Migration And Transformation Of Ceftiofur In The Environment And The Effects On Environmental Microorganisms

At present,as a new type of pollutants veterinary antibiotics have been widely concerned.Because of its large use,many veterinary antibiotics have reached the environment.The large amount of veterinary antibiotics in the environment can not only cause the production and transfer of ARGs in the environment,but also poison the animals and plants in the environment.The third-generation cephalosporin ceftiofur is extensively used in the treatment of bacterial diseases such as pigs,cattle,and sheep because of its strong antibacterial activity and low side effects.Nowadays,it has been reported that ceftiofur has been found in the environment of Taiwan and Turkey.Although the concentration is low,it can still induce the production of drug resistance genes,and its environmental risks cannot be ignored.Because people believed thatβ-lactam antibiotics have low persistence in the environment,so the environmental behavior data of cephalosporins are lack.VICH guidelines proposes that the environmental risk assessment of antibiotics consists of two stages,in stage I,it needs to evaluate the possibility of entering the environment,and in stage II,it needs to evaluate the environmental effects such as behavioral trends and ecological toxicity.In this experiment,ceftiofur was evaluated according to the environmental risk assessment method of veterinary drugs proposed by VICH guidelines.The adsorption-desorption and degradation experiment of ceftiofur in the environment were carried on.Compost and fertilization experiments were used to explore the migration of ceftiofur.Parameters were used to determine the PEC of ceftiofur in soil.The effects of ceftiofur on the structure of environmental microorganism and ARGs were revealed by metagenome sequencing.1.The establishment of UPLC-MS/MS method for ceftiofur in soil and feces samples.Soil samples were extracted by boric acid buffer（p H=9）,after repeated the extraction progress for twice,the supernatant was passed HLB solid phase extraction column,dried with nitrogen,use 1 m L 10%acetonitrile water to reconstituted and then analyzed by UPLC-MS/MS,the external standard method was used to quantification.The feces samples were extracted by phosphoric acid buffer solution（p H=2.5）:acetonitrile（v:v=1:1）,after repeated the extraction progress for twice,the supernatant was dried with nitrogen to remove acetonitrile,the supernatant was passed HLB solid phase extraction column,and dried with nitrogen,then use 1 m L 10%acetonitrile water to reconstituted,and then analyzed by UPLC-MS/MS.The linear range of the method was 1-500μg kg^-1（R²>0.99）means the method has good linear range,and there was no interference near the peak position.The detection limit（LOD）was 0.3μg kg^-1and the quantitative limit（LOQ）was 1μg kg^-1 of the UPLC-MS/MS method.The average recovery of soil and feces samples was 68.6%-89.5%at the concentration of 1μg kg^-1,20μg kg^-1 and 500μg kg^-1.The range of the inter-RSD and the intra-RSD was 3.3%-12.1%.This method has the advantages of simple operation,high recovery rate,strong specificity and good sensitivity,which can meet the requirements of methodological assessment,and can be used as a quantitative detection method for subsequent experiments.2.The adsorption-desorption of ceftiofur in different soilIn the adsorption equilibrium experiment,the initial concentrations of ceftiofur were 0.05,0.2 and 0.5μg m L^-1.Samples were collected at 0 h,2 h,4 h,8 h,12 h,24 h,36 h and 48 h.According to the results,the adsorption equilibrium time was determined as 12 h.The initial concentrations of ceftiofur in the adsorption experiment were 0.05,0.1,0.2,0.5 and 1μg m L^-1.The adsorption constants K_f and K_d were obtained by fitting Freundlich adsorption isotherm equation and linear adsorption equation.The results showed that the K_f range of ceftiofur was 57.43-122.35μg^1-1/n L^1/n kg^-1.The adsorption strength of ceftiofur in three soils was clay>loam>sandy loam.The adsorption strength of ceftiofur was at the medium level compared with other veterinary antibiotics.The results of the desorption experiment showed that ceftiofur was difficult to desorb,the order of desorption intensity was sandy loam>loam>clay,and the range of retardation coefficient H was 1.11-1.36,indicated that ceftiofur had weak hysteresis effect in three kinds of soil.Soil molecules have a weak repelling effect on adsorbed drugs,which also indicates that ceftiofur has the risk of threatening the groundwater environment.3.Research on the degradation and migration of ceftiofur in soil and compostThe degradation of ceftiofur in different initial concentrations,different soil types,different light intensities,different temperatures,different water contents,different amounts of manure addition,and sterilized soil under laboratory conditions was studied.Single-factor experiment method was used,each experimental group only changed one of the conditions,and the entire experiment was carried out in an artificial climate box.Soil samples were collected at 0 d,1 d,2 d,4 d,6 d,8 d,12 d,and 16 d for UPLC-MS/MS detection.The results showed that the degradation of ceftiofur in soil conforms to the first-order degradation kinetic model,and the degradation half-life（DT50）under different conditions was in the range of 0.76-4.31 d,and the degradation rate was fast compared with other antibiotics.The degradation of ceftiofur accelerates with the increase of water content,temperature and light intensity.The degradation of ceftiofur was accelerated with the increase in the amount of feces added.In the sterilized experimental group,the DT50 of ceftiofur was 4.31 d,which was significantly longer than that of the non-sterilized group,indicated that both biodegradation and physical degradation played an important role in the degradation of ceftiofur.Fresh pig manure was collected,part of which was composted using static aerobic composting,and the other part is applied to the surrounding fields.Ceftiofur was added at feces the initial concentration was 15μg g^-1.Samples were collected at 0 d,1 d,2 d,3 d,4 d,5 d,6 d,8 d,12 d,and 16 d,the compost and the soil were sampled to detect the drug concentration.The compost began to heat up the next day,and the maximum temperature reached 60℃or more.The results showed that ceftiofur was rapidly degraded during the high temperature period of composting,the DT50 was 1.12 d,and it was completely degraded on the 12th day,indicated that aerobic composting method can effectively remove ceftiofur in excrement.In the soil fertilization experiment,ceftiofur was detected in the soil on a rainy day,and then gradually dissipated,indicated that the ceftiofur in the feces can migrate into the surrounding soil through rain.4.Prediction of Ceftiofur Exposure ConcentrationAccorded to the exposure prediction model proposed by the European Medicines Evaluation Agency（EMEA）,relevant parameters were used to predict the exposure concentration of ceftiofur in soil.The predicted exposure concentration of the filter level is 136μg kg^-1,and the second level of assessment is required.The predicted exposure concentration of the refined level is 1.73 and 1.09×10^-10μg kg^-1 at 15 days or91 days of composting,indicated that normal composting treatment for more than 15days can effectively remove ceftiofur in excrement,and also indicated that ceftiofur do not need the phase II environmental impact assessment.5.The effect of ceftiofur on microbial community and ARGs in composting and fertilization soilThe compost samples of 0 d,2 d,4 d,and 10 d and the soil samples of 0 d,3 d,4d,6 d,and 16 d were sent to metagenomic sequencing.The results showed that the main phylum in composting samples was Proteobacteria,Actinobacteria,Firmicutes and Bacteroidetes.Pseudomonas and Cellulobacteria were the main microorganism genus.Composting treatment has a certain effect on the removal of ARGs in manure,and we found that the abundance of ARGs increased after adding CEF,indicated that CEF can promote the generation of ARGs in compost.The results of fertilized soil samples showed that the main bacterial phylum in the soil were Proteobacteria,Chioroflexi,Acidobacteria.After ceftiofur migrated into the soil on the third day,a large number of Firmicutes was found to multiply.After the ceftiofur degraded,the microorganism structure was restored.The results of fertilized soil samples showed that the abundance of ARGs increased after ceftiofur entered the soil,and then gradually restored.Among them,multidrug resistance genes,tetracyclines and macrolide resistance genes accounted for the main position.This study revealed the migration and transformation rules of ceftiofur in the environment,evaluated its impact on the structure of environmental microorganism structure and ARGs,providing new information for the environmental safety assessment of ceftiofur,and at the same time in the future,the environmental risk assessment methods of veterinary drugs will provide reference.