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.

Fate of weathered multi-walled carbon nanotubes in an aquatic sediment system.

The widespread application of carbon nanotubes (CNT) in various consumer products leads to their inevitable release into aquatic systems. But only little is known about their distribution among aquatic compartments. In this study, we investigated the partitioning of radiolabeled, weathered multi-walled CNT (14C-wMWCNT) in an aquatic sediment system over a period of 180 days (d). The applied nanomaterial concentration in water phase was 100 µg L-1. Over time, the wMWCNT disappeared exponentially from the water phase and simultaneously accumulated in the sediment phase. After 2 h incubation just 77%, after seven days 30% and after 180 d only 0.03% of applied radioactivity (AR) remained in the water phase. The respective values for the disappearance times DT50 and DT90 were 3.2 d and 10.7 d. Further, minor mineralization of 14C-wMWCNT to 14CO2 was observed with values below 0.06% of AR. In addition, a study was carried out to estimate the deposition of wMWCNT in the water phase with and without sediment in the test system for 28 d. We found no influence of a sediment phase on the sedimentation behavior of wMWCNT in the water phase: After 6.5 d and 7.3 d 50% of the applied wMWCNT subsided in the presence and absence of sediment, respectively. The slow removal of wMWCNT from the water body by deposition into sediment implies that in addition to sediment-dwelling organisms, pelagic organisms are also at risk of exposure to nanomaterials and prone for their take-up.

A trophic transfer study: accumulation of multi-walled carbon nanotubes associated to green algae in water flea Daphnia magna.

Carbon nanotubes (CNT) are promising nanomaterials in modern nanotechnology and their use in many different applications leads to an inevitable release into the aquatic environment. In this study, we quantified trophic transfer of weathered multi-walled carbon nanotubes (wMWCNT) from green algae to primary consumer Daphnia magna in a concentration of 100 µg L-1 using radioactive labeling of the carbon backbone (14C-wMWCNT). Trophic transfer of wMWCNT was compared to the uptake by daphnids exposed to nanomaterials in the water phase without algae. Due to the rather long observed CNT sedimentation times (DT) from the water phase (DT50: 3.9 days (d), DT90: 12.8 d) wMWCNT interact with aquatic organisms and associated to the green algae Chlamydomonas reinhardtii and Raphidocelis subcapitata. After the exposition of algae, the nanotubes accumulated to a maximum of 1.6 ± 0.4 µg 14C-wMWCNT mg-1 dry weight-1 (dw-1) and 0.7 ± 0.3 µg 14C-wMWCNT mg-1 dw-1 after 24 h and 48 h, respectively. To study trophic transfer, R. subcapitata was loaded with 14C-wMWCNT and subsequently fed to D. magna. A maximum body burden of 0.07 ± 0.01 µg 14C-wMWCNT mg-1 dw-1 and 7.1 ± 1.5 µg 14C-wMWCNT mg-1 dw-1 for D. magna after trophic transfer and waterborne exposure was measured, respectively, indicating no CNT accumulation after short-term exposure via trophic transfer. Additionally, the animals eliminated nanomaterials from their guts, while feeding algae facilitated their excretion. Further, accumulation of 14C-wMWCNT in a growing population of D. magna revealed a maximum uptake of 0.7 ± 0.2 µg mg-1 dw-1. Therefore, the calculated bioaccumulation factor (BAF) after 28 d of 6700 ± 2900 L kg-1 is above the limit that indicates a chemical is bioaccumulative in the European Union Regulation REACH. Although wMWCNT did not bioaccumulate in neonate D. magna after trophic transfer, wMWCNT enriched in a 28 d growing D. magna population regardless of daily feeding, which increases the risk of CNT accumulation along the aquatic food chain.