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Direct photolysis of fluoroquinolone antibiotics at 253.7 nm: Specific reaction kinetics and formation of equally-potent fluoroquinolone antibiotics Sebastian Snowberger, Hollie Adejumo, Ke He, Kiranmayi P Mangalgiri, Mamatha Hopanna, Ana Dulce Soares, and Lee Blaney Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01794 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 4, 2016

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Direct photolysis of fluoroquinolone antibiotics at 253.7 nm: Specific reaction kinetics and formation of equally-potent fluoroquinolone antibiotics

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Sebastian Snowberger1, Hollie Adejumo1, Ke He1, Kiranmayi Mangalgiri1, Mamatha Hopanna1,

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Ana Dulce Soares1, Lee Blaney1*

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1:

University of Maryland Baltimore County

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Department of Chemical, Biochemical and Environmental Engineering

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1000 Hilltop Circle, ECS 314

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Baltimore, MD 21250

13 14 15

* Corresponding author:

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Lee Blaney, PhD

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University of Maryland Baltimore County

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Department of Chemical, Biochemical and Environmental Engineering

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1000 Hilltop Circle, ECS 314

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Baltimore, MD 21250 USA

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Tel: +1-410-455-8608

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Fax: +1-410-455-1049

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Email: [email protected]

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Graphical Abstract

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Abstract

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Three fluoroquinolone-to-fluoroquinolone antibiotic transformations were monitored during UV-

30

C irradiation processes. In particular, the following reactions were observed: enrofloxacin-to-

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ciprofloxacin, difloxacin-to-sarafloxacin, and pefloxacin-to-norfloxacin. The apparent molar

32

absorptivity and fluence-based pseudo-first order rate constants for transformation of the six

33

fluoroquinolones by direct photolysis at 253.7 nm were determined for the pH 2-12 range. These

34

parameters were deconvoluted to calculate specific molar absorptivity and fluence-based rate

35

constants for cationic, zwitterionic, and anionic fluoroquinolone species. For a typical

36

disinfection fluence of 40 mJ/cm2, the apparent transformation efficiencies were inflated by 2-

37

8% when fluoroquinolone products were not considered; moreover, the overall transformation

38

efficiencies at 400 mJ/cm2 varied by up to 40% depending on pH. The three product antibiotics,

39

namely ciprofloxacin, sarafloxacin, and norfloxacin, were found to be equally or more potent

40

than the parent fluoroquinolones using an Escherichia coli-based assay. UV treatment of a

41

solution containing difloxacin was found to increase antimicrobial activity due to formation of

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sarafloxacin. These results highlight the importance of considering antibiotic-to-antibiotic

43

transformations in UV-based processes.

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1. Introduction

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The increased use of antibiotics has led to prevalent detection of antimicrobially active

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pharmaceuticals in raw wastewater 1-3 and the corresponding receiving waters 4-6. Trace

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concentrations of antibiotics in wastewater effluent can lead to adverse ecological consequences,

49

including inhibition of microbial species 7-9, development of antimicrobial resistance 10-12, and

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other sub-inhibitory impacts 13-15. The presence of antimicrobial resistant bacteria poses a human

51

health risk 16-18 and expedites the need to develop new antibiotics by reducing the efficacy of

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existing medicines 19, 20. These risks can be partially mitigated by decreasing the load of

53

antibiotics entering environmental systems and, ultimately, drinking water supplies. However,

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because many of the more recalcitrant antibiotic classes demonstrate a high level of structural

55

similarity, a potential exists for antibiotic-to-antibiotic transformation during conventional and

56

advanced water/wastewater treatment processes. Here, we specifically examined transformation

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of a particular suite of fluoroquinolone antibiotics through irradiation with UV light at 253.7 nm.

58 59

Fluoroquinolone antibiotics were investigated due to their high consumption in human and

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veterinary medicine 21, 22, potency at environmentally-relevant concentrations 1, 23, 24, and

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frequent environmental detection 4, 25-29. Several previous studies have examined photolysis

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mechanisms and/or kinetics of fluoroquinolones under natural or simulated solar irradiation 30-36.

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The quantum yields for 296-450 nm of norfloxacin (NOR), ofloxacin, and enrofloxacin (ENR)

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were reported by Wammer et al. 31. Ge et al. 34 reported the solar quantum yield for eight

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fluoroquinolones at 290-420 nm, and proposed a generalized reaction pathway for solar

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irradiation in pure water. Keen, Pereira, and coworkers reported the quantum yield and fluence-

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based first order rate constant for ciprofloxacin (CIP) degradation by low- and medium-pressure

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lamps 37-39; however, those studies did not examine the impacts of pH on photolysis kinetics or

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the identification of transformation products.

70 71

Due to the structural similarity of fluoroquinolone antibiotics, photolytic degradation of specific

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fluoroquinolones may result in formation of other known, and approved, fluoroquinolone

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antibiotics. Note that the pharmacophore, namely the basic unit of the ~30 discovered

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fluoroquinolones, is 6-fluoro-4-oxo-1,4-dihydropyridine-3-carboxylic acid 24 (see Figure S.1 in

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the Supporting Information). Previous studies have shown a tendency for ENR transformation to

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CIP under solar irradiation 7, 30-32, 34. Furthermore, sarafloxacin (SAR) has been reported as a

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primary photoproduct of difloxacin (DIF) exposed to simulated sunlight 34, 40. Photolysis of

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pefloxacin (PEF) has not been previously reported.

79 80

Unlike natural photolysis, the fluence employed during UV disinfection is strictly limited. For

81

this reason, monitoring of fluoroquinolone-to-fluoroquinolone transformations during UV-C

82

irradiation is of particular interest to minimize residual antibiotic concentrations and the

83

corresponding antimicrobial activity. This report examines three antibiotic-to-antibiotic

84

transformations: ENR-to-CIP; DIF-to-SAR; and PEF-to-NOR. The parent compounds (i.e.,

85

ENR, DIF, and PEF) differ from the product compounds (i.e., CIP, SAR, and NOR) by a single

86

methyl- or ethyl- group. Note that all six fluoroquinolones can be “parent” antibiotics; however,

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we specifically use the term “product” to indicate molecules formed by phototransformation of

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other fluoroquinolones. Chemical structures, along with other physicochemical information, for

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the six fluoroquinolones are shown in Table S.1 of the Supporting Information.

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The primary objectives of this study were as follows: (1) to describe the fundamental kinetic

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parameters for direct photolysis of fluoroquinolone antibiotics irradiated with UV light at 253.7

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nm; (2) to monitor fluoroquinolone-to-fluoroquinolone transformations that occur for these

94

conditions, including the effects of pH on product yield; and, (3) to determine the relative

95

potency of parent and product antibiotics, and how the overall residual antimicrobial activity is

96

affected by UV-based treatment.

97 98

2. Experimental Materials and Methods

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2.1 Chemicals

100

ENR (> 98.0%), CIP (98.0%), DIF hydrochloride (> 98%), SAR hydrochloride trihydrate

101

(97.3%), PEF mesylate dihydrate (>97%), and NOR (> 98%) were obtained from Sigma-Aldrich

102

(St. Louis, MO). Anhydrous sodium phosphate (99%) and anhydrous sodium diphosphate (>

103

98%) were acquired from Fisher Scientific (Waltham, MA). Stock solutions of fluoroquinolones

104

were prepared at 100 mg/L by dissolving the appropriate mass into deionized water and

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sonicating the solution until dissolved. To facilitate dissolution, the ENR stock solution included

106

0.1% HCl (Fisher Scientific; Waltham, MA). All fluoroquinolone stock solutions were stored in

107

amber bottles in the dark at 4 °C and were renewed monthly to avoid decomposition.

108 109

2.2 Batch-recycle UV reactor

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A batch-recycle UV reactor, similar to that employed in previous studies 41, was used here. The

111

reactor was comprised of a sampling vessel coupled to a low-pressure, UV-C cell (Trojan

112

Technologies UVMax A; London, Ontario) with Teflon tubing. The net photon flux was

113

4.43×10-7 Einstein/min-cm2, as measured by potassium ferrioxalate actinometry 42.

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Fluoroquinolone stock solutions and concentrated phosphate buffer were added to deionized

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water to generate solutions containing 3 mg/L fluoroquinolone and 10 mM phosphate buffer.

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Experimental solutions were continuously recirculated and 1 mL samples were drawn at pre-

117

determined times. After collection, samples were stored in 2 mL amber glass vials in the dark at

118

4 °C until analysis, which occurred within 72 hours. The solution pH and temperature were

119

constantly recorded using an Accumet pH meter (Fisher Scientific), which was calibrated before

120

each experiment.

121 122

2.3 Analytical Methods

123

2.3.1 Molar absorptivity

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UV-visible spectrophotometry (Evolution 600; Thermo Scientific) was employed to measure the

125

apparent molar absorptivity of fluoroquinolones at 253.7 nm. A 1 cm quartz cuvette was used

126

for these measurements. Fluoroquinolone concentrations ranged from 0 to 5 mg/L, and pH was

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controlled using 10 mM phosphate buffer. The apparent molar absorptivity (εapp) was modeled

128

as a function of pH by assigning each protonated/deprotonated species a specific molar

129

absorptivity (ε0, ε1, and ε2), with the apparent absorptivity being the summation of the

130

absorptivity contributed by each species (Eq. 1). Acid dissociation constants for the diprotic

131

fluoroquinolones were determined by spectrophotometric techniques using the absorbance at

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253.7 nm 43-45. The resulting pKa values are reported in Table S.1 (see Supporting Information);

133

generally, these values compared well to previously reported equilibrium constants for the

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fluoroquinolones of concern 46, 47. The fraction of each species in solution (α0, α1, and α2) was

135

calculated using Equations 2-4 48.

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 =   +   +  

(Eq. 1)

     

(Eq. 2)

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 = 1 +  + 

139 140

 

 =

141



+ 1 +   



(Eq. 3)

142  

 =

143

 

+

 



+ 1



(Eq. 4)

144 145

The apparent molar absorptivity was measured for each fluoroquinolone of concern at distinct

146

pH values ranging from 2 to 12. The specific molar absorptivity for each fluoroquinolone

147

species was fit by minimizing the sum of differences squared between observed and modeled

148

(Eq. 1) apparent molar absorptivity.

149 150

2.3.2 Fluoroquinolone analysis

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Fluoroquinolone concentrations were measured using liquid chromatography tandem mass

152

spectrometry (LC-MS/MS; UltiMate 3000 LC with TSQ Quantum Access Max, Thermo

153

Scientific). Two eluents were employed: (A) acetonitrile and (B) 0.1% formic acid. Samples

154

(50 µL) were injected onto a C18 column (Thermo Accucore; 2.6 µm, 4.6×150 mm) in 5% A,

155

95% B for 8 minutes, then the eluent was linearly ramped to 20% A, 80% B over 5 minutes.

156

These conditions were maintained for 2 minutes, then linearly reverted to the original

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composition (5% A, 95% B) over 3 minutes. To rebalance the column, these conditions were

158

held constant for 5 minutes before injection of the next sample. Others have employed similar

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elution gradients for fluoroquinolone analysis 49. The eluent flow rate was 300 µL/min, and a

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switching valve was employed to flush the phosphate buffer before delivery to the electrospray

161

ionization unit, which was operated in positive mode.

162 163

The MS/MS detector was configured for selected reaction monitoring (SRM). The following

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SRM parameters were optimized: spray voltage, 3 kV; vaporizer temperature, 300 °C; capillary

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temperature, 350 °C; sheath gas, 40 (arbitrary units); auxiliary gas, 15 (arbitrary units); collision

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energy, 18-27 eV; and, tube lens offset, 75-105 V. The final parent ion → product ion mass-to-

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charge ratios (m/z) were as follows: (CIP) 332.310→288.200; (DIF) 400.350→299.200; (ENR)

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360.283→316.200; (NOR) 320.302→302.200; (PEF) 334.329→290.200; and, (SAR)

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386.321→368.200. Similar transitions and operational conditions have been reported elsewhere

170

50-53

171

0.99. All identifications were made using a targeted approach with fluoroquinolone standards.

. Coefficients of determination (R2) for standard calibration curves were always greater than

172 173

2.4 Calculation of apparent rate constants

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To enable comparison with other UV systems, fluence-based pseudo-first order rate constants

175

were reported. Fluence (H´ in mJ/cm2) was calculated as the product of the net photon flux (I0,

176

Einstein/min·cm2), the energy of photons at 253.7 nm (U253.7 = 4.72 × 108 mJ/Einstein), and

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irradiation time (t, min), as indicated in Eq. 5

178 179

  =   . 

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Using the fluence associated with each sampling time, the apparent fluence-based pseudo-first

182

order rate constants were calculated for the batch-recycle reactor using the integrated mass

183

balance shown in Eq. 6.

184 ln "

185

#$,&'()$

#*,&'()$

+ = −-

,



(Eq. 6)

186 187

In Eq. 6, Ct,parent is the concentration of the parent fluoroquinolone at time t (corresponding to

188

fluence H´), C0,parent is the initial concentration of the parent compound, - *,&& is the apparent

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fluence-based rate constant for degradation of the parent fluoroquinolone by UV light at 253.7

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nm. The rate constant was calculated by minimizing the sum of the squared differences for

191

experimental data with Eq. 6. Specific fluence-based rate constants were determined for

192

individual fluoroquinolone species using Eq. 7.

193 -

194

,

=  -

,

+  -

,

+  -

,

(Eq. 7)

195 196

In Eq. 7, the -

197

253.7 degradation of the cationic, zwitterionic, and anionic parent fluoroquinolone species,

198

respectively. The kinetic parameters reported in Section 3.2 stem from these independent

199

experiments. Apparent transformation efficiency was defined as the change in parent compound

200

concentration for a particular fluence.

,

, -

,

, and -

,

terms are the specific fluence-based rate constants for UV

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A fraction of the parent compound degradation was assumed to result in formation of the product

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fluoroquinolone; concurrently, the product also undergoes transformation by UV 253.7.

204

Normalized concentrations for the product fluoroquinolone were, therefore, modeled using Eq. 8.

205 #$,&'./01$

206

#*,&'()$

= 2

4 3&

4 3&

*,&& 4 3& ,&& *,&&

5exp −-

,

′ − exp −-

,

′:

(Eq. 8)

207 208

In Eq. 8, f is the fraction of the parent fluoroquinolone that is transformed into the product

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fluoroquinolone, Ct,product is the concentration of the product fluoroquinolone at time t, and

210

-

211

fluoroquinolone. The rate constant for product transformation and the f value were found by

212

minimizing the sum of the squared differences for experimental data with Eq. 8. Overall

213

transformation efficiency was defined using parent and product fluoroquinolone concentrations.

,

is the apparent fluence-based rate constant for UV 253.7 degradation of the product

214 215

2.5 Antimicrobial activity determination by bioassay

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An Escherichia coli assay was used to quantify the potency of parent antibiotics, as well as

217

residual antimicrobial activity following UV-based treatment. Dose-response curves, or

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inhibition profiles, for the six fluoroquinolones of concern were developed with E. coli ATCC

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25922 (American Type Culture Collection; Manassas, VA) using the standard antibacterial

220

susceptibility protocol 54. A detailed description of assay protocols was previously reported 1.

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Here, the initial fluoroquinolone concentrations in microplate wells was 1000 µg/L and serial

222

dilution (with dilution factor = 0.5 or 0.6, over 18 samples) was employed to achieve final

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concentrations of 0.169 or 0.0076 µg/L. Positive and negative growth controls consisted of

224

deionized water with E. coli inoculum and deionized water with Mueller-Hinton broth,

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respectively. The pH of Mueller-Hinton broth was 7.3±0.1, and that pH was maintained in all

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microplate wells. All conditions were tested in triplicate. Microplates were incubated for 24

227

hours at 160 rpm and 37 °C, before measurement of the optical density at 625 nm using a

228

microplate reader (BioTek Eon; Winooski, VT).

229 230

E. coli growth inhibition data were fit to the Hill equation (Eq. 9).

231  = ; − ; ENR > CIP > SAR > PEF > NOR. The specific molar

272

absorptivity values in Table S.2 (see Supporting Information) can be employed to calculate the

273

apparent molar absorptivity at other pH values using Eq. 1.

274 275

3.2 Fluence-based pseudo-first order rate constants with UV 253.7 nm

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Experiments aimed at analyzing fluoroquinolone phototransformation were conducted at 8-10

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distinct pH values. The corresponding experimental data were analyzed using Eq. 6 to identify

278

fluence-based pseudo-first order rate constants for fluoroquinolone transformation by irradiation

279

with UV light at 253.7 nm. For example, consider Figure 2, which shows transformation

280

kinetics of CIP at six of the tested pH conditions. The slope of these plots corresponds to the

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apparent fluence-based pseudo-first order rate constant; note that all data demonstrate a good

282

linear fit (R2 > 0.99). From Figure 2, it is clear that the apparent rate constant increases from low

283

pH to near-neutral pH, and decreases slightly at high pH. Wammer et al. 31 observed similar

284

trends for solar irradiation of enrofloxacin, norfloxacin, and ofloxacin. These apparent rate

285

constants were deconvoluted to calculate the specific fluence-based rate constants (see Table 1)

286

associated with the cationic, zwitterionic, and anionic species of the six fluoroquinolones of

287

concern for irradiation with UV 253.7 nm. With these rate constants, the apparent

288

transformation efficiency of the six fluoroquinolones of concern can be determined for any

289

water/wastewater pH, providing an estimate of baseline removal in UV-based processes.

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The increasing reactivity of deprotonated fluoroquinolones with UV light at 253.7 nm is due, in

292

part, to the increased molar absorptivity of the zwitterion compared to the cationic and anionic

293

forms (see Figure 1a and Table S.2 in the Supporting Information). The high absorptivity and

294

fluence-based rate constants at near-neutral pH indicate that the photo-efficiency of the

295

transformation reaction, as measured by the quantum yield at 253.7 nm, is higher at pH 7-9.

296

Data from Table S.2 and Table 1 was used to calculate specific quantum yields at 253.7 nm (see

297

Table S.3 in the Supporting Information). In general, the quantum yields determined here were

298

similar to (i.e., within a factor of 1-2) those reported by others for solar irradiation 31, 34.

299 300

The trend in fluence-based reactivity for the six zwitterions is as follows: NOR > PEF > CIP >

301

SAR > DIF > ENR. With the exception of DIF and ENR, this trend is the opposite of that found

302

for the molar absorptivity of the fluoroquinolone zwitterions. NOR and PEF demonstrated the

303

lowest molar absorptivity; however, these fluoroquinolones exhibit the highest fluence-based

304

pseudo-first order rate constants for irradiation with 253.7 nm due to their high quantum yield.

305

On the other hand, DIF and ENR, which absorb the most light at 253.7 nm, showed the slowest

306

photodegradation kinetics due to the lower quantum yield of their zwitterions.

307 308

3.3 Fluoroquinolone-to-fluoroquinolone transformations at UV 253.7 nm

309

Experimental data confirmed the occurrence of three fluoroquinolone-to-fluoroquinolone

310

transformations: ENR-to-CIP; PEF-to-NOR; and DIF-to-SAR. Note that the parent

311

fluoroquinolone for each of these reactions absorbs more UV-C light (Figure 1 and Table S.2 in

312

the Supporting Information), and the products generally undergo faster rates of direct photolysis

313

at 253.7 nm (Table 1). The corresponding reaction mechanism involves loss of the methyl/ethyl

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groups attached to the piperazinyl ring, which is not part of the fluoroquinolone pharmacophore

315

24

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in disinfection processes, namely 40-200 mJ/cm2. In fact, generation of the product

317

fluoroquinolones was maximal in this range, indicating the importance of monitoring

318

fluoroquinolone-to-fluoroquinolone reactions during UV-based processes.

. Formation of product fluoroquinolones is of most interest at the low fluence levels employed

319 320

The apparent transformation efficiencies of the parent fluoroquinolones are reported in Table 2

321

for 40 and 400 mJ/cm2. The 40 mJ/cm2 fluence is the typical design value for disinfection

322

processes, whereas 400 mJ/cm2 reflected an order of magnitude increase in treatment. For these

323

scenarios, the overall removal efficiency, which considers the concentrations of parent and

324

product fluoroquinolones, was also calculated. The results in Table 2 clearly show that removal

325

efficiency is inflated when product fluoroquinolones are not considered. For example, at typical

326

disinfection doses of 40 mJ/cm2, 2-8% differences exist between apparent and overall

327

transformation efficiencies; the overall transformation efficiency varies by up to 10% for

328

different pH conditions. The overall removal efficiencies do not exceed 19%, 12%, and 14% for

329

PEF-to-NOR, DIF-to-SAR, and ENR-to-CIP, respectively. At 400 mJ/cm2, overall

330

transformation efficiencies can vary up to 40% depending on pH. For example, at pH 5.34 and

331

7.06, the ENR-to-CIP reaction results in overall transformation efficiencies of 30.3% and 71.6%,

332

respectively. Regardless, the relative potency of the product fluoroquinolones is also an

333

important variable in assessing treatment efficacy.

334 335

3.4 Effect of pH on formation of fluoroquinolone products

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Above, the effect of pH on fluoroquinolone phototransformation was illustrated for all six

337

compounds of concern. As expected, the rate of transformation for parent fluoroquinolones

338

affects the yield of the corresponding products. Figure 3 shows this phenomenon for each

339

transformation under acidic, near-neutral, and basic conditions. Clearly, the rate of DIF

340

transformation increases from pH 5.6 to 6.7 to 8.9 as expected from the specific rate constants

341

summarized in Table 1. At pH 8.9, 78% DIF transformation was observed for a fluence of 400

342

mJ/cm2, compared to approximately 76% and 52% for pH 6.7 and 5.6, respectively. However,

343

when including the produced SAR at 400 mJ/cm2 treatment, the overall transformation

344

efficiencies were 44%, 69%, and 65%, for pH 5.6, 6.7, and 8.9 respectively. Despite faster

345

transformation of DIF and SAR at pH 8.9, the lower molar absorptivity and quantum yield of

346

SAR at higher pH slows the transformation reaction and results in a higher concentration of

347

fluoroquinolones detected after 400 mJ/cm2 treatment, compared to the same treatment at pH 6.7.

348

The fluence required for 90% transformation ranges from 850 mJ/cm2 at pH 6.7 to 1670 mJ/cm2

349

at pH 5.6. These differences highlight the need for identification of specific rate constants rather

350

than discrete experiments conducted at one particular pH.

351 352

3.5 Residual antimicrobial activity of treated solutions

353

Inhibition profiles for the six fluoroquinolones of concern are shown in Figure 4. The narrow

354

95% confidence bands show that experimental data exhibited a good fit to the Hill equation (Eq.

355

9). From these plots, it can be observed that fluoroquinolone potency (as measured against E.

356

coli) follows the trend: CIP > SAR > ENR > DIF > PEF > NOR. The IC50 and Hill slope values

357

are summarized in Table S.4 in the Supporting Information. IC50 values ranged from 19.8 µg/L

358

for CIP to 89.3 µg/L for NOR. These findings align with previously reported inhibitory

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concentrations for fluoroquinolone antibiotics against a variety of microorganisms 56, 57, where

360

the same trend, namely that CIP is most potent and NOR is least potent, was observed for all but

361

one organism. Furthermore, the magnitude of IC50 values measured here was similar to that

362

found for CIP, ENR, and NOR against Actinobacillus pleuropneumoniae, Actinobacillus suis,

363

Haemophilus parasuis, Pasteurella haemolytica, and Pasteurella multocida 56.

364 365

The transformation of fluoroquinolones into antimicrobially active products, including other

366

fluoroquinolones, suggests that monitoring of specific chemical concentrations may not

367

accurately describe the extent of treatment. Importantly, the IC50 data identified in Figure 4 and

368

Table S.4 (see Supporting Information) highlight that product fluoroquinolones identified in this

369

study are equally, or more, potent than the parent compounds. Consider the magnitude of the

370

IC50 values for the three identified transformations: DIF (57.1 ± 5.1 µg/L) to SAR (21.8 ± 3.1

371

µg/L); ENR (27.4 ± 2.9 µg/L) to CIP (19.8 ± 5.1 µg/L); and PEF (68.7 ± 8.3 µg/L) to NOR (74.6

372

± 5.1 µg/L). From pharmacology literature, this trend is expected due to the identity of the R1

373

and R7 substituents (see Figure S.1 in the Supporting Information) in the fluoroquinolone

374

structures 58-60. The R1 group, namely cyclopropyl (CIP, ENR), fluorinated phenyl (DIF, SAR),

375

or ethyl (PEF, NOR), have been associated with genetic toxicity; in particular, the cyclopropyl

376

and ethyl groups result in the highest and lowest genetic toxicity 58, 59. Substituted piperazinyl

377

moieties also correspond to lower genetic toxicity, which confirms our observations that UV-

378

driven de-methylation and de-ethylation of DIF, ENR, and PEF produces antibiotics that are

379

more potent. Wammer et al. 31 validated increased antimicrobial activity during solar irradiation

380

of ENR due to the production of CIP.

381

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This phenomenon is further highlighted in Figure 5. In Figure 5a, the transformation of DIF (Co

383

= 3 mg/L) and growth inhibition of E. coli (for 20× diluted samples) are plotted as a function of

384

fluence. As expected from above, DIF transformation exhibits pseudo-first order kinetics with

385

approximately 70% transformation at 400 mJ/cm2 of UV 253.7 irradiation. On the contrary, no

386

significant removal of antimicrobial activity occurs until a fluence of greater than 600 mJ/cm2.

387

After 1200 mJ/cm2 of UV-C treatment, the majority of the antimicrobial activity associated with

388

the solution has been removed. The E. coli growth inhibition data are plotted in Figure 5b as a

389

function of DIF concentration. The green curves are the Hill equation, and corresponding 95%

390

confidence bands, found above for DIF standards. Clearly, the UV-irradiated samples exhibit a

391

higher antimicrobial potency, as evidenced by the lower effective IC50 (i.e., 4.59 ± 0.34 µg/L)

392

observed from the data (with black model curves) in Figure 5b. The increased activity stemmed

393

from additive effects caused by the presence of SAR.

394 395

A number of studies have examined removal of large suites of contaminants of emerging

396

concern, including antibiotics, in water and wastewater treatment processes. However, study of

397

specific treatment processes are warranted to provide more insight into the form of removal 61,

398

which often manifests as a transformation reaction (e.g., metabolism and oxidation, among

399

others) or phase change (e.g., adsorption, ion exchange, etc.). UV-based processes are

400

increasingly employed for disinfection of water and wastewater; furthermore, these processes

401

can be configured to generate reactive species through photochemical and photocatalytic

402

reactions. As such, UV processes demonstrate a flexibility for future treatment of contaminants

403

of emerging concern. For that reason, this study focused on determining fundamental parameters

404

dictating the fate of fluoroquinolone antibiotics under irradiation at UV 253.7 nm. Specific

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molar absorptivity and fluence-based pseudo-first order rate constants were determined for the

406

cationic, zwitterionic, and anionic species of six environmentally-relevant fluoroquinolone

407

antibiotics. These parameters can be extended to other UV systems, regardless of

408

water/wastewater pH, to predict baseline transformation efficiency. However, as evidenced

409

above, the formation of antimicrobially active products during UV processes needs to be

410

considered, especially since these products demonstrate high yields at low fluence and exhibit

411

similar or greater potency. Comprehensive evaluation of the full suite of fluoroquinolones and

412

the resulting reaction pathways will further elucidate proper treatment metrics for these

413

important antibiotics of emerging concern.

414 415

4. Supporting Information

416

Supporting Information available. This material is available free of charge via the Internet

417

at http://pubs.acs.org.

418

5. Acknowledgments

419

The authors gratefully acknowledge funding from the NSF Environmental Engineering program

420

(CBET # 1510420), as well as the UMBC startup and undergraduate research award programs.

421

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6. References

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of a wide variety of human and veterinary fluoroquinolone antibiotics in municipal

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wastewater and wastewater-impacted surface water. J. Pharm. Biomed. Anal. 2015, 106,

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Mutiyar, P.; Mittal, A., Occurrences and fate of selected human antibiotics in influents

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and effluents of sewage treatment plant and effluent-receiving river Yamuna in Delhi

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Zhou, L.-J.; Ying, G.-G.; Liu, S.; Zhao, J.-L.; Yang, B.; Chen, Z.-F.; Lai, H.-J.,

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He, K.; Blaney, L., Systematic optimization of an SPE with HPLC-FLD method for fluoroquinolone detection in wastewater. J. Hazard. Mater. 2015, 282, (0), 96-105.

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A., Direct photochemistry of three fluoroquinolone antibacterials: Norfloxacin, ofloxacin,

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and enrofloxacin. Water Res. 2013, 47, (1), 439-448.

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Environmental photochemistry of fluoroquinolones in soil and in aqueous soil

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suspensions under solar light. Environ. Sci. Pollut. Res. 2014, 21, (23), 13215-13221.

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of Fluoroquinolone Antibiotics: Kinetics, Pathways, and Multivariate Effects of Main

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sunlight irradiation: Kinetics, mechanism and toxicity of photolysis products.

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modeling of pharmaceutical compounds in laboratory grade and surface water via direct

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and indirect photolysis at 254 nm. Environ. Sci. Technol. 2007, 41, (5), 1682-1688.

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10485-10486.

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Table 1.

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Specific fluence-based pseudo-first order rate constants (with standard error) for cationic (CDE,FGH.I ), zwitterionic (CDJ,FGH.I ), and anionic (CDF,FGH.I ) fluoroquinolone species, with the modeled apparent fluence-based rate constant at pH 7 (CDFGH.I,KL I ). The apparent rate constant for other pH values can be determined using Eq. 7.

604 Compound

CD

E,FGH.I

CD

J,FGH.I

CD

F,FGH.I

CD

FGH.I,KL I

(cm2/mJ)

(cm2/mJ)

(cm2/mJ)

CIP

5.17 (±0.99) × 10-4

3.94 (±0.72) × 10-3

3.16 (±0.84) × 10-3

2.95 × 10-3

DIF

7.66 (±1.09) × 10-4

2.84 (±0.75) × 10-3

2.73 (±0.46) × 10-3

2.74 × 10-3

ENR

4.18 (±0.08) × 10-4

2.52 (±0.21) × 10-3

4.90 (±0.17) × 10-3

2.39 × 10-3

NOR

7.96 (±0.61) × 10-4

5.55 (±0.68) × 10-3

2.42 (±0.17) × 10-3

3.97 × 10-3

PEF

1.95 (±0.54) × 10-3

5.28 (±1.98) × 10-3

5.47 (±1.73) × 10-3

4.83 × 10-3

SAR

2.77 (±0.64) × 10-4

3.09 (±0.71) × 10-3

1.79 (±0.60) × 10-3

2.11 × 10-3

605

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(cm2/mJ)

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Table 2.

Apparent and overall removal efficiencies for three fluoroquinolone-to-fluoroquinolone transformations at 40 and 400 mJ/cm2 and three pH domains (below pKa1, between pKa1 and pKa2, and above pKa2).

609 Transition

PEF → NOR

DIF → SAR

ENR → CIP

pH 6.53 7.50 8.50 5.65 6.70 8.85 5.34 7.06 8.87

Fluence 40 mJ/cm2 Apparent Overall (%) (%) 14.8 8.2 26.2 18.6 18.4 11.0 7.0 5.7 13.5 11.6 14.1 10.3 5.3 3.5 18.1 13.3 13.9 7.6

Fluence 400 mJ/cm2 Apparent Overall (%) (%) 79.7 60.2 95.1 81.9 87.0 73.5 51.5 44.6 76.5 69.0 78.1 65.0 42.0 30.3 86.5 71.6 77.5 56.5

610

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611 612 613 614 615 616 617

Figure 1.

Effect of pH on the molar absorptivity of (a) ENR, (b) CIP, (c) PEF, (d) NOR, (e) DIF, and (f) SAR. For all six fluoroquinolones, specific molar absorptivities were calculated for the cationic, zwitterionic, and anionic species and used to plot the model curves. Dashed curves represent 95% confidence bands.

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Figure 2.

Pseudo-first order fluence-based phototransformation kinetics of CIP for irradiation at 253.7 nm at different pH values. Note that the fastest reaction kinetics occur between pH 7 and 8.

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624 625 626 627 628 629 630 631 632

Figure 3.

Comparison of fluoroquinolone-to-fluoroquinolone reactions for ENR-to-CIP, PEF-toNOR, and DIF-to-SAR at three different pH domains (i.e., below pKa1, between pKa1 and pKa2, and above pKa2). The black curve in each plot is the additive sum of the modeled parent and product concentrations, which were fit to the corresponding experimental data. Typical UV disinfection processes employ a fluence of 40-200 mJ/cm2.

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Figure 4.

Profiles showing inhibition of E. coli growth as a function of fluoroquinolone concentration for (a) ENR, (b), CIP, (c) PEF, (d) NOR, (e) DIF, and (f) SAR. Note that Hill Equation parameters for all fluoroquinolones are presented in Table S.4 of the Supporting Information.

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639 640 641 642 643 644 645 646

Figure 5.

(a) Transformation of DIF at pH 8 as a function of fluence overlaid with the corresponding residual antimicrobial activity measured by E. coli assay. The residual antimicrobial activity data are plotted in (b) as a function of DIF concentration (with select fluence overlays). The fitted Hill Equation exhibited a shift to lower DIF concentrations, indicating that the SAR produced during phototransformation contributes to the residual activity.

647 648

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