the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Laboratory experiments in ocean alkalinity enhancement research
Maria Debora Iglesias-Rodríguez
Rosalind E. M. Rickaby
Arvind Singh
James A. Gately
Abstract. Recent concern about the consequences of continuing increases in atmospheric CO2 as a key heat-trapping agent (USGCRP, 2017; IPCC, 2021) have prompted ocean experts to come together to discuss how to provide science-based solutions. Ocean alkalinity enhancement (OAE) is being considered not only as a ocean carbon dioxide removal (CDR) approach, but also as a potential way to mitigate ocean acidification. Over the last two decades, inter-laboratory comparisons have proven valuable in evaluating the reliability of methodologies associated with sampling and analysis of carbonate chemistry parameters, which have been routinely used in ocean acidification research (Bockmon and Dickson, 2015). Given the complexity of processes and mechanisms related to ecosystem responses to OAE, consolidating protocols to ensure compatibility across studies is fundamental for synthesis and upscaling analysis. This chapter provides an overview of best practice in OAE laboratory experimentation and facilitates awareness of the importance of applying standardized methods to promote data re- use, inter-lab comparisons, and transparency. This chapter provides the reader with the tools to (1) identify the criteria to achieve the best laboratory practice and experimental design; (2) provide guidance on the selection of response variables for various purposes (physiological, biogeochemical, ecological, evolutionary) for inter-lab comparisons; (3) offer recommendation for a minimum set of variables that should be sampled and propose additional variables critical for different types of synthesis and upscaling; and (4) identify protocols for standardized measurements of response variables.
- Preprint
(984 KB) - Metadata XML
- BibTeX
- EndNote
Maria Debora Iglesias-Rodríguez et al.
Status: final response (author comments only)
-
RC1: 'Comment on sp-2023-7', Anonymous Referee #1, 07 Aug 2023
Review of Laboratory experiments in ocean alkalinity enhancement research
This paper is a chapter within a series of papers about Ocean Alkalinity Enhancement (OAE) research. It is supposed to describe best practices for laboratory experiments in OAE context. I find the paper a mixture of useful and not useful information, with the abstract promising 4 very useful things but then I don’t find I have been given that information on those 4 aspects once I’ve read the whole paper. Especially given it is one chapter as part of a series, and the chapter “Ch. 4.1: Introduction, general considerations – Sam Dupont (lead), Marc Metian https://sp.copernicus.org/preprints/sp-2023-4/” and preceding chapters provide more comprehensive information about many of the areas touched on in this paper (i.e. “4. sources of alkalinity” is covered in chapter 3 and also in chapter 4.1., “6. experimental replication”, “7. Testing impacts on marine oirganisms” and “8. Choice of Species” are covered in part by Ch 4.1) . Overall I think the paper could do with a slight rearrangement and focus to provide the reader with more useful information – perhaps something that follows their Table 1 (thus making table 1 a more important visual representation of the paper) and provides the details that’s in the legend of that table in the text. For example:
1. Introduction
a. Pros and cons to lab experiments
b. Lessons Learned from acidification
2. Seawater media preparation and manipulation of carbonate chemistry (include current sections 3, 4 and 5)
3. Experimental design (include current sections 6, 7, 8, 9, 10)
a. Replication
b. Preliminary experiments
c. Type of experiment (bottle vs container; lab vs ship-board/portable – section 11)
d. Abrupt vs chronic exposures
4. Sampling and analysis (table 2, section 11 and bits of others like second paragraph of section 6)
a. Criteria for key parameters
b. Sampling frequency and timing
c. Reporting key parameters
5. Conclusions and recommendationsSpecific comments:
1. Introduction: I find this a little muddled, with some convoluted sentences. The introduction should be a clear statement about the need for this paper and what the paper sets out to achieve guiding the reader through the remaining sections.
Line 22: change “are intended to be” to “need to be”
Line 35: “The current focus” of what?
Line 38-40: Some of the language could be written more confidently, given this is supposed to be recommendation and best practice. This sentence is a perfect example of this, as it suggests (in a convoluted way) that the authors don’t yet know what the best practice should be. Same for Lines 42 to 45.
Line 42: “The rich insights obtained in ocean acidification research…” sentence is out of place here. It should be the start of section 2.
Line 49: “Offering guidelines provided in this chapter…” this isn’t very good English. The sentence needs re-writing.
2. Lessons Learned from ocean acidification research: This section seems to spend the first paragraph saying how bad acidification research was, in terms of improper reporting, improper replication, or improper conditions (which then are justified in the later sentences of the paragraph). Instead it would be useful to frame OAE in the context of what we have learned from acidification research (and other lab experimental work), pointing towards the OA guide to best practice for a lot of detail on lab and chemistry manipulation information that cannot be covered in this chapter. Highlighting the importance for clear media preparation, consistent manipulation of the chemistry, reporting of this data and experimental design. All of which will then be covered in the following sections. In addition, specific to acidification and OAE, is the second paragraph and third paragraphs about DOM but also the multiple stressors.
Line 57: “In their study…” who’s study?
Line 61-64: Pointing out the choice of treatment conditions is useful, but the way it is done here is not helpful. It would be more useful to state that careful consideration is needed when deciding treatment levels. Learning from acidification research there are two aspects resources and question trying to be answered. If there are limited resources then focus on realistic levels, however if the question is about determining thresholds or determining the underlying physiological mechanisms of a response, then pushing beyond those realistic levels might have more value.
Box 1: This box does not appear to be referred to anywhere in the text other than the last sentence of the portable incubation experiments. If it is meant to be a summary of good standard practise then it needs to be more informative to that point. There is more useful information in Ch4.1 on all of these aspects than is presented in this box. For instance, Reproducibility – experiments need to be considered fully replicable by reporting methods correctly as well as sampling and reporting on all key parameters (see table 2)….
Line 84: “… we have learned that similar approaches can…” what is meant by this?
3. Seawater media preparation: This section, as well as section 4 and 5 could all do with reworking, especially if the authors decide to follow my suggestion in the overall comment. They need to have focussed take away messages – following the lines of Table 1.
4. Sources of alkalinity: As above. Given there is a chapter on this topic, here the authors would benefit from being more specific about how one would manipulate the carbonate chemistry in a lab experiment setting. i.e. the types of alkalinity but also the suitability (pros/cons maybe) for types of experiment (phytoplankton, zooplankton, benthic). Given also the caveat (as explained on line 160) that alkalinity above a certain threshold causes precipitates. Does this need to be avoided? What are the implications?
5. Impacts of impurities: As above.
6. Experimental replication:
Line 229: replace “several approaches can be applied experimentally to address replication” with “Replication is important in order to… …there are a number of experimental designs that can be set up that allow adequate statistical replication”.Line 230: “experimental unit (containing replicates)” is not an experimental unit if it then contains replicates within it! Simple replication involved having more than one experimental unit per treatment where all conditions are manipulated independently but in the same way for that treatment. These experimental units can be considered as independent.
Line 233: “treated as independent experiment units of a treatment” is incorrect. Temporal sampling of the same unit is not independent as there is more chance the response is similar at points closer in time than further away in time (see section 4.4.3 of Guide to best practice for acidification research, or other stats books). However, as long as we have replicate units being sampled through time then we can use repeated measures statistics to account for this non-independence.
Second paragraph (line 243-254) is not about experimental replication but instead is about reducing errors and storage issues. This section should be amended and moved to a section about sampling and analysis.
7. Testing impacts on marine organisms: As per suggestion, parts of this section need to be merged with other sections to form a useful guide to experimental design, and/or sampling and analysis. For instance the first paragraph is about getting the right experimental design (using short term manipulations, or longer term equilibrations), while the second paragraph is about sample collection and preventing errors. The third paragraph here does not provide much useful information in its current form. It should be part of the experimental design considerations for when deciding what type of experiment to choose depending on organism life-history, pre-exposure, and multiple stressors. Probably can be merged with section 8 paragraphs.
Sections 8, 9, 10 and 12 have useful information buried in them, but would benefit from re-ordering with an experimental design context. Choose species, choose experimental duration – stress responses vs other physiological responses vs longer term responses (such as growth, reproduction). Interaction and community experiments. Portable (ship-based) vs non-portable set ups.
Sections 11 and 12 both seem oddly detailed compared to the rest of the sections. Especially section 12, e.g. Line 419 “once the vessels have been placed into the tank, they should be secured…” this is very detailed (unnecessary!) methods compared to most of the other sections on lab set ups.
Figure 1: The sacrificial replication need to illustrate that there are replicates within each of the units that are sacrificed at each time point – and hence time should also be illustrated here. There should be a distinction between repeated sampling through time within the same experimental unit (temporal replication) and sacrificial replication which has much more initial replicates per treatment in order to make sure there are enough to follow through time (e.g. three time points, three replicates, would need 9 initial units at the start, when 3 are sacrificed at T1, leaving 6, then 3 more sacrificed at T2, leaving final 3 for sampling at T3).
Table 2. Please add inorganic nutrients to the basic chemistry variables to be measured as well as temperature and salinity. It seems obvious that these should be measured, but given the lessons we learned from acidification research that often these are inadequately reported and therefore not comparable, maybe they need to be explicitly stated here!
Citation: https://doi.org/10.5194/sp-2023-7-RC1 -
RC2: 'Comment on sp-2023-7', Alex Poulton, 24 Aug 2023
General
The article is a necessary addition to the guidelines for OAE experimental research. It is well written and generally covers the diversity of experimental targets (i.e. organisms) and treatments required for a thorough understanding of OAE biological responses. However, at times the article either needs to recognize this need to cover all organisms and not focus on specifics, or identify that the recommendations presented are more relevant to certain groups (e.g., Table 1). At times the article could also be stronger in its recommendations; reproducibility, representative controls, clear methodology and recognizing inherent limitations are key themes for the article and at times these could be more strongly emphasized to ensure the reader follows them in their future endeavors.
Specific comments
Ln 57: Please make clear that ‘their study’ refers to Cornwall and Hurd (2016), and also what does ‘interdependent’ refer to (replication or treatments)?
Lns 73-80: Resource availability (nutrients, light, prey) is another confounding factor that it would be good to mention here as this will determine the strength of response. It is also an important difference between laboratory and field experiments that needs consideration when scaling from one to the other. This is followed up later in the article but here it would be good to introduce these issues.
Ln 109: Removing ‘as controls’ from this line would avoid confusion with untreated controls.
Ln 116: Suggest adding the caveat that where sterilization is not required for the experimental set up.
Ln 153: Do the authors consider that determining responses to the ‘initial spikes in pH and drops in CO2’ is an important step, or should the focus be on the response under ‘steady state via bubbling’?
Ln 165: Medium preparation is necessary in terms of experimentation with microalgae, what considerations are needed for other organisms (e.g., benthic organisms, zooplankton, fish)?
Ln 385: Whilst the authors focus on 13C methodology for rate determination, would it be advantageous to mention here that treatment-specific determination of DIC concentrations is also recommended for 14C methodology (e.g., in studies looking at photo-physiology, where P v E methods are required, or calcification rates)?
Ln 391: This seems like a weak recommendation (‘we ought to follow these protocols ..’) and stronger wording is suggested.
Ln 422: What about light contamination from the ship – this is an important additional consideration.
Ln 450: Shouldn’t resource availability (prey, nutrients) not be a key variable to measure during the experiments?
Citation: https://doi.org/10.5194/sp-2023-7-RC2 -
RC3: 'Comment on sp-2023-7', Justin Ries, 26 Aug 2023
The manuscript by Iglesias-Rodriguez et al provides a useful framework for designing and implementing experiments aimed at investigating impacts of ocean alkalinity enhancement (OAE) on marine organisms. The authors wisely use the framework for ocean acidification (OA) experiments, which emerged over the past couple of decades of intensive research on the subject, as the starting place for their proposed OAE experimental framework.
My main suggestion for the authors as they revise their manuscript is to avoid being too prescriptive and restrictive in their recommendations. As the authors are aware, some of the foundational studies in the field of OA research came from experiments that did not conform to the style of experiments that most other workers were utilizing. For example, Jury et al (2010) conducted OA experiments on corals where they manipulated carbonate chemistry through the addition of various combinations of acid and alkalinity, rather than through CO2-equilibration. Although this approach was not recommended by the OA Best Practices Guide, it allowed for the deconvolution of the impacts of the various aspects of the seawater carbonate system on coral calcification, ultimately leading to the important finding that coral calcification is most strongly influenced by the bicarbonate ion concentration – a finding that would not have been evident if the experiments were conducted via CO2-equilibration as recommended. It also seems overly prescriptive to limit the upper levels of alkalinity addition to such low levels as described in the manuscript (3000-4000 umol/kg-sw). It is always better to have too much information about how a system works, than too little. If workers looking at higher alkalinity levels produce studies that do not fit perfectly into a meta-analysis because other workers did not evaluate such equally high levels of alkalinity, then the worker conducting the review paper or comparative study can simply restrict their comparison to the range of alkalinities that were tested by all studies under evaluation. Like for OA experiments over the past two decades, OAE experiments will not only provide insight into how marine organisms will respond to applied chemical CDR, it will also provide tremendous insight into the source(s) of carbon for photosynthesis and calcification, thereby improving our understanding of the very mechanisms of tissue and shell production in marine organisms. However, to maximize the insight gained from OAE experiment about these more fundamental processes, we should not unnecessarily restrict the scope and design of these experiments so that they are optimized only for applied CDR via OAE, just as we should not restrict OA experiments so that they are only optimized for year 2100 IPCC CO2 emission scenarios.
Lastly, I would also recommend that the reviewers incorporate information that has been gleaned from quasi-OAE experiments conducted in parallel fields investigating the impact of river liming to combat the effects of acid rain (mainly 1960-1980s) and the effects of seawater buffering (mainly by Na2CO3 addition) utilized by the commercial shellfish industry.
The authors should be commended for their efforts in producing a manuscript that should ultimately prove to be extremely useful in standardizing and advancing the emerging field of experimental OAE research.
Line 25: consider adding ‘meta-analysis’ as reason for standardizing methods
38: invert latter half of sentence for clarity (‘impacts of OAE approaches at various levels of…’
45: consider expanding ‘interlaboratory comparisons’ to include ‘interspecies comparisons’, both of which would benefit from the standardization of methods advocated for herein.
53: In addition to the section on ‘Lessons learned from OA research’, the authors may want to consider including a section on the OAE research that has been conducted to date. There have been numerous studies conducted on the impact of OAE on aqueous organisms from past research by the shellfish industry investigating the utility of so-called ‘sweetening’ the water through addition of mainly soda ash (Na2CO3), a practice utilized in shellfish hatcheries for decades, and also in the academic and industrial fields of ‘river liming’, which dissolved primarily CaCO3 and dolomite in higher latitude watersheds to offset the effects of acid rain (due to NOx and SOx emissions) in the 1960s and 1970, but is still practiced today in Canada and some Scandinavian countries, among other places. Although the goals of increasing the alkalinity of hatchery waters (for shellfish) and river waters (for offsetting acid rain) was not to sequester CO2, many of these studies did evaluate the impacts of these activities on the organisms – which is the subject of the present chapter.
69: Authors mention that DOM ‘contributes’ to alkalinity of seawater. But DOM can also reduce (rather than ‘contribute’ to) alkalinity on a net basis by releasing organic acids. It may be worth mentioning that alkalinity can be both increased or decreased by DOM, depending on what type of DOM is released and what happens to that DOM after release.
70: change ‘to’ to ‘in’, or ‘taken’ to ‘given’
78-79: ‘given the capacity to drive shifts..’; need to include reference the subject in this part of the sentence. i.e., given the capacity of what to drive shifts?
79: consider including ‘physiological outputs, survival, and reproduction’ along with shifts in species abundance, etc., since those former parameters are the ones that will more often be measured in OAE and OA studies.
92: consider adding ‘and precipitation’ after ‘dissolution’, since both are relevant
92: sentence contains a non sequitur
101: sodium is a metal
106 : rates of growth and calcification can also be measured by changes in dry mass or buoyant mass in many species, especially macroinvertebrates and macroalgae (see Davies, and others).
140: For clarity, consider adding ‘and therefore the saturation state of the seawater with respect to CaCO3’ to end of sentence.
145: there is also concern about metals leaching from other sources of alkalinity beyond olivine, such as from basalt, brucite, and even carbonates associated with zones of hydrothermal alteration where metals can be enriched.
147: NaOH is probably not a scalable source of alkalinity for OAE because its production (the chlor-alkali electrochemical process) requires energy (which releases CO2 if derived from fossil fuel) and, more importantly, would produce massive amounts of HCl as a byproduct — which, from an Earth-system mass balance perspective, would eventually cause ocean acidification and re-release of the sequestered CO2. I would avoid advocating for any particular form of alkalinity in this chapter. The jury is still out on that one -- indeed, this is one of the goals of the forthcoming OAE experiments.
147: just because a waste product (HCl) can be used as a ‘cleaning product’ does not make it ‘clean’ in the environmental impact sense. I would not include this line of reasoning here.149: NaOH is indeed mined, it is just mined from the sea rather than from land.
153: specify: a drop in ‘aqueous’ CO2
157: since one of the key goals of OAE research will be to determine the maximum alkalinity that different species can tolerate without deleterious effects, I would strongly recommend against prescribing such a low upper-limit to the OAE experiments as 3000-4000 umol/kg-sw. It is likely that seawater that receives alkalinity or OAE could increase well above 10,000 umol/kg-sw at the instant of addition, and still not cause reprecipitation if diluted/mixed by waves and wind. I would suggest encouraging workers to explore a wide range of alkalinities to maximize inference from their experiments and avoid limiting the applicability of their research in the event that OAE practitioners ends up deploying alkalinity at concentrations greater than 4,000 umol/kg-sw (which they likely will, at least immediately after release). An analogous misstep was also made in the field of OA research, which initially recommended only investigating pCO2 levels within the 400 to 1000 uatm range, as that is what is predicted for the open ocean over the next century, when in reality many of the estuaries, marginal marine environments, and shelf systems that host most of the marine calcifiers in the ocean already experience pCO2 conditions in the 1500-2000 uatm range today due to seasonal eutrophication, upwelling, etc. I would conservatively suggest that OAE experiments investigate alkalinities up to at least 10,000 uatm/kg-sw, which is arguably the maximum that can be maintained without inducing reprecipitation, and – importantly – workers should utilize intermediate treatments (e.g., 2000, 4000, 7000 uatm/kg-sw, rather than just low/high treatments, in order to identify potential nonlinear and even parabolic responses – an important and unexpected outcome of early OA research (e.g., Ries, J.B., Cohen, A.L., McCorkle, D.C. 2009. Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology, 37 (12):1131-1134.).
166: consider specifying at the onset of this section that the formulation of f/2 media, or other nutrient media, would only be applicable to experiments on algae.
213: considering using ‘carbonate precipitation’ instead of ‘carbonate formation’ to differentiate the process of carbonate ion formation from carbonate mineral formation
217-219: sentence should be rephrased for clarity
224: sentence would benefit from clarification of the term ‘tested saturation levels’.
243: ‘sample’ instead of ‘sampling’
302: sentence should also include a statement that prior empirical studies have shown that the Mg/Ca ratio of Mg-calcite producing organisms generally varies proportionally with seawater Mg/Ca (e.g., Ries, J.B. 2004. The effect of ambient Mg/Ca on Mg fractionation in calcareous marine invertebrates: A record of Phanerozoic Mg/Ca in seawater. Geology 32(11):981-984; Ries, J.B. 2006. Mg fractionation in crustose coralline algae: Geochemical, biological, and sedimentological implications of secular variation in the Mg/Ca ratio of seawater. Geochimica et Cosmochimica Acta 70:891-900) – which is key to the logical argument that adding Mg via brucite addition (or Ca via CaCO3 or Ca(OH)2 addition) would locally modify seawater Mg/Ca and, therefore, the Mg content (and solubility) of biomineralized calcite.
303: The Bischoff et al (1987) reference applies mainly to synthetic abiogenic calcite. For a recent reference to support the statement that biogenic calcite dissolves more quickly (i.e., is less stable) when it contains higher Mg/Ca ratios, see: Ries, J.B., Ghazaleh, M.N., Connolly, B., Westfield, I., Castillo, K.D., 2016, Impacts of ocean acidification and warming on the dissolution kinetics of whole-shell biogenic carbonates. Geochimica et Cosmochimica Acta 192: 318-337. doi: 10.1016/j.gca.2016.07.001.
303: I would recommend expanding this section to include discussion of the impacts of the major metals (Mg2+, Ca2+) associated with the alkalinity source on marine calcifiers. For instance, alkalinization with the common alkalinizing minerals Ca(OH)2 (slaked lime), Mg(OH)2 (brucite), CaCO3 (limestone) or (Mg,Ca)CO3 (dolomite) will modify the Mg/Ca of the affected seawater, which has been shown by an extensive body of literature to favor both the biogenic and abiogenic precipitation of low-Mg calcite when seawater Mg/Ca falls within the calcite stability field (seawater mMg/Ca < 2) and the biogenic and abiogenic precipitation of aragonite and high-Mg calcite when seawater Mg/Ca falls within the aragonite stability field (seawater mMg/Ca > 2) (c.f., Ries, J.B., 2010, Geological and experimental evidence for secular variation in seawater Mg/Ca (calcite-aragonite seas) and its effects on marine biological calcification. Biogeosciences 7: 2795-2849.) Thus, modification of local seawater Mg/Ca ratios by OAE has the potential to favor aragonite and high-Mg calcite organisms if seawater Mg/Ca is increased, and low-Mg calcite organisms if seawater Mg/Ca is decreased. This goes beyond the issue of higher-Mg calcite shells being more easily dissolved in seawater than lower-Mg calcite shells that is addressed in this paragraph, and would probably be worth highlighting as an important area of future OAE research.
389: ‘propagation’
Table 2: the references cited for seawater Mg:Ca manipulation studies (Brzezinski 1985 etc) do not include protocols for experimental manipulation of seawater Mg/Ca. A more relevant reference is needed.
Citation: https://doi.org/10.5194/sp-2023-7-RC3
Maria Debora Iglesias-Rodríguez et al.
Maria Debora Iglesias-Rodríguez et al.
Viewed
| HTML | XML | Total | BibTeX | EndNote | |
|---|---|---|---|---|---|
| 253 | 139 | 13 | 405 | 5 | 13 |
- HTML: 253
- PDF: 139
- XML: 13
- Total: 405
- BibTeX: 5
- EndNote: 13
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1