Mar Biol (2007) 151:2053–2062

DOI 10.1007/s00227-007-0634-z

RESEARCH ARTICLE

Domoic acid in benthic XatWsh on the continental shelf of Monterey Bay, California, USA

Veronica L. Vigilant · Mary W. Silver

Received: 18 October 2006 / Accepted: 30 January 2007 / Published online: 20 March 2007© Springer-Verlag 2007

Abstract Within Monterey Bay, California, USA, thefood web transfer of domoic acid (DA), a neurotoxin pro-duced by diatoms of the genus Pseudo-nitzschia, has led tomajor mortality events of marine mammals and birds. Lessvisible, and less well known, is whether invertebrates andWsh associated with the benthos are also aVected by bloomsof DA-producing Pseudo-nitzschia spp. This studyexamines the presence of DA in benthic XatWsh oVshore ofDavenport, California, (37°0�36�N, 122°13�12�W) andwithin Monterey Bay, California (36°45�0�N, 122°1�48�W),including species that feed primarily in the sediment(benthic-feeding) and species that feed primarily in thewater column (benthopelagic-feeding). FlatWsh caughtbetween 10 December 2002 and 17 November 2003 atdepths of 30–180 m had concentrations of DA in theviscera ranging from 3 to 26 �g DA g¡1 of viscera. Althoughthe DA values reported are relatively low, benthic-feedingXatWsh were frequently contaminated with DA, especiallyas compared with the frequency of contamination of XatWshspecies that feed in the water column. Furthermore, on daysin which both benthic-feeding and benthopelagic-feedingXatWsh were collected, the former had signiWcantly higherconcentrations of DA in the viscera. CurlWn turbot, Pleu-ronicthys decurrens, the XatWsh with both the highest leveland frequency of DA contamination, are reported to feed

exclusively on polychaetes, suggesting that these inverte-brates may be an important vector of the toxin in benthiccommunities and may pose a risk to other benthic-feedingorganisms.

Introduction

Increasing concern about economic loss due to harmfulalgal blooms (HABs) (Anderson 1995) has led to a corre-sponding increased interest in the movement and fate ofHAB toxins in the broader marine community. As thesetoxins usually originate from microalgae in the surfacewaters of the ocean, much of this research has understand-ably focused on the transfer of HAB toxins through thepelagic food web and intertidal environments as opposed toHAB toxins in benthic communities. The transfer of someHAB toxins, such as domoic acid (DA), to higher trophiclevels relies on the presence of a short trophic pathway dueto the water-soluble, and hence readily excreted, propertiesof DA (JeVery et al. 2004). In most cases, there is only oneherbivorous species between the toxin-producing organismand the end consumer, and much research has highlightedthe identiWcation of these important herbivorous vectors(Drum et al. 1993; Wekell et al. 1994; McGinness et al.1995; Douglas et al. 1997; Turner and Tester 1997;Lefebvre et al. 1999; Bargu et al. 2002; Powell et al. 2002;Costa et al. 2003, 2004, 2005a, b; Teegarden et al. 2003).

Domoic acid is responsible for the syndromes AmnesicShellWsh Poisoning (ASP) in humans and Domoic AcidPoisoning (DAP) in marine mammals and birds, both ofwhich are classiWed by severe neurological and gastrointes-tinal symptoms and may lead to death (Wright et al. 1989;Work et al. 1993; Silvagni 2003, 2005). DA, an amino acidthat is an analogue of glutamate, exerts its neurological

Communicated by J.P. Grassle.

Electronic supplementary material The online version of this article (doi:10.1007/s00227-007-0634-z) contains supplementary material, which is available to authorized users.

V. L. Vigilant (&) · M. W. SilverOcean Science Department, University of California, Santa Cruz, CA, USAe-mail: vigilant@ucsc.edu

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eVects due to binding to glutamate receptors in the brain(Hampson and Manolo 1998). The toxin has been shown tobe eVectively transferred to humans through consumptionof mussels (Quilliam and Wright 1989; Perl et al. 1990) andto marine mammals and birds through planktivorous Wshand krill (Lefebvre et al. 1999; Bargu et al. 2002; Barguand Silver 2003). In both situations, the vector species isexposed to DA produced by some of the species of diatomswithin the genus Pseudo-nitzschia. Two of these toxicPseudo-nitzschia species, P. australis and P. multiseries,commonly bloom within Monterey Bay (Villac et al. 1993;Trainer et al. 2000). Blooms of these two species have beenthe cause of major mortality events of marine birds andmammals in Monterey Bay and along the entire Californiacoastline (Work et al. 1993; Scholin et al. 2000). Withmuch of the attention drawn to these well-publicized eventsand to contamination of commercially important nearshoreshellWsh, comparatively little is yet known about the fate ofDA in the benthos and its impact on oVshore benthicWsheries.

In order for benthic DA contamination to occur belowthe euphotic zone, toxic algal cells, toxic detritus, or toxicvectors from the euphotic zone source must reach theseaXoor. The delivery of toxin to the seaXoor could resultfrom direct sinking of aggregates of toxic cells, transfer ofthe toxic cells via fecal pellets, or transfer by verticallymigrating organisms. There is evidence that Pseudo-nitzs-chia spp. cells Xocculate and sediment out during and afterblooms (Olivieri 1996; Dortch et al. 1997; Parsons et al.2002) providing one of these transfer mechanisms for DAto reach the benthos. The second transfer mechanism, fecalpellets, can deliver toxin to the seaXoor, as occurs whenDiarhhetic ShellWsh Poisoning and Paralytic ShellWshPoisoning toxins are present in copepod fecal pellets(Maneiro et al. 2000; Guisande et al. 2002). The DA-pro-ducing P. australis appears to be common in midwatercollections of fecal pellets from Monterey Bay, California(Olivieri 1996). Krill, important prey items in the MontereyBay region which exhibit diel vertical migration, are poten-tial vectors of DA (Bargu et al. 2002; Bargu and Silver2003).

Further evidence of the presence of a reservoir of DA inthe benthos is suggested by studies examining DA levels innearshore and intertidal benthic communities. Recently,several nearshore benthic invertebrates in Monterey Baywere found to be contaminated with DA, with levels rang-ing from 2 �g DA g¡1 of tissue in the deposit feeding olivesnail, Olivella biplicata, to 700 �g DA g¡1 in the Wlter-feeding, fat innkeeper worm, Urechis caupo (Goldberg2003). Vale and Sampayo (2001) found high levels of DAin several intertidal bivalves in Portugal, namely Cerasto-derma edule and Scrobicularia plana. The former lives inestuarine sands and mud, and the latter is a deposit feeder.

Similarly, the venus clam, Venus verrucosa, which burrowsin sand and rock habitats, exhibited consistently higher DAlevels than Wlter-feeding mussels (Kaniou-Grigoriadouet al. 2005). DA contamination of invertebrates collected atdepth in the water column have also been reported in theswimming crab, Polybius henslowii, with samples collectedat 351 m oV the coast of Portugal containing DA levels ashigh as 48.5 �g DA g¡1 (Costa et al. 2003). Detectable DAlevels were also found in all crabs caught between thedepths of 43 and 72 m, suggesting that these organismscould be an exposure source of DA to benthic Wsh feedingon the continental shelf. In Monterey Bay, DA has beenreported in nearshore benthic-feeding Wsh species, with lowlevels of DA noted in the viscera of white croaker, Geny-onemus lineatus (Fire and Silver 2005). It is highly likelytherefore, that there are benthic DA reservoirs in the sedi-ment that could pose a threat to oVshore benthic living and/or bottom-feeding Wsh and mammals.

Domoic acid events are common in the Monterey Bayregion and oVshore benthic vertebrates such as XatWsh,which comprise an important portion of the recreationaland commercial benthic Wsheries within Monterey Bay,could be vulnerable to the toxin. There appear to be nopublished reports, however, of oVshore DA contaminationin these important species. Here we present data on the DAcontent of eight species of commercial XatWsh collectedoVshore by trawl within Monterey Bay and near Davenport,California, USA in 2003. Additionally, we present datasupporting the hypotheses that XatWsh with two diVerentmodes of feeding (benthic-feeding vs. benthopelagic-feed-ing) have diVerent DA toxin levels and that DA levels inXatWsh are poorly coupled to toxin and toxic cell presencein overlying surface waters.

Materials and methods

FlatWsh sampling

FlatWsh were collected by trawl from depths of 30–180 mon a monthly basis oVshore of Davenport, California, USA(37°0�36�N, 122°13�12�W) and on a monthly to bimonthlybasis within Monterey Bay (36°45�0�N, 122°1�48�W) from10 December 2002 to 17 November 2003 during groundWshecology cruises of the National Marine Fisheries andScience (NMFS) laboratory in Santa Cruz. FlatWsh speciescollected during the time series included PaciWc sanddab(Citharichthys sordidus), slender sole (Eopsetta exilis),petrale sole (Eopsetta jordani), sand sole (Psettichthysmelanostictus), rex sole (Errex zachirus), Dover sole(Microstomus paciWcus), English sole (Pleuronectesvetulus), and curlWn turbot (Pleuronicthys decurrens).Additionally, one sample of PaciWc halibut (Hippoglossus

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stenolepis) was obtained from a local commercial Wshingvessel on 15 April 2004 (S1). All Wsh were frozen intact(i.e. not eviscerated) after being collected and then storedfor up to 1 month before dissection and subsequent toxinextraction. Fish were thawed during dissection and theviscera from multiple specimens of the same species, often3–20 individuals, pooled (though sometimes only one wasavailable) and weighed (S1). As the viscera of each Wsh wasremoved, gut contents were examined using a dissectingmicroscope, in order to identify possible food items forplacement into broad categories such as “crustaceans” or“small Wsh” and to determine whether sediment was pres-ent. The viscera were then frozen for later DA extractionand analysis.

Water sampling

To determine the relationship between DA in Wsh and thepotential local source of the toxin, phytoplankton sampleswere analyzed from surface water depths (upper 1 m) ofMonterey Bay. Weekly water samples were collected at theMonterey Bay Aquarium Research Institute’s (MBARIs)M1 mooring site in central Monterey Bay (36°45�0�N,122°1�48�W) and analyzed for the toxin-producing species,P. australis and P. multiseries (10-ml water samples), andparticulate DA (500-ml water samples). Additionally, sam-ples from the upper 1 m were available from transectswithin Monterey Bay on monthly NOAA-funded Center forIntegrated Marine Technology (CIMT) cruises. Waters oVDavenport, approximately 20 km north of Monterey Bayand a frequent source of water to the Bay, also were moni-tored during CIMT cruises. Depending on weather condi-tions, up to 11 stations were sampled during the monthlyCIMT cruises. Water from the station corresponding to theM1 mooring site was used to supplement the data in weekswhen M1 water samples were not collected. P. australisand P. multiseries were identiWed and enumerated in watersamples using whole cell molecular probes developed forMonterey Bay clones of toxic Pseudo-nitzschia spp. (Millerand Scholin 1996).

Toxin detection in phytoplankton and Wsh samples

Solvents used for DA extraction and HPLC analysis wereHPLC-grade triXuoroacetic acid (TFA), analytical gradeNaCl, and Fisher Optima methanol (MeOH) and acetoni-trile (MeCN) (Fisher ScientiWc, Pittsburg, Pennsylvania,USA). DACS-1D certiWed DA standard (National ResearchCouncil of Canada, Institute for Marine Biosciences, Hali-fax, Nova Scotia, Canada) and 90% pure DA reagent(Sigma-Aldrich, St Louis, Missouri, USA) were obtainedfor calibration standard preparation and spike and recoverycalculations. Nanopure water was used for preparation of

all the solutions and standards were kept refrigerated in thedark.

Fish viscera samples were extracted within 1 week ofdissection and then immediately cleaned of interferingcompounds using solid-phase extraction columns accordingto HatWeld et al. (1994) and Quilliam et al. (1995) prior tobeing analyzed for DA using an isocratic gradient proWle ona Hewlett-Packard 1050 HPLC equipped with autosampler,oven, quartenary pump, and diode-array detector (DAD) setto 242 nm. The column used was a reverse phase VydacC18 column heated to 30°C with a Vydac guard-column(5-�m particle size). The mobile phase (90/10/0.1 water/MeCN/TFA) was degassed with helium for 15 min prior toanalysis. A 20-�l injection volume was used with an analy-sis time of 15 min and a Xow rate of 0.3 ml min¡1. Datacollection was performed by the HP Chemstation software.

A calibration curve was generated using DACS-1D DAstandards of 1–32 �g DA ml¡1 with a lowest detectablestandard of 0.15 �g DA ml¡1 and a calculated limit ofdetection (three times the standard deviation of the lowestdetectable standard) of 0.16 �g DA ml¡1. Spike and recov-ery of Bakerbond spe column lot # A05554 (CAS no:126850-06-4, J.T. Baker, Phillipsburg, New Jersey, 08865,USA) used in solid-phase extraction using DACS-1Dstandards resulted in an average value of 91% of injectedDA recovered. An average of 74% DA recovered afterextraction and clean-up was calculated for the spike andrecovery of XatWsh viscera Wrst determined to be free ofDA. Values for DA in XatWsh were reported uncorrected forloss of sample in extraction and clean-up.

Water samples for analysis of DA in phytoplankton(particulate DA) were Wltered through GF/F WhatmanWlters and frozen 1–4 weeks before extraction with 10%MeOH. Particulate DA was analyzed according to theHPLC–FMOC method described by Pocklington et al.(1990). The same equipment as described above was usedwith gradient elution proWle and a temperature of 55°C aswas suggested to be optimal by Pocklington et al. (1990).The mobile phase solvents (water/TFA and MeCN/TFA)were degassed with helium for 15 min prior to analysis. A20-�l injection volume was used with an analysis time of40 min and a Xow rate of 0.2 ml min¡1. A calibration curvewas generated using DACS-1D DA standards of 2.5 ng DAml¡1 to 400 ng DA ml¡1.

Data analysis

The frequency of DA levels in XatWsh was calculated as thenumber of days DA was detected versus the number of daysa species was collected and this frequency is reported as“percent occurrence.” The mean values of the percentoccurrence and maximum DA for benthopelagic-feedingXatWsh versus benthic-feeding XatWsh were compared using

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a parametric ANOVA on the original data, using theMicrosoft Excel 2002 graphing program.

Results

All XatWsh species sampled were designated as eitherbenthopelagic-feeding or benthic-feeding based on generalstomach content observations from this study and on resultsfrom previous studies (Table 1). Benthopelagic-feedingspecies, deWned as those XatWsh species feeding in thewater column, included PaciWc sanddab, slender sole,petrale sole, sand sole, and PaciWc halibut. The benthic-feeding XatWsh group, deWned as XatWsh feeding onsediment-dwelling infauna and epifauna, consisted of rexsole, Dover sole, English sole, and curlWn turbot.

DA values in XatWsh viscera rarely surpassed the regula-tory limit of 20 �g DA g¡1 of tissue set for DA in Wsh andshellWsh tissue (with the exception of Dungeness crabviscera, which has a regulatory limit of 30 �g DA g¡1)(Marien 1996), but DA was detected consistently through-out the year in some XatWsh species. Maximum DA valuesin the viscera of XatWsh species during the year 2003 rangedfrom 3 to 26 �g DA g¡1 in PaciWc sanddab and curlWn

turbot, respectively (Table 2). The highest DA concentra-tion of 53 �g DA g¡1 in XatWsh viscera was found outsidethe period of the time-series collection on 15 April 2004 inDover sole (Table 2). The number of sampling days for theeight species of XatWsh collected ranged from 6 to 15 daysand the percent occurrence ranged from 10% for petralesole to 89% for curlWn turbot (Table 2). Both categoriesshow signiWcant diVerences between benthic and bentho-pelagic-feeding Wsh for both frequency of occurrence of thetoxin and average toxin concentrations (P < 0.05) (Table 2).As a group, benthic-feeding XatWsh were more often con-taminated, 75 versus 27% of the dates sampled, and had ahigher maximum DA value, 29.6 versus 7.3 �g DA g¡1,than did benthopelagic-feeding XatWsh (Table 2).

Particulate DA levels and toxic Pseudo-nitzschia spp.concentrations throughout the study period from surfacewaters at the MBARIs M1 mooring site (or the correspond-ing CIMT station) showed three to six bloom periods(Fig. 1). There were four peaks in DA and Pseudo-nitzschiaspp. (DA > 5,000 pg DA ml¡1 and Pseudo-nitzschia spp.concentrations >100 cells ml¡1) and two smaller blooms.DA levels in a spring bloom in April 2003 were substan-tially higher then during other times of the year, with aconcentration of almost 25,000 pg DA ml¡1.

Table 1 Literature and current study observations on feeding habits of eight species of XatWsh in Monterey Bay, California, USA

Based on the feeding habit information available, XatWsh were categorized as either benthic-feeding (those that feed on and in the sediment) orbenthopelagic-feeding (those that feed primarily on invertebrates and Wsh in the water column) (a) Hilaski (1972), (b) US Fish and Wildlife Service(1983), (c) Monterey Peninsual Water Pollution Control Agency (1977), (d) Anderson et al. (1976), (e) Allen et al. (1998), (f) Barry et al. (1996)

Fish species Literature observations Current study observations

Feeding habit/diet Study site(s)/references Feeding habit/diet

PaciWc sanddabCitharichthys sordidus

Opportunistic and benthopelagic: preys on large variety of organisms including small pelagic Wsh, cephalapods, crustaceans, and polychaetes

Monterey Baya,b,c,d

Southern CA BighteBenthopelagic:

crustaceans squid, small Wsh, herring, polychaetes

Slender sole Eopsetta exilis

Benthopelagic Southern CA Bighte Benthopelagic: no observations

Petrale sole Eopsetta jordani

Benthopelagic: anchovies, CA tongue Wsh, dover sole, squid and sardines

Sand sole Psettichthys melanostictus

Highly restrictive diet includes mostly small Wsh and mysids

Monterey Baya,d,f Benthopelagic: small Wsh, squid, crustaceans, anchovies

Rex sole Errex zachirus

Non-visual benthivore Southern CA Bighte Benthic: sediment, crustaceans

Dover sole Microstomus paciWcus

Selective extracting benthivore: diet includes polychaetes, ophiuroids, mollusks, and crustacean prey

NW PaciWcb

Southern CA BighteBenthic: sediment,crustaceans,

polychaetes

English sole Pleuronectes vetulus

Non-selective excavating benthivore: feeds on a great diversity of prey using a “scooping”-type mechanism to obtain benthic invertebrates

Monterey Baya,b,c,d,f

Oregon coastb

Southern CA Bighte

Benthic: sediment in all samples, crustaceans, polychaetes

CurlWn turbot Pleuronicthys decurrens

Highly selective extracting benthivore: restricted diet of polychaetes, speciWcally Nothria sp.

Monterey Bay a,c,d

Southern CA BighteBenthic: some

sediment, frequently polychaetes, on one occasion crustaceans

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The occurrence of DA in XatWsh showed temporal vari-ability, with maximum values occurring in diVerent monthsfor the various species sampled. With the exception of sandsole, the highest DA values in benthopelagic-feeding Wshwere measured during the fall and winter months, with the

highest levels in benthic-feeding Wsh being found in springand summer (Table 2). DA was detected in XatWsh whenthere were few to no toxic cells detected in the surfacewater, with cell detection levels being approximately 1 cellml¡1 (Silver, unpublished data). Furthermore, toxin was

Table 2 Domoic acid (DA) levels in oVshore XatWsh viscera sampled between December 2002 and April 2004 in Monterey Bay, California, USA

FlatWsh are grouped by feeding habit with those XatWsh feeding primarily on invertebrates and Wsh in the water column labeled “benthopelagic”and those feeding primarily in and on the sediment labeled “benthic.” Percent occurrence was calculated as the number of days DA was detectedversus the number of days a species was collected. Percent occurrence was not calculated for PaciWc halibut as there was only one sample obtainedfor this species

Feeding habit Species Number of days sampled

% occurrence Max. DA (�g DA g¡1)

Date of max DA

Benthopelagic-feeding PaciWc sanddab Citharichthys sordidus

15 27 3.4 20 October 2003

Slender sole Eopsetta exillis

8 38 4.9 12 November 2003

Petrale sole Eopsetta jordani

10 10 6.7 18 February 2003

Sand sole Psettichthys melanostictus

6 33 13.2 27 June 2003

PaciWc halibut Hippoglossus stenolepis

1 – 8.4 24 October 2003

Mean benthopelagic-feeding 10 22 9.4

Benthic-feeding Rex sole Errex zachirus

7 71 24.3 15 April 2004

Dover sole Microstomus paciWcus

6 83 53.3 15 April 2004

English sole Pleuronectes vetulus

14 57 15.0 27 June 2003

CurlWn turbot Pleuronectes decurrens

9 89 25.9 27 June 2003

Mean benthic-feeding 11.5 73 20.5

P value for benthopelagic- versus benthic-feeding <0.05 <0.05

Fig. 1 Pseudo-nitzschia spp. concentration and particulate domoic acid (DA) levels collected from surface water in Monterey Bay—at MBARIs M1 mooring site (or the nearby station on cruises)—and DA levels in oVshore XatWsh viscera collected from December 2002 to December 2003 in Monterey Bay, California, USA. FlatWsh data represent the average for all species of a given feeding habit for that date. FlatWsh feeding primarily on invertebrates and Wsh in the water column are labeled “benthopelagic” and those feeding primarily in and on the sediment are labeled “benthic.” Dates marked “nd” represent days on which XatWsh were collected but DA was not detected in the viscera

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sometimes undetectable in XatWsh during months whenhigh numbers of toxic cells were noted in the water (Fig. 1).

Discussion and conclusions

Although XatWsh comprise an important part of thecommercial Wshery, representing 10% of the total catchby weight between 1981 and 2000 in the Monterey BayMarine Sanctuary (Starr et al. 2002), previous DA levelsreported in XatWsh have been limited to shallow nearshoresamples of PaciWc sanddab and petrale sole with DAconcentrations of 2 and 500 �g DA g¡1 of viscera, respec-tively (Lefebvre et al. 2002a; Goldberg 2003). In thepresent study, DA concentrations in the viscera were almostalways below the regulatory levels of 20 �g DA g¡1. Ingeneral, viscera likely have higher toxin concentrationsthan Xesh: for example, in clupeoid Wsh (northern anchovy,Engraulis mordax and PaciWc sardine, Sardinops sagax),DA concentrations are 5–10 times higher than in the Xesh(Lefebvre et al. 2002b).

Although the values for DA in XatWsh viscera sampled inthe present study were relatively low, their high frequencyof occurrence in the XatWsh (Table 2) indicates a phenome-non of potential importance: a frequent exposure of ben-thic-feeding XatWsh to DA. As a water-soluble toxin, DA invertebrates is quickly excreted following exposure to thetoxin and therefore the presence of DA in the viscera ofXatWsh indicates recent dietary exposure to the toxin(Suzuki and Hierlihy 1993; Truelove and Iverson 1994; Sil-vagni 2003; JeVerey et al. 2004). Although to date, no workhas been done on the depuration rates of DA in Wsh, therapid excretion of DA in vertebrates has been reported withhalf-lives of DA ranging from 21 min in rats to 115 and132 min in monkeys and birds, respectively (Suzuki andHierlihy 1993; Truelove and Iverson 1994; Silvagni 2003).Rapid, although varying, depuration rates for a number ofbivalve species have also been reported (Novaczek et al.1991, 1992; WohlgeschaVen et al. 1992; Wekell et al. 1994;Whyte et al. 1995; Douglas et al. 1997; Lund et al. 1997;Blanco et al. 2002a, b). An exception to these rapid depura-tion rates are found in king scallops and razor clams, thelatter of which is known to possess a binding site thatcauses the toxin to be retained and concentrated (Drumet al. 1993; Horner et al. 1993; Blanco et al. 2002a, b;Trainer and Bill 2004; Bogan et al. 2006). As there is noevidence of similar retention of DA in WnWsh, the near-con-sistent presence of DA in the viscera of the collected ben-thic-feeding XatWsh suggests that XatWsh feeding on and/orin the sediment are frequently being exposed to the toxin onthe seaXoor.

The hypothesized route of DA exposure to these benthic-feeding XatWsh is shown in Fig. 2 in the “benthic pathway,”

along with a “pelagic pathway,” the traditionally acceptedroute of exposure for pelagic-feeding organisms, whichwould also apply to benthopelagic-feeding XatWsh. In thepelagic pathway, the benthopelagic-feeding Wsh, whichfeed in the water column, are exposed to the toxin throughtheir diet of DA vectors, such as planktivorous Wsh andinvertebrates (Work et al. 1993; Lefebvre et al. 1999;Bargu et al. 2002; Costa et al. 2003; Costa and Garrido2004; Maneiro et al. 2005). In the benthic pathway, whichhas also been suggested in the transfer of DA to cephalo-pods along the Portuguese coast (Costa et al. 2005b), thebottom-feeding Wsh encounter DA by two major routes:their diet (the infaunal and epifaunal organisms that serveas their primary food source) and/or sediment and associ-ated detritus indirectly ingested while feeding. Some of theinfaunal and epifaunal invertebrates in benthic XatWsh diets,such as bivalves and crustaceans, are known to contain DAin nearshore benthic environments during toxic blooms(WohlgeschaVen et al. 1992; Drum et al. 1993; Horner andPostel 1993; Langlois et al. 1993; Campbell et al. 2001;Ferdin et al. 2002; Goldberg 2003; Kaniou-Grigoriadouet al. 2005), and may contain DA at depth as well if theyare exposed to toxic cells. Meanwhile, other potential vec-tors, such as polychaetes, have received little attention dueto their lack of commercial importance but may also feedon toxic cells and detritus if present in the benthic environ-ment.

In addition to DA indirectly consumed through vectorspecies, XatWsh species that ingest sediment during feedingmay be directly exposed to DA in the benthos. This sedi-ment-associated DA may include intact or disintegratingfecal pellets containing Pseudo-nitzschia spp. cells as wellas aggregates with Pseudo-nitzschia spp. cells that have set-tled from the surface to the seaXoor. Evidence for the sink-ing of Pseudo-nitzschia spp. cells is provided by sedimenttrap studies from various locations (Dortch et al. 1997; Par-sons et al. 2002). In Monterey Bay, Pseudo-nitzschia spp.aggregates of intact cells have been noted in midwater sedi-ment traps and intact and fragmented cells are a commonconstituent of fecal pellets in the water column (Olivieri1996). All the benthic-feeding XatWsh species sampled inthe present study had sediment in their stomachs (Table 2),albeit to varying degrees, indicating exposure to suchpotential DA-containing material in addition to potentiallycontaminated organismal vectors.

The diets of the sampled XatWsh (Table 1) support thetwo hypothetical pelagic and benthic pathways of DA toXatWsh in Fig. 2 and point towards some intriguing possibil-ities as to the speciWc organisms acting as vectors of DA.Those XatWsh grouped as benthopelagic-feeders showed asigniWcantly lower frequency of toxicity and lower DAvalues than those classiWed as benthic-feeders, demonstrat-ing that the less-known benthic pathways may play a more

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important role in transfer of DA than the better knownpelagic pathways. Within the group of benthic-feedingXatWsh, there appears to be a relationship between toxicityfrequency and dietary composition. DA was most fre-quently present in the two species with the most selectivediets of the four bottom-feeders, curlWn turbot and Doversole. CurlWn turbot within Monterey Bay are reported tofeed almost exclusively on polychaetes (Hilaski 1972;Anderson et al. 1976; Allen et al. 1998), which also consti-tute the bulk of the slightly less restrictive diet of Doversole (Allen et al. 1998), suggesting that polychaetes may beacting as an important vector of DA to these XatWsh species.

Since polychaetes comprise a large portion of manybeanthic organisms’ diet, the correlation between highDA levels in the viscera of those XatWsh with diets relyingheavily on polychaetes suggests that other organisms withsimilarly constrained diets may also be exposed. Someexamples of higher trophic levels that feed on polychaetesand/or other benthic invertebrates, and are therefore possi-bly exposed to DA through their diet, given the results of

this study, are shown in Table 3. Listed in the table areexamples of both commercially important species and thosethat play an important role in the ecology of nearshore andoVshore environments.

A complication in predicting the risk of DA exposure tobottom-feeding organisms is the temporal decouplingbetween toxic cell presence at the surface and DA in thebenthos. Sampling limitations make it diYcult to properlyaccount for complicating factors such as patchiness ofPseudo-nitzschia spp. in surface water throughout Monte-rey Bay, as indicated by the several order of magnitudediVerence seen in concentration of cells at the 11 stationssampled throughout the bay on monthly transects of CIMTcruises (M. Silver, personal observation). Additionally, dueto foraging of the mobile species sampled, XatWsh may nothave been feeding at the location in which they werecaptured within the bay, making it diYcult to track the pathof the toxin from the euphotic zone to the benthic XatWsh.

Many physical and biological phenomena aVect thedescent of cells from the surface to depth. The rate of

Fig. 2 Two hypothetical pathways of domoic acid (DA) exposure to oVshore XatWsh, including both a pelagic and a benthic pathway of transfer

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2060 Mar Biol (2007) 151:2053–2062

delivery of cells to the seaXoor depends on the mechanismof sedimentation of toxic cells, whether by cell adhesion tomarine snow, coagulation of toxic cells into aggregates, orthrough packaging into fecal pellets, with cell aggregationand stickiness being promoted by the production of trans-parent exopolymers by the cells (Kiørboe and Hansen1993; Kiørboe et al. 1996; Engel 2000; Passow et al. 2001).In addition, cells at the surface will not settle in a straightvertical trajectory (Siegel et al. 1990), as surface and sub-surfaces eddies, along with seasonal variations in currentspeeds and directions at depth would transport sinking par-ticles in varying directions as they settled through the watercolumn in this complex boundary current of the CaliforniaCurrent system (Collins et al. 2003).

Most research on DA, other than that on bivalves, hasfocused on the pelagic food web and nearshore benthic envi-ronments, with contamination of oVshore benthic food websby DA receiving little attention. Studies on DA in bottom-dwellers to date have mostly focused on shallow nearshoreinvertebrates such as bivalves and crustaceans, which—incontrast to Wsh examined in this study—may be more likelyto have toxic tissue concentrations that correlate temporallywith toxic cell concentrations in their local environment(Drum et al. 1993; Langlois et al. 1993; Altwein et al. 1995;Lund et al. 1997; Amzil et al. 2001; Vale and Sampayo2001; Blanco et al. 2002a, b; Ferdin et al. 2002; Lefebvreet al. 2002a; Kaniou-Grigoriadou et al. 2005). Both the iden-tity of dominant vectors and the extent and residence time ofDA in the sediment oVshore on the continental shelf are notyet well known. However, there is growing evidence thattoxic Pseudo-nitzschia spp. cells and/or organic debrisderived from them, are reaching the seabed during and afterblooms, providing a mechanism for DA to enter the near-and oVshore benthic food web (Dortch et al. 1997; Vale and

Sampayo 2001; Parsons et al. 2002; Costa et al. 2003, 2005a,b; Goldberg 2003). With an apparent increase in intensityand frequency of harmful algal blooms (HallegraeV 1993;Anderson 1995; Anderson et al. 2002; Parsons et al. 2002),contamination of benthic Wsheries and ecosystems maybecome a progressively more serious problem. There is cur-rently no routine practice of analyzing commercial Wsh forDA toxins as there is for bivalves, a practice that may proveof beneWt to public health, as would the early removal of theviscera to prevent diVusion of the toxin into the tissue. Thisstudy has shown that DA is often found in the viscera ofcommercially important XatWsh throughout the year, albeit atlow levels during the study period, and suggests that there isa source of DA in the benthos that may have widespreadimpact on the benthic ecosystem.

Acknowledgments We thank Don Pearson, NMFS-Santa Cruz, forproviding valuable information on XatWsh identiWcation and biologyand NMFS Santa Cruz laboratory for donation of XatWsh samples fromthe groundWsh ecology cruise; Lee Bradford, captain of the R/V John-son, for collection of XatWsh samples; Chris Reeves and Rozalind An-trobus, both of UCSC, for assistance in collection and dissection of Wshsamples; Greg Caillet from Moss Landing Marine Laboratories foradditional information on XatWsh feeding behavior and access to liter-ature on the subject; and MBARI staV for providing water samplesfrom M1. This research was supported by funding from the Friends ofLong Marine Lab and the Meyers Oceanographic and Marine BiologyTrust to V. Vigilant and the NOAA Center for Integrated Marine Tech-nology (CIMT) project (NOAA Award #NA16OC2936-3) and aUniversity of California OYce of the President Award to M. Silver(03T-CEQI¡07-0062).

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Table 3 Selected examples of marine organisms in Monterey Bay, California, USA potentially exposed to domoic acid (DA) in their diet by thepossible vectoring of the toxin through polychaetes, sediment, and miscellaneous benthic invertebrates

Species Potential source of DA Known DA contamination?

Estuarine/migrating birds e.g. black-bellied plover (Pluvialis squatarola)

Polychaetes (and other benthic invertebrates)

No

Benthic sharks and rays e.g. round stingray (Urobatis halleri) e.g. spiny dogWsh shark (Squalus acanthias)

Polychaetes(and other benthic invertebrates)

No

Rock crabs (Cancer spp.) Benthic invertebrates Yes: Cheung et al. (personal communication)—nearshore

Additional XatWsh species e.g. starry Xounder (Platichthys stellatus)

Polychaetes (and other benthic invertebrates)

No

Benthic-feeding Wsh e.g. white croaker (Genyonemus lineatus)

Benthic invertebrates and sediment Yes: Fire and Silver (2005)—nearshore

Grey whales (Eschrichtius robustus) Benthic amphipods and sediment Yes: Ch’ng et al. (2002)

CA sea otter (Enhydra lutris) Benthic invertebrates Yes: Ch’ng et al. (2002)—nearshore

RockWsh (family: Scorpaenidae) Benthic invertebrates or anchovies depending on species and size

Yes: this study, not reported due to egestion of guts during retrieval from depth

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