| Earthworms are the soil animals with the largest biomass in the grassland ecosystem.As an important part of soil microcirculation,they can affect the material circulation and energy flow in the grassland ecosystem,and play a role as a bridge of pollutants transfer in the food chain.Neonicotinoid insecticides have become the fastest growing class of insecticides over the past few decades.As a neurotoxic insecticide,neonicotinoid insecticides have been widely used for crop protection.including these pesticides used to control pests such as grasshopper and grubs in turf,lawn and grassland.However,exposure of such pesticide in the environment may damage the non-target organisms in soil.In the present study,we aimed to investigate the effects of six neonicotinoid insecticides on earthworms(Eisenia fetida)in terms of survival,avoidance behavior.reproduction,detoxification enzyme activity and gene expression using a systematic experimental approach and combining biochemical and molecular biological assays.The major results are as follows:(1)Artificial soil tests were performed to evaluate the acute toxicity of six neonicotinoid insecticides on earthworms.Five or seven concentrations of six neonicotinoid insecticides respectively used in the present experiment.Acute toxicity tests showed that the 14-day median lethal concentration(LC50)value of imidacloprid was 2.26(2.09-2.43)mg a.i.kg-1,thiacloprid was 19.54(17.68-21.70)mg a.i.kg-1,nitenpyram was 4.99(4.71-5.29)mg a.i.kg-1,acetamiprid was 1.55(1.48-1.63)mg a.i.kg-1,clothianidin was 5.71(5.32-6.13)mg a.i.kg-1 and dinotefuran was 2.56(2.36-2.74)mg a.i.kg-1.According to the classification of chemicals.imidacloprid,nitenpyram,acetamiprid.clothianidin and dinotefuran are defined as moderately toxic in soil test(1.0<LC50≤10 mg a.i.kg-1),thiacloprid is defined as low toxic in soil test(LC50>10 mg a.i.kg-1)，and acetamiprid has the highest toxicity to earthworm in all of them.(2)The avoidance response test was conducted in two-chamber rectangular plastic containers.Seven concentrations of six neonicotinoid insecticides respectively used in the present experiment.Avoidance behavior tests showed that the 2-day concentration inducing an avoidance rate of 50%(AC50)value of imidacloprid was 1.34(1.02-1.91)mg a.i.kg-1,thiacloprid was 7.65(6.37-9.79)mg a.i.kg-1.nitenpyram was 1.76(1.43-2.23)mg a.i.kg-1,acetamiprid was 0.67(0.50-0.86)mg a.i.kg-1,clothianidin was 4.93(4.15-6.29)mg a.i.kg-1 and dinotefuran was 1.20(0.97-1.58)mg a.i.kg-1.The results showed that six neonicotinoid insecticides in soil could induce the avoidance behavior of earthworms,and avoidance behavior response of earthworms caused by acetamiprid was the most obvious.(3)The growth rate of earthworms and the number of juveniles were used as the endpoints to evaluate the reproduction toxicity of imidacloprid,nitenpyram and dinotefuran on earthworms.Exposure period was 56 d,and 4 sub-lethal concentrations of three neonicotinoid insecticides respectively used in the present experiment.Reproduction tests showed that the 56-day effect concentration for 50%effect(EC50)value of imidacloprid was 0.87(0.66-1.33)mg a.i.kg-1,nitenpyram was 0.62(0.48-0.77)mg a.i.kg-1 and dinotefuran was 1.08(0.75-1.94)mg a.i.kg-1.In three of them,nitenpyram had the largest effect on earthworm reproduction.In addition,the chronic exposure of imidacloprid,dinitramide and furfuran to earthworm all showed the response of inhibiting the growth and reproduction of earthworm,and there was a positive correlation between the growth rate of earthworms and the number of juveniles in treatments.(4)For the investigation of two detoxification enzyme activities of earthworm in chronic toxicity exposure,earthworms were exposed in 28 days,and four sub-lethal concentrations of six neonicotinoid insecticides respectively used in the present experiment.Detoxification enzyme activity test showed that activities of carboxyl esterase(CarE)and glutathione-S-transferases(GST)in earthworms were disturbed by neonicotinoid insecticides exposure.Moreover,effects of imidacloprid and thiacloprid on the CarE activity in earthworm were more severe than other neonicotinoid insecticides,which exhibited that the CarE activity was inhibited by imidacloprid and thiacloprid exposure up to 14 days.However,on day 21,the CarE activity of imidacloprid and thiacloprid treatments was higher than that of the control group.On day 28,the CarE activity of earthworms in the imidacloprid and thiacloprid treatments were inhibited again.Effects of clothianidin and dinotefuran on the GST activity in earthworm were more severe than other neonicotinoid insecticides,which exhibited that the GST activity was inhibited by clothianidin and dinotefuran exposure up to 7 days.However,on day 14,the GST activity of clothianidin and dinotefuran treatments recovered to level of control group.On day 21,the GST activity of earthworms in the clothianidin treatments showed decreased activity again.(5)In gene expression test,three concentrations of imidacloprid were used for earthworm treatment in chronic toxicity exposure,and earthworms were exposed in 21 days.In the present study,annetocin(ann),calreticulin(crt),transcriptionally controlled tumor protein(tctp),heat shock protein 70(hsp70)and gst were selected as target genes.Gene expression test showed that the expressions of ann and crt at the transcriptional level were decreased upon imidacloprid exposure,reaching the lowest levels of 0.09 fold and 0.16 fold on day 7 and day 14,respectively.tctp,hsp70 and gst exhibited relatively obvious variations(up-regulation or down-regulation)when the exposure duration was extended.Taken together,six neonicotinoid insecticides exerted harmful effects on earthworm E.fetida in terms of survival,avoidance behavior,reproduction,detoxification enzyme activity and gene expression.Furthermore,it will affect the function of earthworm in grassland ecosystem. |
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