A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with ¹⁴C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of ¹⁴C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs.