Units of Measurement

The units of measurement for 15N tracer materials are aimed at expressing the relative abundance of 15N compared to 14n using different notations. There are two main units of measurement; one is commonly used with 15N-enriched materials, while the other approach is more useful when natural variations in 15N abundance are used to trace N.

5.2.2 15N-Enriched Tracers

The unit expresses the isotope abundance as a percentage of 15N

over total N

(Barraclough 1995):

It is also common to express the 15N abundance as a variation with respect to N2 natural abundance, called atom% 15N excess. The 15N/(15N + N) ratio of atmospheric N2 has been determined by Junk and Svec (1958) to be 0.003663 that is 1 15N atom every 272 14N atoms.

5.2.3 15N-Natural Abundance

The unit expresses the variation of the 15N abundance with respect to a standard abundance represented by N2 in air (Bedard-Haughn et al. 2003; Shearer and Kohl 1993). Since the natural variations of 15N abundance are low, typically 1-2% of the atmospheric N2 (Bedard-Haughn et al. 2003; Shearer and Kohl 1993), the unit used to express N natural abundance is d15N (per mil 15N excess, i.e., per mil variation from a standard):

R(standard) R has been alternatively defined as:

The first definition is more useful in source identification studies, while the second is useful for isotope discrimination. The latter is the most commonly used (Bedard-Haughn et al. 2003; Mariotti et al. 1982). At the level of natural abundance, the two definitions are practically identical, as the difference in d15N using the two definitions is much smaller than the error of measurement (Barraclough 1995; Hauck et al. 1994; Shearer and Kohl 1993).

5.3 Tracer Materials

To examine the N source or sink strength, flow rate, or fate in different natural or agricultural ecosystems, two alternative approaches can be used: either natural differences in 15N abundance or alternatively an N source with an artificially altered 15N content applied to produce a substantial difference between the tracer and the surrounding environment (Bedard-Haughn et al. 2003; Knowles and Blackburn 1993).

The natural variation of 15N abundance has been used in many studies (Kerley and Jarvis 1997), although it is limited by the fact that the well-defined natural N-pools of considerably different isotopic composition need to be identified (Gerzabek et al. 2001). Bedard-Haughn et al. (2003) indicated that on average the minimum difference among sources is about 5.9%. Isotope discrimination (Sect. 5.4.1) in the soil N cycle complicates the quantification of N turnover and dynamics by using natural abundance approach (Wagner 1991).

Materials containing unnaturally high or low concentrations of 15N are commonly used when natural differences are too low to be used to trace N, when the natural variability is high enough to cover any difference between sources and sink, or when processes need to be monitored over a prolonged period. In the latter case, a high gradient needs to be maintained over long periods despite a progressive re-equilibration due to N flows. While enriched materials enable the monitoring of many processes that otherwise could not be quantified or detected, natural abundance methods (NAMs) enable systems to be monitored without any disturbance. However, N cycle disturbance is usually not a problem in agro-ecosystems where N fertilization is common (Bedard-Haughn et al. 2003).

When applying artificial tracers, 15N-enriched or 15N-depleted tracers can be used. Depleted sources are cheaper and do not tend to contaminate laboratory equipment. Unfortunately, their tracer value is equivalent to a material containing only 0.7 atom% 15N, thus N can be traced into plants only during the year of application and can be followed into the soil nitrate and organic pools only in soils with a organic N content lower than 1.5 g N kg (Hauck et al. 1994).

Two major 15N-labelling techniques are routinely used to study N transformation in soil-plant systems (1) 15N tracer technique and (2) 15N isotope dilution technique. The first technique is based on 15N labelling of a substrate pool and subsequent monitoring of the isotope's movement through the system over time. The latter technique labels a soil N pool with 15N, and rates are monitored at which the N content of the pool changes and the 15N atom% enrichment of the pool is diluted by 14N influx (Hart and Myrold 1996).

Before beginning a new experiment, it is necessary to decide the difference in atom% needed between sources and sinks to trace N, thus the 15N abundance in the labelled material. This is possible by estimating the likely degree of tracer dilution during the process monitored. For example, with many field crops, the fertilizer derived N in the plant is diluted two- to fourfold with unlabelled N derived from soil (Powlson and Barraclough 1993). Examples of calculation methods can be found in Hauck et al. (1994) and Powlson and Barraclough (1993).

5.3.1 Samples Collection and Processing

The need to collect representative soil and plant samples is greater when conducting field studies with labelled N than with non-labelled N, since a small amount of contamination can have drastic confounding effects on the results (Hauck et al. 1994; Powlson and Barraclough 1993; Shearer and Kohl 1993).

With the application of N isotopes in agricultural studies, plant materials and soil are commonly sampled. Plant sampling usually entails collecting the total biomass of the crop, which is then divided into its constituent plant parts. Separating plants into fractions having a relatively homogeneous N content is recommended, because different 15N abundances can be found in different fractions (see isotope discrimination in Sect. 5.3.5.1). If plant parts are not mixed thoroughly, then there could be errors in the determination of atom% 15N of sampled materials. The atom% 15N of the entire plant can be calculated as a weighted average of the atom% 15N of its individual parts on the quantity of N (kg N ha-1) in the individual parts. If it is not possible to divide the plant into homogeneous tissues, then it is important to sample the heterogeneous mixture only after drying and thoroughly grinding to a particle size lower than 100 mesh (Hauck et al. 1994; Powlson and Barraclough 1993; Shearer and Kohl 1993).

During multi-season studies, special care should be taken to ensure that the sampling or harvesting operations do not cause cross-contamination among plots. Contamination can be particularly serious when large amounts of material are transported across the field (for example, maize stalks) with a relatively high 15N enrichment (Hauck et al. 1994; Powlson and Barraclough 1993).

For soil sampling, a sufficient number of cores need to be mixed to accurately estimate the plot average with a given degree of precision. Examples of number of core estimations are provided by Gomez and Gomez (1984) and Hauck et al. (1994). When exploring different soil horizons, different 15N enrichments are usually found. Soil near the surface usually has a much higher 15N enrichment than deeper soil (Powlson and Barraclough 1993).

Sampling and material processing also need to be executed with extreme care and with suitable equipment to avoid cross-contamination. The most important rules to follow in preparing samples for analysis are (a) quantitative conversion at all steps to avoid isotopic fractionation during incomplete conversion or N loss; (b) avoidance of any contamination, especially of natural abundance samples with enriched material; (c) representative homogeneous sub-samples; (d) replicate samples; and (e) inter-laboratory comparisons. When plant or soil samples need to be ground, disk- or ball-mills are easier to clean between subsequent samples and will usually achieve a finer grinding than hammer- or knife-mills (Powlson and Barraclough 1993).

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