The impact of the long-term historical contamination of the Mrežnica River with textile industry wastewaters on metal/metalloid bioaccumulation in the muscles of the young northern pike (Esox lucius) (0+ to 3+) was evaluated, as well as the association of bioaccumulated metal/metalloid concentrations in the muscle with fish physiology. Increased levels of several elements (As, Bi, Cs, Co, Cu, Zn), bioaccumulated in fish muscle, were occasionally found in front of the former factory, but the obtained metal/metalloid concentrations in muscle were in general either comparable or even lower than in the fish from moderately contaminated freshwaters. Calculated target hazard quotients indicated that the current risk for humans, arising from consumption of the northern pike meat originating from a historically contaminated section of the Mrežnica River, was negligible. The influence of biological factors, especially seasonal physiological changes, on metal/metalloid bioaccumulation was confirmed, indicating the importance of the involvement of these parameters in the freshwater pollution assessment, but also in the estimation of the human health hazard. The increased bioaccumulation of several metals/metalloids in fish muscle at a historically contaminated site pointed to the need for continuous supervision of fish health and biodiversity in freshwaters impacted by currently suspended long-term contamination sources.

Oxidative stress is a prevalent mechanism of physiological stress caused by exposure to pollutants or environmental hypoxia in aquatic organisms. The hyporheic zone (HZ), a transitional area between surface and groundwater with distinct ecological conditions, is highly susceptible to pollution and hypoxia, but the physiological responses of its aquatic inhabitants to these stressors remain poorly understood. The aim of this study was to investigate the effect of wastewater treatment plant (WWTP) effluent as a point source of contamination on the battery of biomarkers of antioxidant defence (catalase, CAT; glutathione reductase, GR), xenobiotic biotransformation (glutathione-S-transferase, GST), and aerobic/anaerobic energy metabolism (pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK; lactate dehydrogenase, LDH) in a stygophilous freshwater amphipod Synurella ambulans from the Sava River HZ. The samplings were conducted in relation to the Sava River's hydrologic regime after low river discharge (in September 2020) and after high river discharge (in April 2021) at one site (Medsave) upstream of the WWTP outflow and at two sites downstream (Podsused and Jarun). S. ambulans populations at sites downstream of the WWTP showed higher CAT, GR, GST and LDH activity than population at the upstream site. PK/PEPCK ratio was significantly lower in the S. ambulans populations at Podsused and Jarun sites compared to Medsave population after low river discharge, indicating lower aerobic capacity and greater reliance on anaerobic metabolic pathways. This study improves the understanding of the physiological responses of crustaceans exposed to chemical pollution and environmental stressors, thus contributing to the ecological assessment of groundwater connected ecosystems.