Table 1.
List of chemicals.
Table 2.
List of emulsions.
Fig 1.
An imaging device was developed, and analysis software was created to evaluate chemical toxicity by quantifying the movements of zebrafish.
(A) The imaging device consists of a computer-controlled z-axis linear slide, camera, spot plate, and flat light box. (B) Image analysis software creates binarized subtracted images from two time-lapse images and computes locomotion activity. (C) Correlation between the calculated average distances of white pixels in binarized images and actual travel lengths of animals (R2 = 0.9854). (D) Difference in locomotion activity between the control (black) and TPT-treated animals (blue) over time.
Fig 2.
Toxicity tests of commercial emulsions showed their various effects on locomotion activity.
(A) Heat map of the locomotion activity of individual larval zebrafish over time. Each row represents a different zebrafish. (B) Effect of emulsions on locomotion activity in larvae (14 dpf). Control: black circle; Oil-A: red circle; Oil-B: red square; Oil-C: red triangle; Oil-D: green circle; Oil-E: green square; Oil-F: blue circle. The locomotion activity of all animals tested under the same condition was recorded as a mean value for 10 minutes. Sample size: n = 5 animals (one animal per well). (C) Effect of emulsions on locomotion activity in adult zebrafish. Control: black circle; Oil-A: red circle; Oil-B: red square; Oil-C: red triangle; Oil-D: green circle; Oil-E: green square; Oil-F: blue circle. Sample size: n = 5 animals. (D) Phenotype observation of animals after three hours of exposure. Yellow arrow: internal hemorrhaging around the heart and gills; Red arrow: skin damage; Blue arrow: eyeball damage; Black arrow: body distortion. Oil-E and Oil-F did not show any noticeable differences.
Fig 3.
Major constituent amines and surfactants of emulsions exhibited various degrees of toxicity.
Zebrafish larvae at 14 dpf were tested at a concentration of 0.5% for amine (v/v) and 0.05% for surfactant. Sample size: n = 4 (one animal per well) for amines; n = 5 for surfactants. In the heat map, the locomotion activity of all animals tested under the same condition was recorded as a mean value over 5 minutes. * p < 0.05; ** p < 0.01; *** p < 0.001; p value, One-way ANOVA with Tukey’s multiple comparison test. The bars and error bars stand for means and standard deviations.
Fig 4.
Chemical structure and concentration affect toxicity.
(A) Difference in locomotion activity according to the three different types of ethanolamines. Primary (1°) amines are MDEA, MIPA, DGA, and AMP95. The secondary (2°) amine is DEA. Tertiary amines (3°) are TEA, MDEA, and BDEA. (B) Effect of the ethoxylate number of polyoxyethylene castor ether (KREL) on locomotion activity. Sample size: n = 10 (one animal per well). The mean locomotion activity of each animal was calculated, and then statistical analysis was conducted. (C) Effect of alkyl chain length of ethanolamines on locomotion activity. ‘-H’, ‘-CH3’, ‘-(CH2)3CH3’, and ‘-C6H6’ indicate DEA, MDEA, BDEA, and CHA, respectively. Zebrafish at 19 dpf were used to identify the more obvious differences in activity value. Sample size: n = 10. (D) Effect of dosage concentration on locomotion activity. * p < 0.05; ** p < 0.01; *** p < 0.001; p value, One-way ANOVA with Tukey’s multiple comparison test. The bars and error bars stand for means and standard deviations.
Fig 5.
Emulsion particle size affects toxicity.
TMP-82.5, which had a bigger emulsion particle size, was more toxic than TMP-72.5 and TMP-77.5. EO/PO-27.5 was a chemical mixture of 72.5% water and 27.5% EO/PO surfactant (w/w). Sample size: n = 10 (one animal per well) for TMP-72.5, TMP-77.5, and TMP-82.5; n = 5 for EO/PO-27.5 surfactant. * p < 0.05; ** p < 0.01; *** p < 0.001; p value, One-way ANOVA with Tukey’s multiple comparison test. The bars and error bars stand for means and standard deviations.