All behavioural testing was performed during the light phase of the 12 h light/dark cycle in a sound-controlled room with diffuse overhead lighting. Testing was conducted in the mornings prior to feeding. Additionally, control testing was interspersed throughout the treatment conditions to account for any time-of-day effects and other confounding variables. Fish were randomly assigned to either a control group or one of the three treatment groups. During testing, a three-sided enclosure of white corrugated plastic encircled the testing arena to minimize exposure to external stimuli. Water temperature was maintained between 26 °C and 28 °C. The arena was placed on a pad heated to 35 °C to reduce heat loss between trials. Luminance in all testing arenas was measured at ~ 32 cd/m3 (cal SPOT photometer; Cooke Corp. CA, USA). A Basler GenICam acA1300-60gc Area Scan video camera (Basler Inc., USA) was suspended approximately 1 m above testing arenas to record zebrafish behaviour. Zebrafish movement was tracked using Noldus EthoVision XT ® tracking software (v. 11.0, Noldus, Wageningen, NL) using differencing settings. Quantification of behaviour began immediately after the fish or shoal was placed in the center of the arena. The time the fish was immobile (immobility) was quantified in EthoVision with a 5% threshold49 (link),50 (link). The individual tests, novel tank dive test (n = 80) and light/dark test (n = 81), consisted of ~ 20 fish per group for each 0, 1, 5, and 15 mg/L concentration of chlordiazepoxide. For the shoaling test (n = 250), each shoal consisted of 5 fish. There were 10 trials conducted in each of the shoaling treatment groups, however, in the control group 20 trials were conducted as they were interspersed throughout the testing trials. Data were only excluded from analyses if the tracking software did not acquire data for the total time spent in arena.
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