The aim of this study was a critical review of the recent methodology applied to evaluate the eco-toxicological state of marine waters and sediments by the use of a biological assay based on the bioluminescence emission by the marine bacterium Vibrio fischeri. In Italy, one of the most used standardized tests is represented by the Microtox® system. The test is based on the evaluation of bioluminescence produced by the bacterium V. fischeri, considering that the luminescence produced is a metabolic expression of the “healthy state” of the bacterium.
The main effect of a toxic substance is the diminution of luminescence produced by V. fischeri, the light emission capacity of the bacteria decrease compared to the toxicity of the chemical compounds.
Freeze-dry bacteria were hydrated and this preparation was in contact with different dilutions of the analyzed sample at a temperature of 15° C. The measurement was compared to a reference blank using a thermostatic luminometer.
The bioluminescence values, recorded at the zero time, after 5, 15, 30 minutes, was used to calculate the loss of bioluminescence related the toxic sample capacity.
Although the system was calibrated on the biological tests of freshwaters, a review of the available literature underlies that in marine water some discordances are present, particularly concerning the use of different diluents that could interfere on the osmotic balance of the bacteria, determining variable results.
In this study, on the basis of the available literature, different tests with the following different diluents were performed:
1) standard diluent for freshwater tests 2) natural marine water, 3) EPA synthetic marine water, 4) ASTM synthetic marine water, 5) phosphate buffer 0.1 M in NaCl (20 g/L) 6) NaCl (35 g/L)
Using each diluents, the trials were performed in replicates (3 times):
1) Evaluation of luminescence emission without the toxic substance and the evaluation of pH and salinity stability during the assay (30 minutes)
2) Three reference toxicants were used: phenol (organic contaminant), copper sulfate and zinc sulfate (inorganic contaminant). Each toxicant was used with each diluent and the EC50 values were calculated.
The results show that the lack of specific standards of reference and intercalibration experiments between laboratories on a wide range of toxic reference, induce to think that it is more appropriate to calculate EC50 values in a 30’ incubation test that showed the higher sensitivity in the trials performed with with ZnSO4 and CuSO4. Experiments performed with the reference toxicants show that the natural seawater and synthetic waters EPA and ASTM appear to be the most appropriate for performing the test. However it could be better to use synthetic sea water instead of the natural seawater, in order to avoid various parameters that could affect the stability of the assay: the location of sampling, other different variables such as salinity, nutrient content, different levels of contamination that should be analyzed every time. In this regard, the EPA water seems to have shown the best features also to the experiments on the stability of the light emission test.