<p>The following text is an excerpt from the <em>Technical Report #15-009,</em> linked below:</p>

<p>The details of the preparation and execution of the hatching experiments are being described in reports in preparation (Preziosi, 2012; Preziosi et al., 2012, 2013; Preziosi, Runge, Christensen and Jones, in prep.). In brief, female zooplankton, freshly caught or reared in the lab with plentiful food, were maintained in a healthy condition and their released eggs separated. When the numbers of eggs required in an experiment were sufficient, the eggs were sorted into individual hatching dishes, each containing natural seawater and 30 eggs. Local seawaters were collected, filtered and used to fill several 20 L hatching tanks. Each tank was consisted of a polycarbonate tank and lid, and an aeration tube into which was bubbled a premixed gas, consisting of 20% oxygen, the preselected concentration of CO2 (ranging from ambient local atmospheric levels to 50000 ppm in the dry gas), and the remainder nitrogen gas (N2). A siphon mounted within the tank allowed for sampling of the waters without opening the tank. A 1-inch diameter hole in the tank lid, normally stoppered, allowed for immersion of the pH and temperature electrodes occasionally throughout the incubation. After the pH stabilized to its quasi-equilibrium value for the bubbled gas, several hatching dishes containing the eggs were immersed in the tank. The tank was closed and hatching allowed to proceed. At an appropriate time, all hatching dishes were removed for determination of hatching success and the experiment then ended. During the incubation, either once (for incubation times less than 2 days) or twice, at the beginning and near the end of the longer 4-6 day experiments, about 2 liters of tank water were withdrawn for collection of the chemistry samples, as described below. The chemical results were used to determine the tank's pH. P-3 <br />
At each sampling event during hatching experiments, about 2 L of the tank waters were siphoned for samples of salinity, titration alkalinity (TA), total carbon dioxide (TCO2), and nutrients. All bottles for TA, TCO2, and nutrients had been previously acid cleaned and dried. Nevertheless, all bottles were rinsed four times with tank water prior to filling. Salinity was stored in tightly capped 0.5 L bottles at room temperature for later measurement. TA and TCO2 samples were drawn in a manner identical to the collection of dissolved oxygen samples. An air bubble of about 1% of the volume of the bottle was left in the top of the bottle and 0.10 ml of a 0.100 mole-Hg/L solution of HgCl2 was added as preservative, yielding a mercury concentration of about 100 μmol/L dissolved in the sample. The sample bottles were tightly capped and stored at room temperature until measurement. Nutrients were collected in plastic vials and frozen at -20°C until analyzed. The temperature of the cold room in which the experiment was being conducted was monitored continuously. All concentrations are reported relative to the weight of the final solution (kg of solution). <br />
In addition to the hatching experiments, we sampled the offshore waters of the Gulf of Maine during a 10-day cruise in autumn of 2012. Seawater was sampled using a vertically profiling CTD with rosette containing 24 30-liter Niskin water sampling bottles. collected. These CTD casts collected waters from the seasurface to within a few meters of the seafloor (depths as great as about 300 m). After the CTD/Rosette had returned from a cast, water from each Niskin was sampled for alkalinity, total carbon dioxide, dissolved oxygen (Carpenter et al., 1965), nutrients, and salinity. Alkalinity and TCO2 were sampled in a manner identical to the collection procedure used for Carpenter oxygen samples. Each alkalinity and total CO2 sample were preserved and stored as described for the hatching experiments. Nutrients and salinities were stored and measured identically to those from the hatching experiments. </p>

<p>For detailed explanation of methods for all chemical determinations and calculations, please refer to <a href="http://dmoserv3.whoi.edu/data_docs/OA_Zoo_Hatch_Chemistry/OAZTCH-9.pdf">... Report #15-009 Green Eyes LLC, Easton MD, 13pp. </a></p>