2.0 Definition
Dugdale and Goering (1967) define new production, described here as nitrate uptake, as “...all primary production associated with newly available nitrogen, for example NO₃ -N and N₂ -N...”. Dinitrogen fixation (N₂-N) has conventionally been viewed as a minor source of nitrogen uptake in ocean waters 1 , therefore, only NO₃-N will be considered here. In view of the importance, conceptually and operationally, of differentiating nitrate uptake and uptake of regenerated forms of nitrogen, it is strongly recommended that regenerated production measurements (NH₄ + uptake, as a minimum) be made in parallel with nitrate uptake measurements.
            NO₃ -N= nmol L^-1 day ^-1

3.0 Principle of Analysis
The measurement of nitrate uptake, as defined above, is based on the incorporation of ‘trace’ additions of 15 N-labelled NO₃ into phytoplankton during incubation experiments, similar in principle to the 14 CO₂ method for measuring photosynthesis.

4.0 Apparatus
Determination of nitrate uptake rates requires knowledge of: (i) the initial substrate (NO₃ ^- ) concentration, (ii) the final concentration of particulate nitrogen and (iii) the final ¹⁵N enrichment of the particulate matter.
For determining substrate concentrations >100 nmol L ^-1 , refer to Chapter 8, “The Determination of Nitrite, Nitrate + Nitrite, Orthophosphate and Reactive Silicate in Sea Water using Continuous Flow Analysis”. For concentrations <100 nmol L ^-1 , Chapter 9, “The Determination of Nitrate in Sea Water”1 . For particulate nitrogen, refer to Chapter 15, “Determination of Particulate Organic Carbon and Particulate Nitrogen”2 .

¹⁵ N enrichment is measured by either emission or mass spectrometry. Generally, the emission spectrometer has the capability of analyzing smaller samples but at the expense of precision and accuracy (Fiedler and Proksch, 1975). Newer mass spectrometers, however, can now match the emission spectrometer in the analysis of small masses of N while retaining their superior analytical precision (Owens and Rees, 1989).

5.0 Reagents
15 N-enriched NO₃ , as NaNO₃ or KNO₃ , is commercially available in dry chemical form at enrichments of 95-99 atom%. Tracer working solutions are made up in distilled/demineralized water. ¹⁵ NH₄ is available at similar enrichments as NH₄Cl or (NH₄ )₂ SO₄ .

6.0 Sampling
Sampling protocols will generally follow those outlined in Chapter 19, “Primary Production by ¹⁴ C”. Sampling depths should be compatible to the extent possible with depths selected for ¹⁴C experiments 3 but additional depths are recommended below the 1% light depth to assess the ‘sub-euphotic zone’ uptake of ¹⁵NO₃ ^- (and ¹⁵NH₄⁺ ).

7.0 Procedures
7.1 Experimentation should be done a minimum of twice daily, once during daylight hours and once during darkness, because N uptake may not be fully light dependant and because bacterioplankton contribute to both NH ₄ + and NO₃ ^- utilization.

7.2 Tracer additions: ¹⁵ NO₃ ^- and ¹⁵NH ₄ ⁺ should be added at ~10% (or less) of ambient concentration where measurable. For waters where ambient concentrations are below the analytical limit of detection, tracer additions should be at the limit of detection, i.e. ~50 nmol L ^-1 when conventional nutrient analysis is employed or ~2 nmol L ^-1 when the new low-level methods are used.

7.3 Incubations: Incubations of 2-4 hr in light-attenuated deck boxes, cooled by flowing surface seawater, are recommended. Surface seawater temperature may be inappropriate for deeper samples where the subsurface chl max is situated within or below the thermocline. Other arrangements for temperature control during the on-deck measurements of “sub-euphotic zone” uptake will be necessary. Acid-cleaned polycarbonate bottles are to be used (see Chapter 19 for bottle washing technique). Extreme caution should be exercised in the use of nitric acid as originally described in the metal-free ‘clean’ techniques of Fitzwater et al. (1982); copious distilled water rinsing is necessary to insure no residual contamination (NO₃ ^- ). Dilute HCl has been employed as an effective substitute, avoiding the nitrogen contaminant problem (HOTS, 1990; BATS, 1991).

7.4 Filtration/storage: After incubation, samples are filtered onto Whatman GF/F glass fiber filters under low vacuum pressure (<70mm Hg) and rinsed with filtered seawater to remove residual ¹⁵NO₃ ^- from filter interstices. Care should be taken not to evacuate the filters to dryness (Goldman and Dennett, 1985). Filters are then immediately dried or frozen at -20^oC (and later dried) for isotope analysis.

7.5 Isotope analysis: Under most circumstances, particulates are converted to N₂ gas by the Dumas combustion method. Isotope ratios are determined either by optical emission spectrometry or by mass spectrometry (Fiedler and Proksch, 1975).

8.0 Calculation and Expression of Results
Nitrate uptake (nmol L^-1 t ^-1 ) = ( 15 Nxs *  PNt ) / (15 Nenr * t)

where:
        t = incubation time in hours
       ¹⁵ Nxs = excess ¹⁵N (measured ¹⁵Nminus ¹⁵N natural abundance, 0.366 atom%) in the post-incubation particulate sample
        PNt = particulate nitrogen content of the sample after incubation in units of nmol L ^{-1
        15} Nenr = ¹⁵N enrichment in the dissolved fraction:
        ¹⁵ Nenr = [(100 * ¹⁵ N)/(¹⁵ N + ¹⁴ N)]-¹⁵Nn
where:
        ¹⁵N = concentration of labelled N, nmol L ^{-1
        14} N = concentration (same units) of unlabeled N
        ¹⁵ Nn = natural abundance of ¹⁵N
Daily (24 hr) nitrate uptake rates (nmol L^-1 d ^-1 )are approximated by multiplying the results from the daylight incubations by the number of daylight hrs (the same for the dark incubations and hrs) and adding the two quantities. The procedure of combining the independent “light” and “dark” period uptake measurements is valid only if one is sampling essentially the same water mass at both times. Otherwise, the estimated
rates must be normalized (for example, to the chlorophyll level) before combining the rates to come up with a daily rate.

9.0 Quality Control
Sample collection and handling should follow the general precautions outlined for other incubation measurements, e.g. ‘clean’ techniques should be employed to minimize metal contamination, exposure of samples during transport to and from incubators to direct sunlight should be avoided to minimize light shock. Additionally, sources of potential nitrogen contamination (e.g. smoking) should be avoided in sample handling, filtration and isotope analysis.

Emission spectra are generally calibrated using commercial N 2 gas standards of known isotope ratios; mass spectra are generally calibrated using commercial standard reference material (NBS, NIST) as primary standards or locally prepared secondary standards which have been calibrated against the primary standards. Under these circumstances, mass spectrometer accuracy should be absolute. Accuracy of emission spectra, however, is problematic since operational samples are not purified gases as are the commercial standards supplied with the instrument. Periodic calibration against a mass spectrometer, therefore, is required. Precision for mass spectrometers should be +/- 0.005 at natural ¹⁵ N abundance level (0.366 atom%); the value is +/- 0.02 for emission spectrometers. Replicate incubations for 15 N uptake rates should fall within 5% of the mean (McCarthy et al. 1977).

10.0 Intercomparison
Intercomparison is desirable but not essential for ¹⁵ N ratio analysis since most instrumentation is calibrated against known commercial standard reference materials, e.g. NBS, NIST standards. Sample collection / incubation particulars, however, are likely to differ, making intercomparisons of the actual nitrate uptake measurements (sample collection/incubation/analysis) on the same water mass by the various researchers highly desirable and to be recommended whenever possible.

11.0 Parameters
Attention has recently been drawn to the need for JGOFS researchers to make a clear distinction between “variables” and “parameters” in process studies (Evans, 1991). With regard to nitrate uptake (and nitrogen utilization in general), daily column-integrated N-fluxes as described above are considered ‘variables’. Parameters of relevance to nitrate uptake fall into three categories: those describing the nitrogen concentration-dependence of uptake, i.e. K s and V max , similar parameters describing the light-dependence of uptake, i.e. K _i , V _max , V _dark , and a parameter describing N interactions, specifically the inhibitory effects of NH₄⁺ on NO₃ ^- uptake. Consideration of the appropriate measurement protocols for these ‘parameters’ is beyond the scope of this report but is judged of some urgency.

12.0 References
BATS (1991). Bermuda Atlantic Time-Series Study. Field and laboratory methods, 2nd edition, March, 1991, Bermuda Biological Station for Research, Inc.
Brzezinski, M.A. (1987). Colorimetric determination of nanomolar concentrations of ammonium in seawater using solvent extraction. Mar. Chem. 20: 277-288.
Carpenter, E.J. and K. Romans (1991). Major role of the cyanobacterium Trichodesmium in nutrient cycling in the North Atlantic Ocean. Science 254: 1356-1358.
Dugdale, R.C. and F.P. Wilkerson (1986). The use of 15 N to measure nitrogen uptake in euphotic oceans; experimental considerations. Limnol. Oceanogr. 31:673-689.
Dugdale, R.C. and J.J. Goering (1967). Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol. Oceanogr. 12: 196-206.
Evans, G.T. (1991). On parameters and variables. U.S. JGOFS News 2(4): 6.
Fiedler, R. and G. Proksch (1975). The determination of nitrogen- 15 by emission and mass spectrometry in biochemical analysis: A review. Anal. Chim. Acta. 17: 1-62.
Fitzwater, S.E., G.A. Knauer and J.H. Martin (1982). Metal contamination and its effects on primary production measurements. Limnol. Oceanogr. 27: 544-551.
Garside, C. (1982). A chemiluminescent technique for the determination of nanomolar concentrations of nitrate and nitrite, or nitrite alone in seawater. Mar. Chem. 11: 159-167.
Goldman, J.C. and M.R. Dennett (1985). Susceptibility of some marine phytoplankton species to cell breakage during filtration and post- filtration rinsing. J. Exp. Mar. Biol. Ecol. 86: 47-58.
HOTS (1990). Hawaii Ocean Time-series Program. Field and laboratory protocols, September, 1990. University of Hawaii.
Jones, R.D. (1991). An improved fluorescence method for the determination of nanomolar concentrations of ammonium in natural waters. Limnol. Oceanogr. 36: 814-819.
Laws, E. A., G.R. DiTullio, P.R. Betzer, D.M. Karl and K.L Carder (1989). Autotrophic production and elemental fluxes at 26 o N, 155 o W in the North Pacific subtropical gyre. Deep-Sea Res. 36: 103-120.
Lohrenz, S. E., D. A. Wiesenburg, C. R. Rein, R. A. Arnone, C. T. Taylor, G. A. Knauer, and A. H. Knap (1992). A comparison of in situ and simulated in situ methods for estimating oceanic primary production. J. Plankt. Res. 14: 201-221.
McCarthy, J.J., W.R. Taylor and J.L. Taft (1977). Nitrogenous nutrition of the plankton in Chesapeake Bay. 1. Nutrient availability and phytoplankton preferences. Limnol. Oceanogr. 22:996-1011.
Owens, N.J.P., and A.P. Rees (1989). Determination of nitrogen-15 at sub-microgram levels of nitrogen using automated continuous-flow isotope ratio mass spectrometry. Analyst 114: 1655-1657.
Raimbault, P., G. Slawyk, B. Coste and J. Fry (1990). On the feasibility of using an automated colorimetric procedure for the determination of seawater nitrate in the 0- 100 nM range: examples from field and culture work. Mar. Biol. 104: 347-351.
Smith, S.L., K. Banse, J.K. Cochran, L.A. Codispoti, H.W. Ducklow, M.E. Luther, D.B. Olson, W.T. Peterson, W.L. Prell, N. Surgi, J.C. Swallow and K. Wishner (1991). U.S.JGOFS: Arabian Sea Process Study, U.S. JGOFS Planning Report No. 13, Woods Hole Oceanographic Institution, Woods Hole, Mass., 164 p.