Fate of methane in canals draining tropical peatlands

Domain: earth_science
Published: 2024-11-11
Editorial outcome: major_revision

Abstract
Tropical wetlands and freshwaters are major contributors to the growing atmospheric methane (CH4) burden. Extensive peatland drainage has lowered CH4 emissions from peat soils in Southeast Asia, but the canals draining these peatlands may be hotspots of CH4 emissions. Alternatively, CH4 oxidation (consumption) by methanotrophic microorganisms may attenuate emissions. Here, we used laboratory experiments and a synoptic survey of the isotopic composition of CH4 in 34 canals across West Kalimantan, Indonesia to quantify the proportion of CH4 that is consumed and therefore not emitted to the atmosphere. We find that CH4 oxidation mitigates 76.4 ± 12.0% of potential canal emissions, reducing emissions by ~70 mg CH4 m−2 d−1. Methane consumption also significantly impacts the stable isotopic fingerprint of canal CH4 emissions. As canals drain over 65% of peatlands in Southeast Asia, our results suggest that CH4 oxidation significantly influences landscape-scale CH4 emissions from these ecosystems.

Introduction
Wetlands and freshwaters contribute ~30–55% of global CH4 emissions, with significant emissions from tropical ecosystems2–4. Rising CH4 emissions from tropical wetlands due to temperature and rainfall anomalies have contributed substantially to the growing atmospheric CH4 burden5–8. In addition to climate change, ongoing disturbances to tropical wetlands like deforestation, drainage, fertilizer application, and slash and burn agriculture, as well as rewetting and restoration efforts, stand to impact their contribution to the global CH4 budget9–14. However, the impact of tropical wetland disturbance on CH4 cycling is not well understood.

In Southeast Asia, wetland disturbance has heavily impacted peatlands, destabilizing the large pool of soil carbon stored in the peat soils of this region15,16. Peatlands in Southeast Asia have undergone extensive drainage for oil palm plantations, timber, and other agriculture through the construction of canals17,18 that lower the water table and therefore lower CH4 emissions from peat soils19–21. Instead, the CH4 produced in peat soils is transported into canals via lateral flow22,23, increasing the relative importance of drainage canals as a source of CH4 emissions24,25. Canals can represent over 50% of peatland CH4 emissions in Southeast Asia26, but estimates of drainage canal CH4 emissions vary by several orders of magnitude27,28. Given that drainage increases the importance of aquatic carbon fluxes from tropical peatlands29, and the large uncertainty around canal CH4 emissions, greater understanding of the key controls and mechanisms driving canal CH4 emissions is needed to constrain their role in tropical peatland CH4 budgets.

One process that strongly influences freshwater CH4 emissions is microbial oxidation of CH4 to carbon dioxide. In other tropical freshwaters (e.g., rivers, lakes) CH4 oxidation attenuates CH4 emissions by 40 to nearly 100%23,30–32. The fraction of CH4 transported into canals from drained peatlands that is oxidized instead of emitted is highly uncertain, as are the factors that mediate CH4 oxidation in drainage canals. For example, both aerobic and anaerobic methanotrophic microbiota are found in tropical freshwaters32–35. As variation in canal water depth and discharge can impact dissolved oxygen in canals36,37, examining the relationship between CH4 oxidation and dissolved oxygen could inform how CH4 oxidation in canal waters may vary over space and time. Constraining the importance of CH4 oxidation in canals draining tropical peatlands is a key step to improving our understanding of the processes controlling CH4 emissions from these ecosystems, especially in the densely drained peatlands of Southeast Asia where canals can have a disproportionate impact on landscape-scale CH4 emissions.

Here, we address how much CH4 transported from drained tropical peatlands into canals is oxidized instead of emitted to the atmosphere. We quantified the percent of CH4 oxidized in 34 canal reaches that drain peat soils under varying land uses across West Kalimantan, Indonesia (Fig. 1, Supplementary Data 1) through shifts in the δ13C composition of CH4 during incubation experiments of canal waters and from field observations of in situ canal CH4 concentration and δ13C-CH4 (Fig. S1). We find that 47.3-91.3% of CH4 transported into canals from drained peatlands is oxidized instead of emitted. The fraction of CH4 that is oxidized is influenced by factors including dissolved oxygen, vegetation, and canal water depth. Overall, our results suggest that CH4 oxidation substantially attenuates CH4 emissions from canals, and as a result, may be a significant control of landscape-level CH4 emissions from drained peatlands in Southeast Asia.

Results
To confirm CH4 oxidation occurs in drainage canals, we incubated water from 13 canals at in situ dissolved CH4 and oxygen concentrations and measured the change in dissolved CH4 and δ13C-CH4 over time. On average, 53.8 ± 25.6% of the initial CH4 was consumed over the incubation period (17.6–99.7%) and δ13C-CH4 increased by 19.8 ± 17.7‰ (2.1–67.8‰, Fig. 2A, Table S1). The increase in δ13C-CH4 observed across incubated waters confirmed the loss of CH4 was from microbial oxidation, as CH4 oxidation leaves residual CH4 enriched in 13C38,39. Methane oxidation rates were variable across incubated waters (0.03–5.6 μmol CH4 L−1 d−1, Table S1) and were strongly influenced by initial CH4 concentration (Fig. S2). Neither CH4 production nor δ13C-CH4 depletion was observed in the canal waters during the incubation period.

From these data we calculated the first empirically derived isotopic fractionation factors for CH4 oxidation40 (αox) in peat-draining freshwaters. Ecosystem-specific values for αox are critical to estimating the percent of CH4 that is oxidized rather than emitted from the natural environment41,42. Mean αox was 1.022 ± 0.009 across the incubated canal waters (range: 1.002–1.039; Fig. 2B). The range of αox encompasses past observations from northern and temperate freshwaters incubated under in situ dissolved CH4 and oxygen concentrations and temperature42,43, as well as results from incubations of soil from subtropical rice paddies44 (αox of 1.025–1.033) that are often used in estimates of CH4 oxidation in tropical freshwaters30,32. While CH4 oxidation rates varied with initial CH4 concentration, we did not observe a correlation between αox and initial CH4 concentration, nor αox and CH4 oxidation rate (Fig. S2). Recent work in temperate lakes identified temperature, pH, and dissolved O2 as potential controls on αox43. Of these factors, αox was only weakly positively correlated with the initial dissolved O2 present in each of the incubated waters (p = 0.07). αox did not vary between surface and bottom waters of the subset of canals sampled at two depths for incubation experiments (1.024 ± 0.006 vs. 1.023 ± 0.012, n = 4 canals). As we did not find significant environmental correlates of αox, we used the mean value to estimate in situ CH4 oxidation as discussed below.

We find that the majority of CH4 transported into canals from drained tropical peatlands is oxidized instead of emitted to the atmosphere. Using the laboratory-derived αox values, measurements of in situ canal water δ13C-CH4 from 34 canal reaches (Supplementary Data 1), and measurements of source porewater δ13C-CH4 (Supplementary Data 2), we estimated that CH4 oxidation consumes 76.4 ± 12.0% of CH4 transported into canals (range: 47.3–91.3%). Considering the standard deviation of αox shifts the mean percent oxidized by ~10%, ranging from 65.5 ± 12.5% to 89.3 ± 8.9% (Figure. S3). Similarly, considering the standard deviation of the porewater source δ13C-CH4 measurements, the mean percent oxidized could range from 68.2 ± 16.1% to 82.4 ± 8.9% (Fig. S3). Our estimate of the fraction of CH4 transported into canals that is oxidized instead of emitted is consistent with Somers et al. (2023), who estimated that 70% of CH4 was oxidized in a canal draining a tropical peatland in Brunei using a reactive transport model. These results are also consistent with past work indicating that oxidation consumes ~80% of CH4 in blackwater rivers in the Amazon32 that have low pH and high concentrations of aromatic-rich, humic-like dissolved organic carbon like the canals in our study region29,45. Compared to previous work in lotic systems that use a similar approach as employed in our study, we find that oxidation mitigates a higher proportion of potential CH4 emissions in drainage canals than in headwater streams in temperate forests46 (55.6 ± 2.8%) or boreal peatlands47 (~60%).

Canal water dissolved CH4 concentration and δ13C-CH4 across our study region supports our finding that CH4 oxidation limits CH4 release from canals. Dissolved CH4 concentration and δ13C-CH4 in canal waters ranged from 0.05 to 31.6 μM and −71.9 to −34.1‰, respectively (Fig. 3B), and dissolved CH4 decreased with increasing δ13C-CH4 (R2 = 0.43, p < 0.001, Fig. S4). Previous observations in tropical river networks32 also observed a negative relationship between the concentration of CH4 in river waters and δ13C-CH4. In these rivers δ13C-CH4 also had a positive relationship with gene markers for methanotrophic bacteria, indicating that variation in CH4 concentration and δ13C-CH4 is influenced by CH4 oxidation. The consistent relationship between CH4 concentration and δ13C-CH4 observed across the drainage canals in our study and these tropical rivers supports the idea that differences in dissolved CH4 concentrations between canal reaches are influenced by CH4 oxidation.

It is unlikely that CH4 concentration in canal waters is dictated only by the amount of CH4 originally transported into canals from the surrounding landscape, including CH4 produced in peat soils and canal sediments. Methane produced in ombrotrophic tropical peat soils is highly depleted in 13C23,48. Unlike in lakes where δ13C-CH4 in littoral sediments and adjacent groundwater can differ by more than 10‰49, porewater δ13C-CH4 has not been shown to differ between canal bottoms and adjacent peat soils22. Porewater δ13C-CH4 collected from 6 profiles (40 to 150 cm depth) located alongside canal waters in our study region had a mean δ13C-CH4 of −85.0 ± 5.9‰, which was consistently more depleted than any observed canal δ13C-CH4 value (Supplementary Data 1, Supplementary Data 2). Porewater δ13C-CH4 varied more between sample depths within each profile than between profiles collected across the landscape, suggesting source δ13C-CH4 is similarly depleted in 13C throughout the study region. Methane production in the water column could also influence canal water CH4 concentration and δ13C-CH4. However, this is unlikely to explain our results because we did not observe net CH4 production in any of the laboratory incubations of canal waters, as CH4 concentration decreased and δ13C-CH4 increased in all incubated waters (Fig. 2A, Table S1). If canal water CH4 concentration were influenced solely by the total amount of CH4 produced and then transported into canal waters, we would expect canal water δ13C-CH4 to be similarly depleted across canals and not vary systematically with dissolved CH4 concentration. Given that CH4 concentrations varied ~600-fold alongside a ~40‰ range in δ13C-CH4, our results indicate that CH4 oxidation has a significant influence on canal water CH4 concentration and δ13C-CH4.

As CH4 oxidation was a major control of canal water CH4 concentration, diffusive CH4 emissions were also strongly influenced by the percent of CH4 oxidized. Diffusive CH4 emissions estimated from dissolved CH4 concentration (Supplemental Text 1) ranged from 1.0 to 761.8 mg CH4 m−2 d−1 (mean = 72.2 ± 151.2; median = 18.0) and decreased as the percent of CH4 oxidized increased (R2 = 0.54, p < 0.001; Fig. 3D). Measurements of CH4 emissions from floating chamber deployments at a subset of study sites (n = 12 canals, mean = 94.9 ± 142.3 CH4 m−2 d−1, median = 33.0, Supplementary Data 1) also indicated a negative relationship between CH4 emissions and the percent of CH4 oxidized (Fig. S5). By back-calculating what diffusive CH4 flux would be in the absence of oxidation, we estimate that CH4 oxidation reduces drainage canal CH4 emissions by a mean of 136.8 ± 154.1 mg CH4 m−2 d−1 (range: 9.9–684.2). Given the skewed distribution of dissolved CH4 concentrations that underlie this estimate, the median value of 72.1 mg CH4 m−2 d−1 (IQR: 36.2–173.6) may be a more robust estimate of the emissions attenuated by CH4 oxidation. Overall, our results provide evidence suggesting that CH4 oxidation mitigates the majority of potential CH4 emissions from canals on the landscape.

Of the studied controls on CH4 oxidation, dissolved oxygen and aquatic vegetation had the most significant influence on the percent of CH4 oxidized in canals as determined by canal water δ13C-CH4. We found that the percent of CH4 oxidized increased and dissolved CH4 concentration decreased with the concentration of dissolved oxygen at the canal water surface (0-10 cm; p < 0.05, Fig. 4A, Table S2). The relationship between dissolved oxygen and CH4 oxidation is consistent with oxidation mediated by aerobic methanotrophic bacteria, as has been observed in other stream and river networks32,46. While all canals had low dissolved oxygen (0.2 to 2.3 mg L-1), methanotrophic bacteria of the order Methylococcales have been shown to have the genetic potential for survival and methanotrophic activity in low oxygen environments50. Abundant Methylococcales have been identified in hypoxic tropical freshwaters where paired measurements of dissolved CH4 concentration and δ13C-CH4 indicate ongoing CH4 oxidation34,35. Our results further support the idea that aerobic CH4 oxidation occurs in tropical freshwaters with low dissolved oxygen. Furthermore, the percent of CH4 oxidized was higher in vegetated canals than those with open water (p = 0.01, Fig. 4B). Vegetation may enhance CH4 oxidation via radial oxygen loss from roots51,52 or via oxidation by epiphytic methanotrophs in submersed plants53. Although we did not observe a significant difference in dissolved oxygen based on the presence of aquatic vegetation (p > 0.05, Table S3), oxygen delivered to the water column by aquatic vegetation is likely rapidly consumed by methanotrophs or by competing aerobic heterotrophs as deposition of more labile organic carbon by aquatic vegetation could stimulate heterotrophic respiration in canal waters29.

Lower CH4 concentrations and more enriched δ13C-CH4 in vegetated canals could alternatively be explained by plant-mediated emissions54, which reduce CH4 concentration and enrich the δ13C of residual CH4 due to the isotopic fractionation of plant-mediated transport55. The deposition of labile organic matter from vegetation could also stimulate acetoclastic methanogenesis, which like CH4 oxidation would contribute towards larger δ13C-CH4 in vegetated canals39. However, acetoclastic methanogenesis likely contributes little to the δ13C-CH4 in vegetated canals because hydrogenotrophic methanogenesis has been identified as the dominant pathway in the ombrotrophic tropical peatlands of Southeast Asia23 and the Americas48,56. Disturbance in peatlands in Southeast Asia has been observed to increase the abundance of plant functional types associated with acetoclastic methanogenesis, like graminoids, but this shift does not appear to increase the abundance of acetoclastic methanogens57. While we cannot rule out the possible influence of acetoclastic methanogenesis on canal water δ13C-CH4, the lower dissolved CH4 concentration in vegetated canals (p = 0.02, Table S3) lends more support to the idea that vegetation enhances CH4 oxidation rather than acetoclastic CH4 production in canals.

Given that higher dissolved oxygen and the presence of aquatic vegetation were observed in canals with a shallower water depth (Fig. S6), canal water depth may indirectly mediate CH4 oxidation in drainage canal waters. Overall, dissolved oxygen in the surface water of canals (0–10 cm) decreased with the depth of water present in the canal (Kendall’s τ = −0.41, p < 0.05, Fig. S6). Dissolved CH4 concentration, and therefore estimated diffusive emissions, also had a weak but significant positive correlation with canal water depth (τ = 0.26, p = 0.03, Table S2). This result contradicts previous findings in drained ditches in temperate peatlands where CH4 emissions had a weak negative correlation with depth58, but these differing results may be explained by how well canal waters are mixed and aerated. For example, while we observed CH4 oxidation in canals where dissolved oxygen is low (<2.5 mg L−1) at the surface, dissolved oxygen may become depleted at depth29,45 to below the concentration needed for aerobic methanotrophs with high oxygen affinity. As such, CH4 oxidation may be limited to the surface waters of deeper canals, while in shallower canals oxidation may occur throughout the water column. Our study also only explicitly considered diffusive emissions. Measurements of CH4 ebullition from canals could further clarify the role of water depth in shaping net canal CH4 emissions, as ebullitive CH4 emissions vary with water depth59. Altogether, our results suggest that shallower, vegetated canals may attenuate a higher percentage of CH4 emission through CH4 oxidation.

Land use and seasonal precipitation cycles can both influence canal water depth and therefore dissolved oxygen. While we did not observe a significant impact of peatland land use on CH4 oxidation nor other parameters including dissolved oxygen (Table S4), peatland water table, which directly influences canal water levels, has been shown to vary significantly between land use types60. Canal water depth also varies 2- to 5-fold throughout the year in response to precipitation (Fig. S7), and reduced precipitation and flow during drier months may facilitate oxygen depletion by limiting turbulent mixing and re-aeration of canal waters27,61. Accordingly, past studies have reported higher canal CH4 emissions during dry periods27,28. While our study was not conducted during pronounced wet or dry periods, the dissolved CH4 and oxygen concentrations measured in our study fall within the range observed across Southeast Asia under varying land uses and seasons22,28,45,62,63 (Table S5). As such, we anticipate that water column CH4 oxidation is prevalent across canals draining degraded peatlands in Southeast Asia.

Our observations of canal CH4 emissions estimated from dissolved CH4 concentration (72.2 ± 151.2 mg CH4 m−2 d−1) and collected using floating chambers (94.9 ± 142.3 CH4 m−2 d−1) are within range of past observations from Indonesia27,28,64 and Malaysia26 where mean emissions range from 2.8 to 1073 mg CH4 m−2 d−1 (Table S6). The IPCC CH4 Emissions Factor for canals in tropical peatlands of 618.9 mg CH4 m−2 d−1 (2259 kg CH4 ha−1 y−1) was based on the only reported data27 at the time of the 2013 Wetlands Supplement65. This emission factor now represents the high end of field estimates to date among a still small number of existing studies and should be reconsidered to more accurately inventory the anthropogenic (e.g., from land use change) component66 of CH4 emissions from degraded tropical peatlands.

Despite high oxidation efficiencies, drainage canals can still emit large amounts of CH4. For example, in canals where ~50% of the CH4 transported from peatlands is oxidized we observe emissions > 200 mg CH4 m−2 d−1 (Figs. 3, S5). The canals in this study were primarily situated in smallholder agricultural systems (Supplementary Data 1), and the mean estimated diffusive CH4 emissions from canals presented here are 30x larger on an area basis than mean peat soil CH4 emissions from smallholder agriculture fields in West Kalimantan67. Thus, while CH4 oxidation plays a critical role in attenuating canal CH4 emissions, canals can still contribute significantly to landscape-level CH4 emissions from drained peatlands in Southeast Asia.

Beyond the rate of emissions, the δ13C signature of CH4 emitted from tropical wetlands and freshwaters are critical for constraining their contribution to the global CH4 budget, as δ13C-CH4 values underpins source partitioning by atmospheric inversion models. Using a floating chamber to capture CH4 emitted from a subset of the studied canals, we found that the mean δ13C-CH4 was -64.7 ± 10.5‰ (Fig. 5A). Canal CH4 emissions generally decreased as emitted δ13C-CH4 increased (Fig. S8), therefore the flux-weighted mean δ13C-CH4 was more negative at -69.0 ± 5.7‰ (Fig. 5A). Past observations of the δ13C signature of tropical wetland CH4 emissions68 indicate a range of -64‰ to -53‰. Our results suggest that the δ13C signature of CH4 emissions from drainage canals, and potentially drained peatlands in Southeast Asia as a whole due to the contribution of canals to landscape-scale CH4 emissions, is more negative than prior measurements from tropical wetlands. As such, implementing a distinct δ13C-CH4 source signature for Southeast Asian peatlands may improve top-down estimates of their CH4 emissions.

Furthermore, we find that the variation in δ13C of CH4 emissions from the canal water surface (−86.9 to -44.3‰) was largely explained by the percent of CH4 oxidized in canal waters (R2 = 0.68, p < 0.05; Fig. 5B). Previous studies have identified oxidation, alongside variation in methanogenic pathways and wetland vegetation, as one potential explanation for latitudinal differences in the δ13C of wetland CH4 emissions68–70. Our results indicate that once CH4 produced in peat soils is transported into canals, both the magnitude and the isotopic signature of canal CH4 emissions are strongly influenced by CH4 oxidation.

In summary, we demonstrate that CH4 oxidation can substantially attenuate CH4 emissions from canals draining peatlands in Southeast Asia. We estimate that CH4 oxidation mitigates > 50% of potential CH4 emissions from canals across West Kalimantan, Indonesia. As landscape-scale measurements of CH4 exchange in drained tropical peatlands indicate that canal networks contribute disproportionately to emissions from these ecosystems20, our results suggest that CH4 oxidation influences emissions not only from drainage canals but from degraded peatlands in Southeast Asia as a whole. Our results also have implications for peatland CH4 emissions in response to land use change, including peatland restoration efforts. For example, we find that oxidation attenuates more CH4 emissions from shallower canals that have higher dissolved oxygen concentrations. As such, efforts to rewet drained peatlands in Southeast Asia through canal blocking may impact CH4 oxidation and therefore canal CH4 emissions through changing canal water depth. Given the extensive networks of drainage canals in Southeast Asia and their substantial contribution to peatland CH4 emissions, land use changes impacting CH4 oxidation in canals will be reflected in the contribution of peatlands in Southeast Asia to the global CH4 budget.

Methods
Field sampling
Drainage canals in lowland peatlands were sampled in Kubu Raya and Mempawah Districts, West Kalimantan, Indonesia. Canals were sampled in Kubu Raya in May 2023 and Mempawah in April 2024. This region has an equatorial rainfall pattern with no clear wet and dry season71. There is heavy rainfall year-round, but the driest months of the year usually occur in July or August. We sampled waters from canals of different sizes (5 to 90 cm water depths, 0.5 to 6 m canal widths), canals with (n=15) and without aquatic vegetation (n=19), and canals situated on peatlands under a variety of land uses. Smallholder mixed agriculture is the most represented land use in this study, but the sampled canals also include areas in smallholder plantations (pineapple and oil palm), industrial oil palm plantations, and open undeveloped land (i.e., deforested and/or burned areas), as well as 1 canal in a degraded forest, to capture the heterogeneity of drainage canals in the region. At each canal, we measured the canal dimensions as well as water temperature (°C), pH, dissolved oxygen (mg L-1), conductivity (μS cm-1), and redox potential (Eh, in mV) using a Hanna Instruments HI9829 multiparameter meter. A summary of the canals included in this study is available in Supplementary Data 1.

To measure the isotopic composition of source CH4, we collected porewater profiles at 6 locations adjacent to a subset of the sampled canals. As shallow porewater is the primary source of discharge to drainage canals22, porewater was collected from 4-5 depths between 40 cm and 150 cm pending water table depth. Porewater was collected using a portable piezometer made of 3/8” stainless steel tubing housing 1/4” polyethylene tubing equipped with a coarse polypropylene screen to prevent collection of coarse debris (SedPoints, M.H.E. Products). Porewater samples were stored in 12 mL glass ExetainerTM vials (Labco Ltd.) without headspace and acidified in the field to a pH of less than 2 using 1.5 M HCl.

Canal CH4 concentration and δ13C
We collected surface water samples for analysis of dissolved CH4 concentration and δ13C-CH4 at all canals. Canal water samples were collected approximately 5 cm below the water surface and stored in 12 mL glass ExetainerTM vials (Labco Ltd.) without headspace. Canal waters collected for assessment of in situ dissolved CH4 concentration and δ13C-CH4 were acidified in the field to a pH of less than 2 using 1.5 M HCl. Canal water samples collected in 2023 (including incubations described below) and all porewater samples were analyzed at the Stable Isotope Facility at UC Davis via a Delta V Plus IRMS following headspace equilibration. Samples collected in 2024 were analyzed at Stanford University via a Picarro G2210-i cavity ring down spectrometer following headspace equilibration. Reference standards with CH4 mixing ratios and δ13C-CH4 of 10 ppm/-45.5‰ and 30 ppm/-69.0‰ were run before and after sample analysis on the Picarro G2210-i to check for accuracy and instrument drift. Dissolved CH4 concentrations were calculated considering the mixing ratio of CH4 in the equilibrated headspace, using the ideal gas law, and in solution, following Henry’s Law, using the neonDissGas package72.

Incubations
We collected canal waters at a subset (n=13) of the drainage canals for incubation experiments. Surface waters (~5 cm) were collected for all canals included in the incubation experiments, and at 5 of the canals we collected water from ~10 cm above the canal bottom using gas-tight tubing and a hand pump. Collecting these deeper canal waters for incubation experiments enabled us to account for any variability in isotopic fractionation of CH4 oxidation with depth in the water column that could impact our estimates of oxidation efficiency. For canal waters collected for incubation experiments, we collected waters as described above but only field acidified samples for the initial incubation time point. Incubations occurred in the dark at room temperature (~25°C) for 3 days. Duplicate samples for each canal (and depth, if applicable) were acidified every ~24 hours to pH < 2 using 1.5 M HCl to stop CH4 oxidation. All incubated waters were analyzed at the Stable Isotope Facility at UC Davis. Dissolved CH4 concentration was calculated as described above.

Dissolved CH4 concentration fell below the limit of quantification within 72 hours, as such incubation results only consider data from the first 2 days. One of the 5 deeper canal waters was omitted as CH4 was not detectable after 24 hours. We calculated potential oxidation rates as the change in CH4 concentration over the total incubation time. We also calculated the fractionation factor of CH4 oxidation, or αox, from the CH4 mixing ratios (in ppm) and δ13C-CH4 of the incubated waters using a simplified Rayleigh model40:

ln(CH4,t / CH4,0) = (αox / 1 - αox) * ln(1000 + δ13C-CH4,t / 1000 + δ13C-CH4,0) (1)

Plotting Eq. (1) with ln(1000 + δ13C-CH4) on the x-axis and ln(CH4) on the y-axis produces a line with a slope of (αox / 1 - αox). As such, we calculated the slope as the difference in ln(CH4) between the initial and final time points over the difference in ln(1000 + δ13C-CH4) over the same time and then solved for αox.

Canal CH4 emissions
We used a floating chamber to manually collect chamber headspace gasses at 12 of the sampled canals to assess CH4 emissions and emitted δ13C-CH4. A 20 cm diameter/2.1 L floating chamber was deployed on the canal water surfaces for 6 minutes in 2023 and 12 minutes in 2024. Chamber deployment time was increased in 2024 to ensure sufficient CH4 accumulation for analysis via Picarro CRDS. The floating chamber was not held in place, but due to low canal water flow (stagnant to ~0.1 m s-1) that chamber didn't travel during flux measurement. Three 15 mL gas samples were collected from the chamber headspace over the deployment time via a sampling syringe and injected into a pre-evacuated 12 mL glass ExetainerTM vial (Labco Ltd.). Floating chamber headspace gas samples from 2023 were analyzed at the Stable Isotope Facility at UC Davis and from 2024 at Stanford University, as described above. Methane emissions were calculated as the linear increase in chamber headspace CH4 mixing ratio over the measurement period and converted from ppm CH4 min-1 to mg CH4 m-2 d-1 using the Ideal Gas Law and the floating chamber dimensions. Fluxes were accepted if the linear increase in CH4 over time met the standards of R2 > 0.9 and p < 0.05. Emitted δ13C-CH4 was determined via a Keeling plot approach, in which the δ13C of CH4 emissions is the y-intercept of a linear regression of the inverse mixing ratio of CH4 versus the δ13C-CH4 of the corresponding sample73,74.

We calculated gas transfer velocity (k, m d-1) using data from the subset of canals where paired floating chamber CH4 fluxes and canal water CH4 concentrations were collected using Eq. (2):

Flux = k(CH4-canal - CH4-eq) (2)

Where CH4-canal is the concentration of CH4 in canal water, CH4-eq is the CH4 concentration at equilibrium the atmosphere (CH4-eq), and flux is the rate of CH4 emissions measured using the floating chamber. We used the median k value from the floating chamber deployments to estimate diffusive fluxes across all sampled (n=34) canals. While applying a uniform value introduces uncertainty into the estimates of diffusive fluxes, conditions across the study region are characterized by high canal water temperature, low canal flow velocity (~0.1 m s-1), and low wind speed. As such, factors that strongly influence CH4 degassing (e.g., solubility and turbulence) should have minimal variation relative to the ~600-fold variation in canal water CH4 concentration across study sites. Values were normalized to k600 for literature comparison. See Supplementary Text 1 for further discussion of approaches to estimate k.

Estimating percent oxidation
We used a simple box model to estimate the percent of CH4 transported from drained peatlands into canals that is oxidized and therefore not emitted to the atmosphere. The model calculated the percent oxidized based on the difference in δ13C between the source CH4 (e.g., peat porewater) and CH4 after oxidation (e.g., in the canal waters) as well as the isotopic fractionation of CH4 oxidation (αox), which we determined via incubations as described above. Oxidation efficiency (fox) was calculated using a Rayleigh model for closed systems42,75:

ln(1 - fox) = [ln(δsource + 1000) - ln(δcanal + 1000)] / [αox - 1] (3)

The closed system approach represents a lower bound on oxidation, as open system models often result in estimates of the percent oxidized > 100%.

The results presented in the main analyses and figures are estimates of the percent oxidized based on mean observed values of αox (1.022 ± 0.009, from incubations) and δsource (-85.0 ± 5.9‰, n=27 measurements from 6 porewater profiles, Supplementary Data 2). To characterize the uncertainty of our estimates due to variability in αox and δsource, we also report how our estimate varies when using ± 1 standard deviation of αox or δsource in Eq. (3). Varying αox or δsource by ± 1 standard deviation changes our estimate of the mean percent oxidized by ~10%.

To estimate the amount of CH4 emissions attenuated by CH4 oxidation, we back-calculated the concentration of CH4 in canal waters based on the fox value for each canal:

Predicted CH4 Concentration = Observed CH4 Concentration / (1 - fox) (4)

Using this predicted concentration, we calculated predicted diffusive CH4 fluxes as described above. We then subtracted the diffusive CH4 fluxes calculated from observed CH4 concentrations from the predicted CH4 fluxes based on the back-calculated concentrations to estimate the CH4 emissions mitigated by CH4 oxidation.

Statistical analysis
Statistical analysis and data visualization were performed in R v4.0.3. Data preparation was conducted using the dplyr package76. Data and analyses were visualized using the ggplot277 and patchwork78 packages. Statistical analyses were performed using the R Core Team stats package. Dissolved CH4 concentration and estimated diffusive emissions were log10-transformed prior to all statistical analysis to improve normality. If there were replicate measurements taken in a canal, the mean value of replicates was used in statistical analysis and data visualization. Summary statistics were calculated using all observations, including spatial replicates, to report the full range of observations. The level of significance for all analyses was 0.05.

We tested relationships between canal water CH4 concentration and δ13C-CH4 and between estimated diffusive CH4 fluxes and the percent of CH4 oxidized using least squares regression. We used Kendall’s rank correlation to assess the strength and direction of monotonic relationships between dissolved oxygen or canal depth and canal water dissolved CH4 concentration, δ13C-CH4, and percent CH4 oxidized. We used a non-parametric correlation for these analyses as relationships between CH4 oxidation and dissolved oxygen are often non-linear due to substrate saturation and potential inhibitory effects of oxygen above the optimal levels for CH4 oxidation in freshwater environments79. We used one-way ANOVA to assess the impact of vegetation and land use on canal properties and CH4 cycling.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a well-written, straight to the point manuscript that investigates aerobic methane oxidation in human-made canals draining tropical peatlands. Even though the aim of the study is relatively simplistic, the results are very relevant for a better understanding of methane cycling in these critical systems. The manuscript’s objective is clear, methodology is sound, and the data presented answer to the proposed questions. Data displays are of high quality. My major concern is a few overstatements along the text such as L73-74: “Overall, our results indicate CH4 oxidation is a major control on drainage canal CH4 emissions in peatlands in Southeast Asia.” One cannot claim that peatlands in Southeast Asia in general behave as the 21 canals sampled in a specific region of Indonesia. I suggest editing those sentences to a *potential* important role of methane oxidation in other peatlands across Southeast Asia. Same for L31-32 and parts of the conclusion.\nI have only a few other comments/questions listed below.\nL88: consider adding “on average”, 53.5%...\nL101-103: this claim depends on incubation temperatures. What was the incubation temperature in this study? In situ temperature?\nL128: remove “in canals”. Not needed as you start with “canal water…”\nL157-159: please add how much the oxidation mitigation represents in terms percentage.\nL173-174: yes, but you have a very narrow range of dissolved oxygen! Your whole range falls into hypoxic.\nL191-192: Why is that? Is it possible that methane oxidation consumes the O2 produced by plants in a cryptic cycle (sensors don't capture the availability of O2)? Please discuss.\nL250-251: what value of fractionation factor was used to calculate percent CH4 oxidized from the d13C-CH4?\nL294: how do the estimates of CH4 flux based on wind data of a meteorological station compares to the floating chamber measurements you’ve done? Are there significant differences in the calculated oxidation mitigation if you use one or the other method of flux estimation?\nL321-322: Can you report the R2 of these relationships? Methane oxidation usually follows a 1st order reaction, meaning that the natural logarithm of CH4 concentration shows a linear relationship with time and the slope of that relationship is the rate constant of oxidation.", "reviewer_2": "The manuscript by Perryman and colleagues addresses methane emissions from peatland ditches in Southeast Asia. They measured methane concentrations and isotopic composition in 20 ditches/canals (once) and conducted incubation experiments for a subset of these ditches. They find that methane oxidation within the water column consumes ~75% of methane, and thus net emissions from the ditches are significantly decreased by this “biofilter”.\nOverall, I enjoyed reading the paper. The writing is good, the figures nicely drawn, and the results support the conclusions. The research topic is important: we know that ditches emit large amounts of CH4 but we lack data from tropical peatlands. Additionally, most studies only consider net emission from the water surface, so process studies such as this add novel information.\nThat said, some parts of the manuscript slightly let it down. I was disappointed not to find raw data linked. Apparently this will be uploaded upon acceptance but it would have been useful to be able to evaluate it during my review.\nAnother issue is the relatively small sample number. The authors measured 20 canals, used 13 for incubation experiments, 5 for a second depth of incubation experiments (was this data reported in the MS?), then 8 for floating chambers. So the experimental design is slightly messy. That’s fine – fieldwork often runs that way. But using this small sample size (measured just once) the authors occasionally make quite sweeping statements: “our finding […] is likely robust across peatland drainage canals in Southeast Asia.” Considering industrial oil palm ditches are not well represented in the data, which focuses mostly on smallholder land, and the lack of temporal replication, this is quite a claim.\nAlso considering the sampling design, it seems that (assuming I interpret correctly) there is some pseudoreplication of sample points in some of the scatter plots, where duplicate measurements are presented separately. This is easily fixed (assuming my interpretation is correct).\nAnother minor issue relates to IPCC accounting. Ditch CH4 emissions are accounted for in the 2019 IPCC Refinement and (more relevant to this study) in the 2013 Wetlands Supplement. The Wetlands Supplement (Table 2.4*) (and associated paper https://doi.org/10.1007/s00027-015-0447-y) highlighted a lack of data from tropical peat ditches but did give an EF. It would be interesting to know how your emissions compare to the IPCC EF (they’re lower I think, if my conversions are correct), and also worth highlighting that ditch emissions are anthropogenic and should be accounted for in inventories.\n*https://www.ipcc.ch/publication/2013-supplement-to-the-2006-ipcc-guidelines-for-national-greenhouse-gas-inventories-wetlands/\nNote that I do not have experience of running isotope/oxidation experiments. I assume methods and analysis here are fine, but cannot comment with any authority myself.\nFollowing revision, I think the manuscript would be acceptable for publication in Nature Communications.\nMike Peacock\nLine comments follow\nThe abstract is concisely written. However, you write:\n“We find that CH4 oxidation mitigates potential canal CH4 emissions by 75.5 ± 12.8%, reducing CH4 emissions by 24.3 ± 32.3 mg CH4 m-2 d-“\nMany people reading will want to know the headline figure of mean CH4 emission without having to do any maths themselves. So you could rephrase to:\n“We find that CH4 oxidation mitigates potential canal emissions by 75.5 ± 12.8%, reducing mean emissions from XX to YY mg CH4 m-2 d-“\nAdditionally, if word limits allow I think you could add a few words into the abstract to say something about how many sites/canals you sampled, and if it was temporally replicated or just a synoptic snapshot.\nAlso, there’s a lot of “CH4 emissions” in this abstract. Once you’ve established that it’s CH4 we’re talking about then it’s fine (and will save you a few words) to just write “emissions”.\nL35. “Wetlands and freshwaters contribute ~30-55% of global CH4 emissions, largely from the tropics”\nIs this perhaps a bit of a stretch? Tropical wetlands are well implicated in rising atmospheric CH4 emissions, but northern waterbodies are also high emitters (as discussed by Rocher-Ros et al, who you cite here). And Rosentreter et al, in their global synthesis of aquatic CH4 say:\n“Despite the global coverage of our data, we did not detect clear latitudinal trends of methane emissions from aquatic ecosystems, except for the emissions from coastal wetlands peaking at 30° N.”\nSo I would suggest toning down this sentence.\nL49. “Given the increased importance of aquatic carbon fluxes in drained tropical peatlands…”\nTo me, this sentence could be clearer. Is this increased importance compared to the past (due to global change), or compared to undrained tropical peatlands, or to peatlands in other climate zones?\nL62. “Constraining the importance of CH4 oxidation in drainage canals in tropical peatlands is a key step to improving CH4 budgets of these ecosystems.”\nIs it? Isn’t simply measuring emissions the key step to improving budgets? What’s oxidised within the water column is irrelevant to the budget (but of course is interesting nonetheless).\nL68. I read the intro first, then methods, and was frustrated to find a lack of info on the canal reaches measured. But then eventually in the results Table S1 is mentioned. This should also be mentioned around L68 (study sites) and L275 (methods) so the reader knows this info is available.\nL68. Here you say 21 canal reaches, but Table S1 shows 20 (presumably because 34 was measured twice). Seems like 20 reaches is the correct one. This also applies to the “field sampling” section of methods.\nL88. “Across incubated waters, 53.5 ± 26.0% of the initial CH4 was consumed over the incubation period (17.6%-99.7%, Fig. 2A).”\nThis is interesting, and the following lines give info on general conc changes, but it would be nice to see a multi-panel figure in the SI showing raw conc changes for each incubation.\nL96. “averaged”. Also L140, 178, L229. Be clear if these are means (presumably) or medians.\nL137. “It is unlikely that CH concentration in canal waters is dictated only by the amount of CH originally transported into canals from the surrounding landscape.”\nAgreed, but in-situ production could also be relevant. Three decades (!) ago Roulet and Moore considered the potential importance of lateral transport vs in-situ production https://cdnsciencepub.com/doi/abs/10.1139/x95-055\nL161. There are 35 data points for 21 canal reaches (and presumably the same for Fig 4a too). It isn’t obvious where this 35 comes from. The “incubations” section mentions sample duplicates but I don’t think the “canal CH4” section does. Also, if you’re plotting sample duplicates as independent data points isn’t this essentially pseudoreplication which will artificially inflate your sample size and therefore statistical power? It seems more honest to calculate these correlations on means of the duplicates.\nL189. “As such, shallow canal water depths likely support more CH4 oxidation.”\nThis is interesting. Other studies, including of ditches (e.g. https://link.springer.com/article/10.1007/s13157-011-0170-y) have found that CH4 emissions are lower in deeper ditches, because there is more scope for oxidation within the water column. There’s also ebullition to consider, which is likely to be higher in shallow canals (because sediments will become warmer in these systems).\nL208. “our finding that the majority of CH4 transported into drainage canals is oxidized rather than emitted is likely robust across peatland drainage canals in Southeast Asia.”\nI acknowledge that it’s always nice to claim that research findings are widely applicable but this is, to me, overreaching. You measured 20 canals, once, in two study regions. I would suggest toning down this sentence.\nL218. Somewhere in the paper, and here seems a good place for it, it would be good to give some mean fluxes from other tropical ditch studies for comparison.\nL276. This says 100cm was your max water depth, Table S1 says 71cm.\nL277. Please add canal widths to Table S1.\nL278. “and canals situated on peatlands under a variety of land uses (smallholder mixed agriculture, smallholder oil palm, and industrial oil palm) to capture the heterogeneity of drainage canals in the region”\nAlthough you only have three in industrial oil palm. Perhaps worth emphasising that your study is biased towards smallholder systems?\nL298. If you’re calculating k solely using windspeed I guess that means these canals aren’t flowing (i.e. there is zero turbulence). If so, best to explicitly say so in the text. But I admit to getting a little hesitant when using wind speed to calculate emissions in these small waterbodies. These relationships between wind speed and k have been tested in lakes, but can you be sure they are appropriate for small, sheltered ditches (e.g. Fig. 1d)? I would suggest:\n1. Including your k values somewhere so the reader can check them. How do they compare to k values from other inland waters and from other ditches (or small waterbodies, e.g. ponds)?\n2. Can you calculate some k values from your floating chamber deployments? Do they compare to your windspeed-derived k values well?\nThere is some data in Fig. S4 where it seems like chambers are giving higher fluxes compared to k values. Is this due to problems with the k method, or are chambers capturing bubble events too? Do you seen any evidence of ebullition in these ditches?\nL307. “We collected canal waters at a subset (n = 13) of the drainage canals for incubation experiments”\nWhich canals? Can you add this into to Table S1.\nL309. How did you collect the deeper water samples? Please state.\nL315. “Duplicate samples for each canal (and depth, if applicable) were acidified every ~24 hours to pH < 2 using 1.5M HCl to stop CH4 oxidation.”\nSo you had two replicates for each measurement and then (presumably) took the mean of both? This is good, but it would be nice to see the reps data; how consistent are they to one another? Considering your small sample size this info would be useful.\nL338. What depth pore water? Please state."}, "author_response": "1 We appreciate the thorough and constructive feedback on our manuscript from all 3 reviewers and feel\n2 our manuscript has been strengthened through the revisions made in response to their suggestions. We\n3 provide a detailed response to all comments below. Here, we summarize the major revisions to our\n4 manuscript. These revisions improved the manuscript, but did not change the key finding that CH\n4\n5 oxidation significantly attenuates CH emissions from canals draining peatlands in the study\n4\n6 region. We feel that the revisions increased the scientific rigor of the work and we are grateful for the time\n7 and care all 3 reviewers invested in helping us improve the manuscript.\n8\n10 data addresses the shared concern amongst all 3 reviewers regarding the sample size and\n11 representativeness of the original dataset. The sample size for the revised manuscript is 34 canal\n12 reaches, compared to 21 canal reaches in the original manuscript. These new data also improve the\n13 spatial coverage and land use representation in our study, as they are from canals in another drained\n14 peatland area (~50-100 km away from the sites presented in the original manuscript) and increase the\n15 representation of industrial oil palm, smallholder plantations, and open undeveloped land. The data\n16 collected at these new sites are consistent with observations presented in the original manuscript. These\n17 include CH\n4\nconcentrations, δ13C-CH\n4\n, basic water chemistry, and canal dimensions for all sites, and\n18 floating chamber CH emissions for a subset of sites. As the expanded dataset shows the same trends in\n4\n4\n20 data further support that CH oxidation has a significant influence on emissions from canals draining\n4\n21 peatlands in Southeast Asia.\n22\n24 implications of our study. While the expansion of the dataset strengthened our results, we acknowledge\n25 that the data only includes sites in West Kalimantan. However, given the similarity in the physical and\n26 chemical properties of canals draining peatlands across Southeast Asia (see response to Reviewer 2 on\n27 line 259-270 of this document), we feel the study sites in our work are representative of the broader\n28 region. We have summarized results of canal CH concentrations and emissions from canals across the\n4\n29 major peatland regions in Southeast Asia in Tables S7-8, showing that our sample sites are similar to\n30 canals across Borneo, Peninsular Malaysia, and Sumatra draining peat soils under varying land uses.\n32 study aims to fill by providing isotopically-enabled insights to CH processing in canal waters.\n4\n33\n35 diffusive CH emissions and have added more methodological detail in the main and supplementary texts.\n4\n37 we determined gas transfer velocity from floating chamber deployments at a subset of study sites. The\n38 revised approach provides a more site- and CH -specific estimate of gas transfer velocity. This revision\n4\n39 increased our estimate of diffusive CH emissions, and the revised diffusive fluxes are in better\n4\n40 agreement with observations from floating chamber deployments at our study sites and prior work in other\n41 regions of Southeast Asia. Supplementary Text 1 of the revised manuscript discusses how approaches to\n42 estimating diffusive fluxes impact our results, and we include a new supplementary table (Table S8)\n43 comparing canal CH emissions across tropical peatlands.\n4\n44\n46 isotopic results, and therefore our estimates of the efficiency of methane oxidation in drainage canal\n47 waters. This includes: potential environmental correlates of isotopic fractionation (L106-113), methane\n48 production in canal sediments and canal waters (L149-168, L217-228), seasonality (L247-259), and\n1\n49 variability in methane production pathways and source methane isotopic composition across the\n50 landscape (L151-159, L217-228).\n51\n53 changes and other reviewer concerns can be found below. We have uploaded the data used to generate\n54 the figures and results presented in the manuscript to a Zenodo repository:\n55 https://doi.org/10.5281/zenodo.11155160\nNumber of canal 21 34 Added new data\nreaches additional sites\nLand use Smallholder mixed Smallholder mixed Added new data\nrepresentation agriculture, industrial oil agriculture, industrial oil additional sites and\npalm, smallholder palm, smallholder land use types\nplantation plantation, open\nundeveloped, degraded\nforest\nEstimated percent 75.5 ± 12.8% 76.4 ± 12.0% Added new data from\noxidized (n = 35 observations (n = 48 observations additional sites\nfrom 21 canal reaches) from 34 canal reaches)\nEstimated diffusive 16.1± 33.2 CH m-2 d-1 72.2 ± 151.2 CH m-2 d- Revised approach to\n4 4\nfluxes (n = 35 observations 1 estimating gas transfer\nfrom 21 canal reaches) (n = 48 observations velocity and added new\nfrom 34 canal reaches) data from additional\nsites\nFloating chamber 98.9 ± 153.7 CH m-2 d- 94.9 ± 142.3 CH m-2 d- Added new data from\n4 4\nfluxes 1 (n = 12 observations 1 additional sites\nfrom 7 canal reaches) (n = 18 observations\nfrom 12 canal reaches)\n59 tempered some statements in the manuscript to avoid overstating the implications of our study. We feel\n60 the new data added during revision strengthen our finding that CH oxidation is a major influence on canal\n4\n4\n62 acknowledge the potential limitations of the findings given the spatial extent of our sampling. We also\n63 modified our approach to calculating oxidation rates from the laboratory incubations in response to their\n64 question about the incubation data. This resulted in a negligible change to the results. Please see below\n65 for detailed responses to all comments raised by Reviewer 1.\n66\n68 human-made canals draining tropical peatlands. Even though the aim of the study is relatively simplistic,\n69 the results are very relevant for a better understanding of methane cycling in these critical systems. The\n70 manuscript’s objective is clear, methodology is sound, and the data presented answer to the proposed\n71 questions. Data displays are of high quality. My major concern is a few overstatements along the text\n72 such as L73-74: “Overall, our results indicate CH4 oxidation is a major control on drainage canal CH4\n2\n73 emissions in peatlands in Southeast Asia.” One cannot claim that peatlands in Southeast Asia in general\n74 behave as the 21 canals sampled in a specific region of Indonesia. I suggest editing those sentences to a\n75 *potential* important role of methane oxidation in other peatlands across Southeast Asia. Same for L31-\n76 32 and parts of the conclusion.\n77\n79 the implications of our findings for CH emissions from canals and the tropical peatlands they drain.\n4\n81\n4\n83 significantly influences landscape-scale CH emissions from these ecosystems.”\n4\n84\n4 4\n86 canals”\n87\n4 4\n89 from canals draining peatlands in Southeast Asia. We estimate that CH oxidation mitigates >50% of\n4\n90 potential CH emissions from canals across West Kalimantan, Indonesia. As landscape-scale\n4\n91 measurements of CH exchange in drained tropical peatlands indicate that canal networks contribute\n4\n92 disproportionately to emissions from these ecosystems18, our results suggest that CH oxidation\n4\n93 influences emissions not only from drainage canals but from degraded peatlands in Southeast Asia as a\n94 whole.”\n95\n97\n100\n101 “On average, 53.8 ± 25.6% of the initial CH was consumed over the incubation period (17.6%-99.7%)\n4\n102 and δ13C-CH increased by 19.8 ± 17.7‰ (2.1-67.8‰, Fig. 2A).”\n4\n103\n105 study? In situ temperature?\n107\n110\n4\n113 presented in the first paragraph of this section of the paper, therefore this information is given earlier in\n114 this section. L124-125:\n115\n116 “...we estimated that CH oxidation consumes 76.4 ± 12.0% of CH transported into canals (range: 47.3-\n4 4\n117 91.3%).”\n118\n120 hypoxic.\n3\n4\n122 aerobic methanotrophs (L196-206):\n123\n124 “We found that the percent of CH oxidized increased and dissolved CH concentration decreased with\n4 4\n125 the concentration of dissolved oxygen at the canal water surface (0-10 cm; p < 0.05, Fig. 4A, Table S4).\n4\n127 aerobic methanotrophic bacteria, as has been observed in other stream and river networks31,45. While all\n128 canals had low dissolved oxygen (0.2 to 2.3 mg L-1), methanotrophic bacteria of the order\n130 in low oxygen environments49. Abundant Methylococcales have been identified in hypoxic tropical\n131 freshwaters where paired measurements of dissolved CH concentration and δ13C-CH indicate ongoing\n4 4\n4 4\n133 freshwaters with low dissolved oxygen.”\n134\n136 cryptic cycle (sensors don't capture the availability of O2)? Please discuss.\n138\n139 “Vegetation may enhance CH oxidation via radial oxygen loss from roots50,51 or via oxidation by epiphytic\n4\n140 methanotrophs in submersed plants52. Although we did not observe a significant difference in dissolved\n141 oxygen based on the presence of aquatic vegetation (p > 0.05, Table S5), oxygen delivered to the water\n142 column by aquatic vegetation is likely rapidly consumed by methanotrophs or by competing aerobic\n143 heterotrophs as deposition of more labile organic carbon by aquatic vegetation could stimulate\n144 heterotrophic respiration in canal waters28.”\n145\n149\n150 “The percent of CH oxidized in drainage canal waters (estimated from dissolved δ13C-CH using α =\n4 4 ox\n151 1.022) versus the δ13C of CH emitted from the corresponding canal.”\n4\n152\n154 floating chamber measurements you’ve done? Are there significant differences in the calculated oxidation\n155 mitigation if you use one or the other method of flux estimation?\n157 than wind speed) in response to feedback from Reviewer 2 and 3. Please see L417-431 for an\n158 explanation of the revised approach:\n159\n160 “We calculated gas transfer velocity (k, m d-1) using data from the subset of canals where paired floating\n161 chamber CH fluxes and canal water CH concentrations were collected using Eqn. (2):\n4 4\n162\n4-canal 4-eq\n164\n4-canal 4 4-eq 4\n166 the atmosphere (CH ), and flux is the rate of CH emissions measured using the floating chamber. We\n4-eq 4\n167 used the median k value from floating chamber deployments to estimate diffusive fluxes across all\n168 sampled (n = 34) canals. While applying a uniform value introduces uncertainty into the estimates of\n169 diffusive fluxes, conditions across the study region are characterized by high canal water temperature,\n4\n170 low canal flow velocity (~0.1 m s-1), and low windspeed. As such, factors that strongly influence CH\n4\n171 degassing (e.g., solubility and turbulence) should have minimal variation relative to the ~600-fold variation\n172 in canal water CH concentration across study sites. Values were normalized to k for literature\n4 600\n173 comparison. See Supplementary Section 1 for further discussion of approaches to estimate k.”\n174\n176 in the revised manuscript are higher, thus so are the estimates of emissions mitigated by CH oxidation.\n4\n177\n179 reaction, meaning that the natural logarithm of CH4 concentration shows a linear relationship with time\n180 and the slope of that relationship is the rate constant of oxidation.\n182 mitigate issues with linearity, we revised our calculations to estimate potential oxidation rates as the\n183 difference in the initial and final CH concentrations (mean of 2 replicates for each time point) divided by\n4\n184 the incubation time. For consistency, we also revised our calculation of α to use just the initial and final\nox\n185 time points. Denfeld et al. (2016; JGR Biogeosciences) previously used initial and final time points to\n186 calculate α in lakes using similar calculations. We have clarified this change in our approach in L388-\nox\n187 397:\n188\n189 “We calculated potential oxidation rates as the change in CH concentration over the total incubation\n4\n190 time. We also calculated the fractionation factor of CH oxidation, or α , from the CH mixing ratios (in\n4 ox 4\n191 ppm) and δ13C-CH of the incubated waters using a simplified Rayleigh model39:\n4\n192\n193 𝑙𝑛(1−𝑓 ) = [𝑙𝑛(𝛿 +1000) −𝑙𝑛(𝛿 +1000)]/[𝛼 −1] (Eqn. 3)\n𝑜𝑥 𝑠𝑜𝑢𝑟𝑐𝑒 𝑐𝑎𝑛𝑎𝑙 𝑜𝑥\n194\n4 4\n196 slope of (α /1-α ). As such, we calculated the slope as the difference in ln(CH ) between the initial and\nox ox 4\n197 final time points over the difference in ln(1000 + δ13C-CH ) over the same time, and then solved for α .”\n4 ox\n198\n200 main findings on our paper are highly dependent on α , the change to the mean α value was negligible,\nox ox\n201 now reporting 1.022 ± 0.009 vs. 1.022 ± 0.008.\n4 ox\nOriginal Draft 0.3 to 6.6 1.022 ± 0.008 (1.002 to 1.035)\nRevised Manuscript 0.3 to 5.6 1.022 ± 0.009 (1.002 to 1.039)\n205 several revisions in response to the concerns raised by Dr. Peacock:\n206\n207 1. We have increased the sample size for estimating the fraction oxidized from 21 canal reaches to\n208 34 canal reaches with the addition of 13 new sample sites. Adding these data also improved the\n209 representation of canals in industrial oil palm plantations, among other land uses, in our dataset\n210 and expanded the spatial coverage of our study.\n5\n211 2. We have revised our statistical approach and conducted statistical analyses using mean values in\n212 cases where replicate measurements were collected. All revised data visualizations were also\n213 produced from mean values.\n214 3. We revised our approach to estimating diffusive CH fluxes. In the revised manuscript we report\n4\n215 fluxes calculated using gas transfer velocity determined through the floating chamber\n216 deployments. We added additional discussion of our approach to estimating fluxes in the main\n217 text (L417-431) and in Supplementary Text 1. Please see further discussion of these revisions in\n218 response to comments from both Dr. Peacock’s and Reviewer 3 comments below.\n219 4. The revised manuscript includes discussion text (L269-277) and a supplementary table (Table\n221 5. The data presented in the paper are now available at: https://doi.org/10.5281/zenodo.11155160\n222\n4\n225 we do feel that the revisions bolstered our efforts to rigorously assess these results. Please find our\n226 detailed responses to all of Dr. Peacock’s comments below.\n227\n230 (once) and conducted incubation experiments for a subset of these ditches. They find that methane\n231 oxidation within the water column consumes ~75% of methane, and thus net emissions from the ditches\n232 are significantly decreased by this “biofilter”.\n233\n235 the conclusions. The research topic is important: we know that ditches emit large amounts of CH4 but we\n236 lack data from tropical peatlands. Additionally, most studies only consider net emission from the water\n237 surface, so process studies such as this add novel information.\n238\n241 during my review.\n243\n245 incubation experiments, 5 for a second depth of incubation experiments (was this data reported in the\n247 often runs that way. But using this small sample size (measured just once) the authors occasionally make\n248 quite sweeping statements: “our finding […] is likely robust across peatland drainage canals in Southeast\n250 on smallholder land, and the lack of temporal replication, this is quite a claim.\n253 manuscript. The revised manuscript includes data from 13 canal reaches in a second peatland area ~50-\n254 100 km north of the 21 canals included in the original manuscript. These data include additional\n255 measurements of CH fluxes and 13C from canals from oil palm plantations, as well as canals from “open\n4\n256 undeveloped” (i.e., deforested but no active land use) areas. These samples were collected in April 2024\n257 vs. the data presented in the original manuscript which came from fieldwork conducted in May 2023.\n258\n6\n260 support our results that CH oxidation is an important control on canal CH emissions. Table S7 and S8\n4 4\n261 report results for dissolved CH and oxygen concentrations and CH emissions from canals in tropical\n4 4\n262 peatlands across Indonesia, Malaysia, and Brunei. Our study canals are within the range observed\n263 across the region for all parameters, including canals from land uses not represented in our work and\n264 sampled during more pronounced wet or dry periods than our study. As we identified dissolved oxygen as\n265 a significant correlate of CH oxidation, we feel there is merit in our assessment that CH oxidation is\n4 4\n266 likely prevalent in canals across the wider region. Furthermore, canals across Southeast Asia are similar\n267 in their physical (depth, width) and chemical (low pH, high DOC concentration and aromaticity, low\n268 dissolved oxygen, etc.) characteristics (Bowen et al., 2024, Nature Geoscience; Extended Data Table 2,\n4\n270 canals are broadly consistent across the study region.\n271\n273 pseudoreplication of sample points in some of the scatter plots, where duplicate measurements are\n274 presented separately. This is easily fixed (assuming my interpretation is correct).\n276 manuscript are based off of the mean values for each canal to avoid pseudoreplication. Summary\n277 statistics (means, ranges, etc.) reported in the revised paper are based on all observations (n = 48\n278 observations from 34 canal reaches for canal water CH measurements) including spatial replicates to\n4\n279 report the full range of observations. We include data files of both the mean values and all spatial\n280 replicates in the files uploaded to our data repository.\n281\n285 lack of data from tropical peat ditches but did give an EF. It would be interesting to know how your\n286 emissions compare to the IPCC EF (they’re lower I think, if my conversions are correct), and also worth\n287 highlighting that ditch emissions are anthropogenic and should be accounted for in inventories.\n288 *https://www.ipcc.ch/publication/2013-supplement-to-the-2006-ipcc-guidelines-for-national-greenhouse-\n289 gas-inventories-wetlands/\n291 anthropogenic (L269-277):\n292\n293 “Our observations of canal CH emissions estimated from dissolved CH concentration (72.2 ± 151.2 mg\n4 4\n4 4\n295 observations from Indonesia25,26,60 and Malaysia24 where mean emissions range from 2.8 to 1073 mg CH\n4\n296 m-2 d-1 (Table S8). The IPCC CH Emissions Factor for canals in tropical peatlands of 618.9 mg CH m-2\n4 4\n297 d-1 (2259 kg CH ha-1 y-1) was based on the only reported data25 at the time of the 2013 Wetlands\n4\n299 small number of existing studies and should be reconsidered to more accurately inventory the\n300 anthropogenic (e.g., from land use change) component62 of CH emissions from degraded tropical\n4\n301 peatlands.”\n302\n304 analysis here are fine, but cannot comment with any authority myself.\n305\n307\n7\n309\n311\n313 “We find that CH4 oxidation mitigates potential canal CH4 emissions by 75.5 ± 12.8%, reducing CH4\n314 emissions by 24.3 ± 32.3 mg CH4 m-2 d-“\n316 any maths themselves. So you could rephrase to:\n317 “We find that CH4 oxidation mitigates potential canal emissions by 75.5 ± 12.8%, reducing mean\n318 emissions from XX to YY mg CH4 m-2 d-“\n320 about how many sites/canals you sampled, and if it was temporally replicated or just a synoptic snapshot.\n322 about then it’s fine (and will save you a few words) to just write “emissions”.\n324 canals (with the addition of new study sites). The abstract now reads:\n325\n326 “Tropical wetlands and freshwaters are major contributors to the growing atmospheric methane (CH )\n4\n327 burden. Extensive peatland drainage has lowered CH emissions from peat soils in Southeast Asia, but\n4\n328 the canals draining these peatlands may be hotspots of CH emissions. Alternatively, CH consumption\n4 4\n329 (oxidation) by methanotrophic microorganisms may attenuate emissions. We used laboratory experiments\n330 and a synoptic survey of the isotopic composition of CH in 34 canals across West Kalimantan, Indonesia\n4\n331 to quantify the proportion of CH that is consumed and therefore not emitted to the atmosphere. We find\n4\n332 that CH oxidation mitigates 76.4 ± 12.0% of potential canal emissions, reducing emissions by ~70 mg\n4\n4 4\n334 emissions. As canals drain over 65% of peatlands in Southeast Asia, our results suggest that CH\n4\n335 oxidation significantly influences landscape-scale CH emissions from these ecosystems.”\n4\n336\n338 tropics”\n340 emissions, but northern waterbodies are also high emitters (as discussed by Rocher-Ros et al, who you\n341 cite here). And Rosentreter et al, in their global synthesis of aquatic CH4 say:\n342 “Despite the global coverage of our data, we did not detect clear latitudinal trends of methane emissions\n343 from aquatic ecosystems, except for the emissions from coastal wetlands peaking at 30° N.”\n346\n347 “Wetlands and freshwaters contribute ~30-55% of global CH emissions1, with significant emissions from\n4\n348 tropical ecosystems2–4.”\n349\n352 change), or compared to undrained tropical peatlands, or to peatlands in other climate zones?\n354\n355 “Given that drainage increases the importance of aquatic carbon fluxes from tropical peatlands…”\n356\n8\n358 to improving CH4 budgets of these ecosystems.”\n360 water column is irrelevant to the budget (but of course is interesting nonetheless).\n362\n363 “Constraining the importance of CH oxidation in canals draining tropical peatlands is a key step to\n4\n364 improving our understanding of the processes controlling CH emissions from these ecosystems…”\n4\n365\n367 measured. But then eventually in the results Table S1 is mentioned. This should also be mentioned\n368 around L68 (study sites) and L275 (methods) so the reader knows this info is available.\n370\n372 twice). Seems like 20 reaches is the correct one. This also applies to the “field sampling” section of\n373 methods.\n375 now reports 34 canal reaches with CH concentration and isotope data needed to estimate the percent\n4\n376 oxidized and diffusive fluxes, as 13 new sites were added during revisions.\n377\n4\n4\n380 included it in the supplemental summary table.\n381\n383 (17.6%-99.7%, Fig. 2A).”\n385 multi-panel figure in the SI showing raw conc changes for each incubation.\n387 table (Table S2) that has the initial and final CH concentrations and δ13C-CH for incubated waters. We\n4 4\n388 have also revised Figure 2, combining percent concentration and isotopic ratio changes into 1 panel to\n389 more clearly show the relationship between these changes:\n390\n391\n393 waters, δ13C-CH increased as the percent of initial CH consumed increased. Each data point shows the mean change over ~50\n4 4\n394 hours of incubation ± standard error of replicates (Table S2). B. Histogram of α values calculated from incubation data.\nox\n395\n9\n398 “Mean α was 1.022 ± 0.009 across the incubated canal waters”\nox\n399\n401 originally transported into canals from the surrounding landscape.”\n403 considered the potential importance of lateral transport vs in-situ production\n404 https://cdnsciencepub.com/doi/abs/10.1139/x95-055\n4\n406 following sections:\n407\n409 “It is unlikely that CH concentration in canal waters is dictated only by the amount of CH originally\n4 4\n410 transported into canals from the surrounding landscape, including CH produced in peat soils and canal\n4\n411 sediments. Methane produced in ombrotrophic tropical peat soils is highly depleted in 13C22,47. Unlike in\n412 lakes where δ13C-CH in littoral sediments and adjacent groundwater can differ by more than 10‰48 ,\n4\n413 porewater δ13C-CH has not been shown to differ between canal bottoms and adjacent peat soils21.”\n4\n414\n416 “Methane production in the water column could also influence canal water CH concentration and δ13C-\n4\n4 4\n418 any of the laboratory incubations of canal waters, as CH concentration decreased and δ13C-CH\n4 4\n419 increased in all incubated waters (Fig. 2A, Table S2).”\n420\n422 obvious where this 35 comes from. The “incubations” section mentions sample duplicates but I don’t think\n423 the “canal CH4” section does. Also, if you’re plotting sample duplicates as independent data points isn’t\n424 this essentially pseudoreplication which will artificially inflate your sample size and therefore statistical\n425 power? It seems more honest to calculate these correlations on means of the duplicates.\n428 for each canal to avoid pseudoreplication. As such, each data point in all scatter plots represents a\n429 separate canal reach.\n430\n4\n432 ranges, etc.) reported in the revised paper are based on all data - including spatial replicates within the\n433 same reach- to report the full range of observations. We include data files of both the mean values and all\n434 spatial replicates in the files uploaded to our data repository.\n437 https://link.springer.com/article/10.1007/s13157-011-0170-y) have found that CH4 emissions are lower in\n438 deeper ditches, because there is more scope for oxidation within the water column. There’s also ebullition\n439 to consider, which is likely to be higher in shallow canals (because sediments will become warmer in\n440 these systems).\n4\n442\n443 “Given that higher dissolved oxygen and the presence of aquatic vegetation were observed in canals with\n444 a shallower water depth (Fig. S6), canal water depth may indirectly mediate CH4 oxidation in drainage\n10\n445 canal waters. Overall, dissolved oxygen in the surface water of canals (0-10 cm) decreased with the\n446 depth of water present in the canal (Kendall’s 𝛕 = -0.41, p < 0.05, Fig. S6). Dissolved CH concentration,\n4\n447 and therefore estimated diffusive emissions, also had a weak but significant positive correlation with canal\n448 water depth (𝛕 = 0.26, p = 0.03, Table S4). This result contradicts previous findings in drainage ditches in\n449 temperate peatlands where CH emissions had a weak negative correlation with depth57, but these\n4\n450 differing results may be explained by how well canal waters are mixed and aerated. For example, while\n451 we observed CH oxidation in canals where dissolved oxygen is low (< 2.5 mg L-1) at the surface,\n4\n452 dissolved oxygen may become depleted at depth28,44 to below the concentration needed for aerobic\n453 methanotrophs with high oxygen affinity. As such, CH oxidation may be limited to the surface waters of\n4\n454 deeper canals, while in shallower canals oxidation may occur throughout the water column. Our study\n4\n455 also only explicitly considered diffusive emissions. Measurements of CH ebullition from canals could\n4\n456 further clarify the role of water depth in shaping net canal CH emissions, as ebullitive emissions vary with\n4\n457 water depth58. Altogether, our results suggest that shallower, vegetated canals may attenuate a higher\n458 percentage of CH emissions through CH oxidation.”\n4 4\n459\n461 is likely robust across peatland drainage canals in Southeast Asia.”\n463 overreaching. You measured 20 canals, once, in two study regions. I would suggest toning down this\n464 sentence.\n466\n467 “As such, we anticipate that water column CH oxidation is prevalent across canals draining degraded\n4\n468 peatlands in Southeast Asia.”\n469\n471 fluxes from other tropical ditch studies for comparison.\n473 to past observations:\n474\n475 “Our observations of canal CH emissions estimated from dissolved CH concentration (72.2 ± 151.2 mg\n4 4\n4 4\n477 observations from Indonesia26,27,63 and Malaysia25 where mean emissions range from 2.8 to 1073 mg CH\n4\n478 m-2 d-1 (Table S8). The IPCC CH Emissions Factor for canals in tropical peatlands of 618.9 mg CH m-2\n4 4\n479 d-1 (2259 kg CH ha-1 y-1) was based on the only reported data26 at the time of the 2013 Wetlands\n4\n481 small number of existing studies and should be reconsidered to more accurately inventory the\n482 anthropogenic (e.g., from land use change) component65 of CH emissions from degraded tropical\n4\n483 peatlands.”\n484\n487\n490\n492 agriculture, smallholder oil palm, and industrial oil palm) to capture the heterogeneity of\n493 drainage canals in the region”\n11\n495 towards smallholder systems?\n497 and canals from open undeveloped (e.g., deforested, no active land use) areas that were not represented\n498 in the original manuscript. To be clear about land use representation in the study we added the following\n499 text to L340-344:\n500\n501 “Smallholder mixed agriculture is the most represented land use in this study, but the sampled canals\n502 also include areas in smallholder plantations (pineapple and oil palm), industrial oil palm plantations, and\n503 open undeveloped land, as well as 1 canal in a degraded forest, to capture the heterogeneity of drainage\n504 canals in the region.”\n505\n507 there is zero turbulence). If so, best to explicitly say so in the text. But I admit to getting a little hesitant\n508 when using wind speed to calculate emissions in these small waterbodies. These relationships between\n509 wind speed and k have been tested in lakes, but can you be sure they are appropriate for small, sheltered\n510 ditches (e.g. Fig. 1d)? I would suggest:\n512 have added this information in L405 and L427.\n513\n514 1. Including your k values somewhere so the reader can check them. How do they compare to k values\n515 from other inland waters and from other ditches (or small waterbodies, e.g. ponds)?\n516 2. Can you calculate some k values from your floating chamber deployments? Do they compare to your\n517 windspeed-derived k values well?\n519 manuscript reports diffusive fluxes estimated using chamber-derived k values (L417-431):\n520\n521 “We calculated gas transfer velocity (k, m d-1) using data from the subset of canals where paired floating\n522 chamber CH fluxes and canal water CH concentrations were collected using Eqn. (2):\n4 4\n523\n4-canal 4-eq\n525\n4-canal 4 4-eq 4\n527 the atmosphere (CH ), and flux is the rate of CH emissions measured using the floating chamber. We\n4-eq 4\n528 used the median k value from the floating chamber deployments to estimate diffusive fluxes across all\n529 sampled (n = 34) canals. While applying a uniform value introduces uncertainty into the estimates of\n530 diffusive fluxes, conditions across the study region are characterized by high canal water temperature,\n531 low canal flow velocity (~0.1 m s-1), and low windspeed. As such, factors that strongly influence CH\n4\n532 degassing (e.g., solubility and turbulence) should have minimal variation relative to the ~600-fold variation\n533 in canal water CH concentration across study sites. Values were normalized to k for literature\n4 600\n534 comparison. See Supplementary Section 1 for further discussion of approaches to estimate k.”\n535\n537 sites to estimates to those from other shallow tropical waters, as well as those from forested ponds\n538 determined via tracer experiments. Please see the response regarding k values to Reviewer 3 for further\n539 discussion of these revisions.\n540\n12\n542 values. Is this due to problems with the k method, or are chambers capturing bubble events too? Do you\n543 seen any evidence of ebullition in these ditches?\n545 quantify the ebullitive component of canal CH emissions for a follow up study. Our revised approach to\n4\n546 estimating diffusive fluxes using chamber-derived k values brings fluxes from the two methods into closer\n547 agreement (see Figure S5 and Table S8). We also identified a minor error in unit conversions in our\n548 estimated flux calculations, that once corrected also increased the estimated fluxes alongside the change\n549 in k value.\n550\n555\n558\n559 “Surface waters (~5 cm) were collected for all canals included in the incubation experiments, and at 5 of\n560 the canals we collected water from ~10 cm above the canal bottom using gas-tight tubing and a hand\n561 pump.”\n562\n564 2 using 1.5M HCl to stop CH4 oxidation.”\n566 good, but it would be nice to see the reps data; how consistent are they to one another? Considering your\n567 small sample size this info would be useful.\n4\n569 δ13C-CH (mean ± standard error) and the incubation time for all incubated waters.\n4\n570\n573 reported in Table S3.\n574\n575 “To measure the isotopic composition of source CH , we collected porewater profiles at 6 locations\n4\n576 adjacent to a subset of the sampled canals. As shallow porewater is the primary source of discharge to\n577 drainage canals21, porewater was collected from 4-5 depths between 40 cm and 150 cm pending water\n578 table depth..”\n580\n582 made in consideration of their major comments. Detailed responses to all comments follow below.\n583\n584 1. In consideration of Reviewer 3’s concerns raised about the use of the boundary layer method\n585 (and comments about flux calculations from the other 2 reviewers), we have revised our approach\n586 to estimating CH fluxes. In the revised manuscript we report fluxes calculated using gas transfer\n4\n587 velocity determined through the floating chamber deployments. We added additional\n13\n588 methodological information about our approach to estimating fluxes in the main text (L417-431)\n589 and in Supplementary Text 1. We discuss these revisions in detail under Reviewer 3’s comments\n590 about flux estimation below.\n591 2. We have added discussion about the potential influence of CH production across the landscape\n4\n592 (e.g., in peat soils L151-159 and L219-225; in canal sediments and waters L149-163 and L217-\n593 228) throughout the manuscript in response to questions raised by Reviewer 3 about the source\n594 δ13C-CH used in the Rayleigh model for estimating the fraction of CH oxidized. We also added\n4 4\n595 more detail about the porewater δ13C-CH data used in our calculations in the results/discussion\n4\n596 (L154-159) and methods sections (L348-355), as well as Table S3. For a variety of reasons\n597 discussed at length below, we elected to not revise the source δ13C-CH value used in our\n4\n598 calculations. In the revised manuscript we report the uncertainty introduced by using a uniform\n599 value for source δ13C-CH (L127-129) and include a new supplementary figure (Fig. S3) showing\n4\n600 how varying source δ13C-CH impacts our estimate of the fraction of CH oxidized vs. emitted.\n4 4\n601 3. Reviewer 3 raised important questions about seasonality, as in climates with large seasonal\n602 variation in temperature (e.g., northern and temperate regions) and rainfall (e.g., monsoonal\n603 climates) there can be large variation in δ13C-CH due to changes in the rate and/or pathway of\n4\n4\n605 heavy rainfall all year; as such we do not anticipate large seasonal variation in δ13C-CH . Variable\n4\n606 temperature, dissolved oxygen, etc. can also impact the isotopic fractionation factor of oxidation,\n607 another critical parameter in our approach to estimating the proportion of the fraction of CH\n4\n608 oxidized. We have added additional discussion of potential controls of the isotopic fractionation\n609 we observed in incubations (L106-113). In short, we did not find any significant environmental\n610 correlates of this isotopic fractionation. Like for source δ13C-CH , we report the uncertainty\n4\n611 introduced to our estimates of oxidation due to the variability in isotopic fractionation in the main\n612 text (L125-127) and Figure S3.\n613\n615 raised above, below. We appreciate their feedback on the manuscript and feel the revisions inspired by\n616 their comments aided us in producing a more comprehensive assessment of our findings.\n617\n620 from Southeast Asia’s tropical peatland drainage canals. The manuscript’s main and most relevant finding\n621 is that CH4 oxidation is an important regulator of CH4 emissions also in tropical peatland drainage\n622 canals. This is reportedly the first study to document isotopic fractionation due to aerobic CH4 oxidation in\n623 these environments, which is crucial for enhancing our understanding of CH4 oxidation's isotopic\n624 fractionation factor in freshwater aquatic settings.\n625\n627 over CH4 oxidation as being significant appears somewhat overstated, considering the weak to moderate\n628 correlations shown in the figures.\n630\n4\n632 most significant influence on the percent of CH oxidized in canals as determined by canal water δ13C-\n4\n4\n634\n636 reproduced. However, some choices may increase the uncertainty of the results and their limitations are\n14\n637 not discussed or well-motivated. The main points here are 1) the choice of estimating CH4 fluxes using\n638 the boundary layer method; 2) the source δ13C-CH4 used in the Rayleigh model for estimating the\n639 fraction of CH4 oxidation; and 3) results not representative of potential seasonal variability of the source\n640 δ13C-CH4 used in calculations and actual seasonality in CH4 oxidation. Detailed comments about these\n641 points can be found below, and in the specific comments.\n642\n644 using floating chambers, which could also be used to calculate site and gas (CH4) specific k600. Flux\n645 estimates would be more robust if using the actual flux measurements and k600 derived from your\n646 measurements instead of wind models developed for CO2 emissions from lakes.\n648 we estimate diffusive CH emissions using gas transfer velocities estimated from chamber deployments\n4\n649 instead of from wind speed (methodological information: L417-431, Supplementary Text 1). This\n650 increases our estimates of diffusive emissions, but they are still within range of the few previous estimates\n651 of canal CH from other major peatland areas in Indonesia and Malaysia (see Table S8).\n4\n652\n654 isotopic signature source in the Rayleigh model. CH4 production may also take place in the canal’s\n655 sediment, and this production may have a heavier isotopic signature attributed to a different CH4\n656 production pathway that could lead to overestimation of results. In addition, the input of CH4 to the water\n657 column may be a mixture of CH4 produced in the canal’s sediment and from groundwater through lateral\n658 flow. The latter may vary seasonally, changing the isotopic signature of the source CH4 influencing the\n659 estimates of CH4 oxidation over seasons.\n4\n662 uncertainties. To capture the uncertainty introduced, we report how our estimate varies in response to\n663 changing the source signature in L127-129:\n664\n665 “Similarly, considering the standard deviation of the porewater source δ13C-CH measurements, the mean\n4\n666 percent oxidized could range from 68.2 ± 16.1% to 82.4 ± 8.9% (Fig. S3).”\n667\n669 pathways to the text (L151-159, 217-228). We appreciate the points raised by reviewer 3, but ultimately\n670 we did not elect to change the source value we used in our calculation of the percent oxidized, but\n671 instead focused on quantifying and discussing the associated uncertainties.\n672\n674 heavy rainfall all year). There is not a markedly wetter or drier season like in monsoonal tropical climates,\n675 but July and August have slightly lower total monthly precipitation (~150-200 mm/month vs. 200-300\n676 mm/month for the rest of the year). Temperatures are consistently warm year-round. This is in contrast to\n677 northern or temperate regions where seasonal variation in temperature and primary production can have\n678 a large influence on the rate and/or pathway of CH production. Extremely limited process-oriented data\n4\n679 on CH cycling in degraded tropical peatlands (e.g., measurements of isotopes, incubations, etc.) exist,\n4\n680 limiting our understanding of potential seasonal variation in these processes. Prior work from a less\n681 heavily degraded site in Brunei that also lacks strong seasonality suggests that the amount of CH\n4\n682 advection from peat soils (due to variability in porewater CH concentration and porewater discharge)\n4\n683 may vary across the year, but porewater δ13C-CH remains depleted year-round (~ -75‰; Somers, Hoyt,\n4\n684 et al., 2023; JGR Biogeosciences).\n685\n15\n687 in Supplementary Tables 7 and 8 (dissolved CH , dissolved oxygen, and CH fluxes) to other studies of\n4 4\n688 canals in regions of Southeast Asia with more pronounced precipitation seasonality. As other studies do\n689 not report δ13C-CH , we cannot contextualize our isotopic results. This emphasizes the need for further\n4\n690 work in this region.\n691\n693 limitations about seasonality. The authors should be careful with extrapolations that may not be\n694 representative for whole year.\n4\n696 discussion above in response to the points raised above about seasonality. We acknowledge the lack of\n697 seasonal representation in the text in L254-257, and also show that relevant data (Table S7; dissolved\n698 oxygen and dissolved CH ) are within range of past observations collected in areas during pronounced\n4\n699 wet or dry seasons:\n700\n701 “While our study was not conducted during pronounced wet or dry periods, the dissolved CH and oxygen\n4\n702 concentrations measured in our study fall within the range observed across Southeast Asia under varying\n703 land uses and seasons21,27,44,60,61 (Table S7).”\n704\n706 ● Some relevant papers about CH4 oxidation based on stable isotopes in tropical aquatic\n707 environments may be relevant in the introduction and/or to further explore in the discussions (e.g.\n709 ○ We have added suggested references where appropriate in the revised\n710 introduction/discussion sections (ref. 29, 31, and 43):\n711 ○ L54-56 :”One process that strongly influences freshwater CH emissions is microbial\n4\n712 oxidation of CH to carbon dioxide. In other tropical freshwaters (e.g., rivers, lakes) CH\n4 4\n713 oxidation attenuates CH emissions by 40 to nearly 100%22,29–31.”\n4\n714 ○ L102-105: “The range of α encompasses…results from incubations of soil from\nox\n715 subtropical rice paddies43 (α of 1.025-1.033) that are often used in estimates of CH\nox 4\n716 oxidation in tropical freshwaters29,31.”\n717 ● Further exploration and discussion of the observed variability of isotopic fractionation would be\n718 relevant since this is one of the most unique results reported. Isotopic fractionation values\n719 estimated for rice paddy environments (Tyler et al. 1997; Zhang et al. 2013) could add some\n720 insights and perhaps is a more similar and relevant type of environment to compare with than\n721 northern lakes.\n722 ○ See responses below in lines 804-823 of this document related to this point. We have\n723 added discussion of the potential drivers of variability in isotopic fractionation and\n724 comparisons to the aforementioned studies conducted in warmer climates to L98-113.\n725 ● Limited number of replicates in incubations might hide potential analytical errors or local variability\n726 of oxidation rates and isotope fractionation in the incubation experiment.\n727 ○ We acknowledge that we have limited replicates. We are limited in the amount of sample\n728 material we can bring back for analysis in the USA due to baggage weight restrictions\n729 when departing Indonesia. We report variability between replicate incubations in Fig. 2A\n730 and Table S2. Overall, the change in dissolved CH and δ13C-CH over the incubation\n4 4\n731 period was larger than variability between replicate samples.\n732 ● Using mean values for the source δ13C-CH4 from groundwater instead of site-specific values\n733 may obscure potential variability between sites that influence the analysis of the controlling\n16\n734 factors. I would suggest using mean values only for sites where you did not measure groundwater\n735 δ13C-CH4.\n736 ○ We discuss this point and revisions made in consideration of it thoroughly in lines 1069-\n737 1191 of this document. In brief:\n738 ○ We appreciate the suggested revision to our calculations, but ultimately we elected not to\n739 revise the source δ13C-CH for a number of reasons. From our porewater observations,\n4\n740 δ13C-CH varied more by depth (up to 20‰ variability between samples from the same\n4\n741 profile) than across the landscape. Furthermore, the canals drain porewater from along\n742 their entire reach; thus a point measurement from one location along the total length of\n743 the canal does not necessarily represent the δ13C of the whole mass of CH advected\n4\n744 into the canal. Given these considerations, and others discussed at length in lines 1069-\n745 1191 of this document, we elected to not revise our approach to selecting source δ13C-\n4\n747 uncertainty introduced by using a uniform source δ13C-CH and discussing factors that\n4\n748 may influence our results (e.g., production pathways and sources of CH )\n4\n749 ○ We acknowledge that using a mean value for source δ13C-CH may not accurately\n4\n750 account for variability between sites and introduce additional uncertainty. We have\n751 revised the manuscript to explicitly report the uncertainty introduced by using a mean\n752 source δ13C-CH value in the main text (L125-129) and Fig. S3.\n4\n753 ● Other relevant papers may also be useful for discussing the isotopic signature of source methane\n754 used as reference for the oxidation calculations (Thottathil and Prairie 2021; Schenk et al. 2021).\n755 ○ We included a reference suggested above in L152-154:\n756 “Unlike in lakes where δ13C-CH in littoral sediments and adjacent groundwater can differ\n4\n757 by more than 10‰48 , porewater δ13C-CH has not been shown to differ between canal\n4\n758 bottoms and adjacent peat soils21.”\n759\n762 the introduction.\n764 of the introduction following the example of other papers in this journal. See examples in recent papers on\n765 tropical peatlands published in Nature Communications: Cooper et al. (2020)\n766 [https://doi.org/10.1038/s41467-020-14298-w] and Hodgkins et al. (2018)\n767 [https://doi.org/10.1038/s41467-018-06050-2].\n768\n4\n771 during incubations”.\n772\n774 would be good to describe discuss the influence of starting concentrations on the results. Extra\n775 supplementary table or figure showing the start and end CH4 concentration and δ13C-CH4 and the time\n776 incubated would be valuable to understand the changes you show in Figure 2.\n4 4\n778 initial CH concentration did not affect the isotopic fractionation observed via incubations. As measuring\n4\n779 the isotopic fractionation of CH oxidation was the primary aim of the incubations, initial CH\n4 4\n780 concentration did not influence downstream results (i.e.; estimates of percent oxidized in situ made using\n781 fractionation factors measured in vitro). To address this point, we have added the following text to L105-\n17\n782 107:\n783\n784 “While CH oxidation rates varied with initial CH concentration, we did not observe a correlation between\n4 4\n785 α and initial CH concentration, nor α and CH oxidation rate (Fig. S2).”\nox 4 ox 4\n786\n4\n788 isotopic fractionation, as well as oxidation rate and isotopic fractionation:\n789\n790\n4 4\n792 (α ) did not vary with initial CH concentration nor CH oxidation rate.\nox 4 4\n794 the initial and final CH concentrations and δ13C-CH and the incubation time for all incubated waters.\n4 4\n795\n797 improve understanding of isotopic fractionation if the variability observed could be associated with some\n798 environmental factors.\n800 2022 cited here show that the fractionation varied with depth and temperature, and although the overall\n801 range be similar that may be local factors controlling the variability of isotopic fractionation that need to be\n802 explored and discussed. Also, the numbers you present from references 37 and 38 are not the correct\n803 overall range reported in these papers combined.\n805 variability of isotopic fractionation. Thank you for catching the error in the numbers from ref. 37-38; (now\n806 ref. 41-42). In the revised text, we now compare the whole range we observed for α (1.002-1.039) in\nox\n807 comparison to that observed by ref. 41-42 (1.0184-1.0208 and 1.004-1.038, respectively).\n808\n809 “From these data we calculated the first empirically derived isotopic fractionation factors for CH\n4\n810 oxidation39 (α ) in peat-draining freshwaters. Ecosystem-specific values for α are critical to estimating\nox ox\n811 the percent of CH that is oxidized rather than emitted from the natural environment40,41. Mean α was\n4 ox\n812 1.022 ± 0.009 across the incubated canal waters (range: 1.002-1.039; Fig. 2B). The range of α\nox\n813 encompasses past observations from northern and temperate freshwaters incubated under in situ\n814 dissolved CH and oxygen concentrations and temperature41,42, as well as results from incubations of soil\n4\n815 from subtropical rice paddies43 (α of 1.025-1.033) that are often used in estimates of CH oxidation in\nox 4\n816 tropical freshwaters29,31. While CH oxidation rates varied with initial CH concentration, we did not\n4 4\n817 observe a correlation between α and initial CH concentration, nor α and CH oxidation rate (Fig. S2).\nox 4 ox 4\n2\n819 α 42. Of these factors, α was only weakly positively correlated with the initial dissolved O present in\nox ox 2\n18\n820 each of the incubated waters (p = 0.07). α did not vary between surface and bottom waters of the subset\nox\n821 of canals sampled at two depths for incubation experiments (1.024 ± 0.006 vs. 1.023 ± 0.012, n = 4\n822 canals). As we did not find significant environmental correlates of α , we used the mean value to\nox\n823 estimate in situ CH oxidation as discussed below.”\n4\n824\n827\n829 relationship and even mention a linear relationship in the caption. I see you use the log scale in the y-axis\n830 but maybe good to make it clearer to the reader that will not find an exponential relationship in the figure.\n832\n834 methanotrophic bacteria and δ13C-CH4”, relates to the negative relationship you have observed?\n836\n837 “Previous observations in tropical river networks31 also observed a negative relationship between the\n838 concentration of CH in river waters and δ13C-CH . In these rivers δ13C-CH also had a positive\n4 4 4\n839 relationship with gene markers for methanotrophic bacteria, indicating that variation in CH concentration\n4\n840 and δ13C-CH is influenced by CH oxidation. The consistent relationship between CH concentration and\n4 4 4\n841 δ13C-CH observed across the drainage canals in our study and these tropical rivers supports the idea\n4\n842 that differences in dissolved CH concentrations between canal reaches are influenced by CH oxidation.”\n4 4\n843\n845 increase if all sites were measured. I would also expect CH4 production in the canal and that this\n846 production would have a less negative δ13C-CH4 because of acetoclastic methanogenesis. Using only\n847 δ13C-CH4 from porewater outside the canal might lead to overestimation of results. See more comments\n848 on this below (comments for L336-338).\n4\n850 from in-canal production in L149-168. See comments below (lines 1127-1154 of this document) for\n851 discussion about the potential role of acetoclastic methanogenesis.\n852\n853 “It is unlikely that CH concentration in canal waters is dictated only by the amount of CH originally\n4 4\n854 transported into canals from the surrounding landscape, including CH produced in peat soils and canal\n4\n855 sediments. Methane produced in ombrotrophic tropical peat soils is highly depleted in 13C22,47. Unlike in\n856 lakes where δ13C-CH in littoral sediments and adjacent groundwater can differ by more than 10‰48 ,\n4\n857 porewater δ13C-CH has not been shown to differ between canal bottoms and adjacent peat soils21.\n4\n4\n859 study region had a mean δ13C-CH of -85.0 ± 5.9‰, which was consistently more depleted than any\n4\n860 observed canal δ13C-CH value (Table S1, S3). Porewater δ13C-CH varied more between sample depths\n4 4\n861 within each profile than between profiles collected across the landscape, suggesting source δ13C-CH is\n4\n862 similarly depleted in 13C throughout the study region. Methane production in the water column could also\n863 influence canal water CH concentration and δ13C-CH . However, this is unlikely to explain our results\n4 4\n864 because we did not observe net CH production in any of the laboratory incubations of canal waters, as\n4\n4 4\n866 water CH concentration were influenced solely by the total amount of CH produced and then\n4 4\n867 transported into canal waters, we would expect canal water δ13C-CH to be similarly depleted across\n4\n868 canals and not vary systematically with dissolved CH concentration. Given that CH concentrations\n4 4\n869 varied ~600-fold alongside a ~40‰ range in δ13C-CH , our results indicate that CH oxidation has a\n4 4\n870 significant influence on canal water CH concentration and δ13C-CH .”\n4 4\n871\n874\n876 distribution) and this is because diffusive fluxes are dependent on CH4 concentrations and % CH4\n877 oxidized dependent on δ13C-CH4. That makes it logical to see a similar pattern and does not necessarily\n878 show that diffusive fluxes are related to oxidation. It would have been interesting to add an extra panel\n879 showing the relationship between measured fluxes (floating chambers) and oxidation rates from\n880 incubations, which would be independent of concentrations and δ13C-CH4.\n882 violin plots of CH and δ13C-CH to show the distribution of these data without having a redundant\n4 4\n883 relationship shown in the same figure. The panel showing the relationship between CH and δ13C-CH\n4 4\n884 has been moved to the supplement (Fig. S4).\n885\n886\n4 4 4 4\n888 emissions. A. Curve showing the relationship between canal water δ13C-CH and estimated percent CH oxidized across the mean\n4 4\n889 (black line) and ± 1 standard deviation (shaded region) of the laboratory derived α value. B-C. Surface water δ13C-CH and\nox 4\n890 dissolved CH concentration across the studied canals (n = 34). D. Estimates of the percent of CH oxidized versus estimated\n4 4\n891 diffusive CH flux across the studied canals. For panels B-D each dot represents a canal. The shaded region of panel D represents\n4\n892 the 95% confidence interval associated with the linear relationship. Dissolved CH concentration and estimated diffusive CH flux\n4 4\n893 are shown on a log scale in panels C and D.\n10\n894\n4\n4\n4 4\n898 from incubations are canals with high initial dissolved CH concentrations and more depleted initial δ13C-\n4\n4 4\n900 determined in incubations had less initial dissolved CH and more enriched initial δ13C-CH , suggesting\n4 4\n20\n901 more oxidation occurs in situ in those waters. As such, comparing chamber CH emissions to incubation\n4\n4 4\n903\n905 only show that %CH4 oxidized in different between open water and vegetation, and not a relationship. I\n906 suggest reformulating and toning down the statement that O2 and vegetation strongly influence the %\n907 oxidation.\n909\n910 “Of the studied controls on CH oxidation, dissolved oxygen and aquatic vegetation had the most\n4\n911 significant influence on the percent of CH oxidized in canals as determined by canal water δ13C-CH .”\n4 4\n912\n914 produced by acetoclastic methanogenesis, which is more likely to happen in vegetated areas.\n915\n917 228:\n918\n919 “The deposition of labile organic matter from vegetation could also stimulate acetoclastic\n920 methanogenesis, which like CH oxidation would contribute towards larger δ13C-CH in vegetated\n4 4\n921 canals38. However, acetoclastic methanogenesis likely contributes little to the δ13C-CH in vegetated\n4\n922 canals because hydrogenotrophic methanogenesis has been identified as the dominant pathway in the\n923 ombrotrophic tropical peatlands of Southeast Asia22 and the Americas47,55. Disturbance in peatlands in\n925 acetoclastic methanogenesis, like graminoids, but this shift does not appear to increase the abundance of\n926 acetoclastic methanogens56. While we cannot rule out the possible influence of acetoclastic\n927 methanogenesis on canal water δ13C-CH , the lower dissolved CH concentration in vegetated canals (p\n4 4\n928 = 0.02, Table S5) lends more support to the idea that vegetation enhances CH oxidation rather than\n4\n929 acetoclastic CH production in canals.”\n4 4\n930\n932 evident in Fig S5.\n934 water depth” to tone down the strength of this finding and better reflect the potential impact of canal water\n935 depth. This paragraph now begins with the following statement (L229-231):\n936\n937 “Given that higher dissolved oxygen and the presence of aquatic vegetation were observed in canals with\n938 a shallower water depth (Fig. S6), canal water depth may indirectly mediate CH oxidation in drainage\n4\n939 canal waters.”\n940\n942 for that could be.\n944 may influence turbulence and/or mixing, causing very recently advected (and therefore more isotopically\n945 depleted) CH to be degassed. Our collaborators have also seen local residents and/or farmers using this\n4\n946 particular canal reach for washing tools and equipment. This canal (#31) was one of the deeper (68 cm)\n947 and more isotopically depleted (canal water δ13C-CH = -65.2 ± 0.1‰), but it had relatively low dissolved\n4\n4\n949 that turbulence from the canal block or recent use of the canal for washing may have degassed CH from\n4\n950 the canal water prior to our sample collection. As transport across the air-water interface results in little to\n951 no isotopic fractionation, the remnant CH would be depleted in 13C (e.g., closer to source value) but CH\n4 4\n952 concentration would be low.\n953\n955 to canals.\n957 338:\n958\n959 “Drainage canals in lowland peatlands were sampled in Kubu Raya and Mempawah Districts, West\n962 year-round, but the driest months of the year usually occur in July or August.”\n963\n965 canal depth, and presence of vegetation.\n4\n967 13C), or vegetation across land uses (Table S6). Our results do not indicate strong land use patterns\n968 across canals, but perhaps a large sampling or synthesis effort could reveal any systematic variation\n969 using a larger dataset.\n970\n972 parameters from Cole and Caraco 5. I wonder how reliable these estimates are considering that the Cole\n973 and Caraco 5 model was created for lakes and CO2. Here, I imagine two sources of uncertainty, 1) lakes\n974 have larger open areas for wind and gusts to develop in comparison to canals that are more sheltered\n975 and canals may be a lotic environment where water velocity could also influence k and is not accounted\n976 by the Cole and Caraco model, and 2) the model was created for CO2 and recent research, Pajala et al.\n977 6 has observed a higher k for CO2 compared with CH4, meaning that the calculated k for CH4 using this\n978 model could overestimate k for CH4 and consequently the fluxes estimates. Why not use the k from the\n979 floating chambers you have deployed and the mean for sites without floating chamber measurements?\n981\n983 which is discussed in more detail below. In response to the reviewer’s specific comments:\n984\n985 1) Flow velocity is very low in these canals. Flow measured at a subset (n = 8) of canals was 0.12 ±\n986 0.03 m s-1. 2) Estimates of k from the chamber measurements (from 12 of 34 sampled canals) are ~2x larger\n987 on average than k values estimated using wind speed from the same canal. As such, it is unlikely\n988 that the flux estimates in the original manuscript were overestimates. The estimated fluxes in the\n989 original manuscript were low compared to previous floating chamber measurements of CH\n4\n990 emissions from canals draining peatlands in other regions of Indonesia and Malaysia.\n991\n994 replicate chamber measurements were made (n = 4 canals) there was considerable variability in k within\n995 the same canal reach. For example, for the same canal reach we estimate k of 0.3 to 3.1 m d-1. Given the\n996 large inter- and intra-canal variability in k estimated from chambers, in the revised manuscript we estimate\n997 diffusive fluxes across all sampled canals using the median k value (k = 1.15 m d-1) from chamber\n600\n998 measurements. We acknowledge that in reality k will not be uniform across sites. However, given the low\n999 wind (often < 0.5 m s-1) and low flow conditions across canals, as well as their relative similarity in size\n1000 and sheltering due to deforestation in the study region, variation in canal water CH concentration across\n4\n1001 canals should be the main factor driving variability in diffusive CH emissions.\n4\n1002\n1004 previously observed from drainage canals in other major peatland regions of Indonesia and Malaysia. We\n1005 have added a supplementary table comparing our estimated diffusive fluxes as well as our chamber\n1006 fluxes to previous observations (Table S8). Supplementary Text 1 also provides discussion of approaches\n1007 to estimating k, how they impact our results, and comparison of k values from our study and others in\n1008 similar environments.\n1009\n1011\n1012 “We calculated gas transfer velocity (k, m d-1) using data from the subset of canals where paired floating\n1013 chamber CH fluxes and canal water CH concentrations were collected using Eqn. (2):\n4 4\n1014\n4-canal 4-eq\n1016\n4-canal 4 4-eq 4\n1018 the atmosphere (CH ), and flux is the rate of CH emissions measured using the floating chamber. We\n4-eq 4\n1019 used the median k value from the floating chamber deployments to estimate diffusive fluxes across all\n1020 sampled (n = 34) canals. While applying a uniform value introduces uncertainty into the estimates of\n1021 diffusive fluxes, conditions across the study region are characterized by high canal water temperature,\n1022 low canal flow velocity (~0.1 m s-1), and low windspeed. As such, factors that strongly influence CH\n4\n1023 degassing (e.g., solubility and turbulence) should have minimal variation relative to the ~600-fold variation\n1024 in canal water CH concentration across study sites. Values were normalized to k for literature\n4 600\n1025 comparison. See Supplementary Section 1 for further discussion of approaches to estimate k.”\n1026\n1029 depth or distance from sediment (e.g. 80 % of the canal depth)?\n1031 deeper samples was not related to a specific depth/distance, but rather as deep as we could sample\n1032 without disturbing the soft peat sediment underlying the canal to minimize disturbance for other ongoing\n1033 measurements at our study sites. In general, this was ~10 cm from the canal bottom. We revised L375-\n1034 377 to be more clear about the sampling approach for incubated waters:\n1035\n1036 “Surface waters (~5 cm) were collected for all canals included in the incubation experiments, and at 5 of\n1037 the canals we collected water from ~10 cm above the canal bottom using gas-tight tubing and a hand\n1038 pump.”\n1039\n1042\n1044 replicates, and it would be good to show this variability. Since you only have two replicates what does not\n1045 make standard deviation very meaningful, you could show the range in Figure 2 or in a supplementary\n1046 table with more information about the incubations as mentioned above.\n4 4\n1048 (mean ± standard error) and the incubation time for all incubated waters (Table S2). The revised Figure\n1049 2A now also shows the standard error of replicate samples for the percent CH consumed and change in\n4\n1050 δ13C-CH over the incubation period.\n4\n1051\n1054\n1056 standard deviation shows a considerable difference in δ13C-CH4 between sites or replicates and if a\n1057 mean was used this would add errors to the results of single sites. Please consider describing in the\n1058 methods what values did you use for the calculations and discuss how this limits the results, especially for\n1059 sites where you did measure porewater. I also wonder about the potential and large variability between\n1060 δ13C-CH4 in porewater outside the canals and in the canal’s sediment. Recent studies 7,8 show large\n1061 variation of δ13C-CH4 in bubbles released from lake sediments attributed to different pathways of CH4\n1062 production that could also be the case for these canals. Not all CH4 may come from groundwater/lateral\n1063 flow and CH4 production may occur in the canal, especially canal with vegetation, where fresh organic\n1064 matter is available and less negative δ13C-CH4 would be expected because of acetoclastic\n1065 methanogenesis. Using different and more negative δ13C-CH4 could largely overestimate the fraction of\n1067\n4\n1069 value. The revised manuscript thoroughly considers these, including additional quantification and\n1070 visualization of the uncertainty introduced into our results by the source δ13C-CH value (#1 below),\n4\n1071 discussion of the variability of the porewater δ13C-CH measurements (#2), methods clarification (#3), and\n4\n1072 text discussing the potential influence of varying methanogenic pathways (#4) and CH production in\n4\n1073 canal sediments and/or waters (#5).\n1074\n1075 1. We acknowledge the variability of our porewater δ13C-CH measurements introduces\n4\n1076 uncertainty into our estimates of the percent oxidized. The revised manuscript quantifies and\n1077 visualizes the uncertainty introduced by the variation in porewater δ13C-CH in L127-129 and Fig.S3:\n4\n1078 “Similarly, considering the standard deviation of the porewater source δ13C-CH measurements, the mean\n4\n1079 percent oxidized could range from 68.2 ± 16.1% to 82.4 ± 8.9% (Fig. S3).”\n1080\n4\n1082 deviation value of our estimate of the isotopic fractionation of oxidation (1.022 ± 0.008). B. Density plot showing estimates of the\n1083 percent of CH oxidized in canal waters using the mean or ± one standard deviation value of our estimate of the source δ13C-CH\n4 4\n1084 (85.0 ± 5.9‰).\n1085\n24\n1086 2. We have added text that describes the variability in porewater δ13C-CH and clarifies our\n4\n1087 porewater sampling scheme:\n1088\n4\n1090 waters in our study region had a mean δ13C-CH of -85.0 ± 5.9‰, which was consistently more depleted\n4\n1091 than any observed canal δ13C-CH value (Table S1, S3). Porewater δ13C-CH varied more between\n4 4\n1092 sample depths within each profile than between profiles collected across the landscape, suggesting\n1093 source δ13C-CH is similarly depleted in 13C throughout the study region.”\n4\n1094\n4\n1096 of the profile, with the most significant increase between the upper 2 samples (top sample ~5 cm below\n1097 water table). The increase in porewater δ13C-CH towards the peat surface coincides with a decrease in\n4\n4\n1099 methanogenic pathway. As such, the more shallow porewater with heavier δ13C-CH contributes much\n4\n1100 less to the total CH pool transported into canals than the deeper porewater with more depleted δ13C-CH\n4 4\n1101 and higher CH concentration. Weighted by concentration - and therefore contribution to the canal CH\n4 4\n1102 pool - the mean porewater δ13C-CH is -84.92‰ (compared to the arithmetic mean of -84.98‰).\n4\n1103\n1105\n1106 “To measure the isotopic composition of source CH , we collected porewater profiles at 6 locations\n4\n1107 adjacent to a subset of the sampled canals. As shallow porewater is the primary source of discharge to\n1108 drainage canals21, porewater was collected from 4-5 depths between 40 cm and 150 cm pending water\n1109 table depth.”\n1110\n1111 3. We appreciate the reviewer's idea to use site-specific δ13C-CH when available. However,\n4\n1112 unfortunately this approach is unable to meaningfully reduce uncertainty in our case. Canals\n1113 drain porewater along their entire length (which can encompass peat soils under a variety of land\n1114 uses and/or vegetation). As such, using a value from a single point is likely a poorer representation of\n1115 the bulk δ13C-CH transported into the canal than using an average value representing a larger area.\n4\n1117 rigorously quantifying uncertainties and discussing factors that could influence source δ13C-CH\n4\n1118 value. We clarified the source δ13C-CH value used in our calculations in L447-451:\n4\n1119\n1120 “The results presented in the main analyses and figures are estimates of the percent oxidized based\n1121 on mean observed values of α (1.022 ± 0.009, from incubations) and δ (-85.0 ± 5.9‰, n= 27\nox source\n1122 measurements from 6 porewater profiles). To characterize the uncertainty of our estimates due to\n1123 variability in α and δ , we also report how our estimate varies when using ± 1 standard deviation\nox source\n1124 of α or δ in Eqn. 3.”\nox source\n1125\n1126 4. While acetoclastic methanogenesis can play an important role in minerotrophic tropical\n1127 peatlands (e.g. Buessecker et al., 2021; Frontiers in Microbiology), we anticipate that\n1128 hydrogenotrophic methanogenesis is the dominant methanogenic pathway across the highly\n1129 disturbed, ombrotrophic peatlands we studied for the following reasons:\n1130 a. Other ombrotrophic peatlands in Borneo, Peru, and Panama (Buessecker et al., 2021; Holmes et\n1131 al., 2015, Global Biogeochemical Cycles; Hoyt, 2017, unpublished PhD thesis) have very\n1132 depleted porewater δ13C-CH reflecting hydrogenotrophic methanogenesis like observed in our\n4\n1133 study. Paired δ13C-CO measurements from these other studies further indicate hydrogenotrophy\n2\n1134 is the dominant methanogenic pathway in these ombrotrophic peatlands.\n25\n1135 b. Tropical peatland disturbance influences the quality of DOM (Gandois et al., 2013,\n1137 derived from decomposed peat carbon vs. fresh plant inputs. As such, disturbed peatlands have\n1138 less labile DOM for acetoclastic methanogenesis.\n1139\n4\n1141 acetoclastic methanogenesis. We address this point in L217-228:\n1142\n1143 “The deposition of labile organic matter from vegetation could also stimulate acetoclastic\n1144 methanogenesis, which like CH oxidation would contribute towards larger δ13C-CH in vegetated\n4 4\n1145 canals38. However, acetoclastic methanogenesis likely contributes little to the δ13C-CH in vegetated\n4\n1146 canals because hydrogenotrophic methanogenesis has been identified as the dominant pathway in the\n1147 ombrotrophic tropical peatlands of Southeast Asia22 and the Americas47,55. Disturbance in peatlands in\n1149 acetoclastic methanogenesis, like graminoids, but this shift does not appear to increase the abundance of\n1150 acetoclastic methanogens56. While we cannot rule out the possible influence of acetoclastic\n1151 methanogenesis on canal water δ13C-CH , the lower dissolved CH concentration in vegetated canals (p\n4 4\n1152 = 0.02, Table S5) lends more support to the idea that vegetation enhances CH oxidation rather than\n4\n1153 acetoclastic CH production in canals.”\n4 4\n1154\n1155 5. We acknowledge that CH production may occur in canal sediments and/or canal waters, but\n4\n1156 do not anticipate this is a large and/or different source of CH to canals for the following\n4\n1157 reasons:\n1158 a. We did not observe net CH production in any of the canal water incubation experiments.\n4\n1159 b. Work from our collaborators in a peatland drainage canal in northern Borneo (Somers et al.,\n1160 2023; JGR Biogeosciences) found that δ13C-CH in peat underlying the canal was -69.2 ± 4.8‰,\n4\n1161 while porewater down to 2.5 m (interval where most CH advected to the canal originates) in the\n4\n1162 ~60 m on either side of the canal (within our sample scheme) had δ13C-CH of -71.7 ± 9.0‰ to -\n4\n1163 68.4 ± 5.1‰, showing these CH sources are isotopically similar.\n4\n1164 c. Overall, the total area of the canal bottom is much smaller than the total area of peatland drained\n1165 by a given canal. Therefore, the proportional contribution of production in underlying sediments to\n4 4 4\n1167 canal sediments had a distinct δ13C-CH , the signature of the peat porewater still would dominate\n4\n1168 due to its larger contribution to the CH pool.\n4\n1169\n4\n1171 in L149-168:\n1172\n1173 “It is unlikely that CH concentration in canal waters is dictated only by the amount of CH originally\n4 4\n1174 transported into canals from the surrounding landscape, including CH produced in peat soils and canal\n4\n1175 sediments. Methane produced in ombrotrophic tropical peat soils is highly depleted in 13C22,47. Unlike in\n1176 lakes where δ13C-CH in littoral sediments and adjacent groundwater can differ by more than 10‰48 ,\n4\n1177 porewater δ13C-CH has not been shown to differ between canal bottoms and adjacent peat soils21.\n4\n4\n1179 study region had a mean δ13C-CH of -85.0 ± 5.9‰, which was consistently more depleted than any\n4\n1180 observed canal δ13C-CH value (Table S1, S3). Porewater δ13C-CH varied more between sample depths\n4 4\n1181 within each profile than between profiles collected across the landscape, suggesting source δ13C-CH is\n4\n1182 similarly depleted in 13C throughout the study region. Methane production in the water column could also\n1183 influence canal water CH concentration and δ13C-CH . However, this is unlikely to explain our results\n4 4\n1184 because we did not observe net CH production in any of the laboratory incubations of canal waters, as\n4\n4 4\n1186 water CH concentration were influenced solely by the total amount of CH produced and then\n4 4\n1187 transported into canal waters, we would expect canal water δ13C-CH to be similarly depleted across\n4\n1188 canals and not vary systematically with dissolved CH concentration. Given that CH concentrations\n4 4\n1189 varied ~600-fold alongside a ~40‰ range in δ13C-CH , our results indicate that CH oxidation has a\n4 4\n1190 significant influence on canal water CH concentration and δ13C-CH .”\n4 4\n1191\n1193 δ13C-CH4 and concentration from each site where it was collected.\n1195\n1197 create isotopic fractionation? Also indicate the model of the piezometer together with the company’s\n1198 name.\n1201 the piezometer (L351-354):\n1202\n1203 “Porewater was collected using a portable piezometer made of 3/8” stainless steel tubing housing 1/4”\n1204 polyethylene tubing equipped with a coarse polypropylene screen to prevent collection of coarse debris\n1205 (SedPoints, M.H.E. Products).”\n1206\n1208 explanation about this in the main text.\n1210\n1211 “Oxidation efficiency (f ) was calculated using a Rayleigh model for closed systems.”\nox\n1212\n1213 f is the fraction oxidized. In the paper we report percent oxidized (fox * 100), as stated in L444-445.\nox\n1214\n1216 accept a flux measurement. Here it would also be nice to describe how much water flows in the canals\n1217 and if the chambers were allowed to follow the flow or if kept on the same position, and if so, who this\n1218 could influence the flux measurement by changing the water turbulence with the chamber. I also wonder\n1219 in shallow places if the edges of the chamber could hit the sediment influencing the measurements.\n1222 “The floating chamber was not held in place, but due to low canal water flow (stagnant to ~0.1 m s-1) that\n1223 chamber did not travel during flux measurement.”\n1224 “Fluxes were accepted if the linear increase in CH over time met the standards of R2 > 0.9 and p < 0.05.”\n4\n1225\n1227\n1229 1. Barbosa, P. M. et al. High rates of methane oxidation in an Amazon floodplain lake.\n1231 2. Sawakuchi, H. O. et al. Oxidative mitigation of aquatic methane emissions in large Amazonian\n1232 rivers. Global Change Biology 22, 1075-1085 (2016). https://doi.org/10.1111/gcb.13169\n27\n1233 3. Tyler, S. C., Bilek, R. S., Sass, R. L. & Fisher, F. M. Methane oxidation and pathways of\n1234 production in a Texas paddy field deduced from measurements of flux, delta C-13, and delta D of\n1236 4. Zhang, G. B. et al. Pathway of CH4 production, fraction of CH4 oxidized, and C-13 isotope\n1237 fractionation in a straw-incorporated rice field. Biogeosciences 10, 3375-3389 (2013).\n1238 https://doi.org/10.5194/bg-10-3375-2013\n1239 5. Cole, J. J. & Caraco, N. F. Atmospheric exchange of carbon dioxide in a low-wind oligotrophic\n1240 lake measured by the addition of SF6. Limnology and Oceanography 43, 647-656 (1998).\n1241 6. Pajala, G. et al. Higher Apparent Gas Transfer Velocities for CO2 Compared to CH4 in Small\n1243 https://doi.org/10.1021/acs.est.2c09230\n1244 7. Schenk, J. et al. Methane in Lakes: Variability in Stable Carbon Isotopic Composition and the\n1246 https://doi.org/10.3389/feart.2021.722215\n1247 8. Thottathil, S. D. & Prairie, Y. T. Coupling of stable carbon isotopic signature of methane and\n1248 ebullitive fluxes in northern temperate lakes. Science of The Total Environment 777, 146117\n1249 (2021). https://doi.org/https://doi.org/10.1016/j.scitotenv.2021.146117\n28"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am satisfied with the responses and changes made to the manuscript.", "reviewer_2": "Overall, the authors have thoroughly considered my original comments and revised their manuscript accordingly. I certainly didn’t require, or expect, additional data to appear, but the new data from 13 canal reaches in another area further strengthen the small dataset and are a welcome addition. I have two small comments on this new draft. Otherwise, I find the manuscript acceptable for publication and look forward to seeing the published version.\nOriginal comment:\nL315. “Duplicate samples for each canal (and depth, if applicable) were acidified every ~24 hours to pH <2 using 1.5M HCl to stop CH4 oxidation.”\nSo you had two replicates for each measurement and then (presumably) took the mean of both? This is good, but it would be nice to see the reps data; how consistent are they to one another? Considering your small sample size this info would be useful.", "reviewer_3": "Comments on the reviewed manuscript NCOMMS-24-34376 “Fate of methane in canals draining tropical peatlands” by Perryman et al.\nPerryman et al. have done an amazing job addressing my concerns in the revised manuscript. All the points raised were carefully considered in the revised text, including new relevant information and clarifications. I don’t have any further considerations about the manuscript and I believe this is a valuable contribution to the field.\nSimilarly to the manuscript, I would like to thank the authors for this well-structured and nice to read response letter."}, "author_response": "Author responses to new reviewer comments are in blue text.\nReviewer #1 (Remarks to the Author):\nI am satisfied with the responses and changes made to the manuscript.\nWe thank Reviewer 1 for their time and constructive feedback which helped improve our manuscript.\nReviewer #2 (Remarks to the Author):\nOverall, the authors have thoroughly considered my original comments and revised their manuscript\naccordingly. I certainly didn’t require, or expect, additional data to appear, but the new data from 13 canal\nreaches in another area further strengthen the small dataset and are a welcome addition. I have two\nsmall comments on this new draft. Otherwise, I find the manuscript acceptable for publication and look\nforward to seeing the published version.\nWe thank Dr. Peacock for his constructive feedback on our work. His suggestions helped us strengthen\nthe manuscript. Please see below for our responses to the 2 minor comments on the revised manuscript.\nOriginal comment:\nL315. “Duplicate samples for each canal (and depth, if applicable) were acidified every ~24 hours to pH\n<2 using 1.5M HCl to stop CH4 oxidation.”\nSo you had two replicates for each measurement and then (presumably) took the mean of both? This is\ngood, but it would be nice to see the reps data; how consistent are they to one another? Considering your\nsmall sample size this info would be useful.\nAuthor response:\nWe have added a supplementary table (Table S2) that has the initial and final CH4 concentrations and\nδ13C-CH4 (mean ± standard error) and the incubation time for all incubated waters.\nNew comment:\nMy original comment asked for the replicate sample data to be included – that is still hidden in Table S2\nby the use of means (although the SEM values give hints). It isn’t in the online data either: the file\nCanal_Water_Incubations_Perryman has the replicate measurements of dissolved CH4 and d13CH4 (or\nare these the replicate *changes* in these parameters?) but this isn’t sufficient. To be clear, I would like to\nsee the raw, replicate data, set out as in Table S2 (CH4 T0, CH4 Tfinal, 13C T0, 13C Tfinal) whereby\neach individual, replicate incubation has its own line (i.e. not averaged together) – unless these data are\nalready hiding somewhere in the SI but if so I don’t see it. It’s potentially important/interesting for the\nreader to see how consistent your reps are.\nThe file “Canal_Water_Incubations_Perryman.xlxs” in the Zenodo repository for this manuscript\n(https://doi.org/10.5281/zenodo.11155160) has the data requested here. Each row reports the CH\n4\nconcentration (in µM) and 13C (in ‰) for the 2 replicate vials for each canal at each time point. For\nexample, the following table in the same format reports the results for canal #10:\nCanal Depth Rep Hours dissolvedCH4 13C\nAt T , CH concentration of the 2 replicates (A and B) was 0.52 and 0.47 µM, and the 13C of those\n0 4\nreplicates -50.5 and -50.0‰. After 53.6 hours of incubation, the T CH concentration and 13C of the 2\nfinal 4\nreplicates was 0.42 and 0.37 µM and -47.9 and -42.7‰, respectively. Table S2 reports the mean ±\nstandard error of the CH concentration and 13C of the 2 replicates.\n0 final\n(preserved after ~50 hours of incubation). Each vial was analyzed for CH concentration and 13C once.\nand the raw data from each time point is in the “Canal_Water_Incubations_Perryman.xlxs” on Zenodo.\nThese data are also in the Source Data file for Figure 2a that will be uploaded alongside final paper\nrevisions.\nWe have revised the description for Table S2 to direct readers to our Source Data File to view the\nindividual (i.e., raw) data points:\n“Table S2. Dissolved CH concentration and δ13C-CH across incubated canal waters. Values are mean ±\n4 4\nstandard error of 2 replicate vials for each time point. Time indicates the incubation length before\ndetermination of the final CH concentrations and δ13C-CH . For depth, S = surface and D = deep. Values\n4 4\nfor the CH concentrations and δ13C-CH of individual replicates are available in the manuscript Source\n4 4\nData file.”\nOne minor comment\nL344. “Open undeveloped land” is slightly vague because to a casual reader it hides human action.\nPerhaps change to “deforested undeveloped land” (or similar)?\n“Open undeveloped land” is a land use classification used in research on peatlands in Southeast Asia.\nOur use of “open undeveloped land” follows the definition of Miettinen et al. (2016, Global Ecology and\nConservation) which includes deforested areas with ferns/low shrub (< 2m) vegetation and ‘clearance’\nareas with no vegetation including recently burned areas.\nWe have revised this sentence to clarify the definition of this classification, but we did not revise the name\nof the group to be consistent with the literature on peatlands in this region (e.g., Bowen et al., 2024,\nNature Geoscience; Dadap et al., 2021, AGU Advances; Deshmukh et al., 2020, Global Change Biology;\nDeshmukh et al., 2021, Nature Geoscience; Miettinen et al., 2017, Environmental Management; Miettinen\net al., 2017, Environmental Research Letters).\nL294-298: “Smallholder mixed agriculture is the most represented land use in this study, but the sampled\ncanals also include areas in smallholder plantations (pineapple and oil palm), industrial oil palm\nplantations, and open undeveloped land (i.e., deforested and/or burned areas), as well as 1 canal in a\ndegraded forest, to capture the heterogeneity of drainage canals in the region.”\nMike Peacock\nReviewer #3 (Remarks to the Author):\nComments on the reviewed manuscript NCOMMS-24-34376 “Fate of methane in canals draining tropical\npeatlands” by Perryman et al.\nPerryman et al. have done an amazing job addressing my concerns in the revised manuscript. All the\npoints raised were carefully considered in the revised text, including new relevant information and\nclarifications. I don’t have any further considerations about the manuscript and I believe this is a valuable\ncontribution to the field.\nSimilarly to the manuscript, I would like to thank the authors for this well-structured and nice to read\nresponse letter.\nWe thank Reviewer 3 for their thorough feedback on the manuscript. The revisions made in response to\ntheir review strengthened the work and we appreciate the time and care taken to provide constructive\nfeedback."}]

Decision letter