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1.
Aquat Toxicol ; 218: 105371, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31790939

RESUMO

Harmful algal bloom (HAB) toxins have severe negative impacts on marine mammals, particularly for Florida bottlenose dolphins (Tursiops truncatus) which frequently experience mass mortality events. Dolphins on the Florida Atlantic coast inhabit a region endemic to two HAB species, Karenia brevis and Pyrodinium bahamense, which produce the neurotoxins brevetoxin (PbTx) and saxitoxin (STX), respectively. Although toxic HABs and associated dolphin mortality events have been reported from this region, there is a lack of available data necessary for comparing toxin exposure levels between bloom ('exposed') conditions and non-bloom ('baseline') conditions. Here we present a 10-year dataset of PbTx and STX concentrations detected in dolphins stranding in this region, and compare the toxin loads from HAB-exposed dolphins to those detected in dolphins recovered in the absence of a HAB. We analyzed liver tissue samples from dead-stranded dolphins (n = 119) recovered and necropsied between 2002-2011, using an enzyme-linked immunosorbent assay (ELISA) modified for use with mammalian tissues. For dolphins recovered during baseline conditions, toxin-positive samples ranged in concentration from 0.27 to 1.2 ng/g for PbTx and from 0.41 to 1.9 ng/g for STX. For K. brevis-exposed dolphins, concentrations of up to 12.1 ng PbTx/g were detected, and for P. bahamense-exposed dolphins, concentrations of up to 9.9 ng STX/g were detected. Baseline PbTx values were similar to those reported in other regions where K. brevis blooms are more frequent and severe, but HAB-exposed PbTx values were considerably lower relative to these other regions. Since no baseline STX dolphin data exist for any region, our data serve as a first step towards establishing reference STX values for potential dolphin mortality events associated with STX-producing blooms in the future. This study demonstrates that although HABs in eastern Florida are only infrequently associated with dolphin mortalities, the presence of toxins in these animals may pose significant health risks in this region.


Assuntos
Golfinho Nariz-de-Garrafa/metabolismo , Monitoramento Ambiental/métodos , Proliferação Nociva de Algas , Toxinas Marinhas/análise , Oxocinas/análise , Saxitoxina/análise , Poluentes Químicos da Água/análise , Animais , Dinoflagelados/crescimento & desenvolvimento , Ensaio de Imunoadsorção Enzimática , Florida , Fígado/química , Fígado/metabolismo , Toxinas Marinhas/toxicidade , Oxocinas/toxicidade , Rios/química , Saxitoxina/toxicidade , Poluentes Químicos da Água/toxicidade
2.
Environ Pollut ; 249: 171-180, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30884396

RESUMO

Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.


Assuntos
Toxinas Marinhas/análise , Frutos do Mar/análise , Poluentes Químicos da Água/análise , Animais , Bivalves/química , China , Cromatografia Líquida/métodos , Dinoflagelados , Furanos/análise , Compostos Heterocíclicos com 3 Anéis/análise , Hidrocarbonetos Cíclicos/análise , Iminas/análise , Ácido Okadáico/análise , Ostreidae/química , Oxocinas/análise , Piranos/análise , Alimentos Marinhos/análise , Frutos do Mar/estatística & dados numéricos , Compostos de Espiro/análise , Espectrometria de Massas em Tandem
3.
Harmful Algae ; 80: 80-87, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30502815

RESUMO

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.


Assuntos
Ciguatoxinas/análise , Monitoramento Ambiental/métodos , Peixes/metabolismo , Toxinas Marinhas/análise , Oxocinas/análise , Animais , Cromatografia Líquida , Ciguatoxinas/metabolismo , Espectrometria de Massas em Tandem
4.
Mar Drugs ; 16(12)2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30545061

RESUMO

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.


Assuntos
Alternativas aos Testes com Animais/métodos , Bioensaio/métodos , Toxinas Marinhas/análise , Intoxicação por Frutos do Mar/prevenção & controle , Frutos do Mar/análise , Alternativas aos Testes com Animais/instrumentação , Animais , Bioensaio/instrumentação , Bivalves , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Reações Falso-Positivas , Toxinas Marinhas/toxicidade , Camundongos , Oxocinas/análise , Oxocinas/toxicidade , Frutos do Mar/toxicidade , Intoxicação por Frutos do Mar/etiologia , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
5.
Harmful Algae ; 78: 9-17, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30196929

RESUMO

The marine dinoflagellate Lingulodinium polyedra is a toxigenic species capable of forming high magnitude and occasionally harmful algal blooms (HABs), particularly in temperate coastal waters throughout the world. Three cultured isolates of L. polyedra from a fjord system on the Skagerrak coast of Sweden were analyzed for their growth characteristics and to determine the effects of a strong salinity gradient on toxin cell quotas and composition. The cell quota of yessotoxin (YTX) analogs, as determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), ranged widely among strains. For two strains, the total toxin content remained constant over time in culture, but for the third strain, the YTX cell quota significantly decreased (by 32%) during stationary growth phase. The toxin profiles of the three strains differed markedly and none produced YTX. The analog 41a-homo-YTX (m/z 1155), its putative methylated derivative 9-Me-41a-homo-YTX (m/z 1169) and an unspecified keto-YTX (m/z 1047) were detected in strain LP29-10H, whereas strain LP30-7B contained nor-YTX (m/z 1101), and two unspecified YTX analogs at m/z 1159 and m/z 1061. The toxin profile of strain LP30-8D comprised two unspecified YTX analogs at m/z 1061 and m/z 991 and carboxy-YTX (m/z 1173). Strain LP30-7B cultured at multiple salinities (10, 16, 22, 28 and 34) did not tolerate the lowest salinity (10), but there was a statistically significant decrease (by 21%) in toxin cell quota between growth at the highest versus lower permissible salinities. The toxin profile for strain LP30-7B remained constant over time for a given salinity. At lower salinities, however, the proportion of the unspecified YTX analog (m/z 1061) was significantly higher, especially with respect to nor-YTX (m/z 1101). This study shows high intra-specific variability in yessotoxin composition among strains from the same geographical region and inconsistency in toxin cell quota under different environmental regimes and growth stages in culture. This variation has important implications for the kinetics of YTX production and food web transfer in natural bloom populations from diverse geographical regions.


Assuntos
Dinoflagelados/química , Dinoflagelados/crescimento & desenvolvimento , Toxinas Marinhas/análise , Oxocinas/análise , Salinidade , Água do Mar/química , Cromatografia Líquida , Estuários , Suécia , Espectrometria de Massas em Tandem
6.
Talanta ; 187: 47-58, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29853065

RESUMO

To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) respectively, as well as the structurally different yessotoxin (YTX), we developed a novel surface-enhanced micro-Raman scattering (micro-SERS) approach to investigate for the first time their micro-SERS signalling in solution and jointly analysed them in conjunction with the normal and toxic mussel tissue. YTX provided the main SERS feature surprisingly similar to DTX-1 and DTX-2, suggesting similar molecular adsorption mechanism with respect to the AgNPs. A fingerprint SERS band at 1017 cm-1 characteristic for the C-CH3 stretching in DTX-1 and DTX-2 and absent in OA SERS signal, allowed direct SERS discrimination of DTX-1,2 from OA. In acid form or as dissolved potassium salt, OA showed reproducible SERS feature for 0.81 µM to 84.6 nM concentrations respectively, while its ammonium salt slightly changed the overall SERS signature. The inherently strong fluorescence of the shellfish tissue, which hampers Raman spectroscopy analysis, further increases when toxins are present in tissue. Through SERS, tissue fluorescence is partially quenched. Artificially intoxicated mussel tissue with DSP toxins and incubated with AgNPs allowed direct SERS evidence of the toxin presence, opening a novel avenue for the in situ shellfish tracking and warning via micro-SERS. Natural toxic tissue containing 57.91 µg kg-1 YTX (LC-MS confirmed) was micro-SERS assessed to validate the new algorithm for toxins detection. We showed that a portable Raman system was able to reproduce the lab-based SERS results, being suitable for in situ raw seafood screening. The new approach provides an attractive, faster, effective and low-cost alternative for seafood screening, with economic, touristic and sustainable impact in aquaculture, fisheries, seafood industry and consumer trust.


Assuntos
Bivalves/química , Toxinas Marinhas/análise , Ácido Okadáico/análise , Oxocinas/análise , Piranos/análise , Animais , Interações Hidrofóbicas e Hidrofílicas , Análise Espectral Raman , Propriedades de Superfície
7.
Toxins (Basel) ; 10(2)2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29419743

RESUMO

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 108 L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14-263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Toxinas Marinhas/imunologia , Oxocinas/imunologia , Animais , Antígenos/análise , Linhagem Celular , Coloides , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Contaminação de Alimentos/análise , Ouro , Hibridomas , Toxinas Marinhas/análise , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Oxocinas/análise , Alimentos Marinhos/análise , Soroalbumina Bovina/imunologia , Frutos do Mar/análise
8.
Toxicon ; 139: 31-40, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28970035

RESUMO

This paper reports a toxic strain of Protoceratium reticulatum, its morphology, phylogeny, yessotoxins (YTXs) production and abundance in northern Yellow Sea of China from 2011 to 2015 was investigated. YTXs in hepatopancreas and edible parts of bottom sowing cultured Japanese scallop Patinopecten yessoensis in this sea area were determined weekly for 5 years. Other potential producers of YTXs, Gonyaulax spinifera and Lingulodinium polyedrum, were also investigated. Results revealed that Protoceratium reticulatum strain from the northern Yellow Sea belongs to a geographically widely distributed species. Motile cells of Protoceratium reticulatum contribute to YTXs in Japanese scallop, and G. spinifera may also be a potential contributor. Resting cysts of Protoceratium reticulatum, G. spinifera, and L. polyedrum in sediments were possibly important origins of YTXs in scallop cultured at sea bottom. YTXs in scallop decreased from 2011 to 2015, most toxins were concentrated in hepatopancreas, while a small portion in edible parts which was safe for consumption the whole year around.


Assuntos
Dinoflagelados/classificação , Oxocinas/análise , Pectinidae/química , Animais , Aquicultura , China , Dinoflagelados/química , Dinoflagelados/citologia , Contaminação de Alimentos/análise , Hepatopâncreas/química , Toxinas Marinhas/análise , Estações do Ano
9.
Rapid Commun Mass Spectrom ; 31(17): 1453-1461, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28582796

RESUMO

RATIONALE: Accurate quantitative analysis of lipophilic toxins by liquid chromatography/mass spectrometry (LC/MS) requires calibration solution reference materials (RMs) for individual toxin analogs. Untargeted analysis is aimed at identifying a vast number of compounds and thus validation of fully quantitative untargeted methods is not feasible. However, a semi-quantitative approach allowing for profiling is still required and will be strengthened by knowledge of the relative molar response (RMR) of analogs in LC/MS with electrospray ionization (ESI). METHODS: RMR factors were evaluated for toxins from the okadaic acid (OA/DTXs), yessotoxin (YTX), pectenotoxin (PTX), azaspiracid (AZA) and cyclic imine (CI) toxin groups, in both solvent standards and environmental sample extracts. Since compound ionization and fragmentation influences the MS response of toxins, RMRs were assessed under different chromatographic conditions (gradient, isocratic) and MS acquisition modes (SIM, SRM, All-ion, target MS/MS) on low- and high-resolution mass spectrometers. RESULTS: In general, RMRs were not significantly impacted by chromatographic conditions (isocratic vs gradient), with the exception of DTX1. MS acquisition modes had a more significant impact, with PnTX-G and SPX differing notably. For a given toxin group, response factors were generally in the range of 0.5 to 2. The cyclic imines were an exception. CONCLUSIONS: Differences in RMRs between toxins of a same chemical base structure were not significant enough to indicate major issues for non-targeted semi-quantitative analysis, where there is limited or no availability of standards for many compounds, and where high degrees of accuracy are not required. Differences in RMRs should be considered when developing methods that use a standard of a single analogue to quantitate other toxins from the same group.


Assuntos
Cromatografia Líquida/métodos , Toxinas Marinhas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida/normas , Proliferação Nociva de Algas , Toxinas Marinhas/química , Ácido Okadáico/análise , Oxocinas/análise , Oxocinas/química , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray/normas , Compostos de Espiro/análise , Compostos de Espiro/química
10.
Chemosphere ; 183: 380-388, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28554022

RESUMO

Some dinoflagellates can produce lipophilic marine toxins, which pose potent threats to seafood consumers. In the Bohai Sea, an important semi-closed inland sea with intensive mariculture industry in China, there is little knowledge concerning lipophilic marine toxins and their potential threats. In this study, net-concentrated phytoplankton samples were periodically collected from 5 typical mariculture zones around the Bohai Sea, including Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD) in 2013 and 2014, and a method using high performance liquid chromatography (HPLC) coupled with a Q-Trap mass spectrometer was applied to analyze seven representative lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), azaspiracid-1 (AZA1), gymnodimine (GYM), and 13-desmethyl spirolide C (desMeC). The method had high sensitivity and repeatability, and exhibited satisfactory recoveries for most of the lipophilic marine toxins (92.1-108%) except for AZA1 (65.8-68.9%). Nearly all the lipophilic marine toxins could be detected in phytoplankton samples from the Bohai Sea. OA, DTX1 and PTX2 were predominant components and present in most of the phytoplankton samples. The maximum content of lipophilic marine toxin in phytoplankton samples concentrated from seawater (OA 464 pg L-1; DTX1 783 pg L-1; YTX 86.6 pg L-1; desMeC 15.6 pg L-1; PTX2 1.11 × 103 pg L-1) appeared in June 2014. Based on toxins present in phytoplankton samples, it is implied that seafood in the Bohai Sea is more likely to be contaminated by OA group and PTX group toxins, and spring is the high-risk season for toxin contamination.


Assuntos
Dinoflagelados/química , Toxinas Marinhas/análise , Fitoplâncton/química , Alimentos Marinhos/normas , Animais , China , Cromatografia Líquida de Alta Pressão/métodos , Furanos/análise , Compostos Heterocíclicos com 3 Anéis/análise , Hidrocarbonetos Cíclicos/análise , Interações Hidrofóbicas e Hidrofílicas , Iminas/análise , Ácido Okadáico/análise , Oxocinas/análise , Piranos/análise , Alimentos Marinhos/análise , Compostos de Espiro/análise , Espectrometria de Massas em Tandem
11.
Harmful Algae ; 63: 173-183, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28366392

RESUMO

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.


Assuntos
Bioensaio/métodos , Dinoflagelados/metabolismo , Toxinas Marinhas/análise , Animais , Intoxicação por Ciguatera , Ciguatoxinas/análise , Ciguatoxinas/toxicidade , Eritrócitos/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Oxocinas/análise , Oxocinas/toxicidade , Filogenia
12.
Anal Bioanal Chem ; 409(1): 95-106, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27830316

RESUMO

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.


Assuntos
Cromatografia Líquida/métodos , Toxinas Marinhas/análise , Espectrometria de Massas/métodos , Mytilus edulis/química , Alimentos Marinhos/análise , Animais , Liofilização , Furanos/análise , Ácido Caínico/análogos & derivados , Ácido Caínico/análise , Ácido Okadáico/análise , Oxocinas/análise , Piranos/análise , Padrões de Referência , Compostos de Espiro/análise
14.
Aquat Toxicol ; 180: 131-140, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27716578

RESUMO

Blooms of Karenia brevis (also called red tides) occur almost annually in the Gulf of Mexico. The health effects of the neurotoxins (i.e., brevetoxins) produced by this toxic dinoflagellate on marine turtles are poorly understood. Florida's Gulf Coast represents an important foraging and nesting area for a number of marine turtle species. Most studies investigating brevetoxin exposure in marine turtles thus far focus on dead and/or stranded individuals and rarely examine the effects in apparently "healthy" free-ranging individuals. From May-July 2014, one year after the last red tide bloom, we collected blood from nesting loggerhead sea turtles (Caretta caretta) on Casey Key, Florida USA. These organisms show both strong nesting and foraging site fidelity. The plasma was analyzed for brevetoxin concentrations in addition to a number of health and immune-related parameters in an effort to establish sublethal effects of this toxin. Lastly, from July-September 2014, we collected unhatched eggs and liver and yolk sacs from dead-in-nest hatchlings from nests laid by the sampled females and tested these samples for brevetoxin concentrations to determine maternal transfer and effects on reproductive success. Using a competitive enzyme-linked immunosorbent assay (ELISA), all plasma samples from nesting females tested positive for brevetoxin (reported as ng brevetoxin-3[PbTx-3] equivalents [eq]/mL) exposure (2.1-26.7ng PbTx-3eq/mL). Additionally, 100% of livers (1.4-13.3ng PbTx-3eq/mL) and yolk sacs (1.7-6.6ng PbTx-3eq/mL) from dead-in-nest hatchlings and 70% of eggs (<1.0-24.4ng PbTx-3eq/mL) tested positive for brevetoxin exposure with the ELISA. We found that plasma brevetoxin concentrations determined by an ELISA in nesting females positively correlated with gamma-globulins, indicating a potential for immunomodulation as a result of brevetoxin exposure. While the sample sizes were small, we also found that plasma brevetoxin concentrations determined by an ELISA in nesting females significantly correlated with liver brevetoxin concentrations of dead-in-nest hatchlings and that brevetoxins could be related to a decreased reproductive success in this species. This study suggests that brevetoxins can still elicit negative effects on marine life long after a bloom has dissipated. These results improve our understanding of maternal transfer and sublethal effects of brevetoxin exposure in marine turtles.


Assuntos
Toxinas Marinhas/toxicidade , Oxocinas/toxicidade , Reprodução/efeitos dos fármacos , Tartarugas/metabolismo , Poluentes da Água/toxicidade , Animais , Cromatografia Líquida de Alta Pressão , Dinoflagelados/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Exposição Ambiental , Ensaio de Imunoadsorção Enzimática , Feminino , Florida , Proliferação Nociva de Algas , Imunidade Inata/efeitos dos fármacos , Toxinas Marinhas/análise , Toxinas Marinhas/sangue , Óvulo/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxocinas/análise , Oxocinas/sangue , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas em Tandem , Tartarugas/crescimento & desenvolvimento , Saco Vitelino/metabolismo
15.
Anal Chim Acta ; 887: 67-74, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26320787

RESUMO

A new signal amplification strategy based on mesoporous carbon-enriched palladium nanostructure (MSC-PdNS) was designed for enzyme-free electrochemical immunoassay of brevetoxin B (BTB) in marine toxins. The assay was carried out on a BTB-bovine serum albumin-functionalized electrode by using monoclonal mouse anti-BTB-labeling MSC-PdNS as the signal-transduction tag. A competitive-type assay protocol was successfully introduced to develop a high-efficiency enzyme-free immunoassay accompanying the doped palladium nanostructure into MSC-PdNS toward reduction of H2O2. Under the optimal conditions, the catalytic current decreased with the increment of BTB concentration in the range from 0.01 to 10 ng mL(-1) with a detection limit (LOD) of 5.0 pg mL(-1) BTB at the 3s(blank) criterion. The selectivity and precision were acceptable. In addition, the methodology was further validated for assaying spiked seafood samples, and consistent results between the electrochemical immunoassay and the referenced enzyme immunoassay were obtained. Importantly, the enzyme-free electrochemical immunoassay provides a promising approach for rapid screening of marine toxin because of its simplicity, low cost, sensitivity, specificity and without the need of sample pretreatment.


Assuntos
Carbono/química , Técnicas Eletroquímicas/métodos , Toxinas Marinhas/análise , Mytilidae/química , Nanoestruturas/química , Oxocinas/análise , Paládio/química , Alimentos Marinhos/análise , Animais , Anticorpos Imobilizados/química , Bovinos , Eletrodos , Peróxido de Hidrogênio/química , Proteínas Imobilizadas/química , Imunoensaio/métodos , Limite de Detecção , Camundongos , Nanoestruturas/ultraestrutura , Oxirredução , Porosidade , Soroalbumina Bovina/química
16.
Methods Mol Biol ; 1308: 277-97, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26108513

RESUMO

Different clinical types of algae-related poisoning have attracted scientific and commercial attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, mainly due to the low sensitivity and the absence of specialized variations. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins. This chapter describes state-of-the-art LC-MS/MS methods for the detection and quantitation of different classes of phycotoxins in shellfish matrices. These classes include the highly hydrophilic paralytic shellfish poisoning (PSP) toxins. Hydrophilic interaction liquid chromatography (HILIC) has been shown to be useful in the separation of PSP toxins and is described in detail within this chapter. Another important class of phycotoxins is diarrhetic shellfish poisoning (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs), and yessotoxins (YTXs). The most recently described shellfish poisoning syndrome, azaspiracid shellfish poisoning (AZP) is caused by azaspiracids, which in turn are diarrhetic, but usually are treated separately as AZP. The last group of regulated shellfish toxins is the amnesic shellfish poisoning (ASP) toxin domoic acid, produced by species of the genus Pseudo-nitzschia.


Assuntos
Toxinas Marinhas/análise , Frutos do Mar/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Ácido Caínico/análogos & derivados , Ácido Caínico/análise , Ácido Okadáico/análise , Oxocinas/análise , Piranos/análise , Compostos de Espiro/análise
17.
Biosens Bioelectron ; 72: 10-7, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25951085

RESUMO

Saxitoxin (STX) and brevetoxin (PbTX-2), which are produced by marine dinoflagellates, are highly-toxic marine toxins targeting separate sites of the α subunit of voltage-dependent sodium channels (VDSCs). In this work, a portable cardiomyocyte-based potential biosensor is designed for rapid detection of STX and PbTX-2. This potential biosensor is constructed by cardiomyocyte and microelectrode array (MEA) with a label-free and real-time wireless 8-channel recording system which can dynamically monitor the multisite electrical activity of cardiomyocyte network. The recording signal parameters, spike amplitude, firing rate and 50% of spike potential duration (SPD50) extracted from extracelluar field potential (EFP) signals of the potential biosensor is analyzed to quantitatively evaluate toxicological risk of STX and PbTX-2. Firing rate of biosensor signals presents high sensitivity to STX with the detection limit of 0.35 ng/ml within 5 min. SPD50 shows high sensitivity to PbTX-2 with the detection limit of 1.55 ng/ml within 5 min. Based on the multi-parameter analysis, cardiomyocyte-based potential biosensor will be a promising tool for rapid detection of these two toxins.


Assuntos
Técnicas Biossensoriais/instrumentação , Dinoflagelados/química , Toxinas Marinhas/análise , Miócitos Cardíacos/citologia , Oxocinas/análise , Saxitoxina/análise , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Desenho de Equipamento , Limite de Detecção , Toxinas Marinhas/metabolismo , Microeletrodos , Miócitos Cardíacos/efeitos dos fármacos , Oxocinas/metabolismo , Ratos Sprague-Dawley , Saxitoxina/metabolismo
18.
Toxicon ; 102: 32-42, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26003796

RESUMO

The presence of phytoplankton responsible for the production of lipophilic marine biotoxins is well recognised throughout parts of South America. To date, the quantitation of lipophilic toxins in Argentinean shellfish has been limited to select and highly focussed geographical studies. This work reports the analysis for lipophilic marine biotoxins in shellfish harvested across five regions of Argentina between 1992 and 2012. LC-MS/MS analysis was used for the quantitation of all regulated lipophilic toxins. High concentrations of okadaic acid group toxins were quantified, with a clear dominance of the parent okadaic acid and more than 90% of the toxin present as esters. Results showed DSP toxins in shellfish from the Buenos Aires Province during 2006 and 2007, earlier than previously described. There was also strong evidence linking the presence of okadaic acid to human intoxications. Other lipophilic toxins detected were yessotoxin, pectenotoxin-2 and 13-desMeC spirolide. With evidence published recently for the presence of azaspiracid producers, this work reports the detection of low concentrations of azaspiracid-2 in shellfish. As such the data provides the first published evidence for yessotoxins and azaspiracids in Argentinean shellfish and further evidence for the continuing presence of lipophilic marine toxins in Argentinean waters.


Assuntos
Contaminação de Alimentos/análise , Toxinas Marinhas/análise , Ácido Okadáico/análise , Fitoplâncton/química , Frutos do Mar/análise , Animais , Argentina , Cromatografia Líquida , Furanos/análise , Humanos , Oxocinas/análise , Piranos/análise , Intoxicação por Frutos do Mar , Compostos de Espiro/análise , Espectrometria de Massas em Tandem
19.
Biosens Bioelectron ; 69: 148-54, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25725463

RESUMO

Brevetoxins (BTXs) are very potent marine neurotoxins that increased in geographical distribution in the past decade causing the illness clinically described as neurological shellfish poisoning (NSP). The ethical problems as well as the technical difficulties associated with the currently employed analysis methods for marine toxins are encouraging the research for suitable alternatives to be applied in a regulatory monitoring regime. Here, we report an electrochemical biosensor platform for BTX-2 detection utilising aptamer as specific receptor. Using in vitro selection, high affinity DNA aptamers to BTX-2 were successfully selected for the first time from a large pool of random sequences. The binding of BTX-2 to aptamer pools/clones was monitored using fluorescence and electrochemical impedance spectroscopy (EIS). The aptamer BT10 exhibited the highest binding affinity to BTX-2, with a dissociation constant of 42nM. The effects of the incubation time, pH and metal ions concentrations on the aptamer-toxin binding were studied. The aptamer BT10 was used to construct a label-free competitive impedimetric biosensor for BTX-2 achieving a detection limit of 106pg/ml. We observed a high degree of cross reactivity of the selected aptamer to the two similar congeners, BTX-2 and -3, whereas no cross reactivity to other marine toxins was obtained. Moreover, the aptasensor was applied for the detection of BTX-2 in spiked shellfish extract showing a very high recovery percentage. We believe that the proposed aptasensor will facilitate the routine detection of BTX-2 in food samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Espectroscopia Dielétrica/instrumentação , Análise de Alimentos/instrumentação , Toxinas Marinhas/análise , Oxocinas/análise , Frutos do Mar/análise , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Contaminação de Alimentos/análise , Toxinas Marinhas/química , Oxocinas/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
20.
Mar Drugs ; 13(4): 1666-87, 2015 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-25815891

RESUMO

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.


Assuntos
Bivalves/química , Contaminação de Alimentos , Inspeção de Alimentos/métodos , Venenos de Moluscos/análise , Neurônios/efeitos dos fármacos , Frutos do Mar/análise , Animais , Oceano Atlântico , Bivalves/crescimento & desenvolvimento , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Estrutura Molecular , Venenos de Moluscos/química , Venenos de Moluscos/toxicidade , Neurônios/citologia , Ácido Okadáico/análogos & derivados , Ácido Okadáico/análise , Ácido Okadáico/química , Ácido Okadáico/toxicidade , Oxocinas/agonistas , Oxocinas/análise , Oxocinas/química , Oxocinas/toxicidade , Piranos/agonistas , Piranos/análise , Piranos/química , Piranos/toxicidade , Frutos do Mar/efeitos adversos , Espanha , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
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