Ecotoxicity test with Aliivibrio (Vibrio) fischeri (UNE 77104: 2014 Standard)

UNE 77104: 2014 establishes a test method to determine the ecotoxicity of a product, by analyzing the acute toxicity with bioluminescent bacteria of the species Aliivibrio (Vibrio) fischeri. This standard applies to all products that can be used in the marine environment to combat accidental pollution by hydrocarbons, including dispersants, absorbers and bioremediation agents. To carry out the toxicity test with absorbents, a previous leaching phase is necessary.

In this method the acute toxicity of a product is determined by studying the inhibition of the fluorescence of Aliivibrio (Vibrio) fischeri due to exposure to the test product for 30 minutes. For this, the initial bioluminescence of Aliivibrio (Vibrio) fischeri bacterial suspensions deposited in vials is measured, and then the different solutions of the product under evaluation or of the control solutions, as appropriate, are added to these vials. The vials are incubated at 15°C ± 1°C for 30 minutes. After the exposure period, the final bioluminescence of each vial is measured and the decrease in bioluminescence during the exposure is evaluated. To assess the toxicity, the mean effective concentration (EC50) is determined, i.e. the concentration of the evaluated product that reduces the luminescence by 50%. In this test, 3 vials with bacterial suspensions should be used for each concentration of product evaluated, and the same number of vials of a negative control, and of a toxic reference product. The solutions of the evaluated product must be prepared by successive dilutions from the stock solution, which according to the standard must be 10 concentrations.

The result of the bioassay of toxicity for dispersants and bioremediation agents is considered negative (non-ecotoxic) when the EC50 of the product at 30 minutes is higher than 100 mg/L, while otherwise it is considered positive (ecotoxic). The result of the toxicity bioassay for sorbents is considered negative (non-ecotoxic) when the EC50 of the leachate at 30 minutes is higher than 1,000 mg/L, while otherwise it is considered positive (ecotoxic).