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 cm diameter on one side at 7 cm from the bottom in each bottle and insert a 13 cm rubber tube (1.3 cm outer and 0.9 cm inner diameter) between the two openings. Seal the openings with hot glue (Figure 2B) and leave to dry for 15 min; put the bottles on a flat surface and fill them with water to verify that there is no leakage.</li>
</ol>
2. Preparation of artificial salt solution
<ol>
	<li>In a glass beaker, prepare an artificial salt solution (brine shrimp salt) at a concentration of 35 g/L. To do this, add 28 g of the salt to 800mL of tap water, according to the manufacturer's instructions. Mix it with a stirring rod until all the salt is thoroughly dissolved. 
	NOTE: Adjust the volume of the prepared saline solution according to the size of the available containers.</li>
</ol>
3. Sample preparation
<ol>
	<li>Prepare all the samples in a microcentrifuge tube by dissolving a suitable amount of extracts (Plectranthus extracts, Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) or compounds 1-5 (two natural compounds [1 and 2] obtained from Plectranthus spp. and three semi-synthetic derivatives [3, 4, 5]; Figure 3) in dimethyl sulfoxide (DMSO)12, so as to obtain a final concentration of 10 mg/mL (if the sample is water-soluble, use of DMSO is not necessary).</li>
	<li>Dilute 10 µL of each sample (and DMSO for the negative control) in a new microcentrifuge tube using 990 µL of artificial saline solution prepared in step 2.1, to obtain a final concentration of 0.1 mg/mL.</li>
	<li>Under a fume hood, in an Erlenmeyer flask, prepare a solution of potassium dichromate (K2Cr2O7) in distilled water at a concentration of 1 mg/mL13,14,15.</li>
</ol>
4. Brine shrimp lethality bioassay
NOTE: This assay is developed from the works of several authors with modifications1,16,17,18,19.
<ol>
	<li>Fill the hatching vessel with the medium prepared in step 2.1 up to the level mark (500 mL) (Figure 2C).</li>
	<li>Place one spoon (approximately 0.75 g) of brine shrimp cysts in the salt solution, and then close the container. Place a lamp (table lamp, 40 W, 230 V, 50 Hz, with a LED light bulb of 8 W, 4,000 K, 830 lm) pointing directly toward the equipment (Figure 2A) and turn it on.</li>
	<li>Attach the air supplier system (3 W output, 50 Hz, 230 V) to the connector placed on the top of the equipment and turn the pump on.</li>
	<li>Keep the room temperature at 25 ± 3 °C. Brine shrimp cysts hatch in the artificial salt solution, under vigorous aeration, continuous lighting, and stable temperature, after 24 h to 48 h. 
	NOTE: Alternatively, a vertical incubator can be used.</li>
	<li>Once the eggs have hatched, turn the air pump off and wait until the nauplii (moving toward the bottom of the funnel) are separated from the empty egg cases (floating at the top).</li>
	<li>In order to separate the unhatched eggs from alive nauplii, open the outlet tap at the bottom and discharge the content of the funnel in one of the containers of the hand-made migration equipment container (described in step 1.2). Ensure that the solution containing the nauplii and the residual unhatched eggs is below the level of the tube. In the second container, add the residual salt solution from step 2.1 above the height of the tube.</li>
	<li>Cover the container with the nauplii and the residual unhatched eggs using aluminum foil. 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 the other (harvesting container), leading to efficient separation between eggs (slowly sedimented to the bottom) and alive Artemia.</li>
	<li>Then, place the equipment in the incubator under the same conditions used in step 4.4 for 4 h (Figure 2E). From the harvesting container, collect 900 µL of saline solution containing 10 to 15 nauplii. Place the saline solution with nauplii in each well of a 24-well plate (Figure 2F); all the samples are tested in quadruplicates.</li>
	<li>Add 100 µL each of the negative control (DMSO), the positive control (K2Cr2O7, potassium dichromate), the artificial salt solution, and each of the samples to respective well (Figure 2F)13,14. 
	NOTE: The samples in each well will be at a concentration of 0.01 mg/mL. The final concentration of the positive control in salt solution will be 0.1 mg/mL, to be sure that all the nauplii in the well are exposed to the toxic effect of potassium dichromate and die. The artificial salt solution will act as blank.</li>
	<li>Incubate the plate at 25 ± 3 °C under illumination for 24 h (Figure 2G). After 24 h, register the number of dead larvae (non-mobile nauplii for 5 s) in each well under a binocular microscope (12x)20 (Figure 2H). Alternatively, use a hand lens.</li>
	<li>Add 100 µL of potassium dichromate solution, to induce the death of the remaining living larvae, and wait for 6 h. Count the total dead larvae in each well under a microscope. Determine the mortality rate according to the following equation. 
	<img alt="Mortality rate formula, arithmetic equation for calculating A. salina mortality percentage." src="/files/ftp_upload/64472/64472eq01.jpg" style="margin: auto;" data-altupdatedat="1762425657000" data-alt="Equation 1"></li>
	<li>Perform all the assay in triplicate. Calculate standard deviations (SD), and express the results as the mean of three independent experiments, each with internal quadruplicates (n = 12), ± SD. As mentioned by Meyer et al., consider crude extracts and pure compounds with LC50&lt; 1,000 µg/mL as toxic; also, take into account that the mortality rate of brine shrimp is proportional to the concentration of the tested samples21.</li>
</ol>
<img alt="Filtration experiment setup; DIY filtration system, beakers, pipette; separation process diagram." class="xfigimg" src="/files/ftp_upload/64472/64472fig02.jpg" data-altupdatedat="1762425657000" data-alt="Figure 2"> 
Figure 2: Artemia salina lethality bioassay method. (A) Commercially available equipment employed for the hatching of brine shrimp cysts; (B) Hand-made migration equipment; (C) Hatching vessel filled with saline solution; (D) Collection of unhatched eggs and nauplii; (E) Hand-made equipment in the incubator during the migration step. The container far from the lamp should be covered with aluminum foil; however, for a better view of the set installation here it was removed; (F) Harvesting of Artemia in wells prior to performing the assay. The compounds should be placed as shown: - refers to the negative control (DMSO), + to the positive control (K2Cr2O7), salt to the artificial salt solution, and 1 to 3 to the samples to test (in this case compounds 1-3); (G) Incubation of the 24-well plate containing Artemia and the selected samples; (H) Artemia count under the binocular microscope. <a href="https://www.jove.com/files/ftp_upload/64472/64472fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chemical structure diagram of sesquiterpene derivatives, five labeled compounds with acetyl groups." class="xfigimg" src="/files/ftp_upload/64472/64472fig03.jpg" data-altupdatedat="1762425657000" data-alt="Figure 3"> 
Figure 3: Structures of selected compounds. Structure of compounds 1-2, extracted from Plectranthus species, and compounds 3-5, obtained by semi-synthesis. <a href="https://www.jove.com/files/ftp_upload/64472/64472fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol><li>Ntungwe, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artemia+species%3A+An+important+tool+to+screen+general+toxicity+samples%5BTitle%5D)+AND+%22Current+Pharmaceutical+Design%22%5BJournal%5D)">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/pubmed?term=((Natural+products%3A+A+continuing+source+of+novel+drug+leads%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+%28BBA%29+-+General+Subjects%22%5BJournal%5D)">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/pubmed?term=((General+toxicity+screening+of+Royleanone+derivatives+using+an+artemia+salina+model%5BTitle%5D)+AND+%22Journal+Biomedical+and+Biopharmaceutical+Research%22%5BJournal%5D)">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/pubmed?term=((secondary+metabolites+as+anticancer+agents%3A+Successes+in+clinical+trials+and+therapeutic+application%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">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/pubmed?term=((The+role+of+natural+products+in+modern+drug+discovery%5BTitle%5D)+AND+%22Anais+da+Academia+Brasileira+de+Ci%C3%AAncias%22%5BJournal%5D)">The role of natural products in modern drug discovery.</a> Anais da Academia Brasileira de Ciências. 91 (3), 1-7 (2019).</li>
<li>Mandrell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+zebrafish+chorion+removal+and+single+embryo+placement%3A+optimizing+throughput+of+zebrafish+developmental+toxicity+screens%5BTitle%5D)+AND+%22Journal+of+Laboratory+Automation%22%5BJournal%5D)">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/pubmed?term=((An+improved+brine+shrimp+larvae+lethality+microwell+test+method%5BTitle%5D)+AND+%22Toxicology+Mechanisms+and+Methods%22%5BJournal%5D)">An improved brine shrimp larvae lethality microwell test method.</a> Toxicology Mechanisms and Methods. 22 (1), 23-30 (2012).</li>
<li>Domínguez-Villegas, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((antioxidant+and+cytotoxicity+activities+of+methanolic+extract+and+prenylated+flavanones+isolated+from+leaves+of+eysehardtia+platycarpa%5BTitle%5D)+AND+%22Natural+Product+Communications%22%5BJournal%5D)">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/pubmed?term=((Toxicological+evaluation+of+the+plant+products+using+Brine+Shrimp+%28Artemia+salina+L.%29+model%5BTitle%5D)+AND+%22Macedonian+Pharmaceutical+Bulletin%22%5BJournal%5D)">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/pubmed?term=((A+review+of+toxicity+testing+protocols+and+endpoints+with+Artemia+spp%5BTitle%5D)+AND+%22Ecological+Indicators%22%5BJournal%5D)">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/pubmed?term=((Cytotoxicity+of+cymbopogon+citratus+%28DC%29+Stapf+fractions%2C+essential+oil%2C+citral%2C+and+geraniol+in+human+leukocytes+and+erythrocytes%5BTitle%5D)+AND+%22Journal+of+Ethnopharmacology%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+the+Artemia+salina+and+Artemia+fransiscana+bioassays+for+toxicity+of+Indian+medicinal+plants%5BTitle%5D)+AND+%22Journal+of+Coastal+Life+Medicine%22%5BJournal%5D)">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/pubmed?term=((Acute+oral+toxicity+and+brine+shrimp+lethality+of+Elaeis+guineensis+Jacq.%2C+%28Oil+Palm+Leaf%29+methanol+extract%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">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/pubmed?term=((Acute+toxicity+impacts+of+Euphorbia+hirta+L+extract+on+behavior%2C+organs+body+weight+index+and+histopathology+of+organs+of+the+mice+and+Artemia+salina%5BTitle%5D)+AND+%22Pharmacognosy+Research%22%5BJournal%5D)">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/pubmed?term=((The+case+of+Artemia+spp.+in+nanoecotoxicology%5BTitle%5D)+AND+%22Marine+Environmental+Research%22%5BJournal%5D)">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/pubmed?term=((Artemia+salina+as+an+animal+model+for+the+preliminary+evaluation+of+snake+venom-induced+toxicity%5BTitle%5D)+AND+%22Toxicon%3A+X%22%5BJournal%5D)">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/pubmed?term=((Artemia+salina+as+a+model+organism+in+toxicity+assessment+of+nanoparticles%5BTitle%5D)+AND+%22DARU+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">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., Mårtensson, L., Bergström, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artemia+salina+as+test+organism+for+assessment+of+acute+toxicity+of+leachate+water+from+landfills%5BTitle%5D)+AND+%22Environmental+Monitoring+and+Assessment%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+toxicity+with+brine+shrimp+assay%5BTitle%5D)+AND+%22Bio-Protocol%22%5BJournal%5D)">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/pubmed?term=((Artemia+salina%3A+A+microcrustacean+to+assess+engineered+nanoparticles+toxicity%5BTitle%5D)+AND+%22Microscopy+Research+and+Technique%22%5BJournal%5D)">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/pubmed?term=((Alternative+approaches+to+vertebrate+ecotoxicity+tests+in+the+21st+century%3A+A+review+of+developments+over+the+last+2+decades+and+current+status%5BTitle%5D)+AND+%22Environmental+Toxicology+and+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Yeasts+as+a+model+for+assessing+the+toxicity+of+the+fungicides+Penconazol%2C+Cymoxanil+and+Dichlofulanid%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">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/pubmed?term=((From+drug+to+protein%3A+using+yeast+genetics+for+high-throughput+target+discovery%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">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/pubmed?term=((Zebrafish+as+a+successful+animal+model+for+screening+toxicity+of+medicinal+plants%5BTitle%5D)+AND+%22Plants%22%5BJournal%5D)">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/pubmed?term=((Toxicity+testing+is+evolving%5BTitle%5D)+AND+%22Toxicology+Research%22%5BJournal%5D)">Toxicity testing is evolving.</a> Toxicology Research. 9 (2), 67-80 (2020).</li>
<li>de Araújo, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+methods+in+toxicity+testing%3A+the+current+approach%5BTitle%5D)+AND+%22Brazilian+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">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/pubmed?term=((A+high-throughput+method+to+measure+the+sensitivity+of+yeast+cells+to+genotoxic+agents+in+liquid+cultures%5BTitle%5D)+AND+%22Mutation+Research%2FGenetic+Toxicology+and+Environmental+Mutagenesis%22%5BJournal%5D)">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/pubmed?term=((Making+waves%3A+New+developments+in+toxicology+with+the+zebrafish%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">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/pubmed?term=((Regular+care+and+maintenance+of+a+zebrafish+%28Danio+rerio%29+laboratory%3A+An+introduction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCunliffe%2C+VT+%22Zebrafish%3A+A+Practical+Approach%22">Zebrafish: A Practical Approach</a>. Nüsslein-Volhard, C., Dahm, R. , Oxford University Press. (2002).</li>
<li>Sitarek, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insight+the+biological+activities+of+selected+Abietane+Diterpenes+isolated+from+Plectranthus+spp%5BTitle%5D)+AND+%22Biomolecules%22%5BJournal%5D)">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/pubmed?term=((Cytotoxic+activity+of+Royleanone+Diterpenes+from+Plectranthus+madagascariensis+Benth%5BTitle%5D)+AND+%22ACS+Omega%22%5BJournal%5D)">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/pubmed?term=((Royleanone+derivatives+from+Plectranthus+spp.+as+a+novel+class+of+P-glycoprotein+inhibitors%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">Royleanone derivatives from Plectranthus spp. as a novel class of P-glycoprotein inhibitors.</a> Frontiers in Pharmacology. 11, (2020).</li>
</ol>

The authors declare no conflicts of interest, financial or otherwise.

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 methods22,23,24,25. All these methods have clear advantages and disadvantages, in consideration of spent money and time, reproducibility of the results, and the type of sample to be analyzed. For example, the in silico approaches represent a standardized low-cost methodology, requiring minimal equipment. Nonetheless, when such studies are focused on a single target, poor quality results are obtained, making this alternative out of the scope for preliminary screenings, as we present in this study25,26. When it comes to microorganisms, Saccharomyces cerevisiae is considered one of the most extensively used models for the evaluation of toxic compounds23. In fact, the use of in vitro yeast is generally fast and easy to carry out; however, it can be expensive, since it requires specific chemical products and equipment, and shows low sensitivity to minimal doses of cytotoxic compounds27. In an alternative approach, human cells can be employed, in a fast, simple, and inexpensive way. Even so, these cannot represent an entire organism; thus, interactions between different cell types and systemic perturbations taking place in physiologic conditions are not appropriately taken into account25,26. On the other hand, in vivo assays have the great advantage to take into consideration the whole organism, but animal models (such as rodents) require more time for the assay execution, are more expensive and imply complex ethical considerations25. Conversely, in vivo assays employing aquatic organisms for toxicity screening are generally well accepted, considering also that the use of marine invertebrates undergoes less ethical concerns, and the methodology is easy to perform, fast, and cheap21,24. In this context, the use of zebrafish for the study of general toxicity often represents the first choice in the field. In fact, compared to other animal tests, it can be considered a low-cost option, as zebrafish develop rapidly and reach the early larval stage around 72 h to 13 days post-fertilization21,28. Nevertheless, maintenance of a zebrafish requires well-trained operators and specific conditions, such as a fish tank with a circulating system, able to aerate and filter the system water, to maintain the overall quality; moreover, several lids and drain covers are necessary, as zebrafish can jump, as well as specific lighting conditions (14 h light, 10 h dark), and pH levels need to be checked daily, among others29,30. All together, these considerations make zebrafish models more time consuming and expensive than the Artemia model reported here, since these microcrustaceans are easier to handle and viable for cultivation in large populations using laboratory methods. This makes A. salina undoubtedly one of the most employed screening tools, mainly used to test the general toxicity of different samples, such as drugs and medicinal plant extracts1.
In the protocol, A. salina nauplii have been reared, collected, and incubated with suitable samples for 24 h, to estimate the nauplii mortality rate induced by each sample. In the first step, the egg hatching is conducted in a commercially available funnel-shaped breeding vessel, in which vigorous bubbling was introduced (Figure 2A). This forced aeration is necessary since it allows the brine shrimp hatching to occur as successfully as possible. The hatching occurs at around 24 h at 25 &#177; 3 &#176;C, whereas up to 48 h could be necessary at lower temperatures. Once the eggs hatch, the pump is turned off to allow the empty egg cases to float on the top, while nauplii and unhatched eggs are easily collected by opening the funnel tap at the bottom. During our experiments, complete egg hatching was never achieved, with the consequent presence of nauplii and non-mature eggs in the collected solution. For this reason, in order to efficiently separate the two forms of Artemia, a hand-made equipment for brine shrimps&#39; migration is prepared (step 1.2). One container is filled up with the harvested organisms, and then covered with aluminum foil, while the other recipient is exposed to the direct light of a lamp. In these conditions, the unhatched eggs tend to settle down, whereas the alive nauplii are attracted by the light hitting the adjacent container, favoring the migration from one side to the other through the bridge connection. After 4 h, almost all live nauplii are inside the container exposed to the light&#160;and ready to be collected for the execution of the assay. At this stage, portions of the saltwater solution, containing ten to fifteen nauplii, are removed and incubated with each of the samples to be tested.
In this work, it has been shown how the method is efficient, economic, and easy to be carried out. However, some critical points should be acknowledged when performing this assay.
The assay is usually conducted with tap water. Depending on geographic and physico-chemical factors, tap water exhibits different hardness distribution, influencing the reproducibility of the assay and, in particular, the egg hatching step. For the same reason, the use of seawater could influence the reproducibility of the test. The varying salt concentration, the presence of contaminants, microplastics and other corpuscles would force the operator to undergo further steps, such as filtrations and other types of purification, that would make the experiment more complex and less easy to handle. All considered, optimal conditions should be settled based on the characteristics and availability of tap water, especially by a careful regulation of the amount of artificial salt added, that creates a suitable environment and acts as nourishment for the nauplii, too.
Wide variations in temperatures can result in lower hatch rates. As a consequence, low levels of living Artemia could be observed, mixed with a high number of unhatched eggs. Clearly, such conditions are not suitable for the development of reliable assays, so a controlled climatization of the room or the use of appropriate vertical incubators should be considered.
Vigorous aeration of the solution within the hatching vessel is required. The presence of continuous bubbling favors the contact between eggs and accelerates the hatching process.
Collecting both unhatched eggs and nauplii during the harvesting can strongly influence the reliability of the assay. This is due to the possibility of hatching during the incubation with the samples. In this case, not all the nauplii will have been exposed for the same amount of time, with consequent erroneous mortality rates. As eggs and nauplii are too small to be efficiently separated, higher incubation times in the hatching equipment or the use of the hand-made migration equipment are needed.
Each well should contain 10-15 nauplii for the incubation step, carefully counted by the use of a binocular microscope (12x magnification). The presence of too many nauplii in the same well can make the final counting difficult, or even erroneous, because of overlapping. On the other hand, small amounts of shrimps would lead to results with no statistical significance.
As is evident, most of the problems of this method are related to the hatching and growth stages, where more attention is required to avoid reliability issues. Even so, the method can tolerate modifications, especially in relation to the most critical points described above. Of course, when one of these parameters is changed, it could be necessary to conduct several attempts to optimize the original method. For example, changing the temperature can help control the overall time of the assay: increasing the temperature up to 28-29 &#176;C will also increase the hatch rate and speed up the entire process.
Some limitations of A. salina bioassay are also related to the stability of the samples to the test conditions and time. The assay can only be carried out using samples that are stable at room temperature and under visible light. Moreover, it must be taken into account that the entire procedure lasts at least 4 days, and if the hatch rate is low, the assay could need an extra day for the hatching step.
Overall, in this study, we highlight two examples of the application of the general toxicity evaluation through the A. salina method. General toxicity has been established for all the samples tested. Extracts from Plectranthus plants and compound 5 displayed no general toxicity, while compounds 1-4&#160;proved to be moderately or even highly toxic on brine shrimps. In particular, the negative effect of compounds 1 and 2 on Artemia was previously demonstrated by Sitarek and Matias by MTT and SRB/MTT assays, respectively31,32. At the same time, the benzoylated analogs, compounds 3 and 4, show higher toxicity values than their precursors 1 and 2, highlighting that the ester functionalization could have an important role in terms of cytotoxicity, as confirmed for compound 4 in human NSCLC MDR cancer cell line by Garcia et al.33. However, other assays are needed to confirm that such a cytotoxic activity could be exploited in cancer therapy. On the other hand, derivative 5, together with all of the assayed extracts, showed no toxicity and appears as the most promising pure compound of the series. However, further studies to evaluate the potential biological activity for cutaneous application are needed.
Here we demonstrated how the use of Artemia salina-based method as a screening tool could allow saving time and money, compared to other known methodologies. It represents an efficient and advantageous way to be exploited in preliminary toxicity contexts. It can be used in the presence of different and complex samples, synthetic, and semi-synthetic drugs, as well as for the bio-guided fractionation of natural product extracts and samples.

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="Microscope image of microcrustaceans, aquatic organisms, highlighting biological sample observation." class="xfigimg" src="/files/ftp_upload/64472/64472fig01.jpg" data-altupdatedat="1762425657000" data-alt="Figure 1"> 
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><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>&amp;nbsp;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>&amp;nbsp;99% purity</td></tr><tr><td>Discartable tips</td><td>Diamond</td><td>F171500</td><td>Volume range: 100 - 1000 &amp;micro;L</td></tr><tr><td>Eppendorf microtubes</td><td>BRAND</td><td>7,80,546</td><td>Microtubes, PP, 2 mL, BIO-CERT&amp;nbsp;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>&amp;nbsp;-</td><td>&amp;nbsp;-</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&amp;nbsp;</td><td>&amp;nbsp;-</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 &amp;micro;L</td></tr><tr><td>Potassium dichromate (K&lt;sub&gt;2&lt;/sub&gt;Cr&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>VWR chemicals</td><td>CAS: 7778-50-9</td><td>&amp;nbsp;99% purity</td></tr><tr><td>Pump ProAir a50</td><td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td><td>&amp;nbsp;-</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>&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/64472/64472eq02.jpg&quot; style=&quot;margin: auto;&quot;/&gt; 6 mm, 250 mm</td></tr><tr><td>Termometer</td><td>VWR chemicals</td><td>620-0821</td><td>0 - 100 &amp;deg;C</td></tr></tbody></table>

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; Figure 3), all described in another work3, were also investigated. Herein, their preliminary evaluation in terms of potential cytotoxic activity is reported.
All the tested extracts showed very encouraging results, with very low mortality rates, comparable to those registered for the blank (salt solution) and the negative control (DMSO; Figure 4). Conversely, among the pure compounds 1-5, only derivative 5 displayed a low mortality rate (2.30% at a concentration of 100 ppm) with no general toxicity (Figure 5).
<img alt="Bar graph of cell viability; K2Cr2O7 induces high mortality, comparing salts and compounds like DMSO." class="xfigimg" src="/files/ftp_upload/64472/64472fig04.jpg" data-altupdatedat="1762425657000" data-alt="Figure 4"> 
Figure 4: Lethality of extracts on Artemia salina. Mortality rate of A. salina (%) after 24 h exposure to four methanolic extracts of Plectranthus spp., at 0.1 mg/mL (Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; Pe- P. ecklonii). All the extracts were acceptable in terms of general toxicity using this assay. Salt corresponds to the salt solution (blank); K2Cr2O7 was used as the positive control and DMSO as the negative control. The results were expressed as the mean of three independent experiments, each with internal quadruplicates (n = 12) ± SD. Comparisons were performed within groups by the analysis of variance, using the one-way ANOVA with Dunnett's post-test. Significant differences between control and experimental groups were assessed using a commercial statistical analysis software. A probability level p &lt; 0.01 was considered to indicate statistical significance. <a href="https://www.jove.com/files/ftp_upload/64472/64472fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar chart of cell viability assay showing % dead across treatments: K2Cr2O7, salt, DMSO, and compounds 1-5." class="xfigimg" src="/files/ftp_upload/64472/64472fig05.jpg" data-altupdatedat="1762425657000" data-alt="Figure 5"> 
Figure 5: Lethality of compounds on Artemia salina. Mortality rate of A. salina (%) after 24 h exposure to five pure compounds at 0.1 mg/mL (1 and 2 are natural products and 3-5 are derivatives prepared from 1 and 2). It is evident that only 5 shows very limited toxicity using this assay. Salt corresponds to the salt solution (blank); K2Cr2O7 was used as the positive control and DMSO as the negative control. The results were expressed as the mean of three independent experiments, each with internal quadruplicates (n = 12) ± SD. Comparisons were performed within groups by the analysis of variance, using the one-way ANOVA with Dunnett's post-test. Significant differences between control and experimental groups were assessed using a commercial statistical analysis software. A probability level p &lt; 0.01 was considered to indicate statistical significance. <a href="https://www.jove.com/files/ftp_upload/64472/64472fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Natural products 1 and 2 showed moderate lethality (36.68% and 30.95% at 100 ppm, respectively), whereas semisynthetic derivatives 3 and 4, obtained from 1 and 2, respectively, were more toxic than the original scaffold (64.02% and 36.64% at 100 ppm, respectively; Figure 5).
Overall data suggested that all the tested extracts, known for their antioxidant activity and showing no general toxicity, can be considered as candidates for further in vitro activity and toxicity studies on selected cell lines. If the absence of toxicity is also demonstrated in cutaneous models, the development of new bioactive products for cutaneous application can be carried out. On the other hand, the toxic effects observed for compounds 1-4 could make them suitable candidates for additional evaluations as anticancer agents.

Watch this Scientific Journal Video about Lethality Bioassay Using Artemia salina L. at JoVE.com

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

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.

Patricia Rijo

Vera M. S. Isca

Epole N. Ntungwe

Marcia Santos Filipe

Natural products (NP) are a historical and valuable source of drugs used for the development of new medicines, due to their high structural diversity and exceptional range of several biological activities. In this special issue, we aim to publish high-quality research procedures within the field of Medicinal Chemistry, namely, natural products chemistry and NP biological activities. This collection covers several procedures related to bioactive natural products, from extraction, isolation, and compound characterization especially to the application of biological activity assays to these extraordinary bioactive compounds.

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Lethality Bioassay Using Artemia salina L.