Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i
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NITRATE, NITRITE and DISSOLVED ORGANIC NITROGEN
SUMMARY: Seawater is collected from known depths using CTD-rosette sampling protocols. Subsamples are carefully drawn and stored in acid-washed polyethylene bottles. Nitrate/nitrite is measured with an azo dye either before (nitrite) or after (nitrite plus nitrate) subsamples are passed through a cadmium reduction column. Dissolved organic nitrogen is determined after quantitative conversion to inorganic N by exposure to UV radiation.
In seawater the forms of dissolved nitrogen of greatest interest are, in order of decreasing oxidation state: nitrate, nitrite, ammonium and organic nitrogen. All these forms of nitrogen, as well as nitrogen gas (N2), are biochemically interconvertible and are components of the biological nitrogen cycle.
In this method nitrate is quantitatively reduced to nitrite in a copperized cadmium reduction column. The nitrite thus produced, along with any nitrite 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 read spectrophotometrically. A second subsample is analyzed without prior reduction in order to determine the nitrite level. Nitrate is calculated by difference between the [nitrate+nitrite] and nitrite concentrations, using standard solutions. For surface water samples (<100 m) where the [nitrate+nitrite] concentration is generally <0.05 µM, we have employed a low-level assay procedure which is based on the production and detection of nitrous oxides.
Total dissolved nitrogen (TDN) is determined by UV oxidation of the sample and subsequent analysis for dissolved inorganic nitrogen (DIN = nitrite + nitrate + ammonia). Dissolved organic nitrogen (DON) is computed from the relationship DON = TDN - DIN, where TDN is total dissolved nitrogen after UV oxidation and DIN is the sum of the dissolved inorganic nitrogen species before UV oxidation. As an alternative to the UV oxidation method, Walsh (1989) has described a high-temperature (1100°C) combustion method which has been applied to open ocean samples collected in the North Pacific Ocean. No significant differences were observed between these two procedures (Walsh, 1989).
Contamination is the primary concern with these samples. This is particularly true with samples collected from the euphotic zone, where inorganic nutrient concentrations are extremely low (<0.2 µM). In order to avoid contamination, sample bottles must be meticulously cleaned with dilute HCl and rinsed with deionized distilled water (DDW) before use. Samples are stored frozen until analysis, generally within 1-2 weeks of sample collection.
3. Sampling Collection and Storage
NOTE: The currently held "dogma" in the oceanographic literature is that seawater samples must be processed fresh and on board ship for high-precision, low-level inorganic nutrient analyses (Morse et al., 1982; Venrick and Hayward, 1985). However, extensive results from automated analyses of nutrients in tropical seawaters (Ryle et al., 1981) and the VERTEX program (D. Karl and S. Moore, unpubl. results) which included direct comparisons of [NO3+NO2], PO4 and SiO3 determinations in fresh vs. frozen samples would suggest otherwise. Provided that caution is taken to collect and store the samples in an environment free of potential contamination, we found no significant treatment effect. A similar conclusion was presented by Walsh et al. (manuscript) following the analysis of a wide range of seawater samples that were either analyzed fresh or frozen and stored for varying periods of time. They conclude that, "Despite published and voiced opinions to the contrary, there appears to be no adequate basis either from the literature or from our experiments for across-the-board dismissal of high-precision nutrient analyses undertaken on properly stored seawater samples." As we are not able to take our autoanalyzer to sea on the HOT program cruises, we have focussed our attention on maintaining a contamination-free environment during collection and storage of nutrient samples.
4. Sample Analysis
5. Calibration, Data Reduction and Calculations
6. Accuracy and Precision
The detection limit for nitrate plus nitrite is approximately 0.03 uM with a coefficient of variation for field-collected replicates of 0.3%. The detection limit for DON is 0.05 with a coefficient of variation of 4%.
POSTSCRIPT: During year 1 of the HOT program, we routinely measured NH4+ concentrations using the standard Berthelot (indophenol) method. Concentrations of NH4+ were consistently at, or below, our detection limits (<0.05 µM) throughout the water column. Although a new method has been described for low-level determinations of NH4+ in seawater (Brzezinski, 1988, Limnology and Oceanography, 33, 1176-1182), we have not yet successfully adapted this procedure for our routine determinations. Until that method, or a suitable alternative, is available we have decided to delete NH4+ measurements from our list of "standard procedures."