Ecotoxicity evaluation of polymeric nanoparticles loaded with ascorbic acid for fish nutrition in aquaculture

Abstract

Background: Ascorbic acid (AA) is a micronutrient essential for the mechanisms of reproduction, growth, and defense in fish. However, the biosynthesis of this micronutrient does not occur in fish, so it must be supplied with food. A difficulty is that plain AA is unstable, due to the effects of light, high temperature, and oxygen, among others. The use of nanoencapsulation may provide protection and preserve the physicochemical characteristics of AA for extended periods of time, decreasing losses due to environmental factors.

Method: This study evaluated the protective effect of nanoencapsulation in polymeric nanoparticles (chitosan and polycaprolactone) against AA degradation. Evaluation was made of the physicochemical stability of the nanoformulations over time, as well as the toxicological effects in zebrafish (Danio rerio), considering behavior, development, and enzymatic activity. For the statistical tests, ANOVA (two-way, significance of p < 0.05) was used.

Results: Both nanoparticle formulations showed high encapsulation efficiency and good physicochemical stability during 90 days. Chitosan (CS) and polycaprolactone (PCL) nanoparticles loaded with AA had mean diameters of 314 and 303 nm and polydispersity indexes of 0.36 and 0.28, respectively. Both nanosystems provided protection against degradation of AA exposed to an oxidizing agent, compared to plain AA. Total degradation of AA was observed after 7, 20, and 480 min for plain AA, the CS nanoparticle formulation, and the PCL nanoparticle formulation, respectively. For zebrafish larvae, the LC₅₀ values were 330.7, 57.4, and 179.6 mg/L for plain AA, the CS nanoparticle formulation, and the PCL nanoparticle formulation, respectively. In toxicity assays using AA at a concentration of 50 mg/L, both types of nanoparticles loaded with AA showed lower toxicity towards the development of the zebrafish, compared to plain AA at the same concentration. Although decreased activity of the enzyme acetylcholinesterase (AChE) did not affect the swimming behavior of zebrafish larvae in the groups evaluated, it may have been associated with the observed morphometric changes, such as curvature of the tail.

Conclusions: This study showed that the use of nanosystems is promising for fish nutritional supplementation in aquaculture. In particular, PCL nanoparticles loaded with AA seemed to be most promising, due to higher protection against AA degradation, as well as lower toxicity to zebrafish, compared to the chitosan nanoparticles. The use of nanotechnology opens new perspectives for aquaculture, enabling the reduction of feed nutrient losses, leading to faster fish growth and improved sustainability of this activity.

Keywords: Chitosan nanoparticles; Ecotoxicity; Polycaprolactone nanoparticles; Vitamin; Zebrafish.

Fig. 1

Size distributions obtained using the DLS technique. A Chitosan nanoparticles loaded with AA, before the spray drying process (NPS_CS_AA) (1) and after spray drying (NPs_CS_AA_SD) (2). B PCL nanoparticles containing AA, before spray drying (NPs_PCL_AA) (1) and after spray drying (NPs_PCL_AA_SD) (2)

Fig. 2

A In vitro release kinetics profiles for ascorbic acid (AA), chitosan nanoparticles containing ascorbic acid (NPs_CS_AA), and PCL nanoparticles containing AA (NPs_PCL_AA), for analyses performed in triplicate, with quantification by HPLC. Fitting of the Korsmeyer-Peppas mathematical model for the release of AA from B NPs_CS_AA and C NPs_PCL_AA

Fig. 3

Oxidation of non-encapsulated ascorbic acid (AA) and ascorbic acid encapsulated in chitosan nanoparticles (NPs_CS_AA) and PCL nanoparticles (NPs_PCL_AA). The oxidation of ascorbic acid was A induced using peroxymonosulfate and B occurred under aquarium conditions simulating the natural environment of fish

Fig. 4

A Mortality rates of embryos and larvae exposed to the different treatments (control, AA, NPs_CS, NPs_CS_AA, NPs_PCL, and NPs_PCL_AA) at different concentrations (3.12, 6.25, 12.25, 25, 50, 100, 200, and 400 mg/L). B–F Hatching rates of embryos exposed to ascorbic acid (AA), chitosan nanoparticles (NPs_CS), chitosan nanoparticles containing AA (NPs_CS_AA), PCL nanoparticles (NPs_PCL), and PCL nanoparticles containing AA (NPs_PCL_AA). For the statistical tests, ANOVA (two-way, significance of p < 0.05) was used to observe the statistical differences between groups with the same concentration, where a indicates a statistically significant difference, compared to the control group

Fig. 5

Hatching rates of embryos and larvae exposed to the treatments at different concentrations (LC₁₀, LC₂₀, LC₃₀, LC₄₀, and LC₅₀): A ascorbic acid (AA), B chitosan nanoparticles (NPs_CS), C chitosan nanoparticles containing AA (NPs_CS_AA), D PCL nanoparticles (NPs_PCL), and E PCL nanoparticles containing AA (NPs_PCL_AA). For the statistical tests, ANOVA (two-way, significance of p < 0.05) was used to identify differences between the groups, where a indicates a statistically significant difference, compared to the control group

Fig. 6

Evaluation of the motor development biomarkers A distance traveled and B speed for zebrafish (Danio rerio) larvae exposed to the different treatments: control, ascorbic acid (AA), CS nanoparticles (NPs_CS), CS nanoparticles containing ascorbic acid (NPs_CS_AA), PCL nanoparticles (NPs_PCL), and PCL nanoparticles containing ascorbic acid (NPs_PCL_AA). The exposures were performed for 96 h at different concentrations (LC₁₀, LC₂₀, LC₃₀, LC₄₀, and LC₅₀). For the statistical tests, ANOVA (two-way, significance of p < 0.05) was used to evaluate differences among the groups, with α, β, δ, σ, and λ indicating statistically significant differences from the control, LC₁₀, LC₂₀, LC₃₀, and LC₄₀ groups, respectively (n = 9)

Fig. 7

Acetylcholinesterase (AChE) activities of zebrafish larvae after exposure for 96 h to different concentrations (LC₁₀, LC₂₀, LC₃₀, LC₄₀, and LC₅₀) of ascorbic acid (AA), CS nanoparticles (NPs_CS), CS nanoparticles containing AA (NPs_CS_AA), PCL nanoparticles (NPs_PCL), and PCL nanoparticles containing AA (NPs_PCL_AA), compared to the control. For the statistical tests, Kruskal-Wallis test (p < 0.05) followed by Dun test was used to evaluate differences among the groups, with α, β, δ, σ, and λ indicating statistically significant differences from the control, LC₁₀, LC₂₀, LC₃₀, and LC₄₀ groups, respectively (n = 9)

Fig. 8

Evaluation of body length A and weight B of zebrafish fed different diets. The control group was fed commercial food (TetraMIN), while the other groups were fed TetraMIN supplemented with different concentrations (25, 50, and 100 mg/kg) of NPs_CS_AA and NPs_PCL_AA. Statistical tests were performed using ANOVA (two-way, significance of p < 0.05) to evaluate differences among the groups, where α, β, and δ indicate statistically significant differences from the control, 25 mg/kg, and 50 mg/kg groups, respectively (n = 10)