Butyltins (BTs), such as tributyltin (TBT) and dibutyltin (DBT), are toxic to aquatic organisms, but the presence of the strong adsorbent, black carbon (BC), can markedly influence BT toxicity and uptake in organisms. In the present study, the acute toxicity and uptake of TBT and DBT in the crustacean, Daphnia magna, were investigated with and without addition of nano-charcoal at different pHs and water hardnesses. The results showed that the toxicity of TBT and DBT increased by lowering the pH from 8 to 6. This reflects a relatively higher toxicity of cationic BT species than of the neutral species. At pH 6, by enhancing the water hardness of the media from 0.6 to 2.5 mM, the toxicity of TBT and DBT consistently decreased due to competitive binding of bivalent cations (Mg²⁺, Ca²⁺) to biotic ligands of D. magna. Furthermore, the toxicity of TBT to D. magna significantly decreased in the presence of nano-charcoal compared with experiments without nano-charcoal at pH 6 and 8, while no significant decrease in toxicity of DBT was observed in the presence of nano-charcoal. This can be attributed to the insignificant decrease of free DBT concentration in the presence of nano-charcoal compared with that for TBT. Conversely, it was observed that more TBT and DBT were taken up in D. magna in the presence of nano-charcoal due to the uptake of TBT or DBT associated with nano-charcoal by Daphnia in gut systems, as seen by light microscopy. This indicated that only free nonadsorbed BTs were toxic to D. magna, at least during short periods of exposure.