Release and degradation of amnesic shellfish poison from

decaying Pseudo-nitzschia multiseries in presence

of bacteria and organic matter

Johannes A. Hagstrom a,*, Edna Graneli a, Isabel Maneiro b,Aldo Barreiro b, Anika Petermann c, Camilla Svensen d

a University of Kalmar, Department of Marine Sciences, SE-39182 Kalmar, Swedenb Dep. de Ecoloxıa e Bioloxıa Animal, Facultade de Ciencias Experimentais, Universidad de Vigo, Aptdo. 874, 36310-Vigo, Spain

c Friedrich-Schiller-Universitat Jena, Department of Food Chemistry, Dornburgerstrasse 25, D-07743 Jena, Germanyd Department of Aquatic Bioscience, Norwegian College of Fishery Science, University of Tromsoe, N-9037 Tromsoe, Norway

Received 27 July 2006; received in revised form 21 August 2006; accepted 23 August 2006

www.elsevier.com/locate/hal

Harmful Algae 6 (2007) 175–188

Abstract

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can

bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most

commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the

cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-

faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures

with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under

quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when

the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at

least 20 days with 1� or 4� bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a

known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site

reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is

important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA

degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that

the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the

mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects

on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a

potential future mitigation technique.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Pseudo-nitzschia multiseries; Domoic acid; Release; Degradation; Bacteria; Nutrient condition

* Corresponding author. Tel.: +46 480 447333;

fax: +46 480 447340.

E-mail address: johannes.hagstrom@hik.se (J.A. Hagstrom).

1568-9883/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.hal.2006.08.002

1. Introduction

The first harmful algal bloom (HAB) caused by a

diatom was observed in 1987, in the Cardigan Bay,

eastern Prince Edward Island, Canada (NW Atlantic

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188176

Ocean). The species Pseudo-nitzschia multiseries Hasle

(Bacillariophyceae) was identified as the source of the

neurotoxin domoic acid (DA) (Bates et al., 1989;

Wright et al., 1989). Contamination of blue mussels

(Mytilus edulis Linnaeus) at aquaculture sites caused

human death (Bates et al., 1989, 1998; Wright et al.,

1989). Mortality of pelicans and other seabirds and

accumulation of DA in anchovy tissues have also been

related to blooms of Pseudo-nitzschia australis Fren-

guelli at Monterey Bay, CA, USA (Buck et al., 1992;

Fritz et al., 1992; Garrison et al., 1992). Transfer of DA

to higher trophic levels has caused mortality and

neurological dysfunction of sea lions along the central

Californian coast, as the toxin was detected both in the

mammal body fluids and in planktivorous fish. This

event was associated with a highly toxic bloom of P.

australis cells (Scholin et al., 2000). The toxin has also

been found in shellfish from French, Portuguese and

Spanish waters; and in Portuguese sardines at levels

above the regulatory limit (Vale and Sampayo, 2001).

Algal isolates of Pseudo-nitzschia spp. and Nitzschia

navis-varingica Lundholm et Moestrup from Danish,

Swedish and Japanese marine or brackish waters also

produce the toxin (Lundholm et al., 1997; Kotaki et al.,

1999, 2004; Lundholm and Moestrup, 2000). Blooms of

Pseudo-nitzschia spp. were first detected in the Galician

Rias (NW Spain) in 1994 (Miguez et al., 1996), and data

from the monitoring of toxic phytoplankton by the

‘‘Centro de Control de Calidade do medio Marino’’

(Xunta de Galicia, Spain) show that blooms have been

reoccurring ever since, with e.g. episodes each year of

contaminated shellfish in Ria de Vigo, Spain, 2002 to

2004. Human consumption of contaminated shellfish

leads to acute sickness, amnesic shellfish poisoning

(ASP), in coastal regions throughout the world.

Permanent short-term memory loss is the most common

effect on humans who have eaten seafood containing the

toxin (Mos, 2001).

Considering the global distribution of ASP events

that cause both ecological and economic losses, it is

important to know the stability of DA, and to understand

the processes that influence degradation of the toxin.

Bates et al. (2003) conclude that photodegradation is the

most likely process by which DA is removed. However,

Stewart et al. (1998) has shown that bacteria isolated

from the blue mussel (M. edulis) and soft shell clam

(Mya arenaria Linnaeus) are capable of degrading the

toxin. Bacteria and enzymes are also known to degrade

toxins, e.g. the cyanobacterial toxin microcystin, and

degradation has been more rapid using bacteria that

were collected from waters where blooms occur

regularly (Jones et al., 1994; Bourne et al., 1996;

Inamori et al., 1998). This suggests that there most

likely are bacterial species associated with P. multi-

series found at elevated numbers during a bloom, that

also have the ability to degrade DA. Guisande et al.

(2002) conclude that the copepod Acartia clausi

Giesbrecht feeding on the paralytic shellfish poisoning

(PSP) producing dinoflagellate Alexandrium minutum

Halim transforms the toxin within its gut. They also

found an increase of dissolved toxins, however,

indicating that toxins were probably released during

feeding. Thus, it seems that several different processes

can degrade DA.

P. multiseries usually proliferates at a time of year

when conditions are unfavourable for other algal

species (Mos, 2001). Blooms of P. multiseries have

been observed in spring and late autumn when

phosphorus (P) and silicate (Si) are depleted in surface

waters, whereas the nitrogen (N) concentration is

elevated due to large amounts of precipitation and

transport of N from land to sea (Bates, 1998; Vale and

Sampayo, 2001). Toxin production by the algal cells has

also been shown to increase under these conditions (Pan

et al., 1996). It is therefore of interest to study the role of

N and P on DA production and algal cell condition, e.g.

cell decay and DA release.

In this work we address the following questions: (1)

Is there a difference in release- and degradation rate of

DA depending on nutrient condition, community

composition (i.e. monocultures vs. mixed planktonic

community mesocosms) during growth or isolation

locale? (2) Does the degradation rate of DA depend on

the number of bacteria present? (3) Do other factors

such as enzymes from mussels and copepods involved

in digestion, and increased bacterial numbers involved

in the decomposition process of organic matter

influence the degradation of DA? In order to determine

if there are spatial differences in the toxin production

and release, two strains of P. multiseries of different

origin were tested, one from the Canadian east coast and

the other from temperate waters of the Brazilian coast.

2. Materials and methods

To study the fate of DA and the importance of

bacteria, both free-living and aggregated with faecal

matter along with possible enzymes associated with

faeces, in the degradation process of the toxin, two

experiments were conducted. In experiment 1, the

release and degradation of DA from cells of two strains

of P. multiseries (from Canada and Brazil), were studied

for 20 days after they were grown in continuous culture

with different nutrient conditions (Fig. 1a). In the

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188 177

Fig. 1. Experimental setup used to study the release and degradation of domoic acid. (a) Experiment 1: Canadian and Brazilian strains of Pseudo-

nitzschia multiseries growing as continuous monocultures under N- or P- deficient and sufficient conditions. Both strains were exposed to the

following treatments: (1) domoic acid standard in autoclaved seawater (SW) (used as control), (2) decaying P. multiseries cells with same bacterial

numbers as during growth and (3) decaying P. multiseries cells with four-fold increase of bacterial density as during growth. Incubation lasted 20

days in complete darkness. (b) Experiment 2: decaying P. multiseries cells (Brazilian strain) from P deficient mesocosms with DA standard and

Whatman GF/C filtered surface water collected in Ria de Vigo, Spain, exposed to the following treatments: (1) domoic acid standard in autoclaved

seawater (SW) (used as control); (2) autoclaved SW (low bacterial abundance); (3) Whatman GF/C filtered SW (representing natural bacterial

abundance); (4) GF/C filtered SW with four-fold concentrated bacteria collected form Ria de Vigo, Spain (high bacterial abundance); (5) copepod

faecal pellets; (6) mussel faecal matter (i.e. a mix of faeces and pseudo-faeces); (7) bottom sediment from Ria de Vigo, Spain.

follow-up experiment 2, the fate of DA was assessed for

75 days from decaying P. multiseries cells (Brazilian

strain) that had been growing in a mesocosm under P-

deficient conditions (Fig. 1b).

2.1. Experiment 1

2.1.1. Phytoplankton cultures

Two non-axenic strains, from the North (Canada)

and South (Brazil) Atlantic Ocean, of P. multiseries

were used. The former was obtained from Provasoli-

Guillard National Center for Culture of Marine

Phytoplankton (CCMP), USA (strain no. 1660, which

had been in culture for 10 years), and the latter was from

the Phytoplankton Laboratory of the Fundacao Uni-

versidade Federal de Rio Grande (FURG), Brazil (strain

PNM1, maintained in culture for 6 months). It should be

noted that the Canada strain was smaller (�32 � 2% in

volume, mean � 1 standard deviation [S.D.]) than the

Brazil strain. The cell volume was calculated according

to Maldonada et al. (2002). The two strains had been

identified using electronic microscopy and 18S rRNA

gene sequence analysis.

The two strains had been growing as continuous

cultures at the Institution of Biology and Environmental

Science, Kalmar University, Sweden in modified f/2

media to get N:P ratios of 16:1 (atomic Redfield ratio),

1:1 and 290:1 (Guillard and Ryther, 1962). The trace

metals stock solution was modified for L1 media

(Guillard, 1995). Si (Na2SiO3�5H2O) was added at a

concentration of 140 mM, higher than in f/2 media, to

avoid limitation. Only N and P concentrations were

altered in the two nutrient-deficient treatments. All

media was prepared using pre-filtered (Whatman GF/C

filters), and autoclaved (20 min, 121 8C) aged Baltic

Sea surface water (SW) adjusted to 31 � 1 psu

(mean � 1 standard deviation [S.D.]) using propor-

tional quantities of the major salts (NaCl, MgCl2�6H2O,

CaCl2�2H2O, Na2SO4 and K2SO4). The pH of the

complete media was adjusted to 8.1 � 0.1, and aseptic

techniques consistently were used.

All treatments (in duplicate), i.e. two algal strains

growing in three nutrient conditions, were maintained in

1 L culture flasks (total of 12 flasks). Both algal strains

in each nutrient treatment were supplied with media

from the same bottle to avoid differences in growth

conditions between the two strains. The algal cultures

were held at 18.0 � 0.5 8C under photosynthetic active

radiation (PAR) of 200 mmol photons s�1 m�2, pro-

vided by cool white fluorescent tubes (Philips TLD

36W/830). The light:dark cycle was 16:8 h. Peristaltic

pumps were used to distribute the media, and all

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188178

cultures were aerated with sterile air (0.2 mm pore size

Sartorius Minisart1 filter) to ensure continuous mixing.

Prior to this experiment, the two P. multiseries strains

were used in other experiments; thus, the cells had been

in continuous culture at 0.2–0.4 dilutions day�1 in the

above media for 150 � 10 days depending on nutrient

condition.

2.1.2. Culture parameters analyzed

Before beginning the DA degradation experiment the

duplicate culture flasks of each algal strain and nutrient

condition were pooled and manually stirred. Samples

were then taken from each of these six flasks for

analyses of cell densities, chlorophyll a (chl a);

particulate organic carbon (POC), nitrogen (PON)

and phosphorus (POP); particulate and dissolved

domoic acid (DA); and bacterial counts. Sub-samples

from the cultures were filtered onto pre-combusted (2 h,

450 8C) glass fibre filters (Whatman GF/C) for POC,

PON and POP analyses. The filters were then dried

(24 h, 60 8C) and analyzed using a CHN elemental auto

analyzer (Fisons Instruments model NA 1500). POP

was measured following Solorzano and Sharp (1980).

Sub-samples collected for DA analyses were filtered

onto Whatman GF/C glass fibre filters. To avoid loss of

DA, the filters with the particulate matter were freeze-

dried prior to shipping for analysis in Germany. The

filtrates of the above samples were collected for

dissolved DA measurements and preserved with sodium

azide (NaN3, final concentration 10 mg L�1), and held

at 4 8C. DA analyses of both the particulate and

dissolved fractions followed Furey et al. (2001), and

used a high-performance liquid chromatograph (HPLC)

coupled to a mass-spectrometer (API 165, Applied

Biosystems/MDS Sciex; Foster City, CA, USA).

Algal cell counts were conducted on samples

preserved with acid Lugol’s solution (Vollenweider,

1974) and cells were counted according to Utermohl

(1958), using a Nikon Diaphot 300 inverted microscope.

Three sub-samples from each culture, and a minimum of

400 cells per sample, were counted. Sub-samples for chl

a determination were filtered through a Gelman Sciences

type A/E glass fibre filter and prepared according to

Jespersen and Christoffersen (1987) with modifications

as follows: the filters with cells were placed in 6 mL 95%

ethanol and sonicated for 15 min in an ice cooled water

bath, followed by at least 2 h extraction at room

temperature; sonication and extraction was done in

complete darkness. Chl a was measured with a Turner

Designs model 10 AU fluorometer. Bacteria were

enumerated by flow cytometry (FACSCalibur flow

cytometer, Becton Dickinson, San Jose, CA, USA)

following Troussellier et al. (1999). For these measure-

ments, 1-mL culture sub-samples (in duplicate) were

fixed with pre-filtered (0.2 mm pore size) formaldehyde

(2% final concentration) and stained for 30 min at room

temperature in darkness with SYTO1 13 (Molecular

Probes), a nucleic acid stain, at a final concentration of

5 mM (Troussellier et al., 1999).

2.1.3. DA release and degradation

After the above samples had been collected, all

culture flasks were placed in 35 8C for 2 h in order to

kill the cells and speed up the lysing process. We did this

since marine diatoms are known to survive long periods

in darkness, and our aim was to determine the

degradation of toxins released from decaying cells

(Smayda and Mitchell-Innes, 1974). The flasks were

manually stirred at 5 min intervals during the heating

period, to make the temperature more homogeneous. A

temperature of 35 8C was used in an attempt to

minimize destruction of enzymes in the algal cells or

death of associated bacteria. After this procedure, the

cells were checked under an epifluorescence micro-

scope to ensure that they were releasing intracellular

material. The microscopical examination showed that

most of the algal cells had lost their chlorophyll red

autoflourescence. The cultures were divided into 35 mL

aliquots in 50 mL Falcon polypropylene tubes together

with 5 mL of either (1) water with the natural bacterial

communities of the culture flasks, or (2) the concen-

trated bacterial community (see below), immediately

after most algal cells had been killed. Domoic acid

standard (Sigma, St. Louis, MO, USA; level of purity

90%) in autoclaved SW at a high initial concentration

(7 mg mL�1), incubated in light or dark conditions, was

used as controls (Fig. 1a).

Bacteria were concentrated from the outflow of the

continuous cultures collected for 2 days before the

experiment, by centrifuging 50 mL aliquots at

10,000 rpm for 20 min, after which the supernatant

was replaced with new water and the tubes were

centrifuged again. This was repeated until the entire 1 L

of effluent had been centrifuged. The resulting pellet

material was re-suspended in the same medium that had

been used for the continuous cultures. All water was

maintained on ice throughout this process, and

centrifugation was conducted at 4 8C.

Controls and all treatments were in triplicate (total of

21 tubes per treatment) and the tubes were incubated in

darkness without further mixing conditions at 16 8C.

The experiment was conducted in darkness because

decaying algal cells will sink through the water column

and hence light will disappear or at least decrease

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188 179

drastically. Furthermore, light as an abiotic factor of

toxin degradation can be ruled out when the experiment

is carried out in complete darkness. Samples for toxin

quantification and bacterial cell counts were collected

for 20 days by sacrificing three tubes at each sampling

point. DA concentrations in the dissolved fraction and

the particulate fraction (i.e., from cells retained on a

Whatman GF/C filter) were analyzed using an HPLC as

described above.

2.2. Experiment 2

A follow-up experiment was conducted in which P.

multiseries cells (Brazil strain) grown in a P-deficient

mesocosm were used. The cells were divided into

20 mL aliquots, placed into glass scintillation vials, and

killed by freezing at �18 8C for 20 min, shaking the

vials at 5 min intervals to ensure that the water did not

freeze solid. HPLC analysis just before the experiment

indicated that these cells contained very low levels of

DA. Therefore, we added DA standard comparable to

the amount that the cells had produced under P

deficiency in experiment 1. It should be noted that

the P. multiseries community, and most other algal

communities, in the mesocosms crashed before reach-

ing steady state. As a result, an unidentified green algal

species dominated the mesocosm phytoplankton com-

munity. We suspect that the decaying cells started to

release P, based on an observed increase of dissolved P

in the water. This is believed to be the reason for the low

DA levels observed in the cells, i.e. unintentionally high

levels of available P and the fact that the P. multiseries

cells never reached steady phase, reduced the cells DA

production (Bates and Trainer, 2006).

To complete further tests regarding the effect of

bacteria on DA degradation, in this experiment we

tested a naturally occurring bacterial community from

surface waters of a site known for P. multiseries blooms

(Ria de Vigo, Spain; 1� and 4�-concentrated densities

as in experiment 1) (Fig. 1b). The collected surface

water (1 L) was pre-filtered through Whatman GF/C

filters. The method of bacterial concentration was the

same as in experiment 1, except that 10 mL aliquots

were centrifuged at 4000 rpm for 20 min, followed by

re-suspension of the bacteria in pre-filtered surface

water. We also included treatments with (1) low

bacterial abundance (P. multiseries cells from the

mesocosm that had been rinsed with, and then

transferred to, filtered and autoclaved SW, i.e. not an

axenic treatment since e.g. bacteria attached to algal

cells were present); (2) 25 copepod faecal pellets in

�0.5 mL SW (see below) with filtered, autoclaved SW

and DA standard to make a final volume of 1.5 mL per

replicate (incubated in sterile Eppendorf tubes); (3)

mussel faecal matter, 4 mL in 10 mL of filtered,

autoclaved SW with DA standard (see below); (4)

10 mL of a 50:50 mix of superficial sediments (top

5 cm) collected, by divers, in cores from Ria de Vigo,

Spain, and filtered and autoclaved SW with DA standard

(Fig. 1b). The copepods producing the faecal pellets

were collected from Ria de Vigo, Spain. The animals

were kept in 25 mL plastic jars for 24 h (12:12 h

light:dark cycle) at 18 8C and fed Tetraselmis suecica

Kylin (Prasinophyceae) prior to the experiment. The

faecal pellets were collected using Pasteur pipettes

under 100� magnification in an inverted microscope.

The mussel faecal matter came from adult Mytilus

galloprovincialis Lamarck that were kept in aquaria

under same conditions as for the copepods. In order to

prevent re-ingestion of the faecal matter the mussels

were placed on a plastic grid (3000 mm mesh size)

above the bottom of the aquaria so that the produced

faeces/pseudo-faeces could fall through the grid, and be

collected using a wide mouth pipette. Both copepod

faecal pellets and mussel faecal matter/pseudo-faeces

had been collected immediately before the experiment

and held in darkness at 4 8C. DA standard (Sigma) in

autoclaved SW was used as controls.

Triplicate vials, from an initial total of 48 per

treatment, were sacrificed after 0–75 days of incubation

in darkness under quiescent conditions at 16 8C, i.e. the

in situ temperature during diatom blooms in Ria de

Vigo, Spain. Sub-samples for determination of bacterial

abundance and chl a/pheopigment concentrations were

collected from each vial and analyzed as above. The

remainder of the sample was used to determine DA

concentrations in the dissolved and the particulate

fractions, as above.

Statistical analyses were performed with STATIS-

TICA (StatSoft Inc.) using a general linear model with

nutrients as a fixed variable, strain as a random variable

(experiment 1), and bacteria as a fixed variable nested

within the interaction of nutrients � strain. Bacteria was

nested with the different combinations of strain and

nutrient condition to adjust error degrees of freedom

since the bacterial triplicates were sub-sampled from the

culture flasks with different nutrient conditions. Time

(days) was used as a continuous covariate. To assess the

homogeneity of slopes in the analysis, we included the

interactions nutrients � days, strain � days, the three-

way interaction nutrients � strain � days, and bacteria

{nutrients � strain} � days. Both response variables

were log-transformed to improve normality. A Tukey

HSD post hoc test was also used in order to determine

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188180

Table 1

Particulate organic nitrogen (PON), carbon (POC) and phosphorus (POP) concentrations and ratios in the diatom Pseudo-nitzschia multiseries,

isolated off the Canadian east coast or Brazil

P-deficient N-deficient NP-sufficient

Canada Brazil Canada Brazil Canada Brazil

PON (pg N cell�1) 8.2 � 0.6 13.7 � 0.5 4.7 � 0.8 8.0 � 0.2 9.4 � 0.9 9.8 � 1.2

POC (pg C cell�1) 53.2 � 4.4 136.6 � 1.2 33.6 � 5.5 88.5 � 6.4 47.2 � 2.8 52.2 � 3.6

POP (pg P cell�1) 1.0 � 0.2 1.1 � 0.2 2.1 � 0.5 3.1 � 0.6 2.4 � 0.4 4.0 � 1.0

N:P (atomic) 18.7 29.9 5.0 5.7 8.7 5.4

C:N (atomic) 7.6 11.6 8.3 12.9 5.9 6.2

The algae were grown as continuous cultures under N- or P-deficient, or NP-sufficient conditions. The two algal strains in each nutrient treatment

were supplied with medium from the same bottle. Sub-samples were collected for a DA release and degradation experiment immediately before the

cells were killed (mean � S.D., n = 4).

which variables were significantly different from one

another.

3. Results

3.1. Experiment 1: cell concentrations, nutrient

conditions and chlorophyll a levels

The Brazil strain of P. multiseries growing under N

or P deficiency had about 2.5-fold higher POC than

corresponding cultures of the Canada strain (Table 1).

The P-deficient Brazil strain also had the highest

PON cell�1 of all cultures, and the highest N:P ratio

(Table 1). The highest cellular PON for the Canada

strain was measured from the NP-sufficient treatment,

which also had the lowest C:N ratio (Table 1).

Cultures of the Brazil strain had higher chl a content

than the Canada strain under P deficiency (Table 2). Total

cell numbers were highest in the N-deficient treatment of

the Canada strain (Table 2). Initial bacterial numbers in

the treatments mostly were �1.3� 0.04 �106 cells

mL�1 (Table 2).

3.2. Degradation of domoic acid

3.2.1. Experiment 1

Domoic acid concentration in both the dissolved and

particulate phase of all treatments began to increase

Table 2

Chlorophyll a, algal cell and bacterial numbers in continuous culture bottl

P-deficient

Canada Brazil

Chl a (mg L�1) 75 � 18 103 � 3

Cell numbers (�103 mL�1) 57.5 � 3.6 61.4 � 2.2

Bacterial numbers (�106 mL�1) 1.0 � 0.1 3.0 � 0.2

The algae had been growing under N- or P-deficient or NP-sufficient nutri

nutrient treatment (means � S.D., n = 3).

within the first few hours after the cells had been killed

(Fig. 2). Although the DA concentration in the

particulate fraction declined, the total amount in the

samples was greater at the end than at the beginning of

the experiment (Fig. 2). The highest total increase in DA

(2.3-fold) during the 20-day incubation was found for

the Canada strain in the N-deficient treatment; all other

treatments increased 1.5–1.9-fold in DA by the end of

the experiment.

Statistical analysis showed that the bacteria � days

interaction was not significant, so it was omitted from

the model. DA release from P. multiseries did not

depend on the initial bacterial abundance ( p > 0.01)

(Tables 3 and 4). DA in the dissolved fraction from the

N-deficient Canada strain increased from 1.7 � 0.1 to

10.8 � 0.3 ng mL�1 in the low-bacteria treatment, and

increased from 1.6 � 0.1 to 12.3 � 0.8 ng mL�1 in the

high-bacteria treatment (Fig. 2a and d). The data

suggest that the bacteria collected from the cultures

were symbionts that were unable to use DA as a

substrate for growth. Corresponding changes of DA in

the particulate fraction of the low- and high-bacteria

treatments were 4.5 � 0.3 to 1.0 � 0.0 and 4.2 � 0.3 to

1.0 � 0.1 ng mL�1, respectively, due to the release of

DA. Most of the other treatments showed the same

initial toxicity and trends over the experimental

duration (Fig. 2). The only treatment that did not show

a continuous increase of DA in the dissolved fraction

es with P. multiseries (Canada and Brazil strains)

N-deficient NP-sufficient

Canada Brazil Canada Brazil

67 � 6 64 � 2 117 � 42 157 � 32

62.2 � 0.5 56.0 � 3.7 82.4 � 18.4 78.0 � 39.7

0.6 � 0.1 0.7 � 0.1 1.3 � 0.04 0.71 � 0.1

ent conditions with one medium bottle supplying both strains of one

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188 181

Fig. 2. The domoic acid (DA) concentration in the dissolved and particulate fraction, i.e. what was retained or not on a Whatman GF/C filter, from

decaying P. multiseries cells isolated off the Canadian east coast or from Brazilian temperate waters and grown as continuous cultures. The three

upper panels (a, b and c) were incubated with the same bacterial numbers as during growth and the three lower panels (d, e and f) were incubated with

four times the bacterial numbers as during growth. Nutrients treatments were: (a) and (d) N deficiency, (b) and (e) P deficiency and (c) and (f) NP

sufficiency. The majority of the cells had been killed by heat (2 h at 35 8C) just prior to the start of the 20 days incubation in darkness under quiescent

conditions at 16 8C. Note the different scales on the Y-axis (n = 3, mean � S.D.; S.D. bars sometimes hidden behind symbol).

was the P-deficient Brazil strain. This treatment also

had the highest initial particulate and dissolved DA

(Fig. 2). Dissolved DA in the low- and high-bacteria

treatments increased from 12.7 � 3.9 to 99.9 � 4.5 ng

mL�1 and from 18.5 � 0.8 to 108.0 � 6.8 ng mL�1,

respectively, during the first 2 days and then levelled off

(Fig. 2b and e). There were significant differences in

both the dissolved and particulate fraction when

comparing the two strains and three nutrient treatments

(Tables 3 and 4). The post hoc test revealed that the

only comparisons of dissolved DA that were not

significantly different were the N-deficient Canada

strain versus the NP-sufficient Brazil strain, and the P-

deficient Canada strain versus the N-deficient Brazil

strain. There were three comparisons in the particulate

fraction that did not show significant differences: N-

deficient Canada strain versus the P-deficient Canada

strain; NP-sufficient Brazil strain versus the P-deficient

Canada strain or the NP-sufficient Brazil strain.

Bacterial numbers increased throughout the experiment

in all treatments, indicating that suitable substrates

for bacterial growth were present (Fig. 3). A short-term

(2-day) assay with high initial DA concentration

showed no difference in DA in light or dark conditions

(7.14 � 0.90 and 7.94 � 1.0 mg DA mL�1, respec-

tively).

3.2.2. Experiment 2

In the follow-up, longer experiment, conducted using

P-deficient P. multiseries (Brazil strain) from meso-

cosms, DA in the controls with autoclaved filtered

seawater was stable; it did not appreciably degrade

during 75 days incubation in darkness despite bacterial

contamination (Fig. 4a and e). DA was rapidly released

from the decaying cells, and the dissolved toxin was

stable under normal (not concentrated) densities of the

natural bacterial community from the bloom site for 35

days (Fig. 4c). After 45 days in darkness, however, DA

concentrations fell below the analytical detection limit,

indicating that the incubation time in experiment 1 had

been too short (Fig. 4c). Furthermore, chl a declined

throughout the incubation in all three treatments with

P. multiseries. Pheopigments decreased rapidly in all

treatments (from �145 to �50 mg L�1), and then

remained at that concentration for the remainder of the

experiment (Fig. 5). The data suggest that the killed P.

multiseries cells released their cellular contents rapidly,

and that cells, which survived the pre-experiment

treatment also survived the incubation. We also found

that a four-fold increase in bacterial densities (from

0.80 � 106 bacteria mL�1 initially to 3.64 � 106

bacteria mL�1) led to a more rapid decline in chl a

than in the other two treatments (Fig. 5). Thus, in the

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188182

Table 3

Summary of general linear model results on dissolved domoic acid concentrations from decaying P. multiseries cells over time

d.f. MS Den. Syn. error d.f. Den. Syn. error MS F p

Nutrients 2 37.08 2.00 92.74 0.40 0.714

Strain 1 4.83 2.00 92.74 0.05 0.841

Nutrients � strain 2 92.74 13.63 0.11 857.27 0.000

Bacteria (nutrients � strain) 6 0.12 234.00 0.09 1.30 0.259

Days 1 54.85 1.00 0.79 69.44 0.076

Nutrients � days 2 0.76 2.00 1.92 0.40 0.717

Strain � days 1 0.79 2.00 1.92 0.41 0.587

Nutrients � strain � days 2 1.92 234.00 0.09 20.90 0.000

Error 234 0.09

Effects from the factors nutrients (N deficient, P deficient and NP sufficient), strain (Canada and Brazil), bacteria (1� and 4� abundance in culture

flasks) and days were tested, as well as factor interactions. The response variable was log-transformed; strain was a random variable and all other

variables were fixed.

Table 4

Summary of general linear model results on particulate domoic acid concentrations from decaying P. multiseries cells over time

d.f. MS Den. Syn. error d.f. Den. Syn. error MS F p

Nutrients 2 6.29 2.00 24.45 0.26 0.796

Strain 1 4.89 2.00 24.45 0.20 0.699

Nutrients � strain 2 24.45 32.29 0.06 439.60 0.000

Bacteria (nutrients � strain) 6 0.04 234.00 0.08 0.48 0.820

Days 1 53.55 1.00 15.75 3.40 0.316

Nutrients � days 2 6.48 2.00 0.01 579.05 0.002

Strain � days 1 15.75 2.00 0.01 1406.74 0.001

Nutrients � strain � days 2 0.01 234.00 0.08 0.14 0.870

Error 234 0.08

Effects from the factors nutrients (N deficient, P deficient and NP sufficient), strain (Canada and Brazil), bacteria (bacterial abundance in culture

flask and four-fold bacterial abundance) and days were tested, along with factor interactions. The response variable was log- transformed; strain was

a random variable and all other variables were fixed.

Fig. 3. The initial and final (after 20 days incubation) bacterial abundances in the test tubes filled with decaying P. multiseries cells from continuous

cultures. The algal cells had been isolated off the Canadian east coast, panels (a) and (b), or from the temperate waters of Brazil, panels (c) and (d).

The initial bacterial abundances in the two left hand panels were unchanged from what found in the continuous culture bottles and the bacterial

abundance in the other treatment (the two right hand panels) had been increased four-fold by centrifuging outflow media collected during culturing

(n = 3, mean � S.D.).

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188 183

Fig. 4. Domoic acid concentration in the dissolved and particulate fraction, i.e. what was retained or not on a Whatman GF/C filter, in presence of

decaying P. multiseries cells from a P deficient mesocosm, is presented in the left hand panels. Most cells were killed by keeping the vials at�18 8Cfor 20 min. The bacterial abundance in the same incubation vial is presented in the adjacent panel. Incubation was conducted under quiescent

conditions in the dark at 16 8C. All vials contained DA standard and Whatman GF/C filtered surface water collected in Ria de Vigo and (a) and (e)

autoclaved SW without P. multiseries cells (control), (b) and (f) autoclaved SW (low bacterial abundance), (c) and (g) Whatman GF/C filtered SW

(representing natural bacterial abundance) and (d) and (h) GF/C filtered SW with four-fold concentrated bacteria collected form Ria de Vigo (high

bacterial abundance) (n = 3, mean � S.D.; S.D. bars sometimes hidden behind symbol).

high-bacteria treatment, DA was more rapidly released

from decaying P. multiseries and/or degraded in the

cells (Fig. 4d). In that treatment, DA ranged from

�2.5 ng mL�1 to below detection after 3 days

(particulate fraction) to 28 days (dissolved fraction).

For the low-bacteria treatment, release and/or degrada-

tion of DA were prolonged to 9 and 45 days in the

particulate and dissolved fractions, respectively (Fig. 4c

and d), indicating that some strains of the naturally

occurring bacteria can degrade DA.

The addition of copepod faecal pellets was the only

treatment in this experiment that did not affect the

concentration of the DA standard, which remained at

�20 ng mL�1 (Fig. 6a). The copepod faeces apparently

did not absorb DA since the toxin concentration in the

particulate fraction remained low (<3 ng mL�1), and

the bacterial abundance was stable throughout the

experiment (Fig. 6a and d). The most rapid degradation

of DA (�1 day) occurred in the presence of mussel

pseudo-faeces or bottom sediment (Fig. 6b and c).

Bacterial numbers followed DA concentrations in most

of the treatments; however, the presence of high organic

matter added with the mussel pseudo-faeces or

sediment caused wider fluctuations in bacterial numbers

(Figs. 4 and 6).

4. Discussion

In this study we have shown that there are significant

differences in release of domoic acid (DA) from

decaying P. multiseries cells grown under different

nutrient conditions, and that the extent of DA release

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188184

Fig. 5. The chlorophyll a and pheopigment concentrations in decay-

ing P. multiseries cells that were collected from a P deficient meso-

cosm, killed (�18 8C for 20 min), and held under quiescent conditions

in darkness at 16 8C in filtered surface water from a known P.

multiseries bloom site. The water had been (a) autoclaved; (b)

untreated (representing the natural bacterial consortium); (c) augmen-

ted with 4� the natural bacterial abundance (means � 1S.D., n = 3).

depends upon the strain and the nutrient regime. These

findings suggest that predictions about impacts and

duration of P. multiseries blooms cannot be generalized.

In both experiments, DA was rapidly released from

decaying P. multiseries cells. When bacterial numbers

from surface waters of known bloom areas were

concentrated four-fold, DA was more rapidly degraded.

This effect was not evident when cultures of P.

multiseries were used as the source of concentrated

bacteria; instead, DA doubled in both the low and high

cultured bacteria treatments. Thus, when testing toxin

degradation by bacteria, it is important to use bacterial

consortia from known bloom areas of Pseudo-nitzschia,

preferably collected during or immediately following

bloom events when specific bacterial taxa involved in

toxin degradation would be expected to be active.

The algal cultures used in the continuous culture

experiment (experiment 1) contained high numbers of

associated bacteria that may have affected algal growth,

cell lysis and toxin production. Bates et al. (2004)

showed that bacteria associated with P. multiseries in

culture could not produce DA independently, although

some bacteria can affect algal toxicity and/or growth.

For instance, higher DA production has been reported in

non-axenic cultures than in axenic cultures (Douglas

and Bates, 1992; Douglas et al., 1993). In addition,

different bacterial groups (e.g. Moraxella and Alter-

omonas sp.) have been identified in cultures of P.

multiseries. Alteromonas sp. was found to be capable of

producing significant amounts of gluconic acid/gluco-

nolactone in the presence of glucose (Stewart et al.,

1997). DA production by P. multiseries, in turn, was

induced by the addition of gluconic acid/gluconolac-

tone when Alteromonas sp. was present, showing the

link between these compounds and toxin production

(Osada and Stewart, 1997). Salomon et al. (2003) found

free-living and attached bacterial communities asso-

ciated with the cyanobacterium Nodularia spumigena

Mertens. These bacteria were able to either stimulate or

depress growth of N. spumigena without strong

algicidal activity. Our results show that lower total

DA is coupled to lower bacterial density, and vice versa,

when the bacteria present are associated with the algal

cells in cultures. For instance, the P-deficient Brazil

strain not only had the highest bacterial density, but also

the highest total DA of all cultures. The data suggest

that the bacteria present in our cultures promoted DA

production; however, we did not attempt to cultivate or

identify these bacteria, so conclusions about their role

can only be speculative.

Although it has been argued that free-living bacteria

with degrading capabilities of DA are rare (Stewart

et al., 1998), it is known that the bacterial consortium

changes during algal blooms. The change could then be

to a consortium with higher abundance of bacterial

species capable of utilizing DA as a substrate for

growth. Such alteration of the bacterial consortium

toward species that can degrade microcystin has been

hypothesized for waters with blooms with the toxigenic

cyanobacterium Microcystis aeruginosa Kutz. emend

Elkin (Jones et al., 1994). Several other authors have

shown a link between bacteria and toxigenic algae; for

example, some bacteria populations with algicidal

properties have been shown to increase toward the end

of a harmful algal bloom (Mayali and Azam, 2004).

This change in the bacterial community can be rapid, in

as little as 1–2 days in mesocosms with decaying

diatoms (Riemann et al., 2000), and up to 4 days in

cultures of the toxigenic dinoflagellate Karenia brevis

(Mayali and Doucette, 2002). The bacteria in both of

those studies may have used the increased DOM

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188 185

Fig. 6. Domoic acid (DA) concentration in the dissolved and particulate fraction, i.e. what was retained or not on a Whatman GF/C filter, is presented

in the left hand panels. The bacterial abundance in the same incubation vial is presented in the adjacent panel. Incubation was conducted under

quiescent conditions in the dark at 16 8C. All vials contained DA standard and Whatman GF/C filtered and autoclaved surface SW collected in Ria de

Vigo with the presence of (a and d) copepod faecal pellets, (b and e) mussel (Mytilus edulis) pseudo-faeces, (c and f) bottom sediment collected form

Ria de Vigo (n = 3, mean � S.D.; S.D. bars sometimes hidden behind symbol).

produced during the algal blooms as substrates for

growth; our data suggest that some members of the

bacterial consortium could also use DA.

In our second experiment, degradation of dissolved

DA began after 28 days in darkness with a naturally

occurring density of a bacterial consortium from a

known P. multiseries bloom area. When these bacteria

were concentrated four-fold, toxin degradation pro-

ceeded more rapidly (beginning after 16 days).

Elevating the bacterial numbers four-fold would have

led to a more rapid reduction in the amount of available

DOM, and may have conferred an advantage to

bacterial taxa that could utilize DA when other

substrates becomes limited. In this experiment, we

also found that the bacterial numbers followed DA

concentration in all treatments.

Regarding the role of nutrients in DA production,

Bates (1998) concluded that N is a crucial component

for toxicity of Pseudo-nitzschia spp., supported by the

fact that DA is an amino acid. Several other authors

have also been able to show a link between P deficiency

in Pseudo-nitzschia and DA production (e.g. Bates,

1998; Pan et al., 1998). Fehling et al. (2004) found that

cultured P-deficient Pseudo-nitzschia seriata (Cleve) H.

Peragallo cells with a C:N ratio of �7 produced

100 ng DA mL�1. Both the C:N ratio and the quantity

of DA in that study were similar to what we observed for

the P-deficient Brazil strain of P. multiseries. Pan et al.

(1996) observed an increase of both intracellular and

released DA into the medium of a P. multiseries culture

under severe P deficiency, strongly supporting our

findings. Both strains in the present experiment showed

highest toxicity in treatments with the highest amount of

PON (i.e., the P-deficient Brazil strain and the NP-

sufficient Canada strain). Also, chl a declined faster and

to a lower concentration in treatments with high

bacterial abundance (4� the natural consortium from

the bloom site) than in treatments without bacterial

additions or with lower bacterial densities. The 4�natural bacteria treatment also was the only treatment of

the three that had higher pheopigment than chl a at the

end of the experiment.

Domoic acid is bioaccumulated by both shellfish and

fish (Douglas et al., 1997; Amzil et al., 2001; Lefebvre

et al., 2002). The depuration rate of DA by shellfish and

fish differs within and among species. Amzil et al.

(2001) found a decontamination period of 1 week in the

blue mussel Mytilus edulis galloprovincialis Lamarck

collected off the French coast whereas Douglas et al.

(1997) observed high levels in the sea scallop

J.A. Hagstrom et al. / Harmful Algae 6 (2007) 175–188186

Placopecten magellanicus Gmelin 14 days after

contamination ceased, or 35 days after the experiment

was initiated. We observed the most rapid degradation

and/or release of DA, both particulate and dissolved, in

the presence of mussel pseudo-faeces and bottom

sediment, whereas copepod faecal pellets did not affect

DA concentrations over a 75-day period. The more

rapid rate of DA degradation in the presence of pseudo-

faeces may have been related to the higher bacterial

numbers and enzymes, both with degrading capabilities,

in the mussel pseudo-faeces that is not present in the

copepod faecal pellets. This finding supports the work

of Stewart et al. (1998), who found that bacteria in the

tissues of some bivalves, especially the blue mussel

Mytilus edulis, quickly degrade DA. In our study,

dissolved DA in the mussel pseudo-faeces began to

increase toward the end of the experiment, and may

reflect the release of toxin from cells within the mucus-

laden material as the pseudo-faeces slowly deteriorated.

We are uncertain as to why the toxin inside the intact

pseudo-faeces could not be detected. Maneiro et al.

(2005) showed that copepods accumulate DA. In

addition, Teegarden and Cembella (1996) demonstrated

that the amount of PSP in the copepods Acartia tonsa

Dana and Eurytemora herdmani Thompson et Scott

followed the toxicity of ingested Alexandrium sp.—

thus, PSP was not degraded inside these microfauna.

Based on these studies and our data, copepods may lack

enzymes and/or associated bacteria with toxin-degrad-

ing capabilities and, so, can function as vectors in toxin

transfer to higher trophic levels. Finally, the DA bound

to sediment particles apparently can be rapidly

degraded by the associated bacterial consortium. This

has been observed to be the case for the well-studied

microcystin: Morris et al. (2000) found that clay

particles effectively bind the microcystin and thereby

reduce the amounts of dissolved toxins.

5. Conclusion

Our experiments demonstrated that P. multiseries

can significantly differ in production and release and/or

degradation of domoic acid, depending upon the strain

and the nutritional status. Associated bacteria in P.

multiseries cultures did not affect DA degradation at 1�or 4� concentrations of bacterial cells, but apparently

did promote toxin production or release. In contrast, a

4� concentration of a bacterial consortium from a

known P. multiseries bloom site degraded DA more

rapidly than the naturally occurring bacterial density.

DA was also degraded rapidly in the presence of mussel

pseudo-faeces or sediment from the bloom site,

suggesting a role of associated bacteria and/or enzymes.

Future studies should aim at isolating bacterial species

that can grow on DA, and should also investigate the

role of enzymes involved. The insights gained from

such research could lead to new mitigation techniques

and reduce the economic loss from blooms of DA-

producing diatoms.

Acknowledgements

We thank P. Dinnetz and R. Engkvist for helping us

with the statistics. We are also grateful to J. Burkholder

for comments on how to improve an early version of this

manuscript. This work was financed by the Swedish

Research Council and the European Commission

(Research Directorate General-Environment Pro-

gramme, Marine Ecosystems), through the FATE project

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

Toxins in European Marine Waters’’ (contract number

EVK3-2001-00050) and the Thresholds of Environ-

mental Sustainability Project (GLOBAL-2, contract

number 003933), contracts holder E. G. The FATE

project is part of the EC EUROHAB cluster.[SES]

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