Dataset: TMs and Nuts from EXPORTS NA Incubations
Deployment: DY131

Incubation set-up

This work was funded by NSF and conducted in collaboration with the NASA EXPORTS campaign at PAP-SO, which sampled the decline of the North Atlantic Spring Bloom (NASB) in May 2021 on board the RRS Discovery (Cruise ID: DY131). All sampling occurred within a retentive eddy named “A2”, with a deep anticyclonic structure. Three types of shipboard incubation experiments were conducted using trace metal clean techniques: Long-Term (LT), Short-Term (ST), and Grow-Out (GO) incubation experiments, for a total of eight incubations. 

Long-Term experiments

Surface water for LT experiments was collected using a trace metal clean towfish (Mellett and Buck 2020) while transiting at a speed of ~6 knots. Four treatments were used for the LT experiments: control (no additions), AllbutSi (+5 nM 57FeCl3, 20 µM nitrate, 1.25 µM phosphate), AllbutFe (+20 µM silicic acid, 20 µM nitrate, 1.25 µM phosphate), and AllbutN (+5 nM 57FeCl3, 20 µM silicic acid, 1.25 µM phosphate). All Fe additions were made with an enriched standard of 57FeCl3 (ISOFLEX) to serve as a tracer of the added Fe. The nitrate and phosphate stocks were chelexed prior to use to remove metal contaminants. The silicic acid stock was made in an acid-cleaned Teflon bottle that was changed periodically to lower dissolved Fe concentrations and minimize metal contamination from this nutrient addition. 

Triplicate 20 L polycarbonate (PC) carboys were used for each treatment. The carboys were filled in three rounds, with the first set of replicate carboys filled first, followed by the subsequent replicates. To facilitate homogenization, the carboys for each replicate round were filled in three passes, with each pass filling them to one-third of their final volume until full to the brim. For each treatment except the control, a 1 L PC bottle was also filled through the same sequence. Once filled, carboys and 1 L bottles were spiked with the treatments outlined above; a second 1 L PC bottle was filled from the treated carboys as a second check on the treatments. The 1 L bottles were sampled for post-spike time-zeroes of the incubation treatments and were identified as “T0.1”; these bottles were uniquely spiked with 10 nM 57FeCl3. The 1 L bottles sampled from the already treated carboys are identified as “T0.2” and had Fe additions of 5 nM 57FeCl3. Both sets of 1 L bottles were sacrificed in full for the time-zero samples. The LT 2 experiments were set up on the same day as the short-term experiment 2 (ST 2), and the time-zero Control treatments were sampled from one set of incubation bottles to represent both experiments. The treated carboys were placed in seawater-flowthrough deckboard incubators, covered with a mesh to allow 40% of surface photosynthetically active radiation (PAR) exposure, and harvested for final timepoint sampling after 6 (LT 1) or 5 (LT 2) days. 

Short-Term experiments

Three short-term experiments were conducted: ST 1A, ST 1B, and ST 2. Surface seawater was collected for the ST experiments following the same approach outlined above for the Long-Term experiments: 20 L PC carboys were filled with the TMC Towfish in three rounds corresponding to each set of replicates, and in each round, the set of carboys were filled to one-third of the volume, consecutively until full to the brim. Each ST experiment had a different set of treatments: ST 1A included Control (no addition), +Fe (+ 5 nM 57FeCl3); ST 1B included Control (no addition), +Si (+20 µM silicic acid); ST 2 included Control (no addition), +N (+20 µM nitrate). T0.1 and T0.2 1 L bottles were collected the same way as described above for the LT experiments: the T0.1 bottles filled from the towfish and then spiked with 10 nM 57FeCl3, and the T0.2 bottles filled from subsampling the post-spike carboys that had already been spiked with 5 nM 57FeCl3. The same stock standards of 57FeCl3, silicic acid, and nitrate were used for these experiments as for the LT experiments. The ST 2 and LT 2 experiments were set up on the same day, and the time-zero Control treatments were sampled from one set of incubation bottles to represent both experiments. The treated carboys were placed in seawater-flowthrough deckboard incubators, covered with a mesh to allow 40% of surface photosynthetically active radiation (PAR) exposure, and harvested for final timepoint sampling after 22 hours for ST 1A, 40 hours for ST 1B, and 25 hours for ST 2.

Grow-Out experiments

Subsurface seawater for the grow-out (GO) experiments was collected with modified x-Niskin bottles (12L Ocean Test Equipment, Inc.) on a trace metal clean rosette (Cutter et al. 2017). Casts to collect the seawater were conducted between 11 am and 12 pm local time on the ship. Seawater was collected at depths of 47% and 12% of the surface photosynthetically active radiation (PAR), for a “High Light” and “Low Light” treatment, respectively. Seawater from the x-Niskins was transferred to 1 L PC incubation bottles that had been acid cleaned, Milli-Q conditioned, and rinsed three times with seawater before filling (Hollister et al., 2020; Burns et al., 2023). Three treatments were conducted for each light level: Control (no addition); +Fe (+5 nM 57FeCl3); +Fe+Si (+5 nM 57FeCl3, 20 µM silicic acid); for GO 3, the Fe addition was +0.5 nM 57FeCl3. The same stock standards of 57FeCl3 and silicic acid were used for these experiments as for the LT and ST experiments. A replicate incubation bottle for all treatments was prepared in filtered (<0.2 µm; Acropak) seawater and incubated wrapped in heavy duty black construction bags to allow for a dark control and an assessment of abiotic changes in trace metals including wall loss and precipitation. 

The PAR intensity (47% and 12%) from the water column was maintained for the incubations by placing the 1-L incubation bottles in custom mesh screen bags designed to achieve the target light levels. All incubation bottles in their respective light treatment bags were placed in the seawater-flowthrough deckboard incubators, and harvested after 67 hours for GO experiment 1, 45 hours for GO experiment 2, and 70 hours for GO experiment 3.

Incubation sampling

All sampling occurred in a positive pressure hood of HEPA filtered air inside a trace metal clean van built into the RRS Discovery. Incubation sampling occurred at two timepoints: time-zero and time-final. For the LT and ST experiments, the T0.1 and T0.2 1 L bottles constituted the time-zeroes of the incubation treatments with additions. For time-final timepoints, the LT and ST experiments were subsampled from the 20 L PC carboys into 1 L PC bottles after carefully mixing. No sub-sampling was required for the GO experiments, which were conducted in 1 L PC bottles. In all cases, the incubation samples were filtered by vacuum using custom filtration rigs with Teflon dual-stage filter holders (Savillex ®) through two consecutive, acid cleaned polycarbonate track etched (PCTE) filters of 5 µm and 0.4 µm (Burns et al., 2023). The <0.4 µm filtrate was collected for dissolved trace metals in acid-cleaned 125 mL low-density polyethylene (LDPE) bottles, and dissolved macronutrients in acid-cleaned 15 mL polypropylene tubes (Falcon). Dissolved trace metal samples were then acidified to 0.024 M with Optima HCl (pH~ 1.8; (Johnson et al., 2007)) and stored at room temperature until returned to the Buck lab at the University of South Florida for analyses. Dissolved macronutrient samples were stored frozen at -20 °C until returned to the Buck lab at the University of South Florida for analyses.

Analysis of dissolved trace metals

Sample analysis for dissolved trace metals was conducted at the University of South Florida (USF) College of Marine Science (CMS), and Florida State University (FSU) National High Magnetic Field Laboratory (MagLab). To prepare for analysis, dissolved trace metal samples were transferred to 30 or 15 mL perfluoroalkoxy (PFA) vials with caps containing a quartz-window to enable UV-oxidization. Samples were UV-oxidized at USF for 90 minutes at ~20 mW cm-2 with an UVO-Cleaner (Jelight; Model No. 5144AX) to liberate organically complexed dissolved Co and Cu (Milne et al. 2010). For cobalt, nickel, and copper, the UV-oxidized and non-UV-oxidized sample results are presented separately.

The automated seaFAST-pico (Elemental Scientific) with a Nobias Chelate-PA1 resin was connected to a High Resolution-Inductively Coupled Plasma-Mass Spectrometer (HR-ICP-MS; Thermo Scientific Element 2 at FSU, Element XR at USF), to preconcentrate and extract trace metals from the seawater samples inline (Lagerström et al. 2013). The reagents and input flow rates for seaFAST were followed from Hollister et al. (2020) and Burns et al. (2023). The elution acid in this study was a solution of 10% HNO3 (Optima) with 1 ppb Indium as the internal standard. Upon loading the buffered sample (pH ~ 6.2 ± 0.2; Burns et al., 2023) in the resin column, the column was rinsed with Milli-Q (≥18.2 MΩ·cm) to wash out seawater salts, and the chelated trace metals were eluted with the elution acid containing the internal standard. The eluent from seaFAST was then drawn by the ICP-MS and analyzed for the natural abundance of Mn, Fe, Co, Ni, Cu, Zn, Cd, and Pb. Samples were analyzed twice, on the Element 2 at FSU, and on the Element XR at USF.

All analytical runs were comprised of seawater samples, instrument-manifold air blanks, quality control (QC) samples, a matrix-matched multielement calibration curve containing all target metals, matrix-matched molybdenum (Mo), two Fe57 calibration curves, including an Fe57 calibration curve made in 5% HNO3 (Optima) and a matrix-matched Fe57 curve, and certified reference materials, including SAFe D2, GEOTRACES GSP, and NASS-7. All seawater calibration curves and reference samples were prepared and treated the same as the seawater samples throughout the analytical runs. 

The in-house QC consisted of 2-L aliquots of filtered (<0.2 µm) PAP-SO surface seawater (49.20469 °N, -14.78163 °E) that was collected in bulk on the 2021 EXPORTS cruise and then subsampled and acidified to 0.024 M HCl (Optima) for each QC aliquot. The matrix-matched curves, including the multielement, Mo, and Fe57 calibration curves were made using the same seawater as the QC to match the matrix of the seawater samples. The matrix-matched multielement calibration curve was made by dilution from a set of working stocks containing the target metals. These working stocks were diluted from ICP primary standards of 1000 µg/mL in 2% HNO3 of Mn (SPEX CertiPrep), Fe, Co, Ni, Cu, Cd (ULTRA Scientific), Zn and Pb (RICCA) diluted with 5% HNO3 (Optima, Fisher) elution acid matrix. The Mo ICP primary standard consisted of 1000 µg/mL Mo in H2O (SPEX CertiPrep), from which a Mo working stock was made by dilution with 5% HNO3 (Optima, Fisher). The Fe57 primary standard consisted of Fe57 oxide enriched to 96.64% (ISOFLEX), which was dissolved in HCl to make a concentrated stock of 57FeCl3. For this work, two working stocks of 186 µM and 10 µM 57FeCl3 were used to achieve Fe additions in 22 L and 1 L incubation bottles. Calibration curves for Fe57 were made with by dilution of the 10 µM working stock in acid-cleaned, 125 mL LDPE bottles.

The analytical runs usually began by conditioning the inline seaFAST-ICP-MS with a few samples of filtered seawater, followed by several air and MQ blanks, calibration curves including the multielement, Mo, and Fe57 curves, certified reference materials (also included in the middle of each analytical run to allow replicate measurements), seawater samples, several QC samples spread throughout the analytical run (every ~15th sample), several additional air blanks, and finalized by air blank samples. Sample trace metal concentrations were determined from calibration curves of the intensity counts against standards of known concentrations and corrected for average air blanks in the sequence as described previously by Hollister et al. (2020) and Burns et al. (2023). 

Analysis of dissolved macronutrients

Macronutrient samples were thawed at room temperature and analyzed following standard colorimetric methods (Parsons et al. 1984; Becker et al. 2020) using a QuAAtro39 AutoAnalyzer (SEAL Analytical) at USF. The analytical runs included seawater samples, calibration curves made in artificial seawater, reagent blanks consisting of the artificial seawater used to make the calibration curves, reference materials including CK, CL, and CO (KANSO TECHNOS), and QC samples, including standards with known concentrations of nitrite and nitrate to check the efficiency of the instrument’s cadmium column, and the lowest and middle-high standards of the calibration curve to check and correct for drift. QC samples were added approximately every 12th sample to assess the quality of the analytical run. Values below these limits of detection are reported as 0 µM with accompanying QC Flag 6. Sample analyses for macronutrients were performed by senior researcher Salvatore Caprara in the Buck lab at the University of South Florida.