When conducting research, reproducibility and correct assessment of the results obtained are important tasks that require the use of modern methods and approaches. To assess the state of biological systems, significance testing of the null hypothesis with respect to the assumed values of the “norm” is often used. To assess the state of biological systems, a common approach is to test the significance of the null hypothesis in relation to the assumed values of the “norm”. Recently, however, the dichotomous approach of confirming or rejecting the null hypothesis has been criticized by data analysts due to the lack of validation of methods for assessing p-level significance and the biological validity of the hypotheses under investigation. Consequently, a novel methodology for identifying the “normal” state of biological systems was proposed and developed into the theory of reference values (RV). Furthermore, the concepts of reference interval (RI) and reference values have become prevalent in various research fields. Our study evaluated the relative intervals of several antioxidant system (AOS) parameters (glutathione S-transferase, catalase, guaiacol-dependent peroxidase, superoxide dismutase) in the liver and muscles of male three-spined stickleback Gasterosteus aculeatus L. in the Seldianaya Inlet of White Sea during the spawning period. The three-spined stickleback is a species that is abundant in the White Sea and has been the subject of numerous studies on population genetics, evolutionary biology, and ecology. AOS plays a role in the defense of the organism against the negative influence of exogenous and endogenous factors causing oxidative stress. Its indicators are used as biomarkers of the physiological state of the organism. The activity of AOS enzymes in stickleback samples from other biotopes of the White Sea (Sukhaya Salma Strait and Koliushkovaya Lagoon) was also compared with each other and relatively calculated RI. Stickleback from the Koliushkovaya Lagoon and the Sukhaya Salma Strait differed in liver superoxide dismutase activity, and the mean values of enzyme activity were included in the RI. To assess the reproducibility of statistical tests, the effect of stickleback sample size on the result of comparing samples was modeled using t-criterion, and the type of dependence and sample sizes at 50% probability of a positive test were determined. The data obtained on biomarkers of oxidative stress in male G. aculeatus may prove useful for monitoring the state of stickleback populations with the aim of developing this species as a model object for ecological studies of both the White Sea ecosystem and the fish population in this reservoir.
