Water record of Crassostrea angulata transgenerational ocean acidification experiment

The basic observed water characteristics (pH, temperature, salinity and total alkalinity (TA)) and calculated water carbonate chemistry in the hatchery located in Zhanjiang, China (21.07°N, 110.74°E). The two-year-old Portuguese oysters Crassostrea angulata were collected from Wuchuan, China (21.37°N, 110.74°E) in 2020. To be specific, about 300 adult oysters were transferred into six black fibreglass tanks (500L each) randomly and each culture tank contained about 50 individuals. Six tanks were independent and completely randomized to be separated into two different pH levels: ambient pH 8.0~8.1 as control (named F0_8.1) and pH 7.4 as ocean acidification (OA) treatment (named F0_7.4) for about one month to produce F1 generation. The F1 larvae were acquired by artificial fertilization of matured F0 generation (F0_8.1 and F0_7.4). Embryos from F0_8.1 were separated into two pH levels: F1_8.1-8.1 and F1_8.1-7.4. Likewise, embryos from F0_7.4 were separated into two pH levels: F1_7.4-8.1 and F1_7.4-7.4. Thus, there were four treatments for F1 generation, and each treatment had three independent 500L tank replicates (A, B, and C). F1 generation continued to be reared to the next year (2021) till most of them reached the first year of gonad maturation to produce F2 generation. The F2 larvae were acquired by artificial fertilization of F1 generation. Embryos from F1_8.1-8.1 were separated into two pH levels: F2_8.1-8.1-8.1 and F2_8.1-8.1-7.4. The embryos from other F1 treatments were under similar processes. There were eight treatments for F2 generation and reared till 2022 to test the transgenerational OA effects on oyster biological responses. 

Low pH was driven by bubbling of CO₂-enriched air mixed with ambient air into filter seawater in the treatment tanks. pH and temperature were monitored daily with pH meter (Mettler Toledo InLab^®, Switzerland) calibrated with NBS standards. Salinity was monitored using a dual-scale refractometer (MingRui, China). The seawater was utterly changed every two days during larval stage and every week for juvenile stage. About 50 mL of seawater was preserved with HgCl₂ for total alkalinity (TA) measurement. The TA was measured using an alkalinity titrator (Mettler Toledo G20S Compact Titrator, Switzerland), and pCO₂, CO₃^2-, saturation states of calcite and aragonite were calculated by CO2SYS program with equilibrium constants K2 and KSO4 (Millero, F. J., Graham, T. B., Huang, F., Bustos-Serrano, H., & Pierrot, D. (2006). Dissociation constants of carbonic acid in seawater as a function of salinity and temperature. Marine Chemistry, 100(1), 80-94. https://doi.org/10.1016/j.marchem.2005.12.001