Effects of the anti-inflammatory drug diclofenac and the beta-blocker metoprolol in brown trout Salmo trutta f. fario and freshwater invertebrates at different levels of biological organization

Abstract

Among the wide range of chemicals released into the environment, pharmaceuticals hold a special position. By design, they are physiologically active at low concentrations and their molecular mode of action is conserved across a broad range of taxa. Because of their increasing consumption, incomplete removal in wastewater treatment plants, and persistence in the environment, trace contaminations are frequently detected in aquatic ecosystems. The project “Eff-Pharm” aimed to establish new in vitro biotests for the monitoring of non-steroidal anti-inflammatory drugs (NSAIDs) and beta-adrenergic receptor blockers (β-blockers). Simultaneous in vivo studies were applied to validate these new tests. In this context, the thesis examined the effects of diclofenac (NSAID) and metoprolol (β-blocker) on embryonic and juvenile brown trout Salmo trutta f. fario, the crustaceans Gammarus fossarum and Daphnia magna, the endobenthic oligochaete Lumbriculus variegatus, and the mudsnail Potamopyrgus antipodarum. Diclofenac reduced the survival of juvenile trout already at concentrations of 100 µg/L and animals exposed to 10 µg/L showed an increased frequency of bite marks on fins. Histological examinations revealed that liver, gill and kidney in the control group were already in a state of cellular reaction, but effects in liver were more pronounced in diclofenac-exposed animals. Brown trout embryos were unaffected by diclofenac up to 100 µg/L. Metoprolol did not affect survival, behavior or developmental parameters in juvenile and embryonic trout in concentrations up to 1000 µg/L. In P. antipodarum, metoprolol increased the level of the stress protein Hsp70 in a laboratory setup, indicating proteotoxic action of the substance. When the same organism was exposed in an artificial indoor stream, Hsp70 levels decreased at the highest test concentration, indicating a breakdown of the Hsp70 system. None of the other examined invertebrates showed clear changes of stress protein or lipid peroxide levels in reaction to any of the tested pharmaceuticals. Brown trout embryos exposed to a wastewater treatment plant effluent containing both test chemicals developed in normal time and without effects on survival. However, they were lighter than control animals and showed reduced levels of lipid peroxides, which is indicative of an energy-consuming oxidative stress response. The results demonstrate the hazard of diclofenac for fish health. At the same time, the β-blocker metoprolol has much lower potential to evoke adverse effects in non-target organisms. Juvenile fish reacted with higher sensitivity than embryos – which should be considered in risk assessment. Changes in the stress protein Hsp70 showed to be a sensitive biomarker for gastropods, but not for the other tested taxa. Finally, the complex mixture of substances present in wastewater treatment plant effluents can evoke adverse effects in developing fish larvae. However, it remains to be investigated how these effects transfer to later life stages and how they will affect the population.