BP-3 and its metabolites have been detected in the aquatic environment with consistency (Bae et al., 2016). BP-3 is commonly found in natural waters, with concentrations ranging between 0.8 and 8 µg L− 1 (Balmer et al. 2005). To date, researchers have evidenced the toxic effects of this sunscreen on several species of fishes, including D. rerio, at concentrations ranging from the ones used in the present study (Coronado et al. 2008, Blüthgen et al. 2012).
For the present study, the mean (± one standard deviation) measured concentrations of BP-3 were 0.9 ± 0.1, 8.7 ± 2.9 µg L− 1, respectively, for nominal concentrations of 1 and 10, µg L− 1, respectively. BP-3 was not detected in the control and solvent control water samples.
Summary statistics of the tested biomarkers are presented in Table 1. The PERMANOVA of apical endpoints indicated no significant differences between treatments (Pseudo-F = 1.12, P(perm) = 0.34, Fig. 1). BP-3 concentrations used in this study did not influence survival or hatching rates. In discordance to our results, Cao et al. (2016) stated that many chemical substances could modify the hatching rate on D. rerio.
Table 1
Summary statistics of biomarkers in zebrafish embryos after 96h exposure to 3-Benzophenone. sd = standard deviation.
Biomarker | Control | Solvent Control | 1 µg L− 1 BP-3 | 10 µg L− 1 BP-3 |
| Mean | Median | sd | Mean | Median | sd | Mean | Median | sd | Mean | Median | sd |
Survival % | 82.56 | 90 | 18.11 | 73.22 | 72 | 20.12 | 78.11 | 86 | 23.25 | 71.33 | 77 | 21.89 |
Hatching rate % | 83.67 | 88 | 18.7 | 77.44 | 83 | 16.58 | 74.22 | 78 | 21.67 | 82.22 | 84 | 10.76 |
AChE nmol/min/mg prot | 93.06 | 83.07 | 18.03 | 72.37 | 72.1 | 6.15 | 70.96 | 70.57 | 3.47 | 64.99 | 62.61 | 6.65 |
AChEgene relative expresion | 1 | 1 | 0 | 0.70 | 0.45 | 0.68 | 3.227 | 1.78 | 2.87 | 2.35 | 2.80 | 1.05 |
CATgene relative expresion | 1 | 1 | 0 | 1.86 | 2.06 | 1.22 | 2.27 | 1.68 | 1.30 | 0.91 | 1.01 | 0.69 |
SODgene relative expresion | 1 | 1 | 0 | 1.19 | 1.35 | 0.84 | 1.52 | 1.21 | 0.58 | 0.79 | 0.73 | 0.56 |
GPXgene relative expresion | 1 | 1 | 0 | 0.76 | 0.38 | 0.76 | 2.89 | 2.64 | 1.80 | 2.07 | 2.24 | 0.88 |
LPO nmol/embryo | 19.64 | 21.25 | 2.99 | 28.86 | 31.81 | 10.93 | 25.51 | 28.32 | 4.99 | 29.22 | 22.37 | 17.26 |
GSH nmol/mg prot | 5.13 | 5.25 | 1.44 | 6.45 | 7.31 | 1.602 | 11.09 | 12.19 | 4.42 | 10.64 | 10.91 | 1.87 |
AChE catalyzes the rupture of the neurotransmitter acetylcholine. Organophosphate pesticides and carbamates are the typical AChE inhibitors, but it has been reported many other compounds that have anti-AChE properties (Colović et al., 2013). The PERMANOVA of AChE data indicated that there were significant differences (Pseudo-F = 3.66, P(perm) = 0.02). Pair-wise tests show significant differences between control and 10 µg L− 1 BP-3 ( P(MC) = 0.037, Fig. 2). There is an increase in AChE gene expression and a diminution of AChE activity as BP-3 concentration augments. Our results are different from previous studies with other animal models that have concluded that AChE is not inhibited by BP-3 (Campos et al. 2017a,b; Muñiz-González and Martínez-Guitarte, 2018). It is essential to note that in these previous studies, the gene expression of AChE was not evaluated. On the other hand, neurotoxicity of BP-3 has been previously reported; Feduik et al. (2010) found that BP-3 decreased cell viability in rat neurons. In accordance, Broniowska et al. (2019) noted in neuroblastoma cell lines reduced cell viability and increased caspase-3 activity. Interestingly, there are also reports of benzophenone-based derivatives that have been proposed as potent and selective AChE inhibitors (Belluti et al., 2011).
Zebrafish embryos exposed to BP-3 did not show significant differences in antioxidant system biomarkers. PERMANOVA indicated no significant differences between treatments (Pseudo-F = 1.60, P(perm) = 0.12 ). The PCO results for the biomarkers of the antioxidant system indicated that the first two axes were able to explain 69.13% of the total variation. Gene expression of CAT and SOD were strongly associated with PCO1, whereas expression of GPX and LPO and GSH concentrations were associated with PCO2 (Fig. 3).
Records about the effect of antioxidant enzymes formed in response to the production of ROS during exposure to pollutants are diverse (Richetti et al. 2011; Blahová et al. 2013; Praskova et al. 2014). In the specific case of BP-3, data are very inconclusive. Rodríguez-Fuentes et al. (2015a) observed a trend toward an increase in GPx gene expression as a significant positive correlation in eleuthero-embryos exposed to BP-3. Still, no significant differences in SOD, CAT, GPx transcription were found. Campos et al. (2017) did not see significant differences in lipid peroxidation or CAT and GST activities; however, they found significant differences in total glutathione levels when they exposed the aquatic insect Sericostoma vittatum to various concentrations of BP-3. Li et al. (2018) report enhanced CAT activity in zebrafish embryos exposed to a mix of UV-filters, including BP-3, that suggest an adaptive response and a compensatory mechanism to avoid oxidative stress.
Differences between studies could be due to several factors, like the life stage and the organisms' biotransformation capacities. Le Fol et al. (2017) mention that biotransformation processes are a critical factor influencing toxic response, but there are significant gaps of information regarding the characterization of functional metabolic capacities expressed in embryo and adult zebrafish. Moreover, Blüthgen et al. (2012) reported that unlike that observed in adults, BP-3 is not metabolized to benzophenone-1 (BP-1) in zebrafish eleuthero-embryos (up to 5 dpf), probably because the required enzymes are not fully active at this stage of fish development. These metabolic differences may influence the biomarker response since an increase in the toxicity of BP-1 compared to BP-3 has been reported due to BP-1 higher hydroxylation (He et al., 2019). It has also been documented a low gene expression of antioxidant enzymes in D. rerio in response to prooxidants because of immature antioxidant and detoxification systems (Rodríguez-Fuentes et al. 2015a). The chorion also plays an essential protective role (Embry et al., 2010; Negro et al., 2014). The chorion is present in the embryo of D. rerio until 48 hpf (Kimmel et al. 1995); therefore, the organisms are directly exposed to the contaminants for a shorter time. In supporting this evidence, Mu et al. (2013) found higher sensitivity to difenoconazole in the larvae of zebrafish than the embryo.
In conclusion, acute exposure to BP-3 in zebrafish embryos did not show differences in survival, hatching rate, or antioxidant system. In contrast, there were significant differences associated with AChE gene expression and activity. It is crucial to expand the information on the effects of organic UV-filters (individual compounds and mixtures) on fish and other organisms at different stages of development, given the increase in the use of these substances worldwide due to their presence in a large number of products like PCPs, textiles, plastics, and paints.