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 drinkin g 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 (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 cri teria are

delineated. Precisio n 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. S ummary 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)eth ylenediamine 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.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 2 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 μ mol nit rite produced per minute at 30°C and pH 7 w ith 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 analys is 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

sulfanilami de (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: D isodium ethylenediaminetetraacetate dihydrate (CAS 60

- 00

- 4 , a heavy

metal chelator.

4. Interferences

4.1. Sample color that absorbs a t wavelengths between 520 and 56 0 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. NOT E:

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.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 3 5. Safety

5.1. General laboratory safe practices should be used in handl ing 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. P otassium 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 i s 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 , o r 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. Proce ss : 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, & 20XTMethod 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 4 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 (KH 2 PO 4 ; CAS 7778

- 77

- 0 ), 0.01 g of disodium ethylenediaminetetraacetate dihydrate (C 10 H 14 O 8 N 2 Na 2 •2H 2 O), 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 polypropyl ene vials and store at

- 20 ° C. Stable for 1 month. NOT E: 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 conditio ns.

7.3. NADH work ing solution: thaw one 1

- mL vial of NADH st ock (refer to section 7 .2) and dilute to 10 mL with phosp hate buffer (refer to section 7 .1). This reagent is stable for about 8 hours. Prepare sufficient NADH working solution for the number of sampl es 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) Re agent (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 fo r about six months when stored in a brown bottle at

20 ° C.

7.5. N

- (1

- naphthyl)ethylenediamine dihydrochloride (NED) solu tion (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.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 5 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 buff er. 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. NOT E: For some NaR forms, high phenol ic co ntent 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

- 4 0°C (Patton and Kryskalla, 2011; 2013) . Humic substa nces at the operation temperatures specified in this test
method do not inh ibit 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 i n use b y analysis of Quality Control checks that approximate the sample matrix.

7.7. Nitrate Solution, St ock (1000 mg/L NO3

- N): Dry potassium nitrate (KNO 3 ) 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 Sol ution, 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 S olution, Stock (1000mg/L NO2

- N): Place about 7 g of potassium nitrite (KNO 2 ) 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 un til 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 commercia lly available through chemical supply vendors and may be used. N OTE : Potassium nitrite is easily oxidized; use only dry, free flowing white, or yellowish white crystalline powder of this reagent.

7.10. Nitrite Sol ution, 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.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 6 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 col lection.

8.1.3. For nitrate

- nitrite: Acidify to pH<2 with H 2 SO 4 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 H 2 SO 4 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 manuf acturer’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 bel ow. 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) : I CR must be determined initially and verified every six months or whenever a significant change in the instrumentMethod 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 7 response is expected or observed. The in itial 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 mu st be reestablished. ICR data must be fitted with a statistics program using either linear or non

- linear regression analysis and a n equation relating instrument response to the nominal values of the standards for the calibration obtained. The regression coefficient (R 2 ) 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% o f 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 nit rite s tandard 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) o f 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 est ablished for the analyte using reagent water (blank) fortified at a concent ration 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 n itra te 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 process ed 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) andMethod 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 8 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 signi ficant 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

- ran ge second source calibrant (SSC) from an external source and different from calibration

standards. The result should fall within ± 10 % of the known conc entration. 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 th is 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 soluti on 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 replic ate 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 C RM. 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, ana lyze 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 th e analytical method including sample preservation and pretreatment. The result obtained for the OPR shall fall withi n ± 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 showsMethod 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 9 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

in terferences 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 co ncentration plus the background concentration of nitrite

- nitrate in water must not exceed the high est 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 s s 

## A V V BV 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, V
s = sample volume (mL) used, and V = added spiking solution volume (mL).

The percen t 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

circumstance s, 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. Indep endent 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 a nd 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 theMethod 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 10 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 e nzyme.

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

calibr ation 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 ± 2 0 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 5 5 μL of NaR (section 7.6) and 5 μL of sa mple. Mix.

NOTE : Larger volumes of samples and reagents may be used while maintai ning the same ratio (See Table 2 ).

11.2.2. Add 1 2 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 (sect ion 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 av ailable, 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).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 11 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 b y 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

- nitrog en, 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 (NO 2

- N), mg N /L;

Nitrate

- Nitrogen (NO 3

- N), mg N /L; and

Combined Nitrate

- Nitrite Nitrogen (NO 3 , NO 2

- N), mg N /L.

13. Method Performance

13.1. T he 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 SummaryMethod 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 12 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. ENVIR ONMENTAL PROTECTION AGENCY. 19 9 3 . Methods for Chemical Analysis of Water and Wastes. Method 353.2. Revision 2.0. U.S. Environmental Protection Agency, Washington, DCMethod for NaR Nitrate Analysis of Drinking Water 1.0

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

Table 1

Example Concentrations of Calibration Standards

NO 3 -

- 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 50Method 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 610Method 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 Cl 2 , 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 + Cl 2 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

0B 

### Lab 1

1B 

### Lab 2

2B 

### Lab 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 3B Nitrate (mg N/L) = (Absorbance Sample

- Intercept) / Slope

4B 

### Slope 0.118

5B 

### Intercept 0.022

6B 

### Not Used in These Analyses

7B 

### R **2** 0.99989

8B 

### Polynomial **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

9B 

### Constant A

- - - - 0.1988 0.0018733

10B 

### Constant B

- - - 15.0219 0.5099

11B 

### Constant C

- - - - 0.5308 0.1713

12B 

### R **2**

- - - 1.00000 0.99993Method 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.

13B 

### Lab 1

14B 

### Lab 2

15B 

### Lab 3*

NaR

- R NaR

- R NaR

- R

16B 

### Nitrate

17B 

### Absorbance **540 nm,**

18B 

### 550 nm,

19B 

### Or **Area/sec** 0.318 0.2080

0.1982

0.1909

0.1929

0.1792 5.4019

9.1622

20B 

### Nitrite

21B 

### Absorbance **540 nm,**

22B 

### 550 nm,

23B 

### Or Area/sec 0.341 0.1963

0.1892

0.1853

0.1881

0.1787 5.7256

8.9972

24B 

### Catalytic

25B 

### Reduction

26B 

### Efficiency

27B 

### (NO **3** 

### /NO **2** 

### )

28B 

### Percent 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)

29B 

### Lab 1

30B 

### Lab 2*

31B 

### Lab 3

NaR

- R NaR

- R NaR

- R

32B 

### IPR

33B 

### Nitrate

- N mg/L 2.01 2.01 2.01

34B 

### Number IPR

35B 

### 4

36B 

### 16 8

37B 

### Mean IPR

38B 

### 2.0705 1.9682 1.9827

39B 

### Mean IPR Recovery (%)

40B 

### 103.0075

41B 

### 97.9201

42B 

### 98.6412

43B 

### OPR

44B 

### Nitrate

- N mg/L 45B 

### 2.01

46B 

### 2.50 2.01

47B 

### Number OPR 7

48B 15

49B 14

50B 

### Mean OPR 2.1388 2.4769 2.4877

51B 

### Standard

Deviation of

Mean 0.03685 0.07204 0.11536

52B 

### Relative

Standard Deviation (%) 1.7233 2.9085 4.6372

53B 

### Mean Recovery (%)

54B 

### 106.4065

55B 

### 99.0740 99.5083Method 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

56B 

### Lab 1

57B 

### Lab 2

58B 

### Lab 3

NaR

- R NaR

- R NaR

- R

59B 

### Analytical Equipment
60B 

### DA

61B 

### DA

62B 

### FIA

63B 

### Spike

64B 

### mg Nitrate

- N/L 65B 

### 0.050

66B 

### 0.025

67B 

### 0.010

68B 

### MDL

69B 

### mg Nitrate

- N/L 0.0097 0.006507 0.009301

70B 

### Ratio

71B 

### Spike/MDL 5.1453 3.8422 1.0751Method 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 0.3072Method 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

0.07 73 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 0.07 64 0.8330

0.0783 0.8633

0.0758 0.8234Method 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 0.0 3393

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 0.11 85
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 0.10 82

0.5539 0.1117

0.5685 0.1192Method 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 101.4001Method 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 97.9394Method 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 Nitrate

N/L Standard

Deviation

mg Nitrate

N/L Relative

Standard

Deviation

% Final

mg Nitrate

N/L Compared

to ERA

Lab 1

- NaRR Auto

diluted

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 Auto

diluted 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%