Effects Of Oxytetracycline On Oxidative Stress And In Vivo And In Vitro Bacterial Communities In Earthworms(Eisenia Fetida)

Antibiotic pollution has become a global environmental issue of widespread concern,with tetracycline antibiotics widely present and causing severe pollution in the environment.Earthworms,as "engineers of soil ecosystem",are relatively sensitive animals to soil pollution.They are listed as model organism for environmental health research,and are widely used to assess the health of agricultural ecosystems.This study used Eisenia fetida as the test organism and oxytetracycline hydrochloride as an exogenous stress substance to explore the impact of oxytetracycline on soil ecosystems.Firstly,the filter paper method was used to conduct an acute toxicity effect test on earthworms,observing the poisoning symptoms and behavior of earthworms under oxytetracycline stress,and determining the effect of oxytetracycline on earthworm growth.Secondly,the artificial soil method was used to measure the oxidative stress indicators in earthworms and conduct experiments on the oxidative stress effects of earthworms.The comprehensive marker response index(IBR)method was used to investigate the toxic effects of oxytetracycline on the anterior and posterior tissues of earthworms under different stress conditions(time and concentration).Simultaneously,16S rRNA technology was used to analyze the succession process of bacterial community structure in and out of earthworms after stress occurred.Finally,a typical correlation analysis model was constructed to explore the relationship between the oxidative stress system and bacterial community in earthworms under tetracycline stress,in order to reveal the response mechanism of earthworms to antibiotic stress.Provide scientific basis and experimental basis for ecological safety risk assessment of antibiotic contaminated soil.The research results are as follows:(1)The filter paper test confirmed that earthworm poisoning response showed good dose-effect relationship and time-effect relationship.(2)Based on the IBR method,After medium to low concentrations(0.3 mg/kg,3 mg/kg,30mg/kg)of oxytotracycline stress,IBR values show the degree of its toxicity to earthworms during the slightly stress phase,The main oxidative stress indicators in earthworms are superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD);When the concentration of oxytetracycline is at a higher concentration(300 mg/kg,600 mg/kg),IBR values showed that earthworms suffered a moderate or greater degree of poisoning,The important oxidative stress indicators in earthworms are malondialdehyde(MDA),glutathione peroxidase(GPX)and glutathione sulfur transfer(GST).(3)According to the analysis of 16S rRNA sequencing results,oxytetracycline stress has an impact on the bacterial community structure: Proteobacteria is the dominant bacteria in the bacterial community after short-term stress of oxytotracycline,among which Acinetobacter bacteria had the highest microbial abundance.At the same time,Bacteroidota in the soil bacterial community and Deinococcota in earthworms are also the dominant bacteria under the stress conditions.After long-term oxytotracycline stress,Firmicutes,Proteobacteria and Bacteria were the dominant bacteria in the internal and external bacterial communities,among which Acinetobacter,Subdoligranulum and Clostridia＿UCG-014 were more abundant.(4)With the help of typical correlation analysis model,the relationship between earthworm oxidative stress effect and bacterial community after stress is analyzed: CAT and SOD are the main indicators of oxidative stress effect under the control effect of bacterial community;under long-term stress,earthworms need to rely on bacterial community to synthesize some essential substances to maintain the basic physiological metabolism.(5)Under short-term oxytotracycline stress,the bacterial microorganisms of Acinetobacter in earthworms can activate the oxidative stress effect;the bacteria in soil are driven by oxidative stress,and resist the stress through immune effect.