To determine the toxicity of the anti-rheumatic drug diclofenac to Daphnia magna, acute toxicity tests according to the OECD guideline 202 were combined with biochemical investigations of the hsp70 level as a biomarker for proteotoxicity. Particular attention was paid to the impact of the solvent DMSO as a confounding factor to diclofenac toxicity by means of testing different variations of producing stock solutions. In the acute immobilisation tests, diclofenac was most toxic as a singular test substance, with indication of a slight antagonistic interaction between the two substances. The highest EC50 values were obtained in those approaches using diclofenac pre-dissolved in DMSO. Thus, the observed antagonism seems to be intensified by pre-dissolution. Hsp70 levels of 12- to 19-days-old D. magna were determined after 48h exposure using a highly reproducible immunological protocol. Hsp70 induction occurred at a LOEC of 30mgl(-1) diclofenac plus 0.6mll(-1) DMSO, and at a LOEC of 40mgl(-1) for diclofenac alone. In summary, DMSO showed only slight confounding effects on diclofenac action in the applied range of concentrations.