Distribution Of Phenanthrene In Earthworms And Its Effects On The Anti-Oxidant Defence System Of Earthworm

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.