Method for NaR Nitrate Analysis of Drinking Water 1.0

Method for Nitrate Reductase Nitrate Analysis of Drinking Water Version 1.0 Revision 2.0 Page 1

Method for Nitrate Reductase Nitrate-Nitrogen Analysis of Drinking Water

Version 1.0 Revision 2.0 1February, 2016

1. Scope and Application

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

in drinking water.

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 (nitratefree) 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. Precision and Bias of the method has been validated to be ±10% for all 

QC procedures and samples of a variety of Drinking Water matrices.

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). This version of the Method is for discrete 

analyzers.

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.
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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).

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 with 

colorimetric detector 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). The product of this 

reaction is a dye that absorbs light at 540 ± 20 nm.

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. 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:

The instrumentation described in this test method may automatically correct.

4.2. Certain ions may cause interferences. 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.
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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

6.1. Automated Discrete Analysis System (see section 3.2).

6.1.1. Discrete Analyzer Characteristics

a. Detectors: Colorimetric detector with optical interference filters, or 

Spectrophotometer (fixed wavelengths, or scanning)

b. Sample delivery system: High-precision syringe or piston pump

c. Heater: Resistant or Peltier-based heater

d. Dilution device: Built-in dilution function

e. Process: Individual cuvette, sequential batch analysis or random-access 

single samples

6.1.2. Equivalent DA instruments include, but are not limited to: 

a. Thermoscientific Instruments see: 

i. AquakemTM Photometric Analyzers: Models 200, 250, & 600

ii. ArenaTM Photometric Analyzers: Models 30, 60, 20, & 20XT
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iii. GalleryTM and GalleryTM Plus Automated Photometric Analyzers

b. Unity Scientific 

i. SMARTCHEM® 200 and SMARTCHEM® 170 ; EASYCHEM® Plus

ii. Westco Scientific Instruments see: http://westcoscientific.com/

iii. SMARTCHEM® 200 and SMARTCHEM® 170 ; EASYCHEM® Plus

c. Astoria-Pacific 

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 snapcap 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 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.
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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 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.

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.
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8. Sample Collection, Preservation, and Storage

8.1. Sample preservation and holding time requirements for drinking water samples are as 

follows:

8.1.1. For nitrate: Chill the sample to ≤4°C and analyze within 48 hours. If the sample 

is chlorinated, the holding time for an unacidified sample kept at 4°C is extended 

to 14 days.

8.1.2. For nitrite: Do not add acid. Chill the sample to ≤4°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. Sample preservation and holding time requirements for wastewater samples or other 

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

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

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

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

analyze within 28 days of collection.

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

9. Quality Control (QC)

9.1. Requirement for QC Analysis: 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 drinking 

water. Follow the instrument manufacturer’s calibration protocols and instructions.

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. Instrument Calibration Range (ICR): ICR must be determined initially and 

verified every six months or whenever a significant change in the instrument 
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response is expected or observed. The initial demonstration of ICR must employ 

a number of standards sufficient to insure that the results are reproducible and 

statistically acceptable. Verification of ICR must use a minimum of a blank and 

three standards. If any verification data exceeds the nominal value of the standard 

by ±10%, ICR must be reestablished. ICR data must be fitted with a statistics 

program using either linear or non-linear regression analysis and an equation 

relating instrument response to the nominal values of the standards for the 

calibration obtained. The regression coefficient (R2) must be 0.999 or greater; if 

it is not, determine the problem and recalibrate the instrument.

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.

9.2.3. Reduction Efficiency (RE): Analyze a nitrite standard (Section 7.10) for 

determining the Reduction Efficiency. The nitrite standard will contain 2.5 mg 

Nitrite-N/L (or other mid-range concentration) and the absorbance at 540 nm (or 

appropriate measure of the instrument response) is compared to the absorbance at 

540 nm (or appropriate measure of the instrument response) of a Certified 

Nitrate-N Standard of the same concentration as the Nitrite-N standard:

RE = [(Nitrate-N Standard A-540)/(Nitrite-N Standard A-540) X 100].

For acceptable results, RE must be 90% or greater. If the RE is unacceptable, the 

source(s) of the problem must be determined and the DA re-calibrated.

NOTE: The likely source of a problem with the RE is - either the NaR solution 

needs to be replaced, or the NADH has expired and needs to be replaced.

9.2.4. 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
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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 reagent 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. Acceptable limits 

are ±10% of the known value.

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 nitritenitrate 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 
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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 nitrite-nitrate 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 highest 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 P A V V BV s s

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).

The percent recovery of the spike shall fall within ±10%, based on the nitrite-nitrate 

concentration in the spike. If the percent recovery is 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 1

specifies suggested calibrants. NOTE: Most discrete analyzers generate calibration 

standards and calibration curves automatically using Certified calibrants. Follow the 
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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.

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 concentration. 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.

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 2).

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).
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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. The performance of this method was validated by an Inter-Laboratory Study (See 

Plan for Inter-Laboratory Test Trial) and results of that study are delineated here.

13.2. Method Performance Data

13.2.1. Table 3. Drinking Water Matrices for Analysis

13.2.2. Table 4. Calibration of Enzymatic Methods Summary

13.2.3. Table 5. Enzymatic Efficiency Summary

13.2.4. Table 6. Initial Performance and Recovery (IPR) Summary and Ongoing 

Performance and Recovery (OPR) Summary

13.2.5. Table 7. Minimum Detection Limit (MDL) Summary

13.2.6. Table 8. DW-1 and DW-1-Cl Tap Water Matrix Nitrate-N Content and Spike 

Analysis

13.2.7. Table 9. DW-2 High TDS Drinking Water Matrix Nitrate-N Content and Spike 

Analysis

13.2.8. Table 10. DW-3 High TOC Drinking Water Matrix Nitrate-N Content and Spike 

Analysis

13.2.9. Table 11. Reference Standard RS-1 Summary
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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
Method for NaR Nitrate Analysis of Drinking Water 1.0

17. Tables, Diagrams, Forms, Flowcharts, and Validation Data

Table 1

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
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 2

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
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 3

Drinking Water Matrices for Analysis

Each Sample Matrix (except DW-1) will be provided to Participating Laboratories as “Ready to Analyze” (samples 

will be filtered with 0.45 μm filter and preserved by acidifying) and in sufficient volume to permit multiple analysis. 

We will supply each lab with Chlorine Standard Solution, 25-30 mg/L as Cl2, 2 mL PourRite Ampules (NIST)

Sample Matrix Identifier Number 

Replicates Spikes* Laboratories

Finished 

drinking water: 

from cold water 

tap of 

participating 

laboratories on 

day of analysis

DW-1 7

3 MS & MSD

At 0.5 mg N/L

At 1.0 mg N/L 

At 2.5 mg N/L

All

Local Tap 

Water with 

2-4 mg/L free 

chlorine

added

DW-1 + Cl2 7

3 MS & MSD

At 0.5 mg N/L

At 1.0 mg N/L 

At 2.5 mg N/L

All

Drinking

water with

high TDS (~

500 ppm)

DW-2 7

3 MS & MSD

At 0.5 mg N/L

At 1.0 mg N/L 

At 2.5 mg N/L

Lab 3

Drinking

water with

high TOC 

(~2 ppm)

DW-3 7

3 MS & MSD

At 0.5 mg N/L

At 1.0 mg N/L 

At 2.5 mg N/L

Lab 2

ERA #698

Standard 

Reference

SR-1** 7 NONE All

*Each MS and MSD sample will be analyzed once. See Appendix D for method of preparing 

spikes and the layout of a set of analysis of typical DW-1 and its spikes.

**SR-1 is Reference Standard of known Nitrate-N mg/L concentration (not acidified) and must

be analyzed by all Participating Laboratories. SR-1 will require dilution with reagent water.
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 4

Calibrants and Standard Calibration Curve Equation for Participating Labs

0BLab 1 1BLab 2 2BLab 3

Calibration NaR-R NaR-R NaR-R

Reagent Blank 0.00 0.00 0.00

Calibrant 1 0.05 0.05 0.05

Calibrant 2 0.25 0.10 0.10

Calibrant 3 0.50 0.25 0.50

Calibrant 4 0.75 0.50 1.00

Calibrant 5 1.25 1.25 2.50

Calibrant 6 2.50 2.50 5.00

Calibrant 7 3.75 5.00

Calibrant 8 5.00

Linear 

Regression 3BNitrate (mg N/L) = (Absorbance Sample - Intercept) / Slope

4BSlope 0.118

5BIntercept 0.022 6BNot Used in These Analyses 

7BR2 0.99989

8BPolynomial 

Regression Nitrate (mg N/L) = (A x Conc x Conc) + (B x Conc) + C for Lab 1

or

= (A x A-550 x A-550) + (B x A-550) + C for Lab 2

Or

= (A x Area x Area) + (B x Area) + C for Lab 3

9BConstant A - - - -0.1988 0.0018733

10BConstant B - - - 15.0219 0.5099

11BConstant C - - - -0.5308 0.1713

12BR2 - - - 1.00000 0.99993
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 5

All Data from All Labs for

Enzymatic and Cadmium Reduction Efficiency

Evaluated using Certified Standards for 2.50 Nitrate-N mg/L and 2.50 Nitrite-N mg/L

Reduction Efficiency of greater than 90% is acceptance criterion.

13BLab 1 14BLab 2 15BLab 3*

NaR-R NaR-R NaR-R

16BNitrate

17BAbsorbance 

540 nm,

18B550 nm,

19BOr Area/sec

0.318

0.2080

0.1982

0.1909

0.1929

0.1792

5.4019

9.1622

20BNitrite

21BAbsorbance 

540 nm,

22B550 nm,

23BOr Area/sec

0.341

0.1963

0.1892

0.1853

0.1881

0.1787

5.7256

8.9972

24BCatalytic

25BReduction

26BEfficiency 

27B(NO3/NO2)

28BPercent

93.2551

105.9603

104.7569

103.0221

102.5518

100.2798

94.3464

101.8339

*Lab 3 ran a series of 10-12 of these tests, which are not shown here.
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 6

All Data Reported by Each Lab for

Initial Performance and Recovery (IPR)

and

Ongoing Performance and Recovery (OPR)

29BLab 1 30BLab 2* 31BLab 3

NaR-R NaR-R NaR-R

32BIPR 

33BNitrate-N 

mg/L

2.01 2.01 2.01

34BNumber IPR 35B4 36B16 8

37BMean IPR 38B2.0705 1.9682 1.9827

39BMean IPR 

Recovery (%) 40B103.0075 41B97.9201 42B98.6412

43BOPR 

44BNitrate-N 

mg/L

45B2.01 46B2.50 2.01

47BNumber OPR 7 48B15 49B14

50BMean OPR 2.1388 2.4769 2.4877

51BStandard 

Deviation of 

Mean

0.03685 0.07204 0.11536

52BRelative 

Standard 

Deviation (%)

1.7233 2.9085 4.6372

53BMean 

Recovery (%) 54B106.4065 55B99.0740 99.5083
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 7

Minimum Detection Limit

7 Replicates

MDL = Standard Deviation x 3.143

Abbreviations: DA, Discrete Analyzer; FIA, Flow Injection Analyzer

56BLab 1 57BLab 2 58BLab 3

NaR-R NaR-R NaR-R

59BAnalytical 

Equipment 60BDA 61BDA 62BFIA

63BSpike

64Bmg Nitrate-N/L 65B0.050 66B0.025 67B0.010

68BMDL 

69Bmg Nitrate-N/L

0.0097 0.006507 0.009301

70BRatio 

71BSpike/MDL

5.1453 3.8422 1.0751
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 8. A. DW-1 and DW-1-Cl - Lab 1 Tap Water Matrix Nitrate-N Content and Spike Analysis

Lab 1 NaR Reduction Method DW1 (Abbreviation: RSD = Relative Standard Deviation; RPD = Relative Percent Difference)

A-540 Nitrate-N

mg/L

Mean ±

Standard

Deviation

Nitrate-N

mg/L

0.5 mg N/L Spike 1.0 mg N/L Spike 2.5 mg N/L Spike

0.059 0.3207

0.3237 ±

0.003698

RSD

1.1424%

A-540 mg N/L

Spike

mg N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD %

0.059 0.3215

0.179 1.3392 0.4859 97.1829

0.9830

0.179 1.3392 1.0155 101.5514

0.3947

0.366 2.9467 2.6230 104.9206

0.4451

0.06 0.3282

0.06 0.3279

0.178 1.3352 0.4907 98.1429 0.178 1.3352 1.0115 101.1514 0.367 2.9584 2.6347 105.3886 0.06 0.3235

0.06 0.3255

0.059 0.3185

Lab 1 NaR Reduction Method DW1-Cl

A-540

Nitrate-N

mg/L

Mean ±

Std. Dev.

Nitrate-N

mg/L

0.5 mg N/L Spike 1.0 mg N/L Spike 2.5 mg N/L Spike

0.058 0.3117

0.3185 ±

0.01625

RSD

5.1020%

A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike

mg N/L

Recovery

% RPD %

0.058 0.3122

0.118 0.8191 0.5006 100.1200

1.4082

0.175 1.3087 0.9902 99.0200

1.6923

0.367 2.9515 2.6330 105.3200

0.3564

0.063 0.3547

0.059 0.3175

0.117 0.8121 0.4936 98.7200 0.177 1.3256 1.0071 100.7100 0.368 2.9609 2.6424 105.6960 0.059 0.3136

0.058 0.3126

0.058 0.3072
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 8. B. DW-1 and DW-1-Cl - Lab 2 Data Set 2 Tap Water Matrix Nitrate-N Content and Spike Analysis

Lab 2 NaR Reduction Method DW1 (Abbreviation: RSD = Relative Standard Deviation; RPD = Relative Percent Difference)

A-550

nm

Nitrate- N

mg/L

Mean ±

Standard

Deviation

Nitrate-N

mg/L

0.5 mg N/L Spike 1.0 mg N/L Spike 2.5 mg N/L Spike

0.0825 0.9304

0.94569 ±

0.016307

RSD

1.7243%

A-550 mg N/L

Spike mg

N/L

Recovery

% RPD % A-550 mg N/L

Spike mg

N/L

Recovery

% RPD % A-550 mg N/L

Spike mg

N/L

Recovery

% RPD %

0.0835 0.9464

0.1151 1.4501 0.5044 100.8837

3.5318

0.1437 1.9045 0.9588 95.8841

4.3690

0.2315 3.2907 2.3451 93.8021

2.4482

0.0828 0.9352

0.0852 0.9735

0.1140 1.4326 0.4869 97.3825 0.1464 1.9474 1.0017 100.1668 0.2352 3.3489 2.4032 96.1270 0.0838 0.9512

0.0823 0.9272

0.0841 0.9560

Lab 2 NaR Reduction Method DW1-Cl

A-550

nm

Nitrate-N

mg/L

Mean ±

Std. Dev.

Nitrate-N

mg/L

0.5 mg N/L Spike 1.0 mg N/L Spike 2.5 mg N/L Spike

0.0820 0.9224

0.8546 ±

0.03249

RSD

3.8017%

A-550 mg N/L

Spike mg

N/L

Recovery

% RPD % A-550 mg N/L

Spike mg

N/L

Recovery

% RPD % A-550 mg N/L

Spike mg

N/L

Recovery

% RPD %

0.0775 0.8505

0.1091 1.3546 0.5000 99.9911

3.8968

0.1361 1.7839 0.9293 92.9273

0.8506

0.2241 3.1744 2.3198 92.7913

2.0549

0.0770 0.8425

0.0773 0.8473

0.0562 0.4791 0.4808 96.1691 0.1366 1.7919 0.9372 93.7211 0.1880 2.3527 2.2726 90.9040 0.0764 0.8330

0.0783 0.8633

0.0758 0.8234
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 8. C. DW-1 and DW-1-Cl - Lab 3 Tap Water Matrix Nitrate-N Content and Spike Analysis

(Abbreviation: RSD = Relative Standard Deviation; RPD = Relative Percent Difference)

Lab 3 NaR Reduction Method DW1 (Abbreviation: RSD = Relative Standard Deviation; RPD = Relative Percent Difference)

Area V/s

540 nm

Nitrate- N

mg/L

Mean ±

Standard

Deviation

Nitrate-N

mg/L

0.5 mg N/L Spike 1.0 mg N/L Spike 2.5 mg N/L Spike

0.53691 0.03439

0.03438

±

0.0006398

RSD

1.8608%

A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD %

0.52026 0.03541

1.4411 0.5674 0.5674 106.6035

2.5541

2.5271 1.1292 1.094818 109.4818

0.3283

5.2217 2.5423 2.507918 100.3167

1.9232

0.53262 0.03376

0.53577 0.03385

1.4678 0.58112 0.5468 109.3615 2.5341 1.1328 1.098418 109.8418 0.367 2.9584 2.6347 105.3886 0.52710 0.03393

0.52747 0.03426

0.53226 0.03509

Lab 3 NaR Reduction Method DW1-Cl (Abbreviation: RSD = Relative Standard Deviation; RPD = Relative Percent Difference)

Area

V/s

A-540

Nitrate-N

mg/L

Mean ±

Std. Dev.

Nitrate-N

mg/L

0.5 mg N/L Spike 1.0 mg N/L Spike 2.5 mg N/L Spike

0.5681 0.1190

0.1166

± 0.004657

RSD

3.9943%

A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD % A-540 mg N/L

Spike mg

N/L

Recovery

% RPD %

0.5703 0.1201

1.4923 0.5938 0.4772 95.44171

0.05210

2.4589 1.0938 0.9772 97.7208

0.05118

5.2544 2.5597 2.4431 97.7243

0.2213

0.5688 0.1194

0.5627 0.1185

1.4875 0.5913 0.4747 94.94571 2.4578 1.0933 0.9767 97.6709 5.2558 2.5543 2.4377 97.5083 0.5471 0.1082

0.5539 0.1117

0.5685 0.1192
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 9 

DW-2 High TDS Drinking Water Matrix Nitrate-N Content and Spike Analysis

Abbreviation: RPD = Relative Percent Difference Lab 3 NaR Reduction Method DW-2

Nitrate-N 

mg/L

Mean 

Nitrate-N 

mg/L

Standard 

Deviation

mg N/L

Relative 

Standard 

Deviation

0.2907

0.2959 0.004749 1.6048%

0.2973

0.3023

0.2985

0.2939

0.2992

0.2893

0.5 mg N/L Spike

A-540 mg N/L Spike 

mg N/L

Recovery 

% RPD %

1.944 0.7900 0.4941 98.8226

0.0486

1.9437 0.7898 0.4939 98.7746

1.0 mg N/L Spike

A-540 mg N/L Spike 

mg N/L

Recovery 

% RPD %

2.8302 1.2489 0.9530 95.3003

0.3875

2.8375 1.2526 0.9567 95.6703

2.5 mg N/L Spike

A-540 mg N/L Spike 

mg N/L

Recovery 

% RPD %

5.8573 2.8417 2.5458 101.8321

0.4251

5.8371 2.8309 2.5350 101.4001
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 10

DW-3 High TOC Drinking Water Matrix Nitrate-N Content and Spike Analysis

Abbreviation: RPD = Relative Percent Difference Lab 2 NaR Reduction Method DW-3

Nitrate-N 

mg/L

Mean 

Nitrate-N 

mg/L

Standard 

Deviation

mg N/L

Relative 

Standard 

Deviation

0.0052

0.003814 0.007267 190.5184%

0.0068

-0.0110

0.0084

0.0036

0.0020

0.0117

0.5 mg N/L Spike

A-540 mg 

N/L

Spike 

mg N/L

Recovery 

% RPD %

0.0621 0.4981 0.4943 98.8571

3.8104

0.0633 0.5173 0.5135 102.6971

1.0 mg N/L Spike

A-540 mg 

N/L

Spike 

mg N/L

Recovery 

% RPD %

0.0934 0.9980 0.9942 99.4186

2.4332

0.0919 0.9741 0.9703 97.0286

2.5 mg N/L Spike

A-540 mg 

N/L

Spike 

mg N/L

Recovery 

% RPD %

0.1836 2.4145 2.4107 96.4274

1.5558

0.1861 2.4523 2.4485 97.9394
Method for NaR Nitrate Analysis of Drinking Water 1.0

Table 11

Standard Reference - SR-1 (Seven Replicates Analyzed)

ERA 698 Certified Value SR-1 = 5.14 ± 1.83% mg Nitrate-N/L

Dilution

Diluted 

Mean

mg 

NitrateN/L

Standard 

Deviation

mg 

NitrateN/L

Relative 

Standard 

Deviation

%

Final

mg 

NitrateN/L

Compared 

to ERA

Lab 1 -

NaRR

Autodiluted 

1:10

5.3989 0.03375 0.6251 5.3989 105.0195%

Lab 1 -

CdR NONE 5.0937 0.07998 1.5701 5.0937 99.0992%

Lab 2 -

NaRR

Autodiluted 1:4 4.9795 0.07552 1.5166 4.9795 96.8783%

Lab 2 -

CdR 1:4 1.0128 0.02132 2.1052 5.0640 98.5214%

Lab 3 -

NaRR 1:2 2.5629 0.005100 0.1990 5.1258 99.7218%

Lab 3 -

CdR 1:2 2.5902 0.01816 0.7011 5.1800 100.7782%