ESR4 Bettina Gro Sørensen

Project: Effect-directed identification of water-extractable toxins in alien species that expand their distribution with climate change

Principal supervisor: Dr. Werner Brack

Intro to project:

“There are currently over 12,000 plants, animals, fungi and micro-organisms in the EU that are alien to their natural environment. Some 15 % of these species are invasive, and their numbers are rapidly growing.” (European commission 20161)

Research and policies on invasive species are most often focused on the loss of biodiversity as the invasive species outcompete the local flora, e.g. in terms of shadowing and nutrient competition. Further, in several cases, the invasive species form dense populations, which sheer mass pose challenges for the anthropogenic use, e.g. when populations of Elodea crowd the otherwise free waters of lakes and makes competitive rowing impossible – without mowing the invasive plant. However, very little research has been performed on the potential toxins that these expanding plants may contain and release into the environment, at risk for both the ecosystem and the anthropogenic use of the water resources. This Ph.D. work thus sets out to assess and identify which plants may potentially cause a risk and to identify whether these plants contain any toxins of concern. The method is effect-directed analysis, pioneered and refined by supervisor Werner Brack, in which one works from the identification of effects of a certain mixture (here plant extracts with an unknown composition of biogenic chemicals) to identification of the causing compounds through an iterative process including fractionation and bio-testing. The objectives of the Ph.D. are thus

  • Literature review on toxins and toxic effects of invasive species
  • Effect-based screening of toxins in extracts of selected invasive species using an array of cell based reporter gene assays addressing endocrine disruption (AR, ER, GR, RAR, RXR), mutagenicity, adaptive stress responses (oxidative stress, inflammatory response and DNA repair) and immunotox effects
  • Identification of new toxins using effect-directed analysis and structure elucidation by LCQExactive MS/MS and in silico methods for prediction of physico-chemical properties, fragmentation, and toxicity

Further, several crops are expanding their distribution in Europe and abroad, as the awareness of global climate change leads populations and governments to look for alternatives to fossil fuels as energy resources. As these by now cover a large percentage of the European landscape, samples of these will be included in order to investigate whether they pose a risk to our drinking water bodies.

Major conclusion
Line of work 1: Toxicity and risk assessment of identified plant secondary metabolites in the water environment
Based on the plant secondary metabolites that have been detected in the water environment in NaToxAq, I identified which ones pose the largest environmental risk in terms of low EC50 values and high Risk Quotients (RQ) / Toxic Units (TU). This can be used to target which compounds should be included in further monitoring and give experimental support for the results of the database-based prioritization of colleagues: The compounds showing the highest RQs are especially Amaryllidaceae alkaloids, and alkaloids generally, also high on the a priori prioritization results. The results are based on Daphnia magna so far, while work on other organisms is in progress.  The results show that most of the detected compounds only have little toxicity, both when compared to predicted baseline toxicity and when calculated as risk quotients, however a few compounds show a significant risk, with risk quotients >1.

2) Line of work 2: Effect-based analysis for the identification of novel compounds of concern in water extracts of invasive species, spreading with climate change
Effect-directed analysis (EDA) is a time-consuming type of work, which includes repeated effect-testing on bioassays and chemical analysis. The work started out with identifying invasive species of potential concern based on (1) proximity to water resources, (2) local abundance (that is, dense plant cover), (3) wide and increasing distribution with climate change and land use, and (4) likelihood of containing toxic compounds. 25 plant species were sampled, and an extraction protocol set up, including optimization of the SPE method using a multilayered-cartridge. Screenings of parent plant extracts on the in vitro reporter gene assays ERa-GeneBLAzer, AREc32 and Danio rerio embryos, as well as the in vivo organism Daphnia magna did unfortunately not show any large distinctions between plant species and no sublethal effects could be identified in the reporter gene assays. It was therefore decided to fractionate several plant extracts through prep-HPLC on a reverse phase C18 column. Repeated testing has shown estrogenic effects of one plant species, as well as cytotoxic effects of several plant species. Work is on-going regarding identification of the causing compounds.

1) http://ec.europa.eu/environment/pdf/13_07_2016_news_en.pdf