| In order to investigate the toxicity effects of maduramycin ammonium on crayfish.In this study,firstly,we established HPLC method for maduramicin ammonium concentration in water.we used crayfish as model animal and researched acute toxicity of maduramycin ammonium to crayfish.Base on the acute toxicity test,determine the sub-lethal toxicity of maduramycin ammonium on oxidative stress,haemolymph parameters,histopathological examination and some genes expression,to explore the sub-lethal toxicity of maduramycin ammonium on crayfish.The details aredivided into three parts as follows:1.Development of HPLC method for determination of maduramicin ammonium in waterTo establish a rapid and sensitive HPLC method with fluorescence detection and pre-column derivatization for determination of maduramicin ammonium in water.After elution by HLB column,vortexing with acetonitrile,glacial acetic acid and dansyl hydrazine were used to derivatize,then was analyzed by HPLC fluorescence detector.The limits of detection(LOD)and quantification(LOQ)in water were 0.05 and 0.1?g·mL-1 respectively.The results showed that the drug in water showed good linear relationship at the range of 0.1?100 ?g·mL-1 and the correlation coefficients were greater than 0.99(R2>0.99),the mean average recoveries of spiked samples ranged from 80.6 to 97.3%with relative standard deviation(RSD)was less thanl5%.The limits of detection(LOD)and quantification(LOQ)in plasma were 0.05 and 0.1?g·mL-1 respectively.This method had high sensitivity,good reproducibility,specificity,so it is suitable for determination of maduramicin ammonium in water.2.Acute toxicity tests of maduramicin ammonium on the Freshwater Crayfish,Procambarus clarkiaAn acute semi-static toxic test was carried,crayfish were exposed to different maduramicin ammonium concentrations.Mortality and toxic reaction were recorded and the maduramicin ammonium concentrations of every groups were measured.The results showed the 96-hour median lethal concentration(96 h LC50)after initiation of crayfish exposure to maduramycin was 67.03 mg·L-1 with a 95%confidence interval of 54.06 to 81.32 mg·L-1,indicating that maduramycin was lowly toxic to crayfish.3.Sub-lethal toxicity impacts of maduramicin ammonium on the freshwater Crayfish,Procambarus clarkiaBase on the acute toxicity test,the freshwater crayfish(Procambarus IVlarkia)were exposed to 3.5,0.7 and 7.0 mg·L-1 for 7,14 and 28 days for sub-lethal toxicity of maduramycin.Histopathological alterations of gills,hepatopancreas and muscles,and alterations in biochemical parameters of haemolymph were analysed as indicators of toxicity.Indices of oxidative stress(malondialdehyde(MDA)),and antioxidant parameters(superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GPx))in crayfish muscles and hepatopancreas were measured.We can see significant differences in histopatological examination to hepatopancreas and muscle after 28 days.Prolonged exposure of maduramycin resulted in biochemical parameters changes.Changes in antioxidant systems were also observed.The results suggest sub-lethal maduramycin exposure affected biochemical profile,and the antioxidant system,caused oxidative stress and histopathological changes in hepatopancreas and muscles of crayfish.4.Effect of maduramicin ammonium on the expression of metabolic and stress-related geneCrayfish were treated with maduramicin ammonium at 7.0 mg·L-1 for 7 days followed by a 7 days clearance period.Total RNA were extracted and the transcription level of genes,namely CYP450?GST?COX1?COX2?HSP70?MT?GST were determined by qRT-PCR.The results showed the expression of these genes in treatment group were upregulated compared with control group,whereas the expression level of these genes was downregulated in clearance period.These results suggest that maduramicin ammonium can induce the expression of CYP450?GST,COX1?COX2?HSP70?MT,GST gene,then effectively responded to the oxidative stress and toxicity effect by maduramicin ammonium. |
PDF Full Text Request