Potent Marine Toxin Research Articles

Development of a Quick and Highly Sensitive Amplified Luminescent Proximity Homogeneous Assay for Detection of Saxitoxin in Shellfish.

Saxitoxin (STX), an exceptionally potent marine toxin for which no antidote is currently available, is produced by methanogens and cyanobacteria. This poses a significant threat to both shellfish aquaculture and human health. Consequently, the development of a rapid, highly sensitive STX detection method is of great significance. The objective of this research is to create a novel approach for identifying STX. Therefore, amplified luminescent proximity homogeneous assay (AlphaLISA) was established using a direct competition method based on the principles of fluorescence resonance energy transfer and antigen-antibody specific binding. This method is sensitive, rapid, performed without washing, easy to operate, and can detect 8-128 ng/mL of STX in only 10 min. The limit of detection achieved by this method is as low as 4.29 ng/mL with coefficients of variation for the intra-batch and inter-batch analyses ranging from 2.61% to 3.63% and from 7.67% to 8.30%, respectively. In conclusion, our study successfully establishes a simple yet sensitive, rapid, and accurate AlphaLISA method for the detection of STX which holds great potential in advancing research on marine biotoxins.

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012.

Tetrodotoxin (TTX), found in various organisms including pufferfish, is an extremely potent marine toxin responsible for numerous food poisoning accidents. Due to its serious toxicity and public health threat, detecting TTX and its analogues in diverse food matrices with a simple, fast, efficient method has become a worldwide concern. This review summarizes the advances in sample preparation and analytical methods for the determination of TTX and its analogues, focusing on the latest development over the past five years. Current state-of-the-art technologies, such as solid-phase microextraction, online technology, novel injection technology, two-dimensional liquid chromatography, high-resolution mass spectrometry, newly developed lateral flow immunochromatographic strips, immunosensors, dual-mode aptasensors, and nanomaterials-based approaches, are thoroughly discussed. The advantages and limitations of different techniques, critical comments, and future perspectives are also proposed. This review is expected to provide rewarding insights to the future development and broad application of pretreatment and detection methods for TTX and its analogues.

An Updated Review of Tetrodotoxin and Its Peculiarities.

Tetrodotoxin (TTX) is a crystalline, weakly basic, colorless organic substance and is one of the most potent marine toxins known. Although TTX was first isolated from pufferfish, it has been found in numerous other marine organisms and a few terrestrial species. Moreover, tetrodotoxication is still an important health problem today, as TTX has no known antidote. TTX poisonings were most commonly reported from Japan, Thailand, and China, but today the risk of TTX poisoning is spreading around the world. Recent studies have shown that TTX-containing fish are being found in other regions of the Pacific and in the Indian Ocean, as well as the Mediterranean Sea. This review aims to summarize pertinent information available to date on the structure, origin, distribution, mechanism of action of TTX and analytical methods used for the detection of TTX, as well as on TTX-containing organisms, symptoms of TTX poisoning, and incidence worldwide.

Dynamics of the genus Ostreopsis (Gonyaulacales, Dinophyceae) in a Mediterranean fish farm.

This study revealed the dynamics of the genus Ostreopsis in the south-western Mediterranean Sea fish farm during the 2016 and 2017 summers. This phytoplankton is known to produce palytoxin-like compounds, listed among the most potent marine toxins known, and can pose a serious concern for humans in the Mediterranean area. Principal component analysis (PCA) explained the significance of temperature, salinity, and dissolved inorganic nitrogen in the proliferation of this toxic dinoflagellate. The peak of the Ostreopsis sp. (6.34 × 103 cells L-1) was recorded at 28.4°C, at a salinity of 38.3 PSU, and the dissolved inorganic nitrogen had a value of 0.60μmolL-1. Our results highlight the importance of monitoring the proliferation of this harmful dinoflagellate in southern Mediterranean waters.

In Vivo Evaluation of the Chronic Oral Toxicity of the Marine Toxin Palytoxin.

Palytoxin (PLTX) is one of the most poisonous substances known to date and considered as an emergent toxin in Europe. Palytoxin binds to the Na+-K+ ATPase, converting the enzyme in a permeant cation channel. This toxin is known for causing human fatal intoxications associated with the consumption of contaminated fish and crustaceans such as crabs, groupers, mackerel, and parrotfish. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Different reports have previously explored the acute oral toxicity of PLTX in mice. Although the presence of palytoxin in marine products is currently not regulated in Europe, the European Food Safety Authority expressed its opinion on PLTX and demanded assessment for chronic toxicity studies of this potent marine toxin. In this study, the chronic toxicity of palytoxin was evaluated after oral administration to mice by gavage during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 µg/kg of PLTX and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 µg/kg for repeated daily oral administration of PLTX were determined. These results indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods, pointing out the need to further reevaluate the levels of this compound in marine products.

Fundamental Studies of the Singlet Oxygen Reactions with the Potent Marine Toxin Domoic Acid.

Domoic acid (DA), a potent marine toxin, is readily oxidized upon reaction with singlet oxygen (1O2). Detailed product studies revealed that the major singlet oxygenation reaction pathways were the [2 + 2] cycloaddition (60.2%) and ene reactions (39.8%) occurring at the Z double bond. Diene isomerization and [4 + 2] cycloaddition, common for conjugated diene systems, were not observed during the singlet oxygenation of DA. The bimolecular rate constant for the DA reaction with 1O2 determined by competition kinetics was 5.1 × 105 M-1 s-1. Based on the rate constant and steady-state concentrations of 1O2 in surface waters, the environmental half-life of DA due to singlet oxygen-induced transformations is between 5 and 63 days. The 1O2 reaction product mixture of DA did not exhibit significant biological activity based on ELISA studies, indicating that singlet oxygenation could be an important natural detoxification process. The characteristic oxidation products can provide valuable markers for the risk assessment of DA-contaminated natural waters.

A new nano biosensor for maitotoxin with high sensitivity and selectivity based fluorescence resonance energy transfer between carbon quantum dots and gold nanoparticles

Maitotoxin (MTX) is a potent marine toxin with critical LD50, which usually causes poisoning because of eating the contaminated marine crustaceans. This poison is a severe threat to neuronal systems and also disables the heart by increasing the calcium ions flow in the cardiovascular. Therefore, the quantitative determination of MTX by using a fast and cost-effective way is essential. Application of carbon dots in the structure of various sensors for the measurement of biological compounds has been developed due to its easy, rapid and inexpensive synthesis. In this study, carbon quantum dots (CQDs) and gold nanoparticles (AuNPs) were synthesized for the determination of MTX with high sensitivity and selectivity. The structure of CQDs and AuNPs were characterized using SEM (Scanning electron microscopy), TEM (Transmission electron microscopy), EDS (Energy-dispersive X-ray spectroscopy) and FT-IR (Fourier-transform infrared spectroscopy) techniques. The Basis of the sensing method is fluorescence resonance energy transfer (FRET) which occurs between energy donor (CQDs) and energy acceptor (AuNPs). Under optimal conditions (excitation wavelength 430 nm and emission wavelength 523 nm), the linear range and detection limit were obtained 1−600 pmol L−1 and 0.3 pmol L-1, respectively. The strong point of this nanosensor is its very fast quenching process. The interference effect of other poisons and some ions on the MTX determination were investigated using CQDs at the constant concentration of MTX50 pmol L-1 and various concentrations of other species. The results show excellent selectivity for the measurement of MTX in the presence of other similar species.

The Microbial Community of Tetrodotoxin-Bearing and Non-Tetrodotoxin-Bearing Ribbon Worms (Nemertea) from the Sea of Japan.

A potent marine toxin, tetrodotoxin (TTX), found in a great variety of marine and some terrestrial species, leaves intriguing questions about its origin and distribution in marine ecosystems. TTX-producing bacteria were found in the cultivable microflora of many TTX-bearing hosts, thereby providing strong support for the hypothesis that the toxin is of bacterial origin in these species. However, metagenomic studies of TTX-bearing animals addressing the whole microbial composition and estimating the contribution of TTX-producing bacteria to the overall toxicity of the host were not conducted. The present study is the first to characterize and compare the 16S rRNA gene data obtained from four TTX-bearing and four non-TTX-bearing species of marine ribbon worms. The statistical analysis showed that different nemertean species harbor distinct bacterial communities, while members of the same species mostly share more similar microbiomes. The bacterial species historically associated with TTX production were found in all studied samples but predominated in TTX-bearing nemertean species. This suggests that deeper knowledge of the microbiome of TTX-bearing animals is a key to understanding the origin of TTX in marine ecosystems.

Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 μg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

Drug Leads Derived from Japanese Marine Organisms.

Many natural products with extraordinary chemical structures and brilliant biological activities have been obtained from marine organisms. We have investigated such fascinating bioactive molecules, exemplified by the potent marine toxin palytoxin and the antitumor molecule halichondrin B, which has been developed as the anticancer drug Halaven®, to explore novel frontiers in organic chemistry and bioscience. Working within the traditional discipline, we have sought to acquire a deeper understanding of biological phenomena. We introduce here our major work along with up-todate topics. We isolated yoshinone A from marine cyanobacteria and completed a gram-scale synthesis. Yoshinone A is a novel polyketide that inhibited the differentiation of 3T3-L1 cells into adipocytes without significant cytotoxicity. The detailed mechanisms of action will be elucidated via further experiments in vitro and in vivo. In this study, we explore the true producers of okadaic acid and halichondrin B by immunostaining of Halichondria okadai with an antibody that was prepared using these natural products as an antigen. We will analyze isolated symbionts and reveal biosynthetic pathways.

Toxicity of crude organic extracts from the zoanthid Palythoa caribaeorum: A biogeography approach

Marine isolates such as palytoxin (PTX) are of concern within the Caribbean region due to their toxicity. PTX for example has been described as a one of the most known potent marine toxins, known to prevent predation from larger species (e.g. vertebrates) as well as the prevention of being overgrown from other coral species. PTX is a polyhydroxylated polyether toxin with a very large and complex chemical structure that possesses both hydrophilic and lipophilic properties. Previous acute toxicity tests using brine shrimp (Artemia salina) and PTX extract had shown it to be moderately toxic. In humans, PTX has been credited to be responsible for extreme symptoms such anaphylactic shock, rapid cardiac failure and eventual death occurring within minutes. Extrapolation for human dose ranges has therefore been suggested to be between 2.3 and 31.5 μg. This study isolates a potentially PTX-enriched extract from Palythoa caribaeorum and examines its organic extract toxicity from a biogeography perspective from a within-colony to a variety of reef sites around Trinidad and Tobago that are popular for marine visitors. This research represents an acute study with a high level of crude organic extract toxicity on A. salina whilst postulating potential factors which may contribute to its extreme toxicity and the risk posed to users of these environments.

Effects of the toxic dinoflagellate Ostreopsis cf. ovata on survival, feeding and reproduction of a phytal harpacticoid copepod

Harmful algal blooms are a source of increasing concern within the health, economic and ecological sectors. In the Mediterranean Sea, severe blooms of the benthic dinoflagellate Ostreopsis cf. ovata have been occurring since the beginning of the century, causing human intoxications by inhalation of bio-aerosols or direct contact with cells. The toxicity of this dinoflagellate is attributed to the presence of palytoxin and several of its analogs called ovatoxins, palytoxin being one of the most potent marine toxins. While mass mortalities of marine invertebrates have already been reported in relation with O. cf. ovata blooms, the toxic effects of this dinoflagellate on benthic organisms is still poorly documented. In the present study, laboratory experiments were performed on a meiobenthic copepod (Sarsamphiascus cf. propinquus), which naturally lives on macrophytes in close contact to O. cf. ovata, in order to assess its potential toxic effects on mortality, fecal pellet production (as a proxy of feeding), as well as fecundity and fertility ratios. Both, O. cf. ovata as well as a non-toxic competitive diatom (Licmophora paradoxa), were used as food in the experiments. Regarding acute toxicity evaluation, this copepod proved to be the most tolerant organism to O. cf. ovata reported to date. Nevertheless, its fecundity and fertility ratios were lower when fed with the toxic dinoflagellate, indicating a possible reprotoxic effect. Moreover, although fecal pellet production decreased significantly when the copepod was fed with a mono-diet of O. cf. ovata, epifluorescence microscopy observations revealed the presence of the toxic cells inside the digestive track, hence suggesting that these primary grazers could be a vector of toxins through the marine food web.

Development of an LC–MS/MS method to simultaneously monitor maitotoxins and selected ciguatoxins in algal cultures and P-CTX-1B in fish

Ciguatera fish poisoning is a serious human health issue that is highly localized to tropical and sub-tropical coastal areas, affecting many of the indigenous island communities intrinsically linked to reef systems for sustenance and trade. It is caused by the consumption of reef fish contaminated with ciguatoxins and is reported as the most common cause of non-bacterial food poisoning. The causative toxins bioaccumulate up the food web, from small herbivorous fish that graze on microalgae of the genus Gambierdiscus into the higher trophic level omnivorous and carnivorous fish predating on them. The number of Gambierdiscus species being described is increasing rapidly and the role of other toxins produced by this microalgal genus in ciguatera intoxications, such as maitotoxin, remains unclear. Ciguatoxins and maitotoxin are among the most potent marine toxins known and there are currently no methods of analysis that can simultaneously monitor these toxins with a high degree of specificity. To meet this need a rapid and selective ultra-performance liquid chromatography tandem mass spectrometry method has been developed to rapidly screen Gambierdiscus cultures and environmental sample device extracts for ciguatoxins and maitotoxins. A fast sample preparation method has also been developed to allow sensitive quantification of the potent ciguatoxin fish metabolite P-CTX-1B from fish extracts, and this method has been subjected to a small validation study. Novel aspects of this approach include the use of alkaline mobile phase for chromatographic separation and specific monitoring of the various toxins. This method has good potential to help evaluate ciguatera risk associated with Gambierdiscus and related microalgal species, and to help promote method development activities for this important and analytically challenging toxin class.

Assessment of cytotoxicity in ten strains of Gambierdiscus australes from Macaronesian Islands by neuro-2a cell-based assays

Within the dinoflagellate genus Gambierdiscus several species are well-known producers of ciguatoxins (CTXs) and maitotoxins (MTXs). These compounds are potent marine toxins that accumulate through the food chain, leading to a foodborne disease known as ciguatera when contaminated fish is consumed. Given the threat that the presence of these toxins in seafood may pose to human health and fisheries, there is an evident necessity to assess the potential toxicity of Gambierdiscus sp. in a particular area. Thus, the purpose of this work was to evaluate the production of CTX and MTX of 10 strains of Gambierdiscus australes isolated from the Selvagem Grande Island (Madeira, Portugal) and El Hierro Island (Canary Islands, Spain). The strains were first characterized by light microscopy and species were confirmed by molecular biology, being identified as G. australes. Following the species identification, CTX and MTX production of G. australes extracts was evaluated at the exponential growth phase using neuro-2a cell-based assays. Additionally, the production of MTX was also investigated in two of the G. australes strains collected at the stationary growth phase. Interestingly, 9 out of 10 strains were found to produce CTX-like compounds, ranging from 200 to 697 fg equiv. CTX1B cell−1. None of the G. australes strains showed MTX-like activity at the exponential phase, but MTX production was observed in two strains at the stationary growth phase (227 and 275 pg equiv. MTX cell−1). Therefore, the presence of G. australes strains potentially producing CTX and MTX in these Macaronesian Islands was confirmed.

Synergistic Effect of Transient Receptor Potential Antagonist and Amiloride against Maitotoxin Induced Calcium Increase and Cytotoxicity in Human Neuronal Stem Cells.

Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results presented here lead to the identification of the TRPC4 channels as contributors to the MTX effects in human neuronal cells. Both, the calcium increase and the cytotoxicity of MTX were either fully (for the calcium increase) or partially (in the case of cytotoxicity) reverted by the blockade of canonical TRPC4 receptors with the selective antagonist ML204. Furthermore, the sodium proton exchanger blocker amiloride also partially inhibited the calcium rise and the cell death elicited by MTX while the combination of amiloride and ML204 fully prevented both the cytotoxicity and the calcium rise elicited by the toxin.

Palytoxin congeners.

Palytoxin, isolated from a zoanthid of the genus Palythoa, is the most potent marine toxin known. Intoxication by palytoxin leads to vasoconstriction, hemorrhage, ataxia, muscle weakness, ventricular fibrillation, pulmonary hypertension, ischemia and death. Palytoxin and its numerous derivatives (congeners) may enter the food chain and accumulate mainly in fishes and crabs, causing severe human intoxication and death following ingestion of contaminated products. Furthermore, toxic effects in individuals exposed via inhalation or skin contact to marine aerosol in coincidence with Ostreopsis blooms, have been reported. Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are a concern in the Mediterranean Sea, since this species produces a wide range of palytoxin-like compounds listed among the most potent marine toxins. Thus, the formerly unsuspected broad distribution of the benthic dinoflagellate Ostreopsis spp. has recently posed a problem of risk assessment for human health. Palytoxin has a strong potential for toxicity in humans and animals, and currently this toxin is of great concern worldwide. This review summarized and discussed the pharmacology and toxicology data of palytoxin and its congeners, including their cytotoxicity, human and animal toxicities. Moreover, the risk assessment and their control strategies including prevention and treatment assays were evaluated.

Efficient Access to the Functionalized Bicyclic Pharmacophore of Spirolide C by Using a Selective Diels–Alder Reaction

AbstractA functionalized bicyclic lactam that was used as a key intermediate in an efficient synthesis of the pharmacophore of potent marine toxin spirolide C was synthesized by using a highly selective Diels–Alder reaction. To improve the reactivity of this transformation without loss of selectivity, substrates that contained a silyl ether or silatrane moiety were elaborately designed and converted into the spirobicyclic core structure with stereochemical control over the two asymmetric centers at the C7 and C29 positions. Moreover, a further C−C bond formation by using a Hiyama cross‐coupling reaction of the vinyl silatrane derivative facilitated versatile modification at the C5 position with an aryl or alkenyl substituent.

Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes.

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

Quantification of saxitoxin in human blood by ELISA

Saxitoxin (STX) is a potent marine toxin that causes paralytic shellfish poisoning (PSP) which can result in significant morbidity and mortality in humans. Low lethal doses, rapid onset of PSP symptoms, and brief STX half-life in vivo require sensitive and rapid diagnostic techniques to monitor human exposures. Our laboratory has validated an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of STX from 0.020 to 0.80 ng/mL in human whole blood and from 0.06 to 2.0 ng/mL in dried human blood which is simple, sensitive, rapid, and cost-effective. To our knowledge, this is the first validated method for the quantitation of saxitoxin in whole blood. Microsampling devices were used in sample collection which allows for standardized collection of blood, stable storage, and cost-efficient shipping. Quality control precision and accuracy were evaluated over the course of validation and were within 20% of theoretical concentrations. No detectable background concentrations of STX were found among fifty whole blood and dried blood convenience samples. Additionally, ten spiked individual whole blood and dried blood samples were tested for accuracy and precision and were within 20% of theoretical concentrations. Gonyautoxins 2&3 (GTX2&3) cross-reacted with this ELISA by 21%, but all other structurally related PSP toxins tested cross-reacted less than two percent. While clinical diagnosis or treatment of PSP would be unaffected by GTX2&3 cross-reactivity by ELISA, to accurately quantify individual PSP toxins, these results should be coupled with high performance liquid chromatography mass spectrometry measurements.

Toxicity screening of 13 Gambierdiscus strains using neuro-2a and erythrocyte lysis bioassays

Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species.This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes, G. balechii, G. caribaeus, G. carpenteri, G. pacificus, G. scabrosus and one strain of an undetermined species (Gambierdiscus sp. Viet Nam), (ii) five strains from the North-Eastern Atlantic Ocean (two G. australes, a single G. excentricus and two G. silvae strains), and (iii) one G. carolinianus strain from the Mediterranean Sea. Cell pellets of Gambierdiscus were extracted with methanol and the crude extracts partitioned into a CTX-containing dichloromethane fraction and a MTX-containing aqueous methanol fraction. CTX-toxicity was estimated using the neuro-2a cytoxicity assay, and MTX-toxicity via a human erythrocyte lysis assay.Different species were grouped into different ratios of CTX- and MTX-toxicity, however, the ratio was not related to the geographical origin of species (Atlantic, Mediterranean, Pacific). All strains showed MTX-toxicity, ranging from 1.5 to 86pg MTX equivalents (eq) cell−1. All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell−1. The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell−1. As was true for CTX, the highest MTX-toxicity was also found in G. excentricus. Thus, the present study confirmed that at least one species from the Atlantic Ocean demonstrates similar toxicity as the most toxic strains from the Pacific, even if the metabolites in fish have so far been shown to be more toxic in the Pacific Ocean.