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Domoic acid in phytoplankton and fish in San Diego, CA, USA
Harmful Algae 5 (2006) 91–101 www.elsevier.com/locate/hal
Domoic acid in phytoplankton and fish in San Diego, CA, USA L.B. Busse a,*, E.L. Venrick a, R. Antrobus b, P.E. Miller c, V. Vigilant b, M.W. Silver c, C. Mengelt d, L. Mydlarz d, B.B. Prezelin d a
Integrative Oceanography Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla CA 92093, USA b Ocean Sciences Department, University of California Santa Cruz, Santa Cruz, CA 95064, USA c Institute of Marine Sciences, University of California Santa Cruz, Santa Cruz, CA 95064, USA d Marine Science Institute, Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106, USA Received 7 January 2005; received in revised form 24 February 2005; accepted 7 June 2005
Abstract We provide the first confirmation of the presence of domoic acid (DA) in phytoplankton and fish in San Diego, California, based on samples collected between 1 October 2003 and 29 September 2004. In February 2004, we detected DA in seawater samples collected off the Scripps Pier and also in coastal samples as far as 120 km to the north. At the same time we observed populations of toxic Pseudo-nitzschia australis and Pseudo-nitzschia multiseries as high as 7.7 104 cells l 1. Elevated concentrations of DA and abundances of the toxic species were also found further north in coastal waters of Orange County and, to a lesser extent, in southern Los Angeles County. DA concentrations in the viscera from four species of fish obtained at or near the Scripps Pier ranged from low to above the critical level for public safety. Samples of mussel tissues from the Scripps Pier analyzed by the State Department of Health Services contained low but detectable amounts of DA. Concomitant sea lion strandings from San Diego to Malibu Beach may be related to the presence of DA. DA in tissue from mussels and fish provides evidence for the local transfer of DA from an algal source to higher trophic levels in San Diego coastal waters. # 2005 Elsevier B.V. All rights reserved. Keywords: Domoic acid; Fish; Mussels; Phytoplankton; Pseudo-nitzschia; San Diego; Toxic algal blooms
1. Introduction
* Corresponding author. Tel.: +1 858 534 4605; fax: +1 858 534 6500. E-mail address: [email protected] (L.B. Busse).
There are a growing number of reports of toxic phytoplankton in coastal waters around the world. The increase in such reports may reflect an enhanced awareness and monitoring of harmful algal blooms, or they may document an increasing frequency of toxic
1568-9883/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2005.06.005
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phytoplankton worldwide. Domoic acid (DA), a naturally occurring but rare amino acid toxic to marine mammals and seabirds, as well as to humans, was first associated with a phytoplankton source in 1987, when over 100 people were sickened and three died after consuming DA-contaminated mussels from eastern Prince Edward Island, Canada (Bates et al., 1989; Wright et al., 1989). At least nine species within the marine diatom genus Pseudo-nitzschia H. Peragallo are now known to produce DA (Bates, 2000). In California, DA was first recognized in September 1991, in Monterey Bay, after a dramatic seabird kill when the deaths of more than 100 brown pelicans and cormorants were linked to DA poisoning (Buck et al., 1992; Fritz et al., 1992; Work et al., 1992). Since then, the toxin has been implicated in other deaths of marine mammals and seabirds between Monterey Bay and San Diego. In 1998, hundreds of sea lions (Zalophus californianus) stranded along Californian beaches. Those along the central Californian Coast were confirmed to be caused by DA poisoning (Scholin et al., 2000; Trainer et al., 2000). The same year, in San Diego, Seaworld reported 170 sick sea lions. Symptoms were consistent with DA poisoning, although samples for neither DA nor toxic Pseudo-nitzschia in local waters were collected. From that time, strandings of sea lions that exhibit symptoms of DA poisoning have continued in northern and central California and now appear to be recurrent but sporadic phenomena along the coastline north of Pt. Conception (Marine Mammal Center, unpubl. data). In 2002, coincident with marine mammal strandings in San Diego, a massive beaching of the pelagic red crab (Pleuroncodes planipes) occurred. Concentrations of DA in the crab tissues reached 374 mg DA g 1 tissue well above the critical level for human safety (California Department of Health Service, 2002). Phytoplankton populations in the waters of San Diego, near the Scripps Pier have been studied for over eight decades (Allen, 1920, 1936, 1938, 1945; Reid et al., 1970, 1985; Lange et al., 1994; Fryxell et al., 1997). In spite of that, the history of toxic Pseudo-nitzschia blooms near the Scripps Pier is poorly known because many species now recognized to be toxic (especially P. australis Frenguelli and P.
multiseries (Hasle) Hasle) have historically been identified as ‘‘Nitzschia seriata (Cleve) Peragallo’’, a composite taxon that may contain toxic and nontoxic species. Species of this complex are known to have occurred off southern California since 1917 (Lange et al., 1994; Fryxell et al., 1997; Thomas et al., 2001). Examination of archival material from the Allen collection with scanning electron microscopy (SEM) identified P. australis in the samples (Lange et al., 1994; this study). In March 1991 a bloom of P. australis (106 cells l 1) was reported at Scripps Pier, but only low concentrations of DA were detected in the mussels at that time (Lange et al., 1994), and no measurements were obtained of DA in phytoplankton or fish. The presence or absence of DA contamination in local food webs may reflect neither the magnitude of the local population of toxic Pseudo-nitzschia nor the local production of DA. First, we do not understand the environmental and oceanographic conditions that stimulate DA production (Buck et al., 1992; Walz et al., 1994). Thus, Pseudo-nitzschia cells may be present with DA levels too low to contaminate planktivores. Second, although depuration of mussels and fish is normally fairly rapid (Scholin et al., 2000; Silvagni, 2003), we cannot completely eliminate the possibility that wide ranging fish, birds or mammals obtain the toxin elsewhere. Since 1991 coast wide monitoring for toxins has been carried out by the California Department of Health Service (CDHS). Analysis for DA has been limited to mussel tissues, the sentinel organism for the state program. CDHS personnel provide qualitative and semi-quantitative assessment of dominant phytoplankton taxa, including Pseudo-nitzschia. Although the agency is not able to discriminate toxic from non-toxic species, toxic species may dominate Pseudo-nitzschia populations in California (Walz et al., 1994). The action levels for DA in shellfish, originally set in Canada, is 20 mg g 1 DA shellfish tissue (=20 ppm; Anderson et al., 2001), and this level is now widely used by other countries, including the U.S. From the toxicity perspective, danger levels for the California food web can be considered 5 104 cells l 1 P. australis, the concentration at which mussels and fish reach average toxin levels considered unfit for human consumption (Silver, unpubl. data).
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Until now, DA production in California has been documented only in Los Angeles and further north. Specific studies of DA and links between DA in phytoplankton, fish and mussels, and sea lion strandings have been focused on the central Californian Coast, between Santa Barbara and Monterey Bay (Scholin et al., 2000; Trainer et al., 2000). Data from CDHS obtained between 1999 and 2002 show that DA levels in mussel tissues from Orange County and San Diego have consistently been below the detection limits, whereas Pseudo-nitzschia spp. have been present in phytoplankton samples most of the time (California Department of Health Service, 1999– 2002). Although DA is thought to be the cause of major sea lion strandings in Orange County and San Diego, prior to the present study, DA was not measured in local seawater or fish, nor were Pseudo-nitzschia population sizes quantified at the time of sea lion strandings. This leaves open the possibility that the sea lions obtained the toxin elsewhere, but succumbed in Southern California. This paper presents the first confirmation in San Diego of DA in phytoplankton during a bloom of P. australis and P. multiseries, both of which are known to produce the toxin in Californian waters. This event was observed from San Diego to Orange County during February 2004. We simultaneously detected DA in two species of pelagic fish (mobile species with potentially wide foraging ranges), in two species of bottom or near-bottom feeding fish (presumed to feed locally), and in mussels incubated below Scripps Pier. At least the latter indicate local transfer of the toxin.
2. Methods 2.1. Study sites The samples for this study were collected from Scripps Pier (San Diego County), Oceanside Pier, San Clemente Pier, Newport Pier (all Orange County), Redondo Pier, and Malibu Pier (both Los Angeles County; Fig. 1), between 1 October 2003 and 29 September 2004. In the following analyses, we use the term ‘‘bloom period’’ to refer to data collected between 3 December 2003 and 28 April 2004. This period encompasses all occurrences of toxic Pseudo-
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Fig. 1. Map of sampling locations.
nitzschia species. Water samples were collected with a bucket from just below the water surface. Net tows were vertical tows with a 20 mm meshed net between about 3 m and the surface. 2.2. Environmental data Rainfall amounts were recorded at San Diego Lindberg Field and are available at: http://cdo.ncdc.noaa.gov/CDO/cdo. Temperature was determined to 0.1 8C from the bucket sample using a digital thermometer. Salinity was measured with a salinometer to 0.003 equiv. psu. Water samples for nitrate, phosphate and silicate were filtered through a GF/F filter and then stored in the freezer until analysis. Analyses were performed at a QuickChem Lachat 8000 Series, Flow Injection Analyzer at the University of California Santa Cruz using standard methods for nutrients. Samples for chlorophyll a were filtered through a Whattman GF/F glass fiber filter, stored in liquid nitrogen until analysis 1–2 months later. Chlorophyll was extracted into 90% acetone and analyzed with a fluorometer. 2.3. Pseudo-nitzschia abundances The abundances of P. australis and P. multiseries were estimated using fluorescent in situ hybridization
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(FISH) in a filter-based whole cell format (Scholin et al., 1996; Miller and Scholin, 1996, 1998, 2000), which utilizes a species-specific molecular probe with a fluorescent tag. Ten ml seawater samples were filtered onto 1.2 mm polycarbonate filters and preserved in saline ethanol (Miller and Scholin, 2000) prior to hybridization reactions. Fluorescently labeled cells were counted on the entire surface of duplicate filters using an epifluorescence microscope. Counts were then averaged for each species. During the peak of the bloom in February 2004 the species P. australis was confirmed as the dominant species using the SEM. 2.4. Domoic acid in phytoplankton and animal tissue DA was measured in material from duplicate 500 ml water samples, each filtered onto a GF/F filter and immediately frozen. DA concentrations were measured by standard FMOC-HPLC methods (Pocklington et al., 1990) at the University of California Santa Barbara. The detection limit is 0.02 mg l 1 DA. Fish species were two pelagic species, Scomber japonicus (chub mackerel) and Trachurus symmetricus (jack mackerel), which were caught by hook and line from the Scripps Pier, and two bottom (or nearbottom) feeding species, Citharichthys sordidus (Pacific sandab) and Zaniolepis latipinnis (longspine combfish), which were caught with a trawl near the pier. All fish were adults. The fish were immediately frozen whole. Measurements for DA in fish viscera were carried out at the University of California Santa Cruz using HPLC-UV methods (Quilliam et al., 1995). The viscera of individuals from each fish species were combined into one sample per species. Mussels (primarily Mytilus californianus, occasionally M. edulis) were collected locally and incubated off the Scripps Pier for 1–2 weeks before harvesting. DA levels in mussels were determined by the CDHS using HPLC-UV regulatory method (Quilliam et al., 1991; Dhoot et al., 1993). All animal samples were measured in mg DA l 1 tissue (wet weight). 2.5. Statistical analyses Statistical analyses are restricted to nonparametric correlations, using Spearman’s r. We use r to screen our data for possible causal mechanisms, recognizing
that, with a single annual cycle, our statistics lack generality. Because of the multitude of correlations (many of which are not shown) and their lack of independence, the probability levels are non-conservative. Many relationships may be obscured by time lags; our data is too sparse to examine these objectively.
3. Results 3.1. Environmental background During the study period (1 October 2003–29 September 2004), near-surface temperature at Scripps Pier ranged between 13.8 8C (7 January) and 24.0 8C (9 September) (Fig. 2a). Between 23 December and 25 February, values were consistently below 15 8C. Salinity during the sampling period was generally correlated with temperature, varying between 33.05 psu on 10 March and 33.71 psu on 22 September (Fig. 2a). In February 2004, storms brought heavier rainfall, totaling 71.37 mm for February (Fig. 2b), which may explain the low salinity at the end of that month. Off Scripps Pier, nitrate concentrations varied between 0–34.8 mg l 1 N, phosphate concentrations between 0 and 29.89 mg l 1 P, and silicate concentrations between 0.029 and 0.345 mg l 1 Si (Fig. 2c). Concentrations tended to be higher at the more northern locations. Over all our samples, nitrate, phosphate and silicate are highly correlated (all p < .001). Nitrate and phosphate tended to reach minimum values in June and July. Between October and February, all nutrients showed frequent, short bursts of elevated concentrations. A relationship between nutrients and rainfall is not clear. There were frequent observations of elevated nutrients in the absence of rainfall. The highest nitrate concentration (30 October 2003) was preceded by at least a month of dry weather. Thus, we conclude that least one source of nutrients other than rainfall must have been present. Over the same period, chlorophyll a varied between 0.53 and 64.3 mg m 3 (Fig. 2d). Highest concentrations were found in October 2003 and were due to a large bloom of the dinoflagellate Lingulodinium polyedrum, which dominated the flora throughout much of the sampling period. Its persistence was unusual and has not been explained.
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3.2. Toxic Pseudo-nitzschia and domoic acid levels in phytoplankton The total abundance of toxic Pseudo-nitzschia (P. australis and P. multiseries) ranged from undetectable to a maximum of 7.7 104 cells l 1 (Fig. 2e and f). Abundances of the two species were highly correlated
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both over all samples and during the bloom period. In samples that contained toxic Pseudo-nitzschia, over 70% of the toxic cells were P. australis, and thus P. australis is probably the main contributor to DA in this study. The sum of P. australis and P. multiseries is hereafter referred to as toxic Pseudo-nitzschia. We assume that most or all of the DA measured in
Fig. 2. Data from Scripps Pier, 1 October 2003–29 September 2004. (a) Sea surface temperature (SST) and salinity; (b) rain; (c) nitrate, phosphate and silicate concentrations; (d) chlorophyll a concentration; (e) abundance of P. australis; (f) abundance of P. multiseries; (g) particulate domoic acid concentration, when available.
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Fig. 2. (Continued ).
particulate material was contained by these two species. No other Pseudo-nitzschia species were dominant in the net tow at that time. Toxic Pseudo-nitzschia first appeared at Scripps Pier on 23 December 2003. Until 4 February 2004 toxic species were seen sporadically and in low abundances (1.8–3.4 103 cells l 1; Fig. 2e and f). At the same time particulate DA concentrations ranged between the detection limit and 0.58 mg l 1 (Fig. 2g). Abundances of toxic Pseudo-nitzschia cells and DA levels showed sudden and sharp increases in the second half of February 2004. At Scripps Pier, highest toxic Pseudo-nitzschia abundances (7.7 104 cells l 1, above the critical level of 5 104 cells l 1) were recorded on 18 February accompanied by DA concentrations of 1.98 mg l 1 (Fig. 2e–g). One week later the abundances decreased below the critical level, but we still measured particulate DA concentrations of 1.48 mg l 1 (Fig. 2e–g). There was no toxic Pseudonitzschia seen after 21 April 2004. DA was close to the detection limit in May, and was not detected in water samples thereafter.
The Pseudo-nitzschia bloom occurred during the winter period when the environment was relatively cool (13.9–14.3 8C) and fresh (33.16–33.22 psu; Fig. 2a). During the bloom period, chlorophyll a concentrations varied between 0.92 and 12.5 mg m 3 (Fig. 2d) and were only weakly correlated with the abundance of toxic Pseudo-nitzschia (Fig. 3a). Although abundant, the Pseudo-nitzschia species never reached high enough numbers to be a major determinant of phytoplankton biomass. During the bloom period, we observed short bursts of elevated nutrients, but the nutrient concentrations were not the most extreme observed, nor were they consistently high. During the bloom period, correlations between the three nutrients and the abundance of toxic Pseudonitzschia were weak (e.g. Fig. 3b). Neither does there seem to be a relationship of with the nutrient ratios (e.g. Fig. 3c). The bloom of toxic Pseudo-nitzschia and elevated concentrations of particulate DA at Scripps Pier prompted us to sample other areas in Orange and Los Angeles Counties to determine the spatial extent of the
Fig. 3. Relationships between environmental variables and toxic Pseudo-nitzschia species. (a) Chlorophyll concentration and the abundance of toxic Pseudo-nitzschia species at all locations during the bloom period; (b) concentration of near-surface nitrate-N and the abundance of toxic Pseudo-nitzschia species at all locations during the bloom period; (c) the relationship of near-surface nitrate-N and phosphate-P at all locations, bloom period indicated as , non-bloom period as closed circle.
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Table 1 Location, sampling date, temperature, salinity, chlorophyll, abundance of toxic Pseudo-nitzschia (sum of P. australis and P. multiseries), and particulate domoic acid concentrations in San Diego County, Orange County, and Los Angeles County; at the peak of the bloom in February 2004; n.d. = no data Location
Sampling date
Temperature (8C)
Salinity (psu)
Chl a (mg m 3)
Toxic Pseudo-nitzschia (cells l 1)
Domoic acid (mg l 1)
Malibu Pier Redondo Pier
20 20 27 20 27 19 27 19 27 11 18 25
13.5 15.0 14.1 15.0 14.4 n.d. 14.7 n.d. 14.6 14.2 13.9 14.3
33.19 33.22 33.92 33.11 30.92 33.13 32.92 33.03 32.28 33.21 33.22 33.16
3.66 1.20 1.52 6.91 4.57 3.97 3.29 4.99 6.57 3.61 3.91 1.78
1300 6950 2250 70500 1250 38000 0 66950 14900 44100 76600 37400
n.d. n.d. n.d. 2.33 0.05 1.24 0 n.d. n.d. 0.22 1.98 1.48
Newport Pier San Clemente Pier Oceanside Pier Scripps Pier
February February February February February February February February February February February February
2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004
Table 2 Domoic acid measurements in mussels and fish (viscera only) Common name
Species name
# Individuals
Sampling date
Domoic acid (mg DA g 1) tissue
Mussels Chub Mackerel Jack Mackerel Pacific sanddab Longspine combfish
M. californianus S. japonicus T. symmetricus C. sordidus Z. latipinnis
27 8 8 11 5
25 February 2004 1 March 2004 1 March 2004 3 March 2004 3 March 2004
5.8 7.3 5.5 50.1 9.7
All were collected at or near the Scripps Pier, San Diego County. All individuals from one species were combined into one sample for domoic acid measurements.
event. At the piers closest to San Diego (Oceanside, San Clemente, and Newport), we found abundances of toxic Pseudo-nitzschia that were comparable to those at Scripps Pier (Table 1). Abundances declined further north in Los Angeles County (Redondo and Malibu Piers). Because of our limited sampling, we cannot eliminate the possibility that the bloom was advecting southward and had been more strongly developed at the two northern sites prior to our sampling. Abundances at all sites decreased between 19/20 February and 27 February. Where data are available, elevated concentrations of DA accompanied the elevated abundances of toxic Pseudo-nitzschia (Table 1). Data from CDHS survey reports show that the relative abundance of total Pseudo-nitzschia (which may include both toxic and non-toxic species) from mid- to late-February increased slightly at sites near Santa Barbara and San Luis Obispo, while sites further north showed only low numbers (California Department of Health Service, 2004). Thus, in
February, the southern California coast had the most dramatic increase in toxic Pseudo-nitzschia. Later in the season, strong blooms with higher cell numbers of P. australis and P. multiseries were observed between Santa Barbara (February–May 2004) and Santa Cruz (April–May 2004; Silver et al., unpubl. data). 3.3. Domoic acid levels in fish and mussels Mussels from Scripps Pier, retrieved on 25 February, showed low, but detectable levels of DA (5.8 mg DA g 1; Table 2). S. japonicus and T. symmetricus, caught on 1 March, also showed low DA (7.3 and 5.5 mg DA g 1 respectively; Table 2). We found higher DA concentrations in near-bottom feeding fish, which were caught on 3 March. These included C. sordidus with DA concentrations of 50.1 mg DA g 1 and Z. latipinnis, with concentrations of 9.7 mg DA g 1.
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3.4. Sea lion strandings During February 2004, California sea lions stranded along southern California beaches, and distressed but live animals were brought into various facilities for treatment: Seaworld (San Diego County: five sea lions; Leger, pers. commun.), Pacific Marine Mammal Center (Orange County: seven sea lions; Hunter, pers. commun.) and Marine Mammal Care Center at Fort MacArthur (Los Angeles County: seven sea lions; Pareti, pers. commun.). All the animals receiving treatment showed one or more symptoms of DA poisoning, which include confusion, disorientation, seizures, excessive scratching and rubbing, and an extremely lethargic or comatose condition (Lefebvre et al., 2001).
4. Discussion Several previous studies off central and southern California (Garrison et al., 1992; Lange et al., 1994; Trainer et al., 2000) suggest that Pseudo-nitzschia blooms are associated with cool, high-nutrient waters that may be related to upwelling events. In other areas, toxic Pseudo-nitzschia blooms have appeared after a period of heavy rainfall resulting in nutrient-rich freshwater runoff (Trainer et al., 2000). At the Scripps Pier, temperatures during the bloom were close to the annual minimum, during a period when short bursts of elevated nutrients were frequent. The start of the bloom preceded the heavy rain. Thus, while fresh water or nutrient addition from runoff may have contributed to the subsequent bloom characteristics, they seem unlikely to have initiated the bloom. Some level of nutrient injection from deeper water seems the more likely explanation. This is the first documentation of DA in phytoplankton and in fish in San Diego. However, the maximum abundance of Pseudo-nitzschia found at Scripps Pier (7.7 104 cells l 1) is relatively low compared to that found in other studies along the coast. Likewise, the highest particulate DA levels found in this study in phytoplankton (2.3 mg l 1) are in the moderate range of DA concentrations. In a major toxic event in June 1998, Trainer et al. (2000) reported maximum abundances of the toxic cells (P. australis and P. multiseries) between San Francisco
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and Monterey Bay ranging from 5 104 to 5 105 cells l 1 and a maximum value of 1.1 107 cells l 1 offshore of Santa Barbara on 4 June 1998. At that time, DA levels up to 7.3 mg l 1 were found in particulate material from water samples, and the event was accompanied by massive marine mammal strandings. Records from Monterey over a 4year period show highest abundances of toxic Pseudonitzschia ranging from 105 to 106 cells l 1, with the higher values usually found offshore (Lefebvre et al., 2001; Silver, unpubl. data). Mammal strandings north of Pt. Conception from 1999 to 2002 occurred when coastal water samples contained particulate DA levels ranging from non-detectible up to 50 mg l 1 (Gulland and Silver, unpubl. data). During February 2004, the DA concentrations within cells of P. australis and P. multiseries varied between 5 and 43 pg cell 1. This is within the range of cellular DA found in samples along the central California coast in 1998 (7–75 pg cell 1; Scholin et al., 2000). Thus, the toxicity of our bloom may have been controlled by population size rather than DA production. A repeat occurrence of blooms of toxic Pseudo-nitzschia as large as those seen in the past (1 106 cell l 1 P. australis in March 1991, Lange et al., 1994) has the potential for a much greater impact. The concentration of DA in mussels at Scripps Pier (5.8 mg DA g 1) is well within the safety guidelines set by the US Food and Drug Administration (alert level: 20 mg DA g 1), but higher than the levels found in May 1992 when the mussels contained 2.2 mg DA g 1 (Lange et al., 1994). The viscera of pelagic fish showed similar DA concentrations (Table 2). In planktivores like anchovies and sardines, which are key vectors of DA to marine birds and mammals, most of the toxin in the fish is found in the gut. Consequently, the toxin is lost when the gut contents are voided and the toxin is usually present only when the fish are feeding or have recently fed (Lefebvre et al., 2002). Generally, toxin burdens in such planktivorous fish correspond approximately to the total toxin in Pseudo-nitzschia populations in the region (Lefebvre et al., 2001). In our study, the viscera of bottom-feeding fish, especially C. sordidus, contained DA concentrations up to 10 times higher than did the viscera of pelagic fish. C. sordidus is a popular food fish, and it has been found to have elevated toxin levels in the viscera at
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other sites along the California coast during blooms of toxic Pseudo-nitzschia (Lefebvre et al., 2002; Goldberg et al., submitted). Some benthic fish and benthic invertebrates contain toxin when DA levels in the water are not measurable and when no toxic Pseudonitzschia are detected (Goldberg et al., submitted). Such an apparent uncoupling of toxin between phytoplankton and benthic animals could result from flocculation of the toxic cells and settlement to the benthos, where the toxic material is consumed directly or indirectly by benthic animals capable of sequestering the toxin (e.g. razor clams; Trainer and Bill, 2004).
5. Conclusions Confirmation of the presence of domoic acid (DA) during elevated abundances of toxic Pseudo-nitzschia and concurrently in fish and mussels provides evidence for the transfer of DA from a local algal source in San Diego to higher trophic levels. Strandings of sea lions with neurological symptoms typically associated with DA poisoning in areas where we found the toxin producing Pseudo-nitzschia suggest further transfer to marine mammals. Although this is the first confirmation of such an event in San Diego, similar events may have occurred in the past as there is evidence that the toxic species have been in the region, at least on occasion, for over eight decades. Previously, DA was thought to be a problem primarily in northern and central California; our findings indicate that DA can occur further south in Orange and San Diego Counties. It is not known whether our observations are merely the result of increased awareness and better detection techniques (i.e. we looked for it) or whether the production of DA in southern Californian waters has intensified recently, possibly spreading south from the central coast of California. Given our findings, a more regular monitoring of DA in phytoplankton and fish is recommended for Orange and San Diego Counties and even further south.
Acknowledgements This project was funded through the University of California Coastal Environmental Quality Initiative
(Grant# 03 T CEQI 07 0062). We thank Gregg Langlois and the California Department of Health Service for the DA analysis of mussel tissue. We also thank Judy St. Leger (Seaworld), Michele Hunter (Pacific Marine Mammal Center) and Jennifer Pareti (Marine Mammal Care Center at Fort MacArthur) for providing the data on marine mammal strandings. Thanks to Eddie Kisfaludy (Scripps Institution of Oceanography) for helping us with the initial set up for our study and for collecting the fish, and to Teresa Kacena (Scripps Institution of Oceanography) for measuring salinity.[TS] References Allen, W.E., 1920. Quantitative studies on inshore marine diatoms and dinoflagellates of Southern California in 1920. Univ. Calif. Publ. Zool. 22, 370–377. Allen, W.E., 1936. Occurrence of marine plankton diatoms in a tenyear series of daily catches in Southern California. Am. J. Bot. 23, 60–63. Allen, W.E., 1938. The Templeton Crocker Expedition to the Gulf of California in 1935 – the phytoplankton. Trans. Am. Microsc. Soc. 76, 328–335. Allen, W.E., 1945. Seasonal occurrence of marine diatoms off Southern California in 1938. Bull. SIO Tech. Ser. 5, 293–334. Anderson, D.M., Andersen, P., Bricelj, V.M., Cullen, J.J., Rensel, J.E., 2001. Monitoring and management strategies for harmful algal blooms in coastal waters. APEC #201-MR-01.1, Asia Pacific Economic Program, Singapore, and Intergovernmental Oceanographic Commission Technical Series No. 59, Paris. Bates, S.S., Bird, C.J., de Freitas, A.S.W., Foxall, R., Gilgan, M., Hanic, L.A., Johnson, G.R., McCulloch, A.W., Odense, P., Pocklington, R., Quilliam, M.A., Sim, P.G., Smith, J.C., Subba Rao, D.V., Todd, E.C.D., Walter, J.A., Wright, J.L.C., 1989. Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shellfish from eastern Prince Edward Island, Canada. Can. J. Fish. Aquat. Sci. 46, 1203–1215. Bates, S.S., 2000. Domoic-acid-producing Diatoms: another genus added. J. Phycol. 36, 978–985. Buck, K.R., Uttal-Cooke, L., Pilskaln, C.H., Roelke, D.L., Villac, M.C., Fryxell, G.A., Cifuentes, L., Chavez, F.P., 1992. Autecology of the diatom Pseudo-nitzschia australis, a domoic acid producer, from Monterey Bay, California (USA). Mar. Ecol. Prog. Ser. 84, 293–302. California Department of Health Service, 1999–2002. Monthly Marine Biotoxin Reports, California Department of Health Service, Berkeley. California Department of Health Service, 2004. Monthly Marine Biotoxin Reports, February/March 2004. California Department of Health Service, Berkeley. Dhoot, J.S., del Rosario, A.R., Appel, B.R., Tamplin, B.R., 1993. An improved HPLC procedure for domoic acid analysis in seafood. Int. J. Environ. Anal. Chem. 53, 261–268.
L.B. Busse et al. / Harmful Algae 5 (2006) 91–101 Fritz, L., Quilliam, M.A., Wright, J.L.C., Beale, A.M., Work, T.M., 1992. An outbreak of domoic acid poisoning attributed to the pennate diatom Pseudonitzschia australis. J. Phycol. 28, 439– 442. Fryxell, G.A., Villac, M.C., Shapiro, L.P., 1997. The occurrence of the toxic diatom genus Pseudo-nitzschia (Bacillariophyceae) on the West Coast of the USA, 1920–1996: a review. Phycologia 36, 419–437. Garrison, D.L., Conrad, S.M., Eilers, P.P., Waldron, E.M., 1992. Confirmation of domoic acid production by Pseudo-nitzschia australis (Bacillariophyceae) cultures. J. Phycol. 28, 604–607. Lange, C.B., Reid, F.M.H., Vernet, M., 1994. Temporal distribution of the potentially toxic diatom Pseudonitzschia australis at a coastal site in Southern California. Mar. Ecol. Prog. Ser. 104, 309–312. Lefebvre, K.A., Dovel, S.L., Silver, M.W., 2001. Tissue distribution and neurotoxic effects of domoic acid in a prominent vector species, the northern anchovy Engraulis mordax. Mar. Biol. 138, 693–700. Lefebvre, K.A., Silver, M.W., Coale, S.L., Tjeerdema, R.S., 2002. Domoic acid in planktivorous fish in relation to toxic Pseudonitzschia cell densities. Mar. Biol. 140, 625–631. Miller, P.E., Scholin, C.A., 1996. Identification of cultured Pseudonitzschia (Bacillariophyceae) using species-specific LSU rRNAtargeted fluorescent probes. J. Phycol. 30, 646–655. Miller, P.E., Scholin, C.A., 1998. Identification and enumeration of cultured and wild Pseudo-nitzschia (Bacillariophyceae) using species-specific LSU rRNA-targeted fluorescent probes and filter-based whole cell hybridization. J. Phycol. 34, 371– 382. Miller, P.E., Scholin, C.A., 2000. Detection of Pseudo-nitzschia (Bacillariophyceae) species using whole cell hybridization: sample fixation and stability. J. Phycol. 26, 1–13. Pocklington, R., Milley, J.E., Bates, S.S., Bird, C.J., de Freitas, A.S.W., Quilliam, M.A., 1990. Trace determination of domoic acid in seawater and phytoplankton by high-performance liquid chromatography of the flourenylmethoxycarbonyl (FMOC) derivative. Int. J. Environ. Anal. Chem. 38, 351–368. Quilliam, M.A., Xie, M., Hardstaff, W.R., 1991. A rapid extraction and clean-up procedure for the determination of domoic acid in tissue samples. NRC Institute for Marine Biosciences, Technical Report #64, NRCC 33001. Quilliam, M.A., Xie, M., Hardstaff, W.R., 1995. Rapid extraction and cleanup for liquid chromatographic determination of domoic acid in unsalted seafood. J. AOAC Int. 78, 543–554. Reid, F.M.H., Fuglister, E., Jordan, J.B., 1970. Phytoplankton taxonomy and standing crop. In: Strickland, J.D.H. (Ed.), The
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Ecology of the Plankton off La Jolla CA in the period of April through September 1967. Bull. SIO Tech. Ser. 17, 51–66. Reid, F.M.H., Lange, C.B., White, M.M., 1985. Microplankton species assemblages at the Scripps Pier from March to November 1983 during the 1982–1984 El-Nin˜o event. Bot. Mar. 28, 443–452. Scholin, C.A., Buck, K.R., Britschgi, T., Cangelosi, G., Chavez, F.P., 1996. Identification of Pseudo-nitzschia australis (Bacillariophyceae) using rRNA-targeted probes in whole cell and sandwich hybridization formats. Phycologia 35, 190–197. Scholin, C.A., Gulland, F., Doucette, G.J., Benson, S., Busman, M., Chavez, F.P., Cordaro, J., DeLong, R., De Vogelaere, A., Harvey, J., Haulena, M., Lefebvre, K., Lipscomb, T., Loscutoff, S., Lowenstine, L.J., Roman III, M., Miller, P.E., McLellan, W.A., Moeller, P.D.R., Powell, C.L., Rowles, T., Silvagni, P., Silver, M., Spraker, T., Trainer, V., Van Dolah, F.M., 2000. Mortality of sea lions along the central California coast linked to a toxic diatom bloom. Nature 403, 80–84. Silvagni, P.A. 2003. Comparative pathology and diagnosis of domoic acid toxicity. PhD Thesis, University of California, Davis, 139 pp. Thomas, W.H., Zavoico, T., Hewes, C., 2001. Historical phytoplankton species time-series data (1917–1939) from the North American Pacific Coast. SIO Ref. No. 01–12. Trainer, V.L., Adams, N.G., Bill, B.D., Stehr, C.M., Wekell, J.C., Moeller, P., Busman, M., Woodruff, D., 2000. Domoic acid production near California coastal upwelling zones. Limnol. Oceanogr. 45, 1818–1833. Trainer, V.L., Bill, B.D., 2004. Characterization of a domoic acid binding site from Pacific razor clam. Aquat. Toxicol. 69, 125– 132. Walz, P.M., Garrison, D.L., Graham, W.M., Cattey, M.A., Tjeerdema, R.S., Silver, M.W., 1994. Domoic acid-producing diatom blooms in Monterey Bay, California: 1991–1993. Nat. Toxins 5, 271–279. Work, T.M, Barr, B., Beale, A.M., Fritz, L., Quilliam, M.A., Wright, J.L.C., 1992. Epidemiology of domoic acid poisoning in brown pelicans (Pelicanus occidentalis) and Brandt’s cormorants (Phalacrocorax penicillatus) in California. J. Zool. Wildl. Med. 24, 54–62. Wright, J.L.C., Boyd, R.K., de Freitas, A.S.W., Falk, M., Foxall, R.A., Jamieson, W.D., Laycock, M.V., McCulloch, A.W., McInnes, A.G., Odense, P., Pathank, V.P., Quilliam, M.A., Ragan, M.A., Sim, P.G., Thibault, P., Walter, J.A., Gilgan, M., Richard, D.J.A., Dewar, D., 1989. Identification of domoic acid, a neuroexciatory amino acid, in toxic mussels from eastern Prince Edward Island. Can. J. Chem. 67, 481–490.
Domoic acid in phytoplankton and fish in San Diego, CA, USA L.B. Busse a,*, E.L. Venrick a, R. Antrobus b, P.E. Miller c, V. Vigilant b, M.W. Silver c, C. Mengelt d, L. Mydlarz d, B.B. Prezelin d a
Integrative Oceanography Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla CA 92093, USA b Ocean Sciences Department, University of California Santa Cruz, Santa Cruz, CA 95064, USA c Institute of Marine Sciences, University of California Santa Cruz, Santa Cruz, CA 95064, USA d Marine Science Institute, Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106, USA Received 7 January 2005; received in revised form 24 February 2005; accepted 7 June 2005
Abstract We provide the first confirmation of the presence of domoic acid (DA) in phytoplankton and fish in San Diego, California, based on samples collected between 1 October 2003 and 29 September 2004. In February 2004, we detected DA in seawater samples collected off the Scripps Pier and also in coastal samples as far as 120 km to the north. At the same time we observed populations of toxic Pseudo-nitzschia australis and Pseudo-nitzschia multiseries as high as 7.7 104 cells l 1. Elevated concentrations of DA and abundances of the toxic species were also found further north in coastal waters of Orange County and, to a lesser extent, in southern Los Angeles County. DA concentrations in the viscera from four species of fish obtained at or near the Scripps Pier ranged from low to above the critical level for public safety. Samples of mussel tissues from the Scripps Pier analyzed by the State Department of Health Services contained low but detectable amounts of DA. Concomitant sea lion strandings from San Diego to Malibu Beach may be related to the presence of DA. DA in tissue from mussels and fish provides evidence for the local transfer of DA from an algal source to higher trophic levels in San Diego coastal waters. # 2005 Elsevier B.V. All rights reserved. Keywords: Domoic acid; Fish; Mussels; Phytoplankton; Pseudo-nitzschia; San Diego; Toxic algal blooms
1. Introduction
* Corresponding author. Tel.: +1 858 534 4605; fax: +1 858 534 6500. E-mail address: [email protected] (L.B. Busse).
There are a growing number of reports of toxic phytoplankton in coastal waters around the world. The increase in such reports may reflect an enhanced awareness and monitoring of harmful algal blooms, or they may document an increasing frequency of toxic
1568-9883/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2005.06.005
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phytoplankton worldwide. Domoic acid (DA), a naturally occurring but rare amino acid toxic to marine mammals and seabirds, as well as to humans, was first associated with a phytoplankton source in 1987, when over 100 people were sickened and three died after consuming DA-contaminated mussels from eastern Prince Edward Island, Canada (Bates et al., 1989; Wright et al., 1989). At least nine species within the marine diatom genus Pseudo-nitzschia H. Peragallo are now known to produce DA (Bates, 2000). In California, DA was first recognized in September 1991, in Monterey Bay, after a dramatic seabird kill when the deaths of more than 100 brown pelicans and cormorants were linked to DA poisoning (Buck et al., 1992; Fritz et al., 1992; Work et al., 1992). Since then, the toxin has been implicated in other deaths of marine mammals and seabirds between Monterey Bay and San Diego. In 1998, hundreds of sea lions (Zalophus californianus) stranded along Californian beaches. Those along the central Californian Coast were confirmed to be caused by DA poisoning (Scholin et al., 2000; Trainer et al., 2000). The same year, in San Diego, Seaworld reported 170 sick sea lions. Symptoms were consistent with DA poisoning, although samples for neither DA nor toxic Pseudo-nitzschia in local waters were collected. From that time, strandings of sea lions that exhibit symptoms of DA poisoning have continued in northern and central California and now appear to be recurrent but sporadic phenomena along the coastline north of Pt. Conception (Marine Mammal Center, unpubl. data). In 2002, coincident with marine mammal strandings in San Diego, a massive beaching of the pelagic red crab (Pleuroncodes planipes) occurred. Concentrations of DA in the crab tissues reached 374 mg DA g 1 tissue well above the critical level for human safety (California Department of Health Service, 2002). Phytoplankton populations in the waters of San Diego, near the Scripps Pier have been studied for over eight decades (Allen, 1920, 1936, 1938, 1945; Reid et al., 1970, 1985; Lange et al., 1994; Fryxell et al., 1997). In spite of that, the history of toxic Pseudo-nitzschia blooms near the Scripps Pier is poorly known because many species now recognized to be toxic (especially P. australis Frenguelli and P.
multiseries (Hasle) Hasle) have historically been identified as ‘‘Nitzschia seriata (Cleve) Peragallo’’, a composite taxon that may contain toxic and nontoxic species. Species of this complex are known to have occurred off southern California since 1917 (Lange et al., 1994; Fryxell et al., 1997; Thomas et al., 2001). Examination of archival material from the Allen collection with scanning electron microscopy (SEM) identified P. australis in the samples (Lange et al., 1994; this study). In March 1991 a bloom of P. australis (106 cells l 1) was reported at Scripps Pier, but only low concentrations of DA were detected in the mussels at that time (Lange et al., 1994), and no measurements were obtained of DA in phytoplankton or fish. The presence or absence of DA contamination in local food webs may reflect neither the magnitude of the local population of toxic Pseudo-nitzschia nor the local production of DA. First, we do not understand the environmental and oceanographic conditions that stimulate DA production (Buck et al., 1992; Walz et al., 1994). Thus, Pseudo-nitzschia cells may be present with DA levels too low to contaminate planktivores. Second, although depuration of mussels and fish is normally fairly rapid (Scholin et al., 2000; Silvagni, 2003), we cannot completely eliminate the possibility that wide ranging fish, birds or mammals obtain the toxin elsewhere. Since 1991 coast wide monitoring for toxins has been carried out by the California Department of Health Service (CDHS). Analysis for DA has been limited to mussel tissues, the sentinel organism for the state program. CDHS personnel provide qualitative and semi-quantitative assessment of dominant phytoplankton taxa, including Pseudo-nitzschia. Although the agency is not able to discriminate toxic from non-toxic species, toxic species may dominate Pseudo-nitzschia populations in California (Walz et al., 1994). The action levels for DA in shellfish, originally set in Canada, is 20 mg g 1 DA shellfish tissue (=20 ppm; Anderson et al., 2001), and this level is now widely used by other countries, including the U.S. From the toxicity perspective, danger levels for the California food web can be considered 5 104 cells l 1 P. australis, the concentration at which mussels and fish reach average toxin levels considered unfit for human consumption (Silver, unpubl. data).
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Until now, DA production in California has been documented only in Los Angeles and further north. Specific studies of DA and links between DA in phytoplankton, fish and mussels, and sea lion strandings have been focused on the central Californian Coast, between Santa Barbara and Monterey Bay (Scholin et al., 2000; Trainer et al., 2000). Data from CDHS obtained between 1999 and 2002 show that DA levels in mussel tissues from Orange County and San Diego have consistently been below the detection limits, whereas Pseudo-nitzschia spp. have been present in phytoplankton samples most of the time (California Department of Health Service, 1999– 2002). Although DA is thought to be the cause of major sea lion strandings in Orange County and San Diego, prior to the present study, DA was not measured in local seawater or fish, nor were Pseudo-nitzschia population sizes quantified at the time of sea lion strandings. This leaves open the possibility that the sea lions obtained the toxin elsewhere, but succumbed in Southern California. This paper presents the first confirmation in San Diego of DA in phytoplankton during a bloom of P. australis and P. multiseries, both of which are known to produce the toxin in Californian waters. This event was observed from San Diego to Orange County during February 2004. We simultaneously detected DA in two species of pelagic fish (mobile species with potentially wide foraging ranges), in two species of bottom or near-bottom feeding fish (presumed to feed locally), and in mussels incubated below Scripps Pier. At least the latter indicate local transfer of the toxin.
2. Methods 2.1. Study sites The samples for this study were collected from Scripps Pier (San Diego County), Oceanside Pier, San Clemente Pier, Newport Pier (all Orange County), Redondo Pier, and Malibu Pier (both Los Angeles County; Fig. 1), between 1 October 2003 and 29 September 2004. In the following analyses, we use the term ‘‘bloom period’’ to refer to data collected between 3 December 2003 and 28 April 2004. This period encompasses all occurrences of toxic Pseudo-
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Fig. 1. Map of sampling locations.
nitzschia species. Water samples were collected with a bucket from just below the water surface. Net tows were vertical tows with a 20 mm meshed net between about 3 m and the surface. 2.2. Environmental data Rainfall amounts were recorded at San Diego Lindberg Field and are available at: http://cdo.ncdc.noaa.gov/CDO/cdo. Temperature was determined to 0.1 8C from the bucket sample using a digital thermometer. Salinity was measured with a salinometer to 0.003 equiv. psu. Water samples for nitrate, phosphate and silicate were filtered through a GF/F filter and then stored in the freezer until analysis. Analyses were performed at a QuickChem Lachat 8000 Series, Flow Injection Analyzer at the University of California Santa Cruz using standard methods for nutrients. Samples for chlorophyll a were filtered through a Whattman GF/F glass fiber filter, stored in liquid nitrogen until analysis 1–2 months later. Chlorophyll was extracted into 90% acetone and analyzed with a fluorometer. 2.3. Pseudo-nitzschia abundances The abundances of P. australis and P. multiseries were estimated using fluorescent in situ hybridization
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(FISH) in a filter-based whole cell format (Scholin et al., 1996; Miller and Scholin, 1996, 1998, 2000), which utilizes a species-specific molecular probe with a fluorescent tag. Ten ml seawater samples were filtered onto 1.2 mm polycarbonate filters and preserved in saline ethanol (Miller and Scholin, 2000) prior to hybridization reactions. Fluorescently labeled cells were counted on the entire surface of duplicate filters using an epifluorescence microscope. Counts were then averaged for each species. During the peak of the bloom in February 2004 the species P. australis was confirmed as the dominant species using the SEM. 2.4. Domoic acid in phytoplankton and animal tissue DA was measured in material from duplicate 500 ml water samples, each filtered onto a GF/F filter and immediately frozen. DA concentrations were measured by standard FMOC-HPLC methods (Pocklington et al., 1990) at the University of California Santa Barbara. The detection limit is 0.02 mg l 1 DA. Fish species were two pelagic species, Scomber japonicus (chub mackerel) and Trachurus symmetricus (jack mackerel), which were caught by hook and line from the Scripps Pier, and two bottom (or nearbottom) feeding species, Citharichthys sordidus (Pacific sandab) and Zaniolepis latipinnis (longspine combfish), which were caught with a trawl near the pier. All fish were adults. The fish were immediately frozen whole. Measurements for DA in fish viscera were carried out at the University of California Santa Cruz using HPLC-UV methods (Quilliam et al., 1995). The viscera of individuals from each fish species were combined into one sample per species. Mussels (primarily Mytilus californianus, occasionally M. edulis) were collected locally and incubated off the Scripps Pier for 1–2 weeks before harvesting. DA levels in mussels were determined by the CDHS using HPLC-UV regulatory method (Quilliam et al., 1991; Dhoot et al., 1993). All animal samples were measured in mg DA l 1 tissue (wet weight). 2.5. Statistical analyses Statistical analyses are restricted to nonparametric correlations, using Spearman’s r. We use r to screen our data for possible causal mechanisms, recognizing
that, with a single annual cycle, our statistics lack generality. Because of the multitude of correlations (many of which are not shown) and their lack of independence, the probability levels are non-conservative. Many relationships may be obscured by time lags; our data is too sparse to examine these objectively.
3. Results 3.1. Environmental background During the study period (1 October 2003–29 September 2004), near-surface temperature at Scripps Pier ranged between 13.8 8C (7 January) and 24.0 8C (9 September) (Fig. 2a). Between 23 December and 25 February, values were consistently below 15 8C. Salinity during the sampling period was generally correlated with temperature, varying between 33.05 psu on 10 March and 33.71 psu on 22 September (Fig. 2a). In February 2004, storms brought heavier rainfall, totaling 71.37 mm for February (Fig. 2b), which may explain the low salinity at the end of that month. Off Scripps Pier, nitrate concentrations varied between 0–34.8 mg l 1 N, phosphate concentrations between 0 and 29.89 mg l 1 P, and silicate concentrations between 0.029 and 0.345 mg l 1 Si (Fig. 2c). Concentrations tended to be higher at the more northern locations. Over all our samples, nitrate, phosphate and silicate are highly correlated (all p < .001). Nitrate and phosphate tended to reach minimum values in June and July. Between October and February, all nutrients showed frequent, short bursts of elevated concentrations. A relationship between nutrients and rainfall is not clear. There were frequent observations of elevated nutrients in the absence of rainfall. The highest nitrate concentration (30 October 2003) was preceded by at least a month of dry weather. Thus, we conclude that least one source of nutrients other than rainfall must have been present. Over the same period, chlorophyll a varied between 0.53 and 64.3 mg m 3 (Fig. 2d). Highest concentrations were found in October 2003 and were due to a large bloom of the dinoflagellate Lingulodinium polyedrum, which dominated the flora throughout much of the sampling period. Its persistence was unusual and has not been explained.
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3.2. Toxic Pseudo-nitzschia and domoic acid levels in phytoplankton The total abundance of toxic Pseudo-nitzschia (P. australis and P. multiseries) ranged from undetectable to a maximum of 7.7 104 cells l 1 (Fig. 2e and f). Abundances of the two species were highly correlated
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both over all samples and during the bloom period. In samples that contained toxic Pseudo-nitzschia, over 70% of the toxic cells were P. australis, and thus P. australis is probably the main contributor to DA in this study. The sum of P. australis and P. multiseries is hereafter referred to as toxic Pseudo-nitzschia. We assume that most or all of the DA measured in
Fig. 2. Data from Scripps Pier, 1 October 2003–29 September 2004. (a) Sea surface temperature (SST) and salinity; (b) rain; (c) nitrate, phosphate and silicate concentrations; (d) chlorophyll a concentration; (e) abundance of P. australis; (f) abundance of P. multiseries; (g) particulate domoic acid concentration, when available.
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Fig. 2. (Continued ).
particulate material was contained by these two species. No other Pseudo-nitzschia species were dominant in the net tow at that time. Toxic Pseudo-nitzschia first appeared at Scripps Pier on 23 December 2003. Until 4 February 2004 toxic species were seen sporadically and in low abundances (1.8–3.4 103 cells l 1; Fig. 2e and f). At the same time particulate DA concentrations ranged between the detection limit and 0.58 mg l 1 (Fig. 2g). Abundances of toxic Pseudo-nitzschia cells and DA levels showed sudden and sharp increases in the second half of February 2004. At Scripps Pier, highest toxic Pseudo-nitzschia abundances (7.7 104 cells l 1, above the critical level of 5 104 cells l 1) were recorded on 18 February accompanied by DA concentrations of 1.98 mg l 1 (Fig. 2e–g). One week later the abundances decreased below the critical level, but we still measured particulate DA concentrations of 1.48 mg l 1 (Fig. 2e–g). There was no toxic Pseudonitzschia seen after 21 April 2004. DA was close to the detection limit in May, and was not detected in water samples thereafter.
The Pseudo-nitzschia bloom occurred during the winter period when the environment was relatively cool (13.9–14.3 8C) and fresh (33.16–33.22 psu; Fig. 2a). During the bloom period, chlorophyll a concentrations varied between 0.92 and 12.5 mg m 3 (Fig. 2d) and were only weakly correlated with the abundance of toxic Pseudo-nitzschia (Fig. 3a). Although abundant, the Pseudo-nitzschia species never reached high enough numbers to be a major determinant of phytoplankton biomass. During the bloom period, we observed short bursts of elevated nutrients, but the nutrient concentrations were not the most extreme observed, nor were they consistently high. During the bloom period, correlations between the three nutrients and the abundance of toxic Pseudonitzschia were weak (e.g. Fig. 3b). Neither does there seem to be a relationship of with the nutrient ratios (e.g. Fig. 3c). The bloom of toxic Pseudo-nitzschia and elevated concentrations of particulate DA at Scripps Pier prompted us to sample other areas in Orange and Los Angeles Counties to determine the spatial extent of the
Fig. 3. Relationships between environmental variables and toxic Pseudo-nitzschia species. (a) Chlorophyll concentration and the abundance of toxic Pseudo-nitzschia species at all locations during the bloom period; (b) concentration of near-surface nitrate-N and the abundance of toxic Pseudo-nitzschia species at all locations during the bloom period; (c) the relationship of near-surface nitrate-N and phosphate-P at all locations, bloom period indicated as , non-bloom period as closed circle.
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Table 1 Location, sampling date, temperature, salinity, chlorophyll, abundance of toxic Pseudo-nitzschia (sum of P. australis and P. multiseries), and particulate domoic acid concentrations in San Diego County, Orange County, and Los Angeles County; at the peak of the bloom in February 2004; n.d. = no data Location
Sampling date
Temperature (8C)
Salinity (psu)
Chl a (mg m 3)
Toxic Pseudo-nitzschia (cells l 1)
Domoic acid (mg l 1)
Malibu Pier Redondo Pier
20 20 27 20 27 19 27 19 27 11 18 25
13.5 15.0 14.1 15.0 14.4 n.d. 14.7 n.d. 14.6 14.2 13.9 14.3
33.19 33.22 33.92 33.11 30.92 33.13 32.92 33.03 32.28 33.21 33.22 33.16
3.66 1.20 1.52 6.91 4.57 3.97 3.29 4.99 6.57 3.61 3.91 1.78
1300 6950 2250 70500 1250 38000 0 66950 14900 44100 76600 37400
n.d. n.d. n.d. 2.33 0.05 1.24 0 n.d. n.d. 0.22 1.98 1.48
Newport Pier San Clemente Pier Oceanside Pier Scripps Pier
February February February February February February February February February February February February
2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004
Table 2 Domoic acid measurements in mussels and fish (viscera only) Common name
Species name
# Individuals
Sampling date
Domoic acid (mg DA g 1) tissue
Mussels Chub Mackerel Jack Mackerel Pacific sanddab Longspine combfish
M. californianus S. japonicus T. symmetricus C. sordidus Z. latipinnis
27 8 8 11 5
25 February 2004 1 March 2004 1 March 2004 3 March 2004 3 March 2004
5.8 7.3 5.5 50.1 9.7
All were collected at or near the Scripps Pier, San Diego County. All individuals from one species were combined into one sample for domoic acid measurements.
event. At the piers closest to San Diego (Oceanside, San Clemente, and Newport), we found abundances of toxic Pseudo-nitzschia that were comparable to those at Scripps Pier (Table 1). Abundances declined further north in Los Angeles County (Redondo and Malibu Piers). Because of our limited sampling, we cannot eliminate the possibility that the bloom was advecting southward and had been more strongly developed at the two northern sites prior to our sampling. Abundances at all sites decreased between 19/20 February and 27 February. Where data are available, elevated concentrations of DA accompanied the elevated abundances of toxic Pseudo-nitzschia (Table 1). Data from CDHS survey reports show that the relative abundance of total Pseudo-nitzschia (which may include both toxic and non-toxic species) from mid- to late-February increased slightly at sites near Santa Barbara and San Luis Obispo, while sites further north showed only low numbers (California Department of Health Service, 2004). Thus, in
February, the southern California coast had the most dramatic increase in toxic Pseudo-nitzschia. Later in the season, strong blooms with higher cell numbers of P. australis and P. multiseries were observed between Santa Barbara (February–May 2004) and Santa Cruz (April–May 2004; Silver et al., unpubl. data). 3.3. Domoic acid levels in fish and mussels Mussels from Scripps Pier, retrieved on 25 February, showed low, but detectable levels of DA (5.8 mg DA g 1; Table 2). S. japonicus and T. symmetricus, caught on 1 March, also showed low DA (7.3 and 5.5 mg DA g 1 respectively; Table 2). We found higher DA concentrations in near-bottom feeding fish, which were caught on 3 March. These included C. sordidus with DA concentrations of 50.1 mg DA g 1 and Z. latipinnis, with concentrations of 9.7 mg DA g 1.
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3.4. Sea lion strandings During February 2004, California sea lions stranded along southern California beaches, and distressed but live animals were brought into various facilities for treatment: Seaworld (San Diego County: five sea lions; Leger, pers. commun.), Pacific Marine Mammal Center (Orange County: seven sea lions; Hunter, pers. commun.) and Marine Mammal Care Center at Fort MacArthur (Los Angeles County: seven sea lions; Pareti, pers. commun.). All the animals receiving treatment showed one or more symptoms of DA poisoning, which include confusion, disorientation, seizures, excessive scratching and rubbing, and an extremely lethargic or comatose condition (Lefebvre et al., 2001).
4. Discussion Several previous studies off central and southern California (Garrison et al., 1992; Lange et al., 1994; Trainer et al., 2000) suggest that Pseudo-nitzschia blooms are associated with cool, high-nutrient waters that may be related to upwelling events. In other areas, toxic Pseudo-nitzschia blooms have appeared after a period of heavy rainfall resulting in nutrient-rich freshwater runoff (Trainer et al., 2000). At the Scripps Pier, temperatures during the bloom were close to the annual minimum, during a period when short bursts of elevated nutrients were frequent. The start of the bloom preceded the heavy rain. Thus, while fresh water or nutrient addition from runoff may have contributed to the subsequent bloom characteristics, they seem unlikely to have initiated the bloom. Some level of nutrient injection from deeper water seems the more likely explanation. This is the first documentation of DA in phytoplankton and in fish in San Diego. However, the maximum abundance of Pseudo-nitzschia found at Scripps Pier (7.7 104 cells l 1) is relatively low compared to that found in other studies along the coast. Likewise, the highest particulate DA levels found in this study in phytoplankton (2.3 mg l 1) are in the moderate range of DA concentrations. In a major toxic event in June 1998, Trainer et al. (2000) reported maximum abundances of the toxic cells (P. australis and P. multiseries) between San Francisco
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and Monterey Bay ranging from 5 104 to 5 105 cells l 1 and a maximum value of 1.1 107 cells l 1 offshore of Santa Barbara on 4 June 1998. At that time, DA levels up to 7.3 mg l 1 were found in particulate material from water samples, and the event was accompanied by massive marine mammal strandings. Records from Monterey over a 4year period show highest abundances of toxic Pseudonitzschia ranging from 105 to 106 cells l 1, with the higher values usually found offshore (Lefebvre et al., 2001; Silver, unpubl. data). Mammal strandings north of Pt. Conception from 1999 to 2002 occurred when coastal water samples contained particulate DA levels ranging from non-detectible up to 50 mg l 1 (Gulland and Silver, unpubl. data). During February 2004, the DA concentrations within cells of P. australis and P. multiseries varied between 5 and 43 pg cell 1. This is within the range of cellular DA found in samples along the central California coast in 1998 (7–75 pg cell 1; Scholin et al., 2000). Thus, the toxicity of our bloom may have been controlled by population size rather than DA production. A repeat occurrence of blooms of toxic Pseudo-nitzschia as large as those seen in the past (1 106 cell l 1 P. australis in March 1991, Lange et al., 1994) has the potential for a much greater impact. The concentration of DA in mussels at Scripps Pier (5.8 mg DA g 1) is well within the safety guidelines set by the US Food and Drug Administration (alert level: 20 mg DA g 1), but higher than the levels found in May 1992 when the mussels contained 2.2 mg DA g 1 (Lange et al., 1994). The viscera of pelagic fish showed similar DA concentrations (Table 2). In planktivores like anchovies and sardines, which are key vectors of DA to marine birds and mammals, most of the toxin in the fish is found in the gut. Consequently, the toxin is lost when the gut contents are voided and the toxin is usually present only when the fish are feeding or have recently fed (Lefebvre et al., 2002). Generally, toxin burdens in such planktivorous fish correspond approximately to the total toxin in Pseudo-nitzschia populations in the region (Lefebvre et al., 2001). In our study, the viscera of bottom-feeding fish, especially C. sordidus, contained DA concentrations up to 10 times higher than did the viscera of pelagic fish. C. sordidus is a popular food fish, and it has been found to have elevated toxin levels in the viscera at
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other sites along the California coast during blooms of toxic Pseudo-nitzschia (Lefebvre et al., 2002; Goldberg et al., submitted). Some benthic fish and benthic invertebrates contain toxin when DA levels in the water are not measurable and when no toxic Pseudonitzschia are detected (Goldberg et al., submitted). Such an apparent uncoupling of toxin between phytoplankton and benthic animals could result from flocculation of the toxic cells and settlement to the benthos, where the toxic material is consumed directly or indirectly by benthic animals capable of sequestering the toxin (e.g. razor clams; Trainer and Bill, 2004).
5. Conclusions Confirmation of the presence of domoic acid (DA) during elevated abundances of toxic Pseudo-nitzschia and concurrently in fish and mussels provides evidence for the transfer of DA from a local algal source in San Diego to higher trophic levels. Strandings of sea lions with neurological symptoms typically associated with DA poisoning in areas where we found the toxin producing Pseudo-nitzschia suggest further transfer to marine mammals. Although this is the first confirmation of such an event in San Diego, similar events may have occurred in the past as there is evidence that the toxic species have been in the region, at least on occasion, for over eight decades. Previously, DA was thought to be a problem primarily in northern and central California; our findings indicate that DA can occur further south in Orange and San Diego Counties. It is not known whether our observations are merely the result of increased awareness and better detection techniques (i.e. we looked for it) or whether the production of DA in southern Californian waters has intensified recently, possibly spreading south from the central coast of California. Given our findings, a more regular monitoring of DA in phytoplankton and fish is recommended for Orange and San Diego Counties and even further south.
Acknowledgements This project was funded through the University of California Coastal Environmental Quality Initiative
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