Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

Water column sampling:
The water column of eight stations in the NE Pacific was sampled to ~1,000 meters (m) in June 2022 aboard the R/V Sikuliaq. Depth profile samples were collected from a combination of the conventional ship rosette (SHIP CTD) and a trace metal clean rosette sampling system (TMC CTD) outfitted with 12-liter (L) modified x-Niskin (OceanTestEquipment) sampling bottles. Surface water (~2 m) samples for the depth profiles were collected using a trace metal clean surface pump "towfish" system (FISH; Mellett and Buck, 2020). Samples for dissolved macronutrients, dissolved trace metals, dissolved iron speciation, labile dissolved nickel concentrations, total alkalinity, pH, and chlorophyll a were collected primarily from the trace metal clean rosette and towfish; additional samples for dissolved macronutrients were also sampled from the conventional rosette. Amber high-density polyethylene bottles were filled directly from the towfish and from the x-Niskin samples immediately after each cast for parallel filtering (< 100 mm Hg) of chlorophyll a on 5-micrometer (µm) membrane (Poretics) filters and on 0.7 µm GF/F (Whatman) filters using a glass and stainless steel Millipore filtration rig in the main lab (William Cochlan lab). Filters for chlorophyll a were frozen at -20 degrees Celsius (°C) in the dark prior to their extraction and analysis at sea.

From the trace metal clean rosette and towfish, samples for dissolved macronutrients and trace metals were filtered (0.2 µm, Acropak) inline and collected inside a trace metal clean and positive pressure sampling bubble. Dissolved macronutrient samples collected from the conventional rosette were syringe filtered (0.2 µm PVDF syringe filters) in the Baltic room of the vessel. All dissolved macronutrient samples were collected into acid-cleaned and thrice sample rinsed 15-milliliter (mL) polypropylene Falcon tubes and stored in zipper bags in the fridge until analyzed shipboard following recommended practices (Becker et al., 2020), typically within 24 hours of collection (Caitlyn Parente, Kristen Buck lab). Samples for dissolved trace metals were collected in acid-cleaned and triple-rinsed narrow mouth low density polyethylene bottles, acidified with 0.024 M ultrapure hydrochloric acid (to pH ~1.8), and stored for shore-based analysis at the University of Nagasaki (Yoshiko Kondo, Shigenobu Takeda). Samples for dissolved iron speciation and labile dissolved nickel concentrations were collected in acid-cleaned, Milli-Q-conditioned, and triple-rinsed narrow-mouth fluorinated high-density polyethylene bottles (Nalgene) and analyzed shipboard for dissolved iron speciation (Lise Artigue, Kristen Buck lab) and labile dissolved nickel concentrations (Calyn Crawford, Kristen Buck lab) before freezing at -20 ºC for additional shore-based speciation analyses at Oregon State University. Samples for pH and total alkalinity were analyzed shipboard (Drajed Seto, Mark Wells lab).

Sample analyses – macronutrients:
Filtered macronutrient samples were analyzed shipboard for phosphate, nitrate+nitrite, silicic acid, and nitrite on a QuAAtro39 AutoAnalyzer (SEAL Analytical) according to standard colorimetric methods (Strickland and Parsons, 1972). All reagents were prepared in dedicated labware with high purity Milli-Q (>18 MΩ cm) water. Working standards were prepared fresh daily in an artificial seawater (ASW; 35 grams per liter (g/L) sodium chloride, 0.5 g/L sodium bicarbonate) matrix using calibrated volumetric pipettes. Nine-point standard curves were analyzed at the beginning of each run with multiple reagent blanks. Quality control checks were analyzed every twelfth sample with ASW blanks and standards. The highest standard from the calibration curve was analyzed approximately every twenty samples to check for drift during the runs. Subsamples of reference material for nutrients in seawater (Kanso) were measured in each run. Detection limits for each parameter were determined from three times the standard deviation of replicate lowest standards. Limits of detection were 0.035 micromolar (µM) for phosphate, 0.048 µM for nitrate+nitrite, 0.051 µM for silicate, and 0.012 µM for nitrite. Values below these limits of detection are reported as 0 µM with accompanying QC Flag 6. Sample analyses for macronutrients were performed by MS student Caitlyn Parente.

Sample analyses – chlorophyll a:
Samples for chlorophyll a were placed in glass test tubes and 8 mL of 100% ethanol was added to each tube (Jespersen and Christoffersen, 1987; Wasmund et al., 2006). The tubes were capped and placed in the dark for the extraction at room temperature. After 12 hours, the fluorescence readings were subsequently measured following the standard acidification protocol (Parsons et al., 1984; Arar and Collins, 1992) using a Turner Designs model 10-AU fluorometer calibrated at the beginning of the cruise with pure chlorophyll a standards (Turner Designs; Anacystis nidulans) following standard JGOFS protocols (Knap et al., 1996).

Sample analyses – dissolved trace metals:
The concentrations of dissolved iron, manganese, nickel, zinc, and copper were analyzed by high-resolution inductively coupled plasma mass spectrometry (Thermo Scientific ELEMENT II) with a preconcentration flow injection system seaFAST-pico (Elemental Scientific Inc., ESI) at Nagasaki University (Yoshiko Kondo and Shigenobu Takeda). Acidified samples were measured without UV-oxidation, and dissolved copper concentrations should be considered ‘reactive Cu’ as recovery may have been hindered by organic complexation in these samples. Briefly, dissolved trace metals in the samples were preconcentrated on a Nobias-chelate PA1 resin, eluted with 2 M HNO3, and quantified by calibration curve prepared with SAFe and GEOTRACES reference samples (S1, GS, and GD) (https://www.geotraces.org/standards-and-reference-materials/).

Sample analyses – labile dissolved nickel:
The concentration of labile dissolved nickel concentrations was measured by competitive ligand exchange-adsorptive cathodic stripping voltammetry using the added ligand dimethylglyoxime (DMG; van den Berg and Nimmo 1987) and following a modification of previously described procedures (Saito et al. 2004; Boiteau et al. 2016). Briefly, seawater sample aliquots were buffered with a borate-ammonium buffer and equilibrated overnight with 200 µM DMG. Following equilibration, the amount of dissolved nickel in the samples that was bound to DMG was measured on a hanging mercury drop electrode and quantified by standard additions of dissolved nickel to the sample. All measurements, of the sample and of the standard additions, were conducted in triplicate. The concentration of labile dissolved nickel was determined from the slope of the standard curve and the triplicate measurements of the initial sample, and the results presented as averages and standard deviations of the three values.

Sample analyses – total alkalinity:
Total alkalinity (TA) was measured using a USB4000 fiber optic spectrometer (Ocean Optics) with bromocresol purple (BCP) as the indicator dye (Hudson-Heck et al., 2021). The spectrometer was calibrated daily using certified reference materials (batch number 189) obtained from the Dickson lab at the Scripps Institution of Oceanography. Calibration was performed at a controlled temperature of 20°C to match the temperature of the spectrophotometric cuvette, which was maintained using a water bath. Seawater samples (2 mL) were pipetted into pre-rinsed (deionized water) and dried spectrophotometric cuvettes and BCP indicator solution was added. The cuvette was capped and purged with N2 gas for 1 minute and the absorbance spectra were recorded at 432, 589, and 700 nanometers (nm). TA was calculated using the absorbance readings with the final absorbance ratio (R ratio) for each sample (Hudson-Heck et al., 2021). All samples were analyzed in triplicate and the results presented as the averages and standard deviations of the three values.

Sample analyses – pH:
pH was measured using a USB4000 fiber optic spectrometer (Ocean Optics) with purified meta-Cresol Purple (mCP) as the pH indicator dye (Liu et al., 2011). The system comprised an open top, flow-thru cell positioned in a temperature-controlled (20 °C) water bath. The cell was zeroed by manually injecting a blank or reference sample (seawater without mCP) and recording the absorbance at 434, 578, and 700 nm (reference). For sample analysis, 1 microliter (µL) of purified mCP indicator solution was drawn into a clean 3 mL syringe followed by 2 mL of seawater sample. The solution was mixed gently to ensure uniform distribution of the indicator while avoiding air bubble formation. The solution then was manually injected into the flow-thru cell (using excess volumes for rinse) and allowed to thermally equilibrate. Once absorbance values had stabilized (1-3 minutes) the values were recorded at 434, 578, and 700 nm. Seawater pH was calculated on the total scale using the absorbance ratio (578/434) according to Liu et al. (2011). All samples were analyzed in triplicate and the results presented as the averages and standard deviations of the three values.

Data Quality Flags:
Data were flagged using the SeaDataNet quality flag scheme recommended by GEOTRACES (https://www.geotraces.org/geotraces-quality-flag-policy/) and described below. Notes specific to the application of these flags to this dataset are noted in brackets […].

0: No Quality Control: No quality control procedures have been applied to the data value. This is the initial status for all data values entering the working archive. [Not used].

1: Good Value: Good quality data value that is not part of any identified malfunction and has been verified as consistent with real phenomena during the quality control process. [Used for analyses that included replicates and/or reference samples].

2: Probably Good Value: Data value that is probably consistent with real phenomena, but this is unconfirmed or data value forming part of a malfunction that is considered too small to affect the overall quality of the data object of which it is a part. [Used when no replicates or reference samples were available to further verify the quality of the data].

3: Probably Bad Value: Data value recognized as unusual during quality control that forms part of a feature that is probably inconsistent with real phenomena. [Not used].

4: Bad Value: An obviously erroneous data value. [Not used].

5: Changed Value: Data value adjusted during quality control. Best practice strongly recommends that the value before the change be preserved in the data or its accompanying metadata. [Not used].

6: Value Below Detection Limit: The level of the measured phenomenon was less than the limit of detection (LOD) for the method employed to measure it. The accompanying value is the detection limit for the technique or zero if that value is unknown. [Values below detection are reported as 0.00 µM for macronutrients].

7: Value in Excess: The level of the measured phenomenon was too large to be quantified by the technique employed to measure it. The accompanying value is the measurement limit for the technique. [Not used].

8: Interpolated Value: This value has been derived by interpolation from other values in the data object. [Not used].

9: Missing Value: The data value is missing. Any accompanying value will be a magic number representing absent data [When sample was not collected the notation ‘na’ for ‘not applicable’ was used; when sample was collected but there is no result for this parameter, the notation ‘nda’ for ‘no data available’ was used].

A: Value Phenomenon Uncertain: There is uncertainty in the description of the measured phenomenon associated with the value such as chemical species or biological entity. [Not used]

NSF Award Abstract:
Iron is an important nutrient for algae in the ocean. Different forms of iron and their availability to algae are influenced by many factors including the acidity of seawater (or pH). This research project focuses on understanding the effects of ocean acidification (low pH) on the associations between iron and chemical substances that bind with iron in seawater. The investigators will work in coastal and oceanic waters of the Pacific Ocean. These waters are characterized by substances that have weak and strong associations with iron. Samples will be collected from coastal waters off Washington State, the northern edge of the North Pacific gyre, and Ocean Station PAPA in the northeast subarctic Pacific. Water samples will be collected to test phytoplankton responses to light, pH, forms of iron, and the composition of the substances that bind with iron. This project will support graduate and undergraduate students. The investigators will participate in a range of education and outreach activities.

This study addresses oceanic responses to rising anthropogenic CO2 and is broadly relevant to ocean biogeochemistry. The investigators will study the role of ocean acidification on iron availability in the Eastern North Pacific Ocean. The study location is characterized as a high nutrient low chlorophyll (HNLC) region of the ocean, where phytoplankton may be particularly sensitive to iron availability. The study region is also characterized by gradients in ligand composition and binding strength. This study will investigate how the associations between iron and different ligands (organic compounds that bind with iron) are influenced by pH and how this, in turn, influences primary production and microbial community structure in the ocean. The investigators will use batch cultures, at pH 8.1 and 7.6, and under high and low light regimes, to examine the iron demand of phytoplankton. Understanding how pH influences iron and its relationship with ligands will provide important information for assessing the impacts of ocean acidification on primary production and biogeochemical processes.