# An unprecedented bloom of Lingulodinium polyedra on the French Atlantic coast during summer 2021

Kenneth Mertens, Michael Retho, Soazig Manach, Maria Laura Zoffoli, Anne Doner, Mathilde Schapira, Gwenael Bilien, Veronique Séchet, Thomas Lacour, Elise Robert, et al.

► To cite this version:

Kenneth Mertens, Michael Retho, Soazig Manach, Maria Laura Zoffoli, Anne Doner, et al. An unprecedented bloom of Lingulodinium polyedra on the French Atlantic coast during summer 2021. Harmful Algae, 2023, 125, 102426 (19p.). 10.1016/j.hal.2023.102426. hal-04204015

HAL Id: hal-04204015

https://hal.science/hal-04204015v1

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1 **An unprecedented bloom of *Lingulodinium polyedra* on the French Atlantic coast**

2 **during summer 2021**

3

4 Kenneth Neil Mertens¹*, Michael Retho², Soazig Manach², Maria Laura Zoffoli³, Anne

5 Doner¹, Mathilde Schapira⁴, Gwenael Bilien¹, Véronique Séchet⁵, Thomas Lacour⁵,

6 Elise Robert⁶, Audrey Duval¹, Aouregan Terre-Terrillon¹, and Amélie Derrien¹, Pierre

7 **Gernez⁷**

8

9 ¹Ifremer, LITTORAL, F-29900 Concarneau, FRANCE.

10 kenneth.mertens@ifremer.fr

11 ²Ifremer, LITTORAL, F-56100, Lorient, FRANCE

12 ³Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine (CNR-ISMAR), 00133,

13 Rome, ITALY

14 ⁴Ifremer, LITTORAL, F-44300, Nantes, FRANCE

15 ⁵Ifremer, PHYTOX, Laboratoire PHYSALG, F-44000 Nantes, France

16 ⁶Ifremer, PHYTOX, Laboratoire GENALG, F-44000 Nantes, France

17 ⁷Nantes Université, Institut des Substances et Organismes de la Mer, ISOMer, UR

18 2160, F-44000 Nantes, FRANCE

19

20 *Corresponding author: Kenneth Neil Mertens

21 E-mail: Kenneth.mertens@ifremer.fr

22 Tel: +33-2-98-10-42-82

23

24 **Abstract**

25 At the end of July 2021, a bloom of *Lingulodinium polyedra* developed along the
French Atlantic coast and lasted six weeks. The REPHY monitoring network and the citizen participation project PHENOMER contributed to its observation. A maximum concentration of 3,600,000 cells/L was reached on the 6th of September, a level never recorded on French coastlines. Satellite observation confirmed that the bloom reached its highest abundance and spatial extension early September, covering about 3200 km² on the 4th of September. Cultures were established, and morphology and ITS-LSU sequencing identified the species as *L. polyedra*. The thecae displayed the characteristic tabulation and sometimes a ventral pore. The pigment composition of the bloom was similar to that of cultured *L. polyedra*, confirming that phytoplankton biomass was dominated by this species. The bloom was preceded by *Leptocylindrus* sp., developed over *Lepidodinium chlorophorum*, and was succeeded by elevated *Noctiluca scintillans* concentrations. Afterwards, relatively high abundance of *Alexandrium tamarense* were observed in the embayment where the bloom started. Unusually high precipitation during mid-July increased river discharges from the Loire and Vilaine rivers, which likely fueled phytoplankton growth by providing nutrients. Water masses with high numbers of dinoflagellates were characterized by high sea surface temperature and thermohaline stratification. The wind was low during the bloom development, before drifting it offshore. Cysts were observed in the plankton towards the end of the bloom, with concentrations up to 30,000 cysts/L and relative abundances up to 99%. The bloom deposited a seed bank, with cyst concentrations up to 100,000 cysts/g dried sediment, particularly in fine-grained sediments. The bloom caused hypoxia events, and concentrations of yessotoxins up to 747 µg/kg were recorded in mussels, below the safety threshold of 3,750 µg/kg. Oysters, clams and cockles also were contaminated with yessotoxins, but at lower concentrations. The established cultures did not produce yessotoxins at detectable levels, although yessotoxins were detected in the sediment. The unusual environmental summertime conditions that
51

triggered the bloom, as well as the establishment of considerable seed banks, provide
important findings to understand future harmful algal blooms along the French coastline.

52

53

54

**Key words:** Seed bank, yessotoxins, Vilaine Bay, southern Brittany, harmful algal blooms

55

56

**Highlights**

57

* An unprecedented bloom of *Lingulodinium polyedra* was observed on the French Atlantic coast during summer 2021.

58

59

* Unusually high precipitation during mid-July led to increased river discharges, which likely triggered the bloom.

60

61

* The bloom deposited a significant seed bank.

62

* The bloom caused hypoxia events, high biomass and increased concentrations of yessotoxins.

63

64

65

**1. Introduction and aims**

66

67

Dinoflagellates are a group of microorganisms that can produce blooms that can be harmful and are called harmful algal blooms (HABs). Blooms are expanding and intensifying globally (Dai et al., 2023), and some of these blooms can cause surface seawater discoloration (i.e., green, red, brown) that can be detected using satellite remote sensing (e.g. Gernez et al., 2023, and references therein). Such massive blooms alter the appearance of coastal waters (Siano et al., 2020) and the remineralisation of the high volume of biomass produced during these phenomena may create hypoxic/anoxic conditions that are deleterious for marine aquaculture and ecosystems (Sournia et al., 1992). For example, green seawater discolorations caused by the marine dinoflagellate

68

69

70

71

72

73

74

75
76 *Lepidodinium chlorophorum* have frequently been observed along Southern Brittany (NE
77 Atlantic, France; Roux et al., 2022, 2023). Other HAB species produce toxins which can
78 accumulate in shellfish and other marine organisms. Most toxigenic dinoflagellates belong
79 to the order Gonyaulacales, either to planktonic genera such as *Alexandrium*,
80 *Lingulodinium*, and *Protoceratium*, or benthic ones such as *Gambierdiscus*, *Ostreopsis* and
81 *Coolia* (Lassus et al., 2016, table 1 on p. 16). The planktonic genera *Gonyaulax*,
82 *Protoceratium* and *Lingulodinium* produce yessotoxins (YTXs; Paz et al., 2008) and these
83 can be potentially produced by *Pentaplacodinium* (Mertens et al., 2018, 2023). YTXs are
84 polycyclic ether-compounds that are considered to be potent cytotoxins, this caused the
85 European Authorities to establish a maximum permitted level in shellfish, which currently
86 is as high as 3.75 mg YTX equivalents/kg (Regulation 786/EC/2013; Rubini et al., 2021).
87 One such YTX-producing species, *Lingulodinium polyedra* (F. Stein) J.D. Dodge
88 1989 is an autotrophic, thecate dinoflagellate that recently was restudied from its type
89 locality, Kiel Bight in Germany (Tillmann et al., 2021). Blooms of this species have been
90 related to enhanced nutrient levels that might be supplied by coastal runoff, upwelling or
91 anthropogenic inputs (Lewis and Hallett, 1997, p. 136), and can cause brown water
92 discoloration (e.g. Peña-Manjarrez et al., 2005, p. 1384). In Europe, blooms are known to
93 occur mostly in southern European waters, and have since long been recorded from the
94 Adriatic, specifically in Kaštela Bay (e.g., Marasovíc and Vukadin, 1982), around Spain
95 (e.g., Margalef, 1956, Caballero et al., 2020) and Portugal (Amorim et al., 2000). A most
96 recent bloom was observed in Odessa Bay (Ukraine, Black Sea) in 2020, with highest ever
97 recorded concentrations up to 56.1 million cells/L (Terenko and Krakhmalny, 2021).
98 Outside Europe, blooms have repeatedly been reported off California (e.g., Eppley and
99 Harrison, 1975; Kahru et al., 2021) and along the Southern (Pitcher et al., 2019) and
100 Northern African coast (e.g., Bennouna et al., 2002, Frehi et al., 2007). For several *L*.
polyedra events, satellite remote sensing made it possible to accurately document the
bloom's spatial and temporal variability, thus usefully complementing field sampling
(Caballero et al., 2020, Kahru et al., 2021).

Production of YTXs by *L. polyedra* has been demonstrated from cultures from NW
Spain (Paz et al., 2004), California (Armstrong and Kudela, 2006) and the U.K. (Stobo et
al., 2003). Elevated YTXs from mussels from the North Adriatic Sea and the Black Sea
have been attributed to *L. polyedra* blooms (Tubaro et al., 1998; Morton et al., 2007;
Rubini et al., 2021), amongst other reports. Mortalities of fish and other organisms have,
however, more often been attributed to high bloom concentrations leading to anoxia
(Lewis and Hallett, 1997, p. 110 and references therein).

*L. polyedra* is a well-known cyst-producer that has been related to the fossil
defined species *Lingulodinium machaerophorum* (e.g. Mertens et al., 2009a). The process
of cyst-formation has been described in detail by Kokinos and Anderson (1995). Cysts are
produced at the end of the bloom (Amorim et al., 2000) and form seed banks that
accumulate preferentially in fine-grained sediments (Lewis, 1988). In general, after the
encystment in autumn and a mandatory dormancy period in winter, excystment occurs in
spring (Lewis and Hallett, 1997, fig. 31). Cysts of *L. polyedra* are widely distributed in
temperate to equatorial regions, with highest relative abundances near upwelling regions or
below river discharge plumes (Zonneveld et al., 2013).

In France, the plankton is monitored through the REPHY network (REPHY, 2022),
and toxins in shellfish through the REPHYTOX network (REPHYTOX, 2022). In the
French Atlantic, HABs are typically related to *Dinophysis* spp., *Alexandrium* and *Pseudo-
nitzschia* (Belin et al., 2021). Cells of *L. polyedra* have been observed in the French
Atlantic since 1964 (e.g. Paulmier, 1965, 1994). One oyster mortality event during summer
1964 was associated with elevated concentrations of this species in the Auray Estuary
126 (Paulmier, 1972). Cysts have been studied rather rarely along the French Atlantic coasts.
127 Cysts of *L. polyedra* were first recorded in 1989 and 1990 in surface sediments from
128 Vilaine Bay by An et al. (1992), whom recorded maximum concentrations of about 110
cysts/g sediment, although none were recorded in surface sediments from Vilaine Bay
129 sampled in 1986 and 1987 by Larrazabal et al. (1990). However, palynological studies
130 show that the cysts have been present for thousands of years along the French Atlantic
131 coast (e.g. Lambert et al., 2020).
132

133 In this study, we report an unprecedented summer bloom of *L. polyedra* from the
134 French Atlantic using in situ and satellite observations. We identify the species morpho-
molecularly, identify its pigments and examine its toxicity. We investigate the potential
135 triggers for the bloom, and discuss its decline. The imprint of a seed bank is investigated
136 by quantifying the cysts in surface sediments.
137

138

139 **2. Material and methods**

140

141 **2.1. Observation area and sampling stations**

142

143 On the French Atlantic Ocean, the Northern Bay of Biscay, and more precisely the
144 coastal zone in southern Brittany (France), is directly influenced by the Vilaine and Loire
Rivers, which have a respective 70 and 850 m³/s mean annual flow (Lazure et al., 2009).
145 Both rivers, and especially the Loire, are the main nutrient sources in the northern Bay of
146 Biscay and play a major role in the eutrophication of coastal waters in southern Brittany
147 (Guillaud et al., 2008; Ménesguen et al., 2018, 2019, Ratmaya et al., 2019). The location
148 and extent of both rivers plumes vary according to the flow of rivers, tidal currents and
149 winds. In winter, the Loire river plume can be oriented towards the northwest along the
150
coast, and in periods of very high flow, the plume extends seaward (Ménesguen and Dussauze, 2015; Ménesguen et al., 2018). The Vilaine river plume generally spreads throughout the Bay before moving westward (Chapelle et al., 1994). The water circulation within the Vilaine Bay is characterized by low tidal and residual currents, and is mainly driven by tide, wind and river flow (Lazure and Salomon, 1991; Lazure and Jegou, 1998). The water residence time in the bay varies from 10-20 days depending on the season and is generally longer during calm periods (Chapelle et al., 1994). Haline stratification is high from February to June in response to high river runoff and relatively low vertical mixing, whereas thermal stratification occurs between May and mid-September (Puillat et al., 2004).

Data from REPHY (Observation and Surveillance Network for Phytoplankton and hydrology in coastal waters; Belin et al., 2021; REPHY, 2022) stations and PHENOMER observations (a citizen monitoring program focused on water discoloration observations, Siano et al., 2020) were used to describe the bloom of *L. polyedra* and the environmental conditions (Fig. 1).

## 2.2. Environmental data

Air temperature data were retrieved from the weather station Vannes-Séné (2.7141°W; 47.6045°N) and daily wind data were retrieved from the weather station Belle Ile - Le Talut (3.2181°W; 47.2942°N) using Météo France observation network (https://donneespubliques.meteofrance.fr/). River discharge data were extracted from the French hydrological database (http://www.hydro.eaufrance.fr/). Daily discharge data were available at the Montjean-sur-Loire gauging station for the Loire River and at the Rieux gauging station for the Vilaine River. Sea water temperature, salinity, and inorganic
nutrients data were provided by the REPHY and the research project LEPIDO-PEN. Data
from three stations were analyzed: "Ouest Loscolo" (OL) and "Nord Dumet" (ND) located
off the Vilaine estuary, and "Basse Michaud" (BM) located off the Loire estuary (Fig. 1).
Samples were collected at these stations fortnightly at high tide (+/- 2h). Vertical profiles
of seawater temperature, salinity, turbidity (FNU) and in vivo fluorescence (FFU) were
performed with a multi-parameter probe type MP6 (NKE, Hennebont, France) from
subsurface to the water-sediment interface (WSI). Water samples were collected using a 5
L Niskin bottle at two depths: subsurface (0–1 m) and 1 m above the WSI. Water samples
aliquots were processed for (i) microphytoplankton identification and enumeration, (ii)
chlorophyll a concentration ([Chl a] mg/m³), and (iii) inorganic nutrients. Analytical
procedure for plankton observations and counts are described in section 2.2.

For Chl a concentrations, water samples (500–1000 ml) were filtered through glass
fiber filters (GF/F 0.7µm, 47 mm; Whatman, Maidstone, UK) and stored at -80°C until
analysis. Chl a was extracted in 10 mL of 90% acetone in the dark at 4°C for 12h and
analysed by monochromatic spectrophotometry (Aminot and Kérouel, 2004).

For inorganic nutrients, 300 mL water samples were pre-filtered through 41 µm
pore silk directly from the Niskin bottle. For dissolved silicate (i.e., DSi = Si(OH)₄)
concentrations, water samples were filtered through 0.45 µm acetate cellulose membrane
and stored at 4°C until analysis. Water samples for the determination of dissolved
inorganic nitrogen (i.e., DIN = NO₃⁻ + NO₂⁻ + NH₄⁺) and phosphorus (i.e., DIP = PO₄³⁻)
were stored directly at -20°C. Samples were analysed using an AutoAnalyzer AA3 (SEAL
Analytic, Mequon, WI, USA) following standard protocols (Aminot and Kérouel, 2007).
The limits of quantification were 0.4 µM for DSi, 0.2 µM for NO₃⁻ + NO₂⁻, and 0.05 µM
for DIP, NH₄⁺, and NO₂⁻. Measurement uncertainty measurement are 12% for DSi, 10%
for NO₃⁻ + NO₂⁻, 15% for DIP, and 27% for NH₄⁺.
In the Vilaine Bay, temperature and salinity at the ND station were also obtained from a high-frequency monitoring buoy, MOLIT, of the French national observation network COAST-HF. Since 2008, this instrumented buoy continuously measures physicochemical parameters in surface and bottom waters at hourly frequency (Retho et al., 2022). Furthermore, dissolved oxygen concentrations in bottom waters (1 m above the WSI) were acquired at the Pont Mahé (PM) station by an autonomous miniDOT sensor (PME, Vista, CA, USA) at high frequency (15 minutes).

208

## 2.2. Plankton observations and cell counts

210

L. polyedra distribution and abundance were assessed using REPHY (REPHY, 2022) and PHENOMER data. Cells of lugol-fixed L. polyedra were counted using an inverted microscope (Zeiss, Axio Observer). Samples were gently homogenized before settling in 10 mL sub-sample for > 12h in counting chambers (Utermöhl, 1958; Hydro-Bios, Alenholz, Germany). The lower limit of quantification was 100 cells/L.

216

## 2.3. Satellite images

218

Satellite observations from the Ocean Land Colour Instrument (OLCI) on-board Sentinel-3 (S3) were used to study the bloom spatial and temporal variability. Since 2016, S3/OLCI acquires radiometric data in 21 spectral bands from 400 – 1020 nm with a revisit time < 2 days and a spatial resolution of 300 m. Images acquired between the 1st of July and 30th of September 2021 were downloaded from EUTMETSAT Earth Observation Portal (https://eoportal.eumetsat.int/). Level2 data, corresponding to water-leaving reflectance, were directly obtained from the portal. A total of 17 images with low cloud
cover were found exploitable for the selected time window in the region of interest

(latitude from 46.75°N – 48°N, and longitude from 1.5°W – 4°W). The concentration of

Chl *a* in the first optical layer (typically < 5 m in coastal waters) was estimated by an

algorithm specifically designed to retrieve [Chl *a*] over a wide range of variability,

including extreme phytoplankton bloom conditions (Smith et al., 2018). Satellite-derived

maps of [Chl *a*] were re-projected in an appropriate coordinate system (i.e., WGS 84 /

UTM zone 30N - EPSG: 32630), and used to compute bloom areal extent. Satellite data

processing and mapping were performed using the Matlab software.

234

## 2.4. Establishment of cultures

236

Living cells were isolated from plankton samples from Port Manec'h (strain IFR-

CC-21-049), Doëlan (strain IFR-CC-21-048) and Le Croisic (strain IFR-LI-01LC) (PMa,

DO, and CR, Fig. 1) using a micropipette, and placed in a single well of a 12-well plate,

together with 500 µl L1 medium. Conditions for growth were 18 °C, 90 µEm⁻²s⁻¹ and a

12:12 h light:dark cycle.

242

## 2.5. Light microscopy and scanning electron microscopy observations

244

A lugol-fixed plankton sample acquired at station KE (Fig. 1) on the 2nd August

was used to isolate cells and cysts of *L. polyedra*. For light microscopy, all measurements

and light photomicrographs were obtained by K.N.M. using an Olympus BX41 with an

Olympus DP72 camera, with 100X oil immersion objectives. All cyst and motile cell

measurements in the species descriptions cite the minimum, average (in parentheses) and

maximum values (µm), in that order.
For scanning electron microscopy (SEM), specimens were first isolated from the sample with a micropipette using an IX51 (Olympus, Tokyo, Japan) or an IM35 (Zeiss, Oberkochen, Germany) inverted microscope and transferred onto polycarbonate membrane filters (Millipore, Billerica, MA, USA; GTTP Isopore, 0.22 µm pore size), following a method proposed by Chomérat and Couté (2008). After 12 to 24 hours of air-drying, the filters were affixed to aluminum stubs with adhesive tabs (Electron Microscopy Sciences, Hatfield, PA, USA). Subsequently, the mounted filters were sputter coated with gold using a 108auto Sputter coater (Cressington Scientific Instruments, UK). The samples were observed at the Station of Marine Biology of Concarneau with a Sigma 300 (Zeiss, Oberkochen, Germany) field-emission SEM equipped with a conventional Everhart-Thornley and in-lens detectors of secondary electrons at 1.5 and 5 kV. Digital images were saved in Tiff format (2048 x 1768 pixels). Photoshop Creative Suite 5 (CS5; Adobe, San Jose, CA, USA) software was used to remove the background while maintaining the integrity of the original image. Labelling of tabulation follows a modified Kofoid system that recognizes homologs (Carbonell-Moore et al., 2021).

266

267 *2.6. Sequencing of culture of L. polyedra*

268

The cultures of *L. polyedra* established from Port Manec'h (IFR-CC-21-049) and Doëlan (IFR-CC-21-048) (PMa and DO, Fig. 1) were extracted with the PCRBIO Rapid Extract PCR Kit (PCR Biosystems Ltd, London, UK) which combines extraction and PCR. In a 1.5ml tube, 1ml of culture were taken and centrifuged for 3 minutes at 14000 rpm. The supernatant was discarded to retain only the pellet. Then the manufacturer's instructions were followed, except for the step dilution where 190µl of PCR grade dH₂O were added instead of 900µl. The pair of primers used for the PCR is ITSFW
276 (GTAGGTGAACCTGCGGAAGG) and D3B (TCGGAGGGAACCAGCTACTA) with a
277 Tm of 60°C.

278 PCR-amplified product was visualized on an agarose gel after electrophoresis and
279 the positive samples were purified using the ExoSAP-IT PCR Product Cleanup reagent
280 (Affymetrix, Cleveland, OH, USA). The Big Dye Terminator v3.1 Cycle Sequencing Kit
281 (Applied Biosystems, Waltham City, MA, USA) was used for sequencing of the amplicon
282 generated. Primers and excess dye-labeled nucleotides were first removed using the Big
283 Dye X-terminator purification kit (Applied Biosystems). Sequencing products were run on
284 an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).

285

286 2.7. Surface sediment sampling for cysts

287

288 Surface sediments were sampled during the dormancy period along the affected
289 coast between February and April 2022, at water depths between 0 and 26 m using a Petite
290 Ponar Grab or just by hand at low tide (Table 1). The upper centimeters were preserved in
291 a labelled falcon tube and kept in the dark until further study.

292 About 1 gram of wet sediment was immersed in filtered seawater and, after one
293 min of ultrasonication using an ultrasonic bath, the sediment was rinsed through a 20 µm
294 metal mesh sieve using filtered seawater. From this residue, the cyst fraction was separated
295 using the heavy-liquid sodium polytungstate (SPT) at a density of 1.4 g/cm (Bolch, 1997).
296 From the total fraction, cyst concentrations were calculated using the volumetric method.
297 Cyst concentrations were also converted into dry weight by calibrating the wet weight with
298 dried weight.

299

300 2.8. Pigment measurements
301

302

Bloom sampling characteristics for pigment analysis are presented in Table S3.

303

Two cultured strains were investigated: a strain previously isolated from station KE in 2003 by S. Fraga (VGO668), and the strain isolated at station CR (IFR-LI-01LC), and compared to published data from Spanish strains (LP4V and LP9V, Zapata et al., 2012).

304

305

306

An aliquot of algal culture (5 mL) or sea water (30–50 mL) was filtered onto glass-fiber filters (GF/F 0.7µm, 25mm; Whatman, Maidstone, UK) and stored at -80°C until analysis.

307

308

Filters were immersed in 2 ml of 95% acetone, sonicated for 10 minutes and stored at -20°C overnight. Acetone extracts were filtered with a 0.2-µm fiberglass filter prior to injection in HPLC. The filtered acetone extracts were analyzed by HPLC-UV-DAD (series 1200 HPLC-UV-DAD; Agilent Technologies, Santa Clara, CA, USA) using an Eclipse XDB-C8 reverse phase column (150 mm × 4.6 mm, 3.5 µm particle size; Agilent Technologies) following the method described by Van Heukelem and Thomas (2001).

309

310

311

312

313

314

315

## 2.9. Toxicity of established cultures, sediments and shellfish

316

317

### 2.9.1. Culture extraction

318

319

The cultures of *L. polyedra* established from Le Croisic (IFR-LI-01LC) and Doëlan

320

(IFR-CC-21-048) (CR and DO, Fig. 1) were extracted according to the protocol described

321

below. Mixer Ball Milling equipment (Mixer Mill MM400, Retsch, Haan, Germany) was

322

used to extract yessotoxin (YTX) and some known analogues from cells. Glass beads

323

(150 mg, 100–250 µm in diameter) were added to the isolated cells. Samples were extracted three times by adding Methanol (Table S1) and shaking at 30 Hz for 10 minutes.

324

325

After centrifugation at 15000 g, the supernatants were pooled and evaporated to dryness
326 with a gentle flow of nitrogen at a temperature of 30°C. The residue was dissolved in
327 300µl of methanol, ultrafiltered (0.20 µm, Nanosep MF, Pall, NY, USA) and transferred
328 into a HPLC vial before LC-MS/MS analyses.

329

330 2.9.2. Sediments extraction

331

332 The sediment was sampled during the dormancy period at Pornichet (PO, Fig. 1) on
333 the 23rd February. Four aliquots were extracted according to the protocol described below.
334 Glass beads (2.5 g, 100–250 µm in diameter + 2.5 g, 750–1000 µm in diameter) were
335 added to 5 g of sediment. Samples were extracted twice by adding 5 ml of Methanol and
336 shaking at 30 Hz for 30 minutes (Mixer Mill MM400, Retsch). After centrifugation at
337 3700 g, the supernatants were pooled and evaporated to dryness with a gentle flow of
338 nitrogen at a temperature of 30°C. The residue was dissolved in 1000µl of methanol,
339 ultrafiltered (0.20 µm, Nanosep MF, Pall) and transferred into a HPLC vial before LC-
340 MS/MS analyses.

341

342 2.9.3. LC-MS/MS analysis

343

344 Culture and sediment samples analyses were performed by LC-MS/MS using a
345 Shimadzu UFLCxr system coupled to a triple quadruple hybrid mass spectrometer Q-Trap
346 (API400QTrap, SCIEX, Framingham, MA) equipped with a heated electrospray ionization
347 (ESI) source. Data acquisitions were performed using negative ion mode and MRM
348 (Multiple Reaction Monitoring) mode. Chromatographic separation was carried out on a
349 reversed-phase column Xbridge BEH C18 (50 x 2.1 mm, 2.5 µm, Waters) equipped with a
350 guard column (5 x 2.1 mm, 2.5 µm, same stationary phase as column). Water (A) and
acetonitrile 90% (B) both containing 6.7 mM of ammonium hydroxide were used as mobile phases at a flow rate of 400 µl.min⁻¹. The following gradient was used: 0 min, 5% B; 1.50 min, 5% B; 4.5 min, 65% B; 5.00 min, 100% B; 7.00 min, 100% B; 7.50 min, 5% B; 12.00 min, 5% B. The oven temperature was 30°C and the injection volume was 5 µL. The LC-MS/MS methods were used to detect 22 toxins (Table S1). The ESI interface operated using the following parameters: curtain gas 20 psi, temperature: 600 °C, gas1 60 psi; gas2 60 psi, ion spray voltage -4500 V. For detection, the parameters were as follows: the declustering potential was -105 V and the entrance potential -10 V. Two collision energies (Q3_01: -46eV; Q3_02: -42eV) and two collision cell exit potentials (Q3_01: -9V; Q3_02: -11V) were applied and the dwell time was 30 ms. The transitions that were used for the MRM mode are presented in Table S2. Quantification was performed relative to the YTX standard (National Research Council Canada, NRCC) with a 6-point calibration curve. The limit of quantification was 0.03 ng/mL for the YTX standard.

### 2.9.4. Toxicity of shellfish

Data were collected within the framework of the French monitoring program for phycotoxins in marine organisms (REPHYTOX, 2022). The data were obtained through the regulatory monitoring operated by the laboratory Inovalys. The REPHY guidelines (Neaud-Masson et al., 2023) define the alert threshold for *L. polyedra* as 10,000 cells/L. Weekly monitoring of phytoplankton in the water and yessotoxins in shellfish was therefore carried out as long as this threshold was exceeded. Analyses of regulated yessotoxins (YTX, homo-YTX, 45-OH-YTX, homo-45-OH-YTX) were carried out on all species of exploited shellfish (mussels, common cockles, clams, Pacific oyster), according
376 to the official method ANSES LSA-INS-0147 (Version 4, February 2018).

377 Briefly, after the shellfish (1 to 2 kg) were washed with tap water, the flesh was
378 removed from the shell and then mixed with a knife blender. Afterwards, (2.00 ± 0.05) g
379 of this homogenate were extracted twice by adding 9 mL of methanol and homogenizing
380 for 2 minutes at approximately 10,000 rpm. After centrifugation, the supernatants were
381 transferred and pooled into a 20 mL volumetric flask completed by adding methanol. After
382 a solid phase extraction step, the extract was filtered and transferred into a HPLC vial
383 before LC-MS/MS analyses at the Inovalys laboratory.

384

385

386 2.10. *Long-term records of L. polyedra*

387

388 Cell counts of *L. polyedra* were extracted from the REPHY dataset since 1990
389 (REPHY, 2022). Observations from the citizen participation project PHENOMER were
390 also used in this study.

391

392 **3. Results**

393

394 **3.1. *Bloom dynamics***

395

396 The first significant number of cells of *L. polyedra* (15,000 cells/L) were observed
397 in Vilaine Bay at OL during summer on the 5th of July (Fig. 2a). The onset of *L. polyedra*
398 occurred during a bloom of diatoms, which was characterized by high numbers of
399 specimens belonging to the *Pseudo-nitzschia delicatissima* complex on the 1st and 5th of
400 July at BM and OL, respectively. The bloom of diatoms was then dominated by
401 *Leptocylindrus* sp., and lasted until the end of July, with maximum numbers around the
402 20th of July at both BM and OL. During the bloom of diatoms, an increase in *L. polyedra*
403 concentration was observed in the Vilaine Bay (Fig. 3). In particular, high numbers were
404 reached at OL (28,000 cells/L) and KE (94,000 cells/L) on the 27th of July.

405 During early August, the microphytoplankton was dominated by dinoflagellates,
406 with high concentrations of *L. polyedra* and/or *L. chlorophorum* (Fig. 2). In the Vilaine
407 Bay, a brownish / reddish seawater discoloration associated with high numbers of *L.*
408 *polyedra* (325,000 cells/L) and *L. chlorophorum* (500,000 cells/L) at OL was observed on
409 the 2nd of August. High concentrations of *L. chlorophorum* were still present on the 10th of
410 August at OL (1,000,000 cells/L). The concentration of *L. chlorophorum* then decreased
411 rapidly from the 17th of August. In contrast, significant increases in *L. polyedra* numbers
412 were seen near OL (2,700,000 cells/L) on the 10th of August, and in ND (2,000,000
cells/L) on the 17th of August. From mid-August, the bloom of *L. polyedra* extended
413 beyond the Vilaine Bay, expanding first southward toward the Loire River estuary, and
414 then north-westward at the end of the month (Fig. 3).

415 Numbers of *L. polyedra* remained high on several sampling points until early
416 September, and the bloom was well visible on aerial photographs (Fig. 4), with highest
417 concentrations on the 6th of September at ND (2,000,000 cells/L), DO (3,600,000 cells/L),
418 and near OL (3,200,000 cells/L). After this period, the numbers declined until a last peak
419 at Port Bélon (PB) (777,000 cells/L), CR (139,600 cells/L), and BM (150,800 cells/L) on
420 the 14th of September. Towards the end of the bloom, *L. polyedra* may have been grazed
421 by *Noctiluca scintillans*, which was first observed on the 17th of August and reached a
422 peak on the 30th of September at OL (see symbol * in Fig. 2). The bloom was terminated
423 by mid-September, and the last observation of *L. polyedra* in field samples was on the 20th
424 of September (Fig. 3). Typical cyst formation of *L. polyedra* was observed at OL in the
425
426 plankton at the end of the bloom, from mid-August to mid-September, with a maximum of
427 30,000 cysts/L on the 7th of September (Fig. 5).

428 *Alexandrium tamarense* occurred in the Vilaine estuary in mid-September.

429 Maximum concentrations of this species were recorded at coastal sites, such as KE on
430 September 13 (300,000 cells/L), where more offshore sites had much lower
431 concentrations, such as OL which had maximum concentrations of 4,100 cells/L on
432 September 28.

433

434 3.2. Satellite images of [Chl a]

435

436 Sentinel-3/OLCI satellite observations made it possible to better appraise the
437 spatial and temporal variability of the bloom, using the remotely-sensed [Chl a] as a proxy
438 of phytoplankton biomass in the first optical layer of the water column (typically < 5 m).
439 During the bloom of diatoms, satellite-derived [Chl a] increased up to ~ 30 mg/m³ around
440 mid-July in the Loire and Vilaine River estuaries (Fig. 6). Though cloud cover hampered
441 satellite observations, it seemed that [Chl a] then progressively decreased until early
442 August. A significant increase in [Chl a] was then observed on the 14th of August, with
443 several patches of [Chl a] > 80 mg/m³ stretching along the coastline from the Vilaine Bay
444 toward the Loire River estuary (Fig. 6), and coinciding with the high numbers of L.
445 *polyedra* observed in situ (Fig. 3). While it is not possible to determine the causative
446 species from satellite measurement, in situ taxonomic observations at both OL and BM
447 support the hypothesis that the patches of high [Chl a] were mainly caused by *L. polyedra*
448 (Fig. 2). Towards the end of August, the bloom progressively extended offshore, with a
449 large patch to the southwest of Noirmoutier Island. The bloom continued to expand
450 spatially during early September, covering a maximum extent of about 3200 km² on the 4th
of September. From the 4th to 6th of September, highly concentrated patches of Chl *a* were observed along more than 100 km of coastline from 47°15' to 47°45'N, as well as offshore the islands of Noirmoutier, Belle-Ile and Groix. The [Chl *a*] within the most concentrated patches was as high as 300 mg/m³. One of the most striking result was the sudden appearance of a large patch of about 500 km² with [Chl *a*] > 50 mg/m³ south to Belle-Ile Island within just a few days between the 31st of August and 4th of September. This large patch did not last long, and was partly dispersed two days later on the 6th of September. The bloom then progressively decreased, with [Chl *a*] dropping below 10 mg/m³ in most parts of the study area on the 12th of September. By mid-September, the bloom had completely vanished.

461

### 3.3. Environmental parameters

463

The monthly river discharge by both the Loire and Vilaine rivers in July 2021 was exceptionally high compared to average July discharge, calculated between 1960 and 2020 for the Loire and between 1970 and 2020 for the Vilaine (Fig. 7). A drop in salinity recorded at the ND station (MOLIT) during the second half of July coincided with the high flows of both rivers, in particular that of the Loire (Fig. 8a). The drop in salinity was accompanied by a sharp rise in sea surface temperature (SST) and air temperature (Fig. 8b). At the ND station, the buoy MOLIT registered a maximum SST of 25.4°C on the 19th of July (Fig. 8b), which is the highest value recorded since its first use in 2008 (Retho et al., 2022). Before this event, high [Chl *a*] was observed at ND in Vilaine Bay (~ 13 mg/m³), and at BM offshore the Loire estuary (~ 16 mg/m³), consistent with the high numbers of diatoms observed during this period (Fig. 8c). At that time, DIP surface concentration was < 0.1 µM. At the beginning of August, DIP and DIN surface
476 concentrations at ND and BM were respectively below 0.1 and 0.5 µM, whereas DSi

477 concentration was high (Fig. 8d, e, f), suggesting a rapid consumption of the nitrogen and

478 phosphorus nutrients brought by the river plumes. During that period, DIP and DIN

479 concentrations in bottom waters were low, but not limiting.

480 In the beginning of August, colored waters of *L. polyedra* were first observed during a

481 neap tide period (tidal range of about 4 m). During that period, the winds were weak with a

482 speed < 10 km/h, and mainly oriented westward. In the Vilaine Bay, the water column was

483 stratified, in terms of temperature and salinity. On the 2nd August, the water temperature

484 difference at OL was around 3°C (surface temperature: 20.7°C; bottom temperature:

485 17.6°C, Fig. 8), and the salinity difference was around 4 (surface salinity: 33.7; bottom

486 salinity: 29.9). The water column remained stratified in temperature throughout the bloom

487 period. The vertical distribution of Chl *a* fluorescence at OL was quite heterogeneous,

488 displaying either a maximum around the thermocline, or a subsurface maximum (Fig. 9).

489 On the 17th of August, a peak in surface [Chl *a*] corresponding to high concentrations of *L.*

490 *chlorophorum* and *L. polyedra* was observed at ND (~ 35 mg/m³) and BM (~ 15 mg/m³).

491 The increase in [Chl *a*] was accompanied by an increase in surface DIP concentration,

492 while NID concentration remained very low, below limits of quantification. Surface DIP

493 concentration became below 0.1 µM again in mid-September. The average wind speed

494 remained < 15 km/h throughout the bloom period. A change in wind direction occurred on

495 the 23rd of August, with easterly winds coinciding with the offshore extension of the bloom

496 (Fig. 6).

497 Two distinct moments of hypoxia could be identified at PM, concomitant with a

498 neap tide: the first at the beginning of August and the second around the 20th of August,

499 with a minimum dissolved oxygen concentration of 2.2 mg/L and 3.2 mg/L, respectively

500 (Fig. 10). Very high concentrations of dissolved oxygen (up to 15 mg/L) were recorded
during the bloom climax between the 4th and 6th September.

502

3.4. *Morphological identification of cells and cysts and molecular identification of culture*

504

Cells of *L. polyedra* displayed the characteristic shape and tabulation (*5', 0a, 6", 6c, 6S, 5"*, 2p, 1""*; Fig. 11; see Carbonell-Moore et al., 2021, their table 2). A ventral pore could also be observed. The plate overlap was as described by Tillmann et al. (2021, their fig. 6). On the 2nd August, in the sample from KE, the cell length was 33.6 (46.3) 59.6 µm (n=20), the cell width was 30.8 (45.2) 62.0 µm (n=20). The transparent, spherical cysts bore the typical tapering, hollow, nontabular processes with spinules. The cyst body diameter, measured in the same sample was 44.6 (45.7) 47.6 µm (n=6), and the process length was 7.9 (12.6) 19.3 µm (n=18).

505

506

507

508

509

510

511

512

513

514

515

516

517

ITS (OQ608805, 540 bp) and LSU (OQ621602, 931 bp) sequences obtained from the cultures IFR-CC-21-048 and IFR-CC-21-049 were identical and are considered indistinguishable from the *L. polyedra* sequences MW267279 from the type locality by Tillmann et al. (2021). It is notable that there were eight ambiguous nucleotides in our LSU sequence, and four in the LSU sequence from the type locality.

518

3.5. *Pigment analysis of cultures and environmental samples*

520

521

522

523

524

525

The bloom was sampled for pigment analysis and *L. polyedra* cell number enumeration in the beginning of September (Table S3). The observed pigments, namely chlorophyll *a* and *c2* (Chl *c2*), β-carotene, diadinoxanthin, dinoxanthin, peridinin, and sometimes diatoxanthin (Fig. 12), were characteristic of dinoflagellates with type 1 chloroplasts (Gonyaulacales, Gymnodiniales, Peridiniales, Prorocentrales,
Thoracosphaerales; Zapata et al., 2012), consistently with the very high number of *L. polyedra* cells. The pigment composition of the field samples was compared with those of *L. polyedra* monospecific cultures (VGO668, LP4V, LP9V, and the new strain IFR-LI-01LC) and no significant differences were found (Fig. 12). In addition, no pigments from other algal groups were detected. These results suggest that most of the observed *Chl a* belonged to *L. polyedra* or possibly other dinoflagellates with type 1 chloroplasts. *Chl a* per *L. polyedra* cell in the bloom (~36 pg/cell) was very similar to what we found in monospecific cultures (~ 41 pg/cell in the bloom strain in culture and 47 pg/cell in VGO668), and consistent with previous studies (20–60 pg/cell in Prézelin and Matlick, 1983). Nitrogen (N) content of *L. polyedra* cells in culture (~45 pmol/cell) was also in the range of published data on *L. polyedra* (35–70 pmol/cell in Prézelin and Matlick, 1983).

537

### 3.6. Toxin concentrations of cultures, sediments and shellfish

539

None of the 22 yessotoxins were detected in both analysed strains IFR-CC-21-048 and IVT-LI-01LC. The detection limits were between 0.0024 and 0.024 fg/cell for these samples. Yessotoxin was however detected in sediment collected at PO in February 2022. The mean YTX concentration was 6.5 ± 0.9 ng/g dry sediment; however, none of the other yessotoxins monitored were detected. In shellfish, the highest YTX concentration was observed in mussels, with 747 µg eq. YTX/kg at Le Maresclé (LM, Fig. 1) during early September. Such level is however far below the regulatory norm of 3.75 mg eq. YTX/kg (Fig. 13). Common cockles (*Cerastoderme edule*), clams (*Ruditapes philippinarum*), Pacific oyster (*Magallana gigas*) were also contaminated, but at a level much lower than in mussels (Fig. 14).

550
551 3.7. *Cyst relative abundances and concentrations in surface sediments*

552

553 During the following winter, cysts of *L. polyedra* were very dominant in the assemblages with relative abundances between 67.1 and 99.3 %. Other remarkable species identified were *Alexandrium minutum* (29.5% of cysts at DO), *A. tamarense* (0.06–0.75% of cysts at several locations), and *Protoceratium reticulatum* (0.06–1.5% of cysts at several locations). *Spiniferites belerius*, *S. lazus*, and *S. membranaceus* were also identified, but at relatively low abundances. Cysts of the autotrophic species *Biecheleria* sp., *Blixaea quinquecorne*, *Gymnodinium aureolum*, *G. impudicum*, *Pentapharsodinium dalei*, *Scrippsiella lachrymosa*, and *Sourniae diacantha* were also encountered throughout the study area. Cysts of the heterotrophic species *Archaeperidinium minutum*, *A. saanichi*, *Dissodinium pseudolunula*, *Dubridinium* sp., *Selenopemphix quanta*, *Trinovantedinium applanatum*, *Votadinium rhomboideum*, and *V. spinosum*, as well as round brown cysts, spiny brown cysts, and cysts of species within the Diplopsaloideae co-occurred in the same samples. Cysts of *L. chlorophorum* were not observed in the sediment samples.

566 Cyst concentrations of living *L. polyedra* were quite variable, with low concentrations of 111 cysts/g dried weight at St. Gildas (SG), and highest concentrations of 94,498 cysts/g dried weight at PO (Figs. 15, 16). There was a clear relationship between cyst concentration and sediment grain-size: fine-grained sediment had the highest concentrations. It is notable that high concentrations of the toxic species *A. minutum* were found at DO (Fig. 1) (10,680 cysts/g dried weight).

572

573 3.8. *Long-term records of cell concentrations*

574

575 Records of *L. polyedra* in the French Atlantic date back to August 1990, where it
576 was identified from Prat-Ar-Coum in the North of Brittany (REPHY, 2022). Since then, *L. polyedra* has occurred in low concentrations, with the exception of two events in August 2003 at Le Halguen in southern Brittany (38,000 cells/L) and September 2017 at Hossegor in the Landes (187,000 cells/L) (REPHY, 2022). This suggests that the bloom reported in this study was unprecedented.

581

## 4. Discussion

583

### 4.1. *Identification of L. polyedra*

585

586 The typical shape and tabulation described by Tillmann et al. (2021) identifies the species 587 morphologically as *L. polyedra*, and the measurements also conform to observations 588 provided by these authors: 37.6–52.5 µm in length and 33.8–47.9 µm in width. The LSU 589 and ITS rDNA sequences confirm this identification. The observed cysts also conform to 590 the description given by Van Nieuwenhove et al. (2020), and their sizes fit the wide ranges 591 reported by the same authors (35–60 µm in body diameter and 0–35 µm in process length). 592

592

### 4.2. *From cells to satellite: importance of observation strategy*

594

595 The synergetic use of taxonomically-accurate point-based field monitoring together with 596 opportunistic citizen seawater discoloration reports and spatially-resolved [Chl *a*] satellite 597 time-series made it possible to provide an accurate picture of the bloom dynamics in terms 598 of phytoplankton composition, concentration, and spatiotemporal variability. While 599 regular, station-based monitoring such as the REPHY program was instrumental to 600 characterize phytoplankton abundance and diversity (Fig. 2), the citizen science project
601 PHENOMER has significantly aided in documenting the bloom. Seven reports of colored
602 water during summer 2021 have enabled to better document the bloom extent and duration
603 (Fig. 3, red stars largely denote PHENOMER observations). This highlights the
604 importance of citizen participation in complement of regular monitoring to study HABs in
605 nearshore waters. In addition, Sentinel-3/OLCI observations made it possible to provide
606 consistent pictures of the bloom up to 100 km off shore, in otherwise under-sampled areas.
607 Due to the considerable size of the bloom, the spatial resolution (300 m) and wide swath of
608 S3/OLCI were appropriate for accurate mapping over a large region (i.e. > 10,000 km²).
609 Whereas blooms of *L. polyedra* generally extend over large scales (e.g. Pitcher et al., 2019,
610 Kahru et al., 2021), seawater discolorations caused by other dinoflagellates may be much
611 smaller, with spatial features < 300 m. In that case, satellite sensors with a higher spatial
612 resolution such as Sentinel-2/MSI (20 m) would be more performant than S3/OLCI, as
613 recently demonstrated for a variety of red tide events worldwide (Gernez et al., 2023),
614 including high-biomass blooms of *L. chlorophorum* (Roux et al., 2022) and *N. scintillans*
615 (Sacilotto Detoni et al., 2023). On the other hand, due to its high temporal resolution,
616 S3/OLCI (1 day) is more suited than S2/MSI (5 days) for short-term temporal studies. In
617 the present study, the high temporal resolution of S3/OLCI made it possible to acquire
618 enough cloud-free images to document the bloom dynamic during a gloomy summer.
619

620

4.3. *Unprecedented concentrations and relation to toxicity and oxygen concentrations*

621

622 Extremely high concentrations of *L. polyedra*, in the range from 2,000,000 to
623 3,600,000 cells/L, were observed in situ at several stations from mid-August to early
624 September. The combined interpretation of both the satellite-derived Chl *a* concentration
625 (up to 300 mg/m³ in the most intense patches during early September) together with the in
626 situ measurement of *L. polyedra* intra-cellular Chl *a* content (36 pg Chl *a*/cell) suggests
627 that *L. polyedra* maximal cell number was of the order of 8,000,000 cells/L during the
628 bloom climax. The occurrence of such high concentrations of *L. polyedra* and the sheer
629 size of the bloom for the first time in over thirty years, is quite impressive, and highlights
630 that a tipping point, an abrupt shift in ecosystem state, was reached because a massive
631 change in the environment has occurred. However, these are not the highest concentrations
632 that have been reached worldwide: highest concentrations ever were one order of
633 magnitude higher: 56,100,000 cells/L as recorded from Odessa Bay by Terenko and
634 Krakhmalny (2022) and similar high numbers have been recorded from Kaštela Bay (e.g.,
635 44,000,000 cells/L; Marasović et al., 1995), California (e.g. 27,700,000 cells/L; Eppley
636 and Harrison, 1975), and Mexico (15,414,000 cells/L; Stohler, 1959).

637 Although no toxicity was detected for the established cultures, toxins were detected
638 in the cyst-containing sediments, and in several shellfish species that were contaminated
639 during and after the bloom, suggesting that *L. polyedra* produces YTXs, but at low
640 concentrations. The cultured *L. polyedra* strains could have lost their toxicity, could have
641 never been toxic, or could produce toxins below detection levels. The other YTX
642 producers, *Protoceratium reticulatum* and *Gonyaulax spinifera*, were rarely present or
643 absent in the plankton and sediment samples, and can thus not explain the presence of
644 YTXs. Toxic episodes that have previously been associated with blooms of *L. polyedra*
645 displayed elevated YTXs concentration in mussels (North Adriatic Sea: Tubaro et al.,
646 1998; NW Spain: Arévalo et al., 2006; Black Sea: Morton et al., 2007).

647 Mortalities have also been associated with such blooms: mortality of abalone in
648 South Africa (Pitcher et al., 2019; although *Gonyaulax membranacea* was co-occurring in
649 the bloom), oyster mortalities during summer 1964 in the Auray Estuary, France
650 (Paulmier, 1972), mussel mortality the fall of 1962 in Alamitos Bay Marina, California
651 (Reish, 1963) and fish and shellfish mortalities in California in 2020 (Anderson and
652 Hepner-Medina, 2020). These mortalities could be related to the toxicity of *L. polyedra*,
653 but have also been related to dissolved oxygen depletion (Reish, 1963; Anderson and
654 Hepner-Medina, 2020). In our study, low oxygen concentrations were recorded in bottom
655 waters by the high frequency monitoring at the PM station. Some concentrations were
656 below 2.9 mg/L, the hypoxic threshold for many benthic invertebrates (Rosenberg et al.,
657 1991; Diaz and Rosenberg, 2008) but no anoxic levels were reached and no mortalities
658 have been observed during the study period. More severe hypoxias might have occurred in
659 deeper and more stratified areas.

660 Although mentioned by Belin et al. (2021) in their review of toxic species along
661 French coasts, *L. polyedra* was not discussed in depth, presumably because of the little
662 variation seen in the spatial, temporal, and abundance trends. The current study documents
663 the accumulation of YTXs in shellfish and hypoxia events caused by *L. polyedra* on the
664 French Atlantic coast.

665

666 **4.4. What triggered and maintained the bloom?**

667

668 It is difficult to understand how all of a sudden such high concentrations were
669 reached. There are two possible explanations as to its origin, which could be autochtonous
670 or allochtonous. Cells or cysts could have been advected from elsewhere. However, there
671 is no evidence of significant blooms of *L. polyedra* further to the south of France (REPHY
672 data) or the North of Spain during this period (Martha Revilla, pers. comm.), which makes
673 it unlikely that the bloom was advected. In addition, 15 surface sediment samples
674 previously screened for cysts in Vilaine Bay during winter 2018/2019, showed low
675 abundances of *L. polyedra* cysts in the area (André, 2019; Schapira et al., 2021; Roux et
al., 2023). It seems thus likely that the right environmental conditions were in place for
bloom development from a small local seed population. *L. polyedra* cysts that have been
stored under anoxic conditions germinate faster than those stored in an oxic environment
(Blanco, 1990). Ratmaya et al. (2022) showed that the concentration of dissolved oxygen
in the sediments of Vilaine Bay decreased rapidly in the first 2 mm and then was
undetectable below this depth. The thickness of the oxidized layer varies according to the
temperature and the intensity of the mineralization of the organic matter (Souchu et al.,
2018). The sediments of Vilaine Bay therefore have favorable characteristics for the rapid
germination of *L. polyedra* cysts. The bay of Vilaine, whose hydro-morphological
characteristics are favorable to the development of phytoplankton, could therefore be the
site of initiation of the *L. polyedra* blooms observed in southern Brittany in 2021. It is also
possible that dredging activities during summer have liberated dormant cysts from the
sediments (e.g. Fernandes et al., 2023), but we have no data that could support such a
claim.

The unusually high river discharge and subsequent water column stratification have
likely contributed to the massive development of *L. polyedra*, although measured nutrient
concentrations were not unusual (cf. Ratmaya et al., 2019). High Chl *a* concentrations have
indeed previously been correlated to higher river discharge in the northern Bay of Biscay
(Gohin et al., 2019). The freshwater input occurred during a period of nutrient depletion,
and coincided with a bloom of diatoms (*Leptocylindrus* spp.). In the Vilaine Bay, blooms
of diatoms are predominantly driven by nutrient inputs associated with freshwater plumes
from the Loire and Vilaine rivers (Ratmaya et al., 2022). The deposited diatom-derived
OM is then mostly directly mineralized and recycled within the water column as
ammonium (NH<sub>4</sub><sup>+</sup>) and organic nitrogen (Ratmaya et al., 2022). These sources of nitrogen
may have benefited to *L. polyedra* (see below). Moreover, in the Vilaine Bay, sediments
represent a substantial DIP source in summer (Ratmaya et al., 2019), and the increase in DIP concentrations observed in seawater around mid-August 2021 could have a benthic origin. Benthic flows of nutrients were all the more important for the development of *L. chlorophorum* and *L. polyedra* that the water mass was stratified during periods of weak winds and/or neap tide. Water column stratification is considered an essential physical condition for the proliferation of dinoflagellates (Margalef, 1978; Smayda, 2002) and it certainly contributed to the development of *L. polyedra* blooms, as suggested by Ruiz-de la Torre et al. (2013). The fluorescence maximum was sometimes recorded at the thermocline, suggesting higher *L. polyedra* abundance at this depth. This species could migrate vertically throughout the water column to use nutrients located below the pycnocline as demonstrated or suggested for other dinoflagellates (Glibert et al., 2016, Roux et al., 2022).

DIN was limiting in surface waters from the beginning of August to the end of September according to the criteria of Fisher et al. (1995), but urea uptake very likely contributed to sustain the growth *L. polyedra*. Kudela and Cochlan (2000) demonstrated that 38% of the nitrogen demand for a *L. polyedra* red tide event in Southern California was supported by urea. *L. polyedra* is also a mixotrophic dinoflagellate (combining phototrophy and phagotrophy), able to ingest a variety of organisms, ranging from picoplankton-sized prey (< 2 µm) up to larger preys (> 30 µm, Jeong et al., 2005).

Mixotrophy is important in nutrient acquisition, growth of harmful algal species, and bloom maintenance in eutrophic habitats (Burkholder et al., 2008). The important growth and persistence of the *L. polyedra* bloom over several weeks is therefore likely due to a combination of both ecophysiological (e.g., mixotrophy) and hydro-meteorological (freshwater inputs followed by a period of low wind and water mass stratification) factors.

The wind played a further role as the change to easterly winds may have contributed to
726 disperse the bloom westwards after mid-August.

727 When such conditions occurred during previous summers in Vilaine Bay, it led to
728 seawater discolorations associated with blooms of *L. chlorophorum* (Siano et al., 2020),
729 such as in 2019 (Roux et al., 2022). In 2019, the spatial extent of the *L. chlorophorum*
730 bloom was however far smaller than the massive bloom of *L. polyedra* documented in the
731 present study. While the exceptional rainfall that occurred in June – July certainly played a
732 role in promoting phytoplankton growth, the reason why *L. polyedra* dominated over other
733 HAB-forming dinoflagellate species remains to be fully elucidated.

734

735 **4.5. What terminated the bloom?**

736

737 Several factors have been documented in the literature to explain bloom termination in
738 dinoflagellates, such as grazing (e.g., Busch et al., 2019), cyst formation (e.g., Wang et al.,
739 2007; Brosnahan et al., 2017), parasite infection (e.g., Velo-Suárez et al., 2013), algicidal
740 bacteria (Doucette et al., 1999), and/or changes in hydrodynamics (e.g., Ralston et al.,
741 2015). Grazing and cyst formation have been observed during this study and are discussed
742 below.

743 *Noctiluca scintillans* is a heterotrophic dinoflagellate that has been described as an
744 effective grazer of large *L. polyedra* blooms in the Southern California Bight (Balch and
745 Haxo, 1984; Howard, 1996; Goldstein, 2011). It could play an important role for *L.*
746 *polyedra* bloom regulation and possibly even termination, as it occurs several weeks after
747 blooms of *L. polyedra* (Busch et al., 2019). In our study area, *N. scintillans* was observed
748 at OL and BM stations around mid-August (Fig. 2b, d). Other grazers that have been
749 related to *L. polyedra* blooms in California are *Polykrikos* sp., *Fragilidinium heterolobum*,
750 as well as rotifers and ciliates (Eppley and Harrisson, 1975), but such taxa have not been
observed in notable numbers in the present study. Zooplankton grazers such as copepods
likely do not have such an effect: *L. polyedra* has a competitive advantage because of its
bioluminescence that can deter copepods (Prevett et al., 2019). It is also notable that a
bloom of (presumably non-toxic) *A. tamarense* occurred in the Vilaine estuary in mid-
September, right after the *L. polyedra* bloom.

Cyst production was also observed during this study in the plankton at the end of
the bloom. The induction of sexuality has been presumed to be related to nutrient
depletion, changes in day length, temperature, light intensity and dissolved gases (Pfiester
and Anderson, 1987, p. 631), but this was not seen in this study.

760

761 4.6. *Seed banks*

762

The very high concentrations of *L. polyedra* cysts frequently found in surface
sediments demonstrate that a significant seed bank has been formed after the bloom.
Concentrations of living *L. polyedra* cysts (between 111 and 100,710 cysts/g dried weight)
are greater than concentrations of living cysts recorded by Lewis (1988) from Loch Creran
(west coast of Scotland) (between 0 and 20,253 cysts/g dried weight), where *L. polyedra*
blooms have been recurrent over many years. They are also much higher than *L. polyedra*
cyst concentrations recorded in palynologically treated surface sediments from the Bay of
Brest, which ranged between 49 and 3,000 cysts/g dried weight (Lambert et al., 2022).

However, the results from the methods used here are not directly comparable to
palynological methods; both methods can introduce a numerical bias and palynological
methods enumerate cysts with cell contents and those without (Mertens et al., 2009b).

High concentrations of *L. polyedra* cysts related to fine grain-size conform to previous
observations by Lewis (1988). The presence of such seed banks suggests that blooms of *L.*
776 *polyedra* will be recurrent in the coming years. It also demonstrates that *L. polyedra*

777 blooms are clearly imprinted in the geological record by an exponential increase in cyst

778 numbers. This result supports the hypothesis of Dale (2009) that *L. polyedra* cysts are

779 useful indicators of increased nutrient supplies.

780 The discovery of cysts of the potentially toxic species *A. minutum* at relatively high

781 concentration at DO not only shows that *L. polyedra* had formed a seed bank, it also

782 highlights that very local seed banks can be formed and pose a risk for local stakeholders

783 (e.g. shellfish farms).

784 The presence of such important seed banks has its implications for regional

785 dredging or trawling activities. Such activities cause resuspension of cysts from sediments,

786 and can provide an inoculum for blooms (e.g., Brown et al., 2013), and therefore are

787 recommended to take place during winter.

788

789 **Declaration of Competing Interest**

790

791 The authors declare that they have no known competing financial interests or

792 personal relationships that could have appeared to influence the work reported in this

793 paper.

794

795 **Acknowledgements**

796

797 We like to thank Sylvain Ballu (CEVA) for providing the aerial photographs. The

798 Sigma 300 FE-SEM used in this study was funded by The Regional Council of Brittany,

799 the General Council of Finistère and the Urban Community of Concarneau-Cornouaille-
800 Agglomération. The authors acknowledge EUTMESAT and ESA for the Sentinel-3
801 observations. Part of this research was supported by the Centre National d'Etudes Spatiales
802 (TOSCA projects LASHA and OSYNICO), Region Pays de la Loire (project LEPIDO-
803 PEN [06582 2019], Agence de l'Eau Loire Bretagne (project EPICE [180408801]). This
804 work benefited from a national funding through the Agence Nationale de la Recherche for
805 France 2030 under the reference ANR-22-POCE-0006.

806

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1165

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1176 Déterminisme et conséquences sur la qualité des masses d'eau côtières. Projet EPICE –

1177 Rapport final. RST/LER/MPL/21.10. https://archimer.ifremer.fr/doc/00724/83598/

1178

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1182

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1196

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1201 Stobo, L.A., Lewis, J., Quilliam, M.A., Hardstaff, W.R., Gallacher, S., Webster, L., Smith, E., McKenzie, M., 2003. Detection of yessotoxin in UK and Canadian isolates of phytoplankton and optimization and validation of LC-MS methods. In: Bates, S. (Ed.), Proceedings of the eighth Canadian workshop on harmful marine algae. Can. Tech. Rep. Fish. Aquat. Sci. 2498, 8-14.

1206

1207 Stohler, R., 1959. The red tide of 1958 at Ensenada, Baja California, Mexico. The Veliger 2, 32-35.

1209

1210 Terenko, G., Krakhmalny, A. 2021. Red tide of *Lingulodinium polyedrum* (Dinophyceae) in Odesa Bay. Turk. J. Fish. & Aquat. Sci. 22, TRJFAS20312.

1212

1213 Tillmann, U., Bantle, A., Krock, B., Elbrächter, M., Gottschling, M., 2021.

1214 Recommendations for epitypification of dinophytes exemplified by *Lingulodinium polyedra* and molecular phylogenetics of the Gonyaulacales based on curated rRNA sequence data. Harmful Algae 104, 101956.

1217

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1223

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1230

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1233 Penaud, A., Radi, T., Rochon, A., 2020. An overview and brief description of common
1234 marine organic-walled dinoflagellate cyst taxa occurring in surface sediments of the
1235 Northern Hemisphere. Mar. Micropaleont. 159, 101814.

1236

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1239 *Alexandrium fundyense* Blooms within a Small Coastal Embayment. PLoS ONE 8,
1240 e81150. doi:10.1371/journal.pone.0081150

1241

1242 Wang, Z.-H., Qi, Y.-Z., Yang, Y.-F., 2007. Cyst formation: an important mechanism for
1243 the termination of *Scrippsiella trochoidea* (Dinophyceae) bloom. J. Plankton Res. 29, 209–
1244 218.

1245

1246 Zapata, M., Fraga, S., Rodríguez, F., Garrido, J.L., 2012. Pigment-based chloroplast types
1247 in dinoflagellates. Mar. Ecol. Prog. Ser. 465, 33–52.
1248

1249 Zonneveld, K. A. F., Marret, F., Versteegh, G. J. M., Bogus, K., Bonnet, S., Bouimetarhan,

1250 I., Crouch, E. et al. 2013. Atlas of modern dinoflagellate cyst distribution based on 2405

1251 data points. Rev. Palaeobot. Palynol. 191, 1–197.

1252

1253 Figure captions

1254

1255 Fig. 1. Map of plankton and sediment sampling stations in southern Brittany as mentioned

1256 in the text. Major rivers and islands are indicated. BM = Basse Michaud, CR = Le Croisic,

1257 DO = Doëlan, KE = Kervoyal, LM = Le Maresclé, ND = Nord Dumet, OL = Ouest

1258 Loscolo, PB = Port Bélon, PM = Pont Mahé, PMa = Port Manec'h, PO = Pornichet Port,

1259 SG = Saint-Gildas, Bathymetry is indicated by grayscale.

1260

1261 Fig. 2. *L. polyedra* cell concentrations and assemblage changes in the plankton observed at

1262 Ouest Loscolo (OL) (a, b) and Basse Michaud (BM) (c, d) throughout the summer of 2021.

1263

1264 Fig. 3. Spatial and temporal changes of *L. polyedra* abundances (cells/L) observed within

1265 the framework of the REPHY network and the PHENOMER project (red stars largely

1266 denote PHENOMER observations).

1267

1268 Fig. 4. Aerial photograph of Clohars-Carnoët showing the presence of the *Lingulodinium*

1269 bloom, taken on 7 September 2021.

1270

1271 Fig. 5. Cyst abundance of *L. polyedra* (cells/L) observed in the surface water at Ouest

1272 Loscolo (OL), where water depth is 10 m.
1273

1274 Fig. 6. Satellite images. High spatial resolution (300 m) Sentinel-3/OLCI derived maps of
1275 [Chl a] computed using an algorithm optimized for highly concentrated phytoplankton
1276 blooms (Smith et al., 2018). Among cloud-free satellite acquisitions, the dates were
1277 selected to (i) concisely depict the bloom development, climax, and decline, and (ii)
1278 complement in situ observations shown in Fig. 3.

1279

1280 Fig. 7. Monthly river discharge (m³/s) by the Loire (a) and Vilaine (b) in 2021 compared
1281 with the mean, percentile 90 and percentile 10 from 1960 to 2020 for the Loire and 1970 to
1282 2020 for the Vilaine.

1283

1284 Fig. 8. Variations of environmental parameters from May to September 2021 (A)
1285 Variations in daily river discharge (m³/s) of the Loire and the Vilaine and salinity recorded
1286 at high-frequency by the MOLIT buoy at Nord Dumet (ND) station. (B) Time-series of
1287 temperature recorded at high-frequency by the MOLIT buoy at ND station, and air
1288 temperature at the weather station Vannes-Séné. (C) Chl a (µg/L) in surface water at ND.
1289 (D–F). DSi, DIP and DIN concentrations (µM) at ND and BM stations. Vertical dashed
1290 lines indicate period when blooms were observed in situ.

1291

1292 Fig. 9. Fluorescence and Temperature profiles at Ouest Loscolo (OL) on August 2nd,
1293 August 17th and September 6th 2021.

1294

1295 Fig. 10. Oxygen concentrations (mg/L) at Pont Mahé (PM), and tidal coefficients from
1296 June to September 2021.

1297
1298 Fig. 11. Scanning electron micrographs of *L. polyedra* from Kervoyal (KE). (A) Apical view. (B) Antapical view. (C) Ventral view and detail of other cell showing presence of ventral pore (denoted by black arrow). (D) Dorsal view. All scale bars = 3 µm.

1301

1302 Fig. 12. Pigment composition of *Lingulodinium polyedra* in the bloom and in monoalgal cultures. (A) Pigment ratios (mol/mol): Peridinin/Chl c2, Peridinin/Chl *a*, Chl c2/Chl *a*, Diadinoxanthin/Chl *a*, Dinoxanthin/Chl *a*. (B) Chl *a* per cell (pg/cell). Each bar is the mean of 12 different samples for the bloom data, the mean of three different strains (LP4V, LP9V from Zapata et al. (2000) and VGO668) for the culture data and the mean of four cultures for the strain IFR-LI-01LC. Error bars represent standard deviations.

1308

1309 Fig. 13. YTX concentrations measured in mussels in Vilaine Bay (Le Maresclé station, LM) and *L. polyedra* concentrations (cells/L) from Ouest Loscolo (OL). Reglementary norm is indicated.

1312

1313 Fig. 14. YTX concentrations (µg/kg) measured in mussels and other shellfish and *L. polyedra* concentrations (cells/L) at Le Croisic (CR). Reglementary norm is indicated.

1315

1316 Fig. 15. Cyst concentrations in surface sediments (cysts/g dried weight) at the end of winter 2021/2022. AP = Anse de Pen Men, CO = Coupelasse, CR = Le Croisic, DO = Doëlan, KE = Kervoyal, LM = Le Maresclé, LO = Lorient harbor, ME = Men er Roué, ND = Nord Dumet, PE = Pénerf, OL = Ouest Loscolo, PM = Pont Mahé, PO = Pornichet Port, SG = Saint Gildas.

1321

1322 Fig. 16. Light microscope image of cysts from surface sediment from Pornichet Port (PO,
1323 23 February 2022). Scale bar = 500 µm.

1324

1325 Fig. 17. Long-term record of *L. polyedra* concentrations (cells/L) recorded along the

1326 French Atlantic and Manche since 1990, extracted from the REPHY dataset (REPHY,

1327 2021).
<figure><img src="image_2.png" /></figure>

Concarneau

FRANCE

Atlantic Ocean

Legend

★ stations

● Weather stations

Bathymetry

-100 m

-80 m

-60 m

-40 m

-20 m

0 25 50 km

Ifremer, SHOM
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>

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

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

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

<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>

L. polyedra

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

<table><tr><td></td><td>Land</td></tr><tr><td>No data</td><td></td></tr></table>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /></figure>
Fluorescence (FFU)

<figure><img src="image_19.png" /></figure>

2 Aug - 3.30 pm

Fluorescence (FFU)

<figure><img src="image_20.png" /></figure>

17 Aug - 1.15 pm

Fluorescence (FFU)

<figure><img src="image_21.png" /></figure>

6 Sep - 1.00 pm
<figure><img src="image_22.png" /></figure>

Dissolved oxygen (mg/L)

Tidal range (m)
A

<figure><img src="image_23.png" /></figure>

B

<figure><img src="image_24.png" /></figure>

C

<figure><img src="image_25.png" /></figure>

D

<figure><img src="image_26.png" /></figure>
<figure><img src="image_27.png" /></figure>

B
<figure><img src="image_28.png" /></figure>

Le Maresclé mussel

reglementary norm YTXs

L. polyedra
<figure><img src="image_29.png" /></figure>
<figure><img src="image_30.png" /></figure>

Source : Ifremer, Google satellite
<figure><img src="image_31.png" /><figcaption>500 µm</figcaption></figure>
<figure><img src="image_32.png" /></figure>
Tables

<table><caption>Table 1: Sampling localities for surface sediment samples obtained for cyst counts, with geographic coordinates, water depth, sampling date and sampling device.</caption><thead><tr><th>Sample name</th><th>Longitude</th><th>Latitude</th><th>Water depth (m)</th><th>Sampling date</th><th>Sampling device</th></tr></thead><tbody><tr><td>Doëlan</td><td>-3.60333</td><td>47.77671</td><td>0.0</td><td>7-Apr-22</td><td>By hand</td></tr><tr><td>Lorient</td><td>-3.36556</td><td>47.71833</td><td>12.0</td><td>1-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Men er Roué</td><td>-3.09375</td><td>47.53477</td><td>9.0</td><td>15-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Anse de Pen Men</td><td>-2.83427</td><td>47.61439</td><td>0.0</td><td>29-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Pénerf</td><td>-2.64783</td><td>47.50798</td><td>6.3</td><td>14-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Port Mahé</td><td>-2.48467</td><td>47.43435</td><td>5.4</td><td>14-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Marescelé</td><td>-2.50060</td><td>47.46197</td><td>5.0</td><td>14-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Estuaire Vilaine</td><td>-2.52568</td><td>47.50985</td><td>4.2</td><td>14-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Ouest Loscolo</td><td>-2.53827</td><td>47.45759</td><td>10.0</td><td>14-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Nord Dumet</td><td>-2.65678</td><td>47.46020</td><td>14.5</td><td>14-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Le Croisic</td><td>-2.53412</td><td>47.27345</td><td>26.0</td><td>28-Mar-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>Pornichet</td><td>-2.34646</td><td>47.25781</td><td>3.0</td><td>23-Feb-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>La Coupelassse</td><td>-2.02635</td><td>47.01644</td><td>0.0</td><td>4-Apr-22</td><td>Petite Ponar<br>Grab</td></tr><tr><td>St Gildas</td><td>-2.32037</td><td>47.17267</td><td>24.0</td><td>29-Mar-22</td><td>Petite Ponar<br>Grab</td></tr></tbody></table>