Dissolved Nitric Oxide in the Lower Elbe Estuary and the Hamburg Port Area

Ingeniero, Riel Carlo O.; Schulz, Gesa; Bange, Hermann W.

doi:https://doi.org/10.5194/egusphere-2023-3009

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https://doi.org/10.5194/egusphere-2023-3009

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21 Dec 2023

| 21 Dec 2023

Dissolved Nitric Oxide in the Lower Elbe Estuary and the Hamburg Port Area

Riel Carlo O. Ingeniero, Gesa Schulz, and Hermann W. Bange

Abstract. Nitric oxide (NO) is an intermediate of various microbial nitrogen cycle processes and the open ocean and coastal areas are generally a source of NO in the atmosphere. However, our knowledge about its distribution and the main production processes in coastal areas and estuaries is rudimentary at best. To this end, dissolved NO concentrations were measured for the first time in surface waters along the lower Elbe Estuary and Hamburg Port area in July 2021. The discrete surface water samples were analyzed using a chemiluminescence detection method. The NO concentrations ranged from below the limit of detection (9.1 × 10⁻¹²mol L⁻¹) to 17.7 × 10⁻¹² mol L⁻¹, averaging at 12.5 × 10⁻¹²mol L⁻¹ and were supersaturated in the surface layer of both the lower Elbe Estuary and the Hamburg Port area, indicating that the study site was a source of NO to the atmosphere during the study period. On the basis of a comprehensive comparison of NO concentrations with parallel nutrient, oxygen, and nitrous oxide concentration measurements, we conclude that the observed distribution of dissolved NO was most likely resulting from microbial nitrogen transformation processes, particularly nitrification in the coastal-brackish and limnic zones of the lower Elbe Estuary and nitrifier-denitrification in the Hamburg Port area.

Received: 13 Dec 2023 – Discussion started: 21 Dec 2023

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Journal article(s) based on this preprint

30 Jul 2024

Dissolved nitric oxide in the lower Elbe Estuary and the Port of Hamburg area

Riel Carlo O. Ingeniero, Gesa Schulz, and Hermann W. Bange

Biogeosciences, 21, 3425–3440, https://doi.org/10.5194/bg-21-3425-2024,https://doi.org/10.5194/bg-21-3425-2024, 2024

Short summary

Riel Carlo O. Ingeniero, Gesa Schulz, and Hermann W. Bange

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-3009', Anonymous Referee #1, 18 Jan 2024
The authors present a recent effort of NO measurement in the Lower Elbe Estuary and the Hamburg Port Area, filling research blanks of this trace gas in coastal areas and estuaries. This manuscript is well-organized, with nice figures. It does provide an important picture of estuarine NO, an active trace gas difficult to measure, showing the distribution, flux, and potential production mechanisms of NO in the study region.
However, I have two major concerns here (also see specific comments below):
This paper is a good case study, but, as a manuscript expected to be published in bg, the text is lacking in the laying out of the scientific issues as well as extrapolation. For example, in the introduction there is a lack of elicitation of the gaps for the current research, and in the discussion, there is a lack of implications of the conclusions for other research in the field (i.e., what is the new knowledge compared to other published NO studies).

The whole discussion section and the present of implications is still weak, e.g., the main conclusions are mainly drawn through correlations, but without sufficient explanation and logic connection between correlation and their conclusion. This problem is particularly evident in section 4.4.

In the present version, I think there are still some gaps away from the publication level, and a major revision would be recommended.

Specific comments:
Introduction
Lines 36-39 This is just a list of past study areas, and the authors should have devoted some space to specifying the major scientific conclusions and advances made by these studies in the marine environment NO.

Line 40 What is the research gap of NO? Where might the behavior of estuarine NO differ from that of the study areas described above, or what is the scientifical importance of studying estuarine NO? These should be briefly described in the Intro section.

Method
Line 51 Define Elbe-km here.

Line 79 Method uncertainty and detect limit should be presented here.

Line 83 The text here says that triplicate NO samples are measured. But I don’t see the error bars in the figures. Uncertainty of NO flux density estimate also need to be added.

Line 128-129 and Fig. S2. I was surprised by the range of data in the figure, which, given the error bars, can range from -5 to 15 µg/m³. I'm a bit curious whether this range of error is primarily from (a) limitations of the detection method, (b) spatial heterogeneity, or (c) temporal variability. If it's from (a), the authors' averaging method may be reasonable, and if it's from (b) and (c), how large is the potential calculation errors? It looks like it might have (up to) an order of magnitude impact on the flux calculations.

Discussion
Section 4.1
The discussion in this section was a bit weak. I really like the summary of NO in Figure 6, but there wasn't much discussion of it in the main text. For example, why is it that estuaries with higher nutrient instead have lower NO concentrations than open ocean/nearshore? This study’ NO is already supersaturated but still on the lower end of all the studies, what is causing the high concentrations (potentially supersaturated several times over) on the other sites? Will some of the correlation patterns in this work appear in whole compile data set? How important are estuarine/oceanic NO emissions relative to terrestrial/human systems based on currently available data? Etc… This may require more work to sort out, but I believe it may expand the scientific value of this paper to be more than just like a case study.

Section 4.2
Because salinity is also an indicator of mixing, the negative correlation with salinity noted here is likely to represent "mixing" for NO (i.e., mixing affects both NO and salinity), not "salinity and freshwater input influencing NO concentrations" (i.e., salinity/freshwater itself influences NO).

Section 4.4
The source/sink of NO is so complex that I would suggest that the authors include a suitable concept fig in an attachment or in the main text to allow more readers to easy follow the processes you describe.

Section 4.4.1
Lines 322-323 Why this statement make sense? Nitrification only contribute minor part of AOU. Some explanations or refences are needed.

Lines 324-325 I can’t follow these sentences. Many ratios (e.g., N₂O/NH₄⁺, NO₂^-/O₂ ...) appear in the correlation diagram. What do these ratios represent? Some background should be provided.

Lines 326-327 How “a significant positive linear relationship exists between N₂O and NO₃⁻” is linked to “These findings point to NO production via nitrification”? I can’t find the logic connection.

Line 331 What “observed trends” refer to?

Line 334 Authors discuss here that nitrification is the SINK of NO. I am a little confused because the whole section discusses about nitrification as SOURCE of NO.

Section 4.4.2
This entire section suffers from a problem like that of section 4.4.1, in that a large amount of the text simply suggests the correlation without explaining it, making the logical chain of support for the author's argument incomplete. For example, almost all of the text in lines 350-365.

Other notes:
Table S2: Why NO flux density (mol m⁻² s⁻¹) have a different unit with N₂O flux density (μmol L⁻¹ d⁻¹)? It also differs from unit in the main text and figure 5 and 6.

Why don't you add NO to the correlation plots of the main text and attachments? I don't see NO in Figure 7 and Figures S4-S6? And if space permits, I suggest you place Fig. S4 (after adding NO) and Fig. S7 into main text.
Citation: https://doi.org/10.5194/egusphere-2023-3009-RC1
- AC1: 'Reply on RC1', Riel Carlo O. Ingeniero, 06 Mar 2024
  
  Publisher’s note: the supplement to this comment was edited on 7 March 2024. The adjustments were minor without effect on the scientific meaning.
  We are grateful to the Reviewer for their insightful and constructive feedback on our manuscript. To address the specific points raised, we have prepared a detailed response, which can be found in the attached PDF document.
  
  Citation: https://doi.org/10.5194/egusphere-2023-3009-AC1
RC2:
'Comment on egusphere-2023-3009', Anonymous Referee #2, 26 Jan 2024

This is an interesting paper providing new data on NO distribution and fluxes and with potential for improving our insight in the complex biogeochemistry of N-transformation in estuarine/riverine environments. The analytical ptocedures for data acquisition are explained in detail and the quality of the data seems very robust.
Next to presenting the estuarine profile of NO concentrations and fluxes together with other physico-chemical parameters (Temp, Sal, O2, nuts, Chl ..) authors proceed with discussing possible processes steering the observed distributions. This is done exclusively based on regression analyses. I found this part of the paper based on a lot of speculation, forcebly as no other tools permitting process identification and process rate assessment were applied. This weakens somewhat the strength of the paper which therefore rests mostly on the quality of the analytical part. Especially N, O isotopic composition measurements of nutrients could possibly confirm/infirm occurrence of nitrification/denitrification and resolve impact of both processes. I can understand such approach was not possible in the present context, but isotopic data for the Elbe have been published by others (Dähnke et al) and some thoughts on how these fit with the present observations might have been a useful addition to the paper. Can authors comment on this ?

Specific comments:
Were any data obtained for the tributary rivers Oste, Meden, Stör ?
In section 4.3 photolysis is mentioned as a source of NO but this is very little discussed further. Can it be a significant process in a turbid estuary ? Are there data for suspended matter load, vertical light profiles?
Lines 313-315: Can presence/absence of anamox activity be confirmed based solely on information of O2 conc.? Could this process possibly proceed inside micro-environments such as aggregates, flocs with low internal O2?
Lines 322-326: As written, the reader gets the impression AOU is solely set by O2 consumption during nitrification. What about respiration?
Lines 335-336: This sentence leaves us asking so what ? Detail the meaning. How does it clarify the foregoing statement?
Lines 361-365: These statements are unclear and the rationale is difficult to follow. Try to clarify.
Lines 366-368: The possible role of suspended particles with low internal O2 is mentioned for the port area. How does this look in the downstream maximum turbidity zone ?
Lines 400 and further (Conclusions): Will a higher temporal resolution and improved sampling strategy be sufficient to get insight into the dynamic interplay of controlling factors? Would adding stable N, O isotopic methodologies be helpful ?

Technical issues:
Figure 1: legend should mention tributaries Oste, Meden, Stör.
Line 336: reference to Fig. 6.. is this correct, or should it be Fig. 5 ?
Line 361: Fig 7g should be Fig S7g
AOU is given without unit

Citation: https://doi.org/10.5194/egusphere-2023-3009-RC2
- AC3: 'Reply on RC2', Riel Carlo O. Ingeniero, 06 Mar 2024
  
  Publisher’s note: the supplement to this comment was edited on 7 March 2024. The adjustments were minor without effect on the scientific meaning.
  We are grateful to the Reviewer for their insightful and constructive feedback on our manuscript. To address the specific points raised, we have prepared a detailed response, which can be found in the attached PDF document.
  
  Citation: https://doi.org/10.5194/egusphere-2023-3009-AC3
RC3:
'Comment on egusphere-2023-3009', Anonymous Referee #3, 13 Feb 2024

Summary
The manuscript titled "Dissolved Nitric Oxide in the Lower Elbe Estuary" by Ingeniero et al. quantified the fluxes of nitric oxide (NO) in relation to other nitrogen cycle parameters in the Elbe River Estuary and Hamburg Port Area. Using a clever chemiluminescent detection method and flow-through sampling system, the authors measured dissolved NO concentrations in surface waters alongside temperature, salinity, pH, and dissolved oxygen (O₂). The authors made concurrent measurements of nitrate, nitrite, and ammonium with an autoanalyzer and nitrous oxide (N₂O) with laser spectroscopy. The authors found that NO was supersaturated in the surface layer of both study areas, so they were both a source of NO to the atmosphere. Based on the concurrent [O₂] and dissolved inorganic nitrogen measurements, the authors conclude that this NO is likely produced via biological processes (nitrification, denitrification, and nitrifier-denitrification), as opposed to the photolysis of nitrite.
General Appraisal
In this paper, the authors present the first-ever measurements of NO in the Elbe River system. NO measurements in the literature are scarce because its short lifetime makes analysis difficult, so this paper represents a substantial contribution to our understanding of NO in the marine environment. Furthermore, the authors measure significant NO supersaturation and fluxes in the surface waters of much of the Elbe River, which is important because NO is a contributor to smog, acid rain, and ozone.
The major strengths of this paper are the presentation of novel, high-resolution NO measurements and the clear relationships that emerge between NO and other inorganic nitrogen species, [O₂], pH, and chlorophyll. The authors present a clean, concise interpretation of these results and the paper is generally straightforward and easy to read.
The major weakness of this paper is that the discussion of temporal variability (day/night and seasonal variations) is not linked to the clear boom-and-bust cycle seen in the Hamburg Port area. The authors have locations with peaks of chlorophyll and [O₂], and other locations with oxygen and pH minima and N₂O and NO maxima. This implies to me that there are some locations where you captured net production and others where they captured net respiration, which draws down [O₂] and creates an ideal environment for N₂O and NO production in sediments or particles. The authors allude to this in the conclusions, but how would day-night temporal variation at each site affects the data? Would blooms in some locations propagate downstream and create pockets of high respiration further downstream? The authors have a paragraph in the conclusions about potential temporal effects, and my suggestion would be to move this paragraph into the discussion and link it more clearly to their results.
The paper is generally well-written. There are only a few grammatical errors and clumsy sentences that I note in the technical corrections.
My primary concern is about the conclusion (and I believe this is only stated in the abstract) that nitrifier-denitrification is the primary source of NO in the Hamburg Port area. While I agree that the lack of correlation between nitrate and apparent oxygen utilization (AOU) in the Hamburg Port area may indicate the presence of denitrification or nitrifier-denitrification, I don’t think you can rule out one or the other. In other words, all you can conclude from this data is that there is a process other than nitrite oxidation that is consuming nitrite. Likewise, if you invoke denitrification and/or nitrifier-denitrification in sediments or particles, I don’t think you can rule out the presence of anammox. In fact, instead of ruling out anammox based on water column [O₂], you should list it as a potential alternative source of NO. The strong correlations between NO, nitrite, and ammonium may indeed be a sign of anammox as a source of NO in the Hamburg Port area. Also, while denitrification and/or nitrifier-denitrification may be present in this zone, the water column [O₂] suggests that the primary source of NO would still be nitrification, and this is supported by the strong correlations in this zone between NO, nitrite, and ammonium.
Specific comments
Line 14: Is the same chemiluminescent optode spot system often used for O₂ (Frey et al., 2023)?
Line 15: Why not write pM instead of 10^-12 mol/L? You do so later in the manuscript.
Line 20: Based on your discussion, this could be nitrifier-denitrification or denitrification. I don't think you can rule out one or the other based on your data.
Line 34: What is the lifetime of NO in seawater/water?
Line 72: The way this equation is written is confusing. Are you multiplying the corrected O₂ by 1.12? Or the uncorrected? What are the units of the intercept? Also, does the intercept of 13.41 mean that the detection limit of the oxygen optodes is 13.41 (units?)?
Line 83: Give us some numbers for what this lifetime is
Line 84: So the calibrator is just an NO source, right?
Line 90: Why do you need the calibrator in addition to the aqueous NO standard solutions?
Line 94: This calculation is to convert the mole fraction you measure in the headspace to the dissolved NO concentration, right? Is there a reason to assume that the headspace is at a pressure of 1 atm? I would assume it would be slightly over pressurized... how would that affect your measurements?
Line 97: Here you use pM. I would stick to this throughout the text.
Lines 102-103: In eqn. (2) you assume the barometric/atmospheric pressure is 1 atm. Is this a reasonable assumption at this time of year, in this part of the world?
Line 125: Same comment as above with setting atmospheric pressure to 1 atm.
Lines 129-130: How was this mean value calculated? Mean of all hourly measurements at all monitoring stations over the study period? Given the short lifetime of NO, doesn't it make sense to calculate a mean c_EQ on a day-by-day or even shorter basis - or do all of the stations look like figure S2, where the hourly concentrations are all within error of the average?
Lines 172-174: Is the variability of [O₂] because of changes in productivity?
Lines 184-185: Report a number for the maximum concentration to give a sense of scale - 200 µM is a lot!
Lines 200-201: It looks like the peaks in N₂O correspond to the minima in [O₂] - if that's the case, worth pointing out here.
Line 225: You should also mention that the peaks in NO in the Hamburg Port area correspond to the peaks in N₂O, NO₂^-, and NH₄⁺!
Line 232: I would reccommend converting these flux values to fM: 0.31-55 fmol cm^-2 s^-1.
Line 238: How do your measurements compare to previous measurements in terms of saturation? If 147-274% saturated is at the low end of marine NO measurements, I'm curious what these higher concentrations correspond to. This would imply that the ocean could be a major source of NO to the atmosphere!
Lines 251-269: I would avoid interpreting a relationship that is not statistically significant. This section is mostly literature review anyways.
Lines 276-278: This is a really important finding: you have much higher NO₃^-, NO₂^-, and NH₄⁺ than previous studies in other rivere and coastal areas, but not higher NO. What is unique to the Elbe river compared to the other rivers cited here?
Lines 291-292: What about the DN₂O/NO₃^- ratio?
Lines 299-302: So the overall trend (which is positive) is driven by the Hamburg Port area, and the overall trend masks the negative relationships in the limnic and coastal-brackish zones. This is a good example of an ecological fallacy.
Lines 307-308: Add citation: Burlacot et al. (2020).
Lines 308-310: It's worth pointing out that the Chl. peaks occurred right before the NO peaks.
Lines 313-315: What about anaerobic process (anammox, denitrification) in the river sediments?
Line 323: You could also look at the relationship of DN₂O/NO₃^- vs. [O2] or DN₂O/AOU vs. O2 (Nevison et al., 2003).
Line 325: In this context, I would actually call NO₂^- a product of nitrification, not a precursor, because NH₄⁺ oxidation to NO₂^- produces N₂O and NO as a byproduct; NO₂^- oxidation does not.
Line 329: The limnic zone correlations in Figure S7 look like they're being driven by two points at either extreme of NO, while the rest of the points cluster in the middle. I would avoid over-interpreting these plots.
Lines 351-352: Elaborate here upon why the lack of a significant relationship between NO₃^- and AOU indicates the presence of denitrification or nitrifier-denitrification.
Line 363-365: If you imply that denitrification could be occurring in the sediments even though the water column oxygen concentrations are too high, I don't think you can rule out anammox based on water column oxygen concentrations.
Line 367: ...or anoxic microsites within particles.
Lines 370-371: I'm really interested in this apparent boom and bust cycle in your data. You have locations with peaks of chlorophyll and oxygen, and other locations with oxygen and pH minima and N₂O maxima. This implies to me that there are some locations where you captured net production and others where you captured net respiration, which draws down O₂ and creates an ideal environment for N₂O and NO production in sediments or particles. You allude to this in the conclusions, but how do you think day-night temporal variation in each of your sites affects your data? Would blooms in some locations propagate downstream and create pockets of high respiration further downstream?
Lines 383-384: You talk very little about photolysis in your discussion so I would remove it here.
Lines 385-397: I would move this paragraph on potential temporal effects into your discussion section (see my previous comment). Then summarize it in your conclusions.
Technical corrections
Line 24: Faulty parallelism: replace "and affecting" with "and affects"
Line 48: replace "Its estuarine part stretches" with "Its estuaries stretch"
Line 83: change to "within 20 minutes OF sampling"
Line 93/eqn. (1): It's confusing to have the letter "x" as the multiplication sign here because you also have an x variable. Use the mathematical symbol you use below or just take them out.
Line 120/eqn. (10): Write e^0.0447T not exp.
Line 124/eqn. (12): p_NO and K_H are quantity symbols - italicize here as you did above.
Figures 2, 3, and 5: I would put the y axis labels (salinity, temperature, etc.) on the left side with the y axis ticks - it's confusing to have them on the opposite side of the plot. You can move the subplot labels ("a", "b") to the upper left corner.
Line 158: Add salinity units.
Figure 6: This is a really nice compilation plot to put your measurements in context. Instead of saying the NO fluxes are x10^-17, just report in units of fmol cm^-2 s^-1.
Table S3: Table S3: instead of superscripts "a", "b" and "c" corresponding to different significance levels, use *, **, and ***, which is the convention.
Figure 7: Use *, **, and *** instead of a, b, c superscripts.
Lines 332-333: I agree with the ammonium limitation idea but rephrase this and the following sentences to improve clarity and flow.
Line 344: Here and elsewhere: "were" not "are", since most of your results are reported in past tense.
Line 348: Remove clause "when the nitrification proceeds" – unnecessary.
Line 350: Remove "therefore" - the support for this statement comes later in this paragraph, not from the preceding one.
Line 355: “correlations” should be plural.
References
Burlacot, A., Richaud, P., Gosset, A., Li-Beisson, Y., and Peltier, G.: Algal photosynthesis converts nitric oxide into nitrous oxide, Proc. Natl. Acad. Sci., 117, 2704–2709, https://doi.org/10.1073/pnas.1915276117, 2020.
Frey, C., Sun, X., Szemberski, L., Casciotti, K. L., Garcia-Robledo, E., Jayakumar, A., Kelly, C. L., Lehmann, M. F., and Ward, B. B.: Kinetics of nitrous oxide production from ammonia oxidation in the Eastern Tropical North Pacific, Limnol. Oceanogr., 68, 424–438, https://doi.org/10.1002/lno.12283, 2023.
Nevison, C., Butler, J. H., and Elkins, J. W.: Global distribution of N2O and the Delta N2O-AOU yield in the subsurface ocean, Glob. Biogeochem. Cycles, 17, 1119, https://doi.org/10.1029/2003GB002068, 2003.

Citation: https://doi.org/10.5194/egusphere-2023-3009-RC3
- AC2:
  'Reply on RC3', Riel Carlo O. Ingeniero, 06 Mar 2024
  
  Publisher’s note: the supplement to this comment was edited on 7 March 2024. The adjustments were minor without effect on the scientific meaning.
  We are grateful to the Reviewer for their insightful and constructive feedback on our manuscript. To address the specific points raised, we have prepared a detailed response, which can be found in the attached PDF document.
  
  Citation: https://doi.org/10.5194/egusphere-2023-3009-AC2
  - AC4: 'Reply on RC3 on Lines 323-333', Riel Carlo O. Ingeniero, 11 Mar 2024
    
    Dear Reviewer,
    Regarding your comment on Lines 332-333: I agree with the ammonium limitation idea but rephrase this and the following sentences to improve clarity and flow.
    Thank you for agreeing to our hypothesis concerning ammonium limitation within the nitrification process in the coastal/brackish zone and limnic zone. In the revised version of our manuscript, we will elaborate on this argument and improve our discussion with additional references. We will aim to enhance both the clarity and the coherence of our argument.
    
    Citation: https://doi.org/10.5194/egusphere-2023-3009-AC4

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-3009', Anonymous Referee #1, 18 Jan 2024
The authors present a recent effort of NO measurement in the Lower Elbe Estuary and the Hamburg Port Area, filling research blanks of this trace gas in coastal areas and estuaries. This manuscript is well-organized, with nice figures. It does provide an important picture of estuarine NO, an active trace gas difficult to measure, showing the distribution, flux, and potential production mechanisms of NO in the study region.
However, I have two major concerns here (also see specific comments below):
This paper is a good case study, but, as a manuscript expected to be published in bg, the text is lacking in the laying out of the scientific issues as well as extrapolation. For example, in the introduction there is a lack of elicitation of the gaps for the current research, and in the discussion, there is a lack of implications of the conclusions for other research in the field (i.e., what is the new knowledge compared to other published NO studies).

The whole discussion section and the present of implications is still weak, e.g., the main conclusions are mainly drawn through correlations, but without sufficient explanation and logic connection between correlation and their conclusion. This problem is particularly evident in section 4.4.

In the present version, I think there are still some gaps away from the publication level, and a major revision would be recommended.

Specific comments:
Introduction
Lines 36-39 This is just a list of past study areas, and the authors should have devoted some space to specifying the major scientific conclusions and advances made by these studies in the marine environment NO.

Line 40 What is the research gap of NO? Where might the behavior of estuarine NO differ from that of the study areas described above, or what is the scientifical importance of studying estuarine NO? These should be briefly described in the Intro section.

Method
Line 51 Define Elbe-km here.

Line 79 Method uncertainty and detect limit should be presented here.

Line 83 The text here says that triplicate NO samples are measured. But I don’t see the error bars in the figures. Uncertainty of NO flux density estimate also need to be added.

Line 128-129 and Fig. S2. I was surprised by the range of data in the figure, which, given the error bars, can range from -5 to 15 µg/m³. I'm a bit curious whether this range of error is primarily from (a) limitations of the detection method, (b) spatial heterogeneity, or (c) temporal variability. If it's from (a), the authors' averaging method may be reasonable, and if it's from (b) and (c), how large is the potential calculation errors? It looks like it might have (up to) an order of magnitude impact on the flux calculations.

Discussion
Section 4.1
The discussion in this section was a bit weak. I really like the summary of NO in Figure 6, but there wasn't much discussion of it in the main text. For example, why is it that estuaries with higher nutrient instead have lower NO concentrations than open ocean/nearshore? This study’ NO is already supersaturated but still on the lower end of all the studies, what is causing the high concentrations (potentially supersaturated several times over) on the other sites? Will some of the correlation patterns in this work appear in whole compile data set? How important are estuarine/oceanic NO emissions relative to terrestrial/human systems based on currently available data? Etc… This may require more work to sort out, but I believe it may expand the scientific value of this paper to be more than just like a case study.

Section 4.2
Because salinity is also an indicator of mixing, the negative correlation with salinity noted here is likely to represent "mixing" for NO (i.e., mixing affects both NO and salinity), not "salinity and freshwater input influencing NO concentrations" (i.e., salinity/freshwater itself influences NO).

Section 4.4
The source/sink of NO is so complex that I would suggest that the authors include a suitable concept fig in an attachment or in the main text to allow more readers to easy follow the processes you describe.

Section 4.4.1
Lines 322-323 Why this statement make sense? Nitrification only contribute minor part of AOU. Some explanations or refences are needed.

Lines 324-325 I can’t follow these sentences. Many ratios (e.g., N₂O/NH₄⁺, NO₂^-/O₂ ...) appear in the correlation diagram. What do these ratios represent? Some background should be provided.

Lines 326-327 How “a significant positive linear relationship exists between N₂O and NO₃⁻” is linked to “These findings point to NO production via nitrification”? I can’t find the logic connection.

Line 331 What “observed trends” refer to?

Line 334 Authors discuss here that nitrification is the SINK of NO. I am a little confused because the whole section discusses about nitrification as SOURCE of NO.

Section 4.4.2
This entire section suffers from a problem like that of section 4.4.1, in that a large amount of the text simply suggests the correlation without explaining it, making the logical chain of support for the author's argument incomplete. For example, almost all of the text in lines 350-365.

Other notes:
Table S2: Why NO flux density (mol m⁻² s⁻¹) have a different unit with N₂O flux density (μmol L⁻¹ d⁻¹)? It also differs from unit in the main text and figure 5 and 6.

Why don't you add NO to the correlation plots of the main text and attachments? I don't see NO in Figure 7 and Figures S4-S6? And if space permits, I suggest you place Fig. S4 (after adding NO) and Fig. S7 into main text.
Citation: https://doi.org/10.5194/egusphere-2023-3009-RC1
- AC1: 'Reply on RC1', Riel Carlo O. Ingeniero, 06 Mar 2024
  
  Publisher’s note: the supplement to this comment was edited on 7 March 2024. The adjustments were minor without effect on the scientific meaning.
  We are grateful to the Reviewer for their insightful and constructive feedback on our manuscript. To address the specific points raised, we have prepared a detailed response, which can be found in the attached PDF document.
  
  Citation: https://doi.org/10.5194/egusphere-2023-3009-AC1
RC2:
'Comment on egusphere-2023-3009', Anonymous Referee #2, 26 Jan 2024

This is an interesting paper providing new data on NO distribution and fluxes and with potential for improving our insight in the complex biogeochemistry of N-transformation in estuarine/riverine environments. The analytical ptocedures for data acquisition are explained in detail and the quality of the data seems very robust.
Next to presenting the estuarine profile of NO concentrations and fluxes together with other physico-chemical parameters (Temp, Sal, O2, nuts, Chl ..) authors proceed with discussing possible processes steering the observed distributions. This is done exclusively based on regression analyses. I found this part of the paper based on a lot of speculation, forcebly as no other tools permitting process identification and process rate assessment were applied. This weakens somewhat the strength of the paper which therefore rests mostly on the quality of the analytical part. Especially N, O isotopic composition measurements of nutrients could possibly confirm/infirm occurrence of nitrification/denitrification and resolve impact of both processes. I can understand such approach was not possible in the present context, but isotopic data for the Elbe have been published by others (Dähnke et al) and some thoughts on how these fit with the present observations might have been a useful addition to the paper. Can authors comment on this ?

Specific comments:
Were any data obtained for the tributary rivers Oste, Meden, Stör ?
In section 4.3 photolysis is mentioned as a source of NO but this is very little discussed further. Can it be a significant process in a turbid estuary ? Are there data for suspended matter load, vertical light profiles?
Lines 313-315: Can presence/absence of anamox activity be confirmed based solely on information of O2 conc.? Could this process possibly proceed inside micro-environments such as aggregates, flocs with low internal O2?
Lines 322-326: As written, the reader gets the impression AOU is solely set by O2 consumption during nitrification. What about respiration?
Lines 335-336: This sentence leaves us asking so what ? Detail the meaning. How does it clarify the foregoing statement?
Lines 361-365: These statements are unclear and the rationale is difficult to follow. Try to clarify.
Lines 366-368: The possible role of suspended particles with low internal O2 is mentioned for the port area. How does this look in the downstream maximum turbidity zone ?
Lines 400 and further (Conclusions): Will a higher temporal resolution and improved sampling strategy be sufficient to get insight into the dynamic interplay of controlling factors? Would adding stable N, O isotopic methodologies be helpful ?

Technical issues:
Figure 1: legend should mention tributaries Oste, Meden, Stör.
Line 336: reference to Fig. 6.. is this correct, or should it be Fig. 5 ?
Line 361: Fig 7g should be Fig S7g
AOU is given without unit

Citation: https://doi.org/10.5194/egusphere-2023-3009-RC2
- AC3: 'Reply on RC2', Riel Carlo O. Ingeniero, 06 Mar 2024
  
  Publisher’s note: the supplement to this comment was edited on 7 March 2024. The adjustments were minor without effect on the scientific meaning.
  We are grateful to the Reviewer for their insightful and constructive feedback on our manuscript. To address the specific points raised, we have prepared a detailed response, which can be found in the attached PDF document.
  
  Citation: https://doi.org/10.5194/egusphere-2023-3009-AC3
RC3:
'Comment on egusphere-2023-3009', Anonymous Referee #3, 13 Feb 2024

Summary
The manuscript titled "Dissolved Nitric Oxide in the Lower Elbe Estuary" by Ingeniero et al. quantified the fluxes of nitric oxide (NO) in relation to other nitrogen cycle parameters in the Elbe River Estuary and Hamburg Port Area. Using a clever chemiluminescent detection method and flow-through sampling system, the authors measured dissolved NO concentrations in surface waters alongside temperature, salinity, pH, and dissolved oxygen (O₂). The authors made concurrent measurements of nitrate, nitrite, and ammonium with an autoanalyzer and nitrous oxide (N₂O) with laser spectroscopy. The authors found that NO was supersaturated in the surface layer of both study areas, so they were both a source of NO to the atmosphere. Based on the concurrent [O₂] and dissolved inorganic nitrogen measurements, the authors conclude that this NO is likely produced via biological processes (nitrification, denitrification, and nitrifier-denitrification), as opposed to the photolysis of nitrite.
General Appraisal
In this paper, the authors present the first-ever measurements of NO in the Elbe River system. NO measurements in the literature are scarce because its short lifetime makes analysis difficult, so this paper represents a substantial contribution to our understanding of NO in the marine environment. Furthermore, the authors measure significant NO supersaturation and fluxes in the surface waters of much of the Elbe River, which is important because NO is a contributor to smog, acid rain, and ozone.
The major strengths of this paper are the presentation of novel, high-resolution NO measurements and the clear relationships that emerge between NO and other inorganic nitrogen species, [O₂], pH, and chlorophyll. The authors present a clean, concise interpretation of these results and the paper is generally straightforward and easy to read.
The major weakness of this paper is that the discussion of temporal variability (day/night and seasonal variations) is not linked to the clear boom-and-bust cycle seen in the Hamburg Port area. The authors have locations with peaks of chlorophyll and [O₂], and other locations with oxygen and pH minima and N₂O and NO maxima. This implies to me that there are some locations where you captured net production and others where they captured net respiration, which draws down [O₂] and creates an ideal environment for N₂O and NO production in sediments or particles. The authors allude to this in the conclusions, but how would day-night temporal variation at each site affects the data? Would blooms in some locations propagate downstream and create pockets of high respiration further downstream? The authors have a paragraph in the conclusions about potential temporal effects, and my suggestion would be to move this paragraph into the discussion and link it more clearly to their results.
The paper is generally well-written. There are only a few grammatical errors and clumsy sentences that I note in the technical corrections.
My primary concern is about the conclusion (and I believe this is only stated in the abstract) that nitrifier-denitrification is the primary source of NO in the Hamburg Port area. While I agree that the lack of correlation between nitrate and apparent oxygen utilization (AOU) in the Hamburg Port area may indicate the presence of denitrification or nitrifier-denitrification, I don’t think you can rule out one or the other. In other words, all you can conclude from this data is that there is a process other than nitrite oxidation that is consuming nitrite. Likewise, if you invoke denitrification and/or nitrifier-denitrification in sediments or particles, I don’t think you can rule out the presence of anammox. In fact, instead of ruling out anammox based on water column [O₂], you should list it as a potential alternative source of NO. The strong correlations between NO, nitrite, and ammonium may indeed be a sign of anammox as a source of NO in the Hamburg Port area. Also, while denitrification and/or nitrifier-denitrification may be present in this zone, the water column [O₂] suggests that the primary source of NO would still be nitrification, and this is supported by the strong correlations in this zone between NO, nitrite, and ammonium.
Specific comments
Line 14: Is the same chemiluminescent optode spot system often used for O₂ (Frey et al., 2023)?
Line 15: Why not write pM instead of 10^-12 mol/L? You do so later in the manuscript.
Line 20: Based on your discussion, this could be nitrifier-denitrification or denitrification. I don't think you can rule out one or the other based on your data.
Line 34: What is the lifetime of NO in seawater/water?
Line 72: The way this equation is written is confusing. Are you multiplying the corrected O₂ by 1.12? Or the uncorrected? What are the units of the intercept? Also, does the intercept of 13.41 mean that the detection limit of the oxygen optodes is 13.41 (units?)?
Line 83: Give us some numbers for what this lifetime is
Line 84: So the calibrator is just an NO source, right?
Line 90: Why do you need the calibrator in addition to the aqueous NO standard solutions?
Line 94: This calculation is to convert the mole fraction you measure in the headspace to the dissolved NO concentration, right? Is there a reason to assume that the headspace is at a pressure of 1 atm? I would assume it would be slightly over pressurized... how would that affect your measurements?
Line 97: Here you use pM. I would stick to this throughout the text.
Lines 102-103: In eqn. (2) you assume the barometric/atmospheric pressure is 1 atm. Is this a reasonable assumption at this time of year, in this part of the world?
Line 125: Same comment as above with setting atmospheric pressure to 1 atm.
Lines 129-130: How was this mean value calculated? Mean of all hourly measurements at all monitoring stations over the study period? Given the short lifetime of NO, doesn't it make sense to calculate a mean c_EQ on a day-by-day or even shorter basis - or do all of the stations look like figure S2, where the hourly concentrations are all within error of the average?
Lines 172-174: Is the variability of [O₂] because of changes in productivity?
Lines 184-185: Report a number for the maximum concentration to give a sense of scale - 200 µM is a lot!
Lines 200-201: It looks like the peaks in N₂O correspond to the minima in [O₂] - if that's the case, worth pointing out here.
Line 225: You should also mention that the peaks in NO in the Hamburg Port area correspond to the peaks in N₂O, NO₂^-, and NH₄⁺!
Line 232: I would reccommend converting these flux values to fM: 0.31-55 fmol cm^-2 s^-1.
Line 238: How do your measurements compare to previous measurements in terms of saturation? If 147-274% saturated is at the low end of marine NO measurements, I'm curious what these higher concentrations correspond to. This would imply that the ocean could be a major source of NO to the atmosphere!
Lines 251-269: I would avoid interpreting a relationship that is not statistically significant. This section is mostly literature review anyways.
Lines 276-278: This is a really important finding: you have much higher NO₃^-, NO₂^-, and NH₄⁺ than previous studies in other rivere and coastal areas, but not higher NO. What is unique to the Elbe river compared to the other rivers cited here?
Lines 291-292: What about the DN₂O/NO₃^- ratio?
Lines 299-302: So the overall trend (which is positive) is driven by the Hamburg Port area, and the overall trend masks the negative relationships in the limnic and coastal-brackish zones. This is a good example of an ecological fallacy.
Lines 307-308: Add citation: Burlacot et al. (2020).
Lines 308-310: It's worth pointing out that the Chl. peaks occurred right before the NO peaks.
Lines 313-315: What about anaerobic process (anammox, denitrification) in the river sediments?
Line 323: You could also look at the relationship of DN₂O/NO₃^- vs. [O2] or DN₂O/AOU vs. O2 (Nevison et al., 2003).
Line 325: In this context, I would actually call NO₂^- a product of nitrification, not a precursor, because NH₄⁺ oxidation to NO₂^- produces N₂O and NO as a byproduct; NO₂^- oxidation does not.
Line 329: The limnic zone correlations in Figure S7 look like they're being driven by two points at either extreme of NO, while the rest of the points cluster in the middle. I would avoid over-interpreting these plots.
Lines 351-352: Elaborate here upon why the lack of a significant relationship between NO₃^- and AOU indicates the presence of denitrification or nitrifier-denitrification.
Line 363-365: If you imply that denitrification could be occurring in the sediments even though the water column oxygen concentrations are too high, I don't think you can rule out anammox based on water column oxygen concentrations.
Line 367: ...or anoxic microsites within particles.
Lines 370-371: I'm really interested in this apparent boom and bust cycle in your data. You have locations with peaks of chlorophyll and oxygen, and other locations with oxygen and pH minima and N₂O maxima. This implies to me that there are some locations where you captured net production and others where you captured net respiration, which draws down O₂ and creates an ideal environment for N₂O and NO production in sediments or particles. You allude to this in the conclusions, but how do you think day-night temporal variation in each of your sites affects your data? Would blooms in some locations propagate downstream and create pockets of high respiration further downstream?
Lines 383-384: You talk very little about photolysis in your discussion so I would remove it here.
Lines 385-397: I would move this paragraph on potential temporal effects into your discussion section (see my previous comment). Then summarize it in your conclusions.
Technical corrections
Line 24: Faulty parallelism: replace "and affecting" with "and affects"
Line 48: replace "Its estuarine part stretches" with "Its estuaries stretch"
Line 83: change to "within 20 minutes OF sampling"
Line 93/eqn. (1): It's confusing to have the letter "x" as the multiplication sign here because you also have an x variable. Use the mathematical symbol you use below or just take them out.
Line 120/eqn. (10): Write e^0.0447T not exp.
Line 124/eqn. (12): p_NO and K_H are quantity symbols - italicize here as you did above.
Figures 2, 3, and 5: I would put the y axis labels (salinity, temperature, etc.) on the left side with the y axis ticks - it's confusing to have them on the opposite side of the plot. You can move the subplot labels ("a", "b") to the upper left corner.
Line 158: Add salinity units.
Figure 6: This is a really nice compilation plot to put your measurements in context. Instead of saying the NO fluxes are x10^-17, just report in units of fmol cm^-2 s^-1.
Table S3: Table S3: instead of superscripts "a", "b" and "c" corresponding to different significance levels, use *, **, and ***, which is the convention.
Figure 7: Use *, **, and *** instead of a, b, c superscripts.
Lines 332-333: I agree with the ammonium limitation idea but rephrase this and the following sentences to improve clarity and flow.
Line 344: Here and elsewhere: "were" not "are", since most of your results are reported in past tense.
Line 348: Remove clause "when the nitrification proceeds" – unnecessary.
Line 350: Remove "therefore" - the support for this statement comes later in this paragraph, not from the preceding one.
Line 355: “correlations” should be plural.
References
Burlacot, A., Richaud, P., Gosset, A., Li-Beisson, Y., and Peltier, G.: Algal photosynthesis converts nitric oxide into nitrous oxide, Proc. Natl. Acad. Sci., 117, 2704–2709, https://doi.org/10.1073/pnas.1915276117, 2020.
Frey, C., Sun, X., Szemberski, L., Casciotti, K. L., Garcia-Robledo, E., Jayakumar, A., Kelly, C. L., Lehmann, M. F., and Ward, B. B.: Kinetics of nitrous oxide production from ammonia oxidation in the Eastern Tropical North Pacific, Limnol. Oceanogr., 68, 424–438, https://doi.org/10.1002/lno.12283, 2023.
Nevison, C., Butler, J. H., and Elkins, J. W.: Global distribution of N2O and the Delta N2O-AOU yield in the subsurface ocean, Glob. Biogeochem. Cycles, 17, 1119, https://doi.org/10.1029/2003GB002068, 2003.

Citation: https://doi.org/10.5194/egusphere-2023-3009-RC3
- AC2:
  'Reply on RC3', Riel Carlo O. Ingeniero, 06 Mar 2024
  
  Publisher’s note: the supplement to this comment was edited on 7 March 2024. The adjustments were minor without effect on the scientific meaning.
  We are grateful to the Reviewer for their insightful and constructive feedback on our manuscript. To address the specific points raised, we have prepared a detailed response, which can be found in the attached PDF document.
  
  Citation: https://doi.org/10.5194/egusphere-2023-3009-AC2
  - AC4: 'Reply on RC3 on Lines 323-333', Riel Carlo O. Ingeniero, 11 Mar 2024
    
    Dear Reviewer,
    Regarding your comment on Lines 332-333: I agree with the ammonium limitation idea but rephrase this and the following sentences to improve clarity and flow.
    Thank you for agreeing to our hypothesis concerning ammonium limitation within the nitrification process in the coastal/brackish zone and limnic zone. In the revised version of our manuscript, we will elaborate on this argument and improve our discussion with additional references. We will aim to enhance both the clarity and the coherence of our argument.
    
    Citation: https://doi.org/10.5194/egusphere-2023-3009-AC4

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

ED: Reconsider after major revisions (11 Mar 2024) by Yuan Shen

AR by Riel Carlo O. Ingeniero on behalf of the Authors (28 Apr 2024) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (30 Apr 2024) by Yuan Shen

RR by Anonymous Referee #1 (07 May 2024)

RR by Anonymous Referee #3 (16 May 2024)

ED: Publish subject to minor revisions (review by editor) (21 May 2024) by Yuan Shen

Dear Mr. Ingeniero,

Thank you for submitting the revised version of your manuscript. Two of the three previous referees have reviewed the revisions and provided overall positive feedback. However, there are still concerns regarding the clarity of the discussion (particularly in section 4.3) and the availability of data. Please review the comments thoroughly and make the necessary revisions.

Additionally, when resubmitting, please include a point-by-point response to the reviewers' comments.

Reviewer #1:
I'm glad the author addressed my previous concerns. And I found the quality of manuscript has been improved. I think the current form is now acceptable for publication.

Only some minor suggestions left:

Main text:
Fig. 7, Fig S4 and S6 The authors replied that they have made separate correlation plots of NO vs other parameters. But I can’t find NO vs salinity plot, NO vs. oxygen plot, and NO vs. AOU plot in other places (e.g., Fig. 8) as I commented on the 1st version manuscript. I would still suggest that NO be added to these figures, as NO is at the center of the discussion in this study.
Line 45: ranging from 0.70 × 10^-17?
Line 360 R2 ammonium monooxygenase should be ammonia monooxygenase.
Line 473 Ludwig Prandtl should be Italic.

Supplementary information (SI):
Line 5 I see that the authors have revised the address in the text, keep consistent in the SI.
Line 50 Table S2 Chlorophyll a (mg L−1) the “)” is incorrectly up-scripted.
Units of Chlorophyll a appeared as (µg L−1) (e.g., Fig. 2) or (mg L−1) (e.g., Table S2) through text and SI. Check and make them consistent.
Ensure that revisions/changes in supplementary information are “accepted” in word/latex/etc. for final publication.

Reviewer #2:
General appraisal
In their revised manuscript and author comments, Ingeniero et al. addressed my major concerns about the paper (namely, the assumption that nitrifier-denitrification was the only reductive process that may be occurring in their study site). While the authors do not spend a lot of time on the implications of their study for global biogeochemical cycles, the measurements are novel and provide another piece of the puzzle of marine NO cycling.

My main criticism of the revised manuscript is that it should be streamlined and revised for clarity. As it is, the discussion is a bit convoluted and difficult to read — especially section 4.3 (see below).

Also, at this stage of publication the data should be deposited in a repository with an associated DOI. Not enough to say it “will be made available.”

Specific comments

Lines 130-131: Why not just use the GSW MATLAB toolbox to calculate density?

Lines 292-303: I would drop these two paragraphs and just say, "Nonetheless, salinity alone is insufficient to explain the uneven distribution of NO at our study site, indicating that other parameters influence NO concentrations along the Elbe estuary." The salinity gradient tells you about mixing but not about the sources of NO, so I think it's sufficient in this section simply to point out that the weak negative correlation between NO and salinity indicates that higher NO concentrations in the Hamburg Port area mix out as your move towards the North Sea.

Line 304/Section 4.3: This section still needs to be streamlined and clarified. Is the main point just that high DIN doesn't necessarily lead to high NO? Or that there isn't much evidence for NO photoproduction in your study area?

Line 336/Table 1: Here, is N2O just the concentration or ∆N2O? Figure 7 is ∆N2O…

Line 360: Specify that this reaction is for ammonia-oxidizing bacteria; the exact pathway and enzymology for archaeal nitrification is still a matter of debate. Also, use the commonly accepted abbreviations for each enzyme to make this figure easier to read. E.g., amo instead of ammonium monooxygenase.

Lines 460-462: Wait, I thought you had a whole section on how your study challenges the assumption that higher concentrations of nitrogen nutrients automatically lead to increased dissolved NO concentration?

Technical corrections

Line 46: global estimates OF oceanic NO emissions
Line 423: should be "these sampling locations"

Best regards,
Yuan Shen

Hide

AR by Riel Carlo O. Ingeniero on behalf of the Authors (11 Jun 2024) Author's response Author's tracked changes Manuscript

ED: Publish as is (17 Jun 2024) by Yuan Shen

AR by Riel Carlo O. Ingeniero on behalf of the Authors (17 Jun 2024) Manuscript

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30 Jul 2024

Dissolved nitric oxide in the lower Elbe Estuary and the Port of Hamburg area

Riel Carlo O. Ingeniero, Gesa Schulz, and Hermann W. Bange

Biogeosciences, 21, 3425–3440, https://doi.org/10.5194/bg-21-3425-2024,https://doi.org/10.5194/bg-21-3425-2024, 2024

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Riel Carlo O. Ingeniero, Gesa Schulz, and Hermann W. Bange

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Our research is the first to measure dissolved NO concentrations in temperate estuarine waters, providing insights into its distribution under varying conditions and enhancing our understanding of its production processes. We found that the lower Elbe Estuary and the Hamburg Port area were a source of atmospheric NO. Our results suggest that NO in the lower Elbe Estuary was produced during nitrification, whereas NO in the Hamburg Port area was produced during nitrifier-denitrification.

Dissolved Nitric Oxide in the Lower Elbe Estuary and the Hamburg Port Area

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