"All adult behavioral testing was conducted between 11:00 AM (2 h post lights-on) and 6:00 PM, with testing time counterbalanced across experimental groups. On days of behavioral testing, the evening feeding was withheld until testing was complete. All larval testing was run between 3:00 PM and 5:00 PM...

2.3. Larval motility assay

After 6-day-old larvae were inspected, they were placed into 96- well plates with glass well inserts each with 0.5 ml of aquarium water (n = 23–28 per exposure condition, over two exposure replicates). Exposure conditions were all represented within each plate and across multiple plates. Plates were then returned to the incubator for an hour before being placed into a DanioVision™ lightbox running EthoVision XT® tracking software (Noldus, Wageningen, The Netherlands). Locomotor activity was tracked during a paradigm in which an initial 10- min acclimation period in the dark (0% illumination) was followed by 2 cycles of 10 min at 100% illumination (5000 lx) and 10 min at 0% illumination. An infrared camera tracked larval locomotion across the 50- min trial. EthoVision XT® was used to calculate the average distance moved in cm per minute for each subject.

2.4. Adult behavioral test battery

6-day-old larvae distinct from those used in the larval motility assay were transferred to the aquarium rack systems and allowed to age normally to 12 weeks of age (n = 24–31 per exposure condition over two exposure replicates). Over approximately the following 2 weeks, the adult fish were run through a series of behavioral tests assessing various behavioral and cognitive functions.

2.5. Novel environment exploration

Adult fish were assessed for novel environment exploration in a test described previously (Bencan and Levin, 2008; Levin et al., 2007). Briefly, fish were placed into 1.5-L tanks filled 10 cm high with aquarium water. A camcorder feeding into EthoVision XT® software was used to track the position of the fish across a 5-min trial and to calculate both the average distance moved in cm per minute and the average distance from the floor of the tank in cm per min of the test.

2.6. Startle habituation

Habituation to a startling stimulus was tested in adult fish via a protocol used previously (Eddins et al., 2010; Sledge et al., 2011). Adult fish were placed into 40 ml of water in one of 8 translucent plastic cups (5.7 cm in diameter) arranged in a 4 × 2 array. Below each cup was a centrally located push solenoid that could be controlled to deliver a sudden tap to the bottom of the cup. A camcorder positioned above the cups was used to record the locomotion of the fish in all 8 cups at once. Following a 5-min acclimation period after the fish were placed in the cups, 10 taps were delivered once per minute over 10 min. EthoVision XT® software was used to control the solenoids and to calculate the average distance moved in cm during the 5 s preceding and following each of the 10 taps. A.N. Oliveri et al. / Neurotoxicology and Teratology 52 (2015) 220–227 221

2.7. Social affiliation

The third test in the behavioral battery, an assessment of social affiliation, utilized a type of behavioral response called shoaling, in which a zebrafish will approach a group of conspecifics (a “shoal”) and engage in a dart/pause locomotor behavior alongside the conspecific group (“shoaling”). Adult fish were singly isolated in 1.5-L tanks surrounded by opaque dividers for 1 h. After the hour, they were placed in a large transparent rectangular tank 45 cm long by 13 cm wide by 12.5 cm deep. On both ends of the tank was positioned a computer monitor for stimulus display. The fish was recorded using a digital video recorder placed above the tank. During the first minute of the 6-min test, each monitor screen displayed a background of static ovals approximately the size of an adult zebrafish and displaying the pattern and colors of a typical zebrafish. At the end of the first minute, one of the two monitors began to display a video recording of a zebrafish shoal for the remaining 5 min. The side of the tank displaying the shoal video was counterbalanced among exposure groups. EthoVision XT® was used to control the two displays, to record the locomotion of the fish, and to calculate both the average distance traveled in cm per minute and the average distance from the tank wall through which the shoal video was visible in cm for each subject across the 6 min of the test.

2.8. Predator escape

The final test involved an assessment of escape and avoidance behavior following exposure to a predator-like stimulus. Adult fish were placed in a 1.5 L tank facing a computer monitor. The 5-min test involved an initial minute of acclimation, followed by 2 rounds of a cycle including a 1- min presentation of a small (1.3 cm diameter) circle appearing in the center of the screen, growing to 30.5 cm in diameter, disappearing, and repeating, to simulate a larger fish swimming toward the tank, followed by 1 min of a blank screen. The test was performed first with a blue dot that grew slowly (4 s), and again several days later with a red dot that grew quickly (1 s). A camcorder positioned above the tank tracked the fish. EthoVision XT® was used to control the display, to record the fish's activity, and to calculate both the average distance traveled in cm per minute and the average distance from the tank wall through which the screen was visible in cm for each subject over the 5 min. The escape/flee response was then defined as the difference between the distance from the tank wall during the minute of stimulus presentation and the distance from the wall during the preceding “blank” 1-min period.

2.9. Statistical analysis

The dependent measures corresponding to each assessment are described above. All statistical analyses were performed with Supernova/ Statview (SAS, Cary, NC, USA) and all graphs were produced with SigmaPlot (SYSTAT Software Inc., Richmond, VA, USA). Type 1 error rate (α) was set at 0.05 for all tests. A mixed-design repeated-measures of analysis (RMANOVA) with organophosphate exposure and dose as the between-subject factor and tap number, session minute, or time condition as the repeated measures was used for all behavioral tests. Dunnett's test was used for all post hoc comparisons of controls to exposed groups. Survival and malformation data were analyzed using an ANOVA. A Huynh–Feldt adjustment was used to control for possible deviations from sphericity. Log transformations of the raw data were performed (as noted) when the distribution of the data was positively or negatively skewed."

Zebrafish (AB* strain) larvae