Pedro Reis Costa

BMAA in shellfish from two Portuguese transitional water bodies suggests the marine dinoflagellate Gymnodinium catenatum as a potential BMAA source

The neurotoxin β-N-methylamino-l-alanine (BMAA) and its putative role in multiple neurodegenerative diseases have been intensely studied since 2005 when the toxin was discovered to be produced by worldwide-distributed cyanobacterial species inhabiting terrestrial, marine, brackish, and freshwater ecosystems. Recently, BMAA production was also associated with one eukaryotic group, namely, diatoms, raising questions about its production by other phytoplanktonic groups. To test for BMAA bioavailability in ecosystems where abundant phytoplanktonic blooms regularly occur, samples of filter-feeding shellfish were collected in two Portuguese transitional water bodies. BMAA content in cockles (Cerastoderma edule) collected weekly between September and November 2009 from Ria de Aveiro and at least once a month from May to November from Ria Formosa, fluctuated from 0.079±0.055 to 0.354±0.066μg/g DW and from below the limit of detection to 0.434±0.110μg/g DW, respectively. Simultaneously to BMAA occurrence in cockles, paralytic shellfish toxins were detected in shellfish as a result of Gymnodinium catenatum blooms indicating a possible link between this marine dinoflagellate and BMAA production. Moreover, considerable high BMAA levels, 0.457±0.186μg/g DW, were then determined in a laboratory grown culture of G. catenatum. This work reveals for the first time the presence of BMAA in shellfish from Atlantic transitional water bodies and consubstantiate evidences of G. catenatum as one of the main sources of BMAA in these ecosystems.

Characterization of Intracellular and Extracellular Saxitoxin Levels in Both Field and Cultured Alexandrium spp. Samples from Sequim Bay, Washington

Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004–2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

A potential vector of domoic acid: the swimming crab Polybius henslowii Leach (Decapoda-brachyura).

The swimming crab Polybius henslowii may play an important role in the movement of the amnesic shellfish toxin, domoic acid (DA), through the marine food chain. High DA concentrations have been determined in crab samples harvested along the Portuguese coast during the summer of 2002, reaching a level of 323.1 microg DA/g crab tissue. Toxin distribution in the different crab organs showed levels as high as 571.6 microg DA/g in the visceral tissues. Levels of toxin 4-12 times lower were detected in the remaining tissues. This crab might be a prominent vector of the toxin to higher trophic levels, including fishes, sea birds and even humans. In Portugal P. henslowii is commercialised during the summer in some local markets. DA concentrations were found close to the legal limit of 20 microg/g in samples purchased at Figueira da Foz market. The crabs are boiled prior to reaching the consumers. The cooking process was evaluated. Determination of toxin losses during the cooking process showed a toxin reduction higher than 50%. DA was determined by HPLC-UV and confirmed by spectra acquired with diode-array detector.

Changes in Tissue Biochemical Composition and Energy Reserves Associated With Sexual Maturation in the Ommastrephid Squids Illex coindetii and Todaropsis eblanae

The aim of this study was to investigate the biochemical changes that occur during sexual maturation of the squids Illex coindetii and Todaropsis eblanae. In both species, amino acids and protein content increased in the gonad throughout maturation, but the allocation of these nitrogen compounds from the digestive gland and muscle was not evident. A significant (P < 0.05) increase in the content of lipids and fatty acids was observed in the gonad and digestive gland. It seems that both species take energy for egg production directly from food, rather than from stored products. Analyses for cholesterol revealed a significant (P < 0.05) increase in the gonad, and the lipid content differences between species are potentially related to different feeding ecologies. The glycogen reserves in the gonad increased significantly (P < 0.05), suggesting that glycogen has an important role in the maturation process. It was evident that sexual maturation had a significant effect upon the gonad energy content, but because the energy variation in the digestive gland and muscle was nonsignificant (P > 0.05), there was no evidence that storage reserves are transferred from tissue to tissue.

Cephalopods as Vectors of Harmful Algal Bloom Toxins in Marine Food Webs

Here we summarize the current knowledge on the transfer and accumulation of harmful algal bloom (HAB)-related toxins in cephalopods (octopods, cuttlefishes and squids). These mollusks have been reported to accumulate several HAB-toxins, namely domoic acid (DA, and its isomers), saxitoxin (and its derivatives) and palytoxin (and palytoxin-like compounds) and, therefore, act as HAB-toxin vectors in marine food webs. Coastal octopods and cuttlefishes store considerably high levels of DA (amnesic shellfish toxin) in several tissues, but mainly in the digestive gland (DG)—the primary site of digestive absorption and intracellular digestion. Studies on the sub-cellular partitioning of DA in the soluble and insoluble fractions showed that nearly all DA (92.6%) is found in the cytosol. This favors the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The available information on the accumulation and tissue distribution of DA in squids (e.g., in stranded Humboldt squids, Dosidicus gigas) is scarcer than in other cephalopod groups. Regarding paralytic shellfish toxins (PSTs), these organisms accumulate them at the greatest extent in DG >> kidneys > stomach > branchial hearts > posterior salivary glands > gills. Palytoxins are among the most toxic molecules identified and stranded octopods revealed high contamination levels, with ovatoxin (a palytoxin analogue) reaching 971 μg kg−1 and palytoxin reaching 115 μg kg−1 (the regulatory limit for PlTXs is 30 μg kg−1 in shellfish). Although the impacts of HAB-toxins in cephalopod physiology are not as well understood as in fish species, similar effects are expected since they possess a complex nervous system and highly developed brain comparable to that of the vertebrates. Compared to bivalves, cephalopods represent a lower risk of shellfish poisoning in humans, since they are usually consumed eviscerated, with exception of traditional dishes from the Mediterranean area.

Biotransformation modulation and genotoxicity in white seabream upon exposure to paralytic shellfish toxins produced by Gymnodinium catenatum.

Fish are recurrently exposed to paralytic shellfish toxins (PSTs) produced by Gymnodinium catenatum. Nevertheless, the knowledge regarding metabolism of PSTs and their toxic effects in fish is scarce. Consequently, the current study aims to investigate the role of phase I and II detoxification enzymes on PST metabolism in the liver of white seabream (Diplodus sargus), assessing ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities. Moreover, the genotoxic potential of PSTs was examined through the erythrocytic nuclear abnormality (ENA) assay. Fish were intracoelomically (IC) injected with a nominal dose (expressed as saxitoxin equivalents) of 1.60 μg STXeq kg⁻¹ semipurified from a G. catenatum cell culture with previously determined toxin profile. Fish were sacrificed 2 and 6 days after IC injection. PST levels determined in fish liver were 15.2 and 12.2 μg STXeq kg⁻¹, respectively, at 2 and 6 days after the injection. Though several PSTs were administered, only dcSTX was detected in the liver after 2 and 6 days. This was regarded as an evidence that most of the N-sulfocarbamoyl and decarbamoyl toxins were rapidly biotransformed in D. sargus liver and/or eliminated. This was corroborated by a hepatic GST activity induction at 2 days after injection. Hepatic EROD activity was unresponsive to PSTs, suggesting that these toxins enter phase II of biotransformation directly. The genotoxic potential of PSTs was also demonstrated; these toxins were able to induce cytogenetic damage, such as chromosome (or chromatid) breaks or loss and segregational anomalies, measured by the ENA assay. Overall, this study pointed out the ecological risk associated with the contamination of fish with PSTs generated by G. catenatum blooms, providing the necessary first data for a proper interpretation of biomonitoring programs aiming to assess the impact of phytoplankton blooms in fish.

In vitro bioaccessibility of the marine biotoxin okadaic acid in shellfish

Okadaic acid (OA) and their derivatives are marine toxins responsible for the human diarrhetic shellfish poisoning (DSP). To date the amount of toxins ingested in food has been considered equal to the amount of toxins available for uptake by the human body. In this study, the OA fraction released from the food matrix into the digestive fluids (bioaccessibility) was assessed using a static in vitro digestion model. Naturally contaminated mussels (Mytilus galloprovincialis) and donax clams (Donax sp.), collected from the Portuguese coast, containing OA and dinophysistoxin-3 (DTX3) were used in this study. Bioaccessibility of OA total content was 88% and 75% in mussels and donax clams, respectively. Conversion of DTX3 into its parent compound was verified during the simulated digestive process and no degradation of these toxins was found during the process. This is the first study assessing the bioaccessibility of OA-group toxins in naturally contaminated seafood. This study provides relevant new data that can improve and lead to more accurate food safety risk assessment studies concerning these toxins.

Distribution, abundance, biology and biochemistry of the stout bobtail squid Rossia macrosoma from the Portuguese coast and

Abstract A study of Rossia macrosoma in Portuguese waters with emphasis on its biogeographical, biological and biochemical characteristics is presented. Approximately 1000 specimens collected in 32 cruises between 1990 and 2003 were used to determine the distribution and abundance of the species and some population biology parameters (weight, length, sex and maturation). The first biochemical characterization of the species at different stages of gonad development was made. The species was found at depths up to 860 m along the whole Portuguese coast. The sex ratio was approximately 1:1 and did not vary with depth or position along the coast (P≫0.05). Females grew larger (84:67 mm mantle length) and heavier (161:100 g total body weight) than males. Mature specimens of both sexes constituted <1% of all records. Immature and maturing specimens were found year round, without significant differences between female monthly distributions (P > 0.05). No significant difference between sexes in the distribution of maturity stages with depth or position along the coast was noted (P≫0.05). During sexual maturation there was a significant increase (P < 0.05) in total amino acid, protein, lipid and fatty acid contents in the gonad, but the allocation of these organic compounds from digestive gland and muscle was not evident. In the three tissues analysed, the major fatty acids were 16:0, 18:0, 18:1, 20:1, 20:4n-6, 20:5n-3 and 22:6n-3, the major essential amino acids were lysine, leucine and arginine and the major non-essential amino acids were glutamic acid, aspartic acid and serine.

Toxin Profile of Gymnodinium catenatum (Dinophyceae) from the Portuguese Coast, as Determined by Liquid Chromatography Tandem Mass Spectrometry

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1–4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

Estimating the contribution of N-sulfocarbamoyl paralytic shellfish toxin analogs GTX6 and C3 + 4 to the toxicity of mussels (Mytilus galloprovincialis) over a bloom of Gymnodinium catenatum

Gymnodinium catenatum, a dinoflagellate species with a global distribution, is known to produce paralytic shellfish poisoning (PSP) toxins. The profile of toxins of G. catenatum is commonly dominated by sulfocarbamoyl analogs including the C3+4 and GTX6, which to date has no commercial certified reference materials necessary for their quantification via chemical methods, such as liquid chromatography. The aim of this study was to assess the presence of C3+4 and GTX6 and their contribution to shellfish toxicity. C3+4 and GTX6 were indirectly quantified via pre-column oxidation liquid chromatography with fluorescence detection after hydrolysis conversion into their carbamate analogs. Analyses were carried out in mussel samples collected over a bloom of G. catenatum (>63×103cellsl-1) in Aveiro lagoon, NW Portuguese coast. Concentration levels of sulfocarbamoyl toxin analogs were two orders of magnitude higher than decarbamoyl toxins, which were in turn one order of magnitude higher than carbamoyl toxins. Among the sulfocarbamoyl toxins, C1+2 were clearly the dominant compounds, followed by C3+4 and GTX6. The least abundant sulfocarbamoyl toxin was GTX5. The most important compounds in terms of contribution for sample toxicity were C1+2, which justified 26% of the PSP toxicity. The lesser abundant dcSTX constitutes the second most important compound with similar % of toxicity to C1+2, C3+4 and GTX6 were responsible for approximately 11% and 13%, respectively. The median of the sum of C3+4 and GTX6 was 27%. These levels reached a maximum of 60% as was determined for the sample collected closest to the G. catenatum bloom. This study highlights the importance of these low potency PSP toxin analogs to shellfish toxicity. Hydrolysis conversion of C3+4 and GTX6 is recommended for determination of PSP toxicity when LC detection methods are used for PSP testing in samples exposed to G. catenatum.