DNA Damage And Effect On Soil Microbial Community Diversity Induced By Endosulfan

Persistent organic pollutants (POPs) are a family of organic pollutants that are persistent, bioaccumulative aneasily cause adverse effects on human and organisms. POPs have been the concern of all over the word. They present immunotoxicity, endocrinetoxicity, reproductive and developmental toxicity, carcinogenesis and so on. The ecotoxicity studieon POPs should be strengthened.Endosulfan is one of the most commonly used organochlorine insecticides. It is being extensively used in crops field due to its broad spectrum of activity and relatively low cost. As a result of its widespread use, endosulfan has been ubiquitously detected in atmosphere, soils, sediments, surface water, rainwaters and foodstuffs.Taking into account the magnitude of use, environmental levels and human and ecological effects of this compound, many countries have recognized the hazards of wide application of this pesticide. Today, Endosulfan has been banned or severely restricted in over 30 countries, but it continues to be widely used in some developing countries like China and Thailand .Studies concerning effects of endosulfan on soil organisms are needed in order to evaluate its toxicity. The aim of the present study was to assess the genotoxic effects of endosulfan in Vicia faba, earthworm (Eisenia foetida), white clover (Trifolium repens L.) and soil microorganism. From molecule level, DNA damage and effects on soil microbial diversity induced by endosulfan were evaluated. Our objective was to get a more comprehensive understanding on the effects of endosulfan and provide more information about the potential ecological risk of this pesticide on the soil ecosystem.The main results are showed as follows:1. Vicia-icronucleus test is applied in most fields, as a detecting means. In order to prove up the influence of endosulfan towards Vicia faba, cell mitosis,chromosomal aberrations and micronucleus were researched. The results showed that when seeds were treated with different endosulfan concentrations, 0.1, 1.0 and 10.0 mg/L, the cell mitosis of seeds were inhibited.With the increasing of endosulfan concentration and poisoned time,the inhibited effect were both enhanced. With the increasing of endosulfan concentration and poisoned time, the frequency of the chromosomal aberration and micronucleus rate increased. There were significant influences on the chromosomal aberrations and micronucleus of seeds treated with endosulfan, which effects were relative to endosulfan concentration and poisoned time.It was very clear that this pesticide with a certain concentration had effects on cytogenetical toxicity of Vicia faba root tip cells.2. The comet assay has been widely used in the fields of genetic toxicology and environmental biomonitoring, which was a new tool for genotoxicity study. In the present study, earthworms were exposed with endosulfan concentrations of 0.1, 1.0 and 10.0 mg/kg in the soil. The white clover roots were immersed in hydroponic pots containing nutrient solutions of different endosulfan concentrations, 0.1, 1.0 and 10.0 mg/L. Tissues of each treatment were collected on the 7th, 14th, 21st and 28th days of treatment process. Significant effects from both concentrations and time of exposure were observed. Under our experimental conditions, the induction of DNA damage in Eisenia foetida and T.repens specimens appeared to be dose-dependent throughout the exposure period. And endosulfan induced DNA damage on earthworm and white clover nuclei.In this research, the potential for DNA damage, as measured by the Comet assay, to act as a biomarker of genetic toxicity in white clover plants was considered. A significant increase of DNA migration was observed on the nuclei obtained from T.repens leaves. During the exposure period, DNA damage increased as the exposure concentrations of the pesticide increased and DNA migration showed a dose-response relationship. The T.repens comet assay is as sensitive as the comet assay using earthworm cells which could be used in genotoxicity study of environmental pollutants. To our knowledge, this is the first report describing the use of T.repens for genotoxicity assessment of environmental pollutants using the comet assay. Based on experimental evidence obtained, we can suggest that DNA damage of leaf cells of T.repens may potentially be used as a bioindicator for detecting genotoxicants in ecosystem.3. In our study, three soil DNA extraction methods which based on different lytic principles for isolation of the total farmland microbial DNA were compared. The results showed that the chemiea-enzymatic method was better than other methods. The chemiea-enzymatic method could obtain the best DNA with the highest molecular weight and purity,which was more propitious to molecular ecologystudy. 4. The PCR method ,as there are"GC clamp"with the primer,was optimeized through the change of Mg2+,dNTP,primers. Primer,The traditional PCR and Nest-PCR strategy were compared. The result showed that with the Nest-PCR strategy,the aim gene fragment was best amplified. It was more suitable for DGGE study.5. DGGE method was applied to analyse the relative genetic complexity of microbial communities in soil treated with different endosulfan concentrations, 0.1, 1.0 and 10.0 mg/kg. The target gene was separated by DGGE,and the cluster analysis wascarried. From the result we can see that there were abundant bacterial communities at the different treated soil. The result showed that there were a certain differences in baeterial communities diversity in soil treated with different concentrations of endosulfan. The results showed that many bacteria were inhibited in soil treated with endosulfan. The Jacarrd indexes among the different samples and control were reduced throughout the exposure period. With the increasing of endosulfan concentration and poisoned time, some bands had been changed. It means that the mutual benefit communities were developed and others were restrained or disappeared. From the result we also can got the conelusion that DNA sequence of soil microorganism was affected by endosulfan.