C Water

Toxic diatoms and domoic acid in natural and iron

enriched waters of the oceanic Paci c

Mary W. Silvera,1, Sibel Bargub, Susan L. Coalea, Claudia R. Benitez-Nelsonc, Ana C. Garciab, Kathryn J. Robertsa,

Emily Sekula-Woodc, Kenneth W. Brulanda, and Kenneth H. Coaled

a

Institute of Marine Sciences, University of California, Santa Cruz, CA 95064; bDepartment of Oceanography and Coastal Sciences, School of the Coast and

Environment, Louisiana State University, Baton Rouge, LA 70803; cMarine Science Program and Department of Earth and Ocean Sciences, University of South

Carolina, Columbia, SC 29208; and dMoss Landing Marine Laboratories, Moss Landing, CA 95039

Edited by Francois M. M. Morel, Princeton University, Princeton, NJ, and approved October 19, 2010 (received for review May 18, 2010)

DA L 1 levels occurring during blooms (11, 12). In contrast, oce-

Near-surface waters ranging from the Paci c subarctic (58 N) to the

anic Pseudo-nitzschia cells are typically smaller and, hence, likely

Southern Ocean (66 S) contain the neurotoxin domoic acid (DA),

contain less DA; for example, a recent study shows the small cos-

associated with the diatom Pseudo-nitzschia. Of the 35 stations

mopolitan P. turgidula from an oceanic NE Paci c site to contain

sampled, including ones from historic iron fertilization experiments

0.1 fg of DA cell 1 in unamended growth containers with local

(SOFeX, IronEx II), we found Pseudo-nitzschia at 34 stations and DA

measurable at 14 of the 26 stations analyzed for DA. Toxin ranged water (13).

from 0.3 fg cell 1 to 2 pg cell 1, comparable with levels found in The special responsiveness of Pseudo-nitzschia to iron additions

similar-sized cells from coastal waters. In the western subarctic, is well known and recently has been associated with their un-

descent of intact Pseudo-nitzschia likely delivered signi cant common ability to store this trace metal (14). This responsiveness

amounts of toxin (up to 4 g of DA m 2 d 1) to underlying meso- is of particular concern because iron fertilization has been pro-

ENVIRONMENTAL

pelagic waters (150 500 m). By reexamining phytoplankton sam- posed in oceanic, high nutrient, low chlorophyll (HNLC) waters

to reduce atmospheric CO2 and moderate ocean acidi cation

SCIENCES

ples from SOFeX and IronEx II, we found substantial amounts of DA

through enhanced carbon xation by phytoplankton (15). Reports

associated with Pseudo-nitzschia. Indeed, at SOFeX in the Antarctic

Paci c, DA reached 220 ng L 1, levels at which animal mortalities of DA from HNLC or truly oceanic waters, either from water

have occurred on continental shelves. Iron ocean fertilization also samples or clonal cultures, are rare: Field grow outs of oceanic

occurs naturally and may have promoted blooms of these ubiqui- P. turgidula, mentioned above, contain DA in situ (13), whereas

tous algae over previous glacial cycles during deposition of iron- earlier studies at the site did not nd measurable DA in that same

rich aerosols. Thus, the neurotoxin DA occurs both in coastal and species (16).

oceanic waters, and its concentration, associated with changes in Here, we address the possible presence of DA in oceanic waters

Pseudo-nitzschia abundance, likely varies naturally with climate at various sites in the Paci c and re-examine archived samples

cycles, as well as with arti cial iron fertilization. Given that iron from mesoscale iron-enrichment experiments for which one of us

fertilization in iron-depleted regions of the sea has been proposed (K.H.C.) was expedition leader.

to enhance phytoplankton growth and, thereby, both reduce at-

mospheric CO2 and moderate ocean acidi cation in surface waters, Results

consideration of the potentially serious ecosystem impacts associ- Station Data and Pseudo-nitzschia Abundance. This study reports

ated with DA is prudent. results from 35 water samples obtained on ve ocean expeditions

in the Paci c (Fig. 1 and Fig. S1). Data are summarized in Table 1

toxicity harmful algal blooms

carbon sequestration

and Table S1, including station positions, oceanic regions, Pseudo-

nitzschia abundance, DA, and nutrients (iron and nitrate) levels.

T he increasing frequency and geographic extent of toxic algal

blooms along populated coastlines is generally attributed to Domoic Acid in Cells and Water. DA was detected in 14 of 26

samples analyzed for DA by cELISA (Table S1). DA was further

human activities (1, 2). Pseudo-nitzschia, a ubiquitous genus of

veri ed, structurally, in a sample collected from IronEx II by using

marine and estuarine diatom, is a relatively recently discovered

contributor to such events and includes 30 species ranging from LC-MS/MS (Methods). Cell toxin quotas, estimated by dividing

tropical to polar waters (3 5). Approximately a dozen Pseudo- cell counts of Pseudo-nitzschia by the particulate DA concentra-

tion (i.e., assumes all species/cells have equal DA levels), were 0.3

nitzschia species are known to produce the amino acid neurotoxin

fg cell 1 to 0.16 pg cell 1 at eastern subarctic stations in the Gulf

domoic acid (DA) (5, 6). Generally, humans discover phytoplank-

of Alaska, whereas those at VERTIGO K2 were 0.4 1.9 pg of

ton toxins, including DA, when consuming contaminated shell sh

DA cell 1 (Table 1). In iron-enrichment studies at SOFeX South,

harvested from coastal waters. In contrast, algal toxins have yet to

DA ranged from 0.7 to 1.0 pg of DA cell 1; DA in the water

be detected in commercial oceanic products (e.g., ocean sh har-

reached 220 ng L 1, the highest level observed in this study (Table

vested for human consumption), leading to the view that toxin-

1). IronEx II samples, collected 12 y before our analyses, had

producing algae occur only in coastal areas, not in truly oceanic

Pseudo-nitzschia toxin quotas of 0.02 0.05 pg cell 1, an order of

waters. In the case of Pseudo-nitzschia, however, some researchers

suggest that all species will be shown to produce DA when tested

with suf ciently sensitive methods (7). Pseudo-nitzschia spp. are

implicated in coastal harmful algal blooms (HABs) worldwide (5, 6) Author contributions: M.W.S., S.B., and S.L.C. designed research; M.W.S., S.B., S.L.C., A.C.G.,

K.J.R., E.S.-W., and K.H.C. performed research; C.R.B.-N., A.C.G., K.W.B., and K.H.C.

and are recognized as contaminating a wide range of animals contributed new reagents/analytic tools; M.W.S., S.B., S.L.C., C.R.B.-N., A.C.G., K.J.R.,

from invertebrates to marine birds and mammals (8 10). E.S.-W., and K.H.C. analyzed data; and M.W.S., S.B., S.L.C., C.R.B.-N., K.J.R., and K.H.C.

With the cosmopolitan nature of many Pseudo-nitzschia (4), and wrote the paper.

its toxin-producing ability well known in continental margin and The authors declare no con ict of interest.

shelf regions, the concern becomes whether DA is present in truly This article is a PNAS Direct Submission.

oceanic and coastal waters. Coastal populations with particularly 1

To whom correspondence should be addressed. E-mail: abqc7c@r.postjobfree.com.

high DA cell quotas (pg of DA cell 1) and cell numbers >104 L 1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

can be associated with animal mortalities, with 102 to 104 ng of 1073/pnas.100*******/-/DCSupplemental.

PNAS Early Edition 1 of 6

www.pnas.org/cgi/doi/10.1073/pnas.100*******

DA L 1 are shown in Tables 1 and 2 and Table S1. There was

a highly signi cant correlation between DA levels and Pseudo-

nitzschia abundance at the Paci c sites, indicating waters with

high concentrations of this diatom genus typically will have

associated high levels of the associated neurotoxin (P

n = 25; Fig. S2).

Pseudo-nitzschia Species. Pseudo-nitzschia was observed at 34 of 35

stations in this study, with 11 species of Pseudo-nitzschia identi ed

by electron microscopy from 12 different stations (Table 2 and

Table S2). Samples usually contained mixed populations of small

oceanic and cosmopolitan Pseudo-nitzschia species. P. turgidula

occurred in six samples, ve of which contained measureable DA,

and was the overwhelming dominant in two of the three samples

with DA levels 0.8 pg cell 1. In samples in which only a single

Pseudo-nitzschia species was observed and toxin was detected, we

attribute toxin quotas to that species: for P.cf turgidula, 0.4 fg 1.9

pg DA cell 1, for P. lineola 0.7 pg cell 1, with the latter implicated

as a possible toxin producer for the rst time.

Fig. 1. Sites of ve major ocean research cruises from which samples were

Daily Sinking Rates of Pseudo-nitzschia Cells. Intact cells (ones with

obtained for measurements of domoic acid (DA) and/or Pseudo-nitzschia

protoplasts) that were sinking into the mesopelagic zone from the

abundance. Samples from GOA-Gulf of Alaska (2007), VERTIGO K2 (2005),

and VERTIGO ALOHA (2004) represent natural oceanic waters (i.e., seaward upper ocean were also collected by using sediment traps on

of the continental shelf in 200m of water), whereas SOFeX (2002) and

VERTIGO cruises. Daily uxes (cells m 2 d 1) at 150 m repre-

IronEx II (1996) samples were obtained from blooms promoted by the arti-

sented from 0.2 to 1% of the overlying euphotic zone Pseudo-

cial addition of Fe, also in deep water ( 1,000m) oceanic regions. The

nitzschia stocks at K2 and from 0.02 0.04% at the more oligotro-

number of stations sampled at each site is given in parentheses. See Table S1

phic ALOHA station (Table 3). In one deployment (VERTIGO

for station locations, geographic descriptions, DA concentrations in cells and

K2D2), sinking cells at 300 and 500 m exceeded those leaving the

water, and Pseudo-nitzschia abundances.

euphotic zone by >10-fold, a phenomenon expected at the end of

a bloom when deeper cells re ect the larger surface populations

magnitude lower (possibly related to sample age: see Methods). from earlier bloom phases (e.g., K2D1).

Pseudo-nitzschia numbers, however, were as high as 106 cells L 1,

and toxin levels reached 45 ng of DA L 1. The highest cell toxin Discussion

levels were encountered at VERTIGO K2 and the second Oceanic Distribution of Pseudo-nitzschia and DA. Our results show

highest at SOFeX. The resulting values for DA cell 1 and that oceanic waters throughout the Paci c contain Pseudo-nitzschia

Table 1. Pseudo-nitzschia abundance, DA cell quotas, and estimated DA concentrations in the waters of six oceanic regions

of the Paci c

Cruise

Measurement Gulf of Alaska VERTIGO ALOHA VERTIGO K2 IronEx II SOFeX (N) SOFeX (S)

Location (no. of NE Subarctic Subtropical NW Subarctic Equatorial Sub-Antarctic Antarctic

Paci c (18) Paci c (7) Paci c (4) Paci c (2) Paci c (1) Paci c (3)

stations sampled)

8.6 103 9.8 101 3.4 103 3.7 1.3 106 1.3 3.7 105 7.5 104

Pseudo-nitzschia

1.3 104 (n = 14) 2.5 102 (n = 14) 103 (n = 11) 106 (n = 2) 1.3 106 (n = 3)

abundance, (n = 1)

cells L 1

b.d.l./4.2 104 b.d.l./9.4 102 7.6 100 3.5 105 to 2.0 103 to

Range Not applicable

to 1.3 104 2.3 106 2.2 105

0.03 0.07 (n = 6) 0.9 0.7 (n = 4) 0.04 0.02 (n = 2) 0.85 0.23 (n = 2)

DA cell quota m.d. m.d.

pg cell 1

0.0003 0.16 0.4 1.9 0.02 0.05 0.69 1.0

Range m.d. m.d.

0.0002 1.4 1.7 3.6 18 45 2.0 220

DA in water m.d. m.d.

range, ng L 1

Average Pseudo-nitzschia abundances, cellular domoic acid (DA) quotas, and water levels of DA are presented here for 35 oceanic stations sampled over 12 y

on ve different research cruises. Ranges are also presented. The designation b.d.l. indicates DA was below the detection limit, which could re ect either the spar-

city of the cells in the water or the possibility that many other species were present in the sample, hence making the contribution of DA, even in cells with reasonably

high DA levels, a small fraction of the total material in that sample and, thus, below the detection limit. Missing data are indicated by m.d. Cell quota averages

are based on samples where DA was detected (i.e., measurements that were below our detections limits were not included in the computation of the average)

(Table S1, legend). It was assumed all species/cells have equal DA levels. DA in the water was then computed by multiplying the cell abundance by the DA cell quotas

(at stations where DA was detected). Values represent means 1 SD and sample replicates (n) appear in parentheses. Of 35 stations sampled, Pseudo-nitzschia was

present at 34 stations. Samples from 26 stations were analyzed for DA with detectable levels found at 14 stations (see Table S1 for complete data set).

2 of 6 www.pnas.org/cgi/doi/10.1073/pnas.100******* Silver et al.

Table 2. Pseudo-nitzschia species and toxin levels for samples from oceanic Paci c locations in this study

Domoic acid, pg cell 1

Cruise/station Location Sample age at time of analysis Dominant Pseudo-nitzschia species

NE Subarctic Paci c

GOA /16 b.d.l. 1 mo P. fraudulenta

P. pseudodelicatissima

NE Subarctic Paci c

GOA /28 b.d.l. 1 mo P. heimii

P. cf granii*

P. turgidula*,

P. turgidula*,

NE Subarctic Paci c

GOA /53 0.003 1 mo

P. pseudodelicatissima

P. in atula

P. turgidula*,

NE Subarctic Paci c

GOA /55 0.0004 1 mo

NE Subarctic Paci c

GOA /58 0.0003 1 mo P. pseudodelicatissima

P. turgidula*,

P.cf lineola

NE Subarctic Paci c

GOA /62 b.d.l. 1 mo

P. turgidula*,

NW Subarctic Paci c

VERTIGO K2 /4 0.84 2y

P. turgidula*,

NW Subarctic Paci c

VERTIGO K2 /76 1.9 2y

Equatorial Paci c P. in atula

IronEx II /240 0.02 12 y

P. delicatissima

P. heimii

P. cuspidata

P. lineola

Equatorial Paci c

IronEx II /331 0.05 12 y

P. roundii

P. turgidula*,

ENVIRONMENTAL

SCIENCES

P. heimii

Antarctic Paci c

SOFeX /36 (South) 1.0 4y P. lineola

P. granii*,

P. pseudodelicatissima

P. cf turgiduloides

Antarctic Paci c

SOFeX /40 (South) 0.69 4y Rare overall

Mostly P. lineola

*Species associated with DA production from grow outs of oceanic phytoplankton samples from station PAPA in the NE Subarctic Paci c and/or from in situ

water samples at that station (16).

Toxin observed from in situ water samples from oceanic stations in the present study.

Species identi cations using electron microscopy (SEM and TEM) were made on samples from 12 sites based on the availability of net tow material and

corresponding DA samples. The Domoic acid, pg cell-1 column presents data showing the average cell quota for the Pseudo-nitzschia in that sample. The

designation b.d.l. indicates DA was below the detection limit, which could re ect either the sparcity of the cells in the water or the possibility that many

other species were present in the sample, hence making the contribution of DA, even in cells with reasonably high DA levels, a small fraction of the total

material in that sample and, thus, below detection limit. The sample age at time of analysis is the time from collecting the sample at sea until the time the

sample was analyzed. Dominant Pseudo-nitzschia species were identi ed from aliquots of the net tows using electron microscopy (Table S2). At 5 of the 12

stations, only 1 species was found and, thus, the DA cell quotas could be assigned to those species, whereas in the 7 other samples 2 4 species were evident

and, thus, the listed pg cell 1 re ects an average for the mix of those species.

Natural Variability in DA Levels of Pseudo-nitzschia. Cellular DA

populations with associated DA, a neurotoxin shown to cause

quotas in eld populations from unfertilized, oceanic waters re-

harm to marine communities in productive coastal waters (11).

ported here (Table 1 and Table S1) are at the low (fg of DA cell 1)

Considering results from 14 eld sites in the Paci c, we nd DA

to moderate (pg of DA cell 1) range of those reported from

concentrations in the water to be signi cantly (P 3 orders of magnitude, i.e., from

High cell abundances of Pseudo-nitzschia (e.g., densities of 104 to 0.4 fg cell 1 in the Northeast (NE) Subarctic (similar to a value

106 cells L 1, or even higher) of the large coastal species that recently described from the region; ref. 13) to 1.9 pg cell 1 in the

produce picogram levels of cellular DA have allowed detection of Northwest (NW) Subarctic Paci c (Tables 1 and 2 and Table S1).

the toxin in natural waters, especially given the advent of highly Similarly, two stations were dominated by P. lineola previously

sensitive methods such as the ELISA. Oceanic Pseudo-nitzschia, not associated with toxin production in oceanic waters and its

however, are typically less abundant in waters overlying the deep cellular DA levels varied from below detection limits (b.d.l.) in the

ocean basins, smaller in size, and with the exception of the recent NE Subarctic Paci c to 0.69 pg cell 1 in the Antarctic Paci c

report by Trick and colleagues (13) of low (femtogram) DA cell (Tables 1 and 2 and Table S1). These results show that, even

quotas and pg of DA L 1 in truly oceanic waters have not been within one morphological species, cell quotas are highly variable

in the eld. Indeed, laboratory studies show that multiple envi-

known to produce this toxin.

PNAS Early Edition 3 of 6

Silver et al.

Table 3. Pseudo-nitzschia standing stocks, daily uxes of intact cells, and DA ux at VERTIGO K2 to mesopelagic

depths

VERTIGO NBST traps

Measurement ALOHA D1 ALOHA D2 K2 D1 K2 D2

Euphotic zone depth 125 m 125 m 60 m 60 m

Euphotic zone standing stocks (cells m 2) 1.1 106 2.2 107 4.2 108 8.6 107

Trap depth, daily cell ux, cells m 2 d 1

0 3.8 9.6 103 4.0 106 2.1 105

150 m

1.6 106 2.3 106

300 m b.d.l. b.d.l.

1.8 106 1.2 106

500 m b.d.l. b.d.l.

Cell ux at 150 m as % of Pseudo-nitzschia standing stock 0.02 0.04 1 0.24

Trap depth, daily DA ux, g m 2 d 1

150 m m.d. m.d. 2.2 0.4

300 m m.d. m.d. 0.9 4.4

500 m m.d. m.d. 1.0 2.3

Pseudo-nitzschia cell uxes were obtained from counts made on material collected from neutrally buoyant sediment traps (NBST)

deployed for 3 5 d at 150, 300, and 500 m on the VERTIGO cruises. DA uxes were calculated based on DA cell quotas that were

obtained from DA measurements on corresponding phytoplankton net tow samples taken during the deployments. Euphotic zone

depths (1% light level) were 125 m for ALOHA and 60 m for K2. Standing stocks of Pseudo-nitzschia were derived from counts on water

samples taken near the surface (25 m at ALOHA, 25 m at K2) and near the base of the euphotic zones (125 m at ALOHA, 40 m at K2).

The designation b.d.l. indicates below detection limit . Missing data are indicated by m.d. (Materials and Methods and SI Methods).

ronmental variables affect cell toxicity in a species, and toxin mental levels become limiting to other local photosynthetic

organisms and likely contributes to the success of Pseudo-nitzschia

quotas in the laboratory may be considerably different (by orders

of magnitude) than those in eld populations dominated by the in oceanic environments where iron additions are intermittent.

Using several assumptions, Trick et al. (13) calculated the diatom

same morphological species, presumably due to such factors as the

biomass increase resulting from Fe additions in HNLC regions

physical and chemical environment, as well as strain differences

between eld populations and laboratory isolates (21). Numerous that was needed to provide C export from the surface mixed layer

to meet commercial carbon credit goals. They assumed that all C

chemical variables affect cell toxicity, including concentrations

uptake was due to the growth of Pseudo-nitzschia, speci cally

of major limiting nutrients (N, P, Si) (22), trace metals (Fe, Cu)

P. turgidula that contained 15 fg of DA cell-1. Their results in-

(23 25), and pH (26), to name a few.

dicated that the responding Pseudonitzschia bloom would result

in 1 2 g DA L 1 and led them to reject iron fertilization as a

Toxin Measurements from Oceanic Iron Fertilization Studies. Given

that archived samples were collected a number of years before the responsible carbon sequestration option.

time of analysis, (12 y for IronEx II and 4 y for SOFeX South), In this present study, we show that high levels of DA have been

toxin estimates presented here may be conservative, if DA de- generated in historic iron enrichment experiments. At IronEx II

graded during storage (Methods and SI Methods). Our ability to and SOFeX South, relatively high Pseudo-nitzschia abundances

(106 and 105 cells L 1 respectively), and moderate DA quotas

measure toxins, even where toxin quotas were low, was due to our

(0.05 and 1 pg of DA cell 1, respectively) led to high toxin levels

concentration of large numbers of cells in net tows, in contrast to

in the water: 45 and 220 ng of DA L 1 respectively. Such DA

Trick et al. (13), who indicated that previous studies might not

have detected DA in oceanic samples because stored, preserved concentrations approach levels associated with harm to marine

samples were analyzed. predators in coastal waters (11, 12). Ecosystem linkages between

DA production and neurotoxin impacts at higher trophic levels,

Relevance to Geo-Engineering Solutions for Atmospheric CO2 Reduc- well known in shelf and coastal regions, have not yet been

tion and Moderation of Ocean Acidi cation. Our ndings have reported in open ocean systems. We show here, however, that

consequences for proposals to enhance phytoplankton production Pseudo-nitzschia populations and their associated DA in iron-

in HNLC oceanic waters by iron fertilization, as a geo-engineering fertilized HNLC regions have reached levels known to pose an

solution to sequester atmospheric CO2 (27). Over a dozen iron- ecosystem threat: An outcome that must be considered when

enrichment studies have been conducted in situ in HNLC envi- weighing the environmental consequences of climate change

(global warming, ocean acidi cation, sea level rise, increased

ronments over more than a decade to test the linkage among iron

supply, glacial increases in marine production, and atmospheric storm severity, and drought) against others (production of marine

CO2 levels. These studies show that Pseudo-nitzschia has fre- toxins and other factors associated with eutrophication).

quently been the major diatom responding to iron fertilization

(15). Iron plays a prominent role in many enzyme reactions (in- DA Transport to Mesopelagic Depths. Our data also suggest that DA

cluding nitrate reductase and chlorophyll synthetase), electron can be delivered to depths below the euphotic zone in HNLC

transport, and biosynthetic pathways (28). DA, likely related to its waters when Pseudo-nitzschia blooms sink from surface waters.

chelation af nity for trace metals (29, 30), has been shown to in- Recent studies have shown that particulate organic matter con-

crease iron uptake by Pseudo-nitzschia (25). Furthermore, a recent taining DA, including Pseudo-nitzschia cells with intact proto-

study (14) shows that Pseudo-nitzschia and a closely related genus plasts, occurs in sediment traps as deep as 800 m on the California

continental margin (31). In that study, the ux was tightly corre-

are unique among diatoms in their use of ferritin, an iron-storage

protein. [Although many other marine diatoms and other algae lated, and slightly time-lagged, with respect to the overlying toxic

have iron storage capabilities (28), the mechanisms remain un- blooms and cell descent was rapid, presumably a consequence of

known.] In low iron regions of the oceans where inputs of iron are surface cell aggregation (32). Our VERTIGO samples from

sediment traps at the oceanic North Paci c site, K2, also con-

episodic, these characteristics may be adaptive. As such, stored

iron becomes available to support further growth when environ- tained intact Pseudo-nitzschia cells from surface populations of

4 of 6 www.pnas.org/cgi/doi/10.1073/pnas.100******* Silver et al.

made on 50 100 mL preserved (4% borate buffered formalin) and DAPI-

DA-containing Pseudo-nitzschia (Table 3). Although the aliquots

stained aliquots in the shore laboratory by using Uterm hl methods (38) (see

available to us from the sediment traps at VERTIGO were too

SI Methods for details).

small to test for DA, the presence of intact cells suggests toxic

material likely was being delivered, possibly 1 2 g m 2 d 1, an

Pseudo-nitzschia Standing Stock and Flux Estimates. On VERTIGO cruises, we

amount 20% of that found on the California borderland (31). measured both euphotic zone stocks and cells with intact protoplasts descending

Such delivery rates into the mesopelagic zone could have physi- to mesopelagic depths (Table 3). Replicate counts were made from the upper

ological consequences for underlying communities, even at the and lower euphotic zone several times during sediment trap deployments. These

present time, under natural conditions. values were then averaged and integrated over the depth of the euphotic zone

[120 m at ALOHA, and 60 m at K2 (39) to estimate Pseudo-nitzschia standing

stocks (cells m 2) during deployment periods (SI Methods)].

Enrichment of Surface Ocean Waters with Iron as a Natural Phenom-

enon. The deposition of iron-containing dust in the ocean is well During the two VERTIGO cruises, at stations ALOHA and K2, we sampled

sinking material from four deployments, using neutrally buoyant sediment

known, and, in part, led to the original suggestion by John Martin

traps (39). Each 3- to 5-d deployment consisted of replicate traps at 150, 300,

(33) that aerosol-derived iron delivery to iron-limited HNLC

and 500 m. Counts of sinking cells were made from settled subfractions of

regions of the ocean could result in reduced atmospheric CO2 the trap material (SI Methods). From deployment duration and trap surface

levels on glacial/interglacial time scales. The addition of iron to collection area, we estimated uxes of Pseudo-nitzschia (i.e., cells m2 d 1). By

waters with otherwise suf cient nutrients to sustain phytoplankton dividing this ux by the integrated Pseudo-nitzschia cell population

growth results in sequestering atmospheric CO2, when the carbon, (standing stock) in the overlying euphotic zone, we estimated the % surface

now trapped as particulate organic matter in the phytoplankton, standing stock of Pseudo-nitzschia reaching trap depths daily (SI Methods).

settles below the surface mixed layer, no longer exchangeable with

Electron Microscopy: Pseudo-nitzschia Studies. Net tows provided material for

the atmosphere. The question is, then, whether periodic iron en-

identi cation of species from selected sites. We examined selected samples

richment by natural aerosols may have resulted in past enhance-

for which we also had abundance estimates and detectable DA. Both scan-

ments of Pseudo-nitzschia growth and, subsequently, in increased

ning (SEM) and transmission (TEM) electron microscopy were used on the

levels of DA in the open ocean. Were that the case, toxin- natural mixes of phytoplankton, using methods slightly modi ed from the

ENVIRONMENTAL

producing phytoplankton may have been a phenomenon not only literature (9, 40) (SI Methods and Table S2).

SCIENCES

of modern coastal waters, but also an alternating presence in

oceanic waters experiencing natural climate variability. Our Collecting and Preparation of Phytoplankton for Domoic Acid Analyses. Based

on the detection limit of the indirect cELISA (ELISA), 10 pg of DA mL 1, and

results, then, raise the possibility that pelagic communities have

already experienced periods of higher neurotoxin concentrations the anticipated low cellular DA level, we recognized the need to harvest

suf cient cells to detect DA, especially because lters might clog from other,

over longer, possibly evolutionary time scales.

more abundant material in the water. Therefore, we concentrated the

In summary, the presence of neurotoxin-producing phyto-

larger phytoplankton, including chain-forming Pseudo-nitzschia, by using

plankton is a common feature in open ocean environments, and

20- to 30- m mesh phytoplankton nets hauled from 10 to 15 m to the sur-

these cells are frequent responders to iron additions. Natural face. Such net tows provided the concentrates used to measure DA on the

additions of iron occur cyclically on millennial time scales (33), ve expeditions.

suggesting an associated variation in Pseudo-nitzschia abundance Net-tow samples for DA analyses were handled differently, depending on

over glacial/interglacial time scales. With the growing acceptance the expedition. On the GOA cruise, a measured volume of the net tow

that anthropogenic activities are profoundly affecting today s concentrate was gently ltered immediately after collection onto 25 mm GF/F

lters and placed in vials in liquid nitrogen for DA analysis at the shore

global ocean, with nearly half of ocean ecosystems strongly and

laboratory (completed within 6 mo of collection). The remaining cell con-

negatively impacted (34), practical, near-term solutions to the

centrates from net tows were xed with 4% hexamine-buffered formalin and

climatological impacts of CO2-driven global warming are both

stored in glass containers in the dark at 4 C for counts (cells ml 1) and EM

timely and necessary. The challenge, however, is to understand, species con rmation. In the laboratory, the frozen lters containing par-

within actionable limits of con dence, the ecosystem conse- ticulate, cellular material that included Pseudo-nitzschia cells were placed in

quences of such purposeful manipulations. This study demon- vials containing 10 mL of 20% methanol. The vials were vortexed, sonicated

strates that the enhancement of Pseudo-nitzschia, which fre- for 2 min (30 40 W) by using a Misonix Sonicator 3000 probe, and centri-

fuged for 10 min at 3000 g to free water-soluble DA possibly remaining in

quently occurs with oceanic iron fertilization, can result in high

the cellular matrix. After mixing, the samples were syringe- ltered through

and potentially problematic levels of the neurotoxin domoic acid

0.2- m polycarbonate membrane lters, and the supernatant diluted 1:10

in such environments. This study also suggests that increased at-

(minimum) with Standard/Sample buffer to minimize interferences.

tention to such ecosystem alterations is needed before iron fer-

On VERTIGO, SOFeX, and IronEx II cruises, DA was measured in archived

tilization can be recommended as a responsible strategy to reduce net-tow samples xed with 4% formalin at sea and stored for periods from 2 y

atmospheric CO2. (VERTIGO) to 12 y (IronEx II) (Table 2). Storage temperatures varied: VERTIGO

samples at 4 C and SOFeX and IronEx II at room temperature. We antici-

Methods pated some leakage of water-soluble DA from cells into the seawater during

storage. Thus, we measured DA both in lter concentrates and ltrate. Fil-

Sample Sources. Euphotic-zone samples of phytoplankton were obtained from

ve oceanic expeditions (Fig. 1 and Fig. S1). Three of these were natural pop- ters were treated as described above (SI Methods).

ulations from the North Paci c, and two were populations that developed

during mesoscale iron-fertilization experiments in the South Paci c. The North ELISA Assay for Domoic Acid. DA was measured routinely by using high-sen-

Paci c Gulf of Alaska (GOA) cruise provided samples at 18 sites. The two VERTIGO sitivity Biosense cELISA kits, using protocols provided by the manufacturer

expeditions (35), also in the North Paci c at ocean observing stations, included (41, 42). cELISA measures both DA and, to some extent, its isomers. We chose

cELISA rather than HPLC for routine measurements because of the former s

one in the northwest Subarctic gyre waters near K2 and the other in subtropical

higher sensitivity and ability to measure DA in formalin- xed samples. HPLC,

central gyre waters near ALOHA. Both expeditions provided euphotic-zone

water as well as sediment trap samples from 150- to 500-m depths. We also reex- in contrast, cannot accurately measure DA in samples with formalin (41, 42).

amined surface water, net-tow samples, and corresponding water samples from Each sample was run in duplicate and at several dilutions. The color intensity

K2 and two mesoscale iron-fertilization experiments: SOFeX (2002, Sub-Antarctic was read by using a standard microplate absorbance reader at 450 nm (see SI

and Antarctic Paci c) (36) and IronEx II (1995, Eastern Equatorial Paci c) (37). Methods for complete method).

(See SI Methods and Table S1 for the complete dataset.)

Structural Con rmation of the Toxin. Net tow material from IronEx II, the

Natural Abundances of Pseudo-nitzschia in the Euphotic Zone. Water from oceanic iron fertilization experiment with high concentrations of Pseudo-

Niskin bottles provided samples of Pseudo-nitzschia at euphotic zone depths nitzschia, was selected for analysis, because higher toxin levels are required

(10 30 m) for cell counts. On the GOA, SOFeX, and IronEx II cruises, counts for structural con rmation of DA as compared with quantization by cELISA

were made from lter preparations. Counts on the VERTIGO samples were methods. We used liquid chromatographic separation coupled with mass

PNAS Early Edition 5 of 6

Silver et al.

spectrometric detection (LC-MS/MS) to con rm DA presence in a formalin- masses (138.00 and 110.00 Da). The detection limit for the ltrate was 1.2 ng

DA. LC-MS/MS DA concentrations in IronEx II averaged 161 30 g of DA L 1,

preserved net-tow sample, mostly using methods described (31). DA was

identi ed and quanti ed from the signal of the parent mass (312.14 Da) and comparable with the 130 g L 1 measured by cELISA in the same sample.

two daughter masses (266.10 and 168.00 Da). Samples were analyzed in the

multiple reaction monitoring mode, using an Agilent 1100 HPLC coupled to ACKNOWLEDGMENTS. Invaluable help was provided by S. Bates, who re-

viewed the manuscript; N. Lundholm, who con rmed Pseudonitzschia spe-

a Micromass-Quattro mass spectrometer equipped with an electrospray ion-

spray, with methods adapted from Burns and Ferry (43). The mobile phase cies in Iron-Ex II and SOFeX samples; K. Buessler for his leading role on

VERTIGO cruises; and S. Tanner, S. Smith, and T. Coale for invaluable assis-

was a mixture of 0.1% aqueous formic acid in deionized water (A) and 0.1%

tance at sea on Vertigo and GOA cruises. We acknowledge helpful com-

aqueous formic acid in acetonitrile (B). The initial condition was 95:5 A/B for

ments of four anonymous reviewers of this manuscript. Funding sources

4 min, followed by a linear gradient over 11 min ending at 5:95 A/B. The ratio

included GOA-NSF OCE-0741481; Vertigo-NSF OCE-0327640; SOFeX-

of A and B was reset to the initial condition over the following 7 min to

National Science Foundation OCE-9911481 (to K.H.C.) and OCE-9530762

reestablish initial conditions. A 4-min solvent diversion was used to avoid salt (to K.H.C.); the Department of Energy DE-FG03-01ER63093 and Subcontract

contamination of the ion source. Additional modi cations included a sample 6491879 (to K.H.C.); IronEx II-US Of ce of Naval Research Grant N00014-94-

injection volume of 20 L and the use of caffeine as an internal standard. 1-0125 (to K.H.C.), and by National Science Foundation Grant OCE-90

Caffeine was monitored from the parent mass (195.10 Da) and two daughter -9217518 (to K.H.C.).

23. Ladizinsky NCThe in uence of dissolved copper on the production of domoic acid by

1. Parsons ML, Dortch Q, Turner RE (2002) Sedimentological evidence of an increase in

Pseudo-nitzschia species in Monterey Bay, California: Laboratory experiments and

Pseudo-nitzschia (Bacillariophyceae) abundance in response to coastal eutrophi-

eld observations)Master s Thesis.

cation. Limnol Oceanogr 47:551 558.

24. Maldonado M, Hughes M, Rue E, Wells MW (2002) The effect of Fe and Cu on growth

2. Hallegraeff GM (1993) A review of harmful algal blooms and their apparent global

increase. Phycologia 32:79 99. and domoic acid production by Pseudo-nitzschia multiseries and Pseudo-nitzschia

australis. Limnol Oceanogr 47:515 526.

3. Hubbard KA, Rocap G, Armbrust EV (2008) Inter-and intraspeci c community

25. Rue E, Bruland K (2001) Domoic acid binds iron and copper: A possible role for the

structure within the diatom genus Pseudo-nitzschia (Bacillariophyceae). J Phycol 44:

toxin produced by the marine diatom Pseudo-nitzschia. Mar Chem 76:127 134.

637 649.

26. Lundholm N, Hansen PH, Kotaki Y (2004) Effect of pH on growth and domoic acid

4. Hasle GR (2002) Are most of the domoic acid-producing species of the diatom genus

Contact this candidate