Assessing the environmental hazard of mixtures of pharmaceuticals: combined acute toxicity of fluoxetine and propanolol to the crustacean Daphnia magna

The aim of the present work is to evaluate the acute toxic response of the crustacean Daphnia magna exposed to individual pharmaceuticals and mixtures. We tested fluoxetine, a selective serotonin reuptake inhibitor widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization tests were performed according to OECD 202 and ISO 6341 guidelines. Less than 24 h old daphnids were exposed for 48 h. Six replicate vessels with five individuals per vessel were tested at each treatment level. Single chemicals were first tested separately; estimated EC50 were 7.0 mg/L for propranolol and 7.8 mg/L for fluoexetine. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design. Five concentrations (from 0.5 to 2 total toxic units) and 5 percentages of each substance in the mixture (0, 25, 50, 75 and 100%) were tested. The MixTox model was applied to analyze the experimental results. This tool evaluates if and how observed data deviates from the CA (Concentration Addition) or IA (independent Action) models, and tests if significantly better descriptions of the observed data can be achieved using a set of deviation functions. These functions allow a differentiation between synergism and antagonism, along with deviations based on the dose-level and chemical ratio dependency. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. This analysis showed a significant deviation from the CA model that indicated antagonism. Concentrations tested were much higher than those detected in the environment; however these results are to be considered as a first step in an ongoing project aimed at assessing chronic ecological effects of mixtures of pharmaceuticals