2.2.2 Elemental analysis by SF-ICP-MS
For elemental analysis by SF-ICP-MS, between 12 and 40 mg of dried material were transferred into clean PFA vials and were digested in a mixture of 8.0M HNO3 (Merck ultrapur) and 2.9M HF (Merck suprapur). Vials were tightly capped and heated to 130°C for 4 hours. The remaining solution was then evaporated to near dryness, then 400 µL of concentrated HNO3 (Merck ultrapur) was added to drive off the fluorides and was then evaporated. Finally, samples were redissolved with 3mL of 3% HNO3 (Merck Ultrapur) and kept in acid-cleaned 15mL polypropylene tubes (Corning®) until analysis by SF-ICP-MS (see details below). This procedure has been proven adequate for digestion of all particulate trace metals (Planquette and Sherrell 2012).
All archive solutions were analyzed by SF-ICP-MS (Element XR) following the method of Planquette and Sherrell (2012). Final concentrations of samples and procedural blanks were calculated from In-normalized data. Analytical precision was assessed through replicate samples (every 10th sample) and accuracy was deduced from analysis of Certified Reference Materials (CRMs) of plankton (BCR-414) and sediments (PACS-3 and MESS-4) (Supplementary Table S1). Dissolved Mn, Fe, Cu, and Co concentrations of the saline solution were determined before deployment and after the recovery in an aliquot collected after the centrifugation, by SF-ICP-MS after preconcentration using the SeaFast (Supplementary Table S2) following the method described previously (Tonnard et al. 2020). Based on these results we calculated the percentage of dissolution of the particulate material within the cups (Supplementary Table S2). We did not correct particulate flux for dissolution as the values are generally low (<10%) with the exception of Mn (23.5%) in cup #11 and Cu in cups #7 (11.4 %), #8 (10.1%) and #11 (30.8%).
2.3 Carbon export fluxes of diatoms and faecal pellets .
Microscopic observations were conducted within four months after recovery of the moorings. For the identification of diatoms, counting and size measurements, we followed the protocol described in Rembauville et al. (2015a) that allows to separately consider full and empty cells. For diatom counting, the samples were processed as follows. Two mL of one-eighth aliquot was diluted with 18 mL of artificial seawater and decanted in a Sedgewick Rafter counting chamber. Full diatoms were enumerated and identified under an inverted microscope with phase contrast (Olympus IX170) at 400x magnification. The morphometric measurements were done using high resolution images (Olympus DP71 camera) and Fiji image processing software. The biovolume was calculated from morphometric measurements (Hillebrand et al. 1999).
The export flux of diatoms (Cell m-2d-1) was calculated using the equation:
\(Cell\ flux=N_{\text{diat}}\times d\times 8\times V_{\text{aliquot}}\times\frac{1}{0.125}\times\frac{1}{11}\times k\)(Eq. 1)
Where Ndiat (cell mL-1) is the number of cells counted in one chamber, d is the dilution factor, 8 relates to measurements being made on a one eighth aliquot of the sample, Valiquot (mL) is the volume of the aliquot, 1/0.125 relates to the trap surface area (in m2), 1/11 relates to the sample collection time (in days), and k is the fraction of the chamber counted.
The diatom flux was then converted to POC flux for each taxon using allometric equations
reported in the literature (Menden-Deuer and Lessard 2000; Cornet-Barthau et al. 2007) and taking into account specific relationships for spores (Rembauville et al. 2015a) (Table S3). The spore and vegetative cell carbon fluxes were then obtained by summing up the contribution of the different taxa. The sum of both fluxes corresponded to the carbon flux associated with diatoms.
To enumerate fecal pellets, an entire one-eighth aliquot of each sample cup was placed in a gridded Petri dish and observed under a stereomicroscope (Zeiss Discovery V20) coupled to a camera (Zeiss Axiocam ERc5s) at 10X magnification. Fecal pellets were classified into three types according to their shape: spherical, cylindrical, ovoid/ellipsoid (Table S3) (Gleiber et al. 2012). Size measurements were used to calculate the volume of each fecal pellet according to their shape that was then converted to carbon using a factor of 0.036 mg C mm-3 (González and Smetacek 1994). The fecal pellets carbon fluxes (Ffp (mg C m-2d-1)) in the different size classes were calculated using the equation:
\(F_{\text{fp}}=C_{\text{fp}}\times 8\times\frac{1}{0.125}\times\frac{1}{11}\)(Eq. 2)
where Cfp (mg C per fecal pellets for each type) is the concentration of carbon in each fecal pellet type. Others terms in the equation have the same definition as in Eq. 1). The Ffpwere finally summed to provide the total fecal carbon fluxes. Although the calculation of total POC flux is associated with large uncertainties (around 50%, (Rembauville et al. 2015a), the linear regression between POCcalculated and POCmeasured was as follows:
POCcalculated =(0.84±0.05) x POCmeasured+ (0.2±0.35) with R2=0.9621 )
2.4 Statistics tools and data visualisation .
Statistical analysis (cross-correlation, Principal Component Analysis (PCA) and Partial least Square Regression (PLSR)) were performed using scikit-learn packages python 2.7. Scipy.stats package python 2.7 was used to conduct ANOVA after checking for homoscedasticity with a levene test. Data visualisation was realised with python 2.7 matplot library.