Projeto de pesquisa

Laboratory Analysis (to be conducted in CEBIMAR)

We have planned to collect around 200 samples that will be analyzed in Aurea Maria Ciotti laboratory at CEBIMAR with the support of the technitian Lúcia. We estimate that these analyses will take about four weeks in total so I plan to work in one (1 month) or two periods (2-3 weeks) between July and September. In parallel to these analyses, I plan to work in the identification of ascidian collected from rhodoliths. These identifications will be done with a stereoscope and a microscope.

We will apply the alkalinity anomaly method to calculate calcification-dissolution rates, assuming that one mole of CaCO₃ precipitation reduces total alkalinity by two moles (Chisholm & Gattuso, 1991). Total alkalinity (TA) will be determined by open-cell potentiometric titration using a Titrino Plus 877, following SOP3 protocols (Dickson et al., 2007), with HCl as titrant (Batch A26). pH (total scale) will be measured using a ROSS Ultra pH/ATC Triode 8107BNUMD electrode calibrated with Tris–HCl buffer from Dickson Lab (Batch 22).

We will calculate carbonate system parameters (DIC, pCO₂, HCO₃⁻, CO₃²⁻, and Ωarag) using the Excel-based CO2SYS program (Pierrot et al., 2006), with constants from Mehrbach et al. (1973) refitted by Dickson & Millero (1987), and parameters from Lueker et al. (2000), Dickson (1990), and Lee et al. (2000). Certified Reference Material (CRM, Batch 139) from the Dickson Lab will be used to validate TA measurements, ensuring analytical errors 0.5% (±10 µmol kg⁻¹ SW).

Experimental Deployment
We will conduct in situ experiments using custom-made acrylic incubation chambers, following the standardized methodology described by Gómez et al. (2023). Photosynthetically Active Radiation (PAR) will be recorded using an Odyssey® underwater irradiance sensor (400–700 nm), calibrated previously with a Li-Cor Quantum Sensor LI-190 to express readings in µmol photons m⁻² s⁻¹.

Rates of calcification will be evaluated through incubations conducted separately for each organism at three time periods: morning (9:00–12:00), afternoon (14:30–17:00), and night (19:00–22:00). Five biological replicates per group will be incubated individually for ~2 hours. Blank chambers containing only seawater will serve to account for background activity (Figueiredo et al., 2012). Water samples will be collected at the beginning (T1) and end of each incubation for analysis of physicochemical parameters. Special care will be taken to avoid bubbles when sealing containers (Dickson et al., 2007).

At both T1 and T2, we will measure temperature, salinity, dissolved oxygen, and pHT (total scale) in all chambers. Instruments to be used include: a handheld refractometer (Vital Sine™) for salinity, an RDO Thermo Scientific Orion Star A223 for temperature and oxygen, and a Thermo Scientific Orion Star A121 pH meter. In-situ temperature will be monitored continuously using a HOBO® Pendant UA-002 sensor. All samples will be preserved in 250 mL high-density polyetilene bottles with 100 µL of saturated HgCl₂ and stored at room temperature until analysis (Dickson et al., 2007).