Comparative lethality of tissue extracts from the malaysian puffer fishes, Lagocephalus lunaris lunaris, L. l. spadiceus and Arothron stellatus

Taxiton, 1973, Vol . l1, pp . 249-254 . Pergai»on Yrcss . Printed in Great Britain

COMPARATIVE LETHALITY OF TISSUE EXTRACTS FROM THE MALAYSIAN PUFFER FISHES, LAGOCEPH.4LUS LUNARIS LUNARIS, L. 1. SPADICEUS AND AROTHRON STELLATUS P. Y. BIERRY and A. Azlz stN HASSAN School of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia

(Acceptedfor publirntion 13 November 1972) Abstract-The lethality of extracts obtained from the muscle, liver, intestine, skin and gonads of specimens of Lagocephalas lurrarls lurwris, L. l. sp~dlceus and Arotlunn stellatus collected at Penang and Port Swettenham is reported . L. 1. lunarLs and A. steUalus from Penang possessed akin, muscle, intestine, liver and gonads which were lethal to mice . A. stellatus from Port Swettenham, however, had only gonads which were lethal whereas the tissues of L. G spadictus were not lethal . Ovaries of A. stelJatus with large conspicuous eggs were more toxic than ovaries with small eggs. INTRODUCTION

poisoning by the ingestion of the tissues of puffer fishes is common all over the Far East, most cases occurring in Japan and China where puffers are considered a delicacy. KAWABATA (1962) reported that in Japan 13 out of 27 known species of puffers (fugu) are consumed, of which all except two species (Liosaccus lunaris Bloch and Schneider and L. cutanus Gunther) are toxic. H~sxlMOTO (1950) found that extracts of the gonads, liver and skin of the puffer Spheroides vermicularis radiatus were highly toxic when injected into white mice. Toxicity of Pacific puffers has been extensively investigated. Fish and Cobb* reported that there are at least 20 toxic species of Pacific puffer fishes. GoE and HALSTF.AD (1953) have found that the Gulf Puffer, Sphoeroides annulatus (Jenyns) collected from Mexico had toxic livers and gonads . The muscles were, however, non-toxic. On the other hand, in the same species collected from San Felipe, California, the muscle and liver both exerted toxic effects. The toxic properties of puffer fishes from the Galapagos Islandst and from the Marshall Islands$ have also been reported . Several workers (LALONE et a1.,1963 ; LAxsoty et a1.,1959,1960; ROHINSON and SCHWARTZ, 1968) have found that the tissues of the North Atlantic puffer, Sphaeroides maculatus (Bloch and Schneider) collected from different localities exhibited different degrees of toxicity . Although the toxicity of puffer fishes from the Pacific and Atlantic oceans has been extensively investigated, puffers from the Indian Ocean have received no scientific investigation. In Malaysia, CANTOR (1849), FOWLER (1938) and Scorr (1959) have recorded at least TETRAODON

'FtsH, O. and Co>sa, M.C . (1954) Research report 36, Fish and Wildlife Service, U.S . Dept . of the Interior, Washington D.C. tH~rsre~n, B. W. and $CHALL, E. (1955) Essays in the Natural Sciences in Honour of Captain Allan Hancock, pp. 147-172 . Univ. of Calif. Press. $Bwit~scx, A. F., Dx~ctrnswrr, R. H. and McF~R~rr, E. F. (1959) Report of a survey of the fish poisoning problem in the Marshall Islands. U.S . Dept . of Health, Ed . and Welfare. 249

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P. Y. BERRY and A. AZIZ BIN HASSAN

16 species of puffers, and although isolated cases of tetraodon poisoning have been reported 1969/1970), no work has been done on the toxicity of Malaysian puffer fishes . Since "trash fishes" in Malaysia and elsewhere (GoE and Hnr..sTEnn, 1953) are receiving increasing attention as potential protein food sources for animals, it was considered advisable to investigate the significance of the toxic puffers which may be included among the "trash fishes". (BERRY,

MATERIALS AND METHODS

A total of 120 specimens of Lagocephalus lunaris spadiceus (Richardson) and 7 specimens of L.1. lunaris (Bloch and Schneider) were collected in March, 1972 from "trash boats" at the fishing villages of Batu Maung and Telok Bahang, Penang . In addition, 10 specimens of Arothron stellatus (Bloch and Schneider) were captured by hook and line at Tanjong Bungah, Penang . Two other lots (28 specimens) of A . stellatus, were captured by hook and line at Port Swettenham, Selangor in February and April, 1972. All specimens were placed in ice immediately after being obtained and were then deep frozen until used. The extracts were prepared and tested according to the method (with slight modification) of LI~LONE et al. (1963) . After allowing the specimens to thaw in running tap water the weight ofthe whole fish was taken and the tissues to be assayed were dissected out, weighed, and 2 ml of distilled water added per g of tissue. The tissues tested were muscle, skin, liver, intestine and ovary or testes. The contents of the gut were Hushed with water before weighing. The tissue mixture was then ground in a manual grinder and filtered through a finemeshed organdy material in order to get rid of the coarser tissue constituents. For the tough skin, sterilized fine sand was added in order to break down the fibers . After filtering through the organdy material, the mixture was homogenized in a blender for approximately 15 min, after which the homogenate was centrifuged for the same length of time. The homogenate was then filtered and one ml of the supernatant injected intraperitoneally into white mice weighing 19 to 25 g (average 201) . Initially, four to eight mice were used in testing each extract. The reactions of the mice were observed continuously for a period of 1 hr and intermittently for a total of 72 hr. The signs and death times were recorded, cessation of all breathing movements being taken as the time of death. For the LD6o determinations, specimens of Lt. lunaris lunaris and A. stellatus from the Penang lots, which were lethal in white mice, were used. The tissues assayed were the muscle, skin, liver, intestine and ovary. After removing the tissue from several puffers, their combined weight was determined and the tissue was ground in a manual grinder and a tissue extract prepared in the same manner as described above. The dose ranges, ratios of mice tested to mice dying and the average death times are listed in Tables 2 and 3. The dose at which 50 per cent of the mice succumbed was then taken as the approximate ~..n 6o. In order to find out if there was any correlation between organ size, gonadal maturity and lethality, LD gp determinations were also carried out for individual puffers (Table 4). For the ovary extract it was necessary to use a 1 J100 dilution of the stock solution because of the high lethality of this extract (Table 4). RESULTS AND DLSCUSSION

Two of the three species of puffer fishes possessed tissues which were lethal to mice. All tissue extracts of L. l. lunaris were lethal . However, tissue extracts of the closely related L. 1. spadiceus (Abe, 1960) collected from the same locality, was not lethal (Table 1). 7bXICON 1973 Vol. Il .

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Tissue extracts of A. stellatus collected from Tanjong Bungah, Penang, demonstrated lethal properties . One mouse which received testis extract died eleven min after injection of the extract (only one male A. stellatus was included in the total catch of ten). By contrast, skin, muscle, liver and intestinal extracts of A. stellatus collected in April, 1972 from Port Swettenham, approximately 200 miles south of Penang were not lethal although the testis and ovary were lethal . Similarly in a second batch collected from the same area in February, 1972 only the gonads were lethal to mice (Table 1). Whether this regional fluctuation is based on a seasonal, dietary or taxonomic basis needs further investigation . Fluctuations in toxicity of puffers taken at different localities have also been demonstrated by GoE and Iini .sTEnn (1953) for Sphoeroides annulatus and by RosrNSOx and SCHWARTZ (1968) for Sphaeroides maculates. The latter workers also found that three different organs, the liver, gut and ovary of S. maculates proved to be toxic, each however on a different date. T~eLE

Dosage (ml/20g) I~0 0~8 0~6 0~4 0~3 0~25 0~2 O~15 0~1

POOLED MUSCLE AND SKIN EXTRACTS OF L. PROM TELOK BAHANG AND B/,TV Mwrra

2. tn  OF

Ratio of mice tested to mice dead Muscle Skin 4~4 4/4 4/4 4/3 8/5" 4/0

S/5 8/8 8/8 8/7 8/5" 8/1 4/0 4/0

!. lunaris

Average death time (min) Muscle Skin 4~0 6~5 7~3 9~0 9~5 -

5~0 7~0 9~5 17~0 24~1 32-0 -

Each ml of extract was obtained from 0~5 g of tissue . "TakCII a3 LD~o.

Employing pooled muscle and skin extracts of L. l. lunaris (Table 2) and pooled liver, intestinal and ovarian extracts of A. stellatus (Table 3), the Ln6o doses for muscle, skin, liver, intestine and ovary were 0" 15, 0"3, 0"12, 0"26 and 0"04 ml per 20 g of body weight, respectively. The average death times for the different dosages are also shown in Tables 2 and 3. In the majority of cases in which death occurred, the time was less than half-anhour after injection and, if the mice lived beyond the one-hour period of direct observation, survival was assured. Signs preceding death were identical irrespective of the extract administered. The mice displayed a marked lethargy immediately after injection. Following the initial lethargic stage, signs of respiratory difficulties devehiped and these were accompanied by restlessness, the mice running around the cages agitatedly for several seconds. Shortly after this, convulsion set in and death immediately followed . Once respiratory difficulties had developed no mice survived. Ovarian extracts of A . stellatus show no correlation between LDs, doses and the weights of the ovaries expressed as percentage of body weight (Table 4) : this matches the findings of GoE and H~tsre~n (1953) with S. annulatus using liver and gonad. Then appeared to be some correlation, however, between lethality and the stage of development of the ovary TOXlCONl973 Vä.ll.

Lethality of Tissues from Puffer Fishes TABLE

3.

Dosage (ml/20 g) 1"0 0"6 0~4 0"3 0~28 0"26 0"25 024 0~22 0"20 0"15 012 0"10 0"08 006 0"04 0-02 001

253

LD,p OF POOLED LIVER, INTESTINE AND OVARY EXTRACTS OF Arothron stellatus FROM TANIONG BUNGAH

Ratio of mice tested to mice dead Liver Intestine Ovary 4/4 4/4 4/3 4/3 4/3 4/3 4/2' 4/0 -

4/4 4/4 4/3 4/4 4/3 4/2' 4/1 4/0 4/0 4/0

8/8 4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/2' 4/1 4/0

Average death time (min) Liver Intestine Ovary 4~8 5"7 14"7 13 "7 11 "3 197 48 "0 -

6~2 6~2 12 "7 16 "0 140 135 150 -

2"3 2~5 2"5 2" 7 2~6 2~7 4" 7 5-0 11 "0 10 "0 -

Each ml of extract was obtained from 0"5 g of tissue. 'Taken as LD,p.

TABLB

4.

Fish no. 1 2 4 S 6 7 1 3 l 3

COMPARISON OF LD,p VALUFS OF TISSUE Ex7RACl'S OF TANIONCI BtJNCiAH

Wt of ßsh (g) Tissue 99 95 67 77 65 45 99 83 99 83

Ovary Ovary Ovary Ovary Skin Skin Muscle Muscle

Wt of tissue (g) 139 137 6" 9 2"4 2"2 3"6 128 14"0 18"1 21 "5

~ of body wt 141 14"3 10"3 3~1 1 3"4 ~ 8"0 12 "9 16 "9 18 "3 26"1

A. stellatus

FROM

LD 

(ml/20 g mouse) 0"01 0012 0"008 0"04 0"2 0"2 0"14 0"25

Fishes S, 6 and 7 had microscopic eggs in the ovary; 1 and 2 had relatively large eggs which are barely visible to the nakod eye and 4 had very large and conspicuous eggs. For the LD determinations each ml was obtained from 0"5 g of tissue. 7~OXICON 1973 Yol. 11 .

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P. Y. BERRY and A. AZIZ BIN HASSAN

(Table 4). Ovaries containing larger eggs were more toxic than ovaries with microscopic eggs. The chemical composition and origin of the toxic substance or substances, and the feeding, reproductive and distributional biology of the puffers needs to be established before one can attempt to explain the variability of toxicity in relationship to place of capture and gonad maturity . Acknowledgements-The authors thank the staff of the Fisheries Division, Glugor, Penang for their assistance in obtaining specimens of Lagocephalus lunaris spadiceus and the staff of the Animal House, Faculty of Medicine, University of Malaya for supplying the white mice. REFERENCES AsE, T. (1960) Taxonomic studies on the puffersfrom Japan and adjacent regions--Corrigenda and Addenda. Part II . Jap. J. Ichthyol . S, 3. BERRY, P. Y. (1969/1970) Puffer fish poisoning. Malay. Scient . S, 42. C~rrroR, T. E. (1849) Catalogue of Malayan fishes . J. Asiat. Soc. Bang., 18, 2, 983. FOWL .FA, H. W. (1938) A list of fishes known from Malaya . Fish . Bull. Slngapon 1, 5. GoE, D. R. and HwtsIFwn, H. W. (1953) A preliminary report of the toxicity of the Gulf Puffer, Sphoeroides anrudatus. Calf Fish Gawk, 39, 229. HeaEUMOro, Y. (1950) On the toxicity of a puffer, "Nashi-Fugu" (Spheroides vrrmicularis radiatus). Bull. Jap. Soc. scient. Fish . 16, 43. KAWAHATA, T. (1962) Fish-home food poisoning in Japan . Part II . FZth as Food 2, 467. LALONE, R. C., DEVn t.EZ, E. D. and LAR.90N, E. (1963) An assay of the toxicity of the Atlantic puffer fish, Spha+oides maculates. Toxicon 1, 159. LeRSON, E., L,etorrE, R. C. and Rlv.~s, L. (1960) Comparative toxicology of the Atlantic puffer fishes of the Benara Spheroides, Lactophrys, Lagocephalus and Chilomycterus. Fedn . Proc. Fadn. Am. Socs. exp. Bio119, 388. LNesotv, E., Rlv~s, L. and LAIANE, R. C. (1959) Toxicology of the Western Atlantic puffer fishes of the Beaus Spheroides. Pharmacologist 1, 70. ROHn~soN, P. F. and Scttw~te~rz, F. J. (1968) Toxicology of the northern puffer, Sphaeroides maculates, in the Chesapeake Bay and lts environs . Chesapeake Sci. 9, 136. Sarrr, J. S. (1959) An Introduction of the Sea Flshes of Malaya. Ministry of Agriculture, Fed. of Malaya .

TOXICON 1973 Vol. 11 .