# Alkaline mineral dissolution can impair embryonic development in the Pacific oyster (Magallana gigas), raising caution for ocean alkalinity enhancement

Fabrice Pernet, Luna Ducoulombier, Hugo Koechlin, Frédéric Gazeau, Cale A Miller

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## To cite this version:

Fabrice Pernet, Luna Ducoulombier, Hugo Koechlin, Frédéric Gazeau, Cale A Miller. Alkaline mineral dissolution can impair embryonic development in the Pacific oyster (Magallana gigas), raising caution for ocean alkalinity enhancement. ICES Journal of Marine Science, 2026, 83 (2), pp.fsag011. 10.1093/icesjms/fsag011. hal-05523139

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# Alkaline mineral dissolution can impair embryonic development in the Pacific oyster (*Magallana gigas*), raising caution for ocean alkalinity enhancement

Fabrice Pernet<sup>1, *</sup>, Luna Ducoulombier<sup>1</sup>, Hugo Koechlin<sup>1</sup>, Frédéric Gazeau<sup>2</sup>, Cale A. Miller<sup>3</sup>

## Abstract

Ocean alkalinity enhancement (OAE) is a promising marine carbon dioxide removal (mCDR) approach aimed at mitigating climate change by increasing oceanic CO₂ uptake. However, its potential biological effects remain poorly investigated. Here, we examined the impact of a wide range of alkalinity levels—from ambient (~2300 µmol kg⁻<fnref n="1" />) to 11 000 µmol kg⁻<fnref n="1" />—achieved through the dissolution of olivine, limestone, ground oyster shell, and sodium carbonate on the embryonic development of Pacific oysters (*Magallana gigas*), a key species in marine ecosystems and aquaculture to defining safe operating limits for various OAE deployment scenarios. We found that relatively modest levels of olivine dissolution, within the ranges feasible for OAE (ca. +600 µmol kg⁻<fnref n="1" /> of total alkalinity), can impair embryonic development of oysters, likely due to the release of trace metals such as nickel. Limestone showed no developmental effects while oyster shell displayed moderate and variable negative effects. Sodium carbonate had no adverse impacts. Our study shows that the careful selection of alkaline materials is key to minimise ecological risks in OAE, emphasising the need to assess biological impacts for sustainable mCDR deployment.

**Keywords** alkalinisation, bivalve, ecotoxicology, limestone, olivine

## Introduction

The latest IPCC report highlights that in order to limit global warming to 1.5°C or 2°C, as outlined in the Paris Agreement, CO₂ emissions must be drastically reduced, with net-zero targets set for 2050 (IPCC 2023). While cutting emissions is crucial, removing existing CO₂ from the atmosphere is also essential to address the current trajectory of emissions (IPCC 2023). To meet these goals, carbon dioxide removal (CDR) methods will need to be scaled up by a factor of 2 by 2030 (from ca. 2 to 4 Gt CO₂) and by 3–4 by mid-century, reaching 7 to 9 Gt CO₂ (Smith et al. 2024). Climate mitigation efforts have generally focused on land-based CDR techniques, but increasing concerns over the limitations of these methods have shifted attention toward novel marine-based CDR (mCDR) (Oschlies et al. 2023).

Ocean alkalinity enhancement (OAE) is a mCDR technique that enhances ocean alkalinity, increasing the imbalance between atmospheric and seawater CO₂ partial pressures, thereby promoting greater oceanic uptake of CO₂. Drawing inspiration from Earth's natural modulation of alkalinity over geological timescales, a prevailing method of OAE is to enhance the natural weathering process by introducing large quantities of finely ground land-based minerals derived from silicate (e.g. olivine) or carbonate rock (e.g. limestone), or their more reactive, human-made derivatives (e.g.

lime) into seawater (Kheshgi 1995, Caldeira and Rau 2000, Harvey 2008, Renforth and Henderson 2017, Gattuso et al. 2018, Bach et al. 2019, NASEM 2022, Oschlies et al. 2023). These materials undergo acid hydrolysis by carbonic acid, converting excess CO₂ into bicarbonate (HCO₃⁻) and carbonate (CO₃<fnref n="2" />⁻), which remain stored in the ocean for thousands of years (Kheshgi 1995, Caldeira and Rau 2000, Harvey 2008, Renforth and Henderson 2017, Gattuso et al. 2018, Bach et al. 2019, NASEM 2022, Oschlies et al. 2023). In addition to generating CDR with theoretical sequestration potential exceeding gigatons of CO₂ per year (Köhler et al. 2013, Renforth and Henderson 2017, Doney et al. 2020), the release of proton-neutralising substances to the surface ocean through OAE counteracts ocean acidification, which is considered an environmental threat for a range of marine ecosystems (Doney et al. 2020).

While the introduction of OAE is technically feasible and shows promise, the potential toxic effects of OAE warrant careful consideration. OAE inevitably alters carbonate chemistry parameters, potentially creating localised increases in pH, DIC, carbonate ions, and omegas near the application site that may exceed the tolerance limits of certain marine organisms.

Additionally, the introduction of alkaline substances may release mineral-derived contaminants into marine environments. For example, alkaline materials such as olivine can contain trace metals such as nickel (Ni), chromium (Cr), iron (Fe), or cobalt (Co),

<footnote n="1"><sup>1</sup>Ifremer, Univ Brest, CNRS, IRD, LEMAR, F-29840 Argenton, France</footnote>

<footnote n="2"><sup>2</sup>Sorbonne Université, CNRS, Laboratoire d'Océanographie de Villefranche, F-06230 Villefranche-sur-Mer, France</footnote>

<footnote n="3"><sup>3</sup>Department of Earth Sciences, Utrecht University, 3584 CB Utrecht, The Netherland</footnote>

<footnote>*Corresponding author. Ifremer, Univ Brest, CNRS, IRD, LEMAR, F-29840 Argenton, France. E-mail: fabrice.pernet@ifremer.fr</footnote>
which may be released during dissolution (Montserrat et al. 2017, Flipkens et al. 2021, Hutchins et al. 2023, Geerts et al. 2025). These metals can be toxic to marine ecosystems, accumulating in the tissue of organisms, and potentially impairing their growth, reproduction, and survival (Mance 2012, Saravanan et al. 2024). Early life stages, such as embryos and larvae, are particularly vulnerable to metal exposure, with even low concentrations capable of disrupting normal development (Calabrese and Nelson 1974, Calabrese et al. 1977, Martin et al. 1981, Markich 2021).

Ultimately, any significant additions of alkaline materials to seawater will inherently alter elemental ratios and nutrient availability, potentially driving ecological imbalances. For instance, OAE achieved through the dissolution of carbonate rocks, which increases both calcium concentration and total alkalinity (TA), is expected to promote the growth of calcifiers, whereas the dissolution of silica-based minerals could benefit diatoms (Bach et al. 2019).

The biological effects of OAE, which are likely influenced by the type of material used, the application method, and the scale of deployment, remain largely unexplored (Bach et al. 2019, NASEM 2022, Dupont and Metian 2023). Existing studies on the impacts of OAE are limited and have primarily focused on phytoplankton (Ferderer et al. 2022, Gately et al. 2023, Hutchins et al. 2023, Guo et al. 2024, Xin et al. 2024, Faucher et al. 2025, Guo et al. 2025, Oberlander et al. 2025) and more marginally on corals (Albright et al. 2016), mollusks (Flipkens et al. 2024, Jankowska et al. 2024), and crustaceans (Flipkens et al. 2023b, Cripps et al. 2013, Flipkens et al. 2024). Existing studies on phytoplankton responses to OAE have yielded mixed results. Some investigations have reported neutral or positive effects, particularly in siliceous phytoplankton like diatoms due to increased availability of silicic acid from olivine dissolution (Hutchins et al. 2023), while others have observed inhibitory effects depending on the OAE method, trace metal release, and carbonate chemistry changes (Faucher et al. 2025, Guo et al. 2025, Oberlander et al. 2025). Regarding crustaceans and bivalves, the few existing studies have reported various effects of adding TA through olivine dispersal on growth, survival, behavior, and reproduction, as well as the elemental composition of tissues (Flipkens et al. 2023b, Flipkens et al. 2024, Jankowska et al. 2024). These species-specific outcomes underscore the need for broader ecological assessments across different taxonomic groups, particularly marine invertebrates.

To date, no empirical research has investigated the biogeochemical and physiological impacts of OAE on economically important marine calcifiers such as bivalves. Their response to OAE presents significant concerns. A negative impact could jeopardise bivalve populations, posing risks not only to global aquaculture and food security, but also to vital ecosystem services. Bivalves indeed play essential roles in coastal areas: they filter and clarify water, enhance nutrient cycling, stabilise sediments, and provide habitat and food for a wide range of marine species (van der Schatte Olivier et al. 2020). Conversely, a positive impact of OAE on bivalve populations, such as enhanced calcification, could paradoxically lower seawater alkalinity and release CO₂, potentially reducing the effectiveness of CDR (Bach et al. 2019). Understanding their response to OAE is therefore critical for addressing both ecological sustainability and socioeconomic stability.

The objective of this study was to evaluate the biological impacts of alkalinity addition on the embryonic development of the Pacific oyster, *Magallana gigas*. This oyster species has been intro-

duced and is now widely established in aquaculture and natural coastal ecosystems across temperate regions worldwide (His et al. 1999), making it particularly well suited for ecotoxicological studies (His et al. 1999, Markich 2021).

We exposed multiple batches of oyster embryos to a wide range of alkalinity levels using four alkaline compounds—olivine, limestone, ground oyster shell, and sodium carbonate—selected to reflect practical, ecological, and economic considerations. Unlike most OAE impact studies—which typically rely on addition of pure chemicals—our approach accounts for the complexity of natural minerals such as olivine and limestone, which can release additional compounds, including trace metals. This study addresses a critical gap by directly testing the biological effects of these materials on development of marine bivalves.

# Materials and Methods

## Alkaline materials

We first tested olivine and limestone, two of the most promising mineral candidates for OAE due to their abundance and alkalinity-generating potential. In addition, we evaluated calcium carbonate derived from ground oyster shells to explore the feasibility of repurposing aquaculture byproducts as a sustainable alternative. Recycling oyster shells not only offers a renewable source of calcium carbonate, but also diverts waste from disposal pathways such as incineration, which emit CO₂, thereby providing an environmentally friendly waste management strategy (Pernet et al. 2025). Finally, we included anhydrous sodium carbonate (Na₂CO₃), a highly soluble and chemically well-characterised compound. Although less viable for large-scale, open-ocean OAE due to its higher cost per unit of alkalinity added, sodium carbonate is commonly used in small-scale applications—such as buffering seawater at bivalve hatchery intakes during upwelling-driven acidification events (Barton et al. 2015). As a pure compound with minimal mineral-derived contaminants, it also served as a control treatment to isolate the biological effects of alkalinity addition from those of mineral dissolution byproducts.

Olivine was sourced from Sibelco, Norway, with a particle diameter of 250–700 µm for 97% of the grains, and a composition of 50.2% MgO, 41.4% SiO₂, and 7.4% Fe₂O₃ by weight, as reported by the manufacturer (reference AFS30 DS). Limestone was from Forschungsgemeinschaft Kalk & Mörtel, Germany, with a particle diameter of less than 100 µm as reported by the manufacturer. Ground oyster shells were from Ovive SA, France, with particle diameter of 200–300 µm as reported by the manufacturer. Anhydrous sodium carbonate (Na₂CO₃) was sourced from TATA Chemicals (NJ, USA).

## Production of alkaline solutions

Our method for alkalinity generation is inspired by the accelerated weathering of limestone (Rau et al. 2007, Eisaman et al. 2023), and involves dissolving minerals under elevated pCO₂ to maximise alkalinity generation (and to address the slow dissolution kinetics of silicate and carbonate minerals in natural seawater), followed by re-equilibration with air bubbling to restore near-ambient pH conditions (Miller and Pernet 2025). This approach allowed us to generate TA levels that exceeded the anticipated range of most OAE applications (reaching up to 11 000 µmol kg⁻¹) and therefore,
to identify tipping points (TPs) and tolerance thresholds (Iglesias-Rodríguez et al. 2023).

We prepared CO₂-saturated seawater by vigorous CO₂-bubbling, lowering the pH to ~5.0 (Table S1). Seawater (600 mL) was first filtered to 1 µm and UV-treated. Minerals were dissolved under these conditions with continuous agitation using a magnetic stirrer. Dissolution lasted 3 h for olivine and 1.5 h for limestone and oyster shell, reflecting differences in mineral dissolution kinetics. The mineral concentrations used were 12% for olivine, 4% for limestone and oyster shell, and 0.2% for sodium carbonate, expressed as weight/volume (w/v). Both the dissolution durations and mineral concentrations were determined and optimised based on findings from preliminary experiments. The resulting solution was re-equilibrated to a pH of approximately 8.0 through vigorous air bubbling (Table S1), then filtered using a 0.45-µm Whatman filter to remove remaining particles. We observed that alkaline stock solutions left undisturbed at room temperature remained stable in terms of pH and TA for several days. However, in a subset of post-incubation samples where pH and TA were re-measured, we occasionally observed a decrease in pH likely due to CO₂ accumulation from embryo respiration under sealed (capped) conditions (data not shown).

An important consideration in OAE experiments is whether alkaline solutions are equilibrated with atmospheric CO₂ or remain non-equilibrated (Dupont and Metian 2023, Eisaman et al. 2023, Iglesias-Rodríguez et al. 2023). In this study, all solutions were adjusted to equivalent pH values, naturally resulting in differences in pCO₂ (as dissolved inorganic carbon and CO₂ concentrations increased with higher TA values, Table S1, Figure S1). In contrast, air-pCO₂ equilibration would have caused mineral precipitation (F. Petron personal observation) and major pH increase. Past research on ocean acidification has shown that Pacific oyster embryos are resilient to hypercapnia with respect to embryonic development (Gazeau et al. 2011, Waldbusser et al. 2015), reducing concerns about confounding effects. However, reductions in seawater pH, which increase proton concentrations, may disrupt the proton gradient between internal cellular compartments and external seawater, impairing pH homeostasis and physiological functions (Melzner et al. 2009, Cyronak et al. 2016, Melzner et al. 2020). Given the lack of information on how decreasing proton concentrations under OAE affects these processes, it may be more appropriate to hold proton concentration constant to specifically investigate the effects of alkalinity addition. Ideally, fully decoupling the various parameters of the carbonate system would allow a more precise assessment of OAE impact, as has been done in ocean acidification research (Melzner et al. 2009, Melzner et al. 2020). However, achieving such decoupling is beyond the scope of this study.

## Alkalinity measurement

After filtration, TA stock solution was measured in 50 mL seawater subsamples by potentiometric titration at 22°C, using a Titrando 888 (Metrohm®) titrator coupled to a glass electrode (ecotrode plus, Metrohm®) and a thermometer (pt1000, Metrohm®). TA was calculated following the protocol described in Dickson et al. (Dickson et al. 2007). The instrument was calibrated before every set of measurements using certified reference material from A. Dickson (Scripps Institution of Oceanography, San Diego). The stock TA solution was then sequentially diluted with ambient seawater (0.45 µm-filtered and UV-treated) to achieve the required TA

concentrations. All TA target values accurately followed linear dilution calculations for proportional mixing giving confidence that high TA values can be appropriately titrated using the open-cell method. At the time of sampling, pH<sub>T</sub> (on the total scale), salinity, and temperature were measured using a multi-parameter portable meter (MultiLine Multi 3630 IDS—WTW: pH electrode SenTix 940, conductivity electrode TetraCon 925, Xylem Analytics, Weilheim in Oberbayern, Germany). The pH electrode was verified weekly using Certipur NBS buffers (pH 4.00, 7.00, and 9.00; Merck, Darmstadt, Germany), and then calibrated using a certified Tris/HCl buffer (provided by A. G. Dickson, Scripps Institution of Oceanography, San Diego, CA, USA). Carbonate chemistry parameters were determined using a modified CO₂SYSv3 function in MATLAB suited for correcting dissociation constant sensitivities to additions of major ions (Ca and Mg) and saturation state sensitivities to changes in the Mg-Ca ratio (Miller and Pernet 2025).

Although the saturation states in our stock solutions exceeded thresholds reported for homogeneous precipitation under controlled conditions (Suitner et al. 2024), we did not observe any visible signs of secondary precipitation, such as cloudiness or sediment formation. This may reflect the quasi-axenic nature of 0.45 µm-filtered and UV-treated seawater used (Flipkens et al. 2023a) and the relatively higher pCO₂ resulting from adjusting the solutions to equivalent pH values rather than allowing equilibration with atmospheric CO₂.

## Oyster embryo production

The oyster embryos were produced from five different spawning events between March and July 2024 (Table 1) following the methodology described by Petton et al. (2015). Four groups of broodstock oysters were collected from their natural habitats and conditioned at the Ifremer facility in Argenton (Brittany, France; 48° 31' 16" N, 4° 46' 2" W) for at least five weeks between February and July 2024 (Table 1). Conditioning was conducted in 400-liter tanks with an open-flow system, maintained at 17°C. During this period, the oysters were fed a diet of microalgae (Chaetoceros sp. and Tisochrysis lutea) in a 1:1 volumetric ratio, cultured at the facility. Once the oysters were reproductively mature, the gonads of these individuals were incised and their gametes were filtered on a 60-µm sieve to remove debris. The oocytes were then collected on a 20-µm sieve, rinsed, and resuspended in filtered seawater in a 2-L glass beaker. Gametes were mixed in a 5-L jar at five spermatozooids per oocyte. After a 2-h contact period, fertilisation rates were assessed, and the resulting embryos were used for the experiments.

## Experimental design

The first experiment consisted of exposing five batches of oyster embryos which were produced sequentially between March and July 2024 to a wide range of TA levels, from ambient (~2300 µmol kg⁻¹) to ~11 000 µmol kg⁻¹. TA levels were initially increased by 700 µmol kg⁻¹ above ambient, followed by eight increments of 1000 µmol kg⁻¹, resulting in a total of 10 levels. In France, oysters are typically found in coastal and estuarine environments where TA ranges from ~1900 to ~2600 µmol kg⁻¹ (Petton et al. 2024). In areas where OAE is directly applied, models suggest that TA could reach 3000–5500 µmol kg⁻¹, depending on the amount of material added and the local ocean dynamics (Ilyina et al. 2013, Renforth
<table><caption>**Table 1** Experimental design.</caption><thead><tr><th>Expt.</th><th>Batch no.</th><th>Fertilisation date</th><th>Broodstock cohort</th><th>Conditioning duration (d)</th><th>Number of male</th><th>Number of female</th><th>Mating Scheme</th><th>Mineral</th><th>Elemental analysis</th></tr></thead><tbody><tr><td rowspan="5">1</td><td>1</td><td>13/03/24</td><td>1</td><td>58</td><td>5</td><td>24</td><td>Pool</td><td>OS</td><td>No</td></tr><tr><td>2</td><td>24/04/24</td><td>2</td><td>71</td><td>4</td><td>4</td><td>Pool</td><td>OS</td><td>No</td></tr><tr><td>3</td><td>28/05/24</td><td>2</td><td>105</td><td>3</td><td>3</td><td>Pool</td><td>O, L, OS, SC</td><td>Yes</td></tr><tr><td>4</td><td>04/06/24</td><td>3</td><td>38</td><td>6</td><td>6</td><td>Pool</td><td></td><td>Yes</td></tr><tr><td>5</td><td>18/06/24</td><td>3</td><td>52</td><td>3</td><td>3</td><td>Pool</td><td></td><td>Yes</td></tr><tr><td>2</td><td>6</td><td>09/07/24</td><td>4</td><td>36</td><td>10</td><td>10</td><td>Biparental families</td><td>O, OS</td><td>No</td></tr></tbody></table>

Characteristics of *M. gigas* broodstock and the resulting embryos used in experiments, experimental design, and analysis. Abbreviations: Expt., Experiment; O, olivine; L, limestone; OS, oyster shell; SC, sodium carbonate.

<figure><img src="image_4.png" /><figcaption>**Figure 1** Influence of olivine dissolution on oyster development. Selected microscopic images of *Magallana gigas* larvae after 48 h of exposure to different alkalinity levels (µmol kg⁻¹) through mineral dissolution of olivine. The lowest alkalinity corresponds to ambient seawater (no olivine dissolution). Normal larvae exhibit characteristic D-shaped shells with straight hinges and smooth, well-defined margins. Abnormal individuals display shell deformities or complete lack of shell development.</figcaption></figure>

and Henderson 2017). By testing this wide alkalinity range, we offer a conservative assessment of the potential ecological risks associated with acute carbonate chemistry perturbation and contribute to defining safe operating limits for various OAE deployment scenarios.

Three to five assays were performed for each alkaline compound during the spring-summer of 2024 (Table 1). After preparing TA-enriched seawater and distributing the alkalinised solutions into 60-mL polypropylene pillboxes (reference number 688262, Dutcher, France), 4 h post-fertilisation embryos were inoculated in each pillbox at a density of 60 individuals mL⁻¹. One pillbox was used for each batch and TA level. Pillboxes were capped and incubated for 48 h at a constant temperature of 22°C, without bubbling or agitation, under a 12:12 light-dark cycle. Then, larvae were collected by filtration through a 20-µm sieve, transferred to Eppendorf tubes with a drop of formalin for preservation, and examined at 40x magnification using a Keyence microscope (Keyence Corporation, Japan) to determine the proportion of normally developed D-hinge (prod isoconch I) and shell length. Normally developed larvae were characterised by D-shaped shells with straight

hinge, smooth curvature along the edge of the valve (Di Poi et al. 2014, Waldbusser et al. 2015). In contrast, abnormal individuals display shell deformities including irregular or absent hinges, malformed or rounded shell outlines, asymmetrical growth, or complete lack of shell (undeveloped individuals). To illustrate developmental morphology, representative images of larvae exposed to different TA levels from olivine dissolution are presented in Fig. 1. Measurements were generally taken from 50 to 200 individuals per pillbox, unless high abnormal development prevented shell length measurement.

A second experiment was performed following the procedure described above with olivine (where all batches from experiment 1 responded to TA increase) and oyster shell (which showed significant TA effects on both % normality and shell length in two batches), while limestone and sodium carbonate was excluded due to the absence of a consistent TA effect across batches (Table 1). We also focused on oyster shells rather than limestone, as they represent a readily accessible, natural source of alkalinity, especially in regions with established bivalve aquaculture. Unlike the initial experiment which prioritised a high resolution of alka-
linity levels, these assays emphasised biological variability by increasing the number of oyster batches while reducing the number of alkalinity levels tested. Specifically, 10 biparental oyster families were produced and exposed to four TA treatments: ambient (~2300 µmol kg⁻¹), 4000 µmol kg⁻¹, 6000 µmol kg⁻¹, and 9000 µmol kg⁻¹.

## Elemental analysis

At the end of the embryonic exposure period in Experiment 1, seawater samples (1 mL) were collected from pillboxes for batches 3, 4, and 5 (n = 3), corresponding to ambient, 5000, and 11 000 µmol kg⁻¹ TA. These samples were subsequently analysed for trace metals and major ions using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Optical Emission Spectrometry (ICP-OES) at Utrecht University's Geosciences Laboratory. Samples were collected in acid-washed polyethylene tubes, acidified immediately to a pH below 2 using ultra-pure nitric acid to stabilise trace metals and prevent mineral precipitation.

Trace metals, including cadmium (Cd), Co, Cr, copper (Cu), Fe, manganese (Mn), Ni, and lead (Pb), were quantified using ICP-MS. Additional trace elements were also analysed (refer to Data File for the complete list). Measurements were conducted using the NexION 2000P from PerkinElmer, equipped with a cooled Peltier conical spray chamber, a micro-concentric Teflon nebuliser, and a PrepFAST autosampler (SC-4 DX), with a sample flow rate of 150 µL min⁻¹.

Major ions, including calcium (Ca), magnesium (Mg), and silicon (Si), were measured using ICP-OES on the AVIO 500 from PerkinElmer. The system featured a conical spray chamber, a micro-concentric Teflon nebuliser, and a PrepFAST autosampler (SC-4 DX), with a sample flow rate of 300 µL min⁻¹. Detection limits ranged from 1 to 100 ppb, depending on the element, solution matrix, and potential spectral interferences. These limits are calculated during each analytical run.

For both ICP-MS and ICP-OES, calibration standards are prepared daily and diluted using the autosampler. Indium is used as an internal standard for all data calculations. Every 12 measurements, a drift standard (DRIFT) and a quality check standard (QC) are analysed. The DRIFT corresponds to the third calibration standard, while the QC is an independently prepared standard with known concentrations.

Daily determinations of the Limit of Detection (LOD) and Limit of Quantification (LOQ) are performed by measuring a blank sample 10 times after calibration. The LOD is calculated as three times the standard deviation (SD) of the blank signal, and the LOQ is three times the LOD.

## Statistical analyses

For experiment 1, % normality and shell length of oysters were first analysed using a Generalised Linear Model, with batch (3–5 levels) as a factor, TA as a covariate (10 levels), and their interaction (two-way ANCOVA) for each alkaline mineral source. When a significant interaction between batch and TA was observed, separate linear regression analyses were conducted for each batch. Based on a visual inspection of the data, where linear regression appeared suboptimal, alternative models, such as segmented regression, were tested and compared using the Bayesian Information Criterion. The dependent variable (% normal development or shell length) was fit with TA using a nonlinear regression model via

the Marquardt algorithm. The model follows the equations:

$$ \% normality or shell length = \begin{cases} \alpha & \text{if } TA < TP \\ \alpha + \beta (TA - TP) & \text{if } TP \le TA \le TP + \delta \\ \alpha + \beta \delta & \text{if } TA > TP + \delta \end{cases} $$

where α represents the mean percentage of normal development before the TP, which is the TA value at which a marked decline begins, β denotes the rate of change in the percentage of normal development as a function of TA between the TP and the stabilisation phase, and δ is the TA range over which the percentage of normal development stabilises after the TP. This segmented nonlinear model effectively captured key shifts in development trends as TA increases, providing insight into thresholds and responses across treatments. The model was parameterised using the following initial search ranges: α ∈ [85, 100] by 1, β ∈ [-0.1, -0.001] by 0.001, D₀ ∈ [3000, 9000] by 200, δ ∈ [100, 6000] by 100.

For experiment 2, the % normal developed larvae were analysed using a Generalised Linear Mixed Model, with mineral type (olivine or oyster shell), TA (4 levels: ambient, 4000, 6000, 9000 µmol kg⁻¹), and their interaction as fixed effects (two-way ANOVA). Oyster family was used as a random factor to account for biological variability (n = 10). Although shell length was measured at the end of the experiment, it was excluded from statistical analysis and is not presented here as embryos failed to develop properly at higher TA levels.

Element concentrations in the seawater across mineral types (four levels) were analysed using two distinct general linear modeling approaches. The first approach tested the interaction effect between TA (two levels, 5000 and 11 000 µmol kg⁻¹) and mineral (four levels), excluding the control TA, to avoid imbalance in the design since the control was shared across all minerals (two-way ANOVA). The second approach evaluated the effect of TA including the control (three levels) within each mineral type (one-way ANOVA or regression).

Pairwise comparisons were conducted using Tukey's Least Square Mean Difference test for element concentrations in the seawater (experiment 1) and normality percentage (experiment 2). Residual plots were examined to ensure normality and homoscedasticity of residuals. Statistical significance was set at P < 0.05. Statistics and graphing were all conducted using the SAS 9.4 software package (SAS Institute, Cary, NC).

## Results

### Mineral dissolution can impair embryonic development

The initial and unequivocal finding was that the percentage of normal embryonic development decreased in high-TA olivine treatments from 85% to 100% to zero (Figs 1 and 2; Tables S2, S3). Further statistical analyses indicated that a segmented regression model provided a better fit to the data, thus revealing TPs (TA thresholds) beyond which even relatively moderate increases in TA (+600 µmol kg⁻¹) led to substantial declines in normal development (Fig. 2; Tables S4 and S5). TPs varied between 2943 and 6400 µmol kg⁻¹, with nearly complete developmental failure observed between 5715 and 9422 µmol kg⁻¹ (Table S4, see TP + δ). High inter-batch variability was evident at intermediate TA levels (3500–6500 µmol kg⁻¹, Fig. 2). Using these segmented models, we
<figure><img src="image_5.png" /><figcaption>**Figure 2** Influence of alkaline material dissolution on oyster development. Percentage of normal development of *M. gigas* embryos as a function of TA levels, where the lowest value corresponds to ambient seawater and higher values result from alkalinity additions through mineral dissolution of olivine, limestone, oyster shell or addition of sodium carbonate. Segmented regression models are plotted where significant. Dotted vertical lines indicate TPs.</figcaption></figure>

estimated the TA levels at which 50% of oyster embryos exhibited developmental failure to be 7710, 4592, and 5256 µmol kg⁻¹ for batches 3, 4, and 5, respectively (mean ± SD: 5853 ± 1642 µmol kg⁻¹).

In contrast, the addition of TA through limestone dissolution had no consistent effect on embryo development. Although one batch (batch 4) exhibited reduced normality (from 0% to 58%, mean 25.6%), this was not consistently related to TA (Fig. 2, Tables S2, S3). For ground oyster shell, a negative effect on embryo development was observed in only one out of five oyster batches. In this specific batch (batch 5), normality remained consistently high (>93%) up to 6906 µmol kg⁻¹, after which it declined sharply. Note that as observed for limestone, the batch 4 exhibited reduced normality (from 0% to 92%, mean 72.6%) unrelated to TA (Fig. 2, Table S3), probably reflecting a batch specific problem.

Finally, the TA addition through sodium carbonate dissolution showed no negative effect on embryo development. Normality remained nearly constant (>86%) across all TA levels, with minimal inter-batch variability. Two TA levels from batch 5 exhibited anonymously low normality in embryo development, likely representing statistical outliers.

In addition to normality, we assessed shell length as a complementary metric to evaluate the effects of TA. Overall, oyster shell length decreased linearly with increasing TA following olivine dissolution, regardless of the oyster batch (Fig. 3; Table S6). However, a significant relationship between shell length and TA was observed in only one batch out of three. Importantly, shell length measurements were unavailable in cases of abnormal development, where no shell formed. This was particularly evident for

<figure><img src="image_6.png" /><figcaption>**Figure 3** Influence of alkaline material dissolution on shell length of oyster. Shell length of *M. gigas* larvae as a function of TA, where the lowest value corresponds to ambient seawater and higher values result from alkalinity additions through mineral dissolution of olivine, limestone, oyster shell, or addition of sodium carbonate. Regression models are plotted where significant.</figcaption></figure>

batches 4 and 5 at TA levels exceeding 7000 µmol kg⁻¹, which limited the statistical power of the regression analyses and likely contributed to the lack of significance.

The addition of TA through the dissolution of limestone had virtually no effect on oyster shell length, except for batch 4 for which shell length decreased with increasing TA (Fig. 3, Tables S6 and S7). This result aligns with the reduced normality observed regardless of TA levels. Shells from batch 4 were noticeably smaller (mean ± SD: 54.5 ± 1.7 µm) compared to other batches (means ± SD: 65.2 ± 0.6 µm for batch 3 and 64.9 ± 1.3 µm for batch 5).

The addition of TA through the dissolution of ground oyster shell negatively affected the shell length of oyster larvae in two out of five batches. This included batch 1 and 5, which were also impacted in terms of normality of development. Additionally, it is noteworthy that the shell length of batch 4 (mean ± SD: 57.1 ± 3.1 µm) was smaller compared to that of batches 3 and 5 (means ± SD: 64.5 µm ± 1.4 µm for batch 3 and 61.9 µm ± 3.1 for batch 5), a pattern similar to what was previously observed with limestone.

Finally, the addition of TA through the dissolution of sodium carbonate had no effect on shell length of oysters (Fig. 3, Tables S6 and S7). Consistent with the observations for normality, the same two TA levels from batch 5 exhibited abnormally small shells (Fig. 3).

Overall, the response of oyster embryo development to increasing TA varied significantly depending on the mineral. Olivine, and to a lesser extent ground oyster shell, exhibit TA-dependent declines in normal development, whereas limestone and sodium carbonate showed no consistent effects with TA. Inter-batch vari-
<figure><img src="image_7.png" /><figcaption>**Figure 4** Influence of alkaline material dissolution on oyster development. Percentage of normal development of *M. gigas* embryos as a function of TA, where the lowest value corresponds to ambient seawater and higher values result from alkalinity additions through mineral dissolution of olivine or oyster shell. Data represent the means of ten biparental oyster families, with shaded bands showing the 95% confidence intervals. Different letters denote statistically significant differences.</figcaption></figure>

ability is particularly pronounced for olivine and oyster shell, likely reflecting biological variability or differences in responses across genetic families.

To explore this further, we investigated the impact of TA—achieved through the dissolution of olivine or oyster shell—on 10 biparental families of oyster embryos (Fig. 4). We found that normality decreased with increasing TA for both olivine and oyster shell treatments, but the patterns differed between the two minerals. For olivine, normality decreased sharply, with significant reductions at a TA level of 4000 µmol kg⁻¹ (47%, range = 3%–95%) and a more pronounced decrease at higher TA levels—reaching 4% normality at 6000 µmol kg⁻¹ (range = 0%–13%) and 0% at 9000 µmol kg⁻¹ TA (range = 0%–3%). For oyster shell, normality decreased progressively, with significant declines observed at 6000 µmol kg⁻¹ (37%, range = 0%–85%). By 9000 µmol kg⁻¹, normality dropped to 6% (range = 0%–21%). Family-level variation was high at intermediate TA, suggesting that genetic or parental background influences tolerance thresholds; however, this variability decreased markedly at higher TA, indicating that elevated alkalinity may override intrinsic differences among lineages.

## Mineral dissolution alters elemental composition of the seawater

We analysed the elemental composition of the seawater at the end of the embryonic assays. The samples consisted of ambient seawater and seawater adjusted to two TA levels (5000 and 11 000 µmol kg⁻¹) through dissolution of olivine, limestone and ground oyster shell, and sodium carbonate (Fig. 5).

Notably, increasing TA from ambient levels to 11 000 µmol kg⁻¹ through the dissolution of olivine led to significant increases in Co, Mn, and Ni concentrations—by factors of 123, 57, and 194, reaching average concentrations of 25, 258, and 791 ppb, respectively (Fig. 5, Tables S8, S9). The positive effect of TA on Ni was specific to olivine, as no other mineral or chemical caused any increase in Ni. By contrast, increasing TA through the dissolution of limestone elevated Co and Mn concentrations, although to a much lesser extent than olivine (Fig. 5).

An interesting observation with increasing TA through limestone dissolution was a rise in Cd levels from undetected to 1.24 ppm.

Increasing TA from ambient levels to 11 000 µmol kg⁻¹ through olivine dissolution significantly elevated Mg and Si concentrations—by factors of 1.072 and 18, reaching average concentrations of 1318 and 5.9 ppm, respectively (Fig. 5, Tables S8 and S9). The increase in Si was unique to olivine. In contrast, increasing TA through the dissolution of oyster shell also raised Mg levels, though to a much lesser extent than olivine (Fig. 5). Notably, increasing TA through limestone and oyster shell dissolution significantly elevated Ca concentrations by factors of 1.40 and 1.46, respectively. Additionally, Ca concentrations were 5.6% higher with oyster shell dissolution compared to limestone (P = 0.022, Table S8).

In contrast, olivine dissolution had minimal effect on Ca levels, with only a slight increase by a factor of 1.02 as TA rose from ambient levels to 11 000 µmol kg⁻¹.

It is noteworthy that raising TA from ambient levels to 11 000 µmol kg⁻¹ through sodium carbonate dissolution had minimal impact on elemental concentrations in seawater, confirming that it is a pure mineral reference. The only detected effect was for Mn, which doubled to an average of 16.7 ppb (Fig. 5, Tables S8 and S9).

## Discussion

In this study, we demonstrate that dissolution of alkaline materials can have an embryotoxic effect on oysters, with varying impacts depending on the type of alkaline compound used. Among the tested compounds, olivine exhibited the most pronounced negative effects, followed by ground oyster shells and limestone, while sodium carbonate had no detectable negative impacts.

### Olivine dissolution induces embryo toxicity likely through trace metal contamination

The fact that the addition of sodium carbonate had no detectable negative impact suggests that elevating TA up to 11 000 µmol kg⁻¹ alone is not sufficient to induce toxicity. This result also suggests that the observed negative effects of olivine and oyster shell are likely due to compound-specific properties rather than alkalinity itself. While sodium carbonate dissolution primarily increases TA without introducing contaminants, olivine dissolution indeed releases other substances, such as Si, Mg, and various trace metals like Fe, Ni, Co, and Cr (Montserrat et al. 2017, Flipkens et al. 2021, Hutchins et al. 2023, Geerts et al. 2025). Among these, Ni is of particular concern due to its known toxicity to marine invertebrates (Calabrese and Nelson 1974, Calabrese et al. 1977, Martin et al. 1981, Markich 2021). Ni substitutes the divalent cations in olivine (Mg²⁺, Fe²⁺), and its content ranges from 0.2 to 1.2 mol % (Montserrat et al. 2017).

Our findings are consistent with existing studies on the embryotoxicity of Ni in *M. gigas* conducted under ambient alkalinity. Previous research reported 50% mortality at 349 ppb Ni (Martin et al. 1981). In our experiment, we observed approximately 50% abnormal D-larvae at a TA of ~5853 µmol kg⁻¹, corresponding to an
<figure><img src="image_8.png" /><figcaption>**Figure 5** Influence of alkaline material dissolution on elemental concentration in the seawater. Elements are presented as a function of TA levels, where the lowest value corresponds to ambient seawater and higher values result from alkalinity additions through mineral dissolution of olivine, limestone, oyster shell, or addition of sodium carbonate. Linear regression trends are plotted regardless of their statistical significance. The shaded bands around the regression lines represent the 95% confidence intervals.</figcaption></figure>

estimated Ni concentration of ~334 ppb (based on the observed relationship between TA and Ni levels)—a very similar concentration. This suggests that the developmental effects observed in our olivine treatments are likely driven primarily by Ni contamination rather than by the increase in alkalinity itself. Although our study was not designed to test whether alkalinity modulates Ni toxicity, the similarity in thresholds points to trace metal exposure as the dominant driver of adverse developmental effects under these conditions. This highlights the need for careful assessment of trace metal release when considering olivine as a strategy for alkalinity enhancement in marine environments.

In addition to Ni, olivine dissolution also led to a concurrent increase in Co and Mn concentrations, two metals that are less toxic

than Ni for bivalves (Markich 2021). In *M. gigas* embryos, the concentration at which 50% of individuals exhibited adverse effects (EC50) was recently determined to be 71.7 ppb for Co and 2700 ppb for Mn (Markich 2021). Since the maximum Co concentration observed in our study (25.0 ± 2.8 ppb) was substantially lower, the risk of toxicity appears lower. Similarly, the highest Mn concentration recorded in our study (258.3 ± 29.2 ppb) was an order of magnitude lower than its reported EC50, suggesting a low likelihood of Mn toxicity.

Finally, although olivine used in this study is primarily composed of Mg-rich forsterite, it contains appreciable amounts of Fe and has the potential to release Cr (Flipkens et al. 2023a, Montserrat et al. 2017, Flipkens et al. 2021). The limited accumulation
of Fe and Cr in the seawater is likely explained by the low solu-
bility of these elements during olivine dissolution. In particular,
Fe²⁺ released during weathering likely underwent rapid oxidation
and precipitated as iron oxyhydroxides (e.g. FeOOH) or remained
bound to partially dissolved minerals, while Cr, mostly present as
chromite (FeCr₂O₄) in olivine, is known to be highly insoluble un-
der oxic conditions. These processes would limit the bioavailabil-
ity of Fe and Cr in solution, despite their presence in the mineral
(Flipkens et al. 2023a, Montserrat et al. 2017, Flipkens et al. 2021).
Overall, seawater concentration of both metals remained low and
well below toxicity thresholds for bivalve embryos (Markich 2021).

Olivine dissolution also led to an increase in Mg and Si, consis-
tent with their natural abundance in its mineral composition. Mag-
nesium is a major component of seawater, naturally ranging from
~1200 to 1400 ppm (Riley and Tongudai 1967), and plays a vital
role in enzyme function, ion regulation, and shell formation. Bi-
valve embryos and larvae are generally well adapted to these lev-
els, and the increase observed in our study due to olivine dissolu-
tion (from 1229 to 1317 ppm) remains within the natural range of
variation, making harmful effects unlikely.

Silicon concentrations, present in solution as orthosilicic acid (H₄SiO₄), increased from 0.32 to 5.92 ppm following olivine dissolution. This reflects that the dissolution of one mole of olivine leads to an increase in silicic acid by one mole. In seawater, Si levels vary significantly across different oceanic regions and depths. In surface waters, Si concentrations are typically low, often below 2 µM (0.19 ppm), due to biological uptake by diatoms and other siliceous organisms. However, the global average Si concentration in seawater is estimated to be around 70 µM (6.73 ppm as silicic acid) (Tréguer et al. 1995). Given that the Si levels observed in our study fall within this natural variability, they are unlikely to pose any toxic effects on oyster embryos, but may be beneficial to diatoms in areas where this nutrient is limiting (Bach et al. 2019).

Overall, the elemental composition analysis suggests that Ni is
likely the primary driver of embryo toxicity resulting from olivine
dissolution, with other contaminants such as Co and Mn either be-
ing less toxic or present in lower concentrations. However, we can-
not exclude the possibility of a cocktail effect, where the combined
influence of multiple contaminants and more broadly the expo-
some may contribute to toxicity (Faust et al. 2001).

Calcium carbonate dissolution can induce
embryo toxicity without a known
mechanism

We observed that embryos exposed to limestone and ground oyster shell, two sources of calcium carbonate, exhibited different responses. Ground oyster shells had a more pronounced effect on developmental normality and shell length compared to limestone. Similar to olivine, limestone dissolution can introduce impurities from its mineral source. However, the composition of contaminants may differ between sources of calcium carbonate, potentially explaining the greater embryotoxicity observed with oyster shell. Nevertheless, this difference is not apparent based on the measured elemental composition of the resulting seawater.

For instance, the only components that differed between sea-
water enriched with limestone and ground oyster shell were Ca
and Cd. The Ca concentration increased at a slightly higher rate
following dissolution from ground oyster shell compared to lime-

stone. However, this is unlikely to explain the greater toxicity, as
the increase was only 5.6% above the high background concen-
tration of Ca. In contrast, Cd levels rose in seawater enriched with
limestone but remained undetectable in seawater enriched with
ground oyster shells. This suggests that other factors, such as trace
contaminants unique to oyster shell-derived limestone, may be re-
sponsible for the observed differences in embryotoxicity.

It is noteworthy that Although Cd levels increased slightly in the
limestone treatment (up to 1.24 ppb), this concentration remains
well below the reported EC50 for *M. gigas* embryos (285 ppb, Mar-
kich 2021), and Cd is generally less toxic to bivalves than other met-
als such as Ni, which helps explain the limited developmental ef-
fects observed despite the increasing Cd concentration.

Overall, this highlights the need for further investigation into
the adverse effects associated with oyster shell while considering
the possible influence of other molecules from the oyster shell ma-
trix, the industrial grinding and transformation processes on its
toxicity.

Implication for OAE deployment and
limitations of the study

Our findings show that alkalinity addition itself is not inherently
harmful to oyster embryos, even at very high TA levels. However,
depending on the mineral source, the addition of TA through min-
eral dissolution can have a significant impact, highlighting the
need for careful material selection in OAE strategies. In our study,
carbonate-based additions were considerably less problematic
than olivine. Olivine dissolution adversely affected oyster embry-
onic development, coinciding with Ni leaching into seawater, un-
derscoring the importance of accounting for trace element release
when evaluating ecological risks. These results suggest that ap-
proaches which increase TA without adding minerals, such as elec-
trochemical methods that remove acidity from seawater to gen-
erate a basic effluent, may be preferable, as they raise alkalinity
without introducing contaminants and are therefore less likely to
cause toxicity to marine organisms.

Field-scale applications such as beach nourishment or direct
mineral dispersion (Flipkens et al. 2023a, Hangx and Spiers 2009,
Köhler et al. 2013, Eisaman et al. 2023), are expected to gener-
ate relatively modest TA increases, typically tens to a few hundred
µmol kg⁻¹, except in confined porewater environments where lo-
cal alkalinity can be substantially higher (Montserrat et al. 2017,
Fuhr et al. 2022, Fuhr et al. 2024, Guo et al. 2025). Since devel-
opmental abnormalities in oyster embryos were only observed
above ~3000 µmol kg⁻¹, such field-scale applications are un-
likely to cause acute toxicity to pelagic early life stages. How-
ever, benthic organisms in direct contact with dissolution sub-
strates may be more vulnerable (Flipkens et al. 2023b, Flipkens et
al. 2024, Jankowska et al. 2024). Conversely, engineered acceler-
ated weathering processes operating at high pCO₂ (Rau et al. 2007,
Eisaman et al. 2023) could generate TA-rich effluents with elevated
metal concentration near discharge points, posing localised risks
if mixing is insufficient.

Our experimental approach, in which seawater was acidified
to dissolve minerals and subsequently re-equilibrated to ambi-
ent pH, most closely represents engineered dissolution systems
where alkalinity-enhanced water is equilibrated prior to release.
This design intentionally minimised changes in proton concentra-
tion in order to isolate the effects of TA alone. Previous work, in-
cluding our sodium carbonate treatment, indicates that modest
increases in pCO₂ do not affect early bivalve development, sup-
porting this rationale (Gazeau et al. 2011, Waldbusser et al. 2015).
However, CO₂ equilibrium state can influence trace metal specia-
tion and bioavailability; therefore, under scenarios in which final
pH and pCO₂ differ, metal toxicity outcomes could vary (Ho et al.
1999, Nys et al. 2016, Mebane et al. 2020), and these interactions
warrant further investigation.

The persistence of elevated TA anomalies in the environment
is expected to depend strongly on hydrodynamics, including mix-
ing, residence time, and release conditions. A recent modeling
study (Bach et al. submitted) suggests that large TA perturbations
(> ~1000 µmol kg⁻¹) are likely to persist only seconds to min-
utes, whereas smaller anomalies (< ~100 µmol kg⁻¹) may persist
from hours to years depending on local conditions and proxim-
ity to release sites. Our 48-h exposure period may therefore repre-
sent a conservative upper bound, providing insight into conditions
where organisms either remain within, or repeatedly encounter,
elevated-alkalinity plumes.

Given the absence of empirical constraints on the magnitude
and duration of TA anomalies expected during real-world OAE im-
plementation, the exposure conditions applied here should be
considered exploratory rather than representative. Consequently,
the value of our findings lies in constraining the biological toler-
ance of early oyster development to elevated TA under conditions
where pH remains environmentally benign. These findings can in-
form the safe operational ranges for alkalinity enhancement.

Acknowledgments

We are particularly grateful to Moussa Diagne for phytoplankton
production, Matthias Huber for zootechnical support, Jaqueline
Legrand and Carole Di Poi for help with image analyses, and An-
dreaz Dupoué and Jeremy Le Luyer for providing oyster families.
We are grateful to four anonymous reviewers for their contribu-
tions to improving this manuscript.

Author contributions

Fabrice Pernet (Conceptualization [lead], Data curation [equal], Formal Analysis [lead], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Validation [equal], Visualization [equal], Writing – original draft [lead], Writing – review & editing [equal]), Luna Ducoulombier (Data curation [equal], Formal Analysis [equal], Investigation [equal], Methodology [equal], Visualization [equal]), Hugo Koechlin (Conceptualization [equal], Data curation [equal], Investigation [equal], Methodology [equal], Project administration [equal], Validation [equal], Writing – review & editing [equal]), Cale A Miller (Conceptualization [equal], Data curation [equal], Formal Analysis [equal], Funding acquisition [lead], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Validation [equal], Visualization [equal], Writing – review & editing [lead]).

**Supplementary material**

Supplementary material is available at *ICES Journal of Marine Science* online.

Conflicts of interest

None declared.

Funding

This paper is funded by the European Maritime and Fisheries Fund (MITIC project) Fondation Prince Albert II De Monaco (REPAIRS project).

Data and materials availability

All data are available in the main text or the supplementary mate-
rials.

References

Albright R, Caldeira L, Hosfelt J et al. Reversal of ocean acidification enhances net coral reef calcification. *Nature* 2016;531:362–5. https://doi.org/10.1038/nature17155

Bach LT, Gill SJ, Rickaby REM et al. CO2 Removal with enhanced weathering and ocean alkalinity enhancement: potential risks and co-benefits for marine pelagic ecosystems. *Front Clim* 2019;1:1–21. https://doi.org/10.3389/fclim.2019.00007

Bach LT, Tyka MD, Wang B *et al*. Lethal by design? Guiding environmental assessments of ocean alkalinity enhancement toward realistic contextualization of the alkalinity perturbation. submitted https://doi.org/10.7021/cdrxiv.2025457.v1

Barton A, Waldbusser GG, Feely RA et al. Impacts of coastal acidification on the Pacific Northwest shellfish industry and adaptation strategies implemented in response. *Oceanography* 2015;28:146–59. https://doi.org/10.5670/oceanog.2015.38

Calabrese A, MacInnes JR, Nelson DA et al. Survival and growth of bivalve larvae under heavy-metal stress. *Mar Biol* 1977;41:179–84. https://doi.org/10.1007/BF00394024

Calabrese A, Nelson DA. Inhibition of embryonic development of the hard clam. *Mercenaria mercenaria*, by heavy metals. *Bull Environ Contam Toxicol* 1974;11:92–7. https://doi.org/10.1007/BF01685034

Caldeira K, Rau GH. Accelerating carbonate dissolution to se-
quester carbon dioxide in the ocean: geochemical implications.
*Geophys Res Lett* 2000;27:225–8. https://doi.org/10.1029/1999
GL002364

Cripps G, Widdicombe S, Spicer JI et al. Biological impacts of enhanced alkalinity in Carcinus maenas. *Mar Poll Bull* 2013;71:190–8. https://doi.org/10.1016/j.marpolbul.2013.03.015

Cyronak T, Schulz KG, Jokiel PL. The Omega myth: what really
drives lower calcification rates in an acidifying ocean. *ICES J
Mar Sci* 2016;73:558–62. https://doi.org/10.1093/icesjms/fsv0
75

Dickson AG, Sabine CL, Christian JR. Guide to best practices for
ocean CO2 measurements. North Pacific Marine Science Orga-
nization, 2007. https://www.nodc.noaa.gov/ocads/oceans/H andbook_2007.html (10 February 2026, date last accessed).

Di Poi C, Evariste L, Serpentini A et al. Toxicity of five antidepres- sant drugs on embryo-larval development and metamorpho- sis success in the Pacific oyster, Crassostrea gigas. *Environ Sci Pollut Res* 2014; **21**:13302–14. https://doi.org/10.1007/s11356 -013-2211-y

Doney SC, Busch DS, Cooley SR et al. The impacts of ocean acidification on marine ecosystems and reliant Human communities. *Annu Rev Env Res* 2020; **45**:83–112. https://doi.org/10.114 6/annurev-environ-012320-083019

Dupont S, Metian M. General considerations for experimental research on ocean alkalinity enhancement. Guide to Best Practices in Ocean Alkalinity Enhancement Research 2023;2- oae2023:4. https://doi.org/10.5194/sp-2-oae2023-4-2023 (10 February 2026, date last accessed).

Eisaman MD, Geilert S, Renforth P et al. Assessing the technical aspects of ocean-alkalinity-enhancement approaches. Guide to Best Practices in Ocean Alkalinity Enhancement Research 2023;2-oae2023:3. https://doi.org/10.5194/sp-2-oae2023-3-2 023 (10 February 2026, date last accessed).

Faucher G, Haunost M, Paul AJ et al. Growth response of Emilia- nia huxleyi to ocean alkalinity enhancement. *Biogeosciences* 2025; **22**:405–15. https://doi.org/10.5194/bg-22-405-2025

Faust M, Altenburger R, Backhaus T et al. Predicting the joint algal toxicity of multi-component s-triazine mixtures at low- effect concentrations of individual toxicants. *Aquatic Toxicol* 2001; **56**:13–32. https://doi.org/10.1016/S0166-445X(01)00187 -4

Ferderer A, Chase Z, Kennedy F et al. Assessing the influence of ocean alkalinity enhancement on a coastal phytoplankton community. *Biogeosciences* 2022; **19**:5375–99. https://doi.org/ 10.5194/bg-19-5375-2022

Flipkens G, Blust R, Town RM. Deriving nickel (Ni(II)) and chromium (Cr(III)) based environmentally safe olivine guidelines for coastal enhanced silicate weathering. *Environ Sci Technol* 2021; **55**:12362–71. https://doi.org/10.1021/acs.est.1c02974

Flipkens G, Dujardin V, Salden J et al. Olivine avoidance behaviour by marine gastropods (Littorina littorea L.) and amphipods (Gammarus locusta L.) within the context of ocean alkalinity enhancement. *Ecotoxicol Environ Saf* 2024; **270**:115840. https: //doi.org/10.1016/j.ecoenv.2023.115840

Flipkens G, Fuhr M, Fiers G et al. Enhanced olivine dissolution in seawater through continuous grain collisions. *Geochim Cos- mochim Acta* 2023a; **359**:84–99. https://doi.org/10.1016/j.gca. 2023.09.002

Flipkens G, Horoba K, Bostyn K et al. Acute bioaccumulation and chronic toxicity of olivine in the marine amphipod *Gammarus locusta*. *Aquatic Toxicol* 2023b; **262**:106662. https://doi.org/10.1016/j.aquatox.2023.106662

Fuhr M, Geilert S, Schmidt M et al. Kinetics of olivine weathering in seawater: an experimental study. *Front Clim* 2022; **4**:1–20. http s://doi.org/10.3389/fclim.2022.831587

Fuhr M, Wallmann K, Dale AW et al. Alkaline mineral addition to anoxic to hypoxic Baltic Sea sediments as a potentially efficient CO2-removal technique. *Front Clim* 2024; **6**:1–16. https://doi.or g/10.3389/fclim.2024.1338556

Gately JA, Kim SM, Jin B et al. Coccolithophores and diatoms re- silient to ocean alkalinity enhancement: a glimpse of hope? *Sci Adv* 2023; **9**:eadg6066. https://doi.org/10.1126/sciadv.adg6066

Gattuso J-P, Magnan AK, Bopp L et al. Ocean solutions to ad- dress climate change and its effects on marine ecosystems. *Front Mar Sci* 2018; **5**:1–18. https://doi.org/10.3389/fmars.2018 .00337

Gazeau F, Gattuso J-P, Greaves M et al. Effect of carbonate chem- istry alteration on the early embryonic development of the Pa- cific oyster (Crassostrea gigas). *PLoS One* 2011; **6**:e23010. https: //doi.org/10.1371/journal.pone.0023010

Geerts LJJ, Hylén A, Meysman FJR. Review and syntheses: ocean alkalinity enhancement and carbon dioxide removal through marine enhanced rock weathering using olivine. *Biogeо- sciences* 2025; **22**:355–84. https://doi.org/10.5194/bg-22-355- 2025

Guo JA, Strzepek RF, Swadling KM et al. Influence of ocean alkalin- ity enhancement with olivine or steel slag on a coastal plank- ton community in Tasmania. *Biogeosciences* 2024; **21**:2335–54. https://doi.org/10.5194/bg-21-2335-2024

Guo JA, Strzepek RF, Yuan Z et al. Effects of ocean alkalinity en- hancement on plankton in the Equatorial Pacific. *Communica- tions Earth & Environment* 2025; **6**:270. https://doi.org/10.1038/ s43247-025-02248-7

Hangx SJT, Spiers CJ. Coastal spreading of olivine to control atmo- spheric CO2 concentrations: a critical analysis of viability. *Int J Greenhouse Gas Control* 2009; **3**:757–67. https://doi.org/10.101 6/j.ijggc.2009.07.001

Harvey LDD. Mitigating the atmospheric CO2 increase and ocean acidification by adding limestone powder to upwelling regions. *J Geophys Res Oceans* 2008; **113**:1–21. https://doi.org/10.1029/ 2007JC004373

His E, Beiras R, Seaman MNL. The assessment of marine pollution—bioassays with bivalve embryos and larvae. In: Southward AJ, Tyler PA, Young CM (eds.), *Advances in Marine Biology*. New York, NY, USA: Academic Press, 1999, 1–178.

Ho KT, Kuhn A, Pelletier MC et al. pH dependent toxicity of five metals to three marine organisms. *Environ Toxi- col* 1999; **14**:235–40. https://doi.org/10.1002/(SICI)1522-7278 (199905)14:2(235::AID-TOX4)3.0.CO;2-J

Hutchins DA, Fu FX, Yang SC et al. Responses of globally important phytoplankton species to olivine dissolution products and im- plications for carbon dioxide removal via ocean alkalinity en- hancement. *Biogeosciences* 2023; **20**:4669–82. https://doi.org/ 10.5194/bg-20-4669-2023

Iglesias-Rodríguez MD, Rickaby REM, Singh A et al. Laboratory ex- periments in ocean alkalinity enhancement research. *Guide to Best Practices in Ocean Alkalinity Enhancement Research* 2023;2-oae2023:5. https://doi.org/10.5194/sp-2-oae2023-5-2 023

Ilyina T, Wolf-Gladrow D, Munhoven G et al. Assessing the potential of calcium-based artificial ocean alkalinization to mitigate ris- ing atmospheric CO2 and ocean acidification. *Geophys Res Lett* 2013; **40**:5909–14. https://doi.org/10.1002/2013GL057981

Jankowska E, Montserrat F, Romaniello SJ et al. Metal bioaccumu- lation and effects of olivine sand exposure on benthic marine invertebrates. *Chemosphere* 2024; **358**:142195. https://doi.org/ 10.1016/j.chemosphere.2024.142195

Kheshgi HS. Sequestering atmospheric carbon dioxide by increas- ing ocean alkalinity. *Energy* 1995; **20**:915–22. https://doi.org/10.1016/0360-5442(95)00035-F

Köhler P, Abrams JF, Völker C et al. Geoengineering impact of open ocean dissolution of olivine on atmospheric CO2, surface ocean
pH and marine biology. *Env Res Lett* 2013;8:014009. https://doi.org/10.1088/1748-9326/8/1/014009

IPCC. Summary for policymakers. In: Lee H, Romero J (eds.), *Climate Change 2023: Synthesis Report Contribution of Working Groups I, II and III to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change*. Geneva, Switzerland: IPCC, 2023, 1-34.

Mance G. Pollution Threat of Heavy Metals in Aquatic Environments. London, United Kingdom: Springer Science & Business Media, 2012.

Markich SJ. Comparative embryo/larval sensitivity of Australian marine bivalves to ten metals: a disjunct between physiology and phylogeny. *Sci Total Environ* 2021;789:147988. https://doi.org/10.1016/j.scitotenv.2021.147988

Martin M, Osborn KE, Billig P et al. Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae. *Mar Poll Bull* 1981;12:305-8. https://doi.org/10.1016/0025-326X(81)90081-3

Mebane CA, Chowdhury MJ, De Schamphelaere KAC et al. Metal bioavailability models: current status, lessons learned, considerations for regulatory use, and the path forward. *Environ Toxicol Chem* 2020;39:60-84. https://doi.org/10.1002/etc.4560

Melzner F, Gutowska MA, Langenbuch M et al. Physiological basis for high CO2 tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny? *Biogeosciences* 2009;6:2313-31. https://doi.org/10.5194/bg-6-2313-2009

Melzner F, Mark FC, Seibel BA et al. Ocean acidification and coastal marine invertebrates: tracking CO2 effects from seawater to the cell. *Annu Rev Mar Sci* 2020;12:499-523. https://doi.org/10.1146/annurev-marine-010419-010658

Miller CA, Pernet F. The alkalinity generation potential of olivine and oyster shell for laboratory experiments: testing the effects of ocean alkalinity enhancement. *Environmental Research Communications* 2025;7:075025. https://doi.org/10.1088/2515-7620/adf0cd

Montserrat F, Renforth P, Hartmann J et al. Olivine dissolution in seawater: implications for CO2 sequestration through enhanced weathering in coastal environments. *Environ Sci Technol* 2017;51:3960-72. https://doi.org/10.1021/acs.est.6b05942

NASEM. *A Research Strategy for Ocean-based Carbon Dioxide Removal and Sequestration*. Washington, DC: The National Academies Press, 2022

Nys C, Janssen CR, Van Sprang P et al. The effect of pH on chronic aquatic nickel toxicity is dependent on the pH itself: extending the chronic nickel bioavailability models. *Environ Toxicol Chem* 2016;35:1097-106. https://doi.org/10.1002/etc.3232

Oberlander JL, Burke ME, London CA et al. Assessing the impacts of simulated ocean alkalinity enhancement on viability and growth of nearshore species of phytoplankton. *Biogeosciences* 2025;22:499-512. https://doi.org/10.5194 bg-22-499-2025

Oschlies A, Bach LT, Rickaby REM et al. Climate targets, carbon dioxide removal, and the potential role of ocean alkalinity

enhancement. *Guide to Best Practices in Ocean Alkalinity Enhancement Research* 2023;2-oae2023:1. https://doi.org/10.5194/sp-2-oae2023-1-2023

Pernet F, Dupont S, Gattuso J-P et al. Cracking the myth: bivalve farming is not a CO2 sink. *Rev Aquac* 2025;17:e12954. https://doi.org/10.1111/raq.12954

Petton B, Boudry P, Alunno-Bruscia M et al. Factors influencing disease-induced mortality of Pacific oysters *crassostrea gigas*. *Aquaculture Env Interact* 2015;6:205-22. https://doi.org/10.3354/aei00125

Petton S, Pernet F, Le Roy V et al. French coastal network for carbonate system monitoring: the CocoriCO2 dataset. *Earth Syst Sci Data Discuss* 2024;2023:1-34. https://doi.org/10.5194/essd-2023-445

Rau GH, Knauss KG, Langer WH et al. Reducing energy-related CO2 emissions using accelerated weathering of limestone. *Energy* 2007;32:1471-7. https://doi.org/10.1016/j.energy.2006.10.011

Renforth P, Henderson G. Assessing ocean alkalinity for carbon sequestration. *Rev Geophys* 2017;55:636-74. https://doi.org/10.1002/2016rg000533

Riley JP, Tongudai M. The major cation/chlorinity ratios in sea water. *Chem Geol* 1967;2:263-9. https://doi.org/10.1016/0009-2541(67)90026-5

Saravanan P, Saravanan V, Rajeshkannan R et al. Comprehensive review on toxic heavy metals in the aquatic system: sources, identification, treatment strategies, and health risk assessment. *Environ Res* 2024;258:119440. https://doi.org/10.1016/j.envres.2024.119440

Smith SM, Geden O, Gidden MJ et al. *The State of Carbon Dioxide Removal*, 2nd edn. 2024. https://doi.org/10.17605/OSF.IT/F85QJ

Suitner N, Faucher G, Lim C et al. Ocean alkalinity enhancement approaches and the predictability of runaway precipitation processes: results of an experimental study to determine critical alkalinity ranges for safe and sustainable application scenarios. *Biogeosciences* 2024;21:4587-604. https://doi.org/10.5194/bg-21-4587-2024

Tréguer P, Nelson DM, Van Bennekom AJ et al. The silica balance in the World Ocean: a reestimate. *Science* 1995;268:375-9. https://doi.org/10.1126/science.268.5209.375

van der Schatte Olivier A, Jones L, Vay LL et al. A global review of the ecosystem services provided by bivalve aquaculture. *Rev Aquac* 2020;12:3-25. https://doi.org/10.1111/raq.12301

Walbusser GG, Hales B, Langdon CJ et al. Saturation-state sensitivity of marine bivalve larvae to ocean acidification. *Nat Clim Chang* 2015;5:273-80. https://doi.org/10.1038/nclimate2479

Xin X, Goldenberg SU, Taucher J et al. Resilience of phytoplankton and microzooplankton communities under ocean alkalinity enhancement in the oligotrophic ocean. *Environ Sci Technol* 2024;58:20918-30. https://doi.org/10.1021/acs.est.4c09838

Handling editor: Brock C. Woodson