The Residue of Tetracycline Antibiotics in Soil and Vegetable and Its Effect on the Diversity of Soil Bacterial Community

[Background] Tetracycline antibiotics (TCs) are a broad-spectrum antibiotic, widely used in livestock and poultry breeding. The residue of tetracycline antibiotics in animal manure may cause changes of soil microbial community. [Methods] On the basis of the investigation and analysis of TCs pollution in the soil of main vegetable bases and the livestock manure of major large-scale farms in Chongqing, China, the effects of tetracycline antibiotics on the structure and diversity of soil microbial community were investigated by high-throughput sequencing. The TCs content in soil was increased by applying livestock manure. The contents of TC, OTC and CTC in the soil under pig manure treatment were 171.07-660.20 μg·kg -1 , 25.38-345.78 μg·kg -1 and 170.77-707.47 μg·kg -1 , respectively. The contents of TC, OTC and CTC in the soil under the treatment of chicken manure were 166.62-353.61 μg·kg -1 , 122.25-251.23 μg·kg -1 and 15.12-80.91 μg·kg -1 respectively. The content of tetracycline antibiotics (TCs) in edible parts of Brassica juncea var. gemmifera was increased after livestock manure treatment, among which pig manure had the greatest inuence on the increase of TCs content, and the larger the dosage of pig manure was, the more signicant the increase of TCs content was. Proteobacteria, Acidobacteria, Actinobacteria, Chioroexi and bacteroidetes under livestock manure treatment were the dominant phyla, accounting for 85.2%-92.4% of the total abundance of soil bacteria.


Background
Farmland is the main place for the release of antibiotics, and the application of livestock manure into farmland as organic fertilizer is considered to be one of the most important ways for antibiotics to enter the soil (Quaik et al. 2020). China consumes 60%-65% of the world's total antibiotics ( Soil microorganisms play a key role in maintaining ecosystem functions (Lladó et al. 2018). The application of livestock manure will affect the microbial community in the soil ecosystem of vegetable elds (Sun et al. 2015;Francioli et al. 2016) by on one hand providing nutrients such as C, N, P and K to the soil to promote the increase of microbial population (Ninh et al. 2015), and on the other hand by bringing harmful substances such as heavy metals and antibiotics into the soil to inhibit microbial growth (Pan and Chu 2017a). Many researchers have linked soil microbial communities to soil nutrient composition, but few have linked them to soil antibiotic content. The content of antibiotics in soil depends on the type and application dosage of livestock manures (Quaik et al. 2020), so different fertilization treatments may have different effects on the diversity of soil microbial community. In recent years, high-throughput sequencing technology has provided a good platform for the assessment of soil microbial diversity and its functional analysis, among which 16S rRNA ampli cation sequencing technology has become an important means to study the microbial community composition in environmental samples (Hess et al. 2011).
It is of great signi cance for current agricultural production to study the effects of tetracycline antibiotics on crops and the environment with manure type and application dosage as variables (Xiong et al. 2018).
On the basis of the investigation and analysis of TCs pollution in the soil of main vegetable bases and the livestock manure of major large-scale farms in Chongqing, the eld experiment was conducted to study the residues of tetracycline antibiotics in brassica juncea var. gemmifera and soil under application of different kinds of livestock manures at different dosages. The effects of tetracycline antibiotics on the structure and diversity of soil microbial community were investigated by high-throughput sequencing.

Materials And Methods
The experimental eld was located in Yuxi vegetable research and development center in Xiguan Village, Determination of tetracycline antibiotics in soil and chicken manure The samples were frozen at -20℃ for 3 days, freeze-dried by a freeze-dryer, ground and screened via a 1mm sieve, and stored at -20℃ for preparation. Soil sample extraction: 2.0000g soil sample was accurately weighed and added into a 50ml centrifuge tube, followed by addition of 10ml of extract, mixed by swirling and shaking for 1min, and then underwent ultrasonic treatment for 10min. The supernatant was collected after centrifugation at 4000r•min -1 for 10min. After that, the supernatant was extracted twice with 8ml and 6ml of extract, respectively. The supernatant extracted three times was combined, and then evaporated to a volume of about 12ml at 40℃. Solid phase extraction: SPE column was activated with 5ml of methanol and 5ml of ultra-pure water in turn, and the extraction solution passed through the column at a ow rate of 1ml·min -1 . Then, the columns were washed with 5ml of ultrapure water and 5ml of 5% methanol solution, vacuumed for 20min, and eluted with 5ml of 0.01mol·L -1 methanol oxalate solution to collect eluent. Subsequently, nitrogen was blown to near dry at 40℃, the volume was xed to 1ml with methanol, and the eluent was stored in the automatic sampler after passing a 0.22μm lter membrane. Sample extraction of chicken manure: 1.0000 g of pig manure/chicken manure sample were weighed and added in a 50ml centrifugal tube, followed by addition of the extraction solution and extract (the process is the same as the soil sample). After that, the extracts were combined, with addition of 5ml of n-hexane for lipid removal, and then evaporated to a volume of about 12ml at 40℃. The SPE column and SAX column were activated with 5ml of methanol and 5ml of ultra-pure water in turn, and the extracted solution passed through the column at a ow rate of 1ml·min -1 . The treatment process of other samples was the same as that of soil samples. The LLOQ of tetracycline, oxytetracycline and aureomycin was 00.007 μg·mL -1 0.014 μg·mL -1 0.021 μg·mL -1 , respectively. The recoveries of three antibiotics were determined by standard additions method.
The average recovery rate of OTC, TC and CTC was 58.3%, 57.1% and 87.4%, respectively, with all variation coe cients less than 11.04%.

Determination of TCs content in plants
Plant samples were analyzed by HPLC-MS/MS. The speci c methods are as follows: Liquid phase: the column temperature was 40℃, the ow rate was 0.4 ml·min -1 , the injection amount was 2μL, the gradient elution procedure:  Characteristics of bacterial community in soil By using high-throughput sequencing technology, through sequence assembly, quality control and removal of chimeric sequences, more than 30,000 reads were obtained for each soil sample. Each sample was attened under 29,555 sequences and clustered at 97% clustering level. A total of 1688 OTUs were generated from 9 samples.
The Venn diagram ( gure 3) shows the amount and intersection of OTUs detected in ve treatments: HPM, HCM, BR, CM and CK. A total of 675 identical OTUs were detected in the soil under the ve treatments. Soil-speci c OTUs (30) of pig manure treatment and soil-speci c OUTs (38) of chicken manure treatment were less than CK (57), BR (58) and CM (61). Table 3 shows the OUT number, richness and diversity of soil bacteria. The number of OTUs under BR treatment was the highest. The number of OTUs under all dosages of pig manure treatment was higher than that under all dosages of chicken manure treatment. There was no signi cant difference in bacterial richness (Chao1) and diversity (Shannon and Simpson) among the treatments. was the highest under MCM treatment, but lower under HPM and LCM treatments. The relative abundance of bacteroidetes was the highest under LCM treatment but lower under HPM and HCM treatments. Different types and application dosages of fertilizer caused differences in the relative abundance of bacteria in each phylum, which indicated that the application dosage and type of fertilizer could affect the structure of soil microbial community.

Correlation analysis between bacterial community and TCs contents in soil
To investigate the effect of tetracycline antibiotics in livestock manure on the structure of bacterial communities (10 dominant bacteria were selected as example), this study carried out Redundancy analysis (RDA) for all sample with the residual amounts of TC, OTC and CTC in soil as the environmental factors. As shown in Fig. 5, contents of TC, OTC, CTC in soil were correlated with Granulicclla, but showed no correlation with other 9 bacteria, indicating that TCs residue in soil had a weak effect on microbial community.

Disscuss
When livestock manure is applied into farmland as organic fertilizer, antibiotics will also be transferred to the soil ( In this study, the contents of tetracycline antibiotics (TCs) in edible parts of brassica juncea var. gemmifera were increased by the treatment of livestock manure, in which the increase of TCs contents in brassica juncea var. gemmifera treated by pig manure was much higher than that under other treatments. Moreover, the TCs contents increased with the increase of the application dosage of livestock manure. The TCs residue in brassica juncea var. gemmifera was lower under chicken manure treatment than that under pig manure treatment. The biogas residue is fermented from the same pig manure sampling point. When the application dosage of biogas residue was equal to and the medium amount of pig manure, the TCs content in brassica juncea var. gemmifera under biogas residue treatment was far lower than that under the treatment of medium amount of pig manure, indicating that manure fermentation can reduce the TCsinduced risk to vegetables.
The correlation analysis showed that the contents of TCs in edible part of brassica juncea var. gemmifera was signi cantly correlated with the residual TCs in soil (P < 0.05). Some studies believe that the absorption process of antibiotics by plants is an active absorption (Kong et al. 2007). The absorption of antibiotics in plant roots is related to the ion trap and electrostatic interaction in cell walls (Miller et al. 2016). After the antibiotics are absorbed by the root system, they enter the above-ground part of the plant through xylem under transpiration action, and most of them are accumulated in the cell uid and the comoplast (Goldstein et al. 2014). The transport factors and enrichment factors of antibiotics are related to lipophilicity. Compounds with high lipophilicity are easy to be absorbed by the intracellular fat, resulting in reduced transport capacity, so tetracycline antibiotics with high water solubility are more likely to be transported and accumulated by plants (Wu et al. 2015). Hu et al.(2010) found that the contents of antibiotics in some vegetable tissues exceeded that in the corresponding soil, which had a enrichment effect on antibiotics. However, in this study, the TCs contents in edible parts of brassica juncea var. gemmifera were generally lower than that in soil, which is consistent with the report by Chi et al.(2018). This may be due to that brassica juncea var. gemmifera has a low enrichment capacity of tetracycline antibiotics.
Soil microbial communities, including bacteria, archaea and fungi, play an important role in maintaining the function and sustainability of soil ecosystems. When applying organic fertilizers to increase crop yield, soil microbial processes can be stimulated, which is mainly due to that the long-term application of organic fertilizers can increase soil organic matter content and improve soil fertility (

Conclusions
The application of livestock manure increased the residual contents of tetracycline antibiotics in soil and in brassica juncea var. gemmifera, where the increment order can be ranked as pig manure > chicken manure > biogas residue. Moreover, with the increase of application dosage of pig manure and chicken manure, the residual content of tetracycline antibiotics in soil and in brassica juncea var. gemmifera both increased. The residual amount of tetracycline and aureomycin was higher in the soil treated by pig manure, the residual of tetracycline and oxytetracycline was higher in the soil treated by chicken manure, and the residual amount of oxytetracycline and oxytetracycline was higher in the soil treated by biogas residue. Soil Proteobacteria, Acidobacteria, Actinobacteria, Chioro exi and bacteroidetes under livestock manure treatment are the dominant phyla. The application of livestock manure changed the diversity and abundance of soil bacterial community. The number of OUTs, richness and diversity of soil bacteria were the highest under biogas residue treatment. The number of OTUs under pig manure treatment was higher than that under chicken manure treatment. Biogas residue after fermentation treatment can effectively reduce the environmental and ecological risks caused by antibiotic residues.

Declarations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The sampling of soil and vegetable did not require permission and the species are not classi ed as endangered, and are not under any protection in any of the sampled areas.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.