Multiple females laid several egg capsules in one large egg cluster after 1–2 days in the holding tank prior to the start of both experimental trials. Each egg capsule may contain between 100–200 fertilized eggs, with considerable inter-capsule variability in the number of eggs per capsule both within and between females [50]. Furthermore, female D. pealeii are able to store sperm from multiple males [51], and their egg capsules are known to contain eggs fertilized by multiple males [52]. Thus, it was likely these egg capsules contained fertilized embryos from multiple males.
The morning after eggs were laid, 2 randomly selected egg capsules from the egg cluster were added to each of 6 containers per trial (3 containers per CO2 concentration per trial). This balanced the need for numerous embryos with the feasibility of measuring paralarvae immediately after hatching. An additional 4 containers were included in the water bath as blanks (2 per CO2 concentration) and contained no eggs throughout the experiment for comparative purposes so that seawater chemistry measurements could be taken independent of potential biotic effects on chemistry parameters.