Journal of Environmental Science and Health, Part B Pesticides, Food Contaminants, and Agricultural Wastes

ISSN: 0360-1234 (Print) 1532-4109 (Online) Journal homepage: https://www.tandfonline.com/loi/lesb20

Antibiotic contamination in a typical water-rich city in southeast China: a concern for drinking water resource safety

Xinyan Guo, Lv Xiaojun, Aiguo Zhang, Zheng Yan, Siyi Chen & Na Wang

To cite this article: Xinyan Guo, Lv Xiaojun, Aiguo Zhang, Zheng Yan, Siyi Chen & Na Wang (2019): contamination in a typical water-rich city in southeast China: a concern for drinking water resource safety, Journal of Environmental Science and Health, Part B, DOI: 10.1080/03601234.2019.1679563 To link to this article: https://doi.org/10.1080/03601234.2019.1679563

Published online: 28 Oct 2019.

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Antibiotic contamination in a typical water-rich city in southeast China: a concern for drinking water resource safety

Xinyan Guoa,b, Lv Xiaojunc, Aiguo Zhanga, Zheng Yand, Siyi Chene, and Na Wanga,b aNanjing Institute of Environmental Science, Ministry of Environmental Protection, , China; bKey Laboratory of Pesticide Environmental Assessment and Pollution Control, Ministry of Environmental Protection of China, Nanjing, China; cAppraisal Center for Environment & Engineering, Ministry of Environmental Protection, Beijing, China; dChinese Society for Environmental Sciences, Beijing, China; eNanjing University of Science and Technology, Nanjing, China

ABSTRACT ARTICLE HISTORY The occurrence and distribution in the aquatic environment of Nanjing of 49 from Received 2 June 2019 seven therapeutic classes were investigated using an improved analytical method developed for Accepted 6 October 2019 multiclass target analysis. The results showed that these antibiotics are widely present in the water KEYWORDS bodies of this city, with a total concentration of up to 1.958 lgL 1. The most abundant class was Antibiotics; drinking tetracyclines, contributing 43.7% to the total antibiotic burden. Lincomycin was detected in all water resource samples, and the detection rate of clindamycin was 90.5%. An exploratory analysis of the data points was performed by unsupervised pattern recognition (hierarchical cluster analysis, HCA) in an attempt to clarify the pollution level in different sampling areas, and robust cluster solutions grouped the data according to their different antibiotic contaminant profiles. The safety of drink- ing water resources was emphasized, and the rivers, as the main receiving water body for treated and untreated wastewater in this city, were more seriously contaminated than the surrounding lakes and reservoir, not only in concentration but also in detection frequency, in our study as well as in similar research studies. A correlation analysis between the presence of antibiotics and the environmental factors was conducted, and it was found that antibiotic contamination and water quality were closely connected; the better the water quality, the lower the antibiotic contamin- ation. Positive correlations existed between the antibiotics and tested heavy metals, as well as between antibiotics and boron and arsenic. However, whether these correlations resulted from their reaction or a common source was difficult to determine, and the mechanism requires further exploration.

Introduction one of the largest economic zones of China. Containing many rivers and lakes, the Nanjing area is known for its Drinking water sources (DWSs) are of great importance to rich water resources, with the water area accounting for human health. However, multiple classes of antibiotics have 11.4% of its total territory. In addition, large quantities of been detected in DWSs in countries such as the USA,[1,2] [3] [4–7] [8–13] antibiotics are widely used in both human medicine and Canada, several European countries, and China. animal husbandry, with a total population in the Nanjing In general, antibiotic concentrations occur at the 1–10 ng 1 area of 8,230,000 (gov.longhoo.net, retrieved 1 October L level, but the concentrations of several antibiotics, such 2016). All of these factors make the Nanjing area a good as and , have exceeded 100 ng case for investigating the contamination pattern of antibiot- 1 [13] L . Antibiotics seem to be persistent contaminants in ics in different water bodies. aquatic environments, whose longest half-life reaches Some studies have previously detected a correlation [14,15] 1,800 days. The presence of various classes of antibiot- between the presence of antibiotics and environmental fac- ics in DWSs has led to an increasing concern about the tors, attempting to determine the behavior, distribution or potential environmental risks and the maintenance and sources of antibiotics. A positive relationship was found spread of antibacterial resistance among microorgan- between dissolved organic carbon (DOC) and the log total – isms,[16 18] which undermines our ability to prevent and concentration of antibiotics in the South Yellow Sea control infectious diseases and exerts a great threat to Estuary[19] and in the Estuary,[20,21] which was human health in the long term. attributed to the binding of antibiotics to DOC, which could Nanjing, with a total land area of 6598 square kilometers, help retain antibiotics in the aqueous phase of environmen- is situated in the heartland of the drainage area of the lower tal matrices.[22] Ammaiyappan et al.[23] studied the correl- reaches of the Yangtze River and in the Yangtze River Delta, ation among livestock populations and aquatic antibiotic

CONTACT Na Wang [email protected] 8 Jiang-Wang-Miao Street, Nanjing 210042, China. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lesb. ß 2019 Taylor & Francis Group, LLC 2 X. GUO ET AL. concentrations for source apportionment in Hong Kong. to collection. The samples were kept at 4 C in a refrigerator Antibiotics do not exist endogenously in the environment. and processed within 24 h. Accordingly, the exploration of the related environmental factors plays an important role in providing insight and dir- ection for the prevention and control of antibiotic pollution. Chemicals and standards Moreover, the multi-residue method for the determin- Antibiotic standards (Table 1) were obtained from Dr. ation of antibiotics in water samples by SPE-LC-MS/MS Ehrenstorfer GmbH (Augsburg, Germany). The internal [24,25] appears to be outdated. It is still important to study standards, tetracycline-d6 and sulfamethazine-d6, were pur- and improve analytical methods for a wider range of target chased from Toronto Research Chemicals (North York, chemicals, making pollution detection more robust, rapid Ontario). Methanol, acetonitrile and formic acid of HPLC and sensitive. grade were acquired from Merck (Darmstadt, Germany), In this study, we improved a simultaneous analytical while ethylenediaminetetraacetic acid disodium salt method to investigate the occurrence and distribution of 49 (Na2EDTA) and sodium hydroxide (NaOH) of analytical antibiotics at 21 sampling sites all over Nanjing. Drinking grade were supplied by Nanjing Chemical Reagent Co., LTD water resources were our focus, including different types of (Nanjing, China). The ultra-pure water used in the experi- rivers, lakes and reservoirs. We also explored the correlation ment was purified from a Milli-Q water purification system between the presence of antibiotics and environmental fac- (Millipore Corporation, Billerica, MA, USA). tors to better understand how antibiotics are affected in the Standard antibiotic stock solutions with concentrations of aquatic environment. These findings advance our fundamen- 1,000 mg L 1 were prepared. Penicillin was prepared in ace- tal understanding of the occurrence, distribution and sources tonitrile:water (1:1, v/v), sulfadiazine and four quinolones of antibiotics in the various types of water bodies in south- were prepared in ammonia:methanol (1:99, v/v), and eastern China. streptomycin was prepared in methanol:water (1:4, v/v). The remaining antibiotics were prepared by dissolving powder of Materials and methods the corresponding standards in methanol. Notably, erythromycin will degrade under acidic condi- Site description and sample collection tions; hence, dehydrated erythromycin was detected to Nanjing, a city from the region exhibiting the largest anti- monitor erythromycin during the experiment. Dehydrated biotic usage out of seven regions of China,[26] was selected erythromycin could be obtained by the following method: a 30% methanol solution was used for the preparation of for this study as a representation of modern urban areas 100 mg L 1 erythromycin, and then the pH was adjusted to with abundant water resources. As reported in the Nanjing 1 City Water Resource Bulletin of 2015, the total city water 3 with H2SO4 (3 mol L ). The solution was stirred at room consumption was 4.024 billion m3, among which 0.1672 bil- temperature for 3 h to ensure that the erythromycin trans- lion m3 was used for drinking. Surrounded by the Yangtze formed completely into dehydrated erythromycin. River, the urban area of the city enjoys a scenic natural environment, and and the lower reaches of Extraction and HPLC-MS/MS methods the Qinhuai River are located in the center of the city. Gucheng Lake and Fangbian Reservoir are DWSs that are A 1 L water sample was filtered through a 0.45 lm glass located in the suburbs of Nanjing. As depicted in Figure 1, microfiber filter, and the pH was adjusted to 11 by 8 mol 1 l some of the samples were taken from the most important L NaOH. Na2EDTA (0.8 g) and 20 L of the internal 1 DWSs for Nanjing, namely, samples from the Yangtze River standard solution (10 lgmL tetracycline-d6 and sulfame- in Nanjing (D1–D4), Fangbian Reservoir (D5) and Gucheng thazine-d6) were added to the solution before solid-phase Lake (D6), which represent a typical river, reservoir and extraction. The filtered water samples were then extracted lake, respectively. All of the DWS sampling sites were using tandem dual SPE cartridges. located within close proximity to municipal drinking water MAX (3 mL, 60 mg, Waters Corporation, USA) cartridges treatment plants. The other samples were taken from were above, and HLB (6 mL, 200 mg Waters Corporation, important groundwater areas in and around Nanjing, USA) were below on the Supelco Visiprep SPE system including the Yangtze River (not from a DWS reserve, (Supelco, USA). All cartridges were preconditioned with R1–R4), the Qinhuai River (W1–W6), Xuanwu Lake 6 mL of methanol, 6 mL of ultra-pure water, and another (W7–W10) and (W11). Three parallel samples 6 mL of ultra-pure water at pH ¼ 11. After loading the sam- were collected for each site during the November 2016 sam- ples at a flow rate of 3–5mL min 1, the MAX cartridges pling campaign. were rinsed with 6 mL of an aqueous 5% ammonia solution, Sampling followed standard examination methods for the HLB cartridges were washed with 6 mL ultra-pure water, drinking water collection and preservation of water sam- and all cartridges then vacuum dried. ples.[27] All samples were surface samples (within 50 cm Subsequently, the MAX cartridges were eluted with under the surface) collected using a water sampler in a fish- 4.5 mL of a solution (formic acid aqueous solution (pH ¼ ing vessel at the center of flow. The samples were immedi- 3):methanol, 1:9, v/v) for water-soluble antibiotics, which ately transferred to a 5-L precleaned amber glass bottle, and was diluted to 5 mL, named Solution I, and then transferred each bottle was rinsed with sample water three times prior to LC vials after filtering through 0.22 lm syringe filters. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 3

Figure 1. Sampling sites in different water bodies of Nanjing city.

Then, the HLB cartridges were eluted with 3 2mL of antibiotics and the rest untreated for the analysis methanol, and the MAX cartridges were eluted again with of penicillin. 3mL of methanol and a 2 2 mL of a methanol solution The UPLC–MS/MS system used comprised an Agilent with 2% formic acid. This procedure was performed for the 1290 Infinity ultrahigh-performance liquid chromatograph poorly water-soluble antibiotics. These combined eluates (Agilent Technologies, USA) and a SCIEX QTRAP 4500 tan- were dried with a gentle stream of nitrogen at 40 Cina dem mass spectrometer (AB SCIEX, USA). The separation water bath. Then, 2 mL of ACN:water, 1:9 (v/v), named was performed with an Agilent EC-C18 column (2.7 lm, Solution II, was added, and the mixture was vortexed for 2.1 75 mm, Agilent, USA), and the column was maintained 2–3 min. The mixtures were filtered through 0.22 lmsyr- at 30 C at a flow rate of 0.4 mL min 1. The injection vol- inge filters and divided into two parts, 1mL (with 1.8 lLof ume was 5 lL. Mobile phase A was an aqueous solution of formic acid added) for analysis of most of the targeted 0.2% formic acid, and mobile phase B was ACN. The elution 4 X. GUO ET AL.

Table 1. Targeted antibiotics. standard were recorded. One was detected directly, and the Antibiotic name Abbreviation Antibiotic name Abbreviation other was spiked with the appropriate amount of a mixed Quinolones Sulfonamides standard solution based on the results of the unspiked ana- NOR Sulfadiazine SDZ Cirofloxain CIP SMZ lysis. The concentrations of the target antibiotics were calcu- ENR SDM lated by the following equation: OFL Sulfamethoxazole SMX Cs Rx FLE STZ Cx ¼ (1) SAR Sulfachloropyridazine SCP Rs Rx LOM Sulfamethazine SMT 1 Balofloxacin BAL SP where Cx is the corrected concentration, mg L ; Cs is the PAZ Sulfamonomethoxine SMM additional concentration of the standard solution, mg L 1; Tetracyclines SMTZ Tetracycline TC Sulfisoxazole SSE Rx is the ratio of the peak areas of the sample and the Oxytetracycline OTC SCM internal standard before the standard solution was added; Chlortetracycline CTC Sulfabenzamide SBM and RS is the ratio of the peak areas of the sample and the Doxycycline DC Lincosamides Macrolides Clindamycin CLN internal standard after the standard solution was added. Erythromycin ERY Lincomycin LNC Recovery tests were prepared by adding appropriate Roxithromycin ROX Penicillins standard solutions of target antibiotics into blank water Josamycin JOS Penicillin G PEN G Spiramycin SPR Penicillin V PEN V samples. The recoveries of the spiked antibiotics ranged Clarithromycin CLR Amoxicillin AMX from 51.74% to 96.30%, and the relative standard deviations Azithromycin AZT Oxacillin OXA (RSD) were 2.19% to 9.67%. Notably, these recoveries were Tylosin TYL Cloxacillin CLO Tilimicosin TLM Nafcillin NAF calculated from both Solution I and Solution II. The linear- Kitasamycin KTS Dicloxacillin DIC ity of the target antibiotics was good in the range of Cephalosporins Ampicillin AMP 0.001–0.5 lgmL 1 (0.01–5 lgmL 1 for streptomycin). The Cephapirin HAP Aminoglycosides Cephalexin LEX Streptomycin STR limits of detection (LODs) and the limits of quantification Cefradine CED (LOQs) were defined as the concentrations corresponding to the signal-to-noise (S/N) ratios of 3 and 10, respectively. – 1 program was initiated with 90% A and 10% B, and then The LODs of the antibiotics were 0.01 3.23 ng L (Solution – 1 mobile phase A was linearly changed to 60% in 7 min and I) and 0.05 3.43 ng L (Solution II). The LOQs were – 1 – 1 further decreased to 40% over 3 min. After that, mobile 0.04 10.75 ng L (Solution I) and 0.17 11.43 ng L phase A was rapidly increased to 90% within 0.01 min and (Solution II). More information is shown in detail in maintained for 1.49 min. The total run time of the elution Table A2. gradient was 11.5 min. Chromatograms of the targeted anti- biotics are shown in Figure A1. Other chemical analyses The mass spectrometric analyses were performed with a positive electrospray ionization (ESIþ) source in multiple The quality parameters of the water samples, including total [28] [29] reaction monitoring (MRM) mode. The source temperature nitrogen (TN), ammonia nitrogen (NH3-N), total was set at 450.0 C, the ion spray voltage (IS) was 4,500 V, phosphorus (TP)[30] and chemical oxygen demand the pressure of the curtain gas (CUR) was 35.0 psi, the ion (CODCr),[31] were analyzed according to the corresponding source gas1 (GS1) was 55.0 psi and gas2 (GS2) was 60.0 psi, Chinese national standards. The concentrations of 22 metals, and the parameter of the collision gas (CAD) was medium. including lithium (Li), beryllium (Be), boron (B), titanium The retention times, parent ions, product ions and other (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron parameters of MS/MS are displayed in Table A1. (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), strontium (Sr), molybdenum (Mo), sil- ver (Ag), cadmium (Cd), antimony (Sb), barium (Ba), thal- Quality assurance/quality control lium (Tl), and lead (Pb), were also quantified, according to [32] To ensure the reliability of the experimental results, a pro- a standard protocol, in this study. cedural blank (batch-to-batch method performance), dupli- cate samples and internal standards were used to perform Statistical analysis the quality control of the analysis process. Sulfonamides and the other antibiotics, except tetracycline, with a retention A statistical evaluation of the data was conducted using time less than 2.5 min were quantified with sulfametha- SPSS version 19.0. Shapiro-Wilks tests were used to deter- zine-d6 (SMT-d6); tetracycline and the antibiotics with a mine whether the dataset (3 < n < 50) belonged to a normal retentiontimemorethan2.5minwerequantifiedwith distribution population. Nonparametric Kruskal–Wallis tests tetracycline-d6 (TC-d6). were conducted to obtain significant testing results. P values The standard addition method was used to reduce the lower than 0.05 were considered to be significant. Pearson influence of the water matrix in this study. The three correlations were conducted to assess the strength of the extracts obtained in Section of Extraction and HPLC-MS/ relationship between environmental factors and residual MS methods were prepared in replicates for detection, and antibiotic concentrations. A clustering analysis was per- the peak areas of the target antibiotics and the internal formed using HemI 1.0.[33] JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 5

Results and discussion target antibiotics, 18 antibiotics from 7 therapeutic classes were detected in the Nanjing aquatic environments, includ- Method development ing quinolones (2/9), sulfonamides (6/13), tetracyclines (3/ The development of multiclass methods for the determination 4), macrolides (3/9), lincosamides (2/2), cephalosporins (2/ of antibiotics in liquid matrices is unsatisfactory with regard 3), and penicillins (0/8). The detection frequency of LNC to many analytical and chemical aspects. The chemical struc- was high, reaching up to 100.0%. The detection frequencies tures of these analytes are disparate; a sample preparation of SCP, SMT, ERY, CLN and CED were between 38.1% and procedure should be versatile enough to ensure the enrich- 90.5%. The other target antibiotics were sporadically < ment of all compounds. On the basis of the literature and the detected with detection frequencies 28.6%. The frequent authors’ experience, HLB cartridges are adequate for retaining detections described above were also shown in previous most of the antibiotics by intermolecular interactions.[24,34,35] studies from other regions. In the Dongjiang River and the However, when confronted with all of the target compounds, Beijiang River of southern China, , norfloxacin the use of only HLB is difficult. Various solid phase extrac- and were detected with detection frequencies of 75–100% under the influence of industrial wastewater and tion cartridges, including HLB, Waters Oasis MAX, Waters [8] Oasis WCX (6 mL, 150 mg, Waters Corporation, USA), domestic sewage. In the Liao River of northeastern China, the detection frequencies of ofloxacin, erythromycin, and Waters Oasis MCX (6 mL, 150 mg, Waters Corporation, roxithromycin in the water were 100%, followed by oxytetra- USA) and CNWBOND SAX (200 mg, 6 mL, Anpel, China) cycline (85.7%), which may originate primarily from agricul- cartridges, were tested, and the general average recoveries are tural activities, animal waste discharge, and human presented in Figure 2. The mixed anion-exchange MAX col- activities.[36] In our study, considering that there were no umn exhibited good enrichment effects for the target antibiot- antibiotic raw material manufacturers located in Nanjing or ics (not including cephalosporins), with recoveries of 60.2%- in adjacent cities in the upper reaches of the Yangtze River, 101.4%. Hence, MAX and HLB columns were set up in tan- the antibiotic residue in the aquatic environment mainly dem for better sample enrichment. The methods of precondi- resulted from their consumption and subsequent excretion. tioning, eluting and reconstituting were optimized for the Frequent detection indicates large usage. To our knowledge, tandem SPE system. A flow diagram of the pretreatment is SCP, SMT, CLN and LNC are mainly consumed by animals presented (Figure 3), and the detailed procedure is described > ( 87% use in animals), and erythromycin-H2O is used in in Section of Extraction and HPLC-MS/MS methods. After both human and animal therapeutics (>25% use in humans) optimization, the range of optimum recoveries was from in China.[10] Cefradine, a cephalosporin, was the largest 51.74% to 96.30%, and the relative standard deviations detected antibiotic intended for human use. Therefore, these (RSDs) ranged from 2.19% to 9.67% (Table A2). antibiotics originate from both human and agricultural activities. Furthermore, the environmental behaviors of anti- > Detection frequency and concentrations of antibiotics in biotics may be responsible for the 90% detection rates. the aquatic environments of Nanjing According to the literature, lincomycin and clindamycin have poor biodegradability because of certain structural moi- A summary of the target antibiotics in the aquatic environ- eties, such as amines, ring structures, aliphatic ethers ments of Nanjing is presented in Figure 4. Among the 49 and sulfur.[37]

Figure 2. The general average recovery of different solid phase extraction columns. 6 X. GUO ET AL.

Figure 3. Flow diagram of pretreatment.

Figure 4. The concentrations of test antibiotics in the aquatic environment of Nanjing. The box represents the 25th- and 75th-percentile values, and the symbol within the box represents the 50th-percentile value (median). The whiskers on the plots represent the maximum and minimum values and the detection frequency (%) of each antibiotic is indicated numerically in brackets.

The total concentrations of antibiotics in the groundwater broad-spectrum antimicrobial activity and are widely used 1 samplesP ranged from 7.9 toP 507.0 ng L . The concentrationsP in veterinary medicine in China. Considering only China, 1 Pof quinolones,P sulfonamides, P macrolides, the concentrations of TCs (n.d.–250.8 ng L ) were found to lincosamides, tetracyclines, and cephalosporins be substantially higher than those in the Huangpu River (defined as the sum of the detected antibiotics of a certain (n.d.–129.5 ng L 1, n.d.–54.3 ng L 1),[38,39] Chaohu Lake class) ranged from 11.1 to 23.8 ng L 1, 7.9 to 177.0 ng L 1, (n.d.–42.3 ng L 1),[40] Jiyun River (n.d.–100 ng L 1),[41] 8.4 to 200.9 ng L 1, 87. 4 to 250.6 ng L 1, 144.4 to 507.0 ng Yellow River (n.d.)[42] and the Pearl River (n.d.).[43] Known L 1, and 21.1 to 15. 4.0 ng L 1, respectively. Tetracyclines from our previous study, TCs are common veterinary drugs were dominant in the groundwater of Nanjing, contributing in livestock farms from province, and they are 43.7% to the total antibiotic burden, followed by lincosa- detected at higher concentrations with higher detection fre- mides (17.3%), sulfonamides (16.4%), macrolides (12.0%), quencies (n.d.–5775.6 mg kg 1, 15.1–45.1%) in animal cephalosporins (8.9%) and quinolones (1.8%). TCs have manure than any other antibiotic,[35] implicating livestock as JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 7

Figure 5. Antibiotic concentrations in sampling sites from different water bodies.

Figure 6. Hierarchical cluster analysis in all the sampling campaigns (a log 10 scale). the main reason for the high TC residues in the aquatic drinking water resources: rivers, lakes and reservoirs. The environment of Nanjing. results revealed that the mean total antibiotic concentrations in the Qinhuai River and in Xuanwu Lake were high 1 1 Spatial variations (116.2 ± 208.1 ng L and 116.7 ± 197.8 ng L ), to which a few high antibiotic pollutant load sites (W4 and W10) con- The sampling strategy was to select two sites representing the tributed greatly (Figure 5). This was followed by the Yangtze river systems (Yangtze River and Qinhuai River), three sites River (92.8 ± 75.0 ng L 1), Shike Lake (47.8 ng L 1), Gucheng representing the lake systems (Gucheng Lake, Shijiu Lake and Lake (3.3 ng L 1) and Fangbian Reservoir (0.59 ng L 1). Xuanwu Lake), and one reservoir (Fangbian Reservoir). The Comprehensive data analysis was conducted to summarize selected sampling sites also contained three typical types of the spatial variations of the Nanjing aquatic systems. A 8 X. GUO ET AL. dendrogram of the sampling points obtained via the hierarch- ical cluster analysis is shown in Figure 6. Two well-differenti- 3.7 4.55 1.25

ated clusters were observed: (I) a cluster containing sampling 4.65 – – – – Three from Tianjin and Tangshan city sites with severe pollution and (II) a cluster formed by two ] 11 subclusters (A and B), characterized by pollution level and [ geographical position. Cluster I, including W4 and W10, always resulted from instantaneous or constant large anti- biotic inputs, the sources of which requires further investiga- tion urgently. Cluster II is larger; the sites with less human activity of Xuanwu Lake and the Qinhai River and the drink- ing water resource sites (the lake and reservoir) merged into subcluster B, exhibiting the lowest pollution levels, and the Our study Sun et al., 2015 Yangtze River sites merged into subcluster A. ] 9 Drinking water resource safety and human health [ risk assessment 1.5 ND 0.69 0.24 35.4 ND NA NA 5.2 0.6 0.35 0.5 4.3 ND 0.25 0.75 – – – According to the “environmental protection guidelines of – centralized drinking water source” [44] released by the Lin et al., 2016 Ministry of Environmental Protection of the People’s ]

Republic of China in 2012, sources of drinking water should 45 [

be protected by a series of measures, including banning sew- Lake Reservoir age outfall in water source protection zones, demolition or 20 NA ND NA NA 12.48 8.7 5.5 1.6 7.1 NA ND NA NA 0.38 NA ND NA NA 1.44 NA ND NA NA – closure construction projects expected for water supplies or – – – – – 2.08 NA ND NA NA protection facilities, banning cage aquaculture, swimming, – fishing and other activities that may contaminate drinking water, etc. These protective measures can also reduce the risk of antibiotic pollution of water to some extent. As

expected, there was little antibiotic residue in the lake and ND 0.662 Our study Zhang et al., 2012 reservoir (Figure 5, black box), where sewage outfalls were more manageable. ] 13 [

Regarding rivers as a drinking water resource, the outfall 3 arrangement is more complicated. Along the Yangtze River, 10

there are drainage outlets of chemical enterprises, sewage 379 ND 0.05 174 ND 0.13 1.34 – 19.2 2.3 2.72 17.8 ND NA ND – – – treatment plants and rainfall runoff. The Yangtze River has – Xue et al., 2013 inevitably become a receiving water body for many indus- tries, agriculture and human activities, threatening the qual- 36.7 NA ND 0.528 40.4 NA ND nd ity and safety of water sources. Seen from our investigation, 35.5 NA ND 4.62 – – – ] 8 drinking water source sites D1–D4 were all more seriously [ contaminated than the control group (R1–R4), not only in concentration but also in detection frequency (Figure 5, red 41.3 nd – box). This result implies that the complexity of water flow 23.1 nd –

makes the pollution unpredictable in Yangtze River DWSs Zhang et al., 2017 despite conventional protective measures. River Currently, there is no drinking water standard or even ] 12 groundwater standard in place outlining the residue limits [ permitted in China. To evaluate the residual antibiotics lev-

els of the drinking water sources, we compared our LOQ); NA: Not analyzed. < 1.7 NA NA nd 13 NA NA NA 1.0 NA 0.8 0.69 NA NA nd – – date with the reported data from other areas nationwide – (Table 2). Although detection was limited by the analytical approach, and a direct comparison was hard to implement, the general pollution trends could be outlined by only the detected concentration. In general, the concentrations of 1 29.4 7.45 antibiotics detected were below 10 ng L for the reservoirs 15.6 21 ± 5 NA NA 6.2 Nanjing Yangtze, Chongqing Dongjiang Beijiang Yongjiang Gucheng Zizhuyuan Taihu Fangbian Baiguishan 59.5 NA NA NA NA ND NA NA ND 1.7 0.92 58.0 NA NA NA 2.9 – – Yangtze, – – and below 35 ng L 1 for the lakes. However, the residual levels were clearly higher in rivers than those in reservoirs or lakes. As the main receiving water body for treated and Nationwide comparison of antibiotic concentrations in drinking water resource (ng/L). untreated wastewater in the city via tributaries and sewage ND: not detected (concentrations CIP ND ND nd Table 2. Type Name OFLNOR ND ND NDNote: ND ND ND nd ROX ND 0.89 SMM 9.1 SDZ ND ND NA NA ND ERY 3.3 LNC 11.3 SMX 11.4 CLR ND 0.35 systems, a new set of protection policy actions should Reference Our study Chang et al., 2010 JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 9 urgently be required for effective pollution isolation in rivers Heavy metal, B and As as a drinking water source. The detection rate seemed lower Anthropogenic activities such as metal mining, indiscrimin- with fewer antibiotic manufacturers and more strict usage ate discharge of untreated sewage, industrial waste, extensive management in Europe, Canada and the United States. mining activity and some agricultural activities are major [2] Focazio et al. from the United States found that antibiotics causes of metals introduced into surface and ground- – were almost not detected in 25 ground- and 49 surface- water.[46 48] Furthermore, antibiotics and metal pollutants water sources of drinking water. However, this observation can be released into aquatic environments simultaneously. cannot be applied to concentration, as the maximum anti- With regard to how metal reacts with antibiotics in aquatic 1 [2,3,16] biotic residues reported ranged from 5 to 300 ng L . environments, there is increasing knowledge that metal coordination modifies antibiotic adsorption and desorption, Human health risk assessment was performed with – – [49 52] photolysis,[53] bio-uptake [54 56] and toxicity. [45,57] residual antibiotics in the DWSs to assess human health risk Additionally, the increasing abundance of ARGs may result and to guarantee the safety of drinking water. The detailed [58–60] information of assessment is shown in Text A1. For detected from the selective pressure of heavy metals and may antibiotics, the age group of 1–3 months had a higher risk have an effect on environmental antibiotic residues in return. (Table A3) due to their lower body weight. Comparatively, Interestingly, in our study (Table 4), there was no lack of balofloxacin was detected and is an antibiotic with a rela- positive correlations between antibiotics and tested heavy tively high RQ. However, no detected antibiotics presented metals or between antibiotics and B and As (Pearson, < < an RQ value higher than 0.0207. These values imply that the P 0.05 or P 0.01). In view of the limitations of this antibiotics detected in DWSs do not pose potential risks to study, it seemed hard to define whether the correlations the health of residents directly. resulted from their reaction, from a common source or from randomness. The mechanism therefore needs further

Antibiotic correlation with environmental factors Table 3. Correlation between concentrations of antibiotics and water quality. Water quality Total Ammonia Total CODcr phosphorus nitrogen nitrogen Water samples were analyzed for chemical oxygen demand OFL 0.96b 0.62b (CODcr), total nitrogen, ammonia nitrogen and total phos- BAL 0.54a b phorus to understand the water quality. The Pearson correl- SMX 0.91 SMT 0.61a ation between the concentrations of antibiotics and the CTC 0.52a 0.68b water quality was analyzed. Strong positive correlations DC 0.51a 0.69b (Pearson, P < 0.05) were observed between the concentra- ROX ERY –0.56a 0.94b 0.46a tions of total antibiotics and total phosphorus, as described CLN –0.43a 0.94b in Table 3. There were many strong positive correlations LNC 0.74b (Pearson, P < 0.05) between the concentration of each com- PCED PQuinolones a pound of antibiotics and the water quality parameters as PSulfonamides 0.58 b well. Clearly, antibiotic contamination and water quality are PTetracyclines 0.82 b b PMacrolides 0.96 0.62 closely connected. In the process of sewage discharge, antibi- a PLincosamides 0.54 b otics always enter the aquatic environment along with other PCephalosporins 0.91 pollutants. On the other hand, less antibiotic-contaminated Antibiotics 0.61a areas, such as D5 and D6, performed well in terms of water Detection rate of SCP, SMM, SMT, ERY, CLN, LNC and CED > 28.5%. quality (Figure A2). aCorrelation is significant at the 0.01 level (2-tailed). bCorrelation is significant at the 0.05 level (2-tailed).

Table 4. Correlation between concentrations of antibiotics and Heavy metal, B and As. B As Li V Cr Mn Fe Co Ni Cu Zn Sr Mo Sb Ba SCP 0.46a 0.51a SMT 0.49a 0.51b 0.52a 0.44a 0.51a 0.47a 0.44a SMM 0.61b 0.44a 0.46a 0.62b 0.43a CLN 0.69b 0.62b 0.65b 0.47a 0.45a 0.56b LNC 0.50a ERY 0.57a 0.5a 0.64b 0.51a a b a PCED 0.62 0.68 0.63 b b b a b b PQuinolones 0.55 0.63 0.55 0.47 0.92 0.65 b a a a a PSulfonamides 0.58 0.46 0.51 0.44 0.47 a a a b a PTetracyclines 0.50 0.50 0.48 0.81 0.52 b a a a b PMacrolides 0.57 0.51 0.44 0.51 0.57 a PLincosamides 0.54 a a b PCephalosporins 0.54 0.45 0.50 Antibiotics 0.62b 0.44a 0.73b 0.50a aCorrelation is significant at the 0.01 level (2-tailed). bCorrelation is significant at the 0.05 level (2-tailed). Detection rate of SCP, SMM, SMT, ERY, CLN, LNC and CED > 28.5%. Detection rate of Li, B, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Sr, Mo, S and Ba > 30.0%. 10 X. GUO ET AL. exploration regarding these three aspects. 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Figure A1. Chromatograms of targeted antibiotics. 14 X. GUO ET AL.

Figure A2. Water quality in sampling sites from different water bodies.

Table A1. MRM parameters for of antibiotics tested. Retention Declustering Entrance Collision cell exit Target antibiotics time (min) Parent ion (m/z) Product ion (m/z) potential (V) potential (V) Collision energy (V) potential (V) Norfloxacin (NOR) 2.26 320.8 277.2a 108.91 4.23 24.26 11.34 233.1 110 10 44 14.08 Cirofloxain (CIP) 2.39 332.8 289.3a 54.03 4.83 22.7 12.86 246 98.08 4.69 32.66 18.36 Enrofloxacin (ENR) 2.76 360.6 317.2a 108.12 4.39 26.91 11.92 245.2 64.87 7.38 37.39 3.15 Ofloxacin (OFL) 2.27 362.1 318.2a 106.83 5.18 25.62 9.03 261.1 104.38 5.14 38.61 6.87 Fleroxacin (FLE) 2.25 370.8 327.2a 111.31 2.91 27.17 3.46 269.9 111.19 4.38 35.84 8.69 Sarafloxacin (SAR) 3.24 386.1 342.1a 95.33 8.72 25.72 8.32 299.3 85.28 9.45 37.24 13.34 Lomefloxacin (LOM) 2.53 352.9 308.7a 94.1 2.85 24.54 4.77 265.3 87.18 11.06 30.19 8.28 Balofloxacin (BAL) 3.8 390 372 110.21 10.24 27.04 5.28 359.3a 84.1 13.17 22.32 1.98 Pazufloxacin (PAZ) 2.27 319.3 301a 79.22 10.38 23.82 2.13 281 78.98 10.38 34.2 2.14 Sulfadiazine (SDZ) 1.40 251.1 155.9a 23.24 4.91 20.48 1.13 185.1 23.24 4.91 24.19 5.79 Sulfamerazine (SMZ) 1.80 264.9 155.9a 67.04 12.54 21.96 5.05 171.6 68.4 12.54 21.06 5.06 Sulfadimethoxine 4.92 310.8 156.1a 72.23 5.4 24.83 4.91 (SDM) 107.9 67.94 3.64 32.29 9.4 Sulfamethoxazole 3.71 254.1 155.9a 74.59 3.16 20.29 2.6 (SMX) 147.2 73.66 3.16 20.48 4.4 Sulfathiazole (STZ) 1.62 256 156 66.29 8.06 18.18 4.22 107.9a 67.2 6.53 33.63 7.96 Sulfachloropyridazine 3.24 285.2 156.2a 71.78 8.46 19.92 5.34 (SCP) 108 68.7 5.28 29.17 7.04 Sulfamethazine 2.18 279.1 124.1 18.07 4.56 31.5 7.99 (SMT) 186a 44.55 4.6 23.77 6.35 Sulfapyridine (SP) 1.37 250.7 156.8 72.33 11.64 23.48 5.92 184.9a 74.38 5.68 24.69 3.28 Sulfamonomethoxine 3.04 281 156a 78.36 4.88 23.21 9.06 (SMM) 215 66.9 4.31 26.1 9.94 Sulfamethizole 2.46 270.9 156a 67.04 4.97 19.52 12.26 (SMTZ) 107.8 65.19 8.37 33.48 17.93 Sulfisoxazole (SSE) 4.07 268.8 155.9a 69.32 6.44 18.17 13.61 113.3 65.12 9.97 22.82 8.73 Sulfacetamide (SCM) 1.26 214.9 156.2a 58.52 11.74 15.47 5.88 107.8 59.79 8.2 29.47 7.92 Sulfabenzamide 4.60 277.2 155.9a 71.48 4.06 16.8 10.51 (SBM) 108.3 72.43 2.8 32.19 7.21 Tetracycline (TC) 2.55 445.2 427.1a 91.36 7.5 13.68 20.74 409.5 93.01 8 18.93 18.1 Oxytetracycline 2.20 461.2 443.3 218.98 9.33 19.23 17.68 (OTC) 426.2a 83.92 9.33 28.49 17.65 381.2 212.52 9.33 35.09 15.28 (continued) JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 15

Table A1. Continued. Retention Declustering Entrance Collision cell exit Target antibiotics time (min) Parent ion (m/z) Product ion (m/z) potential (V) potential (V) Collision energy (V) potential (V) Chlortetracycline 3.86 479.4 444.2a 94.74 8.2 29.24 21.05 (CTC) 154.1 109.7 4.48 36.14 13.71 Doxycycline (DC) 4.32 445.1 428.3a 96.31 11.97 25.92 8.82 154.2 91.98 6.72 36.8 7.44 Erythromycin- 6.44 716.6 158.1 96.31 12.01 36.43 11.74 a H2O (ERY) 558.5 76.3 14.08 20.93 26.89 Roxithromycin (ROX) 7.13 837.4 158.1 7.25 12.05 40.32 9.25 679.7a 42.58 11.17 29.79 2.81 Josamycin (JOS) 7.70 828.6 174.2a 21.91 2.36 36.93 16.98 109.1 17.5 2.36 137.72 7.01 Clindamycin (CLN) 4.11 425.2 126.3a 75.53 6.95 35.5 3.7 377.2 86.58 6.92 27.12 4.29 Lincomycin (LNC) 1.45 407.4 126.2a 19.91 5.68 32.74 9.91 359.2 18.84 4.56 25.29 10.07 Spiramycin (SPR) 3.82 843.7 142.3 177.98 4.52 40.56 8.38 174.1a 185.71 3.22 43.56 15.62 Clarithromycin (CLR) 6.94 748.5 590.4a 117.55 10.94 24.81 29.1 158 106.39 9.55 35.36 11.8 Azithromycin (AZT) 6.98 748.9 590.8a 63.19 10.98 24.96 15.24 158.2 129.14 7.27 34.98 6.48 Tylosin (TYL) 6.06 916.9 174.1a 34.07 12.81 43.83 10.09 101.1 6.41 3.92 86.62 6.27 Tilimicosin (TLM) 4.88 869.5 174a 18.79 6.36 55.76 14.18 132.1 162.12 12.97 92.25 7.09 Kitasamycin (KTS) 6.58 772.4 215.2 180.1 10.06 35.78 1 109a 35.93 10.06 78.32 13.15 Streptomycin (STR) 0.45 614.3 263.2 27.13 8.48 50.68 7.81 582.6a 133.24 12.69 22.53 19.67 Penicillin G (PEN G) 5.44 335.1 160.2 118.93 4.51 21.66 7.46 175.7a 101.95 9.85 19.44 15.27 Penicillin V (PEN V) 6.28 351 160.2 133.11 7.98 20.82 11 192 111.07 7.98 16 2.55 229a 111.88 11.16 22.82 6.64 Amoxicillin (AMX) 0.85 365.9 114.1 22.22 7.13 31.06 7.92 207.9a 42.88 6.76 15.7 7.13 349 35.63 3.12 13.76 17.15 Oxacillin (OXA) 7.17 402.3 159.9 126.16 4.79 23.47 9.56 243a 73.85 12.14 17.93 3.15 Cloxacillin (CLO) 7.85 436.2 159.9 127.59 4.48 26.26 15.25 277.3 129.32 7.45 24.97 8.63 220a 139.15 3.36 22.57 3.5 Nafcillin (NAF) 8.19 415.2 199.1a 121.9 4.1 26.19 6.27 172 74.53 5.27 30.26 11.77 397.4 116.89 3.6 14.94 4.42 Dicloxacillin (DIC) 8.71 470 311a 85.47 12.76 22.94 15.81 160.1 91.86 5.31 20.93 6 Ampicillin (AMP) 1.93 350 192.2 78.96 7.57 22.55 7.99 159.7a 66.21 8.39 20.17 6.47 Cephapirin (HAP) 1.19 424.1 292.3a 55.16 7.65 18.24 12.98 151.7 15 7.53 34.11 42.02 Cephalexin (LEX) 2.00 348 158a 72.15 6.19 12.23 35.19 174.1 68.42 10.78 23.34 4.83 Cefradine (CED) 2.29 350.3 176a 66.83 6.59 17.89 8.12 158 71 8.28 14.63 15.4 Tetracycline-d6 2.55 450.3 415.5a 57.63 6.61 28.38 14.81 (TC-d6) 433.3 52.79 9.59 19.28 17.67 Sulfamethazine-d6 2.18 285.4 186a 19.36 3.21 24.07 5.48 (SMT-d6) 124.1 41.11 4.56 32.29 3.66 aQuantitative ion. 16 X. GUO ET AL.

Table A2. Limits of determination, limits of quantification, recovery and RSD of target antibiotics (n ¼ 5). LOD/ng/L LOQ/ng/L Low level High level Antibiotics Solution I Solution II Solution I Solution II Recovery (%) RSD (%) Recovery (%) RSD (%) NOR 0.35 0.23 1.18 0.75 64.0 8.23 67.2 7.26 CIP 0.32 0.61 1.08 2.04 65.3 7.15 62.8 7.31 ENR 0.44 0.26 1.48 0.87 62.0 6.02 70.0 4.44 OFL 0.10 0.07 0.35 0.23 63.7 6.76 68.7 8.92 FLE 0.25 0.06 0.83 0.20 56.8 8.72 64.3 6.85 SAR 0.49 0.32 1.63 1.08 73.6 5.38 75.3 6.98 LOM 0.55 0.53 1.82 1.75 73.2 9.11 74.7 7.87 BAL 0.28 0.16 0.93 0.53 78.5 5.23 76.8 7.47 PAZ 0.59 0.57 1.96 1.90 62.5 5.09 62.2 6.56 SDE 0.08 0.16 0.28 0.53 87.9 6.73 86.0 8.94 SME 0.03 0.10 0.09 0.32 92.9 3.66 93.6 5.90 SDM 0.02 0.08 0.05 0.26 93.4 8.91 85.4 9.01 SMX 0.09 0.13 0.29 0.44 96.3 7.80 91.9 4.93 STZ 0.02 0.26 0.08 0.87 81.0 5.33 82.4 4.76 SCP 0.03 0.24 0.09 0.80 79.6 5.42 77.5 4.61 SMT 0.01 0.13 0.04 0.44 83.9 6.71 80.3 3.71 SP 0.56 0.51 1.87 1.69 80.3 2.68 79.5 3.20 SMM 0.26 0.53 0.87 1.75 88.3 5.77 91.6 4.89 SMTZ 0.17 0.27 0.57 0.89 53.4 4.25 57.6 8.52 SSZ 0.24 0.41 0.80 1.38 65.8 7.04 67.1 7.58 SCM 0.20 0.43 0.68 1.42 94.8 4.54 93.7 6.28 SBM 0.41 0.48 1.38 1.59 73.3 7.03 75.9 3.82 TC 0.36 0.32 1.21 1.08 82.9 5.77 83.5 3.38 OTC 0.56 0.35 1.88 1.18 94.6 3.49 94.7 3.01 CTC 0.59 0.44 1.96 1.48 78.9 5.71 76.2 5.41 DC 0.69 0.39 2.30 1.29 87.9 7.57 84.3 6.25 ROX 0.04 0.21 0.14 0.70 73.7 4.52 74.7 3.18 TYL 0.40 0.86 1.33 2.86 83.8 4.48 81.1 9.67 JOS 0.29 1.32 0.95 4.40 56.0 7.15 56.0 3.89 CLN 0.28 0.14 0.92 0.46 92.0 3.75 91.9 4.79 LNC 0.03 0.05 0.11 0.17 76.3 3.66 77.2 3.41 ERY 0.07 0.34 0.22 1.14 87.8 5.28 87.3 7.07 TLM 0.71 0.57 2.37 1.89 54.3 4.73 55.4 5.07 SPR 0.66 0.78 2.20 2.60 63.8 6.90 61.3 8.61 KTS 0.64 0.57 2.13 1.89 62.3 8.44 67.9 6.28 CLR 0.02 0.24 0.08 0.80 75.1 6.35 80.9 3.88 AZT 0.18 0.38 0.60 1.27 87.1 8.19 88.4 3.99 STR 3.23 3.43 10.75 11.43 54.3 7.58 51.7 6.76 AMX 0.39 0.34 1.29 1.14 53.1 4.11 54.4 5.36 AMP 0.06 0.26 0.20 0.87 60.2 8.55 56.7 5.07 PEN G 0.52 0.41 1.74 1.38 92.5 2.86 94.6 5.39 PEN V 0.58 0.53 1.94 1.75 95.6 2.19 92.9 4.55 OXA 0.56 0.51 1.87 1.69 59.1 6.31 55.8 5.49 CLO 0.57 0.46 1.89 1.54 54.0 5.21 52.9 4.35 NAF 0.41 0.44 1.38 1.48 53.9 5.26 54.0 4.79 DIC 0.51 0.29 1.69 0.98 57.6 7.37 55.8 6.54 HAP 0.38 0.69 1.26 2.30 55.6 5.68 52.5 7.10 LEX 0.31 0.39 1.03 1.29 81.3 3.51 73.6 7.50 CED 0.57 0.34 1.90 1.14 62.8 7.30 62.9 5.13 Note. Different concentrations (high level and low level) of streptomycin were 100 and 20 lg/L, and the others were 10 and 2 lg/L.

Table A3. Selected age group with respective 50% of body weight and drinking water intakes. Age groups Body weight DWI 0–3 months 5.6 1.15 3–6 months 7.2 1.14 6–12 months 9.4 1.18 1–2 years 12 0.85 2–3 years 13.8 0.83 3–6 years 19 1.16 6–11 years 36 1.55 11–16 years 56 1.90 16–18 years 57 1.77 Adults (18 years) 60 2.04 Note. All the data were referred to CDC (2002) and EPA (2011). JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 17

Table A4. Acceptable daily intake values used in estimation of the respective DWEL. PPCPs ADI (lg/kg.day) Reference Enrofloxacin 0–2 Maximum residue limit of veterinary drugs in 0–20 animal foods. 0–50 Announcement 235 of the Ministry of Agriculture Oxytetracycline/Chlortetracycline/Tetracycline 0–30 of the People’s Republic of China http://www. Erythromycin 0–5 moa.gov.cn/gk/tzgg_1/gg/200302/t20030226_ Lincomycin 0–30 59300.htm