Fig. 1

Setup and timeline of the coiling assay with zebrafish (D. rerio) embryos. A 24-well plate and the Teflon rings were pre-exposed to the respective test solutions for 24 h prior to exposure. Fertilized eggs were raised in crystallized glass dishes containing the respective exposure concentration or control medium until they were transferred into the 24-well plates (5 embryos per well, 20 embryos per concentration per replicate) and placed in the recording setup. Recording was performed at hourly intervals between 21 and 48 hpf, with two light regime changes (at 23.5 and 37.5 hpf). The test solutions were 100% renewed each day
Fig. 2

Effects of acrylamide, DMSO, and ibuprofen on the behavior of zebrafish (D. rerio) embryos during a change in the light regime. Graphs illustrate he difference in mean burst duration [seconds] (a, c, e) and burst count per minute (b, e, f) from 37 to 38 h old zebrafish embryos in the presence of acrylamide (a, b), DMSO (c, d), and ibuprofen (e, f). Data are given as the difference between the two time points ± SD from n = 3 replicates with 20 embryos per concentration/replicate. SC10 and SC50 indicate threshold for 10 and 50% deviation from the control groups, respectively
Fig. 3

Effects of DMSO on spontaneous tail movement (coiling) of zebrafish (D. rerio) embryos during the light/dark cycles of the coiling assay: (a) Mean burst duration [seconds]; (b) normaized burst duration; (c)burst count per minute; (d) normalized burst count between 21 and 47 hpf of zebrafish embryos in the presence of various concentrations of DMSO (n = 3, 20 embryos per concentration/replicate). Normalized data were adjusted to negative controls (water) (b, d), and the 5% DMSO treatment group was excluded for better data visualization, highlighting the applicability of DMSO at ≤ 0.5 % concentration as a solvent in the coiling assay. a, c: Mean ± SD. Top bar: Light cycle pahse (black–dark; white–light). *: Time point and concentration (in corresponding color) of significant difference to controls (for statistical significance of changes over controls, see Supplementary Material 4)
Fig. 4

Effects of acrylamide on spontaneous tail movement (coiling) of zebrafish (D. rerio) embryos during the light/dark cycles of the coiling assay: (a) mean burst duration [seconds], (b) normalized burst duration, (c) burst count per minute, (d) normalized burst count between 21 and 47 hpf of zebrafish embryos in the presence of various concentrations of acrylamide (n = 3; 20 embryos per concentration/replicate). Acrylamide only significantly increased burst count per minute during the second dark phase (after 37.5 hpf) of the trial. a, c mean ± SD; b, d normalized to the untreated control group. Top bar: light cycle phases (black–dark; white–light). *: Time point and concentration (in corresponding color) of significant difference to controls (for statistical significance of changes over controls, see Supplementary Material 4)
Fig. 5

Effects of ibuprofen on spontaneous tail movement (coiling) of zebrafish (D. rerio) embryos during the light/dark cycles of the coiling assay: (a) mean burst duration [seconds], (b) normalized burst duration, (c) burst count per minute, (d) normalized burst count between 21 and 47 hpf of zebrafish embryos in the presence of various concentrations of ibuprofen (n = 3; 20 embryos per concentration/replicate). Ibuprofen exposure significantly reduced the burst count per minute during the entire assay in a concentration and time dependent manner. a, c mean ± SD; b, d normalized to the solvent control group. Top bar: light cycle phases (black–dark; white–light). *: Time point and concentration (in corresponding color) of significant difference to controls (for statistical significance of changes over controls, see Supplementary Material 4)
Fig. 6

Effects of carbaryl on spontaneous tail movement (coiling) of zebrafish (D. rerio) embryos during the light/dark cycles of the coiling assay: (a) mean burst duration [seconds], (b) normalized burst duration, (c) burst count per minute, (d) normalized burst count between 21 and 47 hpf of zebrafish embryos in the presence of various concentrations of carbaryl (n = 3; 20 embryos per concentration/replicate). Carbaryl exposure significantly increased the burst count per minute in early stages of the assay before the onset of hyperactivity (red box) limiting statistical analysis. a, c mean ± SD; b, d normalized to the solvent control group. Top bar: light cycle phases (black–dark; white– light). *: Time point and concentration (in corresponding color) of significant difference to controls (for statistical significance of changes over controls, see Supplementary Material 4). Red box: at least 20% of organisms had to be excluded from the analysis of at least one of the exposure concentrations within this time frame
Fig. 7

Effects of hexachlorophene on spontaneous tail movement (coiling) of zebrafish (D. rerio) embryos during the light/dark cycles of the coiling assay: (a) mean burst duration [seconds], (b) normalized burst duration, (c) burst count per minute, (d) normalized burst count between 21 and 47 hpf of zebrafish embryos in the presence of various concentrations of hexachlorophene (n = 3; 20 embryos per concentration/replicate). No statistically significant behavioral alteration was observed during hexachlorophene exposure, although hyperactivity (red box) was observed, which could not be analyzed. a, c mean ± SD; b, d normalized to the solvent control group. Top bar: light cycle phases (black–dark; white–light). Red box: at least 20 % of organisms had to be excluded from the analysis of at least one of the exposure concentrations within this time frame
Fig. 8

Effects of rotenone on spontaneous tail movement (coiling) of zebrafish (D. rerio) embryos during the light/dark cycles of the coiling assay: (a) mean burst duration [seconds], (b) normalized burst duration, (c) burst count per minute, (d) normalized burst count between 21 and 47 hpf of zebrafish embryos in the presence of various concentrations of rotenone (n = 3; 20 embryos per concentration/replicate). No statistically significant behavioral alteration was observed during rotenone exposure, although hyperactivity (red box) was observed, which could not be analyzed. a, c mean ± SD; b, d normalized to the solvent control group. Top bar: light cycle phases (black–dark; white–light). *: Time point and concentration (in corresponding color) of significant difference to controls (for statistical significance of changes over controls, see Supplementary Material 4). Red box: at least 20% of organisms had to be excluded from the analysis of at least one of the exposure concentrations within this time frame
Acknowledgments
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