the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 11 Mar 2026
Novel in situ CO2 enrichment system reveals seagrass meadows are a refugium against coastal acidification for North Atlantic bivalves
Abstract. While the accumulation of anthropogenic CO2 in the atmosphere is causing a decline in global ocean pH, many eutrophic estuaries are already experiencing acidification due to accelerated respiration driving the consumption of dissolved oxygen (DO) and production of CO2, decreasing available carbonate ions (CO32-) and threatening marine calcifiers. Here, a novel in situ CO2 enrichment system was constructed to examine the effects of coastal acidification on the growth and survival of two species of North Atlantic bivalves (Argopecten irradians and Crassostrea virginica) in two distinct estuarine habitats: a seagrass meadow and an unvegetated sandy bottom in an open water estuary. The in-situ system captured natural diel dynamics as ambient chambers displayed chemistry nearly identical to the surrounding water, while CO2-enriched, acidified chambers maintained a consistent ~Δ 0.3–0.5 pH offset. At the unvegetated sandy bottom site, A. irradians and C. virginica displayed significant reductions in growth and survival in the acidified chambers (pHT = 7.3–7.5; saturation state of aragonite, ΩAr = 0.6–0.9) relative to ambient conditions (pHT = 7.6–7.9; ΩAr = 1.6–2). At the seagrass site, while growth of A. irradians and C. virginica in the acidified treatments (pHT = 7.3–7.7; ΩAr = 0.7) receiving the same delivery of CO2 was, again, significantly slowed compared to the control (pHT = 7.5–8.1; ΩAr = 2 – 2.8), the growth reduction, mortality rates, and levels of acidification were attenuated compared to the sandy bottom experiment, evidencing the ability of seagrass to buffer seawater and serve as a potential acidification refuge for bivalves. Collectively, the novel experimental CO2 enrichment system constructed for this project demonstrates that coastal acidification can have deleterious effects on marine bivalve populations, and that future conditions as well as the habitat refuge offered by seagrasses must be considered when developing management and restoration plans for temperate estuaries.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2026-745', Maxime Daviray, 07 Apr 2026
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AC1: 'Reply on RC1', Ryan Wallace, 19 Sep 2026
General comments
In this manuscript, Wallace et al. started describing spatial distribution of the carbonate chemistry in seawater of the two surveyed coastal areas in August-September (open water and seagrass beds). Then, they presented a novel in situ CO2 enrichment system they used for incubating juvenile bivalves (scallops, Argopecten irradians, and oysters, Crassostrea virginica) in acidified vs ambient conditions (~∆ 0.3 - 0.5 pH) at both sites. They monitored seawater parameters (temperature, salinity, dissolved oxygen and pH) continuously, and measured dissolved inorganic carbon twice a week to reconstruct carbonate system, for about one month. Finally, they estimated the growth rate and mortality of specimens at the end of the incubations, and statistically compared both conditions and both sites (One- and Two-way ANOVA, and pairwise comparison). Through this interesting approach, Wallace et al. showed a seawater buffering in seagrass bed that mitigates significantly the growth reduction and mortality of bivalves under acidified conditions. The methodological approaches are comprehensive and the in-situ incubation system is impressive. The study aims to characterize seawater conditions and relevant biological parameters. However, there is a lack of information on the hydro-sedimentary and bathymetric contexts at both sites. This information is necessary for understanding how the two systems function overall. The authors have extensively studied this area. A brief summary, referencing their previous, more comprehensive studies, would help the reader to better understand these two environments. Some elements lack clarity and the structure of the manuscript is occasionally difficult to follow and should be revised. Occasional redundancies in the description of the results could be avoided, and certain figures should be revised to improve readability, particularly in the results section, where the density of information is high given the number of variables studied. I would also recommend that the authors upload the data used for this publication (statistical tests and figures) to an online repository (i.e., Zenodo), making it accessible to all. Overall, I think this is a robust and very interesting research paper, with a wide variety of high-quality data, which corresponds to the scope of Biogeosciences.
We thank the reviewer for their thoughtful and constructive comments. We have carefully considered each comment below and we will upload the data to an online repository (e.g., Zenodo). We will also add a paragraph describing the hydrodynamic, sediment, and bathymetric context of both sites, with reference to our prior studies of these systems.
Specific comments
Mat & Meth section
Line 115: At what month and what time of day did you take the samples to build the map? Were all the samples collected on the same day, or did you time your campaign over a specific period? I ask because we can expect significant variability in the measurements depending on the month, the time of day and the photosynthetic activity of the seagrass bed. You also specify that the Peconic Estuary map was done in August (l.128), and that you sampled at 3:00 PM during the incubations (l. 238).
All samples were collected on the same day during the afternoon in August. We were also conducting adjacent experimental incubations during this time. We will add this to the manuscript so that there is no confusion regarding the sampling times.
Line 135: You specify that sampling is done one meter below the surface, but how far from the substrate in this case? Why not use the same approach as at the seagrass bed site to obtain truly comparable maps?
These data were collected over a larger area and this is a subset of that survey. The maximum depth at the far northern portion of this figure is ~4 m, but most of this area is < 2 m. We have additional bottom samples that were collected via Niskin bottle although the samples are limited in this specific location (n = 3). This area is well mixed and the samples collected at the surface and bottom are similar. The CO2 range in this area is minimal (< 20 µatm) and aligns with the values observed in the ambient chambers during this period. We used the NDIR data because it provided greater spatial coverage, and this figure illustrates the minimal spatial variability observed in this area.
Lines 165-166: You state that you are targeting a pH of 0.3–0.5 points below ambient conditions to simulate acidified conditions, and that this range reflects the projected pH levels by 2100. However, you also note that these values are “commonly observed in eutrophic NY estuaries during the summer”. In this case, is it possible that future pH projections for this location could be lower than expected globally? Did the available data not enable to simulate a local decrease in pH by 2100?
It’s possible, although these two locations are not considered eutrophic estuaries. We mentioned this to point out that bivalve populations may already be experiencing these conditions in eutrophic systems. We will revise this sentence to clarify that the target offset reflects both projections for 2100 and conditions already observed in eutrophic estuaries, and that neither study site is considered eutrophic.
Line 179: the value of adding a description of these parameters for both sites.
Mean tidal velocity was the same at both locations. The target velocity (~2.9 cm/s) was based on tidal measurements from our prior work (Wallace et al., 2021). We will state this in the manuscript and include it in the new paragraph describing both sites.
Line 210: Why are the experimental periods at the two sites different? This raises the question of whether this difference has an impact on bivalve mortality.
Unfortunately, we had to break down the seagrass experiment early due to an approaching storm. Growth rates were normalized per day and the primary treatment comparisons were made within each site, so the difference in duration does not affect those results. We acknowledge that the shorter seagrass deployment could influence cross-site comparisons of cumulative mortality, and we will note this caveat in the discussion. We will also consider presenting mortality as a daily standardized rate.
Lines 210-212: This information should be included in a paragraph providing context for the two sites.
We agree with the reviewer and will add a paragraph describing both locations.
Line 216: It's a shame there weren't more size categories available for the oysters! It would have been very interesting to include them.
We agree that additional oyster size classes would have been valuable. Unfortunately, only one size class was available from the hatchery at the time of the experiments. We will note this limitation in the manuscript.
Line 228: Have you included dead specimens in the growth rate calculation? If not, please specify (you write l.495: “the growth rates of the survivors”). If so, these specimens should be excluded, as they distort the growth rate, which is calculated over the entire incubation period. It is impossible to determine when their growth ceased due to death rather than the chemical conditions.
We did not include dead bivalves in the growth rate calculations. We will revise both sentences to clarify.
Line 254: Just out of curiosity, why did you choose the dissociation constants from Millero et al. (2010) over those from Cai and Wang (1998)? The latter are also relevant in estuarine environments.
They both work well for estuaries and would not result in large differences. Millero tends to align better with our NDIR CO2 measurements. Additionally, Cai and Wang have a larger uncertainty when salinities are higher. Long Island estuaries have generally high salinities due to minimal freshwater input. The estuaries examined in this study had salinities ~30.
Results section
The results section could be clearer if it were restructured. Section 3.1 presents the spatial distribution of chemical parameters at each site. It would then be logical to present a comparison of these parameters across substrates and treatments (change of scale; Section 3.4), followed by a comparison of treatments for each substrate (first paragraphs of Sections 3.2 and 3.3). Finally, after providing an overview of the carbonate parameters, the responses of the bivalves to the different incubation conditions could be presented (Section 3.5) and detailed for each substrate (second paragraphs of Sections 3.2 and 3.3). This would make the results easier to read by limiting repetition and helping to maintain focus on the objectives of each section of this study.
We thank the reviewer for this comment and agree with their suggestion. We will restructure the Results section accordingly.
Line 296: How many square kilometers does the seagrass bed cover? Is it patchy? This kind of information should be included in a paragraph that describes the context of the site.
There is ~2.5 km2 of seagrass coverage within Shinnecock Bay. We surveyed an area of approximately 35,000 m² surrounding our experimental site. The western portion of our survey was quite dense, whereas the eastern portion was patchy. We provided high resolution drone imagery (see fig 2A) in which the grasses are visible. We agree with the reviewer and will add in total coverage within our survey area. In addition, the values reported in the manuscript were incorrect, and we will correct them accordingly.
Lines 300-306: It is unclear which areas are covered by the seagrass bed and which are not. This information should be shown in Figure 2.
We agree with the reviewer and will add a false color image indicating seagrass and sandy bottom.
Line 311: What about ΩArin the open water site?
Thank you, we will add this to the manuscript.
Lines 333-334: I’m less certain about ΩAr mean value in the seagrass bed under acidic conditions: it appears to have been just saturated during the incubation period. The associated uncertainties are high (± 27 %), suggesting an alternation of under and oversaturated periods. Do you have any insight into this alternation? Could it be a day-night effect? This point is not sufficiently developed in the discussion (lines 469–475).
Yes, this site experienced substantially greater variability, driven primarily by diel cycles, see figure 5. We agree with the reviewer and will add this to the discussion section.
Line 414: You wrote “With respect to mortality, all bivalves demonstrated mortal sensitivity to acidification”, even though, as you pointed out l. 420–421, this is only truly significative for the 5-mm bay scallops.
Thank you, we will revise this sentence to state that a significant effect of acidification on mortality was detected only for the 5 mm bay scallops.
Discussion section
I would also recommend restructuring the discussion to mirror the format of the results section. This would involve beginning by highlighting the 'halo' effect of seagrass beds as potential refuge habitats compared to the open environment, by discussing their buffering capacity and dynamics. The next step would be to move on to the bivalves responses to these different conditions, as well as the ecological and economic vulnerabilities under the tested conditions (acidified treatments). This would make the discussion easier to read, as it would limit repetition between sections and help maintain focus on the objectives of each one.
Thank you. We agree with the reviewer and will restructure the discussion section as such.
Line 444: I completely agree with the authors. The problem is that these factors (diurnal, tidal and seasonal) lack of context in this study. Without this context, it is difficult to fully grasp the results of this otherwise very interesting study, especially given that these factors are critical to the 'halo' effect associated with seagrass beds (Cyronak et al., 2018). Thus, we can assume that the refuge effect no longer exists in winter because the eelgrass loses its leaves. Therefore, the discussion should place greater emphasis on this limited seasonal timeframe.
Thank you, we will discuss the limited seasonal period of this study. Both systems have been extensively monitored through our previous studies, and this timeframe was selected to capture the period when environmental conditions are most extreme. In addition, we do present diel variability in pH, see fig 5.
Line 466: As mentioned earlier, given the magnitude of the associated deviation and the lack of information regarding the sources of this variability, I would be very cautious about saying that ΩAr is saturated, even on average.
We agree with the reviewer and we can clarify that ΩAr was generally higher in the seagrass meadow, but more variable, in relation to the open water site.
Lines 489-490: Based on the results you present, there is no significant effect of the acidified treatment on oyster mortality.
Thank you. We will correct this statement so that the discussion is consistent with the statistical results.
Line 513-519: I'm not sure why you're changing the buffer indicator by introducing the DIC/AT ratio here. Why not apply this to the seagrass substrate as well? Alternatively, why not use this ratio instead of Rf throughout the manuscript? This ratio should then be added to Tables 1 and 2, or at least included in the supplementary material. Also, the sentence about ΩAr (l. 515-516) appears out of the blue when you are actually discussing the buffer capacity of the site; it should come after that as a transition.
The DIC/AT ratio is more indicative of the current state of the carbonate system, and we included it as additional evidence supporting the differences in carbonate chemistry between sites. We do state that the open water site had a higher ratio than seagrass, but we agree with the reviewer that this is out of context in this section. We wouldn’t want to replace Rf throughout the manuscript as this represents the carbonate system capacity to buffer changes in CO2. We will remove the ratio and the ΩAr sentence from the discussion.
Lines 564-566: Do you have any idea what proportion of alkalinization is contributed by sediment compared to seagrasses? Are the carbonate mineral compositions similar between the two sites? It could be assumed that the dissolution of carbonate minerals in the sediment also contributes to the alkalinization of seawater. In that case, the observed substrate effect might not just be a seagrass effect, but rather a combination of the nature of the sediment and the seagrass. It would be interesting to repeat the experiment in winter, when almost all the eelgrass leaves have fallen and photosynthesis has virtually ceased, to see how the bivalves respond to river discharge, which reduce the seawater buffering capacity. This would allow us to determine whether the sediment can then take over from the eelgrass bed as a buffer under these conditions.
We don’t know the proportion, but carbonate mineral compositions are similar (e.g., biogenic shell material) and we do know that the sediment in the seagrass has more organics than the open water site. It is likely that carbonate minerals are contributing to these patterns, and investigating their role would be an interesting experiment and an area worth considering in future work. However, there are no large rivers on Long Island, so it would be unlikely to observe a reduced buffer capacity due to freshwater input. We will add a sentence to the discussion noting the potential contribution of sediment carbonate dissolution to the elevated alkalinity at the seagrass site.
Lines 577-580: Out of interest, are you aware of any seagrass restoration projects in the Long Island area? The same question applies to future shellfish harvesting activities.
Yes, there have been seagrass restoration efforts in Shinnecock Bay. In addition, hard clam restoration projects, the development of oyster reefs, and the expansion of oyster aquaculture in both Shinnecock and Peconic Bays have been quite substantial in recent years.
Figures and Tables
Regarding the figures, I suggest that the authors reduce the number of them by merging some of them (e.g., Fig. 2/Fig. 3, and Fig. 6/Fig. 7/Fig. 8) to make it easier to compare the diversity of conditions, sites and organisms, and to improve clarity when reading the results. Similarly, Tables 3 and 4 should be reorganized by substrate, with one table summarizing growth rates and mortality in seagrass beds and another for open waters. This would follow the structure of the results. I suppose this is due to the document being generated automatically upon submission, but the figures and Tables are not always placed in the corresponding results sections, which makes the document harder to read. This should be corrected.
We agree with the reviewer and will make these changes.
Figure 2: despite the high-resolution imagery, we are unable to identify the seagrass beds in the figure. It would be helpful to add their boundaries to the map. The same applies when adding the black box to maps B and C.
The seagrass beds are the darker colors in the image. We will add a false color image indicating seagrasses vs sandy bottom.
Figure 3: although the resolution is indicated in the caption, a scale bar should be added to images A and B/C, along with the north orientation (or latitude/longitude coordinates, as in Figure 2).
Thank you. We will add this to the figure.
Figure 4: A) I’m not sure if the system diagram is to scale, but it would be helpful to include the dimensions. B) The axis labels are too small to be easily read by everyone.
We include the dimensions in the manuscript and we can add this to the figure, although only the chamber and the raceway path are drawn to scale. We will adjust the axis labels.
Figures 6-7-8: The axis labels are also too small to be easily read. To ensure consistency with the tables and the terminology used in the text to refer to the treatment, I suggest replacing “elevated” with “acidified”.
Thank you, we will make these changes.
Tables 3 and 4: specify again the number of specimens used under each condition. Due to the uncertainty values associated with the mean, it is not very appropriate to keep the decimal point.
Thank you, we will make these adjustments.
Table S1: what is the difference between “true ambient” and “ambient”?
True ambient refers to samples collected outside the chambers, whereas ambient refers to the control chambers. We will clarify this in the table caption.
Technical corrections
As a Latin expression, “in situ” should be written in italic overall the text.
Line 120: Section 2.5 instead of 2.4.
Line 224: please cite the source of the software.
Line 262: please precise that all the statistical test results can be found in the supplementary material.
Line 416: “mortality” instead of “morality”.
Lines 418-420: the two sentences seem the same.
Line 430: “two-month” instead of “two-year”.
Lines 570/581: Sections 4.4 and 4.5 have the same name. I suppose section 4.5 is the conclusion, but I would recommend merging these two sections.
Line 586: I suggest also heat waves?
Thank you, we will make all of the necessary technical corrections.
Citation: https://doi.org/10.5194/egusphere-2026-745-AC1
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AC1: 'Reply on RC1', Ryan Wallace, 19 Sep 2026
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RC2: 'Comment on egusphere-2026-745', Anaïs Richard, 27 Jul 2026
General comments
This article provides valuable insights into the effect of ocean acidification on the growth and survival of commercial bivalves, thanks to innovative in situ experiment. However, some clarifications are needed.
Abstract
L8: You could add « additional » acidification due to accelerated respiration etc. because we have the impression how it phrases that is it the main causes, and not the different concentration between air-water that accelerate the CO2 dissolution.
Introduction
L31-39: I suggest to rearrange a little this paragraph for clarity. The sentence “The chemical response of estuarine environments to acidification is especially difficult to quantify because biological activity may amplify predicted chemical changes resulting from increased atmospheric CO2 (Wallace et al. 2021).” can be place after the first one. Then we would understand better that the main process is the increase of CO2 dissolution, and there are other processes that amplify the acidification, with more local concerns with eutrophication. Please rearrange in that sense, if it was the main idea.
L59-60: If the benefits are those mentioned in the previous sentence, could you establish a connection between the two sentences or rephrase them to reflect that?
L95-98: The objectives could be developed to enhance clarity: is it in a eutrophic site/context? Because it has been introduced in previous paragraphs, but then not mention again; you could mention the two target species already; early life stage is mentioned but later 2 different sizes are used for one species. Please clarify here or detail/justify in the material and method.
Methods
L108-109: It is not clear what was measured during this cruise, the link with drone imagery. Are the parameters cited thereafter measured during the cruise? When happened the two cruises: before/during/after in situ experiment and drone imagery? Please clarify.
L128: same comment as above: when was it? It could be nice to have an overall sentence at the beginning to sum up all methods used (cruise, drone, experiment), when, why, duration etc. for clarification.
L187: Section 2.2 you describe the system, now section 2.3 what you measured during incubation, then section 2.4 the variable related to bivalve, but the duration of the in situ experiment is not mentioned and is missing. As there is a lot of variable measured, please clarify the timeline of everything and experiment duration, even though the dates are finally mentioned L205.
And how many chambers per treatment (acidified and none-acidified), how many replicates?
L229: when water samples were taken during experiment? And once? And no extreme weather event during experiment?
L264 or 268: from drone and/or the cruise?
L266: pH and DO were monitored during in situ experiment?
L275: which non-parametric test? For which variable?
L288: How many bivalves per species and size class were included in analyses?
L293: fixed factors with or without interaction?
I didn’t really understand why one way and two-way anovas were used. It seems redundant as two-way anova will test both substrate and treatment. This is not clear.
Results
Please state what is experiment 1 and 2 in the methods.
Discussion
L430: The in situ experiment lasted 2 years? And not one month as suggest the dates mentioned in the method section? Please clarify.
L431: For how long bivalves in acidified chamber were exposed in this study before the measurement started? Please mentioned this important information, and please discuss the potential impact of the acclimatation or effect on your results.
L434: do you have references or are you stating that seagrass altered the fate of bivalves exposed to acidification based on your experiment?
L460: how is the scale roughly of this halo?
L543: Do you have any understanding of resistance mechanisms?
Citation: https://doi.org/10.5194/egusphere-2026-745-RC2 -
AC2: 'Reply on RC2', Ryan Wallace, 19 Sep 2026
General comments
This article provides valuable insights into the effect of ocean acidification on the growth and survival of commercial bivalves, thanks to innovative in situ experiment. However, some clarifications are needed.
We thank the reviewer for their thoughtful and constructive comments. We have carefully considered each comment below.
Abstract
L8: You could add « additional » acidification due to accelerated respiration etc. because we have the impression how it phrases that is it the main causes, and not the different concentration between air-water that accelerate the CO2 dissolution.
Thank you, we will add additional.
Introduction
L31-39: I suggest to rearrange a little this paragraph for clarity. The sentence “The chemical response of estuarine environments to acidification is especially difficult to quantify because biological activity may amplify predicted chemical changes resulting from increased atmospheric CO2 (Wallace et al. 2021).” can be place after the first one. Then we would understand better that the main process is the increase of CO2 dissolution, and there are other processes that amplify the acidification, with more local concerns with eutrophication. Please rearrange in that sense, if it was the main idea.
We agree with the reviewer and will rearrange this paragraph accordingly.
L59-60: If the benefits are those mentioned in the previous sentence, could you establish a connection between the two sentences or rephrase them to reflect that?
Yes, we will rephrase this sentence to clarify.
L95-98: The objectives could be developed to enhance clarity: is it in a eutrophic site/context? Because it has been introduced in previous paragraphs, but then not mention again; you could mention the two target species already; early life stage is mentioned but later 2 different sizes are used for one species. Please clarify here or detail/justify in the material and method.
The experimental sites examined here are not considered eutrophic systems. We will adjust the paragraph to include more specifics regarding size classes and the habitats examined in this study.
Methods
L108-109: It is not clear what was measured during this cruise, the link with drone imagery. Are the parameters cited thereafter measured during the cruise? When happened the two cruises: before/during/after in situ experiment and drone imagery? Please clarify.
The imagery indicates locations of seagrass beds as this area has patchy seagrass coverage. The darker areas are the seagrass beds. Yes, the parameters stated were measured during the survey. The second cruise mentioned is referring to the open water cruise. We will clarify at the beginning of section 2.1.
L128: same comment as above: when was it? It could be nice to have an overall sentence at the beginning to sum up all methods used (cruise, drone, experiment), when, why, duration etc. for clarification.
Thank you, we will revise this section for clarification.
L187: Section 2.2 you describe the system, now section 2.3 what you measured during incubation, then section 2.4 the variable related to bivalve, but the duration of the in situ experiment is not mentioned and is missing. As there is a lot of variable measured, please clarify the timeline of everything and experiment duration, even though the dates are finally mentioned L205.
We agree with the reviewer. We will adjust these sections to include a timeline of surveys, experiments conducted, and sampling regime.
And how many chambers per treatment (acidified and none-acidified), how many replicates?
Three acidified chambers and three ambient chambers were deployed at each site (n = 3 per treatment). We will state this clearly in the methods.
L229: when water samples were taken during experiment? And once? And no extreme weather event during experiment?
We continuously monitored temperature, dissolved oxygen, and pH, while discrete samples were collected every two to three days. A storm event required us to remove the seagrass chambers approximately ~10 days earlier than anticipated, resulting in an earlier termination of the experiment.
L264 or 268: from drone and/or the cruise?
We are describing interpolation methods and statistics for both surveys, seagrass and open water cruises. We will add a sentence to clarify.
L266: pH and DO were monitored during in situ experiment?
Yes, but this sentence is describing the comparison between seagrass and the sandy bottom within the seagrass survey area.
L275: which non-parametric test? For which variable?
Kruskal-Wallis tests were used when data did not meet the assumptions of normality or homogeneity of variance. This applied to the Revelle factor and the aragonite saturation state in the comparison across substrates and treatments (Section 3.4). We will name the test and the variables explicitly in Section 2.6.2.
L288: How many bivalves per species and size class were included in analyses?
The sample sizes used in the analyses are provided in the Supplemental Materials (Tables S3, S5, and S7). We will add these sample sizes to Section 2.6.3 and to Tables 3 and 4.
L293: fixed factors with or without interaction?
Substrate and treatment were treated as fixed factors, and the models included the substrate × treatment interaction, which allowed for pairwise comparisons among the four substrate and treatment combinations. We will state this in Section 2.6.3 and report the interaction terms in the supplementary tables.
I didn’t really understand why one way and two-way anovas were used. It seems redundant as two-way anova will test both substrate and treatment. This is not clear.
We thank the reviewer for this comment. The two experiments were separate deployments, so one-way ANOVAs were first used within each site to confirm that the acidified chambers differed from the ambient chambers and to test the effect of treatment on bivalves within that habitat. Two-way ANOVAs were then used to compare responses across habitats. We agree that this is partly redundant. In the revised manuscript, we will present the two-way ANOVAs with interaction and the associated pairwise comparisons as the primary analysis and limit the one-way tests to confirming that the ambient chambers matched the surrounding water. This will also reduce repetition in the restructured Results section.
Results
Please state what is experiment 1 and 2 in the methods.
Thank you, we will indicate this in the methods.
Discussion
L430: The in situ experiment lasted 2 years? And not one month as suggest the dates mentioned in the method section? Please clarify.
Thank you, this should be one month.
L431: For how long bivalves in acidified chamber were exposed in this study before the measurement started? Please mentioned this important information, and please discuss the potential impact of the acclimatation or effect on your results.
Thank you, we will add this to the manuscript. Bivalves were acclimated for one week at ambient conditions prior to exposure to the target acidification treatments. We will also briefly discuss the potential influence of the acclimation period and the exposure duration on our results.
L434: do you have references or are you stating that seagrass altered the fate of bivalves exposed to acidification based on your experiment?
We are stating the results of this study. We will revise this sentence.
L460: how is the scale roughly of this halo?
It is difficult to say definitively, but we observed distinct differences in pH and CO₂ approximately 75–100 m outside the dense seagrass beds. We can add a sentence or two to the manuscript describing these observations.
L543: Do you have any understanding of resistance mechanisms?
It’s likely that distinct biomineralization strategies play a role. Schwaner et al. (2024) found that carbonic anhydrase and intracellular pH regulation in oysters contribute to maintaining conditions favorable for calcification under low pH. These were oysters from Long Island waters. In addition, under acidified conditions, juvenile oysters have been found to have positive net calcification when seawater is undersaturated with respect to calcite (Dodd et al., 2021). We can add a few sentences describing these mechanisms.
Schwaner et al.,2024. Probing the role of carbonic anhydrase in shell repair mechanisms in the eastern oyster Crassostrea virginica under experimental acidification stress
Dodd et al., 2021. Juvenile Eastern Oysters More Resilient to Extreme Ocean Acidification than Their Mud Crab Predators
Citation: https://doi.org/10.5194/egusphere-2026-745-AC2
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AC2: 'Reply on RC2', Ryan Wallace, 19 Sep 2026
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General comments
In this manuscript, Wallace et al. started describing spatial distribution of the carbonate chemistry in seawater of the two surveyed coastal areas in August-September (open water and seagrass beds). Then, they presented a novel in situ CO2 enrichment system they used for incubating juvenile bivalves (scallops, Argopecten irradians, and oysters, Crassostrea virginica) in acidified vs ambient conditions (~∆ 0.3 - 0.5 pH) at both sites. They monitored seawater parameters (temperature, salinity, dissolved oxygen and pH) continuously, and measured dissolved inorganic carbon twice a week to reconstruct carbonate system, for about one month. Finally, they estimated the growth rate and mortality of specimens at the end of the incubations, and statistically compared both conditions and both sites (One- and Two-way ANOVA, and pairwise comparison). Through this interesting approach, Wallace et al. showed a seawater buffering in seagrass bed that mitigates significantly the growth reduction and mortality of bivalves under acidified conditions. The methodological approaches are comprehensive and the in-situ incubation system is impressive. The study aims to characterize seawater conditions and relevant biological parameters. However, there is a lack of information on the hydro-sedimentary and bathymetric contexts at both sites. This information is necessary for understanding how the two systems function overall. The authors have extensively studied this area. A brief summary, referencing their previous, more comprehensive studies, would help the reader to better understand these two environments. Some elements lack clarity and the structure of the manuscript is occasionally difficult to follow and should be revised. Occasional redundancies in the description of the results could be avoided, and certain figures should be revised to improve readability, particularly in the results section, where the density of information is high given the number of variables studied. I would also recommend that the authors upload the data used for this publication (statistical tests and figures) to an online repository (i.e., Zenodo), making it accessible to all. Overall, I think this is a robust and very interesting research paper, with a wide variety of high-quality data, which corresponds to the scope of Biogeosciences.
Specific comments
Mat & Meth section
Line 115: At what month and what time of day did you take the samples to build the map? Were all the samples collected on the same day, or did you time your campaign over a specific period? I ask because we can expect significant variability in the measurements depending on the month, the time of day and the photosynthetic activity of the seagrass bed. You also specify that the Peconic Estuary map was done in August (l.128), and that you sampled at 3:00 PM during the incubations (l. 238).
Line 135: You specify that sampling is done one meter below the surface, but how far from the substrate in this case? Why not use the same approach as at the seagrass bed site to obtain truly comparable maps?
Lines 165-166: You state that you are targeting a pH of 0.3–0.5 points below ambient conditions to simulate acidified conditions, and that this range reflects the projected pH levels by 2100. However, you also note that these values are “commonly observed in eutrophic NY estuaries during the summer”. In this case, is it possible that future pH projections for this location could be lower than expected globally? Did the available data not enable to simulate a local decrease in pH by 2100?
Line 179: the value of adding a description of these parameters for both sites.
Line 210: Why are the experimental periods at the two sites different? This raises the question of whether this difference has an impact on bivalve mortality.
Lines 210-212: This information should be included in a paragraph providing context for the two sites.
Line 216: It's a shame there weren't more size categories available for the oysters! It would have been very interesting to include them.
Line 228: Have you included dead specimens in the growth rate calculation? If not, please specify (you write l.495: “the growth rates of the survivors”). If so, these specimens should be excluded, as they distort the growth rate, which is calculated over the entire incubation period. It is impossible to determine when their growth ceased due to death rather than the chemical conditions.
Line 254: Just out of curiosity, why did you choose the dissociation constants from Millero et al. (2010) over those from Cai and Wang (1998)? The latter are also relevant in estuarine environments.
Results section
The results section could be clearer if it were restructured. Section 3.1 presents the spatial distribution of chemical parameters at each site. It would then be logical to present a comparison of these parameters across substrates and treatments (change of scale; Section 3.4), followed by a comparison of treatments for each substrate (first paragraphs of Sections 3.2 and 3.3). Finally, after providing an overview of the carbonate parameters, the responses of the bivalves to the different incubation conditions could be presented (Section 3.5) and detailed for each substrate (second paragraphs of Sections 3.2 and 3.3). This would make the results easier to read by limiting repetition and helping to maintain focus on the objectives of each section of this study.
Line 296: How many square kilometers does the seagrass bed cover? Is it patchy? This kind of information should be included in a paragraph that describes the context of the site.
Lines 300-306: It is unclear which areas are covered by the seagrass bed and which are not. This information should be shown in Figure 2.
Line 311: What about ΩAr in the open water site?
Lines 333-334: I’m less certain about ΩAr mean value in the seagrass bed under acidic conditions: it appears to have been just saturated during the incubation period. The associated uncertainties are high (± 27 %), suggesting an alternation of under and oversaturated periods. Do you have any insight into this alternation? Could it be a day-night effect? This point is not sufficiently developed in the discussion (lines 469–475).
Line 414: You wrote “With respect to mortality, all bivalves demonstrated mortal sensitivity to acidification”, even though, as you pointed out l. 420–421, this is only truly significative for the 5-mm bay scallops.
Discussion section
I would also recommend restructuring the discussion to mirror the format of the results section. This would involve beginning by highlighting the 'halo' effect of seagrass beds as potential refuge habitats compared to the open environment, by discussing their buffering capacity and dynamics. The next step would be to move on to the bivalves responses to these different conditions, as well as the ecological and economic vulnerabilities under the tested conditions (acidified treatments). This would make the discussion easier to read, as it would limit repetition between sections and help maintain focus on the objectives of each one.
Line 444: I completely agree with the authors. The problem is that these factors (diurnal, tidal and seasonal) lack of context in this study. Without this context, it is difficult to fully grasp the results of this otherwise very interesting study, especially given that these factors are critical to the 'halo' effect associated with seagrass beds (Cyronak et al., 2018). Thus, we can assume that the refuge effect no longer exists in winter because the eelgrass loses its leaves. Therefore, the discussion should place greater emphasis on this limited seasonal timeframe.
Line 466: As mentioned earlier, given the magnitude of the associated deviation and the lack of information regarding the sources of this variability, I would be very cautious about saying that ΩAr is saturated, even on average.
Lines 489-490: Based on the results you present, there is no significant effect of the acidified treatment on oyster mortality.
Line 513-519: I'm not sure why you're changing the buffer indicator by introducing the DIC/AT ratio here. Why not apply this to the seagrass substrate as well? Alternatively, why not use this ratio instead of Rf throughout the manuscript? This ratio should then be added to Tables 1 and 2, or at least included in the supplementary material. Also, the sentence about ΩAr (l. 515-516) appears out of the blue when you are actually discussing the buffer capacity of the site; it should come after that as a transition.
Lines 564-566: Do you have any idea what proportion of alkalinization is contributed by sediment compared to seagrasses? Are the carbonate mineral compositions similar between the two sites? It could be assumed that the dissolution of carbonate minerals in the sediment also contributes to the alkalinization of seawater. In that case, the observed substrate effect might not just be a seagrass effect, but rather a combination of the nature of the sediment and the seagrass. It would be interesting to repeat the experiment in winter, when almost all the eelgrass leaves have fallen and photosynthesis has virtually ceased, to see how the bivalves respond to river discharge, which reduce the seawater buffering capacity. This would allow us to determine whether the sediment can then take over from the eelgrass bed as a buffer under these conditions.
Lines 577-580: Out of interest, are you aware of any seagrass restoration projects in the Long Island area? The same question applies to future shellfish harvesting activities.
Figures and Tables
Regarding the figures, I suggest that the authors reduce the number of them by merging some of them (e.g., Fig. 2/Fig. 3, and Fig. 6/Fig. 7/Fig. 8) to make it easier to compare the diversity of conditions, sites and organisms, and to improve clarity when reading the results. Similarly, Tables 3 and 4 should be reorganized by substrate, with one table summarizing growth rates and mortality in seagrass beds and another for open waters. This would follow the structure of the results. I suppose this is due to the document being generated automatically upon submission, but the figures and Tables are not always placed in the corresponding results sections, which makes the document harder to read. This should be corrected.
Figure 2: despite the high-resolution imagery, we are unable to identify the seagrass beds in the figure. It would be helpful to add their boundaries to the map. The same applies when adding the black box to maps B and C.
Figure 3: although the resolution is indicated in the caption, a scale bar should be added to images A and B/C, along with the north orientation (or latitude/longitude coordinates, as in Figure 2).
Figure 4: A) I’m not sure if the system diagram is to scale, but it would be helpful to include the dimensions. B) The axis labels are too small to be easily read by everyone.
Figures 6-7-8: The axis labels are also too small to be easily read. To ensure consistency with the tables and the terminology used in the text to refer to the treatment, I suggest replacing “elevated” with “acidified”.
Tables 3 and 4: specify again the number of specimens used under each condition. Due to the uncertainty values associated with the mean, it is not very appropriate to keep the decimal point.
Table S1: what is the difference between “true ambient” and “ambient”?
Technical corrections
As a Latin expression, “in situ” should be written in italic overall the text.
Line 120: Section 2.5 instead of 2.4.
Line 224: please cite the source of the software.
Line 262: please precise that all the statistical test results can be found in the supplementary material.
Line 416: “mortality” instead of “morality”.
Lines 418-420: the two sentences seem the same.
Line 430: “two-month” instead of “two-year”.
Lines 570/581: Sections 4.4 and 4.5 have the same name. I suppose section 4.5 is the conclusion, but I would recommend merging these two sections.
Line 586: I suggest also heat waves?