Enzyme Based Nitrate Determination Method for Wastewater Reaches First Step in EPA Approval


Source: NECi Superior Enzymes

LAKE LINDEN, MI -- The U.S. Environmental Protection Agency (EPA) has determined that The Nitrate Elimination Company, Inc. (NECi) Method N07-0003 “Method for Nitrate Reductase Nitrate-Nitrogen Analysis” meets all requirements for measurement of nitrate and combined nitrate-nitrite in wastewater.

Enzyme-based nitrate analysis will be recommended for inclusion in the list of 40 CFR Part 136 in the next round of updates.  Until this method is approved and published in Part 136, facilities testing for nitrate in wastewater may seek limited-use approval from their regional authority for use of this method as an alternate test procedure (ATP) in Clean Water Act (CWA) compliance monitoring programs.  NECi's reagents offer a non-toxic alternative to the outdated cadmium reduction method.  This is the first enzyme-based method for regulatory analysis of a primary contaminant in water that has been positively reviewed by EPA as an ATP for use in the United States.  

The cadmium reduction method (EPA Method 353.2) was approved in 1974 when the EPA first established certified methods for water analysis.  It has persisted as the standard despite its dependence on cadmium, a toxic heavy metal, which itself is regulated as a water pollutant by EPA.  Cadmium use requires extra safety precautions in handling and must be disposed as hazardous waste.  Health risks associated with cadmium include impairment of lung function and certain types of kidney disease.  Improper disposal can result in contamination of groundwater.  Now there is a safe and environmentally benign alternative method for nitrate analysis.

Nitrate is one of two contaminants listed by the EPA to “pose an immediate threat whenever levels are exceeded”.  Testing for nitrate is imperative to maintain a healthy population and environment, and it is only logical that the test method used to determine nitrate content be also healthy for the population and the environment.

The recently EPA approved method tests within the standard range of 0.05 to 5 mg/L of nitrate-nitrogen, but may be extended by altering sample dilution or sample volume used.  It is applicable to the determination of nitrate plus nitrite in surface water, saltwater, groundwater, wastewater, and any aqueous solution containing nitrate.  Nitrate Reductase is to be added to a small volume of the sample to be analyzed in a biochemical buffer near neutrality with electron donor NADH.  The reduction of nitrate to nitrite is accomplished within 20 minutes, subsequently followed by the addition of color reagents which is completed within 10 minutes.  Samples are then analyzed using a spectrophotometer measuring absorbance at 540 ± 20 nm.  A standard curve is generated using standards of known nitrate-N content that is used to determine the nitrate-N content of samples.

Nitrate Reductase (NaR) replaces cadmium in the conversion of nitrate to nitrite.  NaR is an enzyme (protein) that binds nitrate as its substrate.  The biological electron donor, or cofactor, NADH provides the electrons required to reduce nitrate to nitrite.  Enzymes offer high specificity for analytical applications: their target substrate fits like a “lock-and-key” due to structure of the three-dimensional active site on the enzyme.  Because of this, they react only with the target to be analyzed, in this case nitrate.  An enzyme can “find” its substrate in a complex sample quickly and efficiently, making for a relatively fast and accurate reaction.  An added benefit of the lock-and-key model is sensitivity, offering low detection limits even in samples containing a complex array of compounds.  This eliminates potential interferences, a problem that other nitrate detection methods sometimes face.  The nitrate reductase reaction runs at temperatures ranging from 10 – 40°C under standard conditions, requiring no heavy metals, solvents, heat, or pressure.  All of the reagents used in this reaction are non-toxic, making this method safe for laboratory personnel, transport, and the environment. 

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