Page 1 of 2 Office of Ground Water and Drinking Water

February 9

, 201 6

MEMORANDUM

SUBJECT: Basis for Expedited Approval of N itrate Elimination Company Inc. (N ECi ) 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 a n 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 dinucleot ide 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 met hod provides a “green er ” 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
reduc tase 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 r eference M ethod 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 M ethod 353.2 of 97.5% to 99.3%. All
collect ed 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 M ethod

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.Page 2 of 2

 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 M ethod 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 specif ied in the approved reference method . Results were comparable to the EPA
reference M ethod 353.2 matrix spike results of 85.9% to 103% (<6.2 %RSD)

.

 Additional test samples evaluated includ ed 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 wi thin acceptable method QC criteria specified in the approved reference method

.

Based on evaluation of the accura cy, 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.