ABSTRACT: Nanoagrochemicals have the potential to increase agricultural productivity while being more environmentally friendly, compared to conventional agrochemicals. However, and given its early days, concerns regarding their risks to human health and environment remain to be investigated. The impact of the commercial nano enabled fertilizer WELGRO® Cu + Zn was previously assessed, and shown to induce a dose- response in the non-target soil living invertebrate Enchytraeus crypticus (Oligochaeta), both in terms of avoidance (2 days, EC50 ca. 300mg WELGRO/kg), and survival/reproduction in longer-term (56 days, EC50 ca. 630mg WELGRO/kg). New Approach Methodologies (NAMs) are on high demand, e.g. omics, allowing to move beyond standard hazards, providing insights in to the mechanism of toxicity of chemicals. The mechanisms of toxicity of WELGRO® are unknown, and in the present study we aimed to investigate it based on high-throughput transcriptomic analysis (4x44K microarray) measuring Differentially Expressed Genes (DEGs). The animals were exposed in the natural soil LUFA 2.2 for 2 and 21 days to WELGRO® (100, 500, 1000 mg WELGRO®/kg), the lower dose corresponding to realistic top soil concentrations, based on the recommended application rates). Results showed that gene transcription was clearly time-dependent. The impacts after immediate exposure (2d) were highest at lowest concentration (100 mg, DEGs = 582) and the opposite occurred for the longer exposure time (21d), at highest concentration (1000 mg, DEGs = 241). Regardless of the exposure period, ABC transporters were shut-down, leading to accumulation of waste products and further endoplasmic reticulum (ER) stress as cause of toxicity. DNA damage seems to also occur as part of the impact. Early exposure (2 days), affected neurotoxicity related pathways, although probably a transient/ reverted impact, as this was no longer observed after 21 days. WELGRO® is probably up-taken (at cellular level) by clathrin-mediated endocytosis, which takes more than 2 days to occur. This study provides insights into the mechanisms of toxicity of a commercially available nano enabled product, based on a realistic exposure scenario for a non-target species.
