Presence of saxitoxin in toxic extracts from Gonyaulax polyedra

Toxicon Vol. 28, No . 9, pp. 1113-1116, 1990. Pri°ted io Grcat Britaio.

0041-0101/90 53.00+ .00 ® 1990 PecSamoo I~ plc

SHORT COMMUNICATIONS PRESENCE OF SAXITOXIN IN TOXIC EXTRACTS FROM GONYAULAX POLYEDRA

MILENA BRUNO, I PAOLA MARGiIERTTA BIANCA GUCCI, 1 ELIO PIERDOMINICI,~ ALFREDO IorroLOZ and LAURA VOLTERRA I 'Department of Eavironmcntal Hygiene and'Department of Veterinarian Medicine, Istituto Superiors di Sanità, Rome, Italy (Accepted for publication 24 April 1990)

M. BRUNO, P. M. B. Guccl, E. Pn?itDO1~IVICI, A. IOPPOLO and L. VOLTERRA . Presence of saxitoxin in toxic extracts from Gonyaulax polyedra . Toxicon 28, 1113-1116, 1990 .-A "red tide" bloom of Gonyaulax polyedra occurred in Italy in Autumn, 1988 . Algal concentrated extracts and undiluted water samples from the bloom were tested both with the Microtox system and a mouse bioassay, revealing the presence of paralytic shellfish poison-like neurotoxins . Saxitoxin levels evaluated on the basis of toxicological and instrumental analysis showed discrepancies. Other toxins could be present in addition to paralytic shellfish poison . INTRODUCTION

Gonyaulax polyedra may cause red tides in the Adriatic sea (FORTUNA et al., 1987). Production of Paralytic Shellfish Poison (PSP}-like toxins is associated with the genus Gonyaulax, and widely demonstrated for G. catenella (SCHANTZ et al., 1966; PROKTOR et al., 1975 ; BATES et al., 1978), G. tamarensis (PRAKASH, 1967 ; ANDERSON and Po-oN-CI-IENG, 1988), G. excavata (BATES et al., 1985). Only Sct-ittADlE and BLISS (1962) have reported a toxic effect on mice for G. polyedra, testing a bloom which occurred along the southern California coast, but their results have been questioned (PATTON et al ., 1967 ; BATES et al ., 1975). Since then, several authors have included G.polyedra among the six species of Gonyaulax documented as being toxic (PRAKASH, 1967 ; SCHMIDT and LOEBLICH, 1975 ; COLLINS, 1978). Until 1987, toxicological analyses performed in our laboratory on algal concentrates containing G. polyedra and collected during bloom episodes, never revealed the presence of toxic strains which were able to produce PSP toxins (FORTUNA et al ., 1987). In October 1988, toxicological experiments carried out on undiluted water samples collected during a red water episode with a prevalence of G. polyedra revealed the presence of algal biotoxins. Further assays ascertained the presence of PSP toxins, which are reported in this paper. In October 1988, a mixed bloom of dinoflagellates, characterized by a phytoplanctonic biomass of 24 .5 x 106 cells/liter with a dominance of 82% of G.polyedra was observed in a harbour of the upper Adriatic sea in Italy. The residual population of the bloom was represented by Scripsiella, Gyrodinium and Peridinium genera . An algal concentrate (1240 mg wet wt) was suspended in 20 ml of filtered marine water. Ten milliliters were 1113

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disrupted with a Polytron homogenizer (10 min), boiled for 5 min, and centrifuged (extract A) and the supernatant tested . The residual 10 ml were boiled for 5 min and then sonicated and centrifuged as previously described (MERILUOTO and ERIIiSSON, 1988) (extract B). Another algal concentrate (450 mg) was suspended on 6 ml of HCl (0.1 I~ and extracted for PSP toxins (AnnMS and MrnsC>E1t, 1989) (extract C). In addition, analyses were performed on water samples . Ten milliliters of an undiluted aqueous extract sample was disrupted with a Polytron homogenizer for 10 min and boiled for 5 min . The three algal extracts, as well as the undiluted aqueous samples, were immediately tested using the Microtox system, following the directions specified by the firm. In particular, the pH of extract C was adjusted to neutrality (pH 7). Extract C (75 mg/ml) and extract A (62 mg/ml) were also tested inoculating i.p. 1 ml of extract in male Swiss white mice of about 19-20 g body weight . Extracts C and B were qualitatively tested on HPLC using a reverse-phase C18 column with 5 ~m diameter spherules, according to the method of RuBlxsorr (1982), using saxitoxin (STX) (EPA, Lot 8, Serial no. 673 U.S. Food & Drug Administration, Cincinnati, OH) as standard. A quantitative analysis was performed for extract B using the spectrofluorometric assay according to the method of SHOPTAUG et al. (1981) with a Turner spectrofluorimeter 430 model, after a calibration curve with STX standard (100 Fig/ml). The undiluted water sample showed a weak toxicity when submitted to the Microtox test (Table 1). More interesting results were obtained from the algal pellet extracts : in these cases the Microtox test exhibited EC50 (at 5 min) values ranging from 1 .740 to 0.101 mg algae (wet wt) /ml, due to the different extraction methods . Adjustment of pH in order to use the Microtox test after the proper PSP extraction (ADAMS and MIESCiER, 1980), decreased the toxicity of the extract. As it is well known, saxitoxin is inactivated at an alkaline pH (ADAMS and MIFSCIER, 1980) . Three male Swiss white mice injected i.p. with 1 ml of extracts A(62 mg/ml) and C(75 mg/ml) died within 3 and 5 min respectively, exhibiting the following symptons : lethargy, convulsive jumping movements, loss of coordination, clonic convulsions and posterior limb paralysis before death . The results supported the presence of a neurotoxin. The limited quantity of the collected algal pellet did not allow us to define an LDSp . In any case, according to the toxicological scheme of SCFIANTZ (1986), extracts A and C might have contained about 0.666 kg/ml and 0.324 pg/ml, respectively, of pure STX . The HPLC results revealed the presence of an STX fraction in extracts B(Fig. 1) and C . Further TABLE 1 . TOXICOLOGICAL AND CHEMICAL DETERA4NATION OF 3AXTfOXIN CONTENT

Amount of STX according to Ec,°

Death time in mice (IIB/m1)

0 .101 1 .151 1 .740

0 .666

(mB/m1) Extract A Extract B Extract C Undiluted sample Pure saxitoxin

290% 0 .00128

0 .324

Fluorometric determinations G;B/~) 0.022

Quantity of the various extracts, and of pure saxitoxin, corresponding to the same Ec,° (Microtox Extinction Coefficient 50 at 5 min) and saxitoxin amount determined from mice bioassay (SCHANTZ, 1986) and from fluorometric determination .

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0.1300

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FIG . I . HPLC ANALYSIS OF EXTRACT B .

Chromatographic conditions : mobile phase was water with 25 mM formic acid and 4 mM pentanesulfonic acid at a flow rate of l .5 ml/min . Injection volume was I50 ul and u .v . absorption was measured at 253 nm .

fluorometric examination of the eluted peaks allowed us to estimate that the amount of STX in extract B, the most toxic obtained by HPLC, was 0.022 pg/ml. The present data can be considered as the first clear experimental evidence of STX production by wild G. polyedra toxic strains. In their work, SCHRADiE and BLtss (1962) had doubtful results: they tested cells from a bloom along the coast of southern California, but the crude extract, purified on ion-exchange resin (IRC-50) was non-toxic. A toxin identical to STX was found, after further purification by paper chromatography . Their conclusions were considered to be the result of a possible contamination of the large amount of STX used as standard (Bn~l~s et al., 1978). Other blooms from the southern Californian coast were successively tested (PAZ-roN et al., 1967; BATES et al., 1978) with no detection of toxicity . In our work, bioassays as well as two chemical assays revealed the presence of STX. Previous experiments with pure STX (FDA standard), to establish its toxic standard curve for the Microtox test, revealed an Ec~ of 1 .281cg/ml. On the basis of such results the content of extracts (A, B, C) is reported in Table 1 . The STX concentration of extract B, ftuorometrically determined, was 100-fold lower: 0.022 pg STX/ml (0.355 ng/mg algae wet wt). The same discrepancy appears when the STX concentrations of extracts A and C are calculated on the basis of a mouse bioassay (ScxArrrz, 1986) (Table 1). Toxins revealed by chemical analyses (HPLC and fluorometry) could be a portion of the entire toxin content contained in the G. polyedra examined and belonging to the PSP group (OMS, 1986). This conclusion is suggested when instrtunental results are compared to those of the bioassays. Further analyses are necessary in order to verify this hypothesis .

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