Planktic foraminifera were collected by towing a 200-\u03bcm-mesh plankton net for 90 min at a target depth within the upper mixed layer. Approximately 90% of the tow material was preserved in a 5-10% pH-buffered formalin solution and stored at 4\u00b0C until sorting for foraminifera (Ren et al., 2012). The remaining 10% was size fractionated and frozen at \u221220\u00b0C for element and isotope analysis of zooplankton. During each tow, bulk (>0.3 \u00b5m) suspended PON and nitrate samples were collected from the underway intake (at 7 m depth) and frozen at \u221280\u00b0C and \u221220\u00b0C, respectively, until isotope analysis. In situ (i.e., tow-depth) nitrate samples and auxiliary hydrographic data come from CTD casts performed on station before the start of each net tow.
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Foraminifera were separated by species, rinsed briefly with Milli-Q and crushed open to expose (non-shell-bound) tissue. Tissue N was converted to nitrate using the persulfate oxidation method (Nydahl, 1978; Knapp et al., 2005). Remnant shell material (having been oxidatively cleaned) was transferred, rinsed six times with Milli-Q and oven dried at 50\u00b0C. Shell samples were then dissolved with hydrochloric acid to release shell-bound organics for persulfate oxidation (Nydahl, 1978; Knapp et al., 2005). For both tissue- and shell-derived nitrate (from persulfate oxidation) as well as seawater nitrate, samples were measured for concentration by chemiluminescence (Braman & Hendrix, 1989) and N isotope composition using the denitrifier method and gas chromatography\u2212isotope ratio mass spectrometry (GC-IRMS) (Sigman et al., 2001; Casciotti et al., 2002; Weigand et al., 2016). The N isotope composition of bulk PON and size-fractionated zooplankton were measured by elemental analyser-IRMS.
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The isotopic composition of N\u2082O was measured either at Princeton University (USA) or the Max Planck Institute for Chemistry (MPIC; Germany) by GC-IRMS using a Thermo MAT 253 mass spectrometer with a purpose-built on-line N\u2082O extraction and purification system. Bulk PON and size-fractionated zooplankton samples were measured at the University of Cape Town using a Thermo Scientific FLASH 2000 elemental analyzer coupled to a Thermo Scientific Delta V Plus mass spectrometer. Hydrographic data were acquired by a Sea-Bird conductivity-temperature depth sensor during each cast.
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See methods section of Smart et al. (2020) for more detail.
At Princeton and MPIC, N isotope measurements were referenced against automated injections of N\u2082O from a gas cylinder, and then calibrated to N\u2082 in air using international reference materials: IAEA-N3 and USGS-34 nitrate standards in the case of seawater samples, or USGS-40 and USGS-41 amino acid standards in the case of foraminifer (oxidized) samples. Oxidized samples were additionally corrected for the contribution of the N blank associated with the persulfate oxidizing reagent.
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At the University of Cape Town, N isotope measurements were referenced to atmospheric N\u2082 using three in-house organic standards: Choc, Merck Gel and Valine.