Methodology:

Sampling and analytical procedures:

Study area and sample collection

Sample collection occurred during the CICLOPS (Cobalamin and Iron Co-Limitation of Phytoplankton Species) expedition (expedition code NBP18-01; chief scientist G.R. DiTullio) aboard the RVIB Nathaniel B. Palmer, December 11, 2017 – March 3, 2018 in the Amundsen Sea and Ross Sea of the Southern Ocean. Water samples were collected using trace metal sampling protocols described previously (Noble et al. 2012). A trace metal clean rosette suspended on a Kevlar line and equipped with twelve 8L X-Niskin bottles (Ocean Test Equipment) was used to collect seawater at depths ranging from 10 – 600 m.

Preparation of plasticware

Polyethylene and polycarbonate sampling and incubation bottles were rigorously cleaned to remove trace metal contaminants before use. Bottles with rinsed with Milli-Q water (Millipore), soaked for 72h in <1% Citranox detergent, rotated, soaked for an additional 72h, and then rinsed five times with Milli-Q water. Bottles were then filled with 10% HCl (Baker instra-analyzed) by volume and soaked for a minimum of one week, rotated, and soaked for another week. Bottles were then rinsed five times with dilute acid (HCl, pH 2) and stored double-bagged in plastic zip bags. All cleaning work was conducted in a Class 100 clean room.

Analyses of total dissolved Cd and Zn using isotope dilution

Samples for the analysis of total dissolved Zn, Cd, Fe, Mn, Cu and Ni concentrations were collected shipboard by pressure-filtering X-Niskin bottles through an acid-washed 142mm, 0.2µM Supor membrane filter (Pall) within 3 hours of rosette recovery using high purity (99.999%) N₂ gas. Total dissolved water samples were collected into 250mL trace metal clean polyethylene bottles and were stored double-bagged in plastic zip bags. Seawater samples for ¹¹⁰Cd and ⁶⁷Zn stable isotope uptake experiments were collected in the same way but without filtering. All sample collection occurred shipboard within a trace metal clean van containing laminar flow hoods and plastic sheeting. Samples for total dissolved metal analysis were acidified to pH 1.7 with high purity HCl (Optima, Fisher Scientific) within 7 months of sampling and were stored acidified at room temperature for over 1 year prior to analysis.

Quantification of dissolved metals in samples and reference seawater was performed for total dissolved Fe, Ni, Cu, Zn, and Cd using isotope dilution. 15mL of acidified seawater sample was spiked with 50μL of a stable isotope spike solution artificially enriched in ⁵⁷Fe, ⁶¹Ni, ⁶⁵Cu, ⁶⁷Zn, and ¹¹⁰Cd. All stable isotopes were received in solid form (Oak Ridge National Laboratory). Initial dissolution and all subsequent dilutions were made using concentrated nitric acid (Optima, Fisher Scientific). Concentrations and spike ratios were verified by ICP-MS using a multi-element standard curve (SPEX CertiPrep). The composition of the isotope spike was made such that the 15mL spiked sample contained the following ratios: ⁵⁷Fe/⁵⁶Fe = 0.7, ⁶¹Ni/⁶⁰Ni = 0.5, ⁶⁵Cu/⁶³Cu = 1, ⁶⁷Zn/⁶⁶Zn = 0.7, and ¹¹⁰Cd/¹¹⁴Cd = 1 and were verified with ICP-MS. These ratios were chosen to minimize the uncertainty introduced by error propagation through the isotope dilution equation (Wu and Boyle 1998; Rudge et al. 2009; Tan et al. 2020). Because it is monoisotopic, total dissolved Mn was calculated using a modified isotope dilution equation. This equation and its details are captured in the Supplemental Files dropdown on this metadata landing page, within the file titled, Modified Isotope Dilution Equation.

Preconcentration of spiked seawater samples for total dissolved metal analysis was performed using the automated solid phase extraction system seaFAST-pico (Elemental Scientific) in offline concentration mode with an initial volume of 15mL and elution volume of 500µL (Bown et al. 2017; Rapp et al. 2017; Jackson et al. 2018; Wuttig et al. 2019). The seaFAST contains a Nobias-chelate PA1 resin column (ethylenediaminetriacete and iminodiacetate) suitable for the simultaneous preconcentration of several trace metals (Fe, Mn, Zn, Cu, Co, Cd, Ni) with high sensitivity and quantitative recovery (Sohrin et al. 2008; Biller and Bruland 2012). Adjusted seaFAST software settings were a 17 second load loop time and a single 10mL load cycle. Process blanks consisted of pH 2 HCl (Optima, Fisher Scientific) and were processed as samples were to account for any contamination introduced by instrument processing.

Reagents consisted of a 1.5M ammonium acetate pH 6.0 buffer made using glacial acetic acid and ammonium hydroxide (20-22%) of the highest purity (Optima, Fisher Chemical), a 1% nitric acid rinse solution (Optima grade, Fisher Chemical), and a 10% nitric acid elution buffer (Optima grade, Fisher Chemical) with 10 ppb indium (¹¹⁵In, SPEX CertiPrep) added as an internal standard. Solutions were prepared with 18.2 Ω Milli-Q water (Millipore). Polypropylene 15mL centrifuge tubes used in sample processing were made trace metal clean by soaking in 10% HCl for 5 days and rinsing with pH 2 HCl prior to use.

Following offline seaFAST preconcentration, multielemental quantitative analysis was performed using an iCAP-Q inductively coupled plasma-mass spectrometer (ICP-MS) (Thermo Scientific). Oxide interference on metal isotopes was minimized through the use of a cooled spray chamber and helium collision gas. Analytes were measured in single quadruple mode (kinetic energy discrimination [KED]). Concentrations of Mn, Fe, Ni, Cu, Zn and Cd were determined using a six-point external standard curve of a multi-element standard (SPEX CertiPrep), diluted to range from 1-10 ppb in 5% nitric acid. An indium standard (SPEX CertiPrep) was similarly added to these standard stocks, diluted to range 1-10 ppb. Instrument injection blanks consisted of 5% nitric acid in Milli-Q. Standard curve R² values were ≥0.98 for all metals monitored. Method accuracy and precision were assessed using the 2009 GEOTRACES coastal surface seawater (GSC) standard which produced values consistent with consensus results.

Uptake experiments: ⁶⁷Zn and ¹¹⁰Cd spiking, incubation, and sample collection

⁶⁷Zn and ¹¹⁰Cd and stable isotope uptake experiments were modelled after those conducted by Cox et. al. 2014. Raw (unfiltered) seawater was collected shipboard over a depth range of 10 – 600m at each station into 250mL trace metal clean polycarbonate incubation bottles. All incubation bottles were filled with minimal headspace such that the total culture volume was ~275mL. Two incubation bottles per depth were filled with raw seawater— one was spiked with ⁶⁷Zn, the other was spiked with ¹¹⁰Cd. The Cd and Zn isotope spikes were prepared by dissolving ¹¹⁰CdO and ⁶⁷ZnO (Oak Ridge National Laboratory) in 5% HNO₃ (Seastar Baseline) and were diluted using Milli-Q water to minimize added acidity. When added to the filled incubation bottles, the total added (spiked) concentration of Cd was 300 pM and the total added concentration of Zn was 2 nM. The chosen total added concentrations were based on the surface ratio of total dissolved Cd (dCd) to total dissolved Zn (dZn) reported previously for the Ross Sea (Fitzwater et al. 2000). Immediately after spiking, incubation bottles were sealed, inverted to mix, and transferred to flow-through on-deck incubators for 24hr. Incubators were shielded by black net neutral density screening to allow 20% ambient light penetration.

Biomass was collected after 24hr by vacuum filtering the entire volume of each incubation sample at 34.5 kPa (5 psi) onto an acid-cleaned 3mm, 50mm Versapore filter (Pall) mounted on an acid-cleaned Teflon (Savillex) filtration rig. Samples were 3mm filtered rather than 0.2mm in order to minimize filtration time (and thus time exposed to potential contamination) and to capture the bulk of eukaryotic phytoplankton biomass typically found in the Southern Ocean. ~1 mL of 0.2mm filtered surface (10m depth) seawater was used to rinse the sample before collecting the filter into an acid-cleaned 2mL cryovial using acid-rinsed plastic forceps. Filter blanks were duplicate 3mm acid-clean Versapore filters that were placed onto the filtration rig, rinsed with filtered surface seawater, collected, stored, and processed as samples were in order to correct for any contaminating metals present on the filters themselves. Blanks were collected at each station. Filters were stored frozen at -80⁰C in acid-cleaned cryovials until analysis. The filtration rig was rinsed with pH 2 HCl between samples. Polycarbonate incubation bottles were cleaned between stations with a 10% HCl rinse and several rinses in Milli-Q water, followed by a brief soak in 10% HCl followed by a pH 2 HCl rinse. All spike addition and sample filtration procedures were completed in a fabricated shipboard positive-pressure clean room environment made of laminar flow hoods and plastic sheeting.

Filter digestion and particulate ICP-MS analysis

All work was performed in a Class 100 clean room under laminar flow hoods. Sample filters were retrieved from storage at -80⁰C, removed from cryovials using plastic acid-washed forceps, and transferred into trace metal clean 15 mL PFA vials with 4 mL of 5% HNO₃ (Optima) containing a 1 ppb Indium (In) internal standard. Filters were digested for ~3.5h at 140⁰C using a HotBlock® heating block (Environmental Express, USA) before the filters were removed and discarded, leaving behind the liquid extract. After evaporating the remaining solution to just dryness, the residue was resuspended in 2 mL of 5% HNO₃(Optima) by light vortexing. Process blank filters were digested and processed as sample filters were. Digests were analyzed in duplicate by ICP-MS using a Thermo ICAP-Q plasma mass spectrometer calibrated to a multi-element standard curve (Spex Certiprep) over a range of 1 – 20 ppb. Natural Cd and Zn isotope abundances of the standards were assumed to calculate concentrations of ¹¹⁰Cd, ¹¹¹Cd, ¹¹⁴Cd, ⁶⁷Zn, ⁶⁶Zn, and ⁶⁸Zn. Digests were analyzed in KED mode after an 85s sample uptake window and element mass windows were scanned 3 times during measurements. The 1 ppb In internal standard was used to correct for variation in sample delivery and plasma suppression between samples. Process blanks were subtracted from measured sample concentrations. Phosphorus concentrations were simultaneously measured by ICP-MS and were calibrated to a standard curve ranging from 100 – 3,200 ppb using a 1 ppm certified P stock (Alfa Aesar Specpure). The equation and its details used for the calculations described above are captured in the Supplemental Files section of this metadata landing page within the file titled, Particulate Rates Metal Concentration Equation.

The Zn spike and Cd spike were also analyzed by ICP-MS using a 1:9 dilution of spike solution into 5% HNO₃ 1 ppb In to determine isotopic compositions and concentrations. When added to filled incubation bottles (275mL total volume), the added concentrations were 288 pM 110Cd, 4.51 pM 111Cd, and 1.69 pM 114Cd for Cd spiked bottles, and were 1.91 nM 67Zn, 0.045 nM 66Zn, and 0.047 nM 68Zn for Zn spiked bottles.

Calculating zinc and cadmium uptake using ⁶⁷Zn and ¹¹⁰Cd

Total Zn and Cd uptake was calculated using Eq. (3) and Eq. (4), respectively. These equations can be found under the Supplemental Files section of this metadata landing page, within the file titled, Metal Uptake Rate Equations. ¹¹⁰Cd_Sample and ⁶⁷Zn_Sampleare the particulate ¹¹⁰Cd and ⁶⁷Zn measured by ICP-MS analysis of the 3μm sample filter, normalized to the total culture volume (275 mL) and 24hr of incubation. ¹¹⁰Cd_Sample and ⁶⁷Zn_Sample already in the particulate fraction (that is, the pCd and pZn that existed in the water column upon collection of the raw seawater samples) was accounted for by subtracting the particulate blank, ¹¹⁰Cd_Blank and ⁶⁷Zn_Blank.¹¹⁰Cd_Blank and ⁶⁷Zn_Blankwill hereafter be referred to as “pre-existing particulate ¹¹⁰Cd and ⁶⁷Zn”. The pre-existing particulate blank value for ¹¹⁰Cd was obtained from incubation bottles that had Zn added, but no Cd spike. Likewise, the pre-existing particulate blank value for ⁶⁷Zn was obtained from incubation bottles that had Cd added, but no Zn spike. The ⁶⁷Zn spike solution was confirmed to contain virtually no ¹¹⁰Cd, ¹¹¹Cd, ¹¹⁴Cd. The ¹¹⁰Cd spike was likewise confirmed to contain virtually no ⁶⁷Zn, ⁶⁴Zn, ⁶⁶Zn. As a result, we assumed that the added ⁶⁷Zn spike did not affect the pre-existing Cd, nor did the ¹¹⁰Cd spike affect the pre-existing Zn. It is assumed that the pre-existing particulate blank was in steady state, i.e. that it represented the Cd or Zn already in the particulate fraction and any possible natural uptake that could occur during incubation for 24h was negligible. The total dissolved pool of each metal isotope (denominator of each equation) is the sum of the dissolved ¹¹⁰Cd or ⁶⁷Zn added as the spike (¹¹⁰Cd_Spike, ⁶⁷Zn_Spike) plus the natural, pre-existing dissolved ¹¹⁰Cd or ⁶⁷Zn that was in the raw seawater (¹¹⁰Cd_Natural, ⁶⁷Zn_Natural) collected at each depth. To calculate ¹¹⁰Cd_Natural and ⁶⁷Zn_Natural, the total dissolved Cd or Zn measured by isotope dilution-ICP-MS (Cd_Total, Zn_Total) was multiplied by the natural abundance of ¹¹⁰Cd and ⁶⁷Zn (12.49% and 4.10%, respectively). Dividing the particulate ¹¹⁰Cd and ⁶⁷Zn by the total dissolved ¹¹⁰Cd and ⁶⁷Zn yields the fraction of these metal isotopes that moved from the dissolved pool to the particulate pool per day.

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