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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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