Reply to Comment on Our Paper ''Comparison of ...

Isotope Pairing and N2:Ar Methods

Reply to Comment on Our Paper ‘‘Comparison of Isotope Pairing and N2:Ar Methods for Measuring Sediment Denitrification’’ BRADLEY D. EYRE1, SøREN RYSGAARD2, TAGE DALSGAARD2, and PETER BONDO CHRISTENSEN2 1

Centre for Coastal Biogeochemistry, Southern Cross University, P. O. Box 157, Lismore 2480, Australia 2 National Environmental Research Institute, Vejlsøvej 25, 8600 Silkeborg, Denmark

In their comment, in response to our paper in Estuaries (Eyre et al. 2002) Kana and Weiss (2004) raise five major issues that we will address individually. 1. Kana and Weiss (2004) report that we ‘‘advocated . . . using the relationship obtained between the O2 and N2 signal to correct N2:Ar ratios back to the O2 concentrations at the start of the incubation’’, and that this approach is invalid. Although we evaluated this approach, we never advocated the use of correction curves and clearly stated in our paper (2002, p. 1081), ‘‘the use of correction curves is not recommended.’’ We found that the reaction of O2 with N2 and the subsequent decrease in N2 was only partly corrected using an O2 correction curve for the relationship between N2 and O2 concentrations. The ‘‘O2 corrected’’ N2: Ar denitrification rates were lower, but still did not match the isotope pairing rates and the variability between replicates was much higher. What we did, and still strongly do, advocate is the use of a copper reduction column heated to 6008C to remove all of the O2 from the sample before MIMS analysis. The justification for this is provided in the following points. 2. Kana and Weiss (2004) report that oxygen has little effect on N2:Ar ratios measured on their MIMS and that the effect is within the normal precision of the technique when measuring replicated samples. They demonstrate this by providing a graph of the relationship between N2:Ar and O2 concentration for an experiment where oxygen was progressively removed. Figure 1 is the same N2: Ar versus O2 relationship as Kana and Weiss’ (2004) Fig. 2, but using the original data set that produced Fig. 2 in Eyre et al. (2002). Figure 1 shows that in our MIMS system oxygen has a very large effect on N2:Ar ratios, with a maximum

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change of about 8.6%, and clearly needs to be removed. Even the change over the first 20% drop in oxygen, which is what we would recommend as a maximum oxygen change in a batch core incubation, N2:Ar ratios change by around 0.5%, which is very large compared to the typical change due to net N2 fluxes from the sediment. What Kana and Weiss’ (2004) data do demonstrate is that, as stated in Eyre et al. (2002, p. 1077) ‘‘the magnitude of this oxygen effect may be instrument specific’’ and as such we recommend that all MIMS users evaluate the oxygen effect on their own instruments. 3. Kana and Weiss (2004) report an oxygen effect on Ar that is proportional to N2 and as such oxygen only has a small effect on N2:Ar ratios on their MIMS system. We analyzed Ar in the original data set that produced Fig. 2 in Eyre et al. (2002), and also found that oxygen has an effect on Ar (Fig. 2), but that the effect was the opposite to that found by Kana and Weiss (2004). Kana and Weiss (2004) found that decreasing O2 resulted in an increase in Ar. In contrast we found that decreasing O2 resulted in a decrease in Ar over most of the oxygen range. The effect of oxygen on Ar also appears to be an important factor in the effect of oxygen on N2:Ar ratios. The oxygen effect on Ar also appears to be instrument-specific. 4. Kana and Weiss (2004) consider that discrepancies in net denitrification rates measured by the N2:Ar method and the isotope-pairing ‘‘are not attributable to an oxygen artefact in the dissolved gas measurement procedure, but are due to unmeasured variables associated with sample collection and incubation that were not adequately controlled in the batch core experiments’’. We strongly disagree. There is an oxygen effect on N2:Ar ratios (see Point 2). We consider it an unlikely coincidence that in nearly every batch core incubation when oxygen is removed net N2 fluxes are less than isotope pairing denitrification rates (the difference being N-fixation as previously demonstrated using acetylene; Eyre et al. 2002), but when oxygen is not removed net N2 fluxes are much larger than isotope pairing denitrification rates. We have recently undertaken numerous more batch incubations using both the N2:Ar with O2 removal and isotope pairing simultaneously and in nearly all cases we get the same result as that reported in Eyre et al. (2002) net N2 fluxes less than the isotope pairing denitrification rates (Eyre unpublished data). 5. Kana and Weiss (2004, p. 7) state that the N2: Ar method ‘‘is a good measure of net denitrification’’. We disagree. As stated in Eyre et al. (2002), the N2:Ar method only measures the net N2 flux (i.e., the difference between N2 production by de-

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Fig. 1. Relationship between N2:Ar and O2 concentration. Data set from Fig. 2 in Eyre et al. (2002).

nitrification and N2 consumption by N-fixation). In the numerous batch incubations we have undertaken, including those reported in Eyre et al. (2002) and more recently using both the N2:Ar with O2 removal and isotope pairing simultaneously, net N2 fluxes (N2:Ar method with O2 removal), in nearly all cases, are significantly less than the isotope pairing denitrification rates (Eyre unpublished data). We are currently undertaking further work to assess if the difference is N-fixation. Conclusions As demonstrated in Eyre et al. (2002), and in this contribution, oxygen has a significant effect on the measurement of N2:Ar ratios on our MIMS and needs to be removed before analysis. Kana and Weiss’ (2004) data support our original conclusion that the magnitude of this effect may be instrument-specific; the oxygen effect on their MIMS was less than on our MIMS. We recommend that all MIMS users evaluate the oxygen effect on their own instruments. Even in the absence of a strong oxygen effect on N2:Ar ratios (e.g., Kana and Weiss 2004), we would still strongly advocate the use of a copper reduction column heated to 6008C to remove all of the O2 from the sample before MIMS analysis for the following four reasons: The removal of oxygen significantly increases the precision of the N2:Ar analysis. As reported in Eyre et al. (2002) coefficients of variation of N2:Ar in triplicate samples (n 5 42) without O2 removal ranged from 0.111% to 0.014% and averaged 0.037%, similar to the variation reported by Kana and Weiss (2004, average 0.030%). When O2 was removed coefficients of variation of N2:Ar in triplicate samples (n 5 21) ranged from 0.032% to

Fig. 2. Relationship between N2:Ar and O2 concentrations. Data set from Fig. 2 in Eyre et al. (2002).

0.003% and averaged 0.010%, a significant improvement in precision, which is important for the measurement of small net N2 fluxes. The removal of oxygen also decreases the time it takes for the signal to stabilize between samples, as the N2 signal is no longer dependent on variations in the O2 concentration that change considerably during sample change-over. This, in turn, increases the through-put of samples; a very important consideration for such a labor-intensive analytical method. The removal of oxygen allows the analysis of 29N2 and 30N2. This, in turn, allows N2:Ar and isotope pairing to be run simultaneously for little additional effort, but a large additional data gain (net N2 flux, denitrification, N-fixation). There is little disadvantage in removing oxygen from the MIMS analysis. Oxygen can be very easily measured by other equally good techniques (e.g., electrochemical probe, Winkler Titration) and the cost of adding the copper reduction column and oven is only about 1% of the MIMS set-up cost. LITERATURE CITED EYRE, B. D., S. RYSGAARD, T. DALSGAARD, AND P. B. CHRISTENSEN. 2002. Comparison of isotope pairing and N2:Ar methods for measuring sediment denitrification rates—Assumptions, modifications and implications. Estuaries 25:1077–1087. KANA, T. M. AND D. L. WEISS. 2004. Comment on ‘‘Comparison of isotope pairing and N2:Ar methods for measuring sediment denitrification’’ By B. D. Eyre, S. Rysaard, T. Dalsgaard, and P. Bondo Christensen. 2002. Estuaries 25:1077–1087.’’ Estuaries 27:173–176. Submitted, November 18, 2003 Accepted, November 18, 2003.