Varian 725 es

Water samples were filtered (GF 6, Whatman, GE Healthcare, Buckinghamshire, UK, pore size < 1 µm) on the day of collection and then frozen (−18°C). Potassium (K, 0.1 mg/L), calcium (Ca, 0.1 mg/L), magnesium (Mg, 0.02 mg/L), phosphorous (P, 0.1 mg/L), sulfur (S, 0.3 mg/L), sodium (Na, 0.1 mg/L), silicon (Si, 0.005 mg/L), iron (Fe, 0.01 mg/L), and manganese (Mn, 0.003 mg/L) concentrations were measured using an inductively coupled plasma optical emission spectrometer (ICP‐OES, Varian 725‐ES, Agilent Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia). Values in brackets give the detection limit of a given element. Nutrient concentrations below the detection limit were arbitrarily set to half of that limit for further calculations. Phosphorous (P) concentrations were below the detection limit in most ecosystem components, and are not presented. Nutrient concentrations were not measured in samples taken on 21 July, 2 August, 26 August, and 9 September 2015.

In this work, in order to estimate the required parameters for the quantitative model, the ion fluxes through the channels and pumps were determined using a flux-based assay with a tracer element. Here, Rb⁺ was used as a tracer of potassium to study the flux through the potassium channels and Na⁺/K⁺ ATPase pumps^{23 (link),24 (link),47 (link)}. In the Rb⁺ assay, cells were incubated with a buffer containing Rb⁺ for 0.5-2 hours. At specific time intervals, cells were washed to remove extracellular Rb⁺ and channel activity was determined by measuring the rubidium concentration of the cell lysate and supernatant using an ion specific tool, inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian 725-ES, (Agilent, Australia)).

The mineral content of empty shells (see above) (for Ca²⁺, Mg²⁺, Sr²⁺, K⁺) of adult individuals of L. littorea exposed for 14 days to different P_CO2 conditions was determined using an optical emission spectrometry technique. Shell fragments were meticulously cleaned of all tissue under low-power magnification (10–50, SZXI6 binocular microscope, Olympus, Tokyo) using only plastic or Teflon-coated dissection tools. Shell fragments were then weighed with a high-precision balance (AT201, Mettler-Toledo; degree of precision (dp) 0.01 mg) before being freeze-dried for 24 h (Modulyo freeze drier, Thermo Electron, Waltham, MA, USA) at −50 °C. The dry mass of each freeze-dried set of shell fragments was determined with a high-precision balance (AT201, Mettler-Toledo; dp 0.01 mg) before being digested in 2 ml nitric acid (79% concentration, trace analysis grade) in a microwave digestion unit (MarsXpress, CEM, Matthews, NC, USA). Digests were then diluted to 10 ml with ultrapure water and analysed for [Ca²⁺], [Mg²⁺] and [Sr²⁺] with an ICP-Optical Emission Spectrometer (Varian 725-ES, Agilent Technologies, Santa Clara, CA). Shell mineral content is expressed as mmol kg⁻¹. Shell mineral content was determined for the populations of Île de Ré, Roscoff, Millport, Trondheim and Tromsø.

To determine macro- and micronutrient content, samples were dried at 70 °C in a drying oven for 72 h and pulverized in a blender (Oster 6832, Milwaukee, WI, USA). Samples were subjected to wet digestion in a mixture of perchloric and nitric acids at a 2:1 (v/v) ratio, according to the protocol described by Alcántar and Sandoval [37 ]. To determine the concentrations of macronutrients (P, K, Ca, Mg, and S) and micronutrients (B, Cu, Fe, Mn, Ni, and Zn), the extracts were analyzed using a coupled plasma induction optical emission spectrometer (ICP-OES, Varian 725-ES, Agilent; Mulgrave, VIC, Australia). The N concentration was determined by the semi-microkjeldahl method according to the protocol described by Bremner [38 ].