Chemosphere 219 (2019) 95e99

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Tetrodotoxin levels of three pufferfish species ( sp.) caught in the North-Eastern Mediterranean sea * € Ali Rıza Kosker a, , Fatih Ozogul a, Deniz Ayas b, Mustafa Durmus a,Yılmaz Ucar a, c, € Joe M. Regenstein d, Yesim Ozogul a a Faculty of Fisheries, Çukurova University, Adana, Turkey b Faculty of Fisheries, Mersin University, Mersin, Turkey c Faculty of Marine Sciences, Ordu University, Ordu, Turkey d Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA highlights graphical abstract

Pufferfish are harmful species that have spread rapidly in the Mediter- ranean Sea recently. TTX contents in pufferfish were determined by using the Q-TOF LC/ MS. This is the first report on the TTX content of L. suezensis and L. spadiceus in the Mediterranean Sea. Regardless of TTX levels, L. sceleratus and L. suezensis are highly toxic puf- ferfish that is dangerous for public health.

article info abstract

Article history: Tetrodotoxin (TTX) levels in Lagocephalus sceleratus (Silverstripe blaasop), Lagocephalus spadiceus (Half- Received 5 September 2018 smooth golden pufferfish) and Lagocephalus suezensis (Suez puffer) caught in Mersin Bay in the North- Received in revised form eastern Mediterranean Sea were analysed using Q-TOF LC/MS. Pufferfish were caught using trawl fishing, 30 November 2018 longlining and fishing line from December 2015 to October 2016. The TTX changes in the gonads, livers, Accepted 1 December 2018 intestines, skins and muscle tissues were evaluated according to sex and season. TTX levels for Available online 3 December 2018 L. sceleratus and L. suezensis for all tissues were in the range of 0.69e35.6 mg/g and 0.67e3.09 mg/g, Handling Editor: J. de Boer respectively. The highest TTX levels were observed in the gonads of female L. sceleratus caught in the autumn, and in the skin of L. suezensis caught in the spring but no quantifiable levels of TTX were found Keywords: for L. spadiceus. In conclusion, L. sceleratus and L. suezensis species caught in the North-Eastern Medi- Marine toxins terranean are toxic, and their consumption is unsafe. Lagocephalus sp. © 2018 Elsevier Ltd. All rights reserved. Pufferfish Tetrodotoxin Q-TOF LC/MS

* Corresponding author. Faculty of Fisheries, Cukurova University, 01330, Adana, Turkey. € E-mail addresses: [email protected] (A.R. Kosker), [email protected] (F. Ozogul). https://doi.org/10.1016/j.chemosphere.2018.12.010 0045-6535/© 2018 Elsevier Ltd. All rights reserved. 96 A.R. Kosker et al. / Chemosphere 219 (2019) 95e99

1. Introduction tetrodotoxin-ab120054 (Lot: APN14294-1-1) was used. Before the Q-TOF LC/MS determination, the TTX standard was diluted with Pufferfish species have been in the news in many Mediterranean 0.01 M acetic acid (Merck, Damstadt, Germany) and 4 dilutions countries (Ayas and Kos€ ¸ ker, 2018) because of their toxicity and wide (0.05, 0.1, 0.5, and 1 mg/mL TTX) were prepared from the stock distribution. Pufferfish consumption is considered to be risky. Most solution and a standard curve (r2 ¼ 0.9992) prepared. TTX standard pufferfish contain tetrodotoxin (TTX), which is one of the strongest solutions were kept at 20 C until used, a maximum of 4 wk. marine toxins. It is a non-protein, low molecular weight toxin. TTX is soluble in water and acidic environments, and is odourless and 2.2. Fish samples, identification and measurements tasteless. There are no known antidotes or antitoxins. TTX poi- sonings, caused by pufferfish consumption, occur regularly in Asia, Pufferfish samples that were trawled in the Northeastern especially in Japan (Hwang and Noguchi, 2007; Anonymus, 2018). Mediterranean from December 2015 to October 2016 to cover the 4 TTX poisoning affects the human nervous and mobility (muscle) seasons were obtained from a commercial vessel (Fig. 1). They were systems resulting in visible symptoms such as paralysis, muscle transported to the laboratory in ice for a maximum of 4 h. coordination disorders, respiratory distress and nausea (Isbister Size-weight measurements are shown in Table 1. The maximum et al., 2002). weight values for female and male individuals of L. sceleratus, L. Pufferfish from of Mediterranean Sea, may also have high spadiceus and L. suezensis were 3660, 2750, 832, 809, 92.8 and enough levels of TTX to potentially be toxic and even fatal (Bentur 96.6 g, respectively. The maximum lengths were 65.2 cm for both et al., 2008; Eisenman et al., 2008; Chamandi et al., 2009; Kheifets genders of L. sceleratus, and 36.6, 37.6, 20.5 and 21.0 cm, respec- et al., 2012). To date most studies on pufferfish toxicity in the tively, for the males and females of the other two species. Only Mediterranean Sea have focused on Lagocephalus sceleratus sexually mature pufferfish were used. Gender was determined with (Katikou et al., 2009; Rodriguez et al., 2012; Kosker et al., 2016; Acar the help of a microscope. For each season, 10 males and 10 females et al., 2017; Rambla-Alegre et al., 2017). Some species that came from each species were selected. into the Mediterranean Sea through the Suez Canal are economi- fi cally valuable, while some like puffer sh are considered invasive, 2.3. Samples preparation and TTX extraction poisonous species (Zenetos et al., 2012). Pufferfish have a harmful fi effect on commercially important sh species in addition to their Samples were dissected to take some dorsal muscle (cautiously potential toxicity (Streftaris and Zenetos, 2006; Bentur et al., 2008). avoiding the breakage of the gastrointestinal tract), the liver, in- Thus, many Mediterranean countries have taken some precautions testines, gonads, and the whole upper and lateral skin from head to fi against the possible risks from puffer sh (Kosker et al., 2018b). tail. A total of 80 composite samples were analysed for each species fi Authorities in the European Union banned the shing, landing and with two genders and 4 seasonal groups from 5 different tissue sale of all species in the family on the assumption parts, each with three replicates. fi fi that all puffer sh contain toxins. Even though all puffer sh are TTX extraction was carried out using the method of Silva et al. fi considered toxic, some puffer sh do not pose a danger to human (2012). For extraction, one g sample from each part was used. health, and are consumed in many countries (Kosker et al., 2015). Three mL of 1% acetic acid was added. Samples were then ho- fi For example, among the puffer sh common in the Mediterranean mogenized using the Ultra Turrax (IKA T25 Digital Ultra Turrax, fi fi Sea, L. spadiceus was the rst invasive puffer sh coming through Staufen, Germany). Afterwards, it was held in an ultrasonic bath the Suez Canal to become established in the Mediterranean (Bandelin Sonorex RK 100, Berlin, Germany) for 10 min. The (Mavruk and Avsar, 2008). This species is consumed in Eastern mixture was held at room temperature (23 C) for 15 min, and then Asian countries like Japan, Thailand etc. but is among the species centrifuged (Hettich Zentrifugen, Universal 32R, Tuttlingen, Ger- banned by both the European Union (EC, 2004) and the Turkish many) at 4500 g for 20 min at 4 C. The supernatant was removed, Ministry of Food, Agriculture and Livestock (Anonymous, 2016). and 3 mL of 1% acetic acid was added to the residue and the However, no reported cases of poisoning associated with extraction procedures were repeated. The supernatants were L. spadiceus were found in the literature. Studies on L. suezensis combined and brought to 7 mL. The final solution was vortex (ZX3, were quite limited. This species spreading to the coasts of Spain VELP, Usmate, Milan, Italy) mixed with the help of a vacuum ~ from the Eastern Mediterranean within a decade (Izquierdo-Munoz manifold device (Supelco, Bellefonte, PA, USA-57030-U). The and Izquierdo-Gomez, 2014). L. sceleratus also spread quickly, but it has had a negative effect on fishing and public health (Streftaris and Zenetos, 2006). L. sceleratus is the best studied Mediterranean pufferfish with respect to the presence of toxin, which has been definitely found in all cases (Katikou et al., 2009; Rodriguez et al., 2012; Kosker et al., 2016; Acar et al., 2017; Rambla-Alegre et al., 2017). There is one study on the TTX levels of T. flavimaculosus (Kosker et al., 2018a). However, no toxin studies were found for L. spadiceus and L. suezensis in the Mediterranean Sea. Therefore, the effects of gender and season on the toxicity of common Medi- terranean pufferfish, L. sceleratus (Silverstripe blaasop), L. spadiceus (Half-smooth golden pufferfish) and L. suezensis (Suez puffer) were analysed for TTX.

2. Materials and methods

2.1. Tetrodotoxin (TTX) standard

The TTX standard was bought from Abcam Biochemicals (Cambridge, UK). For the Q-TOF LC/MS toxin analysis, one mg of Fig. 1. Map of the sampling location. A.R. Kosker et al. / Chemosphere 219 (2019) 95e99 97

Table 1 Length and weight of pufferfish samples.

Seasons N Female Male

Length (cm) Weight (g) Length (cm) Weight (g)

Min-Max Mean ± SD Min-Max Mean ± SD Min-Max Mean ± SD Min-Max Mean ± SD

L. sceleratus Winter 10 49.5e65.2 57.2 ± 5.12 1284.2e3661.0 2518.8 ± 846.3 47.6e65.2 57.0 ± 6.14 1268.2e2746.8 2052.9 ± 549.1 Spring 10 46.2e64.9 55.6 ± 6.12 1152.4e3131.5 2163.7 ± 734.0 44.9e55.9 50.5 ± 3.75 1074.5e2196.6 1635.0 ± 379.5 Summer 10 45.9e54.1 49.8 ± 3.14 943.1e1729.8 1337.4 ± 289.0 43.3e45.2 44.4 ± 0.83 784.8e996.2 891.0 ± 88.5 Autumn 10 48.1e63.1 55.7 ± 5.24 1120.2e3243.2 2121.9 ± 743.7 44.7e60.6 52.7 ± 5.26 1154.1e2274.1 1713.3 ± 394.6 L. spadiceus Winter 10 18.6e23.9 21.3 ± 2.00 128.3e212.5 170.2 ± 27.6 19.5e26.0 22.7 ± 2.07 133.3e306.6 219.3 ± 70.1 Spring 10 25.6e36.6 31.0 ± 3.82 262.2e832.2 546.9 ± 203.7 25.1e37.6 31.4 ± 4.29 277.4e808.7 542.9 ± 195.0 Summer 10 16.6e28.6 20.1 ± 3.70 73.5e316.1 193.7 ± 88.5 15.6e20.5 18.0 ± 1.99 70.2e124.6 97.2 ± 18.8 Autumn 10 14.9e18.7 16.7 ± 1.39 57.5e123.3 89.9 ± 24.8 14.3e19.3 16.7 ± 1.67 77.2e152.5 113.3 ± 23.1 L. suezensis Winter 10 12.2e19.7 16.7 ± 2.14 25.4e92.8 59.6 ± 21.3 15.1e19.1 17.1 ± 1.25 45.0e80.8 62.9 ± 12.7 Spring 10 16.6e20.5 18.6 ± 1.13 64.0e90.5 77.7 ± 8.77 12.8e21.0 17.0 ± 2.38 27.9e96.6 62.0 ± 22.6 Summer 10 13.1e17.0 15.2 ± 1.40 24.27e57.3 41.8 ± 12.2 12.6e16.5 14.1 ± 1.26 27.0e43.6 35.5 ± 5.9 Autumn 10 13.4e16.1 14.7 ± 0.87 29.01e50.2 39.9 ± 8.74 14.4e15.1 14.5 ± 0.42 25.5e34.2 27.8 ± 2.6

X: Mean, SD:Standard deviation, N: number of samples. mixture sample (1 mL) was put onto a C18 solid-phase extraction Mediterranean L. sceleratus had the highest levels of TTX (Katikou (SPE) cartridge (Supelco, Bellefonte, PA, USA) that had been primed et al., 2009; Rodriguez et al., 2012; Kosker et al., 2016; Acar et al., with 6 mL methanol followed by 6 mL water. Following sample 2017; Rambla-Alegre et al., 2017)(Table 3). Generally the highest application, methanol (10 mL) was put through the cartridge. The toxin levels in both males and females were found in autumn and solution was brought to 12 mL with methanol and mixed using the winter as previously observed by Katikou et al. (2009); Rodriguez vortex. The final solution was evaporated (Buchi, Flawil, et al. (2012); and Kosker et al. (2016) (Table 3). Switzerland) and 1 mL methanol was added to the residue. It was The first study on the toxicity of L. sceleratus in Mediterranean then filtered through 0.45 m membrane filters (Isolab, Wertheim, region was carried out by Katikou et al. (2009). Later studies Germany) and moved to vials for Q-TOF LC/MS analysis. (Rodriguez et al., 2012, Kosker et al., 2016; Acar et al., 2017; Rambla- Alegre et al., 2017) have also confirmed that this species is toxic. In 2.4. TTX analysis the previous studies on L. sceleratus in the Mediterranean Sea (Table 3), TTX levels found in gonads, liver, intestines, skin and TTX analysis was done using the method of Kosker et al. (2018a) muscle tissues were 0.17e239, 0.12e87.5, 0.07e178, 0.13e6.63 and on an Agilent 6545 Accurate-Mass Q-TOF LC/MS (Agilent Technol- 0.07e10.2 mg/g, respectively (Katikou et al., 2009; Rodriguez et al., ogies, Inc., Santa Clara, CA, USA) coupled with an Agilent 1260 HPLC 2012; Kosker et al., 2016; Acar et al., 2017; Rambla-Alegre et al., (Agilent Technologies, Inc.). A Poroshell 120 Hilic (3.0 50 mm; 2017). In this study, TTX levels fall within these ranges except for 2.7 mm) column (Agilent Technologies, Inc.) was used. The injection the females in autumn. All these studies show that L. sceleratus is a volume was 10 ml. The limit of detection (LOD; S/N > 3) of the toxic and dangerous L. sceleratus is an invasive species that reached method for TTX was 0.3 mg/g. The limit of quantification (LOQ; S/ the Spanish shores within 10 years of entering the Mediterranean N > 10) was reproducible at 0.6 mg/g of TTX. Method and instru- (Rambla-Alegre et al., 2017). However, more scientific research and ment conditions were given in detail by Kosker et al. (2018a). risk assessments with regards to its toxicity and other potential public health concerns have to be done. 2.5. Statistical analyses 3.3. TTX levels in L. suezensis The data were reported as the mean and standard deviation in triplicates. Duncan's multiple range test comparisons at p < 0.05 The TTX level of L. suezensis were lower than L. sceleratus. TTX were used to determine significant differences, and one-way contents of individual L. suezensis gonads, liver, skin, intestines and analysis of variance (ANOVA) was done using the Statistical Pack- muscle tissues were determined to be in the range of 0.67e3.09 mg/ age for the Social Sciences 18.0 software (Co., City, State, USA) for g(Table 2) during all four seasons. TTX content was found to be multiple comparison. higher in the skin and gonad samples followed by intestines, liver and muscle tissues (Fig. 2) which is different from L. sceleratus. 3. Results and discussion Similarly, while the highest toxicity in L. sceleratus was found in the winter and autumn, the highest TTX levels were found in L. suzensis 3.1. General in the winter and spring, whereas no TTX was found in any sample during the summer. Fishbase (Froese and Pauly, 2017) indicates that The TTX levels of the three pufferfish species, both from the L. suezensis is harmless for humans, but there is no supporting literature and from this study, are shown in Table 3. TTX has not reference and the information may be dangerously wrong as shown been detected in any sample of L. spadiceus caught in the Medi- here. L. suezensis has been shown to be a toxic and dangerous terranean (Table 2). pufferfish species for human consumption.

3.2. TTX levels in L. sceleratus 3.4. TTX levels in L. spadiceus

TTX levels in the tissues of L. sceleratus were between 0.69 and No TTX was found above the detectable levels of 0.6 mg/g. Thus, 35.6 mg/g (Table 2). The highest TTX contents were found in the TTX were found in L. spadiceus. Studies in many other countries gonad tissue followed by livers, intestines, skins and muscles (especially Asian countries) showed that this species is not toxic (Fig. 2). This study confirms that the gonads followed by the liver of and can be consumed (Berry and Bin Hassan, 1973; Yamaguchi 98 A.R. Kosker et al. / Chemosphere 219 (2019) 95e99

Table 2 Sexual and seasonal TTX levels in different tissues of L. sceleratus, L.spadiceus and L. suezensis (mg/g) using Q-TOF LC/MS analysis.

Season Sex N Sex Gonad Liver Muscle Skin Intestine ± ± ± ± ± X SX X SX X SX X SX X SX L. sceleratus Winter \ 10 \ 16.6 ± 0.47bx 2.87 ± 0.86by 3.60 ± 0.83bx 2.20 ± 0.39cx 0.85 ± 0.13cx _ 10 _ 2.93 ± 0.08cy 8.73 ± 0.83bx 4.90 ± 0.02ax 2.90 ± 0.02dx 3.00 ± 0.94ax Spring \ 10 \ 4.91 ± 0.49cx 0.89 ± 0.26bx 0.70 ± 0.07cx 2.58 ± 0.43cx 0.79 ± 0.04cy _ 10 _ 0.69 ± 0.11dy 1.70 ± 0.35cx 1.17 ± 0.32cx 3.58 ± 0.13cx 2.81 ± 0.12ax Summer \ 10 \ 4.15 ± 0.34cx 1.03 ± 0.61by 0.80 ± 0.07cy 3.85 ± 0.01bx 1.83 ± 0.00by _ 10 _ 5.85 ± 1.00bx 12.3 ± 1.66ax 4.32 ± 0.28ax 5.02 ± 0.05ax 2.29 ± 0.04ax Autumn \ 10 \ 35.6 ± 3.75ax 21.1 ± 1.96ax 5.12 ± 0.48ax 11.8 ± 0.29ax 12.5 ± 0.65ax _ 10 _ 11.8 ± 0.89ay 6.99 ± 1.09by 2.61 ± 0.40by 4.28 ± 0.30by 2.96 ± 0.10ay L. spadiceus Winter \ 10 \

Values in same column with different letters (a,b,c,d) are significantly different (p < 0.05). Different letters in the same column (x,y) in every season indicate significant differences for both sexes (p < 0.05). X: Mean, Sx: Standard deviation, the limit of quantification (LOQ): 0.6 (mg/g).

Table 3 TTX levels of Lagocephalus sceleratus (mg/g) from various studies in the Mediterranean countries.

Muscle Gonad Intestine Liver Skin Region

El-Sayed et al. (2003) ND e 27.9 ND e 165 ND e 48.6 ND e 54.1 ND e 26.2 Northwestern Red Sea Katikou et al. (2009) <1.10e10.1 17.05e239 6.31e178 16.12e88 <1.10e6.63 Aegean Sea Rodriguez et al. (2012)

———: No Data, ND: Not detected, NI: Number of individual, LOQ: Limit of quantitation.

However, Azman et al. (2014) reported the TTX levels in the liver, muscles and skin of pufferfish from the species L. spadiceus caught in Malaysia were 8.09, 8.71 and 1.71 mg/g, respectively. This report may represent a real concern but might also be due to the difficulty of differentiating L. spadiceus from L. guentheri, L. wheeleri and L. lunaris all of which are morphologically similar. The con- sumption of L. wheeleri and L. spadiceus is widespread as these species are considered to be non-toxic species (Yamaguchi et al., 2013; Ngy et al., 2008; Yu and Yu, 1997; Brillantes et al., 2003; Chulanetra et al., 2011; Simon et al., 2009). However L. lunaris is a toxic species (Brillantes et al., 2003; Chulanetra et al., 2011). Due to incorrect identification, there have been over 10 TTX poisoning cases in Japan and Taiwan, where consumers thought they are eating L. spadiceus, but were actually consuming L. lunaris (Hwang and Noguchi, 2007; Noguchi and Ebesu, 2001). Fig. 2. TTX levels (mg/g) of female L. sceleratus (a), male L. sceleratus (b), female L. suezensis (c) and male L. suezensis (d) according to tissue and season. More studies on the toxin content of L. spadiceus should be done to establish its safety so that it may be possible in the future to lift the ban on its sale in the European Union and Turkey. et al., 2013; Ngy et al., 2008; Yu and Yu, 1997; Brillantes et al., 2003; Chulanetra et al., 2011). L. spadiceus is often consumed in countries 4. Conclusion like Malaysia, Thailand, China and Japan (Brillantes et al., 2003; Kaewnern et al., 2013; Yu and Yu, 1997; Berry and Bin Hassan, Among the three pufferfish species studied for TTX content, the 1973; Yamaguchi et al., 2013). Over 90% of pufferfish imported highest toxicity was shown by L. sceleratus followed by L. suezensis. into Japan are L. spadiceus (Yamaguchi et al., 2013). This study concluded that these two species should be considered A.R. Kosker et al. / Chemosphere 219 (2019) 95e99 99 dangerous for humans although levels of TTX varied with organ and Version (10/2017). www.fishbase.org. season. On the other hand, L. spadiceus, did not contain any TTX Hwang, D.F., Noguchi, T., 2007. Tetrodotoxin poisoning. Adv. Food Nutr. Res. 52, 141e236. levels that could be quantitated. Thus further studies on the toxicity Isbister, G.K., Son, J., Wang, F., Maclean, C.J., Lin, C.S., Ujma, J., Balit, C.R., Smith, B., of these species in Mediterranean region are needed particularly to Milder, D.G., Kiernan, M.C., 2002. Puffer fish poisoning: a potentially life- e establish the safety of L. spadiceus. threatening condition. Med. J. Aust. 177, 650 653. Izquierdo-Munoz,~ A., Izquierdo-Gomez, D., 2014. First record of Lagocephalus sce- leratus (Gmelin, 1789) (, Tetraodontidae) on the Mediterranean Conflicts of interest Spanish coast. Katsanevakis S et al. (2014), New Mediterranean Biodiversity Records (October, 2014). Mediterr. Mar. Sci. 15 (3), 675e695. Kaewnern, M., Dulyapurk, V., Mookdasnit, J., Jumnongsong, S., 2013. Perception of The authors declare that they have no competing interests. consumers on puffer fish and willingness to pay for consuming puffer fish (Lagocephalus spadiceus). In: Proceedings of the 51st Kasetsart University Acknowledgements Annual Conference, Bangkok, Thailand, 5-7 February 2013. Kasetsart University. Katikou, P., Georgantelis, D., Sinouris, N., Petsi, A., Fotaras, T., 2009. First report on toxicity assessment of the lessepsian migrant pufferfish Lagocephalus sceler- This research was financially supported by the Scientific and atus (Gmelin, 1789) from European waters (Aegean Sea, Greece). Toxicon 54, Technological Research Council of Turkey (TUBITAK); TOVAG- 50e55. ı 115O679. We would like to thank graphic designer Yeter MERCAN Kheifets, J., Rozhavsky, B., Girsh Solomonov ch, Z., Marianna, R., Soroksky, A., 2012. 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