Toxicological Effects Of Soybean Herbicide Acetochlor And Metribuzin On Earthworm Under Combined Stress

Herbicides have been playing an important role in removing weeds and increasing grain yield.However,herbicide dosages are often higher than the maximum required for weed control,resulting in accumulation of large amounts of herbicides in the soil.Earthworms are used as a model organism to measure the toxicity of pollutants to soil organisms,given their important role in terrestrial ecosystems.At present,toxicological studies on herbicides mainly focus on the toxicity mechanism of single pollutants.There are relatively few studies on stress related to compound herbicides,especially on the ecotoxic effects of compound herbicides on the soil pollution ecotoxicological diagnosis model-earthworm.Therefore,in this study,acetochlor and metribuzin,effective components of compound herbicides commonly used in bean fields,were used as target compounds,and earthworms were used as test organisms to explore the toxic effects of combined pollution stress on earthworms and the types of compound effects between pollutants.The physiological responses of earthworm under combined pollution stress were studied,and the effects of combined pollution were fully interpreted at individual level,tissue and cell level,and molecular level.Two methods,comprehensive biomarker response and correlation analysis,were used to screen sensitive biomarkers and identify the relationship between different physiological and biochemical indicators.The main conclusions are as follows:（1）The acute toxicity of acetochlor and metribuzin under single and combined stress was investigated using filter paper contact method and artificial soil method.The filter paper contact method showed that the 24-hour median lethal concentrations（LC50）of acetochlor,metribuzin,and their combination on earthworms were 10.83μg·cm^-2,27.68μg·cm^-2,and 9.13μg·cm^-2,respectively.The above results indicated that for the tested earthworms,the acute toxicity of the combined stress was greater than that of acetochlor alone,which was greater than that of metribuzin alone,demonstrating an enhanced toxic effect of herbicides on earthworms under combined stress.The fa-CI fitting results showed that the CI values were 0.72～0.88 under filter paper contact condition and0.64～0.69 under artificial soil condition,which were always less than 1,indicating a synergistic effect between acetochlor and metribuzin.（2）The individual-level effects of combined stress of acetochlor and metribuzin on earthworms were investigated.According to the test results,with the increase of herbicide concentration and exposure time in artificial soil,the weight inhibition rate of earthworm gradually increased,and the weight inhibition rate under combined stress was higher than that under single stress,indicating that acetochlor,metribuzin and combined stress could inhibit the growth of earthworms,and the combined stress had a more obvious effect on and growth.At the 28th day,when the combined stress concentration of acetochlor and metribuzin reached（8+2）mg·kg^-1,the weight inhibition rate of earthworm reached 42.00%.In the avoidance response test of earthworms,metribuzin did not cause significant avoidance behavior,while the avoidance rate of earthworms under acetochlor and combined stress increased with the increase of stress concentration.When the combined stress concentration of acetochlor and metribuzin reached（16+4）mg·kg^-1,the avoidance rate reached the maximum,approaching 80%,showing a significant toxic effect.（3）The response of physiological and biochemical indexes of earthworm to single/combined stress of acetochlor and metribuzin was investigated by artificial soil method.The results showed that herbicide stress could increase the ROS content in earthworms,with a clear dose-response relationship as the stress concentration increased.Compared to the single stress treatment group,the ROS accumulation amount in the combined stress group was greater.At 28 d,the ROS content in the earthworms treated with 10 mg·kg^-1 combined stress was 167.48 OD·mgpr^-1,which was higher than that in the single herbicide treatment group with the same exposure time.The accumulation of ROS inhibited the activities of antioxidant enzymes（SOD,CAT and POD）,and the inhibitory effect of combined stress treatment group was more obvious than that of single stress treatment group,indicating that the combined stress of acetochlor and metribuzin enhanced the oxidative stress effect on earthworms.The excess accumulation of ROS could lead to an increase of MDA and 8-OHd G contents in earthworms.The contents of MDA and 8-OHd G in both single stress and combined stress treatment groups were significantly higher than that in control treatment,and the combined stress effect was more significant.The ACh E enzyme activity was inhibited except for 7 d.At 14-21 d,the ACh E enzyme activity of earthworms in the combined stress group was more inhibited than that in the single stress group,indicating that the combined stress enhanced the neurotoxicity of pesticides to earthworms.The ACh E enzyme activity was inhibited except for 7 d.At 14-21 d,the ACh E enzyme activity of earthworms in the combined stress group was more inhibited than that in the single stress group,indicating that the combined stress enhanced the neurotoxicity of pesticides to earthworms.Although cellulase activity was inhibited by herbicide,it did not show an obvious rule,indicating that the cellulase activity was affected by other factors.The combined stress of acetochlor and metribuzin aggravated the oxidative stress response,leading to more severe toxicity to earthworms.（4）Biomarkers of acetochlor and metribuzin combined stress were identified.IBR analysis and significance analysis were performed to determine the biomarkers of combined stress.By IBR analysis,it was found that IBRv2 value under combined stress was always higher than that under single stress,which further indicated that acetochlor and metribuzin had synergistic effect on earthworm.Meanwhile,it was found that the activities of CAT enzyme and ACh E enzyme were susceptible to the effects of both single and combined stress of acetochlor and metribuzin.According to the results of significance analysis,the activities of CAT enzyme and ACh E enzyme had significant relationship with various indexes,indicating that CAT and ACh E enzyme could be used as biomarkers.In conclusion,the toxicity effect of acetochlor and metribuzin combined stress on earthworm is synergistic effect.Under acetochlor and metribuzin combined stress,the growth of earthworm is inhibited and it will have obvious avoidance behavior.Combined stress aggravated oxidative damage of acetochlor and metribuzin in earthworm.CAT enzyme activity and ACh E enzyme activity can be used as biomarkers of acetochlor and metribuzin combined stress.