Hach Company TNTplus 835/836 Nitrate Method 10206 

Spectrophotometric Measurement of Nitrate in Water 

and Wastewater 
1

Hach Company TNTplus 835/836 Method 10206 

Revision 2.1 

January 10, 2013 

 

Spectrophotometric Measurement of Nitrate in Water and Wastewater 

1.0 Scope and Application 

1.1 These procedures cover the determination of nitrate in drinking water, surface water, 

domestic and industrial wastes. 

1.2 The method is applicable in the range from 0.20 to 35.0 mg/L NO3

-

 N. 

1.3 This method is equivalent or better in performance to SM 4500-NO3

-

 E, EPA 353.2, and 

EPA 300.0 for the purposes of regulatory reporting of nitrate and nitrate-nitrite. 

2.0 Summary of Method 

2.1 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. Test results are measured at 345 nm. 

3.0 Interferences 

3.1 The items listed in the Interfering substances table have been individually checked up to 

the given concentrations and do not cause interference. The cumulative effects and 

influence of other ions have not been determined. High loads of oxidizable organic 

substances cause the reagent to change color and to give high-bias results. The test can 

thus only be used for wastewater analyses if the chemical oxygen demand (COD) is less 

than 500 mg/L. Measurement results can be verified using sample dilutions or standard 

additions. 

3.2 Nitrite concentrations of more than 2.0 mg/L interfere (high-bias results). Add 50 mg of 

sulfamic acid (amidosulfonic acid) to 5.0 mL of sample, dissolve and wait for 10 

minutes. Analyze the prepared sample as described in the procedure above. 

Interfering 

substance

Interference level 

(mg/L)

Interfering 

substance

Interference level 

(mg/L)

Ag+ 100 Cu2+ 50 

Cl- 500 Ca2+ 50 

Fe3+ 50 NO2

-

 2 

K+ 500 Cd2+ 50 

Na+ 500 Sn2+ 50 

Ni2+ 50 Cr6+ 5 

Pb2+ 50 Fe2+ 10 

Zn2+ 50 Co2+ 10 
2

3.3 Residual chlorine does not cause an interference with this method. 

4.0 Safety 

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely 

determined; however, each chemical should be treated as a potential health hazard. 

Exposure to these chemicals should be reduced to the lowest possible level. It is 

suggested that the laboratory perform personal hygiene monitoring of each analyst using 

this method and that the results of this monitoring be made available to the analyst. 

4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample 

containers should be opened in a hood and handled with gloves to prevent exposure. 

4.3 This method does not address all safety issues associated with its use. The laboratory is 

responsible for maintaining a safe work environment and a current awareness file of 

OSHA regulations regarding the safe handling of any chemicals specified in this method. 

A reference file of material safety data sheets (MSDSs) should be available to all 

personnel involved in these analyses. Additional information on laboratory safety can be 

found in Sections 16.3 and 16.4. 

5.0 Equipment 

Note: Brand names, suppliers, and part numbers are for illustrative purposes only. No 

endorsement is implied. Equivalent performance may be achieved using apparatus and 

materials other than those specified here, but demonstration of equivalent performance 

that meets the requirements of this method is the responsibility of the laboratory. 

5.1 Sampling equipment 

5.1.1 Sample collection bottles – Preferably use polyethylene bottles for collecting and storing 

samples for nitrate analysis. Glass bottles are satisfactory if previously they have not 

contained high-nitrate solutions. 

5.1.2 Cleaning 

5.1.2.1 All glassware used should be washed with hot 1:1 HCl and rinsed with distilled water. 

Preferably, this glassware should be used only for the determination of nitrate and after 

use it should be rinsed with distilled water and kept covered until needed again. If this is 

done, the treatment with 1:1 HCl is only occasionally required. 

 

6.0 Equipment for sample analysis

6.1 Hach Company DR 5000, DR 3800, or DR 2800 spectrophotometer 

6.2 Equipment for standard preparation 

6.2.1 Volumetric flask – Glass, 1000-mL. 

6.2.2 Volumetric pipette – Glass, assorted sizes. 
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7.0 Reagents and Standards 

7.1 Reagent water – Water in which nitrate is not detected at or above the method level of 

this method. Bottled distilled water, or water prepared by passage of tap water through 

ion exchange and activated carbon have been shown to be acceptable sources of reagent 

water. 

7.2 Hach Company TNTplus Nitrate Reagent, Cat. No. TNT835 or TNT836. 

7.3 Hach Company Nitrate Standard Solutions: 100 mg/L as NO3

-

-N (Cat. No. 194749) and 

1000 mg/L as NO3

-

-N (Cat. No.1279249). 

7.4 Method detection limit (MDL) solution 

7.4.1 Prepare 7 or more replicate MDL solutions by diluting 3.0 mL of the 100 mg/L standard 

spiking solution (Section 7.3) to 1000 mL. Final concentration = 0.3 mg NO3

-

-N /L. 

7.5 Initial precision and recovery (IPR) solution 

7.5.1 Prepare 4 or more replicate IPR solutions by diluting 5.0 mL of the 1000 mg/L standard 

spiking solution (Section 7.3) to 1000 mL. Final concentration = 5 mg NO3

-

-N /L. 

8.0 Sample Collection, Preservation and Storage 

8.1 Samples may be collected in clean glass or plastic bottles. 

8.2 Analyze samples as soon as possible. If immediate analysis is not possible, store at ≤ 6o 

C. Samples to be used for measurement of nitrate only must not be preserved with acid 

and must be and analyzed within 48 hours of collection. 

8.3 If longer storage is required (up to 28 days), adjust sample pH to less than 2 with sulfuric 

acid (about 2 mL per liter). Sample refrigeration is still required. If sample is acid 

preserved, results must be reported as nitrate-nitrite, also known as total nitrate. 

9.0 Quality Control 

9.1 It is recommended that each laboratory that uses this method be required to operate a 

formal quality assurance program (16.1). The minimum requirements of this program 

consist of an initial demonstration of laboratory capability and ongoing analyses of 

laboratory prepared water standards as a test of continued performance to assess accuracy 

and precision. Laboratory performance is compared to established performance criteria 

to determine if the results of analyses meet the performance characteristics of the method. 

9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable 

accuracy and precision with this method. This ability is established as described in 

Section 9.2. The laboratory shall, on an ongoing basis, demonstrate through analysis of 

the ongoing precision and recovery sample that the analysis system is in control. 

9.1.2 Accompanying QC for the determination of nitrate is required per analytical batch. An 

analytical batch is a set of samples processed during a contiguous 8-hour period. Each 

analytical batch must be accompanied by a laboratory reagent blank (LRB), an ongoing 

precision and recovery sample (OPR), a matrix spike sample (MS), and a matrix spike 
4

duplicate sample (MSD) resulting in a minimum of five analyses (1 LRB, 1 OPR, 1 

sample, MS, and MSD). 

9.2 Initial demonstration of laboratory capability. 

9.2.1 To establish the ability to detect nitrate the analyst shall determine the MDL per the 

procedure in 40 CFR 136, Appendix B (16.2) using the apparatus, reagents, and standards 

that will be used in the practice of this method. The analyst also shall calculate the 

Minimum Level (ML) of quantitation by multiplying the MDL by 3.18 and rounding to 

the number nearest to (1,2 or 5) x 10n, where n is a positive or negative integer. The 

calculated MDL should be less than or equal to the MDL in Section 13.0 prior to the 

practice of this method. Similarly, the calculated ML should be less than or equal to the 

ML in Section 13.0 

9.2.2 Prepare and measure seven replicates of the MDL standard according to the procedure 

beginning in Section 7.4.1. 

9.3 Initial precision and recovery (IPR) - To establish the ability to generate acceptable 

precision and accuracy, the analyst shall perform the following operations: 

9.3.1 Prepare and measure four samples of the IPR standard according to the procedure 

beginning in Section 7.5. 

 

9.3.2 Using the results of the set of four analyses, compute the average percent recovery (x) 

and the standard deviation of the percent recovery (s) for nitrate. Use the following 

equation for calculation of the standard deviation of the percent recovery: 

 

1

2

2







 

n

n

x

x

s

i

i

 where: 

 n = Number of samples 

 xi = % recovery in sample i 

9.3.2.1 Using the values of x and s calculated in the previous step, calculate the Relative 

Standard Deviation (RSD) using the equation below: 

 *100%

X

s RSD 

9.3.2.2 Compare RSD and x with the corresponding limits for initial precision and recovery in 

Table 1. If RSD and x meet the acceptance criteria, system performance is acceptable and 

analysis of samples may begin. If, however, RSD exceeds the precision limit or x falls 
5

outside the range for recovery, system performance is unacceptable. In this event correct 

the problem, and repeat the test. 

9.4 Laboratory Reagent Blank (LRB) – The laboratory reagent blank (LRB) is an aliquot of 

reagent water that is treated exactly as a sample including exposure to all glassware, 

equipment and reagents that are used with other samples, The laboratory must analyze at 

least one LRB with each batch of samples. Data produced are to asses contamination 

from the laboratory environment. Values that exceed the MDL indicate laboratory or 

reagent contamination should be suspected and corrective action should be taken before 

continuing the analysis. 

9.5 Ongoing precision and recovery (OPR) - To demonstrate that the analysis system is in 

control, and acceptable precision and accuracy is being maintained with each analytical 

batch. An analytical batch is a set of samples processed during a contiguous 8-hour 

period, not to exceed 20 samples. The analyst shall perform the following operations: 

9.5.1 Prepare a precision and recovery standard with each analytical batch. 

9.5.1.1 At the end of each analytical batch of samples, analyze a precision and recovery standard. 

If the recovery is within the acceptable range of 90 -110%, measurement process is in 

control and analysis of samples may proceed. If, however, the recovery is not in the 

acceptable range, the analytical process is not in control. In this event, correct the 

problem, re-analyze analytical batch, repeating the ongoing precision and recovery test. 

9.5.1.2 The laboratory should add results that pass to IPR and previous OPR data and update QC 

charts to form a graphic representation of continued laboratory performance. The 

laboratory should also develop a statement of laboratory data quality for each analyte by 

calculating the average percent recovery (R) and the standard deviation of the percent 

recovery (sr). Express the accuracy as a recovery interval from R - 2sr to R + 2sr. For 

example, if R = 95% and sr = 5%, the accuracy is 85% to 105%. 

9.5.1.3 Depending upon specific program requirements, field replicate spikes may be required to 

assess the precision and accuracy of the sampling and sample transporting techniques.

9.6 Matrix Spike and Matrix Spike Duplicate Precision and Recovery (MS/MSD) – The 

laboratory must, on an ongoing basis, spike at least 5% of the samples from each 

analytical batch as defined in Section 9.4. 

9.6.1. The concentration of the spike in the sample should be determined as follows: 

9.6.1.1 If, as in compliance monitoring, the concentration of nitrate in the sample is being 

checked against a regulatory concentration limit, the spike should be at that limit or one 

to five times higher than the background concentration determined in Section 11, 

whichever concentration would be larger. 

9.6.1.2 If the concentration of nitrate in the sample is not being checked against a a regulatory 

concentration limit, the spike should be at 5 mg/L or one to five times higher than the 

background concentration determined in Section 10, whichever concentration would be 

larger. 
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9.6.2 Analyze one sample aliquot to determine the background concentration (B). 

9.6.3 Analyze the spiked MS and MSD aliquots following the procedure in Section 11. 

9.6.5 Calculate each percent recovery (P) as 100 (A-B)%/T, where A is the concentration of 

 TKN in the spiked samples and T is the known true value of the spike. 

9.6.5.1 Compare the percent recovery (P) TKN with the corresponding QC acceptance criteria 

found in Section 17, Table 3. 

9.6.6 Calculate the relative percent difference (RPD) between two sample results using the 

following equation: 

 x 100

( 1 2)/ 2

1 2

D D

D D

RPD 

 

Where, D1 = Concentration of analyte in the MS, D2 = Concentration of analyte in 

the MSD. 

9.6.6.1 Compare the calculated RPD with the corresponding QC acceptance criteria found in 

Section 17, Table 3. 

10.0 Calibration and Standardization 

10.1 The Hach DR series spectrophotometers have a built-in calibration that is automatically 

used when the TNTplus nitrate vial is inserted into the instrument. No further initial 

calibration is required. However, the instruments have the capability of developing a 

user-calibration. See manufacturer’s manual for instructions. 

10.2 Calibration Verification 

10.2.1 To verify that the instrument is measuring nitrate properly, analyze a 0.3 mg/L and 10.0 

mg/L nitrate nitrogen standard. Results should be within 10 percent of the actual value. 

Perform this calibration verification daily while instrument is in use. 

11.0 Procedure 

11.1 Instrument Setup – follow the instrument manufacturer’s instructions for instrument 

setup. 

11.2 Preparation 

11.2.1 For LR TNT 835: Pipet 1.0 mL of sample into the reagent vial. 

For HR TNT836: Pipet 0.2 mL of sample into the reagent vial. 

11.3 Reaction 

11.3.1 For LR TNT835: Pipet 0.2 mL of Solution A into the vial. 

For HR TNT836: Pipe 1.0 mL of Solution A into the vial. 
7

11.3.2 Cap and invert the reaction tube 2-3 times until no more streaks can be seen in the 

reaction tube solution. 

 

11.3.3 React for 15 minutes. 

11.4 Analysis 

11.4.1 Wipe the vial and insert the prepared vial into the spectrophotometer. The instrument 

 reads the barcode, then selects and performs the correct test. No zero is required. Results 

 are in mg/L NO3

-

-N 

12.0 Data Analysis and Calculations 

12.1 Nitrate concentration is calculated automatically against internal instrument calibration. 

13.0 Method Performance 

Performance of the method was demonstrated in multi-lab studies comparing the method 

against currently promulgated nitrate methods. The method was evaluated in low ionic 

strength (LIS) and high ionic strength (HIS) matrices as well as multiple geographically 

diverse finished drinking water samples obtained from both surface water and ground 

water sources. 

 Acceptance Criterion Section Limit 

 

Method Detection Limit 9.2.1 0.05 mg/L NO3

-

 N

Method Limit 9.2.1 0.20 mg/L NO3

-

 N 

Initial Recovery Range 9.3.1 90% - 110% 

 Initial Precision (RPD) 9.3.1 10% 

 

 Matrix Recovery Range 9.5.1 90- 110% 

14.0 Pollution Prevention 

14.1 Follow guidelines in Section 15. 

15.0 Waste Management 

15.1 It is the laboratory’s responsibility to comply with all federal, state, and local regulations 

governing waste management, particularly the hazardous waste identification rules and 

land disposal restrictions, and to protect air, water, and land by minimizing and control 

all releases from fume hoods and bench operations. Compliance with all sewage 

discharge permits and regulations is also required. 

15.2 For further information on waste management, consult “The Waste Management manual 

 for Laboratory Personnel”, and “Less is Better: Laboratory Chemical Management for 

 Waste Reduction”, both available from the American Society’s Department of 

 Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 

 20036. 
8

16.0 References 

16.1 “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” 

USEPA, EMSL-CI, Cincinnati, OH 45268, EPA-600-4-79-019, March 1979. 

16.2 40 CFR 136, Appendix B. 

16.3 “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational 

Safety and Health Administration, OSHA 2206 (Revised, January 1976) 

16.4 “Safety in Academic Chemistry Laboratories,” American Chemical Society, Committee 

on Chemical Safety, 3rd Edition, 1979. 

16.5 “Water Analysis Handbook,” Hach Company, 5th Edition, 2008. 

17.0 Tables 

17.1 Acceptance Criteria for Performance tests – The QC performance criteria for this method 

 was performed with a Hach Company DR2800 Spectrophotometer and TNTplus 835 

 Reagent. 

 

Table 1. Initial Precision and Recovery Method Performance 

IPR Spike 

Concentration Maximum RSD (%) Lower Limit of Recovery 

(%)

Upper Limit of Recovery 

(%)

5.0 mg/L NO3

-

 N 10 90 110

Table 2. Method Detection Limit (MDL) & Minimum Level (ML) of Quantitation 

 

MDL Test 

Concentration MDL Rounded ML 

0.30 mg/L NO3

-

 N 0.05 0.20 

Table 3. Matrix Spike (MS) / Matrix Spike Duplicate (MSD) QC Acceptance Criteria 

MS/MSD Spike 

Concentration 

Maximum RPD 

(%) 

Lower Limit 

of Recovery 

(%) 

Upper Limit of 

Recovery (%) 

5.0 mg/L NO3

- N 20 90.0 110 
18.0 Glossary of Definitions and Purposes 

The definitions and purposes are specified to this method but have been conformed to common 

usage as much as possible. 

18.1 Units of weight and measure and their abbreviations 

18.1.1 Symbols 

o

C: degrees Celsius 

18.1.2 Alphabetical characters 

mg/L: milligram per liter 

18.2 Definitions, acronyms, and abbreviations 

18.2.1 LRB – Laboratory Reagent Blank 

18.2.2 MDL: Method detection limit 

18.2.3 ML: Minimum level of quantitation 

18.2.4 IPR: Initial precision and recovery 

18.2.5 OPR: On-going precision and recovery 

18.2.6 MS: Matrix spike 

18.2.7 MSD: Matrix spike duplicate