Environmental context Freshwater phytoplankton are involved in the biogeochemical cycling of arsenic within aquatic ecosystems via uptake processes. Rather than determining the mean arsenic content in a population of freshwater phytoplankton, we investigate the heterogeneity of arsenic uptake by single-cell ICP-MS. Our data show that arsenic distribution within a cell population may be highly heterogeneous, measured at the femtogram per cell level, and are affected by species and salinity. Rationale An advanced technique has been developed for analysing intracellular elements at the single-cell level using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Compared with conventional inductively coupled plasma mass spectrometry (ICP-MS) analysis, SC-ICP-MS provides uptake data with greater biological relevance. In this study, the use of SC-ICP-MS enabled the quantification of metal concentrations on an individual cell basis down to the femtogram (fg) per cell level. Methodology Three freshwater phytoplankton cells, namely Staurastrum paradoxum (S. paradoxum), Pediastrum duplex (P. duplex) and Scenedesmus acutus (S. acutus), were incubated in 0.1 µmol L−1 arsenate (AsV) solution for 14 days at varying salinity. Cold trap hydride generator atomic absorption spectrometry (CT-HG-AAS) was used to investigate the biotransformation of arsenate under varying salinity conditions. Results The results reveal that cellular arsenic levels decreased as salinity increased in P. duplex and S. paradoxum but increased in S. acutus. The SC-ICP-MS data, which show uptake of AsV by freshwater phytoplankton, were in good agreement with those produced using ICP-MS analysis. Various arsenic management strategies were seen in the phytoplankton species: P. duplex converted it to methylated forms; S. acutus produced organoarsenicals; and S. paradoxum reduced arsenate (AsV) to arsenite (AsIII) and excreted it. Our study also showed changes in the physiological status of phytoplankton following salt stress and arsenic exposure. Discussion Our results confirm the efficacy of SC-ICP-MS in precisely determining arsenic distribution at the single-cell level and reveal differences in intraspecies mechanisms for arsenic cycling in freshwater ecosystems.