业生产的迅速发展和人民生活水平的不断提高,人类对化学品,尤其是对人工合成有机化合物的依赖程度越来越大。化学农药、除草剂以及工业“三废”如酚、油类、多氯联苯、苯并芘等有机化合物在其中占有重大的比例。有机化合物大都以土壤或水体环境作为其最终的归宿地,且大部分有机化合物易与土壤或水体环境中的沉积物形成结合残留而长期残留于环境中。环境中的结合残留有可能再次释放出来对环境造成一定的危害,且易于通过食物链影响人体的健康。甲磺隆曾是我国“九五”期间广泛应用于麦田的一种磺酰脲类除草剂。我们前期的研究结果表明,甲磺隆较易与土壤有机质等组分结合形成结合残留。残留量一般达引入量的11.4%-55.7%。由于甲磺隆结合残留主要分布于土壤中松结态有机质中,所以我们认为,甲磺隆结合残留在环境中再次释放的可能性极大,很可能存在一定的生态风险。此外,小麦—水稻轮作是我国华南及华东地区较常见的轮作方式,应用于麦田中的甲磺隆极易形成结合残留对后茬水稻产生药害。然而,目前国内外有关这方面的系统报道较少。为此,我们通过室内微型模拟培养试验,并采用分子生物学等手段,对甲磺隆结合残留的环境效应进行了较为系统的评价,并通过室外盆栽试验进行了水稻对土壤中甲磺隆结合残留响应基因型差异及机理的研究,试图为栽种对甲磺隆耐性较强的水稻品种,来减轻除草剂所造成的农业损失提供一定的理论依据。取得的主要研究结果如下:
1.甲磺隆结合残留的生态毒理效应评价
室内培养试验研究结果表明,甲磺隆结合残留在土壤中具有一定的生态风险,对土壤酶活性、土壤微生物量和土壤微生物多样性的影响与其在土壤中的持留时间有关,具体表现为:
(1) 甲磺隆结合残留对土壤酶学指标的影响。甲磺隆结合残留对土壤脲酶、酸性磷酸酶、过氧化氢酶和蔗糖酶的影响较为复杂,但仍有一定的规律。培养第7天,甲磺隆结合残留对土壤脲酶具有激活作用,对土壤过氧化氢酶则有抑制作用,对其他两种酶的影响不显格。培养第14天,甲磺隆结合残留对土壤过氧化氢酶和酸性磷酸酶具有抑制作用。而对土壤脲酶和蔗糖酶的影响则表现为,低浓度时对土壤脲酶和蔗糖酶具有激活作用;高浓度时对土壤脲酶和蔗糖酶具有抑制作用。培养第28天及28天后,甲磺隆结合残留对土壤脲酶、酸性磷酸酶和过氧化氢酶活性具有激活作用以及在0.05-0.281 mg kg~1范围内对土壤蔗糖酶活性具有激活作用。
(2)甲磺隆结合残留对十壤微生物生物量的影响。本试验条件卜,甲磺隆结合残留对
十壤微生物生物量及微生物生物量碳,氮的影响随时间而变化。第7大,低浓度甲磺隆结合
残留处理十壤微生物生物量的含量降低:高浓度甲磺隆结合残留处理十壤微生物生物量的含
举增加。士言养第14大,甲磺隆结合残留显艺一地降低了十壤微生物生物量的含量。第28大,
甲磺隆结合残留能够提高十壤微生物生物量。第56大和第98大,甲磺隆结合残留对十壤微
生物生物量的影响则表现为,低浓度时,甲磺隆结合残留显著地提高十壤微生物生物量:高
浓度时,甲磺隆结合残留显著地降低十壤微生物生物量。另外,培养98天时,甲磺隆结合
残留处理十壤微生物生物量碳/氮显著降低。表明甲磺隆结合残留可能会引起十壤微生物种
群的改变。
(3)甲磺隆结合残留对十壤微生物多样性的影响。通过在不同培养时间和不同浓度甲
磺隆结合残留胁迫卜,对十壤微生物DNA进行DGGE基因多样性指纹图谱分析结果表明,甲
磺隆结合残留对十壤微生物多样性的影响与其在土壤中持留时间有关。培养第14大,低浓
度甲磺隆结合残留对十壤微生物多样性与对照差异不明显;高浓度甲磺隆结合残留对土壤微
生物多样性的影响较为明显,十壤微生物多样性卜降。培养中期(14一56大),甲磺隆结合
残留对十壤微生物的作用为先刺激和后毒害作用,各处理条带数先增加后减少。培养后期
(56一98大),甲磺隆结合残留对十壤部分微生物.n.-有毒害作用,可致使人多数条带的消失,
导致十壤微生物多样性卜降。培养后期,经0.158 ogkg’甲磺隆结合残留处理的土壤DGGE
指纹图谱中部分条带较其他处理颜色加深。说明在一定浓度的甲磺隆结合残留范围内,土壤
中可能存在甲磺隆结合残留专性微生物种。
2.水稻对甲磺隆结合残留响应基因型差异及机理研究
采用生物测定和十_壤盆栽试验法,研究了水稻对甲磺隆及土壤中甲磺隆结合残留响应
的基因刑差异及机理,研究结果表明:
(1)水稻对甲磺隆的耐性存在基因烈差异。浙农952对甲磺隆的耐性较强:秀水63
对甲磺隆的耐性较敏感:两优培九和H优810对甲磺隆的耐性介于浙农952和秀水63之间。
(2)十壤中甲磺隆结合残留对水稻具有约害作用,对水稻生长具有抑制作用。以水稻
根系总长度为指标,十壤甲磺隆结合残留对浙农952、两优培九、H优810和秀水63的ECSO
值分别为13.876一、4.1701、0.2257平11 0.ooxZ林gkg一’。对于同一品种来讲,十壤中甲磺隆
结合残留对水稻根系的抑制作川强丁水稻地卜部。水稻根系总长度、总表面积和根系数量可
以作为评价水稻对十壤中甲磺隆结合残留耐性强弱的指标。
(3)水稻对十壤中甲磺隆结合残留的响应存在基因烈差异,水稻对土壤中甲磺隆结合
残留耐性
Dependence of human beings on chemicals, especially synthetic organic compounds increased gradually with the rapid development of agriculture and industry, improvement of life quality, and the fast growth of population. Organic compounds mainly consisted of pesticides, herbicides, and industrial organic wastes like phenols, oils, polychlorinated biphenyl, benzopyrene. These organic compounds enter into soil or sediments in water at last, while most of these are considered as bound residues in soil or sediments. Bound residues in environment could be released again and pose impacts on plants, animals, or human beings through food chains. Metsulfuron-methyl, one of sulfonylurea herbicides, was widely used in wheat fields during period of the ninth five-year plan in China. Our previous results showed that 11.4 %-55.7 % of metsulfuron-methyl used could tightly bind with organic matter and form bound residues in soils. Bound residues of metsulfuron-methyl in soil were likely to result in some ecological risk, especially in rice fields due to the formation of loose bound residues of metsulfuron-methyl combined with soil organic matter. However, systemic reports about these are very few. The present research illustrates the environmental risk of bound residues of metsulfuron-methyl in soil, and proves the probability of agricultural reduction resulted from use of metsulfuron-methyl. A set of experiments were conducted to investigate effects of bound residues of metsulfuron-methyl on soil enzymes, microbial biomass, and microbial diversities,
and the genotypic different responses of rice to bound residues of metsulfuron-methylin soil. The major results were summarized as follows:1. Ecological risk assessment of bound residues of metsulfuron-methyl in soil.Ecological risk of bound residues of metsulfuron-methyl in soil has been found. Effects of bound residues of metsulfuron-methyl in soil on soil enzymes such as urease, hydrogen peroxidase, acid phosphatase, and sucrase, microbial biomass, and microbial diversity are dependent on the incubation time when bound residues of metsulfuron-methyl exist in soil. (1) Effects of bound residues of metsulfuron-methyl on soil enzymes. At 7 days after treatment (DAT), urease was induced, and hydrogen peroxidase was inhibited significantly by bound residues of metsulfuron-methyl, however sucrase and acid phosphatase were not changed. At 14 DAT, hydrogen peroxidase and acid phosphatase were inhibited significantly by bound residues of metsulfuron-methyl in soil. Urease and sucrase were induced and inhibited significantly by bound residues of metsulfuron-methyl at the levels of 0-0.281 mg kg"1, and 0.50 mg kg'1, respectively. At 28 DAT, soil enzymes such as urease, hydrogen peroxidase, acid phosphatase, and sucrase were induced by bound residues of metsulfuron-methyl in soil. At 56 and 98 DAT, effects of bound residues of metsulfuron-methyl on soil enzymes are similar to that at 28 DAT. (2) Effects of bound residues of metsulfuron-methyl on soil microbial biomass. Effects of bound residues of metsulfuron-methyl in soil on microbial biomass and ratio of microbial biomass carbon to nitrogen changed with time. At 7 DAT, microbial biomass decreased in soil treated with bound residues of metsulfuron-methyl at levels of 0.05 -0.156 mg kg"1 and increased at the levels of 0.281-0.50 mg kg"1 compared with that of control. At 14 DAT, microbial biomass decreased significantly with the increase of bound residues of metsulfuron-methyl in soil compared with that of control. At 28 DAT, microbial biomass increased with the increase of bound residues of metsulfuron-methyl in soil. At 56 and 98 DAT, microbial biomass increased significantly at lower level of bound residues of metsulfuron-methyl and decreased significantly at higher level of bound residues of metsulfuron-methyl in soil. The significant decrease in ratio of microbial biomass carborn to nitrogen has also been
found, indicating that microbial communities changed under the stress of bound residues of metsulfuron-methyl. (3) Effect of bound resi
1.甲磺隆结合残留的生态毒理效应评价
室内培养试验研究结果表明,甲磺隆结合残留在土壤中具有一定的生态风险,对土壤酶活性、土壤微生物量和土壤微生物多样性的影响与其在土壤中的持留时间有关,具体表现为:
(1) 甲磺隆结合残留对土壤酶学指标的影响。甲磺隆结合残留对土壤脲酶、酸性磷酸酶、过氧化氢酶和蔗糖酶的影响较为复杂,但仍有一定的规律。培养第7天,甲磺隆结合残留对土壤脲酶具有激活作用,对土壤过氧化氢酶则有抑制作用,对其他两种酶的影响不显格。培养第14天,甲磺隆结合残留对土壤过氧化氢酶和酸性磷酸酶具有抑制作用。而对土壤脲酶和蔗糖酶的影响则表现为,低浓度时对土壤脲酶和蔗糖酶具有激活作用;高浓度时对土壤脲酶和蔗糖酶具有抑制作用。培养第28天及28天后,甲磺隆结合残留对土壤脲酶、酸性磷酸酶和过氧化氢酶活性具有激活作用以及在0.05-0.281 mg kg~1范围内对土壤蔗糖酶活性具有激活作用。
(2)甲磺隆结合残留对十壤微生物生物量的影响。本试验条件卜,甲磺隆结合残留对
十壤微生物生物量及微生物生物量碳,氮的影响随时间而变化。第7大,低浓度甲磺隆结合
残留处理十壤微生物生物量的含量降低:高浓度甲磺隆结合残留处理十壤微生物生物量的含
举增加。士言养第14大,甲磺隆结合残留显艺一地降低了十壤微生物生物量的含量。第28大,
甲磺隆结合残留能够提高十壤微生物生物量。第56大和第98大,甲磺隆结合残留对十壤微
生物生物量的影响则表现为,低浓度时,甲磺隆结合残留显著地提高十壤微生物生物量:高
浓度时,甲磺隆结合残留显著地降低十壤微生物生物量。另外,培养98天时,甲磺隆结合
残留处理十壤微生物生物量碳/氮显著降低。表明甲磺隆结合残留可能会引起十壤微生物种
群的改变。
(3)甲磺隆结合残留对十壤微生物多样性的影响。通过在不同培养时间和不同浓度甲
磺隆结合残留胁迫卜,对十壤微生物DNA进行DGGE基因多样性指纹图谱分析结果表明,甲
磺隆结合残留对十壤微生物多样性的影响与其在土壤中持留时间有关。培养第14大,低浓
度甲磺隆结合残留对十壤微生物多样性与对照差异不明显;高浓度甲磺隆结合残留对土壤微
生物多样性的影响较为明显,十壤微生物多样性卜降。培养中期(14一56大),甲磺隆结合
残留对十壤微生物的作用为先刺激和后毒害作用,各处理条带数先增加后减少。培养后期
(56一98大),甲磺隆结合残留对十壤部分微生物.n.-有毒害作用,可致使人多数条带的消失,
导致十壤微生物多样性卜降。培养后期,经0.158 ogkg’甲磺隆结合残留处理的土壤DGGE
指纹图谱中部分条带较其他处理颜色加深。说明在一定浓度的甲磺隆结合残留范围内,土壤
中可能存在甲磺隆结合残留专性微生物种。
2.水稻对甲磺隆结合残留响应基因型差异及机理研究
采用生物测定和十_壤盆栽试验法,研究了水稻对甲磺隆及土壤中甲磺隆结合残留响应
的基因刑差异及机理,研究结果表明:
(1)水稻对甲磺隆的耐性存在基因烈差异。浙农952对甲磺隆的耐性较强:秀水63
对甲磺隆的耐性较敏感:两优培九和H优810对甲磺隆的耐性介于浙农952和秀水63之间。
(2)十壤中甲磺隆结合残留对水稻具有约害作用,对水稻生长具有抑制作用。以水稻
根系总长度为指标,十壤甲磺隆结合残留对浙农952、两优培九、H优810和秀水63的ECSO
值分别为13.876一、4.1701、0.2257平11 0.ooxZ林gkg一’。对于同一品种来讲,十壤中甲磺隆
结合残留对水稻根系的抑制作川强丁水稻地卜部。水稻根系总长度、总表面积和根系数量可
以作为评价水稻对十壤中甲磺隆结合残留耐性强弱的指标。
(3)水稻对十壤中甲磺隆结合残留的响应存在基因烈差异,水稻对土壤中甲磺隆结合
残留耐性
Dependence of human beings on chemicals, especially synthetic organic compounds increased gradually with the rapid development of agriculture and industry, improvement of life quality, and the fast growth of population. Organic compounds mainly consisted of pesticides, herbicides, and industrial organic wastes like phenols, oils, polychlorinated biphenyl, benzopyrene. These organic compounds enter into soil or sediments in water at last, while most of these are considered as bound residues in soil or sediments. Bound residues in environment could be released again and pose impacts on plants, animals, or human beings through food chains. Metsulfuron-methyl, one of sulfonylurea herbicides, was widely used in wheat fields during period of the ninth five-year plan in China. Our previous results showed that 11.4 %-55.7 % of metsulfuron-methyl used could tightly bind with organic matter and form bound residues in soils. Bound residues of metsulfuron-methyl in soil were likely to result in some ecological risk, especially in rice fields due to the formation of loose bound residues of metsulfuron-methyl combined with soil organic matter. However, systemic reports about these are very few. The present research illustrates the environmental risk of bound residues of metsulfuron-methyl in soil, and proves the probability of agricultural reduction resulted from use of metsulfuron-methyl. A set of experiments were conducted to investigate effects of bound residues of metsulfuron-methyl on soil enzymes, microbial biomass, and microbial diversities,
and the genotypic different responses of rice to bound residues of metsulfuron-methylin soil. The major results were summarized as follows:1. Ecological risk assessment of bound residues of metsulfuron-methyl in soil.Ecological risk of bound residues of metsulfuron-methyl in soil has been found. Effects of bound residues of metsulfuron-methyl in soil on soil enzymes such as urease, hydrogen peroxidase, acid phosphatase, and sucrase, microbial biomass, and microbial diversity are dependent on the incubation time when bound residues of metsulfuron-methyl exist in soil. (1) Effects of bound residues of metsulfuron-methyl on soil enzymes. At 7 days after treatment (DAT), urease was induced, and hydrogen peroxidase was inhibited significantly by bound residues of metsulfuron-methyl, however sucrase and acid phosphatase were not changed. At 14 DAT, hydrogen peroxidase and acid phosphatase were inhibited significantly by bound residues of metsulfuron-methyl in soil. Urease and sucrase were induced and inhibited significantly by bound residues of metsulfuron-methyl at the levels of 0-0.281 mg kg"1, and 0.50 mg kg'1, respectively. At 28 DAT, soil enzymes such as urease, hydrogen peroxidase, acid phosphatase, and sucrase were induced by bound residues of metsulfuron-methyl in soil. At 56 and 98 DAT, effects of bound residues of metsulfuron-methyl on soil enzymes are similar to that at 28 DAT. (2) Effects of bound residues of metsulfuron-methyl on soil microbial biomass. Effects of bound residues of metsulfuron-methyl in soil on microbial biomass and ratio of microbial biomass carbon to nitrogen changed with time. At 7 DAT, microbial biomass decreased in soil treated with bound residues of metsulfuron-methyl at levels of 0.05 -0.156 mg kg"1 and increased at the levels of 0.281-0.50 mg kg"1 compared with that of control. At 14 DAT, microbial biomass decreased significantly with the increase of bound residues of metsulfuron-methyl in soil compared with that of control. At 28 DAT, microbial biomass increased with the increase of bound residues of metsulfuron-methyl in soil. At 56 and 98 DAT, microbial biomass increased significantly at lower level of bound residues of metsulfuron-methyl and decreased significantly at higher level of bound residues of metsulfuron-methyl in soil. The significant decrease in ratio of microbial biomass carborn to nitrogen has also been
found, indicating that microbial communities changed under the stress of bound residues of metsulfuron-methyl. (3) Effect of bound resi
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