【作者】 史志明；

【导师】 胡锋；

【作者基本信息】 南京农业大学 ， 生态学， 2013， 博士

【摘要】 蚯蚓在陆生生态系统中居于重要地位,是许多动物的食物来源。土壤中疏水性有机污染物,尤其是持久性有机污染物能蓄积在蚯蚓体内,并可能通过食物链的富集和放大作用影响整个土壤生态系统的平衡和健康。然而,对于疏水性有机污染物在蚯蚓体内的归趋并没有完全揭示清楚。本文第一部分(包括第二章和第三章)在评述蚯蚓蓄积疏水性有机污染物的相关研究现状的基础(第一章)上,以菲作为疏水性有机污染物的代表,较为系统地探讨了“污染物在蚯蚓体内如何分布和吸收潜力”等问题,完善了疏水性有机污染在蚯蚓体内归趋的相关知识。蚯蚓是土壤生态毒理测试标准的模式动物之一,蚯蚓生物标志物常用作土壤污染风险评价研究。然而,蚯蚓生物标志物理论并没有在实践中得到广泛应用。本文在第二部分(第四章),在评述蚯蚓蓄积疏水性有机污染物的相关研究现状的基础上(第一章第4节),以蚯蚓体抗氧化防御体系为代表,研究了抗氧化防御体系在蚯蚓生长过程中的变化,在成年蚯蚓亚机体水平上的分布,并进一步探讨了疏水性有机污染物菲对蚯蚓抗氧化防御体系分布的影响。研究显示：1)采用分层次的方法系统研究了菲在蚯蚓体不同分组水平上的分布,菲在蚯蚓体内的分布是异质性的。具体体现在,(a)在亚机体水平上,低浓度处理下,菲在不同组分中的浓度没有表现出显著性差异,但在高浓度水平上,菲浓度分布格局从后部>生殖环节>前部逐渐转变为后部≈生殖环节>前部,而菲在蚯蚓体内的分布比例在所有的处理中均呈现出后部(58～72%)>前部(18～24%)>生殖环节(12～19%)格局；(b)在组织水平上,菲浓度呈现出蚯蚓肠道>体腔液>表皮的规律,而分布比例则呈现出表皮和肠道(分别为38%-43%和34%-47%)>体腔液(15%-22%)的格局；(c)在亚细胞水平上,菲在胞外组分中的浓度是其是胞内组分中浓度的4.68到1.23倍,而分配比例则从胞外组分(51～61%)>胞内组分(39-48%)逐渐变为胞内组分(51-70%)>胞外组分(29～49%)的规律。基于这些结果,我们推测蚯蚓体内的循环系统和分泌系统是菲在亚机体水平上分布差异的原因,而在组织水平和亚细胞水平上,菲从体表到体腔液和从细胞外到细胞内的分配机制为被动扩散。2)蚯蚓对菲的吸附呈现显著线性关系,说明分配作用是土壤溶液中菲进入蚯蚓体的主要机制；推导出的蚯蚓对菲的最大吸收值(410.76和365.12mg kg-1)同Jager等构建机制模型(mechanistic model)预测值(346.08mg kg-1)相一致,支持了该模型所做的前提假设。3)为了解释菲在蚯蚓体内的分布格局,采用批量吸附试验研究了亚机体和组织水平组分对菲的吸附。研究结果表明,在亚机体水平上,蚯蚓各组分对菲的吸附均呈现显著线性关系,但吸附能力呈现后部>生殖环节>前部的格局,这同真实菲在蚯蚓体内的分布格局存在不同,说明了仅仅用分配作用难以解释这种现象,而蚯蚓生命活动如覆盖全身的循环系统能较好的解释；在组织水平上,蚯蚓各组分对菲的吸附亦呈现显著线性关系,且吸附能力呈现出肠道>表皮的格局,同真实的菲在二者间的分布现象一致,说明分配作用能较好的解释菲在蚯蚓组织水平上的分布。4)蚯蚓抗氧化酶系统随着生长过程的不同阶段是变化的,幼年蚯蚓显示出较高的超氧化物歧化酶(SOD)和过氧化物酶(POD)活性,而成年蚯蚓中过氧化氢酶(CAT)活性相对较高,丙二醛(MDA)含量在蚯蚓生长过程中保持相对稳定的含量。表明蚯蚓在成长的不同阶段,遭受不同的活性氧(ROS)胁迫并对活性氧胁迫并具有不同的应对策略,同时也说明,在理想条件下,蚯蚓抗氧化防御体系的功能是有效的,使MDA含量在一个较低的水平。5)抗氧化酶在蚯蚓体内的分布是异质性的,SOD活性分布为生殖环节>前部≈后部,CAT活性分布为后部>前部≈生殖环节,POD活性遵循前部>后部>生殖环节的规律。MDA主要分布在生殖环节处。说明在整个抗氧化防御系统维持低水平的ROS过程中,不同部位不同种类的抗氧化酶起的作用不同。这些结果表明,在蚯蚓生长过程中,蚯蚓的不同部位应对活性氧的策略不同的,这有助于我们全面认识蚯蚓的抗氧化防御体系和更好的应用于土壤污染的风险评价。6)本研究中,也观察到暴露时间是一种环境胁迫因子,因为它不但影响了成年蚯蚓抗氧化酶的活性,而且影响了其分布格局。基于此,我们提出了“胁迫强度(stress magnitude)"的概念,指暴露时间与暴露浓度的共同效应的函数,即胁迫强度=污染物浓度×暴露时间,可以更加全面地反映非生物因子对生物的综合作用。7)我们还通过接触滤纸试验研究了菲对成年蚯蚓抗氧化酶系统分布的影响,结果表明,生殖环节处的SOD酶和POD酶活性对菲的胁迫敏感,可能是一个较好反映菲污染胁迫的指标,而位于蚯蚓前部和后部的CAT活性则对菲的胁迫比其它两种酶敏感。这些结果有利于更好地应用蚯蚓生物标志物反映污染胁迫。本文的研究结果完善了蚯蚓同土壤中疏水性机污染物的相互作用的知识,为从整体上把握蚯蚓蓄积疏水性有机污染物过程及机理,进一步认识蚯蚓对疏水性有机污染物的解毒／致毒机制并保障食物链的安全提供了理论依据。本文蚯蚓生物标志物的研究,对于促进蚯蚓生物标志物的实际应用具有一定的启发作用。更多还原

【Abstract】 Earthworm is an important food resource for many predators and plays a key role in terrestrial ecosystem. Hydrophobic organic contaminants (HOCs), especially persistent organic pollutants (POPs) can accumulate in earthworms and influence the balance and health of soil ecosystem via the enrichment and magnifying effects of food chains. Therefore, comprehensive and complete understanding the interaction between HOCs and earthworms is necessary to secure the food chain. Among the related issues, the fate of HOCs in earthworms especially needs to be further elucidated. In the first part of this paper, we reviewed the processes of earthworm accumulation of hydrophobic organic pollutants and their prediction models in Chapter1. Then we took the phenanthrene (PHE) as a typical representative for polycyclic aromatic hydrocarbons (PAHs) and Eisenia fetida for earthworm to explore "what the distribution of HOCs in earthworms is and how much HOC earthworms are able to accumulate"(Chapters2and3). The results might provide insight into enrich the knowledge of the fate of HOCs in earthworms and the mechanism of taking up and accumulating HOCs by earthworm.Earthworm is recommended test animals in soil ecotoxicological assay and earthworm biomarkers are often employed to assess the risk of soil pollution. However, although earthworm biomarkers studies have been carried on for almost30years, they can still not be used in the practical soil pollution risk assessment and there are several issues need to be elucidated. Therefore, in the second part of this paper, we reviewed the progress of earthworm biomarker study and its application in soil pollutant risk assessment in first chapter. Based on the current stastus, taking the anti-oxidant defense enzymes as the representive of biomarkers, we studied the basal levels of anti-oxidant system at different life stages (juvenile and adult) and the basal distribution in different regions of adult earthworms (pre-clitellum, clitellum and post-clitellum) was studied using filter contact tests. Furthermore, we also investigated effects of PHE at different exposure levels on anti-oxidant enzymes along the earthworm body (Chapter4).Results showed:1) To understand the behavior and fate of HOCs in earthworms, the distributions of phenanthrene (PHE) in Eisenia fetida were studied at sub-organism level (pre-clitellum, clitellum and post-clitellum), tissue level (bodywall, gut and body fluid) and subcellular level (intracellular fraction and extracellular fraction). Earthworms were incubated in soils spiked with PHE (lOmg kg-1, i.e., LC treatment and50mg kg-1, i.e., HC treatment) and sampled at different time intervals. Results showed concentration and relative distribution proportion (DP) of PHE in earthworms varied with the different treatments, overall, a) at sub-organism level, the concentration of PHE appeared no difference between sub-organism fractions at LC treatment, while existed significant difference (p<0.05) among the sub-organism fractions at HC treatment, following post-clitellum> clitellum>pre-clitellum (5days), and gradually reached post-clitellum≈clitellum>pre-clitellum (7and14days). DP followed post-clitellum (58～72%)> pre-clitellum (18～24%)> clitellum (12～19%) in all cases; b) At tissue level, the concentration of PHE followed gut> body fluid> bodywall, and DP of PHE followed bodywall and gut (38%-43%and34%-47%, respectively)> body fluid (15%-22%); c) at subcellular level, the concentrations of PHE in extracellular fraction were4.68to1.23times higher than in the intracellular fraction and DP of PHE followed from extracellular fraction (51～61%)> intracellular fraction (39～48%) to intracellular fraction (51～70%)> extracellular fraction (29～49%) gradually. Based on the results, the possible function of circulatory system at sub-organism level for PHE distribution was discussed and concluded. Partition way (passive diffusion) of PHE between body wall-body fluid-gut and processes of PHE entry inner cellular (passive diffusion) were supported by the results.2) The sorption of phenanthrene by earthworms was investigated using a batch approach. The sorption of phenanthrene could be well described by a liner isotherm (r>0.98), indicating that the partition process is the main mechanism of PHE entry earthworms. The maximum PHE uptake by earthworms is consistent with the prediction of Jajer’s mechanism model, which favored the hypothesis of the model.3) To explain the distribution pattern of PHI in earthworms at sub-organism and tissue level, the sorption of PHE onto different fractions of earthworms was investigated through a batch approach. At sub-organism level, the sorption of phenanthrene could be well described by a liner isotherm and the sorption capacity in different parts followed the post-clitellum> clitellum> pre-clitellum, which was different from the real distribution pattern of PHE in earthworms. The phenomenon indicated that partition theory is not good enough to explain it unless integrated the earthworm’s life activity such as the circle system along the body of earthworms. At tissue level, the sorption isotherm are also linear and the sorption capacity was arranged as the gut> body wall, which is consistent with the real distribution of PHE at tissue level, indicating that partition theory can explain it well.4) Basal level and pattern of anti-oxidant system in different development stages in earthworm were different. Juvenile earthworms show significantly higher superoxide dismutase (SOD) and peroxidase (POD) activity while adult earthworms possess higher catalase (CAT) activity, meaning earthworms inflict different reactive oxidative species (ROS) stress and have different strategies coping with such ROS stress during the growth process. Malondialdehyde (MDA) content stays a relatively stable equal level in both groups and indicates a basal level of cellular lipid peroxide in healthy earthworms.5) Distribution of anti-oxidant system in different regions of adult earthworms is heterogeneous. SOD activity in these regions follows, in descending orders, clitellum> pre-clitellum-post-clitellum, CAT activity, follows, post-clitellum> pre-clitellum≈clitellum, and POD follows pre-clitellum> post-clitellum> clitellum. MDA mainly locates in clitellum region.6) We also concluded that time is an important environmental stress factor, which has been observed impacting not only the anti-oxidant enzyme activity but also their distribution patterns in earthworms. Based on the conclusion, the term "stress magnitude" was defined as a function of exposure concentration and time, i.e. stress magnitude exposure time×exposure concentrations, which might provide a new insight for understanding the stress ecology and ecotoxicology.7) Finally, effects of PHE on distribution of anti-oxidant system imply that SOD and POD activity in clitellum regions might be a good indicator for PHE stress while CAT activity in pre-clitellum and post-clitellum regions is more sensitive to PHE stress. Change of MDA content showed in our study, the stress magnitude threshold for earthworms is exposed to low PHE concentration for48h, and when earthworms suffer a higher PHE stress magnitude (48h exposure in high PHE, concentration condition), the substantial and irreversible damage occurred.To sum up, the paper covered several neglected issues in earthworm ecotoxicology. It provided a global impression for the fate of HOCs in earthworm and therefore enriched the knowledge of interaction between HOCs and earthworms. It is heuristic for promoting the application of earthworm biomarkers in real soil pollution risk assessment.更多还原