Seasonal variation of antibiotics concentration in the aquatic environment: a case study at Jianghan Plain, central China

Science of the Total Environment 527–528 (2015) 56–64

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Seasonal variation of antibiotics concentration in the aquatic environment: a case study at Jianghan Plain, central China Linlin Yao a, Yanxin Wang a,⁎, Lei Tong a, Yonggang Li b, Yamin Deng a, Wei Guo a, Yiqun Gan a a b

School of Environmental Studies, State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, 430074 Wuhan, China Hubei Provincial Center for Disease Control & Prevention, Wuhan 430074, China

H I G H L I G H T S • • • • •

25 antibiotics were detected in surface water and groundwater seasonally. Higher antibiotic residues were observed in groundwater in spring than in winter. Antibiotic residues in groundwater samples commonly decreased with sampling depth. Predominant antibiotics differed in surface water and groundwater. Rivers in the study area were the major sources of antibiotics in groundwater.

a r t i c l e

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Article history: Received 10 January 2015 Received in revised form 24 April 2015 Accepted 24 April 2015 Available online 14 May 2015 Editor: D. Barcelo Keywords: Antibiotics Surface water Groundwater Seasonal variation Vertical infiltration Jianghan Plain

a b s t r a c t 25 antibiotics (macrolides, tetracyclines, fluoroquinolones and sulfonamides) were detected in swine wastewater, river water, rivulet water and in groundwater samples from multi-level monitoring boreholes (with sampling ports, respectively, at 10, 25 and 50 m below the land surface) at Jianghan Plain, central China. Except swine wastewater, the antibiotic concentrations in groundwater, river and rivulet water were higher in spring than those in winter. Nineteen antibiotics were detected at 100% frequencies in all kinds of water samples. In groundwater, fluoroquinolones and tetracyclines were the predominant antibiotics and the total concentrations of 25 antibiotics commonly decreased with the aquifer depth. Most groundwater samples collected in spring had high concentrations of norfloxacin, with average values of 65.27 ng·L−1, 37.28 ng·L−1 and 46.83 ng·L−1, respectively, at 10, 25 and 50 m deep boreholes. By contrast, the concentrations of sulfamethazine and erythromycin were rather low in groundwater, but high in surface water. Groundwater samples collected from sites close to rivers or rivulets had much higher contents of antibiotics than those from other sites, indicating that the dominant source of antibiotics in groundwater should be the contaminated rivers or rivulets, rather than the scattered pig and poultry farms in the study area. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Antibiotics as one kind of emerging contaminants have become a major environmental health concern in recent years. Because of their continuous introduction into the environment, they are also regarded as “pseudopersistent” contaminants in previous studies (Gulkowska et al., 2008; Hernando et al., 2006). In China, up to 180,000 tons of antibiotics were utilized in animal agriculture and medicine in 2009 (Luo et al., 2010). Around 30–90% of antibiotics have been excreted into the environment in the forms of parent or metabolites via urine and feces (Gao et al., 2012; Jiang et al., 2011; Tong et al., 2009; Kim and Carlson, 2007; McArdell et al., 2003; Sarmah et al., 2006), domestic sewage (Brown et al., 2006; Chang et al., 2010; Jia et al., 2012), infiltration of ⁎ Corresponding author. E-mail address: [email protected] (Y. Wang).

http://dx.doi.org/10.1016/j.scitotenv.2015.04.091 0048-9697/© 2015 Elsevier B.V. All rights reserved.

polluted surface water and leaching of manure (Jacobsen et al., 2004; Wei et al., 2011). Although the concentrations of antibiotics are very low in the environment, continual exposure could induce antibioticresistant bacteria or genes which increase health and ecological risks (Al-Bahry et al., 2009; Chee-Sanford et al., 2001; Kuemmerer, 2009; Y.X. Li et al., 2013; Schwartz et al., 2003). In order to understand the adverse effects of antibiotics on environmental ecology (Quinn et al., 2009; Santos et al., 2010; Watkinson et al., 2009), obtaining the distribution and behavior of antibiotics in the environment is the first step. Most efforts have been made to investigate the approaches to improve the antibiotic removal efficiencies in wastewater treatment plants (WWTPs) (Gulkowska et al., 2008; Garcia-Galan et al., 2012; W. Li et al., 2013; Michael et al., 2013; Yan et al., 2014; Zhou et al., 2013) and to assess the antibiotic residual levels in surface water around WWTPs (Al Aukidy et al., 2012; Hernando et al., 2006; Garcia-Galan et al., 2011; McArdell et al., 2003). However, in most

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rural regions of China, there are no centralized sewage treatment facilities, and some livestock farms directly drain wastewater into the environment after simple treatment. Thus livestock wastewater and domestic sewage become sources of antibiotics of groundwater and surface water (Grujic et al., 2009; Milic et al., 2013). Antibiotic residues in shallow groundwater and drinking water have been detected in many countries (Avisar et al., 2009; Bartelt-Hunt et al., 2011; Einsiedl et al., 2010; Schaider et al., 2014). Because of the differences of source, discharge intensity and geochemical behavior of antibiotics, concentrations of specific compounds showed substantial differences in groundwater (Barber et al., 2009; Barnes et al., 2008; Fram and Belitz, 2011; Gottschall et al., 2012; Hannappel et al., 2014; Hu et al., 2010; Lopez-Serna et al., 2013). Fram and Belitz (2011) found the concentration of sulfamethoxazole in groundwater at 0.17 μg·L−1 from a 60 m deep drinking-water well. Hu et al. investigated 11 antibiotics in groundwater samples from local wells at five depths (10, 15, 20, 30, 40 m) below a vegetable field. The concentrations of chloramphenicol (5.8 to 28.1 ng·L−1) and ciprofloxacin (31.8 to 42.5 ng·L−1) in groundwater fluctuated with depth. In particular, the concentrations of both compounds decreased from 10 m to 15 m below the land surface (b.l.s.), but then kept rising with depth, reaching the highest at 40 m (Hu et al., 2010). However, the conclusions about the pattern of variation of antibiotic concentration in groundwater were not convincing due to insufficient data, and their geochemical behavior in the subsurface environment was not fully understood. In this study, the occurrence and distribution of 25 antibiotics (shown in the Supporting Information, Table S1) in surface water and groundwater at Jianghan Plain were investigated. The objectives of this study are: (1) to investigate the vertical distribution of antibiotic residues in aquifers at different depths (10, 25 and 50 m b.l.s.) in the study area, and (2) to characterize the seasonal variations of antibiotic concentrations in waters at the field monitoring site, and (3) to identify the major sources of antibiotics in groundwater at Jianghan Plain. The study provides a comparison between waters affected by human wastewater and those affected by livestock farms. The relative importance of

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each of these has been poorly understood in this region, and management of antibiotic resistance and water resources requires knowledge of the relative importance of these sources. 2. Sampling and analytical work 2.1. Site description and sampling The study area is located at Shahu County, Jianghan Plain of central China; Jianghan Plain is an alluvial plain with subtropical monsoon climate and abundant water resources, which make it one of the nation's major regions of agriculture and aquaculture production. The study area is located at the middle region of the Jianghan Plain and surrounded by four rivers and rivulets: the Tongshun River (TSR) and Dongjing River (DJR), both as tributaries of the Yangtze River, are two main perennial rivers flowing from west to east all the year; the Kuige Rivulet (KGR) and Lvfeng Rivulet (LFR) are two seasonal rivers. Water in LFR and most part of KGR are generally stagnant, and water in KGR would flow to TSR during rainfall period towards the major conjunction between TSR and KGR (at the northeast corner of Fig. 1.). The water table of the study area is commonly 0.5–3.5 m b.l.s. with low hydraulic gradients, and groundwater generally flows from south to north, under the impact of strong interactions between groundwater and surface water (Gan et al., 2014) (Fig. 1.). The main crops in the county are paddy, cotton, peanut and sugarcane, which are irrigated by groundwater. Both manure and fertilizer are used for crop growth. Most of the residents in Shahu County are located along rivers or rivulets, and there is no centralized sewage treatment facility in the area. Domestic sewage and industrial wastewater are drained to the environment, the potential pollutants would either infiltrate into the aquifers or migrate into the rivers and rivulets. In the study area, groundwater and surface water samples were collected from 23 sites in December 2013 (winter, dry season), and April 2014 (spring, rainy season) when there was continuous heavy rain. Groundwater samples (labeled G01 to G13) from 10 m deep boreholes

Fig. 1. Sampling sites and contour map of groundwater table in the study area. The groundwater table was monitored by measuring its elevation above the sea level.

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were obtained in both seasons. In the sampling campaign in April, groundwater samples were collected from five multilevel sampling boreholes (G03, G07, G10, G11, and G12) with sampling ports at three different depths (10, 25, 50 m b.l.s.). The sampling borehole G14 is a 40 m deep pumping well inside the pig farm, from which only water sample in winter was collected because of damage of the well in April. River and rivulet water samples (labeled R01 to R06) were collected from Tongshun River, Dongjing River, Kuige Rivulet and Lvfeng Rivulet. Three wastewater samples (S01, S02 and S03) were obtained along a canal, which collected wastewater from a pig farm with a capacity of hundreds of heads slaughtered every year (within which the well G14 is located). S01 was located at the nearest wastewater outlet of the pig farm, S02 and S03 were later added in spring sampling campaign. Water samples for antibiotic detection were stored in sterile amber glass bottles without head space, and were treated within 24 h after the sample collection. Groundwater samples were collected in a self-designed flow cell and in-situ measured using portable water quality analyzer after 5–10 min purge to get stable readings of physicchemical parameters, including temperature, pH, electrical conductivity (EC), oxidation-reduction potential (ORP) and dissolved oxygen (DO). Surface water samples of 50 mL were collected and added with concentrated HCl after being filtered by 0.45 μm membrane for DOC analysis, and 500 mL unfiltered samples without head space were collected for HCO− 3 titration. Sampling sites and relevant information in study area were labeled in Fig. 1. 2.2. Standards and chemicals All chemicals were of HPLC grade, and the ultra-pure water was prepared with Milli-Q at 18.2 MΩ·cm−1. 25 antibiotics from four classes, sulfonamides (SAs), tetracyclines (TCs), fluoroquinolones (FQs) and macrolides (MLs) were selected based on their high frequency of use in China, including roxithromycin (ROX), azithromycin (AZM), clarithromycin (CAM), erythromycin (ERY), clorotetracycline (CTC), qxytetracycline (OTC), tetracycline (TC), doxycycline (DC), sparfloxacin (SPA), gatifloxacin (GAT), fleroxacin (FLE), ofloxacin (OFL), enrofloxacin (ENR), lomefloxacin (LMX), ciprofloxacin (CIP), enoxacin (ENO), norfloxacin (NOR), sulfamethoxypyridazine (STP), sulfameter (SFM), sulfamethazine (SM-2), sulfamerazine (SMR), sulfathiazole (STZ), sulfamethoxazole (SMZ), sulfadiazine (SDZ) and sulfapyridine (SPD). All of the antibiotic compounds were purchased from Dr. Ehrenstorfer (Augsburg, Germany), simatone was chosen as internal standard (Luo et al., 2011), which was purchased from AccuStandard Inc. (United States). Individual antibiotic standard solutions and internal standard solutions were prepared at concentrations of 1000 mg/L in methanol and stored in dark at − 20 °C. The 25 antibiotic mixtures at different concentrations were prepared by progressively diluting individual stock solutions in methanol on the day before use. All individual stock solutions were renewed every six months.

2.4. Extraction and detection of target antibiotics in water samples 2.4.1. Solid-phase extraction (SPE) The method of SPE was improved based on previous studies (Batt and Aga, 2005; Luo et al., 2011; Tong et al., 2009), which relied on the standards addition method plus a single internal standard (simatone) to analyze multiple antibiotics (details in the Supporting Information). In consideration of different matrices, 1000 mL groundwater, 500 mL river/rivulet water and 100 mL wastewater were passed through cartridges to extract enough antibiotics for detection. Before loading to cartridges, the water samples were filtered through 0.45 μm fiberglass filter to remove particulate matter, their pH values were adjusted to 4 with 1 mol/L hydrochloric acid, and then 10 mL of 5% (v/v) chelating agent Na2-EDTA was added to prevent bonding of tetracycline and divalent cations. After solid phase extraction, simatone is added into final extract solution before LC–MS/MS analysis. 2.4.2. HPLC–MS/MS analysis The 25 target compounds and internal standard were detected by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) using Extend-C18 column (1.8 μm, 2.1 mm i.d. × 100 mm, Agilent, USA). Sample volume of 5 μL was injected to the C18 column, which was maintained at 40 °C. The analytes were gradient eluted by acetonitrile (eluent A) and ultra-pure water with 0.1% Formic Acid and 5 mM ammonium acetate (eluent B) at the flow rate of 0.25 mL/min. The initial percentage of eluent A was 15% and held 2 min, then linearly increased to 90% in 3 min and maintained for 2 min, after that, eluent A returned to 15% in 3 min and held 5 min to complete the whole cycle (15 min in total). The identification of target antibiotics was accomplished by comparing the retention time and two optimized ion pairs with corresponding standard compounds. For each antibiotic, ion pairs with relatively high abundance were picked for quantitative usage. The details of equipment, precursor ion, product ion, fragmentor and collision energy were listed in Table S1, Supporting Information. 2.5. Quantification and method validation The concentrations of target antibiotics in water samples were quantified by using internal standard method based on six points (5, 25, 50, 100, 250, 500 μg/L) calibration curve. The correlation coefficient (R2) of 25 calibration curves was more than 0.9. Standards addition method was used to assess the effect of different water matrices. Three kinds of water (groundwater/GW, wastewater/WW, river water/RW) were spiked with 25 target antibiotics at two concentration levels (10, 100 ng/L for GW; 100, 1000 ng/L for WW; 20, 200 ng/L for RW). Each sample was spiked and analyzed in triplicate (used to calculate the relative standard deviation/RSD), the same non-spiked sample was analyzed in duplicate to measure the background concentrations. Limit of detection (LOD) was determined from a low concentration standard and calculated using a signal-to-noise ratio (S/N) of 3. All the details above could be found in Supporting Information, Table S2.

2.3. Hydrochemical analysis 3. Results and discussion Temperature, pH, EC, ORP and DO were measured in situ using water quality analyzer of HQ40D Field Case.cat (NO. 58258-00, HACH, Colorado, USA) equipped with four probes. The concentrations of 2+ − NH+ and S2 − were also measured in situ using portable 4 , NO2 , Fe spectrophotometer (HACH 2800). HCO− 3 was tested within 24 h by acid–base titration methods. The concentrations of cations, such as Ca2 +, Mg2 +, Na+ and K+, were determined by using ICP-AES (IRIS Intrepid II XSP, USA), while the detection of anions was conducted by ion chromatograph (Dionex 2500, USA), and total organic carbon analyzer (multi N/C 3100, Germany) was used to determine the dissolved organic carbon (DOC) in water with a detection limit of 0.004 mg·L−1 (Gan et al., 2014).

3.1. Hydrochemistry Basic hydrochemical parameters of groundwater and surface water samples were provided in Table S3 in Supporting Information. The pH of groundwater and surface water samples was neutral and weakly alkaline with the mean value of 7.09 and 7.62, respectively. The EC values of groundwater samples ranged from 647 to 1349 μs·cm−1, which were higher than those of river water, but lower than those of swine wastewater. The variation of temperature between winter and spring for surface water was 4 to 5 °C, while the seasonal variation of groundwater temperature was limited, with the mean temperature of

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18.0 °C and 18.3 °C respectively. Groundwater and surface water in the study area belonged to HCO3–CaMg type (Fig. S1. and Table S4, Supporting Information), which corresponded well with our previous data (Gan et al., 2014; Tong et al., 2014). The concentrations of SO2− 4 and Cl− in surface water were higher than those in groundwater, which could be attributed to the impact of anthropogenic activities, including livestock breeding, agricultural fertilizer spreading and factory wastewater discharge (Kim et al., 2009; Ujević et al., 2010). Negative ORP and low DO in groundwater indicated reducing condi2+ tion. So did the elevated concentrations of Fe2+, NH+ and S2− in 4 , Fe groundwater. By contrast, river water showed a distinct oxidizing condition with positive ORP and high DO (details in Supporting Information Table S3). The redox conditions of swine wastewater changed from reducing in winter (ORP = −92.5 mV, DO = 0.42 mg·L−1) to oxidizing in spring (ORP = 63.0 mV, DO = 1.30 mg·L−1), possibly due to the effect of covering of algae or leaves in winter and that of dilution of rainfall in spring. The concentrations of DOC in surface water is higher than those in groundwater (details in Table S3, Supporting Information), and they are commonly higher in spring than in winter in groundwater samples. The continuous heavy rain during spring sampling campaign may have intensified the interaction between surface water and groundwater, and introduced DOC from contaminated surface water to the subsurface (Zhang et al., 2013). 3.2. Occurrence of 25 antibiotics in groundwater and surface water 3.2.1. Groundwater The total concentrations of antibiotics in groundwater samples collected in spring from the 10 m deep boreholes ranged from 0.10 to 0.29 μg·L−1, higher than those in winter (0.11–0.23 μg·L−1), except for the sites G07 and G08 (Fig. 2.). The continuous rainfall in spring might be responsible for the transport of antibiotics into the groundwater. Nineteen antibiotics were detected with the frequencies of 100%, and ROX and CAM were not detected in any groundwater samples in both seasons. FQs and TCs were the predominant compounds in all groundwater samples at 10 m deep boreholes (Fig. 2.). Except for the sites G07 and G08, concentrations for FQs were higher in spring (65.22 to 2.15 × 10 2 ng·L− 1) than in winter (60.65 to 1.56 × 102 ng·L− 1), while for TCs, except the site G13, antibiotics in

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groundwater samples from 10 m depth boreholes had higher residual concentrations in winter (42.53 to 64.04 ng·L−1) than in spring (19.20 to 68.80 ng·L−1), which could be contributed to the significant decrease of CTC during spring. Furthermore, NOR was detected with highest concentration among all 25 target compounds, ranging from 11.16 to 1.14 × 102 ng·L−1 in spring and from 20.24 to 71.76 ng·L−1 in winter. High concentration of NOR in groundwater was also reported by Lopez-Serna et al. (2013). The observed higher concentrations of antibiotics in spring, especially for NOR, could be attributed to the higher frequency of their use both by the local residents and pig farms for disease control in spring (Chen et al., 2012; Sarmah et al., 2006). Besides, the sorption coefficient Kd (ratio between the concentration of the compound in the sorbent and in the aqueous phase) of NOR was lower at higher temperatures (Dorival-Garcia et al., 2013), indicating stronger mobility in spring than in winter. Relatively low concentrations of antibiotics were found at the sites G07, G08, and G10, which are located far apart from rivers or pig farms (Fig. 1.). By contrast, the sampling site G06 which presented the highest average and total antibiotic concentrations in two seasons is located just beside the Tongshun River, suggesting that the contaminated surface water might be an important source of antibiotics for groundwater (Buerge et al., 2009), due to accumulation of these compounds from seeping surface water in the aquifer sediments and subsequent leaching into groundwater (Kulshrestha et al., 2004; Tolls, 2001; Lopez-Serna et al., 2013). The total concentrations of antibiotics in groundwater samples commonly decreased with sampling depth (Fig. 3.), nevertheless, high antibiotic residual concentrations (over 100 ng·L−1) were observed even in the 50 m deep boreholes (except for G03). In contrast to the sites G07 and G10, significant attenuation in antibiotic concentrations was observed at the sites G03, G11, and G12. FQs were detected at the highest concentrations in groundwater samples from boreholes of three different depths, and presented the dominant residual concentrations in aquifers of the study area (Fig. 3.). One similar previous study carried out in Barcelona showed that the concentrations of most drug residues in groundwater decreased with increasing sampling depth, which was attributed to either increase of residence time of the antibiotics in the aquifer or increase of mixing with water from different sources (Jurado et al., 2012). Another study claimed that the infiltration of antibiotics might be partly intercepted or retarded by the degradation and

Fig. 2. The total concentrations of detected antibiotics in groundwater samples collected from the 10 m deep boreholes at the 13 sampling sites for two seasons.

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3.2.2. Surface water

Fig. 3. Concentrations of detected antibiotics in groundwater samples collected from 10, 25, 50 m deep boreholes of the sampling sites G03, G07, G10, G11 and G12 (locations shown in Fig. 1).

adsorption in aquifer (Henzler et al., 2014). The concentrations of NOR were detected at the highest values among 25 antibiotics in all groundwater samples from sites G03, G07, G10, G11, and G12 (except for 50 m deep borehole in G03, Fig. 3.) in spring, ranging from 31.12 to 96.89 ng·L−1, 15.17 to 56.08 ng·L−1 and 2.78 to 73.18 ng·L−1 at the depth of 10, 25 and 50 m, respectively. The concentrations of NOR in groundwater samples from 50 m deep wells were all higher than those from 25 m deep wells (except for G03), and the highest residues were detected at the depth of 50 m instead of 10 m at G07 and G10. The mobility, transport and bioavailability of antibiotics in the environment were dependent either on their sorption behavior (Wegst-Uhrich et al., 2014) or on their usage amount. Although NOR showed apparent tendency for sorption to sediment and low mobility in the aqueous phase in previous studies (Buerge et al., 2009; Pico and Andreu, 2007; Zhang and Dong, 2008), its overuse and continuous release into the environment would exceed sorption capacity and bioavailability in sediment (Wiwattanapatapee et al., 2002) in the top layers, and might be flushed into deeper aquifers during strong rainfall events.

3.2.2.1. Swine wastewater. In swine wastewater sample S01 (the outlet of the pig farm), the total concentrations of detected antibiotics were 12.96 μg·L−1 and 4.25 μg·L−1 in winter and spring, respectively. In winter, the wastewater in canals is almost stagnant, under cold and dry climate conditions. Whereas in rainy season, precipitation dilutes the wastewater and promotes the migration of target compounds, besides, due to rise in temperature, photolysis and microbial activity can be intensified to facilitate the degradation of antibiotics (Alexy et al., 2004; Hatzinger and Alexander, 1997; Huang et al., 2001). All these factors might be the reasons for the observed elevation of antibiotic residues in the wastewater samples in winter. Except for all these speculations, the concentrations of target compounds in wastewater were mostly dependent on their dosage of use in pig farms. Tong et al. (2009) had detected the occurrence of antibiotics in swine effluents in nearby areas in 2009. The results showed that except for the extremely high concentrations of SMR, OFL, and OTC, the concentrations of other compounds were similar to those in this study. Therefore, the decrease of SMR, OFL, OTC concentrations in wastewater could be related to enforced regulation of their use in livestock farming in recent years. The order of concentration levels of target compounds was S01 N S03 N S02 (Fig. 4.). In addition to the impact of rainfall dilution, the distance from the effluent discharge point (S01) also affects the amount of residues. TCs and SM-2 were predominant compounds at S01 with significantly higher concentrations than the other two sites. For TCs, results of a previous study (Luo et al., 2011) showed that the first-order attenuation rate of TCs was the highest among four classes of antibiotics. The concentration of SM-2 changed from being high in S01 (0.46 μg·L−1, spring) to below detection limit in S02, the degradation was quite significant in the canal. Besides, its concentration was much higher in wastewater (S01, 7.68 μg·L−1, winter) than in nearby groundwater sample (G14, 1.90 ng·L−1, winter) (Supporting Information, Table S5 and S6), probably due to its weak vertical transport in the aquifer system. SMR and SMZ of SAs which have properties similar to SM-2, also showed very high concentrations (N10 μg·L−1 and N0.1 μg·L− 1) in wastewater and extremely low (b12 ng·L− 1) in groundwater in previous studies (Heberer et al., 2008; Tong et al., 2009), which could be attributed to degradation and dilution processes.

Fig. 4. Concentrations of detected antibiotics in the three samples collected from the swine wastewater canal in spring.

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Fig. 5. Antibiotic residues in rivers and rivulets around the study area for two seasons.

3.2.2.2. River and rivulet water. Except for ROX, all target antibiotics were detected in four rivers and rivulets during two sampling campaigns. The order of total antibiotic concentrations in the samples was: TSR N DJR N LFR N KGR (Fig. 5). Human activities have strong influence on these four water bodies. TSR and DJR are the two main rivers in the study area and receive wastewater and municipal sewage drained along the river banks. Since there is no large scale livestock farm in upstream and nearby region, the municipal and domestic sewage might be the major source for the observed high residual concentrations of antibiotics in TSR. Along the downstream gradient of TSR, the antibiotic concentrations increased from 2.99 μg·L−1 at R05 to 4.10 μg·L−1 at R01 in spring, indicating accumulation instead of attenuation of antibiotics in the river system. A similar result was reported in the tributary of Liao River in northern China, the concentration of antibiotics in water sample collected from densely polluted urban area in May 2012 was up to 3.97 μg·L−1 (Gao et al., 2012). ERY and NOR were the predominant compounds with average values of 0.51 and 0.11 μg·L−1, respectively, in winter, and 2.42 and 0.23 μg·L− 1, respectively, in spring, which were also reported to be antibiotics of high consumption in China (Chang et al., 2010). Although continuous rainfall during spring does have dilution effect on the concentration levels, intensive use and uncontrolled release into the environment after use can still cause antibiotic contamination of rivers. As a matter of fact, more than 10 years study on antibiotics in the environment has clearly shown that the use of antibiotics for different purposes in different periods can be a main reason for variation of antibiotic concentrations in the environment (Kolpin et al., 2002; Malintan and Mohd, 2006; Milic et al., 2013). 3.3. Distribution of antibiotics in the aquatic environment 3.3.1. Spatial distribution and potential sources The concentrations of antibiotics in the aquatic environment were: swine wastewater N river and rivulet water N groundwater. The major sources for antibiotics in rivers and rivulets include municipal/domestic sewage and wastewaters of livestock/poultry farming. As most of local residents live close to the rivers and without sewage treatment systems,

the partially metabolized antibiotics and unused or expired medications (Ruhoy and Daughton, 2007) were directly excreted to the environment and would thus increase the residual concentrations of antibiotics in the rivers and rivulets. Concentrations of antibiotics in groundwater were slightly higher in the area with lower groundwater table (G04, G05, and G06 in Fig. 1.), suggesting that groundwater flow has positive effect on the horizontal migration and accumulation of antibiotics in groundwater. Furthermore, the residual concentrations of antibiotics in groundwater samples collected from areas close to the rivers or rivulets were much higher than those from other sites (G07, G08, and G10). The seasonal variation pattern (higher in spring) of antibiotic concentrations in rivers/rivulets and groundwater was coincident, while that in swine wastewater showed opposite trend (higher in winter). Thus it can be speculated that the predominant source of antibiotics in groundwater should be the antibiotics-contaminated rivers or rivulets, not the swine wastewater. For aquifers recharged by rivers via natural bank filtration (Lopez-Serna et al., 2013), antibiotics would be transported through the natural bank to groundwater during the process, which could be the main reason for the high antibiotic residues in groundwater samples collected from areas close to rivers. However, accompanying the bank filtration are geochemical processes such as sorption, biodegradation and mixing with non-polluted groundwater (Grunheid et al., 2005), which may account for the observed difference in antibiotic concentrations in surface water and groundwater at Jianghan. 3.3.2. Vertical infiltration from surface water to groundwater G14 and G06 were two sites of groundwater sampling with nearest distance to the surface water sampling sites S01 (the outlet of pig farm) and R01 (Tongshun River), respectively. As shown in Fig. 6A, the predominant compounds in swine wastewater (SM-2, over 60%) were different from those in river water (ERY, over 30%); however, the proportions of antibiotics in groundwater were similar (both were FQs and TCs). In Fig. 6B, the attenuation amplitudes were calculated as follows: the differences of individual antibiotics' concentrations between surface water and groundwater were divided by their concentrations in surface water. The values of attenuation amplitudes significantly associated with the residual levels of individual antibiotics in

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Fig. 6. Percentages of individual antibiotics in water samples (A) and the percentages of attenuation amplitudes from surface water to groundwater (B, points in red rectangle with relative lower values).

groundwater. For example, predominant compounds SM-2 and ERY in S01 and R01, respectively, with the highest attenuation amplitudes were not detected in the nearest groundwater samples (G14 and G06). Among all detected antibiotics in the groundwater samples, most of the compounds with relative lower attenuation amplitudes (labeled with red rectangle in Fig. 6B) belong to FQs and TCs, which made these two classes of antibiotics to be the predominant residues in groundwater. By contrast, higher values of attenuation amplitudes reflect more active physicochemical properties of antibiotics and more complex geochemical reactions during the infiltration process from surface water to the subsurface. ERY would be degraded to anhydroerythromycin (ERYH2O) immediately in the environment (Batt and Aga, 2005; Huang et al., 2001; Yang and Carlson, 2004), while the ERY-H2O was reported to undergo more effective degradation under anoxic conditions (Heberer et al., 2008), so the geochemical reactions in which ERY is involved under reducing conditions in the aquifers may result in its low concentration in groundwater. The high correlations between SM-2 and DO (r = 0.992), SMZ and DO (r = 0.86), respectively, at 0.01 significant level in groundwater, as calculated using SPSS, indicate that these two compounds are sensitive to the redox conditions. 4. Conclusions Twenty-five antibiotics were detected in surface water and groundwater in the study area. The results showed that SM-2 and ERY with highest attenuation amplitudes were detected at high concentrations in surface water and at very low concentrations in groundwater. Under reducing conditions, in groundwater were mainly accumulated TCs and FQs, and the total concentrations of antibiotics decreased with aquifer depths. Higher antibiotic concentrations were detected in swine wastewater in winter; by contrast, in river/rivulet water and groundwater, higher concentrations occurred in spring. According to the pattern of antibiotic occurrence in groundwater and surface water, the predominant source of antibiotics in groundwater was speculated to be rivers or rivulets instead of wastewater from pig farm. Intensive interaction between surface water and groundwater, especially in rainy seasons, would help transport antibiotics into groundwater. The occurrence of antibiotics in the aquatic environment can be attributed

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