Sample Collection
Larvae were produced from adult oysters reared at Whiskey Creek Hatchery (WCH), Netarts Bay, Oregon, USA that were strip-spawned with the resultant sperm and eggs mixed in tanks filled with seawater collected from the bay and thermostatically controlled (to 22 degrees C). After hatching, larvae were grown for ~20 days, with a change in tank water every other day. Two cohorts were sampled, one in May and one in August, 2011. Details on the rearing methods can be found in Waldbusser et al., 2013. A fraction of the larvae and an aliquot of water coming from the bay to fill the rearing tanks were sampled during tank changes. The carbonate chemistry of the tank waters was also measured directly (DIC and PCO2, with calculated alkalinity and pH). Water was filtered at 0.2 um, collected in acid-washed bottles and acidified to pH ~2 with ultra pure HCl. The larvae were collected on a filter, rinsed with MilliQ water, then decanted and freeze-dried for later analyses.

Water carbonate chemistry
Water filling the tanks is pumped directly from Netarts Bay, at an arbitrary time (i.e., not synchronized with tides). The salinity of these waters reflects that in the bay, but temperature is thermostatically-controlled (22 degrees C). The resulting carbonate chemistry is insignificantly modified from that in the bay due to the temperature change, aside from the effects on the carbonic acid equilibrium constants. For carbonate chemistry, all water samples were collected in 350 ml amber glass bottles, and sealed with polyurethane-lined metal crimp caps. PCO2 and DIC analyses were carried out via gas equilibration and stripping, respectively, followed by infrared detection, as in Bandstra et al., 2006 and Hales et al., 2005, but modified for discrete samples. Standards for PCO2 and DIC encompassed the complete range of values in this study, which are outside the range of typical modern-ocean seawater. To compute the complete carbonate chemistry, the investiagors used Millero’s (2010) carbonic acid dissociation constants with temperature and salinity dependencies (which capture the Lueker et al. (2000) seawater constants with improved estimation at intermediate to low salinities), Dickson’s (1990) constants for boric acid, and Millero’s (1995) water dissociation constants. Combined uncertainty in the DIC and PCO2 measurements is estimated at ~0.2% and ~2%, respectively, based on replicate analyses and comparison with Certified Reference Materials (Bockmon and Dickson, 2015). The Mucci (1983) aragonite solubility was used to calculate the aragonite saturation state (Ωa) and pH was reported on the total hydrogen ion scale (pHt).

Water metals chemistry (See tank chemistry dataset)
Samples collected in pre-acid washed polypropylene bottles were analyzed on an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) at the OSU Keck Collaboratory. An IAPSO seawater and NIST 1643e seawater standard were run as external standards at concentrations equivalent to the water samples, and produced 2-sigma standard deviations (n = 5 and n = 2 respectively) of <3% for all elements analyzed (Ca, Mg, Sr). The metals of interest here (Ca, Mg, Sr) are conservative in the oceans, and their abundances measured in the tank water is generally equivalent to that predicted from salinity. The water of Day 13 in August is the only instance where the measured concentrations of Ca, Mg and Sr are significantly (>10%) different than what is expected from salinity. While the investigators' interpretations are based on the measured elemental concentrations, not salinity-based estimates, the generally good comparison indicates that there is no significant alteration of these metals in the transfer of water from the bay to the tanks.

Shell Analyses
We transferred ~2 mg dry weight of freeze-dried larvae into an acid cleaned plastic 1.5 mL centrifuge vial. Our cleaning method was to treat each sample with a 5% (active chlorine) bleach solution. This reagent was added and reacted for 15 minutes, with constant agitation (shaking and sonication) to ensure reaction with all the larvae. The cleaning solution was then removed and the larval shells rinsed through shaking, centrifuging and pipette decanting with 5 repeats of MilliQ water. The cleaned, rinsed shells were then dissolved using ultra-pure 2 M nitric acid. These solutions were analyzed on a Themo XSeries2 quadrupole Inductively Coupled Plasma Mass Spectrometer (ICP-MS), using an In-Re internal standard to correct for instrument drift. The external reproducibility of the analyses, based on replicate analyses of dilute NIST1643e standard was <4% for all analytes, and we report all ratios with a precision of 6%. The investigators further measured carbon and nitrogen content of the cleaned shells post-treatment. These samples were treated the same as described above, then dried in an oven at 60 degrees C before analyses using a Carlo Erba 1500 (Verardo et al., 1990). The precision of analyses for carbon and nitrogen in these carbonate samples is 3%. Finally, a fraction of these treated larval shells were dried for SEM imaging.

References
Bandstra, L., Hales, B. and Takahashi, T. 2006. High-frequency measurements of total CO2: method development and first oceanographic observations. Mar Chem., 100, 24–38. doi: 10.1016/j.marchem.2005.10.009

Bockmon, E.E. and Dickson, A.G. 2015. An inter-laboratory comparison assessing the quality of seawater carbon dioxide measurements. Mar. Chem., 171, 36-43, doi:10.1016/j.marchem.2015.02.002

Dickson, A.G. 1990. Standard potential of the reaction—Agcl(S)+1/2h-2(G) = Ag(S)+HCl(Aq) and the standard acidity constant of the ion Hso4- in synthetic sea-water from 273.15-K to 318.15-K. J Chem Thermodyn., 22, 113–127. doi: 10.1016/0021-9614(90)90074-Z

Hales, B., Chipman, D. and Takahashi, T. 2005. High-frequency measurement of partial pressure and total concentration of carbon dioxide in seawater using microporous hydrophobic membrane contactors. Limnol Oceanogr Methods. 2, 356–364. doi: 10.4319/lom.2004.2.356

Lueker T.J., Dickson A.G. and Keeling C.D. 2000. Ocean pCO2 calculated from dissolved inorganic carbon, alkalinity, and equations for K-1 and K-2: validation based on laboratory measurements of CO2 in gas and seawater at equilibrium. Mar Chem., 70, 105–119. doi: 10.1016/S0304-4203(00)00022-0

Millero F.J. 1995. Thermodynamics of the carbon-dioxide system in the oceans. Geochim Cosmochim Acta., 59, 661–677. doi: 10.1016/0016-7037(94)00354-O

Millero F.J. 2010. Carbonate constants for estuarine waters. Mar Freshwater Res., 61: 139–142. doi: 10.1071/MF09254

Mucci, A. 1983. The solubility of calcite and aragonite in seawater at various salinities, temperatures, and I atmosphere total pressure. Amer. J. Sci., 238, pp. 780–799. doi: 10.2475/ajs.283.7.780

Waldbusser, G.G., Brunner, E.L., Haley, B.A., Hales, B., Langdon, C.J. and Prahl, F.G. 2013. A developmental and energetic basis linking larval oyster shell formation to acidification sensitivity. Geophys Res Lett. 2013; 40: 2171–2176, doi: 10.1002/grl.50449

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