Enzymatic Nitrate Determination Method Approaches Final Stages of EPA Approval
The Nitrate Elimination Co. Inc.
Enzymatic Nitrate Determination Method EPA

EPA recommends non-toxic alternative test procedure to be included in the next round of updates

Laboratories worldwide use chemicals for the purpose of analyzing samples of all types. These chemicals are often toxic, presenting potential health issues for the user and require disposing of the hazardous waste somewhere back into the environment. Whenever applicable, less toxic reagents should be implemented into laboratories to make a safer workplace and environment. One option is to replace toxic reagents with biodegradable compounds that perform essentially the same function.

The Nitrate Elimination Co. Inc. (NECi) was founded in 1993 based on the philosophy that green methods should be implemented in the laboratory for a more sustainable future without compromising valid results. Dr. Bill Campbell, president of NECi and former professor at Michigan Technological University, has studied enzyme biochemistry and molecular biology for over 40 years, specifically the enzyme nitrate reductase. Nitrate reductase can be used to analyze water samples for nitrate, the most persistent pollutant found in water worldwide.

The U.S. Environmental Protection Agency (EPA) has recommended a new method for nitrate testing of wastewater for inclusion in the list of approved methods at 40 CFR Part 136 in the next round of updates. The Office of Science and Technology has determined that NECi Method N07-0003 “Method for Nitrate Reductase Nitrate-Nitrogen Analysis” meets all requirements for measurement of nitrate and combined nitrate-nitrite in wastewater. 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. This is the first enzyme-based method for regulatory analysis of a primary contaminant in water that has been positively reviewed by the EPA as an ATP for use in the U.S.

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

Currently, the most widely used nitrate determination method is the cadmium reduction method (EPA Method 353.2), which 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 the EPA. Cadmium use requires extra safety precautions in handling and must be disposed of as hazardous waste. Health risks associated with cadmium exposure include impairment of lung function and certain types of kidney disease. Improper disposal can result in contamination of groundwater.

NECi has developed a recombinant enzyme that may be used in the place of cadmium or vanadate for the conversion of nitrate to nitrite. This enzyme, nitrate reductase (NaR), is a protein that binds nitrate as its substrate. The biological electron donor, NADH, provides the electrons required to reduce nitrate to nitrite, and is a B-vitamin derivative. Replacing cadmium with NaR creates a safer workplace for laboratory personnel and a greener environment.

There are added benefits in addition to being safe for users and the environment. Enzymes derive their specificity from the “lock-and-key” mechanism of their three-dimensional active site. This means that they react only with the target to be analyzed, a desirable trait for analytical chemistry applications. 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 enzymatic reaction proceeds at temperatures ranging from 10 to 40 degrees Celsius under standard conditions, requiring no heavy metals, solvents, high heat, or pressure. All of the reagents used in this reaction are non-toxic, making this method safe for usage, transport, and disposal.

Nitrate reductase nitrate analysis works on any wet-chemistry photometric instrument, including automated discrete analyzers, micro plate analyzers, and flow injection and segmented flow analyzers. This method is also suitable for manual analysis, including test tube and micro plate formats.

The method is designed to test within the standard range of 0.05 mg/L to 5 mg/L of nitrate-nitrogen, but it may be extended by altering sample dilution or sample volume used. It is applicable to the determination of nitrate plus nitrite in surface water, saline water, groundwater, wastewater, and any aqueous solution containing nitrate. The protocol dictates that Nitrate Reductase 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, with color development within 10 minutes. Samples are then analyzed using a spectrophotometer measuring absorbance at 540 ± 20nm. A standard curve is generated using standards of known nitrate-N content, and is used to determine the nitrate-N content of samples.

In addition to laboratory reagents and kits, a wide range of easy to use test kits have been developed based on these reagents. NECi offers complete nitrate test kits designed for consumers wishing to test their tap water, well water and agricultural water, environmental water in their communities, dry and wet plant samples, soil samples, or any other aqueous sample for nitrate content.

The NECi NaR method has been used for about a decade, but it has been unavailable to laboratories that rely on EPA-approved methods. Comprehensive studies of this method have been published, which include analysis of interferences by the U.S. Geological Survey (USGS) and others. USGS Scientific Investigations Report 2013-5003 demonstrates the equivalency of results obtained using the nitrate reductase method when compared to the cadmium based method. Nitrate Reductase as a reagent in nitrate analysis has been approved by the USGS since 2011, and within the last couple of months has gained approval and is pending publication with ASTM (American Society for Testing and Materials).

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