Ecotoxicology | University of Tübingen

Environmental Toxicology (Ecotoxicology)

Ecotoxicology is a multidisciplinary science that evolved from environmental chemistry and toxicology. It is generally concerned with the effects of chemicals on the living environment. Due to industrialization and the absolute ignorance of the effects on our ecosystems at that time, large amounts of contaminants have been accumulated in different habitats over the years, which still enter our environment through
remobilization from lake sediments and farmlands. Due to today's environmental awareness, laws (REACH) and directives (e.g., EU WFD) should help to protect habitats and stop the loss of biodiversity. Thus, today newly produced chemicals and active substances are ecotoxicologically tested for their acute and chronic effects on different levels (from the cell to the ecosystem). Over time, research within ecotoxicology has developed rapidly, allowing different new branches to emerge, e.g., to understand past effects of contamination, but also to provide insight into future contamination scenarios.

Evolutionary Toxicology within Resurrection Ecology
The resilience of an organism is characterized by a good adaptation and the resulting robustness to constantly changing environmental conditions. Due to increasing anthropogenic influences such as population growth, intensification of agriculture, advancing global industrialization, and the climate change associated with it with increasing evidence, conditions in different ecosystems are changing rapidly, making fast and targeted adaptation difficult for many organisms. Evolutionary toxicology is largely concerned with these adaptation strategies and with the effects of anthropogenic long-term effects on different organisms. These effects can be studied at both the organismal and molecular levels.

Using the model organism Daphnia magna (large water flea), we are now able to look at effects over a period of several hundred years. This evolutionary and unique insight is provided by the very specific reproductive behaviour of the genus Daphnia. Due to an alternation between asexual and sexual reproduction depending on changing conditions in their habitat, they are able to survive over a period of several thousand years in limnic still waters. So-called ephippia (permanent eggs) are produced through initiated sexual reproduction. These ephippia are very resilient and the offspring inside can survive for a very long period of time. The ephippia sink to the sediment of the aquatic habitat after being released by the mother. They remain there until conditions improve and hatch again. If no improvement occurs, the ephippia become inactive and may persist as permanent stages embedded in lake sediments for decades or even centuries. In Resurrection Ecology, these ephippia are recovered from urban lake sediment using a sediment core and the Daphnia within are brought back to life. Radiocarbon dating is used to determine the age of the sediment and thus the age of the resurrected Daphnia layer by layer.

Abbildung 1: Projektdesign mit Betrachtung von unterschiedlichen Endpunkten

Based on this phenomenon of survivability, we are able to travel back in time and look at geno- and the resulting phenotypic changes over a long period of time within evolutionary toxicology. In doing so, we will test the effects of chemical exposure and climate change as multiple stressors on modern and ancient Daphnia clones. With respect to chemical exposure, the polycyclic aromatic hydrocarbon (PAH) phenanthrene will be used. This pollutant is particularly suitable because it is formed by incomplete combustion and spreads ubiquitously through the air in the environment. Furthermore, PAHs have already
been formed in earlier ages and thus also represent a historical pollutant. The project will be structured in such a way that three different stressor states will be investigated. For this purpose, acute, chronic, and genetic effects are to be detected for the clones of different ages at the two sites (Fig.2). Based on this phenomenon of survivability, we are able to travel back in time and look at geno- and the resulting phenotypic changes over a long period of time within evolutionary toxicology. In doing so, we will test the effects of chemical exposure and climate change as multiple stressors on modern and ancient Daphnia clones. With respect to chemical exposure, the polycyclic aromatic hydrocarbon (PAH) phenanthrene will be used. This pollutant is particularly suitable because it is formed by incomplete combustion and spreads ubiquitously through the air in the environment. Furthermore, PAHs have already been formed in earlier ages and thus also represent a historical pollutant. The project will be structured in such a way that three different stressor states will be investigated. For this purpose, acute, chronic and genetic effects are to be detected for the clones of different ages (Fig.1).

Abbildung 2: Ergebnisse einer Proof-of-Concept-Studie der Labore Hollert (Ökotoxikologie) und Rösch (Vegetationsgeschichte, Archäologie) zur Untersuchung von Dioxin-ähnlichen Aktivitäten (bioassay-derived toxicity Äquivalenzquotient; BEQ)

Testing for dioxin-like potency
Dioxins are organic chlorinated compounds that are among the most dangerous known environmental pollutants. Nowadays, 210 PCDD isomers of extreme hazardousness are known. They are formed in all combustion processes in the presence of chlorine and organic carbons (from a combustion temperature of 300-900°C). Dioxins have gained particular importance in the past due to the chemical accident in Seveso (1976), in which the most toxic dioxin 2,3,7,8 tetrachloro-dibenzo-p-dioxin (TCDD) was released into the environment, and due to the use of dioxin-contaminated herbicides (Agent Orange) in the Vietnam War (1961-1971). Nowadays, these substances are now regulated by prohibition orders. However, due to the increased release in the past and the very long half-life of dioxins, they remain present in our ecosystems today as contaminated sites. Due to flood disasters and heavy rain events, they can be remobilized and re-introduced into our environment and/or taken up by organisms.

Within the Bad Waldsee project the dioxin-like potency in different sediment layers of the city lake will be investigated by means of a bioassay for different annual periods. To measure the dioxin load of an environmental sample, the biomarker "EROD" (ethoxyresorufin-O-deethylase) is usually used. EROD is a liver enzyme that is produced by organisms in direct response to contact with dioxin-like substances in order to eliminate them from the body. The enzyme, which belongs to the CYP1A enzyme family, can convert the substance ethoxy resorufin to the fluorescent dye resorufin. Thus, in biological test systems such as the micro-EROD assay, after exposure of a cell culture to the environmental sample, the fluorescence intensity can be directly related to the dioxin-like load of the tested sample. Thus, in summary, dioxin-like potency can be measured within sediments. A proof-of-concept study conducted in 2018 by Hollert et al. has already provided preliminary findings from lake sediments in the Stadtsee (Fig.3). This examined dioxin-like efficacy within sediments for the modern period (likely older than the 18th century AD), the High Middle Ages (10th to 12th centuries AD), and the Iron Age (ca. 1050 BC to 1 BC). Here, the results show the Bioanalytical Equivalents (BEQ) related to the respective sediment depth. Bioanalytical equivalents (BEQs) of mixtures and environmental samples are commonly used to reflect the potential threat of contaminants in the environment and can be determined by bioassays or by chemical analysis in conjunction with relative potencies (REPs).