MARA

A Microbial Array for Ecotoxicity Testing

Contents

This presentation covers 3 areas:

• The drivers behind ecotoxicity testing• Perspective of tests

– Conventional– Micro-scale tests

• MARA – Features– Protocol– Capabilities

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

Legislation

Terrestrial environment• Ecological Risk Assessment

(ERA)– Contaminated Land Regulations

• Landfill– Landfill (Directive) Regulations.

Ecotoxicity Testing

Requires a measure of direct toxicity, taking into account:

• Whole sample toxicity

• Interactive effects

• Unknown chemicals

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

Important Criteria for Micro-Scale Ecotoxicity Tests• simplicity

• speed

• reproducibility

• precision

• sensitivity

• Accuracy

• standardisation

• cost effectiveness

• convenience

• information

Microbial Array

Benefits of using micro-organisms– Rapid growth

– Small size

– Large number tested

– No ethical issues

– Diversity

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

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

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

Protocol

Protocol

Medium& Dye

Freeze-dried micro-organisms

Sample, Medium &Dye

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGH

-Ve Control

Test Control

Inoculate from H, A to G

Discard

Reconstitute

Microbial Fingerprint (effluent)

A microbial fingerprint can be derived

from the analysis

MARA Software – Image Screen

The plate image is copied into the image analysis software

MARA Software – Data Screen

060007005

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

Sample Conc (%)

Gro

wth

In

hib

itio

n(%

)

Sp 1

Sp 2

Sp 3

Sp 4

Sp 5

Sp 6

Sp 7

Sp 8

Sp 9

Sp 10

Sp 11

Species Response

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

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

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

Tests Comparison

MARA has also been compared with the following established tests• Fish • Algal • Invertebrate • Microtox

• Respiration inhibition• Nitrification inhibition

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

Mara Starter Pack

MARA Reagent & Plate Packs

• MARA Plates– 5, 10 or 20 pack sizes

• Reagent Kits– Reagents 1, 2, 3 & 4– 5 or 20 Plate formats

Contact DetailsNCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YATel: +44 (0) 1224 71 11 00, Fax: +44 (0) 1224 71 12 99, Email: mara@ncimb.comSales contact: Carol Phillips, Technical contact: Terry Dando