Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC₅₀, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

We are researchers from University of Tor Vergata in the Italian Institute for Environmental Protection and Research in Rome. We develop tools to evaluate the impact of pollutants in the environment and we identify model organism as bioindicators. We are in the laboratory of ecotoxicology in the Department of Biology of Tor Vergata University.

In this video, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This video covers all the preparation and execution steps of the toxicity testing. In addition to the usual laboratory equipment, prepare the following materials.

1.5 milliliter tubes for serial dilution of bacterial suspensions. 90 millimeters diameter Petri dishes to plate bacteria. Minimum two plates for each test concentration are required including control.

Label the side of the Petri dishes with the test concentration and exposure time. Draw six section on the bottom of the Petri dishes and label with replicate number and dilution factor. 15 milliliters sterile tubes as test containers labeled with the test concentrations.

In this trial, we used 2%sodium chloride sterile solution as exposure medium. Sterilize synthetic or natural seawater can be also used at start concentration ranging from five to 40 gram per liter. Prepare a sufficient amount of tryptic soy agar growth medium with 2%sodium chloride.

Add the sodium chloride considering the amount already present in the medium to reach the 2%final concentration. Sterilize it, and once cool but still liquid, fill the Petri dishes previously labeled. Prepare tryptic soy broth to growth medium with sodium chloride.

Add the sodium chloride to obtain the same salinity of the exposure medium. Always considering the amount of sodium chloride already present in the medium. In this trial, we used tryptic soy broth with 2%sodium chloride.

Sterilize it and leave it to cool before using. In this trial, we use the copper sulfate as a reference toxicant. Prepare a stock solution with double distilled water and store it at four centigrade degrees.

Sterilize the necessary aliquot by using a 0.22 micron syringe filter. In case of environmental samples, prepare an appropriate interval of dilution of the sample. Use 15 milliliters tubes as test containers.

Fill the exposure tubes with exposure medium. Prepare five milliliters negative control. Prepare the exposure tubes with the appropriate amount of exposure medium and copper sulfate stock solution to obtain the testing concentration in a final volume of five milliliters.

This test requires a fresh culture of Vibrio anguillarum. Using a sterile loop, select a single well isolated colony from an agar plate. Inoculate it with that bacterium in the previous days.

Holding the tube at an angle, wrap the loop with the inoculum on the tube surface. Fill it with 10 milliliters of tryptic soy broth. Incubate the bacterial culture at 25 degrees C for 12, 18 hours.

The bacterial concentration of the inoculum is estimated spectrophotometrically. Vortex the inoculum and measure the optical density at 600 nanometers wavelength. Use tryptic soy broth as blank.

To obtain a known bacterial concentration, dilute two milliliters of the vortexed inoculum by adding the amount of tryptic soy broth calculated by this formula. Verify that the optical density of the diluted inoculum is 14 which correspond to the 0.5 point of the McFarland nephelometric standard. Centrifuge the diluted inoculum for 10 minutes at 3, 000 g.

Eliminate supernatant and resuspend the microbial palette in one milliliter of 2%sodium chloride solution. Add 150 microliters of the resuspended bacterial inoculum to each tube. Expose Vibrio anguillarum suspension for six hours up to 25 degrees C under darkness and in continuous shaking to avoid sedimentation.

At the beginning and the end of exposure after six hours, carry out the bacteria counting in all the exposed bacterial suspension by using the colony forming unit counting method. Prepare sterile dilution of the bacterial suspension applying a 10-fold dilution factor up to 10 to minus five. For each bacterial suspension, prepare three replicates of a five-tube set.

Label the tubes with dilution factor and replicate number and fill all the tubes with 900 microliters of 2%sodium chloride solution. Add 100 microliters of the bacterial suspension to corresponding tubes. Proceed with the serial dilution, vortexing at each step.

Plate 10 microliters of the last two dilution onto tryptic soy agar Petri dishes in the corresponding segment and quickly let the drop glide in a small circle by rotating the plate. Then, incubate the plates at 25 degrees C for 48 hours. After 48 hours, count the colonies grown on the Petri dishes.

Plates harboring between five and 50 colonies are optimum for accurate counting. The number of viable bacteria per milliliter in each testing solution can be calculated and the mortality evaluated and compared with the control. For calculation of the colony forming units per milliliter, multiply the average of colonies obtained in the parallel replicates by the known dilution factor N by 100.

The mortality as percentage is obtained by the following formula. Results of the three independent tests show of those dependent mortality. The LC 50, that is the lethal concentration that kills the 50%of bacterial colonies exposed to toxicant is calculated by a known linear regression analysis logit L model using the Benchmark Dose Software.

LC50 of the three independent tests highlight the feasibility and reproducibility of this method. In conclusion, we have described an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This microorganism was chosen because it is halotolerant, ubiquitous, and representative of marine ecosystems.

The new test was successfully applied to assess the toxic effect of copper sulfate with a clear dose response relationship. This protocol if compared to the existing methods with other microorganism can be applied in a wider range of pH and salinity without limitation for samples. So it can be performed on environmental samples too.

Furthermore, this bioassay assess the mortality instead of growth rate or enzymatic activity inhibition. This facilitates the comparison with other acute ecotoxicity test for marine environment which have mortality as endpoint. The test has been designed to evaluate the ecotoxicity of emerging contaminants as nanoparticle and work is in progress on this issue.