Abteilung Umwelttoxikologie

Background
In agricultural areas, insecticides comprise a considerable part of the chemical mixture present in the aquatic environment: they reach water bodies via run-off or leaching after spraying events. While designed to be neurotoxic to insects, insecticides can have adverse effects on a multitude of organisms due to the high conservation of the nervous system among phyla. To assess the risk of insecticides for the environment and estimate their long-term ecological effects, it is important to investigate their impact on non-target organisms such as fish. Toxic effects elicited by insecticides can occur at different organizational levels and may range from easily observable lethal to very subtle behavioral effects. The molecular pathways behind these effects, however, are still largely unknown, but are important to understand in order to develop predictive models, biomarkers and in vitro tests.

Aim
We would like to gain a better understanding of neurotoxic effects in fish by linking observed effects on organism-level to underlying molecular events.

Methods
Due to its exceptional suitability for molecular and developmental studies, the zebrafish, Danio rerio, will be used as a test organism. Based on previous experiments, in which the effects of insecticides on the behavior and neuromuscular structure of zebrafish larvae were investigated, gene expression analysis will be conducted focusing on genes involved in development and functioning of the nervous system and muscles.

The techniques applied include (i) breeding and handling of zebrafish, (ii) design of primers, (iii) RNA isolation, (iv) qPCR, and eventually (v) CRISPR-Cas9 gene editing (if time allows).

Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline and should have a genuine interest in neuroscience and molecular biology.

For further information please contact Colette vom Berg and Sarah Könemann.
This work will be performed at Eawag at the Department of Environmental Toxicology in Dübendorf.

Background

Metals are widespread aquatic contaminants and affect aquatic wildlife in different ways. Copper, for instance, although an element essential to life, can be detrimental in excess. Preliminary studies in our laboratory have shown that zebrafish embryos exposed to sub-lethal concentrations of copper have reduced levels of internal calcium and other elements. Although the interplay of copper and calcium has been documented in many species, the exact mechanisms of copper uptake and calcium reduction are not well understood.

Aim

The aim of this project is to

• Decipher the interplay between uptake of copper and calcium
• Characterize the internal distribution sites and accumulation of these two metals in zebrafish embryos upon copper exposure
• Investigate molecular mechanisms behind lower calcium uptake and consequences of reduction in internal calcium levels

Methods and Techniques applied

Zebrafish maintenance and breeding, zebrafish embryo toxicity assay, histochemical staining to visualize bones and cartilage, microscopy, pharmacological and/or molecular inhibition of transport proteins and channels, qPCR to measure gene expression, ICP-MS to measure metal concentrations

If you are interested in this project, please contact Ksenia Groh, Kristin Schirmer or Colette vom Berg.
This work will be performed at Eawag at the Department of Environmental Toxicology in Dübendorf.

Background:

Tire and Road Wear Particles (TRWP), which are generated by the erosion of tires while driving, have recently been estimated as representing up to 28.3% of all primary microplastics released in the aquatic environment, for an estimated mass of 424 Ktons/year. This finding raises questions regarding the toxicological impact of these TRWP and associated chemicals on aquatic organisms.  
 

Aim:

This study aims at determining the toxicity mechanisms induced by TRWP and associated chemicals to fish using different rainbow trout cell lines (Oncorhynchus mykiss).
 

Methods:

We will use several rainbow trout cells lines (e.g., from gill, gut and brain), which will be subject to different exposure scenarios.

You will gain experience with routine cell culture techniques (how to work in sterile conditions and culture cells). You will moreover learn how to set up and perform different assays: cell viability assays to determine the cytotoxicity of chemicals (seeding cells using different substrates, exposure to chemicals and multi-endpoint viability assay), bioavailability assays to quantify how much of a chemical has entered the cells (mass spectrometry) and gene expression analysis (RT-PCR) to identify molecular mechanisms induced. Finally, you will learn how to produce, interpret and present scientific data.

If you are interested in this particular research topic and are excited to know more about environmental toxicology research in general, please contact William Dudefoi or Kristin Schirmer.

This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.

Preferred starting date: flexible

Background:
It has been demonstrated that permanent cell lines from rainbow trout have the potential to predict the bioaccumulation in fish of uncharged chemicals, such as polyaromatic hydrocarbons. In such an exposure test, the biotransformation rate of the chemical in exposed cell cultures is measured over time. This information serves as an input for toxicokinetic models, which simulate the bioaccumulation in fish. However, whether the cell lines could as well be used to predict biotransformation rates of charged chemicals, such as pharmaceuticals, pesticides or agents in personal care products, is not yet known.

Project:
This project focusses on the bioaccumulation assessment of selected charged chemicals, such as, for example, the primary amine, dodecylamine, in rainbow trout cell lines. The aim is to compare the in vitro-derived biotransformation rates and the modelled bioaccumulation to data available from in vivo studies with rainbow trout. The tasks comprise 1.) the conduction of exposure experiments in rainbow trout cell lines from different tissues (gill, gut and liver) 2.) the determination of the chemical concentrations in the cells and 3.) the modelling and interpretation of data.

Tasks:
You will acquire knowledge and skills in a diverse range of disciplines:

• In vitro cell culture techniques
• Design of kinetic experiments with cells
• Sample preparation and measurement using liquid-chromatography-high resolution tandem mass spectrometry
• Toxicokinetic modelling and data interpretation

Supervision by: Fabian Balk (PhD student), Prof. Dr. Kristin Schirmer, Prof. Dr. Juliane Hollender

Contact: Fabian.balk@cluttereawag.ch, Kristin.Schirmer@cluttereawag.ch, Juliane.Hollender@cluttereawag.ch

Background

In mammals, mechanistic target of rapamycin (mTOR) pathway is known to be a central cellular hub that mediates regulation of cell growth and proliferation in response to a host of endogenous and exogenous stimuli. The mTOR is a serine-threonine kinase that, upon activation, initiates a cascade of phosphorylation events for its downstream targets, which are further involved in regulating transcription, translation, autophagy, metabolism, and cytoskeletal organization, among many other essential cellular processes. Interestingly, exposure to chemicals can influence many of the upstream signals sensed by the mTOR, such as energy levels, oxidative stress, proteotoxic stress, genotoxicity, or inflammation. The role of the mTOR in regulating cellular responses to stress has indeed been recognized, but so far rarely considered with regard to chemical toxicity, in fish even less so than in mammals. Therefore, the research in my group seeks to understand the role of mTOR in regulating fish growth in response to chemical exposures and other environmental factors such as temperature. The project described here will characterize the baseline relationships between the mTOR activity levels and growth responses in a fish cell line.

Aims

This master thesis will investigate the correlation between the mTOR pathway activity and growth in cultured fish cells. For this, fish cells will be exposed to specific pharmacological inhibitors or activators of the mTOR pathway and its activity will be monitored by measuring changes in phosphorylation status of mTOR substrate proteins. In parallel, growth and several other endpoints will be assessed in the exposed cells to understand their correlation with the observed phosphorylation-based signaling dynamics.
 

Methods

Experimental work on the proposed master thesis will provide training in basic cell culture techniques, chemical exposure, fish cell growth assay, and microscopy/imaging. The central analytical task will include performing targeted phosphoproteomic analysis of selected proteins using mass spectrometry. Our phosphoproteomic assays constitute a novel tool to study phosphorylation-based signaling in fish, where this mechanistic aspect has been so far largely neglected due to challenges with obtaining species-specific antibodies for (de)phosphorylated proteins of interest. The workflow consists of protein extraction from cultured cells, protein digestion, phosphopeptide enrichment, and measurement on our state of the art LC-MS/MS instrumentation.

If you are interested in this project please contact: Ksenia Groh.

1 Background
Rainbow trout (Oncorhynchus mykiss) derived cell lines, similar to rainbow trout, can survive in temperature ranges between 4◦C to 24◦C, but are routinely cultured and maintained at 19◦C. However, several lines of evidence suggest that temperature could exert an effect on cellular responses to chemicals in vitro. Firstly, phospho-lipids in membranes adapt to lower temperatures by decreasing permeability, and to higher temperatures by increasing permeability. This can result in temperature-dependent changes in uptake and bioaccumulation of substances. Furthermore, biotransformation of chemicals in cells is effected by enzymes, such as CYP450, whose kinetics are also influenced by temperature.
In order to establish cell lines as a robust in vitro system for environmental risk assessments, it is important to explore whether outcomes can be predicted for complex exposure scenarios, such as with temperature as an additional stressor. Furthermore, understanding the influence of temperature on sensitivity is becoming increasingly important in the context of climate change.

2 Aim
The aim of this Master thesis is to investigate the effect of temperature on the sensitivity and biotransformation capacity of rainbow trout cell lines, and to assess whether trends observed are comparable to those known from experiments with fish in in vivo, and in the environment.

3 Methods
Research comprises training in:
• Basic cell culture techniques
• In vitro cell line based toxicological assays (cell viability assays, biotransformation assays)
• Chemical analysis by Mass Spectrometry (MS)
• Physiologically Based Toxico Kinetic (PBTK) modelling

If you are enthusiastic about exploring the relevance of temperature stress on cellular responses to chemicals, and learning useful skills and methods in environmental toxicology in the process, please contact Kristin Schirmer or Gayathri Jaikumar. This research will be performed at the department of Environmental Toxicology, Eawag, in Duebendorf.

BACKGROUND: One of the main goals in the field of toxicology is the development of Adverse Outcome Pathways (AOPs): an emerging tool aimed at understanding the biochemical and signal transduction pathways that are affected when an organism is exposed to a chemical. An early step in the development of AOPs is the assessment of the toxic action of a chemical at the molecular level. The gene-editing CRISPR/Cas9 technology could help in this field by allowing scientists to specifically modify genes involved in different outcome pathways and observe how the response to the chemical exposure changes.

AIM: The aim of this Master thesis is to establish a CRISPR/Cas9 gene editing system in rainbow trout (Oncorhynchus mykiss) cell lines and use this technology in order to shed light on the molecular processes underlying the chemical action.

METHODS: Research comprises training in: (1) basic molecular biology techniques (e.g. DNA, RNA and plasmid DNA extraction, PCR) and molecular cloning strategies (e.g. Gibson assembly and E. coli transformation), (2) gene editing using the CRISPR/Cas9 system (3) how to culture, transfect and chemically expose fish cell lines (4) fluorescence microscopy and FACS analysis.

If you are eager to learn and implement new technologies and passionate about molecular biology and environmental toxicology, please contact Kristin Schirmer (Kristin.Schirmer@eawag.ch) or Marina Zoppo (marina.zoppo@eawag.ch). This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.

Particulate plastics (microplastics, fibers, and nanoplastics) are produced either intentionally for their commercial use in consumer products or unintentionally as breakdown fragments of larger synthetic materials (e.g. plastic bottles, polyester textiles, rubber tire wear). Currently, there is little information on the fate, transport and bioavailability of  nanoplastics and microplastic fibers in water bodies. Importantly, knowledge on their potential to interact with organisms and to impact food-webs is largely lacking.

AIMS: Here we aim to quantify interactions of nanoplastics and microplastic fibers with stream microbial biofilms, as well as their potential transfer from biofilms to grazers. Focus will be on examining for transgenerational effects on the snail offspring fitness. Unique metal-doped particulate plastics developed in-house will be used through all experiments, which will both expedite analysis times in determining biota-plastic burden and provide more precise and accurate quantification of plastics throughout the exposure system.

METHODS: The candidate can expect to learn (1) how to set up and maintain experimental mesocosms in a laboratory setting, (e.g. growth and sampling of biofilms and snails), (2) how to register changes in biofilm and snail biology (e.g. growth, respiration, reproduction), and (3) how to quantify particulate plastics in various media, including techniques for particle characterization (e.g. Dynamic light scattering, Nanoparticle tracking analysis, Transmission electron microscopy) and analytical chemistry (e.g. Inductively coupled plasma mass spectrometry)).

Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline. For further information please contact Ahmed Tlili (ahmed.tlili@cluttereawag.ch). This work will be performed at Eawag (Dübendorf) in the Department of Environmental Toxicology, and in collaboration with Denise Mitrano (denise.mitrano@cluttereawag.ch) in the Department of Process Engineering.

Stream biofilms are complex communities of algae, bacteria and fungi, which play a fundamental ecological role in aquatic ecosystems. Microorganisms composing biofilms are primary targets for herbicides, which can lead to structural and functional alterations of the community, with potential negative consequences for ecosystem functioning. Yet, molecular processes underlying functional and structural responses of biofilms to herbicides are still largely unknown.

AIM: By establishing a set of genes that are specifically regulated in biofilms upon exposure to herbicides, we aim in this project to (i) examine toxicity and adaptive cellular processes, on the transcriptome level, in aquatic biofilms and  (ii) link these processes to community functional and structural alterations.

METHODS: Candidates will learn (1) how to culture and sample biofilms both in the field and in laboratory mesocosms , (2) various techniques for gene expression (mRNA extraction and quantification, qPCR), molecular and functional diversity analyses (genomic DNA extraction and amplification, DGGE, flowcytometry), and (3) quantification of the herbicides in water and biofilms.

Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline.

For further information please contact Ahmed Tlili (ahmed.tlili@cluttereawag.ch). This work will be performed at Eawag in the department of Environmental Toxicology in Dübendorf.