Philipp Assmy

Pedro Duarte

Jens Dujardin

Mar Fernández-Méndez

Agneta Fransson

Robert Hodgson

Hanna Kauko

Svein Kristiansen

CJ Mundy

Lasse Mork Olsen

Ilka Peeken

Marthe Sandbu

Jago Wallenschus

Anette Wold

Methods

=======

### 1) Inorganic nutrients

Water samples for the inorganic nutrients nitrate and nitrite (NO3 + NO2), phosphate and silicate were were collected in 20 mL scintillation vials,fixed with 0.2 ml chloroform and stored refrigerated until sample analysis approximately 6 months later. Concentrations of these nutrients were measured on a modified Scalar auto-analyzer following Bendschneider & Robinson (1952) and RFA Methodology, for NO3+NO2, and Grasshoff (1965), for PO4 and SiOH4.

Water samples for ammonium were analysed on board right after sampling with the method of Solorzano (1969).

**Measurement of nitrite in seawater**

The method is based on that nitrite reacts colorimetrically with aromatic amin and forms a diazonium ion in acidic medium. The diazonium ion connects to a new aromatic amine and forms an azo. The absorption of the color is measured spectrophotometrically at 540 nm on a modified Alpkem Flow Solution IV autoanalyzer or a modified scalar autoanalyzer (Bendschneider & Robinson 1952 RFA Methodology). The detection limit for nitrites is 0.06 µmol/L (Scalar) and is significant to the second decimal place.

**Measurement of nitrate in seawater**

The method is based on the chemical reduction of nitrate to nitrite by means of cadmium in the presence of copper ions. Then nitrite forms a diazonium ion with an aromatic amine in an acidic environment, which is diverted to a new aromatic amine and an azo dye is formed. Absorption of the color is measured spectrophotometrically at 540 nm on a modified Alpkem Flow Solution IV autoanalyser or a modified scalar autoanalyser (Bendschneider & Robinson 1952 RFA Methodology). The amount of nitrate is calculated as the difference between total reduced nitrite (this method) and NO2 measured without cadmium reduction (see Method U3_3 above). The detection limit for nitrate is 0.4 µmol/L (Scalar) and is significant to the first decimal place.  

**Measurement of phosphate in seawater**

The method is based on the phosphate reacts with molybdate to form phosphomolybdate in acid medium (pH < 1) providing a yellow dye. This dye is reduced by ascorbic acid to a blue dye, and absorbance is measured spectrophotometrically at 810 nm on a modified Alpkem Flow Solution IV autoanalyzer or a modified Scalar autoanalyzer (Grashoff 1965). The detection limit for phosphate is 0.06 µmol/L (Scalar) and is significant to the second decimal place. 

**Measurements of silicate in seawater**

The method is based on the silicate reacts with molybdate to form a silikomolybdat (yellow dye) in an acidic environment (pH = 1.5-2). Oxalic acid is added to the silikomolybdat which then is reduced with ascorbic acid. The result is a blue compound which is measured spectrophotometrically at 810 nm on a modified Alpkem Flow Solution IV autoanalyser or a modified Scalar autoanalyzer (Grashoff 1965). The detection limit for silicate is 0.7 µmol/L (Scalar) and is significant to the first decimal place.  

**Detection limits for nutrients:**

* Ammonium detection limit 0.04 µmol/L. However, the detection limit was 0.5 µmol/L for the period 23 April to 2 May because of contamination in the chemical reagents.

* Nitrate detection limit 0.4 µmol/L

* Nitrite detection limit 0.06 µmol/L

* Phosphate detection limit 0.06 µmol/L

* Silicate detection limit 0.7 µmol/L

### 2) Chlorophyll a (Chl a)

Chl a samples were collected on 25-mm GF/F filters (Whatman), extracted in 100% methanol for 12 h at 5°C on board the ship and measured fluorometrically with an AU10 Turner Fluorometer (Turner Design, Inc.). Phaeopigments were measured by fluorescence after acidification with 5% HCl. Calibration of the Turner fluorometer was carried out following the JGOFS protocol35 (Knap et al., 1996). Chl a measurement uncertainty was estimated from triplicate water samples taken from depths ranging between 5 and 100 m depth and averaged 5.5% of measured values.

### 3) Particulate organic carbon (POC) and nitrogen (PON)

Between 200 - 2000 ml of seawater, depending on particle concentration, were filtered onto pre-combusted 25 mm GF/F filters (the filters were combusted at 450°C for 12 hours and stored in tin foil). After filtration, GF/F filters were placed into Pall filter slides and dried at 60°C in a drying oven and thereafter stored at room temperature. Filter slides from one station were wrapped in tin foil and kept in a labelled ziploc bag.

For each sampling day or event, a MilliQ reference filter was prepared (similar volume than for the samples) and treated in the same way than the samples.

POC/N samples have been analyzed with continuous-flow mass spectrometry (CF-IMRS ) carried out with a roboprep/tracermass mass spectrometer (Europa Scientific, UK) (Lorrain et al., 2003).  All values have been corrected for instrument drift.

Detection limits: 5 µg for C and 2-3 µg for N

### 4) Total particulate silica (TPSi)

Total particulate silica from the water column (ship and on-ice CTD) was only taken during legs 3-6 (with the exception of on-ice CTD 003 and ship CTD 001 during leg 1).Tot part Si samples were filtered on cellulose acetate filters (0.8 µm pore size, 25 mm diameter, Sartorius) during legs 1&2 and on polycarbonate filters (0.4 µm pore size, 25 mm diameter, Whatman) during legs 3-6. After filtration, each filter was directly placed into plastic petri slides and dried at 60°C in a drying oven. When filters were dry, petri dishes were sealed with parafilm and wrapped in tin foil and stored in a labelled ziplock bag. Filters were stored at room temperature until analysis at IMR Bergen in late 2015/early 2016.Cellulose acetate and polycarbonate filter blanks were used to correct tot part Si raw data from legs1&2 and legs3-6 respectively.

The alkaline digestion method used for TPSi is described in detail by Raguenau & Tréguer (1994) and assumes that biological silicate (BSi) is extracted during the first 40 min of the digestion step (Brzezinski & Nelson 1989). If lithogenic silicate (LSi) is present however, the silicate minerals may interfere with the BSi estimate. Therefore, the alkaline silicate extraction described here is termed Total Particulate Silicate (TPSi) when no correction is made for LSi. Briefly, each sample filter was digested in 8 mL of 0.2 M sodium hydroxide (NaOH) for 40 min at +100 °C, before the solution was quickly cooled down and added 2 mL of 1 M hydrochloric acid (HCl) to stop the digestion. Each sample was centrifuged for 10 min at 2500 rpm before 4 mL of the supernatant was removed and analyzed as a dissolved silicate sample on an autoanalyzer. Dissolved silicate `[SiO₄]⁴⁻` reacts with molybdate to form a yellow silico-molybdate solution (pH=1.5-2). The solution is added oxalic acid before it is reduced with ascorbic acid to a blue color complex, that is measured spectrophotometrically at 810 nm using a modified Skalar Autoanalyzer (Grasshoff 1965). Dissolved silicate was corrected for a filterblank, the extraction volume corrected for volume of samplewater filtered, and the amount of TPSi was expressed as µmol/Lol Si L-1 seawater.

### 5) Dissolved organic carbon (DOC) and nitrogen (TDN)

Samples were filled from Niskin bottle directly into pre-rinsed 1 L (or 0.5 L) Duran glass bottles. A pre-rinsed glass filtration unit with pre-combusted (450°C, 5 h) 47 mm GFF filters was used. Before filtration of the final sample, GF/F-filters were pre-rinsed with the water sample (minimal 2 x 150 ml) because DOM adsorbs on the filter material. The sample filtration was carried at low vacuum (max. -30 kPa) to avoid cell leakage. Filtration was always done directly after sampling and the filtration unit covered with aluminum foil during filtration and when filtration unit was not in use (to avoid that dust falls into the sample). The minimal final sample volume was at least 20 ml but in most cases 50 ml. At each station we took two blank samples: 1. MilliQ water directly into acid-washed HDPE bottle, and 2. MilliQ water into acid-washed HDPE bottle after thorough rinsing with MilliQ.Samples were stored at -20°C in 60 ml HDPE bottles.

Dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) concentration were determined by high temperature catalytic oxidation and subsequent non-dispersive infrared spectroscopy and chemiluminescence detection (TOC-VCPN, Shimadzu) (Koch et al., 2014).  Final concentrations are average values of triplicate measurements. If the standard variation or the coefficient of variation exceeded 0.1µM or 1 %, respectively, up to two additional analyses were performed and outliers were eliminated. After each batch of five samples, one reference standard (DOC-DSR, Hansell Research Lab, University of Miami, USA), one ultrapure water blank and one potassium hydrogen phthalate standard were measured.

Detection limits:

The limit of detection (3 sigma of the blank) and quantitation (9 sigma of the blank) was 7 and 21 µmol C L-1, respectively. The accuracy was ±5 %.  

For TDN the limit of detection (LOD) and quantitation (LOQ) are 3 and 10 µmol N L-1, respectively.

LOD indicates the limit at which a substance is still detectable.

LOQ specifies the limit at which a substance is still quantifiable.

### 6) Dissolved oxygen

Dissolved oxygen samples were collected directly from Niskin bottles on either the ship CTD rosette or from bottles on a Hydro-Bios water sampler.

Individual sampling bottles with a nominal capacity of 115 ml were used.The analysis and calculations followed the modified Winkler procedure described in Carpenter [1965]: Sulphuric acid had 50 % volume concentration and the concentration of thiosulphate stock solution was 0.18M. Titrations were carried out in two 50ml aliquots taken from the dissolved oxygen bottles to check the reproducibility of the results. Consecutive titrations led to non-significantly different results. A digital Solarus burette from Hirschmann was used.Standards and blank were determined every time measurements were made. Dissolved oxygen concentrations were calculated in mL L-1.

Detection limit: 0.06 mL L-1

### 7) HPLC pigments

The HPLC method is described in [Tran et al. (2013)]( https://www.biogeosciences.net/10/1909/2013/bg-10-1909-2013.pdf).

Unit ng/L. Header definitions:

Chla - chlorophyll a;

Chl b - chlorophyll b;

Chlc1_Chlc2 - chlorophyll c1 + chlorophyll c2;

Chlc3 - chlorophyll c3;

Phorbida - phorbid a;

Fucox - fucoxanthin;

19But - 19’butanoyloxyfucoxanthin;

19Hex - 19’hexanoyloxyfucoxanthin;

Perid - peridinin;

Gyroxdiester - gyroxanthin diester;

Neox - neoxanthin;

Lutein - lutein;

Prasino - prasinoxanthin;

Allox - alloxanthin;

Violax - violaxanthin;

Zeax - zeaxanthin;

DD - diadinoxanthin;

DT - diatoxanthin;

Astax - astaxanthin;

Echineone - echineone;

Alpha_car - alpha carotene;

Beta_car - beta carotene

## References

Bendschneider, K. & Robinson, R.I. (1952), [A new Spectrophotometric method for the determination of nitrite in Seawater]( http://hdl.handle.net/1773/15938). J. Mar. Res. 2: 87-96.

Brzezinski, M.A. & Nelson, D.M. (1989), [Seasonal changes in the silicon cycle within the Gulf Stream warm-core ring]( https://doi.org/10.1016/0198-0149%2889%2990075-7). Deep-Sea Res. 36: 1009-1030.

Carpenter, J. H. (1965), The Chesapeake Bay Institute technique for the Winkler dissolved oxygen method. Limnol. Oceanogr. 10 (1): 141-143, https://doi.org/10.4319/lo.1965.10.1.0141

Grasshoff, K. (1965), On the Automatic Determination of Phosphate, Silicate and Fluoride in Seawater. ICES Hydrographic Committee Report No. 129.

Koch B.P., Kattner G., Witt M., Passow U. (2014), Molecular insights into the microbial formation of marine dissolved organic matter: Recalcitrant or labile? Biogeosciences, 11: 4173-4190.

Knap, A., Michaels, A., Close, A., Ducklow, H. & Dickson A. (1996), Measurement of Chlorophyll a and Phaeopigments by fluorometric analysis. JGOFS report 19: 118-122.

Lorrain, A., Savoye, N., Chauvaud, L., Paulet, Y. M. & Naulet, N. (2003), Decarbonation and preservation method for the analysis of organic C and N contents and stable isotope ratios of low-carbonated suspended particulate material. Anal. Chim. Acta 491: 125-133.

Ragueneau, O. & Tréguer, P. (1994), Determination of biogenic silica in coastal waters: applicability and limits of the alkaline digestion method. Mar. Chem. 45:43-51.

RFA Methodology, Nitrate+nitrite Nitrogen, A303-S170 Revision 6-89. ALPKEM, a division of OI Analytical, College Station, Texas

Solorzano, L. (1969), Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol Oceanogr. 14:799.

CEOS IDN DIF

9.8.4