This work investigated if the assessment of tolerance to trace metals can depend on plant density in the experimental design. A non-metallicolous and a metallicolous population of Silene paradoxa were hydroponically cultivated at increasing density and in both absence (-Cu conditions) and excess of copper (+Cu conditions). In -Cu conditions, the metallicolous population showed a lower susceptibility to plant density in comparison to the non-metallicolous one, explainable by a higher capacity of the metallicolous population to exploit resources. In +Cu conditions, an alleviationg effect of increasing density was found in roots. Such effect was present at a higher extent in the non-metallicolous population, thus making the populations equally copper tolerant at the highest density used. In shoots, an additive effect of increasing plant density to copper toxicity was reported. Its higher intensity in the metallicolous population reverted the copper tolerance relationship at the highest plant densities used. In both populations, a density-induced decrease in root copper accumulation was observed, thus concurring to the reported mitigation in +Cu conditions. Our work revealed the importance of density studies on the optimization of eco-toxicological bioassays and of metal tolerance assessment and it can be considered the first example of an alleviating effect of increasing plant number on copper stress in a metallophyte.
