LAUSR

Abstract In general, the well-being of aquatic ecosystems is a key parameter reflecting ecosystem wealth. To avoid negative influences on fish physiology, the stress state of species must be monitored. β- d -glucose level is one of the key indicators of acute stress in rainbow trout (Oncorhynchus mykiss) juveniles and has been associated with different environmental conditions, including different amounts of chemicals. In the present study, we explore the possibility of assessing the β- d -glucose level in water as a reliable indicator of acute stress in the fish exposed to chemical contaminants. Amperometric biosensor utilizing immobilized into membrane glucose oxidase Aspergillus niger (GOx) for the measurement of low concentration of β- d -glucose in fish holding water was developed and described. Specific layers of the enzymatic membrane allowed to eliminate interfering signals and the biosensor have been fitted for β- d -glucose measurements in fish-holding water. Besides that, the biosensor exhibited several advantageous functional properties, i.e. a wide linear range (0.005–30 mM), high sensitivity (1.07 ± 0.01 µA mM-1cm−2), good selectivity and long-term stability. It was the combination of these biosensor properties that made the simultaneous measurement of β- d -glucose both at low (in water) and high (in fish blood) concentrations and operating in non-pretreated turbid media feasible. Fish were exposed to the primary complex metal (Zn – 0.1, Cu – 0.01, Cd – 0.0015, Ni – 0.034, Pb – 0.014 and Cr – 0.01 mg·L−1, respectively) mixture (MIX) (prepared at maximum-permissible-concentrations for inland waters in European Union (EU)) and MIX5↓ (5-fold reduced (↓) concentration of each metal in the mixture). The obtained results revealed that the blood-glucose level in fish did not change over time significantly, while the level of β- d -glucose released into water by the fish after 3 h exposure to MIX was found to have significantly increased. However, potential stress was observed in the fish exposed to both MIX and MIX5↓ metal mixtures revealing fish sensitivity and proving suitability of the glucose measurement procedure. No significant relationship was found to exist between blood-glucose and water β- d -glucose levels. The results showed that in contrast to blood-glucose measurements, the proposed non-invasive measurement of β- d -glucose in water was beneficial. Thus, this study provides a new approach for the development of a method for stress indication in fish using the measurement of glucose in holding water. Moreover, this glucose measurement procedure can prove to be another valuable tool for stress evaluation method in aquaculture, fish welfare and toxicological research, especially for studying short-term stress responses induced by chemicals.