Dataset: CO2 x temperature effects on Menidia menidia offspring
Deployment: Mumford_Cove_Subsurface_Buoy

CO2 × temperature manipulations and measurements:
For 2 × 2 and 3 × 2 factorial designs, replicate rearing containers (20 L) were placed into large temperature-controlled water baths. Elevated CO₂ levels were achieved via gas proportioners (ColeParmer®) mixing air with 100% CO₂ (bone dry grade) that was delivered continuously to the bottom of each replicate rearing container via airstone. To counteract metabolic CO₂ accumulation, control CO₂ conditions were achieved by forcing compressed laboratory air through a series of CO₂ stripping units containing granular soda lime (AirGas^®), a particle filter (1 µm), and then to each replicate via airstone. Target pH levels were monitored daily using a handheld pH probes (Orion Ross Ultra pH/ATC Triode with Orion Star A121 pH Portable Meter; Intellical PHC281 pH Electrode with Hach® HQ11D Handheld pH/ORP Meter) calibrated bi-weekly with 2-point pH_NBS references. Continuous bubbling maintained dissolved oxygen saturation (>8 mg/ DO) in rearing vessels. Target treatment temperatures were controlled by thermostats (Aqualogic^®) which powered chillers (DeltaStar®) or glass submersible heaters to maintain water bath temperatures. For 3 × 3 factorial experiments, we developed an automated acidification system composed of nine discrete recirculation units designed for larval fish rearing. We designed a LabView (National Instruments^®) based program to fully automate the control of seawater chemistry. The software interfaces with the recirculating units via a data-acquisition module (NI cDAQ-9184, National Instruments^®), which controls nine sampling pumps (one per tank) and a series of gas and water solenoid valves, while receiving input from a central pH electrode (Hach pHD^® digital electrode calibrated weekly using 2-point pH_NBS references) and dissolved oxygen probe (Hach LDO^® Model 2). The software sequentially assesses the pH conditions in each rearing unit (each tank once per hour) by pumping water for ~450 seconds through the housing of the central pH probe, comparing measured pH levels to set-points and then adjusting levels by bubbling standardized amounts 100% CO₂ (bone dry grade, AirGas^®) or CO₂-stripped air into the sump of each tank. The software also maintains DO saturation (>8 mg/l) by bubbling in CO₂-stripped air. LabView logs current pH, temperature, and DO conditions before cycling to the next unit. Temperatures were controlled by thermostats (Aqualogic^®) that powered submersible heaters or in-line chillers (DeltaStar^®).

Actual treatment CO₂ levels were determined based on measurements of pH, temperature, salinity, and total alkalinity (A_T). Treatment tanks were sampled three times per experiment for measurements of A_T (μmol kg^-1). Seawater was siphoned and filtered (to 10 µm) into 300 ml borosilicate bottles. Salinity was measured at the time of sampling using a refractometer. Bottles were stored at 3˚C and measured for A_T within two weeks of sampling using an endpoint titration (Mettler Toledo^® G20 Potentiometric Titrator). Methodological accuracy (within ±1%) of alkalinity titrations were verified and calibrated using Dr. Andrew Dickson’s (University of California San Diego, Scripps Institution of Oceanography) certified reference material for A_T in seawater. The partial pressure of CO₂ (pCO_2,; μatm) was calculated in CO2SYS (V2.1, http://cdiac.ornl.gov/ftp/co2sys) based on measured A_T, pH_NBS, temperature, and salinity using K1 and K2 constants from Mehrbach et al. (1973)  refit by Dickson and Millero (1987)  and Dickson (1990) for KHSO₄.

Field sampling and experimental designs:
Collections of wild, spawning ripe Atlantic silversides were made during high tide 1-3 days prior to full or new moons during the species spawning season. Adults were caught with a 30 m × 2 m beach seine from local salt marshes and transported live to our laboratory facilities. Ripe adults were held overnight at 20°C in well aerated tanks at low densities with no food and strip spawned the next day.

For each experiment, eggs from 20+ running-ripe females were gently mixed into shallow plastic dishes lined with 1 mm plastic window screening. 20+ males were stripped-spawned together into 500 ml glass beakers, mixed with seawater, stirred, then gently poured into spawning dishes and mixed with eggs for ~15 minutes. Screens were rinsed with seawater to remove unfertilized eggs and then soaked in a 100 ppm buffered iodine (Ovadine^®) solution for 15 minutes to prevent fungal infection. Experiments were initiated within two hours of fertilization when replicate rearing vessels received precisely 100 embryos. Vessels were filled with clean seawater (filtered to 1 µm and UV sterilized). Optimal salinity (27-31) and light conditions (15 h light:9 h dark) for rearing M. menidia were maintained across experiments. Upon hatching larvae were immediately provided ad libitum rations of newly hatched brine shrimp nauplii (Artemia salina, San Francisco strain, brineshrimpdirect.com) and equal rations of powdered weaning diet (Otohime Marine Fish Diet, size A1, Reed Mariculture®). To quantify hatching survival, one day post first hatch larvae were counted by gently scooping small groups into replacement rearing vessels. For initial hatch measurements, random sub-samples (N = 10) from each replicate were preserved in 5% formaldehyde/freshwater solution buffered with saturated sodium tetraborate. All experiments were terminated when larvae reached ~10 mm standard length (SL). At termination, all survivors were counted and measured for standard length (SL, nearest 0.01 mm) via calibrated digital images (Image Pro Premier^® V9.0).