Study On The Single And Combined Toxic Effects Of Copper And Maduramicin On Carassius Auratus

The compound pollution of antibiotics and heavy metals in the current environment is becoming more and more serious,posing a serious threat to environmental ecology and human health.Maduramicin is one of the polyether ionophore antibiotics,mainly used to control chicken coccidiosis.On the other hand,maduramicin is poorly absorbed in chicken and its metabolism process is fast,thus some drugs will be excreted primarily unchanged in the urine and feces.Moreover,maduramicin may be introduced into the soil and water environment when animal manure containing the residual maduramicin were used as fertilizer for farmland.Heavy metal copper is one of the earliest metals discovered by mankind.Copper is widely used in human production activities.Copper poisoning of aquatic animals often occurs.Maduramicin and copper may coexist in the water environment,causing potential harm to aquatic life.In order to evaluate the ecotoxicity effects of single and combined pollution of maduramicin and copper,Carassius auratus was used as a test animal to study the single and joint toxic effects of copper and maduramicin.The specific results are as follows:1 Acute toxicity study of copper single exposure and combined exposure with maduramicin to Carassius auratusBased on the result of preliminary test,semi-static poisoning method was adopted that half of the lethal dose（LC50）of copper to Carassius auratus at 96h was calculated according to Kaber method.The Carassius auratus was randomly placed in 2.5,3.4,4.6,6.2,8.4,11.4,and 15.5mg·L^-1 copper solutions for 96h,and a control group was set up.Observing the symptoms and death status after Carassius auratus copper poisoning.According to the 96h LC50 of Carassius auratus exposed to copper and maduramicin alone,the 96h LC50 of Carassius auratus exposed to the combination of copper and maduramicin was calculated respectively.According to the toxicity of 1:1,set up five joint exposure groups with different concentrations,which were 0.36mg·L^-1 copper+1.12mg·L^-1 maduramicin,0.72mg·L^-1 copper+2.24mg·L^-1 maduramicin,1.45mg·L^-1 copper+4.49mg·L^-1 maduramicin,2.89mg·L^-1 copper+8.98mg·L^-1 maduramicin and 5.79mg·L^-1 copper+17.95mg·L^-1 maduramicin.The results showed that the symptom of copper poisoning of Carassius auratus was increased secretion of mucus in the gills,and some light blue material attached to the gills.The cumulative mortality of Carassius auratus was positively correlated with copper concentration.The 96h LC50 of the single exposure of copper to Carassius auratus was 3.6mg·L^-1 with a 95%confidence interval ranged from 3.31 to 3.95mg·L^-1,and the safe concentration was 0.36mg·L^-1.During the combined exposure of copper and maduramicin,the 96 h LC50 of copper to Carassius auratus was 1.4mg·L^-1 with a 95%confidence interval ranged from 1.16 to 1.73mg·L^-1,and the safe concentration was 0.14mg·L^-1;The 96 h LC50 of maduramicin for Carassius auratus was 4.2mg·L^-1 with a 95%confidence interval ranged from 3.45 to 5.16mg·L^-1,and the safe concentration is 0.42mg·L^-1.Based on the obtained results and the toxicity grading standard of fish,copper was considered highly toxic to Carassius auratus.The combined toxic effects were evaluated according to the Marking additive index method,and the result showed that the combined toxic effects of copper and maduramicin on Carassius auratus were synergistic.2 Study on the effects of single and combined exposure of copper and maduramicin on the antioxidant system and blood biochemistry of Carassius auratusBased on the acute toxicity test,set up single and joint exposure groups of copper and maduramicin respectively,the specific grouping was as follows:0.14mg·L^-1 copper,0.01mg·L^-1 copper,0.42mg·L^-1 maduramicin,0.03mg·L^-1 maduramicin,0.14mg·L^-1 copper+0.42mg·L^-1 maduramicin and 0.01mg·L^-1 copper+0.03mg·L^-1 maduramicin.In addition,a control group was set up.On the days 7 and 21 after exposure,six Carassius auratus were randomly selected from each group.After which,the liver of Carassius auratus were excised and homogenized separately,and the activities of antioxidant enzymes such as SOD,CAT and GPX,as well as the contents of MDA in the livers were determined.Furthermore,the mRNA expression of sod,cat and gpx that involved in the oxidative stress response of Carassius auratus were determined by RT-qPCR.At the same time,on the 21st day of exposure,each group randomly took six Carassius auratus and collected blood from the tail for blood biochemical detection（ALT,AST,ALP）.The results showed that both copper and maduramicin could induce the activities of CAT and GPX on the 7th day after exposure,and gradually showed an inhibitory effect with the prolongation of the dose and exposure time.On the 21st day after exposure,the ALT,AST and ALP activities and the MDA content of each group were higher than the control group.The activities of ALT,AST and ALP and the content of MDA in the combined exposure group were not significantly different from those of single exposure.The results showed that both copper and maduramicin could induce oxidative stress in Carassius auratus.The toxicity of combined infection and single infection cannot be compared by MDA content and blood biochemical indicators.3 Study on genotoxicity of copper and maduramicin single and combined exposure to Carassius auratusBased on the acute toxicity test,set up single and joint exposure groups of copper and maduramicin respectively,the specific grouping was as follows:0.14mg·L^-1 copper,0.01mg·L^-1 copper,0.42mg·L^-1 maduramicin,0.03mg·L^-1 maduramicin,0.14mg·L^-1 copper+0.42mg·L^-1 maduramicin and 0.01mg·L^-1 copper+0.03mg·L^-1 maduramicin.After 21 days of exposure to the poison,six Carassius auratus from each group were taken out immediately,and the fish tail was cut and peripheral blood was collected with heparin sodium.Then,the blood smear was prepared by dropping the blood on glass slide.The blood smear was then stained with giemsa stain and observed under the light microscope.At the same time,the liver of Carassius auratus were excised and digested by trypsase,screened with 200 mesh cell strainer,and then resuspended with PBS to prepare hepatocyte suspension.The degree of DNA damage in hepatocytes was determined by single cell gel electrophoresis.The results showed that both single and joint exposure of copper and maduramicin could increase the number of micronucleus cells in Carassius auratus.The number of micronucleuses was positively correlated with the concentration of copper and maduramicin.In the control group,the DNA comet images of Carassius auratus hepatocytes were regular round.The proportion of the comet tail gradually increased with the increase of copper and maduramicin doses.The micronucleus rate and percentage of hepatocyte tail DNA were positively correlated with drug dose.The micronucleus rate and percentage of hepatocyte tail DNA in the high concentration combined exposure group was significantly higher than that in the single exposure group.Taken together,both copper and maduramicin has genotoxic effects on Carassius auratus,the genotoxic effect of high concentration combined exposure on Carassius auratus was stronger than that of single exposure.4 Effects of maduramicin on hepatic cytochrome P450 enzymes of Carassius auratusSemi-static poisoning method was used to expose Carassius auratus to 0.112,0.224,0.448mg·L^-1 maduramicin for 28 days,and a control group was set up.On the 7th,14th,21st and 28th day after exposure,the liver of Carassius auratus was excised and homogenized,and the mRNA expression of CYP1A,CYP3A and CYP4T in Carassius auratus were determined by RT-qPCR.Furthermore,the activities of cytochrome P450 enzymes such as EROD,APND,AH,ERND and NCCR,as well as the content of CYP450 in the liver were determined.The mRNA expression levels of CYP1A,CYP3A and CYP4T were up-regulated by maduramicin to some degree.Compared with the control group,the content of CYP450 of 0.448mg·L^-1 maduramicin group was significantly reduced on the 21st day.In addition,the activities of APND and NCCR were first inhibited by maduramicin and then induced.On days 14 and 21,the activity of EROD in the liver was significantly inhibited by 0.224 and 0.448mg·L^-1 maduramicin.During the exposure period,the activity of ERND was lower in the experimental groups than in the control group.On the 21st day of exposure,the activity of AH was induced to the highest by maduramicin.Taken together,the inhibition of ERND activity by maduramicin may not be regulated by the regulation of CYP3A transcription.In addition,ERND can relatively stably reflect the toxic effect of maduramicin on Carassius auratus,will be a marker for early detection of maduramicin pollution.