Solar-mediated degradation of linezolid and tedizolid under simulated environmental conditions: Kinetics, transformation and toxicity

Chemosphere 241 (2020) 125111

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Solar-mediated degradation of linezolid and tedizolid under simulated environmental conditions: Kinetics, transformation and toxicity € n b, La szlo  To € lgyesi c, Harald Horn a, d, *, Alexander Timm a, 1, Patrick Abendscho Ewa Borowska a a

Karlsruhe Institute of Technology (KIT), Engler-Bunte-Institut, Water Chemistry and Water Technology, Engler-Bunte-Ring 9a, 76131, Karlsruhe, Germany Hochschule Bonn-Rhein-Sieg, Section 5, von-Liebig-Straße 20, 53359, Rheinbach, Germany Agilent Technologies Sales & Services GmbH and Co. KG, Hewlett-Packard-Straße 8, 76337, Waldbronn, Germany d DVGW Research Laboratories for Water Chemistry and Water Technology, Engler-Bunte-Ring 9a, 76131, Karlsruhe, Germany b c

h i g h l i g h t s
Sunlight is able to degrade oxazolidinone antibiotics.
Sunlight transforms linezolid to transformation products which may have antibacterial efficacy.
Transformation products of tedizolid are proposed to be not antibacterial.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 July 2019 Received in revised form 10 October 2019 Accepted 12 October 2019 Available online 14 October 2019

Linezolid (LIN) and Tedizolid (TED) are representatives of oxazolidinone antibiotics of last resort with a strong efficacy against gram-positive bacteria. This study focused on their solar-mediated degradation to understand better their fate in aquatic environment, for the realistic concentrations in the range of 1 mg/ L. Results showed that both antibiotics (ABs) are degradable by simulated sunlight (1 kW/m2), with halflives of 32 and 93 h in ultrapure water, for LIN and TED, respectively. LIN showed similar photolytic behaviour in pure solution and in surface water, whereas sunlight enhanced the degradation of LIN in pure solutions, but not in surface water. Structure elucidation by liquid chromatography coupled to high resolution mass spectrometry provided information about seven transformation products for LIN and five for TED. The morpholinyl-ring was identified as the target site for most transformation reactions of LIN. TED was prone to oxidation and cleavage of the oxazolidinone ring. Results of a growth inhibition test on Bacillus subtilis exposed to UV light showed antibacterial efficacy of transformation products of LIN and no significant efficacy of degradation products of TED for the concentration range of 100 mg/L10 mg/L of parent compounds. Photolytically treated solutions of the ABs maintained their inhibitory effect on the bioluminescence of Aliivibrio fischeri. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: Klaus Kümmerer Keywords: Photolysis Transformation products Oxazolidinone antibiotics LC-HRMS Simulated sunlight

1. Introduction Due to the intensive use of antibiotics (ABs), their concentrations detected in the environment have reached significant levels (Cars et al., 2001; Carvalho and Santos, 2016; Günther et al., 2003).

* Corresponding author. Karlsruhe Institute of Technology (KIT), Engler-BunteInstitut, Water Chemistry and Water Technology, Engler-Bunte-Ring 9a, 76131, Karlsruhe, Germany. E-mail address: [email protected] (H. Horn). 1 Current address: Institute of Forensic Medicine of the University of Basel, Health Department Basel-Stadt, Pestalozzistrasse 22, CH-4056 Basel, Switzerland. https://doi.org/10.1016/j.chemosphere.2019.125111 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

ABs and their metabolites present in the excretions of patients reach surface and potentially also ground water after incomplete elimination from municipal and hospital effluents in wastewater treatment plants (WWTP) (Kümmerer and Henninger, 2003; Küster et al., 2013). Although currently used treatment may eliminate a large fraction of drugs from the wastewater, ABs such as sulfonamides, macrolides, tetracyclines and chinolones can be still detected in WWTP effluents at concentrations up to low mg/L (Adler et al., 2018; Germap, 2015; Rossmann et al., 2014; Watkinson et al., 2007). The presence of ABs in the environment is often discussed in context of antibiotic resistance. Although this phenomenon is still not fully understood, it has been proven that some ABs in

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concentrations a hundred-fold below the minimum inhibition concentration (MIC), even in the ng/L-range, can modulate the transcription of specific genes (Davies et al., 2006; Gullberg et al., 2011). Each year numerous studies report on the presence of antibacterial substances in various environmental compartments, especially in proximity to discharge points of WWTPs (Akiyama and Savin, 2010; Alexander et al., 2015; Brown et al., 2019; Schwartz et al., 2003). Decrease of efficacy of ABs formed a need to design new classes of ABs, for which the antibiotic resistance mechanisms have not been detected yet. In this group of compounds are oxazolidinone ABs. These ABs act differently against bacteria than conventional ABs, which is the reason for the efficacy in treating infections caused by multi-resistant bacteria. Unfortunately, oxazolidinone-resistant bacteria (especially enterococci) have already been reported (Bi et al., 2018; Mendes et al., 2014; Shaw and Barbachyn, 2011). The 3-(3-fluorophenyl)-1,3-oxazolidin-2-one ring of oxazolidinone ABs is known to inhibit the initiation of protein biosynthesis in ribosomes (Barbachyn and Ford, 2003; Zhanel et al., 2015). In 2000, LIN became the first oxazolidinone AB to be approved for clinical use by the Food and Drug Administration (Norrby, 2001; Pfizer Inc, 2008). In 2014, the authorisation also received TED (Zhanel et al., 2015). Since LIN and TED are relatively novel pharmaceuticals, their application is restricted only to special cases and the information about their consumption and environmental concentrations is very limited. About 30% of applied LIN is excreted in its unmetabolized form (Pfizer Inc, 2008; Slatter et al., 2001). The two main metabolites are inactive carboxylic acids (Slatter et al., 2001). Approximately 97% of TED is excreted as metabolites (Merck Sharp and Dohme Corporation, 2017; Ong et al., 2014). After leaving the human body, pharmaceuticals like LIN and TED as well as their metabolites usually reach WWTPs. In the study performed in United States of America, LIN was detected in wastewater samples at concentrations between 3 and 62 mg/L (Kulkarni et al., 2017). Compared to concentrations of other antibiotics in European wastewater, the concentrations of LIN and other ABs are surprisingly high. However, the high ABs concentrations in that study were not discussed and no concentration values of LIN and TED were available for European waste- or surface water. Since the conventional treatment of wastewater is mainly based on microbial degradation of organic matter, many pharmaceuticals persist the wastewater treatment to some extend and end up in the aquatic environment (BLAC, 2003; Majewsky et al., 2018). After entering the aquatic environment, multiple naturally occurring processes determine the environmental fate of these substances, such as microbial degradation, adsorption on particles, hydrolysis and photolysis by sunlight, and cause the transformation of pharmaceuticals to TPs. Sunlight is the main source of electromagnetic energy in the environment leading to the photolysis of various micropollutants in the upper layer of surface water. Many studies described the direct and indirect photolysis of ABs. Especially sulfamethoxazole is known to be highly photolabile (Bahnmüller et al., 2014; Batchu et al., 2014; Gmurek et al., 2015; Lam and Mabury, 2005). In surface water, dissolved organic matter (DOM) plays an important role in the photolytic degradation of pharmaceuticals. On the one hand, DOM decelerates direct photolysis by the absorption of light. On the other hand, DOM can promote photosensitisation (Fatta-Kassinos et al., 2011; Schwarzenbach et al., 2003; Xu et al., 2011). However, since DOM consists of huge variety of molecules with different photochemical behaviour, the comparison of photolytic processes taking places in different environmental water matrices is difficult. The degradation of micropollutants (MPs) in general does not necessarily lead to changes of their biochemical activity. Some of

the TPs of MPs retain their antimicrobial efficacy, as it was reported for anthracene (Brack et al., 2003), sulfamethoxazole (Gmurek et al., 2015) and naproxen (DellaGreca et al., 2004). Since, as mentioned before, bacteria are exposed to micropollutants and their TPs in the aquatic environment, an evaluation of their potentially toxic effect is essential. Considering all the above, the aim of this study was to assess if sunlight is able to degrade oxazolidinone ABs and if formed TPs exhibit the antibacterial activity. Solar-driven degradation of LIN and TED was therefore investigated with respect to (i) degradation kinetics, (ii) structure elucidation of phototransformation products, and (iii) toxicity of irradiated solutions of ABs against Bacillus subtilis and Allivibrio fischeri. 2. Material and methods 2.1. Chemicals Linezolid, linezolid-d3, tedizolid and tedizolid-d3 of analytical grade were purchased at Toronto Research Chemicals (Canada). Acetonitrile, formic acid, methanol, and ultrapure water were supplied by VWR Chemicals (Germany) in HPLC-grade. For the toxicity tests, casein peptone and sodium chloride from ROTH (Germany) as well as yeast extract from Acros Organics (USA) were used. 2.2. Analytical methods All analytes were quantified with an Agilent 1290 Infinity II UHPLC system equipped with an Agilent ZORBAX Eclipse Plus C-18 column (50  2.1 mm, 1.8 mm particle size). The mobile phase consisted of ultrapure water (eluent A) and methanol acidified with 0.05% formic acid (eluent B). The run started with 10% of eluent B with a linear increase to 100% within 7 min and ended after 8 min. Detection of the analytes was performed with an Agilent 6470 Triple Quad LC/MS system with an Agilent Jet Stream electrospray ionisation source. Table SI 1e2 give the instrumental settings applied for the analysis. Surface water samples were filtered using Chromafil CA-20/25 cellulose acetate filters (0.2 mm). UV/Vis spectra were obtained by a PerkinElmer Lambda XLS photospectrometer. 2.3. Kinetic investigation 50 mL glass beakers filled with aqueous solutions (ultrapure and two types of river water) of LIN and TED in a concentration of 1 mg/L each were continuously irradiated in an Oriel Sol3A Solar Simulator (Newport) equipped with an XBO 1000 W/HS OFR xenon short arc lamp (Osram). Application of an Air Mass 1.5G spectral filter limited the spectrum to wavelengths between 300 and 1400 nm that corresponds the typical solar spectrum (Fig. 1). Irradiance of the samples was set to 1 kW/m2, which is comparable to the light intensity in middle Europe at noon of summer cloudless day (Myers et al., 2000). The temperature was kept at 19  C by an integrated cooling system and the solutions were gently stirred by magnetic stirrers. To differentiate the abatement of ABs caused by light from other processes like hydrolysis, adsorption on particles and microbial degradation, dark control samples were prepared. Two 50 mL beakers with solutions of both ABs in mixture were covered with aluminum foil and placed in the sunlight simulator during the irradiation experiments. Samples of both irradiated and dark control solutions were withdrawn after 0, 6, 12, 24, 48, 72, 96, 120, 144 and 168 h and stored in amber glass HPLC-vials at 4  C until analysis. To compare the susceptibility of photolysis of the ABs in ultrapure and surface

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Fig. 1. UV/Vis spectra of LIN and TED (c ¼ 2 mg/L) and the emission spectrum of simulated sunlight.

water, the experiment was performed with waters from the rivers Rhine and Alb in Karlsruhe, Germany. Experiments using UV254nm light were performed at ambient temperature in a rotating UVreactor (Applied Photolytics LTC) equipped with a low-pressure mercury bulb emitting 710 W/m2 UV254nm light. 2.4. Structure elucidation of transformation products Structure elucidation of the TPs of LIN and TED was performed for two types of processes, namely solar-driven degradation and UV254nm light-driven degradation, in the solutions of non-labelled (LIN and TED) and labelled (LIN-d3 and TED-d3) antibiotics. The structural formulas of LIN-d3 and TED-d3 are shown in Fig. SI 1. By comparing the MS2 spectra of TPs of labelled and non-labelled ABs, the identification of the transformation’s location was facilitated. For the screening of TPs formed by simulated sunlight, a solution of LIN and LIN-d3 (concentration of 2.5 mg/L each) and a solution of TED and TED-d3 (concentration of 2.5 mg/L each) prepared in ultrapure water and exposed to simulated sunlight for 168 h (7 days), were used. For the screening of TPs formed by UV254nm light, nonlabelled and deuterated ABs were irradiated in separate solutions. In addition to that, a control sample of ultrapure water was irradiated and sampled simultaneously to exclude artefacts originated from potential impurities. High resolution mass spectrometry measurements were performed using an Agilent 6545 Quadrupole e Time of flight (QToF) LC/MS system with a nominal mass resolution of 30,000. Full scan MS spectra were acquired in positive and negative ESI mode. Screening of TPs was made with suspect, targetand non-target approach. For the MS2 fragmentation measurements, precursor ions were selected with an isolation window of 1.3 u and ions were fragmented at fixed collision energies of 10, 20 and 40 V. The same chromatographic conditions were applied as for the quantitative analysis. Molecular formula suggestions were generated by the Agilent MassHunter Qualitative Analysis B.07.00 software. Furthermore, the software checked the MS scan data against the theoretical isotope distribution of the proposed formula to determine the MS match score, which implies confidence of the calculated formula. Theoretical atomic masses and m/z values were  calculated using ChemSketch 2015 and chemcalc.org (© 2014 Ecole  de rale de Lausanne). Polytechnique Fe 2.5. Toxicity tests At first, the toxicity was determined as growth inhibition of Bacillus subtilis. The bacterial suspension (B. subtilis DSM 10) was

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freshly prepared in Luria broth medium containing 20 g/L casein peptone, 10 g/L yeast extract, and 20 g/L sodium chloride. The concentration of the bacteria suspension was adjusted in order to obtain an optical density measured at 600 nm (OD600) of 0.04. Solutions of 10 mg/L LIN and 10 mg/L TED were exposed to UV254nm light for the time corresponding to their double (t1/4), quadruple (t1/16), and sextuple (t1/64) half-life by UV254nm irradiation. Afterwards, these irradiated as well as the non-irradiated ABs solutions were diluted with sterile ultrapure water by factors of 2, 4, 8, 16, 32, 64 and 128. 100 mL of the diluted or undiluted ABs solutions and 100 mL of the bacteria suspension were filled into a 96well microtiter plate (Nunclon Delta Surface, Thermo Scientific). After 8 h incubation at 30  C, the OD600 of the bacterial suspension was determined using a Victor3 Wallac 1420 microtiter plate reader. The growth inhibition was calculated by linear regression of blank samples with bacteria (0% growth inhibition) and without bacteria (100% growth inhibition). In addition to this test, toxicity on Aliivibrio fischeri was conducted according to DIN EN ISO 113483 (2007). The samples were irradiated in the same manner as solutions used for the growth inhibition test.

3. Results and discussion 3.1. Kinetic study To evaluate the susceptibility of LIN and TED to simulated sunlight, their UV/Vis spectra were recorded. The ABs showed no significant absorbance at wavelengths higher than 350 nm and an increasing absorbance to shorter wavelengths in the range near 200 nm (Fig. 1). LIN showed two relative absorbance maxima at 252 nm and TED at 299 nm. TED generally exhibited higher absorbance values in the range of sunlight, what indicates better conditions for direct photolysis (Albini, 2016). The pseudo-first order degradation rate constants (Albini, 2016) of LIN and TED under simulated sunlight and in absence of light were determined in ultrapure water and two types of river water. The characteristic parameters like pH and turbidity are given in Table SI 3. Fig. SI 2 shows the UV/Vis-spectra of both surface water samples. Fig. 2 and Table SI 4 show the degradation rate constants in ultrapure water (UW) and in water from the river Alb (RW 1) and Rhine (RW 2). Table SI 4 contains also corresponding half-lives for all investigated processes. In ultrapure water solutions, the photolytic degradation rate constant of LIN was calculated as 0.022 ± 0.004 h1. Despite the higher absorption of TED in the UV-range than LIN (Fig. 1), it abated three times slower with a rate constant of 0.008 ± 0.002 h1. The

Fig. 2. Degradation rate constants of irradiated (bright) and dark control samples of LIN and TED in ultrapure water (UW) solutions and two types of river water (RW) spiked with ABs under simulated sunlight (1 kW/m2, 19  C, c0 ¼ 1 mg/L). Error bars correspond to one standard deviation from the mean value.

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higher photolytic reactivity of LIN may result from the lower stability of the morpholinyl ring, which is absent in TED. LIN’s susceptibility to photolysis has been previously reported (Fasani et al., 2009, 2008; Martin et al., 1999), whereas no reference about the photolysis of TED was found. However, tedizolid phosphate is described to show no significant photodegradation in solid form (Lei et al., 2017). To the authors’ knowledge, there are no reference values for kinetic rate constants given in literature concerning photolytic degradation of oxazolidinone ABs until now. The dark control samples showed no significant decay of LIN with rate constants between 0.0005 and 0.0012 h1. This indicates that LIN is stable in ultrapure water and does not undergo hydrolytic degradation. Much higher rate constant of LIN’s degradation were observed in irradiated solution (Fig. 2) that can be attributed to photolysis. Despite presence of organic matter in river water which is known to absorb UV light (Fig. SI 2, Table SI 3), the observed photolytic degradation rate constants of 0.021 and 0.022 h1 for LIN in river water were in comparable range as in pure solution (0.22 h1). This insignificant difference in the rate constants could be explained by combined effect of light absorption by DOC on one hand, and on the other hand by photosensitisation, which enhances the degradation of LIN. TED showed considerably different behaviour in surface waters compared to LIN. No significant variation in TED abatement between irradiated samples and dark control in the natural water was observed. In river water, rate constants in the dark control solution calculated in the range of 0.0048 and 0.0057 h1 were close to the rate constants observed in irradiated samples of 0.0048 and 0.0049 h1 (Table SI 4). This indicates that sunlight cannot enhance the degradation of TED in river water. In contrast to these values, the degradation rate constant of TED in non-irradiated ultrapure water was 0.0004 h1, meaning that TED is stable to hydrolysis under these conditions. Further environmental factors, which were not simulated during the experiments such as light absorption in deeper water bodies, day-night cycle, weather and seasonal differences, may influence the results significantly, most likely towards higher persistency. Compared to kinetic properties of other ABs investigated under comparable conditions, LIN and TED were degraded rather slowly. For instance, fluoroquinolones exposed to sunlight degraded with rate constants between 0.26 and 0.82 h1 (Batchu et al., 2014; Lam and Mabury, 2005; Prabhakaran et al., 2009). Degradation rate constants for sulfamethoxazole in the comparable conditions were calculated in the range of 1.9e3.1 h1 (Batchu et al., 2014; Lam and Mabury, 2005). b-lactams showed reaction constants ranging from 0.10 to 0.21 h1 (Timm et al., 2019). The highest photostability was observed for macrolides with rate constants

between 0.006 and 0.012 h1 (Batchu et al., 2014) and oxazolidinones with rate constants between 0.008 and 0.022 h1. However, the experimental conditions like light emission differed in these studies. 3.2. Structure elucidation of transformation products LC-QToF-MS scans of LIN and TED solutions exposed to simulated sunlight suggested formation of seven and five TPs, respectively. Form this group 4 and 2 TPs of LIN and TED, respectively, were also detected in the samples irradiated by UV254nm light. Table 1 shows the tentative TPs of LIN and TED after irradiation. All TPs were detected in its non-labelled and deuterated form indicating that the deuterated part of the molecule was not transformed by irradiation. Based on the interpretation of the MS2spectra (Fig. SI 3e32, Table SI 5e20), structures of TPs were proposed and are depicted in Figs. 3e4. Additionally, isotopic distribution provided by the Agilent MassHunter software (Match score) of >87% were considered as a confirmation of the proposed molecular formulas. The assignment of fragments of LIN (Table SI 5, Fig. SI 3) and TED (Table SI 14, Fig. SI 20) are in accordance with published fragmentation pathways (LIN: Tiwari and Bonde, 2012; TED: Lei et al., 2017). LIN underwent three different primary reactions under simulated sunlight, which mainly involved the morpholinyl and fluorophenyl ring. LIN TP1 was formed by the elimination of acetylene leading to the opening of the morpholinyl ring. This structure was already proposed by Tiwari and Bonde (2012) as a TP formed by hydrolysis in basic and neutral conditions. It is also possible, that LIN TP1 is highly sensitive to UV254nm light that results in quick transformation to subsequent TPs. LIN TP1 underwent further defluorination leading to LIN TP2, which was reported by Fasani et al. (2008) and Martin et al. (1999) as photolytic TP of LIN. LIN TP3 is another primary TP of LIN. It is the result of the substitution of fluorine by a hydroxyl group. Formation of this compound was also reported by Fasani et al. (2009). Considering that the fluorophenyl ring belongs to the antibacterial centre together with the oxazolidinone ring, the formation of LIN TP2 and LIN TP3 might potentially decrease the antibacterial activity. Signal assigned as LIN TP4 occurred on chromatograms as two peaks (retention time 2.34 and 2.42 min) with similar MS2-spectra. Until now, the reason for the second peak remains unclear. Since LIN TP4 has only one stereo centre, epimerisation cannot explain the second peak. LIN TP4 can be formed by defluorination of LIN or by dehydrolysis of LIN TP3, both in combination with partial dehydrogenation of the morpholinyl ring. Hypothetically, the

Table 1 Tentative TPs of LIN and TED found in ultrapure water after irradiation. TP

retention time

observed m/z

observed m/z (D3-shift)

difference to Lin/Ted

proposed change

proposed formula

calculated m/z

Error [ppm]

MS Match Score

sunlight

UV254

LIN LIN TP1 LIN TP2 LIN TP3 LIN TP4 LIN TP5 LIN TP6 LIN TP7

3.04 1.96 1.32 2.15 2.34, 2.42 2 1.61 0.46

3.04 e 1.29 2.11 2.34 2 1.61 0.46

338.1516 312.1357 294.1446 336.1554 318.1445 350.135 322.1391 159.0764

341.1705 315.1537 297.1631 339.1739 321.1634 353.1531 325.1584 162.0955

e 26.0159 44.007 1.9962 20.0071 11.9834 16.0125 179.0752

e -C2H2 -C2HF -F, þOH -HF -HF, þo2 -CHF, þo -C10H10FNo

[c16H21FN3o4]þ [C14H19FN3o4]þ [C14H20N3o4]þ [C16H22N3o5]þ [C16H20N3o4]þ [C16H20N3o6]þ [C15H20N3o5]þ [C6H11N2o3]þ

338.1511 312.1354 294.1448 336.1554 318.1448 350.1347 322.1397 159.0764

1.5 1 0.7 0 0.9 0.9 1.9 0

99.94 99.45 95.32 99.89 99.93 99.99 99.99 99.82

TED TED TP1 TED TP2 TED TP3 TED TP4 TED TP5

3.75 3.09. 3.35 3.84 3.35 3.67 2.1

3.74 3.35 e 3.34 e e

371.1263 387.1218 403.1158 369.1309 271.11 206.067

374.1455 390.1397 406.135 372.1493 274.1289 209.0858

e 15.9955 31.9895 1.9954 100.0163 165.0593

e þO þO2 -F, þOH -C4H4O3 -C9H8FNO

[C17H16FN6o3]þ [C17H16FN6o4]þ [C17H16FN6o5]þ [C17H17N6o4]þ [C13H12FN6]þ [C8H3N5o2]þ

371.1262 387.1212 403.1161 369.1306 271.1102 206.0673

0.3 1.5 0.7 0.8 0.7 1.5

99.97 98.3 99.99 95.93 87.04 94.81

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Fig. 3. Photolytic transformation of LIN under simulated sunlight in ultrapure water.

eliminated fluorine or hydroxyl group could adopt one hydrogen atom from the morpholinyl ring and the other hydrogen atom could take place at the phenyl ring. However, this issue needs further verification. This hypothesis could be clarified by an experiment with LIN deuterated at the morpholinyl ring. LIN TP4 was further oxidised to LIN TP5 similarly to a Harries-reaction, in which ozone breaks an alkene bond to two aldehydes (Harries, 1905; Rubin, 2003). Probably, UV-light causes the formation of reactive oxygen species (Boreen et al., 2008; de Jager et al., 2017), which were responsible for the oxidative ring opening of LIN TP4. The further reaction of LIN TP5 was the decarbonylation to LIN TP6. Fasani et al. (2008) and Martin et al. (1999) already described LIN TP5 and LIN TP6 as phototransformation products. Finally, the single oxazolidinone ring was found as LIN TP7. It can be formed from LIN as well as from LIN TP1-6. Fig. 4 shows a proposed phototransformation pathway of TED. Although LIN and TED belong to the same class of ABs, the observed transformations were substantially different. The signal corresponding to TED TP1 was observed as two peaks on chromatogram (with similar MS2-spectra) of samples irradiated by simulated sunlight and as one peak after UV254nm irradiation. TED TP1 resulted from the oxidation of the pyridinyl ring. According to Lei

et al. (2017) the oxidation of the pyridinyl ring takes place at the imine moiety. However, based on MS2 spectra only, this assumption cannot be confirmed. Two peaks registered for TED TP1 after exposition to simulated sunlight could be explained by the oxidation in two positions of the pyridinyl ring resulting in formation of two stereoisomers of TED TP1. Oxidation of the oxazolidinone ring in TED TP1 led to formation of TED TP2. TED TP3 and TED TP4 were primary TPs. Similarly to LIN TP3, TED TP3 was formed by the substitution of fluorine by a hydroxyl moiety. The elimination of the oxazolidinone ring of TED TP3 led to the formation of TED TP4. Finally, TED TP5 was formed by the elimination of the phenyl ring, possibly from TED, TED TP3 and TED TP4. The eliminated oxazolidinone and phenyl ring were not found. Similarly to LIN TP1, TED TP2 and TED TP4-5 not found in samples irradiated by UV254nm light, this might indicate that these TPs may be highly photolabile under UV254nm light. All presented TPs structures are proposed based on MS2-spectra interpretation of both labelled and non-labelled compounds, therefore despite the lack of comparison with reference standards or reference spectra, they demonstrate high confidence level. Since the proposed structures are based on the interpretation of MS2 spectra, the structures are tentative candidates according the level 3 of

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Fig. 4. Photolytic transformation of TED under simulated sunlight in ultrapure water.

confidence in the categorisation of Schymanski et al. (2014). In addition to the structure elucidation, TPs were semiquantified by measuring their peak area during the irradiation by simulated sunlight (Fig. SI 33 and 35, for LIN and TED, respectively), and by UV254nm light. (Fig. SI 34 and 36, for LIN and TED, respectively). In the case of sunlight degradation, the highest intensity of the secondary LIN TP 2 was measured 48 h irradiation (Fig, SI 33), which is 24 h later than the primary LIN TP1. This is in agreement with the proposed transformation pathway, in which LIN TP2 is formed from LIN TP1. Similar to that, the highest signal intensity of LIN TP 4 was measured after 24 h, LIN TP 5 after 48 h and LIN TP 6 after 96 h, which confirms the pathway from the primary LIN TP4 to the tertiary LIN TP 6. Except of LIN TP 7, all TPs of LIN showed photodegradation since the signal intensity decreased after their maxima. LIN TP 7 as well as TED TP2 and TED TP5 did not show photodegradation during 168 h of irradiation by simulated sunlight (Fig. SI 35). TED TP 1, TED TP3 and TED TP4 showed the highest signal intensity after 96 h of irradiation, which indicates a slower degradation of TED compared to LIN.

3.3. Toxicity tests Growth inhibition tests on B. subtilis were performed using dilutions of UV254nm irradiated samples. Fig. 5 shows the effect of post-reactional solutions of LIN and TED collected after various duration of sunlight exposure on growth of Bacillus subtilis. Irradiated solutions of ABs exhibited lower antibacterial efficacy than non-irradiated solutions. Both ABs showed a decline of antibacterial efficacy after irradiation, as presented in Fig. 5. Before irradiation, the undiluted solution of LIN caused a growth inhibition of 84% and after the six fold half-life of irradiation the growth inhibition was 66%. This indicates, that irradiation decreases the efficacy of LIN.

The behaviour of TED was similar to LIN. The growth inhibition before irradiation was 89% and after the six fold half-life this value was at 71%. Since there is a non-linear relationship between initial antibiotic concentration and growth inhibition, the growth inhibition of the irradiated solutions does not indicate that 66% of LIN or 71% of TED was transformed to antibacterial TPs. The remaining growth inhibition effect after UV254nm exposure is coming mainly from the presence of parent compound. Despite the long UV254nm exposure, it is important to remember that initial concentration of both antibiotics was 5 mg/L. That means that for instance after 6*t1/ 2, the remaining concentration of parent compounds is still in the range of 50e100 mg/L, that, generally speaking, is still high enough to efficiently affect bacterial growth. From the results in Fig. 5 and Table SI 21 we can state, the efficacy of LIN solution partially remained after irradiation due to the formation of TPs. This observation can be explained by the TPs, which have structural features responsible antibacterial effect, namely intact oxazolidinone ring, such as LIN TP1. Their presence remained relatively constant during the UV254nm experiment, as depicted in Fig. SI 33. In the case of TED, closer analysis of Fig. 5 and Table SI 22 revealed that efficacy of TED solution was fully eliminated. This statement can be confirmed by Fig. SI 36, in which we can see that the only TPs that based on their structure could exhibit antibacterial effect, namely TED TP1 and TED TP3, are almost fully transformed within 120 min of UV light exposure. Both ABs showed a TP, in which the fluorine atom was replaced by a hydroxyl moiety. Together with the oxazolidinone ring, the fluorine atom contributes to the activity of the AB (Barbachyn and Ford, 2003; Zhanel et al., 2015). This can explain the partial decline of the efficacy of LIN after irradiation. Nonetheless, the differing composition concerning presence of TPs dependence on the irradiation source should be considered

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Fig. 5. Growth inhibition on B. subtilis by LIN (t1/2 ¼ 12 s) (a) and (b) TED (t1/2 ¼ 42 min) before and after different exposition to UV254 irradiation (n ¼ 3, standard deviation < 7%, c0 ¼ 10 mg/L).

when estimating growth inhibition for sunlight treated solutions. Finally, neither TPs were neither quantified nor used for toxicity tests of individual substances. Furthermore, the toxicity was tested as a luminescent bacteria test (ISO 11348-2). LIN and TED both showed an initial bioluminescence inhibition of 49% and 46% respectively, thus they were classified to have medium toxicity. Irradiation of TED samples slightly increased the inhibitory effect up to 55%. Irradiated solutions of LIN showed a decline of toxicity down to 34% after t1/16, implying low toxicity, as presented in Fig. 6 and Table SI 23. Tedizolid phosphate is classified as having chronic aquatic toxicity. LIN and TED both bind to the 23 S rRNA of the 50 S subunit in bacterial ribosomes, preventing protein synthesis and thus inhibiting the bacterial metabolism (Zhanel et al., 2015). Therefore, inhibition of the bioluminescence of A. fischeri was expected. Irradiated samples of LIN or TED did not alter their bioluminescence inhibition and thus toxicity significantly (Table SI 23). Therefore, it is assumed that TPs of both ABs also cause a certain inhibitory effect on bacteria. However, the categories of toxicity based on DIN 32465:2008e11 describe the interference of biomolecular reactions like the biosynthesis of luciferase or luciferin as well as its encymatic oxidation (Madigan et al., 2012) leading to bioluminescence. It does not measure bactericide or bacteriostatic efficacy of a sample.

showed that photolysis of LIN mainly affects the morpholinyl ring and the fluorophenyl ring, while the oxazolidinone ring remains intact. No transformation at the oxazolidinone ring, and only partial modification of fluorophenyl ring, both known to be responsible for the antibiotic’s efficacy, explain the growth inhibition of bacteria exposed to LIN’s TPs. For TED, some TPs with a transformation at the oxazolidinone ring have been found, and for these TPs lower growth inhibitory effect against Bacillus subtilis was observed, despite much higher initial concentration of antibiotic compared to concentrations used in the kinetics study. Since the toxicity tests were performed with a mixture of TPs generated by irradiation of the ABs, it is still unknown, which of the observed TPs are antibacterial potent. To clarify this issue, the toxicity test with reference standards of the individual substances would be necessary. The results of this study contribute in the general understanding of the photochemistry of two representatives of antibiotics of last resort and their potential behaviour in aquatic environment. We pointed out that these substances, due to their limited photodegradability (especially of TED), as well as due to formed phototransformation products exhibiting antibacterial properties, could be a potential thread to receiving waters. More detailed study focusing on the behaviour in natural systems is needed to assess the scale of this thread.

4. Conclusion In this study, the fate of LIN and TED, under sunlight exposure was investigated, with focus on the TPs formation. The results showed that simulated sunlight is able to degrade both oxazolidinone ABs, however, with lower degradation rate constants than for other ABs, such as sulfonamides or fluoroquinolones. Especially TED was highly photostable. We suspect that the in the case of LIN, the photodegradation was driven by direct photolysis and photosensitized degradation transmitted by DOC in surface water. Due to the higher susceptibility of LIN than TED to photodegradation, LIN’s TPs are more likely to occur in the aquatic environment. We

Fig. 6. Bioluminescence inhibition on A. fischeri caused by solutions of LIN and TED before and after UV254 irradiation (c0 ¼ 10 mg/L).

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