Macondo crude oil exposure did not affect cell proliferation but did induce programmed cell death. (A, B) Phospho-Histone H3 labeling of cells in mitosis were unaffected in 30 hpf WAF-treated embryos (B). (C) Quantification of anti-Activated Caspase 3-positive cells in 30 hpf control and WAF-treated embryos over 4 replicates and a WAF-refreshing procedure. The number of apoptotic cells decreased with each successive replicate, but increased following application of freshly-mixed WAF. (D-F) Activated caspase-3 labeled 30 hpf control (D, arrowheads) and WAF-treated embryos (E) from experiments in January 2011, and a WAF-treated embryo from an experiment in March 2011 (F, arrowheads). There was a significant decrease in the number of apoptotic cells in WAF-treated embryos between January (E) and March (F). (G-I) Representative images from the refreshed WAF experiments (arrowheads denote positive anti-Caspase 3 cells). Embryos were either untreated (G), exposed to the same WAF from 3.5 hpf to 30 hpf (H), or exposed to WAF from 3.5 hpf to 15 hpf and then exposed to a fresh WAF solution from 15 hpf to 30 hpf (I). Embryos in the refreshed WAF group (I) partially recovered the cell death phenotype of earlier replicates (E). (A, B, D-I) Lateral trunk views centered on somites 14 to 21. Scale bar 50 μm, A, B, D-I.