Effects of nanomolar cadmium concentrations on water plants - comparison of biochemical and biophysical mechanisms of toxicity under environmentally relevant conditions

Abstract

In this thesis, the effects of the highly toxic heavy metal cadmium (Cd) on the rootless aquatic model plant Ceratophyllum demersum are investigated on the biochemical and biophysical level. The experiments were carried out using environmentally relevant conditions, i.e. light and temperature followed a sinusoidal cycle, a low biomass to water ratio resembled the situation in oligotrophic lakes and a continuous exchange of the defined nutrient solution ensured that metal uptake into the plant was not limited by the nutrient solution, but the capacity of the plant. Above all, Cd concentrations in the nanomolar range were applied and experiments lasted long enough to observe chronic toxicity. The toxicity study revealed that the first site of inhibition was the photosynthetic apparatus. The maximal quantum efficiency of photosystem II (PS II) photochemistry in dark adapted state as well as the PS II operating efficiency in actinic light were the first parameters to be reduced. Only afterwards, an increase in reactive oxygen species (ROS) was observed, indicating that they are the result and not the cause of dysfunctional photosynthesis. For most affected parameters, the respective threshold concentration of inhibition or upregulation due to Cd treatment was 20 nM. This is a much lower concentration than applied in many previous studies. All of the observed effects were more pronounced in plants subjected to Cd stress under high light conditions compared to low light conditions, suggesting a protective role of the comparatively larger antenna system of low light grown plants. Cadmium treatment led to a redistribution of other metals, especially Zn in the tissue of C. demersum as revealed using the non-invasive technique of micro X ray fluorescence (µ XRF) on frozen hydrated leaves. At low Cd concentrations, Zn was found mainly in the epidermis and in the mesophyll. At moderately toxic Cd concentration (20 nM), a higher proportion of Zn was found in the mesophyll. At the highest Cd concentrations, Zn was seemingly stuck in the vein, suggesting that the Zn-exporters were blocked by Cd. As part of the detoxification process, a changing distribution of Cd with increasing Cd concentrations was observed. No Cd was detected in the plants from the control treatment (no Cd added) and a homogenous distribution of Cd all over the leaf section was revealed at low Cd treatment. However, at moderately toxic and highly toxic Cd concentrations, sequestration of Cd into specific organs and tissues was observed. The process of sequestration, transport and storage of the toxic metal is already known from hyperaccumulator plants. It usually results in metal storage in organs and tissues where it interferes least with the sensitive metabolic processes like photosynthesis and respiration, i.e. the vein and the epidermis. Another detoxification mechanism was induced upon Cd treatment in C. demersum: the metal-chelating ligands phytochelatins (PCs) were detected in extracts from the plants. The induction of different PCs was not proportional to the applied Cd concentration, but occurred in a switch-like manner and specifically for each PC species. The most noticeable increase was PC3 at the threshold concentration of 20 nM Cd. A combination of different heavy metals and other factors caused the nearly complete lack of macrophytes in an oligotrophic hard water lake (lake Ammelshain). Within the lake, elevated concentrations of Cd (3 nM), Nickel (300 nM) and reduced Phosphate (75 nM) seemed to be responsible for the lack of aquatic plants and were tested for their inhibitory capability in hard and soft water. While the single treatments with non-toxic concentrations of Cd or slightly toxic concentrations of Ni caused no or only minimal toxicity symptoms in C. demersum, they became highly toxic when applied in combination. This negative effect was even more severe under phosphate-limitation. High concentrations of Calcium and Magnesium in the lake water reduced metal toxicity, indicating additional reasons for the absence of macrophytes in the lake. But regarding other freshwater habitats, these measurements revealed that synergistic metal toxicity may be an important influencing factor for the colonisation of soft waters by water plants. Altogether, the results from this thesis indicate the onset of Cd toxicity and detoxification in a model water plant at a significantly lower level than shown in previous studies.