OECD Workshop on Environmental Biotechnology Rimini, Italy Bioassays and bioreporters

Davide Merulla Jan Roelof van der Meer

Thursday, September 16, 2010 Outline of the presentation

• Current state of the art in use of bioassays • New approaches - promises, advantages • Case study - arsenic bioreporter assays • Implementation - hurdles, difficulties • The way forward?

2 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Bioassays

• Why use bioassays when there is beautiful, precise and expensive chemical analytics?

Pictures: FACEiT consortium

3 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Why bioassays?

Simple reason: Measuring heart beat rates of a crab Picture: Ceri Lewis, Tamara Galloway • Chemical analysis can University of Exeter, UK measure total concentrations • Very difficult to predict on the basis of chemistry: – Concentrations experienced by living organisms – Biological effects of single compounds – Effects of mixtures – Acute - chronic effects

4 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Bioassays come in different forms

• In situ examination of exposed organisms

• Exposure of model organisms under standardized conditions

• Use of cultures, single cell models

• Use of genetically engineered single cell models

• biosensors with purified or biological components

5 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 1) In situ examination of exposed organisms

MSC Napoli, Plymouth January 2007 EXAMPLE

• Local limpets found on shores in- and outside the presumed contaminated area

• Analyzed for a variety of Galloway Tamara Lewis,

molecular and physiological Ceri exposure markers Pictures: Pictures: UK Exeter, of University

6 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Biomarker results - example

Pro’s/Con’s • Some biomarker species typically available at a contaminated site, • But may not be an optimal one • Control samples not trivial to obtain • Typical conflicts with authorities in the first couple of days after an accident

(higher values mean limpets are more healthy) • Chronic effects can be data: Ceri Lewis, Tamara Galloway observed University of Exeter, UK

7 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 2) Exposure of model organisms under standardized conditions

Examples • Daphnia model • Zebrafish - DarT embryo model Pro’s/Con’s • Better interpretable response • No in-situ information • High cost to maintain model organisms • Strict regulation on animal use

8 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Example - DarT Test

• Embryo development over 48 h • Endpoints: heart, muscle, eye, circulation • Alternative to fish acute test • DIN Norm

Exposure to oil on water

Pictures: Rik Eggen, Eawag, Switzerland

9 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 3) In vitro models

In vivo Cell culture

long Time short

high Costs low

large Sample volume small

better Integrative worse effects worse Molecular effects better

more High biological less integration

After: Rik Eggen, Eawag, CH

10 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 4) Use of genetically engineered single cell models • • • Cell cultures

Pro’s/Con’s • Low complexity • Single target • Not live animals • Highest and easiest output

due to ‘reporter’ Data: Bart van der Burg, BDS

11 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Automized GMO cell line exposure analysis

Picture: Bart van der Burg, BDS

12 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Bioreporter bacteria

13 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Case study arsenic bioreporter assays

• Arsenic is a world-wide serious contaminant of potable water sources • Chemical analytics is difficult or requires sophisticated equipment • Which is not easily available in many countries

14 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Arsenic Reporter bacteria

• Use natural (bacterial) defense system against arsenic • Engineered to produce light, color, , electrochemical signal in response to arsenic • Sensitive below drinking water standard of 10 µg/L • ‘Sensor’ production extremely cheap and self- reproducible…

15 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Further research on assay miniaturization

50 µg/L

25 µg/L

10 µg/L Reporter cells in micro!uidics chip 0 µg/L

16 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Implementation efforts

‘business’ Open source • arsenic • 2006 large field study in bioreporter test registered Vietnam - comparison as Arsolux® chemical analytics / • Startup company at UFZ bioreporter luciferase test: Leipzig successful • In process of making 5000 • 2008 field study on test kits - freeze-dried Philippines successful bacteria in glass vials • Multiple occasions: course • Field campaign Autumn instructions to lay people 2010 in Bangladesh and scientists successful • Registration difficult • Business plan fails

17 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Implementation

Success stories Not so success stories Company Biodetection Systems Bacterial bioreporters NL • Only universities, no • GMO cell lines (CALUX®) companies • Method not accredited • In-house in vitro analysis despite multiple proofs of • Accredited method success • Biomedical focus and side • Environmental focus - lack of environmental focus markets • Industry fears GMO production

18 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Future / Prospects

• Synthetic biology: even more easy to produce and tailor bioreporter cells • Micro-engineering: smaller, handier, multi-analyte, contained applications

• Accreditation procedures must become easier • Safety regulations for standardized non pathogenic lab strains as reporter ‘chassis’ could become easier • Finally: science can show it to work / entrepreneurship is needed for market introduction

19 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010