Environmental Protection Agency

Alternate Test Procedure Validation Report:

Enzymatic Reduction Method of Nitrate-N

Analysis in Drinking Water

Compiled and Written by

Wilbur H. (Bill) Campbell

and

Ellen R. Campbell

The Nitrate Elimination Co., Inc. (NECi)

334 Hecla St.

Lake Linden MI 49945

bill@nitrate.com

906-369-9830

ellenr@nitrate.com

906-296-1000

Revised: January 30, 2016
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Acknowledgments:

Dr. Charles J. Patton, retired, Methods R&D Program, U.S. Geological Survey, National Water

Quality Laboratory, P.O. Box 25585, Lakewood, CO 80225-0585, and Dr. William Lipps,

Business Unit Manager - Environmental/Chemical at Shimadzu Scientific Instruments

Baltimore, Maryland, are thanked for their efforts in the development of the Nitrate Reductase 

Nitrate Analysis Method over the past 10 years.

All the laboratories, which are listed in Table 2, are thanked for participating in the

Inter-Laboratory ATP Validation Drinking Water Study.

The following U. S. departments and agencies are thanked for providing funding for research and

development studies carried out at NECi over the past 22 years: Department of Energy (DOE),

Environmental Protection Agency (EPA), National Institutes of Health (NIH), National Science

Foundation (NSF), and Department of Agriculture (USDA).

The State of Michigan is thanked for providing funding to NECi via Michigan Emerging

Technology Fund of the Michigan Economic Development Corporation.
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Table of Contents

Page

Report 1

Section 1 Introduction 2

Section 2 Inter-Laboratory Validation Study 3

Section 2.1 Study Objectives and Plan 3

Section 2.2 Sources of Sample Matrices and Handling of the Samples 3

Section 2.3 Participating Laboratories and Analytical Equipment 4

Section 3 Validation Study Results and Discussion 5

Section 3.1 Summary of Quality Control 5

Section 3.2 Nitrate-N Content and Spike Analysis of Sample Drinking Water Matrices 7

Section 3.3 Evaluation of Accuracy for the Method 8

Section 3.4 Comparison of Enzymatic Reduction and Cadmium Reduction Methods 9

Section 4 Validation

Conclusions 910

Section 5 References and

Glossary 11

Tables

Table 1 List of Drinking Water Sample Matrices

Table 2 List of Participating Laboratories

Table 3 List of Sample and Standards Sent to Participating Labs

Table 4 Calibration of Enzymatic and Cadmium Reduction Methods Summary

Table 5 Enzymatic and Cadmium Reduction Efficiency Summary

Table 6 Initial Performance and Recovery (IPR) Summary and 

Ongoing Performance and Recovery (OPR) Summary

Table 7 Minimum Detection Limit (MDL) Summary

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

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

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

Table 11 Reference Standard RS-1 Summary

Appendices

Appendix A Inter-Laboratory Drinking Water Validation Study Plan

Appendix B Description of the Enzymatic Reduction Method by Discrete Analyzer

Appendix C Description of Enzymatic Reduction Method by Flow Injection Analyzer

Appendix D Certification Sheets for Nitrate and Nitrite Standards used in this study

Appendix E Summary Data Sheets and Bench Sheets for Each Laboratory (Excel® Files)

10

11
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Report
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Section 1 Introduction

This Validation Study Report supports development of Nitrate plus Nitrite Nitrogen by Nitrate

Reductase (Campbell, et al., 2006), also called Enzymatic Reduction Method for Nitrate-N

Analysis, for use as an USEPA Alternate Test Procedure (ATP) for nationwide determination of

nitrate plus nitrite nitrogen in drinking water (EPA, 2015). The enzyme Nitrate Reductase (EC

1.7.1.1/2, NaR; CAS #9029-27-0) catalyzes the reduction of nitrate to nitrite with the reducing 

power provided by the natural reductant, reduced Nicotinamide Adenine Dinucleotide (NADH),

which is a thermodynamically irreversible reaction.

Nitrate + NADH + H+  Nitrite + NAD+ + H2O

This is the basis for a “green”, non-toxic method for nitrate-N analysis.

Eukaryotic NaR is a complex enzyme which contains a polypeptide chain of more than

900 amino acid residues and two metal ions (Fe3+ and Mo6+) and three organic cofactors (Flavin

Adenine Dinucleotide (FAD), Heme, and Molybdopterin) (Campbell, 1999). Since the natural

NaR is of low abundance in plants, algae and fungi, recombinant DNA technology is utilized to

produce the enzyme from a plant NaR gene (Arabidopsis thaliana) in the yeast Pichia pastoris

which is designated AtNaR2 (Campbell et al., 2006). A second form of NaR, which is 

designated YNaR1 (Barbier et al., 2004), is produced from a yeast NaR gene (Pichia angusta, 

recently renamed Ogataea angusta, but originally named Hansenula polymorpha) 

recombinantly expressed in the yeast Pichia pastoris.

Recombinant AtNaR2 and YNaR1 are purified from the Pichia yeast extract to near

homogeneity using immobilized metal ion affinity chromatography via the Histidine-tag built 

into the recombinant gene products. The purified AtNaR2 and YNaR1 are highly stable and

can be stored frozen in a buffered solution at -80°C indefinitely. Furthermore, when the 

AtNaR2 and YNaR1 are freeze-dried and stored, dry and under vacuum in an opaque package,

they can be shipped at room temperature and will remain stable for up to 6 months.

NaR-based Nitrate-N analysis is formulated as a method with a small volume, which is 

ideal for modern instruments such as the automated Discrete Analyzer (DA) and Flow Injection 

Analyzer (FIA). The method’s formulation consists of a biochemical buffer to maintain pH 

near neutrality, the reconstituted NaR (stable for 18 hours), a precise solution of NADH, and the 

small volume of sample to be analyzed for Nitrate-N content. For example, in the discrete

analyzer, the volume of buffered AtNaR2 is 55 μL, NADH 12 μL, and sample 5 μL (Patton and

Kryskalla, 2011; 2013). Compared to EPA method 353.2, where the sample is often 20 mL, the

enzymatic reduction method has obvious advantages in sample and waste handling. After the

reduction of nitrate to nitrite is complete, requiring about 10 min, the nitrite is determined

colorimetrically as in EPA method 353.2, which involves the sequential addition of

sulfanilamide (SAN) and N (1 Naphthyl)ethylenediamine dihydrochloride (NED) and 

measurement at 540 ± 20 nm.
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Section 2 Inter-Laboratory Validation Study

The details of the Inter-Laboratory Validation Study are presented in this section. The Nitrate

Elimination Co., Inc. (NECi), Lake Linden, Michigan, coordinated the Inter-Laboratory

Drinking Water Validation Study of the Enzymatic Reduction Method based on Nitrate

Reductase for determination of nitrate-N and nitrate/nitrite-N. In this study the Enzymatic

Reduction Method for nitrate-N is directly compared to the Cadmium Reduction Method for 

nitrate-N (EPA Reference Method 353.2), in each participating laboratory for each sample 

matrix. 

Section 2.1 Study Objective and Design Plan

The objective of the Inter-Laboratory Study of Nitrate-N Analysis by the Enzymatic Reduction

Method based on Nitrate Reductase was to demonstrate the validity of the Method according to

the Design Plan presented in Appendix A. The Design Plan was developed in accordance with

Protocol for the Evaluation of Alternate Test Procedures for Organic and Inorganic Analytes in 

Drinking Water, Office of Water (MS-140), EPA 815-R-15-007, February, 2015.

The Study Plan was approved, 7July2015, by Dr. Steven Wendelken, Safe Drinking Water Act

ATP Coordinator, U.S. Environmental Protection Agency, (Steven C. Wendelken, PhD, U.S. 

EPA, OGWDW/SRMD/TSC, 26 W. Martin Luther King Dr., Cincinnati, OH 45219).

The Sample Matrices analyzed in the Inter-Laboratory Drinking Water Validation Study are

listed in Table 1. The Enzymatic Reduction Method based on Nitrate Reductase is described

according to EPA format in Appendices B and C, which were incorporated in the Design Plan.

This Method was implemented by the Participating Laboratories which are listed in Table 2, 

and directly compared to EPA Method 353.2. 

The variety of drinking water matrices analyzed, along with direct comparison to the EPA 

Reference Method, validates the Nitrate-N Analysis by the Enzymatic Reduction Method based

on Nitrate Reductase for compliance monitoring under National Primary Drinking Water 

Regulations (NPDWRs).
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Section 2.2 Sources of Sample Matrices and Handling of the Samples

The list of Sample Matrices is presented in Table 1. The sample matrices were shipped in 

certified clean bottles according to the Study Plan and EPA protocols. Each sample Matrix

bottle was labeled with the designated matrix identification (i.e. DW-2, and DW-3). Certified

Nitrate Standards and Calibrants were purchased from ERA in individual, labeled bottles such

that there was no handling of these until they reached the participating Laboratories. The

packaged and sealed Sample Matrices and Nitrate Standards were shipped on “blue ice” to the 

Participating Laboratories by NECi (See Table 3). According to the Design Plan, once the 

Sample Matrices were shipped, the Participating Laboratories were to store them at 4°C until 

they completed the analysis. Certification documents are provided in Appendix D.

Section 2.3 Participating Laboratories and Analytical Equipment

The list of Participating Laboratories is presented in Table 2. Two Participating Laboratories

(Labs 1 and 2) were equipped with automated discrete analyzers (DA), which followed the 

Nitrate Reductase Nitrate-N Method described in Appendix B. One Participating Laboratory 

(Lab 3) used a Lachat Flow Injection Analyzer with off-line nitrate reduction as described in 

Appendix C. Each Participating Laboratory also analyzed the sample matrices using an 

automated Cadmium Reduction Analyzer running EPA Method 353.2.

The discrete analyzers used were: Lab 1, AquakemTM Photometric Analyzer Model 200 

(Thermo-Scientific Instruments); and Lab 2, SMARTCHEM® Model 200 (Unity Scientific). 

Lab 1 reported results as Absorbance @ 540 nm, while Lab 2 reported results as Absorbance @ 

550 nm.

Reagents for the Nitrate analysis, including the enzyme Nitrate Reductase (AtNaR2 or YNaR1),

were supplied to all laboratories by NECi. The enzyme (AtNaR2 or YNaR1) consisted of a

vacuum sealed, opaque pouch with a desiccant and a vial of freeze-dried AtNaR2 or YNaR1

containing one unit of enzyme activity, where the unit of enzyme activity is defined as the 

amount of enzyme catalyzing the NADH-driven reduction of 1 μmol of nitrate to nitrite per min 

at 30°C and pH 7.5. AtNaR2 and YNaR1 when stored in this form at room temperature (~25°C) 

are stable for up to one year. Each laboratory reconstituted the enzyme in phosphate buffer, pH 

7.5, at the time of analysis according to instructions provided with the enzyme packet. For each

type of DA being used in the study, a specific set of Standard Operating Procedures (SOP) has

been developed which condenses the Method in Appendix B.
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Section 3 Validation Study Results and Discussion

The results from the Inter-Laboratory Validation Study are contained in the Excel® files in

Appendix E. The original Excel® files have been provided on a flash drive, which accompany

this Report. Included within the Summary Excel® file are the “bench sheets” where possible 

and, in some cases, as PDF or other files in the directory for each laboratory in the provided 

data sets. An explanation table for error codes used in the KoneLab AquaKem® DA raw data

files is provided in Appendix E. For this section of the Report, summary tables have been

prepared from the original Excel® Summary Data Sheet files (Appendix E).

Section 3.1 Summary of Quality Control

While summary data are present in this report for Quality Control Samples, it is to be understood 

that all analytical analysis done in this study were for data sets that were in compliance with QC 

acceptance standards. This stipulation applies for both the Enzymatic Reduction Method for 

Nitrate Analysis using Nitrate Reductase and Cadmium Reduction Method for Nitrate Analysis 

(Reference Method EPA 353.2).

Statistical analysis of Calibration Curves reported by each laboratory are summarized in Table

4. In many cases, the laboratories ran more than one standard curve and the summary in Table

4 represents selected statistical data. In all cases, the correlation coefficient (r2) = 0.999 or

greater. In each case, the regression fit parameters are utilized to generate an equation relating

the Absorbance @ 540 nm (or 550 nm or Area in volts/sec) to the Nitrate-N content of the 

unknown sample, such that the concentration of Nitrate-N (mg N/L) can be calculated from the 

Absorbance or area of the absorbance peak in the case of FIA.

Enzymatic and cadmium reduction efficiencies for each laboratory are summarized in Table 5

by showing all data for each lab. All labs found enzymatic and cadmium reduction efficiency

of 93% or greater, which meets the acceptance criterion of 90% or greater reduction efficiency.

This establishes the effectiveness of the nitrate reductase-catalyzed enzymatic reduction of

nitrate to nitrite under the conditions of the analytical method, as well as the cadmium reduction 

efficiency under the conditions of the analytical method.

All the Initial Performance and Recovery (IPR) and Ongoing Performance and Recovery (OPR) 

results from all laboratories are summarized in Table 6. IPR certified standard nitrate

concentration was 2.01 mg N/L (see Appendix D). IPR mean recoveries were from 98 to 103%

for NaR Reduction Analysis, and all data met the acceptance criterion of 100 ± 10%. IPR 

individual mean recoveries were from 97 to 99% for Cadmium Reduction Analysis, and all data 

met the acceptance criterion of 100 ± 10%.
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OPR certified standard nitrate concentrations were 2.01, 2.50, and 12.00 mg N/L (see Appendix

D). OPR mean recoveries for all data by all labs in Table 6 were from 99 to 106% which met the

acceptance criterion of 100 ± 10%. All other OPR recoveries, which are not shown in Table 6,

were within the acceptable range of 90 to 110% recovery (Appendix E - Excel® files).

The final Quality Control evaluation is the determination of Minimum Detection Limit (MDL)

for each laboratory’s equipment; these results are summarized in Table 7. All the MDL values

are below the level of the lowest calibrant for these analyzers (Table 4), which indicates that the 

calibration curve for these analyzers is completely valid with respect to detecting nitrate-N at

the lowest level of the calibration. Lab 1 had the highest MDL values found in this study for 

both the Nitrate Reductase Method on the AquaKem Discrete Analyzer, 0.0097 mg N/L, and the 

Cadmium Reduction Method, 0.03436 mg N/L (Table 7). Lab 2 found almost identical MDL 

values for the two methods: Nitrate Reductase Method, 0.006507 mg N/L; and Cadmium 

Reduction Method, 0.006694 mg N/L. Lab 3 running the FIA system found a higher MDL for 

the Nitrate Reductase Method, 0.009301, than the Cadmium Reduction Method, 0.004721 mg 

N/L. Since the MDL is dependent on the analytical equipment used with the Methods, the MDL 

values from the different labs cannot be compared directly.
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Section 3.2 Nitrate-N Content and Spike Analysis of Drinking Water Matrices

Each laboratory participating in the Study analyzed local tap water (DW-1) and the tap water 

spiked with 2 to 4 mg/L free chlorine (DW-1-Cl) using both Methods for Nitrate-N Analysis. 

In all cases the tap water was from a regulated municipal drinking water supply: Lab 1, Chicago 

City Water; Lab 2, East Lyme Water Supply (Southeastern CT), City Water; and Lab 3, 

Loveland, CO, City Water. 

The results from Lab 1, 2, and 3, for determination of the Nitrate-N Content and Spiking 

Studies are presented in Table 8 A, B, and C. For Lab 1 analyzing with the NaR Reduction

Method, the Nitrate Content of the “as is” tap water (DW-1) and chlorinated tap water (DW-1-

Cl), were virtually the same, 0.3237 ± 0.003698 and 0.3185 ± 0.01625 Nitrate-N mg/L, 

respectively, for the mean of 7 replicates and the standard deviation (Table 8A, first page). 

Both DW-1 and DW-1-Cl were spiked in duplicate according to the Study Plan with 3 levels of 

Nitrate-N mg/L, 0.5, 1.0, and 2.5, for analysis with the NaR Reduction Method (Table 8A, first 

page). All the spike recoveries and recovery percentages were within acceptable limits of 90 to 

110% and the Relative Percent Difference between the MS and MSD were also acceptable as 

less than 20%. There was virtually no difference between the results of spiking studies for 

DW-1 and DW-1-Cl, which indicates that chlorination of the tap water did not cause a matrix 

effect in the NaR Reduction Method for these samples.

For Lab 1 applying the Cd Reduction Method, the Nitrate Content of the “as is” tap water 

(DW-1) and chlorinated tap water (DW-1-Cl), were virtually the same, 0.2259 ± 0.008633 and 

0.2186 ± 0.007162 Nitrate-N mg/L, respectively, for the mean of 7 replicates and the standard 

deviation (Table 8A, second page). However, these Nitrate-N contents were lower than found 

by the NaR Reduction Method. Both DW-1 and DW-1-Cl were spiked in duplicate according 

to the Study Plan with 3 levels of Nitrate-N mg/L, 0.5, 1.0, and 2.5, for analysis with the Cd

Reduction Method (Table 8A, second page). All the spike recoveries and recovery percentages 

were within acceptable limits of 90 to 110% and the Relative Percent Difference between the 

MS and MSD were also acceptable as less than 20%. There was virtually no difference 

between the results of spiking studies for DW-1 and DW-1-Cl, which indicates that chlorination 

of the tap water did not cause a matrix effect in the Cd Reduction Method for these samples.

Similar results were found by Lab 2 and Lab 3 for analysis of their tap water with and without 

chlorination. These results are summarized in a Table 8B and 8C in this report. Labs 2 and 3, 

also found that there was no matrix effect for either the NaR Reduction Method or Cd 

Reduction Method for chlorination of their local tap water. Lab 3 reported spike recoveries for 

0.5 mg N/L for chlorinated DW-1 analyzed by cadmium reduction were both less than 90%, 

which are not an acceptable results; however, results for 1.0 and 2.5 mg N/L were acceptable.
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For the DW-2 High TDS Drinking Water Matrix, Lab 3 results for determination of the NitrateN Content and Spiking Studies are presented in Table 9. For the NaR Reduction Method, the 

Nitrate Content of the DW-2 was, 0.2959 ± 0.004749 Nitrate-N mg/L, respectively, for the 

mean of 7 replicates and the standard deviation (Table 9, left column). DW-2 was spiked in 

duplicate according to the Study Plan with 3 levels of Nitrate-N mg/L, 0.5, 1.0, and 2.5, for 

analysis with the NaR Reduction Method (Table 9, left column). All the spike recoveries and 

recovery percentages were within acceptable limits of 90 to 110% and the Relative Percent 

Difference between the MS and MSD were also acceptable as less than 20%. Analysis of the 

Nitrate Content of DW-2 with the Cd Reduction Method yielded 0.2677 ± 0.001071 Nitrate-N 

mg/L (Table 9, right column). DW-2 was spiked in duplicate according to the Study Plan with 

3 levels of Nitrate-N mg/L, 0.5, 1.0, and 2.5, for analysis with the Cd Reduction Method (Table 

9, right column). All the spike recoveries and recovery percentages were within acceptable 

limits of 90 to 110% and the Relative Percent Difference between the MS and MSD were also 

acceptable as less than 20%. Thus, it is clear that the two methods produce very similar results 

for DW-2 and Drinking Water with High Total Dissolved Solids does not produce a matrix 

effect in either method.

For the DW-3 High TOC Drinking Water Matrix, Lab 2 results for determination of the NitrateN Content and Spiking Studies are presented in Table 10 For the NaR Reduction Method, the 

Nitrate Content of the DW-3 was, 0.003814 ± 0. 0.007267 Nitrate-N mg/L, respectively, for the 

mean of 7 replicates and the standard deviation (Table 10, left column). DW-3 was spiked in 

duplicate according to the Study Plan with 3 levels of Nitrate-N mg/L, 0.5, 1.0, and 2.5, for 

analysis with the NaR Reduction Method (Table 10, left column). All the spike recoveries and 

recovery percentages were within acceptable limits of 90 to 110% and the Relative Percent 

Difference between the MS and MSD were also acceptable as less than 20%. Analysis of the 

Nitrate Content of DW-3 with the Cd Reduction Method yielded 0.002450 ± 0.002696 NitrateN mg/L (Table 10, right column). DW-3 was spiked in duplicate according to the Study Plan 

with 3 levels of Nitrate-N mg/L, 0.5, 1.0, and 2.5, for analysis with the Cd Reduction Method 

(Table 10, right column). All the spike recoveries and recovery percentages were within 

acceptable limits of 90 to 110% and the Relative Percent Difference between the MS and MSD 

were also acceptable as less than 20%. Thus, it is clear that the two methods produce very 

similar results for DW-3 and Drinking Water with High Total Organic Carbon does not produce 

a matrix effect in either method. Drinking Water with a high level of humic substances (>2 

mg/L) will not interfere with the NaR Reduction Method when AtNaR2 is used as the catalyst

(Patton and Kryskalla, 2011; 2013; see also section 7.6 Note in Appendix B.
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3.3 Evaluation of the Accuracy of the Method

There was one reference standard analyzed by all the Participating Laboratories in this study

(Table 11). The standard was SR-1 (ERA #698) with certified target value = 5.14 ± 0.094 mg

N/L (see Appendix D). Since this level of Nitrate-N content is greater than the highest calibrant 

for most the Participating Laboratories for either method, the matrix was diluted prior to 

analysis. Lab 1 running the Cd Reduction Method had a calibration range going up to 20 mg 

Nitrate-N/L and did not need to dilute the sample prior to analysis with this method. Each Lab 

found the mean Nitrate-N content SR-1 to be within 10% of the ERA certified value using both 

methods (Table 11). Clearly, both methods are equally accurate for determining Nitrate-N 

content.

3.4 Comparison of the Enzymatic Reduction Method to the Cadmium Reduction Method

Every Drinking Water Sample Matrix analyzed in this Validation Study listed in Table 1 was 

analyzed by both the Nitrate Reductase Reduction and Cadmium Reduction Method, the EPA 

Reference Method (EPA Method 353.2). In general, no significant difference was found 

between the results obtained by the Nitrate Reductase Method and the Cadmium Reduction 

Method in Nitrate-N content in mg/L within the statistical limits of the methods for any of the 

Drinking Water Matrices.

With regard to Matrix Spike analysis, DW-1, DW-1-Cl, DW-2, DW-3 (see Table 1) were all 

analyzed by the Matrix Spike protocol using both the NaR Reduction Method and the Cd 

Reduction Method (Table 8, 9, and 10). No differences were found in the results of these 

studies. The results showed that none of these matrices produced a Matrix Effect on the results 

of either Method by demonstration of completely acceptable Spike Recoveries and acceptable 

comparison between the pairs of each Spike MS and MSD.
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Section 4 Validation Conclusions

The USEPA requires validation of an Alternate Test Procedure (ATP) Method that three

different laboratories analyze the analyte content of one sample of three different finished 

drinking water matrices (EPA, 2015). Thus, the ATP Method will be in compliance with the 

Safe Drinking Water Act (SDWA) and validate the ATP Method for compliance monitoring 

under National Primary Drinking Water Regulations (NPDWRs).

For the Enzymatic Reduction Method for Nitrate-N Analysis, the requirements of nationwide

validation were met by analysis of three different finished drinking water matrices (see Table 1

for list of Drinking Water Sample Matrices analyzed in this study and Tables 8, 9, and 10 for 

details of the analysis by 3 laboratories listed in Table 2). The spiking studies of the Drinking

Water Sample Matrices (spikes of 0.5, 1.0 and 2.5 mg Nitrate-N/L analyzed in duplicate)

indicated that there was no matrix effect on the Method by the three drinking water matrices

(Table 8, 9, and 10); including chlorination of tap water.

Accuracy of the Method was shown to be very high by analysis of the standard reference SR-1 

(Table 11) and the Quality Control results (Tables 6). The MDL evaluation of the equipment used 

in the study demonstrated that the Method has a detection limit of 0.050 mg N/L or less on the DA

and FIA analyzers used in the study (Table 7). Few interfering substances, if any, were discovered

in the present study.

Comparison of the analysis of the three drinking water matrices and the reference standard by

the Enzymatic Reduction Method and the certified Cadmium Reduction Method (EPA Method

353.2) indicated that very similar results were obtained (Table 8, 9, and 10).

In summary, the Enzymatic Reduction Method for Nitrate-N Analysis has been validated by a

robust Inter-Laboratory Study of Drinking Water Sample Matrices. All laboratories analyzing

the Sample Matrices met all Quality Control criteria for valid analyses prior to analyzing the 

samples. The Calibration Curve, Nitrate Reduction Efficiency, and IPR/OPR recoveries (Tables 

4, 5, and 6) were within the acceptable range before analyzing the unknown Sample Matrices. 

Analysis of certified Nitrate Standards indicated that the Method is highly accurate and capable 

of providing definitive analysis of Nitrate-N content of Drinking Water in the field. For

Discrete Analyzers running the Method, the MDL ranged from 0.0066 to

0.0097 mg N/L; while the Flow Injection Analyzer had an MDL = 0.0094 (Table 7). Thus, the 

requirements of the Alternate Test Procedure Protocol have been met for validation of the 

Enzymatic Reduction Method for Nitrate-N Analysis by the Inter-Laboratory Validation Study

reported herein. The Method is ready for compliance monitoring under National Primary 

Drinking Water Regulations (NPDWRs).
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Section 5 References and Glossary

Protocol for the Evaluation of Alternate Test Procedures for Organic and Inorganic Analytes in 

Drinking Water, Office of Water (MS-140), EPA 815-R-15-007, February 2015 

http://water.epa.gov/scitech/drinkingwater/labcert/upload/epa815r15007.pdf

Barbier, GG, JC Joshi, ER Campbell & WH Campbell (2004) Purification and biochemical 

characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris. 

Protein Expression & Purification. 37: 61-71.

Campbell, Wilbur H. (1999) Nitrate Reductase Structure, Function and Regulation: Bridging the

Gap between Biochemistry and Physiology, Annual Review of Plant Physiology and Plant

Molecular Biology 50:277-303.

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,

USGS Scientific Investigations Report 2013-5033 (http://pubs.usgs.gov/sir/2013/5033/).

Glossary:

DA = Automated Discrete Analyzer

ERA = http://www.eraqc.com/

FIA = Flow Injection Analyzer

IPR = Initial Performance and Recovery

OPR = Ongoing Performance and Recovery

MDL = Method Detection Limit

(http://www.gpo.gov/fdsys/pkg/CFR-2012-title40-vol24/pdf/CFR-2012-title40-vol24-

part136- appB.pdf)

MS = Matrix Spike
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MSD = Matrix Spike Duplicate

QA = Quality Assurance 

QC = Quality Control

SDWA= Safe Drinking Water Act

TDS = Total Dissolved Solids

TOC = Total Organic Carbon
Doc = Inter laboratory Validation Study Plan for Drinking Water 4.0.docx

Table 1.

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 1 and 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 and 3

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.
Doc = Inter laboratory Validation Study Plan for Drinking Water 4.0.docx

Table 1, NOTES:

A. Preparation of Chlorinated Tap Water:

Provided Chlorine Standard Solution, 25-30 mg/L as Cl2, 2 mL PourRite Ampules (NIST)

1. In clean tube, pipet 9.0 mL of tap water.

2. Open ampule of Chlorine Standard Solution.

3. Pipet 1.0 mL of Chlorine Standard Solution into tube with tap water.

4. Mix to prepare chlorinated tap water with 2.5 to 3.0 mg/L free chlorine.

5. Analyze the chlorinated tap water as DW-1+CL with 7 replicates by both methods.

6. Also analyzed chlorinated tap water as DW-1+CL spiked at 0.5, 1.0 and 2.5 mg N/L in 

duplicate (MS and MSD). One analysis each for MS and MSD.

B. Material Holding:

1. Sample Matrices, Standard Calibrants, and QC Samples will be held at 4°C until analyzed.

2. The list of materials to be shipped to each laboratory are enumerated in Table 3.

3. The materials will be shipped on “blue ice” (i.e. ~4°C) via overnight shipping.

C. Analysis Window:

1. Each Participating Laboratory will have thirty days (30 days) to complete analysis from the 

day they receive the materials.
Doc = Inter laboratory Validation Study Plan for Drinking Water 4.0.docx

Table 2.

Participating Laboratories

Lab # Lab Name Contact Analytical 

Equipment

1 USEPA Chicago 

Region 5 Lab

Francis Awanya, 

Chemist, 

Group Leader

Discrete Analyzer

And

Cd-Reduction 

Analyzer

2 Unity/Westco 

Scientific

Bill Georgian

Method Development

Discrete Analyzer

And

Cd-Reduction 

Analyzer

3 Lachat Instruments Lynn Egan

Flow Injection 

Analyzer – Both NaR 

and CdR Methods

Lab 1

USEPA Chicago

Francis Awanya

312-886-3682

awanya.francis@epa.gov

536 Clark St.

Chicago, IL 60605

Lab 2

Unity/Westco Sci.

Bill Georgian

billg@westcoscientific.com

Lab 3

Lachat Instruments

Lynn Egan

legan@hach.com
Table 3

List of Materials Shipped to Three (3) Participating Labs

And 

Two (2) Complete Sets Retained by NECi

Certification Documents for Items are in Appendix D

Item Source Preparation Volume (mL) Date Shipped

DW-2 NECi NECi 50

8Jul2015

DW-3 ERA NECi 50

SR-1 ERA NECi 50

Chlorine Standard 

Solution Hach Sealed Ampule 2.0

2.01 mg Nitrate-N/L ERA NECi 50

2.50 mg Nitrate-N/L ERA NECi 50

15.0 mg Nitrate-N/L ERA NECi 50

50.0 mg Nitrate-N/L ERA NECi 50

1000 mg/L Nitrite-N ERA NECi 10
Table 4

Calibrants and Standard Calibration Curve Equation for Participating Labs

0BLab 1 1BLab 2 2BLab 3

Calibration NaR-R Cd-R NaR-R Cd-R NaR-R Cd-R

Reagent Blank 0.00 0.00 0.00 0.00 0.00 0.000

Calibrant 1 0.05 0.10 0.05 0.05 0.05 0.050

Calibrant 2 0.25 0.25 0.10 0.10 0.10 0.125

Calibrant 3 0.50 0.50 0.25 0.25 0.50 0.250

Calibrant 4 0.75 1.00 0.50 0.50 1.00 0.500

Calibrant 5 1.25 5.00 1.25 1.25 2.50 1.000

Calibrant 6 2.50 10.00 2.50 2.50 5.00 2.500

Calibrant 7 3.75 20.00 5.00 5.00 5.000

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 10B--- -0.029 -0.1988 0.4224 0.0018733 .00010392

11BConstant B 12B--- 1.262 15.0219 5.1881 0.5099 0.0472

13BConstant C 14B--- 0.018 -0.5308 -0.1879 0.1713 0.0011335

15BR2 16B--- 0.99996 1.00000 1.00000 0.99993 1.00000
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.

17BLab 1 18BLab 2 19BLab 3*

NaR-R Cd-R NaR-R Cd-R NaR-R Cd-R

20BNitrate

21BAbsorbance 

540 nm,

22B550 nm,

23BOr Area/sec

0.318

0.495

0.491

0.2080

0.1982

0.1909

0.1929

0.1792

0.4915

0.4832

0.4420

0.4018

0.5390

5.4019

9.1622

47.942

28.501

24BNitrite

25BAbsorbance 

540 nm,

26B550 nm,

27BOr Area/sec

0.341 0.527

0.501

0.1963

0.1892

0.1853

0.1881

0.1787

0.5082

0.4981

0.4484

0.4024

0.5777

5.7256

8.9972

49.528

27.381

28BCatalytic

29BReduction

30BEfficiency 

31B(NO3/NO2)

32BPercent

93.2551 93.9279

98.0040

105.9603

104.7569

103.0221

102.5518

100.2798

96.7139

97.0086

98.5727

99.8509

93.3010

94.3464

101.8339

96.7978

104.0904

*Lab 3 ran a series of 10-12 of these tests, which are not shown here.
Table 6

All Data Reported by Each Lab for

Initial Performance and Recovery (IPR)

and

Ongoing Performance and Recovery (OPR)

33BLab 1 34BLab 2* 35BLab 3

NaR-R Cd-R NaR-R Cd-R NaR-R Cd-R

36BIPR 

37BNitrate-N 

mg/L

2.01 2.01 2.01 2.01 2.01 2.01

38BNumber IPR 39B4 40B8 41B16 42B16 8 8

43BMean IPR 44B2.0705 1.9598 1.9682 45B1.9921 1.9827 1.9951

46BMean IPR 

Recovery (%) 47B103.0075 48B97.5044 49B97.9201 99.1091 50B98.6412 99.2562

51BOPR 

52BNitrate-N 

mg/L

53B2.01 54B12.00 55B2.50 56B2.50 57B2.50 58B2.50

59BNumber OPR 7 8 60B15 61B16 62B14 63B13

64BMean OPR 2.1388 11.8393 2.4769 2.5189 2.4877 2.5319

65BStandard 

Deviation of 

Mean

0.03685 0.208999 0.07204 0.1145 0.11536 0.01287

66BRelative 

Standard 

Deviation (%)

1.7233 1.7653 2.9085 4.5475 4.6372 0.5082

67BMean 

Recovery (%) 68B106.4065 69B98.6610 70B99.0740 100.7564 99.5083 101.277

*Lab 2 Cd-R had 1 OPR greater than 110%
Table 7

Minimum Detection Limit

7 Replicates

MDL = Standard Deviation x 3.143

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

71BLab 1 72BLab 2 73BLab 3

NaR-R Cd-R NaR-R Cd-R NaR-R Cd-R

74BAnalytical 

Equipment 75BDA 76BDA 77BDA 78BDA 79BFIA 80BFIA

81BSpike

82Bmg Nitrate-N/L 83B0.050 84B0.050 85B0.025 86B0.020 87B0.010 88B0.010

89BMDL 

90Bmg Nitrate-N/L

0.0097 0.03436 0.006507 0.006694 0.009301 0.004721

91BRatio 

92BSpike/MDL

5.1453 1.4553 3.8422 2.9877 1.0751 2.1182
Table 8

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
Lab 1 Cd Reduction Method DW1

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

0.066 0.2429

0.2259 ±

0.008633

RSD

3.7773%

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 %

0.063 0.2260

0.154 0.7066 0.4807 96.1486

7.1602

0.258 1.2516 1.0257 102.5743

2.1581

0.526 2.6585 2.4326 97.3057

0.7331

0.062 0.2217

0.064 0.2305

0.161 0.7423 0.5164 103.2886 0.253 1.2297 1.0038 100.3843 0.529 2.6764 2.4505 98.0217 0.062 0.2230

0.062 0.2185

0.062 0.2184

Lab 1 Cd 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

0.063 0.2233

0.2186 ±

0.007162

RSD

3.2763%

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 %

0.062 0.2224

0.159 0.7353 0.5167 103.3371

4.0278

0.258 1.2515 1.0329 103.2886

2.6884

0.538 2.7229 2.5043 100.1714

3.4193

0.063 0.2267

0.061 0.2173

0.156 0.7149 0.4963 99.2571 0.252 1.2241 1.0055 100.5486 0.532 2.6387 2.4201 96.8038 0.061 0.2151

0.062 0.2206

0.059 0.2049
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
Lab 2 Cd Reduction Method DW1

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

0.2295 0.9208

0.910579 ±

0.025085

RSD

2.7549%

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 %

0.2195 0.8726

0.3248 1.3804 0.4699 93.9708

1.4507

0.4188 1.8358 0.9252 92.5184

0.2101

0.7264 3.3391 2.4285 97.1403

0.7933 0.2246 0.8972

0.2235 0.8919

0.3234 1.3737 0.4631 92.6174 0.4184 1.8338 0.9232 92.3242 0.7225 3.3199 2.4093 96.3728 0.2327 0.9362

0.2278 0.9126

0.2341 0.9429

Lab 2 Cd 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

0.2256 0.9020

0.9050 ±

0.009564

RSD

1.0567%

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 %

0.2278 0.9126

0.3236 1.3746 0.4696 93.9249

6.1848

0.4213 1.8479 0.9429 94.2891

1.4314

0.7270 3.3420 2.4370 97.4812

1.6897

0.2250 0.8991

0.2237 0.8928

0.3298 1.4046 0.4996 99.9193 0.4241 1.8615 0.9565 95.6484 0.7187 3.3012 2.3962 95.8479 0.2289 0.9179

0.2246 0.8972

0.2280 0.9135
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 Data Set 30Oct2015

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 Data Set 30Oct2015

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
Lab 3 Cd Reduction Method DW1 Data Set 30Oct2015

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

1.7679 0.0849

0.08778 ±

0.001313

RSD

1.4962%

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 %

1.8455 0.0886

11.749 0.5700 0.4822 96.4493

0.1326

21.883 1.0838 0.9960 99.6017

0.6405

50.04 2.6233 2.5355 101.4207

0.0316 1.8438 0.0885

1.8407 0.0884

11.762 0.5707 0.4829 96.5773 21.983 1.0902 1.0024 100.2417 50.026 2.6225 2.5347 101.3887 1.8407 0.0884

1.8363 0.0882

1.8240 0.0876

Lab 3 Cd Reduction Method DW1-Cl Data Set 30Oct2015

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

2.8934 0.1386

0.1387 ±

0.0004249

RSD

0.3063%

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 %

2.9201 0.1390

11.616 0.5634 0.4294 85.8743

0.1350 21.738 1.0763 0.9376 93.7571 0.2135 49.520 2.5933 2.4546 98.1829 0.2978 2.9069 0.1392

2.9061 0.1392

11.722 0.5687 0.4300 85.9903 2.8956 0.1387 21.699 1.0743 0.9356 93.5571 49.392 2.5860 2.4473 97.8909

2.8895 0.1384

2.8831 0.1381
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 

NitrateN 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

Lab 3 Cd Reduction Method DW-2

Nitrate-N 

mg/L

Mean 

NitrateN mg/L

Standard 

Deviation

mg N/L

Relative 

Standard 

Deviation

0.2659

0.2677 0.001071 0.4000%

0.2684

0.2678

0.2679

0.2680

0.2693

0.2669

0.5 mg N/L Spike

A-540 mg 

N/L

Spike 

mg 

N/L

Recovery 

% RPD %

15.418 0.7536 0.4858 97.1697

0.4125

15.386 0.7516 0.4838 96.7697

1.0 mg N/L Spike

A-540 mg N/L Spike 

mg N/L

Recovery 

% RPD %

24.932 1.2425 0.9748 97.4759

0.2050

24.970 1.2445 0.9768 97.6759

2.5 mg N/L Spike

A-540 mg N/L Spike 

mg N/L

Recovery 

% RPD %

53.061 2.7602 2.4925 99.6983

0.5512

52.407 2.7465 2.4788 99.1503
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

NitrateN 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 %

0.062

1

0.498

1

0.494

3 98.8571

3.810

0.063 4

3

0.517

3

0.513

5

102.697

1

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

Lab 2 Cd Reduction Method DW-3

NitrateN mg/L

Mean 

Nitrate-N 

mg/L

Standard 

Deviation

mg N/L

Relative 

Standard 

Deviation

0.0044

0.002450 0.002696 110.0301%

0.0018

0.0057

-0.0021

0.0031

0.0018

0.0025

0.5 mg N/L Spike

A-540 mg 

N/L

Spike 

mg 

N/L

Recovery 

% RPD %

0.1106 0.5147 0.5123 102.4500

3.0723

0.1082 0.4992 0.4968 99.3500

1.0 mg N/L Spike

A-540 mg 

N/L

Spike 

mg N/L

Recovery 

% RPD %

0.1923 1.0417 1.0393 103.9250

1.5373

0.1948 1.0578 1.0554 105.5350

2.5 mg N/L Spike

A-540 mg 

N/L

Spike 

mg N/L

Recovery 

% RPD %

0.4405 2.6422 2.6398 105.5900

3.2766

0.4273 2.5571 2.5547 102.1860
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 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%