Response Against Ethinyl Estradiol and Levonorgestrel of Daphnia Magna: Immobilization and Oxidative Stress Assessment

Abstract

Ethinyl estradiol (EE2) and levonorgestrel (LNG) are synthetic steroids commonly found in contraceptive pills that induce behavioral, physiological and biochemical changes in both human and animal models. The toxic effects of EE2 and LNG are well known, but no reports about their combination exist. Hence, this investigation aims to assess individual and combined effects at low relevant environmental concentrations in a 1:5 ratio similar to that found in most combined birth control pills on a freshwater microcrustacean, Daphnia magna. The acute toxicity of EE2, LNG and their mixture was examined after 24 h and 48 h on daphnia juveniles through the immobilization test, which subsequently enabled the median inhibitory concentration (IC₅₀) to be determined. Additionally, the effects of oxidative stress were examined in D. magna adults over a 48-hour exposure. D. magna immobility was concentration-dependent, decreasing for EE2 and increasing for LNG, while the mixture inhibited motility to a greater extent. In daphnia adults, oxidative stress responses showed a significant decrease in GSH contents and CAT activity, along with an increase in the activity of GST and MDA levels in treated groups compared to the control (p < 0.05). These findings suggest that EE2 and LNG alter the antioxidant system, notably in combination, due to generating reactive oxygen species (ROS) in D. magna.