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Toxicon 49 (2007) 710–716

www.elsevier.com/locate/toxicon

Identification of pectenotoxins in plankton, filter feeders, andisolated cells of a Dinophysis acuminata with an atypical toxin

profile, from Chile

Juan Blancoa,�, Gonzalo Alvarezb, Eduardo Uribeb

aCentro de Investigacions Marinas, Pedras de Coron s/n, apdo 13, 36620 Vilanova de Arousa, Spain.bFacultad de Ciencias del Mar, Departamento de Acuicultura, Universidad Catolica del Norte, Larrondo 1281, Coquimbo, Chile

Received 15 July 2006; received in revised form 16 November 2006; accepted 17 November 2006

Available online 28 November 2006

Abstract

A bloom of Dinophysis acuminata produced, in autumn of 2005, a closure of the scallop harvesting in Bahıa Inglesa, in

the Chilean III region. Isolated cells of this Dinophysis species were shown to contain 180 pg cell�1 of pectenotoxin 2 but

neither okadaic acid nor any of its analogs or derivatives (at least at a detectable level). Examination of plankton and filter-

feeder samples covering an area of ca. 350 km, from the location where the toxicity was recorded to Bahıa Tongoy, showed

that the unique toxin profile found in the first bloom was widespread over that part of Chile and persisted for months. The

analysis were carried out by HPLC-ESI-MS using positive ionization mode, with a detection limit below 2ng of OAmL�1

of methanolic extract. This is the first report of the presence of pectenotoxins in the plankton of the Pacific coast of

America and in the studied filter feeders. This is also the first report of a Dinophysis species containing pectenotoxins and

not any toxin of the okadaic acid group.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Pectenotoxins; Diarrhetic shellfish poisoning; Okadaic acid; Dinophysistoxins; Dinophysis acuminata; Semimytilus algosus;

Argopecten purpuratus; Mulinia edulis; Pyura chilensis; Chile

1. Introduction

Pectenotoxins are polyether lactones (Fig. 1) that,because of their characteristics of solubility andtheir usual co-occurrence with DSP toxins, verylikely interfere frequently with this latter group oftoxins in the mouse bioassay, which is widely usedto monitor shellfish safety. Even though pecteno-toxins are toxic to mice when administered intra-peritoneally and were though to be toxic to

e front matter r 2006 Elsevier Ltd. All rights reserved

xicon.2006.11.013

ng author.

ss: jblanco@cimacoron.org (J. Blanco).

humans—mostly after an outbreak recorded inAustralia (Burgess and Shaw, 2001)—recent studiessuggest that their oral toxicity is very limited (Mileset al., 2004a) and that the Australian outbreak wasprobably due to esters of toxins of the okadaic acidgroup more than to pectenotoxins (Burguess, 2003).

Since the discovery of PTX2 in Dinophysis forti,in Japan (Lee et al., 1989), these toxins have beenfound mostly in Dinophysis acuta (James et al.,1999; Suzuki et al., 2001b; Aune et al., 2002; Pavela-Vrancic et al., 2002; MacKenzie et al., 2002;Fernandez et al., 2003, 2006; Suzuki et al., 2003;Fernandez Puente et al., 2004; Miles et al., 2004b),

.

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Fig. 1. Structures of pectenotoxins.

J. Blanco et al. / Toxicon 49 (2007) 710–716 711

but also, in a few occasions, associated to Dino-

physis acuminata (MacKenzie et al., 2002; Quilliam2003, Miles et al., 2004b; ), Dinophysis norvegica,Dinophysis rotundata (Miles et al., 2004b), Dinophy-

sis sacculus (Pavela-Vrancic et al., 2002) andDinophysis caudata (Fernandez et al., 2003, 2006).At least the photosynthetic species are presumablyproducers of this kind of toxins.

In most cases, when the presence of pectenotoxinsis detected in a bloom, okadaic acid or one orseveral of their analogs and/or derivatives also co-occur (reviewed in Blanco et al., 2005).

This group of toxins has a geographical distribu-tion narrower than okadaic acid analogs, beingrestricted to Europe (both, Atlantic and Mediterra-nean coasts), New Zealand, Japan (reviewed inBlanco et al., 2005) and the North Atlantic coastof Canada (Anonymous, 2003). The presence ofpectenotoxins in plankton from the west coast ofAmerica has not been reported but they seem tohave been found in one occasion in musselsprobably from southern Chile (the available in-formation is reduced to an abstract of a commu-

nication to a conference, (Goto et al., 2000). This is,therefore, the first report of the presence ofpectenotoxins in dinoflagellate species and in anumber of filter feeders from the Pacific coastof America, and more precisely from NorthernChile.

Even though D. acuminata has been shown to bepresent in the north of Chile (Avaria and Munoz,1983, 1985; Avaria et al., 1982), and DSP mousebioassays on molluscs from the area have beencarried out since year 2000, DSP harvesting closuresin the area have not been needed until October2005, when the market of the Chilean Scallop(Ostion del Norte) Argopecten purpuratus had to bebanned. This harvesting closure raised the need ofstudying the toxin profiles of the involved filterfeeders and phytoplanktonic organisms.

In this work, we have studied the first knownbloom of D. acuminata producing pectenotoxinswhich took place in northern Chile at the end of2005, in which pectenotoxins, if they were not theonly toxins present, overwhelmingly dominated thetoxin profile.

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2. Material and methods

2.1. Biological material

Dinophysis acuminata blooms were detected inBahıa Inglesa, Bahıa Salada, Coquimbo and BahıaTongoy (Fig. 2) in the framework of the monitoringprogram ‘‘Sanidad de Moluscos Bivalvos’’ run bySERNAPESCA Chile. Periodic phytoplanktonsamples were obtained, from several locations ofthe North Chilean coast by means of net hauls(20 mm mesh) and a 15-m hose. Samples used forphytoplankton quantification were preserved withlugol (hose) and formaline 4% (net hauls). Phyto-plankton populations in the samples were identifiedand quantified (from hose samples) using themethod of Utermohl under a Olympus light micro-scope with an image processor Image Pro Plus.

2.2. Toxin extraction and analysis

Two 30mL of concentrated water samples (nethaul), containing D. acuminata cells from BahıaInglesa and four from Bahıa Tongoy, were filteredthrough 12 mm Nuclepore filters. The lipophilictoxins were extracted from the filters with aqueous

Fig. 2. Sampling locations.

methanol (80% v/v). An additional sample wasobtained by isolating 100 D. acuminata cells fromBahıa Inglesa, and placing them in aqueousmethanol. The samples were maintained in thoseconditions for about 3 days. After that time theextraction was completed by means of sonicationwith a Branson Sonic Power 250, and the extractwas clarified by centrifugation (10,000g, 8min) andfiltration through 0.22 mm nylon syringe filters.

The soft tissues of the shellfish samples werehomogenized and extracted with aqueous methanol(80% v:v). Subsequently the samples followed thesame processes that those of plankton.

The obtained extracts were directly analysed, but,in the case of plankton and in order to achieve moreresolution for the less abundant toxins, a 1mLaliquot was extracted by means of liquid/liquidpartition with 1mL of dichloromethane twice. Thetwo organic fractions were pooled, evaporated, andfinally, the residue resuspended in 100 mL of 80%aqueous methanol.

The presence of lipophilic toxins was checked byHPLC-MS/MS by means of a Surveyor MS pumpand autosampler, coupled to a Deca XP plus ion trapmass spectrometer (Thermo Electron) with anelectrospray interphase. The chromatographic separa-tion was carried out following the method by(Quilliam, 2003) and that by (Suzuki et al., 2006).The detection of okadaic acid, DTX2 and DTX1 wascarried out in negative ionization mode, using a sprayvoltage of 4000V and a capillar temperature of250 1C. The operation conditions of the mass spectro-meter for the detection of pectenotoxins were set topositive mode and to a spray voltage of 5000V. Forall toxins SIM and MS/MS modes were used.

The possible presence of esterified forms ofokadaic acid or DTX toxins was checked by meansof an alkaline hydrolysis following the procedure by(Vale and Sampayo, 2002a, b) modified by increas-ing the temperature (Suzuki et al., 2004) and by theomission of the hexane washing.

Okadaic acid and pectenotoxins were quantifiedby comparison with reference solutions of okadaicacid and PTX2, obtained from the NRC, Canada.PTX2 seco acid was quantified by reference toPTX2 and its concentration is expressed as equiva-lents of PTX2.

3. Results and discussion

Dinophysis acuminata bloomed in Bahıa Inglesasince the end of October 2005, reaching frequently

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concentrations over 100 cells L�1 (Fig. 3). In thesouthernmost of the studied locations no relevantpopulation of this species developed until January2006, but it maintained an average level of about100 cells L�1 until the end of this study in April 2006(Fig. 3). Bahıa Salada was characterised by lowphytoplankton biomass, and that included D.

acuminata whose populations were always difficultto detect (Fig. 3). In Bahıa Coquimbo, two bloomsof D. acuminata, separated by nearly 2 monthsduring which this species was below the detectionlimit, seem to have occurred (Fig. 3). In the mostsouthern bay, Bahıa Tongoy, D. acuminata startedto bloom at late December, and its populationspersisted in the area until the end of our sampling,reaching maximum concentrations of ca.300 cells L�1, at the end of February and thebeginning of April (Fig. 3).

No trace of okadaic acid, DTX2 or DTX1 wasfound in the analysis of the studied samples neitherof plankton nor of filter feeders. As the limit ofdetection of the technique used is below 2 ng ofOAmL�1 of the injected extract, it can assumed

Fig. 3. Dinophysis acuminata concentration in seawater at the

four locations studied. Symbols indicate when the samples of

plankton and filter feeders were taken.

that, in none of the raw extracts (10-fold lessconcentrated), this compound was in concentrationover 0.2 ngmL�1 (which is equivalent to 0.8 ng g�1

of filter-feeders soft tissue). It can also be assumedthat the two other toxins were also below thatconcentration as their difference of response withelectrospray ionization are probably not large. Thesame situation was found after alkaline hydrolysisof the extracts, to convert the possible esters (diol-esters, DTX4, DTX5, etc.y) or acyl-derivatives(‘‘DTX3’’) to their corresponding free acids.

Contrasting with the toxins of the okadaic acidgroup, pectenotoxins were found to be abundant inplankton samples. Several pectenotoxins were found(Table 1), but the most important one in terms ofconcentration was PTX2. A compound with thesame molecular mass than PTX1 (Fig. 4) was alsofound in the extracts of the plankton filtrate fromBahıa Inglesa, but with a much lower response.PTX1 is considered to be a metabolite of PTX2(Suzuki et al., 1998) and if the compound werePTX1, it would probably be a product of theactivity of the heterotrophic phytoplankton onPTX2. Notwithstanding, the compound could alsobe PTX11, which has the same molecular mass thanPTX1 and has been reported to be produced by D.

acuta (Suzuki et al., 2006). Unfortunately theconcentration in the extracts was too low to allowfor its identification.

The isolated Dinophysis cells were estimated tocontain 180 pg cell�1 of PTX2, which, as far as weknow, is the highest cellular content reported forany Dinophysis species, even when most of them aretypically larger than D. acuminata. If okadaic acidwere present in a concentration just below thedetection limit it would contribute with less than0.8% the concentration of PTX2, so that, atypicallyfor this species, pectenotoxins overwhelminglydominate the toxin profile. Mackenzie et al. (2005)in New Zealand and Miles et al. (2004b) in Norway,reported D. acuminata populations which containedpectenotoxins as main toxins but, in both cases, thecellular toxin content was much smaller than thatfound in the population we have studied (less than25 pg cell�1) and the contribution of the okadaicacid group of toxins to the total cellular toxin wasmuch larger (8–29%, in New Zealand and 6–15% inNorway). In other cases, D. acuminata does notseem to contain any pectenotoxin (Vale andSampayo, 2002a, b; Fernandez et al., 2003).

All filter feeders studied seem to contain PTXsbut, as in the case of plankton, none of them

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Table 1

Samples, toxins detected and relative contribution of PTX2 and PTX2sa to the total amount of PTXs detected. PTX2sa has been

quantified by assuming that it has the same response in the MS detector than PTX2. Relative contributions of the two pectenoxins in the

plankton samples were computed taking into account the amount detected and the detection limit. No concentration per cell is given for

plankton concentrates because cell counts were not available

Sample Species Location Date PTX2, % PTX2sa, %

Isolated cells Dinophysis acuminata Bahıa Inglesa 24/11/05 180 pg/cell n.d.

100 0

Plankton concentrate Dinophysis acuminata+other plankton Bahıa Inglesa 24/11/05 Detected n.d.

425 o75

Plankton concentrate Dinophysis acuminata+other plankton Bahıa Inglesa 24/11/05 Detected n.d.

100 0

Plankton concentrate Dinophysis acuminata+other plankton Bahıa Tongoy 17/03/06 Detected n.d.

100 0

Scallop BS Argopecten purpuratus Bahıa Salada 19/10/05 160 ng g�1 31.2 ng g�1

83.7 16.3%

Scallop BT Argopecten purpuratus Bahıa Tongoy 29/03/06 52 ng g�1 14 ng g�1

78.8 21.2%

Mussel Semimytilus algosus Bahıa Tongoy 29/03/06 4.72 ng g�1 7.6 ng g�1

38.3% 61.7%

Clam Mulinia edulis Coquimbo 27/03/06 3.64 ng g�1 7.2 ng g�1

33.6% 66.4%

Macha clam Mesoderma donacium Coquimbo 27/03/06 4.64 ng g�1 6 ng g�1

43.6% 56.4%

Piure (tunicate) Pyura chilensis Bahıa Salada 10/10/05 2 ng g�1 3.2 ng g�1

38.5% 61.5%

aConcentrations are expressed as equivalents of PTX2, as no certificate material of reference was available for PTX2 seco-acid.

Fig. 4. MS/MS chromatograms and spectra obtained by the method of Quilliam (2003) of the isolated Dinophysis acuminata cells showing

the presence of the ammonium (m/z ¼ 876, main fragment 823) and sodium (m/z ¼ 881, main fragment 837) adducts of PTX2, and the

presence of the sodium adduct of a pectenotoxin with the m/z of PTX1 and PTX11 (m/z ¼ 897, main fragment 853) in one plankton

concentrate from Bahıa Tongoy, containing Dinophysis acuminata.

J. Blanco et al. / Toxicon 49 (2007) 710–716714

okadaic acid or any of its analogs or derivatives(Table 1). In most cases a substantial conversion ofthe PTX2 to its corresponding seco-acid seem tohave occurred (Fig. 5), in sight of the substantially

higher contribution of PTX2sa in the filter feedersthan in the plankton, as it has been observed insome bivalve species (Suzuki et al., 2001a, b;MacKenzie et al., 2002).

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Fig. 5. Chromatogram and spectrum obtained by the method of Suzuki et al (2006) of the extract of scallops from Bahıa Tongoy, showing

the presence of PTX2 seco acid (PTX2sa). The chromatogram was obtained in full scan mode and subsequentely filtered to show the m/z of

the ammonium adduct of PTX2sa (894.5). The spectrum was obtained in data dependent mode.

J. Blanco et al. / Toxicon 49 (2007) 710–716 715

The coincidence of the toxin profile correspond-ing to plankton samples from distant locations(Bahıa Inglesa and Bahıa Tongoy) together withthose of the bivalves collected along the coast,suggest that the D. acuminata with the observedprofile is widely distributed in the area.

This is the first report of pectenotoxins inplankton of the Chilean coast and also of thePacific coast of America. In fact the presence ofpectenotoxin containing Dinophysis has only beenreported from the North Atlantic coast of Canada.This is also the first report of pectenotoxins in filterfeeders from Northern Chile. The presence ofpectenotoxins in bivalves from Southern Chile hasbeen reported (Goto et al., 2000) but no detailedinformation about that finding has been publishedto date.

Ackowledgments

We have to acknowledge to Carmen Marino, InePasten and Helena Martın for their technicalassistance, to Alvaro Rodrıguez, Jenny Rivera yMario Gutierrez (SGS-Chile) for the collection ofthe samples. We are also acknowledge the Asocia-cion de Productores de Ostion y Ostras de Chile(APOOCH) and the Servicio Nacional de Pesca(SERNAPESCA) for their support. This work waspartially funded by Project FDI-CORFO PT-02.

References

Anonymous, 2003. Report of discussion group A. Monitoring

toxins in a regulatory setting. In: Bates, S., (Ed.), Proceedings of

the Eighth Canadian Workshop on Harmful Marine Algae.

Canadian Technical Report of Fisheries and Aquatic Sciences

2498. [Online] http://www.glf.dfo-mpo.gc.ca/sci-sci/cwhma-at-

camn/8th_cwhma_proceedings.pdf [accessed February 2006].

Aune, T., Sorby, R., Yasumoto, T., Ramstad, H., Landsverk, T.,

2002. Comparison of oral and intraperitoneal toxicity of

yessotoxins towards mice. Toxicon 40, 77–82.

Avaria, S., Munoz, P., Uribe, E., 1982. Composicion y Biomasa

del Fitoplancton Marino del Norte de Chile en diciembre de

1980 (Operacion Oceanografica Marchile XI—ERFEN II).

Cienc. y Tec. del Mar CONA 6, 5–36.

Avaria, S., Munoz, P., 1983. Composicion y Biomasa del

Fitoplancton Marino del Norte de Chile en mayo de 1981

(Operacion Oceanografica Marchile XII—ERFEN III).

Cienc. y Tec. del Mar CONA 7, 109–140.

Avaria, S., Munoz, P., 1985. Effects of ‘‘El Nino’’ Phenomenon

on the Marine Phytoplankton off Northern Chile in

December 1982. (Marchile XIV—ERFEN V Cruise). Cienc.

y Tec. del Mar CONA 9, 3–30.

Blanco, J., Morono, A., Fernandez, M.L., 2005. Toxic episodes

in shellfish, produced by lipophilic phycotoxins: an overview.

Rev. Gal. Rec.Mar. (Monog.) 1, 70 [Online] http://www.

recmar-galicia.org/abstract.asp?Idpro=3.

Burguess, V.A., 2003. Toxicology investigations with the

pectenotoxin-2 seco acids. Ph.D. Thesis, Griffith University,

Australia [Online] http://www4.gu.edu.au:8080/adt-root/pub-

lic/adt-QGU20030905.090222/.

Burgess, V., Shaw, G., 2001. Pectenotoxins—an issue for public

health. A review of their comparative toxicology and

metabolism. Environ. Internat. 27 (4), 275–283.

ARTICLE IN PRESSJ. Blanco et al. / Toxicon 49 (2007) 710–716716

Fernandez, M.L., Mıguez, A., Martınez, A., Morono, A.,

Arevalo, F., Pazos, Y., Salgado, C., Correa, J., Blanco, J.,

Gonzalez-Gil, S., Reguera, B., 2003. First report of pecteno-

toxin-2 in phytoplankton net-hauls and mussels from the

Galician Rıas Baixas (NW Spain) during proliferations of

Dinophysis acuta and Dinophysis caudate. In: Villalba, A.,

Reguera, B., Romalde, J., Beiras, R. (Eds.), Molluscan

Shellfish Safety. Consellerıa de Pesca e Asuntos Marıtimos

da Xunta de Galicia and Intergovernmental Oceanographic

Commission of UNESCO, Santiago de Compostela,

pp. 75–83.

Fernandez, M.L., Reguera, B., Gonzalez-Gil, S., Mıguez, A.,

2006. Pectenotoxin 2 in single cells isolates of Dinophysis

caudata and Dinophysis acuta from the Galician Rias (NW

Spain). Toxicon 48, 477–490.

Fernandez Puente, P., Fidalgo Saez, M.J., Hamilton, B., Furey,

A., James, K.J., 2004. Studies of polyether toxins in the

marine phytoplankton, Dinophysis acuta, in Ireland using

multiple tandem mass spectrometry. Toxicon 44 (8), 919–926.

Goto, H., Igarashi, T., Watai, M., Yasumoto, T., Gomez, O.V.,

Valdivia, G.L., Noren, E., Gisselson, L.A., Graneli, E., 2000.

Worldwide occurrence of pectenotoxins and yessotoxins in

shellfish and phytoplankton. In: Hallegraeff, G., (Ed.),

Proceedings of the Ninth International Conference on

Harmful Algal Blooms (Conference Abstracts), 7–11 Febru-

ary, Hobart, Tasmania, Australia, 20p.

James, K.J., Bishop, A.G., Draisci, R., Palleschi, L., Marchia-

fava, C., Ferretti, E., Satake, M., Yasumoto, T., 1999. Liquid

chromatographic methods for the isolation and identification

of new pectenotoxin-2 analogues from marine phytoplankton

and shellfish. J. Chromatogr. A 844 (1+2), 53–65.

Lee, J.-S., Igarashi, T., Fraga, S., Dahl, E., Hovgaard, P.,

Yasumoto, T., 1989. Determination of diarrhetic shellfish

toxins in various dinoflagellate species. J. Appl. Phycol. 1,

147–152.

MacKenzie, L., Holland, P., McNabb, P., Beuzenberg, V.,

Selwood, A., Suzuki, T., 2002. Complex toxin profiles in

phytoplankton and greenshell mussels (Perna canaliculus),

revealed by LC-MS/MS analysis. Toxicon 40 (9), 1321–1330.

Mackenzie, L., Beuzenberg, V., Holland, P., Mcnabb, P., Suzuki,

T., Selwood, A., 2005. Pectenotoxin and okadaic acid-based

toxin profiles in Dinophysis acuta and Dinophysis acuminata

from New Zealand. Harmful Algae 4 (1), 75–85.

Miles, C.O., Wilkins, A.L., Munday, R., Dines, M.H., Hawkes,

A.D., Briggs, L.R., Sandvik, M., Jensen, D.J., Cooney, J.M.,

Holland, P.T., Quilliam, M.A., Mackenzie, A.L., Beuzenberg,

V., Towers, N.R., 2004a. Isolation of pectenotoxin-2 From

Dinophysis acuta and its conversion to pectenotoxin-2 seco

acid, and preliminary assessment of their acute toxicities.

Toxicon 43 (1), 1–9.

Miles, C.O., Wilkins, A.L., Samdal, I.A., Sandvik, M., Petersen,

D., Quilliam, M.A., Naustvoll, L.J., Rundberget, T., Torgersen,

T., Hovgaard, P., Jensen, D.J., Cooney, J.M., 2004b. A novel

pectenotoxin, PTX-12, in Dinophysis spp. and shellfish from

Norway. Chem. Res. Toxicol. 17 (11), 1423–1433.

Pavela-Vrancic, M., Mestrovic, V., Marasovic, I., Gillman, M.,

Furey, A., James, K.J., 2002. DSP toxin profile in the coastal

waters of the central Adriatic Sea. Toxicon 40, 1601–1607.

Quilliam, M.A., 2003. Chemical method for lipophilic shellfish

toxins. In: Hallegraeff, G.M., Anderson, D.M., Cembella,

A.D. (Eds.), Manual on Harmful Marine Microalgae.

UNESCO Publishing, Paris, pp. 211–245.

Suzuki, T., Mitsuya, T., Matsubara, H., Yamasaki, M., 1998.

Determination of pectenotoxin-2 after solid-phase extraction

from seawater and from the dinoflagellate Dinophysis fortii by

liquid chromatography with electrospray mass spectrometry

and ultraviolet detection. Evidence of oxidation of pecteno-

toxin-2 to pectenotoxin-6 in scallops. J. Chromatogr. A 815,

155–160.

Suzuki, T., Mackenzie, L., Stirling, D., Adamson, J., 2001a.

Conversion of pectenotoxin-2 to pectenotoxin-2 seco acid in

the New Zealand scallop, Pecten novaezelandiae. Fish. Sci. 67

(3), 506–510.

Suzuki, T., MacKenzie, L., Stirling, D., Adamson, J., 2001b.

Pectenotoxin-2 seco acid: a toxin converted from pectenotox-

in-2 by the New Zealand greenshell mussel, Perna canaliculus.

Toxicon 39, 1–8.

Suzuki, T., Beuzenberg, V., Mackenzie, L., Quilliam, M.A.,

2003. Liquid chromatography-mass spectrometry of spiroke-

tal stereoisomers of pectenotoxins and the analysis of

novel pectenotoxin isomers in the toxic dinoflagellate

Dinophysis acuta from New Zealand. J. Chromatogr. A 992

(1), 141–150.

Suzuki, T., Beuzenberg, V., Mackenzie, L., Quilliam, M.A., 2004.

Discovery of okadaic acid esters in the toxic dinoflagellate

Dinophysis acuta from New Zealand using liquid chromato-

graphy/tandem mass spectrometry. Rapid Commun. Mass

Spectrom. 18, 1131–1138.

Suzuki, T., Walter, J.A., Leblanc, P., Mackinnon, S., Miles, C.O.,

Wilkins, A.L., Munday, R., Beuzenberg, V., Mackenzie, A.L.,

Jensen, D.J., Cooney, J.M., Quilliam, M.A., 2006. Identifica-

tion of pectenotoxin-11 as 34s-hydroxypectenotoxin-2, a new

pectenotoxin analogue in the toxic dinoflagellate Dinophysis

acuta from New Zealand. Chem. Res. Toxicol. 19 (2),

310–318.

Vale, P., Sampayo, M.A.d.M., 2002a. Pectenotoxin-2 seco acid,

7-epi-pectenotoxin-2 seco acid and pectenotoxin-2 in shellfish

and plankton from Portugal. Toxicon 40, 989–996.

Vale, P., Sampayo, M.A.d.M., 2002b. First confirmation of

human diarrhoeic poisonings by okadaic acid esters after

ingestion of razor clams (Solen marginatus) and green crabs

(Carcinus maenas) in Aveiro Lagoon, Portugal and detection

of okadaic acid esters in phytoplankton. Toxicon 40 (7),

989–996.