Article

pubs.acs.org/JAFC

Distribution of Antibiotics in Wastewater-Irrigated Soils and Their

Accumulation in Vegetable Crops in the Pearl River Delta, Southern
China
Min Pan,† Chris K. C. Wong,‡ and L. M. Chu*,†
†
School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
‡
Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong SAR, China
*
S Supporting Information

ABSTRACT: Wastewater is increasingly being used to irrigate agricultural land in many countries around the world. However,
limited research has examined the occurrence of antibiotics in soil irrigated with wastewater and their accumulation in plants.
This study aimed to determine the distribution of various types of antibiotics in different environmental matrices in the Pearl
River Delta (PRD) region and to evaluate their accumulation and translocation in edible crops. Samples were collected from six
sites in the PRD where either domestic wastewater or fishpond water was used for irrigation. Results showed that fishpond water
irrigated soils had higher concentrations of antibiotics than wastewater-irrigated soils. Different trends were observed in the
accumulation of antibiotics in the different edible parts of various crops. Despite the low human annual exposure to antibiotics
through the consumption of edible crops (1.10 to 7950 μg/y), the potential adverse effects of antibiotics along the food chain
should not be neglected.
KEYWORDS: antibiotics, vegetable crops, plant accumulation, human exposure

■ INTRODUCTION
Wastewater is commonly used to irrigate agricultural land and
recent years.5,17−19 However, these studies either detected the
concentrations of antibiotics in surface water or soil separately
urban greeneries, and replenish surface water and groundwater or restricted their plant uptake experiments to only one type of
in response to the water shortage resulting from climate change, antibiotic under artificially high levels.20,21 Some studies were
regional drought, population growth, and pollution.1,2 Globally, conducted in hydroponic or greenhouse environments, which
at least 20 million hectares of arable land are irrigated with do not reflect plant uptake and accumulation of antibiotics
wastewater that has been treated to various extents.3 In China, under genuine field conditions.19,22 Accordingly, there is
water availability is especially low and unevenly distributed,4 limited knowledge on the distribution and potential effects of
and domestic wastewater and fishpond water have become antibiotics on the terrestrial environment and food quality.
valuable resources for crop irrigation. However, wastewater The Pearl River Delta (PRD) is a major agricultural region in
usually contains toxic inorganic and organic pollutants and southern China, where large quantities of vegetables and edible
pathogens, which are mostly biologically active and create crops are produced and exported to other cities and countries.
further potential risk when they enter into the environment.5−7 Roughly 90% of the vegetables consumed in Hong Kong are
Although some guidelines have been developed in regard to supplied from mainland China,23 mostly from the PRD.
irrigation water quality criteria, they do not take into account However, 20 million hectare of the arable land in China was
the potential risk from trace amounts of organic pollutants,8−10 contaminated by heavy metals and 10 million hectare by
including pharmaceuticals and personal care products (PPCPs) organic pollutants, while 3.3 million hectare was contaminated
in surface water and treated effluent from wastewater treatment as a result of wastewater irrigation.24 Therefore, identifying the
plants.11−13 Despite their low environmental concentrations concentrations of different types of antibiotics in irrigation
(usually in the range of ng/L to μg/L in water), these wastewater, their accumulation in irrigated soil, and their
compounds have the potential to accumulate in irrigated soil or uptake and accumulation in edible crops would allow a more
be taken up by plants when the wastewater is used to irrigate accurate assessment of the risks they pose to the environment
edible crops. Nonionic PPCPs were taken up at higher and human health.
concentrations by root crops,14 and PPCPs were detected in The objectives of this study were (1) to investigate the
the edible tissues of a number of vegetables irrigated with presence and distribution of different types of antibiotics in
treated wastewater and fortified water.15 irrigation water and irrigated soil at different sites in the PRD
Antibiotics include thousands of diverse chemicals that and (2) to comparatively evaluate the uptake and translocation
humans and animals ingest for health or cosmetic reasons.16
These bioactive chemicals eventually enter into the environ- Received: August 11, 2014
ment after usage and as residuals from pharmaceutical Revised: October 28, 2014
manufacturing. There is a growing body of literature on the Accepted: October 30, 2014
occurrence of antibiotic compounds in water, soil, and plants in

© XXXX American Chemical Society A dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 1. Physicochemical Properties of the Antibiotics Investigated in This Studyf

a
Sarmah et al., 2006. bZhang et al., 2009. cYang et al., 2009. dYamanoto et al., 2005. eMeasured in the sorption experiment. f*na: not available. Log
Kow: the logarithm of the octanol/water partition coefficient. Koc: soil adsorption coefficient.

of antibiotics in various crops. Findings from this study would sampled. The region contains more than 11 million hectares of arable
facilitate a better understanding of the transfer of antibiotics to land, and the sampling sites were representative regarding farming
the soil−plant system and the potential ecological and human practice and had long history (>20 y) of wastewater reuse. The
risks through exposure to antibiotic contamination. irrigation water in Huizhou, Foshan, Zhongshan, and Guangzhou was

■
mainly domestic wastewater that was largely untreated and discharged
directly into open ditches or nearby rivers, while fishpond water was
MATERIALS AND METHODS used for irrigation in Dongguan and Shenzhen (Figure 1).
Antibiotics and Chemicals. Target antibiotic compounds were Irrigation water, soil, and crops were sampled in July 2013. Water
selected mainly based on their frequent usage in human and livestock samples were collected in 1 L amber glass bottles, with 50 mL of
in the PRD and other regions as well as their environmental methanol immediately added and pH adjusted to 3.0 using 4 M H2SO4
behaviors.25−27 Tetracycline (TC), sulfamethazine (SMZ), norfloxacin to preserve the aqueous samples (five replicates per sampling site). Soil
(NOR), erythromycin (ERY), and chloramphenicol (CAP), which and crop samples were collected from five random plots (100 × 100
belong to five different antibiotic types, were examined in this study m2) on each sampling site. Soil samples were collected at two depths:
(Table 1). All standards and some internal standards (SMZ-d4, NOR- 0−10 cm and 10−20 cm. An adjacent plot without cultivation served
d5, and ERY-13C2) were obtained from Sigma-Aldrich (USA), CAP-d5 as the control with no antibiotic contamination. Soil samples were
was purchased from Dr Ehrenstorfer GmbH (Germany), and TC-d6 characterized, and their physicochemical properties are given in Table
was obtained from Toronto Research Chemicals (Canada). Oasis HLB S1 in the Supporting Information. Chinese white cabbage, water
extraction cartridges (6 mL, 500 mg) (Waters Corporation, USA) spinach, Chinese radish, corn, and rice were harvested around the soil-
were used for the extraction and purification of the target compounds. sampling sites when the crops reached marketable size. Five whole
All the organic solvents used were of HPLC grade and purchased from crops were randomly collected from each plot, which were pooled to
Merck Corporation (Germany). Individual stock solutions and internal form a bulk sample to result in five replicates per site. Crops cultivated
standards were prepared at 100 mg/L in methanol and stored in an in a greenhouse without wastewater irrigation were used as control
amber glass vial at −20 °C. crops. Once in the laboratory, the water samples were passed through
Sampling Sites. Six locations (Huizhou, Foshan, Zhongshan, 0.7 μm glass fiber filters (Whatman GF/F, U.K.), and extraction was
Guangzhou, Dongguan, and Shenzhen) in the PRD region were carried out within 3 days. Soil samples and fresh crops were rinsed

B dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry Article

Figure 1. Information on the sampling sites in the Pearl River Delta, southern China.

with deionized water and freeze-dried. The dried samples were then Liquid Chromatography Mass Spectrometry Analysis. The
ground to powder and stored at −20 °C until extraction. target antibiotic compounds were analyzed by HPLC−MS/MS
Extraction. Water Samples. The target antibiotics in the water (Agilent Liquid Chromatography 1100 series HPLC system coupled
samples were extracted by solid phase extraction (SPE) using Oasis to an Agilent 6410 triple quadrupole MS) equipped with an
HLB cartridges. The internal standards (100 ng) and 0.2 g of disodium electrospray ionization (ESI) source (Agilent, USA) in multiple-
ethylenediaminetetraacetate (Na2EDTA) were spiked into each water reaction monitoring (MRM) mode. The analyses were performed in
sample (1 L). The SPE cartridges were preconditioned with 10 mL of negative mode for CAP and in positive mode for the other antibiotics.
methanol and 10 mL of Milli-Q water, after which the water samples Nitrogen gas was used as the drying and collision gas. The
were passed through the cartridge at a flow rate of 10 mL/min. The chromatographic column was an Agilent Eclipse XDB-C18 (3.0 ×
analytes were eluted from the cartridge with 10 mL of methanol, 75 mm, 3.5 μm) column with a Poroshell 120 precolumn filter (3.0
concentrated under a gentle nitrogen stream, and then redissolved in 1 mm, 0.2 μm). The column temperature was maintained at 40 °C for
mL of methanol. The final extract was filtered through a nylon syringe the analysis of the antibiotics.
filter into a 2 mL amber glass vial and stored at −18 °C before the For ESI+, 0.01% formic acid was used as mobile phase A and
LC−MS/MS analysis. acetonitrile as mobile phase B. Gradient conditions were set as follows:
Soil Samples. One gram of freeze-dried soil was ground up and 0 min, 20% B; 3 min, 40% B; 6 min, 60% B; 8 min, 80% B; 9 min, 95%
spiked with 100 μL of each internal standard (1.0 mg/L). The soil B and 13 min, 20% B. For ESI−, Milli-Q water was used as mobile
samples were extracted three times with 30 mL of acetonitrile and 0.2 phase A and acetonitrile as mobile phase B (v:v = 15:85) with isocratic
elution. The injection volume was 10 μL, and the flow rates of the
M citric acid buffer (pH 4.4) (v:v = 1:1) by vortex (60 s each time)
mobile phases were 0.3 mL/min and 0.35 mL/min for the positive and
and ultrasonication (15 min each time). The mixture was centrifuged
negative modes, respectively.
in air-cooled conditions at 1370 g for 10 min and concentrated; the Mass spectrometric conditions were optimized using Optimizer
extract for each soil sample was evaporated to near dryness, 0.2 g of (Agilent, USA) for fine-tuning of the fragmentor voltage, collision
Na2EDTA was added, and the mixture was diluted to 200 mL with energy (CE), and multiple reaction monitoring mode (MRM)
Milli-Q water. Each cartridge was preconditioned with 10 mL of transitions for each antibiotic (Table S2 in the Supporting
methanol and 10 mL of Milli-Q water, and the extracts were passed Information). The following optimized parameters were selected:
through HLB cartridges for purification. The analytes were eluted from drying gas temperature 350 °C, drying gas flow rate 9 mL/min (ESI+),
the cartridge with 10 mL of methanol and dried under a gentle 11 mL/min (ESI−), capillary voltage 3500 V. The system was re-
nitrogen stream. Finally, the residue was redissolved in 1 mL of equilibrated for 5 min between runs.
methanol for analysis. Estimation of the Bioconcentration Factor and Dietary
Crop Samples. One gram of each freeze-dried crop tissue was Intake. The ability of a plant to accumulate antibiotics from the soil
ground and spiked with 100 μL of each internal standard (1.0 mg/L). was estimated using the bioconcentration factor (BCF), which was
The crop samples were extracted three times with 30 mL of mixed calculated as the ratio of the chemical concentration in the crop tissue
solution of acidified acetonitrile (pH 3) and acetone (v:v = 1:1) by to the chemical concentration in the soil (all based on dry weight):
vortex mixing (60 s each time) and ultrasonication (15 min each),
followed by centrifugation in air-cooled conditions at 12000g for 15 concentration in crop tissue (μg/kg)
BCF =
min. The combined extractions were pipetted into a 200 mL round- concentration in soil (μg/kg)
bottom flask and evaporated to remove the organic solvent; 0.2 g of
Na2EDTA was added, and the mixture was diluted to 200 mL with An exploratory assessment of the potential human exposure to
Milli-Q water and passed through the preconditioned HLB cartridges antibiotics through the consumption of edible crops was conducted.
for purification. The analytes were then eluted from the cartridge with The level of human exposure was calculated as
10 mL of methanol and dried under a gentle nitrogen stream. Finally,
the residue was redissolved in 1 mL of methanol for analysis. human exposure = C × D × W × T

C dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry Article

where C is the concentration of antibiotics in the edible tissue (ng/g L for SMZ. These results were in general agreement with those
wet weight), D is the average daily consumption of edible crops (g wet for agricultural irrigation water.32,33 The wastewaters in
weight/kg body weight day), W is the body weight (kg) of the person Dongguan and Shenzhen contained significantly higher
consuming the crops, and T is the exposure time (day). The concentrations of antibiotics (ranging 12.9−234.0 ng/L and
concentrations of antibiotic in crop were converted to a fresh weight
basis using the average water content of each crop (95.3% for Chinese 10.5−184.0 ng/L, respectively) than those in the other regions
white cabbage, 91.4% for water spinach, 95.3% for Chinese radish, (p < 0.05). This can be attributed to the common practice of
76.0% for corn, and 11.6% for rice).28 The annual exposure of a 70 kg directly adding antibiotics to water or in medicated feeds to
individual was estimated using the average daily consumption of edible control bacterial diseases in farmed fish.34 In the irrigation
crops (11.8, 0.6, 0.5, 9.2, and 24.0 g wet weight/kg body weight-day waters from Foshan, Zhongshan, and Guangzhou, TC and CAP
for Chinese white cabbage, water spinach, Chinese radish, corn, and were found at relatively high concentrations. The wastewater
rice, respectively).29 used for crop irrigation in Huizhou had the lowest
Quantification and Quality Control. To account for the concentrations of the various antibiotics. The cropland in
potential analyte loss, a reagent blank, method blank, and spiked
matrices were analyzed together with the water, soil, and crop samples.
Huizhou is located on the upper-middle reaches of the Danshui
None of the analytes were detected in the blank controls for water, River and has less interference from human activities.
soil, and crop samples. Quantification of each antibiotic compound Antibiotics in Soils. TC, SMZ, NOR, ERY, and CAP were
was obtained using the internal standard method. Analytical precision all detected in the surface layer (0−10 cm) irrigated soil
was measured by analyzing one sample in triplicate for every 10 samples from the six sites (Table 2). TC, NOR, and CAP in
samples, and the calculated relative standard deviation was less than most cases had significantly higher concentrations in soils
10%.28 The recoveries of the target compounds ranged between 70% irrigated by fishpond water than those by domestic wastewater
and 120% (Table S3 in the Supporting Information). Limit of (p < 0.05). TC, NOR, and CAP showed a similar trend in
detection (LOD) and limit of quantification (LOQ) of the antibiotics concentration in the corresponding irrigation waters (r = 0.93,
were calculated with signal/noise ratios (S/N) of 3 and 10,
respectively. The S/N ratios were obtained by using the software
p < 0.05). All five target antibiotics detected in the irrigation
Masshunter (Agilent, USA). The LOQ of the target compounds water could be found in the irrigated soils. Both the irrigation
ranged from 0.40 to 1.13 ng/L for the water samples, 0.70 to 4.63 ng/g water and irrigated soil samples from Dongguan and Shenzhen
for the soil samples, and 0.80 to 4.40 ng/g for the crop samples (Table had significantly higher concentrations than the other sampling
S3 in the Supporting Information). Ten concentrations (0.5, 1, 2, 5, sites, especially for TC and CAP (p < 0.05), suggesting that the
10, 20, 50, 100, 200, and 500 ng/L) of individual antibiotics were used irrigation water (domestic wastewater and fishpond water) was
to calculate the calibration curves (r2 > 0.999). Recoveries of the study the major contributing source.
compounds were performed by spiking the standard solutions to DI Antibiotics persist in the environment after they are released
water, soil, and crop tissue samples. Ion suppression and enhancement
into the soil from irrigation water.35 Although wastewater is
are considered as the major sources of inaccurate quantitative results in
complex matrices.22,30 Initial recovery was done using the internal also used for irrigation in other countries, few studies have
standards and standard solutions to correct for matrix effects, spiking examined the presence and accumulation of antibiotics in
100 μg/L of each standard into the sample extracts and comparing the irrigated soils and crops.5,36,37 Kinney et al. (2006)36 detected
obtained concentration in matrix to the concentration in solvent. ERY in reclaimed water and wastewater-irrigated soils at
Values of smaller or greater than 100% indicate signal suppression and concentrations of 177−611 ng/L and 0.02−15 μg/kg,
enhancement, respectively.31 respectively. Chen et al. (2011)5 found TC, NOR, and CAP

■ RESULTS AND DISCUSSION

Antibiotics in Irrigation Water. The target antibiotics
in the soils of Hebei, China, under wastewater irrigation for
approximately 50 years. The present study found that the
concentrations of TC, SMZ, NOR, and CAP in soil samples
were detected at concentrations above the LOQ in the were significantly higher in Dongguan than in the other sites (p
irrigation water samples (Figure 2). The highest concentration < 0.05), which corresponded to their higher concentrations in
was 69.3−234.0 ng/L for TC, and the lowest was 4.0−58.2 ng/ the irrigation water. NOR was frequently found at concen-
trations as high as 66.7 μg/kg in the irrigated soils. NOR is one
of the most frequently detected antibiotics in various
environmental media including water, soil, and plants,17,38
which could be related to its extensive medical and veterinary
use. It may also come from animal manure,39 which was
commonly applied in these areas. It is not surprising that NOR
was consistently found at high concentrations in wastewater
irrigated soils in the PRD. TC (5.0−21.9 μg/kg), SMZ (1.3−
4.2 μg/kg), ERY (1.1−4.4 μg/kg), and CAP (3.2−22.3 μg/kg)
were also found in the six sampling sites. TC had higher
concentrations in soil irrigated with fishpond water than that
with domestic wastewater. TC was detected at higher
concentrations in 0−10 cm soils than in 10−20 cm soils,
which is in line with its strong tendency to sorb onto soils due
to their high Koc values (Table 1). SMZ have similarly low
concentrations in the two soil depths. Sorption of SMZ to soils
was influenced by its physicochemical properties and soil pH.
The average soil pH in the six sampling sites was 7.51.
According to the pKa values of SMZ, the fraction of
Figure 2. Concentrations (ng/L) of antibiotics in irrigation water in deprotonated species would be increased, which would have
the Pearl River Delta, China (mean ± SD, n = 5). decreased its sorption to the soil.33,40 Therefore, SMZ is very
D dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 2. Concentrations (μg/kg dw) of Antibiotics Detected in the Irrigated Soils in the Pearl River Delta, China (Mean ± SD,
n = 5)
soil depth (cm) TC SMZ NOR ERY CAP
HZ 0−10 8.9 ± 0.2 1.4 ± 0.1 23.9 ± 2.5 1.1 ± 0.1 4.1 ± 0.9
10−20 <LOQ 1.3 ± 0.1 20.5 ± 2.3 <LOQ <LOQ
FS 0−10 17.1 ± 1.4 3.8 ± 0.6 36.8 ± 1.9 4.4 ± 0.2 10.1 ± 0.7
10−20 10.3 ± 0.3 3.6 ± 0.4 34.9 ± 0.7 3.9 ± 0.2 8.1 ± 1.8
ZS 0−10 15.8 ± 1.1 2.8 ± 0.6 28.5 ± 0.7 3.6 ± 0.1 6.6 ± 1.3
10−20 10.5 ± 0.1 2.5 ± 0.2 25.6 ± 0.6 2.3 ± 0.1 3.2 ± 0.3
GZ 0−10 13.0 ± 2.4 2.1 ± 0.1 25.5 ± 1.1 3.5 ± 0.1 5.5 ± 0.4
10−20 5.0 ± 0.02 2.0 ± 0.1 21.1 ± 1.0 <LOQ <LOQ
DG 0−10 21.9 ± 2.7 4.2 ± 0.2 66.7 ± 1.1 3.4 ± 0.3 22.3 ± 0.8
10−20 19.7 ± 2.3 4.0 ± 0.1 60.8 ± 2.4 3.0 ± 0.1 19.3 ± 1.2
SZ 0−10 18.2 ± 1.5 2.2 ± 0.1 34.4 ± 1.8 3.7 ± 0.2 11.4 ± 1.2
10−20 16.7 ± 0.3 2.3 ± 0.1 27.2 ± 8.8 2.2 ± 0.1 8.9 ± 0.8

mobile and its concentrations were similar in the two soil layers
at all sampling sites. Although most of the antibiotics were
found in the two soil layers, the concentrations were higher at
0−10 cm than 10−20 cm due to their sorption and migration
upon continuous irrigation. Chen et al. (2011)5 detected TC at
higher concentrations in the upper layer soil in wastewater
irrigated plot in Hebei Province. The distribution of the target
antibiotics in the soil profiles indicates that they were preferably
retained in soil, which may lead to further contamination of
plants. Nevertheless, the concentrations of antibiotics in this
study were significantly lower than those reported in other
studies. For example, Hu et al. (2010)41 detected TC in
wastewater-irrigated soils at concentrations of 2.5−105 μg/kg,
which could be attributed to the control of antibiotic use by the
Chinese government in recent years.38,41
Antibiotics in Crops. NOR was consistently found at the
highest concentrations (4.6−23.6 μg/kg) in crop tissues,
followed by CAP (2.6−22.4 μg/kg) and TC (4.0−10.1 μg/
kg) (Table S4 in the Supporting Information), which is similar
to the results of Hu et al. (2010).41 However, SMZ and ERY
were not detected in most of the vegetable crops. The leaves of
Chinese white cabbage and water spinach were found to
contain a variety of antibiotics, e.g., three out of the five
antibiotics with concentrations of 2.6−23.6 μg/kg in Chinese
white cabbage. Three antibiotics were found in water spinach
leaves, though mostly at a lower concentration (5.5−21.8 μg/
kg). SMZ and ERY were only found in the roots of Chinese
radish and Chinese white cabbage. SMZ was not liable to be
retained in the surface soil and absorbed by crops as indicated
by its small Kd value, even though SMZ was present in most of
the irrigated soil samples. Furthermore, ERY was rapidly
adsorbed onto clay particles where protonation occurred.42 It is
difficult for ERY to be absorbed through the cellular Figure 3. Bioconcentration factor (BCF) values of antibiotics in
membranes and translocate into crops as it has a greater different crops (mean ± SD, n = 5).
molecular weight than the other compounds.43 SMZ and ERY
also have a shorter degradation time than the other three.40 The BCFroot values for CAP in the roots of corn and rice
Therefore, these antibiotics are not easily absorbed by crops were 1.33 and 1.36, respectively, and the values for the other
and tend not to accumulate in the plant tissues. Residue compounds were below one. CAP was also found in the shoots
degradation, soil-binding, and leaching are other factors that of Chinese radish, corn, and rice, which had BCFleaf/shoot values
can limit the availability of antibiotics for uptake by crops.41 of 3.86, 1.75, and 1.69, respectively. In the five crops, the values
Bioconcentration Factor of Antibiotics in Crops. The of BCFleaf/shoot for TC, NOR, and CAP were larger than those
mean BCF values for different crops from the six sites are for BCFroot. In contrast, the BCFroot values for SMZ and ERY
shown in Figure 3. The reported BCF values represent the were larger than their BCFleaf/shoot because SMZ and ERY were
minimum values due to biodegradation under the field not detected in most crop tissues. Moreover, the BCFroot and
conditions. BCFleaf/shoot values for TC, SMZ, NOR, and ERY were less than
E dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

one, indicating that they were not readily absorbed and the Table 3. Translocation Factor (TF) Values of Antibiotics in
plants did not accumulate the antibiotics. Different Crops (Mean ± SD, n = 5)a
The uptake and translocation of antibiotics varied among the
TC SMZ NOR CAP
crop species. For example, although CAP was found in the
roots of corn and rice (BCFroot 1.33−1.36), it showed limited Chinese white 0.5 ± 0.1 1.5 ± 0.2 0.8 ± 0.2
cabbage
accumulation in the roots of Chinese white cabbage, water
water spinach 0.4 ± 0.2 1.4 ± 0.4 1.2 ± 0.7
spinach, and Chinese radish (BCFroot 0.03−0.17). In addition, Chinese radish 0.7 ± 0.3 0.2 ± 0.1 1.2 ± 0.03 0.8 ± 0.2
all except ERY were present in the leaves of Chinese radish, and corn 1.2 ± 0.1 1.4 ± 0.3
the BCFleaf/shoot of CAP in Chinese radish was the highest. rice 1.2 ± 0.03 1.4 ± 0.5
Herklotz et al. (2010)22 reported high concentration of a
sulfamethoxazole in cabbage (23.6−3200 ng/g, dry weight) TF for ERY could not be calculated.
under ideal hydroponics conditions. However, another study
only found low concentrations of ionic compounds in crops, (2010)42 found that the concentration of sulfamethoxazole in
probably due to the repulsion of the anions by the negative roots was 135 times that in the leaves of cabbages grown in a
electrical potential of the plant cell membrane.44 The average nutrient solution, and the fraction of sulfamethoxazole in
soil pH was 7.5, and as SMZ existed primarily in anionic form, cabbage roots accounted for 98.4% of the total uptake. The
it was difficult for the crops to absorb. Therefore, SMZ showed translocation of organic compounds within plants is affected by
limited accumulation in both roots and shoots, which is several factors, e.g., hydrophilicity.49 It was found that the TF
comparable to the findings of Wu et al.(2013).7 In all, the was at a maximum for compounds having log Kow from −1.03
differences in the uptake and accumulation of antibiotics are to 0.92, except for SMZ. Ionization could reduce the
governed by the crop species, harvest time, and environmental translocation of ionized organic compounds to shoots.50 This
conditions. may happen in SMZ under the present environmental
Translocation of Antibiotics within Crops. The conditions (existed predominantly as anion) coupled with
distribution of TC, NOR, and CAP in Chinese white cabbage, their low root cell membrane permeability to ionized SMZ.
water spinach, Chinese radish, corn, and rice was in the Nonionic compounds that are highly hydrophilic were less able
descending order of fruit > shoot/leaf > root, which suggests to cross the hydrophobic membranes, while highly lipophilic
that the three antibiotics were transported in the transpiration compounds were less likely to be transferred to shoots because
stream within the xylem of the plants. In contrast, the they were retained in the root lipids.50 In the present study, the
concentrations of SMZ and ERY followed the order of root five antibiotics have similar log Kow that could not distinguish
> shoot/leaf > fruit. The concentration of sulfamethoxazole in the importance of hydrophilicity on the translocation and
roots was about 10 times that in leaves of greenhouse grown accumulation of organic compounds in crops. Moreover,
vegetable (lettuce, cucumber, pepper, and spinach).7 Migliore uptake was species specific, even when the crops shared the
et al. (1998) 45 also found that the concentration of same irrigated soil.20,44 Therefore, antibiotics demonstrate
sulfonamides in roots was higher than that in leaves for different translocation behaviors in different plants according
wheat and corn. The translocation of antibiotics in crops to their physicochemical properties as well as plant species,
depends on the physicochemical properties of antibiotics and growth, and transpiration rates.
crop species as well as the concentrations of antibiotics in soil.46 Human Exposure Implications. On average, the level of
The species type and the growth stage can affect the daily human exposure to the five antibiotics through the
accumulation and distribution of antibiotics in crops.15 consumption of edible crops ranged from 0.003 to 21.8 μg
According to the reported inhibitory concentrations of (Table 4). SMZ and ERY were only detected in Chinese radish,
antibiotics on plants,47 crop growth would not be significantly indicating that there would be negligible risk associated with
affected at the antibiotic concentrations detected in this study. the consumption of these crops. However, the levels of TC,
The translocation factor (TF) represents the translocation of NOR, and CAP were relatively high, especially for corn and rice
antibiotics from the roots to the leaves and/or shoots, and is (daily human exposure was 0.34−2.77 and 11.0−21.8 μg,
calculated simply as the ratio of the leaf to root concentration respectively). The annual human exposure has been estimated
([leaf]/[root]). The uptake by plants is considered to occur by based on the daily consumption of these five edible crops (1.1
passive transport. Once a chemical is taken up by roots, to 7950 μg). Moreover, the United Nations Food and
translocation driven by the transpiration process will distribute Agriculture Organization has established acceptable daily intake
the chemical to other parts of the plant, and the ratio of root to (ADI) levels for TC (0−30 μg/kg bw) and ERY (0.0−0.7 μg/
leaf concentrations is dependent on the amount of water kg bw). Assuming that the body weight of an average adult is 70
transpired and the growth rate of the plants.48 In this study, all kg, the maximum permissible intake of TC is 2100 μg/day
of the antibiotics were found in the leaves and/or shoots, while that for ERY is 49 μg/day. Although the estimated values
except ERY, which was not detected in leaves and/or shoots. of daily human exposure in this study were much lower than
Thus, the TF for ERY cannot be calculated; TF for SMZ could the minimum therapeutic doses (typically in the 20−200 mg
only be calculated in Chinese radish. range) and below the recommended ADI values, antibiotic-
The TF for NOR was generally larger than 1 in all five crops, resistant bacteria or their corresponding resistance genes have
indicating that NOR was preferentially transferred from the been shown to spread to humans via the food chain.51 Thus,
roots to the shoots (Table 3). The TFs for CAP in water further research is needed to assess the accumulation of these
spinach, rice, and corn were also larger than 1. Other antibiotics in the food chain to determine the safe
antibiotics, especially ERY and SMZ, were found to accumulate concentrations for human consumption.
in roots (TF < 1). The translocation of sulfonamides agreed The results of this study indicate that the use of wastewater
with the findings of previous reports for cabbage, lettuce, and fishpond water to irrigate croplands could lead to the
cucumber, and pepper.19,22 For example, Herklotz et al. accumulation of antibiotics in soils and edible crops. However,
F dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 4. Human Exposure to Antibiotics in Chinese White Cabbage, Water Spinach, Chinese Radish, Corn, and Rice,
Calculated from the Concentrations of These Compounds in the Edible Tissues
TC SMZ NOR ERY CAP
human exposure (μg) daily annual daily annual daily annual daily annual daily annual
Chinese white cabbage (leaf) 0.23 84.6 0.59 216 0.21 75.2
water spinach (leaf) 0.01 2.8 0.05 18.5 0.02 6.8
Chinese radish (root) 0.01 2.0 0.01 1.9 0.02 6.8 0.003 1.1 0.004 1.6
corn (fruit) 0.34 125 2.27 830 2.77 1011
rice (fruit) 11 3998 17.2 6266 21.8 7955

significant disparities were observed in the potential root uptake CE, collision energy; LOD, limit of detection; LOQ, limit of
and subsequent accumulation of the different antibiotics in quantification; S/N, signal/noise; Koc, soil adsorption coef-
relation to their physicochemical properties (e.g., ionization, ficient; pKa, a logarithmic measure of the acid dissociation
degradation time, and molecular size) and the environmental constant; BCF, bioconcentration factor; TF, translocation
conditions. SMZ and ERY preferentially accumulated in roots factor

■
as they have difficulties in moving up the crops, while TC,
NOR, and CAP were more prevalent in leaves/shoots and REFERENCES
fruits. More experimental data on other types of antibiotics or
organic compounds are needed for verification. Although the (1) IPCC Intergovernmental Panel on Climate Change. http://www.
ipcc.ch/s (16th July 2014).
levels of antibiotics ingested through the consumption of edible (2) WWAP (World Water Assessment Programme). http://www.
crops were much lower than the minimum therapeutic doses or unesco.org/new/en/natural-sciences/environment/water/wwap/s
ADI levels, the findings from this study provide basic (16th July 2014).
information on the accumulation of antibiotics in edible (3) Raschid-Sally, L.; Jayakody, P. Drivers and Characteristics of
crops and help evaluate human exposure to antibiotics through Wastewater Agriculture in Developing Countries-Results From a Global
crop consumption. Further research on the potential long-term Assessment; International Water Management Institute: Colombo, Sri
health effects of ingesting different types of antibiotics through Lanka, 2008; pp 1−30.
dietary uptake is necessary. (4) Shi, Y.; Gao, L.; Li, W.; Liu, J.; Cai, Y. Investigation of

■
*
ASSOCIATED CONTENT
S Supporting Information
fluoroquinolones, sulfonamides and macrolides in long-term waste-
water irrigation soil in Tianjin, China. Bull. Environ. Contam. Toxicol.
2012, 89, 857−861.
(5) Chen, F.; Ying, G. G.; Kong, L. X.; Wang, L.; Zhao, J. L.; Zhou, L.
A detailed description of the properties of soils from the study J.; Zhang, L. J. Distribution and accumulation of endocrine-disrupting
sites (Table S1), the optimized MRM parameters for the chemicals and pharmaceuticals in wastewater irrigated soils in Hebei,
analysis of antibiotics by HPLC-MS/MS (Table S2), and the China. Environ. Pollut. 2011, 159, 1490−1498.
extraction recoveries of antibiotics in different matrices (Table (6) Papadopoulos, F.; Parissopoulos, G.; Papadopoulos, A.; Zdragas,
S3), as well as the concentrations (μg/kg dw) of antibiotics in A.; Ntanos, D.; Prochaska, C.; Metaxa, I. Assessment of reclaimed
different crop tissues (Table S4). This material is available free municipal wastewater application on rice cultivation. Environ. Manage.
of charge via the Internet at http://pubs.acs.org. 2009, 43, 135−143.

■
(7) Yan, C. X.; Yang, Y.; Zhou, J. L.; Liu, M.; Nie, M. H.; Shi, H.; Gu,
AUTHOR INFORMATION L. J. Antibiotics in the surface water of the Yangtze Estuary:
occurrence, distribution and risk assessment. Environ. Pollut. 2013,
Corresponding Author
175, 22−29.
*Phone: 852-39436378. Fax: 852-26037732. E-mail: (8) USEPA. Guidelines for Water Reuse; U.S. Agency for International
leemanchu@cuhk.edu.hk. Development: Washington, DC, 2004; EPA/625/R-92/004.
Notes (9) SAEPA. South Australian Reclaimed Water Guidelines; Department
The authors declare no competing financial interest. of Human Services and Environmental Protection Agency, Govern-

■
ment of South Australia: Adelaide, Australia, 1999.
ACKNOWLEDGMENTS (10) WELL. Guidelines for wastewater reuse in agriculture and
aquaculture: recommended revisions based on new research evidence;
We thank Ben Yeung (The Chinese University of Hong Kong) Water and Environmental Health: London, 2000; Task No.: 68 Part 1.
for his technical assistance and Dr. Yinge Zhao of the Croucher (11) Gao, L. H.; Shi, Y. L.; Li, W. H.; Niu, H. Y.; Liu, J. M.; Cai, Y. Q.
Institute for Environmental Sciences (Hong Kong Baptist Occurrence of antibiotics in eight sewage treatment plants in Beijing,
University) for her support in laboratory analyses. China. Chemosphere 2012, 86, 665−671.

■ ABBREVIATIONS USED
PRD, Pearl River Delta; PPCPs, pharmaceuticals and personal
(12) Xu, W.; Zhang, G.; Li, X.; Zou, S.; Li, P.; Hu, Z.; Li, J.
Occurrence and elimination of antibiotics at four sewage treatment
plants in the Pearl River Delta (PRD), South China. Water Res. 2007,
41, 4526−4534.
care products; TC, tetracycline; SMZ, sulfamethazine; NOR,
(13) Yang, J. F.; Ying, G. G.; Zhao, J. L.; Tao, R.; Sua, H. C.; Liu, Y. S.
norfloxaxin; ERY, erythromycin; CAP, chloramphenicol; HZ, Spatial and seasonal distribution of selected antibiotics in surface
Huizhou; FS, Foshan; ZS, Zhongshan; GZ, Guangzhou; DG, waters of the Pearl Rivers, China. J. Environ. Sci. Health. 2011, 46,
Dongguan; SZ, Shenzhen; HLB, hydrophilic−lipophilic bal- 272−280.
anced; SPE, solid phase extraction; Na2EDTA, disodium (14) Malchi, T.; Maor, Y.; Tadmor, G.; Shenker, M.; Chefetz, B.
ethylenediaminetetraacetate; HPLC−MS/MS, high perform- Irrigation of root vegetables with treated wastewater: evaluating uptake
ance liquid chromatography double mass spectrometry; ESI, of pharmaceuticals and the associated human health risks. Environ. Sci.
electrospray ionization; MRM, multiple-reaction monitoring; Technol. 2014, 48, 9325−9333.

G dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry Article

(15) Wu, X.; Conkle, J. L.; Ernst, F.; Gan, J. Treated wastewater (35) Ternes, T. A.; Bonerz, M.; Herrmann, N.; Teiser, B.; Andersen,
irrigation: uptake of pharmaceutical and personal care products by H. R. Irrigation of treated wastewater in Braunschweig, Germany: an
common vegetables under field conditions. Environ. Sci. Technol. 2014, option to remove pharmaceuticals and musk fragrances. Chemosphere
48, 11286−11293. 2007, 66, 894−904.
(16) United States Environmental Protection Agency. Pharmaceut- (36) Kinney, C. A.; Furlong, E. T.; Werner, S. L.; Cahill, J. D.
icals and personal care products. http://www.epa.gov/ppcp/basic2.html Presence and distribution of wastewater-derived pharmaceuticals in
(16th July 2014). soil irrigated with reclaimed water. Environ. Toxicol. Chem. 2006, 25,
(17) Yiruhan; Wang, Q. J.; Mo, C. H.; Li, Y. W.; Gao, P.; Tai, Y. P.; 317−326.
Zhang, Y.; Ruan, Z. L.; Xu, J. W. Determination of four (37) Chefetz, B.; Mualem, T.; Ben-Ari, J. Sorption and mobility of
fluoroquinolone antibiotics in tap water in Guangzhou and Macao. pharmaceutical compounds in soil irrigated with reclaimed wastewater.
Environ. Pollut. 2010, 158, 2350−2358. Chemosphere 2008, 73, 1335−1343.
(18) Sabourin, L.; Duenk, P.; Bonte-Gelok, S.; Payne, M.; Lapen, D. (38) Li, Y. W.; Wu, X. L.; Mo, C. H.; Tai, Y. P.; Huang, X. P.; Xiang,
R.; Topp, E. Uptake of pharmaceuticals, hormones and parabens into L. Investigation of sulfonamide, tetracycline, and quinolone antibiotics
vegetables grown in soil fertilized with municipal biosolids. Environ. in vegetable farmland soil in the Pearl River Delta area, Southern
Sci. Technol. 2012, 431, 233−236. China. J. Agric. Food Chem. 2011, 59, 7268−7276.
(19) Wu, X.; Ernst, F.; Conkle, J. L.; Gan, J. Comparative uptake and (39) Zhao, L.; Dong, Y. H.; Wang, H. Residues of veterinary
translocation of pharmaceutical and personal care products (PPCPs) antibiotics in manures from feedlot livestock in eight provinces of
by common vegetables. Environ. Int. 2013, 60, 15−22. China. Sci. Total Environ. 2010, 408, 1069−1075.
(20) Kang, D. H.; Gupta, S.; Rosen, C.; Fritz, V.; Singh, A.; Chander, (40) Thiele-Bruhn, S. Pharmaceutical antibiotic compounds in
soilsa review. J. Plant Nutr. Soil Sci. 2003, 166, 145−167.
Y.; Murray, H.; Rohwer, C. Antibiotic uptake by vegetable crops from
(41) Hu, X.; Zhou, Q.; Luo, Y. Occurrence and source analysis of
manure-applied soils. J. Agric. Food Chem. 2013, 61, 9992−10001.
typical veterinary antibiotics in manure, soil, vegetables and ground-
(21) Watkinsona, A. J.; Murbyd, E. J.; Kolpine, D. W.; Costanzo, S.
water from organic vegetable bases, northern China. Environ. Pollut.
D. The occurrence of antibiotics in an urban watershed: From
2010, 158, 2992−2998.
wastewater to drinking water. Sci. Total Environ. 2009, 407, 2711− (42) Kim, Y. H.; Heinze, T. M.; Kim, S. J.; Cerniglia, C. E.
2723. Adsorption and clay-catalyzed degradation of erythromycin A on
(22) Herklotz, P. A.; Gurung, P.; Vanden Heuvel, B.; Kinney, C. A. homoionic clays. J. Environ. Qual. 2004, 33, 257−264.
Uptake of human pharmaceuticals by plants grown under hydroponic (43) Sanderson, H.; Brain, R. A.; Johnson, D. J.; Wilson, C. J.;
conditions. Chemosphere 2010, 78, 1416−1421. Solomon, K. R. Toxicity classification and evaluation of four
(23) Agriculture, Fisheries and Conservation Department. Agriculture pharmaceuticals classes: antibiotics, antineoplastics, cardiovascular,
in HK. http://www.afcd.gov.hk/english/agriculture/agr_hk/agr_hk. and sex hormones. Toxicology 2004, 203, 27−40.
html (16th July 2014). (44) Calderón-Preciado, D.; Matamoros, V.; Bayona, J. M.
(24) Department of Agriculture of GuangDong Province. http:// Occurrence and potential crop uptake of emerging contaminants
www.gdagri.gov.cn/ (16th July 2014). and related compounds in an agricultural irrigation network. Sci. Total
(25) Sarmah, A. K.; Meyer, M. T.; Boxall, A. B. A global perspective Environ. 2011, 412, 14−19.
on the use, sales, exposure pathways, occurrence, fate and effects of (45) Migliore, L.; Civitareale, C.; Cozzolino, S.; Casoria, P.;
veterinary antibiotics (VAs) in the environment. Chemosphere 2006, Brambilla, G.; Gaudio, L. Laboratory models to evaluate phytotoxicity
65, 725−759. of sulphadimethoxine on terrestrial plants. Chemosphere 1998, 37,
(26) Zhou, L. J.; Ying, G. G.; Zhao, J. L.; Yang, J. F.; Wang, L.; Yang, 2957−2961.
B.; Liu, S. Trends in the occurrence of human and veterinary (46) Chiou, C. T.; Sheng, G. Y.; Manes, M. A partition-limited model
antibiotics in the sediments of the Yellow River, Hai River and Liao for the plant uptake of organic contaminants from soil and water.
River in northern China. Environ. Pollut. 2011, 159, 1877−1885. Environ. Sci. Technol. 2001, 35, 1437−1444.
(27) Jiang, Y. H.; Li, M. X.; Guo, C. S.; An, D.; Xu, J.; Zhang, Y.; Xi, (47) Hillis, D. G.; Fletcher, J.; Solomon, K. R.; Sibley, P. K. Effects of
B. D. Distribution and ecological risk of antibiotics in a typical effluent ten antibiotics on seed germination and root elongation in three plant
- receiving river (Wangyang River) in north China. Chemosphere 2014, species. Arch. Environ. Contam. Toxicol. 2011, 60, 220−232.
112, 267−274. (48) Simonich, S. L.; Hites, R. A. Organic pollutant accumulation in
(28) USEPA. QA/QC guidance for sampling and analysis of sediments, vegetation. Environ. Sci. Technol. 1995, 29, 2905−2914.
water, and tissues for dredged material evaluations; U.S. Agency for (49) Trapp, S.; Legind, C. N., Uptake of organic contaminants from
International Development: Washington, DC, 1995; EPA 823-B-95- soil into vegetables and fruits. In Dealing with Contaminated Sites;
001. Swartjes, F. A., Ed.; Springer: New York, 2011; Vol. 9, pp 369−408.
(29) USEPA. Exposure factors handbook; U.S. Agency for Interna- (50) Tanoue, R.; Sato, Y.; Motoyama, M.; Nakagawa, S.; Shinohara,
tional Development: Washington, DC, 2011; EPA 600-R-090-052F. R.; Nomiyama, K. Plant uptake of pharmaceutical chemicals detected
(30) Hummel, D.; Loffler, D.; Fink, G.; Ternes, T. A. Simultaneous in recycled organic manure and reclaimed wastewater. J. Agric. Food
determination of psychoactive drugs and their metabolites in aqueous Chem. 2012, 60, 10203−10211.
matrices by liquid chromatography mass spectrometry. Environ. Sci. (51) Boehme, S.; Werner, G.; Klare, I.; Reissbrodt, R.; Witte, W.
Technol. 2006, 40, 7321−7328. Occurrence of antibiotic-resistant enterobacteria in agricultural
(31) Grujić, S.; Vasiljević, T.; Lausević, M. Determination of multiple foodstuffs. Mol. Nutr. Food Res. 2004, 48, 522−531.
pharmaceutical classes in surface and ground waters by liquid
chromatography-ion trap-tandem mass spectrometry. J. Chromatogr.,
A 2009, 1216, 4989−5000.
(32) Kummerer, K. Antibiotics in the aquatic environment-a review-
part I. Chemosphere 2009, 75, 417−434.
(33) Boxall, A. B. A.; Kolpin, D. W.; Halling-Sørensen, B.; Tolls, J.
Are veterinary medicines causing environmental risks? Environ. Sci.
Technol. 2003, 37, 286−294.
(34) Luo, Y.; Xu, L.; Rysz, M.; Wang, Y. Q.; Zhang, H.; Alvarez, P. J.
J. Occurrence and transport of tetracycline, sulfonamide, quinolone,
and macrolide antibiotics in the Haihe River Basin, China. Environ. Sci.
Technol. 2011, 45, 1827−1833.

H dx.doi.org/10.1021/jf503850v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX