Summary

微塑料对鸟胚胎发育的生态毒性影响,无需蛋壳孵化

Published: August 14, 2021
doi:

Summary

本文介绍了一种无需使用蛋壳进行微塑料等颗粒污染物毒理学研究的孵化方法。

Abstract

微塑料是一种新兴的全球污染物类型,由于动物组织和器官的吸收和转移,对动物的健康构成巨大威胁。微塑料对鸟类胚胎发育的生态毒性影响尚不清楚。鸟蛋是一个完整的发育和营养系统,整个胚胎发育发生在蛋壳中。因此,在微塑料等污染物的压力下,鸟类胚胎发育的直接记录受到传统孵化中不透明蛋壳的极大限制。在这项研究中,微塑料对卵壳发育的影响通过没有蛋壳的孵化进行视觉监测。主要步骤包括受精卵的清洁和消毒、暴露前的孵化、暴露后的短期孵化以及样品提取。结果表明,与对照组相比,微塑料暴露组的湿重和体长呈统计差异,整个暴露组的肝比例显著增加。此外,我们评估了影响孵化的外部因素:温度、湿度、卵子旋转角度和其他条件。这种实验方法为微塑料的生态毒理学提供了宝贵的信息,是研究污染物对胚胎发育的不利影响的新方法。

Introduction

2015年塑料废物的产量约为6300万吨,其中十分之一被回收利用,其余被焚烧或埋在地下。据估计,到2050年1月1日,约有12,000吨塑料废物将被埋入地下。随着国际社会对塑料废物的关注,汤普森于2004首次提出了微塑料的概念。微塑料(MP)是指颗粒直径小于5毫米的小颗粒塑料。目前,研究人员已经探测到,在各大洲、大西洋群岛、内陆湖泊、北极和深海栖息地海岸线上,议员无处不在。因此,更多的研究人员开始研究议员的环境危害。

生物体可以在环境中摄入议员。在全球233种海洋生物的消化道中发现了MPS(包括100%龟类、36%海豹物种、59%鲸鱼物种、59%海鸟物种、92种海鱼和6种无脊椎动物)8种。此外,议员们可能会阻止生物体的消化系统,积累和迁移在他们的波比9。研究发现,议员可以通过食物链转移,他们的摄入量因栖息地、生长阶段、喂养习惯和食物来源的变化而异。一些研究人员报告说,在海鸟11号的粪便中存在议员,这意味着海鸟充当了议员的载体。此外,摄入议员会影响某些生物体的健康。例如,MP可以纠缠在胃肠道,从而增加鲸目动物的死亡12。

仅议员就对生物体有毒性影响,对具有其他污染物的生物体有联合毒性影响。摄入与环境有关的塑料碎片浓度可能会干扰成年鱼13的内分泌系统功能。微塑料的大小是影响生物体吸收和积累的重要因素之一。小尺寸塑料,特别是纳米大小的塑料,容易与高毒性16、17、18、19等高毒性的细胞和生物体相互作用。虽然纳米颗粒大小的微塑料对生物体的有害影响超过了目前的研究水平,但检测和量化大小小于几微米的微塑料,特别是环境中的亚微子/纳米塑料,仍然是一项巨大的挑战。此外,纳米塑料也对胚胎有一定的影响。聚苯乙烯可以通过调节蛋白质和基因特征20来破坏海胆胚胎的发育。

为了探讨议员对生物体的潜在影响,我们进行了这项研究。由于鸟类胚胎和人类胚胎的相似性,它们通常用于发育生物学研究21,包括血管生成和抗血管生成、组织工程、生物材料植入物和脑肿瘤22、23、24。鸟类胚胎具有成本低、培养周期短、操作方便优点。因此,在这项研究中,我们选择了生长周期短的胚胎作为实验动物。同时,我们可以通过无蛋壳孵化技术直接观察胚胎发育阶段暴露在 MP 的卵壳胚胎的形态变化。使用的实验材料是聚丙烯(PP)和聚苯乙烯(PS)。由于PP和PS27占全世界沉积物和水体中聚合物类型的最大比例,从捕获的海洋生物中提取的最常见的聚合物类型是乙烯和丙烯28。这个实验协议描述了对议员们对暴露在议员面前的胚胎的毒理学影响进行视觉评估的全过程。我们可以很容易地扩展这种方法来检查其他污染物对其他动物胚胎发育的毒性。

Protocol

1. 暴露前的准备 选择同一天出生的受精卵进行暴露测试。 选择重量相似的鸡蛋。每个受精卵大约为10-12克。 完全清洁外部粪便和其他碎屑中的所有受精卵。 用抗生素溶液(青霉素和链霉素,1:1000,室温)对每个预孵化受精卵和要使用的卵子(选择壳形相似的卵子,尤其是蛋尖)进行消毒。用75%的乙醇对孵化器进行消毒。 用牙科钻头的钝端打开鸡蛋,将蛋?…

Representative Results

在实验数据分析中,比较了湿重、体长、胸骨长度以及对照组与6个实验组肝病指数的变化,从宏观角度测量和反映了胚胎的生长发育。我们在每组中检测到六个正常的胚胎。每个胚胎都达到了所需的汉堡和汉密尔顿(HH)阶段。 在 图1中,我们把预孵化的受精卵含量转移到半球蛋壳中,并把它们放入孵化器中。然后,我们记录了胚胎在潜伏期中期的发育…

Discussion

本文通过检测基本发育指标,为评估胚胎发育提供了有效的实验方案。然而,这个实验还是有一些局限性的。

首先,由于无壳孵化,孵化后期胚胎的死亡率较高。在实验过程中,有人为的无法控制的因素,如正常蛋白质比的破坏。我们限制胚胎的暴露时间,以确保实验的准确性。胚胎毒性的研究只能在胚胎发育的早期和中期进行。其次,对胚胎发育的议员们的研究只在基本?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了新疆维吾尔自治区重点研发项目的支持(2017B03014,2017B03014-1,2017B03014-2,2017B03014-3)。

Materials

 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

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Wang, L., Xue, N., Li, W., Wufuer, R., Zhang, D. Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell. J. Vis. Exp. (174), e61696, doi:10.3791/61696 (2021).

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