Nutrients¶

Technicians

Susan Becker (SIO)
Alexandra Fine (AOML/CIMAS)

Summary of Analysis¶

2952 samples from 90 CTD stations
The cruise started with new pump tubes and they were changed twice, before station 033 and station 064.
2 sets of Primary/Secondary standards were made up over the course of the cruise.
The cadmium column efficiency was checked periodically and ranged between 90%-100%.

Equipment and Techniques¶

Nutrient analyses (phosphate, silicate, nitrate+nitrite, and nitrite) were performed on a Seal Analytical continuous-flow AutoAnalyzer 3 (AA3). The methods used are described by Gordon et al [Gordon1992] Hager et al. [Hager1972], and Atlas et al. [Atlas1971]. Details of modification of analytical methods used in this cruise are also compatible with the methods described in the nutrient section of the updated GO-SHIP repeat hydrography manual (Becker et al., 2019, [Becker 2019]_.

Nitrate/Nitrite Analysis¶

A modification of the Armstrong et al. (1967) [Armstrong1967] procedure was used for the analysis of nitrate and nitrite. For nitrate analysis, a seawater sample was passed through a cadmium column where the nitrate was reduced to nitrite. This nitrite was then diazotized with sulfanilamide and coupled with N-(1-naphthyl)-ethylenediamine to form a red dye. The sample was then passed through a 10mm flowcell and absorbance measured at 520nm. The procedure was the same for the nitrite analysis but without the cadmium column.

REAGENTS

Sulfanilamide

Dissolve 10g sulfamilamide in 1.2N HCl and bring to 1 liter volume. Add 2 drops of 40% surfynol 465/485 surfactant. Store at room temperature in a dark poly bottle.

Note: 40% Surfynol 465/485 is 20% 465 plus 20% 485 in DIW.

N-(1-Naphthyl)-ethylenediamine dihydrochloride (N-1-N)

Dissolve 1g N-1-N in DIW, bring to 1 liter volume. Add 2 drops 40% surfynol 465/485 surfactant. Store at room temperature in a dark poly bottle. Discard if the solution turns dark reddish brown.

Imidazole Buffer

Dissolve 13.6g imidazole in ~3.8 liters DIW. Stir for at least 30 minutes to completely dissolve. Add 60 ml of CuSO4 + NH4Cl mix (see below). Add 4 drops 40% Surfynol 465/485 surfactant. Let sit overnight before proceeding. Using a calibrated pH meter, adjust to pH of 7.83-7.85 with 10% (1.2N) HCl (about 10 ml of acid, depending on exact strength). Bring final solution to 4L with DIW. Store at room temperature.

NH4Cl + CuSO4 mix

Dissolve 2g cupric sulfate in DIW, bring to 100 m1 volume (2%). Dissolve 250g ammonium chloride in DIW, bring to 1l liter volume. Add 5ml of 2% CuSO4 solution to this NH4Cl stock. This should last many months.

Phosphate Analysis¶

Ortho-Phosphate was analyzed using a modification of the Bernhardt and Wilhelms (1967) [Bernhardt1967] method. Acidified ammonium molybdate was added to a seawater sample to produce phosphomolybdic acid, which was then reduced to phosphomolybdous acid (a blue compound) following the addition of dihydrazine sulfate. The sample was passed through a 10mm flowcell and absorbance measured at 820nm.

REAGENTS

Ammonium Molybdate H2SO4 sol’n

Pour 420 ml of DIW into a 2 liter Ehrlenmeyer flask or beaker, place this flask or beaker into an ice bath. SLOWLY add 330 ml of conc H2SO4. This solution gets VERY HOT!! Cool in the ice bath. Make up as much as necessary in the above proportions.

Dissolve 27g ammonium molybdate in 250ml of DIW. Bring to 1 liter volume with the cooled sulfuric acid sol’n. Add 3 drops of 15% DDS surfactant. Store in a dark poly bottle.

Dihydrazine Sulfate

Dissolve 6.4g dihydazine sulfate in DIW, bring to 1 liter volume and refrigerate.

Silicate Analysis¶

Silicate was analyzed using the basic method of Armstrong et al. (1967). Acidified ammonium molybdate was added to a seawater sample to produce silicomolybdic acid which was then reduced to silicomolybdous acid (a blue compound) following the addition of stannous chloride. The sample was passed through a 10mm flowcell and measured at 660nm.

REAGENTS

Tartaric Acid

Dissolve 200g tartaric acid in DW and bring to 1 liter volume. Store at room temperature in a poly bottle.

Ammonium Molybdate

Dissolve 10.8g Ammonium Molybdate Tetrahydrate in 1000ml dilute H2SO4. (Dilute H2SO4 = 2.8ml conc H2SO4 or 6.4ml of H2SO4 diluted for PO4 moly per liter DW) (dissolve powder, then add H2SO4) Add 3-5 drops 15% SDS surfactant per liter of solution.

Stannous Chloride

stock: (as needed)

Dissolve 40g of stannous chloride in 100 ml 5N HCl. Refrigerate in a poly bottle.

NOTE: Minimize oxygen introduction by swirling rather than shaking the solution. Discard if a white solution (oxychloride) forms.

working: (every 24 hours) Bring 5 ml of stannous chloride stock to 200 ml final volume with 1.2N HCl. Make up daily - refrigerate when not in use in a dark poly bottle.

Sampling¶

Nutrient samples were drawn into 30 ml polypropylene screw-capped centrifuge tubes. The tubes and caps were cleaned with 10% HCl and rinsed 2-3 times with sample before filling. Samples were analyzed within 4 hours after sample collection, allowing sufficient time for all samples to reach room temperature. The centrifuge tubes fit directly onto the sampler.

Data Collection and Processing¶

Data collection and processing was done with the software provided with the instrument from Seal Analytical (AACE). After each run, the charts were reviewed for any problems during the run, any blank was subtracted, and final concentrations (micro moles/liter) were calculated, based on a linear curve fit. Once the run was reviewed and concentrations calculated a text file was created. That text file was reviewed for possible problems and then converted to another text file with only sample identifiers and nutrient concentrations that was merged with other bottle data.

Standards and Glassware Calibration¶

Primary standards for silicate (Na2SiF6), nitrate (KNO3), nitrite (NaNO2), and phosphate (KH2PO4) were obtained from Johnson Matthey Chemical Co. and/or Fisher Scientific. The supplier reports purities of >98%, 99.999%, 97%, and 99.999 respectively.

All glass volumetric flasks and pipettes were gravimetrically calibrated prior to the cruise. The primary standards were dried and weighed out to 0.1mg prior to the cruise. The exact weight was noted for future reference. When primary standards were made, the flask volume at 20C, the weight of the powder, and the temperature of the solution were used to buoyancy-correct the weight, calculate the exact concentration of the solution, and determine how much of the primary was needed for the desired concentrations of secondary standard. The new standards were compared to the old before use.

All the reagent solutions, primary and secondary standards were made with fresh distilled deionized water (DIW).

Standardizations were performed at the beginning of each group of analyses with working standards prepared every 12-16 hours from a secondary. Working standards were made up in low nutrient seawater (LNSW). Two batches of LNSW were used on the cruise. The first batch of LNSW was treated in the lab. The water was re-circulated for ~8 hours through a 0.2 micron filter, passed a UV lamp and through a second 0.2 micron filter. The actual concentration of nutrients in this water was empirically determined during the standardization calculations.

The concentrations in micro-moles per liter of the working standards used were:

-	N+N (uM)	PO₄ (uM)	SIL (uM)	NO₂ (uM)
0	0.0	0.0	0.0	0.0
3	15.50	1.2	60	0.50
5	31.00	2.4	120	1.00
7	46.50	3.6	180	1.50

Quality Control¶

All final data was reported in micro-moles/kg. NO₃, PO₄, and NO₂ were reported to two decimals places and SIL to one. Accuracy is based on the quality of the standards the levels are:

NO₃	0.05 µM (micro moles/Liter)
PO₄	0.004 µM
SIL	2-4 µM
NO₂	0.05 µM

As is standard ODF practice, a deep calibration “check” sample was run with stations 01-055 to estimate precision within the cruise. The water for the check samples was collected on the test cast from 1500m and was fixed with 1ml of saturated mercuric chloride to precent biological growth and stabilize the nutrient concentration. The deep check samples were discontinued when trouble shooting the issues with cadmium column efficiency since the mercuric chloride may have been contributing to the loss of column efficiency. The data are tabulated below for the first 55 stations.

Parameter	Concentration (µM)	stddev
NO₃	17.75	0.29
PO₄	1.16	0.03
SIL	12.8	0.3

Reference materials for nutrients in seawater (RMNS) were used as a check sample run with every station. The RMNS preparation, verification, and suggested protocol for use of the material are described by [Aoyama2006] [Aoyama2007], [Aoyama2008], Sato [Sato2010] and Becker et al. [Becker 2019]. RMNS batch C0 was used on this cruise, with each bottle being used for all runs in one day before being discarded and a new one opened. Data are tabulated below.

Parameter	Concentration	stddev	assigned conc
-	(µmol/kg)	-	(µmol/kg)
NO₃	15.79	0.16	15.86
PO₄	1.18	0.01	1.177
Sil	34.8	0.16	34.7
NO₂	0.04	0.01	0.04

Analytical Problems¶

There were issues with the cadmium column efficiency for a series of stations/days. The exact issue was never clearly identified but it appears to have been a combination of buffer that was not stable which affected the efficiency and life span of the cadmium reduction columns. The values of the reference material and the deep check samples were used to calculate adjustment factors for the affected stations. The adjusted data for the affected stations was compared to adjacent stations and historical data during the QC checks.

Armstrong1967: Armstrong, F.A.J., Stearns, C.A., and Strickland, J.D.H., “The measurement of upwelling and subsequent biological processes by means of the Technicon Autoanalyzer and associated equipment,” Deep-Sea Research, 14, pp.381-389 (1967).
Atlas1971: Atlas, E.L., Hager, S.W., Gordon, L.I., and Park, P.K., “A Practical Manual for Use of the Technicon AutoAnalyzer in Seawater Nutrient Analyses Revised,” Technical Report 215, Reference 71-22, p.49, Oregon State University, Department of Oceanography (1971).
Aoyama2006: Aoyama, M., 2006: 2003 Intercomparison Exercise for Reference Material for Nutrients in Seawater in a Seawater Matrix, Technical Reports of the Meteorological Research Institute No.50, 91pp, Tsukuba, Japan.
Aoyama2007: Aoyama, M., Susan B., Minhan, D., Hideshi, D., Louis, I. G., Kasai, H., Roger, K., Nurit, K., Doug, M., Murata, A., Nagai, N., Ogawa, H., Ota, H., Saito, H., Saito, K., Shimizu, T., Takano, H., Tsuda, A., Yokouchi, K., and Agnes, Y. 2007. Recent Comparability of Oceanographic Nutrients Data: Results of a 2003 Intercomparison Exercise Using Reference Materials. Analytical Sciences, 23: 1151-1154.
Aoyama2008: Aoyama M., J. Barwell-Clarke, S. Becker, M. Blum, Braga E. S., S. C. Coverly,E. Czobik, I. Dahllof, M. H. Dai, G. O. Donnell, C. Engelke, G. C. Gong, Gi-Hoon Hong, D. J. Hydes, M. M. Jin, H. Kasai, R. Kerouel, Y. Kiyomono, M. Knockaert, N. Kress, K. A. Krogslund, M. Kumagai, S. Leterme, Yarong Li, S. Masuda, T. Miyao, T. Moutin, A. Murata, N. Nagai, G.Nausch, M. K. Ngirchechol, A. Nybakk, H. Ogawa, J. van Ooijen, H. Ota, J. M. Pan, C. Payne, O. Pierre-Duplessix, M. Pujo-Pay, T. Raabe, K. Saito, K. Sato, C. Schmidt, M. Schuett, T. M. Shammon, J. Sun, T. Tanhua, L. White, E.M.S. Woodward, P. Worsfold, P. Yeats, T. Yoshimura, A.Youenou, J. Z. Zhang, 2008: 2006 Intercomparison Exercise for Reference Material for Nutrients in Seawater in a Seawater Matrix, Technical Reports of the Meteorological Research Institute No. 58, 104pp.

Bernhardt1967: Bernhardt, H., and Wilhelms, A., “The continuous determination of low level iron, soluble phosphate and total phosphate with the AutoAnalyzer,” Technicon Symposia, I,pp.385-389 (1967).
Gordon1992: Gordon, L.I., Jennings, J.C., Ross, A.A., Krest, J.M., “A suggested Protocol for Continuous Flow Automated Analysis of Seawater Nutrients in the WOCE Hydrographic Program and the Joint Global Ocean Fluxes Study,” Grp. Tech Rpt 92-1, OSU College of Oceanography Descr. Chem Oc. (1992).
Hager1972: Hager, S.W., Atlas, E.L., Gordon L.I., Mantyla, A.W., and Park, P.K., ” A comparison at sea of manual and autoanalyzer analyses of phosphate, nitrate, and silicate ,” Limnology and Oceanography, 17,pp.931-937 (1972).
Kerouel1997: Kerouel, R., Aminot, A., “Fluorometric determination of ammonia in sea and estuarine waters by direct segmented flow analysis.” Marine Chemistry, vol 57, no. 3-4, pp. 265-275, July 1997.
Sato2010: Sato, K., Aoyama, M., Becker, S., 2010. RMNS as Calibration Standard Solution to Keep Comparability for Several Cruises in the World Ocean in 2000s. In: Aoyama, M., Dickson, A.G., Hydes, D.J., Murata, A., Oh, J.R., Roose, P., Woodward, E.M.S., (Eds.), Comparability of nutrients in the world’s ocean. Tsukuba, JAPAN: MOTHER TANK, pp 43-56.