NITRATE, NITRITE and DISSOLVED ORGANIC NITROGEN
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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.
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1. Principle
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).
2. Precautions
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.
3.1. Sample collection
3.1.1. Rinse the nutrient sample bottle (acid-washed, 125 ml
polyethylene bottle) 3 times before filling. Fill to
approximately 2/3 full, tighten cap and freeze.
3.1.2. Record cruise, cast and Niskin bottle number on the bottle
and data sheet.
4. Sample Analysis
4.1. Standard procedure
Currently, GOFS and WOCE nutrients collected during the Hawaii
Ocean Time Series cruises are analyzed by the Hawaii Institute
of Marine Biology Analytical Facility. Mr. Ted Walsh has
provided us with the following procedure for the analysis of
dissolved N.
4.1.1. Nitrate (NO3-) plus Nitrite (NO2-)
[NO3-+NO2-] analyses are performed on a four-channel
Technicon Autoanalyzer IIR continuous flow system. The
automated wet chemistries generally follow the standard
methods of seawater analysis as given by Technicon (1977),
which involve: (1) reduction of nitrate to nitrite using a
copperized cadmium reduction column, (2) reaction of nitrite
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 15 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 nitrite analysis only. Both
nitrate and nitrite standards are run to check column
efficiency. If speciation is desired, nitrite is determined
separately by omitting the reduction step. Nitrate is
calculated by difference.
4.1.2. Dissolved Organic Nitrogen (DON)
The method for DON involves initial UV digestion of a seawater
sample followed by autoanalysis of the digestion products for
[nitrate+nitrite], as above, and ammonium using the Berthelot
(indophenol) method. The modified photooxidation technique
(Armstrong et al., 1966) utilizes a 24 hour irradiation.
Details are given in Walsh (1989). Periodic calibration
checks of the UV lamp efficiency are made using a dissolved
organic nitrogen (2,2-bypyridyl) standard. As a general rule,
the UV lamp is replaced after approximately 700-800 hr of use.
DON is calculated by difference between the sum of [nitrate+
nitrite+ammonium] before and after UV treatment.
4.2. Low-level procedures for nitrate (NO3-) and nitrite (NO2-) by
chemiluminescence
Nanomolar quantitites of nitrate and nitrite are routinely analyzed
using the chemiluminescence techniques of Cox (1980) and Garside
(1982). This technique relies on the wet chemical reduction of
nitrate and nitrite to nitric oxide in a highly acidic solution
of sulfuric acid, ferrous ammonium sulfate, and ammonium molybdate.
The reduced nitric oxide is carried by an inert carrier gas (argon),
scrubbed of acid and water vapors in a cold finger filled with 6 M
sodium hydroxide solution followed by an anhydrous sodium carbonate
filled drying tube. The gas stream is then routed through a
membrane dryer and the nitric oxide is combined with ozone and
simultaneously exposed to a photomultiplier tube. The nitric
oxide is further oxidized to excited nitrogen dioxide emitting a
photon as it returns to ground state, and the emitted light is
detected by the photomultiplier.
4.2.1. [NO3- + NO2-] determinations
[NO3- + NO2-] analyses are performed on an Antek model 720
nitrogen oxide analyzer. Ten ml of concentrated sulfuric
acid (36 N) plus 2 ml each of ferrous ammonium sulfate (4%,
w/v) followed by ammonium molybdate (2%, w/v) are dispensed
into the reaction tube. The reaction tube is inserted into
the carrier gas line and the reagents degassed. A 10 ml
water sample or standard solution is introduced into the
reaction tube with a syringe through a septum fitted to the
side arm of the reaction tube. Total reaction light emission
is recorded using an automated integrator.
4.2.2. NO3- determinations
Sample analysis is conducted, as above (Chapter 7, section
4.2.1), except that the sample (10 ml) is pretreated with
0.2 ml of sulfanilamide prior to injection into the reaction
tube. Since sulfanilamide quantitatively binds nitrite, the
integral is the result of nitrate only. Therefore, nitrite
can be obtained from the difference of the two analyses.
5. Calibration, Data Reduction and Calculations
5.1. Calibration stocks and regression standards
The calibration of dissolved inorganic nutrients in the autoanalysis
of seawater samples is performed using standard solutions containing
N, P and Si. A nutrient stock solution is prepared by dissolving
dried (65°C, 72 hours) analytical grade reagent chemicals with
distilled-deionized water in 1 liter glass volumetric flasks
containing 1 ml of chloroform. Once dissolved, this stock solution
is immediately transferred into 1 liter amber polypropylene bottles
and stored at 4°C. The reagent chemicals and concentrations are:
phosphate (KH2PO4, 1 mM), nitrate (KNO3, 5 mM) and silica (Na2SiF6,
4 mM).
Working standards are prepared daily by volumetric dilutions of the
stock using glass pipettes and a plastic (polymethylpentene; PMP)
volumetric flask. All pipettes and PMP flasks are acid-washed
(1 M HCl) and gravimetrically calibrated prior to use. The daily
regression standards are prepared by diluting the working standard
with low nutrient natural seawater diluent (SWDIL). The SWDIL is
filtered open ocean surface seawater that is stored in a carboy at
room temperature. By using this technique all standards are
matrix-matched with the seawater samples and any cross-nutrient
interference effect should be accounted for.
Cross-nutrient interference and reagent contamination was evaluated
by preparing separate solutions, as above, but with one of the three
standards omitted. Only phosphate showed a slightly measurable
increase (+0.014 µM) in the presence of 40 µM-NO3 and 160 µM-Si.
The linear regressions of the standards were applied to all seawater
sample peaks for calculating each batch of cruise samples. Typical
correlations produced r2 values that were between 0.9999 and 0.99999.
5.2. Blank corrections
All seawater standard absorbance peaks were corrected for the
absorbance of the seawater diluent (SWDIL). All seawater sample
peaks were corrected for the refractive index absorbance for each
unique nutrient detection system. The refractive index corrections
(in apparent uM units) ranged from approximately 0.13 (for P), 0.23
(for N) to 2.41 (for Si), and represent the increase in absorbance
that is due strictly to the presence of dissolved salts in seawater
when compared to the distilled-deionized water baseline. These
corrections are made running seawater (35 o/oo salinity) through
the autoanalyzer with DDW only in reagent lines. The Levor
surfactant used routinely in the phosphate channel was omitted
from the DDW lines during the refractive index measurement because
Levor reacts erratically with seawater in the absence of the acidic
color reagent.
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%.
7. Equipment/Supplies
Niskin bottles and rosette/CTD unit
acid-washed, 125 ml polyethylene bottles
Autoanalyzer (Technicon Corp.) and accessories
UV oxidizer unit
nitrogen oxide analyzer (Antek model #720, operated in vacuum mode)
reaction tube, cold finger and drying tube glassware array
8. Reagents
glass distilled deionized water (DDW)
1 M HCl for cleaning
concentrated H2SO4 (36 N)
Ammonium Chloride Reagent: Dissolve 10 g of ammonium chloride in DDW,
adjust pH to 8.5 with concentrated ammonium hydroxide and dilute to
1 liter. Add 0.5 ml Brij-35 (Technicon No. T21-0110).
Color Reagent: To approximately 1500 ml of distilled water, add 200
ml of concentrated phosphoric acid and 20 g of sulfanilamide.
Dissolve completely (heat if necessary). Add 1 g of N-1-naphthyl-
ethylenediamine dihydrochloride and dissolve. Dilute to 2 liters.
Add 1.0 ml of Brij-35. Store in a cold, dark place. Stability: one
month.
Cadmium Powder (Technicon No. T11-5063): Use coarse cadmium powder
(99% pure). Rinse the powder once or twice with a small quantity
of clean diethyl ether and 1 M HCl to remove grease and dirt.
Follow with a DDW rinse. Allow the metal to air-dry and store in
a well-stoppered bottle.
Ferrous Ammonium Sulfate (4% w/v): Dissolve 4 g of reagent grade
ferrous ammonium sulfate in 100 ml DDW. Prepare fresh daily.
Ammonium molybdate (2% w/v): Dissolve 2 g reagent grade ammonium
molybdate in 100 ml DDW. Prepare fresh daily.
Sulfanilamide (1% in 10% HCl). Dissolve 1 g reagent grade
sulfanilamide in 100 ml of 10% HCl.
Sodium hydroxide (6 M): Dissolve 240 g of reagent grade sodium
hydroxide and make up to 1 liter with DDW.
Preparation of Reduction Column: See Technicon Industrial System,
1977.
Stock Standard (1000 µM): Dissolve 0.101 g of potassium nitrate
in DDW and dilute to 1 liter. Store in a dark bottle. Add 1 ml
of chloroform as a preservative.
Working Standard (50 µM): Dilute 5 ml of stock standard in a
volumetric flask with DDW or seawater diluent.
9. References
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, 381-389.
Armstrong, F. A. J., P. M. Williams and J. D. H. Strickland.
1966. Photo-oxidation of organic matter in seawater by ultraviolet
radiation, analytical and other applications. Nature, 211 481-483.
Cox, R. D. 1980. Determination of nitrate at the parts per
billion level by chemiluminescence. Analytical Chemistry, 52,
332-335.
Garside, C. 1982. A chemiluminescent technique for the
determination of nanomolar concentrations of nitrate and nitrite
in seawater. Marine Chemistry, 11, 159-167.
Grasshoff, K., M. Ehrhardt, and K. Kremling. 1983. Methods
of Seawater Analysis. Verlag Chemie.
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.
Strickland, J. D. H. and T. R. Parsons. 1972. A Practical
Handbook of Seawater Analysis. Fisheries Research Board of
Canada, 167.
Technicon Industrial Systems. 1977. Nitrate & Nitrite in
Water and Seawater. Autoanalyzer II R Industrial Method No.
155-71W. W. Tarrytown, New York 10591.
Venrick, E. L. and T. L. Hayward. 1985. Evaluation of some
techniques for preserving nutrients in stored seawater samples.
CalCOFI Report, 26, 160-168.
Walsh, T. W. 1989. Total dissolved nitrogen in seawater:
A new high temperature combustion method and a comparison to
photo-oxidation. Marine Chemistry, 26, 295-311.
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."
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