Acute toxicity was determined through commercially developed system mark Microtox© (AZUR Environmental, USA). This system uses Vibrio Vischeri as target bacterium. The test measures the decay on bacteria photoluminescence after 15 min of sample exposure. To facilitate the toxicity assessment, results are expressed as 1/EC50. Samples were kept in a fridge 24 h at 0 °C, before toxicity measurement.