Occurrence,Distribution and Risk Assessment of Five Kinds of Antibiotics in Mattress on Swine Farm use Ectopic Fermentation Systems in Zhejiang Province

Mattress is among the main products of ectopic fermentation system (EFS),however, the research on the data of antibiotic residues in the mattress of EFS and risk assessments of mattress have not been conducted. This study involved a scale survey to assess the levels and distributions of 54 antibiotics residues, including 4 tetracyclines, 19 quinolones, 22 sulfonamides, 3 amphenicols, and 6 macrolides in mattress on 12 swine farm that uses ectopic fermentation systems (EFS) in Zhejiang Province. A total of 25 antibiotics were detected in mattress, and the total residue amount of antibiotics in mattress samples of each farm was 0.77–28.2 g/T. Chlortetracycline had the highest contribution rate, and the residue amount of antibiotics in mattress is not entirely determined by the start-up time of EFS but is related to the use of feed containing antibiotics, medication habits, the level of mattress management, and maintenance methods of EFS. The risk assessments of antibiotics in the mattress were carried out. The results show low risk for soil on swine farm that uses EFS. study aimed to (1) determine the concentrations of 54 common antibiotics, including 4 tetracyclines (TCs), 19 quinolones (QNs), 22 sulfonamides (SAs), 3 amphenicols (APs), and 6 macrolides (MCs) in the mattress of Wenzhou, Lishui, and Quzhou region, Zhejiang Province; (2) analyze the occurrence and distribution of antibiotic residues in mattress of 12 typical farms at 2019; and (3) conduct risk assessments of antibiotic from mattress into absorptive soil along with the guidelines for environmental risk assessment of veterinary drugs. These results provide reliable data support for the safety problem of mattress in EFS.


Introduction
Considering the low utilization rate of swine waste resources, their proper handling has become di cult during swine breeding. The development of scale swine breeding industry experiences important issues on environmental pollution (Oliveira C, 2001;Won S G, 2016). In recent years, ectopic fermentation system (EFS), as A new swine waste treatment technology, was developed for waste treatment by using a complex microbial preparation of functional thermophilic microbes mixed with straw as carrier prior to fermentation (Shen, 2021;Jiang, 2020, Yang, 2018,Guo, 2015). Figure 1 shows the working process of EFS. After dynamic spraying, ploughing, and fermentation, the odor of raw swine waste can be effectively controlled and treated. At present, With the prevention and control of African swine fever, EFS has been developed for waste treatment in a swine farm in Zhejiang Province, and this method has the advantage of separating from the wastes core area, satisfying the requirements of biological protection and convenient maintenance (Guo, 2015;Jiang, 2020).
Antibiotics are used for livestock, and approximately 46.1% of antibiotics produced (~90,000 tons) in China every year are used in animal husbandry (Shen ,2014,;Zhang,2015). In the United States, nearly 50% of antibiotics (~22,700 tons) are used in animals and aquaculture every year (Kümmerer,2009). Van Boecke et al. conservatively estimated that the total consumption antibiotics in animal production in 2010 is 63,151 ± 1,560 tons, and the antibiotic consumption is expected to increase to 67% by 2030 (Van Boeckel,2015). Serious dependence or even overuse of antibiotics will lead to two major problems in safety, namely, antibiotic residues and bacterial resistance, which not only do harm to the sustainable development of animal husbandry, but also seriously threaten food safety, human health, and ecological environment (Ramaswamy, 2010;Hamscher, 2018).
Mattress is among the main products of EFS. In ectopic fermentation, high-temperature-resistant microorganisms are used to degrade feces and urine by aerobic fermentation (Jiang, 2020; Yang, 2018;Shen, 2019). Then, mattresses are used as organic fertilizer for agricultural soil. Antibiotics used in swine farm are mostly excreted in the form of prototypes through feces and urine (Rasschaert ,2020;Wohde, 2016). In the process of EFS, these antibiotics continuously accumulate in the mattress through the spray of feces and urine with prolonged fermentation accumulation time. If the amount exceeds the acceptable range, it may have adverse effects on the living environment of the normal soil and the horizontal transmission of drug resistance genes in the digestion area.
Moreover, antibiotics in mattress have a negative effect on the microbial growth of mattress (Shen 2019). E cient and low-cost treatment of mattress, the environmental safety of antibiotic consumption area, and the quality of mattress products can be ensured by solving the problems on antibiotic residues in matters of EFS and the safety evaluation of mattress.
Previous studies have investigated the ability of thermophiles and improve the fermentation performance in EFS (Shen, 2019;Jiang, 2020;Yang, 2018;Guo 2015). However, the research on the data of antibiotic residues in the mattress of EFS and risk assessments of mattress have not been conducted.
Accordingly, the present study aimed to (1) determine the concentrations of 54 common antibiotics, including 4 tetracyclines (TCs), 19 quinolones (QNs), 22 sulfonamides (SAs), 3 amphenicols (APs), and 6 macrolides (MCs) in the mattress of Wenzhou, Lishui, and Quzhou region, Zhejiang Province; (2) analyze the occurrence and distribution of antibiotic residues in mattress of 12 typical farms at 2019; and (3) conduct risk assessments of antibiotic from mattress into absorptive soil along with the guidelines for environmental risk assessment of veterinary drugs. These results provide reliable data support for the safety problem of mattress in EFS.

Study area of swine farm
The study was conducted in Southern Zhejiang Province. Twelve selected swine farms that use EFS were located in Wenzhou, Lishui and Quzhou region, from which mattress samples were collected in 2019 during the autumn. The locations of 12 swine farm are in Figure 2, and detailed information are shown in Table 1.

Sample collection
Every fermentation bed is divided into nine zones, and the sample has been collected on the center of each area from the top of the bed with a pair of shovels.
Then, nine mattress have been mixed evenly. Approximately 1 kg mattress was obtained from the sealed bag by quartering method. After rapid cooling with liquid nitrogen, the sample was stored at −20 ℃ until analysis.

Sample treatment
Approximately 10 g of the sample was evenly divide into two ball milling tanks, and one steel ball was placed and then precooled with liquid nitrogen for 5 min and smashed with a ball mill at 30 Hz. The sample was placed in a plastic bag at −4 ℃ prior to analysis.
Approximately 2.0 g of the sample was accurately weighed into a 50 mL polypropylene copolymer centrifuge ask. Subsequently, 10 ml of Mcllvaine-Na 2 EDTA buffer (pH 4.5) was added to the sample and swirled for 30 s. The mixture was sonically oscillated for 20 min and then centrifuged for 5 min at 10,000 rpm and −4°C. Thereafter, the supernatant was directly loaded onto anPrime HLB SPE cartridge, and then Prime HLB was eluted using 8 ml of eluent (mixing with 150 ml of methanol, 150 ml of ethyl acetate, and 6 ml of concentrated ammonia). Then, the surplus residues extracted using Mcllvaine-Na 2 EDTA was ultrasonically extracted with 10 ml of acetonitrile repeatedly, and then subjected to acetonitrile extraction. Thereafter, the eluent was collected and evaporated to dryness under N 2 in 40 ℃. The residue was reconstituted in 0.5 mL of starting mobile phase, ltered using a 0.22 µm nylon lter membrane, and transferred into vials until HPLC-MS/MS analysis. The contribution rate of each antibiotics (x i ) was calculated as follows:

LC−MS/MS Analysis
where c i is the mean detectable concentration of each antibiotic, and p i is the detection frequency of each antibiotic.

Risk assessment
Reference For the overall evaluation of total antibiotic risk of each farm, the PEC tatoL for each farm was calculated using the equation .

Distribution and occurrence of antibiotics in mattress of 12 swine farms
All collected mattresses from the 12 farms that use EFS contained 10 or more antibiotic residues. A total of 25 kinds of antibiotic residues in 54 monitored drugs were detected in mattresses, including 4 TCs, 10 QNs, 5 SAs, 2 Aps, and 4 MCs. The detection frequency of antibiotic residues in the 56 mattresses are shown in Figure S1. The frequency percentage of each antibiotics found in different mattress samples is in the following order: DOX>CTC>ENR>TC>OTC>CIP>OFL>TIM>LIN>FF>SDP>TMP>SMZ> SCP>OXO>FLU>LOM>TYL>SAR>SD>CIN>ENO>TAP>NOR>CTM. DOX is the most command antibiotic in mattress (98.4%), and the antibiotics with a detection frequency higher than 90% were OTC, TC, CTC, DOC, and ENR, while CTM was only found once in one mattress; Three antibiotics(SAR,NOR,ENO) were found on only one farm.
The ranges and averages of antibiotic in mattress from the 12 farms that use EFS are presented in Table 2. The concentrations of the antibiotics in all samples were extremely variable, ranging from ND to 30,579 µg/kg. The order of the total average accumulation of 25 antibiotics is as follows: CTC>DOC>TC>OTC>ENR>FF>CIP>TIM>FLU>NOR>TAP>SDP>SCP>SAR>LIN>OFL>SD>SMZ>ENO>TMP>TYL>OXO>LOM>CIN>CTM. CTC had been highest concentration in most farms, except for farm 11, in which the highest concentration is DOX (1,090.6 µg/kg). Nine of the 25 antibiotics showed mean concentrations above 100 µg/kg with a highest concentration of more than 25,243 µg/kg for CTC in farm 8, while other antibiotics had a concentration below 100 µg/kg, and the lowest detectable concentration of antibiotics of 0.63 µg/kg was recorded on CTM in farm 4.   11. The total of TCs was the highest and was nearly 10-100 times of the other four kinds of antibiotic residues. The highest mean concentration (25,243 µg/kg) was recorded on farm 8 (CTC), whereas the lowest mean concentration (6.0 µg/kg) was recorded on farm 7 for OTC. For QNs, the detection rates of enro oxacin and cipro oxacin in farm were 100%, followed by o oxacin (91.7%) and umequine (41.7%), and the six other QNs were only sporadic. Enro oxacin contained high residue (15.2-1,404 µg/kg) for 11 farms, except farm 4 (FLU, 82.6 µg/kg). Another interesting phenomenon is that the cipro oxacin residue in the mattress of other farms was 1/3-1/7 of enro oxacin residue. The highest mean concentration (1,404.5 µg/kg) was recorded on farm 8 (Enr), while the lowest mean concentration (0.9 µg/kg) was recorded on farm for OFL. Florfenicol was detected in 10 of 12 farms. The residues in the nine other samples were less than 60 g/kg. The highest residue of orfenicol in the mattress of farm 1 was 582.8 µg/kg. TAP weas detected in farms 1 and 2 with mean concentrations of 36.8 and 2.4 µg/kg, respectively. For MCs, TIM was detected in 9 bedding samples of 12 farms, and the contribution rate of TIM to the total residue of MCs was 58-100%. Although the detection rate of lincomycin was 83.3%, the residue of LIN (26.3 µ g/kg) was lower than TIM. The highest mean concentration (170.4 µg/kg) was recorded on farm 11 (TIM), while the lowest mean concentration (0.6 µg/kg) was recorded on farm for LIN. The detection rates of SCP, SMM, SDP, and TMP were higher in mattresses in all SAs. The highest mean concentration (33.6 µg/kg) was recorded on farm 9 (SDP), while the lowest mean concentration (1.0 µg/kg) was recorded on farm 11 for TMP.
In general, the total amount of ve kinds of antibiotic residues in the bedding samples of 12 farms ranged from 0.77 g/T to 28 g/T. TCs were the highest among the ve kinds of drugs, and the average order of the total accumulation of these ve kinds of antibiotics is as follows: TCs (81,568.60 µg/kg ) > QNs

Contribution rate
The overall contribution rate of antibiotics is shown in Figure 4. CTC has provided approximately 80.5% of all antibiotics, other antibiotics ,such as TYL and CTM blow 0.005%.

Risk assessment
The PEC tatoL and PEC i of each farm is shown in Table S2.

Source of antibiotics in mattress
The extensive use of antibiotics in animal husbandry is a global concern. The farms in the study area use a complex mixture of antibiotics through feeding or

Antibiotics in mattress, manure, and fertilizer base on swine
Although no relevant data are available to support the detection of antibiotic residues in mattress, the ndings are consistent with the detection results of antibiotic residues in some livestock manure and fertilizers. Table 3      In total, TCs, QNS, and SAs were the most frequently detected antibiotics and exhibited a broad concentration range in animal manure and manure-based fertilizers. However, in our study, TCs and QNS had the highest mattress, which is consistent with the results of manure and fertilizer. However, SAs had the lowest detection rate and concentration. This nding was obtained possibly because of the strong water solubility of SAs, which will be discharged with wastewater in the process of treatment, or the degradation of SAs in the process of EFS. Considering the different degrees of pollution in the treatment process, the actual removal capacity and the removal mechanism of these ESF should be further studied for selected antibiotics.
These results are consistent with the results of feces and fertilizer samples containing 10 or more different antibiotics. The number of antibiotics in mattress is much higher than that in manure and fertilizer. This nding was obtained because the time of treatment cycle for mattress is much longer than manure, and the antibiotics continuously accumulated in the mattress through the spray of feces and urine. Moreover, the amount of antibiotics in mattress in most farms is lower than the amount of manure, possibly because antibiotics may be degraded by microorganisms during EFS or as an effect of prolonged accumulation.
The distribution of antibiotics in the mattress of 12 swine farms has many similarities and differences. Based on our study, the results show that the residue amount of antibiotics in the mattress is not entirely determined by the start-up time of EFS but is related to the use of feed containing antibiotics, medication habits, and the level of management and maintenance methods of mattress in EFS.

Risk assessment
Based on the contribution rate, CTC has the highest contribution to antibiotics of mattress. Therefore, CTC and TCs are the most noteworthy drug in mattress.
Although the contribution rate of other drugs is very low, the toxicity and drug resistance of other antibiotics still need to be focused on.
For maintaining the vitality of the fermentation bed, each farm will take out part of the ripening mattress and supplement new raw material periodically (1 year or half a year). The mattress obtained in EFS was allowed to be applied with organic fertilizer for plant and same vegetable cultivation in Zhejiang with prescribed dosage. Therefore, the use of mattress may introduce antibiotics into agricultural soil. In comparison with previous studies (Li, 2013;Zhang, 2015;Li, 2003), the risk of each antibiotic in manure and manure-base fertilizer was assessed separately. However, the mattress of each farm contains 1 or more antibiotics. Therefore, we used the PEC total of each farm to evaluate the risk of mattress.
Based on VICH, when PEC is greater than or equal to 100 µg/kg, the risk assessment should be started (VICH, 2000). Considering the difference in fertility, the amount of mattress used as fertilizer in soil per unit area is lower than that of manure. Therefore, the ratio of M/A does not refer to the values of relevant studies (6,000 kg/0.67 ha), but uses a permissible level of mattress of each region as fertilizers to soil. According to the calculation results, the PEC total of all farms did not exceed 100 and poses low risk for soil as fertilizers. However, the economic risk of farms with high PEC total value such as farms 8, 10, and 1 (27.38, 19.28, and 13.29, respectively) cannot be ignored, and some measures to control antibiotic residue should be taken.
The results showed that the effect of antibiotic residues in mattress on the environment was acceptable on the premise of supplementing other nutrients and the maximum single use of ectopic fermentation mattress. EFS can effectively solve the problem of fecal antibiotic treatment .Therefore, before applying organic fertilizer, the method of determination of antibiotic content in mattress and the risk assessment of antibiotic for soil can be used as reference standards to ensure the bene t of agricultural production and minimize the ecological risk.

Conclusions
This study involved a scale survey to assess the levels and distributions of antibiotics residues in mattress on 12 swine farms that use EFS in Zhejiang Province. A total of 25 kinds of antibiotics were detected in the mattress of 12 farms. The total residues of bedding samples in each farm ranged from 0.77 g/T to 28 g/T, and CTC had the highest contribution rate among the antibiotics. The amount of antibiotic residues in the mattress material of EFS was not entirely determined by the start-up time of EFS, related to the level of breeding management, medication habits, and maintenance methods of EFS. The results of data and risk show that the effect of antibiotic residues in the mattress on the environment was acceptable on the premise of the prescribed amount of mattress in the planting land. EFS can effectively solve the problem of fecal antibiotic treatment. However, the application of a large number of antibiotics leads to antibiotic residues in the mattress. Therefore, in scale breeding industry, we should strengthen feed management and strictly control the amount of antibiotics to reduce the residues of antibiotics in the environment.

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There arenot Ethical problem.
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