In the present study, the effects of different concentrations of the acrylic resin and its components were separately evaluated during the initial development of the zebrafish. This study showed that 120 hours of exposure to the resin and its components can cause significant changes in heart rate, cognitive deficits, and cellular apoptosis. Exposure to treatments did not cause major morphological abnormalities in developing embryos, such as pericardial and yolk sac edema, caudal deformities, and curvature of the spine, among others, which often occurs in embryos exposed to substances known to be toxic . Despite that, there were discrete and isolated alterations in some measurements, such as interocular distance in animals exposed to MMA, formaldehyde, and DB (Fig. 3). Therefore, it is not possible to conclude that such substances have no teratogenic potential, since they may not have been concentrated enough to express such potential.
The acrylic resin group showed no significant difference for survival and hatching parameters. Regarding the morphological and behavioral parameters, only slight changes were observed. No studies have been found in the literature that have evaluated the toxicity of the acrylic resin in animals, but many studies have evaluated allergic contact reactions. In the current study, the acrylic resin caused a significant deficiency in the escape response at all concentrations (Fig. 1), suggesting that it may cause cognitive deficits in zebrafish, in addition to a significant increase in cellular apoptosis levels (Fig. 4). A decrease in heart rate was also observed, suggesting a potential toxic effect on the cardiac system (Fig. 2).
The MMA group, despite being the main component of the acrylic resin (96%), showed a significant increase in heart rate even at its lowest concentration (Fig. 2), while the deficit in the escape response was significant only at the highest concentration, differently from the resin group which even at its lowest concentration showed significant cognitive deficit (Fig. 1). These differences may suggest an important role of the other components on the resin. Regarding the cellular apoptosis parameter, only the lowest concentration showed no significant difference (Fig. 4). In vitro  and in vivo studies  have shown toxic effects of MMA. It has also been shown to be toxic in human neuron-enriched primary culture, derived from embryonic brain tissue . Developmental defects were observed in methacrylate exposed embryo, as pericardial edema .
Studies in animals have shown the neurotoxic potential of formaldehyde, and that exposure to this compound may induce deficits in spatial learning and memory , symptoms of cognitive dysfunction frequently accompanied by anxiety and depressive disorders , in addition to intensifying aggressive behaviors in rats . Formaldehyde is also responsible for causing toxicity in the reproductive system of rats in short- and long-term exposure  and oxidative stress in cardiac rainbow trout tissue . Studies in humans have shown the neurotoxic potential of this compound and may be an important factor in the induction of Alzheimer's disease, since it may be responsible for loss of memory, cognitive dysfunction, reduction of cholinergic signals and increased acetylcholinesterase activity in humans [43–45]. In this study, formaldehyde showed a significant increase in heart rate (Fig. 2) and cell apoptosis at its highest concentrations (Fig. 4). There was also a decrease in the ability to escape to the aversive stimulus even at its lowest concentration (Fig. 1), as well as in the resin group, which may suggest a great influence of this compound on the toxicity of the acrylic resin for this parameter. These results corroborate the toxicological potential of formaldehyde found in the above-mentioned studies.
In this study, the DB group presented a mortality rate of 100% in its highest concentration, still in the embryonic stage (2o dpf). Many studies have shown that the resin releases by products into the oral cavity [15, 46], so it is possible that the mortality of 100% of the higher dose embryos in the DB group may be related to resin toxicity observed in clinical and in vitro studies. The DB is also responsible for causing reproductive abnormalities, oxidative stress, embryonic toxicity, and neurotoxicity in fish [47–49], rats  and even in children . In this study it was observed that DB, even at its lowest concentration, showed a marked level of cellular apoptosis (Fig. 4) and cognitive loss (Fig. 1), which is considered a toxicological parameter.
The PB group showed no significant difference for the survival and hatching parameters. As with the other groups, there were discrete morphological and behavioral changes and significant alterations in cardiac rhythm and cellular apoptosis in their higher concentration, and a decrease in the escape response only at the highest concentration. The literature is still scarce of toxicological studies with this compound in animals, being more explored in research on dermatitis since it is widely used in the treatment of acne.
All groups, at their highest concentrations, showed changes in heart rate (Fig. 2). The Resin, DB and PB groups showed a decrease in heart rate, while the MMA and formaldehyde groups showed increase. The MMA (increased heart rate) and DB (decreased heart rate) groups showed changes even at their lower concentrations. Even though MMA was the main component of the acrylic resin, it still showed a decrease in the rhythm, which suggests that for this parameter dibutyl phthalate exerted a greater influence than the MMA on the resin group.
In addition to assessing mortality and hatch rates, behavioral tests can also be a simple and rapid method to obtain additional information on the toxic effects of different substances on embryonic and nervous system development . In this study, we observed discrete behavioral changes in zebrafish larvae in all groups tested.
Regarding cognitive abilities, all groups showed a decrease in this capacity in the 5 dpf (Fig. 1). Resin and formaldehyde groups showed a significant decrease in the escape response even at their lower concentrations. Formaldehyde showed a dose dependent response. Although MMA is the main component of the resin, only the highest concentration of MMA showed a decrease in the escape response. Formaldehyde was the only component of the resin that showed a significant reduction of this ability at the lowest concentration, which may suggest a great influence of this component on the resin, since it can be considered more toxic than MMA, requiring more attention .
Cellular apoptosis is an important process that occurs during the development of vertebrates, but the exacerbated loss of cells by this type of programmed cell death can also have irreversible deleterious consequences throughout life . The results of the present study showed that exposure to acrylic resin and all its components significantly increased apoptosis when compared to its controls, suggesting that all its components cause cell death, some at a higher level than others (Fig. 4). MMA showed a dose dependent relationship, which caused more cell death in its higher doses, in comparison to the lower and the control doses. The DB and PB groups also had a dose dependent relationship. Interestingly the controls of these two groups showed a much higher level of cellular apoptosis than in controls of the other groups, which may suggest that these compounds release toxic vapors that may have interfered in their controls, since all treatments were performed on plaques of 6 wells that were closed after treatment changes. These plates were opened again only the other day for further treatment change.
More in vivo research is needed to deepen the knowledge of the toxicological potential that acrylic resin can have since it is a widely used material in dentistry, and where people are exposed for long periods of time either by direct contact, or by inhalation.
Based on the results found, it can be concluded that acrylic resin and all its components present toxicological potential, which can cause cardiac alterations, cognitive losses and increased cellular apoptosis. Although MMA is the main component of acrylic resin, it appeared to have less influence than formaldehyde on cognitive abilities parameters and less influence than dibutyl phthalate on heart rate.
This study showed the toxicological potential of orthodontic acrylic resin on the initial development of zebrafish, reinforcing this animal model as a potential tool for future studies in dentistry.