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Method for Nitrate Reductase Nitrate-Nitrogen Analysis

1. Scope and Application

1.1 This test method is applicable to the determination of nitrate plus nitrite (as nitrogen) in 

surface water, saltwater (saline), ground water, wastewater, and any aqueous solution 

containing nitrate.

1.2 The standard range of this test method is from 0.05 to 5 mg/L of nitrate-nitrogen. 

Samples with greater than 5 mg N/L may be diluted with deionized water (nitrate-free) 

to bring them into the range of the test method. In addition, for samples with nitrate-N 

content lower than 0.05 mg N/L, the sample volume used in the test may be increased 

for a low range test method from 0.01 to 1.0 mg N/L.

1.3 Quality Control and Quality Assurance (QC/QA) procedures and criteria are 

delineated. .

1.4 This description of the test method does not purport to address all safety concerns 

associated with its use. It is the responsibility of the user of this test method to 

establish appropriate safety and health practices and determine the applicability of 

regulatory limitations prior to use.

2. Summary of Method

2.1 Reduction of nitrate in the sample is accomplished by using eukaryotic nitrate 

reductase (EC 1.1.7.1-3; NaR) to catalyze the conversion of nitrate to nitrite in the 

presence of NADH or NADPH as reductant, in a biochemical buffer with a pH near 

neutrality (Campbell et al., 2006).

2.2 The combined nitrite (both original nitrite and that generated by reduction of nitrate to 

nitrite) is reacted with sulfanilamide and subsequent coupling with 

N-(1-napthyl)ethylenediamine dihydrochloride to yield a pink color. The absorbance 

of the chromophore is measured at ~540 nm in a spectrophotometer.

2.3 Calibrants of certified nitrate standard are treated with the reduction step and colorizing 

reaction to yield a standard curve which is used to determine the nitrate-N content of 

samples, expressed as mg/L nitrate N. This step insures that NaR is functioning.

2.4 Nitrite content of the sample (expressed as mg/L nitrite-N) can be determined by 

omitting the reduction step and directly reacting the sample with the color dyes.

2.5 Finally, nitrate-N content can be calculated by subtracting the nitrite content (mg/L 

nitrite N) from the combined nitrate plus nitrite (mg/L nitrate+nitrite N), to yield the 

nitrate content (expressed as mg/L nitrate N).
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3. Definitions

3.1 Nitrate Reductase (NaR): NADH:NaR (EC1.7.1.1 and CAS 9013-03-0) with 1 unit of 

enzyme activity defined as 1 μmol nitrite produced per minute at 30°C and pH 7 with 

NADH (refer to section 3.4) as electron donor. See Note following Section 7.6 for 

information on the type of NaR acceptable for this method.

3.2 Discrete Analyzer: a programmable, computer-controlled instrument that automates 

wet chemical analysis by using one or more robotic arms interfaced to high-precision 

volumetric dispensers to aspirate and dispense samples, standards, diluents and 

reagents. 

3.3 Greiss Reaction: chemical formation of an azo-dye by diazotization of nitrite ion with 

sulfanilamide (CAS 63-74-1), at an acidic pH, and subsequent coupling with 

N-(1-naphthyl)ethylenediamine hydrochloride (CAS 1465-25-4). 

3.4 NADH: β-Nicotinamide adenine dinucleotide, reduced form (CAS 53-54-9), is a 

coenzyme found in all living cells.

3.5 EDTA: Disodium ethylenediaminetetraacetate dihydrate (CAS 60-00-4, a heavy metal 

chelator.

4. Interferences

4.1 Turbid samples should be filtered prior to analysis to eliminate particulate interference. 

4.2 Sample color that absorbs at wavelengths between 520 and 560 nm interferes with the 

absorbance measurements. When color is suspect, analyze a sample blank, omitting the 

N-(1-naphthyl)ethylenediamine dihydrochloride from the color reagent. Note 1: The 

instrumentation described in this test method may automatically correct for some 

turbidity and sample color.

4.3 Certain ions may cause interferences. See Table 1. However, the inclusion of EDTA in 

the system’s phosphate buffer (section 3.6 and 7.1) overcomes most, if not all, of these 

heavy metal interferences.

4.4 Comprehensive studies of this method, which include analysis of interferences, have

been published (Patton and Kryskalla, 2011; 2013).
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5. Safety

5.1 General laboratory safe practices should be used in handling all samples and reagents 

in this test method.

5.2 When preparing the sulfanilamide color reagent in hydrochloric acid (section 7.4), the 

user should handle the concentrated hydrochloric acid with extra caution and wear 

heavy gloves and eye protection. The prepared sulfanilamide color reagent should also 

be handled with caution since it is ~3 N hydrochloric acid.

5.3 Potassium nitrite is toxic if swallowed, and laboratory tests suggest that it may be 

mutagenic or teratogenic. Gloves and safety glasses are used when handling potassium 

nitrite.

5.4 Potassium nitrate, unlike potassium nitrite, is not toxic to humans, unless one is 

chronically exposed to nitrate such as in your drinking water or food. Potassium nitrate 

will not explode on its own. However, it is recommended that standard laboratory 

safety practices such as the use of safety glasses and gloves be used when handling it.

6. Equipment and Supplies

6.1 Automated Discrete Analysis System (see section 3.2).

7. Reagents and Standards

7.1 Phosphate Buffer Solution: Dissolve 3.75 g of potassium dihydrogen phosphate 

(KH2PO4; CAS 7778-77-0), 0.01 g of disodium ethylenediaminetetraacetate dihydrate 

(C10H14O8N2Na2•2H2O), and 1.4 g potassium hydroxide (KOH; CAS 1310-58-3) in 

about 500 mL reagent water contained in a 1000 mL volumetric flask, dilute to the 

mark and mix. Transfer this solution to a screw-cap container and store at 4°C. This 

solution is stable for 6 months.

7.2 β-nicotinamide adenine dinucleotide, reduced form (NADH) stock solution (2mg/mL):

dissolve 0.1 g NADH in 25 mL of reagent water contained in a 50 mL volumetric 

flask, dilute to the mark and mix. Transfer 1-mL aliquots to 1.5 mL snap-cap colorless 

polypropylene vials and store at -20°C. Stable for 1 month. Note: NADH is a 

hygroscopic white powder that is freely soluble in water. The solids are stable if stored 

dry and protected from light. Neutral solutions are colorless and stable for 1 week if 

stored at 4oC, but decompose rapidly under basic or acidic conditions.

7.3 NADH working solution: thaw one 1-mL vial of NADH stock (refer to section 7.2) and 

dilute to 10 mL with phosphate buffer (refer to section 7.1). This reagent is stable for 

about 8 hours. Prepare sufficient NADH working solution for the number of samples 
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and standards to be analyzed. NOTE: NADH inhibits color formation in the Greiss 

reaction (refer to section 3.3). The molar concentration of NADH in the reduction 

medium should be about twice that of the highest calibration standard.

7.4 Sulfanilamide (SAN) Reagent (10g/L): While stirring constantly add 300 mL of 

concentrated hydrochloric acid (HCl, 37% w/v)) and 10 g of sulfanilamide to about 

500 mL reagent water contained in a 1000 mL volumetric flask, dilute to the mark and 

mix. This solution is stable for about six months when stored in a brown bottle at 20°C.

7.5 N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution (1g/L): dissolve 1 g 

NED in about 500 mL reagent water contained in a 1000 mL volumetric flask, dilute to 

the mark and mix. Transfer to a glass or amber screw-cap container. This solution is 

stable for 6 months at 20°C.

7.6 Nitrate Reductase (NaR): follow the manufacturer’s instructions for preparing a 

solution of 1 unit NaR (refer to section 3.1) activity per mL of phosphate buffer (refer 

to section 10.1). Dilute 3 units NaR to 20 mL with phosphate buffer. Store the solution 

at 2-6°C, where it is stable for 8 hours. Prepare sufficient NaR for the total number of 

samples and standards to be analyzed. NOTE: For some NaR forms, high phenolic 

content humic substances ( >2 mg dissolved organic carbon /L) have little effect on the 

NaR activity in the temperature range of 5-15°C, but become increasingly inhibitory in 

the temperature range of 20-40°C (Patton and Kryskalla, 2011; 2013) . Humic 

substances at the operation temperatures specified in this test method do not inhibit 

other forms of NaR, such as recombinant AtNaR2 (Campbell et al., 2006). If humic 

acids are expected to be present, the user must verify reduction efficiency of the NaR in

use by analysis of Quality Control checks that approximate the sample matrix.

7.7 Nitrate Solution, Stock (1000 mg/L NO3-N): Dry potassium nitrate (KNO3) in an oven 

at 105°C for 24 h. Dissolve 7.218 g in water in about 500 mL reagent water contained 

in a 1000 mL volumetric flask, dilute to the mark and mix. This solution is stable for 

up to 2 months with refrigeration. Alternatively, certified nitrate stock solutions are 

commercially available through chemical supply vendors and may be used.

7.8 Nitrate Solution, Standard (10 mg/L NO3-N): Dilute 10 mL of stock nitrate solution 

(7.7) to 1 L with Deionized water and store in a dark bottle. Prepare fresh as needed. 

7.9 Nitrite Solution, Stock (1000mg/L NO2-N): Place about 7 g of potassium nitrite 

(KNO2) in a tared 125-mL beaker and dry for about 24 h to a constant weight in a 

desiccator containing a suitable desiccant. Adjust the weight of the dry potassium 

nitrite to 6.072 g. Add 50 mL of water to the beaker, stir until dissolved, and transfer 

quantitatively to a 1000-mL volumetric flask. Dilute to the mark with deionized water 

and store in a sterilized bottle under refrigeration. Prepare fresh as needed. 

Alternatively, certified nitrite stock solutions are commercially available through 

chemical supply vendors and may be used. NOTE: Potassium nitrite is easily oxidized; 

use only dry, free flowing white, or yellowish white crystalline powder of this reagent.
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7.10 Nitrite Solution, Standard (10 mg/L NO2-N): Dilute 10 mL of stock nitrite solution 

(7.9) to 1 L with water. This solution is unstable; prepare fresh as needed.

8. Sample Collection, Preservation, and Storage

.

8.1 Sample preservation and holding time requirements for wastewater samples or other 

samples to be used for compliance monitoring under the Clean Water Act.

8.1.1 For nitrate: Chill the sample to ≤6 C and analyze within 48 hours of collection.

8.1.2 For nitrite: Chill the sample to ≤6 C and analyze within 48 hours of collection.

8.1.3 For nitrate-nitrite: Acidify to pH <2 with H2SO4 at the time of collection, and 

analyze within 28 days of collection.

8.2. In all cases, samples should be analyzed as soon as possible after collection.

9. Quality Control (QC)

9.1 To be certain that analytical values obtained using the test method are valid and 

accurate within the confidence limits of the test, the following QC procedures must be 

followed when analyzing nitrite-nitrate in water.

9.2 Initial Demonstration of Performance (IDP): IDP is used to characterize instrument 

performance and laboratory performance prior to performing analyses by this test 

method and periodically as specified below. The laboratory must complete the IDP 

tests and demonstrate compliance with the acceptance standards before beginning to 

analyze samples.

9.2.1 Linear Calibration Range (LCR): LCR must be determined initially and 

verified every six months or whenever a significant change in the instrument response 

is expected or observed. The initial demonstration of linearity must employ a number 

of standards sufficient to insure that the results are linear. Verification of linearity 

must use a minimum of a blank and three standards. If any verification data exceeds 

the nominal value of the standard by ±10%, linearity must be reestablished. If any 

portion of the linearity test range is found to be nonlinear, sufficient standards must be 

used to clearly define the linear range.

9.2.2 Quality Control Sample (QCS): QCS is a standard of known concentration 

from an external source and different from calibration standards. QCS must be 

analyzed initially and quarterly or as required to meet QC requirements. If the QCS 

analysis result is not within ±10% of the known concentration, the test has failed. The 

source of the problem must be identified and corrected before continuing with IDP.
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9.2.3 Method Detection Limit (MDL): The MDL must be established for the 

analyte using reagent water (blank) fortified at a concentration near the detection limit 

of the instrument. Perform the MDL study according to “40 CFR Part 136 Appendix 

B”. Samples should be prepared using reagent water fortified with nitrate at a 

concentration between 1 to 5 times the expected MDL. First, calibrate the instrument 

(see section 9.3). MDL is determined by analyses of seven replicates of fortified 

reagent water (spiked) and processed through the entire analytical method. Calculate 

the replicates analyte concentration in system units using the instrument calibration 

(see Section 10). Calculate MDL as follows:

MDL = (t) x (S)

Where, 

t = Student’s t value for a 99% confidence level and a standard deviation estimate 

with n-1 degrees of freedom (t = 3.143 for seven replicates) and

S = standard deviation of the seven replicate analyses.

If the concentration of the sample (spike) used to determine the MDL is greater than 

5X the calculated MDL repeat the MDL determination at a lower concentration for the 

sample (spike) studied. MDL should be determined every six months, when a new 

operator begins work, or whenever there is a significant change in the background or 

instrument response.

9.3 Calibration and Calibration Verification: Prior to the analysis of samples, calibrate 

the instrument using at least four working standards containing concentrations of 

nitrate in water that bracket the expected sample concentration. Before each analysis, 

verify the calibration with a mid-range second source calibrant (SSC) from an external 

source and different from calibration standards. The result should fall within ± 10 % of 

the known concentration. If the SSC is not within ± 10%, determine the problem, and 

recalibrate the instrument.

9.4 Initial Performance and Recovery (IPR): If a laboratory has not performed this test

method before, or if there has been a major change in the measurement system, for 

example, new analyst, new instrument, and so forth, a precision and bias study must be 

performed to demonstrate laboratory capability. Analyze four replicates of a standard 

solution prepared from a Certified Independent Reference Material (CRM) containing a 

mid-range concentration of nitrite-nitrate in water. The matrix and chemistry of the 

solution should be equivalent to the solution used in the collaborative study. Each 

replicate must be taken through the complete analytical test method including any 

sample preservation and pretreatment steps. Calculate the mean and standard deviation 

of the four values and compare to the acceptable ranges provided by the manufacturer 

of the CRM. Do not use this test method to analyze samples unless the IPR recoveries 

are within the accepted limits.
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9.5 Ongoing Performance and Recovery Sample (OPR): To ensure that the test method 

is in control, analyze a OPR containing a mid-range concentration of nitrite-nitrate in 

water with each batch or 10 samples. If large numbers of samples are analyzed in the 

batch, analyze the OPR after every 10 samples. The OPR must be taken through all of 

the steps of the analytical method including sample preservation and pretreatment. The 

result obtained for the OPR shall fall within ± 10% of the known concentration. If the 

result is not within these limits, analysis of samples is halted until the problem is 

corrected, and either all the samples in the batch must be reanalyzed, or the results 

must be qualified with an indication that they do not fall within the performance 

criteria of the test method.

9.6 Method Blank: Analyze a reagent water test blank with each batch or 10 samples. 

The concentration of nitrite-nitrate in water found in the blank should be less than the 

MDL. If the concentration of nitrite-nitrate in water is found above this level, analysis 

of samples is halted until the contamination is eliminated, and a blank shows no 

contamination at or above this level, or the results must be qualified with an indication 

that they do not fall within the performance criteria of the test method. 

9.7 Matrix Spike (MS) and Matrix Spike Duplicate (MSD): To check for interferences 

in the specific matrix being tested, perform MS and MSD on at least one sample from 

each batch by spiking an aliquot of the sample with a known concentration of nitritenitrate in water and taking it through the analytical method. The spike concentration 

plus the background concentration of nitrite-nitrate in water must not exceed the high 

calibration standard. The spike must produce a concentration in the spiked sample that 

is 2 to 5 times the nitrite-nitrate in water concentration in the unspiked sample, or 10 to 

50 times the detection limit of the test method, whichever is greater.

Calculate the percent recovery of the spike (P) using the following formula:

[ ( ) ( )] 100 A V V B V s s P

C V

   

 



In this equation, A = nitrite+nitrate concentration (mg/L) in spiked samples, 

B = nitrite+nitrate concentration (mg/L) in unspiked samples, 

C = nitrite+nitrate concentration (mg/L) in the spiking solution, Vs = sample volume 

(mL) used, and V = added spiking solution volume (mL).

Calculate the relative percent difference (RPD) between two sample results using the 

following equation:

 x 100

( 1 2 ) / 2

1 2

D D

D D

RPD







Where, D1 = Concentration of analyte in the MS, D2 = Concentration of analyte in 

the MSD.
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The percent recovery of the spike shall fall within 90-110% and the RPD shall be ≤ 20 

%, based on the nitrite-nitrate in water concentration. If the percent recovery and RPD

are not within these limits, a matrix interference may be present in the sample selected 

for spiking. Under these circumstances, one of the following remedies must be 

employed: the matrix interference must be removed, all samples in the batch must be 

analyzed by a test method not affected by the matrix interference, or the results must be 

qualified with an indication that they do not fall within the performance criteria of the 

test method. 

9.8 Independent Reference Material (IRM): In order to verify the quantitative value 

produced by the test method, analyze an Independent Reference Material (IRM) 

submitted as a regular sample (if practical) to the laboratory at least once per quarter. 

The concentration of the IRM should be in the concentration mid-range for the method 

chosen. The value obtained must fall within the control limits established by the 

laboratory.

10. Calibration and Standardization

10.1 Using the standard nitrate solution (section 7.8) prepare calibration standards by using 

the automated calibration function of the discrete analyzer (section 3.2). Table 2 

specifies suggested calibrants. NOTE: Most discrete analyzers generate calibration 

standards and calibration curves automatically. Follow the manufacturer’s instructions 

for calibrating with individual calibration standards, if an automatic calibration 

function is not available.

10.2 Prepare at least one calibration standard from the standard nitrite solution (section 

7.10) at the same concentration as one of the nitrate standards to verify the efficiency 

of the reduction. Verify that reduction efficiency is greater than 90% with each batch of 

enzyme. See Table 3 for an example of Reduction Efficiency Analysis.

10.3 Run the calibration analysis as described in section 11 for sample analysis.

10.4 Prepare a standard curve by plotting the absorbance of each processed calibration 

standard against its known concentrations. NOTE: Most discrete analyzers generate 

calibration curves automatically.

11. Procedure

11.1 Removal of Color Interferences: If there is a possibility that the color of the sample 

might absorb in the photometric range from 540 ± 20 nm, determine the background 

absorbance. NOTE: Many discrete analyzers automatically compensate for 

background absorbance and turbidity on each sample. Follow the manufacturer’s 

instructions.
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11.2 Prepare a method in the discrete analyzer software following these specifications:

11.2.1 Dispense 55 μL of NaR (section 7.6) and 5 μL of sample. Mix.

NOTE: Larger volumes of samples and reagents may be used while 

maintaining the same ratio (See Table 4).

11.2.2 Add 12 μL of NADH (section 7.3). Mix and measure the background 

absorbance.

11.2.3 Incubate 600 seconds at 37oC.

11.2.4 Add 25 μL of SAN reagent (section 7.4). Mix and incubate 120 seconds at 

37oC.

11.2.5 Add 25 μL of NED reagent (section 7.5). Mix and incubate 120 seconds at 

37oC.

11.2.6 Measure absorbance at 540 nm, using an optional background subtraction at 

700 nm. If automatic background subtraction is not available, prepare a 

reagent blank (with SAN only added) for background subtraction at 540 nm.

11.3 When determining nitrite alone, replace NaR reagent (section 7.6) with Phosphate 

Buffer (section 7.1).

12. Data Analysis and Calculations

12.1 Determine the concentration of nitrate or nitrite nitrogen in the samples in mg N/L 

using the computer based data handler provided with the automated discrete analyzer 

software. NOTE: The discrete analyzer will automatically calculate the net absorbance 

by subtracting the background absorbance from the measured absorbance of the color 

developed sample. Use the net absorbance to determine the concentration of nitrogen in 

the sample.

12.2 Where separate values are required for nitrite-nitrogen and nitrate-nitrogen, calculate 

the nitrate-nitrogen by subtracting the nitrite-nitrogen from the total nitrate-nitrite 

nitrogen content.

12.3 Report the nitrogen content in mg N/L as:

Nitrite-Nitrogen (NO2-N), mg N/L;

Nitrate-Nitrogen (NO3-N), mg N/L; and 

Combined Nitrate-Nitrite Nitrogen (NO3, NO2-N), mg N/L.

13. Method Performance

13.1 A comparison of results by this method and cadmium reduction method (EPA 353.2) is 

shown in Table 5A and Table 5B. Also Figure 1 presents a graphic showing the 

comparison of the EPA Method 353.2 to NaR Nitrate Method for two enzyme forms 

using environmental water samples.
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13.2 The performance of this method was validated by an Inter-Laboratory Study (See Plan 

for Inter-Laboratory Test Trial) and preliminary results of that study are appended here. 

Comparison of this Method to EPA Method 353.2 are summarized in Table 6. These 

data are plotted in a comparison in Figure 2. The regression line fit for Drinking Water 

matrices shown in Fig. 2A, indicates that the NaR Reduction Method results were

~95% of Cd Reduction Method for the 4 matrices. The linear regression fit for 

wastewatermatrices shown in Fig. 2B, indicates that the NaR Reduction Method results 

were ~98% of Cd Reduction Method for 7 matrices (WW-5 was omitted from graphic). 

Basically, the two methods are equivalent. More detailed analysis of the results of the 

Inter-Laboratory Validation Study of the Nitrate Reductase Reduction Method will be 

published separately.

14. Pollution Prevention

All reagents and standards should be prepared in volumes consistent with laboratory use to 

minimize the generation of waste.

15. Waste Management

All waste should be disposed of in a manner consistent with local regulations. NOTE: 

This method uses very small volumes of samples and reagents and, therefore, less waste is 

generated than with most analysis methods.

16. References

Campbell, Wilbur H., P Song, GG Barbier (2006) Nitrate Reductase for Nitrate Analysis in 

Water. Environmental Chemistry Letters, 4: 69-73.

Patton, C.J., and Kryskalla, J.R., 2011, Colorimetric determination of nitrate plus nitrite in 

water by enzymatic reduction, automated discrete analyzer methods: U.S. Geological 

Survey Techniques and Methods, book 5, chap. B8, 34 p. (Available on line at 

http://pubs.usgs.gov/tm/05b08/).

Patton, C.J., and Kryskalla, J.R., 2013, Analytical properties of some commercially available 

nitrate reductase enzymes evaluated as replacements for cadmium in automated, 

semiautomated, and manual colorimetric methods for determination of nitrate plus nitrite 

in water: U.S. Geological Survey Scientific Investigations Report 2013–5033, 36 p., 

http://pubs.usgs.gov/sir/2013/5033/.

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1993. Methods for Chemical 

Analysis of Water and Wastes. Method 353.2. Revision 2.0. U.S. Environmental 

Protection Agency, Washington, DC
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17. Tables, Diagrams, Forms, Flowcharts, and Validation Data

Table 1 Determination of Nitrate in the Presence of Potential Interferences

Species Concentration 

Added (mg/L)

Unspiked 

Sample 

Result 

(mg N/L)

Spiked 

Sample 

Result

(mg N/L)

Spike 

Added

(mg N/L)

Percent

Recovery

Cl500

0.02 0.23 0.200 105

0.17 2.54 2.50 95

F

-

500 0.01 0.22 0.200 105

Br500

<0.01 0.21 0.200 100

0.15 2.65 2.50 100

SO4

2-

500

<0.01 0.21 0.200 105

0.14 2.53 2.50 96

Fe3+

500 0.17 2.60 2.50 97

1.0

<0.01 0.21 0.200 105

0.168 2.59 2.50 96

Zn2+ 1.0

<0.01 0.22 0.200 110

0.14 2.64 2.50 100

Al3+ 1.0

<0.01 0.21 0.200 105

0.14 2.53 2.50 96

BrO3

-

1.0

<0.01 0.22 0.200 110

0.17 2.64 2.50 99

ClO2

-

1.0

0.01 0.22 0.200 110

0.14 2.54 2.50 96

ClO3

-

1.0 0.23 2.45 2.50 89
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TABLE 2 Example Concentrations of Calibration Standards

NO3

-

-N or

NO 2-N,

mg/L

mL

of 10 mg N/L Standard

per 100 mL final volume

0.01 0.1

0.05 0.5

0.1 1.0

0.5 5.0

1.0 10

2.0 20

3.0 30

5.0 50
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Table 3. Determination of Reduction Efficiency

NO2-N/L NO3-N/L Reduction 

Efficiency

5.19 4.89 94%

5.18 4.99 96%

5.24 4.91 94%
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Table 4. Scaled sample and reagent volumes for 

implementation at larger assay volumes.

Volume (μL) x1 x2 x3 x4 x5

Sample 5 10 15 20 25

AtNaR2 55 110 165 220 275

NADH 12 24 36 48 60

SAN 25 50 75 100 125

NED 25 50 75 100 125

Total Assay 122 244 366 488 610
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Table 5A.

Comparison of Nitrate Analysis by Cadmium Reduction (EPA Method 353.2) and the 

Nitrate Reductase (NaR) Method

The Cadmium Reduction analysis of Nitrate-N plus Nitrite-N in samples was carried out using an automated 

continuous flow analyzer system which meets the specifications defined in EPA Method 353.2 Revision 2.0 

(EPA, 1993). The analysis was done in complete compliance with EPA Method 353.2 by OI Analytical.

The NaR Method was carried out on the OI Analytical Discrete Analyzer using AtNaR2 with a protocol 

modified from the Method described herein to adapt it to this equipment, which basically involves doubling 

of sample and all reagent volumes.

Sample # Analysis by Cd 

Reduction 

(mg NO3+NO2-N/L)

Analysis by NaR 

Reduction

(mg NO3+NO2-N/L)

Analysis by NaR 

Reduction

(mg NO3+NO2-N/L)

Non-Preserved

1 0.96 0.94 0.88

2 0.04 0.05 0.06

3 0.32 0.24 0.55

4 0.68 0.68 0.58

5 10.1 11.6 Lost

6 0.75 0.79 0.77

7* 2.5 3.11 2.88

*Sample #7 contains sulfide. Sulfide reacts with cadmium 

forming cadmium sulfide decreasing the efficiency 

of the reduction
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Table 5B. Comparison of Cadmium Reduction and Nitrate Reductase Reduction Methods.

Data from Table 5 Comparison of Third Party Check Determination for Cadmium Reduction Method by Continuous Flow Analysis (CFA) and

Nitrate Reductase (YNaR1) Reduction Method by CFA (Patton and Kryskalla, 2013), and Table 12 Third Party Check Determination for Nitrate 

Reductase (AtNaR2) Reduction Method by Discrete Analyzer (DA). (Patton and Kryskalla, 2011)

Target 

Concentration

(mg N/L)

Sample 

Identification

Number of 

Analysis

Mean Concentration Found ± Standard Deviation 

(mg N/L)

And (Relative Standard Deviation)

Cadmium Reduction 

by CFA

NaR Reduction 

CFA with YNaR1 DA with AtNaR2

0.20 092 L 63 0.19 ± 0.01 (3.53 %) 0.19 ± 0.01 (3.67 %)

0.05 TPC-L 143 0.49 ± .02 (3.3 %)

2.00 092 M 62 2.00 ± 0.05 (2.26 %) 2.00 ± 0.05 (2.69 %)

2.00 TPC-M 143 1.99 ± 0.06 (3.1 %)

4.00 092 H 63-65* 4.07 ± 0.07 (1.61 %) 4.02 ± 0.11 (2.63 %)

4.00 TPC-H 143 4.03 ± 0.11 (2.7 %)

*For 092 H sample, 65 analyses were done for Cadmium Reduction and 63 analyses for NaR Reduction.
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Nitrate Method for Nitrate Reductase Nitrate Analysis Page 17

Table 6. Comparison of Cadmium Reduction Method to Nitrate Reductase Method.

Cadmium Reduction Method (EPA 353.2) was carried out by Continuous Flow Analyzer (CFA) and the Nitrate 

Reductase (NaR) Method (this document) was done by Discrete Analyzer (DA) in a multiple laboratory study 

involving 10 different laboratories. The samples matrices are identified in the Study Plan and consisted of 4 

Drinking Water matrices (DW), 8 Waste Water matrices (WW), Seawater (SW), and 2 Standard Reference 

Materials (SRM) from the US Geological Survey. Abbreviations: CdR = Cadmium Reduction Method; NaRR = 

Nitrate Reductase Reduction Method; RPD = Relative Percent Difference; and RSD = Relative Standard Deviation.

Matrix Cd Reduction NaR Reduction RPD

Method CFA (mg N/L) DA (mg N/L) CdR - NaRR

Mean ± RSD %

DW2 0.83 0.77 ± 2.3% 7.50

DW3 1.21 1.22 ± 1.8% -0.82

DW4 7.02 6.70 ± 2.5% 4.66

DW5 0.49 0.44 ± 4.5% 10.75

WW1 0.03 0.03 ± 67% 0.00

WW2 8.27 7.7 ± 3.9% 7.14

WW3 0.26 0.23 ±4.3% 12.24

WW4 0.03 0.06 ± 100% -66.67 *

WW5 272.6 273 ± 4.0% -0.15

WW6 4.8 4.9 ± 4.1% -2.06

WW7 0.06 0.02 ± 50% 100.00 *

WW8 14.1 13.9 ± 3.6% 1.43

SW1 0.03 0.02 ± 50% 0.00

SRM-1 0.48 0.44 ± 4.5% 8.70

SRM-2 2.36 2.30 ± 2.6% 2.58

 * RPDs inflated due to concentration levels below QL
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Revision 9.0, March 2014 

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 18

Figure 1. Snapshot of Figure 12 from Patton and Kryskalla, 2011. 

Graphic illustrates the equivalency of Cadmium Reduction and NaR Reduction Methods with real world samples. 

2.50 

2.00 

1.50 

1.00 

0.50 

0 0.50 1.00 1.50 2.00 2.50 

EXPLANATION 

o AtNaR2: 10 minutes, 37°C 

11 YNaRl: 10 minutes, 37"C 

- Une of equal relation 

linear least squares regression analysis 

AtNaR2 nitrate= 0.977 (CdR nitrate I+ 0.017 

Correlation coefficient = 0.999) 

YNaRI nitrate = O.S44 (CdR nitrate!+ 0.051 

:too 3.50 4.00 4.50 

Continous-flow analyzer, cadmium-reduction 

nitrate + nitrite concentration, In milligrams nitrogen per liter 

5.00 

figure 12. Comparison of nitrate+ nitrite concentrations for 115 environmental water samples 

analyzed by automated continuous-flow, cadmium-reduction (CdR) method and automated discrete 

analyzer (DA) enzymatic-reduction method with NAD(P)H:YNaRl nitrate reductase and NADH:AtNaR2 

nitrate reductase. DA determinations were consecutive on November 9, 2005. 
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Nitrate Method for Nitrate Reductase Nitrate Analysis Page 19

Figure 2. Comparison of Cadmium Reduction Method and Nitrate Reductase Method Results. 

y = 0.9548x + 0.003

R2 = 0.9998

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8

Nitrate by NaR Reduction (mg N/L)

Nitrate by CdR Reduction (mg N/L)

A. Drinking Water Matrices

y = 0.9765x - 0.0091

R2 = 0.9988

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16

Nitrate by NaR Reduction (mg N/L)

Nitrate by CdR Reduction (mg N/L)

B. Waste Water Matrices

WW-5 omitted