the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 17 Aug 2023
Biological impact of ocean alkalinity enhancement of magnesium hydroxide on marine microalgae using bioassays simulating ship-based dispersion
Abstract. Increasing the marine CO2 absorption capacity by adding alkaline minerals into the world’s oceans is a promising marine carbon dioxide removal (mCDR) approach to increase the ocean’s CO2 storage potential and mitigate ocean acidification. Still, the biological impacts of dispersion of alkaline minerals needs to be evaluated prior to its field deployment. In this study, the toxicity effect on marine microalgae of two commonly used alkaline minerals, calcium hydroxide (Ca(OH)2) and sodium hydroxide (NaOH), was compared with magnesium hydroxide (Mg(OH)2), by applying the same concentration of hydroxyl radicals (OH-) for each component. Cultures of marine green microalgae Tetraselmis suecica were exposed to NaOH, Ca(OH)2 or Mg(OH)2 in concentrations mimicking dispersion scenarios from a ship which included short-term exposure with high alkaline mineral concentration called “dispersion phase” followed by a dilution and “regrowth” phase over six days. There was no detectable effect of Mg(OH)2 treatment on algae growth either after the dispersion phase or during the regrowth phase, compared to control treatments. The Ca(OH)2 treatment resulted in very few living algal cells after the dispersion phase, but a similar growth rate was observed during the regrowth phase as was for the Mg(OH)2 and control treatments. The NaOH treatment resulted in no surviving algae after the dispersion phase and during the regrowth phase. Standardized whole effluent toxicity (WET) tests were carried out with a range of Mg(OH)2 concentrations using a sensitive marine diatom, Skeletonema costatum, which confirmed the relative low toxicity effect of Mg(OH)2. Similar biological effects were observed on natural microalgae assemblages from a local seawater source when applying the same Mg(OH)2 concentration range and exposure time used in the WET tests. The results suggest that Mg(OH)2 is relatively safe compared to Ca(OH)2 and NaOH with respect to marine microalgae.
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CC1: 'Comment on bg-2023-138', Chris Vivian, 22 Aug 2023
Stephanie,
The dilutions you simulated in section 2.1 lines 104-106 are a significant underestimate of what can be achieved in the wake of a vessel. Consequently, your assessment of the safety of discharging alkaline materials from vessels is overly conservative.
I worked at the Cefas Burnham Laboratory for many years and was responsible for advising the Ministry of Agriculture, Fisheries and Food on the licensing of liquid industrial wastes dumped at sea from 1986-1992, including the setting of discharge rates based on toxicity tests. The UK dumped industrial wastes at sea up until 1992 including high strength alkaline wastes. In order to avoid undesirable effects on the marine environment, the wastes were discharged through twin pipes at the stern of converted tankers and calculated dilutions of up to 10,000 times within 5 minutes could be achieved. The dilutions and discharge rates were determined based on toxicity tests.
There are models for the dilution of wastes in the wake of vessels. Paper MEPC III/7 (copy attached) from the 3rd session of IMO MEPC that provided a method for calculation of dilution capacity in a ship’s wake. The methodology was derived from field experiments carried out by the United States, Netherlands and Norway, as well as an experiment by the Netherlands using a model in the Netherlands Ship Model Basin. This methodology was used by European countries dumping liquid industrial wastes under the Oslo Convention from the 1970’s through to the early 1990’s. There are also models for the discharges from exhaust gas cleaning systems on ships (using the MAMPEC-BW model originally developed for ballast water discharges - https://www.deltares.nl/en/software/mampec/). Also, see the attached paper by Caserini et al. (2021).
In the UK’s case, the MEPC paper methodology was used to ensure that dilution of the waste was such that the 96-hour LC50 concentration derived from toxicity testing of the wastes was achieved within 5 minutes of discharge. In practice, a number of field experiments carried out by the UK found that the methodology in MEPC III/7 underestimated the dilution achieved in practice in these and other experiments (see attached papers). Speed of vessels, waterline length and discharge rate were the most important factors.
Chris Vivian.
Chris.vivian2@btinternet.com
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CC2: 'Reply on CC1', Michael Tyka, 30 Aug 2023
I'll add that it would be quite interesting for the reader to directly see the results of the simulations carried out in section 2.1 (in particular the dilution-vs-time curves) directly compared with the empirical formulas used by IMCO, Chou et al and others. See for example - section 3.3 in https://bg.copernicus.org/articles/20/27/2023/ and Box 2 in https://agupubs.onlinelibrary.wiley.com/doi/full/10.1002/2016RG000533 and also the above-mentioned Caserini et al. (2021). Can we gain any insight from your simulations about the accuracy of these different formulas (which already deviate from each other quite significantly) ?
Citation: https://doi.org/10.5194/bg-2023-138-CC2 -
AC3: 'Reply on CC2', stephanie delacroix, 19 Oct 2023
We thank the reviewer to make us aware of other studies suggesting higher dilution factors. Those references will be added to our paper in the discussion section. We are sorry that we cannot disclose at this stage the details of the BROM model being specifically developed in this project for Mg(OH)2 application due to confidentiality agreement (see above). However, those data will hopefully be described in detail in another publication later, as those model results are not the main subject of this paper focusing exclusively on the toxicity effects on marine algae.
Citation: https://doi.org/10.5194/bg-2023-138-AC3
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AC3: 'Reply on CC2', stephanie delacroix, 19 Oct 2023
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AC2: 'Reply on CC1', stephanie delacroix, 19 Oct 2023
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.
Citation: https://doi.org/10.5194/bg-2023-138-AC2
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CC2: 'Reply on CC1', Michael Tyka, 30 Aug 2023
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RC1: 'Comment on bg-2023-138', Anonymous Referee #1, 07 Sep 2023
Delacroix et al. test the effect of three potential OAE source materials (Mg(OH)2, Ca(OH)2, NaOH) on phytoplankton. They expose Tetraselmis to extremely alkaline conditions (~3.4 mol/kg added alkalinity, pH 13-14) for one hour and test their survival and regrowth. The extremity of the treatment (and other critical issues in the experimental design) impede drawing meaningful conclusions for OAE. The ecotox tests presented alongside the main experiments are potentially informative for OAE as these may reflect protocols used in permitting procedures in environmental agencies. However, those limited tests with just one species seem too slim to justify a stand-alone publication. Due to these shortcomings (further detailed below) I can unfortunately not recommend publication.
Major comments:
- The authors estimated an application rate of 500 kg of alkaline material per second in a ship wake. It is not entirely clear how this number was derived but this is perhaps not the most critical problem. More critically is that this leads to an experimental design where e.g., 141 g of NaOH pellets are added to 1L of Tetraselmis culture for one hour. This seems deadly by design. I am not sure how reliable carbonate chemistry software is under such conditions (i.e., alkalinity = 3.4 mol/kg, DIC = 0.0021 mol/kg) but this translates into a pH of >>13, possibly 14. As such, it is not surprising that even a very robust species like Tetraselmis dies, as the water was essentially sterilized. The fact that Tetraselmis survives the Mg(OH)2 treatment (despite the extremely large amounts added) is most likely due to slower dissolution so that the extreme pH effect cannot fully unfold within an hour of experiment. It could be argued that this is just what happened under such a OAE application and the data is robust (I totally trust the data that all cells died under pH 13-14). However, would anyone consider such application? It would be like doing an ocean acidification experiment under pH 4. Yes, OA reduces pH but pH 4 is still not ocean acidification. Indeed, previous OAE studies have already carefully assessed dosing rates in order to not increase coastal pH beyond a ∆pH of 0.1 on average and pH 11 on the order of seconds assuming a ship wake scenario (He and Tyka, 2023). As such, the way the main experiments are designed will lead to misleading conclusions as what they tested has very little to do with OAE.
- The second major critique is the dilution scenario that follows the 1 hour exposure to extremely alkaline conditions. In the oceans, a small volume directly affected by alkaline substances (where some cells may indeed die) would mix with the same water mass and pH would decline to much lower values (e.g. pH 9) within seconds to minutes (see Fig. 7 in He and Tyka, 2023). The not immediately perturbed water body, with which the highly perturbed water mass is quickly mixed, still contains the same unperturbed phytoplankton community. However, in the experiments presented here, deepwater is used because it is essentially free of phytoplankton. As such, the experiments exclude the natural seed population that has not been affected by extremely high pH for an hour. The conclusions drawn from this experiment can therefore not be used to assess an OAE perturbation in a ship wake.
Other comments:
Title: There seems to be a logic issue. Is the impact from OAE or MgOH2? Also, I would refrain from the word “impact” as it implies bias. Influence?
Line 12: Toxicity effect implies by default detrimental outcomes and thus suggests bias. Influence of XXX on YYY?
Line 13: Title mentions MgOH2 but the study seems to address other alkalinity sources.
Line 14: Hydroxyl radical: Isn’t that hydroxide ions? The former seems to be used in atmospheric sciences.
First part of introduction: The lengthy introduction on OA seems off-topic and is also not tailored towards the subject of the study, i.e., phytoplankton. The three main negative consequences are also not backed by references and the text seems biased towards considering only negative effects of OA. Pretty much all text until line 51 could be condensed to one sentence or fully deleted, since OA is of little relevance for the research here.
Line 53: I would refer to IPCC 2022, I think the relevant working group is either 2 or 3.
Line 55: There are many more, refer to GESAMP 2019.
Line 58: Not all aim to accelerate: Some aim to establish a new C-sink in the earth system that currently does not exist, e.g. seaweed farming.
Line 60: OAE, when done properly and the generated CO2 deficit is matched with atmospheric CO2, has very little influence on ocean pH. This narrative of OA remediation does therefore make little sense.
Line 66/67: Solubility or dissolution? Have not worked with MgOH2 myself but all data I’ve seen suggests rather fast dissolution.
Line 69: Durability is unclear. In principle, all alkalinity should have the same durability.
Line 70ff: As mentioned above, I would refrain from “toxicity” in the context as there are likely pros and cons or different organisms. Toxicity implies everything will suffer or die, which is not what current research suggests.
Line 92: is there a reference that confirms Skeletonema to be more sensitive?
Line 105: Explanation of dilution is unclear. Does that mean a dilutin of 1/1000 after two minutes, 1/7000 after 5 hours, 1/154000 after 10 hours? Please clarify, ideally with an illustrative example.
Line 109: 100g/L appears unrealistically high, which appears to be a critical problem in the study design (see major comment).
Line 174: How was pH calibrated? It seems that best practices for seawater carbonate chemistry measurements (Dickson et al., 2007) were not applied. This may have been necessary but a justification would be required here.
Line 209: Were carbonate chemistry changes through CO2 exchange with the atmosphere considered. These may have altered conditions, especially when cells are grown in beakers.
Line 242: The statistical approach seems to fully counter the experimental design. Why was a gradient established if a range of concentrations were then put together?
Line 292: The WET test results are somewhat unclear.
Line 321: It is quite obvious that pH was similar at the onset of the regrowth phase because the initial water could diluted. The gradient between treatments is still totally as one would expect it, i.e., highest in NaOH and CaOH2.
Line 322: The regrowth conditions were extremely different because in some treatment (those that were inadvertently sterilized with NaOH) all cells were dead, as one would expect from a treatment under pH 13-14. (See major comment 2).
Line 362: This statement lacks any evidence, including in the cited references.
Citation: https://doi.org/10.5194/bg-2023-138-RC1 -
AC5: 'Reply on RC1', stephanie delacroix, 19 Oct 2023
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.
Citation: https://doi.org/10.5194/bg-2023-138-AC5
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RC2: 'Comment on bg-2023-138', Anonymous Referee #2, 25 Sep 2023
In this paper the effect of ocean alkalinity enhancement is tested using different substances simulating discharges from a ship. In the first case Teraselmis sp is exposed to high concentration over a short time, followed by dilution and growth. In addition, further tests with Mg(OH)2 and the diatom Skeletonema costatum and a natural phytoplankton community were done.
All in all, the tests and results are relatively straight forward demonstrating that Mg(OH)2 is compound that affects algal growth the least, but it too will have effects on the survival at high concentrations. As such, it is a nice contribution to the rapidly evolving field of ocean alkalinity enhancement, although not a major breakthrough paper. I noticed the study was funded by a commercial entity and the results were the most favorable for their product, making me wonder if the results had been published if that had not been the case. That is an ethical question to all. I think the transparency about this is good, but would further recommend all of the data to be made fully open in a data repository rather than the 'available upon request' approach that is taken in the present version.
The conclusion is basically a summary, perhaps apart from mentioning further studies in e.g. mesocosms are needed. Given the results, would you be able to make a comparison with alternatives and perhaps draw up the main bottlenecks (or further study needs) for Mg(OH)2 being the OAE mineral of choice?
Minor comments
The first paragraph in the intro is not that relevant and could be cut or shortened.
There are a few typographical errors, have a close read through
Citation: https://doi.org/10.5194/bg-2023-138-RC2 -
AC1: 'Reply on RC2', stephanie delacroix, 19 Oct 2023
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.
Citation: https://doi.org/10.5194/bg-2023-138-AC1
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AC1: 'Reply on RC2', stephanie delacroix, 19 Oct 2023
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CC3: 'Comment on bg-2023-138', Mackenzie Burke, 29 Sep 2023
Delacroix et al. present the outcomes of a set of experiments from which they argue that Mg(OH)2 is the ideal compound choice for OAE over NaOH and Ca(OH)2. We question these conclusions on two grounds. First, the treatments are not equivalent because of differences in solubility in the alkaline materials used; second, the staining method used is likely to be an unreliable way to assess the impact of alkalization.
The hydroxides used in the study were added at equivalent molar concentrations. However, there are significant differences in solubility. We calculate the concentrations of added hydroxide ions in the initial treatment phase as 0.311, 25.1, and 353 mmol L-1 for Mg(OH)2, Ca(OH)2, and NaOH, respectively, using Ksp values collated by UofMass (https://owl.oit.umass.edu/departments/Chemistry/appendix/ksp.html) for the first two and assuming NaOH was fully dissociated. This would give a range of exposures that vary by 3 orders of magnitude as opposed to being equivalent.
The ability of FDA+CMFDA as a means for discriminating between living and dead phytoplankton was tested by MacIntyre and Cullen (2016). It was effective in less than half of the 24 species tested when living cells were compared to killed ones, as recommended in the NSF protocols cited by the authors. An important difference between the approach used by MacIntyre and Cullen and the NSF protocols is the use of flow cytometry rather than microscopy. Cytometry has the advantage of providing a very high number of objective and quantitative estimates of fluorescence intensity. None of the test strains used in this study (Tetraselmis suecica, Phaeodactylum tricornutum, Skeletonema marinoi [costatum]) were tested by MacIntyre and Cullen. However, using the same protocols (flow cytometric analysis of cells that were in balanced growth, i.e., all alive, vs heat-killed), we tested Tetraselmis suecica (CCMP906), obtained from the NCMA (https://ncma.bigelow.org/). Both the living and dead cells had high fluorescein fluorescence compared to cells that had not been exposed to the dyes. However, there was high overlap between the frequency distributions of the staining intensity in live and dead cells (Figure A, B). If the live cells were classified using the 95th percentile of the distribution of heat-killed controls, 60% are not different (i.e., they were false negatives — living cells classified as dead) and only 40% were accurately classified as living.
A general failure of the combined stains to discriminate between living and dead cells would not account for the differences between the treatments presented in Tables 1 and 2, but the pH-dependence of the stains might. Alkalization appears to cause partial hydrolysis of FDA in cell-free seawater, evident as an increase in fluorescence due to the release of no-longer-quenched fluorescein (Figure C). Increased pH-dependent lysis would reduce the concentration of the substrate available for staining the cells. A comparison of staining intensity in control and alkalized Diachronema (formerly Pavlova) lutheri (CCMP1325) in balanced growth is shown in Figure D, E. Classification of the culture in the growth medium (D) and in the same cultures alkalized with a final concentration of 1 mmolar NaOH (E), shows that most living cells have much higher fluorescence than the heat-killed controls. However, even though there is a reduction in the number of cells mis-classified as dead with alkalization (5% vs 13%), the median staining intensity in the alkalized cells is 4x lower than in the controls. In the absence of the appropriate killed controls, these live cells would largely be classified as dead.
Uncertainties over the validity of the means used to classify cells as living or dead could be addressed with additional testing and documentation.
Mackenzie Burke, Jessica Oberlander, Mikaela Ermanovics, Marie Egert, Hugh MacIntyre (Dalhousie University)
MacIntyre HL, Cullen JJ (2016) Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate. J Phycol 52 (4):572-589. doi:10.1111/jpy.12415
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AC4: 'Reply on CC3', stephanie delacroix, 19 Oct 2023
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.
Citation: https://doi.org/10.5194/bg-2023-138-AC4
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AC4: 'Reply on CC3', stephanie delacroix, 19 Oct 2023
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