Distribution of Antibiotics in Wastewater-Irrigated Soils and Their

Oct 30, 2014 - Food Chem. , 2014, 62 (46), pp 11062–11069 ... The Pearl River Delta (PRD) is a major agricultural region in southern China, where la...
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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

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recent years.5,17−19 However, these studies either detected the concentrations of antibiotics in surface water or soil separately or restricted their plant uptake experiments to only one type of antibiotic under artificially high levels.20,21 Some studies were conducted in hydroponic or greenhouse environments, which do not reflect plant uptake and accumulation of antibiotics under genuine field conditions.19,22 Accordingly, there is limited knowledge on the distribution and potential effects of antibiotics on the terrestrial environment and food quality. The Pearl River Delta (PRD) is a major agricultural region in southern China, where large quantities of vegetables and edible crops are produced and exported to other cities and countries. Roughly 90% of the vegetables consumed in Hong Kong are supplied from mainland China,23 mostly from the PRD. However, 20 million hectare of the arable land in China was contaminated by heavy metals and 10 million hectare by organic pollutants, while 3.3 million hectare was contaminated as a result of wastewater irrigation.24 Therefore, identifying the concentrations of different types of antibiotics in irrigation wastewater, their accumulation in irrigated soil, and their uptake and accumulation in edible crops would allow a more accurate assessment of the risks they pose to the environment and human health. The objectives of this study were (1) to investigate the presence and distribution of different types of antibiotics in irrigation water and irrigated soil at different sites in the PRD and (2) to comparatively evaluate the uptake and translocation

INTRODUCTION Wastewater is commonly used to irrigate agricultural land and urban greeneries, and replenish surface water and groundwater in response to the water shortage resulting from climate change, regional drought, population growth, and pollution.1,2 Globally, at least 20 million hectares of arable land are irrigated with wastewater that has been treated to various extents.3 In China, water availability is especially low and unevenly distributed,4 and domestic wastewater and fishpond water have become valuable resources for crop irrigation. However, wastewater usually contains toxic inorganic and organic pollutants and pathogens, which are mostly biologically active and create further potential risk when they enter into the environment.5−7 Although some guidelines have been developed in regard to irrigation water quality criteria, they do not take into account the potential risk from trace amounts of organic pollutants,8−10 including pharmaceuticals and personal care products (PPCPs) in surface water and treated effluent from wastewater treatment plants.11−13 Despite their low environmental concentrations (usually in the range of ng/L to μg/L in water), these compounds have the potential to accumulate in irrigated soil or be taken up by plants when the wastewater is used to irrigate edible crops. Nonionic PPCPs were taken up at higher concentrations by root crops,14 and PPCPs were detected in the edible tissues of a number of vegetables irrigated with treated wastewater and fortified water.15 Antibiotics include thousands of diverse chemicals that humans and animals ingest for health or cosmetic reasons.16 These bioactive chemicals eventually enter into the environment after usage and as residuals from pharmaceutical manufacturing. There is a growing body of literature on the occurrence of antibiotic compounds in water, soil, and plants in © XXXX American Chemical Society

Received: August 11, 2014 Revised: October 28, 2014 Accepted: October 30, 2014

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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.

sampled. The region contains more than 11 million hectares of arable land, and the sampling sites were representative regarding farming practice and had long history (>20 y) of wastewater reuse. The 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 used for irrigation in Dongguan and Shenzhen (Figure 1). Irrigation water, soil, and crops were sampled in July 2013. Water samples were collected in 1 L amber glass bottles, with 50 mL of methanol immediately added and pH adjusted to 3.0 using 4 M H2SO4 to preserve the aqueous samples (five replicates per sampling site). Soil and crop samples were collected from five random plots (100 × 100 m2) on each sampling site. Soil samples were collected at two depths: 0−10 cm and 10−20 cm. An adjacent plot without cultivation served as the control with no antibiotic contamination. Soil samples were characterized, and their physicochemical properties are given in Table S1 in the Supporting Information. Chinese white cabbage, water spinach, Chinese radish, corn, and rice were harvested around the soilsampling sites when the crops reached marketable size. Five whole crops were randomly collected from each plot, which were pooled to form a bulk sample to result in five replicates per site. Crops cultivated in a greenhouse without wastewater irrigation were used as control crops. Once in the laboratory, the water samples were passed through 0.7 μm glass fiber filters (Whatman GF/F, U.K.), and extraction was carried out within 3 days. Soil samples and fresh crops were rinsed

of antibiotics in various crops. Findings from this study would facilitate a better understanding of the transfer of antibiotics to the soil−plant system and the potential ecological and human risks through exposure to antibiotic contamination.

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MATERIALS AND METHODS

Antibiotics and Chemicals. Target antibiotic compounds were selected mainly based on their frequent usage in human and livestock in the PRD and other regions as well as their environmental behaviors.25−27 Tetracycline (TC), sulfamethazine (SMZ), norfloxacin (NOR), erythromycin (ERY), and chloramphenicol (CAP), which belong to five different antibiotic types, were examined in this study (Table 1). All standards and some internal standards (SMZ-d4, NORd5, and ERY-13C2) were obtained from Sigma-Aldrich (USA), CAP-d5 was purchased from Dr Ehrenstorfer GmbH (Germany), and TC-d6 was obtained from Toronto Research Chemicals (Canada). Oasis HLB extraction cartridges (6 mL, 500 mg) (Waters Corporation, USA) were used for the extraction and purification of the target compounds. All the organic solvents used were of HPLC grade and purchased from Merck Corporation (Germany). Individual stock solutions and internal standards were prepared at 100 mg/L in methanol and stored in an amber glass vial at −20 °C. Sampling Sites. Six locations (Huizhou, Foshan, Zhongshan, Guangzhou, Dongguan, and Shenzhen) in the PRD region were B

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Figure 1. Information on the sampling sites in the Pearl River Delta, southern China. Liquid Chromatography Mass Spectrometry Analysis. The target antibiotic compounds were analyzed by HPLC−MS/MS (Agilent Liquid Chromatography 1100 series HPLC system coupled to an Agilent 6410 triple quadrupole MS) equipped with an electrospray ionization (ESI) source (Agilent, USA) in multiplereaction monitoring (MRM) mode. The analyses were performed in negative mode for CAP and in positive mode for the other antibiotics. Nitrogen gas was used as the drying and collision gas. The chromatographic column was an Agilent Eclipse XDB-C18 (3.0 × 75 mm, 3.5 μm) column with a Poroshell 120 precolumn filter (3.0 mm, 0.2 μm). The column temperature was maintained at 40 °C for the analysis of the antibiotics. For ESI+, 0.01% formic acid was used as mobile phase A and acetonitrile as mobile phase B. Gradient conditions were set as follows: 0 min, 20% B; 3 min, 40% B; 6 min, 60% B; 8 min, 80% B; 9 min, 95% B and 13 min, 20% B. For ESI−, Milli-Q water was used as mobile 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 mobile phases were 0.3 mL/min and 0.35 mL/min for the positive and negative modes, respectively. Mass spectrometric conditions were optimized using Optimizer (Agilent, USA) for fine-tuning of the fragmentor voltage, collision energy (CE), and multiple reaction monitoring mode (MRM) transitions for each antibiotic (Table S2 in the Supporting Information). The following optimized parameters were selected: drying gas temperature 350 °C, drying gas flow rate 9 mL/min (ESI+), 11 mL/min (ESI−), capillary voltage 3500 V. The system was reequilibrated for 5 min between runs. Estimation of the Bioconcentration Factor and Dietary Intake. The ability of a plant to accumulate antibiotics from the soil was estimated using the bioconcentration factor (BCF), which was calculated as the ratio of the chemical concentration in the crop tissue to the chemical concentration in the soil (all based on dry weight):

with deionized water and freeze-dried. The dried samples were then ground to powder and stored at −20 °C until extraction. Extraction. Water Samples. The target antibiotics in the water samples were extracted by solid phase extraction (SPE) using Oasis HLB cartridges. The internal standards (100 ng) and 0.2 g of disodium ethylenediaminetetraacetate (Na2EDTA) were spiked into each water sample (1 L). The SPE cartridges were preconditioned with 10 mL of methanol and 10 mL of Milli-Q water, after which the water samples were passed through the cartridge at a flow rate of 10 mL/min. The analytes were eluted from the cartridge with 10 mL of methanol, concentrated under a gentle nitrogen stream, and then redissolved in 1 mL of methanol. The final extract was filtered through a nylon syringe filter into a 2 mL amber glass vial and stored at −18 °C before the LC−MS/MS analysis. Soil Samples. One gram of freeze-dried soil was ground up and spiked with 100 μL of each internal standard (1.0 mg/L). The soil samples were extracted three times with 30 mL of acetonitrile and 0.2 M citric acid buffer (pH 4.4) (v:v = 1:1) by vortex (60 s each time) and ultrasonication (15 min each time). The mixture was centrifuged in air-cooled conditions at 1370 g for 10 min and concentrated; the extract for each soil sample was evaporated to near dryness, 0.2 g of Na2EDTA was added, and the mixture was diluted to 200 mL with Milli-Q water. Each cartridge was preconditioned with 10 mL of methanol and 10 mL of Milli-Q water, and the extracts were passed through HLB cartridges for purification. The analytes were eluted from the cartridge with 10 mL of methanol and dried under a gentle nitrogen stream. Finally, the residue was redissolved in 1 mL of methanol for analysis. Crop Samples. One gram of each freeze-dried crop tissue was ground and spiked with 100 μL of each internal standard (1.0 mg/L). The crop samples were extracted three times with 30 mL of mixed solution of acidified acetonitrile (pH 3) and acetone (v:v = 1:1) by vortex mixing (60 s each time) and ultrasonication (15 min each), followed by centrifugation in air-cooled conditions at 12000g for 15 min. The combined extractions were pipetted into a 200 mL roundbottom flask and evaporated to remove the organic solvent; 0.2 g of Na2EDTA was added, and the mixture was diluted to 200 mL with Milli-Q water and passed through the preconditioned HLB cartridges for purification. The analytes were then eluted from the cartridge with 10 mL of methanol and dried under a gentle nitrogen stream. Finally, the residue was redissolved in 1 mL of methanol for analysis.

BCF =

concentration in crop tissue (μg/kg) concentration in soil (μg/kg)

An exploratory assessment of the potential human exposure to antibiotics through the consumption of edible crops was conducted. The level of human exposure was calculated as human exposure = C × D × W × T C

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where C is the concentration of antibiotics in the edible tissue (ng/g wet weight), D is the average daily consumption of edible crops (g wet weight/kg body weight day), W is the body weight (kg) of the person consuming the crops, and T is the exposure time (day). The concentrations of antibiotic in crop were converted to a fresh weight basis using the average water content of each crop (95.3% for Chinese white cabbage, 91.4% for water spinach, 95.3% for Chinese radish, 76.0% for corn, and 11.6% for rice).28 The annual exposure of a 70 kg individual was estimated using the average daily consumption of edible crops (11.8, 0.6, 0.5, 9.2, and 24.0 g wet weight/kg body weight-day for Chinese white cabbage, water spinach, Chinese radish, corn, and rice, respectively).29 Quantification and Quality Control. To account for the potential analyte loss, a reagent blank, method blank, and spiked matrices were analyzed together with the water, soil, and crop samples. None of the analytes were detected in the blank controls for water, soil, and crop samples. Quantification of each antibiotic compound was obtained using the internal standard method. Analytical precision was measured by analyzing one sample in triplicate for every 10 samples, and the calculated relative standard deviation was less than 10%.28 The recoveries of the target compounds ranged between 70% and 120% (Table S3 in the Supporting Information). Limit of detection (LOD) and limit of quantification (LOQ) of the antibiotics were calculated with signal/noise ratios (S/N) of 3 and 10, respectively. The S/N ratios were obtained by using the software Masshunter (Agilent, USA). The LOQ of the target compounds ranged from 0.40 to 1.13 ng/L for the water samples, 0.70 to 4.63 ng/g for the soil samples, and 0.80 to 4.40 ng/g for the crop samples (Table S3 in the Supporting Information). Ten concentrations (0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 ng/L) of individual antibiotics were used to calculate the calibration curves (r2 > 0.999). Recoveries of the study compounds were performed by spiking the standard solutions to DI water, soil, and crop tissue samples. Ion suppression and enhancement are considered as the major sources of inaccurate quantitative results in complex matrices.22,30 Initial recovery was done using the internal standards and standard solutions to correct for matrix effects, spiking 100 μg/L of each standard into the sample extracts and comparing the obtained concentration in matrix to the concentration in solvent. Values of smaller or greater than 100% indicate signal suppression and enhancement, respectively.31

L for SMZ. These results were in general agreement with those for agricultural irrigation water.32,33 The wastewaters in Dongguan and Shenzhen contained significantly higher concentrations of antibiotics (ranging 12.9−234.0 ng/L and 10.5−184.0 ng/L, respectively) than those in the other regions (p < 0.05). This can be attributed to the common practice of directly adding antibiotics to water or in medicated feeds to control bacterial diseases in farmed fish.34 In the irrigation waters from Foshan, Zhongshan, and Guangzhou, TC and CAP were found at relatively high concentrations. The wastewater used for crop irrigation in Huizhou had the lowest concentrations of the various antibiotics. The cropland in Huizhou is located on the upper-middle reaches of the Danshui River and has less interference from human activities. Antibiotics in Soils. TC, SMZ, NOR, ERY, and CAP were all detected in the surface layer (0−10 cm) irrigated soil samples from the six sites (Table 2). TC, NOR, and CAP in most cases had significantly higher concentrations in soils irrigated by fishpond water than those by domestic wastewater (p < 0.05). TC, NOR, and CAP showed a similar trend in concentration in the corresponding irrigation waters (r = 0.93, p < 0.05). All five target antibiotics detected in the irrigation water could be found in the irrigated soils. Both the irrigation water and irrigated soil samples from Dongguan and Shenzhen had significantly higher concentrations than the other sampling sites, especially for TC and CAP (p < 0.05), suggesting that the irrigation water (domestic wastewater and fishpond water) was the major contributing source. Antibiotics persist in the environment after they are released into the soil from irrigation water.35 Although wastewater is also used for irrigation in other countries, few studies have examined the presence and accumulation of antibiotics in irrigated soils and crops.5,36,37 Kinney et al. (2006)36 detected ERY in reclaimed water and wastewater-irrigated soils at concentrations of 177−611 ng/L and 0.02−15 μg/kg, respectively. Chen et al. (2011)5 found TC, NOR, and CAP 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 significantly higher in Dongguan than in the other sites (p < 0.05), which corresponded to their higher concentrations in the irrigation water. NOR was frequently found at concentrations 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 deprotonated species would be increased, which would have decreased its sorption to the soil.33,40 Therefore, SMZ is very

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RESULTS AND DISCUSSION Antibiotics in Irrigation Water. The target antibiotics were detected at concentrations above the LOQ in the irrigation water samples (Figure 2). The highest concentration was 69.3−234.0 ng/L for TC, and the lowest was 4.0−58.2 ng/

Figure 2. Concentrations (ng/L) of antibiotics in irrigation water in the Pearl River Delta, China (mean ± SD, n = 5). D

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Table 2. Concentrations (μg/kg dw) of Antibiotics Detected in the Irrigated Soils in the Pearl River Delta, China (Mean ± SD, n = 5) HZ FS ZS GZ DG SZ

soil depth (cm)

TC

0−10 10−20 0−10 10−20 0−10 10−20 0−10 10−20 0−10 10−20 0−10 10−20

8.9 ± 0.2