2.4.9.2. Juvenile snook and microme

2.4.9.2. Juvenile snook and micrometric beads. Three groups of ten individuals of juvenile snook where used in the bioassay. Two groups were fed with prepared calcium alginate microbeads containing AST nanoemulsions, and the third group with control pellets (Gemma Wean 0.2 micro-pellets, Skretting). Each group was immersed in a 150 L tank at half capacity with filtered / airy sea water at 14 °C and equipped with a water exchange system allowing continuous aeration of 8 mg/L of oxygen achieving a 96 % of oxygen saturation. Fishes were habituated during three days to the experimental conditions previous to the development of the bioassay that lasted 20 days. Administered dose of the diets (microdiets and control food) corresponded to a 3 % of the total biomass of each group and was divided in two daily rations furnished at 9:00 and 16:00 h. The consumption was analyzed indirectly by collecting the residuals of each tank, two hours after the last administration, and separating the feces from unconsumed diet. Then the collected microdiets and control food were weighed and compared with the administered mass of each diet, thus obtaining an approach of food uptake. The specific growth rate (SGR) was analyzed after the 20 days bioassay period following the equation: SGR = ln(final biomass / initial biomass) / time (1) 3. Results and discussion3.1. Formation and characterization of the nanosystems containing AST. Anionic nanoemulsions were prepared by pouring the organic phase containing AST into water. The obtained nanoemulsions presented nanodroplets of hydrodynamic diameter in the range of 150 to 160 nm of diameter, showing a low PDI ranging between of 0.086 and 0.108, and a negative zeta potential ranging between -27 and -55 mV (n = 4), comparing different batches. The negative charge of the nanodroplets is responsible for the stability of the nanosuspension. The stability of the formulation in aqueous medium at 4 °C corresponding to a single batch has been analyzed during a lapse of 18 days, and the results are shown in Figure 2A, error bars corresponding to the different measurements for a single batch (n = 3). It can be observed that the hydrodynamic diameter of the nanodroplets is conserved, and the zeta potential remains in the negative part of the scale, lower than -20 mV, considered as a threshold beyond which the nanoemulsions lose stability. The formation of the nanosystems has been also corroborated by STEM analyses, as can be seen in Figure 3A, where nanostructures of spherical appearance are seen.Chitosan-coated nanoemulsions were prepared by pouring the organic phase containing AST into a chitosan aqueous solution. Due to that chitosan and some surfactants in the EPIKURON 145 V mixture are sensitive to the pH, the pH was adjusted to different values. The results concerning the hydrodynamic diameter and zeta potential of the formed coated nanoemulsions are shown in Table 1. The error values given correspond to the different measurements for a single batch. Values considering different batches at pH 3.5 differ from each other so that the hydrodynamic diameter for the different coated nanoemulsions have been found to range between 218 and 255 nm (n = 3). For the reported batch in Table 1, it can be seen that the chitosan-coated nanoemulsions