Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

Summary

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Abstract

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

Introduction

The production of plastic waste was about 6300 Mt in 2015, one-tenth of which was recycled, and the rest was burned or buried underground. It is estimated that about 12,000 Mt of plastic waste would be buried underground by 2050¹. With the international community's attention to plastic waste, Thompson first proposed the concept of microplastics in 2004². Microplastics (MPs) refer to small particle plastics with a particle diameter less than 5 mm. At present, researchers have detected the ubiquitous presence of MPs in the coastline of various continents, the Atlantic Islands, inland lakes, the Arctic, and deep-sea hab....

Protocol

1. Preparation before exposure

1. Select fertilized quail eggs born on the same day for the exposure test.
2. Select quail eggs with similar weights. Each fertilized quail egg is about 10-12 g.
3. Fully clean all fertilized quail eggs from external feces and other debris.
4. Sterilize each pre-hatched fertilized quail egg and the eggs to be used (Choose eggs with similar shell shape, especially the tip of the egg) with an antibiotic solution (penicillin and streptomycin, 1:1000, room temperature). Sterilize the incubator with 75% ethanol.
5. Open the eggs with the blunt end of a dental drill, leaving the eggshell at the tip for fur....

Results

For the analysis of experimental data, we compared wet weight, body length, sternum length and the change of hepatosomatic index between the control group and the 6 experimental groups, measuring and reflecting the quail embryos' growth and development from a macro perspective. We detected six normal quail embryos in each group. Each embryo reached the required Hamburger and Hamilton (HH) stage.

In Figure 1, we transferred the pre-hatched fertilized quail egg .......

Discussion

This paper provides an effective experimental scheme to evaluate quail embryo development by detecting the basic development indexes. However, there are still some limitations to this experiment.

First, the mortality of quail embryos in the later stage of hatching is higher because of the shell-less hatching. There are artificially uncontrollable factors such as the destruction of normal protein ratio in the experimental process. We limited the exposure time of embryos to ensure the accuracy .......

Materials

Name	Company	Catalog Number	Comments
Multi sample tissue grinder	Shanghai Jingxin Industrial Development Co., Ltd.	Tissuelyser-24	Grind large-sized plastics into small-sized ones at low temperature
Electronic balance	OHAUS corporation	PR Series Precision	Used for weighing
Fertilized quail eggs	Guangzhou Cangmu Agricultural Development Co., Ltd.		Quail eggs for hatching without shell
Fluorescent polypropylene particles	Foshan Juliang Optical Material Co., Ltd.		Types of plastics selected for the experiment
Incubator	Shandong, Bangda Incubation Equipment Co., Ltd.	264 pc	Provide a place for embryo growth and development
Nanometer-scale polystyrene microspheres	Xi’an Ruixi Biological Technology Co., Ltd.	100 nm, 200 nm, 500 nm	Types of plastics selected for the experiment
Steel ruler	Deli Group	20 cm	Used to measure  length
Vertical heating pressure steam sterilizer	Shanghai Shenan Medical Instrument Factory	LDZM-80KCS-II	Sterilize the experimental articles