Embryotoxicity of silver nanomaterials (Ag NM300k) in the soil invertebrate Enchytraeus crypticus - Functional assay detects Ca channels shutdown

Abstract

Despite that silver (Ag) is among the most studied nanomaterials (NM) in environmental species and Ag's embryotoxicity is well known, there are no studies on Ag NMs embryotoxicity in soil invertebrates. Previous Full Life Cycle (FLC) studies in Enchytraeus crypticus, a standard soil invertebrate, showed that Ag materials decreased hatching success, which was confirmed to be a hatching delay effect for silver nitrate (AgNO₃) and mortality for Ag NM300K. In the present study, we aimed to investigate if the impact of Ag takes place during the embryonic development, using histology and immunohistochemistry. E. crypticus cocoons were exposed to a range of concentrations of Ag NM300K (0-10-20-60-115 mg Ag/kg) and AgNO₃ (0-20-45-60-96 mg Ag/kg) in LUFA 2.2 soil, in an embryotoxicity test, being sampled at days 1, 2, 3 and 6 (3, 4, 5 and 7 days after cocoon laying). Measured endpoints included the number of embryonic structures, expression of transferrin receptor (TfR) and L type calcium channels (LTCC) through histological and immunohistochemistry analysis, respectively. Results confirmed that Ag materials affected the embryonic development, specifically at the blastula stage (day 3). The expression and localization of TfR in E. crypticus was shown in the teloblasts cells, although this transcytosis mechanism was not activated. Ag affected calcium (Ca) metabolism during embryonic development: for AgNO_3, LTCC was initially activated, compensating the impact, for Ag NM300K, LTCC was not activated, hence no Ca balance, with irreversible consequences, i.e. terminated embryonic development. An Adverse Outcome Pathway was drafted, integrating the mechanisms here discovered with previous knowledge.

Keywords: Adverse Outcome Pathway (AOP); Calcium metabolism; Embryonic development; Oligochaete; Transcytosis.