Intra-laboratory evaluation of Microbial Assay for Risk Assessment (MARA) for potential application in the implementation of the Water Framework Directive (WFD)

Abstract

The Microbial Assay for Risk Assessment (MARA) is an innovative system based on an array of 11 different microbial species freeze-dried in a 96-well micro-titre plate format. Developed for testing the toxicity of chemicals, mixtures and environmental samples, the assay employs species of a taxonomically diverse range. In addition to ten prokaryotic species, a eukaryote (yeast) is included in the range. The MARA's innate scope of a multi-dimensional test allows determination of toxicity based on a unique assay fingerprint or index, numerically expressed as the mean Microbial Toxic Concentration (MTC). The most significant potential of the test is in the additional inference that can be conveyed to the toxicity evaluation because of the presence of each of the constituent species. In view of the fact that conventional aquatic bioassays, like fish or cladoceran tests, are expensive and impractical, the MARA could provide a cost-effective solution for routine ecotoxicological testing. The performance of the MARA was evaluated to ascertain its capability and potential scope. Sensitivity to toxicants and different environmental samples was assessed. Evaluation included comparison with other tests: namely Microtox, invertebrate (Daphnia magna and Thamnocephalus platyurus) microbiotests, and respiration-inhibition and nitrification-inhibition tests. The most sensitive invertebrate test was found to be the T. platyurus microbiotest for three of the four metals tested. The LC(50) values for this test for Cd(ii), Cr(vi) and As(iii) were 0.2, 0.018 and 0.3 mg l(-1), respectively; and the corresponding most sensitive MARA species MTC values were 4.4, 2.8 and 17 mg l(-1), respectively.

Application of a battery of biotests for the determination of leachate toxicity to bacteria and invertebrates from sewage sludge-amended soil

“…Ten prokaryotic species and a eukaryote (yeast) constitute the biological indicators of toxicity assessment. In the present work, the microbial species used consisted of ten bacterial species: (1) Microbacterium sp., (2) Brevundimonas diminuta , (3) Citrobacter freundii , (4) Comamonas testosteroni , (5) Enterococcus casseliflavus , (6) Delftia acidovorans , (7) Kurthia gibsonii , (8) Staphylococcus warnerii , (9) Pseudomonas aurantiaca , and (10) Serratia rubidaea , and one yeast species, (11) Pichia anomala (Wadhia et al 2007). The growth of the organisms exposed to the test sample is determined with the reduction of tetrazolium red.…”

“…A scanned image of the microplate obtained using a flatbed scanner is analyzed using Purpose Built software. The MARA test was performed according to the standard protocol described by Wadhia et al (2007). …”

Ecotoxicological Characterization of Waste

“…It would also be prudent to consider the utilisation of other innovative developments such as the Microbial Assay for Risk Assessment (MARA) (Wadhia et al 2007;Blaise et al 2008) to facilitate the toxicity evaluation and classification of wastes.…”

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“…The strains have been selected for their wide diversity and sensitivity to chemicals. The growth of the bacteria is determined after 18‐h incubation at 30°C with the reduction of tetrazolium red dye (Wadhia et al, 2007). Eleven microbial toxic concentration (MTC) values were obtained giving a unique “toxic fingerprint” profile of the test sample.…”