An evidence-based approach to pinpoint genotoxicity in the River Danube

In this study we combined bioassays, chemical analysis, and QSAR modelling to identify genotoxic contaminants in the surface water samples from the River Danube. We started with a screening for the three most bioactive samples using the fish embryo toxicity test (FET). Thereafter we tested for genotoxicity by means of the Ames fluctuation and the micronucleus assays. The chemicals found in the samples after non-target analysis (different study) were then filtered for the genotoxicants based on QSAR modelling and literature data. An artificial mixture of all these potential genotoxicants was tested in Ames and micronucleus, and we could explain nearly 50 % of the genotoxicity with 18 of the substances found in the samples.

Find out more about "Integrating bioassays, chemical analysis and in silico techniques to identify genotoxicants in surface water" (fulltext only with a subscription to the journal, sorry...)

Green Toxicology for a clean environmental - challenging biosurfactants

Ever heard of rhamnolipids? Well, we didn't so much until we were approached by our colleagues from the Institute for Applied Microbiology (iAMB) at RWTH Aachen University. They were interested in an ecotoxicological assessment of those compounds, which they see as future candidate biosurfactants.

Of course, so were we, since this gave us a very nice opportunity to contribute to ensuring that novel compounds are tested well before they get introduced into the market and hence the aquatic environment by using the concept of Green Toxicology. This is the idea that chemicals should and could be thoroughly investigated by means of a set of different methods regarding their environmental impact already during development.

We tested the acute toxicity to the invertebrate Daphnia magna, and to zebrafish embryos (Danio rerio). Microbial and fungicidal effectiveness was also investigated. Furthermore, we determined a potential mutagenicity by means of the Ames fluctuation assay.

We found that mono-rhamnolipids exhibit toxicity to daphnids and zebrafish embryos comparable to or even lower than chemical surfactants. They showed very low toxicity to the germination of Aspergillus niger spores and the growth of Candida albicans. No mutagenicity was observed using the Ames fluctuation assay. Model simulations confirmed our findings regarding no mutagenic potential, and they also indicated that rhamnolipids have no estrogenicity.

Read the whole story about mono-rhamnolipids as an environmentally friendly alternative to chemical surfactants, from an ecotoxicological point of view. (fulltext only with a subscription to the journal, sorry...)

Making things smaller to make them bigger

One of our main research directions is the development and optimisation of bioanalytical tools. In terms of optimisation, among others we seek to make the bioassays more versatile, meaning to include several different effect meaurements and integrate results; to allow for a higher throughput of samples, thus reducing required work force and other resources; and to miniaturise the bioassays where possible, especially with regard to saving sample.

The latter one was the focus of a study we conducted on an assay that detects androgen receptor binding, thus indicating potential for hormon-like activity and disturbance of the endocrine system. Samples containing such substances are often available in only very small amounts, for instance as extracts from water samples. And especially in terms of drinking water the presence of androgen-like chemicals is a great concern.

We succeeded in reducing the medium volume to dilute the sample to one third of what is recommended by the standard protocol, thus also significantly reducing the required amount of sample. Results obtained for standard substances were compared to data obtained using the conventional procedures and found to be similar. This proved that the optimised method can be applied as an alternative to the standard protocol, if limitations by small sample amounts would otherwise prohibit scientifically meaningful investigations, and thus compromise proper risk assessment.

Take a closer look at the "Downscaling procedures reduce chemical use in androgen receptor reporter gene assay". (fulltext only with a subscription to the journal, sorry...)

This is a boring headline about zebrafish embryos exposed to heavy metals

Today I report on a study that was published already a while ago, but tells a nice story about the challenge and necessity to design meaningful experiments. Also, this one deals with heavy metals, which is not common in our research; we mainly focus on organic contaminants.

In this particular study we investgated whether heavy metals spiked into sediments are bioavailable to unhatched zebrafish embryos, and whether it makes a difference if the sediment was a natural one - just taken from, e.g., a riverbed out there - or a formulated, meaning components that typically make up a sediment mixed together. Besides the single heavy metals we also spiked mixtures.

We investigated mortality, several heavy metal-specific proteins and the regulation of a couple of genes that are known to react to heavy metal exposure. What we found was actually not very surprising: the heavy metals in the formulated sediment were better bioavailable than those in the natural one. An artificial mixture of sediment components can never resemble a real "grown" sediment. The matrix in the natural sediment is just much more complex and provides a multitude of possibilities for heavy metals to be trapped, bound, blocked, and thus not able to enter the test organism. Nevertheless, this had to be proven first. Science does not rely on hypotheses. We  have to challenge them to be sure.

However, formulated sediments are widely used in research. And our study shows that using such matrices could largely overestimate bioavailability and thus risk of heavy metal contaminations in sediments. Furthermore, the study revealed that the accumulation of the individual heavy metals from a mixture is relatively lower compared to that in the single metal exposure experiments. Since not all metals show the same toxicity for the embryos this has to be taken into account when doing risk assessment of heavy metal burden.

Last not least we identified some promising biomarkers for low-dose detection of heavy metal exposure. Read the full story on "Bioaccumulation and molecular effects of sediment-bound metals in zebrafish embryos". (fulltext only with a subscription to the journal, sorry...)

Small traces, large impact

Already some time ago we participated in writing an article on Anthropogenic Trace Compounds (ATCs). ATCs are an issue for water quality, since they increased in numbers and concentrations over the last years. This very heterogenous group of chemicals is a major challenge for research and policy making, in regards of identification, detection, monitoring, fate, toxicity and mitigation. They represent a complex network of interrelations of physical-chemical properties and behaviour, and biological activity. Literally, the substances only have in common that they are introduced into the water cycle by humanity, are found at still rather low concentrations, but anyway represent a potential danger for human and environmental health.

Together with our colleagues we reviewed current trends monitoring and management, and identified innovative and effective strategies as well as knowledge gaps and urgent needs. We highlight the lack of generally accepted indicator substances as one major issue that has to be solved for future monitoring. Also, we advocate for the implementation of effect-based tools in water monitoring, and for an appropriate risk assessment of ATCs.

There's a whole lot more to find in this comprehensive review article. Read the entire story about "Anthropogenic Trace Compounds (ATCs) in aquatic habitats — Research needs on sources, fate, detection and toxicity to ensure timely elimination strategies and risk management". And you really can read it, it's Open Access!

Should we drink an drive tea?

Research on biofuels recently suggested compounds that are already known as solvents or flavors to be alternatives for fossil fuels. We investigated the three candidate substances ethyl levulinate, methyltetrahydrofuran, and 2-methylfuran regarding their embryotoxic effectiveness using zebrafish.

Especially ethyl levulinate gave considerable toxicity. Very specifically it tended to reduce head length of zebrafish larvae. As a consequence, we recommended to not further continue developing this substance into a potential biofuel. Moreover, use of ethyl levulinate in, e.g., flavored tea should be critically revised. Read the full story on "Acute embryo toxicity and teratogenicity of three potential biofuels also used as flavor or solvent" (fulltext only with a subscription to the journal, sorry...).

Fish behaviour trajectories converted to usefulness

Zebrafish behaviour is a novel and upcoming endpoint in toxicity assessment of - at large - neuromodulating substances. This includes direct neurotoxicity as well as repellency and any other type of avoidance. Since zebrafish react to contaminants in the water at low nanograms per liter, behaviour measurement has the potential to be used for biological early warning systems.

However, usefulness of such data can be hampered by the independence of the distance moved (as one of the most common endpoints) from the trajectory's shape. A parameter is hence required to identify directed movement as an indication of avoidance behaviour.

In the W3-Hydro project we tested permethrin and cadmium as model substances using a Noldus DanioVision system. Obtained trajectories were converted to polar coordiantes and parametrized. Results showed that by this procedure pure distance-moved-data can be used to identify impact of contamination on zebrafish larvae behaviour. Read more on early detecting water contaminants Ecotoxicology and Environmental Safety (fulltext temporarily free, then limited to subscribers, sorry...).

Why we do not just talk about our science

It is not easy to communicate science, and it is especially difficult from environmental research. Most findings we make are rather directly connected to every day lifes and thus human wellbeing. As a consequence, people tend to assess such research based on their personal feelings, desires, and - in particular - concerns. This can lead to misunderstandings that produce even stronger opinions and are hence even more difficult to solve.

We environmental researchers seem to react with avoidance: before we do something wrong, we do not do anything at all in terms of communicating our science. Thomas Backhaus and I wrote an Editorial in Integrated Environmental Assessment and Management on "Communicating environmental science to the general public", where we shortly analyse this situation and the associated problems and try to encourage our colleagues to anyway take on the challenge. 

Who's afraid of a little picene?

Picenes and alkalyted chrysenes are highly abundant compound classes in lignites. Hence, we found them in high concentrations in a German lignite extract from a former study. Since this extract gave only low PAH content but showed high mutagenicity and dioxin-like activity we decided to separately test these compounds for their toxic potential. Furthermore, we investigated bioavailability of picenes and chrysenes by means of a Lumbriculus variegatus bioaccumulation test.

All experiments yielded data that let us conclude that alkylated chrysenes and picenes, though highly abundant in lignite, pose only a low environmental risk (fulltext access limited to subscribers of the journal, sorry...).

Water quality monitoring of tomorrow - A tools vision

Current water quality monitoring relies mainly on chemical analytics to monitor occurence of a few fistful of priority compounds. This bears a great risk of missing new and emerging pollutants. To account for these the EU-FP7 SOLUTIONS project aims at developing, establishing and recommending (novel) analytical tools, that would be able to identify and also assess yet unknown substances in the aquatic environment.

Our three-pronged approach is to heavily improve target and non-target chemcial analytics, build a strong array of highly sophisticated bioanalytical test systems - i.e. effect-based tools - and further establish effect-directed analysis as a method to reliably identify drivers of toxicity. This whole vision was published in Science of the Total Environment as "Future water quality monitoring — Adapting tools to deal with mixtures of pollutants in water resource management" (fulltext access only through subscription, sorry...).

The EDA guide to surviving environmental analysis

Colleague Werner Brack at the UFZ in Leipzig, Germany is an outstanding expert on effect-directed analysis (EDA). EDA stands for an analytical approach that uses chemical and biological analysis to identify single contaminants as the culprits for toxicity in environmental samples.

It combines the best of both worlds. The ability to detect effects and the power to identify as well as elucidate molecule structures are interconnected by a sophisticated fractionation. The EDA principle is easily explained: (1) samples are tested for their effects in one or more bioassays, (2) effective samples are fractionated and fractions tested again, (3) effective fractions are further fractionated and tested, and finally (4) the substances in the few remaining effective fractions are chemically identified.

While sounding quite easy, EDA can be highly complex effort, due to the multitude of options for experimental design. Which bioassays should be used for screening, which biological effect should be targetted? How to properly fractionate the samples, for a most succesful separation of the compounds of interest? What techniques and methods are suitable for structure identification and elucidation?

These and many other crucial questions form a seemingly uncontrolled network of possibilities, that makes EDA a major challenge in environmental toxicology and chemistry. Werner however set out to tame this beast. He invited a large number of colleagues to write a comprehensive overview article about all aspects involving and impacting experimental design in EDA. We were among the lucky ones he asked to contribute to "Effect-directed analysis supporting monitoring of aquatic environments — An in-depth overview".

A nature protocol on dioxin-like activity

Dioxin-like activity is an important biomarker for the contamination with several of the most prominent pollutants, such as polycyclic aromatic hydrocarbons (PAH), Polychlorinated Biphenyls (PCB), and Polychlorinated Dibenzodioxins and -furans (PCDD/F).

We have longstanding experience with measuring this activity using a variety of different assays. 

Maybe this is why we were asked by nature protocols to write an article about the bioanalysis of dioxin-like activity. Since nature protocols publishes detailed procedures for laboratory techniques, we decided to communicate why and how to use the micro-EROD assay with the H4IIE permanent rat liver cell line.

The main reason for choosing this particular assay was its peak performance in a direct comparison of alternative bioassays we conducted earlier.

The whole exercise led to a nice article about the "Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells".

Zebrafish-directed identification of culprits

The zebrafish has become a standard test species in ecotoxicology, and a versatile tool for effect measurement and assessment. Being a complex vertebrate organism it allows for a multitude of biomarker investigations on various sublethal endpoints. Since such effects are determined after exposure of a whole animal, environmental relevance is higher than for microscale cellbased assays.

Environmental relevance is also the focus of the integrated approach of effect-directed analysis (EDA). This recursive process of sample testing and fractionation helps to seperate the key toxicity drivers for final chemical identification. Thus, the real culprits for toxicity in environmental samples can be revealed and distinct measures taken for mitigation or remediation.

It sounds hence logical to combine zebrafish testing and EDA for environmental risk assessment. For our EDA Emerge project we reviewed the utility of zebrafish in EDA studies to date, described advantages and shortconings as well as further requirenents for method development.

An evidence on failure in sediment risk assessment

Recently, we put out a paper on an ecotoxicological investigation of sediments from an oxbow lake of the River Rhine. What looks just like another research paper about a study of contaminated sediments holds an important finding about standard sediment assessment: it might fail.

We tested the samples using a comprehensive bioassay battery that goes far beyond the regulatory requirements. Besides the commonly employed test on immobilisation of Daphnia magna, we used assays to reveal estrogenicity, teratogenicity, embryotoxicity and cytotoxicity. Surprisingly, all tests gave clear effects of the sediments, except for the standard test with daphnids.

So, are sediments a risk? Read our discussion and conclusions of this important topic in our paper published in the Journal of Soils and Sediments.

Leo Award for TEPHA paper

Leo Award for TEPHA paper at CIRP LCE 2015

Our paper on the ideas and strategy behind the TEPHA project submitted to the 22nd Conference on Life Cycle Engineering (CIRP LCE 2015), Sydney, Australia, received the Leo Award for the best paper.

Read more about "Growing Near Net Shape Components from Renewable Materials".

Passive samplers and bioassays combined for investigation of dissolved organic contaminants

Already some time ago we put out a paper on the investigation of dissolved organic contaminants using extracts of silicone rubber passive samplers in bioassays in Environmental Science and Pollution Research. This study shows the suitability of such an approach for determination of the bioavailable fraction of organic contaminants in the water phase. The study site was the Ythan catchment in Scotland, UK, and we used zebrafish and marine phytoplancton as test organims.

See all details of  "Evaluations of combined zebrafish (Danio rerio ) embryo and marine phytoplankton (Diacronema lutheri ) toxicity of dissolved organic contaminants in the Ythan catchment, Scotland, UK"

What we propose from what we learned

We were invited by Peter Chapman to write a so called Learned Discourse (LD) for the SETAC journal Integrated Environmental Assessment and Management (IEAM). LDs are described as "timely scientific opinions" and give authors the opportunity to communicate their thoughts on a specific topic. Peter is editor for LDs in IEAM. He asked us to write an LD about the Basel session, but we extended the theme to an overall review of our science communication activities. From that we derived "Three propositions for improved science communication from environmental research".

PAHs from coals are ecotoxicologically active

In continuation of the PAHs-from-coals-story, Wiebke Meyer published additional data and findings on the toxic potential of PAHs deriving from different coal types in the journal Science of the Total Environment.

The article titled "Polar polycyclic aromatic compounds from different coal types show varying mutagenic potential, EROD induction and bioavailability depending on coal rank" reports on mutagenicity and dioxin-like activity of coal-derived PAHs. Wiebke found that the toxic properties differ between coal types, and also some are better available to organisms than others, thus might more likely cause adverse effects.

Constant dosing: reliable exposure concepts to reduce "animal" use

Passive dosing is an approach were the partitioning equilibrium of a substance between a solid and a liquid phase is utilised to maintain exposure concentrations in biotesting. This means that test organisms are exposed at defined and constant concentrations, thus reducing experimental variability and uncertainty, which leads to more robust results.

Together with Philipp Mayer and Kilian E.C. Smith, both formerly of NERI in Roskilde, Denmark, and now with Technical University of Denmark and KIST Europe, respectively, we put out a paper "PAH toxicity at aqueous solubility in the fish embryo test with Danio rerio using passive dosing" in Chemosphere reporting on the usage of PDMS cast into glass vials for passive dosing of non-hatched zebrafish embryos (FET biotest). We show for 10 PAHs constant delivery from the PDMS reservoir to the water phase based on equilibrium partitioning, and prove passive dosing applicable in FET. This test design could in future lead to better data and hence reduced use of zebrafish embryos, albeit legally fish larvae up to 120 hours post fertilization are not considered animals. But we are biologists, we care about any organism.

So how about communication now?

The Basel session raised again strong interest. We chaired a lively session with fantastic talks in front of a full-seated room, once more showing how important this topic is for a large number of colleagues. The authors gave practical tips on blogging and social media usage, they dived into the current situation of science communication of environmental research, and they presented approaches on how to properly disseminate activities and results from running research project. This last aspect seemed to us be of wider importance, why we decided to focus our session proposal for the upcoming SETAC Europe annual meeting in Barcelona, Spain, on more of such background and experience.

The session also helped us to further shape a proposal for a SETAC Europe advisory group an science and risk communication, that is currently being prepared and planned to submit this year.

Find a more detailed description of the session and it outcomes in our session brief as a contribution to the June issue of SETAC Globe.