Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang,China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS ⁿ ). The toxicity of nassariid N. semiplicatus showed an ‘M’-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g^?1 of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.     

The Gastropods Possessing TTX and/or PSP

Paralytic gastropod poisoning incidents have frequently occurred in the world. In the outbreaks, the symptoms of victims exhibited quite different patterns depending on the specific outbreak and most of all showed parasthesis with rare fatal cases. The toxin identified was mainly tetrodotoxin (TTX), sometimes with minor paralytic shellfish poison (PSP) and other toxins. Toxic gastropods included Family Nassariidae, Naticidae, Olividae, Muricidae, Buccinidae, Ranellidae, Harpidae, Trochidae, Turbinidae, Burdidae, and Melongenidae. The sources of toxins are from bacteria, dinoflagellate, or biosynthesis. The physiological function of toxin in toxic gastropods acts as defensive and/or attacking agent. The more toxic gastropod has higher preference or palatability preference to TTX and/or PSP.     

Tetrodotoxin in gastropods (snails) implicated in food poisoning in Northern Taiwan

The toxin in the gastropods (snails) Zeuxis sufflatus and Niotha clathrata implicated in a food poisoning incident in northern Taiwan in April 2001 was studied. The symptoms exhibited by four victims were general paresthesia, paralysis of the phalanges and the extremities, paralysis, coma, vomiting, and aphasia. The remaining gastropods were assayed for toxicity in the form of tetrodotoxin (TTX). The ranges of specimen toxicity were 345 to 1,640 mouse units (MU) for Z sufflatus and 190 to 643 MU for N. clathrata. The toxicities of the digestive gland and for other parts of the gastropod were 1,120 +/- 477 MU and 497 +/- 238 MU, respectively, for Z sufflatus and 683 +/- 113 MU and 289 +/- 169 MU, respectively, for N. clathrata. The toxin from the methanolic extract of the gastropods was partially purified by ultrafiltration and Bio-Gel P-2 column chromatography. Cellulose acetate membrane electrophoresis, thin-layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry analyses demonstrated that the toxin consisted of TTX. It was concluded that the causative agent of the food poisoning in question was TTX.     

Tetrodotoxin poisoning due to smooth-backed blowfish Lagocephalus inermis and toxicity of L. inermis caught off the Kyushu coast, Japan

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.     

Contamination of shellfish from Shanghai seafood markets with paralytic shellfish poisoning and diarrhetic shellfish poisoning toxins determined by mouse bioassay and HPLC

This paper reports the results of investigations of shellfish toxin contamination of products obtained from Shanghai seafood markets. From May to October 2003, 66 samples were collected from several major seafood markets. Paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) toxins in shellfish samples were monitored primarily by a mouse bioassay, then analysed by HPLC for the chemical contents of the toxins. According to the mouse bioassay, eight samples were detected to be contaminated by PSP toxins and seven samples were contaminated by DSP toxins. Subsequent HPLC analysis indicated that the concentrations of the PSP toxins ranged from 0.2 to 1.9 µg/100 g tissues and the main components were gonyautoxins 2/3 (GTX2/3). As for DSP, okadaic acid was detected in three samples, and its concentration ranged from 3.2 to 17.5 µg/100 g tissues. Beside okadaic acid, its analogues, dinophysistoxins (DTX1), were found in one sample. According to the results, gastropod (Neverita didyma) and scallop (Argopecten irradians) were more likely contaminated with PSP and DSP toxins, and most of the contaminated samples were collected from Tongchuan and Fuxi markets. In addition, the contaminated samples were always found in May, June and July. Therefore, consumers should be cautious about eating the potential toxic shellfish during this specific period.     

Identification of tetrodotoxin in a marine gastropod (Nassarius glans) responsible for human morbidity and mortality in Taiwan

The toxicity of the gastropod Nassarius glans was investigated. This gastropod was implicated in an incident of food paralytic poisoning on Tungsa Island, Taiwan, in April 2004. Six victims consumed both digestive glands and muscle. These tissues contained high concentrations of toxin; their highest toxicity scores were 2,048 and 2,992 MU/g, respectively, based on the tetrodotoxin (TTX) bioassay. The toxin was purified from these gastropods and analyzed by high-performance liquid chromatography, which revealed TTX and related compounds 4-epi TTX and anhydro-TTX; paralytic shellfish poisons were not found. The urine and blood samples from patients were cleansed using a C18 Sep-Pak cartridge column and 3,000 molecular weight cutoff Ultrafree microcentrifuge filters, and the eluate was filtered and analyzed by liquid chromatography and mass spectrometry. The detection limit for TTX was 1 ng/ml. The standard curves were linear in the range 30 to 600 ng/ml for urine and 1 to 30 ng/ml for blood. TTX was detected in all urine samples but in only three of four blood samples tested. Thus, the causative agent of gastropod food poisoning was identified as TTX.     

Occurrence of tetrodotoxin and paralytic shellfish poisons in a gastropod implicated in food poisoning in southern Taiwan

The toxicity of the gastropod Nassarius papillosus implicated in a food paralytic poisoning incident in Liuchiu Island, Taiwan, in October 2005 is reported. The symptoms of a victim (67 years old) were featured by general paresthesia, paralysis of phalanges and extremities, paralysis, coma, and aphasia. The remaining specimens of shell were assayed for toxicity. The range of specimen toxicity was found to be 63-474 mouse units (MU) per specimen for N. papillosus by a tetrodotoxin (TTX) bioassay. The mean (SD) toxicity of the digestive gland and other portions were 296 ± 120 and 382 ± 156 MU in N. papillosus. The toxin was partially purified from the acidic methanol extract of the gastropod by using a C18 solid-phase extraction column. The eluate was then filtered through a 3000 MW cut-off ultrafree microcentrifuge filter. It was shown that the toxin purified from gastropods analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry contained TTX 42-60 µg g^-1 (about 90%), whereas along with minor paralytic shellfish poisons (PSP) it was 3-6 µg g^-1 (about 10%).     

水产品中麻痹性贝类毒素（PSTs）检测技术的研究进展

麻痹性贝类毒素是一种分布范围广及危害较大的赤潮毒素。可经食物链的富集、传递作用,引发人体麻痹性中毒,大量发生的中毒事件,对人类健康和经济构成了严重威胁。目前,麻痹性贝类毒素常用检测技术主要是小鼠生物法、高效液相色谱法和酶联免疫试剂盒测试法,这些检测方法均有各自的优势,但麻痹性贝类毒素成分多且复杂、结构特殊,毒性又较强,这使得其监管检测较为困难,亟待建立快速简便、灵敏度高、特异性强的分析检测方法。本文基于麻痹性贝类毒素的基本性质,依据检测原理的不同论述了其生物检测技术、仪器分析技术和生化测试技术,并对各类技术的特点进行分析,提出建设性的意见,最后展望了未来麻痹性贝类毒素检测技术的发展趋势,以期为麻痹性贝类毒素检测监管提供借鉴。     

Inter-species variability of okadaic acid group toxicity in relation to the content of fatty acids detected in different marine vectors

Okadaic acid group (OA-group) is a set of lipophilic toxins which are characterised by being produced by species associated with the genera Dinophysis and Prorocentrum. OA-group has been regularly detected in endemic shellfish species from the southern zone of Chile only through the mouse bioassay. The purpose of this work was to determine the variability of OA-group toxins in endemic aquatic organisms (bivalves, crabs, gastropods and fish) and to establish the relationship with the concentration of fatty acids (FAs) detected in the evaluated species. The toxicity of OA-group and the FA profiles were determined using LC-MS/MS and gas chromatography with flame-ionisation detection, respectively. In the study area, the dinoflagellate Dinophysis acuta was detected in densities ≈2000 cells ml^?1 with a toxicity ≈18.3 pg OA equiv cel^?1. The analysis identified OA and dinophysistoxin-1 in shellfish in a range of ≈90 to ≈225 μg OA eq kg^?1, where no toxins in fish were detected. A positive relationship between the FA level and the concentration of OA-group toxins in the digestive glands of bivalves and gastropods was established, noted for high levels of saturated FAs (C14:0 and C16:0). The toxic variability of OA-group toxins determined in the different species allowed us to establish that the consumption of these vectors, regulated by non-analytical methods, can be harmful when consumed by humans, thus suggesting that the sanitary regulations for the control of OA-group in Chile should be updated.     

Mechanism of the decrease of tetrodotoxin activity in modified seawater medium

This study was designed to clarify the mechanism of the decrease of tetrodotoxin (TTX) toxicity during storage in a modified seawater medium (MSWM). When TTX was added to sterilized MSWM, the toxicity of TTX in the medium markedly decreased within 1 day, as determined by a mouse bioassay. HPLC (high-performance liquid chromatography) analysis showed that the peak of TTX was reduced and new unidentified peaks were observed. Omission of the P-1 metal solution from MSWM suppressed the decrease in TTX toxicity and the disappearance of TTX. Further studies indicated that boric acid in the P-1 metal solution triggers this toxicity decrease, indicating that TTX is chemically, not microbiologically, converted to unknown compounds in MSWM.     

Toxins in toxic Taiwanese crabs

Abstract

A total of 459 specimens covering 51 species in 9 families was collected from October 1992 to May 1996 in Taiwan. All specimens were assayed for the presence of tetrodotoxin (TTX) and paralytic shellfish poison (PSP). The specimens of five xanthid crabs Zosimus aeneus, Lophozozymus pictor, Ategatopsis germaini, Atergatis floridus, and De‐mania reynaudi were found to contain potent toxins. Among them, A. germaini showed the highest toxicity. The toxin profile of each toxic crab species was as follows: 82% TTX and 18% PSP in Z. aeneus, 89% TTX and 11% PSP in L. pictor, 3% TTX and 97% PSP inA germaini, 85% TTX and 15% in A. floridus, and 88% TTX and 12% PSP in D. reynaudi. PSP was mainly composed of gonyautoxins (GTXs) 1–4 in Z. aeneus, L. pictor, and A. floridus, but GTX 3 and hydroxysaxitoxin in A. germaini, and neosaxitoxin in D. reynaudi. The PSP‐producing dinoflagellate plankton Alexandrium minutum and TTX‐producing bacteria including Vibrio alginolyticus and Vibrio parahaemolyticus were isolated and considered as the sources of the toxins.     

Comparison of HPLC and Mouse Bioassay Methods for Determining PSP Toxins in Shellfish

Paralytic shellfish poisoning (PSP) toxins in shellfish were analyzed and quantified using both a high pressure liquid chromatographic (HPLC) technique and the standard AOAC mouse bioassay. Good correlation between the two assays was obtained at or below the limit of 80 μg toxin/100g, while at higher levels of toxicity, the HPLC technique tended to underestimate total toxicity slightly. Advantages and disadvantages of both techniques are discussed.     

Paralytic shellfish poisoning (PSP) toxin binders for optical biosensor technology: problems and possibilities for the future: a review

This review examines the developments in optical biosensor technology, which uses the phenomenon of surface plasmon resonance, for the detection of paralytic shellfish poisoning (PSP) toxins. Optical biosensor technology measures the competitive biomolecular interaction of a specific biological recognition element or binder with a target toxin immobilised onto a sensor chip surface against toxin in a sample. Different binders such as receptors and antibodies previously employed in functional and immunological assays have been assessed. Highlighted are the difficulties in detecting this range of low molecular weight toxins, with analogues differing at four chemical substitution sites, using a single binder. The complications that arise with the toxicity factors of each toxin relative to the parent compound, saxitoxin, for the measurement of total toxicity relative to the mouse bioassay are also considered. For antibodies, the cross-reactivity profile does not always correlate to toxic potency, but rather to the toxin structure to which it was produced. Restrictions and availability of the toxins makes alternative chemical strategies for the synthesis of protein conjugate derivatives for antibody production a difficult task. However, when two antibodies with different cross-reactivity profiles are employed, with a toxin chip surface generic to both antibodies, it was demonstrated that the cross-reactivity profile of each could be combined into a single-assay format. Difficulties with receptors for optical biosensor analysis of low molecular weight compounds are discussed, as are the potential of alternative non-antibody-based binders for future assay development in this area.     

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.     

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28–3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g^–1 (S/N = 10) and the limit of detection (LOD) was 0.041 µg g^–1 (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.     

A rapid method for tetrodotoxin (TTX) determination by LC-MS/MS from small volumes of human serum,and confirmation of pufferfish poisoning by TTX monitoring

A simple and rapid detection method for tetrodotoxin (TTX), a powerful sodium channel blocker, in small volumes of the serum of patients with pufferfish poisoning, was developed using an ultrafiltration spin column. The separation and identification of TTX was performed by liquid chromatography (LC) with a multi-mode ODS column and tandem mass spectrometry. TTX and an internal standard (voglibose) were monitored and quantitated using ion transitions: the respective precursor-to-product ion combinations, m/z 320/162 for TTX and 268/92 in MRM mode. The recoveries of TTX and voglibose were 91.0–110.8% and 104.7–107.4%, respectively, and with high accuracy (intra-run, 4.35–5.29%; inter-run, 2.95–5.79%) and linearity (0.5–200 ng/ml serum: r = 0.9994). The lower limit of quantification was 0.5 ng/ml serum. In patients, maximum serum TTX concentrations were 30.2 ng/ml serum for patient 1 on day 0 and 56.1 ng/ml serum for patient 2 on day 1. These results are important for the treatment of patients and for the identification of poisoning as well as for the determination of the cause of the food poisoning.     

鹅膏肽类毒素检测方法的历史与现状

每年因误食毒蘑菇导致中毒死亡事件在世界各国都有发生,也是我国食物中毒事件中导致死亡的重要因素 之一。鹅膏菌属中某些种类含有的肽类毒素是主要的致死原因,快速而有效地检测样品（包括有毒蘑菇子实体、食 物剩余物、呕吐物、中毒患者血液和尿液等）中的毒素对于食物中毒的毒源鉴定和中毒后的针对性治疗具有重要意 义。本文从化学显色反应、生物化学法、物理法、色谱法等4 个方面对鹅膏肽类毒素检测方法的历史和研究进展进 行整理和总结,并对其在我国的应用加以讨论和展望。     

2016-2020年郴州市毒蕈中毒事件流行病学特征分析

目的 通过对2016-2020年郴州市毒蕈中毒事件的流行病学特征进行描述性分析,为制定毒蕈中毒预防控制措施提供科学依据.方法 收集和整理2016-2020年郴州市通过"食源性疾病事件报告系统"上报的毒蕈中毒事件资料,对事件发生的时间、地区、人群特征以及毒蕈来源、种类进行描述性分析.结果 2016-2020年郴州市共报告...     

Determination of the variability of both hydrophilic and lipophilic toxins in endemic wild bivalves and carnivorous gastropods from the Southern part of Chile

The aim of this study was to analyse and determine the composition of paralytic shellfish poisoning (PSP) toxins and lipophilic toxins in the Region of Aysén, Chile, in wild endemic mussels (Mytilus chilensis, Venus antiqua, Aulacomya ater, Choromytilus chorus, Tagelus dombeii and Gari solida) and in two endemic carnivorous molluscs species (Concholepas concholepas and Argobuccinum ranelliforme). PSP-toxin contents were determined by using HPLC with fluorescence detection, while lipophilic toxins were determined by using LC-MS/MS. Mean concentrations for the total of PSP toxins were in the range 55–2505 μg saxitoxin-equivalent/100 g. The two most contaminated samples for PSP toxicity were bivalve Gari solida and carnivorous Argobuccinum ranelliforme with 2505 ± 101 and 1850 ± 137 μg saxitoxin-equivalent/100 g, respectively (p < 0.05). The lipophilic toxins identified were okadaic acid, dinophysistoxin-1 (DTX-1), azaspiracid-1 (AZA-1), pectenotoxin-2 (PTX-2) and yessotoxins (YTX). All analysed molluscs contained lipophilic toxins at levels ranging from 56 ± 4.8 to 156.1 ± 8.2 μg of okadaic acid-equivalent/kg shellfish together with YTX at levels ranging from 1.0 ± 0.1 to 18 ± 0.9 μg of YTX-equivalent/kg shellfish and AZA at levels ranging from 3.6 ± 0.2 to 31 ± 2.1 μg of AZA-equivalent/kg shellfish. Furthermore, different bivalves and gastropods differ in their capacity of retention of lipophilic toxins, as shown by the determination of their respective lipophilic toxins levels. In all the evaluated species, the presence of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods was not identified, in contrast to the identification of PSP toxins, where the profiles identified in the different species are directly related to biotransformation processes. Thus, this study provides evidence that the concentration of toxins in the food intake of the evaluated species (Bivalvia and Gastropoda class) determines the degree of bioaccumulation and biotransformation they will thereafter exhibit.