| In recent years,research on the chronic toxicity of pesticide residues to bee larvae at sublethal doses has become a hot topic.At present,the endpoint for evaluating the chronic toxicity of bee larvae mostly focuses on the mortality rate of the larvae,but solely based on the mortality rate can lead to incomplete risk assessment of chemical pesticides on bee larvae.It’s significant to enrich and expand the theory and methods of risk assessment of pesticides on bees when evaluating the chronic toxicity of pesticide residues to bee larvae using the proboscis extension reflex as the endpoint.This article establishes residue analysis method for 8 herbicides(glyphosate,alachlor,butralin,metolachlor,oxadiazon,quizalofop-p-ethyl,promytryn and ametryn)in honey and beebread samples.Based on the comprehensive testing station of China’s bee industry system,the residues of 8 herbicides in honey and beebread samples has been detected for 2 consecutive years.Using glyphosate as the test pesticide,which has a high detection rate,to explore its effect on the growth and development of bee larvae,evaluated the chronic toxicity of bee larvae with the endpoint of the proboscis extension reflex and explored the toxic effects of glyphosate and deltamethrin on the proboscis extension reflex.The research results have important theoretical guidance and practical application value for strengthening the risk management of pesticide residue pollution in honey source plants and ensuring the health of bee colonies.The main research findings are as follows:1.Establishment of analytical methods for eight herbicides in honey and beebreadAn ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was established for the analysis of glyphosate in honey and beebread.The linear range of the method was 4-1000 μg/L,the correlation coefficient was 0.9977-0.9983,the limit of quantification(LOQ)was 4.0 μg/kg and the detection limit(LOD)was 0.3 μg/kg.The average recoveries of glyphosate in honey were 76.9%-108.1% at the added concentrations of 10-1000 μg/kg,and the relative standard deviations were 7.2%-19.7%.The average recoveries of glyphosate in beebread were 82.4%-106.1% at the added concentrations of 50-1000 μg/kg,and the relative standard deviations were 5.5%-10.7%.The results showed that the method could meet the requirements for the multi-residue analysis of glyphosate in honey and beebread.A gas chromatography-mass spectrometry(GC-MS)method was established for the analysis of seven test herbicides,including alachlor in honey and beebread.The linear range of the method was 10-1000 μg/L,the correlation coefficient was 0.9941-0.9999,the LOQ was 5.0-10.0 μg/kg and the LOD was 0.3-4.4 μg/kg.The average recoveries of the seven herbicides in honey were 64.8%-110.9% at the added concentrations of 10-1000 μg/kg,and the relative standard deviations were 3.1%-11.6%.The average recoveries of the seven herbicides in beebread were 79.1%-112.4% at the added concentrations of 5-2000 μg/kg,and the relative standard deviations were 1.6%-15.4%.The results showed that the method could meet the requirements for the multi-residue analysis of the seven herbicides such as alachlor in honey and beebread.2.Detection of 8 herbicide residues in actual samples of honey and beebreadThe herbicide residue monitoring was conducted on honey(286 samples)and beebread(207 samples)collected from 20 provinces and cities for two consecutive years.The results showed that the detection rate of herbicides in honey samples was 64.3%,with an average detection concentration of 4.9-62.7 μg/kg;the detection rate of herbicides in beebread samples was 49.3%,with an average detection concentration of 11.7-316.6 μg/kg.Among them,the detection rate and average concentration of glyphosate in honey and beebread were relatively high,with detection rates of 47.9% and 32.9% respectively,and the average concentration of detection was 48.2 μg/kg and 316.6 μg/kg respectively.3.Evaluation of the chronic toxicity of glyphosate to bee larvae based on the endpoint of the proboscis extension reflexThe glyphosate was selected as the test pesticide with high detection rate and concentration from honey and beebread samples,and evaluate the chronic toxicity of bee larvae using the proboscis extension reflex as the endpoint.The results showed that when the larvae were exposed to different concentrations of glyphosate for a long time,their survival rate,pupation rate,and eclosion rate decreased with the increase of concentration.When exposed to 1 mg/kg,there was no significant difference in overall survival rate compared to the control group(p>0.05),indicating when concentrations greater than 1mg/kg would have an effect on the growth and development of bee larvae.And on this basis,the treatment concentration of 0.025-1 mg/kg was set to further evaluate the effect of glyphosate on proboscis extension reflex.The proboscis extension reflex rate decreased with the increase of concentrations,when the concentration was 25 μg/kg,there was no significant difference compared to the control group.The results showed that long time exposure of glyphosate to bee larvae caused a decrease in the proboscis extension reflex rate,which had an effect on bee collection behavior.4.Toxic effects of glyphosate and deltamethrin on bee beak extension reactionIn natural environment,honeybees may contact with two or more kinds pesticides.Therefore,based on the comprehensive testing station of China’s bee industry system to detect pesticide residues in bee products,selected the deltamethrin with high detection rate and concentration.The further research was conducted on the effects of glyphosate and deltamethrin on proboscis extension reflex.When honeybees were exposed to glyphosate(25 μg/kg)and deltamethrin(0.3 μg/kg),the proboscis extension reflex rates significantly decreased in 0.1%-30% sucrose solution compared to when treated alone.When the concentration of sucrose solution was 30%,the proboscis extension reflex rates decreased by 21.67% and 23.33% respectively compared to glyphosate and deltamethrin exposed alone.The results showed a synergistic effect between glyphosate and deltamethrin,the combined of two pesticides had an effect on the proboscis extension reflex rates and the collection behavior. |
