Dietary Microplastic Administration during Zebrafish (Danio rerio) Development: A Comprehensive and Comparative Study between Larval and Juvenile Stages

Abstract

One of the main sources of MPs contamination in fish farms is aquafeed. The present study investigated, for the first time through a comparative approach, the effects of different-sized fluorescent MPs included in a diet intended for zebrafish (Danio rerio). A comparison based on fish developmental stage (larval vs. juvenile), exposure time, and dietary MPs' size and concentration was performed. Four experimental diets were formulated, starting from the control, by adding fluorescent polymer A (size range 1-5 µm) and B (size range 40-47 µm) at two different concentrations (50 and 500 mg/kg). Zebrafish were sampled at 20 (larval phase) and 60 dpf (juvenile stage). Whole larvae, intestine, liver and muscles of juveniles were collected for the analyses. Polymer A was absorbed at the intestinal level in both larvae and juveniles, while it was evidenced at the hepatic and muscular levels only in juveniles. Hepatic accumulation caused an increase in oxidative stress markers in juveniles, but at the same time significantly reduced the number of MPs able to reach the muscle, representing an efficient barrier against the spread of MPs. Polymer B simply transited through the gut, causing an abrasive effect and an increase in goblet cell abundance in both stages.

Keywords: fish development; histology; immune response; microplastics; oxidative stress.

Figure 1

Representative images of zebrafish larvae analysed through confocal microscopy. (a) Focus on intestine from a zebrafish larva fed a control diet; (b) polymer B fluorescent microbeads in the gut lumen of a zebrafish larva fed a B500 diet; arrow indicates the direction of the gut tract, from cranial to caudal region; (c) whole larva fed an A50 diet; (d,e) focus on intestine and liver from larvae fed an A50 diet; (f,g) z-stack images of intestine from zebrafish larvae fed A50 and B50 diets, respectively. Asterisks indicate polymer A microbeads; arrowheads indicate polymer B microbeads. Abbreviations: M, muscle tissue; G, gut tract. * indicates microbeads.

Figure 2

Representative images of (a,d) intestine, (b,e) liver, and (c,f) muscle samples of zebrafish juveniles fed Control (a–c) and A500 (d–f) diets. * indicates microbeads.

Figure 3

Examples of histomorphology of the gut tract and liver of zebrafish (a–e) larvae and (f,g) juveniles. (k–o) Examples of Ab+ goblet cells in intestinal mucosal folds. (a,f,k) Control; (b,g,l) A50; (c,h,m) A500; (d,i,n) B50; (e,j,o) B500. Scale bars: (a–e) 200 µm; (f–g) 100 µm; (k–o) 50 µm.

Figure 4

Relative mRNA abundance of genes involved in immune response (a) il1b, (b) il10, and (c) litaf and oxidative stress (d) sod1, (e) sod2, and (f) cat in analysed zebrafish larvae. Control: zebrafish fed a control diet; A50 and A500 groups: zebrafish fed diets containing 50 mg/kg and 500 mg/kg of polymer A (range size: 1–5 µm), respectively; B50 and B500 groups, zebrafish fed diets containing 50 mg/kg and 500 mg/kg of polymer B (range size: 40–47 µm), respectively. Data are reported as mean ± standard deviation (n = 5). ^a,b Different letters denote statistically significant differences among the experimental groups; ns, no significant differences.

Figure 5

Relative mRNA abundance of genes involved in immune response (a) il1b, (b) il10, and (c) litaf and in oxidative stress (d) sod1, (e) sod2, and (f) cat analysed in intestine and liver, respectively, of zebrafish juveniles. Control: zebrafish fed a control diet; A50 and A500 groups: zebrafish fed diets containing 50 mg/kg and 500 mg/kg of polymer A (range size: 1–5 µm), respectively; B50 and B500 groups, zebrafish fed diets containing 50 mg/kg and 500 mg/kg of polymer B (range size: 40–47 µm), respectively. Data are reported as mean ± standard deviation (n = 5). ^a,b Different letters denote statistically significant differences among the experimental groups; ns, no significant differences.