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Nitrogen has two stable isotopes. Natural variation of these isotopes is useful for tracing nitrogen cycling in ecosystems and for tracing the source of nitrogen to surface and ground waters.


Cost of Analysis (return to top)

Isotope Ratio Mass Spectrometry (IRMS): $10 to $20 per sample analysis, and an additional $30 to $50 for sample preparation.
Numerous laboratories analyze for stable isotopes using IRMS;see ISOGEOCHEM (an Internet discussion list) for links to many of them.

New resin sampling:
See the Idaho Stable Isotopes Laboratory web site

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There are two stable isotopes of nitrogen: 14N and 15N. All nitrogen compounds contain both isotopes, but because of isotopic fractionation they are incorporated into compounds in differing ratios depending on the nature of the reactions that produce the compounds. For example, as nitrogen compounds are passed up the food chain, the lighter isotopes are excreted in urine and the heavier isotopes are retained. Nitrogen in animal waste is hydrolyzed to ammonia and then converted to nitrate. During this process more of the heavy isotope is concentrated in the resulting nitrates. When various sources of nitrogen compounds are mixed together in surface runoff or in a body of water, the ratio of light to heavy nitrogen isotopes in the water can be used to estimate the relative contributions of the various sources.

Commercial fertilizers, animal or human waste, precipitation, and organic nitrogen within the soil are common sources of nitrate in groundwater. Each of these nitrate source categories has a distinguishable isotopic signature (i.e., 15N/14N ratio).


(Modified from Hoefs 1997 and Clark and Fritz 1997 with data from Amberger and Schmidt 1987, Böttcher et al. 1990, and Létolle 1980).

Measurement Techniques (return to top)

Typical sampling

Samples can be analyzed for d15N of NH4+, NO3- or gaseous N2. Generally, samples are filtered in the field through 0.1 micron filters, put in rinsed bottles, preserved (with sulfuric acid, mercuric chloride, or chloroform), chilled, wrapped in insulating packing material, and sent to the laboratory in ice chests. Sample-size requirements are in the range of 10-100 µM of N, however sample size requirements also vary between laboratories. In addition to measuring the d15N value of nitrate, a few labs are able to measure the d18O value as well.

See the USGS Water Resources Division, Western Region http://wwwrcamnl.wr.usgs.gov/isoig/guidelines/nitrate/
for detailed information on the collection and recovery of d15N and d18O for analysis.

New resin sampling

Samples with high NO3- concentrations are collected as bulk water samples; however, low concentrations often require the use of ion exchange resins. This alternate method concentrates the NO3- or NH4+ on anion or cation exchange resins. Collection of nitrate on anion exchange resins eliminates the need to send large quantities of chilled water back to the laboratory, eliminates the need for hazardous preservatives, makes it easier to archive samples, and allows analysis of extremely low-nitrate waters.


Nitrate analyses are routinely performed with isotope ratio mass spectrometry (IRMS). IRMS separates the ions of the element (14N/15N) on the basis of their differing mass/charge ratio. Sample preparation consists of converting solid or liquid material to a gas (N2) and isolating the particular gas that must be analyzed.

(See the MDS Sciex website)

(See also our IRMS web page for a further description of the technology)

Hydrological and Other Applications (return to top)

The use of stable nitrogen isotopes in environmental and ecological studies, plant nutrition, and soil fertility has increased considerably during the past three decades. Nitrogen isotopes are often useful in determining sources of nitrate in groundwater and surface water. This is especially true in regions that have sandy, well drained soils (where the d15N of the nitrate has less opportunity to be transformed by biological activity).

Biologically-mediated reactions (e.g., assimilation, nitrification and denitrification) strongly control nitrogen and nitrogen isotopic compositions in both soil and water. Nitrification is a chemical process that produces nitrate (NO3-) through the oxidation of ammonium (NH4+). After gaseous N2, nitrate is the most stable form of nitrogen and is present in most groundwater. The nitrification reaction, below, occurs under aerobic conditions, whereas denitrification occurs under anaerobic conditions.

NH4+ + 2O2 = NO3- + 2H+ + H2O

NO3- + 5/4CH2O = 1/2N2 +5/4HCO3- +1/4H+ +1/2H2O

A bacterium known as Thiobacillus denitrificans is responsible for most of the denitrification in groundwater. However, other types of bacteria can denitrify in the absence of carbon, using electron sources such as Mn2+, Fe2+, sulfide and methane. Fractionation during denitrification causes the d15N of residual nitrate to increase exponentially as nitrate concentrations decrease due to fractionation.

Denitrification processes can also be identified by the amount of gaseous N2. The N2 produced by denitrification results in excess N2 contents in groundwater. The total N2 (which consists of air N2 trapped during recharge plus N2 produced by denitrification) can be collected, analyzed for d15N, and used to estimate the extent of denitrification, initial composition of the nitrate, or the mixing history of the water.

In addition to denitrification, NO3 may be removed from water by plant assimilation, as such plants can assist in the remediation of surface and groundwater, for example. Riparian zones are assumed to buffer NO3- in surface waters. During assimilation, nutrients are incorporated in the plant, where they remain until they are released by mineralization during decay. The plants do not remove N from ecosystem, but increase the residence time of nutrients through a reduction of the mobility of N compounds. However, there are limits, and large amounts of nutrients cause increased plant growth, resulting in eutrophication and anoxia. In this way, increased nutrients lead to a shorter retention time of nutrients in the riparian buffer zone.

The effects of denitrification and assimilation can be distinguished with the use of d15N analyses combined with d18O analysis. If plant uptake alone is responsible for NO3- remediation, the isotopic composition of the remaining NO3- remains unchanged. If both denitrification and assimilation are occurring, the isotopic composition of the residual nitrate is enriched and the overlying plants reflect the isotopic composition of the NO3- source. The isotopic composition of the plants will remain the same and the water will become more enriched if denitrification is the only process occurring.

Analysis of d18O in combination with d15N provides additional information about nitrates in water and soils, specifically on the relative contributions of fertilizers vs. soil NO3 or manure/septic waste, and on the relative contributions of atmospheric NO3 vs. fertilizer, soil NO3, or manure/septic waste.

References and further reading (return to top)

  • Amberger, A. and H.-L. Schmidt, Natürliche isotopegehalte von nitrat als indicatoren für dessen herkunft. Geochimica et Cosmochimica Acta, 51: 2699-2705, 1987.

  • Bohlke, J.K. and J.M. Denver, Combined use of groundwater dating, chemical and isotopic analyses to resolve the history and fate nitrate contamination in two agricultural watersheds, Atlantic Coastal Plain, Maryland, Water Resour. Res. 31(9), 2319-2339, 1995.

  • Böttcher, J., O. Strebel, S. Voerkelius, and H.-L. Schmidt, Using isotope fractionation of nitrate nitrogen and nitrate oxygen for evaluation of denitifcation in a sandy aquifer, J. of Hydrol., 114: 413-424, 1990.

  • Clark, I., and P. Fritz, Environmental Isotopes in Hydrogeology, Lewis Publishers, Boca Raton, 1997.

  • Cook. P.G., and A.L. Herczeg, editors, Environmental Tracers in Subsurface Hydrology, Kluwer Academic Publishers, Boston, 2000.

  • Hoefs, J., Stable Isotope Geochemistry, 4th ed., Springer, Berlin, 1997.

  • Kendall, C., and J.J. McDonell, eds., Isotope Tracers in Catchment Hydrology, Elsevier Science, Amsterdam, 1998.

  • Létolle, R., Nitrogen-15 in the natural environment; chapter 10 in Handbook of Environmental Isotope Cheochemistry, vol. 1: The Terrestrial Environment, ed. by P. Fritz and J.-Ch. Fontes, Elsevier, Amsterdam, 407-433, 1980.

Internet resources (return to top)

Illinois State Water Survey, Nitrogen Cycles Project.


Nolan, B.T.., B.C. Ruddy, K.J. Hitt, and D.R. Helsel, A national Look at nitrate contamination of ground water, Water Conditioning and Purification 39(12), 76-79, 1998. (Republished on USGS Web site)

North Carolina State University, Rivernet Stable Isotope Tracer Program, Annual Report 2001.

Pidwirny, M.J., Fundamentals of Physical Geography, Ch. 9: Introduction to Biogeography and Ecology, The Nitrogen Cycle.

Roadcap, G.S., K.C. Hackley, H.H. Hwang, and T.M. Johnson, Application of nitrogen and oxygen isotopes to identify sources of nitrate, Illinois Groundwater Consortium Conference, 2001,

Roadcap, G.S., K.C. Hackley, H.H. Hwang, and T.M. Johnson, Application of nitrogen and oxygen isotopes to identify denitrification in a shallow aquifer with a variable influx of nitrate, Geological Society of America Annual Meeting 2001.

Rupert, M.G., Nitrate (NO2+NO3-N) in groundwater of the Upper Snake River Basin, Idaho and Western Wyoming, 1991-95, USGS Report.

Sea Grant North Carolina, Study of Excess Nitrogen Sources in Neuse River Estuary 96EP-34.

Teranes, J.L., and S.M. Bernasconi, A century-long record of anthropogenic nutrient loading provided by d15N values in sediment from a eutrophic lake, 9th Annual V.M. Goldschmidt Conference.

US Environmental Protection Agency, U.S. Map: Risk of Groundwater Nitrite Contamination.

USGS, Periodic Table - Nitrogen.

Winter, T.C., J.W. Harvey, O.L. Franke, and W.M. Alley, Ground water and surface water: a single resource, USGS Circular 1139.

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