Method 353.4
Determination of Nitrate and Nitrite in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
Jia-Zhong Zhang, Cooperative Institute for Marine and Atmospheric Studies, Rosenstiel
School of Marine and Atmospheric Science/AOML, NOAA, University of Miami, Miami,
FL 33149
Peter B. Ortner and Charles J. Fischer, Ocean Chemistry Division, Atlantic
Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric
Administration, Miami, FL 33149
Project Officer
Elizabeth J. Arar
Revision 2.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Method 353.4
Determination of Nitrate and Nitrite in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
1.0	Scope and Application
1.1	This method provides a procedure for determining
nitrate and nitrite concentrations in estuarine and coastal
waters. Nitrate is reduced to nitrite by cadmium,13 and the
resulting nitrite determined by formation of an azo dye.4"6
In most estuarine and coastal waters nitrogen is thought
to be a limiting nutrient. Nitrate is the final oxidation
product of the nitrogen cycle in natural waters and is
considered to be the only thermodynamically stable
nitrogen compound in aerobic waters.7 Nitrate in
estuarine and coastal water is derived from rock
weathering, sewage effluent and fertilizer run-off. The
concentration of nitrate usually is high in estuarine waters
and lower in surface coastal waters.
Nitrite is an intermediate product in the microbial
reduction of nitrate or in the oxidation of ammonia. It may
also be excreted by phytoplankton as a result of excess
assimilatory reduction. Unlike nitrate, nitrite is usually
present at a concentration lower than 0.01 mg N/L except
in high productivity waters and polluted waters in the
vicinity of sewer outfalls.
Chemical Abstracts Service
Analyte	Registry Numbers (CASRN)
Nitrate	14797-55-8
Nitrite	14797-65-0
1.2	A statistically determined method detection limit
(MDL)8 of 0.075 |jg N/L has been determined by one
laboratory in seawaters of five different salinities. The
method is linear to 5.0 mg N/L using a Flow Solution
System (Alpkem, Wilsonville, Oregon).
1.3	Approximately 40 samples per hour can be
analyzed.
1.4	This method requires experience in the use of
automated gas segmented continuous flow colorimetric
analyses, and familiarity with the techniques of
preparation, activation and maintenance of the cadmium
reduction column. A minimum of six-months training is
recommended.
2.0	Summary of Method
2.1	An automated gas segmented continuous flow
colorimetric method for the analysis of nitrate
concentration is described. In the method, samples are
passed through a copper-coated cadmium reduction
column. Nitrate in the sample is reduced to nitrite in a
buffer solution. The nitrite is then determined by
diazotizing with sulfanilamide and coupling with N-1-
naphthylethylenediamine dihydrochloride to form a color
azo dye. The absorbance measured at 540 nm is linearly
proportional to the concentration of nitrite + nitrate in the
sample. Nitrate concentrations are obtained by
subtracting nitrite values, which have been separately
determined without the cadmium reduction procedure,
from the nitrite + nitrate values. There is no significant salt
error in this method. The small negative error caused by
differences in the refractive index of seawater and
reagent water is readily corrected for during data
processing.
3.0	Definitions
3.1	Calibration Standard (CAL) - A solution
prepared from the primary dilution standard solution or
stock standard solution containing analytes. The CAL
solutions are used to calibrate the instrument response
with respect to analyte concentration.
3.2	Laboratory Fortified Blank (LFB) - An aliquot of
reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine
whether method performance is within acceptable control
limits, and whether the laboratory is capable of making
accurate and precise measurements. This is a standard
prepared in reagent water that is analyzed as a sample.
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3.3	Laboratory Fortified Sample Matrix (LFM) - An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
3.4	Laboratory Reagent Blank (LRB) - An aliquot
of reagent water that is treated exactly as a sample
including exposure to all labware, equipment, and
reagents that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
the reagents, or apparatus.
3.5	Linear Dynamic Range (LDR) - The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.6	Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified,
measured and reported with 99% confidence that the
analyte concentration is greater than zero.8
3.7	Reagent Water (RW) - Type 1 reagent grade
water equal to or exceeding standards established by
American Society for Testing and Materials (ASTM).
Reverse osmosis systems or distilling units followed by
Super-Q Plus Water System that produce water with 18
megohm resistance are examples of acceptable water
sources. To avoid contamination, the reagent water
should be used the day of preparation.
3.8	Refractive Index (Rl) - The ratio of velocity of
light in a vacuum to that in a given medium. The relative
refractive index is the ratio of the velocity of light in two
different media, such as estuarine or sea water versus
reagent water. The correction for this difference is
referred to as the refractive index correction in this
method.
3.9	Stock Standard Solution (SSS) -A concentrated
solution of method analyte prepared in the laboratory
using assayed reference compounds or purchased from
a reputable commercial source.
3.10	Primary Dilution Standard Solution (PDS) - A
solution prepared in the laboratory from stock standard
solutions and diluted as needed to prepare calibration
solutions and other needed analyte solutions.
3.11	Quality Control Sample (QCS) - A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.12	SYNC Peak Solution - A colored solution used
to produce a synchronization peak in the refractive index
measurement. A synchronization peak is required by the
data acquisition programs to initialize the peak finding
parameters. The first cup in every run must always be
identified as a SYNC sample. The SYNC sample is
usually a high standard, but can be any sample that
generates a peak at least 25% of full scale.
4.0	Interferences
4.1	Hydrogen sulfide at concentrations greater than
0.1 mg S/L can interfere with nitrite analysis by
precipitating on the cadmium column .9 Hydrogen sulfide
in samples must be removed by precipitation with
cadmium or copper salt.
4.2	Iron, copper and other heavy metals at
concentrations larger than 1 mg/L alter the reduction
efficiency of the cadmium column. The addition of EDTA
will complex these metal ions.10
4.3	Phosphate at a concentration larger than 0.1
mg/L decreases the reduction efficiency of cadmium11.
Dilute samples if possible or remove phosphate with ferric
hydroxide12 prior to analysis.
4.4	Particulates inducing turbidity should be removed
by filtration after sample collection.
4.5	This method corrects for small refractive index
interference which occurs if the calibration standard
solution is not matched with samples in salinity.
5.0	Safety
5.1	Water samples collected from the estuarine and
coastal environment are generally not hazardous.
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However, the individual who collects samples should use
proper technique.
5.2 Good laboratory technique should be used when
preparing reagents. Laboratory personnel should obtain
material safety data sheets (MSDS) for all chemicals
used in this method. A lab coat, safety goggles, and
gloves should be worn when handling the concentrated
acid.
6.0	Equipment and Supplies
6.1	Gas Segmented Continuous Flow Autoanalyzer
Consisting of:
6.1.1	Autosampler.
6.1.2	Analytical cartridge with reaction coils for nitrate
analysis.
6.1.3	Open Tubular Cadmium Reactor (OTCR,
Alpkem, OR) or laboratory prepared packed copper-
coated cadmium reduction column (prepared according
to procedures in Section 7.4 - 7.5).
6.1.4	Proportioning pump.
6.1.5	Spectrophotometer equipped with a tungsten
lamp (380-800 nm) or photometer with a 540 nm
interference filter (2 nm bandwidth).
6.1.6	Strip chart recorder or computer based data
acquisition system.
6.1.7	Nitrogen gas (high-purity grade, 99.99%).
6.2	Glassware and Supplies
6.2.1	All labware used in the analysis must be low in
residual nitrate to avoid sample or reagent contamination.
Soaking with lab grade detergent, rinsing with tap water,
followed by rinsing with 10% HCI (v/v) and thoroughly
rinsing with reagent water is sufficient.
6.2.2	Automatic pipetters capable of delivering volumes
ranging from 100 |jl_ to 1000 |jl_ and 1 mL to 10 mL with
an assortment of high quality disposable pipet tips.
6.2.3	Analytical balance, with capability to measure to
0.1 mg, for preparing standards.
6.2.4	60 mL high density polyethylene sample bottles,
glass volumetric flasks and plastic sample tubes.
6.2.5	Drying oven.
6.2.6	Desiccator.
6.2.7	Membrane filters with 0.45 |jm nominal pore size.
Plastic syringes with syringe filters.
6.2.8	A pH meter with a glass electrode and a
reference electrode. A set of standard buffer solutions for
calibration of the pH meter.
7.0	Reagents and Standards
7.1	Stock Reagent Solutions
7.1.1	Stock Sulfanilamide Solution - Dissolved 10 g of
sulfanilamide (C6H8N202S, FW 172.21) in 1 L of 10%
HCI.
7.1.2	Stock Nitrate Solution (100 mg-N/L) -
Quantitatively transfer 0.7217 g of pre-dried (105°C for 1
hour) potassium nitrate (KN03, FW 101.099) to a 1000-
mL glass volumetric flask containing approximate 800 mL
of reagent water and dissolve the salt. Dilute the solution
to the mark with reagent water. Store the stock solution in
a polyethylene bottle in refrigerator at 4°C. This solution
is stable for six months.
7.1.3	Stock Nitrite Solution (100 mg-N/L)
Quantitatively transfer 0.4928 g of pre-dried (105°C for 1
hour) sodium nitrite (NaNOz, FW 68.99) to a 1000 mL
glass volumetric flask containing approximate 800 mL of
reagent water and dissolve the salt. Dilute the solution to
the mark with reagent water. Store the stogksolution in a
polyethylene bottle in a refrigerator at 4°C- Thls solutlon
is stable for three months.
Note: High purity nitrite salts are not available. Assays
given by reagent manufacturers are usually in the range
of 95-97%. The impurity must be taken into account for
calculation of the weight taken.
7.1.4	Low Nutrient Sea Water (LNSW) - Obtain natural
low nutrient seawater from surface water of the Gulf
Stream or Sargasso Sea (salinity 36 %o, < 7 |jg N/L) and
filter it through 0.3 micron pore size glass fiber filters. If
this is not available, commercial low nutrient sea water
(< 7 |jg N/L) with salinity of 35 %o (Ocean Scientific
International, Wormley, U.K.) can be substituted.
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7.2 Working Reagents
7.2.1	Brij-35 Start-up Solution - Add 2 mL of Brij-35
surfactant (ICI Americas, Inc.) to 1000 mL reagent water
and mix gently.
Note: Brij-35 is a trade name for polyoxyethylene(23)
lauiyl ether (C^OCHjCH^OH, FW=1199.57, CASRN
9002-92-0).
7.2.2	Working Sulfanilamide Solution - Add 1 mL of
Brij- 35 solution to 200 mL of stock sulfanilamide solution,
mix gently.
Note: Adding surfactant Brij-35 to sulfanilamide solution
instead of to the buffer solution is to prevent the Brij from
being adsorbed on the cadmium surface, which may
result in decreasing surface reactivity of the cadmium and
reduce the lifetime of the cadmium column.
7.2.3	NED Solution - Dissolve 1 g of NED (N-1-
naphthylethylenediamine Dihydrochloride, C12H14N2.2HCI,
FW 259.18) in 1 L of reagent water.
7.2.4	Imidazole Buffer Solution - Dissolve 13.6 g of
imidazole (C3H4N2, FW 68.08) in 4 L of reagent water.
Add 2 mL of concentrated HCI. Adjust the pH to 7.8 with
diluted HCI while monitoring the pH with a pH meter.
Store in a refrigerator.
7.2.5	Copper Sulfate Solution (2%) - Dissolve 20 g of
copper sulfate (CuS04.5H p, FW 249.61) in 1 L of
reagent water.
7.2.6	Colored SYNC Peak Solution - Add 50 |jL of red
food coloring solution to 1000 mL reagent water and mix
thoroughly. Further dilute this solution to obtain a peak
between 25 to 100 percent full scale according to the
AUFS setting used for the refractive index measurement.
7.2.7	Primary Dilution Standard Solution - Prepare a
primary dilution standard solution (5 mg N/L) by dilution of
5.0 mL of stock standard solutions to 100 mL with reagent
water. Prepare this solution daily.
Note: This solution should be prepared to give an
appropriate intermediate concentration for further dilution
to prepare the calibration solutions. Therefore the
concentration of a primary dilution standard solution
should be adjusted according to the concentration range
of calibration solutions.
7.2.8	Calibration Standards - Prepare a series of
calibration standards (CAL) by diluting suitable volumes
of a primary dilution standard solution (Section 7.2.7) to
100 mL with reagent water or low nutrient seawater.
Prepare these standards daily. The concentration range
of calibration standards should bracket the expected
concentrations of samples and not exceed two orders of
magnitude. At least five calibration standards with equal
increments in concentration should be used to construct
the calibration curve.
If nitrate + nitrite and nitrite are analyzed simultaneously
by splitting a sample into two analytical systems, a nitrate
and nitrite mixed standard should be prepared. The total
concentration (nitrate+nitrite) must be assigned to the
concentrations of calibration standards in the
nitrate+nitrite system.
When analyzing samples of varying salinities, it is
recommended that the calibration standard solutions and
sampler wash solution be prepared in reagent water and
corrections for refractive index be made to the sample
concentrations determined (Section 12.2).
7.2.9	Saline Nitrate and Nitrite Standards - If CAL
solutions will not be prepared to match sample salinity,
then saline nitrate and nitrite standards must be prepared
in a series of salinities in order to quantify the salt error,
the change in the colorimetric response of nitrate due to
the change in the composition of the solution. The
following dilutions of Primary Dilution Standard Solution
(Section 7.2.7) to 100 mL in volumetric flasks with
reagent water, are suggested:
Salinity Volume of Volume of Cone.
(%0) LNSW(mL) PDS(mL) mg N/L
0
0
2
.10
9
25
2
.10
18
50
2
.10
27
75
2
.10
35
98
2
.10
7.3 Open Tubular Cadmium Reactor
7.3.1 Nitrate in the samples is reduced to nitrite by
either a commercial Open Tubular Cadmium Reactor
(OTCR, Alpkem, OR) or a laboratory-prepared packed
copper-coated cadmium reduction column.
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7.3.2 If an OTCR is employed, the following procedures
should be used to activate it.10
Prepare reagent water, 0.5N HCI solution and 2% CuS04
solution in three 50 mL beakers. Fit three 10-mL plastic
syringes with unions. First flush the OTCR with 10 mL
reagent water. Then flush it with 10 mL 0.5N HCI
solution in 3 seconds, immediately followed by flushing
with a couple of syringe volumes of reagent water. Slowly
flush with CuS04 solution until a large amount of black
precipitated copper come out of OTCR, then stop the
flushing. Finally flush the OTCR with reagent water. Fill
the OTCR with imidazole buffer for short term storage.
7.4 Packed Cadmium Reduction Column
The following procedures are used for preparation of a
packed cadmium reduction column.13
7.4.1	File a cadmium stick to obtain freshly prepared
cadmium filings.
7.4.2	Sieve the filings and retain the fraction between 25
and 60 mesh size (0.25-0.71 mm).
7.4.3	Wash filings two times with 10% HCI followed with
reagent water.
7.4.4	Decant the reagent water and add 50 mL of 2%
CuS04 solution. While swirling, brown flakes of colloidal
copper will appear and the blue color of the solution will
fade. Decant the faded solution and add fresh CuS04
solution and swirl. Repeat this procedure until the blue
color does not fade.
7.4.5	Wash the filings with reagent water until all the
blue color is gone and the supernatant is free of fine
particles. Keep the filings submersed under reagent water
and avoid exposure of the cadmium filings to air.
7.4.6	The column can be prepared in a plastic or aglass
tube of 2 mm ID. Plug one end of column with glass wool.
Fill the column with water and transfer Cd filings in
suspension using a 10 mL pipette tip connected to one
end of column. While gently tapping the tube and pipette
tip let Cd filings pack tightly and uniformly in the column
without trapping air bubbles.
7.4.7	Insert another glass wool plug at the top of the
column. If a U- shape tube is used, the pipette tip is
connected to the other end and the procedure repeated.
Connect both ends of the column using a plastic tube
filled with buffer solution to form a closed loop.
7.4.8 If an OTCR or a packed cadmium column has
not been used for several days, it should be reactivated
prior to sample analysis.
7.5 Stabilization of OTCR and Packed Cadmium
Reduction Columns
7.5.1	Pump the buffer and other reagent solutions
through the manifold and obtain a stable baseline.
7.5.2	Pump 0.7 mg-N/L nitrite standard solution
continuously through the sample line and record the
steady state signal.
7.5.3	Stop the pump and install an OTCR or a packed
column on the manifold. Ensure no air bubbles have
been introduced into the manifold during the installation.
Resume the pumping and confirm a stable baseline.
7.5.4	Pump 0.7 mg-N/L nitrate solution continuously
through the sample line and record the signal. The signal
will increase slowly and reach steady state in about 10-15
minutes. This steady state signal should be close to the
signal obtained from the same concentration of a nitrite
solution without the OTCR or packed cadmium column
on line.
7.5.5	The reduction efficiency of an OTCR or a packed
cadmium column can be determined by measuring the
absorbance of a nitrate standard solution followed by a
nitrite standard solution of the same concentration.
Reduction efficiency is calculated as follows:
Absorbance of Nitrate
Reduction Efficiency = 	
Absorbance of Nitrite
8.0	Sample Collection, Preservation and
Storage
8.1	Sample Collection - Samples collected for
nutrient analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Go-Flo or equivalent) that are attached at fixed
intervals to a hydro wire. These bottles are sent through
the water column open and are closed either
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electronically or via a mechanical messenger when the
bottles have reached the desired depth.
8.1.2	In a submersible pump system, a weighted hose is
sent to the desired depth in the water column and water
is pumped from that depth to the deck of the ship for
sample processing.
8.1.3	For collecting surface samples, an acid - cleaned
plastic bucket or a large plastic bottle can be used as a
convenient sampler. Wash the sampler three times with
sample water before collecting samples.
8.1.4	Turbid samples should be filtered as soon as
possible after collection.
8.1.5	60-mL high density polyethylene bottles are used
for sample storage. Sample bottles should be rinsed 3
times with about 20 mL of sample, shaking with the cap
in place after each rinse. Pour the rinse water into the cap
to dissolve and rinse away salt crusts trapped in the
threads of the cap. Finally, fill the sample bottle about 3/4
full, and screw the cap on firmly.
8.2	Sample Preservation - After collection and
filtration, samples should be analyzed as soon as
possible. If samples will be analyzed within 3 hours then
keep refrigerated in tightly sealed, high density
polyethylene bottles in the dark at 4°C until analysis can
be performed.
8.3	Sample Storage - Natural samples usually
contain low concentrations of nitrite (< 14 g N/L) and no
preservation techniques are satisfactory.14 Samples must
be analyzed within 3 hours of collection to obtain reliable
nitrite concentrations.15
Samples containing high concentrations of ammonia or
nitrite may change in nitrate concentration during storage
due to microbial oxidation of ammonia and nitrite to
nitrate. These samples should be analyzed as soon as
possible.
Natural samples containing low concentrations of nitrite
and ammonia ( < 10% of the nitrate concentration ) can
be preserved for nitrate analysis by freezing. A maximum
holding time for preserved estuarine and coastal water
samples for nitrate analysis is one month.16
The results of preservation of natural samples are shown
in Tables 1 and 2 for nitrate and nitrite, respectively.
9.0	Quality Control
9.1	Each laboratory using this method is required to
implement a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of performance, continued analysis of
Laboratory Reagent Blanks (LRB), laboratory duplicates
and Laboratory Fortified Blanks (LFB) with each set of
samples as a continuing check on performance.
9.2	Initial Demonstration of Performance
(Mandatory)
9.2.1	The initial demonstration of performance is used
to characterize instrument performance by determining
the MDL and LDR and laboratory performance by
analyzing quality control samples prior to analysis of
samples using this method.
9.2.2	A method detection limit (MDL) should be
established for the method analytes using a low level
seawater sample containing, or fortified at, approximately
5 times the estimated detection limit. To determine MDL
values, analyze at least seven replicate aliquots of water
which have been processed through the entire analytical
method. Perform all calculations defined in the method
and report concentration in appropriate units. Calculate
the MDL as follows:
MDL = (t)(S)
where, S = the standard deviation of the
replicate analyses
t = Student's t value for n-1 degrees of
freedom at the 99% confidence
limit; t = 3.143 for six degrees
of freedom.
MDLs should be determined every six months or
whenever a significant change in background or
instrument response occurs or a new matrix is
encountered.
9.2.3	The LDR should be determined by analyzing a
minimum of eight calibration standards ranging from
0.002 to 2.00 mg N/L across all sensitivity settings
(Absorbance Units Full Scale output range setting) of the
detector. Standards and sampler wash solutions should
be prepared in low nutrient seawater with salinities similar
to that of samples, therefore a correction factor for salt
error, or refractive index, will not be necessary. Normalize
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responses by multiplying the response by the Absorbance
Units Full Scale output range setting. Perform the linear
regression of normalized response vs. concentration and
obtain the constants m and b, where m is the slope and
b is the y-intercept. Incrementally analyze standards of
higher concentration until the measured absorbance
response, R, of a standard no longer yields a calculated
concentration Cc, that is within 100 ± 10% of known
concentration, C, where Cc = (R-b)/m. That concentration
defines the upper limit of the LDR for the instrument.
Should samples be encountered that have a
concentration that is > 90% of the upper limit of LDR,
then these samples must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1	Laboratory Reagent Blank (LRB) - A laboratory
should analyze at least one LRB with each set of
samples. LRB data are used to assess contamination
from the laboratory environment. Should an analyte value
in the LRB exceed the MDL, then laboratory or reagent
contamination should be suspected. When the LRB value
constitutes 10% or more of the analyte concentration
determined for a sample, duplicates of the sample must
be prepared and analyzed again after the source of
contamination has been corrected and acceptable LRB
values have been obtained.
9.3.2	Laboratory Fortified Blank (LFB) - A laboratory
should analyze at least one LFB with each set of
samples. The LFB must be at a concentration that is
within the daily calibration range. The LFB data are used
to calculate accuracy as percent recovery. If the recovery
of the analyte falls outside the required control limits of
90 -110%, the source of the problem should be identified
and resolved before continuing the analyses.
9.3.3	The laboratory must use LFB analyses data to
assess laboratory performance against the required
control limits of 90-110%. When sufficient internal
performance data become available (usually a minimum
of 20 to 30 analyses), optional control limits can be
developed from the percent mean recovery (x) and
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery
Laboratory Fortified Sample Matrix
(LFM)
9.4.1	A laboratory should add a known amount of
analyte to a minimum of 5% of the total number of
samples or one sample per sample set, whichever is
greater. The analyte added should be 2-4 times the
ambient concentration and should be at least four times
greater than the MDL.
9.4.2	Calculate percent recovery of analyte, corrected
for background concentration measured in a separate
unfortified sample. These values should be compared
with the values obtained from the LFBs. Percent
recoveries may be calculated using the following
equation:
(Cs-C)
R =	x 100
S
where,
R = percent recovery
Cs = measured fortified sample concentration
(background + addition in mg N/L)
C = sample background concentration (mg N/L)
S = concentration in mg N/L added to the
environmental sample.
9.4.3 If the recovery of the analyte falls outside the
required control limits of 90-110%, but the laboratory
performance for that analyte is within the control limits,
the fortified sample should be prepared again and
analyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample
is judged to be the matrix related and the sample data
should be flagged.
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10.0 Calibration and Standardization
CO = (k)(PM)
10.1	At least five calibration standards should be
prepared fresh daily for system calibration. The
calibration concentrations should bracket the
concentrations of samples and the range should not be
over two orders of magnitude.
10.2	A calibration curve should be constructed for
each sample set by analyzing a series of calibration
standard solutions. A sample set should contain no more
than 60 samples. For a large number of samples make
several sample sets with individual calibration curves.
10.3	Analyze the calibration standards, in duplicate,
before actual samples.
10.4	The calibration curve containing five or more data
points should have a correlation coefficient, r, of 0.995 or
better.
10.5	Place a high CAL solution followed by two blank
cups to quantify the carry-over of the system. The
difference in peak heights between two blank cups is due
to the carry over from the high CAL solution. The carry-
over coefficient, k, is calculated as follows:
Pb1 " Pb2
k = 	
Phigh
where,
Phigh = the peak height of the high
nitrate standard
Pb1 = the peak height of the
first blank sample
Pb2 = the peak height of the
second blank sample.
The carry over coefficient, k, for a system should be
measured in seven replicates to obtain a statistically
significant number, k should be remeasured with any
change in manifold plumbing or upon replacement of
pump tubung.
The carry over correction (CO) on a given peak i is
proportional to the peak height of the preceding sample,
Pm-
To correct a given peak height reading, Ph subtract the
carry over correction,1718
Pi c = P; - CO
where Pic is corrected peak height. The correction for
carry over should be applied to all the peak heights
throughout a run. The carry over coefficient should be
less than 5% in this method.
10.6	Place a high standard nitrate solution followed by
a nitrite standard solution of same concentration at the
beginning and end of each sample run to check for
change in reduction efficiency of OTCR or a packed
cadmium column. The decline of reduction efficiency
during a run should be less than 5%.
10.7	Place a high standard solution at the end of each
sample run (60 samples) to check for sensitivity drift.
Apply sensitivity drift correction to all the samples. The
sensitivity drift during a run should be less than 5%.
Note: Sensitivity drift correction is available in most data
acquisition software supplied with autoanalyzers. It is
assumed that the sensitivity drift is linear with time. An
interpolated drift correction factor is calculated for each
sample according to the sample position during a run.
Multiply the sample peak height by the corresponding
sensitivity drift correction factor to obtain the corrected
peak height for each sample.
11.0	Procedure
11.1	If samples are frozen, thaw the samples at room
temperature. If samples are stored in a refrigerator,
remove samples and equilibrate to room temperature.
Mix samples thoroughly prior to analysis.
11.2	Turn on the continuous flow analyzer and data
acquisition components and warm up at least 30 minutes.
11.3	Set up the cartridge according to the type of
cadmium reductor used for nitrate + nitrite analysis
(configuration for OTCR shown in Figure 1 and packed
cadmium column in Figure 2). Configuration for analysis
of nitrite alone is shown in Figure 3.
Note: When a gas segmented flow stream passes
through the OTCR, particles derived from the OTCR
were found to increase baseline noise and to cause
353.4-9
Revision 2.0 September 1997

-------
interference at low level analysis. Packed cadmium
columns are, therefore, preferred for nitrate analysis at
low concentrations.
11.4	Set spectrophotometer wavelength at 540 nm.
11.5	Set the Absorbance Unit Full Scale (AUFS) range
on the spectrophotometer at an appropriate setting
according to the highest concentration of nitrate in the
samples. The appropriate setting for this method is 0.2
AUFS for 0.7 mg N/L.
11.6	Prepare all reagents and standards.
11.7	Begin pumping the Brij-35 start-up solution (Section
7.2.1) through the system and obtain a steady baseline.
Place the reagents on-line. The reagent baseline will be
higher than the start-up solution baseline. After the
reagent baseline has been stabilized, reset the baseline.
NOTE: To minimize the noise in the reagent baseline,
clean the flow system by sequentially pumping the
sample line with reagent water, 1 N HCI solution, reagent
water, 1 N NaOH solution for a few minutes each at the
end of the daily analysis. Make sure to rinse the system
well with reagent water after pumping NaOH solution to
prevent precipitation of Mg(OH)2 when seawater is
introduced into the system. Keep the reagents and
samples free of particulate. Filter the reagents and
samples if necessary.
If the baseline drifts upward, pinch the waste line for a few
seconds to increase back pressure. If absorbance drops
down rapidly when back pressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach
a syringe at the waste outlet of the flowcell. Air bubbles
in the flowcell can often be eliminated by simply attaching
syringe for a few minutes or, if not, dislodged by pumping
the syringe piston. Alternatively, flushing the flowcell with
alcohol was found to be effective in removing air bubbles
from the flow cell.
For samples of varying salinities, it is suggested that the
reagent water used for the sampler wash solution and for
preparing calibration standards and procedures in
Sections 12.2 and 12.3 be employed.
11.8	Check the reduction efficiency of the OTCR or
packed cadmium column following the procedure in
Section 7.5.5. If the reduction efficiency is less than 90%
follow the procedure in Section 7.5 for activation and
stabilization. Ensure reduction efficiencies reach at least
90% before analysis of samples.19
11.9	A good sampling rate is approximately 40 samples
per hour for 60 second sample times and 30 second
wash times.
11.10	Use cleaned sample cups or tubes (follow the
procedures outlined in Section 6.2.2). Place CAL
solutions and saline standards (optional) in sampler.
Complete filling the sampler tray with samples, laboratory
reagent blanks, laboratory fortified blanks, laboratory
fortified sample matrices, and QC samples. Place a blank
after every ten samples.
11.11	Commence analysis.
12.0	Data Analysis and Calculations
12.1	Concentrations of nitrate in samples are
calculated from the linear regression, obtained from the
standard curve in which the concentrations of the
calibration standards are entered as the independent
variable, and their corresponding peak heights are the
dependent variable.
12.2 Refractive Index Correction for Estuarine and
Coastal Samples
12.2.1	If reagent water is used as the wash solution and
to prepare the calibration standard solutions, the operator
has to quantify the refractive index correction due to the
difference in salinity between sample and standard
solutions. The following procedures are used to measure
the relationship between sample salinity and refractive
index for a particular detector.
12.2.2	First, analyze a set of nitrate or nitrite standards
in reagent water with color reagent using reagent water
as the wash and obtain a linear regression of peak height
versus concentration.
Note: The change in absorbance due to refractive index
is small, therefore low concentration standards should be
used to bracket the expected absorbances due to
refractive index.
12.2.3	Second, replace reagent water wash solution with
Low Nutrient Seawater wash solution.
Revision 2.0 September 1997
353.4-10

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Note: In nitrate and nitrite analysis absorbance of the
reagent water is higher than that of the LNSW. When
using reagent water as a wash solution, the change in
refractive index causes the absorbance of seawater to
become negative. To measure the absorbance due to
refractive index change in different salinity samples, Low
Nutrient Seawater must be used as a wash solution to
bring the baseline down.
12.2.4	Replace NED solution (Section 7.2.4) with
reagent water. All other reagents remain the same.
Replace the synchronization sample with the colored
SYNC peak solution (Section 7.2.6).
12.2.5	Prepare a set of different salinity samples with
LNSW. Commence analysis and obtain peak heights for
different salinity samples. The peak heights for the
refractive index correction must be obtained at the same
AUFS range setting and on the same spectrophotometer
as the corresponding standards (Section 12.2.2).
12.2.6	Using Low Nutrient Seawater as the wash water,
a maximum absorbance will be observed for reagent
water. No change in refractive index will be observed in
the seawater sample. Assuming the absolute absorbance
for reagent water (relative to the seawater baseline) is
equal to the absorbance for seawater (relative to reagent
water baseline), subtract the absorbances of samples of
various salinities from that of reagent water. The results
are the apparent absorbance due to the change in
refractive index between samples of various salinities
relative to the reagent water baseline.
12.2.7	For each sample of varying salinity, calculate the
apparent nitrate or nitrite concentrations due to refractive
index from its peak height corrected to reagent water
baseline (Section 12.2.5) and the regression equation of
nitrate or nitrite standards obtained with color reagent
being pumped through the system (12.2.2). Salinity is
entered as the independent variable and the apparent
nitrate or nitrite concentration due to refractive index is
entered as the dependent variable. The resulting
regression allows the operator to calculate apparent
nitrate or nitrite concentration due to refractive index when
sample salinity is known. Thus, the operator would not be
required to obtain refractive index peak heights for all
samples.
12.2.8	An example of typical results follows:
Salinity Apparent concentration (|jg N/L)
(%0)	Nitrate	Nitrite
0.0
0.000
0.000
3.8
0.026
0.015
9.2
0.096
0.040
13.8
0.142
0.055
18.1
0.190
0.086
26.8
0.297
0.153
36.3
0.370
0.187
Note: You must calculate the refractive index correction
for your particular detector. Moreover, the refractive index
must be redetermined whenever a significant change in
the design of flowcell or a new matrix is encountered.
12.2.9 An example of typical linear equations is:
Apparent nitrate (|jg N/L) = 0.01047S
Apparent nitrite (|jg N/L) = 0.00513S
where S is sample salinity. The apparent nitrate and nitrite
concentration due to refractive index so obtained should
be added to samples of corresponding salinity when
reagent water is used as wash solution and standard
matrix.
If nitrate and nitrite concentrations are greater than 100
and 50 |jg N/L respectively, the correction for refractive
index is negligible and this procedure can be optional.
12.3 Correction for Salt Error in Estuarine and
Coastal Samples
12.3.1	When calculating concentrations of samples of
varying salinities from standards and the wash solution
prepared in reagent water, it is common to first correct for
refractive index errors, and then correct for any change in
color development due to the differences in composition
between samples and standards (so called salt error).
12.3.2	Plot the salinity of the saline standards (Section
7.2.9) as the independent variable, and the apparent
concentration of analyte (mg N/L) from the peak height
(corrected for refractive index) calculated from the
regression of standards in reagent water, as the
dependent variable for all saline standards. The resulting
regression equation allows the operator to correct the
353.4-11
Revision 2.0 September 1997

-------
concentrations of samples of known salinity for the color
enhancement due to matrix effect, e.g., salt error.
Following are typical results for the nitrate and nitrite
systems:
27.5
18.6
18.6
18.6
18.6
0.0335 100.1
0.0167
0.0170
0.0229
0.0229
105.8
101.6
106.4
104.5
0.1052
0.0523
0.0534
0.0720
0.0719
Salinity
Apparent concentration (|jg N/L)




(%o)
Nitrate
Nitrite
9.4
0.0222
105.3
0.0698



9.4
0.0229
106.4
0.0720



9.4
0.0197
91.5
0.0620
0.0
569.64
558.15




3.8
570.50
565.50
0.0
0.0260
103.9
0.0817
9.2
572.74
563.00
0.0
0.0306
106.9
0.0961
13.8
568.96
564.94
0.0
0.0160
111.0
0.0501
18.1
566.44
563.00
0.0
0.0248
109.5
0.0780
26.8
558.74
559.06




36.3
559.86
554.67




12.3.3 As shown in above results, salinity has no
systematic effect on the nitrate and nitrite signal and
therefore salt error correction is not recommended.
12.4 Results of sample analyses should be reported
in mg N/L or in |jg N/L.
mg N/L = ppm (parts per million)
|jg N/L = ppb (part per billion)
13.0	Method Performance
13.1	Single Laboratory Validation
13.1.1 Method Detection Limit- A method detection limit
(MDL) of 0.075 |jg N/L has been determined by one
laboratory from LNSW of five different salinities fortified at
a nitrate concentration of 0.28 |jg N/L.
Salinity SD Recovery MDL
(%0) (Mg N/L) (%) (Mg N/L)
13.1.2 Single analyst precision - A single laboratory
analyzed three samples collected from the Miami River
and Biscayne Bay, Florida. Seven replicates of each
sample were processed and analyzed with salinity
ranging from 0.019 to 32.623%o. The results were as
follows:
Sample Salinity
(%o)
Concentration
(M9 N/L)
RSD
t°L\
Nitrate
1
32.623
48.22
2.59
2
13.263
206.41
1.07
3
0.019
276.38
1.99


Nitrite

1
32.623
5.21
1.62
2
13.263
31.03
0.58
3
0.019
54.07
0.49
36.5
0.0234
103.5
0.0734
36.5
0.0298
98.9
0.0935
36.5
0.0148
110.3
0.0464
36.5
0.0261
103.6
0.0819
27.5
0.0203
105.4
0.0638
27.5
0.0321
102.3
0.1009
27.5
0.0314
103.8
0.0986
13.1.3 Laboratory fortified sample matrix - Laboratory
fortified sample matrices were processed in three
different salinities ranging from 0.019 to 32.623 and
ambient nitrate concentrations from 48.22 to 276.38 |jg
N/L. Seven replicates of each sample were analyzed and
the results were as follows:
Revision 2.0 September 1997
353.4-12

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Salinity Concentration RSD Recovery
ambient fortified
(%0)	(Mg N/L)	(%) (%)
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in Section 14.2.
32.623 48.22 139.94 1.50 106.4
13.263 206.41 139.94 1.25 102.6
0.019 276.38 139.94 1.19 102.3
13.2 Multi-Laboratory Validation
Multi-laboratory data is unavailable at this time.
14.0	Pollution Prevention
14.1	Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the agency recommends recycling as the next
best option.
14.2	For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society, Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0	Waste Management
15.1	The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
16.0 References
1.	Morris, A. W. and Riley, J.P., 1963. Determination
of nitrate in sea water. Anal. Chim. Acta. 29:272-
279.
2.	Brewer P. G. and J. P. Riley 1965. The automatic
determination of nitrate in seawater. Deep-Sea
Res., 12:765-772.
3.	Wood, E.O., Armstrong, F.A.J., and Richards, F.A.,
1967. Determination of nitrate in seawater by
cadmium-copper reduction to nitrite. J. Mar. Biol.
Assn. U.K., 47:23-31.
4.	Bendschneider, K. and R. J. Robinson, 1952. A
new spectrophotometric method for the
determination of nitrite in sea water. J. Marine Res.,
11:87-96.
5.	Fox, J.B. 1979. Kinetics and mechanisms of the
Griess reaction. Analytical Chem. 51:1493-1502.
6.	Norwitz, G.,	P.N. Keliher,, 1984.
Spectrophotometric determination of nitrite with
composite reagents containing sulphanilamide,
sulphanilic acid or 4- nitroaniline as the diozotisable
aromatic amine and N-(1-
naphthyl)ethylenediamine as the coupling agent.
Analyst, 109:1281-1286.
7.	Spencer, C.P. 1975, The micronutrient elements.
In Chemical Oceanography (Riley, J. P. and G.
Skirrow, Eds.), Academic Press, London and New
York, 2nd Ed. Vol 2, Chapter 11.
8.	40 CFR, 136 Appendix B. Definition and Procedure
for the Determination of Method Detection Limit.
Revision 1.11.
9.	Timmer-ten Hoor, A., 1974. Sulfide interaction on
colorimetric nitrite determination. Marine Chemistry,
2:149-151.
353.4-13
Revision 2.0 September 1997

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10.	Alpkem Corporation. 1990. RFA Methodology:
Nitrate+Nitrite Nitrogen. Method A303-S170.
Alpkem Corporation, Clackamas, Oregon.
11.	Olson, R.J. 1980. Phosphate interference in the
cadmium reduction analysis of nitrate. Limnol.
Oceanogr., 25(4)758-760.
12.	Alvarez-Salgado, X.A., F.Fraga and F.F.Perez.
1992, Determination of nutrient salt by automatic
methods both in seawater and brackish water: the
phosphate blank. Marine Chemistry, 39:311-319.
13.	Grasshoff, K. 1983, Determination of Nitrate, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp143-150.
14.	Takenaka, N., A.Ueda and Y. Maeda 1992,
Acceleration of the rate of nitrite oxidation by
freezing in aqueous solution. Nature, Vol. 358,
p736-738.
15.	Grasshoff , K. 1983, Determination of Nitrite, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp139-142.
16.	MacDonald, R.W. and F.A. McLaughlin. 1982. The
effect of Storage by freezing on dissolved inorganic
phosphate, nitrate, and reactive silicate for samples
from coastal and estuarine waters. Water
Research, 16:95-104.
17.	Angelova, S, and H.W.Holy. 1983. Optimal speed
as a function of system performance for continuous
flow analyzers. Analytica Chimica Acta, 145:51-58.
18.	Zhang, J.-Z. 1997. Distinction and quantification of
carry-over and sample interaction in gas
segmented continuous flow analysis . Journal of
Automataic Chemistry, 19(6):205-212.
19.	Garside, C. 1993. Nitrate reductor efficiency as an
error source in seawater analysis. Marine
Chemistry 44: 25-30.
Revision 2.0 September 1997
353.4-14

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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Detector
540n
To Waste
Debubbler
/
To Wasted
Sulfanilamide
Sample
OTCR
Nitrogen
Buffer
Manifold
Wash To Sampler—
Pump
mL/min
Reagent Water
or Low Nutrient Seawater
Sample:Wash = 60":30"
Figure 1. Manifold configuration for nitrate + nitrite analysis using an Open Tubular Cadmium Reactor.
353.4-15	Revision 2.0 September 1997

-------
Detector
540n
Debubbler
;tor
o
\/
To Waste
Cd
Column
A
v
10 O-1\
( 8
VL—OH
L
To Waste~f
-G=
JZ_
0
0 5
4
3
~a
0
1
)
Manifold
\
<¦

<-
-j-
\
Wash To Samplei—(r
0.41
0.41
0.10
0.10
0.25
0.32
1.12
1.57
NED
Sulfanilamide
Nitrogen
Sample
Buffer
Reagent Water
or Low Nutrient Seawater
Pump
mL/min
Sample:Wash = 60":30"
Figure 2. Manifold configuration for nitrate + nitrite analysis using a homemade packed copper-coated cadmium
reduction column.
Revision 2.0 September 1997
353.4-16

-------
Detector
540n
Debubbler
:tor
¦0
\/
To Waste

To Wasted
"10 0
-o=J=
8
—-o
0 7
6
0
0 5
4
o
0 3
2 CH
:)
1
!)
Manifold
\
*¦
*
*
Wash To Samplei—(r
0.41
0.10
0.10
0.25
1.01
1.57
NED
Sulfanilamide
Air
Sample
Reagent Water
or Low Nutrient Seawater
Pump
mL/min
Sample:Wash = 60":30"
Figure 3. Manifold configuration for nitrite analysis.
353.4-17
Revision 2.0 September 1997

-------
Table 1 . Percentage recovery of nitrate from natural water samples preserved by freezing and refrigeration.
MethodA
Sample®
Salinity


Time (Day)








0
7
14
21
28
35
46
62
92
25C, P
river
0.019
100
192.5
279
287.3
267.5
262.4
300.7
228.1
260.8

estuary
13.263
100
108.5
106.2
124
103.9
139.3
258.9
188.5
229.1

coast
32.623
100
102
128.8
153.8
93.3
89
44.2
72.4
84.9
25C, G
river
0.019
100
257
294.9
316.4
298.2
225.4
135.4
77.6
66.9

estuary
13.263
100
108.8
108.5
122.5
90.6
79.2
81.5
56.2
128.2

coast
32.623
100
98
135.2
150.9
98.5
84.3
36.9
56.1
66.6
4C,P
river
0.019
100
105
90
111.6
100.7
82.7
112.2
97.3
104.7

estuary
13.263
100
104.5
90.4
107.1
102.6
95.9
109
82.4
101.4

coast
32.623
100
127.6
65.7
149.1
82.3
93.3
43.3
73.5
89.2
4C,G
river
0.019
100
158.2
88.1
108.4
99.4





estuary
13.263
100
103.1
84.5
107.4
95.9





coast
32.623
100
100.9
54.4
123
68.9




4C,P,
river+
0.019
100
105.5
99.2
106.1
96.2
91
114.8
98.4
96.9

estuary+
13.263
100
110.2
116.4
104.8
102.9
93
110.9
85
99.7

coast+
32.623
100
112.7
112.7
103.8
93.3
90.6
102.4
75.4
98.6
4C,G,
river+
0.019
100


105.7
98.3
101
114.5



estuary+
13.263
100


100.1
98
93.3
109.1



coast+
32.623
100


104.4
93.6
90.2
99.5


Fr,P
river
0.019
100
100.5
100.4
103.9
95.8
88.6

85.7
95.9

estuary
13.263
100
114.1
115.5
105.6
97.9
104.6
98.8
72.8
87.6

coast
32.623
100
130.5
100.9
128.2
92.7
98.5
42.2
50.9
87.5
Fr,P,
river+
0.019
100
101.9
103.2
103.1
95.4
91.2
82.5
87.4
90.2

estuary+
13.263
100
102
106.7
102.4
97.4
95
78.5
78
94.7

coast+
32.623
100
103.2
111.1
101.3
91.5
92.1
104.7
69.6
92.3
Revision 2.0 September 1997	353.4-18

-------
Table 2 . Percentage recovery of nitrite from natural water samples preserved by freezing and refrigeration
MethodA
Sample®
Salinity



Time(day)








0
7
14
21
28
35
46
62
92
25C, P
river
0.019
100
220
0.3
0
0
0
0
0
0

estuary
13.263
100
110.6
456.8
920.2
957.8
661.5
58.7
0
0

coast
32.623
100
104.1
92.2
74.1
89.5
74.1
94.6
72.2
0
25C, G
river
0.019
100
182.8
0.3
0
0
0
0
0
0

estuary
13.263
100
108.5
519.1
1026.3
1079.1
867.5
843.1
705.7
209.2

coast
32.623
100
100
87.8
73.8
89.5
73.5
95.9
85.7
66.5
4C,P
river
0.019
100
104.2
88.2
31.8
93.9
0
65
84.1
0

estuary
13.263
100
102.8
101.8
38.9
0
91
17.8
8.5
0

coast
32.623
100
68.4
65.7
33.2
70.5
50.5
0
0
0
4C,G
river
0.019
100
104.9
97.8
99.8
96.7





estuary
13.263
100
104.4
98.8
100.6
91





coast
32.623
100
94.3
87
71.1
97.6




4C,P
river+
0.019
100
47.6
98.9
98.5
97.2
67.8
0
2.2
75.0

estuary+
13.263
100
95.4
21.1
0
0
0
2.7
0
0

coast+
32.263
100
0
0
0
0
0
0
0
0
4C,G
river+
0.019
100


97.9
95.8
84.6
85.9



estuary+
13.263
100


100.6
91.6
94.1
100



coast+
32.623
100


69.5
97.6
65.9
87.6


Fr,P
river
0.019
100
70.6
86.2
98
77.3
68.1

74.9
77.3

estuary
13.263
100
1.3
0.7
0
0
0
96
13.3
57.3

coast
32.623
100
78.6
4.9
0
0
0
8.6
80
27.8
Fr,P
river+
0.019
100
97
87.2
95.4
75.9
75.9
63.1
75.2
69.2

estuary+
13.263
100
103.5
98.6
95.9
52
90.5
74.2
0
77.6

coast+
32.623
100
99.7
95.9
56.5
92.2
67
100.5
80
65.9
Cont'd on
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A Methods of preservation:
25C,P and G: Store the samples in high density polyethylene carboys (P) or glass bottles (G)
at room temperature (~25°C).
4C, P and G: Store samples in high density polyethylene bottles (P) or glass bottles (G) in a
refrigerator (4°C) in the dark.
Fr,P and Fr,P: Freeze the samples in high density polyethylene bottles (P) and store at -20°C
in a freezer in the dark.
Glass and high density polyethylene bottles were used to study the effect of type of sample
bottles on the recovery of nitrite and nitrate from refrigeration.
B For salinity and concentration of nitrate in river, estuary and coast samples see section 13.1.2.
Sample river+, estuary+ and coast+ are the fortified river, estuary and coast samples,
respectively, at nitrate concentrations 139.94 |ig N/L.
Revision 2.0 September 1997
353.4-20

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