Sep 07, 2024

Public workspaceUSDA LTAR Common Experiment measurement: Total nitrogen (TN) and total dissolved nitrogen (TDN) concentration

DOI
dx.doi.org/10.17504/protocols.io.5jyl82rkrl2w/v1
  • Robert W. Malone1,
  • Amy J. Morrow1,
  • Oliva Pisani2,
  • John L. Kovar1,
  • Stephen K. Hamilton3,
  • Kevin J. Cole1
  • 1USDA Agricultural Research Service, National Laboratory for Agriculture and the Environment, Ames, IA;
  • 2USDA Agricultural Research Service, Southeast Watershed Research Laboratory, Tifton, GA;
  • 3Michigan State University, W.K. Kellogg Biological Station, Hickory Corners, MI
Lori J. Abendroth
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82rkrl2w/v1
External link: https://ltar.ars.usda.gov
Protocol Citation: Robert W. Malone, Amy J. Morrow, Oliva Pisani, John L. Kovar, Stephen K. Hamilton, Kevin J. Cole 2024. USDA LTAR Common Experiment measurement: Total nitrogen (TN) and total dissolved nitrogen (TDN) concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82rkrl2w/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 21, 2024
Last Modified: September 07, 2024
Protocol Integer ID: 97104
Keywords: Long-Term Agroecosystem Research, LTAR, crops, dissolved inorganic nitrogen, dissolved organic nitrogen, dissolved organic matter, water quality, groundwater, leaching, ammonium nitrogen, colorimetry, combustion, total Kjeldahl N
Funders Acknowledgement:
United States Department of Agriculture
Grant ID: -
Disclaimer
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.
Abstract
Total dissolved nitrogen (TDN) is composed of dissolved inorganic nitrogen (DIN) and dissolved organic nitrogen (DON). DON is the N-containing component of dissolved organic matter (DOM) and part of the biologically reactive N pool in aquatic ecosystems that can degrade water quality in N-sensitive waters. Evidence suggests that DON may be important in triggering harmful algal blooms, particularly the DON from synthetic urea fertilizers. TDN measurements therefore contribute to an estimate of the N most available to phytoplankton. DIN export from agroecosystems is reasonably well characterized, but the factors that regulate spatial and temporal patterns of DON are not as well understood. Although the export of DON into groundwater has received recognition for more than 100 years and the contribution of DON to total nitrogen (TN) is often significant, leaching losses of N from agricultural systems are often assumed to be dominated by DIN and uninfluenced by DON. The most widely recognized N digestion method is total Kjeldahl N (TKN), whereby organic N is converted to ammonia-N, followed by titrimetric or colorimetric detection. The recommended measurement of TDN at moderate to high concentrations (i.e., >0.5 mg N/L) uses the high-temperature combustion and chemilumniscent detection method.
Before start
Appropriate safety, health, and environmental precautions must be followed based on the selected methods, instrumentation, and workflow. Laboratory supervisors are responsible for knowledge of these precautions and their implementation.

Review the USDA LTAR Common Experiment measurement: Best practices for collection, handling, and analyses of water quality measurements protocol (Pisani et al., 2024) prior to implementing this protocol.
Sample collection and filtration
Sample collection and filtration
Return samples to the laboratory TemperatureOn ice .

Measure total N (TN) on unfiltered samples.
Measure TDN by filtering samples through a 0.22 or 0.45 µm pore-size filter to minimize interference from particulates before chemical analysis.

Note
  • Use a Thikness0.22 µm filter if the more complete removal of the microbial community is desired.
  • Filters can be membrane or glass fiber.
  • Occasional checks of filter blanks by filtering deionized water with the same equipment are always prudent.

Sample storage and preservation
Sample storage and preservation
For TDN measurements, filter the water samples as soon as possible after sample collection.

For TN measurements following TKN digestion, acidify the samples with sulfuric acid to pH < 2 and store them at Temperature4 °C for up to 30 days.

Note
Labs performing alkaline persulfate digestion should not use acid preservation.

If necessary, freezing samples at Temperature-20 °C is an alternative to acid preservation if storing for more than 30 days (USEPA CBP/TRS 6/87).

Archiving
Archiving
Samples for TDN or TN analyses are stored until data certification (QA/QC verification).

Sample analysis
Sample analysis
The most widely recognized N digestion method is total Kjeldahl nitrogen (TKN), which involves the conversion of organic nitrogen into ammonia nitrogen using sulfuric acid, potassium sulfate, and a catalyst followed by titrimetric or colorimetric detection.
Macro- or micro-TKN methods (EPA 351.2, 0.1-20 mg/L TKN; APHA, 2005) enable this conversion.
Obtaining a representative subsample for TKN or TN methods is crucial when particulate matter is present.
Stir samples on a stir plate, and once the slurry is homogeneous, withdraw a subsample while stirring using a large bore pipet tip at a consistent depth.

In contrast to the Kjeldahl method, Total N oxidation methods convert all N-containing compounds into NO3- by oxidation using persulfate digestion followed by colorimetric detection (Water Resources Investigations Report 03-4174; APHA, 2005).

To measure TDN, the high-temperature combustion method with chemiluminescence detection (CLD) has gained popularity because it is typically coupled with the simultaneous analysis of dissolved organic carbon (DOC), allowing high sample throughput with reduced hazardous waste generation.

Covariate metrics to be sampled concurrently
Covariate metrics to be sampled concurrently
Additional primary metrics are described in other USDA LTAR Common Experiment protocols:

  • Dissolved NO3- concentration
  • Dissolved NH3 concentration
  • Total dissolved P (TDP) and Total P (TP)
  • Total suspended solids (TSS)

Calculations
Calculations
Prepare a calibration curve by plotting the area of each standard peak against its respective N concentration. Correction of standards and samples for the blank is necessary; the reagents are usually the source of most of the blank absorbance, although sometimes the water can be high in ammonia. Only the reagent contribution to the blank should be subtracted from sample absorbances, and it can be estimated by running a 2x reagent blank.
Compute the TDN or TN concentration in the sample by comparing the sample peak area to the calibration curve.
Report the concentration as mg N per liter (mg/L).

TDN = DIN (NO3- + NO2- + NH3) + DON (mg N/L)

TN = total inorganic N (NO3- + NO2- + NH3) + total organic N (mg N/L)

The analysis of TN following the TKN digestion method results in the measurement of NH3 plus organic N:

TKN = NH3 + organic N (mg N/L)

TN = TKN + (NO3- + NO2-) (mg N/L)

Recommendations for data collection
Recommendations for data collection
Table 1. Summary of recommendations for the collection and measurement of TKN/TN and TDN concentration.

ABCD
AttributePreferredMinimumComments
Spatial scaleFieldPlot
FrequencyEvent-drivenEvent-drivenMore frequent (weekly) measurements can be preferential when the flow regime can increase seasonally or after precipitation events. Sampling should be event-driven in this protocol to enable cross-site comparisons
Covariate metricsNO3-N, NH3-N, TDP, TSSNO3-N, NH3-N, TDP
Sample preservation and storage TDNFilter with a 0.45 um pore-size filter and freeze as soon as possibleOmit acid preservation for TDN
Sample preservation and storage TKN/TNAcidify to pH <2 with sulfuric acidFreeze as soon as possibleAvoid acid preservation with the TN method
Sample analysis TDNHigh-temperature combustion followed by CLDAlkaline persulfate digestion
Sample analysis TKN/TNTKN digestion followed by colorimetric analysisAcid persulfate digestion followed by colorimetric analysisObtaining a representative sample is crucial
Water quantityDischarge or flow rateDischarge or flow rateCalculate TN or TDN loads by linking this metric to the water quantity metric “flow"
Covariate metrics = other metrics to sample concurrently. NO3--N = nitrate-N; NH3-N = ammonia-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.

Patton CJ, Truitt EP, 2000. Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory-Determination of Ammonium plus Organic Nitrogen by a Kjeldahl Digestion Method and Automated Photometric Finish that includes Digest Cleanup and Gas Diffusion: U.S. Geological Survey Open-File Report 00-170,
http://nwql.usgs.gov/Public/pubs-public.html

Patton CJ, Kryskalla JR, 2003. Methods of Analysis by the U.S. Geological Survey National Water Quality Laboratory- Evaluation of Alkaline Persulfate Digestion as an Alternative to Kjeldahl Digestion for Determination of Total and Dissolved Nitrogen and Phosphorus in Water: Water-Resources Investigation Report 03-4174, http://nwql.usgs.gov/Public/pubs/WRIR03-4174/WRIR03-4174.html

Pisani, O., Lizotte, R., Veum, K., Kovar, J. L., Hamilton, S. K., & Malone, R. W. 2024. USDA LTAR Common Experiment measurement: Best practices for collection, handling, and analyses of water quality measurements. protocols.io

U.S. Environmental Protection Agency, 1986. EPA 600/4-86/043, Development of Preservation Techniques and Establishing of Maximum Holding Times.

U.S. Environmental Protection Agency, 1993. Method 351.2, Revision 2.0: Determination of Total Kjeldahl Nitrogen by Semi-Automated Colorimetry.

U.S. Environmental Protection Agency, 1978. Method 351.3, Methods for Chemical Analysis of Water and Wastes (MCAWW), EPA/600/4-79/020 (accessed through National Environmental Methods Index http://www.nemi.gov).

U.S. Environmental Protection Agency, Results of Comparative Studies of Preservation Techniques for Nutrient Analysis on Water Samples. USEPA CBP/TRS 6/87, Aug 1987.