HACH COMPANY

Validation Study Report for Hach Method 10206, 

TNTplusTM 835/836 Nitrate Using Dimethylphenol 

Chemistry - Measurement of Nitrate in Water and 

Wastewater

Submitted by: 

David MacDonald 

November 2010 
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

2

                                                           

Background 

Purpose of Study 

 The purpose of this study was to evaluate the proposed Hach Company 

Measurement of Nitrate in Water and Wastewater, Method 10206 against the USEPA 

reference nitrate methods (EPA Method 300.1 and SM4500-NO3

-

 E) for approval and 

use in USEPA regulatory drinking water programs. 

Method Summary 

The Hach TNTplus Nitrate chemistry follows classical electrophillic aromatic 

substitution in that nitrate in the presence of sulfuric acid yields a nitronium ion (+NO2) 

and HSO4

-

. Nitronium ions are electrophiles that attack the aromatic ring of the 

dimethylphenol reagent to form intermediate nitro-carbonium ions. The basic HSO4

-

 ion 

then extracts a hydrogen ion from the nitro-carbonium intermediate to yield a stable 

substitution product (o, or p-nitro-dimethylphenol). The nitrodimethylphenol product is a 

highly colored (directly related to the nitro functional group), quantifiable by its visible 

absorption spectra. 

Method Justification 

The current EPA reference methods for the measurement of nitrate nitrogen 

(NO3

-

 -N) and total nitrate-nitrite nitrogen (NO3—NO2 N) (EPA Methods 353.2, SM4500-

NO3—E and F, and ASTM D3867-99(A) (B)) are costly, and contain cadmium which is 

harmful to the environment. A “green” chemistry method that excludes the use of toxic 

metals is desirable that is equally effective in performance in the measurement of nitrate 

and nitrate-nitrite nitrogen. 

Scope of Method 

 The Method 10206 is applicable for use in drinking water, surface and saline 

waters, and domestic and industrial wastewaters. The measurement range for this 

method is in the range from 0.020 to 13.5 mg/L NO3

-

 - N. 

Study Design and Objectives 

 The design and objectives of this validation study are detailed in the Validation 

Study Plan1

 appended to this report. 

1 Revised Hach Validation Study Plan, August 2010
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

3

Study Implementation 

Study Sponsor 

 The organizational sponsor responsible for managing this study is Hach 

Company. 

Study Participants 

 The participants, laboratories, and test matrices were selected from 

geographically diverse regions of the United States and are listed in the following table. 

Matrix Facility Laboratory Source of Treated Test 

Water 

Watertown, Wisconsin Test America-Watertown Surface Water 

Cedar Falls, Iowa Test America-Cedar Falls Surface Water 

Phoenix, Arizona Test America-Phoenix Ground Water 

Tier Study 

 The validation study was performed under Tier II requirements. 

Sample Collection and Distribution 

 Matrix samples for this validation study were collected at the tap for each 

laboratory. Collection followed standard USEPA sampling procedures2

. 

Analysis Description 

 The description of the type and number of analyses are described in the 

Validation Study Plan appended to this report. 

Data Reporting and Validation 

Data Collection, Laboratory Submission, and Validation 

 For each laboratory performing analyses, results from each test measurement 

were read from a DR 2800 spectrophotometer and manually transferred to an electronic 

spread sheet. Each laboratory then submitted the electronic spread sheet to the Hach 

Study Director for review and statistical evaluation. 

Results 

Summary results of the validation study are listed in Tables 1-4. 

                                                           

2 40CFR 141.23
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

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 Table 1 - City of Watertown Treatment Plant Summary Results 

Matrix EPA 353.2 Hach Method 10206 

Method 

Detection 

Limit (mg/L) 

Minimum 

Quantitation 

(mg/L) 

Method 

Detection 

Limit (mg/L) 

Minimum 

Quantitation 

(mg/L) 

Low Ionic 

Strength 

Reagent 

Water 

0.06 0.20 0.10 0.30 

High Ionic 

Strength 

Reagent 

Water 

0.01 0.03 0.04 0.10 

Initial Precision and 

Recovery 

Initial Precision and 

Recovery 

Average 

Percent (%) 

Recovery 

Standard 

Deviation 

Average 

Percent (%) 

Recovery 

Standard 

Deviation 

Low Ionic 

Strength 

Reagent 

Water

108 0.01 100 0.01 

High Ionic 

Strength 

Reagent 

106 0.02 97 0.01 

Matrix Spike Analysis Matrix Spike Analysis 

Average 

Percent (%) 

Recovery 

% Relative 

Difference 

Average 

Percent (%) 

Recovery 

% Relative 

Difference 

Watertown 

Tap

109 0.2 96.0 0.4 
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

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Table 2 – Cedar Falls Treatment Plant Summary Results 

Standard Method 4500-

NO3

-

 E 

Matrix Hach Method 10206 

Method 

Detection 

Limit (mg/L) 

Minimum 

Quantitation 

(mg/L) 

Method 

Detection 

Limit (mg/L) 

Minimum 

Quantitation 

(mg/L) 

Low Ionic 

Strength 

Reagent 

Water 

0.01 0.02 0.09 0.30 

High Ionic 

Strength 

Reagent 

Water 

0.04 0.10 0.08 0.20 

Initial Precision and 

Recovery 

Initial Precision and 

Recovery 

Average 

Percent (%) 

Recovery 

Standard 

Deviation 

Average 

Percent (%) 

Recovery 

Standard 

Deviation 

Low Ionic 

Strength 

Reagent 

Water

99.0 0.05 99.1 0.01 

High Ionic 

Strength 

Reagent 

Water 

102 0.02 90.5 0.003 

Matrix Spike Analysis Matrix Spike Analysis 

Average 

Percent (%) 

Recovery 

% Relative 

Difference 

Average 

Percent (%) 

Recovery 

% Relative 

Difference 

Cedar 

Falls Tap

97.4 3.2 94.3 0.4 
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

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Table 3 – Phoenix Water Treatment Plant Summary Results

Matrix EPA Method 300.0 Hach Method 10206 

Method 

Detection 

Limit (mg/L) 

Minimum 

Quantitation 

(mg/L) 

Method 

Detection 

Limit (mg/L) 

Minimum 

Quantitation 

(mg/L) 

Low Ionic 

Strength 

Reagent 

Water 

0.02 0.10 0.03 0.10 

High Ionic 

Strength 

Reagent 

Water 

0.01 0.10 0.08 0.20 

Initial Precision and 

Recovery 

Initial Precision and 

Recovery 

Average 

Percent (%) 

Recovery 

Standard 

Deviation 

Average 

Percent (%) 

Recovery 

Standard 

Deviation 

Low Ionic 

Strength 

Reagent 

Water

104 0.003 98.6 0.003 

High Ionic 

Strength 

Reagent 

Water 

104 0.002 96.4 0.002 

Matrix Analysis Matrix Analysis 

Average 

Percent (%) 

Recovery 

% Relative 

Difference 

Average 

Percent (%) 

Recovery 

% Relative 

Difference 

Phoenix 

102 0.2 95.1 2.4 

Development of QC Acceptance Criteria

Development of quality control acceptance criteria for method detection limit (MDL) and 

initial precision and recovery (IPR) followed the criteria detailed in Appendix D, Tier 2 of 

Section 9 of the guidance document for EPA approval of new methods3

. A summary of 

the pooled quality control acceptance criteria is described in Table 4. 

                                                           

3 Protocol for EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater and Drinking

Water, March 1999 EPA‐821‐98‐003
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

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Table 4 – Pooled Statistical Results 

Matrix Reference Methods Hach Method 10206 

Method 

Detection 

Limit (mg/L) 

Rounded 

Minimum 

Quantitation 

(mg/L) 

Method 

Detection 

Limit (mg/L) 

Rounded 

Minimum 

Quantitation 

(mg/L) 

Low Ionic 

Strength 

Reagent 

Water 

0.03 0.10 0.06 0.20 

High Ionic 

Strength 

Reagent 

Water 

0.02 0.10 0.05 0.20 

Initial Precision and 

Recovery 

Initial Precision and 

Recovery 

Percent (%) 

Recovery 

Range 

Precision of 

Recovery 

95% 

Confidence 

Percent (%) 

Recovery 

Range 

Precision of 

Recovery 

95% 

Confidence 

Lower 

Limit 

Upper 

Limit 

Lower 

Limit 

Upper 

Limit 

Low Ionic 

Strength 

Reagent 

Water

88 119 

0.06 

97 101 

0.01 

Lower 

Limit 

Upper 

Limit 

Lower 

Limit 

Upper 

Limit 

High Ionic 

Strength 

Reagent 

Water 

95 112 

0.03 

81 108 

0.01 

Matrix Limit Percent (%) 

Recovery Range 

Matrix Limit Percent (%) 

Recovery Range 

Low High Low High 

85.7 120 92.6 97.7 
Data Analysis/Discussion 

MDL Results 

The range of results of the LIS matrix were 0.01 mg/L NO3

-

-N to 0.06 mg/L NO3

-

-N for 

the reference method and 0.03 mg/L NO3

-

-N to 0.10 mg/L NO3

-

-N for the Hach method. 

For the reference HIS matrix, the range was 0.01 mg/L NO3

-

-N to 0.04 mg/L NO3

-

-N and 

0.04 mg/L NO3

-

-N to 0.08 NO3

-

-N for the Hach method. 

The pooled method detection limit results of the LIS matrix yielded 0.03 mg/L NO3

-

-N for 

the reference method and 0.06 mg/L NO3

-

-N for the Hach Method. For the HIS matrix, 

the pooled method detection limit results yielded 0.02 mg/L NO3

-

-N for the reference 

method and 0.05 mg/L NO3

-

-N for the Hach Method. The rounded minimum 

quantitation limit, regardless of the ionic strength yielded 0.10 mg/L NO3

-

-N for the 

reference method and 0.20 mg/L NO3

-

-N for the Hach method. 

Initial Precision and Recovery Results 

The range of recovery results of the LIS matrix were 99% to 108% for the reference 

method and 91% to 100% for the Hach method. For the reference HIS matrix, the 

recovery range was 102% to 106 % and 91% to 97% for the Hach method. For the LIS 

matrix, the range of precision (standard deviation) of the reference method was 0.003 to 

0.05 and 0.003 to 0.01 for the Hach method. For the HIS matrix, the range of precision 

of the reference method was 0.002 to 0.02 and 0.002 to 0.01 for the Hach method. 

The pooled initial precision and recovery limits of the LIS matrix yielded a recovery 

range of 88% to 119 % and a precision of 0.06 for the reference method and 97% to 

101% and a precision of 0.01 for the Hach method. The pooled initial precision and 

recovery limits of the HIS matrix yielded a recovery range of 95% to 112% and a 

precision of 0.03 for the reference method and 81% to 108% and a precision of 0.01 for 

the Hach method. 

Matrix Spike Results 

The matrix spike percent relative standard deviation (%RSD) results ranged from 0.13 

to 2.9 for the reference method and 0.29 to 1.8 for the Hach method. 

The pooled range of recovery was 86% to 120% for the reference method and 93 to 98 

for the Hach method. 
Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

Conclusions 

A validation study was conducted to compare the performance of Hach Method 10206 

for the measurement of NO3

-

-N in drinking water to that of the EPA approved reference 

method. The results of the study indicate that the Hach method (10206) is equally 

effective to the performance of the reference method.

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Hach Company Validation Study Report 

Measurement of Nitrate in Water and Wastewater, Method 10206 

10

Appendices

Appendix A – EMMC formatted Hach Method 10206 (Hach Method 10206.pdf) 

Appendix B – Supporting Data (Supporting Data.xls)