Fig. 3

3 Embryonic damage is seen within 4 hrs of NA/EE-mixture exposure and embryonic movement is stunted from 1 hr of exposure. Zebrafish embryos at 24 hpf were treated with either DMSO (A,C,E) or NA/EE-mixture (B,D,F) for 1 hr (A,B), 2 hr (C,D) or 4 hr (E,F) before fixation. Overall body length, eye area and yolk sac area were measured. Embryos begin to show heart defects (n = 2/5) (black asterisk) and smaller body length (G) and eye area and pigment (H) (grey asterisk) by 4 hr exposure. At 4 hr, there is no change in the area of the yolk sac between DMSO control and NA/EE-mixture treated embryos (I). At 1 hr exposure, it was noted that there was a drastic decrease in movement in NA/EE-mixture treated embryos. This was determined using time lapse recording of embryos for 2 minutes and counting the number of times each embryo moved. The lack of movement can be visualised by comparing the starting and finishing positions of the embryos (J–M). In the DMSO treated well, there has been clear movement between 0 min (J) and 2 min (L), with one embryo having moved out of frame. Black arrows denote embryos in different positions. In comparison, the NA/EE-mixture treated embryos can be observed to have remained in the same position between 0 min (K) and 2 min (M). Black arrowheads show embryos in same position. The average number of movements recorded for DMSO treated embryos was 16 per minute compared to the average 0 per minute in the NA/EE-mixture treated embryos (N).