Corn Leaf Nitrate ReductaseA Nontoxic Alternative to Cadmium for Photometric Nitrate Determinations in Water Samples by Air-Segmented Continuous-Flow Analysis

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method I-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzerconfigured for simultaneous determination of nitrite (0.020−1.000 mg-N/L) and nitrate + nitrite (0.05−5.00 mg-N/L) by the nitrate reductase and cadmium reduction methodswas used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h-1, sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2 - -N/L for nitrite, 0.003 mg of NO3 -+ NO2 - -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3 -+ NO2 - -N/L for nitrate + nitrite by the enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete over the entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously by the two methods was not significantly different from zero at the p = 0.05 level.