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1.
Food Chem Toxicol ; 141: 111386, 2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32376337

RESUMO

Paralytic shellfish poisoning (PSP) episodes cause important economic impacts due to closure of shellfish production areas in order to protect human health. These closures, if are frequent and persistent, can seriously affect shellfish producers and the seafood industry, among others. In this study, we have developed an alternative processing method for bivalves with PSP content above the legal limit, which allows reducing toxicity to acceptable levels. A modification of the PSP detoxifying procedure stablished by Decision 96/77/EC of the European Union in Acanthocardia tuberculata, was developed and implemented for PSP elimination in other bivalves species. The procedure was applied to 6 batches of mussels, 2 batches of clams and 2 batches of scallops, achieving detoxification rates of around 85%. A viable industrial protocol which allows the transformation of a product at risk into a safe product was developed. Although a significant reduction was obtained, in a sample circa 9000 µg STX diHCl equiv/kg, the final toxin level in these highly toxic mussels did not fall below the European limit. The processing protocol described may be applied efficiently to mussels, clams and scallops and it may be a major solution to counteract the closure of shellfish harvesting areas, especially if persistent.

2.
Toxins (Basel) ; 12(5)2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32392808

RESUMO

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species-specific but probably also specific to larger geographic regions.

3.
Food Chem Toxicol ; 140: 111315, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32268157

RESUMO

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many food poisoning incidents and some fatalities. Although mainly associated with the consumption of pufferfish, in recent years, TTX has been found in shellfish, particularly in Europe. In this work, a magnetic bead (MB)-based colorimetric immunoassay was applied to the detection of TTX in Pacific oysters (Crassostrea gigas), razor clams (Solen marginatus) and mussels (Mytilus galloprovincialis). Effective LODs (eLODs) for TTX of 1 µg/kg in oysters and razor clams and 3.3 µg/kg in mussels, significantly below the EFSA guidance threshold (44 µg/kg), were obtained. The strategy was applied to the analysis of naturally-contaminated Pacific oysters (Crassostrea gigas) and mussels (Mytilus edulis) from the Netherlands, and TTX was detected in all samples. The approach, which takes less than 1.5 h, proved to be useful as a rapid and simple method to detect TTX, support shellfish safety and protect consumers.

4.
Anal Chem ; 92(7): 4858-4865, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32133843

RESUMO

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

5.
Aquat Toxicol ; 221: 105427, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32044545

RESUMO

Local population frequently consumes moray eels and dusky groupers from the Canary Islands. These species are top predators and the interactions between them include predation but also, in some cases, collaborative hunting. These fish are well known to cause ciguatera (CFP) outbreaks in several marine areas such as Japan, Hawaii, French Polynesia and Caribe. Groupers have been involved in CFP events in the Canary Islands, however, moray eels have not yet been well studied in this regard. The present research seeks to describe the finding of a black moray in the stomach of a positive dusky grouper during its necropsy, and to clarify the implication of groupers and moray eels in the food webs, accumulating CTXs in the Canarian environment. The study also updates statistics on the presence of toxic groupers in this archipelago. For these purposes, 248 grouper samples from the CFP official control in the Canary Islands (2018-2019) were analysed and 36 moray eels (5 species) were collected under the EuroCigua project and one was obtained during a dusky grouper necropsy. All samples were analysed with the Neuro-2a cell-based assay (CBA) to evidence CTX-like toxicity. Regarding the necropsied grouper and the moray eel found in its stomach content, the LCMS/MS method allowed the identification and quantification of CCTX1 in both fish at similar levels while none of the P-CTXs for which standards were available were detected. Among groupers, 25.4 % displayed CTX-like toxicity with differences between islands. For moray eels 38.9 % showed toxicity, involving 4 species. Black moray exhibited a high proportion of positives (9/12) and a positive correlation was found between CTX-like toxicity quantification and the black moray weight. Regarding the grouper, and the moray eel found in its stomach, the LCMS/MS method allowed the identification and quantification of C-CTX1 in both fish at similar levels. This found suggests a trophic interaction between these species and their role in maintaining CTXs in the Canary waters where local population commonly demand those species for consumption. The island of El Hierro stands out above all the other Canary Islands with the concerning percentage of positive grouper samples and the high CTX toxicity levels obtained in moray eel specimens analysed in this marine area. This is the first report of CTX-like toxicity in flesh of moray eels fished in the Canary archipelago and the confirmation of the presence of C-CTX1 by LCMS/MS in a black moray from this marine area.


Assuntos
Ciguatoxinas/análise , Enguias/metabolismo , Monitoramento Ambiental/métodos , Poluentes Químicos da Água/análise , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Intoxicação por Ciguatera/epidemiologia , Intoxicação por Ciguatera/etiologia , Ciguatoxinas/toxicidade , Cadeia Alimentar , Contaminação de Alimentos/análise , Conteúdo Gastrointestinal/química , Músculos/química , Alimentos Marinhos/análise , Espanha , Poluentes Químicos da Água/toxicidade
6.
Toxins (Basel) ; 12(2)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098095

RESUMO

In the last decade, several outbreaks of ciguatera fish poisoning (CFP) have been reported in the Canary Islands (central northeast Atlantic Ocean), confirming ciguatera as an emerging alimentary risk in this region. Five Gambierdiscus species, G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus, have been detected in macrophytes from this area and are known to produce the ciguatoxins (CTXs) that cause CFP. A characterization of the toxicity of these species is the first step in identifying locations in the Canary Islands at risk of CFP. Therefore, in this study the toxicity of 63 strains of these five Gambierdiscus species were analysed using the erythrocyte lysis assay to evaluate their maitotoxin (MTX) content. In addition, 20 of the strains were also analysed in a neuroblastoma Neuro-2a (N2a) cytotoxicity assay to determine their CTX-like toxicity. The results allowed the different species to be grouped according to their ratios of CTX-like and MTX-like toxicity. MTX-like toxicity was especially high in G. excentricus and G. australes but much lower in the other species and lowest in G. silvae. CTX-like toxicity was highest in G. excentricus, which produced the toxin in amounts ranging between 128.2 ± 25.68 and 510.6 ± 134.2 fg CTX1B equivalents (eq) cell-1 (mean ± SD). In the other species, CTX concentrations were as follows: G. carolinianus (100.84 ± 18.05 fg CTX1B eq cell-1), G. australes (31.1 ± 0.56 to 107.16 ± 21.88 fg CTX1B eq cell-1), G. silvae (12.19 ± 0.62 to 76.79 ± 4.97 fg CTX1B eq cell-1) and G. caribaeus (

7.
Toxins (Basel) ; 11(12)2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835676

RESUMO

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g-1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure-activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

8.
Toxins (Basel) ; 11(10)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554162

RESUMO

Prevalence of marine biotoxins in seafood has been associated with increasing frequency, intensity, and duration of harmful algal blooms, and an increase of the geographical and temporal distribution of harmful algae [...].

9.
Sci Total Environ ; 689: 655-661, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279212

RESUMO

Ostreopsis cf. ovata is a benthic microalga distributed in tropical and temperate regions worldwide which produces palytoxins (PlTXs). Herein, an electrochemical biosensor for the detection of this toxic microalga is described. The detection strategy involves isothermal recombinase polymerase amplification (RPA) of the target using tailed primers and a sandwich hybridisation assay on maleimide-coated magnetic beads immobilised on electrode arrays. The biosensor attained a limit of detection of 9 pg/µL of O. cf. ovata DNA (which corresponds to ~640 cells/L), with no interferences from two non-target Ostreopsis species (O. cf. siamensis and O. fattorussoi). The biosensor was applied to the analysis of planktonic and benthic environmental samples. Electrochemical O. cf. ovata DNA quantifications demonstrated an excellent correlation with other molecular methods (qPCR and colorimetric assays) and allowed the construction of a predictive regression model to estimate O. cf. ovata cell abundances. This new technology offer great potential to improve research, monitoring and management of O. cf. ovata and harmful algal blooms.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Algas/análise , DNA de Protozoário/análise , Dinoflagelados/isolamento & purificação , Técnicas Eletroquímicas/instrumentação , Técnicas Biossensoriais/instrumentação , Proliferação Nociva de Algas
10.
Food Chem Toxicol ; 129: 153-161, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31042590

RESUMO

This study aimed to assess the bioaccessibility of different marine biotoxins in naturally contaminated shellfish and fish gonads using an in vitro digestion methodology. In general, hydrophilic toxins (domoic acid, paralytic shellfish poisoning toxins and tetrodotoxins) showed higher bioaccessibility than lipophilic ones (okadaic acid and azaspiracids). The bioaccessibility of toxins from the okadaic acid group ranged from 69% (raw European razor clams) to 74% (raw donax clams). Regarding azaspiracids, 47% of the initial content was bioaccessible in steamed blue mussel. As for hydrophilic toxins, 100% of the initial content was bioaccessible after digestion in raw shellfish and puffer fish gonads. The total tetrodotoxin bioaccessibility in puffer fish gonads decreased significantly after steaming. The profile of tetrodotoxins changed during the digestion process: TTX and 11-norTTX-6S-ol analogues decreased significantly after digestion, but the 5,6,11-trideoxy TTX analogue increased in both raw and steamed puffer fish gonads. These preliminary findings confirm the need to consider bioaccessibility data in future seafood risk assessment, as such information enables a more accurate and realistic estimation of potential seafood hazards, particularly in what concerns lipophilic toxins, therefore, constituting a crucial tool in the refinement of regulatory limits for the presence of biotoxins in seafood.


Assuntos
Alimentos Marinhos/análise , Água do Mar/química , Toxinas Biológicas/farmacocinética , Animais , Disponibilidade Biológica , Humanos , Interações Hidrofóbicas e Hidrofílicas
11.
Harmful Algae ; 84: 27-35, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31128810

RESUMO

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/µL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.


Assuntos
Colorimetria , Dinoflagelados , DNA , Humanos , Mar Mediterrâneo , Reprodutibilidade dos Testes
12.
Sci Total Environ ; 673: 576-584, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-30999098

RESUMO

This research identifies factors associated with the contamination by ciguatoxins (CTXs) in a population of fish and proposes a predictive score of the presence of CTX-like toxicity in amberjack samples from the official control program of ciguatera in the Canary Islands of the Directorate-General (DG) Fisheries (Canary Government). Out of the 970 samples of fish studied, 177 (18.2%) samples showed CTX-like toxicity. The fish were classified according to the species, amberjack (Seriola dumerili and S. rivoliana) (n = 793), dusky grouper (Epinephelus marginatus) (n = 145) and wahoo (Acanthocybium solandri) (n = 32). The data were separated by species category and statistically examined, resulting in 137 (17.3%) amberjack and 39 (26.9%) grouper samples showing CTX-like toxicity; regarding wahoo species, only 1 toxic sample (3.1%) was found. According to fishing location the contamination rates suggested grouping the islands in four clusters; namely: {El Hierro: HI; La Gomera: LG; La Palma: LP}, {Gran Canaria: GC; Tenerife: TF}, {Fuerteventura: FU} and {Lanzarote: LZ}. For the amberjack species, the multivariate logistic regression showed the factors that maintained independent association with the outcome, which were the warm season (OR = 3.617; 95% CI = 1.249-10.474), the weight (per kg, 1.102; 95% CI = 1.069-1.136) and the island of fish catching. A prediction score was obtained for the probability of contamination by CTX in amberjack fish samples. The area under de curve (AUC) obtained using the validation data was 0.747 (95% CI = 0.662-0.833). Regarding grouper species, the island of fishing was the only factor that showed significant differences associated with the presence of CTX-like toxicity. We provide herein data for a better management and prediction of ciguatera in the Canary Islands, suggesting a review of the minimum limits of fish weight established by the Canary Government for the control program.


Assuntos
Intoxicação por Ciguatera/epidemiologia , Ciguatoxinas/análise , Doenças dos Peixes/parasitologia , Peixes/parasitologia , Alimentos Marinhos/parasitologia , Animais , Ciguatoxinas/toxicidade , Ilhas , Alimentos Marinhos/estatística & dados numéricos , Espanha/epidemiologia
13.
Food Chem ; 290: 255-262, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31000045

RESUMO

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.


Assuntos
Técnicas Eletroquímicas/métodos , Tetraodontiformes/metabolismo , Tetrodotoxina/análise , Animais , Bactérias/isolamento & purificação , Grécia , Separação Imunomagnética/métodos , Oceanos e Mares , Alimentos Marinhos/análise , Alimentos Marinhos/microbiologia , Tetraodontiformes/crescimento & desenvolvimento , Tetrodotoxina/isolamento & purificação
14.
Toxins (Basel) ; 11(4)2019 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-31013948

RESUMO

Ciguatera Fish Poisoning is a worldwide concern caused by the consumption of fish contaminated with ciguatoxins not only in endemic regions in the Pacific Ocean or the Caribbean Sea but also in emerging areas of Macaronesia on the eastern Atlantic. The recent emergence of these toxins in other coastal areas worldwide, prompted the need for the characterization of the risk in these areas. This Ciguatera Fish Poisoning risk has been recently identified as a potential threat in subtropical areas of the Atlantic coast and scientific efforts are being focused in the identification and confirmation of the toxins involved in this potential risk. Neuroblastoma cell assay has been widely used for the evaluation of the toxicity in several marine biotoxin groups, and found to be a very useful tool for toxicity screening. LC-MS/MS has been also used for confirmatory purposes although the main limitation of the advances on LC-MS/MS development is due to commercial unavailability of reference materials and hampers method implementation and validation or even confirmation of the ciguatoxins (CTXs) responsible for the toxic profiles. While neuroblastoma cell assay (N2a) is typically used for toxicity screening as mentioned above, being necessary to confirm this N2a toxicity by LC-MS/MS, this study is designed using N2a as a tool to confirm the toxicity of the fractions obtained corresponding to potential CTXs analogues according to the analysis by LC-MS/MS. With this aim, an amberjack sample (Seriola fasciata) from Selvagen Islads (Portugal) and implicated in Ciguatera Fish Poisoning was analyzed by LC-MS/MS and Caribbean Ciguatoxins were found to be mainly responsible for the toxicity. N2a was used in this work as a tool to help in the confirmation of the toxicity of fractions obtained by HPLC. Caribbean Ciguatoxin-1 was found as the main analogue responsible for the N2a toxicity while three Caribbean Ciguatoxin-1 (C-CTX1) metabolites which contribute to the total toxicity were also identified.


Assuntos
Intoxicação por Ciguatera , Ciguatoxinas/análise , Perciformes , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ciguatoxinas/toxicidade , Camundongos , Espectrometria de Massas em Tandem
15.
Anal Chim Acta ; 1039: 140-148, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30322545

RESUMO

Karlodinium is a dinoflagellate responsible for fish-killing events worldwide. In Alfacs Bay (NW Mediterranean Sea), the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. This work presents a method that combines recombinase polymerase amplification (RPA) with an enzyme-linked oligonucleotide assay (ELONA) to identify, discriminate and quantify these two species. The system was characterised using synthetic DNA and genomic DNA, and the specificity was confirmed by cross-reactivity experiments. Calibration curves were constructed using 10-fold dilutions of cultured cells, attaining a limit of detection of around 50,000 cells/L, far below the Karlodinium spp. alert threshold (200,000 cells/L). Finally, the assay was applied to spiked seawater samples, showing an excellent correlation with the spiking levels and light microscopy counts. This approach is more rapid, specific and user-friendly than traditional microscopy techniques, and shows great promise for the surveillance and management of harmful algal blooms.


Assuntos
Ensaio de Imunoadsorção Enzimática , Toxinas Marinhas/análise , Microalgas/química , Oligonucleotídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Oligonucleotídeos/metabolismo
16.
Food Chem Toxicol ; 112: 188-193, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29292021

RESUMO

In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Doenças Transmitidas por Alimentos/diagnóstico , Toxinas Marinhas/química , Alimentos Marinhos/envenenamento , Tetraodontiformes , Tetrodotoxina/química , Animais , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/sangue , Doenças Transmitidas por Alimentos/urina , Humanos , Toxinas Marinhas/sangue , Toxinas Marinhas/urina , Nova Caledônia , Espectrometria de Massas em Tandem , Tetrodotoxina/análogos & derivados , Tetrodotoxina/sangue , Tetrodotoxina/urina
17.
Mar Environ Res ; 133: 6-14, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29174400

RESUMO

Rapid and cost-effective methods to monitor the presence of diarrhetic shellfish poisoning (DSP) toxins in seawater samples in an easy and reliable manner are required to protect human health and avoid economic losses to shellfish industry. Immunoassays for the detection of okadaic acid (OA) and dinophysistoxin-1 and dinophysistoxin-2 are developed by immobilising OA on self-assembled monothiols or dithiols in an ordered and oriented way, providing an effective limit of detection of ∼1 ng OA equiv./mL seawater. The immunoassays are applied to the analysis of the particulate fraction of seawater samples from two Catalan harbours (NW Mediterranean) and samples collected periodically from the Galician Rias (E Atlantic), as well as a reference mussel sample. Results are in agreement with LC-MS/MS and the certified values. OA concentration in seawater correlates with Dinophysis cell abundance, with a 1-2 weeks lag. The immunoassays provide powerful high-throughput analytical methods potentially applicable as alternative monitoring tools.


Assuntos
Monitoramento Ambiental/métodos , Imunoensaio , Toxinas Marinhas/análise , Ácido Okadáico/análise , Animais , Bivalves , Humanos , Água do Mar/química , Frutos do Mar , Intoxicação por Frutos do Mar
18.
Environ Res ; 161: 392-398, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29197280

RESUMO

Cyclic imines constitute a quite recently discovered group of marine biotoxins that act on neural receptors and that bioaccumulate in seafood. They are grouped together due to the imino group functioning as their common pharmacore, responsible for acute neurotoxicity in mice. Cyclic imines (CIs) have not been linked yet to human poisoning and are not regulated in the European Union (EU), although the European Food Safety Authority (EFSA) requires more data to perform conclusive risk assessment for consumers. Several commercial samples of bivalves including raw and processed samples from eight countries (Italy, Portugal, Slovenia, Spain, Ireland, Norway, The Netherlands and Denmark) were obtained over 2 years. Emerging cyclic imine concentrations in all the samples were analysed on a LC-3200QTRAP and LC-HRMS QExactive mass spectrometer. In shellfish, two CIs, pinnatoxin G (PnTX-G) and 13-desmethylspirolide C (SPX-1) were found at low concentrations (0.1-12µg/kg PnTX-G and 26-66µg/kg SPX-1), while gymnodimines and pteriatoxins were not detected in commercial (raw and processed) samples. In summary, SPX-1 (n: 47) and PnTX-G (n: 96) were detected in 9.4% and 4.2% of the samples, respectively, at concentrations higher than the limit of quantification (LOQ), and in 7.3% and 31.2% of the samples at concentrations lower than the LOQ (25µg/kg for SPX-1 and 3µg/kg for PnTX-G), respectively. For the detected cyclic imines, the average exposure and the 95th percentile were calculated. The results obtained indicate that it is unlikely that a potential health risk exists through the seafood diet for CIs in the EU. However, further information about CIs is necessary in order to perform a conclusive risk assessment.


Assuntos
Iminas , Alimentos Marinhos , Animais , Europa (Continente) , Contaminação de Alimentos , Humanos , Iminas/análise , Iminas/toxicidade , Camundongos , Medição de Risco
19.
Talanta ; 176: 659-666, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917804

RESUMO

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at -20°C and 4°C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health.


Assuntos
Crassostrea , Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Maleimidas/química , Mytilus , Tetrodotoxina/análise , Animais , Anticorpos/imunologia , Limite de Detecção , Reprodutibilidade dos Testes , Extração em Fase Sólida , Tetrodotoxina/química , Tetrodotoxina/imunologia
20.
Anal Chim Acta ; 989: 95-103, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-28915947

RESUMO

The recent detection of tetrodotoxins (TTXs) in European fish and shellfish has emphasized the urgent need to develop specific, selective, rapid and easy-to-use methods for their detection to assess the potential risk posed to human health. For this purpose, a dithiol self-assembled monolayer (SAM)-based immunoassay previously performed on maleimide plates (mELISA) has been adapted to gold electrode arrays for the development of an electrochemical immunosensor for TTX. The electrochemical SAM-based immunosensor designed herein, provided an oriented, stable and spaced sensing platform for the determination of TTX, attaining a limit of detection of 2.6 ng mL-1. The applicability of the biosensor array was demonstrated by the accurate quantifications obtained in the analysis of different tissues of several puffer fish species (Lagocephalus lagocephalus, L. sceleratus and Sphoeroides pachygaster) caught along the Mediterranean coast of Spain. The good agreements found between the TTX concentrations determined by the immunosensor array platforms and those determined by mELISA, surface Plasmon resonance (SPR) immunosensor and liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis, proved the feasibility of the approach. The electrochemical immunosensor enables the determination of TTXs at levels as low as 0.07 mg TTX equiv. kg-1 tissue, thus, well below the Japanese value of 2 mg TTX equiv. kg-1 tissue used as a criterion to consider puffer fish safe for consumption. Compared to the colorimetric SAM-based approach, the immunosensor array described herein shows promise towards the development of disposable, portable and compact analysis tools applicable in monitoring programs for the surveillance of fishery products.


Assuntos
Técnicas Biossensoriais , Imunoensaio , Alimentos Marinhos/análise , Tetraodontiformes , Tetrodotoxina/análise , Tolueno/análogos & derivados , Animais , Cromatografia Líquida , Espectrometria de Massas , Ressonância de Plasmônio de Superfície , Tolueno/química
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