Toxicity Response And The Mechanism Of The Earthworm (Pheretima Aspergillum) Exposed To Pesticide (Chlorpyrifos) In Soil

ObjectiveThe changes in pathophysiology of earthworm(Pheretima aspergillum)when exposed to chlorpyrifos,one of organophosphorus pesticide,in the soil were studied basing on the theory and methods of ecological toxicology.A series of experiments were conducted to investigate the response and its mechanism of earthworm from the level of tissue,cell and molecular.The study aimed to get the basic information about the ecological toxicology effect of chlorpyrifos on earthworms(Pheretima aspergillum)and To provide a indicator of evaluating and a base of management to artificial culture of earthworms on environment control.At the same time,to promote the cultivation industry developing smoothly and healthily,laying a solid foundation for the establishment of a earthworm breeding base of the good agriculture practices(GAP).Methods1.The acute toxicity of chlorpyrifos to earthworms(Pheretima aspergillum)was studied by the standard test methods of natural soil test and filter paper as described in the OECD guidelines.And Calculating the median lethal concentration(LC50)by the improved karber method.The correlation of Mortality rates and concentrations of pesticides was investigated.2.The avoid test examined the influence of Pesticides on earthworm activities by soil(ISO)and the filter paper method.And analysed the correlation of avoid rate and pesticide concentration.3.Pheretima aspergillum was dissected to prepare the tissue pathological slices and observe the toxic injury on microscopic structure of intestinal and somatic,after treated for 28 days with the lethal dose of pesticide.4.The intestinal tissue cells were separated and obtained by the mechanical method after treated for 12 h with the lethal dose of pesticide.The percentage of apoptosis cells was detected by flow cytometry with the Annexin V-FITC/PI double dye cell apoptosis detection kit.5.The front of earthworm body and clitellum were collected to prepare tissue homogenate after treated for 12 h with the lethal dose of pesticide.In order to detect the variation of Pheretima aspergillum antioxidant enzymes activities toxic by means of enzymeactivity.The biochemical indicators,such as the contents of protein and malondialdehyde(MDA)or superoxide dismutase(SOD)activity,were tested in this paper.Results1.The results of acute toxicity indicated that the LC50 of chlorpyrifos for earthworms(Pheretima aspergillum)was 78.34 ?g/cm2(24 h),35.76 ?g/cm2(48 h)with contact filter paper test,291.0 mg/kg(7 d),185.6 mg/kg(14 d)dry weight with natural soil test respectively.So chlorpyrifos was classified as a moderately or toxic pesticide.2.At the test concentration of 7.16 ?g/cm2,the avoidance rate was 86.67%,proving the significant avoidance behavior and the earthworm could perceive and avoid chlorpyrifos.And the CE50(50%effective concentration)was 2.33 ?g/cm2 in filter paper,that of the avoidance response of earthworm(Pheretima aspergillum)to chlorpyrifos.However,the result of the soil method is not ideal,which indicates that it is not applicable to examine the behavior of earthworm(Pheretima aspergillum)away from chlorpyrifos.3.There were some obvious microscopic lesions on intestinal and somatic of earthworm(Pheretima aspergillum)and the damage degree increased with the increase of concentration of chlorpyrifos.The main symptoms were that intestinal epithelial cells sagging,scattered and disordered arrangement,missing or even fall off.The epidermis of somatic appeared cavity,local thinning or sag,even cavitation to local ulcer.4.The detection results of cell apoptosis showed that the early apoptosis rate was on the rise and survival rate decreased significantly, nd these changes have statistical significance.5.Protein contents in the front of earthworm body increased at different levels when concentrations of chlorpyrifos increased to 6 g/cm2.And it could bring out a decrease in the contents of MDA firstly and then an increase on this part.Compared with comparative groups,chlorpyrifos had a significant effect on the contents of protein and SOD activities(P<0.05)located in clitellum,and the most significant change(P<0.05)appered on 4 ?g/cm2,but the MDA content was in a stable state.Relative to the front of earthworm body,antioxidant enzymes of clitellum was more sensitive to chlorpyrifos.And their response performance were not consistent.ConclusionThis study has been clear about the physiological pathology change and response mechanism of Pheretima aspergillum from the behavior,individual,organization,multiple levels,cellular and molecular level,with the stess of chlorpyrifos in soil.Although the results of the acute toxicity test showed that the toxicity of chlorpyrifos is low or medium grade to Pheretima aspergillum,but avoidance test proved that the earthworm could perceive and avoid chlorpyrifos.Further study found that chlorpyrifos have toxic effects on the skin and intestinal of Pheretima aspergillum in the organization,cellular and molecular level.Relative to the skin,the damage on intestinal occurred earlier and more serious under the same conditions.At the same time,The earthworm protected itself from the damage of pesticides relying on the antioxidant enzymes in the body mainly.Therefore,the results of this study suggest that we should pay attention to pesticide residual amount of aquaculture environment.In addition,the research methods have a certain reference value to examine the other environmental influence factors on the healthy life of earthworm in the future.