Exposure of embryonic zebrafish to cell culture medium and nanomaterials. Embryonic zebrafish were staged and dechorionated at 4 h post-fertilization (hpf) and placed in a 96 well plate at 6 hpf. The plates contained various test media and/or the ENMs were sonicated prior to being added to the plate. A total of 12 mortality and morphological effects were examined at 24 and 120 hpf. Figure created with BioRender.com.

Impacts of cell culture medium on zebrafish development. Each circle in the figure represents one animal with an adverse outcome for the indicated endpoint, with statistically adverse outcomes (n = 32; p < 0.05) shown in red. “MO24” describes any observed mortality effects observed at 24 hpf. “MORT” represents any observed mortality effects observed at 120 hpf. “any.effect” represents any adverse effect observed including morphological and mortality at 120 hpf. (A) Embryonic exposures to 0, 20, 40, 60, 80, or 100 % RPMI with 1 % UP and AB. No significant effects were observed at 80 % RPMI with antibiotics at 1 %. (B) Embryonic exposures to FBS solution made of 0, 2, 4, 6, 8, or 10 % FBS with 1 % AB. (C) Embryonic exposures to various combinations of 80 % RPMI, 2 % FBS, and 1 % UP and AB.

Embryonic zebrafish responses to ENMs in cell culture medium. Embryonic zebrafish were exposed to ENMs GO 400 nm x 400 nm, GO 1μm x 1 μm, CeO2, and ZnO where n = 32. Like Fig. 2, each circle represents an adverse outcome, with statistically significant outcomes (p-value < 0.05) shown in red. “MO24” is mortality at 24 hpf. “MORT” is mortality at 120 hpf. “any.effect” is cumulative morphology and mortality at 120 hpf. Fig. 3A, C, E, and G (left side of figure) show effects of ENMs in UP water with no addition of cell culture medium (-). Fig. 3B, D, F, and H (right side of figure) show effects of ENM exposures in the presence of cell culture medium. Nanoparticle concentrations used were 0, 5, 10.7, 23.2, and 50 µg/mL.