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What are Stable Isotopes?
Most elements exist in two or more forms, known as isotopes. Isotopes have the same number of protons but differ in their number of neutrons, resulting in different masses. The lighter form is generally the more common one (Hobson & Wassenaar 2008). This variation in the relative abundance of stable isotopes results from tiny mass differences that cause the isotopes to act differently in chemical reactions and physical processes. The lighter isotope generally forms weaker bonds than the heavier one and tends to react faster. The change in isotopic abundance is called fractionation (Karasov & Martínez del Rio 2007). Different environments are often characterized by predictable isotopic signatures (West et al. 2006).
Stable isotopes are measured as isotopic deviations from international standards and are expressed as delta (δ) values as parts per thousand (Werner & Brand 2001). These values are calculated as follows:
δ X = [(Rsample/Rstandard) – 1] x 1000
where X is the element (such as 13C or 15N), and R is the corresponding isotope ratio (13C/12C or 15N/14N). The quotient of the ratios in the sample relative to the standard is the δ value. The numerical values associated with the isotope ratio (such as δ12C) are the atomic masses of the isotopes and are accounted for by differences in the number of neutrons contained in the atom's nuclei. For example, δ12C contains 6 protons, 6 electrons and 6 neutrons, while its heavier isotope δ13C contains 6 protons, 6 electrons, and 7 neutrons. Therefore, an increase in the δ values denotes an increase in the amount of the heavier isotope component; while a decrease in the values denotes a decrease in the heavy isotope content (Peterson & Fry 1987).
'You Are What You Eat'
Stable isotope analysis is based on the principle ‘you are what you eat.' Stable isotope ratios vary among food webs and are incorporated into an animal's tissue via its diet (Hobson 1999). It is thus sometimes possible to infer the whereabouts of an animal moving between food webs. It is important, however, to choose the appropriate tissue for isotopic analysis, as tissues differ in how metabolically active they are (Rubenstein & Hobson 2004). Keratin-based tissues, such as hair, feather, nail, claw or bill, are metabolically inert after synthesis. They are usually used to study seasonal movements because an isotopic record reflecting the location of tissue synthesis remains unchanged. Conversely, metabolically active tissues provide dietary and source information for a relatively shorter period. For example, blood plasma and liver turnover elements in a matter of days or even hours, but this process takes up to several weeks in muscle and whole blood, and up to several months or even years in bone collagen (Hobson 1999). Studies that examine long-term movements therefore use metabolically inert tissues, while studies on recent movements (e.g., distinguishing between newly arrived and resident individuals) use metabolically active tissues with rapid turnover rates.Stable Isotopes Used in Terrestrial Systems
Carbon (13C/12C)
Carbon isotopic signatures can be used to distinguish plants that use C3, C4 and CAM modes of photosynthesis, as the C4 and CAM pathways lead to lower carbon fractionation than C3 photosynthesis (Karasov & Martínez del Rio 2007). Carbon isotopes can thus be used to reconstruct migratory routes, if the geographical distribution of C3, C4 and CAM plants is known and the diet preferences of the study species (Hobson 1999). Carbon isotopic changes corresponding to dietary changes in a migratory species were first demonstrated in bats (Fleming et al. 1993). It has since been used in other migratory species, including lesser snow geese (Chen caerulescens), in which it was used to infer the wintering origin (Alisaukas et al. 1993). More recently, other stable isotopes, such as nitrogen and strontium, have been added to increase the spatial resolution (Hobson 1999). Carbon isotopes have also been used to infer individual fitness of migratory birds arriving at the breeding ground (Marra et al. 1998). An intriguing avenue for future research is the isotopic discrimination of exhaled breath (CO2), which does not require sacrificing the study animal to obtain a tissue sample (Hobson & Wassenaar 2008).
Nitrogen (15N/14N)
One of the most important applications of nitrogen stable isotopes is its ability to determine the trophic level of a species. Nitrogen undergoes an increase (2-4%) in heavy isotope enrichment with each trophic level and can therefore serve as a tool in determining dietary shifts (Hobson & Wassenaar 2008). A potential problem is that nitrogen is a building block of proteins and therefore may succumb to protein catabolism occurring during migration, which can cause increased nitrogen isotope fractionation that is unpredictable (Hobson & Wassenaar 2008).
Hydrogen (2H/1H)
Deuterium (the heavy hydrogen isotope) has revolutionized stable isotope analysis in the study of animal migration. Unlike other stable isotopes, which require analysis on a species-by-species basis, deuterium signatures can be used to create a continental isotopic map, which is applicable to all migratory species (Figure 1). Deuterium ratios vary strongly with weather conditions, resulting in highly predictable spatial variation across continents (Hobson & Wassenaar 2008). Constructing continental maps that predict deuterium levels is therefore relatively straightforward, given the large amount of existing data on continental weather patterns. These maps detailing variation in deuterium were quickly utilized by biologists to determine migratory origins of species. Deuterium ratios in feathers and claws were shown to be effective indicators of breeding latitude in birds (Hobson & Wassenaar 2008, Bearhop et al. 2003). Determination of migratory origins using hydrogen does not necessarily require sacrificing individuals. Nevertheless, there are several pitfalls when using hydrogen isotope analysis. There are small-scale heterogeneities that are not accounted for in global isotopic maps, including variation with altitude (Hobson & Wassenaar 2008).
Figure 1: Contours of growing season average deuterium (δD) values in precipitation in North America used to link organisms to broad geographic origins.
Filled circles represent the location of sampling sites.
© 2012 Nature Education Modified from Hobson (1999). All rights reserved. 
Stable Isotopes in Aquatic Systems
Carbon (13C/12C)
Carbon isotopes in tissues of aquatic animals reflect the primary producers in the area of feeding and therefore can be used determine diet and movement in space and time, following the same principle used in terrestrial studies. Carbon isotope ratios in marine systems have been utilized to determine movements of migratory species between inshore and offshore ecosystems and dietary changes during migration. They have not yet been applied to migratory species in freshwater systems. Fry (1981) showed that inshore seagrass beds were more enriched in C13 compared to those offshore, and used this patterns to determine life history cycle and migratory movement in several populations of Texas brown shrimp (Penaeus aztecus). Hobson et al. (1994) also used this trend in offshore and inshore carbon isotopic fractionation to distinguish populations of migratory seabirds in British Colombia, Canada. Carbon isotope ratios have also been used to show dietary changes in migrating whales. Schell et al. (1989) determined that oscillations in carbon isotope ratios were due to different zooplankton in the wintering and summer areas of bowhead whale (Balaena mysticetus). Peaks in carbon oscillations were also utilized to determine dietary changes in southern right whale's (Eubalaena australis) migratory route (Best & Schell 1996). The distinct isotopic differences between freshwater and marine food webs, the latter of which is more enriched in C13 were used to identify feeding areas in seabirds, and distinguishing subpopulations in seals (Mizutani et al. 1990, Smith et al. 1996).
Nitrogen (15N/14N)
As mentioned above, nitrogen isotope ratios change when protein catabolism is taking place. This allows distinguishing migratory routes in which animals continue to feed from routes in which they do not. In bowhead whales oscillations in nitrogen isotopic ratios were used to determine that they were in fact feeding during southward migration, but fasted during northward migration (Best & Schell 1996). Likewise, juvenile sooty shearwaters (Puffinus griseus) were shown to be feeding during northward migration while adults did not (Minami & Ogi 1997). Nitrogen fractionation has also been used to distinguish trophic levels in migratory fish in fresh and marine environments (Hobson & Wassenaar 2008).
Oxygen (18O/16O)
Oxygen was the first stable isotope used to determine migratory history in marine environments. Stable isotope analyses using marine oxygen have used barnacle shells attached to the study animal. As barnacles grow, the oxygen stable isotope profiles in the calcite of their shells reflect the salinity and temperature of ambient waters. Therefore, large animals moving through parts of the ocean that differ in temperature and salinity will carry with them information on where they have been in their barnacles. This approach was first used to delineate the life history of California gray whales (Eschritchitius robustus), and was also used in combination with carbon to delineate periods of time where loggerhead turtles (Caretta caretta) spent during migration (Killingley 1980, Killingley & Lutcavage 1983).
Strontium (87Sr/86Sr)
Stable isotope analysis using strontium is effective for analyzing the origin of anadromous fish (those migrating upriver from the sea to breed in freshwater). Otoliths, a component of the inner ear bone of fish, have annual rings that reflect the isotopic profile of the water where the fish spent its growth period, as the strontium isotope ratios in stream water reflect watershed geology and are thus driven by abiotic processes (Kennedy et al. 1997). Otoliths have therefore been very useful in studying the life history of migratory fish (Hobson 1999). Kennedy et al. (1997) also used strontium stable isotope profiles of natal streams to predict the origins of Atlantic salmon (Salmo salar). This approach has since been extended to determining the natal origin of other Atlantic salmon in combination with nitrogen (Harrington et al. 1998).
Sulfur (34S/32S)
Generally, several isotopes are used to determine dietary sources in migratory species in both marine and terrestrial environments. Of these, sulfur is particularly useful, because its isotope ratios are invariant in the ocean and consistently negative in terrestrial ecosystems. Sulfur isotopic variation has been used to distinguish marine feeding from non-marine feeding populations of birds of prey (Casey & Meehna 2003). However, the majority of studies utilizing sulfur to study shifts between marine and terrestrial food sources have focused on non-migratory species. Sulfur ratios have also been used to determine whether salmon are migratory, and to distinguish hatchery from wild salmon (Weber et al. 2002, Godbout et al. 2010).
References and Recommended Reading
Alisaukas, R. T. & Hobson, K. A. The determination of lesser snow goose diets and winter distribution using stable isotope analysis. Journal of Wildlife Management 57, 49-54 (1993).
Bearhop, S. et al. A forensic approach to understanding diet and habitat use from stable isotope analysis of (avian) claw material. Functional Ecology 17, 270-275 (2003).
Best, P. B. & Schell, D. M. Stable isotopes in southern right whale (Eubalaena australis) baleen as indicators of seasonal movements, feeding and growth. Marine Biology 124, 483-494 (1996).
Casey, A. L. & Meehan, T. D. Estimating the latitudinal origins of migratory birds using hydrogen and sulphur stable isotopes in feathers: Influence of marine prey base. Oecologia 134, 505-510 (2003).
Fleming, T. H. et al. Seasonal changes in the diets of migrant and non-migrant nectarivorous bats as revealed by carbon stable isotope analysis. Oecologia 94, 72-74 (1993).
Fry, B. Natural stable isotope tag traces Texas shrimp migrations. Fishery Bulletin 79, 337-345 (1981).
Godbout, L. et al. Sulfur isotopes in otoliths allow discrimination of anadromous and non-anadromous ecotypes of salmon (Oncorhynchus nerka). Environmental Biology of Fishes 89, 521-532 (2010).
Harrington, R. R. et al. N enrichment in agricultural catchments: Field patterns and applications to tracking Atlantic Salmon (Salmo salar). Chemical Geology 147, 281-294 (1998).
Hobson, K. A. Tracing origins and migration of wildlife using stable isotopes: A review. Oecologia 120, 314-326 (1999).
Hobson, K. A. & Wassenaar, L. I. Tracking Animal Migration with Stable Isotopes. London, UK: Academic Press, 2008.
Hobson, K. A. et al. Using stable isotopes to determine seabird trophic relationships. Journal of Ecology 63, 786-798 (1994).
Karasov, W. H. & Martinez del Rio, C. Physiological Ecology: How Animals Process Energy, Nutrients, and Toxins. Princeton, NJ: Princeton University Press, 2007.
Kennedy, B. P. et al. Natural isotope marks in salmon. Nature 387, 776-767 (1997).
Killingley, J. S. Migrations of California gray whales tracked by oxygen-18 variation in their epizoic barnacles. Science 207, 759-760 (1980).
Killingley, J. S. & Lutcavage, M. Loggerhead turtle movements reconstructed from O and C profiles from commensal barnacle shells. Estuarine, Costal and Shelf Science 16, 345-349 (1983).
Marra, P. P., Hobson, K. A. & Holmes, R. T. Linking winter and summer events in a migratory bird using stable carbon isotopes. Science 282, 1884-1886 (1998).
Minami, H. & Ogi, H. Determination of the migratory dynamics of the sooty shearwater in the Pacific using stable carbon and nitrogen isotope analyses. Marine Ecology Progress Series 158, 249-246 (1997).
Mizutani, H. et al. Carbon isotope ratio of feathers reveals feeding behaviour of cormorants. The Auk 107, 400-437 (1990).
Peterson, B. J. & Fry, B. Stable isotopes in ecosystem studies. Ecology, Evolution, and Systematics 18, 293-320 (1987).
Rubenstein, D. R. & Hobson, K. A. From birds to butterflies: Animal movement patterns and stable isotopes. Trends in Ecology & Evolution 19, 256-263 (2004).
Schell, D. M. et al. Bowhead whale (Balaena mysticetus) growth and feeding as estimated by C techniques. Marine Biology 103, 433-443 (1989).
Smith, R. J. et al. Distinguishing between populations of fresh and salt-water harbor seals (Phoca vitulina) using stable-isotope ratios and fatty acids. Canadian Journal of Fisheries and Aquatic Sciences 53, 272-279 (1996).
Weber, P. K. et al. Otolith sulfur isotope method to reconstruct salmon (Oncorhynchus tshawytscha) life history. Canadian Journal of Fisheries and Aquatic Sciences 59, 587-591 (2002).
Werner, R. A. & Brand, W. A. Referencing strategies and techniques in stable isotope ratio analysis. Rapid Communications in Mass Spectrometry 15, 501-519 (2001).
West, J. B. et al. Stable isotopes as one of nature's ecological recorders. Trends in Ecology & Evolution 21, 408-414 (2006).
