Tarek Manasfi

Michel De Méo

Bruno Coulomb

Carole Di Giorgio

Sylvain Ravier

Jean-Luc Boudenne

Development of mutagenic activity following the chlorination of the sunscreen UV filter benzophenone-8 (dioxybenzone) in bromide-rich water

Keywords: Sunscreen UV filters, chlorination, disinfection byproducts, mutagenicity, halogenated tranformation products

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Protection against excessive exposure to ultraviolet (UV) light is recommended as one of the major preventive measures to reduce risks of skin cancer (American Cancer Society, 2016).

Sunscreens are an adjunct to clothing and other physical means of solar UV radiation protection (CDC, 2018). In sunscreens, inorganic and/or organic UV filters are the active ingredients since these compounds act mainly by reflecting and scattering (inorganic) or absorbing (organic) radiation. In sunscreens, usually more than one UV filtering agents are added to achieve a broad-spectrum protection against UV radiation [START_REF] Serpone | Inorganic and organic UV filters: Their role and efficacy in sunscreens and suncare products[END_REF]. The concentrations of UV filters in the final sunscreen product should not exceed certain maximum concentrations, which vary from one country or region to another. In the US, the FDA published a final sunscreen monograph in the 1999, which included 16 sunscreen active ingredients including avobenzone, dioxybenzone, oxybenzone, octocrylene, and octyl methoxy cinnamate (OMC) also known as octinoxate (FDA, 1999). In the European Union, REGULATION (EC) No 1223/2009 on cosmetic products defines UV filters allowed in cosmetic products as well as the maximum concentrations in the final preparations (EC, 2009). Among the allowed UV filters in the EU are: oxybenzone, avobenzone, octocrylene, OMC, but not dioxybenzone. However, the latter has been detected in some swimming pool waters in Europe [START_REF] Ekowati | Occurrence of pharmaceuticals and UV filters in swimming pools and spas[END_REF][START_REF] Lempart | The presence of compounds from the personal care products group in swimming pool water[END_REF]. In addition to their interest for protecting consumers, UV filters are also added to some cosmetic products to confer protection for products against photodegradation.

Although the benefits of use of sunscreens is well established in helping protecting agains sunburns, skin ageing, and skin cancer, there are some concerns about some adverse effects for some UV filter agents. Among the adverse effects or risks for some UV filters: trolamine salicylate risks include the potential for bleeding and increased salicylate toxicity (FDA, 2019), for para-aminobenzoic acid (PABA) risks include allergic and photoallergic skin reactions [START_REF] Fisher | Sunscreen Dermatitis: Para-Aminobenzoic Acid and its Derivatives[END_REF]. Besides, the potential percutaneous absorption of some UV filters and the subsequent induction of systemic effects such as estrogenic and antiandrogenic activity has been one of the major concerns [START_REF] Schlumpf | In vitro and in vivo estrogenicity of UV screens[END_REF][START_REF] Ma | UV filters with antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation assay[END_REF][START_REF] Krause | Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters[END_REF]. In addition, UV filters have been shown to affect the aquatic environment to which they can enter either indirectly in wastewater or directly from recreational activities such as swimming [START_REF] Giokas | UV filters: from sunscreens to human body and the environment[END_REF]. The described environmental effects include coral bleaching [START_REF] Danovaro | Sunscreens cause coral bleaching by promoting viral infections[END_REF], effects on marine bacterioplankton (Danavaro and Corinaldesi, 2003), and endocrine disruption [START_REF] Schreurs | Interaction of polycyclic musks and UV filters with the estrogen receptor (ER), androgen receptor (AR), and progesterone receptor (PR) in reporter gene bioassays[END_REF]klan et al., 2005;[START_REF] Brausch | A review of personal care products in the aquatic environment: environmental concentrations and toxicity[END_REF]. Many UV filters possess high lipohilicity allowing them to bioaccumulate in aquatic organisms [START_REF] Tsui | Occurrence, distribution and ecological risk assessment of multiple classes of UV filters in surface waters from different countries[END_REF]. Another active area of research regarding UV filters is the study of their transformation in the environment or in recreational areas such as swimming pools when exposed to light or oxidants such as chlorine. There are growing concerns regarding the formation of potentially toxic transformation products or byproducts from UV filters [START_REF] Li | Transformation of benzophenone-type UV filters by chlorine: Kinetics, products identification and toxicity assessments[END_REF]. Although UV filters are required to be photostable to fulfill their role in sunscreens, there have been some reports about the photodegradation of certain UV filters (such as OMC) and increased toxicity (Diaz-Cruz et al., 2008). Furthermore, the reaction of UV filters with chlorine added for disinfection of swimming pool water or in wastewater treatment plants has received the attention of researchers since several years. In these studies, halogenated transformation products and disinfection byproducts have been identified for several organic UV filters [START_REF] Santos | The degradation products of UV filters in aqueous and chlorinated aqueous solutions[END_REF][START_REF] Duirk | Reaction of benzophenone UV filters in the presence of aqueous chlorine: kinetics and chloroform formation[END_REF][START_REF] Sakkas | Aqueous photolysis of the sunscreen agent octyl-dimethyl-p-aminobenzoic acid: formation of disinfection byproducts in chlorinated swimming pool water[END_REF][START_REF] Negreira | Study of some UV filters stability in chlorinated water and identification of halogenated by-products by gas chromatography-mass spectrometry[END_REF][START_REF] Manasfi | Degradation products of benzophenone-3 in chlorinated seawater swimming pools[END_REF], Trebše et al., 2016;[START_REF] Kalister | A chromatography-mass spectrometry study of aquatic chlorination of UV-filter avobenzone[END_REF]Manasfi et al., 2017a). Despite evidence of transformation and formation of byproducts, there have been very few studies investigating the toxicity of the formed transformation products. [START_REF] Liu | Acute toxicity formation potential of benzophenone-type UV filters in chlorination disinfection process[END_REF] investigated the transformation and toxicity of 14 benzophenone-type UV filters during chlorination. Toxicity was examined using the photobacterium bioassay method that quantifies the decrease in light emission from the Photobacterium phosphoreum bacteria because of exposure. In this bioassay, only one UV filter (BP-13) was found to induce significant increase of toxicity following chlorination.

However, in another study by [START_REF] Li | Transformation of benzophenone-type UV filters by chlorine: Kinetics, products identification and toxicity assessments[END_REF], enhanced toxicity was observed following the chlorination of several BP-type UV filters. [START_REF] Grbović | Synthesis, characterisation and aquatic ecotoxicity of the UV filter hexyl 2-(4-diethylamino-2-hydroxybenzoyl) benzoate (DHHB) and its chlorinated by-products[END_REF] found that the chlorination byproducts of UV filter hexyl 2-(4-diethylamino-2-hydroxybenzoyl) benzoate (DHHB) induced toxic response in daphnids. Mutagenicity is an endpoint of concern with regard to the toxicity of halogenated transformation products of UV filters in swimming pool water. [START_REF] Nakajima | Aquatic fate of sunscreen agents octyl-4-methoxycinnamate and octyl-4-dimethylaminobenzoate in model swimming pools and the mutagenic assays of their chlorination byproducts[END_REF] detected weak mutagenic activity using Ames test/Salmonella strain TA100 without S9 for the transformation products of OMC in aqueous solution treated with chlorine. [START_REF] Zhang | Chlorination of oxybenzone: kinetics, transformation, disinfection byproducts formation, and genotoxicity changes[END_REF] detected mutagenic activity using SOS/umu test after the chlorination of oxybenzone spiked into ultrapure water. However, despite these few studies, there is a major knowledge gap regarding the toxicity of the formed transformation products and in particular their mutagenic potential. To our knowledge, the mutagenicity of transformation products of UV filters in bromide-rich water (e.g. seawater and brackish water) has not been described so far. In this type of water, upon the addition of chlorine, bromine is formed as a product from the reaction between bromide and chlorine [START_REF] Deborde | Reactions of chlorine with inorganic and organic compounds during water treatment-kinetics and mechanisms: a critical review[END_REF]. Bromine reacts with organic compounds leading to the formation of brominated transformation products and disinfection byproducts (DBPs), known to be far more toxic than their chlorinated analogues [START_REF] Richardson | Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research[END_REF], Manasfi et al., 2017b). To better understand the mutagenicity of halogenated transformation products of UV filters, the mutagenicity of five commonly used UV filters treated with chlorine in seawater was investigated using the Ames test. When a mutagenic activity was detected, to corroborate the findings and understand their origin, chemical analysis using high-resolution mass spectrometry was performed to identify the transformation products responsible for mutagenic activity.

Materials and Methods

Chemicals

Ultrapure water was produced from a Millipore water system (resistivity > 18 MΩ.cm).

Standards of the studied UV filters (purity >98%) were purchased from Sigma-Aldrich benzophenone-d10 was used as an internal standard was also bought from Sigma-Aldrich The byproduct of OXY and DIOXY 2,4,6-tribromo-3-methoxyphenol (TBMP) was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Salicylic acid (purity > 99%) was purchased from Acros Organics (Geel, Belgium). Ammonium formate (UHPLC/MS grade) was purchased from Biosolve Chimie (Dieuze, France).

Methyl tert-butyl ether (MTBE) Chromosolv, HPLC grade, was purchased from Sigma-Aldrich, Germany. Dimethyl sulfoxide (DMSO), Chromasolv plus, ≥ 99.7% was purchased from Sigma-Aldrich, UK.

Stock solutions of chlorine were prepared by diluting a reagent grade commercial solution of sodium hypochlorite (NaOCl, available chlorine 4.00-4.99%, Sigma-Aldrich). Reconstituted seawater was prepared according to the ASTM International standard practice for the preparation of substitute ocean water (method D1141-98, 2013). Dibrominated salicylic acid was produced by chlorinating salicylic acid in artificial seawater according to the same procedure followed for the UV filters (described in the following section).

For Ames test, the Salmonella typhimurium tester strain TA98 was used. This strain was generously supplied by Prof. B.N. Ames (Berkeley, CA, USA). The strain were stored at -80 °C and regularly checked for genetic markers.

Chlorination of UV filters in seawater

Chlorination experiments of the UV filters were carried out at 25 °C. Glass vials with PTFElined screw caps of 65 mL capacities were filled with artificial seawater and placed in a water bath adjusted to 25 °C. Individual UV filters were then spiked at a concentration of 10 µM into separate vials. Chlorine was then added (in the form of NaOCl solution) in a volume corresponding to the desired molar ratio of 1: 10, 1: 100, and 1: 1000 (UV filter : chlorine).

Chlorination reactions were then stopped at different time points by adding sodium thiosulfate as a quenching agent. Sodium thiosulfate was added in 10-fold excess (molar equivalents) with respect to chlorine concentrations. Accordingly, 0.158 g, 1.58 g, and 15.8 g were added to quench reactions at chlorination ratios 1:10, 1:100, and 1:1000, respectively. Sample aliquots (50 mL) were then collected and extracted using liquid-liquid extraction (LLE) with MTBE (5 mL). Organic extracts (4 mL) were then separated from the aqueous phase and divided into two parts. One part (1 mL) was used to perform the chemical analysis using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The other part (3 mL) was treated as described in the following paragraph and used for mutagenicity testing in Salmonella typhimirium (Ames test). To prepare samples for Ames test, the organic extracts (3 mL) were evaporated by placing the container vials in a water bath at 45 ºC and applying a stream of nitrogen gas. After evaporation, DMSO (0.5 mL) was added for solvent exchange. During solvent exchanged, volatile and semi-volatile DBPs (such as bromoform, dibromoacetonitrile) are eliminated, which allows to directly correlate the results of Ames test with the identified non-volatile transformation products of the UV filters. These transformation products are more specific to the UV filters in comparison to the formed DBPs (such as bromoform) which can be produced by diverse precursors including multiple UV filters as described previously by Manasfi et al. (2017).

The same sample treatment was applied to prepare extracts from seawater spiked with UV filters without treatment with chlorine, in order to examine the mutagenicity of the starting products and to ensure that the extraction procedure did not engender any products that could contribute to mutagenicity.

The use of artificial seawater in these experiments was a critical element in the present study, as this ensured that the only organic compound present in solution was the spiked UV filter.

As such, any identified transformation product and observed mutagenicity could be linked to the spiked UV filter without any interference from other organic precursors. This is unlike in natural seawater, where natural organic matter and possibly other present contaminants could also react with the added chlorine contributing to the transformation products and DBPs.

For samples showing mutagenic activity in Ames test, additional chlorination experiments were performed at ratios 1:100 and 1:1000 (UV filter : chlorine) to investigate the fate of transformation products at these higher ratios (as compared to the 1:10 ratio).

Salmonella mutagenicity assay (Ames test)

The mutagenicity assay was carried out according to [START_REF] Maron | Revised methods for the Salmonella mutagenicity test[END_REF], with a modified version of the liquid-incubation technique as described previously [START_REF] De Méo | Optimization of the Salmonella/mammalian microsome assay for urine mutagenesis by experimental designs[END_REF]. The Salmonella tester strain TA98 was used for this study since this strain is capable of detecting frameshift mutations which are more likely to be caused by voluminous compounds (such as polycyclic compounds) [START_REF] Isono | Chemical carcinogens as frameshift mutagens: Salmonella DNA sequence sensitive to mutagenesis by polycyclic carcinogens[END_REF][START_REF] Maron | Revised methods for the Salmonella mutagenicity test[END_REF]. Bacteria were grown for 12 h at 37 °C in Oxoid Nutrient Broth N° 2 containing ampicillin (25 μg/mL) with gentle shaking. After the incubation period, various volumes of extracts of UV filters were added to 0.1 mL of the overnight culture and 0.1 mL of PBS. to test four different sample concentrations. The obtained mixtures were incubated for 60 min in the dark. Melted top agar (2 mL) containing 0.045 mM histidine and biotin was then added. The final mixtures were then poured onto Vogel-Bonner (VB) minimal plates. For each series of experiments, negative controls with DMSO were included to determine the number of spontaneous revertants/plate. 2,4,7-trinitrofluorenone was used as a positive control. After incubation for 48 h, revertant bacterial colonies were counted using an automatic counter (Scan 1200, Interscience, Saint Nom La Bretèche, France). Raw results of Ames test were expressed as number of revertants per plate (rev/plate).

Taking into consideration the relevance of our study to swimming pool water where UV filters are introduced by swimmers to disinfected waters, we did not use S9 mix. Previous investigations showed that disinfected drinking and swimming pool waters were most mutagenic in the absence of S9 mix [START_REF] Takanashi | Surveying the mutagenicity of tap water to elicit the effects of purification processes on Japanese tap water[END_REF][START_REF] Richardson | What's in the pool? A comprehensive identification of disinfection by-products and assessment of mutagenicity of chlorinated and brominated swimming pool water[END_REF][START_REF] Manasfi | Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity[END_REF].

For the interpretation of results of Ames test, analysis was performed in two steps. First, the Dunnett test was carried out to determine if there were a significant difference between the mean number of induced revertants in exposed plates as compared to the mean number of spontaneous revertants in the negative control plates [START_REF] Kim | Statistical methods for the Ames Salmonella assay: a review[END_REF]. If the Dunnett test was positive for at least one dose, a nonlinear regression analysis was performed using an arbitrary model with TableCurve 2D software (version 5.01, Jandel Scientific Software, San Rafael, CA), as described by [START_REF] Kim | Statistical methods for the Ames Salmonella assay: a review[END_REF]. The used model was based on the following equation:

𝑅𝑅𝑅𝑅𝑅𝑅 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑅𝑅 ⁄ = (𝑝𝑝 + 𝑏𝑏 × 𝐷𝐷) × 2 -𝑐𝑐×𝐷𝐷 2
where Rev/plate is the number of revertants by plate, D is the dose, and a, b, and c are calculated coefficients.

Model significance validation was evaluated based on two criteria: (i) model probability (P) being < 0.05 and (ii) error probability (PE) being > 0.05. The mutagenic activity (MA, rev/μg) was defined as the maximal slope of the ascending part of the dose-response curve and was calculated as the first derivative at the origin.

Analytical methods

UPLC-HRMS Analysis. Extracts of field and laboratory samples were analyzed using UPLC system (Acquity, Waters) coupled to quadrupole/time-of-flight mass spectrometry (Q/ToF-MS)

with electrospray ion (ESI) source (Synapt G2 HDMS, Waters, MA, USA). Calibration curves were established for each UV filter by external standard approach using the reference standard.

Benzophenone-d10 was used as surrogate. Calibration standards were prepared by spiking artificial seawater with UV filters and then performing the same procedure as for samples (LLE-UHPLC-MS/MS).

Separations were carried out on a CORTECS UPLC C18 column (2.1×100 mm, 1.6 μm,Waters) at 40 °C. The mobile phase consisted of A: Water with 5 mM ammonium formate (Biosolve, 98%) and B: Methanol (Optima LC-MS, Fischer) with 5 mM ammonium formate. Elution was carried out at a flow rate of 0.4 mL/min with a gradient starting at 15% of solvent B and increasing to reach 100% of solvent B within 6 min and held for 1 min. The sample injection volume was 5 μL.

A solution of sodium formate (HCOONa, Waters Q-ToF product) was infused daily in the ESI source to calibrate the instrument. Optimum ESI conditions were found using a 2.5 kV capillary voltage, 30 V sample cone voltage, 450 °C desolvation temperature, 120 °C source temperature, 20 L/h cone gas flow rate and 800 L/h desolvation gas flow rate. The optimum sample cone voltage for each UV filter has been described previously (Manasfi et al., 2017a).

Identification of transformation products was based on accurate mass measurement (with mass error less than 5 ppm) and MSMS data. For transformation products available commercially in standard form (the case of TBMP), tentative identification was confirmed by comparing retention time and mass spectral data with those of the standard product. For dibrominated salicylic acid, identification was based on accurate mass measurement, MSMS spectral data, as well as comparison to chromatographic and spectral data of 3,5-dibromosalicylic acid.

Results and Discussion

Mutagenicity of UV filters in chlorinated seawater

The mutagenicity of the UV filters (non chlorinated and chlorinated in seawater at ratio 1:10 and 1:1000, reaction time 30 min) was studied in Salmonella typhimirium strain TA98 without S9 mix.

Table 1 presents the raw results of Ames test for the DMSO extracts of the four tested UV filters in sweater without chlorination (samples 1 to 4), with chlorination at UV filter:chlorine ratio 1:100 (Samples 5 to 8), and at ratio 1:1000 (Samples 9 to 12). For each sample extract, the mean reversion (n=3) and standard deviation are presented for each of the four tested doses of each extract. As shown in Table 1, no increased reversion was detected for the non-chlorinated UV filters in comparison to the negative controls. Also, for the chlorinated UV filters, except for DIOXY at chlorination ratio 1:10 (UV filter : chlorine), no increased reversion was detected since reversion levels were similar to negative controls ranging 15 and 29 rev/plate, indicating absence of mutagenic activity. However, for extracts of DIOXY chlorinated in seawater at ratio 1:10, a clear statistically significant mutagenic activity was detected with reversion ranging from 61 to 158 rev/plate. The mutagenic effect increased as a function of doses (4, 6, 8 and 10 μL of the tested extract). An effect-dose relationship for the mutagenic activity of DIOXY at ratio 1:10, reaction time 0.5 h was established (Figure 1). The calculated coefficients of the selected model were: a = 19.9; b = 7.1 and c = -0.01 giving P < 10 -5 , PE = 0.06. The mutagenic activity (MA, rev/μg), defined as the maximal slope of the ascending part of the dose-response curve and calculated as the first derivative at the origin, was 18.7 rev/μL.

To our knowledge, this is the first study to evaluate the mutagenicity of transformation products of UV filters following chlorination in artificial seawater. Previously, the mutagenicity of octyl dimethyl-p-aminobenzoate (ODPABA) and OMC following chlorination in buffered solutions was investigated [START_REF] Nakajima | Aquatic fate of sunscreen agents octyl-4-methoxycinnamate and octyl-4-dimethylaminobenzoate in model swimming pools and the mutagenic assays of their chlorination byproducts[END_REF]. The chlorination intermediates of OMC exhibited weak mutagenic activity in Salmonella typhimirium TA100 strain without S9 mix. Mutagenicity decreased in the presence of S9 mix. Authors reported that the mutagenic activity disappeared within 7 hours following chlorination, suggesting the degradation of the mutagenic intermediate transformation products under the effects of chlorine.

In another study by [START_REF] Zhang | Chlorination of oxybenzone: kinetics, transformation, disinfection byproducts formation, and genotoxicity changes[END_REF], the genotoxicity of chlorination products of oxybenzone was studied using the SOS/umu genotoxicity test. Authors detected genotoxic activity following chlorination and ruled out the contribution of DBPs such as chloroform and haloacetic acids to the observed genotoxicity. In contrast, authors suggested a correlation between the concentrations of trichloromethoxyphenol and the observed genotoxicity.

The discrepancy between our findings and the findings of [START_REF] Zhang | Chlorination of oxybenzone: kinetics, transformation, disinfection byproducts formation, and genotoxicity changes[END_REF] regarding the genotoxicity of oxybenzone might be explained by the difference of employed genotoxicity assays, which detect genotoxic agents acting by different mechanisms. In the Ames test using Salmonella typhimirium TA98, frameshift mutations can be detected. However, the absence of effects using this test does not mean that the tested substance would not produce other types of toxicity in general and other mechanisms of genotoxicity in particular. This emphasizes the need of toxicological studies with different endpoints and different assays for the same endpoint to ensure that toxicants are not overlooked.

Transformation Products in chlorinated DIOXY sample presenting mutagenicity

Extracts of chlorinated DIOXY in seawater were analyzed using UPLC-ESI-QToF-MS and MSMS to identify the transformation products that could be at the origin of the observed mutagenic activity. Analysis was performed in non-target screening and suspect screening modes. Suspect screening was performed based on previously identified transformation products of DIOXY (Manasfi et al., 2017a). In the mutagenic sample, five brominated transformation products namely Dioxy-Br, Dioxy-Br2, Dioxy-Br3, 3,5-dibromosalicylic acid, and TBMP were detected (Table 2). The formation of these brominated transformation products resulted from the reaction of bromine with DIOXY. In bromide-rich water, such as in seawater where bromide levels can reach 69 mg/L, once chlorine is added to water it reacts with bromide ions leading to the formation of bromine, which reacts more rapidly with organic matter in comparison to chlorine [START_REF] Deborde | Reactions of chlorine with inorganic and organic compounds during water treatment-kinetics and mechanisms: a critical review[END_REF].

The chlorination of organic UV filters has been shown to result in the production of compound-specific transformation products as well as non-compound-specific DBPs such as chloroform and bromoform [START_REF] Negreira | Study of some UV filters stability in chlorinated water and identification of halogenated by-products by gas chromatography-mass spectrometry[END_REF][START_REF] Duirk | Reaction of benzophenone UV filters in the presence of aqueous chlorine: kinetics and chloroform formation[END_REF][START_REF] Manasfi | Degradation products of benzophenone-3 in chlorinated seawater swimming pools[END_REF]Manasfi et al., 2017a). Some of the formed volatile and semi-volatile DBPs have been shown to be genotoxic (Richardson et al. 2008;Zhang el al., 2016;Manasfi et al. 2017b). However, in the present study, the preparation of extracts for Ames test involved the evaporation of the initial LLE solvent (MTBE) and solvent exchange with DMSO, which was compatible with the bioassay. This solvent exchange eliminated volatile and semi-volatile DBPs to narrow the mutagenic assessment to the non-volatile products. Therefore, the observed genotoxicity can be considered unrelated to volatile or semi-volatile DBPs, but rather a result of the formation of non-volatile transformation products. Besides, DBPs and specifically bromoform has been shown to be a common byproduct formed from all the UV filters studied here following chlorination in seawater (Manasfi et al. 2017a). However, since mutagenicity in chlorinated seawater was only detected in the case of DIOXY, this further suggests that the observed mutagenicity is not related to the volatile DBP (ones forms by all the studied UV filters) but wa rather related to intermediate non-volatile products specific to the UV filter in question. [START_REF] Zhang | Chlorination of oxybenzone: kinetics, transformation, disinfection byproducts formation, and genotoxicity changes[END_REF] investigated the genotoxicity of chlorination products of oxybenzone using the SOS/umu genotoxicity test. In this study, authors identified several chlorinated transformation products of oxybenzone as well as some DBPs including chloroform. However, when authors tested the genotoxicity of the formed DBPs separately (without the transformation products), no genotoxicity was detected, which allowed them to rule out the contribution of DBPs to the observed genotoxicity. Moreover, authors suggested a correlation between 2,4,6trichloro-3-methoxyphenol (chlorinated analogue of TBMP) and the observed genotoxicity. In our study, it was not possible to assign the observed mutagenicity to a specific transformation product. Further investigations are required to determine the transformation products directly responsible for the mutagenicity. Yet, some preliminary observation can be made by interpreting the mutagenicity results of the different UV filters on the light of previously identified transformation products. Along these lines, since TBMP is a common byproduct between DIOXY and OXY (Manasfi et al. 2017a), and no mutagenicity was detected for chlorinated OXY in seawater (conditions which form TBMP), it can be excluded from the candidate compounds responsible for the observed mutagenic activity of DIOXY chlorination products.

Time course of transformation products at different chlorination ratios

Since DIOXY was the only UV filter shown to produce mutagenic activity in salmonella typhimirium strain TA98 upon chlorination in seawater, it was the subject of further chlorination experiments and analysis using HRMS to follow the kinetics of formation of the transformation products.

The kinetics of degradation of DIOXY and formation of its transformation products were followed as a function of reaction time at different UV filter : chlorine ratios (1:10, 1:100 and l:1000). As shown in Figure 2, DIOXY disappeared rapidly in the presence of excess chlorine (precisely, in seawater the main oxidizing species is bromine) leading to the formation of brominated transformation products. The levels of the transformation products were highest at the lowest chlorination ratio (1:10). At chlorination ratio 1:100, most of the transformation products were not detected, except 3,5-dibromosalicylic acid which was detected at trace levels at 10 min but not anymore at 30 min (the extract tested in Ames test). The absence of the transformation products at ratio 1:1000 explains the absence of mutagenic activity in the same sample and further corroborates the correlation between mutagenicity and the identified transformation products.

These findings show that the formed transformation products are susceptible to to react with bromine leading to their complete disappearance and the formation of bromoform and potentially other DBPs as final products (Manasfi et al., 2017a). The mechanism of cleavage of aromatic rings (e.g. phenols) when reacting with chlorine (and similarly bromine) and the subsequent formation of haloforms as final products has been described previously [START_REF] Rook | Formation of haloforms during chlorination of natural waters[END_REF].

Relevance of our findings to swimming pool context

In real-world scenarios, recommended levels of free chlorine residual in swimming pools differ from one country to another and may vary from 0.3 to 3 mg/L (Ekowati etl al., 2016;Manasfi et al., 2017a). As to UV filters in general, concentrations ranging from sub ng/L up to few tens of μg/L have been reported [START_REF] Ekowati | Occurrence of pharmaceuticals and UV filters in swimming pools and spas[END_REF]Manasfi et al., 2017c). For DIOXY in particular, [START_REF] Ekowati | Occurrence of pharmaceuticals and UV filters in swimming pools and spas[END_REF] detected concentrations up to 21.6 ng/L in swimming pools in Spain. More recently, [START_REF] Lempart | The presence of compounds from the personal care products group in swimming pool water[END_REF] reported levels up to 226.9 ng/L in swimming pools in Poland. Considering the molar mass of DIOXY (244.25 g/mol), the reported molar concentrations would be around 0.08 to 0.8 nmol/L (for 20 to 200 ng/L). For a swimming pool containing 14 μmol/L (or 1 mg/L) of free chlorine (expressed in the form of Cl2), this would mean a ratio ranging from 1:17500 to 1:175000 (DIOXY:chlorine) for the aforementioned DIOXY concentrations. As such, the transformation products detected in the present study at chlorination ratios lower than 1:1000 are likely to disappear rapidly in swimming pools where the UV filter:chlorine ratios are much higher. Nevertheless, investigations in real-case scenarios are still recommended since in swimming pool water, complex mixtures of organic matter (body fluids and personal care products released by swimmers, natural organic matter, etc.) compete for the reactions with chlorine, which may suggest that reactions in such matrices are recommended to rule out the occurrence of the transformation products in the pools.

Conclusion

The present study showed, for the first time to our knowledge, that the UV filter DIOXY could exhibit mutagenic activity when added to chlorinated bromide-rich water. In the mutagenic extracts, several brominated transformation products of DIOXY were detected. These transformation products were unstable, their levels decreased with increasing chlorine doses, and they were not detected at 1000-fold chlorine excess (1:1000, DIOXY:Chlorine). Although excess chlorine (or bromine in seawater) in this case resulted in the elimination of mutagenic activity by eliminating the mutagenic intermediate transformation products, excess chlorine is known to enhance the formation of DBPs, which were not tested in Ames test due to their elimination during solvent exchange of extracts. Some DBPs are known to be mutagenic [START_REF] Richardson | Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research[END_REF]Manasfi et al. 2017a). Therefore, future investigations taking into consideration both intermediate transformation products as well as DBPs are recommended to assess the effects of increasing chlorine doses on the overall mutagenicity (both DBPs and transformation products) in chlorinated/brominated samples. This study highlights the importance of taking into consideration the susceptibility of some UV filters to react with disinfectants in recreational waters when sunscreen formulations are designed.