Fate of 17alpha-ethinylestradiol in the aqueous environment and the associated effects on organisms (UBC)
Prof. Dr. Hans Toni Ratte
Marie Curie Early Stage Research Fellowship of the European Community's 6th Framework Programme (Marie Curie Programm Aquabase)
Prof. Dr. J. Hollender
Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of environmental chemistry, 8600 Duebendorf, Switzerland
Dr. C. Schäfers
Fraunhofer-Institute for Molecular Biology and Applied Ecology, Department of Aquatic Ecology and Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, Germany
In aquatic systems, the bioavailability of a compound is dependent on numerous factors such as partitioning between water, different organisms and solids, biotransformation and food web transfer. This project dealt with the fate of an important environmental xeno-estrogen, 17 -ethinylestradiol (EE2), in the aquatic environment. Therefore, the kinetics of EE2 in indicator species representing the different trophic levels of an ecosystem were assessed. As primary producers, green algae (Desmodesmus suspicatus) were selected. The water flea Daphnia magna and larvae of the midge Chironomus riparius were introduced as primary consumers of the water phase and the sediment, respectively. Finally, water as well as dietary uptake of EE2 were investigated in a target species and secondary consumer: zebrafish (Danio rerio).
In a first series of experiments, uptake of 14C-labelled EE2 (14C-EE2) from the water phase and elimination by the different organisms were investigated over time. In a second test series, both primary consumers were fed 14C-EE2 spiked algae in order to study bioaccumulation. Uptake of 14C-EE2 by chironomid larvae after water and sediment spiking was compared, including sediments of different composition. In a third series of experiments, male fish were short term (48 h) exposed to 14C-EE2 through different routes: by water exposure (WE) and by dietary exposure (DE) via both contaminated daphnids and chironomid larvae. Distribution of 14C-EE2 in the fish was studied by measuring the amount of radioactivity (RA) in the different fish tissues. Additionally, the effect of EE2 on the vitellogenin (Vtg) induction in male fish was compared after WE and DE in a long term (14 d) experiment. The RA in liquid samples was quantified by means of liquid scintillation counting (LSC). Solid samples were subjected to combustion in a biological oxidiser, trapping (14)CO2, measured with LSC. Water and organism extracts were analysed by means of HPLC with a radiodetector, except for algae extracts that were subjected to TLC. Metabolites were identified with GC-MS, high resolution LC-MS and enzymatic hydrolysis followed by HPLC with radiodetection. Metabolites, detected in the water phase, were tested for estrogenic activity by means of YES and ER-CALUX assays.
Accumulation and effects
Of the four organisms mentioned above, bioconcentration of 14C-EE2 was highest in the algae. Whereas the growth rate of D. subspicatus was significantly affected at high EE2 concentrations compared to unexposed algae, EE2 had no acute effects on D. magna and C. riparius. Daphnids showed a higher bioaccumulation potential after exposure via spiked algae. For chironomids, water exposure was the predominant uptake route. The presence of sediment lowered the bioavailability of 14C-EE2 to the larvae after both water and sediment spiking. Nevertheless, uptake was higher when the nutritional quality of the sediment was better. After 24h of exposure, the relative amount of RA taken up by dietary exposed fish, fed chironomid larvae, was larger than by water exposed fish. Daphnid exposed fish took up only negligible amounts. These results were confirmed after evaluation of the long term experiment. Fish exposed via water and contaminated chironomids had highly elevated blood Vtg contents compared to control fish, whereas no effect on fish fed with spiked daphnids was observed.
Metabolism and elimination
Algae metabolised the compounds into two more hydrophobic compounds over time. This biotransformation process was proven to take place under dark/respiration conditions only, and resulted in complete transformation of EE2 within 2 days at high algae cell density. The products were detected in both algae and water and showed a lower estrogenic potency than EE2 in both in vitro assays. After 14C-EE2 exposure of daphnids, several metabolites were detected in the water phase. On the contrary, only untransformed EE2 was present in the water after exposure of both larvae and fish. The amount of radioactivity in daphnids and chironomids during elimination could be described using a one compartment model. Daphnids additionally eliminate a high portion of incorporated RA during moulting and hatching of neonates.
Distribution in fish tissues after exposure via different routes
The highest amount of RA could be detected in the gut and the skin & filet of both WE and DE fish. The relative amount of RA in the gut was higher in DE compared to WE fish. No RA could be detected in the brain of DE fish, whereas the brain of WE fish contained a considerable amount of RA. The most important result of this experiment, is that there was no difference in the relative amount of RA detected in the liver of DE and WE fish, which is the target organ for Vtg induction.
This study shows that the fate of EE2 is influenced by organisms of different trophic levels. Although little research on bioaccumulation and effects of this compound in organisms at the basis of the food chain has been performed, algae highly accumulate the compound and transform it into more lipophilic, but less estrogenic products. Daphnids showed little bioconcentration of EE2, but accumulated more radioactivity after exposure to spiked algae. The bioavailability of EE2 is strongly reduced in presence of sediments, as was tested with chironomid larvae. However, in a system containing only sand as substrate, concentration of EE2 in chironomids was still substantial. Uptake of conjugated EE2 by male fish through a diet of chironomid larvae resulted in similar internal fish concentrations and blood Vtg levels as uptake of EE2 via the water.