Distribution and Accumulative Pattern of Tetracyclines and

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Distribution and Accumulative Pattern of Tetracyclines and Sulfonamides in Edible Vegetables of Cucumber, Tomato, and Lettuce Mohamed Bedair M. Ahmed,†,§ Anushka Upamali Rajapaksha,† Jung Eun Lim,† Ngoc Thang Vu,‡ Il Seop Kim,‡ Ho Min Kang,‡ Sang Soo Lee,*,† and Yong Sik Ok*,† †

Department of Biological Environment and ‡Department of Horticulture Science, Kangwon National University, Chuncheon 200-701, Korea § Department of Food Toxicology and Contaminants, National Research Centre, Cairo, Egypt S Supporting Information *

ABSTRACT: Veterinary antibiotics can be released to environment by the animals’ excretions, which thereby poses human health and ecological risks. Six antibiotics (tetracycline, oxytetracycline, chlortetracycline, sulfamethazine, sulfamethoxazole, and sulfadimethoxine) at three concentrations (5, 10, and 20 mg kg−1 soil) were employed in pots filled with a loamy sand upland soil. Three types of vegetable seedlings, including cucumber (Cucumis sativus), cherry tomato (Solanum lycopersicum), and lettuce (Lactuca sativa), were also cultivated during 45 d in the greenhouse. All antibiotics taken up by tested plants showed negative effects on growth. Relatively high levels of tetracyclines and sulfonamides (SAs) were detected in the nonedible parts, roots, and leaves of cucumber and tomato, but fruit parts accumulated them lower than acceptable daily intake. Indeed, cucumber roots accumulated SAs by up to 94.6% of total addition (at 5 mg kg−1 soil). KEYWORDS: bioaccumulation, emerging contaminant, pharmaceuticals, phytoavailability, risk assessment, veterinary antibiotics

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INTRODUCTION Contamination of agricultural soils with agrochemicals, such as pesticides and fertilizers, and its potential accumulation in the crop tissues are well investigated. Adamo et al.1 studied the uptakes of Zn, Cr, Cu, and Pb by lettuce plant from metalscontaminated soils and reported the order of transfer factor from soil to vegetables, Zn > Cu > Cr ≥ Pb. They revealed that the Pb contents in all collected samples were below the maximum levels of Pb in edible vegetables set by the European Union.2 Babu et al.3 also studied the uptake of organochlorine pesticides (DDT and HCH) by Basmati rice (Oryza sativa) from the soil. Residues of both pesticides were found in all soil and plant samples except for a few grain samples. Maximum and minimum levels of residue were observed in husk and grains, respectively, and the levels of DDT and CHC in grains were quite below the regulation by the government of India and WHO/FAO. Similarly, Waliszewski et al.4 investigated DDT and HCH isomer levels in soil and carrot root and leaf. Their results revealed the organochlorine pesticide diffusion from agricultural soils to growing carrot plants and its vaporadsorption by leaves. Residues and metabolites of antibiotics are very important organic pollutants in our surrounding environments. Antibiotics are widely used for therapeutic purposes to treat or control infectious diseases in humans and animals. Antibiotics in veterinary practice are also used to increase feed efficiency and to promote growth in food producing animals at subtherapeutic levels.5−7 Tetracyclines (TCs) and sulfonamides (SAs) as the most popular antibiotics groups that are active against a broad spectrum of Gram-positive/negative bacteria, some obligate anaerobes, protozoa, parasites, and fungi.8,9 In Korea, TCs and © XXXX American Chemical Society

SAs antibiotics are most commonly used in veterinary medicine, where the Korean Food and Drug Administration10 reported that TCs have been used the most among antimicrobials in veterinary medicine (470.9 t) followed by penicillins (170.7 t) and SAs (157.4 t).11−13 In European countries, including Spain, France, Germany, United Kingdom, Poland, and Italy, TCs are also most commonly used in veterinary medicine followed by penicillins and SAs and show the annual consumptions of 205−658 t of TCs, 83−567 t of penicillins, and 45−174 t of SAs.14 The overuse of antibiotics in the field of livestock industry may lead to occurrence of antibiotics’ residue due to animal excretions and animal products having a high concentration of antibiotics.7,12,15−17 Excretion is the major means by which human and veterinary antibiotics are introduced into the environment. The application of manure to agricultural soils mainly leads to the release of residual antibiotics into soils through leaching and runoff, which thereby threatens groundwater and surface water quality.12 Some leftover livestock feeds that contain therapeutic agents may also be discarded as waste or lost along with the runoff during watering and rainfall episodes.18,19 In addition, significant amounts of antibiotics are discharged into the environment from wastewater treatment facilities, septic systems, and agricultural waste storage structures. Awad et al.11 comprehensively monitored the occurrence and seasonal variations of TCs and SAs in water, sediment, and soil. They Received: July 24, 2014 Revised: December 8, 2014 Accepted: December 15, 2014

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observed higher levels of TCs and SAs in water, sediment, and soil during winter than summer season due to lower degradation rate in winter season. Ok et al.13 also found the highest concentration of TCs and SAs in water, sediment, and soil at a site adjacent to the composting facility than at a site far away from that facility. As a result, the residual antibiotics in soil and water can be taken up by plants.18,20−22 Few studies investigated the uptake of antibiotics by plants. Sulfadimethoxine was found in root and leaves of barley with concentrations of 1.2 and 1.1 mg g−1, respectively (on dry weight basis), when the barley was cultivated in soils treated with 300 mg L−1 sulfadimethoxine.23 Kumar et al.22 reported that the maximum amount of chlortetracycline recovered in plant tops was found in corn followed by cabbage and green onion. The amount of accumulated chlortetracycline in plant tissues was small (0.002−0.017 mg kg−1 fresh weight), but it increased with increased rate of chlortetracycline into the manure. Dolliver et al.21 reported that the highest amount of sulfamethazine was detected in lettuce followed by potato and corn. Michelini et al.24 also evaluated the effects of sulfadiazine on willow (Salix fragilis L.) and maize (Zea mays L.) plants. They found that the high concentration of sulfadiazine caused serious stress in willow (e.g., reduced C/N ratio and total chlorophyll content) and the death of maize, which thereby shows a strong potential to impair plant performance and biomass. Because of consumption of agricultural products containing antibiotics’ residues, public health could be threatened. Especially, continuous exposure to antibiotics could lead to allergic reactions and chemical poisoning by the development of resistant bacterial strains.25,26 Several regulatory authorities had set maximum residue limits (MRLs) for antibiotics in foods of animal origin, for example, the European Union set MRLs for TCs that ranged between 0.1 mg kg−1 in milk and meat and 0.6 mg kg−1 in kidney, while it was set at 0.1 mg kg−1 for SAs in all livestock products.27 However, no limit was set for food crops. Previous studies focused on the presence and prevalence of antibiotics in environmental compartments as well as the impact of antibiotics on seed germination and plant growth. Some studies focused on the uptake of antibiotics by edible and nonedible plants and calculated the antibiotics contents in the different parts of nonripened plants. However, no study was found related to the accumulation and distribution patterns of antibiotics in the ripened plants’ parts, especially the edible parts, through the exposure to different concentrations of antibiotics. Moreover, the previous studies did not focus on the safety assessment of those edible parts for human consumption with the successive increasing of antibiotics’ dosage. Therefore, in this study, we hypothesized that the antibiotics of TCs and SAs accumulated in different parts of cucumber, tomato, and lettuce plants. In addition, the accumulation or plant uptake of antibiotics may show different distribution or patterns depending on the antibiotics’ types or plant species. Consequently, this study was conducted (i) to reveal the effect of TCs and SAs on the growth of cucumber, tomato, and lettuce plants by measuring growth parameters including plant height, number of leaves, chlorophyll content, shoot weight, root weight, total root surface area, and fruit weight; (ii) to quantify the absorbed antibiotics in the different plant parts for food safety assessment; and (iii) to trace the distribution and cumulative patterns of various antibiotics.

Article

MATERIALS AND METHODS

Antibiotics. Target antibiotic compounds were selected based on frequency of use in Korea.10 Target antibiotic standards, including 97.5% tetracycline hydrochloride (referred to as TC in this study), 97% oxytetracycline hydrochloride (OTC), 97% chlortetracycline hydrochloride (CTC), 99% sulfamethazine (SMT), 99.7% sulfamethoxazole (SMX), and 98.5% sulfadimethoxine (SDM), were obtained from Sigma-Aldrich (St. Louis, MO, USA). The physicochemical characteristics of these antibiotics are shown in Table S1 of the Supporting Information. Acetonitrile, methanol, acetone, hexane, formic acid, and water of HPLC-grade were also obtained from LiChrosolv Co. (Merck, Germany). The solid-phase extraction (SPE) cartridges and 3 mL/60 mg of HLB (hydrophilic−lipophillic− balanced) were purchased from Waters Oasis Co. (Milford, MA, USA). Stock solutions of antibiotics were prepared at a concentration of 100 mg L−1 as follows: 10 mg of individual standard (corrected by purity) was accurately weighed, dissolved in small amount of methanol, diluted to 100 mL with deionized water, and then stored at −20 °C. Experimental Procedure. Each of 36 plastic pots (200 mm diameter × 190 mm tall) was filled with a loamy sand upland soil obtained from experimental field at Kangwon National University, Chuncheon, Korea. Each pot contained 3.7 kg of dry soil. Three kinds of plant seedlings, including cucumber (Cucumis sativus, 45 d old), tomatoes (Solanum lycopersicum, 45 d old), and lettuce (Lactuca sativa, 30 d old), were used for the cultivation experiment. Each of 12 pots was used for each cultivation of cucumber, tomato, and lettuce plants. The plants were individually transplanted as one plant in each pot and then irrigated by antibiotics’ free water and kept for 2 weeks for adaptation before the addition of subjected antibiotics. Antibiotics, including TCs and SAs at the levels of 5, 10, and 20 mg kg−1 soil, were collectively spiked through the irrigation water at four doses (one dose per week) along with no antibiotics’ addition as the control. All pots were irrigated twice a week with 50 mL of water containing antibiotics and 100 mL of water containing fertilizers, in order. To ensure the effects of antibiotics on plant growth only, the essential fertilizers of common plants were provided except phosphate fertilizer because of its negative effect on TCs adsorption by plants.28 Plant seedlings were grown in a greenhouse, which was completely controlled at 25 °C and 70% humidity for 45 d of cultivation period. During cultivation, the growth parameters, such as plant height (cm), number of leaves, and leaf chlorophyll content by a chlorophyll meter (Minolta, SPAD-502, Japan), were measured weekly, and other parameters, such as weights of root, shoot, and fruit, and total root surface area, were determined after harvest (or 45 d of cultivation). Plant Samples Preparation. After 45 d of cultivation, all seedlings had completely become ripened. The harvested shoots of each plant were washed with tap water and dried using an adsorbent paper for weighing. The fruits of each plant were also weighed. To remove the moisture completely, the harvested leaves and fruits were chopped and then subjected to a freeze-drier (OPERON, FDB-5503, Korea). For the measurement of root’s surface area, the adhered soil was removed carefully, washed using the tap water, and then dried using an adsorbent paper. The Epson 10000XL scanner equipped with the WIN MAC RHIZO V 2009c program (Regent Instruments Inc., Canada) was employed for the morphological characterization of roots.29 Briefly, the roots were placed in a tray (300 mm width × 400 mm length × 20 mm thickness) with water for separating out roots and moisturizing, and then the surface area of roots was determined. Antibiotics’ Uptake by Plants. The antibiotics’ extraction from plant samples was done using the modified procedures of Migliore et al.23 and Dolliver et al.21 Specifically, 500 mg of dried and crushed plant sample was extracted with 8 mL of methanol:HCl (95:5), mixed for 30 s using a vortex (Vortex-2 genie, Scientific Industries, USA), sonicated for 10 min using an ultrasonic (JAC−1505, Korea), and centrifuged for 10 min at 4500 rpm, and then the supernatant was collected. The residue was extracted again with 5 mL of acetone using the same procedure. The supernatants were mixed and dried under N2 B

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Journal of Agricultural and Food Chemistry Table 1. Differences between Growth Parameters for Control and Antibiotics’ Treated Plants plants tomato

cucumber

lettuce

a

treatments (mg kg−1) control 5 10 20 control 5 10 20 control 5 10 20

plant height (cm) 54.33 30.00 50.33 30.00 66.00 41.33 52.33 40.67 19.67 15.33 18.00 14.33

aa b a b a b ab b a bc ab c

number of leaves 25.00 17.33 19.33 15.33 8.00 8.33 8.33 8.00 11.33 9.00 10.67 8.67

a bc b c a a a a a b a b

chlorophyll content (SPAD) 53.14 49.74 51.08 46.42 59.12 55.21 58.30 54.94 40.77 35.96 38.14 32.60

a b ab c a b a b a b ab c

shoot weight (g) 98.67 68.83 80.17 63.00 72.50 40.93 46.17 32.50 42.23 17.09 23.76 13.69

a bc b c a b b b a c b c

root weight (g) 12.68 6.32 8.92 4.36 10.81 7.77 9.47 7.32 5.36 1.81 3.36 1.62

a c b c a b a b a c b c

total root surface area (cm2) 839.88 683.90 778.35 382.00 919.04 789.12 855.52 685.78 138.94 97.67 126.18 82.16

a c b d a b ab c a b a b

fruit weight (g) 39.83 40.33 40.83 37.67 70.36 55.57 77.35 38.67

a a a a a ab a b

Mean separation within columns by Duncan’s multiple range test at P = 0.05, n = 3.

Figure 1. Tetracyclines (TCs) uptake by (a) cucumber, (b) tomato, and (c) lettuce at 5, 10, and 20 mg kg−1 treatments. C

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Figure 2. Sulfonamides (SAs) uptake by (a) cucumber, (b) tomato, and (c) lettuce at 5, 10, and 20 mg kg−1 treatments. gas at 40 °C. The residue was resuspended in 5 mL of methanol:nanopure water (50:50) and defatted with 5 mL of hexane three times. Hexane (the upper layer) was removed each time after the liquid−liquid partitioning. The remaining liquid extract was dried under N2 gas at 40 °C up to 2.5 mL, which was subjected to the cleanup step using SPE. The SPE cleanup procedure was performed according to Kim and Carlson30 using an OASIS HLB 60 mg cartridge (Waters Corp., Milford, MA, USA). The cartridge was preconditioned with 3 mL of methanol, 3 mL of 0.5 N HCl, and 3 mL of nanopure water. The sample was passed through the cartridge at a flow rate of 2 mL min−1, and then the SPE cartridge was washed twice with 3 mL of nanopure water and dried under depression for 5 min. The antibiotics were then eluted from the cartridge twice with 2.5 mL of methanol. The methanol extract was dried under N2 gas at 40 °C until the volume reached ∼150 μL and was then transferred to a 500-μL degraded Eppendorf tube. The final volume of eluate was completed to 400 μL using the mobile phase A solution (0.1% formic acid in HPLC-grade water, pH 2.6) and then mixed and filtered through a 0.22-μm cellulose acetate filter. The eluate was transferred into an amber vial equipped with 250 μL of a glass insert vial for HPLC−MS analysis. The concentration of antibiotics was determined by the procedures of Kim and Carlson30 using a HPLC−MS (Agilent 1260 series, Agilent Technologies, Palo Alto, CA, USA) equipped with a variable wavelength UV detector. Electrospray ionization (ESI) was used to detect the target antibiotic, and a positive mode was adapted. For the chromatographic separation of antibiotics, the same mobile phases (A,

0.1% formic acid in HPLC-grade water, pH 2.6; and B, 0.1% formic acid in HPLC-grade acetonitrile) of a study by Kim and Carlson30 were used; however, a different HPLC column and pump program for the gradient elution of the mobile phases were employed. The used column was a YMC-Pack Pro C18 RS reversed-phase column (150 mm long × 2.0 mm i.d., 3-μm pore size) instead of an Xterra C18 column (50 mm long × 2.1 mm i.d., 2.5-μm pore size). A C18 guard column (Phenomenex, Torrance, CA, USA) was used to filter any particulates from the sample. The gradient program for the mobile phases started by a volume of 96:4 (A:B), which ramped to 88:12 (A:B) in 15 min and to 80:20 (A:B) in 10 min and then went back to the initial conditions in 5 min and remained isocratic for 5 min. The column temperature was maintained at 25 °C. Injection volume was 20 μL with a flow rate of 0.2 mL min−1. A Thermo Finnigan LCQ Duo ion traps mass spectrometer (Thermo, Woburn, MA, USA), equipped with a heated capillary interface and ESI, was used to perform the mass spectrometric analysis. Ultrapure N2 gas was used for drying and nebulizing. Spray voltage was set to 4.5 kV, and the capillary voltage was autotuned to 3.5 kV. Drying gas temperature was set to 350 °C, and a flow rate was 10.0 L min−1. Average concentration in triplicate of the individual antibiotics was calculated. Calibration curves established for the six antibiotics ranged from 100−5000 μg L−1. When sample concentration exceeded the highest calibration point, the injection volume was reduced. D

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Journal of Agricultural and Food Chemistry Statistics. Data were analyzed using SAS v.9.3 software (SAS Institute Inc., Cary, NC, USA). Mean separations were calculated using Duncan’s multiple range test at a 0.05 significance level.

done to reveal the possible mechanism using more various levels of antibiotics. Antibiotics Uptake by Cucumber, Tomato, and Lettuce. Tetracyclines (TCs). Accumulations of TCs were found in parts of leaves, fruits, and roots for each cucumber, tomato, and lettuce, as shown in Figure 1. For the cucumber, the increasing levels from 5−20 mg kg−1 of OTC, TC, and CTC led to the higher uptake by leaves, fruits, and roots (Figure 1). The accumulation of OTC and CTC was higher in the fruits and roots of cucumber than that of TC in the same parts, whereas the OTC showed the highest accumulation in leaves at 20 mg kg−1. All TCs were highly accumulated in parts of leaves and roots rather than in parts of fruits. The accumulation of OTC was the highest in all parts of tomato over all levels of OTC (Figure 1). Moreover, as the level of OTC increased, its accumulation by tomato was accelerated. No difference in accumulation was found between TC and CTC, and the accumulation of TC and CTC was much less than that of OTC. For the lettuce, CTC showed the highest accumulation in all parts compared to other TCs over all levels of addition (Figure 1). In particular, the roots of lettuce accumulated the highest concentrations of CTC over all levels of addition, which were 0.249, 0.624, and 1.160 mg kg−1 fresh weight at 5, 10, and 20 mg kg−1, respectively. These values of accumulation in the roots indicated up to 5.6-times higher than those in the leaves when the same level of CTC was treated. No difference of TC accumulation in the roots was found, and the OTC accumulation was fluctuated over all levels of addition. In brief summary, the accumulations of TCs by the subjected plants were higher in leaves and roots than in fruits and were gradually increased as the level of addition increased. The accumulations of OTC and CTC were generally higher than that of TC and maximized in tomato and lettuce, respectively. These obtained results were in accordance with Kumar et al.22 who stated that the absorbed level of CTC by corn, cabbage, and green onion increased as the level of CTC increased in the manure−soil mixture. Hu et al.37 also reported that the applied groups of antibiotics, including TCs and SAs, to the cultivated vegetables of radish, rape, celery, and coriander were biologically accumulated in vegetables through water transport and passive absorption. Sulfonamides (SAs). With increasing level of SAs, the accumulating trend was very similar to that of TCs in all parts of tested plants including cucumber, tomato, and lettuce; however, the levels of accumulation were much higher than TCs (Figure 2). For cucumber and tomato, the accumulation of all SAs in leaves and roots was generally higher than that in their fruits, and it was increased as levels of SAs increased to 20 mg kg−1. Especially, the accumulation of SMT was the highest in the leaves of cucumber and tomato over all levels. For the roots of cucumber, no difference of accumulation was found at the same level of SAs; however, as the level of SAs increased, the accumulation was gradually increased. At the level of 5 mg kg−1, the accumulations of SAs in all parts of cucumber were not different, and those were relatively lower than at the higher additive level of SAs. For the fruits of tomato, the accumulation of SDM was the most, and that of SMX was the lowest at all the levels of addition. In other words, the accumulation of SDM was 68.9, 64.7, and 57.2% higher than that of SMX at 5, 10, and 20 mg kg−1, respectively. For lettuce, all types of SAs were highly accumulated in roots rather than leaves. The accumulations of SMT in leaves of lettuce indicated the

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RESULTS AND DISCUSSION Analysis of Antibiotics. Calibration was done for the six antibiotics and showed excellent linearity in the concentration ranges of 100−5,000 μg L−1, with R2 ≥ 0.996 (Table S2 and Figure S1, Supporting Information). The extraction method and HPLC−MS conditions proved to enable a good simultaneous separation for the six antibiotics without neither overlapping between antibiotics nor interfering with the plant matrix as shown in the HPLC−MS chromatograms (Figure S2, Supporting Information). Antibiotics Effects on Plant Growth. The growth parameters, including plant height, number of leaves, leaf chlorophyll content, shoot weight, fruit weight, root weight, and total root surface area, were determined to evaluate the effects of TCs and SAs on plant growth (Table 1). The results revealed that the antibiotics negatively affected all growth parameters for tomato except the fruit weight, which was no different with the control. This result may be due to the low retained quantities of antibiotics in tomato’s fruits, which did not affect the fruit weight of treated plants; meanwhile, the other negatively affected growth parameters concerning roots and leaves of tomato may be due to their high contents of antibiotics as seen in the following results of Figures 1 and 2. The highest level of antibiotics (20 mg kg−1 soil) had the worst effects on the growth parameters of tomato. For cucumber, the antibiotics showed the negative effects on the growth parameters except the values of leaves’ number, because the negative effect for antibiotics on cucumber’s leaves was not observed in leaves’ number but in the leaves sizes, which were reduced compared to the control (leaf size data not shown). Interestingly, the antibiotics’ concentrations of 5 and 20 mg kg−1 had similar and negative effects on the plants’ growth parameters compared to those of 10 mg kg−1. Generally, our results showed that the antibiotics of TCs and SAs had negative effects on the plant growth of cucumber, tomato, and lettuce. Different toxic effects between antibiotic compounds are due to their different behavior in the soil via sorption, degradation, and chelating with metals.35 SAs have been known as antibiotics with higher bioavailability for plants than TCs because of their higher mobility in soils.31−34 Furthermore, SMX and SMT in the SAs group were found to be toxic to plant growth in the soil as reported by Liu et al.35 whereas CTC and OTC in the TCs group enhanced the growth of radish and wheat and also reported no negative effects on the growth of corn.36 Those previous findings support our results concerning the growth negative effects for antibiotics on the three treated plants at the levels of 5 and 20 mg kg−1; this negative effect could be due to the fact that the negative impact of SAs on plants was higher than the enhancing impact for TCs at those levels (5 and 20 mg kg−1). Meanwhile, at the level of 10 mg kg−1, the absorbed amount of TCs may have had a growth enhancing effect equivalent to the growth inhibiting effect for the absorbed amount of SAs. Therefore, some growth parameters for the 10 mg kg−1 treated plants were not negatively affected and statistically equal to those of the control plants. We believe that those obtained results are very important and considered a very interesting phenomenon; however, our data and analysis were limited to explain this phenomenon. Further work should be E

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Figure 3. Distributed ratios of absorbed tetracyclines (TCs) and sulfonamides (SAs) between the different plant parts of roots, fruit, and leaves.

highest values of 1.02 and 2.34 mg kg−1 at 5 and 10 mg kg−1, respectively, among SAs; however, the accumulation of SMT was not extended at 20 mg kg−1 compared to at 10 mg kg−1. SMT and SMX showed rapid and linear increases of their accumulation by adding higher levels in the roots of lettuce, whereas SDM was slightly increased as its level increased. In general, the amounts of accumulated SAs were increased as the levels of each SA increased, and the accumulations of SAs were the least in the fruits for cucumber and tomato in this study. Migliore et al.23 reported that relatively high concentrations of 1.234 and 1.063 mg g−1 SDM were detected in the root and leaves of barley, respectively, grown in the soils treated with 300 mg L−1 of SDM. Dolliver et al.21 found that the highest amount of SMT was taken up by lettuce, followed by potato and corn. On the basis of the results of TCs and SAs uptake by cucumber, tomato, and lettuce in this study, the accumulation of SAs was markedly higher than that of TCs. It can be explained with the low molecular weights of SAs, which are water-soluble and not strongly sorbed to soil particles; therefore, SAs may facilitate the uptake or accumulation by plant tissues.21,31,37,38 On the contrary, because TCs have opposite characteristics to SAs, TCs are relatively stable and strongly sorbed to soil particles and thus not easily taken up by plants from the soil.37,39 Safety Assessment. The acceptable daily intake (ADI) values for TCs and SAs had been established by JECFA40 and are defined as 0−30 μg kg−1 body-weight d−1 for TCs and 0− 50 μg kg−1 body-weight d−1 for SAs. Because body weight is typically assumed to be 60 kg as an adult, the ADI values of TCs and SAs are 1.8 and 3.0 mg d−1, respectively. In this study, the concentrations of TCs in edible parts of all plants were between 0.002 mg kg−1 (OTC in cucumber fruit at 5 mg kg−1) and 0.204 mg kg−1 (CTC in lettuce leaves at 20 mg kg−1). For the SAs, the concentrations in edible parts of all plants were between 0.013 mg kg−1 (SMX in tomato fruits at 5 mg kg−1)

and 3.240 mg kg−1 (SMX in lettuce leaves at 20 mg kg−1). On the basis of the report estimated by WHO,41 a typical adult in the eastern countries consumes fresh vegetables about 233 g d−1 including 81.5, 4.8, and 2.3 g d−1 of tomato, cucumber, and lettuce, respectively. Therefore, the amounts of TCs and SAs accumulated from the edible parts of tested plants were in the safe limit. Distribution of TCs and SAs in Different Parts of Plants. Figure 3 shows the distribution of TCs and SAs in roots, fruits, and leaves of cucumber, tomato, and lettuce. For the cucumber, the total amounts of accumulated TCs to plant bodies (including leaves, fruits, and roots) were 0.574, 1.309, and 3.418 mg kg−1 at the concentrations of 5, 10, and 20 mg kg−1, respectively (Table 2). It was linearly increased as the concentration of TCs into soils increased (Figure S3, Supporting Information). The amount of accumulated TCs in the leaves of cucumber at 5 mg kg−1 was 22.1% of the total accumulated TCs in all parts of plant body, but it was sharply increased to 43.9% at 20 mg kg−1. On the contrary, the Table 2. Total Absorbed Amounts of Tetracyclines by Plants total absorbed amounts (fresh weight basis; mg kg−1) plants

treatments (mg kg−1)

TC

OTC

CTC

total

cucumber

5 10 20 5 10 20 5 10 20

0.089 0.151 0.496 0.199 0.400 1.009 0.077 0.088 0.211

0.175 0.452 1.603 0.590 1.178 3.231 0.035 0.112 0.318

0.310 0.705 1.320 0.231 0.381 0.864 0.346 0.773 1.364

0.574 1.309 3.418 1.021 1.959 5.104 0.459 0.972 1.892

tomato

lettuce

F

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and the distribution on plant, which occurred in the different studies, are due to the limited understanding of the interactions of antibiotic concentrations in manure/soil, antibiotic chemical characteristics, specific crops, plant growth stage, and plant physiology.21 We conclude that the growth of cucumber, tomato, and lettuce was relatively inhibited by the TCs and SAs accumulated from soils spiked at the levels of 5, 10, and 20 mg kg−1. The accumulation of OTC was the highest in the cucumber and tomato, while CTC was the highest accumulated among TCs in the lettuce. The accumulation of SAs was the smallest in a part of fruits for cucumber and tomato that equals < 0.5% and 0.3%, respectively, from the total absorbed amounts of SAs. Moreover, the total amounts of TCs and SAs accumulated in the plant bodies were significantly increased as the level of their additions increased . Adding to that, the descending order for the total TCs contents in plants was tomato > cucumber > lettuce, while for SAs, it was tomato > lettuce > cucumber. In general, the relatively high level of TCs and SAs was detected in the nonedible parts (roots and leaves) of cucumber and tomato, and the fruits of these plants also contained a lower level than the ADI of antibiotics.

amounts of accumulated TCs in the fruits and roots of cucumber were decreased as the level of TCs addition increased (2.3 to 0.8% for fruits and 75.5 to 55.3% for roots). In the case of SAs for the cucumber, the total concentrations of SAs were also linearly increased as the concentrations of SAs into soils increased (Table 3; Figure S4, Supporting Information). Table 3. Total Absorbed Amounts of Sulfonamides by Plants total absorbed amounts (fresh weight basis; mg kg−1) plants cucumber

tomato

lettuce

treatments (mg kg−1)

SMT

SMX

SDM

total

5 10 20 5 10 20 5 10 20

5.359 8.725 16.319 9.573 13.106 42.445 7.813 16.776 25.993

5.633 8.295 11.330 17.193 20.963 38.467 8.582 12.790 30.589

4.924 8.546 12.692 6.113 13.011 20.887 1.773 4.377 7.876

15.916 25.566 40.341 32.879 47.080 101.799 18.169 33.943 64.458

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Moreover, the highest accumulation of SAs was detected in the roots up to 94.6% (at 5 mg kg−1) compared to other parts of plant body. Interestingly, the smallest accumulation of SAs was found in the fruits of cucumber ( leaves > fruits in general. These results agree with a study by Migliore et al.23 who stated that the concentration of antibiotics in the roots was higher than other parts of wheat and corn. Similarly, Liu et al.42 concluded that the distribution of all antibiotics in the wetland plant (Phragmites australis) followed the sequence root > leaf > stem. On the other hand, Hu et al.37 found that the distribution of antibiotics in various tissues of the vegetables was leaves > stems > roots. They also insisted that the types and growth stages of vegetables would affect the distribution of antibiotics. The differences of antibiotics uptake

ASSOCIATED CONTENT

S Supporting Information *

Properties of antibiotics used in the study, analytical performance of the method, calibration graphs of TCs and SAs, chromatograms for antibiotics in positive samples, and total accumulated amounts of TCs and SAs in plants at different treatments. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

This work was supported by the Korea Ministry of Environment as a Geo-Advanced Innovative Action Project (G11200056-0004-0) and by the Ministry of Education, Science and Technology as the Basic Science Research Program through the National Research Foundation of Korea (NRF) (2012R1A1B3001409). Instrumental analyses were supported by the Korea Basic Science Institute, the Environmental Research Institute, and the Central Laboratory of Kangwon National University, Korea. Notes

The authors declare no competing financial interest.

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