This work was funded by the National Natural Science Foundation of China (21777145, 22076170), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R97).

Adult zebrafish are originated from the China Zebrafish Resource Center (Wuhan, China). The experiments were conducted in compliance with the national guide &#34;Laboratory Animal Guideline for Ethical Review of Animal Welfare (GB/T35892-2018).
1. Embryo collection
<ol>
	<li>Maintain fish in 20 L glass tanks with recirculating charcoal-filtered tap water system (pH 7.0 &#177; 0.2) at a constant temperature (28 &#177; 0.5 &#176;C) on a photoperiod of 14:10 h light: dark.</li>
	<li>Feed fish t...

<ol>
	<li>SAPEA (Science Advice for Policy by European Academies). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Scientific Perspective on Microplastics in Nature and Society. , (2019).</li><li>Plastics Europe. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics-the facts 2019. , (2019).</li><li>Andrady, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+the+marine+environment.">Microplastics in the marine environment.</a> Marine Pollution Bulletin. 62, 1596-1605 (2011).</li><li>Arthur, C., Baker, J., Bamford, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proceedings+of+the+International+Research+Workshop+on+the+Occurrence,+Effects+and+Fate+of+Microplastic+Marine+Debris.">Proceedings of the International Research Workshop on the Occurrence, Effects and Fate of Microplastic Marine Debris.</a> National Oceanic and Atmospheric Administration Technical Memorandum. , (2009).</li><li>Ivleva, N. P., Wiesheu, A. C., Niessner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastic+in+aquatic+ecosystems.">Microplastic in aquatic ecosystems.</a> Angewandte Chemie International Edition. 56, 1720-1739 (2017).</li><li>Lu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollutant+toxicology+with+respect+to+microalgae+and+cyanobacteria.">Pollutant toxicology with respect to microalgae and cyanobacteria.</a> Journal of Environmental Sciences. 99, 175-186 (2021).</li><li>Huang, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+microplastics+impairs+digestive+performance,+stimulates+immune+response+and+induces+microbiota+dysbiosis+in+the+gut+of+juvenile+guppy+(Poecilia+reticulata).">Exposure to microplastics impairs digestive performance, stimulates immune response and induces microbiota dysbiosis in the gut of juvenile guppy (Poecilia reticulata).</a> Science of the Total Environment. 733, 138929 (2020).</li><li>Pr&#252;st, M., Meijer, J., Westerink, R. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plastic+brain:+neurotoxicity+of+micro-+and+nanoplastics.">The plastic brain: neurotoxicity of micro- and nanoplastics.</a> Particle and Fibre Toxicology. 17, 24 (2020).</li><li>Jakubowska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+exposure+to+microplastics+of+different+polymer+types+on+early+life+stages+of+sea+trout+Salmo+trutta.">Effects of chronic exposure to microplastics of different polymer types on early life stages of sea trout Salmo trutta.</a> Science of the Total Environment. 740, 139922 (2020).</li><li>Qiang, L., Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+polystyrene+microplastics+impairs+gonads+of+zebrafish+(Danio+rerio).">Exposure to polystyrene microplastics impairs gonads of zebrafish (Danio rerio).</a> Chemosphere. 263, 128161 (2021).</li><li>Hamed, M., Soliman, H. A. M., Osman, A. G. M., Sayed, A. E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidants+and+molecular+damage+in+Nile+Tilapia+(Oreochromis+niloticus)+after+exposure+to+microplastics.">Antioxidants and molecular damage in Nile Tilapia (Oreochromis niloticus) after exposure to microplastics.</a> Environmental Science and Pollution Research. 27, 14581-14588 (2020).</li><li>Burns, E. E., Boxall, A. B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+the+aquatic+environment:+Evidence+for+or+against+adverse+impacts+and+major+knowledge+gaps.">Microplastics in the aquatic environment: Evidence for or against adverse impacts and major knowledge gaps.</a> Environmental Toxicology and Chemistry. 37, 2776-2796 (2018).</li><li>Ma, H., Pu, S., Liu, S., Bai, Y., Mandal, S., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+aquatic+environments:+Toxicity+to+trigger+ecological+consequences.">Microplastics in aquatic environments: Toxicity to trigger ecological consequences.</a> Environmental Pollution. 261, 114089 (2020).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio reio). 4th ed. , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Pikuda, O., Xu, E. G., Berk, D., Tufenkji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+assessments+of+micro-+and+nanoplastics+can+be+confounded+by+preservatives+in+commercial+formulations.">Toxicity assessments of micro- and nanoplastics can be confounded by preservatives in commercial formulations.</a> Environmental Science &amp; Technology Letters. 6, 21-25 (2019).</li><li>Lidster, K., Readman, G. D., Prescott, M. J., Owen, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+survey+on+the+use+and+welfare+of+zebrafish+Danio+rerio+in+research.">International survey on the use and welfare of zebrafish Danio rerio in research.</a> Journal of Fish Biology. 90, 1891-1905 (2017).</li><li>Pitt, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+tissue+distribution,+and+toxicity+of+polystyrene+nanoparticles+in+developing+zebrafish+(Danio+rerio).">Uptake, tissue distribution, and toxicity of polystyrene nanoparticles in developing zebrafish (Danio rerio).</a> Aquatic Toxicology. 194, 185-194 (2018).</li><li>Lin, S. J., Zhao, Y., Nel, A. E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+An+in+vivo+model+for+nano+EHS+studies.">Zebrafish: An in vivo model for nano EHS studies.</a> Small. 9, 1608-1618 (2013).</li></ol>

The author declares no competing or financial interests.

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...

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 &lt;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, microplastics are accumulated in the environment, and have attracted increasing attention from scientists, policymakers and the public1,6. Previous studies documented that microplastics could cause adverse effects in fish, such as gastrointestinal damage7, neurotoxicity8, endocrine disruption9, oxidative stress10 and DNA damage11. However, the toxicity of microplastics has not been fully revealed so far12,13.
Zebrafish embryos offer a lot of experimental advantages, including small size, external fertilization, optical transparency and large clutches, and is considered as an ideal model organism for in vivo studying the effects of pollutants on fish at early life stages. In addition, only limited amounts of test substances are needed for the evaluation of biological responses. Here, zebrafish embryos are exposed to different concentrations of microplastics (0.1, 1, 10 mg/L) for 5 days, and the bioaccumulation and distribution of microplastics in zebrafish embryos/larvae are evaluated. This result will advance our understanding about the toxicity of microplastics to fish, and the method described here can potentially be generalized to determine the accumulation and distribution of other types of fluorescent materials in the early life stages of zebrafish.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon, Japan</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Green fluorescently labeled polystyrene beads</td>
			<td>Phosphorex, USA</td>
			<td>2103A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

accumulation and distribution of fluorescent microplastics in the early life stages of zebrafish

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.

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,...

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

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.

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 ...

Accumulation and distribution of fluorescent microplastics in the early life stages of zebrafish.Introduction. The bioaccumulation of microplastics play a key role in their toxic effects. However, as 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. The experiments were conducted in compliance with the national guide Laboratory animal Guideline for ethical review of animal welfare. One, embryo collection.Adult zebrafish are originated from the China Zebrafish Resource Center. Fish are maintained in 20-liter glass tanks with recirculating charcoal-filtered tap water system at a constant temperature, namely, 28 Celsius degree, on a photoperiod of 14 and 10 hour, light:dark. Fish are fed twice daily with Artemia nauplii.It is recommended that the food is given at maximum 3%fish weight per day, and should be eaten within five minutes every time. Well-developed adult zebrafish are transferred into the spawning tank at a ratio of one male to two females the night before the breeding. The following morning, the fish start to spawn after the onset of the light cycle.Eggs are collected using a Pasteur pipette, rinsed with 10%Hank's solution several times, and then checked for fertilization using a microscope. Fertilized eggs undergo the cleavage period after approximately two-hour post fertilization and can be clearly identified. The fertilized embryos are incubated in a 500-milliliter beaker containing 200-milliliter of 10%Hank's solution with 1%methylene blue for disinfection at 28 Celsius degree.The loading rate is not exceeding one embryo in each 2-milliliter solution. Two, preparation of microplastic suspensions. The stock solution of green fluorescently labeled polystyrene beads with nominal diameter of 500 nanometers are sonicated for 10 minutes.Dilute the stock solution with 10%Hank's solution to produce the desired exposure solutions, namely 0.1, 1, and 10 milligram per liter. The exposure solutions of microplastics are always freshly prepared before exposure. Three, microplastic exposure.Six newly fertilized embryos are randomly selected, and then transferred into each well of six well plate containing five milliliter of microplastic solutions with different concentrations. The control groups containing 10%Hank's solution are included. Triplicate wells, with a total of 18 embryos, are used for each treatment.The embryos are incubated under the same light-dark cycle and temperature as adults, and are observed every 12 hours. The dead are removed immediately. The microplastic solutions are 90%renewed every 24 hours.During the exposure period, the fish are not fed. Generally, the hatching of embryo begins at 48-hour post fertilization and completes at about 72-hour post fertilization. Four, assessment of microplastic distribution.At 24-48-72-96-and 120-hour post fertilization, the embryos and larvae, one from each of the three replicates, are randomly selected and rinsed with 10%Hank's solution. The embryos and larvae are arranged and prepared for observation. Beforehand, the larvae are transferred into a Petri dish, and exposed to 0.016%tricaine for anesthesia.The fish are observed with fluorescence microscope, and imaged with imaging software. The fluorescence intensity in fish is further quantified with ImageJ NIH. Representative results.It is showed in the results that microplastics can bioaccumulate in zebrafish embryos and larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion;while in zebra fish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics.Discussion. This result will advance our understanding about the toxicity of microplastics to fish, and the method described here can potentially be generalized to determine the accumulation and distribution of other types of fluorescent materials in the early life stages of zebrafish.Since the chorion will act as an effective barrier against the particles with large size, the dechorionation process before exposure may be needed. However, the embryo with chorion intact is more recommended to assess the ecotoxicity of pollutants when considering the condition of exposure in the real world. The behavior of microplastics in the solution is critical to the bioavailability as well.The physicochemical characteristics of microplastics should be tracked over the exposure duration.

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4.5K Görüntüleme.  Zhejiang University of Technology. Zebra balıklarının erken yaşam aşamalarında floresan mikroplastiklerin birikmesi ve dağılımı. Giriş. Mikroplastiklerin biyoakümülasyonu toksik etkilerinde önemli bir rol oynar. Bununla birlikte, partikül olarak, biyoakümülasyonları diğer birçok kirleticiden farklıdır.Burada açıklanan, floresan mikroplastikler kullanarak zebra balığı embriyolarında veya larvalarında mikroplastiklerin birikmesini ve dağılımını görsel olarak belirlemek için uygulanabili...