Examining the suitability of molecular and metabolomic-based techniques as tools for assessing the effects of pharmaceuticals in the aquatic environment

Abstract

Pharmaceuticals represent important and indispensable elements in modern society and their usage is considerable. Post consumption and body-elimination, pharmaceuticals are not completely removed in sewage treatment works (STWs) and as such, have been detected at low levels in STW effluents, surface waters, seawaters, ground waters and some drinking waters. Accordingly, pharmaceutical toxicity has been detected in several aquatic organisms. To date, environmental risk assessments (ERA) examine for toxicity using a series of chronic toxicity assays that examine for standard physiological responses in algae, Daphnia and fish and do not address pharmaceutical mode of action. Therefore, using the fathead minnow (Pimephales promelas) and the β-blocking pharmaceutical propranolol as the test-species and test-drug, respectively, the aim of this study was to establish an intelligent targeted 4-phased ERA using molecular, in vivo exposure, metabolomic and quantitative expression analytical techniques. The first phase established that the fathead minnow expressed the β3bi-adrenergic receptor (AR), which is a target receptor for propranolol in humans. The in vivo pair-breeding assay suggested that at 1mgL-1 and 10mgL-1, propranolol levels in fish blood plasma exceeded the human therapeutic concentration and caused 80% and 100% mortality, respectively. The most likely causes of mortality were liver failure and central nervous system toxicity. It was not possible to identify a robust biomarker of propranolol exposure using proton nuclear magnetic resonance (1H NMR) as there was considerable metabolic variation between male liver tissues within the same treatment groups. β3bi¬¬-AR expression was significantly lower at 1mgL-1 in the brain and liver, which was most likely the result of desensitisation in response to elevated levels of epinephrine and cortisol. β3bi¬¬-AR expression was significantly increased in the heart at the environmentally relevant concentration of 0.001mgL 1, however it was not possible to link β3bi¬¬-AR expression to a toxic response. Propranolol is unlikely to pose a threat to the aquatic environment as the concentrations measured in the environment are approximately 1000-fold lower than those that induced a toxic response. The proposed ERA represents a marked improvement over the existing ERA as it addresses pharmaceutical mode of action and both subtle and physiological toxicity responses, however it still requires further validation studies to address both metabolomic and gene expression variation.