Rapid determination of domoic acid in seafood by fluorescence polarization immunoassay using a portable analyzer.

Monitoring phycotoxin accumulation in marine products such as edible shellfish is a regulatory requirement in many countries. Therefore, a simple and rapid onsite quantification method is sought. Herein, we present a fluorescence polarization immunoassay (FPIA), a well-known one-step immunoassay, using a portable fluorescence polarization analyzer for domoic acid (DA), widely referred to as the primary toxin of amnesic shellfish poisoning (ASP). To establish FPIA for DA, the matrix effect of methanol, which is widely used to extract DA from shellfish, on FPIA was investigated. To validate this method, we performed a spike recovery test using oysters containing DA at a concentration equivalent to the regulatory limits of North America and the European Union (20 mg/kg). The recovery rate was found to be 79.4-114.7%, which is equivalent to that of the commercially available enzyme-linked immunosorbent assay (ELISA). We expect that this FPIA system will enable the quantitative onsite analysis of DA and significantly contribute to the safety of marine products.

Selective enrichments for color microplastics loading of marine lipophilic phycotoxins.

Microplastics (MPs) and marine lipophilic phycotoxins (MLPs) are two classes of emerging contaminants. Together, they may exacerbate the negative impacts on nearshore marine ecosystems. Herein, the loading of 14 representative MLPs, closely related to toxin-producing algae, on MPs and their relations with colorful MPs have been explored for the first time based on both field and lab data. The objectives of our study are to explore the roles of multiple factors (waterborne MLPs and MP characteristics) in the loading of MLPs by MPs with the applications of various statistical means, and to further explore the role of the color of MP in the loading of specific MLPs through lab simulation experiments. Our results demonstrated that MPs color determined the loading of some specific MLPs on MPs and green MPs can load much more than other colorful fractions (p < 0.05). These interesting phenomena illustrated that the color effects on the loading processes of MLPs on MPs are a dynamic process, and it can be well explained by the shading effect of MP color, which may affect the growth and metabolism of the attached toxic-producing algae on MPs and hence the production of specific MLPs. Furthermore, loading of MLPs on MPs can be considered as the comprehensive physicochemical and biological processes. Our results caution us that special attention should be paid to explore the real-time dynamic color shading effects on all kinds of bio-secreted contaminants loading on MPs, and highlight the necessary to comprehensive investigate the interaction between biota, organic contaminants and MPs.

Extreme genome diversity and cryptic speciation in a harmful algal-bloom-forming eukaryote.

Harmful algal blooms of the toxic haptophyte Prymnesium parvum are a recurrent problem in many inland and estuarine waters around the world. Strains of P. parvum vary in the toxins they produce and in other physiological traits associated with harmful algal blooms, but the genetic basis for this variation is unknown. To investigate genome diversity in this morphospecies, we generated genome assemblies for 15 phylogenetically and geographically diverse strains of P. parvum, including Hi-C guided, near-chromosome-level assemblies for two strains. Comparative analysis revealed considerable DNA content variation between strains, ranging from 115 to 845 Mbp. Strains included haploids, diploids, and polyploids, but not all differences in DNA content were due to variation in genome copy number. Haploid genome size between strains of different chemotypes differed by as much as 243 Mbp. Syntenic and phylogenetic analyses indicate that UTEX 2797, a common laboratory strain from Texas, is a hybrid that retains two phylogenetically distinct haplotypes. Investigation of gene families variably present across the strains identified several functional categories associated with metabolic and genome size variation in P. parvum, including genes for the biosynthesis of toxic metabolites and proliferation of transposable elements. Together, our results indicate that P. parvum comprises multiple cryptic species. These genomes provide a robust phylogenetic and genomic framework for investigations into the eco-physiological consequences of the intra- and inter-specific genetic variation present in P. parvum and demonstrate the need for similar resources for other harmful algal-bloom-forming morphospecies.

Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis.

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways.

Rat tight junction proteins are disrupted after subchronic exposure to okadaic acid.

Okadaic acid (OA), a lipophilic phycotoxin distributed worldwide, causes diarrheic shellfish poisoning and even leads to tumor formation. Currently, the consumption of contaminated seafood is the most likely cause of chronic OA exposure, but there is a serious lack of relevant data. Here, the Sprague-Dawley rats were exposure to OA by oral administration at 100 µg/kg body weight, and the tissues were collected and analyzed to assess the effect of subchronic OA exposure. The results showed that subchronic OA administration disturbed colonic mucosal integrity and induced colitis. The colonic tight junction proteins were disrupted and the cell cycle of colonic epithelial cells was accelerated. It is inferred that disruption of the colonic tight junction proteins might be related to the development of chronic diarrhea by affecting water and ion transport. Moreover, the accelerated proliferation of colonic epithelial cells indicated that subchronic OA exposure might promote the restitution process of gut barrier or induce tumor promoter activity in rat colon.

First Identification of Amphidinols from Mexican Strains and New Analogs.

The genus Amphidinium has been the subject of recent attention due to the production of polyketide metabolites. Some of these compounds have shown significant bioactivities and could be related to species interactions in the natural benthic microenvironment. Among these compounds, amphidinols (AMs) are suspected to be related to fish kills and probably implicated in ciguatera symptoms associated with the occurrence of benthic harmful algal blooms (bHABs). Here, we present the first report of a variety of AMs produced by cultured strains from several species from the Mexican Pacific, the Gulf of California, and the Gulf of Mexico. Through ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), ten previously known AMs (AM02, -04, -05, -06, -07, -09, -11, -14, -15, and -17), four recently reported AMs (N7, N8/N9, N12, and N13), and three new variants (U1, U2, and U3) were identified. Of the twelve analyzed Amphidinium cultures, five were not AM producers, and the cell quotas of the remaining seven strains ranged from close to nondetectable to a maximum of 1694 fg cell-1, with many intermediate levels in between. The cultures from the Mexican North Pacific coast produced AMs in a higher quantity and variety than those from worldwide locations. This is the first study of AMs from Mexican Amphidinium strains, and our results confirm the relevance of continuing the investigation of the genus bioactive metabolites.

Current Trends and New Challenges in Marine Phycotoxins.

Marine phycotoxins are a multiplicity of bioactive compounds which are produced by microalgae and bioaccumulate in the marine food web. Phycotoxins affect the ecosystem, pose a threat to human health, and have important economic effects on aquaculture and tourism worldwide. However, human health and food safety have been the primary concerns when considering the impacts of phycotoxins. Phycotoxins toxicity information, often used to set regulatory limits for these toxins in shellfish, lacks traceability of toxicity values highlighting the need for predefined toxicological criteria. Toxicity data together with adequate detection methods for monitoring procedures are crucial to protect human health. However, despite technological advances, there are still methodological uncertainties and high demand for universal phycotoxin detectors. This review focuses on these topics, including uncertainties of climate change, providing an overview of the current information as well as future perspectives.

Non-target analysis and stability assessment of reference materials using liquid chromatography‒high-resolution mass spectrometry.

Development and characterization of biological and environmental matrix certified reference materials (CRMs) for organic analytes typically relies heavily on targeted analytical methods, such as liquid chromatography (LC) with triple-quadrupole mass spectrometry detection. LC with high-resolution mass spectrometry (LC‒HRMS) can also provide high quality data for both targeted and non-targeted analytes, with the potential for retrospective data analysis. Here, we demonstrate the utility of non-target analysis (NTA) using LC‒HRMS for profiling and stability assessment of a mussel tissue matrix CRM certified for several classes of marine algal toxins (CRM-FDMT1). First, the NTA method was developed using data-dependent MS/MS acquisition and commercial metabolomics software for data processing. Of 128 toxin analogues previously reported in CRM-FDMT1, 125 were detected by LC-HRMS, with 97 triggered for MS/MS by data dependant acquisition. Automated data processing detected 119 of these compounds and 109 were retained after automated filtering of results for putative toxin analogues. Those analogues not detected were low abundance ions, or poorly resolved isomers. The method was then used to demonstrate new strategies for CRM stability assessment considering the stability of certified analytes, related toxin analogues, and unrelated matrix compounds. Several analogues from each toxin class in CRM-FDMT1 as well as other unrelated matrix compounds were observed to be significantly less stable than the certified toxins. Using this method, no instability was measured for any compounds at conditions ≤4 °C, providing a greater degree of confidence in CRM stability than could be achieved using conventional approaches to stability assessment targeting only the certified analytes. The NTA method and stability assessment approach presented are applicable to future CRM development with other matrices and organic analyte classes.

Potential endocrine correlation with exposure to domoic acid in Southern Right Whale (Eubalaena australis) at the Península Valdés breeding ground.

In waters off Península Valdés (PV), Argentina, southern right whales (SRW, Eubalaena australis) are occasionally exposed to domoic acid (DA), a neurotoxin produced by diatoms of the genus Pseudo-nitzschia. Domoic acid toxicity in marine mammals can cause gastrointestinal and neurological clinical signs, alterations in hematologic and endocrine variables, and can be fatal in extreme cases. In this study, we validated an enzyme immunoassay to quantify fecal glucocorticoid metabolites (fGCm) in 16 SRW fecal samples from live and dead stranded whales in PV from 2013 to 2018 and assessed fGCm levels associated with DA exposure. Overall, fGCm levels were significantly lower in SRWs with detectable fecal DA (n = 3) as compared to SRWs with undetectable fecal DA levels (n = 13). The highest fecal DA was observed in a live lactating female, which had low fGCm compared to the other lactating females studied. The highest fGCm was observed in a lactating female with undetectable DA; interestingly, at the time of sample collection, this female was sighted with two calves, an extremely unusual occurrence in this species. Though the sample size of these exceptionally rare breeding-season fecal samples was unavoidably small, our study provides evidence of potential adrenal alterations in whales exposed to an environmental neurotoxin such as DA.

Recent Advances in Aptasensing Strategies for Monitoring Phycotoxins: Promising for Food Safety.

Phycotoxins or marine toxins cause massive harm to humans, livestock, and pets. Current strategies based on ordinary methods are long time-wise and require expert operators, and are not reliable for on-site and real-time use. Therefore, it is urgent to exploit new detection methods for marine toxins with high sensitivity and specificity, low detection limits, convenience, and high efficiency. Conversely, biosensors can distinguish poisons with less response time and higher selectivity than the common strategies. Aptamer-based biosensors (aptasensors) are potent for environmental monitoring, especially for on-site and real-time determination of marine toxins and freshwater microorganisms, and with a degree of superiority over other biosensors, making them worth considering. This article reviews the designed aptasensors based on the different strategies for detecting the various phycotoxins.

Domoic Acid and Pseudo-nitzschia spp. Connected to Coastal Upwelling along Coastal Inhambane Province, Mozambique: A New Area of Concern.

Harmful algal blooms (HABs) are increasing globally in frequency, persistence, and geographic extent, posing a threat to ecosystem and human health. To date, no occurrences of marine phycotoxins have been recorded in Mozambique, which may be due to absence of a monitoring program and general awareness of potential threats. This study is the first documentation of neurotoxin, domoic acid (DA), produced by the diatom Pseudo-nitzschia along the east coast of Africa. Coastal Inhambane Province is a biodiversity hotspot where year-round Rhincodon typus (whale shark) sightings are among the highest globally and support an emerging ecotourism industry. Links between primary productivity and biodiversity in this area have not previously been considered or reported. During a pilot study, from January 2017 to April 2018, DA was identified year-round, peaking during Austral winter. During an intense study between May and August 2018, our research focused on identifying environmental factors influencing coastal productivity and DA concentration. Phytoplankton assemblage was diatom-dominated, with high abundances of Pseudo-nitzschia spp. Data suggest the system was influenced by nutrient pulses resulting from coastal upwelling. Continued and comprehensive monitoring along southern Mozambique would provide critical information to assess ecosystem and human health threats from marine toxins under challenges posed by global change.

Occurrence and Trophodynamics of Marine Lipophilic Phycotoxins in a Subtropical Marine Food Web.

Marine lipophilic phycotoxins (MLPs) are produced by toxigenic microalgae and cause foodborne illnesses. However, there is little information on the trophic transfer potential of MLPs in marine food webs. In this study, various food web components including 17 species of mollusks, crustaceans, and fishes were collected for an analysis of 17 representative MLPs, including azaspiracids (AZAs), brevetoxins (BTXs), gymnodimine (GYM), spirolides (SPXs), okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), yessotoxins (YTXs), and ciguatoxins (CTXs). Among the 17 target MLPs, 12, namely, AZAs1-3, BTX3, GYM, SPX1, OA, DTXs1-2, PTX2, YTX, and the YTX derivative homoYTX, were detected, and the total MLP concentrations ranged from 0.316 to 20.3 ng g-1 wet weight (ww). The mean total MLP concentrations generally decreased as follows: mollusks (8.54 ng g-1, ww) > crustaceans (1.38 ng g-1, ww) > fishes (0.914 ng g-1, ww). OA, DTXs, and YTXs were the predominant MLPs accumulated in the studied biota. Trophic dilution of the total MLPs was observed with a trophic magnification factor of 0.109. The studied MLPs might not pose health risks to residents who consume contaminated seafood; however, their potential risks to the ecosystem can be a cause for concern.

Three decades of Canadian marine harmful algal events: Phytoplankton and phycotoxins of concern to human and ecosystem health.

Spatial and temporal trends of marine harmful algal events in Canada over the last three decades were examined using data from the Harmful Algal Event Database (HAEDAT). This database contains the most complete record of algal blooms, phycotoxins and shellfish harvesting area closures in Canada since 1987. This 30-year review of 593 Canadian HAEDAT records from 1988 to 2017, together with other Canadian data and publications, shows that recurring harmful algal events have been widespread throughout both the Atlantic and Pacific coastal regions. The 367 paralytic shellfish toxin (PST) reports revealed annual and frequent recurrence throughout both the Atlantic and Pacific regions, including multi-year PST events in the Bay of Fundy, the Estuary and Gulf of St. Lawrence and the Strait of Georgia. The 70 amnesic shellfish toxin (AST) records revealed no recognizable trend, as these events were usually area specific and did not recur annually. The increasing frequency of diarrhetic shellfish toxin (DST) events over the period of this review, in total 59 records, can be at least partially explained by increased sampling effort. Marine species mortalities caused by harmful algae (including diatoms, dictyochophytes, dinoflagellates, and raphidophytes), were a common occurrence in the Pacific region (87 reports), but have been reported much less frequently in the Atlantic region (10 reports). Notable Canadian records contained in HAEDAT include the first detection worldwide of amnesic shellfish poisoning (ASP), attributed to the production of domoic acid (an AST) by a diatom (Pseudo-nitzschia multiseries) in Prince Edward Island in 1987. The first proven case of diarrhetic shellfish poisoning (DSP) in Canada and North America was recorded in 1990, and the first closures of shellfish harvesting due to DST (associated with the presence of Dinophysis norvegica) occurred in Nova Scotia in 1992, followed by closures in Newfoundland and Labrador in 1993. In 2008, mass mortalities of fishes, birds and mammals in the St. Lawrence Estuary were caused by Alexandrium catenella and high levels of PST. During 2015, the Pacific coast experienced a large algal bloom that extended from California to Alaska. It resulted in the closure of several shellfish harvesting areas in British Columbia due to AST, produced by Pseudo-nitzschia australis. Data from the Canadian Arctic coast is not included in HAEDAT. However, because of the emerging importance of climate change and increased vessel traffic in the Arctic, information on the occurrence of harmful algal species (pelagic and sympagic = sea ice-associated) in that region was compiled from relevant literature and data. The results suggest that these taxa may be more widespread than previously thought in the Canadian Arctic. Information in HAEDAT was not always robust or complete enough to provide conclusions about temporal trends. Compilation of spatial and temporal information from HAEDAT and other records is nevertheless important for evaluating the potential role of harmful algae as a stressor on Canadian marine ecosystems, and will support the next step: developing a knowledge gap analysis that will establish research priorities for determining their consequences on human and ecosystem health.

Karmitoxin production by Karlodinium armiger and the effects of K. armiger and karmitoxin towards fish.

The dinoflagellate Karlodinium armiger has a huge impact on wild and caged fish during blooms in coastal waters. Recently, a new toxin, karmitoxin, was chemically characterized from K. armiger and a quantification method was established, thereby allowing investigations of the fish killing mechanism. K. armiger is not able to grow in standard growth media that are based on nitrate as a nitrogen source, and successful cultures of this species have only been achieved in mixotrophic cultures after addition of a prey source. Here we show that addition of ammonium (up to 50 µM) to the growth media is a good alternative, as K. armiger batch cultures achieve growth rates, which are comparable to growth rates reached in mixotrophic cultures. Karmitoxin production (1.9 and 2.9 pg cell-1 d-1) and cellular karmitoxin content (8.72 ± 0.25 pg cell-1 and 7.14 ± 0.29 pg cell-1) were in the same range, though significantly different, in prey-fed cultures and monocultures supplied with ammonium, respectively. Net production of karmitoxin stopped when the K. armiger cultures reached stationary growth phase, indicating no accumulation of karmitoxin in cells or growth media. Toxicity tests towards sheepshead minnow fish larvae indicated rapid death of the fish larvae when exposed to high K. armiger cell concentrations (LT50 of 2.06 h at 44.9 × 103 cells mL-1 cultivated with ammonium). Purified toxins caused the same physical damage to fish larvae as living K. armiger cultures. An exposure of purified karmitoxin to fish larvae and rainbow trout gill cells indicated that the fish larvae were about three times less sensitive than gill cells. When comparing the effect of purified toxins with the effect of whole K. armiger cultures, twice the toxin concentration of the purified toxins was needed to cause the same effect. Although a loss of karmitoxin of twenty percent was observed during the incubation, this could not explain the apparent discrepancy. Other factors, like a direct effect of the K. armiger cells on the fish larvae or other, yet unknown toxins may influence the effect of whole cell cultures. To study the effects of released karmitoxin, fish larvae were exposed to a K. armiger culture that was treated with HP-20 resin, which adsorbs extracellular karmitoxin. The 24 h HP-20 treatment resulted in a K. armiger culture that had 37% less total karmitoxin, without a reduction in cell concentration, and a reduced toxic effect was observed in the HP-20 treated culture, as compared to non-treated controls. Fish larvae that were exposed to HP-20 treated culture were immobilized, but survived during the 12 h exposure, whereas the exposure to non-treated culture led to high mortality of the fish larvae. Direct observations under the microscope revealed no evidence of micropredation of K. armiger on the fish larvae during any of the exposures. Thus, the results presented here, indicate that released karmitoxin is the main cause for fish kills by K. armiger. Finally, we found that juvenile rainbow trout were six times more sensitive than fish larvae towards K. armiger, indicating that juvenile fish are more sensitive to K. armiger in bloom situations than early larval stages.

LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium.

Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM.

The occurrence of lipophilic toxins in shellfish from the Middle Adriatic Sea.

The first survey of the phycotoxin profile in mussels (Mytilus galloprovincialis) from the coastal waters of Bosnia and Herzegovina (The Bay of Neum, Middle Adriatic Sea) in correlation to the Makarska City Bay (Croatia, Middle Adriatic Sea) was conducted in 2017. Throughout the monitoring period, occasions of gymnodimine (GYM) and azaspiracid (AZA2) shellfish toxicity were recorded in concentrations that do not endanger human health. The occurrence of yessotoxins (YTXs), the most common toxins found in the Adriatic Sea, was correlated to the presence of the Gonyaulax species, a potential source of YTX. The DSP group of toxins is represented by the ester-OA. Phytoplankton analysis confirmed the presence of dinoflagellates from the Prorocentrum genus, a species associated with DSP toxicity. Occurrence frequency and variability of toxin composition were investigated in conjunction to physico-chemical parameters in the surrounding sea water. In the central Adriatic Sea, the infestation period ranges in general from June to August. However, the depuration phase extended beyond September in the Bay of Neum, increasing the length of the decontamination period.

Magnetic nanostructures for marine and freshwater toxins removal.

Marine and freshwater toxins contaminate water resources, shellfish and aquaculture products, causing a broad range of toxic effects in humans and animals. Different core-shell nanoparticles were tested as a new sorbent for removing marine and freshwater toxins from liquid media. Water solutions were contaminated with 20 µg/L of marine toxins and up to 50 µg/L of freshwater toxins and subsequently treated with 250 or 125 mg/L of nanoparticles. Under these conditions, carbon nanoparticles removed around 70% of saxitoxins, spirolides, and azaspiracids, and up to 38% of diarrheic shellfish poisoning toxins. In the case of freshwater toxins, the 85% of microcystin LR was eliminated; other cyclic peptide toxins were also removed in a high percentage. Marine toxins were adsorbed in the first 5 min of contact, while for freshwater toxins it was necessary 60 min to reach the maximum adsorption. Toxins were recovered by extraction from nanoparticles with different solvents. Gymnodinium catenatum, Prorocentrum lima, and Microcystis aeruginosa cultures were employed to test the ability of nanoparticles to adsorb toxins in a real environment, and the same efficacy to remove toxins was observed in these conditions. These results suggest the possibility of using the nanotechnology in the treatment of contaminated water or in chemical analysis applications.

Co-culture model of Caco-2/HT29-MTX cells: A promising tool for investigation of phycotoxins toxicity on the intestinal barrier.

Most lipophilic phycotoxins have been involved in human intoxications but some of these toxins have never been proven to induce human gastro-intestinal symptoms, although intestinal damage in rodents has been documented. For investigating the in vitro toxicological profile of lipophilic phycotoxins on intestine, the epithelial Caco-2 cell line has been the most commonly used model. Nevertheless, considering the complexity of the intestinal epithelium, in vitro co-cultures integrating enterocyte-like and mucus-secreting cell types are expected to provide more relevant data. In this study, the toxic effects (viability, inflammation, cellular monolayer integrity, modulation of cell type proportion and production of mucus) of four lipophilic phycotoxins (PTX2, YTX, AZA1 and OA) were evaluated in Caco-2/HT29-MTX co-cultured cells. The four toxins induced a reduction of viability from 20% to 50% and affected the monolayer integrity. Our results showed that the HT29-MTX cells population were more sensitive to OA and PTX2 than Caco-2 cells. Among the four phycotoxins, OA induced inflammation (28-fold increase of IL-8 release) and also a slight increase of both mucus production (up-regulation of mucins mRNA expression) and mucus secretion (mucus area and density). For PTX2 we observed an increase of IL-8 release but weaker than OA. Intestinal cell models integrating several cell types can contribute to improve hazard characterization and to describe more accurately the modes of action of phycotoxins.

Occurrence of Toxigenic Microalgal Species and Phycotoxin Accumulation in Mesozooplankton in Northern Patagonian Gulfs, Argentina.

In the Northern Patagonian gulfs of Argentina (Golfo Nuevo and Golfo San José), blooms of toxigenic microalgae and the detection of their associated phycotoxins are recurrent phenomena. The present study evaluated the transfer of phycotoxins from toxigenic microalgae to mesozooplankton in Golfo Nuevo and Golfo San José throughout an annual cycle (December 2014-2015 and January 2015-2016, respectively). In addition, solid-phase adsorption toxin tracking (SPATT) samplers were deployed for the first time in these gulfs, to estimate the occurrence of phycotoxins in the seawater between the phytoplankton samplings. Domoic acid was present throughout the annual cycle in SPATT samplers, whereas no paralytic shellfish poisoning toxins were detected. Ten toxigenic species were identified: Alexandrium catenella, Dinophysis acuminata, Dinophysis acuta, Dinophysis tripos, Dinophysis caudata, Prorocentrum lima, Pseudo-nitzschia australis, Pseudo-nitzschia calliantha, Pseudo-nitzschia fraudulenta, and Pseudo-nitzschia pungens. Lipophilic and hydrophilic toxins were detected in phytoplankton and mesozooplankton from both gulfs. Pseudo-nitzschia spp. were the toxigenic species most frequent in these gulfs. Consequently, domoic acid was the phycotoxin most abundantly detected and transferred to upper trophic levels. Spirolides were detected in phytoplankton and mesozooplankton for the first time in the study area. Likewise, dinophysistoxins were found in mesozooplankton from both gulfs, and this is the first report of the presence of these phycotoxins in zooplankton from the Argentine Sea. The dominance of calanoid copepods indicates that they were the primary vector of phycotoxins in the pelagic trophic web. Environ Toxicol Chem 2019;38:2209-2223. © 2019 SETAC.

Development of an Indirect Quantitation Method to Assess Ichthyotoxic B-Type Prymnesins from Prymnesium parvum.

Harmful algal blooms of Prymnesium parvum have recurrently been associated with the killing of fish. The causative ichthyotoxic agents of this haptophyte are believed to be prymnesins, a group of supersized ladder-frame polyether compounds currently divided into three types. Here, the development of a quantitative method to assess the molar sum of prymnesins in water samples and in algal biomass is reported. The method is based on the derivatization of the primary amine group and subsequent fluorescence detection using external calibrants. The presence of prymnesins in the underivatized sample should be confirmed by liquid chromatography mass spectrometry. The method is currently only partly applicable to water samples due to the low amounts that are present. The growth and cellular toxin content of two B-type producing strains were monitored in batch cultures eventually limited by an elevated pH. The cellular toxin contents varied by a factor of ~2.5 throughout the growth cycle, with the highest amounts found in the exponential growth phase and the lowest in the stationary growth/death phases. The strain K-0081 contained ~5 times more toxin than K-0374. Further investigations showed that the majority of prymnesins were associated with the biomass (89% ± 7%). This study provides the basis for further investigations into the toxicity and production of prymnesins.