www.elsevier.com/locate/hal

Harmful Algae 7 (2008) 228–234

Okadaic acid accumulation in macrofilter feeders subjected

to natural blooms of Dinophysis acuminata

Sofia Reizopoulou a,*, Evangelia Strogyloudi a, Antonia Giannakourou a,Kalliopi Pagou a, Ioannis Hatzianestis a, Christina Pyrgaki a, Edna Graneli b

a Hellenic Centre for Marine Research, Institute of Oceanography, P.O. Box 712, 19013 Anavyssos, Greeceb Marine Sciences Department, University of Kalmar, S-39182 Kalmar, Sweden

Received 1 February 2007; received in revised form 7 August 2007; accepted 16 August 2007

Abstract

Thermaikos Gulf is a eutrophic area located in the Northwestern part of the Aegean Sea in the Eastern Mediterranean.

Interspecific differences among various filter feeders in their ability to accumulate okadaic acid, were observed during natural

blooms of Dinophysis acuminata in the gulf. Okadaic acid analyses by high performance liquid chromatography (HPLC) were

performed on benthic specimens and D. acuminata cell densities and cell toxin content were estimated in water samples. Seven filter

feeding species were collected in the gulf during two DSP outbreaks in May 2003 and March 2004. The various species showed a

different potential to accumulate okadaic acid in their tissues. The highest concentrations were found in the mussel populations

(Mytilus galloprovincialis and Modiolus barbatus), while among the non-bivalve filter feeders, ascidians were the main

accumulators of okadaic acid. The rest of shellfish populations (Flexopecten proteus, Chlamys varia and Venus verrucosa) were

found to contain toxins only during 2004, when D. acuminata densities were found above 10000 cells l�1. M. galloprovincialis was

proved to be the most appropriate indicator for a safe warning of okadaic acid contamination in Thermaikos Gulf.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Okadaic acid; DSP toxins; Accumulation; Benthos; Dinophysis acuminata

1. Introduction

Harmful algal blooms cause severe economical

losses to aquaculture and adversely impact human

health. Diarrhetic Shellfish Poison (DSP) in humans

causes mainly gastrointestinal symptoms as a result of

eating shellfish contaminated with okadaic acid (OA)

group of toxins. OA and its derivatives are powerful

cytotoxins that can block dephosphorylation of proteins

in mammals and plants, but very little is known

regarding the effect of these compounds on marine

organisms (Bauder et al., 2001). The most important

* Corresponding author.

E-mail address: sreiz@ath.hcmr.gr (S. Reizopoulou).

1568-9883/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.hal.2007.08.001

DSP toxin producers belong to the genus Dinophysis,

which produce toxins consisting mostly of OA and its

derivatives.

Thermaikos Gulf (N. Aegean Sea, Eastern Mediter-

ranean) is enclosed and eutrophic, with reduced water

flux, receiving high nutrient inputs from rivers, urban and

industrial runoff (Gotsis-Skretas and Friligos, 1990;

Balopoulos and Friligos, 1993; Moncheva et al., 2001).

The shellfish farming activity in this area represents

about 85% of the total production in Greece (s10 million

annually; Zanou and Anagnostou, 2001), with 70–80% of

the product exported (Karageorgis et al., 2005). Mussel

rafts increase water stability and water residence time,

and create microhabitats favouring dinoflagellate growth

(Graneli et al., 1998). Many sources of nutrients such as

sewage and animal wastes, agricultural and fertilizer

S. Reizopoulou et al. / Harmful Algae 7 (2008) 228–234 229

runoff, as well as the growing aquaculture industry in

coastal areas, can also contribute to stimulate harmful

algal blooms (Anderson et al., 2002).

In Thermaikos Gulf since 2000, DSP outbreaks have

been confirmed and the causative organism has been

identified as Dinophysis acuminata. The most serious

HAB event was recorded during the winter of 2000,

when D. acuminata reached 8.5 � 104 cells l�1 at the

end of January (Koukaras and Nikolaidis, 2004). During

each bloom period (usually early spring), mussel

cultures remain closed to harvest, resulting in a

substantial socio-economic impact in the area (eco-

nomic losses of �s3 million annually) (Karageorgis

et al., 2005).

As filter feeder animals gather food by filtering the

water, contamination with DSP toxins occurs after their

feeding on toxic Dinophysis cells. The filtering capacity

of the filter feeding communities may play an important

role on the control of phytoplankton blooms (Graneli

et al., 1998; Cloern, 2001). The accumulation of OA,

which D. acuminata mainly produces (Vale et al.,

1998), has been until now studied in shellfish

populations, while for other benthic groups (ascidians

and polychaetes), to our knowledge there is no available

information in the published literature.

Two successive annual DSP toxin outbreaks in

Thermaikos Gulf presented an opportunity to (1) assess,

determine and compare the amounts of free okadaic

acid in different filter feeding species naturally

contaminated by D. acuminata; and (2) identify the

most appropriate species to be used as an indicator of

Fig. 1. Sampling stations

DSP contamination in the Gulf, as a tool in the shellfish

stocks management in DSP-affected areas, such as

Thermaikos Gulf.

2. Material and methods

2.1. Description of the study area

The investigation area, Thermaikos Gulf, is located

in the nortwestern part of the Aegean Sea (Fig. 1). Five

major and several minor rivers flow into the gulf,

introducing significant amounts of particulate matter

and nutrient loads (Lykousis and Chronis, 1989;

Friligos et al., 1997; Karageorgis et al., 2005). The

city of Thessaloniki, with its partly untreated domestic

sewage effluents and adjacent industrial zone, con-

tributes to the eutrophication of the Gulf (Anagnostou

et al., 1997). The bottom relief in the area is smooth and

covered by muddy sediments (Lykousis and Chronis,

1989).

2.2. Sampling methods

Field surveys were performed during two successive

D. acuminata blooms in the Thermaikos Gulf. The first

bloom occurred in May 2003 (D. acuminata >2000 cells l�1) and the second and more prominent

bloom occurred in March 2004 (D. acuminata >10,000 cells l�1).

Phytoplankton samples and benthic macroinverte-

brates were collected from four stations, outside (TP7,

in Thermaikos Gulf.

S. Reizopoulou et al. / Harmful Algae 7 (2008) 228–234230

TP2 and TP6, �5.5, 19 and 8.5 m depth, respectively)

and inside (DA3, 8.5 m depth) the mussel culture area

(Fig. 1). Station DA3 is located between mussel rafts in

the area of Chalastra, near the mouth of Axios River.

Seawater samples, for enumeration of Dinophysis cells

in the water column, were taken from different depths

(2 m, 5 m and also from 10 m in TP2), using Niskin

bottles with teflon-coated springs and O-rings. Samples

were fixed with Lugol solution. Species identification

and cell counts were done with an inverted microscope

(Olympus CK2) after 24 h of sedimentation in 25 ml

settling chambers (Utermohl, 1958). Dinophysis cells

were also collected on GFC glass-fibre filters after

filtering 1 l of seawater. Filters were stored at �18 8Cuntil extraction for toxin analysis.

During the two bloom periods, cultured and natural

filter feeding populations were collected. Cultured

mussels (M. galloprovincialis) from the mussel rafts

(Fig. 1, station DA3) were collected from two different

depths (2 and 5 m). Benthic populations were sampled

from stations TP7, TP2 and TP6 (Fig. 1) by SCUBA

diving and using a rectangular dredge appropriate to

catch large animals. The most abundant filter feeders

collected were bivalves, as the mussel Modiolus

barbatus, scallops (Flexopecten proteus and Chlamys

varia) and clams (Venus verrucosa). The sabellid

polychaete Sabella spallanzanii was mainly found at

station TP7 (Fig. 1).

During the second sampling survey in 2004, solitary

ascidians associated with the mussel cultures and

Mytilus galloprovincialis from the sediments below the

cultures also were collected. M. galloprovincialis is

usually found under the mussel rafts (station DA3), as

animals detach from the rafts and develop benthic

populations.

Pooled samples of 3–5 individuals of each species

with similar body size were prepared, weighed and

stored at �20 8C for whole tissue toxin analyses.

2.3. Okadaic acid determination

Okadaic acid analysis was performed with high

performance liquid chromatography (HPLC)-fluores-

cence detection after pre-column derivatization with

3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one

(BrMB) (Zhou et al., 1999). Fresh homogenized animal

tissue (1.0 g) or a filter containing the phytoplankton

cells was extracted with 4 ml of aqueous 80% methanol.

After adding 60 ml of water the methanolic extract was

further extracted three times with 10 ml of dichlor-

omethane. The extracts were combined, evaporated to

dryness and the residue was redissolved in 0.5 ml of

dichloromethane and cleaned up by solid phase

extraction (SPE). A SPE cartridge (Sep-Pak) containing

1 g of silica, washed with 8.0 ml acetone/water (97:3)

and 10 ml dichloromethane and the extract was then

added. The cartridge was purged with 10 ml of

dichloromethane and 5 ml methanol/acetone (3:97)

and the toxins were eluted with 10 ml of methanol/

acetone (30:70). The eluate was transferred to a glass

vial and derivatized with BrMB (Fluka). The eluate was

dried under nitrogen gas, 50 ml of N-ethyl-diisopropy-

lamine solution (0.1% in acetone), and 50 ml of BrMB

solution (0.1% in acetone) were added, and the mixture

was heated at 70 8C for 20 min. The derivatized mixture

was immediately evaporated to dryness under nitrogen

gas and the residue was redissolved in 100 ml of

acetonitrile for HPLC analysis.

The OA determination was performed with a

Shimadzu HPL chromatograph equipped with a LC-

10AD ternary pump and a RF-10Axl fluorescence

detector. The LC column was a Spherisorb ODS2,

25 cm � 4.6 mm, 5 mm and maintained at 28 8C.

Isocratic conditions were used; the mobile phase was

acetonitrile/water 65:35, and the flow rate was

1.0 ml min�1, and the injection volume was 20 ml.

The fluorescence detector was set to 345 nm excitation

and 440 nm emission. OA concentrations were calcu-

lated using a calibration curve that was based on

injections of standard OA (Calbiochem) solutions

derivatized under the same conditions. The detection

limit (S/N 3:1) for the standard OA solutions was

0.05 ng/injection, corresponding to 0.25 ng filter�1 for

the phytoplankton samples, whereas for tissue samples

the detection limit ranged from 0.8 to 5 ng/g depending

on the substrate. The protocol was tested by analyzing

certified reference material MUS-2, from the Institute

for Marine Biosciences in Halifax, Canada.

3. Results

Fig. 2 illustrates the spatial and vertical distribution

of D. acuminata cell densities and their cellular toxin

content during the blooms of May 2003 and March

2004.

In May 2003, D. acuminata cell densities varied

from 500 cells l�1 in stations DA3 and TP7 to

2200 cells l�1 at station TP2 and the average OA

intracelullar concentrations in D. acuminata varied

from 3.7 to 8.6 pg cell�1 (Fig. 2).

The highest D. acuminata cell densities were

recorded during March 2004. Cell densities ranged

from 600 cells l�1 at station DA3 (mussel culture area)

to 10,700 cells l�1 at station TP2. OA in D. acuminata

S. Reizopoulou et al. / Harmful Algae 7 (2008) 228–234 231

Fig. 2. Cell densities of Dinophysis acuminata and mean (+S.D.)

cellular toxin content during the sampling efforts in 2003 and 2004.

cells was also higher in 2004 compared to 2003, ranging

from 4.4 to 14.0 pg cell�1 (Fig. 2).

Filter feeding species varied in OA accumulation

during each bloom (Table 1).

In 2003, only mussels (Mytilus and Modiolus) were

toxic. The highest concentration of OA was recorded in

M. galloprovincialis and almost 80% of the samples

under examination surpassed the regulatory limit of

Table 1

Minima, maxima, means and standard deviations of OA concentration (ng

blooming period

Species Minima OA

(ng/g ww)

May 2003

Mytilus galloprovincialis

(mussel rafts) (n = 14)

74.9

Modiolus barbatus (n = 30) 11.1

Venus verrucosa (n = 17) nd

Flexopecten proteus (n = 20) nd

Chlamys varia (n = 7) nd

Sabella spallanzanii (n = 5) nd

March 2004

Mytilus galloprovincialis

Mussel rafts (n = 17) 80.8

Sediments (n = 8) 2335.0

Modiolus barbatus (n = 30) 7.7

Venus verrucosa (n = 20) 2.4

Flexopecten proteus (n = 10) 14.9

Chlamys varia (n = 7) 31.4

Sabella spallanzanii (n = 5) 0.9

Ascidiacea (n = 8) 31.7

n: Number of pooled samples (3–5 individuals). nd: Not detected. Detection

verrucosa, F. proteus, C. varia and Ascidiacea and 0.8 ng/g for S. spallanz

200 ng OA g�1 tissue. Within Modiolus populations,

highly toxic and completely toxin-free samples were

found and approximately 30% of the samples sur-

passed the safety threshold. In scallops, clams and

polychaetes, OA was below the analytical detection

limit (Table 1).

The interspecific differences among the various filter

feeder populations in OA accumulation were observed

mainly during the 2004 bloom, when toxin concentra-

tions in animal tissues were higher compared to 2003.

All of the animal tissues examined were toxic except for

clam populations, in which 50% of the samples were

contaminated with OA (Table 1).

Cultured mussels (M. galloprovincialis) consistently

contained the highest OA concentrations, significantly

higher than the regulatory limit, suggesting a high

ingestion rate of Dinophysis cells by this species.

Ascidians collected from the mussel rafts had also high

OA concentrations.

Within harvest populations M. barbatus presented

the highest toxin accumulation levels, again exceeding

the regulatory limit. It is noteworthy that OA con-

centrations within different benthic species exposed to

the same bloom at the same location, indicated that M.

barbatus was the main accumulator of OA. The rest of

the investigated species contained much lower amounts

of OA (Table 1). For example, OA concentrations in

/g ww tissue) detected in tissues of macrofilter feeders during each

Maxima OA

(ng/g ww)

OA average � S.D.

(ng/g ww)

684.1 376.4 � 219.0

351.0 142.1 � 119.8

nd –

nd –

nd –

nd –

2123.4 1201.2 � 742.9

3222.2 2826.9 � 382.5

647.8 198.5 � 141.9

37.9 8.1 � 9.2

148.9 69.3 � 54.2

80.4 55.9 � 23.5

37.0 13.7 � 13.7

340.4 147.5 � 104.8

limits: 5 ng/g for M. galloprovincialis and M. barbatus, 2 ng/g for V.

anii.

S. Reizopoulou et al. / Harmful Algae 7 (2008) 228–234232

scallops consistently were lower than the safety limit for

human consumption.

In each bloom period a high spatial (vertical and

horizontal) intraspecific variation of OA accumulation

was observed, both for natural and cultured populations.

Variations in toxin concentration levels were found in

M. galloprovincialis collected from different depths,

although not statistically significant. Also within the

natural populations there was a spatial variation in toxin

accumulation among the different sampling locations.

However no statistically significant correlation between

animal toxin accumulation levels and Dinophysis cell

abundances was found.

It is important to note that OA levels in suspended

mussels were significantly lower than the levels in

mussels collected from the sediments below the rafts

(t-test, p = 0.0003). OA concentrations were maximal in

the bottom mussel populations exceeding the public

health safety threshold.

4. Discussion

The intense toxic bloom of March 2004 in

Thermaikos Gulf was associated with an increased

OA accumulation in animal tissues and also a higher

number of filter feeding species found to be con-

taminated, in comparison to the toxic bloom of 2003.

During both toxic blooms, D. acuminata cell densities

in the mussel culture area (st. DA3) were generally

comparable, indicating that mussels are effective filter

feeders and their dense populations, are able to reduce/

or control Dinophysis cell densities. The OA concen-

tration in D. acuminata cells found in the present study

was comparable with those of other studies (Lee et al.,

1989; Maneiro et al., 2000; Morono et al., 2003).

Within the filter feeders functional group there was a

marked interspecific variation in the OA accumulation,

indicating that not all species concentrated OA to the

same extent. M. galloprovincialis had the highest OA

accumulation compared to the other filter feeding

species. Sidari et al. (1998) suggested that the mussel is

preferentially selecting dinoflagellates rather than

diatoms and described a selective preference of this

species for the genus Dinophysis. Also the ascidians

associated with cultured mussels contained high OA

concentrations. Knowledge of phytoplankton species

that filter feeders can preferentially remove from the

water column is important for a better understanding of

the degree to which these species can control the toxic

blooms.

Interspecific differences in toxin accumulation can

also be attributed to species-specific metabolism of OA.

Suzuki and Mitsuya (2001) found that mussels have a

higher potential to accumulate OA homologues than

scallops. Differences in animal toxin accumulation may

arise from selective retention or elimination of

individual toxins or from other transformation pro-

cesses (Brijeli and Shumway, 1998). Taleb et al. (2001)

reported that clams accumulate PSP toxins to a lesser

degree than other species; however, members of the

family Veneridae can become very toxic by accumulat-

ing other toxins such as domoic acid (Vale and

Sampayo, 2001). In the present investigation only the

form of free OA was quantified, however, further

research is needed to investigate the presence of

conjugated forms of OA within the various species.

Variations in OA accumulation could also reflect the

different exposure of each species to toxin amounts. The

benthic populations collected outside the mussel culture

area were exposed to particulate material such as

sedimenting toxic algae and algae derived detritus,

depending mainly on the water mass circulation and

particle sinking rates. Among these populations, M.

barbatus was the most contaminated species while the

other species accumulated much less OA, and only

during the toxic bloom of 2004. Scallops (F. proteus and

C. varia) were less contaminated with OA, well below

the regulatory limit and similarly the clam V. verrucosa

presented very low OA levels. Clams can minimize their

exposure to toxins by reducing their feeding activity and

rejecting cells as pseudofaeces (Wikfors, 2005).

Another efficient filter feeding species, such as the

polychaete S. spallanzanii, had only traces of OA.

OA concentrations were maximal in Mytilus

collected from the sediments (>2 mg g�1) under the

mussel rafts, suggesting that these populations were

subjected to a major source of toxins. In addition to

settling phytoplankton and decaying cells, the benthic

mussels were also exposed to high amounts of toxins in

faecal material from the overlying suspended cultured

mussels. Faeces and pseudofaeces may contain high

amounts of toxins, contributing to the transfer of toxins

from the water phase to the sediment (Svensen et al.,

2005). Moreover, high ingestion rates in suspended

mussels would increase digestive activity with faster

elimination of toxins through faecal deposition (Blanco

et al., 1999).

The variation of toxin accumulation could therefore

be attributed to different selection efficiencies among

the various filter feeding species (Shumway and Cucci,

1987), to different species-specific OA metabolism

(Brijeli and Shumway, 1998), but also to the degree of

exposure of each species to different amounts of toxins

within the various habitats.

S. Reizopoulou et al. / Harmful Algae 7 (2008) 228–234 233

Intraspecific spatial variability of OA accumulation

in macrofilter feeding populations reflected the

horizontal and vertical patchiness of the toxic blooms.

The abundance of toxic cells and toxin concentrations in

animal tissues are sometimes not significantly related

(Lassus et al., 1991; Dahl and Johannessen, 2001), as

organisms can be found contaminated without the toxic

algae being detected (MacKenzie et al., 2004). Toxic

cell concentrations can decline while the toxins still

remain in the water column (MacKenzie et al., 2004), or

the toxin effect may be diluted in the presence of

alternative food sources (Dahl and Johannessen, 2001).

In conclusion, among shellfish, mussels (M. gallo-

provincialis and M. barbatus) were the main accumu-

lators of OA and their tissue concentrations exceeded

the levels recommended for consumption. The highest

amounts of OA were detected in M. galloprovincialis,

suggesting this species as the most appropriate

candidate for use as an indicator for DSP toxins in

Thermaikos Gulf. Further investigation on shellfish

toxin accumulation has important implications for

managing bivalve populations for human consumption.

Monitoring of macroinvertebrates is crucial to deter-

mine toxin accumulation in various benthic organisms

that could potentially act as vectors for toxin transfer in

the food web and to explore the dynamics of filtering

capacity of the system, since the dense filter feeding

populations in Thermaikos Gulf strongly influence

matter and energy flow in the coastal ecosystem.

Acknowledgements

We thank I. Varkintzi, A. Gremmenas and S.

Mitsoudi for their help during the field surveys in

Thermaikos Gulf. Financial support was provided by

the European Commission through the FATE project

‘‘Transfer and Fate of Harmful Algal Bloom (HAB)

Toxins in European Marine Waters’’ (contract EVK3-

CT01-00055) as part of the EC-EUROHAB cluster.[SS]

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