## 3 Results and Discussion
### Embryo-Larval Development Assay  
The effects of SiNC, DCOIT, and SiNC-DCOIT on the larval development rate of _P. perna_ are shown in Figure 2 and Table 1. Significant effects were observed as low as at the lowest tested exposure concentration of unloaded SiNC (6.5 \(\mu\)g L\({}^{-1}\)). Additionally, at 4040 \(\mu\)g SiNC L\({}^{-1}\) no developed veliger larvae were found (Figure 2a). The 48 h-EC\({}_{50}\) value was set at 39.8 \(\mu\)g SiNC L\({}^{-1}\), the highest value amongst the three tested compounds. Despite being the less toxic among the tested chemicals, the embryotoxicity caused by SiNC was much higher than expected since silica was expected to be, in principle, inert. According to [PERSON] et al. [12] this effect can be attributed to the cationic surfactant cetyltrimethylammonium bromide (CTAB) used in the synthesis of the mesoporous silica nanocapsules [23]. CTAB is a quaternary ammonium compound (QACs), a class of toxic surfactants towards aquatic species [12; 31]. Besides, residues of this organic component can be detected even after several washes [12] explaining the recently reported effects of SiNC on the fertilization and embryo-larval development of the sea-urchin _Paracentrotus lividus_[32] and the inhibition settlement of the bryozoan _Bugula neriitana_ larvae [33]. This raw nanomaterial is not considered environmentally dangerous when compared with the AF biocides, but our results indicate the importance of knowing its potential toxicity against a wide set of organisms in order to improve the manufacturing process of the engineered nanomaterial, as recently proposed by [PERSON] et al. [34]. Regarding the tested biocide, the development of mussel embryos was significantly affected at 3.3 \(\mu\)g L\({}^{-1}\) of DCOIT (LOEC). Above 33 \(\mu\)g L\({}^{-1}\) (Figure 2b) no tested organisms reached a well-developed veliger larvae stage. The embryotoxicity of DCOIT to _P. perna_ is in agreement with previous findings for other bivalve species from temperate regions, namely, the mussel _Mytitlus edulis_, for which [PERSON] et al. [35] and the USEPA Office of Pesticides Programs [36] reported 48 h-EC\({}_{50}\) values of 10.7 and 2.7 \(\mu\)g L\({}^{-1}\), respectively. Moreover, [PERSON] et al. [10] estimated the DCOIT 48 h-EC\({}_{50}\) at 6.9 \(\mu\)g L\({}^{-1}\) for embryos of the oyster _Crassostrea virginica_. In the SiNC-DCOIT treatment, the embryonic development rates differed significantly from the control at 0.32 \(\mu\)g L\({}^{-1}\) (Figure 2c). Moreover, the estimated 48 h-EC\({}_{50}\) of SiNC-DCOIT was set on 6.77 \(\mu\)g DCOIT L\({}^{-1}\) (Table 1), nearly 2-fold more toxic to the embryos of _P. perna_ than the free form of DCOIT (12.36 \(\mu\)g L\({}^{-1}\)). The present findings are the first evidence that SiNC-DCOIT can exhibit a better antifouling efficacy comparing with the non-nanostructured form of DCOIT in the pre-settlement stage of a fouler organism. This can be justified by the very low and slow release of DCOIT from the nanocapsules through diffusion, during the exposure period (undetectable by HPLC with a high  
Figure 2: Larval development rates observed in _P. perna_ embryos exposed to (**a**) SiNC, (**b**) DCOIT, and (**c**) SiNC-DCOIT. Asterisks (*) indicate significant differences relative to the control (\(p<0.05\)). Data are presented as average \(\pm\) standard deviation (SD). detection limit of 240 \(\upmu\)g DCOIT L\({}^{-1}\)[12]). Interestingly, similar findings were previously reported on settlement inhibition assays of other foulers, such as the bryozoan _Bugula neritina_, and the mussels _Mytitlus galloprovincialis_ and _Brachidontes pharaonis_, in a study comparing the efficacy of other AF nanomaterials containing Zn or Cu pyrithiones (ZnPT and CuPT) and the respective free/dissolved forms [33]. As an example, CuPT immobilized in engineered nanoclays (LDH) can be up to 250-fold more efficient in inhibiting the settlement of the Mediterranean _B. neritina_ larvae than CuPT [33].