取代苯对红裸须摇蚊生物标志物系统的影响
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摘要
取代芳烃类化合物多属有机污染物,随着工业废水、农业径流水、生活污水流人水体,对水生生物的生长、发育和繁殖构成危害,破坏了水生生态系统平衡,而且对人体健康具有潜在危害。摇蚊是一种分布广泛且种类数量众多的底栖生物,常作为水体质量监测的重要指示生物。本文在中国水中优先控制污染物黑名单的基础上,按照取代基的类型、位置及取代基个数的不同筛选出12种取代苯化合物,以红裸须摇蚊(Propsilocerus akamusi Tokunaga)4龄幼虫为研究对象,测定了12种取代苯对其急性毒性、联合毒性以及亚致死剂量(0.4、4、40mg/L)的4种取代苯对摇蚊生长发育和体内6种生物化学标志物(GST、CarE、CAT、POD、SOD)的影响。结果如下：
(1)摇蚊幼虫暴露于取代苯类污染物溶液中表现出典型的中毒症状：甲苯、氯苯、苯酚和对氯苯酚溶液中的幼虫表现出活动减慢和行动迟缓；苯酚溶液中幼虫体表粘性增大；对苯二胺溶液中的幼虫体色变黑,部分体节还会出现炭黑色的点或环状斑。这些现象表明取代苯具有麻痹性、腐蚀性和灼伤性的毒性特点。摇蚊幼虫对取代苯类有机污染物的忍耐性较高,12种取代苯类对摇蚊4龄幼虫24 h LC50范围为90～1100 mg/L,其中最小的是对氯苯酚,LC50为90.07mg/L,最大的是苯胺,LC50为1069.71mg/L。按照有机物的毒性作用机制区分：苯、苯胺、氯苯和邻、间、对-二甲苯为非极性麻醉性化合物；苯酚、对氯苯酚和硝基苯为急性麻醉性化合物；对苯二胺和间苯二胺为反应性有机化合物。取代苯类有机污染物苯环上取代基不同,其毒性不同,基团毒性的贡献为：-NO2>-OH>-C1>-CH3>-NH2;3种二甲苯同分异构体毒性大小依次为邻二甲苯>间二甲苯>对二甲苯；二苯胺同分异构体毒性大小依次为对苯二胺>间苯二胺。
(2)4种指数法对19组二元取代苯联合毒性结果的评价并不完全一致：对联合毒性结果的定性判断上,TU法和MTI法评价结果完全一致,Al法和λ法的评价结果完全一致；对联合毒力强弱程度的判断上TU法与λ法一致,Al法和MTI法一致。4种评价方法对于协同和简单相加毒性效应的评价结果一致,对于部分相加和拮抗作用的评价结果则有所不同。综合4种评价方法结果表明一半以上(52.63%)的二元取代苯在等比毒性配比下的联合作用类型为协同作用；极少数(0.05%)表现为简单相加作用,其余(42.11%)表现为部分相加和拮抗作用。
(3)苯酚影响摇蚊生长发育。亚致死剂量(0.4、4、40 mg/L)苯酚降低摇蚊幼虫干湿重和化蛹率,这表明摇蚊幼虫生长和化蛹过程中对苯酚的胁迫敏感,可将其作为慢性毒性效应指标。摇蚊幼虫体内的生物标志物(SOD、CAT、GST、CarE、ACP、ALP)对于4种亚致死剂量取代苯溶液的胁迫表现一定的时间—剂量效应。对于暴露时间最为敏感的生物化学标志物是SOD和CarE,暴露于苯酚和对氯苯酚6 h,摇蚊体内SOD和CarE就呈现出应答反应。对于浓度最为敏感的指标是SOD,摇蚊体内的SOD能响应0.4mg/L对氯苯酚和对苯二胺的胁迫。对于剂量效应响应最为明显的指标是GST和CarE,摇蚊体内的GST和CarE均随着浓度增加,活性被诱导增强。综合时间-剂量效应及酶响应程度来看,6种生物化学指标对于取代苯胁迫的敏感性大小依次为SOD>CAT>GST>CarE >ALP>ACP。
本研究结果表明取代苯类有机污染物对摇蚊幼虫具有慢性毒性,生长发育指标化蛹率对苯酚胁迫敏感,可以作为慢性毒性效应的标志物之一。摇蚊幼虫体内酶系CAT. SCD、GST和CarE参与了取代苯类有机污染物胁迫的应答反应,并表现出一定时间—剂量效应,可作为水体环境质量监测的早期预测预报。
Substituted aromatic compounds are mostly organic pollutants. With the industrial waste water, agricultural runoff, domestic waste flowing into waters, these organic pollutants with potential hazard to human health damaged the growth, development and reproduction of aquatic organisms, and destroyed the balance of aquatic ecosystems.Chironomids are widely distributed and numerous species of benthic organisms and often as an important indicator organism to monitor water quality. In this study,we selected twelve substituted benzenes according to the type, site and numbers of substituted group based on the list of China prefered controlled pollutants in water. We determined the acute and joint toxicity of twelve substituted benzenes to 4th-instar larval of Propsilocerus akamusi. The effects of sublethal doses of substituted benzenes on weight, pupation rate and six biochemical markers,GST, CarE, CAT, POD,SOD,of 4th-instar larval were also investigated in this study. The results showed as follow:
(1) The typical poisoning symptoms of larval chironomid were showed after chironomid larvae were exposed to substituted benzene. The larvae expressed slowly activity and moving under the treatment of toluene, chlorobenzene, phenol and chlorophenol. The surface viscosity of larvae increased under exposure of phenol. The body color of larvae exposed to p-phenylene-nediamine turned black,and some larvae appeared carbon black point or ring spot in some sections. These phenomena indicated the substituted benzenes have the paralytic, corrosive and burnable toxic characteristics. The chironomid larvae have high tolerance to the organic pollutants of substituted benzenes.The LC50 value range of 12 kinds of substituted benzenes was 90-1100 mg/L. Among them, the substituted benzenes of minium LC50 and maxium LC50 was p-chlorophenol(90.07mg/L)and aniline(1069.71 mg/L), respectively. According to toxicity mechanism of organic compounds, Benzene, aniline, chlorobenzene and 1,(2,3,4)-dimethylbenzene belonged to non-polar narcotic compounds while phenol, p-chlorophenol, nitrobenzene belonged to polar narcotic compounds. The 1,3-dimethylbenzene and p-phenylene diamine belonged to reactive organic compounds. The different substituted group in the benzene ring displayed the different toxicity among all substituted benzenes. The order of toxicity were-NO2>-OH>-C1>-CH3>-NH2. The order of toxicity among three isomers of dimethylbenzene were 1,2-dimethylbenzene>1,3-dimethylbenzen>1,4-dimethylben-en while p-phenylene diamine> p-phenylene diamine.
(2) The joint toxicities of 19 binary substituted benzenes were not completely consistent according to four evaluation methods. The evaluation results of TU were completely consistent with the evaluation results of MTI while the results of AI were completely consistent with of the results ofλ. But the evaluation results of TU were completely consistent with the evaluation results ofλwhile AI was consistent with MTI based on toxic extent to joint toxicity of binary substituted benzenes. The evaluation results of four methods were consistent for the synergim and simple addition but the evaluation results were different for the partial addition and antagonism. The above results showed that 52.63%,0.05% and 42.11% of the toxic effects for binary substituted benzene indicated synergistic, simple addition and partial addition or antagonism, respectively, under mixture of equal proportion.
(3) The phenol had an effect on growth and development of chironomid. The dry weight, fresh weight and pupation percentage of 4th-instar larval chironomid were decreased by treatment of phenol with the sublethal concentration of 0.4,4 and 40 mg/L. These indicators can be used to monitor chronic toxic effect because the larval chironomids were sensitive to phenol in the periods of growth and pupation.The SOD,CAT, GST,CarE,ACP and ALP as biochemical markers in vivo chironomid larval exposed in sublethal dose substituted benzene solution were in a concentration- and time-dependent way with increase of concentration and exposure. The SOD and CarE were most sensitive to exposure time because of they could respond to the stress of phenol and p-chlorophenol under 6 h of treatment. The SOD was most sensitive to the concentration because of it could respond to the stress of 0.4 mg/L of p-chlorophenol and p-phenylene diamine. The GST and CarE were most sensitive to dose because of the enzymatic activities incresed with increase of concentration. According to time-and dose- effect and responsive extent of enzyme activities, the sensibility of six biochemical markers to the stress of substituted benzene were SOD>CAT>GST>CarE.
These results showed that substituted benzene indicated chronic toxicity to larval chironomids.The pupation percentage was sensitive to the stress of phenol and can be as a marker of chronic toxicity effects. The SOD,CAT, GST and CarE among enzymatic system in larval chironomid could respond to the stress of substituted benzene and were in a concentration- and time-dependent way. Therefore, these four biochemical markers can be used to monitor the environmental quality of water.
(1)摇蚊幼虫暴露于取代苯类污染物溶液中表现出典型的中毒症状：甲苯、氯苯、苯酚和对氯苯酚溶液中的幼虫表现出活动减慢和行动迟缓；苯酚溶液中幼虫体表粘性增大；对苯二胺溶液中的幼虫体色变黑,部分体节还会出现炭黑色的点或环状斑。这些现象表明取代苯具有麻痹性、腐蚀性和灼伤性的毒性特点。摇蚊幼虫对取代苯类有机污染物的忍耐性较高,12种取代苯类对摇蚊4龄幼虫24 h LC50范围为90～1100 mg/L,其中最小的是对氯苯酚,LC50为90.07mg/L,最大的是苯胺,LC50为1069.71mg/L。按照有机物的毒性作用机制区分：苯、苯胺、氯苯和邻、间、对-二甲苯为非极性麻醉性化合物；苯酚、对氯苯酚和硝基苯为急性麻醉性化合物；对苯二胺和间苯二胺为反应性有机化合物。取代苯类有机污染物苯环上取代基不同,其毒性不同,基团毒性的贡献为：-NO2>-OH>-C1>-CH3>-NH2;3种二甲苯同分异构体毒性大小依次为邻二甲苯>间二甲苯>对二甲苯；二苯胺同分异构体毒性大小依次为对苯二胺>间苯二胺。
(2)4种指数法对19组二元取代苯联合毒性结果的评价并不完全一致：对联合毒性结果的定性判断上,TU法和MTI法评价结果完全一致,Al法和λ法的评价结果完全一致；对联合毒力强弱程度的判断上TU法与λ法一致,Al法和MTI法一致。4种评价方法对于协同和简单相加毒性效应的评价结果一致,对于部分相加和拮抗作用的评价结果则有所不同。综合4种评价方法结果表明一半以上(52.63%)的二元取代苯在等比毒性配比下的联合作用类型为协同作用；极少数(0.05%)表现为简单相加作用,其余(42.11%)表现为部分相加和拮抗作用。
(3)苯酚影响摇蚊生长发育。亚致死剂量(0.4、4、40 mg/L)苯酚降低摇蚊幼虫干湿重和化蛹率,这表明摇蚊幼虫生长和化蛹过程中对苯酚的胁迫敏感,可将其作为慢性毒性效应指标。摇蚊幼虫体内的生物标志物(SOD、CAT、GST、CarE、ACP、ALP)对于4种亚致死剂量取代苯溶液的胁迫表现一定的时间—剂量效应。对于暴露时间最为敏感的生物化学标志物是SOD和CarE,暴露于苯酚和对氯苯酚6 h,摇蚊体内SOD和CarE就呈现出应答反应。对于浓度最为敏感的指标是SOD,摇蚊体内的SOD能响应0.4mg/L对氯苯酚和对苯二胺的胁迫。对于剂量效应响应最为明显的指标是GST和CarE,摇蚊体内的GST和CarE均随着浓度增加,活性被诱导增强。综合时间-剂量效应及酶响应程度来看,6种生物化学指标对于取代苯胁迫的敏感性大小依次为SOD>CAT>GST>CarE >ALP>ACP。
本研究结果表明取代苯类有机污染物对摇蚊幼虫具有慢性毒性,生长发育指标化蛹率对苯酚胁迫敏感,可以作为慢性毒性效应的标志物之一。摇蚊幼虫体内酶系CAT. SCD、GST和CarE参与了取代苯类有机污染物胁迫的应答反应,并表现出一定时间—剂量效应,可作为水体环境质量监测的早期预测预报。
Substituted aromatic compounds are mostly organic pollutants. With the industrial waste water, agricultural runoff, domestic waste flowing into waters, these organic pollutants with potential hazard to human health damaged the growth, development and reproduction of aquatic organisms, and destroyed the balance of aquatic ecosystems.Chironomids are widely distributed and numerous species of benthic organisms and often as an important indicator organism to monitor water quality. In this study,we selected twelve substituted benzenes according to the type, site and numbers of substituted group based on the list of China prefered controlled pollutants in water. We determined the acute and joint toxicity of twelve substituted benzenes to 4th-instar larval of Propsilocerus akamusi. The effects of sublethal doses of substituted benzenes on weight, pupation rate and six biochemical markers,GST, CarE, CAT, POD,SOD,of 4th-instar larval were also investigated in this study. The results showed as follow:
(1) The typical poisoning symptoms of larval chironomid were showed after chironomid larvae were exposed to substituted benzene. The larvae expressed slowly activity and moving under the treatment of toluene, chlorobenzene, phenol and chlorophenol. The surface viscosity of larvae increased under exposure of phenol. The body color of larvae exposed to p-phenylene-nediamine turned black,and some larvae appeared carbon black point or ring spot in some sections. These phenomena indicated the substituted benzenes have the paralytic, corrosive and burnable toxic characteristics. The chironomid larvae have high tolerance to the organic pollutants of substituted benzenes.The LC50 value range of 12 kinds of substituted benzenes was 90-1100 mg/L. Among them, the substituted benzenes of minium LC50 and maxium LC50 was p-chlorophenol(90.07mg/L)and aniline(1069.71 mg/L), respectively. According to toxicity mechanism of organic compounds, Benzene, aniline, chlorobenzene and 1,(2,3,4)-dimethylbenzene belonged to non-polar narcotic compounds while phenol, p-chlorophenol, nitrobenzene belonged to polar narcotic compounds. The 1,3-dimethylbenzene and p-phenylene diamine belonged to reactive organic compounds. The different substituted group in the benzene ring displayed the different toxicity among all substituted benzenes. The order of toxicity were-NO2>-OH>-C1>-CH3>-NH2. The order of toxicity among three isomers of dimethylbenzene were 1,2-dimethylbenzene>1,3-dimethylbenzen>1,4-dimethylben-en while p-phenylene diamine> p-phenylene diamine.
(2) The joint toxicities of 19 binary substituted benzenes were not completely consistent according to four evaluation methods. The evaluation results of TU were completely consistent with the evaluation results of MTI while the results of AI were completely consistent with of the results ofλ. But the evaluation results of TU were completely consistent with the evaluation results ofλwhile AI was consistent with MTI based on toxic extent to joint toxicity of binary substituted benzenes. The evaluation results of four methods were consistent for the synergim and simple addition but the evaluation results were different for the partial addition and antagonism. The above results showed that 52.63%,0.05% and 42.11% of the toxic effects for binary substituted benzene indicated synergistic, simple addition and partial addition or antagonism, respectively, under mixture of equal proportion.
(3) The phenol had an effect on growth and development of chironomid. The dry weight, fresh weight and pupation percentage of 4th-instar larval chironomid were decreased by treatment of phenol with the sublethal concentration of 0.4,4 and 40 mg/L. These indicators can be used to monitor chronic toxic effect because the larval chironomids were sensitive to phenol in the periods of growth and pupation.The SOD,CAT, GST,CarE,ACP and ALP as biochemical markers in vivo chironomid larval exposed in sublethal dose substituted benzene solution were in a concentration- and time-dependent way with increase of concentration and exposure. The SOD and CarE were most sensitive to exposure time because of they could respond to the stress of phenol and p-chlorophenol under 6 h of treatment. The SOD was most sensitive to the concentration because of it could respond to the stress of 0.4 mg/L of p-chlorophenol and p-phenylene diamine. The GST and CarE were most sensitive to dose because of the enzymatic activities incresed with increase of concentration. According to time-and dose- effect and responsive extent of enzyme activities, the sensibility of six biochemical markers to the stress of substituted benzene were SOD>CAT>GST>CarE.
These results showed that substituted benzene indicated chronic toxicity to larval chironomids.The pupation percentage was sensitive to the stress of phenol and can be as a marker of chronic toxicity effects. The SOD,CAT, GST and CarE among enzymatic system in larval chironomid could respond to the stress of substituted benzene and were in a concentration- and time-dependent way. Therefore, these four biochemical markers can be used to monitor the environmental quality of water.
引文
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