Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis.

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg-1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July-August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

Twenty-Five Years of PSP Toxicity in Galician (NW Spain) Bivalves: Spatial, Temporal, and Interspecific Variations.

Twenty-five years of paralytic shellfish poisoning (PSP) toxicity in Galician bivalves have been studied. PSP was detected in 4785 out of 73,740 samples of the commercially important bivalve species analyzed from 1995 to 2020. Its general prevalence in the area was 6.5%. Only 1.6% of all samples tested were over the regulatory limit (incidence). The maximum level of PSP in the area, 40,800 µg STX 2HCl-eq kg-1, was recorded in raft mussels from Bueu (PON-II, Pontevedra) in December 2005. The highest maximum PSP values were found in mussels, which were mostly affected by Gymnodinium catenatum, but not those of prevalence and incidence which were recorded in clams, mostly affected by Alexandrium. Average levels in mussels were higher than in any other studied species. Spatially, in general, the prevalence, incidence, maximum, and average PSP toxicity during episodes tend to decrease from south to northeast, but some hot points with high levels can be identified. PCA analysis separates the southern rías, associated to G. catenatum blooms, from the middle and northern ones, associated to Alexandrium blooms. Along the year, two main peaks of the four variables are observed, the first one in late autumn-winter and the other in summer, the summer peak being much more important for the infaunal species than for raft mussels. In the seasonal pattern obtained by time series analysis of the average PSP toxicity, the autumn-winter peak was only maintained (and very reduced) in the southern rías, indicating that this peak is seasonally much less important than the summer peak. The observed seasonality is expected based on the timing of the blooms of the two PSP-producing phytoplankton groups present in the area. Over the 25 years of monitoring, large differences in PSP toxicity have been observed. Apart from some special years, an ascending trend in prevalence and incidence seems to be present from 2011 to 2020. No trend seems to exist during the same period for average or maximum toxicity.

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.

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.

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.

Lipophilic Toxins in Galicia (NW Spain) between 2014 and 2017: Incidence on the Main Molluscan Species and Analysis of the Monitoring Efficiency.

Galicia is an area with a strong mussel aquaculture industry in addition to other important bivalve mollusc fisheries. Between 2014 and 2017, 18,862 samples were analyzed for EU regulated marine lipophilic toxins. Okadaic acid (OA) was the most prevalent toxin and the only single toxin that produced harvesting closures. Toxin concentrations in raft mussels were generally higher than those recorded in other bivalves, justifying the use of this species as an indicator. The Rías of Pontevedra and Muros were the ones most affected by OA and DTX2 and the Ría of Ares by YTXs. In general, the outer areas of the Rías were more affected by OA and DTX2 than the inner ones. The OA level reached a maximum in spring, while DTX2 was almost entirely restricted to the fall-winter season. YTXs peaked in August-September. The toxins of the OA group were nearly completely esterified in all the bivalves studied except mussels and queen scallops. Risk of intoxication with the current monitoring system is low. In less than 2% of cases did the first detection of OA in an area exceed the regulatory limit. In no case, could any effect on humans be expected. The apparent intoxication and depuration rates were similar and directly related, suggesting that the rates are regulated mainly by oceanographic characteristics.

Detection and Spatio-Temporal Distribution of Pinnatoxins in Shellfish from the Atlantic and Cantabrian Coasts of Spain.

For the first time, pinnatoxins have been detected in shellfish from the Atlantic and Cantabrian coasts of Spain. High sensitivity LC-MS/MS systems were used to monitor all the currently known pinnatoxins (A-H). Pinnatoxin G (PnTX G) was the most prevalent toxin of the group, but its metabolite PnTX A has also been found at much lower levels. No trend in PnTX G concentration was found in the area, but a hotspot in the Ría de Camariñas has been identified. The maximum concentrations found did not exceed 15 µg·kg-1, being, in most cases, below 3 µg·kg-1. The highest concentrations were found in wild (intertidal) populations of mussels which attained much higher levels than raft-cultured ones, suggesting that the toxin-producer organisms preferentially develop in shallow areas. Other bivalve species had, in general, lower concentrations. The incidence of PnTX G followed a seasonal pattern in which the maximum concentrations took place in winter months. PnTX G was found to be partially esterified but the esterification percentage was not high (lower than 30%).

Presence of azaspiracids in bivalve molluscs from Northern Spain.

Low concentrations of azaspiracids have been found in bivalve molluscs all over the Northern and Northwestern coast of the Iberian Peninsula. The detections started in June 2016 and lasted until March 2017. The observed toxin profile was dominated by AZA2, followed by AZA1 and some other AZAs that were detected only in some samples. Some compounds producing fragments characteristics of AZAs but that do not fit with any of the known ones were also found. The causative agent has not been identified but, in sight of the toxin profile in the bivalves, it seems that it should be a new species or strain. The detections of AZAs in bivalves in the Northern Coast was linked to downwelling or upwelling relaxation and, in the Galician Rías, took place (with only a few exceptions) in the outer (more oceanic) part, suggesting that the responsible species develops at the open sea and that the populations are advected to the shore.

Effect of the industrial canning on the toxicity of mussels contaminated with diarrhetic shellfish poisoning (DSP) toxins.

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels -relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls.

Effect of the industrial steaming on the toxicity, estimated by LC-MS/MS, of mussels exposed for a long time to diarrhetic shellfish poisoning (DSP) toxins.

The effect of industrial steaming on mussels that had been naturally exposed to DSP toxins for a long time was studied using LC-MS/MS. The estimated toxicity increased with steaming by a percentage that cannot be explained by weight loss. The estimated toxin content per mussel increased substantially with the treatment, which can only be explained by an incorrect estimation by the technique (at the extraction or analytical level) or by the presence of unknown derivatives or analogues. Direct alkaline hydrolysis of the mussel meat yielded more toxin than the standard hydrolysis (hydrolysis of the methanolic extracts), suggesting that extraction was, at least in part, responsible for the increase of toxin content. In situations as the one described in this work, it can be expected that mussels with toxicities well below the regulatory limit could easily surpass that level after industrial steaming, thus producing important losses for food processors.

Evaluation of passive samplers as a monitoring tool for early warning of Dinophysis toxins in shellfish.

From June 2006 to January 2007 passive samplers (solid phase adsorbing toxin tracking, SPATT) were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content (liquid chromatography-mass spectrometry, LC-MS) in picked cells of Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week-1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks.