the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 10 Jul 2025
In situ production of hybrid N2O in dust-rich Antarctic ice
Abstract. Nitrous oxide (N2O) is a potent greenhouse gas involved in the destruction of stratospheric ozone. Past atmospheric mixing ratios of N2O are archived in ice cores; however, the presence of in situ N2O production in dust-rich Antarctic ice complicates their accurate reconstruction, especially during glacial periods. This production occurs in extremely cold ice and without sunlight. This study aims to understand the reaction producing N2O in Antarctic ice by identifying the precursors and the reaction pathway. We compared the oxygen and nitrogen bulk and position-specific isotope composition of in situ N2O in ice cores to the isotopic composition of nitrate (NO3-), a possible precursor of N2O. The 15N signature of NO3- is fully transferred into the central N atom (Nα) of in situ N2O, but it is not transferred into the terminal N atom (Nβ), resulting in a 50 % transfer of the 15N signature of NO3- into the bulk 15N isotopic composition. These findings suggest that the in situ N2O production involves two different nitrogen precursors present in ice: the central N atom (Nα) originates from NO3- and the terminal N atom (Nβ) from a different precursor not yet identified. Oxygen isotope analysis shows that NO3- cannot be the only reservoir for the O atom of in situ N2O. Temperature, pH, and absence of sunlight in Antarctic ice point to an abiotic N-nitrosation reaction. The limiting factor of the reaction is probably associated with mineral dust and might be Fe2+, reducing NO3- to NO2- or the precursor of the Nβ atom. The site preference (SP) values of in situ N2O are highly variable between different ice cores and depend on the bulk 15N isotopic composition of N2O, itself depending on the 15N isotopic composition of the NO3- precursor. This finding is unexpected because SP is usually determined by the production pathway through symmetric reaction intermediates that mix the N atoms in α and β positions and average out their isotopic difference. In contrast, our results provide the first evidence of a hybrid N2O production pathway involving an asymmetric intermediate that preserves the distinct 15N signatures of two different precursors – one contributing to the Nα atom and the other to the Nβ atom. This finding has important implications: in this pathway, SP reflects the isotopic difference between the two precursors rather than the pathway itself, challenging how SP is commonly interpreted in environmental studies.
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CC1: 'Comment on egusphere-2025-3108', Reinhard Well, 16 Jul 2025
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AC3: 'Reply on CC1', Lison Soussaintjean, 23 Dec 2025
The paper supplies very interesting data on N2O isotopic values in antarctic ice cores, congratulation. Authors interpret the isotopic fingerprint consisting of d18O , d15Na, d15Nb, SP and d15Nbulk in conjunction with d15N and d18O of NO3. They concluded that there must have been abiotic N2O formation in dust rich sections by a process yielding hybrid N2O, ie were N2O molecules carry N from different precursors in the alpha and beta N position.
Although the previous assumption of biotic production of N2O in ice cores is considered less probable according to the introduction, authors might test whether biotic reduction of N2O to N2 could also explain isotopic patterns (see eg (Yu et al., 2020)), since high SP and d18O values observed in the ice cores (up to or even higher than 100 per mil) have also been found previously from microbial N2O reduction, e.g in groundwater. But a quick look at Fig 3 and Fig. 6d suggests that the data could probably not be explained by microbial N2O reduction, since high SP values were associated with lower d18O and vice versa. In contrast, microbial N2O reduction to N2 would lead to parallel increase in d18O and SP of residual N2O.
Using the measured data authors could evaluate whether biotic reduction can be excluded as a relevant pathway, which would further support their assumption that biotic processes were not relevant. From my view one possible way to address this could be easily done by adding a figure plotting SP against d18O and compare the distribution of the data points with the typical N2O reduction line (see eg, Yu et al 2020). Absence of N2O reduction would not be a strict proof for absence of microbial N2O production, but in terrestrial and aquatic ecosystems, N2O production by bacterial denitrification is almost always associated with N2O reduction to a certain extent.
Moreover, in view of the high relevance of N2O consumption in the stratosphere in in terrestrial and aquatic systems, it might be adequate to address N2O consumption processes in the paper, which has not been done as far as I can see. Thus, even without showing a SP/d18O plot, it might be useful to explain why consumption processes in the ice cores are not probable.
Thank you for this very helpful suggestion. We have now evaluated whether microbial N2O reduction could provide an alternative explanation for the observed isotopic patterns. As you recommended, we generated a dual isotope plot of SP versus δ18O for in situ N2O (Fig. 8, see Supplement file). The data do not follow the characteristic reduction line (slope ≈ 0.36; Yu et al., 2020); instead, SP increases while δ18O decreases, which is opposite to the pattern produced by N2O reduction. We additionally note, as highlighted by Referee #1 (Dominika Lewicka-Szczebak), that the δ15N(NO3-)-δ15Nα(N2Oin situ) relationship exhibits a slope of ~1, whereas N2O reduction would preferentially enrich the α position in 15N and produce a steeper slope. These two lines of evidence show that N2O consumption is not occurring in the ice cores we studied. We have added a paragraph to the manuscript to clarify this point:
“To assess whether microbial N2O reduction to N2 could provide an alternative explanation for the observed isotopic patterns of in situ N2O – such as high SP values correlated with δ15Nbulk or high δ18O values – we examined the relationship between SP and δ18O in our samples (Fig. 8, see Supplement file). Previous studies showed that N2O reduction produces a characteristic increase in both SP and δ18O in the residual N2O, with data falling along a “reduction line” defined by the ratio of the fractionation factors (median slope ≈ 0.36; Lewicka‐Szczebak et al., 2015; Yu et al., 2020). In contrast, in both the Vostok and Taylor Glacier ice cores, SP increases while δ18O decreases, and the data clearly do not fall along the expected reduction line (Fig. 8, see Supplement file). This dual isotope plot for in situ N2O is therefore incompatible with N2O reduction. We note that published reduction lines were derived from studies conducted at warmer temperatures than those in ice cores (Yu et al., 2020). Very low temperatures could modify the fractionation factors; however, colder conditions would be expected to increase the fractionation for both SP and δ18O, still resulting in a positive slope. The mismatch between our data and the reduction line therefore remains regardless of potential temperature effects. A second line of evidence comes from the correlation between δ15N(NO3-) and δ15Nα(N2Oin situ), which shows a slope of ~1. Because N2O reduction preferentially breaks N-O bonds of light isotopes, the residual N2O becomes enriched in 15N at the α position, resulting in a steeper slope than observed here. Taken together, these observations show that microbial N2O reduction cannot explain the isotopic signatures in the ice cores, and that N2O consumption does not occur in the ice.”
References
Lewicka‐Szczebak, D., Well, R., Bol, R., Gregory, A. S., Matthews, G. P., Misselbrook, T., Whalley, W. R., and Cardenas, L. M.: Isotope fractionation factors controlling isotopocule signatures of soil‐emitted N2 O produced by denitrification processes of various rates, Rapid Comm Mass Spectrometry, 29, 269–282, https://doi.org/10.1002/rcm.7102, 2015.
Yu, L., Harris, E., Lewicka‐Szczebak, D., Barthel, M., Blomberg, M. R. A., Harris, S. J., Johnson, M. S., Lehmann, M. F., Liisberg, J., Müller, C., Ostrom, N. E., Six, J., Toyoda, S., Yoshida, N., and Mohn, J.: What can we learn from N2 O isotope data? – Analytics, processes and modelling, Rapid Comm Mass Spectrometry, 34, e8858, https://doi.org/10.1002/rcm.8858, 2020.
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AC3: 'Reply on CC1', Lison Soussaintjean, 23 Dec 2025
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RC1: 'Comment on egusphere-2025-3108', Dominika Lewicka-Szczebak, 05 Sep 2025
Dear authors, this is a very important and interesting study and I’ve read your manuscript with high interest. It is written precisely and clearly with very well-prepared figures. All the data are easily to follow, and your interpretations are clear. You present a very interesting new theory on the asymmetric intermediate in hybrid N2O production, which is well documented with your data and very well presented in your Figure 8.
I have only one major concern about your data, because you have calculated the in-situ N2O isotopic signatures from very low N2O increase in concentration, as low as 20ppb. From my experience such low increases are not possible to be precisely determined for their isotopic signatures because of accumulation of all the uncertainties in the calculations, associated with isotopic signature of the background N2O and analytical errors. While analysing carefully all your data presented for me it is clear, that these lowest N2O samples may be biased, which I point out in below specific comments. This makes your interpretations less robust. I think if you disregard these lowest N2O samples (20ppb) you would get much clearer and reliable picture (see in the comments below).
L 74 (δ15Nα) (δ15Nβ) alpha and beta should be in uppercase, not lowercase
L133-135 – what are the products of the NO3 photolysis, can N2O be also produced in this process? Can the gaseous products be trapped in snow layers? Or is NO2- one of the photolysis products? NO2- could be then the precursor for further chemodenitrification.
L221 add basic details on the instruments used for N2O preparation and isotope measurements, also for SP analysis L227 and for NO3 analysis L305
L310 Fig.3 You calculated and showed isotope values for N2O in situ as low as 20 ppb. These values are probably very unsure due to very small difference between atmospheric background N2O concentration and measured N2O concentration. Taking into account all the uncertainties associated with background N2O values (which are major, as you discuss a lot in the manuscript, which assumptions should be accepted for these!) and measurement errors, these values are associated with large uncertainties. For soil N2O emissions we accept at least 65ppb increase in N2O concentration to calculate the isotopic signatures for in-situ production (for lower increases the calculated propagated error was too large).
(e.g. Buchen et al.,2018, https://doi.org/10.1002/rcm.8132)Please check such calculations of error propagation for your data and decide which minimal N2O in situ makes sense in your case. I expect not lower than 50 ppb. It is also visible on your graph 3B that the lowest in situ N2O concentrations are showing most extreme unrealistic isotope values.
L344 “N2O analyzed in the extracted air” or the calculated in situ N2O isotopic signature? – Fig. 4 shows “N2O in situ” – ok – but be precise in distinguishing these terms in the text
L375, Fig 6b and 6d clearly supports my assumption that for low in-situ N2O you get biased results, your SP values below 0 for very low N2O amount of 20ppm are just calculation artefacts
L385-387 SP values are independent of bulk 15N for microbial N2O production processes, but such relation is typical for N2O reduction where preferentially N-O bonds of light isotopes are broken which results in enrichment in bulk 15N and on alpha position (=increase in SP). The reported slope (Fig. 6d) is within the typical slopes observed for N2O reduction (see Yu et al., 2020, Lewicka-Szczebak et al., 2020).
L391, Fig. 7a – OK – this Figure clearly indicates that N2O reduction plays no role, because due the partial N2O reduction the alpha position would be strongly affected and the slope would be much higher than 1. But I think it would be worth to add a short discussion on N2O reduction in the manuscript, you may indicate that you disregarded this process based on this strong relationship, Fig 7a.
L395 ‘This correlation is robust when excluding the TG data.’ – because TG data are calculated for very low N2O concentration and are simply biased. I believe they should be excluded from any further interpretations.
L430 The d18O values are not really explained, for EDML exchange with water could explain the observed low d18O-N2O values, but for Taylor Glacier the very high d18O-N2O values, largely exceeding the d18O-NO3 values are not plausible – again in my opinion these values are biased and should be excluded from any further interpretations.
L460, Table2: I do not see the point of referring here all the isotope effects for the processes which you do not deal here with (single precursor N2O) after previous review papers, I would only give the data for hybrid N2O.
L465 you mean Table 2 here, I think your description of values for the different pathways in text is enough with respective citations, as suggested above I would shorten the table 2 only to values directly found in your study
L467-468 – a citation for this theory of the common intermediate and the mechanism of N2O formation is missing
L470 – as above – a citation for denitrification N2O formation mechanism is missing
L492 – citation needed
L505 – very good and helpful Figure, your explanation and visualisation of the idea of asymmetric intermediate in hybrid N2O production is very convincing
L542 – I agree that pH probably has the large impact on the extend of O-exchange, do you have data of pH for your samples? If we deal with very high pH it could have completely blocked the O-exchange and would support your theory. The lack of pH values is an important drawback, should be added if available.
Otherwise, without the known differences in pH, I do not believe that the mechanism could be so different to give totally opposite effects, from strongly negative to extremely positive. The story of O-exchange between nitrite and water is very convincing and the reported values are very plausible. However, the very high values in TK are suspicious, even with maximal branching effect they are a bit too high. Importantly, these values are based on 3 points calculated with in-situ N2O production of 20ppb, which is in my opinion to low to calculate representative and true isotope values. I strongly encourage you to critically evaluate this data. I would reduce the calculated in-situ N2O to the higher production only and present and further discuss only the proper values, not associated with possibly large errors. This would vastly improve your manuscript because you wouldn’t need to find theories for the values which are very unsure.
Citation: https://doi.org/10.5194/egusphere-2025-3108-RC1 -
AC1: 'Reply on RC1', Lison Soussaintjean, 23 Dec 2025
Dear authors, this is a very important and interesting study and I’ve read your manuscript with high interest. It is written precisely and clearly with very well-prepared figures. All the data are easily to follow, and your interpretations are clear. You present a very interesting new theory on the asymmetric intermediate in hybrid N2O production, which is well documented with your data and very well presented in your Figure 8.
We sincerely thank you Dominika Lewicka-Szczebak for the careful reading of our manuscript and for the constructive and detailed comments. We appreciate the positive evaluation of the data presentation and the interpretation, as well as the interest expressed in the proposed asymmetric intermediate for hybrid N2O formation. In the following, we address the major concern regarding low in situ N2O concentrations and respond to the individual comments point by point.
I have only one major concern about your data, because you have calculated the in-situ N2O isotopic signatures from very low N2O increase in concentration, as low as 20ppb. From my experience such low increases are not possible to be precisely determined for their isotopic signatures because of accumulation of all the uncertainties in the calculations, associated with isotopic signature of the background N2O and analytical errors. While analysing carefully all your data presented for me it is clear, that these lowest N2O samples may be biased, which I point out in below specific comments. This makes your interpretations less robust. I think if you disregard these lowest N2O samples (20ppb) you would get much clearer and reliable picture (see in the comments below).
We fully agree that the uncertainties for the Taylor Glacier samples with low in situ N2O production (~20 ppb) are large. Still, we want to mention that all sources of uncertainty, including those related to atmospheric N2O estimates and analytical precision, are already incorporated in the error estimates through a Monte Carlo simulation for error propagation.
It is important to note that although in situ N2O production in ice cores is generally small in absolute concentration, it can lead to significant deviations in the measured N2O isotopic composition because the precursor nitrate can be highly fractionated. The latter is especially pronounced for low accumulation sites such as Dome C considered in this study. As even small amounts of in situ production can significantly alter the measured N2O isotopic composition, a characterization of such production is essential for ice core studies. For this reason, mapping the isotopic signatures of in situ N2O across different ice cores is also a key objective of our work. For Taylor Glacier, the only affected samples exhibit low in situ production, so excluding them would prevent us from characterizing this site. As mentioned above, the uncertainties for Talos Dome ice are fully included in the analysis through stringent error propagation.
As a result, the calculated isotopic composition carry large uncertainties, but the general isotopic patterns (high δ18O and low SP) are already apparent directly in the measured N2O data, thus even before applying a mass balance approach. Although we cannot yet fully explain these patterns, we believe it is important to present all available data (together with transparent uncertainty estimates) because they contribute to the broader picture of in situ N2O production in Antarctic ice.
Changes in the text based on the responses to the line-by-line comments:
L331: “The mass balance approach used to calculate the isotopic signature of in situ N2O leads to substantial uncertainties due to error propagation from atmospheric N2O estimates and analytical precision. These uncertainties are not shown in Fig. 3 for readability but are reported in Figs. 4 – 7 and are explicitly taken into account in the comparisons between δ15N(N2Oin situ) and δ15N(NO3-), including the regression analyses. As expected, the uncertainty increases for low in situ N2O concentrations (see Eq. 4).
To avoid too large uncertainties, we excluded samples with calculated in situ N2O concentrations below 20 ppb. This threshold was chosen because a large part of the dataset exhibit in situ concentrations below 50 ppb; applying a higher cutoff would remove a substantial number of samples and significantly limit our ability to investigate in situ N2O production processes. For samples with in situ N2O concentrations close to 20 ppb, the propagated uncertainties are large (up to several tens of per mil). Nevertheless, these samples were included in this study because in situ N2O production in ice, although small in absolute concentration, can significantly alter the measured N2O isotopic composition (Fig. 3a). Characterizing the isotopic signatures of in situ N2O across different ice cores is therefore a key objective of this study. Keeping a large number of data points across multiple ice cores, even with uncertainties of a few tens of per mil, provides valuable information on the potential production mechanisms. For Taylor Glacier, in particular, all affected samples exhibit low in situ N2O concentrations (~20 ppb), so excluding them would prevent us from characterizing this site.”
L 74 (δ15Nα) (δ15Nβ) alpha and beta should be in uppercase, not lowercase
We have changed this as proposed.
L133-135 – what are the products of the NO3 photolysis, can N2O be also produced in this process? Can the gaseous products be trapped in snow layers? Or is NO2- one of the photolysis products? NO2- could be then the precursor for further chemodenitrification.
Thank you for this comment. Nitrate photolysis in snow produces mainly NO2(g) and NO2-, with NO2- accounting for roughly 10% of the photolysis products (Warneck and Wurzinger, 1988; Meusinger et al., 2014). Photochemically driven reactions at the snow surface can, in principle, lead to N2O formation (e.g., hybrid N2O from reactions involving NO3- and NH4+) (Rubasinghege et al., 2011). However, such processes can only occur at the surface of the ice sheet where sunlight penetrates the snow, and where air is not yet enclosed in bubbles, i.e., where air exchange with the atmosphere still takes place. As a result, any N2O produced by photolysis-driven reactions in this zone would be released to the atmosphere and not preserved in the ice core.
Regarding the role of NO2- as a precursor for in situ N2O, this is a plausible pathway in principle. However, our isotopic results do not support a contribution from photolysis-derived NO2-. After NO3- deposition, photolysis enriches the remaining NO3- in 15N, while the produced NO2- is depleted in 15N, with its isotopic composition depending on the fraction of NO3- photolyzed. In that case, the δ15N signature of in situ N2O would depend on both the initial δ15N(NO3-) and the extent of photolysis, and would therefore not be directly proportional to the δ15N values of NO3- archived in the ice.
In contrast, our data show a direct proportionality between δ15N(NO3-) and δ15N(N2Oin situ), which suggests that the precursor of in situ N2O forms deeper in the ice from archived NO3- that already carries its final, post-photolysis isotopic δ15N signature, rather than from NO2- produced in the near-surface snowpack.
Changes in the text:
(L420): “In the case of in situ N2O, the central nitrogen atom (Nα) may indeed originate from NO2- after reduction of NO3-. However, it is unlikely that this NO2- derives directly from NO3- photolysis in the near-surface snowpack. NO3- photolysis produces both gaseous NO2 and NO2- ion, with NO2- accounting for ~10% the photolysis products (Warneck and Wurzinger, 1988; Meusinger et al., 2014). Photolysis occurs at the snow surface only, where sunlight penetrates and where air exchange with the atmosphere is still active. Any N2O produced through photolysis-driven pathways in this zone would therefore be largely released to the atmosphere and not preserved in the ice core record. In addition, photolysis enriches the remaining NO3- in 15N while producing NO2- that is depleted in 15N, with its isotopic composition depending on the extent of photolysis. If photolysis-derived NO2- were a precursor of in situ N2O, the δ15Nα signature of in situ N2O would reflect both the initial δ15N(NO3-) and the extent of photolysis, and would not be directly proportional to the δ15N values of NO3- archived in the ice. Instead, our data show a strong proportionality between δ15N(NO3-) and δ15N(N2Oin situ), indicating that the NO2- precursor must form deeper in the ice from archived NO3- that already carries its final, post-photolysis isotopic signature, rather than from photolysis-derived NO2- produced in the surface snowpack.”
L221 add basic details on the instruments used for N2O preparation and isotope measurements, also for SP analysis L227 and for NO3 analysis L305.
Changes in the text:
For N2O bulk analysis:
“Briefly, the air was extracted by melting the ice core samples with infrared light in a glass vessel under high vacuum. Water vapor was removed from the air sample with a cold trap, CO2 was removed using AscariteTM, and N2O, CH4, and other trace gases were separated from the bulk air components (N2, O2, and Ar) using a cold trap filled with activated carbon. N2O and CH4 were then separated on a gas chromatography column, and N2O was analyzed with an IsoPrime isotope ratio mass spectrometer (IRMS).”
For SP analysis:
“Briefly, the air was extracted by grating the ice core samples at -60°C to open the enclosed air bubbles (Bauska et al., 2016), i.e., a so-called dry extraction device without melting the ice. N2O was purified and pre-concentrated using a series of progressively smaller-volume cold traps and gas chromatography separation of the trapped N2O from residual CO2. Isotopic measurements were performed using a Thermo Delta V Plus IRMS, where m/z 44, 45, and 46 and m/z 30 and 31 were monitored simultaneously for N2O isotopes and N2O fragment (NO) isotopes, respectively.”
For NO3- analysis:
“Briefly, the NO3- concentration was measured by ion chromatography using a ThermoFischer Scientific IRMS system; NO3- in the samples was preconcentrated using an AG 1-X8 anion exchange resin in the chloride form. After the sample was drained and the NO3- ions were quantitatively trapped onto the resin, NO3- was eluted from the resin with 6 mL of 1M NaCl solution in three portions of 2 mL. NO3- was then converted to N2O through bacterial denitrification, using a strain of Pseudomonas aureofaciens. The bacteria were injected in 2 mL aliquots into 20 mL headspace vials. To remove air and dissolved N2O, the vials were purged for 3 h with pure helium. The concentrated NO3- samples were added to the vials in volumes adjusted to obtain 100 nmol of NO3-, and were allowed to denitrify overnight. For isotope analysis, a continuous He flow was used to transfer the produced N2O from the headspace vial and carry it through a purification line. The N2O sample was passed through columns of perchlorate, Ascarite, and Supelco Purge Trap type F to remove water, CO2, and VOCs, respectively. Following this purification step, the purified N2O was decomposed to N2 and O2 on a gold catalyst kept at 850 °C, and the isotopic compositions of the obtained N2 and O2 were measured with a Thermo Fischer MAT 253 IRMS.”
L310 Fig.3 You calculated and showed isotope values for N2O in situ as low as 20 ppb. These values are probably very unsure due to very small difference between atmospheric background N2O concentration and measured N2O concentration. Taking into account all the uncertainties associated with background N2O values (which are major, as you discuss a lot in the manuscript, which assumptions should be accepted for these!) and measurement errors, these values are associated with large uncertainties. For soil N2O emissions we accept at least 65ppb increase in N2O concentration to calculate the isotopic signatures for in-situ production (for lower increases the calculated propagated error was too large).
(e.g. Buchen et al.,2018, https://doi.org/10.1002/rcm.8132)Please check such calculations of error propagation for your data and decide which minimal N2O in situ makes sense in your case. I expect not lower than 50 ppb. It is also visible on your graph 3B that the lowest in situ N2O concentrations are showing most extreme unrealistic isotope values.
Thank you for this thoughtful comment. We agree, and show in our manuscript, that the lowest in situ N2O concentrations carry larger uncertainties. As noted in the manuscript (L263), we already quantified these uncertainties using a Monte Carlo simulation for error propagation. While we did not show the uncertainties in Fig. 3 for readability, they are shown in Figs. 4-7. They are also incorporated into all regression analyses comparing in situ N2O with NO3-, including calculation of the slope and its uncertainty, using the regression method by York et al. (2004). We will clarify this in the revised text.
In our study, the aim is not to determine highly precise isotopic signatures for individual samples with low in situ production, but rather to capture a broad range of environmental conditions and nitrate isotopic compositions to evaluate the resulting patterns in in situ N2O production. For this purpose, keeping a large number of data points across multiple ice cores, even with uncertainties of a few tens of per mil, provides valuable information on the potential production mechanisms. For our analysis, 20 ppb was therefore chosen as a threshold, which is lower than for typical analyses of modern N2O isotope studies, because many samples in our dataset fall below 50 ppb. Using a higher cutoff would therefore remove a substantial part of the data and limit our ability to investigate the production processes. This does result in larger uncertainties for the small enhancements, but as mentioned above we do propagate all these uncertainties into our final results.
L344 “N2O analyzed in the extracted air” or the calculated in situ N2O isotopic signature? – Fig. 4 shows “N2O in situ” – ok – but be precise in distinguishing these terms in the text
Thank you for pointing out the ambiguity. The original wording was indeed confusing. We have revised the text to clearly distinguish between the N2O measured in the extracted air and the calculated in situ N2O isotopic signature.
Changes in the text:
“In this section, we compare the measured isotopic composition of NO3- and the calculated isotopic composition of in situ N2O to test our hypothesis that NO3- is a precursor for in-situ produced N2O. The analyses come from the same ice samples: N2O was measured in the extracted air and subsequently used to calculate the in situ N2O isotopic signature, while NO3- was measured in the meltwater collected after air extraction.”
L375, Fig 6b and 6d clearly supports my assumption that for low in-situ N2O you get biased results, your SP values below 0 for very low N2O amount of 20ppm are just calculation artefacts
Thank you for this comment. We understand the concern regarding the larger uncertainties at low in situ N2O concentrations. However, we respectfully disagree that the low SP values in the Taylor Glacier samples are calculation artefacts. Although the uncertainties are indeed substantial – and we explicitly quantify them – the SP values for Taylor Glacier remain below zero within these uncertainties.
As a simple sanity check, which demonstrates that this is a real signal, low SP values are also visible directly in the measured (total) SP data (Fig. 6c), independent of any mass-balance calculation. While Vostok samples affected by in situ production show total SP values higher than the atmospheric signature, Taylor Glacier samples show the opposite pattern, with total SP values lower than the atmospheric signature. This strongly suggests that the in situ N2O at Taylor Glacier has indeed low SP values.
For this reason, we believe it is valuable to keep these data, as they highlight a difference between the two ice core sites and point to different isotopic signatures of the N2O precursors at Vostok and Taylor Glacier.
L385-387 SP values are independent of bulk 15N for microbial N2O production processes, but such relation is typical for N2O reduction where preferentially N-O bonds of light isotopes are broken which results in enrichment in bulk 15N and on alpha position (=increase in SP). The reported slope (Fig. 6d) is within the typical slopes observed for N2O reduction (see Yu et al., 2020, Lewicka-Szczebak et al., 2020).
L391, Fig. 7a – OK – this Figure clearly indicates that N2O reduction plays no role, because due the partial N2O reduction the alpha position would be strongly affected and the slope would be much higher than 1. But I think it would be worth to add a short discussion on N2O reduction in the manuscript, you may indicate that you disregarded this process based on this strong relationship, Fig 7a.
(Comments on L385-387 and L391) Thank you for this very helpful comment. We agree that N2O reduction should be addressed explicitly, and we have added a short discussion in the revised manuscript. This new section incorporates your argument based on the δ15Nα(N2Oin situ)-δ15N(NO3-) relationship (Fig. 7a), as well as Reinhard Well’s comment regarding the SP-δ18O relationship. These two lines of evidence both show that our data are inconsistent with N2O reduction. We also added the suggested SP-δ18O plot to support this conclusion. The revised paragraph is provided below.
“To assess whether microbial N2O reduction to N2 could provide an alternative explanation for the observed isotopic patterns of in situ N2O – such as high SP values correlated with δ15Nbulk or high δ18O values – we examined the relationship between SP and δ18O in our samples (Fig. 8, see Supplement). Previous studies showed that N2O reduction produces a characteristic increase in both SP and δ18O in the residual N2O, with data falling along a “reduction line” defined by the ratio of the fractionation factors (median slope ≈ 0.36; Lewicka‐Szczebak et al., 2015; Yu et al., 2020). In contrast, in both the Vostok and Taylor Glacier ice cores, SP increases while δ18O decreases, and the data clearly do not fall along the expected reduction line (Fig. 8). This dual isotope plot for in situ N2O is therefore incompatible with N2O reduction. We note that published reduction lines were derived from studies conducted at warmer temperatures than those in ice cores (Yu et al., 2020). Very low temperatures could modify the fractionation factors; however, colder conditions would be expected to increase the fractionation for both SP and δ18O, still resulting in a positive slope. The mismatch between our data and the reduction line therefore remains regardless of potential temperature effects. A second line of evidence comes from the correlation between δ15N(NO3-) and δ15Nα(N2Oin situ), which shows a slope of ~1. Because N2O reduction preferentially breaks N-O bonds of light isotopes, the residual N2O becomes enriched in 15N at the α position, resulting in a steeper slope than observed here. Taken together, these observations show that microbial N2O reduction cannot explain the isotopic signatures in the ice cores, and that N2O consumption does not occur in the ice.”
L395 ‘This correlation is robust when excluding the TG data.’ – because TG data are calculated for very low N2O concentration and are simply biased. I believe they should be excluded from any further interpretations.
Thank you for this comment. We agree that our original wording was unclear. The linear regression in Fig. 7 was calculated using both the Vostok and Taylor Glacier data, and a strong correlation is obtained when all data points are included. What we intended to convey is that this correlation also holds when the Taylor Glacier data are excluded: the Vostok data alone still show a significant positive relationship between δ15Nα(N2Oin situ) and δ¹⁵N(NO₃⁻), even though the isotopic range within a single core is smaller.
We have revised the text to make this point clearer: “In Fig. 7, the δ15Nα and δ15Nβ values of in situ N2O are compared with δ15N values of NO3- measured in the TG and Vostok ice cores. When considering both TG and Vostok samples, δ15Nα(N2Oin situ) shows a strong positive correlation with δ¹⁵N(NO₃⁻) (slope = 1.0 ± 0.1, R2 = 0.97). This relationship also holds when considering the Vostok data alone, indicating that the correlation is robust even within a single ice core. In contrast, δ15Nβ(N2Oin situ) does not show a statistically significant correlation with δ15N(NO3-) for either site”.
L430 The d18O values are not really explained, for EDML exchange with water could explain the observed low d18O-N2O values, but for Taylor Glacier the very high d18O-N2O values, largely exceeding the d18O-NO3 values are not plausible – again in my opinion these values are biased and should be excluded from any further interpretations.
Thank you for this comment. We see two separate issues raised here:
(1) the accuracy of the mass-balance calculation for Taylor Glacier, given the low in situ N2O concentrations and the resulting larger uncertainties. Large uncertainties do not imply a systematic bias toward high δ18O values, but rather increased scatter around the true value.
(2) whether the measured N2O at Taylor Glacier truly shows elevated δ18O values relative to atmospheric N2O.
Without applying the mass-balance calculation, the measured total N2O at Taylor Glacier (Fig. 3a) already shows δ18O values higher than the atmospheric signature in samples affected by in situ production. This indicates that the in situ N2O at Taylor Glacier is indeed enriched in δ18O. A similar pattern is observed in the NGRIP ice core. Importantly, these elevated δ18O values were measured using two different extraction methods and two different IRMS instruments at the University of Bern and Oregon State University, which strengthens our confidence that the signal is real and not an artefact of a single analytical setup.
Excluding these data because the δ18O values are high would therefore require dismissing the measured values themselves, not just the mass balance results. We believe it is important to keep these data points, as they reveal meaningful differences among the ice cores.
L460, Table2: I do not see the point of referring here all the isotope effects for the processes which you do not deal here with (single precursor N2O) after previous review papers, I would only give the data for hybrid N2O.
L465 you mean Table 2 here, I think your description of values for the different pathways in text is enough with respective citations, as suggested above I would shorten the table 2 only to values directly found in your study
(Comments on L460 and L465) We agree that the original version of Table 2 included information not directly relevant to our analysis. Following your recommendation, we have shortened Table 2 to include only the information for hybrid N2O. We have also ensured that the text refers specifically to the revised table.
L467-468 – a citation for this theory of the common intermediate and the mechanism of N2O formation is missing
We have added the appropriate citations: (Heil et al., 2015; Toyoda et al., 2002, 2005, 2017)
L470 – as above – a citation for denitrification N2O formation mechanism is missing
We have added the appropriate citations: (Fehling, 2012; Toyoda et al., 2002)
L492 – citation needed
We have added the appropriate citations: (Frame et al., 2017; Spott et al., 2011; Stieglmeier et al., 2014; Terada et al., 2017)
L505 – very good and helpful Figure, your explanation and visualisation of the idea of asymmetric intermediate in hybrid N2O production is very convincing
Thank you for this positive feedback. We are pleased that the figure and its explanation were found helpful and convincing.
L542 – I agree that pH probably has the large impact on the extend of O-exchange, do you have data of pH for your samples? If we deal with very high pH it could have completely blocked the O-exchange and would support your theory. The lack of pH values is an important drawback, should be added if available.
Otherwise, without the known differences in pH, I do not believe that the mechanism could be so different to give totally opposite effects, from strongly negative to extremely positive. The story of O-exchange between nitrite and water is very convincing and the reported values are very plausible. However, the very high values in TK are suspicious, even with maximal branching effect they are a bit too high. Importantly, these values are based on 3 points calculated with in-situ N2O production of 20ppb, which is in my opinion to low to calculate representative and true isotope values. I strongly encourage you to critically evaluate this data. I would reduce the calculated in-situ N2O to the higher production only and present and further discuss only the proper values, not associated with possibly large errors. This would vastly improve your manuscript because you wouldn’t need to find theories for the values which are very unsure.
Unfortunately, we do not have pH data for these specific samples. Glacial Antarctic ice is generally acidic, but without direct measurements we cannot assess potential pH differences between sites. Moreover, even if pH were measured in the liquid phase (meltwater), it would be difficult to directly infer the pH of the solid ice matrix, as pH is formally defined only for liquid solutions. In addition, the relevant reactions likely occur at the surfaces of dust particles (where the reduction of NO3- to NO2- may take place) rather than in the bulk ice, and the chemical conditions at these micro-environments may differ from those of the surrounding ice.
We agree that, as you point out, we currently lack sufficient information to draw firm conclusions about the mechanism responsible for the δ18O(N2Oin situ) values. We will revise Section 5.4 to make it clear that we are listing possible mechanisms that could explain why the δ18O(NO3-) signature is not transferred to in situ N2O, but that the mechanism at play at Taylor Glacier (and in other ice cores) remains uncertain.
As noted in our responses to other comments, questioning the high δ18O values at Taylor Glacier would imply questioning the measured total N2O data themselves, not only the mass balance calculations. We therefore believe it is more appropriate to keep the TG data and adopt a cautious interpretation, acknowledging that the mechanism is not yet fully understood, rather than excluding data that do not fit the simplest explanation. This approach allows us to provide a more complete picture of in situ N2O production across the different ice cores. We provide the uncertainty of the derived in situ signals in all figures to corroborate our conclusions.
References
Bauska, T. K., Baggenstos, D., Brook, E. J., Mix, A. C., Marcott, S. A., Petrenko, V. V., Schaefer, H., Severinghaus, J. P., and Lee, J. E.: Carbon isotopes characterize rapid changes in atmospheric carbon dioxide during the last deglaciation, Proc. Natl. Acad. Sci. U.S.A., 113, 3465–3470, https://doi.org/10.1073/pnas.1513868113, 2016.
Fehling, C.: Mechanistic Insights from the 15N-Site Preference of Nitrous Oxide utilizing High Resolution Near-Infrared cw Cavity Ringdown Spectroscopy and Density Functional Theory Calculations, PhD Thesis, Kiel University, 130 pp., 2012.
Frame, C. H., Lau, E., Nolan, E. J., Goepfert, T. J., and Lehmann, M. F.: Acidification Enhances Hybrid N2O Production Associated with Aquatic Ammonia-Oxidizing Microorganisms, Front. Microbiol., 7, https://doi.org/10.3389/fmicb.2016.02104, 2017.
Heil, J., Liu, S., Vereecken, H., and Brüggemann, N.: Abiotic nitrous oxide production from hydroxylamine in soils and their dependence on soil properties, Soil Biology and Biochemistry, 84, 107–115, https://doi.org/10.1016/j.soilbio.2015.02.022, 2015.
Lewicka‐Szczebak, D., Well, R., Bol, R., Gregory, A. S., Matthews, G. P., Misselbrook, T., Whalley, W. R., and Cardenas, L. M.: Isotope fractionation factors controlling isotopocule signatures of soil‐emitted N2 O produced by denitrification processes of various rates, Rapid Comm Mass Spectrometry, 29, 269–282, https://doi.org/10.1002/rcm.7102, 2015.
Rubasinghege, G., Spak, S. N., Stanier, C. O., Carmichael, G. R., and Grassian, V. H.: Abiotic Mechanism for the Formation of Atmospheric Nitrous Oxide from Ammonium Nitrate, Environ. Sci. Technol., 45, 2691–2697, https://doi.org/10.1021/es103295v, 2011.
Spott, O., Russow, R., and Stange, C. F.: Formation of hybrid N2O and hybrid N2 due to codenitrification: First review of a barely considered process of microbially mediated N-nitrosation, Soil Biology and Biochemistry, 43, 1995–2011, https://doi.org/10.1016/j.soilbio.2011.06.014, 2011.
Stieglmeier, M., Mooshammer, M., Kitzler, B., Wanek, W., Zechmeister-Boltenstern, S., Richter, A., and Schleper, C.: Aerobic nitrous oxide production through N-nitrosating hybrid formation in ammonia-oxidizing archaea, The ISME Journal, 8, 1135–1146, https://doi.org/10.1038/ismej.2013.220, 2014.
Terada, A., Sugawara, S., Hojo, K., Takeuchi, Y., Riya, S., Harper, W. F., Yamamoto, T., Kuroiwa, M., Isobe, K., Katsuyama, C., Suwa, Y., Koba, K., and Hosomi, M.: Hybrid Nitrous Oxide Production from a Partial Nitrifying Bioreactor: Hydroxylamine Interactions with Nitrite, Environ. Sci. Technol., 51, 2748–2756, https://doi.org/10.1021/acs.est.6b05521, 2017.
Toyoda, S., Yoshida, N., Miwa, T., Matsui, Y., Yamagishi, H., Tsunogai, U., Nojiri, Y., and Tsurushima, N.: Production mechanism and global budget of N2O inferred from its isotopomers in the western North Pacific, Geophysical Research Letters, 29, https://doi.org/10.1029/2001GL014311, 2002.
Toyoda, S., Mutobe, H., Yamagishi, H., Yoshida, N., and Tanji, Y.: Fractionation of N2O isotopomers during production by denitrifier, Soil Biology and Biochemistry, 37, 1535–1545, https://doi.org/10.1016/j.soilbio.2005.01.009, 2005.
Toyoda, S., Yoshida, N., and Koba, K.: Isotopocule analysis of biologically produced nitrous oxide in various environments, Mass Spectrometry Reviews, 36, 135–160, https://doi.org/10.1002/mas.21459, 2017.
York, D., Evensen, N. M., Martı́nez, M. L., and De Basabe Delgado, J.: Unified equations for the slope, intercept, and standard errors of the best straight line, American Journal of Physics, 72, 367–375, https://doi.org/10.1119/1.1632486, 2004.
Yu, L., Harris, E., Lewicka‐Szczebak, D., Barthel, M., Blomberg, M. R. A., Harris, S. J., Johnson, M. S., Lehmann, M. F., Liisberg, J., Müller, C., Ostrom, N. E., Six, J., Toyoda, S., Yoshida, N., and Mohn, J.: What can we learn from N2 O isotope data? – Analytics, processes and modelling, Rapid Comm Mass Spectrometry, 34, e8858, https://doi.org/10.1002/rcm.8858, 2020.
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AC1: 'Reply on RC1', Lison Soussaintjean, 23 Dec 2025
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RC2: 'Comment on egusphere-2025-3108', Anonymous Referee #2, 02 Nov 2025
Title: In-situ production of hybrid N2O in dust-rich Antarctic ice
Authors: Soussaintjean et al.
This manuscript investigated the origin and isotopic compositions of in-situ N2O in Antarctic glacial ice using site-specific 15N isotopomer analysis, combined with d18O of N2O and nitrate. The authors proposed a novel hypothesis of an asymmetric hybrid intermediate mechanism that forms N2O, with α-N derived from nitrate/nitrite and β-N from another unidentified N species likely under low-pH, Fe2+ available conditions.
The manuscript addresses an important topic that may have significant implications for the Earth’s nitrogen cycle, isotope geochemistry, and paleoclimatology. It is well written, logically structured, and generally easy to follow. Although some figure legends are a bit confusing (see minor comments below), the figures clearly present the data. I have one major comment and a few minor suggestions.
Major comment:
I believe that under very low temperature conditions such as those in this study; all reactions occur extremely slowly, which could significantly affect isotopic fractionation. The authors cited several papers to compare SP values (Table 2); however, these references are all based on findings under ambient temperature and pressure conditions. Therefore, I question whether such comparisons are valid for this system.
I recommend including studies that are performed under more relevant temperature conditions, if available. If not, provide a brief theoretical discussion to justify the comparison between the SP values obtained in this study (low temperature, extremely slow reactions) and those of the cited studies (ambient temperature).
Minor comments:
Figure 1. TALDICE ice core values are shown as a solid black line in the figure. So, in the legend, change the black dot to a solid black line.
And are there samples unaffected by in-situ production shown as well? It is difficult to distinguish them. The figure is a bit confusing because EDC, Vostok and EDML are shown as colored dots while the samples affected by in-situ production are marked as stars. If the figure only displays the affected samples, consider using star markers for all three ice cores and deleting the last line in the legend.
Line 71-75: Since SP is a key element of this manuscript, the introduction should provide more background on SP analysis and explain what information it conveys.
Line 106: The main hypothesis should be stated more explicitly.
Line 166: Ca2+ concentrations are cited from different studies. Were they measured using the same analytical methods? Also isn’t there existing data on Fe content? If so, it would be valuable to include them in here.
Line 252: Write Fig. A1 “in Appendix A”. Similarly, for the rest of the manuscript, specify when figures or tables are located in the appendix.
Line 283: should this refer to Section 3.2.2?
Figure 3b. Include a gray cross in the legend.
Line 465: Add the references for the “several studies” mentioned in here.
Citation: https://doi.org/10.5194/egusphere-2025-3108-RC2 -
AC2: 'Reply on RC2', Lison Soussaintjean, 23 Dec 2025
This manuscript investigated the origin and isotopic compositions of in-situ N2O in Antarctic glacial ice using site-specific 15N isotopomer analysis, combined with d18O of N2O and nitrate. The authors proposed a novel hypothesis of an asymmetric hybrid intermediate mechanism that forms N2O, with α-N derived from nitrate/nitrite and β-N from another unidentified N species likely under low-pH, Fe2+ available conditions.
The manuscript addresses an important topic that may have significant implications for the Earth’s nitrogen cycle, isotope geochemistry, and paleoclimatology. It is well written, logically structured, and generally easy to follow. Although some figure legends are a bit confusing (see minor comments below), the figures clearly present the data. I have one major comment and a few minor suggestions.
We thank you for the careful and constructive review of our manuscript and for the positive assessment of its scientific relevance, clarity, and structure. Below, we address the major comment and the minor suggestions in detail.
Major comment:
I believe that under very low temperature conditions such as those in this study; all reactions occur extremely slowly, which could significantly affect isotopic fractionation. The authors cited several papers to compare SP values (Table 2); however, these references are all based on findings under ambient temperature and pressure conditions. Therefore, I question whether such comparisons are valid for this system.
I recommend including studies that are performed under more relevant temperature conditions, if available. If not, provide a brief theoretical discussion to justify the comparison between the SP values obtained in this study (low temperature, extremely slow reactions) and those of the cited studies (ambient temperature).
We thank the reviewer for this important comment. We agree that very low temperatures imply extremely slow reaction rates, which can significantly influence isotopic fractionation and complicate direct comparison with published SP values measured at ambient temperature conditions. To our knowledge, no SP measurements exist under temperature conditions relevant for Antarctic ice. We have therefore added a brief theoretical discussion in the revised manuscript explaining why our comparison focuses on the constancy or variability of SP with respect to the precursor δ15N signature rather than on absolute SP values, and why this comparison remains valid despite the temperature difference.
Changes in the text:
L489 “We note that all published SP datasets used for comparison were obtained at ambient temperatures, whereas in situ N2O production in Antarctic ice occurs at very low temperatures. Such low temperatures imply extremely slow reaction rates, which may alter the magnitude of isotope fractionation and make direct comparison of absolute SP values with ambient-temperature experiments difficult. To our knowledge, no SP measurements exist under such cold conditions. However, our interpretation does not rely on comparing absolute SP values. Instead, we focus on whether SP is constant or variable with respect to the δ15N signature of the precursor; this property should be independent of the absolute magnitude of isotope fractionation. Although lower temperatures may increase kinetic isotope effects and shift absolute SP values, they do not change whether SP remains constant (as in reactions involving symmetrical intermediates) or varies with the precursor isotopic composition (as expected when an asymmetrical intermediate forms). Thus, the comparison of the mechanisms remains valid even without low-temperature experimental data from previous studies.
Several mechanisms could potentially explain why the intermediate of in situ N2O production is asymmetric, even though its exact chemical structure remains unknown. Firstly, the precursor of the β-position N atom could be different from NH2OH. Although most studies on hybrid N2O production report a reaction between NH2OH and NO2-, other nucleophilic precursors have been reported as precursors of hybrid N2O. Hydrazine (N2H4), for example, forms the asymmetrical intermediate HO-N=N-NH2(Perron et al., 1976). A second possibility is that very low temperatures modify the structure or stability of the intermediate normally formed from NH2OH and NO2- at ambient temperature conditions, favoring an asymmetrical species and thereby generating the observed dependence of SP on the precursor δ15N values.”
Minor comments:
Figure 1. TALDICE ice core values are shown as a solid black line in the figure. So, in the legend, change the black dot to a solid black line.
And are there samples unaffected by in-situ production shown as well? It is difficult to distinguish them. The figure is a bit confusing because EDC, Vostok and EDML are shown as colored dots while the samples affected by in-situ production are marked as stars. If the figure only displays the affected samples, consider using star markers for all three ice cores and deleting the last line in the legend.
We have updated the figure legend to use a solid black line for the TALDICE ice core, as recommended. The figure only displays samples affected by in situ N2O production (with the exception of the TALDICE record), and these samples are shown using star markers. We have now adjusted the legend to make this clearer and to avoid any confusion between dots and stars.
Changes in the figure: see Supplement file.
Line 71-75: Since SP is a key element of this manuscript, the introduction should provide more background on SP analysis and explain what information it conveys.
Changes in the text:
“Beyond bulk isotope analyses (δ15N and δ18O), position-specific measurements provide even more detailed insights into N2O production mechanisms. Because N2O is an asymmetric molecule, the nitrogen isotopic composition of the two N atoms can deviate from each other, and this difference can be measured separately at the central N atom (Nα, bonded to oxygen) and the terminal N atom (Nβ). From these values, the site preference (SP = δ15Nα – δ15Nβ) can be calculated. Because SP reflects intramolecular isotope partitioning during N-N bond formation, it is primarily controlled by the reaction mechanism and the structure of the last intermediate rather than by the isotopic composition of the precursor (Frame and Casciotti, 2010; Sutka et al., 2003, 2006; Toyoda et al., 2005). As a result, SP values are often characteristic of specific N2O formation pathways and can remain constant even when precursor δ15N values vary widely. In contrast to bulk δ15N, which integrates source and fractionation effects, SP provides mechanistic information on how N2O is formed and is therefore widely used to discriminate between N2O production pathways; SP values are typically negative for bacterial denitrification and positive for nitrification (Toyoda et al., 2017). Using this tool, Prokopiou et al. (2018) showed that SP values increased since preindustrial times, pointing to a relative shift from denitrification to nitrification, consistent with agricultural emissions playing a major role in the N2O increase. Similarly, Menking et al. (2025) demonstrated that the increase in N2O concentrations during the transition from the Last Glacial Maximum to the Holocene reflected contributions from both nitrification and denitrification, whereas the N2O decrease during the Younger Dryas was driven by reduced nitrification.”
Line 106: The main hypothesis should be stated more explicitly.
Thank you for this suggestion. We have revised the last paragraph of the introduction:
“This study uses isotope analysis to characterize in situ N2O in various ice cores. The background of the study, the extreme environmental conditions in the polar environment, and the potential consequences for the reactions involved are presented in Sect. 2. Based on the strong enrichment in 15N observed in some samples affected by in situ N2O production (Fig. 1), we hypothesize that NO3-, which can also be highly enriched in 15N in ice, is one of the nitrogen precursors for in situ N2O. To test this hypothesis, we measured the isotopic composition of N2O and NO3- in the same ice core samples and calculated the isotopic signature of in situ N2O (Sects. 3 and 4). Position-specific nitrogen isotope analysis of N2O was carried out to further constrain the reaction pathway(s) involved. The potential mechanisms for in situ N2O production are discussed in Sect. 5.”
Line 166: Ca2+ concentrations are cited from different studies. Were they measured using the same analytical methods? Also isn’t there existing data on Fe content? If so, it would be valuable to include them in here.
Yes, the Ca2+ concentrations cited for the different ice cores were all measured using the same analytical technique (continuous flow analysis; Röthlisberger et al., 2000). We will clarify this explicitly in the manuscript.
Regarding Fe content, existing Fe2+ data are indeed available for the EDC and TALDICE ice cores (Spolaor et al., 2013; Traversi et al., 2004). We will incorporate these values into Table 1 in the revised manuscript.
Line 252: Write Fig. A1 “in Appendix A”. Similarly, for the rest of the manuscript, specify when figures or tables are located in the appendix.
We will change this as proposed.
Line 283: should this refer to Section 3.2.2?
Thank you for pointing this out. Yes, the reference should be to Section 3.2.2. We will correct this in the revised manuscript.
Figure 3b. Include a gray cross in the legend.
We will change this as proposed.
Line 465: Add the references for the “several studies” mentioned in here.
We have added the appropriate citations: (Heil et al., 2015; Toyoda et al., 2002, 2005, 2017).
References
Frame, C. H. and Casciotti, K. L.: Biogeochemical controls and isotopic signatures of nitrous oxide production by a marine ammonia-oxidizing bacterium, Biogeosciences, 7, 2695–2709, https://doi.org/10.5194/bg-7-2695-2010, 2010.
Heil, J., Liu, S., Vereecken, H., and Brüggemann, N.: Abiotic nitrous oxide production from hydroxylamine in soils and their dependence on soil properties, Soil Biology and Biochemistry, 84, 107–115, https://doi.org/10.1016/j.soilbio.2015.02.022, 2015.
Menking, J. A., Lee, J. E., Brook, E. J., Schmitt, J., Soussaintjean, L., Fischer, H., Kaiser, J., and Rice, A.: Glacial‐Interglacial and Millennial‐Scale Changes in Nitrous Oxide Emissions Pathways and Source Regions, Global Biogeochemical Cycles, 39, e2024GB008287, https://doi.org/10.1029/2024GB008287, 2025.
Perron, J. R., Stedman, G., and Uysal, N.: Kinetic and product study of the reaction between nitrous acid and hydrazine, J. Chem. Soc., Dalton Trans., 2058, https://doi.org/10.1039/dt9760002058, 1976.
Prokopiou, M., Sapart, C. J., Rosen, J., Sperlich, P., Blunier, T., Brook, E., Van De Wal, R. S. W., and Röckmann, T.: Changes in the Isotopic Signature of Atmospheric Nitrous Oxide and Its Global Average Source During the Last Three Millennia, JGR Atmospheres, 123, https://doi.org/10.1029/2018JD029008, 2018.
Röthlisberger, R., Bigler, M., Hutterli, M., Sommer, S., Stauffer, B., Junghans, H. G., and Wagenbach, D.: Technique for Continuous High-Resolution Analysis of Trace Substances in Firn and Ice Cores, Environ. Sci. Technol., 34, 338–342, https://doi.org/10.1021/es9907055, 2000.
Spolaor, A., Vallelonga, P., Cozzi, G., Gabrieli, J., Varin, C., Kehrwald, N., Zennaro, P., Boutron, C., and Barbante, C.: Iron speciation in aerosol dust influences iron bioavailability over glacial‐interglacial timescales, Geophysical Research Letters, 40, 1618–1623, https://doi.org/10.1002/grl.50296, 2013.
Sutka, R. L., Ostrom, N. E., Ostrom, P. H., Gandhi, H., and Breznak, J. A.: Nitrogen isotopomer site preference of N2O produced by Nitrosomonas europaea and Methylococcus capsulatus Bath, Rapid Comm Mass Spectrometry, 17, 738–745, https://doi.org/10.1002/rcm.968, 2003.
Sutka, R. L., Ostrom, N. E., Ostrom, P. H., Breznak, J. A., Gandhi, H., Pitt, A. J., and Li, F.: Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances, Appl Environ Microbiol, 72, 638–644, https://doi.org/10.1128/AEM.72.1.638-644.2006, 2006.
Toyoda, S., Yoshida, N., Miwa, T., Matsui, Y., Yamagishi, H., Tsunogai, U., Nojiri, Y., and Tsurushima, N.: Production mechanism and global budget of N2O inferred from its isotopomers in the western North Pacific, Geophysical Research Letters, 29, https://doi.org/10.1029/2001GL014311, 2002.
Toyoda, S., Mutobe, H., Yamagishi, H., Yoshida, N., and Tanji, Y.: Fractionation of N2O isotopomers during production by denitrifier, Soil Biology and Biochemistry, 37, 1535–1545, https://doi.org/10.1016/j.soilbio.2005.01.009, 2005.
Toyoda, S., Yoshida, N., and Koba, K.: Isotopocule analysis of biologically produced nitrous oxide in various environments, Mass Spectrometry Reviews, 36, 135–160, https://doi.org/10.1002/mas.21459, 2017.
Traversi, R., Barbante, C., Gaspari, V., Fattori, I., Largiuni, O., Magaldi, L., and Udisti, R.: Aluminium and iron record for the last 28 kyr derived from the Antarctic EDC96 ice core using new CFA methods, Ann. Glaciol., 39, 300–306, https://doi.org/10.3189/172756404781814438, 2004.
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AC2: 'Reply on RC2', Lison Soussaintjean, 23 Dec 2025
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The paper supplies very interesting data on N2O isotopic values in antarctic ice cores, congratulation. Authors interpret the isotopic fingerprint consisting of d18O , d15Na, d15Nb, SP and d15Nbulk in conjunction with d15N and d18O of NO3. They concluded that there must have been abiotic N2O formation in dust rich sections by a process yielding hybrid N2O, ie were N2O molecules carry N from different precursors in the alpha and beta N position.
Although the previous assumption of biotic production of N2O in ice cores is considered less probable according to the introduction, authors might test whether biotic reduction of N2O to N2 could also explain isotopic patterns (see eg (Yu et al., 2020)), since high SP and d18O values observed in the ice cores (up to or even higher than 100 per mil) have also been found previously from microbial N2O reduction, e.g in groundwater. But a quick look at Fig 3 and Fig. 6d suggests that the data could probably not be explained by microbial N2O reduction, since high SP values were associated with lower d18O and vice versa. In contrast, microbial N2O reduction to N2 would lead to parallel increase in d18O and SP of residual N2O.
Using the measured data authors could evaluate whether biotic reduction can be excluded as a relevant pathway, which would further support their assumption that biotic processes were not relevant. From my view one possible way to address this could be easily done by adding a figure plotting SP against d18O and compare the distribution of the data points with the typical N2O reduction line (see eg, Yu et al 2020). Absence of N2O reduction would not be a strict proof for absence of microbial N2O production, but in terrestrial and aquatic ecosystems, N2O production by bacterial denitrification is almost always associated with N2O reduction to a certain extent.
Moreover, in view of the high relevance of N2O consumption in the stratosphere in in terrestrial and aquatic systems, it might be adequate to address N2O consumption processes in the paper, which has not been done as far as I can see. Thus, even without showing a SP/d18O plot, it might be useful to explain why consumption processes in the ice cores are not probable.
Reinhard Well
Yu, L., Harris, E., Lewicka-Szczebak, D., Barthel, M., Blomberg, M.R.A., Harris, S.J., Johnson, M.S., Lehmann, M.F., Liisberg, J., Müller, C., Ostrom, N.E., Six, J., Toyoda, S., Yoshida, N., Mohn, J., 2020. What can we learn from N2O isotope data? – Analytics, processes and modelling. Rapid Communications in Mass Spectrometry 34, e8858.
Yu, L., Harris, E., Lewicka-Szczebak, D., Barthel, M., Blomberg, M.R.A., Harris, S.J., Johnson, M.S., Lehmann, M.F., Liisberg, J., Müller, C., Ostrom, N.E., Six, J., Toyoda, S., Yoshida, N., Mohn, J., 2020. What can we learn from N2O isotope data? – Analytics, processes and modelling. Rapid Communications in Mass Spectrometry 34, e8858.