the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 26 Mar 2026
Methane dynamics along the salinity gradient of the Scheldt estuary
Abstract. Estuaries are important natural sources of methane (CH4) to the atmosphere. These transitional aquatic systems connect freshwater, brackish and fully marine environments. The pronounced salinity gradient, characteristic of estuaries, impose particular challenges to microbes and influence the biogeochemical processes they mediate. Salinity changes can inhibit methanotrophic activity. Here we analysed methane dynamics and microbial communities in the estuary of the Scheldt River, which flows through northern France, western Belgium, and the southwestern Netherlands, and finally discharges into the North Sea. During a research cruise conducted in June 2022 from the river mouth to Antwerp, water samples were collected at ten stations along the estuarine salinity gradient. We investigated water column CH4 inventory and stable carbon isotope dynamics, together with methane oxidation rates. Elevated CH4 concentrations of up to 110 nM with associated δ¹³C -values of about −46 ‰ were found in the freshwater zone of the upper estuary (in the area of the city of Antwerp). Rather than a gradual decrease in CH4 towards the North Sea, we observed a second maximum of 180 nM in the marine zone, with a contrasting isotopic composition of -66 ‰ indicating distinct methanogenic pathways and/or substrates in the estuary. Methane oxidation rates showed no clear relationship with the salinity gradient. In contrast, the composition of the methanotrophic community shifted markedly along the estuary and the salinity gradient, with Methyloparacoccus and Crenothrix prevailing in the freshwater zone Methyloceanibacter and PLTB-vmat-59 dominating in the mixing and marine zones. Our results thus demonstrate that the salinity gradient is not the primary control on estuarine methane oxidation capacity but the community composition of the methane oxidizing bacteria (MOB). In sediments, the MOB community composition mirrored the patterns observed in the overlying water column, indicating that estuarine sediments represent both a key habitat and an important recruitment source sustaining methanotrophic communities in the water column. Despite an active microbial filter, methane oxidation accounted for only a minor fraction of the estuaries’ CH₄ budget. Most methane (about 98 %) was lost through advection and diffusive fluxes to the atmosphere, while a smaller fraction was exported to the North Sea, contributing to sustained methane supersaturation in coastal waters.
Competing interests: At least one of the (co-)authors is a member of the editorial board of Biogeosciences.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, published maps, institutional affiliations, or any other geographical representation in this paper. While Copernicus Publications makes every effort to include appropriate place names, the final responsibility lies with the authors. Views expressed in the text are those of the authors and do not necessarily reflect the views of the publisher.- Preprint
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RC1: 'Comment on egusphere-2026-1582', Anonymous Referee #1, 23 Apr 2026
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AC1: 'Reply on RC1', Annalisa Delre, 03 Jun 2026
We are grateful for the reviewer’s time and effort to check our manuscript
There were no Methylomonas detected? which is rather common in freshwaters ??
No sequences were assigned to the genus Methylomonas in our dataset. However, we detected sequences affiliated with the family Methylomonadaceae. Because these sequences could only be reliably classified at the family level, we did not feel confident assigning them to specific methanotrophic genera, including Methylomonas. To avoid overinterpretation of the taxonomic data, we therefore restricted our discussion to taxa that could be identified with sufficient taxonomic resolution. It is possible that some of the unclassified Methylomonadaceae sequences belong to Methylomonas.
I am a bit sceptical about the floating chamber flux measurements from a ship. The ship and its position toward the wind and waves influences greatly the exchange coefficient k, and in comparison, with flux calculations based on the Wannigkhof equation no corelation between the 2 approaches could be found in our own studies (unfortunately unpublished). Could you comment on this?
Unfortunately we did not have micrometeorological instrumentation with us on board allowing to confidentially apply Wanninkhof equation. We did try this in a back-of-the-envelope-type approach using wind data from Rijkwaterstaat (weather station near Vlissingen), and we also found a weak correlation between the two methods (chamber measurements vs Wanninkhof model). However, the use of the Wanninkhof equation in estuaries may introduce substantial uncertainties because gas transfer velocity is controlled not only by wind speed but also by tidal currents and estuary-specific hydrodynamic processes, as discussed by Abril et al., 2009 (Turbidity limits gas exchange in a large macrotidal estuary). We decided to use latter for our flux measurements.
Concerning the discussion about the influence / relation between salinity and methane concentration a dilution plot would be useful
We will add a figure to show the relation between salinity and methane concentration in the Supplementary section (see also comments to reviewer 2).
To close your budget calculation, methanogenesis from the sediment is calculated. But would your measured methane production rate be sufficient to for this theoretical rate? Can you relate these 2 rates?
While comparing these rates would be interesting, the methane production measurements were conducted on surface sediments only and were primarily intended to investigate isotopic signatures. The do thus not reflect areal methanogenesis rates and as a result, we do not consider them suitable for quantitatively constraining the methane budget at the scale of the estuary. We have therefore chosen not to extrapolate these rates beyond their original purpose.
In relation to the budget calculation: is there no tidal water movement, moving the water back and forth in the estuary? thus the residence time of the water in the estuary is much longer and MOX and emission could work longer…. Can you elaborate?
In our calculations, we used the average freshwater discharge to estimate water transport through the estuary and did not explicitly account for tidal recirculation. We acknowledge that incorporating tidal effects would likely increase the effective residence time and therefore provide more time for methane oxidation to occur. However, the difference between the measured methane oxidation rates and the estimated efflux is sufficiently large that our main conclusion remains unchanged: methane oxidation accounts for only a minor fraction of the overall methane budget, whereas most methane is release into the atmosphere.
Relating to the influence of salinity on MOB community and comparing the Schelde with the Elbe estuary: In the Elbe estuary the river and tidal currents are freely moving along the estuary, but the in the Schelde this might be different as there seem to many locks Thus, if salinity is more or less stable at a certain location this may better allow for a MOB community adapted to this specific salinity...... Could you comment on this?
Similar to the Elbe estuary, the Scheldt remains a highly dynamic tidal system in which water masses and the salinity gradient move along the estuary with the tidal cycle. In the case of the Scheldt, the sediments act as reservoirs for MOB, which are then transferred into the water column. In areas where salinity remains relatively stable over longer timescales, such as the freshwater region near Antwerp, environmental selection may indeed favor MOB taxa adapted to that specific salinity range. This interpretation is consistent with the observed differences in MOB community composition along the estuarine gradient.
P3L18 is the kilometration fixed? I know it the otherway round, km 0 is at the source of the river .....
We are aware that river kilometration is often defined with km 0 located at the river source. However, in this study we focus specifically on the estuarine section of the Scheldt rather than on the river as a whole. For this reason, we chose to define km 0 at the mouth of the estuary and to express distances upstream from this reference point.
P3L18 is not "lock" the more common word for it?
We will change the word to “locks”
P6L1 what does SSU stand for? If this is a universal primer, all RNA is amplifed, not only MOB-RNA ? L24 see comment above, and how can this unversal primer be used to quantify MOBs ?
SSU stands for small subunit ribosomal RNA gene. We used the universal primer pair 515F–806R, which targets both Bacteria and Archaea as already described in the M&M section. We chose this primer set because it minimizes the risk of underestimating specific methanotrophic groups that may not be captured by more targeted primers. This approach allows us to estimate the relative abundance of methanotrophic taxa within the total microbial community based on taxonomic assignment of the obtained sequences. Previous publications could already show that methanotrophic-specific primers strongly underestimate the abundance of MOB (eg. Tavormina et al., 2010).
P9 Fig 3C ??? what is a biological versus a technical replicate ?
Biological replicates refer to independent environmental samples collected from the same site and treated separately throughout the analytical workflow. In contrast, technical replicates are repeated analyses of the same sample and are used to assess methodological precision.
P9L27 methanotrophic ?
In this sentence we are discussing the number of 16S gene copies of the total community.
P11L5 A dilution plot (Mkonz versus salinity) could be helpful here (if only in the supplement)
A figure about methane concentration vs salinity will be added in the Supplementary section.
P11L10 are there any tributaries at the Schelde, near station 3 ? which could contribute high M konz ?
Yes, there is one and in principle it could contribute to high methane concentration. However, in a previous publication (Jacques et al., 2021) they did not find a distinct methane increase related to the influence of the tributary.
P11L10 would your observed methane production rate from the sediment be sufficient to result in the methane increase, could give an estimate here ?
See above. As discussed previously, the methane production measurements were conducted on surface sediments only and were primarily intended to investigate isotopic signatures rather than to quantify in situ methane production. Consequently, we consider it inappropriate to extrapolate these rates to estimate the observed methane increase in the water column. In this section, we therefore focus on the environmental data collected along the estuary.
P13L16 As your cruise was in June, seasonal variations of MOX (related to temperature) could also be taken into account
We agree that seasonal variations, including temperature effects on methane oxidation, are likely to influence the observed patterns. This aspect is already mentioned in the Conclusions section, and we will make this more explicit in the main text.
P15L6 = river discharge
We intentionally used the term volume flux rather than river discharge. While river discharge generally refers to the water discharge at the river mouth, our calculation refers to the total volume of water passing through a specific section of the estuary near Antwerp.
Citation: https://doi.org/10.5194/egusphere-2026-1582-AC1
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AC1: 'Reply on RC1', Annalisa Delre, 03 Jun 2026
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RC2: 'Comment on egusphere-2026-1582', Anonymous Referee #2, 08 May 2026
This submission suffers from a poor presentation of results, incorrect referencing of literature, imprecise wording. This comes as a surprise considering some of the co-authors are widely recognized in the scientific community, and do not seem to have provided the necessary guidance to the first author.
GENERAL COMMENT
This submission suffers from a poor presentation of results, incorrect referencing of literature, imprecise wording.
SPECIFIC COMMENTS
P1 L 10 : “Estuaries are important natural sources of methane (CH4) to the atmosphere”. Word “important” is meaningless. Quantitatively CH4 emissions from estuaries (0.2 TgCH4/yr) are negligible (=0.4%) compared to freshwater wetlands (150 TgCH4/yr) and inland waters (rivers+lakes+reservoirs) (77 TgCH4/yr) according to Rosentreter et al. (2022).
Statement P1 L 13 “Salinity changes can inhibit methanotrophic activity” is an over-simplification of available literature. This statement should be re-phrased or removed from abstract.
A more correct account of literature is given in P2 L33: “Several studies have shown that increasing salinity in estuaries is associated with reduced methane oxidation rates (de Angelis & Scranton, 1993;Osudar et al., 2015; Sherry et al., 2016).”
Osudar et al. (2015) and de Angelis & Scraton (1993) show indeed that specific MOX rates (or turnover time) decreased (or increased) along the salinity gradient of estuaries. However, as discussed by Osudar et al. (2015) this pattern is difficult to interpret because a lot of factors that can affect MOX co-vary with salinity such as nutrients, suspended matter, O2. Experiments of adding salt to a freshwater sample of sediment (Sherry et al., 2016) or of water (De Anglis & Scraton 1993) leads to a decrease of MOX. This is not surprising has there is an enormous osmotic stress to freshwater methanotrophs over a short time-period, as would be the case of any other type of freshwater micro-organism. This cannot be translated by an “inhibition of methanotrophic activity”. Osudar et al. (2017) shows that a gradual increase of salinity allows some of the more halotolerant methanotrophs to overcome the salinity stress, not leading to a collapse of MOX.
Some studies that looked into sediments with stable salinity conditions across sites with variable salinity. These studies do show that community composition of methanotrophs and rates of MOX are indeed different in response to salinity (Zhang et al. 2023).
The real question is if there is an interference at cellular level of salinity on methanotrophs (osmotic stress, enzymatic disruption) or are just marine conditions less favourable than freshwater to methanotrophs, due to dilution of microbial populations, of substrates (CH4), and of other potentially limiting factors (macro- and micro-nutrients)?
P2 L36: The work of Zhang et al. (2023) is mis-cited here. Their Figures 2 and 3 show very important changes with salinity in methanotroph community composition, as well as MOX rates.
P11 L 8 : “our data, together with previous results, challenge the paradigm that CH₄ and salinity are anticorrelated in estuaries.” Such paradigm never existed, and if it did exist, then it was already shattered almost 25 yrs ago by Middelburg et al. (2002) who showed in several European estuaries all kinds of shapes in profiles of CH4 vs salinity. Same comment applies for sentence L11-13. No previous publication has concluded naively that “ CH4 dynamics (...) are only governed by a two-endmember mixing”
P11L 23: The work of Poffenbarger et al. (2011) deals with fluxes of CH4 from sediments to the atmosphere in saltmarshes not MOX, there’s not a single measurement of MOX in this paper.
P11 L23: The work of HO et al. (2018) deals with methanotrophs in rice paddy soils. This seems irrelevant for discussing methanotrophy in the water column of an estuary.
P11 L 25: “In contrast, we found relatively high rates of MOx (up to 5 nM d⁻¹) throughout the entire estuary, apparently independent of salinity” There’s no plot of MOX vs salinity. Figure 3 only shows MOX vs station number. It is necessary to plot variables such as MOX and CH4 vs salinity and not just vs station number.
P11 L 15-33: Here are discussed MOX rates, however, it is well established that MOX depends primarily on CH4 availability (in oxygenated conditions). Hence, MOX and CH4 should co-vary, and it is advisable to normalize for CH4 and look into the variations of specific MOX (MOX:CH4) or turnover (CH4:MOX). For example the work of De Anglis & Scraton (1993) shows a very distinctive decrease of specific MOX as a function of salinity.
Based on the current analysis of MOX, it is difficult to understand what are the drivers of methanotrophy, it is then advisable to plot specific MOX as a function of other potential drivers.
P11 L 31-33: the paper of Zhang et al. (2023) shows very clearly in their Fig. 2 “changes in the composition of the MOB community as a function of salinity”. This is what you do seem to state in P12L7 : “These shifts are likely driven by the differential sensitivity of MOB groups to a range of salinities, which was discussed to shape community composition of MOB (Osudar et al., 2017, Zhang et al., 2023)”
P12L21: “However, despite the differential water column MOB community composition, overall MOx was not substantially affected by salinity (see previous section), but instead followed MOB abundances.” This statement is not supported by the data. Figure 3 shows that MOX rates change by a factor of 10 between stations 3 and 9. It would be useful if you actually plot MOX and specific MOX (MOX:CH4) vs the abundance of methanotrophs.
This would allow to actually test the hypothesis given in the following sentence “This suggest that salinity may indeed exert a selection pressure on the composition of the MOB community but that system-level MOx is determined by the abundance of MOB rather than their identity”.
I actually agree with this statement (CH4 will always be oxidized by whoever is present) but it would be nice you could actually test this with the data by plotting MOX or specific MOX vs the methanotroph abundance.
P12 L 26 : “week” ?
P12 L11 : “MOB abundance primarily governs overall MOx” this is wishful thinking, it is necessary to test this rigorously with the data rather than giving a vague account of patterns in Figure 3.
P12 L 31 “Considering the current velocity of the Scheldt (~95 km d-1, Meire et al., 2005), a typical doubling time of MOB (~9 days, Mayr et al., 2020) and the length of the estuary investigated here (87 km) it seems unlikely that shifts in MOB composition are primarily driven by growth within the water column.” This statement is incorrect. 95km/d should correspond to the average of tidal currents per day (or something equivalent). The residence time of water in this estuary is 1-3 months (de Brye et al. 2012) and not 0.9 days (=87/95).
P12 L35: The sediment and water are tightly linked in such an environment. In the Scheldt, sediment deposition and resuspension are strongly tidally driven, with large amounts of fine sediment alternately eroded, suspended, and deposited during each semidiurnal tide (Baeyens et al. 1997). So there is very active exchange benthic-pelagic of particles (and attached micro-organisms).
A simple way to test this is to plot specific MOX versus TSM.
P12 L 37: “bubbles peculating” ?
Section 4.4. Another consideration is that the sediments might transfer different quantities of CH4 with similar 13C/12C ratios at different sites in the estuary. So the source isotopic signature might be the same but the resulting concentration variable. On top of that there’s fractionation by MOX. This makes the interpretation of water column d13C-CH4 extremely tricky.
P15 L12 : “Similarly, considering the river’s discharge (110 m3s-1, Rijkswaterstaat) and our measured CH4 concentration of 71 nM (station 1), CH4 export into the North Sea (Fout) is ~674 mol CH4 d⁻¹”.
This is not correct and makes little sense. The concentration at the mouth of the estuary results from mixing of mostly seawater and a little bit of upstream water and you cannot simply multiply this concentration by the freshwater discharge to compute the outgoing flux.
Let’s take an element that is only present in seawater and absent in freshwater such as SO42-. The river input is 0. If you apply your computation, then the outflow from the estuary to the ocean would be massive, because the SO42- concentration would be very high at the mouth. But such large outflow does not make sense because the river input is zero, and there’s no generation of SO42- in the estuary. What is really happening is that you have an inflow of SO42- from the ocean to the estuary at the mouth. This can be computed using a simple box model, such as the LOICZ approach (initially developed for phosphate but can be applied to any element).
REFS
Baeyens et al. (1997) General description of the Scheldt estuary. Hydrobiologia 366, 1–14. https://doi.org/10.1023/A:1003164009031
de Brye et al. (2012) Water renewal timescales in the Scheldt Estuary, Journal of Marine Systems, 94, 74-86, https://doi.org/10.1016/j.jmarsys.2011.10.013
Osudar et al. (2017) Effect of salinity on microbial methane oxidation in freshwater and marine environments, Aquatic Microbial Ecology 80(2) https://doi.org/10.3354/ame01845
Rosentreter et al. (2021) Half of global methane emissions come from highly variable aquatic ecosystem sources, Nature Geoscience, 14, 225-230 https://doi.org/10.1038/s41561-021-00715-2
Zhang et al. (2023) Salinity significantly affects methane oxidation and methanotrophic community in Inner Mongolia lake sediments. Front. Microbiol. 13:1067017. https://doi.org/10.3389/fmicb.2022.1067017
Citation: https://doi.org/10.5194/egusphere-2026-1582-RC2 -
AC2: 'Reply on RC2', Annalisa Delre, 03 Jun 2026
Dear reviewer, thank you for taking the time to review our manuscript (plots are in the pdf file).
P1 L 10 : “Estuaries are important natural sources of methane (CH4) to the atmosphere”. Word “important” is meaningless. Quantitatively CH4 emissions from estuaries (0.2 TgCH4/yr) are negligible (=0.4%) compared to freshwater wetlands (150 TgCH4/yr) and inland waters (rivers+lakes+reservoirs) (77 TgCH4/yr) according to Rosentreter et al. (2022).
We will remove the word “important” in the revised version.
Statement P1 L 13 “Salinity changes can inhibit methanotrophic activity” is an over-simplification of available literature. This statement should be re-phrased or removed from abstract.
We will re-phrase the sentence with “Increasing salinity in estuaries has been associated with lower methane oxidation rates”.
Osudar et al. (2015) and de Angelis & Scraton (1993) show indeed that specific MOX rates (or turnover time) decreased (or increased) along the salinity gradient of estuaries. However, as discussed by Osudar et al. (2015) this pattern is difficult to interpret because a lot of factors that can affect MOX co-vary with salinity such as nutrients, suspended matter, O2. Experiments of adding salt to a freshwater sample of sediment (Sherry et al., 2016) or of water (De Anglis & Scraton 1993) leads to a decrease of MOX. This is not surprising has there is an enormous osmotic stress to freshwater methanotrophs over a short time-period, as would be the case of any other type of freshwater micro-organism. This cannot be translated by an “inhibition of methanotrophic activity”. Osudar et al. (2017) shows that a gradual increase of salinity allows some of the more halotolerant methanotrophs to overcome the salinity stress, not leading to a collapse of MOX.
Some studies that looked into sediments with stable salinity conditions across sites with variable salinity. These studies do show that community composition of methanotrophs and rates of MOX are indeed different in response to salinity (Zhang et al. 2023).
The real question is if there is an interference at cellular level of salinity on methanotrophs (osmotic stress, enzymatic disruption) or are just marine conditions less favourable than freshwater to methanotrophs, due to dilution of microbial populations, of substrates (CH4), and of other potentially limiting factors (macro- and micro-nutrients)?
We agree with the comment of the reviewer about the effect of salinity on the activity of methanotrophs. Answering to the last question, our results suggest that increasing salinity does not necessarily lead to a general inhibition of methane oxidation, but rather promotes a shift in methanotrophic community composition towards more halotolerant taxa, while community members which cannot adapt, vanish. This observation is consistent with Osudar et al. (2017), who demonstrated that gradual salinity increases allow certain methanotrophs to adapt to elevated salinity conditions without a complete collapse of methane oxidation activity. Therefore, salinity appears to act primarily as a selective pressure shaping methanotrophic communities rather than as a universal inhibitor of methane oxidation.
P2 L36: The work of Zhang et al. (2023) is mis-cited here. Their Figures 2 and 3 show very important changes with salinity in methanotroph community composition, as well as MOX rates.
We will remove the citation of Zhang et al.,(2023) from that sentence.
P11 L 8 : “our data, together with previous results, challenge the paradigm that CH₄ and salinity are anticorrelated in estuaries.” Such paradigm never existed, and if it did exist, then it was already shattered almost 25 yrs ago by Middelburg et al. (2002) who showed in several European estuaries all kinds of shapes in profiles of CH4 vs salinity. Same comment applies for sentence L11-13. No previous publication has concluded naively that “ CH4 dynamics (...) are only governed by a two-endmember mixing”
We thank the reviewer for this important clarification. We agree that previous studies, including Middelburg et al. (2002), have already demonstrated that methane distributions in estuaries often deviate from simple conservative mixing patterns. Our intention was not to suggest that previous work interpreted estuarine CH₄ dynamics solely in terms of two-endmember mixing, but rather to emphasize the complexity of the processes controlling methane distributions along estuarine gradients. We will therefore adjust the wording throughout the manuscript by replacing “challenge the paradigm” with more neutral phrasing. We will also modify the last sentence, which now reads “Our data further support previous observations that methane dynamics in estuaries are shaped by multiple biogeochemical and hydrodynamic processes beyond simple conservative mixing.”
P11L 23: The work of Poffenbarger et al. (2011) deals with fluxes of CH4 from sediments to the atmosphere in saltmarshes not MOX, there’s not a single measurement of MOX in this paper.
P11 L23: The work of HO et al. (2018) deals with methanotrophs in rice paddy soils. This seems irrelevant for discussing methanotrophy in the water column of an estuary.
We thank the reviewer for the corrections, we will replace the references with more suitable ones in the revised version.
P11 L 25: “In contrast, we found relatively high rates of MOx (up to 5 nM d⁻¹) throughout the entire estuary, apparently independent of salinity” There’s no plot of MOX vs salinity. Figure 3 only shows MOX vs station number. It is necessary to plot variables such as MOX and CH4 vs salinity and not just vs station number.
The sampling stations followed the estuarine salinity gradient, such that, plotting methane concentrations and methane oxidation rates against station number or salinity produces nearly identical spatial patterns. We originally chose station number to facilitate the identification of specific estuarine regions along the transect. However, we agree that this representation may obscure the direct relationship with salinity. We will clarify this point in the text and add plots of CH₄ and MOx versus salinity in the Supplementary section.
P11 L 15-33: Here are discussed MOX rates, however, it is well established that MOX depends primarily on CH4 availability (in oxygenated conditions). Hence, MOX and CH4 should co-vary, and it is advisable to normalize for CH4 and look into the variations of specific MOX (MOX:CH4) or turnover (CH4:MOX). For example the work of De Anglis & Scraton (1993) shows a very distinctive decrease of specific MOX as a function of salinity. Based on the current analysis of MOX, it is difficult to understand what are the drivers of methanotrophy, it is then advisable to plot specific MOX as a function of other potential drivers.
We thank the reviewer for the suggestion. We explored the relationship between methane oxidation and potential environmental drivers, following the reviewer’s recommendation. Based on these data, no significant correlation was observed between CH₄ and MOx (Pearson’s r = -0.84, p = 0.07). When considering first order constant (k’ or “specific MOx”) and salinity alone, a strong negative correlation was observed (Pearson’s , ). However, when accounting for additional environmental variables, the strength of this relationship decreased substantially (partial correlation: r = −0.63, p = 0.25), suggesting that salinity alone may not be the primary control on methanotrophic activity. These results support our interpretation that increasing salinity does not necessarily lead to a general inhibition of methane oxidation, but rather to a shift in methanotrophic community composition. To improve clarity, we will add the statistical results to the Supplementary section.
P11 L 31-33: the paper of Zhang et al. (2023) shows very clearly in their Fig. 2 “changes in the composition of the MOB community as a function of salinity”. This is what you do seem to state in P12L7 : “These shifts are likely driven by the differential sensitivity of MOB groups to a range of salinities, which was discussed to shape community composition of MOB (Osudar et al., 2017, Zhang et al., 2023)”
We are not sure what the reviewer’s concern is here.
P12L21: “However, despite the differential water column MOB community composition, overall MOx was not substantially affected by salinity (see previous section), but instead followed MOB abundances.” This statement is not supported by the data. Figure 3 shows that MOX rates change by a factor of 10 between stations 3 and 9. It would be useful if you actually plot MOX and specific MOX (MOX:CH4) vs the abundance of methanotrophs.
This would allow to actually test the hypothesis given in the following sentence “This suggest that salinity may indeed exert a selection pressure on the composition of the MOB community but that system-level MOx is determined by the abundance of MOB rather than their identity”.
I actually agree with this statement (CH4 will always be oxidized by whoever is present) but it would be nice you could actually test this with the data by plotting MOX or specific MOX vs the methanotroph abundance.
We thank the reviewer for the suggestion. We tested the correlation between MOx and methanotroph abundance. They are strongly positively correlated (Pearson’s , ). In addition, when considering specific methane oxidation rates (k’), the relationship with methanotroph abundance became even stronger in the partial correlation analysis (r = 0.89, p = 0.04). These statistical results will be added in the manuscript.
P12 L 26 : “week” ?
Our apology, this is a typo, the correct word is “weak”
P12 L11 : “MOB abundance primarily governs overall MOx” this is wishful thinking, it is necessary to test this rigorously with the data rather than giving a vague account of patterns in Figure 3
The results of the Pearson’s test are discussed in the comments regarding P12L21. As we are reporting observational data, we obviously cannot make causal inferences, but we can generate hypotheses.
P12 L 31 “Considering the current velocity of the Scheldt (~95 km d-1, Meire et al., 2005), a typical doubling time of MOB (~9 days, Mayr et al., 2020) and the length of the estuary investigated here (87 km) it seems unlikely that shifts in MOB composition are primarily driven by growth within the water column.” This statement is incorrect. 95km/d should correspond to the average of tidal currents per day (or something equivalent). The residence time of water in this estuary is 1-3 months (de Brye et al. 2012) and not 0.9 days (=87/95).
According to Mayr et al., 2020, ~9 days is the average doubling time of MOB. Still, when considering a doubling time of 9 days, it seems unlikely that a community shift is only explained by the growth of the MOB community from whatever is inoculated into the estuary from upstream.
P12 L35: The sediment and water are tightly linked in such an environment. In the Scheldt, sediment deposition and resuspension are strongly tidally driven, with large amounts of fine sediment alternately eroded, suspended, and deposited during each semidiurnal tide (Baeyens et al. 1997). So there is very active exchange benthic-pelagic of particles (and attached micro-organisms).
A simple way to test this is to plot specific MOX versus TSM.
We assume that TSM stands for total suspended matter. If so, unfortunately we do not have these data.
P12 L 37: “bubbles peculating” ?
Our apology, this is a typo, the correct word is “percolating”
Section 4.4. Another consideration is that the sediments might transfer different quantities of CH4 with similar 13C/12C ratios at different sites in the estuary. So the source isotopic signature might be the same but the resulting concentration variable. On top of that there’s fractionation by MOX. This makes the interpretation of water column d13C-CH4 extremely tricky.
We are not completely sure if we understood this comment. We agree that interpreting water-column δ¹³C-CH₄ signatures in estuaries is challenging because the observed isotopic composition reflects the combined effects of source signatures, mixing processes, and isotope fractionation during methane oxidation. However, we argue that differences in CH₄ release rates alone are unlikely to explain the pronounced isotopic differences observed in the estuary.
In our sediment incubation experiments, both sediment types were incubated under identical conditions (same temperature and sterile seawater), yet produced markedly different δ¹³C-CH₄ signatures (−57.8‰ at station 8 and −46.5‰ at station 10). These values closely matched the corresponding environmental observations (−55.2‰ and −46.4‰, respectively), supporting the interpretation that different sedimentary methane sources and/or methanogenic pathways contributed to the observed isotopic variability.
We already considered isotope enrichment caused by methane oxidation. Based on the measured isotopic shift, approximately 50% CH₄ consumption would be required to explain the observed enrichment solely by oxidation. However, the measured methane oxidation rates were too low to support such extensive fractionation. We therefore conclude that methane oxidation likely influenced the isotope signatures but cannot fully explain the observed spatial differences in δ¹³C-CH₄.
P15 L12 : “Similarly, considering the river’s discharge (110 m3s-1, Rijkswaterstaat) and our measured CH4 concentration of 71 nM (station 1), CH4 export into the North Sea (Fout) is ~674 mol CH4 d⁻¹”.
This is not correct and makes little sense. The concentration at the mouth of the estuary results from mixing of mostly seawater and a little bit of upstream water and you cannot simply multiply this concentration by the freshwater discharge to compute the outgoing flux.
Let’s take an element that is only present in seawater and absent in freshwater such as SO42-. The river input is 0. If you apply your computation, then the outflow from the estuary to the ocean would be massive, because the SO42- concentration would be very high at the mouth. But such large outflow does not make sense because the river input is zero, and there’s no generation of SO42- in the estuary. What is really happening is that you have an inflow of SO42- from the ocean to the estuary at the mouth. This can be computed using a simple box model, such as the LOICZ approach (initially developed for phosphate but can be applied to any element).
We considered the concentration of 71nM as a sampling point that reflects the combined influence of upstream riverine input, internal estuarine production and consumption, sediment exchange, and marine mixing. Importantly, even considering lower CH₄ concentrations near the estuarine mouth (e.g. ~50 nM reported by Jacques et al., 2021), the overall conclusion remains unchanged: most CH₄ produced within the estuary is lost to the atmosphere, while only a comparatively small fraction is transported toward the North Sea. We will make this part clearer in the text.
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AC2: 'Reply on RC2', Annalisa Delre, 03 Jun 2026
Status: closed
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RC1: 'Comment on egusphere-2026-1582', Anonymous Referee #1, 23 Apr 2026
The manuscript „Methane dynamics along the salinity gradient of the Scheldt
Estuary“ by Annalisa Delre et al. Is overall well written ms on relevant topic.
I have added minor comments directly into the pdf file and major comments are listed below:
Methods and Results:
- there were no Methylomonas detected? which is rather common in freshwaters ??
- I am a bit sceptical about the floating chamber flux measurements from a ship. The ship and its position toward the wind and waves influences greatly the exchange coefficient k, and in comparison, with flux calculations based on the Wannigkhof equation no corelation between the 2 approaches could be found in our own studies (unfortunately unpublished). Could you comment on this?
- Concerning the discussion about the influence / relation between salinity and methane concentration a dilution plot would be useful
Discussion:
- To close your budget calculation, methanogenesis from the sediment is calculated. But would your measured methane production rate be sufficient to for this theoretical rate? Can you relate these 2 rates?
- In relation to the budget calculation: is there no tidal water movement, moving the water back and forth in the estuary? thus the residence time of the water in the estuary is much longer and MOX and emission could work longer…. Can you elaborate?
- Relating to the influence of salinity on MOB community and comparing the Schelde with the Elbe estuary: In the Elbe estuary the river and tidal currents are freely moving along the estuary, but the in the Schelde this might be different as there seem to many locks Thus, if salinity is more or less stable at a certain location this may better allow for a MOB community adapted to this specific salinity...... Could you comment on this?
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AC1: 'Reply on RC1', Annalisa Delre, 03 Jun 2026
We are grateful for the reviewer’s time and effort to check our manuscript
There were no Methylomonas detected? which is rather common in freshwaters ??
No sequences were assigned to the genus Methylomonas in our dataset. However, we detected sequences affiliated with the family Methylomonadaceae. Because these sequences could only be reliably classified at the family level, we did not feel confident assigning them to specific methanotrophic genera, including Methylomonas. To avoid overinterpretation of the taxonomic data, we therefore restricted our discussion to taxa that could be identified with sufficient taxonomic resolution. It is possible that some of the unclassified Methylomonadaceae sequences belong to Methylomonas.
I am a bit sceptical about the floating chamber flux measurements from a ship. The ship and its position toward the wind and waves influences greatly the exchange coefficient k, and in comparison, with flux calculations based on the Wannigkhof equation no corelation between the 2 approaches could be found in our own studies (unfortunately unpublished). Could you comment on this?
Unfortunately we did not have micrometeorological instrumentation with us on board allowing to confidentially apply Wanninkhof equation. We did try this in a back-of-the-envelope-type approach using wind data from Rijkwaterstaat (weather station near Vlissingen), and we also found a weak correlation between the two methods (chamber measurements vs Wanninkhof model). However, the use of the Wanninkhof equation in estuaries may introduce substantial uncertainties because gas transfer velocity is controlled not only by wind speed but also by tidal currents and estuary-specific hydrodynamic processes, as discussed by Abril et al., 2009 (Turbidity limits gas exchange in a large macrotidal estuary). We decided to use latter for our flux measurements.
Concerning the discussion about the influence / relation between salinity and methane concentration a dilution plot would be useful
We will add a figure to show the relation between salinity and methane concentration in the Supplementary section (see also comments to reviewer 2).
To close your budget calculation, methanogenesis from the sediment is calculated. But would your measured methane production rate be sufficient to for this theoretical rate? Can you relate these 2 rates?
While comparing these rates would be interesting, the methane production measurements were conducted on surface sediments only and were primarily intended to investigate isotopic signatures. The do thus not reflect areal methanogenesis rates and as a result, we do not consider them suitable for quantitatively constraining the methane budget at the scale of the estuary. We have therefore chosen not to extrapolate these rates beyond their original purpose.
In relation to the budget calculation: is there no tidal water movement, moving the water back and forth in the estuary? thus the residence time of the water in the estuary is much longer and MOX and emission could work longer…. Can you elaborate?
In our calculations, we used the average freshwater discharge to estimate water transport through the estuary and did not explicitly account for tidal recirculation. We acknowledge that incorporating tidal effects would likely increase the effective residence time and therefore provide more time for methane oxidation to occur. However, the difference between the measured methane oxidation rates and the estimated efflux is sufficiently large that our main conclusion remains unchanged: methane oxidation accounts for only a minor fraction of the overall methane budget, whereas most methane is release into the atmosphere.
Relating to the influence of salinity on MOB community and comparing the Schelde with the Elbe estuary: In the Elbe estuary the river and tidal currents are freely moving along the estuary, but the in the Schelde this might be different as there seem to many locks Thus, if salinity is more or less stable at a certain location this may better allow for a MOB community adapted to this specific salinity...... Could you comment on this?
Similar to the Elbe estuary, the Scheldt remains a highly dynamic tidal system in which water masses and the salinity gradient move along the estuary with the tidal cycle. In the case of the Scheldt, the sediments act as reservoirs for MOB, which are then transferred into the water column. In areas where salinity remains relatively stable over longer timescales, such as the freshwater region near Antwerp, environmental selection may indeed favor MOB taxa adapted to that specific salinity range. This interpretation is consistent with the observed differences in MOB community composition along the estuarine gradient.
P3L18 is the kilometration fixed? I know it the otherway round, km 0 is at the source of the river .....
We are aware that river kilometration is often defined with km 0 located at the river source. However, in this study we focus specifically on the estuarine section of the Scheldt rather than on the river as a whole. For this reason, we chose to define km 0 at the mouth of the estuary and to express distances upstream from this reference point.
P3L18 is not "lock" the more common word for it?
We will change the word to “locks”
P6L1 what does SSU stand for? If this is a universal primer, all RNA is amplifed, not only MOB-RNA ? L24 see comment above, and how can this unversal primer be used to quantify MOBs ?
SSU stands for small subunit ribosomal RNA gene. We used the universal primer pair 515F–806R, which targets both Bacteria and Archaea as already described in the M&M section. We chose this primer set because it minimizes the risk of underestimating specific methanotrophic groups that may not be captured by more targeted primers. This approach allows us to estimate the relative abundance of methanotrophic taxa within the total microbial community based on taxonomic assignment of the obtained sequences. Previous publications could already show that methanotrophic-specific primers strongly underestimate the abundance of MOB (eg. Tavormina et al., 2010).
P9 Fig 3C ??? what is a biological versus a technical replicate ?
Biological replicates refer to independent environmental samples collected from the same site and treated separately throughout the analytical workflow. In contrast, technical replicates are repeated analyses of the same sample and are used to assess methodological precision.
P9L27 methanotrophic ?
In this sentence we are discussing the number of 16S gene copies of the total community.
P11L5 A dilution plot (Mkonz versus salinity) could be helpful here (if only in the supplement)
A figure about methane concentration vs salinity will be added in the Supplementary section.
P11L10 are there any tributaries at the Schelde, near station 3 ? which could contribute high M konz ?
Yes, there is one and in principle it could contribute to high methane concentration. However, in a previous publication (Jacques et al., 2021) they did not find a distinct methane increase related to the influence of the tributary.
P11L10 would your observed methane production rate from the sediment be sufficient to result in the methane increase, could give an estimate here ?
See above. As discussed previously, the methane production measurements were conducted on surface sediments only and were primarily intended to investigate isotopic signatures rather than to quantify in situ methane production. Consequently, we consider it inappropriate to extrapolate these rates to estimate the observed methane increase in the water column. In this section, we therefore focus on the environmental data collected along the estuary.
P13L16 As your cruise was in June, seasonal variations of MOX (related to temperature) could also be taken into account
We agree that seasonal variations, including temperature effects on methane oxidation, are likely to influence the observed patterns. This aspect is already mentioned in the Conclusions section, and we will make this more explicit in the main text.
P15L6 = river discharge
We intentionally used the term volume flux rather than river discharge. While river discharge generally refers to the water discharge at the river mouth, our calculation refers to the total volume of water passing through a specific section of the estuary near Antwerp.
Citation: https://doi.org/10.5194/egusphere-2026-1582-AC1
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RC2: 'Comment on egusphere-2026-1582', Anonymous Referee #2, 08 May 2026
This submission suffers from a poor presentation of results, incorrect referencing of literature, imprecise wording. This comes as a surprise considering some of the co-authors are widely recognized in the scientific community, and do not seem to have provided the necessary guidance to the first author.
GENERAL COMMENT
This submission suffers from a poor presentation of results, incorrect referencing of literature, imprecise wording.
SPECIFIC COMMENTS
P1 L 10 : “Estuaries are important natural sources of methane (CH4) to the atmosphere”. Word “important” is meaningless. Quantitatively CH4 emissions from estuaries (0.2 TgCH4/yr) are negligible (=0.4%) compared to freshwater wetlands (150 TgCH4/yr) and inland waters (rivers+lakes+reservoirs) (77 TgCH4/yr) according to Rosentreter et al. (2022).
Statement P1 L 13 “Salinity changes can inhibit methanotrophic activity” is an over-simplification of available literature. This statement should be re-phrased or removed from abstract.
A more correct account of literature is given in P2 L33: “Several studies have shown that increasing salinity in estuaries is associated with reduced methane oxidation rates (de Angelis & Scranton, 1993;Osudar et al., 2015; Sherry et al., 2016).”
Osudar et al. (2015) and de Angelis & Scraton (1993) show indeed that specific MOX rates (or turnover time) decreased (or increased) along the salinity gradient of estuaries. However, as discussed by Osudar et al. (2015) this pattern is difficult to interpret because a lot of factors that can affect MOX co-vary with salinity such as nutrients, suspended matter, O2. Experiments of adding salt to a freshwater sample of sediment (Sherry et al., 2016) or of water (De Anglis & Scraton 1993) leads to a decrease of MOX. This is not surprising has there is an enormous osmotic stress to freshwater methanotrophs over a short time-period, as would be the case of any other type of freshwater micro-organism. This cannot be translated by an “inhibition of methanotrophic activity”. Osudar et al. (2017) shows that a gradual increase of salinity allows some of the more halotolerant methanotrophs to overcome the salinity stress, not leading to a collapse of MOX.
Some studies that looked into sediments with stable salinity conditions across sites with variable salinity. These studies do show that community composition of methanotrophs and rates of MOX are indeed different in response to salinity (Zhang et al. 2023).
The real question is if there is an interference at cellular level of salinity on methanotrophs (osmotic stress, enzymatic disruption) or are just marine conditions less favourable than freshwater to methanotrophs, due to dilution of microbial populations, of substrates (CH4), and of other potentially limiting factors (macro- and micro-nutrients)?
P2 L36: The work of Zhang et al. (2023) is mis-cited here. Their Figures 2 and 3 show very important changes with salinity in methanotroph community composition, as well as MOX rates.
P11 L 8 : “our data, together with previous results, challenge the paradigm that CH₄ and salinity are anticorrelated in estuaries.” Such paradigm never existed, and if it did exist, then it was already shattered almost 25 yrs ago by Middelburg et al. (2002) who showed in several European estuaries all kinds of shapes in profiles of CH4 vs salinity. Same comment applies for sentence L11-13. No previous publication has concluded naively that “ CH4 dynamics (...) are only governed by a two-endmember mixing”
P11L 23: The work of Poffenbarger et al. (2011) deals with fluxes of CH4 from sediments to the atmosphere in saltmarshes not MOX, there’s not a single measurement of MOX in this paper.
P11 L23: The work of HO et al. (2018) deals with methanotrophs in rice paddy soils. This seems irrelevant for discussing methanotrophy in the water column of an estuary.
P11 L 25: “In contrast, we found relatively high rates of MOx (up to 5 nM d⁻¹) throughout the entire estuary, apparently independent of salinity” There’s no plot of MOX vs salinity. Figure 3 only shows MOX vs station number. It is necessary to plot variables such as MOX and CH4 vs salinity and not just vs station number.
P11 L 15-33: Here are discussed MOX rates, however, it is well established that MOX depends primarily on CH4 availability (in oxygenated conditions). Hence, MOX and CH4 should co-vary, and it is advisable to normalize for CH4 and look into the variations of specific MOX (MOX:CH4) or turnover (CH4:MOX). For example the work of De Anglis & Scraton (1993) shows a very distinctive decrease of specific MOX as a function of salinity.
Based on the current analysis of MOX, it is difficult to understand what are the drivers of methanotrophy, it is then advisable to plot specific MOX as a function of other potential drivers.
P11 L 31-33: the paper of Zhang et al. (2023) shows very clearly in their Fig. 2 “changes in the composition of the MOB community as a function of salinity”. This is what you do seem to state in P12L7 : “These shifts are likely driven by the differential sensitivity of MOB groups to a range of salinities, which was discussed to shape community composition of MOB (Osudar et al., 2017, Zhang et al., 2023)”
P12L21: “However, despite the differential water column MOB community composition, overall MOx was not substantially affected by salinity (see previous section), but instead followed MOB abundances.” This statement is not supported by the data. Figure 3 shows that MOX rates change by a factor of 10 between stations 3 and 9. It would be useful if you actually plot MOX and specific MOX (MOX:CH4) vs the abundance of methanotrophs.
This would allow to actually test the hypothesis given in the following sentence “This suggest that salinity may indeed exert a selection pressure on the composition of the MOB community but that system-level MOx is determined by the abundance of MOB rather than their identity”.
I actually agree with this statement (CH4 will always be oxidized by whoever is present) but it would be nice you could actually test this with the data by plotting MOX or specific MOX vs the methanotroph abundance.
P12 L 26 : “week” ?
P12 L11 : “MOB abundance primarily governs overall MOx” this is wishful thinking, it is necessary to test this rigorously with the data rather than giving a vague account of patterns in Figure 3.
P12 L 31 “Considering the current velocity of the Scheldt (~95 km d-1, Meire et al., 2005), a typical doubling time of MOB (~9 days, Mayr et al., 2020) and the length of the estuary investigated here (87 km) it seems unlikely that shifts in MOB composition are primarily driven by growth within the water column.” This statement is incorrect. 95km/d should correspond to the average of tidal currents per day (or something equivalent). The residence time of water in this estuary is 1-3 months (de Brye et al. 2012) and not 0.9 days (=87/95).
P12 L35: The sediment and water are tightly linked in such an environment. In the Scheldt, sediment deposition and resuspension are strongly tidally driven, with large amounts of fine sediment alternately eroded, suspended, and deposited during each semidiurnal tide (Baeyens et al. 1997). So there is very active exchange benthic-pelagic of particles (and attached micro-organisms).
A simple way to test this is to plot specific MOX versus TSM.
P12 L 37: “bubbles peculating” ?
Section 4.4. Another consideration is that the sediments might transfer different quantities of CH4 with similar 13C/12C ratios at different sites in the estuary. So the source isotopic signature might be the same but the resulting concentration variable. On top of that there’s fractionation by MOX. This makes the interpretation of water column d13C-CH4 extremely tricky.
P15 L12 : “Similarly, considering the river’s discharge (110 m3s-1, Rijkswaterstaat) and our measured CH4 concentration of 71 nM (station 1), CH4 export into the North Sea (Fout) is ~674 mol CH4 d⁻¹”.
This is not correct and makes little sense. The concentration at the mouth of the estuary results from mixing of mostly seawater and a little bit of upstream water and you cannot simply multiply this concentration by the freshwater discharge to compute the outgoing flux.
Let’s take an element that is only present in seawater and absent in freshwater such as SO42-. The river input is 0. If you apply your computation, then the outflow from the estuary to the ocean would be massive, because the SO42- concentration would be very high at the mouth. But such large outflow does not make sense because the river input is zero, and there’s no generation of SO42- in the estuary. What is really happening is that you have an inflow of SO42- from the ocean to the estuary at the mouth. This can be computed using a simple box model, such as the LOICZ approach (initially developed for phosphate but can be applied to any element).
REFS
Baeyens et al. (1997) General description of the Scheldt estuary. Hydrobiologia 366, 1–14. https://doi.org/10.1023/A:1003164009031
de Brye et al. (2012) Water renewal timescales in the Scheldt Estuary, Journal of Marine Systems, 94, 74-86, https://doi.org/10.1016/j.jmarsys.2011.10.013
Osudar et al. (2017) Effect of salinity on microbial methane oxidation in freshwater and marine environments, Aquatic Microbial Ecology 80(2) https://doi.org/10.3354/ame01845
Rosentreter et al. (2021) Half of global methane emissions come from highly variable aquatic ecosystem sources, Nature Geoscience, 14, 225-230 https://doi.org/10.1038/s41561-021-00715-2
Zhang et al. (2023) Salinity significantly affects methane oxidation and methanotrophic community in Inner Mongolia lake sediments. Front. Microbiol. 13:1067017. https://doi.org/10.3389/fmicb.2022.1067017
Citation: https://doi.org/10.5194/egusphere-2026-1582-RC2 -
AC2: 'Reply on RC2', Annalisa Delre, 03 Jun 2026
Dear reviewer, thank you for taking the time to review our manuscript (plots are in the pdf file).
P1 L 10 : “Estuaries are important natural sources of methane (CH4) to the atmosphere”. Word “important” is meaningless. Quantitatively CH4 emissions from estuaries (0.2 TgCH4/yr) are negligible (=0.4%) compared to freshwater wetlands (150 TgCH4/yr) and inland waters (rivers+lakes+reservoirs) (77 TgCH4/yr) according to Rosentreter et al. (2022).
We will remove the word “important” in the revised version.
Statement P1 L 13 “Salinity changes can inhibit methanotrophic activity” is an over-simplification of available literature. This statement should be re-phrased or removed from abstract.
We will re-phrase the sentence with “Increasing salinity in estuaries has been associated with lower methane oxidation rates”.
Osudar et al. (2015) and de Angelis & Scraton (1993) show indeed that specific MOX rates (or turnover time) decreased (or increased) along the salinity gradient of estuaries. However, as discussed by Osudar et al. (2015) this pattern is difficult to interpret because a lot of factors that can affect MOX co-vary with salinity such as nutrients, suspended matter, O2. Experiments of adding salt to a freshwater sample of sediment (Sherry et al., 2016) or of water (De Anglis & Scraton 1993) leads to a decrease of MOX. This is not surprising has there is an enormous osmotic stress to freshwater methanotrophs over a short time-period, as would be the case of any other type of freshwater micro-organism. This cannot be translated by an “inhibition of methanotrophic activity”. Osudar et al. (2017) shows that a gradual increase of salinity allows some of the more halotolerant methanotrophs to overcome the salinity stress, not leading to a collapse of MOX.
Some studies that looked into sediments with stable salinity conditions across sites with variable salinity. These studies do show that community composition of methanotrophs and rates of MOX are indeed different in response to salinity (Zhang et al. 2023).
The real question is if there is an interference at cellular level of salinity on methanotrophs (osmotic stress, enzymatic disruption) or are just marine conditions less favourable than freshwater to methanotrophs, due to dilution of microbial populations, of substrates (CH4), and of other potentially limiting factors (macro- and micro-nutrients)?
We agree with the comment of the reviewer about the effect of salinity on the activity of methanotrophs. Answering to the last question, our results suggest that increasing salinity does not necessarily lead to a general inhibition of methane oxidation, but rather promotes a shift in methanotrophic community composition towards more halotolerant taxa, while community members which cannot adapt, vanish. This observation is consistent with Osudar et al. (2017), who demonstrated that gradual salinity increases allow certain methanotrophs to adapt to elevated salinity conditions without a complete collapse of methane oxidation activity. Therefore, salinity appears to act primarily as a selective pressure shaping methanotrophic communities rather than as a universal inhibitor of methane oxidation.
P2 L36: The work of Zhang et al. (2023) is mis-cited here. Their Figures 2 and 3 show very important changes with salinity in methanotroph community composition, as well as MOX rates.
We will remove the citation of Zhang et al.,(2023) from that sentence.
P11 L 8 : “our data, together with previous results, challenge the paradigm that CH₄ and salinity are anticorrelated in estuaries.” Such paradigm never existed, and if it did exist, then it was already shattered almost 25 yrs ago by Middelburg et al. (2002) who showed in several European estuaries all kinds of shapes in profiles of CH4 vs salinity. Same comment applies for sentence L11-13. No previous publication has concluded naively that “ CH4 dynamics (...) are only governed by a two-endmember mixing”
We thank the reviewer for this important clarification. We agree that previous studies, including Middelburg et al. (2002), have already demonstrated that methane distributions in estuaries often deviate from simple conservative mixing patterns. Our intention was not to suggest that previous work interpreted estuarine CH₄ dynamics solely in terms of two-endmember mixing, but rather to emphasize the complexity of the processes controlling methane distributions along estuarine gradients. We will therefore adjust the wording throughout the manuscript by replacing “challenge the paradigm” with more neutral phrasing. We will also modify the last sentence, which now reads “Our data further support previous observations that methane dynamics in estuaries are shaped by multiple biogeochemical and hydrodynamic processes beyond simple conservative mixing.”
P11L 23: The work of Poffenbarger et al. (2011) deals with fluxes of CH4 from sediments to the atmosphere in saltmarshes not MOX, there’s not a single measurement of MOX in this paper.
P11 L23: The work of HO et al. (2018) deals with methanotrophs in rice paddy soils. This seems irrelevant for discussing methanotrophy in the water column of an estuary.
We thank the reviewer for the corrections, we will replace the references with more suitable ones in the revised version.
P11 L 25: “In contrast, we found relatively high rates of MOx (up to 5 nM d⁻¹) throughout the entire estuary, apparently independent of salinity” There’s no plot of MOX vs salinity. Figure 3 only shows MOX vs station number. It is necessary to plot variables such as MOX and CH4 vs salinity and not just vs station number.
The sampling stations followed the estuarine salinity gradient, such that, plotting methane concentrations and methane oxidation rates against station number or salinity produces nearly identical spatial patterns. We originally chose station number to facilitate the identification of specific estuarine regions along the transect. However, we agree that this representation may obscure the direct relationship with salinity. We will clarify this point in the text and add plots of CH₄ and MOx versus salinity in the Supplementary section.
P11 L 15-33: Here are discussed MOX rates, however, it is well established that MOX depends primarily on CH4 availability (in oxygenated conditions). Hence, MOX and CH4 should co-vary, and it is advisable to normalize for CH4 and look into the variations of specific MOX (MOX:CH4) or turnover (CH4:MOX). For example the work of De Anglis & Scraton (1993) shows a very distinctive decrease of specific MOX as a function of salinity. Based on the current analysis of MOX, it is difficult to understand what are the drivers of methanotrophy, it is then advisable to plot specific MOX as a function of other potential drivers.
We thank the reviewer for the suggestion. We explored the relationship between methane oxidation and potential environmental drivers, following the reviewer’s recommendation. Based on these data, no significant correlation was observed between CH₄ and MOx (Pearson’s r = -0.84, p = 0.07). When considering first order constant (k’ or “specific MOx”) and salinity alone, a strong negative correlation was observed (Pearson’s , ). However, when accounting for additional environmental variables, the strength of this relationship decreased substantially (partial correlation: r = −0.63, p = 0.25), suggesting that salinity alone may not be the primary control on methanotrophic activity. These results support our interpretation that increasing salinity does not necessarily lead to a general inhibition of methane oxidation, but rather to a shift in methanotrophic community composition. To improve clarity, we will add the statistical results to the Supplementary section.
P11 L 31-33: the paper of Zhang et al. (2023) shows very clearly in their Fig. 2 “changes in the composition of the MOB community as a function of salinity”. This is what you do seem to state in P12L7 : “These shifts are likely driven by the differential sensitivity of MOB groups to a range of salinities, which was discussed to shape community composition of MOB (Osudar et al., 2017, Zhang et al., 2023)”
We are not sure what the reviewer’s concern is here.
P12L21: “However, despite the differential water column MOB community composition, overall MOx was not substantially affected by salinity (see previous section), but instead followed MOB abundances.” This statement is not supported by the data. Figure 3 shows that MOX rates change by a factor of 10 between stations 3 and 9. It would be useful if you actually plot MOX and specific MOX (MOX:CH4) vs the abundance of methanotrophs.
This would allow to actually test the hypothesis given in the following sentence “This suggest that salinity may indeed exert a selection pressure on the composition of the MOB community but that system-level MOx is determined by the abundance of MOB rather than their identity”.
I actually agree with this statement (CH4 will always be oxidized by whoever is present) but it would be nice you could actually test this with the data by plotting MOX or specific MOX vs the methanotroph abundance.
We thank the reviewer for the suggestion. We tested the correlation between MOx and methanotroph abundance. They are strongly positively correlated (Pearson’s , ). In addition, when considering specific methane oxidation rates (k’), the relationship with methanotroph abundance became even stronger in the partial correlation analysis (r = 0.89, p = 0.04). These statistical results will be added in the manuscript.
P12 L 26 : “week” ?
Our apology, this is a typo, the correct word is “weak”
P12 L11 : “MOB abundance primarily governs overall MOx” this is wishful thinking, it is necessary to test this rigorously with the data rather than giving a vague account of patterns in Figure 3
The results of the Pearson’s test are discussed in the comments regarding P12L21. As we are reporting observational data, we obviously cannot make causal inferences, but we can generate hypotheses.
P12 L 31 “Considering the current velocity of the Scheldt (~95 km d-1, Meire et al., 2005), a typical doubling time of MOB (~9 days, Mayr et al., 2020) and the length of the estuary investigated here (87 km) it seems unlikely that shifts in MOB composition are primarily driven by growth within the water column.” This statement is incorrect. 95km/d should correspond to the average of tidal currents per day (or something equivalent). The residence time of water in this estuary is 1-3 months (de Brye et al. 2012) and not 0.9 days (=87/95).
According to Mayr et al., 2020, ~9 days is the average doubling time of MOB. Still, when considering a doubling time of 9 days, it seems unlikely that a community shift is only explained by the growth of the MOB community from whatever is inoculated into the estuary from upstream.
P12 L35: The sediment and water are tightly linked in such an environment. In the Scheldt, sediment deposition and resuspension are strongly tidally driven, with large amounts of fine sediment alternately eroded, suspended, and deposited during each semidiurnal tide (Baeyens et al. 1997). So there is very active exchange benthic-pelagic of particles (and attached micro-organisms).
A simple way to test this is to plot specific MOX versus TSM.
We assume that TSM stands for total suspended matter. If so, unfortunately we do not have these data.
P12 L 37: “bubbles peculating” ?
Our apology, this is a typo, the correct word is “percolating”
Section 4.4. Another consideration is that the sediments might transfer different quantities of CH4 with similar 13C/12C ratios at different sites in the estuary. So the source isotopic signature might be the same but the resulting concentration variable. On top of that there’s fractionation by MOX. This makes the interpretation of water column d13C-CH4 extremely tricky.
We are not completely sure if we understood this comment. We agree that interpreting water-column δ¹³C-CH₄ signatures in estuaries is challenging because the observed isotopic composition reflects the combined effects of source signatures, mixing processes, and isotope fractionation during methane oxidation. However, we argue that differences in CH₄ release rates alone are unlikely to explain the pronounced isotopic differences observed in the estuary.
In our sediment incubation experiments, both sediment types were incubated under identical conditions (same temperature and sterile seawater), yet produced markedly different δ¹³C-CH₄ signatures (−57.8‰ at station 8 and −46.5‰ at station 10). These values closely matched the corresponding environmental observations (−55.2‰ and −46.4‰, respectively), supporting the interpretation that different sedimentary methane sources and/or methanogenic pathways contributed to the observed isotopic variability.
We already considered isotope enrichment caused by methane oxidation. Based on the measured isotopic shift, approximately 50% CH₄ consumption would be required to explain the observed enrichment solely by oxidation. However, the measured methane oxidation rates were too low to support such extensive fractionation. We therefore conclude that methane oxidation likely influenced the isotope signatures but cannot fully explain the observed spatial differences in δ¹³C-CH₄.
P15 L12 : “Similarly, considering the river’s discharge (110 m3s-1, Rijkswaterstaat) and our measured CH4 concentration of 71 nM (station 1), CH4 export into the North Sea (Fout) is ~674 mol CH4 d⁻¹”.
This is not correct and makes little sense. The concentration at the mouth of the estuary results from mixing of mostly seawater and a little bit of upstream water and you cannot simply multiply this concentration by the freshwater discharge to compute the outgoing flux.
Let’s take an element that is only present in seawater and absent in freshwater such as SO42-. The river input is 0. If you apply your computation, then the outflow from the estuary to the ocean would be massive, because the SO42- concentration would be very high at the mouth. But such large outflow does not make sense because the river input is zero, and there’s no generation of SO42- in the estuary. What is really happening is that you have an inflow of SO42- from the ocean to the estuary at the mouth. This can be computed using a simple box model, such as the LOICZ approach (initially developed for phosphate but can be applied to any element).
We considered the concentration of 71nM as a sampling point that reflects the combined influence of upstream riverine input, internal estuarine production and consumption, sediment exchange, and marine mixing. Importantly, even considering lower CH₄ concentrations near the estuarine mouth (e.g. ~50 nM reported by Jacques et al., 2021), the overall conclusion remains unchanged: most CH₄ produced within the estuary is lost to the atmosphere, while only a comparatively small fraction is transported toward the North Sea. We will make this part clearer in the text.
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AC2: 'Reply on RC2', Annalisa Delre, 03 Jun 2026
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The manuscript „Methane dynamics along the salinity gradient of the Scheldt
Estuary“ by Annalisa Delre et al. Is overall well written ms on relevant topic.
I have added minor comments directly into the pdf file and major comments are listed below:
Methods and Results:
Discussion: