Photodegradation of uoroquinolones in aqueous solution under light conditions relevant to surface waters, toxicity assessment of photoproduct mixtures

Sarka Klementova (  sklement@jcu.cz ) University of South Bohemia Faculty of Science: Jihoceska Univerzita v Ceskych Budejovicich Prirodovedecka Fakulta https://orcid.org/0000-0003-3646-5945 Martina Poncarová University of South Bohemia: Jihoceska Univerzita v Ceskych Budejovicich Helena Langhansová University of South Bohemia Faculty of Science: Jihoceska Univerzita v Ceskych Budejovicich Prirodovedecka Fakulta Jaroslava Lieskovská University of South Bohemia Faculty of Science: Jihoceska Univerzita v Ceskych Budejovicich Prirodovedecka Fakulta David Kahoun University of South Bohemia Faculty of Science: Jihoceska Univerzita v Ceskych Budejovicich Prirodovedecka Fakulta Pavla Fojtíková University of South Bohemia Faculty of Science: Jihoceska Univerzita v Ceskych Budejovicich Prirodovedecka Fakulta


Introduction
Antibiotics have been increasingly detected in surface waters in the past few decades, and are thus classi ed as pollutants of emerging signi cance, though there is limited understanding of their environmental fate and toxicological effects (Van Doorslaer et al., 2014).
Among antibiotics, uoroquinolones represent a group widely used in both human and veterinary medicine. According to WHO, uoroquinolones are considered "highest priority critically important antibiotics" (Roth et al., 2019). An estimation of quinolone production and usage from the year 2007 shows an amount of around 50 tons in the form of proprietary products and 70 tons in the form of generic quinolones for the USA, EU, Japan and South Korea, while with respect to China the estimate reached almost 2000 tons (Sukul & Spiteller;2007). The review article of Frade et al. (2014) provides an overview of environmental concentrations of uoroquinolones found in surface waters, waste water treatment plants in uents and e uents and hospital e uents in European countries, the USA, Japan and China -the concentration range is from tens of ng/l to several mg/l for surface waters as well as for waste-water treatment plant in uents. The higest concentrations, reaching hundreds of mg/l were found in hospital e uents.
The objectives of this study were: (1) To test whether three widely used uoroquinolone representants, namely cipro oxacin, enro oxacin, and nor oxacin, may undergo photochemical degradation under irradiation relevant to natural short-wavelength conditions and at neutral and mildly alkaline pH values common in natural waters. (2) To reveal whether the degradation pathways and products formed during degradation processes depend on pH. (3) To ascertain whether this degradation leads to complete or partial loss of antibacterial activity.

Materials And Methods
Photodegradation procedure Sample preparation: Cipro oxacin, enro oxacin, and nor oxacin (Sigma-Aldrich, all ≥ 98%, HPLC) solutions were prepared by the dissolution of 5 mg of the compound in 4 ml HCl (0.1 mol/l), then diluted to 100 ml with Milli-Q ® water. The pH values were adjusted by 10 % NaOH, for neutral pH to a range of 7.0 -7.13, for alkaline pH to 8.13 -8.8. Irradiation: Samples containing 3 ml of an antibiotic solution in 1-cm glass cuvettes with PTFE lids were irradiated in a Rayonet reactor with RPR 3000 Å lamps emitting light at a wavelength range of 254-350 nm -light below 300 nm was ltered out by optical glass to imitate short-wavelength solar radiation that reaches the Earth's surface. Radiant ux was measured using a Lutron UV A light metre, the total power of all the electromagnetic radiation within the wavelength range 320 -390 nm (the range detected by the Lutron metre) emitted per unit time was calculated for the irradiated area, the value being 4.5 W.
Analyses of samples: The extent of uoroquinolones photodegradation was determined by HPLC (ThermoScienti c Dionex Ultimate, column Phenomenex Kinetex ® 5 mm EVO C18, 30 ×2.1 mm, mobile phase 0.1 % HCOOH in water and acetonitrile 1:1) with PDA 3000RS spectrophotometric and FLD 3000RS uorescence detectors. The identi cation of products was performed using a HPLC-MS system (Thermo Scienti c Ultimate 3000 Rapid Separation Quaternary System coupled with Velos Pro mass spectrometer with dual-pressure linear ion trap). The details of HPLC-MS measurement are summarized in Tab. 1. Ion chromatography (Dionex ISC 3000, Dionex Ion Pack AS11-HC column 250 mm, mobile phase KOH from EG C III KOH eluent generator with AERS 500 suppressor) was applied for the detection of low-molecular charged products.
Antibacterial activity tests: Escherichia coli Seattle 1946 (ATCC 25922) and Staphylococcus epidermidis RP62A (ATCC 35984) bacterial strains were grown in LB medium overnight at 37 °C with constant agitation. Then the bacteria were diluted to 200 000 cells per ml of DMEM culture medium supplemented with 10 % fetal calf serum and seeded into 96-well plate. Undiluted, 10-fold diluted and 100-fold diluted samples were added to the bacteria and cultivated at 37 °C and 5 % CO 2 for 3 hours. Bacterial viability was quanti ed by measuring uorescence upon excitation and emission wavelengths of 550 nm and 590 nm, respectively, after an additional 3 and 6 hours of cultivation using alamarBlue™ Cell Viability Reagent (ThermoFisher Scienti c). The values from photoproduct-containing samples were normalized to control (antibiotics without photoproducts), i.e. the metabolic activity of the control samples (as a function of bacterial proliferation) is set to 100 %. Data are represented as means and standard error of mean (SEM). Differences between samples were evaluated using 2-way analysis of variance followed by Tukey posthoc test.

Results
The photoinitiated degradation of the three studied substances (cipro oxacin, enro oxacin and nor oxacin) followed rst order kinetics. Their kinetic characteristics are summarized in Tab.2. The values of rate constants are of the same order of magnitude, nevertheless cipro oxacin differed from the other two compounds since it has a higher reaction rate at neutral pH while enro oxacin and nor oxacin were degraded more quickly at alkaline pH. The half-lives of the uoroquinolones under the experimental conditions were in the range of 0.9 to 2.7 min.
The HPLC analyses revealed the formation of a signi cant amount of intermediates and nal products in all reaction systems. Thus, reaction mixtures at irradiation times providing the most complex reaction mixtures were chosen for HPLC-MS and for antibacterial activity tests.
The reaction schemes of cipro oxacin in neutral and alkaline solution are demonstrated in Fig. 1 and 2, respectively, The observed m/z values of products and their proposed structures are summarised in Tab.
3 for neutral pH and in Tab. 4 for alkaline pH. Tab. 4 contains additional three low-molecular ions ( uoride, formate and acetate) that were detected in the reaction mixture using ion chromatography. The scheme for neutral pH (Fig. 1) contains the pathways for formation of 13 products, The main degradation mechanisms are de uorination, hydroxylation, decarboxylation, and the opening and further degradation of cyclic structures. In alkaline pH, 26 products were documented, for 22 of them their chemical structure has been proposed. Under these conditions, the double hydroxylated product 1 and protonated structure of product 23 are the main sources for a series of further product formation.
The photoinitiated degradation of the three studied substances (cipro oxacin, enro oxacin and nor oxacin) followed rst order kinetics. Their kinetic characteristics are summarized in Tab.2. The values of rate constants are of the same order of magnitude, nevertheless cipro oxacin differed from the other two compounds since it has a higher reaction rate at neutral pH while enro oxacin and nor oxacin were degraded more quickly at alkaline pH. The half-lives of the uoroquinolones under the experimental conditions were in the range of 0.9 to 2.7 min.
The HPLC analyses revealed the formation of a signi cant amount of intermediates and nal products in all reaction systems. Thus, reaction mixtures at irradiation times providing the most complex reaction mixtures were chosen for HPLC-MS and for antibacterial activity tests.
The reaction schemes of cipro oxacin in neutral and alkaline solution are demonstrated in Fig. 1 and 2, respectively, The observed m/z values of products and their proposed structures are summarised in Tab. 3 for neutral pH and in Tab. 4 for alkaline pH. Tab. 4 contains additional three low-molecular ions ( uoride, formate and acetate) that were detected in the reaction mixture using ion chromatography. The scheme for neutral pH (Fig. 1) contains the pathways for formation of 13 products, The main degradation mechanisms are de uorination, hydroxylation, decarboxylation, and the opening and further degradation of cyclic structures. In alkaline pH, 26 products were documented, for 22 of them their chemical structure has been proposed. Under these conditions, the double hydroxylated product 1 and protonated structure of product 23 are the main sources for a series of further product formation.
Enro oxacin at neutral pH (Fig. 3) has the lowest number of products -only nine were found in the reaction mixture, their structures are shown in Tab. 5. In alkaline medium, 19 products were detectable, for 16 of them structures are proposed (Tab. 6). The degradation pathways are presented in Fig. 4. In all cases, the degradation in alkaline medium lead to a signi cantly greater amount of products, nevertheless the degradation pathway (de uorination, hydroxylation, decarboxylation, and the opening and further degradation of cyclic structures) were common under all conditions tested.
Antibacterial ativity was tested with paternal compounds and mixtures of photoproducts, i.e. irradiated samples with developed intermediates and products pro les, using the same irradiation times as in the HPLC-MS analyses. Results are summarised in Fig. 7.
As can be seen from Fig. 7, the inhibitory activities against the two tested bacteria strains were signi cantly higher for photoproducts of cipro oxacin and enro oxacin in comparison to the parental antibiotics (numbers in the presence of the respective parental antibiotic represents control column in Fig. 7). Cipro oxacin photoproducts exhibited higher inhibition towards E. coli when irradiated at neutral pH, the effect being more pronounced with increasing cultivation time; a lesser signi cant inhibitory effect was observed in the reaction mixture of cipro oxacin irradiated at alkaline pH. The inhibitory effects towards S. epidermidis were on the other hand evidently higher in photoproduct mixtures irradiated at mildly alkaline pH.
Enro oxacin photoproducts exhibit an extremely signi cant decrease in the numbers of bacteria towards both tested species with the effect being more noticeable with regard to photoproducts that were formed by photochemical degradation at mildly alkaline pH.
Regarding nor oxacin, no differences in the antibacterial activity of photoproduct mixture when compared to nor oxacin itself were discerned towards either tested species.

Discussion
The phototranformation of the studied substrates followed rst-order kinetics, the values of reaction rate constants are similar to those attained by Babić et al. (2013) and Wammer et al. (2013) for cipro oxacin and enro oxacin. They observed a faster photodegradation of nor oxacin, but this may have been caused by a higher concentration of antibiotics in this study in comparison to their experiment, since Babić et al. (2013) noticed a decrease in reaction rate in relation to increasing concentration.
The degradation mechanism of the studied uoroquinolone antibiotics depends strongly on the pH value of the irradiated reaction mixture. Even though the main degradation pathways involve in all cases processes such as de uorination, hydroxylation, decarboxylation, and the opening and further degradation of the cyclic structures under all conditions tested, the number of products was noticeably higher when the photodegradation was carried out at alkaline pH. The ndings are in agreement with the results of Zhichao Zhang et al. (2019) who revealed the in uence of chemical speciation on photochemical transformation of three uoroquinolones (lome oxacin, nor oxacin and o oxacin). In their study, they also observed de uorination, decarboxylation and direct piperazinyl ring oxidation. Generally it can be concluded that at alkaline pH, hydroxylation prevails due to abundant hydroxyl groups. Nevertheless, the hydroxylated products depend on the character of the antibiotics: with cipro oxacin and nor oxacin, a substitution of the OH group for the F atom and a direct hydroxylation of the piperazinyl ring and its futher oxidation and breakage were observed, whereas in enro oxacin the OH group was bound to the ethyl group on the piperazinyl group. Enro oxacin photodegradation provided cipro oxacin in both neutral and alkaline pH; the production of cipro oxacin from enro oxacin was mentioned in the study of Babić et al. (2013), though their subsequent products differ from the products presented in this study, since their reaction mixture contained humic acid which may affect degradation pathways. The products No 7 and 8 (m/z = 374 and 358, respectively) of enro oxacin photodegradation at neutral pH were noticed by Wammer et al. (2013). Two of the products of enro oxacin degraded at alkaline pH were reported by Li et al.(2011), product No 2 (m/s = 316.2 in + ESI) and product No 6 (m/s =372.2 in + ESI). Product No 13 (m/s = 294.1) was found by Ahmad et al. (2015).
Antibacterial activity tests performed in the study against two bacterial strains came from the assumption (null hypothesis) that photochemical degradation would result in a decrease or complete loss of antibacterial activity. The loss of antibacterial activity was shown to be true for nor oxacin since the mixture of nor oxacin photoproducts exhibited the same inhibitory effect as the antibiotic itself; since the dose of photoproducts mixture for the test was calculated to contain the same amount of parental antibiotics as the antibiotic sample, the result shows conclusively that nor oxacin photoproducts do not contribute to inhibition of the bacterial growth in either tested species. This corresponds with the results of bioluminescent inhibition tests on Vibrio scheri performed by Zhichao Zhang et al. (2019) in which they observed a signi cant decrease in toxicity for nor oxacin and o oxacin.
The null hypothesis about the decrease of antibacterial activity through photochemical degradation must be rejected for the two remaining compounds tested in this study, cipro oxacin and enro oxacin. Taking into account the statistical evaluation presented in Fig. 7, only for the pair cipro oxacin/alkaline photoproducts tested on E. coli were the p-values less than or equal to 0.05 and 0.01 for 6 and 9 hour incubation, resp; nevertheless even these values of p-value are considered to be signi cant for rejecting null hypothesis. In all other cases, the pairs control/photoproducts p-values were 0.0001, which means there is an extreme statistical signi cance that photoproduct toxicity to bacteria is notably higher than those of parent antibiotics themselves. The difference between photoproducts formed at neutral and mildly alkaline pH and the extremely signi cant higher toxicity of photoproducts formed in alkaline solution were proven with regard to enro oxacin toward both tested species. With regard to cipro oxacin, photoproducts formed at neutral pH exhibited extremely signi cant higher antibacterial activity against E. coli; against S. epidermidis, though no signi cant difference was observed after longer incubation time. This is in agreement with the results of Sturini et al. (2012) who found that the irradiation of four uoroquinolone antibiotics (cipro oxacin, dano oxacin, levo oxacin and moxi oxacin) dissolved in untreated river water under solar light led to photoproducts that possessed residual antibacterial activity towards E. coli and S. aureus. The authors attributed the activity manifestation to the products conserving the fundamentals of the uoroquinolone structure. Since a signi cant portion of photoproducts detected in our study retain the basic uoroquinolone structure, the hypothesis that this structure is responsible for the antibacterial activity may be considered viable. Nevertheless, the increased antibacterial effect in some photoproduct mixtures observed in this study suggests that some modi cations of the base structure lead to augmentation of the antibacterial effect when compared to the original antibiotic. Thus, though photodegradation may represent a feasible degradation pathway for uoroquinolone antibiotics in surface waters, there is even the possibility of production substances possessing enhanced antibacterial activity which may pose a threat to microorganism populations. Photodegradation pathways of cipro oxacin at alkaline pH (pH = 8.5). Numbers represent individual degradation products according to Tab. 4.