1. Stable Isotopes Isotopes are atoms of the same atomic weight but different atomic mass. Most elements of biological importance (C, H, O, N, and S) have two or more stable isotopes - with the lightest in much greater abundance. Most of the heavy isotopes are in abundance of 1% or less. Stable isotope geochemistry focuses on the variations of the isotopic compositions of light elements arising from chemical fractionations rather than nuclear processes. The most commonly studied stable isotopes are H, Li, B, C, N, O, Si, S, and Cl.

2. 12 C - 12 amu - 98.89% 13 C - 13 amu- 1.11%

3. These elements have several common characteristics: 1)They have low atomic mass; 2)The relative mass difference between the isotopes is large; 3)They form bonds with a high degree of covalent character; 4)They exist in more than one oxidation state, form a wide variety of compounds; 5)The abundance of the rare isotope is sufficiently high (at least tenths of a percent) to facilitate analysis. Terrestrial Abundance of Stable Isotopes: ElementIsotopeAbundance % Hydrogen 1 H99.985 2 H0.015 Carbon 12 C98.89 13 C1.11 Nitrogen 14 N99.63 15 N0.37 Oxygen 16 O99.759 17 O0.037 18 O0.204 Sulfur 32 S95.00 33 S0.76 34 S4.22 36 S0.014

4. Measurement Notation: Results from environmental and agricultural studies using isotopically enriched tracers report in units of atom percent (At %). This value gives the absolute number of atoms of a given isotope in 100 atoms of total element: At% 15 N =  15 N/ 14 N + 15 N  (100 At%) At% 13 C = ( 13 C/ 12 C + 13 C + 14 C) (100 At%) Note: For the At% 13 C calculation of the amount of naturally present 14 C is usually treated as negligible and the sum of 12 C and 13 C is taken to be total C.

5. The  Notation: Studies examining stable isotopes at or near natural abundance levels are usually reported as delta, a value in parts per thousand or per mil (% o ) to make into larger numbers.

6. Delta values are not absolute isotope abundance but differences between sample readings and one of another of the widely used natural abundance standards (air for Nitrogen, Pee Dee Belemnite for C). Pee Dee Belemnite (Cretaceous marine fossil, Belemnitella americana from South Carolina) has a higher 13 C/ 12 C ratio than nearly all other carbon based substances for convenience it is assigned a delta 13 C value of zero giving almost all other naturally occurring samples negative delta values. PDB now used up so NBS-21 graphite carbon has replaced it. Absolute ratios (R) are measured for sample and standard, and the relative measure delta is calculated:

7. For example, if a leaf sample is found to have a 15 N/ 14 N ratio R greater than the standard’s by 5 parts per thousand, this value is reported as delta 15 N = 5 delta % o Isotopic Fractionation: Isotopic fractionation can originate from both kinetic (evaporation, diffusion, and dissociation reactions) and equilibrium (energy of a molecule such a vibration motion) effects. (Temperature-dependent equilibrium isotope fractionations arise from quantum mechanical effect in vibrational motions). Kinetic effects might intuitively be expected since lighter isotopes will diffuse faster than heavier ones. 16 O will react about 15% faster than 18 O in oxygen molecules.

8. Oxygen Isotopes and Paleoclimate Change: 16 O will evaporate more quickly than 18 O: water vapor above ocean is typically  18 O of around -13 per mil. Temp. effects on fractionation of isotopes (Urey, 1941). Heavier isotope is enriched in the evaporation process. Thus, atmospheric water vapor is depleted in heavy isotopes relative to the sea water from which is evaporates

9. Interglacial Scenario High sea-level, little ice at the poles, and relatively little storage of light oxygen isotope in the ice caps leads to relatively depleted heavy oxygen containing seawater. Forams living in the ocean will fractionate this high 18 O proportion in their carbonate shells. Clouds contain high proportion of light isotope because of its higher vapor pressure

10. Glacial Scenario Sea-level lowered (ca. 120 m), more ice at poles. Polar ice stores more of the light isotope and sea water will contain higher proportions of the heavy isotope. This proportion will be mirrored by forams that live here and fractionate oxygen into their carbonate shells. Clouds contain high proportion of the light because of it’s higher vapor pressure

11. Fractionation in the Biosphere: Autotrophs and heterotrophs can fractionate isotopes. The most important process producing isotopic fractionation of carbon is photosynthesis. Early work by Park and Epstein (1960) - several steps. Stomatal conductance correlated with  13 C of plant species - indicating that diffusion is an important process.

13. Aquatic : 4.4 % o difference between diffusion coefficients ( 12 C is faster) - so a -4.4 % o fractionation is expected. Marine algae and aquatic plants can utilize either dissolved CO 2 of HCO 3 for photosynthesis. Atmospheric CO 2 at about -7 per mil At equilibrium fractionation of +9 per mil is associated with dissolution (+7 to +12 depending on temp.) occurs during hydration and dissolution of CO 2. C 3 and C 4 pathways: Most plants use enzyme called ribulose bisphosphate carboxylase (RuBP) to catalyze reactions whereby RuBP reacts with one molecule of CO 2 and water to produce 2 molecules of 3-phosphoglycerate (carboxylation).

14. Such plants are called C 3 plants this process is called the Calvin-Benson Cycle. C 3 plants constitute about 90% of all plants including alage, autotrophic bacteria, cultivated plants (wheat, rice etc.). Kinetic fractionation associated with carboxylation is about -- -29.4 % o in higher terrestrial plants. Bacterial carboxylation is about -20 % o. C 4 or Hatch-Slack Cycle : Plants use phosphoenol pyruvate carboxylase (PEP) to initially fix CO 2 and form oxalacetate - compound with 4 carbons. A fractionation of about -2 to -2.5 % o occurs here. C 4 plants have an average  13 C of -13 % o. Occur in dry climates (grasses, marshgrass, corn, sugarcane) -efficient use of water.

15. C 4 photosynthesis: high WUE, low photorespiration, high ATP cost, low leaf N, hot climate, high light conditions. A third group of plants, the CAM plants, have a unique metabolism called the Crassulacean acid metabolism. They generally use the C 4 pathway but can use the C 3 under certain conditions. Arid plants (pineapple and many cacti). CAM: day- stomates close, C 4 acids decarboxylated, fixed by normal C 3 pathway (RuBp); night stomates open, CO 2 fixed by PEP carboxylase, C 4 acids stored in vacuoles C 3 plants: Higher plants  13 C of avg. -27 % o ; algae and lichens  13 C -12 to 23 % o. C 4 plants have an average  13 C of -13 % o CAM:  13 C of -14 % o

16. Fractionation in surface and deep oceanic waters

19. Short-term Carbon Cycle and Anthropogenic Inputs  13 C of fossil fuel has varied from -24 % o in 1850 to -27.3 % o in 1980 as coal has been replaced by oil and gas. Thus, we might expect to see a decrease in the  13 C of atmopheric CO 2. Based on measurements in tree rings and ice cores, the  13 C of atmospheric CO 2 has declined by about 1.5 since 1800.  13 C in Antarctic Ice Core 1700-1850 range from -6.2 to -6.6 % o 1900-1950 range from -6.7 to -6.9 % o Recent air samples from South pole : 1980-2000 range from -7.4 to -7.8 % o

20. There is a -19 equilibrium fractionation in the conversion of NH 3 to NH 4. NH 3 is generally the form used, most natural waters have NH 4 as the dominant form. Ammonium assimilation and fractionation by two principle reactions (Fogel and Cifuentes, 1993) : 1) Formation of glutamate from alpha-ketoglutarate via glutamate dehydrogenase and; 2) formation of glutamine from glutamate via the enzyme glutamine synthetase A positive fractionation (enrichment 15 N) occurs for both reactions (about 2-4 % o ) - this is common in reversible reactions because the heavy N is concentrated in the molecule with the strongest bond (glutamate).

21. Passive Diffusion Model when enzyme or diffusion limited (Fogel and Cifuentes, 1993):  = E q + D + (C i /C o )(E enz + D) where, E q is the equilibrium fractionation between NH3 and NH4; D is the fractionation between associated with diffusion in and out of the cell, C i /C o is the ratio of concentration of ammoinia inside to outside the cell, and E enz is the fractionation associated with enzymatic fixation of by either glutamine synthetase or glutamate dehydrogenase.

22. Nitrogen Isotopes: Forms of inorganic nitrogen: N 2, NO 3, NO 2, NH 3 and NH 4. Equilibrium and kinetic occur between these five forms of N. Ammonia is dominant form utilized by plants. N 2 can be taken up through N fixation (reduction) in some plants (legumes) and cyanobacteria. NO 3 and NO 2 can be utilized as well, in these cases N must be reduced by the action of reductase enzymes.  15 N fractionations of 0 to -24 % o have been measured for assimilation of NO 3. Fractionation of 0 to -20 % o has been measured for assimilation of NH 4. Fractionations of -3 to +1 % o have been measured for fixation of N 2.

23. Nitrogen Isotopes in Organic Matter Atmospheric =  15 N 0 % o Non-fixing plankton or macroalgae:  15 N -3 to + 18 % o Terrestrial sources:  15 N -6 to + 18 % o Cyanobacteria: -2 to +4 % o and N-fixing terrestrial plants -6 to +6 % o