Assessing the toxic impact of chemicals using bacteria

Abstract: There is a considerable backlog in the testing of new chemical compounds for their ecotoxic properties, mostly due to the lack of appropriate testing methods. In addition, there is a vivid debate today about the test endpoint for many of the existing methods. This thesis describes the Microbial Assay for Risk Assessment, MARA, a new method for ecological risk assessment and toxicity testing. MARA is based on the simultaneous exposure of eleven microbial strains to a concentration gradient of the chemical compound to be tested. The eleven strains have been selected so that different strains exhibit different sensitivities to chemicals. A growth inhibition pattern - the toxic fingerprint - can be thus detected on the microplate in which the test is performed. The toxic fingerprint, rather than the eleven individual growth inhibitory concentrations, is the result from the test. It can be compared to toxic fingerprints from other tested chemicals in a database and thereby generate more information regarding the type of toxic effect and species specificity of the tested compound than a singlevalue result would. The toxic fingerprint for two groups of chemicals, disinfectants and chlorophenols, were compared. It was concluded that MARA can differentiate between different types of toxic effects and also between chemicals with similar molecular structure. MARA is comparable to other bacteria based tests both regarding sensitivity and reproducibility. The toxic fingerprint is heavily dependent on the selection of the strains on which the assay is based. They must give a diversified answer to large groups of chemicals, and as bacteria have a larger genetic diversity than higher organisms, it would plausibly be advantageous with a high genetic diversity among the strains. It was shown that the strains should preferably belong to different genera, but not necessarily to different phyla, to yield the most differentiated response to different chemicals. In order to make MARA as user-friendly as possible, a new method for detection of microbial growth/inhibition of growth in microplates using a flat-bed scanner was developed. The results obtained with the scanner were highly correlated to results obtained with a spectrophotometer, the classical device used for instant quantification of microbial growth, when using TTC (triphenyl tetrazolium chloride, tetrazolium red) or MTT (chelating tetrazole) as growth indicators.