Trace element:calcium ratios in shell of juvenile Perna canaliculus raised under pCO2 of 400 µatm and 1050 µatm.

This study was designed to examine how predicted future oceanic CO₂ conditions may affect the trace elemental composition of bivalve shell. Individuals were spawned and raised under two different CO₂ conditions, those existing today and those predicted to exist in the future.

Individual male and female mussels were randomly selected from unrelated F2 families raised on long-line aquaculture facilities in the Marlborough Sounds (South Island, New Zealand) as part of the Cawthron Perna canaliculus selective breeding programme. Thermal shock was used to induce spawning; eggs were diluted to 1000 mL^-1 in either 400 µatm CO₂ (ambient) or 1050 µatm CO₂ (elevated) seawater and fertilized with sperm at 200 egg^-1, prior to transfer to 160 L conical incubation tanks containing 5 µm-filtered seawater and 12 µM EDTA at 16°C, maintained at 400 µatm CO₂ or 1050 µatm CO₂ using gentle aeration (air or CO₂ enriched via a gas mixer/analyser (WMA-5, PP Systems, Annesbury, MA). After 48 h incubation the embryos had formed the prodissoconch I shell, entering the feeding veliger stage. Larvae were transferred to a continuous-flow culture system. Triplicate 2.5 L tanks for each pCO₂ condition with 200 larvae mL^-1 received filtered seawater enriched with dietary microalgae (Tisochrysis lutea + Chaetoceros calcitrans, 40 cells µL^-1). After 3 weeks, pediveligers were offered coir string as a settlement substrate and allowed to metamorphose. Juveniles were harvested after 3 months, rinsed in deionised water, snap frozen, and stored (-20 °C) prior to analysis.

Each tank was sampled to determine temperature, salinity and total pH, and measured colorimetrically using m-cresol purple. Every 3-4 weeks 1.0 L water samples were fixed with 100 μL saturated HgCl₂ and analysed by potentiometric titration to determine total alkalinity (TA) and dissolved inorganic carbon (DIC) content, calibrated against certified reference material (CRM126 AIMS or CRM118) at the University of Otago. TA was stable over time and therefore combined with colorimetric pH_T to calculate pCO₂ and calcium carbonate saturation state (Ω) using CO2SYS.

Laboratory Methods

Mussels were defrosted the shells, split open and the flesh removed using stainless steel forceps. Twenty seven individuals raised at 400 µatm CO₂ and thirty two raised at 1050 µatm CO₂ were examined. We selected one valve from each individual at random and fixed it to a glass microscope slide using double sided adhesive tape. To determine the elemental composition of shell material we performed laser ablation inductively coupled plasma mass spectroscopy (LA-ICP-MS) using a New Wave deep ultra violet (193nm) laser ablation system (Electro Scientific Industries) coupled to an Agilent 7700 ICP-MS (Agilent Technologies). Seventeen trace element:calcium ratios were monitored (Li:Ca; B:Ca; Mg:Ca; Al:Ca; Ti:Ca; V:Ca; Mn:Ca; Co:Ca; Ni:Ca; Cu:Ca; Zn:Ca; Sr:Ca; Y:Ca; Ba:Ca; La:Ca; Pb:Ca; U:Ca). We analysed two locations within the shells; one was located approximately 200 µm from the most recently formed shell edge, and the other was located on the shell umbo. These locations were selected to represent shell formed early in the individuals life, although we did not intend to analyse larval shell formed prior to transfer to the continuous flow culture system, and as close to the end of the experiment as possible. Backgrounds were monitored for 30 seconds prior to each analysis. The laser operated with a spot size of 50 µm, a repetition rate of 5 Hz, and a dwell time of 40 seconds. We analysed NIST610 and NIST612 standards every 20 spots for standardisation and calibration purposes. Laser power was 45% and the fluence was between 7 and 7.5 Jcm^-2. All LA-ICP-MS analyses were performed at The University of Auckland Plasma Mass Spectrometry Centre. Gas flows and voltages were optimised to maximise sensitivity and signal stability whilst maintaining an oxide reduction ratio of less than 2% this was monitored using the thorium:thorium oxide ratios.

In order to remove contaminants we used a pre-ablation technique in which the first five seconds of the laser dwell time were not included in the data reduction process. We used only the next 10 seconds of data to determine the elemental composition of the shell material in order to reduce the chances of laser burn through to lower layers within shell material. We background corrected data by subtracting background average counts from the ablation average counts. We then converted background corrected counts to trace element:calcium (TE:Ca) ratios and standardised them using the most recently published NIST610 values. Internal precision was calculated from the NIST612 standard. Finally, TE:Ca ratios were converted to µmol:mol ratios which we used for all statistical analyses.