Seafood Toxins - American Chemical Society

14 Paralytic Shellfish Toxins in Tropical Waters Y A S U K A T S U OSHIMA , TAKESHI YASUMOTO

Y U I C H I ΚΟΤΑΚI ,

1

1

Downloaded by CORNELL UNIV on May 19, 2017 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch014

2

2

TAKAKO

HARADA ,

and

1

1

Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Tsustsumi-dori, Sendai 980, Japan S h o k e i Women's Junior College, Hachiman, Sendai 980, Japan

The d i n o f l a g e l l a t e Pyrodiniun bahamense var. compressa and bivalves collected at Palau contained saxitoxin, neosaxitoxin, gonyautoxins V and VI and an unidentified toxin code-named PBT. Chemical structures of gonyau toxins V, VI and PBT were confirmed to be carbamoyl-N -sulfοsaxitoxin, carbamoyl-N-sulfoneosaxitoxin and decarbamoylsaxiton, respectively. Occurrence of para l y t i c s h e l l f i s h toxins was also evidenced i n ten spe cies of crabs belonging to four different f a m i l i e s , two turban shells and two top s h e l l s collected at Ishigaki Island, Japan. Analyses of the representative species confirmed the presence of saxitoxin, neosaxi toxin, decarbamoylsaxitoxin, gonyautoxins I-III and a new toxin code-named TST. A calcareous red alga Jania sp. was proved to produce gonyautoxins I-III and was assigned as the primary source of the toxins i n the crabs and gastropods.

Paralytic s h e l l f i s h toxins i n the dinoflagellate Protogonyaulax (=Gonyaulax) spp. and bivalves of temperate waters have been the subjects of extensive studies. In contrast, information on the occurrence of these toxins i n tropical waters has been scarce. Maclean reported the occurrence of poisonings resembling paralytic s h e l l f i s h poisoning i n Papua New Guinea and Borneo and associated the incidence with the concurrent red tide of the d i n o f l a g e l l a t e Pyrodiniwn bahamense (1), which was later amended to Pyrodiniwn bahamense var. compressa (2). However, no chemical evidence has been presented to prove the occurrence of p a r a l y t i c s h e l l f i s h toxins i n tropical bivalves u n t i l Kamiya and Hashimoto detected saxitoxin (STX) in Palauan bivalves (3). STX was also found i n animals of e n t i r e l y different feeding habit and habitat. Hashimoto and his colleagues found that three xanthid crabs, Zosimus a.eneus Atergatis floridus and Platipodia granulosa^ were highly poisonous i n certain areas and i d e n t i f i e d STX i n Z. aeneus ( 4 ^ ) . STX was also detected i n a green turban s h e l l by Yasumoto and Kotaki (2)· However, d e t a i l s of toxin composition and the primary source of toxin were l e f t unsolved. 3

0097-6156/ 84/ 0262-0161 $06.00/ 0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

162

SEAFOOD TOXINS

In this paper we summarize our recent findings on p a r a l y t i c s h e l l f i s h toxins i n t r o p i c a l waters (8-15) with new assignment of a component previously unreported. It includes the confirmation of paralytic s h e l l f i s h toxins i n the d i n o f l a g e l l a t e Pyrodiniwn bahamense var. compressa and bivalver exposed to the organism (8), with struc tural elucidation of three components (9,10). The detailed analyses of the toxin composition of crabs (11-13) and marine s n a i l s (13,14) and confirmation of a calcareous red alga Jania sp. as the primary source of the toxins (15) are also described. Materials and Methods Materials. The d i n o f l a g e l l a t e Pyrodiniwn bahamense var. compressa; bivalves Spondylus butleri, Tridacna crocea, Lopha cristagalli Saxostrea mordax, Modiolus sp. and Barbatia sp.; and a top s h e l l Tectus sp. were collected at Palau, Western Calorine Islands, i n 1980 and 1981. Eleven species of crab were collected at Kabira reef, Ishigaki Island, Japan, over the period from 1980 to 1982: family Xanthidae Zosimus aeneus, Atergatis floridus, Platipodia granulosa^ Pilmnus vespertilioy Actaea polyacantha, Neoxanthias impressus, Eriphia scabrioula, Actaeodes tomentosus; family Grapsidae Percmon planissimwn; family Majidae Schizpphrys aspera; and family Portunidae Thalamita sp. Turban s h e l l s Turbo marrnorata and Turbo argyrostoma and top s h e l l s Tectus pyramis and Tectus nilotica maxima were collected at Shiraho, Ishigaki Island, i n 1980. Eighteen species of alga were collected at Kabira i n A p r i l and May, 1981: unidentified five species of Cyanophyta; Chlorophyta Halimeda opuntia and Boodles sp.; Phaeophyta Sargasswn sp.and Dictiota sp.; Rhodοphyta Jania s p . - l , Jania sp.-2 Gelidiella acerosa, Eucheuma serra, Centrocearas clavulatum, Leveillea jungermunnioides> Hypnea sp., Laurencia sp. and Ceramiwn sp. To examine seasonal v a r i a t i o n i n t o x i c i t y additional samples of Jania s p . - l were collected at the same place i n August and December, 1981 and February, 1982. The Jania specimens were shaken vigorously i n sea water immediately after c o l l e c t i o n to be freed of sands and other p a r t i c l e s . Contaminating algae were then eliminated by forceps to ensure the homogeneity of the specimens.


3

3

Bioassay. Toxicity of the materials was measured by the standard mouse bioassay f o r p a r a l y t i c s h e l l f i s h toxins and expressed by mouse unit (MU) as defined by the method (16). For testing the low toxin levels of algal specimens, extracts were treated with a charcoal column p r i o r to i n j e c t i o n into mice. Analysis of toxins. The a n a l y t i c a l methods were e s s e n t i a l l y the same as were used f o r the toxins of Protogonyaulax tamarensis (17). Toxins were extracted with 0.1 N HC1 or 75% EtOH a c i d i f i e d to pH 2 and treated with successive columns of charcoal, Bio-Gel P-2 and Bio-Rex 70. Toxins separated by the last column were i d e n t i f i e d by t i c and electrophoresis. Relative abundance of each toxin was deter mined by monitoring the eluate from Bio-Rex 70 column by mouse assay. A fluorometric p a r a l y t i c s h e l l f i s h toxin analyzer was applied to samples which were too small to be followed by mouse assay. Toxins separated by the ion exchange column (Hitachi gel 3011C) were contin uously aromatized by t^rt-butylhydroperoxide and monitored by the fluorometer (18).


14.

O S H I M A ET A L .

163

Paralytic Shellfish Toxins in Tropical Waters

i J

i

NMR spectra. C NMR and H NMR spectra of toxins were taken with JEOL FX-100 and FX-400 spectrometers i n D 0. Chemical s h i f t s are expressed i n ppm downfield from TMS with the use of dioxane (61.4 ppm) and tert-butanol (1.23 ppm) as internal standards, respectively, for the C and ^Ti NMR analyses. 2

1 3


Results Toxicity of the organisms. Dinoflagellate Pyrodinium bahamense var. compressa was p r o l i f e r a t i n g i n Arumizu Bay, Koror Island, 450 cells/ml at the maximum count. Pyrodinium comprised more than 97% in the plankton samples collected by net. Toxicity levels of plankton specimens and molluscs i n the same area are shown i n Table I. Toxin productivity of Pyrodinium was comparable to that reported f o r Protogonyaulax tamarensis from Ofunato Bay, Japan (19). Consistent with the d i s t r i b u t i o n of the d i n o f l a g e l l a t e , a l l the s h e l l f i s h e s were highly contaminated by the toxins as shown by an extraordinarily high toxic scores of 1100 MU/g i n a specimen of Spondylus butleri (8).

Table I.

Toxicity of the Dinoflagellate and Molluscs i n Palau

Specimens Pyrodinium Pyrodinium

bahamense var.compressa ** bahamense var.compressa

Spondylus butleri Tridacna crocea Lopha cri stagalii Saxostrea mordax Modiolus sp. Barbatia sp. Septifer bilocularis** Tectus sp.

Toxicities* 1.5 χ ID" 1.4 χ 10"

4

4

1100 96 130 210 160 140 48 5.3

* Dinoflagellate: MU/cell, s h e l l f i s h : MU/g. ** Collected i n December, 1980. A l l other specimens were collected i n May, 1981.

Toxic levels of crabs and marine s n a i l s are summarized i n Table H. Toxins were detected i n ten species of crab out of eleven tested. Z. aeneus, A. floridus and P. granulosa were, s i g n i f i c a n t l y more toxic than others, though individual t o x i c i t y varied greatly from 3.6 to 660 MU/g. Considerably high t o x i c i t y (80-130 MU/g) was also found i n A. tomentosus, E. sebana and Thalamita sp. Toxic scores of other four species were less than 10 MU/g (12). The turban shells and top shells contained toxins i n varied degree up to 20 MU/g (14). Among algal specimens two calcareous algae, H. opuntia and Jania sp. tentatively coded as type-1, were toxic to mice. However, the


164

SEAFOOD TOXINS Table IE. Toxicity of Crabs and Marine Snails


Toxicities**

Zosimus aeneus Platipodia granulosa Atergatis floridus Neoxanthias impressus Aataeodes tomentosus Eriphia scabricula Pilmnus vespertilio Actaea polyacantha Schizophrys aspera Thalamita Percmon planissimum Gastropods Turbo marmorata Turbo argyro stoma Teotus pyramis Tectus nilotica maxima

8/8 111 5/5 2/3 3/5 4/8 1/2 1/1 1/1 4/5 2/2

660 110 490 10 130 180 6.1 ND 2.3 80 7.4

2/3 21k 1/1 2/2

4.2 20 19 5.0

Number of toxic specimens against those tested. The maximum t o x i c i t y expressed by MU/g. ND refers below 2.0

MU/g.

toxin i n H. opuntia was judged to d i f f e r from p a r a l y t i c s h e l l f i s h toxins on the basis of the symptoms i n mice and chromatographic properties. On the other hand, toxic components of Jania s p . - l were indistinguishable from reference toxins i n both symptomatology and chromatographic behaviors. Increased t o x i c i t y of Jania specimens after elimination of contaminants indicated that the toxins were genuine products of Jania s p . - l . Seasonal observation of t o x i c i t y of Jania s p . - l revealed low t o x i c i t y through August to December (0.04-0.17 MU/g), moderate i n February (0.13-0.69 MU/g) and the highest i n A p r i l and May (1.3-1.5 MU/g). Presence of this alga was confirmed i n the stomach of two crabs Z. aeneus and A. floridus, and four toxic gastropods (15). Toxins i n the organisms. The compositions and r e l a t i v e abundance of toxins i n the organisms tested are shown i n Table I I . The similar data for cultured Protogonyaulax tamarensis are also included i n the table as comparison (17). Pyrodinum contained STX and neosaxitoxin (neoSTX), gonyautoxins V and VI ( G T X 5 ) and toxin coded PBT, which was later i d e n t i f i e d as decarbamoylsaxitoxin (dcSTX). Unlike Protogonyaulax, the d i n o f l a g e l l a t e lacked 11-0-sulfate derivatives such as gonyautoxins I-IV (GTX^_4). In the bivalves, r e l a t i v e r a t i o of neoSTX, G T X 5 and G T X 5 were lower than i n the causative d i n o f l a g e l l a t e , indicating Bioconversion or preferential accumulation of toxins. The c h a r a c t e r i s t i c feature of the toxin p r o f i l e s of crabs i s the predominance of neoSTX, STX i n contrast to the small amount of dcSTX and GTXi_3 (11-13). Toxin compositions of gastropods were characterized by the abundance of STX and a new toxin code-named TST (13>, 14). Investigation of toxins i n Jania s p . - l by the p a r a l y t i c s h e l l 6



Palau Palau Palau

Ishigaki Ishigaki Ishigaki Ishigaki Ishigaki Ishigaki

Ishigaki Ishigaki Ishigaki

Ishigaki

Decapods Zosimus aeneus Atergatis floridus Platipodia granulosa Eriphia scabricula Pilumnus vespertilio Thalamita sp.

Gastropods Turbo marmorata Turbo argyro stoma Tectus pyramis

Rhodophyta Jania s p . - l

Ofunato

Palau

Locality

Pelecypods Spondylus butleri Tridacna crocea Septifer bilocularis

Protogonyaulax tamarensis

Dinoflagellate Pyrodinium bahamense var.compressa

Organisms

—

+

+

+ +

+ + _

+ + -

+ -

+

—

+

+

+

+

+

GTX3

_

2

_

-

4h-

GTX

-

±

GTX

GTX4

+ +

GTX5 6

+ + +

-+f

+ +

-Hf

+H-

-H-

GTX neoSTX STX

+ + +

+ + +

dcSTX

Table ΠΙ. Composition of P a r a l y t i c S h e l l f i s h Toxins i n Organisms of T r o p i c a l Waters


TST

166

SEAFOOD TOXINS

f i s h toxin analyzer confirmed the presence of GTX^, GTX and the r a t i o 73:31:6 and the absence of STX and neoSTX (15).


2

GTX3

in

Structures of G T X 5 , GTX^ and PBT. The chemical structures of G T X 5 and G T X 6 have not been known despite the frequent occurrence i n organisms from cold waters (20). The s p e c i f i c a c t i v i t i e s of G T X 5 and G T X 6 were 136 and 108 MU/;umol, corresponding to 1/15 of STX and neo STX, respectively. Upon hydrolysis i n mild condition, they were converted to STX and neoSTX by releasing 1 mol of s u l f a t e . As shown in Table IV, NMR spectra of G T X 5 and GTX^ were e s s e n t i a l l y the same as those of STX and neoSTX except for the s l i g h t difference i n chemical s h i f t s for 13-H protons or C-13 and C-14 carbons. This confirms that the conjugation s i t e for the sulfate i n both toxins i s the carbamoyl nitrogen. Thus G T X 5 and G T X 6 were proved to carbamoylN-sulfοsaxitoxin and carbamoyl-N-sulforteosaxitoxin, respectively (Fig. 1)(9_). They correspond to the toxins coded Bl and B2 isolated from Protogonyaulax by Koehn et al. (21). PBT was proved to be decarbamoylsaxitoxin by the lack of C-14 signal i n l^C NMR spectra as well as by the complete agreement of a l l spectral and chromatographic properties with dcSTX prepared from STX by acid hydrolysis. Production of STX by carbamoylation with chlorosulfonylisocyanate also evidenced the i d e n t i t y between PBT and dcSTX (10). Discussion Present work provides a firm evidence that s h e l l f i s h poisoning involving toxins of STX family occurs i n the t r o p i c a l areas, and Pyrodinium bahamense var. compressa i s the toxin progenitor. Wide d i s t r i b u t i o n of this species i n areas such as Papua New Guinea, Brunei, Sabah and Palau and the actual occurrence of poisoning i n F i j i (22) and India (23) indicate that the real and potential threat of p a r a l y t i c s h e l l f i s h poisoning i n t r o p i c a l waters i s as great as in the northern waters. M u l t i p l i c i t y of the toxin composition was observed i n a l l specimens tested. The predominance of strongly basic toxins over 11-0-sulfated toxins i s also a prominent feature common to a l l tropical specimens, except for Jania s p . - l . The wide d i s t r i b u t i o n of dcSTX i s also a d i s t i n c t i o n of toxin composition of t r o p i c a l specimens. The present study i s the f i r s t to demonstrate the production of paralytic s h e l l f i s h toxins by a macroalga Jania sp. and transmission of toxins to crabs and marine s n a i l s through food chain. It i s debatable whether Jania sp. i s the sole source of the toxins found i n crabs and gastropods. However, the p o s s i b i l i t y of the presence of a planktonic toxic d i n o f l a g e l l a t e such as Pyrodinium sp. or Protogonyaulax sp. i s ruled out because plankton-feeding bivalves c o l l e c t ed from the same area were found to be nontoxic (11). Our extensive survey for epibenthic d i n o f l a g e l l a t e led to a discovery of a number of toxic species. However none of them produced p a r a l y t i c s h e l l f i s h toxins (24,25). Blue-green algae were throughly collected and tested for the toxins because a fresh water species, Aphanizomenon frosaquae, i s known to produce p a r a l y t i c s h e l l f i s h toxins (26). Yet none of them contained toxins. A l l these r e s u l t s support our conclusion



(s) (s) (s) (s) (d) (d) (t)

33.0 (t) 98.6 (s) 63.3 (t)

159.1 158.1 156.2 82.6 57.1 53.2 42.8

C NMR

4.07 (11,5) 4.33 (11,9)

(1) (9,5,1) (10) (10) (m)

H NMR

X

1 3

(s) (s) (s) (s) (d) (d) (t)

98.6 (s) 64.0 ( t )

154.1 i:i8.1 156.1 82.6 57.2 53.0 42.9

C NMR

4.12 (12,5) 4.42 (12,9)

*

(1) (9,5,1) (10) (10)

NMR

4.76 3.88 3.81 3.61

%

GTX5

(s) (6,6) (10) (10)

4.21 (11,6) 4.44 (11,6)

*

4.83 4.13 3.78 3.58

Ε NMR

λ

neoSTX

5

%

1 3

(s) (s) (s) (d) (d) (t)

Ε

λ

32.9 (t) 98.7 (s) 61.4 (t)

158.0 156.1 82.5 56.5 55.7 42.9

C NMR

(1) (m) (10) (10) (m) 3.63 (m) 3.63 (m)

4.61 3.63 3.77 3.54 2.39

NMR

PBT (dcSTX)

and PBT (dcSTX)

(s) (6,6) (10) (10)

6

4.26 (11,6) 4.52 (11,6)

4.83 4.13 3.78 3.58

NMR

GTX$

C NMR and H NMR Chemical Shifts of STX, GTX , neoSTX, GTX

4.71 3.90 3.81 3.61 2.41

X

1 3

* Unobserved due to deuterium replacement at C - l l .

11 12 13

14 8 2 4 5 6 10

1 3

STX

Table IV.



168

SEAFOOD TOXINS

13

R2'

R1

R2

Η

STX : RI N ^

6

\ ^ ' \

+

neoSTX: GTX : 5

-H

- C O N H

2

-OH

- C O N H

2

-H

-CONHSO3

GTXe : - O H OH

12 Fig. 1.

O UH

dcSTX:

- H

-CONHSO3 -H

Structures of toxins i n Pyrodinium bahamense var. compressa.


14. OSHIMA ET AL.

Paralytic Shellfish Toxins in Tropical Waters

169

that on coral reefs, Jania sp. is the main source of paralytic shell fish toxins found in crabs and gastropods. Since Jania sp.-l shows spotty distribution, the individual and regional variation observed in the toxicity of animals might be the reflection of the abundance of Jania sp. in their habitat. Different preference of crabs and gastropods to the alga might also cause the variation. The discre pancy in the toxin composition between Jania sp.-l and crabs and marine snails suggests that a reductive cleavage of 11-0-sulfate of l - 3 takes place in the animals. In preliminary tests, incubation of GTXi_4 with the homogenate of the digestive organs of crabs and gastropods indicated the conversion of these toxin to STX. Since the conversion was deterred by the addition of bacteriostatic substances, bacterial role in the conversion of the toxins was suggested. Further investigation is under way to elucidate the mechanism of conversion. Downloaded by CORNELL UNIV on May 19, 2017 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch014

G T X

Acknowledgments Present study was supported by a Grant-in-Aid from the Toyota Foundation. Literatures Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Maclean, J . L. In "Toxic Dinoflagellate Blooms", Taylor, D. L.; Seliger, Η. Η., Eds.; Elsevier North-Holland: New York, 1979; pp. 173-178. Steidinger, Κ. Α.; Tester, L. S.; Taylor, F. J . R. Phycologia 1980, 19, 329-37. Kamiya, H . ; Hashimoto, Y. Toxicon 1978, 16, 303-6. Hashimoto, Y . ; Konosu, S.; Yasumoto, T . ; Inoue, Α . ; Noguchi, T. Toxicon 1976, 5, 85-90. Konosu, S.; Noguchi, T . ; Hashimoto, Y. Bull. Jpn. Soc. Sci. Fish. 1970, 36, 715-9. Noguchi, T . ; Konosu, S.; Hashimoto, Y. Toxicon 1969, 7, 325-6. Yasumoto, T . ; Kotaki, Y. Bull. Jpn. Soc. Sci. Fish. 1977, 43, 207-11. Harada, T . ; Oshima, Y . ; Kamiya, H . ; Yasumoto, T. Bull. Jpn. Soc. Sci. Fish. 1982, 48, 821-5. Harada, T . ; Oshima, Y . ; Yasumoto, T. Agric. Biol. Chem. 1982, 46, 1861-4. Harada, T . ; Oshima, Y . ; Yasumoto, T. Agric. Biol. Chem. 1983, 47, 191-3. Yasumoto, T . ; Oshima, Y . ; Konta, T. Bull. Jpn. Soc. Sci. Fish. 1981, 47, 957-9. Yasumoto, T . ; Oshima, Y . ; T a j i r i , M.; Kotaki, Y. Bull. Jpn. Soc. Sci. Fish. 1983, 49, 633-6. Yasumoto, T . ; Oshima, Y . ; Kotaki, Y. Toxicon 1983, Suppl. 3, 513-6. Kotaki, Y . ; Oshima, Y.; Yasumoto, T. Bull. Jpn. Soc. Sci. Fish. 1981, 47, 957-9. Kotaki, Y . ; T a j i r i , M.; Oshima, Y . ; Yasumoto, T. Bull. Jpn. Soc. Sci. Fish. 1983, 49, 283-6. Horwitz, W., Ed., In "Official Methods of Analysis of Associ ation of Official Analytical Chemists" 13th Ed.; A. O. A. C.; Washington, D. C., 1980, 298-9.


SEAFOOD TOXINS

170

17. 18. 19. 20. 21. 22.


23. 24. 25. 26.

Oshima, Y . ; Hayakawa, T . ; Hashimoto, M.; Kotaki, Y . ; Yasumoto, T. Bull. Jpn. Soc. Sci. Fish. 1982, 48, 851-4. Oshima, Y . ; Machida, M.; Sasaki, K.; Tamaoki, Y . ; Yasumoto, T. Submitted to Agric. Biol. Chem. Kodama, M.; Fukuyo, Y . ; Ogata, T . ; Igarashi, T . ; Kamiya, H . ; Matsuura, F. Bull. Jpn. Soc. Sci. Fish. 1982, 48, 657-71. Shimizu, Y. In "Toxic Dinoflagellate Blooms"; Taylor, D. L.; Seliger, Η. Η., Eds.; Elsevier North-Holland; New York, 1979; pp. 321-326. Koehn, F. E.; Hall, S.; Wichmann, C. F . ; Schnoes, H. K.; Reichardt, P. B. Tetrahedron Lett. 1982, 23, 2247-8. Raj, U. Presentation at the Symposium on Seafood Toxins in Tropical Regions, held at Kagoshima, in September 1981. Bhat, R. V. "Report of Food and Drug Toxicology Research Centre" Hyderabad, India, September 1981. Yasumoto, T . ; Oshima, Y . ; Nakajima, I . ; Bagnis, R.; Fukuyo, Y. Bull. Jpn. Soc. Sci. Fish. 1980, 46, 327-31. Nakajima, I.; Oshima, Y . ; Yasumoto, T. Bull. Jpn. Soc. Sci. Fish. 1981, 47, 1029-33. Alam, M.; Shimizu, Y.; Ikawa, M.; Sasner, J . J . Jr. J. Environ. Sci. Health 1978, 13, 439-9.

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