Toxicological Study Of Exogenous Nitrogen On Physiological And Biochemical Indexes Of Earthworm

The increasing global nitrogen pollution caused by fossil fuels and nitrogen fertilizer use is bound to have an important impact on soil food webs in forest ecosystems.Earthworm is an important regulator of subsoil food web and can respond quickly to soil environmental stress.In addition,the ecological function of earthworm mainly depends on its internal enzymes and intestinal microorganisms.However,the response of earthworm enzyme activity and intestinal microbial diversity to exogenous nitrogen supplementation is not clear,and further studies are needed.Most of the simulated nitrogen deposition tests are implemented by the addition of exogenous nitrogen.Our previous study found that exogenous nitrogen treatment resulted in a significant reduction in the number of earthworms.Therefore,Eisenia fetida was selected as the study object,and the control(N0:0g Nm^-2a^-1),nitrogen(N5:5g Nm^-2a^-1,N10:10g Nm^-2a^-1,N15:15g Nm^-2a^-1,N30 were simulated in the laboratory.30 g Nm^-2a^-1)5 treatments with 3 replicates per treatment.The enzyme activities of earthworms were measured on the 5th,10th,15th,20th and 30th days,respectively,and the intestinal microorganisms of earthworms after 30 days were analyzed by high-throughput sequencing to analyze the effects of nitrogen addition on enzyme activities and intestinal bacterial community structure and diversity of earthworms.The main research results are as follows:（1）The relative growth rate and relative weight gain rate of earthworms decreased with the increase of nitrogen concentration,and the relative growth rate and relative weight gain rate of earthworms in N10 and N15 treatments were significantly lower than those in the control treatment,but there was no significant difference in the relative growth rate and relative weight gain rate among nitrogen addition treatments.In addition,soil p H value,total phosphorus and ammonium nitrogen contents decreased significantly with nitrogen application,while available phosphorus,total nitrogen and nitrate nitrogen contents increased significantly with no significant change in soil water content.（2）The activities of catalase（CAT）and glutathione S-transferase（GST）,as well as the content of total protein（TP）,in earthworms showed an initial increase followed by a decrease with increasing nitrogen addition levels.Superoxide dismutase（SOD）and peroxidase（POD）activities fluctuated,while malondialdehyde（MDA）content gradually increased.Except for day 10,the POD activity continuously decreased.Compared to the control,nitrogen addition treatment significantly increased CAT activity,GST activity,POD activity,MDA content,and TP content in earthworms.Among them,N15 and N30reached highly significant levels.At the same time,it significantly decreased POD enzyme activity,reaching highly significant levels under the N30 treatment.Different treatment concentrations,time points,and their interactions had a significant impact on earthworm enzyme activities.with enzyme activities on day 10,15,and 30 being higher than on day 5.（3）Different nitrogen addition treatments had no significant effect on the number of intestinal OTUs of earthworms,while the Chao and Shannon-Wiener indices showed a trend of first increasing and then decreasing with the increase of nitrogen concentration,and the diversity index was the lowest under N30 treatment,but there was no significant difference with the control.Simpson index increased with the increase of nitrogen concentration,and Simpson index in different nitrogen concentration groups was higher than that in control treatment,and reached the maximum under N30 treatment.The dominant population at the phylum,class and genus level of the intestinal bacterial community of earthworms accounted for 90%,73%and 11%of the relative abundance of the entire bacterial community,that is,the relative abundance did not change significantly at the phylum and class level,but changed significantly at the genus level.（4）There were significant differences between earthworm SOD activity and TP content and soil physical and chemical factors,but there were significant differences between earthworm SOD activity and soil physical and chemical factors.There was no significant difference between soil physical and chemical factors and diversity index,and soil physical and chemical properties had little effect on soil bacterial community structure.In addition to the soil total nitrogen and nitrate nitrogen,soil physical and chemical factors and Isosphaera,unclassified＿f＿Thermomonosporaceae there is strong correlation between the two kinds of bacteria genera.（5）In conclusion,exogenous nitrogen has great influence on earthworm enzyme activity.In general,the activities of CAT,GST,SOD and CL and the contents of TP and MDA were promoted by nitrogen addition treatment,while the activities of POD were inhibited.At the same time,nitrogen addition had no significant effect on intestinal microbial community structure and diversity of earthworm.The results of this study help to reveal the response mechanism of earthworm to exogenous nitrogen,and provide a theoretical basis for the sustainable and healthy development of forest ecosystem in the future.