Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Incubation Setup:

Surface water was collected for shipboard incubations in June 2022 aboard the R/V Sikuliaq using a trace metal clean surface pump “towfish” system (Mellett and Buck, 2020). Filtered (<0.2 µm, Acropak) seawater from the towfish was homogenized in three acid-cleaned and seawater rinsed 50-L carboys that were filled round-robin style (Burns et al., 2023). Each carboy was then bubbled overnight with a custom CO2-air mixture to achieve the target pH levels of pH 8.1, 7.6, and 7.1, which was verified with shipboard spectrophotometric pH analyses using the Byrne MICA system (Adornato et al., 2016). Unfiltered surface seawater was then collected and homogenized in a fourth acid-cleaned and seawater-rinsed carboy using the trace metal clean towfish. Trace metal clean polycarbonate incubation bottles were then filled two-thirds with filtered seawater and one-third unfiltered seawater, amended for the nutrient and/or iron treatment, sealed with the caps/threads wrapped in parafilm and electrical tape, and delivered to deckboard flow-through seawater incubators that were covered in screening to mimic surface light levels. Once all incubation bottles were in the incubators, the time-zero sampling of each incubation began. Six incubations were conducted, two each at a coastal upwelling station (Inc 1, 2; 40.112 ºN, 125.56 ºW), in the oligotrophic central North Pacific (Inc 3, 4; 35 ºN, 145 ºW), and at Ocean Station PAPA (Inc 5, 6; 50 ºN, 145 ºW) in the subarctic North Pacific. For incubations 1-4, all incubation bottles were spiked with chelexed stocks of nitrate and phosphate, and aged (for trace metal cleanliness) silicic acid stocks, to target additions of 10 µM nitrate, silicic acid, and 0.8 µM phosphate; no macronutrients were added to Incs 5 and 6, which were already macronutrient replete. Replicates of pH treatment were additionally spiked with 1 nM ⁵⁷FeCl₃ as a dissolved iron addition. Incubation bottles were labeled according to treatment and were the same across light bottles all incubations: A = pH 8.1, B = pH 8.1 + Fe, C = pH 7.6, D = pH 7.6 + Fe, E = pH 7.1, F = pH 7.1 + Fe. Replicates of each treatment were also incubated in heavy duty black contractor bags to serve as dark controls (G = A, H = B, I = C, J = D, K = E, L = F), which were sampled on day final only.

Incubation sampling:

Triplicate bottles from each incubation were sampled daily over the course of the experiments. Incubation bottles were brought in from the incubators into a clean lab bubble in the ship, where they were washed down with Milli-Q and transferred into a clean hood. After gently inverting to mix, each was subsampled for pH, chlorophyll a, and particulate organic nutrients. The remaining contents of each bottle were filtered in the bubble clean hood on a custom acrylic filtration rig outfitted with dual stage Teflon filtration holders (Savillex) that allows the filtrate to go directly into sample bottles after passing through consecutive 5 µm and 0.4 µm acid-cleaned polycarbonate track-etched (PCTE; Whatman) filters. 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). Samples for dissolved iron and nickel speciation 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) before freezing at -20 ºC for shore-based dissolved nickel speciation analyses at Oregon State University (Matthew Koteskey, Kristen Buck lab). Samples for pH were analyzed shipboard (Drajed Seto, Mark Wells lab).

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 total 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/). 

A subset of incubation samples were analyzed for total dissolved nickel concentrations by competitive ligand exchange-adsorptive cathodic stripping voltammetry following UV-oxidation of remaining volume in nickel speciation samples. Frozen seawater samples were thawed at room temperature in the lab at Oregon State University and UV-oxidized in Teflon jars (Savillex) with quartz lids for at least ninety minutes in a Jelight model 342 UVO cleaner. Following UV-oxidation, seawater sample aliquots were buffered with a borate-ammonium buffer and amended with 200 µM dimethylglyoxime to complex the dissolved nickel in the sample and form an electroactive complex, which was then measured by standard addition on a hanging mercury drop electrode (BioAnalytical Systems, Inc.). 

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 – 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 a 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 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 min) 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 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. [Used when value was flagged high].
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. [Not used].
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]

* Created ISO Date-Time column from original DATE_SHIP and TIMESTART_SHIP and TIMESTOP_SHIP
* Added latitude & longitude of stations to dataset
* Converted pH row 285 fron 7,58 to 7.58

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.