This study investigates the impact of ozonation on the concentration of two androgen steroid hormones, testosterone and androstenedione, and on androgenic activity as measured using the YAS assay. While these compounds are remarkably similar in chemical structure and showed comparable removal profiles in the presence and absence of a hydroxyl scavenger (isopropanol), large differences between percent removal of target compounds using chemical analysis and percent removal of bioactivity was observed when small ozone doses were applied. The dynamic change and initial increase in bioactivity during ozonation can be attributed to transformation products. These results demonstrate the importance of combining chemical analysis and bioanalytical techniques not only to perform a comprehensive assessment of environmental risks but also as a tool to optimize treatment technologies proposed as a mean to mitigate the impact of contaminants of emerging concern. (C) 2017 Elsevier B.V. All rights reserved.