2.1 Material and general methodology
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Multi-core GS07-150-24 was collected on board the RV G.O. Sars at a depth of 2412 m offshore of north-eastern Brazil (3∘46.474′ S, 37∘03.849′ W; Fig. 3). Following sub-sampling, the top of the core was washed over a >63 µm sieve, dried overnight, before being dry sieved over a 150 and 500 µm mesh. Regardless of the research question, each specimen underwent the same methodological protocol which aims to reduce uncertainty (e.g. specimen misidentification; anomalous or abnormal features) within single-shell stable isotope analysis by cataloguing morphology and physical features of specimens prior to destructive analysis. After picking, the selected specimens were given a unique identifier, imaged in the umbilical position (Fig. 2) using a Nikon Digital Research microscope with a prior motorized stage. The motorized stage enables multiple images to be taken at pre-determined intervals in µm. These images were then combined using Nikon Digital Research D software into an extended depth of focus (EDF) image. Each EDF image was then used to measure the diameter and surface area of both the final chamber and the whole shell, using the same programme. Groups of specimens were imaged together, with little impact upon the resolution (1 pixel, depending on the magnification, is equal to 0.3 to 1.5 µm) and placed into individual slides in order to generate a high throughput. After imaging, specimens were weighed individually in tin capsules using a Mettler-Toledo UMT microbalance (manufacturers precision 0.1 µg). In total 207 specimens of T. sacculifer were picked, weighed and measured for size. Following these measurements, specimens selected for research questions 1 (δ18O difference between F and <F) and 2 (δ18O difference between size) underwent additional steps, outlined in Sect. 2.2 (dissection of chambers) and Sect. 2.3 (size fractions), prior to stable isotope analysis.
For δ18O and δ13C analysis, shells and/or single chambers between 5 and 70 µg were placed in a 4.5 mL borosilicate exetainer vial, whereas shells between 20 and 145 µg were placed in larger 12 mL borosilicate exetainer vials (Breitenbach and Bernasconi, 2011; Feldmeijer et al., 2015; Metcalfe et al., 2015). Each vial was sealed with a cap with a pierce able septum, placed in a heated block (45 ∘C), before being flushed with helium for 3 or 5 min to remove the ambient air (flow rate >100 mL min−1) depending on the size of the vial. Each sample was reacted with a few drops of phosphoric acid (H3PO4) for 160 min, transferred using a continuous flow of helium into a GasBench II preparation device, in which impurities were removed, before being introduced into a Thermo Delta+ mass spectrometer. Results were reported as δ values in per mil (‰), following voltage correction of the amplitude of mass 44 using grains of 150-180 µm of Vrije Universiteit Internal Carbonate Standard (VICS: δ18O=-5.44 ‰; δ13C=1.35 ‰) in order to be placed on the V-PDB scale. The precision of within-run international standards of IAEA-CO-1 and IAEA-CO-603 (minimum n=10), placed to book-end every 6 samples, was better than 0.14 ‰ for both δ18O and δ13C. Shell size, weight and stable isotope data are available online (Pracht et al., 2018).
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