## 3 Results and Discussion  
### Fertilization Assay  
All tested treatments exhibited significant effects compared to the negative control (Figure 1; Table 1). The 40 min-EC\({}_{50}\) value of DCOIT (free form) was estimated at 0.063 ug L\({}^{-1}\) (Table 1), roughly 137-fold more toxic than the nanostructured form SiNC-DCOIT (8.6 ug DCOIT L\({}^{-1}\)) and 4 orders of magnitude more toxic than the unloaded SiNC (161 ug L\({}^{-1}\)). The high difference between toxicity of SiNC-DCOIT in comparison with DCOIT may be related to the biocidal controlled release property of SiNC-DCOIT, which occurs gradually in time and by predefined stimuli [12; 13].  
These results stress the high toxicity of the conventional form of DCOIT by inhibiting the fertilization of _P. perna_ gametes even at very low and environmentally relevant exposure concentrations. As a comparison, the EC\({}_{50}\) reported for the sea urchin _Paracentrotus lividus_ fertilization assay was 198 ug L\({}^{-1}\)[30]. However, it is worth pointing out that the experiment was slightly different since sperm was pre-exposed for 45 min, and the subsequent fertilization was carried out in artificial seawater without DCOIT. In the present study, the fertilization was carried out in the same test-solutions that sperm was exposed for 40 min.  
Previous studies demonstrate that DCOIT was more toxic (i.e., efficient) towards temperate target species, namely the bacterium _Vibrio fischeri_ (IC\({}_{50}\) = 299 ug L\({}^{-1}\) of free DCOIT vs. IC\({}_{50}\) = 459 ug DCOIT L\({}^{-1}\) for SiNC-DCOIT) and the diatom _Phaeodactylum tricontum_, involved in the biofilm formation  
\begin{table}
\begin{tabular}{c c c c c c} \hline  & **Parameter** & **NOEC** & **LOEC** & **EC\({}_{50}\)** & **95\% CI** \\ \hline \multirow{4}{*}{SiNC} & Fertilization & \(<\)250.0 & 250.0 & 161.3 & 139.3–186.8 \\  & Embryotoxicity & \(<\)6.5 & 6.5 & 39.8 & 10.5–150.8 \\  & Byssus threads & 1000 & \(>\)1000 & 1323 & 218.2–8019 \\  & Air survival capacity & 1000 & \(>\)1000 & nd & – \\ \hline \multirow{4}{*}{DCOIT} & Fertilization & \(<\)1.0 & 1.0 & 0.063 & 0.016–0.255 \\  & Embryotoxicity & 1.0 & 3.3 & 12.4 & 9.9–15.4 \\  & Byssus threads & 81.0 & \(>\)81.0 & 96.1 & 6.3–470 \\  & Air survival capacity & \(<\)0.810 & 0.810 & nd & – \\ \hline \multirow{4}{*}{SiNC-DCOIT} & Fertilization & \(<\)24.7 & 24.7 & 8.6 & 4.9–15.0 \\  & Embryotoxicity & 0.064 & 0.320 & 6.8 & 2.7–16.9 \\ \cline{1-1}  & Byssus threads & 100.0 & 1000 & 305.5 & 124.2–751.5 \\ \cline{1-1}  & Air survival capacity & \(<\)10.0 & 10.0 & nd & – \\ \hline \end{tabular}
\end{table}
Table 1: NOEC, LOEC, EC\({}_{50}\) values and respective confidence intervals (95%) of DCOIT, SiNC-DCOIT, and SiNC on gametes fertilization, embryo-larval development,byssus threads production, and survival-in-air of _P. perna_. Units are given in ug L\({}^{-1}\). SiNC-DCOIT values correspond to the concentration of encapsulated DCOIT (ug DCOIT L\({}^{-1}\)). “nd”: not determined.  
Figure 1: Fertilization rates observed for _P. perna_ gametes 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).  
(IC\({}_{50}\) = 4 \(\mu\)g L\({}^{-1}\) and 7 \(\mu\)g L\({}^{-1}\) for free DCOIT and SiNC-DCOIT, respectively [12]). Nevertheless, in such study, the sibling nanomaterial containing also silver (SiNC-DCOIT-Ag), demonstrated the opposite being much more efficient than the dissolved forms of DCOIT or Ag towards the tested target species [12].