Published online 12 March 2007
Published in Soil Sci Soc Am J 71:355-361 (2007)
DOI: 10.2136/sssaj2006.0266
© 2007 Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
SOIL BIOLOGY & BIOCHEMISTRY
Nitrogen-15 and Oxygen-18 Natural Abundance of Potassium Chloride Extractable Soil Nitrate Using the Denitrifier Method
Luc Rock* and
Benjamin H. Ellert
Agriculture & Agri-Food Canada, Lethbridge Research Centre, 5403 1st Ave. South, P.O. Box 3000, Lethbridge, AB, T1J 4B1, Canada
* Corresponding author (lrock{at}ucalgary.ca).
 |
ABSTRACT
|
|---|
In agroecosystems, most isotopic investigations of NO3– involve the use of tracers that are artificially enriched in 15N. Although the dual isotope composition of NO3–— 
15N and 18O is especially beneficial for understanding the origin and fate of NO3–, its use for KCl-extractable soil NO3– has been hampered by the lack of a suitable analytical technique. Our objective was to test whether the denitrifier method, whereby NO3– is reduced to N2O before mass spectrometric analysis, can be used to determine the N and O isotopic composition of NO3– from 2 M KCl soil extracts. Several internationally accepted NO3–standards were dissolved in 2 M KCl, the conventional extractant for soil inorganic N, and inoculated with the bacterial strain Pseudomonas aureofaciens (ATCC no. 13985). The standard deviation of the NO3– standards analyzed did not exceed 0.2
for 15N and 0.3 for 18O values. After appropriate corrections, differences between our measured and consensus 15N and 18O values of standard NO3– generally were within the standard deviations given for the consensus values. Both 15N and 18O values were reproducible among separate analytical runs. The method was also tested on genuine 2 M KCl extracts from unfertilized and fertilized soils. Depending on N fertilization, the soils had distinct 15N and 18O values, which were attributed to amendment with NH4NO3 fertilizer. Hence, our data indicate that the denitrifier method provides a fast, reliable, precise, and accurate way of simultaneously analyzing the natural abundances of 15N and 18O in KCl-extractable soil NO3–.
|
INTRODUCTION
|
|---|
The natural abundance of 15N in NO3– is rarely used to investigate N dynamics in agroecosystem studies. Instead, most of these studies use 15N-enriched substances. Furthermore, the O isotopic signature on soil NO3– has received even less attention, probably because suitable analytical techniques are lacking.
Kohl et al. (1971) were among the first to study whether N isotopes could be used to identify the origin of aqueous N in surface water. Their study sparked insightful discussions on the pros and cons of using natural 15N abundance to study agricultural N dynamics. Taken alone, however, 15N values fail to distinguish among various NO3– sources, because the 15N values overlap and isotopic fractionation by various N transformations may further obscure the original source signature(s) (e.g., Kendall, 1998). When Amberger and Schmidt (1987) introduced the measurement of 18O values, however, a new technique became available to better differentiate between NO3– sources and understand NO3– transformations. The 18O values in conjunction with 15N values of NO3– have been used extensively and successfully in hydrological studies to address NO3– sources and transformations (e.g., Aravena et al., 1993; Wassenaar, 1995; Durka et al., 1994; Chang et al., 2002; Rock and Mayer, 2002, 2004), although rarely in soil–plant studies. Högberg (1997) noted that the combined use of 15N and 18O values may provide a powerful tool to elucidate soil N sources and transformations.
Various techniques are available to determine the N isotopic composition of NO3–. Diffusion techniques (e.g., Sebilo et al., 2004) are the most common; however, they do not enable measurement of 18O values of NO3–. To measure the N isotopic composition of NO3–, Stevens and Laughlin (1994) used an abiotic conversion of NO3– to N2O, but they focused on 15N-enriched samples, and did not assess the O isotopic composition of NO3–. Currently, the most widely used method to determine the O isotopic composition of NO3– is based on extracting aqueous NO3– using an anion exchange resin, and subsequent purification as AgNO3 (Silva et al., 2000). This method, however, is not directly applicable to highly saline solutions, such as 2 M KCl extracts from soils, as such solutions interfere with the ion exchange processes.
Recently, a new technique, the denitrifier method (Sigman et al., 2001; Casciotti et al., 2002), was developed to determine the N and O isotopic composition of NO3– in freshwater and seawater. The latter has total dissolved solids (TDS) of about 33 000 mg L–1, whereas 2 M KCl soil extracts have TDS at least five times higher. Our aim was to test whether the denitrifier method can be used to determine the N and O isotopic composition of NO3– from 2 M KCl extracts.
|
MATERIALS AND METHODS
|
|---|
The denitrifier method (Sigman et al., 2001; Casciotti et al., 2002) is based on bacterial reduction of NO3– to N2O via a bacterium that lacks nitrous oxide reductase, so further reduction to N2 does not occur. This enables simultaneous determination of both 15N and 18O values of the sample NO3– by measuring 15N and 18O values of the produced N2O. In contrast, the conventional ion exchange method may require separate mass spectrometric analyses of 15N and 18O values in purified AgNO3. A detailed description of the denitrifier method for determining the 15N value of NO3– can be found in Sigman et al. (2001), and for the 18O value of NO3– in Casciotti et al. (2002).
In brief, we used the bacterial strain Pseudomonas aureofaciens (ATCC no. 13985) to reduce NO3– to N2O. A fresh culture was prepared for each sample set by inoculating a tryptic soy broth medium (BD Diagnostics, Sparks, MD) amended with KNO3, KH2PO4, and NH4Cl in 125-mL serum bottles. After 7 d of growth, the bacteria were harvested and 2.25-mL aliquots were distributed among 20-mL headspace vials for sample analysis. Before injecting the samples, sample incubation vials containing bacteria plus medium were crimp sealed with septa caps, flushed with N2, and an antifoaming agent (Antifoam B, R06436, EMD Chemicals, Gibbstown, NJ) was added. We compared two addition times for the antifoaming agent: one during medium preparation and one just before crimp sealing the sample incubation vials. This test was done because it was noted that certain samples for which the antifoam was added during medium preparation still produced quite a bit of foam during sample analysis. This, in turn, posed a technical challenge, as liquid may enter the capillaries used to extract the N2O. After sample injection, the vials were inverted and incubated overnight at room temperature to achieve complete reduction of NO3– to N2O. Knowing the sample NO3– concentrations, the injected sample volumes were adjusted such that 50 nmol of NO3– (producing 25 nmol of N2O) would be analyzed for every sample. This is essential for using the correction procedure, which is discussed below, of the raw (idem measured) values. We were able to analyze smaller amounts (e.g., 10 nmol, data not shown), as previously demonstrated by Sigman et al. (2001). We chose 50 nmol, as this resulted in a clear and strong signal of about 6 V for Mass 44 on the isotope ratio mass spectrometer that we used. Analyses were performed in the Isotope Science Laboratory (ISL) at the University of Calgary, Calgary, AB, Canada.
The instrumentation used to analyze isotopes of N2O produced in the incubated sample vials included the following in-line components (all from Thermo Electron Co., Bremen, Germany, except where indicated): a modified PreCon trace gas concentrator, an HP6890 gas chromatograph with an HP-PLOT U column (Agilent Technologies, Palo Alto CA), a GC/C-TC interface, and a Delta Plus XL mass spectrometer. In essence, continuous flow isotope ratio mass spectrometry was used to determine the 15N and 18O values of N2O derived from reduction of NO3–. The PreCon was modified through the addition of a Nafion gas drier (Perma Pure LLC, Toms River, NJ) to ensure thorough dehumidification, two pieces of Bev-A-Line IV tubing to which stainless steel needles were attached that had different lengths, and a holder for 20-mL headspace vials in place of the 100-mL flasks normally used for the PreCon.
To test whether the denitrifier method (Sigman et al., 2001; Casciotti et al., 2002) can be used to determine the N and O isotopic composition of NO3– from 2 M KCl soil extracts, four international NO3– standards and one internal laboratory standard were dissolved in 2 M KCl. Some of these standards were used as references to correct the isotope measurements, and others were treated as samples for an independent assessment of analytical performance. Blanks, which were composed of the solvent 2 M KCl, were also analyzed during each run. In addition to the NO3– standards and blank KCl, we also analyzed 2 M KCl extracts of surface soil samples (0–15 cm) from unfertilized and fertilized plots and the applied NH4NO3 fertilizer (prills crushed and dissolved in 2 M KCl) to further assess the method. The extraction protocol was as follows: 10 g of air-dry soil and 50 mL 2 M KCl were added to 125-mL polypropylene bottles; these were placed horizontally on a reciprocating shaker (Eberbach Corp., Ann Arbor, MI) for 1 h, after which the samples were filtered using Fisherbrand Q2 filter paper.
The soil samples had been collected shortly after fertilization in May 2006 from plots under two contrasting management regimes in a field experiment with three replicates. Thus, for this study, six plots under two contrasting management regimes were sampled. Two soil cores (each 3.7-cm diameter) were combined by depth for each plot. For this study, the 0- to 15-cm depth increment was air dried and crushed to pass a 2-mm sieve. The experimental plots are part of the irrigated cropping systems study designated Rotation U and located at Agriculture & Agri-Food Canada's Lethbridge Research Centre (AAFC-LRC). Nitrate-N and NH4+–N concentrations in the soil extracts and in the dissolved fertilizer prills were determined colorimetrically via segmented continuous flow (AutoAnalyzer 3, Bran+Luebbe GmbH, Norderstedt, Germany) at AAFC-LRC.
The international standards used were IAEA-NO3, USGS-34, USGS-35, and USGS-32. The internal laboratory standard was ISL-KNO3. The standards were used to prepare solutions of 2 M KCl containing NO3– at a concentration ([NO3–]) of 3.07 mg L–1 for each standard, except USGS-35 for which [NO3–] was 3.65 mg L–1. The given 15N and 18O values of the NO3– standards used are listed in Table 1, which is based in part on data obtained from IAEA (2004). The values are defined as follows:
()=
where R is the 15N/14N or 18O/16O ratio of the sample or a standard. The 15N values are reported relative to air N2, and 18O values relative to Vienna Standard Mean Ocean Water.
During each analysis, the N2O produced from the various samples was compared with laboratory N2O reference gas introduced via the bellows of the dual inlet connected to the Delta Plus XL. The absolute 15N and 18O values of the laboratory N2O reference gas are immaterial, because, as will be discussed below, some of the international standards were used as absolute references to correct the other analyses.
|
RESULTS AND DISCUSSION
|
|---|
First, results obtained from two distinct bacterial batches (bat1 and bat2) are presented for analysis of NO3– standards. Note that bat2 was subdivided into two sets (bat2a and bat2b) to compare the two addition times for the antifoaming agent. The agent was added during medium preparation for bat2a, and just before the vials were crimp sealed for bat2b. Comparisons among the three batches enabled assessment of the precision, accuracy, and reproducibility of the denitrifier method for 15N and 18O values of NO3– in 2 M KCl. Second, results obtained for analysis of 2 M KCl-extractable NO3– in fertilized and unfertilized soils and of the added fertilizer are discussed.
The continuous flow isotope ratio mass spectrometry system used to extract and analyze the N2O produced from reduction of sample NO3– by the bacterial strain Pseudomonas aureofaciens (ATCC no. 13985) provided excellent chromatographic resolution of the N2O (data not shown). Samples were analyzed individually, and each analysis took about 17 min. Hence, with our current system using manual sample changing, 22 samples (including instrument checks) can be analyzed in a typical 8-h workday. Manual changing of samples contained in septum-capped 20-mL headspace vials involved inserting first a long inlet needle that was submerged in the liquid, and second a short outlet needle that remained in the headspace above the liquid (and foam). Integrating an autosampler would significantly increase sample throughput.
Precision
The precision of the denitrifier method was assessed with bat1. This batch included three blanks (2 M KCl), three samples of IAEA-NO3 and USGS-34, five samples of USGS-35 and ISL-KNO3, and six samples of USGS-32. Note that the raw (or measured) 15N and 18O values given below have already been corrected for mass interference due to the contributions of 17O to Masses 45 and 46 using the same ISODAT 2.0 software as used for instrument control, data capture, and signal processing.
Nitrogen-15 Natural Abundance Values
The raw 15N values were 4.5 ± 0.1 (n = 3) for IAEA-NO3, –1.8 ± 0.1 (n = 3) for USGS-34, 3.8 ± 0.1 (n = 5) for USGS-35, 175.3 ± 0.2 (n = 6) for USGS-32, and 4.5 ± 0.1 (n = 5) for ISL-KNO3 (Table 2). For blank KCl, the mass 44 peak areas were very small ( < 200 mV), and amounted to 
2.3% of the overall average mass 44 peak areas recorded for the NO3– standards (Table 2). The analytical precision for 15N values of NO3– in 2 M KCl determined by the denitrifier technique is excellent, as the standard deviation (
) of the NO3– standards analyzed did not exceed 0.2.
View this table:
[in this window]
[in a new window]
|
Table 2. Averages and corresponding standard deviations of amplitude of Mass 44, area of Mass 44, and raw 15N and 18O values of NO3– standards prepared with bacterial batch bat1.
|
|
Oxygen-18 Natural Abundance Values
The measured raw 18O values were 64.7 ± 0.2 (n = 3) for IAEA-NO3, 12.8 ± 0.2 (n = 3) for USGS-34, 96.4 ± 0.1 (n = 5) for USGS-35, 64.9 ± 0.3 (n = 6) for USGS-32, and 61.5 ± 0.2 (n = 5) for ISL-KNO3 (Table 2). Based on the results obtained, it can be concluded that the precision for 18O values of NO3– from 2 M KCl extracts determined by the denitrifier technique is excellent, as the of the samples analyzed did not exceed 0.3.
Accuracy
The accuracy of the denitrifier method was assessed with bat1 and bat2. The latter included the same sample set as bat1, except that only one sample of each standard was prepared with bat2a and bat2b. To obtain the "true" 15N and 18O values of the sample NO3– based on the raw (or measured) 15N and 18O values of the analyzed N2O, different corrections need to be applied to the 15N and 18O values.
Nitrogen-15 Natural Abundance Values
The only correction needed for the 15N values is a blank correction, as the conversion of NO3– to N2O is complete after an overnight incubation (Sigman et al., 2001). The IAEA-NO3 was used as the absolute reference for 15N analysis. Hence, to correct the raw 15N values, first the 15N value of the blank was estimated based on the given and measured 15N values of IAEA-NO3, and measured areas of Mass 44 with the following equation:
 | [1] |
where AM44 = area of Mass 44.
Then to calculate the blank-corrected 15N value for a specific sample, the following equation was used:
 | [2] |
where meas = raw (or measured) values for the sample.
The corrected and given or consensus 15N values of USGS-34, USGS-32, and ISL-KNO3 were basically identical, except for USGS-35 (Table 3). The difference between the corrected and given 15N values (
15N) of the three former standards was within the standard deviation of the given 15N values (Table 3). For both bat1 and bat2, 15N was ±0.1 for USGS-34 with a given of 0.2, –0.6 to –0.9 for USGS-32 with a given of 1.0, and 0.0 to 0.2 for ISL-KNO3. The only exception was USGS-35, for which the 15N was consistently between 1.1 to 1.3 relative to its given 15N value, which has a of 0.2. This difference between the given and corrected 15N values of USGS-35 was also observed when we used deionized water as a solvent (data not shown). The USGS-35 sample, an atmospherically derived NO3– salt, has a significant mass-independent 17O anomaly (Böhlke et al., 2003). Sigman et al. (2001) noted that the 15N values of atmospheric NO3– may be overestimated by 1 to 2 with the denitrifier method if it is assumed that there is no mass-independent 17O anomaly. This explains the discrepancy observed for the 15N of USGS-35 relative to the other NO3– standards, as our software assumed a mass-dependent relationship to correct Mass 45 for 17O contribution. Coplen et al. (2004) discussed in detail the determination of 15N values of NO3– containing mass-independent 17O, such as atmospherically derived NO3–. Otherwise, accuracies were consistently good and similar for bat2a and bat2b: the denitrifier method provided accurate 15N values of NO3– derived from 2 M KCl extracts.
Oxygen-18 Natural Abundance Values
In addition to a blank correction like that applied to the 15N values, the raw 18O values require further corrections (Sigman et al., 2001; Casciotti et al., 2002). These are due to: (i) isotopic fractionation during reduction of NO3– to N2O, as only one of every six O atoms within the sample NO3– is preserved within the produced N2O, and (ii) exchange of O atoms between sample water and intermediate N compounds produced during NO3– reduction to N2O (Sigman et al., 2001; Casciotti et al., 2002). The ATCC no. 13985 strain of Pseudomonas aureofaciens was selected to reduce NO3– to N2O because it results in the lowest O exchange between the intermediate N compounds and sample water (Casciotti et al., 2002).
To correct the measured 18O values, two approaches may be taken, as described by Casciotti et al. (2002). One approach involves the use of one absolute reference, namely IAEA-NO3, whereas the other approach involves the use of two absolute references. In the former, "exchange" and "blank" are determined separately and involve the analysis of additional samples produced with several distinct 18O-enriched waters. The latter, however, enables the determination of a correction factor including all the factors influencing the analysis of the 18O values. Although Casciotti et al. (2002) felt the second approach might be better, they used the first approach because two NO3– standards with well-characterized 18O values were not yet available. We opted to use the second approach, and chose IAEA-NO3 and USGS-34 as the two absolute references. To calculate the correction factor the following equation was used, which was derived from an equation in Casciotti et al. (2002):
 | [3] |
To use Eq. [3], one has to ensure that the same amount of N2O is produced from each sample, as this was the assumption used when this equation was derived (Casciotti et al., 2002). Another point to note is that the correction factor varies among bacterial batches (observed both for NO3– dissolved in deionized water [data not shown] or 2 M KCl). The correction factors calculated for the three bacterial batches (idem analytical runs) used in this study, bat1, bat2a, and bat2b, were 1.030954, 1.004506, and 1.029916, respectively. Thus this correction requires that IAEA-NO3 and USGS-34 standards are included in every sample set or run. This concurs with Casciotti et al. (2002), who noted that each batch of bacteria behaves slightly differently.
To calculate the corrected 18O value for a specific sample, the following equation was used, which is based on Eq. [4] in Casciotti et al. (2002):
 | [4] |
The corrected and given 18O values of USGS-35, and USGS-32 were nearly identical, but those for ISL-KNO3 differed (Table 3). The difference between the corrected and given 18O values (18O) for the first two standards generally was within the of the given 18O values (Table 3). For both bat1 and bat2, 18O was –0.1 to 0.7 for USGS-35 with a given of 0.6, and –0.2 to 0.1 for USGS-32 with a given of 0.4. The only exception was ISL-KNO3, for which the 18O was consistently between 2.6 to 3.2 relative to the given 18O value with = 1.2. Note that this difference between the given and corrected 18O values of ISL-KNO3 was also observed when deionized water was used as the solvent (data not shown). As for the 15N values, no difference in the accuracy was observed between bat2a and bat2b. Hence, based on the results obtained, the denitrifier method provides accurate 18O values of NO3– derived from 2 M KCl extracts.
Reproducibility
The reproducibility of the denitrifier method was assessed by comparing the corrected 15N and 18O values obtained from bat1 and bat2. As can be seen from Table 3, both the 15N and 18O values were reproducible between different runs for which distinct batches of the bacterial strain Pseudomonas aureofaciens (ATCC no.13985) were used. Furthermore, adding the antifoam agent either during medium preparation or just before the vials were crimp sealed did not affect the results, as no difference was observed in the corrected values obtained from bat2a and bat2b.
Example: Comparison of Fertilized and Unfertilized Soils
After testing the denitrifier technique on NO3– standards (with given 15N and 18O values) dissolved in 2 M KCl, we used 2 M KCl to extract NO3– from soil samples collected from the 0- to 15-cm layer under three replicate plots of two contrasting treatments: unfertilized and NH4NO3 fertilized (100 kg N ha–1) silage corn (Table 4). In addition, three samples of blank 2 M KCl used for the extraction and three samples of the NH4NO3 fertilizer applied to the soil were analyzed. Included within the sample run were also the international standards IAEA-NO3 and USGS-34 to correct the raw values obtained, and USGS-35 to assess the accuracy–precision–reproducibility of the run. The international standards were dissolved in 2 M KCl, and then treated the same way as the soil extracts during sample preparation.
View this table:
[in this window]
[in a new window]
|
Table 4. Concentrations of NH4+–N and NO3––N, volume added to 20-mL headspace analysis vial, area of Mass 44, and corrected 15N and 18O values of NO3– for the unfertilized soil, fertilized soil, applied fertilizer, blank (or 2 M KCl solution), and international NO3– standards.
|
|
The average NO3––N concentration was 6.9 ± 0.1 mg L–1 (n = 3) for the fertilizer, 8.2 ± 1.5 mg L–1 (n = 3) for the fertilized soil extracts, and 2.3 ± 0.5 mg L–1 (n = 3) for the unfertilized soil extracts (Table 4). The average NH4+–N concentration was 8.4 ± 0.1 mg L–1 (n = 3) for the fertilizer, 2.8 ± 1.0 mg L–1 (n = 3) for the fertilized soil extracts, and 0.7 ± 0.0 mg L–1 (n = 3) for the unfertilized soil extracts (Table 4).
Based on the NO3– concentrations, the sample or extract volume added to the 20-mL headspace vials was adjusted to obtain 50 nmol of NO3–, as discussed above. In turn, the sample volume added ranged from 0.08 to 0.41 mL (Table 4), which represented 3.5 to 18.2% of the bacterial medium added to each vial. Considering the very small volumes of soil extract and fertilizer solution added to each sample incubation vial, there was good agreement among the average Mass 44 peak areas for these samples as well as those for the analyzed international NO3– standards (Table 4). The blank 2 M KCl solution (after dispensing, shaking, filtering, etc.) used for extraction had an average Mass 44 peak area of 0.369 ± 0.025 Vs (n = 3). This represented <1.5% of the average area Mass 44 measured for all the soil extracts (Table 4). Hence, any trace of NO3– present within the 2 M KCl solution used for the extraction is negligible, and did not affect the isotopic measurements.
The average 15N and 18O values were 3.3 ± 0.1 (n = 3) and 25.4 ± 0.3 (n = 3), respectively, for the applied fertilizer; –0.1 ± 1.3 (n = 3) and 11.0 ± 2.2 (n = 3), respectively, for the fertilized soil; and 6.4 ± 0.6 (n = 3) and –6.2 ± 0.4 (n = 3), respectively, for the unfertilized soil (Table 4). The USGS-35 standard had average 15N and 18O values of 3.7 and 57.3 (n = 2, Table 4), respectively, which agree with the values shown in Table 3. The 15N and 18O values of NO3– determined for the applied fertilizer and the unfertilized and fertilized soil extracts are consistent with published 15N and 18O values for synthetic fertilizers and NO3– derived from soil organic N (Fig. 1
, after Mengis et al. [2001] and references therein). The range given in Fig. 1 for NO3– derived from soil organic N is based on the analysis of soil water samples. Note that Mayer et al. (2004) determined 15N values of synthetic NO3– fertilizer used in Alberta that were as high as 3.2, and Kendall (1998) mentioned that the 18O values of NO3– derived from soil nitrification may be as low as –10. On a 18O vs. 15N plot, the data for each group of samples (fertilizer, fertilized soil, and unfertilized soil) form distinct clusters (Fig. 1). A mean comparison using the all-pairs Tukey–Kramer test between the three groups indicates that all three groups are characterized by significantly different 15N and 18O values of NO3– at the 0.05 significance level (Table 4). The greater variability observed in the 15N and 18O values for the fertilized soil compared with the unfertilized soil was also observed in the NO3––N and NH4+–N concentrations (Table 4). This may be attributed to spatial variability in the distribution of the fertilizer, which had been applied only 2 d before soil sampling.
Interpretations of the 15N and 18O values of the KCl-extractable soil NO3– may be compromised by the presence of soil microbes or other soil constituents that were potentially extracted together with the NO3–. For instance, denitrifiers are abundant within the soil microbial community, and thus there might be enough N2O reductase to reduce some of the NO3––derived N2O to N2. This could increase 15N and 18O values in the residual N2O and thus lead to errors in estimating the isotopic signatures of the sample NO3–. On closer observation, however, potentially extracted soil microorganisms did not appear to appreciably affect the determination of the 15N and 18O values of KCl-extractable soil NO3– using the denitrifier method. First, the volume of soil extract solution added to an analysis vial was much lower than the bacterial medium solution within the vial. Second, the Mass 44 peak areas for the soil extracts were nearly identical to those recorded for the dissolved standards. This suggests that the entire amount of NO3– from each soil extract (50 nmol) was converted to N2O, and was not further reduced to N2. If the latter had occurred, then Mass 44 peak areas for the soil extracts should have been smaller than those for the international NO3– standards. Third, the 15N and 18O values obtained are consistent with published data (Fig. 1). Fourth, excellent reproducibility was observed among the values for replicate plots, considering inherent variability among soil properties and microbial processes in the field (e.g., Parkin, 1993). Fifth, a clear difference in the N and O isotopic composition of NO3– in unfertilized and fertilized soils was observed. The only major difference between the treatments was the addition or omission of synthetic fertilizer.
Another potential negative effect on the determination of the isotopic composition of NO3– using the denitrifier method might be the presence of NH4+ within a sample and its interaction with the bacterial medium. A test was made in which the international standard IAEA-N1, an ammonium sulfate compound, was analyzed as an individual sample and in a mixture with IAEA-NO3 (data not shown). The IAEA-N1 did not yield a signal, and the 15N and 18O values of the mixture were what would be expected for IAEA-NO3 alone. Thus, the presence of NH4+ in a sample does not compromise the performance of the denitrifier method for NO3–.
Ideally the 15N and 18O values obtained for the soil extracts and fertilizer would have been compared with data obtained using an independent method. Unfortunately, we are unaware of an alternate method for reliably estimating the 18O values of KCl-extractable soil NO3–. The denitrifier method has, however, been compared with the AgNO3 method using a set of shallow groundwater samples, whose water type ranged from fresh to brackish, from the same study site (unpublished data, 2006). The data from that study indicated that both methods yielded similar results for the 15N and 18O values of groundwater NO3–.
Our results provide an example of how synthetic fertilizer might influence the N and O isotopic composition of KCl-extractable soil NO3–. Addition or omission of the NH4NO3 fertilizer was the main treatment difference. The shift toward lower 15N and higher 18O values in the fertilized compared to the unfertilized soil can be attributed to fertilization. The data from the fertilized soil fell within the range of values expected for NO3– derived from NH4+ and NO3– in NH4NO3. In addition to nitrification of fertilizer NH4+, various soil microbial processes may also have contributed to the observed changes in the 15N and 18O values, but the overriding contributor simply was addition of the fertilizer itself. A detailed analysis of processes, such as mineralization–immobilization turnover (Jansson and Persson, 1982), may become important in the longer term but was beyond the scope of this study.
Finally, this short example confirms that the denitrifier technique can be used to simultaneously determine both the 15N and 18O values of soil NO3– extracted by 2 M KCl, and can potentially provide essential information on soil N dynamics.
|
Conclusions
|
|---|
Our aim was to test whether the denitrifier method can be applied to 2 M KCl soil extracts to determine the 15N and 18O values of NO3–. For NO3– standards, the standard deviations of the means for replicate analyses did not exceed 0.2 for 15N or 0.3 for 18O values. After appropriate corrections, differences between the measured and given 15N and 18O values of the NO3– standards generally were within the standard deviations for the assigned values. Both 15N and 18O values were reproducible among contrasting analytical runs. Furthermore, the denitrifier method was successfully applied to NO3– in genuine 2 M KCl extracts from fertilized and unfertilized soils. Both soils were characterized by distinct 15N and 18O values that reflected the addition of the inorganic fertilizer. Hence, the data from this study indicate that the denitrifier method provides a fast, reliable, precise, and accurate way of simultaneously analyzing the natural abundances of 15N and 18O in 2 M KCl-extractable soil NO3–.
|
ACKNOWLEDGMENTS
|
|---|
We are very grateful to: S. Wankel and S. Silva from the USGS at Menlo Park for helpful discussions and for providing a starting culture of Pseudomonas aureofaciens; S. Taylor from the Isotope Science Laboratory at the University of Calgary, B. Buziak and A. Grigoryan from the Department of Biological Sciences at the University of Calgary; J. Erickson, L. Kremenik, and E. Nakonechny from Agriculture and Agri-Food Canada's Lethbridge Research Centre for technical help; and B. Mayer at the University of Calgary for access to the isotope science laboratory. We acknowledge the support of R.L. Desjardins and funding from the Panel for Energy Research and Development via the program on Enhancement of Greenhouse Gas Sinks in Agroecosystems for a postdoctoral fellowship to L. Rock.
Received for publication July 26, 2006.
|
REFERENCES
|
|---|
- Amberger, A., and H.L. Schmidt. 1987. Natürliche Isotopen Gehalte von Nitrat als Indikatoren für dessen Herkunft. Geochim. Cosmochim. Acta 51:2699–2705.[CrossRef][Web of Science]
- Aravena, R., M.L. Evans, and J.A. Cherry. 1993. Stable isotopes of oxygen and nitrogen in source identification of nitrate from septic systems. Ground Water 31:180–186.[CrossRef][Web of Science]
- Böhlke, J.K., S.J. Mroczkowski, and T.B. Coplen. 2003. Oxygen isotopes in nitrate: New reference materials for 18O:17O:16O measurements and observations on nitrate–water equilibration. Rapid Commun. Mass Spectrom. 17:1835–1846.[CrossRef][Web of Science][Medline]
- Casciotti, K.L., D.M. Sigman, M. Galanter Hastings, J.K. Böhlke, and A. Hilkert. 2002. Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method. Anal. Chem. 74:4905–4912.[Medline]
- Chang, C.C.Y., C. Kendall, S.R. Silva, W.A. Battaglin, and D.H. Campbell. 2002. Nitrate stable isotopes: Tools for determining nitrate sources among different land uses in the Mississippi River Basin. Can. J. Fish. Aquat. Sci. 59:1874–1885.
- Coplen, T.B., J.K. Böhlke, and K.L. Casciotti. 2004. Using dual-bacterial denitrification to improve 15N determinations of nitrates containing mass-independent 17O. Rapid Commun. Mass Spectrom. 18:245–250.[CrossRef][Web of Science][Medline]
- Durka, W., E.D. Schulze, and G. Gebauer. 1994. Effects of forest decline on uptake and leaching of deposited nitrate determined from N-15 and O-18 measurements. Nature 372:765–767.
- Högberg, P. 1997. Tansley review no. 95: 15N natural abundance in soil–plant systems. New Phytol. 137:179–203.
- IAEA. 2004. IAEA analytical quality control services reference materials catalogue 2004–2005. IAEA, Vienna, Austria.
- Jansson, S.L., and J. Persson. 1982. Mineralization and immobilization of soil nitrogen. p. 229–252. In F.J. Stevenson (ed.) Nitrogen in agricultural soils. Agron. Monogr. 22. ASA, Madison, WI.
- Kendall, C. 1998. Tracing nitrogen sources and cycling in catchments. p. 519–576. In C. Kendall and J.J. McDonnell (ed.) Isotope tracers in catchment hydrology. Elsevier, Amsterdam.
- Kohl, D.A., G.B. Shearer, and B. Commoner. 1971. Fertilizer nitrogen: Contribution to nitrate in surface water in a Corn Belt watershed. Science 174:1331–1334.[Abstract/Free Full Text]
- Mayer, B., L. Rock, and C. Ryan. 2004. Assessment of the fate of fertilizer nitrogen using isotope techniques. AARI project no. 2001J493. Alberta Agric. Res. Inst., Edmonton.
- Mengis, M., U. Walther, S.M. Bernasconi, and B. Wehrli. 2001. Limitations of using 18O for the source identification of nitrate in agricultural soils. Environ. Sci. Technol. 35:1840–1844.
- Parkin, T.B. 1993. Spatial variability of microbial processes in soil—A review. J. Environ. Qual. 22:409–417.
- Rock, L., and B. Mayer. 2002. Isotopic assessment of sources and processes affecting sulfate and nitrate in surface water and groundwater of Luxembourg. Isot. Environ. Health Stud. 38:191–206.
- Rock, L., and B. Mayer. 2004. Isotopic assessment of sources of surface water nitrate within the Oldman River Basin, Southern Alberta, Canada. Water Air Soil Pollut. Focus 4:545–562.
- Sebilo, M., B. Mayer, M. Grably, D. Billiou, and A. Mariotti. 2004. The use of the ‘ammonium diffusion’ method for 15N–NH4+ and 15N–NO3– measurements: Comparison with other techniques. Environ. Chem. 1:99–103.
- Sigman, D.M., K.L. Casciotti, M. Andreani, C. Barford, M. Galanter, and J.K. Böhlke. 2001. A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. Anal. Chem. 73:4145–4153.[Medline]
- Silva, S.R., C. Kendall, D.H. Wilkison, A.C. Ziegler, C.C.Y. Chang, and R.J. Avanzino. 2000. A new method for collection of nitrate from fresh water and the analysis of nitrogen and oxygen isotope ratios. J. Hydrol. 228:22–36.
- Stevens, R.J., and R.J. Laughlin. 1994. Determining nitrogen-15 in nitrite or nitrate by producing nitrous oxide. Soil Sci. Soc. Am. J. 58:1108–1116.
- Wassenaar, L.I. 1995. Evaluation of the origin and fate of nitrate in the Abbotsford Aquifer using the isotopes of 15N and 18O in NO3–. Appl. Geochem. 10:391–405.
TOP
ABSTRACT
INTRODUCTION
