Challenges in measuring nitrogen isotope signatures in inorganic nitrogen forms: An interlaboratory comparison of three common measurement approaches

Abstract

Rationale: Stable isotope approaches are increasingly applied to better understand the cycling of inorganic nitrogen (N_i ) forms, key limiting nutrients in terrestrial and aquatic ecosystems. A systematic comparison of the accuracy and precision of the most commonly used methods to analyze δ¹⁵ N in NO₃ ^- and NH₄ ⁺ and interlaboratory comparison tests to evaluate the comparability of isotope results between laboratories are, however, still lacking.

Methods: Here, we conducted an interlaboratory comparison involving 10 European laboratories to compare different methods and laboratory performance to measure δ¹⁵ N in NO₃ ^- and NH₄ ⁺ . The approaches tested were (a) microdiffusion (MD), (b) chemical conversion (CM), which transforms N_i to either N₂ O (CM-N₂ O) or N₂ (CM-N₂ ), and (c) the denitrifier (DN) methods.

Results: The study showed that standards in their single forms were reasonably replicated by the different methods and laboratories, with laboratories applying CM-N₂ O performing superior for both NO₃ ^- and NH₄ ⁺ , followed by DN. Laboratories using MD significantly underestimated the "true" values due to incomplete recovery and also those using CM-N₂ showed issues with isotope fractionation. Most methods and laboratories underestimated the at%¹⁵ N of N_i of labeled standards in their single forms, but relative errors were within maximal 6% deviation from the real value and therefore acceptable. The results showed further that MD is strongly biased by nonspecificity. The results of the environmental samples were generally highly variable, with standard deviations (SD) of up to ± 8.4‰ for NO₃ ^- and ± 32.9‰ for NH₄ ⁺ ; SDs within laboratories were found to be considerably lower (on average 3.1‰). The variability could not be connected to any single factor but next to errors due to blank contamination, isotope normalization, and fractionation, and also matrix effects and analytical errors have to be considered.

Conclusions: The inconsistency among all methods and laboratories raises concern about reported δ¹⁵ N values particularly from environmental samples.

FIGURE 1

A heatmap illustrating the accuracy, precision, and specificity of the methods used by variable laboratories in this interlaboratory comparison to analyze δ¹⁵N in inorganic nitrogen (N) forms. Methods tested were the chemical method transforming inorganic N forms to N₂O (CM‐N₂O), the chemical method transforming inorganic N forms to N₂ (CM‐N₂), the microdiffusion (MD) method, and the bacterial denitrifier (DN) method. Results are presented for natural abundance standards of NO₃ ⁻ (NA‐SN) and NH₄ ⁺ (NA‐SA) and for labeled standards of NO₃ ⁻ (LA‐SN) and NH₄ ⁺ (LA‐SA). Color codes were normalized to the maximum value found for each category, where the dark green color (closer to 0) indicates highest accuracy, precision, and lowest nonspecificity (demonstrating better performance of the method utilized by one lab) and the red color (closer to 1) indicates lowest accuracy, precision, and lowest nonspecificity. Yellow colors indicate intermediate values. Accuracy is given as the difference of δ¹⁵N value of NA standard from true δ¹⁵N value as ∆δ¹⁵N [n] and as at% offset for LA standards. Data are mean and median values and standard deviation (SD) including lower (25%) and upper (75%) quartile ranges. Precision is given as the mean SD (± relative SD; in ‰ for NA and in at% ¹⁵N for LA) of results received from each laboratory (intralaboratory precision). Nonspecificity was calculated assuming that a conversion of nontarget N species would result in 100% contamination (more details in the main text). The ∆δ¹⁵N results of S1, S2, and S3 (NA) and S4 and S5 (LA), which were characterized by different isotope signatures (NA) or isotopic enrichment (LA), respectively, were pooled. Asterisks indicate significant differences at P < 0.05 [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

The δ¹⁵N results from isotopic analysis of environmental samples by different laboratories (A, δ¹⁵N of NO₃ ⁻; B, δ¹⁵N of NH₄ ⁺). The solid line indicates the average of all the results, plus minus standard deviation (±SD; shaded area). Red arrows indicate results from laboratories showing high precision and selectivity when analyzing standards, and the dotted line indicates the SD when calculating the average from those results. P1S, P2S, and HWS are soil extracts, and DR and DL are river samples (for more information, refer to the text and Table S3 [supporting information]). Black circles (L1–L3) = results from CM‐N₂O; semi‐filled diamonds (L4–L5) = results from CM‐N₂; gray circles (L6–L9) = results from MD; crossed squares (L10–L13) = results from DN. For abbreviations of the methods, see the legend of Figure 1. Note that not all laboratories participating in this intercomparison analyzed ¹⁵N from environmental samples (but only from standards, e.g., L3, L12) [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Method recommendation for samples with high and low nitrogen content and for natural abundance (NA) and labeled samples for analysis of δ¹⁵N of inorganic nitrogen forms (NO₃ ⁻, NH₄ ⁺). Best‐practice guidelines (standard operating procedures and optimization procedures) are included. Note that all methods require proper calibration and normalization procedures. Note also that for labeled samples, it is recommended to use labeled standards and to include “dummies” to avoid memory effects. The check mark and green color mean “recommended,” the cross and red color mean “not suitable,” and the exclamation mark and yellowish color mean “not recommended, only if.” For more details, read the information in the boxes [Color figure can be viewed at wileyonlinelibrary.com]