Toxic Effects And Mechanism Of Single And Combined Pollution Of Imidacloprid And Cadmium On Earthworms In Soil

Neonicotinoid insecticides occupy an important position in the global insecticide market due to their novel and efficient mode of action and insecticidal activity.Imidacloprid is the first generation of neonicotinoid pesticides to be developed and sold for the first time.Because of the huge amount of imidacloprid used,it will inevitably remain in the environment,which will have a negative impact on non-target organisms.Due to the rapid development of modern industry,agriculture and animal husbandry,the potential accumulation of heavy metals in soil has also become a global concern.Among them,cadmium(Cd),a heavy metal with great environmental risks,exists for a long time in soil and is difficult to be degraded,which may accumulate in soil organisms and have toxic effects on it.Therefore,imidacloprid,a typical neonicotinoid pesticide with high residues in soil,and Cd,a heavy metal with high ecological risk,may coexist in the soil environment.However,there are few reports on the toxicity and mechanism of their combined pollution on soil organisms.Therefore,it is of great significance to study the toxic effects and mechanism of their single and combined pollution.In this study,the standard test organism of toxicology,Eisenia fetida,was used as the soil test organism.The single and combined pollution of imidacloprid(0.1 mg/kg,0.5 mg/kg,1mg/kg)and Cd(10 mg/kg)were determined for 28 days.The effects of individual level,physiological and biochemical level of oxidative stress and enzyme activity,gene level,intestinal tissue influence,and quantitative and characterization of pollutants in artificial soil were studied.Pathological tissue section technology,reverse transcription polymerase chain reaction(RT-PCR)technology,microbial 16 S r DNA sequencing,transcriptome sequencing,UV-vis absorption spectrometry,high performance liquid chromatography,Fourier transform infrared spectroscopy and other methods were used in this study.It was used to explore the toxic effects of single and combined pollution of imidacloprid and Cd on earthworms in artificial soil and the mechanism of action of pollutants.Biomarker deviation indices,the second-generation integrated biological response(IBR)indices,and redundancy analyses were used to explore sensitive biomarkers and assess the combined toxicity risk of single and compound contamination.The main findings are as follows:(1)At the individual level of Eisenia fetida,the growth of earthworms was inhibited to varying degrees during the whole exposure period of single and combined pollution of imidacloprid and Cd.The growth inhibition effect was more significant in the later period of exposure.The damage on the surface of earthworms was also more serious after 28 days of exposure.The vacuoles in the epidermis of earthworms treated with imidacloprid continued to expand compared with day 14.In the combined treatment group of 1 mg/kg imidacloprid and10 mg/kg Cd,the annulus muscle on the body surface became thinner and the longitudinal muscle structure changed.With the prolongation of exposure time,the self-repair function of earthworms declined,and the damage was aggravated.The reproduction inhibition rate of earthworms and the number of unhatched cocoons were greatly affected by imidacloprid,and the average weight of cocoons was positively correlated with the average number of larvae.According to the IBR index,it was found that 1 mg/kg of imidacloprid alone and its combined pollution with Cd had the highest ecological risk.The combined pollution of 0.5 mg/kg imidacloprid and Cd showed antagonistic effects on the growth inhibition rate and reproduction inhibition rate of earthworms,while the combined pollution of 1 mg/kg imidacloprid and Cd showed a synergistic effect on the growth and reproduction inhibition rate and average cocoon weight.(2)In terms of oxidative stress on earthworms caused by imidacloprid and Cd alone and their combined pollution,the reactive oxygen species(ROS)and malondialdehyde(MDA)contents of earthworms in most of the treatment groups were higher than those in the control group during the exposure period.There was a significant positive correlation between ROS and MDA content.The excessive ROS produced by the organism under the stress of imidacloprid and Cd causes oxidative pressure on itself,leading to the activity of lipid peroxidation of cell membrane in earthworm.In terms of earthworm enzyme activity,catalase(CAT)activity in most of the treatment groups was inhibited throughout the exposure period.Glutathione S-transferase(GST)activity was activated,and there was a time-effect relationship.Combined pollution makes the increase of GST activity more significant.Superoxide dismutase(SOD)activity was positively correlated with acetylcholinesterase(ACh E)activity.The comprehensive toxicity risk at the physiological and biochemical level of earthworms increased with the increase of the single stress concentration of imidacloprid.(3)Regarding the genotoxicity of single and combined pollution of imidacloprid and Cd on earthworms,the concentration of 8-hydroxydeoxyguanosine(8-OHd G)in each treatment group of earthworms increased continuously and showed a time-effect relationship in the later stage of pollutant stress.The overall change of 8-OHd G concentration was consistent with the change of ROS content,and a large amount of ROS can cause DNA damage in the body.Cd alone and its combined stress with imidacloprid caused significant up-regulation of metallothionein(MT)genes.Heat shock protein 70(HSP70)gene was up-regulated with the increase of exposure time.The annetocin(ANN)gene of earthworms was positively correlated with the HSP70 gene on the 14 th day of exposure.Excessive ROS induced abnormal expression of two functional genes.Significant upregulation of the translationally controlled tumor protein(TCTP)gene was associated with damage to the body surface tissue of earthworms.The combined toxicity risk increased with increasing imidacloprid combined concentrations.The combination of 0.1 mg/kg and 0.5 mg/kg imidacloprid with Cd showed antagonistic effect respectively,and the combination of 1 mg/kg imidacloprid and Cd showed synergistic effect.(4)The intestinal tissues of earthworms were damaged by exposure to imidacloprid and Cd in soil,and the damage degree increased with the increase of exposure time.On the 28 th day,under the stress of Cd alone and combined with imidacloprid,the villi shedding of intestinal epithelial cells was aggravated,and the tearing and shedding of the chloragogenous tissue was aggravated.Imidacloprid and Cd caused damage to the body surface and intestinal tissue of earthworms,which in turn affected the growth of earthworms.The cellulase activity in the gut of earthworms decreased significantly with the exposure time,and the stress of pollutants affected the body's digestion and decomposition of food.The abundance of Proteobacteria in the gut of earthworms increased under the single stress of imidacloprid and Cd,but decreased slightly under the combined effect compared with the control group.The abundance and diversity of microbial species in the soil where earthworms lived were significantly higher than those in the gut microbes.The combined pollution of imidacloprid and Cd decreased the species abundance of gut microbes and increased the species abundance in the soil.Under the stress of imidacloprid alone and combined with Cd,the pathways of intestinal differential genes were significantly enriched by Cytochrome P450(Cytochrome P450)and Glutathione metabolism.Pollutants induced oxidative stress and detoxification in intestine of earthworm.(5)The single and combined effects of imidacloprid and Cd were quantitatively analyzed by simulating the concentration ratio of imidacloprid and Cd in soil.Compared with the aqueous solution of single imidacloprid and single Cd,when the concentration ratio of imidacloprid and Cd was 1:20,the concentration of imidacloprid and Cd decreased significantly.Fourier transform infrared spectroscopy analysis showed that the C-H bond of imidacloprid under the composite action was obviously shifted,the relative intensity of the hydroxyl functional group increased,the characteristic absorption peaks of pyridine ring and aryl chloride disappeared.It shows that the pyridine ring and the Cl atom on the ring are the contact sites for hydroxylation and Cd compounding,and the Cd and the hydroxyl group may chemically bond with the adsorbent by substituting the Cl atom.The IBR index at the individual level and genotoxicity level of earthworms also showed that the combined pollution of 0.5 mg/kg imidacloprid and 10mg/kg Cd exhibited antagonistic effects.The concentrations of imidacloprid and Cd decreased under the combination of these concentrations,and the toxic effects on the growth,reproduction and gene expression of earthworms also decreased.In this study,the toxic effects and mechanism of single and combined pollution of imidacloprid and Cd on earthworms in artificial soil were evaluated from five aspects:individual level,oxidative stress and enzyme activity,gene level,intestinal damage of earthworms,and the quantification and characterization of pollutants.The toxic effects and mechanism on earthworms were evaluated,in order to contribute to soil environmental ecological risk assessment and protection of biodiversity and human health,and to provide scientific basis for the formulation of corresponding environmental standards.