## 3 Results and Discussion
### Short-Term Exposure Assay with Juvenile Mussel Stages  
The parameters estimated for the studied chemicals on byssus threads production are presented in Table 1. The number of byssus threads secreted by mussels exposed to the different treatments showed no significant differences comparing to organisms from the experimental control, except for SiNC-DCOIT (at 1000 \(\upmu\)g DCOIT L\({}^{-1}\)), in which the number was significantly lower. However, increasing concentrations of DCOIT and SiNC-DCOIT reduce byssus production (Figure 3b,c, respectively). The 72 h-EC\({}_{50}\) value of DCOIT was estimated at 96.1 \(\upmu\)g L\({}^{-1}\), being 3-fold more toxic than the SiNC-DCOIT (305.5 \(\upmu\)g DCOIT L\({}^{-1}\)) and one order of magnitude more toxic than the unloaded SiNC (1323 \(\upmu\)g L\({}^{-1}\)). These data confirm the high antifouling efficacy of free DCOIT, which inhibit the attachment of _P. perna_. In temperate and subtropical regions, mussels have been successfully used as a model system to test the antifouling activity of both free-forms and nanostructural biocides by determining their continued attachment by byssus threads [33].  
Regarding to evaluation of the capacity of organisms to survive to aerial exposure after the chemical exposure in water, mussels from the control group survived for approximately 53 h (Table 1). Mussels previously exposed to SiNC treatments survived in air, in average, \(40\pm 17\) h at 10 \(\upmu\)g L\({}^{-1}\), \(40\pm 21\) h at 100 \(\upmu\)g L\({}^{-1}\) and \(36\pm 23\) h at 1000 \(\upmu\)g L\({}^{-1}\). The air survival capacity of mussels exposed to unloaded SiNC did not differ significantly concerning the experimental control. SiNC-DCOIT impaired the mussels physiological capacity to survive to a prolonged aerial exposure, set on \(33\pm 12\) h at 10 \(\upmu\)g L\({}^{-1}\) and \(31\pm 11\) h at 100 \(\upmu\)g DCOIT L\({}^{-1}\) treatments (not possible to measure on the highest SiNC-DCOIT tested concentration (1000 \(\upmu\)g DCOIT L\({}^{-1}\)) due to the high lethality during the aqueous exposure). Juvenile _P. perna_ exposed to dissolved DCOIT presented the lowest survival-in-air rate. In average, the air survival was \(31\pm 17\), \(29\pm 14\), and \(24\pm 13\) h for animals previously exposed to 0.81, 8.1, and 81 \(\upmu\)g DCOIT L\({}^{-1}\), respectively, being significantly different from the control. It was possible to conclude that the average survival was reduced by less than 50% for mussels exposed to  
Figure 3: Byssus threads produced by juvenile mussels exposed for 72 h in aqueous solution/dispersion of (**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).  
81 \(\upmu\)g DCOIT L\({}^{-1}\) (Figure 4). Since _P. perna_ mussels live mainly at the intertidal level and are periodically exposed to air during tidal cycles, less ability to survive may indicate limited physiological capacity.  
Overall findings indicate that the biocide's encapsulation protected the mussels from significant effects compared with the DCOIT exposure. Similarly, the acute toxicity of DCOIT, in its free form, in the mussel _Mytilus galloprovincialis_, from the temperate region was much higher than the nanostructured form SiNC-DCOIT (72 h-EC\({}_{50}\) = 1270 \(\upmu\)g DCOIT L\({}^{-1}\) and 38,500 \(\upmu\)g DCOIT L\({}^{-1}\), respectively; [12]). According to the authors, these mussels close the valves as a defense mechanism to prevent the animals' exposure to contaminants. Thereby, the survival of mussels during the 72 h exposure to free DCOIT solutions can also be related to this mechanism to avoid exposure. In this sense, these individuals possibly remained without oxygenation of gills for a longer time, characterizing physiological effects that reduced their survival capability in a dry environment.