We thank the reviewer for pointing out that our study design is rather conservative. The dilution factors suggested by the model in our manuscript were indeed based on minimum estimations. We will also point this out in the discussion, by adding information that other models suggest higher dilution factors, resulting in conservative toxicity data in our study.

We are in the situation that we cannot go more in details about the model, due to confidentiality agreement. Thus, we cannot share information regarding the model. If the editor thinks that this is a central information needed for being able to publish the part regarding how we chose our exposure concentrations and times, then we will remove this model part from our manuscript. However, we think it is valuable information, especially if the results from the model are related to expected outcomes from other models in the discussion of the paper.

We are also aware of the MAMPEC model, as you can see in our paper from 2013 (Delacroix S., Vogelsang C., Tobiesen A. and Liltved H. 2013. Disinfection by-products and ecotoxicity of ballast water after oxidative treatment – results and experiences from seven years of full-scale testing of ballast water management systems. Marine Pollution Bulletin 73, 24-36). However, in our present study, we were rather interested in the alkaline mineral concentration which didn’t have any toxicity effect on algal species (i.e. NOEC as presented in Table 2) rather than LC50 or PEC/PNEC ratio to be <1 as per MAMPEC. This, again to be more conservative by suggesting doses that won’t have any impact on the aquatic environment rather than doses that might have “acceptable” effect on the aquatic environment.

According to our 18 years of experience in toxicity risk assessment for the aquatic environment of treated ballast water discharge, phytoplankton species are the most sensitive organisms, in addition to being a critical primary producer for the ocean ecosystem and the most abundant and widely spread organisms in the oceans at global scale, compared to crustacean species and fish species for which the toxicity data is indeed also required prior to commercialization permit at global scale. Therefore, we focused only on phytoplankton in this first indicative study. Still, we agree in that the next step would be a larger scale mesocosm study including several trophic levels. In fact, this is pointed out in the conclusions in the manuscript. In addition, we want to point out that regarding the effects of MgOH₂, the results conducted with a single marine species, were indeed confirmed with an assemblage of different local marine algal species in our study.

Regarding the experimental design concerns, we want to point out that the main purpose of our study was to investigate the temporary extreme water quality conditions change at the local contact point of the large amount of dumped alkaline mineral from a moving vessel. This because, virtually all scenarios of OAE require the initial addition of alkaline materials at high concentrations. To mimic this, our experimental was designed to study the short-term toxicity effect on the first phytoplankton exposed to the alkaline mineral discharge in addition to the following long-term exposure effect of a highly diluted alkaline mineral.

Regarding the comment related to ~3.4 mol/kg added alkalinity and pH 13-14, this might apply to the use of NaOH (this part of the study is now omitted from the manuscript, see further down) as we observed a maximum pH of 9.5 only for example when we used Mg(OH)2. And as mentioned in our article, Hartmann et al. (2022) demonstrated that Mg(OH)2 was 2.4 times more effective in alkalinity enhancement of seawater compared to Ca(OH)2. Therefore, the results of our study might indeed be conservative as our study was aiming in direct toxicity effect comparison when adding a same amount of alkaline mineral only. We agree that further toxicity studies must be conducted when considering equivalent alkalinity increase effect, implying a larger amount of Ca(OH)2 than used in our study and thus probably higher toxicity effect than showed in our study, as we mentioned in our discussion in this manuscript.

Regarding the first major comment:

While we agree on the application of such amount of NaOH are not recommended for OAE application due to toxicity effect, the double amount of Ca(OH)2 added in our study is recommended according to Hartmann et al., (2012) to achieve the same alkalinity enhancement effect than Mg(OH)2. Therefore, those results might still be a valuable demonstration for those who will need such knowledge with Mg(OH)2 or Ca(OH)2 applications. We agree with the reviewer on removing the results from the NaOH experiment in this manuscript as we couldn’t find any available data on the alkalinity increase performance of NaOH.

Regarding the second major comment:

We thank the reviewer for elucidating concerns regarding dispersion of NaOH by the simulated scenario and have decided to exclude the NaOH part of the study from the manuscript. However, we still think that using “sterile” autoclaved deep water from the Olsofjord, for study resilience of the algae after OAE dispersal, adds important information regarding the biological impact of the OAEs. Still, we agree that additional studies, including resilience in a scenario where both temporal and spatial effects on natural phytoplankton communities are investigated, are needed for investigating the biological impact effects of OAEs.

Regarding the other comments:

Title: Much of the carbon fixation of OAEs is related to how the OAEs influence the biology of the ecosystem. Because this paper focuses on potential negative effects of OAE dispersal, we think impact is the right wording. However, we agree in that “Biological impact of ocean alkalinity enhancement of magnesium hydroxide on marine microalgae using bioassays simulating ship-based dispersion” has some logic short comings, and suggest a new title:  “The biological potential impact of magnesium hydroxide ocean alkalinity enhancement on marine microalgae using bioassays simulating ship-based dispersion”.

Line 12: See above reply to concerns regarding the title.

Line 13: Yes, we chose to keep Mg(OH)2 in the title although we also mentioned two other commonly used alkaline minerals, as the results of the study was verified by additional experiments using Mg(OH)2 only:

• The WET tests on skeletonoma
• The test with assemblage of ambient algae from local seawater.  

Line 14: The hydroxyl radical is the terminology which is commonly used for disinfection of water as it is the oxidative and reactive group of each alkaline mineral used in this study. We agree in replacing “hydroxyl radical” by “hydroxyl ions”.

First part of introduction: OK, we will shorten the introduction.

Line 53: Ok, this citation will be updated to the IPCC 2022 WG 3 report as suggested: M. Pathak, R. Slade, P.R. Shukla, J. Skea, R. Pichs-Madruga, D. Ürge-Vorsatz,2022: Technical Summary. In: Climate Change 2022: Mitigation of Climate Change. Contribution of Working Group III to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [P.R. Shukla, J. Skea, R. Slade, A. Al Khourdajie, R. van Diemen, D. McCollum, M. Pathak, S. Some, P. Vyas, R. Fradera, M. Belkacemi, A. Hasija, G. Lisboa, S. Luz, J. Malley, (eds.)]. Cambridge University Press, Cambridge, UK and New York, NY, USA. doi: 10.1017/9781009157926.002.

Line 55: Yes, we mentioned “at least” when listing the 7 main approaches as reported by Siegel et al., 2021 and NASEM, 2021. GESAMP 2019 will be added as a citation and the sentence will be revised to indicate that there are many approaches, among which include OAE.

Line 58: Right, we meant it for the OAE application, we will reformulate this although technically, seaweed farming is accelerating natural biological processes (seaweed growth) and C sink to the deep sea (biological pump).

Line 60: While CO2 returns to initial conditions, pH is slightly higher and omega aragonite and calcite are elevated - this is the remediation part.

Line 66/67: We meant indeed “solubility” in this sentence. It is indeed the benefit of using an alkaline mineral with low solubility that it is possible to add it in large amount to still get the immediate pH and alkalinity increase effect without inducing precipitation (when added in the right form) and neither toxicity effect on the local marine algae.

Line 69: Different alkaline materials might dissolve or react differently, thus resulting in varying degrees of durability of an OAE approach.

Line 70ff: We used the standard GESAMP terminology for WET tests that includes different gradient of toxicity effect depending on the applied concentration of the studied substance; with for example NOEC as non-observed effect concentration. A sentence on the potential of positive impacts can be added.

Line 92: The reason that Skeletonoma is mentioned in the ISO standard reference method 10253 is because Skeletonoma is known as highly sensitive species to contaminants or substances/mixtures contained in seawater or environmental water samples (effluents, elutriates, etc.). More references can be added to support this.

• ISO 10253:2016. Water quality – Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum. ISO/TC 147/SC 5 Biological methods, ICS: 13.060.70, 19 p.

Line 105: Yes, it is correct and we can rephrase our description accordingly.

Line 109: According to the modelist team’s calculations, one of the most economically and realistic scenario was the addition of 500 kg/s of alkaline mineral, resulting in injection of approximately 100 g/L at the speed and ship’s length, etc… of this scenario, to be further explained in a forthcoming article. We choose a high concentration to simulate the worst case to get conservative results. If no or little toxicity effect is observed at this high concentration, we can then safely assume that lower concentrations won’t induce any significant toxicity effects either; which might be helpful results for later or further studies.

Line 174: Yes, we usually use spectrophotometric pH measurements according to Dickson et al. (2007) in our studies requiring detailed carbonate chemistry at our laboratory, but it wasn’t not necessary in this study where we focused only on the toxicity effect of alkaline mineral. In this study, we used pH-meters calibrated with NBS buffers prior to each test and will add the calibration method description in the material and methods section of this manuscript.

Line 209: To simulate properly the dispersion of alkaline mineral in open ocean, we chose to not cover the beakers and CO2 exchange was not considered other than that there was ample atmospheric CO2 in the culture room for CO2 uptake by the culture media.

Line 242: Because the part of the study with natural assemblage of local algal species was conducted only once, we divided the concentrations in two groups being constituted each by three low or three high concentrations.

Line 292: The analysis reports from the laboratory related to the WET tests can be added as supplements in appendix of the manuscript for better clarity and transparency.

Line 321: Yes, we agree if one thinks about the toxicity related to the pH increase alone, being higher for Ca(OH)2 than for Mg(OH)2. However, it could have been other intrinsic toxic effects on the algae that is impossible to verify without conducting the study.

Line 322: The part regarding NaOH will now be omitted from the study, see answer to Major comment No.2.

Line 362: This sentence will be revised to reflect that previous studies have mainly focused on the impact of OAE on organisms with calcium carbonate containing parts.

Thanks for your comments. Yes, we agree on improving the manuscript by shortening the introduction, correcting the typographical errors, adding the raw data related to our tests in an appendix of the article for total transparency, adding information about needs for further study to confirm Mg(OH)2 as alkaline mineral of choice for OAE application in the conclusion. 

We are aware that alkaline metals have different solubility, resulting in different temporal concentrations of hydroxide ions. However, as we state in the introduction; “there will be considerable local/regional effects of dispersion of alkaline minerals. Increased cation levels (Mg2+ and Ca2+), increased bicarbonate and carbonate ions, temporary local pH increase, or temporary local decrease of dissolved carbon dioxide might cause perturbation hotspots”. Thus, in the chosen scenario, also including tonnage capacity and operating costs of a ship, we do not think it is correct to compensate for differences in solubility in the initial concentration. This is because a lower initial concentration will affect the cost efficiency of the dispersion of the alkaline metals. The study design was based on some degree of realism related to dispersion of alkaline materials. Furthermore, as mentioned in the discussion, Hartmann et al. (2022) demonstrated that double the amount of Ca(OH)₂ compared to Mg(OH)₂ had to be dispersed onto the ocean’s surface to reach the same alkalinity increase effect. Considering this, the toxicity results of Ca(OH)₂ in our study is conservative as double amount of Ca(OH)₂ than Mg(OH)₂ should actually have been added.

The double staining method FDA/CMFDA is the only viability method that is currently recognized by the international regulators for approval of ballast water discharge from 70,000 commercial ships at global scale (USCG, 2012, USEPA, 2010, IMO, 2018), based on the validation work of US Navy Research Laboratory (Drake et al., 2010) to distinguish between living and dead cells after treatment. 

With regard to lack of discrimination between live and dead Tetraselmis suecica cells after heat treatment, this is an observation that our group has also observed. The heat treatment procedure of McIntyre and Cullen (2016) consists in heating the cells  to 50 deg C for 10 minutes and then staining the cells with FDA/CMFDA after lowering temperature to 18 deg C after an additional 20 minutes. The reason for the observed inconsistency is that the heating of cells to 50 deg C , while it does kill the cells, it does not immediately destroy cell walls and cell membranes. The ester enzymes are therefore still kept within the cell and activate the stains upon entry into the cells. T. suecica has, in addition to a cell membrane, also a cellulose cell wall. The membranes of heat killed cells will eventually (after a few hours) lose their integrity and then the ester enzymes will be lost to the surroundings and the cells will no longer appear stained. In our present study, we looked at the viability of T. suecica and other algae species over several days after treatment during the regrowth period and therefore algae observed with fluorescent stain are clearly alive and capable of growth as indicated by increasing cell densities with time in our tests.

With regard to lysis of stain because of high pH in test water, this is not relevant in our study as our procedure incorporated addition of HCl to sample prior to staining of cells (as indicated in Line 168 in our manuscript), bringing the pH back to around pH 8.

References:

• Drake, L.A., M.K. Steinberg, S.H. Robbins, S.C. Riley, B.N. Nelson, and E.J. Lemieux. Development of a method to determine the viability of organisms ≥ 10 µm and < 50 µm (nominally protists) in ships’ ballast water: a combination of two vital, fluorescent stains.  NRL Letter Report 6130/1011 , Washington, D.C.

• International Maritime Organization (IMO). 2018. RESOLUTION MEPC.300(72) CODE FOR APPROVAL OF BALLAST WATER MANAGEMENT SYSTEMS (BWMS CODE).

• United States Coast Guard (USGC). 2012. Ballast Water Management System Specifications and Approvals. Title 46—Shipping. Subpart 162.060.

• S. Environmental Agency Protection (EPA). 2010. Generic protocol for the verification of the ballast water treatment technology (ETV). EPA/600/R-10/146.