Optimisation of the Solid-Contact Test with Arthrobacter globiformis (7 pp)

Background, Aim and Scope   A number of biotests are available for the characterisation of solid matters such as soil or sediment. Among these, bacterial biotests using single test species often analyse the toxicity of water-soluble contaminants in aquatic extracts, but there is also a need for a fast and inexpensive bacterial solid-contact test. In this study, a solid contact test with added bacteria (Arthrobacter globiformis) was optimised (through miniaturization) for the development of a test kit with conserved bacteria. As in other tests, the results can be influenced by natural soil factors, often masking anthropogenic impacts. For this reason, a further goal of this study was the investigation of the influence of natural soil characteristics on the result of the solid contact test. The project is part of the joint research project 'Optimization of ecotoxicological test methods for routine use' (abbreviated as ERNTE-Forschungsvorhaben: 0330305). Materials and Methods: This method is based on an existing German standard (DIN 38412 L 48) using Arthrobacter globiformi for testing whole soils and sediments. The test principle is the measurement of the dehydrogenase activity of the test organism A. globiformis after an incubation time of two hours with the solid material. To attain the miniaturization in microplates, dye measurement was changed from spectrophotometrical determination of the substrate resazurine to the fluorimetric measurement of the product resorufin. A second step towards optimisation was the use of freeze-dried bacteria. Seven selected uncontaminated soils were tested in order to determine the influence of natural soil characteristics on the results of the solid contact test with A. globiformis. Freshly spiked and polluted field soils were analysed in order to obtain information about the sensitivity of the test. Results: It is possible to perform the contact test in microplates. The fluorimetric dye measurement can be carried out in the presence of the solid material, so the work-intensive step of centrifugation and filtration is no longer necessary. The measurement in the optimised contact test is based on the kinetics of the enzyme reaction. The investigation showed that conserved bacteria have the same activity and sensitivity as cultivated bacteria. Discussion: The study of the uncontaminated soils demonstrated the influence of various soil characteristics on the results of the solid contact test. This information is the basis for the selection of the control and reference soils and is crucial for setting the threshold value in toxicity testing. The investigation of freshly spiked and contaminated soils showed a different sensitivity dependent on the kind of the contamination. Conclusions: The solid contact test was successfully optimised using microplates, whereas now less than six hours are necessary for the analysis. The optimised test is rapid and sensitive, requiring small samples and no stock culture of the bacteria A. globiformis if using freeze-dried bacteria. In this study, the effect of natural soil factors such as pH-value was shown. This information is used to define the threshold value for toxicity. Therefore, the optimised contact test can be used for an efficient assessment of soil or soil substrates. Further studies will clear up if this optimisation is also valid for aquatic sediments and waste. Recommendations and Perspectives: Due to its short analysis time, the test is suitable for screening different kinds of solid matter and can be used for on-site analysis. - The optimised contact test with freeze-dried bacteria as part of a battery of tests is appropriate for the assessment of contaminated soils, sediments and waste.