NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 1 

### 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

esta blish 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 st andard 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 sampl e (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).NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 2 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 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: D isodium 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 a t wavelengths between 520 and 56 0 nm interferes with the absorbance measurements. When color is suspect, analyze a sample blank, omittin g 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. Howeve r, 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 C omprehensive stud ies of this method, which include analysis of interferences, ha ve been published (Patton and Kryskalla , 2011 ; 2013 )

.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 3 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 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 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 (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 month s.

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 polyp ropylene 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 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. Prep are sufficient NADH working solution for the number of samplesNECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 4 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 for 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.

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 standar ds to be analyzed. NOTE : For some NaR forms, high phenol ic 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

- 4 0°C (Patton and Kryskalla, 2011; 2013) . Humic substances 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 pres ent

, the user must verify reduction efficiency of the NaR i n use by 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 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 throu gh 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 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. N OTE : Potassium nitrite is easily oxidized; use only dr y, free flowing white, or yellowish white crystalline powder of this reagent.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 5 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.

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 al l 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 proce dures 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 stan dards 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 concent ration 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.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 6 9.2.3 Method Detection Limit (MDL): The MDL must be established for the analyte using reagent water (blank) fortified at a concent ration near the detection limit

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

B ” . Samples should be prepared using reagent water fortified with nitra 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) and

S = standard deviation of the seven replicate analy ses.

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

op erator 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

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

source and different from calibration standards. Th e 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 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 solution prepar ed 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 n ot use this test method to analyze samples unless the IPR recoveries are within the accepted limits.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 7 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 ni trite

- 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 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 rea nalyzed, 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

- nitra te 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 thi s 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 o f 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 l imit of the test method, whichever is greater.

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

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).

Calculate the relative percent difference ( RPD ) between two sample resu lts using the following equation:

Where, D 1 = Concentration of analyte in the MS, D 2 = Concentration of analyte in

the MSD.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 8 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 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 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

ca libration 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

.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 9 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 maintaining the same ratio (See Table 4).

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 (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 availab le, 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

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

- nitroge n, 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 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.NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 10 13.2 The performance of this method was validated by an Inter

- Laboratory Study (See Plan

for In ter

- 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

m atrices 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

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

w ere ~9 8 % of Cd Reducti on 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 sepa rately

.

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, DCNECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 11 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

Cl 500 0.02 0.23 0.200 105

0.17 2.54 2.50 95

F - 500 0.01 0.22 0.200 105

Br 500 <0.01 0.21 0.200 100

0.15 2.65 2.50 100

SO 4 2

- 500 <0.01 0.21 0.200 105

0.14 2.53 2.50 96

Fe 3+ 500 0.17 2.60 2.50 97

1.0 <0.01 0.21 0.200 105

0.168 2.59 2.50 96

Zn 2+ 1.0 <0.01 0.22 0.200 110

0.14 2.64 2.50 100

Al 3+ 1.0 <0.01 0.21 0.200 105

0.14 2.53 2.50 96

BrO 3 - 1.0 <0.01 0.22 0.200 110

0.17 2.64 2.50 99

ClO 2 - 1.0 0.01 0.22 0.200 110

0.14 2.54 2.50 96

ClO 3 - 1.0 0.23 2.45 2.50 89NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 12

TABLE 2 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 50NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 13 Table 3. Determination of Reduction Efficiency

NO 2 - N/L NO 3 - N/L Reduction Efficiency

5.19 4.89 94%

5.18 4.99 96%

5.24 4.91 94%NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 14

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 610NECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 15 Table 5 A

. 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 i n EPA Method 353.2 Revision 2.0 (EPA, 1993 )

. The analysis was done in complete c ompliance 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 NO 3 +NO 2 - N/L) Analysis by NaR

Reduction

(mg NO 3 +NO 2 - N/L) Analysis by NaR

Reduction

(mg NO 3 +NO 2 - 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 t he efficiency

of the reductionNECi Method N07

- 0003 Revision 9.0, March 2014

Nitrate Method for Nitrate Reductase Nitrate Analysis Page 16

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 ( Y NaR 1 ) Reduction Method by CFA (Patton and Kryskalla, 2013) , and Table 12 Third Party Check Determination for Nitrate Reductase ( At NaR 2 ) Reduction Method by Discrete Analyzer (DA). (Patton and Kryskalla, 2011 )

Target

Concentration

(mg N/L) Sample

Identification Number of

Analysis Mean Concentration Found ± St andar d Deviation

(mg N/L)

And (Relative St an d ard 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.NECi Method N07

- 0003 Revision 9.0, March 2014

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 sample s 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 level s below QLNECi Method N07-0003 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.

Gra phic 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 rela tion

linear least squares regression analysis

AtNaR2 nitrate= 0.977 (CdR nitrate I+ 0.017

Correlation coefficient = 0

. 999)

YNaRI nitrate = O

. S44 (CdR nitr ate!+ 0

. 051



### :too 3

. 50 4

. 00 4

. 50

Continous-flow analyzer, cadmium-reduction
nitrate + nitrite concentration, In milligrams nitroge n 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:YNaR l nitrate reductase and NADH : AtNaR2

nitrate reductase. DA determinations were consecutive on November 9, 2005

.NECi Method N07

- 0003 Revision 9.0, March 2014

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