Large amounts of carbon nanotubes are already being manufactured, which will lead to increasing concentrations of nanomaterials in the environment through accidental spills, product use and degradation, or known releases. Previous research has used acute toxicity tests to assess the potential toxicity of CNTs to freshwater invertebrates and fish, yet few chronic studies have been conducted to assess nonlethal effects. We studied chronic effects of 14C-labeled multi-walled carbon nanotubes (MWNTs) on Ceriodaphnia dubia, an aquatic invertebrate. For chronic exposures 14C- MWNTs were solubilized in moderately hard water (MHW) by four different methods: bath sonication, probe sonication, bath sonication followed by addition of Sewanee River natural organic matter (NOM, 2.3 mg C/L), and by stirring nanotubes overnight in NOM dissolved in MHW. Standard 3-brood chronic toxicity tests were conducted following EPA protocol. Reproductive effects (number and size of broods) and
accumulation of nanotubes by adults and neonates were measured in replicated experiments for each solubilization treatment.

Ceriodaphnia exposed to bath-sonicated MWNTs had significantly smaller brood numbers and size at the 2.5 mg/L concentration (LOEC), compared with controls. Exposures with probe-sonicated nanotubes showed less reproductive toxicity, with a LOEC of 5 mg/L. No reproductive toxicity was observed for nanotube exposures with added NOM (LOEC > 5 mg/L). Reproductive toxicity of the bathsonicated nanotubes may be related to association of the MWNTs onto the body surfaces of the adults,
which likely interfered with molting and prevented neonate release (Fig. 1).

We hypothesize that the surface association and reproductive toxicity of the bath-sonicated nanotubes is related to the larger nanotube size and higher degree of aggregation in these treatments. Probe sonicated MWNT solutions were more transparent and may have contained smaller-sized nanotubes. Solubilization of MWNTs in NOM likely increased their water solubility, reducing their potential to associate with adult body surfaces. However, neonates exposed to the NOM-solubilized nanotubes accumulated 14C-nanotubes in a concentration-dependent manner, suggesting ingestion of these more soluble nanotubes. Neonates and adults exposed to bath-sonicated MWNTs randomly accumulated 14C-MWNTs, likely by adherence of larger aggregates to body surfaces. This is one of the first known studies to document reproductive effects and accumulation of MWNTs in aquatic invertebrates and emphasizes the need to assess chronic, non-lethal effects of nanomaterials in aquatic organisms.