Photochemical degradation of the carbapenem antibiotics imipenem and meropenem in aqueous solutions under solar radiation

Accepted Manuscript Photochemical degradation of the carbapenem antibiotics imipenem and meropenem in aqueous solutions under solar radiation Alejandro Cabrera Reina, Ana B. Martínez-Piernas, Yannis Bertakis, Christina Brebou, Nikolaos P. Xekoukoulotakis, Ana Agüera, José Antonio Sánchez Pérez PII:

S0043-1354(17)30882-5

DOI:

10.1016/j.watres.2017.10.047

Reference:

WR 13304

To appear in:

Water Research

Received Date: 27 July 2017 Revised Date:

19 October 2017

Accepted Date: 21 October 2017

Please cite this article as: Reina, A.C., Martínez-Piernas, A.B., Bertakis, Y., Brebou, C., Xekoukoulotakis, N.P., Agüera, A., Sánchez Pérez, José.Antonio., Photochemical degradation of the carbapenem antibiotics imipenem and meropenem in aqueous solutions under solar radiation, Water Research (2017), doi: 10.1016/j.watres.2017.10.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Photolysis of imipenem 1.0 2.0 1.5

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Photochemical degradation of the carbapenem antibiotics imipenem and meropenem in

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aqueous solutions under solar radiation

3 Alejandro Cabrera Reinaa, Ana B. Martínez-Piernasb, Yannis Bertakisd, Christina Breboud, Nikolaos

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P. Xekoukoulotakisd*, Ana Agüerab**, José Antonio Sánchez Pérezb,c

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a

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EUDIM, Escuela Universitaria de Ingeniería Mecánica, Universidad de Tarapacá, Av. General Velásquez 1775, Arica, Chile

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CIESOL, Joint Centre University of Almería-CIEMAT, Almería, Spain

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Chemical Engineering Department, University of Almería, Spain

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Department of Environmental Engineering, Technical University of Crete, Polytechneioupolis, GR-73100 Chania, Greece

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* Corresponding author: e-mail: [email protected]; Tel.: +302821037772; Fax:

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+302821037848

** Corresponding author: e-mail: [email protected]; Tel. +34950015531, +34628188352

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Abstract

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This paper deals with the photochemical fate of two representative carbapenem antibiotics, namely

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imipenem and meropenem, in aqueous solutions under solar radiation. The analytical method

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employed for the determination of the target compounds in various aqueous matrices, such as

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ultrapure water, municipal wastewater treatment plant effluents, and river water, at environmentally

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relevant concentrations, was liquid chromatography coupled with hybrid triple quadrupole-linear

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ion trap-mass spectrometry. The absorption spectra of both compounds were measured in aqueous

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solutions at pH values from 6 to 8, and both compounds showed a rather strong absorption band

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centered at about 300 nm, while their molar absorption coefficient was in the order from 9×103 to

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104 L·mol–1·cm–1. The kinetics of the photochemical degradation of the target compounds was

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studied in aqueous solutions under natural solar radiation in a solar reactor with compound

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parabolic collectors. It was found that the photochemical degradation of both compounds at

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environmentally relevant concentrations follows first order kinetics and the quantum yield was in

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the order of 10–3 mol·einsten−1. Several parameters were studied, such as solution pH, the presence

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of nitrate ions and humic acids, and the effect of water matrix. In all cases, it was found that the

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presence of various organic and inorganic constituents in the aqueous matrices do not contribute

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significantly, either positively or negatively, to the photochemical degradation of both compounds

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under natural solar radiation. In a final set of photolysis experiments, the effect of the level of

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irradiance was studied under simulated solar radiation and it was found that the quantum yield for

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the direct photodegradation of both compounds remained practically constant by changing the

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incident solar irradiance from 28 to 50 W·m−2.

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Keywords: carbapenem antibiotics, imipenem, meropenem, solar radiation, photolysis, quantum

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yield

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1. Introduction

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In recent years, considerable attention has been given to the occurrence and fate of various

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pharmaceutical compounds in the aquatic environment, and in particular to antibiotics (Michael et

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al., 2013; Luo et al., 2014; Carvalho and Santos, 2016; Richardson and Kimura, 2016). Antibiotics

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used in human medicine are either excreted (metabolized or not) by patients or dumped down the

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drain, thus reaching municipal wastewater treatment plants (MWWTPs). However, conventional

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MWWTPs are not intentionally designed for the removal of micropollutants, including antibiotics.

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Therefore, human antibiotics are only partially eliminated in MWWTPs, and they are released into

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the receiving aquatic bodies and, eventually, end up in the aquatic environment (Michael et al.,

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2013; Carvalho and Santos, 2016). Indeed, several studies have been performed on the occurrence

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and fate of antibiotics in environmental aqueous matrices, and various classes of antibiotics have

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been detected in low concentrations, typically in the order of ng·L−1 to µg·L−1 (Michael et al., 2013;

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Li, 2014; Luo et al., 2014; Carvalho and Santos, 2016).

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The most adverse effect attributed to the presence of antibiotics in the environment is the

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development of both genes and bacteria which are resistant to antibiotics, thereby reducing the

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therapeutic capacity of antibiotics to prevent and treat diseases (Davies and Davies, 2010; Rizzo et

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al., 2013; Manaia et al., 2016). Numerous national and global agencies and organizations, including

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the European Union, and the World Health Organization, have recognized antibiotic resistance as

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one of the most critical challenges of our time. In particular, MWWTPs have been identified as

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hotspots for the spread of antibiotic resistant bacteria and genes into the environment (Rizzo et al.,

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2013).

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There are several classes of natural, synthetic or semi-synthetic antibiotic compounds, such as β-

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lactams, tetracyclines, macrolides, aminoglycosides, sulfonamides, and quinolones, among others

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(Katzung et al., 2012). Bicyclic β-lactams, i.e. compounds containing the four-membered 2-

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azetidinone ring (or β-lactam ring) fused with another five- or six-membered heterocyclic ring

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containing sulfur, such as penicillins and cephalosporins, were among the first antimicrobial agents

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available for the therapy of various infectious diseases. Moreover, β-lactam antibiotics are currently

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the most used class of antibacterial agents for the treatment of various infectious diseases (Katzung

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et al., 2012).

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However, problems related to the development of resistance to penicillins and cephalosporins led to

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the development of more effective β-lactam antibiotics, such as carbapenems (Papp-Wallace et al.,

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2011). Carbapenems are synthetic antibiotics that contain a β-lactam ring fused with a

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dihydropyrrole ring, as well as a hydroxyethyl and a sulfur-containing side chain. Representative

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carbapenems are imipenem, meropenem, doripenem and ertapenem. Carbapenems possess the

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broadest spectrum of activity and the greatest potency against Gram-positive and Gram-negative

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bacteria (Papp-Wallace et al., 2011; Katzung et al., 2012). As a result, carbapenems are often used

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as “last-line agents” or “antibiotics of last resort” when patients with infections become gravely ill

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or are suspected of harboring resistant bacteria (Papp-Wallace et al., 2011). Unfortunately, recently

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it has been discovered that some Gram-negative bacteria have developed resistance to carbapenems

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(Kumarasamy et al., 2010), which is now spreading throughout the world, and seriously threatens

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the efficiency of this essential class of life-saving antibiotics (Papp-Wallace et al., 2011).

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Organic micropollutants, including pharmaceuticals, undergo several transformation processes in

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the aquatic environment, including biotic (such as biodegradation and bioaccumulation) and abiotic

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(such as hydrolysis, photolysis, oxidation, and adsorption) transformations (Wang and Lin, 2012;

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Schwarzenbach et al., 2017). Photochemical degradation induced by solar radiation allows natural

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attenuation of pharmaceuticals, and many other micropollutants, both in surface waters and in

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wastewaters, thus diminishing their detected concentrations in the aquatic environment (Challis et

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al., 2014; Yan and Song, 2014). In general, it has been found that the effectiveness of

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photodecomposition depends on the integrative effects of photon flux, the structure of the target

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molecule and the composition of the water matrix (Challis et al., 2014; Yan and Song, 2014).

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Several recent studies have been performed on the photolysis of various pharmaceutical compounds

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in the aquatic environment (Challis et al., 2014; Yan and Song, 2014), including β-lactam

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antibiotics (Andreozzi et al., 2004; Jiang et al., 2010; Xu et al., 2011; Carlos et al., 2012; Wang and

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Lin, 2012; Wang and Lin, 2014; Li and Lin, 2015). Moreover, recently, meropenem has been found

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in MWWTPs influents and effluents (Tran et al., 2016a; Tran et al., 2016b), while imipenem was

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found in hospital wastewater effluents (Szekeres et al., 2017).

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Based on the above, the aim of the present work was to study the photolytic degradation under solar

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radiation of two representative carbapenems, namely imipenem and meropenem, in aqueous

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solutions, since solar photodegradation is an important natural attenuation process, both in surface

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waters and in wastewaters. The effect of various parameters was investigated, such as initial

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antibiotics concentration, pH of the solution, the addition of nitrates and dissolved organic matter

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(DOM), water matrix, and level of irradiance. The analysis of the kinetic data obtained from the

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above experiments made it possible to calculate the quantum yield of the photolysis of both

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compounds in aqueous solutions under solar radiation. It should be noted that, to the best of our

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knowledge, the photolytic degradation of carbapenem antibiotics in aqueous solutions under solar

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radiation has not been reported in the literature yet.

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2. Materials and methods

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2.1. Reagents and materials

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Reagents and materials used in the present work are given in the supplementary material. The

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chemical structures of imipenem monohydrate and meropenem trihydrate are shown in Figure 1.

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For the development of the analytical methods, individual stock standard solutions of each

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antibiotic were prepared in methanol at a concentration of 1000 mg·L−1 and stored in amber screw-

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capped glass vials at −20 °C and in the dark, for a maximum period of three weeks.

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2.2. Aqueous matrices and solutions preparation

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Experiments were carried out in three aqueous matrices, namely: (i) ultrapure water (UPW); (ii)

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river water (RW) collected from Andarax River, near to Padules, Almería, Spain; and (iii) simulated 5

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MWWTP effluent (WW). The detailed composition of the WW and the main characteristics of each

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aqueous matrix are given in the supplementary material. For the photolysis experiments, aqueous

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solutions were prepared daily by dissolving the appropriate amounts of both antibiotics in the

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corresponding aqueous matrix. The exact initial concentrations of the resulting aqueous solutions

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were measured by liquid chromatography (vide infra), and they were in the range from a few µg·L−1

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to a few hundred µg·L−1 for both compounds. These concentrations are considered as

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environmentally relevant since several pharmaceutical compounds have been identified in the

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aquatic environment in this range of concentrations (Luo et al., 2014; Richardson and Kimura,

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2016). It should be emphasized that no organic solvents were used for the preparation of the

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aqueous solutions used for the irradiation experiments, to avoid any complications resulting from

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the presence of organic solvents that may quench photogenerated transient species (Challis et al.,

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2014).

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2.3. Photolysis experiments under natural solar radiation

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Photolysis experiments under natural solar radiation were carried out in a solar reactor with

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compound parabolic collectors (CPC), as shown in Figure 2. This pilot plant consists of two twin

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photoreactors, each one made of two Pyrex glass tubes (length: 1.5 m, inner diameter: 45 mm,

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thickness: 2.5 mm) fitted onto the focus of two CPC mirrors, each with a 0.21 m2 illuminated

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surface (i.e. the total illuminated surface of each photoreactor is equal to 0.42 m2). Moreover, the

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illuminated volume of each photoreactor is equal to 4.77 L. It should be emphasized that CPC are

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non-concentrating devices, that is, they are one-sun equivalent, and they are employed to distribute

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solar radiation on the surface of the tubes evenly. Modules are facing south and tilted at 37° from

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the horizontal (local latitude). Aqueous solutions are driven by a centrifugal pump (Pan World NH-

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40PX model) and maintained in recirculation. The Crison 5335 sensors were used for pH

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monitoring, while incident UVA irradiance (in W·m−2) was measured employing a global UV

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radiometer (Delta Ohm, LP UVA 02 AV) with a spectral response range from 327 nm to 384 nm,

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mounted on a platform tilted at 37º. The UVA irradiance measurements were acquired throughout

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the entire experiments using a data acquisition card (LabJack U12) connected to a computer.

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Moreover, the incident solar spectral irradiance was measured employing a spectroradiometer

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(AvaSpec-ULS2048-2 purchased from Avantes). Chemical actinometry was used for the calculation

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of the effective path length of radiation of the photoreactor, employing p-nitroanisole and pyridine

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(Dulin and Mill, 1982; Laszakovits et al., 2017), as described in detail in the supplementary

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material (Text S5).

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Experiments under natural solar radiation were carried out during September 2014 from

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approximately 10 a.m. to 3 p.m. in sunny days and under clear sky conditions. As expected, the

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incident UVA irradiance varied almost every minute and from day to day. Consequently, for each

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experimental run, the average UVA irradiance was calculated by integrating the values recorded

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over the time of the experiments. In all cases where the effect of various parameters on the

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photolytic degradation of both compounds was studied, experiments were performed either in

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parallel or on successive days at about the same time. In all cases, it was found that the average

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UVA irradiance of each experimental run did not differ significantly from one experiment to the

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other.

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In a typical experimental run, 7 L of the corresponding water matrix containing the target

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compounds at the desired initial concentration were loaded into the recirculation tanks of the CPC

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reactor, while the CPC system was covered and kept in the dark. After 15 minutes of recirculation

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in the dark for achieving perfect mixing conditions, the CPC system was uncovered and at the same

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time started the acquisition of UVA irradiance data. Samples periodically taken were analyzed for

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residual imipenem and meropenem concentration. Control experiments were performed in the dark

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to assess the possible hydrolysis of the target compounds by stirring aqueous solutions of both

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compounds in the aqueous matrices for more than 5 hours at room temperature. In all cases, it was

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found that the hydrolysis of the target compounds was rather negligible after 5 hours stirring in the

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dark.

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2.4. Photolysis experiments under simulated solar radiation

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Additional photolysis experiments by varying incident irradiance at predetermined levels were

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performed employing a SunTest CPS+ solar simulator purchased from Atlas. This solar simulator

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employs a Xenon Lamp with an emission range from 250 to 765 W·m−2 (complete emission

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spectrum) and an emission wavelength from 300 to 800 nm. Experiments were carried out in a

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jacketed stirred tank reactor placed inside the SunTest CPS+ solar box, which allowed water

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temperature to be controlled, as shown in Figure 3. The volume of this reactor was 0.500 L and the

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liquid depth was 7.0 cm, while the surface area exposed to radiation was 7.14×10−3 m2. UVA

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irradiance inside the solar box was measured with a PMA2100 radiometer, employing a UVA

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sensor, with spectral response in the 320 to 400 nm range, purchased from Solar Light Company.

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The temperature of the reaction mixture was kept constant at 25.0 ± 0.2 °C using a thermostatic

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bath (thermo Scientific NESLAB RTE7).

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2.5. Analytical measurements

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The concentration of both micro-contaminants in UPW was monitored by HPLC-UV, employing a

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C18 (1.8 µm, 4.6 × 50 mm) analytical column, and the injection volume was 100 µL. For the

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determination of the analytes in more complex samples (i.e., in RW and WW) or at lower initial

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concentrations, a more sensitive LC-MS system was employed, consisting of a HPLC-QTRAP-MS

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system. The detailed description of the analytical methods is given in the supplementary material

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(Text S2).

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3. Results and discussion

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3.1. Optimization and validation of the LC-MS/MS analytical method

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A careful optimization of the LC-MS/MS parameters was performed, to obtain appropriate

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selectivity and sensibility for the analysis of target compounds in WW and RW. As both target 8

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analytes are amphoteric and polar, the pH of the mobile phases represented a critical factor in their

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chromatographic analysis. Different mobile phases and pH values were tested. The best results

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regarding intensity, resolution and peak shape were found with water (0.001% v/v formic acid) and

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methanol. Moreover, the method was validated in terms of linearity, sensitivity, expressed as limit

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of detection (LOD) and limit of quantification (LOQ), matrix effects and precision. LOQs obtained

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were 2-3 orders of magnitude lower than that achieved by HPLC-UV. Matrix matched calibration

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was selected as calibration procedure to minimize the signal suppression effects observed, mainly in

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the case of imipenem in WW. The detailed description of the optimization and validation of the LC-

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MS/MS analytical method is given in the supplementary material (Text S3).

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3.2.1. Molar absorption coefficients of imipenem and meropenem

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The molar absorption coefficient, ε(λ) (in L·mol–1·cm–1), expresses the ability of a compound to

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absorb radiation at a specific wavelength λ. Therefore, the molar absorption coefficients of

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imipenem and meropenem were measured according to the Beer-Lambert law, in the UV region of

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the electromagnetic spectrum, in aqueous solutions and at pH values from 6 to 8 adjusted by 20

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mmol·L–1 phosphate buffers, and the results are shown in Figure 4. Moreover, Figure 4 also shows

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the incident solar spectral irradiance, as it was measured by the spectroradiometer on the 14th of

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June 2017 at 14:14 local time, in Almeria, Spain. As seen, the absorption spectrum of both

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compounds extends to the UVA region of the electromagnetic spectrum. More specifically, they

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both show a rather strong absorption band centered at about 300 nm, as well as a relatively strong

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continuous absorption band below approximately 240 nm.

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Also, as seen in Figure 4, the pH of the solution in the range from 6 to 8 has a rather marginal effect

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on the absorption spectrum of both compounds. More specifically, as seen in Figure 4a, imipenem

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shows a rather strong absorption band centered at 299 nm, and the molar absorption coefficient

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slightly decreases from 9300 to 9000 L·mol–1·cm–1 by increasing the pH of the solution from 6 to 8.

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On the other hand, meropenem shows a small bathochromic shift (i.e. red shift) and a hyperchromic

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effect, and the wavelength of maximum absorption increases from 297 to 301 nm by increasing the

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pH of the solution from 6 to 8, while the molar absorption coefficient slightly increases from 10700

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to 11000 L·mol–1·cm–1. These results can be explained if we take into account the ionization

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constants of both compounds. As seen in Figure 1, both compounds have two pKa values. The first

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one is at about 4.3 for both compounds and corresponds to the acidic hydrogen of the carboxyl

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group of both molecules, while the second one is at about 10.6 and 8.3 for imipenem and

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meropenem, respectively, and corresponds to an additional acidic hydrogen. Based on these pKa

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values, the fractional composition of the various molecular forms of both compounds was

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calculated as a function of the pH of the solution, and the results are shown in Figure S1 and S2 in

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the supplementary material. As seen, in the pH range from 6 to 8 imipenem exists predominately

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(i.e., more than 98%) in the single ionized form (i.e., HImi−), while in the pH range from 6 to 7

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meropenem exists predominately (i.e., more than 95%) in the single ionized form (i.e., HMer−), and

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at pH 8 it exists at about 67% in the single ionized form and 33% in the double ionized form (i.e.,

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Mer2−). Consequently, by changing solution pH from 6 to 8, no significant differences were

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observed in the absorption spectra of imipenem as the ionization of the molecule remains practically

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constant, while for meropenem, the two ionized forms (i.e., HMer− and Mer2−) are likely to have

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almost similar absorption spectra, and hence relatively small differences were also observed in its

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absorption spectra.

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Based on the above absorption spectra of both compounds and on the fact that the solar radiation

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reaching the surface of the Earth contains UV radiation in the UVA and UVB region of the

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electromagnetic spectrum (i.e. as can be seen in Figure 4, for practical purposes, radiation with

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wavelengths higher than 300 nm (Challis et al., 2014)), it is concluded that direct photolysis of both

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compounds under solar irradiation is possible. In addition, the average values of the molar

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absorption coefficient for each compound, εaver, were calculated for the λ interval from 300 to 350

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nm (i.e. to the maximum wavelength in which they absorb in the UVA region of the

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electromagnetic spectrum) using the following equation 1: λmax

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∑ ε( λ)∆λ

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where λmax is the maximum wavelength in which they absorb in the UVA region of the

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electromagnetic spectrum (i.e. 350 nm), and ∆λ = 1 nm is the wavelength interval used in the

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spectrophotometer for the measurement of ε(λ). The average values of the molar absorption

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coefficient of both compounds are given in Table S5 in the supplementary material, and they will be

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used for the calculation of the quantum yield of their photolysis under solar radiation (vide infra).

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3.2.2. Direct photolysis under natural solar radiation

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In an initial set of direct photolysis experiments, aqueous solutions of imipenem and meropenem in

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UPW at inherent solution pH (i.e. approximately 6.6) and in various initial concentrations were

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irradiated in the CPC system under natural solar radiation. It should be noted that the pH of the

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solution was left uncontrolled and it was practically stable during the course of the reaction.

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Experiments were conducted by varying the initial concentration in the range from 0.0224 µmol·L−1

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(i.e. 7.12 µg·L−1) to 1.016 µmol·L−1 (i.e. 322.4 µg·L−1) for imipenem, and from 0.0136 µmol·L−1

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(i.e. 5.96 µg·L−1) to 0.7367 µmol·L−1 (i.e. 322.3 µg·L−1) for meropenem, and the results are shown

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in Figure 5a and 5b for imipenem and meropenem, respectively.

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As seen, both compounds were readily degraded photochemically under natural solar radiation,

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even at the relatively higher concentrations employed in the present work. More specifically, the

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photodegradation of meropenem was slightly higher (i.e. approximately 93%) than the

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corresponding photodegradation of imipenem (i.e. approximately 88%) after 300 min irradiation

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under natural solar radiation. It should be noted that control experiments were performed in the

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dark, and it was found that both compounds were rather stable after 5 hours stirring of their aqueous

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solutions in the dark. Therefore, the above results indicate that the observed degradation of both 11

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compounds can be attributed to their direct photolysis under natural solar radiation. The above

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results show that photodegradation under natural solar radiation is a factor that may contribute

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significantly to the fate of these compounds when they are released to the aquatic environment.

277 3.2.3. Photodegradation kinetics

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As seen in Figure 5a and 5b, the rate of the photochemical degradation of both compounds under

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natural solar radiation remained practically constant, within the limits of experimental error, by

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varying the initial concentration from 0.0224 µmol·L−1 (i.e. 7.12 µg·L−1) to 1.016 µmol·L−1 (i.e.

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322.4 µg·L−1) for imipenem, and from 0.0136 µmol·L−1 (i.e. 5.96 µg·L−1) to 0.7367 µmol·L−1 (i.e.

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322.3 µg·L−1) for meropenem. These results indicate that, at the experimental conditions employed

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in the present work, the direct photolysis of both compounds in aqueous solutions in UPW under

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natural solar radiation follows first-order kinetics.

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It has been well documented in the literature that the rate of the direct photochemical degradation of

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organic micropollutants in the aqueous phase at wavelength λ, r(λ) (in mol·L–1·s–1), is given by the

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following equation (Zepp, 1978; Leifer, 1988; Beltrán et al., 1995):

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 dC  0 −[α ( λ )+ε ( λ ) C ]l r (λ ) =  − }{ε ( λ)C/[α( λ) + ε ( λ)C ]}Φ(λ )  = En,p,o (S/V ){1 − 10  dt  λ

(2)

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where C is the reactant concentration (in mol·L–1); En0,p,o is the incident photon fluence rate on a

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chemical amount basis, defined as the total number of moles of photons (i.e. einsteins) incident

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from all directions onto a small sphere, divided by the cross-sectional area of the sphere and per

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time interval (in mol·m−2·s−1 or einstein·m−2·s−1) (Bolton and Stefan, 2002; Braslavsky, 2007); S is

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the surface area of the photochemical reactor exposed to radiation (in m2), and V is the volume of

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the photochemical reactor (in L); α(λ) is the attenuation coefficient of the medium (i.e. solvent) at

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wavelength λ (in cm–1); ε(λ) is the molar absorption coefficient of the reactant at wavelength λ (in

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L·mol–1·cm–1); l is the effective radiation absorption path length of the photochemical reactor (in

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cm); and Φ(λ) is the quantum yield of the direct photochemical degradation of the micro-pollutant

299

at wavelength λ (dimensionless or mol·einstein−1).

300

However, in dilute aqueous solutions when radiation is weakly absorbed by the system (i.e. when

301

the absorbance is lower than 0.04), equation 2 simplifies to (Zepp, 1978; Leifer, 1988; Beltrán et al.,

302

1995):

 dC  0 r (λ ) =  −  = 2.303En,p,o ( S/V )lε ( λ)Φ(λ )C  dt  λ

303 304

Then, if we set:

k1 (λ ) = 2.303En0,p,o ( S/V )lε( λ)Φ(λ )

(4)

equation 3 can be written as:

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(3)

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305

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298

 dC  −  = k1 (λ )C  dt  λ

307

(5)

which corresponds to a first-order kinetic rate law. Therefore, in dilute aqueous solutions where the

309

reactant weakly absorbs radiation, the direct photolysis of the reactant follows first-order kinetics

310

(Zepp, 1978; Leifer, 1988; Beltrán et al., 1995). Equation 5 after integration at the boundary

311

conditions yields the well-known expression of the first-order rate law:

ln

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312

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308

C = −k1 (λ )t C0

(6)

where C0 is the initial concentration of the reactant (in mol·L–1) and k1(λ) = 2.303 En0,p,o

314

(S/V)lε(λ)Φ(λ) is the first-order rate constant of the direct photolysis of the reactant (in s–1).

315

The insets in Figure 5 show the plot of –ln(C/C0) versus time for both compounds, while Table 1

316

shows the corresponding values of the first-order rate constants and the correlation coefficients, R2.

317

As seen in Table 1, under the experimental conditions employed in the present work, the first-order

318

rate constants for the direct photolysis of imipenem in UPW under natural solar radiation were in

319

the range from 1.12×10−4 to 1.17×10−4 s–1, while the corresponding values for meropenem were in

320

the range from 1.42×10−4 to 1.60×10−4 s–1. Moreover, the corresponding correlation coefficients, R2,

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were always higher than 0.98, thus showing the relative good fitting of the experimental results to

322

the first-order kinetic rate law for both compounds. In addition, as seen in Table 1, for each

323

compound, the first-order rate constants obtained from the experiments conducted at various initial

324

concentrations have minimal variance, and the average values of the first-order rate constant were

325

1.15×10−4 and 1.52×10−4 s–1 for imipenem and meropenem, respectively, while percent relative

326

standard deviation (%RSD) was 2.3% and 6.0%, for imipenem and meropenem, respectively.

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327 3.2.4. Determination of the quantum yield

329

The first-order rate constants of both compounds determined in the previous section mainly depend

330

on the experimental conditions employed in the photodegradation experiments, such as the

331

geometry of the photochemical reactor and the incident solar irradiance on the photochemical

332

reactor. Therefore, a more fundamental photochemical parameter is needed, such as quantum yield,

333

to describe more adequately the photochemical fate of the compounds under investigation (Challis

334

et al., 2014). The quantum yield at wavelength λ, Φ(λ), determines the efficiency of a

335

photochemical reaction and is defined as the number of moles of a compound that are transformed

336

per number of moles of photons (i.e. einsteins) that are absorbed by the compound (Braslavsky,

337

2007). In the present work, the quantum yield of the direct photolysis of both compounds in

338

aqueous solutions in UPW under natural solar radiation was determined using equation 4. More

339

specifically, by solving equation 4 with respect to the quantum yield, equation 4 can be written as

340

follows (Zepp, 1978):

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328

Φ(λ ) =

k (λ ) 2.303E ( S/V )lε ( λ) 1 0 n ,p,o

(7)

342

It should be noted that in the above equation, En0,p,o is the incident photon fluence rate in the

343

wavelength range from 300 to 350 nm, while the molar absorption coefficient for each compound is

344

the average value calculated for the λ interval from 300 to 350 nm using equation 1 and given in

345

Table S5 in the supplementary material. For each experimental run, En0,p,o was calculated using the 14

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average incident UVA irradiance measured by the radiometer as described in detail in Text S4 in

347

the supplementary material. Moreover, the average effective path length of radiation of the CPC

348

photochemical reactors was equal to l = 0.93 cm, as described in detail in Text S5 in the

349

supplementary material.

350

By substituting these values into the above equation 7, as well as the values of the first-order rate

351

constants determined in the previous section, the quantum yields of the direct photolysis of both

352

compounds were determined, and the values are shown in Table 1. As seen, the average values of

353

the quantum yield were 5.5×10−3 and 6.5×10−3 mol·einsten−1 for imipenem and meropenem,

354

respectively, while the corresponding %RSD was 1.0% and 7.1%. These values of the quantum

355

yield are rather moderate to high since the quantum yields of the direct photolysis under solar

356

radiation of various pharmaceutical compounds, including β-lactam antibiotics, have been typically

357

reported to be in the order from 10−6 to 10−2 mol·einstein−1 (Carlos et al., 2012; Wang and Lin,

358

2012; Challis et al., 2014). These results confirm that indeed photochemical degradation of both

359

compounds is an important factor determining their fate in the aquatic environment. It should be

360

emphasized that quantum yields are fundamental photochemical parameters of the studied

361

antibiotics, and they were obtained under controlled conditions in a CPC photoreactor to quantify

362

radiation absorption and change in target compound concentration better. Therefore, results are

363

applicable under real environmental conditions whenever radiation absorption can be evaluated,

364

such as in wastewaters and in surface waters, including river waters.

365

At this point, it should be noted that for the calculation of the quantum yield, it would be more

366

accurate to measure the incident solar spectral irradiance for every experimental run. However, for

367

routine measurements, incident solar spectral irradiance cannot be measured very easily and

368

accurately, since the whole spectroradiometer system (and more importantly, the fiber optic cable)

369

is very sensitive to positioning, and light displacements and may give different readings if not

370

properly mounted. On the other hand, the Delta Ohm radiometer measuring global irradiance in the

371

wavelength region from 327 nm to 384 nm is ideal for routine measurements, since it can be

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15

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mounted on the platform of the CPC photoreactor very easily and it gives very accurate and

373

reproducible readings. Therefore, incident UVA irradiance measured by the Delta Ohm radiometer

374

was correlated with the incident solar spectral irradiance measured by the spectroradiometer for the

375

λ interval from 300 to 350 nm, as described in detail in the supplementary material (Text S4), and

376

then the quantum yield was calculated, as explained in the previous paragraphs. This method,

377

although not entirely accurate, it gives a reasonably representative value of the quantum yield.

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378 3.2.5. Effect of solution pH

380

In further photolysis experiments under natural solar radiation, the effect of solution pH was

381

investigated, as it can affect the photolysis process changing the molecule configuration and,

382

consequently, the light absorbing properties of the compounds (Challis et al., 2014). Experiments

383

were conducted at two pH values, namely, 6 and 8, adjusted by 5 mmol·L–1 phosphate buffers,

384

while the initial concentration of imipenem and meropenem was 1.575 µmol·L−1 (i.e. 500 µg·L−1)

385

and 0.114 µmol·L−1 (i.e. 50 µg·L−1), respectively. This tight range of pH values was selected

386

because natural waters do not often present values outside these limits. Experiments were

387

performed in the CPC system, and the results are shown in Figure S10 in the supplementary

388

material. As seen, by varying solution pH from 6.0 to 8.0 the first-order rate constants of the direct

389

photolysis of both compounds under natural solar radiation remained practically unchanged, within

390

the limits of experimental error.

391

Then, the quantum yield was calculated using the approach discussed in detail in the previous

392

section. It was found that the values of the quantum yield obtained from the experiments carried out

393

at pH 6 and 8 did not show any noticeable differences to the corresponding values obtained from

394

the direct photolysis of both compounds in UPW. More specifically, for imipenem, the quantum

395

yield was 6.2×10−3 and 6.4×10−3 mol·einsten−1 for the experiments carried out at pH 6 and 8,

396

respectively, while the corresponding average value for the experiments performed in UPW was

397

5.5×10−3 mol·einsten−1 (Table 1). On the other hand, the quantum yield for the direct photolysis of

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meropenem at pH 6 and 8 was 5.1×10−3 and 4.1×10−3 mol·einsten−1, respectively, while in UPW the

399

corresponding average value was 6.5×10−3 mol·einsten−1 (Table 1). These results indicate that

400

solution pH has a rather marginal to negligible effect on the direct photolysis of both compounds

401

under natural solar radiation, in the range of values studied in the present work. These results can be

402

explained by taking into account the discussion mentioned in section 3.2.1. By changing solution

403

pH from 6 to 8, the ionization of imipenem is practically unaffected, and the same is true for

404

meropenem in the pH range from 6 to 7. Also, at pH 8 the two ionized forms of meropenem (i.e.,

405

HMer− and Mer2−) are probably photolyzed with the same rate, and therefore no significant changes

406

were observed in the photolysis of the compound in the pH range from 6 to 8.

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407 3.2.6. Effect of nitrates and humic acids

409

In additional photolysis experiments under natural solar radiation, the effect of several constituents

410

of the aqueous matrix was investigated, such as nitrates and DOM. Among natural water

411

constituents that are radical producers and scavengers, nitrates have been recognized to have a

412

significant effect on photolysis. Indeed, several authors have found that the presence of nitrates

413

accelerates photolysis removal rates of many organic pollutants, due to nitrate excitation under UV

414

irradiation, that results in the formation of various reactive species, including hydroxyl radicals and

415

nitrogen reactive species (Gligorovski et al., 2015; Vione et al., 2014).

416

Based on the above, further photolysis experiments of both compounds were performed in UPW in

417

the presence of NO3– at an initial concentration of 0.484 mmol·L–1 (i.e. 30.0 mg·L–1). It should be

418

noted that nitrate concentration in natural waters is highly dependent on geographic location and

419

human agriculture activity and has been reported to lie in the range from 10−5 to 10−3 mol·L–1 (Mao

420

et al., 2011). Two photochemical experiments under natural solar radiation were run in parallel in

421

the CPC system in UPW in the absence and the presence of 0.484 mmol·L–1 (i.e. 30.0 mg·L–1) NO3–

422

, while the initial concentration of imipenem and meropenem was 1.350 µmol·L−1 (i.e. 428.6

423

µg·L−1) and 0.127 µmol·L−1 (i.e. 55.5 µg·L−1), respectively, and the results are shown in Figure S11

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17

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in the supplementary material. As seen, negligible differences were found for the experimental runs

425

carried out with and without nitrates for both carbapenem antibiotics. More specifically, for

426

imipenem, the first-order rate constant was 9.67×10−5 s−1 for the experiment carried out in the

427

presence of nitrates, while the corresponding value for the parallel experiment performed in UPW

428

was 1.28×10−4 s−1. In addition, the first-order rate constant for the photolysis of meropenem in the

429

presence of nitrates was 1.94×10−4 s−1, while in UPW the corresponding value was 1.76×10−4 s−1.

430

The above results show that the presence of nitrate ions does not have any significant impact on the

431

direct photolysis of both compounds in UPW under natural solar radiation.

432

In addition, solar irradiation of natural waters containing DOM results in the photochemical

433

generation of various transient species, such as hydroxyl radicals, DOM in its triplet state, and

434

singlet oxygen, among others (Vione et al., 2015). These photogenerated transient species usually

435

play a significant role in the photochemical transformations of organic pollutants in surface waters,

436

through various indirect photochemical degradation pathways (Challis et al., 2014; Vione et al.,

437

2014; Yan and Song, 2014). However, at the same time, DOM is a very efficient hydroxyl radical

438

scavenger, and it absorbs solar radiation very efficiently, thus acting as a radiation filter in natural

439

waters. Therefore, DOM can have either a positive or a negative impact on the photochemical

440

transformation of various organic pollutants in surface waters, depending on its chemical structure

441

and its concentration.

442

In the present study, further photolysis experiments were carried out in UPW in the presence of HA

443

at an initial concentration of 5.0 mg·L–1. More specifically, two photochemical experiments under

444

natural solar radiation were run in parallel in the CPC system in UPW in the absence and the

445

presence of 5.0 mg·L–1 HA, while the initial concentration of imipenem and meropenem was 1.439

446

µmol·L−1 (i.e. 456.8 µg·L−1) and 0.122 µmol·L−1 (i.e. 53.5 µg·L−1), respectively, and the results are

447

shown in Figure S12 in the supplementary material. As seen, no significant differences were

448

observed between the experiments carried out with and without HA. More specifically, the first-

449

order rate constant of the photodegradation of imipenem was 1.15×10−4 s−1 for the experiment

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18

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conducted in the presence of HA, while the corresponding value for the parallel experiment

451

performed in UPW was 9.69×10−5 s−1. In addition, the first-order rate constant for the

452

photodegradation of meropenem in the presence of HA was 9.22×10−5 s−1, while in UPW the

453

corresponding value was 1.27×10−4 s−1.

454

The above results show that the presence of HA does not have any significant impact on the direct

455

photolysis of both compounds in aqueous solutions under natural solar radiation. Overall, from the

456

experiments conducted in the presence of nitrates and HA, it can be concluded that the elimination

457

of both compounds in aqueous solutions under natural solar radiation takes place preferably, or

458

almost exclusively, through direct photochemical degradation pathways, rather than through

459

indirect photochemical processes (Challis et al., 2014; Vione et al., 2014; Yan and Song, 2014).

460

However, it should be noted that in natural surface waters, where the water column depth might be

461

in the order of a few meters, thus resulting in reduced penetration depth of UV radiation, direct

462

photolysis can be strongly inhibited by CDOM, as well as by other dissolved organic compounds

463

and ions. In such cases, indirect photochemical reaction pathways triggered by various water

464

components may play a significant role.

465

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450

3.2.7. Effect of water matrix

467

Additional photolysis experiments of imipenem and meropenem were carried out in two aqueous

468

matrices, namely in RW and WW, and the results were compared with those obtained in UPW.

469

More specifically, photochemical experiments under natural solar radiation were run in the CPC

470

system at an initial concentration of 0.158 µmol·L−1 (i.e. 50.0 µg·L−1) and 0.114 µmol·L−1 (i.e. 50.0

471

µg·L−1) for imipenem and meropenem, respectively, and the results are shown in Figure S13 in the

472

supplementary material. As seen, no significant differences were observed for the experiments

473

carried out either in UPW or in RW and WW.

474

More specifically, for imipenem, the first-order rate constant was 6.55×10−5 s−1 for the experiment

475

carried out in UPW, while the corresponding value for the experiments conducted in RW and WW

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were 7.28×10−5 and 8.72×10−5 s−1, respectively. In addition, the first-order rate constant for the

477

direct photolysis of meropenem in UPW was 7.31×10−5 s−1, while in RW and WW the

478

corresponding values were 6.33×10−5 and 6.98×10−5 s−1, respectively. Overall, the above results

479

show that the presence of various organic and inorganic constituents in the aqueous matrices do not

480

contribute significantly, either positively or negatively, to the photochemical degradation of

481

imipenem and meropenem under natural solar radiation. However, once again it should be noted

482

that when the water column depth is in the order of a few meters, several indirect photochemical

483

reaction pathways initiated by various dissolved constituents in the aqueous phase may play a

484

significant role.

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476

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485 3.3. Photolysis under simulated solar radiation

487

In a final set of photolysis experiments, the effect of UVA irradiance on the photodegradation of

488

imipenem and meropenem was investigated in aqueous solutions in UPW and under simulated solar

489

radiation. Experiments were carried out in the SunTest CPS+ solar simulator so that the irradiance

490

could be controlled and at the same time to keep the temperature constant at about 25 ºC. The

491

irradiance levels tested were in the range from 28 to 50 W·m−2, because the UVA irradiance at mid-

492

latitude is in the order of a few tens W·m−2 (Cabrera Reina et al., 2014; Vione at al. 2014).

493

Moreover, the initial concentration of imipenem and meropenem was 1.281 µmol·L−1 (i.e. 406.4

494

µg·L−1) and 0.101 µmol·L−1 (i.e. 44.0 µg·L−1), respectively, and the results are shown in Figure 6.

495

As seen, the first-order rate constants of the direct photolysis of both compounds increased by

496

increasing UVA irradiance, thus indicating that higher degradation rates were obtained for both

497

compounds when increasing incident solar irradiance.

498

Moreover, the quantum yield for each experimental run and each compound was calculated using

499

the approach discussed in detail in section 3.2.4, and the results are shown in Figure 7. As seen, for

500

both compounds, the photodegradation quantum yield remained practically constant by changing

501

the incident solar irradiance from 28 to 50 W·m−2. More specifically, the quantum yield was in the

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20

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range from 1.2×10−3 to 1.3×10−3 mol·einsten−1 and from 1.4×10−3 to 1.6×10−3 mol·einsten−1, for

503

imipenem and meropenem, respectively, in the above range of irradiance values. It should be noted

504

that these values of the quantum yield calculated under simulated solar radiation were of the same

505

order of magnitude as the corresponding values calculated under natural solar radiation (Table 1),

506

but slightly smaller, i.e. approximately 4 times smaller for both compounds. Such a discrepancy can

507

be characterized as rather low to moderate (Challis et al., 2014). Similar discrepancies have been

508

reported in the literature and have been attributed to several factors, including small differences in

509

the wavelength distribution between natural and simulated (i.e. artificial) solar radiation (Challis et

510

al., 2014). Another reason for these discrepancies is linked with the calculations that were made in

511

the present work by considering the average values of the molar absorption coefficients of the target

512

pollutants.

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502

513 Conclusions and outlook

515

The conclusions drawn from the present work can be summarized as follows:

516

•

TE D

514

The photochemical degradation under solar radiation of imipenem and meropenem in aqueous solutions at environmentally relevant concentrations follows first-order kinetics.

518

The quantum yields of their direct photolysis are in the order of 10−3 mol·einstein−1. These

519

relatively high values of the quantum yields show that the photochemical degradation of

520

both compounds is a very critical factor which contributes significantly to their fate in the

521

aquatic environment. •

AC C

522

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517

The photochemical degradation of both compounds in aqueous solutions under solar

523

radiation is not influenced significantly by various parameters, such as solution pH, and the

524

presence of nitrates, humic acids, as well as various organic and inorganic constituents of

525

the aqueous matrix.

526 527

•

The photochemical degradation of both compounds in aqueous solutions under solar radiation takes place preferably, or almost exclusively, through direct photochemical 21

ACCEPTED MANUSCRIPT

528

degradation pathways, whereas indirect photolysis reactions due to various photogenerated

529

transient chemical species appear to be rather insignificant.

530

•

Upon irradiation of both compounds in aqueous solutions under simulated solar radiation at various levels of incident solar irradiance in the range from 28 to 50 W·m−2, the

532

photodegradation quantum yield remained practically constant.

533

•

RI PT

531

The quantum yields of the direct photolysis of both compounds under simulated solar radiation were of the same order of magnitude as the corresponding values obtained under

535

natural solar radiation, but approximately 4 times smaller. Such a discrepancy can be

536

attributed to small differences in the wavelength distribution between natural and artificial

537

solar radiation.

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534

It should be noted that further work is under way to identify the phototransformation products of

539

both compounds, employing liquid chromatography coupled with high-resolution mass

540

spectrometry. Moreover, since it has been reported that photochemical degradation of various β-

541

lactam antibiotics results in higher toxicity (Wang and Lin, 2012; Wang and Lin, 2014; Li and Lin,

542

2015), efforts will be made to assess the toxicity of the irradiated solutions of imipenem and

543

meropenem.

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Acknowledgements

546

The authors wish to thank the SFERA-II Programme for financial support. A.C. Reina wishes to

547

thank FONDAP/15110019. The authors wish to thank Dr. José Luis García Sánchez from the

548

Department of Chemical Engineering of the University of Almería and CIESOL for his invaluable

549

help on the measurements of the incident solar spectral irradiance. Moreover, the authors would like

550

to thank M.Sc. Virginia Papadosifou from TUC for performing the chemical actinometry

551

experiments during her stay in CIESOL in June 2017.

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Captions of Figures

Figure 1. Chemical structures of imipenem monohydrate and meropenem trihydrate. The ionization constants, pKa, of both compounds were obtained from the Advanced Chemistry Development (ACD/Labs) software (ACD/Labs, I-Lab 2.0, https://ilab.acdlabs.com/iLab2/). Acidic hydrogen

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atoms are marked in red. Figure 2. Experimental setup under natural solar radiation: outdoors solar detoxification pilot plant with compound parabolic collectors (CPC).

Figure 3. Experimental setup employing the SunTest CPS+ solar simulator (solar box).

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Figure 4. Molar absorption coefficient, ε (in L·mol−1·cm−1), of (a) imipenem and (b) meropenem in

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UPW and at pH values from 6 to 8 adjusted by 20 mmol·L–1 phosphate buffers. Right axis: solar spectral irradiance (in µW·cm−2·nm−1) recorded on the 14th of June 2017 in Almeria, Spain, at 14:14 local time under clear sky conditions.

Figure 5. Direct photolysis of (a) imipenem and (b) meropenem in UPW under natural solar

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radiation, at inherent solution pH (i.e. approximately 6.6) and in various initial concentrations. Experiments were conducted in the CPC system. The inset figures show the plot of –ln(C/C0) versus time for each experimental run.

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Figure 6. Effect of irradiance on the direct photolysis of (a) imipenem and (b) meropenem in UPW at inherent solution pH under simulated solar radiation. Experimental conditions: [imipenem]0 =

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1.281 µmol·L−1 (i.e. 406.4 µg·L−1); [meropenem]0 = 0.101 µmol·L−1 (i.e. 44.0 µg·L−1). Experiments were conducted at the SunTest CPS+ solar simulator. The inset figures show the plot of –ln(C/C0) versus time for each experimental run. Figure 7. Effect of irradiance on the quantum yield of the photodegradation of imipenem and meropenem in UPW at inherent solution pH under simulated solar radiation. Experimental conditions: [imipenem]0 = 1.281 µmol·L−1 (i.e. 406.4 µg·L−1); [meropenem]0 = 0.101 µmol·L−1 (i.e. 44.0 µg·L−1). Experiments were conducted at the SunTest CPS+ solar simulator. Dashed lines are plotted to guide the eye.

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Table 1. First-order rate constants, k1, coefficients of determination, R2, and quantum yields, Φ, of the direct photolysis of imipenem and meropenem in UPW at inherent solution pH (i.e. approximately 6.6) under natural solar radiation at various initial concentrations, C0. Φ×103 (mol·einsten−1) 6.1 6.4 7.0 6.5

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Φ×10 C0 (mol·einsten−1) (µmol·L−1) 5.6 0.0136 5.5 0.134 5.5 0.737 5.5 average % relative 1.0% standard deviation

Meropenem k1×104 R2 −1 (s ) 1.42 0.980 1.54 0.989 1.60 0.999 1.52 6.0%

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C0 (µmol·L−1) 0.0224 0.170 1.016 average % relative standard deviation

Imipenem k1×104 R2 −1 (s ) 1.16 0.992 1.17 0.989 1.12 0.998 1.15

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OH

NH HH N

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HO pKa,1 = 4.29±0.40

Imipenem monohydrate

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H2O

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S O

HO

N

3H2O

O NH pKa,2 = 8.31±0.10

pKa,1 = 4.27±0.60

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Photo-degradation determines the fate of the target pollutants in the environment

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Degradation in aqueous matrices under solar radiation follows first-order kinetics

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Decomposition takes place preferably through direct photo-degradation pathways

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The constituents of the aqueous matrix have a minimal effect on the photolysis

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