Stability of 6PPD-quinone in fish water. The stability of the 20 ng/L (A), 200 ng/L (B) and 2000 ng/L (C) experimental solutions was determined after 6 and 24 h of incubation at 28 °C with a 12D:12 L photoperiod. The dashed lines show the stability of the solutions over time, while solid show the decrease in 6PPD-quinone during the 24 h of exposure (larva + experimental solution). Percent decreases in the 6PPD-quinone concentration during the first 6 h and the following 18 h are indicated.

Effects of 24 h of exposure to environmental concentrations of 6PPD-quinone on the behavior and neurotransmitter profile of zebrafish larvae. Behavioral endpoints analyzed include the basal locomotor activity (A), visual motor response (B), vibrational startle response (C) and habituation time to a series of vibrational stimuli (D). The neurotransmitters analyzed in the larval heads (E) include catecholamines (dopamine, norepinephrine, and epinephrine), serotonin, acetylcholine, GABA and glycine. Behavioral data are shown as scatter plots with the median (n = 55–181 for basal locomotor activity, n = 133–188 for visual motor response, n = 60–210 for vibrational startle response, and n = 133–295 for habituation; Kruskal–Wallis test with Bonferroni correction). Neurotransmitter results are presented as the mean ± SE (n = 4–6 pools, 15 heads each; one-way ANOVA with Dunnett's multiple comparison test) *p < 0.05, * *p < 0.01, * **p < 0.001; data are from 2 to 3 independent experiments.

Effects of 24 h of exposure to environmental concentrations of 6PPD-quinone on the zebrafish larvae wake/sleep cycle. (A) Locomotor activity of zebrafish larvae, both control and those exposed to the three concentrations of 6PPD-quinone, entrained and tested under 12:12 h light–dark conditions. The period in which activity was recorded, from 7 to 8 dpf, corresponds to the 24 h exposure period. The bars at the bottom of the graph on the left indicate the light/dark/light periods (white bar on the left (D1): 2 pm to 8 pm/black bar in the middle (N): 8 pm to 8 am/white bar on the right (D2): 8 am to 2 pm). The results are presented as the mean ± SE. (B) Total distance moved during the daytime (D1 + D2) and N periods. Data are shown as scatter plots with the median (n = 34–35; one-way ANOVA with Dunnett's multiple comparison test). (C) Time spent by the larvae in the sleep state per hour over 24 h. (D) Time spent by the larvae in the sleep state during D1 (6 h), N (12h), and D2 (6 h). Data are shown as scatter plots with the median (D1: n = 31, Student's t-test; N and D2: n = 31, Mann–Whitney U test). (E) Number of sleep bouts during the D1, N, and D2 periods (n = 31, Student's t-test). (F) Sleep latency at night (n = 30, Mann–Whitney U test). (G) 6PPD-quinone leads to significant changes in the expression of the circadian clock genes per1a, per3 and cry3. Log2 ΔΔCT values are shown as a scatter plot with the median (n = 8 pools of 9 larvae; one-way ANOVA with Dunnett's multiple comparison test). *p < 0.05, * *p < 0.01, * **p < 0.001; data are from 2 independent experiments.

Effects of 24 h of exposure to three environmental concentrations of 6PPD-quinone on the heart rate and oxygen consumption in zebrafish larvae. (A) Effect of 6PPD-quinone on heart beat frequency (n = 15–21; one-way ANOVA with Dunnett's multiple comparison test). (B) Oxygen consumption rate in 8-day post fertilization zebrafish larvae, both control and those exposed for 24 h to 6PPD-quinone (n = 14–19; one-way ANOVA with Dunnett's multiple comparison test). *p < 0.05, * *p < 0.01, * **p < 0.001; data are from 2 independent experiments.