SOP# 516NYA/WQL-DLL/15.02.A Laboratory Manager Approval: DLo Quality Assurance Officer Approval: DLo

Revision # A000000

Effective Date: 08/13/2016 Date: 08/13/2016 Date 08/13/2016

NITROGEN-NITRITE (NO2--N) SPECTROPHOTOMETRIC STANDARD OPERATING PROCEDURE 1.0 Scope and Application 1.1 This method may be used to analyze samples containing nitrite ion (NO2--N) from 0.004 to 0.300mg/L in drinking, surface, saline waters, and domestic and industrial wastes. 1.2 Please consult SOP# 516NYA/WQL-DLL/MIS-05 for Initial Demonstration of Capability (IDC) and SOP# 516NYA/WQL-DLL/MIS-06 for Method Detection Level (MDL). 2.0 Summary of Method 2.1 Nitrite (NO2-) in water is determined spectrophotometrically by the formation of a pink/red azo dye measured at a wavelength of 540 nm. The color is produced by the reaction of diazotized sulfanilamide with N-(1-napthyl)-ethylenediaminedihydrochloride (NED dihydrochloride). The azo dye is produced at pH 2.0 to 2.5. 3.0 Definitions 3.1 Please consult SOP# 516NYA/WQL-DLL/MIS.01. 4.0 Interferences 4.1 Chemical incompatibility makes it unlikely that NO 2-, free chlorine, and nitrogen trichloride (NCl 3) will coexist. NCl3 imparts a false red color when color reagent is added. 4.2 Sb3+, Au3+, Bi3+, Fe3+, Pb2+, Hg2+, Ag2+, chloroplatinate (PtCl62-), and metavanadate (VO32-) cause precipitation. 4.3 Cupric ion may cause low results by catalyzing decomposition of the diazonium salt. 4.4 Very high levels of nitrate (>100mg/L NO3--N) appear to undergo a slight amount of reduction to nitrite. 4.5 Remove suspended solids by filtration. 4.6 High alkalinity (>600mg/L) will give low results due to a shift in pH. 5.0 Apparatus and Materials 5.1 HACH DR6000 UV-Vis Spectrophotometer for use at 540nm. 5.2 1-Inch 10mL sample cell with appropriate cell adapter. 5.3 30mL mixing vials with caps. 5.4 100, 500mL volumetric flasks. 5.5 25mL graduated cylinders. 5.6 Single-channel pipette 0.1-1.0mL. 5.7 Single-channel pipette 1.0-10.0mL. 6.0 Reagents 6.1 Reagent Quality (DI) Water: DI water should be monitored for impurities. 6.2 Buffer-color reagent: Dissolve in a 500mL volumetric flask 5.00g sulfanilamide and 0.50g N-(1napthyl)-ethylenediamine dihydrochloride (NED dihydrochloride) into 250mL DI water + 105mL HCl mix. Add 136.00g sodium acetate (CH3COONa•3H2O). Stir until dissolved and dilute to 500mL with DI water. Store in a brown bottle for up to 1 month at 4oC. 6.3 Stock Nitrite Solution (1000mg/L NO2--N), commercially purchased or laboratory made: 6.3.1 NO2- is oxidized readily in the presence of moisture. Keep bottles tightly stoppered against the free access of air when not in use. Good for only 6 months when stored at 4oC. 6.3.2 Anhydrous ACS grade Sodium Nitrite (oven dried for 1-2hours at 110oC): Dissolve 4.928g NaNO2 in DI water. Add 1mL of chloroform to prevent bacterial growth. Dilute to 1000mL in a 1L volumetric flask. Mix well on a stir plate. 6.3.3 Anhydrous ACS grade Potassium Nitrite (oven dried for 1-2hours at 110oC): Dissolve 6.072g KNO2 in DI water. Add 1mL of chloroform to prevent bacterial growth. Dilute to 1000mL in a 1L volumetric flask. Mix well on a stir plate 6.3.4 NOTE: The above amounts are based on 100% purity. Use the following calculation to determine C, the exact amount needed when purity is not 100%:

VIRGINIA AQUARIUM & MARINE SCIENCE CENTER 15.02.A-1

SOP# 516NYA/WQL-DLL/15.02.A Laboratory Manager Approval: DLo Quality Assurance Officer Approval: DLo

Revision # A000000

Effective Date: 08/13/2016 Date: 08/13/2016 Date 08/13/2016

C = 100 / A x B , where A is the % purity, B is the amount required based on 100% purity. 6.4 Standard Nitrite Solution (100mg/L NO2--N): Dilute 10mL stock nitrite solution (6.3) to 100mL with DI water in a volumetric flask. Mix well. 6.5 Quality Control Standard (0.100mg/L NO2--N): This standard is to be made daily to insure accuracy and precision. Add 0.100mL of 100mg/L standard nitrite solution (6.4) to DI water in a 100mL volumetric flask. Bring to volume and mix well. 7.0 Sample Collection, Preservation, and Handling 7.1 Collect samples in an acid-washed glass or HDPE containers. Follow strict acid-washing procedure (see SOP# 516NYA/WQL-DLL/MIS.03). 7.2 Analyze promptly to prevent bacterial conversion of NO2- to NO3- or NH3. 7.3 For preservation for up to 48hours store at 4oC unacidified. Bring to room temperature before analyzing. 8.0 Procedure 8.1 If the sample has a pH greater than 10 or a total alkalinity in excess of 600mg/L, adjust to approximately pH 6 with 1:3 HCl. 8.2 If necessary, filter the sample through a 0.45µm pore size filter using the first portion of filtrate to rinse the filter flask. 8.3 Color development and measurement: 8.3.1 Transfer 25mL of the sample to be tested to a 30mL test tube. 8.3.2 Add 1.0mL buffer-color reagent solution (6.2) into each test tube. Mix well. 8.3.3 Record results after 15 minutes but no more than 2 hours. 8.3.4 Prepare an instrument blank (IB) and a reagent blank (RB) using DI water as a reference. Follow the procedure above. 8.3.5 Analyze and record at least two Quality Control Standard results (as described above). 8.4 Using spectrophotometer: 8.4.1 On HACH DR6000 UV-Vis Spectrophotometer select USER PROGRAM and then select the stored program for Nitrogen-Nitrite. Press START. The wavelength (λ), 540nm, is automatically selected. 8.4.2 Use instrument blank to zero the instrument. The display will show: 0.000mg/L. 8.4.3 Rinse the sample cell well with DI water. For each sample, transfer the content of the test tube into 1-inch absorption cell and measure its absorbance at 540nm. Record the result as NO2--N. 8.4.4 Calculate nitrite concentration as follows (if needed): mg/L NO2- = 3.285 x mg/L NO2--N. 8.5 Preparation of calibration curve: 8.5.1 Using Nitrite Stock Solution, generate a curve with standard concentrations ranging from 0.004 to 0.300mg/L NO2--N. Create quantitative limit around 0.020 abs. Create using at least five calibration standard points with r2 ≥ 0.995 in the first (linear) order. 8.5.2 Treat the nitrite standards by the same procedure as the sample. 8.5.3 Review HACH DR6000 UV-Vis Spectrophotometer manual to create a User Method. 8.6 Quality Control: 8.6.1 Employ a minimum of one reagent blank per sample batch to determine if contamination is occurring. 8.6.2 Run one matrix spike replicate or one replicate sample every 20 samples. 8.6.3 Second Source Standards: 8.6.3.1 When a standard curve is manually prepared (as opposed to a factory-set calibration), it is required to analyze one known standard in addition to calibration VIRGINIA AQUARIUM & MARINE SCIENCE CENTER 15.02.A-2

SOP# 516NYA/WQL-DLL/15.02.A Laboratory Manager Approval: DLo Quality Assurance Officer Approval: DLo

Revision # A000000

Effective Date: 08/13/2016 Date: 08/13/2016 Date 08/13/2016

standards each day samples are analyzed to document accuracy. This standard must be prepared from materials obtained from a source independent from the one used for preparing the calibration standards (often referred to as a second source standard or external reference standard). A second source standard may be: 8.6.3.1.1 a quality control standard obtained from a vendor, 8.6.3.1.2 a standard prepared from primary standards obtained from a second vendor, or 8.6.3.1.3 a standard made from primary standards from the same vendor but from a different lot number (i.e., an independent lot) as those used to make the calibration standards. 8.6.3.2 Laboratory control standards are evaluated to assess whether the lab is in control of the processes involved in the preparation and analysis of specific tests. Laboratory control standards must be similar in composition to the environmental samples. They must contain known concentrations of all analytes of interest and undergo the same preparatory and determinative procedures as the environmental samples. 8.6.3.3 Second source standards must be evaluated using one of the following: vendor supplied criteria, method-defined acceptance criteria, in-house calculated acceptance limits that are statistically-derived from historical data based on three standard deviations from the mean in the detectable range or other statistically viable evaluation protocol. If the results fall outside of acceptance limits, the analysis is out of control. The analysis must be terminated and the problem corrected prior to sample analysis. 8.7 Data Assessment and Corrective Actions: 8.7.1 Please consult SOP# 516NYA/WQL-DLL/MIS-02. 9.0 Safety 9.1 Good safety habits and laboratory techniques should be used throughout the procedure. 9.2 All containers storing reagents, standards, controls blanks, and waste used in the laboratory must be properly identified through appropriate labeling and hazard definition. 9.3 Consult the Material Safety Data Sheet for information specific to the reagents used. 10.0 References 10.1 Method 354.1: Nitrogen, Nitrite (Spectrophotometer), U.S.EPA. 10.2 Method 353.2: Determination of Nitrate-Nitrite Nitrogen by Automated Colorimetry, U.S.EPA. 10.3 Standard methods for the Examination of Water and Wastewater, 20th Ed.: Method 4500NO2 B. p. 4-112 – 4-114 (1998). 10.4 Strickland, J.D.H., and Parsons, T.R., A Practical Handbook of Seawater Analysis, Method II.7, p. 77-80 (1977). 10.5 Parsons, T.R., Maita, Y., and Lalli, C.M., A Manual for Chemical and Biological Methods for Seawater Analysis, 1984. 10.6 Bower, Carol E. and Holm-Hansen, Thomas. A Simplified Hydrazine-Reduction Method for Determining High Concentrations of Nitrate in Recirculated Seawater. Aquaculture, 21 (1980), p. 281-286.

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SOP# 516NYA/WQL-DLL/15.02.A Laboratory Manager Approval: DLo Quality Assurance Officer Approval: DLo

Revision # A000000

Effective Date: 08/13/2016 Date: 08/13/2016 Date 08/13/2016

Appendix I: Revisions In accordance with the requirement to produce unequivocal, accurate records that document all laboratory activities (1VAC30-45-630, NELAC 2003 5.4.12), changes to SOPs should be documented with a Change Log that accompanies the current version and captures the timeline and content of changes.

Date: 08/13/16

Rev. #: A00000

Summary of Changes: Original SOP

Submitted By: DLo

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Approved By/Date: DLo 08/13/16

Effective Date: 08/13/16

SOP# 516NYA/WQL-DLL/15.02.A Laboratory Manager Approval: DLo Quality Assurance Officer Approval: DLo

Revision # A000000

Effective Date: 08/13/2016 Date: 08/13/2016 Date 08/13/2016

Appendix II: Additional Notes

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