Skip to main content

Biological screening of selected Pacific Northwest forest plants using the brine shrimp (Artemia salina) toxicity bioassay


The brine shrimp (Artemia salina) bioassay was used to screen 211 methanol extracts from 128 species of Pacific Northwest plants in search of general cytotoxic activity. Strong toxicity (LC50 < 100 µg/ml) was found for 17 extracts from 13 species, with highest activity observed for Angelica arguta roots at <10 µg/ml. Notably, four species of cedar trees and one of juniper in the family Cupressaceae dominated this group with LC50 for heartwood extracts ranging from 15 to 89 µg/ml. Moderate toxicity (LC50 100–500 µg/ml) was found in 38 extracts from 27 species, while weak toxicity (LC50 500–1000 µg/ml) was detected for 17 extracts in 16 species. There were 139 extracts from 99 species that were non-toxic (LC50 > 1000 µg/ml). Our subsequent studies of conifer heartwoods with strong activity confirm the assay’s value for identifying new investigational leads for materials with insecticidal and fungicidal activity.


The forests and rangelands of Washington and Oregon are diverse ecosystems ranging from the temperate rainforests of the Olympic Peninsula in Washington to the semiarid shrub-steppe of southeastern Oregon (Franklin and Dyrness 1988). Across this region, fir, pine and cedar species are basic foundations to industries producing lumber and structural wood products. Native Americans have long used many forest plants for foods, medicines and handmade materials to improve daily life (Gunther 1973; Forlines et al. 1992). There remains an interest in the herbal remedies (Moore 1993), and many of the plants still have potential for development of new, natural sources of medicines and insecticides.

The brine shrimp toxicity bioassay is a simple method of screening crude plant extracts for cytotoxicity (Meyer et al. 1982; McLaughlin et al. 1991) and is an indicator of potential antitumor, insecticidal, and fungicidal activity (Michael et al. 1956; Harwig and Scott 1971; McLaughlin et al. 1998). The mode of action causing toxicity is unknown, but the results typically correlate with more specific bioactivity tests. The brine shrimp bioassay has also been used to guide the isolation of bioactive compounds, testing of water quality, and detection of fungal toxins (Nguta et al. 2011; Arcanjo et al. 2012; Gadir 2012). This method is an attractive pre-screen for such activities as it is relatively simple and inexpensive to test large numbers of crude plant extracts in a relatively short time. Most surveys of this type have been carried out on traditional medicinal plants of various cultures from around the world (Pimentel et al. 2002; Krishnarajua et al. 2005; Rahman et al. 2008; Moshi et al. 2010; Ved et al. 2010; Bussmann et al. 2011; Nguta et al. 2011; Oryema et al. 2011; Arcanjo et al. 2012; Gadir 2012; Nguta et al. 2012; Biradi and Hullatti 2014; Khatun et al. 2014). A few studies have targeted forest and savannah plants (Horgen et al. 2001; Adouom 2009; Rizwana et al. 2010; Soonthornchareonnon et al. 2012; Ravikumar et al. 2014).

In this paper we report survey results for some forest plants from the Pacific Northwest to gain a preliminary understanding of which ones may merit further, more specific testing with potential for developing new medicines and pesticides to benefit future generations.


Plant materials

Plants were collected during their active growing seasons in western Washington, western and central Oregon. Voucher specimens were deposited at the Oregon State University Herbarium.

Preparation of extracts

Plant materials were air-dried, ground and then extracted at room temperature for 48 h with methanol. The methanol was analytical grade and freshly distilled prior to use. Extracts were evaporated under vacuum on a rotary evaporator and the residue briefly freeze dried under high vacuum to remove traces of solvent and water, then stored at −20 °C until tested.

Brine shrimp toxicity bioassay

Bioassays of the crude extracts were carried out as described by Meyer et al. (1982) and McLaughlin et al. (1991) on freshly hatched brine shrimp (Artemia salina Leach). Triplicate samples of each extract were tested initially at concentrations of 10, 100 and 1000 ppm (µg/mL) in vials containing 5 mL of brine solution and 10 shrimp. Survivors were counted after 24 h and the median lethal concentration (LC50) with 95 % confidence intervals calculated using Probit Analysis.


Results of the brine shrimp cytotoxicity screening are shown in Table 1. Extracts with LC50 values >1000 µg/ml are considered non-toxic (Meyer et al. 1982). Values between 500 and 1000 µg/ml are considered weakly toxic, those between 100 and 500 µg/ml as moderately toxic, and those <100 µg/ml as strongly toxic (Nguta et al. 2012). A total of 211 crude methanol extracts from 128 species, 116 genera, and 49 families are represented. Strong cytotoxic activity was found in 17 extracts from 13 species (Table 2), moderate toxicity in 38 extracts from 27 species, weak activity for 17 extracts in 16 species, and 139 non-toxic extracts from 99 species. The proportions of all extracts by activity category are shown in Fig. 1.

Table 1 Brine shrimp toxicity at 24 h exposure to plant extracts
Table 2 Plant species and tissues with strong, <100 µg/ml LC50, brine shrimp toxicity at 24 h exposure to plant extracts
Fig. 1
figure 1

The percentage of extracts within the four categories of cytotoxic activity


There were more than twice as many extracts with moderate activity than there were with strong activity. Moderately active extracts need not be dismissed as unimportant, since Bussmann et al. (2011), Nguta et al. (2012) and others have noted that toxicity can vary significantly due to harvest time, collection location, plant organ or tissue, and solvent used for extraction. Alcohol or organic solvent extracts are often more toxic than aqueous ones, but not always. Extracts from genera and species with the strongest bioactivity can also exhibit a wide range in their levels of activity for the same reasons, thus varying among experiments and research groups. Given this natural variability and our extensive list of genera and species we decided not to attempt cross comparing levels of activity with those observed by others, as it is beyond the scope of this report.

Tissues identified with LC50 < 100 µg/ml cytotoxicity have served us as leads for further studies of bioactive extracts and compounds from heartwoods of yellow, incense, and Port-Orford cedars, and western juniper against mosquitoes (Aedes aegypti), ticks (Ixodes scapularis), fleas (Xenopsylla cheopis) or microbes influencing animal and forest health (Johnston et al. 2001; Panella et al. 2005; Dietrich et al. 2006; Manter et al. 2006, 2007; Dolan et al. 2007, 2009). It is worthwhile noting that three of the compounds in yellow or incense cedar heartwoods have different modes of action than other commercially available mosquito adulticides currently in use (McAllister and Adams 2010). New modes of action are particularly relevant in the search for compounds to overcome resistance to existing pesticides.


Natural products from Pacific Northwest forest resources can offer alternative biocides and repellent compounds with activities comparable to synthetic pesticides for control of arthropods of public health concern and forest microbial pathogens. Other bioactive extracts from our brine shrimp screening need to be investigated further. In addition, other forest plants from this region need to be pre-screened by this method as well to provide a more complete understanding of the potential value for all our forest and rangeland resources.


  • Adouom OA (2009) Determination of toxicity levels of some savannah plants using brine shrimp test (BST). Bayero J Pure Appl Sci 2:135–138

    Google Scholar 

  • Arcanjo DDR, Albuquerque ACM, Melo-Neto B, Santana LCLR, Medeiros MGF, Citó AMGL (2012) Bioactivity evaluation against Artemia salina L. each of medicinal plants used in Brazilian northeastern folk medicine. Braz J Biol 72:505–509

    Article  Google Scholar 

  • Biradi M, Hullatti K (2014) Screening of Indian medicinal plants for cytotoxic activity by brine shrimp lethality (BSL) assay and evaluation of their total phenolic content. Drug Dev Ther 5:139–144

    Article  Google Scholar 

  • Bussmann RW, Malca G, Glenn A, Sharon D, Nilsen B, Parris B, Dubose D, Ruiz D, Saleda J, Martinez M, Carillo L, Walker K, Kuhlman A, Townesmith A (2011) Toxicity of medicinal plants used in traditional medicine in northern Peru. J Ethnopharm 137:121–140

    Article  Google Scholar 

  • Dietrich G, Dolan MC, Peralta-Cruz J, Schmidt J, Piesman J, Eisen RJ, Karchesy JJ (2006) Repellent activity of fractionated compounds from Chamaecyparis nootkatensis essential oil against Nymphal Ixodes scapularis (Acari: Ixodidae). J Med Entomol 43:957–961

    Article  Google Scholar 

  • Dolan MC, Dietrich G, Panella NA, Montenieri JA, Karchesy JJ (2007) Biocidal activity of three wood essential oils against Ixodes Scapularis (Acari: Ixodidae), Xenopsylla cheopis, and Aedes aegypti. J Econ Entomol 100:622–625

    Article  Google Scholar 

  • Dolan MC, Jordan RA, Schulze TL, Schulze CJ, Manning MC, Ruffolo D, Schmidt JP, Piesman J, Karchesy JJ (2009) Ability of two natural products, nootkatone and carvacrol, to suppress Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in a lyme disease endemic area of New Jersey. J Econ Entomol 102:2316–2324

    Article  Google Scholar 

  • Forlines DR, Tavenner T, Malan JCS, Karchesy JJ (1992) Plants of the Olympic coastal forests: ancient knowledge of materials and medicines and future heritage. Basic Life Sci 59(Plant Polyphenols):767–782

    Google Scholar 

  • Franklin JF, Dyrness CT (1988) Natural vegetation of Oregon and Washington. USDA Forest Service Oregon State University Press, Corvallis

    Google Scholar 

  • Gadir SA (2012) Assessment of bioactivity of some Sudanese medicinal plants using brine shrimp (Artemia salina) lethality assay. J Chem Pharm Res 4:4148–5145

    Google Scholar 

  • Gunther E (1973) Ethnobotany of western Washington. Univ. Washington Press, Seattle

    Google Scholar 

  • Harwig J, Scott PM (1971) Brine shrimp (Artemia salina L.) larvae as a screening system for fungal toxins. Appl Microbiol 21:1011–1016

    Google Scholar 

  • Horgen FD, Edrada RA, de los Reyes G, Agcaoili F, Madulid DA, Wongpanich V, Angerhofer CK, Pezzuto JM, Soejarto DD, Farnsworth NR (2001) Biological screening of rain forest plot trees from Palawan Islant (Philippines). Phytomedicine 8:71–81

    Article  Google Scholar 

  • Johnston WH, Karchesy JJ, Constantine GH, Craig AM (2001) Antimicrobial activity of some Pacific Northwest woods against anaerobic bacteria and yeasts. Phytother Res 15:586–588

    Article  Google Scholar 

  • Khatun A, Rahman M, Haque T, Rahman M, Akter M, Akter S, Jhumur A (2014) Cytotoxicity potentials of eleven Bangladeshi medicinal plants. The Sci World J. Article ID 913127, p 7. doi:10.1155/2014/913127

  • Krishnarajua AV, Raoa TVN, Sundararajua D, Vanisreeb M, Tsayb H-S, Subbarajua GV (2005) Assessment of bioactivity of Indian medicinal plants using brine shrimp (Artemia salina) lethality assay. Int J Appl Sci Eng 3:125–134

    Google Scholar 

  • Manter DK, Karchesy JJ, Kelsey RG (2006) The sporidical activity of yellow-cedar heartwood, essential oil and wood constituents towards Phytophthora ramorum in culture. For Pathol 36:297–308

    Google Scholar 

  • Manter DK, Kelsey RG, Karchesy JJ (2007) Antimicrobial activity of extractable conifer heartwood compounds toward Phytophthora ramorum. J Chem Ecol 33:2133–2147

    Article  Google Scholar 

  • McAllister JC, Adams MR (2010) Mode of action for natural products isolated from essential oils of two trees is different from available mosquito adulticides. J Med Entomol 47:1123–1126

    Article  Google Scholar 

  • McLaughlin JL, Chang CJ, Smith DL (1991) “Bench-top” bioassays for the discovery of bioactive natural products: an update. In: Rahman A (ed) Studies in natural product chemistry, vol 9. Elsevier, Amsterdam, pp 383–409

    Google Scholar 

  • McLaughlin JL, Rogers LL, Anderson JE (1998) The use of biological assays to evaluate botanicals. Drug Inform J 32:513–524

    Google Scholar 

  • Meyer BN, Ferrigini RN, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med 45:31–35

    Article  Google Scholar 

  • Michael AS, Thompson CG, Abramovitz M (1956) Artemia salina as a test organism for bioassay. Science 123:464

    Article  Google Scholar 

  • Moore M (1993) Medicinal plants of the Pacific West. Red Crane Books, Santa Fe

    Google Scholar 

  • Moshi MJ, Innocent E, Magadula JJ, Otieno DF, Weisheit A, Mbabazi PK, Nondo RSO (2010) Brine shrimp toxicity of some plants used as traditional medicines in Kagera Region, northwestern Tanzania. Tanzania J Health Res 12:63–67

    Google Scholar 

  • Nguta JM, Mbaria JM, Gakuya DW, Gathumbi PK, Kabasa JD, Kiama SG (2011) Biological screening of Kenyan medicial plants using A. salina L. (Artemiidae). Pharmacologyonline 2:458–478

    Google Scholar 

  • Nguta JM, Mbaria JM, Gakuya DW, Gathumbi PK, Kabasa JD, Kiama SG (2012) Evaluation of acute toxicity of crude plant extracts from Kenyan biodiversity using brine shrimp, Artemia salina L. (Artemiidae). Open Conf Proc J 3:30–34

    Article  Google Scholar 

  • Oryema C, Ziraba RB, Odyek O, Omagor N, Opio A (2011) Phytochemical properties and toxicity to brine shrimp of medicinal plants in Erute county, Lira district, Uganda. J Med Plants Res 5:5450–5457

    Google Scholar 

  • Panella NA, Dolan MC, Karchesy JJ, Xiong Y, Peralta-Cruz J, Khasawneh M, Montenieri JA, Maupin GO (2005) Use of novel compounds for pest control: insecticidal and acaricidal activity of essential oil components from heartwood of Alaska yellow cedar (Chamaecyparis nootkatensis). J Med Entomol 42:352–358

    Article  Google Scholar 

  • Pimentel AB, Pizzolatti MG, Costa IM (2002) An application of the brine shrimp bioassay for general screening of Brazilian medicinal plants. Acta Farm Bonaerense 21:175–178

    Google Scholar 

  • Rahman MS, Begum B, Chowdhury R, Rahman KM, Rashid MA (2008) Preliminary cytotoxicity screening of some medicinal plants of Bangladesh. Dhaka Univ J Pharm Sci 7:47–52

    Google Scholar 

  • Ravikumar AR, Madgaonkar V, Venkatesha RT, Bharathi R, Murthy VK (2014) Potential cytotoxic drug effects of secondary metabolites derived from selected medicinal plants of Savanadurga forest in Karnataka. Int J Pharm Pharm Sci 6:238–241

    Google Scholar 

  • Rizwana JN, Nazlina I, Razehar ARM, Noraziah AZS, Ling CY, Muzaimah SAS, Farina AH, Yaacob WA, Ahmad IB, Din LB (2010) A survey on phytochemical and bioactivity of plant extracts from Malaysian forest reserves. J Med Plants Res 4:203–210

    Google Scholar 

  • Soonthornchareonnon N, Wiwat C, Chuakul W (2012) Biological activities of medicinal plants from mangrove and beach forests. Mahidol Univ J Pharm Sci 39:9–18

    Google Scholar 

  • Ved CH, More NS, Bharate SS, Bharate SB (2010) Cytotoxicity screening of selected Indian medicinal plants using brine-shrimp lethality bioassay. Adv Nat Appl Sci 4:389–395

    Google Scholar 

Download references

Authors’ contributions

YMK collected plant material, prepared extracts, conducted the bioassays and processed the data. RGK collected some plants, prepared some extracts and co-wrote the manuscript. GC assisted with the bioassays. JJK conceived the study, collected some of the plants, and co-wrote the manuscript. All authors read and approved the final manuscript.


The authors thank Richard Halse, Oregon State University Herbarium, for assistance with plant identification.

Competing interests

The authors declare that they have no competing interests.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Rick G. Kelsey.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Karchesy, Y.M., Kelsey, R.G., Constantine, G. et al. Biological screening of selected Pacific Northwest forest plants using the brine shrimp (Artemia salina) toxicity bioassay. SpringerPlus 5, 510 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: