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Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment
Science of the Total Environment 521–522 (2015) 101–107
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Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment Cheng Li a,b, Jiayi Chen a,b, Jihua Wang a,b, Zhihong Ma a,b, Ping Han a,b, Yunxia Luan a,b, Anxiang Lu a,b,c,⁎ a b c
Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China Risk Assessment Lab for Agro-products (Beijing), Ministry of Agriculture, Beijing 100097, China Collaborative Innovation Center for Key Technology of Smart Irrigation District in Hubei, Yichang 443002, China
H I G H L I G H T S • • • •
High levels of antibiotics were found in soils from vegetable greenhouses. Concentrations of antibiotics were higher in greenhouse soils than in open fields. Manure application was the primary source of antibiotics in greenhouse soils. Several antibiotics in greenhouse soils pose a high ecological risk.
a r t i c l e
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Article history: Received 15 November 2014 Received in revised form 18 March 2015 Accepted 18 March 2015 Available online xxxx Editor: Kevin V. Thomas Keywords: Antibiotics Greenhouse vegetable production base Manure Risk assessment Soil
a b s t r a c t The occurrence of 15 antibiotics in soil and manure samples from 11 large-scale greenhouse vegetable production (GVP) bases in Beijing, China was investigated. Results showed that the greenhouse soils were ubiquitously contaminated with antibiotics, and that antibiotic concentrations were significantly higher in greenhouses than in open field soils. The mean concentrations of four antibiotic classes decreased in the following order: tetracyclines (102 μg/kg) N quinolones (86 μg/kg) N sulfonamides (1.1 μg/kg) N macrolides (0.62 μg/kg). This investigation also indicated that fertilization with manure and especially animal feces might be the primary source of antibiotics. A risk assessment based on the calculated risk quotients (RQs) demonstrated that oxytetracycline, chlortetracycline, norfloxacin, ciprofloxacin and enrofloxacin could pose a high risk to soil organisms. These results suggested that the ecological effects of antibiotic contamination in GVP bases and their potential adverse risks on human health need to be given special attention. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Antibiotics have been widely used to treat bacterial infections and promote the growth of livestock animals for several decades (Thiele-Bruhn, 2003). China is the world's largest producer and consumer of antibiotics. An estimated 210,000 t of antibiotics is produced every year, of which 48% are used in the agricultural and livestock industries. Most animals cannot completely metabolize the antibiotics they receive, and the animals excrete them into the environment as intact bioactive substances or metabolites. Researchers have reported that antibiotic residues in the environment can affect terrestrial organisms, ⁎ Corresponding author at: Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China. E-mail address: [email protected] (A. Lu).
http://dx.doi.org/10.1016/j.scitotenv.2015.03.070 0048-9697/© 2015 Elsevier B.V. All rights reserved.
alter microbial activity and community composition in soil, and promote the development of antibiotic resistant genes (ARGs), which represent a risk to human and animal health (Ashbolt et al., 2013). Previous studies have frequently reported high concentrations of antibiotics in animal manure. Martínez-Carballo et al. (2007) reported that the concentrations of chlortetracycline, oxytetracycline and tetracycline in 30 pig manure of Austria were up to 46 mg/kg, 29 mg/kg and 23 mg/kg, respectively, whereas the concentrations as high as 225 and 1420 mg/kg of norfloxacin and enrofloxacin were found in chicken manures from China (Zhao et al., 2010). Current environmental legislation fails to cover these antibiotics; the antibiotics could lead to undesirable effects on ecosystems (Li et al., 2012a). Furthermore, the high concentrations of antibiotics in manure can be associated with an increased presence of resistant bacteria in environment, which is a major public health concern, due to the increased occurrence
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of associated clinical infections (Ashbolt et al., 2013; Luo et al., 2010). Most livestock manure is directly or indirectly applied on agricultural land as fertilizers, which constitute the main source of the dissemination of these chemicals in soil environments (Zhou et al., 2013). Hamscher et al. (2002) detected concentrations of tetracyclines up to 0.3 g/kg in an agricultural field fertilized with liquid manure. In addition, Li et al. (2011) found high antibiotic concentrations in vegetable farmlands affiliated with livestock farms and detected maximum concentrations of tetracyclines, sulfonamides, and quinolones of 242.6, 321.4, and 1537.4 μg/kg, respectively. Market demands and economic incentives have resulted in intensified greenhouse vegetable production (GVP), particularly in developing countries; this intensive form of agriculture has expanded rapidly worldwide. China accounts for more than 90% of the global GVP operations, where GVP area had expanded to almost 4.67 million ha by 2010 (Chang et al., 2013). GVP can result in improved vegetable yields by extending the growing seasons and intensifying agriculture compared with conventional vegetable production. However, heavy fertilization strongly increases production and is always adopted under GVP conditions to achieve high vegetable production. In addition, growers of organic vegetables primarily use manure in GVP systems. Most GVP involves large and repeated amounts of animal manure application and probably increases the risk of antibiotic contamination, which could also have adverse effects to human health due to the consumption of these vegetables contaminated with antibiotics and antibioticresistant bacteria. Presently, no regulations address the presence of antibiotics in soil as well as in the manure applied to certified vegetable production systems. This has created an urgent problem related to the contamination of antibiotics in GVP systems. Beijing, the capital of China, supports a resident population of about 20 million. In recent years, large-scale GVP bases have been widely developed on the outskirts of Beijing, to a total area of 1531.49 ha by 2011. With China's emphasis on food security and sustainability, an increasing demand exists for organic vegetables from GVP bases, where most fertilizer comes from manure. This cultivation technique may elevate the contamination of foods by antibiotics relative to conventional farming methods. To date, limited information is available on the occurrence of antibiotics in the GVP bases of Beijing. Therefore, the objective of this study was to improve our understanding of the general status of antibiotic pollution in the GVP bases. The data collected can also be used to evaluate the ecological risks created by antibiotics in GVP systems. 2. Materials and methods 2.1. Sampling sites and sample collection Eleven large-scale GVP bases were selected in the suburbs of Beijing based on geographic location, cultivated area, history and environment. Bases are defined as large-scale areas with numerous greenhouses; sample sites are defined as the various sample points within those bases. The sampling bases were distributed across four districts, including Changping, Daxing, Shunyi and Yanqing, which covered a relatively large area. These vegetable bases were registered with the local Department of Agriculture, and the vegetable products from these bases were transported throughout of Beijing area. Therefore, the selected bases could reflect the whole situation of GVP bases in Beijing. The GVP bases were generally operated in the form of enterprises and only manure was applied in the process of production. And, groundwater was used for irrigation. Sampling work occurred in May–July 2013 (Fig. 1). Fifty-six vegetable greenhouses were selected from these GVP bases, with estimated areas ranging from 20 to 66.7 ha. Three to nine samples were evenly sampled in each GVP base, the exact number depending on the total area of each base. Fig. 1(B) shows Site YQ1 as an example showing the layout of plots. In addition, soils from the open fields near each GVP base were also collected as controls. These open fields were applied to plant seasonal vegetables and cultivated by local farmers. A
combination of chemical fertilizers and organic fertilizers was generally used in the open fields. Topsoil was sampled at a depth of 0–20 cm using a stainless-steel auger. Five sampling sites were distributed in an S-shaped pattern in each greenhouse; these were mixed as a single sample. Furthermore, manure samples (N = 17) that were actively being applied in the studied GVP bases, were also gathered for potential source analysis. The categories of the manure include chicken, duck, pig, and cow manure as well as commercial organic fertilizers which were produced through microbial fermentation using the mixture of livestock dungs and some farm waste such as corn stalks or wheat shafts as raw materials. Soil and manure samples were air dried at ambient temperature, ground and homogenized by sieving through a stainless steel sieve (60-mesh) after removing stones and residual roots; samples were then sealed in brown glass bottles and stored at −20 °C prior to analysis. 2.2. Chemicals and standards The antibiotics analyzed here were selected based mainly on the extent of their use in animal production in China. These drugs belonged to four different antibacterial families. Six sulfonamides (SAs) included sulfamerazine (SMR), sulfamethazine (SMZ), sulfadiazine (SDZ), sulfameter (SM), sulfadimethoxine (SDM), and sulfamethoxazole (SMX). Three tetracyclines (TCs) included tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC). Four quinolones (QNs) included norfloxacin (NFX), ciprofloxacin (CIP), enrofloxacin (ENR) and lomefloxacin (LOM). Two macrolides (MLs) included erythromycin (ETM) and roxithromycin (RTM). Standards for SDM, SMX, TC, CTC, LOM and RTM with purities of N 98% were obtained from SigmaAldrich (St. Louis, MO, USA), while the other antibiotic standards, with purities of N98% were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Simeton (J & K Chemical Ltd., USA) was used as the internal standard to enhance the analytical precision. A stock solution (1.0 mg/mL) of Simeton was prepared in methanol solution, stored in at − 20 °C prior to use. Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (New Jersey, USA). Formic acid (98%) was purchased from Fluka (Bucks, Switzerland). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). Disodium ethylenediamine tetraacetate, citric acid and sodium citrate of analytical grade were obtained from Yaohua Chemical Reagent Factory (Tianjin, China). Citric acid buffer (pH 4) was prepared according to the procedure described by Zhou et al. (2011). Strong anion exchange (SAX) cartridges (6 mL, 500 mg) were provided by Agilent Technologies (Wilmington, DE, USA) and Oasis HLB cartridges (6 mL, 500 mg) were purchased from Waters (Milford, MA. USA). All other reagents were of analytical reagent grade. 2.3. Sample extraction and clean-up One half-gram of each freeze-dried manure sample and 2 g of each soil sample were extracted with 10 mL acetonitrile and 10 mL citric acid buffer (pH = 4) in a 50 mL polypropylene centrifuge tube. The sample was vortexed for 1 min and treated ultrasonically for 15 min, followed by centrifugation. This extraction process was repeated twice. The extract was combined into a round-bottom flask, concentrated with a rotary evaporator at 50 °C to remove the organic solvent, and diluted to 100 mL with ultrapure water to make sure the organic solvent in solution had a concentration of less than 5%. SAX and HLB cartridges were set up in tandem for cleanup of extracts. Prior to the SPE cleanup, 0.2 g of disodium ethylenediamine tetraacetate was added into each aqueous extract to chelate with metal cations. The cartridges were pre-treated with 10 mL methanol and 10 mL ultrapure water; the diluted extract was passed through the cartridges at a loading rate of about 5 mL/min. After loading the entire amount of extract, the SAX cartridge was removed and the HLB
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Fig. 1. Map of the sampling bases in the 11 greenhouse vegetable production (GVP) bases of Beijing. Two inset maps provide A) the study area within Beijing, and B) a map showing the layout of sampling locations in Site YQ1 as an example. GVP base names include CP1, CP2, CP3, CP4, CP5, DX1, SY1, SY2, SY3, SY4, SY5, and YQ1 (CP = Changping; DX = Daxing; SY = Shunyi; YQ = Yanqing).
cartridge was rinsed with 10 mL ultrapure water, air dried for 10 min, and then eluted with 10 mL methanol. The eluate was dried under a gentle nitrogen stream at 50 °C. Simeton (10 μL, 10 μg/mL) was spiked as an internal standard and methanol/aqueous solution (1:1 v/v) was used to bring the final purified extract volume up to 1 mL. After filtration through a 0.22 μm membrane to remove particles, the final extract was transferred to a 2 mL amber vial and stored at − 18 °C until ultrahigh performance liquid chromatography tandem mass spectrometric (UPLC–MS/MS) analysis could be performed.
determined for matrix-matched calibration curves using five different concentrations (5–200 μg/kg) for both soil and manure. The correlation coefficients (R) were all N0.99. Analyte recoveries were in the range of 80.2–104.6% for soil and 70.5–128.2% for manure samples, with the relative standard deviation (RSD) b 10%. The limits of detection (S/N = 3) were 0.23–5 μg/kg for soil and 0.35–13 μg/kg for manure samples. Statistical analysis was performed using SPSS (IBM SPSS Statistics 20). One-way analysis of variance (ANOVA) was used for statistical comparisons and the Pearson coefficient was used for correlation analysis. 3. Results and discussion
2.4. Instrumental analysis 3.1. Occurrence and distribution of antibiotics in greenhouse soils The UPLC–MS/MS system consisted of an ACQUITY UPLC system (Waters) and a Xevo TQ triple quadrupole mass spectrometer (Waters). Separation was performed using an ACQUITY UPLC BEH C18 column (1.7 μm; 2.1 × 50 mm), operated at 35 °C, with a mobile phase flow rate of 0.35 mL/min. The mobile phase contained A (H2O with 0.1% formic acid) and B (methanol). The solution transitions for the gradient system were as follows: at time 0 min 20% B; 1–5 min 20% B → 80% B; 5–6 min 80% B; 6–7 min 80% B → 20% B and 7–8 min 20% B, with a sample injection volume of 5 μL. For MS detection, the instrument operated performed in the positive electrospray ionization mode (ESI +) with multiple reaction monitoring (MRM). Argon and nitrogen were used as the collision and nebulizer gases, respectively. Supplementary Table S1 lists more parameters of MS/MS and the pair. 2.5. Quality analysis and quality control Field and laboratory blanks were collected and analyzed routinely, and the values were found lower than the limit of detection of the required methods for different kinds of samples. To compensate for matrix effects, the linearity of the response (the peak area ratio of the antibiotic to internal standard versus antibiotic concentration) was
Table 1 presents a summary of the fifteen antibiotics found in greenhouse soils, open field soils and manures. Across all studied bases, the studied antibiotics were detected in 100% of the greenhouse soils, and total concentrations ranged from 28 μg/kg to 1051 μg/kg, indicating that extensive antibiotic pollution has occurred in the GVP bases. Thirteen antibiotics, including five sulfonamides (SDZ, SMZ, SM, SDM and SMX), three tetracyclines (TC, OTC and CTC), four quinolones (NFX, CIP, ENR and LOM) and one macrolides (RTM) were detectable. Concentrations of total ∑SAs, ∑TCs, ∑QNs and ∑MLs ranged from not detectable (ND) to 13 μg/kg, from 6.1 to 430 μg/kg, from ND to 649 μg/kg and from ND to 5.7 μg/kg, respectively. Significant differences in the spatial distribution of antibiotics were observed in the greenhouse soil samples among the 11 GVP bases (p b 0.01). The highest total antibiotic concentrations were found in Base SY4, with a mean concentration of 432 μg/kg, while Base SY3 had the lowest level of detected antibiotics, with a mean concentration of 78 μg/kg (Fig. 2). This variation in the distribution of antibiotics may have been caused by numerous factors, such as the amounts of fertilizers applied and application methods, density of cultivation, soil properties, or the cultivation age of a greenhouse (Hu et al., 2010; Li et al., 2013; Wu et al., 2014).
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Table 1 Concentrations of antibiotics in soil and manures (μg/kg) from GVP bases of Beijing, China. Compounds
SDZ SMR SMZ SM SDM SMX ∑SAs TC OTC CTC ∑TCs NFX CIP ENR LOM ∑QNs ETM RTM ∑MLs
Open field soil (N = 11)
Greenhouse soils (N = 56)
Manure (N = 17)
Freq.
Mean
Med.
Max
Freq.
Mean
Med.
Max
Freq.
Mean
Med.
Max
32.1 0 23.2 16.1 8.9 21.4 69.6 96.4 100 83.9 100 64.3 69.6 89.3 57.1 91 0 39.3 39.3
0.11 ND 0.37 0.08 0.48 0.06 1.1 5.2 80 17 102 13 23 47 2.3 86 ND 0.62 0.62
ND ND ND ND ND ND ND 4.2 36 6.8 53 11 13 14 2.7 51 ND ND ND
0.60 ND 1.7 1.2 13 1.2 13 22 423 120 430 69 253 389 10 649 ND 5.7 5.7
9.1 0 27.3 0 0 0 27.3 72.7 100 45.5 100 27.3 27.3 72.7 36.4 100 0 0 0
0.04 ND 0.05 ND ND ND 0.10 2.6 13 3.9 20 4.3 2.9 6.3 1.2 15 ND ND ND
ND ND ND ND ND ND ND 3.1 7.0 ND 13 ND ND 6 ND 7.7 ND ND ND
0.40 ND 0.30 ND ND ND 0.60 5.4 42 14 58 24 21 23 4.4 71 ND ND ND
41.2 0 58.8 11.8 5.9 29.4 88.2 88.2 100 94.1 100 52.9 76.5 94.1 41.2 94.1 0 29.4 29.4
3.9 ND 7.5 0.18 0.05 13 23 413 2082 2504 4976 692 1313 1248 7.5 3220 ND 1.7 1.7
ND ND 1.2 ND ND ND 7.2 29 187 107 531 24 76 89 ND 168 ND ND ND
22 ND 61 1.8 0.90 102 102 3580 23,271 26,218 28,317 4187 9342 8684 38 16,736 ND 6.7 6.7
GVP: greenhouse vegetable production; N: number of samples analyzed; Freq.: frequency (%); Med.: median (μg/kg); Max: maximum (μg/kg); ND: not detected. SDZ: sulfadiazine; SMR: sulfamerazine; SMZ: sulfamethazine; SM: sulfameter; SDM: sulfadimethoxine; SMX: sulfamethoxazole; ∑SAs: total concentrations of six sulfonamides; TC: tetracycline; OTC: oxytetracycline; CTC: chlortetracycline; ∑TCs: total concentrations of three tetracyclines; NFX: norfloxacin; CIP: ciprofloxacin; ENR: enrofloxacin; LOM: lomefloxacin; ∑QNs: total concentrations of four quinolones; ETM: erythromycin; RTM: roxithromycin; ∑MLs: total concentrations of two macrolides.
The concentrations varied greatly among different classes of antibiotics. TCs and FQs were the predominate antibiotics in soils from all the GVP bases and on average contributed 53.9% and 45.2% of the total concentrations (Table 1 and Fig. 2). In contrast, SAs and MLs contributed little to the total antibiotic concentrations (0.58% and 0.33%). This may be explained by the fact that TCs and QNs were more extensively used for treating diseases and as feedstuff additives in the livestock industry (Li et al., 2013), while the SAs and MLs were less commonly used in the concentrated animal feeding operations. In addition, the chemical prosperities of the different antibiotics may have caused variations in the amounts found here. Compared with most SAs and MLs, the TCs and QNs are known to be more strongly absorbed by soil particles or sediment, which can promote their persistence in the environment (Hu et al., 2010; Li et al., 2012b; Shi et al., 2012). In the class of TCs, the detection frequencies were 96.4% (with the mean concentrations of 5.2 μg/kg) for TC, 100% for OTC (mean concentrations of 80 μg/kg) and 83.9% for CTC (mean concentrations of 17 μg/kg). The concentrations of TCs in this study were much higher than those of previous studies. For example, soils that were analyzed from wastewater irrigation
Fig. 2. Average concentrations and distribution of antibiotics in greenhouse soils collected from 11 greenhouse vegetable production bases. SAs: sulfonamides; TCs: tetracyclines; QNs: quinolones; MLs: macrolides.
areas in Beijing and Tianjin had the median concentrations for OTC of 3.9 μg/kg (Chen et al., 2014). These were also higher than concentrations for OTC in farmland soil from Fuyang, Zhejiang Province, China (mean: 23 μg/kg) (Wu et al., 2013). However, the findings of the present study were comparable to those found in vegetable soil of the Pearl River Delta Area, China (mean, 84.8 μg/kg) (Li et al., 2011) and in Fujian, China (median: 68.4 μg/kg) (Huang et al., 2013). The steering committee of the Veterinary International Committee on Harmonization sets 100 μg/kg as the trigger value for soil antibiotics based on the ecotoxic effects of antibiotic compounds on a range of organisms. If the antibiotic concentrations in agricultural soil are higher than the trigger value, some toxic effects on soil communities can be expected (Huang et al., 2013; Karcı and Balcıoğlu, 2009). In the present study, the concentrations of 23% ∑ TCs and 19% of individual compounds in the greenhouse soils were higher than the trigger value, indicating that a potential risk exists for the class of TCs analyzed here. In the class of QNs, the detection frequencies were 64.3% for NFX, 69.6% for CIP, 89.3% for ENR and 57.1% for LOM. The average concentrations of individual compounds decreased in the order of ENR (47 μg/kg) N CIP (23 μg/kg) N NFX (13 μg/kg) N LOM (2.3 μg/kg). The total concentration of these four QNs in greenhouse soils varied from ND to 649 μg/kg and about 22% of the FQ concentrations were N100 μg/kg. It is remarkable that the maximum concentrations of ENR and CIP were up to 389 μg/kg and 253 μg/kg, respectively, because this can pose a probable ecological risk to soils. In Pearl River Delta Area, Li et al. (2011) reported levels of QNs in vegetable soil with mean concentrations of 195.3 μg/kg. Wu et al. (2014) investigated the residues of QNs in soils from the organic vegetable farms in Guangzhou; their concentrations ranged from ND to 42 μg/kg. Shi et al. (2012) investigated the quinolones in soils that had received long-term wastewater irrigation in Tianjin; the total QNs ranged from ND to 274.81 μg/kg (mean, 33.56 μg/kg). Furthermore, high levels of CIP were detected with the concentrations up to 370 μg/kg in soil samples fertilized with manures in Austria (Martínez-Carballo et al., 2007), and ENR was also found in the manure-amended agricultural soil samples of Malaysia ranging from 36 to 378 μg/kg (Ho et al., 2014). The concentrations of QNs in this study were higher than those in Guangzhou and Tianjin, China, and were comparable to those in the soils fertilized with manure in Austria and Malaysia, but were lower than those in farmland of the Pearl River Delta.
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The six SAs were present in 69.6% of the greenhouse soil samples, but all SAs were found at very low levels (ND to 13 μg/kg), most of which were less than 10 μg/kg. This finding agrees with findings from soil of organic vegetable bases of Tianjin, where SMZ concentrations were only in the range of 0.03–0.9 μg/kg (Hu et al., 2010). However, these concentrations were much lower than those in vegetable soil of the Pearl River Delta Area (mean, 114.8 μg/kg) (Li et al., 2011). For the class of MLs, only RTM was detected and only with low concentrations in the range of ND to 5.7 μg/kg (mean, 0.62 μg/kg). These results are also comparable to those in soil from the Tianjin area, where RTM was the most frequently detected ML, and concentrations of RTM were lower than 8.16 μg/kg (Shi et al., 2012). For comparison purposes, we collected the open field soils from the studied local farmlands near each GVP base as controls. Of the antibiotics examined, the detected antibiotic concentrations ranged from ND to 0.6 μg/kg for SAs, 5.1 to 58 μg/kg for TCs, and ND to 71 μg/kg for QNs, while no MLs were detectable in the open field soils (Table 1). These concentrations were significantly lower than those in the greenhouse soils. The average concentrations of TCs and QNs in greenhouse soil were 5.2 and 5.9 times higher, respectively, than those collected from the open fields for the major groups of antibiotics detected in this study. Statistical analysis also revealed that there were significant differences (p b 0.05) in concentrations of TCs and QNs between the GVP soils and the open fields. These results clearly demonstrated that GVP agricultural activities are associated with elevated antibiotic concentrations in soils because of the intense cultivation strategy and higher fertilization frequency. A similar situation has also been found in the case of heavy metal residues. For example, Yang et al. (2013) reported that greenhouse production practices resulted in an increased accumulation of trace metals in soils, particularly Cd, Zn, and Cu because of the large amounts of manure applied in that setting. Yang et al. (2014) also suggested that the levels of total Cd, Cu, Pb and Zn were generally higher in GVP than in open fields. 3.2. Occurrence of antibiotics in manure The use of manure as fertilizer created one of the major sources of antibiotic residues in soil (Ho et al., 2014; Li et al., 2011; Martínez-Carballo et al., 2007). To better understand the antibiotic status in manure applied as fertilizer of the GVP system, 17 manure samples collected from the studied GVP bases were analyzed. The pollution levels of antibiotics in the manures were very high; total concentrations of 15 antibiotics ranged from 43 μg/kg to 29,435 μg/kg (mean, 8221 μg/kg), which were several orders of magnitude higher than those in soils (Table 1). TCs were the most frequently detectable compounds followed by QNs, with mean concentrations of 4976 μg/kg and 3220 μg/kg, respectively. In contrast, much lower detection concentrations were obtained for SAs and MLs, with mean values of 23 μg/kg and 1.7 μg/kg. This distribution pattern of groups of antibiotic agreed with those found in greenhouse soils. Furthermore, Pearson correlation analysis indicated that the mean concentrations of individual antibiotics in greenhouse soils were positively correlated with those in the applied manures (R = 0.76, p b 0.05). These significant correlations also demonstrated that the transfer of antibiotics from manure to soil could be responsible for the occurrence of antibiotics in the greenhouse soils. Vast differences in antibiotic concentrations were observed among the various manure categories. The mean concentrations of total antibiotics in chicken, duck, pig, and cow manure, as well as commercial organic fertilizer were 23,179 μg/kg, 17,321 μg/kg, 15,701 μg/kg, 43 μg/kg and 459 μg/kg, respectively. These residual characteristics were consistent with the fact that antibiotics were widely used in the livestock industry, especially for the poultry and swine industries and a large portion of these compounds had been excreted as feces. In contrast, the commercial organic fertilizer may have been subjected to various innocuous treatment processes, which can accelerate the decomposition of antibiotic residues.
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3.3. Estimation of the antibiotic concentrations that migrated from manure to soil The antibiotic migration from manure to soil is a complex process. Therefore, the concentrations of antibiotics were difficult to calculate in practical, after antibiotics from manure to soil (Hu et al., 2010). For preliminary assessment of the potential environmental risk of these manures on soils, in this work the estimated concentrations of antibiotics transferred from manure to soil were calculated using Formula (1) (refer to the methods of Hu et al. (2010)): EC s ¼
CmM ; As H s ρs
ð1Þ
where ECs is the estimated concentration transferred from manure to soil; Cm is the antibiotic concentration in manure; M is the annual applied manure amount (6000 kg); As is the area where manure was applied (0.067 ha); Hs was the thickness of the ploughed soil layer (20 cm); and ρs is the density of the soil; note that the soil bulk density changes with soil texture, so an arbitrary average value of 1500 kg/m3 was used (Spaepen et al., 1997). The intermediate processes that occur when antibiotics migrate from fertilizer to soil, such as degradation, plant uptake and runoff were not considered. The calculations indicated that the estimated concentrations in soils were in the same order of magnitude with those in the greenhouse samples, indicating that this formula could simply estimate the migration of antibiotics from manure to soil (Fig. 3). Note that about 23.5%, 29.4% and 50% of the estimated soil concentrations for TCs, QNs, and total antibiotics, respectively, exceeded the trigger values of 100 μg/kg; this suggests that these antibiotics pose a potential ecological risk to the associated organisms. These data are instructive and can be used to determine how organic fertilizers should be used rationally in GVP bases. The findings of this study are also applicable to measures that must be taken to control the use of fertilizers in GVP bases, especially as they relate to the use of the animal feces that may introduce a great amount of antibiotic contaminants into soils. 3.4. Potential environmental risk assessment High levels of antibiotics were frequently found in the greenhouse soil samples, especially for the TCs and QNs. The adverse ecological impacts of these contaminants need to be identified. In the present study, the potential environmental risks of TCs and QNs in greenhouse soil samples were assessed on the basis of the risk quotient (RQ) values (European Commission, 2003), which were calculated through the measured environmental concentration (MEC) divided by the “predicted no
Fig. 3. Estimated concentrations (ECs) of antibiotics in soils transferred from applied manures. SAs: sulfonamides; TCs: tetracyclines; QNs: quinolones; MLs: macrolides.
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effect concentration” (PNEC). PNEC values are derived from data related to the acute toxicity of antibiotics or short-term data divided by an assessment factor (Chen and Zhou, 2014; Zhang et al., 2013). The value of assessment factor is 1000 if the risk assessment is conducted by an acute toxicity test or 100 if based on a chronic toxicity test (Li et al., 2013). For TCs, the values of PNEC were obtained from available literature (Thiele-Bruhn and Beck, 2005; Vaclavik et al., 2004). Only limited information is available on the risk that QNs pose to soil. In our study, the PNEC of QNs in soil was obtained based on an equilibrium partitioning method reported by Wu et al. (2014), in which PNEC values in soils (PNECsoil) were derived from PNEC values in water (PNECwater) by the formula: PNECsoil = PNECwater × Kd, where Kd is the soil–water partition coefficient. The predicted no-effect concentrations of the antibiotics were listed in the in Supplementary Table S2. To better elucidate the risk levels created by antibiotics in farm soil, the RQ values were classified into three levels of risk: 0.01–0.1, low; 0.1–1, medium; and N 1 high risk (Li et al., 2012b; Park and Choi, 2008; Xu et al., 2013). OTC, CTC, NFX, CIP and ENR could pose a high risk to soil organisms and the proportions were 39.3%, 10.7%, 10.7%, 17.9%, and 41.1%, respectively, in greenhouse soils (Fig. 4). For the TC, all the RQs were lower than 0.1; 92% of the samples posed a low risk. As to the LOM, only one sample posed a medium risk and 55% of the samples posed a low risk to the soil organisms. Based on these calculations, it should be noted that several antibiotics are likely to cause adverse toxic effects to soil microorganisms in the GVP system, and the risks of these pollutants in soils should be given special attention. This risk assessment approach can provide useful guidance for antibiotic control in GVP systems. However, the risk assessment in this study has not considered the mixed toxicity of different antibiotics and other coexisting pollutants, which can be more significant than individual effects (Chen and Zhou, 2014). 4. Conclusions The present study investigated the levels of residue antibiotics in soil and manure samples collected from the GVP bases of Beijing. Soil from greenhouses had significantly higher antibiotic concentrations than soils from open fields. TCs and QNs were the predominate antibiotics found in greenhouse soil and contributed more than 99% of the total concentrations. The contents of these drugs in some samples have exceeded the ecotoxic effect trigger value (100 μg/kg) set by the Steering Committee of the Veterinary International Committee on Harmonization. Composition of antibiotics in greenhouse soils was consistent to those in the manure applied, suggesting that fertilization with manure might be the most important input source of antibiotics in the
Fig. 4. Calculated risk quotients (RQ) for the tetracyclines and quinolones detected in the 56 greenhouse soil samples. TC: tetracycline; OTC: oxytetracycline; CTC: chlortetracycline; NFX: norfloxacin; CIP: ciprofloxacin; ENR: enrofloxacin; LOM: lomefloxacin.
GVP system. Risk assessment based on the RQ values revealed that several antibiotics presented high ecological risk to soil microorganisms. These results suggest that regular monitoring of antibiotic pollutants in GVP bases is needed, and some control measures are urgently required to prevent the associated risks to humans from the excessive use of antibiotics. Acknowledgments This research was funded by the National Natural Science Foundation of China (No. 41401540), the Youth Scientific Funds of Beijing Academy of Agriculture and Forestry Sciences (No. QNJJ201531) and the Innovation and the Capacity-building Projects of Beijing Academy of Agriculture and Forestry Sciences (No. KJCX20140302). Appendix A. Supplementary material Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2015.03.070. References Ashbolt, N.J., Amezquita, A., Backhaus, T., Borriello, P., Brandt, K.K., Collignon, P., et al., 2013. 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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment Cheng Li a,b, Jiayi Chen a,b, Jihua Wang a,b, Zhihong Ma a,b, Ping Han a,b, Yunxia Luan a,b, Anxiang Lu a,b,c,⁎ a b c
Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China Risk Assessment Lab for Agro-products (Beijing), Ministry of Agriculture, Beijing 100097, China Collaborative Innovation Center for Key Technology of Smart Irrigation District in Hubei, Yichang 443002, China
H I G H L I G H T S • • • •
High levels of antibiotics were found in soils from vegetable greenhouses. Concentrations of antibiotics were higher in greenhouse soils than in open fields. Manure application was the primary source of antibiotics in greenhouse soils. Several antibiotics in greenhouse soils pose a high ecological risk.
a r t i c l e
i n f o
Article history: Received 15 November 2014 Received in revised form 18 March 2015 Accepted 18 March 2015 Available online xxxx Editor: Kevin V. Thomas Keywords: Antibiotics Greenhouse vegetable production base Manure Risk assessment Soil
a b s t r a c t The occurrence of 15 antibiotics in soil and manure samples from 11 large-scale greenhouse vegetable production (GVP) bases in Beijing, China was investigated. Results showed that the greenhouse soils were ubiquitously contaminated with antibiotics, and that antibiotic concentrations were significantly higher in greenhouses than in open field soils. The mean concentrations of four antibiotic classes decreased in the following order: tetracyclines (102 μg/kg) N quinolones (86 μg/kg) N sulfonamides (1.1 μg/kg) N macrolides (0.62 μg/kg). This investigation also indicated that fertilization with manure and especially animal feces might be the primary source of antibiotics. A risk assessment based on the calculated risk quotients (RQs) demonstrated that oxytetracycline, chlortetracycline, norfloxacin, ciprofloxacin and enrofloxacin could pose a high risk to soil organisms. These results suggested that the ecological effects of antibiotic contamination in GVP bases and their potential adverse risks on human health need to be given special attention. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Antibiotics have been widely used to treat bacterial infections and promote the growth of livestock animals for several decades (Thiele-Bruhn, 2003). China is the world's largest producer and consumer of antibiotics. An estimated 210,000 t of antibiotics is produced every year, of which 48% are used in the agricultural and livestock industries. Most animals cannot completely metabolize the antibiotics they receive, and the animals excrete them into the environment as intact bioactive substances or metabolites. Researchers have reported that antibiotic residues in the environment can affect terrestrial organisms, ⁎ Corresponding author at: Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China. E-mail address: [email protected] (A. Lu).
http://dx.doi.org/10.1016/j.scitotenv.2015.03.070 0048-9697/© 2015 Elsevier B.V. All rights reserved.
alter microbial activity and community composition in soil, and promote the development of antibiotic resistant genes (ARGs), which represent a risk to human and animal health (Ashbolt et al., 2013). Previous studies have frequently reported high concentrations of antibiotics in animal manure. Martínez-Carballo et al. (2007) reported that the concentrations of chlortetracycline, oxytetracycline and tetracycline in 30 pig manure of Austria were up to 46 mg/kg, 29 mg/kg and 23 mg/kg, respectively, whereas the concentrations as high as 225 and 1420 mg/kg of norfloxacin and enrofloxacin were found in chicken manures from China (Zhao et al., 2010). Current environmental legislation fails to cover these antibiotics; the antibiotics could lead to undesirable effects on ecosystems (Li et al., 2012a). Furthermore, the high concentrations of antibiotics in manure can be associated with an increased presence of resistant bacteria in environment, which is a major public health concern, due to the increased occurrence
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of associated clinical infections (Ashbolt et al., 2013; Luo et al., 2010). Most livestock manure is directly or indirectly applied on agricultural land as fertilizers, which constitute the main source of the dissemination of these chemicals in soil environments (Zhou et al., 2013). Hamscher et al. (2002) detected concentrations of tetracyclines up to 0.3 g/kg in an agricultural field fertilized with liquid manure. In addition, Li et al. (2011) found high antibiotic concentrations in vegetable farmlands affiliated with livestock farms and detected maximum concentrations of tetracyclines, sulfonamides, and quinolones of 242.6, 321.4, and 1537.4 μg/kg, respectively. Market demands and economic incentives have resulted in intensified greenhouse vegetable production (GVP), particularly in developing countries; this intensive form of agriculture has expanded rapidly worldwide. China accounts for more than 90% of the global GVP operations, where GVP area had expanded to almost 4.67 million ha by 2010 (Chang et al., 2013). GVP can result in improved vegetable yields by extending the growing seasons and intensifying agriculture compared with conventional vegetable production. However, heavy fertilization strongly increases production and is always adopted under GVP conditions to achieve high vegetable production. In addition, growers of organic vegetables primarily use manure in GVP systems. Most GVP involves large and repeated amounts of animal manure application and probably increases the risk of antibiotic contamination, which could also have adverse effects to human health due to the consumption of these vegetables contaminated with antibiotics and antibioticresistant bacteria. Presently, no regulations address the presence of antibiotics in soil as well as in the manure applied to certified vegetable production systems. This has created an urgent problem related to the contamination of antibiotics in GVP systems. Beijing, the capital of China, supports a resident population of about 20 million. In recent years, large-scale GVP bases have been widely developed on the outskirts of Beijing, to a total area of 1531.49 ha by 2011. With China's emphasis on food security and sustainability, an increasing demand exists for organic vegetables from GVP bases, where most fertilizer comes from manure. This cultivation technique may elevate the contamination of foods by antibiotics relative to conventional farming methods. To date, limited information is available on the occurrence of antibiotics in the GVP bases of Beijing. Therefore, the objective of this study was to improve our understanding of the general status of antibiotic pollution in the GVP bases. The data collected can also be used to evaluate the ecological risks created by antibiotics in GVP systems. 2. Materials and methods 2.1. Sampling sites and sample collection Eleven large-scale GVP bases were selected in the suburbs of Beijing based on geographic location, cultivated area, history and environment. Bases are defined as large-scale areas with numerous greenhouses; sample sites are defined as the various sample points within those bases. The sampling bases were distributed across four districts, including Changping, Daxing, Shunyi and Yanqing, which covered a relatively large area. These vegetable bases were registered with the local Department of Agriculture, and the vegetable products from these bases were transported throughout of Beijing area. Therefore, the selected bases could reflect the whole situation of GVP bases in Beijing. The GVP bases were generally operated in the form of enterprises and only manure was applied in the process of production. And, groundwater was used for irrigation. Sampling work occurred in May–July 2013 (Fig. 1). Fifty-six vegetable greenhouses were selected from these GVP bases, with estimated areas ranging from 20 to 66.7 ha. Three to nine samples were evenly sampled in each GVP base, the exact number depending on the total area of each base. Fig. 1(B) shows Site YQ1 as an example showing the layout of plots. In addition, soils from the open fields near each GVP base were also collected as controls. These open fields were applied to plant seasonal vegetables and cultivated by local farmers. A
combination of chemical fertilizers and organic fertilizers was generally used in the open fields. Topsoil was sampled at a depth of 0–20 cm using a stainless-steel auger. Five sampling sites were distributed in an S-shaped pattern in each greenhouse; these were mixed as a single sample. Furthermore, manure samples (N = 17) that were actively being applied in the studied GVP bases, were also gathered for potential source analysis. The categories of the manure include chicken, duck, pig, and cow manure as well as commercial organic fertilizers which were produced through microbial fermentation using the mixture of livestock dungs and some farm waste such as corn stalks or wheat shafts as raw materials. Soil and manure samples were air dried at ambient temperature, ground and homogenized by sieving through a stainless steel sieve (60-mesh) after removing stones and residual roots; samples were then sealed in brown glass bottles and stored at −20 °C prior to analysis. 2.2. Chemicals and standards The antibiotics analyzed here were selected based mainly on the extent of their use in animal production in China. These drugs belonged to four different antibacterial families. Six sulfonamides (SAs) included sulfamerazine (SMR), sulfamethazine (SMZ), sulfadiazine (SDZ), sulfameter (SM), sulfadimethoxine (SDM), and sulfamethoxazole (SMX). Three tetracyclines (TCs) included tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC). Four quinolones (QNs) included norfloxacin (NFX), ciprofloxacin (CIP), enrofloxacin (ENR) and lomefloxacin (LOM). Two macrolides (MLs) included erythromycin (ETM) and roxithromycin (RTM). Standards for SDM, SMX, TC, CTC, LOM and RTM with purities of N 98% were obtained from SigmaAldrich (St. Louis, MO, USA), while the other antibiotic standards, with purities of N98% were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Simeton (J & K Chemical Ltd., USA) was used as the internal standard to enhance the analytical precision. A stock solution (1.0 mg/mL) of Simeton was prepared in methanol solution, stored in at − 20 °C prior to use. Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (New Jersey, USA). Formic acid (98%) was purchased from Fluka (Bucks, Switzerland). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). Disodium ethylenediamine tetraacetate, citric acid and sodium citrate of analytical grade were obtained from Yaohua Chemical Reagent Factory (Tianjin, China). Citric acid buffer (pH 4) was prepared according to the procedure described by Zhou et al. (2011). Strong anion exchange (SAX) cartridges (6 mL, 500 mg) were provided by Agilent Technologies (Wilmington, DE, USA) and Oasis HLB cartridges (6 mL, 500 mg) were purchased from Waters (Milford, MA. USA). All other reagents were of analytical reagent grade. 2.3. Sample extraction and clean-up One half-gram of each freeze-dried manure sample and 2 g of each soil sample were extracted with 10 mL acetonitrile and 10 mL citric acid buffer (pH = 4) in a 50 mL polypropylene centrifuge tube. The sample was vortexed for 1 min and treated ultrasonically for 15 min, followed by centrifugation. This extraction process was repeated twice. The extract was combined into a round-bottom flask, concentrated with a rotary evaporator at 50 °C to remove the organic solvent, and diluted to 100 mL with ultrapure water to make sure the organic solvent in solution had a concentration of less than 5%. SAX and HLB cartridges were set up in tandem for cleanup of extracts. Prior to the SPE cleanup, 0.2 g of disodium ethylenediamine tetraacetate was added into each aqueous extract to chelate with metal cations. The cartridges were pre-treated with 10 mL methanol and 10 mL ultrapure water; the diluted extract was passed through the cartridges at a loading rate of about 5 mL/min. After loading the entire amount of extract, the SAX cartridge was removed and the HLB
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Fig. 1. Map of the sampling bases in the 11 greenhouse vegetable production (GVP) bases of Beijing. Two inset maps provide A) the study area within Beijing, and B) a map showing the layout of sampling locations in Site YQ1 as an example. GVP base names include CP1, CP2, CP3, CP4, CP5, DX1, SY1, SY2, SY3, SY4, SY5, and YQ1 (CP = Changping; DX = Daxing; SY = Shunyi; YQ = Yanqing).
cartridge was rinsed with 10 mL ultrapure water, air dried for 10 min, and then eluted with 10 mL methanol. The eluate was dried under a gentle nitrogen stream at 50 °C. Simeton (10 μL, 10 μg/mL) was spiked as an internal standard and methanol/aqueous solution (1:1 v/v) was used to bring the final purified extract volume up to 1 mL. After filtration through a 0.22 μm membrane to remove particles, the final extract was transferred to a 2 mL amber vial and stored at − 18 °C until ultrahigh performance liquid chromatography tandem mass spectrometric (UPLC–MS/MS) analysis could be performed.
determined for matrix-matched calibration curves using five different concentrations (5–200 μg/kg) for both soil and manure. The correlation coefficients (R) were all N0.99. Analyte recoveries were in the range of 80.2–104.6% for soil and 70.5–128.2% for manure samples, with the relative standard deviation (RSD) b 10%. The limits of detection (S/N = 3) were 0.23–5 μg/kg for soil and 0.35–13 μg/kg for manure samples. Statistical analysis was performed using SPSS (IBM SPSS Statistics 20). One-way analysis of variance (ANOVA) was used for statistical comparisons and the Pearson coefficient was used for correlation analysis. 3. Results and discussion
2.4. Instrumental analysis 3.1. Occurrence and distribution of antibiotics in greenhouse soils The UPLC–MS/MS system consisted of an ACQUITY UPLC system (Waters) and a Xevo TQ triple quadrupole mass spectrometer (Waters). Separation was performed using an ACQUITY UPLC BEH C18 column (1.7 μm; 2.1 × 50 mm), operated at 35 °C, with a mobile phase flow rate of 0.35 mL/min. The mobile phase contained A (H2O with 0.1% formic acid) and B (methanol). The solution transitions for the gradient system were as follows: at time 0 min 20% B; 1–5 min 20% B → 80% B; 5–6 min 80% B; 6–7 min 80% B → 20% B and 7–8 min 20% B, with a sample injection volume of 5 μL. For MS detection, the instrument operated performed in the positive electrospray ionization mode (ESI +) with multiple reaction monitoring (MRM). Argon and nitrogen were used as the collision and nebulizer gases, respectively. Supplementary Table S1 lists more parameters of MS/MS and the pair. 2.5. Quality analysis and quality control Field and laboratory blanks were collected and analyzed routinely, and the values were found lower than the limit of detection of the required methods for different kinds of samples. To compensate for matrix effects, the linearity of the response (the peak area ratio of the antibiotic to internal standard versus antibiotic concentration) was
Table 1 presents a summary of the fifteen antibiotics found in greenhouse soils, open field soils and manures. Across all studied bases, the studied antibiotics were detected in 100% of the greenhouse soils, and total concentrations ranged from 28 μg/kg to 1051 μg/kg, indicating that extensive antibiotic pollution has occurred in the GVP bases. Thirteen antibiotics, including five sulfonamides (SDZ, SMZ, SM, SDM and SMX), three tetracyclines (TC, OTC and CTC), four quinolones (NFX, CIP, ENR and LOM) and one macrolides (RTM) were detectable. Concentrations of total ∑SAs, ∑TCs, ∑QNs and ∑MLs ranged from not detectable (ND) to 13 μg/kg, from 6.1 to 430 μg/kg, from ND to 649 μg/kg and from ND to 5.7 μg/kg, respectively. Significant differences in the spatial distribution of antibiotics were observed in the greenhouse soil samples among the 11 GVP bases (p b 0.01). The highest total antibiotic concentrations were found in Base SY4, with a mean concentration of 432 μg/kg, while Base SY3 had the lowest level of detected antibiotics, with a mean concentration of 78 μg/kg (Fig. 2). This variation in the distribution of antibiotics may have been caused by numerous factors, such as the amounts of fertilizers applied and application methods, density of cultivation, soil properties, or the cultivation age of a greenhouse (Hu et al., 2010; Li et al., 2013; Wu et al., 2014).
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Table 1 Concentrations of antibiotics in soil and manures (μg/kg) from GVP bases of Beijing, China. Compounds
SDZ SMR SMZ SM SDM SMX ∑SAs TC OTC CTC ∑TCs NFX CIP ENR LOM ∑QNs ETM RTM ∑MLs
Open field soil (N = 11)
Greenhouse soils (N = 56)
Manure (N = 17)
Freq.
Mean
Med.
Max
Freq.
Mean
Med.
Max
Freq.
Mean
Med.
Max
32.1 0 23.2 16.1 8.9 21.4 69.6 96.4 100 83.9 100 64.3 69.6 89.3 57.1 91 0 39.3 39.3
0.11 ND 0.37 0.08 0.48 0.06 1.1 5.2 80 17 102 13 23 47 2.3 86 ND 0.62 0.62
ND ND ND ND ND ND ND 4.2 36 6.8 53 11 13 14 2.7 51 ND ND ND
0.60 ND 1.7 1.2 13 1.2 13 22 423 120 430 69 253 389 10 649 ND 5.7 5.7
9.1 0 27.3 0 0 0 27.3 72.7 100 45.5 100 27.3 27.3 72.7 36.4 100 0 0 0
0.04 ND 0.05 ND ND ND 0.10 2.6 13 3.9 20 4.3 2.9 6.3 1.2 15 ND ND ND
ND ND ND ND ND ND ND 3.1 7.0 ND 13 ND ND 6 ND 7.7 ND ND ND
0.40 ND 0.30 ND ND ND 0.60 5.4 42 14 58 24 21 23 4.4 71 ND ND ND
41.2 0 58.8 11.8 5.9 29.4 88.2 88.2 100 94.1 100 52.9 76.5 94.1 41.2 94.1 0 29.4 29.4
3.9 ND 7.5 0.18 0.05 13 23 413 2082 2504 4976 692 1313 1248 7.5 3220 ND 1.7 1.7
ND ND 1.2 ND ND ND 7.2 29 187 107 531 24 76 89 ND 168 ND ND ND
22 ND 61 1.8 0.90 102 102 3580 23,271 26,218 28,317 4187 9342 8684 38 16,736 ND 6.7 6.7
GVP: greenhouse vegetable production; N: number of samples analyzed; Freq.: frequency (%); Med.: median (μg/kg); Max: maximum (μg/kg); ND: not detected. SDZ: sulfadiazine; SMR: sulfamerazine; SMZ: sulfamethazine; SM: sulfameter; SDM: sulfadimethoxine; SMX: sulfamethoxazole; ∑SAs: total concentrations of six sulfonamides; TC: tetracycline; OTC: oxytetracycline; CTC: chlortetracycline; ∑TCs: total concentrations of three tetracyclines; NFX: norfloxacin; CIP: ciprofloxacin; ENR: enrofloxacin; LOM: lomefloxacin; ∑QNs: total concentrations of four quinolones; ETM: erythromycin; RTM: roxithromycin; ∑MLs: total concentrations of two macrolides.
The concentrations varied greatly among different classes of antibiotics. TCs and FQs were the predominate antibiotics in soils from all the GVP bases and on average contributed 53.9% and 45.2% of the total concentrations (Table 1 and Fig. 2). In contrast, SAs and MLs contributed little to the total antibiotic concentrations (0.58% and 0.33%). This may be explained by the fact that TCs and QNs were more extensively used for treating diseases and as feedstuff additives in the livestock industry (Li et al., 2013), while the SAs and MLs were less commonly used in the concentrated animal feeding operations. In addition, the chemical prosperities of the different antibiotics may have caused variations in the amounts found here. Compared with most SAs and MLs, the TCs and QNs are known to be more strongly absorbed by soil particles or sediment, which can promote their persistence in the environment (Hu et al., 2010; Li et al., 2012b; Shi et al., 2012). In the class of TCs, the detection frequencies were 96.4% (with the mean concentrations of 5.2 μg/kg) for TC, 100% for OTC (mean concentrations of 80 μg/kg) and 83.9% for CTC (mean concentrations of 17 μg/kg). The concentrations of TCs in this study were much higher than those of previous studies. For example, soils that were analyzed from wastewater irrigation
Fig. 2. Average concentrations and distribution of antibiotics in greenhouse soils collected from 11 greenhouse vegetable production bases. SAs: sulfonamides; TCs: tetracyclines; QNs: quinolones; MLs: macrolides.
areas in Beijing and Tianjin had the median concentrations for OTC of 3.9 μg/kg (Chen et al., 2014). These were also higher than concentrations for OTC in farmland soil from Fuyang, Zhejiang Province, China (mean: 23 μg/kg) (Wu et al., 2013). However, the findings of the present study were comparable to those found in vegetable soil of the Pearl River Delta Area, China (mean, 84.8 μg/kg) (Li et al., 2011) and in Fujian, China (median: 68.4 μg/kg) (Huang et al., 2013). The steering committee of the Veterinary International Committee on Harmonization sets 100 μg/kg as the trigger value for soil antibiotics based on the ecotoxic effects of antibiotic compounds on a range of organisms. If the antibiotic concentrations in agricultural soil are higher than the trigger value, some toxic effects on soil communities can be expected (Huang et al., 2013; Karcı and Balcıoğlu, 2009). In the present study, the concentrations of 23% ∑ TCs and 19% of individual compounds in the greenhouse soils were higher than the trigger value, indicating that a potential risk exists for the class of TCs analyzed here. In the class of QNs, the detection frequencies were 64.3% for NFX, 69.6% for CIP, 89.3% for ENR and 57.1% for LOM. The average concentrations of individual compounds decreased in the order of ENR (47 μg/kg) N CIP (23 μg/kg) N NFX (13 μg/kg) N LOM (2.3 μg/kg). The total concentration of these four QNs in greenhouse soils varied from ND to 649 μg/kg and about 22% of the FQ concentrations were N100 μg/kg. It is remarkable that the maximum concentrations of ENR and CIP were up to 389 μg/kg and 253 μg/kg, respectively, because this can pose a probable ecological risk to soils. In Pearl River Delta Area, Li et al. (2011) reported levels of QNs in vegetable soil with mean concentrations of 195.3 μg/kg. Wu et al. (2014) investigated the residues of QNs in soils from the organic vegetable farms in Guangzhou; their concentrations ranged from ND to 42 μg/kg. Shi et al. (2012) investigated the quinolones in soils that had received long-term wastewater irrigation in Tianjin; the total QNs ranged from ND to 274.81 μg/kg (mean, 33.56 μg/kg). Furthermore, high levels of CIP were detected with the concentrations up to 370 μg/kg in soil samples fertilized with manures in Austria (Martínez-Carballo et al., 2007), and ENR was also found in the manure-amended agricultural soil samples of Malaysia ranging from 36 to 378 μg/kg (Ho et al., 2014). The concentrations of QNs in this study were higher than those in Guangzhou and Tianjin, China, and were comparable to those in the soils fertilized with manure in Austria and Malaysia, but were lower than those in farmland of the Pearl River Delta.
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The six SAs were present in 69.6% of the greenhouse soil samples, but all SAs were found at very low levels (ND to 13 μg/kg), most of which were less than 10 μg/kg. This finding agrees with findings from soil of organic vegetable bases of Tianjin, where SMZ concentrations were only in the range of 0.03–0.9 μg/kg (Hu et al., 2010). However, these concentrations were much lower than those in vegetable soil of the Pearl River Delta Area (mean, 114.8 μg/kg) (Li et al., 2011). For the class of MLs, only RTM was detected and only with low concentrations in the range of ND to 5.7 μg/kg (mean, 0.62 μg/kg). These results are also comparable to those in soil from the Tianjin area, where RTM was the most frequently detected ML, and concentrations of RTM were lower than 8.16 μg/kg (Shi et al., 2012). For comparison purposes, we collected the open field soils from the studied local farmlands near each GVP base as controls. Of the antibiotics examined, the detected antibiotic concentrations ranged from ND to 0.6 μg/kg for SAs, 5.1 to 58 μg/kg for TCs, and ND to 71 μg/kg for QNs, while no MLs were detectable in the open field soils (Table 1). These concentrations were significantly lower than those in the greenhouse soils. The average concentrations of TCs and QNs in greenhouse soil were 5.2 and 5.9 times higher, respectively, than those collected from the open fields for the major groups of antibiotics detected in this study. Statistical analysis also revealed that there were significant differences (p b 0.05) in concentrations of TCs and QNs between the GVP soils and the open fields. These results clearly demonstrated that GVP agricultural activities are associated with elevated antibiotic concentrations in soils because of the intense cultivation strategy and higher fertilization frequency. A similar situation has also been found in the case of heavy metal residues. For example, Yang et al. (2013) reported that greenhouse production practices resulted in an increased accumulation of trace metals in soils, particularly Cd, Zn, and Cu because of the large amounts of manure applied in that setting. Yang et al. (2014) also suggested that the levels of total Cd, Cu, Pb and Zn were generally higher in GVP than in open fields. 3.2. Occurrence of antibiotics in manure The use of manure as fertilizer created one of the major sources of antibiotic residues in soil (Ho et al., 2014; Li et al., 2011; Martínez-Carballo et al., 2007). To better understand the antibiotic status in manure applied as fertilizer of the GVP system, 17 manure samples collected from the studied GVP bases were analyzed. The pollution levels of antibiotics in the manures were very high; total concentrations of 15 antibiotics ranged from 43 μg/kg to 29,435 μg/kg (mean, 8221 μg/kg), which were several orders of magnitude higher than those in soils (Table 1). TCs were the most frequently detectable compounds followed by QNs, with mean concentrations of 4976 μg/kg and 3220 μg/kg, respectively. In contrast, much lower detection concentrations were obtained for SAs and MLs, with mean values of 23 μg/kg and 1.7 μg/kg. This distribution pattern of groups of antibiotic agreed with those found in greenhouse soils. Furthermore, Pearson correlation analysis indicated that the mean concentrations of individual antibiotics in greenhouse soils were positively correlated with those in the applied manures (R = 0.76, p b 0.05). These significant correlations also demonstrated that the transfer of antibiotics from manure to soil could be responsible for the occurrence of antibiotics in the greenhouse soils. Vast differences in antibiotic concentrations were observed among the various manure categories. The mean concentrations of total antibiotics in chicken, duck, pig, and cow manure, as well as commercial organic fertilizer were 23,179 μg/kg, 17,321 μg/kg, 15,701 μg/kg, 43 μg/kg and 459 μg/kg, respectively. These residual characteristics were consistent with the fact that antibiotics were widely used in the livestock industry, especially for the poultry and swine industries and a large portion of these compounds had been excreted as feces. In contrast, the commercial organic fertilizer may have been subjected to various innocuous treatment processes, which can accelerate the decomposition of antibiotic residues.
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3.3. Estimation of the antibiotic concentrations that migrated from manure to soil The antibiotic migration from manure to soil is a complex process. Therefore, the concentrations of antibiotics were difficult to calculate in practical, after antibiotics from manure to soil (Hu et al., 2010). For preliminary assessment of the potential environmental risk of these manures on soils, in this work the estimated concentrations of antibiotics transferred from manure to soil were calculated using Formula (1) (refer to the methods of Hu et al. (2010)): EC s ¼
CmM ; As H s ρs
ð1Þ
where ECs is the estimated concentration transferred from manure to soil; Cm is the antibiotic concentration in manure; M is the annual applied manure amount (6000 kg); As is the area where manure was applied (0.067 ha); Hs was the thickness of the ploughed soil layer (20 cm); and ρs is the density of the soil; note that the soil bulk density changes with soil texture, so an arbitrary average value of 1500 kg/m3 was used (Spaepen et al., 1997). The intermediate processes that occur when antibiotics migrate from fertilizer to soil, such as degradation, plant uptake and runoff were not considered. The calculations indicated that the estimated concentrations in soils were in the same order of magnitude with those in the greenhouse samples, indicating that this formula could simply estimate the migration of antibiotics from manure to soil (Fig. 3). Note that about 23.5%, 29.4% and 50% of the estimated soil concentrations for TCs, QNs, and total antibiotics, respectively, exceeded the trigger values of 100 μg/kg; this suggests that these antibiotics pose a potential ecological risk to the associated organisms. These data are instructive and can be used to determine how organic fertilizers should be used rationally in GVP bases. The findings of this study are also applicable to measures that must be taken to control the use of fertilizers in GVP bases, especially as they relate to the use of the animal feces that may introduce a great amount of antibiotic contaminants into soils. 3.4. Potential environmental risk assessment High levels of antibiotics were frequently found in the greenhouse soil samples, especially for the TCs and QNs. The adverse ecological impacts of these contaminants need to be identified. In the present study, the potential environmental risks of TCs and QNs in greenhouse soil samples were assessed on the basis of the risk quotient (RQ) values (European Commission, 2003), which were calculated through the measured environmental concentration (MEC) divided by the “predicted no
Fig. 3. Estimated concentrations (ECs) of antibiotics in soils transferred from applied manures. SAs: sulfonamides; TCs: tetracyclines; QNs: quinolones; MLs: macrolides.
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effect concentration” (PNEC). PNEC values are derived from data related to the acute toxicity of antibiotics or short-term data divided by an assessment factor (Chen and Zhou, 2014; Zhang et al., 2013). The value of assessment factor is 1000 if the risk assessment is conducted by an acute toxicity test or 100 if based on a chronic toxicity test (Li et al., 2013). For TCs, the values of PNEC were obtained from available literature (Thiele-Bruhn and Beck, 2005; Vaclavik et al., 2004). Only limited information is available on the risk that QNs pose to soil. In our study, the PNEC of QNs in soil was obtained based on an equilibrium partitioning method reported by Wu et al. (2014), in which PNEC values in soils (PNECsoil) were derived from PNEC values in water (PNECwater) by the formula: PNECsoil = PNECwater × Kd, where Kd is the soil–water partition coefficient. The predicted no-effect concentrations of the antibiotics were listed in the in Supplementary Table S2. To better elucidate the risk levels created by antibiotics in farm soil, the RQ values were classified into three levels of risk: 0.01–0.1, low; 0.1–1, medium; and N 1 high risk (Li et al., 2012b; Park and Choi, 2008; Xu et al., 2013). OTC, CTC, NFX, CIP and ENR could pose a high risk to soil organisms and the proportions were 39.3%, 10.7%, 10.7%, 17.9%, and 41.1%, respectively, in greenhouse soils (Fig. 4). For the TC, all the RQs were lower than 0.1; 92% of the samples posed a low risk. As to the LOM, only one sample posed a medium risk and 55% of the samples posed a low risk to the soil organisms. Based on these calculations, it should be noted that several antibiotics are likely to cause adverse toxic effects to soil microorganisms in the GVP system, and the risks of these pollutants in soils should be given special attention. This risk assessment approach can provide useful guidance for antibiotic control in GVP systems. However, the risk assessment in this study has not considered the mixed toxicity of different antibiotics and other coexisting pollutants, which can be more significant than individual effects (Chen and Zhou, 2014). 4. Conclusions The present study investigated the levels of residue antibiotics in soil and manure samples collected from the GVP bases of Beijing. Soil from greenhouses had significantly higher antibiotic concentrations than soils from open fields. TCs and QNs were the predominate antibiotics found in greenhouse soil and contributed more than 99% of the total concentrations. The contents of these drugs in some samples have exceeded the ecotoxic effect trigger value (100 μg/kg) set by the Steering Committee of the Veterinary International Committee on Harmonization. Composition of antibiotics in greenhouse soils was consistent to those in the manure applied, suggesting that fertilization with manure might be the most important input source of antibiotics in the
Fig. 4. Calculated risk quotients (RQ) for the tetracyclines and quinolones detected in the 56 greenhouse soil samples. TC: tetracycline; OTC: oxytetracycline; CTC: chlortetracycline; NFX: norfloxacin; CIP: ciprofloxacin; ENR: enrofloxacin; LOM: lomefloxacin.
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