Aquaculture Europe 2023

September 18 - 21, 2023


Add To Calendar 19/09/2023 15:45:0019/09/2023 16:00:00Europe/ViennaAquaculture Europe 2023DIETARY MICROPLASTICS EXPOSURE IN DIFFERENT LIFE-CYCLE STAGES: A STUDY ON ZEBRAFISH Danio rerio PHYSIOLOGICAL RESPONSES AND WELFARE FROM LARVAE TO JUVENILESSchubert 4The European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982


N. Cattaneo* , M . Zarantoniello , F. Conti, A. Frontini, G. Cardinaletti, G. Gioacchini  and I . Olivotto


Department of Life and Environmental Sciences, Marche Polytechnic University, via Brecce Bianche, 60131 Ancona, Italy




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.