# Mineral UV filters and their effects on Pocillopora damicornis metabolome

Claire Guillier, Maeva Giraudo, Fanny Clergeaud, Evane Thorel, Leïla Chapron, Lionel Marcon, Philippe Lebaron, Emeline Houël, Didier Stien

► To cite this version:

Claire Guillier, Maeva Giraudo, Fanny Clergeaud, Evane Thorel, Leïla Chapron, et al. Mineral UV filters and their effects on Pocillopora damicornis metabolome. Science of the Total Environment, 2025, 991, pp.179961. 10.1016/j.scitotenv.2025.179961. hal-05133154

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1 Mineral UV filters and their effects on *Pocillopora damicornis* metabolome

2

3 Claire Guillierª, Maeva Giraudoª, Fanny Clergeaudª, Evane Thorelª, Leïla Chapronᵇ, Lionel Marconª,

4 Philippe Lebaronª, Emeline Houëlª, Didier Stienª,*

5 ª Sorbonne Université, Université de Perpignan Via Domitia, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes, LBBM, F-66650 Banyuls-sur-Mer, France.

7 ᵇ Sorbonne Université, CNRS, Laboratoire d'Ecogéochimie des Environnements Benthiques, LECOB, F-66650 Banyuls-sur-Mer, France.

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10 * Corresponding author: didier.stien@cnrs.fr, Tel.: +33 4 30 19 24 76

11 Abstract

12 The rising use of sunscreens and cosmetics containing ultraviolet (UV) filters has increased their 13 presence in marine ecosystems. UV filters encompass a wide range of organic and mineral compounds 14 with diverse behaviors and properties in aquatic environments. The mineral filters titanium dioxide 15 (TiO₂) and zinc oxide (ZnO) are commonly found in cosmetic products as particles or nanoparticles 16 (NPs) and are increasingly used as alternatives to organic UV filters. In this study, the effects of a 17 coated form of TiO₂ (Solaveil™ XTP₁, 60 nm, hydrophobic) and uncoated ZnO particles from two 18 different sizes (Zano®10 and Zano®M, 60 and 250 nm, hydrophilic) were assessed on the symbiotic 19 tropical coral *Pocillopora damicornis* using an untargeted metabolomic approach. Corals were 20 exposed for seven days to environmentally relevant particle concentrations (5 to 1000 µg.L⁻¹), and 21 their metabolomes were analyzed using UHPLC-HRMS/MS. While TiO₂ exposure did not induce 22 significant metabolomic changes, both sizes of ZnO particles caused shifts in the metabolome of the 23 coral's symbiotic dinoflagellates, leading to a noticeable decrease in the relative concentrations of 24 symbiont lipids and pigments. A size-dependent toxicity of ZnO was observed: ZnO NPs triggered 25 signs of bleaching at concentrations as low as 300 µg.L⁻¹, whereas larger ZnO particles exhibited only 26 mild effects at the highest concentration tested (1000 µg.L⁻¹). This underscores the critical role of

ATR: Attenuated Total Reflection; BP-3: Oxybenzone; CD: Compound Discoverer™; DGCC: 1,2-Diacylglyceryl-3-O-carboxy-(hydroxymethyl)-choline; DGDG: Digalactosyldiacylglycerol; DMSO: Dimethyl sulfoxide; DNP: Dictionary of Natural Products; EHMC: Octinoxate; FASW: Filtered Artificial Sea Water; FDR: False discovery rate; FTIR: Fourier Transform InfraRed spectroscopy; GNPS: Global Natural Products Social Molecular Networking; GSL: Glycosphingolipid; LDGCC: Lyso-diacylglycerylcarboxy-hydroxymethylcholine; Log₂ FC: Log₂(fold-change); LPC: Lyso-phosphatidylcholine; MGDG: Monogalactosyldiacylglycerol; MoNa: MassBank of North America; NP: Nanoparticle; OC: Octocrylene; PEG: Polyethylene glycol; PSII: Photosystem II; PRM: Parallel Reaction Monitoring; ROS: Reactive Oxygen Species; SEM: Scanning electron microscopy; TEM: Transmission electron microscopy; TiO₂: Titanium Dioxide; UV: Ultraviolet; ZnO: Zinc Oxide; ZP: Zeta Potential
particle size in determining toxicity. This research highlights the contrasting effects of different mineral UV filters on symbiotic corals and aims to inform cosmetic companies in selecting mineral filters that minimize harmful impacts on coral reefs.

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31 **Keywords:** coral bleaching, metabolomics, inorganic sunscreen, nanoparticles, Symbiodiniaceae

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## 1. Introduction

Over recent decades, coral reefs have experienced a persistent decline, characterized by frequent and severe bleaching events (Suggett and Smith, 2020). Coral bleaching occurs when the symbiotic relationship between corals and their endosymbiotic dinoflagellates, commonly referred to zooxanthellae (*Symbiodinium* spp.), breaks down (Nielsen et al., 2018). This disruption leads to pigment degradation and/or the loss of zooxanthellae, which can ultimately result in coral mortality (Bridge et al., 2014). Together with the coral host and its associated microbiota, these symbionts form the coral holobiont — a complex symbiotic system whose stability is crucial for reef health and resilience (Berkelmans and Van Oppen, 2006). The integrity of this symbiotic relationship is increasingly threatened by global climate change, which alters sea temperatures and ocean acidity (Cameron et al., 2022), as well as by localized stressors, including the influx of sunscreen-derived chemicals into coastal waters (Tovar-Sánchez et al., 2013). While sunscreens play a critical role in protecting human health from the adverse effects of ultraviolet (UV) radiations, their widespread use driven by rising tourism, has led to the accumulation of UV filters in marine ecosystems, raising concerns about their impact on aquatic organisms (Sánchez-Quiles and Tovar-Sánchez, 2015). Among these UV filters, certain organic compounds such as benzophenone-3 (or oxybenzone, BP-3), ethylhexyl methoxycinnamate (or octinoxate, EM), and octocrylene (OC), have received particular attention due to their adverse effects on marine organisms, especially coral (ANSES, 2022). Several studies have demonstrated that BP-3 and EM can disrupt the coral microbiome equilibrium, induce coral bleaching and promote viral proliferation, potentially leading to coral death (Conway et al., 2021; Danovaro et al., 2008; Yang et al., 2023). OC has been shown to accumulate in coral tissues, both in its initial form and as derivatives, and trigger deleterious processes such as oxidative stress and mitochondrial dysfunction (He et al., 2019; Stien et al., 2019; Thorel et al., 2022; Tsui et al., 2015).
56 2017). In response to these findings, several countries have implemented strict regulations or outright bans on sun care products containing these three UV filters. For example, OC has been banned in Palau (2018), the Marshall Islands (2019), and the Virgin Islands (2020). Similarly, both EM and BP-3 have been banned in Key West (2019), Thailand (2021) and Hawaii (2021), reflecting a growing international commitment to protect marine ecosystems from sunscreen pollution. Consequently, sun care formulations are undergoing significant reformulation to meet both human health standards and environmental sustainability goals.

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64 This shift has led to the increased adoption of mineral UV filters, primarily titanium dioxide (TiO₂) and zinc oxide (ZnO), as alternatives to organic UV filters in many sun care products (Schneider and Lim, 2019). These mineral filters are often incorporated in the form of nanoparticles (NPs), whose UV-protected efficiency is closely tied to their physicochemical characteristics, including particle size, shape, surface charge and surface coatings. These features enhance formulation stability and broaden UV protection compared to their organic counterparts (Smijs and Pavel, 2011). In this context, there is growing interest in understanding the fate and behavior of mineral UV filters once released into aquatic environments. However, limited quantitative data exist on their environmental concentrations largely due to the difficulty in distinguishing anthropogenic inputs from natural particulate matter.
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73 Some measurements from Mediterranean coastal areas have been reported: Tovar-Sánchez et al. (2013) found TiO₂ concentrations ranging from 12-37 µg.L⁻¹ and ZnO from 0.8-10.8 µg.L⁻¹ in surface waters off the coast of Majorca, while organic filters were detected at concentrations in the ng.L⁻¹ range. Similarly, Thallinger et al. (2023) reported higher TiO₂ levels compared to organic filters, with concentrations between 20-296 µg.L⁻¹ in Palavas-les-Flots, France. In Marseille, Labille et al. (2020) observed TiO₂ concentrations of 100-900 µg.L⁻¹ and ZnO levels between 20-50 µg.L⁻¹ in surface water, with lower values of 10-15 µg.L⁻¹ and 1-3 µg.L⁻¹, respectively, detected in deeper water layers.

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81 The distribution, persistence and bioavailability of mineral UV filters are governed by their intrinsic properties (Mitjans et al., 2023; Samei et al., 2019; Yung et al., 2017), as well as by environmental parameters such as temperature, pH, salinity, and UV radiation (Lewicka et al., 2011; Pagnout et al., 2012; Li et al., 2013). These factors ultimately influence particle-organism interactions and associated
toxicity mechanisms. The size-dependent toxicity of metal oxide particles, such as TiO₂ and ZnO, has been documented in various aquatic species. Nano-sized dispersed TiO₂ and ZnO have been shown stronger inhibitory effects on algal growth (e.g., *Nitzschia closterium* and *Dunaliella tertiolecta*) compared to their bulk counterparts (Manzo et al., 2013; Xia et al., 2015). In aquatic invertebrates such as *Daphnia magna*, smaller TiO₂ and ZnO agglomerates accumulate more readily in the digestive tract (Kim et al., 2010; Lopes et al., 2014). In *Danio rerio*, TiO₂ NPs at 50 mg.L⁻¹ induced significant gill cell damage over 96 hours, whereas bulk TiO₂ caused negligible effects (Xiong et al., 2011).

By contrast, the role of surface coatings in modulating toxicity remains underexplored. These coatings — either mineral (e.g., Al₂O₃ or SiO₂) or organic (e.g., stearic acid, silane, polyacrylic acid) — affect particle properties such as wettability, dispersion, and texture, facilitating their integration into formulations by altering their hydrophobicity/hydrophilicity (Slomberg et al., 2021).

Coatings may also enhance particles stability by reducing undesired photocatalytic reactions, which may also promote their persistence into the environment after release (Mbanga et al., 2022; Thallinger et al., 2023). While coatings may mitigate toxicity by altering aggregation, solubility or dispersion in water (Lai et al., 2021; Yung et al., 2017), they can also influence particle uptake and depuration in aquatic organisms (Connolly et al., 2022; Lai et al., 2021).

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The impact of inorganic UV filters on corals remains insufficiently understood. However, emerging evidence suggests that these filters may contribute to coral bleaching through symbiont loss and reduced photosynthetic efficiency (Fel et al., 2019; Jovanović and Guzmán, 2014), as well as by promoting microbial enrichment following ZnO exposure (Corinaldesi et al., 2018). Still, many of these studies lack detailed physicochemical characterization of the NPs used, which is essential for accurately assessing their toxicity and ensuring comparability between studies. Based on previous studies focusing on corals, it can be hypothesized that ZnO particles will exhibit greater toxicity than TiO₂ particles due to their higher solubility and potential for Zn²⁺ release. Additionally, nanoparticles are expected to cause more pronounced biological effects compared to larger particles of the same composition, as smaller particles have higher surface area-to-volume ratios and typically higher dissolution rates. To overcome such limitations, metabolomics has emerged as a promising and integrative approach for exploring stress responses in complex biological systems such as the coral
114 holobiont. While primarily used to investigate stress-specific responses to coral bleaching (Pei et al., 2022; Roach et al., 2021; Sogin et al., 2016), metabolomics has also proven valuable in evaluating the effects of organic UV filters (Clergeaud et al., 2023; Stien et al., 2020). By analyzing the complete set of metabolites in an organism, untargeted metabolomics can detect subtle, early biochemical changes induced by nanoparticle exposure, providing light on toxicity mechanisms at a molecular level.

119 The present study aims to further elucidate the effects of TiO₂ and ZnO particles and NPs on the symbiotic tropical scleratinian coral *Pocillopora damicornis*. This ubiquitous Indo-Pacific species thrives in shallow waters (Bramanti et al., 2015). As a fast-growing, reef building (hermatypic) coral, *Pocillopora damicornis* plays a fundamental ecological role by facilitating the settlement of various other species, making it an ideal model for assessing risks to coral reef ecosystems (Brener-Raffalli et al., 2018). In this study, corals were exposed to increasing concentrations (5, 50, 300 and 1000 µg.L⁻¹) of one stearic acid-alumina coated TiO₂ (Solaveil™ XTP₁) and two ZnO nanopowders of different sizes (Zano10® and ZanoM®) to evaluate metabolomic shifts associated with stress induced by these widely used mineral UV filters.

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## 2. Materials and Methods

### 2.1. Mineral UV filters characterization

#### 2.1.1 Mineral UV filters

132 Solaveil™ XTP₁ alumina stearic acid coated nanoparticles (TiO₂ NPs) were purchased from Croda (Croda Personal Care, Montigny-le-Bretonneux, France). Zano®10 and Zano®M (EverCare, Netherlands, Eijsden) uncoated zinc oxide particles (ZnO) were purchased from Comercial Química Massó (Barcelona, Spain). Particles specific surface area given by the supplier for Zano®10 and Zano®M are respectively of 29.1 m²/g and 9 m²/g (BET technical measurements), and not available for Solaveil™ XTP₁. Purity of each mineral UV filter tested is greater than 98% according to the data provided by suppliers.

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#### 2.1.2 Fourier transform infrared spectrometry analysis (FTIR)

141 The aim of FTIR analysis was to confirm the presence or absence of surface coatings on the mineral UV filters and to identify characteristic functional groups that may influence their environmental
behavior and interaction with biological systems. The analysis was performed on the pristine powder form of each sample (Solaveil™ XTP1, Zano®10, and Zano®M), without any pre-treatment, in order to assess their chemical surface composition as supplied by the manufacturer.

All spectra were recorded using a handheld Fourier transform infrared (FTIR) spectrometer (Agilent 4300, Agilent Technologies, USA) equipped with a single-reflection attenuated total reflectance (ATR) sensor. Measurements were conducted in the wavenumber range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹, and 68 scans were averaged per spectrum. This method allows for non-destructive analysis of surface-bound organic or inorganic moieties, such as stearate or carboxylate groups, potentially indicative of surface coatings or synthesis residues.

## 2.2.2. Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM, S-3400N, HITACHI, Japan) was used to assess the morphology and size range of Solaveil™ XTP₁, Zano®10 and Zano®M primary particles. To do so, a brush was dipped into UV filters powders, and the particles were sprayed over a glow-discharged copper grid. The primary particle size distribution was pictured under an operating voltage set at 80 kV and determined by measuring the length of approximately 300 particles, by a manual point-to-point measurement on AMT software (512 version 5.42.544 Q, USA).

## 2.2.3 Scanning Electron microscope (SEM)

Similar to TEM sample preparation, a few micrograms of particles were sprayed onto carbon tape attached to an aluminum support for scanning electron microscopy (SEM, JSM-IT800 SHL LV, Jeol, Japan). Particle images were collected at a working distance of 4 mm, with an accelerating voltage set at 2 kV for Solaveil™ XTP₁ and Zano®10, and 5 kV for Zano®M.

## 2.2.4. Zeta potential and hydrodynamic size measurements

Electrophoretic mobility and hydrodynamic diameters of TiO₂ and ZnO particles were measured using a Malvern Zetasizer® Nano-ZS instrument based on Dynamic Light Scattering (DLS). Measurements were performed at 25 °C on 1 mg·L⁻¹ suspensions prepared in filtered artificial seawater (FASW), using the same dispersion protocols as for coral exposures. Hydrophobic particles (e.g., Solaveil™ XTP1) were first dispersed in a DMSO solution containing 0.1% Emulium® Illustro,
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then diluted 1:1000 in FASW. All suspensions were sonicated (10–30 min) and vortexed prior to
measurement.

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Each sample was measured in triplicate (8 runs per measurement), and the cuvettes were re-agitated between runs to prevent sedimentation. Hydrodynamic diameters were determined using the Mie theory algorithm provided by the instrument software. For each particle type, we report volume-weighted mean diameters, considered more representative for polydisperse samples, and additionally include intensity-weighted diameters and polydispersity indices (PDI) in the supplementary material (Table Sx). The residuals of curve fitting were within acceptable limits, and measurement quality was validated by the analytical platform.

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Zeta potential (ZP) measurements yielded values between -6.3 and -9.6 mV for particle suspensions, similar to the background of the FASW control (-6 mV), indicating low electrostatic repulsion and a tendency to form aggregates.”

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## 2.3. Rearing of *Pocillopora damicornis*

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Colonies of *P. damicornis* were maintained at the Banyuls Observatory nursery since their collection from Oman in 2014 (CITES permit 37/2014). They are cultured in FASW (salinity = 36 g.L⁻¹, pH = 8, and temperature maintained at 24 ± 1 °C) prepared using reverse osmosis-purified water mixed with TropicMarin™ Salt (Tropic Marin PRO REEF salt, Wartenberg, Germany). The lighting is provided by Ecotech Marine Radion G5 lamps (model XR15 Pro, Bethlehem, PA, USA), set to deliver a photosynthetic photon flux density of 250 µmol.m⁻².s⁻¹ under a 10:14-hour light:dark photoperiod. Prior exposure, small coral fragments (about 5 cm) were cut from a mother colony and suspended to a fishing line (nylon) for a one-month acclimation period to ensure a complete tissue repair. Colonies and suspended fragments were fed daily with brine shrimps *Artemia salina* (approximatively 800 shrimps per liter in culture tank).

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## 2.4. Exposure to Zano®10, Zano®M and Solaveil™ XTP₁

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The concentrations of the tested suspensions were defined on the basis of a preliminary acute toxicity test and were set at 1000 µg.L⁻¹ for Solaveil™ XTP₁ and 5, 50, 300 and 1000 µg.L⁻¹ for Zano®10 and Zano®M. Stock suspensions of Zano®10 and Zano®M were prepared at concentrations of 5, 50, 300
and 1000 mg.L⁻¹ in MilliQ water. For each exposure condition, 200 µL of the stock suspension was dispersed into beakers containing 200 mL of FASW freshly collected from the culture aquarium to achieve the nominal concentrations of 5, 50, 300 and 1000 µg.L⁻¹ of Zano®10 and Zano®M suspensions. Due to the insolubility of Solaveil™ XTP₁ in water, solutions were prepared in DMSO containing 0.1% emulsifier (Emulium® Illustro, Gattefossé, Saint-Priest, France).

Each coral nubbin was immersed in individual exposure beakers (N = 5 per condition) and exposed for seven days. Unexposed controls were placed in 200 mL of FASW only, while solvent controls (DMSO+0.1% emulsifier) were used for Solaveil™ XTP₁ exposure. These solvent controls were subjected to the same metabolomic analysis as the test conditions and showed no significant differences from FASW controls (data not shown). The exposure medium was renewed daily, maintaining identical physicochemical parameters than acclimation period (salinity = 36 g.L⁻¹; pH = 8; temperature = 24 ± 1 °C), oxygen saturation was maintained by gentle air flux, and the lighting conditions were kept identical to those in coral nurseries. Daily photographs of the coral pieces were taken at a consistent time to monitor polyps retraction (SI, figures S1–S5). Corals were not fed during the exposure.

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## 2.5. Extraction and metabolomic profiling

Metabolome extraction was performed at the end of the seven days of exposure as described previously (Thorel et al., 2022). Briefly, after methanol/acetonitrile 1:1 extraction, extracts (1 mg.mL⁻¹, 2 µL injection in column) were analyzed using an Ultimate 3000™ UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Orbitrap MS/MS FT Q-Exactive focus mass spectrometer as previously described (Stien et al, 2019). Parallel Reaction Monitoring (PRM) analyses were conducted to obtain missing MS/MS data. Retention times were checked before targeted analyses. The resolution was 35.000, AGC target 5.10⁴, isolation window 0.4 Da, and stepped normalized collision energy 15/30/45 was used with 15 s dynamic exclusion. Blank injections were performed to remove irrelevant chemical background. The retention time window was set to 2–17 min. The maximum shift for alignment was 0.1 min, the maximum mass tolerance was 2 ppm, and the minimum peak intensity was 2.10⁶. For Zano®10 differential analysis, only 4 replicates were used due to a strong polyethylene glycol (PEG) derivative contamination of one of the replicates (Zano®10, 50
230 µg.L⁻¹), confirmed by comparison to a commercial standard (Polyoxyethylene glycol (10) tridecyl ether, CAS: 78330-21-9, Sigma-Aldrich). Annotation was carried out by assessing molecular formulas 231 accurately calculated on the basis of high-resolution mass spectrometry (Freestyle software 1.5, 232 Thermo Fisher Scientific) and comparing them to databases (Dictionary of Natural Products – DNP 233 (CHEMnetBASE, Taylor&Francis, Informa, London, UK), LipidMaps (https://www.lipidmaps.org/), 234 LOTUS (https://lotus.naturalproducts.net/) and SciFinderⁿ (American Chemical Society)) to offer 235 candidate compounds. MS/MS spectra were then submitted to databases for comparison (Global 236 Natural Products Social Molecular Networking – GNPS (https://gnps.ucsd.edu) (Wang et al., 2016), 237 MassBank of North America – MoNa (https://mona.fiehnlab.ucdavis.edu), and SIRIUS software (5.8.3 238 version, Lehrstuhl Bioinformatik, Jena), (Dührkop et al., 2019) or manually elucidated with 239 fragmentation pattern and structural similarities. For annotation, standards L-tryptophan (CAS: 73-22- 240 3), β-sitosterol (CAS: 83-46-5) and β-carotene (CAS: 7235-40-7) were acquired from Sigma-Aldrich, 241 and injected as 5 µg.mL⁻¹ methanolic solutions in the same conditions for comparison of retention time 242 and MS/MS spectra. 243

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245 **2.6 Statistical analysis**

246 Data processing and visualization were performed using Compound Discoverer™ software (CD 2.1, 247 ThermoFisher, Villebon, France). The relative concentrations of ions in exposed groups were 248 compared to those in control groups (as FASW condition for Zano®10 and Zano®M, and DMSO + 249 0.1% emulsifier condition for Solaveil™ XTP₁) at each tested concentration to calculate Log₂(fold 250 change) (Log₂(FC)). For Solaveil™ XTP₁, *p*-values for differences in relative ion concentrations were 251 determined using a t-test. For Zano®10 and Zano®M, *p*-values were determined using analysis of 252 variance (ANOVA) followed by Tukey's HSD post hoc test. Ions with a Log₂ (FC) > ±1 and *p*-value < 253 0.05 were initially flagged as candidates for annotation. Each selected feature was then manually 254 inspected for signal quality, annotation confidence, and consistency across replicates. This expert- 255 based approach prioritizes biologically coherent patterns across related metabolites (e.g., carotenoids, 256 lipids), which we find more robust than relying solely on statistical corrections. 257

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3. Results
259 **3.1 Mineral UV filters characterization**

260 FTIR analysis of SolaveilTM XTP₁ revealed a broad peak centered around 3400 cm⁻¹, attributed to
261 hydroxyl groups present on the alumina surface (Figure 1). Strong peaks observed at 2853 and 2920
262 cm⁻¹ correspond to the symmetric and asymmetric vibrations of CH₂ groups (Ahmad et al., 2021; Feng
263 et al., 2018). Intense absorption bands at 1435 and 1574 cm⁻¹ were assigned to the symmetric and
264 asymmetric stretching modes of bidentate carboxylate groups, characteristic of aluminum stearate
265 moieties (Egerton et al., 2005). Furthermore, the two intense peaks centered at 1535 and 1380 cm⁻¹
266 could be interpreted in two ways: they might correspond to the bending of hydroxyl residues caused
267 by moisture and the hygroscopic nature of ZnO, or alternatively to the stretching vibrations of surface-
268 bound carboxylate groups (COO⁻). The presence of carboxylate peaks on uncoated ZnO has been
269 reported in the literature and is often attributed to residual carbon-containing species from the
270 synthesis process, as shown by Xiong et al. (2006), who observed similar FTIR features on plasma-
271 synthesized ZnO nanoparticles. In our case, both Zano®10 and Zano®M are uncoated according to the
272 manufacturer, and such surface-bound carboxylates likely arise from synthesis residues or adsorption
273 from ambient air. Additionally, bands in the 400-700 cm⁻¹ region were attributed to Ti-O stretching
274 and Ti-O-Ti bridging stretching modes (Praveen et al., 2014). FTIR spectra of Zano®M and Zano®10
275 also exhibited a broad absorption band around 3400 cm⁻¹ associated with the stretching vibrations of
276 hydroxyl groups. The bands within the 400-600 cm⁻¹ region were assigned to the Zn-O stretching
277 vibrations (Mahalakshmi et al., 2020). Furthermore, two intense peaks centered at 1535 and 1380 cm⁻¹
278 could be interpreted in two ways: they may correspond to the bending vibration of hydroxyl groups
279 due to moisture absorption-reflecting the hygroscopic nature of ZnO-or, alternatively, to the stretching
280 vibrations of carboxylate (COO⁻) groups, depending on the particle synthesis method (Xiong et al.,
281 2006).
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282
<figure><img src="image_2.png" /><figcaption>Figure 1. Normalized FTIR spectra of Solaveil™ XTP1 (black), Zano®M (red), and Zano®10 (blue) recorded in powder form. For Solaveil™ XTP1, characteristic peaks at 2853 and 2920 cm⁻¹ indicate CH₂ stretches of stearate groups, while bands at 1435 and 1574 cm⁻¹ correspond to bidentate carboxylates from aluminum stearate coating. Peaks between 400–700 cm⁻¹ reflect Ti–O and Ti–O–Ti vibrations. Zano®10 and Zano®M show broad OH stretching bands (~3400 cm⁻¹) and Zn–O stretching bands in the 400–600 cm⁻¹ range.</figcaption></figure>

The morphology and size range of primary particles were determined using SEM/TEM imaging (Figure 2); results are synthesized in Table 1. Solaveil™ XTP₁ NPs has a cylindrical rounded shape, with a measured mean size of 64 ± 22 nm. Zano®M can be classified as a micromaterial with measured mean size around 244 ± 100 nm. Its exhibits various morphologies, including cylindric, hexagonal and cubic shapes. Zano®10 particles were more heterogeneous, consisting of two populations as a mix of cylindrical-shaped NPs and much larger spikes-shaped particles clearly visible on TEM and SEM images. The measured mean size around 62 ± 24 nm. DLS analysis provided hydrodynamic diameter and ZP values of suspended particle agglomerates in FASW at the highest concentration. The suspended particles of Zano®10 and Solaveil™ XTP1 exhibited similar volume-weighted hydrodynamic sizes, with mean diameters of 238 ± 19 nm and 250 ± 16 nm, respectively, while
300 Zano®M particles formed larger agglomerates of 411 ± 52 nm. The corresponding polydispersity indices (PDI) were 1 for Zano®10, 0.86 for Zano®M, and 0.33 for Solaveil™ XTP1, reflecting the highly polydisperse nature of the ZnO suspensions and the more moderate heterogeneity of the TiO₂ suspension. These values are consistent with the known aggregation behaviour of uncoated ZnO particles in seawater and support the interpretation of the DLS size data in the context of strongly polydisperse systems.

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<figure><img src="image_3.png" /><figcaption>Figure 2. Transmission electron microscopy (TEM, left) and scanning electron microscopy (SEM, right) images of Solaveil™ XTP1 (a), Zano®M (b), and Zano®10 (c) primary particles. Solaveil™ XTP1 appears as homogeneous cylindrical-rounded nanoparticles with a mean diameter of ~64 nm. Zano®M exhibits a heterogeneous morphology, including cylindrical, hexagonal, and cubic micrometric particles (~245 nm average). Zano®10 presents a bimodal distribution: small cylindrical nanoparticles (~60 nm) and larger spiky-shaped particles are clearly distinguishable. All samples show evidence of agglomeration, particularly for Zano®M and Zano®10. Scale bars represent 100 nm; magnifications: TEM ×50,000 (a,b) and ×40,000 (c); SEM ×50,000 (a,b) and ×35,000 (c).</figcaption></figure>

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<table><caption>Table 1. Mineral filters characteristics</caption><thead><tr><td>Mineral filter</td><td>Primary size (nm)</td><td>Hydrodynamic size (nm)</td><td>ZP (mV)</td><td>Morphology</td></tr></thead><tbody><tr><td>Solaveil™ XTP<sub>1</sub></td><td>64 ± 22</td><td>250 ± 16</td><td>- 6.3</td><td>Cylindrical rounded</td></tr><tr><td>Zano®10</td><td>62 ± 24</td><td>238 ± 19</td><td>- 9.6</td><td>Mix of cylindric and spike</td></tr></tbody></table>
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## 3.2 Compared effect of mineral filters on coral metabolomes

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Differential metabolomic analysis comparing exposed and control *P. damicornis* extracts revealed 44 ions with significantly altered relative concentrations (*p* < 0.05, Log₂(FC) > 1 or < -1) after seven days of exposure to TiO₂ and ZnO. These included 2 ions for SolaveilTM XTP₁, 18 ions for Zano®M, and 24 ions for Zano®10 exposure. All MS and MS/MS data used for metabolite annotations are provided as Supplementary Data (SI, figures S6-S69). Exposure to TiO₂-based product SolaveilTM XTP₁ induced only minimal changes in the coral metabolome, with the relative concentration of a single compound, namely monogalactosyldiacylglycerol (MGDG) C₁₈:₁/C₁₆:₀, showing a modest increase (*p* = 2.6 × 10⁻², Log₂(FC) = 1.82) (SI, Figures S6–S8). In contrast, Zano®10 exposure led to more pronounced effects: 24 altered ions were manually annotated as 15 distinct metabolites, detailed in Table 2.

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Compounds **1-9** were annotated as pigments. The fragmentation patterns of compounds **1** and **2** were characteristic of carotenoids and matched the MS/MS fragmentation of a β-carotene standard (SI, figure S69). These compounds were annotated as two isomers of pyrrhoxanthin, with the molecular formula C₃₉H₄₈O₆ (calculated *m/z* for C₃₉H₄₉O₆⁺ 613.3524, 16 double-bond equivalents), as previously described by Clergeaud et al. (2023). The structural similarity of pyrrhoxanthin to peridinin, along with comparable fragmentation profiles (Venn et al., 2006), suggests these compounds may be structurally related to peridinin.

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Compounds **3-5** ionized as radical cations with a molecular formula of C₄₀H₅₄O₂ (calculated *m/z* for C₄₀H₅₄O₂⁺ 566.4118, 14 double-bond equivalents). According to the DNP database, this formula corresponds to several natural compounds, including β-carotene derivatives, diatoxanthin, and eschscholtzxanthin. Based on compound class proposals from SIRIUS and CANOPUS (classification scores of **77%**, 54% and 66%), these ions were annotated as three isomeric xanthophylls. Their ionization as cation radicals in ESI⁺ mode and the successive loss of methyl radicals in the MS/MS spectra (e.g., *m/z* 566.411 to 551.388, and *m/z* 474.347 to 459.324) indicate the presence of methyl substituents near a conjugated π system, consistent with observation by Crupi et al. (2010).
Compounds **6-9** exhibited MS/MS patterns indicative of chlorophyll-derivatives. Each showed a phorbine
core (with four nitrogen atoms) and prominent losses of methyl and carbonyl groups. These patterns
suggested a dephytylated chlorophyl structure, further supported by matches to molecular formulas in
the DNP database. The only known natural compound matching the molecular formula of compound 6
is pyrophaeoporphyrin c', a derivative of chlorophyll c' described by Harradine and Maxwell (1998).
Compound 7 shared its formula with 13²,17³-cyclopheophorbide a enol, previously isolated from the
zooxanthellae of Montipora digitata (Suzuki et al., 2015). Isomeric compounds 8 and 9 had the same
molecular formula as chlorophyllone a, a recognized chlorophyll a degradation product (Porphyrin
91% CANOPUS classification score) (Sakata et al., 1990). Based on their fragmentation patterns and
structural predictions by SIRIUS, compounds 6-9 were annotated as chlorophyll degradation products.

The second group of annotated compounds (10-12) was identified as membrane lipid metabolites
based on their molecular formulas and manual interpretation of their MS/MS fragmentation patterns.
Compounds 10 and 11 were manually annotated as a ceramide and a glycosphingolipid (GSL)
respectively, based on characteristic fragmentations of their fatty acid chains and dehydration events.
Compound 10 (C₃₈H₇₁NO₃, calculated m/z for C₃₈H₇₁NO₃Na⁺ 612.5326, 4 degrees of unsaturation) was
annotated as follows: the collision-induced dissociation (CID) spectrum of [10+H–H₂O]⁺ at m/z
572.5403 revealed a second water loss yielding a product at m/z 554.5308 (Figure S25). The same
precursor ion also generated a fragment at m/z 312.3265, corresponding to a protonated C₂₀:₀ amide,
and another at m/z 278.2482 (C₁₈H₃₂NO⁺), indicative of the loss of the C20:0 chain. This ion
subsequently lost water (m/z 260.2371, C₁₈H₃₀N⁺) and ammonia (m/z 260.2371, C₁₈H₂₇⁺) confirming
the annotation of compound 10 as a ceramide featuring a C₁₈:₃;O₂ sphingoid base and a C₂₀:₀;O fatty
acid. Similarly, compound 11 was identified as a GSL based on a visible hexose loss in its MS/MS
spectrum (m/z 770.6182 to 590.5520, – C₆H₁₂O₆), along with two specific products at m/z 328.3219
(C₂₀H₄₂NO₂⁺) and 280.2635 (C₁₈H₃₄NO⁺) attributed to an N-linked hydroxylated C₂₀:₀;O₂ moiety and a
dehydrated C₁₈:₂ sphingoid base, respectively. It was therefore annotated as HexCer C₁₈:₂;O/C₂₀:₀;O₂.

An ion at m/z 397.3828 (calculated. for C29H47+, 397.3829) was found at higher relative concentration
in exposed coral. The MS/MS spectrum matched that of β-sitosterol in the GNPS database. To
investigate further, the MS/MS spectrum of compound 12 was compared with that of a protonated
dehydrated β-sitosterol standard (SI, Figure S30). Despite a strong spectral similarity, the shorter
retention time of compound **12** suggested it be a more polar β-sitosterol derivative. Additionally, no adduct of the molecular ion was observed in the mass spectrum, limiting the accuracy of its structural annotation. Compounds **13-15** exhibited fragmentation patterns similar to those of compound **12** and the β-sitosterol standard, suggesting they were also steroids. Their molecular formulas matched several known sterol derivatives. This hypothesis is supported by previous reports on ergosterol derivatives in *Pocillopora damicornis* with comparable MS/MS spectra (Stien et al., 2020).

The fragmentation of these compounds was dominated by successive losses of water molecules. Compound **13**, with a molecular formula of C₂₉H₅₀O₄ (calcd. for C₂₉H₅₀O₄Na⁺, 485.3601, 5 degrees of unsaturation), exhibited three dehydration steps, as evidenced by fragments at m/z 445.3678 [M+H−H₂O]⁺, m/z 427.3572 [M+H−2H₂O]⁺, and m/z 409.3457 [M+H−3H₂O]⁺. Interestingly, a polyhydroxylated steroid with the same formula was previously isolated from the soft coral *Litophyton mollis* (Zovko Končić et al., 2016).

Compound **14** (C₃₅H₆₀O₉, calcd. m/z for C₃₅H₆₀O₉Na⁺ 647.4130, 6 degrees of unsaturation) is likely a heterosidic derivative of compound **13**. Its protonated molecular ion fragmented in the source to yield a product ion at m/z 445.3677, corresponding to the [M−C₆H₁₂O₆+H]⁺ ion. This ion exhibited the same collision-induced fragmentation spectrum as [13−H₂O+H]⁺. Compound **15** (C₂₉H₅₀O₅, calcd. m/z for C₂₉H₄₉O₄⁺ 461.3626, 5 degrees of unsaturation) displayed two dehydration sites, as indicated by the ions at m/z 461.3626 [M+H−H₂O]⁺ and m/z 443.3520 [M+H−2H₂O]⁺. Given its formula and dehydration pattern, compound **15** was also considered as a steroid, consistent with known natural products.

The relative concentration of all annotated pigments (**1-9**) significantly decreased following exposure to Zano®10 exposure at 300 µg.L⁻¹ and above, with Log₂(FC) values ranging from - 1.6 to - 4.5. Membrane lipids metabolites (**10-12**) also showed moderate decreases starting at 1000 µg.L⁻¹, with Log₂(FC) values ranging from -1.1 to -2.1. In contrast, steroids **13-15** showed significantly increased relative concentrations at the highest Zano®10 concentrations tested, with Log₂(FC) values ranging from 4.50 to 5.36.
<table><caption>Table 1. List of P. damicornis metabolites with significantly altered relative concentrations after 7 days of exposure to 5, 50, 300, 1000 µg.L⁻¹ of Zano®10, compared to control conditions (Log₂(FC) > 1 or < -1, p < 0.05).</caption><thead><tr><th rowspan="2">Cmpd.<br>No.</th><th colspan="4">Log₂(FC) (µg.L⁻¹)ª</th><th rowspan="2">RT<br>(min)</th><th rowspan="2">Adduct</th><th rowspan="2">Exp. m/z</th><th rowspan="2">Th. m/z</th><th rowspan="2">Molecular<br>formula</th><th rowspan="2">Compound<br>class</th><th rowspan="2">Annotationᵇ</th></tr><tr><th>5</th><th>50</th><th>300</th><th>1000</th></tr></thead><tbody><tr><td colspan="12"><strong>Pigments</strong></td></tr><tr><td>1</td><td>-0.72</td><td>-1.13</td><td>-4.23</td><td>-4.2</td><td>11.06</td><td>[M+Na]⁺</td><td>635.335</td><td>635.3343</td><td>C₃₉H₄₈O₆</td><td>Carotenoid</td><td>Pyrrhoxanthin<br>or isomer</td></tr><tr><td>2</td><td>-0.91</td><td>-0.75</td><td>-3.69</td><td>-3.64</td><td>10.93</td><td>[M+H]⁺</td><td>613.352</td><td>613.3524</td><td>C₃₉H₄₈O₆</td><td>Carotenoid</td><td>Pyrrhoxanthin<br>or isomer</td></tr><tr><td>3</td><td>-0.57</td><td>-0.62</td><td>-1.81</td><td>-1.65</td><td>12.07</td><td>[M]⁺</td><td>566.4113</td><td>566.4118</td><td>C₄₀H₅₄O₂</td><td>Carotenoid</td><td>Xanthophyll</td></tr><tr><td>4</td><td>-0.62</td><td>-0.54</td><td>-1.95</td><td>-1.71</td><td>12.46</td><td>[M]⁺</td><td>566.412</td><td>566.4118</td><td>C₄₀H₅₄O₂</td><td>Carotenoid</td><td>Xanthophyll</td></tr><tr><td>5</td><td>-0.75</td><td>-0.72</td><td>-1.64</td><td>-1.77</td><td>12.65</td><td>[M]⁺</td><td>566.4119</td><td>566.4118</td><td>C₄₀H₅₄O₂</td><td>Carotenoid</td><td>Xanthophyll</td></tr><tr><td>6</td><td>-0.55</td><td>-1.6</td><td>-4.49</td><td>-3.95</td><td>11.32</td><td>[M+H]⁺</td><td>529.2235</td><td>529.2234</td><td>C₃₃H₂₈N₄O₃</td><td>Chlorophyll<br>derivative</td><td>n.d.</td></tr><tr><td>7</td><td>-0.74</td><td>-1</td><td>-2.55</td><td>-2.87</td><td>15.28</td><td>[M+H]⁺</td><td>561.2494</td><td>561.2496</td><td>C₃₄H₃₂N₄O₂</td><td>Chlorophyll<br>derivative</td><td>n.d.</td></tr><tr><td>8</td><td>-1.09</td><td>-1.3</td><td>-3.34</td><td>-3.36</td><td>10.45</td><td>[M+H]⁺</td><td>533.2547</td><td>533.2547</td><td>C₃₃H₃₂N₄O₃</td><td>Chlorophyll<br>derivative</td><td>n.d.</td></tr><tr><td>9</td><td>-1.08</td><td>-1.24</td><td>-3.19</td><td>-3.44</td><td>10.61</td><td>[M+H]⁺</td><td>533.2548</td><td>533.2547</td><td>C₃₃H₃₂N₄O₃</td><td>Chlorophyll<br>derivative</td><td>n.d.</td></tr><tr><td colspan="12"><strong>Membrane lipids</strong></td></tr><tr><td>10</td><td>-0.58</td><td>-0.48</td><td>-0.35</td><td>-1.05</td><td>16.22</td><td>[M+Na]⁺</td><td>612.5324</td><td>612.5326</td><td>C₃₈H₇₁NO₃</td><td>Ceramide</td><td>Cer<br>C₁₈:₃;O₂/C₂₀:₀;O<br>HexCer</td></tr><tr><td>11</td><td>-1.34</td><td>-0.56</td><td>-0.73</td><td>-2.08</td><td>14.12</td><td>[M+Na]⁺</td><td>792.5955</td><td>792.596</td><td>C₄₄H₈₃NO₉</td><td>GSL</td><td>C₁₈:₂;O/C₂₀:₀;O₂<br>β-sitosterol<br>derivative</td></tr><tr><td>12</td><td>-0.15</td><td>0.32</td><td>0.03</td><td>-1.81</td><td>11.43</td><td>Frag. (C₂₉H₄₇⁺)</td><td>397.3828</td><td>397.3829</td><td>n.d.</td><td>Steroid</td><td></td></tr><tr><td colspan="12"><strong>Steroids (putative ergosterol analogs)</strong></td></tr><tr><td>13</td><td>-0.96</td><td>2.67</td><td>1.75</td><td>4.49</td><td>10.67</td><td>[M+Na]⁺</td><td>485.3603</td><td>485.3601</td><td>C₂₉H₅₀O₄</td><td>Steroid</td><td>n.d.</td></tr><tr><td>14</td><td>-0.53</td><td>2.25</td><td>1.2</td><td>4.5</td><td>8.56</td><td>[M+Na]⁺</td><td>647.4131</td><td>647.4130</td><td>C₃₅H₆₀O₉</td><td>Steroid</td><td>n.d.</td></tr><tr><td>15</td><td>0.2</td><td>2.27</td><td>1.04</td><td>5.36</td><td>9.62</td><td>[M+H-H₂O]⁺</td><td>461.3625</td><td>461.3626</td><td>C₂₉H₅₀O₅</td><td>Steroid</td><td>n.d.</td></tr></tbody></table>

ªA color gradient is applied to the Log₂(FC) values, ranging from dark green to yellow for metabolites whose relative concentration decreased (Log₂(FC) < -1), and from light orange to dark red for those whose relative concentration increased (Log₂(FC) > +1) after exposure to Zano®10. The color is only applied at exposure concentrations that have induced significant variation (p < 0.05). ᵇFor the ceramide and the glycosphingolipid (GSL), chain lengths were visible in the MS/MS spectra but the position of double bonds in the sphingoid base remained speculative and is therefore deliberately omitted in the annotations. n.d.: not determined.

A total of 12 metabolites with altered relative concentration after exposure to Zano®M were annotated and are listed in Table 3. These include pyrrhoxanthins **1-2** and **GSL 11** already annotated above. Compound **16** MS/MS spectrum was similar to that of compound **11**, allowing for the annotation of **16** as the GSL HexCer C₁₉:₂;O/C₁₆:₀;O₂. Compound **17** was annotated as the DGDG C₁₈:₂/C₁₆:₀ as in previous work (Marcellin-Gros et al., 2020). Compounds **18-20** were manually annotated based on their characteristic fragments at m/z 104.1070 and 132.1021 visible in the [M+H]⁺ MS/MS spectrum
as well as the visible loss of C₃H₉N in the [M+Na]⁺ MS/MS spectrum (Marcellin-Gros et al., 2020).

Eventually, compound **18** was annotated as the lyso-DGCC C₂₂:₆, **19** as the DGCC C₂₂:₆/C₂₂:₆, and **20** as the DGCC C₁₆:₀/C₁₈:₄. Compound **21** matched to LPC C₂₂:₆ in the GNPS database. This assertion is corroborated by the observation of a phosphocholine ion at *m/z* 184.0736 in the MS/MS fragmentation spectrum of its protonated molecular ion (Stien et al., 2020). Compound **22** was annotated as L-tryptophan. The annotation was confirmed by comparison with an analytical standard (SI, Figures S59-60). Compounds **23** and **24** remained unidentified but appeared to be analogs of each other. Overall, the coral metabolome was significantly affected by exposure to Zano®M only at the highest tested concentration of 1000 µg.L⁻¹.

Lastly, the only compound whose relative concentration was affected following exposure to Solaveil™ XTP₁ was annotated as MGDG C₁₈:₁/C₁₆:₀, already observed in a previous publication (Marcellin-Gros et al., 2020). As the alteration of a single metabolite does not constitute a significant response signal, it can be concluded that Solaveil™ XTP₁ had no measurable effect on the metabolome of adult corals at 1000 µg.L⁻¹ exposure.

<table><caption>Table 2. List of P. damicornis metabolites with significantly altered relative concentrations after 7 days of exposure to 5, 50, 300, 1000 µg.L⁻¹ of Zano®M, (Log₂(FC) > 1 or < -1, p < 0.05).</caption><thead><tr><th rowspan="2">Cmpd.<br>No.</th><th colspan="4">Log₂(FC) (µg.L⁻¹)ª</th><th rowspan="2">RT<br>(min)</th><th rowspan="2">Adduct</th><th rowspan="2">Exp. m/z</th><th rowspan="2">Th. m/z</th><th rowspan="2">Molecular<br>formula</th><th rowspan="2">Compound<br>classᵇ</th><th rowspan="2">Annotationᶜ</th></tr><tr><th>5</th><th>50</th><th>300</th><th>1000</th></tr></thead><tbody><tr><td colspan="12"><strong>Pigments</strong></td></tr><tr><td>1</td><td>0.87</td><td>0.03</td><td>-1.61</td><td>-1.55</td><td>10.66</td><td>[M+H]⁺</td><td>613.3523</td><td>613.3524</td><td>C₃₉H₄₈O₆</td><td>Carotenoid</td><td>Pyrrhoxanthin or<br>isomer</td></tr><tr><td>2</td><td>0.66</td><td>0.09</td><td>-1.08</td><td>-2.58</td><td>10.48</td><td>[M+H]⁺</td><td>613.3523</td><td>613.3524</td><td>C₃₉H₄₈O₆</td><td>Carotenoid</td><td>Pyrrhoxanthin or<br>isomer</td></tr><tr><td colspan="12"><strong>Membrane lipids</strong></td></tr><tr><td>11</td><td>-0.27</td><td>-0.21</td><td>-0.24</td><td>-1.25</td><td>15.04</td><td>[M+Na]⁺</td><td>792.5963</td><td>792.596</td><td>C₄₄H₈₃NO₉</td><td>GSL</td><td>HexCer<br>C₁₈:₂;O/C₂₀:₀;O₂</td></tr><tr><td>16</td><td>-1.24</td><td>-0.21</td><td>-0.4</td><td>-1.02</td><td>12.14</td><td>[M+Na]⁺</td><td>750.5475</td><td>750.549</td><td>C₄₁H₇₇NO₉</td><td>GSL</td><td>HexCer<br>C₁₉:₂;O/C₁₆:₀;O₂</td></tr><tr><td>17</td><td>2.98</td><td>2.93</td><td>1.08</td><td>3.66</td><td>13.28</td><td>[M+Na]⁺</td><td>939.6013</td><td>939.602</td><td>C₄₉H₈₈O₁₅</td><td>DGDG</td><td>DGDG C₁₈:₂/C₁₆:₀</td></tr><tr><td>18</td><td>-1.18</td><td>-0.79</td><td>-0.53</td><td>-1.17</td><td>7.06</td><td>[M+H]⁺</td><td>562.3738</td><td>562.374</td><td>C₃₂H₅₁NO₇</td><td>LDGCC</td><td>LDGCC C₂₂:₆</td></tr><tr><td>19</td><td>-0.61</td><td>-0.6</td><td>-0.18</td><td>-1.03</td><td>11.99</td><td>[M+H]⁺</td><td>872.6042</td><td>872.604</td><td>C₅₄H₈₂NO₈</td><td>DGCC</td><td>DGCC C₂₂:₆/C₂₂:₆</td></tr><tr><td>20</td><td>-0.07</td><td>0.1</td><td>0.3</td><td>1.38</td><td>12.73</td><td>[M+H]⁺</td><td>748.5729</td><td>748.572</td><td>C₄₄H₇₇NO₈</td><td>DGCC</td><td>DGCC C₁₆:₀/C₁₈:₄</td></tr><tr><td>21</td><td>-0.81</td><td>-0.49</td><td>-0.83</td><td>-1.05</td><td>7.45</td><td>[M+H]⁺</td><td>568.3398</td><td>568.340</td><td>C₃₀H₅₀PNO₇</td><td>LPC</td><td>LPC C₂₂:₆</td></tr><tr><td colspan="12"><strong>Amino acid</strong></td></tr><tr><td>22</td><td>-0.72</td><td>-0.45</td><td>-0.42</td><td>-1.03</td><td>4.22</td><td>[M+H]⁺</td><td>205.0971</td><td>205.097</td><td>C₁₁H₁₂N₂O₂</td><td>Amino acid</td><td>L-Tryptophane</td></tr><tr><td colspan="12"><strong>Unidentified</strong></td></tr><tr><td>23</td><td>-0.63</td><td>-0.39</td><td>0.02</td><td>-1.5</td><td>14.52</td><td>[M+Na]⁺</td><td>571.4331</td><td>571.433</td><td>C₃₄H₆₀O₅</td><td>n.d</td><td>n.d</td></tr></tbody></table>
A color gradient is applied to the $Log_2(FC)$ values, ranging from dark green to yellow for metabolites whose relative concentration decreased ($Log_2(FC) < -1$), and from light orange to dark red for those whose relative concentration increased ($Log_2(FC) > +1$) after exposure to Zano®M. The color is only applied at exposure concentrations that have induced significant variation (p < 0.05). DGDG: digalactosyldiacylglycerol, LDGCC: lysodacylglycerol carboxyhydroxymethylcholine, DGCC: diacylglycerol carboxyhydroxymethylcholine, LPC: lysophosphatidylcholine, GSL: glycosphingolipid, nd: non determined. For DGDG, LDGCC, DGCC, GSL and LPC, chain lengths were visible in the MS/MS spectra but the position of double bonds remained speculative and is, therefore, deliberately omitted in the annotations.

## 4. Discussion

The present study investigated the effects of three mineral UV filters using an untargeted metabolomic approach, complemented by detailed particle characterization, to provide a more comprehensive understanding of their potential mechanisms of toxicity. Each mineral filter showed a tendency to form agglomerates with low negative zeta potential (ZP) values, approximating those of the FASW medium, indicating weak electrostatic repulsion and a high likelihood of aggregation. The formation of agglomerates in FASW was further confirmed by hydrodynamic size measurements, with agglomerate diameters exceeding 200 nm for Solaveil™ XTP₁ and Zano®10 NPs, and 400 nm for Zano®M. The fate of these agglomerates may vary depending on their intrinsic physical and chemical properties.

For Solaveil™ XTP₁ NPs, it is likely that the agglomerates did not interact substantially with the exposed corals, as no polyp retraction was observed after seven days of exposure (SI, figure S1). Polyp retraction is a morphological colony-scale response observed in corals under stress, acting as a protective mechanism to limit contact with adverse environmental conditions (Bejarano et al., 2022). This behavior has also been reported in previous studies involving exposure to UV filters (Miller et al., 2022; Stien et al., 2019). This limited interaction is further corroborated by the negligible impact of Solaveil™ XTP₁ on the coral metabolome. The minimal effect may be attributed to the buoyant properties of the particles in FASW. Slomberg et al. (2021) reported that $TiO_2$ particles coated with aluminum and stearic acid remain at the water surface due to their hydrophobic coating. Consistently,
464 TiO₂ quantification in marine ecosystems has shown peak concentrations near the surface (Labille et al., 2020; Thallinger et al., 2023; Tovar-Sánchez et al., 2013). These findings suggest that Solaveil™ 465 XTP₁ may not reach coral nubbins for seven days exposure, explaining the limited metabolic and 466 behavioral responses observed. In contrast, Zano®10 and Zano®M exposure resulted in marked coral 467 responses with full polyp retraction observed within 24 hours at 1000 µg.L⁻¹ (SI, Figures S2-S3). Once 468 dispersed in FASW, these agglomerates likely sediment through the water column, possibly causing 469 mechanical stress via direct physical contact with the coral surface or through ingestion, ultimately 470 reaching the gastrodermal tissues (Tang et al., 2017; Yuan et al., 2023). In addition, these particles 471 may release Zn²⁺ ions into the water, contributing to chemical toxicity (Freitas Neto and Espósito, 472 2023). 473

474

475 Untargeted metabolomics analysis allowed the annotation of various metabolites affected by the two 476 ZnO particles, revealing distinct trends. Significant metabolome shifts were mainly observed for 477 Zano®10 which led to a decrease in the relative abundance of several pigments including carotenoids, 478 xanthophylls and chlorophylls metabolites at 300 and 1000 µg.L⁻¹. Most of these pigments originate 479 from Symbiodiniaceae and are essential for photosynthetic energy production (Kopp et al., 2015). 480 Variations in photosynthetic pigment levels are commonly used as reliable indicators of 481 environmentally induced stress in corals (McDougall et al., 2006; Scheufen et al., 2017). The major 482 light-harvesting pigments in Symbiodiniaceae include peridinin and chlorophylls, which bind to 483 antennae proteins to form Peridinin-Chlorophyll Proteins (PCPs) (Jiang et al., 2012). Chlorophyll in 484 particular was widely used as a bioindicator to assess the density of unicellular photosynthetic 485 organisms such as *Symbiodinium* spp. (Ramsby et al., 2014; Zhou et al., 2022). Furthermore, 486 decreased levels of pyrrhoxanthin, a structural analog of peridinin, were previously reported in *P. 487 damicornis* exposed to 300 and 1000 µg/L of avobenzone (butyl methoxydibenzoylmethane), 488 indicating potential disruption of symbiont integrity (Clergeaud et al., 2023). In the present work, the 489 marked loss of photosynthetic pigments following Zano®10 exposure suggested a direct damage to the 490 PCP complex or a reduction in Symbiodiniaceae density, suggesting impairment of the photosynthetic 491 machinery. In contrast, Solaveil™ XTP₁ had no detectable impact on the pigmental composition. A 492 decrease in pyroxanthin derivatives was also observed in corals exposed to 1000 µg.L⁻¹ Zano®M.
These results are consistent with literature reporting stronger impacts of ZnO particles compared to coated TiO₂. Corinaldesi et al. (2018) showed that exposure to 6.3 mg.L⁻¹ of uncoated ZnO NPs (20 to 200 nm) induced massive expulsion of damaged zooxanthellae in *Acropora* spp. within 24 hours. Conversely, lesser effects were reported with coated TiO₂ NPs (Eusolex® T2000 and Optisol™), where only minor zooxanthellae release and no visible bleaching were observed after 48 hours.

Fel et al. (2019) also observed reduced photosynthetic efficiency in *Stylophora pistillata* exposed to 100 µg.L⁻¹ ZnO for 35 days, suggesting damage to photosystem II (PSII), although the particle characteristics were not fully defined. Similar observations were made by Chen et al. (2025), who investigated the acute toxicity of ZnO (800 µg.L⁻¹, 20–40 nm) and TiO₂ (30 mg.L⁻¹, 40–60 nm) NPs on *Galaxea fascicularis* coral for 48 hours, revealing a significant impairment of Symbiodiniaceae functions, characterized by a decrease in chlorophyll content and a reduction in photosynthetic capacity for both NPs. These effects were associated with oxidative stress-induced damage, with ZnO NPs exhibiting a more pronounced deleterious impact despite their lower concentration. In this study, the metabolomic profile of corals exposed to ZnO®10 indicate early signs of bleaching, reinforcing the idea that nanoparticle size plays a critical role in toxicity. ZnO®M, being larger particles, was comparatively less toxic. The relationship between size and toxicity has been well-documented in photosynthetic organisms such as algae: smaller particles have a larger surface area, which may facilitate interaction with or uptake into cell membranes, leading to cellular deformation and increased permeability (Chen et al., 2012; Djearamane et al., 2019), reduced pigment contents (Djearamane et al., 2018), and metabolic deficiency (Elrefaey et al., 2021), all of which can precede potential damage to the photosynthetic apparatus. Moreover, the dissolution rate of uncoated metal oxide NPs like ZnO is size-dependent, with smaller particles dissolving faster and releasing more bioavailable Zn²⁺ ions (Wu et al., 2019). Smaller NPs dissolve more quickly, release bioavailable Zn²⁺ ions into the medium, and affect the photosynthetic homeostasis of exposed organisms. Trace concentrations of Zn²⁺ ions are essential enzyme cofactors and play a key role in photosynthetic efficiency of corals symbionts (Ferrier-Pagès et al., 2005), while excessive concentrations can adversely affect this biological function and enhance bleaching process. Kuzminov et al. (2013) demonstrated that Zn accumulation can lead to electron transport and growth inhibition in *Symbiodinium* spp. after two days of exposure to 100 µM of Zn. Zn²⁺ poisoning has also had a detrimental effect on the pigment composition of
marine microalgae, as evidenced by a decrease in chlorophyll a content in *Chlorella marina*, *Isochrysis galbana*, *Tetraselmis* sp., *Nannochloropsis* sp. and *Dunaliella salina* following 15 days exposure to 500 ppm of dissolved Zn (Dinesh et al., 2014). In symbiotic cnidarians, zinc is primarily absorbed by the zooxanthellae, as shown by Hardefeldt and Reichelt-Brushett (2015) in the sea anemone *Exaiptasia pallida*, and by Reichelt-Brushett and McOrist (2003) in the hermatypic coral *Acropora tenuis*. This partitioning acts as a metal regulation mechanism but may also contribute to bleaching via metabolic deterioration of cells or the enhanced expulsion of Zn²⁺ contaminated zooxanthellae. Both particulate and ionic forms of zinc can trigger the formation of reactive oxygen species (ROS) in photosynthetic organisms, leading to oxidative damage that impairs cellular components, and compromises structural integrity (Vicente et al., 2018). Chen et al. (2025) observed a significant increase in ROS activity in *Galaxea fascicularis* after 48 hours of exposure to both 800 µg·L⁻¹ ZnO nanoparticles and 30 mg·L⁻¹ TiO₂ in coral tissues, suggesting a potential contribution of ROS to the bleaching effects observed for both tested NPs. ROS production is a well-documented factor in coral bleaching, particularly in response to thermal stress (Lesser, 2019; Strohecker et al., 2021), may contribute to the observed changes in pigment or lipid profiles.

Zano®M exhibited a weaker effect on coral metabolome, with only moderate variations observed at the highest concentration tested (1000 µg·L⁻¹). The metabolomic analysis showed that exposure primarily affected the relative concentrations of membrane lipids in Symbiodiniaceae. In addition to pigments, corals possess a diverse and complex lipid profile, with varying structures and ratios depending on the taxa of both symbiotic zooxanthellae and coral host (Reddy et al., 2023). For Zano®10 exposure, the few membrane lipids annotated as symptomatic of exposure were one ceramide, one GSL, and four phytosteroids. In contrast, Zano®M exposure induced a broader variation in lipid metabolites (11-21), involving metabolites specifically associated with the *Symbiodinium* spp. membrane lipidome. Symbiont membrane properties and integrity are strongly influenced by lipid composition, which can be modulated in response to environmental pressures, especially during coral bleaching events (Ermolenko and Sikorskaya, 2021; Sikorskaya et al., 2020; T. Botana et al., 2022; Tang et al. 2014a), but this phenomenon has also been observed as an acclimatization response in scleractinian corals under chemical stress (Tang et al. 2014b). Variation in betaine lipids, such as DGCCs, may indicate a
reorganization of the symbiont's membrane lipids. These metabolites are often associated with the thermotolerance capacity of different zooxanthellae clades (Rosset et al. 2019; Roach et al. 2021), particularly by modulating the structure of the cellular membrane (Pei et al., 2022). In a context of particles exposure, Tang et al. (2017) highlighted cellular adaptation to mechanical disturbances in the coral *Seriatopora caliendrum* exposed to uncoated ZnO NPs (50–70 nm). They observed changes in phosphatidylcholine content at 50 µg.L⁻¹, but only in response to particles exposure and not to dissolved Zn²⁺ ions. Additionally, they identified a variation in LPC 22:6 (compound **21**) relative concentration, a metabolite found in tissues of various species, including corals (Tang et al., 2014b; Williams et al., 2024), where it may influence membrane fluidity and permeability, particularly in response to NP-induced physical damages. Unlike Zano®10 NPs, which significantly affected pigment content, Zano®M particles seem to primarily interacted mechanically with the coral surface, accounting for the observed variation in lipid relative concentrations. The larger agglomerates formed by Zano®M may have reduced the contact area between the particles and the coral while also promoting sedimentation, thereby decreasing their bioavailability.

Other metabolites were annotated in this study, including three polar ergosterol derivatives, whose relative concentration increased following Zano®10 exposure. Symbiotic cnidarians acquire sterols from their symbionts and/or diet, which are essential for cell maintenance and immune signaling (Hambleton et al., 2019; Lu et al., 2020). Ergosterol derivatives are widespread across the animal kingdom, including in soft corals (Savić et al., 2022), and play diverse roles due to their highly variable structures and involvement in multiple metabolic pathways. However, interpreting their biological significance remains challenging, as sterol functions in corals are not yet well understood, and their precise structures have not been identified. Increased steroids relative concentration was previously associated to an inflammatory stress response of *P. damicornis* exposed to the UV filter ethylhexyl salicylate (Stien et al., 2020). Notably, this study did not identify the same steroids or other metabolites previously associated with an inflammatory response in corals. This suggests that Zano®10 toxicity does not affect the same partner within the coral symbiosis. Based on the variations in the relative concentrations of pigments, it appears that Zano®10 toxicity primarily targets Symbiodiniaceae.
Finally, a slight decline in the relative concentration of the amino acid L-tryptophan was observed following Zano®M exposure. Tryptophan biosynthesis is primarily associated with the Symbiodiniaceae (Liu et al., 2018). In photosynthetic organisms, tryptophan is commonly described as a precursor to the phytohormone indole-3-acetic acid, which plays a key role in growth and development (Palacios et al., 2016). While a single metabolite within a class provides only a weak signal, this observation suggests potential disruptions in Symbiodiniaceae metabolism.

## Conclusion

The bleaching potential of mineral filters such as ZnO and TiO₂ particles has been well-documented in several studies. The present findings provide new insights into the potential toxicity mechanisms of these filters in relation to their physicochemical properties. This study revealed distinct effects of the mineral filters Solaveil™ XTP₁ (TiO₂ NPs), Zano®10 (ZnO NPs) and Zano®M (ZnO particles) on *P. damicornis* after seven days of exposure, as evidenced by alterations in the metabolite profile of the symbiotic zooxanthellae. Solaveil™ XTP₁ induced minimal changes in the coral metabolome, likely due to its hydrophobic properties that may have limited its interaction with the corals. In contrast, Zano®10 exhibited the highest toxicity, significantly reducing the relative concentration of several photosynthetic pigments at 300 and 1000 µg.L⁻¹. Zano®M, on the other hand, caused only mild effects, primarily indicative of cell membrane remodeling at 1000 µg.L⁻¹.

Further investigations into the functional biological effects of ZnO, particularly on the symbiont, could provide deeper insights into its toxicity mechanisms and contribute to assessing the safety of these compounds. While nominal concentrations were used to reflect realistic exposure scenarios, we acknowledge that quantifying the actual concentrations of dissolved Zn²⁺ or total particulate matter in the medium would have provided deeper insights into the relative contributions of ionic versus particulate toxicity. Dissolved zinc analysis typically relies on ultrafiltration followed by inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS), sometimes after dialysis or centrifugal separation (Li et al., 2013; Wu et al., 2019). However, the implementation of such techniques requires labor-intensive protocols and specialized equipment, especially when handling dynamic nanoparticle suspensions in complex media such as seawater. Given the already
609 broad scope of this study, we suggest addressing this question in future work to better distinguish the
610 roles of ZnO particles and dissolved zinc in coral ecotoxicology. Given the anticipated rise in the use
611 of mineral filters, continued investigations into their effects, especially considering their
612 physicochemical characteristics, remains crucial.

613

614 **Data availability**

615 The mass spectrometry data can be accessed on the European research data repository Zenodo at
616 https://zenodo.org/uploads/15167165, which is publicly available.

617 Acknowledgments.

618 This work was carried out with the financial support of the Agence Nationale de la Recherche, grant
619 number ANR21-CE34-0027, SPOC project (Sunscreen Pollution on Coral). Preliminary work was
620 financed by Pierre Fabre Dermo Cosmétique as part of their commitment to sunscreens that protect the
621 skin and are more respectful of the oceans.

622 Authors thank the BIO2MAR (http://bio2mar.obs-banyuls.fr), BioPIC (https://www.embrc-france.fr)
623 and EnRMAT (https://enrmat.univ-perp.fr) platforms for providing technical support and access to
624 instrumentation for metabolomic profiling and particle characterization; the Softmat platform
625 (https://softmat.fr) for DLS analysis; the European Marine Biological Resource Center (EMBRC) at
626 the Observatoire Océanologique de Banyuls, France, for providing access to the Banyuls aquarium
627 facilities. Finally, the authors also would like to thank Rémi Pillot and Pascal Romans (Sorbonne
628 Université) for their help in coral rearing.

629 Author contributions.

630 CG: Investigation, Formal analysis, Writing – Original draft, Writing – Review & Editing

631 MG: Supervision, Validation, Writing – Original draft, Writing – Review & Editing

632 FC: Investigation

633 ET: Investigation

634 LC: Investigation – Review & Editing
635 LM: Writing – Review and Editing

636 PL: Conceptualization, Supervision, Project administration

637 EH: Supervision, Validation, Writing – Original draft, Writing – Review & Editing

638 DS: Conceptualization, Methodology, Validation, Supervision, Original draft, Review & Editing,

639 Project administration

640 All authors contributed to and approved the final manuscript.

641

642 Competing interests

643 Preliminary work reported in this article was by Pierre Fabre Dermo Cosmétique as part of their

644 commitment to sunscreens that protect the skin and are more respectful of the oceans. It was neither

645 supervised nor audited by Pierre Fabre Laboratories. The interpretations and views expressed in this

646 manuscript do not reflect those of the company.

647

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