Microtox Toxicity Testing using bioluminescent bacteria
What is microtox toxicity testing
Microtox is an in vitro testing system which utilises the bioluminescent bacteria Aliivibrio fischeri to detect toxic substances in different substrates.
Aliivibrio fischeri are non-pathogenic, marine, bacteria that luminesce as a natural part of their metabolism. When exposed to a toxic substance, the respiratory process of the bacteria is disrupted, reducing light output. Aliivibrio fischeri have demonstrated high sensitivity across a wide variety of toxic substances. Response to toxicity is observed as a change in luminescence, which is a by-product of cellular respiration.
This change can be used to calculate a percent inhibition of Aliivibrio fischeri that directly correlates to toxicity ALS has a range of tools to assess whether chemicals, effluents or wastewaters discharged into water will be toxic to aquatic biota. These tools include sensitive chemical analyses and bioassay based toxicity test- Microtox.
The principle behind microtox
The Microtox toxicity test exposes Aliivibrio fischeri to a sample for analysis. Aliivibrio fischeri has the property of emitting part of the energy released in the metabolic reaction (ascorbic acid cycle) as light. Any disruption in this metabolic reaction as a result of the presence of toxic substances will result in a change in the amount of light emitted. The inhibition of light emission correlates with the degree of toxicity.
The reduction in light can be quantitatively determined using a luminescence meter (Microtox Model 500 Analyser). The toxicity of a sample is expressed as the concentration at which 50% reduction in luminescence occurs relative to the blank (EC50).
Microtox is an in vitro test system based on changes in light production of the marine bioluminescent bacterium Aliivibrio fischeri.
Toxicity reduces enzyme activity of this bacteria and thus reduces light output. The Microtox photometer, a temperature controlled analyser, measures the light levels before and after addition of the sample. The reduction in intensity of light emitted from the bacteria is measured along with standard solutions and control samples. The change in light output and concentration of the toxicant produces a dose/response relationship. EC50 – effective concentration at which a 50 % reduction of the light emission was observed after a certain exposure time, is calculated based on dose response curve.
Different chemicals affect living organisms at different rates, reflecting modifications in the mechanism of action and producing distinctly different Light Level-Time Response curves. For some chemicals (like organic compounds) the effect on light output is complete in 5 minutes whereas decay rate of light is slower with heavy metals compounds. Due to different response curves, multiple light readings (5, 15 & 30 minutes) are taken for sample analysis. Different dilutions of sample are tested to determine EC50 values.
This test approach is rapid, sensitive, uses small sample size and has large sample throughput capabilities. Statistically, the Microtox system gives better results because the response of a larger number of organisms, about one million bacteria is observed.
This in vitro bioassay provides an alternate to traditional & complex methods and detects unknown pollutants and effects of mixtures (synergistic effects).
Microtox toxicity sampling requirements
|Holding Time||48 hrs for unrefrigerated samples; one week for samples stored at 3°C ± 2°C|
|Turnaround time||Standard: 5 days (<24hrs available if required)|
|Sample volume||100mL amber bottle|
|Sample shipping and storage||Transport sample on ice or in a refrigeration unit.|
For further information or to order sample containers please contact your local ALS client services team.
Water industry applications
Microtox can be applied to a variety of matrices including drinking water, storm water run-off, effluents, aqueous extracts from sediments and industrial discharges.
A sample pre-treatment step also allows the toxicity analysis of turbid and coloured samples.
A number of water industry applications are:
- Wastewater treatment plants effluent testing for protection of receiving water and influent testing for protection of activated sludge;
- Toxicity Reduction Evaluations (TREs) and Toxicity Identification Evaluations (TIEs);
- Surface water monitoring for identification of point source and non-point source pollution;
- Monitoring of remediation process;
- Biocide monitoring of industrial processed waters;
- Drinking water surveillance for regular monitoring at strategic points.