Palytoxin-Analogues Accumulation in Natural Mussel Banks during an Ostreopsis cf. ovata Bloom

: Intense blooms of the toxic dinoflagellate Ostreopsis producing palytoxin (PlTX) analogs, mainly ovatoxins (OVTXs), have been a recurrent phenomenon along several Mediterranean coasts. Although there is evidence of seafood contamination by these toxins, the dynamics of their bioaccumulation during Ostreopsis blooms is not yet clear. Toxin concentrations in wild mussels at two sites in the Conero Riviera, along the northern Adriatic Sea (Portonovo and Passetto), were analyzed from August to October 2021, throughout an Ostreopsis cf. ovata bloom, to investigate their relationships with the bloom pattern and abundances. Contaminated mussels showed the typical toxin profile dominated by OVTX-a and -b, with levels lower than the threshold considered unsafe for human consumption (30 µg PlTX-equivalent kg − 1 soft tissue). The toxin content reached 36.4 µg PlTX kg − 1 soft tissue only in a single sampling date. A clear correlation between the bioaccumulation of OVTXs in mussels and the abundance of Ostreopsis was observed. Our results highlighted, however, that bioaccumulation in the mussels is not affected merely by the abundance of toxic microalgae, since higher toxin levels were recorded at Portonovo, where the cell abundances were lower than at Passetto. The results from this study indicate that the Italian guidelines for the management of Ostreopsis blooms in bathing waters are also useful in managing the risks of human intoxication through ingestion, as mussel contamination was detected only during the alert phase (10,000–30,000 cells L − 1 ).

Several species of this genus produce toxins, mainly ovatoxins (OVTXs), belonging to the group of palytoxin (PlTX) analogs ( Figure 1) [20][21][22][23][24][25][26][27][28]. In tropical areas, Ostreopsis (i.e., O. siamensis) is strongly suspected (although without confirmation) to be involved in some events of clupeotoxism. This intoxication is characterized by a symptomatology similar to that of ciguatera but with a much higher mortality rate, due to the consumption of contaminated crustaceans and finfish (Fam. Clupeidae) [29,30]. In temperate areas, such as the Mediterranean Sea, there are no reports of intoxications due to the ingestion of contaminated seafood [31]. At these latitudes, however, Ostreopsis blooms (mainly produced by O. cf. ovata) cause problems related to human health, mostly due to the inhalation of seawater droplets containing aerosolized toxins and/or fragments of microalgal cells, or cutaneous contact with cells, causing mainly dermatitis and ocular irritation/conjunctivitis [32][33][34]. PlTX analogs have been detected in a wide range of marine animals, such as finfish, echinoderms, gastropods, crustaceans, and cephalopods, without any apparent sign of harm to these organisms [35,36]. Ostreopsis blooms have been often associated with anomalous arm positions in starfish and the loss of spines in sea urchins in Mediterranean waters (northern Tyrrhenian Sea) [37], as well as mass mortalities of sea urchins along Brazilian [38] and New Zealand coasts [39]. In laboratory experiments, O. cf. ovata was shown to be harmful to the larvae of jellyfish [40], invertebrates, and finfish [41,42]. Impairments observed consisted of sperm fertility reduction [43] and abnormal growth in embryos produced via the fertilization of sea urchins during blooms [44,45]. In mussels, high concentrations of PlTX analogs may cause an evident decrease in metabolic rates in hepatopancreas and mantle cells [46] or increase the percentage of phagocytizing immunocytes [47]. Mussel susceptibility to Ostreopsis was shown in the inhibition of the Na + /K + ATPase and the alteration of other enzymatic activities [48], as well as in immunopathological signals [49]. Mussels exposed to Ostreopsis activated an inflammatory response, mainly characterized by digestive tubule damage and hemocyte aggregates, preventing further feeding and food assimilation [50].
In particular, in the Mediterranean Sea, PlTX analogs were detected in shellfish collected from the northern Aegean Sea, the French Mediterranean coasts, the northern Adriatic Sea, and the Gulf of Naples, with maximum concentrations ranging from 100 to 600 µg kg −1 [51][52][53][54]. These concentrations are much higher than the threshold of 30 µg kg −1 indicated by the European Food Safety Authority [55]. Therefore, although there is evidence of shellfish contamination by Ostreopsis toxins in temperate areas, the dynamics of their bioaccumulation during the entire period of an Ostreopsis bloom are not clear yet. Consequently, the potential health risks related to mussel consumption in the course of a bloom are also poorly understood.
The aim of this study was to investigate the accumulation of PlTX analogs in natural mussel banks during an Ostreopsis cf. ovata bloom that occurred in late summer/early fall in 2021 in an area considered a hot-spot for the blooms of this toxic dinoflagellate (Conero Riviera, northern Adriatic Sea, Italy) [18]. The results of this study will help to better define the risks associated with the consumption of contaminated mussels during the occurrence of these phenomena.

Sampling
The study was carried out along the Conero Riviera (northern Adriatic Sea) at two stations, Passetto (43°37'09" N, 13°31'54" E) and Portonovo (43°33'41" N 13°36'06" E) ( Figure 2). The shallow part of the sea bottom (1-5 m) at these stations is rocky and gently sloping, partially covered by cobbles and coarse gravel. The Passetto station is a sheltered site, whereas Portonovo is affected by moderate hydrodynamics. Both stations are affected by moderate human effects, as the shore is a popular area for summer holidays. There, mussels from natural banks are collected for human consumption by swimmers in summer and autumn.
Samples of wild mussels were collected at both stations. Samples for Ostreopsis analysis were collected only at the Passetto station.
Mussel (Mytilus galloprovincialis) samples were collected with approximately weekly frequency from natural banks from August to October 2021, i.e., during the O. cf. ovata bloom. In consideration of the bioaccumulation time of toxins, mussel sampling was carried out 4-6 days after each Ostreopsis sampling. About thirty mussels of commercial size (5-7 cm in length) were collected manually via scuba diving and were transported to the laboratory. Sampling of Ostreopsis cf. ovata was conducted monthly from May to July 2021 and weekly from August to October 2021 (i.e., when the bloom ended) at the Passetto station both on benthic substrata (macroalgae) and in the water column. A CTD, Model 30 Handheld Salinity, Conductivity and Temperature System, YSI (Yellow Spring, OH, USA), was used to measure sea surface temperature and salinity and wave height was recorded according to the Douglas scale. For nutrient analysis, water samples were collected near the sampled substratum, avoiding resuspension phenomena, in polyethylene bottles (50 mL); immediately filtered through GF/F Whatman filters (25 mm); and stored in triplicate in 4 mL polyethylene bottles at −22 °C until analysis. Fragments of macroalgal thalli, i.e., Alsidium corallinum, Hypnea musciformis (Florideophyceae, Rhodophyta), Dictyota dichotoma, and Dictyopteris polypodioides (Phaeophyceae, Ochrophyta) were collected in three replicates at a depth of 0.5 m, following the protocol recommended by Totti et al. [56]. On each sampling date, samples of surface seawater (1 replicate) were collected in polyethylene bottles (500 mL) near the sampled macroalgae to analyze the abundance of Ostreopsis cf. ovata in the water column.

Mussel Sample Treatment
The mussel samples were prepared within 24 h of arrival in the laboratory to prevent the processes of metabolizing toxins. The valves were opened, cleaned, and any residual sand and solids were removed with running water. The mollusk flesh was separated from the shell by removing it with a metal scalpel and was then placed in a large mesh sieve to remove excess water. The pulp was then finely chopped with a blade homogenizer, obtaining a representative sample of 100 g from about 20 specimens used. The homogenate was transferred to a plastic jar and stored at −20 °C until analysis.

Ostreopsis Sample Treatment
In the laboratory, macroalgae were immediately treated following the method of Totti et al. [56]. Briefly, in the field, samples of macroalgae were vigorously shaken in wide-necked HDPE sample bottles in ~100 mL of seawater to dislodge the epiphytic cells. The samples were preserved with 0.8% neutralized formaldehyde [57] and stored in the dark at 4 °C until microscopic observation. Finally, the macroalgae were weighed to assess their fresh and dry weights, and their area was calculated using a conversion factor obtained from the ratio of fresh weight/surface, following the method of Accoroni et al. [54]. Some subsamples without the addition of any fixative were used for strain isolation (see below).

Microscopic Analysis of Ostreopsis
The estimation of Ostreopsis abundances was performed using an inverted microscope (Zeiss Axiovert 135) equipped with phase contrast, at 200× magnification. Sub-samples (1-25 mL) were settled in counting chambers after homogenization, according to the Utermöhl sedimentation method [58]. Cells of O. cf. ovata were identified by observing samples in epifluorescence after staining with a fluorochrome (Calcofluor White). Counting was performed on 10-30 random fields, 1-2 transects, or the whole sedimentation chamber, to count a representative cell number. The final data were expressed as cells g −1 fw/dw and cells cm −2 of macroalga, and as cells L −1 for the water column.

Strain Isolation, DNA Extraction, Amplification, and Sequencing
Ostreopsis cell isolation was conducted following the capillary pipette method [59]. After an initial growth period on microplates, cells were cultured with a 12:12 h L:D photoperiod and an irradiance of 90-100 µmol m −2 s −1 at 21 °C ± 0.1 °C in a modified, silicafree, f/4 medium [60] prepared with autoclaved natural seawater (salinity 35). Trace metals, vitamins (H, B1, and B12), and HEPES pH 7.1 were added at levels corresponding to the f/2 medium. Two strains of Ostreopsis sp. were isolated.
A 30 mL culture of each strain in the exponential growth phase was used to extract genomic DNA. The sample was centrifuged at 4000× g for 15 min and DNA was extracted using the CTAB (N-cetyl-N,N,N-trimethylammonium bromide) buffer (2% CTAB, 1 M Tris pH 8.0, 0.5 M EDTA pH 8.0, 5 M NaCl, 1%) modified from the method of Doyle and Doyle [61]. Extracted DNA was amplified by means of a polymerase chain reaction (PCR) with a SimpliAmp TM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). PCR amplification of region D1-D3 of the LSU rDNA gene was performed following the method of Accoroni et al. [62].
Sanger sequencing was carried out by Macrogen Europe using the same couple of universal primers used for PCR amplification (D1R-D3Ca [63]). Sequences were compared to the NCBI database via a BLAST search with default settings (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 17 December 2021), confirming the identification of Ostreopsis cf. ovata (showing >99% of identity with Ostreopsis cf. ovata LSU sequences from GenBank).
A matrix-matched standard of PlTX, at five levels of concentration (0.080, 0.125 0.250, 0.5, and 1.5 µg mL −1 ), was prepared to build calibration curves for quantitative analyses. A mussel tissue reference material, certified as negative control (CRM-Zero_Mus), was purchased from the National Research Council Certified Reference Materials Program (Institute for Marine Biosciences, Halifax, NS, Canada) and used to prepare the calibration curves.
The PlTX standard was used to quantify PlTX analogs, assuming an equimolar response.

PlTX Analog Extraction from Mussels
The extraction of PlTX analogs from mussels was performed following the procedure described by Ciminiello et al. [64] with some modifications to improve sensitivity.
Briefly, 10.0 g ± 0.5 g of mussel homogenate was weighted in a 50 mL PP tube and added to 30 mL of MeOH/H2O (80:20 v/v). The sample was then homogenized using an Ultra Turrax T25 mixer for 2 min at 9500 rpm and centrifuged for 15 min at 2000× g (20 °C). The supernatant was transferred in a 100 mL evaporation flask and the solid residue was re-extracted twice times in the same conditions described above. The supernatants were collected, obtaining a final volume of 90 mL and evaporated to dryness (T = 50 °C-60 °C) via rotary evaporation (R-114 Büchi, Flawil, Switzerland).
The residue was reconstituted in 5 mL of MeOH/H2O (80:20 v/v) and filtered through a 0.2 µm syringe filter for LC-MS/MS analysis.

LC-MS/MS Analysis
LC-MS/MS analyses were performed on a 3200 QTRAP mass spectrometer (AB Sciex, Darmstadt, Germany) equipped with an electrospray ionization (ESI) interface, coupled to a 1200 series HPLC system (Agilent, Palo Alto, CA, USA), including a solvent reservoir, inline degasser, quaternary pump, refrigerated autosampler, and column oven.
The method was performed following the conditions described previously [54,64], with slight modifications. LC separation was obtained by means of a Gemini NX-C18 column (2 mm × 100 mm, 3 µm particle size; Phenomenex, Torrance, CA, USA), thermostated at 40 °C, using a flowrate of 0.2 mL min −1 and an injection volume of 10 µL. Mobile phases A and B were H2O and MeACN/H2O (95:5 v/v), respectively, both containing 30 mM of CH3COOH. Gradient elution was performed as follows: from 0% to 50% B in 20 min, from 50% to 100% B in 10 min, hold for 5 min, return to the original conditions at 40 min, and hold for 10 min before the next injection.
Multiple Relative retention times (RRTs) were also used to identify PlTX analogs using the PlTX standard. The method was validated in house in order to obtain good performances in terms of linearity, recovery, matrix effect, repeatability (data not shown), the limit of quantification (LOQ), and the limit of detection (LOD). The LOQ, calculated assuming a signal/noise (S/N) ratio of 10, was 13 µg kg −1 , whereas the LOD (S/N ratio of 3) was 5 µg kg −1 .

UHPLC-HRMS 2 Analysis
UHPLC-HRMS 2 analysis was performed using a hybrid quadrupole-Orbitra Q-Exactive mass spectrometer, working in tandem mass spectrometry mode (Thermo Fisher). The mass spectrometer was coupled to a UHPLC system (Thermo Fisher) equipped with a solvent reservoir, a quaternary pump, a refrigerated autosampler, and a column oven.
LC separation was performed using a Luna PFP 2 column (2 mm × 150 mm, 3 µm particle size from Phenomenex) at 40 °C, at a 0.2 mL min −1 flow rate and a 10 µL injection volume. The mobile phases were (A) Water and (B) Acetonitrile/Water 95/5 in volume, both containing 0.1% (v/v) of CH3COOH. The elution was performed with a linear gradient: from 20% to 70% B in 25 min, from 70% to 98% B in 0.5 min, hold for 5 min, then down to 20% B at 35 min. HRMS 2 analysis was performed in negative ion mode (ESI-), setting the ion spray source parameters as follows: sheath gas flow rate-50 psi, auxiliary gas flow rate-15 psi, auxiliary gas heater temperature-325 °C, ion spray voltage-3.20 kV, resolution-70000, and capillary temperature-275 °C.
For each toxin, the molecular precursor ion and 1 or 2 product ions were monitored, with a mass accuracy greater than 5 ppm, as indicated in Table 1. The method was based on the quantification of palytoxins by means of a calibration line with a matrix standard, in a measuring range ≥ 1 µg kg −1 of the sample as is (LOQ). By analyzing the trend of the product ions detected in the MS/MS spectrum characteristic of molecules similar to palytoxin, it was possible to identify and quantify them.

Nutrient Analysis
The colorimetric method of Strickland and Parsons [65] was used to perform the analysis of nitrates, nitrites, ammonium, phosphates and silicates, using an Autoanalyzer QuAAtro Axflow. Detectability limits were 0.02 µmol L −1 for nitrates, nitrites, ammonia, and silica and 0.03 µmol L −1 for orthophosphates. The dissolved inorganic nitrogen (DIN) concentration represents the sum of NO2 − , NO3 − , and NH4 + concentrations

Statistical Analysis
Comparisons between Spearman's (rs) and Pearson's (r) correlations were undertaken to determine both monotonic and linear relationships between (i) the abundances of Ostreopsis cf. ovata recorded in the water column and on benthic substrate and (ii) the abundances of Ostreopsis cf. ovata and OVTX concentrations recorded in the soft tissue of mussels at Passetto station. The statistical analyses were conducted using Statistica (StatSoft) software.

Ostreopsis Bloom
The first cells of Ostreopsis cf. ovata on the benthic substrata were recorded on 19 August and the bloom developed reaching the maximum abundances approximately one month later, i.e., on 16 September (4.9 × 10 3 ± 2.3 × 10 3 cells cm −2 , corresponding to 1.2 × 10 5 ± 5.9 × 10 4 cells g −1 fw and 9.9 × 10 5 ± 4.6 × 10 5 cells g −1 dw). During this period of maximum proliferation, the characteristic brownish mucilaginous biofilm produced by Ostreopsis covered all the macroalgae and the other benthic substrata. Then, around mid-September the abundances rapidly decreased to values around 10 3 cells cm −2 , although the bloom ended only in late October (Figure 3). The abundance pattern of Ostreopsis cf. ovata in the water column closely followed that of the Ostreopsis abundances on macroalgae; the first cells in the water column were recorded on 2 September and the highest abundances were recorded on 16 September (as for the macroalgae) with 1.3 × 10 4 cells L −1 . Then, abundances decreased until early October and O. cf. ovata cells were no longer recorded in the water column by mid-October ( Figure  3). The correlation between the abundance of O. cf. ovata on macroalgae and its abundance in the water column was positive and significant (n = 16; r = 0.7595; rs = 0.8944; p < 0.001).

Environmental Parameters and Their Relationships with Blooms
During the bloom period, the sea surface temperature ranged from 26.5 °C to 16.4 °C, and salinity from 36.4 to 30.7. At the start of the bloom, the water temperature reached the highest values (26.5 °C), whereas at the bloom peak (mid-September 2021) the temperature was 24.2 °C. Then, when the temperature decreased to values below 20 °C, the bloom rapidly declined (Figure 4). Concentrations of DIN ranged from 0.978 to 13.324 µmol L −1 (2 September and 15 October, respectively). NH4 + and NO3 − were the main contributors to DIN ( Figure 5). The concentrations of PO4 3− ranged from 0.03 to 0.467 µmol L −1 . The three highest PO4 3− values were detected around the start of the bloom (0.162 µmol L −1 ), at the bloom peak (0.151 µmol L −1 ), and near the end of the bloom (0.467 µmol L −1 ), whereas PO4 3− values generally did not exceed 0.100 µmol L −1 during the rest of the bloom ( Figure  5). . Temporal variability of the N:P ratio (bars, left y-axis) and water temperature (line, °C, right y-axis) at the Passetto station. The horizontal line represents both (i) the upper N:P ratio threshold, below which better nutrient conditions are supplied to Ostreopsis cf. ovata growth, and (ii) the lower temperature threshold, permitting the bloom's onset [66]. * denotes dates when sea conditions could potentially disturb the development of the bloom (Douglas scale ≥ 2, i.e., wave heights from 0.1 to 2.5 m, ranging from smooth to moderate). The ratios of inorganic N:P ranged from 19 to 164 during the bloom. Values above 50 were recorded by the end of September, when the O. cf. ovata bloom was approaching its end. N:P values close to 16 (i.e., the Redfield ratio) were recorded just before the bloom's onset (13)(14) and during the bloom's peak (19) (Figure 4). The bloom dynamics were affected by hydrodynamism. Sea conditions were calm until the end of August, (i.e., at the time of the bloom's onset) and during the bloom's peak (Figure 4), whereas in mid-September the sea conditions became rougher (Douglas scale ≥ 2). At this time, a sharp decrease in abundance was recorded on the substrata, not always followed by a proportional decrease in the water column (e.g., on 23 September). The bloom declined rapidly by the end of September, in correspondence with moderate-high hydrodynamic conditions (Douglas scale ≥ 2, i.e., wave height from 0.1 to 2.5 m, from smooth to moderate), although sporadic cells were recorded until the second part of October.

Temporal Trend of OVTXs Detected in Mussels and its Relationship with Ostreopsis cf. ovata Blooms
OVTX was the most abundant toxin (71%-74%) among the PlTX analogs detected in mussels, followed by OVTX-b (26%-29%) (Figure 6). At Passetto, OVTX was first recorded in the mussels sampled on 20 September (i.e., 32 days after the appearance of O. cf. ovata on macroalgae and 18 days after the first record in the water column) with a concentration of 15.8 and 15.7 µg kg −1 of soft tissue (LC-MS/MS and UHPLC-HRMS 2 results, respectively). These represented the highest values of OVTXs detected in this station (Figure 7). This peak of bioaccumulation in mussels was recorded 4 days after the recording of the bloom peak on 16 September. At the subsequent sampling date (27 September), OVTX concentrations were drastically lower, i.e., <LOD (5.0 µg/kg and 1.0 µg/kg for LC-MS/MS and UHPLC-HRMS 2 , respectively). The same pattern was observed for the abundances of Ostreopsis (4 days before, abundances of O. cf. ovata were 456 cells cm −2 and 8.9 x 10 3 cells L −1 on macroalgae and in the water column, respectively). After 14 consecutive days without records of O. cf. ovata in seawater, OVTXs were no longer detected in mussels. A significant positive Pearson's correlation was found between O. cf. ovata abundances on macroalgae and OVTXs in mussels (n = 7; r= 0.8336; p < 0.05). A similarly positive correlation was found between O. cf. ovata abundances in the water column and OVTXs recorded in mussels (n = 7; r= 0.8574; p < 0.05).
Moreover, comparisons between Pearson's and Spearman's correlations of O. cf. ovata abundances in the water column and OVTXs recorded in mussels showed a monotonic rather than linear relationship (n = 7; rs= 0.8846; p < 0.01). Conversely, no significant Spearman's correlation was observed between O. cf. ovata abundances on macroalgae and OVTXs recorded in mussels (n = 7; 0.6742; p > 0.05).
At Portonovo, OVTXs were first recorded in mussels on 20 September using the LC-MS/MS method and on 14 September using the UHPLC-HRMS 2 method. Maximum concentrations were recorded in mussels collected on 20 September (36.4 and 25.5 µg kg −1 of soft tissue, LC-MS/MS, and UHPLC-HRMS 2 results, respectively).

Discussion
Although blooms of Ostreopsis cf. ovata have been constantly reported along the Conero Riviera in the late summer/early autumn period since 2006 [54,56,67], the bioaccumulation of PlTX analogs produced during this phenomenon in edible invertebrates has been poorly studied. The European Legislation, with Regulation No. 853/2004 and subsequent amendments, limits the commercialization of bivalve mollusks for human consumption when contaminated by phycotoxins, setting a maximum level for each toxin except for some compounds called 'emerging marine biotoxins', e.g., tetrodotoxins, cyclic imines, and palytoxins, for which an EU regulation has not yet been established. In 2006, the European Food Safety Authority (EFSA) received a request from the European Commission (EC) to develop a series of scientific opinions concerning these marine biotoxins. The EFSA indicated 30 µg kg -1 as threshold value for PlTX, above which PlTX-contaminated mollusks would be unsafe for consumers [55].
In this study, we showed that, in the Conero Riviera, wild mussels bioaccumulated PlTX-equivalent concentration at levels generally lower than the EFSA threshold. The only exception was recorded for Portonovo on 20 September 2021, with 36.4 µg kg −1 of soft tissue (LC-MS/MS). This finding highlights the necessity of characterizing the hazard posed by oral exposure to these toxins, which still represents a major gap in our knowledge [31]. The observed toxin profile fits well with the profile previously detected in field samples of O. cf. ovata populations in the same study area [68]. This is the most common toxin profile of O. cf. ovata strains from the Mediterranean area [69], dominated by OVTX-a, followed by OVTX-b, -d, -e, -c, and isobPlTX, in decreasing order of percentage. However, in our samples the contributions of OVTX, -d, -e, -c, and isobPlTX were undetectable, probably because such toxins represented a very minor component of the profile, as suggested by Accoroni et al. [67].
The finding that contaminated mussels showed the same toxin profile as that of the toxigenic microalga blooming in the area was not so obvious. It is well known that phycotoxins can be subjected to biotransformation, and/or to a differential accumulation/excretion equilibrium related to the molecular properties of each compound after their ingestion by invertebrates. Such processes have been described for paralytic shellfish toxins (PSTs [70]), diarrhoetic shellfish toxins [71], and azaspiracids [72,73].
The results of this study support the concept that the bioaccumulation of toxins in wild mussels does not depend only on the abundances of toxic microalgae. Indeed, although higher abundances of Ostreopsis have been recorded in Passetto than in Portonovo since 2006 (https://www.arpa.marche.it/balneazione-nuovo/ostreopsis-cf-ovata, accessed on 18 April 2022), mussels bioaccumulated higher OVTX concentrations at the latter site, exceeding the EFSA threshold. Several biotic and abiotic factors (intrinsic or non-intrinsic to the vector organisms) play a certain role in this process. First, algal growth and toxin production (and, therefore, algal abundances and toxin content) are not necessarily correlated, as they are influenced by different environmental parameters and by the physiological status/life cycle stage of the alga [68,74,75]. Moreover, these two sites have different characteristics, which, regardless of the abundance of Ostreopsis, might affect the assimilation/detoxification rate in mussels. Bioaccumulation depends on several boundary conditions, such as the abundance and quality of the whole phytoplankton communities that affect the filtration rates [76], the nature of the contaminating toxins, and the physiology of the shellfish [77] and its history of exposure to toxins [78]. Indeed, the modulation of filtration (and/or rejection) capacity is a behavioral response that is common in bivalves exposed to toxic microalgae (e.g., PST-producing species [79]). In particular, in laboratory experiments carried out on mussels fed on O. cf. ovata, Sardo et al. [51] observed that the ratio between mussel size and cell concentration, rather than the simple cell densities, affected the filtration rates of the invertebrates. Moreover, Gorbi et al. [49] observed inhibition of the feeding activity in mussels exposed to Ostreopsis cf. ovata. Therefore, as mussels from Passetto are generally exposed to higher Ostreopsis abundances than those from Portonovo [54,56], paradoxically they could accumulate fewer toxins, being inhibited in their filtration rate.
Previous papers have extensively discussed the role of hydrodynamics, temperature, nutrients, and depth on the dynamics of Ostreopsis cf. ovata blooms in the Conero Riviera. This area is a shallow coast, which is strongly phosphorus limited [80]. Calm sea conditions, temperatures >25 °C, and the increased availability of inorganic phosphorus leading to N:P ratios reaching the Redfield ratio are known to be ideal conditions for the onset of Ostreopsis blooms. These conditions also favor the blooms' development, as long as sea surface temperatures remain >20 °C [66]. The results of this study confirm once again this bloom mechanism: bloom onset occurred at 26.5 °C (i.e., the yearly maximum), after days with low hydrodynamism, and a high level of inorganic P and an N:P ratio close to the Redfield ratio. High levels of P and optimal N:P values were also recorded during the bloom peak.
A clear and crucial role of the hydrodynamic conditions in the development of Ostreopsis blooms has been previously recognized. Blooms occur even in exposed sites, but higher abundances are detected under sheltered conditions [39,56,[81][82][83]. Higher abundances have been historically observed in Passetto, the sheltered station, than in the more exposed Portonovo station [54,56] (https://www.arpa.marche.it/balneazione-nuovo/ostreopsis-cf-ovata, accessed on 18 April 2022). Moreover, intense hydrodynamic events may influence the correlation between the abundances of O. cf. ovata in the water column and on benthic substrata. The O. cf. ovata biofilm is merely attached to the substrate and is easily resuspended in the water column. Therefore, strong hydrodynamism can either increase abundances in the water column via resuspension (as happened in this study on 23 September) or, if a hydrodynamic event is very intense, it can decrease them via dilution (as happens each year at the end of the bloom). This also explains why correlations of benthic and planktonic abundances showed a monotonic rather than linear relationship. Given that Ostreopsis cells exhibit predominantly benthic behaviour, they can be transferred to the water column only under certain hydrodynamic conditions [5].
In this study, it was possible to recognize a clear correlation between the abundances of O. cf. ovata and the bioaccumulation of OVTXs' in mussels. The fact that this toxic species is benthic rather than planktonic may make it relatively unavailable as a food resource for filter-feeding shellfish except during periods when cells are resuspended in the water column and/or when the biofilm of benthic cells extends onto the mollusc shells. Indeed, although this correlation is observable with abundances of O. cf. ovata both on benthic substrates and in the water column, it is more evident with the latter, where the monotonic relationship suggests that other factors in addition to the abundances of Ostreopsis cells in water influence the bioaccumulation of toxins in filter-feeding shellfish.
In Italy, Ostreopsis blooms are managed following specific guidelines to protect human health in bathing waters affected by this phenomenon. These guidelines consider three phases based on Ostreopsis abundances in the water column [84]: routine (≤10,000 cells L −1 ), alert (10,000-30,000 cells L −1 ), and emergency (>30,000 cells L −1 ) phases. Looking at the monitoring data of Ostreopsis cf. ovata bloom collected by the Regional Environmental Protection Agency of Marche (ARPAM) at the Passetto and Portonovo stations in the summer/fall of 2021, a similar pattern was observed in both stations and in agreement with the values obtained in this study (Table S1). At both stations, mussels accumulated an appreciable amount of OVTXs only when the abundance of Ostreopsis in the water column reached the alert phase. Conversely, when its abundance in the water column declined to the routine phase, a few days later the mussels showed much lower bioaccumulation of OVTXs. This indicates that the Italian guidelines to protect human health in bathing waters affected by O. cf. ovata blooms are also useful in managing the risk of human intoxication through the ingestion of contaminated mussels.