The seasonal toxicological profile of four puffer fish species collected along Bengal coast, India

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1 Indian Journal of Marine Sciences Vol 33(3), September 2004, pp The seasonal toxicological profile of four puffer fish species collected along Bengal coast, India Somiranjan Ghosh, Alok K. Hazra, Shivaji Banerjee & Biswapati Mukherjee* S. N. Pradhan Centre for Neurosciences, University of Calcutta, 244B, Acharya J. C. Bose Road, Calcutta , India *[ impuffer@cal2.vsnl.net.in] Received 17 November 2003, revised 29 June 2004 Toxicological profiles of the livers and ovaries of four Indian marine puffer fish species viz. Chelonodon patoca, Takifugu oblongus, Lagocephalus lunaris, Lagocephalus inermis collected along coastal Bengal of Digha-Talsari region were evaluated by mouse bioassay. Toxicity expressed in MU (mouse unit)/g in ovaries of all four species were high in monsoon ( MU/g) and postmonsoon ( MU/g) seasons during annual reproductive cycle. However, toxicity of livers was much lower ( MU/g) in all the species with seasonal variation. Chelonodon patoca specimens were found to be most toxic and Lagocephalus inermis were least toxic in comparison to other species under investigation. Therefore, health hazard due to puffer fish consumption could be minimised by the information given in this study regarding lethality. [Key words : Puffer, toxicity, Chelonodon patoca, Takifugu oblongus, Lagocephalus inermis, Lagocephalus lunaris, fish] [IPC Code: Int. Cl. 7 G01N 33/12] Introduction Evidence of puffer fish intoxication has been reported in different geographical locales viz., Japan 1, USA 2, Taiwan 3, Mexico 4, Malaysia 5, Bangladesh 6 and also in India 7. Jones 8 reported deaths due to ingestion of cooked roe of puffer Chelonodon patoca for the first time in India in Although the occurrence of puffer fish poisoning is sporadic in India, there is no systematic documentation of their toxicological profiles. The toxicological profiles of wild puffer fishes differ widely amongst different species 9, time of collection 10 and the geographical region of occurrence 11. Even the anatomical distribution of toxicity varies from species to species 12. The present work was undertaken to find out the toxicological profiles of puffer fishes with seasonal variation collected along Bengal coast, India and to assess the toxicity. Materials and Methods Four species of marine puffers, viz. Chelonodon patoca Hamilton (Class:Actinopterygii, Order: Tetraodontiformes, Family:Tetraodontidae), Takifugu oblongus Bloch (Class:Actinopterygii, Order: Tetraodontiformes, Family:Tetraodontidae), Lagocephalus lunaris Bloch & Schneider (Class:Actinopterygii, Order:Tetraodontiformes, Family:Lagocephalidae), Lagocephalus inermis Temminck & Schlegel (Class:Actinopterygii, Order: Tetraodontiformes, Family:Lagocephalidae) were collected regularly from Digha-Talsari region of Bengal, eastern coast of India (latitude N, longitude E) during April 2000 March The voucher specimens of different puffer fishes have been submitted to National Marine Repository, National Institute of Oceanography, Goa, India. The catch was segregated into three prominent seasons, i.e. premonsoon (March June, summer; temp o C, rainfall cm), monsoon (July October, rainy; temp o C, rainfall cm) and postmonsoon (November February, winter; temp o C, rainfall cm). The puffer species collected from the coastal waters were immediately transferred on ice to the laboratory. After random sampling ten fish specimens of each species were chosen for toxicity assessment. Livers and ovaries of each different puffer species, according to the respective seasons were dissected out and kept in vacuum-sealed containers below -20 C. The liver (10g) and ovary (10g) of each specimen of different puffer fish species were separately homogenized for 5 min with 3 volumes of 1% acetic

2 GHOSH et al : TOXICOLOGICAL PROFILE OF PUFFER FISH 277 acid in methanol and centrifuged (3000 g) for 15 min. The residue was extracted twice in the same manner. The supernatants were combined, concentrated under reduced pressure, the residue dissolved in 0.1% aqueous acetic acid and defatted 3 times with dichloromethane. The aqueous layer was ultrafiltered with Amicon YM-1 membrane to cut off substances of more than 1000 daltons. The filtrate was freeze-dried and examined for toxicity by mouse bioassay. Mouse toxicity assay, similar to the method developed for PSP (paralytic shellfish poisoning) toxin monitoring 14, has been applied for toxicity determination among puffers. The mouse bioassay was standardized for tetrodotoxin (TTX) in puffers after Kawabata 13 and was expressed as mouse unit (MU) per gram of material, where one MU is the equivalent amount of TTX (0.22 µg) that kills a mouse of ddy strain weighing 20 g in 30 minutes. The lyophilized extract from each puffer specimen was dissolved in 1ml saline solution for mouse toxicity assay. Injections were made intraperitoneally to Swiss albino mice (19-21 g) (obtained from Central Drug Laboratory, Kolkata). A group of three mice were used for each experiment. The lethal potency was calculated as the time required for killing the mice and was expressed in mouse unit (MU) 13. All the experiments were statistically analyzed and expressed in Mean ± SD. Results Anatomical distribution of toxicity in the puffer Chelonodon patoca is shown in Table 1A. The size of all fishes collected was almost same and the body weight in postmonsoon season (1121± g) was nearly double with respect to other seasons. Female fishes were more abundant except in monsoon season when male and female fishes were found in equal proportion. The hepatosomatic inedx (liver weight percent with respect to body weight, H I ) increased gradually from premonsoon (8.0±1.2) to monsoon (10.5±0.8) and postmonsoon (14.0±1.6). Gonadosomatic index (gonadal weight percent with respect to their body weight, G I ) in monsoon (9.72±2.9), postmonsoon (6.29±2.6) indicated abundance of gorged ovaries full of matured eggs compared to premonsoon (0.44±0.21). From toxic ratio and frequency of toxicity it was observed that almost all the species of C. patoca were toxic. The toxicity of liver in premonsoon, monsoon and postmonsoon seasons were 9.5±3.7, 15.0±3.9, 18.5±9.3 MU/g respectively indicating slight increment in toxicity through seasons. Matured ovaries were not found in premonsoon season and the toxicity was less than 4 MU/g. However, during monsoon, fish species with matured ovaries were plenty as evidenced from G I (9.72±2.9) and toxicity was 80.2 ± 12.9 MU/g. The toxicity of ovaries in postmonsoon season was even more, 136.4±20.3 MU /g. The body weight and length of T. oblongus fish specimen were almost same in all three seasons (Table 1B). Female fishes are abundant throughout the year. Liver weight in monsoon season (15.0±3.25) was higher than premonsoon (10.0±2.90) and postmonsoon (11.0 ± 3.10), as evidenced from hepatosomatic index. The gonadosomatic index increased abruptly in monsoon (7.45±1.8) and postmonsoon (5.91±1.6) compared to the premonsoon (0.56±0.3). Frequencies of toxicity in T. oblongus specimens were almost cent per cent. The toxicity in livers viz., 4.8±2.8, 12.0±4.2, 9.2±1.7 MU/g in premonsoon, monsoon and postmonsoon seasons respectively indicated that it was more toxic in monsoon seasons. Ovaries developed substantial toxicity in monsoon (24.5±4.6) and in postmonsoon (22.0±5.9) compared to premonsoon. Lagocephalus lunaris were found plenty throughout seasons (Table 1C). The body weight varied slightly, although the lengths were same in three seasons. Female fishes were abundant as observed in C. patoca (Table 1A) and T. oblongus (Table 1B). Hepatosomatic index in premonsoon, monsoon and postmonsoon were 4.6±1.00, 7.9±1.50, 6.0±1.90 respectively which indicated increased liver size in monsoon season. Mature ovaries were found in monsoon and postmonsoon as evident from gonadosomatic index 8.05±2.1, 5.56±2.7 respectively. Frequency of toxicity among specimens were almost same (84.37, 85.18, 77.78) throughout the seasons. Liver toxicity was highest in monsoon 7.2±1.8 MU/g compared to premonsoon (4.9±1.9) and postmonsoon (6.8±1.6). Ovaries were toxic in monsoon (23.2±6.9) and postmonsoon (8.9±2.5). Lagocephalus inermis fishes were larger in premonsoon (21.35±3.1) than monsoon (18.83±2.9) and postmonsoon (18.74±2.4). Similar trend was reflected in their average body weight (Table 1D). Female specimens were available more than male

3 278 INDIAN J. MAR. SCI., VOL. 33, No. 3, SEPTEMBER 2004 Table 1 Anatomical distribution of toxicity in the puffer fishes collected from Bengal coast during March 2000 February 2001 ( - Mar-June, July-Oct, Nov-Feb) Seasons (n=19 d ) (n= 15 d ) (n=16 d ) (n=15 d ) (n= 13 d ) (n=16 d ) (n=13 d ) (n= 17 d ) (n=17 d ) Body weight(g) Length (cm) M/F ratio H I a G I b Toxic c ratio Frequency of toxicity (%) Toxicity (MU/g) Liver Ovary (A) Chelonodon patoca 447.5± ±3.2 3 : 4 8.0± ± / ±3.7 e ND f 516.0± ±2.9 1 : ± ±2.9 15/ ±3.9 e 80.2±12.9 e ± ±4.6 1 : ± ±2.6 16/ ±9.3 e 136.4±20.3 e (B) Takifugu oblongus 354.0± ±3.1 1 : ± ±0.3 14/ ±2.8 e ND f 362.0± ±2.5 1 : ± ±1.8 13/ ±4.2 e 24.5±4.6 e 423.9± ±4.2 3 : ± ±1.6 16/ ±1.7 e 22.0±5.9 e (C) Lagocephalus lunaris 142.3± ±4.2 2 : 3 4.6± ±0.7 11/ ±1.9 e ND f 189.7± ±2.7 3 : 7 7.9± ±2.1 15/ ±1.8 e 23.2±6.9 e 216.5± ±3.4 3 : 7 6.0± ±2.7 13/ ±1.6 e 8.9±2.5 e (D) Lagocephalus inermis ± ±3.1 1 : 4 3.1± ±0.6 9/ ND f ND f (n=12 d ) ± ±2.9 3 : 7 9.0± ±3.2 11/ ±1.2 e 13.9±2.7 e (n= 14 d ) ± ±2.4 2 : 3 7.6± ±1.9 16/ ±0.8 e 14.2±2.2 e (n=19 d ) a = Hepatosomatic index (%), b = Gonadosomatic index (%), c = Specimen toxicity in terms of liver and gonad, d = number of specimens, e = Means±SD (n=2), f = ND means less than 4 MU/g fishes. Hepatosomatic index in monsoon season was highest (9.0±2.3) indicative of larger liver size. Gonadosomatic index in monsoon and postmonsoon seasons were 6.44±3.2 and 4.28±1.9 respectively. This was quite higher than gonadosomatic index in premonsoon season (0.98±0.6). Frequency of toxicity is almost same through seasons. Livers were feebly toxic in monsoon (4.6±1.2 MU/g) and postmonsoon (4.9±0.8 MU/g). Similar trend was found in case of gonadal toxicity. Interestingly during premonsoon season the fishes were non-toxic. All four-puffer species viz., C. patoca, T. oblungus, L. lunaris and L. inermis followed similar trend with respect to their average body weight, average length and abundance of female fishes throughout seasons (Table 1). The livers were bulky in monsoon seasons compared to other seasons as evident from hepatosomatic index (H I ) with similar species variation. Toxicities of liver were comparatively higher during monsoon than premonsoon in all species. The gonadosomatic index was much higher during monsoon and postmonsoon compared to premonsoon which indicated the enlargement of ovaries in female fishes and roes consisting of mature eggs in all puffer species. It was observed that they had spawning seasons, twice in a year from June- August and December to February. Toxicity in ovaries sharply increased from premonsoon to monsoon with a concurrent increase in gonadosomatic index. Among the puffers C. patoca showed maximum toxic potency of 136 MU/g in their ovary. The lethal potency of ovaries was more than their respective livers in all puffer species. Discussion Puffers are toxic due to the occurrence of tetrodotoxin (TTX) in the tissues, a neurotoxin and

4 GHOSH et al : TOXICOLOGICAL PROFILE OF PUFFER FISH 279 TTX was found in greater concentration in their vital organ like liver and ovary 15. Often food poisoning was reported from different geographical regions due to puffer fish ingestion and the lethality was dependent on the concentration of TTX 16 present in consumed fish tissues. The mouse bioassay method has been successfully employed to monitor and assess toxic potential in puffers. Reports available from the Japanese market indicate that the severity of the clinical poisoning in humans depends upon both the concentration of toxin and quantity of toxic organ ingested 17. Therefore, the consumption of puffers is regulated in Japan and it is considered to be unsafe for human consumption if the toxicity 13 is > 10 MU/g. Wild puffers were found toxic although their anatomical distribution of toxicity has wide species variation 18. Lethal potency also showed seasonal variation in a single puffer species 10. Present investigation showed C. patoca is the most toxic species in comparison to other puffers collected along Bengal coast (Table 1). Moreover, the matured ovaries of C. patoca showed maximal lethal potency (136 MU/g). A toxicity study from Okinawa, Japan has shown that of the tissues tested of muscle, skin, liver, gonad and intestine, extremely high toxicity was in the muscle of C. patoca (520 MU/g) 19. Lethality of extracts obtained from the anatomical organs of 6 species of southern puffer from Tropics were studied. In most of the specimens of C. patoca the highest level of toxicity was detected in skin and ovaries 20. Lethality increased as the fishes approached to their spawning season and it might be expected that the total amount of toxin would also increase with the maturation of the ovaries. Tani 21 clearly showed that the concentration of toxin per gram ovary increased with the increase in the weight of the ovaries. The concentration of toxin in liver also increased in a parallel way even though the liver weight did not change appreciably. There were also some species variations in the relative concentration of toxin in the ovary 18,19. The toxicological profile of T. oblongus (Table 1B) showed similar seasonal variation as in C. patoca (Table 1A). They had comparatively less lethal potential than C. patoca, whereas L. lunaris and L. inermis showed much decrease in their lethal potency. It was reported 9,22 that L. lunaris having highest toxicity in their ovary (460 MU/g) followed by intestine (380 MU/g) and muscle (100 MU/g), whereas, the highest score in T. oblongus was in the liver (2000 MU/g) followed by ovary (770 MU/g). Toxicological investigation on puffers in this study revealed that the ovaries showed maximum lethal potency in their spawning season when ovaries were found with full of matured eggs. Livers and ovaries were mainly tested as these are the organs where the toxin is concentrated more in most cases and mainly responsible for clinical poisoning while ingested 23. There is a proportional relationship between toxicity and gonadosomatic index suggesting that the increased toxin is a product of embryos as postulated by Matsumura 24. It has been observed that increased weight of liver was due to the fat deposition 25. However, the livers of the four species had a tendency to be lethal in the reproductive cycle (Table 1). The distribution of toxicity in organs of four puffer species showed a prominent seasonal pattern as reported in puffers collected along Hong Kong coastal waters 10. This investigation also indicated that L. inermis is least toxic (Table 1D). This study also reveals that four species of puffer collected along Bengal coast were less toxic than the same species collected in other coastal waters 22,26. However, this investigation may help reduce the health hazard due to consumption of puffer fishes in the coastal region of Bengal by identifying the season when they are dangerously toxic. Acknowledgement This work was supported by the research grant from Department of Ocean Development, Govt. of India. The Marine Fish Division, Zoological Survey of India, Calcutta, assisted in identifying the fishes. References 1 Yoshikawa-Ebesu, J.S.M., Hokama, Y. & Noguchi, T., Tetrodotoxin, in: Foodborne disease handbook, Vol.4, edited by Hui, Y.H., Kitts, D. & Stanfield, P.S. (Marcel Dekker Inc., New York) 2001, pp Lange, W.R., Puffer fish poisoning, Am. Fam. Physician, 42 (1990) Yang, C.C., Liao, S.C. & Deng, J.F., Tetrodotoxin poisoning in Taiwan: an analysis of poison center data, Vet. Hum. 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5 280 INDIAN J. MAR. SCI., VOL. 33, No. 3, SEPTEMBER 2004 substances, in: Nutrients and biocative substances in aquatic organism, edited by Devadasan K, Mukundan P D, Antony P G, Nair V, Penigreen P A & Joseph J, (Paico Printing Press, Cochin, Inida) 1993, pp Jones, S., Some deaths due to fish poisoning (Icthyosarcotoxism) in India. Indian J. Med. Res., 42 (1956) Few, H.D., Noguchi, T., Arakawa, O., Abe, T. & Hasimoto, K., Toxicological studies on several species of puffer in Taiwan, Bull. Jap. Soc. Fish., 54 (1988) Yu, C.F. & Yu, P.H., The annual toxicological profiles of two common puffer fish, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected along Hong Kong coastal waters, Toxicon, 40 (2002) Matsui, T., Hamada, S. & Yamamori, K., Local variation of toxicity of the puffer fish Fugu niphobles, Bull. Jap. Soc. Sci. Fish., 48 (1982) Fuchi, Y., Hoashi, K., Akaeda, H., Makino, Y. & Noguchi, T., Anatomical distribution and seasonal variation of toxicity of puffer fish, Hoshifugu Arothron firmamentum specimen collected from the Bungo Channel, Oita, J. Food Hygn. Soc. Jap. 39 (1998) Kawabata, T., Puffer toxin, in: The manual for methods of food sanitation tests, Vol. 2, (Environmental Health Bureau, Japan Food Hygienic Association, Tokyo) 1978, pp Anon, Official methods of analysis of AOAC International, Vol. II, edited by Cunniff PA (AOAC International, Washington DC, U.S.A.) 1995, pp Kao, C.Y., Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomenon, Pharmacol. Rev., 18 (1966) Chou, S.S., Tsai, Y.H., Chang, P.C. & Hwang, D.F., Tetrodotoxin-associated food poisoning due to ingesting fish, J. Food Drug Anal., 2 (1994) Murata, E.F., Stabile, D.E. & Wills, J.H., Some pharmacological effects of puffer poison, J. Pharmacol. Exptl. Therap., 22 (1958) Endo, R., Toxicological studies on puffer fishes: comparison of toxicities in the various species, J. Toxicol. Sci., 1 (1984) Khora, S.S., Isa, J. & Yasumoto, T., Toxicity of puffers from Okinawa, Japan, Bull. Jap. Soc. Fish., 57 (1991) Khora, S.S. & Yasumoto, T., Toxicity of Soutrhern puffers, in: Nutrients and biocative substances in aquatic organism, edited by Devadasan K, Mukundan P D, Antony P G, Nair V, Penigreen P A & Joseph J (Society of Fisheries Technologists (India), Paico Printing Press, Cochin, India) 1993, pp Tani, I., A study of the toxicity of Japanese Fugu (Teikoku Tosho Co., Tokyo, Japan.) 1945, pp Hwang, D.F., Chung, H.M., Lin, M.C., Mock, H.K. & Jeng, S.S., Food poisoning due to ingestion of the puffer Lagocephalus lunaris, J. Fish. Soc. Taiwan, 16 (1989) Hwang, D.F., Chung, C.R., Ling, L.C. & Jeng, S.S., Tetrodotoxin-associated food poisoning due to ingesting fish roe, J. Biomed. Lab. Sci., 4 (1989) Matsumura, K., Production of tetrodotoxin in puffer fish embryos, Env. Toxicol. Pharm., 6 (1998) Hazra, A.K., Ghosh, S., Banerjee, S. & Mukherjee, B., Studies on lipid and fatty acid compositions of puffer livers from Indian coastal waters with seasonal variation, J. Am. Oil Chem. Soc., 75 (1998) Mahmud, Y., Tanu, M.B., Takatani, T., Asayama, E., Arakawa, O. & Noguchi, T., Chelonodon patoca, a highly toxic marine puffer in Japan, J. Nat. Toxins, 10 (2001)

(Graduate School of Marine Science and Engineering,Nagasaki University: 1-14, Bunkyomachi, Nagasaki , Japan)

(Graduate School of Marine Science and Engineering,Nagasaki University: 1-14, Bunkyomachi, Nagasaki , Japan) Original First Occurrence of a Food Poisoning Incident Due to Ingestion of Takifugu oblongus, Along with a Toxicological Report on Three Marine Puffer Species in Bangladesh Yahia MAHMUD, Mohosena (Received