Name: Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs (Video) | JoVE Journal
Uploaded: 2016-03-18T13:00:00.000+00:00
Duration: 6 min 29 s
Description: Learn a high-throughput toxicity assay using medaka eggs to assess silver nanocolloid effects across freshwater, brackish, and seawater conditions. This method advances ecotoxicology research.

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5 minutes

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://scholar.google.com/scholar?hl=en&safe=off&q=%22OECD+Guidelines+for+the+Testing+of+Chemicals%2C+Section+2+Effects+on+Biotic+Systems%22">OECD Guidelines for the Testing of Chemicals, Section 2 Effects on Biotic Systems</a>. , OECD. Available from:
 <a target="_blank" href="http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-effects-on-biotic-systems_20745761">http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-effects-on-biotic-systems_20745761</a> (2015).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22National+Coastal+Condition+Report%22">National Coastal Condition Report</a>. , Environmental Protection Agency, Office of Water and Office of Research and Development. Washington, DC. (2001).</li>
<li>Sakaizumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+inorganic+salts+on+mercury-compound+toxicity+to+the+embryos+of+the+Medaka%2C+Oryzias+latipes%5BTitle%5D)+AND+%22J.+Fac.+Sci.+Univ.+Tokyo%22%5BJournal%5D)">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/pubmed?term=((Medaka+%28Oryzias+latipes%29+Embryo+toxicity+of+treated+leachate+from+waste-landfill+sites%5BTitle%5D)+AND+%22J.+Jpn.+Soc.+Waste+Manage.+Exp%22%5BJournal%5D)">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/pubmed?term=((Distribution+of+Nanoparticles+in+the+See-through+Medaka+%28Oryzias+latipes%29%5BTitle%5D)+AND+%22EHP%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aIwamatsu%2C+T+%22The+Integrated+Book+for+the+Biology+of+the+Medaka%22">The Integrated Book for the Biology of the Medaka</a>. , University Education Press. Japan. (2006).</li>
<li>Miyamoto, T., Machida, T., Kawashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+environmental+salinity+on+the+development+of+chloride+cells+of+freshwater+and+brackish-water+medaka%2C+Oryzias+latipes%5BTitle%5D)+AND+%22Zoo.+Sci%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22XSERIES+2+ICP-MS+Getting+Started+Guide+Revision+B+-+121+9590%22">XSERIES 2 ICP-MS Getting Started Guide Revision B - 121 9590</a>. , Thermo Fisher Scientific Inc.. Available from:
 <a target="_blank" href="http://202.127.146.37/eWebEditor/uploadfile/20130314161434190.pdf">http://202.127.146.37/eWebEditor/uploadfile/20130314161434190.pdf</a> (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/pubmed?term=((Silver+nanocolloids+disrupt+medaka+embryogenesis+through+vital+gene+expressions%5BTitle%5D)+AND+%22ES+%26+T%22%5BJournal%5D)">Silver nanocolloids disrupt medaka embryogenesis through vital gene expressions.</a> ES & T. 46 (11), 6278-6287 (2012).</li>
<li>Iwamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stages+of+normal+development+in+the+medaka+Oryzias+latipes%5BTitle%5D)+AND+%22Mech.+Dev%22%5BJournal%5D)">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/pubmed?term=((Salinity+increases+the+toxicity+of+silver+nanocolloids+to+Japanese+medaka+embryos%5BTitle%5D)+AND+%22Environ.+Sci.%3A+Nano%22%5BJournal%5D)">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/pubmed?term=((Revised+effective+ionic+radii+and+systematic+studies+of+interatomic+distances+in+halides+and+chalcogenides%5BTitle%5D)+AND+%22Acta+Cryst%22%5BJournal%5D)">Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides.</a> Acta Cryst. 32, 751-767 (1976).</li>
<li>Pore size chart. , Spectrum Laboratories, Inc.. Available from:
 <a target="_blank" href="http://jp.spectrumlabs.com/dialysis/PoreSize.html">http://jp.spectrumlabs.com/dialysis/PoreSize.html</a> (2015).</li>
<li>Wakamatsu, Y. Medaka Book, 6.1: Preparation of hatching enzyme. , National BioResource Project (NBRP) Medaka. Available from:
 [cited 2015] <a target="_blank" href="https://shigen.nig.ac.jp/medaka/medakabook/index.php?6.1%20preparation%20of%20hatching%20enzyme">https://shigen.nig.ac.jp/medaka/medakabook/index.php?6.1%20preparation%20of%20hatching%20enzyme</a> (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-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−1)-exposed embryos. These phenotypic biomarkers revealed salinity-dependent SNC toxicity.
Heart rates ranged from 29.6 to 32.2 beats/15 sec th...

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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JoVE Journal

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