部分硝基苯类和双酚类有机物及其混合物对发光菌的毒性研究
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摘要
有毒化学品的毒性测试与生态风险评价历来是环境污染领域的研究重点。选择两类环境中常见的有毒有机污染物作为研究对象,采用微板毒性测试方法测试毒物对发光菌的发光抑制效应。这两类有毒有机污染物分别是硝基苯类物质和双酚类物质,这两类物质都属于高毒、高生物富集性、难降解有毒化学品,且生产量大,使用广泛,在环境中普遍存在,严重的威胁着生物体和人类的健康,因此从方法学上探讨单个有毒化学品及其混合物的生态毒性的测试方法以及模型预测方法有很大的研究意义。
本文采用的菌种是一种淡水发光菌——青海弧菌Q67,以96孔微板为载体在SpectraMaxM5光度测试仪上同时测试12个浓度梯度的发光抑制效应。将测试结果进行处理,得到各毒物的浓度.效应关系,应用非线性最小二乘法将数据拟合到logit和weibull函数,并由此计算各个效应下的效应浓度如EC50等。
在环境中,不存在纯的有毒化合物,各毒物都是以复杂混合物的形式作用于生物体,因此由对单个物质的研究逐步扩展到对混合体系的研究是生态毒理学研究的必然趋势。目前对于混合毒性的研究多集中在两两混合阶段,而且混合比例固定,不能很好地反映实际环境体系中的混合作用机制,本文将混合物研究扩展到三种以上多组分的混合,并且引入均匀实验设计探索更大混合空间内的浓度配比,使选取的实验点更具代表性,更全面地反映混合毒性作用特性。
首先对硝基苯及其6种一元取代物进行了发光菌的单一急性毒性测试,得出这7种毒物的pEC_(50)值,其大小关系是:对硝基苯胺(4.22)>对硝基甲苯(3.39)>间氯硝基苯(3.30)>对氯硝基苯(3.29)>邻氯硝基苯(3.22)>间硝基苯胺(2.94)>硝基苯(2.66)。接下来,测试了10种双酚物质对发光菌的急性毒性,由于在实验中遇到溶解性问题,因此最后考虑将测试物质分为两组,一组配制成低浓度的水溶液,另一组加入二甲亚砜作助溶剂配制成20%二甲亚砜水溶液,同时考察了二甲亚砜作助溶剂对毒性测试结果的影响。结果表明,五种双酚物质水溶液的pEC_(50)值及其大小关系是:BPA(5.17)>BPE(5.13)>BPAF(4.29)>BPF(4.25)>BPS(3.69)。BPA的水溶液、BPA的5%DMSO水溶液和BPA的20%DMSO水溶液的pEC_(50)值分别是:5.18、4.97、4.84,可以看出有机溶剂对结果影响很小。五种难溶双酚物质的20%DMSO/水溶液的pEC_(50)值分别是:BPC=4.33、BPZ=5.3、BPM=4.13、BPP=4.32、BPAP=4.27。
此外,对这两类物质的混合毒性进行了初步探索。采用的是结构相似的同类组分的混合,并且引入CA、IA模型对混合物的毒性进行预测,并将实验测试结果与预测结果进行对比,研究CA、IA模型的预测能力,并对混合毒性作用机制进行初步分析。首先按照传统的等效应浓度比混合设计3个5组分的双酚物质的混合物和5个7组分硝基苯类物质的混合物,研究发现实验测试结果和模型预测结果具有很高的吻合性,但是CA、IA预测线没有很好的分开,预测结果相差不大,可以说CA和IA模型对该混合物都具有较好的预测能力,其作用机制还有待进一步探索。为了进一步研究多组分混合物的整个浓度空间的不同配比情况,设计了一个7因素,5水平,包括10次实验的均匀设计表,按照均匀设计的方法安排了10种浓度配比的混合物,结果发现,与等效应浓度比混合不同的是10个均匀设计混合物的DRC能够很好的分散,不再像等效应浓度比混合那样集成一束,所以均匀设计混合能够在更大组分浓度配比空间内考察多组分混合物毒性作用类型。并且CA、IA模型对均匀设计安排的混合物也具有很强的预测能力。
Toxicity test and ecologic risk assessment for the toxic chemicals are always the important parts in the environmental pollution research area. Choosing two kinds of typical toxic organic pollutants as the study objective, using microplate toxicity test method, we tested the luminescent-inhibition effecting of toxicants to the photobacterium. The two kinds of toxic organic pollutants were nitrobenzene and bisphenol compounds, which were the toxic chemicals with high toxicity, high biologic concentration and difficult to degrade.
The test bacterium is the only kind of freshwater photobacterium-Vibrio Qinghaiensis sp.. The luminescent-inhibition effecting to the photobacterium was tested on the SpectraMax M5 device. After data processing, the dose-effect relations of toxicants were got, and then were transformed into function expression and related parameters through regression analysis with least square method. Different from the usual liner analysis which only could get the dose-effect relations in the limited range; this nonlinear analysis could get the whole dose-effect relations of toxicants.
It don't exist pure toxic compound in the nature environment, and every toxic compound affected the living body by complex mixture forms. So, from the single compound research extended to mixture system research is the inevitable trend of eco toxicology development. At present, the mixture toxicity research major concentrated in two-two action steps, which could not reflect the mixture toxicity action in the actual environment.
The luminescent-inhibition effecting of nitrobenzene and 6 kinds of its substitution to the photobacterium were determined. The results showed that the pEC_(50) values of this 7 toxicants were: 4-Nitroaniline(4.22)>4-Nitrotoluene(3.39) >1-Chloro-3-nitrobenzene(3.30)>1-Chloro-4-nitrobenzene(3.29)>1-Chloro-2-nitrobe nzene(3.22 )>3-Nitroaniline (2.94 )>Nitrobenzene(2.66). Then, 10 kinds of bisphenol compounds were tested by microplate toxicity test method. Because of solution problem, the test compounds were separated into two groups. The toxicants in the first group were dissolved in distilled water, and the toxicants in the second group were dissolved in 20%DMSO/water. In the meanwhile, research the influence of cosolvent-DMSO. The pEC_(50) values of toxicants in the first group were: BPA(5.17)> BPE(5.13) >BPAF(4.29)> BPF(4.25) >BPS(3.69). The pEC_(50) values of toxicants in the second group were: BPC=4.33、BPZ=5.3、BPM=4.13、BPP=4.32、BPAP=4.27.
Choosing the same kind compounds with the similar structure as mixture components, introducing CA/IA model to predict the mixture toxicity, a preliminary exploration of mixture toxicity of nitrobenzene and bisphenol compounds was made. 3 mixtures with 5 bisphenol components and 5 mixtures with 7 nitrobenzene components were designed by the traditional equivalent-effect concentration ratio method. For the further research of different concentration ratio in the whole space of mixture, a uniform table with 7 factors, 5 levels and 10 lines was designed. 10 kinds of concentration ratio mixture were arranged based on the uniform table. It is found the CA/IA model prediction results had a high reliability. The DRC of 10 uniform design mixtures were scattered and don't assemble together as equivalent-effect concentration ratio mixtures, which mean uniform design could see about the mixture toxicity of multi component mixture in a greater scope.
本文采用的菌种是一种淡水发光菌——青海弧菌Q67,以96孔微板为载体在SpectraMaxM5光度测试仪上同时测试12个浓度梯度的发光抑制效应。将测试结果进行处理,得到各毒物的浓度.效应关系,应用非线性最小二乘法将数据拟合到logit和weibull函数,并由此计算各个效应下的效应浓度如EC50等。
在环境中,不存在纯的有毒化合物,各毒物都是以复杂混合物的形式作用于生物体,因此由对单个物质的研究逐步扩展到对混合体系的研究是生态毒理学研究的必然趋势。目前对于混合毒性的研究多集中在两两混合阶段,而且混合比例固定,不能很好地反映实际环境体系中的混合作用机制,本文将混合物研究扩展到三种以上多组分的混合,并且引入均匀实验设计探索更大混合空间内的浓度配比,使选取的实验点更具代表性,更全面地反映混合毒性作用特性。
首先对硝基苯及其6种一元取代物进行了发光菌的单一急性毒性测试,得出这7种毒物的pEC_(50)值,其大小关系是:对硝基苯胺(4.22)>对硝基甲苯(3.39)>间氯硝基苯(3.30)>对氯硝基苯(3.29)>邻氯硝基苯(3.22)>间硝基苯胺(2.94)>硝基苯(2.66)。接下来,测试了10种双酚物质对发光菌的急性毒性,由于在实验中遇到溶解性问题,因此最后考虑将测试物质分为两组,一组配制成低浓度的水溶液,另一组加入二甲亚砜作助溶剂配制成20%二甲亚砜水溶液,同时考察了二甲亚砜作助溶剂对毒性测试结果的影响。结果表明,五种双酚物质水溶液的pEC_(50)值及其大小关系是:BPA(5.17)>BPE(5.13)>BPAF(4.29)>BPF(4.25)>BPS(3.69)。BPA的水溶液、BPA的5%DMSO水溶液和BPA的20%DMSO水溶液的pEC_(50)值分别是:5.18、4.97、4.84,可以看出有机溶剂对结果影响很小。五种难溶双酚物质的20%DMSO/水溶液的pEC_(50)值分别是:BPC=4.33、BPZ=5.3、BPM=4.13、BPP=4.32、BPAP=4.27。
此外,对这两类物质的混合毒性进行了初步探索。采用的是结构相似的同类组分的混合,并且引入CA、IA模型对混合物的毒性进行预测,并将实验测试结果与预测结果进行对比,研究CA、IA模型的预测能力,并对混合毒性作用机制进行初步分析。首先按照传统的等效应浓度比混合设计3个5组分的双酚物质的混合物和5个7组分硝基苯类物质的混合物,研究发现实验测试结果和模型预测结果具有很高的吻合性,但是CA、IA预测线没有很好的分开,预测结果相差不大,可以说CA和IA模型对该混合物都具有较好的预测能力,其作用机制还有待进一步探索。为了进一步研究多组分混合物的整个浓度空间的不同配比情况,设计了一个7因素,5水平,包括10次实验的均匀设计表,按照均匀设计的方法安排了10种浓度配比的混合物,结果发现,与等效应浓度比混合不同的是10个均匀设计混合物的DRC能够很好的分散,不再像等效应浓度比混合那样集成一束,所以均匀设计混合能够在更大组分浓度配比空间内考察多组分混合物毒性作用类型。并且CA、IA模型对均匀设计安排的混合物也具有很强的预测能力。
Toxicity test and ecologic risk assessment for the toxic chemicals are always the important parts in the environmental pollution research area. Choosing two kinds of typical toxic organic pollutants as the study objective, using microplate toxicity test method, we tested the luminescent-inhibition effecting of toxicants to the photobacterium. The two kinds of toxic organic pollutants were nitrobenzene and bisphenol compounds, which were the toxic chemicals with high toxicity, high biologic concentration and difficult to degrade.
The test bacterium is the only kind of freshwater photobacterium-Vibrio Qinghaiensis sp.. The luminescent-inhibition effecting to the photobacterium was tested on the SpectraMax M5 device. After data processing, the dose-effect relations of toxicants were got, and then were transformed into function expression and related parameters through regression analysis with least square method. Different from the usual liner analysis which only could get the dose-effect relations in the limited range; this nonlinear analysis could get the whole dose-effect relations of toxicants.
It don't exist pure toxic compound in the nature environment, and every toxic compound affected the living body by complex mixture forms. So, from the single compound research extended to mixture system research is the inevitable trend of eco toxicology development. At present, the mixture toxicity research major concentrated in two-two action steps, which could not reflect the mixture toxicity action in the actual environment.
The luminescent-inhibition effecting of nitrobenzene and 6 kinds of its substitution to the photobacterium were determined. The results showed that the pEC_(50) values of this 7 toxicants were: 4-Nitroaniline(4.22)>4-Nitrotoluene(3.39) >1-Chloro-3-nitrobenzene(3.30)>1-Chloro-4-nitrobenzene(3.29)>1-Chloro-2-nitrobe nzene(3.22 )>3-Nitroaniline (2.94 )>Nitrobenzene(2.66). Then, 10 kinds of bisphenol compounds were tested by microplate toxicity test method. Because of solution problem, the test compounds were separated into two groups. The toxicants in the first group were dissolved in distilled water, and the toxicants in the second group were dissolved in 20%DMSO/water. In the meanwhile, research the influence of cosolvent-DMSO. The pEC_(50) values of toxicants in the first group were: BPA(5.17)> BPE(5.13) >BPAF(4.29)> BPF(4.25) >BPS(3.69). The pEC_(50) values of toxicants in the second group were: BPC=4.33、BPZ=5.3、BPM=4.13、BPP=4.32、BPAP=4.27.
Choosing the same kind compounds with the similar structure as mixture components, introducing CA/IA model to predict the mixture toxicity, a preliminary exploration of mixture toxicity of nitrobenzene and bisphenol compounds was made. 3 mixtures with 5 bisphenol components and 5 mixtures with 7 nitrobenzene components were designed by the traditional equivalent-effect concentration ratio method. For the further research of different concentration ratio in the whole space of mixture, a uniform table with 7 factors, 5 levels and 10 lines was designed. 10 kinds of concentration ratio mixture were arranged based on the uniform table. It is found the CA/IA model prediction results had a high reliability. The DRC of 10 uniform design mixtures were scattered and don't assemble together as equivalent-effect concentration ratio mixtures, which mean uniform design could see about the mixture toxicity of multi component mixture in a greater scope.
引文
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