Prepared by the U.S. Geological Survey Office of Water Quality, National Water Quality Laboratory

Colorimetric Determination of Nitrate Plus Nitrite in 

Water by Enzymatic Reduction, Automated Discrete 

Analyzer Methods

Chapter 8

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis

U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 5–B8
Cover photographs. A, Arabidopsis thaliana, a small plant, perhaps about 4 or 5 inches tall, also known as wall cress. 

B, Charles J. Patton and Jennifer R. Kryskalla. C, U.S. Geological Survey National Water Quality Laboratory Nutrients Unit Analysts, 

October 2011.

A

B

C
Colorimetric Determination of Nitrate Plus 

Nitrite in Water by Enzymatic Reduction, 

Automated Discrete Analyzer Methods

By Charles J. Patton and Jennifer R. Kryskalla

Chapter 8

Section B, Methods of the National Water Quality Laboratory 

Book 5, Laboratory Analysis

Techniques and Methods 5–B8

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2011

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U.S. Government.

Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

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.
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Purpose and Scope .......................................................................................................................................2

Analytical Methods........................................................................................................................................2

 1. Application......................................................................................................................................2

 2. Method Summaries and Analytical Considerations ................................................................3

 3. Interferences and Temperature Effects.....................................................................................3

 4. Instrumentation..............................................................................................................................4

 5. Apparatus........................................................................................................................................7

 6. Reagent Preparation.....................................................................................................................7

 7. Calibrants and Quality-Control Solutions ..................................................................................8

 8. Sample Preparation ....................................................................................................................10

 9. Instrument Performance ............................................................................................................10

10. Calibration.....................................................................................................................................10

11. Procedure and Data Evaluation ................................................................................................10

12. Calculations..................................................................................................................................12

13. Reporting Results.........................................................................................................................12

14. Detection Limits, Precision, Spike Recovery, and Bias.........................................................12

Analytical Performance and Comparative Results ................................................................................14

Background Information....................................................................................................................14

Effects of Temperature and Dissolved Organic Matter on AtNaR2 Activity.............................14

Reagent Stability.................................................................................................................................20

Interference by Anionic and Cationic Sample Matrix Constituents...........................................20

Demonstration of Method Capability...............................................................................................23

Comparison of AtNaR2 and YNaR1 Reagents in Standard- and Low-Level 

DA Nitrate + Nitrite Assays ........................................................................................23

Graphical Comparison of Standard- and Low-Level DA-AtNaR2 and 

CFA-CdR Assays ..........................................................................................................24

Equivalence of Low-Level DA-AtNaR2 Assay Response to Nitrate and Nitrite..............24

Statistical Comparisons of Nitrate + Nitrite Results Determined by CFA-CdR and 

DA-AtNaR2 Methods ...................................................................................................27

Conclusions...................................................................................................................................................30

Acknowledgments.......................................................................................................................................31

References Cited..........................................................................................................................................32
iv

Figures

1. Graph showing kinetics effects of diverse metal ions and perchlorate on the 

Griess reaction colorimetric nitrite assay ................................................................................5

2. Graphs showing effect of temperature on kinetics and yield of the Griess 

reaction nitrite assay ...................................................................................................................5

3. Functional diagram of the Aquakem 600TM automated discrete analyzer...........................6

4. Graph showing typical calibration graphs for standard- and low-level 

concentration range enzymatic-reduction nitrate determination methods by 

automated discrete analyzer....................................................................................................11

5. Graph showing control chart of third-party-check sample results for 

standard-level automated discrete analyzer nitrate + nitrite methods using 

soluble AtNaR2 nitrate reductase............................................................................................14

6. Graph showing activity of different nitrate reductases in relation to reaction 

temperature for 5 milligrams nitrogen per liter nitrate solutions with and without 

added Suwannee River humic acid.........................................................................................18

7. Kinetics plots showing nitrate to nitrite reduction rates by AtNaR2 nitrate 

reductase at 37 degrees Celsius for nitrate concentrations of 0.05 to 

5.00 milligrams nitrogen per liter for each experiment.........................................................19

8. Graph showing nitrate recoveries from deionized water solutions containing 

0.25 milligrams nitrogen per liter (mg-N/L), 2.50 mg-N/L, and 5.00 mg-N/L and 

incrementally increasing Suwannee River humic acid concentrations for 

automated discrete analyzer nitrate assays that differed only in the nitrate 

reductase (YNaR1 or AtNaR2) used to reduce nitrate to nitrite .........................................19

9. AtNaR2 storage stability plots..................................................................................................20

10. Graph showing standard-level discrete analyzer AtNaR2 assay recovery of 

2.50 milligrams nitrogen per liter nitrate from deionized water amended with 

various anions at 1, 10, and 100 times their National Water Quality Laboratory 

annual median concentrations.................................................................................................21

11. Graph showing standard-level discrete analyzer AtNaR2 assay recovery of 

2.50 milligrams nitrogen per liter nitrate from deionized water amended with 

various metal ions at 1, 10, and 100 times their National Water Quality 

Laboratory annual median concentrations ............................................................................22

12. Graph showing comparison of nitrate + nitrite concentrations for 

115 environmental water samples analyzed by automated continuous-flow, 

cadmium-reduction method and automated discrete analyzer enzymaticreduction method with NAD(P)H:YNaR1 nitrate reductase and NADH:AtNaR2 

nitrate reductase ........................................................................................................................23

13. Graph showing comparison of nitrate + nitrite concentrations of 

101 environmental water samples analyzed by standard-level automated 

continuous-flow analyzer, cadmium-reduction method and standardlevel automated discrete analyzer, AtNaR2-nitrate-reductase reduction 

method..........................................................................................................................................24

14. Graph showing comparison of nitrate + nitrite concentrations for 

67 environmental water samples analyzed by low-level automated continuousflow analyzer, cadmium-reduction method and low-level automated discrete 

analyzer, AtNaR2-nitrate-reductase reduction method.......................................................25

15. Graph showing the relation between nitrate + nitrite and nitrite concentrations 

for surface-water (medium code WS) and groundwater (medium code WG) 

samples analyzed at the National Water Quality Laboratory in 2010 with nitrite 

concentrations of 0.05 milligrams nitrogen per liter or more..............................................26
v

16. Box plots showing concentration distributions for the population of 

476 standard-level nitrate + nitrite concentrations determined in 

surface water and groundwater by continuous-flow-analysis, cadmiumreduction and discrete analysis, nitrate-reductase-reduction methods 

for validation experiments spanning November 2005 through May 2008 .........................29

Tables

1. Laboratory, parameter, and method codes for U.S. Geological Survey automated 

discrete analyzer, enzymatic reduction, standard-level (I-2547-11) and low-level 

(I-2548-11) nitrate + nitrite determination methods.................................................................3

2. Aquakem 600TM automated discrete analyzer operational protocols, termed test 

flows by the vendor, for standard-level and low-level AtNaR2-reduction 

nitrate + nitrite determination methods ....................................................................................7

3. Nitrate Stock II calibrants used for automated discrete analyzer calibration...................9

4. Standard-level concentration range nitrate working calibrants used for 

automated discrete analyzer calibration..................................................................................9

5. Low-level concentration range nitrate working calibrants used for automated 

discrete analyzer calibration ......................................................................................................9

6. Second-source, certified nitrate and nitrite solution volumes needed to prepare 

standard-level-assay third-party-check samples in 100-mL quantities ..............................9

7. Second-source, certified nitrate solution volumes needed to prepare nitrate-only, 

standard-level-assay third-party-check samples in 100-mL quantities ..............................9

8. Second-source, certified nitrate and nitrite solution volumes needed to prepare 

low-level-assay third-party-check samples in 100-mL quantities......................................10

9. Second-source, certified nitrate solution volumes needed to prepare nitrate-only, 

low-level-assay third-party-check samples in 100-mL quantities......................................10

10. U.S. Geological Survey National Water Quality Laboratory laboratory codes 

and standard operating procedure numbers for cadmium-reduction and 

enzymatic-reduction nitrate determination methods referenced in this report ..............11

11. Data and calculations used to estimate method detection limits for 

nitrate + nitrite determination with soluble AtNaR2 nitrate reductase by 

automated discrete analysis ....................................................................................................13

12. Third-party-check sample nitrate + nitrite data summary for automated 

discrete analysis with soluble AtNaR2 nitrate reductase ...................................................13

13. Within-day and between-day replicate agreement for nitrate + nitrite analyses of 

surface-water samples and groundwater samples analyzed by the 

AtNaR2-reduction automated discrete analyzer method (National Water Quality 

Laboratory laboratory code 3156)............................................................................................15

14. Between-day replicate agreement for nitrate + nitrite analyses of surface-water 

samples and groundwater samples by the low-level AtNaR2-reduction 

automated discrete analyzer method......................................................................................15

15. Recovery of replicate spikes in reagent-water, surface-water, and groundwater 

matrices by standard-level AtNaR2-reduction nitrate + nitrite assay (National 

Water Quality Laboratory laboratory code 3156) ..................................................................16

16. Recovery of replicate spikes in reagent-water, surface-water, and groundwater 

matrices by low-level AtNaR2-reduction nitrate + nitrite assay (National 

Water Quality Laboratory laboratory code 3157) ..................................................................17
vi

17. Nominal concentrations of anions tested for possible interference in the discrete 

analyzer AtNaR2 nitrate assay .................................................................................................21

18. Metal ions tested for possible inhibition of AtNaR2 enzyme and Griess-reaction 

interference .................................................................................................................................22

19. Summary statistics for nitrite concentrations determined in surface water and 

groundwater during calendar year 2004 at the National Water Quality 

Laboratory by automated continuous-flow analyzer methods ...........................................25

20. Summary statistics for nitrite concentrations determined in surface water and 

groundwater during calendar year 2010 at the National Water Quality 

Laboratory by the automated discrete analyzer method.....................................................26

21. Data demonstrating near equivalent response of low-level AtNaR2 

nitrate + nitrite assay (National Water Quality Laboratory laboratory code 3157) to 

nitrate and nitrite individually and combined.........................................................................27

22. Summary statistics for nitrate + nitrite concentrations determined in 

filtered-water samples by continuous-flow analyzer, cadmium-reduction 

method and discrete analyzer nitrate-reductase-reduction method ................................28

23. Two-population paired t-test results for nitrate + nitrite concentrations 

determined in filtered-water samples by continuous-flow analyzer, 

cadmium-reduction method and discrete analyzer nitrate-reductasereduction method........................................................................................................................29

24. Wilcoxon signed-rank test results for nitrate + nitrite concentrations 

determined in filtered-water samples by continuous-flow analyzer, cadmiumreduction method and discrete analyzer nitrate-reductase-reduction 

method..........................................................................................................................................30
vii

Conversion Factors

[SI to Inch/Pound]

Multiply By To obtain

Length

centimeter (cm) 3.94 × 10–1 inch

micrometer (μm) 3.94 × 10–5 inch

millimeter (mm) 3.94 × 10–2 inch

nanometer (nm) 3.94 × 10–8 inch

Mass

gram (g) 3.53 × 10–2 ounce, avoirdupois

milligram (mg) 3.53 × 10–5 ounce, avoirdupois

Volume

liter (L) 2.64 × 10–1 gallon

liter (L) 3.38 × 101 ounce, fluid

microliter (μL) 2.64 × 10–7 gallon

milliliter (mL) 2.64 × 10–4 gallon

Concentration

milligrams nitrate nitrogen per 

liter (mg NO3

–

-N/L)

7.14 × 101 micromoles nitrate per liter 

(NO3

–

, μM)

milligrams nitrite nitrogen per 

liter (mg NO2

–

-N/L)

7.14 × 101 micromoles nitrite per liter 

(NO2

–

, μM)

molar (M, moles/L) 1.00 × 103 millimolar (mM)

molar (M, moles/L) 1.00 × 106 micromolar (μM)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

 °F = 9/5 (°C) + 32

Acronyms and Abbreviations

≈ Approximately equal to

≥ Greater than or equal to

ASTM American Society for Testing and Materials

AtNaR2TM Recombinant nitrate reductase from Arabidopsis thaliana (Enzyme 

 Commission # EC 1.7.1.1) expressed in Pichia pastoris

BQS USGS Branch of Quality Systems

CdR Cadmium reduction

CFA Air-segmented continuous-flow analysis (or analyzer)

DA Automated discrete analysis (or analyzer)

DI (water) Deionized water piped throughout the NWQL. For purposes of nutrient 

 analysis, NWQL DI water is equivalent to ASTM type I DI.

DOC Dissolved organic carbon

DOM Dissolved organic matter
viii

DTPA Diethylenetriaminepentaacetic acid

EC Enzyme Commission; Swiss organization that assigns unique numerical 

 identifiers to enzymes

EDTA Ethylenediaminetetraacetic acid and its di-sodium salt

EPA U.S. Environmental Protection Agency

FCA Bottle type for nutrient samples that are filtered (0.45 μm), amended with 

 sulfuric acid, and chilled at collection sites. FCA samples are shipped and 

 stored at a nominal temperature of 4°C.

FCC Bottle type for nutrient samples that are filtered (0.45 μm) and chilled at 

 collection sites. FCC samples are shipped and stored at a nominal 

 temperature of 4°C.

FW Formula weight

HA High-phenolic-content humic acid

IRL NWQL interim reporting level (or limit)

LC NWQL laboratory code

LCL Lower control limit

LL Low-level analytical range

LRL NWQL laboratory reporting limit

LT-MDL NWQL long-term method detection level (or limit)

M Molar (molarity); unit of concentration equal to moles solute per liter of 

 solution

MC USGS NWIS sample medium code used to identify sample matrix types

MDL Method detection level (or limit)

MOPS 3-N-morpholino-propansulfonic acid; pKa = 7.20

MPV Most probable value

MRL Method reporting level (or limit)

NAD(P)H:NaR Bispecific forms of nitrate reductase that can use either NADH or NADPH as 

 their cofactor

NADH Nicotinamide adenine dinucleotide in reduced form, a natural cofactor of 

 YNaR1 and AtNaR2

NADH:NaR Forms of nitrate reductase that can use only NADH as their cofactor

NADPH Nicotinamide adenine dinucleotide phosphate in reduced form, the other 

 natural cofactor of bispecific YNaR1 and of nitrate reductase from Aspergillus 

 sp.

NADPH:NaR Forms of nitrate reductase that can use only NADPH as their cofactor

NaR Nitrate reductase in the generic sense

NaR1TM Nitrate reductase purified from corn (Zea mays) by NECi

NECi The Nitrate Elimination Company, Lake Linden, MI 49945

NED N-(1-Naphthyl)ethylenediamine; coupling reagent in Griess-reaction nitrite 
ix

 assays

NWIS USGS National Water Information System

NWQL USGS National Water Quality Laboratory

pKa -log10 Ka, where Ka is the dissociation constant of a weak acid

QC Quality control; used as an adjective in this report for materials analyzed to 

 assure analytical results are within specified limits

RSD Relative standard deviation

SAN Sulfanilamide; diazotizing reagent in Griess-reaction nitrite assays

SL Standard-level analytical range

SOP Standard operating procedure 

SR HA Suwannee River humic acid isolate; a high-phenolic-content HA

TPC Third-party check sample. A QC sample prepared with a certified stock 

 analyte solution obtained from a source different from the one used 

 to prepare calibrants.

UCL Upper control limit

USGS U.S. Geological Survey

v/v Volume-to-volume

WCA Bottle type for whole-water nutrient samples that are amended with 

 sulfuric acid and chilled at collection sites. WCA samples are shipped and 

 stored at a nominal temperature of 4°C.

WG USGS NWIS medium code for groundwater (formerly medium code 6)

WS USGS NWIS medium code for surface water (formerly medium code 9)

YNaR1TM Recombinant nitrate reductase from Pichia angusta (Enzyme Commission 

 # EC 1.7.1.2) expressed in Pichia pastoris and purified to near homogeneity

[AtNaR2, NaR1, and YNaR1 are trade names of the Nitrate Elimination Company, Inc., Lake 

Linden, MI 49945]
x
Colorimetric Determination of Nitrate Plus Nitrite in 

Water by Enzymatic Reduction, Automated Discrete 

Analyzer Methods

By Charles J. Patton1 and Jennifer R. Kryskalla2

Abstract

This report documents work at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) to 

validate enzymatic reduction, colorimetric determinative methods for nitrate + nitrite in filtered water by automated discrete 

analysis. In these standard- and low-level methods (USGS 

I-2547-11 and I-2548-11), nitrate is reduced to nitrite with 

nontoxic, soluble nitrate reductase rather than toxic, granular, 

copperized cadmium used in the longstanding USGS automated continuous-flow analyzer methods I-2545-90 (NWQL 

laboratory code 1975) and I-2546-91 (NWQL laboratory code 

1979). Colorimetric reagents used to determine resulting nitrite 

in aforementioned enzymatic- and cadmium-reduction methods are identical. The enzyme used in these discrete analyzer 

methods, designated AtNaR2 by its manufacturer, is produced 

by recombinant expression of the nitrate reductase gene from 

wall cress (Arabidopsis thaliana) in the yeast Pichia pastoris. 

Unlike other commercially available nitrate reductases we 

evaluated, AtNaR2 maintains high activity at 37°C and is not 

inhibited by high-phenolic-content humic acids at reaction 

temperatures in the range of 20°C to 37°C. These previously 

unrecognized AtNaR2 characteristics are essential for successful performance of discrete analyzer nitrate + nitrite assays 

(henceforth, DA-AtNaR2) described here.

Method detection levels (or limits; MDL) estimated for 

standard- and low-level DA-AtNaR2 nitrate + nitrite methods 

were 0.02 milligrams nitrogen per liter (mg-N/L) and 0.002 

mg-N/L, respectively, which are comparable to 2010 NWQL 

long-term MDLs of the continuous-flow analyzer, cadmiumreduction methods (henceforth, CFA-CdR) they replace. Typically, reagent-water blanks for standard- and low-level DAAtNaR2 nitrate + nitrite methods are one half MDL or less. 

Nitrate + nitrite concentration differences for between-day 

replicates were 3 percent or less at or above 5 times the MDL 

and were as great as 35 percent near the MDL. Typically, 

1U.S. Geological Survey, National Water Quality Laboratory, Denver, Colo.

2Veterans Health Administration, VISN 21 Pharmacy Benefits Management 

Group, Reno, Nev.

nitrate spike recoveries from reagent water, surface water, 

groundwater, and high-phenolic-content, humic-acid-amended 

reagent water were 100±20 percent.

In addition to operational details and performance benchmarks for these new DA-AtNaR2 nitrate + nitrite assays, this 

report also provides results of interference studies for common inorganic and organic matrix constituents at 1, 10, and 

100 times their median concentrations in surface-water and 

groundwater samples submitted annually to the NWQL for 

nitrate + nitrite analyses. Paired t-test and Wilcoxon signedrank statistical analyses of results determined by CFA-CdR 

methods and DA-AtNaR2 methods indicate that nitrate concentration differences between population means or sign ranks 

were either statistically equivalent to zero at the 95 percent 

confidence level (p ≥ 0.05) or analytically equivalent to zero—

that is, when p < 0.05, concentration differences between 

population means or medians were less than MDLs.

Introduction

Nitrate (NO3

-

) is one of the most universally determined 

anions in natural water and drinking water because it can 

promote eutrophication and is toxic to fetuses and young of 

livestock and humans at concentrations that exceed about 

10 milligrams nitrogen per liter (mg-N/L) (U.S. Environmental Protection Agency, 1995). A thorough review of detection 

and determination methods for nitrate and nitrite (NO2

-

) in 

a variety of matrices is available elsewhere (Moorcroft and 

others, 2001). Some important references not cited in Moorcroft’s review include one describing reduction of nitrate 

to nitrite with trivalent vanadium (Miranda, 2001), another 

on optimizing cadmium-reduction assays (Gal and others, 

2004), a third documenting ferrous iron interference in the 

Griess colorimetric indicator reaction (Colman and Schimel, 

2010a, b), and several pertaining to nitrate-reductase-based 

nitrate assays (Senn and Carr, 1976; Guevara and others, 

1998; Mori 2000, 2001; Patton and others, 2002; MacKown 

and Weik, 2004; Pinto and others, 2005; Campbell and others, 2006).
2 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

Cadmium in various forms—such as electrolytically precipitated, “mossy” or “spongy,” filings, granules, and filings or 

granules washed with solutions of mercury (II), silver (I), or 

copper (II) ions (Nydahl, 1976; Davison and Woof, 1978)—

has long been the reducing agent of choice for colorimetric 

nitrate determinations. For example, copper-washed (copperized) cadmium granules packed into small columns (Wood and 

others, 1967) are prescribed in the longstanding U.S. Geological Survey (USGS) and U.S. Environmental Protection 

Agency (EPA) continuous-flow analyzer, cadmium-reduction 

(CFA-CdR) methods I-2545-90 and 353.2, respectively. Wirein-tube cadmium reactors (Stainton, 1974; Willis, 1980; Willis 

and Gentry, 1987; Patton and Rogerson, 2007) and open-tubular cadmium reactors (Patton, 1983; Elliot and others, 1989; 

Zhang and others, 2000) are well known and effective alternatives to packed-bed reactors. A definitive study on continuousflow cadmium reactors (Nydahl, 1976) demonstrated that 

reaction-stream pH in the range of 7.0 to 8.5 is required for 

near-quantitative reduction of nitrate to nitrite with only minor 

(less than 3 percent) reduction of nitrite to lower oxidation 

species. Long-term reactor stability also depends critically on 

including reagents in the analytical stream that form strong 

complexes with cadmium (II) ions—imidazole or ammonium 

chloride, typically. Without such reagents, cadmium (II) ions 

formed during reactions between cadmium and nitrate, dissolved oxygen, or both would precipitate as hydroxides on 

cadmium surfaces and deactivate them.

Despite their long predominance as reducing agents 

of choice for colorimetric nitrate determinations in water, 

flow-through cadmium reactors are difficult to prepare and 

activate, pose health risks to analysts and waste stream processors, increase waste stream disposal costs, and are incompatible with discrete analyzers. These drawbacks motivated 

the National Water Quality Laboratory (NWQL) to explore 

commercially available nitrate reductase enzymes as soluble, 

nontoxic replacements for cadmium. Success of preliminary 

work (Patton and others, 2002) provided motivation and 

continued institutional support for further studies in which we 

investigated and validated two other nitrate reductase enzymes 

as direct replacements for cadmium in USGS-approved colorimetric nitrate + nitrite assays. The automated discrete analyzer 

standard- and low-level enzymatic-reduction, colorimetric 

nitrate + nitrite assays described in the sections that follow are 

the end products of this multiyear research effort.

Purpose and Scope

This report describes new, enzymatic-reduction methods 

for colorimetric nitrate + nitrite determinations in surface 

water and groundwater on automated discrete analyzer (DA) 

instrument platforms. In this report, we provide the following information to NWQL customers and other USGS data 

users who interpret or report nitrate concentration data and to 

analysts at the NWQL and elsewhere who need to implement 

these methods and routinely operate them:

1. Graphical and statistical analysis of paired analytical 

data demonstrating equivalence of nitrate + nitrite 

concentrations determined by these new methods 

(I-2547-11 and I-2548-11) and by time-honored 

USGS CFA-CdR methods I-2545-90 and I-2546-91; 

2. Operational details and performance benchmarks 

for these new discrete-analyzer, AtNaR2-reduction 

(DA-AtNaR2) methods, including method detection 

levels (or limits, MDLs), blank levels, between-day 

precision, and spike recovery from reagent water, 

surface water, groundwater, and high-phenolic-content, humic-acid-amended, reagent water, and;

3. Summaries of experiments demonstrating negligible 

interference in enzymatic and colorimetric assay 

reaction steps by common surface-water and groundwater matrix constituents such as major and minor 

ions and humic substances over a reaction temperature range of 5°C to 37°C.

This report focuses on development and validation of 

standard- and low-level discrete analyzer nitrate + nitrite assays 

using AtNaR2 nitrate reductase (Skipper and others, 2001; 

Campbell and others, 2006) and its cofactor, β-nicotinamide 

adenine dinucleotide, reduced form (NADH). The NWQL analytical services sample stream was the source of seasonally and 

geographically diverse surface-water and groundwater samples 

that we used to demonstrate capability and validate these new 

methods. These samples had nitrate concentrations ranging from 

hundredths to tens of milligrams nitrogen per liter. Specifically, 

we used these new enzymatic reduction methods to analyze 

nitrate + nitrite in subsets of samples originally submitted to the 

NWQL for analysis by USGS-approved cadmium-reduction 

methods, and we then compared results of the new analyses with 

the previous results. This approach is practical, cost effective, 

and would clearly indicate bias, if any, in nitrate + nitrite concentrations determined in real samples during routine operation by 

USGS-approved methods and new methods. Data and statistical analysis supporting established 30-day holding times for 

nitrate + nitrite in filtered and filtered-acidified water samples are 

published elsewhere (Patton and Truitt, 1995; Patton and Gilroy, 

1998).

Analytical Methods

1. Application

The subject new methods listed in table 1 are suitable for 

determination of nitrate + nitrite in filtered (FCC bottle type) 

and filtered-acidified (FCA bottle type) water samples. They 
Analytical Methods 3

Table 1. Laboratory, parameter, and method codes for U.S. Geological Survey automated discrete analyzer, enzymatic 

reduction, standard-level (I-2547-11) and low-level (I-2548-11) nitrate + nitrite determination methods.

[FCC, filtered chilled container; μm, micrometer; FCA, filtered, chilled, acidified container; mL, milliliter]

Description

 

Codes Bottle 

Laboratory Parameter Method type

Nitrate + nitrite, as N, colorimetry, DA, enzymatic reductiondiazotization, filtered (method I-2547-11)

Nitrate + nitrite, as N, colorimetry, DA, enzymatic reductiondiazotization, filtred, low-level (method I-2548-11)

Nitrate + nitrite, as N, colorimetry, DA, enzymatic reductiondiazotization, filtered acidified (method I-2547-11)

3156

3157

3222 

00631

00631

00631

RED01

RED02

RED03 

FCC 

FCC 

FCA

1

1

2

1

FCC samples must be processed through 0.45-μm filters and chilled at collection sites.

2

FCA samples must be processed through 0.45-μm filters, chilled, and amended with 1 mL of 4.5 N H SO2 4

water-quality field supply number Q438FLD) per 120 mL of sample at collection sites.

 solution (U.S. Geological Survey

also are applicable to whole-water-acidified (WCA bottle type) In accordance with the colorimetric reaction scheme 

Samples that are laboratory filtered prior to analysis. They are below, resultant nitrite plus any nitrite present in the sample 

direct replacements for longstanding USGS and EPA colori- prior to enzymatic reduction diazotizes with sulfanilamide at 

metric nitrate + nitrite methods and differ from them only in pH ≈ 1. The p-diazonium sulfanilamide thus formed subsethe reagents used to reduce nitrate to nitrite (nontoxic, soluble quently reacts with N-(1-Naphthyl)ethylenediamine (Brattonnitrate reductase replaces toxic, granular, copperized cad- Marshall variant of the Griess reaction) to form a pink, azo 

mium) prior to colorimetric nitrite determination with Griess dye with an absorption maximum at 543 nm (Bratton and Marreagents. Like cadmium-reduction methods, these enzymatic- shall, 1939; Bendschneider and Robinson, 1952; Fox, 1979, 

reduction methods are intended for surface-water and ground- 1985; Pai and others, 1990).

water matrices (NWIS medium codes WG and WS, formerly 

6 and 9). Seawater, brines, leachates, potassium chloride soil NH

extracts, landfill effluents, and other nonconforming matrices 2

HN

should not be submitted for analyses without prior consulta- N

tion with the NWQL. Such matrices do not match those of NH2

NH N+ 2 HN

calibrants and quality-control samples and therefore might 

produce incorrect analytical results. Nominal analytical ranges NO -

2 + N

for standard- and low-concentration methods are 0.04 to 5.00 H+

N

mg-N/L and 0.008 to 1.00 mg-N/L, respectively. SO2NH2 SO2NH2

Sulfanilamide p-diazonium N-(1-Napthyl)-

2. Method Summaries and Analytical (SAN) sulfanilamide ethylenediamine

(NED)

Considerations SO2NH2

Azo chromophore (λmax = 543 nm)

NADH:nitrate reductase, Enzyme Commission number 

EC 1.7.1.1, hereafter designated AtNaR2 (Skipper and others, 

2001; Campbell and others, 2006), requires NADH as its elec- 3. Interferences and Temperature Effects

tron donating reagent (cofactor). AtNaR2 is produced through Large buffer-to-sample ratios used in methods I-2547- recombinant expression of the nitrate reductase gene from 11 and I-2548-11 mitigate the potential reduction-step and a land plant commonly known as “wall cress” (Arabidopsis colorimetric-step interferences listed below. thaliana) in the yeast Pichia pastoris. AtNaR2 is a proprietary 

product of the Nitrate Elimination Company (NECi), Lake 3.1 Any particles in assays (turbidity) introduced by samples, 

Linden, Mich. In phosphate or 3-N-morpholino-propansul- reagents, or both scatter light during photometric measurefonic acid (MOPS) buffers in the pH range of 7 to 8, AtNaR2 ments. Such turbidity contributes to chromophore absorbance 

quantitatively reduces nitrate (NO -) to nitrite (NO -), as shown and can cause high bias in analytical results. Discernible 3 2

in equation 1. turbidity in samples or colorimetric reagents, therefore, should 

be removed by filtration (0.45-μm or 0.2-μm polyethersulfone

NO− + NADH + H+ AtNaR2 NO−

= N +

3 pH 7−8 + A H2O or nylon) prior to analytical determinations. ( ) 2 D + (1)
4 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

3.2 High concentrations of certain transition- and heavy-metal 

ions can inhibit nitrate reductase to varying extents. Ethylenediaminetetraacetic acid (EDTA) forms strong complexes with 

many metal ions and effectively minimizes interference by 

these potential sample matrix constituents that might otherwise hinder quantitative reduction of nitrate to nitrite. Eight of 

the more abundant metal ions in water analyzed at the NWQL 

at up to 100 times their median annual concentrations affected 

nitrate recovery by less than ±2 percent. See the section titled 

“Analytical Performance and Comparative Results” for additional details.

3.3 Sulfate, chloride, and bromide at up to 100 times their 

median concentration in samples analyzed at the NWQL annually affected nitrate recovery by less than ±2 percent. Perchlorate at concentrations up to 5 mg/L affected nitrate recovery by 

less than 2 percent. See the section “Analytical Performance 

and Comparative Results” for additional details.

3.4 High-phenolic-content humic substances (HAs) are matrix 

constituents in perhaps 15 percent of water samples received 

for nitrate analysis at the NWQL annually. Concentrations 

of HA up to 20 mg/L do not inhibit AtNaR2 in the reduction 

reaction temperature range of 10°C to 37°C. However, for 

other commercially available nitrate reductase enzymes that 

we evaluated, HA inhibition was negligible only in the temperature range of 10°C to 20°C. Above 20°C, HA inhibition 

increased continuously in direct proportion to reduction reaction temperature and HA concentration (see section “Effects 

of Temperature and Dissolved Organic Matter on AtNaR2 

Activity”).

3.5 NADH inhibits the Griess indicator reaction (Patton and 

others, 2002, table 1; Moody and Shaw, 2006). Quantitative 

reduction of nitrate to nitrite with minimum Griess reaction 

inhibition occurs when initial NADH concentration is in 

two-fold molar excess to that of a method’s maximum nitrate 

concentration in the reaction medium. Methods I-2547-11 

and I-2548-11 conform to this initial NADH concentration 

condition.

3.6 AtNaR2 and other nitrate reductases we evaluated promote oxidation of NADH to NAD+ even in the absence of 

nitrate. Separate AtNaR2 and NADH reagents used in assays 

described here eliminate the possibility of this potentially 

reagent-limiting side reaction. If a mixed AtNaR2-NADH 

reagent were required—because of analytical platform 

limitations, perhaps—its useful lifetime would be less than 

2 hours.

3.7 Norwitz and Keliher (1985, 1986) systematically 

assessed inorganic and organic interferences for the Griess 

indicator reaction. Colman and Schimel (2010a, b) recently 

reported that Fe (II) at or above 10 mg/L suppresses the 

Griess reaction. According to these authors, replacing ethylenediaminetetraacetic acid (EDTA) with diethylenetriaminepentaacetic acid (DTPA) in nitrate assay buffers eliminates 

this interference. Although Fe (II) concentrations of 10 mg/L 

or more are unlikely to occur in surface water and groundwater, analysts applying these methods to high-iron soil extracts, 

acid mine drainage water, or pore water from low-oxygen bed 

sediments should be aware of this potential interference and 

its remedy.

3.8 A number of metal cations are minor nitrite indicator reaction inhibitors (see fig. 1). Group II (alkaline earth) 

cations produce the largest effects. Calcium ions reduce the 

yield of indicator reaction chromophore the most—about

5 percent at NWQL-median concentrations—but barium ions 

are the most potent indicator reaction suppressor on a molar 

basis.

3.9 The inverse relationship between reaction temperature 

in the range of 10°C to 50°C and formation rate and yield of 

the Griess indicator reaction chromophore evident in figure 2

results from thermal instability of nitrous acid and diazonium 

intermediates in the Griess reaction (Noller, 1966).

4. Instrumentation

We developed automated DA soluble AtNaR2-reduction 

nitrate + nitrite methods using a Kone Aquakem 600TM 

analyzer (Thermo Fisher Diagnostics, Fremont, Calif.). Basic 

operation of the Aquakem 600TM DA can be understood with 

reference to figure 3 and the text that follows.

On startup, cuvette segments—linear arrays of 12 cells 

in which individual tests or dilutions take place and through 

which absorbance is measured—move from the cuvette loader 

into available incubator slots. The incubator hub rotates to 

align cuvette segments with sample- or reagent-dispensing 

alleys as appropriate during the analytical cycle. Sample 

and reagent “disks” that hold sample segments and reagent 

containers are thermostatted at 10°C and 4°C, respectively. 

Precisely controlled rotation of these disks aligns the appropriate sample or reagent with dispensing arms during operation. 

As cuvette segments move sequentially through sample- and 

reagent-dispensing alleys, individual cells are aligned with 

high-precision dispensers attached to robotic arms. Stirrers 

on another set of robotic arms mix cell contents after each 

dispense cycle. The robotic arms return dispenser needles 

and stirrer blades to wash stations for thorough rinsing after 

each dispense/mix operation. Between dispensing operations, 

cuvette segments return to the incubator where programmed 

reaction times up to 60 minutes occur. The incubator, dispensing alleys, and photometer module are thermostatted at 

37°C. Liquids dispensed into cells equilibrate to 37°C during 

the course of analyses. At the end of the three programmed 

incubations (see table 2), cuvette segments exit the incubator 

one at a time and enter the photometer module where sequential measurements of absorbance in each cell occurs. The DA 

ejects cuvette segments into a waste compartment positioned 

below the photometer after absorbance measurements are 

complete.
Analytical Methods 5

EXPLANATION

1.00

0.75

0.50

0.25

0

0 0.250 0.500 0.750 1.000

Time, in minutes

Griess reaction absorbance at 540 nanometers

Ca(II), 908.18 μM

Sr(II), 3.04 μM

Mn(II), 0.19 μM

Li(I), 1.02 μM

CI04

−

Mg(II), 341.56 μM

Ba(II), 0.3 μM

Fe(III), 0.14 μM

Cr(III), 0.005 μM

No amendment

0.975

0.950 

0.925

0.900

0.150 0.300 0.450 0.600 0.750 0.900

Figure 1. Kinetics effects of diverse metal ions and perchlorate on the Griess reaction 

colorimetric nitrite assay. (μM, micromolar)

Figure 2. Effect of temperature on kinetics and yield of the Griess reaction nitrite assay. (°C, degrees 

Celsius)

EXPLANATION

0.900

Griess reaction chromophore absorbance at 540 nanometers

0.750

0.600

0.450

0.300

0.150

0

Time after N-(1-Naphthyl)ethylenediamine reagent addition, in seconds

0 15 30 45 60 75 90 105 120

10°C

15°C

20°C

25°C

30°C

35°C

37°C

40°C

50°C
6 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

The Aquakem 600TM measures absorbance with a “dualbeam-in-time” filter photometer (Ingle and Crouch, 1988). 

This photometer design compensates for wavelength-dependent light-source intensity and detector sensitivity as well as 

light source flicker and drift. Aquakem 600TM software provides two methods to record and correct minor contributions 

to assay absorbance caused by turbidity and cuvette imperfections. The first, termed side-wavelength correction, involves 

photometric measurement of finished assays at the wavelength 

where chromophore absorbance is maximum (λmax ≈ 540 nm) 

and at a second wavelength where chromophore absorbance 

is negligible (700 nm). The difference between absorbance 

measured at 540 nm and 700 nm yields chromophore absorbance corrected for light scattering effects. The correction 

works because light scattering in the wavelength range of 

540–700 nm is nearly constant. The second method, termed 

reagent-blank correction, involves (1) measuring the intermediate assay absorbance after adding and mixing the first 

reagent sulfanilamide (SAN) at λmax, (2) adding the second, 

color forming reagent N-(1-Naphthyl)ethylenediamine (NED), 

and (3) measuring finished assay absorbance again at λmax after 

chromophore formation is complete. Here, correction and 

analytical absorbance measurement wavelengths are the same, 

but the small volume difference between measurements might 

slightly overcorrect scattering effects. In practice, analytical 

results obtained with either correction method are the same 

within assay precision limits. We used side-wavelength correction during method development and validation. We have 

since implemented reagent-blank correction because the Kone 

software generates automatic warnings, report flags, and conditional branching from operator specified reagent-blank upper 

and lower absorbance limits. This functionality is not provided 

for side-wavelength absorbance corrections.

See table 2 for DA operational protocols (test flows) used 

for standard- and low-level concentration AtNaR2-reduction 

nitrate + nitrite determinations. Because standard-level (SL) 

and low-level (LL) methods are identical except for a fivefold 

sample-volume increase in the latter, we combined their test 

flows in table 2. In this table, “extra” refers to aspirated volumes 

that are not delivered into cuvette cells. According to the DA 

manufacturer, dispensing with “extra” minimizes sample and 

reagent dilution during dispensing operations and thus improves 

precision of analytical results. “Extra” sample and reagent 

volumes are purged from dispensing needles during rinse cycles 

and collected in the analyzer waste stream. Additional details of 

Aquakem 600TM hardware and software are in the manufacturer’s operation manual and NWQL Technical Operations Manual 

for Kone Aquakem 600TM DA (Schwab and others, 2009).

Figure 3. Functional diagram of the Aquakem 600TM automated discrete analyzer. (Image courtesy of Thermo 

Scientific.)

Sample dispensing alley Reagent dispensing alley

REAGENT DISK
Analytical Methods 7

5. Apparatus

We used EDP-plusTM electronic, digital pipets (Rainin 

Instruments, Oakland, Calif.) fitted with 10–100-μL, 100–

1,000-μL, and 1,000–10,000-μL liquid ends as appropriate for 

most precision dispensing.

We configured the purpose-built, thermostatted, continuous flow reaction monitor used for interference studies and 

enzyme reaction rate experiments from components in our 

laboratory, including an OB-1 large-platform autosampler 

(Oregon Manufacturing Support, Malin, Oreg.), RFA-300 

continuous flow analyzer modules (no longer in production), 

and a model TLC 40 temperature-controlled cuvette holder 

equipped with a magnetic stirring accessory (Quantam Northwest, Spokane, Wash.).

6. Reagent Preparation

This section provides detailed instructions for preparing 

enzymatic and colorimetric reagents used for standard- and 

low-level discrete analyzer assays. All references to deionized 

(DI) water refer to DI water piped throughout the NWQL. For 

purposes of nutrient analysis, NWQL DI water is comparable 

to ASTM type I DI water (American Society for Testing and 

Materials, 2001, p. 107–109). We triple rinsed all volumetric 

glassware and containers for reagent and calibrant storage with 

dilute (≈ 5 percent v/v) hydrochloric acid and DI water just 

prior to use. We also triple rinsed reagent and calibrant storage 

containers with small portions of the solutions they were to 

contain before we filled them.

6.1 Enzymatic reagents

6.1.1 Di-sodium ethylenediaminetetraacetic acid (EDTA), 

25 millimolar (mM).—Dissolve 9.3 g EDTA (FW = 

372.24, Ultrapure grade) in approximately 800 mL DI 

water contained in a 1-L volumetric flask. Dilute the 

resulting solution to the mark with DI water, mix it well, 

and transfer it to a bottle where it is stable at room temperature for one year.

6.1.2 Phosphate buffer (pH = 7.5).—Dissolve 3.75 g 

potassium di-hydrogen phosphate (KH2

PO4, FW = 136.1) 

and 1.4 g potassium hydroxide (KOH, FW = 56.11) in 

about 800 mL of DI water contained in a 1-L volumetric 

flask. Add 1 mL 25 mM EDTA and dilute the resulting 

solution to the mark with DI water; mix it well. Transfer 

this solution to a bottle where it is stable at room temperature for one year.

6.1.3 Nitrate reductase from Arabidopsis thaliana, 

AtNaR2, EC #1.7.1.1.—Remove the cap from a vial 

containing 3 units of freeze-dried AtNaR2 and add to it 

about 1 mL of the proprietary reconstitution buffer supplied with the enzyme. Alternatively, substitute 1 mL of 

pH 7.5 phosphate buffer (6.1.2). Recap the vial and invert 

it several times over the course of 30 minutes to speed 

dissolution of the freeze-dried enzyme.

NOTE: According to the enzyme manufacturer, 3 units 

of AtNaR2 dissolved in ≈1 mL of their proprietary reconstitution buffer are stable at or below -15°C for several 

months. NECi includes a squeezable plastic ampoule 

containing about 1 mL of this buffer with each 3-unit vial 

of AtNaR2.

6.1.4 Working AtNaR2 reagent.—Quantitatively transfer 

and dilute the dissolved enzyme concentrate in a 20-mL 

Kone reagent tube as follows:

• Carefully pour the dissolved enzyme concentrate 

from the vial in which it was reconstituted into the 

reagent tube.

• Use a digital pipet to dispense 1,000 μL of pH 

7.5 phosphate buffer (6.1.2) into the empty enzyme 

vial.

• Recap the vial and invert it several times.

• Before removing the cap, tap it sharply with your 

finger to dislodge adherent droplets.

• Remove the cap and pour the resulting rinse solution 

into the reagent tube.

• Repeat steps 2–5 two more times, after which the 

reagent tube should contain 4 mL of enzyme concentrate in phosphate buffer.

Table 2. Aquakem 600TM automated discrete analyzer 

operational protocols, termed test flows by the vendor, for 

standard-level (SL) and low-level (LL) AtNaR2-reduction nitrate + 

nitrite determination methods.

[μL, microliter; s, second; nm, nanometers; extra, aspirated volumes that are 

not delivered into reaction cells; NADH, nicotinamide adehine dinucleotide in 

reduced form; SAN, sulfanilamide; NED, N-(1-Naphthyl)ethylenediamine]

Dispensed 

liquid

Volume/extra 

(μL)

Incubation 

time (s)

Wavelength (nm)

Analytical Side*

AtNaR2 55/10

Sample, SL 5/25

Sample, LL 25/25

NADH 12/15

600

SAN 25/10

120

Reagent blank 540

NED 25/10

120

540 700

*Optional.
8 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

• Add 16.0 mL of phosphate buffer (dispensing 8 mL

twice from a digital pipet equipped with a 10-mL 

liquid end works well) into the reagent tube and recap

it. Then mix the working reagent gently by repeated

inversion. Working AtNaR2 enzyme reagent 

is stable at 2°C to 8°C for about 18 hours.

If a 20-mL batch of this reagent, which is sufficient for 

about 330 assays, cannot be used within a day, prepare a 

smaller volume—for example, 250 μL AtNaR2 concentrate 

diluted to 5 mL with pH 7.5 buffer—and store remaining 

750 μL of AtNaR2 concentrate at or below -15°C for future 

use. Alternatively, remove remaining working AtNaR2 

reagent from the analyzer and freeze it at or below -15°C.

6.1.5 β-Nicotinamide adenine dinucleotide, reduced form, 

disodium salt (NADH) stock solution.—Dissolve 0.100 g 

of NADH (FW = 709.4, product number N 8129, Sigma, 

St. Louis, Mo., ≈98 percent) in approximately 40 mL of 

DI water contained in a 50-mL volumetric flask. Dilute 

the resulting solution to the mark with DI water and mix 

it well. Use a digital pipet to transfer 1-mL aliquots of 

stock NADH reagent into 1.7-mL snap-cap vials (VWR, 

Cat. No. 20170-650) and store them in a freezer at -20°C 

where NADH thus prepared is stable for 6 weeks.

6.1.6 NADH working solution.—Remove one vial of 

stock NADH from the freezer and allow it to thaw at 

ambient temperature (about 20 minutes is required) while 

AtNaR2 reconstitutes. Then quantitatively transfer the 

stock NADH solution into in a 20-mL DA reagent tube as 

follows:

• Carefully pour the thawed NADH concentrate into 

the working reagent tube.

• Use a digital pipet to dispense 1,000 μL of phosphate 

buffer into the empty snap-cap vial.

• Recap the vial and invert it several times.

• Before flipping the cap up, tap it sharply with your 

finger to dislodge adherent droplets.

• Use a digital pipet equipped with a 10-mL liquid 

end to dispense 8.0 mL of phosphate buffer into the 

reagent tube and mix the contents well. This 10-mL 

volume of working NADH reagent, which is sufficient for 330 assays, is stable at 2°C to 8°C for at 

least 24 hours.

NOTE: The NWQL has found it convenient to use 

reagent kits (NECi product number DA-ARK-1) that contain a vial of freeze-dried AtNaR2 (3 units), an ampoule 

of reconstitution buffer, and a vial of freeze-dried NADH 

(2 mg). Use the procedure for frozen NADH concentrate 

described in 6.1.6 to prepare freeze-dried NADH.

6.2 Colorimetric reagents

6.2.1 Sulfanilamide reagent (SAN).—Slowly add 150 mL 

concentrated hydrochloric acid (HCl, ≈ 12M) to about 

250 mL deionized water contained in a 500-mL volumetric flask. While the solution is still warm, add 5.0 g sulfanilamide (C6

H8N2O2S, FW = 172.2) to the flask. Swirl 

the flask gently to dissolve the SAN. Dilute this reagent to 

the mark with deionized water and mix it well. Store SAN 

at room temperature in a clear glass or translucent plastic 

500-mL bottle where it is stable for 6 months.

6.2.2 N-(1-Naphthyl)ethylenediamine reagent (NED).—

Dissolve 0.5 g NED (C12H14N2•2HCl, FW = 259.2) in 

about 400 mL of DI water contained in a 500-mL volumetric flask. Dilute this reagent to the mark with DI 

water and mix it well. Store NED at room temperature 

in an amber, 500-mL glass bottle where it is stable for 

6 months.

7. Calibrants and Quality-Control Solutions

7.1 Use digital pipets and Class-A volumetric flasks to 

prepare secondary (Stock II) calibrants from a commercially 

obtained, certified 1,000 mg-N/L primary nitrate calibrant 

as indicated in table 3. In its current configuration, the DA 

prepares working calibrants for these assays by serial dilution 

of Stock II calibrants as indicated in tables 4 and 5. It is possible to calibrate the DA with individual, manually prepared 

working calibrants, but typically there is no advantage to this 

labor-intensive practice. Prepare Stock II calibrants monthly, 

transfer them to screw-cap, glass media bottles, and store them 

at 4°C. Follow vendor-specified storage temperatures and shelf 

lives for primary calibrants.

7.2 Laboratory control samples and spike solutions

7.2.1 The purpose of third-party-check (TPC) samples 

is to confirm and document the accuracy of instrument 

calibration. It is necessary, therefore, to prepare them 

from certified nitrate and nitrite solutions different from 

those used to prepare calibrants. Use digital pipets, ClassA volumetric flasks, and second-source, certified nitrate 

and nitrite solutions to prepare working TPC samples 

for standard- and low-level nitrate assays as indicated 

in tables 6 and 8, respectively. Tables 7 and 9 provide 

preparation guidelines for nitrate-only TPCs. Nominal concentrations of low-level assay TPC samples in 

tables 8 and 9 are slightly different from those used during validation experiments to simplify their preparation 

and to make their concentrations proportional (5-times 

less) to standard-level assay TPCs. Prepare TPCs 

monthly, transfer them to screw-cap glass media bottles, 

and store them at 4°C.
Analytical Methods 9

Table 3. Nitrate Stock II calibrants used for automated discrete analyzer calibration.

[ID, identifier; μg-N/μL, microgram nitrogen per microliter; μL, microliter; mL, milliliter; mg-N/L, milligram nitrogen per liter; 

NO3

–

-N, nitrate nitrogen] 

Stock II 

calibrant 

ID

Analyte

Stock I 

concentration 

μg-N/μL

Stock I 

dispensed 

volume 

(μL)

Stock II 

volume 

(mL)

Nominal 

concentration 

(mg-N/L)

S6 NO3

–

-N (low-level) 1.0 400 100 4.00

S7 NO3

–

-N (low-level) 1.0 60 100 0.60

S8 NO3

–

-N 1.0 2,000 100 20.00

S9 NO3

–

-N 1.0 300 100 3.00

Table 5. Low-level concentration range nitrate working 

calibrants used for automated discrete analyzer (DA) calibration.

[ID, identifier; mg NO3

–

-N/L, milligram nitrate nitrogen per liter] 

Stock II 

calibrant 

ID

DA 

dilution 

factor notation

Dilution 

factor

Nominal 

concentration 

(mg NO3

–

-N/L)

S6 S6: 1+3 4 1.000

S6 S6: 1+4 5 0.800

S6 S6: 1+ 6.5 0.615

S6 S6: 1+9 10 0.400

S6 S6: 1+19 20 0.200

S6 S6: 1+39 40 0.100

S7 S7: 1+11 12 0.050

S7 S7: 1+59 60 0.010

Table 4. Standard-level concentration range nitrate working 

calibrants used for automated discrete analyzer (DA) calibration.

[ID, identifier; mg NO3

–

-N/L, milligram nitrate nitrogen per liter] 

Stock II 

calibrant 

ID

DA 

 dilution 

factor notation

Dilution 

factor

Nominal 

concentration 

(mg NO3

–

-N/L)

S8 S8: 1+3 4 5.00

S8 S8: 1+4 5 4.00

S8 S8: 1+5.5 6.5 3.08

S8 S8: 1+9 10 2.00

S8 S8: 1+19 20 1.00

S8 S8: 1+39 40 0.50

S9 S9: 1+11 12 0.25

S9 S9: 1+74 75 0.04

Table 6. Second-source, certified nitrate and nitrite solution 

volumes needed to prepare standard-level-assay third-partycheck (TPC) samples in 100-mL quantities.

[mL, milliliter; ID, identifier; μL, microliter; mg-N/L, milligram nitrogen 

per liter; NO3

–

, nitrate; NO2

–

, nitrite] 

Kone 

TPC 

ID

Second-source 

1,000-mg 

NO3

–

-N/L 

calibrant 

(μL)

Second-source 

100-mg 

NO2

–

-N/L 

calibrant * 

(μL)

Nominal concentration 

(mg-N/L)

NO3

–

-N NO2

–

-N NO3

–

+NO2

–

-N

TPC_Low 48 20 0.48 0.02 0.50

TPC_Med 192 80 1.92 0.08 2.00

TPC_High 384 160 3.84 0.16 4.00

* If a certified 100 mg-N/L solution is not available commercially, prepare 

it from certified 1,000 mg-N/L solution after 1+9 dilution with deionized 

water.

Table 7. Second-source, certified nitrate solution 

volumes needed to prepare nitrate-only, standardlevel-assay third-party-check (TPC) samples in 100-mL 

quantities.

[mL, milliliter; ID, identifier; μL, microliter; mg-N/L, milligram 

nitrogen per liter] 

Kone 

TPC 

ID

Second-source 

1,000 mg-N/L 

calibrant 

(μL)

Nominal nitrate 

concentration 

(mg-N/L)

TPC_Low 50 0.500

TPC_Med 200 2.000

TPC_High 400 4.000
10 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

7.2.2 Continuing calibration verification (CCV) samples 

are used to confirm and document that instrument calibration is maintained within specified limits during the course 

of analyses. Nitrate solutions prepared at concentrations 

50 to 75 percent of assays’ upper calibration limits are suitable. Stock II calibrants S9 and S7 (table 3) are convenient 

to use as standard- and low-level CCVs, respectively. 

Prepare CCVs monthly from the same certified nitrate 

solution used to prepare calibrants, transfer them to screwcap glass media bottles, and store them at 4°C.

7.2.3 Spike solutions.—Prepare spike solutions for 

standard- and low-level nitrate assays with digital pipets, 

Class-A volumetric flasks, and the same certified nitrate 

solution used to prepare calibrants. Separately dilute 

2.5 mL (standard level) and 0.5 mL (low level) of certified 

1,000 mg-N/L nitrate solution to 50 mL with DI water. 

Resulting standard- and low-level nitrate spike solutions 

contain 0.05 μg-N/μL and 0.01 μg-N/μL, respectively. 

To increase sample or DI water blank concentration by 

0.5 mg-N/L or 0.1 mg-N/L, dispense 10 μL of the appropriate spike solution into the conical well of a standard 

2-mL analyzer cup, add 990 μL of sample or blank to 

it, and mix. This procedure dilutes spiked solutions by 

1 percent. Prepare spike solutions monthly, transfer them 

to screw-cap glass media bottles, and store them at 4°C.

8. Sample Preparation

The DA enzymatic nitrate + nitrite methods require analysts to rinse and fill analyzer cups or tubes with well-shaken 

samples, place them into appropriate racks, and load racks into 

the sampler compartment. No other manual sample preparation 

is required.

9. Instrument Performance

The DA used to validate enzymatic reduction standardand low-level nitrate assays has a nominal analysis rate of 

600 tests per hour. However, for multistep assays such as 

these—four reagent additions and a total incubation time 

of about 14 minutes—the analysis rate is substantially less 

(300 tests per hour, perhaps) and is further reduced by samples 

that require dilution and by incidents of failed quality-control 

(QC) samples. Standard- and low-level assay volumes (sample 

+ reagents) are 122 μL and 142 μL, respectively. For comparison, analysis rates for a single-channel, third generation 

continuous-flow (CF) analyzer performing similar assays was 

90 tests per hour and per test sample and reagent volumes 

exceeded those of DA assays by about five times (Patton and 

others, 2002). Based on the 2011 price for NECi DA-ARK-1 

reagent kits ($75.00; see note at the end of section 6), the perassay cost of AtNaR2 and NADH for standard- and low-level 

methods is about 25 cents.

10. Calibration

Calibration functions for standard- and low-level 

assays are linear with linear least squares fit (Draper and 

Smith, 1966) correlation coefficients (r2

) equal to or greater 

than 0.999 as shown in figure 4. Calibration functions take 

the form y = a + bx, where y is the reagent-blank-corrected 

absorbance at 540 nm, x is the nitrate + nitrite concentration in 

mg-N/L, and a and b are the y-intercept and slope parameters. 

If there is slight bend off at higher concentrations, a secondorder polynomial least-squares calibration function in the form 

y = a + bx + cx2 might provide a better fit.

11. Procedure and Data Evaluation

Except as noted in sections 6 and 7, procedures for standard- and low-level assays were as specified in NWQL SOP 

INCF0452.2 (Gupta and others, 2011). Table 10 identifies NWQL 

standard operating procedures (SOPs) that provide complete 

procedural details of USGS CFA-CdR methods against which we 

validated soluble DA-AtNaR2-reduction nitrate methods.

Table 9. Second-source, certified nitrate solution volumes 

needed to prepare nitrate-only, low-level-assay third-partycheck (LLTPC) samples in 100-mL quantities.

[mL, milliliter; ID, identifier; μL, microliter; mg-N/L, milligram nitrogen per liter] 

Kone 

TPC 

ID

Second-source 

100 mg-N/L 

calibrant * 

(μL)

Nominal nitrate 

concentration 

(mg-N/L)

LLTPC_Low 100 0.100

LLTPC_Med 400 0.400

LLTPC_High 800 0.800

* If a certified 100 mg-N/L solution is not available commercially, 

prepare it from certified 1,000 mg-N/L solution after 1+9 dilution with 

deionized water.

Table 8. Second-source, certified nitrate and nitrite solution 

volumes needed to prepare low-level-assay third-party-check 

(LLTPC) samples in 100-mL quantities.

[mL, milliliter; ID, identifier; μL, microliter; mg-N/L, milligram nitrogen per 

liter; NO3

–

, nitrate; NO2

–

, nitrite] 

Kone 

TPC 

ID

Second-source 

100-mg 

NO3

–

-N/L 

calibrant * 

(μL)

Second-source 

100-mg 

NO2

–

-N/L 

calibrant * 

(μL)

Nominal concentration 

(mg-N/L)

NO3

–

-N NO2

–

-N NO3

–

+NO2

–

-N

LLTPC_Low 80 20 0.08 0.020 0.100

LLTPC_Med 320 80 0.32 0.080 0.400

LLTPC_High 640 160 0.64 0.160 0.800

* If certified 100 mg-N/L nitrate and nitrite solutions are not available 

commercially, prepare them from certified 1,000 mg-N/L solutions after 1+9 

dilution with deionized water.
Analytical Methods 11

Nominal nitrate + nitrite concentration, in milligrams nitrogen per liter

EXPLANATION

0.800

0.700

0.600

0.500

0.400

0.300

0.200

0.100

0

0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Absorbance at 540 nanometers

Absorbance = 0.5824 (Nominal nitrate + nitrite) + 0.0067

Correlation coefficient = 0.9995

Absorbance = 0.1344 (Nominal nitrate + nitrite) + 0.0077

Correlation coefficient = 0.9996

AtNaR2 nitrate calibration: 04 June 2010, LC 3156

AtNaR2 LL nitrate calibration: 04 June 2010, LC 3157

Linear (AtNaR2 nitrate calibration: 04 June 2010, LC 3156)

Linear (AtNaR2 LL nitrate calibration: 04 June 2010, LC 3157)

Figure 4. Typical calibration graphs for standard- and low-level concentration range enzymaticreduction nitrate determination methods by automated discrete analyzer. (LL, low level; LC, National 

Water Quality Laboratory laboratory code)

Table 10. U.S. Geological Survey National Water Quality Laboratory (NWQL) laboratory codes and standard operating procedure 

(SOP) numbers for cadmium-reduction (CdR) and enzymatic-reduction (AtNaR2) nitrate determination methods referenced in this 

report.

Method name Laboratory

code

NWQL SOP

number

Nitrate + nitrite, CdR, automated continuous-fl ow 1975 ID0163.6-1

Nitrate + nitrite, CdR, automated continuous-fl ow, low-level 1979 ID0200.2-1

Nitrate + nitrite, AtNaR2-reduction, automated discrete analyzer, standard-level concentration 

range

3156 INCF0452.2

Nitrate + nitrite, AtNaR2-reduction, automated discrete analyzer, low-level concentration range 3157 INCF0452.2
12 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

12. Calculations

12.1 We used vendor supplied software to acquire and process data from CFA (fASPacTM version 3.3, Astoria-Pacific, 

Clackamas, Oreg.) and DA (Aquakem 600TM versions 6.5, 7.0, 

and 7.2, Thermo Fisher Diagnostics, Fremont, Calif.) instrument platforms and to convert them into concentration units. 

Calibration functions for cadmium-reduction CFA methods 

were quadratic, linear least-squares fits (Draper and Smith, 

1966) of the form y = a + bx + cx2 (see section 10). Calibration 

functions for AtNaR2-reduction DA methods were typically 

linear least-squares fits.

12.2 We used Microsoft Office 2003 ExcelTM to compile data 

acquired from instrument-specific software packages, to perform arithmetic and linear least-squares regression parameter 

calculations, and to prepare most graphical representations 

of data in this report. We used Microcal Origin Pro 8.0TM to 

perform Kolmogorov-Smirnov statistical tests of normality on 

spike recovery datasets and t-tests and Wilcoxon signed-rank 

tests on populations on paired CFA-CdR and DA-AtNaR2 

nitrate + nitrite concentration data for surface water and 

groundwater.

12.3 Software packages identified in Section 12.1 provide 

for automatic application of dilution factors—the number by 

which a measured concentration must be multiplied to obtain 

the analyte concentration in the sample prior to dilution. Automatic, online dilution was not possible with the CFA equipment we used for work reported here, so we diluted off-scale 

samples manually using electronic pipets. The DA software 

also provides entry fields for offline dilution factors, but 

because this instrument was capable of up to 120-fold online 

sample dilution, manual offline sample dilution was rarely 

necessary. When both offline and online dilution factors are 

associated with the same sample, the dilution factor applied is 

the product of the two. Although dilution factors are applied 

identically by CFA and DA software applications, the factors 

are entered differently. CFA software requires entering the 

sum of 1 part sample + n parts diluent. For example, entering 

values of 2, 5, and 10 into the CFA software dilution factors 

fields indicate sample-to-diluent proportions of 1+1, 1+4, and 

1+9—that is, two-, five-, and tenfold dilutions. DA software 

requires entry only of the parts of diluent added to 1 part of 

sample, and dilution factor entry fields always appear as 1+n. 

Therefore, entering values of 1, 4, and 9 into DA software 

dilution factor fields result in two-, five-, and tenfold dilutions.

12.4 Control limits for TPC solutions were calculated according to protocols developed by the USGS Branch of Quality 

Systems (BQS) for use with their Inorganic Blind Sample 

Program (IBSP). BQS control limit estimates use a robust 

(median-based) statistic equivalent to mean-based standard 

deviation, which BQS denotes ƒσ. BQS suggests three methods 

for estimating ƒσ.

12.4.1 Use regression equations of analyte concentration 

in relation to ƒσ

, which are tabulated on the BQS Web site 

(http://bqs.usgs.gov/ibsp/) by year.

12.4.2 ƒσ = 75 percent of LT-MDL or provisional MDL 

(set at 0.04 mg-N/L and 0.008 mg-N/L for standard- and 

low-level DA-AtNaR2 assays during the first year of 

operation following Office of Water Quality approval), or

12.4.3 ƒσ = 5 percent of each nominal TPC concentration.

The BQS suggests estimating ƒσ by all three methods and 

selecting the largest of the three for each nominal concentration. By NWQL Nutrients Unit convention, upper and 

lower control limits (UCL and LCL) are set at 1.5 times ƒσ

± the nominal TPC concentration.

13. Reporting Results

13.1 Reporting units for nitrate + nitrite and nitrite concentrations are milligrams nitrogen per liter (mg-N/L) in accordance 

with longstanding USGS conventions. A table at the front of 

this report provides factors necessary to convert these units 

into several other commonly used concentration units.

13.2 We report concentrations such that the rightmost digit 

(called the least significant digit) represents the uncertainty in 

the analytical result (Novak, 1985; Hansen, 1991; U.S. Geological Survey, 2002). The least significant digit is determined 

using guidance outlined by the American Society for Testing 

and Materials (1999). Presently (2011), the NWQL reports 

results in the national database to the least significant digit 

plus one additional digit.

14. Detection Limits, Precision, Spike Recovery, 

and Bias

14.1 As listed in table 11, MDLs for standard- and low-level 

DA-AtNaR2 nitrate + nitrite assays were 0.02 mg-N/L and 

0.002 mg-N/L, respectively, which we calculated in accordance with EPA guidelines (U.S. Environmental Protection 

Agency, 1997). By NWQL consensus, interim reporting limits 

(IRLs) for standard- and low-level DA-AtNaR2 nitrate + 

nitrite assays will be set at 0.04 mg-N/L and 0.008 mg-N/L, 

respectively, during the first year of routine operation.

14.2 Table 12 lists the average, standard deviation, and other 

information related to TPC samples that we analyzed along 

with standard-level environmental water samples during DAAtNaR2 assay validation work between November 30, 2007, 

and August 8, 2008. Table 12 also contains the same parameters for low-level TPCs that we analyzed during a single day 

on February 15, 2007. Inspection of table 12 reveals that over 
Analytical Methods 13

a period of about 9 months, standard-level assay TPC results 

at high, medium, and low concentrations agree within about 

3 percent. Again with reference to table 12, within-day results 

for the low-level AtNaR2 nitrate assay at high, medium, and 

low TPC concentrations agree within about 1 percent. Figure 5 

provides a plot of standard-level assay TPC data obtained 

between November 30, 2007, and August 8, 2008, in control 

chart format. Upper and lower control limits in figure 5 correspond to those listed in table 12, which are in accordance with 

IBSP guidance as described in section 12.4.

Table 13 provides within-day and between-day precision summaries of AtNaR2-reduction nitrate + nitrite assays performed 

on water samples between May and July of 2007. With reference to table 13, within-day precision was about ±1 percent 

but decreased as concentrations approached the detection 

limit. Between-day precision was remarkably good (on the 

order of ±5 percent) considering that on the second analysis 

date most samples were past their 30-day holding time limit. 

Table 14 provides within-day precision summaries for lowlevel AtNaR2-reduction nitrate + nitrite assays. With reference 

to table 14, within-day precision was about ±3 percent but 

decreased as concentrations approached the detection limit.

Tables 15A–C and 16A–C list recoveries of nitrate spiked into 

reagent water, surface water, and groundwater at about 5 times 

and 50 times standard- and low-level MDLs for standard- and 

low-level DA-AtNaR2 assays, respectively. With reference to 

these tables, recoveries typically were 100±20 percent, which 

are well within NWQL-specified criteria for accepted analytes 

Table 11. Data and calculations used to estimate method 

detection limits (MDL) for nitrate + nitrite determination with 

soluble AtNaR2 nitrate reductase by automated discrete analysis 

(DA).

[mg-N/L, milligram nitrogen per liter; NO3

–

, nitrate; NO2

–

, nitrite; SL, standard level; LL, low level; LT, long term] 

Nitrate + nitrite concentration (mg-NO3

–

-N + NO2

–

-N/L)

Target concentration 

SL (LL)

Concentration found

AtNaR2 

SL

AtNaR2 

LL

0.08 (0.020) 0.0767 0.0190

0.08 (0.020) 0.0748 0.0189

0.08 (0.020) 0.0732 0.0180

0.08 (0.020) 0.0870 0.0186

0.08 (0.020) 0.0746 0.0187

0.08 (0.020) 0.0731 0.0180

0.08 (0.020) 0.0770 0.0201

0.08 (0.020) 0.0826 0.0181

Average 0.0774 0.0187

Standard deviation 0.0049 0.0007

Number of values 8 8

Degrees of freedom 7 7

t-value (1-sided, 99 percent) 2.998 2.998

MDL 0.02 0.002

2011 CFA-CdR (LT-MDL) 0.02 0.008

Table 12. Third-party-check (TPC) sample nitrate + nitrite data summary for automated discrete 

analysis with soluble AtNaR2 nitrate reductase. 

[Standard-level (SL) data were collected between November 30, 2007, and August 8, 2008. Low-level (LL) data were collected on February 15, 2007. NO2

–

, nitrite; NO3

–

, nitrate; mg-N/L, milligram nitrogen per liter; ID, identifier; H, high; M, 

medium; L, low] 

SL NO2

–

 + NO3

–

 (mg-N/L) LL NO2

–

 + NO3

–

 (mg-N/L)

Reference sample ID TPC-H TPC-M TPC-L TPC-H TPC-M TPC-L

Most probable value 4.00 2.00 0.50 0.750 0.500 0.125

Upper control limit 4.33 2.17 0.54 0.816 0.544 0.136

Lower control limit 3.67 1.83 0.46 0.684 0.465 0.114

Average concentration 4.03 1.99 0.49 0.783 0.536 0.129

Standard deviation (SD) 0.11 0.06 0.02 0.005 0.003 0.002

Relative SD, percent 2.7 3.1 3.3 0.6 0.6 1.2

Number of points 143 143 143 10 10 10
14 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

(Green and Foreman, 2005, table 1). Each seven-replicate 

dataset used to estimate spike recoveries was normally distributed at the p = 0.05 probability level on the basis of Kolmogorov-Smirnov statistical tests of normality (Pollard, 1979). 

Predictably, recovery of nitrate spiked near the standard-level 

assay MDL concentration (see table 15A) was more variable. 

See section 7.2.3 for details of how to prepare the seven replicate spikes at each concentration level in the three matrices.

Statistical and graphical comparisons of paired nitrate results 

obtained with USGS-approved CFA-CdR and DA-AtNaR2 

methods provided in the section “Analytical Performance and 

Comparative Results” that follows permit direct assessment of 

between-method bias.

Analytical Performance and 

Comparative Results

Background Information

During initial development of DA enzymatic reduction 

nitrate + nitrite assays, we were surprised to observe that analytical results obtained did not compare as well with CFA-CdR 

reference assay results as those obtained with CFA-enzymatic 

reduction assays that were developed previously (Patton and 

others, 2002). This was the case not only for nitrate reductase 

purifi ed from corn seedlings (NaR1TM), but also for two other 

commercially available nitrate reductases—Aspergillus sp.

NADPH:nitrate reductase (product number N 7625, Sigma, 

St. Louis, Mo.) and recombinant, bispecifi c NAD(P)H:nitrate 

reductase from Pichia angusta (YNaR1TM, Nitrate Elimination 

Company, Lake Linden, Mich.). We eventually discovered 

that these performance issues had two distinct, temperaturedependent causes: (1) high-phenolic-content humic acid (HA) 

irreversibly inhibits these enzymes at reaction temperatures 

greater than 20°C, and (2) except for NaR1, temperatures 

above about 25°C decrease the activity of these enzymes. 

In the sections that follow, we present descriptions and results 

of experiments with these three enzymes and recombinant 

NADH:nitrate reductase from Arabidopsis thaliana (AtNaR2TM, 

Nitrate Elimination Company, Lake Linden, Mich.) that 

demonstrate the latter’s particular suitability as an analytical 

reagent for routine analysis of nitrate in environmental water 

samples on DA platforms.

Effects of Temperature and Dissolved Organic 

Matter on AtNaR2 Activity

Our initial characterization of AtNaR2 as an analytical 

reagent began with a replication of experiments we had performed earlier to elucidate the effects of reaction temperature 

and HA concentration on YNaR1 activity. A major fi nding of 

this research was that AtNaR2 activity remains high—suffi cient for quantitative reduction of 5 mg NO3

–

-N/L to nitrite 

in less than 10 minutes—at reaction temperatures ranging 

from 10°C to 37°C and at HA concentrations up to 20 mg/L. 

We similarly assessed the susceptibility of Aspergillus sp. 

Figure 5. Control chart of thirdparty-check (TPC) sample results 

for standard-level automated 

discrete analyzer nitrate + nitrite 

methods using soluble AtNaR2 

nitrate reductase. (MPV, most 

probable value; UCL, upper 

control limit; LCL, lower control 

limit)

4.50

4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0

TPC_High

TPC_Med

TPC_Low

High-MPV

Med-MPV

Low-MPV

High-UCL

Med-UCL

Low-UCL

High-LCL

Med-LCL

Low-LCL

11/16/2007 1/5/2008 2/24/2008 4/14/2008 6/3/2008 7/23/2008

AtNaR2 nitrate concentration, in milligrams nitrogen per liter

Date

EXPLANATION
Analytical Performance and Comparative Results 15

Table 13. Within-day and between-day replicate agreement for nitrate + nitrite analyses of surface-water samples and groundwater 

samples analyzed by the AtNaR2-reduction automated discrete analyzer method (NWQL laboratory code 3156).

[NWQL, National Water Quality Laboratory; ID, identifier; mg-N/L, milligram nitrogen per liter]

NWQL 

sample 

ID

First 

analysis 

date

Result-1 Result-2

Within-day 

difference 

 (mg-N/L)

Within-day 

difference 

(percent)

Second 

analysis 

date

Days 

between 

analyses

Result-3

Between-day 

difference 

(mg-N/L)

Between-day 

difference 

(percent)

20081580131 6/6/2008 0.05 0.05 0.00 –0.7 7/25/2008 49 –0.02 0.07 149.3

20081560054 6/6/2008 0.06 0.05 0.01 8.8 7/25/2008 49 0.02 0.04 69.0

20081570105 6/6/2008 0.07 0.07 0.00 4.0 7/25/2008 49 0.03 0.04 54.5

20081340114 5/16/2008 0.35 0.35 0.00 0.2 7/25/2008 70 0.32 0.03 8.8

20081340115 5/16/2008 0.35 0.35 0.00 1.3 7/25/2008 70 0.22 0.13 36.5

20081580042 6/6/2008 0.49 0.49 0.00 –0.4 7/25/2008 49 0.47 0.02 4.7

20081570106 6/6/2008 0.58 0.59 0.00 –0.3 7/25/2008 49 0.55 0.03 5.3

20081370088 5/16/2008 1.19 1.20 –0.01 –1.0 7/25/2008 70 1.18 0.01 0.8

20081580126 6/6/2008 1.36 1.35 0.00 0.3 7/25/2008 49 1.28 0.07 5.3

20081970033 7/18/2008 1.71 1.70 0.01 0.4 7/25/2008 7 1.72 –0.01 –0.7

20081970032 7/18/2008 1.71 1.71 0.00 0.1 7/25/2008 7 1.73 –0.02 –1.1

20081480025 6/6/2008 2.56 2.52 0.04 1.4 7/25/2008 49 1.68 0.88 34.4

20081580041 6/6/2008 2.67 2.68 0.00 –0.1 7/25/2008 49 2.61 0.06 2.3

20081350062 5/16/2008 2.83 2.86 –0.03 –0.9 7/25/2008 70 2.85 –0.02 –0.8

20081370089 5/16/2008 3.08 3.07 0.00 0.1 7/25/2008 70 3.00 0.07 2.4

20081580124 6/6/2008 3.14 3.10 0.04 1.3 7/25/2008 49 3.05 0.09 2.9

Table 14. Between-day replicate agreement for nitrate + nitrite analyses of surface-water samples and groundwater samples 

by the low-level AtNaR2-reduction automated discrete analyzer method. Units for cadmium reduction (CdR) and nitrate reductase 

(AtNaR2) reduction methods are milligrams nitrate + nitrite nitrogen per liter.

[NWQL, National Water Quality Laboratory; ID, identifier; LC, NWQL laboratory code; SD, standard deviation; RSD, percent relative standard deviation; 

MC, U.S. Geological Survey National Water Information System sample medium code; WS, surface-water medium code; --, no data; WG, groundwater 

medium code] 

NWQL 

sample ID

CdR 

(LC 1979)

AtNaR2 

trial 1

AtNaR2 

trial 2

AtNaR2 

trial 3

AtNaR2 

average

SD RSD MC

20060820056-2 2.792 2.779 2.758 2.907 2.815 0.081 2.9 WS

20060820058-2 1.949 1.951 1.931 2.024 1.969 0.049 2.5 WS

20060820080-4 1.299 1.293 1.277 1.308 1.293 0.015 1.2 WS

20060820082-4 1.042 1.036 1.040 1.058 1.045 0.012 1.1 WS

20060820077-4 0.652 0.657 0.681 0.667 0.668 0.012 1.9 WS

20060860110-2 0.171 0.193 0.186 -- 0.190 0.005 2.8 WS

20060830065-2 0.133 0.135 0.129 0.133 0.132 0.003 2.3 WS

20060820079-4 0.120 0.122 0.110 0.116 0.116 0.006 5.1 WS

20060820078-4 0.089 0.097 0.093 0.094 0.095 0.002 2.1 WS

20060820075-4 0.083 0.086 0.087 0.087 0.087 0.001 0.8 WS

20060820074-4 0.080 0.080 0.077 0.080 0.079 0.002 2.3 WS

20060860113-5 0.053 0.060 0.059 -- 0.060 0.000 0.7 WS

20060860111-2 0.040 0.043 0.043 -- 0.043 0.000 0.2 WS

20060830066-2 0.023 0.020 0.027 0.024 0.024 0.004 15.6 WS

20060860108-2 0.013 0.016 0.010 -- 0.013 0.004 34.9 WS

20060830067-2 0.007 0.007 0.012 0.009 0.009 0.003 28.0 WS

20060880004-4 1.078 1.074 1.095 -- 1.084 0.015 1.3 WG

20060870028-2 0.241 0.263 0.252 -- 0.258 0.008 2.9 WG

20060880003-4 0.002 0.004 0.004 -- 0.004 0.000 5.1 WG
16 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

Table 15. Recovery of replicate spikes in reagent-water, surface-water, and groundwater matrices by standard-level (SL)

AtNaR2-reduction nitrate + nitrite assay (NWQL laboratory code 3156).

[NWQL, National Water Quality Laboratory; n = 7 unless otherwise indicated; spike composition = 10 microliter (μL) spike solution + 990 μL 

sample; NO3

–

-N, nitrate nitrogen; ID, identifier; MC, U.S. Geological Survey medium code; LL CdR, NWQL laboratory code 1979; mg-N/L, 

milligram nitrogen per liter; CdR, NWQL laboratory code 1975; DI, deionized; NA, not applicable; --, no data; <, less than; ±, plus or minus; 

WS, surface water; WG, groundwater]

A. Recovery of replicate 0.02 mg NO3

–

-N/L spikes. 

NWQL 

laboratory 

ID

MC

Approved cadmiumreduction (CdR) methods Standard-level AtNaR2 nitrate-reductase reduction method

LL CdR 

(mg-N/L)

CdR 

(mg-N/L)

Unspiked 

(mg-N/L)

Spike added 

(mg-N/L)

Found 

(mg-N/L)

Recovery 

(percent)

DI water NA -- -- < 0.02 0.02 0.01 ± 0.03 59

20110700114 WS 0.100 -- 1 0.09 ± 0.00 0.02 0.11 ± 0.00 97

20110760169 WS 0.519 -- 0.52 ± 0.00 0.02 0.54 ± 0.01 114

20110770085 WS 2 2.32 -- 2.15 ± 0.03 0.02 2.16 ± 0.03 32

20110750033 WG -- 0.10 1 0.10 ± 0.00 0.02 0.11 ± 0.00 103

20110750036 WG -- 0.56 0.58 ± 0.00 0.02 0.60 ± 0.01 123

20110770052 WG -- 2.61 2.54 ± 0.02 0.02 2.52 ± 0.02 –55

1 n = 6. 2Diluted 1+4.

C. Recovery of replicate 0.50 mg NO3

–

-N/L spikes. 

NWQL 

laboratory 

ID

MC

Approved cadmiumreduction (CdR) methods Standard-level AtNaR2 nitrate-reductase reduction method

LL CdR 

(mg-N/L)

CdR 

(mg-N/L)

Unspiked 

(mg-N/L)

Spike added 

(mg-N/L)

Found 

(mg-N/L)

Recovery 

(percent)

DI water NA -- -- < 0.02 0.50 0.51 ± 0.01 101.7

20110700114 WS 0.100 -- 1 0.09 ± 0.00 0.50 1 0.59 ± 0.03 100.0

20110760169 WS 0.519 -- 0.52 ± 0.00 0.50 1 1.06 ± 0.05 108.0

20110770085 WS 2 2.32 -- 2.15 ± 0.03 0.50 2.68 ± 0.05 105.0

20110750033 WG -- 0.10 1 0.10 ± 0.00 0.50 0.63 ± 0.01 106.0

20110750036 WG -- 0.56 0.58 ± 0.00 0.50 1.12 ± 0.01 107.0

20110770052 WG -- 2.61 2.54 ± 0.02 0.50 3.03 ± 0.03 98.9

1 n = 6. 2Diluted 1+4.

B. Recovery of replicate 0.10 mg NO3

–

-N/L spikes. 

NWQL 

laboratory 

ID

MC

Approved cadmiumreduction (CdR) methods Standard-level AtNaR2 nitrate-reductase reduction method

LL CdR 

(mg-N/L)

CdR 

(mg-N/L)

Unspiked 

(mg-N/L)

Spike added 

(mg-N/L)

Found 

(mg-N/L)

Recovery 

(percent)

DI water NA -- -- < 0.02 0.10 0.10 ± 0.005 98

20110700114 WS 0.100 -- 1 0.09 ± 0.00 0.10 0.20 ± 0.00 113

20110760169 WS 0.519 -- 0.52 ± 0.00 0.10 0.62 ± 0.01 107

20110770085 WS 2 2.32 -- 2.15 ± 0.03 0.10 2.26 ± 0.03 109

20110750033 WG -- 0.10 1 0.10 ± 0.00 0.10 0.21 ± 0.00 110

20110750036 WG -- 0.56 0.58 ± 0.00 0.10 0.69 ± 0.01 111

20110770052 WG -- 2.61 2.54 ± 0.02 0.10 2.62 ± 0.03 82

1 n = 6. 2Diluted 1+4.
Analytical Performance and Comparative Results 17

Table 16. Recovery of replicate spikes in reagent-water, surface-water, and groundwater matrices by low-level (LL) AtNaR2-

reduction nitrate + nitrite assay (NWQL laboratory code 3157).

[NWQL, National Water Quality Laboratory; n = 7 unless otherwise indicated; spike composition = 10 microliter (μL) spike solution + 990 μL sample; 

NO3

–

-N, nitrate nitrogen; ID, identifier; MC, U.S. Geological Survey medium code; LL CdR, NWQL laboratory code 1979; mg-N/L, milligram 

nitrogen per liter; CdR, NWQL laboratory code 1975; DI, deionized; NA, not applicable; --, no data; ±, plus or minus; WS, surface water; WG, 

groundwater]

A. Recovery of replicate 0.02 mg NO3

–

-N/L spikes. 

NWQL 

laboratory 

ID

MC

Approved cadmium-reduction 

(CdR) methods Low-level AtNaR2 nitrate-reductase reduction methods

LL CdR 

(mg-N/L)

CdR 

(mg-N/L)

Unspiked 

(mg-N/L)

Spike added 

(mg-N/L)

Found 

(mg-N/L)

Recovery 

(percent)

DI water NA -- -- 0.002 ± 0.000 0.020 0.021 ± 0.003 95.0

20110700114 WS 0.100 -- 0.100 ± 0.001 0.020 0.121 ± 0.002 104.0

20110760169 WS 0.519 -- 0.517 ± 0.008 0.020 0.533 ± 0.003 79.4

20110770085 WS 1 2.32 -- off-scale -- -- --

20110750033 WG -- 0.10 0.101 ± 0.001 0.020 0.125 ± 0.006 119

20110750036 WG -- 0.56 0.606 ± 0.005 0.020 0.626 ± 0.011 100

20110770052 WG -- 2.61 off-scale -- -- --

1Diluted 1+4.

B. Recovery of replicate 0.10 mg NO3

–

-N/L spikes. 

NWQL 

laboratory 

ID

MC

Approved cadmium-reduction 

(CdR) methods Low-level AtNaR2 nitrate-reductase reduction methods

LL CdR 

(mg-N/L)

CdR 

(mg-N/L)

Unspiked 

(mg-N/L)

Spike added 

(mg-N/L)

Found 

(mg-N/L)

Recovery 

(percent)

DI water NA -- -- 0.002 ± 0.000 0.100 0.102 ± 0.006 100.0

20110700114 WS 0.100 -- 0.100 ± 0.001 0.100 0.202 ± 0.002 101.0

20110760169 WS 0.519 -- 0.517 ± 0.008 0.100 0.615 ± 0.004 98.0

20110770085 WS 1 2.32 -- off-scale -- -- --

20110750033 WG -- 0.10 0.101 ± 0.001 0.100 0.217 ± 0.004 116.0

20110750036 WG -- 0.56 0.606 ± 0.005 0.100 0.713 ± 0.006 106

20110770052 WG -- 2.61 off-scale -- -- --

1Diluted 1+4.

C. Recovery of replicate 0.50 mg NO3

–

-N/L spikes. 

NWQL 

laboratory 

ID

MC

Approved cadmium-reduction 

(CdR) methods Low-level AtNaR2 nitrate-reductase reduction methods

LL CdR 

(mg-N/L)

CdR 

(mg-N/L)

Unspiked 

(mg-N/L)

Spike added 

(mg-N/L)

Found 

(mg-N/L)

Recovery 

(percent)

DI water NA -- -- 0.002 ± 0.000 0.500 0.499 ± 0.006 99.0

20110700114 WS 0.100 -- 0.100 ± 0.001 0.500 1 0.58 ± 0.03 97.0

20110760169 WS 0.519 -- 0.517 ± 0.008 0.500 off-scale --

20110770085 WS 2 2.32 -- off-scale -- -- --

20110750033 WG -- 0.10 0.101 ± 0.001 0.500 0.65 ± 0.01 110.0

20110750036 WG -- 0.56 0.606 ± 0.005 0.500 off-scale --

20110770052 WG -- 2.61 off-scale -- -- --

1 n = 6. 2Diluted 1+4.
18 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

NADPH:nitrate reductase to HA inhibition in relation to reaction temperature.

Figure 6 provides a graphical summary of results from 

these kinetics experiments. Points plotted in fi gure 6 are 

proportional to nitrite concentrations recorded after about 

9 minutes of enzymatic reaction time. In fi gure 6, nitrite concentrations determined in 5 mg-N/L nitrate solutions spiked 

with 20 mg/L HA are plotted with hollow symbols; those for 

5 mg-N/L nitrate solutions not containing HA are plotted with 

solid symbols. To enhance clarity in this fi gure, only points 

from the highest temperature tested in nitrate solutions not 

containing HA are shown. With reference to fi gure 6, apparent 

activity of AtNaR2 was little affected by 20 mg/L HA at reaction temperatures ranging between 10°C and 37°C. However, 

apparent activities of the other three nitrate reductases tested 

began to decrease precipitously when reaction temperatures 

exceeded 20°C. As expected from prior experiments, apparent activity of AtNaR1 and YNaR1 were comparable at 10°C. 

Furthermore, at 37°C for nitrate solutions not containing 

HA, apparent activities of YNaR1 and Aspergillus sp. nitrate 

reductases were substantially less than that of nitrate reductase purifi ed from corn, which approached that of AtNaR2. 

In summary, apparent activities of the four nitrate reductases 

tested in solutions containing 5 mg NO3

-

-N/L and 20 mg HA/L 

at the DA reaction zone temperature of 37°C were as follows: 

AtNaR2 >> NaR1 > NADPH:NaR ≈ YNaR1.

Data shown in fi gure 7 demonstrate that AtNaR2 and 

NADH concentrations equal to those optimized in previous 

work with NaR1 and YNaR1 were suffi cient to reduce nitrate 

to nitrite quantitatively within 10 minutes at 37°C for nitrate 

concentrations in the range of 0.05 to 5.0 mg-N/L. Nitrate 

solutions used in these experiments did not contain HA.

We next confi rmed that YNaR1 and AtNaR2 reactivity at 37°C for nitrate solutions spiked with HA were the 

same on the DA platform as on the CFA kinetics platform. 

For this work, we prepared three series of nitrate standards 

in deionized water—fi ve each at concentrations of 0.25 mgN/L, 2.5 mg-N/L, and 5.0 mg-N/L—and amended each 

concentration series with 0, 5, 10, 15, and 20 mg/L HA. 

We then analyzed these solutions successively on the DA, 

fi rst with YNaR1 and then with AtNaR2 enzyme reagent. 

Figure 8 provides graphical summaries of these experimental results. As expected, apparent nitrate concentrations for 

test solutions decreased as HA concentration increased with 

YNaR1 reagent but remained high and constant with AtNaR2 

reagent.

Nitrate reductase reaction temperature, in degrees Celsius

EXPLANATION

4.50

4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

Peak height, in volts

NADH:AtNaR2 (lot #2), 20 mg SR DOC/L

NADH:AtNaR2 (lot #2), 0 mg SR DOC/L

NADH:AtNaR2 (lot #1), 20 mg SR DOC/L

NADH:AtNaR2 (lot #1), 0 mg SR DOC/L

NADH:YNaR1, 20 mg SR DOC/L

NADH:YNaR1, 0 mg SR DOC/L

NADH:NaR (corn), 20 mg SR DOC/L

NADH:Na(corn), 0 mg SR DOC/L

NADPH:NaR, 20 mg SR DOC/L

NADPH:NaR, 0 mg SR DOC/L

Figure 6. Activity of different nitrate reductases in relation to reaction temperature for 5 milligrams 

nitrogen per liter nitrate solutions with and without added Suwannee River (SR) humic acid (SR DOC in 

explanation). NADPH:NaR is from Aspergillus species. Data were collected from late August through 

November 2005. (mg, milligram; L, liter; NADH, nicotinamide adenine dinucleotide in reduced form; NADPH, 

nicotinamide adenine dinucleotide phosphate in reduced form)
Analytical Performance and Comparative Results 19

EXPLANATION

4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0

0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0

Griess reaction response, in arbitrary units

AtNaR2 nitrate reductase reaction time at 37 degrees Celsius, in minutes

5.00

3.75

2.50

1.25

0.25

0.05

Figure 7. Nitrate to nitrite reduction rates by AtNaR2 nitrate reductase at 37 degrees Celsius for 

nitrate concentrations of 0.05 to 5.00 milligrams nitrogen per liter for each experiment.

Suwannee River humic acid DOC concentration, in milligrams per liter

EXPLANATION

Nitrate concentration, in milligrams nitrogen per liter

5.00

4.00

3.00

2.00

1.00

0

0 5 10 15 20

AtNaR2: 5.00 mg-N/L

AtNaR2: 2.50 mg-N/L

AtNaR2: 0.25 mg-N/L

YNaR1: 5.00 mg-N/L

YNaR1: 2.50 mg-N/L

YNaR1: 0.25 mg-N/L

Nominal nitrate concentration (mg-N/L)

Figure 8. Nitrate recoveries from deionized water solutions containing 0.25 milligrams nitrogen per 

liter (mg-N/L), 2.50 mg-N/L, and 5.00 mg-N/L and incrementally increasing Suwannee River humic acid 

concentrations for automated discrete analyzer nitrate assays that differed only in the nitrate reductase 

(YNaR1 or AtNaR2) used to reduce nitrate to nitrite. Humic acid concentrations are expressed as 

milligrams dissolved organic carbon (DOC) per liter. Nominal temperature was 37 degrees Celsius 

for the enzymatic reduction reaction and the Griess reagent indicator reaction. Reaction time was 

10 minutes for the enzymatic reduction step.
20 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

Reagent Stability

Experimental results summarized in fi gure 9 established 

that the useful lifetime for working AtNaR2 reagent is about 

18 hours in the 4°C environment of the DA’s reagent compartment. We found, however, that preparing working AtNaR2 

reagent in 0.05 M, pH 7.5 MOPS buffer increased its useful 

lifetime at 4°C to about 3 days (see fi g. 9). Despite the longer 

AtNaR2 working reagent storage life afforded by MOPS buffer, we opted to continue using phosphate buffer to maintain 

continuity with our previous work. Addition of 25 percent 

glycerol to either phosphate or MOPS assay buffers stabilized 

working AtNaR2 reagent somewhat, but it also reduced assay 

sensitivity by 10–15 percent. With fi nal reference to fi gure 9, 

NADH reagents prepared in either phosphate or MOPS buffers remained stable at 4°C for the 4-day duration of these 

experiments.

Interference by Anionic and Cationic Sample 

Matrix Constituents

As shown in fi gure 10, chloride, bromide, and sulfate at 

up to 100 times NWQL median concentrations—1,515 mg/L, 

15 mg/L, and 2,287 mg/L, respectively (see table 17)—had 

negligible effect on recovery of 2.5 mg NO3

–

-N/L in relation to 

the anticipated NWQL interim reporting limit for the standardlevel AtNaR2 assay of 0.04 mg-N/L. In fi gure 10, error bars 

indicate the standard deviation for three replicate nitrate 

determinations in each anion-amended test solution. The 

leftmost column, labeled DI, indicates the average concentration measured for 2.5 mg NO3

–

-N/L in DI water that was not 

amended with anions. Median perchlorate concentrations were 

not available, and those selected coincide with 10 percent, 

50 percent, and 100 percent of full-scale nitrate concentrations 

in the standard-level DA-AtNaR2 assay.

Days since reagent preparation

EXPLANATION

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

5 mg-N/L nitrate response, absorbance

0 1 2 3 4

AtNaR2 in MOPS buffer (NADH fresh daily in

MOPS buffer)

AtNaR2 in phosphate buffer (NADH fresh daily

in phosphate buffer)

AtNaR2 in MOPS buffer + glycerol (NADH fresh

daily in MOPS buffer)

AtNaR2 in phosphate buffer + glycerol (NADH

fresh daily in phosphate buffer)

NADH in MOPS buffer (AtNaR2 fresh daily)

NADH in phosphate buffer (AtNaR2 fresh daily)

AtNaR2 and NADH (fresh daily in phosphate

buffer)

Figure 9. AtNaR2 storage stability plots. In-use and between-day storage for all reagent solu tions was at 

4 degrees Celsius. (mg-N/L, milligram nitrogen per liter; NADH, nicotinamide adenine dinucleotide in reduced 

form; MOPS, 3-N-morpholino-propansulfonic acid)
Analytical Performance and Comparative Results 21

Companion figure 11 provides a graphical summary of 

the effects of metal ions at concentrations equal to the NWQL 

median, 10 times the median, and 100 times the median (see 

table 18) on recovery of 2.5 mg-N/L nitrate solutions. Error 

bars in figure 11 indicate the standard deviation of three replicate nitrate determinations by the DA-AtNaR2 nitrate assay in 

each metal-ion-amended test solution. The leftmost column, 

labeled DI, indicates the average concentration measured for 

2.5 mg-N/L nitrate in DI water that was not amended with 

metal ions. With the exception of nitrate recovery at a calcium 

concentration 100 times greater than the NWQL median, other 

cations tested had only minor effects (less than ±2 percent) 

on nitrate recovery in relation to the metal-free 2.5 mg-N/L 

nitrate test solution. Additional experiments suggested that low 

nitrate recovery (about 85 percent) from calcium test solutions 

at concentrations 100 times the median (prepared with calcium 

chloride) resulted from AtNaR2 inhibition by chloride counter 

ions (≈ 6,400 mg/L).

EXPLANATION

Median concentration

10x Median concentration

100x Median concentration

Nitrate found, in milligrams nitrogen per liter

2.60

2.50

2.40

2.30

2.20

2.10

2.00

DI Chloride Bromide Sulfate Perchlorate

Figure 10. Standard-level discrete analyzer AtNaR2 assay recovery of 2.50 milligrams nitrogen per 

liter (mg-N/L) nitrate from deionized (DI) water amended with various anions at 1, 10, and 100 times their 

National Water Quality Laboratory annual median concentrations as listed in table 17. The leftmost column, 

labeled DI, indicates recovery of 2.50 mg-N/L nitrate from DI water not amended with anions. Column 

heights and associated error bars at tops of columns indicate the average and standard deviation of nitrate 

recovery for three replicate determinations of each test solution.

Table 17. Nominal concentrations of anions tested for possible interference in the 

discrete analyzer AtNaR2 nitrate assay.

[FW, formula weight; mg/L, milligram per liter] 

Constituent FW Concentration (mg/L)

Median 10x Median 100x Median

Chloride 35.45 15.15 151.5 1,515

Bromide 79.90 0.15 1.5 15

Sulfate 96.06 22.87 228.7 2,287

Perchlorate* 99.45 0.5 2.5 5.0

*Perchlorate concentrations coincide with 10 percent, 50 percent, and 100 percent of full-scale nitrate 

concentrations for the standard-level nitrate assay.
22 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

DI Ba (II) Ca (II) Cr (III) Fe (II) Fe (III) Li (I) Mg (II) Mn (II) Sr (II)

EXPLANATION

Nitrate found, in milligrams nitrogen per liter

2.60

2.50

2.40

2.30

2.20

2.10

2.00

Median concentration

10x Median concentration

100x Median concentration

Figure 11. Standard-level discrete analyzer AtNaR2 assay recovery of 2.50 milligrams nitrogen per liter 

(mg-N/L) nitrate from deionized (DI) water amended with various metal ions at 1, 10, and 100 times their 

National Water Quality Laboratory annual median concentrations as listed in table 18. The leftmost column, 

labeled DI, indicates recovery of 2.50 mg-N/L nitrate from DI water not amended with metal ions. Column 

heights and associated error bars at tops of columns indicate the average and standard deviation of nitrate 

recovery for three replicate determinations of each test solution.

Table 18. Metal ions tested for possible inhibition of AtNaR2 enzyme and Griess-reaction interference.

[μg/L, microgram per liter; μM, micromole per liter]

Metal ion

Median

concentration

(μg/L)

Atomic

weight

Median

concentration

(μM)

10x Median

concentration

(μM)

100x Median

concentration

(μM)

Ba2+ 4.07 × 101 137.34 2.96 × 10–1 2.96 × 100 2.96 × 101

Ca2+ 3.64 × 104 40.08 9.08 × 102 9.08 × 103 9.08 × 104

Cr3+ 2.60 × 10–1 52.00 5.00 × 10–3 5.00 × 10–2 5.00 × 10–1

Fe2+ + Fe3+ 7.93 × 100 55.85 1.42 × 10–1 1.42 × 100 1.42 × 101

Li+ 7.11 × 100 6.94 1.02 × 100 1.02 × 101 1.02 × 102

Mg2+ 8.33 × 103 24.31 3.43 × 102 3.43 × 103 3.43 × 104

Mn2+ 1.07 × 101 54.94 1.95 × 10–1 1.95 × 100 1.95 × 101

Sr2+ 2.7 × 102 87.62 3.04 × 100 3.04 × 101 3.04 × 102
Analytical Performance and Comparative Results 23

Demonstration of Method Capability

With the properties of AtNaR2 as an analytical reagent 

for reducing nitrate to nitrite fully characterized, we began 

a four-part demonstration of capability for standard- and 

low-level DA nitrate + nitrite assays using the soluble 

AtNaR2:NADH reagent system. The DA, enzymatic-reduction 

assay results were in all cases compared with corresponding 

USGS-approved CFA-CdR assays. Part 1 confi rms that for 

typical DA assays thermostatted at 37°C, AtNaR2 is a better reagent than YNaR1 for quantitatively reducing nitrate 

to nitrite. Part 2 is a graphical demonstration that analytical 

results for representative samples obtained with standard- and 

low-level DA-AtNaR2 and CFA-CdR assays are equivalent. Part 3 demonstrates that DA-AtNaR2 assay analytical 

response to nitrate and nitrite is equivalent. Part 4 provides 

results of paired t-tests and nonparametric Wilcoxon signrank tests that demonstrate equivalence of analytical results 

obtained by CFA-CdR and DA-AtNaR2 methods.

Comparison of AtNaR2 and YNaR1 Reagents in 

Standard- and Low-Level DA Nitrate + Nitrite 

Assays

We analyzed a set of 115 samples—72 surface water, 

32 groundwater, and 11 blind fi eld and laboratory QC—on 

the DA platform, fi rst with YNaR1 and then with AtNaR2 as 

the enzyme reagent. Figure 12 provides a graphical comparison of nitrate concentrations resulting from nitrate-reductase 

methods in relation to those from USGS-approved CFA-CdR 

methods. The CFA-CdR method (NWQL laboratory code 

1975) concentration data were products of routine operations 

in the NWQL Nutrients Unit. The inset in fi gure 12 pertains to 

two surface-water samples and fi ve groundwater samples with 

nitrate + nitrite concentrations greater than the 5 mg-N/L calibration limit of CFA and DA assays, therefore requiring manual 

(CFA) or online automatic (DA) dilution prior to reanalysis. In 

fi gure 12, nitrate + nitrite concentrations determined by the reference CFA-CdR method (x-axis) and by DA-AtNaR2 methods 

AtNaR2: 10 minutes, 37°C

YNaR1: 10 minutes, 37°C

Line of equal relation

EXPLANATION

15.0

12.5

10.0

7.5

5.0

5.0 7.5 10.0 12.5 15.0

Continous-flow analyzer, cadmium-reduction

 nitrate + nitrite concentration, in milligrams nitrogen per liter

 Discrete analyzer, nitrate-reductase-reduction

nitrate + nitrite concentration, in milligrams nitrogen per liter

4.00 4.50 5.00

AtNaR2 nitrate = 0.977 (CdR nitrate) + 0.017

Correlation coefficient = 0.9990

YNaR1 nitrate = 0.944 (CdR nitrate) + 0.051

Correlation coefficient = 0.9796

Linear least squares regression analysis

5.00

4.00

4.50

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0

0 0.50 1.00 1.50 2.00 2.50 3.00 3.50

Figure 12. Comparison of nitrate + nitrite concentrations for 115 environmental water samples 

analyzed by automated continuous-flow, cadmium-reduction (CdR) method and automated discrete 

analyzer (DA) enzymatic-reduction method with NAD(P)H:YNaR1 nitrate reductase and NADH:AtNaR2 

nitrate reductase. DA determinations were consecutive on November 9, 2005.
24 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

(y-axis) are plotted about the line of equal relation (slope = 1). 

As predicted from prior experimental results, nitrate concentrations determined by the DA method using AtNaR2 reagent 

(blue circles) agreed more closely with those determined by 

CFA-CdR reference method than did those determined by the 

DA method using YNaR1 reagent (red triangles). This was also 

the case for diluted samples (see inset graph, fi g. 12).

Graphical Comparison of Standard- and LowLevel DA-AtNaR2 and CFA-CdR Assays 

We analyzed data plotted in fi gure 13 in May 2008. 

As identifi ed in the fi gure legend, these data are noteworthy 

because of their wide concentration distribution and matrix 

diversity. Agreement between nitrate + nitrite concentrations determined by standard-level DA-AtNaR2 and USGSapproved CFA-CdR methods are excellent as indicated by 

regression parameters in the fi gure inset.

Figure 14 shows that nitrate + nitrite concentrations 

determined by the low-level DA-AtNaR2 method also compare well with those determined by the corresponding USGSapproved low-level CFA-CdR method. Four of the eight blind 

blanks for the DA-AtNaR2 method included in this fi gure 

had concentrations near the anticipated 0.008 mg-N/L interim 

reporting limit, but they are within the NWQL Nutrients Unit 

blank concentration criteria of ±1 interim or long-term MDL.

Equivalence of Low-Level DA-AtNaR2 Assay 

Response to Nitrate and Nitrite

As with the CFA-CdR assays they replace, DA-AtNaR2 

assays measure the sum of nitrate and nitrite. Nitrate concentrations, therefore, are calculated by subtraction of independently determined nitrite concentrations. Summary statistics 

for nitrite concentrations determined at the NWQL in 2004 

and 2010 provided in tables 19 and 20, respectively, demonstrate that nitrite concentrations typically are small. For 

example, with reference to table 20, 95 percent of surfacewater and groundwater samples analyzed for nitrate at the 

NWQL in 2010 had nitrite concentrations less than 0.06 mgN/L. Furthermore, 21 percent of the 8,418 surface-water 

samples and 69 percent of the 2,719 groundwater samples 

analyzed had nitrite concentrations less than the 0.001 mg-N/L 

MDL. Figure 15 demonstrates that the small population of 

samples analyzed at the NWQL in 2010 containing nitrite concentrations of 0.05 mg-N/L or more, nitrate was predominant 

without exception and nitrite concentrations did not exceed 

0.2 mg-N/L up to the 5.0 mg-N/L dilution limit of the standard-level nitrate + nitrite assay. Because of the demonstrated 

low nitrite concentration in surface water and groundwater, 

we evaluated response of the low-level DA-AtNaR2 nitrate + 

nitrite assay to nominally identical concentrations of nitrate 

and nitrite. In one experiment, we prepared DI water solutions 

5.0 mg-N/L

25 mg-N/L

0.04 mg-N/L Regression parameters

DA-AtNaR2 nitrate = 1.0004 (CFA-CdR

 nitrate) - 0.0092

Correlation coefficient = 0.9998

Kone run # 0134B on May 14, 2008

Continuous-flow analyzer CdR nitrate concentration, in milligrams nitrogen per liter

Discrete analyzer AtNaR2 nitrate concentration, 

in milligrams nitrogen per liter

EXPLANATION

100

10

1

0.1

0.01

0.01 0.1 1 10 100

Surface water (medium code WS)

Groundwater (medium code WG)

Artificial matrix sample (medium code OA)

Interstitial water sample (medium code WI)

Surface-water QA sample (medium code WSQ)

Groundwater QA sample (medium code WGQ)

Line of equal relation for CFA-CdR and DA-AtNaR2

 methods

Interim reporting limit (IRL)

Set point for automatic 1+4 dilution

Set point for prompt to enter a larger dilution

 factor 

Figure 13. Comparison of nitrate + nitrite concentrations of 101 environmental water samples analyzed by 

standard-level automated continuous-flow analyzer, cadmium-reduction method (CFA-CdR) and standardlevel automated discrete analyzer, AtNaR2-nitrate-reductase reduction method (DA-AtNaR2). (QA, quality 

assurance)
Analytical Performance and Comparative Results 25

Figure 14. Comparison of nitrate + nitrite concentrations for 67 environmental water samples 

analyzed by low-level (LL) automated continuous-flow analyzer, cadmium-reduction method (CFA-CdR) 

and low-level automated discrete analyzer, AtNaR2-nitrate-reductase reduction method (DA-AtNaR2). 

One point (0.994, 1.043) included in linear regression analysis is not shown. (MDL, method detection 

limit; IRL, interim reporting limit)

Low-level continuous-flow analyzer, CdR nitrate concentration, in milligrams nitrogen per liter

Low-level discrete analyzer, AtNaR2 nitrate

concentration, in milligrams nitrogen per liter

EXPLANATION

4.0

3.5

3.0

2.5

2.0

1.5

1.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0 Linear regression, all data

Surface water

Groundwater

Blind blanks

Line of equal relation

0.001 0.01 0.1

0.1

0.01

0.001

1

1

Correlation coefficient = 0.994

LL DA-AtNaR2 nitrate = 1.030 (LL CFA-CdR

 nitrate) + 0.002

LL CFA-CdR MDL and

 LL DA-AtNaR2 IRL

Table 19. Summary statistics for nitrite concentrations determined in surface water and groundwater during calendar year 2004 at 

the National Water Quality Laboratory (NWQL) by automated continuous-flow analyzer methods.

[MDL, method detection limit; mg NO2

–

-N/L, milligram nitrite nitrogen per liter; DL, dilution limit; WG, groundwater medium code; WS, surface water 

medium code; <, less than; ≤, less than or equal to]

NWQL laboratory code (test name) 1973 (Standard-level nitrite) 1977 (Low-level nitrite)

MDL (mg NO2

–

-N/L) 0.004 0.001 

DL (mg NO2

–

-N/L) 1.000 0.200

Medium code WG WS WG WS

Total samples analyzed 3,842 6,439 210 1,932

Counts: Concentration < MDL (percent of total) 3,100 (81) 2,583 (40) 116 (55) 458 (24)

Counts: MDL ≤ Concentration ≤ DL (percent of total) 739 (19) 3,845 (60) 94 (45) 1,471 (76)

Counts: Concentration > DL (percent of total) 3 (<1) 11 (<1) 0 3 (<1)

50th percentile concentration (mg NO2

–

-N/L) 0.012 0.014 0.002 0.003

75th percentile concentration (mg NO2

–

-N/L) 0.030 0.033 0.004 0.007

95th percentile concentration (mg NO2

–

-N/L) 0.169 0.124 0.007 0.025

Maximum concentration (mg NO2

–

-N/L) 1.29 6.61 0.025 0.045
26 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

EXPLANATION

WG: Nitrite ≥ 0.05 mg-N/L; n = 45

WS: Nitrite ≥ 0.05 mg-N/L; n = 425

LC 3156 dilution limit

LC 3157 dilution limit 

2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0

0.1 1 10 100

Nitrite concentration, in milligrams nitrogen per liter

Nitrate + nitrite concentration, in milligrams nitrogen per liter

Table 20. Summary statistics for nitrite concentrations determined in surface water and groundwater 

during calendar year 2010 at the National Water Quality Laboratory (NWQL) by the automated discrete 

analyzer method.

[MDL, method detection limit; mg NO2

–

-N/L, milligram nitrite nitrogen per liter; DL, dilution limit; WG, groundwater 

medium code; WS, surface water medium code; <, less than; ≤, less than or equal to]

NWQL laboratory code (test name) 3117 (Nitrite)

MDL (mg NO2

–

-N/L) 0.001

DL (mg NO2

–

-N/L) 0.200

Medium code WG WS

Total samples analyzed 2,719 8,418

Counts: Concentration < MDL (percent of total) 1,880 (69) 1,793 (21)

Counts: MDL ≤ Concentration ≤ DL (percent of total) 839 (31) 6,623 (79)

Counts: Concentration > DL (percent of total) 0 2 (<0.1)

50th percentile concentration (mg NO2

–

-N/L) 0.002 0.005

75th percentile concentration (mg NO2

–

-N/L) 0.008 0.014

95th percentile concentration (mg NO2

–

-N/L) 0.055 0.059

Maximum concentration (mg NO2

–

-N/L) 0.778 2.018

Figure 15. The relation between nitrate + nitrite and nitrite concentrations for surface-water (medium 

code WS) and groundwater (medium code WG) samples analyzed at the National Water Quality 

Laboratory in 2010 with nitrite concentrations of 0.05 mg-N/L or more. (mg-N/L, milligram nitrogen per 

liter; n, number of samples; LC, laboratory code)
Analytical Performance and Comparative Results 27

of nitrite in the concentration range of 0.00 mg-N/L to 

0.90 mg-N/L and analyzed them on the DA platform for nitrite 

(NWQL laboratory code 3117) and low-level nitrate + nitrite 

(NWQL laboratory code 3157). In another, we prepared DI 

water solutions containing nitrite and nitrate in six concentration ratios that nominally summed to 0.50 mg-N/L and again 

analyzed them on the DA for nitrite and nitrate + nitrite by 

NWQL laboratory codes 3117 and 3157, respectively. Calibrants for the low-level DA-AtNaR2 nitrate + nitrite assay 

contained only nitrate. Table 21 provides nominal concentrations of test solutions and analytical results for both sets of 

experiments, which demonstrate near equivalent response of 

the low-level DA-AtNaR2 nitrate assay to nitrate and nitrite 

individually and in combination.

Statistical Comparisons of Nitrate + Nitrite 

Results Determined by CFA-CdR and DA-AtNaR2 

Methods

Summary statistics for nitrate + nitrite concentrations 

determined in filtered-water samples by CFA-CdR and DAAtNaR2 methods appear in table 22. Box plots in figure 16

show distributions of standard-level nitrate + nitrite concentrations determined by CFA-CdR and DA-AtNaR2 methods in surface water (WS) and groundwater (WG) for 476 

samples included in validation experiments between November 2005 and May 2008. Paired t-test analysis (Pollard, 

1979) of all standard-level data (539 data pairs; see table 23) 

indicates that the difference between means of cadmium- and 

AtNaR2-reduction method nitrate concentrations are not 

statistically different from zero at the 0.05 or 0.01 probability 

level. Furthermore, differences between population means 

for surface-water, groundwater, and “other” subsets of these 

data are less than the NWQL MDL for the standard-level 

CFA-CdR nitrate method (NWQL laboratory code 1975) and 

are therefore analytically insignificant. When paired t-test 

analyses are restricted to “in-range” data (454 data pairs with 

concentrations less than or equal to 5.00 mg NO3

–

-N/L), differences between population means for “all data” and associated surface water and groundwater subsets are statistically 

different from zero at the 0.05 and 0.01 probability levels. 

However, differences between population means are not analytically significant. It is also noteworthy that DA-AtNaR2 

assay population means were equal to or slightly greater 

than nitrate concentrations measured by USGS-approved 

CFA-CdR reference assays. This result is in sharp contrast to 

nitrate concentrations measured by DA-YNaR1 assays that 

on average were biased low in relation to CFA-CdR assays 

(see figs. 8 and 12). Nonparametric Wilcoxon signed-rank 

test (Pollard, 1979) results for these data (see table 24) are 

in general agreement with paired t-test results; that is, differences in nitrate concentration populations measured by 

CFA-CdR and DA-AtNaR2 assays are statistically significant at the 0.05 and 0.01 probability levels, but calculated 

differences between population medians (table 22) are less 

than the 0.02 mg-N/L MDL and are therefore not analytically 

significant.

Table 21. Data demonstrating near equivalent response of low-level (LL) AtNaR2 nitrate + nitrite 

assay (National Water Quality Laboratory laboratory code 3157) to nitrate and nitrite individually 

and combined. Calibrants for the LL DA-AtNaR2 nitrate + nitrite assay contained only nitrate.

[mg-N/L, milligram nitrogen per liter; NO2

–

, nitrite; NO3

–

, nitrate; SD, standard deviation; n, number of samples] 

Nominal (mg-N/L) LL NO3

–

 + NO2

–

 (mg-N/L) NO2

–

 (mg-N/L)

NO2

–

-N NO3

–

-N Found SD (n = 3) Found SD (n = 3)

0.00 0.00 0.01 0.001 0.001 0.0000

0.05 0.00 0.06 0.010 0.050 0.0003

0.10 0.00 0.10 0.001 0.101 0.0004

0.15 0.00 0.15 0.000 0.152 0.0002

0.30 0.00 0.29 0.001 0.293 0.0053

0.60 0.00 0.58 0.004 0.600 0.0003

0.90 0.00 0.87 0.009 0.912 0.0015

0.00 0.50 0.50 0.005 0.001 0.0000

0.05 0.45 0.49 0.001 0.050 0.0003

0.10 0.40 0.49 0.001 0.098 0.0002

0.15 0.35 0.49 0.002 0.151 0.0002

0.20 0.30 0.49 0.003 0.190 0.0027

0.25 0.25 0.49 0.003 0.242 0.0045
28 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

Table 22. Summary statistics for nitrate + nitrite concentrations determined in filtered-water samples by continuous-flow 

analyzer, cadmium-reduction (CFA-CdR) method and discrete analyzer, nitrate-reductase-reduction (DA-AtNaR2) method.

[mg-N/L, milligram nitrogen per liter; n, number of samples; MC, U.S. Geological Survey sample medium code; WG, groundwater sample medium 

code (formerly 6); WS, surface-water sample medium code (formerly 9); NWQL, National Water Quality Laboratory] 

n Minimum 25th percentile Median 75th percentile Maximum

Standard-level CFA-CdR (NWQL laboratory code 1975) and DA-AtNaR2 nitrate methods

All CdR 539 –0.003 0.367 0.922 2.815 77.3

All AtNaR2 539 –0.028 0.368 0.922 2.854 75.5

MC WG CdR 238 –0.003 0.527 1.281 4.126 77.3

MC WG AtNaR2 238 –0.028 0.525 1.289 4.353 75.5

MC WS CdR 238 0.000 0.415 0.731 1.986 14.3

MC WS AtNaR2 238 –0.025 0.440 0.732 1.987 14.9

MC (other) 1

 CdR 63 –0.002 0.011 0.043 1.65 14.2

MC (other) 1

 AtNaR2 63 –0.027 0.002 0.031 1.72 14.4

Standard-level CFA-CdR and DA-AtNaR2 nitrate methods (in-range results) 2

All CdR 454 –0.003 0.225 0.714 1.49 5.00

All AtNaR2 454 –0.028 0.222 0.714 1.52 4.96

MC WG CdR 185 –0.003 0.347 0.912 1.92 5.00

MC WG AtNaR2 185 –0.028 0.338 0.922 2.00 4.96

MC WS CdR 212 0.000 0.370 0.653 1.31 4.97

MC WS AtNaR2 212 –0.025 0.371 0.639 1.32 4.82

MC (other) 1

 CdR 57 –0.002 0.009 0.018 1.02 4.09

MC (other) 1

 AtNaR2 57 –0.027 0.0015 0.016 1.04 4.21

Low-level CFA-CdR (NWQL laboratory code 1979) and DA-AtNaR2 nitrate methods

All CdR 67 –0.002 0.010 0.070 0.205 3.78

All AtNaR2 67 –0.011 0.010 0.074 0.229 3.99

MC WG CdR 9 0.001 0.054 0.241 1.88 3.78

MC WG AtNaR2 9 0.004 0.048 0.263 1.928 3.99

MC WS CdR 46 0.004 0.014 0.075 0.200 2.79

MC WS AtNaR2 46 –0.011 0.016 0.077 0.223 2.78

MC (other) 1

 CdR 12 –0.002 0.002 0.004 0.109 0.616

MC (other) 1

 AtNaR2 12 0.002 0.004 0.0075 0.121 0.673

1Typically laboratory-blind quality control samples and blanks.

2Samples with nitrate + nitrate concentrations greater than 5 mg-N/L that required dilution are not included.
Analytical Performance and Comparative Results 29

 CdR (WG) AtNaR2 (WG) CdR (WS) AtNaR2 (WS)

90th percentile 90th percentile

90th percentile 90th percentile

75th percentile 75th percentile

75th percentile 75th 

 percentile Median Median

Median Median

25th percentile 25th percentile 25th percentile 25th 

 percentile

10th percentile

10th percentile 10th percentile

10th percentile

n = 238 n = 238 n = 238 n = 238

Nitrate + nitrite concentration, in milligrams nitrogen per liter 100

10

1

0.1

0.01

Figure 16. Concentration 

distributions for the population 

of 476 standard-level nitrate + 

nitrite concentrations determined 

in surface water (WS) and 

groundwater (WG) by cadmiumreduction (CdR), continuous-flow 

analysis and nitrate reductase 

(AtNaR2), discrete analysis for 

validation experiments spanning 

November 2005 through May 

2008. Square and triangle symbols 

in each box plot indicate mean 

and maximum concentrations, 

respectively. (n, number of samples)

Table 23. Two-population paired t-test results for nitrate + nitrite concentrations determined in filtered-water samples by 

continuous-flow analyzer, cadmium-reduction (CFA-CdR) method and discrete analyzer, nitrate-reductase-reduction (DA-AtNaR2) 

method.

[mg-N/L, milligram nitrogen per liter; n, number of samples; MC, U.S. Geological Survey sample medium code; WG, groundwater sample medium code 

(formerly 6); WS, surface water sample medium code (formerly 9); NWQL, National Water Quality Laboratory] 

n

Nitrate + nitrite concentration (mg-N/L)

Mean Difference Standard deviation Significant 1

CdR AtNaR2 CdR – AtNaR2 CdR AtNaR2 pcalc p0.05 p0.01

Standard-level CdR (NWQL laboratory code 1975)

All 539 2.715 2.721 –0.005 6.47 6.35 0.564 no no

MC WG 238 4.073 4.085 –0.012 9.18 8.99 0.555 no no

MC WS 238 1.675 1.671 0.004 2.31 2.29 0.504 no no

MC (other) 2 63 1.514 1.523 –0.018 2.85 2.90 0.119 no no

Standard-level CdR (in-range results) 3

All 454 1.085 1.100 –0.015 1.15 1.17 0.0000 yes yes

MC WG 185 1.31 1.34 –0.024 1.25 1.29 0.0000 yes yes

MC WS 212 0.986 0.994 –0.008 1.004 1.003 0.0010 yes yes

MC (other) 2 57 0.722 0.729 –0.007 1.175 1.208 0.3455 no no

Low-level CdR (NWQL laboratory code 1979) 

All 67 0.332 0.344 –0.012 0.710 0.731 0.0040 yes yes

MC WG 9 0.925 0.962 –0.038 1.374 1.442 0.1635 no no

MC WS 46 0.282 0.289 –0.007 0.552 0.555 0.0104 yes no

MC (other) 2 12 0.078 0.087 –0.009 0.178 0.194 0.0762 no no

1 pcalc is the probability that population means of nitrate + nitrite concentrations determined by the CdR- and YNaR1-reduction methods are the same—that 

is, difference between the population means is statistically equivalent to zero—on the basis of calculated paired t-tests. Difference between population means 

is significant at the 95 percent confidence level (p0.05) when pcalc is less than 0.05 and at the 99 percent confidence level (p0.01) when pcalc is less than 0.01.

2Typically laboratory-blind quality control samples and blanks.

3Samples with nitrate + nitrate concentrations greater than 5 mg-N/L that required dilution are not included.
30 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

Conclusions

Numbered conclusions in the list that follows correspond 

to the list of objectives in the section “Purpose and Scope.”

1. Paired statistical and graphical analyses of nitrate 

+ nitrite concentrations determined in more than 

500 seasonally, geographically, and compositionally diverse surface-water and groundwater samples 

demonstrate the comparability of analytical results 

determined by standard- and low-level continuousflow analyzer, cadmium-reduction (DFA-CdR) and

discrete analyzer, nitrate-reductase-reaction (DAAtNaR2) methods. Effects on nitrate + nitrite concentration trend analysis across this method-change 

boundary should be negligible.

a. Paired t-test statistical analyses of results fromCFA-CdR and DA-AtNaR2 methods (see table 23)

indicate that the difference between population means of nitrate + nitrite concentrations 

determined by the two methods was statistically 

equivalent to zero at the 0.05 probability level. 

Nonparametric Wilcoxon signed-rank statistical 

analyses (see table 24) indicate that the concentration distributions of the same population are not 

equally distributed at the 0.05 probability level. 

With reference to summary statistics in table 22

and box plots in figure 16, however, the difference between population medians is less than the 

method detection limit (MDL) (0.02 mg-N/L) and 

therefore not analytically significant. Although

the difference between means for the subset of 

surface-water samples that did not require dilution (n = 212) was statistically different than zero, 

the calculated difference (-0.008 mg-N/L) is not 

analytically different from zero. The difference 

Table 24. Wilcoxon signed-rank test 1 results for nitrate + nitrite concentrations determined in filtered-water samples 

by continuous-flow analyzer, cadmium-reduction (CFA-CdR) method and discrete analyzer, nitrate-reductase-reduction 

(DA-AtNaR2) method.

[mg-N/L, milligram nitrogen per liter; n, number of samples; MC, U.S. Geological Survey sample medium code; WG, groundwater sample 

medium code (formerly 6); WS, surface water sample medium code (formerly 9); NWQL, National Water Quality Laboratory; <, less than] 

AtNaR2-CdR Positive ranks Negative ranks Methods results populations different? 2

p p p calc 0.05 0.01

Standard-level CdR samples (NWQL laboratory code 1975)

All MCs

MC WG

MC WS

MC (other) 3

348

148

170

30

191

90

68

33

< 0.0001

0.0002

< 0.0001

0.8011

yes

yes

yes

no

yes

yes

yes

no

Standard-level CdR samples (in-range results) 4 

All MCs

MC WG

MC WS

MC (other) 3

304

116

162

26

150

69

50

31

< 0.0001

0.0007

< 0.0001

0.5962

yes

yes

yes

no

yes

yes

yes

no

Low-level CdR samples (NWQL laboratory code 1979)

All MCs

MC WG

50

7

17

2

< 0.0001

0.1797

yes

no

yes

no

MC WS

MC (other) 3

33

10

13

2

0.0051

0.0386

yes

yes

yes

no

1

 The paired-sample, Wilcoxon signed-rank test is a nonparametric alternative to the paired-sample t-test. It can be used to examine whether 

or not two paired sample populations have the same distribution. Unlike the paired-sample t-test, this function does not require either test 

population to be normally distributed.

2

 pcalc is the probability that population distributions of nitrate + nitrite concentrations determined by the CdR- and YNaR1-reduction methods 

are the same on the basis of the paired sample, Wilcoxon signed-rank test. The difference between population distributions is significant at

the 95 percent confidence level (p0.05) when pcalc is less than 0.05 and at the 99 percent confidence level (p0.01) when pcalc is less than 0.01.

3

 Typically laboratory-blind quality control samples and blanks.

4

 Samples with nitrate + nitrate concentrations greater than 5 mg-N/L that required dilution are not included.
Acknowledgments 31

between means for the subset of groundwater 

samples that did not require dilution (n = 185) also 

was statistically different than zero, and the calculated difference (-0.024 mg-N/L) is analytically 

significant. In this case, however, the population 

mean difference is negative—that is, DA-AtNaR2 

assay nitrate + nitrite concentrations were on average slightly greater than those for the CFA-CdR 

assay (see table 23). Trends evident in graphical 

analysis and calculated linear least-squares regression parameters within the body of this report 

support these results.

b. Paired t-test statistical analyses of results from 

low-level CFA-CdR and DA-AtNaR2 methods 

(see table 23) indicate the difference between 

nitrate concentration population means determined 

by the two methods were statistically different 

from zero at the 0.05 probability level. This statistically significant difference between populations means (-0.012 mg-N/L), however, is negative—that is, low-level DA-AtNaR2 assay nitrate 

+ nitrite concentrations were on average slightly 

greater than those for the low-level CFA-CdR 

assay. This was also the case for nonparametric 

Wilcoxon signed-rank statistical analyses (see 

table 24), which indicate that the concentration 

distributions of the two populations are not equally 

distributed at the 0.05 probability level. With reference to table 22, differences between population 

medians are not analytically significant. Differences in calculated population means (table 23) 

and medians (table 22) for groundwater samples 

(-0.038 mg-N/L and -0.022 mg-N/L, respectively) 

were statistically and analytically significant. 

This might be due in part to the small number of 

samples in this population (n = 9), but it might 

also reflect better tolerance to reduced metals and 

sulfides by the enzymatic-reduction assays than 

the cadmium-reduction assays. As was the case for 

standard-level population mean differences, lowlevel method differences were negative—that is, 

AtNaR2-reduction method nitrate concentrations 

on average were slightly greater than cadmiumreduction method nitrate concentrations (see table 

23). Trends evident in graphical analysis and calculated linear least-squares regression parameters 

within the body of this report support these results.

2. Complete operational details (preparation of 

reagents, calibrants, and QC solutions) and performance benchmarks for these new methods (MDLs, 

blank levels, between-day precision, and spike 

recovery) are provided for analysts at the NWQL 

and elsewhere who need to implement these methods 

and operate them routinely.

3. Experimental results provided in this report demonstrate negligible interference in either enzymatic 

or colorimetric assay reaction steps by common 

surface-water and groundwater matrix constituents, 

such as major and minor ions and humic substances, over a reaction temperature range of 5°C 

to 37°C.

a. Anions and cations at concentrations up to 

100 times their median concentrations in typical 

freshwater matrices have negligible effects on 

the activity of AtNaR2 nitrate reductase. Group 

II cations suppress formation of Griess reaction 

chromophore. Calcium ions at NWQL-median 

concentrations exert the greatest suppression,

although barium ions are more potent indicator reaction suppressors on the basis of molar 

concentration. Due to thermal instability of 

nitrous acid and diazonium intermediates (Noller, 

1966), both the yield of Griess indicator reaction 

chromophore and its formation rate are inversely 

proportional to reaction temperature in the range 

of 10°C to 50°C.

b. High-phenolic-content humic acids (HAs) do not 

inhibit AtNaR2 at reaction temperatures ranging between 5°C and 37°C. This unique property 

makes AtNaR2 the reagent of choice for DA 

nitrate determination methods described in this 

report and other natural water nitrate assays that 

are most easily performed at or above typical ambient laboratory temperatures (72–78°F, 

22–26°C).

Acknowledgments

We are grateful to Ms. Christine McEvoy for technical 

assistance in summer 2003. At that time, she was a chemistry 

major at the College of Wooster in Wooster, Ohio, and her 

participation in our research was supported through the USGS 

Summer Student Intern Program. Authors Kryskalla and Patton thank the Nitrate Elimination Company in Lake Linden, 

Mich., for partially funding their 2003 and 2004 research at 

the NWQL through a U.S. Department of Agriculture Phase-II 

small business innovation research (SBIR) grant (USDA SBIR 

# 2002-33610-12300, Environmentally benign automated 

nitrate analysis) and for continuing collaborative support and 

providing NADH:nitrate reductase from Arabidopsis thaliana

in 2005 and 2006. We also acknowledge Harold Ardourel, 

Eric Schwab, Mary Olson, Chris Klimper, Colleen Gupta 

(retired), and other members of the NWQL Analytical Services 

Nutrients Unit for their interest and technical support during 

development of automated DA nitrate methods described in 

this report.
32 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

The report also benefited from reviews by Nancy Simon 

at the USGS National Research Program in Reston, Va., Carl 

Zimmermann and Carolyn Keefe at the University of Maryland Chesapeake Biological Laboratory in Solomons, Md., and 

others at the USGS Office of Water Quality and the NWQL.

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Manuscript approved for publication August 5, 2011

 Report approved by Keith Lucey

Prepared by the U.S. Geological Survey Science Publishing Network

USGS Publishing Staff

 Edited by Melanie Parker, Denver Publishing Service Center, Denver, 

Colorado

 Design and layout by Felicia D. Dodd, Rolla Publishing Service Center, 

Rolla, Missouri

Technical Reviews

 Nancy S. Simon, USGS, Water Science Center, Reston, Virginia

 Harold D. Ardourel, USGS, National Water Quality Laboratory, 

Denver, Colorado

 Jeff W. McCoy, USGS, National Water Quality Laboratory, Denver, 

Colorado

 Doug Stevenson, USGS, National Water Quality Laboratory, Denver, 

Colorado

 Carl Zimmerman, University of Maryland, College Park, Maryland

For additional information concerning the research in this report, 

contact:

National Water Quality Laboratory

US. Geological Survey

P.O. Box 25585

Denver Federal Center

Lakewood, CO 80225–0585

(303) 236–2000

Or visit the National Water Quality Laboratory Web site at:

http://nwql.usgs.gov

Patton and Kryskalla—Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods—Techniques and Methods 5–B8