Review of the OAE impact study on isopods and sea hares by Jones et al.

This study examines the biological effect of NaOH on two invertebrate species (isopod and sea hare) in a 4-day long experiment testing three different pH levels. The study presents the results that are indictive of high sensitivity of both species under elevated pH (aligned with stronger OAE treatment). There are serious data missing on the carbonate chemistry and the overlap in conditions among the experimental treatments shows that the analyses have to be conducted in a different way (see the text below).

The introduction addressed OA and OAE and provides a rational for the OAE testing. However, it does provide an insufficient background on the OAE effects across different species. It also fails to provide OA effects (low pH) on the two examined species, which would give the background on their sensitivity. This is pertinent to more detailed explanation in lines 92-95.

Importantly, these are the sub-tidal species inhabiting the eel grass habitat but no data on the pH variability in the Puget sound and specifically within the eelgrass ecosystem is provided. Such information is essential for us to understand the diel variability these species encounter, as well as the pH max- namely, if subjected to high pH conditions in situ in this temporal period (Aug-Sept), then we might be expecting some natural acclimation/adaptation capacity in these species, that might be impacting the experimental results. Also, are there any changes in the month-long Aug-Sept period?

Methodology is the section where most of the clarifications are needed to evaluate the correctness of the exp design.

It needs to be noted that while the invertebrates living in the eelgrass are exposed to pH variability, none of the experiments incorporated this pattern in the exposed. IN addition, it is not provided how much of the eelgrass is sufficient for their nutrition and if the added eelgrasses sufficed or not- could there be some malnutrition occurring in the experiments? How was the diatom concentration determined, what equal concentration distributed within all the experimental jars? Are these two specific invertebrates feeding on diatoms and what is the heir C intake from eelgrass vs diatom?

Provide pH variability data as this represents a baseline to what these species are exposed to- lines 112-113.

Why was unfiltered water used in experiments? Line 126

What is the uncertainty of the YSI probe? Line 127

How does the variability of pH (7.8 +/- 0.3) impact the amount of NaOH that needs to be added? Is it possible that water with higher initial pH, where less NaOH was added, could induce less negative effects?

Was NaoH actually produced in the electrodialysis or was it commercially available?  line 133-134.

All OA/OAE biological studies need to report on the whole carbonate chemistry parameters and none of this is provided, not for the baseline chemistry and not for the amended (treated with NaOH) water. This absolutely needs to be added.  In the same way, all the changes are only reported in pH, while we need to understand also the change in TA to understand how much NaOH was added to different treatments. Provide a clear and methodical way of representing missing data.  Lines 177-180 are not solid justification and this needs to be amended.

Figure 1 is not clear and could be improved: 3 acclimation tanks with four treatments? How was this really conducted? Also missing is the chemical control with no NaOH added to demonstrate how the diel variability impacts diel changes in seawater before added NaOH.

Why was pCO2 then measured if full carb chem was not calculated and provided (the variability in such big pH changes should still be theoretically lower than the uncertainty of the measurements).

A major drawback is reporting pooled data on water chemistry as well as on the experimental results. You should provide unpooled data and conduct analyses on this? How else was the variability determined that the within the treatment levels?

Please, explain table 1 in more detail, I do not understand how min, max and average pH/T/DO levels determined the treatments? Why are there three values- is it form the diel variability? If so, why was the water not always taken at the same time to avoid this variability? How does this impact NaOH additions. The variability on the initial pH levels is almost as high as between treatment 1 and 2.

Results section:

Why are the changes in pCO2 and pH are not a major part of the experiments in linking chemistry to the biological data? Strongly consider removing after all the parameters have been provided.

Why was there such an increase in pCO2? Line 201-204 inaccurate statements

From the Figure 2 is appears that in most rounds the treatments 8.3, 8.8 and 9.3 were the same (accounting for the variability) and there were no statistical differences between them, only 7.8 was different (not in Round 1 for no hares present, and in Round 1 and 2 for hares present). The same issue of the treatment overlap was present in the isopod treatments. This likely is the consequence of a not properly tight system. Can you provide pH data for all the rounds?

Given the overlap in experimental treatments, it is impossible to separate the effects on the treatment levels. First, the levels need to be examined to really understand which of them are sufficiently different and only then examine biological differences. Right now section 3.2 is not valid. As it seems, the combined 8.3, 8.8 and 9.3 could be one treatment, which could be compared to 7.8, which means a 2-treatment design (at the best).

What is mortality (LC50) or LOEC? This is an ecotox study so present all data, i.e. NOEC; LOEC, EC50 on the combined graphs (not only LC50).

I am not sure why all the regression models (Table 3) were built with multiple parameters 8salinity, DO, temp) if only the pH (CO2) was intentionally (artificially) changed?

Until these changes are introduced, the text from line 380 is not applicable (I did not continue from here onwards).

Why is data discussed in the Discussion (growth and reproductive behaviors) not presented in the Results?

Discussion should be expanded!

General comment

The manuscript ‘Biological response of eelgrass epifauna, Taylor’s Sea hare (Phyllaplysia taylori) and eelgrass isopod (Idotea resecata), to elevated ocean alkalinity’ by Jones et al. is an interesting study on the topic of ecological safety of one of the promising marine carbon dioxide removal (mCDR) approaches Ocean Alkalinity Enhancement (OAE). This study aimed to analyze the survivability of two ecologically significant eelgrass epifauna to OAE-induced seawater chemistry changes, specifically increased pH. They observed the epifauna isopoda species Idotea resecata to be more resilient to elevated pH than the studied sea hare species Phyllaplysia taylori.

Although this incubation study might contribute to the baseline information in understanding OAE impacts on marine ecosystems, the manuscript contains substantial flaws making it unsuitable for publication in its current form. The main reasons for rejection are as follows:

1. Missing total alkalinity measurements and complete focus on pH alone
2. Lack of proper statistical analysis
3. There is no point in plotting the three experimental rounds separately
4. Short and limited discussion

Specific comments

33        The author should concise the overall OA impact on marine life. They could then consider adding specific information on OA's impact on the eelgrass ecosystem or the influence on their epifauna.

39        The first and second paragraphs do not naturally fit together. The author starts by introducing Ocean acidification (OA) and its impact on marine life, then abruptly shifts to mCDR and OAE. To bridge the gap, the author should add a line stating that OAE could counteract OA, making the connection between these topics more obvious.

42        Alkalinity enhancement is more than that, this is one possible method. This should be clarified.

55        acclimate to what?

72        Scientific name of eelgrass should be provided.

80        Specify what is synergistic with what to avoid ambiguity.

84        The authors mentioned why these two epifauna species are important for the eelgrass ecosystem. They only connected the importance of the isopoda species with higher trophic levels as prey for Pacific salmon. Could the author mention similar for the sea hare species to support the importance of studying this particular species of gastropod? The authors might consider adding information if this species is prey for fish, crustaceans, or sea birds. Right now, it seems this sea hare species is only contributing to reducing the epiphyte load from the eelgrass blade.

89        In line 85, it is mentioned that Idotea sp. feeds on eelgrass blades, while here it is stated they feed on epiphytes. This inconsistency needs clarification. If line 85 means that they physically sit on the blades then the sentence is not well formulated.

96        Why was the study conducted in an environment with such a large natural pH variation? The organisms might have already acclimated to large pH variations.

110      I found it difficult to understand the experimental setup, specifically the details regarding the number of organisms and replications. Iwould recommend revising the methods section to help readers easily understand howthe experiments wereperformed.

118      Why do animals need nutrients?

135      The manuscript highlighted the pH increase as the seawater chemistry change of OAE. If this study is about biological responses to elevated ocean alkalinity, it should at least mention the initial total alkalinity (TA) value and the TA values after NaOH addition. Also, I didn't understand the reason for targeting a specific pH instead of targeting alkalinity for the treatment manipulation.

150      Fig 1 needs to be improved with additional information to simplify the complex setup.

156      rather stressful for the animals, or not?

161      I would not classify reproduction as unusual behavior.

166      Why there are ranges for all physio-chemical paraments? 

180      This is a pity. At least in the starting water total alkalinity should have been measured, and also at least a few times in the treatments. We have no idea about total alkalinity now, and whether there were risks of precipitation (no indication either whether this was observed).

186      mortality was noted every day, how was this dealt with?

200      The pCO₂ values of the controls are very high, why?

245      Presumably the CO₂ in the animal containers comes from respiration, but if the animals played such a large role in the pCO₂, how relevant was the treatment?

245      In Fig 2, the y-axis title is not included in all plots. If the authors want to show the y-axis title for the left plot only, then they should do the same for all figures. The y-axis title is labelled as CO₂, but it displays the pCO₂ data. Why are the symbols for pH 7.8 and 8.3 different in rounds 1 to 2 and 3?

322      Here and in other places, I do not understand why the analysis was not done using all rounds in a more sophisticated analysis, it is not so relevant that in round 1 this happened and in round 3 something else, unless you can explain. The general picture is more important, though. So, I would not present the rounds separately, but as a composite picture

345      In Fig 4 authors may want to consider adjusting the maximum limit of the y-axis to 10.

345      Table 2, again. It is not so interesting to see the rounds separately. The reader would like an overall picture. If you want to present put Figs 1-4 in the supplementary material.

375      I do not understand the word t-test in the caption of Fig 5. What was exactly tested, I hope not everything is against everything, using t-tests, would be incorrect as it would involve multiple testing with inflated type 2 error. The data needs to be analyzed better.

375      The quality of the figures needs improvement. The picture quality of Figures 4 and 5 is much better than that of the other figures. Additionally, it's preferable to keep the font size of the axis labels, titles, and legend text consistent across all plots.

379      Was there enough variation in any of these variables to warrant an inclusion in the model, probably not.

390      x-axis title is missing in Fig 6. LC50 describes a concentration. I do not see a concentration in this figure.

408      Do the observed reproduction and growth rates remain consistent across all pH levels?

412      Essentially your tests state that it is not, so there is no suggestion here it is the outcome of the study.

414      It is also possible that growth and reproduction were done by those animals that had energy intake.

414      The author conducted the study using starvation due to standards for acute toxicity tests with macroinvertebrates. The observed responses in the survivability of sea hares might have intensified due to starvation, leading to an overstretched result from an OAE perspective.

466      In the discussion, the author should include the pH ranges of the mentioned previous OAE studies.

466      Overall the discussion is very short and lacks focus on the specific findings of this study. There is a significant lack of discussion regarding the high mortality observation in sea hares. Specifically, if the results are so contrasting with the OA study by Hughes et al. (2017), the authors should at least mention what may have influenced the outcomes in their study. The discussion part needed information on how pH imbalance might have impacted physiological functions in sea hares, which could have contributed to the high mortality. If there is no information regarding this particular species, then discuss the effect of pH change on other sea hares or other gastropod species.