The authors are grateful for the financial support by the National Natural Science Foundation of China (21876135 and 21876136), the National Major Science and Technology Project of China (2017ZX07502003-03, 2018ZX07701001-22), the Foundation of MOE-Shanghai Key Laboratory of Children&#39;s Environmental Health (CEH201807-5), and Swedish Research Council (No. 639-2013-6913).

The protocol is in accordance with guidelines approved by the Animal Ethics Committee of Tongji University.
1. Zebrafish embryo collection
<ol>
	<li>Put two pairs of healthy adult Tubingen zebrafish into the spawning box on the night before exposure, keeping the sex ratio at 1:1.</li>
	<li>Remove the adult fish back to the system 30-60 min after daylight the next morning.</li>
	<li>Remove the embryos out of the spawning box.</li>
	<li>Rinse the embryos with system water.</li>
	<li>Transfer the embryos into a glass Petri dish (9 cm diameter) with enough system water.</li>
	<li>Observe the embryos under the microscope and select healthy embryos for later exposure. 
	NOTE: Healthy embryos are usually transparent with light golden color under the microscope. The unhealthy embryos are usually pale and clumped together as observed under the microscope.</li>
</ol>
2. Preparation before exposure
<ol>
	<li>Prepare the Hanks&#39; solution according to the guidelines of the zebrafish book31. 
	NOTE: The Hanks&#39; solution includes 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3.</li>
	<li>Dilute the Hanks&#39; solution to 10% Hanks&#39; solution using sterile water.</li>
	<li>Add 1 mL of DMSO into 999 mL of 10% Hanks&#39; solution to make a control solution of 10% Hanks&#39; solution including 0.1% DMSO. 
	NOTE: The next steps use BDE-47 as an example of an exposure solution.</li>
	<li>Dissolve 5 mg of the neat BDE-47 in 1 mL of 100% DMSO to make a standard exposure solution of 5 mg/mL.</li>
	<li>Vortex the 5 mg/mL solution for 1 min to completely dissolve the BDE-47 in the DMSO.</li>
	<li>Transfer 10 &#181;L of the 5 mg/mL solution to a 12 mL brown glass bottle.</li>
	<li>Add sterilized water to a final volume of 10 mL to make the concentration of BDE-47 exposure solution 5 mg/L and DMSO ratio at 0.1%, then vortex for 1 min.</li>
	<li>Transfer 10 &#181;L and 100 &#181;L of the 5 mg/L solution into two 100 mL volumetric flasks respectively.</li>
	<li>Add 10% Hanks&#39; solution including 0.1% DMSO (prepared in step 2.3) to 100 mL to make the final concentrations of the BDE-47 exposure solutions 5 &#181;g/L and 50 &#181;g/L, respectively.</li>
	<li>Transfer the solutions into 100 mL brown glass bottles and store them at 4 &#176;C.</li>
</ol>
3. Exposure of embryos
<ol>
	<li>Transfer ~50 embryos into each of the three glass Petri dishes (6 cm diameter) 3-5 hours post fertilization (hpf).</li>
	<li>Use a 1 mL pipette tip to blot the system water around the embryos.</li>
	<li>Use a pipette to transfer the control and two BDE-47 exposure solutions (control, 5 &#181;g/L, 50 &#181;g/L) into the three glass Petri dishes, respectively.</li>
	<li>Shake the glass Petri dishes gently one by one to make the embryos disperse in the bottom of the plate.</li>
	<li>Put the glass Petri dishes into the light incubator under 28.5 &#176;C.</li>
	<li>Renew half of the exposure solutions every 24 h until 5 dpf.</li>
	<li>Check the dead embryos of every group on 1 dpf and 2 dpf and calculate the death rate.</li>
	<li>Check the incubated embryos of every group on 2 dpf and 3 dpf and calculate the hatchability.</li>
	<li>Check the deformity of the larvae every day after they come out from the chorion and calculate the deformity rate of every group. 
	NOTE: The deformity indicators include pericardial cyst, spinal curvature, tail curvature, among others factors32.</li>
</ol>
4. Preparation for the behavior test
<ol>
	<li>Prepare a 48 well microplate for the locomotion and path angle test and three 6 well microplates for the social activity test on the morning of 5 dpf.</li>
	<li>Transfer 800 &#181;L of exposure solution into every well of the 48 well microplate. 
	NOTE: Use 16 wells for every group (i.e., the control solution, 5 &#181;g/L, and 50 &#181;g/L group).</li>
	<li>Use a 1 mL pipette tip to transfer 200 &#181;L of exposure solution with one larva from the glass Petri dish into one well of the 48 well microplate.</li>
	<li>Transfer 4 mL of exposure solution into every well of the 6 well microplate. 
	NOTE: Use one 6 well microplate for every group.</li>
	<li>Use a 1 mL pipette tip to transfer 200 &#181;L of exposure solution with two larvae into each well of the 6 well microplate. 
	NOTE: Every group has six repeating groups.</li>
	<li>Make sure the temperature of the test room is 28 &#176;C 2 h before the test. 
	NOTE: Behavioral tests are usually performed in the afternoon.</li>
</ol>
5. Behavioral test
<ol>
	<li>Locomotion and path angle test
	<ol>
		<li>Click the launcher icon on the computer desk to open the software (see Table of Materials) that controls the high-throughput monitoring enclosure to start the program.</li>
		<li>Choose the &#34;Tracking, Rotations, Path Angles&#34; module to enter the operating interface.</li>
		<li>Transfer the 48 well microplate prepared in step 4.3 to the recording platform and pull down the cover.</li>
		<li>Click the &#34;File&#34; &#34;Generate Protocol&#34; button in turn in the software to begin generating a new protocol.</li>
		<li>Input &#34;48&#34; in the &#34;Location count&#34; position and click the &#34;OK&#34; button.</li>
		<li>Click the &#34;Parameters&#34; &#34;Protocol Parameters&#34; &#34;Time&#34; button in turn in the software. Set the experiment duration for 1 h and 10 min and set the integration period to 60 s33.</li>
		<li>Draw the detected areas.
		<ol>
			<li>Select the elliptical shape and draw the first circle around the first top left well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Top-Right Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the top right well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Bottom Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the bottom right well.</li>
			<li>Click the &#34;Build&#34; &#34;Clear marks&#34; button in turn. 
			NOTE: The system will automatically draw every other well of the plate. The newly created areas should perfectly fit each well between the actual fish and its reflection on the side of the well.</li>
		</ol>
		</li>
		<li>Click the &#34;Draw Scale&#34; button, draw a calibration line on the screen (a diagonal path or parallel to the side of the microplate), enter its length and set the &#34;Unit&#34;. Then click the &#34;Apply to group&#34; button.</li>
		<li>Set the animal color at &#34;Black&#34; in the software.</li>
		<li>Set the detection threshold at 16-18 to allow the detection of the animals.</li>
		<li>Input inactive/small and small/large speed at 0.5 cm/s and 2.5 cm/s respectively.</li>
		<li>Set the path angle classes. Input &#34;-90, -30, -10, 0, 10, 30, and 90&#34; to make path angle classes from -180&#176;-+180&#176;.</li>
		<li>Set the light conditions
		<ol>
			<li>Click the &#34;Parameters&#34; &#34;Light driving&#34; &#34;Uses one of the 3 triggering methods below&#34; &#34;Enhanced stimuli&#34; button in turn to set the light conditions.</li>
			<li>Choose the &#34;Edge&#34; button, then set a dark period of 10 min, followed by three cycles of alternating 10 min light and dark periods.</li>
		</ol>
		</li>
		<li>Save the protocol and turn down the light of the test room.</li>
		<li>Acclimate the larvae in the system for 10 min and click the &#34;Experiment&#34; &#34;Execute&#34; button in turn, then choose the folder where the experiment files are saved and enter the result name.</li>
		<li>Click the &#34;Background&#34; &#34;Start&#34; button in turn to start the test.</li>
		<li>Click the &#34;Experiment&#34; &#34;Stop&#34; button in turn to stop the experiment when the test ends. 
		NOTE: The system shows the data tested when the system stops. The data include the tracked distance at three speed classes and path angle numbers at eight angle classes of every minute. For the locomotion test in the presented example, the total distance in every light period (10 min) is calculated and the difference between the control group and the treatment groups compared.</li>
		<li>Transfer the 48 well microplate back to the light incubator for other experiments.</li>
	</ol>
	</li>
	<li>Social activity test
	<ol>
		<li>Click on the launcher icon on the computer desk to open the software that controls the high-throughput monitoring enclosure to start the program.</li>
		<li>Choose the &#34;Social Interactions&#34; module to enter the operating interface.</li>
		<li>Transfer the prepared 6 well microplate (control group) in step 4.4 to the recording platform and pull down the cover.</li>
		<li>Click the &#34;File&#34; &#34;Generate Protocol&#34; button in turn in the software to begin generating a new protocol.</li>
		<li>Input &#34;6&#34; in the &#34;Location count&#34; position and click the &#34;OK&#34; button.</li>
		<li>Click the &#34;Parameters&#34; &#34;Protocol Parameters&#34; &#34;Time&#34; button in turn in the software. Set the experiment duration for 1 h and 10 min and set the integration period to 60 s.</li>
		<li>Draw the detected areas.
		<ol>
			<li>Select the elliptical shape and draw the first circle around the first top left well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Top-Right Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the top-right well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Bottom Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the bottom right well.</li>
			<li>Click the &#34;Build&#34; &#34;Clear marks&#34; button in turn. 
			NOTE: The newly created areas should fit each well perfectly and between the actual larvae and its reflection on the side of the well.</li>
		</ol>
		</li>
		<li>Click the &#34;Draw Scale&#34; button, draw a calibration line in the screen (a diagonal path or parallel to the side of the microplate), enter its length and set the &#34;Unit&#34;. Then click the &#34;Apply to group&#34; button.</li>
		<li>Set the detection threshold at 16-18 to allow the detection of the animals.</li>
		<li>Click the &#34;Black animal&#34; button in the software.</li>
		<li>Choose the &#34;Distance Threshold&#34; button and input &#34;5&#34; in the software.</li>
		<li>Set the light conditions.
		<ol>
			<li>Click the &#34;Parameters&#34; &#34;Light driving&#34; &#34;Uses one of the 3 triggering methods below&#34; &#34;Enhanced stimuli&#34; button in turn to set the light conditions.</li>
			<li>Choose the &#34;Edge&#34; button, then set a dark period of 10 min, followed by three cycles of alternating 10 min light and dark periods.</li>
		</ol>
		</li>
		<li>Save the protocol and turn down the light of the room.</li>
		<li>Acclimate the larvae in the system for 10 min and click the &#34;Experiment&#34; &#34;Execute&#34; button in turn, then choose the folder where the experiment files are saved and enter the result name.</li>
		<li>Click the &#34;Background&#34; &#34;Start&#34; button in turn to start the test.</li>
		<li>Click the &#34;Experiment&#34; &#34;Stop&#34; button in turn to stop the experiment in the software when the test ends. 
		NOTE: The system shows the data tested when the system stops.</li>
		<li>Transfer the 6 well microplate (control group) back to the light incubator for other experiments.</li>
		<li>Transfer the 6 well microplates (5 &#181;g/L and 50 &#181;g/L groups) in turn to the recording platform and repeat the steps from 5.2.4 to 5.2.17 by ordinal.</li>
	</ol>
	</li>
</ol>
6. Data analysis
<ol>
	<li>Open the spreadsheet file in the locomotion and path angle results.</li>
	<li>Select the three distance columns (inadist, smldist, lardist) and add them up. 
	NOTE: The data of inadist, smldist, and lardist mean different distances recorded by the system in different speed classes (inactive/small/large), respectively.</li>
	<li>For every 10 minute light period sum up the distance of every well, calculate the average distance of 16 wells, and compare the data of the three groups under light stimuli.</li>
	<li>For every 10 minute light period sum up the angle number of every well in every light duration from cl01 to cl08 in turn, and compare the data of the three groups under light stimuli. 
	NOTE: The data of columns from cl01 to cl08 mean different path angle numbers recorded by the system in different path angles, respectively.</li>
	<li>Open the spreadsheet file in the social activity results.</li>
	<li>Select the contct and contdur columns, and for every 10 min light period sum up the social times and their duration for every well.</li>
	<li>Calculate the average social times and duration of one group in every light duration and compare the data of the three groups under light stimuli.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+exposure+to+bisphenol+A+and+replacement+bisphenol+S+induces+precocious+hypothalamic+neurogenesis+in+embryonic+zebrafish.">Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (5), 1475-1480 (2015).</li><li>Javed, I., 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=Inhibition+of+amyloid+beta+toxicity+in+zebrafish+with+a+chaperone-gold+nanoparticle+dual+strategy.">Inhibition of amyloid beta toxicity in zebrafish with a chaperone-gold nanoparticle dual strategy.</a> Nature Communications. 10 (1), 1-14 (2019).</li><li>Green, J., 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=Automated+high-throughput+neurophenotyping+of+zebrafish+social+behavior.">Automated high-throughput neurophenotyping of zebrafish social behavior.</a> Journal of Neuroscience Methods. 210 (2), 266-271 (2012).</li><li>Tytell, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamics+of+eel+swimming+II.+Effect+of+swimming+speed.">The hydrodynamics of eel swimming II. Effect of swimming speed.</a> Journal of Experimental Biology. 207 (19), 3265-3279 (2004).</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+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> The Zebrafish Book. 4, (2000).</li><li>Ying, L., Jiang, L., Bo, P., Yong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogenic+effects+of+embryonic+exposure+to+pretilachlor+on+the+larvae+of+zebrafish.">Teratogenic effects of embryonic exposure to pretilachlor on the larvae of zebrafish.</a> Journal of Agro-Environment Science. 36 (3), 481-486 (2017).</li><li>Macphail, R. C., 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=Locomotion+in+larval+zebrafish:+Influence+of+time+of+day,+lighting+and+ethanol.">Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol.</a> Neurotoxicology. 30 (1), 52-58 (2009).</li><li>Kais, B., 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=DMSO+modifies+the+permeability+of+the+zebrafish+(Danio+rerio)+chorion-implications+for+the+fish+embryo+test+(FET).">DMSO modifies the permeability of the zebrafish (Danio rerio) chorion-implications for the fish embryo test (FET).</a> Aquatic Toxicology. 140, 229-238 (2013).</li><li>Truong, L., Harper, S. L., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drug Safety Evaluation. , 271-279 (2011).</li><li>Peeters, B. W., Moeskops, M., Veenvliet, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+preference+in+Danio+rerio:+effects+of+age+and+anxiolytic+treatments.">Color preference in Danio rerio: effects of age and anxiolytic treatments.</a> Zebrafish. 13 (4), 330-334 (2016).</li><li>Barba-Escobedo, P. A., Gould, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+social+preferences+of+lone+zebrafish+in+a+novel+environment:+strain+and+anxiolytic+effects.">Visual social preferences of lone zebrafish in a novel environment: strain and anxiolytic effects.</a> Genes, Brain and Behavior. 11 (3), 366-373 (2012).</li><li>Blaser, R., Penalosa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+affecting+zebrafish+(Danio+rerio)+behavior+in+the+light/dark+preference+test.">Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test.</a> Physiology &amp; Behavior. 104 (5), 831-837 (2011).</li><li>Blaser, R. E., Rosemberg, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+anxiety+in+zebrafish+(Danio+rerio):+dissociation+of+black/white+preference+and+novel+tank+test.">Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test.</a> PloS One. 7 (5), e36931 (2012).</li><li>Weichert, F. G., Floeter, C., Artmann, A. S. M., Kammann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+ecotoxicity+of+potentially+neurotoxic+substances-Evaluation+of+a+behavioural+parameter+in+the+embryogenesis+of+Danio+rerio.">Assessing the ecotoxicity of potentially neurotoxic substances-Evaluation of a behavioural parameter in the embryogenesis of Danio rerio.</a> Chemosphere. 186, 43-50 (2017).</li></ol>

The authors have nothing to disclose.

This work provides a detailed experimental protocol to evaluate the neurotoxicity of environmental pollutants using zebrafish larvae. Zebrafish go through the process from embryos to larvae during the exposure period, which means that good care of the embryos and larvae is essential. Anything that affects the development of the embryos and larvae can influence the final result. Here the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the whole assay.
<p class=...

In recent years more and more environmental pollutants have been proved neurotoxic1,2,3,4. However, the assessment of neurotoxicity in vivo after exposure to environmental pollutants is not as easy as that of endocrine disruption or developmental toxicity. In addition, early exposure to pollutants, especially at environmentally relevant doses, has attracted increasing attention in toxicity studies5,6,7,8.
Zebrafish is being established as an animal model fit for neurotoxicity studies during early development after exposure to environmental pollutants. Zebrafish are vertebrates that develop faster than other species after fertilization. The larvae do not need to be fed because the nutrients in the chorion are enough for sustain them for 7 days postfertilization (dpf)9. Larvae come out from the chorion at ~2 dpf and develop behaviors such as swimming and turning that can be observed, tracked, quantified, and analyzed automatically using behavior instruments10,11,12,13 starting at 3-4 dpf14,15,16,17,18. In addition, high-throughput tests can also be realized by behavior instruments. Thus, zebrafish larvae are an outstanding model for the neurobehavioral study of environmental pollutants19. Here, a protocol is offered using high-throughput monitoring to study the neurobehavioral toxicity of environmental pollutants on zebrafish larvae under light stimuli.
Our lab has studied the neurobehavioral toxicity of 2,2&#39;,4,4&#39;-tetrabromodiphenyl ether (BDE-47)20,21, 6&#39;-Hydroxy/Methoxy-2,2&#39;,4,4&#39;-tetrabromodiphenyl ether (6-OH/MeO-BDE-47)22, deca-brominated diphenyl ether (BDE-209), lead, and commercial chlorinated paraffins23 using the presented protocol. Many labs also use the protocol to study the neurobehavioral effects of other pollutants on larvae or adult fish24,25,26,27. This neurobehavioral protocol was used to help provide mechanistic support showing that low-dose exposure to bisphenol A and replacement bisphenol S induced premature hypothalamic neurogenesis in embryonic zebrafish27. In addition, some researchers optimized the protocol to perform corresponding studies. A recent study eliminated the toxicity of amyloid beta (A&#946;) in an easy, high-throughput zebrafish model using casein-coated gold nanoparticles (&#946;Cas AuNPs). It showed that &#946;Cas AuNPs in systemic circulation translocated across the blood-brain barrier of zebrafish larvae and sequestered intracerebral A&#946;42, eliciting toxicity in a nonspecific, chaperone-like manner, which was supported by behavioral pathology28.
Locomotion, path angle, and social activity are three neurobehavioral indicators used to study the neurotoxicity effects of zebrafish larvae after exposure to pollutants in the presented protocol. Locomotion is measured by the swimming distance of larvae and can be damaged after exposure to pollutants. Path angle and social activity are more closely related with the function of the brain and the central nervous system29. The path angle refers to the angle of the path of animal motion relative to the swimming direction30. Eight angle classes from ~-180&#176;-~+180&#176; are set in the system. To simplify the comparison, six classes in the final outcome are defined as routine turns (-10&#176; ~0&#176;, 0&#176; ~+10&#176;), average turns (-10&#176; ~-90&#176;, +10&#176; ~+90&#176;), and responsive turns (-180&#176; ~-90&#176;, +90&#176; ~+180&#176;) according to our previous studies21,22. Two-fish social activity is fundamental of group shoaling behavior; here a distance of &#60; 0.5 cm between two larvae valid is defined as social contact.
The protocol presented here demonstrates a clear process for studying neurobehavioral effects on zebrafish larvae and provides a way to reveal the neurotoxicity effects of various substances or pollutants. The protocol will benefit researchers interested in studying the neurotoxicity of environmental pollutants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48-well-microplate</td>
			<td>Corning</td>
			<td>3548</td>
			<td>Embyros housing</td>
		</tr>
		<tr>
			<td>6-well-microplate</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Embyros housing</td>
		</tr>
		<tr>
			<td>BDE-47</td>
			<td>AccuStandard</td>
			<td>5436-43-1</td>
			<td>Pollutant</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>67-68-5</td>
			<td>Cosolvent</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SZX 16</td>
			<td>Observation instrument</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000267</td>
			<td>Transfer solution</td>
		</tr>
		<tr>
			<td>Zebrabox</td>
			<td>Viewpoint</td>
			<td>ZebraBox</td>
			<td>Behavior instrument</td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>Shanghai FishBio Co., Ltd.</td>
			<td>Tubingen</td>
			<td>Zebrafish supplier</td>
		</tr>
		<tr>
			<td>ZebraLab</td>
			<td>Viewpoint</td>
			<td>ZebraLab</td>
			<td>Behavior software</td>
		</tr>
	</tbody>
</table>

studying neurobehavioral effects of environmental pollutants on zebrafish larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

Here, we describe a protocol for studying the neurobehavioral effects of environmental pollutants using zebrafish larvae under light stimuli. The locomotion, path angle, and social activity tests are defined in the introduction. The setup of the microplates in the locomotion and path angle tests and the images of the software are shown below. In addition, our own research results are presented as examples. Two studies present the locomotion and path angle effects after exposure to BDE-47 ...

Watch this Scientific Journal Video about Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae at JoVE.com

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

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5.8K Views.  Tongji University. A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Video: Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study evaporation under various surface boundary conditions to improve our current understanding and characterization of this multiphase phenomenon as well as to validate numerical heat and mass transfer theories that couple Navier-Stokes flow in the atmosphere and Darcian flow in the porous media. Experimental data were collected using a unique soil tank apparatus interfaced with a small climate controlled wind tunnel. The experimental apparatus was instrumented with a suite of state of the art sensor technologies for the continuous and autonomous collection of soil moisture, soil thermal properties, soil and air temperature, relative humidity, and wind speed. This experimental apparatus can be used to generate data under well controlled boundary conditions, allowing for better control and gathering of accurate data at scales of interest not feasible in the field. Induced airflow at several distinct wind speeds over the soil surface resulted in unique behavior of heat and mass transfer during the different evaporative stages.

A protocol for the design and construction of a soil tank interfaced to a small climate controlled wind tunnel to study the effects of atmospheric forcings on evaporation is presented. Both the soil tank and wind tunnel are instrumented with sensor technologies for the continuous in situ measurement of environmental conditions.

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: &#34;There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region&#34;, where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: &#34;There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region&#34;.

The protocol focuses on the collection of sea turtle samples, describing all the steps from the animal recovery and necropsy to the classification and quantification of ingested marine litter. Moreover, the representative results show how to use the collected data to elaborate the possible thresholds for Good Environmental Status.

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the ...

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...