Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 7

Section 5: Specific Procedures for Testing Chemicals

This section gives particular instructions for testing chemicals, in addition to the procedures 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 the concentration of the major ingredients, water solubility, vapour pressure, chemical stability, dissociation constants, n-octanol:water partition coefficient, and biodegradability. Data-sheets on safety aspects of the test substance(s) (e.g., Material Safety Data Sheets) should be consulted, if available. Where aqueous solubility is in doubt or problematic, acceptable procedures used previously for preparing aqueous solutions of the chemical(s) should be obtained and reported, and/or chemical solubility in test water should be determined experimentally.Footnote 70 Other available information such as structural formula, degree of purity, nature and percentage of significant impurities and additives, handling precautions, and estimates of toxicity to humans and/or aquatic organisms, should be obtained and recorded.Footnote 71 An acceptable analytical method for the chemical in water at concentrations intended for the test should also be known, together with data indicating the precision and accuracy of the analysis.

Chemical containers must be sealed and coded or labelled upon receipt to indicate at least the chemical name, supplier, and date received.

Storage conditions are to be dictated by the nature of the chemical, and often include temperature restrictions and the need for protection from light. Standard operating procedures for chemical handling and storage should be followed.

5.2 Preparing Test Solutions

Test solutions of the chemical should be prepared if possible, by adding aliquots of a stock solution made up in control/dilution water.Footnote 72 If deionized water, distilled water, or fresh water was used to make the stock solution, commercially-available dry ocean salts, reagent-grade salts, or hypersaline brine (HSB) at 90 ± 1 g/kg salinity should be added as necessary to adjust the salinity of each test solution to within the acceptable range (i.e., 30 ± 2 g/kg). For aqueous samples (e.g., chemical formulations in water), test solutions may also be prepared by adding appropriate quantities of commercially available dry ocean salts, reagent-grade salts, or HSB (or deionized water, if necessary) to the sample or each of the test solutions (see Sections 2.3.4 and 4.3.2). Alternatively, for strong solutions or large volumes, weighed (analytical balance) quantities of chemical may be added to control/dilution water to give the nominal strengths for testing. Nominal test concentrations must be prepared and reported in consideration of any salinity adjustments. A set of controls comprised solely of HSB and deionized water (i.e., HSB control) must be included in any test in which HSB is added to the sample or test solutions (see Section 4.1.1). Likewise, a set of controls comprised solely of commercially-available dry ocean salts or reagent-grade salts, and deionized water (i.e., salt controls) must be included in any test in which these salts are added to the sample or test solutions (see Section 4.1.1).

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 73 Organic solvents, emulsifiers, or dispersants should not be used to increase 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 same concentration of solubilizing agent as in the most concentrated solution of the test chemical. Such agents should be used sparingly, and should not exceed 0.1 mL/L in any test solution. If solvents are used, the preferred ones (USEPA, 1985; ASTM, 1990) are triethylene glycol and dimethyl formamide. Methanol, ethanol, and acetone could be used but are more volatile.

5.3 Control/Dilution Water

Control/dilution water may be reconstituted (artificial) seawater, the laboratory’s supply of natural “uncontaminated” seawater, or a sample of particular receiving water if there is special interest in a local situation. The choice of control/dilution water depends on the intent of the test (see Section 3.4).

Reconstituted seawater should be used if a high degree of standardization is required, such as for measuring toxicity of a chemical relative to values derived elsewhere for this chemical and others. The salinity of the control/dilution water used for such comparative tests should be common to all tests and used for all dilutions. This salinity should be within the range 28 to 32 g/kg. Additionally, the salinity of all test concentrations should be within 1 g/kg of the controls.

If the toxic effect of a chemical on a particular receiving water is to be assessed, sample(s) of the receiving water could be used as the control/dilution water by taking them from an area that was not contaminated by the chemical. Examples of such situations include appraisals of the toxic effect of chemical spills (real or potential) or intentional applications of a chemical (e.g., spraying of a pesticide) on a particular waterbody. If a sample of receiving water is to be used as control/dilution water, a separate control solution must be prepared using the control/dilution water that is normally used for the echinoid fertilization test and is able to achieve valid test results on a routine basis (see Section 4.5.1).

The laboratory supply of uncontaminated natural seawater, or reconstituted seawater, may also be used to appraise the toxic effect of a chemical on a particular receiving water, especially if there is already an interfering toxicity in the receiving water, or its collection and use is impractical (see Section 4.1.1). The laboratory seawater in which adults are held is also appropriate for use in other instances (e.g., preliminary or intra-laboratory assessment of chemical toxicity).

If information is desired on the influence of salinity on toxicity of the chemical under investigation, separate tests should be conducted concurrently at two or more salinities. However, it should be kept in mind that salinities outside the 28 to 32 g/kg range might in themselves affect success of fertilization.Footnote 74 Control/dilution water for such tests should be from a single source, either reconstituted seawater (Section 2.3.4) or natural seawater adjusted for salinity as necessary using hypersaline brine, dry salts, deionized water, distilled water, or an uncontaminated fresh water.

5.4 Test Observations and Measurements

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

During preparation, 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 aliquots of test solutions be analyzed to determine the concentrations of chemicals to which gametes are exposed, in at least the high, medium, and low test concentrations, and the control(s).Footnote 75

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 good reason to believe that the chemical measurements are not accurate. In making calculations, each test solution should be characterized by the geometric average of the measured concentration to which the gametes are exposed.

5.5 Test Endpoints and Calculations

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

If a solvent control is used, the test is rendered invalid if the fertilization success in this control is decreased significantly from that for the control using only water.

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