the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 22 Apr 2026
Shell hash promotes growth in Pacific littleneck clams (Leukoma staminea) by altering pore water chemistry
Abstract. Bivalves that build calcium carbonate skeletons are at particular risk from ocean acidification, and mitigation strategies will be needed to keep coastal populations healthy. It can be energetically costly for organisms like clams and mussels to build their shells under low pH conditions, and acidification can lead to shell dissolution. Adding crushed shells (shell hash) to beach sediments, a practice used by some Indigenous communities and aquaculturists, may mitigate the negative effects of ocean acidification by altering the chemistry of the pore fluids they live in. We tested the hypothesis that mixing shell hash into the sediment improves the growth and physiology of infaunal Pacific littleneck clams (Leukoma staminea). Juvenile clams (pre-sexual maturity) were raised for 90 days under four conditions: control seawater with sediment, acidified seawater with sediment, control seawater with sediment plus shell hash, and acidified seawater with sediment plus shell hash. Pore water and overlying seawater were sampled three times a week for pH, alkalinity, salinity, temperature, and dissolved oxygen. Clam shell weight, soft tissue weight, and new shell growth were measured, and mantle tissue RNA was collected for gene sequencing after three months. Our results demonstrate that the addition of shell hash increased the pH of porewater relative to the control, and animals exposed to acidified water plus shell hash grew larger than animals exposed to acidified water alone. Gene expression profiling suggests that animals in acidified seawater with shell hash were largely indistinguishable from animals in non-acidified water. Our experimental results suggest that adding shell hash to sediments alters the chemistry of pore fluids, thus buffering against acidic conditions that can negatively affect the growth of economically and culturally important shellfish like littleneck clams.
- Preprint
(2408 KB) - Metadata XML
-
Supplement
(810 KB) - BibTeX
- EndNote
Status: open (until 03 Jun 2026)
- RC1: 'Comment on egusphere-2026-1930', Lennart de Nooijer, 12 May 2026 reply
-
RC2: 'Comment on egusphere-2026-1930', Fabrice Pernet, 31 May 2026
reply
In this paper, the authors evaluated the impact of adding shell hash to sediment on the pH and alkalinity of pore fluids, as well as on the growth of Pacific littleneck clams. The clams were raised in four experimental conditions for 90 days: a control condition; an acidified condition; a control condition with shell hash; and an acidified condition with shell hash. The pH, alkalinity and saturation state of the pore water and the overlying seawater were analyzed throughout the experiment. The weights of the clam shells and soft tissues were measured, and mantle tissue RNA was collected for gene sequencing at the end of the experiment. The main finding was that adding shell hash increased the pH of the pore water, and that clams exposed to acidified water plus shell hash grew faster than those exposed to acidified water alone.
The paper is interesting and well-written, providing an adaptation strategy to mitigate the effects of ocean acidification on bivalve fisheries and aquaculture.
That said, I have some major comments, mainly regarding the experimental design and the statistics, that prevent me from accepting the paper for publication.
- Lack of replication for the pH treatment
The experimental design is very limited. The structure is as follows:
- 1 ambient-pH header tank supplying 4 buckets
- 1 acidified-pH header tank supplying 4 buckets
The problem here is that pH and header tank are completely confounded. Any difference between ambient and acidified treatments could be due to pH itself, or some other/unmeasured characteristic of the header tank (microbial community, chemistry, etc.). Because there is only one header tank per pH level, there is no way to separate these effects statistically. This is particularly concerning because the seawater in the header tanks was replaced every three weeks (line 153), which may have allowed water quality to evolve differently between tanks and deteriorate independently of pH, thereby confounding the interpretation of treatment effects.
Strictly speaking, substrate effect is testable, but pH effect is not formally testable because pH has only one experimental unit (header tank) per level. The pH × substrate interaction is also problematic because pH lacks replication at the level at which it was applied. Overall, shell ash was replicated among buckets and its effect can be evaluated. In contrast, pH was manipulated at the header-tank level with only one header tank per treatment, so pH effects and pH × substrate interactions should be interpreted as treatment-level patterns rather than formally replicated tests.
See (Hurlbert 1984) for a full discussion and (Riebesell et al. 2011) for application to ocean acidification experiments (chapter 4).
- Low replication of the shell ash treatment
Another important limitation of the experiment is that there are only 2 buckets per treatment combination. So the effective replication for testing pH and substrate effects is low. The confidence intervals and p-values should be interpreted cautiously because they are based on only 8 experimental units.
- The statistical models used are unclear and probably unappropriated
Line 253. The statistical approach does not appear to reflect the hierarchical structure of the experimental design. The authors compared several ANOVA models, including additive and interaction models with tank ID treated as a blocking factor, and selected the preferred model based on AIC. However, tank is not a blocking factor but rather the experimental unit to which treatments were applied. Individual clams within a tank are therefore not independent observations.
Given the nested structure of the data (clams within tanks), the analysis should be based on a general linear mixed-effects model (GLMM), with pH and shell-ash treatment specified as fixed effects and tank included as a random effect to account for the non-independence of observations within tanks. Model selection among different ANOVA formulations does not address this issue and may lead to pseudoreplication. Line 260: For whatever reason, the variable 'new growth' was analyzed using GLMM, which seems appropriate.
More broadly, the statistical analyses are not presented consistently across response variables, making it difficult to evaluate the strength of the evidence and compare results among parameters. In addition, the manuscript does not provide statistical analyses for the carbonate chemistry parameters measured in the overlying water, a summary table for carbonate chemistry parameters in pore water, or the full test statistics associated with the growth responses.
I recommend that the authors provide a comprehensive summary table reporting the statistical results for all measured variables. At a minimum, this table should include the model used, sources of variation, numerator and denominator degrees of freedom, effect estimates and test statistics (F-values and p-values). Such a table would greatly improve transparency, facilitate interpretation of the results, and allow readers to assess the consistency of treatment effects across response variables.
In the same vein, the statistical treatment of the carbonate chemistry parameters could be improved. Rather than conducting separate analyses for pore water and overlying water, the authors could consider fitting a single model including water type as an additional factor. Such an approach would allow formal testing of differences between water compartments while accounting for the hierarchical structure of the experiment. Depending on the sampling design, water type could be treated as a repeated measure or as a factor nested within tank.
In addition, it is unclear how temporal replication was handled in the analyses. If measurements collected at different sampling dates were treated as independent replicates, this would constitute pseudoreplication because observations through time within the same tank are not independent. Instead, sampling date should be incorporated explicitly into the model, for example as a repeated-measures factor (or fixed effect), with an appropriate covariance structure to account for temporal dependence. The authors should clarify how temporal replication was handled and revise the analyses if repeated observations were treated as independent replicates.
Finally, I do not understand how to interpret Table 4. Also, I cannot figure out which treatments differed from which. Could you please add different letters to indicate significant differences directly in Figure 4?
- Growth parameters and husbandry conditions are difficult to appreciate
As a bivalve biologist, I was struck by the small increments in shell size observed after 90 days at a temperature of 15°C. Is this normal for this species? I would appreciate a discussion about this specific point. How does this growth compare to what happens under natural field conditions?
- Discussion needs further development
The discussion is far too short. I would very much appreciate some thoughts on the potential of using shell ash to cope with ocean acidification. Do you think this is feasible? Is it scalable? Would it be acceptable to growers and fishermen?
Furthermore, given the limitations of the experimental design, I would be grateful for a statement on this in the discussion.
- Few other minor comments
- Please respect the following variable order in the manuscript text, figures and tables: pH, TA and omega
- Figure 3 plots pHtot in pore waters as a function of time for each treatment. This is the only variable presented with such detail. Why is that? Please consider doing the same for other carbonate chemistry parameters, at least in a supplementary file. If not, please provide a justification. It is likely that the TA will increase over time in the shell ash treatment, particularly under OA conditions. In any case, time should be investigated and considered a repeated measure in the statistical model (see point 3).
- Add some description of the shell ashes composition, grain size, …
- F-values in the text are not well reported. The author reported as F(numerator df) while it should be F(numerator df, denominator df).
- References
Hurlbert SH. Pseudoreplication and the design of ecological field experiments. Ecol Monogr 1984; 54: 187–211.
Riebesell U, Fabry VJ, Hansson L, et al. Guide to best practices for ocean acidification research and data reporting. Luxembourg: Office for Official Publications of the European Communities, 2011
Citation: https://doi.org/10.5194/egusphere-2026-1930-RC2
Data sets
Shell images H. L. Kempf and D. A. Gold https://doi.org/10.7910/DVN/DL8BY0
Model code and software
Code for porewater chemistry analysis and RNA-Seq H. L. Kempf and D. A. Gold https://github.com/DavidGoldLab/2025_Leukoma_RNA-Seq
Viewed
| HTML | XML | Total | Supplement | BibTeX | EndNote | |
|---|---|---|---|---|---|---|
| 286 | 70 | 16 | 372 | 19 | 14 | 16 |
- HTML: 286
- PDF: 70
- XML: 16
- Total: 372
- Supplement: 19
- BibTeX: 14
- EndNote: 16
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1
Dear editor,
Â
after carefully reading the manuscript by Kempf and co-workers (egusphere-2026-1930) on the effect of shell hash in mitigating the effects of acidification on growth of a clam species, I recommend publication after moderate revisions. The paper is very well written and all methods are clearly explained. Presentation of the results is very adequate and I have a number of relatively small suggestions for improvement added to the enclosed pdf. Two additional, major issues stand out and need special attention.
First, it should be clear where in the artificial sediment the littleneck clams (L. staminea) live. Pore water chemistry was determined at a depth of 8 cm (Line 172) and compared in the results section with samples taken from the overlying water. Since (carbonate chemistry) parameters are different for these two samples, the sediment likely contains a gradient of conditions. It should be clear where the clams exactly lived (even if it varies between specimens, between conditions or in time) to indicate what conditions they experienced. Now it is implied that they all lived at a depth of 8 cm and that those pore water conditions were the ones experienced by the clams.
More importantly, the Discussion lacks the necessary depth. I suggest the authors extend the discussion by comparing their results with earlier experiments with bivalves under OA-conditions. Is reduced growth under acidified conditions reported before and were responses comparable? Or do they seem to vary between species? What does this mean for natural environments in which the sediment is rich/ poor in carbonates? Is the applied level of acidification representative for a near-future scenario? How fast does the shell hash dissolve? And how does this fit on the growing literature on alkalinity enhancement to mitigate acidification?
I am looking forward to reading a broader discussion on these topics and an evaluation of the results presented here with earlier reports.
Sincerely,
Â
Lennart de Nooijer