international mobility fund activity ... - cawthron · pdf filecawthron institute ... and...

REPORT NO. 2249

INTERNATIONAL MOBILITY FUND ACTIVITY REPORT: BROADER USES OF THE ZEBRAFISH MODEL IN ECOLOGICAL RISK ASSESSMENT (ERA)

CAWTHRON INSTITUTE | REPORT NO. 2249 OCTOBER 2012

This report is available for public release:

INTERNATIONAL MOBILITY FUND ACTIVITY REPORT

Contract Number: FRG12-04

Project Title: Broader uses of the zebrafish model in ecological risk assessment (ERA).

Commencement Date: 20-31 August 2012

Funding provided: $6,000

NZ Principal Investigator: Louis Tremblay (PhD) Environmentalist Toxicologist Cawthron Institute Nelson New Zealand

Collaborators: Professor Dr Thomas Braunbeck University Of Heidelberg Germany

Prepared for Royal Society of New Zealand

CAWTHRON INSTITUTE 98 Halifax Street East, Nelson 7010 | Private Bag 2, Nelson 7042 | New Zealand Ph. +64 3 548 2319 | Fax. +64 3 546 9464 www.cawthron.org.nz

REVIEWED BY:

Robert Matheson

APPROVED FOR RELEASE BY:

Rowan Strickland

ISSUE DATE: 3 October 2012

RECOMMENDED CITATION: Tremblay LA 2012. International Mobility Fund Activity Report: Broader Uses of the Zebrafish Model in Ecological Risk Assessment (ERA). Prepared for Royal Society of New Zealand Cawthron Report No. 2249. 7 p.

© COPYRIGHT: Apart from any fair dealing for the purpose of study, research, criticism, or review, as permitted under the Copyright Act, this publication must not be reproduced in whole or in part without the written permission of the Copyright Holder, who, unless other authorship is cited in the text or acknowledgements, is the commissioner of the report.


v

TABLE OF CONTENTS

1. INTRODUCTION ........................................................................................................... 1

2. PROJECT AND ACTIVITY REPORT ............................................................................. 1

3. BENEFITS ..................................................................................................................... 4

4. PUBLICATION INTENTIONS ......................................................................................... 5

5. INTERNATIONAL OPPORTUNITIES............................................................................. 5

6. INTERNATIONAL RECOGNITION ................................................................................ 5

7. MAINSTREAM FUNDING .............................................................................................. 6

8. FUTURE COLLABORATION ......................................................................................... 6

9. HIGHLIGHTS ................................................................................................................. 6

10. DECLARATION ............................................................................................................. 7


1

1. INTRODUCTION

The previous visit to Dr Thomas Braunbeck’s Ecotoxicology Laboratory at the

University of Heidelberg in August 2011 provided the opportunity to familiarise myself

with the research group and was mainly focused on technical aspects and learning

the zebrafish fish embryo toxicity (FET) assay financed by FRG11-26. This second

visit was to continue to build the partnership with Dr Braunbeck but also to meet with

other key organisations with ecotoxicology expertise and capability around the

Heidelberg region. The main objectives of the visit were:

Meet with potential PhD candidate Svenja Böhler to confirm her intentions to

conduct a NZ/Germany based research project and to develop a funding

strategy.

Visit key organisations from local and national government authorities, industry

and consultancies involved in ecotoxicology and sustainable management.

Discuss the results from the tests conducted on New Zealand sediment samples.

Identify and discuss opportunities to support future New Zealand and Germany

collaborative ecotoxicology research projects.

2. PROJECT AND ACTIVITY REPORT

Dr Braunbeck and I had meetings with potential PhD candidate Svenja Böhler to

discuss and confirm her intention to conduct a research project based in New Zealand

and Germany. Svenja is fully committed to undertake the research and has very

suitable experience for the project. Her MSc research was based on developing the

fish embryo toxicity (FET) test in the fathead minnow. This is strongly aligned with our

collaboration with Heidelberg University. The research would include a comparative

assessment of embryos from two New Zealand fish species, the estuarine triplefin

(Forsterygion sp) and the freshwater common bully (Gobiomorphus cotidianus), to

determine whether the zebrafish FET model can be predictive of effects in our native

species. There is also an option to include American fish species from collaboration

with colleagues from Florida. We are developing the proposal for submission to

various funding sources. Svenja is in the process of developing an expression of

interest (EOI) for enrolment at the University of Auckland so that she can apply for

funding in New Zealand. Svenja will accompany Dr Braunbeck on his second New

Zealand visit to Cawthron.

We visited the chemical company, BASF (in Ludwigshafen) that has the biggest

industrial sewage treatment plant in Europe. It is also one of the best operated

systems as there is extensive real-time toxicity monitoring to ensure that unwanted

toxic spills are detected early and the effluent can be diverted to emergency ponds

OCTOBER 2012 REPORT NO. 2249 | CAWTHRON INSTITUTE

2

until treated suitably for release to the main system. We met with Dr Sabine Zok, head

of Ecology, Product Safety, Regulations, Toxicology and Ecology at BASF who gave

an overview of their impressive toxicity testing capability. The BASF ecotoxicology

laboratories play a major role in activities on sustainable and clean production of

chemicals. The tests are done under Good Laboratory Practices (GLP), a highly

regulated framework that oversees how the tests are conducted and information

archived. All toxicity tests meet International Organization for Standardization (ISO)

protocols which are accepted in the EU. Dr Zok suggested that tests used in New

Zealand should meet those standards to allow easy comparisons between results

generated with different test species.

We visited the Bundesanstalt für Gewässerkunde in Koblenz in the Rhine Valley. BFG

is the federal laboratory in charge of monitoring water quality in large rivers in

Germany. The bulk of their research is focussed on sediment toxicity, particularly to

manage the safe handling of dredging material that can be very toxic in hot spots of

Germany. Dr Georg Reifferscheid is the lead ecotoxicologist and showed us around

their facility. BFG has the range of more traditional toxicity testing capability with

algae, bacteria, and invertebrate protocols. They are also working to extend the

capability of their tests to include more mechanistic models to assess specific

activities such as endocrine disruption and genotoxicity. Dr Thomas Ternes is an

environmental chemist. His research is addressing the multiple challenges of

extracting contaminants from the complex matrix of sediments. His group has

developed impressive sediment extraction methodologies that are continuously being

improved and validated. Dr Ternes is also one of the world leaders in the new

research area of Emerging Contaminants. We are currently in the process of

developing a New Zealand National Strategy to manage Emerging Contaminants and

Dr Ternes has agreed to provide inputs/comments in specific areas around his

expertise and to share his European experience.

I had a meeting with Dr Raino Winkler from the Office of Environmental Protection,

Trade Supervision and Energy at the City of Heidelberg. Dr Winkler is a geologist by

training but has a position to promote sustainability initiatives and to ensure public

engagement. Heidelberg is an interesting city as it has a population of 140,000

including 30,000 students. It is mainly a touristic destination with no heavy industry

and a very wealthy demographic profile. The city is surrounded by forests and the

river Neckar is flowing in the middle. Dr Winkler explained that there is a strong move

to engage the population on sustainability issues. He was also interested in

establishing partnerships with key stakeholders to develop solutions. For example,

they have an impressive collaboration with Mercedes around the development of new

technologies to reduce the noise of trucks used in urban areas. Mercedes developed

new technologies that significantly reduced noise emission and the City bought those

trucks. Dr Winkler was impressed with the high level of multi-expertise research

projects we have that involve engagement of the community, especially Maori.


3

We visited Drs Runar Eberhardt and Thomas Berbner from Knoell Consult based in

Mannheim. Knoell are providing services to assist industry deal with the various

aspects of the European Union Registration, Evaluation, Authorisation and Restriction

of Chemicals (REACH) regulation. REACH is a world-leading regulation that will have

implications for industry and our NZ EPA. Knoell are dealing with all aspects of

REACH from gathering information, dealing with the data and identifying data gaps for

developing files for the registration of chemicals. They don’t have laboratories

themselves but have a good network of test labs around the world that can provide

the tests required for REACH. They have clients all around the world including

Australia. They have developed an interesting approach of working in close

partnership with their clients and often have a presence directly on the company site.

For instance, they have a permanent presence on the campus of BASF to assist them

with the major task of registering their chemicals.

We visited Ibacon, based in Rossdorf. Ibacon is an ecoxicology consultancy

established in the 1990s that now has 110 staff. Dr Ralf Petto (CEO) and Dr Melanie

Boettcher (Section Head Terrestrial Ecotoxicology) gave an overview of the company

and a tour of the ecotoxicology facility. Ibacon provides a wide range of services to

assess the risk of pesticides and does work mainly for agrochemical companies to

meet all aspects of the EU REACH. In addition to basic terrestrial and aquatic toxicity

testing, they also research the fate of chemicals using approaches such as carbon 14

(14C). The laboratories are well designed, particularly for the soil tests and bee testing

facility. Dr Maria Meinerling (Section Head of Analytical Chemistry) gave me the tour

of their facility and discussed their analytical capability. For a private laboratory they

have impressive equipment and can develop analytical methods for just about any

analytes as requested by clients. It was a very interesting visit and Ibacon staff were

very generous with their time. We also discussed the possibility of them attending the

Society of Environmental Toxicology and Chemistry Australasia Chapter (SETAC-AU)

that we will host in Nelson in March 2015.

I visited the Institute of Toxicology and Genetics (ITG) at the Karlsruhe Institute of

Technology (KIT) in Karlsruhe. KIT is a leading German interdisciplinary research

institute. The ITG is led by Professor Uwe Strähle who unfortunately was on holiday

so Dr Sepand Rastegar coordinated the visit. The main area of research at ITG is the

study of molecular genetics and toxicology of the vertebrate nervous system and

muscle development using zebrafish as a model. They have an impressive research

facility and are now the EU repository for holding zebrafish mutants. Research labs

particularly from Europe but also other parts of the world will be able to access the

mutants for their projects. PhD candidate Jens Otte discussed his research on

sediment extracts from German rivers using microarray and rtPCR in exposed

zebrafish embryo. His results so far using this approach showed that it is challenging

to identify specific physiological pathways that are being modulated by the very

complex extracts. He has recently moved in a different direction and is now using a

single chemical, Bisphenol-A, and sophisticated imagery technologies to monitor the


4

effects of the chemical on cell movement in the developing embryo. This approach is

showing promising results. I had a very stimulating meeting with Dr Ravindra Peravali

who was trained as a mathematician but has an interest in biological systems. His

research combines robotics with image analysis to quantify behavioural patterns in the

Japanese medaka (Oryzias latipes), another commonly used fish model. He has

developed software that can analyse and quantify behavioural patterns. These

methodologies offer a great opportunity to study the effects of endocrine disruptors on

sexual behaviour. Finally I met Dr Ralf Mikut, Team Leader of the Institute for Applied

Computer Science attached to the KIT. The latest technologies such as the new

generation sequencing methodologies and image analysis generate vast amounts of

data and there is a significant need for biometrician expertise. Dr Mikut’s group

provide this highly valuable expertise with a good understanding of the biological and

physiological questions being asked.

We had meetings with Professor Braunbeck’s laboratory group to discuss the results

from tests conducted on extracts from New Zealand estuarine sediment samples. This

extends on a previous research project where we collected information about the level

of organic micro-contaminants at Napier and Invercargill sites. PhD candidate Ruben

Strecker and student Markus (?) conducted the zebrafish Fish Embryo Toxicity (FET)

tests on six samples and found toxicity. We are now extracting more sediment to

provide more mechanistic information.

PhD candidate Britta Kais will use the extracts to assess the induction of the

detoxifying enzyme cytochrome P450 in an in situ method in the zebrafish using

ethoxyresorufin, a fluorescent substrate. The extracts will also be tested by MSc

candidate Patrick Heinrich who works with the rainbow trout hepatocyte cell line RTW-

1 to look at cytotoxicity and the expression of P450 in a cell system.

3. BENEFITS

The development of more ethically acceptable alternative methodologies for toxicity

testing continues to be the main outcome of the collaboration. The visit provided

scope to expend to other very useful areas including:

Building a collaboration with key ecotoxicology expertise at the University of

Heidelberg.

Establishing a support network in relation to the EU REACH regulation and help

New Zealand comply with this very important legislation.

Establishment of links with some of the best European experts in the field of

Emerging Contaminants.


5

PhD candidate Svenja Böhler has confirmed that she wants to undertake a

research project to consolidate the link between Germany and New Zealand. It is

likely that she will enrol at the University of Auckland.

4. PUBLICATION INTENTIONS

The results from the testing of the New Zealand estuarine sediment extracts using the

various tests conducted in Dr Braunbeck’s laboratory will be combined with results

from the New Zealand partners and a scientific manuscript will be submitted to a peer-

reviewed journal.

5. INTERNATIONAL OPPORTUNITIES

The zebrafish fish embryo toxicity (FET) test is likely to be confirmed as an OECD

guideline for its multiple advantages over traditional methodologies and its low ethical

cost. To have a partnership with one of the main laboratories involved in the

development of the protocol is a great advantage for New Zealand. This is particularly

important in view of the implications of EU REACH regulation requirements on our

export industry. The links with German groups with extensive expertise dealing with

REACH will become increasingly valuable as the regulation starts to influence other

jurisdictions.

New Zealand is currently developing a New Zealand National Strategy to manage the

risk of Emerging Organic Contaminants. Dr Thomas Ternes from the Bundesanstalt

für Gewässerkunde is a world expert and has agreed to provide priority setting advice

to address the families of chemicals with higher risk.

6. INTERNATIONAL RECOGNITION

New Zealand is a country but has the population the size of a medium city. This

provides us with the unique opportunity to engage most endusers with interest in

environment management. This is a real advantage for developing management plans

that are endorsed by the community. The German colleagues I talked to about the

approach we use in our research based on partnership with community groups and in

particular Maori were impressed by the process. They view it as a very efficient

framework to ensure a high level of acceptance of the management solutions.


6

Once again it was suggested by Professor Braunbeck that New Zealand is unique in

the developed world as it still has large areas of relatively unspoiled ecosystems. This

provides great opportunities to select reference sites where species are exposed to

varying contaminant levels for inclusion into ecotoxicological studies. This is a growing

problem for environmental studies in Europe where relatively pristine environments

that receive low levels of anthropogenic stressors suitable as reference sites are hard

to find.

7. MAINSTREAM FUNDING

To finance the project of PhD candidate Svenja Böhler, we are applying for a

fellowship from the University of Auckland (where Dr Tremblay is a Senior Lecturer).

Dr Braunbeck will also explore German opportunities such as the German Academic

Exchange Service (DAAD) and regional sources of funding such as the region of

Baden-Würrtemberg-Stipendium. We will discuss other funding opportunities during

Dr Braunbeck’s next visit to New Zealand in March-April 2013.

We are exploring the possibility of a collaborative proposal in the 2013 Ministry of

Business, Innovation and Employment (MBIE) investment round.

8. FUTURE COLLABORATION

The discussions over the two weeks generated multiple ideas for collaboration. Of

particular interest is the opportunity to collaborate with Dr Ravindra Peravali from the

KIT Institute of Toxicology and Genetics (ITG) on an endocrine disrupting chemicals

(EDCs) project. Dr Peravali has developed a programme that can analyse fish

behaviour that can be quantified. He can measure sexual behaviour in the Japanese

medaka (Oryzias latipes) model. This provides a methodology to assess the effect of

EDCs with estrogenic activity as it has been demonstrated that they can reduce

sexual behaviour and therefore have the potential to affect reproductive fitness. We

have agreed to maintain contact and look for options to fund the research which may

be a pilot study to begin with.

9. HIGHLIGHTS

Overall I felt very welcome by each group I visited. They were all very open and

generous with their time and we had many very collegial discussions. I found the

German colleagues to be very generous with their time and their offers to assist and

interact with New Zealand. It was great to see that everyone was very enthusiastic


7

about their research and conscious of the importance of their contribution to the

protection of the German environment. The main aim of this collaboration is the

development of a fish-based toxicity testing capability with high ethical acceptability.

The research will be maintained by the involvement of a German PhD candidate

enrolled at either Heidelberg or Auckland University working on the validation of the

zebrafish FET in New Zealand species. This will ensure the completion of the aim and

sustain the collaboration. This second visit to the University of Heidelberg was a very

productive visit that enabled me to meet people from many organisations dealing with

various aspects of ecotoxicology in environmental management. Germany is a leader

of sustainability but it was rewarding to find that in terms of community engagement,

New Zealand is doing extremely well.

10. DECLARATION

I confirm that the grant provided was fully expended on the costs associated with my

IMF application.

Signed: __ Date ___27 September 2012

international mobility fund activity ... - cawthron · pdf filecawthron institute ... and...

Documents