This study quantifies nitrous oxide (N2O) concentrations in the sea surface microlayer (SML, the upper 1 mm of the water column) and the underlying water (ULW) during a phytoplankton bloom in a mesocosm experiment. The authors also estimate N2O fluxes and discuss potential pathways for N2O production in the SML, including microbial nitrification, release from phytoplankton, and photochemodenitrification. Overall, this work addresses an important and understudied topic. It provides valuable data, as no previous research has measured N2O concentrations in the SML. Understanding this layer is critical because it may play a key role in regulating fluxes of this potent greenhouse gas.

My primary concern is the statistical analysis, which currently appears insufficient to fully address the research questions. A more rigorous examination of the dataset is necessary to reveal potential dynamics in N2O concentrations. The manuscript states that there are no temporal trends in N2O within the ULW and SML (line 201), that mean concentrations in both layers do not differ significantly, and that no diurnal patterns were detected (lines 201–210). However, beyond a t-test, the methods used to assess these trends are not described in detail. A simple comparison of averages is not adequate to rule out differences or identify underlying patterns. I strongly recommend that the authors apply more robust statistical approaches to explore these relationships and potential drivers among all measured variables. For example, Figure 1 shows notable changes in temperature and salinity during the experiment, could these influence N2O concentrations? Similarly, what about chlorophyll a or other parameters, such as surfactants? That might indicate the potential role of phytoplankton.

Temporal trends (including diel trends) may not be visually apparent but could emerge when covariates are incorporated into the analysis. Besides, “samples to measure N2O concentration at the SML were taken every three days alternating either 30 minutes past sunrise or 10 hours past sunrise.”. Whether the sample was taken 30 minutes past sunrise or 10 hours past sunrise could be relevant for the temporal trend. Strengthening the statistical framework would significantly enhance the manuscript’s contribution and provide deeper insights into the processes governing N2O dynamics at the sea surface.

My second concern relates to the Methods section. While I understand that many details of the incubation setup are described in Bibi et al. (2025), the current manuscript still lacks essential information needed to fully understand the experimental design. Readers should not have to rely entirely on another source to grasp the methodology. For example, the depth at which ULW samples were collected and the sampling procedure should be clearly stated. A brief explanation of the glass plate method for SML sampling may be beneficial, too. A brief description of the mesocosm facility is also necessary at the beginning, including the total volume of the setup, and if the setup has a mixing system.

In line 82, the authors mention that nutrients were added to trigger a phytoplankton bloom; the exact amounts or concentrations should be provided here rather than referring to the previous article. Additionally, the manuscript notes that “Jade Bay water was replenished with 4.5 L per day to replace the water removed by sampling.” The potential impact of this replenishment on N2O concentrations should be discussed, as it could influence the interpretation of the results. I wonder if the addition of water could cause mesocosm mixing or if it may add some N2O or dilute it.

Since the estimated rates of photochemodenitrification are derived from nitrite concentrations, the authors should provide a description of the analytical method used to measure nitrite, including its detection limit and sensitivity. Additionally, it would be important to discuss whether the nitrite detection limit could constrain the ability to identify diel variations in photochemodenitrification rates.

SML sampling method: The use of the glass plate technique for collecting N2O samples from the sea surface microlayer is not ideal, as it may underestimate N2O concentration. However, the discussion provided in Section 3.1 is valuable and helps address these concerns.

Some minor comments below:

Please check the section numbering.

Figures may need some work. See the asterisks on the axes titles, carefully check figure captions, and the dimensioning issue in Fig 2. It might be a problem during the preprint editing, but in my version of the manuscript, I see ULW samples as yellow circles rather than open circles.

The detailed explanation provided in sections 1.3 – 1.6, which includes equations that are often omitted in manuscripts, is excellent. The community will appreciate that the authors included this information.

Did the authors check for outliers in the dataset? If the maximum concentration of 16.6 nM is an outlier, this should be explicitly stated rather than repeatedly highlighted throughout the manuscript (e.g., lines 202, 207, …).

I recommend that the authors verify the N2O gas exchange calculations and, if possible, compare the approach used (based on Liss and Merlivat, 1986) with alternative parameterizations. The estimated flux values appear unusually high (up to 4.8 nmol N2O L^-1 h^-1).

Table 1. I suggest authors specify if the values from McLeod et al 2021 were cultures exposed to natural sunlight or if they were UV irradiated.

Note that lines 307 and 308 are missing in the preprint pdf.

Stoltenberg et al detail measurements of nitrous oxide (N2O) in the sea surface microlayer (SML) and underlying water (ULW) during a phytoplankton bloom in a mesocosm study using coastal North Sea waters. The results show no significant SML enrichment in the SML relative to the ULW - although as the authors note the technique used for measuring this is not optimal – and also increasing N2O supersaturation in both SML and ULW over the course of a 2-month long experiment. The interpretation of the data is somewhat cursory, with limited interpretation that relies on the unproven assumption that N2O is lost from the glass plate during SML measurement, and the identification of N2O source leans heavily on other studies with only limited analysis of ancillary data. I recommend the addition of a) more details on the mesocosm set up and operations, b) further investigation/discussion re glass plate use for dissolved gas measurement in the SML and c) further analysis of the light, temperature and nitrite data to determine whether these support the interpretation of the N2O source (or not).

1. Does the paper address relevant scientific questions within the scope of BG?

Yes

1. Does the paper present novel concepts, ideas, tools, or data?

Measuring N2O in the SSM is novel

1. Are substantial conclusions reached?

No

1. Are the scientific methods and assumptions valid and clearly outlined?

No, more methodological details are required for the mesocosm; it is insufficient to only refer to another publication. For example, “fleece filtration and protein skimming” are mentioned, but there are no further details and no references.  Conversely, there is repetition in the limited details provided on mesocosm set up, and also in 1.3 N2O sampling.

1. Are the results sufficient to support the interpretations and conclusions?

No, data for ancilliary parameters are not used sufficiently in the interpretation of the source of N2O

1. Is the description of experiments and calculations sufficiently complete and precise to allow their reproduction by fellow scientists (traceability of results)?

No, see comments re limited information provided on mesocosm set up

1. Do the authors give proper credit to related work and clearly indicate their own new/original contribution?

Yes

1. Does the title clearly reflect the contents of the paper?

Yes

1. Does the abstract provide a concise and complete summary?

Generally yes, but the abstract contains statements that are not supported by the data (see comments below)

1. Is the overall presentation well structured and clear?

Yes

1. Is the language fluent and precise?

Some typos and repetition. Final sentence in Discussion incomplete

1. Are mathematical formulae, symbols, abbreviations, and units correctly defined and used?

Yes

1. Should any parts of the paper (text, formulae, figures, tables) be clarified, reduced, combined, or eliminated?

See comments below

1. Are the number and quality of references appropriate?

References lacking in the discussion of uncertainties associated with the glass plate method

1. Is the amount and quality of supplementary material appropriate?

None provided

Comments

Major

Abstract

Line 16 - “However, the enrichment of N2O in the SML was most probably underestimated due to the loss of N2O during sampling with the glass plate method”. This may be the case  - and the authors would have been aware of this from the outset and could have considered alternative SML sampling techniques  - but no evidence is presented in the paper so it should be removed from the abstract or rephrased to say the effectiveness of the glass sampling was not tested.

“was most probably driven by photochemodenitrification”. This is also speculative in the absence of supporting evidence and also potential evidence to the contrary (see below). This would benefit from further analysis if this sentence is to remain in the abstract.

“Our results indicate that the role of the SML for N2O cycling in the surface ocean and its emissions to the atmosphere has been overlooked so far”. Although N2O has not been previously measured in the SML, the results and interpretation do not show significant N2O cycling in the SML so this sentence overstates their importance.

Methods

Line 135-139. There is an assumption that the overlying air in the mesocosm facility reflects that measured at the clean air site at Mace Head on the west coast of Ireland, whereas Jade Bay is surrounded by urban and industrial development. Is there any published evidence that local atmospheric N2O is consistent with that measured at Mace Head? It would have been better if N2O was measured in local air during the experiment. As the mesocosm roof was closed at night (and during rain events) this could also have influenced N2O in the overlying air. As the atmospheric N2O value is used to the saturation further justification is required here.

Line 160. “Fase was set to 0 when the roof of the mesocosm facility was closed”. The water was still being circulated by pumps so there would still have been some exchange particularly as the water supersaturated relative to the overlying air

Results

Line 190 & Figure 2. It should be noted that nitrite is higher in the SML than the ULW throughout most of the experiment. This seems to counter the interpretation that photochemodenitrification is the source of the N2O? This should be discussed

Lines 214-218. There appears to be a difference in N2O saturation between experiment phases with greater deviation between the SML and ULW in Phase 3. This should be mentioned and considered in the Discussion in relation to bloom phase and phytoplankton source of N2O

Line 219. As photochemodenitification rate is calculated using nitrite concentration, the change in the nitrite concentration between experiment days and phases, and between night and day, should be considered.

Discussion

Line 230. “The overall average N2O enrichment factor indicated an enrichment of N2O in the SML.” Clarify here that this enrichment is not statistically significant

Lines 235. The surfactant concentration also influences the amount of water retained on the glass plate, and so arguably the N2O concentration.

Lines 239. “the film of SLM water on the glass plate is exposed to enhanced wind speeds”. Its not so much a different windspeed, as the angle of exposure to the air movement and shear are different.

Line 242 “Overall, there seems be no constant loss factor …” Its unclear what this sentence is referring to, as there have been no previous studies of N2O in the SML but also there is no analysis of loss in this paper (other than qualitatively in the previous sentences).

Line 245. “we measured supersaturations of N2O in the SML despite the occurrence of surfactants in the SML which counteract the N2O release…”. Why “despite”? Wouldn’t suppression of gas exchange by surfactants enhance N2O supersaturation in the SML?

Line 247. “we conclude that there must have been a significant enrichment of N2O in the SML during the course of the mesocosm study.” Its not clear what this conclusion is based on other than assumption of loss from the glass plate. There are studies for other gases that have compared recovery of the glass plate with other SML techniques (see references below)

Line 248. “This is in line with the suggestion of a UV light-driven photochemical production of N2O (i.e. photochemodenitrification) which should be more enhanced in the SML because the SML is directly exposed to the sunlight.”

Line 279 “the in-situ production of N2O during the mesocosm study was most likely resulting from photochemodenitrification”,

Line 302 that “photochemical production…. occurs during day time only because they are light-dependent”.

Contrary to these statements (above) there doesn’t appear to be any evidence of a trend in N2O or nitrite concentration with experiment day or day-night variation in light in the SML? Further analysis comparing N2O, nitrite and light availability is required to support this interpretation and conclusion that photochemodenitrification is the source

Line 252. “This supersaturation of dissolved N2O was obviously resulting from a net in-situ production of N2O in the water.” This seems a reasonable interpretation based on the increase in N2O concentration in Figure 3; however, N2O saturation will also be affected by warming of the water from 16 to 24^oC during the experiment. For example, N2O supersaturation could increase if the mesocosm water was ventilating at a slower rate than it was warming; this can be tested using the N2O gas exchange rate in Line 297.

Line 282. “The time-series of N2O concentrations in both the SML and ULW showed no temporal trends”. There is a suggestion in Figure 3 that N2O concentration in the ULW decreased during the C. closterium bloom relative to the rest of the experiment, and it would be interesting to examine this statistically.

Conclusions

Line 312. “Consequently, a significant enrichment of N2O in the SML results in an enhanced N2O release to the atmosphere.” No evidence for this is presented so this needs to be rewritten.

Line 327. “we plea to develop a sampling method which minimizes the loss of dissolved trace gases…from the SML”. SML methods are compared for non-gaseous substances in the Cunliffe & Wurl (2014) Best Practise, and there have been comparisons of SML techniques for dissolved gases largely focussed on DMS (Yang et al 2001). For example, Saint-Macary et al (2023) show that the glass plate measures lower DMS enrichment in the SML than a permeable tubing technique which could support the interpretation here and in the opening paragraph of the Discussion.

Minor

Abstract

“Jade Bay (southern North Sea) water” – specify if this is coastal water and also location (Germany)

Introduction

Line 52 “release of N2O FROM the water side”

Line 55-56 “with the inherent problems of gas loss with the usually applied SML sampling methods (i.e. the glass plate and related methods).” Provide more information & references here

Methods

Line 72 How do small pumps reduce biofilm formation?

Fig 3. The y-axis scale for N2O concentration is too large; it can be reduced by half so that the variability in the data is more evident. Its also unclear why chlorophyll is shown in Fig 3 when theres no corresponding trend in N2O, and chlorophyll would be better combined in Figure 2 with nitrate and nitrite.

Line 268 “C. closteriuma” typo

Line 295 “Rough estimates” - honest, but perhaps not the best terminology in a publication

Line 307. The last sentence finishes abruptly - “because sources and the….”

References

Saint-Macary, A.D., Marriner, A., Barthelmeß, T., Deppeler, S., Safi, K., Costa Santana, R., Harvey, M. and Law, C.S., 2023. Dimethyl sulfide cycling in the sea surface microlayer in the southwestern Pacific–Part 1: Enrichment potential determined using a novel sampler. Ocean Science, 19(1), pp.1-15.

Yang, G.P., Watanabe, S. and Tsunogai, S., 2001. Distribution and cycling of dimethylsulfide in surface microlayer and subsurface seawater. Marine Chemistry, 76(3), pp.137-153.