## 3 Results and Discussion  
### Danilo rerio  
3.1.1. Fish Embryo Acute Toxicity (FET) Test  
FOS caused no significant effects on the organisms' survival during 96 h of exposure (_p_ > 0.05, Figure 1A). In terms of hatching, organisms did not hatch at 200 mg/L FOS (_p_ < 0.05, Figure 1B). FOS did not cause significant malformations' induction and did not affect the heartbeat rate (_p_ > 0.05, Figure 1C,D).  
Based on the mentioned results, FOS showed low toxicity to zebrafish embryos. No study was found about the effects of FOS to zebrafish. A LC\({}_{50}\) of 428 mg/L was reported for the fish rainbow trout after 96 h exposure to FOS [37]. FOS has a period for 50% dispersion (DT\({}_{50}\)) of 3 d in surface waters [10]. Thus, the obtained effects on hatching may be mainly due to the parental compound FOS and not its metabolites: phosphonic acid and ethanol. A 20% effect concentration (EC\({}_{20}\)) to hatching of _Pimephales promelas_ was determined as 0.44 mg of active substance (FOS WG80 of Bayer CropScience)/L, for a chronic exposure [10]. Comparing both freshwater fish (_P. promelas_ versus _D. rerio_), the fungicide FOS WG80 was more toxic than the FOS used in the current study. Based on the literature, EFSA (2018) considered FOS as a compound with lower acute/chronic risk to aquatic organisms [10]. Regarding FOS metabolites' effects, in a study using disodium phosphonate (related to phosphonic acid), a LC\({}_{50}\) > 61.26 mg/L for _Daplinia magna_ (48 h, static exposure) was calculated [38], which showed a greater (severe) effect in comparison to the parental compound (FOS).  
Considering the effects of other pesticides to zebrafish embryos, a LC\({}_{50}\) of 0.235 mg/L was found after 24 h exposure to sumithion [12]. Increasing sumithion concentrations also decreased hatching success and induced several malformations on the organisms [12]. Triazole fungicides [13] and the insecticide diazinon [14] induced a strong inhibition of hatching in zebrafish embryos. From five tested pesticides, chlorpyrifos, \(\lambda\)-cyhalothrin, and butachlor had the highest toxicities to \(D\). _ rerio_ with a LC\({}_{50}\) value ranging from 0.28 to 0.45 mg/L [22]. Comparing the results from studies including other pesticides [12; 22] with the ones found in our study (malformations, heartbeat, hatching, and survival), FOS induced less toxicity for zebrafish embryos than the other referred pesticides.  
The organisms' hatching occurs from the activity of the hatching enzyme chorinase, muscle contraction, perivitelline pressure, and water uptake by the embryo [14]. FOS may disturb directly these processes, e.g., through the inhibition of the chorinase or less energy could be available for hatching since exposed organisms assign part of their energy to metabolic pathways for FOS elimination. Inhibition of hatching is generally classified as a sub-lethal effect; however, it can result in death or delay in the organism development/maturation if it is not reversed in a short term. Thus, hatching inhibition may cause a negative impact at the population level. There is a data gap concerning the involvement of hormones in the hypothesized mode of action of FOS as an endocrine disruptor. A study using a mixture of fungicides (without FOS) showed impairments in the reproductive performance of _Hyalella azteca_ (amphipod) [39]. It will be important to explore this issue, applying a multigenerational approach where multi-level hierarchical responses can be elucidated (e.g., at molecular, biochemical, and population level).