Indian Journal of Marine Sciences Vol 33(3), September 2004, pp. 276-280

The seasonal toxicological profile of four puffer fish 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 – 700 020, India *[E-mail : [email protected]] 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 (13.9 – 80 MU/g) and postmonsoon (8.9 – 136 MU/g) seasons during annual reproductive cycle. However, toxicity of livers was much lower (3.2 – 18.5 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 (Class:, Order:, Evidence of puffer fish intoxication has been reported Family:Lagocephalidae), Lagocephalus inermis in different geographical locales viz., Japan1, USA2, Temminck & Schlegel (Class:Actinopterygii, Order: Taiwan3, Mexico4, Malaysia5, Bangladesh6 and also in Tetraodontiformes, Family:Lagocephalidae) were India7. Jones8 reported deaths due to ingestion of collected regularly from Digha-Talsari region of cooked roe of puffer Chelonodon patoca for the first Bengal, eastern coast of India (latitude 21°40′N, time in India in 1956. Although the occurrence of longitude 87°30′E) during April 2000 – March 2001. puffer fish poisoning is sporadic in India, there is no The voucher specimens of different puffer fishes have systematic documentation of their toxicological been submitted to National Marine Repository, profiles. The toxicological profiles of wild puffer National Institute of Oceanography, Goa, India. The fishes differ widely amongst different species9, time catch was segregated into three prominent seasons, of collection10 and the geographical region of i.e. premonsoon (March–June, summer; temp 29- 11 occurrence . Even the anatomical distribution of 35oC, rainfall 0.7-19 cm), monsoon (July–October, 12 toxicity varies from species to species . The present rainy; temp 28-33oC, rainfall 8.2-10.6 cm) and work was undertaken to find out the toxicological postmonsoon (November–February, winter; temp 21- profiles of puffer fishes with seasonal variation 28oC, rainfall 0.8-5.8 cm). The puffer species collected along Bengal coast, India and to assess the collected from the coastal waters were immediately toxicity. transferred on ice to the laboratory. After random sampling ten fish specimens of each species were Materials and Methods chosen for toxicity assessment. Livers and ovaries of Four species of marine puffers, viz. Chelonodon each different puffer species, according to the patoca Hamilton (Class:Actinopterygii, Order: respective seasons were dissected out and kept in Tetraodontiformes, Family:), Takifugu vacuum-sealed containers below -20°C. oblongus Bloch (Class:Actinopterygii, Order: The liver (10g) and ovary (10g) of each specimen Tetraodontiformes, Family:Tetraodontidae), of different puffer fish species were separately Lagocephalus lunaris Bloch & Schneider homogenized for 5 min with 3 volumes of 1% acetic GHOSH et al : TOXICOLOGICAL PROFILE OF PUFFER FISH 277

acid in methanol and centrifuged (3000 × g) for 15 postmonsoon seasons were 9.5±3.7, 15.0±3.9, min. The residue was extracted twice in the same 18.5±9.3 MU/g respectively indicating slight manner. The supernatants were combined, increment in toxicity through seasons. Matured concentrated under reduced pressure, the residue ovaries were not found in premonsoon season and the dissolved in 0.1% aqueous acetic acid and defatted 3 toxicity was less than 4 MU/g. However, during times with dichloromethane. The aqueous layer was monsoon, fish species with matured ovaries were ultrafiltered with Amicon YM-1 membrane to cut off plenty as evidenced from GI (9.72±2.9) and toxicity substances of more than 1000 daltons. The filtrate was was 80.2 ± 12.9 MU/g. The toxicity of ovaries in freeze-dried and examined for toxicity by mouse postmonsoon season was even more, 136.4±20.3 bioassay. MU /g. Mouse toxicity assay, similar to the method The body weight and length of T. oblongus fish developed for PSP (paralytic shellfish poisoning) specimen were almost same in all three seasons 14 toxin monitoring , has been applied for toxicity (Table 1B). Female fishes are abundant throughout determination among puffers. The mouse bioassay the year. Liver weight in monsoon season (15.0±3.25) was standardized for tetrodotoxin (TTX) in puffers was higher than premonsoon (10.0±2.90) and after Kawabata13 and was expressed as mouse unit postmonsoon (11.0 ± 3.10), as evidenced from (MU) per gram of material, where one MU is the hepatosomatic index. The gonadosomatic index equivalent amount of TTX (0.22 µg) that kills a increased abruptly in monsoon (7.45±1.8) and mouse of ddY strain weighing 20 g in 30 minutes. postmonsoon (5.91±1.6) compared to the premonsoon The lyophilized extract from each puffer specimen was dissolved in 1ml saline solution for mouse (0.56±0.3). Frequencies of toxicity in T. oblongus toxicity assay. Injections were made intraperitoneally specimens were almost cent per cent. The toxicity in to Swiss albino mice (19-21 g) (obtained from Central livers viz., 4.8±2.8, 12.0±4.2, 9.2±1.7 MU/g in Drug Laboratory, Kolkata). A group of three mice premonsoon, monsoon and postmonsoon seasons were used for each experiment. The lethal potency respectively indicated that it was more toxic in was calculated as the time required for killing the monsoon seasons. Ovaries developed substantial mice and was expressed in mouse unit (MU)13. All the toxicity in monsoon (24.5±4.6) and in postmonsoon experiments were statistically analyzed and expressed (22.0±5.9) compared to premonsoon. in Mean ± SD. Lagocephalus lunaris were found plenty throughout seasons (Table 1C). The body weight Results varied slightly, although the lengths were same in Anatomical distribution of toxicity in the puffer three seasons. Female fishes were abundant as Chelonodon patoca is shown in Table 1A. The size of observed in C. patoca (Table 1A) and T. oblongus all fishes collected was almost same and the body (Table 1B). Hepatosomatic index in premonsoon, weight in postmonsoon season (1121± 110.0 g) was monsoon and postmonsoon were 4.6±1.00, 7.9±1.50, nearly double with respect to other seasons. Female 6.0±1.90 respectively which indicated increased liver fishes were more abundant except in monsoon season size in monsoon season. Mature ovaries were found in when male and female fishes were found in equal monsoon and postmonsoon as evident from proportion. The hepatosomatic inedx (liver weight gonadosomatic index 8.05±2.1, 5.56±2.7 respectively. percent with respect to body weight, HI) increased Frequency of toxicity among specimens were almost gradually from premonsoon (8.0±1.2) to monsoon same (84.37, 85.18, 77.78) throughout the seasons. (10.5±0.8) and postmonsoon (14.0±1.6). Liver toxicity was highest in monsoon 7.2±1.8 MU/g Gonadosomatic index (gonadal weight percent with compared to premonsoon (4.9±1.9) and postmonsoon respect to their body weight, GI) in monsoon (6.8±1.6). Ovaries were toxic in monsoon (23.2±6.9) (9.72±2.9), postmonsoon (6.29±2.6) indicated and postmonsoon (8.9±2.5). abundance of gorged ovaries full of matured eggs Lagocephalus inermis fishes were larger in compared to premonsoon (0.44±0.21). From toxic premonsoon (21.35±3.1) than monsoon (18.83±2.9) ratio and frequency of toxicity it was observed that and postmonsoon (18.74±2.4). Similar trend was almost all the species of C. patoca were toxic. The reflected in their average body weight (Table 1D). toxicity of liver in premonsoon, monsoon and Female specimens were available more than male 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 (Premonsoon - Mar-June, Monsoon – July-Oct, Postmonsoon – Nov-Feb)

a b c Seasons Body Length M/F HI GI Toxic Frequency Toxicity (MU/g) weight(g) (cm) ratio ratio of toxicity (%) Liver Ovary (A) Chelonodon patoca Premonsoon 447.5±58.9 31.22±3.2 3 : 4 8.0±1.2 0.44±0.21 18/19 94.73 9.5±3.7 e ND f (n=19d) Monsoon 516.0±78.2 25.59±2.9 1 : 1 10.5±0.8 9.72±2.9 15/15 100.00 15.0±3.9 e 80.2±12.9 e (n= 15 d) Postmonsoon 1121.5±110.0 36.14±4.6 1 : 9 14.0±1.6 6.29±2.6 16/16 100.00 18.5±9.3 e 136.4±20.3 e (n=16 d) (B) Takifugu oblongus Premonsoon 354.0±60.0 26.03±3.1 1 : 4 10.0±2.90 0.56±0.3 14/15 93.34 4.8±2.8 e ND f (n=15d) Monsoon 362.0±72.2 25.46±2.5 1 : 9 15.0±3.25 7.45±1.8 13/13 100.00 12.0±4.2 e 24.5±4.6 e (n= 13 d) Postmonsoon 423.9±48.9 26.25±4.2 3 : 5 11.0±3.10 5.91±1.6 16/16 100.00 9.2±1.7 e 22.0±5.9 e (n=16 d) (C) Lagocephalus lunaris Premonsoon 142.3±13.4 21.70±4.2 2 : 3 4.6±1.00 1.46±0.7 11/13 84.61 4.9±1.9 e ND f (n=13d) Monsoon 189.7±29.8 19.93±2.7 3 : 7 7.9±1.50 8.05±2.1 15/17 88.23 7.2±1.8 e 23.2±6.9 e (n= 17 d) Postmonsoon 216.5±50.2 20.30±3.4 3 : 7 6.0±1.90 5.56±2.7 13/17 76.47 6.8±1.6 e 8.9±2.5 e (n=17 d) (D) Lagocephalus inermis Premonsoon 202.80±17.8 21.35±3.1 1 : 4 3.1±0.9 0.98±0.6 9/12 75.00 ND f ND f (n=12d) Monsoon 167.21±32.8 18.83±2.9 3 : 7 9.0±2.3 6.44±3.2 11/14 78.57 4.6±1.2 e 13.9±2.7 e (n= 14 d) Postmonsoon 182.51±26.2 18.74±2.4 2 : 3 7.6±1.7 4.28±1.9 16/19 84.21 4.9±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 variation. Toxicities of liver were comparatively highest (9.0±2.3) indicative of larger liver size. higher during monsoon than premonsoon in all Gonadosomatic index in monsoon and postmonsoon species. The gonadosomatic index was much higher seasons were 6.44±3.2 and 4.28±1.9 respectively. during monsoon and postmonsoon compared to This was quite higher than gonadosomatic index in premonsoon which indicated the enlargement of premonsoon season (0.98±0.6). Frequency of toxicity ovaries in female fishes and roes consisting of mature is almost same through seasons. Livers were feebly eggs in all puffer species. It was observed that they toxic in monsoon (4.6±1.2 MU/g) and postmonsoon had spawning seasons, twice in a year from June- (4.9±0.8 MU/g). Similar trend was found in case of August and December to February. Toxicity in gonadal toxicity. Interestingly during premonsoon ovaries sharply increased from premonsoon to season the fishes were non-toxic. monsoon with a concurrent increase in gonadosomatic All four-puffer species viz., C. patoca, T. oblungus, index. Among the puffers C. patoca showed L. lunaris and L. inermis followed similar trend with maximum toxic potency of 136 MU/g in their ovary. respect to their average body weight, average length The lethal potency of ovaries was more than their respective livers in all puffer species. and abundance of female fishes throughout seasons (Table 1). The livers were bulky in monsoon seasons Discussion compared to other seasons as evident from Puffers are toxic due to the occurrence of hepatosomatic index (HI) with similar species tetrodotoxin (TTX) in the tissues, a neurotoxin and GHOSH et al : TOXICOLOGICAL PROFILE OF PUFFER FISH 279

TTX was found in greater concentration in their vital Toxicological investigation on puffers in this study organ like liver and ovary15. Often food poisoning revealed that the ovaries showed maximum lethal was reported from different geographical regions due potency in their spawning season when ovaries were to puffer fish ingestion and the lethality was found with full of matured eggs. Livers and ovaries dependent on the concentration of TTX16 present in were mainly tested as these are the organs where the consumed fish tissues. The mouse bioassay method toxin is concentrated more in most cases and mainly has been successfully employed to monitor and assess responsible for clinical poisoning while ingested23. toxic potential in puffers. Reports available from the There is a proportional relationship between toxicity Japanese market indicate that the severity of the and gonadosomatic index suggesting that the clinical poisoning in humans depends upon both the increased toxin is a product of embryos as postulated concentration of toxin and quantity of toxic organ by Matsumura24. It has been observed that increased ingested17. Therefore, the consumption of puffers is weight of liver was due to the fat deposition25. regulated in Japan and it is considered to be unsafe for However, the livers of the four species had a tendency human consumption if the toxicity13 is > 10 MU/g. to be lethal in the reproductive cycle (Table 1). The Wild puffers were found toxic although their distribution of toxicity in organs of four puffer species anatomical distribution of toxicity has wide species showed a prominent seasonal pattern as reported in variation18. Lethal potency also showed seasonal puffers collected along Hong Kong coastal waters10. variation in a single puffer species10. This investigation also indicated that L. inermis is least toxic (Table 1D). This study also reveals that Present investigation showed C. patoca is the most four species of puffer collected along Bengal coast toxic species in comparison to other puffers collected were less toxic than the same species collected in along Bengal coast (Table 1). Moreover, the matured other coastal waters22,26. However, this investigation ovaries of C. patoca showed maximal lethal potency may help reduce the health hazard due to (136 MU/g). A toxicity study from Okinawa, Japan consumption of puffer fishes in the coastal region of has shown that of the tissues tested of muscle, skin, Bengal by identifying the season when they are liver, gonad and intestine, extremely high toxicity was 19 dangerously toxic. in the muscle of C. patoca (520 MU/g) . Lethality of extracts obtained from the anatomical organs of 6 Acknowledgement species of southern puffer from Tropics were studied. This work was supported by the research grant In most of the specimens of C. patoca the highest from Department of Ocean Development, Govt. of 20 level of toxicity was detected in skin and ovaries . India. The Marine Fish Division, Zoological Survey Lethality increased as the fishes approached to their of India, Calcutta, assisted in identifying the fishes. spawning season and it might be expected that the total amount of toxin would also increase with the References maturation of the ovaries. Tani21 clearly showed that 1 Yoshikawa-Ebesu, J.S.M., Hokama, Y. & Noguchi, T., Tetrodotoxin, in: Foodborne disease handbook, Vol.4, edited the concentration of toxin per gram ovary increased by Hui, Y.H., Kitts, D. & Stanfield, P.S. (Marcel Dekker with the increase in the weight of the ovaries. 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L. inermis showed much decrease in their lethal 5 Loke, Y.K. & Tam, M.H., A unique case of tetrodotoxin potency. It was reported9,22 that L. lunaris having poisoning, Med. J. Malaysia, 52 (1997) 172-174. highest toxicity in their ovary (460 MU/g) followed 6 Mahmud, Y., Arakawa, O. & Noguchi, T., An epidemic by intestine (380 MU/g) and muscle (100 MU/g), survey on fresh water puffer poisoning in Bangladesh, J. Nat. Toxins, 9 (2000) 319-326. whereas, the highest score in T. oblongus was in the 7 Ghosh, S., Hazra, A.K., Mitra, S.K. & Mukherjee, liver (2000 MU/g) followed by ovary (770 MU/g). B.,Poisonous fishes: Potential begetter of bioactive 280 INDIAN J. MAR. SCI., VOL. 33, No. 3, SEPTEMBER 2004

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