Experimental setup:

These four species were grown in four pH treatments (~7.4, 7.6, 7.8, and ambient conditions with a pH=8.0) at three temperature treatments (~6, 9, 12° C). There was one tank at each pH for T=6 and 12° C and two tanks at each pH for T=9° C.

Specimens were randomly assigned to tank conditions with roughly equal numbers of each specimen in each tank condition.

Specimens were in the tank conditions for 20.5 weeks before removed with the following exception. Half of the P. magellanicus were removed in April 2022.

Environmental Conditions:

pH and salinity were measured weekly with a YSI probe. pH was controlled within each tank condition by the Apex control system. Tanks with lower pH conditions than ambient were obtained by bubbling CO2 from compressed cylinders into the water of a mixing chamber before leading to each of the three sets of controlled pH tanks.

Temperature was measured with two Hobo Tidbit devices in each tank throughout the experiment. Temperatures were measured every minute throughout the 20.5 weeks. Temperatures were maintained within the tanks using Inkbird controllers and two (500W/800W) heaters per tank.

All pH measurements are on the total pH scale (pHT).

The reported measurements for temperature, salinity, and pHT are measured tank averages.

Specimen measurements:

All study specimens were measured for maximum height, dry (live) weight, and buoyant weight at the start and end of the experiment. For the P. magellanicus removed in April 2022, height, dry (live) weight, buoyant were measured after removal from the tanks. Photos were taken of each specimen at the conclusion of the experiment.

After the conclusion of the experiment, all specimens were dissected, shells were rinsed with DI water and allowed to air dry prior to sampling for boron isotopes.

Shell sampling:

Organic matter was removed with burring bit (Brasseler #835.11.010) and hand milling with a Dremel tool prior to sampling of the shell. 8 mg of shell was sampled (3-4 mg for P. magellanicus) for boron isotopes with the same bit and Dremel tool.

Boron samples were collected on the shells from the portion of the shell that grew from approximately week 2 to week 10 of the experiment (prior to the spring freshening and second calcein stain line in April 2022) for all species except the adult A. islandica. Due to much slower growth in the adult A islandica, the portion of the shell that grew during the entire tank experiment was sampled.

For most shells, one sample was collected from each shell. There were two exceptions: a comparison was done between the two halves of the shell for three juvenile A. islandica (g5, g32, g48) and a comparison of the outer shell layer (both inner and outer layers of the outer shell) and the inner shell layer on two juvenile A. islandica (g40 and g52). The samples selected for these comparisons were randomly chosen.

Sample preparation:

All powdered shell samples were subject to two 15-minute leaches in warm 10% (by volume) H2O2 (80 °C; buffered in NH4OH) and a weak acid leach (0.0005 M HNO3) before powders were dissolved in distilled 0.5 M HNO3. Dissolved samples were centrifuged (1 min at 13k rpm), and if there was any visible undissolved non-carbonate material at the bottom of the microcentrifuge tube the dissolved sample was transferred to a clean vial, rejecting the small undissolved fraction.

The burring bit was used to remove all visible organic matter from the outside of the shells. The leaching in H2O2 was performed to remove the remaining organic matter.

Chemical Processing:

All trace element and boron isotope analyses were performed at the University of Bristol. An aliquot of the dissolved sample was analyzed by Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) using well-characterized, matrix-matched, synthetic standard solutions to give B/Ca and U/Ca ratios. Samples and standards were introduced in 0.5 M HNO3 and an acid wash solution of 0.5 M HNO3 matrix and 0.3 M HF was utilized between samples/standards to aid B wash out.

The remaining sample containing between 4 and 40 ng of B was separated from the carbonate matrix using 20 μl micro-columns containing Amberlite IRA 743 boron-specific anionic exchange resin. The boron separation from the matrix was performed only on the day preceding Multi Collector-ICP-MS (MC-ICP-MS) analysis. All samples, blanks, and standard solutions were introduced to the instrument in a 0.5 M HNO3 and 0.3 M HF acid matrix again to ensure optimal B wash out.

A total of 108 samples were prepared for boron isotope analysis. There were two samples that did not produce useful results due to blocked columns (one adult A. islandica and one juvenile P. magellanicus).

See Thatcher et al., (2026; GCA) and McMahon et al. (2024; PLOS Climate) for full details on the methods.