Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 5


Section 5: Specific Procedures for Testing Chemicals

This section gives particular instructions for testing chemicals, in addition to the procedures outlined in Section 4.

5.1 Properties, Labelling, and Storage of Sample

Information should be obtained on the properties of the chemical, formulated product, or chemical mixture to be tested, including concentration of the major ingredients and impurities, water solubility, vapour pressure, chemical stability, dissociation constants, toxicity to humans and aquatic organisms, and biodegradability. Data sheets on safety aspects of the substance(s) (e.g., Material Safety Data Sheets) should be consulted, if available. Where aqueous solubility is in doubt or problematic, acceptable procedures previously used for preparing aqueous solutions of the chemical(s) should be obtained and reported, and/or chemical solubility in test water should be determined experimentally. Other available information such as structural formulae, nature and percentage of significant impurities, presence and amounts of additives, and n-octanol:water partition coefficient, should be obtained and recorded.Footnote 62 Water solubility and vapour pressure can be used to calculate Henry’s Law Constant, which will indicate if significant losses of the test substance during the test period are likely. This will help signify whether steps to control such losses should be taken (Organization for Economic Cooperation and Development (OECD), 1998, 2002) (see Section 5.2). An acceptable analytical method should be available for the chemical in water at concentrations intended for the test, together with data indicating the precision and accuracy of the analysis.

Chemical containers must be sealed and coded or labelled upon receipt. Required information (chemical name, supplier, date received, grade or purity, person responsible for testing, etc.) must be indicated on the label and/or recorded on a separate data sheet dedicated to the sample, as appropriate. Storage conditions (e.g., temperature, protection from light) are frequently dictated by the nature of the chemical. Standard operating procedures of the laboratory, or else those recommended by manufacturers, by a Material Safety Data Sheet, or by similar advisory information should be followed for handling and storage of a chemical.

5.2 Preparing Test Solutions

Solutions of the test chemical are usually prepared by adding aliquots of a stock solution made up in control/dilution water (Swedish Standard [SIS] growth medium, modified Steinberg medium, or modified American Public Health Association (APHA) medium; see Section 5.3). Volumetric flasks should be used to prepare stock and test solutions. Stock solutions should normally be prepared by dissolving the test substance(s) in test medium. For some substances or materials (e.g., pesticides), a foliar application (spray) of the test substance directly onto the fronds might be applicable, if this is considered to be the most likely exposure scenario (Lockhart et al., 1989; Boutin et al., 1993; OECD, 1998, 2002). Alternatively, for strong solutions or large volumes, weighed (analytical balance) quantities of chemical may be added to control/dilution water (e.g., SIS or modified Steinberg medium) to give the nominal strengths for testing. Regardless of how test solutions are prepared, the concentration, solubility, and stability of the chemical in the test medium under test conditions should be determined before the test is initiated. Stock solutions subject to photolysis should be shielded from light, and unstable solutions must be prepared as frequently as necessary to maintain concentrations for each test solution renewal.

The water solubility of the test substance should not be exceeded in any test concentration (OECD, 1998, 2002).Footnote 63 For chemicals that do not dissolve readily in water, stock solutions may be prepared using the generator column technique (Billington et al., 1988; Shiu et al., 1988) or, less desirably, by ultrasonic dispersion.Footnote 64 Organic solvents, emulsifiers, or dispersants should not be used to assist chemical solubility except in instances where they might be formulated with the test chemical for its normal commercial purposes. If used, an additional control solution must be prepared containing the highest concentration of the agent used in the test. Solubilizing agents should be used sparingly, and should not exceed 0.1 mL/L in any test solution; the type and final concentration used must be reported. If solvents are used, the preferred ones are triethylene glycol and dimethyl formamide (American Society for Testing and Materials (ASTM), 1991; OECD, 1998).Footnote 65 Methanol, ethanol, and acetone could also be used but are more volatile and can stimulate the undesirable growth of microorganisms (ASTM, 1991).

The static test is recommended for use with stable chemicals, commercial products, and mixtures of known substances. However, for tests where the concentration of the test substance is not expected to remain within ±20% of the nominal concentration (or a preliminary stability test shows that the concentration of the test substance or one or more of its biologically active ingredients falls below 80% of the measured initial concentration) over the duration of the test (7 days), the static-renewal procedure must be followed (OECD, 1998, 2002). In the static-renewal test, Lemna minor colonies must be transferred to new test solutions on at least two occasions during the test (e.g., Days 3 and 5) (see Section 4.3). More frequent renewals might be necessary to maintain concentrations (≥80%) of highly unstable or volatile substances (USEPA, 1996; OECD, 1998, 2002).

5.3 Control/Dilution Water

For tests designed to assess toxicity of a chemical to L. minor, either the modified Swedish Standard (SIS) medium (OECD, 1998, 2002), the modified Steinberg medium (ISO, 2005), or receiving water spiked with SIS or modified Steinberg nutrient stock solutions (nutrient-spiked receiving water) should be used as the control/dilution water.Footnote 66 Where appropriate (e.g., for testing metals), modified APHA medium, which contains no EDTA, or receiving water spiked with modified APHA nutrient stock solutions, may be used as control/dilution water (see Section 6.3).

The control/dilution water recommended for standard use for tests with chemical samples is either the SIS medium or the modified Steinberg medium. The SIS medium consists of seven stock solutions, as outlined in Table 4. Stock solutions are prepared in distilled water, or equivalent, using reagent grade chemicals. Stock solution VII (MOPS (4-morpholinepropane sulphonic acid) buffer) is only used for testing substances or materials in which additional pH control is required.Footnote 67 Stock solutions I to V are sterilized by autoclaving at 120°C for 15 minutes or by membrane filtration (0.2 µm pore size). Stock solutions VI and VII (optional) are sterilized by membrane filtration (0.2 µm pore size) only (they should not be autoclaved), and then they are aseptically added to the remaining stock solutions.

To prepare 1 L of SIS test medium, the following are added to 900 mL of glass-distilled, deionized water (or equivalent):

  • 10 mL of stock solution I,
  • 5 mL of stock solution II,
  • 5 mL of stock solution III,
  • 5 mL of stock solution IV,
  • 1 mL of stock solution V, and
  • 5 mL of stock solution VI.

If buffer is required, 1 mL of stock solution VII (optional) is also added. The pH is adjusted to 6.5 ± 0.2 with either 0.1 or 1 N HCl or NaOH, and adjusted to 1 L with distilled water (OECD, 1998, 2002).

Sterile stock solutions should be stored under cool and dark conditions. Stock solutions I to V have a shelf life of 6 months, whereas stock solutions VI and VII should be discarded after 1 month. The medium is stored in the dark to preclude possible (unknown) photochemical changes. Under these conditions, the prepared medium has a shelf-life of approximately 6 to 8 weeks; however, it is recommended that fresh medium be prepared for use in a test. The SIS medium should be prepared 1 to 2 days before use to allow the pH to stabilize, although it is advisable to check the pH of the medium before use. If the pH lies outside the specified range (6.5 ± 0.2), it may be readjusted by adding NaOH or HCl as previously described (OECD, 1998, 2002).

The modified Steinberg medium can also be used as control/dilution water for tests with chemical samples, as recommended in the draft ISO L. minor growth inhibition test (ISO, 2005). This medium consists of eight stock solutions as outlined in Table 11 of Appendix D. Details on how to prepare the medium are found in the draft ISO standard (ISO, 2005).

In instances where the toxic effect of a chemical in a particular receiving water is to be appraised, the recommended control/dilution water is the receiving water itself, spiked with the same nutrients as those used to prepare the SIS medium or the modified Steinberg medium (nutrient-spiked receiving water), whichever is being used (see footnote 40 and Table 4). Examples of such situations would include appraisals of the toxic effect of chemical spills or intentional applications of chemicals (e.g., pesticide) on a water body.

If a sample of upstream receiving water is to be used as dilution and control water, a separate control solution must be prepared using the SIS medium or the modified Steinberg medium, depending on which medium is used in the test (see Section 4.1).Footnote 68 Test medium might be used for all dilutions and the control when a high degree of standardization is required for testing (e.g., if the toxicity of a chemical is to be determined and compared at a number of test facilities), or when the collection and use of receiving water is impractical (e.g., too expensive).

Table 4 Chemical Composition of Nutrient Stock Solutions for Preparing SIS Medium and Nutrient-Spiked Receiving Water, for Testing Chemical Samples Using Lemna minor
Stock Substance Concentration
Stock Solution
(g/L)
Concentration
MediumFootnote a
(mg/L)
I NaNO3 8.50 85
I KH2PO4 1.34 13.4
II MgSO4 · 7H2O 15.0 75
III CaCl2 · 2H2O 7.20 36
IV Na2CO3 4.00 20
V H3BO3 1.00 1.00
V MnCl2 · 4H2O 0.200 0.200
V Na2MoO4 · 2H2O 0.010 0.010
V ZnSO4 · 7H2O 0.050 0.050
V CuSO4 · 5H2O 0.005 0.005
V Co(NO3)2 · 6H2O 0.010 0.010
VI FeCl3 · 6H2O 0.168 0.84
VI Na2EDTA · 2H2O 0.280 1.40
VII MOPS (buffer)Footnote b 488 488

5.4 Test Observations and Measurements

In addition to the observations on toxicity described in Section 4.4, there are certain additional observations and measurements to be made while testing with chemicals.

During the preparation of solutions and at each of the prescribed observation times during the test, each solution should be examined for evidence of chemical presence and change (e.g., odour, colour, opacity, precipitation, or flocculation of chemical). Any observations should be recorded.

It is desirable and recommended that test solutions be analyzed to determine the chemical concentrations to which L. minor are exposed.Footnote 69 If chemicals are to be measured in a static test, sample aliquots should be taken from all replicates in at least the high, medium, and low test concentrations, and the control(s). Separate analyses of the aliquots should be performed on samples taken immediately before the start of the initial exposure and at the end of the test, as a minimum. If chemicals are to be measured in a static-renewal test, sample aliquots should be taken from at least the high, medium, and low test concentrations, and the control(s). As a minimum, separate analyses should be performed with samples taken at the beginning and end of each renewal period and on the first and last days of the test.

All samples should be preserved, stored, and analyzed according to proven methods with acceptable detection limits for determining the concentration of the particular chemical in aqueous solution. Toxicity results for any tests in which concentrations are measured should be calculated and expressed in terms of those measured concentrations, unless there is a good reason to believe that the chemical measurements are not accurate.Footnote 70 In making calculations, each test solution should be characterized by the geometric average of the measured concentration to which the organisms are exposed.

At the start of the test, frond and colony numbers in the test vessels are recorded. Frond numbers and the appearance of the colonies must be observed at the beginning and at the end of the test. Two additional observations of frond number (e.g., on Days 3 and 5) should be made if the average specific growth rate or area under the curve is the preferred statistical endpoint (see footnote 46 and Section 4.5.5). Any changes in plant development, frond size, appearance, necrosis, or chlorosis should be noted as well as additional observations of root length, atypical appearance of the test media (e.g., presence of undissolved material), or any other abnormalities.

5.5 Test Endpoints and Calculations

The ICp is the statistical endpoint recommended for a multi-concentration test performed using a chemical (see Section 4.5.2).

If a solvent control is used to maintain the test substance in solution, there must be assurance that the solvent itself does not cause undue effects. Such a test is rendered invalid if Lemna growth in the solvent control (or untreated control) does not meet the criteria for test validity specified in Section 4.5.1.

When a solvent or other chemical is used, it becomes the control for assessing the effect of the toxicant. Data for the solvent control must not be pooled with those for the control/dilution water. Pooling the controls could bias endpoint calculations; the control/dilution water lacks an influence that could act on organisms in the other concentrations (i.e., the solvent).

Average specific growth rate (i.e., relative growth rate) and/or area under the curveFootnote 71 can also be calculated based on frond number data. Calculation of either of these two optional endpoints requires additional observations at intervals (e.g., Days 3 and 5) during the test (see Sections 4.5.5 and 5.4).

5.6 Interpretation of Results

For any test which uses a water source other than SIS medium, modified Steinberg medium or, where appropriate, modified APHA medium as the control/dilution water, particular attention should be given to a comparison of Lemna growth in the control/dilution water with that in the standard controls using test medium (SIS, Steinberg, or APHA). This comparison is necessary to determine whether the control/dilution water is phytotoxic. Any enhanced growth in test solutions, relative to that in the control solutions, must be reported and considered when interpreting the findings (see Sections 4.5.2 and 4.5.4).

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