We are grateful to Ms. Kaori Shimizu and Mr. Masaki Takasu of the Graduate School of Life Sciences, Toyo University, for their technical support. This project was supported by research grants from the Special Research Foundation and Bio-Nano Electronics Research Centre of Toyo University (to SK); by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to SK); by the New Project Fund for Risk Assessments, from the Ministry of Economy, Trade and Industry (to SK); by a Grant-in-Aid for Challenging Exploratory Research (award 23651028 to SK); by a Grant-in-Aid for Scientific Research (B) and (C) (award 23310026 and 26340030 to SK); and by a Grant-in-Aid for Strategic Research Base Project for Private Universities (award S1411016 to SK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

The Japanese medaka used in this study were treated humanely in accordance with the institutional guidelines of Toyo University, with due consideration for the alleviation of distress and discomfort.
1. Silver Nanocolloids (SNCs)
<ol>
	<li>Purchase purified SNCs (20 mg/L&#8722;1, 99.99% purity, particle mean diameter about 28.4 &#177; 8.5 nm suspended in distilled water).</li>
	<li>Validate the purity and concentration of the silver by inductively coupled plasma mass spectrometry ...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD Guidelines for the Testing of Chemicals, Section 2 Effects on Biotic Systems. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> National Coastal Condition Report. , (2001).</li><li>Sakaizumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+inorganic+salts+on+mercury-compound+toxicity+to+the+embryos+of+the+Medaka,+Oryzias+latipes.">Effect of inorganic salts on mercury-compound toxicity to the embryos of the Medaka, Oryzias latipes.</a> J. Fac. Sci. Univ. Tokyo. 14 (4), 369-384 (1980).</li><li>Sumitani, K., Kashiwada, S., Osaki, K., Yamada, M., Mohri, S., Yasumasu, S., 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=Medaka+(Oryzias+latipes)+Embryo+toxicity+of+treated+leachate+from+waste-landfill+sites.">Medaka (Oryzias latipes) Embryo toxicity of treated leachate from waste-landfill sites.</a> J. Jpn. Soc. Waste Manage. Exp. 15 (6), 472-479 (2004).</li><li>Kashiwada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Nanoparticles+in+the+See-through+Medaka+(Oryzias+latipes).">Distribution of Nanoparticles in the See-through Medaka (Oryzias latipes).</a> EHP. 114 (11), 1697-1702 (2006).</li><li>Iwamatsu, T. <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 Integrated Book for the Biology of the Medaka. , (2006).</li><li>Miyamoto, T., Machida, T., Kawashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+environmental+salinity+on+the+development+of+chloride+cells+of+freshwater+and+brackish-water+medaka,+Oryzias+latipes.">Influence of environmental salinity on the development of chloride cells of freshwater and brackish-water medaka, Oryzias latipes.</a> Zoo. Sci. 3 (5), 859-865 (1986).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> XSERIES 2 ICP-MS Getting Started Guide Revision B - 121 9590. , (2007).</li><li>Kashiwada, S., Ariza, M. E., Kawaguchi, T., Nakagame, Y., Jayasinghe, B. S., Gartner, K., 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=Silver+nanocolloids+disrupt+medaka+embryogenesis+through+vital+gene+expressions.">Silver nanocolloids disrupt medaka embryogenesis through vital gene expressions.</a> ES &amp; T. 46 (11), 6278-6287 (2012).</li><li>Iwamatsu, T. <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+normal+development+in+the+medaka+Oryzias+latipes.">Stages of normal development in the medaka Oryzias latipes.</a> Mech. Dev. 121, 605-618 (2004).</li><li>Kataoka, C., Ariyoshi, T., Kawaguchi, H., Nagasaka, S., Kashiwada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salinity+increases+the+toxicity+of+silver+nanocolloids+to+Japanese+medaka+embryos.">Salinity increases the toxicity of silver nanocolloids to Japanese medaka embryos.</a> Environ. Sci.: Nano. 2, 94-103 (2014).</li><li>Shannon, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+effective+ionic+radii+and+systematic+studies+of+interatomic+distances+in+halides+and+chalcogenides.">Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides.</a> Acta Cryst. 32, 751-767 (1976).</li><li>Wakamatsu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Medaka Book, 6.1: Preparation of hatching enzyme. , (2015).</li></ol>

Medaka is a freshwater fish that is highly tolerant to seawater; it is not well known that the original natural habitat of this fish was saltwater off the Japanese coast6. Hence, medaka fish have well-developed chloride cells7. This unique property provides scientists with a new way to test the toxicity of chemicals in the environment as a function of salinity (freshwater to seawater) by using only a single species of fish.
To obtain medaka eggs at stage 21, eggs must be ...

Since the establishment of the Organisation for Economic Co-operation and Development (OECD) test guidelines for testing chemicals in 1979, 38 test guidelines have been published in Section 2 of the guidelines, Effects on Biotic Systems1. All of the aquatic organisms tested have been from freshwater habitats, namely freshwater plants; algae; invertebrates such as daphnia and chironomids; and fishes such as medaka, zebrafish, and rainbow trout. Compared to saltwater environments, freshwater environments are more directly affected by human economic and industrial activities. Therefore, freshwater environments have been prioritized for testing because they are at higher risk from pollution.
In coastal areas, including estuaries, salinities vary between brackish water and seawater conditions, and these areas are often polluted by industrial activity2. Coastal areas and their associated wetlands are characterized by high ecological biodiversity and productivity. Coastal ecosystems must therefore be protected from chemical pollution. However, there has been limited ecotoxicological research in brackish water and seawater habitats.
Sakaizumi3 studied the toxic interactions between methyl mercury and salinity in Japanese medaka eggs and found that increasing the osmotic pressure of the test solution enhanced the toxicity of the methyl mercury. Sumitani et al.4 used medaka eggs to investigate the toxicity of landfill leachate; they found that the osmotic equivalency of leachate to the eggs was the key to inducing abnormalities during embryogenesis. In addition, Kashiwada5 reported that plastic nanoparticles (39.4 nm in diameter) easily permeated through the medaka egg chorion under brackish conditions (15x embryo rearing medium (ERM)).
A typical small fish model, the Japanese medaka (Oryzias latipes) has been used in basic biology and ecotoxicology6. Japanese medaka can live in conditions ranging from freshwater to seawater because of their highly developed chloride cells7. They are therefore likely to be useful for testing in conditions with a wide range of salinities.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Silver nanocolloids</td>
			<td style="width:188px;">Utopia Silver Supplements</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">NaCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31319-45</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">KCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">28513-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2&#183;2H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">06730-15</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">21002-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3&#160;</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31212-25</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgNO3</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31018-72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>HORIBA, Ltd.</td>
			<td style="width:119px;">F-51S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balance</td>
			<td>Mettler-Toledo International Inc.</td>
			<td style="width:119px;">MS204S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka (Oryzias latipes) orange-red strain&#160;</td>
			<td colspan="2">National Institute for Environmental Studies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka flow-through culturing system</td>
			<td>Meito Suien Co.</td>
			<td>MEITOsystem</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Artemia salina nauplii eggs</td>
			<td>Japan pet design Co. Ltd</td>
			<td>4975677033759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aeration pomp</td>
			<td>Japan pet design Co. Ltd</td>
			<td>non-noise w300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otohime larval &#946;-1</td>
			<td style="width:188px;">Marubeni Nissin Feed Co. Ltd</td>
			<td style="width:119px;">Otohime larval &#946;-1</td>
			<td>Artificial dry fish diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td style="width:188px;">Leica microsystems</td>
			<td style="width:119px;">M165FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micrometer</td>
			<td>Fujikogaku, Ltd.</td>
			<td>10450023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator</td>
			<td>Nksystem</td>
			<td>TG-180-5LB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">shaker</td>
			<td style="width:188px;">ELMI Ltd.</td>
			<td style="width:119px;">Aizkraukles 21-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plastic plates</td>
			<td>Greiner CELLSTAR</td>
			<td style="width:119px;">M8562-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum foil</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">6-713-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stopwatch</td>
			<td>DRETEC Co. Ltd.</td>
			<td style="width:119px;">SW-111YE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-kDa membrane filter</td>
			<td>EMD Millipore Corporation</td>
			<td colspan="2">0.5-mL centrifugal-type filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-mL Teflon beaker</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">33431097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-538</td>
			<td>For internal standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-622</td>
			<td>For external standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ultrapure nitric acid</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">28163-5B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">hydrogen peroxide&#160;</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">18084-1B</td>
			<td>for atomic absorption spectrometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ICP-MS</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Thermo Scientific X Series 2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hot plate</td>
			<td>Tiger Co.</td>
			<td>CRC-A300</td>
			<td></td>
		</tr>
	</tbody>
</table>

salinity-dependent toxicity assay of silver nanocolloids using medaka eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

The effect of salinity on SNC toxicity was very obvious: the induction of deformity or death was salinity dependent (Figures 1 and 2). We measured phenotypic biomarkers (heart rate, eye size, full body length, and hatching rate) in SNC (10 mg/L&#8722;1)-exposed embryos. These phenotypic biomarkers revealed salinity-dependent SNC toxicity.
Heart rates ranged from 29.6 to 32.2 beats/15 ...

Watch this Scientific Journal Video about Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs at JoVE.com

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

The overall goal of this experiment is to test the salinity dependent toxicity of chemicals to aquatic organisms. This method can help answer key questions in the Ecotoxicology field, such as assessment of toxicity on the fresh water, brackish water, and sea water conditions. The main advantage of this technique, is that medaka are never asked to test toxicity of chemicals in different salinities.Additionally, the implication of this technique extend to other therapy or diagnosis of chemicals toxicity. Preparation of commerical silver nanocolloids in solution, at various salinities, is covered in the text protocol. As is medaka culturing and egg collection.Begin with rinsing six collections of Stage 21 medaka eggs, three times in test solution. Do this using ERMs, at a different concentration for each set of eggs. Now, from each rinsed collection, transfer 15 eggs to a different well of test solution, in a six-well plastic plate.Set this experiment up in triplicate, washing every group separately. Wrap the plates in foil, and incubate them at 25 degrees Celcius, in the dark. Until the eggs hatch, or for 14 days, whichever comes sooner.During the incubation, observe the eggs daily for any biological changes. And for any dead eggs. Change the test solution daily, along with making the observations.On the sixth day of observation, count the heart rate of each embryo over 15 seconds. Also on the sixth day, measure the diameter of each embryo's eyes, in each experimental condition, using a micrometer. Whenever the embryos hatch into larvae, measure their full body lengths, using a micrometer.Also, over the 14 days, keep count of the total eggs that hatch. To measure silver bioaccumulation, on the sixth day, collect the eggs'chorions, using the standard method. The dechorionated eggs can be analyzed, just as the SNC solution is analyzed, as described in the following section.To isolate the soluble silver from each SNC solution. first, filter the mixture of colloids and soluble silver out, using a three kilodalton membrane. Then, spin down the loaded filter at 14, 000 Gs, for 10 minutes, in a refrigerated centrifuge.From 50 microliters of the filtrate, measure the silver concentration by ICP-MS analysis. Load the aliquot into a 50 millileter Teflon beaker, and add two milliliters of ultrapure nitric acid to the beaker. Then, heat the mixture on a hot plate, at 110 degrees Celcius.Over about three hours of heating, let the solution nearly dry out. Once the beaker has cooled, completely dissolve the organic solids. Add two millilters of ultrapure nitric acid, and 0.5 milliliters of hydrogen peroxide, to the beaker.Then, heat the mixture again, until it has nearly dried out again. Now, dissolve the remaining residue in four milliliters of one percent ultrapure nitric acid, made in ultrapure water. Transfer the solution to a 15 milliliter centrifuge tube.Repeat the process of dissolving the remaining solids in the Teflon beaker, two more times, cooling the three collections into a single tube. Now, measure the silver concentration in the sample, using ICP-MS. Use the manufacturer's recommended protocol to analyze the samples.Be sure to use internal and external standards. The effect of salinity on SNC toxicity was easily apparent. The induction of deformity or death was clearly salinity-dependent.Various physiological measurements were made. Heart rates were unaffected until the salinity reached 30 X ERM. Body length was significantly shorter at salinities of 15 and 30 X ERM.Eye diameter dropped as well, consistent with body length. Hatching was less likely at 15 X ERM, and only occurred for two percent of eggs in 30 X ERM. Under the experimental solutions, water soluble metal complexes formed more readily as the salt concentration increased.Examination of the metal complexes in the embryos after six days showed that they increased in concentration in the embryos as they increased in concentration in the solution. While attempting this procedure, it's important to remember to adjust the pH of the test conditions, because silver ion release is facilitated by acidic condition.

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7.9K Views.  Toyo University. The overall goal of this experiment is to test the salinity dependent toxicity of chemicals to aquatic organisms. This method can help answer key questions in the Ecotoxicology field, such as assessment of toxicity on the fresh water, brackish water, and sea water conditions. The main advantage of this technique, is that medaka are never asked to test toxicity of chemicals in different salinities.Additionally, the implication of this technique extend to other therapy or diagnosis of ch...