## 4 Discussion  
### Lethal Effect and Inhibition of the Hatching Rates by C10 toward Zebrafish Embryos  
The ecotoxicological toxicity of plant essential oils (EO) and their monoterpenoids in aquatic organisms, including various fish species, has been studied. Three EO derived from _Aloysia triphyllla_, _Lippi graclis_, and _Piper aduncum_ showed lethal effects on juvenile tambaqui (_Colossoma macroporum_) with LC\({}_{50}\) values (4 h of exposure) of 109.57, 41.63, and 48.17 mg.L\({}^{-1}\) for _A. triphyllla_ EO, _L. gracilis_ EO, and _P. aduncum_ EO, respectively [17]. These EOs caused severe damage to the gills of the tested fish larvae. However, the 96 h-LC\({}_{50}\) value for EO of thyme plant, thymol, EO of cumin seeds, and EO of caraway seeds was reported using a 30-day-old rainbow trout as 6.6, 2.6, 35, and 14 mg.L\({}^{-1}\), respectively [18]. Similarly, two monoterpenes, namely, thymol and 1,8-cineol, were assessed to determine their LC\({}_{50}\) values toward the insectivorous guppy fish _Poecilia reticulate_[19]. The 24 h-LC\({}_{50}\) values of thymol toward female and male guppy fish were 12.51 and 10.99 mg.L\({}^{-1}\), respectively, whereas the LC\({}_{50}\) values of 1,8-cineol toward female and male guppy fish were 3997.07 and 1701.93 mg.L\({}^{-1}\), respectively [19]. These two monoterpenes showed 319.5-fold and 154-9-fold differences for female guppy and male guppy, respectively [19].  
Figure 5: Graphs of the expression levels of cardiac development-related genes in C10 massoia lactone-treated zebrafish embryos at 96 hpf. C10 massoia lactone was treated as 250, 500, and 1000 \(\upmu\)g.L\({}^{-1}\) against the embryos. Data analyses were undertaken using a one-way ANOVA, and the differences in the means between control group and the treated groups were analyzed using post hoc Tukey tests (a \(>\) b \(>\) c, \(p\) \(<\) 0.05). Lowercase letters indicate a significant difference between the control and the C10 massoia lactone-treated groups; a \(>\) b \(>\) c (\(p\) \(<\) 0.05). The genes _nppa_, _myl7_, _myh6_, _tmt2a_, _myl7_, _gata4_, _nkx-2.5_, and _cacna1c_ are responsible for encoding natriuretic peptide A, myosin heavy chain 7, myosin heavy chain 6, troponin T type 2a (cardiac), myosin heavy chain 7, GATA binding protein 4, NK2 homeobox 5, and calcium voltage-gated channel subunit alpha 1C, respectively.  
In our study, C10 massoia lactone (C10) showed 100% mortality at a treated dose of 2 mg\(\cdot\)L\({}^{-1}\), and 100% paralyzed larvae were observed at 1 mg\(\cdot\)L\({}^{-1}\). However, no lethal effect on zebrafish embryos was found until 96 h exposure at 1 mg\(\cdot\)L\({}^{-1}\). This result was not directly different from the LC\({}_{50}\) value in previous reports because of the use of different fish organisms and their developmental stages, but this lactone exhibited strong mortality toward zebrafish embryos at a lower treatment concentration to kill 100% tested organisms. Thus, _Zingiber cassumunar_ EO showed 100% mortality at 100 mg\(\cdot\)L\({}^{-1}\) against zebrafish embryos [20], and Port Orford cedar heart oil showed 100% mortality toward _O. mykiss_ at 5.0 mg\(\cdot\)L\({}^{-1}\)[21]. Therefore, C10 was stronger than EO of _Z. cassumunar_ on zebrafish embryos for 96 h exposure.  
In the case of hatching rates, no direct comparison is performed between C10 and other EOs and their constituents using zebrafish embryos. However, there are many reports on effect of pesticides on hatching rates using the same organism. Fipronil (FIL), a phenylpyrazole insecticide, exhibited about 70% mortality and 85% hatchability in zebrafish embryos treated with 5.0 mg\(\cdot\)L\({}^{-1}\) of FIL [22]. In our study, 100% mortality was achieved in zebrafish embryos exposed to 2.0 mg\(\cdot\)L\({}^{-1}\) of C10, and at the same treatment concentration, hatchability was completely suppressed. In this regard, C10 caused a stronger inhibitory effect on hatchability when compared with the two insecticides, CPM, and FIL.