Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Curation Notes
  • Problem Description
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

Adult Mytilus trossulus (44–75 mm shell length) were collected in September 2023 (n = 131) and December 2023 (n = 73) from Penn Cove Shellfish Farm in Penn Cove, WA, USA. Once they arrived at UC Davis Bodega Marine Lab, they were acclimated for 10 days in flow-through seawater tanks maintained at approximately 13°C prior to experimentation. The following methods are based on and derived from the protocol described in Ninokawa et al. (2024) and Carlson et al. (2025). Experimental incubations were done in a temperature-controlled room at 13°C using airtight 1 L glass vessels containing approximately 0.86 kg seawater and either a live mussel, a de-fleshed and sterilized shell, or only seawater (blank controls to account for any background changes in seawater chemistry). 

The experiment consisted of three complementary carbonate chemistry manipulation approaches intended to separate the effects of pH, bicarbonate concentration ([HCO3−]), calcium carbonate saturation state (Ω), and calcium concentration ([Ca2+]) on calcification and dissolution rates. To make each treatment, 20 L sumps of filtered seawater were first acidified with Hydrochloric acid (HCl), reducing total alkalinity and shifting dissolved inorganic carbon (DIC) equilibrium toward dissolved CO2. The seawater was then aerated for ~48hrs with Tetra Whisper AP300 Air Pumps to remove excess CO2 through off-gassing. Following aeration, a gradient-based treatment design targeted a continuum of carbonate chemistry conditions, achieved through varying additions of DIC stock solution, HCl, Sodium hydroxide (NaOH), Calcium chloride (CaCl2), or Sodium chloride (NaCl). Carbonate chemistry targets were planned using the R package seacarb.

For the first trial, we assessed how pH and DIC influence calcification processes using a batch-treatment design of nine manipulated carbonate chemistry treatments spanning low-to-high pH (7.1–8.2) and [HCO3−] (878.5–4256.8 µmol/kg) conditions, along with one ambient seawater control treatment. For this portion of the experiment, each treatment consisted of a 20L seawater chemistry batch that was distributed among nine replicate live-mussel incubation vessels. 

A second trial quantified abiotic shell dissolution across a broad calcium carbonate saturation state (aragonite) gradient (0.05–8.24) using de-fleshed mussel shells that had been sterilized by drying at 60°C for approximately 24 h prior to incubation. These shell-only incubations (n = 81) allowed the disaggregation of net calcification from dissolution in order to estimate gross calcification rates (gross calcification = net calcification + dissolution). In contrast to the live-mussel incubations, shell-only incubations isolated abiotic calcium carbonate dissolution from physiological processes such as respiration and ammonium excretion. To increase the magnitude of alkalinity changes associated with shell dissolution, dissolution incubations done in December 2023 and February 2024 were extended to 42–44 h, compared to the 2–4 incubations done in September 2023.  

In the third trial, we independently manipulated calcium concentration to decouple Ω from pH. This experiment used a 2 × 2 factorial design with two target pH conditions (approximately 7.8 and 8.1) and two calcium treatments: ambient Ω (“low Ca2+”, adjusted with NaCl) and elevated Ω (“high Ca2+”, adjusted with CaCl2). Addition of CaCl2 increased Ω without substantially altering pH to differentiate whether calcification responses were more strongly associated with Ω or pH under similar bicarbonate conditions. Each calcium treatment combination contained six replicate live-mussel incubation vessels.

For all experiments, seawater chemistry measurements were collected before and after incubations. Measurements included total alkalinity, pH, dissolved oxygen, salinity, temperature, and ammonia concentration; calcium concentration was additionally measured for calcium-manipulation treatments. Initial and final alkalinity were analyzed using a Metrohm robotitrator. A salicylate spectrophotometric assay measured ammonia concentration to correct alkalinity anomaly calculations for any alkalinity changes caused by ammonium production rather than calcification. 

Net calcification rates were determined using the ammonia-corrected alkalinity anomaly technique and normalized to incubation duration, seawater mass (kg), and tissue dry mass (Gazeau et al 2015). Abiotic shell dissolution was quantified from parallel incubations of cleaned, sterilized shells and added to net calcification rates to determine rate of gross calcification. Data processing was done with R statistical software, version 4.3.3. Carbonate system parameters were calculated using the package seacarb and equilibrium constants from Lueker et al 2000.

* Loaded Mytilus_trossulus_carbonate_decoupling_incubation_data.csv as table res1, using "", "NA", and "nd" as missing values.
* Removed a blank-named column.
* Renamed columns for consistency, including mussel.number to mussel_number, h2o.kg to h2o_kg, date.cat to date_cat, and treatment to sample_type.
* Added scientific_name, assigning Mytilus trossulus where applicable.
* Added the WoRMS identifiers for Mytilus trossulus: AphiaID 140482 and LSID urn:lsid:marinespecies.org:taxname:140482. These values were hard-coded because live API lookups were unavailable.
* Reordered the columns into the final specified order.
* Set column data types, including integers for mussel_number and aphia_id, numbers for measurement fields, and strings for descriptive and categorical fields.
* Added metadata—including descriptions, standard names, and units—for all columns. Units included µmol/kg, unitless, degrees C, hours, and g, as applicable.
* Saved the final data file as 1007055_1_data.csv.

NSF Award Abstract:
This research is exploring the capacity of coastal organisms to cope with alterations in seawater chemistry driven by both freshwater inputs and absorption of carbon dioxide into the world's oceans (ocean acidification). The project focuses on calcification responses and behavioral impairments of shoreline animals under altered seawater chemistry, and forefronts a common mussel species (the California mussel), and a common snail (the black turban snail), each abundant on rocky shores along the west coast of North America. The target species operate as exemplar organisms for characterizing the responses of marine invertebrates more generally. Methods involve experimental decoupling of multiple components of the carbonate system of seawater to isolate drivers that are difficult to separate otherwise. Broader impacts include transfer of scientific information to policy-makers, including legislators, as well as training and skill-set development of future generations of scientists and citizens. One Ph.D. student is supported, as are UC Davis undergraduates conducting mentored research. The project also provides research internships for undergraduates from a local community college (Santa Rosa Junior College), many of whom are from underrepresented groups. The latter project component substantially bolsters an ongoing program at Bodega Marine Laboratory that includes efforts in diversity, equity, and inclusion. Data and interpretations from the project are feeding into an existing educational program that links to local K-12 schools and reaches ~10,000 members of the public each year.

Overall, the research of the project is dissecting drivers of calcification and behavioral disruption in key shoreline invertebrates, across present-day and future carbonate system conditions appropriate to coastal marine environments. Efforts are exploring the extent to which calcification depends on one versus multiple parameters of the seawater carbonate system. In particular, existing conceptual models emphasize the importance of calcium carbonate saturation state (Ω) and/or the ratio of bicarbonate to hydrogen ion concentrations ([HCO3-]/[H+]), and the project is examining these mechanisms as well as the possibility that more than one driver acts simultaneously. It is doing so both in bivalves and in gastropods to test for generality across mollusks. The project is additionally examining whether pH is the only carbonate system factor contributing to known patterns of behavioral impairment in marine invertebrates. Leading explanations for debilitating behaviors induced by ocean acidification involve altered ion channel function, but discussion in the literature continues, and studies that explicitly decouple the carbonate system are necessary.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.