Sampling and analytical procedures:
Underway samples were collected from the flow-through seawater system aboard the R/V/I/B Nathanial B. Palmer between September 10 and October 11 of 2016. Samples for macronutrients were filtered through 0.2 um syringe filters and collected in 50 mL Falcon tubes that had been rinsed with distilled water (DIW), soaked overnight in 10% hydrocholoric acid (HCl, Fisher, Trace Metal Grade), rinsed three times with DIW again, dried and rinsed three times with sample prior to filling. Samples were analyzed shipboard, typically within 24 hours, for nitrate+nitrite, phosphate, silicate, and occasionally nitrite. Until analyzed shipboard, samples were stored sealed at 4 ºC in the dark and, following analyses, samples were frozen at -20 ºC and shipped back to the University of South Florida for laboratory-based analyses of nitrite and ammonium, and in some cases again for nitrate+nitrite, phosphate and silicate.

Analytical methodology was based on established methods (Parsons 1984; Gordon et al. 1993) as described for the Lachat 8500 QuickChem in the Lachat QuickChem methods manuals and for the Technicon AAII in the CARIACO methods manual. All labware were either glass or high density polyethylene and were cleaned by an initial distilled water (DIW) rinse, followed by an overnight 10% hydrochloric acid (Fisher, Trace Metal Grade) soak, then rinsed three times with DIW and three times with solvent, analyte or sample prior to fill. Dedicated glassware was used for reagents and standards to avoid cross contamination issues. All reagents were made in high purity Milli-Q (>18 MΩ cm) water.

An artificial seawater matrix was used as the carrier for the analyses on the Lachat QuickChem 8500 at sea and on the Technicon AAII in the lab. 4L batches of artificial seawater were made by dissolving pre-weighed salts (128.50 g NaCl, 28.50 g MgSO4*7H2O, 0.6714 g NaHCO3) in Milli-Q to match Southern Ocean seawater salinity and adjusted to match Southern Ocean seawater pH with 10% hydrochloric acid.

Five-point standard curves were analyzed in duplicate at the beginning and end of each run with duplicate reagent blanks, and quality control checks every seventh sample. Quality control check consisted of either an international reference sample or a quality control sample. The certified nutrient reference samples used, lots CC and CD, were purchased from Kanso Technos in Osaka, Japan. The quality control sample was made from a 20-L homogenized surface Southern Ocean filtered and autoclaved seawater sample. The midpoint standard from the calibration curve was also analyzed every fourteenth sample to check for drift during the runs.

Detection limits for all five parameters on the two instruments used were determined from three times the standard deviation of replicate artificial seawater blanks (n>6). On the Lachat QuickChem 8500, limits of detection were 0.01 uM for nitrate+nitrite, 0.02 uM for phosphate, 0.03 uM for silicate, 0.05 uM for nitrite and 0.5 uM for ammonium. On the Technicon AAII, limits of detection were 0.06 uM for nitrate+nitrite, 0.02 uM for phosphate, 0.2 uM for silicate, 0.01 uM for nitrite, and 0.05 uM for ammonium.

Sample analyses for macronutrients were performed by William Abbott (USF).

Gordon, L.I., Jennings, J., J.C., Ross, A.A. and Krest, J.M., 1993. A suggested protocol for continuous flow automated analysis of seawater nutrients (phosphate, nitrate, nitrite and silicic acid) in the WOCE Hydrographic Program and the Joint Global Ocean Fluxes Study, Methods Manual WHPO 91-1. WOCE Hydrographic Program Office.

Parsons, T.R., Maita, Y. and Lalli, C.M., 1984. A manual of chemical and biological methods for seawater analysis. Pergammon Press, Oxford, 173 pp.