Name: lethality bioassay using artemia salina l.
Uploaded: 2022-10-11T13:40:00
Description: Watch this Scientific Journal Video about Lethality Bioassay Using Artemia salina L. at JoVE.com

In memory of&#160;Professor Amilcar Roberto.
This work was financially supported by Fundac&#807;a&#771;o para a Cie&#770;ncia e a Tecnologia (FCT, Portugal) under projects UIDB/04567/2020 and UIDP/04567/2020 attributed to CBIOS and PhD grant SFRH/BD/137671/2018 (Vera Isca).

1. Equipment preparation
<ol>
	<li>Acquire commercially available hatching equipment. Select a suitable place to set up the hatching equipment (Figure 2A). Place the funnel-shaped container in the black support (included in the set) and turn the funnel in a suitable direction to see the level mark and the tap.</li>
	<li>To make hand-made migration equipment, cut the top of two 0.5 L (5.8 cm diameter) plastic bottles to obtain a final height of 12 cm. Create a hole of 1.5 c...

<ol>
	<li>Ntungwe, N. E., 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=Artemia+species:+An+important+tool+to+screen+general+toxicity+samples.">Artemia species: An important tool to screen general toxicity samples.</a> Current Pharmaceutical Design. 26 (24), 2892-2908 (2020).</li><li>Cragg, G. M., Newman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products:+A+continuing+source+of+novel+drug+leads.">Natural products: A continuing source of novel drug leads.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1830 (6), 3670-3695 (2013).</li><li>Ntungwe, E., 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=General+toxicity+screening+of+Royleanone+derivatives+using+an+artemia+salina+model.">General toxicity screening of Royleanone derivatives using an artemia salina model.</a> Journal Biomedical and Biopharmaceutical Research. 18 (1), 114 (2021).</li><li>Seca, A., Plant Pinto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=secondary+metabolites+as+anticancer+agents:+Successes+in+clinical+trials+and+therapeutic+application.">secondary metabolites as anticancer agents: Successes in clinical trials and therapeutic application.</a> International Journal of Molecular Sciences. 19 (1), 263 (2018).</li><li>Calixto, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+natural+products+in+modern+drug+discovery.">The role of natural products in modern drug discovery.</a> Anais da Academia Brasileira de Ci&#234;ncias. 91 (3), 1-7 (2019).</li><li>Mandrell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17 (1), 66-74 (2012).</li><li>Zhang, Y., Mu, J., Han, J., Gu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+brine+shrimp+larvae+lethality+microwell+test+method.">An improved brine shrimp larvae lethality microwell test method.</a> Toxicology Mechanisms and Methods. 22 (1), 23-30 (2012).</li><li>Dom&#237;nguez-Villegas, V., 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=antioxidant+and+cytotoxicity+activities+of+methanolic+extract+and+prenylated+flavanones+isolated+from+leaves+of+eysehardtia+platycarpa.">antioxidant and cytotoxicity activities of methanolic extract and prenylated flavanones isolated from leaves of eysehardtia platycarpa.</a> Natural Product Communications. 8 (2), 177-180 (2013).</li><li>Hamidi, M. R., Jovanova, B., Panovska, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+evaluation+of+the+plant+products+using+Brine+Shrimp+(Artemia+salina+L.)+model.">Toxicological evaluation of the plant products using Brine Shrimp (Artemia salina L.) model.</a> Macedonian Pharmaceutical Bulletin. 60 (01), 9-18 (2014).</li><li>Libralato, G., Prato, E., Migliore, L., Cicero, A. M., Manfra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+toxicity+testing+protocols+and+endpoints+with+Artemia+spp.">A review of toxicity testing protocols and endpoints with Artemia spp.</a> Ecological Indicators. 69, 35-49 (2016).</li><li>Mendes Hacke, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+cymbopogon+citratus+(DC)+Stapf+fractions,+essential+oil,+citral,+and+geraniol+in+human+leukocytes+and+erythrocytes.">Cytotoxicity of cymbopogon citratus (DC) Stapf fractions, essential oil, citral, and geraniol in human leukocytes and erythrocytes.</a> Journal of Ethnopharmacology. 291, 115147 (2022).</li><li>Thangapandi, V., Pushpanathan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Artemia+salina+and+Artemia+fransiscana+bioassays+for+toxicity+of+Indian+medicinal+plants.">Comparison of the Artemia salina and Artemia fransiscana bioassays for toxicity of Indian medicinal plants.</a> Journal of Coastal Life Medicine. 2 (6), 453-457 (2014).</li><li>Syahmi, A. R. M., 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=Acute+oral+toxicity+and+brine+shrimp+lethality+of+Elaeis+guineensis+Jacq.,+(Oil+Palm+Leaf)+methanol+extract.">Acute oral toxicity and brine shrimp lethality of Elaeis guineensis Jacq., (Oil Palm Leaf) methanol extract.</a> Molecules. 15 (11), 8111-8121 (2010).</li><li>Sasidharan, 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=Acute+toxicity+impacts+of+Euphorbia+hirta+L+extract+on+behavior,+organs+body+weight+index+and+histopathology+of+organs+of+the+mice+and+Artemia+salina.">Acute toxicity impacts of Euphorbia hirta L extract on behavior, organs body weight index and histopathology of organs of the mice and Artemia salina.</a> Pharmacognosy Research. 4 (3), 170 (2012).</li><li>Libralato, 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+case+of+Artemia+spp.+in+nanoecotoxicology.">The case of Artemia spp. in nanoecotoxicology.</a> Marine Environmental Research. 101, 38-43 (2014).</li><li>Okumu, M. O., 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=Artemia+salina+as+an+animal+model+for+the+preliminary+evaluation+of+snake+venom-induced+toxicity.">Artemia salina as an animal model for the preliminary evaluation of snake venom-induced toxicity.</a> Toxicon: X. 12, 100082 (2021).</li><li>Rajabi, S., Ramazani, A., Hamidi, M., Naji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+salina+as+a+model+organism+in+toxicity+assessment+of+nanoparticles.">Artemia salina as a model organism in toxicity assessment of nanoparticles.</a> DARU Journal of Pharmaceutical Sciences. 23 (1), 20 (2015).</li><li>Svensson, B. -. M., Mathiasson, L., M&#229;rtensson, L., Bergstr&#246;m, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+salina+as+test+organism+for+assessment+of+acute+toxicity+of+leachate+water+from+landfills.">Artemia salina as test organism for assessment of acute toxicity of leachate water from landfills.</a> Environmental Monitoring and Assessment. 102 (1), 309-321 (2005).</li><li>Banti, C., Hadjikakou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+toxicity+with+brine+shrimp+assay.">Evaluation of toxicity with brine shrimp assay.</a> Bio-Protocol. 11 (2), 3895 (2021).</li><li>Pecoraro, R., 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=Artemia+salina:+A+microcrustacean+to+assess+engineered+nanoparticles+toxicity.">Artemia salina: A microcrustacean to assess engineered nanoparticles toxicity.</a> Microscopy Research and Technique. 84 (3), 531-536 (2021).</li><li>Lillicrap, A., 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=Alternative+approaches+to+vertebrate+ecotoxicity+tests+in+the+21st+century:+A+review+of+developments+over+the+last+2+decades+and+current+status.">Alternative approaches to vertebrate ecotoxicity tests in the 21st century: A review of developments over the last 2 decades and current status.</a> Environmental Toxicology and Chemistry. 35 (11), 2637-2646 (2016).</li><li>Ribeiro, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeasts+as+a+model+for+assessing+the+toxicity+of+the+fungicides+Penconazol,+Cymoxanil+and+Dichlofulanid.">Yeasts as a model for assessing the toxicity of the fungicides Penconazol, Cymoxanil and Dichlofulanid.</a> Chemosphere. (10), 1637-1642 (2000).</li><li>Armour, C. D., Lum, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+drug+to+protein:+using+yeast+genetics+for+high-throughput+target+discovery.">From drug to protein: using yeast genetics for high-throughput target discovery.</a> Current Opinion in Chemical Biology. 9 (1), 20-24 (2005).</li><li>Modarresi Chahardehi, A., Arsad, H., Lim, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+successful+animal+model+for+screening+toxicity+of+medicinal+plants.">Zebrafish as a successful animal model for screening toxicity of medicinal plants.</a> Plants. 9 (10), 1345 (2020).</li><li>Fischer, I., Milton, C., Wallace, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+testing+is+evolving.">Toxicity testing is evolving.</a> Toxicology Research. 9 (2), 67-80 (2020).</li><li>de Ara&#250;jo, G. L., 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=Alternative+methods+in+toxicity+testing:+the+current+approach.">Alternative methods in toxicity testing: the current approach.</a> Brazilian Journal of Pharmaceutical Sciences. 50 (1), 55-62 (2014).</li><li>Toussaint, M., 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=A+high-throughput+method+to+measure+the+sensitivity+of+yeast+cells+to+genotoxic+agents+in+liquid+cultures.">A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures.</a> Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 606 (1), 92-105 (2006).</li><li>Horzmann, K. A., Freeman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+waves:+New+developments+in+toxicology+with+the+zebrafish.">Making waves: New developments in toxicology with the zebrafish.</a> Toxicological Sciences. 163 (1), 5-12 (2018).</li><li>Avdesh, A., 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+An+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: An introduction.</a> Journal of Visualized Experiments. (69), e4196 (2012).</li><li>Cunliffe, V. T., N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: A Practical Approach. , (2002).</li><li>Sitarek, P., 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=Insight+the+biological+activities+of+selected+Abietane+Diterpenes+isolated+from+Plectranthus+spp.">Insight the biological activities of selected Abietane Diterpenes isolated from Plectranthus spp.</a> Biomolecules. 10 (2), 194 (2020).</li><li>Matias, D., 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=Cytotoxic+activity+of+Royleanone+Diterpenes+from+Plectranthus+madagascariensis+Benth.">Cytotoxic activity of Royleanone Diterpenes from Plectranthus madagascariensis Benth.</a> ACS Omega. 4 (5), 8094-8103 (2019).</li><li>Garcia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Royleanone+derivatives+from+Plectranthus+spp.+as+a+novel+class+of+P-glycoprotein+inhibitors.">Royleanone derivatives from Plectranthus spp. as a novel class of P-glycoprotein inhibitors.</a> Frontiers in Pharmacology. 11, (2020).</li></ol>

During the last years, the scientific community has increased its attention toward alternative models for toxicity screenings21. Beside A. salina lethality bioassay, other methodologies are usually performed for the evaluation of sample tolerability and include vertebrate bioassays (such as rodents), invertebrates (such as zebrafish), in vitro methods using yeast strains or cells, and in silico methods22,23,...

Natural products from plants, animals, or microorganisms have been a growing area of interest over the years in the development of new bioactive molecules because of their varied range of biological and pharmacological activities1. However, the associated side effects, drug resistance, or inadequate specificity of the agents, especially when used as anticancer drugs, represent the major factors that can lead to ineffective treatment1,2.
Over the last few decades, several plant-derived cytotoxic agents have been discovered, some of them used as anticancer agents1,2,3. In this context, paclitaxel is reported as one of the best-known and most active chemotherapeutic drugs of natural origin3,4. Currently, it is estimated that more than 35% of all medicines on the market are derived from or are inspired by natural products5. The potential high toxicity of these compounds requires consideration during all of the study phases, since different types of contaminants or even metabolic components of the plant itself can cause toxic effects. For this reason, pharmacological and toxicological profiles should be undertaken in the preliminarily phase, to assess the biological activity and safety of new potential plant-based treatments. To evaluate the toxicity of new bioactive samples, invertebrate animals can be considered as the best models to study. They demand minimal ethical requirements and allow preliminary in vitro assays, to prioritize the most promising products for the next round of testing in vertebrates1,6.
Commonly known as brine shrimp, A. salina is a small halophilic invertebrate belonging to the genus Artemia (family Artemiidae, order Anostraca, subphylum Crustacea; Figure 1). In marine and aquatic saline ecosystems, brine shrimps play an important nutritional role as they feed on microalgae and are constituents of the zooplankton used to feed fish. Moreover, their larvae (known as nauplii) are widely used in the assessment of general toxicity during preliminary studies1,3,7.
Artemia spp. are widely used in lethality studies and are also a convenient starting point for toxicity assessments, by tracking the toxicity of potentially bioactive compounds based on their ability to kill nauplii grown in the laboratory1,8. For this reason, the use of A. salina gained attraction in general toxicity studies, because it is a very efficient and easy-to-use method, compared to other tests on animal models9.
Owing to their simple anatomy, tiny size and short life cycle, a vast number of invertebrates can be studied in a single experiment. As such, they combine genetic amenability and low-cost compatibility with large-scale screenings1. In this context, the use of brine shrimp in a general toxicity assay shows several advantages, such as fast growth (28-72 h is needed from hatching to the first results), cost-effectiveness, and long shelf-life of commercial eggs, that can be used all year round3,10. On the other hand, since invertebrates have a primitive organ system and lack an adaptive immune system, they do not represent a perfect and reliable model for human cells1.
However, it provides a preliminary evaluation method for the general toxicity of selected samples. Since it is widely used as a lethality assay, it can provide provisional indications about the toxic effects of potential anticancer agents. It is often also used to obtain feedback about the general toxicity of compounds endowed with any other biological activities for which it is essential to show the lowest mortality rate possible among the Artemia shrimps.
In an ongoing study from our group, different extracts from Plectranthus species showed antioxidant and antimicrobial activities (unpublished results). In parallel, isolated compounds were obtained by purification of the extracts and were then chemically modified. The extracts, pure compounds, and semisynthetic derivatives were then tested in terms of general toxicity. In this context, the present work aims to give an overview of the use of the Artemia lethality bioassay for the evaluation of general toxicity and potential cytotoxic activity of bioactive extracts and isolated compounds from different plants of the genus Plectranthus11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64472/64472fig01.jpg" /> 
Figure 1: Artemia salina under the microscope. Newly hatched nauplii of A. salina as seen under the microscope (magnification 12x). <a href="https://www.jove.com/files/ftp_upload/64472/64472fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific, Denmark</td>
			<td>174899</td>
			<td>Thermo Scientific Nunc Up Cell 24 multidish</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Albal</td>
			<td>-</td>
			<td>Can be purchased in supermarket</td>
		</tr>
		<tr>
			<td>Artemio Set</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>61066000</td>
			<td>Can be purchased in pet shops</td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Ceti, Belgium</td>
			<td>&#160;1700.0000</td>
			<td>Flexum-24AED, 220-240 V, 50 Hz</td>
		</tr>
		<tr>
			<td>Bottles</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 L Diameter: 5.8 cm; Height: 12 cm</td>
		</tr>
		<tr>
			<td>Brine shrimp cysts</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>3090700</td>
			<td>Can be purchased in pet shops</td>
		</tr>
		<tr>
			<td>Brine shrimp salt</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>3090600</td>
			<td>Can be purchased in pet shops</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>VWR chemicals</td>
			<td>CAS: 67-68-5</td>
			<td>&#160;99% purity</td>
		</tr>
		<tr>
			<td>Discartable tips</td>
			<td>Diamond</td>
			<td>F171500</td>
			<td>Volume range: 100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Eppendorf microtubes</td>
			<td>BRAND</td>
			<td>7,80,546</td>
			<td>Microtubes, PP, 2 mL, BIO-CERT&#160;PCR QUALITY</td>
		</tr>
		<tr>
			<td>Erlenmeyer flask</td>
			<td>VWR chemicals</td>
			<td>4,47,109</td>
			<td>volume: 100 mL</td>
		</tr>
		<tr>
			<td>Glass beaker</td>
			<td>Normax</td>
			<td>3.2111654N</td>
			<td>Volume: 1000 mL</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Guantes Luna</td>
			<td>GLSP3</td>
			<td>-</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, San Diego, CA, USA</td>
			<td>-</td>
			<td>GraphPad Prism version 5.00 for Windows, www.graphpad.com, accessed on 5 February 2021; commercial statistical analysis software</td>
		</tr>
		<tr>
			<td>Home-made A. salina Grower</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Home made: two plastic bottles connected by a hose</td>
		</tr>
		<tr>
			<td>Hot glue</td>
			<td>Parkside</td>
			<td>PHP500E3</td>
			<td>230 V, 50 Hz, 25 W</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heidolph Instruments, Denmark&#160;</td>
			<td>&#160;-</td>
			<td>One Heidolph Unimax 1010 equipment and one Heidolph Inkubator 1006</td>
		</tr>
		<tr>
			<td>Light</td>
			<td>Roblan</td>
			<td>SKYC3008FE14</td>
			<td>LED light bulb</td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>VWR chemicals</td>
			<td>613-5265</td>
			<td>Volume range: 100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Potassium dichromate (K2Cr2O7)</td>
			<td>VWR chemicals</td>
			<td>CAS: 7778-50-9</td>
			<td>&#160;99% purity</td>
		</tr>
		<tr>
			<td>Pump ProAir a50</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>&#160;-</td>
			<td>Included in the Artemio Set+1 kit</td>
		</tr>
		<tr>
			<td>Rubber tube</td>
			<td>-</td>
			<td>-</td>
			<td>1.3 cm outer and 0.9 cm inner diameter</td>
		</tr>
		<tr>
			<td>Stirring rod</td>
			<td>VWR chemicals</td>
			<td>441-0147</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/64472/64472eq02.jpg" style="margin: auto;" /> 6 mm, 250 mm</td>
		</tr>
		<tr>
			<td>Termometer</td>
			<td>VWR chemicals</td>
			<td>620-0821</td>
			<td>0 - 100 &#176;C</td>
		</tr>
	</tbody>
</table>

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.

The general toxicity of some natural products recently studied by our group was evaluated through the brine shrimp lethality bioassay. Four extracts (Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) from Plectranthus genus, known for their antioxidant activity (unpublished results), were tested. Additionally, two natural compounds (1 and 2) obtained from Plectranthus spp., and three semi-synthetic derivatives (3, 4, 5; ...

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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.

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 ...

This protocol is a very useful tool for a preliminary general toxicity evaluation on different types of samples, such as extracts, fractions, and compounds from different natural or synthetic sources. The main advantage of our protocol is that it's very easy and cheap and can be used on many samples at the same time. Moreover, the handmade equipment improves the reliability of the site.In this technique stage, we do a screening to select the samples based on their preliminary general toxicity profile that will undergo further specific tests, such as expansive cell lines or even in-vivo tests. This method is mainly exploited in medicinal and other products chemistry fields to screen biotic samples. Our protocol can also be used for other kinds of samples such as nanosystems.While performing for the first time, be patient because this needs fine observation skills and a lot of attention to all the parameters reported Marcia Filipe and Vera Isca, the PhD students from our lab will help to demonstrate this procedure. Begin by placing the funnel shaped container in the black support and turning the funnel in a suitable direction to see the level mark and the tap. To make the handmade migration equipment cut the top of two 0.5 liter plastic bottles to obtain a final height of 12 centimeters.Create a hole of 1.5 centimeter diameter on one side and seven centimeters from the bottom of each bottle and insert a 13 centimeter rubber tube between the two openings. Seal the openings with hot glue and leave them to dry for 15 minutes. Put the bottles on a flat surface and fill them with water to verify there is no leakage.Add 28 grams of the salt to 800 milliliters of tap water to prepare the artificial salt solution, or brine shrimp salt at a concentration of 35 grams per liter. Mix it with a stirring rod until all the salt is thoroughly dissolved. To prepare the samples dissolve a suitable amount of each extract and compound dimethylsulfoxide, or DMSO, at a final concentration of 10 milligrams per milliliter.Then dilute 10 microliters of each sample in a new micro centrifuge tube using 990 microliters of the artificial saline solution to obtain a final concentration of 0.1 milligrams per milliliter. Under a fume hood in an Erlenmeyer flask, prepare a solution of potassium dichromate in distilled water at a concentration of one milligram per milliliter. Fill the hatching vessel with the medium up to the 500 milliliter mark and add one spoon, or approximately 0.5 grams of brine shrimp cysts to the salt solution.Close the container. Place a lamp pointing directly toward the equipment and turn it on. Attach the air supplier system to the connector placed on top of the equipment and turn the pump on.Brine shrimp cysts hatch in the artificial salt solution under vigorous aeration, continuous lighting, and stable temperature. After 24 to 48 hours, when the eggs have hatched, turn the air pump off and wait until the nauplii are separated from the empty egg cases. To separate the unhatched eggs from the alive nauplii, open the outlet tap at the bottom and discharge the content of the funnel in one of the containers of the handmade migration equipment container.Ensure that the solution containing the nauplii and the residual unhatched eggs is below the tube level. Place the equipment in the incubator for four hours, then in the second container add the residual salt solution above the height of the tube. Cover the container with nauplii and the residual unhatched eggs using aluminum foil and place the lamp on the second container with just the salt solution.The brine shrimp will be attracted by the light and migrate from one container to another, leading to efficient separation between eggs and alive artemia. From the harvesting container, collect 900 microliters of saline solution containing 10 to 15 nauplii and place the solution in one well of a 24 well plate. All the samples are tested in quadruplicates.Add 100 microliters of each negative control. Positive control, the artificial salt solution, and each of the samples to the wells and incubate the plate under illumination for 24 hours. After 24 hours, register the number of dead larvae in each well under a binocular microscope at 12 x magnification.Add 100 microliters of potassium dichromate solution to induce the death of the remaining living larvae and wait for six hours. Count the total dead larva in each well under a microscope and determine the mortality rate according to the equation shown on the screen. Perform all the tests in triplicates and calculate the standard deviations.Finally, express the results as the mean of three independent experiments each with internal quadruplicates plus, minus standard deviations. The structure of the selected compounds extracted from Plectranthus species and obtained by semi-synthesis are shown in this figure. A newly hatched nauplii of Artemia Salina as seen under the microscope is shown in this image.This graphical image represents the mortality rate of Artemia Salina after 24 hours of exposure to four methanol extracts of Plectranthus species at 0.1 milligrams per milliliter. All the extracts were acceptable in terms of general toxicity using this assay. Here salt corresponds to the salt solution or blank.Potassium dichromate was used as a positive control and DMSO was used as a negative control. All the tested extracts showed very encouraging results with very low values of mortality percentages comparable to those registered for the blank and the negative control. The mortality rate of Artemia Salina after 24 hours of exposure to the five pure compounds at 0.1 milligrams per milliliter is presented in this figure.From this data, it is evident that only five shows minimal toxicity using this assay You need to remember three steps in particular, hatching, migration and collection. Consequently, we have other shrimp separated from and the numbers to work with. The preliminary site is a simple and low cost model channel toxicity compared to other sites like cell culture or zebra fish.Further, more expensive cytotoxicity sites can then be exploited.

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