Acute Toxicity And Enzyme Activity Of Pyrethroid Pesticides On Apis Mellifera Ligustica And Apis Cerana Cerana

Honeybees,as the main pollinators of crops,play an important role in the global ecosystem.Affected by the Colony collapse disorder(CCD),the honeybee population was declining in the world.The extensive use of chemical pesticides may be one of the important reasons for CCD.In this paper,a multi-residue analysis method,which used some new purification material(including PSA/GCB)based on GC-MS(Gas chromatography-mass spectrum),has been established for monitoring 12 kinds of pesticide residues in 214 honeybee samples collected from five major honey source regions in China depended on 13 National Bee Industry System Test Stations.Then the acute oral toxicity of four pyrethroid insecticides with high exposure risk in honeybee samples were tested on both Apis mellifera ligustica and Apis cerana cerana.Beta-cypermethrin was further studied for its analysis of enzyme activity and survival risk of A.mellifera ligustica and A.cerana cerana.The main results are presented as follows:1.TheEstablishment of GC-MS analysis method for 12 pesticide residues such as beta-cypermethrin in honeybeesThe bee samples were extracted by 1%acetonitrile acetate solution,purified by PSA/GCB solid phase extraction column,and detected by GC-MS.The results of recovery experiments which 12 pesticides in honeybee at three added levels showed that,the average recoveries of chlorothalonil,lambda-cyhalothrin,beta-cypermethrin,boscalid,quizalofop-p-ethyl and deltamethrin pesticides ranged 74 to 112%with added concentrations of 10-1000 ng/g,and RSDs were between 3 to 14%;the average recoveries of alachlor,oxadiazon,butralin and fenpropathrin pesticides ranged from 88 to 108%with added concentrations of 5-500 ng/g,and RSDs were between 2 to 9%.The average recoveries of metolachlor and bifenthrin ranged 81 to.108%with added concentrations of 2.5-250 ng/g,and RSDs were between 2 to 7%.The LOQ of 12 pesticides in the honeybee matrixes were ranged from 5-20 ng/g.The accuracy and sensitivity of this residual analysis method all suit the requirements of pesticide residue analysis.2.Detection of beta-cypermethrin and other pesticide residues in bee samples from 13 test stations in ChinaThe 214 honeybee samples,collected from 13 National Bee Industry System Test Stations in China in 2016 and 2017,were collected and analyzed by using the established GC-MS pesticide residue analysis methods.Five pesticides were detected.The detection rates were fenpropathrin(14.5%),bifenthrin(4.7%),beta-cypermethrin(3.7%),and lambda-cyhalothrin(1.9%),chlorothalonil(1.4%),respectively.The mean,median,and maximum values of the detected concentrations were different among different pesticides.The highest concentration pesticide was fenpropathrin(835.4 ng/g),and the pesticide with the highest average concentration and the median value was lambda-cyhalothrin.The median values of the detected concentrations were as follows:lambda-cyhalothrin(144.5 ng/g),beta-cypermethrin(94.4 ng/g),bifenthrin(63.9 ng/g),chlorothalonil(27.2 ng/g),fenpropathrin(14.5 ng/g).3.The effects of four pyrethroid pesticides on acute toxicities in A.mellifera ligustica and A.cerana ceranaFour pyrethroid pesticides with high detection rate in bee samples were selected for oral feeding to A.mellifera ligustica and A.cerana cerana.The results showed that the four pyrethroid insecticides were highly toxic to both A.mellifera ligustica and A.cerana cerana.These pesticides toxicity were ranked from high to low were beta-cypermethrin,fenpropathrin,lambda-cyhalothrin and bifenthrin.The LD50 values of the four insecticides on the A.mellifera ligustica were higher than the LD50 values of the A.cerana cerana.The LD5O values of fenpropathrin,beta-cypermethrin,lambda-cypermethrin and bifenthrin on the A.mellifera ligustica at 48 h were 0.2774μg/bee,0.1509 μg/bee,0.2815 μg/bee,and 0.3289 μg/bee,respectively,which were 3.95,9.20,1.67,and 1.78 times than A.cerana cerana.Beta-cypermethrin has the greatest difference in toxicity between A.mellifera ligustica and A.cerana cerana.4.The effects of beta-cypermethrin on activities of four enzymes in A.mellifera ligustica and A.cerana ceranaThe effects of beta-cypermethrin on the activities of four enzymes such as AChE,GST,PPO and SOD in A.mellifera ligustica and A.cerana cerana were determined under laboratory conditions.The results indicated that the changes of four enzymes activities were different during the detoxification of beta-cypermethrin to A.mellifera ligustica and A.cerana cerana.With the exposed time increasing(0-48 h),the activities of AChE in the A.mellifera ligustica first declined then increased,reaching a maximum at 48 h(0.1983 U/mgprot).While the AChE activities of the A.cerana cerana first increased then decreased,and reached the maximum value(0.3880 U/mgprot)at 12 h.The activities of GST in A.mellifera ligustica increased continuously,reaching the maximum at 48 h(32.2454 U/mgprot),which was 2.048 times of the control group.The activities of GST in A.cerana cerana was first increased then declined,the maximum value was reached at 24 h(97.8175 U/mgprot),which was 5.211 times of the control group.The activities of PPO and SOD in the two species bees were first increased and then decreased.After treatment with different doses of beta-cypermethrin for A.mellifera ligustica and A.cerana cerana,the activities of AChE in A.mellifera ligustica may be inhibited by low doses,while induced by high doses.The activities of AChE in A.cerana cerana may be induced by low doses,while inhibited by high doses.The activities of GST in A.mellifera ligustica and A.cerana cerana may be induced by low doses,while inhibited by high doses,and reached the maximum in LD25.The activities of PPO and SOD in A.mellifera ligustica and A.cerana cerana may be induced by low doses,while inhibited by high doses.5.Analysis of the survival risk of beta-cypermethrin on A.mellifera ligustica and A.cerana ceranaThe average concentration,median concentration and maximum concentration of beta-cypermethrin were continuously fed to A.mellifera ligustica and A.cerana cerana for 10 days.The results showed that the higher the concentration of beta-cypermethrin fed,the lower the survival rate of the two species bees.Moreover,under the same concentration treatment,the survival rate of A.cerana cerana was significantly lower than that of A.mellifera ligustica.After 10 days of feeding with the highest concentration(0.2906 mg/L)of beta-cypermethrin,the survival rate of the A.mellifera ligustica was 40.0%,while the survival rate of the A.cerana cerana was only 18.9%.A.cerana cerana are more sensitive than A.mellifera cerana,and A.cerana cerana are more threatened than A.mellifera ligustica.