Assessment of combined toxic and genotoxic effects of soil metal pollutants: a laboratory and a field experiment using the test plant Trifolium repens L.

The use of efficient early warning bioindication systems represents a powerful approach for assessing and interpreting the impact of natural or anthropogenic perturbations in soil ecosystems preventing environmental alteration and human disease. Living organisms provide information on the cumulative effects of environmental stressors and as such bioindication is complementary to direct physical and chemical measurements (Heger et al., 2012). Trifolium repens is a pollutant-sensitive plant, suitable for biomonitoring campaigns. Specifically, its environmental exposition followed by a DNA analysis with molecular markers allows the detection of sublethal levels of genotoxic compounds in the environment (Piraino et al., 2006). However, given the limited information available on the joint genotoxic-effect of chemicals, the interpretation of biomonitoring results is often difficult. There is then a clear need to improve the knowledge about the combined effects of stressors on bioindicators. Starting from these considerations, the objective of the first part of my PhD research was to study the combined toxic and genotoxic effects of soil Cd and As, two of the most dangerous compounds for both environment and human health, whose joint action is still unknown. The results that I obtained showed that individual and joint toxicity and genotoxicity were related to the concentration of Cd and As measured in plant organs and that As concentration was the most relevant variable. Joint effects on plant growth were additive or synergistic, whereas joint genotoxic effects were additive or antagonistic. The interaction between Cd and As occurred at both soil and plant level. in soil the presence of As limited the bioavailability of Cd, whereas the presence of Cd increased the bioavailability of As. Nevertheless only the As biovailability was linearly correlated with the amount of As absorbed by plants. These results revealed that the simultaneous presence in soil of Cd and As, although producing an additive or synergistic toxic effect on Trifolium repens L. growth, generates a lower DNA damage. I have supposed that growth reduction was due to both toxic effects of Cd and As and to plant response to the high DNA damage, which led to a temporary arrest of cell cycle providing a longer time for DNA repair and for free radical scavenger production. This would be consistent with the antagonistic genotoxic effect that I observed in most of the combined treatments. Nevertheless I am also aware that the antagonistic interaction of Cd and As that I observed can be also associated to the similar genotoxic mechanisms own of the two metal(loid)s. In the second part of my PhD, I used the information and the techniques that I learned during the first period to assess the genotoxicity of soils in Lombardy Region (Italy). I analyzed a total of 67 samples of surface soil (0-30 cm in depth) which were collected in 7 different agricultural areas of concern, within Lombardy region. Results showed that in general soils did not affect the survival of the test plants, excepted for the soils CR3 and CR6 (from the area of Treviglio) and O1 (from the Origgio area). Furthermore, no statistically significant change was observed in the growth of seedlings (measured in terms of dry weight), except for some soils from the Treviglio area (CR2, CR3, CR6 and CR14) and for IT5 soil from the Broni area (PV). The other soils from this last area, although induced a reduction of root growth, were considered not potentially toxic as the shoot growth of the text plants was not affected and the soil characteristics were not appropriate for white clover development. Regarding the soil genotoxicity, all soils except those from Broni area were found to contain genotoxic bioavailable compounds and were classified as "moderately genotoxic”.