Mussel larvae were obtained and cultured as described in Frieder et al. 2014, EST. At the end of each experiment, the larvae were frozen then cleaned from organic material using trace-metal-clean techniques in a clean room (described in Frieder et al. 2014, EST).
Once clean and mounted, larvae were individually analyzed for a combination of elements (Mg, Cu, Zn, Sr, Ba, Pb, U with respect to Ca) at UC Santa Barbara using a New Wave research UP-213 laser ablation unit coupled with a Thermo Element 2 single-collector inductively coupled plasma mass spectrometer operating in low resolution mode.
As described in the supplemental material for Frieder et al. 2014 (EST) "three standards were used to ensure proper calibration of the instrument: a solution-based dissolved CaCO3 reference material (OTO, [Spex certified primary standard solutions]), and two solid standards, NIST 612 and USGS MACS3 CaCO3 standard. All reference materials, both solution- and solid-based, were analyzed at the beginning and the end of each run; mounting medium[, solution standards] and instrument blanks were run multiple times during a sequence. For ablations, laser settings were a single, 30-µm-long by 80-µm-wide line at 40% power, 10 Hz, and 8 µm s-1 scanning speed. These settings usually consumed the entire larval shell. The tape used to mount the shells had less than 5% of the average shell value for each element."
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