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Microplastics (MP) are suitable substrates for the colonization of harmful microalgal cells and the adsorption of their lipophilic compounds including phycotoxins. Moreover, such interactions likely change as physical-chemical characteristics of the MP surface are gradually modified during plastic degradation in aquatic environments. Using a combination of innovative laboratory experiments, this study systematically investigated, for the first time, the influence of various MP characteristics (polymeric composition, shape, size, and/or surface roughness) on its capacity to carry both living harmful algal cells and dissolved phycotoxins. Cell colonization by the dinoflagellate Prorocentrum lima started early (within 24 h) on particles of all shapes tested. However, cell colonization was much more intense on polystyrene ∼800 µm microspheres (0.63-46.4 cells mm - ²; mean=11.7) and 500 × 1000 μm cuboid fragments (0.64-28.3 cells mm - ²; mean=7.0), compared to polypropylene 11,000 × 50 µm microfibers of equivalent surface area (0.01-0.64 cells mm - ²; mean=0.28), which were probably too narrow and light to interact with these benthic cells. Similar to lipophilic pollutants, adsorption of the diarrhetic toxin okadaic acid (OA) was greater on smaller MP particles (50 µm), attaining up to 8.0 pg mm² after 168 h of exposure. Moreover, in the short term (24 h), OA adsorption was significantly higher on aged MP, whose surface was modified following common degradation processes (abrasion, UV-photodegradation or microbial biodegradation), relative to virgin particles. During benthic P. lima blooms, the presence of aged MP covered by toxic cells and/or their dissolved compounds are expected to make diarrhetic toxins available to a greater diversity of organisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

The Magellan Subantarctic ecoregion (MSE) in the Southern Hemisphere (47°-56°S; 71°-73°W) is a unique natural laboratory subject to persistent and accelerated glacial ice melt, generating a complex system of environmental gradients (e.g., salinity and temperature) that influence the ecological patterns of marine biodiversity. However, the factors influencing marine epiphytic microalgal assemblages are still poorly understood. In this context, we characterized the richness and structure of epiphytic assemblages in different benthic macroalgal hosts (Acrosiphonia arcta, Ectocarpus siliculosus, and Leptosiphonia brodiei) in sites with glaciers and estuarine characteristics (Yendegaia Bay and Fouquet Estuary) and sites without glaciers and oceanic characteristics (Batchelor River and Offing Island) of the MSE, revealing how sites, host, and environmental variables influence variation of epiphytic assemblages. In 36 samples, 67 genera of epiphytes were recorded. The dominant divisions were Bacillariophyta (50 genera), Dinophyta (7 genera) and Cyanophyta (6 genera). We observed significantly high diversity in epiphytic assemblages, with contrasting patterns of variation depending on site and/or host macroalgae. Host specificity was not evident for most epiphytes. The most factor influencing the variation of the epiphythic assemblage was the marked environmental gradient (changes in temperature, salinity, nutrients, among others) between sites with and without glacial influence. Additionally, our research identified potentially toxic and/or harmful epiphytic microalgae belonging to the divisions Dinophyta (dinoflagellates) and Cyanophyta (cyanobacteria). The data on ecological patterns of epiphyte assemblages provides valuable insights into the current state of a poorly understood microscopic biodiversity, shaped by diverse environmental factors at different sites. Under current and future climate change scenarios in the MSE, environmental gradients may become more pronounced, with important positive and/or negative consequences on epiphyte assemblages. In light of these findings, we present a baseline for future research to further develop our understanding and facilitate the monitoring and conservation of epiphytic microalgae in the MSE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Algal toxins are involved in the mortality and/or illness of marine organisms via consumption of contaminated prey, or upon direct exposure to toxic cells. In this study, the presence of potentially toxic microalgal cells was investigated within the digestive tract contents of a threatened species of green turtle (Chelonia mydas). Additionally, lipophilic toxins were determined by LC-MS/MS in tissue samples (liver, stomach and/or intestine) of selected animals (n = 39 individuals) found dead-stranded in southern Brazil, from winter/2015 to autumn/2016. Thirteen potentially toxic species of microalgae (both benthic and planktonic), including seven dinoflagellates, six cyanobacteria and one diatom, were found in the digestive tract contents of green turtles. Among them, dinoflagellates belonging to the Dinophysis acuminata species complex were the most frequent (36%) and abundant (maximum average abundance of 566 cells g -1 in spring/2015). Moreover, 23% of the examined sea turtles exhibited detectable levels of the diarrhetic shellfish toxin okadaic acid (OA) in washed digestive tissues. Seven individuals accumulated OA in their intestines (max. 24.1 ng g -1 ) and two in the stomachs (max. 7.4 ng g -1 ). Toxin levels in the tissues were directly and significantly (r = 0.70, p < 0.025) associated with the cell abundance of OA-producing D. acuminata and Prorocentrum lima species complexes within the digestive contents of green turtles. Although OA concentrations were relatively low, possible chronic exposure might deteriorate general health conditions of exposed sea turtles, increasing the risk for diseases. Okadaic acid has been regarded as a tumor-promoting compound and an environmental co-factor in the incidence of fibropapillomatosis, a frequent disease in juvenile green turtles inhabiting this geographic region. Even though, only one green turtle containing OA in the digestive tissues (out of six examined) also presented fibropapillomatosis in this study. Notwithstanding, sea turtles are sentinels of ocean health. Monitoring the accumulation of algal toxins and their negative effects on these organisms contributes to conserving biodiversity and marine habitats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Public awareness about Benthic Harmful Algal Blooms (BHABs) and their negative impacts has increased substantially over the past few decades. Even so, reports of BHABs remain relatively scarce in South America (SA). This paper provides a comprehensive overview of the current state of knowledge on BHABs in the continent, by integrating data from published articles, books, and technical reports. We recorded ∼300 different occurrences of potentially toxic BHAB species over the Caribbean, Atlantic and Pacific coasts, mostly in marine (>95%) but also in estuarine areas located from 12⁰36' N to 54⁰53' S. Over 70% of the data was published/released within the past 10 years, and ∼85% were concentrated in Brazil, Venezuela, Ecuador and Colombia. Benthic species were mainly associated with macroalgae, seagrass and sediment. Incidental detection in the plankton was also relevant, mainly in places where studies targeting BHAB species are still rare, like Argentina, Uruguay, Chile and Peru. The study listed 31 infrageneric taxa of potentially toxic benthic dinoflagellates and eight of estuarine cyanobacteria occurring in SA, with the greatest species diversity recorded in the equatorial-tropical zone, mainly in northeastern Brazil (Atlantic), Venezuela and Colombia (Caribbean), and the Galapagos Islands, Ecuador (Pacific). Local strains of Amphidinium, Gambierdiscus, Coolia and Prorocentrum spp. produced toxic compounds of emerging concern. Prorocentrum lima species complex was the most common and widely distributed taxon, followed by Ostreopsis cf. ovata. In fact, these two dinoflagellates were associated with most BHAB events in SA. Whereas the former has caused the contamination of multiple marine organisms and cases of Diarrhetic Shellfish Poisoning in subtropical and temperate areas, the latter has been associated with faunal mortalities and is suspected of causing respiratory illness to beach users in tropical places. Ciguatera Poisoning has been reported in Colombia (∼240 cases; no deaths) and Venezuela (60 cases; two deaths), and may be also a risk in other places where Gambierdiscus spp. and Fukuyoa paulensis have been reported, such as the Galapagos Islands and the tropical Brazilian coast. Despite the recent advances, negative impacts from BHABs in SA are intensified by limited research/training funding, as well as the lack of official HAB monitoring and poor analytical capability for species identification and toxin detection in parts of the continent., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Several species of microalgae can produce potent phycotoxins that negatively affect aquatic organisms and their consumers following different exposure routes, as well as toxicokinetic (TK) and toxicodynamic (TD) processes. Benthic organisms are especially vulnerable as they are exposed to both benthic and planktonic species causative of harmful algal blooms (HABs). While benthic algae can come into direct contact with annelids during substrate remobilization, planktonic cells can settle to the bottom mostly during senescence and/or encystment stages, and in shallow and calm waters. We performed a systematic, qualitative review of the literature on the phycotoxin TK and TD processes in marine annelids, summarizing the most relevant findings and general trends. Besides, by using innovative analytical/statistical approaches, we were able to detect patterns and gaps in the current literature, thus pointing to future research directions. We retrieved and analyzed studies involving diarrhetic shellfish toxins (DSTs), paralytic shellfish toxins (PSTs), brevetoxins (PbTXs), domoic acid (DA), as well as palytoxin and its congeners, the ovatoxins (treated together as PLTXs). It is worth mentioning that studies evaluating other phycotoxins (e.g., ciguatoxins, yessotoxins) were not found in the literature. The absence of data on PbTXs, PSTs and DA is the largest gap hampering TK assessment in annelids, although some relevant information on TD is already available. Whereas lethal effects from DSTs have not been reported, more potent toxins like PbTXs, PSTs, DA and those grouped as PLTX-like compounds can cause mortality and/or marked decrease in annelid abundance. In addition, phycotoxins have been linked to sublethal effects on annelid cells. Although very sparse, field and laboratory studies offer strong evidence that annelids may be reliable indicators of toxin exposure and their negative effects during both early and later stages of HABs in marine environments. Besides quickly responding to these compounds at both organismic and suborganismic levels, annelids are easily found in areas affected by HABs. The use of annelids in future investigations evaluating the action mechanisms of toxic microalgae on marine invertebrates should be thus encouraged. In this case, the choice for widely dispersed and numerically dominant species of annelids would strengthen the validation and extrapolation of results from risk assessments in areas affected by HABs worldwide., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A new marine benthic toxic Prorocentrum species is described from the tropical/subtropical regions of the Atlantic (Colombian Caribbean Sea and Northeast Brazil) and Pacific (Southern Japan) oceans. Morphological cell structures were examined using light (LM) and scanning electron (SEM) microscopy. Prorocentrum porosum sp. nov. was characterized by 35.9-50.2 μm long and 25.4-45.7 μm deep cells, covered by broadly ovoid symmetric thecal plates. The surface of both thecal plates is smooth and covered by randomly scattered kidney-shaped pores (n = 102-149), rounder towards the center, absent in the central part, and surrounded by a conspicuous marginal ring of about 69-92 evenly spaced pores. Broad V-shaped periflagellar area exhibiting flagellar and accessory pores. The molecular phylogenetic position of P. porosum sp. nov. was inferred using partial LSU rRNA gene (rDNA) and rDNA ITS sequences. This new species branched with high support in a Prorocentrum clade including P. caipirignum, P. hoffmannianum and P. cf. lima (P. lima morphotype 5 sensuZhang et al., 2015). Pairwise comparison of ITS1 and ITS2 transcripts with these closest relatives revealed the presence of compensatory base changes (CBCs), with the exception of P. cf. lima (P. lima morphotype 5), which only showed in ITS2 a hemi-CBC (HCBC) and two base changes that possibly induce a structural modification. Toxin analyses performed in two Colombian and Brazilian strains in the present study detected the presence of low amounts of okadaic acid., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The diarrhetic shellfish toxins (DSTs) okadaic acid (OA) and its analogues - the dinophysistoxins (DTXs) - are produced by dinoflagellates such as Prorocentrum lima and can bioaccumulate in filter-feeding organisms as they are transferred through the food web. Although there is no assessment of the harmful effects of these toxins on the fish's immune system, this study developed a primary culture protocol for kidney cells from marine fish Centropomus parallelus and evaluated the immunotoxic effects to P. lima extracts containing DSTs. The cells were obtained by mechanical dissociation, segregated with Percoll gradient, and incubated for 24 h at 28 °C in a Leibovitz culture medium supplemented with 2% fetal bovine serum and antibiotics. The exposed cells were evaluated in flow cytometry using the CD54 PE antibody. We obtained >5.0 × 10 6 viable cells per 1.0 g of tissue that exhibited no cell differentiation. Exposure to 1.2 or 12 ng DST mL -1 stimulated the immune system activation and increased the proportion of activated macrophages and monocytes in 48 to 52% and in 127 to 146%, respectively. The protocol proved to be an alternative tool to assess the immunotoxic effects of DST exposure on fish's anterior kidney cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Lipophilic toxins were investigated in different benthic compartments of Paranaguá Bay, southern Brazil. Relatively low concentrations of okadaic acid (OA) were frequently reported in macrophyte extracts (maximum 0.64 ng g -1 ), mainly in sheltered estuarine areas, correlated with the cell abundance of toxigenic benthic dinoflagellates, Prorocentrum lima . Non-conjugated OA was also detected in benthic marine animals, mostly during summer-autumn 2016. Higher OA concentrations were found in the viscera of Genidens genidens fish (24.3 ± 3.7 ng g -1 ) and in soft tissues of Mytella guyanensis bivalves (21.3 ± 2.13 ng g -1 ), whereas lower levels were less frequently reported in many other benthic feeders, including cephalopods, gastropods, crustaceans and fishes. Although OA concentrations were below the levels associated with acute human intoxication, possible chronic effects to benthic animals and their consumers cannot be disregarded due to the frequent presence of this biotoxin in various fishery resources. Human health risks are substantially enhanced for small-scale fishing communities and other socioeconomically vulnerable populations.

We evaluated the accumulation of okadaic acid (OA), a diarrhetic toxin, and the antioxidant responses in the marine annelid Laeonereis acuta exposed to the benthic toxigenic dinoflagellate Prorocentrum cf. lima. Nontoxic Tetraselmis sp. was used as a control diet. Living cells of the two algae were supplied as food to animals kept in agar medium for 72 h. To assess the significance of the observed effects, our experimental design treated the algal species (diet), algal cell densities, and exposure time as fixed factors. Responses of the organisms were assessed through oxidative stress biomarkers (glutathione-S-transferase [GST], catalase [CAT], reduced glutathione [GSH] and lipid peroxidation [LPO]). Toxin accumulation was measured by LC-MS/MS in whole-body homogenates after 12, 24 and 72 h of exposure. Worms exposed to the toxigenic dinoflagellate gradually accumulated OA, with toxin levels directly related to the cell density of Prorocentrum cf. lima. Worms fed with Prorocentrum cf. lima exhibited decreased CAT activity, increased LPO levels - both interactively affected by algal species and time - and decreased GSH levels, which were interactively affected by algal species and cell density. Higher LPO levels, along with the inhibition of CAT and GSH, clearly indicated an oxidative stress situation in worms exposed to the toxigenic dinoflagellate. Laeonereis acuta accumulated moderate OA levels and may act as a vector of OA to food webs in estuarine areas under high Prorocentrum cf. lima abundance., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The marine dinoflagellate Vulcanodinium rugosum produces powerful paralyzing and cytotoxic compounds named pinnatoxins (PnTX) and portimines. Even though, no related human intoxication episodes following direct exposure in seawater or the ingestion of contaminated seafood have been documented so far. This study aimed at investigating a dinoflagellate bloom linked to acute dermatitis cases in two recreational beaches in Cienfuegos Bay, Cuba. We used epidemiological and clinical data from 60 dermatitis cases consisting of individuals in close contact with the bloom. Seawater physical-chemical properties were described, and the microorganism causing the bloom was identified by means of light and scanning electron microscopy. Morphological identification was confirmed genetically by sequencing the internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA region. Toxic compounds were identified from a bloom extract using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS), and their concentrations were estimated based on low-resolution tandem mass spectrometry (LC-MS/MS). Sixty people who had prolonged contact with the dinoflagellate bloom suffered acute dermal irritation. Most patients (79.2%) were children and had to be treated with antibiotics; some required >5-day hospitalization. Combined morphological and genetic characters indicated V. rugosum as the causative agent of the bloom. rDNA sequences of the V. rugosum genotype found in the bloom aligned with others from Asia, including material found in the ballast tank of a ship in Florida. The predominant toxins in the bloom were portimine, PnTX-F and PnTX-E, similar to strains originating from the Pacific Ocean. This bloom was associated with unusual weather conditions such as frequent and prolonged droughts. Our findings indicate a close link between the V. rugosum bloom and a dermatitis outbreak among swimmers in Cienfuegos Bay. Phylogenetic evidence suggests a recent introduction of V. rugosum from the Pacific Ocean into Caribbean waters, possibly via ballast water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prorocentrum lima is a cosmopolitan benthic dinoflagellate capable of producing the diarrhetic shellfish toxins (DSTs) okadaic acid (OA) and dinophysistoxin (DTX). These compounds may cause oxidative stress and accumulate in bivalve tissues, which become vectors of intoxication to human consumers. We investigated DST accumulation, detoxification and oxidative stress biomarkers in clams (Anomalocardia flexuosa) experimentally exposed to P. lima cells or their compounds. Experimental diets consisted of 6000 cells mL -1 of the non-toxic chlorophyte Tetraselmis sp. (C; control condition), and combinations of C with 10 P. lima cells mL -1 (T10), 100 P. lima cells mL -1 (T100), or to a toxin concentration of ∼4 μg OA L -1 and ∼0.65 μg DTX-1 L -1 (T100d). Clams were exposed to these diets for 7 days (uptake phase), followed by a 7-day depuration period. No DSTs were detected in clams exposed to treatments C (control) nor to T100d (dissolved compounds) during either uptake or detoxification phase. Conversely, clams exposed to T10 or T100 accumulated, on average, up to 2.5 and 35 μg DST kg -1 in their whole bodies at the end of the uptake phase. These concentrations are ∼64 and ∼4.5 times lower than the regulatory level of 160 μg OA kg -1 , respectively. Accumulated OA quotas were 12-22 times higher in the digestive gland (DG) than in remaining tissues over the uptake phase. Quick toxin transformation was indicated by the early detection of conjugated compounds - DTX-1 and OA esters - in the DG after 6 h of exposure, with OA-ester representing the main compound (30 - 100 %) in that tissue over the experiment. During the depuration period, detoxification rates represented 0.024 h -1 , 0.04 h -1 and 0.052 h -1 for OA, DTX-1 and OA-ester, respectively. The activities of catalase, glutathione S-transferase, glutathione peroxidase and the levels of oxidative stress by lipoperoxidation varied similarly in the DG of A. flexuosa individuals subjected to T100, T100d and the control condition. However, contrasting antioxidant responses were measured in those exposed to T10. These findings indicate that no oxidative stress was primarily induced by DST-producing dinoflagellates in this clam species under laboratory conditions representative of toxic bloom situations. Even though, possible interactions should be considered under multistressor scenarios., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Coolia is a genus of marine benthic dinoflagellates which is widely distributed in tropical and temperate zones. Toxicity has been reported in selected Coolia species, although the identity of causative compounds is still controversial. In this study, we investigated the taxonomical and toxicological aspects of Coolia species from Brazil. Since light- and electron microscopy-based morphology was not enough to distinguish small-celled species, ITS and LSU D1-D3 phylogenetic analyses were used for species definition. Cultures of Coolia palmyrensis and Coolia santacroce were established from samples collected along the northeastern Brazilian coast, the first record of both species in South Atlantic waters. Cultures of Coolia malayensis and Coolia tropicalis were also established and exhibited acute in vivo toxicity to adults of Artemia salina , while C. palmyrensis and C. santacroce were non-toxic. The presence of 30 yessotoxin analogues, 7 metabolites of Coolia and 44 Gambierdiscus metabolites was screened in 14 strains of Coolia . 44-methyl gambierone (formerly referred to as MTX3) and a new isomer of this compound were detected only in C. tropicalis , using both low- and high-resolution LC-MS/MS. To our knowledge, this is the first report of gambierone analogues in dinoflagellates other than Gambierdiscus ; the role of C. tropicalis in ciguatera poisoning thus deserves to be considered in further investigations.

: Recurrent blooms of Ostreopsis cf. ovata have been reported in Brazil and the Mediterranean Sea with associated ecological, and in the latter case, health impacts. Molecular data based on the D1-D3 and D8-D10 regions of the LSU rDNA and ITS loci, and the morphology of O . cf. ovata isolates and field populations from locations along the Brazilian tropical and subtropical coastal regions and three oceanic islands are presented. Additional ITS sequences from three single cells from the tropical coast are provided. Toxin profiles and quantities of PLTX and their analogues; OVTXs; contained in cells from two clonal cultures and two field blooms from Rio de Janeiro were investigated. Morphology was examined using both light and epifluorescence microscopy. Morphometric analysis of different strains and field populations from diverse locations were compared. Molecular analysis showed that six of the seven sequences grouped at the large "Atlantic/Mediterranean/Pacific" sub-clade, while one sequence branched in a sister clade with sequences from Madeira Island and Greece. The toxin profile of strains and bloom field samples from Rio de Janeiro were dominated by OVTX-a and -b, with total cell quotas (31.3 and 39.3 pg cell -1 ) in the range of that previously reported for strains of O . cf. ovata ., Competing Interests: The authors declare no conflict of interest.

On June 2016, a major bloom of Dinophysis acuminata complex was noticed over the coast of Paraná State (PR), southern Brazil, an area unprotected by any official monitoring program. Here we report the results of an extensive sampling effort that ultimately led PR authorities to issue the first State shellfish-harvesting ban due to multi-species okadaic acid (OA) contamination. During its peak, the bloom covered an area of 201 km 2 (∼2.0-3.5 × 54.0 km), attaining unprecedentedly high cell densities along the shallow (<15 m) continental shelf (mean 2.2 × 10 5 , maximum 2.1 × 10 6 cells L -1 ) and adjacent sandy beaches (mean 2.8 × 10 5 , maximum 5.2 × 10 6 cells L -1 ). Only OA was detected in suspension (max. 188 ng L -1 ). Toxin levels measured in bivalves were several times greater than the regulatory limit of 160 ng g -1 , reaching up to 3600 ng g -1 in Crassostrea gasar, by far the highest OA concentrations ever reported in oysters worldwide, 7700 ng g -1 in brown mussels, Perna perna, and lower levels in clams, Anomalocardia brasiliana, and mangrove mussels, Mytella spp. Nine cases of human intoxication were officially reported and five people were hospitalized with typical symptoms of Diarrhetic Shellfish Poisoning linked to the consumption of contaminated bivalves. All bivalves quickly converted most of the OA into its esterified form, DTX-3, and eliminated the toxins only a few weeks following the bloom, with C. gasar being the slowest-detoxifying species. Lower OA levels were accumulated in zooplankton, gastropods and several novel toxin vectors, including benthic organisms such as sand dollars Mellita quinquiesperforata and the ghost-shrimp Callichirus major, which may act as a good indicator of the presence of toxins in sandy beaches, and pelagic fish species that can serve as potential alternative sources of OA to humans (Chaetodipterus faber and Mugil liza). Monitoring toxin contamination in seafood other than bivalves is thus recommended to ensure comprehensive human health protection during massive Dinophysis blooms. Additionally, since OA was also present at low concentrations in the liver of Guiana dolphins Sotalia guianensis and penguins Spheniscus magellanicus, exposure to biotoxins should be considered in conservation actions involving threatened and near-threatened marine organisms in this region., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ostreopsis cf. ovata is a toxic marine benthic dinoflagellate responsible for harmful blooms affecting ecosystem and human health, mostly in the Mediterranean Sea. In this study we report the occurrence of a summer O. cf. ovata bloom in Currais, a coastal archipelago located on the subtropical Brazilian coast (~25° S). This bloom was very similar to Mediterranean episodes in many aspects: (a) field-sampled and cultivated O. cf. ovata cells aligned phylogenetically (ITS and LSU regions) along with Mediterranean strains; (b) the bloom occurred at increasing temperature and irradiance, and decreasing wind speed; (c) cell densities reached up to 8.0 × 10 4 cell cm -2 on fiberglass screen and 5.6 × 10 5 cell g -1 fresh weight on seaweeds; (d) and toxin profiles were composed mostly of ovatoxin-a (58%) and ovatoxin-b (32%), up to 35.5 pg PLTX-eq. cell -1 in total. Mussels were contaminated during the bloom with unsafe toxin levels (up to 131 µg PLTX-eq. kg -1 ). Ostreopsis cells attached to different plastic litter, indicating an alternate route for toxin transfer to marine fauna via ingestion of biofilm-coated plastic debris.

Dinophysis spp. are a major source of diarrheic toxins to marine food webs, especially during blooms. This study documented the occurrence, in late May 2016, of a massive toxic bloom of the Dinophysis acuminata complex along the southern coast of Brazil, associated with an episode of marked salinity stratification. The study tracked the daily vertical distribution of Dinophysis spp. cells and their ciliate prey, Mesodinium cf. rubrum , and quantified the amount of lipophilic toxins present in seston and accumulated by various marine organisms in the food web. The abundance of the D. acuminata complex reached 43 &times; 10⁴ cells&middot;L &minus;1 at 1.0 m depth at the peak of the bloom. Maximum cell densities of cryptophyceans and M. cf. rubrum (>500 &times; 10⁴ and 18 &times; 10⁴ cell&middot;L &minus;1 , respectively) were recorded on the first day of sampling, one week before the peak in abundance of the D. acuminata complex. The diarrheic toxin okadaic acid (OA) was the only toxin detected during the bloom, attaining unprecedented, high concentrations of up to 829 &micro;g&middot;L &minus;1 in seston, and 143 ± 93 pg&middot;cell &minus;1 in individually picked cells of the D. acuminata complex. Suspension-feeders such as the mussel, Perna perna , and barnacle, Megabalanus tintinnabulum , accumulated maximum OA levels (up to 578.4 and 21.9 &micro;g total OA&middot;Kg &minus;1 , respectively) during early bloom stages, whereas predators and detritivores such as Caprellidae amphipods (154.6 &micro;g&middot;Kg &minus;1 ), Stramonita haemastoma gastropods (111.6 &micro;g&middot;Kg &minus;1 ), Pilumnus spinosissimus crabs (33.4 &micro;g&middot;Kg &minus;1 ) and a commercially important species of shrimp, Xiphopenaeus kroyeri (7.2 &micro;g&middot;Kg &minus;1 ), only incorporated OA from mid- to late bloom stages. Conjugated forms of OA were dominant (>70%) in most organisms, except in blenny fish, Hypleurochilus fissicornis , and polychaetes, Pseudonereis palpata (up to 59.3 and 164.6 &micro;g total OA&middot;Kg &minus;1 , respectively), which contained mostly free-OA throughout the bloom. Although algal toxins are only regulated in bivalves during toxic blooms in most countries, including Brazil, this study indicates that human seafood consumers might be exposed to moderate toxin levels from a variety of other vectors during intense toxic outbreaks.

This study evaluated an 8-year dataset (2007 to 2015, except 2008) in the attempt to identify the most susceptible periods for the occurrence of diarrheic shellfish poisoning (DSP) episodes associated with the presence of toxigenic dinoflagellates, Dinophysis spp., in the mussel farming area of Babitonga Bay (southern Brazil). Dinophysis acuminata complex was the most frequent (present in 66% of the samples) and abundant (max. 4100 cells L -1 ) taxon, followed by D. caudata (14%; max. 640 cells L -1 ) and D. tripos (0.9%; max. 50 cells L -1 ). There was a marked onset of the annual rise in Dinophysis spp. abundance during weeks 21-25 (early winter) of each year, followed by a second peak on week 35 (spring). Mussel (Perna perna) samples usually started testing positive in DSP mouse bioassays (MBA) in late winter. Positive results were more frequent in 2007 and 2011 when the mean D. acuminata complex abundance was ~ 500 cells L -1 . Although positive DSP-MBA results were observed in only 11% of the samples during the studied period, the toxin okadaic acid (OA) was present in 90% of the analyzed mussels (max. 264 μg kg -1 ). MBA results were positive when D. acuminata complex cell densities exceed 1200 ± 300 cells L -1 , while trace toxin amounts could be detected at cell densities as low as 150 ± 50 cells L -1 (free OA) to 200 ± 100 cells L -1 (conjugated OA). Low salinity and the meteorological conditions triggered by La Niña events were the main factors associated with both Dinophysis abundance and OA accumulation in mussels.

Pseudo-nitzschia is a diatom genus capable of producing the neurotoxin domoic acid (DA), which has been related to mortalities of marine vertebrates, and the amnesic shellfish poisoning (ASP) in human consumers of contaminated bivalves. This study reports DA production among Pseudo-nitzschia strains isolated from shellfish farming areas in southern Brazil. Twenty-seven cultures of potentially toxigenic Pseudo-nitzschia species were established. Growth, stepped-chain formation, and DA production were evaluated in static, intermittently illuminated (12:12 photoperiod) batch cultures for 12 selected strains, and under continuous light and/or turbulence for a single Pseudo-nitzschia calliantha strain. Growth rate ranged from 0.16 to 0.39 day -1 among the 12 strains. Only P. calliantha and P. cf. multiseries yielded detectable levels of intracellular DA, reaching up to 0.054 fg cell -1 in late exponential and 0.15 pg cell -1 in early stationary phase, respectively. Continuous light impaired cell growth, and turbulence enhanced step-chain formation by threefold during exponential growth phase, but no DA production was detected under both conditions. The effect of turbulence on chain formation should be further evaluated in the field, once particle size is expected to affect the ingestion of toxic cells and thus toxin accumulation by certain DA vectors. The low cell toxicity revealed herein under laboratory conditions is in accordance with the low frequency of DA contamination episodes in south Brazilian waters. Nevertheless, monitoring of Pseudo-nitzschia abundance and DA presence in farming areas is continuously required to assure the quality of local shellfish products.

In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries , static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (μ = 0.45-0.73 d -1 ) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2-4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50-70% reduction in cell density and 70-90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species., Competing Interests: The authors declare no conflict of interest.

Toxigenic Dinophysis spp. are obligate mixotrophic dinoflagellates that require a constant supply of prey-Mesodinium rubrum-to achieve long-term growth by means of kleptoplasty. Mesodinium rubrum is, however, a fast moving, jumping ciliate exhibiting an effective escape response from suspensivorous predators. In the present study, a series of laboratory experiments evaluating the motility and survival of M. rubrum in the presence of Dinophysis cells and/or substances contained in their culture medium was designed, in order to assess the mechanisms involved in prey capture by Dinophysis spp. Cell abundance of M. rubrum decreased in the presence of Dinophysis cf. ovum cells producing okadaic acid (OA; up to 7.94±2.67pgcell -1 ) and smaller amounts of dinophysistoxin-1 (DTX-1) and pectenotoxin-2 (PTX-2). Prey capture was often observed after the ciliate had been attached to adhesive "mucus traps", which only appeared in the presence of Dinophysis cells. Before being attached to the mucus traps, M. rubrum cells reduced significantly their swimming frequency (from ∼41 to 19±3 jumps min -1 ) after only 4h of initial contact with D. cf. ovum cells. M. rubrum survival was not affected in contact with purified OA, DTX-1 and PTX-2 solutions, but decreased significantly when the ciliate was exposed to cell-free or filtered culture medium from both D. cf. ovum and D. caudata, the latter containing moderate concentrations of free eicosapentaenoic acid and docosahexaenoic acid. The results thus indicate that Dinophysis combines the release of toxic compounds other than shellfish toxins, possibly free PUFAs, and a "mucus trap" to enhance its prey capture success by immobilizing and subsequently arresting M. rubrum cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study investigates the occurrence of diarrhetic shellfish toxins (DSTs) and their producing phytoplankton species in southern Brazil, as well as the potential for toxin accumulation in co-occurring mussels (Perna perna) and octopuses (Octopus vulgaris). During the spring in 2012 and 2013, cells of Dinophysis acuminata complex were always present, sometimes at relatively high abundances (max. 1143 cells L-1), likely the main source of okadaic acid (OA) in the plankton (max. 34 ng L-1). Dinophysis caudata occurred at lower cell densities in 2013 when the lipophilic toxins pectenotoxin-2 (PTX-2) and PTX-2 seco acid were detected in plankton and mussel samples. Here, we report for the first time the accumulation of DSTs in octopuses, probably linked to the consumption of contaminated bivalves. Perna perna mussels were consistently contaminated with different DSTs (max. 42 µg kg-1), and all octopuses analyzed (n = 5) accumulated OA in different organs/tissues: digestive glands (DGs) > arms > gills > kidneys > stomach + intestine. Additionally, similar concentrations of 7-O-palmytoyl OA and 7-O-palmytoly dinophysistoxin-1 (DTX-1) were frequently detected in the hepatopancreas of P. perna and DGs of O. vulgaris. Therefore, octopuses can be considered a potential vector of DSTs to both humans and top predators such as marine mammals.

Multiple species in the toxic marine diatom genus Pseudo-nitzschia have been identified in the Northwestern Atlantic region encompassing the Gulf of Maine (GOM), including the Bay of Fundy (BOF). To gain further knowledge of the taxonomic composition and toxicity of species in this region, Pseudo-nitzschia isolates (n=146) were isolated from samples collected during research cruises that provided broad spatial coverage across the GOM and the southern New England shelf, herein referred to as the GOM region, during 2007-2008. Isolates, and cells in field material collected at 38 stations, were identified using electron microscopy (EM). Eight species ( P. americana, P. fraudulenta, P. subpacifica, P. heimii, P. pungens, P. seriata, P. delicatissima and P. turgidula ), and a novel form, Pseudo-nitzschia sp. GOM, were identified. Species identity was confirmed by sequencing the large subunit of the ribosomal rDNA (28S) and the internal transcribed spacer 2 (ITS2) for six species (36 isolates). Phylogenetic analyses (including neighbor joining, maximum parsimony, and maximum likelihood estimates and ITS2 secondary structure analysis) and morphometric data supported the placement of P . sp. GOM in a novel clade that includes morphologically and genetically similar isolates from Australia and Spain and is genetically most similar to P. pseudodelicatissima and P. cuspidata . Seven species (46 isolates) were grown in nutrient-replete batch culture and aliquots consisting of cells and growth medium were screened by Biosense ASP ELISA to measure total domoic acid (DA) produced (intracellular + extracellular); P. americana and P. heimii were excluded from all toxin analyses as they did not persist in culture long enough for testing. All 46 isolates screened produced DA in culture and total DA varied among species (e.g., 0.04 to 320 ng ml -1 for P. pungens and P . sp. GOM isolates, respectively) and among isolates of the same species (e.g., 0.24 - 320 ng ml -1 for P . sp. GOM). The 15 most toxic isolates corresponded to P. seriata, P . sp. GOM and P. pungens , and fg DA cell -1 was determined for whole cultures (cells and medium) using ELISA and liquid chromatography (LC) with fluorescence detection (FLD); for seven isolates, toxin levels were also estimated using LC - with mass spectrometry and ultraviolet absorbance detection. Pseudo-nitzschia seriata was the most toxic species (up to 3,500 fg cell -1 ) and was observed in the GOM region during all cruises (i.e., during the months of April, May, June and October). Pseudo-nitzschia sp. GOM, observed only during September and October 2007, was less toxic (19 - 380 fg cell -1 ) than P. seriata but more toxic than P. pungens var. pungens (0. 4 fg cell -1 ). Quantitation of DA indicated that concentrations measured by LC and ELISA were positively and significantly correlated; the lower detection limit of the ELISA permitted quantification of toxicity in isolates that were found to be nontoxic with LC methods. The confirmation of at least seven toxic species and the broad spatial and temporal distribution of toxic Pseudo-nitzschia spp. have significant implications for the regional management of nearshore and offshore shellfisheries resources.

Toxin accumulation by suspension-feeding qualifier depends on a balance between processes regulating toxin uptake (i.e. ingestion and absorption of toxic cells) and elimination (i.e. egestion, exchange among tissues, excretion, degradation and/or biotransformation) during exposure to toxic blooms. This laboratory study compares the size-specific uptake and elimination kinetics of domoic acid (DA) from Pseudo-nitzschia multiseries in two co-occurring bivalves, the oyster Crassostrea virginica and the mussel Mytilus edulis. Domoic acid concentrations were measured in visceral and non-visceral tissues of different-sized oysters and mussels during simultaneous long-term exposure to toxic P. multiseries cells in the laboratory, followed by depuration on a non-toxic algal diet. Mussels attained 7-17-fold higher DA concentrations than oysters, depending on the body size and exposure time, and also detoxified DA at higher rates (1.4-1.6 d(-1)) than oysters (0.25-0.88 d(-1)) of a comparable size. Small oysters attained markedly higher weight-specific DA concentrations (maximum=78.6 μg g(-1)) than large, market-sized individuals (≤ 13 μg g(-1)), but no clear relationship was found between body size and DA concentration in mussels (maximum=460 μg g(-1)). Therefore, differential DA accumulation by the two species was, on average, approximately 3-fold more pronounced for large bivalves. An inverse relationship between DA elimination rate and body size was established for oysters but not mussels. Elimination of DA was faster in viscera than in other tissues of both bivalves; DA exchange rate from the former to the latter was higher in oysters. The contribution of viscera to the total DA burden of mussels was consistently greater than that of other tissues during both uptake (>80%) and depuration (>65%) phases, whereas it rapidly decreased from 70-80% to 30-40% in oysters, and this occurred faster in smaller individuals. Residual DA concentrations (≤ 0.25 μg g(-1)) were detected at later depuration stages (up to 14 d), mainly in viscera of oysters and non-visceral tissues of mussels, suggesting that a second, slower-detoxifying toxin compartment exists in both species. However, a simple exponential decay model was found to adequately describe DA elimination kinetics in these bivalves. The lower capacity for DA accumulation in oysters compared to mussels can thus only be explained by the former's comparatively low toxin intake rather than faster toxin elimination., (2010. Published by Elsevier B.V. All rights reserved.)

The neurotoxin domoic acid (DA), produced by diatoms Pseudo-nitzschia spp., is transferred to humans via consumption of contaminated bivalves. This study examines feeding mechanisms, namely reduced filtration, pre-ingestive rejection and poor absorption, that might explain the comparatively low DA levels commonly found in oysters during toxic Pseudo-nitzschia blooms. Clearance rate (CR), absorption efficiency (AE) of organic matter and selective rejection in pseudofeces of oysters (Crassostrea virginica) and mussels (Mytilus edulis) were investigated in relation to the DA levels accumulated during 2-wk, simultaneous exposure to toxic Pseudo-nitzschia multiseries. Effects of temperature and P. multiseries cell size were also tested to identify conditions, if any, under which oysters can accumulate unsafe DA levels. Oysters accumulated 3.0-7.5x less DA than mussels from a short-celled P. multiseries clone (length=24microm) at 12 degrees C. This was related to the 7.4-8.5x lower CRs determined for oysters relative to mussels at this temperature. Exposure to a longer-celled P. multiseries clone (81microm) resulted in up to 70x lower toxin levels in oysters compared to mussels, which was attributed to differential feeding selectivity. Mussels were unable to discriminate between long- and short-celled P. multiseries clones from a mixed suspension, whereas oysters were previously shown to preferentially reject long cells (>70microm) in pseudofeces. Both bivalves selectively rejected P. multiseries cells from mixed suspensions containing a flagellate but not another diatom. AE of organics from P. multiseries cells by oysters and mussels was comparably low (42 and 39%, respectively) and thus unlikely to explain their differential DA accumulation. CR and DA uptake by oysters were negligible at

Quantitation of trace levels of domoic acid (DA) in seawater samples usually requires labour-intensive protocols involving chemical derivatization with 9-fluorenylmethylchloroformate and liquid chromatography with fluorescence detection (FMOC-LC-FLD). Procedures based on LC-MS have been published, but time-consuming and costly solid-phase extraction pre-concentration steps are required to achieve suitable detection limits. This paper describes an alternative, simple and inexpensive LC method with ultraviolet detection (LC-UVD) for the routine analysis of trace levels of DA in seawater without the use of sample pre-concentration or derivatization steps. Qualitative confirmation of DA identity in dubious samples can be achieved by mass spectrometry (LC-MS) using the same chromatographic conditions. Addition of an ion-pairing/acidifying agent (0.15% trifluoroacetic acid) to sample extracts and the use of a gradient elution permitted the direct analysis of large sample volumes (100 microl), resulting in both high selectivity and sensitivity (limit of detection=42 pg ml(-1) by LC-UVD and 15 pg ml(-1) by LC-MS). Same-day precision varied between 0.4 and 5%, depending on the detection method and DA concentration. Mean recoveries of spiked DA in seawater by LC-UVD were 98.8% at 0.1-10 ng ml(-1) and 99.8% at 50-1000 ng ml(-1). LC-UVD exhibited strong correlation with FMOC-LC-FLD during inter-laboratory analysis of Pseudo-nitzschia multiseries cultures containing 60-2000 ng DA ml(-1) (r(2)>0.99), but more variable results were obtained by LC-MS (r(2)=0.85). This new technique was used to confirm the presence of trace DA levels in low-toxicity Pseudo-nitzschia spp. isolates (0.2-1.6 ng ml(-1)) and in whole-water field samples (0.3-5.8 ng ml(-1)), even in the absence of detectable Pseudo-nitzschia spp. cells in the water column.