To investigate hydrophobic test compounds in toxicological studies, solvents like dimethylsulfoxide (DMSO) are inevitable. However, using these solvents, the interpretation of test compound-induced responses can be biased. DMSO concentration guidelines are available, but are mostly based on acute exposures involving one specific toxicity endpoint. Hence, to avoid solvent-toxicant interference, we use multiple chronic test endpoints for additional interpretation of DMSO concentrations and propose a statistical model to assess possible synergistic, antagonistic or additive effects of test compounds and their solvents. In this study, the effects of both short- (1day) and long-term (2weeks) exposures to low DMSO concentrations (up to 1000 mu ll(-1)) were studied in the planarian Schmidtea mediterranea. We measured different biological levels in both fully developed and developing animals. In a long-term exposure set-up, a concentration of 500 mu ll(-1) DMSO interfered with processes on different biological levels, e.g. behaviour, stem cell proliferation and gene expression profiles. After short exposure times, 500 mu ll(-1) DMSO only affected motility, whereas the most significant changes on different parameters were observed at a concentration of 1000 mu ll(-1) DMSO. As small sensitivity differences exist between biological levels and developmental stages, we advise the use of this solvent in concentrations below 500 mu ll(-1) in this organism. In the second part of our study, we propose a statistical approach to account for solvent-toxicant interactions and discuss full-scale solvent toxicity studies. In conclusion, we reassessed DMSO concentration limits for different experimental endpoints in the planarian S. mediterranea. Copyright (c) 2014 John Wiley & Sons, Ltd. The solvent dimethylsulfoxide (DMSO) is used to investigate the toxicity of hydrophobic test compounds, but can bias the interpretation of test compound-induced responses. We assessed the effects of low DMSO concentrations on the planarian Schmidtea mediterranea, paying special attention to long-term exposures and stem cell responses, two important parameters in carcinogenicity assays. Above 500 mu l(-1), DMSO affected different biological levels. We propose to avoid higher DMSO concentrations, or statistically take into account DMSO-induced effects and solvent-toxicant interactions.