Biol Res 31: 375-386 (1998) 3 7 5

Microalgal blooms: A global issue with negative impact in Chile*

N É S T O R L A G O S * *

Labora to r io B ioqu ímica de M e m b r a n a , Inst i tu to de Cienc ias B i o m é d i c a s , Facul tad de Medic ina , Univers idad de Chi le , San t iago , Ch i l e

Ecological and health problems posed by microalgal blooms (red tides) occurring in the Southern part of Chile are reviewed. Out of the six human illnesses provoked by microalgal toxins, paralytic shellfish poisoning is the most important, because of its high mortality rate and the high levels of phycotoxins found in contaminated molluscs. Saxitoxin and its-analogues bind to a receptor in the voltage-gated sodium channel of neural membranes. The most important toxin-producer microalgae are Alexandrium catenella and Dinophysis acuta. Phycotoxins become concentrated by filter-feeding shellfish, like Mytilus chilensis. Highly sensitive methods available for detection of microalgal toxins are analyzed.

Key-terms: Alexandrium catenella, microalgal blooms, paralytic shellfish poisoning, phycotoxins, red tides, saxitoxin, toxins detection

INTRODUCTION

T h e e v e r - i n c r e a s i n g n u m b e r of t o x i c m i c r o a l g a e s p e c i e s c o u p l e d w i t h h i g h i n c i d e n c e of o u t b r e a k s of t he se s p e c i e s presents a cons tan t threat to publ ic health w o r l d w i d e . B l o o m s of tox ic or ha rmfu l mic roa lgae , c o m m o n l y called "red t ides" , represent a s ignif icant and expand ing threat to h u m a n hea l th and f i sher ies r e s o u r c e s t h r o u g h o u t t h e w o r l d ( W h i t e , 1 9 8 8 ; S m a y d a , 1 9 9 2 ; Ha l legraef f , 1993) . T h e impac t of these p h e n o m e n a ranges from il lness and death of human consumers of she l l f i sh or fish tha t h a v e a c c u m u l a t e d algal t ox ins to e c o s y s t e m a l te ra t ion and m a s s i v e m o r t a l i t y of f i sh , s e a b i r d s and m a r i n e m a m m a l s ( G e r a c i et al, 1 9 8 9 ; Anderson , 1989; Anderson & Whi t e , 1989; S m a y d a , 1992; Hallegraeff, 1993).

Microa lgae play an important role in the m a r i n e b i o l o g i c a l s y s t e m . W i t h t h e i r

pho tosynthe t ic abil i ty, they are the major p r o d u c e r s of b i o m a s s a n d o r g a n i c compounds in the ocean. In most cases , the p ro l i f e ra t ion of p l a n k t o n a lgae , up to a m i l l i o n of c e l l s p e r l i t e r , is t h e r e f o r e beneficial for aquacul ture and wild fisheries. Never theless , in some c i rcumstances , algal b l o o m s p r o d u c e nega t ive effects, caus ing s e v e r e e c o n o m i c l o s s e s to a q u a c u l t u r e , f i s h e r i e s a n d t o u r i s m , w i t h m a j o r envi ronmenta l and health problems.

A m o n g the 5,000 to 10,000 species of m i c r o a l g a e d e s c r i b e d in t h e l i t e r a t u r e (Sourn ia et al, 1 9 9 1 ; Sh imizu , 1993), about 320 species had deve loped in an intensity enough to c h a n g e the co lor of sea waters (red t ides) . Fu r the rmore , a round 45 species , i n c l u d i n g c y a n o p h y t e s in i n l and w a t e r s , have p roduced harmful algal b looms due to the i r c a p a c i t y to p r o d u c e p o t e n t t o x i n s (phyco tox ins ) , that th rough shellf ish, fish and jus t f reshwater can reach human beings

Manuscript based on a presentation delivered in a Workshop on Physiology and Environment, held during the 13th Annual Meeting of the Chilean Society of Physiological Sciences. Quinamávida, Chile, 5-8 April 1998.

** C o r r e s p o n d e n c e t o : Dr Néstor Lagos, Laboratorio Bioquímica de Membrana, Instituto de Ciencias Biomédicas, Facul­tad de Medicina, Universidad de Chile. Casilla 70005, Santiago 7, Chile. Phone: (56-2) 678-6309. Fax: (56-2) 777-6916. E-mail: nlagos@machi.med.uchile.cl

3 7 6 Biol Res 31: 375-3X6 (199S)

who thus become intoxicated (Smayda, 1992; Hal legraeff , 1993 ; H a w k i n s et al, 1 9 8 5 ; C a r m i c h a e l & F a l c o n e r , 1 9 9 3 ; Fa lconer , 1996).

It is not c lear why these mic roo rgan i sms p roduce phyco tox ins . They are secondary m e t a b o l i t e s , k n o w n as n a t u r a l p r o d u c t s w i t h n o e x p l i c i t r o l e in t h e i n t e r n a l e c o n o m y of t h e o r g a n i s m tha t p r o d u c e them, but wi th very specif ic and p o t e n t act ivi t ies on fish, b i rds and m a m m a l s . If t h e i r p r o d u c e r s ( d i a t o m s a n d d i n o f l a g e l l a t e s ) u s e t h e m as a w a y to c o m p e t e for s p a c e , f igh t p r e d a t i o n , o r defend themse lves from the overgrowth of other o rgan i sms , are still open ques t ions . On t h e o t h e r h a n d , t h i s is a ve ry o ld p h e n o m e n o n with which mankind has been dea l ing with ever s ince. The first known writ ten ment ion of a harmful algal b loom is d e s c r i b e d in the O ld T e s t a m e n t , in the Bib le : "... all the water that were in the r iver were turned to b lood. And the fish that was in the r iver died; and the river stank, and the Egyp t i ans could not dr ink of the water of the r iver" (Exodus 7: 20 -21 ) .

SYNDROMES PROVOKED BY M I C R O A L G A L POISONING

Until now, six h u m a n i l lnesses associated with mic roa lgae toxins have been descr ibed (Hal legraeff , 1993 ; Y a s u m o t o & Mura ta , 1 9 9 3 ; Y a s u m o t o et al, 1 9 9 5 ; F a l c o n e r , 1996) (Table I ) :

- Para ly t ic Shellf ish Po i son ing (PSP) - Diar rhe ic Shellf ish Po i son ing (DSP) - A m n e s i c Shellf ish Po ison ing (ASP) - Neuro tox ic Shel lf ish Po i son ing (NSP) - C igua te ra Po i son ing (CP) - Cyanobac te r i a l Po i son ing ( C N P )

Cyanobacterial poisoning is not a harmful m a r i n e i s s u e , bu t s o m e s p e c i e s of cyanobacter ia also known as freshwater blue-green algae are producers of extremely toxic phycotoxins often associated with poisoning in h u m a n s and a n i m a l s ( C a r m i c h a e l & Fa lconer , 1993; Fa lconer , 1996; S ivonen, 1996; Carmichael , 1996; Lagos et al, 1997). Freshwater cyanobacter ia may accumulate in s u r f a c e w a t e r s u p p l i e s a s b l o o m s and

Table I

Syndromes associated to microalgal poisoning

Syndrome Paralytic Diarrheic Amnesic Neutotoxic Ciguatera Cyanobacterial Shellfish Shellfish Shellfish Shellfish Poisoning Poisoning Poisoning Poisoning Poisoning Poisoning

Causative microalga

Alexandrium sp. G. catenation P. bahamense

Dinophysis sp. P. lima

Pseudo-nitzclna sp. pungens f. multiseries australis; seriata, pseudodelicalissima

G. breve G. cf breve

G. toxicus Cyanobactcnas

Vectors Shellfish Shellfish Shellfish Shellfish Fish Freshwater Haemodialysis

Toxins Saxitoxin and analogous

Okadaic acid DTXl DTX2 DTX3

Domoic acid and isomers

Brevetoxins Ciguatoxins Maitotoxin Gambiertoxins

Anatoxin-a Anatoxin-a(s) Microcystins Nodulann Cylindrospermopsin PSP toxins

Detection HPLC Mouse assay Binding assay Cytotoxic assay

HPLC PP2A assay Mouse assay

HPLC Binding assay CE-UV

HPLC EL1SA Mouse assay

HPLC ELISA

HPLC ELISA Immunoassay PP2A assay

Mortality 13 % 0 % 3 % 0 % < 1 % i

Biol Res 3 1 : 375-386 (1998) 3 7 7

concen t r a t e on the surface as b lue -g reen s c u m . T h e y p r o d u c e t o x i n s w h i c h a r e classified according to their mode of action i n t o h e p a t o t o x i n s (e.g., m i c r o c y s t i n s , nodularin), neurotoxins (e.g., anatoxins), PSP t o x i n s (e.g., s a x i t o x i n s and a n a l o g o u s ) ( J a c h i m & G e n t i l e , 1 9 6 8 ; M a h m o o d & C a r m i c h a e l , 1986; H u m p a g e et al, 1994; Negri & Jones, 1995; Onodera et al, 1997; Lagos et al, 1997), skin irritants and other toxins (e.g., cyl indrospermopsin) (Ohtani et al, 1 9 9 2 ; S i v o n e , 1 9 9 6 ) . In C h i l e , c y a n o b a c t e r i a b l o o m s wi th p r e s e n c e of mic rocys t ine L-R and mass ive fish death have already been reported (Penaloza et al, 1990; G a e t e ^ a / , 1994).

T h e global d i s t r ibu t ion of harmful algal b looms is shown in F igure 1. This f igure , u p d a t e d to 1997 , s h o w s the p r e s e n c e of t h e f o u r m a j o r p o i s o n i n g s y n d r o m e s re la ted to m a r i n e tox ins . T w o of t hem are p resen t in Ch i l e ( P S P and D S P ) , a third one ( A S P ) is po ten t ia l ly a threat to Ch i l e , s ince the d i a tom Nitzschia pseudoseriata

(Pseudonitzschia australes F r e n g u e l l i ) , one of the pos tu l a t ed c a u s a t i v e o rgan i sms t h a t p r o d u c e d o m o i c a c i d , h a s b e e n d e s c r i b e d f r e q u e n t l y in p h y t o p l a n k t o n s amp l ing in Ch i l ean wa te r s ( L e m b e y e et al, 1975; M u n o z et al, 1992; Hallegraeff , 1993) . P S P and D S P are wel l d o c u m e n t e d in C h i l e (Zao et al, 1993 ; Ur ibe , 1993; L e m b e y e , 1 9 9 2 ; L a g o s et al, 1 9 9 6 ; C o m p a g n o n et al, 1998 ) a n d v i r t u a l l y w o r l d w i d e (Hal legraeff , 1993 ; Y a s u m o t o & M u r a t a , 1 9 9 3 ; P r o e n c a et al, 1997) .

Due to the ex tens ion of the topic , this rev iew will be focused on de te rmina t ion of P S P toxins and its p resence in the Southern part of Chi le .

PARALYTIC SHELLFISH POISONING

T h i s p o i s o n i n g p o s e s t h e m o s t s e r i o u s th rea t to pub l i c hea l th and the shel lf ish i n d u s t r y in C h i l e , b e c a u s e of i ts h igh mor ta l i ty rate (Tab le II) and the h ighes t

• PSP 0 DSP NSP ASP

Fig 1. Global distribution of four human illnesses associated with marine toxic algal blooms: Paralytic Shellfish Poisoning (PSP). Diarrheic Shellfish Poisoning (DSP), Amnesic Shellfish Poisoning (ASP) and Neurotoxic Shellfish Poisoning (NSP).

378 Biol Res 31 : 375-386 l l W

Tabic II

Human illness and fatalities associated to paralytic shellfish poisoning (by A. catenella) in Chile since 1972. (Source: Ministerio de Salud, S.S.R. Magallanes).

Date Locality Illness Death Bivalve sp. M. Bioassay

XII Region MU

22 Oct 1972 Bahia Bell 3 3 A. ater 96.000

20 Feb 1981 Seno Unión 26 2 A. ater W/O

16 Apr 1989 Estero Núñez 8 0 A. ater 1,555

29 Mar 1991 Bahía Nash 95 2 M. chilensis 9,855 18 Nov 1991 Seno Unión 125 2 A. ater W/Q 26 Die 1991 Seno Nevado 2 2 A. ater 1 1,448

13 Jan 1992 Bahía Wodsworth 14 6 M. chilensis 12,636 13 Jan 1992 Estero Asia 5 0 M. chilensis 70,157 ¡6 Feb 1992 Km 49 Sur 3 0 M. chilensis 7,560 01 Mar 1992 Caleta La Olla 1 1 M. chilensis W/Q 06 May 1992 Paso Nuevo 3 0 A. ater W/Q 18 Jul 1992 Isla Vancouver 1 0 A. ater W/Q 20 Jul 1992 Paso Schoal 3 0 A. ater 6.795 27 Die 1992 Puerto Williams 6 1 M. chilensis 14.544

02 Jan 1994 San Juan 8 0 M. chilensis 10,800 02 Jan 1994 Los Ñires 1 0 M. chilensis 4,104 02 Jan 1994 Punta Arenas 2 1 M. chilensis 2.6 lit 03 Jan 1994 Punta Arenas 1 0 M. chilensis 2.412 04 Feb 1994 Punta Arenas 1 0 M. chilensis W/Q 1 I Apr 1994 Bahía G. Grande 1 0 A. ater W/Q 27 Jun 1994 Seno Ringdove 1 1 A. ater 8.415

02 Jan 1995 Chabunco 4 0 M. chilensis 1.896 09 Feb 1995 Seno Profundo 1 0 A. ater W/Q

06 Jan 1997 Isla Isabel 2 0 A. ater 1.627 10 Jan 1997 Fuerte Bulnes 1 0 A. ater 12.330

23 Jan 1997 Santa María 1 0 A. ater 2,602

Total XII Region 319 20

XI Region HPLC Hg/100g

0 5 J u n 1995 Isla Toto 8 , M. chilensis 38.554

28 Nov 1995 Puerto Aguirre 1 1 A. ater W/Q

24 Feb 1996 1 1 W/Q Total XI Region 10 3

TOTAL 329 23

W/Q = without quantitation 'mportant: 3 more fatalities occurred in XI Region during Spring 1987-Summer 1998 period.

toxici ty found in its mol luscs (Lagos et al, 1996; C o m p a g n o n et al, 1998).

Para ly t ic shellfish po i son ing toxins act by revers ibly b ind ing to a receptor on the v o l t a g e - g a t e d s o d i u m c h a n n e l , b l o c k i n g n e u r o n a l t r a n s m i s s i o n ( K a o , 1 9 6 6 ; H e n d e r s o n et al, 1973 ; S t r ichar tz , 1984; M o c z y d l o w s k i et al, 1984, 1986; Guo et al, 1987; Hall et al, 1990; L o n g et al, 1990;

St r ichar tz et al, 1995) . Saxi toxin , was the f i r s t o n e k n o w n a n d t h e m o s t s t u d i e d c o m p o n e n t of P S P tox ins ; it is hydrophi l ic (posi t ively charged at neutral pH) , low mol wt (298 Da) and a non-pro te in toxin. Due to its high affinity to its b ind ing site, it has b e e n a very useful tool to quant i fy the sod ium channe l dens i ty , to purify and study the sod ium channel p roper t ies . Until know,

Biol Res 3 1 : 375-386 (1998) 379

it h a s b e e n d e s c r i b e d m o r e t h a n 2 6 a n a l o g u e s of S a x i t o x i n w h i c h o c c u r n a t u r a l l y ( F i g 2) ( H a r a d a et al, 1 9 8 2 ; O s h i m a et al, 1 9 8 9 ; O s h i m a , 1 9 9 5 ; O n o d e r a et al, 1997).

T h e o r g a n i s m s that are r ecogn ized as pr imary sources of P S P toxins inc lude three m o r p h o l o g i c a l l y d i s t i n c t g e n e r a o f d i n o f l a g e l l a t e s , Alexandrium sp., Pvrodinium sp. and Gymnodinium sp. (Hall et al, 1990; Hallegraeff, 1993; Y a s u m o t o et

al, 1995; O s h i m a , 1995), as well as four species of b lue-green alga, Aphanizomenon

flos-aquae, Anabaena circinalis, Lyngbya wollei and Cylindrospermopsis raciborskii ( M a h m o o d & C a r m i c h a e l , 1986; H u m p a g e et al, 1994; Negri & Jones , 1995; Falconer , 1996; S ivonen , 1996; O n o d e r a et al, 1997; L a g o s et al, 1997).

Toxic algal b looms have been detected in t h e t h r e e S o u t h e r n m o s t r e g i o n s of the c o u n t r y , w i t h Dinophysis acuta and

OH R2 R3

RI R2 R3 R4 R5 STX H H H COONH 2 OH

neoSTX OH H H COONH2 OH dcSTX H H H OH OH

dcneoSTX OH H H OH OH doSTX H H H H OH GTX1 OH oso 3

H COONH2 OH GTX2 H oso 3 H COONH 2 OH GTX3 H H OS03- COONH 2 OH GTX4 OH H OS03" COONH 2 OH GTX5 H H H COONHS03 OH GTX6 OH H H COONHS03" OH

dcGTXl OH oso 3" H OH OH dcGTX2 H OSOJ H OH OH dcGTX3 H H OS03" OH OH dcGTX4 OH H OS03- OH OH doGTX2 H OS03" H H OH doGTX3 H H OS03 H OH

CI H OS03- H COONHS03" OH C2 H H OS03 COONHS03 OH C3 OH OSO," H COONHS03" OH C4 OH H OS03- COONHS03" OH

1 H H OS03" COOCH3 H 2 H H OS03 C O O C H 3 OH 3 H OS03 H COOCH3 OH 4 H H H H H 5 H H H COOCH3 OH 6 H H H COOCH3 H

Fig 2. Chemical structures of paralytic shellfish toxins.

3 8 0 Biol Res 31: 375-386 ( 1998

Alexandrium catenella being t h e most i m p o r t a n t t ox i c s p e c i e s ( G u z m a n et al, 1975a,b; Munoz et al, 1992; Lembeye, 1992; Lagos et al, 1996; Compagnon et al, 1998).

Pa ra ly t i c shellf ish po i son ing has been recognized for over a century as a cl inical enti ty in this far part of South Amer ica . In 1908, Segers documen ted the intoxicat ion and p e r i o d i c m a s s p o i s o n i n g of n a t i v e people due to musse ls consumpt ion in the proximi ty of Ushua ia (Beagle Channe l ) in 1886. This is the earliest report of P S P in this a rea . S ince then , the p r e v e n t i o n of human intoxicat ion due to the ingest ion of toxic shellfish has been an impor tan t issue of m u t u a l i n t e r e s t to p u b l i c hea l th and fishery author i t ies of the country .

Alexandrium catenella p r o d u c e s P S P t o x i n s w h i c h c o n t a i n s s ax i t ox in and its de r iva t ives . This d inof lage l la te is main ly found in the Sou the rnmos t reg ions of Chi le , a d i s t a n c e c o v e r e d by 1,200 km l o n g be tween 4 3 ° 4 5 ' 0 0 " - 5 6 ° 0 0 ' 0 0 " Lat S (Fig 3) ( L a g o s et al, 1996 ) . S i n c e 1 9 7 2 , t ox i c b looms of A. catenella had resul ted in the death of 26 persons in Chi le (Table II), all of them intoxicated by the consumpt ion of s h e l l f i s h h a r v e s t e d in t h e XI a n d X I I R e g i o n s ( L e m b e y e , 1 9 9 2 ; M o n t e b r u n o , 1 9 9 3 ; m e e t i n g p r e s e n t a t i o n s of off ic ia ls b e l o n g i n g to Servicios Régionales de Salud). Also, the highest P S P con tamina ted shellfish had been repor ted in this part of S o u t h A m e r i c a ( B e n a v i d e s et al, 1995 ; C o m p a g n o n et al, 1998). S ince 1993, the appea rance of P S P is cons idered endemic in this part of the world (Lembeye , 1992; U r i b e , 1 9 9 3 ; L a g o s et al, 1 9 9 6 ; C o m p a g n o n et al, 1998).

TOXINS DETECTION

A l t h o u g h t h e f i r s t t o x i c b l o o m of A. catenella was documen ted in the Mage l l an Strait (XII Reg ion) in 1972 ( G u z m a n et al, 1975a,b) , is well known that P S P is a lso p r e s e n t in t h e XI R e g i o n s i n c e 1992 ( M u n o z et al, 1992). In both cases , until 1996, all the moni to r ing of P S P in survey p r o g r a m s and dur ing ou tb reaks has been d o n e us ing the mouse bioassay to es t imate the total toxicity in shellfish ( S o m m e r &

56° •

Fig 3. Geographical location of the two Southern regions of Chile, where paralytic shellfish poisoning is occurring All samples were collected from these regions.

M e y e r , 1937) . Th i s b ioassay has several d r a w b a c k s : l a c k of s p e c i f i c i t y a n d prec is ion , i nconven ience and difficulty of m a i n t a i n i n g l a r g e s tock of a n i m a l s , no in format ion abou t toxin compos i t i on and signif icant er rors when test ing samples of low toxic i ty . T h u s , an a l ternat ive method u s i n g a p o s t - c o l u m n d e r i v a t i z a t i o n incorpora ted with high per formance liquid ch roma tog raphy ( H P L C ) and f luorescence de tec t ion was in t roduced ( O s h i m a et al, 1 9 8 9 ; O s h i m a , 1 9 9 5 ) . W i t h t h i s n e w analyt ica l tool , the P S P toxin profi les of Ch i l ean shel l f ish and d inof lage l l a tes had been known for the first t ime (Lagos et al. 1996; C o m p a g n o n et al, 1998).

A s c h e m a t i c i l l u s t r a t i o n of the P S P toxins ana lyzer used in our laboratory is s h o w n in F i g u r e 4 . T h e r a d i o c h e m i c a l l iquid sc int i l la t ion coun t ing f low- through moni to r coup led on- l ine also a l lows us to fol low the r ad ioac t ive tox ins ((3 emi t t e r s like 3 H or 1 4 C ) in real t ime display of both

Biol Res 31 : 375-386 (1998) 381

HPLC Pumpl ) • I • — 1

Injector

Mobile phase

( PC Computer

I DATA COLLECTION

Fluorescence

monitor

Water bath (65°C) 1 Column

monitor (] D.0000 ] )

UV-VIS

Drain

I Pump 3 S Pump 2 J

Acid reagent w Oxidizing reagent

H 3 P 3 2 C

Radiochem Detector

Split Sample Liquid Scintillation Cocktails

^Fraction Collector,

Fig 4. Schematic illustration of PSP toxins analyzer with fluorescent and radiochemical detection on line.

rad ioac t ive and f luorescent s ignals . Us ing t h i s m e t h o d , a h i g h s e n s i t i v i t y w i t h d e t e c t i o n l i m i t s of 1 0 0 - 2 0 0 fmo l w a s at ta ined. F igures 5A and 5B show typical c h r o m a t o g r a m s of P S P t o x i n s . T h e s e c o r r e s p o n d to p u r e t o x i n s i s o l a t e d and purif ied from Chi lean highly con tamina ted s h e l l f i s h u s i n g p r e p a r a t i v e l i q u i d c h r o m a t o g r a p h y . Ident i f icat ion and puri ty de te rmina t ion of these toxins were done by H P L C with on- l ine f luorescence detect ion ( H P L C - F D L ) a n d H P L C c o u p l e d m a s s s p e c t r o m e t r y ( H P L C - M S ) . F o r r o u t i n e a n a l y s i s of s h e l l f i s h o r d i n o f l a g e l l a t e s

s amp le s , s tandard ca l ib ra ted so lu t ions of P S P t o x i n s as r e f e r e n c e m a t e r i a l w e r e prepared ( F O N D E C Y T 196! 122).

H P L C - F D L is an analyt ical method that has the abi l i ty to quant i fy each toxin in crude samples of small size (20 pi), which yield a s ingle toxin concen t ra t ion . It is also a p o w e r f u l t oo l in t h e r e s e a r c h f ie ld , special ly for analys is of toxins product ion by d i n o f l a g e l l a t e s f r o m p h y t o p l a n k t o n c o l l e c t e d in s e a w a t e r s or in c u l t u r e d samples . The high sensi t ivi ty of the system enable us to e luc ida te the comple te toxin profi les of A. catenella c lones in cul ture

382 Biol Res 31 : 375-3X6 ( 1 W K >

col lected in both regions and also, for the first t ime, to measure the amoun t of P S P toxins/cel l p roduced by these c lones of A. catenella (Fig 6) .

Pure toxins of known concent ra t ion are essent ia l for this type of analys is . Mos t of t h e P S P t o x i n s a r e n o t c o m m e r c i a l l y avai lable . Our laboratory is the only one in S o u t h A m e r i c a a n d o n e of t h e f e w w o r l d w i d e to h a v e t h e m . T o x i n concen t ra t ions of samples were resolved by c o m p a r i n g the peaks areas of each toxin with those of the s tandards (Osh ima , 1995; Lagos et al, 1996; C o m p a g n o n et al, 1998).

TOXINS PROFILES

Using the above method , the analys is of mussel Mytilus chilensis samples -col lec ted in 1994 in the XII Region and in 1994-95 in the XI Reg ion- a l lowed us to de te rmine and c o m p a r e the ave rage profi le of both

r e g i o n s ( L a g o s et al, 1996 ) . Af te r the a n a l y s i s of m o r e than 100 s a m p l e s , we found a d i s t inc t ive toxin profi le for each r e g i o n . F i g u r e 7 s h o w s b o t h a v e r a g e p r o f i l e s ( t o p and c e n t e r ) . T h e y d i sp l ay e l e v e n of t h e m o s t k n o w n P S P t o x i n s a l ready descr ibed in o ther musse l samples wor ldwide ( O s h i m a et al, 1989; Cembe l l a et al, 1987; O s h i m a , 1995). Never the less , both average profi les undoub ted ly showed different a m o u n t s of each toxin.

The samples from XII Region (nearbv the Mage l l an Strait , 52°00 '00" -56°00 '00" ' ) showed the G T X 1 / G T X 4 ep imeres as the mos t abundan t ones . In contras t , samples from XI Region showed the G T X 2 / G T X 3 ep imeres as the major ones . The presence of G T X S was also un ique in these samples and in the s ax i tox ins g roup ( S T X s ) , the S T X c o n t e n t w a s h i g h e r than n e o S T X . B o t h p r o f i l e s s h o w e d the g o n y a u t o x i n s g roup as the mos t abundan t in the extract samples , be ing in both cases around 8 0 % of

Biol Res 31 : 375-386 (1998) 3 8 3

A .

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Fig 6. PSP toxins produced by A. catenella clones in culture ( femtomole/cel l : Means ± SD; N=4). A. Clone AAC7 from XI Region. B. Clone MACS from XII Region. Data from MI Bahamonde ' s Thesis as Marine Biologist, performed in our laboratory (June 1997).

50-

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the total con ten t . In the Ch i l ean musse l s a m p l e s , g o n y a u t o x i n s ( G T X S ) a re the toxins that account for the high toxici ty of the shellfish in both regions (Lagos et al, 1996; C o m p a g n o n et al, 1998).

W e have found ev idence that the toxin p r o f i l e s in f i l t e r - f e e d i n g s h e l l f i s h l i k e Mytilus chilensis, are mainly a reflection of the tox in p ro f i l e s of the d i n o f l a g e l l a t e s occur r ing in each region, and subsequent ly mark the event ou tbreak of d iverse s trains or c lones of A. catenella in Chi lean waters . S imi lar ev idence had been found in Japan ( O s h i m a et al, 1989; Osh ima , 1995) and in the Eas tern coas t of C a n a d a (Cembe l l a et al, 1987; Bricel j et al, 1991).

At the bo t tom of F igure 7, an average t o x i n p r o f i l e of s ix m u s s e l s a m p l e s col lec ted in 1992 is shown. These samples

X h-

Fig 7. Average PSP toxin composition (mole %) found in Mytilus chilensis extracts of samples collected in 1994-95 from XI Region (top), in 1994 from XII Region (center) and in 1992 from XII Region (bottom).

w e r e k e p t f r o z e n by Dr L u i s V e r g a r a , former chief of Departamento Programas sobre el Ambiente, Servicio de Salud Regional de Magallanes. T h i s p r o f i l e s h o w s a t o t a l d i f f e r e n t P S P t o x i n s c o m p o s i t i o n . H e r e , t h e m o s t a b u n d a n t toxins were S T X and n e o S T X , which are the most toxic of P S P tox ins . These two P S P t o x i n s s h o w , by m o u s s e b i o a s s a y , specific toxic i t ies of 2 ,483 and 2,295 M L 7 pmol , respect ive ly (Osh ima , 1995).

3 8 4 Biol Res 3 1 : 3 7 5 - 3 X 6 ( I W M

The average percentage of toxicity (MU %) by g roup of P S P toxins of these samples is s h o w n in F i g u r e 8. D e f i n i t e l y , t h e p redominan t one cor responds to the S T X s group (STX, n e o S T X and d c S T X ) , which represents 6 4 % of the total toxicity of the s a m p l e s . Th i s f ea tu re e x p l a i n s the high m o r t a l i t y r a t e tha t o c c u r r e d d u r i n g the S p r i n g - S u m m e r pe r i od ( f rom N o v e m b e r 1991 to March 1992). Dur ing this outbreak, 10 out of 150 i n t o x i c a t e d p a t i e n t s d ied (Table II) . Dur ing that season, Chi le had t h e h i g h e s t r e g i s t e r e d n u m b e r of in toxicated peop le and fatalit ies associa ted to a P S P outbreak of our his tory. Moreover , in t h e s a m e S u m m e r of 1 9 9 2 , an e x c e p t i o n a l b l o o m of A. catenella w a s r e p o r t e d in the N o r t h - E a s t e r n s h o r e of B e a g l e C h a n n e l ( 5 5 ° 0 0 ' 0 0 " S ) , w h e r e musse l s a m p l e s co l l ec ted on Janua ry 20 reached a m a x i m u m of 127,200 pg S T X eq/ 100 g, by m o u s s e b i o a s s a y . Th i s is the h i g h e s t m u s s e l t o x i c i t y r e p o r t e d in the l i terature (Benav ides et al, 1995).

W e be l ieve that in the sed iment of our S o u t h e r n fjords in XI and XI I R e g i o n s , different strains of Alexandrium sp. exist . T h e y s u r v i v e as d o r m a n t Alexandrium cys ts , and every year - d u r i n g the Southern S p r i n g - S u m m e r s e a s o n - these cysts c o m e b a c k as g e r m i n a t i v e c e l l s , p r o d u c i n g ou tbreaks . This resul ts in the con tamina t ion of the c o m p l e t e food chain of the fjord, s t a r t i ng wi th the f i l t e r - f eed ing she l l f i sh ( C o m p a g n o n et al, 1998).

The fjord system in the Southern part of Chi le , most unspoi led land and distant from c i t i e s w i t h h i g h p o p u l a t i o n d e n s i t i e s , presents one of the highest levels of natural b ioaccumula t ion of P S P toxins by shellfish in the wor ld ( C o m p a g n o n et al, 1998). The presence of A. catenella is endemic in both regions and, every S p r i n g - S u m m e r season, ou tbreaks of different intensity and toxici ty ar ise , one in the Spr ing and other in late S u m m e r ( D r L u i s V e r g a r a , p e r s o n a l c o m m u n i c a t i o n ; L a g o s et al, 1 9 8 6 ; C o m p a g n o n et al, 1998).

T r a d i t i o n a l l y , o n l y f i l t e r - f e e d i n g molluscs that concentra te these toxic algae are considered in moni tor ing programs for s h e l l f i s h p o i s o n s . H o w e v e r , i n c r e a s i n g a t t e n t i o n is b e i n g pa id to h i g h e r o r d e r

Fig 8. Toxicity contribution by groups of PSP toxins (MU obtained from average toxin composition and each toxin specific toxicity of 6 samples collected in 1992. Value (•)'/< in group of N-su l focarbamoyl - l l -hydroxysuI fa le toxins (C1-C4 0.03 MU %) means that it contributes less than 1'.; to total sample toxicity.

consumers , such as carnivorous gastropods a n d c r u s t a c e a n s , d u e to t h e t h e i r accumula t ion capaci ty of PSP toxins and to b e f r e q u e n t l y c o n s u m e d by h u m a n s (Shumway , 1995; C o m p a g n o n et al, 1 9 9 8 ) . T h e c a r n i v o r o u s g a s t r o p o d s Concholepas concholepas (loco) a n d Argobuccinum ranelliformes (caracol del sur), which are popular domest ic and commercia l sea foods in Chi le , had been repor ted to be highly c o n t a m i n a t e d for m o n t h s a f t e r an A. catenella outbreak (Compagnon et al, 1 9 9 8 ) .

In t h e p a s t , in C h i l e , t h e p r i n c i p a l i m p e d i m e n t s to a n a l y z e P S P t o x i n s in nat ive mol luscs , phy top lank ton (sea water sample) and d inof lagel la tes in cul ture , were t h e l a c k of a s i m p l e , a u t o m a t i c , r e p r o d u c i b l e m e t h o d w i t h s u f f i c i e n t sens i t iv i ty to d e t e c t p i c o g r a m quan t i t i e s and the ana ly t ica l s t anda rds requi red for such ana lys i s . Both imped imen t s have been o v e r c o m e by our labora tory .

A C K N O W L E D G E M E N T S

W e t h a n k D r Y a s u k a t s u O s h i m a . L a b o r a t o r y of B i o o r g a n i c C h e m i s t r y . T o h o k u U n i v e r s i t y , S e n d a i , J a p a n , for i n t r o d u c i n g us in th is f ield and for his suppor t . He init ial ly p rov ided us with his P S P t o x i n s a n a l y t i c a l s t a n d a r d s , w h i c h a l lowed us to begin with this method and to ca l ibra te our s tandards .

W e are also grateful to Dr Luis Vergara . Departamento de Programas sobre el

Biol Res 31 : 375-386 (1998) 385

Ambiente, Servicio de Salud Regional de Magallanes, for p rov id ing us with the six samples from the 1992 b loom and also for shar ing with us his amaz ing exper ience in para lyt ic shellfish po i son ing .

F ina l ly , we are thankful to the Sociedad Chilena de Ciencias Fisiológicas, fo r invi t ing the author of this review to present it as a lec ture dur ing its Annual Mee t ing (Qu inamáv ida , Chi le , April 6, 1998).

The work of the author of this review has been suppor ted by Gran ts F O N D E F 2-3 7 , F O N D E C Y T 1 9 6 1 1 2 2 a n d by Fundación Andes.

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