ECOHAB PNW Cruise IV Report

ECOHAB PNW 4 CRUISE REPORT
R/V Atlantis AT11-30
July 7- July 27, 2005

B. Hickey, W. Cochlan, E. Lessard, V. Trainer, C. Trick, and A. MacFadyen

Area of Operations
Itinerary
Participating Organizations
Chief Scientist
Personnel
Cruise Objectives and Sampling Scheme
Operations
Samples Collected
Cruise Summary

Introduction

1. Regional Surveys (ECOHAB PNW team)

2. Drift Studies (MacFadyen, Hickey, drifters; whole team for water/nutrients)

3. Drifter Deployments (MacFadyen, Hickey, Falkner, Geier)

4. Satellite Imagery (Woodruff, Stumpf)

5. Laboratory Analyses

a) Lessard Group (Lessard, Olson, Foy, DeAmicis)
b) RTC/SFSU Research Activities (Cochlan, Herndon, Auro, Betts)
c) University of Western Ontario Research Group (Trick, McClintock, Beall, Doran, Whiston)
d) Trainer Group (Trainer, Baugh, Nance, Day, Bill, Sarich, Ohana-Richardson, Hendrickson)
e) Wells Group (Roy, Hughes)

6) Moored Sensor Arrays: (Hickey, Thomson, Geier, Juhasz, Johnson)

Acknowledgements

List of Tables and Figures

ECOHAB PNW 4
CRUISE REPORT
R/V Atlantis AT11-30
July 7-27, 2005
B. Hickey, W. Cochlan, E. Lessard, V. Trainer, C. Trick, and A. MacFadyen

Area of Operations

Itinerary

Participating Organizations

Chief Scientist

Personnel

Principle Investigators

Dr. William Cochlan, Romburg Tiburon Center, San Francisco State University
Dr. Evelyn Lessard, University of Washington
Dr. Vera Trainer, NOAA/Northwest Fisheries Science Center
Dr. Charlie Trick, University of Western Ontario

Staff

Liza McClintock, University of Western Ontario
Mike Foy, University of Washington
Susan Geier, University of Washington
Julian Herndon, San Francisco State University
Denis Costello, North High School/San Francisco State Universitty
William Nitsche, University of Washington
James Falkner, University of Washington
Keri Baugh, NOAA/Northwest Fisheries Science Center
Sheryl Day, NOAA/Northwest Fisheries Science Center
Brian Bill, NOAA/Northwest Fisheries Science Center
Shelly Nance, NOAA/Northwest Fisheries Science Center
Alan Sarich, Washington State Dept. of Fish and Wildlife
Margaret Hughes, University of California, Santa Cruz

Students

Amy MacFadyen, University of Washington
Ben Beall, University of Western Ontario
Brady Olson, University of Washington
Eric Roy, University of Maine
Maureen Auro, San Francisco State University
Julia Betts, San Francisco State University
Sean Doran, University of Western Ontario
Caroline Whiston, University of Western Ontario
Jessica Hendrickson, Hampshire College
Andrew Ohana-Richardson, University of Oregon
Stacey DeAmicis, University of Washington

Cruise Objectives and Sampling Scheme

The purpose of this cruise was to determine the physical, chemical and physiological conditions under which diatoms of the genus Pseudo-nitzschia (PN) produce the neurotoxin domoic acid (DA), and the ecophysiological conditions which promote cellular release of toxin to the surrounding environment. We attempted to observe the conditions under which toxic cells advect towards the coast of Washington where they are consumed by shellfish. Such occurrences lead to closure of beaches to razor clam collection to avoid outbreaks of amnesic shellfish poisoning.

Sampling was organized around a comprehensive grid of stations, sampled repeatedly as environmental conditions changed. Continuous surface water measurements included: temperature, salinity and in vivo fluorescence, discrete surface samples for planktonic community analysis and species identification with net tows. Property profiles were obtained with a CTD (conductivity, temperature, depth) including additional sensors that measured in vivo fluorescence, photosynthetically active radiation (PAR), beam attenuation (light transmission), and oxygen concentration. During CTD casts discrete samples were collected with Niskin water samplers for chlorophyll, nutrient analyses, species and community identification (via FlowCAM and flow cytometer analyses), particulate and dissolved DA. A trace metal clean, underway sampling system was employed to measure iron concentrations on board, and to collect samples for multi-element determination (including copper) ashore. On-deck incubations of phytoplankton assemblages were conducted for growth and grazing experiments, and shipboard analyses of the plankton were routinely conducted using both traditional (microscopic) and advanced (FlowCAM image and flow cytometric analyses) methods. Satellite-tracked drifters were released in the Strait of Juan de Fuca, near the Juan de Fuca eddy and off the coast of Washington. One drifter was followed, sampling the water properties with CTD and Niskin water samples at 1-2 hour intervals. The cruise was diverted to Neah Bay on July 13 to exchange personnel. The overall ship track and CTD stations are shown in Figure 1.

Operations

ADCP lines: ~3000 km
Flow-Through system track with T,S,FL sensors: ~2000 km
CTD casts: 225
Satellite-tracked buoy deployments: 11

Samples Collected

Chlorophyll samples: >200 stations and deck-board experiments (~1500 samples)
Nutrient samples: >150 stations and deck-board experiments (>2000 samples)
¹⁴C Uptake (P vs. E) rates: 50 experiments (~1000 samples)
Heterotrophic Bacterial Productivity: 50 experiments (350 samples and controls)
Flow Cytometry samples (nanoplankton, cyanobacteria, bacteria): >125 Stations and Deckboard incubation experiments (~700 samples)
HPLC Pigment samples: >125 Stations at 5 m depth
Dilution growth and grazing experiments: 17
Microplankton samples (preserved): ~13 stations and 17 dilution experiments
FlowCAM samples: ~ 500 stations and experiments
Phytoplankton/DA samples: 228 stations
Surface (4 m) samples for Fe determination and samples for analysis of other bioactive trace metals (Zn, Co, Cu, Ni, Cd) ~120 samples
Sinking Rates: 50 stations
Particulate DA: 1010 samples (650 survey, 360 grow out experiments)
Dissolved DA: 1010 samples (650 survey, 360 grow out experiments)
Preserved net tow samples (650 survey samples) for scanning electron microscopy
Whole water samples (650 survey samples) for PN cell counts

Cruise Summary

Introduction

The ECOHAB-PNW 4 cruise has provided an important contrast to the 2003 and 2004 cruises. During ECOHAB-PNW 4, approximately 95% of our normal study area was devoid of PN cells producing measurable amounts of DA, and concentrations of both the diatom and the toxin DA never reached maximal levels experienced in previous cruises. Perhaps equally interesting, the cruise took place during a summer with anomalously weak upwelling. The lack of upwelling and media reports of a crashing ecosystem were reported in a number of prominent West Coast newspapers (e.g., Seattle Times, San Francisco Chronicle) as well as on National Public Radio. Our research team was able to measure the system in the reduced upwelling state, but also were able to follow its recovery in a strong, persistent upwelling period that lasted for ~2 weeks of the cruise period.

The study obtained multi-disciplinary data from a large scale grid (Section 1), sampling water properties and plankton while following a drifter (Section 2), deployment of surface drifters (Section 3), satellite imagery (Section 4), and on-board laboratory studies using water/plankton collected at selected sites (Section 5).

The setting of cruise sampling events with respect to wind direction (upwelling or downwelling-favorable) is shown in Figure 2. For simplicity we have characterized the wind patterns into two periods: predominantly downwelling (1) and strong, persistent upwelling (2). All data sections and maps on the website are grouped into these two periods.

Over 200 water column profiles were obtained. Satellite imagery [Sea surface temperature (SST) and chlorophyll] was limited during the first week of the cruise (one useable image). However a number of very good surface fluorescence (Chl a) and turbidity images were obtained during the upwelling period during the second half of the cruise. Cruise activities were recorded in a sequential “Event” log (Table 1) from which summary tables discussed below were derived.

Our cruises to date have been highly successful—moreover we have been able to sample sufficiently different biological, chemical and physical conditions in our study area to allow a comparative analysis of various environmental factors and their effects on PN, growth, grazing, nutrient pathways and DA production. In particular, with the new information obtained during this cruise we have shown that:

The Juan de Fuca eddy is more eddy-like during periods of downwelling winds.
Thus development of a large bloom in the eddy such as in September 2004 likely requires a substantial period of downwelling.
Phytoplankton (including PN) and their associated toxins escape from the eddy primarily during periods of upwelling.
In contrast with the nearshore coastal regions, the Juan de Fuca eddy region has substantial surface macronutrients (nitrate, silicate) even during extended periods of no coastal upwelling. Thus plankton in the eddy experience a different nutrient environment (N and Si present at saturating concentrations for uptake and growth of diatoms) than the plankton communities near the coast where sub-saturating concentrations are more prevalent.

Four problems were encountered on the cruise: 1) freezers (both -20 and -70 C) were turned off without notice before the cruise left, leading to a meltdown of important reagents and nutrient (non-radioactive) stocks prior to leaving University of Washington; 2) the nutrient autoanalyzer malfunctioned due to severe organic contamination of water used for baseline and reagent preparation. Two days were spent troubleshooting the instrument. The problem was finally traced to substandard Milli-Q water on the R/V Atlantis. The filter set was changed (despite indicator readings suggesting ‘clean’ water) and the problem was eventually solved within another 2 days; 3) the light on the ECOHAB mooring near the eddy center (EH3) was found to be out. A new light was purchased, brought to the ship at Neah Bay and subsequently installed by Amy MacFadyen (UW) and Julian Herndon (SFSU); 4) one of the two recirculating water baths used to regulate water temperature for the P vs E incubators malfunctioned, and could not be repaired despite assistance from the Ship’s Engineering Department and the manufacturer; this necessitated a reduction in scheduled number of P vs E experiments completed during the cruise.

1. Regional Surveys (ECOHAB PNW team)

The large scale survey grid was designed to include areas influenced by the Strait of Juan de Fuca, the Juan de Fuca eddy region and the coastal upwelling region off the Washington coast (Fig. 3). Data collected on surveys included conductivity (C), temperature (T), light transmission, PAR, oxygen and fluorescence (Fl) profiles, and bottle samples for phytoplankton biomass (Chlorophyll a), whole cell fluorescent molecular probe assays, particulate DA, dissolved DA, FlowCAM and Flow cytometry samples, samples for scanning electron microscopy of PN species, plankton (including PN cell counts) and macronutrients, all at selected depths in the water column. Surface net tows for qualitative community assessment were taken at all survey stations. Water samples containing PN were placed in medium for isolation and culturing in the laboratory. Underway data included T, S and Fl pumped from a depth of about 4 m as well as ADCP current profiles from a 150 kHz narrowband RDI ADCP. An ISUS nitrate sensor was tested during this cruise.

In addition to surveys, a time series at a fixed location (LAB3) was obtained. The time series was taken to determine whether the enhanced upper water column upwelling previously observed at this station was due to tidal forcing. The series was continued for 25 hours, CTD profiles were taken every 2 hours.

A list of CTD stations organized by sample line and including bottle sample types taken is given in Table 2. Lines were sampled in whichever direction made best use of ship time. CTD profiles were taken to 500 m where possible. Deeper data were taken on the OZ line only once and the section occurred during the downwelling period. For the first time in our ECOHAB-PNW studies, the northern line LD was sampled. Several calibration stations were taken at the three mooring sites.

Chlorophyll a (size-fractionated samples: >5 µm and GF/F filters), particulate and dissolved DA and plankton samples (for both microscope and molecular probe analysis) were taken near the surface (~0 m), 5 m and 10 m (and at 15 m at stations after the initial grid survey). DA samples were also taken at 15, 30 and 50 m at other stations, in particular, at Juan de Fuca Strait stations. Flow cytometry and HPLC pigment samples were taken at 5 m depth. Macronutrients (nitrate + nitrite, silicate) were taken generally at the surface, 5 m, 10 m, 15 m, 30 m, 50 m, 100 m, 200 m, 500 m and ~5-10 m above bottom if the bottom was less than 500 m deep. In the Juan de Fuca Strait survey, samples were taken also at 150 m. Whole water samples (4 L) from these deep stations were concentrated through a 20 micron mesh and plankton were visualized through the FlowCAM. On transects, macro-nutrients were taken in most cases at the two stations closest to shore on a line and then every other station on each line. Chlorophyll samples were taken at all stations at 3-4 depths (0, 5 10 m, and chlorophyll maximum depth)

The ISUS nitrate sensor, mounted on the instrumented rosette can be used for vertical profiling, with appropriate calibration. Zero values had an offset of 5-8 µm, and low values were not reliable as the offset diminished with magnitude, but not linearly. These data could be improved with some careful comparison with bottle samples. Above values of ~15 µm nitrate, the sensor was very accurate (plus/minus 1 µm) during this cruise. Missing casts are due to battery failure.

More emphasis was placed this year on determining the source of cells to the Juan de Fuca eddy. At a few stations, 4 L of water was concentrated from the bottom boundary layer to investigate this issue. These samples were both placed into f/2 medium for culturing (Trainer) and analyzed with the FlowCAM (Lessard). Sediment grabs were taken at two stations. A drop of sediment was added to f/2 medium to determine what types of cells are still viable in the sediment surface layers. We hypothesize that the silica cell walls of PN (and therefore other diatoms as well) will assist their survival in the upper layers of sediment.

Upper water column iron samples were taken at selected stations (Tables 1 and 2). These samples were obtained by flying the trace-metal sampler “FISH” below the surface (~4 m). Samples were taken as the ship approached station (within 10-min). Water was pumped for roughly 10 minutes (20 min prior to station location) to flush the lines thoroughly before samples were taken. In addition to FISH sampling for trace metals, all physiological measures and growth rates (phytoplankton and bacteria) were obtained from the FISH, as well as samples for deckboard incubation experiments.

The CTD data were partially edited onboard ship. Shipboard editing included replacing downcast data with upcast data at one station where the CTD pump was inadvertently not turned on in the upper water column. The shipboard data were used to construct the preliminary maps and sections appended to the report. Following the cruise, salinity calibration will be performed and more detailed editing completed (Hickey group). Although water property spatial patterns are likely robust, actual values may change slightly following the final editing which we hope to complete this fall. ADCP set up was performed by Susan Geier onboard ship. ADCP data require more extensive processing and will be provided later this year (Foreman group). Preliminary water property maps and sections obtained from CTD data are given on the ECOHAB-PNW website (T, S, O2, Chl, Fl maps at selected depths; T, S, density, Fl, O2 transects versus depth for all transects, 0-100 m and 0-500 m scales). Maps of relative abundance of PN at the surface are also included.

The CTD data are organized into two groups: Survey 1 (July 7-13) and Survey 2 (July 13-26). Grid stations sampled in each period are shown in Figures 4a-c. Survey 2, which took place during persistent and strong upwelling-favorable winds, was the only complete survey (Fig. 4b). However, sufficient CTD and underway sampling were done in Survey 1 to define the large scale patterns during downwelling reasonably well.

Underway data should be treated with caution. Improvements were made over last cruise. Sensors are available at 4 m from mid ship at 4 m (SBE) and at 5 m from the standard location near the bow thruster. Even so, we noticed that because of the draft of the ship, underway data were sometimes compromised when we were on station and the pycnocline was shallow: the ship mixed up deeper water. However the underway data did provide reasonable maps for both sample periods when some smoothing was incorporated into the plotting routines. We did not perform a systematic evaluation of underway sensors.

This is the first year we have been able to sample a very strong and prolonged period of downwelling and also a prolonged period of upwelling. In prior years winds have been much more variable. The data thus provide important end points in terms of water properties as well as plankton distributions and physiology.

Only one drift study was performed on the cruise. This was primarily because PN were present only in low numbers and DA levels were also low. The focus of this cruise was to obtain systematic patterns of trace-metal ambient concentrations, water properties, phytoplankton physiological “health” and growth and grazing rates, and bacterial productivity over the entire study area; this had not been attempted in prior cruises. The one drift, which began at station LAB9 was undertaken primarily to investigate changes in mixed layer depth. The drift lasted about 22 hours.

CTD/nutrient transects were made along and across the Strait of Juan de Fuca during the weak downwelling period and neap tides (Fig. 4b) and again during strong upwelling and spring tides (Fig. 4c). In each case the along-strait survey started near the mouth during max ebb and the tide was ebbing during much of the survey. The first near cross-strait section was performed on ebb, the second on flood tide. No chlorophyll data were taken on the last cross-strait section as the ship was headed in and there was not time to process samples. Deeper nutrients as well as ammonium and urea samples were collected on these sections. In the first strait survey, sections were also made across the mouth and midway down the strait.

Some Preliminary Results:

The first survey clearly captured the coastal downwelling that was occurring during the sampling period (see web site surface maps). Fresher water was observed along the Washington coast; this is the signature of a plume from the Columbia River, which flows northward and hugs the coast during downwelling-favorable wind events. Fresher water was also observed off the Vancouver Island coast: this is the signature of water emanating from the strait, forming the Vancouver Island Coastal Current that typically hugs the Vancouver Island coast. In spite of the lack of coastal upwelling, relatively high macronutrients were observed in the eddy region. However, along the Washington coast surface nitrate was below detection (< 0.1 µM).

The second survey successfully captured the change to upwelling-favorable conditions. The survey was started several days after the onset of upwelling—the time needed for freshwater to move offshore and permit upwelling on the Washington coast. High nutrients were observed along the coast and well out into the eddy.

The surface fluorescence during the period showed higher fluorescence in the region corresponding to the eddy and near the Washington coast in the Columbia plume water in survey 1. Thus in spite of the lack of upwelling and media claims that the ecosystem is failing, plankton persist in the eddy and near the Washington coast in reasonable numbers. In survey 2, fluorescence increased dramatically in the eddy and along the coast, during and following the change from downwelling to upwelling-favorable winds. Areas of high fluorescence spread well offshore along the coast, in the eddy region, and north of the eddy region (from satellite images as well as survey data).

PN cell numbers and particulate DA were extremely low, especially during survey 1. Numbers of cells increased in the second period along with the generally increasing biomass (perhaps doubled). During survey 2, toxin levels were low but detectable, similar in value to estimates made in June 2003. In general highest particulate DA was observed on the fringes of the eddy. Some PN were also observed near the Washington coast—these did not appear to have associated DA. Molecular probe work indicated that P. australis and P. pungens were present both in the Juan de Fuca Strait and the eddy region.

2. Drift Surveys (Amy MacFadyen, Barbara Hickey, drifters; whole team for water samples)

One drift study was performed, following a water patch with a satellite-tracked Brightwaters drifter. To avoid regions of high current shear, no drogues were used—the drifters average the top meter of the flow. Deployment and recovery times and deployment location are listed in Table 3. Note that drifts are being numbered sequentially beginning each ECOHAB year. CTD profiles and bottle casts were taken at 1-2 hourly intervals (1 hour around noon and after midnight). Nutrients were taken at the usual depths plus 20 and 40 m every 2 hours, starting on the third cast.

The drifter (#3938) was deployed at LAB8 on July 23. The purpose was to study possible effects of atmospheric surface heating and cooling. Dr. Hickey had noted that deep mixed layers were observed at stations at the ends of transects and that these stations were always taken late at night when surface cooling might be significant. The drifter moved southwest in the large scale coastal flow well offshore of the shelf break (Fig. 5). The drift continued for 20 hours.

3. Drifter Deployments (Amy MacFadyen, Barbara Hickey, James Falkner, Sue Geier)

Several Davis-type Brightwaters drifter were deployed to delineate patterns and speeds of surface flows in the eddy area, as well as to determine the ultimate fate of eddy water. Drifter deployment and recovery times and deployment locations are given in Table 3.

Data were stored at the University of Washington and also available online on the ship as the ship had web access. Drifter location and water temperatures are available at 30 minute intervals during deployment periods. Four drifters will continue to collect data until about the end of August. All drifters were deployed at the surface (i.e. no drogues were used).

Three drifters were deployed at the beginning of the cruise: one (#3819) just inside the strait; a second (#3861) near the expected center of the eddy; a third (#3885) near the southwest edge of the eddy (Fig. 6). All three drifters were deployed prior to any real time information about the eddy location. Only the drifter deployed inside the strait (#3819) escaped the eddy during the cruise period: this drifter apparently entered the Vancouver Island Coastal Current. It was recovered after passing Barkley Sound. The drifter deployed near the center of the eddy (#3861) made a complete circuit and a half of the eddy during the downwelling wind period, finally escaping the eddy on its western side on July 19 after 11 days of deployment (note dates are GMT).

The drifter deployed near the southwest edge of the eddy (#3885) also made a complete circuit of the eddy during the downwelling-favorable wind period. It was on the north side of the eddy starting to pass Barkley Sound when winds changed to upwelling-favorable (July 13). The drifter immediately turned southward, then traveled southeastward passing along the south side of the eddy. The drifter subsequently joined the coastal current over the slope and continued south along the coast to Oregon (Fig. 6).

When the cruise began it was unknown whether a southeastward slope current would exist, given the lack of seasonal upwelling. Thus a drifter was deployed in the northwest region at station LC10 on July 13 (#3819) and moved southeast, confirming the existence of the seasonal slope current. This drifter continued southeast throughout the cruise, traveling persistently about 200 km in about 14 days (about 15 km per day).

To determine the location of the coastal jet over the shelf, several drifters were deployed just south of the eddy region (#3775 on July 10, #3917 on July 8, #3901 on July 9). Two of the drifters turned north, joining the eddy rather than joining the coastal jet. The third drifter barely moved in the first 3 days. When the winds changed to upwelling-favorable all 3 drifters turned southward immediately joining the coastal jet on the shelf. #3917 was recovered on July 18 just south of the GH line. #3775 was also recovered. However #3901 passed through the survey grid and went south to Oregon.

To study very nearshore flow direction under upwelling conditions, one drifter (#3861) was deployed at LP1 on July 19. It headed southward along the isobaths but, perhaps surprisingly, did not move offshore. This could be an example of nearshore Ekman layer shutdown.

To contrast with drifter paths deployed during downwelling conditions, two drifters were again deployed at EH1 (#3818, July 20) and on the north side of the eddy (#3917, July 22). Both of the drifters joined the eddy rather than the Vancouver Island Coastal Current. However they both escaped the eddy on its south side and joined the coastal jet, where they were recovered. Thus, during upwelling winds, more water appears to be exported to the eddy rather than the coast of Vancouver Island, and also water can escape the eddy more readily than during downwelling, especially on the west and south sides.

4. Satellite Imagery (Dana Woodruff, Rick Stumpf)

Satellite imagery during the cruise was provided by two groups who sent data to the ECOHAB PNW ftp site—Dana Woodruff from Battelle Northwest Laboratory provided SST imagery and surface chlorophyll and turbidity imagery was provided by Rick Stumpf at NOAA. The available imagery and an assessment of its quality are listed in Table 4. In general, because of the predominantly downwelling wind conditions, few good images were available during the downwelling period. Good chlorophyll images were obtained during the upwelling period. Those chlorophyll and temperature images illustrated a filament of high biomass moving down the coast away from the eddy.

5. Laboratory Analyses

a) Lessard Group (Evelyn Lessard, Brady Olson, Mike Foy, Stacey DeAmicis)

The main goal of this component of ECOHAB PNW is to determine the role of grazers in PN population dynamics and DA production. We used the dilution technique to experimentally alter grazing rate and nutrient recycling to determine the effects of grazers on the net growth rate of the whole and size fractionated phytoplankton community, specific species and groups of phytoplankton, and the production of dissolved and particulate DA. These experiments also provide estimates of the in situ growth rates of PN and other phytoplankton. We took FlowCAM and fixed samples to follow the in situ spatial and temporal changes in the protist grazing community in relation to PN and hydrography. We also conducted an experiment to determine the fate of dissolved DA in seawater.

On this cruise, we performed the following:

17 dilution growth and grazing experiments: These experiments are part of the Ph. D. research of Brady Olson. In these experiments, we followed changes in <5 µm, >5 µm and total chlorophyll, particulate DA, dissolved DA, PN species, and macronutrients (including ammonium in association with Cochlan group). Samples were also preserved and processed onboard for microscopic enumeration of major phytoplankton species later in the laboratory. Chlorophylls were analyzed onboard as well as macronutrients (measured by Cochlan’s group), dissolved and particulate DA (Trainer’s group), and cyanobacteria (Trick’s group). Seventeen microplankton dilution experiments were conducted during this cruise. Of these experiments, 13 were done in regions devoid of PN. This is in stark contrast to 2004, and will provide data necessary to determine the selective constraints imposed on PN, both biotic and abiotic.
High frequency abundance estimates of PN and other plankton with the FlowCAM: Discrete samples from ~3 depths as well as the Fe pump at all inshore stations and select offshore stations along all transect lines during the survey were analyzed with the FlowCAM. Most initial and final samples from the dilution experiments were also analyzed with the FlowCAM. The data files were stored and will be edited and calibrated in the lab to obtain quantitative counts. Replicate fixed samples were taken for microscopic enumerations and calibration of the FlowCAM. During surveys, the FlowCAM proved particularly useful for a quick assessment of PN abundance and community composition at the surface and at depth. In addition, the FlowCAM was used to analyze 10 bottom boundary layer samples taken with the CTD (in collaboration the with Trainer’s group).
Vertical profiles of micro- and nanoplankton: We took preserved plankton samples at thirteen stations on the large scale survey for microscopic determination of autotrophic and heterotrophic nanoplankton, and heterotrophic/mixotrophic dinoflagellates and ciliates.
Dissolved DA degradation experiment: We conducted a controlled experiment (in collaboration with Trainer’s group) to determine degradation rates and the ultimate fate of dissolved DA. Four treatments consisting of whole seawater, <0.2 µm seawater (light and dark), and <0.8 µm seawater were run in parallel with a control. All treatments were spiked with ~50 nM dissolved DA. Four time points were taken and the concentration of dissolved DA will be analyzed later.

b) RTC/SFSU Research Group (Bill Cochlan, Julian Herndon, Maureen Auro, Julia Betts)

The primary objective of this component of ECOHAB-PNW is to examine the relationship between elevated concentrations of the pennate diatom PN and its toxin DA, and ambient concentrations of macro-nutrients and phytoplankton biomass. In addition bioassays (grow-out experiments described below) were conducted to determine the relationship between copper, iron and DA production. At each station of the survey sampling grid, size-fractionated phytoplankton biomass levels were estimated from Chlorophyll a (Chl a) concentrations determined using in vitro fluorometry (aboard ship) after extraction for 24 h with 90% acetone. Chl a samples generally were collected at three depths (0, 5, 10 m) and, at an additional depth corresponding to the chlorophyll maximum layer, when present. Size-fractionated biomass estimates were conducted as follows: total planktonic community was collected on Whatman GF/F filters (nominal pore-size of 0.7 µm), and cells >5 µm in size were collected on Poretics silver membranes. At every second station, dissolved inorganic nutrients were collected at 0, 5, 10, 15, 30, 50, 100, 200 m and near bottom) and analyzed using appropriate colorimetric methods for determination of nitrate plus nitrite, and silicate with a Lachat Instruments QuickChem 8000 Series Flow Injection Automated Ion Analyzer. Both Chl a and nutrients were determined at the two most-shoreward stations of each sampling line. Vertical profiles of dissolved inorganic nutrients were also determined at the drifter stations, during deep canyon profiles, and at a series of 6-7 vertical stations in the Strait of Juan de Fuca on both of the strait transits. Samples from the Juan de Fuca transit were also analyzed for ammonium and urea, in addition to the standard inorganic nutrients. Dissolved nutrients were determined at the beginning (time-zero) and end (time-final) of all of the dilution experiments performed by Lessard’s research group.

A series of 45 shipboard incubation experiments (termed ‘grow-outs’) were designed to assess the role of trace metal (Cu and Fe) availability on the growth of PN and DA production. These multi-day experiments were conducted with water collected from the surface mixed layer (~ 4 m) using the trace-metal clean sampling system (FISH; Wells Group) at stations throughout the sampling grid, with particular emphasis in regions previously found to harbor elevated concentrations of PN and DA. This is the first such extensive survey of the ECOHAB-PNW study area, and it also will provide estimates of the spatial and temporal variability of autotrophic and heterotrophic productivity in relation to the physical and chemical water mass properties of the study area. During all grow-out experiments, samples were analyzed onboard for bacterial and picoplankton abundance by the University of Western Ontario team using flow cytometry (Becton Dickinson, FACSCalibur), and will be used to generate specific rates of bacterial productivity from the bacterial protein synthesis estimates (³H-leucine method). Photosynthetic-irradiance (P-E) curves were generated from short-term (1h) ¹⁴C uptake experiments using photosynthetrons at the initiation of all grow-out experiments and at selected stations in the Strait of Juan de Fuca; these results will be used to describe the efficiency and capacity of phytoplankton photosynthesis with respect to light intensity. P-E curves were generated for all shipboard incubation experiments at the beginning of the 3 to 4-day grow-out incubations. Phytoplankton biomass estimates (as previously described) were determined for all metal and macronutrient treatments at the initiation and termination of each incubation experiments. These measures, together with draw-down rates of macronutrients, will be used to estimate the growth response (including DA production) of the phytoplankton community to copper and iron amendments. Other biological measurements conducted during the grow-out experiments included: microscopic taxonomy, sinking rates (Trick Group), total and dissolved DA (Wells and Trainer Groups), trace metals (Wells Group), and cellular fluorescence capacity (CFC; as measured using the inhibitor DCMU).

Expected Results:

Dissolved Nutrients: Over 60% of collected samples were analyzed onboard and final, processed concentration data made available. This enabled working maps of nutrients to be developed that helped guide further sampling strategies. The remainder will be available by December 1, 2005 using automated and manual colorimetric methods. Inadequate clean water supply aboard the R/V Atlantis precluded complete analysis of all samples collected during the 3-week cruise.
Phytoplankton Biomass: All initial survey grid samples, drifter profiles and onboard deck experiments were analyzed onboard, and are currently available in draft form.
Photosynthetic Efficiency: Radio-isotope samples (¹⁴C) were prepared on board for liquid scintillation counting ashore at RTC; P-E curves should be generated by December 1.
Cellular Fluorescence Capacity: All samples analyzed onboard and are available in draft form.
Bacterial Productivity: Radio-isotope samples (³H) were prepared on board for liquid scintillation counting ashore at NWFSC; rates should be generated by September 1, 2005.

c) Trick Research Group (Charlie Trick, Liza McClintock, Benjamin Beall, Sean Doran, Caroline Whiston)

Our contribution to the ECOHAB project is two-fold: 1) to provide flow cytometric analysis (FCM) and HPLC pigment analysis to characterize the community assemblage; and 2) to provide experimental evidence of factors that either increase the competitive ability of PN or increase the level of DA per cell. Samples for FCM were collected at all survey stations at 5 m and 10 m depths and at 5 m and 30 m in the Strait of Juan de Fuca surveys. HPLC samples were collected at the 5 m depth throughout the grid and Strait of Juan de Fuca surveys. This will allow for quantitative analysis of bacteria, cyanobacteria, and nanoplankton communities, complemented by pigment analysis to characterize the phytoplankton assemblage, which will be performed using HPLC isolation-and-characterization methods. This method uses the presence or absence of the taxon-specific pigments (often referred to as the “minor or accessory” pigments) in relation to the ubiquitous photosynthetic pigments (chlorophyll) to describe the phytoplankton community structure. Our analysis by HPLC will establish the composition of the communities before and after the presence of the diatom communities, thus serving as an important oceanographic descriptor. These samples will be analyzed within ~ 1-2 months since they preserve poorly. Maps of reconstructed photosynthetic communities will be available soon thereafter.

In our second major contribution to the cruise mandate, the personnel from the Cochlan, Wells and Trick labs carried out deckboard incubation growth experiments. All labs offered their expertise to the common goal of all growth experiments (biomass formation, nutrient drawdown measurements, DA analysis (particulate and dissolved), community structure changes, bacterial and phytoplankton productivity, sinking rates and photosynthetic efficiency and capacity). The overall foundation of these grow-out experiments was aimed at elucidating the factors that influence the initiation, formation and/or maintenance of PN blooms or DA levels (either cellular or extracellular). The working hypothesis for this set of experiments was that PN benefits from producing DA because DA serves as an iron and/or copper chelator. Thus in the presence of macronutrients (either in upwelling sites or in the areas of high nutrients associated with the Juan de Fuca eddy) DA would act as an iron chelator, ensuring that the cells would have a supply of iron as iron concentrations diminish, either through colloid formation or utilization. Alternatively DA could serve as a copper chelator, reducing the levels of cupric ion to less inhibitory levels, allowing PN to fully utilize the macro-nutrients and grow effectively.

d) Trainer Group (Vera Trainer, Keri Baugh, Shelly Nance, Sheryl Day, Brian Bill, Alan Sarich, Andrew Ohana-Richardson, Jessica Hendrickson)

At each survey and drift station, samples were routinely taken at 0, 5, 10 m for measurement of particulate and dissolved levels of DA, whole cell counts of PN, enumeration of PN size classes, and scanning electron microscopy for species determination in selected samples. A net tow was taken at every station to rapidly determine the presence or absence of PN and their relative abundance. At Juan de Fuca Strait stations, depth profiles of cells and toxins were done at some of the following depths: 0, 5, 10, 20, 30, 50 m.

Particulate DA was analyzed by filtering 1 L seawater through a Nucleopore HA filter (0.45 micron pore size). Filters were minced in 5 ml distilled water with a thin metal spatula and sonicated for 2 h in a bath sonicator to lyse cells. An aliquot of each sample was analyzed using a receptor binding assay in 96-well plate format using a multiwell harvester and Top Count scintillation counter. The receptor binding assay tests the displacement of [³H]kainate by DA in a sample from a cloned glutamate receptor. Each plate of samples is compared to known DA standards analyzed on the same plate. Endogenous glutamate was digested prior to sample analysis using glutamate dehydrogenase.

Whole cell hybridization assay: Approximately 15 ml sample was filtered and fixed with saline-ethanol for 2 hours. Then specific P. australis (auD1) P. multiseries (muD2) and P. pungens (puD1) probes (fluorescein labeled) were incubated with samples from stations with abundant PN (assessed by surface net tows) taken at several depths. Fluorescence intensity was compared to uniC (positive universal species control) and uniR (negative control) probes. Positively labeled cells on each filter were counted using fluorescence microscopy. Slides were kept in the dark for cell counting in our land-based laboratory.
Dissolved DA: These samples were filtered through a 0.45 mm syringe filter and refrigerated until analysis. Selected samples from grow out experiments were tested using a commercially available enzyme-linked immunosorbent assay (ELISA) with picomolar sensitivity (Beacon Analytical System). This ELISA was developed using antibodies produced at NWFSC, therefore kits can be produced by Beacon at great savings ($100 per kit) over the Biosense ELISA kits (>$300 per plate).
PN culturing: At stations throughout the cruise where PN cells were present, a drop of sample was placed in f/2 medium for isolation and culturing upon return to the lab. PN cells will be allowed to grow in artificial seawater medium and growth and toxin production will be determined for several isolates. This will allow us to understand the relative levels of dissolved and particulate toxin each species is contributing to our cruise samples. Monoclonal isolates from the eddy and nearshore regions will be used to assess the genetic diversity among certain PN species using microsatellite DNA markers. This information will be used to make a preliminary determination of the relationship between PN populations in the eddy and nearshore regions (Nicolaus Adams, Master’s thesis).

e) Wells Group (Eric Roy, Peggy Hughes)

The University of Maine component of the ECOHAB PNW cruise had two primary goals: to collect seawater samples from the study area for trace metal analysis and to optimize and field test a flow injection based method for iron analysis. Roughly 110 surface samples from the survey station grid and the Strait of Juan de Fuca were collected underway, through a trace-metal clean sampling fish. The collected samples will be later analyzed at the University of Maine by high resolution Inductively Coupled Plasma Mass Spectrometry to observe spatial and tidal variability of trace metals, and to serve as means for comparison with the shipboard iron method.

The shipboard method for iron provided preliminary iron concentrations to guide various grow-out experiments conducted by Cochlan’s and Trick’s research team. As a general pattern, total iron concentrations were high (1-2 nM) at nearshore stations and decreased with distance offshore. Elevated iron concentrations were observed at the seaward end of several survey lines.

Acknowledgements

We would like to thank the captain and crew of the R/V Atlantis for their support and extra effort that made the July 2005 cruise successful. We thank the crew and officers of CCGS J.P Tully and the IOS/OSAP/UW mooring team of Susan Geier, Jim Johnson, Tom Juhasz, Dave Spears and Rick Thomson for their help in mooring deployment in May and recovery in October. This research was supported through the Ecology and Oceanography of Harmful Algal Blooms program by National Oceanographic and Atmospheric Administration/Coastal Ocean Program Award No. NA17OP2789 and National Science Foundation Award No. 0234587. Mooring recovery on the Tully was made possible by Canadian support to Richard Thomson at the Institute of Ocean Sciences.

List of Tables and Figures

Table 1 Event log
Table 2 CTD stations organized by sample line and date, showing types of bottle samples taken as well as associated surface iron samples.
Table 3 Drifter deployment locations and times
Table 4 Dates and file name of available satellite imagery
Table 5 Mooring locations, bottom depths, deployment times and satellite PTT ID

Fig. 1 Cruise track with sampling stations.
Fig. 2 Time series of buoy vector winds
Fig. 3 Theoretical survey grid and locations of moored arrays
Fig. 4 CTD cast numbers for Period 1 (Survey 1) and Period 2 (Survey 2+, Final Stations)
Fig. 5 Drifter tracks during Drift A with CTD cast numbers
Fig. 6 Trajectories of drifters deployed on the cruise
Fig. 7 Mooring schematic