Detailed methodology and results are described in following publication:
Hettinger, A., E. Sanford, T.M. Hill, E.A. Lenz, A.D. Russell, and B. Gaylord. 2013. Larval carry-over effects from ocean acidification persist in the natural environment. Global Change Biology 19: 3317-3326. doi:10.1111/gcb.12307

Briefly (excerpted from above):
Larval culturing:
Adult Olympia oysters (n = 105) were collected in September 2010 from Tomales Bay, California (38° 06' 57.01"N, 122° 51' 14.39" W) and transported to Bodega Marine Laboratory (BML). Adults were cleaned of epiphytes and distributed evenly among three 100 L cylinders. Adults were fed microalgal food to encourage larval release. After 72 h, larvae were released in one of the cylinders, and subsets of these individuals were distributed by pipette among 4.5 L glass jars (n = 1000/jar) used for culturing. Larvae were reared through their entire planktonic duration in a culturing facility at BML. The target seawater pCO2 concentration in larval treatment cultures was 1000 uatm. The accompanying control seawater pCO2 target was 400 uatm. After filling culture jars with carboy water, pCO2 concentrations were maintained in the jar seawater by pumping the same CO2 gas mixtures into sealed air spaces above the free surfaces of the seawater in the culture jars ("headspaces") shared by 6 replicate jars per pCO2 concentration. There were three headspaces for each pCO2 concentration, and 6 jars associated with each headspace (n = 2 pCO2 concentrations x 3 headspaces x 6 replicate jars = 36 jars).

On day 11 of the experiment, prior to the commencement of settlement and metamorphosis, larvae were transferred into new glass "substrate" jars. The surface of each new jar base was abraded with sandpaper to encourage larval settlement. The underside of each new jar base was scored to facilitate separation into four tiles after larvae had metamorphosed into benthic juveniles. These tiles were suitable for outplanting juvenile oysters to the field.

Seawater chemistry:
Seawater pH(NBS) and temperature were quantified using a potentiometric pH/temperature meter (Accumet Excel XL60), salinity was determined using a YSI 6600V2 multi-parameter instrument, and total alkalinity (TA, umol per kg of seawater) was measured using automated Gran titration with duplicates (Metrohm 809). A subset of samples was analyzed for dissolved inorganic carbon (DIC, umol per kg of seawater) at the University of Georgia’s infrared CO2 analysis facility (Cai & Wang, 1998). Both TA and DIC measurements were standardized using certified reference material from A. Dickson at Scripps Institute of Oceanography (La Jolla, California). Calcite and aragonite saturation states (Ωcalcite, Ωaragonite) and seawater pCO2 were calculated using the carbonate system analysis software, CO2SYS (Lewis & Wallace, 1998).

Metamorphosis and field outplants:
Settlement of larvae and metamorphosis into benthic juveniles was assessed daily starting at day 11 when larvae were transferred into substrate jars. Each jar base was divided into four 50 cm2 tiles for outplanting. Tiles from a given pCO2 concentration with similar juvenile densities were arranged into four groups, each composed of six replicate tiles. The four groups were then randomly assigned to one of the two field sites and one of the two shore levels. The overall design was: n = 2 larval pCO2 concentrations x 2 sites x shore levels x 6 tiles = 48 tiles. Tiles were outplanted to the field on the same day as settlement (day 14 post larval release).

A shoreline region half-way along the Tomales Bay estuary was selected for the two replicate field sites (38° 09' 01.01"N, 122° 53' 19.19"W). The replicate sites were 40 m apart in the alongshore direction, and were similar in substrate type, solar exposure, and bottom slope. At each replicate site, six PVC ‘T’ stakes, placed 0.5 m apart and each holding two tiles, were driven into the substrate such that the tiles were situated at either 0 or 0.3 m above MLLW (i.e., 'low' and 'mid' shore levels, respectively). Tiles deployed on four of the six stakes per site and shore level were outfitted with temperature loggers (iButton, Maxim, Sunnyvale, CA, USA). A YSI 6600V2 multi-parameter instrument was used to measure temperature, salinity, and pH(NBS) at each of the outplant sites approximately weekly up to day 52 postsettlement, and then monthly up to day 127 postsettlement.

Juvenile survival on each tile was calculated as the percentage of initial juveniles that were alive on each sampling date (day 6, 13, 27, 127 postsettlement). Each tile was examined for live juvenile oysters under a dissecting microscope (Leica M125 with DC290 camera) at BML. Juvenile growth rates were estimated from photographs of juveniles randomly sampled on each tile using a random number table to select squares on the gridded tile. Juvenile growth rates were calculated on days 6, 13, and 27 postsettlement as the change in the total projected area of the shell between the sample date and when the larvae metamorphosed, divided by the intervening number of days (mm2 per day). Thus, these growth rates represent the average growth rate over the full benthic life stage to the age examined, not an age-specific growth rate characteristic of the period between assay dates.

size_at_settlement.csv