## Environmental Protection Agency

Alternat e T est 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. d epartments and a gencies 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 M ichigan E conomic D evelopment C orporation.

## 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 E valuation 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 1 1

Tables

Table 1 List of Sample Matrices

Table 2 List of Participating Laboratories

Table 3 Calibration of Enzymatic Reduction Method Summary

Table 4 Enzymatic Redu ction 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 Nitrat e

Reductase Method , and by Cadmium Reduction Method

Appendices

– Appendices are not included contact NECi if needed

Appendix A Inter

- Labo r atory 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 S tandards utilized in t his 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 ReportNECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 2 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 US EPA Alternate Test Procedure (ATP) for Tier 3 level (nationwide)

determination of nitrate plus nit rite 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 + + H 2 O

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 (Fe 3 + and Mo 6 + ) and three organic cofactors (Flavin

Adenine Dinuc leotide (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 pa storis

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 sta ble 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 Nitrate

N content. For example, in the discrete analyzer, the volume of buffered AtNaR2 is 55 μL,

NADH 12 μ L, 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, requ iring

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 Inte r

- 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 n itrate

- N is not directly compared to t he 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 matric es 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 method s for de termining 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 N itrate

- 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 Analyt es (USEPA, 1999) in order to validate the

Method for Tier 3 (nationwide) status for both Drinking and Wastewater

. The Study P lan 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 t his 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

accord ing to EPA format in Appendix B, which was incorporated in the Design Pla n. 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 statu s 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 designat ed matrix identification (i.e. WW

- 1, W W

- 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 certi fication for the Nitrite S tandard 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 o f a vacuum sealed,

opaque p ouch 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. Fo r each type of DA being used in

the study, a specific set of Standard Operating Procedures (SOP) has been developed which

condenses th e Method in Appendix B. As an example, the SOP for the OI Analytical DA ha s

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 , whichNECi 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 with in 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 Ap pendix F

. Supplemental Excel ® File for the

MDL Study by Laboratory 5 i s provided in Appendix F, which is supplied b y 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 Curve s reported by each laboratory are summarized in Table

3 A . 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 (r 2 ) = 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 r 2

= 0.9999 and 0.9991, for labs 7 and 10, respectively. In each

case, the parameters for the slope and intercept are utiliz ed 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 t o described the relationship of Absorbance to Nitrate

- N content,

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

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

information i s 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 oth er labs using a

discrete analyzer used 0 .05 mg N/L (Lab 7 used 0.04 mg N /L ).

Enzymatic reduction efficiencies for each la boratory 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 labor atories are s ummarized in Table 5 and 6 , respectively. IPR certified standard

nitrate concentrations were 2.0 0 , 2.5 0 , and 2.7 0 mg N/L ( see Appendix D)

. IPR mean recoveries

for the sele cted 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 2.0 0, 2.5 0 , and 2.60 mg N/L (see Appendix D)

. O PR meanNECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 6 recoveries for the selected data in Table 6 were from 96 to 10 4 % 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 11 0% recov ery (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 th e mea n 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 0.5 2 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 T able 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 calibr ation.

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 apparen tly 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 Reduc tion 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 differenc es 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 re sults 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 spikedNECi 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 matric es which were diluted to bring them within range were : WW

- 2, WW

- 5 , WW

- 6

,

and WW

- 8. Relat ive 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 o f 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 s piking 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

Mat rix 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, result ing in data that were not comparable to those generated by

the other laboratories.

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

nitrate

- N analysis by the Enzymatic Reduction Method , which indicates the me thod 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 co ntent 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 Nitrate

N determination in seawa ter 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

T here were three reference standards analyzed by the participating lab oratories 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 98.20 59 %. The two other reference standards were from the USGS

(S RM

- 1 and SRM

- 2). SRM

- 1 was analyzed by 7 laboratories for a total 36 analyses and theNECi 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 reporte d 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 Nitrate

N 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 Appe ndix 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 we re also analyzed by the cadmium reduction method for

Nitrate

- N content (EPA Method 353.2). T hese 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 analyz ed 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 footno ted 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 cl ear 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 con tent 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 Alternat e 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 Cle an Wate r 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 i n 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 Sa mple Matrices (spikes of 0.5 mg N/L analyzed six times)

indicated that there was little or no mat rix 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 ni trate and analyzed 6 times, was found on

the Nitrate

- N content determined f or 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 Quali ty 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. 0 5 0 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 Enzym atic Reduction Method for Nitrate

- N Analysis has been validated by a

robust Inter

- Laboratory Study of Wastewater and Seaw ater Sample Matrices. All laboratories

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

analyzing the samples. T he 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 indicate d that the Method is

highly accurate and capable of providing definitive analysis of N itrate

- N content of WastewaterNECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 10 in the field. For Discrete Analyzer s running the Method, the MDL ranged from 0.008 to 0. 0 46

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

Wa ter. 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 au tomated, semi

automated, and manual colorimetric methods for determination of nitrate plus nitrite in water,

USGS Scientific Investiga tions 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 L imit

MS = Matrix S pike

MSD = Matrix S pike D uplicate

QA = Q uality A ssurance

QC = Quality C ontrolNECi 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 N o

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 NoNECi 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 U niv. 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 Summar y

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; ꜟ Polynomial 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 5.00NECi 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 nmNECi 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 95.85 101.50NECi 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 blankNECi 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

(mg

N/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 2.7849 278.4896 WW

- 5 MS 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 2.4732 4.9463 WW

- 6 MS 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 0.0070 0.0070 WW

- 7 MS

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 2.9017 14.5083 WW

- 8 MS

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 0.0094 0.0094 SW

- 1 MS

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

1.1698NECi 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 #

Anal yses Concentration Result

(mg N/L) Recovery (%) RSD

ERA

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

6.80

±0.12

(1.8 2 %) 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 M ost Probable Value from USGS*)

Sample Target Lab #

Anal yses 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 M ost Probable Value from USGS*)

Sample Target Lab #

Anal yses 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 DeviationNECi 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.