353.4-1

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
Revision 2.0 September 1997 353.4-2

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, and the 1-3 

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. Nitrate in

7

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 0.01mg 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) of 0.075 μg N/L has been determined by one

8

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.
 353.4-3 Revision 2.0 September 1997

3.3 Laboratory Fortified Sample Matrix (LFM) - An 3.10 Primary Dilution Standard Solution (PDS) - A

aliquot of an environmental sample to which known solution prepared in the laboratory from stock standard 

quantities of the method analytes are added in the solutions and diluted as needed to prepare calibration

laboratory. The LFM is analyzed exactly like a sample, solutions and other needed analyte solutions.

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background 3.11 Quality Control Sample (QCS) - A solution of

concentrations of the analytes in the sample matrix must method analytes of known concentrations which is used

be determined in a separate aliquot and the measured to fortify an aliquot of LRB or sample matrix. The QCS is

values in the LFM corrected for background obtained from a source external to the laboratory and

concentrations. different from the source of calibration standards. It is

3.4 Laboratory Reagent Blank (LRB) - An aliquot prepared test materials.

of reagent water that is treated exactly as a sample

including exposure to all labware, equipment, and 3.12 SYNC Peak Solution - A colored solution used 

reagents that are used with other samples. The LRB is to produce a synchronization peak in the refractive index

used to determine if method analytes or other measurement. A synchronization peak is required by the

interferences are present in the laboratory environment, data acquisition programs to initialize the peak finding

the reagents, or apparatus. parameters. The first cup in every run must always be

3.5 Linear Dynamic Range (LDR) - The absolute usually a high standard, but can be any sample that

quantity or concentration range over which the instrument generates a peak at least 25% of full scale. 

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 (RI) - 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.

used to check laboratory performance with externally

identified as a SYNC sample. The SYNC sample is

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 . Hydrogen sulfide 9

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 cadmium .

11

Dilute samples if possible or remove phosphate with ferric

hydroxide prior to analysis. 12

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.
Revision 2.0 September 1997 353.4-4

However, the individual who collects samples should use 6.2.4 60 mL high density polyethylene sample bottles,

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 coppercoated 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% HCl (v/v) and thoroughly

rinsing with reagent water is sufficient.

6.2.2 Automatic pipetters capable of delivering volumes

ranging from 100 μL to 1000 μL and 1mL 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.

glass volumetric flasks and plastic sample tubes.

6.2.5 Drying oven.

6.2.6 Desiccator.

6.2.7 Membrane filters with 0.45 μm 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 (C6H8N2O2S, FW 172.21) in 1 L of 10%

HCl. 

7.1.2 Stock Nitrate Solution (100 mg-N/L) -

Quantitatively transfer 0.7217 g of pre-dried (105 C for 1 o

hour) potassium nitrate (KNO , FW 101.099) to a 1000- 3 

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 o

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 o

hour) sodium nitrite (NaNO2 , 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 stock solution in a

polyethylene bottle in a refrigerator at 4

oC. This solution

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 μg 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 μg N/L) with salinity of 35 %o (Ocean Scientific

International, Wormley, U.K. ) can be substituted.
 353.4-5 Revision 2.0 September 1997

7.2 Working Reagents 7.2.8 Calibration Standards - Prepare a series of

7.2.1 Brij-35 Start-up Solution - Add 2 mL of Brij-35

surfactant (ICI Americas, Inc.) to 1000 mL reagent water 100 mL with reagent water or low nutrient seawater.

and mix gently. Prepare these standards daily. The concentration range

Note: Brij-35 is a trade name for polyoxyethylene(23) concentrations of samples and not exceed two orders of

lauryl ether (C H (OCH CH ) OH, FW=1199.57, CASRN magnitude. At least five calibration standards with equal 12 25 2 2 23

9002-92-0). increments in concentration should be used to construct

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, C H N .2HCl, 12 14 2

FW 259.18) in 1 L of reagent water. 

7.2.4 Imidazole Buffer Solution - Dissolve 13.6 g of

imidazole (C H N , FW 68.08) in 4 L of reagent water. 3 4 2

Add 2 mL of concentrated HCl. Adjust the pH to 7.8 with

diluted HCl 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 (CuSO .5H O, FW 249.61) in 1 L of 4 2

reagent water. 

7.2.6 Colored SYNC Peak Solution - Add 50 μL 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. 

calibration standards (CAL) by diluting suitable volumes

of a primary dilution standard solution (Section 7.2.7) to

of calibration standards should bracket the expected

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 Conc.

(%o) 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.
Revision 2.0 September 1997 353.4-6

7.3.2 If an OTCR is employed, the following procedures Connect both ends of the column using a plastic tube

should be used to activate it.10

Prepare reagent water, 0.5N HCl solution and 2% CuSO4

solution in three 50 mL beakers. Fit three 10-mL plastic not been used for several days, it should be reactivated

syringes with unions. First flush the OTCR with 10 mL prior to sample analysis.

reagent water. Then flush it with 10 mL 0.5N HCl

solution in 3 seconds, immediately followed by flushing 7.5 Stabilization of OTCR and Packed Cadmium

with a couple of syringe volumes of reagent water. Slowly Reduction Columns

flush with CuSO4 solution until a large amount of black

precipitated copper come out of OTCR, then stop the 7.5.1 Pump the buffer and other reagent solutions

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% HCl followed with

reagent water.

7.4.4 Decant the reagent water and add 50 mL of 2%

CuSO solution. While swirling, brown flakes of colloidal 4

copper will appear and the blue color of the solution will 7.5.5 The reduction efficiency of an OTCR or a packed

fade. Decant the faded solution and add fresh CuSO4

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 Absorbance of Nitrate 

particles. Keep the filings submersed under reagent water Reduction Efficiency = 

and avoid exposure of the cadmium filings to air. Absorbance of Nitrite 

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.

filled with buffer solution to form a closed loop.

7.4.8 If an OTCR or a packed cadmium column has

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.

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:

______________________________

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
 353.4-7 Revision 2.0 September 1997

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 o

be performed.

8.3 Sample Storage - Natural samples usually

contain low concentrations of nitrite (< 14 g N/L) and no

preservation techniques are satisfactory. Samples must 14 

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
Revision 2.0 September 1997 353.4-8

responses by multiplying the response by the Absorbance The optional control limits must be equal to or better than

Units Full Scale output range setting. Perform the linear the required control limits of 90-110%. After each 5 to 10

regression of normalized response vs. concentration and new recovery measurements, new control limits can be

obtain the constants m and b, where m is the slope and calculated using only the most recent 20 to 30 data

b is the y-intercept. Incrementally analyze standards of points. Also the standard deviation (S) data should be

higher concentration until the measured absorbance used to establish an ongoing precision statement for the

response, R, of a standard no longer yields a calculated level of concentrations included in the LFB. These data

concentration C , that is within 100 ± 10% of known must be kept on file and be available for review. c

concentration, C, where C = (R-b)/m. That concentration c 

defines the upper limit of the LDR for the instrument. 9.4 Assessing Analyte Recovery -

Should samples be encountered that have a Laboratory Fortified Sample Matrix

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

(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:

 (C - C) s

R = x 100 ____________

 S

where,

R = percent recovery

C = measured fortified sample concentration s

 (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.
 353.4-9 Revision 2.0 September 1997

10.0 Calibration and Standardization

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 carryover coefficient, k, is calculated as follows:

 Pb1 - Pb2

k = __________________

 Phigh

where,

P = the peak height of the high 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,

P . i-1

CO = (k)(P ) i-1

To correct a given peak height reading, Pi, subtract the

carry over correction,17,18

Pi,c = Pi - CO

where P is corrected peak height. The correction for i,c

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
Revision 2.0 September 1997 353.4-10

interference at low level analysis. Packed cadmium stabilization. Ensure reduction efficiencies reach at least

columns are, therefore, preferred for nitrate analysis at 90% before analysis of samples.

low concentrations.

11.4 Set spectrophotometer wavelength at 540 nm. per hour for 60 second sample times and 30 second

11.5 Set the Absorbance Unit Full Scale (AUFS) range

on the spectrophotometer at an appropriate setting 11.10 Use cleaned sample cups or tubes (follow the

according to the highest concentration of nitrate in the procedures outlined in Section 6.2.2). Place CAL

samples. The appropriate setting for this method is 0.2 solutions and saline standards (optional) in sampler.

AUFS for 0.7 mg N/L. Complete filling the sampler tray with samples, laboratory

11.6 Prepare all reagents and standards. fortified sample matrices, and QC samples. Place a blank

11.7 Begin pumping the Brij-35 start-up solution (Section

7.2.1) through the system and obtain a steady baseline. 11.11 Commence analysis.

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 HCl 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 

19

11.9 A good sampling rate is approximately 40 samples

wash times.

reagent blanks, laboratory fortified blanks, laboratory

after every ten samples.

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.
 353.4-11 Revision 2.0 September 1997

Note: In nitrate and nitrite analysis absorbance of the _______________________________________

reagent water is higher than that of the LNSW. When Salinity Apparent concentration (μg N/L)

using reagent water as a wash solution, the change in (% ) Nitrate Nitrite

refractive index causes the absorbance of seawater to _______________________________________ 

become negative. To measure the absorbance due to

refractive index change in different salinity samples, Low 0.0 0.000 0.000

Nutrient Seawater must be used as a wash solution to 3.8 0.026 0.015

bring the baseline down. 9.2 0.096 0.040

12.2.4 Replace NED solution (Section 7.2.4) with 18.1 0.190 0.086

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:

o

13.8 0.142 0.055

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 (μg N/L) = 0.01047S

Apparent nitrite (μg 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 μg 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
Revision 2.0 September 1997 353.4-12

concentrations of samples of known salinity for the color 27.5 0.0335 100.1 0.1052

enhancement due to matrix effect, e.g., salt error.

Following are typical results for the nitrate and nitrite 18.6 0.0167 105.8 0.0523

systems: 18.6 0.0170 101.6 0.0534

_______________________________________ 18.6 0.0229 104.5 0.0719

Salinity Apparent concentration (μg N/L)

(%o) Nitrate Nitrite 9.4 0.0222 105.3 0.0698

_______________________________________ 9.4 0.0229 106.4 0.0720

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 and Biscayne Bay, Florida. Seven replicates of each

therefore salt error correction is not recommended. sample were processed and analyzed with salinity

12.4 Results of sample analyses should be reported follows:

in mg N/L or in μg N/L.

mg N/L = ppm (parts per million)

 μg N/L = ppb (part per billion) Sample Salinity Concentration RSD

13.0 Method Performance

13.1 Single Laboratory Validation

13.1.1 Method Detection Limit- A method detection limit

(MDL) of 0.075 μg N/L has been determined by one

laboratory from LNSW of five different salinities fortified at

a nitrate concentration of 0.28 μg N/L.

____________________________________

Salinity SD Recovery MDL

(% ) (μg N/L) (%) (μg N/L) o

_____________________________________

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

18.6 0.0229 106.4 0.0720

9.4 0.0197 91.5 0.0620

13.1.2 Single analyst precision - A single laboratory

analyzed three samples collected from the Miami River

ranging from 0.019 to 32.623‰. The results were as

______________________________________

 (% ) (μg N/L) (%) o

______________________________________

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

______________________________________

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 μg

N/L. Seven replicates of each sample were analyzed and

the results were as follows:
 353.4-13 Revision 2.0 September 1997

_______________________________________ waste identification rules and land disposal restrictions.

Salinity Concentration RSD Recovery

 ambient fortified Personnel, available from the American Chemical Society

(% ) (μg N/L) (%) (%) o

______________________________________

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 naphthyl)ethylenediamine as the coupling agent.

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

For further information on waste management consult

The Waste Management Manual for Laboratory

at the address listed in Section 14.2.

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-

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.
Revision 2.0 September 1997 353.4-14

10. Alpkem Corporation. 1990. RFA Methodology: 18. Zhang, J.-Z. 1997. Distinction and quantification of

Nitrate+Nitrite Nitrogen. Method A303-S170. carry-over and sample interaction in gas

Alpkem Corporation, Clackamas, Oregon. segmented continuous flow analysis . Journal of

11. Olson, R.J. 1980. Phosphate interference in the

cadmium reduction analysis of nitrate. Limnol. 19. Garside, C. 1993. Nitrate reductor efficiency as an

Oceanogr., 25(4)758-760. error source in seawater analysis. Marine

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.

Automataic Chemistry, 19(6):205-212.

Chemistry 44: 25-30.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold

Sample

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 60":30"

540nm

1.12 Buffer

Nitrogen

0.32

0.10 Sulfanilamide

0.10 NED

8

0.41

OTCR

 353.4-15 Revision 2.0 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Figure 1. Manifold configuration for nitrate + nitrite analysis using an Open Tubular Cadmium Reactor.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold

Sample

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 60":30"

540nm

1.12 Buffer

Nitrogen

0.32

0.10 Sulfanilamide

0.10 NED

8

0.41

Cd

Column

Revision 2.0 September 1997 353.4-16

Figure 2. Manifold configuration for nitrate + nitrite analysis using a homemade packed copper-coated cadmium

reduction column.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 60":30"

540nm

1.01 Sample

Air

0.10 Sulfanilamide

0.10 NED

8

 353.4-17 Revision 2.0 September 1997

Figure 3. Manifold configuration for nitrite analysis.
Revision 2.0 September 1997 353.4-18

Table 1 . Percentage recovery of nitrate from natural water samples preserved by freezing and refrigeration.

Method Sample Salinity Time (Day) A B

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
 353.4-19 Revision 2.0 September 1997

Table 2 . Percentage recovery of nitrite from natural water samples preserved by freezing and refrigeration

Method Sample Salinity Time(day) A B

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

 next page
Revision 2.0 September 1997 353.4-20

Cont’d

Methods of preservation: A

25C,P and G: Store the samples in high density polyethylene carboys (P) or glass bottles (G)

at room temperature (~25 C). o

4C, P and G: Store samples in high density polyethylene bottles (P) or glass bottles (G) in a 

refrigerator (4 C) in the dark. o

Fr,P and Fr,P: Freeze the samples in high density polyethylene bottles (P) and store at -20 C

o

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.

For salinity and concentration of nitrate in river, estuary and coast samples see section 13.1.2. B

Sample river+, estuary+ and coast+ are the fortified river, estuary and coast samples,

 respectively, at nitrate concentrations 139.94 μg N/L.