## Hach Company TNTplus 835/836 Nitrate Method 10206

Spectrophotometric Measurement of Nitrate in Water

and Wastewater1 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 NO 3 - N.

1.3 This method is equivalent or be tter in performance to SM 4500-NO 3 - E, EPA 353.2, and EPA 300.0 for the purposes of regulatory repo rting of nitrate a nd nitrate-nitrite.

2.0 Summary of Method

2.1 The Hach TNTplus Nitrate chemistry follo ws classical electrophillic aromatic substitution in that nitrate in the presence of sulfuric acid yields a nitronium ion ( + NO 2 )

and HSO 4 - . Nitronium ions are electrophiles that attack the aromatic ring of the dimethylphenol reagent to form intermedia te nitro-carbonium ions. The basic HSO 4 - ion then extracts a hydrogen ion from the nitro- carbonium intermediate to yield a stable substitution product (o, or p- nitro-dimethylphenol). The ni trodimethylphenol product is a highly colored (directly relate d 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 de termined. 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 veri fied using sample dilutions or standard

additions.

3.2 Nitrite concentrations of more than 2.0 mg/L interfere (hig h-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 Cu 2+ 50

Cl - 500 Ca 2+ 50

Fe 3+ 50 NO 2 - 2

K + 500 Cd 2+ 50

Na + 500 Sn 2+ 50

Ni 2+ 50 Cr 6+ 5

Pb 2+ 50 Fe 2+ 10

Zn 2+ 50 Co 2+ 102 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 possi ble level. It is suggested that the laboratory perform persona l hygiene monitoring of each analyst using

this method and that the results of this m onitoring be made availa ble to the analyst.

4.2 Unknown samples may contain high concentrat ions 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 e nvironment 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. Additi onal 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. Equi valent performance may be ac hieved using apparatus and materials other than those specified here, but demonstration of e quivalent performance that meets the requirements of this method is the respons ibility 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 ar e 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 onl y 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.3 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 wate r 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 Reag ent, Cat. No. TNT835 or TNT836.

7.3 Hach Company Nitrate Standa rd Solutions: 100 mg/L as NO 3 - -N (Cat. No. 194749) and 1000 mg/L as NO 3 - -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 NO 3 - -N /L.

7.5 Initial precision and recovery (IPR) solution

7.5.1 Prepare 4 or more replicat e 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 NO 3 - -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 i mmediate analysis is not possible, store at ≤ 6o C. Samples to be used for measurement of n itrate 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 refrigerat ion is still required. If sample is acid preserved, results must be repor ted 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). Th e minimum requirements of this program

consist of an initial demonstration of la boratory capability and ongoing analyses of laboratory prepared water standards as a test of continued performance to assess accuracy and precision. Laboratory performance is co mpared to established performance criteria

to determine if the results of analyses meet the performanc e characteristics of the method.

9.1.1 The analyst shall make an initial demonstr ation 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 th rough analysis of the ongoing precision and recovery sample that the analysis system is in control.

9.1.2 Accompanying QC for the determination of n itrate is required per analytical batch. An analytical batch is a set of samples proce ssed 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 matr ix spike sample (MS), and a matrix spike4 duplicate sample (MSD) resulting in a minimu m 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 nitrat e the analyst shall determine the MDL per the procedure in 40 CFR 136, Appendix B (16.2) usi ng the apparatus, reag ents, and standards that will be used in the pr actice of this method. The analyst also shall calculate the Minimum Level (ML) of quantitation by mu ltiplying the MDL by 3.18 and rounding to the number nearest to (1,2 or 5) x 10n, where n is a positive or ne gative integer. The

calculated MDL should be less than or equal to the MDL in Secti on 13.0 prior to the practice of this method. Similarly, the calculate d ML should be less than or equal to the

ML in Section 13.0

9.2.2 Prepare and measure seven replicates of th e MDL standard according to the procedure beginning in Section 7.4.1.

9.3 Initial precision and recovery (IPR) - To es tablish the ability to generate acceptable precision and accuracy, the analyst shall perform the following operations:

9.3.1 Prepare and measure four samples of th e IPR standard according to the procedure beginning in Section 7.5.

9.3.2 Using the results of the set of four analys es, 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

x i = % recovery in sample i

9.3.2.1 Using the values of x and s calculated in the previous step, ca lculate the Relative Standard Deviation (RSD) using the equation below:

% 100 * X s RSD 

9.3.2.2 Compare RSD and x with the corresponding lim its for initial precision and recovery in Table 1. If RSD and x meet the acceptance crit eria, system performance is acceptable and analysis of samples may begin. If, however , RSD exceeds the precision limit or x falls5 outside the range for recovery, system performa nce is unacceptable. In this event correct the problem, and repeat the test.

9.4 Laboratory Reagent Blank (LRB) – The laborat ory 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 ot her 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 a nd corrective action should be taken before continuing the analysis.

9.5 Ongoing precision and recovery (OPR) - To de monstrate that the anal ysis system is in control, and acceptable precision and accuracy is being maintained with each analytical batch. An analytical batch is a set of sa mples processed during a contiguous 8-hour period, not to exceed 20 samples. The analys t shall perform the following operations:

9.5.1 Prepare a precision and recovery stan dard 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 samp les may proceed. If, however, th e recovery is not in the acceptable range, the analytical process is not in control. In this event, correct the problem, re-analyze analytical batch, repea ting the ongoing precision a nd 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 devi ation of the percent recovery (sr). Express the accuracy as a recove ry 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, fi eld 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 concen tration 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 samp le 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 Sect ion 10, whichever concentration would be larger.6 9.6.2 Analyze one sample aliquot to dete rmine the background concentration (B).

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

9.6.5 Calculate each percent recove ry (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 w ith 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:

100 x 2 / ) ( 2 1 2 1

D D D

D RPD

 



Where, D 1 = Concentration of analyte in the MS, D 2 = Concentration of analyte in

the MSD.

9.6.6.1 Compare the calculated RPD with the corr esponding 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 insert ed into the instrument. No further initial calibration is required. However, the instru ments 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 instru ment is measuring nitrate properly, analyze a 0.3 mg/L and 10.0 mg/L nitrate nitrogen standard. Results shou ld be within 10 percent of the actual value.

Perform this calibration verification da ily while instrument is in use.

11.0 Procedure
11.1 Instrument Setup – follow the instrument manufacturer’s instruc tions 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 th e prepared vial into the spect rophotometer. The instrument reads the barcode, then selects and performs the correct test. No zero is required. Results are in mg/L NO 3 - -N

12.0 Data Analysis and Calculations

12.1 Nitrate concentration is cal culated automatically against internal in strument calibration.

13.0 Method Performance

Performance of the method was demonstrated in multi-lab studies comparing the method against currently promulgated nitrate met hods. The method was evaluated in low ionic strength (LIS) and high ionic strength (HIS) matrices as well as multiple geographically diverse finished drinking water sample s obtained from both surface water and ground

water sources.

Acceptance Criterion Section Limit

Method Detection Limit 9.2.1 0.05 mg/L NO 3 - N

Method Limit 9.2.1 0.20 mg/L NO 3 - 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 fe deral, state, and local regulations governing waste management, particularly th e hazardous waste iden tification rules and land disposal restrictions, and to protect air, water, and land by minimizing and control

all releases from fume hoods and bench operations. Complian ce with all sewage discharge permits and regulat ions is also required.

15.2 For further information on waste management , consult “The Waste Management manual for Laboratory Personnel”, and “Less is Be tter: Laboratory Chemical Management for Waste Reduction”, both avai lable from the American Society’s Department of Government Relations and Science Policy, 1155 16 th 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, Ge neral Industry,” (29 CFR 1910), Occupational Safety and Health Administrati on, OSHA 2206 (Revised, January 1976)

16.4 “Safety in Academic Chemistry Laboratorie s,” American Chemical Society, Committee on Chemical Safety, 3 rd Edition, 1979.

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

17.0 Tables
17.1 Acceptance Criteria for Performance tests – The QC performance criteria for this method was performed with a Hach Compa ny 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 NO 3 - 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 NO 3 - 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 NO 3 - N 20 90.0 11018.0 Glossary of Definitions and Purposes
The definitions and purposes are specified to this method but ha ve 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 precisi on and recovery

18.2.5 OPR : On-going precision and recovery

18.2.6 MS : Matrix spike

18.2.7 MSD : Matrix spike duplicate