Experimental seawater collection: For shipboard bioassay experiments, whole seawater and resident biota were collected from ~4 m depth whilst underway at ~5 knots, using a trace-metal clean towfish system (Sedwick et al., 2011). This seawater was used to fill two 60 L polyethylene carboys in parallel inside a shipboard trace-metal clean container laboratory, after passing through pre-cleaned 180 µm nylon screen to exclude larger organisms, then subsequently used to fill the experimental incubation bottles (see below). Three bioassay experiments were performed (Sedwick et al., 2018), for which seawater was collected during three separate deployments of the towfish system.

For bioassay experiment 1, seawater was collected on 1 August, 2014, between 16:55 and 17:35 local time, between 38.6053°N, 72.2534°W (start) and 38.5692°N, 72.2354°W (end). Single determinations of iron and macronutrient concentrations in seawater from the towfish that was filtered in-line through a 0.8/0.2 µm AcroPak Supor filter capsule (Pall) yielded the following results:

Dissolved iron (DFe): 0.55 nM (start), 0.43 nM (end)

Dissolved nitrate+nitrite (NO3+NO2): 0.07 µM (start) 0.08 µM (end)

Dissolved phosphate (PO4): 0.19 µM (start), 0.20 µM (end)

Dissolved ammonium (NH4): not determined

For bioassay experiment 2, seawater was collected on 4 August, 2014, between 11:10 and 12:10 local time, between 38.3800°N, 72.4743°W (start) and 38.3847°N, 72.4761°W (end). Single determinations of iron and macronutrient concentrations in seawater from the towfish that was filtered in-line through a 0.8/0.2 µm AcroPak Supor filter capsule (Pall) yielded the following results:

Dissolved iron (DFe): 0.33 nM (start), 0.32 nM (end)

Dissolved nitrate+nitrite (NO3+NO2): 0.07 µM (start) 0.07 µM (end)

Dissolved phosphate (PO4): 0.19 µM (start), 0.20 µM (end)

Dissolved ammonium (NH4): 0.01 µM (start), 0.01 µM (end)

For bioassay experiment 3, seawater was collected on 9 August, 2014, between 15:19 and 15:54 local time, between 35.5305°N, 72.2760°W (start) and 35.5165°N, 72.2703°W (end). Single determinations of iron and macronutrient concentrations in seawater from the towfish that was filtered in-line through a 0.8/0.2 µm AcroPak Supor filter capsule (Pall) yielded the following results:

Dissolved iron (DFe): 0.89 nM (start), 0.90 nM (end)

Dissolved nitrate+nitrite (NO3+NO2): 0.05 µM (start) 0.07 µM (end)

Dissolved phosphate (PO4): not determined

Dissolved ammonium (NH4): 0.03 µM (start), 0.01 µM (end)

Bioassay experiment protocols: The shipboard bioassay experimental protocols are described by Sedwick et al. (2018). For each experiment there were 6 different incubation treatments (control, iron, nitrate, nitrate+iron, nitrate+iron+phosphate, rainwater), with triplicate bottles for each treatment sampled at each of three timepoints. Each bottle was completely subsampled for measurements of nutrients (NO3+NO2, NH4), chlorophyll-a and primary productivity. For the initial (time = 0) measurements, the seawater that remained in the 60 L polyethylene carboys after filling the incubation bottles was transfered into a 20 L polyethylene carboy from which subsamples were taken for measurements of NO3+NO2 after filtration through 0.8 µm pore size AcroDisc Supor syringe filters (Pall), for chlorophyll-a after filtration on to combusted 0.7 µm pore size GF/F filters (Whatman), and for incubation with carbon-13 labeled bicarbonate for estimation of primary production. For initial (t = 0) NH4 concentrations, we use mean values measured in seawater sampled from the towfish outlet after in-line filtration (see above).

Analytical procedures:

DFe: Filtered seawater samples were acidified at-sea to pH ~1.8 with Fisher Optima grade ultrapure hydrochloric acid, and then stored at room temperature until post-cruise analysis at Old Dominion University. Dissolved iron was determined by flow injection analysis with colorimetric detection after in-line preconcentration on resin-immobilized 8-hydroxyquinoline (Sedwick et al., 2015), using a method modified from Measures et al. (1995). Analyses were performed on a volumetric basis, so concentrations are reported in units of nanomole liter-1 (nM). Analytical precision is estimated from multiple (separate-day) determinations of the SAFe seawater reference materials, which yield uncertainties (expressed as one relative standard deviation on the mean, or one sigma) of ~15% at the concentration level of SAFe S seawater (0.090 nM), and ~10% at the concentration level of SAFe D2 seawater (0.90 nM). The analytical limit of detection is estimated as the DFe concentration equivalent to a peak area that is three times the standard deviation on the zero-loading blank (manifold blank), which yields an estimated detection limit below 0.04 nM (Bowie et al., 2004). Blank contributions from the ammonium acetate sample buffer solution (added on-line during analysis) and hydrochloric acid (added after collection) are negligible.

NO3+NO2: Dissolved nitrate and nitrite was determined at sea using an Astoria Pacific nutrient autoanalyzer using standard colorimetric methods with an estimated detection limit of 0.14 µM (Parsons et al., 1984; Price and Harrison, 1987). In surface waters, nitrate and nitrite were determined using the same autoanalyzer equipped with a liquid waveguide capillary cell (World Precision Instruments) (Zhang, 2000) to achieve an estimated detection limit of 0.02 µM.

PO4: Dissolved phosphate was determined at sea using an Astoria Pacific nutrient autoanalyzer using standard colorimetric methods with an estimated detection limit of 0.03 µM (Parsons et al., 1984; Price and Harrison, 1987).

NH4: Dissolved ammonium was determined at sea using the manual orthophthaldialdehyde method (Holmes et al., 1999), with an estimated detection limit of 10 nM.

Chl-a: Chlorophyll-a was determined at sea using the non-acidification method with a Turner 10-AU fluorometer (Welschmeyer, 1994).

PP: Primary production was measured using carbon stable istopes (Mulholland et al., 2006).

Missing data identifiers:
ND = not determined (single measurement)
NR = not reported (contamination likely, only used for NH4 data)