Microplastics ( size < 5 mm; MPs) have been found in almost every environment including oceans and represents a widely diffused pollutant for aquatic organisms. Several studies have demonstrated the presence of MPs in marine animals , from plankton to higher trophic levels. Furthermore, sea-farmed aquatic species have been shown similar rates of MPs accumulation as those evidenced by wild specimens. More recently, the problem of MPs contamination in farmed fish has been found to also affect the land aquaculture sector. In this context, the main MPs source is represented by aquafeeds since conventional marine-derived ingredients used for their formulation derived from caught fish . Particularly, it has been shown that the concentration of MPs in fish meal is higher than that found in the raw materials since the processing procedures of the ingredients and packaging methods significantly contribute to increase the final amount of MPs in the aquafeed (polyethylene is one of the most widely used materials to produce "storage bags" for fishmeal). Dietary MPs contamination can have negative effects on farmed fish during different life-cycle stages. The larval development is one of the most critical phases because of the fast morphological and behavioural changes that increase the risk of mortality. During this phase: (i) possible obstruction of the gastrointestinal tract; (ii) a reduced predatory activity caused by an apparent feeling of satiety; (iii) a decrease in growth and swimming capacity; (iv) the activation of inflammatory responses in gut and other organs because of potential translocation processes can occur. However, despite the large number of publications on the presence of MP s in fish, not much is known about the long-term MPs exposure and the effects on the different life cycle stages and developmental phases of fish . In this regard, t he present study aims to compare the effects of dietary MPs exposure in zebrafish ( Danio rerio ) larval and juvenile stages , monitoring MPs translocation among organ and tissues and the effects on fish growth and welfare through a multidisciplinary laboratory approach.
Materials and Methods
Five experimental diets were used in this present study. A c ontrol diet was prepared according to a commercially available standard diet for zebrafish (Zebrafeed, Sparos ltd, Portugal). The four experimental diets containing MPs were prepared by adding during the preparation of Control diet the fluorescent polymers A and B at two different concentrations, as follows: (i) 50 mg/kg feed of polymer A (diet A50); (ii) 500 mg/kg feed of polymer A (diet A500); (iii) 50 mg/kg feed of polymer B (diet B50); (iv) 500 mg/kg feed of polymer A (diet B500). The microbeads were purchased from Cospheric LLC (Goleta, CA, USA) and their features were: (i) polymer A: amino formaldehyde polymer, 1-5 µm of dimensional range, peak of emission at 636 nm when excited at 584 nm; (ii ) polymer B: polyethylene, 40-47 µm of dimensional range, peak of emission at 607 nm when excited at 575 nm . After hatching, zebrafish larvae were initially reared in fifteen 20 L tanks (3 tanks per experimental group; 500 larvae per tank). After 20 days post fertilization (dpf), fish of each tank were transferred in 100 L tanks (3 tanks per experimental group). Zebrafish were fed the experimental diets two times a day (daily dose corresponding to the 3% of the body weight) from 5 to 60 dpf. The required amount of fish were sampled at both 20 dpf (in which whole larvae were collected for each analyses) and at 60 dpf (in which samples of liver, intestine, and muscle were collected from juveniles). For both larvae and juveniles, the following parameters were evaluated: (i) survival and specific growth rate (SGR%); (ii) the absorption of the fluorescent MPs microbeads at intestinal level and their potential translocation to liver and muscle through a Nikon A1R confocal microscope (Nikon Corporation, Tokyo, Japan) ; (iii) the MPs microbeads quantification in whole larvae or in the target organs of juveniles through chemical digestion followed by a vacuum filtration on 0.7 µm pore-size fiber-glass filters and consequent analyses of the filters through a fluorescence microscope (Zeiss Axio Imager.A2 ; Zeiss, Oberkochen, Germany); (iv) possible structural alteration of the intestin al epithelium and the hepatic parenchyma through the application of a series of histopathological indexes and stains; (v) the relative expression of genes involved in immune (il1b, il10 , and litaf ) and oxidative stress response (sod1 , sod2, and cat) starting from total RNA extraction from whole larvae or liver and intestine samples for juveniles.
Results and Discussion
No significant differences in survival and specific growth rates were detected among the experimental groups for both zebrafish larvae and juveniles . The ingestion of the two polymers used in this study was confirmed both by confocal microscopy and MPs quantification in the zebrafish larvae and juveniles. However, only the polymer A (size 1-5 µm) was absorbed at intestinal level in both larvae and juveniles. The presence of polymer A microbeads was detected in liver and muscle samples only in juveniles suggesting a time-related translocation from the gastrointestinal tract. Furthermore, the MPs quantification in both whole larvae and in intestine and liver samples of juveniles highlighted a dose dependent accumulation of polymer A. Regarding polymer B (size 40-47 µm), no absorption was detected, but the transit through the intestinal tract caused a reduction of mucosal folds length and an increase in goblet cells relative abundance in B50 and B500 groups, suggesting a higher intestine lubrication.
The absorption or the simple transit of both MPs in groups A and B did not cause inflammatory events at intestinal level nor alteration in the expression of immune markers in both larvae and juveniles. However, the accumulation of polymer A microbeads in liver samples of juveniles caused the upregulation of the oxidative stress marke rs.
Results of the present study suggest the presence of biological barriers against the ingested MPs in zebrafish that are related to the polymer size, dietary concentration, and time of exposure , able to reduce the number of MPs reaching the muscle which is the edible part of a fish . This result is extremely interesting for the aquaculture sector and needs further studies performed on finfish species.
Bhagat, J., Zang, L., Nishimura, N., Shimada, Y., 2020. Zebrafish: An emerging model to study microplastic and nanoplastic toxicity. Science of the Total Environment, 728.