Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean.

Dinoflagellates of the genus Gambierdiscus have been associated with ciguatera, the most common non-bacterial fish-related intoxication in the world. Many studies report the presence of potentially toxic Gambierdiscus species along the Atlantic coasts including G. australes, G. silvae and G. excentricus. Estimates of their toxicity, as determined by bio-assays, vary substantially, both between species and strains of the same species. Therefore, there is a need for additional knowledge on the metabolite production of Gambierdiscus species and their variation to better understand species differences. Using liquid chromatography coupled to mass spectrometry, toxin and metabolomic profiles of five species of Gambierdiscus found in the Atlantic Ocean were reported. In addition, a molecular network was constructed aiming at annotating the metabolomes. Results demonstrated that G. excentricus could be discriminated from the other species based solely on the presence of MTX4 and sulfo-gambierones and that the variation in toxin content for a single strain could be up to a factor of two due to different culture conditions between laboratories. While untargeted analyses highlighted a higher variability at the metabolome level, signal correction was applied and supervised multivariate statistics performed on the untargeted data set permitted the selection of 567 features potentially useful as biomarkers for the distinction of G. excentricus, G. caribaeus, G. carolinianus, G. silvae and G. belizeanus. Further studies will be required to validate the use of these biomarkers in discriminating Gambierdiscus species. The study also provided an overview about 17 compound classes present in Gambierdiscus, however, significant improvements in annotation are still required to reach a more comprehensive knowledge of Gambierdiscus' metabolome.

Lipophilic Shellfish Poisoning Toxins in Marine Invertebrates from the Galician Coast.

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5-75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg-1) and Tellina donacina (63 µg kg-1), respectively. However, mean concentrations for the bivalve group were shallow (2-6 µg kg-1). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6-44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg--1, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions.

First Report of Two Gymnodimines and Two Tetrodotoxin Analogues in Invertebrates from the North Atlantic Coast of Spain.

Gymnodimine D (GYM D), 16-desmethyl gymnodimine D (16-desmethyl GYM D), and two tetrodotoxin analogues have been found in invertebrates obtained from the north Atlantic coast of Spain from May 2021 to October 2022. It is the first report of GYMD and 16-desmethyl GYM D in invertebrates worldwide and of the tetrodotoxin analogues, 5,6,11 trideoxy tetrodotoxin (5,6,11 trideoxy TTX) and its isomer (referred to as 5,6,11 trideoxy-epi-TTX), in the north Atlantic Coast of Spain. In this study, we also report for the first time the detection of tetrodotoxin (TTX) in three species (the cnidaria Calliactis parasitica, an unidentified species, and the bivalve Tellina donacina). The prevalence was medium for GYM D and 16-desmethyl GYM D and low for TTXs overall. The concentrations recorded were variable, with maximum values of GYM D in the bivalve Cerastoderma edule (8.8 µg GYM A equivalents kg-1), of 16-desmethyl GYM D in the bivalve Magellana gigas (10 µg GYM A equivalents kg-1) and of TTX and 5,6,11 trideoxy TTX in the cnidaria C. parasitica (49.7 and 233 µg TTX equivalents kg-1, respectively). There is very scarce information about these compounds. Therefore, the reporting of these new detections will increase the knowledge on the current incidence of marine toxins in Europe that the European Food Safety Authority (EFSA), in particular, and the scientific community, in general, have. This study also highlights the importance of analyzing toxin analogues and metabolites for effective monitoring programs and adequate health protection.

Enzymatic Biotransformation of 13-desmethyl Spirolide C by Two Infaunal Mollusk Species: The Limpet Patella vulgata and the Cockle Cerastoderma edule.

The presence of a 13-desmethyl Spirolide C isomer (Iso-13-desm SPX C) is very common in some infaunal mollusks in Galicia contaminated with this toxin. Its possible origin by biological transformation was investigated by incubating homogenates of the soft tissues of limpets and cockles spiked with 13-desmethyl Spirolide C (13-desm SPX C). The involvement of an enzymatic process was also tested using a raw and boiled cockle matrix. The enzymatic biotransformation of the parent compound into its isomer was observed in the two species studied, but with different velocities. The structural similarity between 13-desm SPX C and its isomer suggests that epimerization is the most likely chemical process involved. Detoxification of marine toxins in mollusks usually implies the enzymatic biotransformation of original compounds, such as hydroxylation, demethylation, or esterification; however, this is the first time that this kind of transformation between spirolides in mollusks has been demonstrated.

Okadaic Acid Depuration from the Cockle Cerastoderma edule.

The cockle Cerastoderma edule is a commercially important species in many European Countries. It can accumulate okadaic acid (OA) and other toxins in its group, which makes it unsuitable for human consumption, producing harvesting bans to avoid intoxications. The duration of those bans depends in part on the depuration kinetics of the toxin in this species. In this work, this kinetics was studied by means of fitting different models to depuration data experimentally obtained, using naturally contaminated cockles. Cockles depurated OA faster than most other bivalve species studied. Models that include Michaelis-Menten kinetics describe the depuration better than those using a first order exponential decrease to describe the first (or the only) compartment. One-compartment models were not able to describe the final part of the depuration curve, in which OA was depurated very slowly. Therefore, two-compartment models were needed. Esters were depurated at a much faster rate than the free form of the toxin; however, no significant esterification was detected during the process. The slow depuration rate suggests that other bivalve species could be used as sentinels to monitor cockle populations, but caution should be taken when toxin concentrations are very high.

Spirolides in Bivalve Mollusk of the Galician (NW Spain) Coast: Interspecific, Spatial, Temporal Variation and Presence of an Isomer of 13-Desmethyl Spirolide C.

Spirolides are cyclic imines whose risks to human health have not been sufficiently evaluated. To determine the possible impact of these compounds in Galicia (NW Spain), their presence and concentration in bivalve mollusk were studied from 2014 to 2021. Only 13-desmethyl spirolide C (13desmSPXC) and an isomer have been detected, and always at low concentrations. Mussel, Mytilus galloprovincialis, was the species which accumulated more spirolides, but the presence of its isomer was nearly restricted to cockle, Cerastoderma edule, and two clam species, Venerupis corrugata and Polititapes rhomboides. On average, the highest 13desmSPXC levels were found in autumn-winter, while those of its isomer were recorded in spring-summer. Both compounds showed decreasing trends during the study period. Geographically, the concentration tends to decrease from the southern to the north-eastern locations, but temporal variability predominates over spatial variability.

Latitudinal Variation in the Toxicity and Sexual Compatibility of Alexandrium catenella Strains from Southern Chile.

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5-55° S) 50 years ago and is responsible for most of the area's cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell-1), with the more toxic strains occurring in the north (-40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000-1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.

Development of a Fast Liquid Chromatography Coupled to Mass Spectrometry Method (LC-MS/MS) to Determine Fourteen Lipophilic Shellfish Toxins Based on Fused-Core Technology: In-House Validation.

Prevalence and incidence of the marine toxins (paralytic, amnesic, and lipophilic toxins) including the so-called emerging toxins (these are, gymnodimines, pinnatoxins, or spirolides among others) have increased in recent years all over the world. Climate change, which is affecting the distribution of their producing phytoplankton species, is probably one of the main causes. Early detection of the toxins present in a particular area, and linking the toxins to their causative phytoplankton species are key tools to minimize the risk they pose for human consumers. The development of both types of studies requires fast and highly sensitive analytical methods. In the present work, we have developed a highly sensitive liquid chromatography-mass spectrometry methodology (LC-MS/MS), using a column with fused-core particle technology, for the determination of fourteen lipophilic toxins in a single run of 3.6 min. The performance of the method was evaluated for specificity, linearity, precision (repeatability and reproducibility) and accuracy by analysing spiked and naturally contaminated samples. The in-house validation was successful, and the limit of detection (LOD) and quantification (LOQ) for all the toxins were far below their regulatory action limits. The method is suitable to be considered in monitoring systems of bivalves for food control.

Twenty-Five Years of Domoic Acid Monitoring in Galicia (NW Spain): Spatial, Temporal and Interspecific Variations.

Prevalence, impact on shellfish resources and interspecific, spatial, and temporal variabilities of domoic acid (DA) in bivalves from Galicia (NW Spain) have been studied based on more than 25 years of monitoring data. The maximum prevalence (samples in which DA was detected) (100%) and incidence (samples with DA levels above the regulatory limit) (97.4%) were recorded in Pecten maximus, and the minimum ones in Mytilus galloprovincialis (12.6 and 1.1%, respectively). The maximum DA concentrations were 663.9 mg kg-1 in P. maximus and 316 mg kg-1 in Venerupis corrugata. After excluding scallop P. maximusdata, DA was found (prevalence) in 13.3% of bivalve samples, with 1.3% being over the regulatory limit. In general, the prevalence of this toxin decreased towards the North but not the magnitude of its episodes. The seasonal distribution was characterized by two maxima, in spring and autumn, with the later decreasing in intensity towards the north. DA levels decreased slightly over the studied period, although this decreasing trend was not linear. A cyclic pattern was observed in the interannual variability, with cycles of 4 and 11 years. Intoxication and detoxification rates were slower than those expected from laboratory experiments, suggesting the supply of DA during these phases plays an important role.

Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity, Toxin Profile, and Co-Occurrence with Cyclic Imines.

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC-MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg-1 for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg-1 for the LC-MS/MS method, with a good correlation between both methods (r2 = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.

Characterization of the Domoic Acid Uptake Mechanism of the Mussel (Mytilus galloprovincialis) Digestive Gland.

Cultures of the mussel Mytilus galloprovincialis are frequently affected by accumulation of the amnesic shellfish poisoning toxin domoic acid (DA). This species is characterized by a fast uptake and release of the toxin. In this work, the main characteristics of the uptake mechanism have been studied by incubation of digestive gland thin slices in media with different composition and DA concentration. DA uptake seems to follow Michaelis-Menten kinetics, with a very high estimated KM (1722 µg DA mL-1) and a Vmax of 71.9 µg DA g-1 h-1, which is similar to those found for other amino acids in invertebrates. Replacement of NaCl from the incubation media by Cl-choline (Na+-free medium) did not significantly reduce the uptake, but replacement by sorbitol (Na+-free and Cl--depleted medium) did. A new experiment replacing all chlorides with their equivalent gluconates (Na+- and Cl--free medium) showed an important reduction in the uptake that should be attributed to the absence of chloride, pointing to a Na+-independent, Cl- (or anion-) dependent transporter. In media with Na+ and Cl-, neither decreasing the pH nor adding cyanide (a metabolic inhibitor) had significant effect on DA uptake, suggesting that the transport mechanism is not H+- or ATP-dependent. In a chloride depleted medium, lowering pH or adding CN increased the uptake, suggesting that other anions could, at least partially, substitute chloride.

Gymnodimine A in mollusks from the north Atlantic Coast of Spain: Prevalence, concentration, and relationship with spirolides.

Gymnodimine A has been found in mollusks obtained along the whole northern coast of Spain from April 2017 to December 2019. This is the first time that this toxin is detected in mollusks from the Atlantic coast of Europe. The prevalence of the toxin was, in general, low, being detected on average in approximately 6% of the obtained samples (122 out of 1900). The concentrations recorded were also, in general, low, with a median of 1.3 µg kg-1, and a maximum value of 23.93 µg kg-1. The maxima of prevalence and concentration were not geographically coincident, taking place the first at the easternmost part of the sampled area and the second at the westernmost part. In most cases (>94%), gymnodimine A and 13-desmethyl spirolide C were concurrently detected, suggesting that Alexandrium ostenfeldii could be the responsible producer species. The existence of cases in which gymnodimine A was detected alone suggests also that a Karenia species could also be involved. The geographical heterogeneity of the distribution suggests that blooms of the producer species are mostly local. Not all bivalves are equally affected, clams being less affected than mussels, oysters, and razor clams. Due to their relatively low toxicity, and their low prevalence and concentration, it seems that these toxins do not pose an important risk for the mollusk consumers in the area.

First Report of Paralytic Shellfish Toxins in Marine Invertebrates and Fish in Spain.

A paralytic shellfish poisoning (PSP) episode developed in summer 2018 in the Rías Baixas (Galicia, NW Spain). The outbreak was associated with an unprecedentedly intense and long-lasting harmful algal bloom (HAB) (~one month) caused by the dinoflagellate Alexandrium minutum. Paralytic shellfish toxins (PSTs) were analyzed in extracts of 45 A. minutum strains isolated from the bloom by high-performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-PCOX-FLD). PSTs were also evaluated in tissues from marine fauna (invertebrates and fish) collected during the episode and in dolphin samples. The analysis of 45 A. minutum strains revealed a toxic profile including GTX1, GTX2, GTX3 and GTX4 toxins. With regard to the marine fauna samples, the highest PSTs levels were quantified in bivalve mollusks, but the toxins were also found in mullets, mackerels, starfish, squids and ascidians. This study reveals the potential accumulation of PSTs in marine invertebrates other than shellfish that could act as vectors in the trophic chain or pose a risk for human consumption. To our knowledge, this is the first time that PSTs are reported in ascidians and starfish from Spain. Moreover, it is the first time that evidence of PSTs in squids is described in Europe.

Rapid Domoic Acid Depuration in the Scallop Argopecten purpuratus and Its Transfer from the Digestive Gland to Other Organs.

Domoic acid (DA), the main toxin responsible for Amnesic Shellfish Poisoning, frequently affects the marine resources of Chile and other countries across the South Pacific, thus becoming a risk for human health. One of the affected resources is the scallop Argopecten purpuratus. Even though this species has a high commercial importance in Northern Chile and Peru, the characteristics of its DA depuration are not known. In this work, the DA depuration was studied by means of two experiments: one in controlled (laboratory) and another in natural conditions. All organs of A. purpuratus depurated the toxin very quickly in both experiments. In some organs, an increase or a very small decrease of toxin was detected in the early depuration steps. Several models were used to describe this kinetics. The one that included toxin transfer between organs and independent depuration from each organ was the model that best fit the data. It seems, therefore, that the DA in this species is quickly transferred from the digestive gland to all other organs, which release it into the environment. Physiological differences in the two experiments have been shown to have some effect on the depuration from each organ but the actual reasons are still unknown.

Toxicity Characterisation of Gambierdiscus Species from the Canary Islands.

In the last decade, several outbreaks of ciguatera fish poisoning (CFP) have been reported in the Canary Islands (central northeast Atlantic Ocean), confirming ciguatera as an emerging alimentary risk in this region. Five Gambierdiscus species, G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus, have been detected in macrophytes from this area and are known to produce the ciguatoxins (CTXs) that cause CFP. A characterization of the toxicity of these species is the first step in identifying locations in the Canary Islands at risk of CFP. Therefore, in this study the toxicity of 63 strains of these five Gambierdiscus species were analysed using the erythrocyte lysis assay to evaluate their maitotoxin (MTX) content. In addition, 20 of the strains were also analysed in a neuroblastoma Neuro-2a (N2a) cytotoxicity assay to determine their CTX-like toxicity. The results allowed the different species to be grouped according to their ratios of CTX-like and MTX-like toxicity. MTX-like toxicity was especially high in G. excentricus and G. australes but much lower in the other species and lowest in G. silvae. CTX-like toxicity was highest in G. excentricus, which produced the toxin in amounts ranging between 128.2 ± 25.68 and 510.6 ± 134.2 fg CTX1B equivalents (eq) cell-1 (mean ± SD). In the other species, CTX concentrations were as follows: G. carolinianus (100.84 ± 18.05 fg CTX1B eq cell-1), G. australes (31.1 ± 0.56 to 107.16 ± 21.88 fg CTX1B eq cell-1), G. silvae (12.19 ± 0.62 to 76.79 ± 4.97 fg CTX1B eq cell-1) and G. caribaeus (

Simple Diffusion as the Mechanism of Okadaic Acid Uptake by the Mussel Digestive Gland.

Okadaic acid (OA) and other toxins of the diarrheic shellfish poisoning (DSP) group are accumulated and transformed mainly in many bivalves, inside the digestive gland cells. In this work the absorption of okadaic acid by those cells has been studied by supplying the toxin dissolved in water and including it in oil droplets given to primary cell cultures, and by checking if the uptake is saturable and/or energy-dependent. Okadaic acid was found to be absorbed preferentially from the dissolved phase, and the uptake from oil droplets was substantially lower. The process did not require energy and was non-saturable, indicating that it involved a simple diffusion across the cellular membrane. Some apparent saturation was found due to the quick biotransformation of OA to 7-O-acyl esters.

Esterification of okadaic acid in the mussel Mytilus galloprovincialis.

Okadaic acid and other toxins of the diarrheic shellfish poisoning (DSP) group are transformed mainly to their acyl-derivatives in bivalves. Some recent studies suggest that bacteria present in the bivalve gut could contribute substantially to the acylation of the toxins. By feeding microcapsules containing okadaic acid to mussels we have shown unequivocally that the ingested okadaic acid is nearly completely transformed to its fatty acid esters (acyl-derivatives). Treating mussels with antibiotics did not have any significant effect on the acylation of the supplied okadaic acid, suggesting that bacteria do not play any significant role in this process. The microsomal and mitochondrial subcellular fractions of the cells of the digestive gland have been shown to have contain enzymes that are able to transfer a fatty acid molecule from Coenzyme A to okadaic acid (so, that have Acyl-CoA:OA acyltransferase activity). This activity was related to that of the enzyme Cytochrome C reductase (NADPH), a marker of endoplasmic reticulum, suggesting that this organelle is the main responsible for the acylation process. Acylation of DSP toxins seems to be a key step in the depuration of these toxins from mussels, as these compounds are found in feces as acyl-derivatives. This is probably true for most bivalves. The proportion of acyl-derivatives accumulated can point to the key process of the depuration: acylation or excretion of acylated derivatives. In the mussels Mytilus galloprovincialis, Mytilus edulis and in Donax trunculus, the first process seems to be the most important, but in most bivalve species it seems to be the second one. Other aspects of the relationship between depuration and acylation are also discussed.

Microencapsulation of okadaic acid as a tool for studying the accumulation of DSP toxins in mussels.

The possibility and effectiveness of microencapsulation of okadaic acid (OA) in gelatin-acacia microcapsules has been studied. The encapsulation efficiency was higher than 33%. The microcapsules were shown to be very stable, not leaching more than 9% of the encapsulated OA in a 20-h period. OA from the microcapsules was absorbed by the mussels very efficiently, accumulating--after 3 days of feeding and one of depuration--65% of the OA in microcapsules and 22% of the total OA used at the beginning of the microencapsulation process. These efficiencies and the possibility of encapsulating single DSP toxins and derivatives constitute a valuable tool for the study of the accumulation and biotransformation of DSP toxins in bivalves.

Automated on-line solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry for determination of lipophilic marine toxins in shellfish.

Automated on-line solid-phase extraction (SPE) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for fast determination of lipophilic marine toxins in shellfish samples. The direct coupling of an on-line SPE column to LC-MS/MS was accomplished using column switching techniques. Suitable chromatographic separation was performed on a reversed-phase C18 column under alkaline conditions (pH 11). The selected reversed-phase C18 SPE column allowed rapid and efficient on-line desalting of hydrolysed shellfish samples, avoiding signal suppression during mass spectrometry detection. Furthermore, the on-line SPE procedure allowed reducing matrix effects observed in raw and hydrolysed shellfish extracts. The proposed method was evaluated in terms of linearity, precision, accuracy and limits of detection (LODs). Quantitative recovery (97-102%) and satisfactory inter-day precision (RSD<8%) were achieved for all target compounds. LODs in the sub-µgkg-1 level (0.37-0.68µgkg-1) were obtained for all toxins except for okadaic acid, which showed a value of 2.75µgkg-1. Several mussel samples from North-western Spain were finally analysed in order to demonstrate the applicability of the method. Okadaic acid was the predominant toxin in all samples, although other lipophilic toxins were also detected.

Subcellular distribution of okadaic acid in the digestive gland of Mytilus galloprovincialis: first evidences of lipoprotein binding to okadaic acid.

The subcellular distribution of okadaic acid, the main diarrhetic shellfish poisoning (DSP) toxin, in the cells of the digestive gland of the mussel Mytilus galloprovincialis was studied. By means of differential centrifugation, ultrafiltration and extraction with methanol, it was found that most okadaic acid was stored in the cytosol. Notwithstanding only a small proportion of the total toxin was found to be in free form, being most of it bound to a soluble cellular compound with a molecular mass which ranged from 30 to 300 kDa. A series of fractionations of samples digested with a protease, a lipase, and amylase suggested that the component to which okadaic acid is bound is a high density lipoprotein. A new fractionation after digestion with a protein lipase additionally supports the previous conclusion.