Biological test method for toxicity tests using early life stages of rainbow trout: chapter 5

5.1 Test Options
5.2 Properties, Labelling, and Storage of Sample
5.3 Preparing and Aerating Test Solutions
5.4 Control/Dilution Water
5.5 Test Observations and Measurements
5.6 Test Endpoints and Calculations

This Section gives particular instructions for testing chemicals, in addition to the procedures in Section 4. A multiple-concentration test is usually performed, to determine the endpoints of the E, EA, or EAF test.

5.1 Test Options

Depending on objectives and regulatory requirements, a rainbow trout early life-stage test to evaluate the toxicity of chemical sample(s) may be undertaken using the embryo (E) test, the embryo/alevin (EA) test, or the embryo/alevin/fry (EAF) test (Sections 4.3.1 and 4.3.6). Assessments required under regulations for registering a pesticide or similar category of chemical, or for other regulatory assessments of chemicals, might be most suitably performed as an EAF test. The EAF test might also be used in research studies concerned with providing a definitive assessment of a chemical's toxicity toward rainbow trout. The EA test might be used for such purposes as comparative screening of several chemicals for relative toxicity to rainbow trout, while the E test might be used for frequent monitoring. Selection of the most suitable test will require consideration of the physicochemical characteristics, as well as the mode of toxic action, of the substance being tested.

At the time that an EA or EAF test is set up, it is recommended that a multi-concentration E test be established and run concurrently using the test chemical and fertilized eggs from the same pool of test organisms. The findings of the E test will provide insight into the fertilization success rate for controls in the EA or EAF test, and will be useful in appraising the relative sensitivity to the test substance for the acute (E) and longer (EA or EAF) test options.

Before any frequent or "routine" use of the E test for regulatory or other programs involving the screening of chemicals for toxicity, initial comparison of its sensitivity with that of the more definitive EAF test is recommended, to confirm that results will be sufficiently protective for the purpose intended.

5.2 Properties, Labelling, and Storage of Sample

Information should be obtained on the properties of the chemical to be tested, including water solubility, vapour pressure, chemical stability, dissociation constants, and biodegradability. Datasheets on safety aspects of the test substance should be consulted, if available. Where aqueous solubility is in doubt or problematic, acceptable procedures used previously for preparing aqueous solutions of the chemical should be obtained and reported. Other available information, such as structural formula, degree of purity, nature and percentage of significant impurities, presence and amounts of additives, and n-octanol:water partition coefficient, should be obtained and recorded.^Footnote 35 An acceptable analytical method should also be known for the chemical in water at concentrations intended for the test, together with data on precision and accuracy.

Chemical containers must be sealed and coded or labelled upon receipt (e.g., chemical name, supplier, date received). Storage conditions (e.g., temperature, protection from light) are frequently dictated by the nature of the chemical. Standard operating procedures should be followed for handling and storage of a chemical, or else those recommended by the manufacturer, by a Material Safety Data Sheet, or by similar advisory information.

5.3 Preparing and Aerating Test Solutions

Solutions of the chemical are usually prepared by adding aliquots of a stock solution made up in control/dilution water. 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. If stock solutions are used, the concentration and stability of the chemical in the solution should be determined before the test. 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 renewal of test solutions.

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. Ultrasonic dispersion can produce droplets that differ in size and uniformity, some of which might migrate towards the surface of the liquid, or vary in biological availability creating variations in toxicity. 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 following are preferred: dimethyl formamide, triethylene glycol, methanol, ethanol, and acetone (USEPA, 1985b).

Upon preparation of each test solution including the control(s), its dissolved oxygen content should be measured. Thereafter, the test organisms should be exposed to the solutions, or else each test solution should be pre-aerated (see Section 4.3.4). In most instances, the pre-aeration (before fish exposure) and aeration (during fish exposure) of chemical solutions is not necessary nor warranted, and should be avoided unless dissolved oxygen levels go outside the range 60% to 100% saturation at any time (see Section 3.3 including footnotes 6 to 8).

Any test performed without aeration should use a flow-through setup (see Sections 3.3 and 4.3.2, and Figure 3C), to enable a continuous circulation of test solutions around the developing embryos or alevins. If pre-aeration or aeration is appropriate (e.g., dissolved oxygen in one or more test solutions is <60% or >100% of air saturation), the guidance given in Section 4.3.4 should be followed.

5.4 Control/Dilution Water

Control/dilution water may be natural groundwater, surface water, reconstituted water, dechlorinated municipal water (as a last choice, if necessary; see Section 3.4), or a particular sample of receiving water if there is special interest in a local situation. The choice of control/dilution water depends on the intent of the test.^Footnote 36

If a high degree of standardization is required for comparative purposes, soft reconstituted water should be prepared and used for the control and all dilutions (hardness 40 to 48 mg/L as CaCO₃, pH 7.2 to 7.5)^Footnote 37 (USEPA, 1985b; EC, 1990b).

If the toxic effect of a chemical in a particular body of wateris to be appraised, sample(s) of the receiving water could be taken from a place that was isolated from influences of the chemical, and used as the control/dilution water.^Footnote 38 Examples of such situations include appraisals of the toxic effects of real or potential chemical spills or intentional applications of spray or pesticide on a particular waterbody. The laboratory supply of water may be used for this purpose, especially if the collection and use of receiving water is impractical. Normal laboratory water is also appropriate for preliminary or intralaboratory assessment of a chemical's toxicity.

5.5 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 during tests with chemicals.

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

It is desirable and recommended that test solutions be analyzed to determine the concentrations of chemicals to which embryos, alevins, and fry are exposed.^Footnote 39 If chemicals are to be measured, sample aliquots should be taken from all replicates in at least the high, medium, and low concentrations, and the control(s). Separate analyses of the aliquots should be performed, preferably on samples taken immediately before the start of the initial exposure, and at weekly intervals thereafter until the test is completed. On sampling days, separate aliquots should be taken from static-renewal tests at the beginning and end of the renewal periods; in flow-through tests, aliquots should be taken twice per day, at least six hours apart.

If chemical measurements indicate that concentrations declined by more than 20% during the test, the toxicity of the chemical should be re-evaluated by a test in which solutions are renewed more frequently, using either the static-renewal or flow-through mode. If there is rapid disappearance or decline of toxicant, it might be possible to use a high-volume flow-through test to maintain stable concentrations of chemical in solution (perhaps decreased, but stable) (McKim, 1985).

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 the calculations, each test solution should be characterized by the geometric average measured concentration. The intent of sampling intervals and averaging should be to obtain a realistic and time-balanced average concentration to which organisms were exposed.

5.6 Test Endpoints and Calculations

The endpoints for tests performed with chemicals will usually be the standard ones, i.e., the EC50 and EC25 for nonviability at various stages of development in the E, EA, and EAF tests, and additionally in EAF, the 30-day LC50 and 30-day IC25 for average attained weight of swim-up fry. Other narrative statements on delayed development, deformities, and behaviour are required in the longer tests, and additional (optional) observations can be detailed, as described in Section 4.5.

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 the solvent control (or the untreated control) does not meet the criteria for test validity specified in Section 4.6.

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

Footnotes

Footnote 35

Knowledge of the properties of the chemical will help to identify any special precautions or requirements for handling and testing it (e.g., a well ventilated facility, or the need for solvent). Information regarding chemical solubility and stability in fresh water will also be useful in interpreting results.

Return to footnote35 Referrer

Footnote 36

Volume requirements, based on the choice of test option or type (E, EA, or EAF, and static-renewal or flow-through), might also have a bearing on the choice of control/dilution water (see also Section 6.1 and its footnotes).

Return to footnote36 Referrer

Footnote 37

Because the pH, hardness, and other characteristics of the dilution water can markedly influence the toxicity of the test substance, use of a standard reconstituted water provides results that can be compared in a meaningful way with results for other chemicals and from other laboratories. It is desirable to test in reconstituted water, although that requires making up large volumes of water. In some laboratories, that might be feasible, at least for static-renewal tests. Soft, reconstituted water is recommended, and is prepared by adding the following quantities of reagent-grade salts to carbon-filtered, deionized water or glass-distilled water (ASTM, 1991b):

Salt	mg/L
Sodium bicarbonate (NaHCO₃)	48
Calcium sulphate (CaSO₄·2H₂O)	30
Magnesium sulphate (MgSO₄)	30
Potassium chloride (KCl)	2

The reconstituted water should be aged several days (USEPA, 1985b) and aerated intensely before use. It can be expected to have a total hardness of 40 to 48 mg/L and a pH of 7.4 ± 0.2.

Return to footnote37 Referrer

Footnote 38

Contaminants already in the receiving water might add toxicity to that of the chemical being tested. In such cases, uncontaminated dilution water (natural, reconstituted, or dechlorinated municipal) would give a more accurate estimate of the individual toxicity of the chemical of concern, but not necessarily of the total impact at the site.

If the intent of the test is to determine the effect of a specific chemical on a specific receiving water, it does not matter if that receiving water modifies sample toxicity by the presence of additional toxicants, or conversely by the presence of substances that reduce toxic effects, such as humic acids. In the case of toxicity added by the receiving water, it would be appropriate to include a second control using laboratory water as a minimum and, as a maximum, another series of concentrations using laboratory water as diluent.

Tests using receiving water for the control and dilution would require transport of large volumes of water to the laboratory. That might be reasonable for the E test, but it might not be feasible for the EA or EAF tests. If not, consideration should be given to conducting EA or EAF tests adjacent to the site of interest, by pumping upstream receiving water into a mobile testing facility.

A compromise would be to adjust the pH and hardness of the laboratory water supply, or reconstituted water, to that of the receiving water, using reagent-grade salts (ASTM, 1991b; USEPA, 1985b). Depending on the situation, the adjustment might be to seasonal means, or to values measured at a particular time.

Return to footnote38 Referrer

Footnote 39

Such analyses need not be undertaken in all instances, due to analytical limitations, cost, or previous technical data indicating chemical stability in solution under conditions similar to those in the test.

Chemical analyses are particularly advisable if: the test solutions are aerated; the test substance is volatile, insoluble, precipitates out of solution, or is known to sorb to the material(s) from which the test vessels are constructed; or if a flow-through system is used (USEPA, 1985b). Some situations (e.g., testing of pesticides for purposes of registration) might require the measurement of chemical concentrations in test solutions.

Return to footnote39 Referrer

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Date modified:: 2014-08-26

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