Fisheries Science (2020) 86:181–186 https://doi.org/10.1007/s12562-019-01375-3

ORIGINAL ARTICLE

Food Science and Technology

Occurrence of tetrodotoxin in three gastropod in Khanh Hoa Province, Vietnam

Ha Viet Dao1 · Ky Xuan Pham1 · Ben Xuan Hoang1 · Masato Tanioka2 · Ryuichi Watanabe3 · Toshiyuki Suzuki3

Received: 5 August 2019 / Accepted: 10 October 2019 / Published online: 12 November 2019 © Japanese Society of Fisheries Science 2019

Abstract Neurotoxic poisonings with fatal symptoms caused by consumption of Nassarius gastropods have been reported in Vietnam but the causative toxins have not been confrmed. In the present study, Nassarius conoidalis, N. glans and N. pullus, which are considered to be common causative species, were collected in Khanh Hoa province, Vietnam in May 2016 for toxin analysis using LC/MS/MS. The results confrmed that TTX is a dominant toxin in these gastropods. Among them, N. glans exhibited the highest toxicity (412.7 ± 107.3 MU/g), followed by N. conoidalis (144.8 ± 82.0 MU/g) and N. pullus (19.6 ± 9.8 MU/g). This is the frst report confrming TTX in these species. All specimens in this study were hightly toxic, indicating that the frequency of toxic specimens of the three Nassarius species is extremely high. Further, their toxicities were all beyond the regulatory level of consumption (10 MU/g) for pufer(fsh) as recommended in Japan. The present data indicates that N. conoidalis, N. glans and N. pullus are not suitable for food in Vietnam.

Keywords LC/MS/MS · Tetrodotoxin · Gastropods · Nassarius · Vietnam

Introduction these gastropods have revealed that paralytic shellfsh toxins (PSTs) and/or tetrodotoxin (TTX) were the causative toxins It has been reported that food poisonings due to consumption in the cases of poisoning (Narita et al. 1984; Hwang et al. of marine gastropods have occurred sporadically in Asian 2002, 2004, 2007; Liu et al. 2004; Taniyama et al. 2009, countries such as Taiwan, Japan and China (Hwang et al. 2013). 1995; Yang et al. 1995; Shiu et al. 2003; Sui et al. 2002, There are more than 15 Nassarius species found along 2003; Hwang et al. 2005; Takatani et al. 2005). They belong the Vietnamese coasts (Hylleberg and Kilburn 2003; Nghi to several families such as , Naticidae, Olividae, 2005) and they are one of many local popular seafoods. The Turbinidae, Trochidae, Charonidae, Babylonidae, Tutufadae, poisonings with fatal symptoms by consumption of Nas- and Niothadae (Noguchi et al. 2011). Analysis of toxins in sarius gastropods had been reported in Khanh Hoa province, Vietnam (Dao and Sato 2010). Victims showed neurotoxic symptoms (Dao and Sato 2010; Dao unpublished observa- Electronic supplementary material The online version of this tion), but causative toxins have not been confrmed, except article (https​://doi.org/10.1007/s1256​2-019-01375​-3) contains for TTX detected by HPLC in one N. papilosus specimen supplementary material, which is available to authorized users. remaining implicated in a food poisoning case in 2006 (Dao * Ha Viet Dao and Sato 2010). In 2015, we screened toxins in fve com- [email protected] mon Nassarius species collected in Khanh Hoa province,

1 Vietnam (Dang et al. 2015). As the preliminary result, a Institute of Oceanography, Vietnam Academy of Science N. conoi- and Technology, 01 Cau Da Street, Nha Trang, certain level of mouse toxicity was detected in Khanh Hoa Province, Vietnam dalis, N. glans and N. pullus. However, causative toxins in 2 College of Bioresource Sciences, Nihon University, 1866 these species could not be confrmed due to lack of available Kameino, Fujisawa‑shi, Kanagawa 252‑0880, Japan equipment, and the frequency of toxic specimens as well as 3 National Research Institute of Fisheries Science, 2‑12‑4 individual variation in the toxicities was not investigated. Fukuura, Kanazawa, Yokohama 236‑8648, Kanagawa, Japan

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The present study describes toxin confrmation in N. Kanto Chemicals (Tokyo, Japan). Milli-Q water was used conoidalis, N. glans and N. pullus collected from Khanh throughout the experiment. The standards of PSTs (C1/2, Hoa province, Vietnam in May 2016. The frequency of GTX1-5, dcGTX2/3, NEO, dcSTX) were provided by Prof. toxic specimens and the individual toxicities were also Oshima, Tohoku University. Tetrodotoxin citrate, 1 mg, was documented. purchased from Tocris Bioscience (Bristol, UK). Ultra high- performance liquid chromatography was used (Shimadzu NexeraXR, Tokyo, Japan) with an upper pressure of 620 bar. Materials and methods The mass spectrometer used was a SCIEX QTRAP 4500 instrument (Tokyo, Japan). Specimen collection Analysis of tetrodotoxin and paralytic shellfsh The gastropods used in the analysis were N. conoidalis, N. toxins glans and N. pullus. In May 2016, 30 specimens each of these species (Fig. 1) were collected from near-shore mud As these species are small gastropods and people often eat bottom sediment in Van Ninh District, Khanh Hoa Province all their soft tissue, all the soft tissue was collected for toxin where food poisonings by consumption of small gastro- analysis. The toxins in the soft tissue of the gastropods were pods had been recognized (Dao unpublished observation). extracted according to the method of Supapun et al. (2003). They were transferred into the laboratory of the Institute of The extracts were treated using an ENVI-Carb SPE car- Oceanography under cool conditions. Specimen photos were tridge 250 mg/3 ml (Sigma Aldrich Japan, Tokyo, Japan), taken and identifed by a mollusk specialist at the Institute of the eluates were four-fold diluted with acetonitrile and then Oceanography, Vietnam. After having been cleaned outside PSTs and TTX in the eluates were analyzed by UHPLC/MS/ and deshelled, the whole soft tissues were collected indi- MS according to the method of Boundy et al. (2015). The vidually and stored at – 20 °C. UHPLC separation was carried out with an Acquity UPLC BEH amide column (2.1 mm I.D. × 150 mm, 1.7 µm) with Reagents and instruments a gradient elution of A1 and B1 at 60 °C. Sample injection volume was 1 μl. Ion source parameters for the MS spec- Formic acid and acetic acid were purchased from Wako trometer were as follows: curtain gas (CUR), 30 psi; ion pure chemicals (Osaka, Japan). Ammonium hydroxide spray voltage (IS), 4500 V for positive mode and − 4500 V of 25% as LC–MS additive was purchased from Sigma- for negative mode; heater temperature (TEM), 600 °C; Aldrich (Tokyo, Japan). Acetonitrile was purchased from source gas (GS1), 50 psi; turbo gas (GS2), 80 psi; entrance

Fig. 1 Photos of the three gastropod species in the study. a, b Nassarius glans; c, d N. conoidalis; and e, f N. pullus

1 3 Fisheries Science (2020) 86:181–186 183 potential (EP), 10 V for positive mode and − 10 V for nega- 14.2 nM, dcSTX: 2.0 nM, TTX: 3.4 nM. The MS/MS frag- tive mode. Multiple reaction monitoring (MRM) mode was mentation spectra obtained from the peak with exactly performed in both positive (ESI+) and negative (ESI−) elec- the same retention time as TTX in LC monitored at m/z trospray ionization. A minimum of two transitions were used 320.0 in the MRM was indistinguishable from those of for each PST analog. For each target ion, MRM ion channels standard TTX, indicating that the peak is unambiguously were selected for specifc product ions generated from the TTX (Fig. 3). The dominant MS/MS spectra observed for selected precursor ion (Table 1). To confrm identifcation of the TTX standard were m/z 162, 178, 256, 284, 302 and TTX in the gastropods, MS/MS spectra were obtained with 320. The characteristic fragment spectrum at m/z 162 was 40 eV of collision energy for the precursor of m/z 320.0 with assumed to be a mixture of 2-aminohydroxyquinazolines a range of m/z 50–m/z 350. arising from the guanidium moiety of TTX (Shoji et al. The TTX toxicities were calculated from UHPLC-MS/ 2001). These fragment spectra were observed from the MS data and expressed in mouse units (MU/g) according extract of Nassarius gastropod species. In contrast, no to Nakamura and Yasumoto (1985), in which 1 mg TTX peak corresponding to standard PSTs was detected in any corresponds to 4500 MU, 1 mg 4-epiTTX corresponds to extract of these gastropods (Online Resource). The present 709 MU and 1 mg 4,9-anhydroTTX corresponds to 92 MU. results indicate that TTX is a dominant toxin in N. conoi- One MU is the dose of toxin that will kill a 20-g male mouse dalis, N. glans and N. pullus in Vietnam. (ddY) in 30 min. Table 2 shows the mean and range of total toxicities (MU/g) calculated based on the specifc mouse toxic- ity of each toxin component (Nakamura and Yasumoto Results 1985) in three gastropod species. Among them, N. glans is the most toxic (412.7 ± 107.3 MU/g), then N. conoida- In the UHPLC/MRM chromatogram, the retention time lis (144.8 ± 82.0 MU/g), and fnally N. pullus (19.6 ± 9.8 (Rt) of TTX and 4-epiTTX was 4.3 and 3.8 min, respec- MU/g). The range of toxicity in each species was also quite tively (Fig. 2). Peaks with the same retention time of TTX wide: 187.0–787.0 MU/g in N. glans, 71.0–292.0 MU/g and 4-epiTTX were observed in all extracts of N. conoi- in N. concoidalis, and 10.0–46.7 MU/g in N. pullus. The dalis, N. glans and N. pullus. Limits of detection (LOD) highest TTX toxicity score (787 MU/g) was observed in in the UHPLC-MS/MS method for PSTs and TTX stand- N. glans while the lowest toxicity score was detected in ards were as follows; C1: 8.1 nM, C2: 0.4 nM, GTX1: N. pullus (10.0 MU/g). It is shown in Table 2 that 100% 4.3 nM, GTX2: 2.0 nM, GTX3: 17.6 nM, GTX4: 65.8 nM, of specimens in this study exhibited a certain level of GTX5: 0.8 nM, dcGTX2: 5.7 nM, dcGTX3: 0.7 nM, NEO: toxicity.

Table 1 MRM transition and Analog ESI+ transition ESI− transition collision energy of PST and + − TTX analogs for ESI­ and ­ESI TTX 320.1 > 302.1, 162.1 (25, 38) mode STX 300.1 > 204.1, 138.0 (24, 30) NEO 316.1 > 126.1, 298.1 (15, 24) dcSTX 257.1 > 126.1, 222.0 (19, 22) dcNEO 273.1 > 126.1, 225.1 (20, 18) doSTX 241.1 > 60.0, 206.1 (23, 22) GTX2 316.1 > 148.0 (20) 394.1 > 351.1, 333.1 (− 16, − 22) GTX3 396.1 > 298.1 (20) 394.1 > 333.1, 351.1 (− 22, − 16) GTX1 332.1 > 314.1 (20) 410.1 > 367.1, 349.1 (− 15, − 22) GTX4 412.1 > 314.1 (20) 410.1 > 367.1, 349.1 (− 15, − 22) GTX5 380.1 > 300.1 (16), 300.1 > 204.1 (24) 378.1 > 122 (− 25) GTX6 396.1 > 316.1 (15), 316.1 > 298.1 (15) 394.1 > 122 (− 25) dcGTX2 273.1 > 126.1 (20) 351.1 > 164.0, 333.1 (− 30, − 17) dcGTX3 353.1 > 255.1 (18) 351.1 > 333.1, 164.0 (− 17, − 30) C1 396.1 > 298.1 (20) 474.1 > 122.0, 351.1 (− 30, − 25) C2 396.1 > 298.1 (20) 474.1 > 351.1, 122.0 (− 25, − 30) C3 412.1 > 332.1 (10) 490.1 > 410.1 (− 20) C4 412.1 > 314.1 (20) 490.1 > 392.1 (− 20)

The numbers in parentheses are collision energy (eV)

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Fig. 2 Extracted ion mass chromatograms (m/z 320 > 302) of tetrodotoxin in the small gastropods: standard of tetrodotoxin at 0.5 μg/ml, Nas- sarius conoidalis, N. glans, N. pullus

Table 2 Total tetrodotoxin toxicities determined by UHPLC/MS–MS in three Nassarius species (n = 30) collected in Khanh Hoa Province, Vietnam in May 2016

Species Total toxicities (MU/g) Frequency of toxic specimens Mean ± SD Range (%)

N. conoidalis 144.8 ± 82.0 71.0–292.0 100.0 N. glans 412.7 ± 107.3 187.0–787.0 100.0 N. pullus 19.6 ± 9.8 10.0–46.7 100.0

as the horseshoe crab Carcinoscorpius rotundicauda (Dao et al. 2009a), the blue-ringed octopus Hapalochlaeta nulu- nata (Dao et al. 2009b), the goby Yongeichthys nebulosus (Dao 2009) and marine pufers (Dao et al. 2012). This result again indicates that a dominant toxin causing most of poisoning cases by consumption of marine toxic organ- isms in Vietnam is TTX. The toxicity levels in the specimens were all beyond the safe level of consumption (10 MU/g) for pufer(fsh) as sug- Fig. 3 MS/MS spectra of tetrodotoxin standard and the extract from gested in Japan (Kodama and Sato 2005) and comparable to Nassarius pullus. MS/MS spectra were acquired with a collision our previous report (Dang et al. 2015), strongly suggesting energy of 40 eV for the precursor of 320.0 that all specimens of three Nassarius species in Vietnam are highly toxic. On the other hand, no PSTs were detected in any specimen. Thus, the food poisoning due to the consump- Discussion tion of these small gastropods in Vietnam could be caused by TTX. Several marine organisms implicated in food poisonings The highest TTX toxicity observed in N. glans was still in Vietnam were found to be TTX-bearing species, such lower than that reported in Taiwan (2992 MU/g) (Hwang et al. 2005) and in Japan (4290 MU/g) (Taniyama et al.

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2009); however, consumption of only 12–13 g of soft tis- References sue from N. glans (equivalent to 5–6 specimens) contain- ing maximum toxicity may cause death in people, as the Boundy MJ, Selwood AI, Harwood DT, McNabb PS, Turner AD minimum lethal dose (MLD) for humans is estimated to (2015) Development of a sensitive and selective liquid chro- matography-mass spectrometry method for high throughput be approximately 10,000 MU (Noguchi et al. 2011). In analysis of paralytic shellfsh toxins using graphitized carbon contrast, according to this study, the toxicity of N. conoi- solid phase extraction. J Chromatogr A 1387:1–12 dalis in Vietnam (166.99 ± 146.94 MU/g) was much higher Nghi BQ (2005) Gastropod species in Khanh Hoa province. The than that from some other areas such as Chiating, Kaoh- proceedings of the 1st National Conference on “Ecology and Biology”. Agricultural Publishing House, Ha Noi, pp 172–185 siung, Taiwan (13.1 ± 5.9 MU/g) (Hwang and Jeng 1992) (in Vietnamese) or Enshunada, Japan (5.1 ± 2.2 MU/g) (Jeon et al. 1984). Dang QM, Pham XK, Dao VH, Le HKH, Nguyen TH, Phan BV, N. conoidalis and N. glans have been reported as being Doan TT (2015) Tetrodotoxin and saxitoxin in some Nassarius Nassarius TTX-bearing species from other contries such as Japan species ( Duméril, 1806) collected in Khanh Hoa waters. Coll Mar Res Works 21(2):70–79 (in Vietnamese with and Taiwan (Narita et al. 1984; Taniyama et al. 2009); but English abstract) neither toxin nor poisoning cases due to consumption of N. Dao VH, Sato S (2010) Toxicity of some marine snails responsi- pullus have been reported. Although lower TTX toxicity ble for recent food poisonings in Vietnam. 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Hwang PA, Tsai YH, Deng JF, Cheng CA, Ho PH, Hwang DF (2005) This should be an interesting subject for futher investiga- Identifcation of tetrodotoxin in a marine gastropod (Nassarius tion. The present results, together with previous results, glans) responsible for human morbidity and mortality in Tai- show that N. glans, N. conoidalis and N. pullus in Viet- wan. J Food Protect 68:1696–1701 Hwang PA, Tsai YH, Lin SJ, Hwang DF (2007) The gastropod pos- nam need to be paid more attention as potential causes sessing TTX and/or PSP. Food Rev Int 23:321–340 of poisoning. Also, this is an additional record that these Hylleberg J, Kilburn RN (2003) Marine molluscs of Vietnam, Poly- gastropods are now on the list of marine toxic organisms placophora, , Cephalopoda, Bivalvia, Scaphopoda. causing food poisoning in Vietnam. Annotations, voucher material, and species in need of verifca- tion. Phuket Mar Biol Cent Spec Publ 28:5–300 Acknowledgements Jeon JK, Narita H, Nara M, Noguchi T, Maruyama J, Hashimoto K This work is the result of the VAST project (1984) Occurrence of tetrodotoxin in a gastropod mollusk, "Ara- KHCBBI.02/18-20. The authors would like to give sincere thanks to regai" Niotha clathrata. Nippon Suisan Gakkaishi 50:2099–2102 Japan Funds in Trust (JFIT) for the IOC/WESTPAC-TMO project. The Kodama M, Sato S (2005) Pufer toxin. Shyokuhin Eiseikensasisin authors would also like to thank Prof. Oshima, Tohoku University, (The Manual for Food Sanitation Test). In: Ministry of Health, Japan, for providing the PST reference materials, and Mr. Bui Quang Labour and Welfare (ed). Japanese Hygienic Association, Nghi, Oceanographic Museum, Institute of Oceanography, Vietnam, Tokyo, pp 661–666 (in Japanese) for species identifcation and photos. 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