Effect Of Carbendazim On Biomarkers Of Oxidation And Immune Process In Misgurnus Anguillicaudatus And Its Reversibility Analysis

Carbendazim（BCM）is a fungicide pesticide.The principle is to interfere with the formation of spindles in the mitotic process of pathogenic bacteria to achieve control effect.It is mainly used to prevent and control plant diseases such as wheat,rice,cotton,fruits and vegetables,flowers and so on.BCM is widely used,and the ecological pollution caused by its frequent use has aroused widespread concern at home and abroad.BCM can flow into the surrounding waters through surface runoff,which is highly toxic to a variety of aquatic organisms and poses a potential threat to the water environment and human health.At present,the researches on BCM at home and abroad mainly focus on the residual aspects of water and soil,and the toxic effects of BCM on aquatic animals are few.In order to comprehensively evaluate the effect of BCM on related biomarkers of aquatic organisms and analyze its reversibility,Misgurnus anguillicaudatus was selected as the test animal.Stress 10,20 and 30 were detected by semi-static subacute toxicity method under four BCM concentration gradients(0.020,0.075,0.282 and 1.060 mg·L^-1)The contents of malondialdehyde（MDA）and 8-hydroxy-deoxyguanosine（8-OHd G）,the activities of superoxide dismutase（SOD）and catalase（CAT）,the activities of 7-ethoxy-3-isooxazolone-deethylase（EROD）and glutathione mercaptotransferase（GST）,the activities of acid phosphatase（ACP）and alkaline phosphatase（AKP）,Cu/Zn-sod,cat3,CYP1A1,GST,tumor necrosis factor-α（tnf-α）,interferon-γ（ifn-γ）,cysteine aspartic acid specific protease 3（caspase3）,caspase9 and B-cell lymphoma-2（bcl2）m RNA expression levels were then re-detected 30 days after the recovery of natural water,and the toxicity effect of BCM was comprehensively evaluated by Integrated Biomarker Responses index.The main results are as follows:1.The semi-static water test showed that the 24,48,72 and 96 h semi-lethal concentration(LC₅₀)values of BCM for Misgurnus anguillicaudatus were 23.53,17.66,6.54 and 3.58 mg·L^-1,respectively,and the 24 h safe concentration（SC）of BCM for Misgurnus anguillicaudatus was 3.98 mg·L^-1,indicating that BCM was toxic to Misgurnus anguillicaudatus.Subacute toxicity test showed that the growth rate of death of Misgurnus anguillicaudatus under long-term BCM stress gradually slowed down,but the overall death number continued to increase,and there was a certain correlation between the death number of Misgurnus anguillicaudatus and stress concentration.Under long-term stress,the external morphology of Misgurnus anguillicaudatus is curved and deformed,and the color of liver tissue is abnormal.2.Under BCM stress,MDA content in the liver of Misgurnus anguillicaudatus continued to increase in the middle and late stages of stress,and was still significantly increased in the recovery stage.The toxicity effect was significant in the high concentration group,and the content of 8-OHd G was actively induced in both the exposure cycle and the recovery stage.SOD activity and Cu/Zn-sod expression level were significantly induced in the early stage of exposure,CAT activity was significantly inhibited,while cat3 expression level was significantly increased.Antioxidant enzyme activities were significantly induced in the recovery stage,and antioxidant enzyme activities and expression levels were not completely synchronized.EROD activity and CYP1A1 gene expression were induced at short exposure,but were inhibited to varying degrees at long exposure and recovery stage.GST activity and GST expression were significantly induced at exposure stage,and the induction effect was weakened with the extension of stress time,while they were significantly inhibited at recovery stage.The activities of ACP and AKP in the liver of Misgurnus anguillicaudatus under BCM stress were induced in the middle stage of stress,but were inhibited to varying degrees in the late stage of stress,and there was no significant difference in the recovery stage.The expression of tnf-αgene was significantly induced in the early stage of stress,but was inhibited in the long-term stress.The expression of ifn-γgene was inhibited in the early and middle stages of stress,and induced in the late stage of stress.The expressions of tnf-αand ifn-γwere significantly induced in the recovery stage.The expression levels of caspase3 and caspase9 genes were induced in different degrees in the stress stage,but only in the high concentration group in the recovery stage.The expression level of bcl2gene did not change significantly in the early and middle stages,but was significantly inhibited in the late stage of stress.3.IBR analysis was performed on the changes of MDA,8-OHDG,SOD,EROD,GST and ACP in the 4 BCM concentration treatment groups under different stress time,and it was found that the IBR value of Misgurnus anguillicaudatus liver under BCM stress was 10 days<20 days<30 days.The IBR index of high concentration group reached the expected maximum at 30 days of stress.The liver IBR index of Misgurnus anguillicaudatus after 30 days of reversible recovery was lower than that at BCM stress stage.There was a certain correlation between IBR index and BCM stress duration and concentration.The IBR index decreased slightly in the recovery period,but the toxic damage effect was still significant.To sum up,the toxicity of BCM to Misgurnus anguillicaudatus belongs to poisoning.A certain dose of BCM stress will have a serious impact on biological processes such as anti-oxidase system,detoxification metabolic enzyme system,non-specific immunity and apoptosis,and induce a series of toxic stress reactions.The comprehensive toxic effect of the liver is obvious with certain biological toxicity,causing partial irreversible damage to the body.Even after a period of domestication,it has not recovered to the normal level,so BCM pollution should be widely concerned.