Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

Summary

Zebrafish embryos/larvae develop externally and are optically transparent. The bioaccumulation of microplastics in fish at early life stages is readily assessed with fluorescently labeled microbeads.

Abstract

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The bioaccumulation of microplastics plays a key role in their toxic effects; however, as a particulate, their bioaccumulations are different from many other pollutants. Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. Embryos are exposed to different concentrations (0.1, 1, and 10 mg/L) of fluorescent microplastics with a diameter of 500 nm for 120 h. It is shown in the results that microplastics can bioaccumulate in zebrafish embryos/larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion; while in zebrafish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics. The results demonstrate the uptake and internalization of microplastics in zebrafish at early life stages, which will provide basis for better understanding the impact of microplastics on aquatic animals.

Introduction

Since first synthesized in the 1900s, plastics are widely used in various fields, resulting in rapid growth of global production¹. In 2018, approximately 360 million tons of plastics were produced worldwide². The plastics in the natural environment will degrade to fine particles due to chemical, physical or biological processes³. Generally, fine plastic particles <5 mm in size are defined as microplastics⁴. Microplastics are also engineered for specific applications, such as microbeads from cosmetic products⁵. As near-permanent contaminants, micropla....

Protocol

Adult zebrafish are originated from the China Zebrafish Resource Center (Wuhan, China). The experiments were conducted in compliance with the national guide "Laboratory Animal Guideline for Ethical Review of Animal Welfare (GB/T35892-2018).

1. Embryo collection

1. Maintain fish in 20 L glass tanks with recirculating charcoal-filtered tap water system (pH 7.0 ± 0.2) at a constant temperature (28 ± 0.5 °C) on a photoperiod of 14:10 h light: dark.
2. Feed fish twice daily with Artemia nauplii. It is recommended that the food is given at max. 3% fish weight per day and should be eaten within 5 min eve....

Results

The distribution and accumulation of fluorescent microplastics are shown in Figure 1 and Table 1. No visible fluorescence is observed in the unexposed group (control). However, an accumulation of fluorescence is found surrounding the chorion after exposure to different concentrations of microplastics (24 hpf). Green fluorescence is also detected in larvae, and the fluorescence levels appear to increase in a concentration- and time-dependent manner. The yolk sac, pericardium,.......

Discussion

According to the guideline on the protection of animals used for scientific purposes, such as EU Directive 2010/63/EU, animal ethics permission is not mandatory for an experiment with early life-stages of zebrafish until the stage of being capable of independent feeding (5 days post fertilization)¹⁷. However, best welfare practice is important for optimizing the use of zebrafish, and, for example, the humane methods of anesthesia and euthanasia should be of concern. Ethyl 3-aminobenzoate methanesu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluorescent microscope	Nikon, Japan	Eclipse Ti-S
Green fluorescently labeled polystyrene beads	Phosphorex, USA	2103A
Tricaine	Sigma-Aldrich, USA	A5040