Two phenomena usually concern isotope flows into the environment and potentially affect the goodness of rate estimates: isotope discrimination and added N interaction.
Isotope discrimination (also called isotope fractionation) consists in a differential reaction rate between 15N and 14N in biochemical or physical processes, with biotic processes generally showing a higher variability of intensity (Bedard-Haughn et al.
2003; Shearer and Kohl 1993). Usually, 14N transformation is faster, leading to an 15N enrichment of the reagents or original forms. Of all the consequences, the natural variability of 15N abundance is a valuable example of the isotope discrimination effect. Usually, isotope discrimination is not a problem when using 15N enriched materials because the variation is small compared to the difference between sources. Instead, isotope discrimination should be considered when using natural abundance, since the isotope shift is likely to be of the same order of magnitude as the difference between N sources (Barraclough 1995; Hauck et al. 1976).
Isotope fractionation can be caused by both the equilibrium isotope effect and kinetic isotope effect. The equilibrium isotope effect occurs when the transformation rates determining the equilibrium are affected by isotopic composition. They can make a considerable contribution to variations in 15N abundance in components of the N cycle. One very important example is the equilibrium between NH3 and NH4+, where N abundance in NH3 results approximately 20% lesser than in NH4+. As a consequence, ammonia volatilization from terrestrial and aquatic ecosystems enriches residual NH4+ and may contribute substantially to the general elevation of 15N in soil compared to atmospheric N2.
The kinetic isotope effect occurs when differences in the reaction rates of molecules bearing different isotopes result in differences in 15N abundance between substrates and products. Since reactions are linked, some are not reversible, some are influenced by secondary substrates, enzymes and cofactors, and more than one reaction can compete for the same substrate, the isotope effect of a process in certain conditions may vary according to environmental conditions and be expressed only partially. The overall isotope effect of a certain process is commonly known as the "Overall Observed Isotope Effect" of the entire reaction (bobs). In the soil, the combined effects of the kinetic isotopic fractionation of NH4+ nitrification and NO3- denitrification can lead to an increase in the 15N abundance of soil organic N. In fact, nitrification leads to NH4+ enrichment (NO3- has a lower isotopic ratio than the NH4+ from which it derives); denitrification leads to a NO3-enrichment (more 14N is lost) (Bedard-Haughn et al. 2003; Shearer and Kohl 1993). Plants usually reflect the variability of soil N, although other factors such as genotype and mycorrhizal associations also influence plant 15N (Bedard-Haughn et al. 2003). Microbial processes can usually alter the isotope N composition (Macko and Estep 1984).
The isotopic technique can also lead to an added nitrogen interaction (ANI). ANI consists in an under- or overestimation of the transformation rate of added 15N into the monitored sink. It is caused by processes that lead to a substitution of the original 14N by added 15N in different pools from the monitored sink. Such substitution results in an 15N dilution due to a greater amount of 14N made available (Jenkinson et al. 1985; Powlson and Barraclough 1993). Since a lower amount of labelled N is actually available than the amount distributed, a lower transformation rate is estimated than actually occurs. ANI can be caused by processes whose rate is not determined by N availability, but by other factors. Examples are immobilization and, sometimes, denitrification. If the extent of the process is not determined by the N availability, the amount of N involved in the process is constant and will be distributed between the labelled and unlabelled N on the basis of their relative abundance. If the rate is determined by N availability, the amount of N involved in the process will be a fraction of the total available N (for example in leaching). Consequently, the amount of N subtracted will be a constant fraction of each labelled and unlabelled pool, and no variation in the relative abundance of labelled and unlabelled fertilizers will occur (Giusquiani et al. 1994; Jenkinson et al. 1985; Powlson and Barraclough 1993).
An example of ANI in the inorganic N pool of soil consists in the difference between the quantity of unlabelled inorganic nitrogen remaining in soil in fertilized and unfertilized situations. Assuming that a fixed quantity of N is immobilized, the labelled inorganic fertilizer N takes the place of the unlabelled inorganic N that would have been otherwise immobilized, thus increasing the proportion of unlabelled inorganic N remaining available in the soil for plant uptake. If the plant uptake of applied fertilizer is quantified by an 15N technique, an underestimation will occur due to the greater uptake of unlabelled N. The consequence of labelled N is that its quantity retained in the total plant plus soil system will remain unaltered, while the relative amount in either plants or soil will change (Powlson and Barraclough 1993).
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