E. A. Sar et. al. - DSP toxins in mollusks from Buenos Aires (Argentina)
Bol. Soc. Argent. Bot. 47 (1-2) 2012
ISSN 0373-580 X
Bol. Soc. Argent. Bot. 47 (1-2): 5-14. 2012
División Ficología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Argentina;
Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Argentina.
Departamento de Toxinas Marinas, Laboratorio Regional Mar del Plata, Centro Regional Buenos Aires Sur, SENASA.
Dirección de Pesca, Ministerio de Asuntos Agrarios de la Provincia de Buenos Aires. Argentina.
Laboratorio Bioquímica de Membrana, Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad de
* Author to whom correspondence should be addressed: firstname.lastname@example.org
EUGENIA A. SAR
*, INÉS SUNESEN
, ALEJANDRA B. GOYA
, ANDREA S. LAVIGNE
, CARLOS GARCÍA
y NÉSTOR LAGOS
Aires (Argentina) asociado con Dinophysis spp.: evidencia de Ácido Okadaico, Dinophysistoxina-1 y
sus acyl-derivados. En enero de 2010, los dinoflagelados productores de toxinas Dinophysis acuminata
y D. caudata (10
) fueron detectados en Mar Azul durante un monitoreo rutinario de fitoplancton
realizado en aguas costeras de la Provincia de Buenos Aires, Argentina. Mesodesma mactroides (almeja
amarilla) y Donax hanleyanus (berberecho) del intermareal de Mar Azul, que son parte de la dieta de los
habitantes del lugar y de turistas, dieron resultado positivo para toxinas lipofílicas mediante bioensayo
ratón. Este trabajo está focalizado en la detección de Toxinas Diarreicas de Moluscos (DSP) en muestras
colectadas durante el evento de toxicidad usando un HPLC-FLD con procedimiento de derivatización
precolumna. Los datos evidenciaron contaminación de los moluscos con toxinas DSP y un perfil
compuesto por Ácido Okadaico (OA), Dinophysistoxina-1 (DTX-1), Acyl-Dinophysistoxina-1 (Acyl-DTX-1)
y Acyl-Ácido Okadaico (Acyl-OA). Las toxinas DSP encontradas en este estudio producen síntomas de
diarrea consistentes con los experimentados por los pacientes que habían ingerido moluscos cocinados
en enero. Este es el primer reporte de Acyl-derivados en muestras de moluscos procedentes del Atlántico
Sudamericano y de OA en muestras de moluscos procedentes de Argentina.
Palabras clave: DSP, Ácido Okadaico, Dinophysistoxina-1, Acyl-derivados, Dinophysis spp.
Summary: In January 2010, the toxin-producing dinoflagellates Dinophysis acuminata and D. caudata
) were detected in Mar Azul during routine plankton monitoring in Buenos Aires Province
coastal waters, Argentina. Wild clams Mesodesma mactroides and Donax hanleyanus from Mar Azul
intertidal beach, which are part of the diet for local inhabitants and tourists, tested positive with the official
lipophilic mouse bioassay. This paper focuses on the detection of Diarrhetic Shellfish Poison (DSP) toxins
in these samples using a HPLC-FLD pre column derivatization procedure. The data showed that shellfish
were contaminated with complex DSP toxin profiles composed of Okadaic Acid (OA), Dinophysistoxin-1
(DTX-1), Acyl-Dinophysistoxin-1 (Acyl-DTX-1) and Acyl-Okadaic Acid (Acyl-OA). The DSP toxins found in
this study produce diarrhea symptoms consistent with those experienced by patients who had ingested
cooked shellfish in January. This is the first report of Acyl-derivatives in South American Atlantic shellfish
samples and of OA in Argentinean shellfish samples.
Key words: DSP, Okadaic Acid, Dinophysistoxin-1, Acyl-derivatives, Dinophysis spp.
Bol. Soc. Argent. Bot. 47 (1-2) 2012
The first documented episode of gastrointestinal
distress and diarrhea in humans consistent with
Diarrhetic Shellfish Poisoning occurred in Argentina
was reported from the Gulfs San José and Nuevo,
Patagonia Argentina in 2001 (Gayoso et al., 2002).
The Diarrhetic Shellfish Poison (DSP) toxin vectors
were mussels Aulacomya ater Molina and Mytilus
which was present in the plankton, epiphyte on
macroalgae and in the stomach contents of the
bivalves, was Prorocentrum lima (Ehrenberg)
Dodge. The only DSP toxin described in that report
was Dinophysistoxin-1 (DTX-1).
Recently, Sar et al. (2010) reported another
episode of human gastrointestinal illness associated
with consumption of cooked wedge clams Donax
hanleyanus Philippi collected in Villa Gesell
(Buenos Aires Province) that gave positives for
DSP by mouse bioassay. In that event, the DSP toxin
vectors were two bivalve species: wedge clams and
mussels Brachydontes rodriguezii d’Orbigny. The
analyzed phytoplankton community consistently
contained the toxigenic species Dinophysis
acuminata Claparède et Lachmann and D. caudata
as Okadaic Acid (OA) producer based on bloom
samples collected at Le Havre, France and Tokyo
Bay, Japan by using High Performance Liquid
Chromatography with fluorescence detection
(HPLC-FLD) (Lee et al., 1989). In 2001 the toxin
content of D. acuminata from the Galician Rías
Bajas, showed OA as the major toxin and a very
small peak with retention time corresponding to
Dinophysistoxin-2 (DTX-2) (Fernández et al.,
2001), then in 2005 D. acuminata collected in New
Zealand showed a DSP toxin profile including OA,
DTX-1, Pectenotoxin 2 (PTX-2) and Pectenotoxin
11 (PTX-11) (MacKenzie et al., 2005). More
recently, Hackett et al. (2009) established unialgal
cultures of D. acuminata from Woods Hole,
USA, maintained this culture by using a two step
feeding system described in (Park et al., 2006).
Through chemical analysis of the cell culture
extracts by Liquid Chromatography tandem Mass
Spectrometry (LC-MS/MS) and Ultra Performance
Liquid Chromatography (UPLC), they reported the
detection of OA, diol ester of OA (OA D8), DTX-
1, PTX-2 and hydroxylated PTX-2.
producer, based on picked cells from Johor Strait,
Singapore (Holmes et al., 1999) and similarly
based on picked cells from the Galician Rías of
Vigo and Pontevedra and analyzed by HPLC-
FLD and LC-MS (Fernández et al., 2003). Picked
cells from Sapian Bay, Panay Islands, Philippines,
analyzed by HPLC-FLD determined content of OA
and DTX-1 (Marasigan et al., 2001) and later cell
extracts from Galician Rías Bajas analyzed by LC-
MS/MS have shown that the species may have high
levels of PTX-2 (Fernández et al., 2006).
To date DSP toxins in Chile are restricted to
the shellfish of the southern regions (García et
as the most probable source of DTX-1 detected
in Mytilus chilensis Hupe collected in a Southern
Chilean Magellanic fjord (Uribe et al., 2001)
and Dinophysis acuta Ehrenberg was the species
commonly associated with DSP outbreaks in
Northern Chilean Patagonian fjords (Lembeye et
al., 1993; Zhao et al., 1993). More recently, there
are two reports that described strains of Dinophysis
spp. from Chile that produced only pectenotoxins,
one corresponding to D. acuminata collected in the
North of Chile (Blanco et al., 2007) and the other
to Dinophysis sp. (Fux et al., 2011).
There is little information about the DSP toxins
in the Atlantic coastal waters of South America.
detection of the diarrhetic toxin OA in mussels
from Santa Catarina, Brazil, during the winter
of 1995 by HPLC-FLD (Proença et al., 1999)
and Dinophysis acuminata and D. caudata were
associated with the detection of diarrhetic toxins by
mouse bioassay in clams, wedge clams and mussels
during the summers-falls of 1992, 1994 and 1996
from several localities of Uruguay (Méndez &
Ferrari, 2002). Extracts of D. acuminata and D.
waters, analyzed by HPLC-FLD showed OA as the
only DSP toxin (Méndez & Ferrari, 2002).
Based on previous results (Sar et al., 2010), we
carried out the present study with the purposes of
analyzing the phytoplankton composition from
Mar Azul between January and April of 2010,
checking lipophilic shellfish toxins in bivalve
mollusks by mouse bioassay and determining the
diarrhetic shellfish toxins profiles by HPLC-FLD.
Bol. Soc. Argent. Bot. 47 (1-2) 2012
The phytoplankton samples analyzed in this
paper were collected at the Buenos Aires Province
coastal waters between early January and middle
April of 2010 from Mar Azul (37º 21’ 25’’ S-57º
01’ 49’’ W) at about 10 to 20 m from the shoreline,
from the surface layer of the water column (0 and 5
meters depth). Qualitative samples were collected
with 30 µm net hauls and immediately fixed with
4% formalin; quantitative samples were collected
with Van Dorn bottle and fixed with 0.4% formalin.
Microscopic observations were made with a
light microscope (LM), Nikon Microphot FX,
using phase contrast and with a scanning electron
microscope (SEM) Jeol JSM 6360 LV. The
microphotographs were taken with the Jeol JSM
6360 LV microscope. The quantitative analyses
of phytoplankton were carried out using the
Sedgewick-Rafter chamber and the cell counts
were made in triplicate. Data were processed with
Statistical Software for Windows 7.0. The results
were expressed as mean ± standard deviation.
Qualitative and quantitative samples and
permanent slides correlatively labeled were
incorporated into the Herbarium, deposited at the
División Ficología (LPC index Herbariorum),
Facultad de Ciencias Naturales y Museo,
Universidad Nacional de La Plata under the
numbers LPC 11182, 01/04/2010; LPC 11186,
01/28/2010; LPC 11286, 02/12/2010; LPC11190,
02/26/2010; LPC11194, 03/09/2010; LPC 11198,
03/26/2010 and LPC 11202, 04/12/2010.
Shellfish samples were collected at low tide
in the same time and area as the phytoplankton
samples, along the beach from the intertidal fringe,
by digging in the sand. Collected species were
wedge clam (Donax hanleyanus Philippi) and/
or yellow clam (Mesodesma mactroides Reeves),
depending on which population was accessible.
In each sampling, two subsamples of shellfish
around 500 g were obtained and frozen at -18 ºC
for checking DSP toxicity by mouse bioassay in
the Departamento de Toxinas Marinas, Regional
Mar del Plata of the SENASA and by HPLC-
FLD analysis in the Laboratorio Bioquímica de
Membrana, Facultad de Medicina, Universidad de
Chile. The sample corresponding to early January
A sample of cooked wedge clam from a locality
neighboring Mar Azul (Villa Gesell, 37º 16’
48’’S-56º 58’ 57’’W), ingested by patients who
experienced diarrhea, nausea and abdominal
cramps, all symptoms consistent with DSP
intoxication, was also checked by mouse bioassay
and by analytical methods.
The procedure employed for sample preparation
prior to mouse bioassay was described in Yasumoto
et al. (1984) and modified in Fernández et al.
(2002). The Japanese mouse assay for DSP toxins
with modifications is the official method accepted
by the Argentinean Officials. Extracts for bioassay
were prepared from whole soft tissues of shellfish
and mice were intraperitoneal injected with 1 mL
Tween 60 1% equivalent to 25 g shellfish tissues.
Experimental animals were albino mice strain CF1
of 20 ± 1 g weight. According to the Decision
2002/225/CEE appeared in the Official Journal
of the European Communities and followed
by the Servicio Nacional de Sanidad y Calidad
Agroalimentaria from Argentina, this method uses
mouse survival time for determination of DSP
toxicity and two of three mouse deaths in less than
24 hours as criterion for a positive test.
High Performance Liquid Chromatography with
Fluorescence detection (HPLC-FLD)
A portion of 1 g of homogenate prepared from
ten individuals of each shellfish sample was
extracted twice with 4 mL of methanol-water (8:2
v/v) following Lee et al. (1987). The methanolic
phase was centrifugated, 2.5 mL of the supernatant
diluted with water to a final 26.6% methanol and
cleaned up by liquid/liquid partitioning according
to García et al. (2003, 2010). This eluate was
evaporated to dryness under reduced pressure in
a Speed Vac Plus (Savant, SC 210A). The dried
extraction was resuspended in 100 microliters of
The alkaline hydrolysis of Acyl-DSP toxins was
performed as described in García et al. (2004a).
Briefly, aliquots of 2.5 mL of 80% methanol extract
of each shellfish sample were treated with 2.5 mL
of 0.5 N NaOH in 90% methanol solution. The
methanol was evaporated. The aqueous layer was
acidified with 2.5 mL of 0.5 N HCl, extracted twice
with 5 mL diethyl ether, evaporated to dryness
and dissolved with 2.5 mL of 80% methanol.
The methanolic solution was treated with 1 mL
of 0.2% acetic acid, extracted twice with 4 mL of
dichloromethane and evaporated to dryness under
Clean and dry extracts and standards were
derivatized with 9-anthryl-diazomethane (ADAM,
Molecular Probe, USA) freshly prepared solution
of 0.1% ADAM in 100 µl of acetone and 400 µl
of methanol. The mixture was kept to 25º for 60
minutes and then evaporated to dryness. Residues
were diluted in 200 µl of dichloromethane/hexano
The derivatized samples were cleaned up on a
solid-phase extraction device, silica column Sep-
Pak cartridge (Water CO.) (García et al., 2003)
and analyzed by HPLC using isocratic conditions
with mobile phase of acetonitrile/methanol/water
(8:1:1 v/v), and column with reversed phase,
Supelcosil LC (C18, 4 × 250 mm, 5 µm). The
chromatographic separation was performed on a
Liquid Chromatograph System equipped with a
pump (Shimadzu LC-6A), a Rheodyne injector
(7725i Rheodyne) and in-line fluorescence detector
(Jasco FP-2020 Plus). The fluorescence detector
was set for 365 nm excitation and 415 nm emissions.
Peaks in the resulting chromatograms were
identified by comparison with the retention times
of toxins analytical standards. Certified reference
materials containing Dinophysistoxin-1 (CRM-
DSP-Mus-b) and Okadaic acid (NCR-CRM-OA-c)
were obtained from National Research Council
Canada. The detection limits for the DSP toxins as
ADAM esters were 0.02 ng of DSP toxins detected
in the chromatogram meaning a signal that it was
the double amount of the base signal noise in the
recorded equipment (García et al., 2010).
Temporal distribution and density of the species
of Dinophysis in Mar Azul
The concentration of Dinophysis cells was
quite low in Buenos Aires coastal waters in the
years 2008 and 2009 and the occurrence from one
year to the next varied considerably (unpublished
data). Nevertheless, in early January 2010, there
occurred an unusual proliferation of Dinophysis
acuminata (Figure 1A) that reached a maximum
number of cells in Mar Azul, 26,000 cells·l
with a 2.41% relative contribution to the total
concentrations of Dinophysis acuminata declined
to 9,000 cells·l
, Dinophysis caudata (Figure 1B)
, and the phytoplankton
and Rhizosolenia imbricata Brightwell, as well
as additional nanoplanktonic centric diatoms and
phytoflagellates of indeterminate identity (Table 1).
From February to early March the density
of Dinophysis acuminata tended to diminish,
varying between 8,675 and 333 cells·l
into the total phytoplankton assemblage also
decreased (0.43 to 0.01% respectively) (Table 1).
The opposite situation was found in reference to
the cell concentration of the total phytoplankton
population that increased between 2.0·10
and Dinophysis caudata was
(Table 1). In later March the abundance of
and D. caudata appeared at concentration
Bol. Soc. Argent. Bot. 47 (1-2) 2012
Table 1. Density of the total phytoplankton assemblage and relative density of the potentially toxigenic
species of dinoflagellates and of the more abundant species, from Mar Azul, in the samplings from
January to April.
01/04/10 01/28/10 02/12/10 02/26/10 03/09/10 03/26/10 04/12/10
Undeterminated centric diatoms
Undeterminated pennate diatoms
of 333 cells·l
(Figure 2). The relative contributions
increased (0.55% and 0.12% respectively), the
density of the total phytoplankton was the lowest
of all the sampled periods, 2.4·10
indeterminate phytoflagellates and nanoplanktonic
centric diatoms. In April both toxigenic species
disappeared or only scattered cells persisted in
Shellfish extracts toxicity tested by mouse bioassay
From all the shellfish samples tested for DSP
toxicity using the mouse bioassay, only one,
dated in 12 April 2010, tested negative with one
of three mice died. In general, mice presented
prostration and weakness as common symptoms
in most of the samples. Details about survival
times post inoculation and death rate in relation
with cells concentrations of Dinophysis spp. in the
phytoplankton are shown in Table 2.
All shellfish sample extracts including the
one that tested negative by mouse bioassay, were
analyzed by HPLC-FLD as described above. The
search of each DSP toxin was performed on all
sample extracts derivatized with ADAM.
The chromatographic runs performed by HPLC-
FLD analysis of the sample yellow clam (03/26/10)
as examples of fluorescent chromatograms
performed in this study, are shown in Figure 3. In
the top chromatogram (labeled Methanol Sample)
the peaks displayed correspond to OA and DTX-1
respectively. The second chromatogram (labeled
Hydrolyzed Sample), that corresponds to an extract
hydrolyzed with NaOH, shows the same peaks (OA
and DTX-1) but an increase in signal was observed
due to the hydrolysis of the Acyl-derivatives.
The third chromatogram (labeled STD) shows
the analytical standards of OA and DTX-1. In the
bottom of Figure 3, the last chromatogram (labeled
DOCA) shows the run internal standard which
corresponded to Deoxicholic acid derivatized
with ADAM. The alkaline hydrolysis revealed the
chemical transformation of Acyl-DTX-1 into DTX-
1 and the Acyl-OA into OA. In fact, this is the only
way to detect the Acyl derivatives (Acyl-DTX-1
and Acyl-OA) by HPLC-FLD. This procedure was
repeated with every sample extract and the results
are shown in Table 3.
The sample that tested negative by mouse
bioassay showed no peaks corresponding to OA or
DTX-1 for the extract not hydrolyzed with NaOH
and showed small peaks corresponding to OA and
DTX-1 for the hydrolyzed extract.
During routine phytoplankton monitoring carried
out in Buenos Aires Province coastal waters, some
shellfish samples tested positive with the official
DSP mouse bioassay when D. acuminata and D.
caudata were detected in parallel water samples
(Sar et al., 2010). As an extension of this work the
present study allowed us to determine in shellfish
sample extracts that the DSP toxin profiles are
composed of OA, DTX-1, Acyl-OA and Acyl-
DTX-1. This is the first report of Acyl-derivatives
compounds in the South American Atlantic Ocean
and of OA in Argentinean waters. Acyl derivatives
were previously reported in shellfish extracts from
the Southern Chile by García et al. (2004b; 2006;
2010). The metabolic transformation of OA and
DTX-1 in their respective Acyl-derivatives is a
biochemical way of self protection from these toxic
compounds that are chemically lipophilic and very
hard to eliminate as has been recently reviewed by
Rossignoli et al. (2011).
period 01/25-28/10 – 04/12/10 from Atlantic coast of Buenos Aires Province, Argentina. Yellow clam:
01/28/10 Wedge clam Mar Azul
Between 1h and 1h 15min.
01/25/10 to 01/28/10
Cooked wedge clam
Between 1h and 1h 5min.
02/12/10 Wedge clam Mar Azul
Between 1h 55min
and 2h 10min.
a and b
a) 3:3 (100%)
b) 3:3 (100%)
a) Between 4 h and 12 h
b) Between 8h and 18h
03/09/10 Wedge clam
Between 5h 40
min and 21 h
Between 10 and 20 h
Less than 24 h
derivatized with ADAM. OA, Okadaic acid; DTX-1, Dinophysistoxin-1; DOCA, Deoxicholic acid; STD,
Table 3. DSP toxins presence in bivalve samples. Ten individuals of each shellfish sample (whole flesh)
were used to obtain homogenate. Yellow clam:
Nd: no detectable.
Bol. Soc. Argent. Bot. 47 (1-2) 2012
We express our gratitude to A. Franetovich, to J.
D. Novero, of the Ministerio de Asuntos Agrarios of
the Buenos Aires Province, and to G. Meléndez, of
the SENASA, for technical and financial assistance,
and to Christian Maurs for his collaboration in
mollusk sampling. This study was supported by
the Consejo Federal Pesquero from Argentina
Grant CFP 15/09, the CONICET Grant PIP 0067
and FONDECYT Chile Grant 1070706 and Grant
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Recibido el 13 de noviembre de 2011, aceptado el 1 de
febrero de 2012.