Photodegradation of metoprolol in the presence of aquatic fulvic acids. Kinetic studies, degradation pathways and role of singlet oxygen, OH radicals and fulvic acids triplet states

Journal Pre-proof Photodegradation of metoprolol in the presence of aquatic fulvic acids. Kinetic studies, degradation pathways and role of singlet oxygen, OH radicals and fulvic acids triplet states Olga M.S. Filipe, Eduarda B.H. Santos, Marta Otero, Elsa A.C. Gonc¸alves, M. Grac¸a P.M.S. Neves

PII:

S0304-3894(19)31477-3

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121523

Reference:

HAZMAT 121523

To appear in:

Journal of Hazardous Materials

Received Date:

1 June 2019

Revised Date:

5 October 2019

Accepted Date:

21 October 2019

Please cite this article as: Filipe OMS, Santos EBH, Otero M, Gonc¸alves EAC, Neves MGPMS, Photodegradation of metoprolol in the presence of aquatic fulvic acids. Kinetic studies, degradation pathways and role of singlet oxygen, OH radicals and fulvic acids triplet states, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121523

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Photodegradation of metoprolol in the presence of aquatic fulvic acids. Kinetic studies, degradation pathways and role of singlet oxygen, OH radicals and fulvic acids triplet states

Olga M.S. Filipe(a)*, Eduarda B.H. Santos(b,c)*, Marta Otero(b,d), Elsa A.C. Gonçalves(c), M. Graça P. M. S. Neves(e)

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(a) CERNAS – Research Centre for Natural Resources, Environment and Society, College of Agriculture, Polytechnic Institute of Coimbra, Bencanta, 3045-601 Coimbra, Portugal

(b) CESAM - Centre for Environmental and Marine Studies, University of Aveiro, 3810-193

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Aveiro, Portugal

(c) Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

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(d) Department of Environment and Planning, University of Aveiro, 3810-193 Aveiro,

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Portugal

(e) QOPNA & LAQV-REQUIMTE and Department of Chemistry, University of Aveiro,

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3810-193 Aveiro, Portugal

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*corresponding authors

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Graphical abstract

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Highlights

Metoprolol indirect photodegradation occurs in presence of aquatic fulvic acids, FA



The photosensitizer effect of aquatic FA is mainly attributed to OH



Involvement of other reactive species, 1O2 and/or some 3FA*was assessed



Formation mechanisms were proposed for the several metoprolol photoproducts



Relation between reactive species and photoproducts was established

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

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Abstract

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Metoprolol is a pharmaceutical used for the treatment of cardiovascular diseases and disorders, whose frequent detection in surface waters raises concern. Indirect

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photodegradation is an important degradation pathway in waters and dissolved organic matter has a major role as photosensitizer. In this study, metoprolol photodegradation, in the absence and in the presence of fulvic acids extracted from the Vouga River (Portugal) (VRFA), was assessed under simulated sunlight. While metoprolol direct photodegradation was deniable, indirect photolysis occurred under the presence of VRFA. It followed a pseudo-first order kinetics and after 72 h of irradiation there was a 2

decrease of metoprolol concentration of ~80%. The OH radical (OH) was verified to be the main reactive species (RS) responsible for the photosensitized degradation of metoprolol, but other RS are also involved, probably triplet excited states of FA (3FA*) and singlet oxygen (1O2), as demonstrated by the higher inhibition of the photodegradation in presence of sodium azide than in presence of 2-propanol. Based on a previous identification of photoproducts, tentative degradation mechanisms were here proposed. Photoproducts analysis after 24 h irradiation in the absence and presence of

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scavengers, shown that different RS are involved in the formation of different products/intermediates.

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Keywords: Emerging contaminants; Indirect photolysis; Humic substances; Reactive

Introduction

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species; Degradation mechanism;

Pharmaceuticals, whose global consumption is increasing due to the population growth and aging, are considered contaminants of emerging concern and their presence in natural

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waters is well documented [1]. One of the most frequently detected pharmaceuticals in aquatic systems is metoprolol [2-10], which belongs to the class of β-blockers and is used for

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the treatment of hypertension and heart diseases. In their recent review on the occurrence,

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ecotoxicological effects and risks of antihypertensives in the aquatic environment, Godoy et al. [11], revealed that metoprolol, together with the β-blockers atenolol and propranolol, were the antihypertensives with the highest concentrations and risks for biota in surface waters. Once they reach surface waters, organic contaminants are subject to several transformation, degradation and removal processes. Indeed, engineered wastewater treatments usually mimic or build on these natural processes. In this sense, UV-driven advanced

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oxidation processes point to the role of photolysis in the fate of this sort of contaminants, some authors having highlighted the importance of assessing matrix effects, degradation pathways, transformation products (TPs) and removal kinetics [12-16]. In the specific case of metoprolol, its low biodegradability [17] and unremarkable direct photolysis (it does not absorb in the wavelength range of solar radiation that reaches Earth’s surface) [18,19], explain its persistence in the aquatic medium [11]. However, photo-induced transformation has been shown to account for a significant fraction of metoprolol elimination both in natural waters

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[18,20] and during wastewater treatment [21]. Indirect photodegradation of organic

contaminants may be induced by common photosensitizers in natural waters such as dissolved organic matter (DOM) and nitrate. Using natural freshwater, both the works of Peuravuori and

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Pihlaja [18] and Liu et al. [20] highlighted the photosensitizing effect of DOM towards

indirect photo-transformation of metoprolol and other β-blockers. Since humic substances

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(HS), whose soluble fractions in water are humic acids (HA), fulvic acids (FA) and XAD-4

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fraction [22], are the major component of natural DOM, their effect on the photodegradation of β-blockers has been investigated using synthetic solutions. The sensitizing effect of HS on

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the photodegradation of contaminants can be due either to direct reaction with excited triplet states of these substances (3HS*) or to reaction with reactive species (e.g. •OH or 1O2) that are

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photo-chemically produced in HS solutions under solar irradiation [23]. The indirect photolytic processes induced by HS depend on their origin and composition, as well as on the

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structural features of the contaminant [23]. The photosensitizing effect of HS has been investigated for some -blockers, namely metoprolol [24,25] and atenolol [24-26]. According to Chen et al. [24], who studied the effect of commercial FA on the photodegradation of metoprolol and atenolol, direct reaction with 3HS* is the predominant route for the photosensitized degradation of these -blockers. However, FA used by these authors were extracted from weathered coal and do not represent aquatic fulvic acids in surface waters. 4

Differently, Wang et al. [25] and Zeng et al. [26] studied the sensitizing effect of Suwannee River fulvic acids (SRFA) obtained from the International Humic Substances Society (IHSS) on the photodegradation of atenolol, but their results are contradictory. While Wang et al. [25] attributed a major role to 3HF*, Zeng et al. [26] showed that OH had a major role. The above described context evidences that, more studies are needed to clarify the pathways of the photodegradation of -blockers, particularly metoprolol, in presence of aquatic FA. Thus, using FA extracted from the Vouga River (VRFA), the two main goals of

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this work were: • to increase the knowledge on the role of natural photosensitizers, namely aquatic FA, on the degradation of metoprolol in surface waters.

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• to contribute to the establishment of the mechanism of formation of metoprolol

photoproducts in the presence of aquatic FA and to evaluate the role of reactive oxygen

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species (RS), namely 1O2 and OH, and triplet state of FA (3HF*) on the photodegradation

2 2.1

Experimental

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process.

Chemicals and solutions

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Metoprolol tartrate salt (≥ 98%), 2-propanol and sodium azide were acquired from Sigma-Aldrich; acetonitrile (HPLC grade) was purchased from Lab-Scan Analytical Sciences.

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Formic acid (HCO2H, 98%) was obtained from Panreac. Aquatic FA were extracted from

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Vouga River (Portugal) (VRFA), at Carvoeiro sampling site. The extraction procedure and structural characterization of these FA were described elsewhere [27,28]. Stock solutions of metoprolol tartrate (~500 or 1000 mg L-1) and VRFA (~250 mg L-1) were prepared in ultrapure water.

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2.2

Irradiation of aqueous solutions Irradiations were carried out in 20 cm quartz tubes with an internal diameter of 1.8 cm.

Samples were irradiated under simulated solar radiation using a Solarbox 1500 (Co.fo.me.gra, Italy) equipped with a 1500W arc xenon lamp and outdoor filters that restrict the transmission of light with wavelengths below 290 nm. Solutions containing metoprolol tartrate and VRFA ([FA] = 10 mg L-1, ~5 mg C/L of dissolved organic carbon) were irradiated. Concentrations of 1.46 × 10-4 mol L-1 and 7.30 ×

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10-4mol L-1 of metoprolol were used for the kinetic studies and for the detection of degradation products, respectively. Solutions containing the same concentrations of

metoprolol and RVFA and also 2-propanol or sodium azide were also prepared and irradiated

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to study the role of OH and 1O2 in the metoprolol indirect photodegradation. Concentrations of 20 mmol L-1 of 2-propanol and 0.5 mmol L-1 of sodium azide were used to study the

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scavenger’s effect on the kinetics of the photodegradation. To study their effect on the formation of metoprolol degradation products, the concentration of 2-propanol was

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maintained, but that of sodium azide was increased to 5 mmol L-1 (please, see the justification for this increase in section 3.3).

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In all cases, the metoprolol concentration in solution was determined by HPLC-UV [29]. More details about on instrumentation and analytical procedures, as well as about on

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preparation of solutions and the irradiation conditions can be found in the Supplementary

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Material.

2.3

Fluorescence spectroscopy

Excitation emission matrices (EEMs) fluorescence were recorded using a spectrofluorometer FluoroMax4 (Horiba Scientific, USA) with a 1 cm path quartz cuvette, run in sample emission to lamp reference mode (S/R). The EEMs were measured using excitation

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wavelengths (λexc) from 240 to 500 nm and emission wavelengths (λem) from 290 to 600 nm, both in 5 nm increments. Excitation and emission slit widths were set to 10 nm, and the integration time was 0.1 s. Fluorescence data were corrected following the procedure described in Supplementary Material.

2.4

Data analysis For the kinetic studies, the concentration of metoprolol remaining in solution after a

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certain time of irradiation (C) was normalized relatively to the metoprolol initial concentration (C0), as C/C0, where C0 and C were determined by HPLC analysis before and

𝐶 = 𝑒 −𝑘𝑡 𝐶0

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after irradiation, respectively. The first order kinetics equation:

(Eq. 1)

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where k is the first order rate constant and t is the irradiation time, was fitted to the

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experimental data (C/C0 versus irradiation time) by non-linear regression, using the software GraphPadPrism trial version (https://www.graphpad.com/, access 28th May 2019).

3.1

Results and discussion

Effect of fulvic acids on metoprolol degradation under simulated solar radiation

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Results on C/C0 vs. irradiation time are shown in Figure 1A for metoprolol in the

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absence and in the presence of VRFA. In the absence of VRFA, no significant differences

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were found between the C/C0 values at different times (ANOVA, p=0.52, 3 replicates and 7 irradiation times from zero to 48 h). However, in the presence of VRFA, C/C0 decreased with time. These results confirm that metoprolol does not suffer direct photolysis under solar light, as already referred by other authors [19], and that VRFA act as photosensitizers promoting its indirect photodegradation [24]. The metoprolol photodegradation follows a pseudo-first order kinetics in the presence of VRFA, as confirmed by the good fitting of the first order kinetic

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equation (Eq. 1) to the experimental data shown in Figure 1A. Figure S1 in the Supplementary Material represents the HPLC-chromatograms of the metoprolol solution in the absence of FA after 24 h of irradiation and in presence of FA after 0 and 24 h of irradiation

3.2

Evaluation of the role of different reactive species (RS) on metoprolol degradation photosensitized by fulvic acids Considering that different pathways may be involved in the sensitizing effect of FA,

𝑘=𝑘

•

𝑂𝐻,𝑚𝑒𝑡 [

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the k for metoprolol degradation in the presence of FA is given by the following equation:

𝑂𝐻 ]𝑠𝑠 + 𝑘 1𝑂2,𝑚𝑒𝑡 [ 1𝑂2 ]𝑠𝑠 + 𝑘 3𝐹𝐴∗ ,𝑚𝑒𝑡 [ 3𝐹𝐴∗ ]𝑠𝑠 + 𝑘𝑜𝑡ℎ𝑒𝑟

•

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where,

(Eq. 2)

[ •𝑂𝐻 ]𝑠𝑠 , [ 1𝑂2 ]ss, [ 3𝐹𝐴∗ ]ss are the steady-state concentrations of OH, 1O2, 3FA* in the •

𝑂𝐻,𝑚𝑒𝑡

, 𝑘 1𝑂2,𝑚𝑒𝑡 , 𝑘 3𝐹𝐴∗,𝑚𝑒𝑡 are the second-order rate constants for the

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solution of FA; 𝑘

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reactions between metoprolol and those reactive species; and kother corresponds to the contributions of other pathways to the k.

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The involvement of each RS (OH, 1O2, 3FA*) can be tested through the addition of scavengers that promote a decrease of the steady-state concentration of these RS and an

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effective reduction in the k of the photosensitized metoprolol degradation. The effect of the scavenger depends on the importance of the RS in the mechanism (importance of the

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corresponding term in Eq. (2)) and also on the scavenger concentration and on the second order rate constant for its reaction with the RS. According to the literature, several probes are used to assess the contribution of each RS. For example, 2-propanol is usually used as scavenger of OH [25,29], but the specificity of sodium azide as scavenger is not so clear. Although some authors use sodium azide as a scavenger specific for 1O2 [30], it is well known

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that sodium azide is also a scavenger of OH [31,32]. Furthermore, some authors demonstrated that sodium azide can also act as a quencher of triplet states of HS [33]. In the present work, some experiments were conducted in the presence of sodium azide and 2-propanol. Figure 1B shows that the photodegradation of metoprolol sensitized by VRFA was less efficient in the presence of the scavengers 2-propanol and sodium azide, but the effect of sodium azide was more remarkable than that of 2-propanol. The degradation follows a pseudo first order kinetics, both in the presence and in the absence of the selected

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scavengers (Figure 1B). A summary of the kinetic parameters obtained for the photodegradation of metoprolol in the presence of VRFA, with and without the addition of

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scavengers, is shown in Table 1.

Table 1 – Kinetic parameters obtained for the photodegradation of 1.46 × 10-4 mol L-1

k (× 10-2 h-1) 2.79 ± 0.09 0.67 ± 0.04 0.38 ± 0.04

t1/2 (h) 24.8 ± 0.8 102 ± 5 183 ± 17

r2 0.9980 0.9545 0.8441

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Scavenger Absence (n=7) 2-propanol (n=2) Sodium azide (n=2)

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metoprolol in the presence of 10 mg L-1 VRFA, with and without the addition of scavengers.

Since the concentration of 2-propanol (20 mM) is enough to scavenge almost all OH

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in solution (~96%, as estimated in the section S2 of the Supplementary Material) then the difference between the rate constants without and with 2-propanol addition corresponds to a

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pseudo-first order rate constant for metoprolol reaction with OH (𝑘

•

𝑂𝐻,𝑚𝑒𝑡 [

•

𝑂𝐻 ]𝑠𝑠 ) of 2.12

× 10-2 h-1 (~76% contribution of OH to the overall photodegradation in the VRFA solution). Since the OH scavenging by sodium azide is lower (scavenger efficiency ~74%, as shown in section S2), the decrease of the 𝑘

•

𝑂𝐻,𝑚𝑒𝑡 [

•

𝑂𝐻 ]𝑠𝑠 in the presence of sodium azide is expected

to be 1.57 × 10-2 h-1 (74% of the decrease induced by 2-propanol). Thus, from the total decrease of k observed in the presence of sodium azide (2.41 ×10-2 h-1), a decrease of 0.84 × 9

10-2 h-1 (30% of the k value) can be attributed to the scavenging of other RS. If this 30% decrease could be attributed to 1O2, then it would correspond to a 60% contribution of 1O2 to k, since only about 50% of the total 1O2 produced was expected to be scavenged (Table S2). However, the sum of the contribution of OH and 1O2 would exceed 100%. Thus, it is possible that the azide anion is also scavenging other RS, probably, some triplet states of VRFA, namely those associated to moieties containing aromatic carbonyls and quinones [33]. Thus, the obtained results suggest a high contribution of OH and a lower, but also relevant,

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contribution of 1O2 and/or triplet states of FA in the indirect photodegradation of metoprolol in the presence of VRFA.

As far as we know, there are only two studies [24,25] concerning the RS involved in the

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sensitizing effect of HS on the photodegradation of metoprolol. Chen et al [24] used FA

extracted from weathered coal while Wang at al. [25] used Standard Suwannee River FA

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(SRFA). Chen et al [24] did not observe any effect of the scavengers 2-propanol and sodium azide and attributed a major role to 3FA*; Wang at al. [25] attributed a major role to 3FA*

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(66%) and a contribution of ~27% to OH.

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Some authors have related the origin and structural characteristics of natural organic matter with its tendency to produce different RS [23,34,35]. The structural characterization of

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VRFA, namely elemental analysis, carboxyl content and FTIR and NMR spectra have been

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presented elsewhere [27,28] and additional data are presented in Supplementary Material (Figure S2 and Table S2). The 13C-NMR spectra of SRFA and VRFA were compared in [28] and suggest a higher aromaticity of the latter, which has been positively correlated to the tendency to produce OH [34]. Besides, Batista et al. [34] observed that SRFA produced less 

OH than predicted by their aromaticity. This is in agreement with a higher involvement of



OH in the photosensitizing effect of VRFA. However, there is some contradiction in the

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literature about the importance of OH in the sensitizing effect of SRFA on the photodegradation of another -blocker (atenolol): while Wang et al. [25] only attributed a contribution of ~7% to OH, Zeng et al. [26] considered that OH contributes 100% to the degradation. The tendency to produce 1O2 has been positively correlated to the terrestrial origin of HS and inversely correlated to their protein and carbohydrate content and structures [23,34]. VRFA are mainly of terrestrial origin, with fluorescence spectra dominated by peaks

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characteristic of humic-like moieties and a very low content of protein-like fluorophores (Figure. S2). The comparison of RMN spectra of VRFA and SRFA [28] has evidenced a

lower content of carbohydrate and protein structures in VRFA, which suggests that some of

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the difference between the effect of azide and 2-propanol on the sensitizing effect of VRFA can be attributed to 1O2. As previously referred, some authors have shown that sodium azide

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can also scavenge 3FA* associated to moieties containing aromatic carbonyls and quinones

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[33]. It is worth to notice that the comparison of 13C-NMR spectra revealed a higher content of this type of structures in VRFA than in SRFA [28]. In what concerns the products of metoprolol indirect photodegradation sensitized by

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HS, Chen et al [24] identified only one product (product XVI, referred in the section 3.3), which is similar to the photoproduct of atenolol identified by Wang et al [25]; both of

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products have been attributed to the reaction with 3FA*. In order to investigate the

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involvement of different RS in the formation of different degradation products, the scavengers’ effect was studied.

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3.3

Metoprolol degradation photoproducts in the presence of natural FA: possible formation pathways In a previous work [36] we observed the formation of several degradation products of

metoprolol under simulated solar radiation, in the presence of the same FA used in the present work. These degradation products were separated and tentatively identified by HPLC-UVESI-MSn. Table S3 in the Supplementary Material depicts the sixteen compounds identified (numbered according

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to their retention times). Considering the three structural domains of metoprolol identified in Scheme 1, the degradation products can be divided into three classes:

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1) compounds with modifications in the phenoxy moiety: products with hydroxyl groups in the aromatic ring and open ring derivatives;

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2) compounds with modifications in the aminopropanol moiety;

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3) compounds with modifications only in the ether moiety or in the ether and

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phenoxy moieties.

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Scheme 1 – Metoprolol molecular structure.

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In order to investigate the role of OH and other RS in the formation of each degradation product, the concentrations of 2-propanol and sodium azide (20 mM and 5 mM, respectively) were chosen so that their effect on [OH]ss was the same (𝑘

•

𝑂𝐻,𝑆𝑐

 [Sc] = 3.8 ×

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107 s-1). Thus, a significantly higher decrease of a chromatographic peak in the presence of sodium azide than in the presence of 2-propanol may indicate the involvement of 1O2 or other

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RS scavenged by sodium azide in the pathway of formation of the corresponding degradation

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product. In this way, based on the involvement of these RS and on the structure of the previously identified compounds [36] we propose plausible pathways for metoprolol

3.3.1

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photodegradation in the presence of VRFA.

Compounds with structural changes in the phenoxy moiety (hydroxylated and

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open ring compounds): proposed pathways involving 1O2

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The formation of compounds XIV, X and III with two, three and four hydroxyl groups can be easily justified by the involvement of OH hydroxylation of the aromatic ring following the pathways previously reported by other authors [38,39]. However, these compounds can also be formed by a [4+2] cycloaddition reaction involving 1O2 and the aromatic ring (nonhydroxylated and already hydroxylated), followed by cleavage of the endoperoxide and isomerization as proposed in Scheme 2 [37]. When the [4+2] process involves the substituted

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carbons of the aromatic ring, the formation of the open-ring compound VII can be easily explained as proposed in Scheme 3A (via i). Alternatively, the formation of dioxetanes involving those two carbons followed by their cleavage can also justify compound VII appearance (via ii). On the other hand, further reaction of 1O2 with the hydroxylated aromatic compounds (for example compound III) by a [2+2] cycloaddition reaction followed by cleavage of the dioxetane intermediates can explain the formation of compounds I and II (Scheme 3B).

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The effect of scavengers could be studied only for compounds VII and X, since the chromatographic peaks of the other compounds above mentioned are too small or masked by other peaks. The involvement of 1O2 in the formation pathway of compound VII was

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confirmed by its almost complete disappearance in the presence of azide, while the effect of 2-propanol was negligible (Figure 2). The possible involvement of OH in the formation of

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compound X could be confirmed by the decrease of its peak in the presence of 2-propanol.

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The larger decrease of the peak in the presence of azide than in the presence of 2-propanol (Figure 2) is in agreement with the involvement of 1O2, according to the mechanism proposed in Scheme 2.

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Open ring compounds I and II and the hydroxylated compound X were also detected in our previous work [28] concerning the metoprolol photodegradation sensitized by

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5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (H2TF5PP), which occurred mainly via 1O2,

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without involvement of OH, thus corroborating the possibility of their formation via 1O2. Thus, both OH and 1O2 can originate hydroxylation of the phenoxy moiety, but only

1

O2 seems to be involved in the formation of open ring compounds. It is interesting to notice

that the formation of open ring compounds was also observed by other authors [40] during the photodegradation of metoprolol in the presence of TiO2 as catalyst, but their formation was attributed to the superoxide radical and not to OH. 14

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Scheme 2

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Proposed mechanism for the hydroxylation of metoprolol molecule in the aromatic ring

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Scheme 3

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Proposed mechanism for the open ring compounds with 1O2: (A) formation of compound VII from metoprolol; (B) formation of compounds I and II from a hydroxylated metoprolol derivative (compound III).

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3.3.2 Metoprolol derivative with structural changes in the aminopropanol moiety Compound XVI is the only one with modifications in the aminopropanol moiety. It has been identified by several authors as a degradation product of metoprolol, formed by solar light irradiation in natural waters [20] or in solutions containing dissolved HS [24]. The initiation step for its formation has been considered to be an electron transfer from the nitrogen of the amine group to 3HS* [24]. This pathway has also been proposed for the photosensitized degradation of other amine compounds in presence of humic matter [41]. The

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complete pathway for the formation of compound XVI is presented in Scheme 4. Despite the irreproducibility in the areas of this peak, the results show that it does not decrease in the presence of OH or 1O2 scavengers (Figure 2). This means that the 1O2 and OH are not

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involved in its formation, which is in agreement with the proposal of a 3FA* as reactive

species in the initiation step of the reaction mechanism, as shown in Scheme 4 and proposed

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by Chen et al. [24].

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It is interesting to notice that the chromatographic peak of compound XVI, not only does not decrease, but even seems to increase in the presence of both scavengers (Figure 2). FA can also suffer direct and self-sensitized photodegradation and their photobleaching

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induces alterations of their sensitizing properties [42]. Thus, a tentative explanation for the increase of compound XVI in the presence of 1O2 and OH scavengers is the involvement of

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these ROS in the degradation of the VRFA moieties whose triplet state (3FA*) is the reactive

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species involved in the formation of compound XVI. The addition of scavengers would keep [3FA*]ss higher. Indeed, it is known that the aromatic moieties in FA are more reactive towards OH [43] and the triplet involved can be associated to some of these moieties. Another possible contribution to the higher increase of the product XVI in presence of 𝑁3− is the side reaction of this ion with FA, which gives rise to the azide radical, due to

electron transfer from 𝑁3− to the triplet state of DOM [43]. Some authors observed that the 18

addition of 𝑁3− increased the photodegradation rate of 2,4,6-trimethylphenol (TMP) [44] and of the pharmaceutical mefenamic acid [45] in the presence of DOM isolated from natural waters. Cawley et al [44] attributed the enhancement of the oxidation of TMP to its reaction with the azide radical. The capacity of this radical to accept an electron from certain phenols had been demonstrated before [30]. In the present work, the enhancement effect of azide was only observed for the formation of compound XVI and not for the global kinetic degradation rate, which can be attributed to the existence of several degradation pathways, some of them

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involving RS, which are scavenged by 𝑁3− .

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Scheme 4 Proposed mechanism of N-dealkylation of metoprolol molecule (compound XVI).

3.3.3 Compounds with structural changes in the ether moiety

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The structures of compounds resulting from the oxidation of the aliphatic ether moiety

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suggest an autoxidation process, which is a complex chain reaction initiated by H abstraction from one of the three carbons of this moiety [46,47]: RH + S

R + SH

(reaction 1)

where S is a reactive species such as the OH [46,47] or organic reactive species formed upon irradiation of FA, such as 3FA* or FA radicals [48,49]. The radicals R react with O2 affording

20

the alkylperoxy radicals ROO (reaction 2); the alkylperoxy radicals can also abstract weakly bound hydrogen atoms affording alkyl hydroperoxides ROOH and radicals R (reaction 3):

Then, reaction (3) is followed by reaction (2) and the chain reaction is established. Finally, a termination step can occur, namely by self-reaction of two alkylperoxy radicals affording

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stable compounds like alcohols and ketones (reaction 4).

Three groups of compounds with structural changes in the ether moiety of metoprolol

a) terminal methyl group (methoxy carbon);

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can be considered, depending on the carbon from which occurs the H abstraction:

b) carbon in  position (relatively to the benzene ring);

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c) carbon in  position (relatively to the benzene ring).

3.3.3.1 Compounds whose formation is initiated from the terminal methyl group

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(compounds VIII, IX, XII)

The mechanisms proposed for the formation of these compounds are shown in Scheme

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5 and OH is proposed as the reactive species S in reaction 1. The OH involvement is corroborated by the decrease of the area of the chromatographic peaks of these compounds in

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presence of 2-propanol (Figure 2). It is not possible to have a definitive conclusion about a higher effect of azide on the peak area of compound XII, due to the very high uncertainty of the effect of 2-propanol. However, the peaks of compounds IX and VIII decrease even more in the presence of azide, than in the presence of 2-propanol. Thus, other reactive species, which are scavenged by azide, such as 1O2 or some triplets of VRFA, are also involved. In the case of compound VIII, this additional effect of azide can be explained by the involvement 21

of 1O2 in the hydroxylation of the aromatic ring, following a mechanism similar to those described in Scheme 2 of section 3.3.1 (formation of compound X by hydroxylation of compound XIV). In case of compound IX, the RS is probably a 3FA*. However, a possible pathway for the involvement of 1O2 was also considered as shown in Scheme 5 (formation of the hydroperoxide through the insertion of 1O2). Indeed, 1O2-mediated selective C–H bond hydroperoxidation in -positions of ethereal hydrocarbons has been recently demonstrated by Sagadevan et al. [50]. Besides, this pathway via 1O2 is in agreement with the detection of two

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of these products, IX and XII, in our previous work concerning the metoprolol

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ur

na

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photodegradation sensitized by H2TF5PP, which occurs mainly via 1O2 [28].

22

f

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na l

Pr

e-

pr

oo

Scheme 5 Proposed autoxidation mechanism pathways for the formation of the compounds VIII, IX and XII.

23

3.3.3.2 Compounds whose formation is initiated by H abstraction from the carbon in  position relatively to the benzene ring (compounds XIII and XV) The mechanism for the formation of compounds XIII and XV is presented in Scheme 6. The effect of scavengers on the peak of compound XIII could not be assessed, but the chromatographic peak of compound XV is not influenced by the addition of •OH or 1O2 scavengers (Figure 2). Thus, an organic reactive species S, formed upon irradiation of FA, is

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proposed to be involved in the initiation step (reaction 1) of the autoxidation mechanism. This organic reactive species can be a 3FA* or a FA radical. Experimental and theoretical evidence of the formation of phenoxyl radicals of FA upon irradiation at 355 nm has been presented recently [49]. These radicals can be formed by photoionization of FA or as a result of charge-

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transfer reactions between donor groups of FA (for example: polyphenol moieties) and

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acceptor moieties (for example: triplet states of quinones). Phenoxyl radicals are able to react

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na

disregarded [49].

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with many compounds [51] but their involvement in the degradation of contaminants has been

24

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Scheme 6 Proposed autoxidation mechanism pathways for the formation of the compounds XIII and XV.

3.3.3.3 Compounds whose formation is initiated by H abstraction from the carbon in

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position relatively to the benzene ring (compounds IV, V, VI and XI) The potential pathways for the formation of compounds IV, V, VI and XI are

3

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presented in Scheme 7. The reactive species S involved in the H abstraction (reaction 1) was FA* or a FA radical, since the addition of scavengers did not influence the formation of

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compound XI (Figure 2). As the peaks corresponding to compounds IV and VI were too small and/or not well resolved, it was not possible to conclude about the effect of scavengers. In the case of compound V, it was observed that its chromatographic peak is not influenced by the addition of 2-propanol (as expected if 3FA* is the S reactive species) but the peak decreases in the presence of 𝑁3− . This effect is attributed to the involvement of 1O2 in the oxidation of compound XI giving rise to compound V. Indeed, the oxidation by 1O2 of 25

alcohols in position relatively to a phenyl group has been referred by other authors [52,53]. In our previous work [36], the formation of compound V by oxidation of compound XI was proposed based on their structures and on the profiles of the areas of their chromatographic peaks versus the irradiation time. Finally, the formation of compound IV can be justified based on the Hock rearrangement [54] (Scheme 7); this process is also initiated with the formation of the hydroperoxide in the position (relatively to the benzene ring). The protonation of this intermediate is followed by the migration of the phenyl group to the

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adjacent oxygen with elimination of a water molecule. Then, the stabilized carbocation reacts with water affording the hemiacetal that is responsible by the formation of phenol and

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ur

na

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aldehyde.

26

f

Jo ur

na l

Pr

e-

pr

oo

Scheme 7 Proposed mechanism pathways for the formation of the compounds IV, V, VI and XI, included Hock rearrangement for the formation of compound IV.

27

4

Conclusions The sensitizing effect of Vouga River Fulvic Acids (VRFA) on the photodegradation

of metoprolol in water was investigated. Metoprolol did not suffer direct photodegradation under simulated sunlight, but it was extensively degraded (80%) in the presence of VRFA after 72 h and its indirect photodegradation followed a pseudo-first order kinetics. The high decrease (75%) of the first order rate constant in the presence of 2-propanol suggests that OH has a major role in the photosensitizing effect of VRFA. The further decrease of the

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degradation rate constant in presence of sodium azide, relative to the decrease caused by 2propanol, suggests the involvement of other reactive species besides OH, probably 1O2 and/or some triplet states of RVFA that are scavenged by sodium azide

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Several of the identified degradation products involve structural modifications in the ether moiety of metoprolol and their formation can be explained by a chain reaction initiated

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by H abstraction from one of the three carbons of this moiety. The addition of the above

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mentioned scavengers only decreased the formation of the products that involve the carbon from the terminal methyl group, which suggests that both OH and other RS scavenged by

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azide, namely, some triplet states of RVFA, are abstracting H from this carbon. The possible involvement of 1O2 in the formation of these products would be explained by hydroperoxidation of the terminal methyl group. Other reactive species, namely RVFA

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triplets not scavenged by azide, are abstracting H from the other carbons of this moiety.

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Further studies are needed to clarify which reactive species are involved. Another group of photoproducts result from modifications of the aromatic ring of

metoprolol. It was demonstrated that OH is involved in the hydroxylation of the aromatic ring and the formation of open ring products was attributed to 1O2. The OH participation in the formation of other products not detected under the analytical procedures here used,

28

namely very polar products that are eluted at the dead volume, cannot be excluded, and would contribute to such an important role of OH. Only one of the identified degradation products involves modifications in the aminopropyl unit of metoprolol. This product was identified in previous works as being formed in the presence of other samples of FA. An electron transfer from nitrogen to a triplet state of FA has been proposed as the initiation reaction for its formation. The results of the present work are in agreement with this mechanism.

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This work will allow contribute to predict which degradation products of metoprolol will be favored by the photosensitizing effect of different humic substances, knowing their

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relative tendency to produce different reactive species.

Declaration of interests

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Acknowledgements

Authors acknowledge National Foundation for Science and Technology / Ministério

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da Educação e Ciência-FCT/MEC (POPH/FSE) for the financial support to QOPNA

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(UID/QUI/00062/2013) and CERNAS (UID/AMB/00681/2013) through national funds (PIDDAC) and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement and Compete 2020, and also to the Portuguese NMR Network. Thanks are due for the financial support to CESAM (UID/AMB/50017/2019), to FCT/MCTES through national funds. Marta Otero thanks support from the FCT Investigator Program (IF/00314/2015).

29

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Figure captions

Figure 1 – Normalized concentration of metoprolol (C/C0) along irradiation time for metoprolol (1.46 × 10-4 mol L-1) (A) in the absence and in the presence of VRFA (10 mg L-1); (B) in the presence of VRFA and absence of scavengers (•), in presence of VRFA together with 20 mmol L-1 of 2-propanolol (○), and in presence of VRFA together with 0.5 mmol L-1 of azida (). The lines are the graphic representation of the first order equation (Eq. (1)) fitted to the C/C0 values vs. time. Note: In presence of VRFA but absence of scavengers, mean values of the experimental data are represented together with error bars standing for the standard deviation (n = 7). In presence of VRFA and scavengers, experimental results are represented (n = 2).

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Figure 2– Effect of scavengers on the area of the chromatographic peaks corresponding to the photodegradation products of metoprolol depicted in Table S3 ([Met]= 7.30 × 10-4 mol L-1, [VRFA] = 10 mg L-1, tirradiation= 24 h).

38

A

1.0

0.8

C/C0

0.6

0.2

presence of FA absence of FA 0.0 0

10

20

30

40

50

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0.4

60

70

B

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1.0

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0.8

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C/C0

0.6

0.4

0.0

10

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0

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absense of scavenger -1 2-propanol 20 mmol L -1 azide 0.5 mmol L

0.2

20

30

40

50

60

70

Time (h)

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Figure 1 – Normalized concentration of metoprolol (C/C0) along irradiation time for metoprolol (1.46 × 10-4 mol L-1) (A) in the absence and in the presence of VRFA (10 mg L-1); (B) in the presence of VRFA and absence of scavengers (•), in presence of VRFA together with 20 mmol L-1 of 2-propanolol (○), and in presence of VRFA together with 0.5 mmol L-1 of azida (). The lines are the graphic representation of the first order equation (Eq. (1)) fitted to the C/C0 values vs. time. Note: In presence of VRFA but absence of scavengers, mean values of the experimental data are represented together with error bars standing for the standard deviation (n = 7). In presence of VRFA and scavengers, experimental results are represented (n = 2).

39

300000

250000

Absence of scavengers -1 2-propanol 20 mmol L -1 sodium azide 5 mmol L

Area

200000

150000

100000

ro of

50000

0

I I V II II III V X X XI ak eak V eak V eak V eak I eak eak eak X eak X ak XV pe p p p e p p p p p p

-p

Figure 2– Effect of scavengers on the area of the chromatographic peaks corresponding to the

Jo

ur

na

lP

[VRFA] = 10 mg L-1, tirradiation= 24 h).

re

photodegradation products of metoprolol depicted in Table S3 ([Met]= 7.30 × 10-4 mol L-1,

40