Krill: A potential vector for domoic acid in marine food webs

Abstract

Over the past decade, blooms of the domoic acid (DA) producing diatom Pseudonitzschia have been responsible for numerous deaths of marine mammals and birds in Monterey Bay, California. Euphausiids (krill) are important members of the local zooplankton grazer community and comprise the primary diet of squid, baleen whales, and many seabirds. Krill are thus a key potential vector for the transfer of DA to higher trophic level organisms in Monterey Bay. A better understanding of the quantitative trophic interactions and body burden of DA in krill is required to predict whether they tan act as an effective vector for this neurotoxin. Here we report results of toxin analyses and gut content examinations of krill Euphausia pacifica collected from Monterey Bay in 2000. Corresponding counts of toxic Pseudo-nitzschia species in the water and their cellular DA concentrations were also obtained at the collection sites. Toxin analysis by receptor binding assay demonstrated that DA in krill tissue varied between 0.1 to 44 mug DA equiv. g(-1) tissue (confirmed by tandem mass spectrometry), with levels corresponding to the abundance of toxic Pseudo-nitzschia species present in the water. The occurrence of Pseudo-nitzschia australis frustules in the digestive tract of E. pacifica verified that a toxic species of this diatom was an important part of their diet and thus implicated this phytoplankter as the source of DA. These findings provide compelling evidence for the role of krill as a potential transfer agent of the phycotoxin DA to higher trophic levels in marine food web.

Full text

MARINE ECOLOGY PROGRESS SERIES
Mar Ecol Prog Ser
Vol. 237: 209–216, 2002 Published July 18
INTRODUCTION
Blooms of toxic phytoplankton are becoming more
frequent and better documented in the world’s oceans
(Smayda 1989, Hallegraeff 1993, Van Dolah 2000).
Although there is great concern about the effects of
phycotoxins on higher organisms, including humans,
marine mammals and birds, the links between toxic
phytoplankton and these organisms are still not clear.
The direct and rapid transfer of these toxins to the
highest levels of the food chain requires herbivores
that consume the toxic species. Therefore, identifica-
tion of herbivorous vectors that can transfer phycotox-
ins to higher trophic levels becomes essential to under-
standing the fate of the toxins in marine ecosystems.
Monterey Bay, California, has been a region im-
pacted by toxic phytoplankton populations over the
last decade (Walz et al. 1994, Fryxell et al. 1997,
Scholin et al. 2000). As is typical of many productive
upwelling ecosystems, food chain linkages to top-level
predators in Monterey Bay can be very short, incorpo-
rating large herbivores such as krill, anchovies and
sardines as key intermediates (Ryther 1969). These
planktivores thus represent a direct mechanism for the
potentially rapid transfer of phycotoxins from toxic
phytoplankton to higher-level consumers.
Recent events in Monterey Bay have demonstrated
the transfer of the neurotoxin domoic acid (DA) from
© Inter-Research 2002 · www.int-res.com
*E-mail: sibelbargu@hotmail.com
Krill: a potential vector for domoic acid in marine
food webs
Sibel Bargu
1,
*
, Christine L. Powell
2
, Susan L. Coale
1
, Mark Busman
2
,
Gregory J. Doucette
2
, Mary W. Silver
1
1
Department of Ocean Sciences, University of California at Santa Cruz, 1156 High Street, Santa Cruz, California 95064, USA
2
Marine Biotoxins Program, NOAA/NOS Center for Coastal Environmental Health & Biomolecular Research,
219 Fort Johnson Road, Charleston, South Carolina 29412, USA
ABSTRACT: Over the past decade, blooms of the domoic acid (DA) producing diatom Pseudo-
nitzschia have been responsible for numerous deaths of marine mammals and birds in Monterey Bay,
California. Euphausiids (krill) are important members of the local zooplankton grazer community and
comprise the primary diet of squid, baleen whales, and many seabirds. Krill are thus a key potential
vector for the transfer of DA to higher trophic level organisms in Monterey Bay. A better understand-
ing of the quantitative trophic interactions and body burden of DA in krill is required to predict
whether they can act as an effective vector for this neurotoxin. Here we report results of toxin analy-
ses and gut content examinations of krill Euphausia pacifica collected from Monterey Bay in 2000.
Corresponding counts of toxic Pseudo-nitzschia species in the water and their cellular DA concen-
trations were also obtained at the collection sites. Toxin analysis by receptor binding assay demon-
strated that DA in krill tissue varied between 0.1 to 44 µg DA equiv. g
–1
tissue (confirmed by tandem
mass spectrometry), with levels corresponding to the abundance of toxic Pseudo-nitzschia species
present in the water. The occurrence of Pseudo-nitzschia australis frustules in the digestive tract of E.
pacifica verified that a toxic species of this diatom was an important part of their diet and thus impli-
cated this phytoplankter as the source of DA. These findings provide compelling evidence for the role
of krill as a potential transfer agent of the phycotoxin DA to higher trophic levels in marine food web.
KEY WORDS: Krill · Pseudo-nitzschia · Domoic acid · Harmful algal blooms · Toxic algae · Trophic
transfer · Food webs
Resale or republication not permitted without written consent of the publisher

Mar Ecol Prog Ser 237: 209–216, 2002
toxigenic species of the diatom Pseudo-nitzschia to sea
lions and seabirds. The first major event occurred
in 1991, when more than 200 brown pelicans and
Brandt’s cormorants were found dead on the beaches
of Monterey Bay (Fritz et al. 1992, Work et al. 1993).
Similarly, Monterey Bay was the site of another major
DA poisoning incident in 1998, when sea lions were
observed dying on the beaches suffering from neuro-
logical symptoms (Lefebvre et al. 1999, Scholin et al.
2000). In all DA poisoning events in this region,
Pseudo-nitzschia australis was determined to be the
primary source of the toxin, and filter-feeding anchovies
Engraulis mordax the apparent vector. Anchovies are
not the only potential filter-feeding vectors of DA in the
Monterey Bay system. For example, zooplankton are
generally key consumers of phytoplankton. Nonethe-
less, while the link between zooplankton and toxic
dinoflagellates has been confirmed (e.g. White 1981,
Huntley et al. 1986, Ives 1987, McClatchie 1988, Uye
&Takamatsu 1990, Turriff et al. 1995, Bagoien et al.
1996, Teegarden & Cembella 1996, Shaw et al. 1997,
Turner & Tester 1997, Tester et al. 2000, Turner et al.
2000), there has been less attention focused on toxin
transfer between toxic diatoms and zooplankton
grazers (Windust 1992, Tester et al. 2001, Lincoln et al.
2001).
Euphausiids (krill) are important members of the
zooplankton grazer community in the world’s oceans.
As filter feeders, they directly link the base of the food
chain to higher trophic levels. They can be consumed
by large predators because of their relatively large
size. This intermediary position in the pelagic food
chain gives them a potentially critical role in toxin
transfer. Euphausia pacifica and Thysanoessa spini-
fera are the most abundant euphausiids in the Califor-
nia Current system and are important indicators of
oceanic and neritic water masses in the North Pacific
Ocean, respectively (Ponomareva 1966, Schoenherr
1991). They are a common prey for many marine
organisms ranging from fish to whales. Pelagic fishes
such as Pacific herring, salmon and the Pacific hake
(Yamamura et al. 1998), and seabirds such as Cassin’s
auklet and ashy storm petrel (Ainley et al. 1996) can
feed heavily on krill in Monterey Bay. In addition to
pelagic predators, demersal fish such as Pacific cod
and rockfish feed directly on E. pacifica, which is found
normally at greater depths during the day (Chess et al.
1988). Because krill can be found both near the surface
and at depth, they can potentially introduce DA into
both shallow and deep-dwelling predators. Addition-
ally, krill fecal pellets sink at high rates (Komar et
al. 1981), and could rapidly convey toxin-containing
material to deep water and the benthos.
To better evaluate and understand the role of krill as
a DA vector, we collected krill and water samples in
Monterey Bay throughout 2000. Additionally, we mea-
sured DA in krill and examined their digestive tract for
toxic Pseudo-nitzschia species. Our results present
clear evidence of the role of krill as a potential vector
for the neurotoxin DA.
MATERIALS AND METHODS
Sample collection. Samples of the euphausiid Eu-
phausia pacifica were collected opportunistically be-
tween March and August 2000 offshore of Monterey
Bay, CA, USA, using 0.7 m BONGO nets fitted with
333 µm mesh (Ocean Instruments). Samples were
frozen immediately after collection, transferred to the
UCSC laboratory packed on ice, and stored at –20°C
until analyzed for the presence of toxic Pseudo-
nitzschia spp. in gut contents by scanning electron
microscopy (SEM) and for the presence of DA by
receptor binding assay. For DA analyses, samples
were split and sent via overnight carrier on dry ice to
the NOAA/NOS Charleston Laboratory. Water sam-
ples were also obtained to identify toxic Pseudo-
nitzschia spp. co-occurring with krill, as well as to
determine their cell numbers and cellular DA toxic-
ity. Approximately 15 ml aliquots of water were pre-
served in 10% Lugol’s (10% KI, 5% I
2
and 10%
glacial acetic acid in 100 ml deionized water) for
identification of toxic Pseudo-nitzschia species pre-
sent and confirmation by SEM. Another 15 to 30 ml
of water was collected for cell enumeration using
rRNA probes. For cellular DA determinations, sam-
ples of 500 ml water were filtered through 25 mm
Whatman GF/F filters, immediately frozen, trans-
ferred to the UCSC laboratory packed on ice, and
stored at –80°C until analysis. A summary of both
krill and water samples is given in Fig. 1 with loca-
tion and collection dates plotted on a map of Mon-
terey Bay.
Krill preparation for DA toxin analysis. Homo-
genization and extraction: Whole krill were used in
the extraction process, because predators consume the
entire organism. A minimum of 25 individual 15 to
28 mm specimens (equiv. to 2 g wet wt) from each col-
lection date was placed into a 50 ml conical tube. An
equal weight of Milli-Q water (Millipore) was added
and blended using a polytron (PowerGen 700; Fisher
Scientific) equipped with 10 mm × 195 mm sawtooth
generator probe at 10 000 rpm until the krill became a
smooth homogenate (ca. 2 min).
DA was extracted from krill according to the method
of Quilliam et al. (1995) with modifications as de-
scribed by Powell et al. (2002). Krill extracts were
cleaned using a strong anion exchange (SAX)-solid
phase extraction as described by Hatfield et al. (1994).
210

Bargu et al.: Krill as vector for domoic acid in marine food webs
DA extraction efficiency from krill: The DA extrac-
tion efficiency for krill was tested by performing spike-
recovery experiments. Control krill, Thysanoessa
spinifera, obtained from Monterey Bay during non-
bloom periods, were used and verified by receptor-
binding assay to contain <0.01 µg DA equiv. g
–1
tissue
following the protocol described above. Subsequently,
individual krill were injected with a known amount of
DACS-1C, a certified DA reference standard (Institute
for Marine Biosciences, National Research Council of
Canada, NS, Canada), into the ventral side of each krill
abdomen with 4 replicate samples of 25 individuals
each. An additional recovery test was performed by
spiking 4 replicate krill homogenates with DACS-1C
standard. Extracts were analyzed by receptor binding
assay and percent recoveries determined.
DA toxin analysis of krill using receptor binding
assay. Prior to testing on the assay, naturally occurring
glutamate was removed from SAX-cleaned krill
extracts by incubating 50 µl of sample with 40 µl citrate
buffer (10 mM citrate, pH 5.0; 2 mM pyridoxal 5-phos-
phate; 200 mM NaCl) and 10 µl glutamate decarboxy-
lase (100 units ml
–1
in 10 mM citrate, pH 5.0) for 30 min
at 4°C. Receptor assays were performed in 96-well
microtiter filtration plates (Millipore) and employed a
cloned rat GluR6 glutamate receptor expressed in SF9
insect cells using a bacculovirus expression system
(Van Dolah et al. 1997). The endpoint of this competi-
tive binding assay was determined by microplate scin-
tillation counting (Microbeta 1450; Wallac). Additional
details of the domoic acid receptor binding assay are
provided in Lefebvre et al. (1999).
LC-MS/MS for DA. Selected krill extracts were ana-
lyzed by liquid chromatography coupled with tandem
mass spectrometric detection (LC-MS/MS) for the
absolute confirmation of DA. Liquid chromatographic
separation was performed on a Vydac 201TP52 C
18
col-
umn (2.1 mm × 250 mm; Separations Group) with an
elution gradient of 1 to 95 % aqueous MeOH in 0.1%
trifluoroacetic acid (TFA) run over 35 min at a flow rate
of 0.2 ml min
–1
. Following separation, the eluent was
introduced into a PE SCIEX API-III triple quadrupole
mass spectrometer (SCIEX Instruments) operating in
positive ion mode and using compressed air as the
nebulization gas. Confirmation of DA was by selective
ion monitoring based on the MS/MS fragmentation
pattern for this toxin given by Quilliam (1996), includ-
ing the parent ion (312 m/z) and 2 diagnostic fragment
ions produced by collisionally induced dissociation
(161 and 266 m/z).
DA toxin analysis of water samples. Cellular DA
concentrations in microalgae were determined using
the FMOC-HPLC method described by Pocklington et
al. (1990), which involves pre-column derivatization of
DA in cell extracts with 9-fluorenylmethylchlorofor-
mate (FMOC) reagent to form the fluorescent FMOC-
derivative of DA. Prior to analysis, particulate samples
collected on Whatman GF/F filters were extracted in
2.5 ml 10% aqueous MeOH. For derivatization, 200 µl
of extract were vortexed with 50 µl 1 M borate buffer
(pH 6.2), 10 µl dihydrokainic acid (DHKA) internal
standard (100 µg ml
–1
DHKA in 10% aqueous MeOH)
for 10 s, and then 250 µl FMOC-Cl reagent were added
and vortexed again for 45 s. Three ethyl acetate
washes were performed and the aqueous layer was
removed for HPLC analysis. A 5 µl sample was injected
into a Hewlett-Packard HP1090M HPLC equipped with
an HP1046A fluorescence detector set for excitation at
264 nm and emission at 313 nm with a mobile phase
flow rate of 0.2 ml min
–1
comprised of 40% aqueous
MeCN, 0.1% TFA. Isocratic separations were per-
formed on a reverse phase C
18
column (2.1 mm ×
25 mm, Vydac 201TP52; Separations Group) heated to
40°C. A calibration curve of 5, 10, 25, 50, 100 and
250 ng ml
–1
in 10% aqueous MeOH was generated
using the DACS-1C standard (r
2
= 0.99).
Toxic Pseudo-nitzschia cell counts. Water column
abundance of toxic Pseudo-nitzschia australis and P.
multiseries was determined using whole cell hybrid-
ization with species-specific large subunit (LSU)
rRNA-targeted fluorescent probes (Miller & Scholin
1996). Between 5 and 30 ml aliquots of seawater were
filtered onto 1.2 µm isopore polycarbonate filters (Mil-
lipore) and preserved with a saline ethanol solution for
at least 1 h. After rinsing with hybridization buffer
(5× SET [
1
⁄
5
dilution of 3.75 M NaCl, 25 mM EDTA,
0.5 M Tris HCl, pH 7.8], 0.1% [v/v] IGEPAL-CA630
211
Fig. 1. Map of Monterey Bay, CA, USA, showing sites of krill
and water sample collections. Positions of numbered study sites
indicate collection dates (given as mm/dd/yy) and locations of
krill and water samples: 1 (03/22/00, 122° 02.18’ W, 36° 46.26’ N),
2 (05/15/00, 121° 51.69’ W, 36° 46.78’ N), 3 (07/03/00, 122° 00’ W,
36° 47.79’ N), 4 (08/07/00, 121°58.37’ W, 36°47.82’ N), 5
(08/24/00, 121° 57.4’ W, 36° 31.3’ N), 6 (08/27/00, 122° 30.25’ W,
36° 53.72’ N), 7 (08/29/00, 122°22.30’ W, 36°39.81’ N)
37°
00’’
–122°
1
2
3
4
5
6
7
km
0 5 10
36°
45’’
36°
30’’

Mar Ecol Prog Ser 237: 209–216, 2002
[Sigma], 25 µg ml
–1
polyadenylic acid [Sigma]), each
sample was incubated with species-specific probes for
P. australis, P. multiseries, a positive eukaryote control
probe and a negative control probe designed for the
dinoflagellate Alexandrium tamarense. A third control
consisted of a sample with no probe added. After 1 h,
filters were rinsed and placed on microscope slides.
Intact cells that retained the fluorescein labeled probe
were then counted on a Zeiss Standard 18 compound
microscope, equipped with epifluorescence (micro-
scope illuminator 100, Zeiss). The entire area of each
filter was counted. Results are given in cells l
–1
.
Scanning electron microscopy. Scanning electron
microscopy (SEM) was used to verify the species com-
position of Pseudo-nitzschia in krill digestive tracts
and seawater using methods slightly modified from
Miller & Scholin (1998). The gut contents of Euphausia
pacifica were dissected from specimens under a
dissecting microscope, removed from the body, and
gently discharged into scintillation vials. All the gut
samples were preserved in 5% buffered formalin
solution until being processed for SEM.
Dissected krill gut contents and water samples
(10 ml) were concentrated onto 1.2 µm pore size iso-
pore polycarbonate membranes (Millipore). Salt was
removed from samples by rinsing with DI water under
low vacuum pressure (150 mm Hg). To remove organic
material, saturated KMnO
4
was added until the filters
were covered and allowed to digest the samples for
15 min (krill gut contents) or 5 min (water). 12 N HCl
(3 ml) was then added to the water samples until the
color became clear. Krill gut contents were held for
30 min in 3 ml of 12 N HCl to complete the oxidation
process. Samples were then vacuumed gently and
rinsed with DI water. This process was repeated 2
times, and filters removed from the filter tubes and
fixed to aluminum stubs. Filters were air-dried in a
dessicator for 24 h and then mounted onto SEM stubs
with double-sided tape and sputter coated with gold
palladium. All micrographs were taken with an ISI
WB-6 electron microscope at 10 kV.
RESULTS
Protocol development
The extraction methods used for domoic acid (DA)
detection in animals can be adjusted depending on the
size and physical structure of the animal. Since krill is
a new matrix for DA measurement, testing the effi-
ciency of the toxin extraction protocol was essential
before running any field samples. Therefore, we con-
ducted a spike-recovery test on control krill, Thysano-
essa spinifera, samples obtained from Monterey Bay
during non-bloom periods, verified by receptor bind-
ing assay to contain <0.01 µg DA equiv. g
–1
tissue.
Samples of krill injected individually with a known
amount of DA yielded a recovery of 98 ± 7.8%, while
the addition of DA to previously homogenized animals
gave a recovery of 99 ± 10.4%. Both treatments
showed very high recovery for DA. Although differ-
ences between the 2 methods were not significant (p >
0.05, t-test), injection of individual animals may better
represent natural conditions.
Field krill samples
DA in krill Euphausia pacifica collected from Mon-
terey Bay between March and August 2000 ranged
from 0.1 to 44 µg DA equiv. g
–1
tissue, as determined
by receptor binding assay. DA content of krill varied
according to the abundance of toxic Pseudo-nitzschia
species in the water (Table 1), as demonstrated by a
highly significant, positive correlation between these 2
212
Table 1. Field measurements of domoic acid in krill (n = 28 to 56, µg DA equiv. g
–1
tissue) determined by receptor binding assay.
In addition, values as shown for the corresponding abundance of toxic Pseudo-nitzschia (cells l
–1
) obtained using species-specific
LSU rRNA-targeted fluorescent probes and for DA concentration per cell (pg DA equiv. cell
–1
) determined by HPLC-
FMOC. Both krill and water samples were obtained from Monterey Bay, CA, between March and August 2000
Sample collection 10
5
toxic Pseudo-nitzschia cells l
–1
Cellular DA DA in krill
dates (mm/dd/yy) (P. australis + P. multiseries) (pg DA equiv. cell
–1
(µg DA equiv. g
–1
Pseudo-nitzschia) tissue)
03/22/00 0.68 3 0.27
05/15/00 0 0 <LD
a
07/03/00 0 0 <LD
a
08/07/00 0.54 14 0.34
08/24/00 10 24 44
08/27/00 0.0046 11 0.10
08/29/00 0.31 16 0.31
a
<LD = samples are below the limits of detection for receptor binding assay (<0.01 µg DA equiv. g
–1
)

Bargu et al.: Krill as vector for domoic acid in marine food webs
variables (p << 0.01, r
2
= 0.998, Pearson Product
Moment Correlation). The highest DA concentra-
tion in krill of 44 µg DA equiv. g
–1
was obtained
during the August 2000 bloom event when P. aus-
tralis reached 10
6
cells l
–1
. Confirmation of DA in
this particular sample was obtained by tandem
mass spectrometry, which identified the parent
ion of DA (312 m/z) as well as its 2 diagnostic
daughter ions (161 and 266 m/z) (Fig. 2). In addi-
tion to this sample, all other krill samples yielding
positive DA-like activity on the receptor binding
assay (see Table 1) were later confirmed by mass
spectrometry to contain DA.
SEM analysis demonstrated that stomach con-
tents of Euphausia pacifica were dominated by
toxic Pseudo-nitzschia spp. when their cell num-
bers exceeded 10
5
cells l
–1
, but in the presence of
lower cell numbers, assemblages of diatom frus-
tules were mixed. E. pacifica shown to contain
the highest concentrations of DA were found to
have digestive tract contents dominated by Pseudo-
nitzschia australis, thus implicating this phytoplankter
as the most likely source of the toxin (Fig. 3). In con-
trast, when DA in E. pacifica was found in the range of
0.3 µg DA equiv. g
–1
tissue, concentrations of toxic
Pseudo-nitzschia spp. in the water were low and P.
australis occurred in more mixed assemblages with
small numbers of P. multiseries, another toxin-produc-
ing species as well as non-toxic diatom species, includ-
ing P. pseudodelicatissima and P. pungens, Thalas-
siosira sp. cf. minima Gaarder, Skeletonema costatum,
and Chaetoceros sp. Finally, no Pseudo-nitzschia frus-
tules were observed in the stomachs of E. pacifica
when toxic Pseudo-nitzschia were absent from the
water, and correspondingly no DA was detected in
either water or krill samples.
DISCUSSION
Food web contamination by DA may be widespread
during blooms of toxic Pseudo-nitzschia species. After
the first DA poisoning event in Monterey Bay, CA, in
1991, with northern anchovies Engraulis mordax sus-
pected as the DA vector, researchers identified addi-
tional DA vectors on the US West Coast: mussels
Mytilus californianus, razor clams Siliqua patula, dun-
geness crabs Cancer magister, and mole crabs Emerita
analoga (Loscutoff 1992, Wohlgeschaffen et al. 1992,
Langlois et al. 1993, Horner & Postel 1993, Lund et al.
1997, Lefebvre et al. 1999). Very few studies, however,
have focused on DA in zooplankton, specifically on
copepods or euphausiids, and these 2 are the dominant
phytophagous macrozooplankton groups in the Mon-
terey Bay area. Both are efficient filter feeders of
diatoms and comprise most of the zooplankton biomass
in pelagic ecosystems, implicating them as potentially
effective vectors for DA. Copepods have been found to
accumulate DA in earlier studies (Windust 1992, Lin-
coln et al. 2001, Tester et al. 2001). The only previous
213
Fig. 2. LC-MS/MS chromatogram of krill extract taken from
Monterey Bay, CA, on August 24, 2000, during Pseudo-
nitzschia australis bloom event. The bottom chromatogram
establishes the presence of the DA parent ion (312 m/z) and
the top panels show 2 diagnostic daughter ion fragments (161
and 266 m/z) according to Quilliam et al. (1996)
Fig. 3. (A) Scanning electron micrograph of DA-contaminated krill
gut containing Pseudo-nitzschia australis frustules. (B) Higher
magnification view of one of the frustules from (A) Arrow indicates
the presence of 2 rows of large poroids within the striae. A central
nodulus is absent. (C) Scanning electron micrograph of several P.
australis frustules from water samples. Both krill and water sam-
ples were obtained during the August 2000 P. australis bloom
event in Monterey Bay, CA

Mar Ecol Prog Ser 237: 209–216, 2002
report implicating krill as a DA vector is our own pre-
liminary field observation showing that the digestive
tract of krill contains both toxic and non-toxic Pseudo-
nitzschia spp. during blooms of these diatoms (Bargu &
Silver in press). However, we could not perform toxin
measurements on krill at the time due to technical dif-
ficulties. Therefore the present study is the first report
confirming DA in krill during a bloom of toxin-produc-
ing diatom Pseudo-nitzschia.
DA concentrations in krill varied depending on the
densities of toxic Pseudo-nitzschia cells in the water.
Similar results were found for filter-feeding mussels
(Silvert & Rao 1991). The maximum level of DA in
krill for this study was 44 µg DA equiv. g
–1
tissue (3 µg
DA equiv. krill
–1
), somewhat higher than that found in
dungeness crab (average 13 µg DA g
–1
) and mole
crabs (5 µg DA g
–1
) on the US West Coast (Wekell et
al. 1994, Powell et al. 2002). Since the highest DA
concentration measurement for krill exceeded the
upper limit allowed for human consumption (i.e. 20 µg
DA g
–1
for shellfish), toxin levels in krill may also
represent a danger for marine mammals that feed on
krill.
Since DA was measured in Euphausia pacifica only
when toxic Pseudo-nitzschia spp. were present in the
water column, we considered the possibility that krill
do not retain and distribute the toxins in their tissues
and organs but rather have toxin only pass in mater-
ial within the gut. Because we did not separate the
stomach contents from the body for toxin analyses,
we could not test for the partitioning of DA into the
various organs and tissues. Instead, we calculated
whether it was possible that all of the measured DA
in an individual krill came from P. australis material
present in the krill’s digestive tract. Considering the
highest DA content per P. australis cell during the
bloom was 24 pg cell
–1
(see Table 1), the 44 µg DA
equiv. g
–1
tissue means that a typical 70 mg E. paci-
fica (wet wt) would contain approximately 10
5
cells.
With cell concentrations of 10
6
P. australis cells l
–1
at
the site with highest measured cell concentration (see
Table 1), the DA measured in E. pacifica suggests
that these animals were retaining cells removed from
approximately 100 ml of seawater. Since filtering
rates of E. pacifica have been measured at ~150 ml
h
–1
(S.B. unpubl. data) and gut residency time mea-
sured at ~2 h (S.B. unpubl. data) such gut concentra-
tions of P. australis in krill appear reasonable. The
results of these calculations suggest that krill are
retaining (i.e. assimilating) relatively little, if any,
toxin from their food source. Thus, the high toxin lev-
els found in individual specimens suggest that krill
may serve as vectors only when toxic cells are in their
digestive tract. Furthermore, the relationship be-
tween body burden of DA in field-collected krill and
DA in Pseudo-nitzschia available in the water (Table
1) corroborates this hypothesis. Nevertheless, con-
trolled laboratory measurements of krill feeding and
depuration are clearly needed for confirmation. For
example, filtering rates may decline in the presence
of toxic species, and the high toxin levels in krill
could therefore represent retention from a previous
exposure to the toxin.
The link from phytoplankton to euphausiids to blue
whales is possibly the shortest food chain involving a
large marine mammal. A single blue whale can con-
sume up to 2 tons of krill each day (D. Croll pers.
comm.), the equivalent of 20 million krill. Thus, krill
could convey DA levels up to 62 g to a blue whale
d
–1
, based on our maximum DA concentration mea-
sured in krill. This amount of DA would yield a dose
of 0.62 mg kg
–1
for an average blue whale weighing
100 000 kg, less than one-sixth of the LD
50
reported
for mice at 3.8 mg kg
–1
(Iverson et al. 1989). Although
this dosage is far less than what is considered to be
lethal for mice, whales may have physiological factors
that make them more susceptible to lower doses of
DA. In 1987, 14 humpback whales died in Cape Cod,
MA, USA, after consuming Atlantic mackerel con-
taining saxitoxin (STX), a neurotoxin produced by
dinoflagellates responsible for paralytic shellfish poi-
soning (Geraci et al. 1989). The calculated dosage of
STX believed to be delivered to these humpbacks
was 3.2 µg per kg of body weight, several times less
than what has been reported as the lethal oral dose
for humans. Nonetheless, the authors suggested that
the mammalian diving adaptation, which channels
blood (and thus toxin) to the heart and brain and
away from organs involved in detoxification (i.e. kid-
ney and liver), rendered the whales more susceptible
than usual to STX contained in the mackerel. A simi-
lar scenario is clearly possible with DA exposures in
other cetaceans.
To date, assessment of the effect of toxic Pseudo-
nitzschia blooms on higher-level consumers has
depended on evidence from beach strandings. Un-
doubtedly, the consequences for consumers living off-
shore are difficult to measure, since considerable mor-
tality may go unnoticed. Krill are common in both
neritic and oceanic environments of Monterey Bay,
and play a key role in the food chain as a link between
phytoplankton and large predators such as baleen
whales and seabirds. Our results demonstrate that dur-
ing toxic Pseudo-nitzschia blooms, krill accumulate
DA and thus may act as an effective vector for the
direct transfer of DA to higher trophic levels. However,
there is still much to learn about toxin transmission by
krill, including whether DA affects krill behavior or
mortality, before the role of krill is better understood
in these events.
214

Bargu et al.: Krill as vector for domoic acid in marine food webs
Acknowledgements. We thank Dr. Baldo B. Marinovic, Dr.
Christopher A. Scholin, Roman Marin and Dr. Francisco P.
Chavez for their help on krill sample collections. We are
grateful to Dr. Jonathan M. Krupp for his assistance with the
scanning electron microscopy and Dr. Greta A. Fryxell for
identifying Thalassiosira sp. cf. minima Gaarder. We also
thank Dr. Don Croll for his valuable insights on whale feeding
behavior. This study was supported by NOAA Award no.
NA960P0476 (ECOHAB) to M.W.S. and by NOAA Coastal
Ocean Program project no. ECO 99-184 to G.J.D. Funding
was also provided by the Dr. Earl Myers and Ethel Myers
Oceanographic and Marine Biology Trust to S.B.
Disclaimer: The National Ocean Service (NOS) does not
approve, recommend, or endorse any product or material
mentioned in this publication. No reference shall be made to
NOS, or to this publication furnished by NOS, in any adver-
tising or sales promotion which would indicate or imply that
NOS approves, recommends, or endorses any product or
material mentioned herein or which has as its purpose any
intent to cause directly or indirectly the advertised product to
be used or purchased because of NOS publication.
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216
Editorial responsibility: Michael Landry (Contributing Editor),
Honolulu, Hawaii, USA
Submitted: August 13, 2001; Accepted: January 10, 2002
Proofs received from author(s): July 9, 2002