Overview:  The goal of these laboratory experiments were to determine the degree to which lowered seawater pH would inhibit egg hatching success in marine zooplankton and specifically in the copepod, Calanus finmarchicus.  To do this, adult female zooplankton were collected at an open coastal site.  The eggs from these females were then collected and incubated for several days in several tanks of seawater bubbled with premixed gas at a specific CO2 content.  The proportion of eggs which hatched were determined in replicate.  While the eggs were being incubated, waters in each tank were monitored with sensors for temperature, salinity, pH, and dissolved oxygen.  Once or twice during an experiment, 2 L of seawater were withdrawn for measurement of the carbonate system parameters (salinity, alkalinity, total carbon dioxide) and nutrient concentrations.  From these high-precision carbonate measurements, the total pH and other carbonate system parameters were calculated.  Thus, inhibition of hatching success was related to the pH of the tanks.

Egg Collection:  Zooplankton were collected from an open coastal site  (43.7474 N, 69.5010 W) nearby the University of Maine's Darling Marine Center, using a 1-m 300 m mesh ring net towed from 100 m to the surface.  Sufficient quantities of seawater was also collected from 30-40 m depth for most experiments.  The copepod, Calanus finmarchicus, was studied in experiments 1-3 and 6-13, while the euphausid, Meganyctiphanes norvegica, was used in experiments 4 and 5.  Females were separated from the catch and allowed to release their eggs for about a day.  On occasional experiments, insufficient numbers of females were collected to supply the numbers of eggs required in an experiment.  In this case, C. finmarchicus eggs from females maintained in the laboratory were used instead.  These females were maintained at 6C and were fed twice daily at concentrations of >500 g C L-1 with an equal mixture of Gymnodinium sp., Rhodomonas sp. and Oxyrrhis marina.  For experiments with the laboratory-maintained females, waters used in the experimental tanks were those pumped from the Marine Center's dock into the marine aquarium building, which housed our laboratory.  The estuary's seawater was only slightly less saline (salinities of 30.2-31.1) than the open coastal waters (31.1-32.4).  All seawaters used were filtered through 1.0 m filters.

Incubations at differing pH:  After obtaining sufficient numbers of eggs, the eggs were separated in replicate Petri dishes with 30 eggs per dish.  The dishes were specially modified with Nitex screen to allow exchange between the interior of the dish and the surrounding seawater.  Between 3 and 5 tanks containing 12.5 L of seawater had been bubbled with specialty-purchased gas mixtures of 20% oxygen, a preselected CO2 concentration (between 913 and 15,290 ppm), and the remainder nitrogen.  One experiment (experiment 7) used two gases to reach a very low pH, one gas cylinder containing 50,000 ppm CO2 in N2, and one containing 913 ppm CO2 in 20% O2 and the remainder N2.  In this tank, the dissolved oxygen content in the tank remained about the same level as the other tanks in the experiment, above 70% of saturation.  Each experiment was begun by placing up to 8 replicate Petri dishes with the eggs into each tank.  The tanks were maintained in this state for about 24 hours past the hatching time determined by McLaren et al. (1969) for the particular temperature.  The experiment ended by removing the replicate Petri dishes and manually counting the number of nauplii contained within each petri dish.  Hatching success was the percentage of nauplii counted relative to the number of eggs initially in the dish.