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Occurrences and regional distributions of 20 antibiotics in water bodies during groundwater recharge
Science of the Total Environment 518–519 (2015) 498–506
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Occurrences and regional distributions of 20 antibiotics in water bodies during groundwater recharge Yeping Ma, Miao Li, Miaomiao Wu, Zhen Li, Xiang Liu ⁎ School of Environment, Tsinghua University, Beijing 100084, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Antibiotic pollution was investigated nationwide in China. • Regional differences in antibiotics pollution were observed. • SMZ, OFL and ERY were the top three antibiotics with high ecotoxicological risk.
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 27 February 2015 Accepted 27 February 2015 Available online 13 March 2015 Editor: Adrian Covaci Keywords: Antibiotics Reclaimed water Groundwater Recharge Occurrences Ecotoxicological risk
a b s t r a c t To develop a better understanding of the pollution conditions of antibiotics during the groundwater recharge process, a nation-wide survey was conducted across China for the first time. Overall, 15 recharge sites employing reclaimed water located in different humid, semi-humid and semi-arid regions were selected for analysis of the presence of the 20 most commonly used antibiotics, including tetracyclines (TCs), fluoroquinolones (FQNs), sulfonamides (SAs) and macrolides. All types of antibiotics were detected at concentrations of 212–4035 ng/L in reclaimed water and 19–1270 ng/L in groundwater. FQNs were the predominant antibiotics in reclaimed water samples (38%), followed by SAs (34%). In the SAs group, sulfamethoxazole (SMZ) and sulfamonomethoxine together with trimethoprim accounted for 78% of the total, while ofloxacin (OFL) and norfloxacin accounted for 90% of the FQNs, and doxycycline and oxytetracycline accounted for 82% of the TCs. The concentrations in groundwater were generally 1–2 orders of magnitude lower than in reclaimed water. The three most common antibiotics were OFL, erythromycin (ERY) and SMZ. Similar occurrences of different group antibiotics might be evidence of the influence of groundwater recharge by reclaimed water. FQNs were predominant in northern China, while SAs were predominant in the south. Ecotoxicological risk assessment showed that SMZ, ERY and OFL had the top three hazard quotient values, indicating they should receive preferential treatment before recharging. Overall, these results provide a theoretical basis for development of a recharge standard in China. © 2015 Elsevier B.V. All rights reserved.
Abbreviations: TCs, tetracyclines; FQNs, fluoroquinolones; SAs, sulfonamides; SMZ, sulfamethoxazole; OFL, ofloxacin; ERY, erythromycin. ⁎ Corresponding author at: School of Environment, Tsinghua University, Beijing 100084, China. E-mail address: [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.scitotenv.2015.02.100 0048-9697/© 2015 Elsevier B.V. All rights reserved.
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1. Introduction
2.2. Chemicals
Groundwater is the most important source of water in China, especially in the north, which contains many arid and semi-arid climate zones. According to a study released by the State Council in China, 400 out of 657 cities use groundwater for drinking water in northern China, and 65% of all drinking water in this region originates from groundwater. The large-scale overuse of groundwater has led to a serious shortage of groundwater, resulting in investigation of the potential for groundwater recharge using reclaimed water. Owing to the extremely high water quality required for direct aquifer injection, studies have investigated the use of surface percolation to recharge aquifers with reclaimed water. However, during recharge, certain contaminants that pose risks to the environment and human health may also enter groundwater systems. Indeed, many contaminants, including antibiotics, hormones, and polycyclic aromatic hydrocarbons have been detected in groundwater, especially in areas disturbed by anthropogenic activity. Antibiotics have been extensively used during the past few decades; however, 70% of these are excreted unchanged into wastewater on average (Kümmerer, 2009). Their frequent use and continual input has led to accumulation of antibiotics in the environment (Gao et al., 2012). The amount of human antibiotics used by the general population is greater than that used in hospitals (Thomas et al., 2007); thus, wastewater treatment plants (WWTPs) have become hotspots of antibiotics. Additionally, veterinary antibiotics can enter the environment through manufacturing plants, process effluents, overland flow runoff, and transport from fields to which agricultural waste has been applied (Heberer, 2002). WWTPs play important roles in gathering and eliminating of antibiotics. Unfortunately, many existing water treatment units that employ flocculation, sedimentation and active sludge treatment, as well as some advanced systems that use disinfection and ultrafiltration do not effectively remove antibiotics (Peng et al., 2006); therefore, they are still released into aquatic systems at levels as high as tens to thousands of nanograms per liter (Jiang et al., 2013; Manzetti and Ghisi, 2014; Verlicchi et al., 2012; Zhang et al., 2013). In cases of bank filtration, contaminated river water can flow into groundwater systems, resulting in their contamination. In this investigation, typical groundwater recharge sites in 15 cities across China were investigated and basic information was collected. In addition, samples of reclaimed water and groundwater were collected to detect the concentrations of 20 antibiotics to obtain a general understanding of antibiotic pollution in groundwater throughout China. High risk antibiotics that require treatment before discharge were then screened by the risk quotient method. The results presented herein provide a theoretical basis for development of a guideline for antibiotic controls during groundwater recharge projects.
Twenty antibiotics were selected for this study based on known antibiotics use in China. These antibiotics could be classified into four groups, tetracyclines group (TCs), including tetracycline (TC), chlortetracycline (CTC), doxycycline (DO) and oxytetracycline (OTC); fluoroquinolones group (FQNs), including ciprofloxacin (CIP), OFL, norfloxacin (NOR), enrofloxacin (ENR), lomefloxacin (LOM), and difloxacin (DIF); sulfonamides group (SAs), including trimethoprim (TMP), sulfadiazine (SD), sulfamerazine (SM1), sulfamethazine (SM2), sulfisoxazole (SIZ), SMZ, sulfamonomethoxine (SMM), sulfachlorpyridazine (SCP) and sulfathiazole (STZ); and macrolides group (MCs), including ERY and azithromycin (AZM). Since TMP is usually used as a SA drug synergist, it was categorized into the SAs group. The properties of targeted antibiotics are listed in Table 2. All antibiotics standards were purchased from Dr Ehrenstorfer (Augsburg, Germany). The chemicals were of N 98% purity and used directly in experiments. Standard antibiotic-mix stock solutions with concentrations up to 500 mg/L were prepared in methanol (HPLC grade) and stored in the dark at − 20 °C before use. Working solutions with different concentrations were prepared by diluting the stock solutions before each analytical run.
2. Materials and methods 2.1. Sampling sites Fifteen typical cities in China that use reclaimed water for groundwater recharge were selected owing to their different climatic and geological conditions. For the climate parameter, temperature and precipitation were considered. Locations and detailed information regarding the sites are shown in Fig. 1 and Table 1. Cities C1–C4 were located in Region 1, which is semi-humid, while C12–C15 were located in Region 3, which is semi-arid. Both of these regions are in northern China. Cities C5–C11 were located in Region 2, which is humid and has a relatively higher annual precipitation and temperature. In each city, satisfactory sampling sites were selected based on the presence of reclaimed water reused as a groundwater replenishment source through infiltration by rivers or lakes, available groundwater monitoring wells, and river and lake beds that were not lined with concrete and retained a natural permeability.
2.3. Sample collection and processing All samples were collected between April and early July of 2013. Reclaimed water samples were collected from the outlet of reclaimed water treatment plants and groundwater samples were collected from monitoring wells. Duplicate samples (1 L each) for detection of antibiotics were collected into brown glass bottles. Samples were pretreated immediately upon arrival in the laboratory. 2.4. Quantification of antibiotics Analytical procedures for the 20 antibiotics in reclaimed and groundwater samples were optimized based on EPA Method 1694 (EPA, 2007), with some modifications. Briefly, water samples were filtered through a muffle furnace-burned glass fiber filter (47 mm in diameter with a 0.7 μm pore size) (Whatman, USA). Next, 500 mg Na2EDTA was added to the filtrate to complex divalent cations and the pH value was then adjusted to 3 with HCL (6 mol/L). An antibiotic mix standard solution was then spiked into one of the duplicate filtrates to monitor the pretreatment loss. Solid phase extraction (SPE) was conducted using the Supelco Visiprep SPE system (Supelco, USA), and oasis hydrophilic–lipophilic balance (HLB) cartridges (6 mL/500 mg, Waters, UK) were used to gather antibiotics. The cartridges were pretreated with 5 mL methanol followed by 3 × 5 mL ultrapure water, after which samples were passed through at a loading rate of 1–2 mL min−1. After all samples were loaded, the HLB cartridges were washed with 10 mL ultrapure water and then lyophilized (Labconco Freeze Dry System, USA) for at least 8 h before being eluted with 2 mL of methanol three times. The final eluate was collected into a glass tube and evaporated to dryness using a pressure gas blowing concentrator (Anpel, China). Next, 1 mL methanol containing 100 ng of each internal standard substance was added to redissolve the target antibiotics. Here, sulfamethoxazole (SMZ)-13C6, CIP-13C15 3 N, erythromycin (ERY)-13C2 and d3-thiabendazole were selected for calibration of SAs, FQNs, MCs and TCs group antibiotics according to the EPA method (EPA, 2007). Experiments were performed on an ultraperformance liquid chromatography system and tandem mass spectrophotometer (Waters, UK) equipped with a Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, particle size 1.7 μm). Mass analysis was carried out on a Quattro Premier XE tandem quadrupole mass spectrometer operating in positive ion electrospray mode (ESI(+)) (Waters, USA). The procedure was described in detail in the Supplementary Information.
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Fig. 1. Map of sample sites in China.
Recovery experiments were performed by spiking standard solutions into 1 L of water to give a final concentration of 100 ng/L for each antibiotic. The method recoveries for reclaimed and groundwater were 63–107% and 71–103%, respectively. The instrumental detection limits were determined by direct injection of standard solutions at low concentrations. Method detection limits and method quantitation limits (MQLs) were defined as the concentrations that yielded signalto-noise ratios of 3:1 and 10:1, respectively. The MQLs are listed in Table S3 (See Supplementary Information).
2.5. Hazard quotients and ecotoxicological risk assessment Most approaches for antibiotic ecotoxicological risk assessment were based on the European Medicines Agency (EMA) guidelines, in which ecotoxicological risks are expressed as ratios between the predicted environmental concentrations (PECs) and the predicted no-effect concentrations (PNECs) (Grung et al., 2008). HQs higher or equal to 1 suggest that the particular substances had the potential to cause adverse ecological effects. PECs are generally estimated using calculation models based
Table 1 Description of sampling locations. Climatic conditions
Recharge information Distancec (km)
Type of the aquifer
Matrix
Surrounding land use
20 260
0.03 0.5
Unconfined aquifer Confined aquifer
Farmland Residential area
18 10 10
28 30 5
0.4 5.4 3.0
Unconfined aquifer Unconfined aquifer Unconfined aquifer
13 4.5 7.0 8.5 11 13 6.0 6.0
10 8 1.5 10 20 10 8.5 4.76
15 15 1 5 3 12 10 10
0.3 0.4 0.1 0.02 0.85 2.5 0.6 0.4
Unconfined aquifer Confined aquifer Vadose zone Vadose zone Vadose zone Unconfined aquifer Unconfined aquifer Unconfined aquifer
6.5 3.0
10 0.46
70 6
1.0 0.45
Confined aquifer Unconfined aquifer
Sandy soil Loam and sandy loam Loam Sandy clay Silt and sandy loam Loam Loam Loam Loam Loam Sandy loam Sandy clay Sandy loam soil Loam Sandy soil
City
Region
Precipitation (mm)
Temperature zone
C1 C2
Nanyang Tianjin
1
400–800
Warm temperate zone
6.5 9.5
C3 C4 C5
Jinan Beijing Nantong
Subtropical zone
11.5 7.5 10
C6 C7 C8 C9 C10 C11 C12 C13
Chengdu Hefei Shanghai Changzhou Guangzhou Nanning Xining Taiyuan
Middle temperate zone
C14 C15
Xi'an Baotou
2
3
800–1600
200–400
Geological conditions Depthb (m)
No.
Recharge historya (year)
Note: a As of June 2013. b Depth of the sample well. c Distance between sampling locations of reclaimed water and groundwater.
Daily recharge quantity (104 m3) 1 2.38
Residential area Parkland Industrial land Landscape Residential area Riverway Riverway Residential area Residential area Residential area Landscape Parkland Farmland
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Table 2 Physicochemical properties of targeted antibiotics. Groups
Chemicals
Abbreviation
CAS #
Mol. weight
Water solubility (mg/L at 25 °C)a
Log Kowa
Koc (L/kg)a
PNEC (μg/L)
Tetracyclines (TCs)
Tetracycline hydrochloride Chlortetracycline hydrochloride Doxycycline Oxytetracycline hydrochloride Ciprofloxacin hydrochloride Ofloxacin Norfloxacin Enrofloxacin Lomefloxacin hydrochloride Difloxacin hydrochloride Trimethoprim Sulfadiazine Sulfamerazine Sulfamethazine Sulfisoxazole Sulfamethoxazole Sulfamonomethoxine Sulfachloropyridazine Sulfathiazole Erythromycin Azithromycin
TC CTC DO OTC CIP OFL NOR ENR LOM DIF TMP SD SM1 SM2 SIZ SMZ SMM SCP STZ ERY AZM
64-75-5 64-72-2 24390-14-5 2058-46-0 93107-08-5 82419-36-1 70458-96-7 93106-60-6 98079-52-8 91296-86-5 738-70-5 68-35-9 127-79-7 57-68-1 127-69-5 723-46-6 38006-08-5 80-32-0 72-14-0 114-07-8 83905-01-5
480.90 515.30 512.90 496.90 367.80 361.37 319.30 359.40 387.81 435.90 290.30 250.30 264.30 278.33 267.30 253.27 320.20 284.74 255.32 733.90 748.98
2.31 × 102 8.49 × 104 3.13 × 102 3.13 × 102 3.00 × 104 2.83 × 104 1.78 × 105 3.40 × 103 2.72 × 104 1.33 × 103 4.00 × 102 7.70 × 101 1.49 × 104 1.50 × 103 2.60 × 103 6.10 × 102 4.03 × 103 7.00 × 103 3.73 × 102 5.17 × 10−1 6.20 × 10−2
−1.30 −3.60 −0.02 −0.90 0.28 0.35h −1.03 0.70 −0.30 0.89 0.91 −0.09 0.14 0.19 1.01 0.89 0.70 0.31 0.05 3.06 4.02
4.00 × 103 b 3.23 × 101 4.94 × 101 6.79 × 104b 6.10 × 104b 1.22 × 101 5.67 × 102 1.49 × 101 –i 5.53 × 102 1.68 × 103–3.99 × 103b 3.70 × 101–1.25 × 102b 1.19 × 102 8.20 × 101–2.08 × 102b 4.32 × 102 2.58 × 102 4.76 × 101 1.19 × 102 1.61 × 102 5.67 × 102 3.14 × 103
9.00 × 10−2 c 5.00d 3.00 × 10−1 c 2.07 × 10−1 c, 3.10 × 10−1d 9.38 × 102 c 1.60 × 10−2 c 15.0c, 20 e 4.90 × 10−2f 1.30 × 102 e 4.35 × 102a 2.60 c 1.35 × 10−1 c, 2.20 e 1.59 × 103 a 2.02 × 102g, 1.28 × 102d 4.35 × 102a 2.70 × 10−2 c 1.72 × 103 a 2.64 × 101 c 8.54 × 101 c, 4.44 × 101d 2.00 × 10−2 c, 2.00 × 10−1d 1.50 × 10−1 c
Fluoroquinolones (FQNs)
Sulfonamides (SAs)
Macrolides (MCs)
Note: a Water solubility, log Kow and Koc were obtained from Estimation Program Interface (EPI) Suite Version 4.11 ed. (USEPA (2012)). b Sarmah et al. (2006). c Verlicchi et al. (2012). d Ji et al. (2012). e Zheng et al. (2012). f Montforts (2005). g Yang et al. (2011). h Tolls (2001). i Not available.
on production/use volume of antibiotics or a maximum daily dose of 1% of the population consuming the pharmaceutical. However, these data are scarce or have a high level of uncertainty in China. In addition, it has been reported that PECs were always overestimated (Liebig et al., 2006). In such instances, determination of measured environmental concentrations (MECs) instead of PECs using Eq. (1) has been shown to be a reliable method of minimizing the adverse impact (Santos et al., 2007). In this study, the highest MECs and the lowest PNECs of each antibiotic were used to conduct a conservative assessment. The values of PNECs were collected from the literature and, if not available, the lowest values of EC50 or LC50 obtained from EPI (EPA, 2012) were used to calculate PNECs by Eq. (2). A safety factor of 1000 was used to divide EC50 or LC50. HQ ¼ MECs=PNECs
ð1Þ
PNECs ¼ ðEC50 or LC50 Þ=1000:
ð2Þ
3. Results and discussion 3.1. Occurrence and distributions of selected antibiotics in reclaimed water and groundwater The frequency of detection and concentrations of 20 target antibiotics in reclaimed water and groundwater are presented in Table 3, while the distributions of the four groups of antibiotics are shown in Fig. 2. Composition percentages of each group are displayed in Fig. 3. All types of antibiotics were detected in both the reclaimed water and groundwater samples, indicating an alarming antibiotic residue problem in water environments. The detection frequencies of 15 antibiotics were higher than 50% in reclaimed water samples, with median values that ranged from not
detected (ND) to 185 ng/L. The total concentrations of TCs, SAs, FQNs and MCs were 1162, 7438, 6608 and 4316 ng/L, respectively. The composition distributions in reclaimed water showed that FQNs were the primary antibiotics (38.1%), followed by SAs (33.8%). Specifically, ofloxacin (OFL), ERY and SMZ were the three most abundant antibiotics in reclaimed water, with mean concentrations of 343, 190 and 144 ng/L, respectively. In each group, certain types of antibiotics that accounted for the majority of the group might be considered representative compounds indicative of antibiotic pollution. SMZ, sulfamonomethoxine (SMM) and TMP accounted for 78% of the SAs, while OFL and norfloxacin (NOR) accounted for 90% of the FQNs and doxycycline (DO) and oxytetracycline (OTC) accounted for 82% of the TCs. Since only two types of MCs were detected, more data are needed to identify priority compounds. However, ERY was present at higher concentrations than any other antibiotics, except OFL, in reclaimed water in this study. The level of antibiotic pollution in reclaimed was higher in the present study than in previous studies. For example, although a lower concentration of FQNs was observed for CIP in this study, the total concentrations remained high and many more types of FQNs were observed, including enrofloxacin (ENR), lomefloxacin (LOM) and difloxacin (DIF). The concentration of FQNs in the final wastewater effluents from a densely populated region in Switzerland ranged from 36 to 106 ng/L (Golet et al., 2002). In the United States, CIP and OFL were frequently detected in effluent at concentrations of 80 to 2000 ng/L (Renew and Huang, 2004; Bhandari et al., 2008). NOR and ENR were reported to be present at mean concentrations of 120 and ND ng/L (Kolpin et al., 2002). In Canada, CIP, NOR and OFL were also detected in the final effluent of eight WWTPs, with maximum concentrations as high as 0.12 mg/L for CIP and ~110 ng/L for NOR and OFL (Miao et al., 2004). In Europe, OFL was detected at concentrations of 330–510 ng/L in France, 290–580 in Italy and 460 ng/L in Greece (Andreozzi et al., 2003). In China, Peng et al. (2006) detected SMZ, SM1, OFL and three other antibiotics in the secondary and tertiary
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Table 3 Occurrence of antibiotics in reclaimed water and groundwater of China. Compounds
Reclaimed water (n = 15) Frequency (%)
Tetracyclines TC CTC DO OTC
Max (ng/L)
Groundwater (n = 15) Med (ng/L)
Min (ng/L)
Frequency (%)
Max (ng/L)
Med (ng/L)
Min (ng/L)
46.7 46.7 66.7 80.0
39 66 191 80
NDa ND 9.5 9.7
ND ND ND ND
60.0 66.7 46.7 60.0
48 76 39 39
ND 1.4 ND 2.9
ND ND ND ND
Fluoroquinolones CIP OFL NOR ENR LOM DIF
46.7 100.0 53.3 66.7 86.7 66.7
99 1119 703 45 83 23
ND 185 11 ND 1.8 0.4
ND 7.8 ND ND ND ND
40.0 93.3 26.7 86.7 86.7 66.7
155 80 503 49 159 35
ND 14 ND 1.3 1.0 ND
ND ND ND ND ND ND
Sulfonamides SM1 SM2 SIZ SMZ SMM SCP STZ TMP
53.3 53.3 40.0 100.0 86.7 46.7 53.3 100.0
242 469 17 492 909 171 128 736
3.0 2.4 80 15 ND 2.3 ND 58
ND ND ND 2.4 ND ND ND 3.3
66.7 46.7 33.3 93.3 66.7 26.7 26.7 80.0
15 49 8.4 250 29 117 32 40
1.1 ND ND 15 ND ND ND 9.4
ND ND ND ND ND ND ND ND
83.3 80.0
833 248
72 86
ND ND
93.3 73.3
143 73
16 4.8
ND ND
Macrolides ERY AZM a
Below the MDLs.
effluents of two WWTPs in Guangzhou. In their study, OFL was found at a concentration of 0.86 ng/L in the secondary and 0.74 ng/L in the tertiary effluent, which was lower than the levels observed in the present study. The detection frequencies of selected antibiotics were about the same in groundwater samples as those of reclaimed water, with most types of TCs and ENR, LOM and DIF in FQNs group having even higher detection frequencies in groundwater. Additionally, the frequencies of 13 antibiotics were higher than 50%, implying that groundwater is a potential reservoir of antibiotics in water environments. The concentration of antibiotics in groundwater was 1–2 orders of magnitude lower than that in the reclaimed water, with a mean concentration between 4.4 and 57 ng/L. SMZ, NOR and ERY were predominant in the groundwater, being present at 57, 35, 34 ng/L, respectively. A few papers have reported the occurrence of antibiotic residues in groundwater of different
regions. Hirsch et al. (1999) reported SMZ in groundwater wells close to the irrigation area with a concentration of 470 ng/L and SM2 deriving from veterinary applications with concentrations of 80–160 ng/L. Sacher et al. (2001) reported the occurrence of SMZ (up to 410 ng/L) and dehydro-erythromycin (up to 49 ng/L) in groundwater samples in Baden-Württemberg, Germany. Heberer found that, under recharge conditions, polar PhACs could leach through the subsoil and that they have also been detected at measurable (but low) concentrations in several groundwater samples in Germany (Heberer et al., 2008, 2004; Heberer, 2002). In addition, groundwater samples from bank-filtration sites in Nebraska in the United States were analyzed for 24 antibiotics and only SMZ and TMP were detected at concentrations of less than 1 μg/L (Heberer et al., 2001). When compared with these findings, more types of antibiotics, especially FQNs group, were found in China at similar or higher concentrations. However, the distributions of TCs,
Fig. 2. Distributions of four groups of antibiotics and relative proportions in reclaimed water and groundwater. (a) Reclaimed water and (b) Groundwater. Areas of the circle represent the relative concentrations between reclaimed water and groundwater.
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Fig. 3. Composition of each antibiotic group in reclaimed water and groundwater. (a) TCs, (b) FQNs, (c) SAs, and (d) MCs. RW, reclaimed water; and GW, ground water.
FQNs and SAs in groundwater differed from those in reclaimed water. The distribution became somewhat more even in groundwater (Fig. 3). CTC (37%) became the dominating compound of the TCs group, followed by TC, and the percentage of DO decreased from 53% to 9%. The even distributions were clearer in the FQNs group, with NOR, LOM, OFL and CIP accounting for nearly the same percentage (around 18–25%). SMZ (56%) was still the predominant SAs antibiotic in groundwater, followed by TMP, SCP, sulfamethazine (SM2), STZ, SMM and SM1 in descending order from 11% to 5%. The therapeutic ranges and environmental behaviors of the four selected groups of antibiotics varied owing to their physicochemical properties. Among all groups, FQNs have been recognized as potent antibiotics for the last four decades owing to their broad spectrum activity against Gram positive and negative bacteria. Certain compounds, such as ERN, were developed as veterinary medicines, while CIP, NOR and OFL are primarily used to treat humans (Alfredo et al., 2001), and may therefore be present in higher concentrations in reclaimed water. Several studies have reported that no FQN residues were detected in groundwater (Vazquez et al., 2012); however, in the present study, the concentration of FQNs in groundwater was up to tens to hundreds of nanograms. These findings may reflect the high regional consumption. In China, the annual quinolone consumption for humans and animals was about 1350 and 470 tons, respectively (WHO, 1998), which would cause high accumulation in the environment. Although some FQN compounds have a tendency to bind to soil (Hektoen et al., 1995; Sukul and Spiteller, 2007), once they enter the groundwater FQNs showed lower degradation than under aerobic conditions. SAs have been widely used for veterinary purposes. Most SAs have high solubility and low binding to soil, which enables them to enter groundwater easily. SMZ was the dominant antibiotic in this group owing to its large use. A total of 16 SAs of seven groundwater bodies
in Spain were analyzed and SMZ was detected most frequently (56.4%), with an average concentration of 0.2 ng/L (Garcia-Galan et al., 2011). Batt et al. (2006) examined six private water wells to assess the impact of a nearby animal feeding operation and found SM2 at 76–220 ng/L. A landfill survey conducted in Denmark revealed that compounds with high water-solubility and low octanol–water distribution coefficients could be highly mobile and migrate with the same velocity as groundwater in the aquifer (Holm et al., 1995). However, the concentrations found in this study were more likely associated with the consumption of medicine. In contrast, TCs are always strong chelators with the potential to combine with soils (Lindsey et al., 2001); therefore, they may not occur in groundwater. However, in our investigation, all four TC antibiotics were detected in groundwater, with CTC (37%) being the dominant compound, followed by TC. Nevertheless, the percentage of DO in reclaimed water and groundwater decreased from 53% to 9%. The water solubility showed a remarkable difference between CTC and other TC antibiotics, which might explain the larger percentage of CTC in groundwater. Moreover, the average concentration of CTC in groundwater was higher than that in reclaimed water, indicating that the infiltration showed a limited effect to cut down CTC, but had the potential to leach existing contaminants from soils. Lindsey et al. (2001) investigated the presence of three TCs in groundwater in Washington, but detected none. Hu et al. (2010) analyzed the residual antibiotics in groundwater of an organic vegetable base in northern China and found that only TC was detected at 5.2 ng/L. MCs, which act as inhibitors of ribosomal protein synthesis, are mainly active against Gram-positive bacteria. These compounds are generally not metabolized to a large extent, but are primarily excreted with bile and feces (Göbel et al., 2005). To the best of our knowledge, few studies have reported the concentrations of MCs in groundwater.
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However, high concentrations of ERY and AZM were detected in this study. Accordingly, studies of their environmental behavior should be conducted to better explain the occurrence and distribution of antibiotics in groundwater. 3.2. Regional distributions of selected antibiotics Different climate conditions in China lead to an uneven water distribution; therefore, diverse patterns of water use are applied among different regions. For instance, in semi-humid areas, the effluent of WWTPs is always reused as a supplemental source of groundwater or scenic environmental water. Thus, it is important to determine the antibiotic distribution in different climate regions of China. In this study, a series of reclaimed water and groundwater samples were collected from 15 cities located in semi-humid (C1–C4, Region 1), humid (C5– C11, Region 2) and semi-arid (C12–C15, Region 3) regions. The distribution of the added concentrations of the antibiotic groups of different regions is shown in Fig. 4. The average concentrations of antibiotics in reclaimed water did not differ significantly, with levels of about 1300 ng/L being present in all regions. However, the groundwater concentrations differed obviously, with 527 ng/L being present in the semi-humid region, 345 ng/L in the humid region and 154 ng/L in the semi-arid region. The regional differences suggested that climate conditions may influence groundwater quality during the infiltration process. Nine of the 15 cities showed levels above 1000 ng/L in reclaimed water, with C5 (Nanyang) having an extremely high concentration of 4035 ng/L. Take C4 (Beijing) for instance, water from Wenyu River, which mainly composed of WWTP effluent, was further treated in a membrane bio-reactor to produce
reclaimed water. Similar distributions, that SAs and FQNs were the dominant groups of antibiotics were found in both Wenyu River and reclaimed water (Zhang et al., 2014). The variations in antibiotics in reclaimed water could be due to many factors, including antibiotic consumption patterns, effects of WWTPs scale, and different treatment technologies. In groundwater, the total concentrations ranged from 19 (C2, Tianjin) to 1270 (C3, Jinan). C3 had the largest daily recharge quantity of 1.8 × 105 m3 and longer recharge histories than most recharge sites, which may explain the higher concentration in groundwater of C3. Previous studies have reported that higher concentrations of inorganic materials (Ca2+ and Mg2+) could decrease the removal efficiency of some specific antibiotics (Li and Zhang, 2011). In the present study, the total hardness of the samples was detected and a weak positive correlation between concentrations of antibiotics and total hardness was shown in groundwater (Table S4; Supplementary Information). Further studies are needed to explain these results. The compositions of antibiotics also differed among cities. In regions 1 and 3, FQNs comprised the majority of antibiotics in reclaimed water, with levels as high as 650 ng/L accounting for 45.5% of the total in region 1 to 52.2% in region 3. In region 2, SAs were the dominant group, being present at 677 ng/L and accounting for 54.7% of the total. However, the composition in groundwater was not the same as that in reclaimed water. In region 1, FQNs accounted for 61.6% of the total and were present at 325 ng/L. In region 2, FQNs and SAs accounted for 65.2% of the antibiotics (in almost equal proportion), followed by TCs and MCs. In region 3, all types of antibiotics were detected at lower levels than in other regions, with a total concentration of 154 ng/L, and SAs accounting for 59.8% of the total. Overall, the difference in concentrations in reclaimed water between areas were believed to be related to antibiotic usage, wastewater treatment processes and concentrations in groundwater, which might have been affected by the parameters of recharge sites and geological conditions. 3.3. Ecotoxicological risk assessment Since WWTPs cannot completely remove all the contaminants, several types of antibiotics may enter the groundwater via water infiltration. However, the risk of contamination may be reduced for some antibiotics that are adsorbed to soil particles or subject to biodegradation. Conversely, leaching or transformation might amplify the risks. To prevent groundwater from being polluted and control the recharge process, priority antibiotics need to be screened. However, recharge sites show varying hydrogeological condition and recharge histories; thus, specific assessments should be considered. Ecotoxicological risks were expressed by hazard quotients (HQs) as the ratios between MECs and PNECs. Here, the maximum concentrations of each antibiotic found in 15 groundwater and reclaimed water samples was used as the MEC. HQs for both groundwater and reclaimed water were calculated and are shown in Fig. 5. In groundwater samples, SMZ, ERY, OFL and ENR were found to have HQs larger than 1. Four other antibiotics (TC, AZM, OTC and DO) that had HQs between 0.1 and 1 may pose a mild risk to groundwater. In reclaimed water, similar results were observed, with AZM showing the fourth highest risk. The risks associated with ENR, TC and SMZ were not clearly reduced during the infiltration process, which is in accordance with the results of our pervious study (Li et al., 2014). Residues of antibiotics in aquatic environments are believed to increase antibiotic resistance and influence human health; therefore, much more detailed work regarding the risk of antibiotics is needed. 4. Conclusions
Fig. 4. Occurrence and distribution of antibiotics in different cities. (a) Concentrations of reclaimed water, and (b) concentrations of groundwater.
In this study, a nationwide survey of China was conducted to evaluate antibiotic pollution during groundwater recharge. Fifteen recharge sites were investigated and the occurrence of antibiotics in both reclaimed water and groundwater was measured. The detection
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Fig. 5. Hazard quotients (HQs) of antibiotic residues in reclaimed water and groundwater.
frequencies of 20 types of antibiotics were 100%, and the total concentrations of TCs, SAs, FQNs and MCs were 212–4035 ng/L in reclaimed water and 19–1270 ng/L in groundwater. Obvious regional differences in composition distributions were observed, with FQNs and SAs being the predominant antibiotics in northern and southern China, respectively. The daily recharge quantity, recharge histories, total hardness and climate conditions may help explain the occurrence and distributions. Overall, SMZ, ERY, and OFL showed higher HQ values and concentrations and should therefore be focused on before conducting groundwater recharge. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgments The authors thank the National Natural Science Foundation of China (No. 51378287), Major Science and Technology Program for Water Pollution Control and Treatment (No. 2012ZX07301-001), national 863 plans (No. 2013AA065205) and the Special Environmental Research Funds for Public Welfare (No. 201209053) for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2015.02.100. References Alfredo, C.A., Christa, S.M., Eva, M.G., Slavica, I., Eva, M., Norriel, S.N., Walter, G., 2001. Am. Chem. Soc. 56–69. Andreozzi, R., Raffaele, M., Nicklas, P., 2003. Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment. Chemosphere 50 (10), 1319–1330. Batt, A.L., Snow, D.D., Aga, D.S., 2006. Occurrence of sulfonamide antimicrobials in private water wells in Washington County, Idaho, USA. Chemosphere 64 (11), 1963–1971. Bhandari, A., Close, L., Kim, W., Hunter, R., Koch, D., Surampalli, R., 2008. Occurrence of ciprofloxacin, sulfamethoxazole, and azithromycin in municipal wastewater treatment plants. Pract. Period. Hazard. Toxic Radioact. Waste Manag. 12 (4), 275–281. EPA, U., 2007. Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS. EPA, U., 2012. Estimation Program Interface (EPI) Suite Version 4.11.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Occurrences and regional distributions of 20 antibiotics in water bodies during groundwater recharge Yeping Ma, Miao Li, Miaomiao Wu, Zhen Li, Xiang Liu ⁎ School of Environment, Tsinghua University, Beijing 100084, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Antibiotic pollution was investigated nationwide in China. • Regional differences in antibiotics pollution were observed. • SMZ, OFL and ERY were the top three antibiotics with high ecotoxicological risk.
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 27 February 2015 Accepted 27 February 2015 Available online 13 March 2015 Editor: Adrian Covaci Keywords: Antibiotics Reclaimed water Groundwater Recharge Occurrences Ecotoxicological risk
a b s t r a c t To develop a better understanding of the pollution conditions of antibiotics during the groundwater recharge process, a nation-wide survey was conducted across China for the first time. Overall, 15 recharge sites employing reclaimed water located in different humid, semi-humid and semi-arid regions were selected for analysis of the presence of the 20 most commonly used antibiotics, including tetracyclines (TCs), fluoroquinolones (FQNs), sulfonamides (SAs) and macrolides. All types of antibiotics were detected at concentrations of 212–4035 ng/L in reclaimed water and 19–1270 ng/L in groundwater. FQNs were the predominant antibiotics in reclaimed water samples (38%), followed by SAs (34%). In the SAs group, sulfamethoxazole (SMZ) and sulfamonomethoxine together with trimethoprim accounted for 78% of the total, while ofloxacin (OFL) and norfloxacin accounted for 90% of the FQNs, and doxycycline and oxytetracycline accounted for 82% of the TCs. The concentrations in groundwater were generally 1–2 orders of magnitude lower than in reclaimed water. The three most common antibiotics were OFL, erythromycin (ERY) and SMZ. Similar occurrences of different group antibiotics might be evidence of the influence of groundwater recharge by reclaimed water. FQNs were predominant in northern China, while SAs were predominant in the south. Ecotoxicological risk assessment showed that SMZ, ERY and OFL had the top three hazard quotient values, indicating they should receive preferential treatment before recharging. Overall, these results provide a theoretical basis for development of a recharge standard in China. © 2015 Elsevier B.V. All rights reserved.
Abbreviations: TCs, tetracyclines; FQNs, fluoroquinolones; SAs, sulfonamides; SMZ, sulfamethoxazole; OFL, ofloxacin; ERY, erythromycin. ⁎ Corresponding author at: School of Environment, Tsinghua University, Beijing 100084, China. E-mail address: [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.scitotenv.2015.02.100 0048-9697/© 2015 Elsevier B.V. All rights reserved.
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1. Introduction
2.2. Chemicals
Groundwater is the most important source of water in China, especially in the north, which contains many arid and semi-arid climate zones. According to a study released by the State Council in China, 400 out of 657 cities use groundwater for drinking water in northern China, and 65% of all drinking water in this region originates from groundwater. The large-scale overuse of groundwater has led to a serious shortage of groundwater, resulting in investigation of the potential for groundwater recharge using reclaimed water. Owing to the extremely high water quality required for direct aquifer injection, studies have investigated the use of surface percolation to recharge aquifers with reclaimed water. However, during recharge, certain contaminants that pose risks to the environment and human health may also enter groundwater systems. Indeed, many contaminants, including antibiotics, hormones, and polycyclic aromatic hydrocarbons have been detected in groundwater, especially in areas disturbed by anthropogenic activity. Antibiotics have been extensively used during the past few decades; however, 70% of these are excreted unchanged into wastewater on average (Kümmerer, 2009). Their frequent use and continual input has led to accumulation of antibiotics in the environment (Gao et al., 2012). The amount of human antibiotics used by the general population is greater than that used in hospitals (Thomas et al., 2007); thus, wastewater treatment plants (WWTPs) have become hotspots of antibiotics. Additionally, veterinary antibiotics can enter the environment through manufacturing plants, process effluents, overland flow runoff, and transport from fields to which agricultural waste has been applied (Heberer, 2002). WWTPs play important roles in gathering and eliminating of antibiotics. Unfortunately, many existing water treatment units that employ flocculation, sedimentation and active sludge treatment, as well as some advanced systems that use disinfection and ultrafiltration do not effectively remove antibiotics (Peng et al., 2006); therefore, they are still released into aquatic systems at levels as high as tens to thousands of nanograms per liter (Jiang et al., 2013; Manzetti and Ghisi, 2014; Verlicchi et al., 2012; Zhang et al., 2013). In cases of bank filtration, contaminated river water can flow into groundwater systems, resulting in their contamination. In this investigation, typical groundwater recharge sites in 15 cities across China were investigated and basic information was collected. In addition, samples of reclaimed water and groundwater were collected to detect the concentrations of 20 antibiotics to obtain a general understanding of antibiotic pollution in groundwater throughout China. High risk antibiotics that require treatment before discharge were then screened by the risk quotient method. The results presented herein provide a theoretical basis for development of a guideline for antibiotic controls during groundwater recharge projects.
Twenty antibiotics were selected for this study based on known antibiotics use in China. These antibiotics could be classified into four groups, tetracyclines group (TCs), including tetracycline (TC), chlortetracycline (CTC), doxycycline (DO) and oxytetracycline (OTC); fluoroquinolones group (FQNs), including ciprofloxacin (CIP), OFL, norfloxacin (NOR), enrofloxacin (ENR), lomefloxacin (LOM), and difloxacin (DIF); sulfonamides group (SAs), including trimethoprim (TMP), sulfadiazine (SD), sulfamerazine (SM1), sulfamethazine (SM2), sulfisoxazole (SIZ), SMZ, sulfamonomethoxine (SMM), sulfachlorpyridazine (SCP) and sulfathiazole (STZ); and macrolides group (MCs), including ERY and azithromycin (AZM). Since TMP is usually used as a SA drug synergist, it was categorized into the SAs group. The properties of targeted antibiotics are listed in Table 2. All antibiotics standards were purchased from Dr Ehrenstorfer (Augsburg, Germany). The chemicals were of N 98% purity and used directly in experiments. Standard antibiotic-mix stock solutions with concentrations up to 500 mg/L were prepared in methanol (HPLC grade) and stored in the dark at − 20 °C before use. Working solutions with different concentrations were prepared by diluting the stock solutions before each analytical run.
2. Materials and methods 2.1. Sampling sites Fifteen typical cities in China that use reclaimed water for groundwater recharge were selected owing to their different climatic and geological conditions. For the climate parameter, temperature and precipitation were considered. Locations and detailed information regarding the sites are shown in Fig. 1 and Table 1. Cities C1–C4 were located in Region 1, which is semi-humid, while C12–C15 were located in Region 3, which is semi-arid. Both of these regions are in northern China. Cities C5–C11 were located in Region 2, which is humid and has a relatively higher annual precipitation and temperature. In each city, satisfactory sampling sites were selected based on the presence of reclaimed water reused as a groundwater replenishment source through infiltration by rivers or lakes, available groundwater monitoring wells, and river and lake beds that were not lined with concrete and retained a natural permeability.
2.3. Sample collection and processing All samples were collected between April and early July of 2013. Reclaimed water samples were collected from the outlet of reclaimed water treatment plants and groundwater samples were collected from monitoring wells. Duplicate samples (1 L each) for detection of antibiotics were collected into brown glass bottles. Samples were pretreated immediately upon arrival in the laboratory. 2.4. Quantification of antibiotics Analytical procedures for the 20 antibiotics in reclaimed and groundwater samples were optimized based on EPA Method 1694 (EPA, 2007), with some modifications. Briefly, water samples were filtered through a muffle furnace-burned glass fiber filter (47 mm in diameter with a 0.7 μm pore size) (Whatman, USA). Next, 500 mg Na2EDTA was added to the filtrate to complex divalent cations and the pH value was then adjusted to 3 with HCL (6 mol/L). An antibiotic mix standard solution was then spiked into one of the duplicate filtrates to monitor the pretreatment loss. Solid phase extraction (SPE) was conducted using the Supelco Visiprep SPE system (Supelco, USA), and oasis hydrophilic–lipophilic balance (HLB) cartridges (6 mL/500 mg, Waters, UK) were used to gather antibiotics. The cartridges were pretreated with 5 mL methanol followed by 3 × 5 mL ultrapure water, after which samples were passed through at a loading rate of 1–2 mL min−1. After all samples were loaded, the HLB cartridges were washed with 10 mL ultrapure water and then lyophilized (Labconco Freeze Dry System, USA) for at least 8 h before being eluted with 2 mL of methanol three times. The final eluate was collected into a glass tube and evaporated to dryness using a pressure gas blowing concentrator (Anpel, China). Next, 1 mL methanol containing 100 ng of each internal standard substance was added to redissolve the target antibiotics. Here, sulfamethoxazole (SMZ)-13C6, CIP-13C15 3 N, erythromycin (ERY)-13C2 and d3-thiabendazole were selected for calibration of SAs, FQNs, MCs and TCs group antibiotics according to the EPA method (EPA, 2007). Experiments were performed on an ultraperformance liquid chromatography system and tandem mass spectrophotometer (Waters, UK) equipped with a Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, particle size 1.7 μm). Mass analysis was carried out on a Quattro Premier XE tandem quadrupole mass spectrometer operating in positive ion electrospray mode (ESI(+)) (Waters, USA). The procedure was described in detail in the Supplementary Information.
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Fig. 1. Map of sample sites in China.
Recovery experiments were performed by spiking standard solutions into 1 L of water to give a final concentration of 100 ng/L for each antibiotic. The method recoveries for reclaimed and groundwater were 63–107% and 71–103%, respectively. The instrumental detection limits were determined by direct injection of standard solutions at low concentrations. Method detection limits and method quantitation limits (MQLs) were defined as the concentrations that yielded signalto-noise ratios of 3:1 and 10:1, respectively. The MQLs are listed in Table S3 (See Supplementary Information).
2.5. Hazard quotients and ecotoxicological risk assessment Most approaches for antibiotic ecotoxicological risk assessment were based on the European Medicines Agency (EMA) guidelines, in which ecotoxicological risks are expressed as ratios between the predicted environmental concentrations (PECs) and the predicted no-effect concentrations (PNECs) (Grung et al., 2008). HQs higher or equal to 1 suggest that the particular substances had the potential to cause adverse ecological effects. PECs are generally estimated using calculation models based
Table 1 Description of sampling locations. Climatic conditions
Recharge information Distancec (km)
Type of the aquifer
Matrix
Surrounding land use
20 260
0.03 0.5
Unconfined aquifer Confined aquifer
Farmland Residential area
18 10 10
28 30 5
0.4 5.4 3.0
Unconfined aquifer Unconfined aquifer Unconfined aquifer
13 4.5 7.0 8.5 11 13 6.0 6.0
10 8 1.5 10 20 10 8.5 4.76
15 15 1 5 3 12 10 10
0.3 0.4 0.1 0.02 0.85 2.5 0.6 0.4
Unconfined aquifer Confined aquifer Vadose zone Vadose zone Vadose zone Unconfined aquifer Unconfined aquifer Unconfined aquifer
6.5 3.0
10 0.46
70 6
1.0 0.45
Confined aquifer Unconfined aquifer
Sandy soil Loam and sandy loam Loam Sandy clay Silt and sandy loam Loam Loam Loam Loam Loam Sandy loam Sandy clay Sandy loam soil Loam Sandy soil
City
Region
Precipitation (mm)
Temperature zone
C1 C2
Nanyang Tianjin
1
400–800
Warm temperate zone
6.5 9.5
C3 C4 C5
Jinan Beijing Nantong
Subtropical zone
11.5 7.5 10
C6 C7 C8 C9 C10 C11 C12 C13
Chengdu Hefei Shanghai Changzhou Guangzhou Nanning Xining Taiyuan
Middle temperate zone
C14 C15
Xi'an Baotou
2
3
800–1600
200–400
Geological conditions Depthb (m)
No.
Recharge historya (year)
Note: a As of June 2013. b Depth of the sample well. c Distance between sampling locations of reclaimed water and groundwater.
Daily recharge quantity (104 m3) 1 2.38
Residential area Parkland Industrial land Landscape Residential area Riverway Riverway Residential area Residential area Residential area Landscape Parkland Farmland
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Table 2 Physicochemical properties of targeted antibiotics. Groups
Chemicals
Abbreviation
CAS #
Mol. weight
Water solubility (mg/L at 25 °C)a
Log Kowa
Koc (L/kg)a
PNEC (μg/L)
Tetracyclines (TCs)
Tetracycline hydrochloride Chlortetracycline hydrochloride Doxycycline Oxytetracycline hydrochloride Ciprofloxacin hydrochloride Ofloxacin Norfloxacin Enrofloxacin Lomefloxacin hydrochloride Difloxacin hydrochloride Trimethoprim Sulfadiazine Sulfamerazine Sulfamethazine Sulfisoxazole Sulfamethoxazole Sulfamonomethoxine Sulfachloropyridazine Sulfathiazole Erythromycin Azithromycin
TC CTC DO OTC CIP OFL NOR ENR LOM DIF TMP SD SM1 SM2 SIZ SMZ SMM SCP STZ ERY AZM
64-75-5 64-72-2 24390-14-5 2058-46-0 93107-08-5 82419-36-1 70458-96-7 93106-60-6 98079-52-8 91296-86-5 738-70-5 68-35-9 127-79-7 57-68-1 127-69-5 723-46-6 38006-08-5 80-32-0 72-14-0 114-07-8 83905-01-5
480.90 515.30 512.90 496.90 367.80 361.37 319.30 359.40 387.81 435.90 290.30 250.30 264.30 278.33 267.30 253.27 320.20 284.74 255.32 733.90 748.98
2.31 × 102 8.49 × 104 3.13 × 102 3.13 × 102 3.00 × 104 2.83 × 104 1.78 × 105 3.40 × 103 2.72 × 104 1.33 × 103 4.00 × 102 7.70 × 101 1.49 × 104 1.50 × 103 2.60 × 103 6.10 × 102 4.03 × 103 7.00 × 103 3.73 × 102 5.17 × 10−1 6.20 × 10−2
−1.30 −3.60 −0.02 −0.90 0.28 0.35h −1.03 0.70 −0.30 0.89 0.91 −0.09 0.14 0.19 1.01 0.89 0.70 0.31 0.05 3.06 4.02
4.00 × 103 b 3.23 × 101 4.94 × 101 6.79 × 104b 6.10 × 104b 1.22 × 101 5.67 × 102 1.49 × 101 –i 5.53 × 102 1.68 × 103–3.99 × 103b 3.70 × 101–1.25 × 102b 1.19 × 102 8.20 × 101–2.08 × 102b 4.32 × 102 2.58 × 102 4.76 × 101 1.19 × 102 1.61 × 102 5.67 × 102 3.14 × 103
9.00 × 10−2 c 5.00d 3.00 × 10−1 c 2.07 × 10−1 c, 3.10 × 10−1d 9.38 × 102 c 1.60 × 10−2 c 15.0c, 20 e 4.90 × 10−2f 1.30 × 102 e 4.35 × 102a 2.60 c 1.35 × 10−1 c, 2.20 e 1.59 × 103 a 2.02 × 102g, 1.28 × 102d 4.35 × 102a 2.70 × 10−2 c 1.72 × 103 a 2.64 × 101 c 8.54 × 101 c, 4.44 × 101d 2.00 × 10−2 c, 2.00 × 10−1d 1.50 × 10−1 c
Fluoroquinolones (FQNs)
Sulfonamides (SAs)
Macrolides (MCs)
Note: a Water solubility, log Kow and Koc were obtained from Estimation Program Interface (EPI) Suite Version 4.11 ed. (USEPA (2012)). b Sarmah et al. (2006). c Verlicchi et al. (2012). d Ji et al. (2012). e Zheng et al. (2012). f Montforts (2005). g Yang et al. (2011). h Tolls (2001). i Not available.
on production/use volume of antibiotics or a maximum daily dose of 1% of the population consuming the pharmaceutical. However, these data are scarce or have a high level of uncertainty in China. In addition, it has been reported that PECs were always overestimated (Liebig et al., 2006). In such instances, determination of measured environmental concentrations (MECs) instead of PECs using Eq. (1) has been shown to be a reliable method of minimizing the adverse impact (Santos et al., 2007). In this study, the highest MECs and the lowest PNECs of each antibiotic were used to conduct a conservative assessment. The values of PNECs were collected from the literature and, if not available, the lowest values of EC50 or LC50 obtained from EPI (EPA, 2012) were used to calculate PNECs by Eq. (2). A safety factor of 1000 was used to divide EC50 or LC50. HQ ¼ MECs=PNECs
ð1Þ
PNECs ¼ ðEC50 or LC50 Þ=1000:
ð2Þ
3. Results and discussion 3.1. Occurrence and distributions of selected antibiotics in reclaimed water and groundwater The frequency of detection and concentrations of 20 target antibiotics in reclaimed water and groundwater are presented in Table 3, while the distributions of the four groups of antibiotics are shown in Fig. 2. Composition percentages of each group are displayed in Fig. 3. All types of antibiotics were detected in both the reclaimed water and groundwater samples, indicating an alarming antibiotic residue problem in water environments. The detection frequencies of 15 antibiotics were higher than 50% in reclaimed water samples, with median values that ranged from not
detected (ND) to 185 ng/L. The total concentrations of TCs, SAs, FQNs and MCs were 1162, 7438, 6608 and 4316 ng/L, respectively. The composition distributions in reclaimed water showed that FQNs were the primary antibiotics (38.1%), followed by SAs (33.8%). Specifically, ofloxacin (OFL), ERY and SMZ were the three most abundant antibiotics in reclaimed water, with mean concentrations of 343, 190 and 144 ng/L, respectively. In each group, certain types of antibiotics that accounted for the majority of the group might be considered representative compounds indicative of antibiotic pollution. SMZ, sulfamonomethoxine (SMM) and TMP accounted for 78% of the SAs, while OFL and norfloxacin (NOR) accounted for 90% of the FQNs and doxycycline (DO) and oxytetracycline (OTC) accounted for 82% of the TCs. Since only two types of MCs were detected, more data are needed to identify priority compounds. However, ERY was present at higher concentrations than any other antibiotics, except OFL, in reclaimed water in this study. The level of antibiotic pollution in reclaimed was higher in the present study than in previous studies. For example, although a lower concentration of FQNs was observed for CIP in this study, the total concentrations remained high and many more types of FQNs were observed, including enrofloxacin (ENR), lomefloxacin (LOM) and difloxacin (DIF). The concentration of FQNs in the final wastewater effluents from a densely populated region in Switzerland ranged from 36 to 106 ng/L (Golet et al., 2002). In the United States, CIP and OFL were frequently detected in effluent at concentrations of 80 to 2000 ng/L (Renew and Huang, 2004; Bhandari et al., 2008). NOR and ENR were reported to be present at mean concentrations of 120 and ND ng/L (Kolpin et al., 2002). In Canada, CIP, NOR and OFL were also detected in the final effluent of eight WWTPs, with maximum concentrations as high as 0.12 mg/L for CIP and ~110 ng/L for NOR and OFL (Miao et al., 2004). In Europe, OFL was detected at concentrations of 330–510 ng/L in France, 290–580 in Italy and 460 ng/L in Greece (Andreozzi et al., 2003). In China, Peng et al. (2006) detected SMZ, SM1, OFL and three other antibiotics in the secondary and tertiary
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Table 3 Occurrence of antibiotics in reclaimed water and groundwater of China. Compounds
Reclaimed water (n = 15) Frequency (%)
Tetracyclines TC CTC DO OTC
Max (ng/L)
Groundwater (n = 15) Med (ng/L)
Min (ng/L)
Frequency (%)
Max (ng/L)
Med (ng/L)
Min (ng/L)
46.7 46.7 66.7 80.0
39 66 191 80
NDa ND 9.5 9.7
ND ND ND ND
60.0 66.7 46.7 60.0
48 76 39 39
ND 1.4 ND 2.9
ND ND ND ND
Fluoroquinolones CIP OFL NOR ENR LOM DIF
46.7 100.0 53.3 66.7 86.7 66.7
99 1119 703 45 83 23
ND 185 11 ND 1.8 0.4
ND 7.8 ND ND ND ND
40.0 93.3 26.7 86.7 86.7 66.7
155 80 503 49 159 35
ND 14 ND 1.3 1.0 ND
ND ND ND ND ND ND
Sulfonamides SM1 SM2 SIZ SMZ SMM SCP STZ TMP
53.3 53.3 40.0 100.0 86.7 46.7 53.3 100.0
242 469 17 492 909 171 128 736
3.0 2.4 80 15 ND 2.3 ND 58
ND ND ND 2.4 ND ND ND 3.3
66.7 46.7 33.3 93.3 66.7 26.7 26.7 80.0
15 49 8.4 250 29 117 32 40
1.1 ND ND 15 ND ND ND 9.4
ND ND ND ND ND ND ND ND
83.3 80.0
833 248
72 86
ND ND
93.3 73.3
143 73
16 4.8
ND ND
Macrolides ERY AZM a
Below the MDLs.
effluents of two WWTPs in Guangzhou. In their study, OFL was found at a concentration of 0.86 ng/L in the secondary and 0.74 ng/L in the tertiary effluent, which was lower than the levels observed in the present study. The detection frequencies of selected antibiotics were about the same in groundwater samples as those of reclaimed water, with most types of TCs and ENR, LOM and DIF in FQNs group having even higher detection frequencies in groundwater. Additionally, the frequencies of 13 antibiotics were higher than 50%, implying that groundwater is a potential reservoir of antibiotics in water environments. The concentration of antibiotics in groundwater was 1–2 orders of magnitude lower than that in the reclaimed water, with a mean concentration between 4.4 and 57 ng/L. SMZ, NOR and ERY were predominant in the groundwater, being present at 57, 35, 34 ng/L, respectively. A few papers have reported the occurrence of antibiotic residues in groundwater of different
regions. Hirsch et al. (1999) reported SMZ in groundwater wells close to the irrigation area with a concentration of 470 ng/L and SM2 deriving from veterinary applications with concentrations of 80–160 ng/L. Sacher et al. (2001) reported the occurrence of SMZ (up to 410 ng/L) and dehydro-erythromycin (up to 49 ng/L) in groundwater samples in Baden-Württemberg, Germany. Heberer found that, under recharge conditions, polar PhACs could leach through the subsoil and that they have also been detected at measurable (but low) concentrations in several groundwater samples in Germany (Heberer et al., 2008, 2004; Heberer, 2002). In addition, groundwater samples from bank-filtration sites in Nebraska in the United States were analyzed for 24 antibiotics and only SMZ and TMP were detected at concentrations of less than 1 μg/L (Heberer et al., 2001). When compared with these findings, more types of antibiotics, especially FQNs group, were found in China at similar or higher concentrations. However, the distributions of TCs,
Fig. 2. Distributions of four groups of antibiotics and relative proportions in reclaimed water and groundwater. (a) Reclaimed water and (b) Groundwater. Areas of the circle represent the relative concentrations between reclaimed water and groundwater.
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Fig. 3. Composition of each antibiotic group in reclaimed water and groundwater. (a) TCs, (b) FQNs, (c) SAs, and (d) MCs. RW, reclaimed water; and GW, ground water.
FQNs and SAs in groundwater differed from those in reclaimed water. The distribution became somewhat more even in groundwater (Fig. 3). CTC (37%) became the dominating compound of the TCs group, followed by TC, and the percentage of DO decreased from 53% to 9%. The even distributions were clearer in the FQNs group, with NOR, LOM, OFL and CIP accounting for nearly the same percentage (around 18–25%). SMZ (56%) was still the predominant SAs antibiotic in groundwater, followed by TMP, SCP, sulfamethazine (SM2), STZ, SMM and SM1 in descending order from 11% to 5%. The therapeutic ranges and environmental behaviors of the four selected groups of antibiotics varied owing to their physicochemical properties. Among all groups, FQNs have been recognized as potent antibiotics for the last four decades owing to their broad spectrum activity against Gram positive and negative bacteria. Certain compounds, such as ERN, were developed as veterinary medicines, while CIP, NOR and OFL are primarily used to treat humans (Alfredo et al., 2001), and may therefore be present in higher concentrations in reclaimed water. Several studies have reported that no FQN residues were detected in groundwater (Vazquez et al., 2012); however, in the present study, the concentration of FQNs in groundwater was up to tens to hundreds of nanograms. These findings may reflect the high regional consumption. In China, the annual quinolone consumption for humans and animals was about 1350 and 470 tons, respectively (WHO, 1998), which would cause high accumulation in the environment. Although some FQN compounds have a tendency to bind to soil (Hektoen et al., 1995; Sukul and Spiteller, 2007), once they enter the groundwater FQNs showed lower degradation than under aerobic conditions. SAs have been widely used for veterinary purposes. Most SAs have high solubility and low binding to soil, which enables them to enter groundwater easily. SMZ was the dominant antibiotic in this group owing to its large use. A total of 16 SAs of seven groundwater bodies
in Spain were analyzed and SMZ was detected most frequently (56.4%), with an average concentration of 0.2 ng/L (Garcia-Galan et al., 2011). Batt et al. (2006) examined six private water wells to assess the impact of a nearby animal feeding operation and found SM2 at 76–220 ng/L. A landfill survey conducted in Denmark revealed that compounds with high water-solubility and low octanol–water distribution coefficients could be highly mobile and migrate with the same velocity as groundwater in the aquifer (Holm et al., 1995). However, the concentrations found in this study were more likely associated with the consumption of medicine. In contrast, TCs are always strong chelators with the potential to combine with soils (Lindsey et al., 2001); therefore, they may not occur in groundwater. However, in our investigation, all four TC antibiotics were detected in groundwater, with CTC (37%) being the dominant compound, followed by TC. Nevertheless, the percentage of DO in reclaimed water and groundwater decreased from 53% to 9%. The water solubility showed a remarkable difference between CTC and other TC antibiotics, which might explain the larger percentage of CTC in groundwater. Moreover, the average concentration of CTC in groundwater was higher than that in reclaimed water, indicating that the infiltration showed a limited effect to cut down CTC, but had the potential to leach existing contaminants from soils. Lindsey et al. (2001) investigated the presence of three TCs in groundwater in Washington, but detected none. Hu et al. (2010) analyzed the residual antibiotics in groundwater of an organic vegetable base in northern China and found that only TC was detected at 5.2 ng/L. MCs, which act as inhibitors of ribosomal protein synthesis, are mainly active against Gram-positive bacteria. These compounds are generally not metabolized to a large extent, but are primarily excreted with bile and feces (Göbel et al., 2005). To the best of our knowledge, few studies have reported the concentrations of MCs in groundwater.
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However, high concentrations of ERY and AZM were detected in this study. Accordingly, studies of their environmental behavior should be conducted to better explain the occurrence and distribution of antibiotics in groundwater. 3.2. Regional distributions of selected antibiotics Different climate conditions in China lead to an uneven water distribution; therefore, diverse patterns of water use are applied among different regions. For instance, in semi-humid areas, the effluent of WWTPs is always reused as a supplemental source of groundwater or scenic environmental water. Thus, it is important to determine the antibiotic distribution in different climate regions of China. In this study, a series of reclaimed water and groundwater samples were collected from 15 cities located in semi-humid (C1–C4, Region 1), humid (C5– C11, Region 2) and semi-arid (C12–C15, Region 3) regions. The distribution of the added concentrations of the antibiotic groups of different regions is shown in Fig. 4. The average concentrations of antibiotics in reclaimed water did not differ significantly, with levels of about 1300 ng/L being present in all regions. However, the groundwater concentrations differed obviously, with 527 ng/L being present in the semi-humid region, 345 ng/L in the humid region and 154 ng/L in the semi-arid region. The regional differences suggested that climate conditions may influence groundwater quality during the infiltration process. Nine of the 15 cities showed levels above 1000 ng/L in reclaimed water, with C5 (Nanyang) having an extremely high concentration of 4035 ng/L. Take C4 (Beijing) for instance, water from Wenyu River, which mainly composed of WWTP effluent, was further treated in a membrane bio-reactor to produce
reclaimed water. Similar distributions, that SAs and FQNs were the dominant groups of antibiotics were found in both Wenyu River and reclaimed water (Zhang et al., 2014). The variations in antibiotics in reclaimed water could be due to many factors, including antibiotic consumption patterns, effects of WWTPs scale, and different treatment technologies. In groundwater, the total concentrations ranged from 19 (C2, Tianjin) to 1270 (C3, Jinan). C3 had the largest daily recharge quantity of 1.8 × 105 m3 and longer recharge histories than most recharge sites, which may explain the higher concentration in groundwater of C3. Previous studies have reported that higher concentrations of inorganic materials (Ca2+ and Mg2+) could decrease the removal efficiency of some specific antibiotics (Li and Zhang, 2011). In the present study, the total hardness of the samples was detected and a weak positive correlation between concentrations of antibiotics and total hardness was shown in groundwater (Table S4; Supplementary Information). Further studies are needed to explain these results. The compositions of antibiotics also differed among cities. In regions 1 and 3, FQNs comprised the majority of antibiotics in reclaimed water, with levels as high as 650 ng/L accounting for 45.5% of the total in region 1 to 52.2% in region 3. In region 2, SAs were the dominant group, being present at 677 ng/L and accounting for 54.7% of the total. However, the composition in groundwater was not the same as that in reclaimed water. In region 1, FQNs accounted for 61.6% of the total and were present at 325 ng/L. In region 2, FQNs and SAs accounted for 65.2% of the antibiotics (in almost equal proportion), followed by TCs and MCs. In region 3, all types of antibiotics were detected at lower levels than in other regions, with a total concentration of 154 ng/L, and SAs accounting for 59.8% of the total. Overall, the difference in concentrations in reclaimed water between areas were believed to be related to antibiotic usage, wastewater treatment processes and concentrations in groundwater, which might have been affected by the parameters of recharge sites and geological conditions. 3.3. Ecotoxicological risk assessment Since WWTPs cannot completely remove all the contaminants, several types of antibiotics may enter the groundwater via water infiltration. However, the risk of contamination may be reduced for some antibiotics that are adsorbed to soil particles or subject to biodegradation. Conversely, leaching or transformation might amplify the risks. To prevent groundwater from being polluted and control the recharge process, priority antibiotics need to be screened. However, recharge sites show varying hydrogeological condition and recharge histories; thus, specific assessments should be considered. Ecotoxicological risks were expressed by hazard quotients (HQs) as the ratios between MECs and PNECs. Here, the maximum concentrations of each antibiotic found in 15 groundwater and reclaimed water samples was used as the MEC. HQs for both groundwater and reclaimed water were calculated and are shown in Fig. 5. In groundwater samples, SMZ, ERY, OFL and ENR were found to have HQs larger than 1. Four other antibiotics (TC, AZM, OTC and DO) that had HQs between 0.1 and 1 may pose a mild risk to groundwater. In reclaimed water, similar results were observed, with AZM showing the fourth highest risk. The risks associated with ENR, TC and SMZ were not clearly reduced during the infiltration process, which is in accordance with the results of our pervious study (Li et al., 2014). Residues of antibiotics in aquatic environments are believed to increase antibiotic resistance and influence human health; therefore, much more detailed work regarding the risk of antibiotics is needed. 4. Conclusions
Fig. 4. Occurrence and distribution of antibiotics in different cities. (a) Concentrations of reclaimed water, and (b) concentrations of groundwater.
In this study, a nationwide survey of China was conducted to evaluate antibiotic pollution during groundwater recharge. Fifteen recharge sites were investigated and the occurrence of antibiotics in both reclaimed water and groundwater was measured. The detection
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Fig. 5. Hazard quotients (HQs) of antibiotic residues in reclaimed water and groundwater.
frequencies of 20 types of antibiotics were 100%, and the total concentrations of TCs, SAs, FQNs and MCs were 212–4035 ng/L in reclaimed water and 19–1270 ng/L in groundwater. Obvious regional differences in composition distributions were observed, with FQNs and SAs being the predominant antibiotics in northern and southern China, respectively. The daily recharge quantity, recharge histories, total hardness and climate conditions may help explain the occurrence and distributions. Overall, SMZ, ERY, and OFL showed higher HQ values and concentrations and should therefore be focused on before conducting groundwater recharge. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgments The authors thank the National Natural Science Foundation of China (No. 51378287), Major Science and Technology Program for Water Pollution Control and Treatment (No. 2012ZX07301-001), national 863 plans (No. 2013AA065205) and the Special Environmental Research Funds for Public Welfare (No. 201209053) for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2015.02.100. References Alfredo, C.A., Christa, S.M., Eva, M.G., Slavica, I., Eva, M., Norriel, S.N., Walter, G., 2001. Am. Chem. Soc. 56–69. Andreozzi, R., Raffaele, M., Nicklas, P., 2003. Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment. Chemosphere 50 (10), 1319–1330. Batt, A.L., Snow, D.D., Aga, D.S., 2006. Occurrence of sulfonamide antimicrobials in private water wells in Washington County, Idaho, USA. Chemosphere 64 (11), 1963–1971. Bhandari, A., Close, L., Kim, W., Hunter, R., Koch, D., Surampalli, R., 2008. Occurrence of ciprofloxacin, sulfamethoxazole, and azithromycin in municipal wastewater treatment plants. Pract. Period. Hazard. Toxic Radioact. Waste Manag. 12 (4), 275–281. EPA, U., 2007. Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS. EPA, U., 2012. Estimation Program Interface (EPI) Suite Version 4.11.
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