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    Brine Shrimp Lethality Assay

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    mario puntaca
    This document describes the brine shrimp lethality assay, which is used to assess the toxicity of bioactive compounds. It involves exposing brine shrimp larvae to varying concentrations of test compounds and determining the concentration that kills 50% of the shrimp (LC50) through probit analysis. The assay is rapid, inexpensive, and useful for preliminary screening of compounds isolated from natural products. It characterizes the dose-response relationship, which can be sigmoidal and transformed to a linear relationship using probit analysis to calculate the LC50.

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    Brine Shrimp Lethality Assay

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    mario puntaca
    107 views2 pages
    This document describes the brine shrimp lethality assay, which is used to assess the toxicity of bioactive compounds. It involves exposing brine shrimp larvae to varying concentrations of test compounds and determining the concentration that kills 50% of the shrimp (LC50) through probit analysis. The assay is rapid, inexpensive, and useful for preliminary screening of compounds isolated from natural products. It characterizes the dose-response relationship, which can be sigmoidal and transformed to a linear relationship using probit analysis to calculate the LC50.

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    This document describes the brine shrimp lethality assay, which is used to assess the toxicity of bioactive compounds. It involves exposing brine shrimp larvae to varying concentrations of test compounds and determining the concentration that kills 50% of the shrimp (LC50) through probit analysis. The assay is rapid, inexpensive, and useful for preliminary screening of compounds isolated from natural products. It characterizes the dose-response relationship, which can be sigmoidal and transformed to a linear relationship using probit analysis to calculate the LC50.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    107 views2 pages

    Brine Shrimp Lethality Assay

    Uploaded by

    mario puntaca
    This document describes the brine shrimp lethality assay, which is used to assess the toxicity of bioactive compounds. It involves exposing brine shrimp larvae to varying concentrations of test compounds and determining the concentration that kills 50% of the shrimp (LC50) through probit analysis. The assay is rapid, inexpensive, and useful for preliminary screening of compounds isolated from natural products. It characterizes the dose-response relationship, which can be sigmoidal and transformed to a linear relationship using probit analysis to calculate the LC50.

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    of 2

    Activity 7: Brine Shrimp Lethality Assay Arcenas, Sanchez

    Toxicodynamics Correlative relationship between the characteristics of


    Toxicodynamics exposure and the spectrum of toxic effects
    ● Describes the dynamic interactions of a toxicant
    with a biological target, and its biological effects Individual dose-response relationship
    ● Spectrum of undesired effects of some ● Describes response of an individual organism to
    chemicals may be broad or not varying doses of a chemical
    ● Often referred to as “graded” response because
    Side effects measured effect is continuous over a range of
    Some are never desirable and are always deleterious to doses
    the well-being of humans. Some of these side effects ● Response vs concentration/dose = usually
    may be desired for another therapeutic indication. results to hyperbolic curve
    ● Adverse ○ Results to sigmoidal curve when plotted
    ● Deleterious against logarithmic dose
    ● Toxic ○ Better fit of data to a straight line
    Quantal-dose relationship
    Toxic effects ● Characterizes distribution of individual responses
    Not produced by a chemical agent unless that agent or to different doses in a population of individual
    its metabolites reach appropriate sites in the body at a organisms which are quantal
    concentration and for a length of time sufficient to ● “All or none” in nature
    produce a toxic manifestation ● Particular toxic response (e.g. death) vs log dose
    Characterization of potential hazard of a chemical administered
    ● Effect ○ Individual is either a responder or a
    ● Dose required nonresponder
    ● Exposure ○ Sigmoidal curve
    ● Disposition by the subject ● In toxicology: provides widely used statistical
    Two major factors regarding exposure situation are the: approach for estimating response of a population
    ● Route of exposure to a toxic exposure in the “Toxic Dose” or TD
    ● Duration and frequency of exposure ○ Midpoint or 50%: TD50
    ● When death is measured as endpoint, TD is
    referred as Lethal Dose or LD
    Route of Ingestion (GIT)
    Exposure Inhalation (Lungs)
    Topical, Percutaneous, BRINE SHRIMP LETHALITY ASSAY
    Dermal (Skin) • Considered a useful tool for preliminary assessment of
    Parenteral Route toxicity of bioactive compounds as they are often
    considered to be toxic to shrimp larvae
    Duration and Acute Exposure (Less than • Suggested for screening pharmacological activities of
    Frequency of 24 hrs)
    bioactive compounds during isolation of natural products
    Exposure Subacute Exposure
    • Rapid, inexpensive, in-house, general bioassay
    (Repeated exposure for 1
    month or less) developed for screening, fractionation and monitoring of
    Sub-chronic Exposure physiologically active natural products
    (Repeated exposure for 1 to
    3 months) Brine shrimp
    Chronic Exposure (Repeated • Eggs bought as fish food in pet stores
    exposure for more than 3 • Hatch within 48 hours once placed in artificial sea water
    months)
    (40g/L salt),
    o Partition with perforation- allow brine shrimp to
    Dose-Response Relationship move between 2 environments
    o Light - Attracts nauplii
    Activity 7: Brine Shrimp Lethality Assay Arcenas, Sanchez

    o Dark Chamber- simulate night time conditions 0% MORTALITY


    =100*(0.25/N)
    4. Take the log of the concentrations.
    5. Graph the probits versus the log concentration and fit
    a line of regression.
    6. Find the LC50 by finding the dose with a probit of 5 in
    the y-axis.

    Probit analysis
    • Type of regression used to analyze binomial response
    variables
    • Used to determine relative toxicity of chemicals to living
    organisms
    • In brine shrimp lethality assay:
    o Response is always binomial (death/no death)
    o Relationship always sigmoidal
    • Probit analysis acts as transformation from sigmoid to
    linear then runs a regression on the relationship
    • LC50/LD50
    o Most commonly used outcomes
    o Concentration at which 50% of the population
    dies

    METHODS
    Solvent-solvent partitioning
    • Partitioned with n-hexane and ethyl acetate

    Assay
    • Artificial sea water – 40g sea salt/L dH2O
    • Nauplii – shrimp larvae
    o Illumination attracts them
    o 48 h, 22-29°C to hatch
    • DMSO limit in solubilizing water-insoluble fractions:
    50μL/5mL sea water
    • 24 hours of exposure to fractions

    DATA ANALYSIS
    1. Count number of dead brine shrimp
    2. Average and round up
    % Mortality =​ 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑏𝑟𝑖𝑛𝑒 𝑠ℎ𝑟𝑖𝑚𝑝 ​ x 100
    𝑠𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑏𝑟𝑖𝑛𝑒 𝑠ℎ𝑟𝑖𝑚𝑝𝑠
    3. Convert % mortality to probits using Finney’s table.
    3.1 In case of 100% mortality use correction
    CORRECTION FOR 100% MORTALITY
    =100*((N-0.25)/N)

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