Detailed methodology for sampling and determination of iron
Surface seawater sample collection and processing
Filtered and unfiltered underway surface samples were pumped through acid washed low density
polyethylene (LDPE) tubing from a trace metal towed fish which was deployed on the port side
of the ship to a depth of ~ 5 m. The fish consisted of an epoxy coated, brass body with a PTFE
proboscis that drew water from 20 cm forward of the nose. Water was pumped using a
SandpiperII™ (UK) double diaphragm pump that contained only plastic components, powered by
a separate air compressor. The underway sampling system was cleaned by pumping 0.5 M HCl
through the whole system, soaked for ~ 24 h and then flushed with seawater for >12 h. Unfiltered
samples for TDFe and filtered (< 0.2 μm) surface samples for dFe and aluminium and dissolved
aluminium (dAl) were collected directly from an outlet opposite a laminar flow hood in the clean
container. Underway sample filters were Sartobran™ cartridges (0.2 μm cut off, Sartorius)
connected in-line to the supply. These were soaked with 1% HCl (Romil-SpA™) for 24 h and
rinsed with 100 L of seawater before use and changed after ~ 500 L of seawater had been filtered.
Sub-surface seawater sample collection and processing
Sub-surface samples were collected using a titanium alloy CTD frame deployed on a new
stainless steel wire. The bottom 10 m of the wire was plastic coated and great care was taken to
minimise the time the sample bottles were exposed on the deck and to observe each cast in case
of contamination from deck operations and rain water. The hydrocast water samplers were 10 L
Ocean Test Equipment™ sample bottles (Model 110, PVC body, external adapted with plastic
coated spring closures and polytetrafluoroethylene (PTFE) spigots). Six of the 24 sample bottles
were assigned to use for trace metal work only and these were stored in the clean laboratory
container between casts. These were initially cleaned by soaking in 10% HNO 3 and rinsed
thoroughly with DI water. At sea, the samplers were then filled with surface sea water and
acidified with HCl (final concentration 1%, Aristar™ Grade, VWR), soaked for ~12-14 h and
finally rinsed inside and out with DI water. Between CTD casts, samplers were stored opposite a
laminar flow hood in a clean container. Generally, no obvious sources of contamination were
detected and mixed layer concentrations for samples collected underway and during the casts
compared well. Bottle leakages were checked regularly with salinity and nutrient measurements.
The filters used for cast samples for dFe and dAl were 0.2 μm pore size, PTFE membrane syringe
filters (GD/X ™, Whatman) The filters were connected to a peristaltic pump (8-channel,
Minipuls2™, Gilson, France) using PTFE tubing (0.8 mm I.D., Fisher, UK) and were cleaned by
first activating the membrane with 2 mL of methanol (SpA™ grade, Romil, UK), followed by
soaking in 1 M HCl (SpA™ grade, Romil, UK) for 4 h and rinsed with >50 mL of DI water. The
filters were finally conditioned with 20 mL of sample seawater before collection was made
For sample filtration, 250 mL aliquots were collected in LPDE bottles (stored in 1% HCl and
rinsed before use) from the 10 L samplers and transferred to the laminar flow hood. Six samples
were then simultaneously filtered by pumping at 1.5 mL min -1 through the pre-washed syringe
filters. Before sample collection in 30 mL LDPE sample bottles, 30 mL of each sample was
collected and used as a final rinse for the filters and the sample bottles.
Selected sub-samples of filtered surface water (0.2 μm cut off) were collected for sFe and were
filtered again at a flow rate of ca. 1 mL min-1 through pre-cleaned Anotop™ syringe filters (25
mm, 0.02 µm pore size, Whatman) connected in-line to a 6-channel peristaltic pump (Gilson
Minipuls 3™). The filters and the peristaltic pump tubing (Elkay) and PTFE tubing (1/32” ID,
Cole Palmer) used to transfer the sample were cleaned before use with 0.01 M HCl (20 min) and
DI water (1 h) and then conditioned with seawater sample (20 min). After each filtration, the lines
were rinsed with 1M HCl followed by DI water and the filters were replaced.
Determination of iron species in seawater
Iron in size fractionated seawater samples (sFe dFe and TDFe) was determined using flow
injection
with
chemiluminescence
detection
(FI-CL).
This
used
luminol
as
the
chemiluminescence reagent and employed a sulfite reduction step (spiked addition of sodium
sulfite to make a final concentration of 0.1 mM, 12 h reaction time) prior to determination of
Fe(II) [Bowie et al., 1998]. A 20.0 mM ammonium iron(II) sulphate (hexahydrate, 99.997%,
Sigma) stock standard solution was made weekly in 0.1 M HCl and working standards were made
daily from this standard. Calibrations were performed daily by standard additions of a 0.200 µM
Fe(II) standard (pH 3.0) to an acidified surface seawater sample taken from the same region as
the batch of samples analysed on that day.
The blank subtractions were the sum of the blanks of all reagents added to the sample. These
included the injection blank (from the flow injection analyser), an acid blank (iron in bottles
stored filled with DI water at pH 1.9) and a sulfite blank. The injection blank was defined as the
mean of four replicate analyses, where the sample line of the FI-CL manifold was closed and the
pump tubing removed from the peristaltic pump, hence only residual Fe(II) in the FI system
(including buffers and rinsing) was measured. The acid blank included the storage (or sample
bottle) blank and was determined by spiking two bottles containing DI water with HCl
(ROMIL™, UpA) to make 0.005 and 0.015 M concentrations and doing standard additions to
determine dFe in each. Typically the acid blank was negligible (< ca. 25 pM) and hence only the
contribution from the bottle was considered (i.e. the analytical signal itself rather than the
difference between the two solutions). The sulfite blank was assessed daily by analysing 0.1 mM
(concentration used for reduction of Fe(III)) and 0.3 mM solutions of sodium sulfite
(SigmaUltra™ grade, Sigma). Typically the difference between the signal of the two solutions
was negligible (< 12 pM) as the sulfite had been cleaned through a 10 cm column containing 8-
hydroxyquinoline (8-HQ) resin.
The between batch consistency of dFe analysis and the within batch drift was checked by
analysing the IRONAGES reference seawater [Bowie et al., 2006] and an in-house reference
seawater with each batch of samples. Analysis of SAFe S1 and D2 seawater reference materials
gave 0.18 ± 0.02 nM and 0.93 ±0.04 nM, respectively (consensus values were 0.097 ±0.043 nM
and 0.91 ±0.17 nM, respectively [Johnson et al., 2007]). Two blind intercomparisons with an
ICP-MS method were also performed using Atlantic open ocean and shelf samples and gave good
agreement within the range 0.2-2.0 nM [Bowie et al., 2007; Ussher et al., 2010].
References
Bowie, A. R., E. P. Achterberg, R. F. C. Mantoura, and P. J. Worsfold (1998), Determination of
sub-nanomolar levels of iron in seawater using flow injection with chemiluminescence detection,
Analytica Chimica Acta, 361(3), 189-200.
Bowie, A. R., E. P. Achterberg, P. L. Croot, H. J. W. de Baar, P. Laan, J. W. Moffett, S. Ussher,
and P. J. Worsfold (2006), A community-wide intercomparison exercise for the determination of
dissolved iron in seawater, Mar. Chem., 98(1), 81-99.
Bowie, A. R., S. J. Ussher, W. M. Landing, and P. J. Worsfold (2007), Intercomparison between
FI-CL and ICP-MS for the determination of dissolved iron in Atlantic seawater, Environmental
Chemistry, 4(1), 1-4.
Johnson, K. S., et al. (2007), Developing standards for dissolved iron in seawater, Eos Trans.
AGU, 88(11), 131.
Ussher, S. J., I. Petrov, C. R. Quétel, and P. J. Worsfold (2010), Validation of a portable flow
injection–chemiluminescence (FI-CL) method for the determination of dissolved iron in Atlantic
open ocean and shelf waters by comparison with isotope dilution–inductively coupled plasma
mass spectrometry (ID-ICPMS), Environmental Chemistry, 7(2), 139-145.