Occurrence and fate of antibiotics and antibiotic resistance genes in typical urban water of Beijing, China

Accepted Manuscript Occurrence and fate of antibiotics and antibiotic resistance genes in typical urban water of Beijing, China Xiaohui Liu, Guodong Zhang, Ying Liu, Shaoyong Lu, Pan Qin, Xiaochun Guo, Bin Bi, Lei Wang, Beidou Xi, Fengchang Wu, Weiliang Wang, Tingting Zhang PII:

S0269-7491(18)33295-0

DOI:

https://doi.org/10.1016/j.envpol.2018.12.005

Reference:

ENPO 11945

To appear in:

Environmental Pollution

Received Date: 17 July 2018 Revised Date:

1 December 2018

Accepted Date: 2 December 2018

Please cite this article as: Liu, X., Zhang, G., Liu, Y., Lu, S., Qin, P., Guo, X., Bi, B., Wang, L., Xi, B., Wu, F., Wang, W., Zhang, T., Occurrence and fate of antibiotics and antibiotic resistance genes in typical urban water of Beijing, China, Environmental Pollution (2019), doi: https://doi.org/10.1016/ j.envpol.2018.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Occurrence and fate of antibiotics and antibiotic resistance genes in typical urban

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water of Beijing, China

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Xiaohui Liua,b, Guodong Zhangc,Ying Liua, Shaoyong Lua*, Pan Qina, Xiaochun Guoa,

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Bin Bia, Lei Wanga, Beidou Xia, Fengchang Wua, Weiliang Wangc,Tingting Zhangd

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a

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Lake Dongtinghu (SEPSORSLD), National Engineering Laboratory for Lake

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Pollution Control and Ecological Restoration, State Key Laboratory of Environmental

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Criteria an Risk Assessment, Research Centre of Lake Environment, Chinese Research

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Academy of Environmental Sciences, Beijing 100012, People's Republic of China.1

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State Environmental Protection Scientific Observation and Research Station for

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School of Environment, Tsinghua University, Beijing 100084, China.

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School of geography and environment, Shandong Normal University, Jinan,

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Shandong, 250358, China;

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d

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Beijing, 100029, China

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Capsule： ：

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Occurrence of antibiotics and ARGs in diverse water shows a danger signal,

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especially in groundwater.

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Abstract: The pollution of antibiotics and antibiotic resistance genes (ARGs) has

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been highlighted on a global scale because of their serious threats to the environment

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and human health. Typical urban water in cities with high population density are ideal

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School of Chemical Engineering, Beijing University of Chemical Technology,

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*Corresponding author: Tel.: +86 10 84935064 E-mail: [email protected](S. Lu) 1

ACCEPTED MANUSCRIPT mediums for the acquisition and spread of antibiotics and ARGs. The pollution level

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of a broad range of antibiotics and ARGs in hospital wastewater, groundwater and the

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Wenyu River, and their fates through three sewage treatment plants (STPs) were

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investigated in this study. The concentrations of the 11 detected antibiotics ranged

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from not detected (ND)-16800 ng L-1 in diverse water samples from Beijing, and

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fluoroquinolones were detected at the highest concentration, especially in the hospital

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samples. The maximum concentrations of antibiotics in STPs and hospital were 1-3

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orders of magnitude higher than those in the surface water from Wenyu River and

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groundwater. Good removal efficiencies by treatment processes were observed for

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tetracyclines and quinolones, and low removal efficiencies were observed for

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sulfonamides and macrolides. These results also revealed that the sulfonamide

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resistance genes (sul1, sul2) and macrolide resistance genes (ermB) were detected at

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the highest relative abundances (7.11×10-2-1.18×10-1) in the water bodies of Beijing.

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It was worth noting that sul1 abundance was the highest in groundwater samples. The

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relative abundance of most ARGs in STPs exhibited a declining trend in the order of

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influent > secondary effluents > effluent. However, the relative abundance of sul 1,

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sul 2 and tetC in the effluent was higher than those in the influent. The incomplete

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removal of antibiotics and ARGs in STPs poses a serious threat to the receiving rivers,

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and affects ecosystem security. Overall, our findings provide favorable support for a

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further investigation of the spread and risk of antibiotics and ARGs from diverse

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sources (e.g., STPs and hospitals) to the aquatic environment.

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Key words: Antibiotics; ARGs; Occurrence; typical urban water

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1 Introduction Antibiotics are widely used and often abused for the therapeutic treatment of

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infectious diseases in humans and protection of health or promoter of the growth for

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animals. However, 25-75% of antibiotics are excreted as parent compounds or

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metabolites in feces (Luo et al., 2011), which resulted in their frequent detection in

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rivers, lakes and groundwater that originated mainly from STPs (as they are not

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completely removed) surface runoff or aquacultural activities (Arikan et al., 2008;

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Rosal et al., 2010; Xu et al., 2014; Rodriguez-Mozaz et al., 2015). Antibiotics into

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water bodies pose a significant risk to human health and ecological environment, even

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at low concentrations (Kümmerer, 2009).

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Greater concern stems from the fact that studies have confirmed that there were

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significant positive correlations between antibiotic resistance genes (ARGs) and

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corresponding antibiotics, and the dispersion of antibiotics in the environment can

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contribute to the development and dissemination of antibiotic ARGs on a global scale

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(Pruden et al., 2006; Martínez, 2008; Chen et al., 2013; Huerta et al., 2013;

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Rodriguez-Mozaz et al., 2015; Larson, 2015; Zheng et al., 2018). ARGs in the water

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environment have frequently been detected in many countries in recent years, such as

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the USA (Pruden et al., 2012), diverse European countries (Carvalho and Santos.,

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2016), Australia (Stoll et al., 2012), Canada (Drudge et al., 2012) and China (Zhang et

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al., 2015). China is a disaster zone of resistance gene pollution, as ARGs have been

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detected in many rivers or lakes (Jiang et al., 2013, Zhu et al. 2013, Zhou et al., 2014,

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Yao, 2016, Yang et al., 2017, Dang et al., 2017). This phenomenon was identified as a

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global public health crisis by the World Health Organization (WHO, 2014). ARGs,

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which can pass microbial reproduction to their descendants and spread between

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non-pathogens and pathogens, and even distantly related organisms through

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ACCEPTED MANUSCRIPT horizontal gene transfer, are organic pollutants with characteristics that are different

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from those of the single generation source of other pollutants (Liu et al., 2018a; Zhu

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et al., 2015). Sewage treatment plants (STPs), hospitals, livestock and poultry farms

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and aquaculture are important sources in addition to the intrinsic resistance of

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microbes in the environment (Qiao et al., 2017; Zhu et al., 2017; Sui et al., 2017),

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especially STPs for urban water bodies. Besides domestic domestic sewage, industrial

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and hospital wastewater are also collected into STPs (Rodriguez-Mozaz et al., 2015).

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In particular, hospital effluents as point sources for antibiotics and pathogens,

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including multi-antibiotic resistant bacteria, are highly hazardous because of their

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high toxic characteristics (Escher et al., 2011). However, ARGs and antibiotics cannot

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be completely removed using the current processing technology, and the ratio of

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ARGs to total bacteria in STP effluents can even increase after processing (Chen and

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Zhang., 2013, Qiao et al., 2017). A common problem with STPs is the potential risk of

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the increase in the abundance of ARGs in the effluent (Zhang et al., 2018) which can

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threaten receiving rivers or lakes. Thus, it is important to understand the occurrence of

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antibiotics in sewage treatment plants and hospitals and analyze their reduction rules.

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The high population density in cities results in large usage and emission of

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antibiotics. ARGs take advantage of this ideal habitat, which can pose a significant

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threat to the public health of the residents. A recent study showed that the transfer of

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ARGs from the pig farms to global patients was a cause for concern regarding

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antibiotic resistance in human (Wang et al., 2018). A large number of antibiotics, ARB

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and ARGs can are released into the environment in form of farming wastes through

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food chain, direct or indirect contact, and mutual interchange of ARB between human

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and animal sources. ARGs can be transferred in various environmental media such as

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soil, water, groundwater, etc. Meanwhile, they can also be integrated into mobile gene

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diffuse among bacteria through gene horizontal transfer (Wright, 2010). Thus, ARGs

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might be transferred from STPs, hospitals, groundwater (drinking water) or rivers to

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the human populations in cities. However, limited data exist on the variability of

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antibiotics and the occurrence of ARGs in typical urban water in cities, which

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underscores the need for complementary studies. Beijing, is the capital of China, and

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is the second most densely populated city with a large population of 21.73 million and

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a population density of 1324 people/km2 (Ma et al., 2017), resulting in the highest

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antibiotics consumption rate in the world, and one of the most heavily populated cities

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of antibiotics (Dai et al., 2015). Most studies have demonstrated the occurrence of

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antibiotics in STPs (Xu et al., 2015; Gao et al., 2012), urban rivers or lakes (Li et al.,

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2015; Xu et al., 2016), while none of them have valuated simultaneously the

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occurrence and response relationship of antibiotics and ARGs in STPs, hospital, rivers

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and groundwater of Beijing. This study characterizes the occurrence of antibiotics

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residues (sulfadiazine (SD), sulfamethoxazole (SMX), sulfamethazine (SMT),

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trimethoprim (TMP), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR),

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ofloxacin (OFLO), sarafloxacin (SFLO), tetracycline (TC), oxytetracycline (OTC),

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chlortetracycline (CTC), erythromycin-H2O (ERM-H2O), roxithromycin (ROM) ) and

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ARGs (sulfonamide resistance genes (sul1, sul2), five tetracycline resistance genes

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(tetA, tetB, tetC, tetM, tetW), one quinolone resistance genes (qnrS), one macrolide

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resistance genes (ermB) and Class I Integron (int1)) in typical urban water (STPs,

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hospital, rivers and groundwater) and explores the response relationship of antibiotics,

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ARGs and environmental factors. To the best of our knowledge, this is the first study

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to systematically analyze the abundance and distribution of ARGs and explore the

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correlation among antibiotics, ARGs and environmental factors in typical urban water.

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ACCEPTED MANUSCRIPT The results will be helpful to understand the pollution level of antibiotics and ARGs in

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typical urban water of modern metropolis.

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2 Materials and Methods

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2.1 Sampling sites

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Surface water was collected from the Wenyu River. Sampling section is mainly

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located in important bridges across the river and main the estuary of main tributaries.

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Seven sampling points from WHY1 to WYH7 are Shahe floodgate→Mafang bridge

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→Xisishang village →Wenyu bridge→Yigezhuang bridge→Wenyu river Bridge→

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Beiguan floodgate according to the water flow. The distance from WYH1 to WYH2 is

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8.8km, 10.8km from WYH2 to WYH3, 10.2km from WYH3 to WYH4, 8.6km from

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WYH4 to WYH5, 3.9km from WYH5 to WYH6 and 4.2km from WYH6 to WYH7.

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Groundwater was collected from five wells (G1, G2, G4 and G5 with the depth of 29

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m, G3 is a multi-level monitoring well with the depth of 6.79 m, 11.64 m and 22.53 m)

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which were located in Shunyi District. Influent (MW1, HW1 and CW1),

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secondary effluents (MW2, HW2 and CW2) and effluents (MW3, HW3 and CW3)

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were collected from three sewage treatment plants from Miyun District(MW) ,

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Haidian District (HW) and Chaoyang District (CW). Hospital wastewater containing

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an influents (YY1) and effluent (YY2) from a hospital in Haidian District were

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selected to study the antibiotic and ARG pollution. Detailed information on the

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Wenyu River, STPs, wells and the hospital is provided in Figure 1 and Table S1. The

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water samples were collected in triplicate in January 2018, then kept in brown glass

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bottles at 4 °C before laboratory analysis. The water samples were treated within 24 h

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after being transported to the laboratory.

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Figure 1 Sampling site

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2.2 Quantification of antibiotics Surface water (1 L), groundwater (2 L) and other water samples (500 mL) were

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pretreated as previously described (Liu et al., 2018b) and the details are provided in

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the Supporting Information. The antibiotics were analyzed by an ultra-high

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performance liquid chromatography (Ultimate3000 HPLC system, Dionex, USA)

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coupled with an electrospray ionization tandem mass spectrometry (ESI-MS/MS,

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API3200, AB Sciex, USA) operated in positive or negative mode and equipped with a

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Waters BEH- C18 column (3.0×150 mm, 3.5 µm). The column was maintained at

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40 °C during sample analysis. The mobile phase consisted of eluent A (0.01% formic

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acid in ultrapure water) and eluent B (acetonitrile). The separation of the antibiotics

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was achieved with the following gradient program: 0-7 min, 3-15% B; 7-9 min, 15%

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B; 9-12min, 15-30% B; 12-13 min, 30% B; 13-18min, 30-42% B; 18-19 min, 42% B;

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19-21min 42-3% B; 21-29 min, 3% B. The results of the mass spectrometric analyses

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are provided in Table S2 and Figure S1. The antibiotic concentrations were

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determined by quantification using an internal standard (Ciprofloxacin (CIP)-D8,

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sulfamethoxazole (SMX) 13C6, roxithromycin (ROM)-D7, tetracycline (TC) D4). The

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calibration curves (50-10000 µg L-1 concentrations for TC, OTC and CTC, 5-1000 µg

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L-1 concentrations for other antibiotics) for the antibiotic detection exhibited good

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linear relationships (R2 > 0.99). The recoveries of the antibiotics from the surface

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water samples ranged from 83% to 117.01% (Table S3). The limit of quantification

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(LOQ) calculated with a signal/noise ratio of 10 was 0.66-7.92 ng L-1.

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2.3 Quantification of ARGs

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influent (100 mL), secondary effluent (200 mL) and effluent (200 mL) samples were

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filtered through a 0.22 µm membrane before DNA extraction. The process of DNA

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extraction and treatment was performed according to a previous study (Liu et al.,

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2018c), and the details are provided in the Supporting Information. The extractions of

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the 16S RNA gene, sul1, sul2, tetA, tetB, tetC, tetM, tetW, qnrS, ermB, and int1 were

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conducted using a Biometra TGradient Thermal Cycler (Biometra Company,

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Germany). Primer sequences and their annealing temperatures targeting the different

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genes are provided in Table S4. Real-time PCR was carried out in triplicate based on

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the process according to Mao et al. (2015). The calibration curves for plasmid

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standard curves presented good linear relationships (R2 = 0.99). The PCR

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amplification efficiencies ranged from 95% to 110%, which indicated that the

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efficiency of qPCR met the requirements.

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2.4 Statistical analysis

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The statistical significance of the differences was evaluated by ANOVA, which

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was considered significant at p < 0.05. The correlation analysis of antibiotics, ARGs

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and environmental factors was carried out using SPSS 20.0 and Canoco 4.5 software.

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Cluster analysis of ARGs and sampling sites was carried out using MATLAB

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software.

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3 Results and discussion

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3.1 Occurrence of antibiotics

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The concentrations of 12 antibiotics in different water samples from Beijing are 9

ACCEPTED MANUSCRIPT provided in Table 1 and 2. The concentrations of antibiotics ranged from ND-16800

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ng L-1 in diverse water samples from Beijing. The maximum concentrations of

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antibiotics in STPs and hospital are 1-3 orders of magnitude higher than those in the

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surface water of the Wenyu River and groundwater. Despite their high consumption

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levels, the concentrations of tetracycline antibiotics were relatively low level in the

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different water samples from Beijing because of their high distribution coefficient

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(e.g., OTC = 420-1030 L kg-1, TC = 1140-1620 L kg-1 and CTC = 401 L kg-1) and the

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chemical instability (Luo et al., 2011; Ding et al., 2017), and these were similar to

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those in most rivers or lakes (Liu et al., 2018a). In the hospital and STPs samples, low

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pollution levels of tetracycline antibiotics were also observed, which might be slightly

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related to the seasonal differences of antibiotic usage and types and mainly affected

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by the above-mentioned reasons (Hu et al., 2010; Pean et al., 2010; Pan et al., 2011).

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In cities of the high population density, the domestic sewage and hospital wastewater

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are main sources of antibiotics, which are directly related to human. In winter as a

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worst case scenario, antibiotic usage will increase with the increase of incidence of a

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disease, especially the spread of influenza. Although the concentration of different

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antibiotics have seasonal differences, most studies showed a predominant presence of

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sulfonamides or quinolones in most water bodies (Liu et al., 2018a).

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In the Wenyu River and groundwater, SAs and QNs showed a predominant

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presence with the highest concentration of 256 ng L-1 (SD) for SAs and 1270 ng L-1

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(OFLO) for QNs in the Wenyu River and 17.6 ng L-1 (SD) for SAs and 13.2 ng L-1

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(OFLO) for QNs in groundwater. The highest frequencies of these antibiotics were 10

ACCEPTED MANUSCRIPT 100% because of the high solubility, chemical stability or high consumption (Hari et

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al., 2005; Luo et al., 2011), and these frequencies were similar to those in most rivers

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or lakes in China (Liu et al., 2018a). However, the results that indicated the

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predominant presence of SAs and QNs in the Wenyu River are different from those

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found in a precious study (the predominant presence of SAs and TCs) (Xu et al.,

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2016), which was probably because of seasonal differences. The small kd values of

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SAs and QNs may result in high mobility from soil to groundwater (Hu et al., 2010;

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Liu et al., 2018a). The concentrations of antibiotics in the Wenyu River were 1-2

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orders of magnitude higher than those in groundwater. The migration of antibiotics

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was hindered because of the natural soil infiltration layer, which can remove most

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antibiotics from rainfall, irrigation, and other human activities and result in low

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residues (Sukul et al., 2008). However, the trace levels of antibiotics in groundwater

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had a great potential risk to human health, which is a situation that should be

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highlighted. In addition, obvious spatial difference in different sampling sites was

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observed, but no obvious regularity along the water flow was found (Figure 2). The

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accumulative concentration in site WYH 1-9 ranged from 245.06 to 1657.47 ng L-1

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with the mean value of 21.08-138.12 ng L-1. The highest concentration was observed

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in WYH 2 with 76.76% contribution of OFLO, followed by WYH 7 > WYH 1>

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WYH 4 > WYH 6 > WYH 3 > WYH 5, which antibiotics attenuation didn't seem to

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follow first-order kinetics (Luo et al., 2011). The spatial distribution in sampling sites

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can be effected by many factors, which need further investigation. According to the

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risk quotient (RQ) method, SMX and OFLO were predominant risk factors in the

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ACCEPTED MANUSCRIPT Wenyu River and groundwater due to their very low PNECs and high consumptions

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(displayed in Table 1), especially in the Wenyu River (RQ values of SMX and OFLO

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were 4.74 and 7.06, respectively, indicating that they might present significant

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environmental risks). Due to their trace residues, the RQs for most antibiotics were

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below 0.01 in groundwater, showing low or no environmental risk to the groundwater

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environment. However, low or no environmental risk of detected antibiotics should

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not be ignored. In European countries (EMA, 2006), an ERA of the environmental

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concentrations greater than 10 ng L-1 need to be carried out immediately.

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Unfortunately, there is not a uniform risk assessment method for antibiotics in the

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water environment in China until now. In addition, the establishment of

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water quality criteria for antibiotics is urgent.

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Fluoroquinolones were detected at the highest concentration in STPs and

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hospital,, especially in hospital influent and effluent samples, and these results are

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similar to the results from the main hospital in Girona, Spain (Rodriguez-Mozaz et al.,

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2015) and Chongqing, China (Chang et al., 2010). Among fluoroquinolones, CIP and

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OFLO were detected at the highest concentration (8480 ng L-1 for CIP and 16800 ng

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L-1 for OFLO). This common phenomenon indicated that CIP and OFLO are

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frequently used in hospital practice to treat infections, resulting in their high medical

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consumption (MacDougall et al., 2005; Chang et al., 2010; Rodriguez-Mozaz et al.,

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2015; Kwon et al., 2017). In addition, the levels of SD, SMX and TMP in STPs and

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hospital wastewater were also high. Trimethoprim (TMP) is commonly used in

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combination with SMX and SDZ, at a ratio of 1:5(TMP: SMX or SDZ), resulting in

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ACCEPTED MANUSCRIPT high residues (Kong et al., 2017). Interestingly, the pollution levels of macrolides in

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STPs had the highest concentrations of 1280 ng L-1 for ERM-H2O and 1640 ng L-1 for

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ROM, which were 1-2 orders of magnitude higher than those in the hospital samples

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(240.8 ng L-1 for ERM-H2O and 40.89 ng L-1 for ROM). This finding illustrates that

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macrolide consumption is more widespread in households than in clinical settings

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(Kümmerer and Henninger, 2003).

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3.2 Removal efficiency of antibiotics in STPs and hospital

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Of course, the concentrations of most antibiotics in STPs influent can be reduced,

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but the removal efficiency of some antibiotics are not always satisfactory. In the

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hospital samples, the removal efficiencies of detected antibiotics ranged from 8-73%.

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Chlorination is the only wastewater treatment process, which limits the

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removal efficiency for most antibiotics in medical wastewater, probably because of

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the high pollution load and complex composition. Compared to the hospital samples,

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the removal efficiency of antibiotics in STPs were relatively higher with a total

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removal efficiencies of 17-100%. Overall, the removal efficiencies of tetracyclines

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and quinolones were relatively high and are similar to the removal efficiencies of

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other STPs (Xu et al., 2007; Vieno et al., 2007; Gao et al., 2010; Chen et al., 2012),

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because quinolones and tetracyclines are susceptible to photodegradation or sorption

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(abiotic) onto sludge (Lindberg et al., 2006; Xu et al., 2007). Compared with

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fluoroquinolones and tetracyclines, the removal efficiencies of sulfonamides and

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macrolides were lower, especially macrolides (17-51%), and these efficiencies were

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similar to those reports in four STPs in the Pearl River Delta (Xu et al., 2007), eight

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ACCEPTED MANUSCRIPT STPs in Beijing (Gao et al., 2012), one STP in Chongqing, China (Chang et al., 2010)

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and STPs in Italy (Castiglioni et al., 2006).This phenomenon might be explained by

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two main reasons. Some studies confirmed that sulfonamides are highly water soluble

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and have negligible sorption to sludge biomass (Batt et al., 2007). Macrolides tend to

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be sorbed to sludge, however, conjugated metabolites and adsorption behavior may

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easily be altered by changing physicochemical parameters during the treatment

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process, which results in the secondary release of macrolides from sludge to water or

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de-conjugation (Xu et al., 2007; Gao et al., 2012). In addition, the concentrations of

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SD and SMX in the effluent were higher than those in the influent, which may

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indicate that the metabolites of SD and SMX were translated into their parent (Göbel

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et al., 2005; Göbel et al., 2007). Thus, these results provide a good insight into the

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mechanism of mutual transition between metabolites and the parents of some

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antibiotics. In STPs, the removal of antibiotics depends on the biological systems and

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chlorination plays a subsidiary role. The elimination of antibiotics at various STPs is a

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complex process, that can be affected by many factors (hydraulic retention time

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(HRT), solids retention time (SRT), temperature and pollution load) (Xu et al., 2007;

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Battl et al., 2007). Therefore, elimination rates can vary significantly in different STPs.

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The STPs in Haidian District possessed the best removal capabilities for sulfonamides

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(72%) and macrolides (42%), which were lower than those of the STPs in Beijing.

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STP in Chaoyang District possessed the best removal capabilities for quinolones

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(92%) and tetracyclines (100%), which might be related to the low pollution load. The

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worst removal capabilities were found at the STP in Chaoyang District for

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ACCEPTED MANUSCRIPT sulfonamides (28%) and macrolides (25%), which were slightly lower than those of

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the STP in Miyun District. Compared with cyclic activaled sludge technolohy (CAST)

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(STP in Chaoyang District) and A2/O (STP in Miyun District), A/O+MBR technology

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may be more effective for the removal of antibiotics, especially sulfonamides and

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macrolides. Zhao (2017) reported that A/O+MBR technology can result in a higher

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removal efficiency of SMX than that of NOR. As a currently rare sewage treatment

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process, CAST technology showed the high removal efficiencies for quinolones and

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tetracyclines.

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The maximum removal efficiencies for sulfonamides (70%) in the STPs of this

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study were higher than those (48%) of the STP in Chongqing (Chang et al., 2010),

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similar to the value (65%) by the STPs in the Pearl River Delta (Xu et al., 2007) and

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Taiwan (82%) (Lin et al., 2009) and lower than those (100%) of a STP in the USA

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(Karthikeyan and Meyer, 2006). The maximum removal efficiencies (100%) of

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quinolones were higher than those of a STP in Madrid (57%) (Rosal et al. 2010),

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similar to a STP in the USA (100%) (Karthikeyan and Meyer, 2006) and slightly

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higher than those of the STPs in Finland (96%) (Vieno et al., 2007) > in Sweden

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(90%) (Zorita et al., 2009) > in Switzerland (87%) (Golet et al., 2002) > in the Pearl

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River Delta (81.88%) (Xu et al., 2007) > in Chongqing (81%) (Chang et al., 2010) >

330

in Taian (80%) (Lin et al., 2009). The maximum removal efficiencies of tetracyclines

331

were 100% in the STPs in this study, and these values were 100% in Chongqing

332

(Chang et al., 2010) 100% in the USA (Karthikeyan and Meyer, 2006) and > 91% in

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Taiwan. Thus, high removal efficiencies of tetracyclines are common in most STPs.

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study>80% in several STPs in the USA (Karthikeyan and Meyer, 2006) > 77% in

336

Taiwan (Lin et al., 2009) > 76.23% in the STPs in the Pearl River Delta (Xu et al.,

337

2007) > 39% in Chongqing (Chang et al., 2010) > 4% in Madrid (Rosal et al., 2010).

338

These results reflected the large difference in the removal efficiencies of macrolides.

339

Overall, the removal capabilities of antibiotics were lower than those found in a

340

previous study on the STPs in Beijing (Gao et al., 2012), especially the removal

341

efficiencies of sulfonamides and macrolides, which may indicate that lower

342

temperatures in winter resulted in ow bioactivity of microorganisms.

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343 344

Table 1 Occurrence profile and risk quotients of the target antibiotics in Wenyu River and

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groundwater of Beijing a

-1

PNEC (ng L )

SD SMX SMT TMP NOR CIP ENR OFLO SFLO TC OTC CTC

135 27 1277 16000 16 112.3 81.7 713.6 28.1 87.9 90.3 142.5

-1

MEC(ng L )

DF (%)

RQ

MEC(ng L-1)

DF (%)

RQ

1.57-256.6 32.5-128 ND-4.11 ND-73 ND-113 ND-36.9 ND 80.9-1270 ND ND-16.15 ND-6.24 ND-6.12

100 100 57 43 71 86 0 100 0 43 14 29

1.90 4.74 <0.01 <0.01 7.06 0.33 <0.01 1.78 <0.01 0.18 0.07 0.04

ND-17.6 ND-9.41 ND-1.67 ND ND-3.6 ND-4.1 ND ND-13.2 ND ND ND ND

86 86 43 0 57 71 0 57 0 0 0 0

0.13 0.35 <0.01 <0.01 0.225 0.04 <0.01 0.02 <0.01 <0.01 <0.01 <0.01

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Compounds

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ERM-H2O

624.8

ND-84

86

0.13

ND-1.21

57

<0.01

ROM

314.2

ND-69

86

0.22

ND

0

<0.01

346 347 348 349 350

PNEC: predicted no effect concentration MEC: measured environmental concentrations DF: detection frequency RQ: Risk quotient a

Liu et al., (2018a) 16

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Figure 2 Accumulative concentration of antibiotics in each sampling site in Wenyu River.

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NOR 910 354 61 113 68 88 99.8 ND 100 172.36 20.62 88 5.1 75 97 607 331 45

CIP 98.4 ND 100 ND 78.7 ND 100 51.6 6.5 87 ND 100 8480 3784.4 20

ENR 63.99 ND 100 ND 41.9 6.73 84 ND ND ND ND ND -

18

OFLO SFLO 1740.3 ND 800.6 ND 54 519.27 ND 35 70 796 ND 117.22 ND 85 1340.35 ND 498.2 ND 63 381.44 ND 23 72 16800 ND 10430 ND 38 -

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TMP 560.8 229.5 59 140.37 39 75 143 60.98 57 300.77 106.15 65 91.26 14 70 844.77 779.15 8

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SMT 40.33 20.21 50 21.19 -5 47 ND ND ND ND ND ND ND -

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SMX 460 483.55 -5 582.09 20 -27 70.01 81.53 -16 1020 881 54 640 27 67 2490.3 1546.61 46

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SD 1010.6 518.91 49 585 -13 42 370 207.6 43.89 526 136.6 74 107.6 21 80 ND ND -

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Compounds influent Secondary effluents STPs Removal(%) ( Miyun effluent District) Removal(%) Total removal(%) STPs influent ( Chaoyang effluent District) Total removal(%) influent Secondary Effluents STPs Removal(%) ( Haidian effluent District) Removal(%) Total removal(%) Hospital influent(YY1) (Haidian effluent(YY2) Distric) Removal(%)

Table 2 Occurrence profile and fate of the target antibiotics in STPs and hospital of Beijing, ng L-1

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TC 490.3 79.49 84 10.69 87 98 30.17 ND 100 57.1 20.99 63 ND 100 76.33 20.81 73

OTC 16.5 6.78 59 6.32 7 62 7.14 ND 100 88.75 ND 100 ND ND ND -

CTC 9.25 ND 100 ND 9.6 ND 100 24.9 ND 100 ND ND ND -

ERM-H2O 1280 1100 14 976 17 24 362.19 241.1 33 1030 697 32 509.61 27 51 240.8 190.1 21

ROM 879 610 30 482 21 45 1640 1368 17 990 761.4 23 657.45 14 34 56.33 40.89 27

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3.3 Occurrence of AGRs To minimize the variance caused by different background abundances of bacteria

356

and DNA manipulation efficiencies, the relative abundances (normalized to 16S

357

rRNA) of all nine selected ARGs, including two sulfonamide resistance genes (sul1,

358

sul2), five tetracycline resistance genes (tetA, tetB, tetC, tetM, tetW), one quinolone

359

resistance gene (qnrS), one macrolide resistance gene (ermB) and Class I Integron

360

(int1), were calculated in the different water samples (Wenyu River, groundwater,

361

hospital, STPs) (Figure 3). Among the tested ARGs, sul1, sul2 and qnrS had 100%

362

detection frequencies in typical urban water of Beijing, followed by tetA (95.83%),

363

tetM (95.83%), ermB (95.83%), tetW (87.50%), tetC (83.33%), and tetB (79.17%).

364

sul1 had the highest relative abundance (1.77×10-3-1.18×10-1), followed by sul2

365

(6.04×10-5-7.11×10-2), ermB (2.53×10-5-4.19×10-2), tetW (1.76×10-5-1.23×10-2), tetA

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(6.46×10-5-1.08×10-2), tetC (1.31×10-4-5.35×10-3), tetM (9.83×10-5-7.97×10-3), qnrS

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(1.67×10-6-9.25×10-4), and tetB (4.31×10-7-3.92×10-4).

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The differences in ARG abundances were highly significant. The relative

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abundance of sul1 was significantly higher than that of other ARGs (p<0.01) in

370

typical urban water of Beijing, especially in groundwater, was 1-4 orders of

371

magnitude higher than that of other ARGs. The relative abundance of sul 2 was

372

significantly higher than that of other ARGs (P<0.05) except for erm B and tet C

373

(P>0.05). This results showed a predominant presence of sulfonamide resistance

374

genes. In addition, the sul1 gene encodes dihydropteroate synthase and is generally

375

harbored in int1 (Antunes et al., 2005; Luo et al., 2010; Ramesh Kumar et al., 2017).

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between sul1 and int1 were observed (R = 0.9887) (Figure 3). Thus, these results may

378

provide good insight for the establishment of a quantitative model of sul1 and int1,

379

and the reliability of the model can be proven by a large number of additional

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detection data.

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Overall, a higher relative abundance of ARGs was detected in hospital

382

wastewater and STP sewage samples than in the other water samples, and these

383

results are similar to those observed in Girona, Spain (Rodriguez-Mozaz et al., 2015).

384

STPs and hospitals are highly favorable environments for the selection of

385

antibiotic-resistant bacteria (ARB) or the horizontal gene transfer and propagation of

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ARGs because of their high microbial densities (Chen and Zhang, 2013; Qiao et al.,

387

2017; Wang et al., 2017). STPs and hospitals have been identified as important

388

sources of ARG pollution in the water environment (Chang et al., 2010; Marti and

389

Balcázar, 2013). A predominant presence of ermB was observed in hospital

390

wastewater. Curiously, macrolide consumption is more widespread in households

391

than in clinical settings (Kümmerer and Henninger, 2003). The abundances of

392

tetracycline resistance genes appeared to be higher than those of other ARGs in STPs.

393

However, no predominant presence of tetracyclines was observed in the STPs. Thus,

394

the abundance of ARGs did not appear to be correlated with antibiotic usage, and

395

co-selection or cross-selection likely occurred (Di et al., 2016). Di et al. (2016)

396

reported that the co-selection of heavy metal resistance genes and ARGs in water

397

environment can increases the complexity of the ecological role of ARGs, which may

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ACCEPTED MANUSCRIPT reduce the effectiveness of control actions. The relative abundances of ARGs in

399

hospital effluents were higher than or not significantly different (p < 0.05) from those

400

in hospital influents. This phenomenon was observed for all ARGs except for tetA,

401

which indicated that chlorination did not have an obvious effect on ARG removal

402

from hospital wastewater under the current process parameters. However, the relative

403

abundances of most ARGs in the STPs exhibited a declining trend in the order of

404

influent > secondary effluents > effluent. Zhuang et al. (2015) reported that

405

chlorination was better than ultraviolet and ozonation disinfection for ARG removal

406

in STPs. However, Jia et al. (2015) reported that the relative abundances of ARGs in

407

drinking water increased after chlorination, and chlorine disinfection could not

408

destroy ARGs (Furukawa et al., 2017). The mechanisms of the responses of ARGs to

409

chlorination in different wastewaters require further study. In addition, it was

410

common for the relative abundances of sul1, sul2, tetC and int1 in the effluent to be

411

significantly higher than those in the influent in three STPs (p < 0.05), which might be

412

explained by the fact that either some microbes proliferated during the process of

413

sewage treatment or moving components that carried sul1, sul2, tetC and int1 were

414

amplified or related to the spread of some ARGs among bacterial cells in activated

415

sludge. This phenomenon also was observed and proved by some studies

416

(Szczepanowski et al., 2009; Rizzo et al., 2013; Rodriguez-Mozaz et al., 2015), which

417

indicted that ARGs removal need further study and can pose a great threat to

418

receiving rivers or lakes.

419

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Although some ARGs were not detected in groundwater (detection frequencies 21

ACCEPTED MANUSCRIPT of 100% in other water bodies), sul1 had the highest relative abundance in

421

groundwater compared to other water bodies or ARGs, which was related to the high

422

relative abundance of int1 (Figure 2). The relative abundances of other ARGs were

423

low, which might be related to the purification of the soil filtration system. In addition,

424

there were no significant differences in most ARGs under different water levels

425

(p>0.05) except for sul1 (p<0.05), which exhibited the order of middle water level (12

426

m) > low water level (31.5 m) > high water level (6.5 m). Tang et al. (2015) reported

427

that a positive correlation between the abundance of ARGs and soil depths was

428

observed, which may result in the differences observed in the different water levels.

429

The effect of water level on the relative abundance of ARGs requires further

430

investigation. Groundwater pollution poses a serious threat to drinking water safety.

431

However, studies on ARG migration and transformation in groundwater are limited,

432

which should be highlighted.

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Overall, the relative abundances of all ARGs except for sul2 in the Wenyu River

434

were low. According to a previous investigation (Liu et al., 2018a), unlike

435

tetracyclines, the concentrations of sulfonamides in water bodies were high due to the

436

widespread use of the corresponding antibiotics and high solubility and chemical

437

stability in the environment, which likely resulted in the high abundance of sul2.

438

Sulfonamide resistance genes have a broad host range and can be carried by strains in

439

different environments, resulting in high detection frequencies (Zhang et al., 2009). In

440

addition, the relative abundance of ARGs at the WYH2 site was higher than that of

441

other sites. This phenomenon might be explained by two main reasons. First, some

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443

antibiotics at the WYH2 site. Second, the flow velocity of water bodies is very slow,

444

which provides an ideal environment for ARG enrichment.

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Figure 3 Relatively abundance of ARGs in surface water in Wenyu river,Groundwater,

447

Medical wastewater and Sewage in STPs The heat map generated from MATLAB’s clustergram function gives a visual

449

representation of the sul1, sul2, tetA, tetB, tetC, tetM, tetW, qnrS, ermB and int1

450

concentrations in typical urban water in Beijing (Figure 4). The figure shows that sul1

451

and int1 are clustered in one group (Class 1), which further confirmed that int1

452

abundance in typical urban water in Beijing may play a vital role in the distribution of

453

the sul1 gene. tetB and qnrS (Class 2), ermB and tetW (Class 3) and tetC and sul2

454

(Class 4) were clustered, indicating that there is the possibility of the coexistence of

455

tetB and qnrS, ermB and tetW and tetC and sul2 in typical urban water in Beijing.

456

tetC and sul2 (Class 4) were clustered in one group, which was similar to the patterns

457

observed in Honghu Lake and East Dongting Lake (Yang et al., 2016). tetA and tetM

458

were clustered in one group, probably because of independently induced mechanisms

459

or existing modes. In addition, the sampling sites in typical urban water in Beijing

460

were mainly clustered into three main groups. Overall, class 1 mainly comprised the

461

groundwater samples; class 2 comprised the water samples from the Wenyu River;

462

class 3 comprised the water samples from hospital and STPs. This result indicated that

463

there were differences in the ARG abundances in typical urban water in Beijing.

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Figure 4 Heat map constructed for the cluster of sul1, sul2, tetA, tetB, tetC, tetM,

467

tetW, qnrS, ermB, int1and sampling sites in Beijing 3.4 Correlation between concentrations of antibiotics and corresponding ARGs

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Most studies have confirmed that antibiotics play a vital role in the process of

470

inducing ARGs (Rodriguez-Mozaz et al., 2015; Chen et al., 2016; Qiao et al., 2017;

471

Yang et al., 2018). However, whether there is an inevitable correlation between

472

antibiotics and ARGs has not reached a uniform conclusion. Rodriguez-Mozaz et al.

473

(2015) found that there were significant positive correlations between the

474

concentrations of antibiotics and their corresponding ARGs in hospital, urban

475

wastewater and surface water samples. However, Gao et al. (2012) found that the

476

abundances of tet genes in a municipal wastewater treatment plant appeared to be not

477

significantly correlated with the concentration of tetracyclines. Positive correlations

478

were observed between the total concentrations of SAs and sulfonamide resistance

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genes in the Haihe River (Luo et al., 2010), but there was no correlation with

480

sulfonamide resistance genes in a sewage treatment plant and its effluent-receiving

481

river of Beijing (Xu et al., 2015). In this study, significant positive correlations between tetA and total TC

483

concentrations (R=0.5788, p=0.002), tetM and total TC concentrations (R=0.6576,

484

p=0.001), and tetW and total TC concentrations (R=0.6345, p=0.001) were observed

485

(Figure 5). The correlations between tetW and total TC concentrations (R=0.4843,

486

p=0.016) and ermB and total MC concentrations (R=0.4003, p=0.043) were found to

487

be considerably weak. sul1 was negatively correlated (R=-0.6518, p=0.001) with total

488

SA concentrations. There were no positive correlations between sulfonamide

489

resistance genes and total SA concentration, and these findings are similar to the

490

results of Xu et al. (2015), probably because of the longer use and existence of

491

complex cross-induction

492

between other ARGs and their corresponding ARGs (p>0.05). However, significant

493

positive correlations between the concentrations of antibiotics and their corresponding

494

ARGs were observed if abnormal points were deleted except for qnrS and total QN

495

concentrations. Overall, the current relevance between the concentrations of

496

antibiotics and their corresponding ARGs has achieved satisfactory results due to the

497

complexity of the typical urban water. This study indicates that most ARGs increase

498

with the increase in the concentration of antibiotics, which is consistent with the

499

results of previous studies (Czekalski et al., 2012; Rodriguez-Mozaz et al., 2015;

500

Chen et al., 2016; Wang et al., 2016; Qiao et al., 2017;Yang et al., 2018).

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(Zhang et al. 2015). No significant differences were found

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Figure 5 Correlations between the concentrations of antibiotics and their corresponding ARGs. Light gray lines show 95% confidence intervals. 4. Conclusions This study reported the occurrence of antibiotics and ARGs in the surface water of

506

the Wenyu River, groundwater and hospital samples, as well as their fates in the STPs.

507

The results show a predominant presence of fluoroquinolones, especially in the

508

hospital samples. Fluoroquinolones and tetracyclines were removed relatively more

509

efficiently than sulfonamides and macrolides in the STPs, resulting in a high relative

510

abundance of ARGs. Sulfonamide resistance genes were detected at the highest

511

relative abundance and highest frequencies. Most ARGs in the STPs were reduced in

512

the effluent except for sul1, sul2 and tetC. The removal of antibiotics and ARGs by

513

STPs is incomplete, and these antibiotics are discharged into the environment, which

514

contributes to their persistence and dissemination in aquatic environments. Given this

515

situation, the sources and fates of ARGs and their potential hazards to the aquatic

516

ecosystem and human health need further investigation.

517

Acknowledgements

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This work was financially supported by Ministy of Science and Technology of

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China (No.2015FY110900) and the National Natural Science Foundation of China

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(No.41877409).

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ACCEPTED MANUSCRIPT Highlights: This study firstly explored the occurrence and fate of antibiotics and antibiotic resistance genes in diverse water samples of Beijing.

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The quinolone antibiotics were predominant risk and pollution factors, especially in the hospital wastewater.

Higher relative abundance of ARGs were detected in hospital wastewater and

SC

STPs sewage samples.

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It was worth noting that sul1 abundance was the highest in groundwater.

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Negative correlation between sul1 with total SAs concentrations were observed.