汞与溴苯腈复合污染对赤子爱胜蚓及球肾白线蚓的生态毒理效应研究
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摘要
多种化合物的复合污染在土壤中十分常见。汞是环境中广泛存在的重金属之一，溴苯腈是最常使用的除草剂之一，因此在污灌区及矿区周围土壤中常同时检测到汞与溴苯腈。目前，二者的复合污染效应研究相对较少。
本研究从生物学四个层次探讨了汞与溴苯腈暴露对赤子爱胜蚓的毒性效应，筛选出不同暴露剂量的生物标志物,初步探讨了汞与溴苯腈暴露对蚯蚓生态毒性的效应机制。以我国本土化物种球肾白线蚓作为试验生物，研究了汞与溴苯腈暴露对球肾白线蚓的急性毒性效应，并进行了三种场地土壤的毒性评估.探讨了球肾白线蚓为土壤污染试验生物的可行性，并初步建立了球肾白线蚓为试验生物的毒性评估方法。
主要研究结果如下：
1.个体水平上，研究了汞与溴苯腈暴露对赤子爱胜蚓和球肾白线蚓的急性毒性。
（1）汞与溴苯腈对受试生物有急性毒性；污染物与致死率之间存在着剂量-效应关系和时间-效应关系；汞与溴苯腈复合污染的毒性效应关系既有协同作用又有拮抗作用；死亡率可以作为生物标志物来评估致死剂量范围的土壤生态风险；汞与溴苯腈暴露对赤子爱胜蚓的14天LC50分别为143.67mg/kg和41.75mg/kg，对球肾白线蚓的LC50分别为3.87mg/kg和2.41mg/kg；球肾白线蚓比赤子爱胜蚓敏感，在较低低剂量暴露的环境中，球肾白线蚓适合作为监测环境的受试生物。（2）比较了OECD推荐的参比物氯乙酰胺对两种受试生物的毒性效应，球肾
白线蚓对氯乙酰胺急性毒性反应更灵敏。2.组织水平上，研究了汞与溴苯腈暴露对赤子爱胜蚓体内抗氧化物酶系的毒
性效应。汞与溴苯腈暴露均能诱导SOD、CAT和GST的活力。汞与溴苯腈暴露对酶活力影响随时间变化的规律不同，CAT酶活力随暴露时间的变化规律相似，CAT可作为汞与溴苯腈毒性评估的生物标记物。3.细胞水平上，优化了体腔细胞提取方法，研究了汞与溴苯腈暴露对赤子爱胜蚓体腔细胞溶酶体膜稳定性以及体腔细胞凋亡率的影响。
（1）中性红停留时间（NRRT）随着汞与溴苯腈暴露时间的延长、暴露浓度的增加而下降。汞与溴苯腈复合暴露的交互作用体现为协同作用，溴苯腈是复合污染的主效应因子。NRRT作为生物标志物的检测方法重现性好、准确，能够对汞与溴苯腈引起的环境进行早期监测。
（2）体腔细胞的早期凋亡率与体腔细胞质膜损伤密切相关。随着汞与溴苯腈暴露浓度的增加，体腔细胞凋亡率上升。溴苯腈是复合污染的主效应因子。蚯蚓体腔细胞早期凋亡率可作为污染早期预警的生物标志物。
（3）溶酶体膜稳定性的降低对应着细胞凋亡率的升高，表明溶酶体-线粒体途径是汞与溴苯腈暴露引起蚯蚓体腔细胞凋亡的机制之一。
4.分子水平上，运用单细胞凝胶电泳技术（SCGE）研究了汞与溴苯腈暴露对赤子爱胜蚓体腔细胞DNA损伤的遗传毒性。随着汞与溴苯腈暴露浓度的增加，DNA损伤加剧。溴苯腈是复合污染的主效应因子；OTM比TL更适合作为DNA损伤的生物标志物标志。
5.以球肾白线蚓作为受试生物，评估了三个场地土壤的生态毒性效应。结果显示，球肾白线蚓的死亡率随暴露时间的延长而增加，随土壤污染物浓度的增加，死亡率上升。球肾白线蚓的死亡率能作为污染场地的评价指标。试验建立了球肾白线蚓作为受试生物的场地毒性评价方法,为场地土壤的毒性评估和生态修复提供了参考。
Co-exposures to complex mixtures are common in soil. Mercury (Hg) is widely distributed and bromoxynil (BX) is one of the most common used herbicide, which detected in the soil irrigated by waste water. The combined pollution of mercury and bromoxynil are less explored.
The ecotoxicological effects of Eisenia fetida (E. fetida) co-exposure to mercury and bromoxynil were studied. Some appropriate biomarkers were selected and the mechanism of the toxic effects were investigated. Meanwhile, Fridericia bulbosa(F. bulbosa) as a local organism was chosen to compare with E.fetida by an acute toxicity test and to assess the toxicity of the pollutants in three site soil. The possibility of the F. bulbosa as a tested organism for soil ecotoxicity assessment was evaluated and the assessment method for eco-toxicological risk was provided in soil.
The results were as follows:
1. The acute toxic effect of Hg and BX on F. fetida and F. Bulbosa were tested individual.
(1) The toxic effect of the tested pollutant were detected on the E. fetida and F. bulbosa exposure to Hg and BX. A dose-response relationship and a time-response relationship between the pollutant and the mortality were observed. Not only synergism but also antagonism were induced by the co-exposure course to the mixture. The morality is a suitable biomarker to assesses the risk of pollutant in soil at a range of lethal doses. The14d LC50of E. fetida exposure to Hg and BX were 143.67mg/kg and41.74mg/kg, respectively. The14d LC50of F. bulbosa exposure to Hg or BX were3.87mg/kg and2.41mg/kg. The response of F. bulbosa was more sensitive, which means F. bulbosa is available to monitor the risk of pollutant in the soil environment, especially in the soil contaminated with low dose.
(2) F. bulbosa was more sensitive than F. fetida exposure to the chloroacetamide (the reference chemical recommended by OECD) by a competitive trials by two organism.
2. The toxicity of Hg and BX on the enzymatic activities of F. fetida was investigated in tissue. The activity of Superoxide Dismutase (SOD) Catalase (CAT) and Glutathione-S-Transferase (GST) of E.fetida were not only induced by Hg but also by BX. The changes of the enzyme activity on earthworms during the exposure course were different with each other. The changes of the CAT activity influenced by Hg and BX were similar and therefore CAT was proposed as a biomarker.
3. The extracted method of the coelomocyte of earthworms was optimized. The changes of the neutral red retention time (NRRT) and the apoptotic rate of the coelomcytes on E. fetida exposure to Hg and BX were investigated.
(1)An opposite relationship was observed not only betweenthe NRRT and the time of exposure,but also between the NRRT and the concentration of pollutants. Synergism were observed on earthworms exposure to the mixtures with BX as the main factor. Due to the accuracy and repeatability of the detected method, NRRT could be used as a biomarker to detect the early pollution.
(2) The apoptotic rate was relatively higher to the damage of the cytoplasmic membrane. It was raised with the Hg and BX exposure dose increased. BX was the main factor in combined pollution. The ratio of the apoptotic could be considered as a biomarker to early detection of contamination.
(3) The decrease of lysosome stability corresponds with the increase of apoptotic rate, which revealed that the lysosome-mitochondria pathway may be involved in the mechanism of the apoptosis.
4. The genotoxicity of Hg and BX on E. fetida were investigated by means of the single cell microgel electrophoresis (SCGE). The DNA damage had been raised by Hg and BX along with the pollutants exposure dose increased. BX was the main factor of contribution on DNA damage. OTM was a comprehensive biomarker and more suitable to estimate the DNA damage.
5. Using F. Bulbosa as the test organism, acute toxic experiment had been performed to assess the toxicity in three soil samples from contaminated site. The mortality had been raised during the exposure time, which shows again that the mortality of F. bulbosa was a sensitive index to assess on the risk in contaminated site. The assessed method of F. bulbosa in soil was established.
本研究从生物学四个层次探讨了汞与溴苯腈暴露对赤子爱胜蚓的毒性效应，筛选出不同暴露剂量的生物标志物,初步探讨了汞与溴苯腈暴露对蚯蚓生态毒性的效应机制。以我国本土化物种球肾白线蚓作为试验生物，研究了汞与溴苯腈暴露对球肾白线蚓的急性毒性效应，并进行了三种场地土壤的毒性评估.探讨了球肾白线蚓为土壤污染试验生物的可行性，并初步建立了球肾白线蚓为试验生物的毒性评估方法。
主要研究结果如下：
1.个体水平上，研究了汞与溴苯腈暴露对赤子爱胜蚓和球肾白线蚓的急性毒性。
（1）汞与溴苯腈对受试生物有急性毒性；污染物与致死率之间存在着剂量-效应关系和时间-效应关系；汞与溴苯腈复合污染的毒性效应关系既有协同作用又有拮抗作用；死亡率可以作为生物标志物来评估致死剂量范围的土壤生态风险；汞与溴苯腈暴露对赤子爱胜蚓的14天LC50分别为143.67mg/kg和41.75mg/kg，对球肾白线蚓的LC50分别为3.87mg/kg和2.41mg/kg；球肾白线蚓比赤子爱胜蚓敏感，在较低低剂量暴露的环境中，球肾白线蚓适合作为监测环境的受试生物。（2）比较了OECD推荐的参比物氯乙酰胺对两种受试生物的毒性效应，球肾
白线蚓对氯乙酰胺急性毒性反应更灵敏。2.组织水平上，研究了汞与溴苯腈暴露对赤子爱胜蚓体内抗氧化物酶系的毒
性效应。汞与溴苯腈暴露均能诱导SOD、CAT和GST的活力。汞与溴苯腈暴露对酶活力影响随时间变化的规律不同，CAT酶活力随暴露时间的变化规律相似，CAT可作为汞与溴苯腈毒性评估的生物标记物。3.细胞水平上，优化了体腔细胞提取方法，研究了汞与溴苯腈暴露对赤子爱胜蚓体腔细胞溶酶体膜稳定性以及体腔细胞凋亡率的影响。
（1）中性红停留时间（NRRT）随着汞与溴苯腈暴露时间的延长、暴露浓度的增加而下降。汞与溴苯腈复合暴露的交互作用体现为协同作用，溴苯腈是复合污染的主效应因子。NRRT作为生物标志物的检测方法重现性好、准确，能够对汞与溴苯腈引起的环境进行早期监测。
（2）体腔细胞的早期凋亡率与体腔细胞质膜损伤密切相关。随着汞与溴苯腈暴露浓度的增加，体腔细胞凋亡率上升。溴苯腈是复合污染的主效应因子。蚯蚓体腔细胞早期凋亡率可作为污染早期预警的生物标志物。
（3）溶酶体膜稳定性的降低对应着细胞凋亡率的升高，表明溶酶体-线粒体途径是汞与溴苯腈暴露引起蚯蚓体腔细胞凋亡的机制之一。
4.分子水平上，运用单细胞凝胶电泳技术（SCGE）研究了汞与溴苯腈暴露对赤子爱胜蚓体腔细胞DNA损伤的遗传毒性。随着汞与溴苯腈暴露浓度的增加，DNA损伤加剧。溴苯腈是复合污染的主效应因子；OTM比TL更适合作为DNA损伤的生物标志物标志。
5.以球肾白线蚓作为受试生物，评估了三个场地土壤的生态毒性效应。结果显示，球肾白线蚓的死亡率随暴露时间的延长而增加，随土壤污染物浓度的增加，死亡率上升。球肾白线蚓的死亡率能作为污染场地的评价指标。试验建立了球肾白线蚓作为受试生物的场地毒性评价方法,为场地土壤的毒性评估和生态修复提供了参考。
Co-exposures to complex mixtures are common in soil. Mercury (Hg) is widely distributed and bromoxynil (BX) is one of the most common used herbicide, which detected in the soil irrigated by waste water. The combined pollution of mercury and bromoxynil are less explored.
The ecotoxicological effects of Eisenia fetida (E. fetida) co-exposure to mercury and bromoxynil were studied. Some appropriate biomarkers were selected and the mechanism of the toxic effects were investigated. Meanwhile, Fridericia bulbosa(F. bulbosa) as a local organism was chosen to compare with E.fetida by an acute toxicity test and to assess the toxicity of the pollutants in three site soil. The possibility of the F. bulbosa as a tested organism for soil ecotoxicity assessment was evaluated and the assessment method for eco-toxicological risk was provided in soil.
The results were as follows:
1. The acute toxic effect of Hg and BX on F. fetida and F. Bulbosa were tested individual.
(1) The toxic effect of the tested pollutant were detected on the E. fetida and F. bulbosa exposure to Hg and BX. A dose-response relationship and a time-response relationship between the pollutant and the mortality were observed. Not only synergism but also antagonism were induced by the co-exposure course to the mixture. The morality is a suitable biomarker to assesses the risk of pollutant in soil at a range of lethal doses. The14d LC50of E. fetida exposure to Hg and BX were 143.67mg/kg and41.74mg/kg, respectively. The14d LC50of F. bulbosa exposure to Hg or BX were3.87mg/kg and2.41mg/kg. The response of F. bulbosa was more sensitive, which means F. bulbosa is available to monitor the risk of pollutant in the soil environment, especially in the soil contaminated with low dose.
(2) F. bulbosa was more sensitive than F. fetida exposure to the chloroacetamide (the reference chemical recommended by OECD) by a competitive trials by two organism.
2. The toxicity of Hg and BX on the enzymatic activities of F. fetida was investigated in tissue. The activity of Superoxide Dismutase (SOD) Catalase (CAT) and Glutathione-S-Transferase (GST) of E.fetida were not only induced by Hg but also by BX. The changes of the enzyme activity on earthworms during the exposure course were different with each other. The changes of the CAT activity influenced by Hg and BX were similar and therefore CAT was proposed as a biomarker.
3. The extracted method of the coelomocyte of earthworms was optimized. The changes of the neutral red retention time (NRRT) and the apoptotic rate of the coelomcytes on E. fetida exposure to Hg and BX were investigated.
(1)An opposite relationship was observed not only betweenthe NRRT and the time of exposure,but also between the NRRT and the concentration of pollutants. Synergism were observed on earthworms exposure to the mixtures with BX as the main factor. Due to the accuracy and repeatability of the detected method, NRRT could be used as a biomarker to detect the early pollution.
(2) The apoptotic rate was relatively higher to the damage of the cytoplasmic membrane. It was raised with the Hg and BX exposure dose increased. BX was the main factor in combined pollution. The ratio of the apoptotic could be considered as a biomarker to early detection of contamination.
(3) The decrease of lysosome stability corresponds with the increase of apoptotic rate, which revealed that the lysosome-mitochondria pathway may be involved in the mechanism of the apoptosis.
4. The genotoxicity of Hg and BX on E. fetida were investigated by means of the single cell microgel electrophoresis (SCGE). The DNA damage had been raised by Hg and BX along with the pollutants exposure dose increased. BX was the main factor of contribution on DNA damage. OTM was a comprehensive biomarker and more suitable to estimate the DNA damage.
5. Using F. Bulbosa as the test organism, acute toxic experiment had been performed to assess the toxicity in three soil samples from contaminated site. The mortality had been raised during the exposure time, which shows again that the mortality of F. bulbosa was a sensitive index to assess on the risk in contaminated site. The assessed method of F. bulbosa in soil was established.
引文
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