Environ Sci Pollut Res DOI 10.1007/s11356-014-3632-y
RESEARCH ARTICLE
Occurrence and distribution of sulfonamides, tetracyclines, quinolones, macrolides, and nitrofurans in livestock manure and amended soils of Northern China Jie Hou & Weining Wan & Daqing Mao & Chong Wang & Quanhua Mu & Songyan Qin & Yi Luo
Received: 29 June 2014 / Accepted: 18 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A feasible and rapid analysis for the simultaneous determination of sulfonamides (SAs), tetracyclines (TCs), fluoroquinolones (FQs), macrolides (MACs) and nitrofurans (NFs) in livestock manure and soils was established by solidphase extraction (SPE)-ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS). A total of 32 manure and 17 amended soil samples from the Liaoning and Tianjin areas in Northern China were collected for analysis. The largest detected frequencies and concentrations in manure samples were those of TCs (3326.6±12,302.6 μg/kg), followed by FQs (411.3 ± 1453.4 μg/kg), SAs (170.6 ± 1060.2 μg/kg), NFs (85.1±158.1 μg/kg), and MACs (1.4± 4.8 μg/kg). In general, veterinary antibiotics (VAs) were detected with higher concentrations in swine and chicken manure than in cattle manure, reflecting the heavy usage of VAs in swine and chicken husbandry in the studied area. Furthermore, higher residues of antibiotics were found in piglet and fattening swine manure than in sow manure. In addition, TCs
were the most frequently (100 %) detected antibiotics in amended soil with higher concentrations (up to 10,967.1 μg/ kg) than any other VAs. The attenuation of SAs was more obvious than TCs in amended soil after fertilization, which can most likely be attributed to the stronger sorption of TCs than SAs to soil organic matter through cation exchange. This study illustrated the prevalence of TCs detected in both animal manure and fertilized agricultural soils in Northern China, which may increase the risk to human health through the food chain. Thus, TCs should be given more attention in the management of veterinary usage in livestock husbandry. Keywords Animal husbandry . Veterinary antibiotics (VAs) . Animal manure . SPE-UPLC-MS/MS . Pollution profile . Amended soil
Introductions Responsible editor: Philippe Garrigues Jie Hou and Weining Wan contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3632-y) contains supplementary material, which is available to authorized users. J. Hou : W. Wan : D. Mao (*) : C. Wang : Q. Mu College of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China e-mail:
[email protected] Y. Luo (*) Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071, China e-mail:
[email protected] S. Qin School of Environmental Science and Safety Engineering, Tianjin University of Technology, Tianjin 300384, China
Antibiotics are used extensively for the treatment of bacterial infections in humans and animals, and the worldwide use of antibiotics varies from 100,000 to 200,000 t per annum (Kummerer 2009). In recent years, with the rapid development of animal husbandry, veterinary antibiotics comprised 70 % of the world’s antibiotic consumption for the purposes of illness prevention and growth promotion (Hirsch et al. 1999; Kummerer 2009). In the USA, approximately 9000 to 13,000 t of veterinary antibiotics were used from 2002 to 2004 (Sarmah et al. 2006). In the EU, it was estimated that over 13,000 t of veterinary antibiotics were applied in animal husbandry in 1999. In addition, approximately 93 t of antibiotics were used for veterinary purposes in New Zealand in 2000, accounting for 57 % of the total antibiotic consumption (Sarmah et al. 2006).
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China is the worldwide leader in antibiotic consumption in the livestock-breeding industry. It was estimated that the use of veterinary antibiotics as feed additives for growth promotion had reached approximately 6000 to 8000 t annually in China (Zhao et al. 2010; Ben et al. 2008; Zhou et al. 2008). The heavy uses of veterinary antibiotics generated high levels of antibiotic residues in China. For instance, chlortetracycline was detected in swine manure with the highest concentration of 754.4 mg/kg in Shandong Province, and the highest concentrations for quinolones such as norfloxacin and enrofloxacin were 225.45 and 1420.76 mg/kg, respectively, in eight provinces of China (Pan et al. 2011; Zhao et al. 2010). In addition, up to 90 % of veterinary antibiotics (VAs) could not be assimilated by animals and might be excreted unchanged from the bodies via urine or feces (Kemper 2008). As a result, the antibiotics consumed by animals could be introduced into different environmental compartments through manure and lagoon water application to farmlands as fertilizer. Moreover, overflow or leakage also likely contributes to antibiotic transfer from manure storage to surface water and groundwater or adjacent soil environments (Sarmah et al. 2006). The majority of previous studies have observed the occurrences of veterinary antibiotics in water or sediment environments. The concentrations of sulfamethoxazole in ground water (Sacher et al. 2003), surface water (Hirsch et al. 1999; Yan et al. 2012), and drinking water (Muckter 2008) were 410, 480, and 66 ng/L, respectively. In soils, the concentrations of tetracycline, oxytetracycline, and chlorotetracycline were detected to be as high as 0.2, 0.3, and 0.039 mg/kg, respectively (Hamscher et al. 2002; Boxall et al. 2006; Hamscher et al. 2005). Therefore, animal manure, as an important source of veterinary antibiotics entering soil and water environments, should be given more attention. In addition, the existence of antibiotics in the environment was also of great concern because they could threaten ecosystems and favor the development of resistant bacteria and resistance genes (Luo et al. 2010). Northern China has developed areas of animal husbandry and an intensive distribution of concentrated animal feeding operations (CAFOs) sites throughout the regions. Livestock and aquaculture accounted for 57 % of Tianjin’s gross agricultural output, whereas the national average level was only 30 %. In Liaoning Province, animal husbandry was also in rapid development; by 2005, the amount of breeding swine and chickens accounted for 44.0 % and 38.4 %, respectively, of the total livestock farming in the northeast region. Both Tianjin and Liaoning Province were developed areas of animal husbandry in Northern China, but information on the residue levels of antibiotics in the manure and the surrounding environments in these regions was scarce. The objective of this study is to investigate the current polluted situation of 17 typical veterinary antibiotics (VAs) that belong to five classes, i.e., tetracyclines (TCs),
sulfonamides (SAs), fluoroquinolones (FQs), macrolides (MACs), and nitrofurans (NFs), in typical livestock farming in the Liaoning and Tianjin areas of Northern China. A solidphase extraction (SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used for the simultaneous determination of 17 VAs in animal manure samples that were collected from CAFOs and the amended soils in the studied areas. This study collects data to make an integrated profile of the various types of antibiotics in livestock-farming feedlots in Northern China, thereby providing a reference for the management of antibiotic usage in livestock farming.
Materials and methods Chemicals Sulfadiazine (SD), sulfamethoxazole (SMX), sulfadimidine (SM2), sulfamonomethoxine (SMN), sulfachlorpyridazine (SCP), enrofloxacin (ENR), ciprofloxacin (CIP), ofloxacin (OFL), and furazolidone (FUR) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxycycline (DOXY), chlortetracycline (CTC), and tylosin (TYL) were obtained from Dr. Ehrenstorfer (Augsburg, Germany). Tetracycline (TC), oxytetracycline (OTC), erythromycin (ERY), roxithromycin (ROX), and trimethoprim (TMP) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). In addition, sulfadiazine-phenyl-13C6 (Cambridge Isotope Laboratories; Andover, MA), caffeine-trimethyl-13C3 (J&K Chemical LTD.; Beijing, China), lomefloxacin, and meclocycline (LOMX&MECL; Sigma-Aldrich, St. Louis, MO, USA) were used as surrogates to compensate for matrix effects during sample pretreatment and to monitor the performance of the sample preparation process. Simeton, the internal standard used for quantification, was purchased from AccuStandard, Inc. (New Haven, CT, USA). Sampling A total of 32 manure samples and 17 amended soil samples (June, 2013) were collected from Ninghe (S1b, C1b), Dongli (S2b, C2b), and Jinnan (S3b, C3b) in Tianjin and from Shenyang (B3a, B4a, B5a, B6a, B7a, B8a, S3a, C3a), Fushun (S4a, S5a), and Tieling (B1a, B2a, S1a, S2a, C1a, C2a, C4a) in Liaoning Province for investigation (Fig. 1), including 16 swine, 8 chicken, and 8 cattle livestock farms. Manure samples were taken from daily fresh excrement heaps that were stockpiled adjacent to the fields in which the soil samples were obtained. Soil samples were collected from farmlands adjacent to CAFOs and amended with the animal manure in Tianjin (S1b2∼S1b4, S3b1, S3b3) and in Liaoning Province
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(B1a∼B6a, S1a, S2a, S4a, C1a, C2a, C4a). For each sampling site, three subsamples were collected from the top 0 to 15 cm of the surface soil using a multiple-point sampling method and then mixed together to obtain the composite samples. Each sample was placed into a plastic container and immediately chilled in an icebox. All samples were transported under cooled conditions to the laboratory, lyophilized for at least 48 h, homogenized and sieved (diameter ≤0.5 mm), and then stored in the dark at −18 °C until analysis. Sample pretreatment and solid-phase extraction In addition to using standard additions for quantification (see “Analytical method validation” section below), meclocycline, sulfadiazine-phenyl- 13 C 6 , lomefloxacin, and caffeinetrimethyl-13C3 (1 mL of 50 μg/L each) were added to manure and soil samples as surrogates for TCs, SAs, FQs, and other compounds (NFs and MACs), respectively. Simaton was also used as internal standard to enhance analytical precision. During pretreatment, 1 g of lyophilized and sieved manure sample was added to a 50-mL polytetrafluoroethylene (PTFE) centrifuge tube and spiked with standards (1 mL of 50 μg/L each) of meclocycline, sulfadiazine-phenyl- 1 3 C 6 , lomefloxacin, and caffeine-trimethyl-13C3 as surrogates. Then, 10 mL of Na2EDTA–McIlvaine buffer (pH 4.0) was added. The mixtures were vortexed at 2500 rpm for 30 s, ultrasonicated for 10 min (50 kHz, 300 W), and centrifuged at approximately 4500 rpm for 15 min. The supernatants were decanted into 50-mL PTFE tubes. The extraction was repeated twice more with another 5 mL methanol-acetonitrile-acetone (v/v/v=2:2:1), as described above. The supernatants were combined and then degreased by 10 mL n-hexane. After centrifuging at 4500 rpm for 10 min, the supernatant was adjusted with deionized sterile water to 250 mL, filtered through 0.45-μm glass fiber filters, and adjusted to pH 4. To avoid interference from dissolved organic matter, samples were also pretreated with Strata strong anion exchanger (SAX) cartridges (3 mL/200 mg, Waters, Taunton, MA, USA) followed by extraction with Oasis hydrophilic–lipophilic balance (HLB) cartridges (6 mL/500 mg, Waters, Taunton, MA, USA). The detailed extraction procedure applied to the soil samples was developed based on a previous description (Luo et al. 2011). After adding 0.1 g NaF as the ion exchanger and 50 ng of each surrogate (meclocycline, sulfadiazine-phenyl-13C6, lomefloxacin, and caffeine-trimethyl-13C3), 1 g lyophilized and sieved soil was extracted by 5 mL methanol-EDTAcitrate buffer (V/V/V=3:2:1), vortexed for 30 s at 2500 rpm, ultrasonicated for 15 min, and centrifuged for 5 min at 4000 rpm. The procedure was repeated three times. The supernatant mixture was combined and degreased by n-hexane, and it was then diluted to 250 mL with deionized sterile water, filtered through 0.45-μm glass fiber filters and adjusted
to pH 4. The extract was cleaned upon a SAX-HLB solidphase column. Oasis HLB cartridges were combined with SAX cartridges in series. The cartridges were pretreated with methanol (5 ml) and deionized sterile water (5 ml). The diluted manure and soil extract were passed through the cartridges at a flow rate of 3– 5 mL/min. After extraction, the SAX cartridges were removed, and the HLB cartridges were washed with 10 ml deionized sterile water, vacuum-dried for 30 min, and then eluted with 5 ml of methanol-acetone (V:V=80:20). The SPE eluent was reduced to approximately 100 μL under a gentle stream of nitrogen at 50 °C and reconstituted to approximately 1 mL with a solvent mixture of 30 % methanol in water (V:V). The extract was spiked with 10 μL of 10 mg/L simeton as an internal standard, filtered through 0.22-μm cellulose acetate syringe filters, and stored at −18 °C until UPLC-MS/MS analysis. Ultra-performance liquid chromatography-tandem mass spectrometry analysis UPLC-MS/MS instrumental determinations were performed using a Waters Acquity UPLC system (Waters, USA) equipped with a Waters 2777C autosampler system and Waters Xevo TQ MS (Waters, USA) detector with an electrospray ionization (ESI) source. A Waters Acquity UPLC BEH C18 column (2.1 mmol/L×100 mm, 1.7 μm) (Waters, USA) was used for separations and was thermostated at 40 °C with injection volumes of 10 μL. With acetonitrile as mobile phase A and 0.1 % formic acid in water (V/V) as mobile phase B for gradient conditions with a total flow rate of 0.45 mL/min, the gradient elution was set as follows: 0 min, 10 % A; 0∼1.5 min, 10∼16 % A; 1.5∼2.0 min, 16∼18 % A; 2.0∼3.0 min, 18∼22 % A; 3.0∼4.0 min, 22∼35 % A; 4.0∼4.5 min, 35∼60 % A; 4.5∼5.0 min, 60 % A; 5.0∼6.5 min, 60∼95 % A; 6.5∼7.0 min, 95 % A; 7.0∼7.1 min, 95∼10 % A; and 7.1∼10.0 min, 10 % A. The total run time was 10 min, and the last 2.9 min was used to re-equilibrate the column before the next injection. For the MS detection, the instrument was operated in the positive ion mode and multiple reaction monitoring (MRM) function. To optimize the MS parameters, 0.1 mg/L individual standard solutions of all compounds were infused directly into the MS from an integrated syringe pump, together with a mobile phase of 0.1 % formic acid in water/acetonitrile (90:10) at 0.3 mL/min under combined mode, and the flow rate was 20 μL/min. Nitrogen was used as the cone and desolvation gas, while argon was used as collision gas. The other instrument parameters were set as follows: capillary voltage 3.5 kV, capillary temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow 600 L/h, cone gas flow 50 L/h, and collision gas flow of 0.15 mL/min. Table S1 listed the optimal conditions for each compound.
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C4a
B8a
Fig. 1 Map of the studied area and sampling locations in Tianjin (located in the Haihe River basin) and Liaoning Province (located in the Liaohe River basin). B represents beef cattle, S represents swine, and C represents broiler chickens
Analytical method validation Recoveries of the 17 target compounds were determined for manure and soil using the standards addition method. The simeton was used to compensate for the variation in the volumes of the final extracts and to monitor the instrumental performance. Calibration curves were established by plotting the ratio of the analyte quantifying ion response to the internal standard (IS) ion response and then against the spiked concentrations. Nine concentration levels (0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, and 1000.0 μg/L) were chosen for quantification, and they were prepared by subsequently diluting the stock solutions with 30 % methanol in water (V/V). The correlation coefficients (r2) for all the antibiotics were typically above 0.99, indicating that in the selected concentration range, fine linearity was achieved for the calibration curves of all target compounds. For method validation in real samples, the recoveries of the analytes were determined by applying the standard addition method. The recovery (RE) and matrix effect (ME) were calculated based on the method of Matuszewski et al. (2003). Both manure and soil
samples were spiked with different antibiotic concentrations (1.0, 10.0, 50.0, 100.0, 500.0, 1000.0, 5000.0, and 10,000.0 μg/kg) and analyzed using the method developed in this study. In the manure samples, the recoveries of target compounds at the three spiked levels ranged from 59.2 %–73.7 % for TCs, 63.1–79.6 % for SAs, 54.1–68.8 % for FQs, 42.3–63.6 % for MACs, and 52.3–62.1 % for furazolidone. Meanwhile, the recoveries were 65.6–90.0 % for SAs, 60.1–81.6 % for TCs, 61.3–73.2 % for FQs, 47.3–73.4 % for MACs, and 58.2 %–67.4 % for furazolidone in the soils. The recoveries of the four surrogates ranged from 60.0 to 79.1 %. The limits of detection (LOD) and limits of quantification (LOQ), defined as the lowest concentration levels corresponding to a signal-to-noise (S/N) ratio of 3 and signal-to-noise (S/N) ratio of 10, respectively, were also determined. The relative standard deviations (RSD) ranged from 1.3 to 5.4 % for manure and from 0.6 to 4.7 % for soil samples. The method showed high sensitivity and accuracy, as applied in the determination of residual antibiotics in both manures and soils. The data from the analytical method validation are shown in Tables S2 and S3 (Supplementary Information).
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Results and discussion Occurrence and distribution of various VAs in animal manure Tianjin and Liaoning Province were selected as sampling districts because both regions were highly developed with concentrated animal farming operations (CAFOs) in Northern China. TCs comprised the highest detected frequencies (81.3– 90.6 %) and concentrations (3326.6±12,302.6 μg/kg) in the manure samples, followed by FQs (411.3±1453.4 μg/kg), SAs (170.6±1060.2 μg/kg), NFs (85.1±158.1 μg/kg), and MACs (1.4±4.8 μg/kg) (Fig. 2). Among the four TCs, CTC was the most predominant antibiotic, detected with an average concentration of 8060.6±23,079.9 μg/kg and a peak concentration of 124,579.1 μg/kg and a detection frequency of 90.6 %. OTC was also a prevalent antibiotic in the manure samples from livestock farming, with an occurrence of 87.5 % and a concentration of 2875.0±5233.8 μg/kg. The residues of CTC and OTC in this studied area were significantly higher compared with previous studies in chicken and turkey manure from Austria but relatively lower than those of swine manure detected in the Shandong Province of China, reflecting the more serious overuse of TCs in the animal feeding industry in China compared with other countries (Martínez-Carballo et al. 2007; Pan et al. 2011). In this study, the most frequently detected SAs in animal husbandry were SMX (18.9 ± 20.1 μg/kg) and SCP (34.9±21.6 μg/kg), both of which were obtained with a relatively high occurrence of 100 % but lower residue levels compared with SM2 (884.6±2497.8 μg/kg on average, with a peak concentration of 9356.2 μg/kg) (Fig. 2).
1600 1400
concentration frequency
a
soil
Concentration (µg/kg)
150 100 50 0 SD
8000 7500 3500 3000 2500 2000 1500 1000 500 0
SMX SMN SM2 SCP TMP
TC
CTC OTC DOXY ENR
CIP
OFL ROX ERY
b
SD
TYL
FUR
manure
SMX SMN SM2 SCP TMP
TC
CTC OTC DOXY ENR
Antibiotics
CIP
OFL ROX ERY
TYL
100 90 80 70 60 50 40 30 20 10 0
FUR
100 90 80 70 60 50 40 30 20 10 0
Frequency (%)
Fig. 2 Detection frequencies (DFs) and concentrations of 17 target antibiotics in soil (a) and animal manure (b) samples. Error bars represent the standard deviation of various antibiotics
Our results were significant lower than those in swine manure detected in Turkey (35.5 mg/kg for SCP) and northwestern Germany (1–2 mg/kg for SM2) (Karcı and Balcıoğlu 2009; Christian et al. 2003). As for FQs, the average concentration of CIP was 808.3± 2307.5 μg/kg (with a peak concentration of 10,111.1 μg/kg), which exhibited higher detection frequencies (81.3 %) than the other two FQs, but was much lower than the residual level (up to 45.6 mg/kg) in animal manure from Southeast China (Zhao et al. 2010). This finding reflects the heavier use of FQs in Southeast China than in the northern regions. However, the concentration of FQs (411.3±1453.4 μg/kg) was equivalent to the concentration level (0.13–0.75 mg/kg) in animal manure samples collected in Austria (Martínez-Carballo et al. 2007). In comparison, MACs were not detectable or quantifiable in most of the manure samples, with detected frequencies less than 20 % and low residue levels (0.0–96.3 μg/kg), indicating that MACs were not the dominant veterinary drug used in animal feeding operations in Northern China. In addition, tylosin was detected at low concentration levels (0.0–37.1 μg/kg) in the animal manure in our studies, which was comparable with Liguoro’s study in Italy (2003). The significance of the differences in the antibiotic concentrations in the manure samples between sampling locations (Tianjin and Liaoning) was determined using the independent sample t test (Table S9, Supplementary Information). No statistically significant difference was observed between the Tianjin and Liaoning areas for SAs (p>0.05), indicating the similar application of the six SAs in these two regions. For
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TCs, no statistically significant difference was observed, with the exception of OTC and CTC (p
