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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 habitats^3,4,5,6,7. Therefore, more researchers have begun to study the environmental hazards of MPs.

Organisms could ingest MPs in the environment. MPs were found in the digestive tract of 233 marine organisms worldwide (including 100% turtle species, 36% seal species, 59% whale species, 59% seabird species, 92 kinds of sea fish, and 6 kinds of invertebrates)⁸. Moreover, MPs may block the organisms' digestive system, accumulate, and migrate in their bobies⁹. It has been found that MPs can be transferred via the food chain, and their intake differs with the changes of habitat, growth stage, feeding habits, and food sources¹⁰. Some researchers reported the existence of MPs in the droppings of seabirds¹¹, which means that seabirds act as the carrier of MPs. In addition, ingestion of MPs can affect health of some organisms. For example, MPs can be entangled in the gastrointestinal tract, thus increasing the mortality of cetaceans¹².

MPs alone have toxic effects on organisms as well as joint toxic effects on organisms with other pollutants. Ingestion of environmental-related concentrations of plastic debris may disturb the endocrine system function of adult fish¹³. The size of microplastics is one of the important factors that affect their uptake and accumulation by organisms^14,15. The small-size plastics, especially the nanosize plastics, are prone to interaction with cells and organisms with high toxicity^16,17,18,19. Although the harmful effects of nano-particle size microplastics on organisms exceed the current research level, the detection and quantification of microplastics with sizes less than several micrometers, especially the submicron/nano-plastics in the environment, is still a great challenge. In addition, nano-plastics also have some effects on embryos. Polystyrene can damage the development of sea urchin embryos by regulating protein and gene profiles²⁰.

To explore the potential impact of MPs on organisms, we conducted this study. Due to the similarity between bird embryos and human embryos, they are usually used in developmental biology research²¹ including angiogenesis and antiangiogenesis, tissue engineering, biomaterial implant, and brain tumors^22,23,24. Bird embryos have the advantages of low cost, a short culture cycle and easy operation^25,26. Therefore, we chose quail embryos with a short growth cycle as the experimental animal in this study. Simultaneously, we can directly observe the morphological changes of quail embryos exposed to MPs during the embryonic development stage using an eggshell-free hatching technology. The experimental materials used were polypropylene (PP) and polystyrene (PS). Because PP and PS²⁷ account for the largest proportion of polymer types obtained in sediments and water bodies worldwide, the most common polymer types extracted from captured marine organisms are ethylene and propylene²⁸. This experimental protocol describes the whole process for visual evaluation of toxicological effects of MPs on quail embryos exposed to MPs. We can easily extend this method to examine other pollutants' toxicity to embryo development of other oviparous animals.

Protokół

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 further use. Before transferring the fertilized eggs, the contents of the eggs are poured out. This is to keep the moisture of the eggshell. The opening diameter of the egg was about 3 cm.
   NOTE: To reduce the damage to the quail embryo, use a dental drill to open the blunt end of the egg and make the crack as smooth as possible.
6. After sterilization, place the fertilized quail eggs in a 38 °C incubator with 60% humidity for 24-48 h. Ensure that the blunt end of the quail egg faces up.
7. During the incubation of fertilized quail eggs, sterilize the tools needed in the subsequent experiments in a sterilization pot. These tools include plastic wrap, a beaker, sterile water, pipette tips, surgical straight scissors, tweezers, and a spoon.
   NOTE: Use a film with a temperature tolerance high enough to avoid problems with the high-temperature sterilization.

2. Hatching the quail egg without a shell

1. Transfer the pre-hatched fertilized quail eggs from the incubator to a clean bench and lay them flat on the container to stabilize them for about 1-2 min.
2. Use scissors (12.5 cm surgical straight scissor) to poke a small hole (diameter 3 mm) in the central axis of the pre-hatched fertilized quail eggs and to cut 1-2 cm small opening. Carefully transfer the egg white and yolk of the fertilized quail eggs to the cut eggshell.
   NOTE: When cutting a small opening with scissors, avoid touching of the yolk of quail eggs.
3. Add the control solution (without MPs) and the exposed solution of different masses (0.1, 0.2, and 0.3 mg) of microplastics with three particle sizes (100, 200, and 500 nm) to the egg contents by pipette. At the same time, add 1 drop of penicillin and 1 drop of streptomycin with a 1 mL syringe.
4. Cover the opening of the eggshell with the sterilized film (step 1.6).
5. According to step 2.1-2.4, treat all the fertilized quail eggs.
6. Place the transferred quail embryos into the 38 °C incubator with 60% humidity for the necessary period. In this experiment, use an egg rotation angle of ±30°. Turn the eggs once an hour.
   ​NOTE: The transfer should be as fast as possible, which requires more practice at the early stage.

3. Sample collection

1. After seven days of culture, remove well-developed embryos observed by the naked eye from the yolk and wash with phosphate buffered solution (PBS).
2. Dry the surplus solution outside the cleaned embryo with absorbent paper and weigh in a clean Petri dish.
3. Open the whole chest cavity, separate the liver and the heart from the viscera with needle-nose pliers, and place in 1.5 mL centrifuge tubes immediately after clearing.
4. Quickly record the weight on an electronic balance and calculate the hepatosomatic index (HIS = liver weight / body weight x 100). Measure the length of the sternum and body.
5. Based on the above indicators, evaluate the impact of MPs on embryonic development.
   ​NOTE: Embryo quality here refers to the quality of yolk removal.

4. Data analysis

1. Report the experimental data in the form of mean ± standard error (SEM).
2. Use single-factor analysis of variance to compare the means of multiple groups of samples. The significant difference value was α = 0.05.

Wyniki

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 ...

Dyskusje

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 ...

Materiały

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