Accepted Manuscript

Title: Detection of a wide variety of human and veterinary

fluoroquinolone antibiotics in municipal wastewater and
wastewater-impacted surface water

Author: Ke He Ana Dulce Soares Hollie Adejumo Melissa

McDiarmid Katherine Squibb Lee Blaney

PII: S0731-7085(14)00547-0
DOI: http://dx.doi.org/doi:10.1016/j.jpba.2014.11.020
Reference: PBA 9807

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 13-8-2014

Revised date: 7-11-2014
Accepted date: 10-11-2014

Please cite this article as: K. He, A.D. Soares, H. Adejumo, M. McDiarmid,
K. Squibb, L. Blaney, Detection of a wide variety of human and veterinary
fluoroquinolone antibiotics in municipal wastewater and wastewater-impacted
surface water, Journal of Pharmaceutical and Biomedical Analysis (2014),
http://dx.doi.org/10.1016/j.jpba.2014.11.020

This is a PDF file of an unedited manuscript that has been accepted for publication.
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HIGHLIGHTS

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• FQs inhibit growth of the Escherichia coli indicator organism at 1-10 µg/L

• Eight FQ antibiotics in raw wastewater at inhibitory levels (2-3 µg/L)

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• Several less-prescribed FQs (DIF, FLE, MOX, and ORB) were detected

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• FQ removal negatively correlated to BOD removal, positively correlated to PO4 removal
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Detection of a wide variety of human and veterinary fluoroquinolone antibiotics in municipal wastewater

and wastewater-impacted surface water

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Ke He1, Ana Dulce Soares1, Hollie Adejumo1, Melissa McDiarmid2, Katherine Squibb2, Lee Blaney1*

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1: University of Maryland Baltimore County

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Department of Chemical, Biochemical and Environmental Engineering

1000 Hilltop Circle, ECS 314

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Baltimore, MD 21250
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2: University of Maryland School of Medicine

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Division of Occupational and Environmental Medicine

Department of Medicine

11 South Paca Street, Suite 200

Baltimore, MD 21201

* Corresponding author:

Lee Blaney, PhD

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University of Maryland Baltimore County
Department of Chemical, Biochemical and Environmental Engineering
1000 Hilltop Circle, ECS 314
Baltimore, MD 21250 USA

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Tel: +1-410-455-8608
Fax: +1-410-455-1049
Email: blaney@umbc.edu

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ABSTRACT

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As annual sales of antibiotics continue to rise, the mass of these specially-designed compounds entering municipal wastewater treatment

systems has also increased. Of primary concern here is that antibiotics can inhibit growth of specific microorganisms in biological processes of

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wastewater treatment plants (WWTPs) or in downstream ecosystems. Growth inhibition studies with Escherichia coli demonstrated that solutions

containing 1-10 µg/L of fluoroquinolones can inhibit microbial growth. Wastewater samples were collected on a monthly basis from various

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treatment stages of a 30 million gallon per day WWTP in Maryland, USA. Samples were analyzed for the presence of 11 fluoroquinolone

antibiotics. At least one fluoroquinolone was detected in every sample. Ofloxacin and ciprofloxacin exhibited detection frequencies of 100% and

98%, respectively, across all sampling sites. Concentrations of fluoroquinolones in raw wastewater were as high as 1900 ng/L for ciprofloxacin
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and 600 ng/L for ofloxacin. Difloxacin, enrofloxacin, fleroxacin, moxifloxacin, norfloxacin, and orbifloxacin were also detected at appreciable
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concentrations of 9-170 ng/L. The total mass concentration of fluoroquinolones in raw wastewater was in the range that inhibited E. coli growth,

suggesting that concerns over antibiotic presence in wastewater and wastewater-impacted surface water are valid. The average removal efficiency
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of fluoroquinolones during wastewater treatment was approximately 65%; furthermore, the removal efficiency for fluoroquinolones was found to

be negatively correlated to biochemical oxygen demand removal and positively correlated to phosphorus removal.

Keywords: fluoroquinolones; ciprofloxacin; ofloxacin; antibiotics; wastewater; pharmaceuticals

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ABBREVIATIONS

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ATCC American Type Culture Collection
BOD5 5-day Biochemical Oxygen Demand
CIP Ciprofloxacin
CLSI Clinical Laboratory Standards Institute

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DFI Denitrification Filter Influent
DI Deionized Water
DIF Difloxacin
ENR Enrofloxacin

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EPA Environmental Protection Agency
FLD Fluorescence Detection
FLE Fleroxacin
FQ Fluoroquinolone
GAT Gatifloxacin
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HPLC High Performance Liquid Chromatography
LOM Lomefloxacin
MGD Million Gallons per Day
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MOX Moxifloxacin
NH3-N Ammonia Concentration as N
NO3-N Nitrate Concentration as N
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NOR Norfloxacin
OD625 Optical Density at 625 nm
OFL Ofloxacin
ORB Orbifloxacin
PE Population Equivalent
PO4-P Phosphate Concentration as P
RAS Return Activated Sludge
SAR Sarafloxacin
SPE Solid-Phase Extraction
TSS Total Suspended Solids
UV Ultraviolet
UV254nm Ultraviolet Absorbance at 254 nm
WWTP Wastewater Treatment Plant

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1. INTRODUCTION

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The presence of pharmaceutical compounds in surface and ground water resources has instigated public health concerns since the mid-1980s

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as numerous studies of the fate and effects of these chemicals in aquatic environments have been reported [1-6]. Although concentrations of

pharmaceuticals in surface water are normally quite low (i.e., less than 1 µg/L), the current widespread, and rapidly increasing, use of

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pharmaceuticals in both veterinary and human healthcare is of particular concern, due primarily to elevated usage of 1) steroids for growth

promotion and 2) antibiotics for prophylaxis in livestock grown for food production. Together, these uses contribute to drug burdens that threaten

both sensitive aquatic ecosystems and human health through contaminated drinking water sources [7-10]. As agribusinesses grow, surface water
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runoff from animal farms will continue to deposit higher amounts of active drugs directly into local rivers and streams. In addition, the many
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pharmaceuticals used in human healthcare which enter municipal wastewater treatment plants (WWTPs), including those from hospitals, are a

significant point source contributor. Importantly, WWTPs are not currently designed to remove these trace organic compounds, but rather the bulk
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biochemical oxygen demand (BOD), nutrients, and microorganisms. For this reason, antibiotics are discharged to the environment.

Given the urgency of this threat to water quality, WWTPs are currently being studied in greater depth to better characterize their ability to

decrease the release of active pharmaceuticals into surface water. Information on the total mass of a particular pharmaceutical released into a river

or stream is useful for conducting risk assessments based on calculated hazard quotients associated with individual drugs, and for establishing

management of specific pharmaceuticals on impacted ecosystems [1, 11]. Within the US, the task of managing pharmaceutical waste lies with the

Environmental Protection Agency (EPA), which has taken regulatory action against several hundred hospitals in the last decade using its

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regulatory authority for managing hazardous waste [12]. Stepped up action by the EPA is likely to be enhanced due to a recent report from the

Office of the Inspector General citing the need for more regulatory action by the agency, since lack of action by the EPA may result in unsafe

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disposal of pharmaceuticals determined to be hazardous compounds [13]. Regardless, no regulations currently exist for acceptable levels of

pharmaceuticals in hospital or municipal wastewater.

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Antibiotics are one of the most frequently observed pharmaceutical classes in wastewater effluent due to their ubiquity of use in both

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animal and human healthcare. Many environmental studies have focused on the presence of the same blockbuster antibiotics in wastewater and

surface water supplies, among other environmental compartments. While this information is useful, a need exists for understanding the
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concentration and load of more peripheral antibiotics, and more importantly, classes of antibiotics. Therefore, in the present study, we examined a

group of eleven fluoroquinolone (FQ) antibiotics in wastewater and downstream surface water supplies. The FQ class was chosen for study due to
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the relatively high potency of these antibiotics against pathogens, as well as the completely synthetic nature of these chemicals (i.e.,

fluoroquinolones were not isolated from microorganisms). The removal of FQ antibiotics during wastewater treatment was correlated with
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removal of background wastewater quality parameters. The reported antibiotic concentrations were contextualized through measurement of the

growth inhibition of Escherichia coli, a common environmental indicator organism, in the presence of ciprofloxacin (CIP), which is the

blockbuster drug of the fluoroquinolone class.

2. MATERIAL AND METHODS

2.1 Chemicals and materials

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The FQ antibiotics investigated in this study include the following: CIP, difloxacin (DIF), enrofloxacin (ENR), fleroxacin (FLE), gatifloxacin

(GAT), lomefloxacin (LOM), moxifloxacin (MOX), norfloxacin (NOR), ofloxacin (OFL), orbifloxacin (ORB), and sarafloxacin (SAR); the

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veterinary-use compounds are DIF, ENR, ORB, and SAR. Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO USA) or Fisher

Scientific (Pittsburgh, PA USA). The purity of all compounds was at least 97.3%. Except for SAR (50 mg/L), stock solutions for all FQs were

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prepared at 200 mg/L in a water and methanol mixture (1:1, v/v) containing 0.2% hydrochloric acid. The pH of stock solutions was ~2 and these

solutions were stable for one month at -20 °C. Working solutions containing 100-1000 µg/L FQs were prepared weekly in 20 mM phosphate

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buffer (pH 2.4) and stored at 4 °C. Physical and chemical properties of the 11 FQs of concern are provided in Table 1.
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2.2 Antimicrobial activity assay

Antimicrobial activity of CIP was measured with a growth inhibition assay using E. coli (ATCC 25922). The assay protocol was adapted from
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a standard method for measurement of antibiotic susceptibility [14]. E. coli was grown in Mueller-Hinton Broth (Thermo Scientific) to generate a

high-strength culture, which was mixed 1:1 (v/v) with a 50% glycerol solution and deposited as seeds into cryovials for storage at -80 °C. The E.
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coli inoculum had the same optical density at 625 nm as a 0.5 McFarland Standard (i.e., 0.7-0.8 cm-1). The inoculum (100 µL) and antibiotic

standards (100 µL) were added to 96-well microplates, incubated at 37 °C for 20 hours, and measured using a microplate reader (BioTek Eon;

Winooski, VT). All samples were run in triplicate, including three positive/negative growth controls. After the 20 hour incubation period,

microplates were manually aspirated, shaken in the microplate reader, and the optical density at 625 nm was recorded for each sample. The

shaking and recording process was repeated three times to ensure homogeneity within wells. Mean measurements were used for analysis. Optical

density was converted to inhibition of E. coli growth using Eq. 1.

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(Eq. 1)

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In Eq. 1, I is the percent inhibition, OD625 is the optical density at 625 nm; pos, neg, and exp indicate the positive growth control, negative growth

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control, and experimental samples, respectively. Inhibition data was fit to the Hill Equation (Eq. 2).
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(Eq. 2)
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In Eq. 2, Imax is the maximum inhibition, Imin is the minimum inhibition, IC50 is the CIP concentration that results in 50% inhibition of E. coli

growth, C is the CIP concentration in the well, and H is the Hill slope. The Hill model was fit to experimental data using the GraphPad Prism

software package (La Jolla, CA); additionally, the 95% confidence intervals on IC50 and H were obtained using this software.

2.3 Wastewater and surface water collection

Wastewater samples were collected from three anonymous WWTPs in Maryland, USA. These plants can be characterized as small (3 million

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gallons per day (MGD)), medium (30 MGD), and large (180 MGD). The majority of sampling was completed for the medium-sized plant;

however, data from the small and large WWTPs were used to validate FQ concentrations. Samples were collected from the medium-sized plant

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once per month for a 17-month period stretching from February 2013 to July 2014. Using the BOD algorithm (i.e., 54 mg BOD/person-d)

described by von Sperling and de Lemos Chernicharo [15], the population equivalent (PE) for the 30 MGD plant is 266,000. Raw wastewater

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samples were collected from the grit chamber located in the primary treatment train of the WWTP. Grab samples were collected from the return

activated sludge (RAS) and denitrification filter influent (DFI) lines to better understand FQ concentrations within the plant. Effluent samples

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were collected after UV disinfection. The hydraulic residence time and solids retention time were approximately 3 hours and 10 days,

respectively. To quantify FQ levels in the environment, surface water (river) samples were collected 50 m downstream of the discharge site for
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the medium-sized WWTP; no surface water samples were collected for the small and large WWTPs. In addition, a location upstream of the

medium-sized WWTP was also sampled. The wastewater and surface water samples were collected in 1 liter amber glass bottles and immediately
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transported to the lab for analysis. Particulates were removed by filtering samples with 1.2 µm glass fiber filters (Millipore; Billerica, MA).

Hydrochloric acid (0.1%, v/v) was added to samples to minimize biological activity and shift FQ speciation chemistry. Samples were refrigerated
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(4 °C) in the dark until extraction which typically happened within 48 hours.

The WWTPs provided wastewater quality data for sampling days. These data included flow rate, 5-day BOD (BOD5), total suspended solids

(TSS), ammonia concentration (NH3-N), nitrate concentration (NO3-N), and phosphate concentration (PO4-P). In addition, the UV-visible

absorption spectra and pH were recorded for all samples. As the UV absorbance at 254 nm (UV254 nm) is correlated to the presence of aromatic

rings, UV254 nm was extracted from the absorbance spectra as a surrogate estimate of the dissolved organic carbon concentration. Average

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concentrations (± standard deviation) of these wastewater quality parameters for raw wastewater and wastewater effluent for the medium-sized

WWTP are provided in Table 2. The TSS, BOD5, and NH3-N of raw wastewater were approximately 103 mg/L, 205 mg/L, and 21.8 mg/L,

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respectively; these values are representative of a medium-strength wastewater, which is expected for plants that serve residential and light

commercial users [16].

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2.4 Analytical methods

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Fluoroquinolone antibiotics were measured using offline solid phase extraction (SPE) and high performance liquid chromatography with

fluorescence detection (HPLC-FLD). The method has been reported elsewhere [17]; the literature has shown SPE with HPLC-FLD to be an
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effective means of measuring FQ antibiotics in environmental media [17-20]. Briefly, FQs were extracted from wastewater and environmental

samples using weak cation exchange SPE cartridges (Oasis WCX 150 mg, 6 cc). FQs are eluted from the SPE cartridge using methanol,
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acetonitrile, and 2% formic acid (pH 3). Samples were analyzed in triplicate. Mean recovery was generally greater than 80%. Extracts were dried

under a gentle stream of nitrogen gas, and reconstituted in 20 mM phosphate buffer (pH 2.4) for HPLC-FLD analysis. Samples were injected into
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an UltiMate 3000 HPLC equipped with a four channel UV detector and fluorescence detector from Dionex Corporation (Sunnyvale, CA).

Antibiotics were chromatographically separated on a pentafluorophenyl column (150×4.6 mm, 2.6 μm) at 50 °C using a gradient elution method

with methanol, acetonitrile, and 20 mM phosphate buffer (pH 2.4). The FQs were quantified by fluorescence detection, which was optimized for

individual antibiotics [17]. Coefficients of determination for calibration curves were generally greater than 0.999.

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3. RESULTS AND DISCUSSION

3.1 Antimicrobial potency of fluoroquinolones

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To better understand the ability of wastewater and wastewater-impacted surface water to exert selective pressures on microbial populations, an

inhibition profile for CIP was constructed for E. coli (Figure 1). The IC50 and Hill slope were 7.92 (±0.46) µg/L and 5.21 (±1.72), respectively; the

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values in parentheses are 95% confidence intervals. The corresponding IC10 value is approximately 5 µg/L. For this reason, antibiotic

concentrations in the 1-10 µg/L range will impact microbial populations. Furthermore, the potency of FQs against other microorganisms is

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expected to vary by at least an order of magnitude (i.e., IC10 = 0.5-50 µg/L). As antibiotics have demonstrated a number of effects at sub-

inhibitory concentrations [21, 22], trace-level concentrations of FQ antibiotics in wastewater and impacted surface waters may also have other,
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unknown, impacts. Below, the concentrations for a suite of FQs are determined for a medium-strength wastewater to better characterize whether
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wastewater exerts antimicrobial activity.

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3.2 Detection of fluoroquinolones in wastewater and wastewater-impacted surface water

Ciprofloxacin (10 ng/L) and ofloxacin (9 ng/L) were detected in samples collected upstream of the medium-sized WWTP. In general,

fluoroquinolones were widely detected in wastewater and wastewater-impacted surface waters. Eight of the 11 FQs included in this study were

detected; however, ORB was only detected once in the raw wastewater for the small WWTP. GAT, LOM, and SAR were not present in any

sample. The detection frequency for the other FQ antibiotics ranged from 15% for DIF to 100% for OFL. Four compounds (i.e., CIP, FLE, MOX,

and OFL) were detected in almost every wastewater sample; the corresponding detection frequencies were 98%, 85%, 69%, and 100%;

respectively. While a number of previous reports have detected CIP and OFL [23-26], such high detection frequency for FLE and MOX was not

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expected as these compounds have not been previously reported for US surface water and wastewater. FLE and MOX are both human-use

antibiotics. FLE is a tri-fluorinated second-generation FQ that is used for treatment of urinary tract, respiratory tract, and skin infections;

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gonorrhea; typhoid fever; and traveler’s diarrhea [27]. On the other hand, MOX is a fourth-generation FQ antibiotic that was only introduced to

the United States in 1999 [28]; MOX is an effective treatment for bronchitis, pneumonia, and sinusitis [29].

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The concentration of FQ antibiotics varied for individual compounds and between water sources. As expected, FQ concentrations were

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highest in raw wastewater and lowest in surface water samples. Minimum concentrations were measured in surface water at 1-10 ng/L, which was

close to the limits of quantitation (see Table S1 in the Supplementary Data); however, most measured concentrations were in the 10-1000 ng/L
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range. The maximum recorded FQ concentration was 1920 ng/L CIP in raw wastewater from the small WWTP; the highest total FQ mass

concentration (i.e., 2239 ng/L) was also measured in the small WWTP. These concentrations, while slightly lower than the IC10 identified for CIP
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(i.e., 5 µg/L), are within the same order of magnitude. This finding validates the hypothesis that wastewater containing antibiotics can exhibit

antimicrobial activity and affect microbial populations.

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As indicated above, the four most frequently detected fluoroquinolones were CIP, FLE, MOX, and OFL. While FLE was detected, some

samples demonstrated a high level of interference from unidentified FQs or background organic matter; therefore, the FLE concentrations were not

quantified. The concentrations of the other FQs in raw wastewater, wastewater effluent, and downstream surface water (for the medium WWTP)

are presented in box-and-whisker diagrams in Figure 2. The figure shows that FQ concentrations decrease through treatment and upon discharge

into receiving water bodies. The apparent removal efficiency for the antibiotics through treatment was as follows: OFL (49%), MOX (64%), and

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CIP (74%).

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The data in Figure 2 also show that the concentration ranges of FQ antibiotics in raw wastewater and wastewater effluent are fairly narrow for

the medium-sized WWTP. For instance, the 10-90th percentiles for the total FQ mass concentration in raw wastewater, wastewater effluent, and

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downstream surface water are 992-1880 ng/L, 345-584 ng/L, and 8-53 ng/L, respectively. Raw wastewater concentrations of CIP and OFL

corresponding to the 10-90th percentiles were 500-1260 ng/L and 290-566 ng/L; the surface water concentrations for CIP and OFL ranged from

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6.5-31 ng/L and 9.4-39 ng/L, respectively. The upper whiskers in the surface water samples in Figure 2 stem from high detections in October

2013, for which a total FQ mass concentration of 145 ng/L was recorded. FQ concentrations in wastewater effluent were also elevated on that
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sampling day; furthermore, the BOD5, PO4-P, and UV absorbance at 254 nm were higher than average. These findings suggest a direct correlation

of background wastewater quality and antibiotic concentrations.

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The FQ concentrations in Figure 2 align with those reported by others [18, 23-26, 30-32]. For example, Leung et al. [26] measured CIP and
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OFL concentrations of 700-5640 ng/L and 140-7900 ng/L in raw wastewater in Hong Kong. In another study, CIP and OFL concentrations in

effluent wastewater were reported as 10-15 and 50-175 ng/L [23]. In wastewater effluent-impacted surface water from New Jersey, USA, Gibs et

al. [25] measured 10-80 ng/L CIP and 50-900 ng/L OFL. Maximum CIP and OFL levels in eight Chinese rivers were reported as 34 ng/L and 127

ng/L, respectively [24]. In this study, no significant differences in fluoroquinolone concentrations were apparent between the small-, medium-,

and large-WWTPs (Figure 2). These observations suggest that not only are antibiotic concentrations ubiquitous in wastewater, but that the

corresponding concentrations are within the same order of magnitude, regardless of location.

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ENR (18%) and NOR (27%) were detected frequently and at concentrations similar to previous studies [23-26]. The other detected FQs,

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especially DIF, FLE, MOX, and ORB, represent novel findings. The 10-90th percentiles for MOX in raw wastewater was 39-95 ng/L,

respectively; the corresponding range for wastewater effluent was 18-32 ng/L. FLE was detected, but not quantified (see above), in the majority of

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surface water samples. MOX was detected in two surface water samples at 36% of the concentration present in wastewater effluent. The other

compounds, namely DIF and ORB, are used in veterinary applications [33, 34]; therefore, their low detection frequency is probably associated

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with the use of antibiotics in dogs, cats, and other domestic animals, rather than humans. DIF was detected in four raw wastewater samples and

three wastewater effluent samples. Recall from above, that ORB was only detected once in the raw wastewater at the small WWTP.
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3.3 Removal of fluoroquinolones through wastewater treatment
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FQ concentrations from five sampling points corresponding to the medium-sized WWTP are plotted in Figure 3. While the antibiotic

concentrations in given samples vary, the figure demonstrates that mean concentrations steadily decrease through treatment. For example, the
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mean total FQ mass concentrations (i.e., the summation of FQ mass concentrations detected in this study) in raw wastewater, the RAS stream, the

DFI line, wastewater effluent, and downstream surface water were 1378, 966, 497, 474, and 51 ng/L, respectively. This trend was expected due to

removal of FQs during treatment through sorption to solids, biodegradation, and other transformation processes; furthermore, FQs in wastewater

effluent were diluted upon discharge to the receiving water body. Recall that physicochemical properties of the FQ antibiotics are provided in

Table 1. The low values of log Dow indicate that partitioning of FQs into wastewater solids is not preferred, and so FQs are expected to remain in

the aqueous phase; in addition, previous research has indicated that biodegradation is not favorable for typical wastewater operating parameters

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[35, 36]. FQ antibiotics are somewhat susceptible to phototransformation [37, 38]; however, less than 5% transformation is expected for

disinfection-level UV doses. Note that this expected transformation aligns fairly nicely with the observed mean concentrations upstream and

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downstream of the UV disinfection unit, which exhibit a FQ removal efficiency of 4.6%.

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Figure 4 shows the fraction of each FQ for various water compartments. From this plot, it is clear that OFL and CIP account for more than

90% of the total FQ mass concentration. The relative fraction of NOR, MOX, and DIF are fairly constant throughout the treatment process, and

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do not correspond to a significant percentage of the total FQ concentration. As treatment proceeds, the fraction of OFL increases, while CIP

decreases. During treatment, the ENR fraction increases. These data indicate that CIP is preferentially removed compared to OFL and ENR.
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Interestingly, CIP and ENR have fairly similar chemical structures, with the only difference being the presence of an ethyl group on the

piperazinyl functionality. The presence of this group shifts the second acid dissociation constant for ENR to ~6.7 compared to 8.7 for CIP. While
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the chemical structures of ENR and OFL differ, the acid dissociation constants are similar (e.g., pKa2,OFL = 6.2). Ultimately, these chemical

properties result in lower fractions of the zwitterionic form of ENR and OFL in solution compared to CIP. For a pH of 7.5 (consistent with raw
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wastewater from the medium WWTP), approximately 92%, 13%, and 5% of CIP, ENR, and OFL, respectively, are present as zwitterions.

In general, compounds that existed in the zwitterionic state demonstrated higher removal efficiencies. In particular, CIP and MOX are

predominantly zwitterionic at pH 7.5 and demonstrated high removal (66-75%); DIF and OFL, which are predominantly anionic at pH 7.5, were

removed to a lesser extent (52-57%). Plots of log Dow for these four compounds, along with the other FQs detected in this study, are presented in

Figure S1 in the Supplementary Data. Interestingly, CIP and MOX exhibit lower values of log Dow compared to DIF and OFL, which would

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suggest that OFL is better removed through partitioning into activated sludge. As the opposite trend was observed, the zwitterionic characteristic

may facilitate sorption to mineral surface sites, thereby increasing removal efficiency.

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3.4 Correlations to background wastewater quality

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To assess potential correlations of antibiotic presence with background wastewater quality, the total FQ mass concentrations were plotted as a

function of the flow rate, BOD5, NO3-N, PO4-P, TSS, NH3-N, pH, and UV absorbance at 254 nm (Figure S2 in the Supplementary Data). These

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plots provide some context for understanding the change in FQ concentration as a function of other wastewater quality parameters. A shift from

the upper right hand corner of these plots (e.g., high FQ concentration, high BOD) to the lower left hand corner is expected to occur during
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treatment. This trend is observed for all parameters with the obvious exceptions of flow rate and pH, which remain fairly constant, and NO3-N,

which is produced (not removed) during nitrification. More importantly, FQ removal was plotted against the removal efficiency for wastewater
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quality parameters to determine whether correlations exist between treatment of FQs and traditional parameters like BOD.
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Positive correlations were observed for removal of FQs with removal of PO4-P and UV254; however, negative correlations were determined for

BOD5 and TSS (Figure 5). These trends indicate that as phosphorus and UV254 removal efficiency increase, FQ treatment efficiency improves.

On the contrary, marginal improvements in BOD5 and TSS removal efficiency (i.e., 98 → 99%) appear to decrease FQ treatment efficiency. No

effective correlation was observed for NH3-N removal, suggesting that nitrifying bacteria play a minimal role in degradation of FQs. FQ removal

was also considered as a function of the mass flow of solids in the RAS stream; the resulting correlation was positive indicating that FQ sorption to

biological solids does occur. While these trends offer insight, a larger data set across multiple plants and wastewater composition is needed to

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validate trends and optimize removal of traditional wastewater quality parameters, as well as emerging contaminants like FQs.

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3.5 Calculation of population-normalized loads

Total FQ loads into the medium-sized WWTP were approximately 100 g/day with an effluent load of 34 g/day. Taking the population

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equivalents into account, the corresponding loads were 365 and 125 µg/PE-day, respectively. These results can be extrapolated to other WWTPs

serving similar populations (i.e., residential and light commercial) to predict the expected load of FQ antibiotics in raw and treated wastewater. As

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suggested above, CIP comprises the majority of this load: 220 µg/PE-day in raw wastewater and 55 µg/PE-day in wastewater effluent. Similarly,

the OFL load was high in raw wastewater (117 µg/PE-day) and wastewater effluent (56 µg/PE-day). Overall, CIP and OFL account for ~92% of
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the FQ load in raw wastewater and ~88% of the FQ load in wastewater effluent. While CIP and OFL represented the highest detected

concentrations, detection of multiple FQ antibiotics indicates that targeted detection campaigns focused only on blockbuster drugs may not be
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sufficient. In this case, we advocate measurement of individual classes of compounds, especially as potential for antibiotic-to-antibiotic

concentrations exists [37-39]. As others have reported [40, 41], FQs are also present in biosolids; therefore, the reduced FQ load does not
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necessarily remove the potential for FQ introduction into the environment. Furthermore, the half-life of FQs in environmental settings has been

shown to be longer than other PPCPs [42], increasing exposure of environmental organisms to FQs.

Although 30 years have passed since initial reports of pharmaceuticals in surface water [2], information on the fate and biological effects

of these chemicals in humans and aquatic ecosystems is still limited [1]. Other early studies have examined the toxicity of seven FQs in five

different aquatic organisms, with overall toxicity ranging from 7.9 to 23,000 µg/L; cyanobacteria was the most sensitive organism [11]. Note that

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the lower toxicity threshold is in the same order of magnitude as FQ concentrations detected in raw wastewater samples from this study. To better

understand the toxicity of specific drugs and their transport in the aquatic environment, additional information of this type is needed for individual

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classes of compounds. A key result of this study is that the presence and fate of individual FQ antibiotics in wastewater treatment differ

significantly. This information can be used in tandem with structure-based toxicity models to enable risk assessments based on expected FQ

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concentrations in wastewater and wastewater effluent.

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4. CONCLUSIONS

In summary, we have identified concentrations of a wide variety of FQ antibiotics in wastewater. These results contribute to a growing body
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of literature on emerging contaminants in water, a situation which may compromise drinking water quality and aquatic ecosystems. Early toxicity

studies have primarily examined qualitative data; however, quantitative data is necessary to better understand the impacts of low-level, continuous
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exposure of emerging contaminants in water and wastewater. In this study, we demonstrated that 125 µg/PE-day is discharged from a WWTP

serving residential and light commercial users; the PE-corrected load is 34 g/day. These population-normalized loads provide a starting point for
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characterizing dose-response relationships for sensitive organisms with a variety of emerging contaminants. For example, the antimicrobial

activity relationship determined above for CIP shows that wastewater-level FQ concentrations may exert selective pressures on microbial

populations. A greater research focus on similar dose-response relationships will facilitate better characterization of the toxicological threat of

fluoroquinolones and other emerging contaminants.

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5. ACKNOWLEDGMENTS

The authors gratefully acknowledge the Waters Academic Grant program for supporting this research. We also thank anonymous individuals

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from the Maryland Environment Service and operators at regional WWTPs for collecting wastewater samples and providing water quality and

operational information.

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REFERENCES

[1] P. Bottoni, S. Caroli, A.B. Caracciolo, Pharmaceuticals as priority water contaminants, Toxicological & Environmental Chemistry, 92 (2010)

an
549-565.

[2] M.L. Richardson, J.M. Bowron, The fate of pharmaceutical chemicals in the aquatic environment, Journal of Pharmacy and Pharmacology, 37

M
(1985) 1-12.

ed
[3] I.H. Rogers, I.K. Birtwell, G.M. Kruzynski, Organic extractables in municipal wastewater, Vancouver, British Columbia, Water Pollution

Research Journal of Canada, 21 (1986) 187-204.

pt
[4] K.V. Thomas, M.J. Hilton, The occurrence of selected human pharmaceutical compounds in UK estuaries, Marine Pollution Bulletin, 49

(2004) 436-444.
ce

[5] L. Quirós, R. Céspedes, S. Lacorte, P. Viana, D. Raldúa, D. Barcelò, B. Piña, Detection and evaluation of endocrine-disruption activity in
Ac

water samples from Portuguese rivers, Environmental Toxicology and Chemistry, 24 (2005) 389-395.

[6] D.W. Kolpin, E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, H.T. Buxton, Pharmaceuticals, hormones, and other organic

wastewater contaminants in U.S. streams, 1999-2000: A national reconnaissance, Environmental Science & Technology, 36 (2002) 1202-

1211.

[7] G.L. Cromwell, Antimicrobial and promicrobial agents, Swine nutrition, 2 (2001) 401-426.

[8] NRC, The Use of Drugs in Food Animals: Benefits and Risks, The National Academies Press, 1999.

Page 21 of 30
 i
cr
us
[9] O.A.H. Jones, N. Voulvoulis, J.N. Lester, Human pharmaceuticals in the aquatic environment a review, Environmental Technology, 22 (2001)

1383-1394.

an
[10] Á. Tölgyesi, Z. Verebey, V.K. Sharma, L. Kovacsics, J. Fekete, Simultaneous determination of corticosteroids, androgens, and progesterone

in river water by liquid chromatography–tandem mass spectrometry, Chemosphere, 78 (2010) 972-979.

M
[11] A.A. Robinson, J.B. Belden, M.J. Lydy, Toxicity of fluoroquinolone antibiotics to aquatic organisms, Environmental Toxicology and

Chemistry, 24 (2005) 423-430.

ed
[12] D. Swenson, Going green: Helping hospitals be friendlier to the environment, Pharmacy Times (2011).

[13] C. Copper, S. Hanna, R. Batni, T. Bhattacharyya, B. Shull, EPA inaction in identifying hazardous waste pharmaceuticals may result in unsafe
pt
disposal, US Environmental Protection Agency, Office of Inspector General, 2012.
ce

[14] NCCLS, Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Approved Standard 6th Edition ed., Wayne, Pennsylvania,

USA, 2004.
Ac

[15] M. von Sperling, C.A. de Lemos Chernicharo, Biological wastewater treatment in warm climate regions, IWA publishing, 2005.

[16] G. Tchobanoglous, F.L. Burton, H.D. Stensel, Metcalf, Eddy, Wastewater Engineering: Treatment and Reuse, McGraw-Hill Education, 2003.

[17] K. He, L. Blaney, Systematic optimization of an SPE with HPLC-FLD method for fluoroquinolone detection in wastewater, Journal of

Hazardous Materials, In Press (2014).

Page 22 of 30
 i
cr
us
[18] E.M. Golet, A.C. Alder, A. Hartmann, T.A. Ternes, W. Giger, Trace determination of fluoroquinolone antibacterial agents in urban

wastewater by solid-phase extraction and liquid chromatography with fluorescence detection, Analytical Chemistry, 73 (2001) 3632-3638.

an
[19] H. Nakata, K. Kannan, P.D. Jones, J.P. Giesy, Determination of fluoroquinolone antibiotics in wastewater effluents by liquid

chromatography–mass spectrometry and fluorescence detection, Chemosphere, 58 (2005) 759-766.

M
[20] F.J. Lara, M. del Olmo-Iruela, A.M. García-Campaña, On-line anion exchange solid-phase extraction coupled to liquid chromatography with

fluorescence detection to determine quinolones in water and human urine, Journal of Chromatography A, 1310 (2013) 91-97.

ed
[21] C. Ding, J. He, Effect of antibiotics in the environment on microbial populations, Applied Microbiology and Biotechnology, 87 (2010) 925-

941.
pt
[22] D.I. Andersson, D. Hughes, Microbiological effects of sublethal levels of antibiotics, Nature Reviews Microbiology, 12 (2014) 465-478.
ce

[23] L.-J. Zhou, G.-G. Ying, S. Liu, J.-L. Zhao, B. Yang, Z.-F. Chen, H.-J. Lai, Occurrence and fate of eleven classes of antibiotics in two typical

wastewater treatment plants in South China, Science of The Total Environment, 452–453 (2013) 365-376.
Ac

[24] W. Xu, W. Yan, X. Li, Y. Zou, X. Chen, W. Huang, L. Miao, R. Zhang, G. Zhang, S. Zou, Antibiotics in riverine runoff of the Pearl River

Delta and Pearl River Estuary, China: Concentrations, mass loading and ecological risks, Environmental Pollution, 182 (2013) 402-407.

[25] J. Gibs, H.A. Heckathorn, M.T. Meyer, F.R. Klapinski, M. Alebus, R.L. Lippincott, Occurrence and partitioning of antibiotic compounds

found in the water column and bottom sediments from a stream receiving two wastewater treatment plant effluents in Northern New

Jersey, 2008, Science of The Total Environment, 458–460 (2013) 107-116.

Page 23 of 30
 i
cr
us
[26] H.W. Leung, T.B. Minh, M.B. Murphy, J.C.W. Lam, M.K. So, M. Martin, P.K.S. Lam, B.J. Richardson, Distribution, fate and risk

assessment of antibiotics in sewage treatment plants in Hong Kong, South China, Environment International, 42 (2012) 1-9.

an
[27] J. Balfour, P. Todd, D. Peters, Fleroxacin, Drugs, 49 (1995) 794-850.

[28] FDA, Avelox (moxifloxacin hydrochloride) IV label. Accessdata FDA, NDA 021085, 1999.

M
[29] R. Wise, A review of the clinical pharmacology of moxifloxacin, a new 8-methoxyquinolone, and its potential relation to therapeutic efficacy,

Clinical Drug Investigation, 17 (1999) 365-387.

ed
[30] A. Jia, Y. Wan, Y. Xiao, J. Hu, Occurrence and fate of quinolone and fluoroquinolone antibiotics in a municipal sewage treatment plant,

Water Research, 46 (2012) 387-394.

pt
[31] L. Gao, Y. Shi, W. Li, H. Niu, J. Liu, Y. Cai, Occurrence of antibiotics in eight sewage treatment plants in Beijing, China, Chemosphere, 86
ce

(2012) 665-671.
Ac

[32] R. Andreozzi, M. Raffaele, P. Nicklas, Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment,

Chemosphere, 50 (2003) 1319-1330.

[33] Boehringer, Difloxacin hydrochloride product label information [package insert]. Fort Dodge, IA: Boehringer Ingelheim Vetmedica, Inc.,

2010.

[34] Intervet, Orbax (orbifloxacin) product label information [package insert]. Intervet Inc., Friesoythe, Germany; 2010.

Page 24 of 30
 i
cr
us
[35] N. Dorival-García, A. Zafra-Gómez, A. Navalón, J. González, J.L. Vílchez, Removal of quinolone antibiotics from wastewaters by sorption

and biological degradation in laboratory-scale membrane bioreactors, Science of The Total Environment, 442 (2013) 317-328.

an
[36] B. Li, T. Zhang, Biodegradation and adsorption of antibiotics in the activated sludge process, Environmental Science & Technology, 44

(2010) 3468-3473.

M
[37] K.H. Wammer, A.R. Korte, R.A. Lundeen, J.E. Sundberg, K. McNeill, W.A. Arnold, Direct photochemistry of three fluoroquinolone

antibacterials: Norfloxacin, ofloxacin, and enrofloxacin, Water Research, 47 (2013) 439-448.

ed
[38] L. Blaney, S. Snowberger, K. He, Determination of fluoroquinolone antibiotics in wastewater and transformation by UV and UV-H2O2

processes: Proceedings of the Water Environment Federation Technical Exhibition and Conference, Chicago, IL, 2013.
pt
[39] M.C. Dodd, H.P.E. Kohler, U. von Gunten, Oxidation of antibacterial compounds by ozone and hydroxyl radical: elimination of biological
ce

activity during aqueous ozonation processes, Environmental Science & Technology, 43 (2009) 2498-2504.

[40] A.L. Spongberg, J.D. Witter, Pharmaceutical compounds in the wastewater process stream in Northwest Ohio, Science of The Total
Ac

Environment, 397 (2008) 148-157.

[41] K. McClellan, R.U. Halden, Pharmaceuticals and personal care products in archived U.S. biosolids from the 2001 EPA national sewage

sludge survey, Water Research, 44 (2010) 658-668.

[42] N. Gottschall, E. Topp, C. Metcalfe, M. Edwards, M. Payne, S. Kleywegt, P. Russell, D.R. Lapen, Pharmaceutical and personal care products

in groundwater, subsurface drainage, soil, and wheat grain, following a high single application of municipal biosolids to a field,

Chemosphere, 87 (2012) 194-203.

Page 25 of 30
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[43] ChemAxon, www.chemicalize.org. Accessed on June 26, 2013., 2013.

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FIGURE CAPTIONS

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Figure 1. Inhibition profile of CIP against E. coli. (Error bars indicate 95% confidence intervals.)

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Figure 2. Box-and-whisker diagram of FQ concentrations in raw wastewater (red), wastewater effluent (blue), and downstream surface

water (green). (Black diamonds indicate mean concentrations; triangles indicate FQ concentrations in raw wastewater and

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wastewater effluent from the large WWTP; circles indicate FQ concentrations in raw wastewater from the small WWTP; WW =

wastewater; SW = surface water)

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Figure 3. Average FQ concentrations measured in various compartments of the medium-scale WWTP. (Bars are standard deviation.)
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Figure 4. Normalized column plot showing the relative fraction of each FQ present in different wastewater compartments. (Aggregate

concentrations from sampling campaign were used to find the fractional composition; the averaged total mass concentration of FQs is
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indicated above each column.)

Figure 5. Percent removal of FQ antibiotics plotted against removal of (a) BOD5, (b) TSS, (c) NH3-N, (d) PO4-P, (e) UV254 nm and as a

function of RAS mass flow (f).

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Table 1. Physical-chemical properties of the FQ antibiotics.
Abbreviated Chemical Mol. wt. log Dow a Structure
Compound CAS number pKa a
name formula (g/mol) (pH 7) (zwitterionic) b
O O

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F -
pKa1 = 5.76 O
Ciprofloxacin CIP 85721-33-1 C17H18FN3O3 331.34 -0.81
pKa2 = 8.68 N N
+
H2N

O O

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F -
O
pKa1 = 5.64 H N N
Difloxacin DIF 98106-17-3 C21H19F2N3O3 399.39 1.96 N
+
pKa2 = 6.45 H3C

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O O
F -
pKa1 = 5.69 O
Enrofloxacin ENR 93106-60-6 C19H22FN3O3 359.39 0.89 H N N
pKa2 = 6.68 +
H3C N
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O O
F -
O
pKa1 = 5.44
Fleroxacin FLE 79660-72-3 C17H18F3N3O3 369.34 0.47 H +
N N
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pKa2 = 6.06 N F
H3C F

O O
F -
pKa1 = 5.69 O
Gatifloxacin GAT 112811-59-3 C19H22FN3O4 375.39 -0.58 H3 C
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N N
pKa2 = 8.73 H2N
+
O
H3C

O O
F -
O
pKa1 = 5.64
Lomefloxacin LOM 98079-51-7 C17H19F2N3O3 351.35 -0.39 +
N N
pKa2 = 8.70 H2N F
CH3
CH3

O O
F -
O
pKa1 = 5.69 H H
Moxifloxacin MOX 186826-86-8 C21H24FN3O4 401.43 -0.50 H N
+ N N
pKa2 = 9.42 O
H3C
H

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O O
F -
pKa1 = 5.77 O
Norfloxacin NOR 70458-96-7 C16H18FN3O3 319.33 -0.92 N N
pKa2 = 8.68 +
H2N
CH3

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O O
F -
pKa1 = 5.45 O
Ofloxacin OFL 82419-36-1 C18H20FN3O4 361.37 0.07
pKa2 = 6.20 H + N N
N O
H3C CH3

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F O O
F -
O
pKa1 = 5.49 H3C
Orbifloxacin ORB 113617-63-3 C19H20F3N3O3 395.38 0.25 +
N N
pKa2 = 8.77 H2N F

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CH3

O O
F -
O
pKa1 = 5.74 N N
Sarafloxacin SAR 91296-87-6 C20H17F2N3O3 385.36 0.56 H 2N
+
pKa2 = 8.68
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F

a: Information collected from www.chemicalize.org [43]

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b: Note that FQs are often drawn as neutral species (e.g., deprotonated amine functionality and protonated carboxylic acid group); however,
these species are never dominant in the aquatic environment. The chemical differences between a seemingly neutral species and the actual
zwitterionic species have important consequences in relation to the ultimate environmental fate of FQs. For this reason, structures were
drawn for the zwitterionic state.
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1 Table 2. Mean (± standard deviation) properties of raw wastewater and wastewater effluent.
Parameter Raw wastewater Wastewater effluent
Flow rate (MGD) 19.2 (±1.6) 19.4 (±1.8)
pH 7.41 (±0.32) 7.32 (±0.09)
BOD5 (mg/L) 205 (±32) 2.46 (±0.45)
NH3-N (mg/L) 21.8 (±2.5) 0.56 (±0.76)
NO3-N (mg/L) 0.28 (±0.30) 0.43 (±0.30)
PO4-P (mg/L) 3.85 (±0.62) 0.28 (±0.27)

t
TSS (mg/L) 103 (±14) 1.22 (±0.27)

ip
UV254 (cm-1) 0.26 (±0.04) 0.12 (±0.01)
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