• BioTox luminometric toxicity tests
• Introduction
• Sinilevämyrkkyjen pikatesti

• BioTox flash test
• Toxkit microbiotests
• Mercury kit + Arsenic kit / Biological heavy metal assay kits

Bioluminescent Vibrio fischeri has been widely used for measuring the acute toxicity of water samples and chemical substances. The test is standardised (EN ISO 11348) and there is several commercial test systems available (Microtox, BioTox, LumiStox, ToxAlert etc.). The sample is incubated in contact with the bacteria for 15 or 30 minutes and the luminescence intensity after the incubation is compared to the luminescence intensity of pure bacteria. Reduction in light production is regarded as toxicity. The method is rapid and it is known to be especially sensitive for organic contaminants. The main disadvantage is that colour and turbidity present in most of the natural samples scatter the light and are being seen as toxicity. The methods for correcting the effects of colour and turbidity are time consuming, inconvenient and somewhat unreliable.

The Flash test is an improved BioTox^TM test, originally designed for solid samples. The method takes account the colour and turbidity of the sample during the whole measurement. The measurement is accomplished in kinetic mode with automated luminometer capable of dispensing and measuring at the same time. In the Flash method the V. fischeri bacteria are dispensed into the sample and the signal is recorded continuously for 30 seconds. The maximum signal received immediately after dispensing (Ipeak) is compared to the signal after 30 seconds incubation period (I30s). With most of the chemicals, especially organic ones, the toxic effect (reduction in light production) can be seen already after few seconds of incubation. However, the effect of some chemicals can be seen only after longer contact times. Thus, kinetic data from samples after 15 or 30 min gives an additional dimension for obtaining reliable results.

The features described above makes the colour/turbidity correction methods unnecessary and facilitates the toxicity screening of majority of the samples within few seconds. The sensitivity of the Flash test with 30 s contact time is slightly reduced when compared to the standard method using 15 or 30 minutes contact times. When same contact times are used, the sensitivities of standard and Flash-methods are of same order of magnitude. The coefficient of variation is excellent being normally below 1%. Sample volume needed for one test is less than 1 ml and the capacity of the method is 40 to 80 samples/hour depending on the contact time used. NEW! Software and calculation for a PLATE READER LUMINOMETER!

Sirius luminometer (Berthold D.S.) for Flash measurement.	Kinetic data obtained from non-toxic and toxic samples.

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The Toxkit microbiotests (Microbiotests. Inc)

Toxkits is the generic name of the new generation of simple, practical and low cost microbiotests, as alternatives to conventional bioassays, the performance of which is restricted to specialized laboratories. Toxkits are developed by the Laboratory for Biological Research in Aquatic Pollution (renamed in 1999 "Laboratory for Environmental Toxicology and Aquatic Ecology" = LETAE) at the University of Ghent in Belgium.

The major characteristic (and assett) of these new microbiotests, besides their "miniaturization" in small kits, is their total independence of culturing or maintenance of test biota. The principle Toxkits microbiotests, is the use of dormant or immobilized stages of selected test species, as the starting biological material from which the test organisms can easily be hatched or reactivated at the time of performance of the bioassays.

Several Toxkit microbiotests make use of the same species as the conventional toxicity tests prescribed by environmental legislations at national and international level; the Toxkit testing procedures have been structured such that they comply with the test methodologies of regulatory bioassays. Besides the "resting" biological material, Toxkits contain all the components (test containers, micropipettes, solutions) for performance of several bioassays. All Toxkit microbiotests require but modest laboratory equipement for their implementation.

A whole range of Toxkit microbiotests, with test species belonging to diverse phylogenetic groups (micro-algae, protozoans, rotifers, crustaceans) are now commercially available and used in an increasing number of laboratories worldwide. In Finland the Toxkit microbiotests are sold by Aboatox Oy.

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See also:
Microbiotests Inc. www.microbiotests.be

Sinilevämyrkkyjen (mikrokystiinit) pikatesti
Virkistyskäyttöön tarkoitetut vesistöt

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Aboatox Biotox heavy Metal Assay Kits

Luminescence-based biosensors for the detection of heavy metals:

Aboatox has commercialised genetically modified organisms capable of sensing heavy metals in environmental samples. These bacterial strains give a totally new meaning for the word bioavailability, because now it is possible to measure toxic compounds in sub toxic concentrations. The test is very rapid compared to standard methods and it is possible to test hundreds of samples daily. There are tests available for Mercury and Arsenic at the moment, and more to come in the future. The method can be used with a simple tube luminometer or with a plate reader.

Introduction:

• Whole cell biosensors detect the bioavailable fraction of the heavy metal from different samples; water samples, leachate samples, sediment and soil samples
• Bioavailability means the concentration of a metal available for biological systems
• Important factor in determination of metal toxicity
• Not equal to solubility
• Difficult to measure with traditional chemical methods, but easily measured by using biosensors
• Extremely sensitive method, much more sensitive than traditional toxicity tests

Measurement principle:

A change in the environmental conditions of the bacteria
  --> Metabolic response
    --> Measurable signal
      --> Measure (luminescence)
        -->

1. No heavy metal: luciferase synthesis is repressed, and bacteria emit low level of light
2. Heavy metal present: luciferase synthesis is activated, and bacteria emit high level of light

Advantages:

• Luminescence can be quantified very sensitively by a simple luminometer within a wide dynamic range
• Luminescence can be measured in vivo from intact cells
• Normal advantages of the microbial biosensor

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Measurement protocol:

• Pipette sample suspension into the cuvette or micro titer plate
• Add sensor bacteria suspension
• Incubate 2 h at 37 °C
• Add substrate D-luciferin
• Incubate 30 min
• Measure

Conclusions:

• Simple to perform: the sensor organism works well in kit format (lyophilized)
• High signal vs. background -> no need for expensive luminometers
• Portable luminometer, tube luminometer, plate luminometer and scintillation counter
• Can be used as a screening tool with big amount of samples
• Very cost effective

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