1. MARA A Microbial Array for Ecotoxicity Testing

2. Contents This presentation covers 3 areas: The drivers behind ecotoxicity testing Perspective of tests –Conventional –Micro-scale tests MARA –Features –Protocol –Capabilities

3. Legislation Aquatic environment –EC Directives Specific uses e.g. Freshwater Fish Directive Industry sectors e.g. TiO2 Directive Substances e.g. Dangerous substances Directive Products e.g. Detergents Directive Water Framework Directive (SWIFT – WFD) –Direct Toxicity Assessment / Integrated Pollution Prevention and Control

4. Legislation Terrestrial environment Ecological Risk Assessment (ERA) –Contaminated Land Regulations Landfill –Landfill (Directive) Regulations.

5. Ecotoxicity Testing Requires a measure of direct toxicity, taking into account: Whole sample toxicity Interactive effects Unknown chemicals

6. Micro-Scale Ecotoxicity Tests The acceptability of micro- scale ecotoxicity tests is well established, with a wide range available commercially. These include: Algae Invertebrates Higher Plants

7. Important Criteria for Micro-Scale Ecotoxicity Tests simplicity speed reproducibility precision sensitivity Accuracy standardisation cost effectiveness convenience information

8. Microbial Array Benefits of using micro-organisms –Rapid growth –Small size –Large number tested –No ethical issues –Diversity

9. What is MARA? MARA (Microbial Assay for Risk Assessment) The MARA product is a multi-species toxicity test based on an array of 11 micro-organisms that –is simple to use –provides a battery of tests within a test –produces data for 11 test results –can be used to produce toxic fingerprints of –chemicals or environmental samples –requires small sample volumes –is particularly suitable for use in environmental toxicity testing

10. MARA Species The MARA test includes genetically diverse organisms from the following groups: Prokaryotes Gram +ve Gram –ve –α-proteobacteria –β-proteobacteria –γ-proteobacteria Eukaryote yeast

11. Microbial Toxic Concentration Microbial Toxic Concentration (MTC) is a measurement developed for MARA which is used to express growth inhibition and is equivalent to an EC50 value. The benefits of this are: –Reduces complexity and simplifies interpretation –Knowledge of the shape of the growth curves is not required –Calculation uses area above and below total curve –Eliminates interpolation errors of classic EC50 determination –Sensitive, repeatable and reproducible

12. Protocol

13. Medium& Dye Freeze-dried micro-organisms Sample, Medium &Dye 1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH ABCDEFGH -Ve Control Test Control Inoculate from H, A to G Discard Reconstitute Serial Dilution Decreasing Sample Conc.

14. Microbial Fingerprint (effluent) A microbial fingerprint can be derived from the analysis

15. MARA Software – Image Screen The plate image is copied into the image analysis software

16. MARA Software – Data Screen

17. Species Response When the data generated by the software is plotted, each of the species shows a different response to the toxicant.

18. MARA Evaluation Summary Excellent repeatability Highly reproducible Sensitivity is comparable to other tests on the market Works well with organics Can indicate mode of action of chemicals Has been tested using confounding variables

19. Application of MARA MARA is capable of determining the toxicity of a wide range of environmental samples, including –Effluents –Waters Raw Final –Soils –Landfill leachates –Sewage sludges MARA also has significant application in the chemical and pharmaceutical industries, process monitoring and QC testing

20. Tests Comparison MARA has also been compared with the following established tests Fish Algal Invertebrate Microtox Respiration inhibition Nitrification inhibition

21. Benefits of MARA Multi-species Simple protocol Observations readily made Easy image storage Extensive testing feasibility Minimal space requirement Proprietary software – easy inference of results

22. Mara Starter Pack

23. MARA Reagent & Plate Packs MARA Plates –5, 10 or 20 pack sizes Reagent Kits –Reagents 1, 2, 3 & 4 –5 or 20 Plate formats

24. Contact Details NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Tel: +44 (0) 1224 71 11 00, Fax: +44 (0) 1224 71 12 99, Email: mara@ncimb.commara@ncimb.com Sales contact: Carol Phillips, Technical contact: Terry Dando