All solutions were filtered through 0.22 μM syringe filters (Fisher Scientific). Within each experiment, drug treatment was performed as a two-fold serial dilution with a starting concentration of 1 mM in the water column. These dilutions were then spilt further and tested at three different water columns for each solution (top, middle, and bottom) for a total of 12 perinatal exposures. Water columns were changed between treatments and half-filled with fresh water and stirred to mix. Corals were exposed in 8- or 24-well cell culture plates (Corning Costar or Greiner Bio-One). Each treatment plate was seeded with 25 polyps. All plates were acclimated overnight at room temperature before use. Before use, polyps were picked individually from each plate and placed in a culture dish containing 2-4 ml of sterile water. Polyps were monitored for 1 h to ensure that they were responsive and acclimated before the exposure experiment. Behind the treated polyps, a duplicate dish with 100 % acclimated polyps was placed as a measure of control. Additional coral tissue was collected from each euthanized polyp to assess survival and general health. Each treatment was replicated three times and each replicate was tested in two cell plates (8-well or 24-well).
Before filtering coral tissue in saline, polyps were kept in a 50-ml falcon tube on ice for 2 h to allow the gills to effervesce and bleed. Polyps were then transferred to a 50-ml tube containing 500 μl saline. After 5 min of vortexing, the supernatant was removed and the remaining tissue spun down in a microfuge for 5 min at 2500 rpm. The supernatant was decanted off, the gel spun down and discarded, and then the cells were collected into nine 1.5 ml tubes (~10-20 cells per tube) containing 100 μl solutions of each compound in HM. Each tube was immediately vortexed and spun down. After 5 min of vortexing, the supernatant was decanted off, and the remaining cells were spun down in a micorfuge for 5 min at 2500 rpm, the supernatant was decanted, the glass vortexed and left to stand for 10 min. d2c66b5586