Environmental Protection

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Enzymatic Method for Determining Nitrate in Wastewater Reaches First Step Toward EPA Approval

Enzyme-based nitrate analysis will be recommended for inclusion in the list of approved methods at 40 CFR Part 136 in the next round of updates.

The U.S. Environmental Protection Agency has determined that The Nitrate Elimination Company, Inc. 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 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 NECi's reagents offer a non-toxic alternative to the outdated cadmium reduction method.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 news about the nitrate reductase method is extraordinary. I can't wait until every lab around the world is using this environmentally friendly technique," said a Thermo Fisher Scientific team member about the NECi method.

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.

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 to 40 degrees 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.

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 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.

Customers have reliably used the method for about a decade, but it has been unavailable to labs that rely on EPA-approved methods. EPA's review of the method as an ATP and recommendation for inclusion in a future regulatory action that would add this to the list of approved methods for use in Clean Water Act compliance monitoring programs will enable many more labs to move their nitrate analysis into the 21st century and away from the problems and costs of cadmium-based analysis.

Comprehensive studies of this method have been published, which include analysis of interferences by the U.S. Geological Survey and others. USGS Scientific Investigations Report 2013-5033 demonstrates the equivalency of results obtained using the nitrate reductase method when compared to the cadmium based method. NECi's NaR reagents have had Standard Method status with the U.S. Geological Survey since 2011. EPA approval will encourage a broader spectrum of laboratories across the globe to turn to enzyme-based nitrate analysis.

Nitrate Reductase Nitrate Analysis works on any wet-chemistry photometric instrument, including automated discrete analyzers, microplate analyzers, flow injection and segmented flow analyzers, manual test tube format, and can be adapted to use with almost any automated laboratory equipment. NECi recombinant Nitrate Reductase (NaR) is manufactured according to strict quality control standards, ensuring lot-to-lot reproducibility. This warrants that NECi NaR is accurate and reliable. The enzyme is in a freeze-dried protein glass form and is stable for six months stored at room temperature, longer at colder temperatures. Reagents are available in a variety of test kits and reagent packs, or the NaR enzyme can be purchased separately. NECi manufactures two types of superior stock recombinant Nitrate Reductase, YNaR and AtNaR. Both are Immobilized Metal Affinity Chromatography (IMAC) purified eukaryotic Nitrate Reductase forms.

Dr. Bill Campbell, NECi's president, has more than 40 years' experience studying Nitrate Reductase biochemistry and molecular biology. He was a professor at Michigan Technological University in Houghton, Mich., before founding NECi in 1993 with his wife and NECi vice president, Ellen R. Campbell.

NECi's R&D has received support from the Small Business Innovation Research programs of the U.S. Department of Agriculture, the National Institutes of Health, and the National Science Foundation.

About the Authors

Ellen Campbell is vice president and co-founder of The Nitrate Elimination Company. She has the lead role in business development and financial management at NECi and is involved in experimental design and performance, supervision of lab and administrative personnel, contract management, and proposal preparation. She is principal investigator or co-PI on NECi's competitive research awards and grants. Campbell earned a B.S. in Chemistry from the State University of New York College of Environmental Science and Forestry, Syracuse, N.Y., and has been involved in biotechnology research since 1982.

Anna-Marie Davidson attended Western Michigan University in Kalamazoo, where she obtained by B.S. in Biomedical Sciences with minors in art and chemistry in December 2012. She began working at The Nitrate Elimination Company, Inc. in November 2013 as a laboratory technician and marketing assistant.

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