| With the rapid economic development, more and more pollutants enter the soil environment. It is estimated that more than 90% of the total burden of pollutants resides in the surface soils. Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are frequently found together as contaminants in soils and have attracted much attention in recent decades. However, they are mostly concentrated on the effect of a single organic pollutants and heavy metals. In fact, contaminated soils usually contain mixtures rather than a pure substance in the real environment. It is very important to investigate the compound pollution between heavy metal and organic pollutant for the management and diagnosis of soil pollution.Earthworms and enchytraeid are the representative organisms in soil animals, which have become a bioindicator of soil contamination. Therefore the acute and chronic tests of Cadmium (Cd) and Phenanthrene (Phe) to earthworms and enchytraeid were performed in this study in order to know the toxicity and mechanism of toxicosis of these typical heavy metal and typical organic pollutant on environmental organisms. In this paper, quantitative expression and effect of combined pollution between Cd and Phe were studied. The results are showed as follow:1. The acute toxicity of Cd and Phe to earthworms (Eisenia andrei) was studied by the methods of artificial soil test. The results showed that the single toxic sequence of Cd and Phe was Phe > Cd. The values of their LC50 were 788.71 mg kg-1,42.51mg kg-1, respectively. The difference may depend on the biological mechanisms of the earthworm population. Cd at all concentration, it could decrease the toxicity of Phe, and the toxicity of Phe decreased with the decrease of Cd concentration. Phe at low and medium concentration,it could decrease the toxicity of Cd, and the toxicity of Cd decreased with the decrease of Phe concentration, at high Cd concentration, it could increase the toxicity of Phe. Therefore,the component and different concentration of combined pollutant are the significant factors to determine the toxicity of combined pollutant. Phe was the main factor of contribution of acute toxicity on earthworms (Eisenia andrei).. The acute toxicity may be used to express the combined pollution between Cd and Phe. The toxic effects of single and joint stress of Cd (cadmium) and Phe (phenanthrene) on enchytraeid (Fridericia bulbosa) were investigated by natural soil tests. Mortality of single and joint stress of Cd and Phe was significant (p<0.01) except the lowest concentration of single pollutant. There was a positive correlation between mortality and the concentration of pollutant added to soil. Also, the more exposed to single or joint Cd and Phe, the more morality. For joint pollution, Cd was the main contributive factor of toxic effects, the different concentration and interaction of Cd and Phe significantly influenced mortality (p<0.01). The joint effects of Cd and Phe were additive, after a 14-d exposure.When Phe was 0.1 mg kg-1 and 1.6 mg kg-1, the interactive effects between Cd and Phe were synergistic. When Phe was 25.6 mg kg-1, the interactive effects were antagonistic. It suggested that morality may be considered as a valuable and sensitive biomarker to diagnose adverse effects of Cd or Phe in soil environment.2. Enzymatic activities are regarded as fast and prognostic indices of individual reaction to the environmental stress, and should allow prediction of the consequences of pollution at population and ecosystem levels. Our objective was to investigate whether their catalase (CAT),superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were influenced by Cd and Phe at different concentrations and exposure times. Three replicates of adult earthworms (Eisenia andrei)were exposed to exposure times ranging from 2-d to 64-d. Results indicated that each change of Cd and Phe in treatments was significantly different compared with the control at the same duration of exposure times. CAT activity was not shown the significant order all the experimentation. In the experiments of combined pollution, the activity of SOD was weaker than single experiment but the activity of GSH-PX was more activate than single experiment. The different concentration of Cd and Phe was significantly influence the activity of SOD and GSH-PX, and their interaction was significantly influence the activity of CAT and GSH-PX. It suggested that GSH-PX may be considered as a valuable and sensitive biomarker to diagnose adverse effects of Cd or Phe in soil environment.3. Four different coelomcytes extraction methods for earthworms (Eisenia andrei) were evaluated. They are fiber needle extraction, ultrasound protocol, electrical extraction and combined method with fiber needle extraction and ultrasound protocol. The effect of them was compared based on the total of coelomcytes and viability of coelomcytes. The results showed that the combined method with fiber needle extraction and ultrasound protocol is most effective. It has higher total and viability of coelomcytes. It could be found a good base for the tests about the neutral red retention time (NRRT) and single cell gel electrophoresis (SCGE). Lysosome is used as the biomarker. NRRT of single and joint stress of Cd and Phe on earthworms (Eisenia andrei) were investigated by natural soil tests. The results showed there was an opposite relation between NRRT and the concentration of pollutant added to soil. Also, the more exposed to single Cd, the lower NRRT. For joint pollution, the joint effects of Cd and Phe were synergistic. After the 7-d or 14-d exposure,Cd was the main contributive factor of toxic effects. It suggested that NRRT may be considered as a valuable and sensitive biomarker to diagnose adverse effects of combined Cd and Phe in soil environment. The integrality of plasmolemma of single and joint stress of Cd (cadmium) and Phe (phenanthrene) on coelomcytes of Earthworms (Eisenia andrei) were investigated by natural soil tests. The results showed there was an opposite relation between integrality of plasmolemma and the concentration of pollutant added to soil. It founds a good theoretical base for the mechanism of stride membrane of Cd and phenanthrene. Moreover, the rate of early apoptotic coelomcytes could be considered as a valuable and sensitive biomarker to diagnose adverse effects of combined Cd and Phe in soil environment. Using FCM to detect the subpopulations of coelomcytes is feasible to indicate the combined pollution of heavy metal and PAHs.4. The DNA-damaging effects of the combined pollution of heavy metal and organic contamination on earthworms were evaluated by SCGE assay. Earthworms (Eisenia andrei) were exposed to single or combined test compounds in different doses of cadmium (Cd) 5, 10, 50 mg kg-1 and phenanthrene (Phe) 0.5, 2.5, 12.5 mg kg-1 with a treatment of 14 days. In SCGE assay, isolated coelomcytes and electrophoresis were employed to determine DNA damage degree after a 14-d treatment by test compounds. The results showed that there was significant statistical difference between earthworms treated with Cd combined Phe with them treated alone with Cd or Phe. The Olive tail moment (OTM) of SCGE assay using earthworm coelomcytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds. These tests also revealed that the interaction between Cd and Phe to DNA damaging effects was negative, and was strongly dependent on the concentration of pollution. This study corresponds to exploratory phase in order to reveal interaction effects of heavy metal and organic contamination on earthworms and then to set up more in-depth analysis to increase progressively the understanding of the genotoxicity mechanisms involved.
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