SUMMARY: Seawater is collected from known depths using CTD-rosette sampling procedures. Subsamples are drawn and stored frozen (-20°C) in acid-washed, HDPE bottles. Nitrate+nitrite is measured from subsamples passed through a cadmium reduction column and reacted with aromatic amines to form an azo dye which is analyzed colorimetrically.
In seawater the forms of dissolved nitrogen of greatest interest are, in order of decreasing oxidation state: NO3-, NO2-, NH4+ and organic-N. All these forms of N, as well as N2, are biochemically interconvertible and are components of the biological N-cycle.
In this method NO3- is quantitatively reduced to NO2- in a copperized cadmium reduction column. The NO2- thus produced, along with any NO2- present in the original sample, is coupled with an aromatic amine, which in turn is reacted with a second aromatic amine to produce an azo dye. The extinction due to the dye is then measured spectrophotometrically. For surface water samples (<100 m) where the [NO3-+NO2-] is generally less than 0.05 μM, we have employed a high-sensitivity assay procedure that is based on the production and detection of nitric oxide.
Contamination is a primary concern in oligotrophic ocean [NO3+NO2] determinations. This is particularly true for samples collected from the euphotic zone, where inorganic nutrient concentrations are extremely low. In order to avoid contamination, sample bottles must be meticulously cleaned with dilute HCl (10%) and rinsed with DIW before use. Samples are stored frozen (-20°C) until analysis. These sampling and storage procedures have been shown to be adequate even for oligotrophic ocean samples (Dore et al., 1996).
Rinse the nutrient sample bottle (acid-washed, 125 ml HDPE bottle) 3 times before filling. Fill to approximately ⅔ full, cap tightly and freeze upright. Record cruise, cast and bottle number on the bottle and data sheet.
[NO3-+NO2-] analyses are performed using a 4-channel SEAL Analytical AA3 continuous flow system. The automated wet chemistries generally follow the standard methods of seawater analysis provided by the manufacturer. These involve: (1) reduction of NO3- to NO2- using a copperized cadmium reduction column, (2) reaction of NO2- with sulfanilamide for diazotization and (3) coupling with N-1-napthylethylenediamine dihydrochloride (NED) forming a purple azo dye (Armstrong et al., 1967). The dye absorbance is read through a 10 mm pathlength flowcell at 550 nm. The reduction column is looped in line using a Hamilton 4-way valve and-can be by-passed for NO2- analysis only. Both NO3- and NO2- standards are run to check column efficiency.
| 5.1. | Calibration stocks and regression standards |
The calibration of dissolved inorganic nutrient determinations in the autoanalysis of seawater samples is performed using standard solutions containing dissolved N, P and Si salts. A nutrient stock solution is prepared by dissolving dried (65°C, 72 hr) analytical grade reagent chemicals with DIW in 1 L glass volumetric flasks containing 1 ml of chloroform. Once dissolved, this stock solution is immediately transferred into 1 L HDPE bottles and stored at room temperature in the dark (see Reagents section for details). The daily regression standards are prepared by diluting the stock standards with low nutrient natural seawater (LNSW). LNSW is 0.2 µm filtered open ocean surface seawater stored in a carboy at room temperature in the dark for at least 6 months prior to use. This technique provides a mixed standard solution of N, P and Si that is matrix-matched with the seawater samples and any cross-nutrient interference effect should be accounted for. | |
| 5.2. | Blank corrections |
All seawater standard absorbance peaks were corrected for the absorbance of the seawater (LNSW). All seawater sample peaks were corrected for the refractive index absorbance for each unique nutrient detection system. The refractive index corrections represent the increase in absorbance that is due strictly to the presence of dissolved salts in seawater when compared to the DIW baseline. These corrections are determined by running alternating sample cups of DIW and LNSW through the autoanalyzer with only non color developing reagents on-line. The difference in peak height between DIW and LNSW is calculated and an average is taken to obtain the refractive index correction. This refractive index run is done once a week. |
The detection limit for [NO3-+NO2-] is approximately 0.05 µM with a coefficient of variation for field-collected replicates of 0.046 ± 0.041 µmol L-1 based on 2019 data (9 cruises).
acid-washed, 125 ml high-density polyethylene bottles
SEAL Analytical AA3 and accessories
pipettes and volumetric flasks
DIW
10% Hydrochloric Acid
Ammonium Chloride Reagent: Dissolve 60 g of NH4Cl in DIW, adjust pH to 8.7 with concentrated NH4OH and dilute to 1 L. Add 30%w/v Brij-35 as needed (approximately 5 small drops in 500 ml).
Color Reagent: To approximately 1500 ml of distilled water, add 200 ml of concentrated phosphoric acid and 20 g of sulfanilamide. Dissolve completely. Add 1 g of N-1-naphthylethylenediamine dihydrochloride and dissolve. Dilute to 2 L. Store in a cold, dark place.
Cadmium 30-80 mesh granules (Sigma 414891-25g)
Copper Sulfate Solution: Dissolve 20 g of ACS grade Cupric Sulfate Pentahydrate in ~800 ml DIW in a 1 L volumetric flask. Dilute to 1 L.
Stock Standard (10,000 µM): Dissolve 1.0120 g of KNO3 in ~800 ml DIW containing 1 ml of chloroform. Dilute to 1 L.
Nitrite Check Standard (10,000 µM): Dissolve 0.6900 g of ACS grade sodium nitrite into ~800 ml DIW containing 1 ml of chloroform. Dilute to 1 L.
Armstrong, F. A. J., C. R. Sterns and J. D. H. Strickland. 1967. The measurement of upwelling and subsequent biological processes by means of the Technicon Autoanalyzer and associated equipment. Deep-Sea Research, 14(3), 381-389. doi: 10.1016/0011-7471(67)90082-4.
Dore, J. E., T. Houlihan, D. V. Hebel, G. Tien, L. M. Tupas and D. M. Karl. 1996. Freezing as a method of seawater preservation for the analysis of dissolved inorganic nutrients in seawater. Marine Chemistry, 53(3-4): 173-185. doi: 10.1016/0304-4203(96)00004-7.
Morse, J. W., M. Hunt, J. Zullig, A. Mucci, and T. Mendez. 1982. A comparison of techniques for preserving dissolved nutrients in open ocean seawater samples. Ocean Science and Engineering, 7, 75-106.
Ryle, V. D., H. R. Mueller, and P. Gentien. 1981. Automated analysis of nutrients in tropical sea waters. Australian Institute of Marine Science Technical Bulletin, Oceanography Series No. 3.
Standard Methods for the Examination of Water and Wastewater, 15th Edition, by American Public Health Association, AWWA, WPCF.
Venrick, E. L. and T. L. Hayward. 1985. Evaluation of some techniques for preserving nutrients in stored seawater samples. CalCOFI Report, 26, 160-168.
analyte-compound class: nitrate plus nitrite [NO3-+NO2-]
method: automated, colorimetric analysis
precision: - laboratory: 0.025 ± 0.049 µmol L-1 based on 2019 data (9 cruises, 309-317) - field: 0.046 ± 0.041 µmol L-1 based on 2019 data (9 cruises, 309-317)
accuracy: See QC Charts
reference standard: - primary: Potassium Nitrate KNO3 (10,000 µM) - secondary: WAKO, OSIL, SCOR-JAMSTEC and sodium nitrite check standard
analysis history for HOT program:
HOT 1-12 cruise samples only were filtered through combusted GF/F filters prior to storage.
Up until February 2000, analyses were conducted at room temperature on a four-channel Technicon Autoanalyzer II continuous flow system at the University of Hawaii Analytical Facility.
Between 2001 and 2005, the HOT nutrient program underwent substantial changes, including switching analysts twice, eventually establishing an analytical nutrient laboratory centered around a 4-channel SEAL Analytical AA3.
During this transition period, samples from > 200 m depth from HOT 127-166 were shipped to Oregon State University (OSU) for analyses. The OSU nutrient facility uses an AutoAnalyzer II manifold with 5 cm flow cell for PO4 analyses, and an Alpkem RFA 300 system for analyses of NO3+NO2.
Notes, comments or additions: