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

.......................................................................................................................................... 24 14. Graph showing comparison of nitrate + nitrite concentrations for67 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 insurface 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 concentrationsdetermined 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 × 10 1 ounce, fluid microliter (μL) 2.64 × 10 –7 gallon milliliter (mL) 2.64 × 10 –4 gallon Concentration milligrams nitrate nitrogen per

liter (mg NO 3 – -N/L) 7.14 × 10 1 micromoles nitrate per liter (NO 3 – , μ M ) milligrams nitrite nitrogen per

liter (mg NO 2 – -N/L) 7.14 × 10 1 micromoles nitrite per liter (NO 2 – , μ M ) molar ( M , moles/L) 1.00 × 10 3 millimolar (m M

) molar ( M , moles/L) 1.00 × 10 6 micromolar (μ M ) 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; pK a = 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 (Z ea 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 pK a -log 10 K a , where K a 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. Patton 1 

### and Jennifer R. Kryskalla 2



# Abstract
This report documents work at the U.S. Geological Sur

vey (USGS) National Water Quality Laboratory (NWQL) to validate enzymatic reduction, colorimetric determinative meth

ods for nitrate + nitrite in filtered water by automated discret e 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 auto

mated 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 meth

ods 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 success

ful 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 NWQLlong-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,

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

2 Veterans 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, thisreport 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 fornitrate + nitrite analyses. Paired t-test and Wilcoxon signedrank statistical analyses of results determined by CFA-CdRmethods 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 (NO 3 - ) 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. Environmen

tal Protection Agency, 1995). A thorough review of detection and determination methods for nitrate and nitrite (NO 2 - ) in a variety of matrices is available elsewhere (Moorcroft and others, 2001). Some important references not cited in Moor

croft’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 oth

ers, 2006).**2 Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods** 
Cadmium in various forms—such as electrolytically pre

cipitated, “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 colorimetricnitrate determinations. For example, copper-washed (copperized) cadmium granules packed into small columns (Wood andothers, 1967) are prescribed in the longstanding U.S. Geological Survey (USGS) and U.S. Environmental Protection Agency (EP A) continuous-flow analyzer, cadmium-reduction(CFA-CdR) methods I-2545-90 and 353.2, respectively. Wirein-tube cadmium reactors (Stainton, 1974; Willis, 1980; Willisand 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) ionsformed 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 andactivate, 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 enzymesas 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 assayreaction steps by common surface-water and groundwater matrix constituents such as major and minorions 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 ana

lytical 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 concen

trations determined in real samples during routine operation by USGS-approved methods and new methods. Data and statisti

cal 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**Anal yt**  **ica l**  **M et**  **h od**  **s 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 type**  **Laboratory Parameter**  **Method**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

FC A 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 SO 2 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, azomium) 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 NH 2 NH N +

2 HN calibrants and quality-control samples and therefore might produce incorrect analytical results. Nominal analytical ranges N O - 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. SO 2 NH 2 SO 2 NH 2
Sulf anil amide p-diazonium N -( 1-N apth yl)- 

## 2. Me **th od**  **Su mm**  **ar ie**  **s and**  **Anal yt**  **ica l**  (SAN) sulfanilamide eth yl ened iamine (NED) 

## C **on s**  **ide r**  **ation s**  SO 2 NH 2 Az o chrom opho re ( λ m a x = 5 4 3 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. **Inte rfe**  **rence s**  **and Tem**  **pera t**  **ure Effe**  **cts** 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 + At NaR 2 NO −

= N + 3 pH 7

− 8 + A H 2 O 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-metalions can inhibit nitrate reductase to varying extents. Ethylenediaminetetraacetic acid (EDTA) forms strong complexes with many metal ions and effectively minimizes interference bythese 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 addi

tional details. 3.3 Sulfate, chloride, and bromide at up to 100 times theirmedian 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 thatwe evaluated, HA inhibition was negligible only in the temperature range of 10°C to 20°C. Above 20°C, HA inhibitionincreased 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 pro

mote 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 ethyl

enediaminetetraacetic acid (EDTA) with diethylenetriamine

pentaacetic 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 groundwa

ter, 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 indica

tor 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 programmedreaction 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 incubatorone 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 minutesGriess reaction absorbance at 540 nanometers Ca(II), 908.18 μ M Sr(II), 3.04 μ M Mn(II), 0.19 μ M Li(I), 1.02 μ M CI0 4 − Mg(II), 341.56 μ M Ba(II), 0.3 μ M Fe(III), 0.14 μ M Cr(III), 0.005 μ M No amendment

0.975

0.950

0.925

0.900 0.150 0.450 0.600 0.750 0.900 0.300

**Figure 1.** Kinetics effects of diverse metal ions and perchlorate on the Griess reaction colorimetric nitrite assay. (μ M , micromolar)

**Figure 2.** Effect of temperature on kinetics and yield of the Griess reaction nitrite assay. (°C, degrees Celsius) **EXPLANATION** 0.900Griess 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 pro

vides two methods to record and correct minor contributionsto 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 (λ
max ≈ 540 nm) and at a second wavelength where chromophore absorbance is negligible (700 nm). The difference between absorbancemeasured 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, termedreagent-blank correction, involves (1) measuring the intermediate assay absorbance after adding and mixing the first reagent sulfanilamide (SAN) at λ
max , (2) adding the second, color forming reagent N -(1-Naphthyl)ethylenediamine (NED), and (3) measuring finished assay absorbance again at λ
max 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 samewithin 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 con

ditional 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 manufactur

er’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- plus TM 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, continu

ous 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 holderequipped 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 ( m

M). —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 (KH 2 PO 4 , 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 m M

EDTA and dilute the resulting solution to the mark with DI water; mix it well. Transferthis 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 itabout 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 buf fer 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 w orking 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-mLvolume 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 usereagent 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, ≈ 12 M ) to about 250 mL deionized water contained in a 500-mL volumetric flask. While the solution is still warm, add 5.0 g sul

fanilamide (C 6 H 8 N 2 O 2 S, 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 (C 12 H 14 N 2 •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 fromthose 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. Nomi

nal concentrations of low-level assay TPC samples in tables 8 and 9 are slightly different from those used dur

ing 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; NO 3 – -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 NO 3 – -N (low-level) 1.0 400 100 4.00 S7 NO 3 – -N (low-level) 1.0 60 100 0.60 S8 NO 3 – -N 1.0 2,000 100 20.00 S9 NO 3 – -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 NO 3 – -N/L, milligram nitrate nitrogen per liter]

**Stock II calibrant**  **ID DA**  **dilution factor notation**  **Dilution factor**  **Nominal concentration**  **(mg NO 3**  **– -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 NO 3 – -N/L, milligram nitrate nitrogen per liter]

**Stock II calibrant**  **ID DA**  **dilution factor notation**  **Dilution factor**  **Nominal concentration**  **(mg NO 3**  **– -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 solutionvolumes 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; NO 3 – , nitrate; NO 2 – , nitrite]

**Kone TPC**  **ID Second-source**  **1,000-mg NO**  **3 –**  **-N/L calibrant** 

**(μL) Second-source**  **100-mg NO**  **2 –**  **-N/L calibrant**  ***** **(μL) Nominal concentration**  **(mg-N/L)**

**NO 3**  **– -N NO**  **2 –**  **-N NO 3**  **– +NO**  **2 –**  **-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 solutionvolumes 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 calibra

tion 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 suit

able. 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 nitratesolution 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 ana

lysts 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-1reagent 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 (r 2 ) 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 + cx 2 might provide a better fit.



## 11. **Procedure and Data Evaluation**
Except as noted in sections 6 and 7, procedures for stan

dard- 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 volumesneeded 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 nitro

gen 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; NO 3 – , nitrate; NO 2 – , nitrite]

**Kone TPC**  **ID Second-source**  **100-mg NO**  **3 –**  **-N/L calibrant**  ***** **(μL) Second-source**  **100-mg NO**  **2 –**  **-N/L calibrant**  ***** **(μL) Nominal concentration**  **(mg-N/L)**

**NO 3**  **– -N NO**  **2 –**  **-N NO 3**  **– +NO**  **2 –**  **-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 5.00Absorbance 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 + cx 2 (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 per

form 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 obtainthe 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 ƒ
σ . BQS suggests three methods for estimating ƒ
σ . 12.4.1 Use regression equations of analyte concentration in relation to ƒ
σ , which are tabulated on the BQS Web site ( http://bqs.usgs.gov/ibsp/ ) by year.
12.4.2 ƒ
σ = 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 ƒ
σ = 5 percent of each nominal TPC concentration. The BQS suggests estimating ƒ
σ by all three methods andselecting 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 ƒ
σ ± 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 inthe 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 and0.002 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 alongwith 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 sum

maries of AtNaR2-reduction nitrate + nitrite assays performedon 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; NO 3 – , nitrate; NO 2 – , nitrite; SL, standard level; LL, low level; LT, long term]
**Nitrate + nitrite concentration (mg-NO 3**  **– -N + NO**  **2 –**  **-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. NO 2 – , nitrite; NO 3 – , nitrate; mg-N/L, milligram nitrogen per liter; ID, identifier; H, high; M, medium; L, low]

**SL NO 2**  **– + NO**  **3 –**  **(mg-N/L) LL NO**  **2 –**  **+ NO 3**  **– (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-replicatedataset 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 reductionnitrate + 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 (NaR1 TM ), 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 (YNaR1 TM , Nitrate Elimination Company, Lake Linden, Mich.). We eventually discoveredthat 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 (AtNaR2 TM , 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 analyticalreagent 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 ofthis research was that AtNaR2 activity remains high—suffi cient for quantitative reduction of 5 mg NO 3 – -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 7/23/2008AtNaR2 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; NO 3 – -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 NO 3 – -N/L spikes.

**NWQL laboratory**  **ID MC**  **Approved cadmium reduction (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. 2 Diluted 1+4.

**C.** Recovery of replicate 0.50 mg NO 3 – -N/L spikes.

**NWQL laboratory**  **ID MC**  **Approved cadmium reduction (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. 2 Diluted 1+4. **B.** Recovery of replicate 0.10 mg NO 3 – -N/L spikes.

**NWQL laboratory**  **ID MC**  **Approved cadmium reduction (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. 2 Diluted 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; NO 3 – -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 NO 3 – -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 --

-- --

1 Diluted 1+4. **B.** Recovery of replicate 0.10 mg NO 3 – -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

-- -- --

1 Diluted 1+4.
**C.** Recovery of replicate 0.50 mg NO 3 – -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. 2 Diluted 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 , apparentactivity 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 temperaturesexceeded 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. nitratereductases 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 NO 3 - -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 standardsin 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 40.0Peak 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 9.0 10.5 12.0 7.5Griess 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 about18 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 longerAtNaR2 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 NO 3 – -N/L in relation tothe 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. Theleftmost column, labeled DI, indicates the average concentration measured for 2.5 mg NO 3 – -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

05 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 figur e 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 concentrationNitrate 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; μ M , micromole per liter]

**Metal ion Median**  **concentration (μg/L)**  **Atomic weight**  **Median concentration**  **(μ M**  **) 10x Median**  **concentration (μ**  **M )**  **100x Median concentration**  **(μ M**  **)** Ba 2+ 4.07 × 10 1 137.34 2.96 × 10 –1 2.96 × 10 0 2.96 × 10 1 Ca 2+ 3.64 × 10 4 40.08 9.08 × 10 2 9.08 × 10 3 9.08 × 10 4 Cr 3+ 2.60 × 10 –1 52.00 5.00 × 10 –3 5.00 × 10 –2 5.00 × 10 –1 Fe 2+ + Fe 3+ 7.93 × 10 0 55.85 1.42 × 10 –1 1.42 × 10 0 1.42 × 10 1 Li+ 7.11 × 10 0 6.94 1.02 × 10 0 1.02 × 10 1 1.02 × 10 2 Mg 2+ 8.33 × 10 3 24.31 3.43 × 10 2 3.43 × 10 3 3.43 × 10 4 Mn 2+ 1.07 × 10 1 54.94 1.95 × 10 –1 1.95 × 10 0 1.95 × 10 1 Sr 2+ 2.7 × 10 2 87.62 3.04 × 10 0 3.04 × 10 1 3.04 × 10 2**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- andlow-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 providesresults 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 withnitrate + 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 literDiscrete analyzer, nitrate-reductase-reductionnitrate + nitrite concentration, in milligrams nitrogen per liter

5.00 4.00 4.50 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 matrixdiversity. 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 concentrationsdetermined 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-AtNaR2assays 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 theNWQL 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 ofsamples 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 exceed0.2 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 literDiscrete 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 bystandard-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 literLow-level discrete analyzer, AtNaR2 nitrateconcentration, 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 NO 2 – -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 NO 2 – -N/L) 0.004 0.001 DL (mg NO 2 – -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 NO 2 – -N/L) 0.012 0.014 0.002 0.003 75th percentile concentration (mg NO 2 – -N/L) 0.030 0.033 0.004 0.007 95th percentile concentration (mg NO 2 – -N/L) 0.169 0.124 0.007 0.025 Maximum concentration (mg NO 2 – -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 100Nitrite 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 NO 2 – -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 NO 2 – -N/L) 0.001 DL (mg NO 2 – -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 NO 2 – -N/L) 0.002 0.005 75th percentile concentration (mg NO 2 – -N/L) 0.008 0.014 95th percentile concentration (mg NO 2 – -N/L) 0.055 0.059 Maximum concentration (mg NO 2 – -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 DIwater 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 concentrationsdetermined 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 concen

trations determined by CFA-CdR and DA-AtNaR2 meth

ods in surface water (WS) and groundwater (WG) for 476 samples included in validation experiments between Novem

ber 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 NO 3 – -N/L), dif

ferences between population means for “all data” and associ

ated 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 ana

lytically 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, dif

ferences in nitrate concentration populations measured by CFA-CdR and DA-AtNaR2 assays are statistically signifi

cant 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; NO 2 – , nitrite; NO 3 – , nitrate; SD, standard deviation; n, number of samples]
**Nominal (mg-N/L) LL NO**  **3 –**  **+ NO 2**  **– (mg-N/L)**  **NO 2**  **– (mg-N/L)**  **NO 2**  **– -N NO**  **3 –**  **-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

1 Typically laboratory-blind quality control samples and blanks.

2 Samples 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 = 238Nitrate + 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) andgroundwater (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 p calc**  **p 0.05**  **p 0.01**  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 p calc 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 (p 0.05 ) when p calc is less than 0.05 and at the 99 percent confidence level (p 0.01 ) when p calc is less than 0.01.

2 Typically laboratory-blind quality control samples and blanks.

3 Samples with nitrate + nitrate concentrations greater than 5 mg-N/L that required dilution are not included.**30 Co lo**  **r imet**  **ric Det**  **e rm**  **in ati**  **on o f N**  **it rat**  **e Pl**  **us Ni tri**  **te i n**  **W at**  **er b y**  **En zym**  **at ic**  **R educ**  **t io**  **n, Au**  **to ma**  **ted D**  **iscr et**  **e An**  **al yzer**  **Me t**  **hod s** 



# C **on clu**  **s ion**  **s**
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 than500 seasonally, geographically, and compositionally diverse surface-water and groundwater samples demonstrate the comparability of analytical resultsdetermined by standard- and low-level continuousflow analyzer, cadmium-reduction (DFA-CdR) anddiscrete 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 ofsurface-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 2**  **Methods results populations different? 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 p calc 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 (p 0.05 ) when p calc is less than 0.05 and at the 99 percent confidence level (p 0.01 ) when p calc 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) alsowas 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-AtNaR2assay nitrate + nitrite concentrations were on average slightly greater than those for the CFA-CdR assay (see table 23 ). Trends evident in graphicalanalysis 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 differentfrom zero at the 0.05 probability level. This statistically significant difference between popula

tions 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 CF A-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. Differ

ences 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 forstandard-level population mean differences, lowlevel method differences were negative—that is, AtNaR2-reduction method nitrate concentrationson average were slightly greater than cadmiumreduction method nitrate concentrations (see table 23 ). Trends evident in graphical analysis and cal

culated linear least-squares regression parameters within the body of this report support these results.
2. Complete operational details (preparation ofreagents, 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 dem

onstrate 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 sub

stances, 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 indica

tor 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 notinhibit 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 thatare 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 USGSSummer 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., CarlZimmermann 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.govPatton and Kryskalla**—Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods—**Techniques and Methods 5–B8