Toxicity test in which the water is renewed constantly in the test chambers, the chemical under test being transported with the water used to renew the test medium.
1.6.1. Reagents
1.6.1.1. Solutions of test substances
Stock solutions of the required strength are prepared by dissolving the substance in deionised water or water according to 1.6.1.2.
The chosen test concentrations are prepared by dilution of the stock solution. If high concentrations are tested, the substance may be dissolved in the dilution water directly.
The substances should normally only be tested up to the limit of solubility. For some substances (e.g. substances having low solubility in water, or high P
ow
, or those forming stable dispersion rather than true solution in water), it is acceptable to run a test concentration above the solubility limit of the substance to ensure that the maximum soluble/stable concentration has been obtained. It is important, however, that this concentration will not otherwise disturb the test system (e.g. film of the substance on the water surface preventing the oxygenation of the water, etc.).
Ultrasonic dispersion, organic solvents, emulsifiers or dispersants may be used as an aid to prepare stock solutions of substances with low aqueous solubility or to help to disperse these substances in the test medium. When such auxiliary substances are used, all test concentrations should contain the same amount of auxiliary substance, and additional control fish should be exposed to the same concentration of the auxiliary substance as that used in the test series. The concentration of such auxiliaries should be minimised, but in no case should exceed 100 mg per litre in the test medium.
The test should be carried out without adjustment of the pH. If there is evidence of marked change in the pH, it is advised that the test should be repeated with pH adjustment and the results reported. In that case, the pH value of the stock solution should be adjusted to the pH value of the dilution water unless there are specific reasons not to do so. HCl and NaOH are preferred for this purpose. This pH adjustment should be made in such a way that the concentration of test substance in the stock solution is not changed to any significant extent. Should any chemical reaction or physical precipitation of the test compound be caused by the adjustment, this should be reported.
1.6.1.2. Holding and dilution water
Orinking-water supply (uncontaminated by potentially harmful concentrations of chlorine, heavy metals or other substances), good-quality natural water or reconstituted water (See Appendix 1) may be used. Waters with a total hardness of between 10 and 250 mg per litre (as CaCO
3
) and with a pH from 6,0 to 8,5 are preferred.
1.6.2. Apparatus
All apparatus must be made of chemically inert material:
— automatic dilution system (for flow-through test),
— oxygen meter,
— equipment for determination of hardness of water,
— adequate apparatus for temperature control,
— pH meter.
1.6.3. Test fish
The fish should be in good health and free from any apparent malformation.
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