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 (ICP-MS) analyses according to operating manual8. The pretreatment method for ICP-MS analyses is described in section 7.</li>
</ol>
2. Preparation of SNC Solutions (Mixtures of Silver Colloids and Ag+) with Different Salinities
<ol>
	<li>Prepare 60&#215; ERM consisting of 60 g NaCl, 1.8 g KCl, 2.4 g CaCl2&#183;2H2O, and 9.78 g MgSO4&#183;7H2O in 1 L of ultrapure water; adjust the pH to 7.0 with 1.25% NaHCO3 in ultrapure water.</li>
	<li>Stir the ERM solution at 25 &#176;C overnight.</li>
	<li>Mix SNCs with diluted ERM. Prepare 40 ml of each SNC-ERM mixed solution. The final concentration is 10 mg/L&#8722;1 of SNCs in different concentrations of ERM (1x, 5x, 10x, 15x, 20x, or 30x).</li>
	<li>Adjust pH of the SNC-ERM mixed solution to 7.0 with 0.625% NaHCO3 in ultrapure water. pH adjustment is very important in preparing the SNC solution, because Ag+ release is facilitated by acidic conditions9.</li>
	<li>Use AgNO3 as a reference compound for SNCs.
	<ol>
		<li>Mix AgNO3 with diluted ERM. Prepare 40 ml of AgNO3-ERM mixed solution at an AgNO3 concentration of 15.7 mg/L-1 (10 mg/L&#8722;1 silver) in different concentrations of ERM (1x, 5x, 10x, 15x, 20x, or 30x). 
		Note: To examine silver colloid toxicity, AgNO3 solution, which is a source of soluble silver, is used as a reference compound for SNCs, which are a mixture of silver colloids and soluble silver.</li>
	</ol>
	</li>
</ol>
3. Medaka Culture and Egg Harvesting
<ol>
	<li>Obtain the medaka (O. latipes) (orange-red strain) (60 males and 60 females).</li>
	<li>Culture medaka as groups (20 males and 20 females as one group) in 1x ERM in 3 L tanks by using a medaka flow-through culturing system.
	<ol>
		<li>Culture at the following conditions: 
		pH range of the culture medium: 6.2 to 6.5&#160;&#160; 
		light:dark cycle: 16:8 hr 
		temperature of the culture medium: 24 &#177; 0.5 &#176;C 
		osmotic pressure of the culture medium: 257 mOsm</li>
	</ol>
	</li>
	<li>Feed medaka on Artemia salina nauplii at 10:00 (once a day) and feed an artificial dry fish diet at 09:00, 11:00, 13:00, 15:00, and 17:00 (five times a day).
	<ol>
		<li>Obtain A. salina nauplii.</li>
		<li>Prepare 5 L of a 3.0% salt solution in a plastic beaker.</li>
		<li>Add 30 g of brine shrimp eggs to the salt solution in the beaker.</li>
		<li>Incubate the eggs at 25 &#176;C for 48 hr with bubbling (4 L/min&#8722;1) using an aeration pump.</li>
		<li>After 48 hr, stop the bubbling.</li>
		<li>Allow the solution to stand for 5 to 10 min to separate the hatched A. salina nauplii (lower part of the solution) from the unhatched eggs and eggshells (upper part of the solution).</li>
		<li>Remove the upper layer of the solution by decantation.</li>
		<li>Filter the lower portion of the solution through a sieve with openings of 283 &#181;m, and collect the nauplii that pass through on a net with openings of 198 &#181;m.</li>
		<li>Feed the nauplii to the medaka within 6 hr.</li>
	</ol>
	</li>
	<li>After the female medaka have spawned, remove the external egg clusters gently from the females&#39; bodies or collect the eggs from the bottom of the fish tank by using a small net (net size 5 cm x 5 cm, hole size 0.2 mm x 0.2 mm).</li>
	<li>Rinse the egg cluster with flowing tap water for 5 sec.</li>
	<li>Add all of the rinsed egg clusters to 30x ERM solution.</li>
	<li>Remove the clusters from the solution after 1 min and place the egg clusters between dry paper towels and roll gently.</li>
	<li>Put the eggs back into the 30x ERM.</li>
	<li>Select fertilized eggs under a dissecting microscope.</li>
	<li>Place selected 810 eggs in 1x ERM in six-well plastic plates by using forceps.</li>
	<li>Incubate the eggs at 25 &#177; 0.1 &#176;C in an incubator until developmental stage 21. (Developmental stages of the medaka embryos were defined from the work of Iwamatsu10.)</li>
	<li>Pick out incubated eggs at developmental stage 21 under a dissecting microscope.</li>
	<li>Rinse selected eggs with 1x ERM.</li>
	<li>Subject the rinsed eggs to exposure experiments (section 4).</li>
</ol>
4. Toxicity Testing of SNCs or AgNO3 at Different ERM Salinities
<ol>
	<li>Rinse medaka eggs (stage 21) three times with test solution [SNCs (10 mg/L&#8722;1) or AgNO3 (15.7 mg/L&#8722;1 as 10 mg/L&#8722;1 silver) at each concentration of ERM (1x, 5x, 10x, 15x, 20x, or 30x) at pH 7]. As controls, use eggs in 1&#215; to 30&#215; ERM at pH 7.</li>
	<li>Add 15 rinsed eggs to 5 ml of each test solution in six-well plastic plates. (Perform the exposure experiments three times for SNC or AgNO3 toxicity testing using each test solution.)</li>
	<li>Wrap the plates in aluminum foil.</li>
	<li>Incubate the wrapped plates at 25 &#176;C in the dark until hatching or for 14 days.</li>
	<li>Observe the exposed eggs every 24 hr for biological changes and dead eggs (Figures 1 and 2).</li>
	<li>Exchange the test solutions every 24 hr.</li>
	<li>Perform observations as follows.
	<ol>
		<li>On day 6 of exposure, count the heart rate (per 15 sec) of medaka embryos under a dissecting microscope by using a stopwatch (Figure 3a).</li>
		<li>On day 6 of exposure, measure the eye size (diameter) of medaka embryos under a dissecting microscope by using a micrometer (Figure 3b).</li>
		<li>On hatching day, measure the full body lengths of larvae under a dissecting microscope by using a micrometer (Figure 3c).</li>
		<li>Count the total number of exposed eggs that hatch over the 14 days (Figure 3d).</li>
	</ol>
	</li>
</ol>
5. Isolation of Soluble Silver from SNC Solution, and Silver Analysis
<ol>
	<li>Isolate soluble silver from each SNC solution (a mixture of silver colloids and soluble silver) by filtering through a 3 kDa membrane filter at 14,000 x g and 4 &#176;C for 10 min. Use a 3 kDa membrane filter to isolate soluble silver from the SNCs, because the reported mean diameter of aggregated SNCs in 1x ERM is 67.8 nm11 and that of Ag+ is 0.162 nm12; the 3 kDa membrane excludes particles with diameters of 2 nm or more13.</li>
	<li>Measure the silver concentration in 50 &#181;l of filtered solution (= the soluble silver concentration) by ICP-MS analysis (Figure 3e) according to the ICP-MS operating manual8. The pretreatment method for the ICP-MS analyses is described in section 7.</li>
</ol>
6. Measurement of Silver Bioaccumulation in Medaka Embryos
<ol>
	<li>Expose medaka eggs (stage 21) to SNCs or AgNO3 as described in section 4.</li>
	<li>On day 6 of exposure, remove chorion from the egg (i.e., dechorion) by using medaka hatching enzyme according to the protocol described in the Medaka Book14.</li>
	<li>Measure the silver concentration of the dechorioned eggs by ICP-MS analysis according to the ICP-MS operating manual8 (Figure 3f). The pretreatment method for the ICP-MS analyses is described in section 7.</li>
</ol>
7. Measurement of Silver Concentration by ICP-MS Analysis
<ol>
	<li>Add samples [50 &#181;l of silver solution (for validation of the silver concentration; section 1); three dechorionated embryos (section 5); or 50 &#181;l of filtered solution (section 5)] to a 50 ml Teflon beaker.</li>
	<li>Add 2.0 ml of ultrapure nitric acid to the 50 ml beaker.</li>
	<li>Heat the mixture on a hot plate at 110 &#176;C until just before it dries out (about 3 hr).</li>
	<li>To dissolve the organic matter completely, add 2.0 ml of ultrapure nitric acid and 0.5 ml of hydrogen peroxide to the beaker.</li>
	<li>Heat the mixture again on the hot plate until just before it dries out (about 3 hr).</li>
	<li>Dissolve the residue in 4 ml of 1.0% ultrapure nitric acid solution.</li>
	<li>Transfer 4 ml of solution to a centrifuge tube.</li>
	<li>Repeat 7.6 to 7.7 twice (a total of three times). The final volume is 12.0 ml.</li>
	<li>Measure the silver concentration of the sample (dissolved in 1.0% ultrapure nitric acid) by using ICP-MS analysis according to the operating manual8.
	<ol>
		<li>Use an internal and an external standard solution (See Materials List) to quantify the silver concentration. The internal and external standard solution is accredited by American Association for Laboratory Accreditation (A2LA). Detection limits of silver were 0.0018 ng/ml&#8722;1 (solution) and 0.016 ng mg-weight&#8722;1 (embryo body).</li>
	</ol>
	</li>
</ol>

<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>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>Silver nanocolloids</td><td>Utopia Silver Supplements</td><td></td><td></td></tr><tr><td>NaCl</td><td>Nacalai Tesque, Inc.</td><td>31319-45</td><td>For making ERM</td></tr><tr><td>KCl</td><td>Nacalai Tesque, Inc.</td><td>28513-85</td><td>For making ERM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Nacalai Tesque, Inc.</td><td>06730-15</td><td>For making ERM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Nacalai Tesque, Inc.</td><td>21002-85</td><td>For making ERM</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Nacalai Tesque, Inc.</td><td>31212-25</td><td>For making ERM</td></tr><tr><td>AgNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Nacalai Tesque, Inc.</td><td>31018-72</td><td></td></tr><tr><td>pH meter</td><td>HORIBA, Ltd.</td><td>F-51S</td><td></td></tr><tr><td>Balance</td><td>Mettler-Toledo International Inc.</td><td>MS204S</td><td></td></tr><tr><td>medaka (&lt;em&gt;Oryzias latipes&lt;/em&gt;) orange-red strain</td><td>National Institute for Environmental Studies</td><td></td><td></td></tr><tr><td>medaka flow-through culturing system</td><td>Meito Suien Co.</td><td>MEITOsystem</td><td></td></tr><tr><td>Artemia salina nauplii eggs</td><td>Japan pet design Co. Ltd</td><td>4975677033759</td><td></td></tr><tr><td>aeration pump</td><td>Japan pet design Co. Ltd</td><td>non-noise w300</td><td></td></tr><tr><td>Otohime larval &amp;beta;-1</td><td>Marubeni Nissin Feed Co. Ltd</td><td>Otohime larval &amp;beta;-1</td><td>Artificial dry fish diet</td></tr><tr><td>dissecting microscope</td><td>Leica microsystems</td><td>M165FC</td><td></td></tr><tr><td>micrometer</td><td>Fujikogaku, Ltd.</td><td>10450023</td><td></td></tr><tr><td>incubator</td><td>Nksystem</td><td>TG-180-5LB</td><td></td></tr><tr><td>shaker</td><td>ELMI Ltd.</td><td>Aizkraukles 21-136</td><td></td></tr><tr><td>6-well plastic plates</td><td>Greiner CELLSTAR</td><td>M8562-100EA</td><td></td></tr><tr><td>aluminum foil</td><td>AS ONE Co.</td><td>6-713-02</td><td></td></tr><tr><td>stopwatch</td><td>DRETEC Co. Ltd.</td><td>SW-111YE</td><td></td></tr><tr><td>3 kDa membrane filter</td><td>EMD Millipore Corporation</td><td>0.5 ml centrifugal-type filter</td><td></td></tr><tr><td>50 ml Teflon beaker</td><td>AS ONE Co.</td><td>33431097</td><td></td></tr><tr><td>Custom claritas standard</td><td>SPEXertificate</td><td>ZSTC-538</td><td>For internal standard</td></tr><tr><td>Custom claritas standard</td><td>SPEXertificate</td><td>ZSTC-622</td><td>For external standard</td></tr><tr><td>ultrapure nitric acid</td><td>Kanto Chemical Co.</td><td>28163-5B</td><td></td></tr><tr><td>hydrogen peroxide&amp;nbsp;</td><td>Kanto Chemical Co.</td><td>18084-1B</td><td>for atomic absorption spectrometry</td></tr><tr><td>ICP-MS</td><td>Thermo Scientific</td><td>Thermo Scientific X Series 2&amp;nbsp;</td><td></td></tr><tr><td>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 ...

salinity-dependent-toxicity-assay-silver-nanocolloids-using-medaka

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Developmental Biology

7.9K Views.  Toyo University. 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.

Video: Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Environment

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal toxicity test is frequently used in regulatory risk assessment of chemicals. In order to achieve high reliability and reproducibility the development of standardized guidelines is vital. For algal toxicity testing, the guidelines require stable and uniform conditions of parameters such as pH, temperature, carbon dioxide levels and light intensity. Nanomaterials and other so-called difficult substances can interfere with light causing a large variation in results obtained hampering their regulatory acceptance. To address these challenges, we have developed LEVITATT (LED Vertical Illumination Table for Algal Toxicity Tests). The setup utilizes LED illumination from below allowing for a homogenous light distribution and temperature control while also minimizing intra-sample shading. The setup optimizes the sample volume for biomass quantification and does at the same time ensure a sufficient influx of CO2 to support exponential growth of the algae. Additionally, the material of the test containers can be tailored to minimize adsorption and volatilization. When testing colored substances or particle suspensions, the use of LED lights also allows for increasing the light intensity without additional heat generation. The compact design and minimal equipment requirements increase the possibilities for implementation of the LEVITATT in a wide range of laboratories. While compliant with standardized ISO and OECD guidelines for algal toxicity testing, LEVITATT also showed a lower inter-sample variability for two reference substances (3,5-Dicholorophenol and K2Cr2O7) and three nanomaterials (ZnO, CeO2, and BaSO4) compared to Erlenmeyer flasks and microtiter plates.

We demonstrate algal toxicity testing for difficult substances (e.g., colored substances or nanomaterials) using a setup illuminated vertically with an LED.

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

Chemistry

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Biology

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Medicine

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Neuroscience

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

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Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

Lethality Bioassay Using Artemia salina L.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System