Study On The Combined Toxicity Of Azoxystrobin And Exogenous Selenium On Zebrafish At Different Life Stages

Azoxystrobin,a representative species of methoxyacrylate fungicides,is widely used in the agricultural environment and is highly toxic to aquatic organisms such as Daphnia magna,fish and algae;selenium(Se)is an essential trace element for humans and animals but excessive intake can cause adverse effects and can lead to stunted development of aquatic organisms.In recent years the discharge of industrial selenium-containing wastewater and excessive application of selenium fertilizers in agriculture have brought attention to safe environmental selenium concentrations,while the effects of pesticides on environmental organisms have also received high attention.The toxic effects of single exposure of pyrimethamine and selenium on aquatic organisms have been studied more frequently,but the toxic effects of combined exposure of both have not been reported.In this thesis,the toxic effects of combined exposure of Azoxystrobin and three currently commonly used exogenous selenium sources:inorganic selenium(sodium selenite),organic selenium(selenomethionine),and nanoselenium were studied using zebrafish as a typical representative of aquatic organisms,and the following results were obtained.The acute toxicity of sodium selenite,selenomethionine and nano-selenium to zebrafish at different life stages was determined by semi-static method and OECD standard,respectively.By comparing the LC₅₀(lethal intermediate concentration)at 96 h,it was clear that the sensitivity trends of zebrafish at different life stages to sodium selenite and selenomethionine were the same,and they were all the same for the young(sodium selenite:1.11 mg Se·L^-1,selenomethionine)>embryo(sodium selenite:1.48 mg Se·L^-1,selenomethionine:1.03 mg Se·L^-1)>adult(sodium selenite:13.05 mg Se·L^-1,selenomethionine:9.36 mg Se·L^-1);however,the sensitivity trends to selenium nanoparticles were different,showing as:adult(0.48 mg Se·L^-1)>larve(1.67 mg Se·L^-1)>embryo(4.32 mg Se·L^-1).The acute toxicity of Azoxystrobin to zebrafish at different life stages was determined and the results showed that the acute toxicity of Azoxystrobin to zebrafish adults,juveniles and embryos were time-effect correlated and all showed toxic levels and above,and the toxicity of Azoxystrobin to zebrafish at 96 h was high at all life stages.The toxicity of Azoxystrobin in combination with three exogenous selenium sources(LC₅₀:1:1)at different life stages of zebrafish was evaluated by summation index,toxicity unit and mixed toxicity index methods.The summation index method and the toxicity unit method calculated that the combined toxicity of Azoxystrobin and the three exogenous selenium sources was antagonistic at different life stages of zebrafish,while the mixed toxicity index method showed partial summation for adults and antagonism for both embryos and larves.The effects of four combined exposures of azoxystrobin and selenomethionine on the short-term development of zebrafish embryos were studied according to OECD standards.The results showed that the combined exposure of Azoxystrobin and selenomethionine could affect the development of zebrafish embryos by affecting hatching rate,mortality,heart rate and body length,disrupting the antioxidant system and disrupting the mitochondrial respiratory function of zebrafish fry,as follows.1)The toxic effects of Azoxystrobin and selenomethionine on zebrafish embryos were significantly correlated with the exposure dose when the mixture was exposed simultaneously at isotoxicity ratios,and the toxic effects increased with increasing exposure dose.Moreover,the visible significant effect concentrations of combined exposure on embryonic hatching rate,mortality,heart rate and body length at 96 h were 0.25 mg·L^-1 AZO+0.5 mg Se·L^-1Se Met.In addition,embryonic CAT enzyme activity was inhibited and low concentrations induced mitochondrial complex III enzyme-promoting effects,which may cause mitochondrial dysfunction.2)The toxic effects of the mixture on zebrafish embryos did not show a significant correlation with the exposure dose when exposed to Azoxystrobin and selenomethionine in unequal ratios,but were more pronounced when the toxicity ratio of Azoxystrobin to selenomethionine was 1/2 LC₅₀ azoxystrobin:1/5LC₅₀ selenomethionine.Hatching rate of zebrafish embryos was delayed in the combined exposure groups at all concentrations,and the tail of the young showed signs of deformity,but the growth of the larve was not affected and the body length was not significantly different from that of the blank control zebrafish.the CAT enzyme and mitochondrial complex III enzyme activities showed the same trend as the isotoxicity ratio treatment,but the SOD enzyme was induced to activate at the low exposure.3)The toxic effect of staggered exposure to azoxystrobin followed by selenomethionine did not increase with increasing exposure dose,but was more significant when the dose ratio of Azoxystrobin to selenomethionine was 1/2 LC₅₀ azoxystrobin:2/5 LC₅₀ selenomethionine and 1/2 LC₅₀ azoxystrobin:4/5LC₅₀ selenomethionine.Zebrafish embryos in the treated group showed symptoms of pericardial cysts and caudal deformities.The CAT enzyme and SOD enzyme activities were inhibited in the larve,and the mitochondrial complex III enzyme was induced to promote lower activity with increasing concentration.4)The toxic effects of staggered exposure of selenomethionine followed by azoxystrobin also did not increase with increasing selenium exposure concentration.Rather,zebrafish embryo hatching rate was most significantly affected when the toxicity ratio of Azoxystrobin to selenomethionine toxicity was 1/2LC₅₀ Azoxystrobin:2/5 LC₅₀ selenomethionine.CAT enzyme was inhibited in the litter of each treatment group,and mitochondrial complex III enzyme was induced to promote higher activity with increasing concentration.Taken together,the dose ratio of the two agents when selenomethionine and azoxystrobin are jointly exposed is likely to be an important factor affecting the toxic effects.Therefore,it is recommended that the dose ratio of selenomethionine and azoxystrobin should be considered comprehensively when using selenium as an exogenous supplement and when using selenomethionine for crop disease control to avoid adverse effects on aquatic organisms.This thesis has positive implications for the scientific use of azoxystrobin and selenium fertilizers to control the risk of both on the water environment.