Ecotoxicity - Freshwater fishes, Brachidario rerio: Acute toxicity test (EN ISO 7346-1: 1996 standard or OECD 203: 2019 guideline). Static method.

This test can be performed in accordance with the specifications of ISO 7346-1 standard or OECD 203 guideline 203. Both guidelines assess the toxic effect of a substance on fishes. For this, specimens of the species Brachidario rerio (zebra fish) are used, which are exposed to different concentrations of the test substance for 96 hours, recording mortality at 24, 48, 72 and 96 hours. In addition, during exposure, dissolved oxygen, pH, salinity, hardness, and water temperature should be measured daily.

In accordance with the ISO 7346-1 standard, the test is carried out in two phases: 1) a preliminary test, to determine the range of concentrations to be used later in the final test, using a geometric series of 5 concentrations (for example 1,000 mg /L, 100 mg/L, 10 mg/L, 1 mg/L and 0.1 mg/L) and three fish per test concentration, and 2) a definitive test, which is performed by exposing 7 fish to a minimum of five concentrations in geometric series, preferably with a factor not exceeding 2.2 (preferably 1.6 or 1.8). With the results of the test, the lethal dose 50, the concentration that causes the mortality of 50% of the test individuals, is calculated (LD50: Lethal dose 50%). To do this, the lowest concentration that causes 100% mortality and the highest non-lethal concentration determined in the preliminary test must be included among the test concentrations. The tests also include a positive and negative control.

The OECD 203 guideline establishes that, if the client has prior information on the toxicity of the substance and knows the range of concentrations in which the LD50 is found, they can directly carry out the definitive test by selecting the 5 test concentrations, since it is not mandatory to determine the maximum lethal concentration and the minimum non-lethal concentration in a preliminary test. However, if the toxicity of the test substance and the range of concentrations to be selected are unknown, you can request to perform the preliminary test.

Both guides also include the possibility of carrying out a limit test, for substances that are not expected to show toxicity, which must be carried out by exposing 10 fishes to a single test concentration (100 mg/L or the solubility limit of the test substance in test conditions) and to the test controls.

To request the test, the client must indicate: the guide to be followed, the test to be selected (definitive, preliminary and definitive test or limit test), the test concentration or concentrations (expressed in mg of product/L), the nature and water solubility of the test substance. The substances to be tested are dissolved in the dilution water. The use of solvents other than water should be avoided. In the event that other solvents must be used, a control of the toxicity of the solvent must be included.