Spectrophotometric determination of nitrate in hypersaline waters after optimization based on the Box-Behnken design

Monitoring dissolved nitrate (NO3-) concentrations is essential for conservation efforts in aquatic ecosystems. Spectrophotometric methods are a widely accepted approach for NO3- analysis. They detect NO3- as a colored diazo complex after reduction to nitrite (NO2-) and its consequent reaction with the so-called Griess reagent. This method is commonly used for freshwater and saline water samples, even though it requires applying a heavy metal in powder form (cadmium) or high concentrations of heavy metal salts (vanadium-III), as a reductant. There has been little discussion about applying these methods for hypersaline samples. This study optimizes an existing method for use in high saline conditions based on the Griess reaction. Five factors were studied: incubation temperature, reaction time, concentration of EDTA, concentration of trisodium citrate, and concentration of reductant (VCl3). Optimal conditions were obtained by using the Box-Behnken design and included using VCl3 17.5 mM, trisodium citrate 70 mM, and an incubation temperature of 60 degrees C for 40 min. These conditions provided a linear range from 0.55 mu M to 50 mu M NO3-. The method showed a moderate precision (ranging from 4.3% to 15.4%). The proposed protocol was tested with hypersaline natural samples and showed recovery rates between 92.6% and 100.1%. This protocol for NO3- determination is the first specifically described for hypersaline samples.