Justina Salomé

Julie Julien

Justina Kévin

Leïla

Léo

Maxence Lucas

UV filters are the active components of sun protection products and are used as stabilizing agents in cosmetics and industrial products. Several studies reported the worldwide occurrence of UV filters in coastal waters and demonstrated their bioaccumulation and toxic effects on aquatic organisms.

Marine bacteria dominate the marine biomass and are crucial for biogeochemical cycles.

Notwithstanding their essential roles, the toxicity of UV filters has never been assessed on microorganisms. This thesis aims to explore the effects of the most occurrent organic UV filters, taking into account the physiological, morphological, and molecular response, on environmentally relevant heterotrophic bacteria, isolated from the bay of Banyuls (NW Mediterranean Sea, France).

First, we demonstrated the dose-dependent bactericidal effects of four organic UV filters, octinoxate being the most toxic compound. In order to address the impact of solar radiation on UV filters toxicity, their combined effects were assessed, using a solar simulator. We showed that solar light modulated the effects of UV filters and that physiological state was a key parameter in the cell response.

Epibacterium mobile, and environmentally relevant -proteobacteria were shown to be sensitive to the controversial oxybenzone. The physiological and molecular responses were assessed via transmission electron microscopy, enzymatic activity monitoring, and label-free quantitative proteomics. While cellular morphology and membrane integrity seemed intact, the enzymatic activity was affected in a dose-dependent way. Proteomics revealed differential regulation of 56 proteins involved in major cellular pathways.

Genes involved in UV filters resistance are still unknown. In order to fill the gaps in the understanding of the link between genome and resistance phenotype, in silico comparative genomic between UV filters sensitive and resistant bacteria were conducted. The genomes of two Bacteroidetes and three Firmicutes species were compared through pairwise comparisons. Resistant Firmicutes displayed multiple unique genes that could explain the differences between phenotypes. Genes involved in relevant mechanisms in the context of xenobiotic resistance were shown to be exclusive to resistant species. Overall, this thesis provides a unique insight into the effects of UV filters on marine bacteria, and demonstrates that bacteria are suitable for ecotoxicological investigations.

II

ACKNOWLEDGMENTS

I would like to acknowledge the people who made this thesis not only an exciting scientific but also an amazing human experience. A huge thank you to my supervisor Sabine Matallana-Surget, that provided the best supervising I could expect. Words are missing to describe how available you were and how valuable were your advice and numerous feedbacks on every aspect of the project. You have been a real mentor to me throughout these years, and working with you, your enthusiasm and constant good mood, has been a real pleasure.

I would like to thank Philippe Lebaron for

List of figures

List of tables CHAPTER 1

Table 1.1. Prokaryotic population size in various habitats. Adapted from [START_REF] Whitman | Prokaryotes: The Unseen Majority[END_REF] ....... Table 1.2. Terrestrial and marine prokaryotic biomass and primary productivity. Adapted from [START_REF] Whitman | Prokaryotes: The Unseen Majority[END_REF]

List of abbreviations

Role of Microorganisms in the Ocean

The ocean covers approximately 70% of the Earth's surface and represents around 1.27 10 18 m 3 of water [START_REF] Orcutt | Microbial Ecology of the Dark Ocean above, at, and below the Seafloor[END_REF]. Multiple habitats exist under the water column such as oceanic crust, marine sediments, and hydrothermal vents. It makes it the widest habitable environment on Earth. One milliliter of sea water contains several million cells of prokaryotic and eukaryotic microorganisms. Bacteria are abundant with more than 10 30 cells in oceanic water [START_REF] Whitman | Prokaryotes: The Unseen Majority[END_REF]) and between 10 5 to 10 8 cells per milliliter in the photic zone, i.e. the depth of water exposed to sunlight (approximately 100 meters [START_REF] Ahmad | Euphotic Depth Zone Variation in Peninsular Malaysia Maritime[END_REF]). Subseafloor sediment also represents a major bacterial reservoir that accounts for at least 2.9 10 29 cells corresponding to 4.1 petagrams (Pg) C and ∼0.6% of Earth's total living biomass [START_REF] Kallmeyer | Global Distribution of Microbial Abundance and Biomass in Subseafloor Sediment[END_REF]. Considerable biomass involves key ecological functions, most of which still unknown.

Bacterial communities are phylogenetically diverse as they are ramified into more than 53 phyla [START_REF] Keller | Tapping into Microbial Diversity[END_REF], some of them can be divided into more than 30 orders, containing hundreds of different species [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. Bacteria able to grow in a petri dish represent 2% of the total diversity [START_REF] Wade | Unculturable Bacteria -The Uncharacterized Organisms That Cause Oral Infections[END_REF]. This major concern for Microbiology -called "the great plate count anomaly"is due to Microbiologists failing to replicate essential aspects of their environment. The combination of parameters that need to be set up in a culture medium is considerable, making the task challenging. For instance, the SAR11 clade of -proteobacteria is a widespread group of ubiquist bacteria, which was for many years unculturable [START_REF] Stewart | Growing Unculturable Bacteria[END_REF]. To solve this problem, new approaches have been developed. Promising techniques include coculture with a helper strain [START_REF] Vartoukian | Strategies for Culture of 'unculturable' Bacteria[END_REF], in vitro microenvironment, microfluidic one cell culture system, long-term culture [START_REF] Stewart | Growing Unculturable Bacteria[END_REF]) and dilution to extinction methods [START_REF] Rappé | Cultivation of the Ubiquitous SAR11 Marine Bacterioplankton Clade[END_REF][START_REF] Song | Improved Culturability of SAR11 Strains in Dilution-to-Extinction Culturing from the East Sea , West Pacific Ocean[END_REF].

Molecular biology allows the characterization, identification, and study of unculturable species. 16S rRNA gene is an ubiquist prokaryotic gene made of several invariable regions and variable regions that are species-specific. The 16S rRNA gene is approximately 1500 nucleotides long and the 16S rRNA represents often over 5% of the cell dry weight. Additionally, it is not subjected to horizontal gene transfer [START_REF] Tian | Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S RRNA Gene[END_REF]. These characteristics make it the perfect phylogenetic marker. Hence, the amplification of the 16S rRNA gene followed by sequencing allows for species identification.

The microbial loop

In the marine ecosystem, the microbial loop plays a critical role in all biogeochemical cycles and interspecies regulation [START_REF] Azam | Microbial Structuring of Marine Ecosystems[END_REF]. As depicted in Figure 1.1, phytoplankton releases POM, leading to DOM formation that is used by heterotrophic bacteria. Protozoa feed on Bacteria and archaea while they are preyed on by Zooplankton. Inorganic microelements (C, N, P, Si, Fe) are consumed by phytoplanktonic species and autotrophic bacteria. Consumption of DOM through respiration results in inorganic CO2 release.

Figure 1.1. Microbial structure of a marine ecosystem. DOM, Dissolved Organic Matter; DMS, dimethylsulfide; hv, light; POM, particulate organic matter [START_REF] Azam | Microbial Structuring of Marine Ecosystems[END_REF]. Phytoplanktonic algae are unicellular or multicellular eukaryotic organisms performing photosynthesis. As they need light for their metabolism, algae are found in the photic zone of the water column. Our ecosystem's stability strongly relies on the metabolism of algae as they are major producers of dioxygen with at least 70% of Earth's global O2 and 50% of the world's primary production, along with Cyanobacteria. In spring, a phytoplanktonic bloom occurs with diatoms dominating, followed by grazers like dinoflagellates and green flagellates. Protozoa are abundant in marine ecosystems and feed mainly on bacteria, followed by algae, other protists, and organic matter. Protozoa are known to decrease the average size of bacteria and maintain the bacterial population at early growth phase, promoting faster nutrients assimilation in the system [START_REF] Hahn | Grazing of Protozoa and Its Effect on Populations of Aquatic Bacteria[END_REF]. Viruses, along with protozoa, are the main oceanic actors leading to bacterial mortality. Highly abundant, they contribute to the regulation of bacterial and phytoplanktonic communities. Lysis by viruses induces the release of DOM [START_REF] Azam | Microbial Structuring of Marine Ecosystems[END_REF] and contributes to the low bacterial biomass relative to phytoplankton in eutrophic systems [START_REF] Cotner | Small Players, Large Role: Microbial Influence on Biogeochemical Processes in Pelagic Aquatic Ecosystems[END_REF].

Bacteria are at the core of the microbial loop as they can be both autotrophic or heterotrophic. This diversity of metabolism highly contributes to the assimilation and subsequent transfer of biomass to higher organisms. Heterotrophic bacteria are the main decomposers of DOM that can originate from primary production (by algae and cyanobacteria), or cell lysis induced by viral infection of other bacteria, algae, and protists. To support this DOM uptake, bacteria hydrolyze polymers and particles using ectoenzymes (protease, glucosidase, lipase, phosphatase, nuclease, and chitinase) [START_REF] Delong | Genomic Perspectives in Microbial Oceanography[END_REF]. Bacteria represent the most dominant biotic surface in the marine environment. One cubic meter of seawater contains between 0,1 and 1 m² of bacterial surfaces, that can interact with DOM.

Bacterial motility structures make them mobile along the oceanic DOM gradients and the different nutrient hotspots (chemotaxis). The ability of bacteria to assimilate and degrade DOM makes them the main oceanic decomposers. Indeed, detritus from dead material contribute to the pool of DOM that is assimilated by the bacterial community [START_REF] Delong | Genomic Perspectives in Microbial Oceanography[END_REF]. Autotrophic bacteria, including both photosynthetic and non-photosynthetic cells, take up inorganic nutrients to produce organic material.

As bacteria take up a great variety of organic substrates and inorganic nutrients, they are central organism in geochemical cycling in the Oceans.

Diversity and abundance of marine prokaryotes

Prokaryotes are the most abundant and diverse microorganisms. These prokaryotic unicellular organisms are characterized by a simple nucleus-free structure, which enables them to adapt to a wide range of living conditions. Oceanic bacteria are taxonomically classified into more than 16 major clades [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. Each phylum displays a differential abundance, regarding different biomes (Figure 1.2). Planctomycetes are mainly found in the neritic (coastal) mesopelagic zone while other phyla (Euryarchaeota, Thaumarchaeota, Fibrobacteres, and Firmicutes) are completely absent from this area.

Alpla, Beta, Delta and Gammaproteobacteria are the most abundant clade and remain present throughout the entire water column. [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. The bubble size indicates relative abundance, scaled from 0, the minimum, to 1 representing the maximum abundance.

Clade abundance changes depending on the location and the depth in the marine environment.

If we consider the vertical repartition of 8 bacterial phyla in the water column in three different Environments: North Pacific Ocean, Tropical South China, and Costa Rica Dome [START_REF] Jing | Vertical Profiles of Bacteria in the Tropical and Subarctic Oceans Revealed by Pyrosequencing[END_REF], Proteobacteria remains the most abundant phyla in all environments followed by Actinobacteria and Bacteroidetes (Figure 1.3). Cyanobacteria, Actinobacteria and Planctomycetes are less common as they need specific conditions. For example, Cyanobacteria are common in euphotic areas while they are less present in aphotic environments. Autotrophs use ATP, along with inorganic compounds to achieve their anabolic processes. The resulting organic material serves as a substrate for heterotrophs, which in turn generate ATP, CO2, and inorganic substrate, utilized by autotrophs. The relation between organisms with complementary metabolisms suggests that a decline or an increase of a population will impact on the other.

Marine environments are relatively limited in nutrients. Therefore, microorganisms adapt their metabolism and morphology to nutrient depletion. Environments characterized by low nutrient availability, and organisms able to develop in such a system, are named oligotrophs [START_REF] Kuznetsov | Biology of Oligotrophic Bacteria[END_REF]). Bacteria can be strictly oligotrophic, i.e. they cannot grow in nutrient-rich environments, or adapt to that last by expressing new enzymes [START_REF] Schutl | Oliogotrophy and Pelagic Marine Bacteria: Facts and Fiction[END_REF]. Oligotrophic systems are characterized by a high proportion of dissolved nutrients in organic form, favoring the presence of heterotrophs. Indeed, the major component of the microbial biomass in oligotrophic waters are prokaryotic heterotrophs and prokaryotic photoautotrophs such as Prochlorococcus and Synechococcus [START_REF] Cotner | Small Players, Large Role: Microbial Influence on Biogeochemical Processes in Pelagic Aquatic Ecosystems[END_REF].

In contrast, eutrophic environments are characterized by a high concentration of particulate organic carbon and inorganic nutrients, with a decreased relative importance of heterotrophs [START_REF] Cotner | Small Players, Large Role: Microbial Influence on Biogeochemical Processes in Pelagic Aquatic Ecosystems[END_REF]. Organisms able to survive in eutrophic systems are named copiotrophs.

Heterotrophs, also named chemoorganotrophs, take up their energy source in organic compounds. In the euphotic layer of oligotrophic waters, bacterial carbon use tends to exceed net primary production (NPP), meaning a heterotrophic dominance [START_REF] Duarte | The Oligotrophic Ocean Is Heterotrophic[END_REF]). Heterotroph's metabolism plays a significant role in the carbon cycle as respiration by heterotrophic bacteria is the main source of CO2 in estuaries and coastal waters [START_REF] Moran | Resourceful Heterotrophs Make the Most of Light in the Coastal Ocean[END_REF], participating strongly in the carbon balance. Major ecological functions such as nitrate and nitrite reduction (Ward et al. 2009), organic carbon degradation (Quaiser et al. 2008) including cellulose, hemicellulose and chitin [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF], and phosphate uptake [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]) can be attributed to heterotrophic bacteria.

Marine microenvironments

Microenvironments such as marine snow and the surface micro layer are major marine microhabitats that inhabit complex communities. Because their functioning can be affected by anthropogenic factors, marine snow and the surface micro layer are described. Marine snow is an aggregate of organic and inorganic material combined with bacteria, protists and other microorganisms.

The density of microorganisms in these micro-ecosystems is 2-5 orders of magnitude higher than in the surrounding seawater. The bacterial biomass in marine snow provides food for bacterivorous flagellates and other protozoans whereas dissolved organic matter released from marine snow particles is a substrate for free-living bacteria (Figure 1.5) [START_REF] Stal | The Marine Microbiome The Marine Microbiome[END_REF]. Marine snow can comprises several phyla, for example: Bacteroidetes, Cyanobacteria (Synechococcus), Gammaproteobacteria (Alteromonas and Pseudoalteromonas), Alphaproteobacteria (Roseobacter) and Planctomycetes (Thiele et al. 2014). Sinking marine snow is a food source for benthic invertebrates. Previous studies highlighted that marine snow sorbed polycyclic aromatic hydrocarbons (PAH) [START_REF] Bacosa | Marine Snow Aggregates Are Enriched in Polycyclic Aromatic Hydrocarbons ( PAHs ) in Oil Contaminated Waters : Insights from a Mesocosm Study[END_REF][START_REF] Eenennaam | Ecotoxicological Benthic Impacts of Experimental Oil-Contaminated Marine Snow Deposition[END_REF]) and TiO2 nanoparticles [START_REF] Doyle | Behavior of Titanium Dioxide Nanoparticles in Three Aqueous Media Samples: Agglomeration and Implications for Benthic Deposition[END_REF], making it a contamination source from small invertebrates towards higher trophic level organisms. Another major marine microenvironment is the sea surface microlayer (SML). The SML constitutes the 1000 µm thick top layer of the ocean and represents the interface mediating the exchange of gases, and inorganic and organic matter between the hydrosphere and the atmosphere. The bacterial communities inhabiting the SML are called the bacterioneuston. Previous research reported that while bacterial abundance was similar in the SML and underlying water, the fraction of particle-attached cells were two to three times greater in the SML [START_REF] Dias | Relation between Bacterial Activity in the Surface Microlayer and Estuarine Hydrodynamics ˜[END_REF]). In addition, bacterial respiration was significantly higher in the SML [START_REF] Reinthaler | Dissolved Organic Matter and Bacterial Production and Respiration in the Sea -Surface Microlayer of the Open Atlantic and the Western Mediterranean Sea[END_REF], suggesting a greater carbon bacterial uptake and remineralization into CO2. Recently, [START_REF] Martinez-Varela | Large Enrichment of Anthropogenic Organic Matter Degrading Bacteria in the Sea-Surface Microlayer at Coastal Livingston Island ( Antarctica )[END_REF] demonstrated that the relative abundance of specific SML-enriched bacterial groups was correlated to the concentration of hydrophobic anthropogenic pollutants, namely perfluoroalkyl substances. UV filters, known as highly hydrophobic, were more abundant in the SML than the water column [START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF]Tovar-Sánchezet al. 2019). Anthropogenic contaminants accumulating in the SML can modulate and alter the functioning of the marine SML microbiome, pivotal for biogeochemical cycles.

Biogeochemical cycles

Anthropogenic inputs were shown to impact biogeochemical cycles. The enrichment of aquatic biota by organic pollutants, metals, and excessive concentration of microelements such as phosphorus and nitrogen are responsible for eutrophication and acidification [START_REF] Krishnamurthy | Impacts of Increasing Anthropogenic Soluble Iron and Nitrogen Deposition on Ocean Biogeochemistry[END_REF][START_REF] Moiseenko | Evolution of Biogeochemical Cycles under Anthropogenic Loads : Limits Impacts[END_REF]. With more than 6000 tons discharged yearly, emerging contaminants such as UV filters could possibly alter the functioning of microbial communities, resulting in disrupted nutrients cycling.

Carbon cycle

The amount converted from CO2 to carbohydrate during photosynthesis is known as gross primary production (GPP). Net Primary Production (NPP) represents the difference between photosynthesis and autotrophic respiration. In the Ocean, the carbon mass is fifty times greater than in the atmosphere. CO2 exchange between the atmosphere and surface Ocean is about 90 PgC.yr -1 . This exchange is mediated by simple molecular diffusion across the air-sea interface. CO2 is represented in the Ocean under three forms: non ionic CO2 (1%), bicarbonate ions (HCO3 -) (91%) and carbonate ions (CO3 2-) (8%). Oceanic phytoplankton gross primary production is assured by algae and cyanobacteria.

The carbon fixed by GPP returns to dissolved inorganic matter (DIM) through autotrophic respiration.

Prokaryotic microorganisms (Bacteria and Archaea) represent a huge biomass in marine habitats (Table 1.1), marine heterotrophs account for 11.8 10 29 cells in the water column while autotrophs are 2.9 10 27 .

It is noteworthy to underline that the majority of bacterial communities lives in sea subsurface (sediment), with 4.9 10 30 cells [START_REF] Whitman | Prokaryotes: The Unseen Majority[END_REF]. Between 14 and 30% of NPP occurs in coastal areas [START_REF] Gattuso | Carbon and Carbonate Metabolism in Coastal Aquatic Ecosystems[END_REF]. Most of the organic carbon from NPP is consumed by zooplankton to be released as dissolved organic carbon (DOC) (Figure 1.6). Detritus and dead organisms represent an organic carbon reservoir. Sinking of this particulate organic carbon is named "vertical carbon transfer".

Nitrogen cycle

Nitrogen is the most abundant gas in the atmosphere (80%) but is not directly assimilable by living organisms. The Nitrogen cycle (Figure 1.7) starts from the nitrogen fixation process supported by diazotrophs (Cyanobacterium, Rhizobium and Azotobacter sp.) [START_REF] Montoya | High Rates of N 2 Fixation by Unicellular Diazotrophs in the Oligotrophic Pacific Ocean[END_REF]). This step consists of reducing N2 to ammonium ion (NH4 + ) assimilable by the microbial biomass. Ammonium can be assimilated by autotrophic bacteria, plants or algae, that convert it into organic nitrogen, or undergo nitrification. The latter is mainly supported by Archaea belonging to the Crenarchaeota clade and Nitrospina bacterial species [START_REF] Könneke | Isolation of an Autotrophic Ammonia-Oxidizing Marine Archaeon[END_REF][START_REF] Wuchter | Archaeal Nitrification in the Ocean[END_REF], that convert ammonium in NO2 - and oxidize NO2 -to NO3 -, assimilable by some plants, algae or Cyanobacteria. Ammonia is incorporated into organic compounds within the cell via the glutamine synthetase and glutamate synthase pathways.

This organic nitrogen can be decomposed by heterotrophic bacteria back into ammonium (ammonification). In the aquatic system, nitrate can undergo denitrification under anaerobic conditions, resulting in a gaseous form (N2, N2O or NO2). This process is carried out by Foraminifera and several bacteria, e.g. Planctomycetes [START_REF] Kuypers | Anaerobic Ammonium Oxidation by Anammox Bacteria in the Black Sea[END_REF].

Phosphorus cycle

Phosphorus is a functional and structural component of all organisms. It is a major constituent of DNA and RNA as it provides the phosphate-ester backbone. In addition, it provides energy via the ATP molecules. Many cellular structures rely on phosphorus, such as phospholipids and phosphoproteins, to properly ensure their functions. In the Ocean, sediments are the main repository of P, provided by sinking particulate matter [START_REF] Paytan | The Oceanic Phosphorus Cycle[END_REF]. Oceanic P exists in dissolved and particulate forms. Both forms contain inorganic P (orthophosphate, pyrophosphate, polyphosphate, and minerals)

and organic P (P-esters, P-diesters, phosphonates) compounds. Dissolved inorganic phosphorus, i.e.

HPO4 2-and PO4 3-is assimilated by phytoplankton which synthesizes organic phosphorus compounds (Figure 1.8). When zooplankton feeds on phytoplankton, a large fraction of the organic phosphorus is excreted as dissolved inorganic P. Organic P can be released in aquatic media by phytoplanktonic cell lysis. Bacterial enzymes can hydrolyze organic P before subsequent assimilation.

UV filters from the regulatory aspects to environmental issues

Physicochemical properties

Sunscreen products are composed of UV filters, preservative molecules such as parabens derivatives, coloring agents (ammonium sulfate, copper, ferric ammonium ferrocyanide, iron and zinc oxides etc.), film-forming agents (acrylate or acrylamide), surfactants, chelators, viscosity controllers and fragrances [START_REF] Tovar-Sánchez | Sunscreen Products as Emerging Pollutants to Coastal Waters[END_REF]. Ultraviolet filters are divided into two types: organic and inorganic. Most organic UV filters are aromatic molecules able to absorb UVB (280-315 nm), UVA (315-400 nm), or both, while inorganic filters can scatter, reflect or absorb UV rays [START_REF] Shaath | Ultraviolet Filters[END_REF]. The UV absorption mechanism operates as follows: a UV photon excites an electron that will transit from the highest occupied molecular orbital (HOMO) to the lowest unoccupied orbital (LUMO). The obtained singlet excited state can be deactivated by a return to the ground state through fluorescence, energy transfer, vibrational relaxation or a photochemical reaction that can lead to a photoproduct (Figure 1.9) [START_REF] Shaath | Ultraviolet Filters[END_REF]. Log Kow of benzophenone compounds is heterogeneous (Table 1.3), varying from 0.993 to 3.79 while their solubilities range from 0.1 to 11 g.L -1 . Benzophenones show the highest solubility among all UV filters. However, other widespread UV filters are poorly soluble. Ethylhexyldimethyl PABA (OD-PABA), Ethylhexyl methocycinnamate (EHMC), Butylmethoxydibenzoylmethane (BMDBM) and Octocrylene (OC) show a solubility of 4.7 10 -3 , 6.4 10 -3 , 4.3 10 -3 and 3.6 10 -4 g.L -1 , and a Log Kow of 5.4, 5.8, 4.19 and 6.88, respectively. Generally, the highest the Log Kow, the lower the solubility.

Considering these parameters is important for in vitro toxicological analysis. Indeed, organic UV filters are poorly soluble. Therefore, the use of solubilizers is mandatory during in vitro analysis in liquid media. Because UV filter naming strongly differs from one publication to another, the main names of UV filter approved in EU and US are given in Appendix 1. The risk quotient (RQ) is obtained by dividing the environmental concentration by the PNEC.

If the PNEC is lower than the environmental concentration (RQ>1), a risk is presumed for the compound of interest.

Occurrence of UV filters in aquatic biota

Freshwater ecosystem

The occurrence of UV filters has been investigated in freshwater habitats. Their presence has been reported in the water and sediments of rivers, lakes as well as in fishes, worldwide. In Swiss lakes and rivers, 4-MBC and BP3 are the most prevalent with 28 and 35 ng.L -1 , respectively [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF]. [START_REF] Fent | Widespread Occurrence of Estrogenic UV-Filters in Aquatic Ecosystems in Switzerland[END_REF] has detected BP3, 4-MBC, and EHMC in the Glatt river (Switzerland) between 6 and 68 ng.L -1 . In Japanese rivers, BP, BP3, Benzylhydroxybenzoate (BZS), EHMC and octyl salicylate (OS) concentrations ranged from 7 to 266 ng.L -1 . Higher concentrations were found in sediments, ranging from 1.3 to 1735 ng.g -1 [START_REF] Kameda | Occurrence and Profiles of Organic Sun-Blocking Agents in Surface Waters and Sediments in Japanese Rivers and Lakes[END_REF]. Interestingly, BP3 has not been detected in samples of sediments, perhaps because of its relatively low Log Kow compared to other organic compounds. This finding is confirmed by Langford and colleagues (2015), who investigated the occurrence of BP3 and EHMC in WTP sludge and sediment in Norway. They showed that BP3 concentration was significantly lower compared to EHMC in a solid matrix.

UV filters were found in WTP influents and effluents. In Switzerland, concentrations of 4-methylbenzylidene camphor (4-MBC), benzophenone-3 (BP3), ethylhexyl methoxycinnamate (EHMC) and octocrylene (OC) varied from 0.1 to 19µg.L -1 with an elimination efficacy ranging from 18 to almost 100% [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF]. The concentration of seven UV filters was measured in fish flesh for perch (Perca fluviatilis) and roach (Rutilus rutilus) from Maarfelder Lake (Eifel, Germany) with total concentrations of approximately 2000 ng.g -1 lipid and 500 ng.g -1 lipid, respectively (see review of [START_REF] Gago-Ferrero | An Overview of UV-Absorbing Compounds (Organic UV Filters) in Aquatic Biota[END_REF]. When collected downstream of a WTP discharge, the UV filter concentration in fish (Salmo trutta fario) is greater, with 4-MBC and OC concentrations of 1800 and 2400 ng.g -1 lipid, respectively [START_REF] Gago-Ferrero | An Overview of UV-Absorbing Compounds (Organic UV Filters) in Aquatic Biota[END_REF], meaning that removal efficacy in WTP could still be improved. Fishes from Swiss lakes contain much lower concentrations with 4-MBC and BP3 at 44 and 123 ng.g -1 lipid [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF]. Bioaccumulation of UV filters in marine organisms will be discussed in section 2.5.

Marine ecosystem

Commonly used organic UV filters such as BP3, 4-MBC, OD-PABA and EHMC have been found in marine samples collected in different locations: Ionanian sea [START_REF] Giokas | Determination of Residues of UV Filters in Natural Waters by Solid-Phase Extraction Coupled to Liquid Chromatography-Photodiode Array Detection and Gas Chromatography-Mass Spectrometry[END_REF]),

Mediterranean sea [START_REF] Tovar-Sánchez | Sunscreen Products as Emerging Pollutants to Coastal Waters[END_REF][START_REF] Sánchez Rodríguez | Occurrence of Eight UV Filters in Beaches of Gran Canaria (Canary Islands). An Approach to Environmental Risk Assessment[END_REF], Pacific [START_REF] Trissa | Occurrence, Distribution, and Fate of Organic UV Filters in Coral Communities[END_REF][START_REF] Allinson | Occurrence and Assessment of the Risk of Ultraviolet Filters and Light Stabilizers in Victorian Estuaries[END_REF]) and Atlantic oceans [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF] [START_REF] Allinson | Occurrence and Assessment of the Risk of Ultraviolet Filters and Light Stabilizers in Victorian Estuaries[END_REF]. We recommend the review of Pintado-Herrera and coworkers (2020), along with the study published by [START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF], for a comprehensive overview of UV filters occurrence in different water bodies.

Overall, BP3 is the most studied compound with concentrations varying greatly among biota [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] Giokas | Determination of Residues of UV Filters in Natural Waters by Solid-Phase Extraction Coupled to Liquid Chromatography-Photodiode Array Detection and Gas Chromatography-Mass Spectrometry[END_REF][START_REF] Tovar-Sánchez | Sunscreen Products as Emerging Pollutants to Coastal Waters[END_REF][START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF].

Interestingly, a discrepancy in the published data was noted as concentrations at the same location varied from the nanogram (Mitchelmore et al. 2019) to the milligram range [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF]. Chemical properties of UV filters make their accurate quantification a challenging task. Several parameters need to be taken into account: their partitioning between the different interfaces (surface microlayer, water column, sediment) [START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF], their photochemistry (see section 4.2 below), their spatial and monthly and intraday temporal variations [START_REF] Sankoda | Seasonal and Diurnal Variation of Organic Ultraviolet Filters from Personal Care Products Used Along the Japanese Coast[END_REF], and their accumulation and subsequent metabolization [START_REF] Stien | Metabolomics Reveal That Octocrylene Accumulates in Pocillopora Damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction[END_REF].

Bioaccumulation of UV filters in marine biota

Bioaccumulation studies provide a better indicative measure of the spread of UV filters in the Ocean and are pivotal to better understand to which extent marine organisms are exposed to UV filters.

Two types of studies can be identified: those that measure the concentration of different compounds from organisms collected in situ and those that expose the organism to a given concentration in vivo, in order to measure their bioaccumulated fraction.

Organic UV filters are poorly soluble compounds, characterized by a high octanol-water partition coefficient (Log Kow) (Apel et al. 2018;[START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF] The fate of sunscreen agents in species of higher trophic levels has been poorly documented. To date, three studies were carried out on dolphins, being the first assessments of UVF in marine mammals.

Dolphins accumulate organic pollutants and are good candidates as sentinel species for Ocean health, due to biomagnification [START_REF] Bossart | Marine Mammals as Sentinel Species for Oceans and Human Health[END_REF][START_REF] Tanabe | Global Contamination by Persistent Organochlorines and Their Ecotoxicological Impact on Marine Mammals[END_REF]. Benzotriazole UV stabilizers were identified in blubbers of finless porpoises in Japan with a bioaccumulation factor as high as 33 300, which was ten times higher than those reported for small fish [START_REF] Nakata | Detection of Benzotriazole UV Stabilizers in the Blubber of Marine Mammals by Gas Chromatography-High Resolution Mass Spectrometry (GC-HRMS)[END_REF]. Alonso and coworkers (2015) demonstrated maternal transfer of UV filters in Franciscana Dolphin, revealing that 4-MBC, EMC, OD-PABA and OC are transplacental contaminants. Overall, these studies suggest that UV filters biomagnify through the marine food web. Further analyses are needed to better apprehend the biomagnification of these emerging pollutants.

Toxicity of UV filters

The toxicity of UV filters has been widely documented for diverse organisms: invertebrates, vertebrates, aquatic and terrestrial species. Toxicity is studied through the use of different biological parameters, called endpoints. The main endpoints will be introduced below. This section documents the data found in the literature regarding the effect of UV filters on aquatic species living in freshwater or marine environments.

Effects on marine organisms

Common endpoints have been studied on a recurrent basis. Oxidative stress, highlighted by the increase in antioxidative enzymes, was monitored in mussels [START_REF] Barmo | In Vivo Effects of n-TiO2 on Digestive Gland and Immune Function of the Marine Bivalve Mytilus Galloprovincialis[END_REF][START_REF] Ciacci | Immunomodulation by Different Types of N-Oxides in the Hemocytes of the Marine Bivalve Mytilus Galloprovincialis[END_REF][START_REF] D'agata | Enhanced Toxicity of 'bulk' Titanium Dioxide Compared to 'Fresh' and 'Aged' Nano-TiO2 in Marine Mussels (Mytilus Galloprovincialis)[END_REF]Giraldo et al. 2017;[START_REF] Hanna | Impact of Engineered Zinc Oxide Nanoparticles on the Individual Performance of Mytilus Galloprovincialis[END_REF][START_REF] Huang | Hemocyte Responses of the Thick Shell Mussel Mytilus Coruscus Exposed to Nano-TiO2 and Seawater Acidification[END_REF][START_REF] Libralato | Embryotoxicity of TiO2 Nanoparticles to Mytilus Galloprovincialis (Lmk)[END_REF][START_REF] Wang | Immune Toxicity of TiO2 under Hypoxia in the Green-Lipped Mussel Perna Viridis Based on Flow Cytometric Analysis of Hemocyte Parameters[END_REF] and algae (M. [START_REF] Bhuvaneshwari | Toxicity and Trophic Transfer of P25 TiO2 NPs from Dunaliella Salina to Artemia Salina: Effect of Dietary and Waterborne Exposure[END_REF]Giraldo et al. 2017;[START_REF] Miller | TiO2 Nanoparticles Are Phototoxic to Marine Phytoplankton[END_REF][START_REF] Paredes | Ecotoxicological Evaluation of Four UV Filters Using Marine Organisms from Different Trophic Levels Isochrysis Galbana, Mytilus Galloprovincialis, Paracentrotus Lividus, and Siriella Armata[END_REF][START_REF] Sendra | Effects of TiO2 Nanoparticles and Sunscreens on Coastal Marine Microalgae: Ultraviolet Radiation Is Key Variable for Toxicity Assessment[END_REF][START_REF] Seoane | Flow Cytometric Assay to Assess Short-Term Effects of Personal Care Products on the Marine Microalga Tetraselmis Suecica[END_REF][START_REF] Thiagarajan | Chemosphere Diminishing Bioavailability and Toxicity of P25 TiO2 NPs during Continuous Exposure to Marine Algae Chlorella Sp[END_REF][START_REF] Wong | Temperature-Dependent Toxicities of Nano Zinc Oxide to Marine Diatom, Amphipod and Fish in Relation to Its Aggregation Size and Ion Dissolution[END_REF][START_REF] Xia | Interaction of TiO2 Nanoparticles with the Marine Microalga Nitzschia Closterium : Growth Inhibition , Oxidative Stress and Internalization[END_REF]. Growth inhibition was assessed for organisms such as bacteria, amphipods, bivalves and algae [START_REF] Deng | Biological Effects of TiO2 and CeO2 Nanoparticles on the Growth, Photosynthetic Activity, and Cellular Components of a Marine Diatom Phaeodactylum Tricornutum[END_REF][START_REF] Fabrega | Sequestration of Zinc from Zinc Oxide Nanoparticles and Life Cycle Effects in the Sediment Dweller Amphipod Corophium Volutator[END_REF][START_REF] Li | Comparative Toxicity of Nano ZnO and Bulk ZnO towards Marine Algae Tetraselmis Suecica and Phaeodactylum Tricornutum[END_REF][START_REF] Manzo | Toxic Effects of ZnO Nanoparticles towards Marine Algae Dunaliella Tertiolecta[END_REF]; [START_REF] Sendra | Effects of TiO2 Nanoparticles and Sunscreens on Coastal Marine Microalgae: Ultraviolet Radiation Is Key Variable for Toxicity Assessment[END_REF][START_REF] Wang | Environmental Microbial Community Proteomics: Status, Challenges and Perspectives[END_REF][START_REF] Xia | Interaction of TiO2 Nanoparticles with the Marine Microalga Nitzschia Closterium : Growth Inhibition , Oxidative Stress and Internalization[END_REF]. Mortality was a common parameter for Crustaceans [START_REF] Paredes | Ecotoxicological Evaluation of Four UV Filters Using Marine Organisms from Different Trophic Levels Isochrysis Galbana, Mytilus Galloprovincialis, Paracentrotus Lividus, and Siriella Armata[END_REF][START_REF] Wong | Toxicities of Nano Zinc Oxide to Five Marine Organisms: Influences of Aggregate Size and Ion Solubility[END_REF]Wong and Leung, 2013). Toxicity to coral species was evaluated using specific endpoints such as bleaching, polyp retraction, mortality [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] Downs | Toxicological Effects of the Sunscreen UV Filter, Benzophenone-2, on Planulae and in Vitro Cells of the Coral, Stylophora Pistillata[END_REF][START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF]) and metabolic alterations [START_REF] Stien | Metabolomics Reveal That Octocrylene Accumulates in Pocillopora Damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction[END_REF]. DNA damage was studied on bivalves, lugworm, coral and algae [START_REF] D'agata | Enhanced Toxicity of 'bulk' Titanium Dioxide Compared to 'Fresh' and 'Aged' Nano-TiO2 in Marine Mussels (Mytilus Galloprovincialis)[END_REF][START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] Galloway | Sublethal Toxicity of Nano-Titanium Dioxide and Carbon Nanotubes in a Sediment Dwelling Marine Polychaete[END_REF][START_REF] Jovanović | Effects of Titanium Dioxide (TiO2) Nanoparticles on Caribbean Reef-Building Coral (Montastraea Faveolata)[END_REF][START_REF] Wang | Trophic Transfer of TiO2 Nanoparticles from Marine Microalga (Nitzschia Closterium) to Scallop (Chlamys Farreri) and Related Toxicity[END_REF]. Mortality, behavioral and morphological abnormalities were the most studied parameters for Fish [START_REF] Araújo | Effects of UV Filter 4-Methylbenzylidene Camphor during Early Development of Solea Senegalensis[END_REF]Barone et al. 2019;[START_REF] Cong | The Embryotoxicity of ZnO Nanoparticles to Marine Medaka, Oryzias Melastigma[END_REF]. Table 1.7 summarizes the results of toxicity studies for organic UV filters on marine organisms. UV filters are discussed in alphabetical order for clarity purposes. et al. 2011). Its toxicity was also shown for sole [START_REF] Araújo | Effects of UV Filter 4-Methylbenzylidene Camphor during Early Development of Solea Senegalensis[END_REF]) and a marine copepod [START_REF] Chen | Multigenerational e Ff Ects of 4-Methylbenzylidene Camphor ( 4-MBC ) on the Survival , Development and Reproduction of the Marine Copepod Tigriopus Japonicus[END_REF]) at 25 and 10 µg/L, respectively.

Benzophenones

Benzophenone derivatives are by far the most studied compounds, with the greatest diversity of parameters studied. Among the compounds investigated: BP1, BP2, BP3, BP4, and BP8; BP4 was the only one that showed no toxicity to different organisms (algae, mussels, sea urchins, crustaceans, fish and coral) [START_REF] Paredes | Ecotoxicological Evaluation of Four UV Filters Using Marine Organisms from Different Trophic Levels Isochrysis Galbana, Mytilus Galloprovincialis, Paracentrotus Lividus, and Siriella Armata[END_REF][START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF]Barone et al. 2019). BP3 induced bleaching in coral species such as Seriatopora caliendrum, Styolphora pistillata and Acropora sp. [START_REF] Danovaro | Sunscreens Cause Coral Bleaching by Promoting Viral Infections[END_REF][START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF][START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF]) from 33 µg/L. [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF] reported increased bleaching in the presence of light, as well as DNA damage from 2.8 µg/L, and mortality from 22.8 µg/L, concentrations in the same range as reported in the literature for environmental water samples (S. Kim and Choi 2014).

Ethylhexyl methoxy cinnamate

EHMC toxicity was studied for algae, mussels, sea urchins, and a crustacean by [START_REF] Paredes | Ecotoxicological Evaluation of Four UV Filters Using Marine Organisms from Different Trophic Levels Isochrysis Galbana, Mytilus Galloprovincialis, Paracentrotus Lividus, and Siriella Armata[END_REF]. EC50 for algae was 74.73 µg/L. EHMC showed a species-dependent effect on corals species.

While it did not increase mortality in P. damicornis from 0.1 to 1000 µg/L, EHMC increased the mortality of S. caliendrum and induced bleaching at 1 mg/L [START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF]). Polyp retraction was observed from 10 µg/L for the same species [START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF]). 91% of bleaching was observed at concentrations of 33 µg/L and 50 µg/L for Acropora sp. Comparatively, occurrence study highlighted its presence up to 4 µg/L [START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF].

Octocrylene

OC was detected in seawater at concentrations up to 7 µg/L [START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF][START_REF] Langford | Environmental Occurrence and Risk of Organic UV Filters and Stabilizers in Multiple Matrices in Norway[END_REF]. Several studies have highlighted detrimental effects against marine organisms from 40 µg/L. Its effect on coral species appeared to be nuanced [START_REF] Danovaro | Sunscreens Cause Coral Bleaching by Promoting Viral Infections[END_REF][START_REF] Fel | Photochemical Response of the Scleractinian Coral Stylophora Pistillata to Some Sunscreen Ingredients[END_REF]). However, He et al. ( 2019) demonstrated revealed polyp retraction at 1 mg/L and increased mortality in P. damicornis if combined with EHMC. A recent metabolomic study showed that P. damicornis accumulated OC in fatty acid-conjugated forms from 5 µg/L and was associated with mitochondrial dysfunctions at 50 µg/L, whereas polyps were only retracted at 300 µg/L [START_REF] Stien | Metabolomics Reveal That Octocrylene Accumulates in Pocillopora Damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction[END_REF]). This study showed that usual ecotoxicological parameters such as mortality or polyps state may result in an underestimation of compound toxicity.

Other organic UV filters

Other organic UV filters such as TDSA, BM, DT, ES, and ET have received little attention, with only one or two studies on coral species [START_REF] Danovaro | Sunscreens Cause Coral Bleaching by Promoting Viral Infections[END_REF][START_REF] Fel | Photochemical Response of the Scleractinian Coral Stylophora Pistillata to Some Sunscreen Ingredients[END_REF]. The toxicity of approximately 45 globally authorized sunscreen UV filters has not been assessed on marine biota.

Therefore, it is difficult to conclude about their effects and the risk associated with their use.

Noteworthy, while homosalate is common in cosmetic formulation, no ecotoxicological investigation has been conducted so far.

Effects on freshwater organisms

Camphor derivatives

Camphor derivatives are well-known compounds. They inhibited the growth of two algal species, namely D. subspicatus [START_REF] Sieratowicz | Acute and Chronic Toxicity of Four Frequently Used UV Filter Substances for Desmodesmus Subspicatus and Daphnia Magna[END_REF]S. vittatum (D. Campos, Gravato, Quintaneiro, et al. 2017), and demonstrated toxicity to fishes in vitro [START_REF] Kunz | Comparison of in Vitro and in Vivo Estrogenic Activity of UV Filters in Fish[END_REF][START_REF] Li | Effects of 4-Methylbenzylidene Camphor (4-MBC) on Neuronal and Muscular Development in Zebrafish (Danio Rerio) Embryos[END_REF][START_REF] Torres | Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban[END_REF], from 435 µg/L, concentration higher than those reported in the literature for environmental samples [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF].

Benzophenones

Benzophenones toxicity studies dominate the research in freshwater environments. Multiple studies highlighted its toxicity on different trophic levels, including daphnid, fish, worm or algae from 10 µg/L while its concentration reached the nanogram per liter in freshwater environments [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF][START_REF] Fent | Widespread Occurrence of Estrogenic UV-Filters in Aquatic Ecosystems in Switzerland[END_REF][START_REF] Tang | Concentrations and Tissue-Specific Distributions of Organic Ultraviolet Absorbents in Wild Fish from a Large Subtropical Lake in China[END_REF]

Ethylhexyl methoxy cinnamate

Similar studies have been performed with EHMC with the only exception of [START_REF] Christen | Effects of the UV-Filter 2-Ethyl-Hexyl-4-Trimethoxycinnamate (EHMC) on Expression of Genes Involved in Hormonal Pathways in Fathead Minnows (Pimephales Promelas) and Link to Vitellogenin Induction and Histology[END_REF], who investigated the response of P. promelas at the transcripts level. The authors highlighted differential expression of several genes, including ESR1, coding for an estrogen receptor also present in humans.

Octocrylene

Only a few studies were performed on octocrylene. To the best of our knowledge, only three organisms were investigated (see table below). Blüthgen and coworkers (2014) did a similar transcripts analysis as described above for zebrafish (D. rerio). The authors showed that OC dysregulates most genes related to the hormone system from 22 µg/L.

Effects on bacteria

Inorganic UV filters, also known as mineral UV filters, have been broadly advertised as ecofriendly. Nevertheless, several studies reported their toxicity on marine biota (Lozano et al. 2020).

Because they are not at the core of this thesis, the effect of inorganic UV filters won't be investigated nor discussed apart from this section. Titanium dioxide (TiO2) shows antimicrobial activity against several ubiquist Enterobacteriaceae strains with minimal inhibitory concentration (MIC) ranging from 40 to 80 µg.ml -1 [START_REF] Rajakumar | Fungus-Mediated Biosynthesis and Characterization of TiO2nanoparticles and Their Activity against Pathogenic Bacteria[END_REF]. Another inorganic filter, Zinc oxide (ZnO) nanoparticles, inhibit strongly the growth of E. coli and are internalized by bacterial cells [START_REF] Brayner | Toxicological Impact Studies Based on Escherichia Coli Bacteria in Ultrafine ZnO Nanoparticles Colloidal Medium[END_REF]).

Oxybenzone demonstrates toxicity on the freshwater cyanobacterium: Microcystis aeruginosa [START_REF] Mao | Effects of Benzophenone-3 on the Green Alga Chlamydomonas Reinhardtii and the Cyanobacterium Microcystis Aeruginosa[END_REF], and the marine bioluminescent bacteria: Vibrio fischeri [START_REF] Zhang | Evaluation of Ecotoxicological Effects of Benzophenone UV Filters: Luminescent Bacteria Toxicity, Genotoxicity and Hormonal Activity[END_REF]) and

Photobacterium phosphoreum [START_REF] Liu | Acute Toxicity of Benzophenone-Type UV Filters for Photobacterium Phosphoreum and Daphnia Magna: QSAR Analysis, Interspecies Relationship and Integrated Assessment[END_REF]. A decrease in the chl-a content and an elevation of carotenoid production have been reported for Microcystis aeruginosa [START_REF] Mao | Effects of Benzophenone-3 on the Green Alga Chlamydomonas Reinhardtii and the Cyanobacterium Microcystis Aeruginosa[END_REF]). 4-MBC and its degradation by-products, when tested on V. Fischeri cultures, demonstrated acute toxicity at 400 µg.L - 1 [START_REF] Sakkas | TiO2/H2O2mediated Photocatalytic Transformation of UV Filter 4-Methylbenzylidene Camphor (4-MBC) in Aqueous Phase: Statistical Optimization and Photoproduct Analysis[END_REF]. In microcosms experiments, sunscreen mixtures strongly increased the viral to bacteria ratio with a number of viral cells 20-40 times higher than in control. In addition, even if the bacterial cell number was not affected, enzymatic activities were modulated by sunscreen exposure (higher glucosidase, aminopeptidase and phosphatase activity) ( [START_REF] Danovaro | Sunscreen Products Increase Virus Production through Prophage Induction in Marine Bacterioplankton[END_REF]. It is important to underline that these results cannot be attributed to UV filters as the experiments have been performed with a commercial mixture.

The toxicity of benzophenone derivatives against bacteria was correlated with the number of hydroxyl groups on the benzene rings [START_REF] Liu | Acute Toxicity of Benzophenone-Type UV Filters for Photobacterium Phosphoreum and Daphnia Magna: QSAR Analysis, Interspecies Relationship and Integrated Assessment[END_REF][START_REF] Zhao | Substituent Contribution to the Genotoxicity of Benzophenone-Type UV Filters[END_REF]. Additionally, the position of the hydroxy groups influences molecular polarity which induces increasing bacterial genotoxicity, by causing mutations. Indeed, bacteria were found to be more sensitive to molecules with higher dipole moments, i.e. polar compounds [START_REF] Zhao | Substituent Contribution to the Genotoxicity of Benzophenone-Type UV Filters[END_REF]). In addition, the introduction of the methoxy group at the 4-position and/or the chloric group at the 5-position increases the compound toxicity whereas the addition of a sulfonic acid group at the 5-position decreases toxicity.

A linear relationship has been established between n-octanol/water partition coefficient (log Kow) and toxicity against V. fischeri (Figure 1.10) [START_REF] Zhang | Evaluation of Ecotoxicological Effects of Benzophenone UV Filters: Luminescent Bacteria Toxicity, Genotoxicity and Hormonal Activity[END_REF]. Similarly, a positive relation between toxicity and chemical hydrophobicity (R²=0.862) has been established (H. [START_REF] Liu | Acute Toxicity of Benzophenone-Type UV Filters for Photobacterium Phosphoreum and Daphnia Magna: QSAR Analysis, Interspecies Relationship and Integrated Assessment[END_REF].

Solar radiations and UV filters 4.1 Solar radiations in the marine environment

Sunlight radiations that reach the earth's surface comprise UVB, UVA, visible light (400-700 nm)

and infrared (>700 nm). UVC (100-280 nm) are absorbed by the stratospheric ozone (Figure 1.11). UVB are strongly absorbed by the stratospheric ozone layer but still reach the earth's surface. UVA, visible light and infrared reach the earth's surface without attenuation. The fraction of UV rays that penetrate through natural water cannot be given as a stable measure as it strongly depends on weather and water composition [START_REF] Booth | The Penetration of UV into Natural Waters[END_REF]. In the Ocean, particulate components such as dissolved matter vary daily and affect light penetration. Depending on the regarded study, UVB penetration range from 1 to 15 meters while UVA penetration can reach a hundred meters (Figure 1.11) [START_REF] Fleischmann | The Measurement and Penetration of Ultraviolet-Radiation Into Tropical Marine Water[END_REF][START_REF] Tedetti | Penetration of Ultraviolet Radiation in the Marine Environment. A Review[END_REF]. The light that reaches the deepest part of the euphotic zone, i.e. 100 m, is made of higher wavelength photons. Per photon, UVB is more biologically harmful than UVA.

However, the higher photon fluxes and penetration of UVA into the water column make it the strongest If compared to other planktonic organisms, bacterioplankton are more affected by UV radiation, because of the lack of protective pigments, microsporine like amino acid, scytonemin or their large surface:volume ratio [START_REF] Garcia Pichel | A Model for Internal Self-Shadingvin Planktonic Organisms and Its Implications for the Usefulness of Ultraviolet-Sunscreen[END_REF][START_REF] Karentz | Ultraviolet Tolerance Mechanisms in Antarctic Marine[END_REF]. The composition of bacterial communities is affected by UV radiation as bacteria show differential resistance patterns toward UV exposure [START_REF] Winter | Impact of UV Radiation on Bacterioplankton Community Composition †[END_REF]. For instance, UVR and PAR inhibit SAR11 clade while Gammaproteobacteria and Bacteroidetes are more resistant [START_REF] Ruiz-González | Seasonal Patterns in the Sunlight Sensitivity of Bacterioplankton from Mediterranean Surface Coastal Waters[END_REF]. Intra-phyla differential pattern of resistance has been reported [START_REF] Agogué | Resistance of Marine Bacterioneuston to Solar Radiation[END_REF], meaning that sensitivity against UV radiation is species-dependent. This observation was confirmed later by Santos et al. (2012), who demonstrated via a microcosm experiment that UVB preferentially affected the bacterial abundance and DNA synthesis of bacterioplanktonic communities, compared to the bacterioneuston, i.e. bacteria from the surface microlayer, even if the latter are more subjected to UV radiations. While UV radiations are known to induce direct biological damages to bacteria, their impact combined with anthropogenic input, known as indirect effects, is not well understood.

UV filter photochemistry

Because UV filters can undergo photodegradation processes, the understanding of their photochemistry is essential. Photoproducts can be less, equally, or more toxic than initial chemicals.

The following compounds remain stable under direct light exposure in experimental conditions: BP3 [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF][START_REF] Liu | Photostability of the UV Filter Benzophenone-3 and Its Effect on the Photodegradation of Benzotriazole in Water[END_REF], OC [START_REF] Herzog | Photostability of UV Absorber Systems in Sunscreens[END_REF][START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF], Tinosorb S [START_REF] Herzog | Photostability of UV Absorber Systems in Sunscreens[END_REF]) and 4-MBC [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]). On the other hand, EHMC [START_REF] Jentzsch | Photodegradation of the UV Filter Ethylhexyl Methoxycinnamate under Ultraviolet Light: Identification and in Silico Assessment of Photo-Transformation Products in the Context of Grey Water Reuse[END_REF], IAMC (MacManus-Spencer et al. 2011), OD-PABA [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]), Ethylhexyl triazone (EHT) [START_REF] Damiani | Assessment of the Photo-Degradation of UV-Filters and Radical-Induced Peroxidation in Cosmetic Sunscreen Formulations[END_REF][START_REF] Herzog | Photostability of UV Absorber Systems in Sunscreens[END_REF], BMDBM [START_REF] Damiani | Assessment of the Photo-Degradation of UV-Filters and Radical-Induced Peroxidation in Cosmetic Sunscreen Formulations[END_REF] and Benzotriazole (BT) [START_REF] Liu | Photostability of the UV Filter Benzophenone-3 and Its Effect on the Photodegradation of Benzotriazole in Water[END_REF] demonstrate a lower photostability. In addition, the influence of photosensitizers in the medium has been demonstrated, distinguishing direct or indirect photodegradation process [START_REF] Li | Effects of 4-Methylbenzylidene Camphor (4-MBC) on Neuronal and Muscular Development in Zebrafish (Danio Rerio) Embryos[END_REF]). The environmental relevance of these studies must be taken with caution. Indeed, strong variations between experimental setup (e.g. lamp spectrum, exposure time, aqueous solution, single or combined compound, etc.) have been observed (Table 1.9). These differences depend on the context of the study.

For instance, the stability in phosphatidylcholine liposomes or mixed with other compounds aims to assess the photo-stability in a cosmetic product formulation. Studies that investigate the environmental fate of UV filters have been performed in sea or freshwater collected in situ, under simulated sunlight exposure. Finally, the assessment of UV filter stability under UVC exposure aims to mimic the degradation process realized in WTP. Table 1.9 compiles all UV filters photochemistry studies. For clarity, details will be provided below for compounds studied in this thesis only. BP3 is photostable under both UVC (254nm) irradiation (9h) and artificial solar light (290 to 800nm) exposure (50 days) [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF][START_REF] Liu | Photostability of the UV Filter Benzophenone-3 and Its Effect on the Photodegradation of Benzotriazole in Water[END_REF]. Under Artificial Solar Light (ASL) exposure, the presence of NaCl, Cu 2+ and Fe 3+ slightly increased the photodegradation. The presence of humic acid in the medium significantly increased BP3 photodegradation (31% loss with 50 mg.L -1 humic acid). [START_REF] Liu | Photostability of the UV Filter Benzophenone-3 and Its Effect on the Photodegradation of Benzotriazole in Water[END_REF]. In pure water, BP3 and 4-hydroxybenzone, its human derived metabolite, were resistant to direct photolytic degradation in their neutral form (Hg lamp). Only anionic forms underwent direct photodegradation at alkaline pH [START_REF] Li | Effects of 4-Methylbenzylidene Camphor (4-MBC) on Neuronal and Muscular Development in Zebrafish (Danio Rerio) Embryos[END_REF]. Indirect photodegradation by environmental photosensitizers (e.g. excited triplet state Dissolved Organic Matter ( 3 DOM), 1 O2, and hydroxyl radical (•OH)), i.e. compounds that absorb radiation and transfer it to adjacent molecules can occur. 3 DOM was responsible for BP3 and 4-hydroxybenzone (4-OH-BP3) indirect photodegradation in sea water while 3 DOM and •OH were responsible for indirect photodegradation in freshwater [START_REF] Semones | Photodegradation of UV Filters Oxybenzone and Sulisobenzone in Wastewater Effluent and by Dissolved Organic Matter[END_REF][START_REF] Li | Effects of 4-Methylbenzylidene Camphor (4-MBC) on Neuronal and Muscular Development in Zebrafish (Danio Rerio) Embryos[END_REF][START_REF] Gago-Ferrero | An Overview of UV-Absorbing Compounds (Organic UV Filters) in Aquatic Biota[END_REF].

OC demonstrated strong stability under irradiation for 72h with a halogen lamp (200 to 800nm) [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]. Using the plate method, i.e. a technique that consists of plating UV absorbers in realistic formulation on quartz plates and analyzing the mixture via HPLC after irradiation, OC remained photo-stable under UV exposure (from 290 to 400nm) [START_REF] Herzog | Photostability of UV Absorber Systems in Sunscreens[END_REF]. Under halogen lamp exposure, 4-MBC remained stable but (E) to (Z) photo-isomerization was reported and assumed to be a way to reduce the absorbed UV energy [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF][START_REF] Moneyron | Photochemical E ⇄ Z Isomerization of Some Benzylidene Camphor and Benzylidene Tetrahydrofuranone Derivatives[END_REF].

Under irradiation for 72h with a halogen lamp (200 to 800nm) EHMC and IAMC were photodegraded (half-life: 21h and 59h, respectively). In addition, both compounds underwent dimerization (MacManus-Spencer et al. 2011) and fast (E) to (Z) photo-isomerization [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]. Under UVA exposure into a phosphatidylcholine liposome (a technique used to determine photostability under conditions of usage in a cosmetic product), its absorbing capabilities decreased [START_REF] Damiani | Assessment of the Photo-Degradation of UV-Filters and Radical-Induced Peroxidation in Cosmetic Sunscreen Formulations[END_REF]. When irradiated by a Hg lamp, covering UVA, UVB and UVC range (200-400nm), EHMC parent compounds were completely removed via UV treatment after less than 64min, leading to six different photoproducts [START_REF] Jentzsch | Photodegradation of the UV Filter Ethylhexyl Methoxycinnamate under Ultraviolet Light: Identification and in Silico Assessment of Photo-Transformation Products in the Context of Grey Water Reuse[END_REF].

Combined effects of UV radiation and UV filters

Toxicity studies combining exposure of organic compounds and UV radiation are scarce. In addition, the few studies available investigated the effect of compounds after light exposure and not both at the same time. Because of the environmental condition in the photic zone of the oceans, experiments combining light and compound exposure are necessary to understand the mechanisms involved in an environmentally relevant fashion. Indeed, organic compounds can undergo structural modification under light exposure. As a consequence, the toxicity of the molecule can be affected, either negatively or positively. In order to appreciate the ecotoxicology of UV filters, it is essential to investigate the effect of these molecules, combined with light exposure.

Photomutagenicity of three benzylidene camphor derivates has been tested at different intensities of UVA/UVB exposure for an Escherichia coli strain. None of the molecules induced photomutagenicity, regardless of the UV intensity [START_REF] Dean | Development of Assays for the Detection of Photomutagenicity of Chemicals during Exposure to UV Light. II. Results of Testing Three Sunscreen Ingredients[END_REF]). However, experimental details are scarce, and time of exposure is not mentioned. 2-Phenylbenzimidazole (PBI) did not induce bacterial death alone at high concentrations (625 µM). Light exposure under a solar simulator (Hg lamp, 30 min, 18.6 J.cm -2 ) prior to incubation increased cytotoxic effects depending on light exposure duration (0 to 30 mn) [START_REF] Mosley | Light-Induced Cytotoxicity and Genotoxicity of a Sunscreen Agent, 2-Phenylbenzimidazole in Salmonella Typhimurium TA 102 and HaCaT Keratinocytes[END_REF]). Rodil and coworkers (2009) assessed the toxicity of IAMC, OD-PABA, and EHMC after ASL exposure times (from 0 to 72h) on S. vacuolatus, a freshwater green algae. Light exposure decreased the toxicity for all compounds, indicating that toxicity is correlated with the concentration of the parent substances [START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]). The authors also demonstrated that OD-PABA lost its methyl groups after light exposure. These results are consistent with a study from [START_REF] Zhao | Substituent Contribution to the Genotoxicity of Benzophenone-Type UV Filters[END_REF], reporting that the methyl group is involved in the compound toxicity against bacteria. Conversely, a more recent study highlighted that EHMC toxicity increased after UV light exposure on three toxicological models, including Aliivibrio fischeri (microtox® test) [START_REF] Gackowska | Estimation of Physicochemical Properties of 2-Ethylhexyl-4-Methoxycinnamate (EHMC) Degradation Products and Their Toxicological Evaluation[END_REF].

The non-organic UV filter: titanium dioxide, combined with light exposure, demonstrated strong bactericidal effects for several strains (E. coli, S. typhimurium, P. aeruginosa, E. cloacae) when combined with UVA [START_REF] Ibáñez | Photocatalytic Bactericidal Effect of TiO2on Enterobacter Cloacae. Comparative Study with Other Gram (-) Bacteria[END_REF]. Under solar light, TiO2 nanoparticles, present in sunscreen, produced hydrogen peroxide that affects the growth of phytoplankton (Sánchez-Quiles and Tovar-Sánchez 2014; [START_REF] Sendra | Effects of TiO2 Nanoparticles and Sunscreens on Coastal Marine Microalgae: Ultraviolet Radiation Is Key Variable for Toxicity Assessment[END_REF].

Proteomics for environmental research

Proteomics consists of the study of the proteome, i.e. all proteins in a given time and set of conditions. Recently, proteomics has emerged as a powerful tool allowing for bacterial species identification, biomarker discovery, the understanding of unraveled molecular pathways, and organismal response under different conditions (natural, pathological, or toxicological). Noteworthy, while "expression" is the term used to describe the number of genes transcribed into mRNA, "regulation" is more suitable to discuss the changes in protein abundance. Indeed, the cellular protein concentration implies four processes: (i) protein synthesis, (ii) protein processing, (iii) protein secretion, and (iv)

protein degradation/turn-over.

Mass spectrometry (MS) involves the separation of ionized molecules based on their mass-tocharge ratio. Recently designed mass spectrometers are highly resolutive, permitting the discrimination between molecules with close masses (Allison et al. 2008;[START_REF] Schymanski | Identifying Small Molecules via High Resolution Mass Spectrometry: Communicating Confidence[END_REF]. MS allows for relative protein quantification and identification. Compared to gel-based techniques, mass spectrometry allows for higher throughput and a greater number of identified molecules in a single run. Proteomics is used to compare conditions between biological states (treated vs control; healthy vs disease; mutant vs wild type). To do so, labeling techniques -such as Isobaric tags for relative and absolute quantitation (iTRAQ)were developed [START_REF] Evans | Profiling the Secretome of the Marine Bacterium Pseudoalteromonas Tunicata Using Amine-Specific Isobaric Tagging (ITRAQ)[END_REF]). However, these methods are complex and timeconsuming, hardly reproducible, and expensive. Thanks to high-resolution mass spectrometers and powerful software, label-free approaches have emerged, permitting the profiling of a large number of proteins for multiple different conditions.

In proteomic research, two approaches can be distinguished: in vitro studies assessing the response of an organism under a set of conditions as described in chapter 4, and metaproteomic studies, assessing the response of natural communities (see preliminary results described in chapter 6).

Proteomics and ecotoxicological applications

Non-targeted shotgun proteomics is helping us to discover biomarkers in multiple conditions, including pollutant exposure. Several proteomic studies highlighted differential proteome regulations in fish cells exposed to a wide range of pollutants, such as flame retardants, steroids, sodium fluoride, and heavy metals [START_REF] Sanchez | Review of Recent Proteomic Applications in Aquatic Toxicology[END_REF]. Most articles cited in this review involved a gel-based approach, namely 2D gel electrophoresis -MALDI TOF. [START_REF] Tomanek | Environmental Proteomics: Changes in the Proteome of Marine Organisms in Response to Environmental Stress, Pollutants, Infection, Symbiosis, and Development[END_REF] reviewed proteomic studies assessing the effect of pollutants and environmental stressors on multiple species. Overall, these studies allowed the identification and/or quantification of proteins that are specific to culture conditions, and provided insights into the adaptation strategies employed by the studied organisms.

Several proteomic studies addressed the response of marine bacteria exposed to changing salt concentrations and culture media components (Christie-Oleza et al. 2011), cold stress [START_REF] Isabelle | The Effect of Cold Stress on the Proteome of the Marine Bacterium Pseudomonas Fluorescens BA3SM1 and Its Ability to Cope with Metal Excess[END_REF], copper [START_REF] Favre | Metabolomic and Proteomic Changes Induced by Growth Inhibitory Concentrations of Copper in the Biofilm-Forming Marine Bacterium[END_REF], UV radiation (Matallana- [START_REF] Matallana-Surget | The Response of the Marine Bacterium Sphingopyxis Alaskensis to Solar Radiation Assessed by Quantitative Proteomics[END_REF], heat shock, and variation in metal concentration (Matallana-Surget 2018). However, proteomic studies exploring the bacterial response toward anthropogenic contaminant exposure are scarce [START_REF] Fajardo | Transcriptional and Proteomic Stress Responses of a Soil Bacterium <i>Bacillus Cereus<i/> to Nanosized Zero-Valent Iron ( NZVI ) Particles[END_REF][START_REF] Spalla | Metabolomic and Proteomic Investigations of Impacts of Titanium Dioxide Nanoparticles on Escherichia Coli[END_REF]. Quantitative proteomics is on the rise with shotgun proteomics allowing the study of the whole proteome using LC-MS/MS analysis. Conventional DDA MS implies the scanning of all precursor peptide ions during the survey scan (MS1), followed by the selection of a predefined number of precursor ions of subsequent fragmentation (MS2). This approach, involving sequential selection/fragmentation, provides the peptide sequence of the precursor ions. The technical improvement in the MS instruments resulted in the rise of a next-generation proteomic known as Dataindependent analysis (DIA), or SWATH (Sequential window acquisition of all Theoretical Mass Spectra).

Data

In contrast to DDA where only a predefined set of precursor ions are fragmented, DIA is based on the fragmentation of all precursor ions identified in the MS1 survey scan where fragment ions are accumulated in a fixed number of wide isolation windows that span the entire mass-to-charge ratio (m/z) range. In DIA, because all precursor ions from the survey scan are fragmented, there is a need to interrogate the resulting MS data with a spectral library, which is a database generated from a DDA analysis, containing mass spectrometric and chromatographic parameters such as precursor and fragment m/z value, fragment type, and elution time for each peptide. Hence, the proteome coverage of a DIA analysis depends on the coverage obtained from the DDA run performed to generate the spectral library. In order to decrease time and cost related to spectral library generation, public spectral library databases have been employed but are only available for a limited number of organisms (see [START_REF] Krasny | Molecular Omics Data-Independent Acquisition Mass Spectrometry ( DIA-MS ) for Proteomic Applications in Oncology[END_REF]).

Figure 1.12. Typical workflow for Data-independent acquisition (DIA) proteomics.

Metaproteomics

In the last decades, community proteomics, also called metaproteomics have emerged, permitting to consider the physiological and ecological functions of an entire community, rather than a single organism (see Figure 1.13 for the experimental workflow). While information from metagenomics is limited to the identification of the presence of genes, taxa, and their inferred functional capacity, the introduction of metaproteomics enables a thorough functional understanding of microbial communities [START_REF] Wang | Environmental Microbial Community Proteomics: Status, Challenges and Perspectives[END_REF]. The 2D-LC-MS/MS approach has demonstrated that 36% and 17% of the proteins from Oregon coast microbial plankton belong to the SAR11 and the Rosebacter clades, respectively [START_REF] Sowell | Environmental Proteomics of Microbial Plankton in a Highly Productive Coastal Upwelling System[END_REF]). The most abundant proteins were amino acid transporters, taurine, polyamine, and glutamine

synthetase. An LC-MS/MS study has demonstrated that nitrification by Thaumarchaeota and Nitrospina was a dominant metabolic strategy [START_REF] Colatriano | Metaproteomics of Aquatic Microbial Communities in a Deep and Stratified Estuary[END_REF]. MS techniques have also been used to investigate the microbial response under exposure to pollutants (uranium, hydrocarbon, cadmium) [START_REF] Matallana-Surget | Comparative Metaproteomics to Study Environmental Changes[END_REF][START_REF] Wang | Environmental Microbial Community Proteomics: Status, Challenges and Perspectives[END_REF]. These studies helped to decipher the pathways involved in the community response and the bacterial fraction responsible for it.

Project aims

Organic UV filters occur worldwide in marine environments from the surface microlayer to sediments. As described in section 2 of this manuscript, while ecotoxicological studies have reported their detrimental effects on multiple organisms, bacteria, that constitute a major portion of the biomass, have been largely omitted from the picture. Heterotrophic marine bacteria play pivotal roles in biogeochemical cycles. Hence, this thesis aims to expand our comprehension of the impact of UV filters, on environmental bacterial species. Chapter 2 describes the effects of five common organic UV filters on 27 heterotrophic strains, isolated from the bay of Banyuls (NW Mediterranean Sea). Because UV radiations modulate UV filters chemistry, the combined effects of solar light and UV filters are investigated.

Epibacterium mobile is a well-studied Alpha-proteobacteria from the abundant Roseobacter clade that displayed sensitivity under oxybenzone exposure (Chapter 2). Consequently, the latter is used to broaden our knowledge on the bacterial molecular response toward oxybenzone exposure. In Chapter 3, enzymatic activity is measured and cell physiology is assessed via transmission electronic microscopy (TEM). In Chapter 4, the proteome response after oxybenzone exposure at 350 µg/L (the EC50 determined in Chapter 2) is assessed via label-free quantification proteomics. Chapter 5 describes a genomic pairwise comparison between UV filter sensitive and resistant strains. This in silico analysis aims to decipher the bacterial features that could explain the resistant phenotypes. Lastly, Chapter 6

provides an overview of the main take-home messages and suggests areas for future research. A preliminary method development, critical thinking on the current research state of the research and an insight into the potential solutions are presented.

Introduction

Organic UV filters are chemicals that absorb UVB (280-315nm) and/or UVA (315-400nm) [START_REF] Shaath | Ultraviolet Filters[END_REF]). Due to their absorbing properties, UV filters are the main components of sunscreen, and are also found in industrial products such as paints and plastics as stabilizing agents. UV filters are of emerging concern due to their large production volumes, the increasing coastal tourism, their persistence related to their chemical properties, and their toxicity [START_REF] Rainieri | Occurrence and Toxicity of Musks and UV Filters in the Marine Environment[END_REF] Our study investigates for the first time the toxicity of commonly used UV filters, namely BP3, OC, 4-MBC, EHMC and homosalate (HS) on marine heterotrophic bacteria, sampled from the Mediterranean Sea (Banyuls sur Mer, France). For this purpose, twenty-seven environmentally relevant bacteria were tested for their response against the above-mentioned compounds. Furthermore, dose response analysis was carried out for sensitive strains, in order to better understand the range of UV filters toxicity. Interestingly, UV filters toxicity can also be modulated by other key intrinsic or extrinsic parameters that must be taken into account. For instance, both the physiological state [START_REF] Eng | Bactericidal Effects of Antibiotics[END_REF];

Jaishankar and Srivastava 2017) and sunlight can alter the bacterial susceptibility to bactericidal compounds and anthropogenic contaminants [START_REF] Pelletier | PHOTOTOXICITY OF INDIVIDUAL POLYCYCLIC AROMATIC HYDROCARBONS AND PETROLEUM TO[END_REF][START_REF] Petersen | Phototoxicity of Pyrene Affects Benthic Algae and Bacteria from the Arctic[END_REF][START_REF] Sakkas | TiO2/H2O2mediated Photocatalytic Transformation of UV Filter 4-Methylbenzylidene Camphor (4-MBC) in Aqueous Phase: Statistical Optimization and Photoproduct Analysis[END_REF]. Therefore, we also aimed at assessing the bacterial response to UV filters, harvested at both exponential and stationary phases and combined with solar radiation exposure. Overall, this paper presents a comprehensive investigation of the impact of five common UV filters on diverse marine bacterial species, spanning a range of different cellular parameters and UV exposure.

Materials and methods

Chemicals

Five organic UV filters, namely, benzophenone-3 (CAS-No. 131-57-7), ethylexyl methoxy cinnamate (CAS-No. 5466-77-3), octocrylene (CAS-No. 6197-30-4), 4-methylbenzylidene camphor (CAS-No 36861-47-9) and homosalate (CAS-No 118-56-9), were purchased from Sigma-Aldrich (Steinheim, Germany). Stock solutions of UV filters were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, purity >99%) at a concentration of 750 mg L -1 and stored in the dark at room temperature.

Bacterial strains

A total of 27 bacterial strains from the Banyuls Bacterial Culture Collection (BBCC, https://collection.obs-banyuls.fr/catalogue.php) was used in this study (Table 2.2). Species were selected to ensure environmentally relevant diversity. Bacterial strains were kept at -80°C in marine broth 2216 (DIFCO, United States) with 35% glycerol. Bacteria were grown on marine agar plates. After 24-48 hours incubation, colonies were suspended and grown aerobically on a rotary shaker (110 rpm) at 25ºC in artificial seawater with 3 mM D-glucose, vitamins, and trace elements (ASW-G) [START_REF] Eguchi | Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas Sp. Strain RB2256[END_REF].

Cultures were performed in triplicates.

Toxicity of UV filters

The response of 27 bacterial strains (Table 2.2) against UV filters (1000 µg L -1 ) was assessed in minimum growth medium ASW-G. Bacterial growth was monitored in a 24 well plate for 48 hours using a microplate reader (Paradigm, Molecular Device, United States) in presence of UV filters, with 1% of DMSO, to enhance the UV filters solubility. UV filters free and DMSO free controls were performed.

Dose response on bacterial growth

Dose response was further investigated for strains that showed sensitivity to UV filters at 1000 µg L -1 . The tested concentrations of UV filters were the following: 0, 100, 200, 500, 1000, 2000 and 4000 µg L -1 , in ASW-G with 1% DMSO.

Combined effect of UV filter and solar radiation

The combined effect of solar radiation and UV filter was assessed on sensitive strains. Exposure to artificial solar light was performed using an Oriel (United States) solar simulator, equipped with Hg 1600W sunlight full spectrum lamp (280-800nm).

The toxicity of the irradiated and non-irradiated UV filters was assessed on bacteria in stationary phase and exponential phase (Figure 2.1). Stationary phase cells were maintained in ASW while growing cells were cultivated in ASW-G (supplemented with 3mM D-glucose, trace metals and vitamins). Solar radiation exposure was performed in quartz flasks.

Effect of irradiated UV filters on stationary phase culture

Bacteria were cultured in ASW-G, until stationary phase was reached. Cultures were centrifuged for 10 min at 3000 g and the cell pellets were washed in ASW to remove traces of carbon source. Cells were suspended in ASW with 1000 µg L -1 UV filters, pre-irradiated for 5 hours under solar light. Non irradiated molecule and UV filters free medium were used as controls. Flasks were incubated and plated for CFU counting after 0, 5 and 24 hours of incubation at 25°C.

Effect of irradiated UV filters on exponential phase culture

UV filters (1000 µg L -1 ) in ASW were exposed to artificial solar radiation for 5 hours. It is noteworthy to mention that vitamins and glucose were added after exposure to solar radiation in order to prevent UV-induced photoproducts from the growth medium components. Media were inoculated with fresh precultures and the optical density (OD) at 620 nm was monitored from 48 to 72 hours. Non irradiated medium and UV filters free medium were used as controls.

Direct effect of solar radiation and UV filters on stationary phase culture

Stationary phase cultures were prepared as described in 2.5.1. Bacteria were submitted to four treatments: (1) ASW + UV filters 1000 µg L -1 in dark condition, (2) ASW + UV filters 1000 µg L -1 exposed to artificial solar light, (3) ASW in dark condition, (4) ASW exposed to artificial solar radiation.

Plating for CFU counting was performed after 0, 2.5 and 5 hours of exposure to the different treatments.

Direct effect solar radiation and UV filters on exponential phase culture

Bacterial cultures were grown in Erlenmeyer flasks. Bacteria were harvested at an OD (620 nm) of 0.15 and subsequently transferred into quartz flasks. Bacteria were subjected to four treatments: (1) ASW + UV filters 1000 µg L -1 in dark condition; (2) ASW + UV filters 1000 µg L -1 + solar radiation;

(3) ASW in dark condition; (4) ASW + solar radiation. OD (620 nm) was monitored for 5 hours.

Statistical analysis

All experiments were performed in triplicate. The normality of the data was verified using Kolmogorov-Smirnov test. One-way analysis of variance (ANOVA) and post-hoc Tukey HSD tests were used to assess the significance of the toxicity between different treatments (p-value < 0.05).

Results and discussion

. Our study addressed for the first time, the combined impact of five common UV filters with solar radiation on diverse marine bacteria. In addition to the well-studied UV filters such as octinoxate, oxybenzone, and octocrylene, we provided the original toxicity assessment of homosalate on marine organisms.

Toxicity of UV filters

We first analyzed the bacterial response to UV filters at a concentration of 1000 µg L -1 . For this purpose, we studied a diversity of bacteria from different Phyla, a panel composed of nine γ-Proteobacteria, seven α-Proteobacteria, five Bacteroidetes, three Actinobacteria, and three Firmicutes.

A total of 7 out of 27 bacterial species showed sensitivity to one, or more UV filters at 1000 µg L -1 , representing 26% of all tested bacteria (Table 2.3). OC was toxic to one species. BP3 and HS were deleterious to two species and EHMC was the most toxic affecting five bacterial species. Although 4-MBC toxicity was previously reported [START_REF] Campos | Ecotoxicity of Two Organic UV-Filters to the Freshwater Caddis Fly Sericostoma Vittatum[END_REF][START_REF] Sieratowicz | Acute and Chronic Toxicity of Four Frequently Used UV Filter Substances for Desmodesmus Subspicatus and Daphnia Magna[END_REF][START_REF] Torres | Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban[END_REF], no sensitivity was observed in this study. It is interesting to note that BP3 only affected gram negative species (Bacteroidetes and Proteobacteria), while EHMC and HS showed toxicity against both gram negative and gram positive bacteria (Bacteroidetes, Proteobacteria and Firmicutes), thus suggesting that UV filters could target different membrane structures according to their physicochemical properties. As previously demonstrated with antibiotics, bactericidal compounds can alter multiple cellular processes, such as DNA replication, by targeting DNA gyrase [START_REF] Lewin | Proteinand RNA-Synthesis Independent Bactericidal Activity of Ciprofloxacin That Involves the A Subunit of DNA Gyrase[END_REF], membrane synthesis, by interfering in the lipid organization of the cell membrane [START_REF] Müller | Daptomycin Inhibits Cell Envelope Synthesis by Interfering with Fluid Membrane Microdomains[END_REF], or protein synthesis, through binding to ribosomal subunits (Greulich et al. 2015b).

Therefore, if we hypothesized that UV filters altered the above-mentioned mechanisms, further investigation would be needed to confirm these hypotheses.

Proteobacteria are the most abundant class as they can reach 50% abundance in coastal ecosystem [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF]. They support multiple functions such as nitrate reduction, denitrification and carbon fixation [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]). Among all proteobacteria tested, none of the -proteobacteria was sensitive to any UV filter and 2 out of 7 α-proteobacteria were sensitive to BP3, including Epibacterium mobile, from the Roseobacter clade. Bacteria from the Roseobacter clade account for 20% of bacterioplanktonic communities in coastal waters [START_REF] Buchan | MINIREVIEW Overview of the Marine Roseobacter Lineage †[END_REF], and display versatile metabolic activities [START_REF] Azam | Microbial Structuring of Marine Ecosystems[END_REF]. Bacteroidetes are the second most abundant phyla [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF] and play an important role in denitrification and organic carbon degradation [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF].

Algoriphagus ornithivorans was the only one out of five Bacteroidetes to be reported as sensitive to HS.

Actinobacteria are involved in phosphate uptake and the degradation of cellulose, hemicellulose and chitin [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. Within this phylum, Arthrobacter species are known to degrade PAH [START_REF] Sawulski | Effects of Polycyclic Aromatic Hydrocarbons on Microbial Community Structure and PAH Ring Hydroxylating Dioxygenase Gene Abundance in Soil[END_REF], polyethelyene [START_REF] Balasubramanian | High-Density Polyethylene (HDPE)-Degrading Potential Bacteria from Marine Ecosystem of Gulf of Mannar, India[END_REF], and metabolize pesticide [START_REF] Kundu | Defining Lower Limits of Biodegradation: Atrazine Degradation Regulated by Mass Transfer and Maintenance Demand in Arthrobacter Aurescens TC1[END_REF]. The growth of two out of three Actinobacteria, including one Arthrobacter species, was altered by UV filters. Lastly, similar trends were observed for Firmicutes that are often found in the gut of marine invertebrates [START_REF] Li | Elevated Seawater Temperatures Decrease Microbial Diversity in the Gut of Mytilus Coruscus[END_REF]. Taken together, these results suggest that the studied UV filters might affect the growth of key players within coastal microbial communities where UV filters can reach high concentrations [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] Kim | Occurrences, Toxicities, and Ecological Risks of Benzophenone-3, a Common Component of Organic Sunscreen Products: A Mini-Review[END_REF][START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF]). that the observed inhibition of growth did not increase as we increased the concentrations of the compounds (Figure 2.2). Monotonic and non-monotonic responses were observed for the same given compound, showing the response was species-specific.

The growth of P. halotolerans was inhibited by BP3 from 100 to 4000 µg L -1 (Figure 2.2). The strain responded to BP3 in a non-monotonic manner with no difference between concentrations from 1000 to 4000 µg L -1 . Similarly, non-monotonic effects were noticed for A. aurescens with OC as well as for D. maris and P. glucanolyticus with EHMC (Figure 2.2). Interestingly, a non-monotonic effect was reported when assessing the toxicity of a biocide on Daphna magna [START_REF] Souza Machado | Low-Dose Effects: Nonmonotonic Responses for the Toxicity of a Bacillus Thuringiensis Biocide to Daphnia Magna[END_REF]. Indeed, the growth inhibition of D. magna was only observed for a given range of lower concentrations, as no effect was reported for the highest concentrations.

In contrast, A. ornithinivorans, H. dabanensis and E. mobile responded in a monotonic dose response manner to HS and BP3, with EC50 of 772, 1000 and 364 µg L -1 , respectively (Figure 2.3). The dose-response was found to be species and compound-dependent. Indeed, while BP3 exhibited a dosedependent growth inhibition of E. mobile (BBCC 367), a non-monotonic response was observed for P.

halotolerans (BBCC 52) (Figure 2.2), at similar concentrations than previously reported [START_REF] Mao | Effects of Benzophenone-3 on the Green Alga Chlamydomonas Reinhardtii and the Cyanobacterium Microcystis Aeruginosa[END_REF]). BP3 and HS inhibited the growth of E. mobile and A. ornithinivorans, respectively, whereas HS only delayed the growth of H. dabdanensis, regardless of the concentration. It should be noted that the concentration of UV filters was not monitored throughout the experiments due to the inherent difficulties of measuring the concentration of UV filters in such a small volume.

Combined effect of solar radiation and UV filters

Previous studies demonstrated that UV filters could undergo photo-induced modifications that could alter their toxicity. BP3 displayed phototoxicity on human skin cells (H. J. [START_REF] Kim | Phosphodiesterase 4B Plays a Role in Benzophenone-3-Induced Phototoxicity in Normal Human Keratinocytes[END_REF]) and the UVexposed EHMC showed enhanced toxicity to Aliivibrio fischeri, Daphnia magna and Artemia salina [START_REF] Gackowska | Estimation of Physicochemical Properties of 2-Ethylhexyl-4-Methoxycinnamate (EHMC) Degradation Products and Their Toxicological Evaluation[END_REF]. In this study, two strategies were used to investigate the combined effect of solar radiation and UV filters: (i) indirect effects: bacterial viability was monitored in the dark with UV filters previously exposed to solar radiation during 5 hours (Figure 2.4 and 2.5) (ii) direct effects: bacteria were simultaneously exposed to both solar radiation and UV filters (Figure 2.6 and 2.7). In order to understand whether the cellular physiological state plays a role in the bacterial response, experiments were performed on bacteria harvested in stationary (Figures 2.4 and 2.6) and exponential (Figures 2.5

and 2.7) phases.

Regarding the indirect effects of solar radiation on the bacterial response, different patterns were observed depending on the bacterial growth phase. Viability of bacteria harvested in stationary phase was not affected by pre-irradiated UV filters (Figure 2.4). On the contrary, growing bacteria were found to be more impacted by the presence of UV filters (Figure 2.5). The toxicity of BP3 was not altered by prior exposure to solar radiation (Figure 2.4 and 2.5). In agreement with these results, former studies indicated high photostability of BP3 under artificial light exposure [START_REF] Liu | Photostability of the UV Filter Benzophenone-3 and Its Effect on the Photodegradation of Benzotriazole in Water[END_REF][START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]).

Pre-irradiation treatments of HS and EHMC were found to reduce their toxicity. Rodil and coworkers (2009) demonstrated that light exposure lowered the toxicity of EHMC on algal culture. To the best of our knowledge, the photostability of HS has never been reported. We provided evidence that its toxicity slightly decreased after exposure to solar radiation, suggesting a photodegradation of the parent compound. Irradiated EHMC was found to produce photoproducts resulting from degradation, isomerization, and dimerization reactions [START_REF] Jentzsch | Photodegradation of the UV Filter Ethylhexyl Methoxycinnamate under Ultraviolet Light: Identification and in Silico Assessment of Photo-Transformation Products in the Context of Grey Water Reuse[END_REF][START_REF] Macmanus-Spencer | Aqueous Photolysis of the Organic Ultraviolet Filter Chemical Octyl Methoxycinnamate[END_REF][START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]). In our study, irradiated EHMC did not present any enhanced toxicity in comparison to the non-irradiated molecule. It is noteworthy to emphasize that the viability of Hallobacillus dabanensis showed a 10-fold increase after 24 hours of exposure while the bacterial cells were suspended in glucose free ASW. This growth was consistently observed using a total of 6 replicates in independent experiments and this could be explained by spore-forming characteristics of Halobacillus species [START_REF] Liu | Halobacillus Dabanensis Sp. Nov: And Halobacillus Aidingensis Sp. Nov., Isolated from Salt Lakes in Xinjiang, China[END_REF]). Interestingly, it was recently reported that under nutrient limitation, spore-forming Bacillus subtilis would secrete extracellular factors that kill surrounding cells in order to feed on released nutrients [START_REF] Gonzalez-Pastor | Cannibalism by Sporulating Bacteria[END_REF]. Hence, H. dabanensis might leave its stationary state and reenter a growth phase by metabolizing nutrients from lysed cells. This consistent latter result needs to be further investigated. Bacterial growth subjected to combined solar light and UV filters was monitored for 5 hours.

Fold changes of optical density measured after and before the combined treatment (ODT5/ODT0) were compared for each condition (Figure 2.7). The toxicity of BP3 on E. mobile slightly increased with light exposure. However, no significant difference was observed between the control without BP3 exposed and the condition with BP3 exposed. EHMC displayed no enhanced phototoxicity on H. dabanensis.

HS displayed greater toxicity on both H. dabanensis and A. ornithinivorans (Figure 2.7), while pretreatment to solar radiation lowered its effect (Figure 2.6). These observations emphasized that the effect of a compound can be enhanced by environmental parameters such as solar radiation.

Overall, our results suggest that bacteria in stationary phase were more resistant to UV filters than in exponential phase. Bacterial resistance in stationary phase was reported for a broad spectrum of antibiotics: fluoroquinolones, beta-lactams [START_REF] Eng | Bactericidal Effects of Antibiotics[END_REF][START_REF] Spoering | Biofilms and Planktonic Cells of Pseudomonas Aeruginosa Have Similar Resistance to Killing by Antimicrobials[END_REF], or aminoglycosides (Greulich et al. 2015b) that target DNA gyrase, penicillin binding proteins, and ribosomes, respectively. Bacteria entering stationary phase have evolved a well-regulated process allowing to cope with different stresses such as nutrient starvation, temperature change, acidic pH, UV damage or high osmolarity [START_REF] Hengge | Stationary-Phase Gene Regulation in Escherichia Coli[END_REF]. It was previously reported that the entry in stationary phase induced the expression of key transcription factors, such as stationary phase sigma factor in E. coli [START_REF] Jaishankar | Molecular Basis of Stationary Phase Survival and Applications[END_REF] and Salmonella species [START_REF] Testerman | The Alternative Sigma Factor Controls Antioxidant Defences Required for Salmonella Virulence and Stationary-Phase Survival[END_REF], leading to the resistance to multiple stressors.

Conclusion

This study is the first to assess the toxicity of UV filters on numerous heterotrophic marine bacteria. Our results demonstrated that 26 % of all studied marine bacteria were sensitive to at least one organic UV filter commonly used in sunscreen products. HS toxicity was, for the first time, observed on a marine organism. BP3 was shown to exclusively affect gram negative while EHMC and HS impacted both gram positive and gram negative species. EHMC, also known as octinoxate, was the most toxic compound in our study, affecting five bacterial species. Phototoxicity analyses revealed a lower toxicity for HS and EHMC after pre-treatment to solar radiation while combined exposure highlighted an increase in HS toxicity. None of the UV filters showed any toxicity at 1000 µg L -1 on stationary phase cells, demonstrating that physiological state is a key parameter in the bacterial response. In terms of environmental risk characterization, our results revealed that the increasing use of sun blockers could have detrimental impacts on bacterioplanktonic communities in coastal areas.

Introduction

Oxybenzone is one of the most studied UV filters. Its occurrence and toxicity have been emphasized on a wide range of organisms, including species from multiple trophic levels [START_REF] Ghazipura | Exposure to Benzophenone-3 and Reproductive Toxicity : A Systematic Review of Human and Animal Studies[END_REF][START_REF] Kim | Occurrences, Toxicities, and Ecological Risks of Benzophenone-3, a Common Component of Organic Sunscreen Products: A Mini-Review[END_REF]. However, only a few studies have investigated its impact at the molecular scale to determine the cause and the underlying mechanisms of the toxicity. Oxybenzone altered metabolites involved in amino acid synthesis and lipid storage from 50 µg/L, in the liver of gilt-head bream (Ziarrusta et al. 2018) and increased DNA abasic sites from 22.8 µg/L in coral [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF].

Bactericidal activity of oxybenzone, also known as benzophenone-3 (BP3), has recently been demonstrated on heterotrophic (Lozano et al. 2020) and autotrophic [START_REF] Mao | Effects of Benzophenone-3 on the Green Alga Chlamydomonas Reinhardtii and the Cyanobacterium Microcystis Aeruginosa[END_REF]) marine bacteria.

BP3 was shown to increase the activities of superoxide dismutase, glutathione reductase, and glutathione S-transferase in Microcystis aerugunosa with dose-dependent effects [START_REF] Mao | Antioxidant Responses in Cyanobacterium Microcystis Aeruginosa Caused by Two Commonly Used UV Filters , Benzophenone-1 and Benzophenone-3 , at Environmentally Relevant Concentrations[END_REF]. A benzophenone derivative exhibiting a similar chemical structure, namely 2,2,4-Trihydroxybenzophenone, showed antimicrobial properties toward poultry pathogens, by targeting the bacterial cell wall [START_REF] Aranda | Antimicrobial and Synergistic Activity of 2,2′,4-Trihydroxybenzophenone Against Bacterial Pathogens of Poultry[END_REF].

The present study aims to investigate the physiological and molecular response induced by BP3, on a representative species of the Roseobacter clade, a prevalent clade in the World's Ocean, namely Epibacterium mobile. Growth media composition was shown to impact the results of toxicological investigations [START_REF] Leersnyder | Influence of Growth Media Components on the Antibacterial Effect of Silver Ions on Bacillus Subtilis in a Liquid Growth Medium[END_REF]. Therefore, the influence of medium components in the cellular response was addressed in order to qualitatively assess oxybenzone toxicity. Esterase is an enzyme involved in the phospholipid turnover as it uses organic ester as substrates [START_REF] Dorsey | Rapid Analytical Technique for the Assessment of Cell Metabolic Activity in Marine Microalgae[END_REF]).

Hence, esterase activity is used as toxicity endpoint as it reflects the cellular metabolic activity [START_REF] Hampel | Acute Toxicity of LAS Homologues in Marine Microalgae: Esterase Activity and Inhibition Growth as Endpoints of Toxicity[END_REF]). In the present study, we performed a preliminary morphological examination and enzymatic activity assay using flow cytometry. Finally, in support of the cytometry results, the impact of BP3 on the cellular shape, size, and membrane integrity of E. mobile was analyzed by TEM.

Materials and methods

Impact of culture media components on oxybenzone toxicity

E. mobile was precultured in artificial seawater (ASW), supplemented with 3mM glucose, vitamins, and trace metal [START_REF] Eguchi | Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas Sp. Strain RB2256[END_REF]. Precultures were used to inoculate several culture media with and without 2000 µg/L BP3 : (1) marine broth (MB), (2) ASW, (3) ASW + tryptone (0.1, 1, or 5 g/L) and

(4) ASW + Yeast extract (0.1, 1 or 5 g/L). Cultures were performed in 96 well plates at 25 °C and optical density (570nm) was monitored in a plate reader (Paradigm, Molecular Devices).

Flow cytometry and esterase activity assay

Esterase was used as a marker of cellular activity as it is known to assume a broad range of cellular processes [START_REF] Holt | The Esterase and Lipase Activity of Aerobic Skin Bacteria[END_REF][START_REF] Matthews | Biochemical Characterisation of the Esterase Activities of Wine Lactic Acid Bacteria[END_REF][START_REF] Oterholm | Glycerol Ester Hydrolase Activity of Lactic Acid Bacteria[END_REF] and has an activity correlated to cellular viability [START_REF] Parthuisot | Evaluation of ChemChrome V6 for Bacterial Viability Assessment in Waters[END_REF]. To measure the esterase activity of bacteria, 100 µl of bacterial culture exposed to BP3 was added to 890µl of ChemSol B26 buffer (BioMérieux, France) and labeled with 10µl of ChemChrom V6 (CV6) (bioMérieux, France) after 24 hours, 72 hours and 7 days of culture. Samples were incubated for 20 min at room temperature in dark conditions before analysis with a CytoFlex flow cytometer (Beckman Coulter). A total of 10000 events per sample were acquired.

Transmission electronic microscopy (TEM) analysis

Cultures of E. mobile (BBCC 367) were prepared in 200ml Erlenmeyer flasks in three conditions:

Control with 0.046% DMSO, 350 µg/L and 1000 µg/L BP3. Bacterial cultures were centrifuged 15 min at 3000 rpm in early exponential phase (0.15 OD620nm) in order to observe bacteria in the same physiological state for all conditions. Cells were fixed with glutaraldehyde in their culture medium at a final concentration of 1% and were concentrated by centrifugation for 30 min at 2000 g. The centrifuged material was then mixed rapidly with 40 μl of 1 % low melting point agarose at 37°C using a microrepetor (SMI, Emerville, CA, USA). Once the agarose in the disposable glass micropipette had solidified, a "noodle" containing the cells was obtained and treated as a piece of agar, fixed for 16 h at 4 °C in 2.5 % glutaraldehyde with one volume of 0.1 M cacodylate buffer. The noodle was then washed 3 times for 30 min in a buffer medium made of one volume 0.1 M cacodylate buffer. Postfixation was carried out in 1% OsO4 in 0.1 M cacodylate at 4°C for 1h. After two washes in 0.1 M cacodylate, the noodle was cut into small pieces, dehydrated in a series of ethyl alcohol, and embedded in Epon 812.

Thin sections were stained with uranyl acetate and lead citrate before examination on a 7500 Hitachi transmission electron microscope. A total of 10 TEM images per condition were recorded in order to perform statistical analysis of the cell morphological parameters. ImageJ software was used to calculate the length and the number of granules per cell.

Results and discussion

Importance of culture media formulation for toxicity testing

The impact of culture media composition on the toxicity of oxybenzone was assessed. Different concentration of tryptone and yeast extract, previously shown to drastically enhance bacterial growth [START_REF] Leersnyder | Influence of Growth Media Components on the Antibacterial Effect of Silver Ions on Bacillus Subtilis in a Liquid Growth Medium[END_REF][START_REF] Olmos-Dichara | Growth and Lactic Acid Production by Lactobacillus Casei Ssp . Rhamnosus in Batch and Membrane Bioreactor : Influence of Yeast Extract and Tryptone Enrichment[END_REF], was used to supplement ASW [START_REF] Eguchi | Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas Sp. Strain RB2256[END_REF]. Both yeast extract and tryptone increased the bacterial growth from 1 g/L (Figures 3.1 and 3.2).

Optical density reached maxima of 0.34, 1.38 and 1.49 in ASW, ASW + 5 g/L tryptone and ASW + 5 g/L yeast extract, respectively. Interestingly, oxybenzone inhibited bacterial growth except in Marine Broth (Figure 3.2). In all conditions, the addition of tryptone and yeast extract decreased the toxicity of oxybenzone in a dose-dependent manner. While E. mobile started its exponential phase in ASW with 2000 µg/L BP3 after 34 h of growth, the culture reached stationary phase when the media were supplemented from 1 g/L of yeast extract or tryptone. Similarly, increased bacterial tolerance to silver (De [START_REF] Leersnyder | Influence of Growth Media Components on the Antibacterial Effect of Silver Ions on Bacillus Subtilis in a Liquid Growth Medium[END_REF]) and mercury [START_REF] Chang | Interaction of Mercuric Ions with the Bacterial Growth Medium and Its Effects on Enzymatic Reduction of Mercury[END_REF]) due to the addition of yeast extract and tryptone was reported.

Two mechanisms were described to explain the increase in bacterial tolerance. First, the medium components adsorb the compound of interest, resulting in the loss of toxicity due to sequestration and decreased interaction with cells. This scenario is hypothesized in our experimental setup since hydrophobic contaminants were shown to interact with organic matter [START_REF] Ahmed | Interaction of Polar and Nonpolar Organic Pollutants with Soil Organic Matter: Sorption Experiments and Molecular Dynamics Simulation[END_REF]. Secondly, bacterial metabolism was more efficient, allowing cells to effectively cope with the chemical. [START_REF] Smith | The Nature of the Stimulation of the Growth of Streptococcus Lactis by Yeast</i> Extract[END_REF] demonstrated that yeast extract stimulated the growth of Streptococcus lactis [START_REF] Smith | The Nature of the Stimulation of the Growth of Streptococcus Lactis by Yeast</i> Extract[END_REF]. The authors fractionated yeast extract and emphasized that amino acids were the most stimulatory fraction for bacterial growth. Efficient protein synthesis might play a critical role in the synthesis of bacterial structures such as ABC transporter, DNA repair systems, or multidrug efflux pump that are critical to bacterial stress response. Varik and coworkers (2016) reported that the re-growth of a bacterial strain post antibiotic exposure was strongly affected by the choice of the culture medium. Because ASW alone was the most environmentally relevant medium for ecotoxicological investigations, it was retained for all subsequent analyses.

Inhibition of enzymatic activity

Esterase activity was assessed for E. mobile after 24h of culture with concentrations of BP3 ranging from 0 to 4000µg/L. Esterase activity decreased significantly in the presence of BP3 from 500 µg/L compared to the control (Figure 3.3). Growth inhibition was reported at similar concentrations in chapter 2 (Figure 2.3, p60).

Morphological analysis

A previous study highlighted that cell wall disruption by antimicrobial compounds was detectable via flow cytometry [START_REF] Surre | Strong Increase in the Autofluorescence of Cells Signals Struggle for Survival[END_REF]). Hence, cytometry analysis was performed to identify morphological changes induced by oxybenzone to better understand the toxicity mechanisms. No increase in FSC and SSC signal intensity, associated with size and granulometry, respectively, was observed with bacteriostatic concentrations of oxybenzone (Figure 3.4). Species characterization reported that Epibacterium scottomollicae was shown to hold poly-βhydroxybutyrate granule while E. mobile did not [START_REF] Penesyan | Epibacterium." Bergey's Manual of Systematics of Archaea and Bacteria[END_REF]). Nonetheless, our data indicated the presence of granules that are so far uncharacterized. Polyhydroxyalkanoates granules are microbial polyesters accumulated by prokaryotes. Protective functions of polyhydroxyalkanoates towards multiple stressors including temperature change, osmotic shock, UV ray and oxidative stress were reported [START_REF] Obruca | Novel Unexpected Functions of PHA Granules[END_REF]. Overall, the dose-dependent increase in granule number corroborates that they could play a role in the cell response toward BP3 exposure.

Conclusion

We investigated the impact of growth media composition on oxybenzone toxicity.

Subsequently, enzymatic activity and cellular morphology were assessed in ASW. Culture medium composition was a key characteristic as oxybenzone toxicity decreased in both tryptone and yeast extract enriched medium. These results highlighted that careful medium optimization should be conducted prior to antimicrobial toxicity testing. Cytometry and microscopy analyses revealed that cell morphology was not impaired by oxybenzone. TEM analysis revealed an increased number of storage granules per cell, which might be cell response toward oxidative stress. Overall, these data suggest that molecular processes such as impaired metabolism, protein or DNA synthesis might be the cause of oxybenzone toxicity.

CHAPTER 4 Unraveling the molecular effects of oxybenzone on the proteome of the marine bacterium, Epibacterium mobile

Chapter published in 2021: Lozano, C.; Lee, C.; Wattiez, R.; Lebaron, P.; Matallana-surget, S.; Unraveling the molecular effects of oxybenzone on the proteome of an environmentally relevant marine bacterium. Sci. Total Environ. 2021, 793, 148431. https://doi.org/10.1016/j.scitotenv.2021.148431

Introduction

The toxicity of organic UV filters in aquatic biota is well-documented (Lozano et al. 2020).

Benzophenone-3, also known as oxybenzone, is one of the most controversial UV filters found to be toxic to freshwater (D. [START_REF] Campos | Ecotoxicity of Two Organic UV-Filters to the Freshwater Caddis Fly Sericostoma Vittatum[END_REF][START_REF] Mao | Effects of Benzophenone-3 on the Green Alga Chlamydomonas Reinhardtii and the Cyanobacterium Microcystis Aeruginosa[END_REF][START_REF] Sieratowicz | Acute and Chronic Toxicity of Four Frequently Used UV Filter Substances for Desmodesmus Subspicatus and Daphnia Magna[END_REF] and marine (Barone et al. 2019b;[START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF][START_REF] Paredes | Ecotoxicological Evaluation of Four UV Filters Using Marine Organisms from Different Trophic Levels Isochrysis Galbana, Mytilus Galloprovincialis, Paracentrotus Lividus, and Siriella Armata[END_REF][START_REF] Seoane | Flow Cytometric Assay to Assess Short-Term Effects of Personal Care Products on the Marine Microalga Tetraselmis Suecica[END_REF]) organisms. Its use has been prohibited in Hawaii and the Republic of Palau in 2020 because of its adverse effects on coral reefs. Oxybenzone was detected worldwide from the nanogram (S. [START_REF] Kim | Occurrences, Toxicities, and Ecological Risks of Benzophenone-3, a Common Component of Organic Sunscreen Products: A Mini-Review[END_REF][START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF] to the milligram per liter range [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF]. Oxybenzone toxicity toward marine organisms, in particular, has been described for mussels [START_REF] Paredes | Ecotoxicological Evaluation of Four UV Filters Using Marine Organisms from Different Trophic Levels Isochrysis Galbana, Mytilus Galloprovincialis, Paracentrotus Lividus, and Siriella Armata[END_REF], algae [START_REF] Seoane | Flow Cytometric Assay to Assess Short-Term Effects of Personal Care Products on the Marine Microalga Tetraselmis Suecica[END_REF], fish (Barone et al. 2019b;Ziarrusta et al. 2018), corals (Roberto Danovaro et al. 2008;[START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF], and more recently bacteria (Lozano et al. 2020).

Marine bacteria prevail in the biomass [START_REF] Bar-On | The Biomass Distribution on Earth[END_REF]) and the support of major functions in marine ecosystems [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. Because they are the heart of biogeochemical cycles, it is essential to comprehend the effect of emerging pollutants on these microorganisms. Several studies addressedthrough proteomic based approachthe response of marine bacteria exposed to changing salt concentrations and culture media components (Christie-oleza et al. 2011), cold stress [START_REF] Isabelle | The Effect of Cold Stress on the Proteome of the Marine Bacterium Pseudomonas Fluorescens BA3SM1 and Its Ability to Cope with Metal Excess[END_REF], copper [START_REF] Favre | Metabolomic and Proteomic Changes Induced by Growth Inhibitory Concentrations of Copper in the Biofilm-Forming Marine Bacterium[END_REF], UV radiation (Matallana- [START_REF] Matallana-Surget | The Response of the Marine Bacterium Sphingopyxis Alaskensis to Solar Radiation Assessed by Quantitative Proteomics[END_REF], heat shock, and variation in metal concentration [START_REF] Surget | Proteogenomic Analysis of Epibacterium Mobile BBCC367, a Relevant Marine Bacterium Isolated from the South Pacific Ocean[END_REF]. Bacteria displayed a core proteome and an accessory proteome involved in the response to environmental and anthropogenic changes. This approach provides comprehensive information on cellular physiological adaptations toward bactericidal agents, and their mode of action [START_REF] Sivaranjani | Deciphering the Antibacterial Mode of Action of Alpha-Mangostin on Staphylococcus Epidermidis RP62A Through an Integrated Transcriptomic and Proteomic Approach[END_REF]) on environmentally relevant species. Studies evaluating the effect of emerging pollutants (manufactured nanoparticles, personal care products) on axenic culture through proteomic analysis are scarce [START_REF] Fajardo | Transcriptional and Proteomic Stress Responses of a Soil Bacterium <i>Bacillus Cereus<i/> to Nanosized Zero-Valent Iron ( NZVI ) Particles[END_REF][START_REF] Spalla | Metabolomic and Proteomic Investigations of Impacts of Titanium Dioxide Nanoparticles on Escherichia Coli[END_REF].

Epibacterium mobile, formerly known as Ruegeria mobilis [START_REF] Wirth | Phylogenomic Analyses of a Clade within the Roseobacter Group Suggest Taxonomic Reassignments of Species of the Genera Aestuariivita, Citreicella, Loktanella, Nautella, Pelagibaca, Ruegeria, Thalassobius, Thiobacimonas and Tropicibacter, and the Proposal[END_REF], and R. pelagia [START_REF] Lai | Ruegeria Pelagia Is a Later Heterotypic Synonym of Ruegeria Mobilis[END_REF]K. Lee et al. 2007), was isolated from the Sargasso Sea in the Atlantic Ocean.

E. mobile is a member of the Roseobacter clade, that belongs to the Alphaproteobacteria, the most abundant phylum in marine biota [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. It was shown to be sensitive to oxybenzone from 100 µg L -1 , with an EC50 of 364 µg L -1 (Lozano et al. 2020). E. mobile BBC367 strain showed high tolerance to UV radiation [START_REF] Matallana-Surget | Response to UVB Radiation and Oxidative Stress of Marine Bacteria Isolated from South Pacific Ocean and Mediterranean Sea[END_REF]) alone and combined with UV filters (Lozano et al. 2020). A proteogenomic analysis of E. mobile BBCC367 cultured under 16 conditions, such as thermal and oxidative stresses, revealed that 81% of proteins belonged to the adaptative proteome, allowing the bacterium to effectively cope with its environment [START_REF] Surget | Proteogenomic Analysis of Epibacterium Mobile BBCC367, a Relevant Marine Bacterium Isolated from the South Pacific Ocean[END_REF]).

In the present study, we investigated, through label-free quantitative proteomics, the response of the marine Roseobacter, Epibacterium mobile exposed to oxybenzone in order to decipher the molecular adaptation mechanisms. Quantification, using mass spectrometry in data-independent analysis (DIA), was conducted twice on five biological replicates, to ensure results robustness. To our knowledge, this is the first UV filter toxicity investigation involving quantitative proteomics. Organic solvents are common in ecotoxicology studies when assessing highly hydrophobic compounds such as UV filters.

The use of solvents in stock solutions is mandatory to vehicle a low concentration of the compound of interest. However, the solvent impact on the studied organism is rarely questioned. DMSO is often utilized with concentrations ranging from 0.1 to 4 % [START_REF] Liu | Acute Toxicity of Benzophenone-Type UV Filters for Photobacterium Phosphoreum and Daphnia Magna: QSAR Analysis, Interspecies Relationship and Integrated Assessment[END_REF][START_REF] Mosley | Light-Induced Cytotoxicity and Genotoxicity of a Sunscreen Agent, 2-Phenylbenzimidazole in Salmonella Typhimurium TA 102 and HaCaT Keratinocytes[END_REF][START_REF] Sharma | Different DNA Damage Response of Cis and Trans Isomers of Commonly Used UV Filter after the Exposure on Adult Human Liver Stem Cells and Human Lymphoblastoid Cells[END_REF]. Therefore, DMSO effect was evaluated alone, aiming to provide a comprehensive overview of the proteome response of E. mobile.

Materials and methods

Chemicals and bacterial cultures

Benzophenone-3 (CAS-No. 131-57-7) was purchased from Sigma-Aldrich (Steinheim, Germany).

The stock solution of Benzophenone-3 was prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich, purity >99%) at a concentration of 750 mg L -1 and stored in the dark at room temperature. Bacterial strains were kept at -80°C in marine broth 2216 (DIFCO, United States) with 35%

glycerol. Bacteria were grown on marine agar plates. After 24-48 hours of incubation, colonies were suspended and grown aerobically on a rotary shaker (110 rpm) at 25ºC in artificial seawater with 3 mM D-glucose, vitamins, and trace elements (ASW-G) [START_REF] Eguchi | Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas Sp. Strain RB2256[END_REF]. Bacterial cultures of 70 ml were prepared in Erlenmeyer flasks under three conditions: (1) seawater control (2) the solvent control including 0.0467% DMSO and (3) 350 µg L -1 BP3 + 0.0467% DMSO. Five replicates per condition were performed. When an optical density (620 nm) of 0.15 was reached, cultures were centrifuged at 8,000 g for 15 min, and cell pellets were stored at -80 °C until processing.

Assessment of BP3 toxicity

Bacterial cultures of E. mobile were prepared as described above in the same conditions, namely control, DMSO, and BP3. Optical density (620 nm) was monitored for 67 h. Three replicates per condition were performed and growth curves were generated using Excel (Microsoft Office).

Protein extraction

Protein extraction was performed as described in Matallana- [START_REF] Surget | Proteogenomic Analysis of Epibacterium Mobile BBCC367, a Relevant Marine Bacterium Isolated from the South Pacific Ocean[END_REF]. Briefly, the cell pellet was re-suspended in one pellet volume of lysis buffer (6 M guanidine chloride), and cells were sonicated on ice (5 cycles of 1 min with tubes on ice, amplitude 30%, 0.5 pulse rate). Sonicated cells were centrifuged at 16,000 g at 4 °C for 15 min. Protein reduction was conducted with 25 mM dithiothreitol (DTT) at 56 °C for 30 min and alkylated with 50 mM iodoacetamide at room temperature for 30 min. Proteins were precipitated with cold acetone overnight at -80 °C, with an acetone/aqueous protein solution ratio of 4:1. The protein pellet was dissolved in 100 mM phosphate buffer (pH 8) containing 2 M urea. For LC-MS/MS analysis, tryptic digestion (sequencing grade modified trypsin, Promega) was performed overnight at 37 °C, with an enzyme/substrate ratio of 1:25.

LC-MS analysis

Protein samples were analyzed on an ultra-high-performance liquid chromatography-highresolution tandem mass spectroscopy (UHPLC-HRMS/MS) Eksigent 2D Ultra and AB Sciex TripleTOF 5600 system. Two micrograms of peptides were analyzed using acquisition parameters previously reported [START_REF] Meur | Genetic Plasticity and Ethylmalonyl Coenzyme A Pathway during Acetate Assimilation in Rhodospirillum Rubrum S1H under Photoheterotrophic Conditions[END_REF]. In order to generate the spectral library, analyses were conducted with the instrument operating in data-dependent acquisition (DDA). MS/MS spectra were acquired in the 100 -1,800 m/z range. Mass spectrometry runs were conducted twice: in micro (40 min LC separation) and nano injection (120 min LC separation) modes in order to increase the number of quantified and identified proteins. as the reference spectral library for subsequent data-independent acquisition (DIA). Resulting .wiff files were processed using the AB Sciex PeakView 2.1 software and the SWATH™ Acquisition MicroApp considering up to 6 peptides (with at least 99% confidence) and 6 transitions per peptide. The XIC peak area was extracted and exported in AB Sciex MarkerView™ 1.2 software for normalization using Total Area Sums. Principal component analyses (PCA) were performed on normalized data using MarkerView™. The significance of the relative abundance (fold change) observed between our experimental conditions was determined through Student's t-test with a statistical threshold set at 5% (P-value < 0.05). The cut-off for significant differential regulation (fold change) was set to 2 for upregulated proteins or below 0.5 for downregulated proteins.

The list of differentially regulated proteins was obtained by combining the quantification results from nano and micro injections: quantified proteins that met the significance criteria present in the macro injection and absent from the nano injection were added in order to increase the number of quantified proteins. Subsequently, protein functions were manually annotated. The protein list including fold change and p-values for each protein is provided in Supplementary file S7. Raw data files for both identification (exported from ProteinPilot) and quantification (exported from MarkerView) were provided in Supplementary files S1 -S6 and S8 -S9 respectively. Mass spectrometry data were submitted to the iProx repository (project ID: IPX0002836000).

Results and discussion

Effect of DMSO and benzophenone-3 on the bacterial growth

The effect of BP3 was assessed by culturing E. mobile under three conditions: control, DMSO, and BP3 (Figure 4.2). No difference was observed between the control and the DMSO conditions. BP3 delayed the bacterial growth, the exponential phase starting after 39h of culture versus 15 hours in the DMSO and control conditions. Stationary phase was reached after 39h of culture in control and DMSO conditions, while 63h were needed under BP3 exposure. Noteworthy, the slope in the exponential phase and the optical density reached in stationary phase were similar in all conditions, meaning that E. mobile

Protein identification

DDA dataset was used to generate a protein list per condition. An average of 901, 887, and 890 proteins were identified in the Control, DMSO, and BP3 conditions, respectively (Supplementary file S10). The number of identified proteins was not significantly different between conditions. Overall, 1182 proteins were identified, representing a proteome coverage of 27.4% of its theoretical proteome.

Label-free protein quantification

Label-free data-independent acquisition (DIA) proteomics was performed to compare the relative concentrations of proteins within the proteome of bacteria cultured under BP3 and solvent

Effect of DMSO on protein regulation

In our study, DMSO concentration was reduced to a minimal level, i.e. 0.046 %, in order to evaluate cells in optimal physiological fitness. Hence, the proteomes cultured in artificial seawater with DMSO (solvent control) and without (control) were compared.

When comparing solvent control and control conditions, downregulated and upregulated proteins accounted for 12 and 11, respectively. Transporter was the most upregulated functional category, with 4 proteins, followed by general metabolism with 3 upregulated proteins (Figure 4.4B).

Among the downregulated proteins, Energy and DNA processing were the most affected functional category, with 5 and 4 downregulated functions, respectively (Figure 4.4B). RdgB/HAM1 family non-canonical purine NTP pyrophosphatase was the most upregulated protein (Table 4.2). Involved in nucleotide metabolism, RdgB was shown to prevent noncanonical purine dNTPs from incorporating into DNA, hence protecting the cell from mutation [START_REF] Burgis | Substrate Specificity of RdgB Protein , a Deoxyribonucleoside Triphosphate Pyrophosphohydrolase *[END_REF]. Interestingly, the iron-related proteins downregulated in presence of BP3, were upregulated with DMSO, when compared to the control condition (Figure 4.4). The Fe 3+ ABC transporter substrate-binding protein displayed a fold change of 2.43 and the hemin uptake protein (HemP) was second most upregulated (5.68 fold). DMSO was reported to form complexes with cations, including Fe 3+ [START_REF] Neklyudov | Solvation State of Iron ( III ) in Aqueous Solutions of Dimethyl Sulfoxide . Complex Formation Ability of Iron ( III ) with Respect to Derivatives of Sym -Triazine and Bis ( Hydrazinocarbonylmethyl ) Sulfoxide[END_REF][START_REF] Shvartsburg | Fragmentation Chemistry of DMSO Complexes of Metal Dications[END_REF]. The upregulation of proteins involved in iron uptake might be a cell response to counteract the iron starvation induced by the formation of DMSO-Fe 3+ complexes. Other proteins, involved in general metabolism and housekeeping functions such as oxidoreduction, peptidase activity, and phosphorylation displayed dysregulation

(Figure 4.4)
Overall, our work highlighted that even at a low concentration, DMSO did not alter bacterial growth (Figure 4.2), but induced a differential protein regulation at the molecular level (Figure 4.3).

Since the solvent concentration is rarely debated, our results corroborate that reducing the concentration to a minimum is the best strategy to adopt in ecotoxicological studies.

Effect of benzophenone-3 on protein regulation

Downregulated and upregulated proteins accounted for 13 and 43, respectively, with protein level variation ranging from 0.2 to 8.38 fold between BP3 and DMSO conditions (Table 4.3). Among the upregulated proteins, the most represented function groups were transporters (13 proteins), followed by putative & unknown functions (9 proteins, Figure 4.3A). Interestingly, transporter was also the most downregulated functional category with 10 proteins (Figure 4.3A). Other functions involved in general metabolism, energy, motility and oxidative stress response were affected. antibiotic resistance through their export [START_REF] Goodall | ABC Transporters of the Wheat Pathogen Mycosphaerella Graminicola Function as Protectants against Biotic and Xenobiotic Toxic Compounds[END_REF][START_REF] Nishino | Critical Review Role of Xenobiotic Transporters in Bacterial Drug Resistance and Virulence[END_REF][START_REF] Wasi | ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of Candida Auris : A Genome-Wide Characterization of ATP-Binding Cassette ( ABC ) Transporter Genes[END_REF].

The TMAO reductase system periplasmic protein TorT was the most upregulated protein (8.38 fold) in presence of BP3. TorT is a periplasmic molecular sensor, able to bind trimethylamine N-oxide (TMAO), and subsequently, overexpress the torCAD operon [START_REF] Baraquet | TorT , a Member of a New Periplasmic Binding Protein Family , Triggers Induction of the Tor Respiratory System upon Trimethylamine N -Oxide Electron-Acceptor Binding in Escherichia Coli * □[END_REF][START_REF] Carey | Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Article Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Change[END_REF].

In anaerobic conditions, TMAO can be used as a final electron acceptor. Upregulation of the torT transcript was described in anaerobic conditions [START_REF] Carey | Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Article Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Change[END_REF]. This up-regulation could be explained by a BP3 driven sequestration of the aerobic respiration pathways, or an energy deficit, forcing the cell to trigger all respiration routes available.

A total of 4 proteins involved in the response and signaling of oxidative stress were upregulated (Figure 4.5). Peroxiredoxindisplaying a 3.11 fold change (Table 4.3)is a core member of the antioxidant system in both eukaryotic and prokaryotic organisms. This thiol specific peroxidase can reduce various substrates such as H2O2, alkyl hydrogen peroxides, and peroxynitrite [START_REF] Wakita | The Significance of Type II and PrxQ Peroxiredoxins for Antioxidative Stress Response in the Purple Bacterium[END_REF].

5-oxoprolinase hydrolyzes 5-oxoprolinea toxic intermediate of the glutathione metabolism [START_REF] Niehaus | Discovery of a Widespread Prokaryotic 5-Oxoprolinase That Was Hiding in Plain Sight[END_REF])to form glutamate. This enzyme was upregulated (3.68 fold) along with allophanate (5.40 fold). Genes coding for these proteins are known to cluster in the genome (pxpABC genes cluster)

suggesting a functional relationship [START_REF] Niehaus | Discovery of a Widespread Prokaryotic 5-Oxoprolinase That Was Hiding in Plain Sight[END_REF]. Such variation might be resulting from an increased activity of the glutathione detoxification system, to counteract BP3 induced oxidative stress.

PrkA family serine protein kinasea widely distributed kinasedisplayed a 2.55 fold change. PrkA assumes diverse functions in signal transduction and regulation through phosphorylation of multiple substrates [START_REF] Cousin | Protein-Serine/Threonine/Tyrosine Kinases in Bacterial Signaling and Regulation[END_REF]. [START_REF] Lima | Serine / Threonine Protein Kinase PrkA of the Human Pathogen Listeria Monocytogenes : Biochemical Characterization and Identification of Interacting Partners through Proteomic Approaches[END_REF] revealed, through the investigation of the Listeria monocytogenes proteome, that PrkA interacted with 62 proteins involved in 19 functional categories.

Previous studies showed PrkA upregulation in the presence of low oxygen, high heat, and two microbicides: chlorhexidine gluconate and benzalkonium chloride [START_REF] Knapp | The Effect of Cationic Microbicide Exposure against Burkholderia Cepacia Complex ( Bcc ); the Use of Burkholderia Lata Strain 383 as a Model Bacterium[END_REF].

Hydroperoxidase I (HPI)another important enzyme for cell homeostasis, produced by bacteria under peroxide stress [START_REF] Li | The Glutathione-Glutaredoxin System in Rhodobacter Capsulatus: Part of a Complex Regulatory Network Controlling Defense against Oxidative Stress[END_REF])displayed a 2-fold change, in response to BP3. Lastly, the transcriptional repressor LexA, involved in DNA damage response, showed a 3.10 fold change. This upregulation suggests that BP3 induced DNA damage through oxidative stress. Two proteins involved in oxidative stress response were downregulated. Pyridoxine 5'-phosphate synthase (0.49 fold), involved in the synthesis of vitamin B6, is known to provide resistance against oxidative stress by quenching ROS [START_REF] Dick | Vitamin B6 Biosynthesis Is Essential for Survival and Virulence of Mycobacterium Tuberculosis[END_REF]. A previous study showed that a Mycobacterium tuberculosis pyridoxine 5'phosphate synthase mutant showed a decreased viability due to the subsequent vitamin B6 auxotrophy [START_REF] Dick | Vitamin B6 Biosynthesis Is Essential for Survival and Virulence of Mycobacterium Tuberculosis[END_REF]. Furthermore, the YchF ATPase, known to mitigate oxidative stress through the regulation of ATPase activity under stress conditions [START_REF] Hannemann | Redox-Activation of the Universally Conserved ATPase YchF by Thioredoxin 1[END_REF], was downregulated (0.49 fold).

Upregulation of a biotin-dependent carboxyltransferase family protein was characterized (6.39 fold). This multisubunit enzyme is known to play key roles in fatty acid synthesis and polyketide synthesis pathways [START_REF] Salaemae | The Role of Biotin in Bacterial Physiology and Virulence: A Novel Antibiotic Target for Mycobacterium Tuberculosis[END_REF]. The upregulation of this enzymealong with the upregulation of an acyl-CoA dehydrogenase family proteincould be a response from the cell to restore damaged membranes.

Motility was one of the most represented functions in upregulated proteins with 3 flagellins and 1 flagellar hook-basal body complex protein that displayed fold change ranging from 2.24 to 2.58. Du and coworkers (2011) demonstrated that flagellar gene expression was induced in hyperosmotic stress in Salmonella enterica to survive in the human intestine [START_REF] Du | RpoE May Promote Flagellar Gene Expression in Salmonella Enterica Serovar Typhi Under Hyperosmotic Stress[END_REF]. To the best of our knowledge, the upregulation of flagellin in the presence of xenobiotics in marine bacteria has never been reported.

Known as a part of the chemotaxis component, the upregulation of these proteins could be linked to a defense mechanism, such as negative chemotaxis, towards benzophenone-3.

Changes in the regulation of proteins involved in amino acid metabolism were reported.

Succinyl-diaminopimelate desuccinylase (DapE) was the most downregulated protein (0.2 fold) in BP3exposed E.mobile. DapE is a major component of lysine biosynthesis through the succinylase pathway, the only one in most bacterial species [START_REF] Gillner | Lysine Biosynthesis in Bacteria : A 123 Metallodesuccinylase as a Potential Antimicrobial Target[END_REF]. Because lysine, an essential amino acid, is only available through succinylase pathway, antimicrobial compounds have been designed to target DapE [START_REF] Lobo-Galo | Interaction of N -Succinyl-Diaminopimelate Desuccinylase with Flavonoids[END_REF], hence inhibiting protein synthesis. Among the most downregulated proteins, citramalate synthase and the RidA family protein displayed a 0.29 and 0.34 fold change, respectively. Both proteins are involved in L-isoleucine biosynthesis. Conversely, the 2isopropylmalate synthase, involved in L-Leucine biosynthesis was upregulated. A previous study demonstrated that leucine containing peptides inhibited the growth of E. coli, while the addition of isoleucine reversed toxicity [START_REF] Tavori | Toxicity of Leucine-Containing Peptides in Escherichia Coli Caused by Circumvention of Leucine Transport Regulation[END_REF]. The authors stipulate that leucine accumulation impaired isoleucine biosynthesis. This imbalance in isoleucine/leucine ratios could therefore be a side-effect of BP3 toxicity. Furthermore, RidA was shown to protect cells from damaging reactive intermediate such as 2-aminoacrylate [START_REF] Irons | RidA Proteins Protect against Metabolic Damage by Reactive Intermediates[END_REF], reinforcing that its downregulation could be detrimental to cell integrity.

Interestingly, three proteins involved in iron uptake and metabolism, namely the iron-sulfur cluster assembly accessory protein (0.41 fold), the Fe 3+ ABC transporter substrate-binding protein (0.47 fold), and the hemin uptake protein (HemP) (0.32 fold), were downregulated. Since Iron is a cofactor of multiple enzymes [START_REF] Kashmiri | Free Radicals and Oxidative Stress in Bacteria[END_REF], dysregulation in its provisioning could severely hamper vital cellular processes.

Conclusion

Our study deciphered for the first time how UV filter toxicity affected marine bacteria at a proteomic level. We demonstrated that E. mobile responded to BP3 by upregulating proteins involved in export, detoxification, cell motility and fatty acid synthesis. We also reported the downregulation of proteins involved in amino acid metabolism and iron uptake. Our research stressed that BP3 toxicity implied complex processes, altering multiple cellular functions, with an emphasis on oxidative stress and amino acid synthesis. Lower proteomic variations were found between the control and solvent control groups.

(23 vs 56 affected proteins). Our work suggested that DMSO induced disequilibrium in iron homeostasis, emphasizing the importance of reducing solvent concentration in ecotoxicological studies.

Introduction

UV filters are active ingredients of sunscreens products, accounting for up to 30% in sun lotion formulation. Increasing tourism results in their direct and indirect discharge in aquatic biota, through bathing and wastewater treatment plants (Tovar-Sánchez et al. 2019). Their occurrence was reported in freshwater and marine environments from the surface microlayer [START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF]), water column [START_REF] Langford | Environmental Occurrence and Risk of Organic UV Filters and Stabilizers in Multiple Matrices in Norway[END_REF][START_REF] Sánchez Rodríguez | Occurrence of Eight UV Filters in Beaches of Gran Canaria (Canary Islands). An Approach to Environmental Risk Assessment[END_REF] to sediments (Apel et al. 2018;[START_REF] Apel | Environmental Occurrence and Distribution of Organic UV Stabilizers and UV Filters in the Sediment of European North and Baltic Seas[END_REF][START_REF] Ramos | Advances in Analytical Methods and Occurrence of Organic UV-Filters in the Environment -A Review[END_REF], along with their bioaccumulation and biomagnification properties (Lozano et al. 2020). UV filters toxicity was demonstrated on a broad range of organisms (Barone et al. 2019b;[START_REF] Danovaro | Sunscreens Cause Coral Bleaching by Promoting Viral Infections[END_REF][START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] He | Comparative Toxicities of Four Benzophenone Ultraviolet Filters to Two Life Stages of Two Coral Species[END_REF][START_REF] Seoane | Flow Cytometric Assay to Assess Short-Term Effects of Personal Care Products on the Marine Microalga Tetraselmis Suecica[END_REF][START_REF] Stien | A Unique Approach to Monitor Stress in Coral Exposed to Emerging Pollutants[END_REF]Ziarrusta et al. 2018), including bacteria (Lozano et al. 2020), yet UV filters toxicity mechanisms remain unclear. Bacteria are an interesting ecotoxicological model as they are easy to handle, constitute a major part of the marine biomass (Bar-On, Phillips, and Milo 2018), and support essential functions in marine ecosystems [START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]. In a former study, marine bacteria from the major phyla, namely Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, were sensitive to UV filters from 200 µg/L (Lozano et al. 2020). Environmentally relevant bacteria investigated in this study belong to the Bacteroidetes, the most abundant phyla after Proteobacteria in marine environments [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF], and the Firmicutes, widespread symbionts in marine organisms [START_REF] Li | Elevated Seawater Temperatures Decrease Microbial Diversity in the Gut of Mytilus Coruscus[END_REF][START_REF] Math | Metagenomic Characterization of Oyster Shell Dump Reveals Predominance of Firmicutes Bacteria[END_REF].

A growing number of bioinformatic tools designed to analyze and compare microbial genomes are available [START_REF] Karp | A Comparison of Microbial Genome Web Portals[END_REF]. They implement genome annotation tools, e.g. RAST, allowing for genome categorization and gene function identification. Functional genomic comparison is widely used to investigate variations between genomes or to depict particular traits [START_REF] Kube | Genome Sequence and Functional Genomic Analysis of the Oil-Degrading Bacterium Oleispira Antarctica[END_REF][START_REF] Wagner | The Composition and Functional Protein Subsystems of the Human Nasal Microbiome in Granulomatosis with Polyangiitis : A Pilot Study[END_REF]). For instance, [START_REF] Kube | Genome Sequence and Functional Genomic Analysis of the Oil-Degrading Bacterium Oleispira Antarctica[END_REF] provided an extensive functional analysis of the bacterium Oleispira antarctica, corroborating its capacity for alkane degradation and cold adaptation [START_REF] Kube | Genome Sequence and Functional Genomic Analysis of the Oil-Degrading Bacterium Oleispira Antarctica[END_REF]. Our study aims to perform a genomic comparison of UV filters sensitive and resistant species, using the RAST platform, and the subsystems hierarchical annotation scheme implemented in SEED.

Bactericidal compounds can alter numerous cellular functions such as DNA replication, by targeting DNA gyrase [START_REF] Lewin | Proteinand RNA-Synthesis Independent Bactericidal Activity of Ciprofloxacin That Involves the A Subunit of DNA Gyrase[END_REF], membrane synthesis, by competing with lipid constituting the cell membrane [START_REF] Müller | Daptomycin Inhibits Cell Envelope Synthesis by Interfering with Fluid Membrane Microdomains[END_REF], or protein synthesis, through binding to ribosomal subunits (Greulich et al. 2015a). Although multiple genes might be responsible for bacterial resistance, the targets of UV filters or the membrane permeability towards these compounds are undetermined.

Untargeted pairwise comparisons were conducted between resistant and sensitive species, in order to identify genes that could be involved in UV filters resistance and bridge the gap between genotypes and phenotypic traits. Comparisons were performed to address relevant functions, such as membrane biosynthesis, cell signaling, stress response and xenobiotic degradation.

Materials and Methods

Bacterial strain genomes

Genomes of bacterial species screened for sensitivity [START_REF] Clément | Toxicity of UV Filters on Marine Bacteria : Combined Effects with Damaging Solar Radiation[END_REF] were

Genome comparison

Bacterial genomes were uploaded on the RAST platform (https://rast.nmpdr.org). Subsequently, pairwise comparisons were conducted between sensitive and resistant bacteria belonging to the same Phylum (table 5.1), using the SEED functional classification. Average nucleotide identity (ANI) between the compared species were calculated using the EZbiocloud platform (https://www.ezbiocloud.net) to provide a view of the species relativeness.

Results and discussion

Bacterial genome size as an indicator of bacterial resistance

Bacteroidetes selected in this study -belonging to the Algoriphagus genera -exhibited a smaller genome compared to Firmicutes (

Comparative genomic analysis of H. dabanensis and B. megaterium revealed relevant resistance features

The cell wall constitutes the first barrier against xenobiotics. Therefore, genes classified in this functional category were compared in this study. B. megaterium displayed 14 unique genes belonging to the cell wall category while H. dabanensis had only one. Among them, the subsystems "Rhamnose containing glycans" and "sialic acid metabolism" were the most represented. Rhamnose is a six-carbon deoxy hexose, mostly incorporated in cell wall anchored polysaccharides, glycoproteins, and the capsule of many bacteria [START_REF] Mistou | Bacterial Glycobiology : Rhamnose-Containing Cell Wall Polysaccharides in Gram-Positive Bacteria[END_REF]. It is essential for cell viability, environmental adaptation, and biofilm formation [START_REF] Mäki | Biosynthesis of 6-Deoxyhexose Glycans in Bacteria[END_REF][START_REF] Michael | Biofilm Plasmids with a Rhamnose Operon Are Widely Distributed Determinants of the ' Swim-or-Stick ' Lifestyle in Roseobacters[END_REF]. The presence of genes involved in rhamnose metabolism and the gene coding for the capsular polysaccharide biosynthesis protein strongly suggests that B. megaterium holds a capsule on its surface (see Figure 5.2 for a schematic representation). While phospholipid bilayers are hypothetically permeable to lipophilic compounds such as homosalate, the capsule, made of a polysaccharides layer could prevent these compounds from entering the cell. Sialic acids are nine carbon sugar acids derivatives mainly incorporated into glycan chains. Common on the surface of pathogenic bacteria, they provide camouflage from the immune system by imitating eukaryotic structures. pathway, that has an aromatic structure (benzene) common to with UV filters [START_REF] Díaz | Bacterial Promoters Triggering Biodegradation of Aromatic Pollutants[END_REF].

Once the substrate binds to CatR, the promoter regulating the transcription of the catabolic genes is activated and the substrate is degraded into tricarboxylic acid intermediates [START_REF] Díaz | Bacterial Promoters Triggering Biodegradation of Aromatic Pollutants[END_REF] (Figure 5.2).

Comparative genome analysis of H. dabanensis and Paenibacillus glucanolyticus

Compared to the UV filter sensitive H. dabanensis, P. glucanolyticus harbored more genes coding for proteins involved in pyridoxine (vitamin B6), cobalamin (vitamin B12), and thiamin (vitamin B1) biosynthesis. Vitamins, such as vitamin B12 allows tolerance to oxidative stress in bacteria belonging to the Nitrospira phylum [START_REF] Ferrer | Cobalamin Protection against Oxidative Stress in the Acidophilic Iron-Oxidizing Bacterium Leptospirillum Group II CF-1[END_REF]. Similarly to B. megaterium, P. glucanolyticus coded for Hyaluronan synthase and all genes belonging to the Rhamnose containing glycans subsystem, involved in capsule synthesis -a structure known to mediate antibiotic resistance [START_REF] Campos | Capsule Polysaccharide Mediates Bacterial Resistance to Antimicrobial Peptides[END_REF].

A total of 11 unique genes categorized in subsystem related to antibiotic and metal stress response were identified in the genomes of P. glucanolyticus. Among them, the Multidrug resistance transporter (Bcr/CflA family), reported being involved in bicyclomycin, fosfomycin, kanamycin, and sulfathiazole resistance [START_REF] Smith | Identification, Cloning, and Functional Characterization of EmrD-3, a Putative Multidrug Efflux Pump of the Major Facilitator Superfamily from Vibrio Cholerae O395[END_REF]. Transcriptomic analyses highlighted that this transporter was overexpressed (4.35 fold change) in the presence of toluene [START_REF] García | Functional Analysis of New Transporters Involved in Stress Tolerance in Pseudomonas Putida DOT-T1E[END_REF] in Pseudomonas putida, hence revealing that this gene could play a pivotal role in xenobiotic resistance.

Both bacteria harbored DNA repair systems such as Rec, SOS response, and mutL-mutS system.

P. glucanolytics expressed the ADA regulatory protein, involved in 6-O-Methylguanine demethylation, a mechanism known to bypass G:C to A:T due to guanine methylation (Figure 5.2). P. glucanolyticus genome contained multiple genes involved in response against oxidative stress such as superoxide dismutase, HtrA protease/chaperone protein, Glutaredoxin, and the outer membrane stress sensor protease DegS. HtrA protease/chaperone protein was shown to be involved in high temperature and oxidative stress tolerance in Campylobacter jejuni [START_REF] Baek | Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter Jejuni Stress Tolerance and Physiology[END_REF]. Mutation induced lack of glutathione-glutaredoxin in a Rhodobacter strain resulted in decreased growth rates and high sensitivity to oxidative stress [START_REF] Li | The Glutathione-Glutaredoxin System in Rhodobacter Capsulatus: Part of a Complex Regulatory Network Controlling Defense against Oxidative Stress[END_REF]). In addition, proteomic analyses revealed that glutaredoxin was upregulated in Rhodobacter sp. exposed to artificial UVB treatment [START_REF] Pérez | Bacterial Survival under Extreme UV Radiation : A Comparative Proteomics Study of Rhodobacter Sp ., Isolated from High Altitude Wetlands in Chile Bacterial Isolation and Growth[END_REF]). Overall, we can postulate that among Firmicutes, both P. glucanolyticus, and B. megaterium were better fitted to cope with the potentially harmful effect of xenobiotics, including UV filters, than H. dabanensis.

Algoriphagus displayed fewer genomics differences but different phenotypes

The UV filter resistant A. mannitolivorans showed a chromosomic Doc/Phd toxin-antitoxin system, known to help cells maintaining genome integrity over generations, control cell growth, and face environmental stresses, by entering into persistent states [START_REF] Liu | Bacterial Addiction Module Toxin Doc Inhibits Translation Elongation through Its Association with the 30S Ribosomal Subunit[END_REF]. Interestingly, A.

mannitolivorans genome harbored the gene coding for the antitoxin ParD without the corresponding DNA gyrase targeting toxin, ParE. Therefore, we could hypothesize that ParD can compete for other DNA gyrase binding molecules. DNA repair systems were similar between the two species. However, A. ornithinivorans showed 5 genes belonging to the CRISPR system, involved in bacterial prophage immunity.

A. ornithinivorans and A. mannitolivorans showed genes involved in the resistance against Betalactam, Bacitracin, Fosfomycin, fluoroquinolones, and metal such as cadmium, cobalt, and copper.

Genes coding for multiple multidrug resistance efflux pumps were present in both species. Minor differences were observed between genes classified into the cell wall and capsule category. No genes involved in xenobiotics resistance were identified in the two strains. As discussed earlier, these two species displayed only a few unique proteins (Figure 5.1A). Overall, these observations suggested that only minor genomic differences could explain different phenotypes.

Conclusion

Phenotypes variations were supported by genomic pairwise comparisons between Firmicutes and Bacteroidetes species. Overall, the analysis of Bacteroidetes genomes displayed two distinctive features, i.e. the antitoxin and CRISPR systems. On the other hand, the analysis of Firmicutes genomes allowed for the identification of multiple genes that could be involved in UV filters resistant mechanisms, coding for multidrug transporters, ROS responsive elements, periplasmic stress response regulons, or capsule components. Interestingly, bacteria with the bigger genomes held the resistant phenotypes, corroborating previous observations that organisms with larger genomes are more adaptable to environmental and anthropogenic perturbations. Further functional experiments such as transcriptomic and proteomic analyses would be needed to confirm the xenobiotic susceptibility and resistance biomarkers.

Finding summary

Anthropogenic inputs in the marine environment are an increasing concern as they are responsible for detrimental impacts on primary production, nutrient cycling, and microbial diversity.

Organic UV filters, the core component of sunscreen products, were shown to be damaging, in experimental conditions, for multiple species from all trophic levels. Until now, the effects of UV filters on heterotrophic marine bacteria have been overlooked, while they are pivotal for biogeochemical cycles and hence, essential for life. The aims of this thesis were to assess the effects of the most abundant and controversial organic UV filters on heterotrophic marine bacteria. Physiological, genomic, and proteomic approaches were used to decipher their effects at cellular and molecular levels.

As discussed in the photochemistry section (Chapter 1, section 4.2), UV filters can undergo chemical modifications after exposure to solar radiation. Research focusing on the toxicity of photoproducts is scarce. In Chapter 2, we assessed the combined effects of solar light and five of the most common UV filters: enzacamene, homosalate, octocrylene, octinoxate, and oxybenzone; on 27 bacterial strains, isolated from the bay of Banyuls (NW Mediterranean Sea). Dose-dependent effects were reported for octinoxate, homosalate and oxybenzone while no toxicity was noticed for enzacamene.

None of the tested -proteobacteria were sensitive while the Actinobacterium, Arthrobacter aurescens was found to be sensitive to two UV filters (octinoxate and octocrylene). Interestingly, bacteria in stationary phase were resistant to UV filters, suggesting a better adaptation to oxidative stress. On the other hand, prior exposure to solar light was shown to decrease the toxicity of octinoxate and homosalate on growing cells while combined exposure slightly increased the toxicity of homosalate.

Epibacterium mobile, an oxybenzone-sensitive -proteobacteria, was used as a model for further investigation. In order to provide a better insight into the physiological and molecular response of this bacterium, TEM, esterase activity monitoring (Chapter 3), and label-free quantitative proteomics (Chapter 4) were performed. Flow cytometry analysis revealed that oxybenzone decreased esterase activity, a biomarker of metabolic health, in a dose-dependent way. As per the morphology analysis conducted by cytometry, TEM did not highlight variations in cell size. On the other hand, oxybenzone induced an increase in the number of storage granules per cell (Chapter 3) and altered the regulation of 56 proteins involved in vital cellular pathways (Chapter 4). We also showed that culture media components should be optimized with care as they modulated the bacterial response under UV filter exposure (Chapter 3). In addition, while the use of 1% DMSO had no impact on bacterial growth (Chapters 2, 3, and 4), proteomics revealed that low concentrations of DMSO (0.046%) caused significant changes in the regulation of 23 proteins, emphasizing the need to minimize the solvent concentration in ecotoxicological studies (Chapter 4).

We demonstrated that the bacterial response was UV filter-dependent (Chapter 2) and involved morphological (Chapter 3), and molecular (Chapter 4) adaptations. In Chapter 5, we deciphered the link between genotypes and phenotypes via an in silico analysis aiming at comparing genomes of sensitive and resistant Bacteroidetes and Firmicutes. For both phyla, bacteria holding the greater genomes displayed the resistant phenotypes, but considering the small number of comparisons, this has to be confirmed. While Bacteroidetes genomes were similar (approximately 30 unique proteins), resistant Firmicutes harbored from 359 to 801 unique proteins that might be pivotal for UV filter resistance. This study allowed us to draw a bacterial model presenting potential protective cellular structures, involved in the protection and/or the response to UV filters.

Effects of UV filters on natural communities: method development

Meta-omic studies exploring the impact of UV filters on natural communities could provide a better understanding of their impact on the ecological functions of bacterioplankton. Preliminary data resulting from the comparison of a UV filter-contaminated versus a pristine area are presented below.

Seawater samples were collected in June, July, and August 2019, in the NW Mediterranean Sea.

For each month, samplings were performed at 3 pm, at a one-day interval on two sampling sites: (1) the SOLA station located approximately 500 m offshore (42°29'N, 03°08'E), considered as our control site,

(2) the beach of Banyuls Sur Mer (France) (42°28'56"N 3°07'48.7"E), a highly frequented beach in the summer season, considered as our polluted site. Solid-phase extraction followed by UPLC-MSMS analysis was conducted on each sample to get relative concentrations of UV filters (Table 6.1). For most compounds, pic area fold changes gradually increased from June to August, suggesting a positive relationship between summer-related touristic activities and UV filters concentration during Summer as previously reported [START_REF] Bachelot | Organic UV Filter Concentrations in Marine Mussels from French Coastal Regions[END_REF][START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF]. In June, values ranged from 0.7 to 42.4, showing that UV filters concentration was greater in coastal waters before the drastic increase in beachgoers that occurred in July, suggesting that some UV filters remain stable throughout the year.

The greatest changes were observed for BMDBM (avobenzone) and DHHB (uvinul A+) with concentrations 268.4 ± 233.0 and 322.9 ± 21.1 greater at the central beach, respectively. Subsequently, diversity analysis was performed using Illumina diversity assay. Diversity analysis at the Phylum level revealed that Proteobacteria were the most abundant at all locations regardless of the month, accounting for 70.8 to 83.1 % of the total reads, followed by Bacteroidetes (Figure 6.1). At the class level, Alphaproteobacteria dominated the bacterial biomass, gradually increasing from 40.1 % in June to 61.7 % in August. Looking at the family level, major contributors were the Pelagibacteraceae.

In July, minor changes were observed as Sphingobacteriaceae were less abundant at the polluted site.

Seasonal variations driven by temperature and changes in dissolved organic matter were documented [START_REF] Bunse | Marine Bacterioplankton Seasonal Succession Dynamics[END_REF][START_REF] Mestre | Seasonal Variation of Bacterial Diversity Along the Marine Particulate Matter Continuum[END_REF]. [START_REF] Fuhrman | Annually Reoccurring Bacterial Communities Are Predictable from Ocean Conditions[END_REF] reported repeatable temporal patterns over 4.5 years, predictable via abiotic and biotic factors characterization. On the other hand, anthropogenic polycyclic aromatic hydrocarbons (PAH) -hydrophobic contaminants with chemical properties comparable to UV filters -were found to modulate bacterial communities [START_REF] Sauret | Influence of PAHs among Other Coastal Environmental Variables on Total and PAH-Degrading Bacterial Communities[END_REF].

Metatranscriptomics revealed that hydrophobic pollutants favored taxa including organic pollutant degraders and triggered a transcriptional response in ubiquist Flavobacterria and -proteobacteria, including detoxifying mechanisms, lipidic membrane synthesis, and transporters (Cerro-gálvez et al.

2019).

These preliminary results provide a substantial foundation for further research. Concentrations of UV filters should be monitored several years consecutively to acknowledge the most persistent compounds and investigate their impact on marine biota. Combining in situ and in vitro ecotoxicoproteomic approaches, by performing metaproteomics on natural and artificial communities exposed to UV filters would step up our understanding of how UV filters impact the functioning of bacterial communities.

Discrepancies in occurrence studies

As discussed in section 2.4 (Chapter 1) of this manuscript, UV filters occurrence was investigated worldwide, in multiple matrices including surface microlayer, water column, sediments, and living organisms. While most studies noted the presence of UV filters in the nanogram range, a few have found UV filters in the microgram range, mostly focusing on a single section of the water column.

To the best of our knowledge, only one study considered the three water bodies (microlayer, water column, and sediments), providing valuable information on the environmental partitioning of UV filters [START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF]. Moreover, some UV filters were shown to generate photoproducts under light

The use of bacteria in ecotoxicology

Microbial ecotoxicology is an emerging discipline since the last decade, allowing for the understanding of microbial communities' adaptation and response toward multiple anthropogenic contaminants in soil and aquatic environments [START_REF] Cravo-Laureau | Microbial Ecotoxicology Microbial Ecotoxicology[END_REF]). More recently, ecotoxicoproteomics was discussed as a valuable tool for risk assessment and environmental monitoring [START_REF] Gouveia | Ecotoxicoproteomics: A Decade of Progress in Our Understanding of Anthropogenic Impact on the Environment[END_REF]. The use of bacteria in aquatic toxicology is limited to the Microtox® test (ISO 11348), based on luminescence monitoring in Vibrio fischeri; and the Photobacterium phosphoreum growth inhibition test . As discussed by [START_REF] Menz | Chemosphere Toxicity Testing with Luminescent Bacteria -Characterization of an Automated Method for the Combined Assessment of Acute and Chronic Effects[END_REF], the Microtox test is not adapted to substances with delayed effects due to very short exposure time. In a study from [START_REF] Liu | Acute Toxicity of Benzophenone-Type UV Filters for Photobacterium Phosphoreum and Daphnia Magna: QSAR Analysis, Interspecies Relationship and Integrated Assessment[END_REF], BP3 displayed an EC50 of 14.27 mg/L using the luminescence assay with P. phosphoreum, which is approximately 40 times greater than the EC50 obtained from E. mobile (Chapter 2, Lozano et al.

(2020)), emphasizing that the current tests are not adapted to such pollutants.

In this work, we showed that bacteria were suitable as an ecotoxicology model. Most heterotrophic species used reached plateau within 72h growth in ASW, thus making it a rapid, cheap, and easy to handle model with little technical expertise required compared to more complex organisms such as vertebrates and holobionts. Our results showed that a combination of several species is needed to get an unbiased overview of compound toxicity. Hence, a microbial toxicity testing kit, aiming to perform a rapid toxicity screening in 96 well plates, could be developed. A combination of bacterial strains would be used, with glucose-supplemented and glucose-free ASW provided, to assess acute and chronic toxicity, respectively.

Environmental relevancethe key to convince policy makers

As described in section 4.3 (Risk assessment), the risk quotient (RQ) of a chemical is the factor of the environmental concentration and the PNEC (predicted no effect concentration). A chemical is considered at risk if RQ is above 1. In Chapter 2, PNEC of HS, BP3, and EHMC of 1000, 364 and 200 ng/L, respectively, were obtained. Based on the literature, these compounds have been found at greater concentrations at different locations [START_REF] Kim | Occurrences, Toxicities, and Ecological Risks of Benzophenone-3, a Common Component of Organic Sunscreen Products: A Mini-Review[END_REF][START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF], suggesting a potential environmental risk (Figure 6.3). Most UV filter toxicological investigations, including ours, were conducted in vitro. Therefore, it is challenging to extrapolate our conclusions to environmental conditions. Combined approaches confronting in vitro studies and biological investigation conducted in situ, such as metagenomic and metaproteomic, are promising to understand the functional response of natural communities toward anthropogenic contaminants.

UV filter pollutiontoward solutions

As discussed in the previous chapters of this thesis, UV filters occurrence and detrimental effects were reported worldwide. While touristic activities are responsible for direct discharge, indirect route via wastewater treatment plant (WTP) is also a major environmental concern. UV filters removal efficacy in WTP varies from 30 to 70%, depending on the molecule and the technology implemented [START_REF] Li | Occurrence and Behavior of Four of the Most Used Sunscreen UV Filters in a Wastewater Reclamation Plant[END_REF][START_REF] Tsui | Occurrence, Distribution and Ecological Risk Assessment of Multiple Classes of UV Filters in Surface Waters from Different Countries[END_REF]. Consequently, UV filters concentrations in water bodies downstream of the WTP are greater than upstream [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF]. The improvement of UV filters removal and degradation technologies in WTP would reduce indirect release in aquatic biota. Removal strategies such as ozonation-based degradation combined with ultrasound [START_REF] Hopkins | Ozonation of the Oxybenzone , Octinoxate , and Octocrylene UV-Filters : Reaction Kinetics , Absorbance Characteristics , and Transformation Products[END_REF]) and hydrogen peroxide mediated photocatalytic transformation by UV irradiation [START_REF] Sakkas | TiO2/H2O2mediated Photocatalytic Transformation of UV Filter 4-Methylbenzylidene Camphor (4-MBC) in Aqueous Phase: Statistical Optimization and Photoproduct Analysis[END_REF][START_REF] Zúñiga-Benítez | Heterogeneous Photocatalytic Degradation of the Endocrine-Disrupting Chemical Benzophenone-3: Parameters Optimization and by-Products Identification[END_REF] were promising.

Another strategy toward a reduction of risks associated with the use of toxic UV filters is the synthesis of innovative eco-friendly compounds. Organic naturally sourced UV filters derived from Meldrum's acid were developed. The resulted compounds displayed good absorption properties in the UVA but not in the UVB range, which is the most damaging [START_REF] Peyrot | Innovative Bio-Based Organic UV-A and Blue Light Filters from Meldrum's Acid[END_REF]. [START_REF] Klocking | Development of an Innovative Peat Lipstick Based on the UV-B Protective Effect of Humic Substances[END_REF] reported that humic acid couple with castor oil was a suitable alternative to UV protective lipstick with efficient UVB protection. Recently, UV filters based on furan moieties, absorbing in the UVB and UVA region, were synthesized [START_REF] Giacomo | Development of an Innovative and Eco-Friendly UV Radiation Absorber, Based on Furan Moieties[END_REF]. Badea and coworkers (2015) 4,4'-[[6-[[4-[[(1,1-dimethylethyl) 4-[[4,6-bis[[4-(2-ethylhexoxy-oxomethyl)

CHAPTER 1 Figure 1 . 1 .Figure 2 . 1 .

 11121 Figure 1.1. Microbial structure of a marine ecosystem.. ........................................................................ Figure 1.2. Relative abundance of bacterial phyla across biomes .......................................................... Figure 1.3. Relative abundance of the bacterial taxonomic groups in three different areas. .................. Figure 1.4. Patterns of operational taxonomic units (OTU) richness within bacterial phyla. ................ Figure 1.5. Schematic representation of marine snow ............................................................................ Figure 1.6. Carbon cycle in the Ocean. ................................................................................................ Figure 1.7. Schematic representation of the nitrogen cycle and key players. ....................................... Figure 1.8. Oceanic phosphorus cycle. ................................................................................................. Figure 1.9. Electron absorption and subsequent energy release pathways. .......................................... Figure 1.10. Correlation between log Kow and logEC50 ....................................................................... Figure 1.11. Penetration of solar radiation through the stratospheric ozone layer and the water column. ............................................................................................................................................................... Figure 1.12. Typical workflow for Data-independent acquisition (DIA) proteomics. ......................... Figure 1.13. Typical workflow for microbial community proteomic analysis. ....................................

Figure 2 . 2 .

 22 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV filters and solar radiation on exponential (A) and stationary phase bacteria (B). .................................Figure 2.2. Bacterial growth curves with different concentrations of UV filters.. ...............................Figure 2.3. Bacterial growth curves with different concentrations of UV filters. ................................Figure 2.4. Indirect effect of solar radiation. ........................................................................................ Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters .............Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure ......................................................................Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ..............................................................................................................................................

Figure 2 . 3 .

 23 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV filters and solar radiation on exponential (A) and stationary phase bacteria (B). .................................Figure 2.2. Bacterial growth curves with different concentrations of UV filters.. ...............................Figure 2.3. Bacterial growth curves with different concentrations of UV filters. ................................Figure 2.4. Indirect effect of solar radiation. ........................................................................................ Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters .............Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure ......................................................................Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ..............................................................................................................................................

Figure 2 . 4 .

 24 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV filters and solar radiation on exponential (A) and stationary phase bacteria (B). .................................Figure 2.2. Bacterial growth curves with different concentrations of UV filters.. ...............................Figure 2.3. Bacterial growth curves with different concentrations of UV filters. ................................Figure 2.4. Indirect effect of solar radiation. ........................................................................................ Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters .............Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure ......................................................................Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ..............................................................................................................................................

Figure 2 . 5 .

 25 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV filters and solar radiation on exponential (A) and stationary phase bacteria (B). .................................Figure 2.2. Bacterial growth curves with different concentrations of UV filters.. ...............................Figure 2.3. Bacterial growth curves with different concentrations of UV filters. ................................Figure 2.4. Indirect effect of solar radiation. ........................................................................................ Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters .............Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure ......................................................................Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ..............................................................................................................................................

Figure 2 . 6 .

 26 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV filters and solar radiation on exponential (A) and stationary phase bacteria (B). .................................Figure 2.2. Bacterial growth curves with different concentrations of UV filters.. ...............................Figure 2.3. Bacterial growth curves with different concentrations of UV filters. ................................Figure 2.4. Indirect effect of solar radiation. ........................................................................................ Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters .............Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure ......................................................................Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ..............................................................................................................................................

Figure 2 . 7 .Figure 3 . 1 .

 2731 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV filters and solar radiation on exponential (A) and stationary phase bacteria (B). .................................Figure 2.2. Bacterial growth curves with different concentrations of UV filters.. ...............................Figure 2.3. Bacterial growth curves with different concentrations of UV filters. ................................Figure 2.4. Indirect effect of solar radiation. ........................................................................................ Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters .............Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure ......................................................................Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ..............................................................................................................................................

Figure 3 . 4 .

 34 Figure 3.1. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of tryptone ......................................................................................................Figure 3.2. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of yeast extract. ............................................................................................... Figure 3.3. Fluorescence intensity of 10,000 cells of E. mobile cultured in ASW-G with different concentrations of BP3, labeled with CV6.. ...........................................................................................Figure 3.4. Cytograms of E. mobile exposed to BP3 at 0, 1000 and 4000 µg/L. .................................Figure 3.5. TEM images of E. mobile treated with DMSO (solvent control) and 1000 µg/L oxybenzone. ...........................................................................................................................................Figure 3.6. Average length of E. mobile cells (A) and number of granules per cell (B) ......................

Figure 3 . 5 .

 35 Figure 3.1. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of tryptone ......................................................................................................Figure 3.2. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of yeast extract. ............................................................................................... Figure 3.3. Fluorescence intensity of 10,000 cells of E. mobile cultured in ASW-G with different concentrations of BP3, labeled with CV6.. ...........................................................................................Figure 3.4. Cytograms of E. mobile exposed to BP3 at 0, 1000 and 4000 µg/L. .................................Figure 3.5. TEM images of E. mobile treated with DMSO (solvent control) and 1000 µg/L oxybenzone. ...........................................................................................................................................Figure 3.6. Average length of E. mobile cells (A) and number of granules per cell (B) ......................

Figure 3 . 6 .Figure 4 . 1 .

 3641 Figure 3.1. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of tryptone ......................................................................................................Figure 3.2. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of yeast extract. ............................................................................................... Figure 3.3. Fluorescence intensity of 10,000 cells of E. mobile cultured in ASW-G with different concentrations of BP3, labeled with CV6.. ...........................................................................................Figure 3.4. Cytograms of E. mobile exposed to BP3 at 0, 1000 and 4000 µg/L. .................................Figure 3.5. TEM images of E. mobile treated with DMSO (solvent control) and 1000 µg/L oxybenzone. ...........................................................................................................................................Figure 3.6. Average length of E. mobile cells (A) and number of granules per cell (B) ......................

Figure 4 . 2 .

 42 Figure 4.2. Growth curves of Epibacterium mobile in artificial seawater only (dark circle), with 0.047 % DMSO (white circle), or with 350 µg L -1 BP3 and 0.046 % DMSO (square) ...................................Figure 4.3. Principal component analysis of E.mobile proteomes. ......................................................Figure 4.4. Number of upregulated (green bars) and downregulated (red bars) proteins induced by the presence of BP3 (A) and DMSO (B), classified by functional category. ..............................................Figure 4.5. Schematic representation of up and downregulated proteins within the proteome of benzophenone 3 exposed E. mobile (350 µg/L), compared to the solvent control condition. ...............

Figure 4 . 3 .

 43 Figure 4.2. Growth curves of Epibacterium mobile in artificial seawater only (dark circle), with 0.047 % DMSO (white circle), or with 350 µg L -1 BP3 and 0.046 % DMSO (square) ...................................Figure 4.3. Principal component analysis of E.mobile proteomes. ......................................................Figure 4.4. Number of upregulated (green bars) and downregulated (red bars) proteins induced by the presence of BP3 (A) and DMSO (B), classified by functional category. ..............................................Figure 4.5. Schematic representation of up and downregulated proteins within the proteome of benzophenone 3 exposed E. mobile (350 µg/L), compared to the solvent control condition. ...............

Figure 4 . 4 .

 44 Figure 4.2. Growth curves of Epibacterium mobile in artificial seawater only (dark circle), with 0.047 % DMSO (white circle), or with 350 µg L -1 BP3 and 0.046 % DMSO (square) ...................................Figure 4.3. Principal component analysis of E.mobile proteomes. ......................................................Figure 4.4. Number of upregulated (green bars) and downregulated (red bars) proteins induced by the presence of BP3 (A) and DMSO (B), classified by functional category. ..............................................Figure 4.5. Schematic representation of up and downregulated proteins within the proteome of benzophenone 3 exposed E. mobile (350 µg/L), compared to the solvent control condition. ...............

Figure 4 . 5 .Figure 5 . 1 .Figure 6 . 1 .

 455161 Figure 4.2. Growth curves of Epibacterium mobile in artificial seawater only (dark circle), with 0.047 % DMSO (white circle), or with 350 µg L -1 BP3 and 0.046 % DMSO (square) ...................................Figure 4.3. Principal component analysis of E.mobile proteomes. ......................................................Figure 4.4. Number of upregulated (green bars) and downregulated (red bars) proteins induced by the presence of BP3 (A) and DMSO (B), classified by functional category. ..............................................Figure 4.5. Schematic representation of up and downregulated proteins within the proteome of benzophenone 3 exposed E. mobile (350 µg/L), compared to the solvent control condition. ...............

  ............................................................................................................................ Table 1.3. Common organic UV filters and their physiochemical properties (Gago-Ferrero et al. 2012) ............................................................................................................................................................... Table 1.4. UV filters authorized in sunscreen by European and FDA regulation and related maximum concentration in cosmetics ....................................................................................................................

  Physiochemical properties of tested UV filters. .................................................................. Table 2.2. List of bacterial strains used in this study. ........................................................................... Table 2.3. Sensitivity of marine bacteria to different UV filters at 1000 µg L -1 . .................................. Physiochemical properties of benzophenone-3. .................................................................. Table 4.2. Identification, function, and fold change of proteins differentially regulated between E. mobile exposed to 0.046% DMSO and artificial seawater only. ........................................................... Table 4.3. Identification, function, and fold change of proteins differentially regulated between E. mobile exposed to 350 µg/L BP3 + 0.046% DMSO and 0.046% DMSO only. ................................... Pairwise compared species and respective UV filters sensitivity. ....................................... Table 5.2. Structures of bacterial genomes.. ......................................................................................... Pic area fold change between polluted (central beach) and control (SOLA) sites at different time points in summer 2019. ...............................................................................................................

Figure 1 . 2 .

 12 Figure 1.2. Relative abundance of prokaryotic major clades across biomes[START_REF] Yilmaz | Expanding the World of Marine Bacterial and Archaeal Clades[END_REF]). The bubble size indicates relative abundance, scaled from 0, the minimum, to 1 representing the maximum abundance.

Figure 1 . 3 .

 13 Figure 1.3. Relative abundance of the bacterial taxonomic groups in three different areas (Jing et al. 2013). SNP: subarctic North Pacific; SCS: South China Sea; CRD: Costa Rica Dome.

Figure 1 . 4 .

 14 Figure 1.4. Patterns of operational taxonomic units (OTU) richness within bacterial phyla. (Ladau et al. 2013) Microorganisms are classified as phototrophs, organotrophs, or lithotrophs, depending on whether their energy comes from light, organic compounds or inorganic nutrients, respectively.

Figure 1 . 5 .

 15 Figure 1.5. Schematic representation of marine snow[START_REF] Azam | Microbial Structuring of Marine Ecosystems[END_REF]

Figure 1 . 6 .

 16 Figure 1.6. Carbon cycle in the Ocean. DIC: Dissolved inorganic carbon; DOC: Dissolved organic carbon; GPP: Gross primary production; NPP: Net primary production

Figure 1 . 7 .

 17 Figure 1.7. Schematic representation of the nitrogen cycle and key players.

Figure 1 . 8 .

 18 Figure 1.8. Oceanic phosphorus cycle. PIP: particulate inorganic P; POP: particulate organic P; DIP: dissolved inorganic P; DOP: dissolved organic P

Figure 1 . 9 .

 19 Figure 1.9. Electron absorption and subsequent energy release pathways. (adapted from Shaath 2010) About 55 of ultraviolet filters are approved for cosmetic use, with only 10 approved unanimously worldwide (Shaath 2010). As highly lipophilic compounds, UV filters are characterized by high Log Kow value (the ratio of the concentration of a solute between octanol and water in a biphasic solution). Consequently, they accumulate in aquatic organisms and have been detected at various trophic levels. Many studies have demonstrated the accumulation of UV filters into a broad range of organisms.

  NOECs from at least three species (normally fish, Daphnia and algae) representing three trophic levels 10

  photosynthetic efficiency

Figure 1 .

 1 Figure 1.10. Correlation between log Kow and logEC50 (Zhang et al. 2017).The effect of 26 UV filters was investigated on Vibrio fischeri[START_REF] Baumgartner | Bioactivity-Based Analysis of Sunscreens Using the Luminescent Bacteria Vibrio Fischeri[END_REF]). They measured the luminescence of Vibrio cells as it is a marker of viability. All filters <400 Da had an inhibiting effect on bacterial luminescence. The non-toxicity of UV filters >400 Da may be due to the difficulty for those compounds to cross a biological barrier such as the cell membrane. It is interesting to note that every compounds >400 Da regarded in this paper are only approved in the EU; namely Camphor Benzalkonium methosulfate, Mexoryl SX, Mexoryl XL, Tinosorb S, Bidisulizole disodium, Bisoctrizole, Uvasorb HEB and Ethylhexyl triazone.

  source of biological damage (Ruiz-González et al. 2013).

Figure 1 .

 1 Figure 1.11. Penetration of solar radiation through the stratospheric ozone layer and the water column. UV-C, B, A: Ultraviolet C, B, A; PAR: Photosynthetically Active Radiation.

  -independent acquisition mass spectrometry (DIA-MS), a step forward label-free quantification DIA-MS, an emerging technology allowing for accurate and reproducible label-free quantification (Krasny et al. 2018; Krasny and Huang 2020), was used in Chapter 4. Therefore, the theoretical basis for its understanding and the difference with Data-dependent analysis (DDA) are provided below.

Figure 1 . 13 .

 113 Figure 1.13. Typical workflow for microbial community proteomic analysis (adapted from Wang et al. 2016).

Figure 2 . 1 .

 21 Figure 2.1. Schematic drawing of the experimental design used to study the combined effect of UV

Following

  the guidelines supported by the Regulation (EC) No 1907/2006 regarding the registration, evaluation, authorization and restriction of chemicals (REACH), we achieved environmental risk characterization. Predicted no effect concentration (PNEC), based on EC50 for HS and BP3, and on NOEC for EHMC, divided by arbitrarily defined constants (1000), are respectively 1000, 364 and 200 ng L -1 . Taking into account that these values were lower than the ones encountered in the environment (Kim and Choi 2014; Tsui et al. 2014), a potential ecological risk should be considered.

Figure 2 . 2 .

 22 Figure 2.2. Bacterial growth curves with different concentrations of UV filters. Only species that showed non monotonic response were presented. (average ± standard deviation, n=3).

Figure 2 . 3 .

 23 Figure 2.3. Bacterial growth curves with different concentrations of UV filters. Only species that displayed a dose dependent response were presented. (average ± standard deviation, n=3).

Figure 2 . 4 .

 24 Figure 2.4. Indirect effect of solar radiation. Viability of stationary phase bacteria submitted to irradiated or non irradiated UV filters at 1000 µg L -1 , expressed as mean ± standard deviation (n=3).

Figure 2 . 5 .

 25 Figure 2.5. Growth of bacterial strains in presence of irradiated or non irradiated UV filters, expressed as optical density (620nm) mean ± standard deviation. (n=3).

Figure 2 . 6 .

 26 Figure 2.6. Direct effect of solar radiation. Viability of stationary phase bacteria exposed to UV filters at 1000 µg L -1 in dark condition or under light exposure, expressed as mean ± standard deviation. (n=3).Regarding the direct combined effect of solar radiation and UV filters on stationary phase cells,

Figure 2 . 7 .

 27 Figure 2.7. Bacterial growth expressed as optical density fold change between 0 and 5 hours of incubation ± standard deviation, exposed to UV filters in dark or light condition. Asterisk (*) and double asterisk (**) highlight significant differences with the control without UV filterdark, and exposed, respectively (ANOVA post-hoc Tukey's test p<0.05)

Figure 3 . 1 .Figure 3 . 2 .

 3132 Figure 3.1. Growth of E. mobile exposed to 2000 µg/L oxybenzone in ASW supplemented with different concentrations of tryptone (n=3).

Figure 3 . 3 .

 33 Figure 3.3. Fluorescence intensity of 10,000 cells of E. mobile cultured in ASW-G with different concentrations of BP3, labeled with CV6. Bar charts represent fluorescence intensity in FL1/FITC chanel. The values represent the mean ± standard deviation (n=3). * highlights significant differences with the control (ANOVA post-hoc Tukey's test p b .05).

Figure 3 . 4 .

 34 Figure 3.4. Cytograms of E. mobile exposed to BP3 at 0, 1000 and 4000 µg/L.

Figure 3 . 5 .

 35 Figure 3.5. TEM images of E. mobile treated with DMSO (solvent control) and 1000 µg/L oxybenzone.

Figure 3 . 6 .

 36 Figure 3.6. Average length of E. mobile cells (A) and number of granules per cell (B) based on the observation of 10 TEM images.

  Protein searches were performed with ProteinPilot (ProteinPilotSoftware 5.0.1; Revision: 4895; Paragon Algorithm: 5.0.1.0.4874;AB SCIEX, Framingham, MA, United States) (Matrix Science,London, United Kingdom; v. 2.2). Paragon searches were conducted using LC MS/MS Triple TOF 5600 System instrument settings. Other parameters used for the search were as follows: Sample Type: Identification, Cys alkylation: Iodoacetamide,Digestion: Trypsin, ID Focus: Biological Modifications andAmino acid substitutions, Search effort: Thorough ID, DetectedProtein Threshold [Unused ProtScore (Conf )]>: 0.05. Proteins identified at a false-discovery rate of below 1% were used

  metabolism was notably delayed by BP3 but not fully inhibited at 350 µg L -1 . Previous work highlighted that BP3 concentrations from 2000 µg L -1 were necessary to show bactericidal effects (Lozano et al. 2020).

Figure 4 . 2 .

 42 Figure 4.2. Growth curves of Epibacterium mobile in artificial seawater only (dark circle), with 0.047 % DMSO (white circle), or with 350 µg L -1 BP3 and 0.046 % DMSO (square) (average ± standard deviation, n =3).

  control conditions. Principal component analyses (PCA) including the data of all replicates, in micro injection (Figure 4.3A) and nano injection mode (Figure 4.3B) were conducted. All replicates clustered by conditions, meaning there is a statistical difference among treatment groups, and proteome consistency throughout the replicates, regardless of the injection mode. PCA revealed that the control and solvent control conditions were closer compared to the BP3 condition, mainly differentiated through the PC1 axis. In addition, these results indicate that the three treatments led to significantly different proteomes, suggesting phenotypic adaptations to DMSO and BP3, to a greater extent.

Figure 4 . 3 .

 43 Figure 4.3. Principal component analysis of E.mobile proteomes obtained from Nano-LC analysis (A) and Micro-LC analysis (B)

Figure 4 . 4 .

 44 Figure 4.4. Number of upregulated (green bars) and downregulated (red bars) proteins induced by the presence of BP3 (A) and DMSO (B), classified by functional category. ABC transporters, the largest family of ATP-dependent transporters, were upregulated and, to a

Figure 4 . 5 .

 45 Figure 4.5. Schematic representation of up and downregulated proteins within the proteome of benzophenone 3 exposed E. mobile (350 µg/L), compared to the solvent control condition.

Figure 5 . 1 .

 51 Figure 5.1. Venn diagrams representing the number of unique and shared proteins between (A) A. mannitolivorans and A. ornithinivorans, (B) B. megaterium and P. glucanolyticus, (C) B. megaterium and H. dabanensis.

  Both species harbored genes involved in bacitracin response. B. megaterium possessed multiple stress response genes such as cadmium-transporting ATPase, multicopper oxidase and multidrug resistance transporter (Bcr/CflA family), the cytoplasmic copper homeostasis protein CutC, the mercuric resistance operon regulatory protein, and the membrane fusion protein of RND family multidrug efflux pump, known to confer resistance against multiple hydrophobic substrates[START_REF] Nikaido | Mechanisms of RND Multidrug Efflux Pumps[END_REF]. Several gene copies belonging to the choline and betaine uptake and biosynthesis subsystem were observed in the genome of B. megaterium, including four choline specific ABC transporters. Choline is a precursor of glycine betaine, known as a potent osmoprotectant[START_REF] Kiene | Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters[END_REF]. Furthermore, B. megaterium presented genes coding for flavodoxin, a small soluble protein belonging to the nonenzymatic antioxidant molecules, known to confer resistance against herbicides to soil bacteria (De la Pena et al. 2013) (Figure5.2). Among the genes categorized in the regulation and cell signaling category, the aromatic hydrocarbon utilization transcriptional regulator CatR (LysR family) was the single unique gene belonging to B. megaterium. CatR regulates the expression of catechol and phenol degradation

Figure 5 . 2 .

 52 Figure 5.2. Cell diagram showing the hypothetical protective structures against UV filters, obtained from genomic functional comparison.

Figure 6 . 1 .

 61 Figure 6.1. Diversity analysis at the (A) phylum, (B) class, and (C) family level for the different sampling points.

  exposure (see section 4.2, Chapter 1). To the best of our knowledge, no occurrence study has conducted UV filter photoproducts quantification in environmental matrices. Recently, microplastics were shown to adsorb UV filters in the nanogram per gram range, adding to the complexity of UV filters environmental partitioning[START_REF] Santana-Viera | UV Filters and UV Stabilisers Adsorbed in Microplastic Debris from Beach Sand[END_REF]. Finally,[START_REF] Stien | Metabolomics Reveal That Octocrylene Accumulates in Pocillopora Damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction[END_REF] revealed that octocrylene was metabolized, in vitro, into fatty acid conjugates by the coral species Pocillopora damicornis[START_REF] Stien | Metabolomics Reveal That Octocrylene Accumulates in Pocillopora Damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction[END_REF].[START_REF] Labille | Assessing UV Fi Lter Inputs into Beach Waters during Recreational Activity : A Fi Eld Study of Three French Mediterranean Beaches from Consumer Survey to Water Analysis[END_REF] demonstrated that UV filters in the water column displayed intraday spatial and temporal variations. The authors carried out a social survey to estimate the predicted concentration based on the type and amount of sunscreen products used by beachgoers. Quantification was performed to compare the predicted versus actual values. While inorganic UV filters, i.e. TiO2 and ZnO displayed 49 and 31.7% recovery, respectively; avobenzone, octocrylene and oxybenzone displayed a 0.15, 0.02 and 0.3% recovery[START_REF] Labille | Assessing UV Fi Lter Inputs into Beach Waters during Recreational Activity : A Fi Eld Study of Three French Mediterranean Beaches from Consumer Survey to Water Analysis[END_REF]. Overall, taking into account the lack of consideration for partitioning, abiotic degradation, spatiotemporal variations, biotic accumulation and metabolization (Figure6.2), we can assume that UV filters occurrence studies tend to underestimate their actual concentrations, which could bias subsequent environmental risk characterization.

Figure 6 . 2

 62 Figure 6.2 Schematic drawing highlighting the complexity of UV filter partitioning in the marine environment. Arrows indicate the UV filters fluxes within the biota. "+" represents the relative concentration of UV filters in the different water bodies.

Figure 6 . 3 .

 63 Figure 6.3. Schematic representation of the risk assessment performed using the data obtained for BP3 (chapter 2) on Epibacterium mobile.

  amino]carbonyl]phenyl]amino]-1,3,5triazine-2,4-diyl]diimino]bis-, bis(2

Table 1 .

 1 5. Assessment factors to derive a PNEC (adapted from Technical Guidance Document on Risk Assessment, REACH) ................................................................................................................... Table 1.6. In situ summary of UV filter bioaccumulation studies (Lozano et al., 2020)...................... Table 1.7. Summary of organic UV filter toxicity studies on marine organisms. ................................ Table 1.8. Summary of organic UV filter toxicity studies on freshwater organisms............................ Table 1.9. Photostability of UV filters under different light sources and intensities. Text in orange represents studies in which the UV filters were tested in cosmetic product formulation or liposome. .

Table 1 . 1 .

 11 Prokaryotic abundances size in various habitats. Adapted from[START_REF] Whitman | Prokaryotes: The Unseen Majority[END_REF] 14% of the total biomass[START_REF] Parkes | A Review of Prokaryotic Populations and Processes in Sub-Seafloor Sediments, Including Biosphere: Geosphere Interactions[END_REF], and 2.2 PgC in the water column. That last assumes a NPP of 51 PgC.yr -1 in marine habitat while the terrestrial plant biomass (560 PgC) assumes 48 Pg.yr -1 , meaning a much greater NPP under biomass ratio in marine ecosystems (Table1.2).

	Habitat

Table 1 .3.

 1

Table 1 .4. UV

 1 Regulation related to cosmetic products influences the composition of sunscreens and their relative concentration of UV filters. Consequently, implemented regulations shape the environmental occurrence of UV filters. This section describes the regulatory aspects in the EU and the US regarding UV filters in sunscreen products. UV filter compounds are submitted to different regulatory authorities depending on the regarded country. In Europe, cosmetic products are regulated by the Cosmetic Products Regulation (EC/1223/2009). Annex VI of the regulation presents the list of UV filters allowed in cosmetic products and this accounts for 28 compounds. Before listing by the above-mentioned regulatory boards, chemicals, including UV filters must be compliant with the Registration, Evaluation, filters authorized in sunscreen by European and FDA regulation and related maximum concentration in cosmetics.

	Authorization and restriction of CHemicals (REACH) regulation, established in 2006. Technical and
	administrative aspects of the REACH regulation are managed by the European Chemicals Agency
	(ECHA, https://echa.europa.eu/). Companies must provide ECHA a registration dossier including
	multiple assessments (persistence, bioaccumulation and toxicity assessment) to introduce a new
	compound on the market. For example, in October 2015, Zinc Oxide has been approved for cosmetic
	use, by the European Commission. In the United States, cosmetic products are regulated under FDA
	standard 21 CFR part 352 subpart B with 19 chemicals listed. Only 12 UV filters are approved by both
	agencies, and authorized maximum concentration can vary (Table 1.4). For instance, avobenzone
	maximum concentration is 5% in Europe while it is 3% in USA. Oxybenzone maximum concentration
	is also different between Europe and USA (10 vs 8%); and products containing oxybenzone must display
	the label "contains oxybenzone" in the US; and "contains benzophenone-3" in the EU.
	Sobek and colleagues (2013) reported inconsistency in the application of EU regulations. They
	noticed that classification, labeling, and packaging of substances and mixtures (CLP) must be
	determined for UV filters in industrial chemical products whereas sunscreens are exempted from CLP.
	Almost 50% of the investigated UV filters approved for use in cosmetic products on the European
	market met the CLP classification as being hazardous to the aquatic environment. The authors stressed
	that some sunscreen products could be labeled with the environmental hazard pictogram if the CLP
	regulation was applicable.

Common organic UV filters and their physiochemical properties (Gago-Ferrero et al. 2012) a INCI (International Nomenclature for Cosmetic Ingredient) established by CTFA and COLIPA b In water at 25 °C c Calculated by use of Advanced Chemistry Development (ACD/Labs) Software V11.02 (1999-2011 ACD/Labs) d Experimental values from database of physicochemical properties; Syracuse Research Corporation: http://www.syrres.com/esc/physdemo.htm

2.2 Regulatory aspects

.3 Risk assessment

	Table 1.5. Assessment factors to derive a PNEC (adapted from Technical Guidance Document on
	Risk Assessment, REACH).
	Environmental risk characterization of emerging pollutants is supported by the REACH
	(registration, evaluation, authorization, and restriction of chemicals) guidelines and the Regulation (EC)
	No 1907/2006. For a better understanding of the following section, common toxicology terms are
	described. First, the No observed effect concentration (NOEC) is defined as "the highest concentration
	tested at which the measured parameter shows no significant inhibition" (OECD 201, 1984a) or the test
	concentration immediately below the lowest observed effect concentration (LOEC) (OECD 210,
	1984g). The Predictive no effect concentration (PNEC) is the concentration we expect no effect from
	the pollutant. The PNEC is obtained by dividing L(E)C50 (or NOEC if not available) by arbitrary
	constants defined based on the nature of the test (Table 1.5).

Table 1 .6. In

 1 situ summary of UV filter bioaccumulation studies(Lozano et al., 2020).

	Reference	Organism model	Sunscreen	Concentration range	Location
			components
			(abbreviations)
	Alonso et al. 2015	Dolphin	EMC	ND-250 ng/g lw	South Atlantic
		(Pontoporia blainvillei & Sotalia guianensis)	4-MBC	ND-855 ng/g lw	Ocean
			OC	ND-11,130 ng/g lw
			OD-PABA	ND-1385 ng/g lw
	Bachelot et al. 2012	Mussels	EMC	3-256 ng/g dw	French Atlantic,
		(Mytilus edulis & Mytilus galloprocincialis)	OC	1-200 ng/g dw	Mediterranean Sea
			OD-PABA	ND
	Barbosa et al. 2018	Gilt-head bream,	ES	5-12 μg/kg ww	North Sea,
		mackerel, salmon, cod, monkfish and mussels.	HS	>LOD-10 μg/kg ww	Atlantic Ocean, Mediterranean Sea
		(Seafood species, wild	BP6	20-110 μg/kg ww
		and farmed origins.)	OC	4-28 μg/kg ww
			BP1	9-27 μg/kg ww
			4MBC	ND (detected after steaming)
			DHHB	ND (detected after steaming)
	Castro et al. 2018	Mussels	ES	ND-59.2 ng/g dw	North Atlantic Ocean
		(Mytilus galloprovincialis & Mytilus edulis)	BP3	ND-622.1 ng/g dw
			4MBC	ND-88.3 ng/g dw
			IMC	ND-43.1 ng/g dw
			EMC	ND-181.8 ng/g dw
	Cunha et al. 2015	Mussels, mullet, clam &	BP1	ND	North Atlantic
		flounder	BP3	ND -<MQL (ng/g dw)	Ocean, North Sea, Mediterranean Sea
			OD-PABA	ND
			HS	ND
			DHMB	ND -<MQL (ng/g dw)
			IMC	ND
			OC	ND -<LOQ (ng/g dw)
			EMC	ND -<LOQ (ng/g dw)
			ES	ND
			DHHB	ND
			4-MBC	ND
	Cunha et al. 2018	Mackerel, tuna, salmon,	BP1	ND-98.9 µg/kg dw	Mediterranean Sea,
		seabream, cod, monkfish, crab, shrimp, octopus,	BP3	ND-98.7 µg/kg dw	North Sea, North Atlantic
		plaice, hake, mussel &	ES	ND-72.1 µg/kg dw	Ocean, Pacific
		sardine (Seafood species, wild,	4-MBC	ND-56.2 µg/kg dw	Ocean
		farmed and industrial origins)	EMC	ND-74.4 µg/kg dw
			IMC	ND-66.7 µg/kg dw
			DHMB	ND-90.7 µg/kg dw
			OD-PABA	ND
			OC	ND-103.3 µg/kg dw
			DHHB	ND
	Gago-Ferrero et al.	Dolphin	OC	89-782 ng/g lw	South Atlantic
	2013	(Pontoporia blainvillei)			Ocean
	Horricks et al. 2019	Lionfish	4MBC	2.11 µg/kg ww	Caribbean Sea
		(Pterois volitans)
			EMC	ND or below LOD
			BP3	0.12-2.90 µg/kg ww

Abbreviations: dw,dry weight; ww, wet weight; lw, lipid weight; ND, not detected; LOD, limit of detection; MQL, method quantification limit. Abbreviations for organic UV filters: 4-MBC, 4-Methylbenzylidene camphor; BM, Butyl methoxydibenzoylmethane; BP1, Benzophenone-1; BP3, Benzophenone-3; BP4, Benzophenone-4; BP6, Benzophenone-6; BP8, Benzophenone-8; EMC, Ethylhexyl methoxycinnamate; OC, octocrylene; OD-PABA, Ethylhexyl dimethyl PABA; IMC, Isoamyl p-methoxycinnamate; DHHB, Diethylamino hydroxybenzoyl hexyl benzoate; PABA, para-Aminobenzoic acid; ES, Ethylhexyl salicylate; MBBT, Methylene bis-benzotriazolyl tetramethylbutylphenol * Benzotriazole derivatives used in sunscreens ** Benzophenone metabolites

Table 1 .

 1

	UV filter	Species Model [Common name]	Endpoint	Tested conc. range	Toxic Effects [Conc with toxic effect]	Reference
	4-Methylbenzilidene	Acropora pulchra	bleaching	50 μl/L	95% bleaching rate over 62 hours	Danovaro et al. 2008
	camphor (4-MBC)	[Staghorn coral]			[50 µg/L]
		Acropora sp.	bleaching	33 μl/L	63% bleaching rate over 48 hours	Danovaro et al. 2008
		[Stoney coral]			[33 µg/L]
		Isochrysis galbana	growth rate	N/A	EC50 = 171.45 µg/L over 72h	Paredes et al. 2014
		[Unicellular algae]
		Mytilus	morphologies		changes in D-larvae, EC50 = 587.17
		galloprovincialis			µg/L over 48h
		[Mediterranean
		Mussel]
		Paracentrotus lividus	larval length	0.32-5000	5% decrease in larval length [2	Torres et al. 2016
		[Purple sea urchin]		µg/L	µg/L]
			morphological	0.32-5000
			abnormalities	µg/L

7. Summary of organic UV filter toxicity studies on marine organisms. 20% of deformed larvae [0.8 µg/L] larval growth N/A EC50 = 854 µg/L over 48h Paredes et al. 2014 Senegalese sole [Sole flatfish] enzymatic activity 25-216 µg/L increase of AChe activity [85 µg/L] Araújo et al. 2018 Benzophenone-8 (BP8) Pocillopora damicornis [Cauliflower coral] mortality 0.1-1000 μg/L mortality 100% on day 3 (nubbin) [1000 µg/L] He et al. 2019 bleaching rate 0.1-1000 μg/L 100% bleaching (nubbin) [1000 µg/L] Seriatopora caliendrum mortality 0.1-1000 μg/L mortality 30% on day 9 (larvae) [1000 µg/L] Ethylhexyl Triazone (ET) Stylophora pistillata [Hood coral] photosynthetic efficiency 10-5000 µg/L increased photosynthetic efficiency Fel et al. 2018 Octocrylene (OC) Acropora sp. [Stoney coral] bleaching 33 μl/L no bleaching. Sunscreen formulations were tested and demonstrated different effects (see article) Danovaro et al.

Table 1 .8.

 1 Summary of organic UV filter toxicity studies on freshwater organisms.

	UV filter	Species Model [Common name]	Endpoint	Tested conc. range	Toxic Effects [Conc with toxic effect]	Reference
	3-benzylidene camphor	Desmodesmus	growth inhibition	0.5 -8 mg.L -1	IC50 = 6.99 mg.L -1	Sieratowicz et al.
	(3-BC)	subspicatus				(2011)
		[Unicellular algae]
		Pimephales	VTG induction	10 -1000 µg.L -	VTG induction at 435 and 953	Kunz et al. (2006)
		promelas [fathead		1	µg.L -1
		minnow]
		Xenopus laevis	Metamorphosis	1 -50 µg.L -1	No effect on metamorphosis	Kunz et al. (2004)
		[African clawed frog]	rate		and sex ratio
	3-(4'-methylbenzylidene)-	Chironomus	Growth inhibition	0.25 -25	Decreased growth [0.75	Campos et al.
	camphor	riparius [Harlequin		mg.kg -1	mg.kg -1 ]	(2017)
	(4-MBC)	fly]
			Electron transport		Increase of 61.85 % [14.13
			system		mg.kg -1 ]
			Catalase activity		22.84 % decrease [14.13
					mg.kg -1 ]
			glutathione-S-		Increase [14.13 mg.kg -1 ]
			transferase activity
			Development time		Delayed for female only [2.05
					mg.kg -1 ]

Table 1 .

 1 9. Photostability of UV filters under different light sources and intensities. Text in orange represents studies in which the UV filters were tested in cosmetic product formulation or liposome.

	UV Filter common name (Abbreviation)	Exposure device	Lamp spectrum (nm)	Analytical device	Time of exposure	Solvant (experimental details)	Results	Reference
	3-(4'-	Halogen lamp	290-800	SBSE	72h	Ultrapure water	(E) to (Z) photo-	Rodil et al. (2009)
	methylbenzylidene)-			GC-MS			isomerization but no
	camphor (4-MBC)						degradation
	Avobenzone (BMDBM)	Metal halide lamp	290-400	HPLC	3h	ethanol +	Photodegraded	Herzog et al. (2009)
						capric/caprylic
						triglyceride (plate
						method)
	Avobenzone	Philips UVA sun lamp	315-400	Spectrophotometer	15mn	(phosphatidylcholine	Reduction of absorbing	Damiani et al.
						liposome)	abilities	(2010)
	Benzophenone-3 (BP3)	Hg lamp	290-436	Spectrophotometer	-	Pure water, freshwater	Stable against direct	Li et al (2016)
				HPLC		or seawater (presence	photodegradation in pure
						of 3DOM, 1O2, and	water
						hydroxyl radical	Indirect photodegradation
						scavengers)	in seawater ( 3 DOM) and
							freshwater ( 3 DOM and
							hydroxyl radical)
	Benzophenone-3	Halogen lamp	290-800	SBSE	72h	Ultrapure water	High stability	Rodil et al. (2009)
				GC-MS
	Benzophenone-3	UV Lamp	254	HPLC	9h	-	High stability	Liu et al. (2011)
				GC-MS
	Benzophenone-3	Artificial sunlight	290-800	HPLC	50 days	-	Photostable alone	Liu et al. (2011)
				GC-MS			Photodegradation
							increased by 31% with
							humic acid at 50mg.L-1
		Xe lamp	250-800	HPLC	-	Buffered H2O + DOM	Indirect photodegradation	Semones et al.
				spectrophotometer			induced by the presence	(2017)
							of OH . inducers
		Xe lamp	250-800	HPLC	24h	-	High stability	Gago-Ferrero et al.
				MS/MS				(2012)
	Benzotriazole (BT)	UV Lamp	254	HPLC	9h		Unstable (45% of	Liu et al. (2011)
				GC-MS			degraded molecules)
	Ethylhexyl-methoxy-	Halogen lamp	290-800	SBSE	72h	Ultrapure water	(E) to (Z) photo-	Rodil et al. (2009)
	cinnamate (EHMC)			GC-MS			isomerization
							Photodegraded
		Metal halide lamp	290-400	HPLC	3h	ethanol +	Photodegraded	Herzog et al. (2009)
						capric/caprylic
						triglyceride (plate
						method)
		Hg lamp	200-400	HPLC	256mn		Photodegraded (6	Jentzsch et al.,
				ion trap MS			photoproducts)	2016
		Philips UVA sun lamp	315-400	Spectrophotometer	15mn	(phosphatidylcholine	Reduction of absorbing	Damiani et al.
						liposome)	abilities	(2010)
	Iso-amylmethoxy-	Halogen lamp	290-800	SBSE	72h	Ultrapure water	(E) to (Z) photo-	Rodil et al. (2009)
	cinnamate (IAMC)			GC-MS			isomerization
							Photodegraded
	Octocrylene (OC)	Halogen lamp	290-800	SBSE	72h	Ultrapure water	High stability	Rodil et al. (2009)
				GC-MS
	Octocrylene	Metal halide lamp	290-400	HPLC	3h	ethanol +	Photostable	Herzog et al. (2009)
						capric/caprylic
						triglyceride (plate
						method)
	Padimate O (OD-PABA)	Halogen lamp	290-800	SBSE	72h	Ultrapure water	Two dealkylation	Rodil et al. (2009)
				GC-MS			degradation products and
							one methylated derivate
	Tinosorb S (BEMT)	Metal halide lamp	290-400	HPLC	3h	ethanol +	Photostable	Herzog et al. (2009)
						capric/caprylic

Table 1 .

 1 9. Photostability of UV filters under different light sources and intensities. Text in orange represents studies in which the UV filters were tested in cosmetic product formulation or liposome.

	UV Filter common name (Abbreviation)	Exposure device	Lamp spectrum (nm)	Analytical device	Time of exposure	Solvant (experimental details)	Results	Reference
						triglyceride (plate
						method)
	Uvinul T 150 (EHT)	Metal halide lamp	290-400	HPLC	3h	ethanol +	Photodegraded	Herzog et al. (2009)
						capric/caprylic
						triglyceride (plate
						method)
	Uvinul T 150	Philips UVA sun lamp	315-400	Spectrophotometer	15mn	(phosphatidylcholine	Reduction of absorbing	Damiani et al.
						liposome)	abilities	(2010)

  [START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF][START_REF] Langford | Environmental Occurrence and Risk of Organic UV Filters and Stabilizers in Multiple Matrices in Norway[END_REF], coastal waters[START_REF] Apel | Environmental Occurrence and Distribution of Organic UV Stabilizers and UV Filters in the Sediment of European North and Baltic Seas[END_REF][START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF][START_REF] Sánchez Rodríguez | Occurrence of Eight UV Filters in Beaches of Gran Canaria (Canary Islands). An Approach to Environmental Risk Assessment[END_REF][START_REF] Sankoda | Seasonal and Diurnal Variation of Organic Ultraviolet Filters from Personal Care Products Used Along the Japanese Coast[END_REF][START_REF] Tovar-Sánchez | Sunscreen Products as Emerging Pollutants to Coastal Waters[END_REF][START_REF] Tsui | Organic Ultraviolet (UV) Filters in the South China Sea Coastal Region: Environmental Occurrence, Toxicological Effects and Risk Assessment[END_REF]) and sediments[START_REF] Fagervold | Occurrence and Environmental Distribution of 5 UV Filters During the Summer Season in Different Water Bodies[END_REF] Kameda,

	Kimura, and Miyazaki 2011; Mitchelmore, He, Gonsior, Hain, Heyes, Clark, Younger, Schmitt-
	Kopplin, et al. 2019)) but also demonstrated their bioaccumulation and toxic effects on several aquatic
	organisms (algae (Mao, Yiliand, and Karina 2018; Seoane et al. 2017), coral (He et al. 2019;
	Mitchelmore, He, Gonsior, Hain, Heyes, Clark, Younger, Schmitt-Kopplin, et al. 2019; Tsui et al. 2017),
	benthic mollusks (Giraldo, Montes, Rodil, Quintana, Vidal-linan, et al. 2017), fishes (Araújo et al.
	2018), dolphin (Alonso et al. 2015; Gago-Ferrero et al. 2013)), considering a wide variety of
	toxicological endpoints. Organic UV filters showed variable photostability. While benzophenone-3
	(BP3), octocrylene (OC) and 4-methylbenzilidene camphor (4-MBC) were described as photostable

. Previous studies reported the occurrence of UV filters ranging from nano to microgram per liter in different biotopes (rivers

[START_REF] Fent | Widespread Occurrence of Estrogenic UV-Filters in Aquatic Ecosystems in Switzerland[END_REF][START_REF] Kameda | Occurrence and Profiles of Organic Sun-Blocking Agents in Surface Waters and Sediments in Japanese Rivers and Lakes[END_REF]

, lakes

[START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF][START_REF] Herzog | Photostability of UV Absorber Systems in Sunscreens[END_REF] Y. S. Liu et al. 2011;[START_REF] Rodil | Photostability and Phytotoxicity of Selected Sunscreen Agents and Their Degradation Mixtures in Water[END_REF]

, cinnamate and triazone derivatives were found to produce photoproducts

[START_REF] Damiani | Assessment of the Photo-Degradation of UV-Filters and Radical-Induced Peroxidation in Cosmetic Sunscreen Formulations[END_REF][START_REF] Jentzsch | Photodegradation of the UV Filter Ethylhexyl Methoxycinnamate under Ultraviolet Light: Identification and in Silico Assessment of Photo-Transformation Products in the Context of Grey Water Reuse[END_REF][START_REF] Macmanus-Spencer | Aqueous Photolysis of the Organic Ultraviolet Filter Chemical Octyl Methoxycinnamate[END_REF]

and their toxicity is yet to be explored. While many publications emphasized the detrimental impact of UV filters on our Oceans, until now, there has been a limited number of studies addressing the toxicity of those emerging pollutants on microorganisms. Marine bacteria are dominant organisms on Earth and play a vital role in marine ecosystems

[START_REF] Bar-On | The Biomass Distribution on Earth[END_REF][START_REF] Whitman | Prokaryotes: The Unseen Majority[END_REF]

). In addition, they are symbiotic partners of multiple organisms such as coral

[START_REF] Reshef | The Coral Probiotic Hypothesis[END_REF]

,sea weed

[START_REF] Egan | The Seaweed Holobiont: Understanding Seaweed-Bacteria Interactions[END_REF]

, algae

[START_REF] Ramanan | Algae-Bacteria Interactions: Evolution, Ecology and Emerging Applications[END_REF]

) and sponge

[START_REF] Lee | Microbial Symbiosis in Marine Sponges[END_REF]

. Only a limited number of studies have reported the toxicity of oxybenzone on bacteria. A decrease in Chl-a content and an elevation of carotenoid production have been reported for Microcystis aeruginosa

[START_REF] Mao | Effects of Benzophenone-3 on the Green Alga Chlamydomonas Reinhardtii and the Cyanobacterium Microcystis Aeruginosa[END_REF]

.

Zhang and coworkers (2017)

showed that benzophenone derivatives inhibit the growth of Vibrio fischeri. The authors observed a linear relationship between the LogKow and the toxicity of the UV filter. Similarly, the toxicity of benzophenones on Photobacterium phosphoreum was shown to be correlated with their polarity (H.

[START_REF] Liu | Acute Toxicity of Benzophenone-Type UV Filters for Photobacterium Phosphoreum and Daphnia Magna: QSAR Analysis, Interspecies Relationship and Integrated Assessment[END_REF]

. Therefore, there is an urgent need to understand to what extent marine bacteria are affected by those chemicals.

Table 2 . 1 .

 21 Physiochemical properties of tested UV filters.

	Name	CAS n°	Structure	Molecular weight	Log Kow
	Benzophenone-3 (BP3)	131-57-7		228.243	3.52
	Ethylhexyl methoxy	5466-77-3		290.397	5.80
	cinnamate (EHMC)
	Octocrylene (OC)	6197-30-4		361.477	6.88
	4-methyl benzylidene	36861-47-9		254.367	5.92
	camphor (4-MBC)
	Homosalate (HS)	118-56-9		262.344	6.16

Table 2 .

 2 2. List of bacterial strains used in this study.

	Phyla	Bacterial species	BBCC number
	Actinobacteria	Arthrobacter aurescens	172
	Actinobacteria	Brachybacterium sacelli	164
	Actinobacteria	Dietzia maris	167
	Bacteroitedes	Algoriphagus mannitolivorans	266
	Bacteroitedes	Algoriphagus ornithinivorans	48
	Bacteroitedes	Maribacter dokdoensis	57
	Bacteroitedes	Olleya marilimosa	14
	Bacteroitedes	Sabulilitoribacter multivorans	185
	Firmicutes	Bacillus megaterium	240
	Firmicutes	Halobacillus dabanensis	119
	Firmicutes	Paenibacillus glucanolyticus	237
	α-proteobacteria Epibacterium mobile	367
	α-proteobacteria Erythrobacter citreus	2
	α-proteobacteria Erythrobacter nanhaisediminis	234
	α-proteobacteria Paracoccus hibiscisoli	192
	α-proteobacteria Pelagibacterium halotolerans	52
	α-proteobacteria Phaeobacter inhibens	654
	α-proteobacteria Pseudoalteromonas agarivorans	182
	γ-proteobacteria Alteromonas genovensis	151
	γ-proteobacteria Alteromonas marina	54
	γ-proteobacteria Enterovibrio calviensis	113
	γ-proteobacteria Paraglaciecola mesophila	6
	γ-proteobacteria Pseudoalteromonas hodoensis	177
	γ-proteobacteria Pseudomonas kunmingensis	268
	γ-proteobacteria Rheinheimera baltica	75
	γ-proteobacteria Vibrio aestuarianus	280
	γ-proteobacteria Vibrio azureus	222

Table 2 . 3 .

 23 Sensitivity of marine bacteria to different UV filters at 1000 µg L -1 . Black colored cells represent toxic UV filters, inducing bacterial growth inhibition.

	Phyla	Species	BBCC number BP3 EHMC OC 4MBC HS
	Actinobacteria	Arthrobacter aurescens	172
	Actinobacteria	Brachybacterium sacelli	164
	Actinobacteria	Dietzia maris	167
	Bacteroitedes	Algoriphagus mannitolivorans	266
	Bacteroitedes	Algoriphagus ornithinivorans	48
	Bacteroitedes	Maribacter dokdoensis	57
	Bacteroitedes	Olleya marilimosa	14
	Bacteroitedes	Sabulilitoribacter multivorans	185
	Firmicutes	Bacillus megaterium	240
	Firmicutes	Halobacillus dabanensis	119
	Firmicutes	Paenibacillus glucanolyticus	237
	α-proteobacteria Epibacterium mobile	367
	α-proteobacteria Erythrobacter citreus

2 α-proteobacteria Erythrobacter nanhaisediminis 234 α-proteobacteria Paracoccus hibiscisoli 192 α-proteobacteria Pelagibacterium halotolerans 52 α-proteobacteria Phaeobacter inhibens 654 γ-proteobacteria Alteromonas genovensis 151 γ-proteobacteria Alteromonas marina 54 γ-proteobacteria Enterovibrio calviensis 113 γ-proteobacteria Paraglaciecola mesophila 6 γ-proteobacteria Pseudoalteromonas hodoensis 177 γ -proteobacteria Pseudoalteromonas agarivorans 182 γ-proteobacteria Pseudomonas kunmingensis 268 γ-proteobacteria Rheinheimera baltica 75 γ-

proteobacteria Vibrio aestuarianus 280 γ-proteobacteria Vibrio azureus 3.2 Dose response on bacterial growth

  Bacteria showing sensitivity to UV filters at 1000 µg L -1 , colored in black in Table2.3, were further studied for their dose responses with concentrations ranging from 100 to 4000 µg L -1 . The dose response of seven bacteria to different UV filters was tested as follows: Arthrobacter aurescens(BBCC 172)

	against EHMC and OC; Algoriphagus ornithinivorans (BBCC 48) against HS; Dietzia maris (BBCC
	167) against EHMC; Epibacterium mobile (BBCC 367) against BP3; Halobacillus dabanensis (BBCC
	119) against HS and EHMC; Paenibacillus glucanolyticus (BBCC 237) against EHMC and
	Pelagibacterium halotolerans (BBCC 52) against BP3 and EHMC (Figures 2.2 and 2.3). We observed
	different profiles of dose responses. Some strains showed a non-monotonic dose response, which means

Table 4 . 1 .

 41 Physiochemical properties of benzophenone-3.

	Name	CAS n°	Structure	Molecular weight	Log Kow
	Benzophenone-3 (BP3)	131-57-7		228.243	3.52

Table 4 .

 4 2. Identification, function, and fold change of proteins differentially regulated between E. mobile exposed to 0.046% DMSO and artificial seawater only. Proteins are classified from the most downregulated to the most upregulated. Color code match the function group highlighted in Figure 3. Proteins included displayed a p-value < 0.05, bold in the fold change column indicate p-value <0,01.

	Accession number Protein name	Function	Fold Change
	WP_126138581.1 SDR family oxidoreductase	Oxidoreductase	0.22
	WP_126138243.1 KpsF/GutQ family sugar-phosphate isomerase	Carbohydrate metabolism	0.29
	WP_005651340.1 succinate dehydrogenase iron-sulfur subunit	Carbohydrate metabolism	0.38
	WP_005612958.1 polyphosphate kinase 2	Kinase	0.42
	WP_005618566.1 NADH-quinone oxidoreductase subunit NuoF	Oxidoreductase	0.48
	WP_126136393.1 glutamate--tRNA ligase	tRNA ligase	0.36
	WP_005634757.1 H-NS histone family protein	Histone	0.42
	WP_005652398.1 Lrp/AsnC family transcriptional regulator	Transcription regulator	0.49
	WP_005608877.1 ribosome-associated translation inhibitor RaiA	Translation regulator	0.49
	WP_126136968.1 carboxypeptidase M32	Peptidase	0.29
	WP_126137747.1 penicillin-insensitive murein endopeptidase	Peptidase	0.37
	WP_126136244.1 NAD(P)(+) transhydrogenase (Re/Si-specific) subunit beta Metabolism of cofactors and vitamins 0.38
	WP_126138751.1 ABC transporter ATP-binding protein	ABC transporter	2.36
	WP_009177819.1 Fe(3+) ABC transporter substrate-binding protein	ABC transporter	2.43
	WP_009177251.1 ABC transporter ATP-binding protein	ABC transporter	2.53
	WP_126136253.1 ABC transporter substrate-binding protein	ABC transporter	4.55
	WP_126136458.1 peptidase	Peptidase	2.69
	WP_126136458.1 peptidase	Peptidase	3.54
	WP_005628449.1 hemin uptake protein HemP	Hemin uptake	5.68
	WP_126136289.1 hypothetical protein	Unknown function	2.11
	WP_172599654.1 GTP-binding protein	Putative function	2.11
	WP_126135976.1 RdgB/HAM1 purine NTP pyrophosphatase	Nucleotide metabolism	8.58
	WP_126137866.1 NAD(P)-dependent oxidoreductase	Carbohydrate metabolism	2.81

Table 4 .

 4 3. Identification, function, and fold change of proteins differentially regulated between E. mobile exposed to 350 µg/L BP3 + 0.046% DMSO and 0.046% DMSO only. Proteins are classified from the most downregulated to the most upregulated. Color code matching the functional category highlighted in Figure 4.3. Proteins included displayed a p-value < 0.05, bold in the fold change column indicate p-value <0.01.

	Accession number	Protein name

Table 5 .

 5 1. Pairwise compared species and respective UV filters sensitivity.

	Phyla	Species	Sensitive/resistant	ANI Score (%)
			(UV filters)
	Bacteroidetes Bacteroidetes	Algoriphagus mannitolivorans Algoriphagus ornithinivorans	R S	(HS)	72.13
	Firmicutes Firmicutes	Paenibacillus glucanolyticus Halobacillus dabanensis	R (EHMC, HS) S	65.32
	Firmicutes Firmicutes	Bacillus megaterium Halobacillus dabanensis	R S	(EHMC, HS)	68.02
	2.3 Protein comparison
	Protein FASTA files were downloaded from the RAST platform and uploaded on the Orthovenn
	online tool (https://orthovenn2.bioinfotoolkits.net/) to generate Venn diagrams.

  Table5.2). Similarly, the number of rRNA, tRNA, and proteins was lower in the Bacteroidetes species. Among Firmicutes, H. dabanensis displayed the smaller genome with approximately 1000 fewer proteins than B. megaterium and P. glucanolyticus. Noteworthy, UV filters resistant species for both Firmicutes and Bacteroidetes held the greater genome. A correlation between the appearance of resistance mechanisms and genome size has already been discussed earlier[START_REF] Projan | Genome) Size Matters[END_REF]. Similarly, a high tRNA number has been correlated with temperature resistance (Siddhartha Sankar, Malay, and Suvendra Kumar 2010).

Table 5 .

 5 2. Structures of bacterial genomes. Asterisks (*) indicate UV filters resistant bacteria.

	Species	chromosome plasmid size (Mb) GC% Genes rRNA tRNA Proteins
	Algoriphagus mannitolivorans* 1	0	4.15	42.7	3636	7	39	3564
	Algoriphagus ornithinivorans	1	0	4.09	39.5	3,557 5	35	3,500
	Paenibacillus glucanolyticus* 1	0	5.9	49.2	5,380 24	74	5,174
	Halobacillus dabanensis	1	0	4.14	41.7	4,135 18	67	3,971
	Bacillus megaterium*	1	6	5.34	38.1	5,541 41	123	5,268
	A. mannitolivorans and A. ornithinivorans shared 2823 proteins and displayed 34 and 28 unique
	proteins, respectively (Figure 5.1A). Among these proteins, A. mannitolivorans displayed 2 protein
	counts included in the "cellular aromatic compound metabolic process" cluster, identified as the 4,5-
	DOPA dioxygenase extradiol, an enzyme involved in betalain biosynthesis. P. glucanolyticus and H.
	dabanensis shared 1719 proteins (Figure 5.1B). Interestingly, P. glucanolyticus showed a fewer number
	of unique proteins with 586 against 797. It is noteworthy to underline that the Orthovenn tool only
	considers proteins included in clusters, which could explain why P. glucanolyticus had fewer unique
	proteins even though it had a greater genome. B. megaterium and H. dabanensis shared 2157 proteins
	and exhibited 455 and 359 unique proteins, respectively (Figure 5.1C).

Table 6 .

 6 1. Pic area fold change between polluted (central beach) and control (SOLA) sites at different time points in summer 2019.

		BMDBM	TM	EHT	DHHB	IS	EHMC	BP3	TS	OC
	June	16.1 ± 3.2	2.8 ± 1.4	4.8 ± 0.6	42.4 ± 17.1 5.6 ± 1.3	0.7	1.6	1.6	4.3
	July	116.8 ± 25.1 21.1 ± 5.0 48.9 ± 8.2 163.2 ± 33.6 41.9 ± 12.2 1.5	16.2	14.1	35.3
	August 268.4 ± 233.0 32.1 ± 10.2 73.8 ± 6.67 322.9 ± 21.1 47.4 ± 12.9 2.5	1.4	ND	84.0

  developed a

	Iscotrizinol	Diethylhexyl Butamido Triazone	-	Uvasorb HEB

Octinoxate ETHYLHEXYL METHOXYCINNAMATE or Octyl methoxycinnamate EHMC or OMC Neo Heliopan®AV 2-Ethylhexyl 4-methoxycinnamate

					phenyl]amino]-1,3,5-triazin-2-
					yl]amino]benzoic acid 2-ethylhexyl ester
	Ensulizole	phenylbenzimidazole sulfonic acid	-	-	2-Phenyl-3H-benzimidazole-5-sulfonic acid
	Enzacamene	4-METHYLBENZYLIDENE CAMPHOR	4-MBC	Eusolex 6300 or Parsol 5000	(3E)-1,7,7-Trimethyl-3-[(4-methylphenyl)methylene]-2-norbornanone
	Homosalate	Homosalate	HMS	Neo Heliopan®HMS	3,3,5-Trimethylcyclohexyl 2-hydroxybenzoate
	Menthyl anthranilate	Meradimate	-	Neo Heliopan®MA	(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-aminobenzoate
	Meradimate	Menthyl anthranilate	-	Neo Heliopan®MA	(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-aminobenzoate
	Octocrylene	Octocrylene	OC	Neo Heliopan®303	2-Cyano-3,3-diphenyl acrylic acid, 2-ethylhexyl ester
	Octisalate	ETHYLHEXYL SALICYLATE OR Octyl	-	Neo Heliopan®OS	2-ethylhexyl 2-hydroxybenzoate
		salicylate
	Oxybenzone	Benzophenone-3	BP3	Milestab 9, Eusolex 4360,	(2-Hydroxy-4-methoxyphenyl)-phenylmethanone
				Escalol 567, KAHSCREEN BZ-3
	Padimate O	ETHYLHEXYL DIMETHYL PABA	OD-PABA	Escalol 507	2-Ethylhexyl 4-(dimethylamino)benzoate
	PEG-25 PABA	Ethoxylated Ethyl-4-Aminobenzoate	-	Unipabol U-17 OR Uvinul P25 Polyoxyethylene ethyl-4-aminobenzoate
	POLYACRYLAMIDOMETHYL	POLYACRYLAMIDOMETHYL	-	Mexoryl SW	Polymer of N-{(2 and 4)-[(2-oxoborn-3-
	BENZYLIDENE CAMPHOR	BENZYLIDENE CAMPHOR			ylidene)methyl]benzyl}acrylamide

Abstract

Organic UV filters are of emerging concern due to their occurrence and persistence in coastal ecosystems. Because marine bacteria are crucial in the major biogeochemical cycles, there is an urgent need to understand to what extent these microorganisms are affected by those chemicals. This study deciphers the impact of five common sunscreen UV filters on twenty-seven marine bacteria, combining both photobiology and toxicity analysis on environmentally relevant species. Seven bacteria were sensitive to different organic UV filters at 1000 µg L -1 , including octinoxate and oxybenzone. This is the first report demonstrating inhibition of bacterial growth from 100 µg L -1 . None of the UV filters showed any toxicity at 1000 µg L -1 on stationary phase cells, demonstrating that physiological state was found to be a key parameter in the bacterial response to UV filters. Indeed, non growing bacteria were resistant to UV filters whereas growing cells exhibited UV filter-dependent sensitivity. Octinoxate was the most toxic chemical at 1000 µg L -1 on growing cells. Interestingly, photobiology experiments revealed that the toxicity of octinoxate and homosalate decreased after light exposure while the other compounds were not affected. In terms of environmental risk characterization, our results revealed that the increasing use of sun blockers could have detrimental impacts on bacterioplanktonic communities in coastal areas. Our findings contribute to a better understanding of the impact of the most common UV filters on bacterial species and corroborate the importance to consider environmental parameters such as solar radiation in ecotoxicology studies.

CHAPTER 3

Physiological investigation: the effects of oxybenzone on Epibacterium mobile, an environmentally relevant bacterium

Abstract

Oxybenzone is one of the most controversial organic UV filters, being the first banned in Hawaii and the Republic of Palau due to detrimental effects on coral reefs. Our previous study showed that Epibacterium mobile, a marine Alphaproteobacterium, is sensitive to oxybenzone in a dose-dependent fashion. This study aims to further explore the effect of oxybenzone on Epibacterium mobile. Different media were tested to determine the best formulation for ecotoxicological applications. The effects of oxybenzone on cellular integrity and enzymatic activity were assessed via flow cytometry and transmission electronic microscopy (TEM). Artificial sea water (ASW) was more adapted than tryptone or yeast extract-supplemented culture media to assess the toxicity of oxybenzone at 1000 µg/L. Flow cytometry analysis revealed a dose-dependant decrease in esterase activity from 100 µg/L oxybenzone.

Microscopy showed that cell membranes were not affected whilst the average number of granules per cell increased under oxybenzone exposure.

Abstract

The use of Benzophenone-3 (BP3), also known as oxybenzone, a common UV filter, is a growing environmental concern in regard to its toxicity on aquatic organisms. Our previous work stressed that BP3 is toxic to Epibacterium mobile, an environmentally relevant marine Alphaproteobacteria. In this study, we implemented a label-free quantitative proteomics workflow to decipher the effects of BP3 on the E. mobile proteome. Furthermore, the effect of DMSO, one of the most common solvents used to vehicle low concentrations of lipophilic chemicals, was assessed to emphasize the importance of limiting solvent concentration in ecotoxicological studies. Data-independent analysis proteomics highlighted that BP3 induced changes in the regulation of 56 proteins involved in xenobiotic export, detoxification, oxidative stress response, motility, and fatty acid, iron and amino acid metabolisms. Our results also outlined that the use of DMSO at 0.046% caused regulation changes in proteins related to transport, iron uptake and metabolism, and housekeeping functions, underlining the need to reduce the concentration of solvents in ecotoxicological studies. CHAPTER 5 Shedding lights on the bacterial response to toxic UV filters: a comparative genomic study Abstract UV filters are toxic to marine bacteria that dominate the biomass in marine planktonic ecosystems.

Ecotoxicological studies often measure broad physiological responses but rarely integrates the toxicity mechanisms at the molecular level. In this study, in silico comparative genomic between UV filters sensitive and resistant bacteria were conducted in order to unravel the genes responsible for a resistance phenotype. The genomes of two environmentally relevant Bacteroidetes and three Firmicutes species were compared through pairwise comparison. Larger genomes were carried by bacteria exhibiting a resistant phenotype, favoring their ability to adapt to environmental stresses. While the antitoxin and CRISPR systems were the only distinctive features in resistant Bacteroidetes, Firmicutes displayed multiple unique genes that could support the difference between sensitive and resistant phenotypes.

Several genes involved in ROS response, vitamin biosynthesis, xenobiotic degradation, multidrug resistance, xenobiotic degradation, and lipophilic compound permeability were shown to be exclusive to resistant species. Our investigation contributes to a better understanding of UV filters resistance phenotypes, by identifying pivotal genes involved in key pathways.

CHAPTER 6 General discussion

photoprotective formulation using vegetable oils that reached an SPF of 5.1. However, oils are often not suitable for cosmetic use due to unpleasant texture. While the authors of the above-mentioned studies advertised their UV filters as eco-friendly, toxicity investigations are yet to be conducted.

UV resistance in marine bacteria was reported [START_REF] Agogué | Resistance of Marine Bacterioneuston to Solar Radiation[END_REF][START_REF] Matallana-Surget | Response to UVB Radiation and Oxidative Stress of Marine Bacteria Isolated from South Pacific Ocean and Mediterranean Sea[END_REF].

In Chapter 2, we showed that E. mobile was surprisingly resistant to solar radiation, displaying 73% survival after 5 hours of exposure while other species showed <1% survival. While mycosporine-like amino acids confer UV resistance to cyanobacteria [START_REF] Dzeha | UV Resistance of Bacteria from the Kenyan Marine Cyanobacterium Moorea Producens[END_REF], studies characterizing molecules involved in UV resistance in heterotrophs are scarce. Overall, our results suggest that E. mobile is a promising source of bio-based UV protective compounds. Chromatographic separation of cellular components followed by UV absorption characterization of the different fractions would allow for the isolation of the UV-resistant compound.

Concluding remarks

This project demonstrated the species and compound-dependent bactericidal effects of common organic UV filters on marine bacteria and contributed to a better understanding of the cellular response induced by UV filter exposure. We showed, via morphological investigation and quantitative proteomics, that bacteria exhibited a variety of stress-related molecular responses, emphasizing that bacteria are suitable for ecotoxicological studies. Future meta-omic research on natural and artificial communities will enable us to assess the impact of anthropogenic inputs on the functions of microbial communities.

Appendix