The nano-form of copper (Cu-NPs) is already extensively used. In this paper the toxic effect of Cu in the worm Enchytraeus crypticus (Oligochaeta: Enchytraeidae) was assessed following exposure to (1) Cu-salt: freshly spiked soil with copper-nitrate, (2) Cu-NPs: freshly spiked soil with Cu nanoparticles (80nm), and (3) Cu-field: historically Cu contaminated soil (80years ago). Our main aims were to compare the three different exposure regimes and respective toxicity, and to determine how the oxidation state of the Cu and dissolution state of the particles differed. Characterization of in situ-exposure included identification of oxidation states with synchrotron generated X-ray absorption near-edge spectroscopy (XANES) analysis, activity of free Cu(2+) in soil-solution (Ion Selective Electrode), and the relative distribution of the labile Cu-fractions (Sequential Extraction). Freshly spiked Cu-salt was the most toxic for reproductive output of the worms, followed by Cu-NPs and then Cu-field. XANES indicated only one oxidation state (II) in Cu-salt and Cu-field soil, whereas in Cu-NPs soil it was present in all oxidation states (0, I and II). The partial oxidation of the Cu-NPs (in soil) was evident and with limited dissolution.