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Office of Ground Water and Drinking Water

February 9, 2016

MEMORANDUM

SUBJECT: Basis for Expedited Approval of Nitrate Elimination Company Inc. (NECi) Method for 

Nitrate Reductase Nitrate-Nitrogen Analysis of Drinking Water

FROM: William A. Adams, Chemist and Steve Wendelken, Chemist

OGWDW/SRMD/TSC

TO: The Record

The NECi nitrate reductase method was submitted as an alternate test procedure to EPA Method 

353.2 for nitrate analysis. EPA Method 353.2 is approved in the national primary drinking water 

regulations at 40 CFR 141.23(k)(1) for the determination of nitrate by automated cadmium reduction.

The NECi nitrate reductase method uses a eukaryotic nitrate reductase (i.e. an enzyme) to catalyze 

the conversion of nitrate to nitrite in the presence of nicotinamide adenine dinucleotide as a 

reductant in a buffer with a near neutral pH. The combined nitrite (both the original and reduced 

nitrate) is reacted with sulfanilamide and N-(1-napthyl) ethylenediamine dihydrochloride to produce 

a chromophore. The combined nitrite concentration is then measured at ~540 nm using a discrete 

analyzer. The approved EPA Method 353.2 also relies on reduction of nitrate to facilitate

colorimetric detection, but the method employs toxic cadmium metal as the reducing agent. The 

NECi nitrate reductase method provides a “greener” non-toxic approach to nitrate-nitrogen analysis 

and only requires a fraction the sample volume used in the approved EPA method.

A multi-laboratory validation study was conducted to compare the performance of the NECi nitrate 

reductase method to EPA Method 353.2. Multiple drinking water matrixes were used in the 

validation study. Precision and accuracy data (for fortified matrixes and IPR/OPR samples) and 

detection limit data were collected from the drinking water matrixes fortified with varying 

concentrations of nitrate standards. Briefly, the results are as follows:

 The NECi NaR-R Nitrate method yielded detection limits calculated as MDLs ranging from 

0.0065 mg/L to 0.0097 mg/L compared to EPA reference Method 353.2 of 0.0047 mg/L to 

0.034 mg/L.

 The mean initial precision and recovery (IPR) data for the NECi NaR-R method ranged 

from 97.9% to 103% compared to the EPA reference Method 353.2 of 97.5% to 99.3%. All 

collected data were within the QC requirements of the reference method.

 The mean ongoing precision and recovery (OPR) data for the NECi NaR-R method ranged 

from 99.1% to 106% with a %RSD of 1.7 to 4.6, compared to the EPA reference Method 

353.2 of 98.7% to 101% with a %RSD of 0.51 to 4.5. All collected data were within the QC 

requirements of the reference method.
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 For matrix spike samples (finished tap water from each of the participating laboratories), 

the NECi NaR-R method recovery ranged from 93.8% to 110% (<4.4 %RSD), which is 

within the acceptable QC criteria specified in the approved reference method. Results 

were comparable to the EPA reference Method 353.2 matrix spike results of 92.3% to 

103% (<7.2 %RSD).

 For chlorinated matrix spike samples (finished tap water from each of the participating 

laboratories with 2–4 mg/L free chlorine added), the NECi NaR-R method recovery 

ranged from 90.9% to 106% (<3.9 %RSD), which is within the acceptable QC criteria

specified in the approved reference method. Results were comparable to the EPA 

reference Method 353.2 matrix spike results of 85.9% to 103% (<6.2 %RSD).

 Additional test samples evaluated included drinking water samples with ~500 ppm total 

dissolved solids (TDS), drinking water samples fortified with ~2 ppm TOC, and analysis of 

a standard reference sample (5.14 mg/L). The results of all samples were within 

acceptable method QC criteria specified in the approved reference method.

Based on evaluation of the accuracy, precision and sensitivity determined in the multi-laboratory

validation study, the NECi Method for Nitrate Reductase Nitrate-Nitrogen Analysis of Drinking Water is

equally as effective as EPA Method 353.2 in determining the nitrate plus nitrite (as nitrogen)

concentration in drinking water.