Aliivibrio fischeri Toxicity Tests (formerly Vibrio Fischeri)

Toxicity of environmental samples is an important aspect of pollution monitoring and risk assessment. Commonly employed chemical parameters for analysis are accurate and sensitive for specific molecules under investigation, but give no information regarding biological effects within the ecosystem. These methods also ignore synergistic effects from compound mixtures and can underestimate toxic potential of a sample. Evaluation of biological effects using rapid, simple and cost effective methods can provide information on whole sample effects and incorporate these important toxicity parameters into regulatory frameworks. 

One important method for assessing toxicity quickly and cost effectively is a bioluminescence inhibition assay that employs a marine gram negative bacterium, Aliivibrio fischeri (formerly Vibrio fischeri) as the test species. Light production is directly proportional to the metabolic activity of the bacterial population, and any inhibition of enzymatic activity due to toxicity or cell death causes a corresponding decrease in the bioluminescence produced by the colony. Test samples are exposed to rehydrated bacteria for a pre-determined period and the degree of light emission inhibition is compared to a negative control. In this way, the assay provides a measure of sub-lethal response as well as lethality through the degree of inhibition. 

EBPI is currently carrying several preparations of high quality Aliivibrio fischeri test reagents from Finnish manufacture Aboatox to conduct rapid toxicity assessments in a wide range of environmental samples. The BioTox™ line of reagents and kits are designed to function independently and include all necessary components. The reagents and diluents can also be combined with other commercially available detector systems to produce results. Several different methodologies are available to fit project budget or laboratory setup, and can be used for analyzing various sample sizes, both clean and turbid water, and rapid determination of single tests. The reagents are standardized for use with 

ISO 11348-3: Water quality – Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (luminescent bacteria test).

ISO 21338:2010 Water quality — Kinetic determination of the inhibitory effects of sediment, other solids and coloured samples on the light emission of Vibrio fischeri (kinetic luminescent bacteria test)

The BioTox™ reagents perform comparably to several similar commercial test systems (Microtox, LumiStox, ToxAlert) when conducted according to the ISO standards mentioned above. The test organisms are extremely sensitive to organic contaminants and show excellent correlation with toxicity assessments performed with other aquatic species like the fathead minnow and Daphnia magna. 

The following BioTox™ products are available for purchase according to various methodology:

 

1243-1000 BioTox WaterTox Standard Kit

The WaterTox STD (Standard) kit is packaged to provide users with a complete setup to replace traditional vibrio fischeri acute toxicity tests (Microtox® users). All required reagents and solutions are included with the kit to permit facile adoption using current detectors like the M500.

 

1243-500 BioTox WaterTox EVO Kit

The WaterTox EVO (Evolution) is packaged as a cost effective alternative to the 1243-1000 Water Toxicity Kit. Bacterial reagents are packaged to permit less reagent waste for smaller sample numbers. The operation protocol has also been simplified to reduce pipetting steps and a salt tablet is included instead of OAS solution and sample diluent to reduce costs in shipping. Results can be interpreted by any approved ISO luminometer. 

 

1243-700 BioTox LumoPlate Ultimate Matrix Kit

This kit is provides complete solutions for clients requiring a more accurate and robust assay system that employs the kinetic method for determining toxicity. This method accounts for sample coloration and turbidity and can be used for direct measurement of soil and sediment samples without time consuming correction procedures or filtration. Bacterial reagents are packaged to accommodate 1 entire 96-well plate. Cooling block and luminometer manufactured by Labrox is also offered to provide a complete set up. Individual replacement kit components can be purchased and reagent lease options are available for qualified clients.

 

1243-70 BioTox LumoStix Instant Kit

This kit includes reagent, diluent and LumoStix™ devices to conduct 20 individual measurements. Bacteria are packaged to permit 4 determinations per vial and the LumoStix™ devices are manufactures for use in the portable Kikkoman PD30 luminometer. Replacement kit components can be bought individually to meet testing requirements.

Traditional Method

This method is based on ISO 11348-3: Water quality – Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (luminescent bacteria test). The assay works very well for clear water samples and is the most widely used method internationally. Toxicity is measured by preparing a dilution series and determining differences in light output after incubations of 5 to 30 minutes using a luminometer. EC50’s are calculated based on the amount of inhibition for different concentrations manually or by using provided computer software programs. Any approved luminometer in the ISO standard can be employed to read the light emission which can save on cost for the system and portable models can take assessments from the lab into the field.  

The main advantages to this method are:

• Widely used and popular. Many laboratories have the technology to conduct the assay already
• Results can be acquired rapidly (5-30 minutes) according to published international standards 
• Luminometer can be benchtop or portable model.
• Low overall cost per sample compared to other bioassays

Kinetic Method

This method, originally designed for solid samples is an improved method for determining toxicity data using luminescent bacteria according to ISO 21338:2010. Colour and turbidity of the sample are accounted and matrix effects can be largely eliminated. The method employs an automated luminometer capable of dispensing reagent and measuring luminescence at the same time and a microplate which permits up to 96 sample wells. Signal measurement immediately prior to reagent addition as well as continuously after reagent addition permits a fluid evaluation of changes in luminescence that automatically corrects for background effects. Initial signal intensity is compared to the signal intensity after the incubation period using a kinetic curve. Most compounds of interest will produce a toxic effect immediately after contact with the bacteria is initiated results can be seen immediately. Depending on sample composition, the software can be employed to correct for interferences automatically in the calculation. Contact times can also be varied to observe effects of some chemicals that can only be seen after longer contact times.

This method contains several benefits including

• Reduced operator time as toxicity results are gathered immediately over short incubations times
• Greatly reduced analysis costs per sample
• High throughput capabilities as 96-well plates are used in the analysis
• Small sample volumes required for 96-well plates
• Sample dilution made directly to the 96 well plates; saves lots of waste associated in the test
• Colour turbidity and matrix effects are corrected for automatically using software
• Cost of system purchase and setup is comparable to Microtox® assay 
• Automated result acquisition and EC50 calculations improve accuracy and reduce operator error.  
• Any sample type can be analyzed in the same way without method adjustment or extra procedural steps. 
• Between 10 to 20 samples/hour can be analyzed depending on the contact time used.

Rapid Screening Method

This method employs a novel, patent pending sampling and test device called the LumoStix™. Swabs at each end of the device provide both a sampling and test location. A single sample is taken directly off a surface using the swab. The prepared BioTox™ reagent is measured into one end of the device and the initial luminescence is measured. The device is then flipped and the reagent moves through the device into the end containing the sample swab. After an appropriate incubation time the luminescence is again measured to determine toxicity of the sample from direct contact. This assay is extremely easy to use and can be implemented for individual samples. The method is also extremely useful for taking measurements directly off solid surfaces and can detect residues from cleaning products as well as chemical contamination in environmental samples. Several benefits to this method include

• True field measurements of toxicity
• Quick screening applications for environmental monitoring and regulatory compliance
• Single samples can be tested, minimal reagent waste
• Cooling block not required
• Low cost per sample