Isotope Dilution Analysis of Combined Nitrogen in Natural Waters. III. Nitrate and nitrite

Abstract

The measurement of nitrate uptake is of major importance to our investigations of primary production and nitrogen regeneration in the photic zone. Endogenous nitrate production (nitrification), by diluting the enrichment of added 15 N-nitrate, can lead to underestimation of nitrate uptake. Although nitrification is assumed to be minimal in the photic zone, this needs to be confirmed by experiment. We have developed a specific isotope dilution method based on the analysis of a volatile derivative of the azo dye, 1-phenylazo-2-naphthol (Sudan-1). Sea water is spiked to approximately 10% of the nitrate pool with a known quantity of 15 N-nitrate. An aliquot is filtered immediately. A second aliquot is incubated in situ for a fixed period and is then filtered. Nitrate concentration is estimated by isotope dilution analysis of the first filtrate. Nitrification rate is estimated from the change in 15 N enrichment between the first and second filtrate. Nitrate from a 10 mL aliquot is first reduced to nitrite. Sudan-1 is formed by diazotization with aniline and coupling to 2-naphthol. It is extracted by solid phase extraction using C-18 cartridges, with ethyl acetate as eluant. 4-Phenylazophenol is added as internal standard and the samples are dried. They are analysed as tertbutyldimethylsilyl derivatives, using gas chromatography/mass spectrometry in selected ion recording mode. The intense M-57 fragment ion formed under electron impact ionization is used for 15 N analysis. The procedure is precise, sensitive and, unlike most previous methods, it is free from interference by other forms of nitrogen. This method compliments our existing isotope dilution methods for the analysis of ammonium and dissolved free amino acids. Here, the new method has been used to confirm that our estimates of nitrate uptake in the photic zone of the Southern Ocean are not compromised by high nitrification rates.

Balance of assimilative and dissimilative nitrogen processes in a diatom-rich tidal flat sediment

“…To distinguish the 15 N in organic matter from ammonium that was bound to sediment particles, a subsample of sediment was transferred to a 2 M KCl solution, shaken vigorously and then centrifuged (1500 g, 15 ) (Rich et al, 2008). The supernatant was removed, and the pellet was washed three times with Milli-Q water before the sediment was freeze-dried for later isotope analysis.…”

“…15 N enrichment of nitrite and nitrate was measured by the indophenol extraction method based on Preston et al (1998). When referring to labelled 15 NO − 3 in the following, this accordingly includes nitrite.…”

“…Samples were then left to react for 3 h at room temperature on a horizontal shaker at 130 rpm. This step was followed by isotope analysis of nitrate and nitrite as described in detail by Preston et al (1998). In brief, nitrite was converted to the colour complex Sudan-I by diazotization with 2-naphthol and aniline sulfate.…”

Isolation of NO3‐N as 1‐phenylazo‐2‐naphthol (Sudan‐1) for measurement of δ15N

“…We found that the naphthol colour development step was complete after only 2.5 min, which is one-quarter of the time previously reported for this step. 10,11 At high concentrations, the dye absorbed onto the glassware if the reaction was allowed to proceed for longer than 2.5 min. This was prevented by using diluted NO 3 À solutions, adding methanol after 2.5 min and extracting the dye as quickly as possible.…”

Isolation of ammonium‐N as 1‐sulfonato‐iso‐indole for measurement of δ15N

“…We successfully used the isolation of nitrate as 1‐phenylazo‐2‐naphthol to measure the δ 15 N of nitrate,6 adapting the method of Preston et al 7 The success of this method lies in using solid‐phase extraction (SPE) to concentrate and purify an organic derivative containing the target nitrate‐N. Preston et al 7 used a similar approach for ammonium, converting it to the indophenol derivative, which can also be purified by SPE. This derivatisation does not involve the addition of non‐ammonium nitrogen to the final product.…”