# Comparative toxicity of Karlodinium veneficum from France and New Zealand on fish gill cells and oyster gametes

Anne Rolton, Guillaume Barnouin, Hannah Greenhough, Audrey Duval, Karthiga Kumanan, Nelly Quéré Le Goïc, Lucy Thompson, Damien Réveillon, Fabienne Hervé, Andrew Selwood, et al.

► To cite this version:

Anne Rolton, Guillaume Barnouin, Hannah Greenhough, Audrey Duval, Karthiga Kumanan, et al. Comparative toxicity of Karlodinium veneficum from France and New Zealand on fish gill cells and oyster gametes. Harmful Algae, 2025, 150, pp.102991. 10.1016/j.hal.2025.102991. hal-05336755

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1 Comparative toxicity of *Karlodinium veneficum* from France and New Zealand on fish gill cells and oyster gametes

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4 Anne Rolton¹*, Guillaume Barnouin²,³, Hannah Greenhough¹, Audrey Duval², Karthiga Kumanan¹,

5 Nelly Quéré Le Goïc³, Lucy Thompson¹, Damien Réveillon⁴, Fabienne Hervé⁴, Andrew I. Selwood¹,

6 Hélène Hégaret³, Kirsty F. Smith¹, Malwenn Lassudrie²

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8 ¹ Cawthron Institute, 98 Halifax Street East, Nelson, 7010, New Zealand

9 ² Ifremer, COAST, LER-BO, Concarneau, F-29900, France

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11 ³ Laboratoire des Sciences de l'Environnement Marin, UMR 6539 LEMAR CNRS, UBO, IRD, Ifremer,

12 Plouzané F-29280, France

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14 ⁴ Ifremer, PHYTOX, Metalg, Nantes, F-44000, France

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16 *Corresponding author : anne.vignier@cawthron.org.nz

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33 Abstract

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35 Harmful algal blooms (HABs) can cause mass mortalities of marine fauna, leading to major economic losses in aquaculture and fisheries worldwide. Toxicity varies widely among strains of the same HAB species, making broad assumptions of toxicity inaccurate. This study investigated the ichthyotoxic activity of *Karlodinium veneficum*, a HAB species of risk for aquaculture in France (FR) and New Zealand (NZ). Five *K. veneficum* strains (3 FR, 2 NZ) were cultured and extracted under standardized conditions. Following inter-laboratory assay comparison, crude (culture) and methanolic intra- and extracellular extracts were tested on *in vitro* models: rainbow trout gill cells (RTgill-W1) and Pacific oyster, *Crassostrea (=Magallana) gigas*, sperm.

43 Crude and methanolic extracts showed toxicity to both cell types, with variation between intra-(IN) and extracellular (EX) fractions. Of the crude extracts, only CAWD93 IN was toxic to gill and sperm cells, inducing 44% and 81% mortality, respectively, at 8.75 x 10⁴ equiv. cells mL⁻¹. Most methanolic extracts were toxic; MAR1F7 EX and CAWD93 IN induced the highest mortality rates, with EC₅₀'s reaching cell concentrations equivalent to high environmental bloom levels.

48 Karlotoxin analysis of methanolic extracts revealed distinct profiles by country. Karlotoxins were detected only in the three most toxic strains (MAR1F7, IFR-CC-20-44, CAWD93) and cytotoxicity was only weakly correlated with karlotoxin concentration, suggesting involvement of additional toxic compounds.

52 This study highlights strain-specific toxicity in *K. veneficum*, underscoring the importance of testing local HAB strains. The bioassays used offer a rapid, cost-effective, high-throughput, and ethical approach to evaluating HAB toxicity—crucial as ichthyotoxic blooms become more frequent globally.

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57 Keywords

58 Harmful algal blooms, KmTx, ichthyotoxicity, bioassays, RTgill-W1, bivalves, aquaculture

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66 Highlights

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68 • Toxicity of 5 *Karlodinium veneficum* strains from NZ and FR were assessed *in vitro*.

69 • Crude and methanolic algal extracts caused RTgill-W1 and oyster sperm mortalities.

70 • There was significant variation in the toxicity of extracts from different strains.

71 • Compounds additional to karlotoxins are likely involved in toxicity towards cells.

72 • These methods allow rapid, cost-effective assessment of the toxic effects of HABs.

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# 1. Introduction

Coastal harmful algal blooms (HABs) are increasing worldwide (Dai et al., 2023). They can affect human health via the consumption of shellfish or fish that have accumulated toxins in their flesh, can cause skin irritation through direct contact or, respiratory problems via the production of toxic aerosols (Lassus, 2016). Other species of microalgae, regardless of their toxicity to humans, can be ichthyotoxic (i.e. toxic to marine fauna) and blooms can cause mortality in marine vertebrates and invertebrates, leading to considerable economic losses to the fishing and aquaculture sectors (i.e. Hallegraeff et al., 2018; Rolton et al., 2022). For example, in 2016, the “Godzilla Red Tide”, an ichthyotoxic bloom of various HAB species including *Pseudochattonella verruculosa* and *Alexandrium* spp. caused mass mortalities of farmed salmon in Chile, resulting in an estimated $800 million USD loss (Hallegraeff et al., 2018, Mardones et al., 2021). A recent review summarizes the major mechanisms and physiological impacts of ichthyotoxic species on marine organisms (Hallegraeff et al, 2023); however, little is still known about the ichthyotoxic mechanisms of microalgae and the toxicity of many species (Lassus, 2016, Rasmussen et al 2017). Moreover, as the climate continues to change, the frequency of HABs may increase (Brandenburg et al., 2019; Gobler, 2020; Moore et al., 2008). Studying microalgal toxicity enables risk assessment to mitigate the effects of future HABs on human and environmental health, while also advancing understanding of the socio-economic risks that may result from blooms.

HAB events are common in key aquaculture regions of France and New Zealand (NZ), where they have caused both lethal and sublethal effects on commercially important fish and bivalve species (Chauvaud et al., 1998; Nézan et al., 2014, Paulmier, 1994, Rolton et al., 2022 and references therein). One HAB genus of particular concern in both countries is *Karlodinium* (Rolton et al., 2022; Nézan et al., 2014). The species *K. veneficum* is recurrently detected at low concentrations along both the Atlantic and Mediterranean coasts of France (Nézan et al., 2014; REPHY, 2023) and has bloomed on the French-Spanish Mediterranean border, causing mortality of vertebrate and invertebrate species (Garcès et al., 2006; REPHY, 2023). Globally, *K. veneficum* blooms, which typically reach 10⁴–10⁷ cells L⁻¹, have been linked to mass mortalities in fish and molluscs (Place et al., 2012; Kempton et al., 2002; Hall et al., 2008). For instance, a four-week bloom in the Mediterranean Sea in summer 2007, reaching >1.6 × 10³ cells mL⁻¹, coincided with high mortalities of the razor clam, *Ensis ensis* (Fernandez-Tejedor et al., 2010). In NZ, *K. veneficum* has been recorded in the northeastern Hauraki Gulf and southern Marlborough Sounds (Rhodes et al., 2020).

*Karlodinium veneficum* produces karlotoxins (KmTxS) and possibly other bioactive molecules with hemolytic, cytotoxic and allelopathic effects (Adolf et al., 2015; Deeds et al., 2006; Place et al., 2012; Beesoo et al., 2025). In vertebrate cells, it has been shown to induce pores in the plasma
membranes (Deeds et al 2006, 2015), but the mechanisms of action on invertebrate cells remain unexplored.

Genetic and phenotypic diversity within a single microalgal species can facilitate adaptive responses to environmental fluctuations (Brandenburg et al., 2018; Burkholder and Glibert, 2006; Hadjadji et al., 2020, Calbet et al 2011), potentially resulting in variation in toxin production (Brandenburg et al., 2018). Notably, strain-specific differences in toxicity have been observed in *K. veneficum* (Yang et al., 2019), with some strains reported to be non-toxic (Place et al., 2012; Beesoo et al., 2025). Therefore, assessing the intra-specific diversity of toxin profiles and toxicities of *K. veneficum* is essential.

Assessing the effects of HABs on whole animals is costly, time-consuming, and raises ethical concerns. In the past decade, *in vitro* cellular bioassays have emerged as an alternative to live animal testing for evaluating ichthyotoxicity (Hallegraeff et al., 2023). One promising approach involves the RTgill-W1 gill cell line established from rainbow trout, *Oncorhynchus mykiss*, which has previously been used to assess microalgal ichthyotoxicity (Beesoo et al., 2025; Dorantes-Aranda et al., 2011, 2015; Mardones et al., 2020; Prause et al., 2025). As gills are the primary interface with the aquatic environment, critical for gas exchange, osmoregulation, pH balance, and nitrogen excretion, they are highly susceptible to bioactive compounds produced by toxic microalgae (Deeds et al., 2006; Mardones et al., 2018). RTgill-W1 cells are relatively robust, tolerating varying osmotic conditions, grow well at room temperature (Lee et al., 2009), and an assay targeting these cells have been standardized for aquatic contaminant testing (ISO 21115:2019, OECD Test No. 249). No standardized method exists for marine phycotoxins, however, most studies adapting the protocol of Dorantes-Aranda et al. (2011).

Beyond fish, HAB toxins also pose risks to bivalve reproduction. The Pacific oyster, *Crassostrea gigas*, a broadcast spawner, releases gametes into the water where they may be exposed to toxins for extended periods. Toxins from dinoflagellates such as *Karenia* spp., *Alexandrium* spp. and *Dinophysis* spp., have been shown to affect bivalve sperm, with implications for gamete quality, larval development and spat recruitment (Greenhough et al., 2024; Rolton et al., 2015; Castrec et al., 2021; Gaillard et al., 2020). Notably, seasonal oyster spawning often coincides with times when HAB events often occur, increasing the risk of reproductive failure and population declines.

This study aimed to evaluate the toxic activity of several *K. veneficum* strains from France and NZ using RTgill-W1 fish gill cell- and pacific oyster sperm-bioassays, and to assess the reliability of both methods across different laboratories. Microalgae were cultured in similar conditions in both countries. Intra- and extra-cellular fractions were collected either as crude fractions or processed into chemical (methanolic) extracts. These were then tested against both bioassays. Firstly, the same methanolic extracts were tested in respective laboratories to enable inter-laboratory
bioassay comparison and calibration. Next, toxicity was compared across *K. veneficum* strains using both cell types to assess intraspecific variation. Finally, comparing the toxicity of chemical extracts with crude fractions helped determine whether known compounds such as KmTxs, accounted for observed effects or whether other bioactive compounds were involved in microalgal toxicity.

## 2. Methods

### 2.1 *Karlodinium veneficum* cultures

Three French (FR) and two New Zealand (NZ) strains of *K. veneficum* were cultured for toxicity assessments (Table 1).

To confirm species identification DNA sequences (large subunit ribosomal RNA gene, D1-D3 region) from each strain were downloaded from GenBank (Table 1) and aligned with sequences from other *Karlodinium* spp., *Takayama* spp., and *Karenia* spp. from GenBank in Geneious Prime® v2019.2.3 (Kearse et al. 2012) using ClustalW (Thompson et al. 2003). Bayesian analyses were carried out in Geneious using MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001) using the evolutionary model (general time reversible with gamma-distributed rate variation across sites and a proportion of invariable sites, GTR + I + G). Analyses of alignments were carried out in two simultaneous runs with four chains each 2.1 10⁶ generations, sampling every 1000 trees. A 50% majority-rule consensus tree was drawn from the last 1000 trees. All final split frequencies were <0.01.

For each strain, three culture replicates were grown under standardized conditions. All cultures were grown in autoclaved seawater (which had either been collected from Concarneau, Atlantic Ocean for FR-grown strains or, from Tasman Bay, Pacific Ocean for NZ-grown strains) to which L1 nutrients (Guillard and Hargraves, 1993) had been added aseptically. The salinity of the FR and NZ grown cultures were 34-35 and 32-34 respectively. Cultures were grown at a temperature of 17 ± 1°C under 400-700nm LED lights (Aquatlantis EasyLed 2.0 – 6800K, FR; Cree CR 190 3000K, NZ). The light intensity was between 70-100µmol photons⁻² m s⁻¹ and cultures were grown on a 12:12h light:dark cycle.

Microalgae were grown in 0.5L glass flasks in a final volume of 330mL and 280mL for FR and NZ *K. veneficum* respectively. Microalgal growth was assessed every 2-3 days using Guava® easyCyte™ 5HT flow cytometer (Luminex Corporation, FR strains) or a Sedgewick rafter (NZ strains). Growth curves for all cultures are available in Supplementary Figure 1.
<table><caption>Table 1: Summary table of *Karlodinium veneficum* strains studied and their origins. FR = France, NZ = New Zealand.</caption><thead><tr><th>Strain</th><th>Location where cultured</th><th>Geographic origin</th><th>Date of isolation</th><th>GenBank accession number</th></tr></thead><tbody><tr><td>MAR1F7</td><td>FR</td><td>Le Maresclé, Morbihan, Brittany, Atlantic coast, FR</td><td>2019</td><td>PV853837</td></tr><tr><td>IFR-CC-20-44</td><td>FR</td><td>Urbino pond, Corsica, Mediterranean coast, FR</td><td>2020</td><td>PV853839</td></tr><tr><td>CBC7</td><td>FR</td><td>Concarneau Bay, Finistère, Brittany, Atlantic coast, FR</td><td>2018</td><td>PV853838</td></tr><tr><td>CAWD84</td><td>NZ</td><td>Kawau Bay, Hauraki Gulf, NZ</td><td>1994</td><td>AY947665</td></tr><tr><td>CAWD93</td><td>NZ</td><td>Ponui Island, Hauraki Gulf, NZ</td><td>1995</td><td>AY947666</td></tr></tbody></table>

## 2.2 Preparation of *K. veneficum* extracts

Cultures of *K. veneficum* were harvested at the start of the stationary phase (Figure 1, Supplementary Figure 1, Supplementary Table 1). Cultures were swirled gently to ensure uniform mixing of cells, before 250mL of culture were poured into 5 X 50mL falcon tubes. The falcon tubes were centrifuged at 1000 x g for 5 mins at 4°C to separate intracellular and extracellular fractions (one falcon for crude extract, the other four for methanolic extracts) before being kept protected from light on ice.

### 2.2.1 Crude extracts

Crude extracellular and intracellular fractions were obtained from 50mL of culture centrifuged as described above (one falcon tube per replicate). The supernatant ("crude extracellular" fraction) was transferred to glass vials, protected from light and immediately frozen at -80°C (Figure 1). The remaining pellet from this 50mL sample ("crude intracellular" fraction) was immersed in liquid nitrogen to inhibit enzymatic reactions, then resuspended in 50mL L1 medium in a glass container, protected from light, and lysed using either a 19kHz (FR strains) or 36kHz (NZ strains) ultrasonic probe for 5 mins. This "crude intracellular" fraction was then observed under a light microscope to check for cell lysis and transferred to amber glass vials before being stored at -80°C.

### 2.2.2 Methanolic extracts

The un-lysed pellets from 200mL culture (four falcon tubes per replicate) were immersed in liquid nitrogen and stored at -80°C, along with the remaining 200mL of culture supernatant stored in glass containers, protected from light, for later methanolic extraction.
After thawing the culture supernatant, C18 SPE cartridges (BondElut LRC C18 200mg, Agilent, 224 12mL) were used to extract karlotoxins from this extracellular fraction. The SPE cartridge was 225 activated with 5mL methanol followed by 5mL L1 medium, and the 200mL of previously frozen 226 supernatant was passed through the cartridge. The cartridge was then washed with 5mL MilliQ 227 water, dried and eluted twice with 1mL methanol to generate a final “methanolic extracellular” 228 extract of 2mL (Figure 1). 229

For *K. veneficum* intracellular fractions, culture pellets were resuspended in 1mL 100% methanol, 230 homogenized, ultrasonicated for 10 min, then centrifuged at 4000 x g for 5 min to remove debris. 231 This procedure was repeated twice to have a final “methanolic intracellular” extract of 2mL (Figure 232 1).

All methanol extracts were stored in amber glass vials at -80°C before use. They are hereafter 234 referred to as "Methanolic extracts". 235

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<figure><img src="image_2.png" /></figure>

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**Figure 1:** Harvest and extraction of *Karlodinium veneficum* cultures. The three culture replicates 239 were harvested at stationary phase and each were divided into 5 X 50mL tubes and centrifuged. 240 One 50mL tube was processed and stored at -80°C representing “crude extracellular” and “crude 241 intracellular” fractions. The remaining four tubes were processed, underwent extraction and were 242
stored at -80°C, representing the “methanolic extracellular” and “methanolic intracellular” fractions.

## 2.3 Karlotoxin determination

Karlotoxin analyses of the methanolic extracts were performed on an Ultra-Fast Liquid Chromatography (model UFLC, Shimadzu) coupled to a triple-quadrupole mass spectrometer (4000QTrap, Sciex) equipped with a turboV® ESI source, based on Krock et al. (2017). The chromatographic separation was carried out on a Kinetex C18 column (2.6µm, 150 × 2.1 mm, Phenomenex) equipped with a guard column (2.6µm, 5 × 2.1mm). The mobile phases were water (eluent A) and acetonitrile/water (95:5, v/v, eluent B), both containing 2.0mM ammonium formate and 50mM formic acid. The flow rate was 0.2mL min⁻¹ and the injection volume was 10µL. Column and sample temperatures were 40°C and 4°C, respectively. A gradient elution was used, starting with 5% B for 1 min, followed by a linear increase to 100% B in 8 min held for 6 min, then decreasing to initial conditions at 5% B in 0.1 min, held for 5 min to equilibrate the system.

In the absence of commercially available standards, and to properly detect and identify KmTxs, samples were iteratively analyzed in positive ionization using three different liquid chromatography-tandem mass spectrometry (LC-MS/MS) acquisition modes. For each method, mass spectrometric parameters are detailed in Supplementary material; Supplementary Table 2; and Beesoo et al. (2025). All the MS/MS spectra that were acquired, showing the KmTx-like structural characteristics, are provided as a supplementary pptx file.

## 2.4 Bioassay preparation

### 2.4.1 RTgill-W1 line culture

The RTgill-W1 cell line (CRL-2523, ATCC, USA), derived from *Oncorhynchus mykiss* rainbow trout gill cells, was grown under the same standardized conditions for experiments performed both in NZ and FR. The culture method followed ATTC recommendation and Dorantes-Aranda et al. (2011) description. Briefly, cells were grown in 75cm² tissue-culture treated flasks, in complete L15 growth medium (Leibovitz 15 (2.05mM L-Glutamine, HyClone™ SH30525.01), with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (A5955, Sigma) added). Cells were incubated at 19°C in the dark, culture medium was changed every 2-3 days and cells were passaged every ~10 days, at ~80% confluency. During passages, cells were rinsed with a sterile saline solution in phosphate buffer (PBS 1X) and then detached using 0.25% trypsin-0.02% EDTA solution (Dorantes-Aranda et al., 2011). Cell concentration and viability were determined after staining with 0.02% trypan blue (final concentration) either using an automated cell counter (CellDrop FL, DeNovix, Wilmington, DE, USA) or hemocytometer (FR and NZ, respectively).
276 2.4.2 Oyster broodstock

277 For the experiments performed in FR, 100 18-month-old *Crassostra (=Magallena) gigas* oysters
278 were produced and conditioned for gonadal maturation at the IFREMER Plateforme
279 Expérimentale des Mollusques Marins in Bouin, FR. Once mature, oysters were transferred to
280 LEMAR facilities (Plouzané, FR) for the duration of the experiments (one month), oysters were kept
281 in 100L tanks at 16°C (around 50 oysters per tank) with open flow seawater filtered at 1µm in the
dark. They were fed with a concentrated phytoplankton mixture (Sustainable Aquatics Live Marine
282 Phytoplankton - Premium Reef Blend, containing *Nannochloropsis oculata*, *Phaeodactylum*
283 tricornutum and *Chlorella* sp., 1mL per day). Oyster size averaged 77.3 ± 10.1g (mean ± SD).
284

285 For the experiments performed in NZ, two-year old NZ Pacific oysters *C. gigas* were obtained from
286 the field (Marlborough Sounds and Mahurangi, NZ, n = 100 respectively) and kept at the Cawthron
287 Aquaculture Park in Nelson for 8-10 weeks prior to spawning. During this time, oysters were fed
288 *ad-libitum* with the natural microalgae present in production ponds and maintained at a
289 temperature of 21 ± 1 °C to bring them into spawning condition. Oyster size averaged 71.9 ± 12.9g
290 (mean ± SD, Marlborough) and 71.1 ± 11.6g (mean ± SD, Mahurangi).

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292 2.5 Bioassays

293 i) Inter-laboratory assay comparison using the same algal samples

294 Before comparing the toxicity of different *K. veneficum* strains, methanolic extracts from the three
295 FR *K. veneficum* strains were tested on fish gill and oyster sperm cells in both FR and NZ to
296 evaluate the consistency and repeatability of the assays.

297 ii) Toxicity comparison of different algal strains

298 Following this assay assessment, a comparison of the toxicity of the different *K. veneficum* strains
299 was carried out. Crude and methanolic extracts from all five *K. veneficum* strains (three FR and
300 two NZ) were screened on fish gill and oyster sperm cells.

301 iii) Dose-response assays on each cell model

302 For each *in vitro* model, a dose-response assay was then performed for a selected *K. veneficum*
303 strain that induced more than 60% mortality.

304 2.5.1 Exposure concentrations

305 i & ii) Assay comparison and toxicity screening
To ensure viability results of the inter-laboratory assay comparison and extract screening were comparable, for each bioassay (fish gill cells and oyster sperm, exposed to either crude or methanol extracts), extracts were prepared as detailed below.

Firstly, all *K. veneficum* samples were adjusted to similar concentration (1.75 x 10⁵ equivalent cells mL⁻¹ for crude fraction, or 100x concentrated for methanolic extract, Supplementary Table 1), in L1 media or 100% methanol, respectively. Samples were further diluted according to target cells tolerance to the solvent (L1 medium or methanol) as described Table 2 and following assays described thereafter. For methanolic extracts, 1% and 0.5% final methanol concentration were shown to have no toxic effects on fish gill cells and oyster sperm, respectively (data not shown). For crude extracts L1-seawater diluted 4X into L15/ex media did not show any toxicity to fish gill cells for 2h (data not shown). The final equivalent *K. veneficum* exposure concentrations (in equivalent cells mL⁻¹) for each bioassay are given in Table 2.

Table 2: Exposure concentrations of *Karlodinium veneficum* crude and methanolic extracts in microalgal cell equivalents per mL for fish gill cell (RTgill-W1) or oyster spermatozoa bioassays. Percent values in brackets indicate the corresponding concentration of solvent (L1 media or methanol).

<table><thead><tr><th rowspan="2">Bioassay<br>Extract</th><th colspan="2">RTgill-W1</th><th colspan="2">Oyster sperm</th></tr><tr><th>Crude (25% L1)</th><th>Methanolic (1%)</th><th>Crude (50% L1)</th><th>Methanolic (0.5%)</th></tr></thead><tbody><tr><td>Equivalent<br>microalgal<br>exposure<br>concentration<br>(cells mL<sup>-1</sup>)</td><td>4.38 x 10<sup>4</sup></td><td>FR: 4.38 x 10<sup>4</sup><br>NZ: 1.75 x 10<sup>5</sup></td><td>8.75 x 10<sup>4</sup></td><td>8.75 x 10<sup>4</sup></td></tr></tbody></table>

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iii) Dose-response assays on each cell model

For each *in vitro* bioassay model, we selected methanolic extracts from one *Karlodinium* strain identified to be toxic from screening exposures (inducing >60% mortality), which were then used to obtain dose-response curves. Fish gill cells were thus exposed to serial dilutions of methanolic extracts from the NZ strain (CAWD93) and oyster sperm were exposed to methanol extracts of the FR strain (MAR1F7). Concentrations inducing 50% cell mortality (EC₅₀) were then estimated.

2.5.2 Fish gill cell line (RTgill-W1) viability assay

Bioassays were conducted in 96-well flat-bottom microplates (167008, Thermo Fisher Scientific) following Beesoo et al. (2025), adapted from Dorantes-Aranda et al. (2011).

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Briefly, cells at 80-90% confluence and >90% viability were diluted to 1.5 x 10⁵ cells mL⁻¹ in complete L15 medium. Each well received 100µL of the cell suspension, while border wells were filled with PBS. After 24 h at 19°C in the dark, cells were rinsed with PBS and exposed to 100 µL of *K. veneficum* extracts (Section 2.2 & 2.4.1), diluted in L15/ex: 25% for crude and 1% for methanol extracts. Exposures lasted 2h (crude) or 24h (methanol extracts, Figure 2A) at 19 °C in the dark. Cells were then rinsed with PBS, incubated 30 min with 5% PrestoBlue (Invitrogen™) in L15/ex in the dark, and fluorescence was measured (Ex: 550 ± 10nm, Em: 590 ± 10nm) on FLUOstar® Omega (FR) or EnSpire (NZ) plate readers. Viability (%) was calculated as: (sample – blank) / (negative control – blank) × 100. Controls included: positive (saponin, 50µg mL⁻¹), negative (L15/ex), solvent (25% L1 or 1% MeOH in L15/ex), and blanks (no cells). Five well-replicates were used for each sample, control and blank and solvent control viability was confirmed >90% and comparable to the L15/ex negative control.

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 2:** Schematic of the bioassay methods, showing exposure of RTgill-W1 cells (A) and oyster, *Crassostrea gigas*, sperm cells (B) to either crude or methanolic extracts of *Karlodinium veneficum* for either screening or, to obtain a dose-response curve.</figcaption></figure>
2.5.3 Oyster spermatozoa assays

Sperm was stripped from mature oysters and examined under a light microscope; dense, motile spermatozoa were selected from 4 (NZ) and 5 (FR) males for assays. Gametes were collected in 0.22µm-filtered sterile seawater (FSW), sieved through 100µm mesh to separate sperm from debris and sperm concentration was determined by flow cytometry (Le Goïc et al., 2013). Sperm was diluted to 10⁶ cells mL⁻¹ in FSW.

Test solutions (1mL total volume; 50% sperm, 50% extract or control) were prepared in eppendorf tubes and incubated in the dark at 20°C for 2h (Figure 2B). Controls were L1 medium for crude extracts and 1% MeOH for methanol extracts. Viability and ROS production were assessed on spermatozoa using a Guava® easyCyte™ 5 HT flow cytometer (Guava Technologies, Millipore) equipped with a 488nm argon laser and three fluorescence detectors: green (525 ± 15nm), yellow (583 ± 13nm) and red (680 ± 15nm).

2.4.3.1 Viability assessment

After 1h50 of exposure of sperm, 200µL of the homogenised test solution was transferred to 96-well plates. Propidium iodide and SYBR14 were added at final concentration of 10µg mL⁻¹ and 1µM respectively and samples were incubated for 10 min at 20 °C in the dark. Viability was determined from relative red (dead) or green (live) fluorescence according to Le Goïc et al. (2013).

2.4.3.2 Reactive oxygen species (ROS) production assessment

After 1h of exposure, 200µL of the test solution was transferred to 96-well plates and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to the sample at 10µM final concentration. Plates were incubated at 20°C in the dark for 1h. Oxidation of DCFH-DA to fluorescent DCF enabled relative ROS quantification (green fluorescence).

2.5 Statistics and modelling of dose-response curves

Statistical and graphical analyses were produced using R version 4.4.0 (R Core Team, 2024). Percentage viability data were transformed as appropriate (arcsine or log) and normality of the ANOVA residuals was checked using the Shapiro-Wilk test and homogeneity of variance with Levene's test. If the framework was parametric, a one-factor ANOVA followed by a Tukey HSD post-hoc test was performed, and if non-parametric, a Kruskal-Wallis test followed by a Dunn test for multiple comparison were performed and differences were considered significant if the p-value was less than 0.05. On one occasion, data was normal but non-homogenous and a Welch's ANOVA followed by Games-Howell post-hoc test was used. Significant differences between culture replicates were checked; culture replicate 1 of IFR-CC-20-44 was different from other replicates and was not grouped with the other replicates for analysis (beginning of decay phase, Supplementary Figure 1).
384 To model the relationship between viability and microalgal cell concentration, a series of 385 nonlinear dose-response models were fitted using the drm() function from the {drc} package in R. 386 Candidate models included several commonly used sigmoidal functions.

387 Model selection was based on the Akaike Information Criterion (AIC), with the model exhibiting 388 the lowest AIC considered the best fit to the data. The model obtained was then compared with 389 the null model using a Fisher test and then validated if the p-value was less than 0.05.

390 Correlations between both the inter-laboratory assays and the total karlotoxin content versus 391 biological toxicity were performed and Pearson's correlation coefficient (r) and the associated p- 392 value were calculated.

393 **3 Results**

394 Phylogenetic analyses of the five strains from FR and NZ showed that all clustered with other K. 395 *veneficum* DNA sequences with high support (Supplementary Figure 2).

396 **3.1 Inter-laboratory assay comparison using the same algal strains**

397 The mean viabilities of fish gill- and oyster sperm-cells which had been grown or attained in each 398 respective country (FR and NZ) and exposed to the same methanol extracts of *K. veneficum*, were 399 compared (Figure 3). The RTgill-W1 line which had been grown in each respective country and 400 exposed to the same three French *K. veneficum* strains showed an R² value of 0.56 (Figure 3A). 401 The viability results from oyster sperm which had been stripped from oysters from either NZ or 402 France and exposed to the same methanol extracts, were highly correlated, with an R² value of 403 0.93 (Figure 3B).
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

**Figure 3:** Comparison of the mean viability of chemical extracts from three different strains (each with two fractions) of *French Karlodinium veneficum*, tested on French (FR) and New Zealand (NZ) RTgill-W1 cells (A) and *Crassostrea gigas* sperm (B). % values were normalised to the control for each assay and mean control viability was > 90% for all assays. Screening exposures were 4.38 x 10⁴ cells mL⁻¹ for gill cells and 8.75 x 10⁴ cells mL⁻¹ for oyster sperm (Table 2). n = 12 (NZ oyster sperm) and n = 15 (FR oyster sperm and fish gill cells).

## 3.2 Inter-laboratory extract screening of *Karlodinium veneficum* strains

Following the assay comparison, all the extracts from *K. veneficum* strains were screened to assess their toxicity. For this, fish gill cells and oyster sperm were exposed to intracellular and extracellular crude- and methanolic-extracts in each respective country at the exposure concentrations shown in Table 2.
417 **3.2.1 Crude extracts**

418 RTgill-W1

419 Only the intracellular crude extract of strain CAWD93 induced significant mortality of RTgill-W1

420 cells, with the mean viability dropping to 66% compared with 99.7% in the controls (p = 0.007,

421 Figure 4).

422

<figure><img src="image_7.png" /></figure>

423

424

425 **Figure 4:** Effect of crude intracellular and extracellular *Karlodinium veneficum* fractions (tested at 4.38 x 10⁴ equivalent cells mL⁻¹) and 25% L1 media control on gill cell line (RTgill-W1) viability.

426 Results are presented as median with interquartile range (central line and box), whiskers represent the minimum and maximum values and outliers are points beyond the whisker.

427 Different letters indicate significant differences between groups (p < 0.05). Tests on gill cells
428 were repeated 5 times (n = 5 well replicates) for each of the three culture replicates per strain,
429 i.e. n = 15 for each strain. Nb: only strain replicates 2 and 3 were tested for IFR-CC-20-44, as the
430 replicate 1 culture was in decline, not stationary phase.

433

434 *Oyster sperm*
Of the four crude extracts from different strains of *K. veneficum* tested (MAR1F7, CBC7, CAWD84 and CAWD93), only the extra- and intracellular fractions of CAWD93 induced significant mortality of oyster sperm, inducing 63% and 81% mortality respectively (p < 0.0001, Figure 5). There was no significant effect of any of the *K. veneficum* crude 'extracts' on sperm ROS production.

439

<figure><img src="image_8.png" /></figure>

440

441

Figure 5: Effect of crude intracellular and extracellular *K. veneficum* 'extract' fractions (tested at 8.75 x 10⁴ equivalent cells mL⁻¹) and L1 media control on *Crassostrea gigas* sperm viability.

Results are presented as median with interquartile range (central line and box), whiskers represent the minimum and maximum values and outliers are points beyond the whisker.

Letters indicate significant differences between groups. N = 5 for FR for one culture per strain and n = 4 for NZ for each of the three culture replicates per strain, i.e. n = 12-15 for each strain.

### 3.2.2 Methanolic extracts

Methanolic extracts from all five *K. veneficum* strains were toxic to both fish gill and sperm cells (Figures 6 and 7).

#### RTgill-W1

Although not screened at the same concentration (Table 2), The MAR1F7 extracellular (4.38 x 10⁴ cells mL⁻¹) and CAWD93 intracellular (1.75 x 10⁵ cells mL⁻¹) fractions were particularly toxic to fish gill cells, causing >90% mortality (MAR1F7: p-value < 0.0001; CAWD93: p-value < 0.0001, Figure
455

6). The extracellular fraction of CBC7, intracellular fraction of IFR-CC-20-44 and both fractions of CAWD84 did not induce significant toxicity in gill cells (p> 0.05, Figure 6).

456

457

<figure><img src="image_9.png" /></figure>

458

459

460

**Figure 6:** Effect of intracellular and extracellular *Karlodinium veneficum* methanol extracts (tested at $4.38 \times 10^4$ (FR) and $1.75 \times 10^5$ (NZ) equivalent cells mL$^{-1}$) and 1% MeOH control in L15/ex medium on gill cell line (RTgill-W1) viability. Results are presented as median with interquartile range (central line and box), whiskers represent the minimum and maximum values and outliers are points beyond the whisker. Different letters indicate significant differences between groups (p<0.05). Tests on gill cells were repeated 5 times (n = 5 well replicates) for each of the three culture replicates per strain, i.e. n = 15 for each strain. Nb: only strain replicates 2 and 3 were tested for IFR-CC-20-44 as the replicate 1 culture was in decline, not stationary phase. FR= France, NZ= New Zealand.

461

462

463

464

465

466

467

468

469

470

### Oyster sperm

471

The same extracellular methanol extract of *K. veneficum* MAR1F7 was also highly toxic to oyster sperm cells (p-value <0.0001), inducing ~65% mortality (Figure 7). Except for MAR1F7, intracellular fractions induced higher sperm mortality than extracellular fractions. No effect was observed on ROS production for any of the methanolic *Karlodinium* extracts.

472

473

474
<figure><img src="image_10.png" /></figure>

476

**Figure 7:** Effect of methanol intracellular and extracellular *K. veneficum* extract fractions (at 8.75 X 10⁴ equivalent cells mL⁻¹) and 0.5% MeOH control in L1 medium on *Crassostrea gigas* sperm cell line viability. Results are presented as median with interquartile range (central line and box), whiskers represent the minimum and maximum values and outliers are points beyond the whisker. Letters indicate significant differences between groups. N = 5 for France and n = 4 for New Zealand for all three culture replicates. Nb: only strain replicates 2 and 3 were tested for IFR-CC-20-44 as the replicate 1 culture was in decline, not stationary phase.

477

478

479

480

481

482

483

484

485

## 3.3 Dose-response assays on each cell model

486

### RTgill-W1

487

488

489

490

491

Fish gill cells were exposed to serial dilutions of methanolic extracts from strain CAWD93, which had been identified as toxic during screening. The equivalent concentration of CAWD93 inducing 50% mortality of the cells (EC₅₀) was 1.21 ± 0.10 x 10⁵ cells mL⁻¹ for the intracellular fraction and was 1.20 ± 0.08 x 10⁵ cells mL⁻¹ for the extracellular fraction (Figure 8A and B, mean ± standard error, SE).

492

493
<figure><img src="image_11.png" /><figcaption>**Figure 8:** Dose-response curves for RTgill-W1 cells exposed to A) Intracellular and B) Extracellular fractions of CAWD93 methanol extracts. Three culture replicates were tested against up to 5 wells for each algal concentration, n =15. A two-parameter log-logistic model was run, curves are displayed with the 95% confidence interval uncertainties in grey, abscissa axis with logarithmic scale. For A) Fisher test with null model (p-value = 2.2 x 10⁻¹⁶), EC₅₀ = 1.21 ± 0.10 x 10⁵ cells mL⁻¹, for B) Fisher's test with null model (p-value = 2.2 x 10⁻¹⁶), EC₅₀ = 1.20 ± 0.08 x 10⁵ cells mL⁻¹.</figcaption></figure>

### Oyster sperm

Oyster sperm were exposed to methanol extracts of strain MAR1F7. The EC₅₀ of the MAR1F7 intracellular methanolic extract was 4.56 ± 8.56 x 10⁵ cells mL⁻¹, mean ± SE, Figure 9A). The
estimated EC₅₀ for the extracellular methanolic extract of MAR1F7 was 8.65 ± 0.35 x 10⁴ cells mL⁻¹.

¹, mean ± SE, Figure 9B).

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

**Figure 9:** Dose-response curve for oyster sperm mortality for the A) intracellular fraction and B) extracellular fraction of MAR1F7 methanolic extracts. A mixture of the three culture replicates

were tested against up to 6 oysters for each algal concentration, n = 3-6. Curves are displayed

with the 95% confidence interval uncertainties in grey, abscissa axis with logarithmic scale. A)

Four-parameter log-logistic model, fisher test with null model (p-value= 0.6), EC₅₀ = 4.56 ± 8.56 x 10⁵ mean ± SE, cells mL⁻¹.

B) Three-parameter log-logistic model, fisher's test with null model (p-value = 2.2 x 10⁻¹⁶), EC₅₀ = 8.65 ± 0.35 x 10⁴, mean ± SE, cells mL⁻¹.
518

519

520 3.4 Karlotoxin quantification of methanol extracts

521 Methanolic extracts were analysed for the presence of karlotoxins and only compounds 522 exceeding 1% of the overall profile of each sample are reported (Figure 10). The toxin content 523 varied greatly between strains. Karlotoxins and karlotoxin-like compounds were detected only in 524 cultures MAR1F7, IFR-CC-20-44 and CAWD93, where they were significantly more abundant in 525 the extracellular fractions (MAR1F7= 5.3 vs 0.6 area cell⁻¹, p<0.0001; IFR-CC-20-44= 1.9 vs 0.5 526 area cell⁻¹, p< 0.004; CAWD93= 3.7 vs 1.1 area cell⁻¹, p< 0.0001, Figure 10). Known karlotoxins, 527 KmTx-5 and a KmTx-5 analogue, were present only in French cultures MAR1F7 and IFR-CC-20-44, 528 accounting for approximately 55-80% of the toxin profile in each culture. Nearly 100% of the toxin 529 present in CAWD93 was from an unknown toxin analogue, 1307>895 - RT 7.4 (Figure 10).

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

530

531 **Figure 10:** Concentrations of karlotoxin and karlotoxin-like compounds identified in methanolic 532 extracts from multiple strains of *Karlodinium veneficum*. Only compounds exceeding 1% of the 533 overall profile of each sample are shown. Compounds identified were 1325>895 - RT 7.6 (KmTx-5), 1325>895 - RT 7.5 (KmTx-5 analogue), 1303>891 - RT 7.7, 1274>895 - RT 7.3, 1383>877 - RT 7.5, 534 1383>877 - RT 7.6, 1395>877 - RT 7.2, 1427>877 - RT 7.5, 1307>895 - RT 7.5, 1307>895 - RT 7.4, 535 1307>895 - RT 6.8, 1341>911 - RT 7.5. Red dots represent mean ± SD of total concentration, 536
537 expressed in Area. cell⁻¹. N=3 culture replicates, except for IFR-CC-20-44 where N=2 culture
538 replicate.

539
540 3.5 Correlation between karlotoxin and biological toxicity
541 The correlation between the total karlotoxin content and biological toxicity for each of the
542 bioassays was low to moderate, with Pearson's correlation coefficient (r) values between 0.09 and
543 -0.54 (Table 3, Supplementary Figure 3). Although total KmTx in the methanolic extracts were
544 significantly correlated with fish gill cell toxicity (p = 0.0033), R² was still low (0.3, Table 3).

545 **Table 3:** Pearson correlation coefficient (r), p-values and R² of the Pearson correlation test
546 between biological toxicity (mean cell viability, %) and total KmTx content (area cell⁻¹) for RTgill-
547 W1 cells and *Crassostrea gigas* sperm exposed to crude and methanolic extracts of *Karlodinium*
548 veneficum.

<table><thead><tr><th>Extract</th><th>Bioassay</th><th>r</th><th>p-value</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td rowspan="2">Crude</td><td>RTgill-W1</td><td>0.09</td><td>0.71</td><td>0.008</td></tr><tr><td>Oyster sperm</td><td>-0.44</td><td>0.069</td><td>0.19</td></tr><tr><td rowspan="2">Methanolic</td><td>RTgill-W1</td><td>-0.54</td><td>0.0033</td><td>0.3</td></tr><tr><td>Oyster sperm</td><td>-0.34</td><td>0.14</td><td>0.12</td></tr></tbody></table>

549
In this study, we used *in vitro* bioassays to compare the toxicity of *Karlodinium veneficum* strains isolated from France (FR) and New Zealand (NZ). Both laboratories used standardised growth conditions and protocols for culturing the microalgal strains and preparing extracts to enable comparisons of results. A strong correlation was observed between laboratories when exposing cells to the same methanolic extracts, indicating consistency across assays. Screening five different strains of *K. veneficum*, showed that toxicity was highly dependent on the strain of *K. veneficum*, the type of extract (crude or methanol), the culture fraction (intracellular or extracellular) and targeted cell-type (fish or oyster) tested. Biological toxicity was poorly correlated with total KmTx. Furthermore, dose-response assays on extracts identified as more toxic following screening, enabled determination of the equivalent cell concentration that induced 50% cell mortality (EC₅₀).

At screening concentrations, all tested strains of *K. veneficum*, except CAWD84 from NZ, induced significant toxicity to at least one of the exposed cell types. While the specific screening concentrations varied depending on the bioassay and extract used, all were ≥ 4.38 x 10⁴ equivalent cells mL⁻¹. Dose-response assays further confirmed that microalgal concentrations causing 50% cell death also fell within the 10⁴–10⁵ cells mL⁻¹ range. These concentrations align with those typically observed during *K. veneficum* blooms, which often occur in shallow, stratified systems and reach levels between 10⁴ and 10⁶ cells mL⁻¹ (Place et al., 2012; Kempton et al., 2002; Hall et al., 2008). The negative effects observed in the present study at these environmentally relevant concentrations are therefore not unexpected, especially considering the microalgal densities previously associated with fish and shellfish mortalities (Kempton et al., 2002; Hall et al., 2008; Place et al., 2012; Calbet et al., 2011). For instance, Hall et al. (2008) documented *K. veneficum* bloom concentrations exceeding 2 x 10⁵ cells mL⁻¹ in North Carolina, USA, which resulted in fish kills affecting species such as trout, flounder, and bass. Similarly, Glibert et al. (2007) reported nearly 95% deformities—an early indicator of mortality—in *Crassostrea gigas* (Pacific oyster) embryos exposed for 48 hours in the lab to *K. veneficum* at just 10⁴ cells mL⁻¹.

To assess toxicity, cell viability was measured using the PrestoBlue® reagent for RTgill-W1 cells and dual staining with SYBR14 and PI for oyster sperm cells. PrestoBlue® detects a reduction of a resazurin-based compound to resorufin inside the gill cells (Ahmed et al., 1994; O'Brien, Wilson, Orton, & Pognan, 2000) suggesting that the cytotoxic compounds from the various *K. veneficum* extracts tested disrupt the metabolic activity in the cells. This is in keeping with previous studies, which have shown that toxic compounds associated with *K. veneficum* have many effects including, targeting the gills of fish, especially the chloride cells responsible for osmoregulation (Deeds et al., 2006). They have also been shown to be hemolytic and have allelopathic effects
585 (Adolf et al., 2015; Place et al., 2012) and in vertebrate cells, the toxic compounds have been
586 shown to make pores in the plasma membranes (Deeds et al 2006, 2015). Other strains of K.
587 *veneficum* have also been reported to be toxic to RTgill-W1 cells (Dorantes-Aranda et al., 2015;
588 Beesoo et al., 2025). On oyster sperm, the fluorescent dye, PI, penetrates cells when cell
589 membranes are damaged, and the cytotoxicity measured in the present study suggests that toxic
590 compounds resulted in pores developing in the sperm membrane.

591 Contrastingly, none of the extracts tested in the present study resulted in a change in sperm ROS
592 production, suggesting that the strains and extracts tested do not induce oxidative stress in the
593 target oyster cells. Studies of other fish and shellfish species, however, show that *K. veneficum*
594 does induce ROS production, which is hypothesised to form part of their fish killing mechanism
595 (Dorantes-Aranda et al., 2011; Galimany et al., 2008; Marshall et al., 2005; Mooney et al., 2007).

596 Although almost all the algal strains were toxic to some degree, there were considerable
597 differences in toxicity. Methanol and crude extracts of NZ strain CAWD93 induced significant
598 toxicity on both fish gill and oyster sperm cells, as did methanol extracts of French strains MAR1F7
599 and IFR-CC-20-44. In contrast, CBC7 (FR) and CAWD 84 (NZ) were much less toxic, inducing
600 negligible effects on fish and oyster cells. Variation in the toxicity of *K. veneficum* populations is
601 evident in the wild, where blooms reaching 10³ cells mL⁻¹, have coincided with high mortalities of
602 the razor clam, *Ensis ensis* (Fernandez-Tejedor et al., 2010) yet large blooms approaching 10⁶ cells
603 mL⁻¹ have been reported in the USA with no observed mortalities (Place et al., 2012). Variability of
604 physiological traits among strains of *K. veneficum* from a same bloom also was evidenced, and is
605 believed to be important for bloom formation (Calbet et al., 2011). Karlotoxin analysis of the
606 methanolic extracts also showed strains CAWD93, MAR1F7 and IFR-CC-20-44 contained
607 karlotoxins and karlotoxin-like compounds, whereas strains CBC7 and CAWD84 did not contain
608 detectable levels of KmTx. A high degree of toxin variation has been recorded between strains of
609 *K. veneficum* (Bachvaroff et al., 2009; Krock et al 2017) from genetically distinct populations
610 (Place et al., 2012). Toxin production ranges from non-detectable, to picograms per cell and many
611 strains show some toxin production (Place et al., 2012). Toxin production of *K. veneficum* could
612 correspond to a competition strategy with other microalgal species or, defence against grazers
613 (Adolf et al., 2009; Wang et al., 2020). *K. veneficum* is also a mixotrophic species, and toxin
614 production may be related to predation to immobilize and kill its prey (Berge et al., 2012; Sheng et
615 al., 2010).

616 Karlotoxins have been shown to cause plasma membrane perforations, having lytic, cytotoxic and
617 ichthyotoxic effects (Deeds et al., 2015; Waters et al., 2015). FR strain MAR1F7, contained more
618 karlotoxins and karlotoxin-like compounds in the extracellular methanolic fraction and was
619 significantly more toxic than the intracellular fraction when tested on both bioassays, suggesting
a causal link between bioactivity and KmTx contents. However, IFR-CC-20-44 and CAWD94 resulted in similar levels of biological toxicity to both cell types despite CAWD94 containing almost twice as much karlotoxin per cell. Indeed, biological toxicity and total KmTx content were only weakly correlated. However, the differences in biological toxicity may be due to different karlotoxins having different potencies. Indeed, the toxicity of KmTx's appears to be driven by e.g. the length of the lipophilic arm, the chlorination/sulfatation or the degree of saturation (Place et al., 2012). Van Wagoner et al. (2010) for example, reported that KmTx 1 and the chlorinated congener 64-E-chloro-KmTx 3 were approximately 2X more potent than KmTx 3, while KmTx 2 was nearly 10X less potent than either KmTx 1 or KmTx 3 to rainbow trout red blood cells. In the present study, none of these karlotoxins were detected in methanolic extracts, however; NZ strain CAWD93 contained nearly exclusively a previously unknown toxin analogue 1307>895 - RT 7.4, whereas FR strains MAR1F7 and IFR-CC-20-44 contained mostly KmTx-5 and the KmTx-5 analogue, which may be more potent. Further studies should investigate the potencies of these different analogues.

Differences in biological toxicity may also be due to undetected karlotoxin-like compounds. Strain CAWD93, such as MAR1F7, contained more karlotoxins in the extracellular methanolic fraction; however, unlike MAR1F7, CAWD93 exhibited greater biological toxicity in the intracellular fractions across both bioassays. Similarly, toxic effects were observed in a treatment that did not contain detectable karlotoxins (CBC7 intracellular methanolic extract on the RT-W1gill cells). The detection and quantification of karlotoxins remains time-consuming and are limited by both the lack of sensitivity and the absence of commercial standards, highlighting the possibility that novel, yet uncharacterized, compounds may be contributing to the observed toxicity. Indeed, the detection of new candidate analogues here, as already suggested in a previous study (Beesoo et al. 2025), may confirm that the chemodiversity of KmTxs is much higher than current knowledge.

Additional toxicity may arise from compounds other than karlotoxins. For instance, the methanolic intracellular fraction of CBC7 was toxic to fish gill (RTgill-W1) cells, despite no detectable karlotoxins (KmTx) in this extract. This suggests the presence of unknown bioactive metabolites alongside or independent of karlotoxins. Indeed, *Karlodinium* is known to produce a diverse array of bioactive compounds, as highlighted by Assunção et al. (2022), though many remain poorly characterized. Dinoflagellates are known to produce a vast array of diverse secondary metabolites. For example, *Alexandrium* spp., can not only produce paralytic shellfish toxins, but also a suite of bioactive compounds, of which the activity can vary significantly with species and strains (Long et al., 2021). To highlight the presence of other bioactive metabolites in these *K. veneficum* strains, a bioguided fractionation approach should be implemented in future studies, such as recently conducted to discover the leadbeaterins produced by *Chrysochromulina leadbeateri* (Wang et al., 2024).
656 Methanolic extracts may increase the apparent bioavailability of these compounds, as crude
657 extracts can contain additional substances—such as extracellular matrix components or other
658 cellular debris—that might buffer or mask toxicity. In the case of CAWD93 however, crude extracts
659 induced greater sperm mortality than did methanolic extracts. It is possible that some of the
660 toxicity observed in crude extracts may be attributable to metabolites that would not have been
661 extracted in methanol, from algal origin, or also possibly from bacterial origin. Indeed, the
662 extracts were derived from non-axenic cultures and microalgae naturally co-exist with bacteria,
663 often forming mutualistic associations (Ramanan et al., 2016).

664 It is important to note that for RTgill-W1 cells however, the different cytotoxicities recorded
665 between crude extracts and methanolic extracts could be partly explained by the exposure time
666 and concentration. Crude extracts were tested for 2 hours at an equivalent exposure of 4.38 x 10⁴
667 cells mL⁻¹ (due to saline conditions becoming toxic to gill cells after this time), whereas
668 methanolic extracts were tested over 24 hours at a higher concentration equivalent to 1.75 x 10⁵
669 cells mL⁻¹.

670 Similarly, although most methanolic extracts were toxic to fish gill cells and were less toxic to
671 oyster sperm cells (with only MAR1F7, IFR-CC-20-44 and the intracellular fraction of CAWD93
672 being toxic to oyster spermatozoa), suggesting an effect specific to the target cell type, this could
673 also be due to the different exposure time of the assay (24 h for RTgill-W1 vs 2 h for oyster sperm)
674 and that sperm were exposed to different concentrations, both higher and lower than those of the
675 fish gill cells (sperm= 8.75 x 10⁴ cells mL⁻¹ equivalent vs fish = 4.38 x 10⁴ (FR) and 1.75 x 10⁵ (NZ)
676 cells mL⁻¹).

677 Despite the differences between each bioassay making direct comparison of toxicity *between*
678 each target species difficult; for each target species, the same culture and bioassay protocols
679 were followed, enabling comparison of toxic algal strains between laboratories.

680 To further ensure comparability between the assays performed in the two laboratories, RTgill-W1
681 cells were attained and grown under the same conditions in FR and NZ and exposed following the
682 same protocols to the same methanol extracts of FR *K. veneficum* strains. These protocols were
683 a variant of the OECD guidelines (Test No. 249: Fish Cell Line Acute Toxicity - The RTgill-W1 cell
684 line assay method) and of the method developed by Dorantes-Aranda et al. (2011) for the study of
685 toxic microalgae. Pacific oyster, *C. gigas*, sperm were obtained from stocks present in each
686 country and gametes were stripped and exposed under standardized protocols as described
687 previously. We used oyster spermatozoa, as they are a sensitive cell type, relatively easy to handle
688 and have been used in many studies on the effects of HAB species (e.g. Castrec et al., 2021; Le
689 Goïc et al., 2013; Rolton et al., 2015; Greenhough et al., 2024).
Comparing the toxicity of the same methanolic extracts of FR strains MAR1F7, IFR-CC-20-44 and CBC7 on the two bioassays in each country showed relatively similar results. However, the linear regression between oyster sperm assays in both countries was better (higher R²) than for the RTgill-W1 assay. This is surprising, given that for the fish bioassay, the same RTgill-W1 cell line was exposed whereas, for the oyster bioassay, there would have been considerable genetic variation associated to oyster provenance. Although every attempt was made to ensure assays were completed within a few months of preparing the algal extracts, testing the FR methanolic extracts on NZ cells happened 3 months after exposure of FR cells and it is possible there was a loss of toxicity during this time. It is therefore recommended that bioassays should be done closer in time to each other. Furthermore, comparison of gill-line sensitivity against a whole animal model is needed to clarify the consequence of such observations on entire fishes in real-world scenarios. Several studies highlighted similar results between EC₅₀ of RTgill-W1 *in vitro* assay and LC₅₀ of *in vivo* assays on different fish species and testing different chemicals, following the OECD Test No. 249 protocol (Tanneberger et al. 2013; Natsch et al. 2018). However, Mooney et al. (2011) demonstrated that the RTgill-W1 line was more sensitive than larvae of the sheepshead minnow, *Cyprinodon variegatus*, upon exposure to fatty acids produced by *K. veneficum*, *K. conicum* and *Karenia mikimotoi*, highlighting the need for further studies on microalgae ichthyotoxicity.

## 5 Conclusion

The sensitivity of oyster sperm and fish gill cell lines to certain *K. veneficum* strains underscores potential risks to fisheries and aquaculture in both FR and NZ. Identifying whether toxins are intracellular or extracellular provides insight into potential impacts: if the toxins were released extracellularly, their effects would be most pronounced at the peak of a bloom. Conversely, if the compounds were intracellular, their impact would be greater toward the end of a bloom when microalgal lysis occurs. Strains like MAR1F7 and CAWD93, from each country, are therefore of particular concern, as they are toxic to both oyster sperm and fish gill cells and the toxic compounds are detected both intracellularly and extracellularly.

This study also demonstrates that standardized methods allow global comparison of *K. veneficum* toxicity and identification of harmful algal concentrations. The observed variation among strains highlights the importance of testing local, recent HAB isolates and *in vitro* methods like RTgill-W1 and oyster sperm assays offer fast, high-throughput, and ethical toxicity screening, providing insights into toxic mechanisms. These techniques can also be adapted for other cell lines or bivalve species.

## Acknowledgements

We thank Fernando Ramos Queiroga and Charlotte McDonald for support in the laboratory, Véronique Séchet and Liliane Carpentier for contribution to algal cell isolation, and Gwenael
725 Bilien for genetic identification of French strains. This work was made possible through French
726 ANR-22-CE20-0024 HABIS project, a Royal Society Te Apārangi Catalyst Seeding grant (CSG-
727 CAW2102) and was further funded by the New Zealand Ministry of Business, Innovation and
728 Employment (CAWX2207, CAWX1801 and CAW2438). Guillaume Barnouin's internship was
729 funded by Ifremer, and Hannah Greenhough's travel was supported by the Marjorie McCallum
730 Award and a University of Otago PhD scholarship.

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Supplementary figure 1: Growth curves of the five strains of *Karlodinium veneficum*. Three replicate cultures were grown for each strain. Dotted line is the rolling average between data points for each replicate culture. At the last data point, cultures were harvested for storage and extraction.

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

975
976 Supplementary figure 2: Phylogenetic tree showing the *Karlodinium veneficum* strains used in the current study (bold) aligned with sequences from other *Karlodinium veneficum* strains, *Karlodinium* spp., *Takayama* spp., and *Karenia* spp. from GenBank in Geneious Prime® v2019.2.3 (Kearse et al. 2012) using ClustalW (Thompson et al. 2003).

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990 Supplementary table 1: The cell concentrations of *Karlodinium veneficum* at harvest in cells mL⁻¹

991 ¹.

<table><thead><tr><th>Strain</th><th>Replicate</th><th>Harvest cell concentration, cells mL<sup>-1</sup></th></tr></thead><tbody><tr><td rowspan="3">MAR1F7</td><td>a</td><td>223000</td></tr><tr><td>b</td><td>249000</td></tr><tr><td>c</td><td>186000</td></tr><tr><td rowspan="3">IFR-CC-20-44</td><td>a</td><td>181000</td></tr><tr><td>b</td><td>239000</td></tr><tr><td>c</td><td>226000</td></tr><tr><td rowspan="3">CBC7</td><td>a</td><td>202000</td></tr><tr><td>b</td><td>175000</td></tr><tr><td>c</td><td>232000</td></tr><tr><td rowspan="3">CAWD 84</td><td>a</td><td>180667</td></tr><tr><td>b</td><td>240000</td></tr><tr><td>c</td><td>220000</td></tr><tr><td rowspan="3">CAWD 93</td><td>a</td><td>242667</td></tr><tr><td>b</td><td>269333</td></tr><tr><td>c</td><td>236667</td></tr></tbody></table>

992
Supplementary material: Strategy of the LC-MS analyses of *Karlodinium veneficum* extracts or karlotoxins

First, the *m/z* values corresponding to the [M+Na]⁺ ions of the known KmTxs were analyzed by Single Ion Monitoring (SIM) mode, and some other ions in the mass range 1200-1500 and retention time window of KmTxs were also searched (Supplementary Table 3).

Then, for each chromatographic peak detected in SIM mode between 6.2 and 7.8 min, an MS/MS spectrum was obtained by EPI (Enhanced Product Ion mode), from *m/z* 500 to *m/z* of the precursor ion, in unit resolution. This way, in addition to some KmTxs already reported (Krock et al., 2017) (e.g. KmTx-5), many other compounds with an MS/MS spectrum showing a similar fragmentation pattern to known karlotoxins were observed. More specifically, many of these KmTx-like molecules had fragmentation patterns with mass shifts of 2, 4, 16, 18, 28 and/or chlorinated or complex isotopic patterns compared to the reference fragmentations described by Krock et al. (2017) for groups 1, 2 and 3 (they were referred to as group 1, 2 or 3-like). Due to the lack of analytical standards and the limited availability of samples, it was not possible to confidently identify these compounds in the current study. These compounds were named considering their major Q1>Q3 transition and their retention time in minutes (e.g. Q1>Q3-RT, 1307>895-7.5).

Finally, in addition to the method described in Krock et al. (2017), six other Multiple Reaction Monitoring (MRM) methods were constructed (transitions Q1>Q3 reported in Supplementary Table 4), based on the KmTxs profiles of five French (Ifremer collection) and two American (CCMP 2936, CCMP 2064) strains of *K. veneficum*, as well as two strains of *K. armiger* (K-0668 and one from Ifremer collection). Semi-quantitative results were expressed in area cell⁻¹, by using the major MRM transition, and only compounds contributing to ≥1% of KmTx-like areas were considered.

Acquisition and data processing were performed using Analyst 1.7.2 (Sciex) software.
<table><caption>Supplementary Table 2: Mass spectrometric parameters (4000 QTrap, Sciex) for the detection of karlotoxins in the three acquisition modes (single ion monitoring, enhanced product ion and multiple reaction monitoring.</caption><thead><tr><th>Parameter</th><th>Single Ion<br>Monitoring<br>(SIM)</th><th>Enhanced<br>Product Ion<br>(EPI)</th><th>Multiple Reaction<br>Monitoring<br>(MRM)</th></tr></thead><tbody><tr><td>Curtain gas (psi)</td><td>20</td><td>20</td><td>20</td></tr><tr><td>Collision activated dissociation gas<br>(CAD)</td><td>high</td><td>high</td><td>high</td></tr><tr><td>Ion spray voltage (V)</td><td>5500</td><td>5500</td><td>5500</td></tr><tr><td>Temperature (°C)</td><td>550</td><td>550</td><td>550</td></tr><tr><td>Nebulizer gas (psi)</td><td>40</td><td>40</td><td>40</td></tr><tr><td>Auxiliary gas(psi)</td><td>60</td><td>60</td><td>60</td></tr><tr><td>Declustering potential (V)</td><td>151</td><td>151</td><td>151</td></tr><tr><td>Entrance potential (V)</td><td>10</td><td>-</td><td>10</td></tr><tr><td>Collision energy(V)</td><td>-</td><td>110</td><td>100*<br>*: For Q1&gt;Q1<br>transitions, CE=5V</td></tr></tbody></table>
<table><caption>Supplementary Table 3: Ions (m/z) searched in Single Ion Monitoring Mode</caption><thead><tr><th>Compound</th><th>Molecular Weight</th><th>Ion</th><th>Parent Ion (m/z) Q1</th></tr></thead><tbody><tr><td>KmTx-1 / hydroxy KmTx 1-3</td><td>1338.8</td><td>[M+Na]+</td><td>1361.8</td></tr><tr><td>KmTx-2 / 6-oxo-KmTx-2 / KmTx-8</td><td>1344.8</td><td>[M+Na]+</td><td>1367.8</td></tr><tr><td>KmTx-3 / hydroxy KmTx 1-1</td><td>1324.8</td><td>[M+Na]+</td><td>1347.8</td></tr><tr><td>KmTx-4</td><td>1208.8</td><td>[M+Na]+</td><td>1231.8</td></tr><tr><td>KmTx-5</td><td>1302.8</td><td>[M+Na]+</td><td>1325.8</td></tr><tr><td>KmTx 1-1 / KmTx-7</td><td>1308.8</td><td>[M+Na]+</td><td>1331.8</td></tr><tr><td>KmTx 1-3 / KmTx-6</td><td>1322.8</td><td>[M+Na]+</td><td>1345.8</td></tr><tr><td>KmTx-9 / KcTx-1</td><td>1316.8</td><td>[M+Na]+</td><td>1339.8</td></tr><tr><td>Cand. KmTx-10</td><td>1280.7</td><td>[M+Na]+</td><td>1303.8</td></tr><tr><td>Cand. KmTx-11</td><td>1356.8</td><td>[M+Na]+</td><td>1379.8</td></tr><tr><td>Cand. KmTx-12</td><td>1298.8</td><td>[M+Na]+</td><td>1321.8</td></tr><tr><td>Cand. KmTx-13</td><td>1332.8</td><td>[M+Na]+</td><td>1355.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1239.8</td></tr><tr><td>59-E-Chloro-KmTx-4</td><td>1242.8</td><td>[M+Na]+</td><td>1265.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1274.8</td></tr><tr><td>4.5-dihydro-KmTx-2</td><td>1346.8</td><td>[M+Na]+</td><td>1369.8</td></tr><tr><td>64-E-Chloro-KmTx-3</td><td>1358.8</td><td>[M+Na]+</td><td>1381.8</td></tr><tr><td>44-OH-KmTx-2</td><td>1360.8</td><td>[M+Na]+</td><td>1383.8</td></tr><tr><td>Karmitoxin</td><td>1385.8</td><td>[M+H]+</td><td>1386.8</td></tr><tr><td>65-E-Cl-KmTx-1</td><td>1372.8</td><td>[M+Na]+</td><td>1395.8</td></tr><tr><td>Karmitoxin</td><td>1385.8</td><td>[M+Na]+</td><td>1408.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1411.8</td></tr><tr><td>Cand. Sulfo-KmTx-10</td><td>1400.8</td><td>[M+Na]+</td><td>1423.8</td></tr><tr><td>10-O-sulfo-KmTx-3</td><td>1404.8</td><td>[M+Na]+</td><td>1427.8</td></tr><tr><td>10-SO4-KmTx-1</td><td>1418.8</td><td>[M+Na]+</td><td>1441.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1307.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1337.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1333.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1341.8</td></tr><tr><td>/</td><td>/</td><td>/</td><td>1342.8</td></tr></tbody></table>
<table><caption>Supplementary Table 4: Multiple Reaction Monitoring (MRM) methods</caption><thead><tr><th>Method<br>MRM ID</th><th>Parent<br>Ion m/z<br>(Q1)</th><th>Product Ions<br>m/z<br>(Q3)</th><th>ID</th></tr></thead><tbody><tr><td rowspan="4">1</td><td>1361.8</td><td>1361.8 / 937.4 / 877.4 / 951.4 / 965.4 / 931.4 / 947.4 / 895.4</td><td>/</td></tr><tr><td>1367.8</td><td>1367.8 / 937.4 / 877.4 / 921.4 / 919.4 / 861.4 / 934.4 / 895.4</td><td>/</td></tr><tr><td>1347.8</td><td>1347.8 / 937.4 / 877.4 / 859.4 / 861.4 / 933.4 / 917.4 / 922.4 / 895.4</td><td>/</td></tr><tr><td>1231.8</td><td>1231.8 / 877.4 / 895.4 / 937.4</td><td>/</td></tr><tr><td rowspan="4">2</td><td>1325.8</td><td>1325.8 / 895.4 / 921.4 / 893.4 / 877.4 / 919.4 / 937.4</td><td>/</td></tr><tr><td>1331.8</td><td>1331.8 / 934.4 / 937.4 / 921.4 / 877.4 / 898.5 / 859.4 / 861.4</td><td>/</td></tr><tr><td>1345.8</td><td>1345.8 / 921.4 / 912.4 / 896.4 / 933.4 / 861.4 / 873.4</td><td>/</td></tr><tr><td>1339.8</td><td>1339.8 / 937.4 / 924.4 / 965.4 / 966.5 / 934.4 / 895.4 / 877.4 / 691.4</td><td>/</td></tr><tr><td rowspan="4">3</td><td>1303.7</td><td>1303.7 / 891.4 / 873.4 / 895.4 / 877.4 / 919.4 / 859.4 / 921.4 / 861.4</td><td>/</td></tr><tr><td>1321.8</td><td>1321.8 / 933.4 / 895.4 / 877.4 / 891.4 / 937.4 / 919.4 / 939.4 / 924.4 / 889.4</td><td>/</td></tr><tr><td>1355.8</td><td>1355.8 / 966.5 / 965.4 / 937.4 / 922.4 / 895.4 / 877.4</td><td>/</td></tr><tr><td>1239.8</td><td>1239.8 / 895.8 / 877.8 / 937.4</td><td>/</td></tr><tr><td rowspan="4">4</td><td>1265.8</td><td>1265.8 / 893.4 / 875.4 / 895.4 / 877.4 / 921.4 / 861.4</td><td>/</td></tr><tr><td>1379.8</td><td>1379.8 / 949.4 / 933.4 / 877.4 / 921.4</td><td>/</td></tr><tr><td>1274.8</td><td>1274.8 / 896.5 / 878.4 / 895.4 / 877.4</td><td>/</td></tr><tr><td>1369.8</td><td>1369.8 / 937.4 / 935.4 / 922.5 / 921.4 / 877.4 / 861.4 / 895.4</td><td>/</td></tr><tr><td rowspan="4">5</td><td>1381.8</td><td>1381.8 / 949.4 / 933.4 / 921.4 / 937.4 / 877.4 / 875.8 / 893.8</td><td>/</td></tr><tr><td>1383.8</td><td>1383.8 / 877.4 / 953.4 / 937.4 / 951.4 / 950.4 / 934.4 / 920.4 / 919.4 / 921.4 / 893.4 / 895.4</td><td>/</td></tr><tr><td>1386.8</td><td>1386.8 / 877.4 / 953.4 / 895.4</td><td>/</td></tr><tr><td>1395.8</td><td>1395.8 / 877.4 / 965.5 / 937.4 / 962.5 / 905.4 / 901.4</td><td>/</td></tr><tr><td rowspan="4">6</td><td>1408.8</td><td>1408.8 / 1142.4 / 717.4 / 877.4 / 977.4 / 961.4 / 919.5 / 937.4 / 895.4 / 860.4</td><td>/</td></tr><tr><td>1411.8</td><td>1411.8 / 1143.8 / 717.4 / 965.4 / 962.4 / 937.4 / 895.4 / 905.4 / 902.4 / 877.4</td><td>/</td></tr><tr><td>1423.8</td><td>1423.8 / 1303.8 / 933.4 / 873.4 / 877.4 / 895.4 / 973.4 / 937.4 / 921.4 / 965.4</td><td>/</td></tr><tr><td>1427.8</td><td>1427.8 / 937.4 / 921.4 / 877.4 / 859.4 / 1161.4 / 717.4 / 1307.4</td><td>/</td></tr><tr><td rowspan="4">Krock et<br>al., 2017</td><td>1441.8</td><td>1441.8 / 919.5 / 859.4 / 877.4 / 875.4 / 857.4 / 965.4 / 905.4</td><td>/</td></tr><tr><td>1307.8</td><td>1307.8 / 895.4 / 877.4 / 875.4 / 833.5</td><td>/</td></tr><tr><td>1337.8</td><td>1337.8 / 947.8 / 887.5 / 962.5 / 933.4 / 938.4 / 924.5 / 921.4 / 877.4 / 863.4 / 873.4</td><td>/</td></tr><tr><td>1333.8</td><td>1333.8 / 937.4 / 877.5 / 903.4 / 922.4 / 861.4</td><td>/</td></tr><tr><td rowspan="4"></td><td>1341.8</td><td>1341.8 / 937.4 / 877.4 / 911.4</td><td>/</td></tr><tr><td>1342.8</td><td>1342.8 / 938.5 / 911.4 / 895.4</td><td>/</td></tr><tr><td>1361.8</td><td>1361.8 / 937.4 / 877.4</td><td>KmTx-1</td></tr><tr><td>1367.8</td><td>1367.8 / 937.4 / 877.4</td><td>KmTx-2</td></tr><tr><td rowspan="4"></td><td>1347.8</td><td>1347.8 / 937.4 / 877.4</td><td>KmTx-3</td></tr><tr><td>1383.8</td><td>1383.8 / 937.4 / 877.4</td><td>44 oxo KmTx-2</td></tr><tr><td>1325.8</td><td>1325.8 / 895.4 / 691.4</td><td>KmTx-5</td></tr><tr><td>1303.8</td><td>1303.8 / 877.4 / 615.4</td><td>KmTx-10</td></tr></tbody></table>
<table><tr><td>1321.8</td><td>1321.8 / 895.4 / 633.4</td><td>KmTx-12</td></tr><tr><td>1355.8</td><td>1355.8 / 895.4 / 677.4</td><td>KmTx-13</td></tr><tr><td>1379.8</td><td>1379.8 / 877.4 / 615.4</td><td>KmTx-11</td></tr><tr><td>1423.18</td><td>1423.8 / 1303.8 / 877.4</td><td>Sulfo-KmTx-10</td></tr><tr><td>1339.8</td><td>1339.8 / 1121.7 / 719.6</td><td>KcTX-1</td></tr><tr><td>1408.8</td><td>1408.8 / 1142.4 / 717.4</td><td>Karmitoxin</td></tr></table>

<figure><img src="image_16.png" /><figcaption>Supplementary Figure 3: Correlation between biological toxicity and total KmTx concentration for A) *Crassostrea virginica* sperm exposed to crude extracts of *K. veneficum*, B) RT-W1gill cells exposed to crude extracts of *Karlodinium veneficum*, C) *C. gigas* sperm exposed to methanolic</figcaption></figure>
extracts of *K. veneficum* and D) RT-W1gill cells exposed to methanolic extracts of *K. veneficum*.
Mean viability (%) and KmTx content (area cell⁻¹) are presented for each replicate culture.