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.