May 28, 2025

Screening natural products against Artemia salina

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm9q35l3p/v1
Screening natural products against Artemia salina
  • 1Universidade de São Paulo
Camila Manoel Crnkovic
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm9q35l3p/v1
Protocol Citation: Jaewon Y, Heloísa Amorim, Letícia ilva Andrade, Maria Gabriela Silva Bueno, Camila Manoel Crnkovic 2025. Screening natural products against Artemia salina. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9q35l3p/v1
Manuscript citation:
Campos, T.G.V., Gama, W.A., Geraldes, V., Yoon, J., Crnkovic, C.M., Pinto, E., Jacinavicius, F.R. (2024) New records on toxic cyanobacteria from Brazil: Exploring their occurrence and geography. Science of The Total Environment 931:172689. doi:10.1016/j.scitotenv.2024.172689

Médice, R. V. (2023). Caracterização e bioprospecção da biomassa de cianobactérias e microalgas presentes em reservatórios de água visando seu potencial biotecnológico (Thesis (phD)). Universidade de São Paulo, São Paulo. https://www.teses.usp.br/teses/disponiveis/9/9143/tde-12122023-113247/
Médice, R. V., Arruda, R. S., Yoon, J., Borges, R. M., Noyma, N. P., Lürling, M., Crnkovic, C. M., Marinho, M. M., & Pinto, E. (2024). Unlocking biological activity and metabolomics insights: Primary screening of cyanobacterial biomass from a tropical reservoir. Environmental Toxicology and Chemistry, 43(10), 2222–2231. https://doi.org/10.1002/etc.5962

Weiss, M. B. (2023). Quimioprospecção de cianobactérias brasileiras utilizando metabolômica e ensaios biológicos. Dissertação de Mestrado, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo. doi:10.11606/D.9.2023.tde-08032024-115334
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 21, 2025
Last Modified: May 28, 2025
Protocol  Integer ID: 118812
Keywords: Artemia salina, Brine shrimp, Bioassay, Bioactivity, natural products against artemia salina, brine shrimp bioassay, using artemia salina, bioassay method, artemia salina, effective tool for toxicity screening, screening natural product, brine shrimp, toxicity screening, analyses of natural product extract, natural product extract, preliminary cytotoxicity testing, cytotoxicity
Funders Acknowledgements:
FAPESP
Grant ID: 2020/07710-7
FAPESP
Grant ID: 2022/02872-4
Abstract
This protocol outlines a bioassay method using Artemia salina (brine shrimp) for the analyses of natural product extracts and fractions. The brine shrimp bioassay serves as a simple and cost-effective tool for toxicity screening and/or for preliminary cytotoxicity testing.

Materials
Solvents and solutions

  • Artemia salina cysts
  • Dimethyl sulfoxide (DMSO)
  • Deionized water
  • Iodized NaCl salt
  • Potassium dichromate (K₂Cr₂O₇)

Equipment

  • 96-well plate
  • Air pump
  • Incubator
  • Light source
  • Pipette (1-10 and 1-100 μL)
Background
Artemia salina is a microcrustacean adapted to high salinities that, under appropriate conditions, can hatch in 24 to 36 hours, becoming a nauplius without complete formation of the digestive tract. Due to its easy of handeling, rapid development, and low cost, it is adopted as a model for natural product screening and ecotoxicological tests. It is utilized for detecting toxic substances (Campos et al., 2024; Médice et al., 2024) and for preliminary cytotoxicity testing (Albuquerque et al., 2014; Solis et al., 1993). Toxicity is one of the main factors leading to the abandonment of drug candidates. Therefore, the use of preliminary methods for its assessment is essential in bioprospecting, helping to prevent the discontinuation of costly studies. From a different perspective, preliminary cytotoxicity results may justify additional, more complex studies in anticancer drug discovery programs. Limitations of this assay include the requirement for a saline environment, which can lead to issues related to sample solubility, and low sensivity (Göethel et al., 2022). Nevertheless, when conducted under optimal conditions, it is a highly reproducible model and an effective test for preliminary toxicity results (Salay et al., 2024).
Day 01
Preparing brine shrimp for hatching

  • In a 250 mL Erlenmeyer flask, add 250 mL of deionized water, 4.8 g of iodized NaCl salt, and 0.25 g of dehydrated Artemia cysts.
  • Incubate for 24 hours at 25 °C with constant agitation by aeration and lighting.




Sample preparation and controls

  • Samples: solubilize samples in DMSO, according to final test concentration. Note: Our results suggest that A. salina nauplii are resistant up to 1% DMSO in final test concentration.
Suggested test concentration (final concentration in well) for preliminary, single point concentrations tests:
- Natural product extracts: 1000 μg/mL;
- Natural product fractions: 100 μg/mL.
  • Positive control: dissolve K2Cr2O7 in deionized water to obtain a concentration of 20 mg/mL (final concentration in well: 200 μg/mL).
  • Negative control: DMSO 100% (final concentration in well: 1%).
Day 02
Exposing Artemia salina nauplii to test samples and controls

  • Sterilize a clean 96-well plate under UV light.
  • Transfer 99 µL of solution containing 15-20 nauplii, without cysts, in each well of the 96-well plate.
  • Transfer 1 µL of sample solution (or controls) to each well.
Every sample is tested in triplicate and controls in quintuplicate
Negative control: DMSO 100%
Positive control: K2Cr2O7
  • Count and register dead Artemia salina that are eventually found in wells prior to incubation.
  • Incubate in the absence of light at 25 ºC for 24 hours.

Day 03
Assessing mortality

  • Count the number of dead A. salina nauplii in each well. A nauplius is considered dead if it remains completely motionless for at least 10 seconds. Use an optical microscope or magnifier lense to assist in observation.
  • After recordingthe number of dead individuals, add a few drops of ethanol to kill the remaining live nauplii. This step ensures an accurate count of total number of organisms.
  • Finally, count and record the total number of nauplii in each well.
  • Be sure to exclude from the final calculations any nauplii that were found dead on Day 2, prior to the start of incubation.
  • Calculate mortality using the formula below:





References
Albuquerque, L. P. D., Pontual, E. V., Santana, G. M. D. S., Silva, L. R. S., Aguiar, J. D. S., Coelho, L. C. B. B., Rêgo, M. J. B. D. M., Pitta, M. G. D. R., Silva, T. G. D., Melo, A. M. M. D. A., Napoleão, T. H., & Paiva, P. M. G. (2014). Toxic effects of Microgramma vacciniifolia rhizome lectin on Artemia salina, human cells, and the schistosomiasis vector Biomphalaria glabrata. Acta Tropica, 138, 23–27. https://doi.org/10.1016/j.actatropica.2014.06.005 Campos, T. G. V., Gama, W. A., Geraldes, V., Yoon, J., Crnkovic, C. M., Pinto, E., & Jacinavicius, F. R. (2024). New records on toxic cyanobacteria from Brazil: Exploring their occurrence and geography. Science of The Total Environment, 931, 172689. https://doi.org/10.1016/j.scitotenv.2024.172689 Elshafie, H. S., Caputo, L., De Martino, L., Gruľová, D., Zheljazkov, V. Z., De Feo, V., & Camele, I. (2020). Biological investigations of essential oils extracted from three Juniperus species and evaluation of their antimicrobial, antioxidant and cytotoxic activities. Journal of Applied Microbiology, 129(5), 1261–1271. https://doi.org/10.1111/jam.14723 Fadholly, A., Ansori, A. N. M., Jayanti, S., Proboningrat, A., Kusala, M. K. J., Putri, N., Rantam, F. A., & Sudjarwo, S. A. (2019). Cytotoxic effect of allium cepa l. Extract on human colon cancer (Widr) cells: In vitro study. Research Journal of Pharmacy and Technology, 12(7), 3483. https://doi.org/10.5958/0974-360X.2019.00591.2

Göethel, G., Augsten, L. V., Neves, G. M.; Gonçalves, I. L., Souza, J. P. S., Garcia, S. C., Eifler-Lima, V. L.a. The Role of Alternative Toxicological Trials in Drug Discovery Programs.The Case of Caenorhabditis elegans and Other Methods. Current Medicinal Chemistry, 29(32), 5270-5288, 2022. http://dx.doi.org/10.2174/0929867329666220329190825.

Médice, R. V., Arruda, R. S., Yoon, J., Borges, R. M., Noyma, N. P., Lürling, M., Crnkovic, C. M., Marinho, M. M., & Pinto, E. (2024). Unlocking biological activity and metabolomics insights: Primary screening of cyanobacterial biomass from a tropical reservoir. Environmental Toxicology and Chemistry, 43(10), 2222–2231. https://doi.org/10.1002/etc.5962

Salay, G.; Lucarelli, N.; Gascón, T. M.; Carvalho, S. S.; Veiga, G. R. L.; Reis, B. C. A. A.; Fonseca, F. L. A.. Acute Toxicity Assays with the Artemia salina Model: assessment of variables. Alternatives To Laboratory Animals, 52(3), 142-148, 2024. http://dx.doi.org/10.1177/02611929241242443. Solis, P., Wright, C., Anderson, M., Gupta, M., & Phillipson, J. (1993). A Microwell Cytotoxicity Assay using Artemia salina (Brine shrimp). Planta Medica, 59(03), 250–252. https://doi.org/10.1055/s-2006-959661

Protocol references
Metcalf, J. S., Lindsay, J., Beattie, K. A., Birmingham, S., Saker, M. L., Törökné, A. K., & Codd, G. A. (2002). Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins. Toxicon, 40(8), 1115-1120.