Toxicity of double-walled carbon nanotubes to algae, macro-invertebrates and fish

PhD. (Chemistry)This project assessed the toxicity of double-walled carbon nanotubes to three aquatic organisms belonging to different trophic levels, namely Pseudokirchneriella subcapitata (algae), Daphnia pulex (macro-invertebrate) and Poecilia reticulata (fish). Prior to the toxicity testing, the dry DWCNTs were characterised using scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy and Raman spectroscopy. Dynamic light scattering was used to characterise DWCNT suspensions. Natural water parameters such as increased ionic strength (Ca2+ and Na+) and increased humic acid affected the agglomeration potential of DWCNTs in aquatic medium. Increased ionic strength increased the agglomeration of DWCNTs while humic acid decreased agglomeration. The study explored the lethal/effective concentrations and sublethal effects of DWCNTs on the three organisms. The LC50/EC50 of DWCNTs for the three organisms differed in order of magnitude with D. pulex being the most sensitive and P. reticulata being the least sensitive. The LC50 for D. pulex was 2.81 and 4.45 mg/L for pristine and oxidised DWCNTs, respectively. Pseudokirchneriella subcapitata had an average EC50 of 10.01 mg/L and 10.93 mg/L for pristine and oxidised DWCNTs, respectively. Poecilia reticulata had an LC50 of 113.64 mg/L and 214.0 mg/L for pristine and oxidised DWCNTs, respectively. Exploring the effects of natural water parameters such as humic acid and ionic strength revealed that the acute toxicity of DWCNTs to D. pulex and P. reticulata was increased with increasing humic acid concentrations in exposure media, but increased ionic strength decreased the toxicity of both pristine and oxidised DWCNTs. However, these water parameters all decreased the toxicity of DWCNTs to P. subcapitata. The acute toxicity of DWCNTs was found to be directly linked to their agglomeration state in aquatic systems. Humic acid decreased the hydrodynamic sizes of DWCNT agglomerates making the engineered nanomatrials (ENMs) more available to the organisms while the cations increased the hydrodynamic sizes of DWCNT agglomerates, thereby reducing the probability of interactions with organisms. Time-based survival plots revealed that for P. reticulata and P. subcapitata, there were steady mortality/growth inhibitions throughout the duration of the exposures. For D. pulex, however, the plots revealed that there was a high initial die-off, whereafter mortalities proceeded at different rates. An assessment of whether DWCNTs cause oxidative stress in the three organisms revealed that DWCNTs caused significantly high oxidative stress in D. pulex and P. reticulata but not in P. subcapitata. In D. pulex and P. reticulata, DWCNTs were found to also cause DNA damage. The sublethal toxicity of DWCNTs was affected differently by the humic acid and increased cation concentration in exposure experiments. The sublethal effects were linked to the mode of interaction between DWCNTs and organisms. In P. subcapitata, the interaction was mainly physical with DWCNTs entrapping the algal cells in agglomerates and depriving the algal cells of light for normal photosynthesis to take place. For the other two organisms, the interaction was through intestinal cells as the organisms ingested DWCNTs and through accumulation of nanotubes on the exterior or organisms. The intestinal cell/DWCNT interaction resulted in the excessive generation of reactive oxygen species (ROS) and led to the death of the organism. Humic acid induced the highest antioxidant responses in both D. pulex and P. reticulata and this led to increased DNA damage in both organisms. Increased ionic strengths induced increased antioxidant responses at some DWCNT concentrations but the DNA damage was not significantly increased. These results suggested that with humic acid, the ROS production was excessive and sustained and had an effect on the DNA. The ROS production in increased ionic strengths was not excessive and was not prolonged, reducing their impact on DNA. The use of three organisms to assess the toxicity of DWCNTs provided comprehensive information on the potential effects of these ENMs in the aquatic food chain. Moreover, a multi-tier approach provided information on the potential effect of DWCNTs on populations at sublethal concentrations