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Silver (Ag) is amongst the most well studied nanomaterials (NMs), although most studies have only dealt with a single AgNM at a time and one biological endpoint. We here integrate the results of various testing-tools (endpoints) using a terrestrial worm, the standard ecotoxicological model organism Enchytraeus crypticus. Exposure spanned both water and soil exposure, it covered all life stages (cocoons, juveniles and adults), varying exposure durations (1-2-3-4-5-21 days), and covered 5 biological endpoints: hatching success, survival, reproduction, avoidance and gene expression (qPCR target genes GABA and Acetyl cholinesterase). We tested 4 Ag materials: PVP coated (PVP-AgNM), non-coated (NC-AgNM), the JRC reference Ag NM300K and AgNO3. Results showed that short-term exposure via water to assess impact on cocoons’ hatching predicted longer term effects such as survival and reproduction. Moreover, if we extended the exposure from 11 to 17 day this allowed discrimination between hatch delay and impairment. Exposure of juveniles and adults via water showed that juveniles were most sensitive with survival affected. Across materials the following toxic ranking was observed: AgNO3 ≥ Ag NM300K ≫ NC-AgNM ≥ PVP-AgNM. E. crypticus avoided AgNO3 in a dose-response manner, avoiding most during the first 24 h. Avoidance of Ag NM300K and NC-AgNM only occurred during the first 24 h and the PVP coated AgNM were not avoided at all. The up-regulation of the GABA triggering anesthetic effects, indicated the high ecological impact of Ag materials in soil: Ag affects the GABAergic system hence organisms were not able to efficiently avoid and became intoxicated, this caused impacts in terms of survival and reproduction. Multi-endpoint approach advances the understanding of effects of (nano)materials: towards a mechanistic based risk assessment strategy.

Avoidance behaviour offers a highly relevant information as it reveals the ability to avoid (or not) possible toxic compounds in the field, hence it provides information on reasons for the presence/absence in the field. The earthworm Eisenia fetida was used to study avoidance behaviour to four silver forms (three nanomaterials (NMs) and one salt) over four time points (24, 48, 72 and 96 h), using OECD standard soil. Avoidance behaviour depended on both exposure material and concentration, but in general changed little with exposure duration. Avoidance was highest for the salt (AgNO3) for all exposure durations and showed a continuous higher avoidance with time (based on EC50 values). The AgNMs avoidance was in the order NM300K

This study assessed and monitored 40 consecutive reproduction tests - multigenerational (MG) - of continuous exposure to Cd (at 2 reproduction Effect Concentrations (EC): EC10 and EC50) using the standard soil invertebrate Folsomia candida, in total 3.5 years of data were collected. Endpoints included survival, reproduction, size and metallothionein (MTc) gene expression. Further, to investigate adaptation to the toxicant, additional standard toxicity experiments were performed with the MG organisms of F6, F10, F26, F34 and F40 generations of exposure. Exposure to Cd EC10 caused population extinction after one year, whereas populations survived exposure to Cd EC50. Cd induced the up-regulation of the MTc gene, this being higher for the higher Cd concentration, which may have promoted the increased tolerance at the EC50. Moreover, EC10 induced a shift towards organisms of smaller size (positive skew), whereas EC50 induced a shift towards larger size (negative skew). Size distribution shifts could be an effect predictor. Sensitivity increased up to F10, but this was reverted to values similar to F0 in the next generations. The maximum Cd tolerance limits of F. candida increased for Cd EC50 MG. The consequences for risk assessment are discussed.