# Ranking the cytotoxic potency of Ostreopsis cf. ovata, Ostreopsis cf. siamensis (O. sp. 9), and purified ovatoxins in human keratinocytes

Loïc Plessis, Korian Lhaute, Fabienne Hervé, Madeleine Thimann, Veronique Séchet, Liliane Carpentier, Ariana B. Moutinho, Tiago Simões, Ana Amorim, Kevin Henry, et al.

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## To cite this version:

Loïc Plessis, Korian Lhaute, Fabienne Hervé, Madeleine Thimann, Veronique Séchet, et al. Ranking the cytotoxic potency of Ostreopsis cf. ovata, Ostreopsis cf. siamensis (O. sp. 9), and purified ovatoxins in human keratinocytes. Harmful Algae, 2025, 150, 103000 (11p.). 10.1016/j.hal.2025.103000. hal-05409900

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# Ranking the cytotoxic potency of *Ostreopsis* cf. *ovata*, *Ostreopsis* cf. *siamensis* (O. sp. 9), and purified ovatoxins in human keratinocytes

Loïc Plessis<sup>a,b,*</sup>, Korian Lhaute<sup>a</sup>, Fabienne Hervé<sup>a</sup>, Madeleine Thimann<sup>a</sup>, Véronique Séchet<sup>a</sup>, Liliane Carpentier<sup>a</sup>, Ariana B. Moutinho<sup>c</sup>, Tiago Simões<sup>c</sup>, Ana Amorim<sup>c</sup>, Kevin Henry<sup>d</sup>, Jorge L.B. Neves<sup>e,f</sup>, Léana Gorse<sup>g</sup>, Margaux Paradis<sup>g</sup>, Stéphanie Brédif<sup>b</sup>, Philipp Hess<sup>a,1</sup>, Damien Réveillon<sup>a,1</sup>

## ARTICLE INFO

### Keywords:

Ovatoxins

Palytoxin

Skin model

### In vitro

Risk assessment

## ABSTRACT

Ovatoxins (OVTXs), a family of marine polyketides structurally similar to palytoxin (PLTX), are suspected to cause respiratory symptoms and skin irritation among coastal users during blooms of the dinoflagellate *Ostreopsis* cf. *ovata*. To investigate their role in cutaneous toxicity, crude extracts from six *O. cf. ovata* strains (OVTX(+)) and OVTX(-)) and three *O. cf. siamensis* (*O. sp.* 9) strains (OVTX(-)), along with six purified OVTX analogs, were tested for cytotoxicity (MTT assay) and cell death phenotypes (microscopy) in human keratinocyte models. Interestingly, cytotoxicity was driven by OVTX content, while OVTX(-) extracts were, on average, 150-fold and >1000-fold less potent for *O. cf. siamensis* and *O. cf. ovata*, respectively. Nevertheless, the latter two induced PLTX-like cell death, suggesting the presence of unidentified PLTX-like toxins. Purified OVTXs exhibited sub-nanomolar cytotoxicity, 3.5- to 12.5-fold lower than PLTX (IC₅₀ = 0.036-0.642 nM vs. 0.011-0.049 nM), ranking in potency: PLTX > OVTX-d > OVTX-b > OVTX-a > OVTX-c > OVTX-e >>> OVTX-h (non-toxic, likely due to ring-opening). This study demonstrates the high toxic potency of OVTXs, close to that of PLTX, while providing a comprehensive cytotoxicity assessment of OVTX analogs, which could be useful for assessing the risks of human exposure.

## 1. Introduction

Harmful algal blooms (HABs) are a growing global concern. Extensively studied for their toxins, which can accumulate in shellfish and cause food poisoning when consumed, HABs also pose direct threats to humans through exposure to toxins and cell debris via skin contact or

inhalation of marine aerosols (Berdal et al., 2023; Gorse et al. 2025; Lassus et al. 2016; Lim et al. 2023; Moreira-Gonzalez et al. 2021). A prominent example is the benthic dinoflagellate *Ostreopsis* cf. *ovata* (*O. cf. ovata*) (Fukuyo 1981), widely present in the Mediterranean, whose blooms have regularly been associated with respiratory symptoms (Durando et al. 2007; Illoul et al. 2012; Vila et al. 2016) and skin

**Abbreviations:** HABs, Harmful algal blooms; OVTXs, Ovatoxins; OVTX+, OVTX-producing or OVTX-containing strain/extract; OVTX-, Non-OVTX-producing or OVTX-free strain/extract; OVTX-mix, OVTX mixture of OVTX-a to -e; PLTX, Palytoxin; PLTX eq, PLTX equivalents; OSTs, Ostrocins; McTXs, Mascarenotoxins; LGTXs, Liguriatoxins; RVTXs, Livieratoxins; HaCaTs, Human adult low Calcium Temperature keratinocytes; NHEKs, Normal Human Epidermal Keratinocytes; N/TERTs, NHEK immortalized with Telomerase Reverse Transcriptase; LC-MS/MS, Liquid Chromatography coupled to tandem Mass Spectrometry; LOD, limit of detection; LOQ, limit of quantification; FC, Flash chromatography; SPE, Solid-phase extraction.

* Corresponding author.

E-mail address: loic.plessis28@gmail.com (L. Plessis).

¹ These authors contributed equally as senior authors to this work.

<footnote><sup>a</sup> Ifremer, PHYTOX Physiologie et Toxines des Microalgues Toxiques et Nuisibles, F-44000, Nantes, France</footnote>

<footnote><sup>b</sup> Groupe Rocher, Research-Innovation & Development Department, Issy-les-Moulineaux, France</footnote>

<footnote><sup>c</sup> MARE-Marine and Environmental Sciences Centre, ARNET-Aquatic Research Network Polytechnic of Leiria, Peniche, Portugal</footnote>

<footnote><sup>d</sup> Institut Louis Malardé, UMR EIO, Laboratoire des Biotoxines Marines, Papeete, Tahiti, French Polynesia</footnote>

<footnote><sup>e</sup> Instituto de Engenharia e Ciências do Mar, Universidade Técnica do Atlántico, Mindelo, São Vicente, Cabo Verde</footnote>

<footnote><sup>f</sup> Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR-LA), University of Porto, Matosinhos, Portugal</footnote>

<footnote><sup>g</sup> Institute of Pharmacology and Structural Biology (IPBS), University of Toulouse, CNRS, Toulouse, France</footnote>
irritations (Tichadou et al. 2010; Tubaro et al. 2011). Beyond human health, adverse ecological impacts have also been reported, including mortality of marine organisms (reviewed in Pavaux et al. (2020)). With the ongoing impacts of climate change, blooms of O. cf. *ovata* in the Mediterranean Sea are expected to increase in both intensity and duration (Fabri-Ruiz et al. 2024), amplifying the risk and frequency of outbreaks and potentially leading to significant socio-economic repercussions (Beralet et al. 2022). Additionally, like other di-noflagellates, this species is anticipated to expand its distribution to higher latitudes (Leite et al. 2022; Tester et al. 2020). Supporting this possibility, an unprecedented bloom of O. cf. *ovata* co-occurred with O. cf. *siamensis* (O. sp. 9) in 2021 along the French Basque coast (Atlantic), resulting in 674 reported cases of respiratory and cutaneous symptoms among beachgoers (Chomérat et al. 2022; Paradis et al. 2024). Although O. cf. *ovata* is suspected to be the principal contributor to this incident, the roles and implications of both species remain unclear, and the potential involvement of O. cf. *siamensis* cannot be ruled out (Chomérat et al. 2022). This incident underscores the potential for O. cf. *ovata* blooms to emerge as a significant health and ecological concern beyond the Mediterranean, emphasizing the need for risk assessment—particularly through in-depth toxicological studies—monitoring and management strategies (Anses 2023).

Ovatoxins (OVTXs) were the first toxins identified in O. cf. *ovata* and suspected to be responsible for the reported health issues associated with its blooms (Ciminiello et al. 2010; Ciminiello et al. 2008). These compounds belong to the same molecular family as palytoxin (PLTX) (Fig. S1), one of the largest and most potent natural non-proteinaceous toxins, which is known to induce symptoms similar to those observed during O. cf. *ovata* blooms (Patocka et al. 2018; Tartaglione et al. 2016). First isolated in 1971 from the soft coral Palythoa toxica (Moore and Scheuer 1971), PLTX has been implicated in severe respiratory, skin, and eye symptoms in aquarists following exposure to aquarium corals from the genera Palythoa and Zoanthus (Pelin et al. 2016a; Schulz et al. 2019). Thanks to the availability of a commercial PLTX standard (Wako Chemicals), numerous toxicological studies have been conducted, including *in vivo* experiments and *in vitro* assays reporting LD₅₀ and IC₅₀ values in the nanomolar to picomolar range, providing valuable insights into PLTX's mechanism of action (Bellocci et al. 2011; Hammond and Roy 2024; Lanceleur et al. 2024; Pelin et al. 2016a). Mechanistically, PLTX binds to a specific site on the Na⁺/K⁺ ATPase pump (Pirahanchi et al. 2024; Skou 1957), a transmembrane protein essential for maintaining ion homeostasis within the cell (Gillman et al. 2023; Habermann 1989; Scheiner-Bobis et al. 1994). Upon binding, the pump loses its regulatory function and transforms into a non-selective open cation channel, causing both K⁺ outflow and Na⁺ influx (Artigas and Gadsby 2004). This disruption dissipates the membrane potential and triggers a myriad of downstream effects (e.g., pro-inflammatory activity, activation of protein kinases, ribotoxic stress, and various cell death cascades), ultimately leading to cell death (Cheng et al. 2022; Crinelli et al. 2012; Pelin et al. 2018; Rossini and Bigiani 2011; Rozario et al. 2024).

Contrary to PTLX, the lack of available standardized materials challenges OVTX-related studies, both in toxicological research and in their quantification by LC-MS methods. Currently, OVTXs are expressed as PLTX equivalents (PLTX eq) based on the strong assumption that they exhibit a similar molar response (Ciminiello et al. 2012a). However, several difficulties complicate the purification process of OVTXs: i) the labor-intensive and time-consuming collection of *Ostreopsis* biomass, ii) the significant evaporation-related losses during purification, and iii) degradation or irreversible adsorption depending on storage conditions (Brissard et al. 2014; Brissard et al. 2015; Gémin et al. 2022; Mazzeo et al. 2021; Miele et al. 2024). Lastly, isolating individual OVTX analogs presents an additional hurdle due to their structural similarity (Fig. S1) and nearly identical chromatographic behavior (Brissard et al. 2015). Nevertheless, despite these constraints and the low recovery yields reported (ranging from 1.5 % to 20 %), OVTX-a and OVTX-d have been successfully purified (Ciminiello et al. 2012a; Gémin et al. 2022), with

recent major improvement (Miele et al. 2024).

Purified OVTXs (OVTX-a and -d) have been used in toxicological studies, enabling comparisons of their activity across various bioassays with the PLTX reference material and other PLTX-like congeners, including 42-OH-PLTX derived from *Palythoa toxica* (Ciminiello et al. 2009; Poli et al. 2018) and Ostreocin-D (OST-D) from Japanese *Ostreopsis siamensis* (O. sp. 6) (Pelin et al. 2016b; Ukena et al. 2001; Usami et al. 1995). *In vitro* cytotoxicity evaluations using HaCaT cells, an immortalized human keratinocyte line derived from adult skin (Boukamp et al. 1988), have yielded varying potency estimates. Pelin et al. (2016b) initially reported that OVTX-a was approximately 100-fold less potent than PLTX but 20-fold more potent than OST-D. Conversely, Lanceleur et al. (2024) found that OVTX-a, OVTX-d, and PLTX exhibited IC₅₀ values within the same order of magnitude on the same cell line. However, while some toxicological data are available for OVTX-a and OVTX-d (Gémin et al., 2022; Lanceleur et al., 2024; Pelin et al., 2016b; Poli et al., 2018), no data are currently available for other analogs (e.g., OVTX-b, -c, -e, -f), which also contribute to the OVTX profiles reported in O. cf. *ovata* strains. The most common profile, for instance, comprises OVTX-a to -e (Profile #1, Tartaglione et al., 2017). Overall, significant gaps remain in our understanding of OVTX toxicity, as well as the toxicity of *Ostreopsis* species, whether or not they produce OVTXs.

To address this lack of toxicological data, this study investigates the following using three model human keratinocyte cell types: i) the relationship between OVTX content and O. cf. *ovata* extract cytotoxicity, ii) the cytotoxicity and the cell death phenotype induced by O. cf. *siamensis* extracts, and iii) the individual cytotoxicity of purified OVTX analogs (-a to -e and -h). Ten *Ostreopsis* strains (O. cf. *ovata* and O. cf. *siamensis*) from various geographical regions (Table 1 and Fig. S2), including both OVTX-producing (OVTX+) and non-producing (OVTX−) strains (i.e., within LC-MS/MS detection limits), were assessed. A fit-for-purpose purification strategy was then developed to isolate six OVTX analogs, providing the first comparative cytotoxicity data for multiple OVTX analogs (-a to -e and -h). All extracts and purified toxins used in the

<caption>**Table 1**</caption>

Locations, ovatoxin content, and profiles of *Ostreopsis* strains studied. *dry weight.

<table><thead><tr><th>Strain ID<br>Year of<br>isolation</th><th>Species</th><th>Geographical origin</th><th>OVTX<br>content<br>(mg g<sup>-1</sup>*)</th><th>OVTX profile<br>(OVTX+/<br>OVTX−)</th></tr></thead><tbody><tr><td>IFR-OST-<br>03V<br>2008</td><td>O. cf. <i>ovata</i></td><td>Rochambeau Beach<br>(France)</td><td>2.23</td><td>OVTX-a to -e<br>(OVTX+)</td></tr><tr><td>IFR-OST-<br>01La<br>2018</td><td>O. cf. <i>ovata</i></td><td>Laginha Beach (Cape<br>Verde)</td><td>1.25</td><td>OVTX-a to -e<br>(OVTX+)</td></tr><tr><td>IFR-OST-<br>01P<br>2022</td><td>O. cf. <i>ovata</i></td><td>Parlementia Beach<br>(France)</td><td>1.42</td><td>OVTX-a to -e<br>(OVTX+)</td></tr><tr><td>IFR-OST-<br>08E<br>2022</td><td>O. cf. <i>ovata</i></td><td>Erromardie Beach<br>(France)</td><td>1.61</td><td>OVTX-a to -e<br>(OVTX+)</td></tr><tr><td>IFR-OST-<br>23E<br>2023</td><td>O. cf. <i>ovata</i></td><td>Erromardie Beach<br>(France)</td><td>1.72</td><td>OVTX-a to -e<br>+ -h<br>(OVTX+)</td></tr><tr><td>IFR-OST-<br>12E<br>2022</td><td>O. sp. 9<br>(O. cf.<br><i>siamensis</i>)</td><td>Erromardie Beach<br>(France)</td><td>&lt;LOD</td><td>&lt;LOD<br>(OVTX−)</td></tr><tr><td>IO96-01<br>2008</td><td>O. sp. 9<br>(O. cf.<br><i>siamensis</i>)</td><td>Marina de Sines<br>(Portugal)</td><td>&lt;LOD</td><td>&lt;LOD<br>(OVTX−)</td></tr><tr><td>IO96-17<br>2017</td><td>O. sp. 9<br>(O. cf.<br><i>siamensis</i>)</td><td>Dona Ana Beach<br>(Portugal)</td><td>&lt;LOD</td><td>&lt;LOD<br>(OVTX−)</td></tr><tr><td>NH1810<br>2018</td><td>O. cf. <i>ovata</i></td><td>Hatiheu Bay (French<br>Polynesia)</td><td>&lt;LOD</td><td>&lt;LOD<br>(OVTX−)</td></tr><tr><td>NH1811<br>2018</td><td>O. cf. <i>ovata</i></td><td>Hatiheu Bay (French<br>Polynesia)</td><td>&lt;LOD</td><td>&lt;LOD<br>(OVTX−)</td></tr></tbody></table>
bioassays were compared to the PLTX reference material and re-
quantified via LC-MS/MS prior to testing, due to the susceptibility of
OVTXs to degradation during storage.

## 2. Materials and methods

### 2.1. Obtention of crude extracts from O. cf. ovata and O. cf. siamensis (O. sp. 9)

Strains of *Ostreopsis* (O. cf. *ovata* and O. cf. *siamensis*) from diverse geographical origins, including both OVTX+ and OVTX- strains, were used in this study (Table 1; a corresponding map is provided in Fig. S2). OVTX+ strains exhibited comparable toxin content of 1.25–2.23 mg OVTX per gram of freeze-dried biomass and displayed similar OVTX profiles (OVTX-a to -e, Profile #1 (Tartaglione et al. 2017)), except for strain IFR-OST-23E, which additionally produced OVTX-h (Fig. S3). To our knowledge, this unusual analog and OVTX profile has only been reported once before, in strain IFR-OST-03 V (Brissard et al. 2015), which no longer produces OVTX-h in culture. In contrast, neither the two O. cf. *ovata* strains from French Polynesia nor the three O. cf. *siamensis* strains from the Atlantic produced detectable OVTXs or other known PLTX-like compounds (i.e., within LC-MS/MS detection limits). The origin, culture, and harvesting methods for each strain are detailed the Supplementary Material (S1, S2, and S3).

Crude extracts were obtained from 10 mg of each *Ostreopsis* strain through a two-step extraction process using 500 µL of MeOH, with vortexing and ultrasonication in an ice bath (25 kHz, sweep mode, 15 min, Transsonic TI-H-15, Elma, Germany). Following centrifugation (3500 g, 15 min, 4 °C), the resulting extracts were ultra-filtered (3500 g, 30 s, 4 °C) using 0.2 µm modified nylon Nanosep® filters (ODM02C34, Pall, USA) and stored at -20 °C until further processing. All crude extracts were used in the cell-based bioassays, with the exception of the IFR-OST-23E strain, which was used exclusively for the purification of OVTX-h.

To attempt the detection of known PLTX-like compounds in the O. cf. *siamensis* IO96-01 and IO96-17 strains, additional extracts were prepared at a 20-fold higher concentration (200 mg of dry biomass per mL instead of the 10 mg mL⁻¹). In complement, the injection volume in the LC-MS/MS method was doubled (10 µL instead of 5 µL), providing an additional concentration factor of 2, resulting in an overall theoretical 40-fold increase in sensitivity. Despite these adjustments, no known PLTX-like compounds were detected in these strains (Fig. S4).

### 2.2. LC-MS/MS analysis

Toxin analyses were conducted using the same instrument and chromatographic conditions described by Chomérat et al. (2022). Three methods were employed with Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) and a Diode Array Detector, enabling the detection of two PLTX analogs (PLTX and 42-OH PLTX), twelve OVTXs (-a to -k), and nine other PLTX-like compounds, including ostreocins (OST-A, -B, -D, and -E1), mascarenotoxins (McTX-A, -B, and -C), and ostreotoxins 1 and 3. The precursors, product ions, and wavelengths used for detection are detailed in Table S1.

Quantification was performed relative to the only available standard, PLTX, using a 9-point calibration curve and assuming similar molar responses between the standard and PLTX-like molecules (Ciminiello et al. 2012a). Instrument control, data processing, and analysis were carried out using Analyst software version 1.7.2. The limits of detection (LOD) and quantification (LOQ) for the PLTX standard were 20 ng mL⁻¹ and 40 ng mL⁻¹, respectively. The chemicals and reagents used are detailed in the Supplementary Material (S4).

### 2.3. Purification of ovatoxins

A methanolic extract was obtained from the dried biomass of

*Ostreopsis* strain IFR-OST-03V and subjected to a four-step purification process (Fig. 1). Briefly, the extract was first fractionated by C18 reversed-phase flash chromatography, and OVTX-containing fractions were identified by LC-MS/MS. The resulting aqueous-organic fractions were then subjected to solid-phase extraction (SPE, Strata-X cartridges, Phenomenex) to recover the toxins in MeOH, followed by concentration under N₂. This workflow yielded an OVTX mixture (OVTX-mix, 5 mL) at 187 µg OVTX mL⁻¹, corresponding to a 42 % recovery. From this OVTX-mix, individual OVTX analogs were isolated using analytical reversed-phase chromatography, with concentrations of 1.4, 0.88, 0.49, 0.48, and 0.33 µg mL⁻¹ for OVTX-a to -e, respectively (Fig. S5A–E). In addition, OVTX-h was directly isolated from the extract of *Ostreopsis* strain IFR-OST-23E, also using analytical chromatography, yielding a purified analog solution at a concentration of 0.36 µg mL⁻¹ (Fig. S5F). The purity percentage of each OVTX analog ranged from 71.2 % to 100 % depending on the toxin (Fig. 1 and Fig. S5G). These values reflect only the proportions of known OVTX analogs targeted by the LC-MS/MS method and do not account for other possible contaminants. A complete and detailed description of the purification process is provided in the Supplementary Material (S5, Fig. S6 and Fig. S7).

### 2.4. MTT cytotoxicity assay

HaCaT cells (Human adult low Calcium Temperature) (Boukamp et al. 1988) were cultured in a tissue-culture flask (734-0050, VWR, France) in DMEM containing 4.5 g L⁻¹ glucose, supplemented with 5 % FBS (Fetal Bovine Serum), 30 U mL⁻¹ penicillin and 30 µg mL⁻¹ streptomycin, 0.05 mg mL⁻¹ gentamicin and 1 mM sodium pyruvate. NHEK-Neo (Neonatal Normal Human Epidermal Keratinocytes), from a single donor (00192907, Lonza, Switzerland) were cultured in a Corning® CellBIND® flask (003290, Dominique Dutscher, France) in KGM™ Gold Keratinocyte Growth Medium BulletKit™. Both cultures were maintained at 37 °C and 5 % CO₂ in a culture chamber (MCO-170AIC, Dominique Dutscher, France). The chemicals and reagents used are detailed in the Supplementary Material (S4).

Cells were seeded in a 96-well tissue culture-treated plate (734-4058, VWR, France) at 20,000 cells per well in 200 µL of medium. NHEKs were treated 3 h post-seeding, while HaCaTs were treated after 24 h. For exposure, 6 µL of toxins or sample extracts (final concentration <3 % of the total well volume), were added in a 9-point serial dilution. Untreated cells, and solvents (i.e. methanol for *Ostreopsis* extracts and ACN/water/ 0.2 % acetic acid for purified OVTXs) at concentrations <3 %, were used as negative controls. Each condition was tested in triplicate (n = 3) on the plate. Cell viability was assessed 24 h post-treatment using the MTT colorimetric assay. After medium removal, 50 µL of MTT solution (0.8 mg mL⁻¹ in PBS) was added to each well. The MTT incubation times were 40 min for HaCaT and 3 h for NHEK. After incubation, the MTT solution was removed, and the formazan dye solubilized in 100 µL of DMSO. Optical density was measured at 540 nm using a microplate reader (CLARIOstar PLUS, BMG Labtech, France). To ensure accurate quantification of OVTX content, given their susceptibility to degradation during storage (Brissard et al. 2015; Mazzeo et al. 2021), all extracts and purified toxins were quantified by LC-MS/MS within the same week as their bioassay testing. The tested concentrations of purified OVTXs were determined based on LC-MS/MS quantification of the major OVTX analog present in each fraction (e.g., OVTX-d or OVTX-e in Fig. S5G).

GraphPad Prism software (version 10.1.2) was used to plot sigmoid curves from at least 2 independent experiments of 3 biological replicates. The half maximal inhibitory concentration (IC₅₀), was determined using nonlinear regression analysis (curve fitting) with the "[Inhibitor] vs. response – Variable slope (four parameters)" model. Comparison of IC₅₀ values was performed using one-way ANOVA followed by Tukey's post hoc test for multiple pairwise comparisons.
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Purification process of OVTXs from 1 g of *Ostreopsis* cf. *ovata* freeze-dried biomass. Recovery yields for each step are expressed as percentages, along with the amount of OVTX (determined by LC-MS/MS as PLTX eq). Purity percentages of purified OVTXs are determined solely by their relative OVTX analog profile using targeted LC-MS/MS analysis (Figure S5G). \*OVTX content of the initial biomass was estimated based on the 10 mg mL⁻¹ extract (Table 1).</figcaption></figure>

## 2.5. Microscopic imaging of keratinocytes

Immortalized N/TERT keratinocytes (Dickson et al. 2000), provided by H. Rheinwald (material transfer agreement), were cultured in tissue-culture flasks (734-0050) using Keratinocyte Serum-Free Medium, supplemented with 294.4 ng L⁻¹ EGF (Epidermal Growth Factor), 25 mg L⁻¹ BPE (Bovine Pituitary Extract), and 300 µM CaCl₂. Cells were seeded in a 96-well tissue culture-treated plate (734-4058) at 30,000 cells per well in 200 µL of medium. After 24 h of incubation, the medium was replaced with 180 µL of Opti-MEM™ culture medium supplemented with 500 nM SYTOX™ Green Nucleic Acid Stain. Cells were then treated with 20 µL of extracts, toxins, PMA (phorbol 12-myristate 13-acetate) or methanolic control diluted in Opti-MEM™. Cell imaging was performed 3- and 24-h post-treatment using an EVOS™ M7000 imaging system (AMF7000, Invitrogen, USA), with both brightfield and fluorescence microscopy (470/525 nm) at 20X magnification.

# 3. Results

## 3.1. Cytotoxic effect of the extracts in keratinocytes (HaCaT)

Three distinct patterns were observed depending on strain characteristics (Fig. 2): (1) OVTX+ O. cf. *ovata* strains with the lowest IC₅₀ values around 0.15 µg mL⁻¹; (2) OVTX− O. cf. *siamensis* strains with IC₅₀ values approximately 150-fold higher, around 22 µg mL⁻¹, and comparable toxicity levels between the Portuguese (IO96-01/IO96-17) and French (IFR-OST-12E) strains; and (3) OVTX− O. cf. *ovata* strains from French Polynesia, with IC₅₀ values exceeding 100 µg mL⁻¹, showing some degree of toxicity at the highest concentrations tested (≈300 µg

mL⁻¹).

When the concentrations of extracts from OVTX+ O. cf. *ovata* strains (i.e., strains from the first pattern; Fig. 2) were expressed as PLTX eq, their dose-response curves in the MTT assay were closely aligned across strains, with IC₅₀ values ranging from 211 to 323 pg PLTX eq mL⁻¹ (Fig. 3) and overlapping 95 % confidence intervals (95 % CIs). The OVTX-mix, which had a similar OVTX profile to the extracts (i.e., OVTX-a > OVTX-b >> OVTX-c/-d/-e, Profile #1 (Tartaglione et al. 2017); Fig. S8), exhibited an IC₅₀ value slightly lower than those of the crude extracts. However, it remained within the same order of magnitude, with overlapping 95 % CIs observed between the OVTX-mix and some of the extracts, such as strains IFR-OST-01P and IFR-OST-01La.

## 3.2. PLTX-like effect in keratinocytes (N/TERT)

A similar stress response was observed in N/TERT keratinocytes exposed to PLTX, the OVTX-mix, purified OVTX analogs (OVTX-a to -d; OVTX-e not tested due to limited quantity), OVTX+ and OVTX− O. cf. *ovata* extracts, as well as O. cf. *siamensis* extracts (Fig. 4, S9, S10 and S11). Under brightfield microscopy, this response was characterized by homogeneous cell swelling and a rounded morphology, distinct from untreated cells and from the cellular stress induced by PMA. Staining with SYTOX™ Green dye at 24 hours post-treatment revealed important plasma membrane permeabilization induced by all crude *Ostreopsis* extracts, purified OVTXs (-a to -d), and PLTX. Only OVTX-h did not induce this type of cellular stress and appeared similar to the solvent control, even after 24 hours of treatment (Fig. S11).

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Cytotoxicity of *Ostreopsis* cf. *ovata* and O. cf. *siamensis* methanolic extracts in HaCaT cells after 24 h of exposure (n = 2 independent experiments, each with 3 biological replicates, mean ± standard deviation (error bars)). No confidence interval could be calculated. It should be noted that concentrations were expressed as mg of freeze-dried extracted biomass per mL of culture medium for comparison purpose.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Cytotoxicity of OVTX+ (-a to -e) O. cf. *ovata* extracts and a purified OVTX-mix (-a to -e) in HaCaT cells after 24 h of exposure (n = 2 independent experiments, each with 3 biological replicates, mean ± standard deviation (error bars)). It should be noted that OVTX concentrations were expressed as PLTX eq (determined by LC-MS/MS).</figcaption></figure>

### 3.3. Cytotoxic effects of the toxins on human keratinocytes (HaCaT & NHEK)

At the tested concentrations, all purified OVTX analogs, except OVTX-h, induced a dose-dependent cytotoxicity in both keratinocyte types (Fig. 5 and Table 2). HaCaT cells demonstrated approximately 5-fold higher sensitivity to the toxins compared to NHEK, with IC₅₀ values ranging from 30 to 296 pg mL⁻¹ (11–111 pM) for HaCaT versus from 132 to 1738 pg mL⁻¹ (49–642 pM) for NHEK.

Based on the calculated IC₅₀ values and ANOVA box plot analysis (Fig. S12), the same cytotoxicity ranking was observed for both keratinocyte types (HaCaT/NHEK): PLTX (11/49 pM) > OVTX-d (36/258 pM) > OVTX-b (53/260 pM) > OVTX-a (61/380 pM) > OVTX-c (91/642 pM) > OVTX-e (111 pM/no data) >>> OVTX-h (no IC₅₀). However, it is important to note that overlapping 95 % CIs were observed between certain OVTX analogs, such as OVTX-a and OVTX-b (between 138 and 168 pg mL⁻¹) or OVTX-c and OVTX-e (between 226 and 294 pg mL⁻¹) in HaCaT cells, as well as OVTX-b and OVTX-d (between 595 and 834 pg mL⁻¹) in NHEK cells. Purified OVTXs (-a to -e) exhibited sub-nanomolar cytotoxicity, ranging from 3.5- to 12.5-fold lower than PLTX (IC₅₀ = 36–642 pM vs. 11–49 pM), depending on the analog and cell type. Finally, PLTX was 3- to 6-fold more toxic than the OVTX-mix across both keratinocyte models, with IC₅₀ values of 30 vs. 175 pg (PLTX eq) mL⁻¹ in HaCaT and 132 vs. 453 pg (PLTX eq) mL⁻¹ in NHEK.

The OVTX-mix exhibited IC₅₀ values in HaCaT cells consistent with those of the individual OVTX analogs, reflecting an average of their toxicity. A theoretical IC₅₀, calculated based on the individual IC₅₀s of the OVTX analogs present in the OVTX-mix, was 160 pg (PLTX eq) mL⁻¹, closely aligning with the observed IC₅₀ of 175 pg (PLTX eq) mL⁻¹ for the OVTX-mix (Table S2 and Table 2). This theoretical value also closely matched the IC₅₀ of OVTX-a (161 pg (PLTX eq) mL⁻¹), which constitutes 56 % of the OVTX-mix (Fig. S8). However, this calculation did not hold as well for NHEK cells, where the theoretical IC₅₀ (901 pg (PLTX eq) mL⁻¹) was approximately two-fold higher than the observed IC₅₀ of 453 pg (PLTX eq) mL⁻¹ in the bioassay.

## 4. Discussion

### 4.1. Purification strategy for the OVTX-mix and purified OVTXs

To increase the knowledge and fill the gap about the cytotoxicity of OVTXs, the current study relied on a fit-for-purpose purification strategy that provided i) a mixture of OVTXs (OVTX-mix) representative of the most common OVTX profile in the Mediterranean (OVTX-a to -e), and ii) six purified OVTX analogs (OVTX-a to -e and -h).

The first purification step, flash chromatography (FC), resulted in a recovery of 63 %, consistent with the 46–60 % OVTX recovery yields reported by Miele et al. (2024) using a similar technique on a different

scale. The observed OVTX losses may be attributed to vacuum evaporation of the *Ostreopsis* extract onto the sorbent prior to FC, as evaporation steps (e.g. prolonged or complete) are critical in OVTX purification (Gémin et al. 2022; Mazzeo et al. 2021). As a result, we successfully implemented an SPE step, inspired by previous studies that used SPE on spiked seawater to recover PLTX or OVTX (Riccardi et al. 2017; Tartaglione et al. 2016; Ternon et al. 2022b; Zendong et al. 2014). Our Strata-X SPE method achieved a remarkable OVTX recovery of 89 % (Fig. 1), providing a practical and efficient alternative to the time-consuming and potentially damaging evaporation of aqueous fractions when purifying OVTXs. Nevertheless, evaporation of SPE eluates remained unavoidable, causing a 25 % loss after pooling, which confirms that evaporation is a major source of OVTX loss during purification (Gémin et al. 2022; Mazzeo et al. 2021).

Finally, due to the complexity of isolating individual OVTX analogs arising from their similar chromatographic behavior (Brissard et al. 2015; Ciminiello et al. 2010), the OVTX-mix was directly subjected to analytical chromatography to obtain purified OVTXs, as it provided satisfactory resolution between analogs (Fig. S8). This approach proved effective for our study, which required only minimal sample volumes and concentrations, given their effects in keratinocytes at picomolar concentrations (Fig. 5 and Table 2). However, it may not be directly transposable for purifying larger quantities of OVTXs, as detailed in previous studies (Ciminiello et al. 2012a; Gémin et al. 2022; Miele et al. 2024; Pelin et al. 2016b).

### 4.2. Cytotoxic activity of *Ostreopsis* cf. *ovata* extracts

To investigate the relationship between OVTX content and the cytotoxicity of O. cf. *ovata* extracts, we compared OVTX+ and OVTX– strains, along with the purified OVTX-mix, using a cytotoxic bioassay and microscopic imaging of keratinocytes (HaCaT and N/TERT cells, respectively). The OVTX+ extracts showed strong and comparable cytotoxicity, with IC₅₀s of 211–323 pg PLTX eq mL⁻¹ (Figs. 2 and 3), within the same order of magnitude (<2-fold difference) as the OVTX-mix (IC₅₀ = 175 pg PLTX eq mL⁻¹). This aligns with Lanceleur et al. (2024), who reported that a comparable OVTX-mix was slightly more toxic than the crude extract, with a 2- to 6-fold difference depending on the cell line. Altogether, these results, along with the observation that OVTX– extracts were at least three orders of magnitude less toxic (Fig. 2), suggest that the cytotoxic effects observed in HaCaT and other human skin cell types may be predominantly driven by the OVTX content in O. cf. *ovata* extracts.

Microscopic imaging further supported this hypothesis, revealing that *Ostreopsis* cytotoxic extracts, the OVTX-mix, and purified OVTX analogs (-a to -d) induced the same cellular response observed with PLTX (Fig. 4, S9, S10 and S11). This response was characterized by rapid swelling, the formation of rounded “balloon-shaped” cells, and plasma
N/TERT keratinocytes
(24 hours post treatment)

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Microscopic imaging of N/TERT keratinocytes treated with *Ostreopsis* extracts (1 mg freeze-dried extracted biomass mL⁻¹; O. cf. *ovata* IFR-OST-03V and O. cf. *siamensis* IO96-17), PLTX (1 µg mL⁻¹), OVTX-mix (18.7 µg PLTX eq mL⁻¹), and PMA (phorbol 12-myristate 13-acetate; 10 µg mL⁻¹). Images were captured at 24 hours post-treatment, with membrane permeabilization visualized using SYTOX™ Green dye. Additional treatment conditions, including OVTX analogs and other *Ostreopsis* extracts, are available in the Supplementary Material (Figure S9, S10 and S11).</figcaption></figure>

membrane permeabilization (observed with SYTOX™ Green), consistent with previous observations on other cell types exposed to PLTX (Amir et al. 1997; Rozario et al. 2024; Valverde et al. 2008a; Valverde et al. 2008b). Interestingly, at a 3-fold higher concentration than the highest used in the MTT assay, the presumed OVTX– extracts induced similar cellular patterns (Fig. S10), suggesting that these strains (NH1810 and NH1811) may produce trace amounts of PLTX-like compounds below the LOD.

Finally, while our study shows a strong correlation between OVTX content and the cytotoxicity of *O. cf. ovata* extracts, the potential presence of other cytotoxic compounds cannot be excluded. Cytotoxic but non-PLTX-like compounds, such as liguriatoxins (LGTXs), rivieratoxins (RVTXs), and ostreols, have also been reported in *O. cf. ovata* (Hwang et al. 2018; Hwang et al. 2013; Ternon et al. 2022a). While LGTXs and RVTXs exhibit strikingly lower cytotoxicity than PLTX (IC₅₀ = 0.68–3.1

µg mL⁻¹ vs. 45 pg mL⁻¹ on a human lung cell line) and ostreol B shows moderate cytotoxicity (IC₅₀ = 0.1–4.8 µM across three cell lines), their contribution to the overall cytotoxicity of *O. cf. ovata* remains to be clarified.

4.3. *Cytotoxic activity of purified ovatoxins*

This study provides the first comprehensive cytotoxicity evaluation of individual OVTX analogs (-a to -e) from *O. cf. ovata*, offering new insights into their relative potency and advancing our understanding of their toxicity on human keratinocytes. Immortalized HaCaT cells were used for comparison with previous studies (Table 3), while primary NHEKs were included due to their closer resemblance to *in vivo* keratinocytes, regarding cellular responses and differentiation (Boukamp et al. 1988; Lehman et al. 1993; Seo et al. 2012). An approximately 6-fold
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

**Fig. 5.** Cytotoxicity of purified OVTX analogs (A-F), the reference PLTX material (G), and a purified OVTX mixture (OVTX-mix) (H) in HaCaT (lighter tones/circles) and NHEK (darker tones/triangles) after 24 h of exposure (n = 2-5 independent experiments, each with 3 biological replicates, mean ± standard deviation (error bars)). Note that due to the relatively low purity (71.2 %, Figure S5G), and limited quantity, OVTX-e was only tested in HaCaT cells.
<caption>**Table 2**</caption>

IC₅₀ values of purified OVTX analogs, PLTX, and a purified OVTX mixture (OVTX-mix) in keratinocytes (HaCaT and NHEK), after 24 h of exposure (n = 2–5 independent experiments, each with 3 biological replicates, mean with 95 % confidence intervals (95 % CI)).

<table><caption>**Table 3**</caption><thead><tr><th rowspan="2">Toxin</th><th colspan="2">Cell type</th></tr><tr><th>HaCaT<br>IC<sub>50</sub> (95 % CI) pg mL<sup>-1</sup><br>IC<sub>50</sub> (95 % CI) pM</th><th>NHEK<br>IC<sub>50</sub> (95 % CI) pg mL<sup>-1</sup><br>IC<sub>50</sub> (95 % CI) pM</th></tr></thead><tbody><tr><td>OVTX-a</td><td>161 (138–187)<br>61 (52–70)</td><td>1007 (906–1119)<br>380 (342–423)</td></tr><tr><td>OVTX-b</td><td>144 (123–168)<br>53 (46–63)</td><td>700 (562–866)<br>260 (209–322)</td></tr><tr><td>OVTX-c</td><td>247 (201–294)<br>91 (74–108)</td><td>1738 (1586–1951)<br>642 (586–720)</td></tr><tr><td>OVTX-d</td><td>97 (77–122)<br>36 (29–46)</td><td>687 (595–834)<br>258 (223–313)</td></tr><tr><td>OVTX-e</td><td>296 (226–374)<br>111 (85–140)</td><td>Not tested<br>Not tested</td></tr><tr><td>OVTX-h</td><td>&gt; 10,000<br>&gt; 4000</td><td>&gt; 10,000<br>&gt; 4000</td></tr><tr><td>PLTX</td><td>30 (26–36)<br>11 (10–13)</td><td>132 (115–148)<br>49 (43–55)</td></tr><tr><td>OVTX-mix</td><td>175 (143–216)</td><td>453 (365–558)</td></tr></tbody></table>

IC₅₀ values for OVTX-a, OVTX-d, and PLTX obtained from the HaCaT MTT assay in previous studies, compared with those obtained herein (SD = standard deviation).

<table><thead><tr><th>Toxin</th><th>HaCaT IC<sub>50</sub><br>IC<sub>50</sub> (95 % CI) / ± SD pM</th><th>Exposure time</th><th>Study</th></tr></thead><tbody><tr><td>OVTX-a<br>PLTX</td><td>1100 (700–1700)<br>18 (10–34)</td><td>4h</td><td>Pelin et al. (2016b)</td></tr><tr><td>OVTX-a<br>OVTX-d<br>PLTX</td><td>1130 ± 450<br>4030 ± 1290<br>2880 ± 1270</td><td>24h</td><td>Lanceleur et al. (2024)</td></tr><tr><td>OVTX-a<br>OVTX-d<br>PLTX</td><td>61 (52–70)<br>36 (29–46)<br>11 (10–13)</td><td>24h</td><td>This study</td></tr></tbody></table>

difference in sensitivity was observed between the two cell types, with IC₅₀s ranging from 30 to 296 pg mL⁻¹ (11–111 pM) for HaCaTs and 132 to 1738 pg mL⁻¹ (49–642 pM) for NHEKs (Fig. 5 and Table 2). This difference may result from the immortalized nature of HaCaTs, which can alter cellular responses, e.g., their resistance to H₂O₂-induced oxidative stress (Liu et al. 2012).

Despite this variability, a consistent cytotoxicity ranking across both cell types was observed: PLTX > OVTX-d > OVTX-b > OVTX-a > OVTX-c > OVTX-e >>> OVTX-h (Fig. S12 and Table 2). However, due to the inherent variability of cell-based bioassays and limitations in PLTX eq quantification, and the relative purity of OVTX-d (93.5 %) and OVTX-e (71.2 %) (Fig. 1), this ranking should be considered indicative. Notably, Lanceleur et al. (2024) reported different rankings for PLTX, OVTX-a, and OVTX-d across cell types, including HaCaTs, indicating that such differences may depend on the cell type or assay conditions.

A comparison of IC₅₀ values for HaCaTs (Table 3) revealed inconsistencies between studies: PLTX values matched between Pelin et al. (2016b) and our study but not Lanceleur et al. (2024), while OVTX-a values aligned between Lanceleur et al. (2024) and Pelin et al. (2016b) but differed from ours. Nevertheless, Lanceleur et al. (2024) reported that IC₅₀ values for PLTX, OVTX-a, and d remained within the same order of magnitude in the same cell type, a pattern also observed in our study with HaCaTs and NHEKs. In contrast, Pelin et al. (2016b) reported a ~2-order-of-magnitude difference between OVTX-a and PLTX in HaCaTs. These findings confirm that OVTX-a's cytotoxicity may be closer to PLTX than previously thought and that, while a ranking is

proposed here, OVTX analogs (except OVTX-h) exhibit cytotoxicity within the same order of magnitude, at least in keratinocytes. We hypothesize that the observed inconsistencies between studies may, in part, be driven by the high susceptibility of PLTX-like compounds to degradation during storage (Brissard et al. 2015; Mazzeo et al. 2021), which could significantly affect the accuracy of cytotoxicity assessments. Therefore, to ensure reliable and reproducible results, we strongly recommend re-quantifying these compounds via LC-MS/MS prior to bioassays, as done in our study. These differences between studies also highlight the need for certified reference standards for both PLTX and OVTXs.

Unlike the other OVTX analogs, OVTX-h showed no cytotoxicity at the tested concentrations and did not induce a PLTX-like response during microscopic imaging (Fig. 5, Table 2 and Fig. S11). Its apparent lack of toxicity may result from the hypothesized ring opening between carbons C42 and C49 in its chemical structure (Brissard et al. 2015) (Fig. S1), altering its spatial conformation and binding affinity to the Na⁺/K⁺ ATPase pump. These observations suggest that several structural elements can considerably modulate the cytotoxic potency PLTX-like compounds, and that further work is required to better understand their structure-activity relationship, such as binding assays, X-ray crystallography, and computational modeling (Gillman et al. 2023).

### 4.4. Cytotoxic and PLTX-like activity of Ostreopsis cf. siamensis (O. sp. 9) extracts

Only a few toxicological studies have focused on O. cf. siamensis, likely because its blooms have not been associated with human health issues. However, its co-occurrence with O. cf. ovata during the 2021 outbreak along the French Basque coast (Atlantic) (Chomérat et al. 2022), prompted us to analyze its potential toxicity on human keratinocytes. The three tested O. cf. siamensis extracts exhibited similar cytotoxicity in HaCaTs, making them approximately 150-fold less potent (IC₅₀s ~20 µg mL⁻¹) than the OVTX+ O. cf. ovata extracts (Fig. 2). Interestingly, at high concentration (i.e., 1 mg freeze-dried extracted biomass mL⁻¹), they triggered a similar cellular response (i.e., rapid swelling and membrane permeabilization) in N/TERTs as observed with O. cf. ovata extracts and PLTX (Fig. 4 and Fig. S10), suggesting a low level of PLTX-like toxicity. These findings align with previous studies (Cagide et al. 2009; Penna et al. 2005) that reported PLTX-like effects of O. cf. siamensis extracts on human erythrocytes (RBCs) and neuroblastoma (BE (2)-M17) cells.

Atlantic and Mediterranean O. cf. siamensis (O. sp. 9) are taxonomically distinct from the tropical O. siamensis (O. sp. 6) (Chomérat et al. 2022; Chomérat et al. 2020; Nguyen-Ngoc et al. 2021), which produces PLTX-like compounds known as Ostreocins (OSTs). Neither OVTXs nor any known PLTX-like compounds (e.g., PLTXs, OSTs, McTXs, and ostreotoxins) were detected in our O. cf. siamensis extracts by LC-MS/MS (Fig. S4), consistent with other studies (Chomerat et al. 2022; Ciminiello et al. 2013; Drouet et al. 2024), even under our enhanced analysis conditions (S6). Only Ciminiello et al. (2013) had reported traces of PLTX in one strain, but no appreciable toxicity was observed in mouse bioassays. Given the wide diversity of PLTX-like analogs reported across Ostreopsis species (Pavaux et al. 2020), O. cf. siamensis may produce analogs undetectable by current targeted analytical methods. Identifying these compounds may require a bio-guided fractionation approach that could combine the sensitivity of keratinocyte assays with LC-MS/MS and HRMS analyses.

### 4.5. Contribution to risk assessment for O. cf. ovata and O. cf. siamensis (O. sp. 9)

Growing concerns about Ostreopsis-related health issues highlight the need for a better assessment of the risks associated with Ostreopsis proliferation (Berdalet et al. 2022). Although aerosol inhalation is considered the principal exposure route, direct ingestion of water
containing PLTX-like compounds, as well as cutaneous or ocular contact, and indirect intoxication through the consumption of contaminated seafood cannot be excluded (Anses 2023). In this context, the toxicological data obtained in this study can contribute to risk assessment, particularly regarding cutaneous exposure.

This study provides the *in vitro* toxic potency of individual OVTX analogs, addressing questions raised by Tartaglione et al. (2017) regarding how different *O. cf. ovata* toxin profiles may pose varying levels of risk to humans. OVTXs showed a similar high cytotoxicity, reaching the sub-nanomolar range in *in vitro* bioassays (this study; Lanceleur et al., 2024), and our results suggest that cytotoxicity is likely influenced more by the total quantity of OVTXs than by the relative proportions of individual analogs (except for OVTX-h). Based on the correlation between the IC₅₀ of the OVTX-mix and those of individual OVTX analogs in HaCaTs (Table S2 and Table 2), a predictive IC₅₀ could be calculated for Mediterranean OVTX profiles #1 to #3, which differ in their proportions of OVTX-a to -e (Tartaglione et al. 2017). Notably, these OVTX profiles are also found in strains from the Atlantic (Brazil, France) and Pacific (Japan) regions (Chomérat et al. 2022; Miele et al. 2024; Tibiriçá et al. 2019). However, this theoretical prediction may not apply to Profile #4, which comprises 50 % OVTX-f (Ciminiello et al. 2012b), an analog absent in our strains and for which no toxicological data are currently available.

Second, as reported in previous studies (Lanceleur et al. 2024; Pelin et al. 2016b), we observed that PLTX-like effects could appear within just a few hours of exposure (e.g., after 3 hours, Fig. S9), depending on toxin concentration.

Third, *O. cf. siamensis* extracts showed a PLTX-like effect, albeit 150 times less toxic than *O. cf. ovata* in the tested conditions. This observation may explain why *O. cf. siamensis* alone has not been linked to any symptoms, especially along the Basque coast, where no *Ostreopsis*-related health issues were reported until the emergence of *O. cf. ovata* (Chomérat et al. 2022). Thus, distinguishing and quantifying the two species separately appears necessary to assess the overall risk, which could ultimately lead to refine the alert levels based on cell concentrations in regions with co-occurring blooms (Anses 2023).

Future research should clarify the distribution, concentration, bioavailability and stability of toxins during *O. cf. ovata* blooms to better estimate exposure levels. Given our results, special attention should be paid to dissolved OVTXs in seawater, but LGTXs and RVTXs can be found in much higher concentrations and should not be disregarded (Ternon et al. 2024). However, this will be highly challenging as *Ostreopsis* toxins can be found across various environmental compartments (i.e., water column, surface microlayer, and aerosols) (Ciminiello et al. 2014; Medina-Pérez et al. 2021; Ternon et al. 2024), as well as within micro-algal cells, debris, or mucus, which may be suspended in seawater, attached to substrates (e.g., macroalgae), or forming floating aggregates known as "sea flowers" (Berdalet et al. 2022).

## 5. Conclusion

This study provides new insights into the cytotoxic potency of two *Ostreopsis* species, namely *O. cf. ovata* and *O. cf. siamensis* (O. sp. 9), on human keratinocytes. The toxicity of *O. cf. ovata* extracts was closely linked to their OVTX content, with IC₅₀ values comparable to those of purified OVTX analogs (OVTX-a to -e). While PLTX was more potent, our findings suggest that OVTXs exhibit cytotoxicity closer to PLTX than previously thought. In contrast, *O. cf. siamensis* isolates were approximately 150-fold less cytotoxic than *O. cf. ovata* but still induced a similar stress response in keratinocytes, suggesting the possible presence of PLTX-like compounds. These findings highlight the need for further research into the chemodiversity and toxicology of *Ostreopsis* species, across a broader range of biological systems to better understand their health and ecological impacts. Future studies could focus on the purification and characterization of other PLTX-like compounds, such as OVTX-f, additional OVTXs, OSTs, or McTXs. Such efforts would

contribute to a deeper understanding of the mechanisms underlying this toxin family, which, despite their structural similarities, exhibits subtle variations that may influence toxicity, as demonstrated by the open-ringed OVTX-h in our study.

## CRediT authorship contribution statement

**Loïc Plessis:** Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. **Korian Lhaute:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Fabienne Hervé:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Madeleine Thimann:** Investigation, Formal analysis. **Véronique Séchet:** Writing – review & editing, Resources, Methodology. **Liliane Carpentier:** Resources, Methodology. **Ariana B. Moutinho:** Writing – review & editing, Resources. **Tiago Simoes:** Writing – review & editing, Resources. **Ana Amorim:** Writing – review & editing, Resources. **Kevin Henry:** Writing – review & editing, Resources. **Jorge L.B. Neves:** Writing – review & editing, Resources. **Léana Gorse:** Resources, Methodology. **Margaux Paradis:** Resources, Methodology. **Stéphanie Brédif:** Writing – review & editing, Supervision, Funding acquisition. **Philipp Hess:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. **Damien Rêveillon:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors acknowledge the contributions of all lab members for their guidance and support in this project. They would like to thank H. David from the MARE (Marine and Environmental Sciences Centre) for the isolation and identification of the initial Portuguese *Ostreopsis* cf. *siamensis* strains, as well as M. Chinain from the ILM (Institut Louis Malardé) for kindly providing algal biomasses of Polynesian *Ostreopsis* cf. *ovata*. The authors also acknowledge E. Meunier's team from the IPBS (Institute of Pharmacology and Structural Biology) for their help and resources, specifically in microscopic imaging work. Finally, they acknowledge N. Chomérat from Ifremer's COAST unit for his taxonomic assistance with *Ostreopsis* species. The authors acknowledge funding provided to Loïc Plessis through the Ifremer–Groupe Rocher convention and the ANRT (CIFRE n°2021/1382), as well as the ANR-INFLAMATOX grant (ANR-23-CE34–0007), Ifremer core funding (PHYTOX-R306–01) and the EMERTOX project (grant 734748), funded by H2020-MSCA-RISE 2016. This work was also supported by the OSS64 research program ‘Ostreopsis survey in the Basque Country’, co-financed by the Basque Country Agglomeration Community and the Adour Garonne Water Agency grants. The authors would like to thank in particular E. Antajan for managing the OSS64 research program. The Graphical abstract and Fig. 1 included in this manuscript were created with BioRender (www.BioRender.com).

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.hal.2025.103000.

## Data availability

Data will be made available on request.
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