The development of the embryos from blastulas to early life stages at defined times was observed with a stereomicroscope (×8 to × 50). The endpoints used for assessing developmental toxicity were recorded and described for embryos in both the control and treated groups [30]. The observation times Tideglusib ic50 were at 4, 8, 12, 16, 24, 36, 48, 72, and 96 hpf. Lethal and sublethal endpoints were used for determining the combined toxicological effects, including embryo

survival, coagulated eggs, malformation, no extension of tail at 24 hpf, no spontaneous movements within 20 s, no heartbeat, no blood circulation and weak pigmentation, heart sac edema, spine deformation, and hatching rate. Determination of dispersed ABT263 TiO2-NPs concentrations in exposure solutions During processes of the embryo exposure, dispersed TiO2-NPs concentrations were monitored using an UV–VIS spectrophotometer (UV-2550, Shimadzu Corporation, Kyoto, Japan).

Spectral scans of the sonicated TiO2-NPs suspensions (200 to 700 nm) gave the typical profile with find more a peak at about 329 nm. The absorbance spectra from dispersed TiO2-NPs are shown in Figure 2A, which shows an example of 60 mg/L TiO2 solution after sonicating for 30 min compared to 20 mg/L BPA solution and dilution water. Water samples were analyzed against 0 to 60 mg/L TiO2-NPs standards. The equation for the standard curve is y = 0.0149x − 0.0217, r 2 = 0.9892. Percentages of dispersed TiO2-NPs concentrations at 0, 6, 12, and 24 h after dosing the embryos are shown in Table 1. Figure 2 Absorbance spectra (A), standard curve of BPA (B), and chromatograms of BPA 5 mg/L + TiO 2 10 mg/L (C, D). Table 1 Percentages of dispersed

TiO 2 -NPs concentrations Exposure dose (mg/L) Percentages of dispersed TiO 2-NPs concentrations in exposure FER solutions (%) 0 h 6 h 12 h 24 h T2.5 99 96 93 88 T5.0 97 96 94 89 T10 99 98 92 87 T20 99 97 83 81 T40 99 97 88 79 B0.5 + T10 99 96 89 87 B1.0 + T10 99 95 90 84 B2.0 + T10 99 95 89 82 B5.0 + T10 99 98 91 85 B10 + T10 99 95 89 82 B20 + T10 99 97 91 85 Statistical analysis All data were obtained from the toxicological endpoints and were analyzed by type and severity. Significant differences between each exposure group and the control group were determined by one-way ANOVA within the same treatment group. For different treatments, a chi-square test was used to compare the BPA alone-exposed group with the mixture-exposed groups. A p value <0.05 was considered statistically significant. The graphs were compiled using ORIGIN 7.0 (OriginLab Corp., Northampton, MA, USA). Results Changes in BPA concentration before and after mixture exposure in vitro In this test, we determined that the BPA concentrations of the supernatants decreased after exposure to the BPA and TiO2-NPs mixture. The equation for the standard curve of BPA is Y = 29,221.8X + 1945.1 (a = 29,221.8, b = 1945.1, r 2 = 0.9998) (Figure 2B).