The Nitrate Reductase method is a green, cadmium-free alternative to EPA method 353.2 for the reduction of nitrate to nitrite prior to analyses. Method N07-003 was designated as an EPA Alternative Test Protocol for Safe Drinking Water Act (SDWA) for nitrate determinations using discrete analyzers in July 2016.  On August 7th, 2017, the EPA granted NECi's Nitrate Reductase ATP status for Clean Water Act applications as part of the 2016 Methods Update Rule.  Regulation 40 CFR Part 136 allows method flexibility: the assay is compatible with virtually any type of colorimetric detection, for anything except drinking water.  The method is a green' alternative to the other approved methods which use cadmium, a known carcinogen, for the reduction of nitrate to nitrite prior to analyses. NECi Method N07-003 uses automated discreet analysis, flow injection analysis, or manual reagent additions and spectrophotometry to determine concentrations of nitrate and nitrite, combined or separately in wastewater. The method involves the following steps:

• Enzymatic reduction of nitrate in a sample to nitrite using eukaryotic nitrate reductase;

• Diazotizing the nitrite originally in the sample plus the reduced nitrate with sulfanilamide followed by coupling with N-(1-napthyl)ethylenediamine dihydrochloride under acidic conditions to form a highly colored azo dye;
   
• Colorimetric determination in which the absorbance of color at 546 nm is directly proportional to the concentration of the nitrite plus the reduced nitrate in the sample;
   
• Measurement of nitrite separately, if needed, by analysis of the sample while eliminating the reduction step;
   
• Subtraction of the nitrite value from that of the combined nitrate -nitrite value to measure nitrate separately if needed.

Contact your regulators or auditors for information on how you can start using the method now.

Reagent packs are available for discrete or flow injection analyzers as well as manual addition. A separate reagent kit formulated especially for soil analysis is also available. For field screening, there is a portable handheld photometer operated via an Android app. 

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