Photos by Daniel Boone, NAU
 
 

What are stable isotopes?

Isotopes are atoms of the same element that differ in atomic mass, due to differences in the number of neutrons contained in the atoms' nuclei. For example, the three most abundant isotopes of carbon are carbon-12 (12C), which contains 6 protons, 6 electrons, and 6 neutrons; carbon-13 (13C), which also has 6 protons and electrons, but has 7 neutrons; and carbon-14 (14C), which also contains 6 protons and electrons, but has 8 neutrons. Having too few or too many neutrons compared to protons causes some isotopes, such as 14C, to be unstable. These unstable 'radioisotopes' will decay to stable products. Other isotopes, such as 12C and 13C do not decay, because their particular combinations of neutrons and protons are stable. These are referred to as stable isotopes.
 

How do you measure stable isotopes

Stable isotopes of carbon, nitrogen, sulfur, oxygen, and hydrogen - those most commonly used in ecological and environmental research - are measured by gas isotope-ratio mass spectroscopy. The sample is converted into a gas (such as CO2, N2, SO2, or H2), the gas molecules are ionized in the Ion Source (Figure 1) which strips an electron from each of them (causing each molecule to be positively charged), and then the charged molecules enter a flight tube. The flight tube is bent, and a magnet is positioned over it such that the charged molecules separate according to their mass, with molecules containing the heavier isotope bending less than those containing the lighter isotope. Faraday collectors are present at the end of the flight tube to measure the intensity of each beam of ions of a given mass, after they've been separated by the magnet.
 
For CO2, three faraday collectors are set to collect ion beams of masses 44, 45, and 46. Several masses are collected simultaneously, so that the ratios of these masses can be determined very precisely.
In the flight tube, the magnet causes the ions to be deflected, with a radius of deflection that is proportional to the mass-to-charge ratio of the ion. Heavier ions are deflected less (larger radius) than lighter ions. For example, for CO2, mass 46 has the largest radius of deflection, mass 44 has the smallest, and mass 45 is intermediate. Charge also affects the radius of deflection, but - for the most part - this is held constant because the Ion Source strips only 1 electron from most molecules.
 
Stable isotope abundances are expressed as the ratio of the two most abundant isotopes in the sample compared to the same ratio in an international standard, using the 'delta' (δ) notation. Because the differences in ratios between the sample and standard are very small, they are expressed as parts per thousand or 'per mil' () deviation from the standard. For example, for carbon:
 
δ13Csample = {(13C/12C sample) / (13C/12C standard) - 1} x 1000
 
The standard is defined as 0. For carbon, the international standard is Pee Dee Belemnite, a carbonate formation, whose generally accepted absolute ratio of 13C/12C is 0.0112372. Materials with ratios of 13C/12C > 0.0112372 have positive delta values, and those with ratios of 13C/12C < 0.0112372 have negative delta values. The table below shows the international standards and their absolute isotope ratios for the 5 environmental isotopes. Some elements, such as oxygen and hydrogen, have more than one international standard.
 
 

 
To view published guidelines for reporting stable-isotope ratio data, please refer to the following articles...
 
Werner, R.A., and Brand, W.A. 2001. Referencing strategies and techniques in stable isotope ratio analysis. Rapid Communications In Mass Spectrometry 15: 501-519.
 
Coplen, T.B. 1996. New guidelines for reporting stable hydrogen, carbon, and oxygen isotope-ratio data. Geochimica et Cosmochimica Acta 60: 3359-3360.
 

How are stable isotopes used in ecological and environmental research?

Some of the most exciting advances in ecology and environmental sciences in the past decade have relied on stable isotopes.  Stable isotopes can be used to address many questions in ecology and environmental sciences, including: 

    -What is the base of this food web ( 13C, 34S)?  
    -How long is this food chain ( 15N)? 
    -Where does this nitrate and sulfate pollution come from ( 15N, 34 S, 18O)? 
    -How efficiently do these plants use water ( 13C)? 
    -Where in the soil do these plants get their water ( 18O, 2H)?  
    -How much nitrogen did a plant get from nitrogen fixation vs. NH 4 + or NO 3 - uptake ( 15 N)? 
    -What are the rates of carbon and nitrogen turnover in this soil ( 13C, 15N)?  
    -Was this CO2 produced by plant roots or by soil microorganisms ( 13C, 18O)? 
    -Was this N2O produced by nitrifiers or denitrifiers ( 15N, 18O)? 
    -Where did this butterfly migrate from ( 18O, 2H)?  
    -Did this prehistoric human society rely on corn as a food source ( 13C)?  

Stable isotope techniques allow the investigation of these questions quantitatively and non-intrusively (without the environmental hazards of radioisotopes), and thus offer considerable advantages over other techniques.  Indeed, many of these questions can only be addressed by using stable isotopes. 

Many of these techniques rely on natural differences in the ways that 'heavy' and 'light' isotopes are processed in the environment through chemical, biological, and physical transformations. These are referred to as natural abundance isotope techniques. For example, plants preferentially take up carbon dioxide containing the lighter carbon isotope ( 12C-CO2 ) in photosynthesis, but the degree of preference depends on water availability and on the photosynthetic pathway, which is a major distinguishing characteristic of plants from hot, xeric environments versus more mesic environments.  Thus, the 13C composition of plants provides a time-integrated measure of the efficiency with which plants use water.  It is also possible to tell how much a plant depends on surface water compared to deep sources of water by measuring the 2H and 18O composition of water in a plant's stem, because 1) 'heavier' water will evaporate at the soil surface more slowly than 'lighter' water, causing surface soil water to be isotopically enriched compared to deeper water, such as groundwater, and 2) the rain and snow that falls during winter (precipitation that contributes to deeper water sources in the soil) will be isotopically 'lighter' than summer precipitation. 

Other stable isotope techniques rely on adding trace amounts of compounds that are artificially enriched in the rare (heavy) isotope of the element of interest.  These are referred to as isotope tracer techniques. For example, without isotopes, measuring independently the processes of microbial production of ammonium (NH4+) through mineralization and the consumption of NH4+ through immobilization and nitrification was not possible, because all these processes occur simultaneously. By adding 15NH4+ to soil and monitoring the rate at which it is 'diluted' by the more abundant 14NH4+ , one gets a measure of the rate of mineralization of soil organic matter, a rate that is independent of nitrification and immobilization (the NH4+ -consuming processes).  By adding 15N as NH4+ or NO3 - and monitoring both 15N and 14N in the soil, it is possible to quantify each of these microbial transformations, in situ. 

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