【作者】 郑师梅；

【导师】 周启星；

【作者基本信息】 南开大学 ， 环境科学， 2013， 博士

【摘要】 本论文探讨了甲苯、乙苯和二甲苯等苯系物对我国典型腹足纲动物铜锈环棱螺的急性和亚慢性毒害效应,同时利用本课题组的实验数据和搜集到的甲苯、乙苯和二甲苯的毒性数据,对我国苯系物的水质基准展开了尝试性的研究。目前,脊椎动物中的鱼类和无脊椎动物中的节肢动物是国内外发展的主要实验生物,但是却缺少了无脊椎动物很重要的一部分——软体动物,特别是其中的腹足纲动物,因此其也极少被应用于水质管理质量基准等保护政策的制定中。我国正在发展适合我国生态系统的水质基准,而面临的最大困难之一是缺少本土的模式受试物种,因此,开发我国本土的腹足纲测试物种将能为我国水质基准的制定提供技术的支持,同时也避免了其他国家在制定水质基准时对软体动物不重视的问题。本研究选取了我国本土腹足纲动物——铜锈环棱螺(Bellamya aeruginosa)为受试生物,探讨甲苯、乙苯和二甲苯三种苯系物对其的毒害效应。急性毒性试验研究发现,甲苯、乙苯和二甲苯未造成铜锈环棱螺的死亡现象,而对铜锈环棱螺的行为有明显的影响。因此,甲苯、乙苯和二甲苯对铜锈环棱螺的急性毒性试验是以其对螺行为的影响,而不是以死亡为研究指标。甲苯、乙苯和二甲苯对螺的急性行为毒性是引起了不良反应,且具有显著的剂量-效应关系,甲苯、乙苯和二甲苯对螺不良反应的96-h EC50分别为37.2、13.3和8.9mg/L。同时发现,以乙苯为例,不良反应螺的AChE活性受抑制的程度要大于正常反应螺的,而其他生化指标,包括超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽硫转移酶(GST)、还原型谷胱甘肽(GSH)和脂质过氧化(丙二醛,MDA)与螺的行为变化未呈现出明显的关联性。在其他的研究中发现,抑制AChE的活性是与行为变化有相关性的,因此,本研究认为甲苯、乙苯和二甲苯对铜锈环棱螺的行为变化与AChE的活性受到抑制有关联。本论文重点研究了乙苯亚慢性低剂量暴露对铜锈环棱螺的影响,实验分为两个阶段：21天的暴露阶段和17天的恢复阶段。其中,对铜锈环棱螺的影响主要是从两个方面研究的,一方面是生化指标的变化情况,包括AChE、肝胰脏和肾脏组织中的SOD、CAT、GST、GSH和MDA的活性和含量变化；另一方面是利用碱性彗星实验检测肝胰脏组织中的DNA损伤。研究结果表明,乙苯低剂量的暴露亦能对铜锈环棱螺的行为产生影响,造成了铜锈环棱螺的收缩反应,且收缩反应的比例随着暴露时间的增加而增加,但是未有明显的剂量-效应关系。铜锈环棱螺的6种生理生化指标对乙苯胁迫的灵敏度各不相同,且生理生化指标在两种不同行为的铜锈环棱螺中并未呈现出规律性的差异,生理生化指标与铜锈环棱螺行为的相关性不明确。碱性彗星实验的结果表明,乙苯胁迫显著性的引起了螺肝胰脏细胞的DNA损伤,且DNA损伤程度有明显的剂量-效应和时间-效应关系,表明乙苯可能对铜锈环棱螺存在潜在的基因毒性。同时发现,不同行为铜锈环棱螺的DNA损伤没有差异性。在恢复阶段的早期5天内,DNA损伤持续的增加,这应该是由于对DNA损伤的修复造成的,例如,碱基切除修复或者核苷酸切除修复。在恢复阶段的17天之后,DNA损伤的修复还没有完成,表明DNA损伤的修复还需要更多的时间。同时,本论文还研究了铜锈环棱螺对乙苯的富集和净化能力,实验分为两个阶段：23天的暴露阶段和17天的净化阶段。结果表明,正常反应螺对乙苯的生物富集因子(BCF)从2.24到80,收缩反应螺对乙苯的BCF从2.05到70,由此可见,正常反应螺对乙苯的富集能力要大于收缩反应螺的。在净化阶段发现,两种不同行为螺对乙苯的净化速率均遵循一级动力学规律,两种不同行为铜锈环棱螺净化乙苯的平均半衰期分别为8.5天和8.0天,两者之间没有明显的差异性,而且5个处理组之间在净化速率上同样也不存在显著的差异性。在净化阶段17天之后,两种不同行为螺体内均还能检测到乙苯,说明螺完全净化干净乙苯还需要更多的时间。为了制定符合我国水生态系统特征的苯系物水质基准,本研究利用来自本课题组的苯系物毒性数据,同时还搜集了苯系物相关的毒理数据,采用美国制定水质基准的毒性百分数排序法推导了我国甲苯、乙苯和二甲苯的淡水生物的双值水质基准。毒性百分数排序法得到的基准值可以为水生生物起到应有的保护作用。采用毒性百分排序法得出甲苯、乙苯和二甲苯的基准最大浓度推荐值分别为4.49、0.89和1.15mg/L,基准连续浓度推荐值分别为0.81、0.25和0.41mg/L。该研究将为科学制订苯系物地表水环境质量标准以及排放标准提供重要的理论依据,也为我国今后系统开展水质基准的科学研究提供了示范。更多还原

【Abstract】 As a consequence of their wide usage, monocyclic aromatic hydrocarbons toluene, ethylbenzene, and xylene have been detected frequently in the aquatic environment, which could lead to harmful effects to the aquatic systems and human. In this paper, acute and subchronical toxicity of toluene, ethylbenzene, and xylene on an aquatic gastropod were investigated, and we attempted to tentatively enact the Water Quality Criteria for toluene, ethylbenzene, and xylene for protecting the aquatic organisms in Chinese aquatic ecosystem.As far as we know, the model testing organisms are primarily confined to fish in vertebrates and arthropods in invertebrates. Molluscs also are of great importance in the kingdom of invertebrates, whereas they are absent in the model testing organisms, especially gastropods. So, they are seldom considered in the protect policy of water standard and criteria. Nowadays, China is embarking on the development of its own national water quality system fitting for Chinese aquatic ecosystem, and one of the biggest difficulties is short of the local model testing organisms. Therefore, developing our country own gastropod testing organisms will provide technical support to establish Water Quality Criteria for our country and will also avoid the neglected problem of gastropod in establishing the Water Quality Criteria in the other countries. A local gastropod, Bellamya aeruginosa, was selected as the test organism in the present toxicity study of toluene, ethylbenzene, and xylene, and it is further to discuss the practicability of B. aeruginosa as a potential model testing organism in the present study.In the acute study of toluene, ethylbenzene, and xylene, it was found that no mortality occurred, but behavioral alteration, i.e. distress syndrome, was observed in the exposed snails, thus, here the acute effect of toluene, ethylbenzene, and xylene on the snails was evaluated through the behavioral toxicity, and coupled with biochemical responses, including acetylcholinesterase (AChE) in the whole body, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferases (GST), reduced glutathione (GSH), and lipid peroxidation (malonyldialdehyde, MDA) in the hepatopancreas and kidney. Distress syndrome had clear dose-and time-dependent effects (p<0.05), and96-h EC50values for the distress syndrome of toluene, ethylbenzene, and xylene were37.2,13.3, and8.9mg/L in the tested snails. Taken ethylbenzene as example, AChE activity of the distressed snail was all inhibited more than45%, while the inhibition of AChE activity in the nondistressed snail was all less than30%, and the oxidative biomarkers (SOD, CAT, GST, GSH, and MDA) showed no clear correlation with the distress syndrome. The inhibition in AChE activity is found to be related with behavioral alterations in many studies; therefore, more than45%inhibition in AChE activity induced by ethylbenzene was thought to be connected with the distress syndrome in the tested snails. The findings showed that toluene, ethylbenzene, and xylene caused the inhibition on AChE activity, and eventually led to the behavioral alteration occurred in the snails.The focus of present work was performed to elucidate the sublethal effects of ethylbenzene using the freshwater snail, B. aeruginosa (Reeve). The experiment was divided into two periods:a21-day exposure period followed by a17-day recovery period. AChE, SOD, CAT, GST, GSH, and MDA were used as the biochemical biomarkers to evaluate oxidative stress in hepatopancreas and kidney of snails. In addition, alkaline comet assay was applied to determine the genotoxicity of ethylbenzene in hepatopancreas of snails. The results showed that behavioral alteration, namely retraction response, was also observed during the exposure period, and the proportion of retracted snails increased under each treatment as the exposure time prolonged but there was no linear relationship between the retracted proportion and the exposure dose. These six biomarkers exhibited various responses to ethylbenzene in the tested snails, and the differences in these biochemical biomarkers between the two different behavioral snails were not showed same trend, indicating that the correlation between these biochemical biomarkers and the behavioral alteration (retraction response) was not clear. Alkaline comet assay showed that ethylbenzene could significantly induce DNA damage in hepatopancreas of snails, and there was a good dose-and time-response in DNA damage, indicating potential genotoxicity of ethylbenzene on snails. Moreover, the levels of DNA damage had no difference between the two different responses of snails. At the first five days of the recovery period, the increased DNA damage may be due to the repair of DNA damage, such as base or nucleotide excision repair. At the end of the recovery period, the repair of DNA damage was not yet completed, showing that DNA repair requires more time.Meanwhile, the capability of the freshwater gastropod B. aeruginosa to take up and depurate ethylbenzene was studied in the subchronical study; the snail was subjected to two treatments, a23-day exposure period followed by a17-day depuration period. The results showed that ethylbenzene uptake in unretracted snails was greater than in retracted snails, the bioconcentration factor (BCF) in the unretracted snails ranged from2.24to80, and BCF in the retracted snails ranged from2.05to70. The depuration rates followed the first order kinetics for both different behavior snails, and the average half-lives of ethylbenzene depuration were8.5d and8.0d, indicating that the depuration abilities in the two different responses of snails had no significant difference from each other. There were also no significant differences among the five treatments in terms of depuration rate for each group. Because of the limited capability of snails to detoxify ethylbenzene, the depuration was mainly through a slow excretion process, and ethylbenzene was still present in the tissue of snail after the17-day depuration. So, the snails need more time to depurate ethylbenzene completedly.In order to derive Water Quality Criteria for toluene, ethylbenzene, and xylene that can protect the freshwater ecosystem and biota system in China, all available toxicity data of toluene, ethylbenzene, and xylene to Chinese representative species in freshwater were collected. The toxicity percentile rank method, used for criteria derivation in America was used to derive aquatic life criteria for the three substances. The toxicity percentile rank method was suitable to derive Water Quality Criteria to protect the aquatic organisms. The results from the toxicity percentile rank method showed that, the criteria maximum concentrations of toluene, ethylbenzene and xylene were4.49,0.89, and1.15mg/L, respectively, and the criteria continuous concentrations were0.81,0.25, and0.41mg/L, respectively.更多还原