Study On The Toxic Effects And Metabolism Of Acetamiprid In Tadpoles Of Rana Nigromaculat

Acetamiprid,a chlorinated nicotine compound,is an important neonicotinoid insecticide commonly used for the control of Delphacidae and other insect pests in paddy fields,but due to its properties such as good water solubility and not easily hydrolyzed,it poses a potential threat to non-target organisms in rice systems.Rana nigromaculata,the most widely distributed rice frog in China.Its larvae（Rana nigromaculata tadpoles）grow fast,have highly permeable skin,and live in polluted rice fields for a long time,in addition to going through a metamorphic developmental stage（at which exogenous pollutant disturbances are more sensitive）.In this paper,the acute toxicity,growth inhibition effect,biochemical parameters,metabolomic and bioconcentration-elimination of acetamiprid on the Rana nigromaculata tadpoles were carried out.The main research contents and conclusions are as follows:1.Acute toxicity test of acetamiprid on Rana nigromaculata tadpoles was carried out.The results showed that the LC₅₀ values of acetamiprid on tadpoles at 24,48,72and 96 h were 51.49,43.57,30.81 and 18.49 mg/L,respectively.The 96 h LC₅₀ for acetamiprid was 18.49（>10）mg/L,and based on the test guidelines on environmental safety assessment for chemical pesticides（Part 18:Amphibian acute toxicity test）of a toxicity categorization,acetamiprid exerted low toxicity to Rana nigromaculata tadpoles.2.Based on the results of acute toxicity,the sub-chronic toxicity of acetamiprid to Rana nigromaculata tadpoles was carried out to investigate the growth inhibition effect,biochemical parameters and metabolomics of acetamiprid on Rana nigromaculata tadpoles.The results showed that after 28 days of exposure to 0.185 and 1.85 mg/L acetamiprid,no significant effects on growth morphological parameters（body length,tail length,body weight and eye spacing）and thyroid-related hormones（TSH,TH,T3and T4）were observed in the tadpoles compared with the control group,indicating that aminopyralid did not inhibit the growth of tadpoles.The tadpoles showed significant changes in biochemical parameters,including CAT,SOD,GR,GST and MDA levels increased,while causing a decrease in Ach E and protein content,and the changes in these biochemical parameters were positively correlated with acetamiprid concentrations.In metabolomics studies,acetamiprid significantly affected metabolites such as nucleotides and analogs,organic acids and their derivatives,organic nitrogen compounds,organic oxygenated lipids and lipid-like molecules in tadpoles.Significantly different metabolites were found in the low and high concentration treatment groups,indicating that high and low concentrations of acetamiprid produced different toxic effects on tadpoles.The pathway enrichment analysis of the differential metabolites showed that the Valine,leucine and isoleucine biosynthesis,Glycine,serine and threonine metabolism,Unsaturated biosynthesis of unsaturated fatty acids,Sphingolipid metabolism,Linoleic acid metabolism,and Histidine metabolism.Combined with the biochemical parameters and metabolomic studies in tadpoles,indicated the existence of strong oxidative stress in tadpoles during sub-chronic exposure,which was positively correlated with the acetamiprid concentration.3.A liquid chromatography tandem mass spectrometry（LC-MS/MS）method was developed for the determination of acetamiprid in tadpoles and water.The results showed that the limits of detection（LOD）of acetamiprid in tadpoles and water were0.003 and 0.002 mg/kg,and the limits of quantification（LOQ）were 0.001 and 0.0005mg/kg,respectively.The concentrations of acetamiprid in solvent and matrix had a good linear relationship between the concentration and peak area of acetamiprid in solvent and matrix（R²>0.99）,the spiked recovery experiments revealed that the average recoveries in tadpole matrix were 87.00-102.76%with RSDs of 2.12-4.74%,and the average recoveries in water were 87.13-105.11%with RSDs of 5.91-7.72%.These relust showed that the method of sensitivity,accuracy and stability could meet the analytical requirements of the experiment.Based on the assay method,a bioconcentration-degradation experiments of acetamiprid in Rana nigromaculata tadpoles were carried out.The results showed that there was a dose effect relationship between the concentration of acetamiprid in tadpoles and the concentration of exposed culture in the comparative experiment of exposure enrichment at different concentrations,the enrichment of acetamiprid in tadpoles showed rapid uptake,with the bioconcentration factor（BCF）ratios were 0.56 and 0.39（<1）in 0.185 and 1.85mg/L exposed solutions.In the elimination experiments,acetamiprid showed rapid elimination with half-lives of 0.44 and 0.65 days for 0.185 and 1.85 mg/L,respectively.