Concentration?response curves used for the calculations of the D. rerio: (a) mortality, (b) abnormality, and (c) unhatching rate after 96 h of exposure to CAT, GUA, 4NC, 5NG, and 4,6DNG. Error bars indicate standard deviations (SD). Dotted lines represent 95% confidence intervals. Dose?response curves were generated using GraphPad Prism software version 6.0

Graphical view of ECOSAR predicted and experimentally obtained toxicity values within the toxicity gradient according to the criteria set by the European Union³⁴ (Very toxic < 1 mg/L, Toxic 1 < LC₅₀ < 10 mg/L, Harmful 10 < LC₅₀ < 100 mg/L, Not harmful LC₅₀ > 100 mg/L). Predicted values were obtained from ECOSAR v2.0 software (https://www.epa.gov/tsca-screening-tools/ecological-structure-activity-relationships-ecosar-predictive-model), while GraphPad Prism software version 6.0 (https://graphpad-prism.software.informer.com/6.0/) was used for data presentation.

Recorded sublethal morphological effects in D. rerio embryos/larvae after 48, 72, and 96 h of exposure to CAT, 4NC, GUA, 5NG, and 4,6DNG. Negative control: normally developed embryo at (a) 48, (b) 72, and (c) 96 hpf. During exposure period alterations were manifested as: (d) yolk sac edema (arrow); (e) pericardial edema (asterisk), undeveloped tail region (arrow); (f) hatched fish with malformed spine (arrow); (g) underdeveloped tail and necrosis of its apical part (dashed arrow), rare pigments; (h) pericardial edema (asterisk), scoliosis (arrow), necrosis of the apical part of the tail (dashed arrow), rare pigments, not hatched; (i) scoliosis (arrows), blood accumulation in the brain region (dashed arrow); (j) pericardial edema (asterisk), yolk sac edema (arrow), scoliosis (dashed arrow); (k, l) pericardial edema (asterisk); (m) underdeveloped embryo: underdeveloped head (arrow), tail not detached (asterisk), delay or anomaly in the absorption of the yolk sac; (n) pericardial edema (asterisk), blood accumulation (arrow), not hatched; (o) pericardial edema (asterisk), blood clotting (arrow), not hatched; (p) blood accumulation at the yolk sac (arrow); (r) hatched fish with malformed spine; (s) pericardial edema (black asterisk), blood accumulation above the yolk sac (arrow), swelling of the yolk sac (white asterisk), yolk sac edema (dashed arrow), mild scoliosis. Developmental abnormalities were recorded using LAS EZ 3.2.0 digitizing software (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-ez/).

Morphometric measurements of D. rerio larvae after 96-h exposure to tested compounds (CAT, 4NC, GUA, 5NG, and 4,6DNG) and control (C). (a) Lateral view showing eye area (EA), head height (HH), yolk sac area (YSA), and pericardial sac area (PSA). Scale bar = 1000 µm. Morphometric parameters are presented by their mean value (b–e; n = 15). The symbol * indicates a significant difference between tested samples and negative control (*p < 0.05; **p < 0.01; ***p < 0.001). Mean values sharing common letters indicate significant differences among different tested compounds (*p < 0.05). A line within the box represents the median value, while the boundaries of box-plot indicate 25th and 75th percentiles. Whiskers above and below the box indicate 10th and 90th percentiles. Statistical analysis and data presentation were prepared using GraphPad Prism software version 6.0 (https://graphpad-prism.software.informer.com/6.0/), while visualization and morphometric measurements were performed using Microsoft AnalySIS Soft Imaging System software (https://www.olympus-lifescience.com/en/support/downloads/).

Representation of the interactions between (a) GUA and (b) 4,6DNG with the active site D. rerio CA II. Structure of D. rerio CA II (UniProt entry: Q92051) is modeled by I-TASSER, coordinates for Zn cation, and active site water molecule (oxygen atom in red) were taken from the structure of human CA deposited under PDB ID 1fql, whereas docking pose for GUA was obtained by SwissDock server. The same pose was obtained by AutoDock vina. Representation was made in PyMOL software (https://pymol.org/2/).