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A toxin profile for shellfish involved in an outbreak of paralytic shellfish poisoning in India
0041-0101j90 $3 00
To,;"," Vol. lX, No 7, pp X6X-X70, 1990 Printed in Great Britain.
I{ 'I
1990 Pergumon
+
Prcs~
,00
pIc
A TOXIN PROFILE FOR SHELLFISH INVOLVED IN AN OUTBREAK OF PARALYTIC SHELLFISH POISONING IN INDIA IDDYA KARUNASAGAR,l INDRANI KARUNASAGAR, I YASUKATSU OSHIMA 1 and TAKESHI YASUMOT01 'Department of Fishery Microbiology, College of Fisheries, University of Agricultural Sciences, Mangalore575 002, India, and 'Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidori Amamiya, Sendai 981, Japnn
(Accepted/or publication 8 February 1990)
1. KARUNASAGAR, 1. KARUNASAGAR, Y. OSHIMA and T. YASUMOTO. A toxin profile for shellfish involved in an outbreak of paralytic shellfish poisoning in India. Toxicon 28, 868-870, 1990.-Toxin profiles of clams and oysters involved in the outbreak of paralytic shellfish poisoning in India in 1983 were studied by a liquid chromatographic technique. Gonyautoxins I, 2, 3, 4 and 8, and l1-epigonyautoxin 8 appeared to be the major toxins along with small amounts of saxitoxin, neosaxitoxin, decarbamoylsaxitoxin, decarbamoylgonyautoxins 2 and 3, C3 and C4. Toxin profile suggests the involvement of Alexandrium spp. in this outbreak, A NUMBER of incidents of paralytic shellfish poisoning (PSP) have been reported from the Indo-Pacific region (MACLEAN, 1984). In India, BHAT (1981) reported illness in some people due to consumption of mussels from Tamilnadu. However, shellfish were not subjected to mouse bioassay nor chemical analysis for the toxins. In 1983, another outbreak of shellfish poisoning occurred in Mangalore on the West coast of India, which was the first incident confirmed in India by mouse bioassay (KARUNASAGAR et al.. 1984). Clams involved in the outbreak showed toxicity as high as 180 MU jg, although no phytoplankton bloom was observed in the affected area. In this paper, we report the toxin profile for the shellfish involved in the 1983 outbreak, Clams (Meretrix casta) and oysters (Crassostrea cucullata) collected from the site of the outbreak (Kumble estuary) during 1983 were extracted according to the standard mouse bioassay (WILLIAMS, 1984) and the extracts were kept frozen at - 20°C until the chemical analysis was carried out in 1989. The thawed extracts were purified by passing through a cartridge column (Sep-Pak C18, Waters) and a 10,000 mol. wt cut-off membrane filter (Ultrafree C3GC, Millipore) prior to HPLC analysis. The analytical conditions were essentially the same as reported previously (OSHIMA et al., 1987) except for slight modification in mobile phases (OSHIMA et al., 1989). Chromatograms of clam toxins are shown in Fig. 1 and toxin compositions of the two species are summarized in Table 1. Gonyautoxins 1 to 4 (GTX 1-4), II-epigonyautoxin 8 (epiGTX8), saxitoxin (STX) were the major toxins in both clams and oysters with smaller amounts of GTX8 and neosaxitoxin (neoSTX). It is noteworthy that both specimens contained decarbamoylgonyautoxins 2, 3 (dcGTX2, dcGTX3) and decarbamoylsaxitoxin (dcSTX). Toxin levels of the extracts as determined by the mouse bioassay as well as HPLC were approximately 5 times higher than those measured immediately after collec868
869
Short Communications
(Al
-
1O~
-
20 ~
(B l
~8
r: FC4
C3
x 40
-
10 ~
GTX1
dcGTX3 dcGTX2
20 ~ 30
(el 10
GTX8
GTX3 GTX2
~
§-
20 ~
min
r
=
r":m]
STX
x 20
FIG. 1. CHROMATOGRAMS OF TOXINS IN Meretrix casta. HPLC was carried out on a Develosil C8-S column (4.6 x 250 mm) with mobile phases of (A) I mM tetrabutylammonium phosphate in acetate bufTer (pH 5.8), (B) I mM I·heptanesulfonic acid in 10 mM sodium phosphate buffer (pH 7.2), and (C) I·heptanesulfonic acid in 30 mM phosphate buffer (pH 7.1): acetonitrile (100:6). Eluate was continuously oxidized by mixing with 7 mM periodic acid in sodium phosphate buffer (pH 9.0) at 65°C and resultant fluorescent derivatives were monitored by a fluoromonitor at 390 nm with excitation wavelength 330 nm, after acidifying the reaction mixture with 0.5 M acetic acid. An extract equivalent to 0.25 mg of clam meat was applied for each analysis.
TABLE
1.
TOXIN COMPOSITION OF SHELLFISHES FROM INDIA
Meretrix casta Toxins STX neoSTX dcSTX GTXI GTX2 GTX3 GTX4 dcGTX2 dcGTX3 epiGTX8 GTX8 C3 C4 Total
Species
Toxin content* (mole %) (nmole/g) 4.2± 0.1 0.6± 0.1 0.2± 0.01 270 ± 6 211 ± 8 69.5± 2.2 148 ± 5 21.2± 0.8 13.8± 004 331 ±13 110 ± 4 3.7 ± 1.0 1.7 ± 0.9 1185
(004) (0.1) (0.0) (22.8) (17.8) (5.9) (12.9) (1.8) ( 1.2) (27.9) (9.3) (0.3) (0.1)
±32
*Resulls are shown as mean ± S.D. (II = 3).
Crassostrea clicct/llata
Toxin content* (mole %) (nmole/g) 2.2±0.1 N.D. 0.2±0.02 45.0±0.9 175 ±7 33.6±0.9 15·stO.7 I 1.7 ±O.3 2.3±0.1 39.0± 1.0 8.3±0.2 N.D. N.D. 333 ±II
(0.7) (0.0) (13.5) (52.5) (10.1 ) (4.7) (3.5) (0.7) (11.7) (2.5)
870
Short Communications
tion (KARUNASAGAR et al., 1984). This indicated that the shellfish originally contained larger amounts of N-sulfocarbamoyl-ll-hydroxysulfate toxins (epiGTX8, GTX8, C3 or C4) which might have been hydrolyzed to the more potent carbamate toxins during 6 years of storage in acidic conditions (approximately pH 2). When comparing toxin profiles of two species, clams contained more GTXl and GTX4 than oysters and neoSTX, C3 and C4 were below detection levels in oysters. Along with difference in toxicity level. the discrepancy might be a reflection of physiological properties of toxin catabolism in the bivalves. PSP in tropical areas are mainly attributed to the dinoflagellate Pyrodinium bahamense var. compressa (MACLEAN, 1984), with some exceptional occurrence of Alexandrium spp. in Thailand (FUKUYO et al., 1988). Toxin profiles of the Pyrodinium species and contaminated shellfish were characterized and showed a complete absence of ll-hydroxysulafate toxins (HARADA et al., 1982; OSHIMA et al., 1990), while those of A. cohorticula and the shellfish from Thailand consisted of mainly ll-hydroxysulfate toxins (FUKUYO et al., 1988). The toxin profiles in the present study were similar to that of A. cohorticula and also to some isolates of Alexandrium from Japan (OSHIMA et al., 1990) and Canada (CEMBELLA et al., 1987), indicating Alexandrium sp. as the most probable cause of PSP in the west coast of India. The finding of cysts morphologically similar to those of A. cohorticula (smooth round shape with gelatinous coverage) at the affected area (unpublished data) also support the hypothesis. Investigation of the causative organism is now underway. Acknowledgements-This work was partly supported by funds from United States Department of Agriculture under the Cooperative Agriculture Research Program.
REFERENCES BHAT, R. V. (1981) A report on an outbreak of mussel poisoning in coastal Tamilnadu, India. Hyderabad, National Institute of Nutrition, Indian Council of Medical Research. CEMBELLA, A. D., SULLIVAN, J. S., BOYER, G. L., TAYLOR, F. J. R. and ANDERSEN, R. J. (1987) Variation in paralytic shellfish toxin composition within the Protogonyaulax tamarensisfcatenella species complex; red tide dinoflagellates. Biochem. System. Ecol. 15, 17 1-186. FUKUYO, Y., YOSHIDA, K., OGATA, T., ISHIMARU, T., KODAMA, M., PHOLPUNTHIN, P., WISESSANA, S., PHANICHYAKARN, V. and PIYAKARNCHANA, T. (1988) Suspected causative dinoflagellates of paralytic shellfish poisoning in the Gulf of Thailand. In: Red Tides: Biology, Environmental Science and Toxicology, pp. 403-406 (OKAICHI, T., ANDERSON, D. M. and NEMOTO, T., Eds). New York: Elsevier. HARADA, T., OSHIMA, Y., KAMIYA, H. and YASUMOTO, T. (1982) Confirmation of paralytic shellfish toxins in the dinoflagellate Pyrodinium bahamense var. compressa and bivalves in Palau. Nippon Suisan Gakkaishi 48, 821-825. KARUNASAGAR, I., GOWDA, H. S. V., SUBBURAJ, M., VENUGOPAL, M. N. and KARUNASAGAR, I. (1984) Outbreak of paralytic shellfish poisoning in Mangalore, West Coast of India. Curro Sci, 53, 247-249. MACLEAN, J. L. (1984) Indo-Pacific toxic red tide occurrences 1972-1984. In: Toxic Red Tides and Shellfish Toxicity in Southeast Asia, pp,92-98 (WHITE, A. W., ANRAKU, M. and Hom, K. K., Eds). Singapore: Southeast Asian Fisheries Development Center. OSHIMA, Y., HASEGAWA, M., YASUMOTO, T., HALLEGRAEFF, G. and BLACKBURN, S. (1987) Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish. Toxicon 25, l105-lllI. OSHIMA, Y., SUGINO, K. and YASUMOTO, T. (I 989) Latest advances in HPLC analysis of paralytic shellfish toxins. In: Mycatoxin and Phycotoxin '88, pp. 319-326 (NATORI, S., VENO, Y. and HASHIMOTO, K., Eds). New York: Elsevier. OSHIMA, Y., SUGINO, K., ITAKURA, H., HIROTA, M. and YASUMOTO, T. (\990) Comparative studies on paralytic shellfish toxin profile of dinoflagellates and bivalves. In' Toxin Marine Phytoplankton, pp. 391-396 (GRANELl, E., SUNDSTROM, B., EDLER, L. and ANDERSON, D. M., Eds). New York: Elsevier. WILLIAMS, S. (Ed.) (1984) Paralytic shellfish poison. In: Official Methods of Analysis, pp.344-346. Arlington: Association of Official Analytical Chemists.
To,;"," Vol. lX, No 7, pp X6X-X70, 1990 Printed in Great Britain.
I{ 'I
1990 Pergumon
+
Prcs~
,00
pIc
A TOXIN PROFILE FOR SHELLFISH INVOLVED IN AN OUTBREAK OF PARALYTIC SHELLFISH POISONING IN INDIA IDDYA KARUNASAGAR,l INDRANI KARUNASAGAR, I YASUKATSU OSHIMA 1 and TAKESHI YASUMOT01 'Department of Fishery Microbiology, College of Fisheries, University of Agricultural Sciences, Mangalore575 002, India, and 'Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidori Amamiya, Sendai 981, Japnn
(Accepted/or publication 8 February 1990)
1. KARUNASAGAR, 1. KARUNASAGAR, Y. OSHIMA and T. YASUMOTO. A toxin profile for shellfish involved in an outbreak of paralytic shellfish poisoning in India. Toxicon 28, 868-870, 1990.-Toxin profiles of clams and oysters involved in the outbreak of paralytic shellfish poisoning in India in 1983 were studied by a liquid chromatographic technique. Gonyautoxins I, 2, 3, 4 and 8, and l1-epigonyautoxin 8 appeared to be the major toxins along with small amounts of saxitoxin, neosaxitoxin, decarbamoylsaxitoxin, decarbamoylgonyautoxins 2 and 3, C3 and C4. Toxin profile suggests the involvement of Alexandrium spp. in this outbreak, A NUMBER of incidents of paralytic shellfish poisoning (PSP) have been reported from the Indo-Pacific region (MACLEAN, 1984). In India, BHAT (1981) reported illness in some people due to consumption of mussels from Tamilnadu. However, shellfish were not subjected to mouse bioassay nor chemical analysis for the toxins. In 1983, another outbreak of shellfish poisoning occurred in Mangalore on the West coast of India, which was the first incident confirmed in India by mouse bioassay (KARUNASAGAR et al.. 1984). Clams involved in the outbreak showed toxicity as high as 180 MU jg, although no phytoplankton bloom was observed in the affected area. In this paper, we report the toxin profile for the shellfish involved in the 1983 outbreak, Clams (Meretrix casta) and oysters (Crassostrea cucullata) collected from the site of the outbreak (Kumble estuary) during 1983 were extracted according to the standard mouse bioassay (WILLIAMS, 1984) and the extracts were kept frozen at - 20°C until the chemical analysis was carried out in 1989. The thawed extracts were purified by passing through a cartridge column (Sep-Pak C18, Waters) and a 10,000 mol. wt cut-off membrane filter (Ultrafree C3GC, Millipore) prior to HPLC analysis. The analytical conditions were essentially the same as reported previously (OSHIMA et al., 1987) except for slight modification in mobile phases (OSHIMA et al., 1989). Chromatograms of clam toxins are shown in Fig. 1 and toxin compositions of the two species are summarized in Table 1. Gonyautoxins 1 to 4 (GTX 1-4), II-epigonyautoxin 8 (epiGTX8), saxitoxin (STX) were the major toxins in both clams and oysters with smaller amounts of GTX8 and neosaxitoxin (neoSTX). It is noteworthy that both specimens contained decarbamoylgonyautoxins 2, 3 (dcGTX2, dcGTX3) and decarbamoylsaxitoxin (dcSTX). Toxin levels of the extracts as determined by the mouse bioassay as well as HPLC were approximately 5 times higher than those measured immediately after collec868
869
Short Communications
(Al
-
1O~
-
20 ~
(B l
~8
r: FC4
C3
x 40
-
10 ~
GTX1
dcGTX3 dcGTX2
20 ~ 30
(el 10
GTX8
GTX3 GTX2
~
§-
20 ~
min
r
=
r":m]
STX
x 20
FIG. 1. CHROMATOGRAMS OF TOXINS IN Meretrix casta. HPLC was carried out on a Develosil C8-S column (4.6 x 250 mm) with mobile phases of (A) I mM tetrabutylammonium phosphate in acetate bufTer (pH 5.8), (B) I mM I·heptanesulfonic acid in 10 mM sodium phosphate buffer (pH 7.2), and (C) I·heptanesulfonic acid in 30 mM phosphate buffer (pH 7.1): acetonitrile (100:6). Eluate was continuously oxidized by mixing with 7 mM periodic acid in sodium phosphate buffer (pH 9.0) at 65°C and resultant fluorescent derivatives were monitored by a fluoromonitor at 390 nm with excitation wavelength 330 nm, after acidifying the reaction mixture with 0.5 M acetic acid. An extract equivalent to 0.25 mg of clam meat was applied for each analysis.
TABLE
1.
TOXIN COMPOSITION OF SHELLFISHES FROM INDIA
Meretrix casta Toxins STX neoSTX dcSTX GTXI GTX2 GTX3 GTX4 dcGTX2 dcGTX3 epiGTX8 GTX8 C3 C4 Total
Species
Toxin content* (mole %) (nmole/g) 4.2± 0.1 0.6± 0.1 0.2± 0.01 270 ± 6 211 ± 8 69.5± 2.2 148 ± 5 21.2± 0.8 13.8± 004 331 ±13 110 ± 4 3.7 ± 1.0 1.7 ± 0.9 1185
(004) (0.1) (0.0) (22.8) (17.8) (5.9) (12.9) (1.8) ( 1.2) (27.9) (9.3) (0.3) (0.1)
±32
*Resulls are shown as mean ± S.D. (II = 3).
Crassostrea clicct/llata
Toxin content* (mole %) (nmole/g) 2.2±0.1 N.D. 0.2±0.02 45.0±0.9 175 ±7 33.6±0.9 15·stO.7 I 1.7 ±O.3 2.3±0.1 39.0± 1.0 8.3±0.2 N.D. N.D. 333 ±II
(0.7) (0.0) (13.5) (52.5) (10.1 ) (4.7) (3.5) (0.7) (11.7) (2.5)
870
Short Communications
tion (KARUNASAGAR et al., 1984). This indicated that the shellfish originally contained larger amounts of N-sulfocarbamoyl-ll-hydroxysulfate toxins (epiGTX8, GTX8, C3 or C4) which might have been hydrolyzed to the more potent carbamate toxins during 6 years of storage in acidic conditions (approximately pH 2). When comparing toxin profiles of two species, clams contained more GTXl and GTX4 than oysters and neoSTX, C3 and C4 were below detection levels in oysters. Along with difference in toxicity level. the discrepancy might be a reflection of physiological properties of toxin catabolism in the bivalves. PSP in tropical areas are mainly attributed to the dinoflagellate Pyrodinium bahamense var. compressa (MACLEAN, 1984), with some exceptional occurrence of Alexandrium spp. in Thailand (FUKUYO et al., 1988). Toxin profiles of the Pyrodinium species and contaminated shellfish were characterized and showed a complete absence of ll-hydroxysulafate toxins (HARADA et al., 1982; OSHIMA et al., 1990), while those of A. cohorticula and the shellfish from Thailand consisted of mainly ll-hydroxysulfate toxins (FUKUYO et al., 1988). The toxin profiles in the present study were similar to that of A. cohorticula and also to some isolates of Alexandrium from Japan (OSHIMA et al., 1990) and Canada (CEMBELLA et al., 1987), indicating Alexandrium sp. as the most probable cause of PSP in the west coast of India. The finding of cysts morphologically similar to those of A. cohorticula (smooth round shape with gelatinous coverage) at the affected area (unpublished data) also support the hypothesis. Investigation of the causative organism is now underway. Acknowledgements-This work was partly supported by funds from United States Department of Agriculture under the Cooperative Agriculture Research Program.
REFERENCES BHAT, R. V. (1981) A report on an outbreak of mussel poisoning in coastal Tamilnadu, India. Hyderabad, National Institute of Nutrition, Indian Council of Medical Research. CEMBELLA, A. D., SULLIVAN, J. S., BOYER, G. L., TAYLOR, F. J. R. and ANDERSEN, R. J. (1987) Variation in paralytic shellfish toxin composition within the Protogonyaulax tamarensisfcatenella species complex; red tide dinoflagellates. Biochem. System. Ecol. 15, 17 1-186. FUKUYO, Y., YOSHIDA, K., OGATA, T., ISHIMARU, T., KODAMA, M., PHOLPUNTHIN, P., WISESSANA, S., PHANICHYAKARN, V. and PIYAKARNCHANA, T. (1988) Suspected causative dinoflagellates of paralytic shellfish poisoning in the Gulf of Thailand. In: Red Tides: Biology, Environmental Science and Toxicology, pp. 403-406 (OKAICHI, T., ANDERSON, D. M. and NEMOTO, T., Eds). New York: Elsevier. HARADA, T., OSHIMA, Y., KAMIYA, H. and YASUMOTO, T. (1982) Confirmation of paralytic shellfish toxins in the dinoflagellate Pyrodinium bahamense var. compressa and bivalves in Palau. Nippon Suisan Gakkaishi 48, 821-825. KARUNASAGAR, I., GOWDA, H. S. V., SUBBURAJ, M., VENUGOPAL, M. N. and KARUNASAGAR, I. (1984) Outbreak of paralytic shellfish poisoning in Mangalore, West Coast of India. Curro Sci, 53, 247-249. MACLEAN, J. L. (1984) Indo-Pacific toxic red tide occurrences 1972-1984. In: Toxic Red Tides and Shellfish Toxicity in Southeast Asia, pp,92-98 (WHITE, A. W., ANRAKU, M. and Hom, K. K., Eds). Singapore: Southeast Asian Fisheries Development Center. OSHIMA, Y., HASEGAWA, M., YASUMOTO, T., HALLEGRAEFF, G. and BLACKBURN, S. (1987) Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish. Toxicon 25, l105-lllI. OSHIMA, Y., SUGINO, K. and YASUMOTO, T. (I 989) Latest advances in HPLC analysis of paralytic shellfish toxins. In: Mycatoxin and Phycotoxin '88, pp. 319-326 (NATORI, S., VENO, Y. and HASHIMOTO, K., Eds). New York: Elsevier. OSHIMA, Y., SUGINO, K., ITAKURA, H., HIROTA, M. and YASUMOTO, T. (\990) Comparative studies on paralytic shellfish toxin profile of dinoflagellates and bivalves. In' Toxin Marine Phytoplankton, pp. 391-396 (GRANELl, E., SUNDSTROM, B., EDLER, L. and ANDERSON, D. M., Eds). New York: Elsevier. WILLIAMS, S. (Ed.) (1984) Paralytic shellfish poison. In: Official Methods of Analysis, pp.344-346. Arlington: Association of Official Analytical Chemists.