Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Scribd Logo
Upload

Welcome to Scribd!

  • 19 views

    Nanotechnologies - Assessment of Nanomaterial Toxicity Using Dechorionated Zebrafish Embryo

    Uploaded by

    Cappucino Ningning
    This technical specification provides a method for assessing nanomaterial toxicity using dechorionated zebrafish embryos from 0-120 hours post fertilization (HPF). It describes removing the protective chorion layer from the embryos using either mechanical or enzymatic dechorionation. The enzymatic method uses the enzyme pronase to gently remove the chorion in 5-6 minutes followed by rinsing. The specification provides materials, equipment, and detailed step-by-step procedures for chorion removal and conducting the nanomaterial toxicity assessment tests on the dechorionated zebrafish embryos.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd

    Nanotechnologies - Assessment of Nanomaterial Toxicity Using Dechorionated Zebrafish Embryo

    Uploaded by

    Cappucino Ningning
    19 views3 pages
    This technical specification provides a method for assessing nanomaterial toxicity using dechorionated zebrafish embryos from 0-120 hours post fertilization (HPF). It describes removing the protective chorion layer from the embryos using either mechanical or enzymatic dechorionation. The enzymatic method uses the enzyme pronase to gently remove the chorion in 5-6 minutes followed by rinsing. The specification provides materials, equipment, and detailed step-by-step procedures for chorion removal and conducting the nanomaterial toxicity assessment tests on the dechorionated zebrafish embryos.

    Original Title

    1-2-1

    Copyright

    © © All Rights Reserved

    Available Formats

    DOCX, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    This technical specification provides a method for assessing nanomaterial toxicity using dechorionated zebrafish embryos from 0-120 hours post fertilization (HPF). It describes removing the protective chorion layer from the embryos using either mechanical or enzymatic dechorionation. The enzymatic method uses the enzyme pronase to gently remove the chorion in 5-6 minutes followed by rinsing. The specification provides materials, equipment, and detailed step-by-step procedures for chorion removal and conducting the nanomaterial toxicity assessment tests on the dechorionated zebrafish embryos.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    19 views3 pages

    Nanotechnologies - Assessment of Nanomaterial Toxicity Using Dechorionated Zebrafish Embryo

    Uploaded by

    Cappucino Ningning
    This technical specification provides a method for assessing nanomaterial toxicity using dechorionated zebrafish embryos from 0-120 hours post fertilization (HPF). It describes removing the protective chorion layer from the embryos using either mechanical or enzymatic dechorionation. The enzymatic method uses the enzyme pronase to gently remove the chorion in 5-6 minutes followed by rinsing. The specification provides materials, equipment, and detailed step-by-step procedures for chorion removal and conducting the nanomaterial toxicity assessment tests on the dechorionated zebrafish embryos.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 3

    TECHNICAL SPECIFICATION ISO/TS 22082:2020(E)

    Nanotechnologies — Assessment of nanomaterial toxicity


    using dechorionated zebrafish embryo

    1 Scope
    This document specifies a method for rapidly assessing nanomaterial toxicity (fish early life stage,
    0 HPF to 120 HPF). It includes information on the importance of acellular chorion removal, detailed
    chorion removal procedures, and a complete protocol for the toxicity assessment of nanomaterials
    using dechorionated zebrafish embryos. The focus of this document is on testing nanomaterial toxicity.

    2 Normative references
    The following documents are referred to in the text in such a way that some or all of their content
    constitutes requirements of this document. For dated references, only the edition cited applies. For
    undated references, the latest edition of the referenced document (including any amendments) applies.
    ISO/TS 12805, Nanotechnologies — Materials specifications — Guidance on specifying nano-objects
    ISO/TR 13014, Nanotechnologies — Guidance on physico-chemical characterization of engineered
    nanoscale materials s for toxicologic assessment
    ISO/TS 17200, Nanotechnology — Nanoparticles in powder form — Characteristics and measurements
    ISO/TR 18196, Nanotechnologies — Measurement technique matrix for the characterization of nano-
    objects ISO 22412, Particle size analysis — Dynamic light scattering (DLS)

    ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects


    OECD. Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals.
    Section 2. OECD Publishing, Paris, 2013

    3 Terms and definitions


    For the purposes of this document, the terms and definitions given in ISO/TS 12805, ISO/TR 13014,
    ISO/TS 17200, ISO/TS 80004-1, ISO/TS 80004-2, OECD TG 236 and the following apply.
    ISO and IEC maintain terminological databases for use in standardization at the following addresses:
    — IEC Electropedia: available at http://www.electropedia.org/
    — ISO Online browsing platform: available at https://www.iso.org/obp
    3.1
    solubilizing agent
    solvent or dispersant that can disperse and stabilize nanomaterials in solution
    3.2
    spawning
    releasing the eggs into the water for fertilization

    © ISO 2020 – All rights reserved 1


    TECHNICAL SPECIFICATION ISO/TS 22082:2020(E)

    3.3
    positive control
    any well-characterized material or substance that, when evaluated by a specific test method,
    demon-strates the suitability of the test system to yield a reproducible, appropriately positive or
    reactive re-sponse in the test system
    [SOURCE: ISO 10993-10:2010, 3.14]
    3.4
    range-finding test
    abbreviated acute test that exposes test organisms to a broad range of nanomaterial testing
    solutions to establish the range of concentrations to be used in the definitive test
    Note 1 to entry: The test includes at least five concentrations of the nanomaterial and untreated
    control.
    [SOURCE: OECD TG 236:2013, modified]
    3.5
    LC50
    concentration of a toxic substance that is lethal to half of a group of test organisms (50 %)
    Note 1 to entry: Usually, the exposure to the substance is continuous and the LC50 is defined by
    reference to a specified exposure period.
    [SOURCE: ISO 6107-3:1993, 39, modified]

    4 Abbreviated terms

    DPF day post fertilization


    EM embryo media

    HPF hour post fertilization

    NOAEL no observed adverse effect level


    DO dissolved oxygen

    5 Materials

    5.1 Organism (zebrafish, Danio rerio).


    Zebrafish (Danio rerio, see Figure 1) is a tropical freshwater teleost belonging to the Cyprinidae,
    natu-rally distributed throughout India, Pakistan, Bangladesh, Nepal and Burma. The average
    size of an adult is 2 cm to 3 cm and has blue stripes, similar to a zebra on the side of the body.
    The adult male zebrafish has a thinner body, with blue and gold hue stripes. The female has a
    whitish belly, larger than male and silver stripes instead of gold. This species can be kept and
    bred in aquaria. The lifespan of zebrafish is approximately 2 to 3 years. Organogenesis of
    zebrafish is completed within 5 DPF and it is an adult within approximately 3 months.

    © ISO 2020 – All rights reserved 2


    TECHNICAL SPECIFICATION ISO/TS 22082:2020(E)

    3.1 Dechorionation of embryos

    7.3.1 Methods
    There are two types of dechorionating methods commonly used: mechanical and
    enzymatic dechorionation. Embryos can be mechanically dechorionated using two
    forceps under a stereomicroscope. In general, mechanical dechorionation requires
    only short preparation time and it does not require the use of enzymes. However, it is
    both time and labour intensive, which substantially limits the number of embryos
    that can be dechorionated at one time. It is particularly challenging to remove
    chorion of early life stage embryos (0 HPF to 10 HPF) as manual manipulations can
    cause unforeseen damage leading to defects later in life. Enzymatic dechorionation
    generally uses a protease extracted from bacteria species and allows for simultaneous
    dechorionating large numbers of embryos.

    7.3.2 Enzymatic dechorionation method

    Preparation of chemicals, supplies and equipment.


    — For 1 l of EM, dissolve 5,0 mM NaCl (0,292 g), 0,17 mM KCl (0,013 g), 0,33 mM CaCl
    (0,044 g), 0,33 mM MgSO4 (0,081 g) in distilled water and adjust pH to ~7,4 using
    0,01 N NaOH or 0,01 N HCl.
    — Collect fertilized embryos and separate as many as needed (include 20 % more
    than needed) all at the same 4 HPF developmental stage.
    — Prepare the enzyme [Pronase (EC 3.4.24.4; CAS# 9036-06-0), which is a mixture
    enzyme of several non-specific endo proteases and exoproteases that digest
    proteins down to single amino acids, from Streptomyces griseus] stock solution on
    ice using distilled water and into single-use microtubes in small volumes (~100
    µl) and store in ‒80 °C until ready for use.
    — Treat chorionated embryos (~1 000) with the enzyme at 19,1 U into 25 ml (0,764
    U/ml) of EM in a 90 mm glass petri dish.
    — Gently agitate for 5 min or 6 min followed by 6 min rinse with at least a total of
    500 ml of EM, then a final mix (without pronase) for 3 min.
    — After the rinse, allow the embryos to rest for 20 min to 30 min at (27 ± 1) °C.
    Following the rest, the weakened chorions left in a petri dish are removed by
    another rinse cycle (for 6 min) with EM.
    — Do not let the embryo contact air or plastic during the process of dechorionation.
    The use of glass wide bore polish pipettes is recommended.
    The method is shown in Figure 2.

    © ISO 2020 – All rights reserved 3

    You might also like