Environmental Protection Agency

Alternate Test Procedure Validation Report 

for Enzymatic Reduction Method of Nitrate-N Analysis

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

Revised: 20 February 2014
NECi ATP Validation Study Report 3.0

NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page ii

Acknowledgments:

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

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

OI Analytical Co., are thanked for their assistance with the design of the Inter-Laboratory ATP 

Validation Study and preparation of the Validation Study Report.

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

Inter-Laboratory ATP Validation 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 20 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.
NECi ATP Validation Study Report 3.0

NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page iii

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 4

Section 3.1 Summary of Quality Control 5

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

Section 3.3 Evaluation of Accuracy for the Method 7

Section 3.4 Comparison of Enzymatic Reduction and Cadmium Reduction Methods 8

Section 4 Validation Conclusions 9

Section 5 References and Glossary 11

Tables

Table 1 List of Sample Matrices

Table 2 List of Participating Laboratories

Table 3 Calibration of Enzymatic Reduction Method Summary

Table 4 Enzymatic Reduction Efficiency Summary

Table 5 Initial Performance and Recovery (IPR) Summary

Table 6 Ongoing Performance and Recovery (OPR) Summary

Table 7 Minimum Detection Limit (MDL) Summary

Table 8 Nitrate-N Content and Spike Analysis of Sample WastewaterWastewater Matrices

Table 9 Detailed Data Supporting Table 8

Table 10 Standard Reference Materials Summary

Table 11 Comparison of Nitrate-N Content for the Sample Matrices Determined by Nitrate 

Reductase Method, and by Cadmium Reduction Method

Appendices – Appendices are not included contact NECi if needed

Appendix A Inter-Laboratory Validation Study Plan

Appendix B Description of the Enzymatic Reduction Method by Discrete Analyzer

Appendix C FACET Protocol for Sample Handling and Shipping

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

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

Appendix F Summary Data Sheets for MDL (Excel® Files)
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 1

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 Tier 3 level (nationwide) 

determination of nitrate plus nitrite nitrogen in wastewater, and other aqueous solutions (EPA, 

1999). The enzyme Nitrate Reductase (EC 1.7.1.1, NaR) 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 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). Recombinant AtNaR2 is purified from the 

yeast extract to near homogeneity using immobilized metal ion affinity chromatography via the 

Histidine-tag built into the recombinant gene product. The purified AtNaR2 is highly stable and 

can be stored in a buffered solution at -80°C indefinitely. Furthermore, when the AtNaR2 is 

freeze-dried and stored, dry and under vacuum in an opaque package, it 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. 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 NitrateN 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. 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 3

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 

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 not directly compared to the Cadmium Reduction Method for nitrate-N (EPA 

Reference Method 353.2), since the automated discrete analyzers used by the participating 

laboratories do not run the Reference Method. However, one participating laboratory analyzed 

the study’s sample matrices using the Cd Reduction Method running on a continuous flow 

analyzer and this provided a comparative data set. In addition, two published studies have 

compared the Enzymatic Reduction Method to the Cd Reduction Method and demonstrated the 

equivalence of the two methods for determining nitrate-N and nitrate/nitrite-N in aqueous 

samples (Patton and Kryskalla, 2011; Patton and Kryskalla, 2013).

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 

ATP Protocol for Organic and Inorganic Analytes (USEPA, 1999) in order to validate the 

Method for Tier 3 (nationwide) status for both Drinking and Wastewater. The Study Plan was 

approved by Lemuel (Lem) Walker, Jr., Clean Water Act ATP Coordinator, U.S. Environmental 

Protection Agency, Office of Science and Technology, Engineering and Analysis Division 

(EAD) on 14June2013 (see Appendix A). Subsequently, the scope of the Inter-Laboratory Study 

was narrowed to just the Wastewater and Seawater Matrices, but the Study Plan was not altered 

to reflect this change. The Sample Matrices analyzed in the Inter-Laboratory Validation Study 

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

according to EPA format in Appendix B, which was incorporated in the Design Plan. This 

Method was implemented by the Participating Laboratories which are listed in Table 2. The 

variety of wastewater matrices to be analyzed brings the study into compliance with the Clean 

Water Act (CWA).

Section 2.2 Sources of Sample Matrices and Handling of the Samples

The list of Sample Matrices is presented in Table 1. All matrices were collected on site and 

processed by filtration and preservation by addition of 4.5 Normal sulfuric acid to lower the pH 

to below 2 and cooled to <6°C, according to the Design Plan. The preservation status of the 

sample matrices is shown (Table 1).
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 4

The sample matrices were shipped in bulk to FACET Analytical Services and Technology, LLC, 

Indianapolis, IN 46217. FACET LLC filtered, acidified and aliquoted the sample matrices to 

certified clean bottles according to the Study Plan and EPA protocols (Appendix C). Each 

sample Matrix bottle was labeled with the designated matrix identification (i.e. WW-1, WW-2, 

etc.). 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 according to the plan developed by FACET and approved by 

NECi as shown in Appendix C. According to the Design Plan, once the Sample Matrices were 

shipped, the Participating Laboratories were to store them at 4°C and they had to complete the 

analysis within 30 days. The deadline for analyses completion was July 17, 2013. Certification 

documents for the calibrants and nitrate standards are provided in Appendix D. Each laboratory 

provided their own nitrite standard and the certification for the Nitrite Standard utilized by one 

laboratory is also included in Appendix D.

Section 2.3 Participating Laboratories and Analytical Equipment

The list of Participating Laboratories is presented in Table 2. A more detailed list of information 

on the Participating Laboratories is presented in Table 2 in the Study Plan (Appendix A). 

Laboratory 6 dropped out of the study after the samples had been shipped to them and did not 

complete the analysis of the samples. All the Participating Laboratories were equipped with 

automated discrete analyzers (DA).

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

supplied to all laboratories by NECi. The enzyme (AtNaR2) consisted of a vacuum sealed, 

opaque pouch with a desiccant and a vial of freeze-dried AtNaR2 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 

when stored in this form at room temperature (~25°C) is stable for up to six months. 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. As an example, the SOP for the OI Analytical DA has

been added to the Method in Appendix B.

Section 3 Validation Study Results and Discussion

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

Appendix E and F. The original Excel® files have been provided on a flash drive, which 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 5

accompanied the original version of this Report and have not been included with the current 

version of 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 

previously provided data sets. An explanation table for error codes used in the KoneLab 

AquaKem® DA raw data files is provided in Appendix F. Supplemental Excel® File for the 

MDL Study by Laboratory 5 is provided in Appendix F, which is supplied by email attachment. 

For this section of the Report, summary tables have been prepared from the original and 

supplemental data in the Excel® Summary Data Sheet files Appendix E and F).

Section 3.1 Summary of Quality Control

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

3A. In many cases, the laboratories ran more than one standard curve and the summary in Table 

3 represents selected statistical data. In all cases, the correlation coefficient (r2

) = 0.999 or 

greater, except for Lab 7 and 10. However, both of these labs used a polynomial fit to the 

calibration results and found r2= 0.9999 and 0.9991, for labs 7 and 10, respectively. In each 

case, the parameters for the slope and intercept are utilized to generate an equation relating the 

Absorbance @ 540 nm (or 550 nm) 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. In cases where 

a polynomial equation is used to described the relationship of Absorbance to Nitrate-N content, 

the slope is replaced by constants “A” and “B”, which are not listed in the table.

Table 3B catalogs the nitrate concentrations of the standards used in the calibration. This 

information is useful in comparing the results from different labs, since some labs used as many 

as 8 calibration concentrations, while some used as few as 5 calibration concentrations. In 

addition, Lab 12 used 0.10 mg N/L as the lowest calibrant, while most of the other labs using a 

discrete analyzer used 0.05 mg N/L (Lab 7 used 0.04 mg N/L).

Enzymatic reduction efficiencies for each laboratory are summarized in Table 4. All labs found 

enzymatic reduction efficiency of 95 % or greater. This establishes the effectiveness of the 

nitrate reductase-catalyzed enzymatic reduction of nitrate to nitrite under the conditions of the 

analytical method.

Initial Performance and Recovery (IPR) and Ongoing Performance and Recovery (OPR) results 

from all laboratories are summarized in Table 5 and 6, respectively. IPR certified standard 

nitrate concentrations were 2.00, 2.50, and 2.70 mg N/L (see Appendix D). IPR mean recoveries 

for the selected data in Table 5 were from 96 to 106%, which meets the acceptance criterion of 

100 ± 10%. Other IPR analysis by the labs, which are not shown in Table 5, also fell within the 

acceptance criteria of 90 to 110% recovery (Appendix E - Excel® files). OPR certified standard 

nitrate concentrations were 2.00, 2.50, and 2.60 mg N/L (see Appendix D). OPR mean 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 6

recoveries for the selected data in Table 6 were from 96 to 104% which meets 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 IPR and OPR results provide measures of the precision and accuracy of the Enzymatic 

Reduction Method of Nitrate-N Analysis. For IPR, five labs used the ERA 2.00 ± 0.02 mg N/L 

certified nitrate standard and obtained the mean value = 1.985 ± 0.048; RSD = 2.4391. Four labs 

used an 2.50 mg N/L certified nitrate standard and obtained the mean value = 2.573 ± 0.058 mg 

N/L; RSD = 2.2418 % and. Only one lab used the 2.70 mg N/L certified nitrate standard and 

found the mean value = 2.730 ± 0.026 mg N/L; RSD = 0.9463 %. Thus, there was less than a 

2% difference between the values determined and the known values of the certified nitrate 

standards. The OPR values yielded RSD from 0.52 to 5.66 % (Table 6). Thus, the Enzymatic 

Reduction Method is highly precise and accurate based on these IPR and OPR results.

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 3B), 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.

Three published studies evaluated the MDL for Enzymatic Reduction Method for Nitrate-N 

Analysis (Patton et al., 2002; Patton and Kryskalla, 2011; Patton and Kryskalla, 2013). When 

the Method was run on an Air-segmented Continuous Flow Analyzer (Patton et al., 2002), the 

MDL was reported to be 0.006 mg N/L. When the Method was run on a Discrete Analyzer 

(Patton and Kryskalla, 2011), the MDL was reported to be 0.02 mg N/L. When the MDL of the 

reference method, EPA Method 353.2, was determined on an Air-segmented Continuous Flow 

Analyzer (Patton et al., 2002; Patton and Kryskalla, 2013), it was found to be 0.003 mg N/L. 

The lower MDL for the reference method is apparently due to differences in the analyzer 

equipment: Air-segmented Continuous Flow Analyzer uses the same cuvette for analyzing all 

samples and blanks; and the Discrete Analyzer uses a different cuvette for every sample and 

blank. Indeed, the Enzymatic Reduction Method for Nitrate-N Analysis, has a lower MDL for 

the Air-segmented Continuous Flow Analyzer than the Discrete Analyzer. Although the 

Discrete Analyzer uses a correction for background absorbance, it apparently does not correct for 

all the differences between the cuvettes (Patton and Kryskalla, 2011).

Section 3.2 Nitrate-N Content and Spike Analysis of Wastewater and Seawater Matrices

The Wastewater Matrices were analyzed for Nitrate-N content and spiked with 0.5 mg N/L and 

analyzed 6 times. These results are summarized in Table 8 and more detailed presentation of 

these results are presented in Table 9. Seawater was analyzed for Nitrate-N content and spiked 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 7

with 0.5 mg N/L and analyzed 6 times. These results are also summarized in Table 8 and 9. The 

Nitrate-N content of the matrices varied greatly and some had to be diluted before analysis. The 

sample matrices which were diluted to bring them within range were: WW-2, WW-5, WW-6, 

and WW-8. Relative Standard Deviations were less than 10%, except for three matrices. These 

three matrices with Relative Standard Deviations greater than 10% were ones that had Nitrate-N 

content less than the lowest calibrant of the Standard Curves: WW-1; WW-7; and SW-1. 

Nitrate-N content determined below the lowest calibrant of the Standard Curves may not be valid 

and would be expected to have greater error in the determination than ones falling within the 

range of the standard curves.

For the diluted matrices, the spiking was carried out with the diluted matrix. The acceptance 

criterion for spike recovery in the Wastewater and Seawater matrices was 77 to 121%. The 

acceptance criterion for the Relative Percent Difference (RPD) of the concentration results of the 

Matrix Spike (MS) and Matrix Spike Duplicate (MSD) pair was 22%.As shown in Table 8 and 9, 

all of the spiking results yielded data within the acceptance criteria for mean spike recovery and 

RPD. The mean spike recoveries varied from 85.0 to 112.4%. The RPD varied from 0.08 to 

9.6%. No data from Laboratory 8 is in Table 8 or 9, since the spiking protocol specified in the 

Study Plan was not followed. Specifically, this laboratory did not spike samples at the specified 

concentration level for all pairs, resulting in data that were not comparable to those generated by 

the other laboratories.

Overall, none of the Wastewater Matrices, nor the Seawater Matrix, yielded a “matrix effect” on 

nitrate-N analysis by the Enzymatic Reduction Method, which indicates the method is not biased 

by the constituents of the sample matrices listed in Table 1. It is noteworthy that Nitrate-N 

content was successfully determined in seawater by the DA Enzymatic Reduction Method; 

however, the seawater matrix had such a low Nitrate-N content that the results were not valid, 

but the 0.5 mg N/L spikes were accurately determined. Thus, the salt content of seawater does 

not bias the Method even when no salt is included in the calibrants. A previous study of NitrateN determination in seawater by the Enzymatic Reduction Method also found that valid results 

were found compared to the cadmium reduction method; however, the calibrants in this study 

were prepared in synthetic seawater (Ringuet et al., 2011).

3.3 Evaluation of the Accuracy of the Method

There were three reference standards analyzed by the participating laboratories in this study 

(Table 10). The first standard was SR-1 (ERA #608) and it was analyzed by 6 laboratories 

yielding a total of 38 analyses. The mean Nitrate-N content of SR-1 was 6.6780 ± 0.0414 mg 

N/L (RSD = 0.6199 %), which compared to the certified target value = 6.80 ± 0.12 mg N/L. The 

mean recovery of SR-1 was 98.2059 %. The two other reference standards were from the USGS 

(SRM-1 and SRM-2). SRM-1 was analyzed by 7 laboratories for a total 36 analyses and the 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 8

mean Nitrate-N content found was 0.4413 ± 0.0103 mg N/L (RSD = 2.3340 %). The target 

value was the Most Probable Value found by multiple laboratory analysis and reported on the 

USGS website (see Table 9 footnote for the website URL; also see Appendix D for the USGS 

SRM1 data) was 0.443 ± 0.011 mg N/L (RSD = 2.4831 %). The mean recovery of SRM-1 was 

99.6163 %. SRM-2 was analyzed by 7 laboratories for a total 25 analyses and the mean NitrateN content found was 2.2868 ± 0.0691 mg N/L (RSD = 3.0217 %). The target value was the 

Most Probable Value found by multiple laboratory analysis and reported on the USGS website 

(see Table 9 footnote for the website URL; also see Appendix D for the USGS SRM2 data) was 

2.300 ± 0.009 mg N/L (RSD = 0.3913 %). The mean recovery of SRM-1 was 99.4261 %.

From the results of the analysis of the 3 standard references (Table 9) and the certified nitrate 

standards analyzed for Quality Control (see section 3.1), it is clear that the Enzymatic Reduction 

Method of Nitrate-N Analysis yields highly accurate and precise results.

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

The samples matrices listed in Table 1 were also analyzed by the cadmium reduction method for 

Nitrate-N content (EPA Method 353.2). These results are compared to results from the 

Enzymatic Reduction Method in Table 10. For most of the matrices, the Enzymatic Reduction 

Method resulted in determination of a higher Nitrate-N content than the Cadmium Reduction 

Method, but two matrices were greater by CdR and two were the same for the two methods. 

Most of the matrices analyzed were within the range of the standard curves of the Methods (or 

diluted to bring them into range), but four matrices were below the lowest calibrant used to 

prepare the standard curves. These 4 matrices were WW-1, WW-4, WW-7, and SW-1, which 

are footnoted in Table 10. These matrices had RPD greater than 10%, which is not unexpected 

since the determined values are not really valid. One matrix with low nitrate content (WW-1) 

had an acceptable RPD. All the other matrices had RPD less than 10%.

It is clear from the results in Table 10, that the Enzymatic Reduction Method gave comparable 

results to the certified EPA reference method. In addition, extensive studies comparing the 

Enzymatic Reduction Method and Cadmium Reduction Method for the Nitrate-N content of 

natural water samples at the USGS have shown the two methods yield comparable results (Patton 

et al., 2002; Patton and Kryskalla, 2011; 2013). A less extensive study comparing the two 

methods found “no differences in variability” meaning that the standard deviation of the analysis 

of analyte nitrate in double-distilled water were not significantly different (p < 0.05) (Ringuet et 

al., 2011).
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 9

Section 4 Validation Conclusions

The USEPA requires for Tier 3 (nationwide) validation of an Alternate Test Procedure (ATP)

Method that nine different laboratories analyze the analyte content of one sample of eight 

different wastewater matrices, and one sample matrix of ocean water (EPA, 1999). Thus, the 

ATP Method will be in compliance with the Clean Water Act (CWA) and validate the ATP 

Method for nationwide application as a regulatory method. 

For the Enzymatic Reduction Method for Nitrate-N Analysis, the requirements of Tier 3 

validation were met by analysis of eight wastewater matrices and one seawater matrix by ten 

laboratories (see Table 1 for list of Wastewater Sample Matrices analyzed in this study and 

Tables 8 and 9 for details of the analysis by 9 of the 10 laboratories listed in Table 2). The 

spiking studies of the Wastewater Sample Matrices (spikes of 0.5 mg N/L analyzed six times)

indicated that there was little or no matrix effect on the Method by the eight Wastewater matrices 

(Table 8 and 9). Seawater (ocean water) was also analyzed (Table 8 and 9). No significant 

effect of the seawater matrix spiked with 0.5 mg N/L nitrate and analyzed 6 times, was found on 

the Nitrate-N content determined for seawater (Table 8 and 9).

Accuracy of the Method was shown to be very high by analysis of 3 reference standards (Table 

10) and the precision of the Quality Control results (Tables 5 and 6). The MDL evaluation of the 

equipment used in the study demonstrated that the Method has a detection limit of less than 

0.050 mg N/L on the DA analyzers used in the study (Table 7). Few interfering substances, if 

any, were discovered in the present study. Previous analysis of interferences with specific 

compounds showed that there was little interference with the Method (Patton and Kryskalla, 

2011; Patton and Kryskalla, 2013). See also data on interferences in The Method description in 

Appendix B. 

Comparison of the analysis of the eight wastewater and seawater matrices and three reference 

standards by the Enzymatic Reduction Method and the certified Cadmium Reduction Method 

(EPA Method 353.2) indicated that very similar results were obtained (Table 11). Previous 

studies have also found the two methods yielded similar results (Patton et al., 2002; Patton and 

Kryskalla, 2011; 2013; Ringuet et al., 2011).

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

robust Inter-Laboratory Study of Wastewater and Seawater 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 3, 4, 5, 6, and 7) 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 Wastewater
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 10

in the field. For Discrete Analyzers running the Method, the MDL ranged from 0.008 to 0.046

mg N/L (Table 7). Thus, the requirements of the Tier 3 level 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.
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 11

Section 5 References and Glossary

Protocol for EPA Approval of Alternate Test Procedures for Organic and Inorganic Analytes in 

Wastewater and Drinking Water, USEPA, 1999.

http://water.epa.gov/scitech/methods/cwa/atp/upload/2007_02_06_methods_atp_EPA821B98003.pdf

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 CJ, AE Fischer, WH Campbell & ER Campbell (2002) Corn leaf nitrate reductase: A 

nontoxic alternative to cadmium for photometric nitrate determinations in water samples by 

air-segmented continuous-flow analysis. Environmental Science and Technology, 36: 729-

35.

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/).

Ringuet, R., L. Sassano, and Z. L. Johnson (2011) A Suite of microplate reader-based 

colorimetric methods to quantify ammonium, orthophosphate, and silicate concentrations for 

aquatic nutrient monitoring. Journal of Environmental Monitoring, 13: 370-376.

Glossary:

DA = Automated Discrete Analyzer

IPR = Initial Performance and Recovery

OPR = Ongoing Performance and Recovery

MDL = Method Detection Limit

MS = Matrix Spike

MSD = Matrix Spike Duplicate

QA = Quality Assurance

QC = Quality Control
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 12

Table 1. List of Sample Matrices for Analysis

Sample 

Number Sample Type Matrix

Identifier Filtered Acidified

Waste Water Samples 

1 Denver area treatment plant 

Influent wastewater WW-1 Yes Yes

2 Denver area treatment plant 

Wastewater effluent #1 WW-2 Yes Yes

3 Denver area treatment plant 

Wastewater effluent #2 WW-3 Yes Yes

4 Michigan paper mill waste stream 

effluent WW-4 Yes Yes

5 Denver area metal finisher waste 

stream effluent WW-5 Yes Yes

6 Denver area Commercial laundry 

waste stream effluent WW-6 Yes Yes

7

Environmental Resources 

Associates #507 Hardness 

WasteWatR reference material

WW-7 Yes Yes

8

Michigan Confined Animal 

Feeding Operation (CAFO) 

effluent from tiled field

WW-8 Yes Yes

Other Sample

9 Low-nutrient seawater (collected 

offshore Hawaii) SW-1 Yes No

Reference Standards

ERA # 608 Reference Standard SR-1 Yes Yes

USGS PE N-116

(low nutrient-fortified river water)

SRM-1 Yes No

USGS PE N-115

(high nutrient-fortified river water)

SRM-2 Yes No
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 13

Table 2 Participant Laboratories

Lab Number Lab Name Contact Analytical Method

2 USGS/NWQL Charles Patton Discrete Analyzer #1

3 USGS/NWQL Charles Patton Discrete Analyzer #2

4 OI Analytical William Lipps Discrete Analyzer

5 ThermoFisher Stephen White Discrete Analyzer #1

6* ThermoFisher Stephen White Discrete Analyzer #2

7 ThermoFisher Stephen White Discrete Analyzer #3

8 Univ. of Maryland/Solomons Jerry Frank Discrete Analyzer

9 Klamath Tribes Kris Fischer Discrete Analyzer

10 Geochemical Testing Tim Boergstresser Discrete Analyzer

11 Unity/Westco Scientific Bill Georgian Discrete Analyzer

12 Astoria-Pacific Winston Pavitt, CEO Discrete Analyzer

*Laboratory 6 dropped out of the study. 

Laboratory 1 employed a different analytical procedure than that used by the other laboratories 

(Lab Numbers 2-12). Therefore, those data have not been included as part of this final study report. 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 14

Table 3 Calibration of Enzymatic Reduction Method Summary

A. Statistics for the Calibration Curve for Each Laboratory

NaR Method Slope A-540 nm

Correlation 

Coefficient

A-540 per mg N/L Intercept r

2

Lab 2 DA 0.1392 0.0027 0.9999

Lab 3 DA 0.1468 0.0019 0.9999

Lab 4 DA 0.0954 0.0026 0.9999

Lab 5 DA 0.1159 -0.0006 0.9994

Lab 7 DA 0.1420 -0.0048 0.9963 (0.9999)ꜟ

Lab 8 DA 0.1361 0.0148 0.9994

Lab 9 DA 0.1352 -0.0015 0.9997

Lab 10 DA 0.1328 0.0014 0.9980 (0.9991) ꜟ

Lab 11* DA 0.0643 0.0027 0.9986

Lab 12 DA 0.0530 0.0084 0.9999

*Lab 11 used Absorbance @ 550 nm; ꜟPolynomial Fit.

B. Calibration Nitrate Concentrations (mg N/L) used.

Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 10 Lab 12

0.05 0.05 0.05 0.05 0.04 0.05 0.05 0.05 0.05 0.10

0.25 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.10 0.50

0.50 0.50 1.00 0.50 0.75 0.5 0.75 0.50 0.25 1.00

0.75 0.75 2.50 0.75 1.00 0.75 2.50 0.75 0.50 2.50

1.25 1.25 5.00 1.25 1.25 1.25 5.00 1.25 1.00 5.00

2.50 2.50 2.50 2.50 2.5 2.50 2.50

3.75 3.75 3.75 3.75 3.75 3.75 5.00

5.00 5.00 5.00 5.00 5 5.00
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 15

Table 4 Enzymatic Reduction Efficiency Summary

Acceptance Standard is 90% or greater reduction efficiency.

2nd Source = Nitrate Standard in mg N/L and Nitrite Standard = mg N/L

A-540 nm mg N/L Reduction 

Efficiency

Lab 2 2nd Source 2.50 0.35217 2.51 102.1% Nitrite Standard 2.50 0.34507 2.46

Lab 3 2nd Source 2.50 0.36664 2.48 101.3% Nitrite Standard 2.50 0.36190 2.45

Lab 4 2nd Source 2.50 0.2456 2.548

107.2% Nitrite Standard 2.50 0.2292 2.376

Lab 5 2nd Source 2.50 0.295 2.59 116.6% Nitrite Standard 2.50 0.252 2.22

Lab 7 2nd Source 2.50 0.33422 2.3576 94.9% Nitrite Standard 2.50 0.3514 2.4833

Lab 8 2nd Source 2.50 0.37078 2.557 103.8% Nitrite Standard 2.50 0.35867 2.464

Lab 9 2nd Source 2.50 2.506 103.7% Nitrite Standard 2.50 2.417

Lab 10 2nd Source 2.50 0.358 2.4872

102.3% Nitrite Standard 2.50 0.350 2.4310

Lab 

11* 2nd Source 3.04 0.2127 3.00 98.6%

Nitrite Standard 3.04 0.2151 3.04

Lab 12 2nd Source 2.50 0.146 2.598

105.0% Nitrite Standard 2.50 0.140 2.475

*Lab 11 used Absorbance @ 550 nm
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 16

Table 5 Initial Performance and Recovery (IPR) Summary

Lab # Analyses

Spike 

Conc.

(mg/L)

Mean 

Recovery 

(%)

RSD (%)

Minimum 

Recovery 

(%)

Maximum 

Recovery 

(%)

2 4 2.5 100.67 0.71 99.67 101.30

3 4 2.5 101.82 0.54 101.29 102.40

4 4 2.0 96.16 2.23 93.50 98.70

5 4 2.5 106.38 0.73 105.82 107.52

7 4 2.0 101.79 0.78 100.63 102.37

8 4 2.5 102.76 0.92 101.77 103.93

9 4 2.0 100.50 1.13 98.97 101.66

10 4 2.0 98.45 0.66 97.77 99.21

11 4 2.7 101.11 0.95 100.17 102.43

12 4 2.0 99.34 2.45 95.85 101.50
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 17

Table 6 Ongoing Performance and Recovery (OPR) Summary

RSD = Relative Standard Deviation

Lab OPR

mg N/L

# 

Analyses*

Mean 

Recovery 

%

RSD

%

Minimum 

Recovery 

(%)

Maximum 

Recovery 

(%)

2 2.50 13 97.50 2.74 94.67 101.23

3 2.50 16 96.38 1.48 94.36 99.74

4 2.50 11 101.04 2.12 98.00 105.00

5** 2.50 4 104.49 0.68 103.48 105.48

5** 2.00 9 96.30 5.66 89.74 107.41

7 2.00 31 102.89 1.06 100.54 104.58

8 2.50 22 99.75 0.52 98.43 102.41

9 2.50 39 103.26 1.61 99.76 106.43

10 2.00 27 102.95 1.11 100.54 104.58

11 2.60 13 101.34 1.69 98.17 104.42

12 2.00 11 99.60 5.49 92.65 108.45

*Some Labs did more OPR than are summarized here for reasons of repeated analyses.

** Lab 4 initially used OPR = 2.50 mg N/L and then switched to OPR = 2.00 mg N/L.
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 18

Table 7 Method Detection Limit (MDL) Summary

Abbreviations: DA = Discrete Analyzer; NA = Not Analyzed.

Lab Method MDL Replicates Spike Spike/MDL

mg N/L mg N/L Ratio

2 DA 0.0079 8 0.040 5.068

3 DA 0.0148 8 (7)* 0.040 2.701

4 DA 0.0130 7 0.050 3.832

5 DA 0.0055 7 0.025 4.522

7 DA 0.0226 7 0.050 2.215

8 DA 0.0310 7 0.050 1.615

9 DA 0.0260 7 0.075 2.881

10 DA NA

11 DA 0.0060 7 0.045 7.541

12 DA 0.0463 7 0.100 2.160

*One replicate discarded due to issue with blank 
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 19

Table 8 Nitrate-N Content and Spike Analysis of Waste Water and Seawater Matrices

Abbreviations: RSD = Relative Standard Deviation; RPD = Relative Percent Difference

Laboratory Matrix

Unspiked Sample Summary Spiked Sample Summary

Nitrate-N 

(mg N/L)

Standard 

Deviation 

(mg N/L)

RSD (%) Dilution

Final 

Nitrate-N 

(mg N/L)

Mean 

Spike 1 % 

Recovery 

(%RPD)

Mean 

Spike 2 

% 

Recovery 

(%RPD)

Mean 

Spike 3 

% 

Recovery 

(%RPD)

2 WW-6 2.4732 0.0249 1.0080 2X 4.9464 100.1677 

(1.0138)

103.2587 

(0.7319)

99.9327 

(0.6115)

3 WW-8 2.9017 0.01752 0.6037 5X 14.5083 112.3524 

(0.8002)

108.9310 

(0.8933)

111.1024 

(0.3202)

4 WW-7 0.0070 0.00872 124.5429 None 0.0070 96.1024 

(4.7179)

91.9024 

(4.5016)

90.3024 

(0.6543)

5 WW-5 2.7849 0.07318 2.6276 100X 278.4896 98.4712 

(2.4315)

98.9524 

(1.0010)

94.3448 

(0.5755)

9 WW-4 0.0400 0.0010 2.5000 None 0.0400 107.0000 

(2.4348)

105.6000 

(3.5211)

102.3000 

(1.2693)

10 WW-3 0.2251 0.0114 5.0473 None 0.2251 103.0767 

(0.3376)

103.4267 

(2.2096)

100.1567 

(1.3639)

11 WW-2 4.0667 0.0139 0.3410 2X 8.1333 99.3867 

(0.1534)

98.6767 

(0.0768)

85.0667 

(1.6296)

12 WW-1 0.0403 0.0307 76.0286 None 0.0403 95.9400 

(1.5385)

95.9400 

(9.6154)

92.1400 

(1.5968)

7 SW-7 0.0094 0.0139 148.4678 None 0.0094 100.4180 

(2.4635)

101.3640 

(1.9760)

104.6390 

(1.1698)
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 20

Table 9 Detailed Data Supporting Table 8

Data in this table are summarized from each Laboratory's Summary Excel File in Appendix F.

Standard Curve Data are in Table 3. Spiking was done at 0.5 mg N/L

A-540 Nitrate-N Nitrate-N Spike 1 Spike 2 Spike 3

Matrix ID Lab Dilution 

Factor

Matrix 

Replicates

Measured 

(mg-N/L)

Final 

(mg-N/L)

Spike 

A-540

Spiked 

(mgN/L)

Spike 

Recovery 

%

Spike 

A-540 Spiked 

(mg-N/L)

Spike 

Recovery 

%

Spike 

A-540 Spiked 

(mg-N/L)

Spike 

Recovery %

WW-1 12 1

0.012

0.009

0.012

0.0403 0.0403

WW-1 MS

WW-1 MSD 0.037

0.036

0.524

0.516

96.7400

95.1400

0.038

0.035

0.545

0.495

100.9400

90.9400

0.036

0.035

0.505

0.497

92.9400

91.3400

WW-1 Mean Spike Recovery (%)

Relative Percent Difference (%)

95.9400

1.5385

95.9400

9.6154

92.1400

1.5968

Lab

11

A-550

WW-2 2 0.2635

0.2630

0.2646

4.0667 8.1333 WW-2 MS

WW-2 MSD 0.2962

0.2957

4.5671

4.5601

100.0867

98.6867

0.2958

0.2956

4.5618

4.5583

99.0267

98.3267

0.2937

0.2889

4.5286

4.4554

92.3867

77.7467

WW-2 Mean Spike Recovery (%)

Relative Percent Difference (%)

99.3867

0.1534

98.6767

0.0768

85.0667

1.6296

Lab

10

A-540

WW-3 1 0.041

0.039

0.038

0.2251 0.2251 WW-3 MS

WW-3 MSD 0.116

0.115

0.7417

0.7392

103.3267

102.8267

0.115

0.117

0.734

0.7504

101.7867

105.0667

0.114

0.113

0.7308

0.7209

101.1467

99.1667

WW-3 Mean Spike Recovery (%)

Relative Percent Difference (%)

103.0767

0.3376

103.4267

2.2096

100.1567

1.3639

Lab

9

A-540

WW-4 1 0.0053

0.0055

0.0054

0.0400 0.0400 WW-4 MS

WW-4 MSD 0.0787

0.0768

0.582

0.568

108.4000

105.6000

0.0781

0.0754

0.578

0.558

107.6000

103.6000

0.0750

0.0741

0.555

0.548

103.0000

101.6000

WW-4 Mean Spike Recovery (%)

Relative Percent Difference (%)

107.0000

2.4348

105.6000

3.5211

102.3000

1.2693

Lab

5

A-540

WW-5 100 0.2992

0.3463

0.3228

WW-5 MS 2.7849 278.4896 0.3752

0.3845

3.2374

3.3171

90.5024

106.4400

0.3820

0.3782

3.2961

3.2632

102.2354

95.6694

0.3764

0.3785

3.2472

3.2660

92.4706

96.2190 WW-5 MSD

WW-5 Mean Spike Recovery (%)

Relative Percent Difference (%)

98.4712

2.4315

98.9524

1.0010

94.3448

0.5755

Lab

2

A-540

WW-6 2 0.35647

0.35077

0.34998

WW-6 MS 2.4732 4.9463 0.42141

0.42569

2.9589

2.9891

97.1527

103.1827

0.42419

0.42730

2.9785

3.0004

101.0707

105.4467

0.42467

0.42209

2.9819

2.9637

101.7507

98.1147 WW-6 MSD

WW-6 Mean Spike Recovery (%)

Relative Percent Difference (%)

100.1677

1.0138

103.2587

0.7319

99.9327

0.6115

Lab

4

A-540

WW-7 1 -0.0005

0.0008

0.0017

WW-7 MS 0.0070 0.0070 WW-7 MSD

0.0454

0.0476

0.4760

0.4990

93.8024

98.4024

0.0455

0.0435

0.4770

0.4560

94.0024

89.8024

0.0439

0.0436

0.4600

0.4570

90.6024

90.0024

WW-7 Mean Spike Recovery (%)

Relative Percent Difference (%)

96.1024

4.7179

91.9024

4.5016

90.3024

0.6543

Lab

3

A-540

WW-8 5 0.40495

0.40953

0.40572

WW-8 MS 2.9017 14.5083 WW-8 MSD

0.4873

0.4834

3.4773

3.4496

115.1238

109.5810

0.4808

0.4851

3.4309

3.4617

105.8524

112.0095

0.48528

0.48373

3.4627

3.4516

112.2095

109.9952

WW-8 Mean Spike Recovery (%)

Relative Percent Difference (%)

112.3524

0.8002

108.9310

0.8933

111.1024

0.3202

Lab

7

A-540

SW-1 1 -0.00121

-0.00448

-0.00476

SW-1 MS 0.0094 0.0094 SW-1 MSD

0.0735

0.0717

0.5178

0.5052

101.6780

99.1580

0.0726

0.0740

0.5111

0.5213

100.3440

102.3840

0.0761

0.0752

0.5357

0.5295

105.2620

104.0160

SW-1 Mean Spike Recovery (%)

Relative Percent Difference (%)

100.4180

2.4635

101.3640

1.9760

104.6390

1.1698
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 21

Table 10 Standard Reference Materials Summary

Labs 5 and 8 did not analyze these matrices.

Sample Matrix ERA #698 - Finished drinking water (Target value from ERA)

Sample Target Lab # 

Analyses

Concentration Result 

(mg N/L) Recovery (%) RSD

ERA

#698

mg N/L Mean Min. Max Mean Min. Max %

6.80 

±0.12 

(1.82%)

1 7 6.7809 6.7316 6.8038 99.72 98.99 100.06 0.38

2 7 6.6154 6.5942 6.6497 97.28. 96.97 97.79 0.31

3 7 6.5852 6.4661 6.6443 96.84 95.09 97.71 0.94

4 0

6 7 6.3445 6.2929 6.4189 93.30 62.54 94.39 0.66

7 7 6.8857 6.8000 6.9700 101.26 100.00 102.50 1.00

9 3 6.8565 6.8295 6.8885 100.83 100.43 101.30 0.43

10 0

N = 38 Mean = 6.6780 ± 0.62% Mean Recovery = 98.20%

Sample Matrix SRM-1 - USGS PE Sample N-116

(Target Most Probable Value from USGS*)

Sample Target Lab # 

Analyses

Concentration Result 

(mg N/L) Recovery (%) RSD

SRM-1

mg N/L Mean Min. Max Mean Min. Max %

0.443 

±0.011 

(2.44%)

1 3 0.4477 0.4476 0.4477 100.60 100.59 100.60 0.01

2 3 0.4605 0.4581 0.4626 103.48 102.95 103.96 0.49

3 7 0.4190 0.3520 0.4420 94.16 79.10 99.33 7.28

4 3 0.4046 0.4023 0.4064 90.92 90.41 91.32 0.51

6 7 0.4304 0.4269 0.4335 96.72 95.93 97.42 0.56

7 10 0.4579 0.4430 0.4710 102.90 99.56 105.26 2.86

9 0

10 3 0.4690 0.4410 0.4950 105.39 99.10 105.26 7.65

N = 36 Mean = 0.4413 ± 2.34% Mean Recovery = 99.62%
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 22

Table 10 Continued

Sample Matrix SRM-2 - USGS PE Sample N-115

(Target Most Probable Value from USGS*)

Sample Target Lab # 

Analyses

Concentration Result 

(mg N/L) Recovery (%) RSD

SRM-2

mg N/L Mean Min. Max Mean Min. Max %

2.300 

±0.009 

(0.38%)

1 3 2.2653 2.2209 2.3022 99.36 97.41 100.97 1.82

2 3 2.3215 2.3019 2.3407 101.82 100.96 102.66 0.84

3 3 2.2647 2.2590 2.2680 99.33 99.08 99.47 0.22

4 3 2.1676 2.0896 2.2353 95.07 91.65 98.04 3.39

6 7 2.3783 2.3497 2.4028 104.31 103.06 105.39 0.78

7 3 2.3267 2.2700 2.4000 102.05 99.56 105.26 2.86

9 0

10 3 2.2833 2.1210 2.4680 100.15 93.03 108.25 7.65

N = 25 Mean = 2.2868 ± 3.02% Mean Recovery = 99.43%

*Data from US Geological Survey (see link below and Appendix D of the Final Report)

(http://bqs.usgs.gov/srs_study/reports/round_details.php?PHPSESSID=vifh972qads22dirctsvevi841)

For SRM-1, which is USGS N-116,

25 analysis were reported by colorimetric methods with the following results (units = mg N/L):

MPV = 0.443; Fps = 0.016 (Fps = StDev/1.4826) therefore, StDev = 0.011 (2.44%)

For SRM-2, which is USGS N-115,

25 analysis were reported by colorimetric methods with the following results:

MPV = 0.230; Fps = 0.013; StDev = 0.009 (0.38%)

MPV = Most Probable Value; Fps = F-pseudo-sigma or MAD = Median Absolute Deviation
NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 23

Table 11 Comparison of Nitrate-N Content of Sample Matrices and Standard References

Determined by Discrete Analyzer Enzymatic Reduction Method (NaR) and

Cadmium Reduction Air-Segmented Continuous Flow Analyzer Method (CdR)

Abbreviations: RPD = Relative Percent Difference for NaR-CdR (+ = NaR higher; - = CdR higher)

Note: NaR and CdR Method were done by Laboratory 2 on 19Jul2013 and values are mean of 3 replicates.

Sample Matrix NaR CdR RPD

mg N/L mg N/L %

WW-1* 0.03 0.03 0.0000

WW-2 7.8 7.6 +2.5974

WW-3 0.23 0.26 -12.7656

WW-4* 0.04 0.03 +28.5714

WW-5 270.8 272.6 -0.6625

WW-6 4.8 4.8 0.0000

WW-7* 0.05 0.06 -18.1818

WW-8 13.77 14.1 -2.3681

SW-1* 0.027 0.030 -10.5263

SR-1 6.80 7.02 -3.1838

SRM-1 0.45 0.48 -6.4516

SRM-2 2.28 2.36 -3.4188

*These values at or below the lowest calibrant of the method standard curve.