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Cobalt and cobalt nanoparticles have many applications, for example, in the hard metal industry and in tires. The assessment of long term effects is crucial, as these materials are persistent. For many organism groups, multigenerational (MG) exposure is a highly relevant scenario for persistent materials. In this study, the biological effect of CoCl2 (salt) and Tungsten Carbide Cobalt nanoparticles (WCCo NPs) exposure was assessed in an MG test (4 generations in spiked + 2 generations in clean soil) using the OECD/ISO standard soil test species Enchytraeus crypticus. To ensure transgenerational survival, sublethal concentrations were used to assess the MG impact. MG exposure did not increase toxicity (survival, reproduction). There was an increase in reproduction at low concentrations of Co. Materials were characterized in the exposure media and the organisms in terms of Co content. Uptake of Co-occurred from exposure to both CoCl2 and WCCo, although without toxicity for WCCo. Cobalt from CoCl2 exposure seemed to be stored, whereas for WCCo it was eliminated.

The current testing of nanomaterials (NMs) via standard toxicity tests does not cover many of the NMs specificities. One of the recommendations lays on understanding the mechanisms of action, as these can help predicting long-term effects and safe-by-design production. In the present study, we used the high-throughput gene expression tool, developed for Enchytraeus crypticus (4 × 44k Agilent microarray), to study the effects of exposure to several copper (Cu) forms. The Cu treatments included two NMs (spherical and wires) and two copper-salt treatments (CuNO3 spiked and Cu salt field historical contamination). To relate gene expression with higher effect level, testing was done with reproduction effect concentrations (EC20, EC50), using 3 and 7 days as exposure periods. Results showed that time plays a major role in the transcriptomic response, most of it occurring after 3 days. Analysis of gene expression profiles showed that Cu-salt-aged and Cu-nanowires (Nwires) differed from CuNO3 and Cu-nanoparticles (NPs). Functional analysis revealed specific mechanisms: Cu-NPs uniquely affected senescence and cuticle pattern formation, which can result from the contact of the NPs with the worms' tegument. Cu-Nwires affected reproduction via male gamete generation and hermaphrodite genitalia development. CuNO3 affected neurotransmission and locomotory behavior, both of which can be related with avoidance response. Cu salt-aged uniquely affected phagocytosis and reproductive system development (via different mechanisms than Cu-Nwires). For the first time for Cu (nano)materials, the adverse outcome pathways (AOPs) drafted here provide an overview for common and unique effects per material and linkage with apical effects.

In the present study, the main goal was to compare the effects of ionic copper versus copper nanoparticles in Enchytraeus albidus assessing the effect at the biomarker level, testing different concentrations and exposure times. Measured parameters were lipid peroxidation (LPO), total, reduced and oxidized glutathione content (TG, GSH and GSSG), the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferases (GSTs) and cholinesterases (ChEs). Results showed that both salt- and nano-copper caused oxidative stress and damage to E. albidus, as confirmed by LPO levels, and effects could be discriminated between the copper forms. Nevertheless and despite the visible discrimination between nano and the salt form (time and exposure dependent), there was no single or a set of biomarkers that provided the best discrimination.