Daniovision chamber

Twelve-well plates with basket inserts were used. Five larvae (6 days post-fertilization) were placed in each basket/well containing 1.5 ml of system water (Fig. 2A). The well plate was then placed in the Noldus DanioVision chamber for 1 h so larvae could dark adapt. Following dark adaptation, a visual-motor response assay (VMR) coupled with a vibrational stimulus was used to assess larval state. Larvae were exposed to 30 s of lights-on (12% intensity in Noldus, 1200 lx) and 20 s of lights-off conditions followed by a tap (at intensity 5) and left in the dark for another 10 s. This cycle was repeated for five minutes. Next, larvae were transferred to increasing concentrations of anesthetic, the stimulation protocol repeated, and the proportion of larvae responding was recorded. The proportion responding in the 5 s before and after tap/light responses during 4th and 5th VMR cycles at each dose was subsequently fitted with a sigmoidal dose–response curve using Prism (GraphPad) as in^{22 (link)}. For tricaine, the concentrations tested were 76.5 µM, 95.7 µM, 114.8 µM, 153.1 µM, 191.4 µM, 306.2 µM, 421 µM, and 765.4 µM. For ketamine, the concentrations tested were 50 µM, 100 µM, 500 µM, 1 mM, 5 mM, and 10 mM. For propofol the concentrations tested were 0.05 µM, 1 µM, 2 µM, 4 µM, 6 µM, 8 µM, and 10 µM.

To investigate the effects of LCCs on the behavior of BPA-treated zebrafish, 6 days-post-fertilization (dpf) larvae from the four treatment groups were randomly selected on a 24-well plate and placed in the DanioVision chamber (Noldus, Wageningen, the Netherlands). After acclimation for 10 min, free swimming activity in the continuous visible light (25 min) and in the dark (25 min) was monitored. EthoVision (Noldus, Wageningen, the Netherlands) was used to analyze the locomotor behavior and average speed during moving time.