Pteropod shell dissolution in natural and high-CO2 environments from samples collected on RRS James Clark Ross cruise JR177 in the Scotia Sea, Southern Ocean from 2007-2008 (NCEI Accession 0278115)

Acquisition Description:
Sample collection:
Pteropods were collected from upper ocean water down to a maximum depth of 200 m from various locations across the Scotia Sea using a combination of vertically and obliquely towed Bongo nets and MOCNESS nets during the JR177 research cruise. Oblique tows were carried out at speeds of less than 1 knot.

Experimental conditions:
A fraction of the captured specimens was preserved immediately in 70% ethanol to act as controls for comparison with those exposed to raised pCO2 conditions. A further fraction of specimens was incubated at various levels of pCO2 to test the effect on shell dissolution. Two liter bottles containing filtered sea water (0.7 um filters) were bubbled with air/CO2 mixtures of 500 ppm, 750 ppm, and 1200 ppm, until the required xCO2 was reached. An average of 30 live pteropod of Limacina helicina ant. were incubated in each experimental container and maintained for 4, 8, and 14 days before extraction and immediate preservation in 70% ethanol. The majority of specimens were juvenile stages of Limacina helicina ant ., but the incubations were also carried out on adult stages of both Limacina helicina ant . and Clio pyramidata f . ant.

Omega was assessed from measurements of DIC (dissolved inorganic carbon) and total alkalinity (TA) at the start and end of each incubation experiment. DIC and TA were measured using VINDTA instrument (Versatile INstrument for the Determination of Titration Alkalinity, Marianda, Kiel, Germany) following the Standard Operating Procedures for oceanic CO 2 measurements (Dickson et al. 2007) with a Certified Reference Material (CRM) analysed in duplicate at the beginning and end of each sample analysis day. Other carbonate chemistry parameters (total pH and Omega-aragonite) were calculated from all discrete samples using DIC, TA, temperature, salinity, pressure and macronutrient concentrations using the CO2SYS programme (Lewis and Wallace 1998) with thermodynamic dissociation constants for K1 and K2 by Mehrbach et al. (1973) refitted by Dickson & Millero (1987).

Shell dehydration:
Dehydration was undertaken using 2,2-Dimethoxypropane (DMP; chemical formula: (CH3)2C(OCH3)2), and 1,1,1,3,3,3-hexamethyldisilazane (HMDS; chemical formula: (CH3)3SiNHSi(CH3)3). Before starting dehydration with DMP, the shells were transferred to 50% methanol for two 5 min washes then transferred to 85% methanol (10 min). Complete tissue dehydration was accomplished by immersion in DMP: two changes at 15-20 min each. It was important not to let the shells air dry at this stage, so they were transferred to a 1:1 mixture of DMP and HMDS for about 10 min, followed by 100% HMDS for 20–25 min twice. The HMDS was subsequently allowed to evaporate allowing the shells to dry completely (Figure 2 of Bednarsek et al., 2012). The moderate vapor pressure and very low surface tension of HMDS allowed the shells to dry without distortion or loss of shell integrity.

SEM:
Scanning Electron Microscopy (SEM) was done using a JEOL JSM 5900LV fitted with a tungsten filament at an acceleration voltage of 15 kV and a working distance of about 10 mm. Analysis of SEM photos enabled observation of the shell surface and identification of shell dissolution. Refer to Bednarsek et al. (2012) for more information on dissolution types.