Differential histological, cellular and organism-wide response of earthworms exposed to multi-layer graphenes with different morphologies and hydrophobicity

Abstract

The growing use of graphene-based nanomaterials (GBNs) for various applications increases the probability of their environmental releases and calls for a systematic assessment of their potential impacts on soil invertebrates that serve as an important link along terrestrial food chains. Here, we investigated the response of earthworms (Eisenia fetida) to three types of multi-layer graphenes (MLGs) (G1, G2 and G3 with 12–15 layers) with variable morphology (lateral sizes: 7.4 ± 0.3, 6.4 ± 0.1 and 2.8 ± 0.1 μm; thicknesses: 5.0 ± 0.1, 4.2 ± 0.1 and 4.0 ± 0.2 nm, respectively) and hydrophobicity ((O + N)/C ratios: 0.029, 0.044 and 0.075; contact angles: 122.8, 118.8 and 115.1°, respectively). Exposure to these materials was conducted for 28 days (except for 48-h avoidance test) separately in potting or farm soil at 0.2% and 1% by weight. Earthworms avoided both soils when amended with 1% of the smaller and more hydrophilic MLGs (G2 and G3), leading to a decreased trend in worm cocoon formation. The smallest and most hydrophilic MLG (G3), which was easier to assimilate, also significantly inhibited the viability (20.2–56.0%) and mitochondrial membrane potential (32.0–48.5%) of worm coelomocytes in both soils. In contrast, oxidative damage (indicated by lipid peroxides) was more pronounced upon exposure to more hydrophobic and larger graphenic materials (G1 and G2), which were attributed to facilitated adhesion to and disruption of worm membranes. These findings highlight the importance of MLG morphology and hydrophobicity in their potential toxicity and mode of action, as well as ecological risks associated with incidental and accidental releases.