Multiple contamination with radionuclides and heavy metals: characterizing plant stress responses using duckweed Lemna sp. (PhD)

Certain radionuclides and heavy metals are naturally present at trace amounts in the environment. Anthropogenic activity has, however, lead to enhanced levels of many of these components leading to environments containing toxic levels of one or a number of these contaminants. The need for investigating potential risks induced by these contaminants focusing on the environment in a general sense, on non-human biota and on ecosystems, is now internationally recognized.

Radionuclides are rarely released as sole contaminant in the environment but co-occur in a mixture of pollutants for example metals, pesticides, organics. Typically radioactive elements such as ²³⁸U, ²²⁶Ra or ²³²Th and non-radioactive pollutants such as Cu, Cd, Zn, Ni and As are simultaneously present (van der Graaf, 2007, Appl Radiat Isot 65, 619-633). For chemical toxicants governmental institutes and regulators use experimentally derived screening values in risk assessment to set discharge levels for the industry. At this moment these values are based on one stressor-one organism experiments and at best, for situations where more than one stressor is present, additive interactions are considered. Research has, however, indicated that in multiple contaminant exposure conditions additive but also synergistic or antagonistic effects can occur. As such we showed that exposure of Arabidopsis plants to a combination of ²³⁸U and Cd had different effects on nutrient profiles and on expression of the antioxidative enzyme ascorbate peroxidase compared to the single stressor situation (Vanhoudt pers. com.). Using Lemna aequinoctialis, a tropical Lemna species, Charles and coworkers (2006, Chemosphere 62, 1224-1233) were able to show antagonistic effects on growth in a combined Cu-²³⁸U situation.

Based on the current knowledge of toxic effects of contaminants simultaneously present in the environment the hypothesis is build that current screening values for radionuclides might be underestimating the actual risks and need to be validated in a multiple stress situation. In order to understand and to be able to model multiple stressors, knowledge on mechanisms governing these multiple stressor effects needs to be assembled.

This research proposal aims at analyzing biological effects in Lemna minor induced by two natural occurring radionuclides ²³⁸U and ²³²Th and the heavy metals Cd added either as single pollutant or combined as binary mixtures. Special focus will be on possible mixture effects resulting from exposure to a combination of these pollutants. We will aim at  (i) Modeling of effects of multiple stressors: possible synergistic additive reactions and (ii) Validating existing screening levels in a multi-pollution set-up or as influenced by changing environmental parameters and (iii) at delivering new information on mechanisms underlying the toxicity of radionuclides and heavy metals in Lemna minor. A major part of this project will be to develop a stress-responsive Lemna specific microarray that will enable us to understand the mechanisms and mode of action of the radionuclide and heavy metal toxicants.

 Research proposal

Lemna sp. (Lemnaceae, duckweeds) was chosen as model system. Lemna sp. are monocotylodenous aquatic macrophytes consisting of floating plant bodies (fronds) and submerged greenish roots. A Lemna growth inhibition test (OECD guideline 221) has been optimized and is internationally recognized as an ecotoxicological test. It has been assigned as a more sensitive bioassay test for assessment of heavy metal ecotoxicological risks than human cells, protozoans, bacteria and fish and ideal for testing the impact of aquatic pollutions (Fenske, 2006, Int J Hyg Environ Health 209, 275-284). In addition Lemnaceae are known hyper-accumulators to a number of heavy metals and the toxicity of Lemna to a range of heavy metals has been tested (Naumann, 2007, J Plant Physiol 164, 1656-1664). As such they can potentially be used for phytoremediation or rhizofiltration programs.

Following the growth inhibition ecotoxicologial test the concentration of the individual contaminants (²³⁸U, ²³²Th, and Cd ) resulting in 10, 30 or 50% growth inhibition (EC_10, EC₃₀ en EC₅₀ ) on the different growth parameters of the Lemna colonies (frond number, frond area, total area, fresh and dry weight) will be determined and bioaccumulation of the compounds in the plant tissue will be assayed. In addition to the one-stressor experiments, Lemna plants will be exposed to mixtures of the different radionuclides and heavy metals each at their EC_10, EC₃₀ or EC₅₀ concentration. These data will be fed in existing models to estimate possible synergistic/antagonistic interactions and dose ratio or dose level dependent effects between the different compounds as described by Jonker and co-workers (2005, Environ Toxicol Chem 24, 2701-2713).

To understand the mechanism of the stress reactions and to be able to more accurately implement it in models the response of Lemna colonies exposed to  ²³⁸U, ²³²Th, Cd will be analyzed in more detail. A custom made cDNA-microarray will be developed containing oxidative stress responsive genes using suppression subtractive hybridization PCR as has been described for Daphnia (Moens et al 2007, Comp Bioch Physiol (D) 2:18-33). Once developed this array will give mechanistic insights of the toxicological responses. Genomic data will be complemented with a physiological and biochemical characterization on organism/tissue level (morphogenetic responses, photosynthesis) as well as on a sub-cellular level for example as the induction oxidative stress.

The data obtained in this project will help to understand fundamental process regulating stress response mechanisms in higher plants and adapting themselves in to complex environmental conditions. In addition, this project will contribute to set up and optimize existing reference levels and screening values for radionuclides and heavy metals present in industrial effluents.