This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
Author's personal copy
Harmful Algae 8 (2009) 493–503
Contents lists available at ScienceDirect
Harmful Algae journal homepage: www.elsevier.com/locate/hal
A toxic Pseudo-nitzschia bloom in Todos Santos Bay, northwestern Baja California, Mexico Ernesto Garcı´a-Mendoza a,*, David Rivas b, Aramis Olivos-Ortiz c, Antonio Almaza´n-Becerril f, ˜ a-Manjarrez e ˜ eda-Vega d, Jose´ Luis Pen Carolina Castan a
Departamento de Oceanografı´a Biolo´gica, CICESE, Km. 107 carr. Tij-Ens, Ensenada, Baja California, Mexico College of Oceanic and Atmospheric Sciences, Oregon State University, 104 COAS Administration Building, Corvallis, OR, USA Facultad de Ciencias Marinas, Universidad de Colima, Km. 20 carr. Mnazanillo-Barra de Navidad, Manzanillo, Colima, Mexico d Facultad de Ciencias, UABC, Km. 103 carr. Tij-Ens, Ensenada, Baja California, Mexico e Centro de Estudios Tecnolo´gicos del Mar en Ensenada, Km. 6.5 carr. Ens-Tij, Ensenada, Baja California, Mexico f Centro para Estudios del Agua, CICY. Calle 8, No. 39, Mz. 29, S.M. 64 Cancu´n, Quintana Roo, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 May 2008 Received in revised form 7 October 2008 Accepted 7 October 2008
A toxic Pseudo-nitzschia spp. bloom in the Todos Santos Bay area (31.88N), Mexico, is described. This is the southernmost report of the presence of domoic acid (DA) in the California Current System and it is also the first report of the distribution of toxic Pseudo-nitzschia species and DA on the Baja California west coast. The maximum cell abundance of Pseudo-nitzschia was 3.02 105 cells L 1 and the maximum concentration of DA in particulate matter (pDA) was 0.86 mg L 1. P. australis constituted the major proportion of cells identified as Pseudo-nitzschia. The environmental conditions associated with wind-driven upwelling were the cause for the accumulation of toxic cells. Maximum pDA and cell concentration were detected around 14 8C. The ratio of the concentration of macronutrients seemed to be the important factor for the accumulation of P. australis. The highest cell abundance was detected in areas with a high Si(OH)4 to N ratio in the entire water column. Therefore, the relative increase of silicate concentration related to upwelling conditions was the probable cause for the accumulation of P. australis. Maximum photosystem II (PSII) quantum efficiency of charge separation (Fv/Fm) was negatively correlated to the pDA to fucoxanthin ratio. This ratio was used in this work as an index of cellular DA content. Therefore, the photosynthetic competence of the cells might be an important factor that affected their DA cellular content. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Baja California Domoic acid Pseudo-nitzschia australis Toxic bloom Upwelling
1. Introduction One of the main toxic syndromes caused by marine microalgae in the California Current System is amnesic shellfish poisoning, also known as domoic acid poisoning (DAP; GEOHAB, 2005). DAP events are caused by the accumulation of high levels of domoic acid (DA) in marine organisms. DA is a potent neurotoxin that can have deleterious effects on several marine species and represents an important risk to public health. The presence of DA in the environment is associated with blooms of diatoms of the genus Pseudo-nitzschia Peragallo. Eleven species of this genus have been identified as potential producers of this toxin (Moestrup and Lundholm, 2007). DA is accumulated in tissue of shellfish, sardines, anchovies and euphausiids after consuming toxic Pseudo-nitzschia cells (Bargu et al., 2002; Lefebvre et al., 1999, 2002) and this toxin
* Corresponding author at: Oc. Biologica/CICESE, P.O. Box 430222, San Diego, CA 92143-0222, USA. Tel.: +52 646 175 0500; fax: +52 646 175 0587. E-mail address:
[email protected] (E. Garcı´a-Mendoza). 1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2008.10.002
could be transferred to higher trophic levels through consumption of these vector species. DA intoxication was the cause of three human deaths in 1987 in Prince Edward, Canada (Bates et al., 1989). No human intoxications have been reported since this event but DA has been the cause of massive mortalities of marine mammals and sea birds, specifically in the central California area (Scholin et al., 2000; Trainer et al., 2000). The characterization of the biogeographical distribution of toxic species is, among others, a key element to understand the phenomenology of harmful algae blooms (GEOHAB, 2003). Blooms of Pseudo-nitzschia species and high DA concentrations in phytoplankton samples are common in the southern part of British Columbia in Canada, in northern coastal areas of Washington State and in the coasts of central California in the USA (Fritz et al., 1992; Horner et al., 1997; Trainer et al., 1998; Taylor and Trainer, 2002; Anderson et al., 2006; Trainer et al., 2008). The species responsible for the accumulation of DA in these areas are Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle, P. multiseries (Hasle) Hasle, P. pungens (Grunow ex P.T. Cleve) Hasle and P. australis Frenguelli (Fryxell et al., 1997; Hasle, 2002; Trainer
Author's personal copy
494
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
et al., 2007). There is less evidence of the presence of DA in the southern part of the California Current System, but two recent reports indicate that high concentrations of DA toxin are also present in this region (Busse et al., 2006; Schnetzer et al., 2007). Specifically, in San Pedro Channel and in Los Angeles harbor, high concentrations of DA in particulate matter were detected in 2003– 2004, and the highest concentration (12.1 mg DA L 1) of all reported values was found inside Los Angeles harbor (Schnetzer et al., 2007). South from this location, DA was found in molluscan shellfish, fish and in particulate matter collected from the San Diego area (Busse et al., 2006). The high levels of DA were associated with the presence of P. australis and P. multiseries, with the former reaching much higher cell abundances (Busse et al., 2006). Further south, there is scarce information about the distribution of Pseudo-nitzschia toxic species (Herna´ndez-Becerril, 1998; Go´mez-Aguirre et al., 2004) and DA concentration in Mexican Pacific waters. The only report of a DAP event in Mexican waters was the massive mortality of seabirds that occurred at the tip of the Baja California Peninsula in 1996 (Sierra-Beltra´n et al., 1997). The presence of DA in tissue of a dead pelican was confirmed but the species responsible for the production of the toxin could not be identified in the stomach content of sardines or in water samples (Sierra-Beltra´n et al., 1997). The DA concentration in waters of the Baja California west coast has not been monitored. This region is part of California Current System and presents prominent oceanographic mesoscale structures such as meanders, eddies, filaments (Soto-Mardones et al., 2004) and strong upwelling events along the coastline (Espinosa-Carreo´n et al., 2004). These oceanographic features have been associated with the blooming of toxic species in northern regions (Scholin et al., 2000; Trainer et al., 2000; Taylor and Trainer, 2002; Anderson et al., 2006). Given that the conditions for the growth of toxic diatom species are present along the Baja California west coast, we hypothesize that there is the potential for the occurrence of toxic diatom blooms in the region. These might be triggered by environmental conditions associated with mesoscale oceanographic processes (mainly upwelling events) as in northern regions. Therefore, we characterized the concentration of DA in particulate matter and Pseudo-nitzschia cell abundances during a period in which upwelling events are present (April) near the Todos Santos Bay (TSB) area (northern region of the Baja California Peninsula). 2. Material and methods 2.1. Study area and hydrographical data collection Hydrographic data and water samples were collected during the cruise BTS0407 aboard the B/O Francisco de Ulloa (CICESE). Samples were collected during April 23–24, 2007 in the Todos Santos Bay area (Fig. 1), northwestern Mexico. Twenty-six stations were sampled. At each station, salinity, temperature, chlorophyll a (Chl a) fluorescence and dissolved oxygen concentration were measured with a Sea Bird SBE911 plus CTD. Water samples were collected from different depths at each station with 2.5 L Niskin bottles to measure phytoplankton pigment concentration, particulate DA and the photosystem II (PSII) maximum quantum efficiency of charge separation (Fv/Fm). Fv/Fm is an indicator of the optimal photochemical efficiency of PSII (Kromkamp and Forster, 2003) and has been used as a proxy to measure nutrient stress in the phytoplankton community (Almaza´n-Becerril and Garcı´aMendoza, 2008). The collecting depths (3–5 sample points depending to the depth of the station) were chosen according to the vertical distribution of the Chl a concentration as determined by the CTD fluorescence profile.
2.2. Phytoplankton pigments Pigment quantification was as in Van Heukelem and Thomas (2000) with modification described in Almaza´n-Becerril and Garcı´a-Mendoza (2008). Water from each sample depth (maximum 1 L) was filtered through 25 mm diameter GF/F filters. The filters were frozen immediately in liquid nitrogen. Extraction of the pigments was done by mechanical disruption (Mini-BeadBeaterTM, Biospec Inc., USA) of the filters with 0.5 mm diameter zirconia/ glass beads in 1.5 mL pre-cooled 100% acetone. The homogenate was left for at least 2 h at 20 8C in darkness. Sample debris was removed by two centrifugation steps (15,000 g 5 min, 4 8C). Pigment quantification was performed by high performance liquid chromatography (HPLC) using a Shimadzu AV-10 series instrument equipped with a Zorbax Eclipse XDB-C18 reverse-phase column (150 mm 4.6 mm internal diameter and 3.5 mm size particles). The absorption detector was set at 436 nm. The protocol was calibrated with 16 pigment standards (DHI, Inc., Sweden). 2.3. Phytoplankton cell enumeration and identification Samples from the surface and from the deep chlorophyll maximum (DCM) were taken at each station to characterize the phytoplankton community. Samples were placed in dark plastic bottles and fixed with a Lugol-acetate solution. The Utermo¨hl technique (Hasle, 1978) was used to count and identify phytoplankton cells. P. australis was identified by its cell dimensions (68–80 mm length, 5–6 mm width; Herna´ndezBecerril, 1998). Scanning electron microscopy (SEM) and whole-cell hybridization (WCH) techniques were used to identify Pseudo-nitzschia cells to species level. SEM was performed according to the protocol described by Miller and Scholin (2000). SEM was performed on two samples only, i.e. from the deep chlorophyll maximum of stations 9 and 10. WCH was applied in samples from the DCM of selected stations (12 in total). The WCH protocol was as in Miller and Scholin (2000) using the auD1 probe raised against P. australis isolated from Monterey Bay, California (Scholin et al., 1996). 2.4. Particulate DA concentration A modified protocol of the HPLC-UV methodology proposed by Quilliam (2003) was used to measure DA in phytoplankton samples (pDA). Sample collection and extraction procedures were similar to the phytoplankton pigment determination protocol. However, 50% MeOH was used as the extraction solvent. Filter debris and broken cells were removed by two centrifugation steps. To increase the detection limit, an injection volume of 150 mL of the clean extract was injected into the HPLC system. This sample loading did not produce peak deterioration (data not shown). The system was equipped with a Zorbax Eclipse XDB-C18 reversephase column (250 mm 4.6 mm internal diameter, 3.5 mm size particles). The elution gradient was (%B, min): 10, 0; 50, 10; 50, 12; 10, 13. Solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was acetonitrile with 0.1% TFA. Commercial DA (Sigma, Co.) was used to calibrate the system. The detection limit achieved with this protocol was 0.02 mg mL 1 of extract. The practical pDA detection limit depends on the filtered volume and in this work was 0.02 mg L 1 of seawater filtered. 2.5. Nutrient concentration Concentrations of nitrates plus nitrites (N), ammonium (NH4+), phosphates (PO43 ) and silicates (Si(OH)4) were measured using a segmented flow autoanalyzer (Skalar SanPlus II, Skalar Analytical,
Author's personal copy
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
495
Fig. 1. Map of the study area and sampling grid. The sampling campaign was conducted during 23–24 April 2007. The map shows the bathymetry of the study area in meters and also the three transects (T1, T2 and T3) in which the vertical properties are analyzed (see Section 3).
The Netherlands) with a precision of 0.01 mM, using the techniques described by Strickland and Parsons (1972). 2.6. Maximum PSII quantum efficiency of charge separation (Fv/Fm) PSII emission was measured with a pulse amplitude modulated fluorometer (XE-PAM; Heinz Walz, Effeltrich, Germany) as in Almaza´n-Becerril and Garcı´a-Mendoza (2008). PSII maximum quantum efficiency was calculated as Fv/Fm, in which Fv is the difference between the maximum (Fm) and the minimum (F0) Chl a emission. F0 is the fluorescence in darkness excited only by the modulated measuring beam pulsed at 2 Hz (Xenon lamp with a Schott BG39 filter) and Fm represents the maximum fluorescence measured in dark adapted (nonphotochemical quenching relaxed) samples when all the photochemical quenching is suppressed by a short saturating pulse of light (0.8 s). The fluorescence signal was measured in samples that were maintained in darkness for at least 30 min. 2 mL of the sample were placed in a quartz cuvette mounted in the ED-101US optical unit of the fluorometer. Three saturating light pulses were applied every 30 s and these measurements were performed three times for each sample. Baseline correction of each measurement was done by subtracting the background fluorescence signal of GF/F-filtered seawater sample corresponding to the sample depth.
2.7. Data processing and statistical analysis Ocean Data View software (http://odv.awi.de/) was used to generate the distribution of hydrographic properties in the studied area. Software Statistica (StatSoft Inc., Tulsa, OK, USA) was used for statistical analysis. 3. Results 3.1. Hydrographic and environmental conditions The distribution of mesoscale hydrographic properties (Fig. 2) shows that there were upwelling favorable conditions during the sampling period close to the Todos Santos Bay area. Although the circulation close to the coastline is probably not well resolved by the altimetry-drifter-derived maps, Fig. 2a suggests a surface inshore flow at 32–338N which bifurcates in poleward and equatorward flows, this latter one affecting the TSB area. This flow-bifurcation region has been referred to as the Ensenada Front (Venrick, 2000). Also, surface currents surrounding TSB are clearly linked to the ‘‘permanent meanders’’ of the California Current System recently discussed by Centurioni et al. (2008). The synoptic wind field (Fig. 2b) shows a mean wind stress of 0.08 Pa in the southward direction for the 24 April 2007. Surface circulation and
Author's personal copy
496
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
Fig. 2. Mesoscale surface features offshore of Todos Santos Bay. The location of the study area is enclosed in a circle. (a) Sea surface height (SSH; cm) and its corresponding geostrophic velocity (m s 1). Panel (a) is a combination of gridded satellite-merged sea-level anomaly available at AVISO website (http://www.jason.oceanobs.com) and drifter-derived mean dynamic topography available at the 1992–2002 MDOT website (http://apdrc.soest.hawaii.edu/projects/DOT). (b) Wind stress (in Pa) taken from the gridded QuickScat daily wind data available at the IFREMER website (http://ifremer.fr/cersat/en/data/download/gridded/mwfqscat.htm). (c) Sea surface temperature (SST; 8C), and (d) chlorophyll (in 10-base logarithmic scale) for 24 April 2007 taken from the weekly averaged Level-3 Standard Mapped Images of Chlorophyll and 11 m-day SST available at the Aqua-MODIS website (http://oceancolor.gsfc.nasa.gov/cgi/level3.pl). Except for the wind data in Panel (b), the data were linearly interpolated for 24 April 2007 using the maps immediately before and after this date.
the wind field produced upwelling conditions in the area close to TSB as seen by the low sea surface temperature (Fig. 2c) and the high chlorophyll concentration (Fig. 2d) in the coastal area from 32.88N to 30.68N. Local hydrographic properties confirmed the presence of upwelling conditions in the study zone. There was a well-defined area with surface temperatures around 14 8C and a higher salinity as compared to surrounded water (Fig. 3a and b) in the Salsipuedes Bay coastal zone (stations 7–9) and in the north part of TSB (stations 10 and 18; Fig. 3b). The lowest surface temperature was detected at station 7 (13.4 8C) while the highest temperature was measured at station 1, located 9 km offshore (Fig. 3a). At this station, the lowest surface concentration of nitrate (2.4 mM) was also detected (Fig. 3c). The surface concentration range of this macronutrient was from 2.4 to 14.8 mM. In most of the TSB area, nitrate concentration was above 8 mM and relatively low values (5 mM) were present close to the Salsipuedes Bay coastline (Fig. 3c). In this area of low temperature and high salinity, the concentration range of silicates (Si(OH)4) was from 7 to 8.5 mM, which were relatively high values as compared to the concentrations found in offshore stations (stations 1–3). The surface Si(OH)4 concentration was 4.05 mM
at these stations (Fig. 3d). Inside TSB, the mean Si(OH)4 concentration was 5.5 mM with areas of high concentrations located in front of Punta San Miguel and in the southern part of the bay (stations 23 and 24). Surface concentrations of NH4 ranged from 1.4 to 8.5 mM and for PO43 from 0.7 to 3.9 mM. The distribution of these nutrients did not present a clear pattern (data not shown). Upwelling detected in the coastal area of Salsipuedes Bay affected surface water properties only few kilometers offshore. However, the upwelled water had a strong influence in a large area of TSB as seen by the hydrographic properties inside the Bay (Fig. 3). 3.2. Phytoplankton biomass, Pseudo-nitzschia cell abundance and particulate DA concentration Phytoplankton biomass was distributed heterogeneously. Surface Chl a was above 2 mg L 1 in most of the study area (Fig. 4a). Chl a concentrations lower than this value were detected only in the northwest part of the study zone. The area with the highest surface Chl a concentration was located in the north part of TSB in front of Punta San Miguel and near El Sauzal Port. Here, surface Chl a reached a concentration of 16 mg L 1. The highest Chl a
Author's personal copy
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
497
Fig. 3. Distribution of hydrographic properties in the study area. Surface (a) temperature (8C), (b) salinity, concentration (mM) of (c) nitrates, and (d) silicates.
concentration (45 mg L 1) was detected at 7 m depth at station 18 located in front of El Sauzal Port (Fig. 4b). Pigment analysis shows that the concentration of fucoxanthin (Fuc) presented a linear relationship with Chl a concentration up to 10 mg L 1 (Fig. 5A). Above this concentration, the relationship was no longer linear (Fig. 5A). In contrast, the peridinin (Per) to Chl a relationship was linear at all Chl a concentrations above 2 mg L 1 (Fig. 5B). Peridinin concentration was minimal below 2 mg L 1 of Chl a (Fig. 3b). This analysis revealed that the phytoplankton community in the study area was dominated by diatoms and dinoflagellates since the concentration of other carotenoids (pigment markers for other phytoplankton groups) was low compared to Fuc and Per. Although the high phytoplankton biomass accumulation (above 10 mg Chl a L 1) was associated mainly with the presence of dinoflagellates (station 18 and surrounding area; Fig. 4), diatoms were an important component of the phytoplankton community in the study area. Below 10 mg Chl a L 1, diatoms contributed the most to total phytoplankton biomass. Diatom assemblages were dominated by species of the genus Pseudo-nitzschia. In most surface and depth chlorophyll maximum samples, Pseudo-nitzschia cells represented more than 60% of the diatom cell abundance. Chaetoceros was the
other important genus but its relative abundance was no more than 20%. At stations 9 and 10 Pseudo-nitzschia cells constituted more than 90% of the total diatom biomass. The area with the highest Pseudo-nitzschia cell abundance was detected close to the coastline (stations 7–10) in the north part of the study zone (Fig. 6a). Cell abundances at stations 9 and 10 were 2.41 105 and 2.61 105 cells L 1 respectively while the highest abundance (3.02 105 cells L 1) was detected at 7 m depth at station 9. Domoic acid in particulate matter (pDA) presented a widespread distribution in the study area. pDA was below the detection limit only at stations from the northwest part of the study area (stations 1–3 and 6; Fig. 6b). In contrast, high surface pDA concentrations were detected at Salsipuedes Bay coastal stations (stations 7–9) and in front of Punta San Miguel (stations 10–12). Here, the highest pDA concentration (0.87 mg L 1) was measured at station 10 (Fig. 6b). 3.3. Distribution of DA and toxic content of Pseudo-nitzschia cells in relation to environmental conditions pDA concentration was inversely correlated to water temperature (Pearson’s r = 0.64). High concentrations of pDA were
Author's personal copy
498
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
Fig. 5. Relationship of chlorophyll a concentration to (A) peridinin and (B) fucoxanthin concentrations. Solid lines represent the best fit to a linear regression model in the case of peridinin and to an exponential function in the case of fucoxanthin. Also presented (broken line) is the fit to a linear regression model of fucoxanthin to chlorophyll a concentration below 10 mg L 1.
Fig. 4. Phytoplankton biomass distribution (as chlorophyll a concentration in mg L 1) at (a) surface and (b) 10 m depth in the study area.
present in the areas with surface temperatures around 14 8C (Figs. 3a and 6b) and were lower in areas with higher temperatures. pDA was associated with the accumulation of toxic Pseudo-nitzschia cells. However, the relationship between pDA and cell abundance was not apparent when comparing the surface distribution of both variables (Fig. 6). However, two groups were distinguished when exploring the relationship between cell abundance and pDA of surface and DCM samples (Fig. 7A). Fig. 7 shows the separation of the data into two groups which indicates that cells with different DA content were likely present in the study area. One group reached (empty circles in Fig. 7A) a high cell abundance and presented a mean DA concentration of 2.73 pg DA cell 1 (slope of the pDA vs. cell abundance relationship; r = 0.78, Fig. 7A) while the other group (grey circles in Fig. 7A) presented a DA concentration 15 times higher (m = 42 pg DA cell 1, r = 0.48). P. australis represented more than 80% of the organisms enumerated as Pseudo-nitzschia (Fig. 4). P. australis cells were recognized by light microscopy as they have a lower length to width ratio than other species present in the samples of similar length. SEM analysis confirmed that P. australis was the dominant
species in the samples analyzed. This species was identified according to the morphological characteristics described by Hasle and Syvertsen (1996) and Herna´ndez-Becerril (1998). The presence of P. australis in the study zone was further confirmed with the WCH technique. Positive results using the auD1 probe were obtained; however, the estimation of cell abundances with this technique differed from the optical light microscopy results. P. australis abundances estimated by WCH were no more than 20% of the ones estimated by light microscopy (data not shown). The presence of pDA in the environment must be related to the accumulation of toxic species and its toxin content. A cross correlation analysis between pDA and DA per cell (n = 49) with different environmental variables was performed. The correlation coefficients, although significant in some cases, were low (data not shown). Perhaps, a non-linear relationship between environmental variables and pDA and DA per cell reduces the statistical power of this analysis. Therefore, a synoptic analysis of the distribution of variables was performed to recognize some factors that affected the presence of pDA in the study area. The vertical distribution of pDA and the pDA to fucoxanthin ratio (pDA:Fuc) was analyzed. It is assumed that the concentration of Fuc in the samples was principally associated with the presence of P. australis. Therefore, the concentration of this pigment could be associated to the abundance of this species. The correlation coefficient between pDA and fucoxanthin concentration was 0.66 (n = 116) and increased to 0.75 when data from the upper 10 m were considered (n = 52; Fig. 7B). The vertical distribution of variables was analyzed in three transects parallel to the coastline that represented contrasting conditions (Fig. 1). Analysis of transects from a south to north direction was done according to the circulation pattern presented
Author's personal copy
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
499
Fig. 7. Relationship between particulate domoic acid (pDA) concentration and the abundance of (A) Pseudo-nitzschia cells and (B) fucoxanthin. Data in panel (A) were divided into two groups (grey and empty circles) for the linear regression analysis. The data point indicated with an arrow was not considered for the regression analysis (outlier). Filled symbols in panel (B) represent data from surface samples (upper 10 m). The regression line in panel (B) includes all data in the plot.
Fig. 6. Surface distribution of (a) Pseudo-nitzschia cell abundance (Cells L 1 103) and (b) domoic acid in particulate matter (pDA; mg L 1) in the study area.
in Fig. 2. We assumed that the properties detected in the north part of the study area would propagate southward, affecting TSB. This notion is consistent with the numerical results of Chhak and DiLorenzo (2007), which suggest that the water upwelled around the study area comes from northern regions. Domoic acid was mainly present in the first 10 m of the water column but relatively high amounts of this toxin were found down to 20 m depth in some areas of the study zone (Fig. 8). The distribution of this toxin indicates that the origin of the toxic bloom was localized in the inshore area of Salsipuedes Bay and propagated offshore (transects 1–3, Fig. 8) and southward into the TSB area (stations 7–26, transect 1; Fig. 8). The pDA:Fuc ratio followed the widespread distribution of the pDA (Fig. 8). However, areas with a high pDA:Fuc ratio were detected. Specifically, at station 9 of transect 1 (Salsipuedes Bay coastal area) a high pDA:Fuc ratio was detected from the surface down to 20 m depth (transect 1; Fig. 8). An important observation is that relatively high pDA:Fuc ratios were present deeper (down to 40 m depth) in stations 8 and 7 (transect 1; Fig. 8). In transect 2, the high pDA:Fuc
values were found at station 11 close to the surface and at station 15 at 20 m depth (transect 2; Fig. 8). pDA and pDA:Fuc ratios were correlated to the vertical distribution of peridinin to Chl a (Per:Chl), silicates to N (Si(OH)4:N ratio) ratios and to Fv/Fm. Specifically, the distribution of the Si(OH)4:N ratio defined the different environmental conditions in the study area for the accumulation (growth) of toxic Pseudonitzschia. The Si(OH)4:N distribution followed the propagation of properties associated with upwelling conditions detected in the Salsipuedes Bay coastal area. For example, in transect 1 a Si(OH)4:N ratio higher than 1.5 was present in the entire water column in the Salsipuedes Bay coastal area (stations 9–7, transect 1; Fig. 8) concomitantly with a high pDA accumulation. The relatively high Si(OH)4:N ratio close to the surface was also detected in transect 2 but not in the offshore stations (transect 3; Fig. 8). In contrast, a Si(OH)4:N ratio lower than 1.5 was present at the surface along transect 3 (Fig. 8) and principally in the shallow environments of TSB (transect 1 and 2; Fig. 8). Here, lower pDA concentrations were measured compared to the areas with a higher Si(OH)4:N ratio and a high Per:Chl ratio was present, which indicates that dinoflagellates constituted an important component of the phytoplankton community in these areas (Fig. 8). This analysis shows that nutrient conditions defined the phytoplankton community response and influence greatly the distribution and accumulation of toxic P. australis. Furthermore, the DA content of P. australis seems to have been related to the photosynthetic physiology of the cells. The distribution of the pDA:Fuc ratio in the area of high Pseudo-nitzschia cell abundances was negatively correlated to Fv/
Author's personal copy
500
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
Fig. 8. Vertical distribution of particulate domoic acid concentration (pDA) and its relation to fucoxanthin (pDA:Fuc ratio) in three transects parallel to the coastline (see Fig. 1). The vertical distributions of ratios of peridinin to chlorophyll a (Per:Chl), silicate to nitrate (Si(OH)4:N) and the maximum PSII quantum efficiency (Fv/Fm) are also presented.
Fm (stations 7–9, transect 1; Fig. 8). Relatively high values of the pDA:Fuc ratio were present when Fv/Fm was below 0.5. This was also observed in transect 3. The high pDA:Fuc values detected in transect 3 were related to low Fv/Fm values at stations 21 and 16 (Fig. 8). 4. Discussion The environmental conditions associated with wind-driven upwelling in the Todos Santos Bay area (31.88N) caused the accumulation of DA-producing Pseudo-nitzschia cells. This is the southernmost report of a DA-producing Pseudo-nitzschia bloom in the California Current System and it is also the first report of the distribution of DA in Baja California west coast. The lack of information about the distribution and abundance of potentially toxic Pseudo-nitzschia species and DA does not imply that Baja California coastal waters are free of DAP events. Potentially toxic Pseudo-nitzschia species are part of the phytoplankton assemblages in the Southern California Bight (Villac et al., 1993; Lange et al., 1994; Fryxell et al., 1997; Hasle, 2002) and specifically P. australis
seems to have a wide distribution range since it has been identified in samples from the southern part of the Baja California Peninsula (Herna´ndez-Becerril, 1998). If potentially toxic species are present along the Baja California coast, their blooms will depend on suitable environmental conditions that could promote their growth and cell accumulation. Nutrient fertilization during upwelling events is a key condition that promotes the development of toxic blooms in the area of central California (Scholin et al., 2000; Trainer et al., 2000; Taylor and Trainer, 2002; Anderson et al., 2006). Transitional stages from strong to weak upwelling periods seem to promote the formation of toxic Pseudo-nitzschia blooms (GEOHAB, 2005). It has been also proposed that coastal toxic blooms are formed after an upwelling event and during wind relaxation when cells are transported to inshore areas (Trainer et al., 2000). The west coast of Baja California presents mesoscale oceanographic features that could offer the environmental conditions for the growth of toxic diatom species. Upwelling conditions occur in late spring and in summer close to the study area (Pe´rez-Brunius et al., 2006). Specifically, the documented toxic bloom in the present work was related to weak
Author's personal copy
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
upwelling conditions present in Salsipuedes Bay (probably further north). Upwelling events in the area could affect surface hydrographic properties 20 km offshore (Pe´rez-Brunius et al., 2006) while the signal of the event documented here was detected only 5 km offshore. The weak upwelling conditions originated in the northern part of the study zone affected TSB and had an important impact on the phytoplankton community of the region. Nutrient conditions allowed the accumulation of diatoms together with a high biomass of dinoflagellates in specific areas in the northern part of the bay. Particularly, a Ceratium divaricatum (Lemmermann) Kofoid (Dinoflagellata) bloom developed a week after the sampling date with cell abundances as high as 4 106 cells L 1. This bloom covered a great expanse of the study area and extended southward from TSB (data not shown). This indicates that our sampling date coincided with the transition from upwelling to more stratified conditions, which could be a factor that promoted the accumulation of toxic Pseudo-nitzschia cells. Maximum Pseudo-nitzschia cell abundances (3.02 105 cells L 1) and maximum pDA concentration (0.86 mg L 1) measured in BTS are within the reported values for northern toxic blooms (see Table 2 in Schnetzer et al., 2007). However, important differences were found when compared to the blooms described closer to the study area of this work. Cell abundances were higher than in the San Diego and Los Angeles areas but maximum pDA concentrations were lower than in San Diego (2.3 mg L 1; Busse et al., 2006) and Los Angeles blooms (12.7 mg L 1; Schnetzer et al., 2007). On the other hand, the common characteristic between these blooms is that P. australis was the principal species responsible for the accumulation of DA in the environment. Particular environmental characteristics in which the blooms occur might be the reason for the different toxic cell abundances and pDA concentrations. Also, physiological differences among Pseudo-nitzschia strains of the regions might contribute to this variability. Therefore, it is difficult to predict a geographical pattern of potential DA toxicity along the distribution range of P. australis in the southern part of the California Current System. 4.1. Accumulation of toxic species related to environmental factors P. australis have the highest toxic potential of all DA-producing diatoms, given their large cell volume (Bates, 2000). The specific factors that promote the dominance of P. australis over other diatom species are not well understood. We found that water temperature was an important factor for the distribution and accumulation of this species in the TSB area. Maximum pDA and high cell abundances were found around 14 8C. Blooms of P. australis in northern areas were also found around 14 8C (Buck et al., 1992; Trainer et al., 2000; Anderson et al., 2006). This suggests an optimal growth temperature for P. australis in the field. Alternatively and most likely, the environmental conditions associated with the physical phenomena that bring depth water close to surface are important for the growth of P. australis. Specifically, the injection of nutrients to upper layers and an optimal nutrient ratio associated with upwelling conditions are key factors for the accumulation of P. australis. For example, in a bloom reported for the Santa Barbara Channel the abundance of P. australis was negatively correlated to the Si(OH)4:N ratio (Anderson et al., 2006). We detected also that the macronutrient ratio instead of the absolute concentration seemed to be an important factor for the accumulation of P. australis. However, high cell abundances were detected in an area with a high Si(OH)4:N ratio in the entire water column. This is in contradiction with the assumption that low silicate requirements of Pseudo-nitzschia species allow them to outcompete other diatoms when Si(OH)4 values drop after an upwelling condition (Marchetti et al., 2004).
501
This could be true for smaller Pseudo-nitzschia species with different physiological demands but not for P. australis. Perhaps nutrient requirements of this species are higher as compared to smaller Pseudo-nitzschia cells. Also, it should be considered that dynamic environments (vertical mixing) could be important for the dominance of P. australis. It has been reported that phytoplankton growth is optimal when there are relaxation periods (3–7 days) after the injection of nutrients associated with upwelling conditions (Wilkerson et al., 2006). Si(OH)4:N values in the area of high P. australis abundances reflect the injection of silicates to upper layers of the water column (see transect 1; Fig. 8), which indicates that low stratification of the water column was present in this area. This condition perhaps restricted the growth (accumulation) of other diatoms, but not P. australis. 4.2. Toxic potential of P. australis in relation to environmental conditions DA content per cell (cDA) was comparable to the reported values for blooms dominated by P. australis in the Monterey Bay, Los Angeles and San Diego areas (Walz et al., 1994; Scholin et al., 2000; Busse et al., 2006; Anderson et al., 2006; Schnetzer et al., 2007). However, there is a high variability in the documented cDA in the field and values from 0 to 117 pg DA cell 1 have been reported (Schnetzer et al., 2007). We also observed a high variability in cDA. Two groups of cells with different cDA content were detected (Fig. 7A). Since P. australis represented the major proportion of all Pseudo-nitzschia species in the samples, the different DA content between the two groups could be associated with a differential physiological status of the cells (see below). Another explanation is that there were different strains of P. australis with different toxic potentials. The discrepancy between the estimation of cell abundances with the WCH technique and optical microscopy might be related to this explanation. If the auD1 probe cross reacted only with a certain proportion of the P. australis population, this implies that there are some genetic differences between the Monterey Bay strain from which the probe was raised and strain(s) from TSB. These differences (if present) might be reflected on a differential physiological response of the strains and perhaps on DA production. However, problems that could bias this comparison have to be considered (Parsons et al., 1999) and the use of molecular probes developed for different isolates from our region has to be validated. This is beyond the scope of this work. The synoptic analysis of the distribution of properties was important to infer which conditions might control P. australis DA content. We found that photosynthetic competence reflected by the maximum efficiency of charge separation of PSII (Fv/Fm) was inversely correlated to the DA:Fuc ratio. Relatively low values of Fv/ Fm matched high DA:Fuc ratios. This is clearly seen in the area with the highest abundance of toxic cells (stations 7–9, transect 1; Fig. 8). A relatively high cellular DA content together with a low photosynthetic competence, has been reported for P. australis growth under Si(OH)4 stress conditions (Kudela et al., 2003). Furthermore, enhancement of cDA under low Si(OH)4 levels has been documented in field surveys (Anderson et al., 2006). In the case of the bloom reported in this work, nutrient stress seems not to be the cause of the relatively low Fv/Fm values and a high DA:Fuc ratio (used as a proxy of cDA). Low Fv/Fm values have been associated with nutrient stress conditions in phytoplankton communities (Kromkamp and Forster, 2003). However, a reduced photosynthetic competence in an area in which a high Si(OH)4:N ratio was present could be explained by the lack of acclimation of the phytoplankton community (dominated by P. australis) to a specific environmental condition. Physiological responses when cells are transferred from one condition into another might reduce
Author's personal copy
502
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503
transitionally the photosynthetic potential of microalgae. Lowlight acclimated cells transferred to high-light and to different nutrient conditions show transitional low Fv/Fm values that will be permanent if the cells are incapable of acclimatize to the new conditions (Parkhill et al., 2001). Perhaps, low Fv/Fm values can be related to the transport of deeper (low-light acclimated) cells to surface or near to the surface layers due to vertical mixing and not necessarily to nutrient stress. The relatively high pDA:Fuc values detected in deeper samples from stations 7 and 8 (transect 1; Fig. 8) indicate that there may have been cells of P. australis down to 40 m depth (Fuc concentration at this depth was 0.35 mg L 1) that might be transported to upper layers (distribution of pDA:Fuc in station 9). The presence of cells below the euphotic zone that retains its toxicity has been also reported near Point Conception upwelling region (Trainer et al., 2000). Our data show that the amount of DA in the environment will depend on the abundance of the toxic species but principally on the physiological status of the cells. Therefore, there is not a direct link between the density of toxic cells and toxin concentration in the environment. As a consequence, it is difficult to assess the potential of occurrence of a DA poisoning event by only identifying and enumerate potentially toxic species (Trainer et al., 2000; Anderson et al., 2006). Therefore, DA concentrations in the environment should be measured routinely in order to assess the potential of a DAP event. For the bloom reported in this work, there were no reports of sea mammal standings close to the sampling area, which indicates that the amount of DA or the time in which the toxin was present was not sufficient to cause intoxications at higher trophic levels. In conclusion, toxic blooms of Pseudo-nitzschia should not been seen as extraordinary events on the Baja California west coast. Potentially DA-producing species are part of phytoplankton community of the region and oceanographic features that could create the environmental conditions that promote the blooms of these species are also present. Macronutrient ratios and specifically the Si(OH)4:N ratio were important for the accumulation of DA-producing species. In particular, P. australis accumulation was detected in areas of high Si(OH)4:N ratios and water surface temperatures close to 14 8C related to a wind-driven upwelling event. For P. australis, the photosynthetic competence of the cells might influence DA production. Acknowledgements Part of this research was financed by CICESE internal project number O0F047 and COSNET project number 910.06P. Also, the oceanographic campaign was only possible with the financial support of Lorax Consultores S.A. de C.V. DR has been supported by the National Science Foundation (NSF) Science and Technology Center for Coastal Margin Observation and Prediction (CMOP), NSF Award #0424602. We are grateful to Melissa Carter for helping with the WCH technique. We thank Israel Gradilla for the help with SEM analysis and Guadalupe Cabrales and Fatima Castro for the help in the phytoplankton cell counts and pigment analysis. Observations and comments on the manuscript of an anonymous reviewer and Dr. Peter Strutton are greatly appreciated.[SS] References Almaza´n-Becerril, A., Garcı´a-Mendoza, E., 2008. Maximum efficiency of charge separation of photosystem II of the phytoplankton community in the Eastern Tropical North Pacific off Mexico: a nutrient stress diagnostic tool? Cienc. Mar. 34, 29–43. Anderson, C.R., Brzezinski, M.A., Washburn, L., Kudela, R., 2006. Mesoscale circulation effects on a toxic diatom bloom in the Santa Barbara Channel, California. Mar. Ecol. Prog. Ser. 327, 119–133.
Bargu, S., Powell, C., Coale, S., Doucette, G., Silver, M., 2002. Domoic acid detection in krill: a potential vector in marine food webs. Mar. Ecol. Prog. Ser. 237, 209–216. Bates, S.S., 2000. Domoic-acid-producing diatoms: another genus added! J. Phycol. 36, 978–983. 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. Buck, K.R., Ut-tal-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. Mar. Ecol. Prog. Ser. 84, 293–302. Busse, L.B., Venrick, E.L., Antrobus, R., Miller, P.E., Vigilant, V., Silver, M.W., Mengelt, C., Mydlarz, L., Prezelin, B.B., 2006. Domoic acid in phytoplankton and fish in San Diego, CA, USA. Harmful Algae 5, 91–101. Chhak, K., DiLorenzo, E., 2007. Decadal variations in the California Current upwelling cells. Geophys. Res. Lett. 34, L14604, doi:10.1029/2007GL030203. Centurioni, L.R., Ohlman, J.C., Niiler, P.P., 2008. Permanent meanders in the California Current System. J. Phys. Oceanogr. 38, 1690–1710. Espinosa-Carreo´n, T.L., Strub, P.T., Beier, E., Ocampo-Torres, F., Gaxiola-Castro, G., 2004. Seasonal and interannual variability of satellite-derived chlorophyll pigment, surface height, and temperature off Baja California. J. Geophys. Res. 109, 304–315. 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. GEOHAB, 2003. Global Ecology and Oceanography of Harmful Algal Blooms, Science Plan. In: Glibert, P., Pitcher, G. (Eds.), SCOR and IOC, Baltimore and Paris. 87 pp. GEOHAB, 2005. Global Ecology and Oceanography of Harmful Algal Blooms, GEOHAB Core Research Project: HABs in Upwelling Systems. In: Pitcher, P., Moita, T., Trainer, V.L., Kudela, R., Figueiras, P., Probyn, T. (Eds.), IOC and SCOR, Paris and Baltimore. 82 pp. Go´mez-Aguirre, S., Licea, S., Go´mez, S., 2004. Blooms of Pseudo-nitzschia spp. (Bacillariophyceae) and other phytoplankton species at Bahia de Mazatla´n, Mexico. Rev. Biol. Trop. 52, 69–73. Hasle, G.R., 1978. The inverted-microscope method. In: Sournia, A. (Ed.), Phytoplankton Manual. UNESCO, Paris, pp. 88–96. Hasle, G.R., 2002. Are most of the domoic acid-producing species of the diatom genus Pseudo-nitzschia cosmopolites? Harmful Algae 1, 137–146. Hasle, G.R., Syvertsen, E.E., 1996. Marine diatoms. In: Tomas, C. (Ed.), Identifying Marine Diatoms and Dinoflagellates. Academic Press, San Diego, pp. 5–385. Herna´ndez-Becerril, D.U., 1998. Species of the planktonic diatom genus Pseudonitzschia of the Pacific coast of Mexico. Hydrobiologia 379, 77–84. Horner, R.A., Garrison, D.L., Plumley, F.G., 1997. Harmful algal blooms and red tide problems on the U.S. west coast. Limnol. Oceanogr. 42, 1076–1088. Kromkamp, C.J., Forster, R.M., 2003. The use of variable fluorescence measurements in aquatic ecosystems: differences between multiple and single turnover measuring protocols and suggested terminology. Europ. J. Phycol. 38, 103–112. Kudela, R., Cochlan, W., Roberts, A., 2003. Spatial and temporal patterns of Pseudonitzschia spp. in central California related regional oceanography. In: Steidinger, K.A., Landsberg, J.H., Tomas, C.R., Vargo, G.A. (Eds.), Harmful Algae 2002. Florida and Wildlife Conservation Commission, Florida Institute of Oceanography, and Intergovernmental Oceanographic Commission of UNESCO, pp. 347–349. Lange, C.B., Reid, F.M., Vernet, M., 1994. Temporal distribution of the potentially toxic diatom Pseudonitzschia australis at a coastal site in Southern California. Mar. Ecol. Prog. Ser. 104, 309–412. Lefebvre, K.A., Powell, C.L., Busman, M., Doucette, G.J., Moeller, P.D.R., Silver, J.B., Miller, P.E., Hughes, M.P., Singaram, S., Silver, M.W., Tjeerdema, R.S., 1999. Detection of domoic acid in northern anchovies and California sea lions associated with an unusual mortality event. Nat. Toxins 7, 85–92. Lefebvre, K.A., Silver, M.W., Coale, K.H., Tjeerdema, R.S., 2002. Domoic acid in planktivorous fish in relation to toxic Pseudo-nitzschia cell densities. Mar. Biol. 140, 625–631. Marchetti, A., Trainer, V.L., Harrison, P.J., 2004. Environmental conditions and phytoplankton dynamics associated with Pseudo-nitzschia abundance and domoic acid in the Juan de Fuca eddy. Mar. Ecol. Prog. Ser. 281, 1–12. Miller, P.E., Scholin, C.A., 2000. On detection of Pseudo-nitzschia (Bacillariophyceae) species using whole cell hybridization: sample fixation and stability. J. Phycol. 36, 238–250. Moestrup, Ø., Lundholm, N., 2007. IOC Taxonomic Reference List of Toxic Plankton Algae Diatoms. http://www.bi.ku.dk/ioc/group1.asp. Parkhill, J.P., Maillet, G., Cullen, J.J., 2001. Fluorescence-based maximal quantum yield for PSII as a diagnostic of nutrient stress. J. Phycol. 37, 517–529. Parsons, M.L., Scholin, C.A., Miller, P.E., Doucette, G.J., Powell, C.L., Fryxell, G.A., Dortch, Q., Soniat, T.M., 1999. Pseudo-nitzschia species (Bacillariophyceae) in Louisiana coastal waters: molecular probe field trials, genetic variability, and domoic acid analyses. J. Phycol. 35, 1368–1378. Pe´rez-Brunius, P., Lopez, M., Pineda, J., 2006. Hydrographic conditions near the coast of northwestern Baja California: 1997–2004. Cont. Shelf. Res. 26, 885–901. Quilliam, M.A., 2003. Chemical methods for domoic acid the amnesic shellfish poisoning (ASP) toxin. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D.
Author's personal copy
E. Garcı´a-Mendoza et al. / Harmful Algae 8 (2009) 493–503 (Eds.), Manual on Harmful Marine Microalgae, Monographs on Oceanographic Methodology, vol. 11Intergovernmental Oceanographic Commission (UNESCO), Paris, pp. 247–266 (Chapter 9). Schnetzer, A., Miller, P.E., Schaffner, R.A., Stauffer, B.A., Jones, B.H., Weisberg, S.B., DiGiacomo, P.M., Berelson, W.M., Caron, D.A., 2007. Blooms of Pseudo-nitzschia and domoic acid in the San Pedro Channel and Los Angeles harbor areas of the Southern California Bight, 2003–2004. Harmful Algae 6, 372–387. 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., Cordaro, J., Delong, E.F., Vogelaere, A.D., Harvey, J., Haulena, M., Lefebvre, K., Lipscomb, T., Loscutoff, S., Lowenstine, L.J., Marin III, R., Miller, P.E., McLellan, W.A., Moeller, P.D.R., Powell, C.L., Rowles, T., Silvagni, P., Silver, M.W., Spraker, T., Trainer, V.L., Dolah, F.M.V., 2000. Mortality of sea lions along the central California coast linked to a toxic diatom bloom. Nature 403, 80–84. Sierra-Beltra´n, A., Palafox-Uribe, M., Grajales-Montiel, L., Cruz-Villacorta, A., Ochoa, J.L., 1997. Sea bird mortality at Cabo San Lucas, Mexico: evidence that toxic diatom blooms are spreading. Toxicon 35, 447–453. Soto-Mardones, L., Pare´s-Sierra, A., Garcı´a, J., Durazo, R., Hormazabal, S., 2004. Analysis of the mesoscale structure in the IMECOCAL region (off Baja California) from hydrographic. ADCP and altimetry data. Deep Sea Res. Part II 51 (6–9), 785–799. Strickland, J.D.H., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis. Bull. 167, second ed. Fish. Res. Bd., Ottawa, Canada. Taylor, F.J.R., Trainer, V.L., 2002. Harmful Algal Blooms in the PICES Region of the North Pacific. PICES Sci. Rep. No. 23, 152 pp.
503
Trainer, V.L., Adams, N.G., Bill, B.D., Anulacion, F., Wekell, J.C., 1998. Concentration and dispersal of a Pseudo-nitzschia bloom in Penn Cove, Washington, USA. Nat. Toxins 6, 113–126. 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, June 1998. Limnol. Oceanogr. 45, 1818–1833. Trainer, V.L., Cochlan, W.P., Erickson, A., Bill, B.D., Cox, F.H., Borchert, J.A., Lefebvre, K.A., 2007. Recent domoic acid closures of shellfish harvest areas in Washington State inland waterways. Harmful Algae 6, 449–459. Trainer, V.L., Hickey, B., Bates, S.S., 2008. Toxic diatoms. In: Walsh, P.J., Smith, S.L., Fleming, L.E., Solo-Gabriele, H., Gerwick, W.H. (Eds.), Oceans and Human Health: Risks and Remedies from the Sea. Elsevier Science Publishers, New York, pp. 219–237. Van Heukelem, L., Thomas, C.S., 2000. Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J. Chromatogr. A 910, 31–49. Venrick, E.L., 2000. Summer in the Ensenada Front: the distribution of phytoplankton species, July 1985 and September 1988. J. Plankton Res. 22, 813–841. Villac, M.C., Roelke, D.L., Chavez, F.P., Cifuentes, L.A., Fryxell, G.A., 1993. Pseudonitzschia australis Frenguelli and related species from the west coast of the U.S.A.: occurrence and domoic acid production. J. Shellfish Res. 12, 457–465. 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 2, 271–279. Wilkerson, F.P., Lassiter, A.M., Dugdale, R.C., Marchi, A., Hogue, V.E., 2006. The phytoplankton bloom response to wind events and upwelled nutrients during the CoOP–WEST Study. Deep Sea Res. Part II 53, 3023–3048.
