Environ Sci Pollut Res (2013) 20:9066–9074 DOI 10.1007/s11356-013-1905-5
RESEARCH ARTICLE
Occurrence and distribution of veterinary antibiotics and tetracycline resistance genes in farmland soils around swine feedlots in Fujian Province, China Xu Huang & Chaoxiang Liu & Ke Li & Feng Liu & Derun Liao & Lin Liu & Gefu Zhu & Jie Liao
Received: 22 March 2013 / Accepted: 3 June 2013 / Published online: 28 June 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Six antibiotics, tetracyclines (TCs), and quinolones (QNs) in farmland soils from four coastal cities in Fujian Province of China were investigated. Oxytetracycline was most frequently detected, followed by enrofloxacin, ciprofloxacin, chlorotetracycline, ofloxacin, and tetracycline, with maximum concentrations of 613.2, 637.3, 237.3, 2668.9, 205.7, and 189.8 μg kg−1, respectively. Samples from Putian City contained the highest maximum concentration of ∑TCs (3,064.2 μg kg−1), whereas those from Fuzhou City contained the highest maximum concentration of ∑QNs (897.8 μg kg−1). It is noteworthy that the ∑TCs and ∑QNs in 46.4 and 28.6 % of samples exceeded the ecotoxic effect trigger value (100 μg kg−1), respectively. The concentrations of these antibiotics and five tetracycline resistance genes in four soil plots at depth profiles were quantified thereafter. In most cases, both antibiotics and resistance genes decreased with the increase of depth. Some antibiotics can be detected at a depth of 60– 80 cm where the abundance of tetO, tetM, and tetX reached Responsible editor: Leif Kronberg Electronic supplementary material The online version of this article (doi:10.1007/s11356-013-1905-5) contains supplementary material, which is available to authorized users. X. Huang : C. Liu (*) : F. Liu : D. Liao : L. Liu : G. Zhu Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China e-mail:
[email protected] K. Li Zhejiang Entry-Exit Inspection and Quarantine Bureau, Hangzhou 310016, China J. Liao College of Environmental Science and Engineering, Xiamen University of Technology, Xiamen 361005, China
up to 107 copies g−1. Additionally, the sum of all tet genes (normalized to 16S rRNA genes) correlated with ∑TCs significantly (r=0.676). Our results suggest that resistance determinants can migrate to deeper soil layers and would probably contaminate groundwater by vertical transport. Keywords Tetracyclines . Quinolones . Tetracycline resistance genes . Farmland soils . Swine feedlots . Depth profiles
Introduction Antibiotics have been widely used as feed additives, and for prophylactic, metaphylactic, and therapeutic purposes in animal husbandry (Hamscher et al. 2005). Globally, approximately 39.5 % of antibiotic products were used in animal husbandry in 2006 (Luo et al. 2010). In China, over 8,000 t of antibiotics are used as feed additives every year (Ben et al. 2008). However, because they are weekly absorbed and not completely metabolized in the animal's gut, the majority of applied antibiotics are excreted through feces and urine in unchanged form (Shi et al. 2012). Furthermore, many antibiotics cannot be entirely removed by anaerobic digestion in lagoon (Campagnolo et al. 2002). Therefore, agricultural soils will receive residual antibiotics after repeated manure application (Karci and Balcioglu 2009) and wastewater irrigation (Shi et al. 2012; Gatica and Cytryn 2013). Although the Chinese government has set maximum residue limits of some veterinary antibiotics in animal foodstuff (Wei et al. 2011), regulations for controlling usage of antibiotic-containing manure and wastewater have not yet been established. Soil becomes the primary sink of antibiotics because some classes of antibiotics such as tetracyclines (TCs) and
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quinolones (QNs) can adsorb to soil particles strongly and are resistant to biodegradation. The total contents of four QNs were detected to be up to 1,537.4 μg kg−1 in vegetable farmlands affiliated with livestock farms in the Pearl River Delta Area, southern China (Li et al. 2011). In northern Turkey, oxytetracycline was detected in all the manured agricultural soil at a maximum concentration of 500 μg kg−1 (Karci and Balcioglu 2009). Residual antibiotics may reduce soil phosphatase activity, change microbial community and function, give rise to phytotoxicity, and develop resistance genes (Li et al. 2011). Thus, it is of special importance to investigate the concentrations of residual antibiotics in agricultural soils. In China, a few studies investigated residual antibiotics in agricultural soils in Tianjin (Shi et al. 2012), Shanghai (Ji et al. 2012), and Pearl River Delta Area (Li et al. 2011). Fujian Province, located in the western of Taiwan Strait, is a typical stock-breeding region in China. Data in the statistical yearbook of Fujian Province show that in 2011, there were in total 19 million fattened pigs in Fujian. At present, there is a lack of information on residual antibiotics in farmland soils in different cities of Fujian Province. Extensive use of veterinary antibiotics is receiving more attention because they can select for resistant bacteria in the gut of animals, providing a potential opportunity for dissemination of resistant bacteria into the environment (Mackie et al. 2006). Afterward, transfer of resistance genes from fecal microorganisms to indigenous environmental bacteria occurs under selection pressure of antibiotics driven by the mechanism of horizontal gene transfer (HGT) (Pruden et al. 2006). In soil, transfer of resistance determinants to pathogens may reduce the efficiency of antibiotics on both human and animals. Recently, antibiotics resistance genes (ARG) were considered as new emerging contaminants (Pruden et al. 2006). Diversity and abundance of various ARGs have been investigated in swine feces (Chen et al. 2010; Barkovskii and Bridges 2012) and environments surrounding swine feedlots, including soils with manure application (Schmitt et al. 2006; Wu et al. 2010; Ji et al. 2012; Zhu et al. 2013) and waters from storage lagoons (Chen et al. 2010; Barkovskii and Bridges 2012; Koike et al. 2007) and rivers (Barkovskii and Bridges 2012; Zhang et al. 2012). Besides, a large amount of tetracycline-resistant genes (tet genes) was detected in groundwater adjacent to swine feedlots (Mackie et al. 2006; Koike et al. 2007). Whether ARGs can migrate from agricultural soils to deeper soil layers or groundwater still remains unknown, and it is necessary to investigate the behavior and fate of ARGs and correlative antibiotics in agricultural soils at depth profiles to understand this. The objectives of this study were to: (1) investigate the concentrations of three TCs and three QNs in agricultural soils around swine feedlots in four cities of Fujian Province and (2) understand the concentrations of antibiotics and tet genes, and their correlations in the soils profiles of representative
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farmlands. Five different tetracycline resistance determinants were determined in this study, including representatives of three classes of tet genes (encoding: tetracycline efflux (tetA), ribosomal protection (tetO, tetW, and tetM), and tetracycline transformation (tetX)) (Diehl and LaPara 2010).
Materials and methods Soil sampling and pretreatment Soil samples were collected from farmlands and vegetable fields adjacent to seven swine production facilities located in four cities, Fuzhou, Putian, Quanzhou, and Xiamen, in Fujian Province between January 2011 and August 2011 (Fig. 1). For each plot, four subsamples were collected from the top 0 to 15 cm of the surface soil and were mixed to form one composite sample. All samples were freeze-dried and ground into 100-mesh particles (diameter≤0.15 mm), and stored at −20 °C before extraction and analysis of antibiotics. In June 2012, four plots were selected to determine antibiotics and antibiotic resistance genes at different depths (0–20, 20–40, 40–60, and 60–80 cm). These plots were found to contain high concentrations of antibiotics in the surface soils in 2011. Of these, two plots are planted with grape (G-1 and G2); the third and fourth plots are planted with paddy rice (PR) and sweet potato (SP), respectively. Samples were collected using a stainless steel corer. All samples were lyophilized and homogenized as above. Subsamples were stored at −70 °C until DNA extraction. Total organic carbon (TOC) was determined by a TOC-VCPH SSM-5000A elemental analyzer (Shimadzu, Japan). Soil pH was determined with a soil-towater ratio of 1:3. Extraction and analysis of antibiotic residues Extraction of antibiotics from soil samples Antibiotics were extracted in duplicate using the method described by Zhang et al. (2011) with some modifications. Briefly, 1.00 g of each soil sample was weighed into 50-mL centrifuge tubes. Na2EDTA (0.2 g) and 5 mL of the mixed solution containing phosphate buffer (pH=3.0) and acetonitrile (1:1, v/v) were then added into each tube. Tubes were shaken at 250 rpm for 20 min, sonicated for 10 min, and centrifuged at 6,000 g for 5 min. The supernatant was transferred to a 500-mL beaker. This extraction process was repeated three times. The supernatants combined in the beaker were then diluted to 300 mL with Milli-Q water and acetonitrile was kept at a concentration of 2.5 % (v/v). The diluted supernatants were passed through Oasis HLB (200 mg, 6 mL) cartridges that had been sequentially preconditioned with 6.0 mL of methanol and 6 mL Milli-Q water at a flow rate of approximately
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Environ Sci Pollut Res (2013) 20:9066–9074
Fig. 1 Map of the study area and the sampling sites in Fujian Province, China
5 mL min−1. After that, the cartridges were rinsed with 6 mL of Milli-Q water, dried under a vacuum air pump for 30 min and eluted with 6.0 mL methanol. For each eluate, after the methanol was evaporated under a gentle nitrogen stream, the remainder in the tube was finally redissolved in 20 % methanol– water solution to a final volume of 1.0 mL. The final extracts were stored at −20 °C before LC–MS/MS analysis. Detection of antibiotic residues Six target antibiotics (Bio Basic Inc., Canada), including tetracycline (TC), oxytetracycline (OTC), chlorotetracycline (CTC), ciprofloxacin (CIP), ofloxacin (OFL), and enrofloxacin (ENR), were determined by a LC–MS/MS system (ABI 3200 Q-TRAP, Applied Biosystems, USA). Separations were performed on an Inertsil® ODS-SP column (4.6 mm×150 mm, 5 μm, GL Science Inc., Japan) with stable column temperature at 40 °C. The mobile phase was composed of 0.5 % formic acid water solution (v/v) (A) and pure methanol (B). The flow rate was set at 0.8 mL min−1. The elution gradient was as follows: phase A:phase B was 85:15 at 0 min, 80:20 at 1 min, 70:30 at 3 min, 15:85 at 6.5 min and maintained for 3.5 min, and 85:15 at 10.1 min and maintained for 2.9 min. Each separation was completed in 13 min. The optimal MS/MS condition for each antibiotic was listed in Table S1 (supporting information). Quality assurance and quality control External standard calibration curves including six points (10, 20, 50, 100, 200, and 500 μg L−1) were established to quantify
antibiotics in the soil samples. Results indicate that good linearity (r2 >0.99) was achieved for the calibration curves of all selected antibiotics. Control samples and spiked samples (1.00 g of sample spiked with 200 ng multiple antibiotics) in triplicate were determined to obtain the recoveries of antibiotics. Recoveries were calculated as the percentages of the mean concentration of spiked samples minus that of control samples in comparison to the spiked concentration. Limits of quantification (LOQ) of antibiotics were calculated with signal/noise (S/N) of 10. Table S1 shows the recoveries and limits of quantification of antibiotics in detail. Quantification of tet genes DNA extraction Genomic DNA was extracted from 0.5 g of soil using E.Z.N.A® soil DNA kit (OMEGA Bio-Tek, USA) following the manufacturer's instructions. The concentration and quality of the extracted DNA was determined by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and gel electrophoresis. Quantitative real-time PCR Five tet genes (tetO, tetW, tetM, tetA, and tetX) and 16S rRNA gene were quantified using ABI 7500 Real-time PCR system (Applied Biosystems, USA) and SYBR Green qPCR mix (Takara Biotechnology, Dalian, China). Primers targeting these genes were described previously (He et al. 2007; Aminov et al. 2001; Ng et al. 2001; Ghosh et al. 2009),
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68.4 UQ 28.5 55.6 52.5 29.3 4.7 15.3 85.7 67.9 96.4
32.1 100 75
8.1 UQ 7.2 ND ND ND ND ND 473.6 58.1 219.1 240.0 229.2 111.6 41.8 126.8 113.3 UQ 46.6 68.6 96.2 60.2 5.6 12.5 100 85.7 100
42.9 100 57.1
45.3 UQ 44.7 ND 36.9 27.6 UQ 10.1 932.6 45.4 23.1 864.0 109.1 35.5 13.9 66.2 72.4 UQ 12.8 59.6 20.6 11.8 5.0 6.4 71.4 71.4 100
28.6 100 85.7
10.0 UQ 8.3 UQ 15.3 UQ UQ 3.4 3,064.2 189.8 613.2 2,668.9 579.0 138.3 205.7 235.0 322.0 23.8 34.5 287.5 114.2 40.6 ND 58.4 100 71.4 100
0 100 71.4
ND not detected, UQ unquantified concentration, ENRMax >OTCMax >CIPMax >OFLMax >TCMax; the median concentrations for individual antibiotics were CTCMed > CIPMed > OTCMed > ENRMed > OFL Med > TCMed. Thus, CTC is a predominant antibiotic, whereas TC is a minor component of antibiotics in the collected samples. Table S3 shows that the maximum concentration of CTC in this study is highest among recently reported literatures. The concentrations of TC and OTC in this study were comparable to those in farmlands of Pearl River Delta (Li et al. 2011), north Turkey (Karci and Balcioglu 2009), and Shandong (Yin et al. 2012), but lower than those in Shanghai (Ji et al. 2012). QNs were less frequently investigated than TCs (Table S3). The concentrations of three QNs detected in current study were also comparable to those in Pearl River Delta (Li et al. 2011) and Shandong (Yin et al. 2012). Table 1 further indicates that based on the maximum concentrations and median concentrations of ∑TCs, samples from Putian City are most seriously polluted by TCs while those from Fuzhou City on the contrary. The maximum
Antibiotics
Occurrence of residual antibiotics in the soils from four cities
Table 1 Detection frequency (Freq, %) and concentrations (μg kg−1) of antibiotic residues in farmland soils from four cities
Results and discussion
Med
Statistical analysis Statistical analyses were conducted using SPSS Version 16.0 (SPSS Inc., USA). For correlation analysis, Shapiro–Wilks's test were used to determine whether the dataset (3
