Dec 04, 2024
USDA LTAR Common Experiment measurement: Dissolved nitrate (NO3-) concentration
- Richard Lizotte1,
- Oliva Pisani2,
- Kristen S. Veum3,
- John L. Kovar4,
- Robert W. Malone4,
- Kevin J. Cole4
- 1USDA Agricultural Research Service, Water Quality and Ecology Research, Oxford, MS;
- 2USDA Agricultural Research Service, Southeast Watershed Research Laboratory, Tifton, GA;
- 3USDA Agricultural Research Service, Cropping Systems and Water Quality Research, Columbia, MO;
- 4USDA Agricultural Research Service, National Laboratory for Agriculture and the Environment, Ames, IA
External link: https://ltar.ars.usda.gov
Protocol Citation: Richard Lizotte, Oliva Pisani, Kristen S. Veum, John L. Kovar, Robert W. Malone, Kevin J. Cole 2024. USDA LTAR Common Experiment measurement: Dissolved nitrate (NO3-) concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qm1pl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 12, 2024
Last Modified: December 04, 2024
Protocol Integer ID: 97167
Keywords: Long-Term Agroecosystem Research, LTAR, USDA LTAR, Common Experiment, crops, nitrate, surface water, groundwater, drinking water standard, nitrogen, nitrate nitrogen, colorimetry, concentration nitrate, nitrate reductase, cadmium with nitrate reductase, nitrogen, ionized form of nitrogen, colorimetric cadmium reduction method, nitrite, mg no3, nutrient, hypoxia of surface water, photosynthesis in aquatic autotroph, dissolved organic matter, essential nutrient for growth, presence of cadmium, essential nutrient, cadmium reduction, contributes to eutrophication, high levels in some surface water, colored azo dye, flow analyzer method with cadmium reduction, drinking water standard, identical colorimetric quantification reaction, cadmium method, copperized cadmium, groundwater, cadmium, photosynthesis,
Funders Acknowledgements:
United States Department of Agriculture
Grant ID: -
Abstract
Nitrate (NO3-) is an ionized form of nitrogen (N) that is of interest in surface and groundwater. Nitrate often occurs in trace amounts (<0.01 mg/L) in rivers and lakes but can reach high levels in some surface water and groundwater. In excessive amounts, it can elicit an illness known as methemoglobinemia in infants, and the drinking water standard is limited to 10 mg NO3--N/L to prevent this illness. In addition, NO3- is an essential nutrient for growth and photosynthesis in aquatic autotrophs, and elevated NO3- loading contributes to eutrophication and hypoxia of surface waters, particularly in coastal marine ecosystems. The recommended methods for measuring NO3- concentration in water include the colorimetric cadmium reduction method, whereby NO3- is reduced quantitatively to nitrite (NO2-) in the presence of cadmium. Following the addition of appropriate reagents, a highly colored azo dye is produced and measured colorimetrically. An alternative enzymatic method utilizes “greener” chemistry by replacing the toxic copperized cadmium with nitrate reductase (specifically, AtNaR2) in combination with two coenzymes (nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate) to reduce NO3- to NO2-, followed by the identical colorimetric quantification reaction used in the cadmium method. This enzyme reduction method overcomes interferences from phenolics and humic substances noted in earlier enzymatic methods. The method detection levels (or limits; MDL) for standard and low NO3- + NO2- concentrations with enzyme reduction are comparable to the long-term MDLs of the continuous-flow analyzer method with cadmium reduction. In addition, NO3- concentration differences were statistically equivalent to zero or analytically equivalent to zero (i.e., concentration differences were less than MDLs [Patton and Kryskalla, 2011]). Recent advances in field sensor technology have afforded commercial sensors that allow for in situ NO3- measurement using an optical sensor. These sensors can record values over short durations (seconds to minutes) and use an ultraviolet spectrophotometer (Johnson and Coletti, 2002). Although these sensors provide high-resolution data, their accuracy can degrade because of background interference from colored dissolved organic matter (DOM) and high turbidity. Thus, sensor readings must be validated at least monthly by conducting water sample collection and lab analyses.
Troubleshooting
Safety warnings
Follow appropriate safety, health, and environmental precautions based on the selected methods, instrumentation, and workflow. Laboratory supervisors are responsible for knowledge of these precautions and their implementation.
Sample collection and filtration
Return samples to the laboratory On ice and filter them on collection day if possible.
For NO3- measurements, filter water samples through a 0.45 µm pore-size filter to minimize interference from particulates before chemical analysis. If desired, remove the microbial community using a 0.22 µm filter. Filters can be membrane or glass fiber, and may require pre-washing to prevent contamination.
Occasional checks of filter blanks by filtering deionized water with the same equipment are always prudent. The use of a blank is described in the USDA LTAR Common Experiment measurement: Best practices for collection, handling, and analyses of water quality samples protocol (Pisani et al., 2024b).
Sample storage and preservation
Analyze samples as soon as possible after collection.
If storage is required, maintain filtered samples at 4 °C (not frozen) for up to 48 hours (US EPA, 1993). If lengthier storage is required, prepare samples with concentrated sulfuric acid to a pH < 2, and maintain them at 4 °C .
Hold acidified samples for up to 28 days in clean (N-free) plastic bottles. For acidified samples, NO3- and NO2- cannot be determined as individual species but only as total inorganic N.
Archiving
The common practice is to store water samples for NO3- analysis at 4 °C (not frozen) until data certification (QA/QC verification).
Storage of preserved water samples should not exceed 28 days.
Sample analysis
Using the cadmium reduction method, determine NO3- in water by passing a filtered sample through a column containing granulated copper–cadmium to reduce NO3- to NO2-.
The sum of NO2- initially present plus the reduced NO3- is measured colorimetrically (APHA, 2005; USEPA, 1993).
The sum of NO3- + NO2- is sometimes called NOx-.
To obtain separate NO3- and NO2- values, perform the procedure with the reduction step (total NOx-) and then without the reduction step (NO2- only), and calculate NO3- by difference.
The NO2- is usually, but not always, a relatively small component of the total.
To reduce NO3- to NO2- using the enzymatic reduction method, nitrate reductase (specifically, AtNaR2) is used in combination with two coenzymes (nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate). Enzymatic reduction is an alternate method to cadmium reduction.
This effort is followed by the identical colorimetric quantification reaction used in the cadmium method (ASTM, 2014; Patton and Kryskalla, 2011), as described in Step 10.
Covariate metrics to be sampled concurrently
Dissolved ammonia (NH3), total dissolved N (TDN), total dissolved phosphorus (TDP), and total suspended solids (TSS). These metrics are described in the following protocols:
- USDA LTAR Common Experiment measurement: Dissolved ammonia (NH3) concentration
- USDA LTAR Common Experiment measurement: Total nitrogen (TN) and total dissolved nitrogen (TDN) concentration
- USDA LTAR Common Experiment measurement: Total phosphorus (TP) and total dissolved phosphorus (TDP) concentration
- USDA LTAR Common Experiment measurement: Total suspended solids (TSS)
Calculations
Prepare a calibration curve by plotting the blank-corrected area of each standard peak against its respective NO3- or NOx- (NO3- + NO2-) concentration. The laboratory blank is used for this step.
Compute the NO3- or NOx- concentration of the sample by comparing the sample peak area to the calibration curve. Report the concentration(s) as mg NOx--N/L.
Recommendations for data collection
Table 1. Summary of recommendations for the collection and measurement of dissolved NO3- concentration.
| A | B | C | D | |
| Attribute | Preferred | Minimum | Comments | |
| Spatial scale | Field | Plot | ||
| Frequency | Event-driven | Event-driven | More frequent (weekly) measurements may be preferred when the flow regime varies seasonally or following precipitation events. Sampling in this protocol is event-driven to enable cross-site comparisons | |
| Covariate metrics | NH3-N, TDN, TDP, TSS | NH3-N, TDN, TDP | ||
| Sample preservation and storage | Filter with a 0.45 μm pore-size filter (membrane or glass fiber), refrigerate as soon as possible, and analyze within 48 hours | Filter with a 0.45 μm pore-size filter (membrane or glass fiber), freeze as soon as possible, and analyze within 28 days | Alternatively, use a 0.7 μm GF/F. Use a 0.2 μm pore-size filter to exclude bacteria from samples but at much slower flow rates | |
| Sample analysis | Nitrate reductase with colorimetric analysis | Cadmium reduction with colorimetric analysis | Manually set up reduction columns or use them as part of an auto-analyzer system | |
| Water quantity | Discharge or flow rate | Discharge or flow rate | Calculate NO3-N loads by linking this metric to the water quantity metric “flow” |
Covariate metrics = NH3-N = ammonia-N; TDN = total dissolved N; TDP = total dissolved phosphorus; TSS = total suspended solids.
Protocol references
American Public Health Association (APHA), (2005). Standard Methods for the Examination of Water and Wastewater, 21st ed. Washington, DC: American Public Health Association, American Water Works Association, and Water Environment Federation.
American Society for Testing and Materials (ASTM) International, (2014). Standard Test Method for Nitrite-Nitrate in Water by Nitrate Reductase. Standard ASTM-D7781-14.
Dalzell, B. & Pisani, O. (2024). USDA LTAR Common Experiment measurement: Total suspended solids (TSS). protocols.io dx.doi.org/10.17504/protocols.io.261ge5pjog47/v2
Hamilton, S. K., Pisani, O., Kovar, J. L., Malone, R. W., Morrow, A. J., & Cole, K. J. (2024). USDA LTAR Common Experiment measurement: Total phosphorus (TP) and total dissolved phosphorus (TDP) concentration. protocols.io dx.doi.org/10.17504/protocols.io.8epv5r7m6g1b/v1
Johnson, K. S. & Coletti, L. J. (2002). In situ ultraviolet spectrophotometry for high resolution and long-term monitoring of nitrate, bromide and bisulfide in the ocean. Deep Sea Research Part I: Oceanographic Research Papers, Vol 49, Issue 7, Pages 1291-1305.
Malone, R. W., Morrow, A. J., Pisani, O., Kovar, J. L., Hamilton, S. K., & Cole, K. J. (2024). USDA LTAR Common Experiment measurement: Total nitrogen (TN) and total dissolved nitrogen (TDN) concentration. protocols.io dx.doi.org/10.17504/protocols.io.5jyl82rkrl2w/v1
Patton, C. J. & Kryskalla, J. R. (2011). Colorimetric determination of nitrate plus nitrite in water by enzymatic reduction, automated discrete analyzer methods: U.S. Geological Survey Techniques and Methods, Book 5, Chapter B8, Page 34.
Pisani, O., Kovar, J. L., Malone, R. W., Morrow, A. J., & Cole, K. J. (2024a). USDA LTAR Common Experiment measurement: Dissolved ammonia (NH3) concentration. protocols.io dx.doi.org/10.17504/protocols.io.j8nlk8b61l5r/v1
Pisani, O., Lizotte, R., Veum, K. S., Kovar, J. L., Hamilton, S. K., & Malone, R. W. (2024b). USDA LTAR Common Experiment measurement: Best practices for collection, handling, and analyses of water quality samples. protocols.io dx.doi.org/10.17504/protocols.io.q26g71z68gwz/v1
US Environmental Protection Agency (US EPA), (1993). Method 353.2, Revision 2.0: Determination of Nitrate-Nitrite Nitrogen by Automated Colorimetry.
Acknowledgements
This research is a contribution from the Long-Term Agroecosystem Research (LTAR) network. LTAR is supported by the United States Department of Agriculture. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.