Name: long-term lethal toxicity test with the crustacean artemia franciscana
Uploaded: 2012-04-14T14:00:00
Description: Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

Produced by Loredana Manfra of Institute for environmental protection and research (ISPRA-Department of Environmental Quality Monitoring) and Federica Savorelli of Regional Agency for Environmental Protection (Emilia-Romagna, Italy).
Reference Members for the sub-working Group on the Artemia crustacean within the Biological Methods Group &#x2013; Saltwater/Brackish Water and Sediment of the UNICHIM Commission on Water Quality (the institute for technical standardization operating in the chemical sector).
Videography and editing by Marco Pisapia of ISPRA (Web Unit). 
Production Coordination by Anna Maria Cicero and Erika Magaletti of ISPRA (Department of Environmental Quality Monitoring).
We would like to thank the Regional Agency for Environmental Protection of Emilia-Romagna, Ferrara Branch, the Biological Methods Group - Saltwater /Brackish Water and Sediment of the UNICHIM Commission on Water Quality and Luciana Migliore, Tor Vergata University (Rome) for their cooperation; Rossella Boscolo and Massimo Gabellini of ISPRA (Department of Prevention and Mitigation of Impacts) for their financial support; Fabio Matassa and the London School of Languages in Rome for their linguistic support.

1. Method Standard 
Our testing involves exposing Artemia larvae to an interval of substance concentrations or a test sample for the purpose of determining the concentration or dilution levels that would cause the death of 50% of the organisms (LC50) exposed over 14 days and meeting the conditions defined by this method. If necessary and possible, the following may also be determined: a) the concentration level that causes the death of 20 % of organisms exposed (LC20</su...

<ol>
	<li>Togulga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Short-Term+Toxicity+of+Two+Toxicants+to+Artemia+Nauplii.">The Short-Term Toxicity of Two Toxicants to Artemia Nauplii.</a> Tr. J. of Zoology. 22, 259-266 (1998).</li><li>Persoone, G., Wells, P. G., Sorgeloos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+in+aquatic+toxicology:+a+review.">Artemia in aquatic toxicology: a review.</a> Artemia Research and its Applications. Morphology, Genetics, Strain characterization Toxicology. , 259-275 (1987).</li><li>Nunes, B. S., Carvalho, F. D., Guilhermino, L. M., Stappen, G. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+genus+Artemia+in+ecotoxicity+testing.">Use of the genus Artemia in ecotoxicity testing.</a> Envir. Poll. 144, 453-462 (2006).</li><li>APAT IRSA-CNR. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metodi analitici per le acque Manuali e Linee guida 29/2003. Terzo Metodo, 8060 (2003).</li><li>Sorgellos, P., Wielen, C. R. e. m. i. c. h. e. -. V. a. n. D. e. r., Persoone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Artemia+nauplii+for+toxicity+tests+â€“+a+critical+analysis.">The use of Artemia nauplii for toxicity tests â€“ a critical analysis.</a> Ecotox. Env. Saf. 2, 249-255 (1978).</li><li>Savorelli, F., Palazzi, D., Gorbi, G., Invidia, M., Sei, S., Magaletti, E., Manfra, L., Gelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Set+up+of+a+standard+methodology+for+14-day+bioassay+on+Artemia+franciscana+and+A.+parthenogenetica.">Set up of a standard methodology for 14-day bioassay on Artemia franciscana and A. parthenogenetica.</a> Biol. Amb. 21, 27-36 (2007).</li><li>Brix, K. V., Cardwell, R. D., Adams, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+toxicity+of+arsenic+to+the+Great+Salt+Lake+brine+shrimp,+Artemia+franciscana.">Chronic toxicity of arsenic to the Great Salt Lake brine shrimp, Artemia franciscana.</a> Ecotox. Environ. Saf. 54, 169-175 (2003).</li><li>Brix, K. V., Deforest, D. K., Cardwell, R. D., Adams, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+chronic+site-specific+water+quality+standard+for+selenium+in+the+Great+Salt+Lake,+Utah,+USA.">Derivation of a chronic site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA.</a> Environ. Toxicol. Chem. 23, 606-612 (2004).</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=Qualit&#225;+dell'acqua+-+Determinazione+della+tossicit&#225;+letale+a+14+giorni+con+Artemia+franciscana+(Crustacea:+Anostraca).">Qualit&#225; dell'acqua - Determinazione della tossicit&#225; letale a 14 giorni con Artemia franciscana (Crustacea: Anostraca).</a> Commissione UNICHIM "Qualit&#225; dell'acqua" Gruppo di Lavoro "Metodi Biologici" Sottogruppo "Acque salate/salmastre e sedimenti". , (2010).</li><li>Varo, I., Navarro, J. C., Amat, F., Guilhermino, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+cholinesterases+and+evaluation+of+the+inhibitory+potential+of+chlorpyrifos+and+dichlorvos+to+Artemia+salina+and+Artemia+parthenogenetica.">Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica.</a> Chem. 48, 563-569 (2002).</li></ol>

Artemia is one of the most valuable test organisms available for ecotoxicity testing and research done so far allows us to state that it is possible to sustain several options related to Artemia use in Toxicology and Ecotoxicology. The characteristics that turn this organism into a suitable species for bioassays are: a wide geographic distribution, high adaptability to adverse environmental conditions and varied nutrients, relatively simple laboratory culture and maintenance, resistance to manipulation,...

Produced by Loredana Manfra of Institute for environmental protection and research (ISPRA-Department of Environmental Quality Monitoring) and Federica Savorelli of Regional Agency for Environmental Protection (Emilia-Romagna, Italy).
Reference Members for the sub-working Group on the Artemia crustacean within the Biological Methods Group &#x2013; Saltwater/Brackish Water and Sediment of the UNICHIM Commission on Water Quality (the institute for technical standardization operating in the chemical sector).
Videography and editing by Marco Pisapia of ISPRA (Scientific Advisory and Documentation Services). 
Production Coordination by Anna Maria Cicero and Erika Magaletti of ISPRA (Department of Environmental Quality Monitoring).
We would like to thank the Regional Agency for Environmental Protection of Emilia-Romagna, Ferrara Branch and the Biological Methods Group &#x2013; Saltwater /Brackish Water and Sediment of the UNICHIM Commission on Water Quality for their cooperation; Rossella Boscolo and Massimo Gabellini of ISPRA (Department of Prevention and Mitigation of Impacts) for their financial support; and the London School of Languages in Rome for their linguistic support.
<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Product number</td>
 </tr>
 <tr>
 <td>Sodium Dodecyl Sulfate </td>
 <td>Sigma-Aldrich </td>
 <td>L4390 </td>
 </tr>
 </tbody>
</table>
Appendix
The algal media has to be prepared using analytical quality reagents. 
A liter of nutrient solution is prepared by dissolving 300 g of NaNO3, 30 g of KH2PO4 and 20 g of NH4Cl in distilled water. 
Four initial solutions (A, B, C, D) of minerals are prepared as follows:
<ul>
 <li> A liter of solution "A" containing 30 g of ZnSO4 H2O, 25 g of CuSO4 5 H2O, 30 g of CoSO4 7 H2O and 20 g of MnSO4 H2O in distilled water;
 </li>
 <li> A liter of solution "B" containing 50 g of FeCl3 &bull;6 H2O in distilled water; 
 </li>
 <li> A liter of solution "C" containing 25 g of Na2MoO4 &bull;2 H2O in distilled water;
 </li>
 <li> A liter of solution "D" containing 50 g di Na2EDTA &bull;2 H2O in distilled water.
 </li>
</ul>
A liter of a final solution of minerals (solution "E") is obtained adding aliquots of the above-mentioned solutions to distilled water. The following order of addition is recommended: 100 ml of solution "D", 10 ml of solution "C", 10 ml of solution "B" and 10 ml of solution "A".
Three initial solutions (F, G, H) of vitamins are prepared as follows:
<ul>
 <li> A liter of solution "F" containing 100 mg of biotin in distilled water;
 </li>
 <li> A liter of solution "G" containing 10 g of thiamine in distilled water;
 </li>
 <li> A liter of solution "H" containing 100 mg of cobalamin in distilled water.
 </li>
</ul>
A liter of a final solution of vitamins (solution "I") is obtained adding 10 ml of the three initial solutions to distilled water.
A liter of the algal media is prepared by dissolving 1 ml of the nutrient solution, 1 ml of the mineral final solution (solution "E") and 1 ml of the vitamin final solution (solution "I") in seawater. This latter may be obtained by dissolving recognized analytical quality reagents or a commercially available formulation in distilled or de-ionized water. Salt blends for creating the ideal saltwater are commercially available, e.g. the Instant Ocean mixtures. It is recommended the using of a seawater with salinity of 30 (&plusmn;2) PSU and temperature of 20 (&plusmn;1) &deg;C.

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.

Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

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.

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 ...

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