The Toxicity Of Two Insecticides On Earthworm(Eisenia Fetida) At Sublethal Doses

Systemic pesticides have broad-spectrum and efficient insecticidal and bactericidal effects.Among them,neonicotinoid insecticides and triazole fungicides are the most widely used at home and abroad.As an important animal in soil,earthworms can improve the structure and fertility of soil.Nevertheless,due to earthworms’excavation and swallowing activities,their exposure to soil pollutants would be greatly increased.At present,earthworm is the most widely used indicator of soil ecotoxicology.In recent years,concern of stress response of earthworms to heavy metals,antibiotics and persistent organic pollutants in soil has been increasing,while there are few studies on the effects of pesticides,especially neonicotinoid insecticides and triazole fungicides on earthworms.Although a few studies have demonstrated the negative effects of systemic pesticides on earthworms under elevated concentrations of soil pollutants in simulation tests,most of the studies have focused on a single pollutant,and there is a lack of research on the long-term stress of sublethal pesticides and their combined exposure on earthworms,which lacks a reliable basis for revealing the toxicity of actual agricultural production activities to soil animals.We selected thiamethoxam（THIM）and difenoconazole（DIF）as representative pesticides of neonicotiniod insecticides and triazole fungicides,respectively.Earthworms were used as the research object.Purple soil was collected to simulate contaminated soil to study the toxicity of THIM and DIF to earthworms.By analyzing the acute toxicity of two systemic pesticides to earthworms,the lethal concentrations(LC₅₀)of THIM and DIF to earthworms were determined,and the acute toxicity grade of THIM and DIF to earthworms was evaluated.Based on the recommended application rates in agricultural uses,the dosing concentrations of THIM and DIF were set at 3.60×10^-4 mg/kg and 2.64×10^-5 mg/kg,respectively.We then added 0.1 times and 10 times concentrations as the lower and higher dosing concentrations to create a real-field exposure range.By analyzing the effects of two pesticides on the avoidance behavior of earthworms,the toxic effects of THIM and DIF on the activity behavior of earthworms at sublethal doses were studied,and whether there was synergistic toxicity when earthworms were exposed to THIM and DIF simultaneously.At the same time,the feasibility of introducing earthworm lysosomal neutral red retention time（NRRT）and mitochondrial DNA copy number（mtDNAcn）as biomarkers in the evaluation of the toxicity of THIM and DIF in earthworms was demonstrated.The research results are as follows:（1）The acute toxicity of THIM and DIF to earthworms was determined by filter paper contact method and natural soil method.The results showed that in the filter paper contact test,the 48h-LC₅₀values of THIM and DIF were>786.35μg/cm² and 173.57（165.95-181.55）μg/cm²,respectively,suggesting the toxicity grade to earthworms is non-toxic.In the natural soil method,the 14d-LC₅₀values of THIM and DIF were>3000 mg/kg and 101.03（96.06-106.26）mg/kg respectively,suggesting the toxicity levels to earthworms were non-toxic.The results showed that there were differences in the acute toxicity of different pesticide varieties to earthworms,and the acute toxicity of DIF to earthworms was greater than that of THIM.（2）The avoidance behavior of earthworms in THIM,DIF and THIM&DIF mixture was measured by two chamber avoidance test device.The results showed that the avoidance effect of the three treatments on earthworms was lower than 7d-LC₅₀ within the concentration range of the experimental setting,indicating that the avoidance behavior of earthworms sensitively reflect the degree of soil contamination.Within the concentration range of the experiment,the effect of THIM on the behavior of earthworms is not obvious.In the DIF treatment group,the avoidance effect of DIF on earthworms is greater than that of THIM,but it also has a little short-term stress effect on earthworms.In the mixtrue group,when the concentrations of THIM and DIF were 0.1,1,10 and 100times of the recommended dosage,the 48h-NR of earthworms were 13.33±0.12%,26.67±0.12%,46.67±0.12%and 60.00±0.20%,respectively.The results showed that the NR values of earthworms in the mixtrue treatment groups were significantly higher than those in the single treatment group（p<0.05）,and the avoidance effect of mixture is greater than that of single dose,indicating that DIF has great subacute toxicity to earthworms,and the mixed application may increase its toxicity.There was no significant difference between the 48-h avoidance rate and the 7-d avoidance rate,indicating that earthworms have certain degree of endurance to systemic pesticides,and can survive in the soil contaminated by systemic pesticides for a long time.（3）Based on the recommended dosage,and the treatment groups of THIM,DIF and mixed application were set to determine the changes of NRRT and mtDNAcn of earthworms during 56 days of exposure.The results showed that the three treatments caused varying degrees of damage to the two biomarkers of earthworms after 56 days,which prove that the two biomarkers are feasible and sensitive to the toxicity of THIM and DIF.The NRRT of earthworms was significantly correlated with the duration of exposure.After 56 days of exposure to the recommended dose of THIM,DIF and the ratio NRRT in the mixed treatment group were 0.23±0.03,0.47±0.01 and 0.09±0.01 respectively.At the same time,the NRRT of earthworms in the 3.60×10^-5、3.60×10^-4、3.60×10^-3 mg/kg groups after 28 days were 24.57±2.13 min,22.57±2.56 min and 18.86±2.17 min,respectively,indicating that the NRRT was also affected by the dosages.Under the treatment of the same pesticide,the increased dosage corresponded with the decrease of NRRT values.mtDNAcn of earthworms increased with the extension of exposure time,and the rise of mtDNAcn in the mixed treatment group was faster than that in the single dose treatment group,indicating that there may be synergistic toxicity of THIM and DIF.In the Thiamethoxam（THIM）groups,the linear regression equations of mtDNAcn of earthworms in the 3.60×10^-5、3.60×10^-4、3.60×10^-3 mg/kg dose group were y=0.05x+0.77y=0.13x+0.54 and y=0.28x+0.05 respectively,indicating that mtDNAcn increased significantly with the increase of dose.However,in the DIF and mixed treatment group,the change trend of earthworm mtdnacn was not related to the exposure dose It is necessary to extend the test cycle and carry out intergenerational test in the follow-up study.There was a negative correlation between NRRT and mtDNAcn at p<0.01,indicating that there may be some interaction between the two organelles,but the interaction mechanism needs to be further elucidated.In conclusion,we have demonstrated THIM,DIF and THIM&DIF mixture at the range of recommended doses have toxicological effects on earthworms.Therefore,in the actual agricultural production,it is necessary to control its application concentration and frequency to avoid irreversible damage to the soil environment and non target organisms,such as earthworms.In order to further study the toxicity of systemic pesticides,such as THIM and DIF,to non target organisms at actual environmental concentrations,it is suggested that:（1）extend the test cycle to understand the long-term accumulation of pesticides in soil and their intergenerational transmission of damage to earthworms;（2）combined with the results of NRRT and mtDNAcn,the mitochondrial ultrastructure,cell metabolic mechanism and related gene expression of earthworm were further analyzed to clarify the internal damage mechanism of pesticides and the interaction mechanism of organelles,so as to provide data support for the development of more targeted biomarkers and provide theoretical basis for the protection of non-targeted organisms in soil.