Review Reports - Effects of Low pH and Low Salinity Induced by Meltwater Inflow on the Behavior and Physical Condition of the Antarctic Limpet, <i>Nacella concinna</i>

Round 1

Reviewer 1 Report

This manuscript details an experiment that considered the effects of low pH and low salinity on the responses of an Antarctic limpet, specifically righting behaviour, mortality, condition factor, and shell surface dissolution. Given the potential for increasing meltwater to alter these conditions, understanding their effects is of interest.

General comments

A quick note: This manuscript included “track changes” in the document. For the purposes of the review I read the manuscript as though all of those changes had been accepted.

Overall the manuscript is presented clearly, with a good structure in which each section supports the others. However, there could be some improvement to the clarity of language used and further editing would be of benefit. (I have identified a number of these edits in the “specific comments” section below.)

In terms of the methods used, they are generally well-described, but some further detail could be provided. Specifically, how many experimental tanks were used for each of the treatments (lines 148-155)? Similarly, when talking about the statistical analysis (lines 228-240), it would be worth specifying if individual limpets were held in their own tanks and so able to use them as replicates. Or was some smaller number of tanks used?

In terms of the treatments, it is stated these are “close to the habitat environmental conditions of experimental limpets” (lines 140-141). Later in the manuscript detail is provided of what the salinity is, it would be useful to also have an indication of what the pH conditions are (lines 148-155).

It is stated that salinity and DO were measured when the sea water was replaced (lines 144-145) – was the salinity and DO of the water that was removed measured, or the water that was added? If it was the water that was added, this may not reflect the conditions the animals experienced. Are there any measurements showing the salinity and DO of the water added and removed was the same?

In reporting the water chemistry conditions, I found Figure 2 confusing. I assume the bottom four lines on the Figure are the pH conditions, while the top two are salinity (also that the top two represent the four treatments, but that the elevated and ambient treatments were so similar that the lines overlapped)? If so, it might be useful to use a different symbol to represent the salinity lines, and to mention that it is also reported in the figure legend. Alternatively, this figure could be split into two panels, one for the pH and another for the salinity.

In Figure 2 it also looks like there is one point per day – does that represent the mean of the day and night measurements? How many tanks were there to be measured?

It would be useful to have additional details on the titration method used to determine TA (lines 183-184). That is, was this done manually or using an automated titrator?

In the Results section, it would be useful to retain the statements regarding the statistical effects on pH and salinity currently marked for deletion (lines 245-249), although I think if DO is not important, this could be deleted (lines 251-252), as can the second reference to the table (lines 252-253).

I am not sure if I understand the approach used to measure the condition factor correctly, and extra details could be added to clarify this. Specifically, I am wondering if the “tissue wet weight” mentioned in line 208 is the “tissue mass” that is used in the calculation in line 206? If so, the language should be standardised to avoid confusion.

Also, I am assuming that tissue mass is a measurement of just the flesh. How was this measured? Is it possible to measure this on the same organism repeatedly, or is it necessary to separate the shell from the flesh? (Given the later result comparing the before and after exposure measurements, it appears possible to measure it repeatedly for the same individual.)

For Figure 4 it might be clearer to plot and do analysis on the response as change in modified condition factor (i.e. the difference between the before and after exposure measurements for each individual). This could complement the figure currently included.

It would be useful for all figures to follow similar formatting and language as used through the manuscript – e.g. that for figure 6 could be changed to have L, LS, LP, LPLS along the x-axis as in Figure 4, rather than the indication of pH and salinity levels.

Overall, I think the Discussion is well-written and clearly structured, although I am not sure about the significance of the paragraph that runs from line 322 to line 330. I suggest either adding an opening statement regarding why the information that follows is important, or removing it from the manuscript.

Similarly, I find the explanation for the lack of difference between different salinity treatments confusing and difficult to follow (lines 351-358); this should either be rephrased or removed. I also suggest that the paragraph break between this paragraph and the next could be removed.

Throughout the manuscript, the referencing is typically appropriate, although I do think there are some statements that could use support or further detail that is currently not provided, specifically:

Lines 35-36 regarding the ability of calcifying organisms and their capacity to maintain skeleton or shells, suggest removing or adding reference

Lines 45-46 regarding studies that have determined the effect of meltwater inflow on Southern Ocean acidification, suggest adding references for those that have

Lines 81-83 regarding the study that has considered the effect on a pteropod, suggest adding a brief summary of what was found

Lines 87-88 regarding warming and glacial shrinking are fast occurring, suggest adding a reference to support this statement of change

Specific comments

Line 21: Replace “impairing shell formation system” with “enhancing shell dissolution”

Line 32: Replace “year 2030, no later than 2038” with “year 2030, and no later than 2038,”

Lines 37-39: Replace “concentration of Total Alkalinity (TA) and Dissolved Inorganic Carbon (DIC), which contribute to carbonate undersaturation compared to sea water” with “lower Total Alkalinity (TA) and concentration of Dissolved Inorganic Carbon (DIC) compared to seawater, which contribute to carbonate undersaturation”

Line 45: Replace “little” with “few”

Line 48: Replace “ecosystem” with “ecosystems”

Line 50: Delete “in the WAP”

Line 55: Replace “of summer months being accompanied by massive” with “of the summer months and is being accompanied by a massive”

Line 56: Replace “Fresh water” with “Freshwater”

Line 59: Replace “had fairly” with “has relatively”

Line 63: Replace “fluctuated” with “fluctuates”

Line 65: Replace “fields although” with “fields, although”

Line 68: Replace “Cove and (b)” with “Cove and (c)”

Line 78: Replace “faunas” with “fauna”

Line 80: Replace “Even the pH and salinity are related, and those” with “The pH and salinity are related and these”

Line 81: Replace “by” with “due to”

Lines 82-83: Replace “effect of mortality and swimming behaviour on” with “effect on mortality and swimming behaviour of”

Line 88: Replace “proceeding in the resent decades. It occurs” with “occurring. This species is found” (if this is what is meant, I was not sure what “It occurs” refers to)

Line 90: Replace “increased as the depth decreased” with “increases as the depth decreases”

Line 91: Replace “species observed by naked eyes” with “species to be observed by the naked eye”

Line 93: Replace “mediator connecting” with “mediating role connecting”

Line 109: Replace “level” with “levels”, “can” with “should”

Line 113: Replace “will” with “would”

Line 115: Replace “items” with “response variables”

Line 117: Delete “for investigating the impacts of low pH and low salinity in short-term exposure”

Line 120: Replace “getting worse by” with “worsened under”

Line 121: Replace “will” with “would”

Line 146: Replace “was” with “were”

Line 148: Replace “mixing” with “crossing”, and “made” with “considered”

Line 157: Replace “an experiment” with “this experiment”

Line 163: Replace “Seawater” with “Before being used in the experiment, seawater”

Line 166: Replace “was melted” with “was then melted”

Line 185: Replace “the physical” with “the behavioral response and physical”

Line 190: Replace “behavior” with “behaviors”

Line 195: Replace “experiment on” with “measurement of”

Line 196: Replace “their” with “the”

Line 200: Replace “in case they” with “in the cases where they”

Line 268 and Figure 3: Replace “Probability” with “Proportion”

Line 332: Replace “by only low” with “by low”, replace “factors” with “factor”

Line 339: Replace “limpets expose” with “limpets are exposed”

Line 340: Replace “Therefore,” with “In this experiment, however,”

Line 343: Replace “contents” with “content”, and “Even the” with “Even though the”

Lines 349, 351, 352: Remove “variable”

Line 362: Replace “the other” with “another”

Lines 366-367: Replace “to the treatment” with “to the pH treatment”

Author Response

General comments

A quick note: This manuscript included “track changes” in the document. For the purposes of the review I read the manuscript as though all of those changes had been accepted.

Response: Each individual was kept in the separate 250 ml transparent PET container which was a reused cup. Therefore there were 12 replicates for each treatments. Two plastic tanks (80 × 45 × 20 cm, W × L × H) were used to hold the containers to maintain the temperature. Temperatures were maintained using ice cubes. (lines 149-153)

Response: “close to the habitat environmental conditions of experimental limpets” means pH 8.00, and salinity 34.0 psu. (lines 157-160)

Response: Both the water that was removed and the water that was added were measured.

Response: The figure 2 was replaced as suggested.

In Figure 2 it also looks like there is one point per day – does that represent the mean of the day and night measurements? How many tanks were there to be measured?

Response: The pH and salinity of replicates were measured when the experimental sea water was replaced. And the point represent the mean of the day when the experimental sea water was replaced.

It would be useful to have additional details on the titration method used to determine TA (lines 183-184). That is, was this done manually or using an automated titrator?

Response: TA was determined with an automated titrator. (Lines 203-204)

Response: The statement is retained on lines 280-288.

Response: Yes, it is same in this paper. We united the words as ‘Tissue mass’ which is used for formula (Fulton' s condition factor) (line 233)

Response: The tissue wet weight was calculated as (whole body mass)-(shell mass). Shell mass was measured by separating the shell from the flesh after finishing the experiment (Lines 235-237).

Response: Thank you for the suggestion. However, we conclude that showing the before and after exposure data separately show the results well.

Response: We revised the figure 6 as commented.

Response: This paragraph emphasizes why we determine the effect of ocean acidification and freshening on N. concinna which lived in Marian Cove where the spatial and temporal variability is huge.

Response: This explanation is meaningful to explain the result of shell dissolution. This kind of result was also shown on the other study (Cho et al., unpublished data)

Lines 35-36 regarding the ability of calcifying organisms and their capacity to maintain skeleton or shells, suggest removing or adding reference

Response: The reference was added on line 44

Lines 45-46 regarding studies that have determined the effect of meltwater inflow on Southern Ocean acidification, suggest adding references for those that have

Response: There was no reference for the effect of meltwater on Southern Ocean acidification, But there was reference for the effect of meltwater on Arctic acidification. And this reference was added on line 57.

Lines 81-83 regarding the study that has considered the effect on a pteropod, suggest adding a brief summary of what was found

Response: We revised it in lines 92-94.

Lines 87-88 regarding warming and glacial shrinking are fast occurring, suggest adding a reference to support this statement of change

Response: We revised it in line 99.

Specific comments

Line 21: Replace “impairing shell formation system” with “enhancing shell dissolution”

Response: We revised it in line 25.

Line 32: Replace “year 2030, no later than 2038” with “year 2030, and no later than 2038,”

Response: We revised it in line 38.

Response: We revised it in lines 44-46.

Line 45: Replace “little” with “few”

Response: We revised it in line 54.

Line 48: Replace “ecosystem” with “ecosystems”

Response: We revised it in line 57.

Line 50: Delete “in the WAP”

Response: We revised it in line 59.

Line 55: Replace “of summer months being accompanied by massive” with “of the summer months and is being accompanied by a massive”

Response: We revised it in lines 64-65.

Line 56: Replace “Fresh water” with “Freshwater”

Response: We revised it in line 66.

Line 59: Replace “had fairly” with “has relatively”

Response: We revised it in line 69.

Line 63: Replace “fluctuated” with “fluctuates”

Response: We revised it in line 74.

Line 65: Replace “fields although” with “fields, although”

Response: We revised it in line 77.

Line 68: Replace “Cove and (b)” with “Cove and (c)”

Response: We revised it in lines 604 and 629.

Line 78: Replace “faunas” with “fauna”

Response: We revised it in lines 88.

Line 80: Replace “Even the pH and salinity are related, and those” with “The pH and salinity are related and these”

Response: We revised it in lines 90-91.

Line 81: Replace “by” with “due to”

Response: We revised it in line 91.

Lines 82-83: Replace “effect of mortality and swimming behaviour on” with “effect on mortality and swimming behaviour of”

Response: We removed it.

Line 88: Replace “proceeding in the resent decades. It occurs” with “occurring. This species is found” (if this is what is meant, I was not sure what “It occurs” refers to)

Response: We revised it in lines 98-99.

Line 90: Replace “increased as the depth decreased” with “increases as the depth decreases”

Response: We revised it in lines 100-101.

Line 91: Replace “species observed by naked eyes” with “species to be observed by the naked eye”

Response: We revised it in line 102.

Line 93: Replace “mediator connecting” with “mediating role connecting”

Response: We revised it in line 104.

Line 109: Replace “level” with “levels”, “can” with “should”

Response: We revised it in lines 123-124.

Line 113: Replace “will” with “would”

Response: We revised it in line 128.

Line 115: Replace “items” with “response variables”

Response: We revised it in line 130.

Line 117: Delete “for investigating the impacts of low pH and low salinity in short-term exposure”

Response: We revised it in line 132.

Line 120: Replace “getting worse by” with “worsened under”

Response: We revised it in line 134.

Line 121: Replace “will” with “would”

Response: We revised it in line 136.

Line 146: Replace “was” with “were”

Response: We revised it in line 167.

Line 148: Replace “mixing” with “crossing”, and “made” with “considered”

Response: We revised it in line 170.

Line 157: Replace “an experiment” with “this experiment”

Response: We revised it in lines 605 and 632.

Line 163: Replace “Seawater” with “Before being used in the experiment, seawater”

Response: We revised it in line 179.

Line 166: Replace “was melted” with “was then melted”

Response: We revised it in line 183.

Line 185: Replace “the physical” with “the behavioral response and physical”

Response: We revised it in line 207.

Line 190: Replace “behavior” with “behaviors”

Response: We revised it in line 213.

Line 195: Replace “experiment on” with “measurement of”

Response: We revised it in line 219.

Line 196: Replace “their” with “the”

Response: We revised it in line 220.

Line 200: Replace “in case they” with “in the cases where they”

Response: We revised it in line 225.

Line 268 and Figure 3: Replace “Probability” with “Proportion”

Response: We revised it in lines 608, 638 and fig3.

Line 332: Replace “by only low” with “by low”, replace “factors” with “factor”

Response: We revised it in line 355.

Line 339: Replace “limpets expose” with “limpets are exposed”

Response: We revised it in line 363.

Line 340: Replace “Therefore,” with “In this experiment, however,”

Response: We revised it in line 365.

Line 343: Replace “contents” with “content”, and “Even the” with “Even though the”

Response: We revised it in line 369.

Lines 349, 351, 352: Remove “variable”

Response: We revised it in lines 375-378.

Line 362: Replace “the other” with “another”

Response: We revised it in line 389.

Lines 366-367: Replace “to the treatment” with “to the pH treatment”

Response: We revised it in line 394.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

First of all, I would like to appreciate the opportunity to review your work. Within the submitted work, the authors addressed the effects of low pH and/or low salinity on the righting behavior, mortality, condition factor, and shell
dissolution of the Antarctic limpet Nacella concinna. Specific comments are annotated on the revised PDF file. The submitted work adds information to existing state-of-the-art but considerable shortcomings are present. All the sections should be considerably revised and improved, with special emphasis but not restricted to "Discussion". In this last section, I did not provide comments once certain details in previous ones (i.e. M&Ms, Results) should be included/explained in order for data to be properly addressed. Minor details include: i) further references/info should be inserted and provided, in order to substantiate the general outcome of the submitted work; ii) adding a figure to the manuscript, which could graphically show experimental design, would be important for the overall quality and output of the manuscript; iii) the submitted paper should be further read and revised by native English speakers, in order to improve the overall output.

Sincerely yours,

The reviewer

Comments for author File: Comments.pdf

Author Response

Thank you very much for the comments.

Our responses to the comments are attached.

Best wishes,

Sin et al.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have done a nice job in addressing most of my comments on the last version of the manuscript, but there are two that I feel could use more detail.

- As adding ice does not seem to me to be an approach that would keep temperature particularly stable, I think the manuscript would benefit from some information regarding how much, and how rapidly, this varied over time during the experimental period

- It is noted that salinity and DO were measured before and after water changes – it would be useful to indicate in the manuscript if they were different at these times. Similarly, were pH and temperature different before and after water changes?

Author Response

The authors have done a nice job in addressing most of my comments on the last version of the manuscript, but there are two that I feel could use more detail.

Response:

The 4 treatments were shared the water bath, and the experimental animals (replicates) were randomly located in the water bath (see below figure). Therefore the water temperature of each treatment was not significant different between groups (line 276-278, table1).

This study determined the effect of low pH and salinity, not the temperature effect. Therefore the water temperature was just checked whether it was different between groups or not.

And the melted water from the added ice acted as a buffer preventing the rapid temperature change.

Response:

The water quality of experimental sea water was shown on supplemental table S2. The pH, salinity, and DO of seawater were not much different between just before and after replacement (Supplemental Table S2). Please see Lines 184-185.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

I have checked your review of the comments that were performed.

In this sense and according to the answers/corrections that were given/performed, I do not have any more comments to perform.

Sincerely yours,

The reviewer

Author Response

Dear authors,

I have checked your review of the comments that were performed.

In this sense and according to the answers/corrections that were given/performed, I do not have any more comments to perform.

Sincerely yours,

The reviewer

Response:

Thank you for the previous helpful comments that made this manuscript better.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript presents the result of an experiment testing the impact of salinity and pH on an Antarctic limpet in the context of meltwater inflow. The experimental design is good (but see below concerns regarding the carbonate chemistry) and the data interesting. However, some changes are needed before publication including some of the statistics, the general background and conceptual framework, and the interpretation of the data.

GENERAL COMMENTS

Better description of the natural context and justification of the experimental design

The article is developed around the context of meltwater inflows consequences on the seawater chemistry. However, there is not enough information to understand the dynamics of the process. For example:

What variability is observed at the sampling site for pH and salinity at the relevant temporal scale (“weather”)? What are the extremes observed today (including the influence of the tides and the meltwater)
What is the temporal scale of this variability? (including tides and meltwaters, revise line 136-138).

This information is critical both for the justification of the design and the interpretation of the data.

How is the experimental design (e.g. 35days exposure to constant conditions) relevant in the context of the present natural variability?
Local adaptation to present environmental conditions (range of pH and salinity and dynamics and variability in itself) is critical to build up a hypothesis. An organism living in a tidal zone and experiencing regulation fluctuation is then expected to be resilient to change. Different processes are expect to modulate the biological response depending on how the tested scenarios relate to present natural variability (plasticity within the present range vs. selection outside; e.g. Thor & Dupont 2015 for an example on pH). How does the tested scenarios relate to the present environmental variability? (revise line 148-149).
Link to this last point, it is likely that the tested population (tidal, high variability) is adapted to change and must be quite resilient to low pH and salinity (e.g. see Vargas et al. 2017). Revise lines 79-80, section starting line 97.

Some information is provided in Supplementary Material but it is rather unclear (and methods are not provided). Overall, it does not have the needed resolution to have a proper idea of the situation at the sampling site.

Link between carbonate chemistry changes and calcification/dissolution

The conceptual framework linking changes in carbonate chemistry and calcification/dissolution is outdated. There is no physiological mechanism that is directly related to saturation states and the only direct effect of decrease saturation states and net calcification (as suggested in this paper) are in species with exposed calcium carbonate structures (e.g. corals or pteropods) or with very poor acid-base regulation capabilities (e.g. first 2 days of development for bivalves). Most organisms have acid-base machineries to control the carbonate chemistry at the calcification site as well as mechanisms to protect the shells from dissolution. The effect of changes in carbonate chemistry is more an effect of energy allocation. This should be revised. For example, see Roleda et al. 2012 or Cyronak et al. 2016.

This includes revisions of statements at lines 21-22, 34-37, 119-120; line 332-333

Carbonate chemistry measurements

It is not possible with the available information to evaluate if the experiment if following best practices for the measurement and calculation of the carbonate chemistry. There is mention of 3 “determination” (not sure what that means as it is not possible to measure saturation state) per replicate (line 169) for TA, DIC and omega and once a day for temperature, salinity and pH.

What method was used for temperature?

For pH, what buffers were used? What is the scale used (total scale, nbs?)
From the Figure 2, salinity is very constant. That sounds unlikely. What is the quality of the measurements?
Was DIC measured? If yes, how?
What was used for the calculation of the other parameters of the carbonate system? (temperature, pH, salinity and TA)? What were the dissociation constant used?
In the Table 1, precise what was measured and what was calculated. The N does not add up to the description in the methods.

Statistics and analyses

- Line 63, Figure S1: it is claimed that there is a “moderate positive correlation” between pH and salinity. However, this is not supported by the data. The regression is strongly biases by a few data (attraction effect). If extremes are removed, there is no trend. This should be removed.

- Water chemistry (start line 226). The statistics are not appropriates. As there are multiple measurement per replicates, an ANOVA 3 model should be performed for all the measured and calculated parameters to test the effect of salinity, pH and replication. Depending on the tested parameters, some significant differences should be observed (and no interactions).

- It is unclear why two different analyses are performed for the righting response (before and after). All data should be included into a single model (with a time effect – 4 levels).

- The mortality data does not have enough resolution to make any statistics. No need for statistics and a quantitative description is enough.

- For the CF, add “time” as a factor in the ANOVA model.

- Figure 6 and 7 are presenting the same set of data. As 4 points are weak to perform a serious regression, I would keep the Figure 7 and an ANOVA design for the analysis. More omega points would be needed to prove a relationship between the two parameters. I would tone down that part in the manuscript (see previous comment).

The description of the statistics should be corrected in the Methods accordingly.

The discussion should be revised based on previous comments. Based on the available information, the tested population must be adapted to variability and the lack of strong negative response is then expected..

SPECIFIC COMMENTS

Line 39. “Natural” should be changed. Meltdown water influence is also “natural”.

Figure 1. Provide more information on the legend (example, reference to the different panels)

Figure 2. Provide more information on the legend: What is the color code for the different experimental regimes? What symbol correspond to what parameter (salinity or pH)?

Line 159. Seawater conditions in such experimental system is influenced by the presence of the organisms (respiration, food, calcification, dissolution). Was it constant between the water changes? Provide information.

Line 190. How many animals were used for the CF (some died over the course of the experiment).

Line 197. For the shell dissolution, only living animals should have been considered. Please precise.

Line 211. How can 48 limpets be used for all analysis while some died during the experiment. Be more specific on the number of individuals used for each analysis and each treatment.

Figure 3. How was the “probability of righting” calculated? This is not clear how it relates with the text in the Methods. Add the “before” data and explain the abbriviations (figures should be self-explanatory).

Figure 5. There is no information in the legend and it is impossible to know what we are looking at.

Reviewer 2 Report

All comments are addressed in the text file.

Comments for author File: Comments.pdf