© 2022 The Institution of Chemical EngineersFe2+ in ferrocene facilities the oxidation. Based on this phenomenon, increased iron (Fe) level in freshwater ecosystems is thought as an important environmental problem in many geographic regions. In addition to increased mobilization of Fe from sediment due to changes in land use, mining, industrial activity, and elevated acid deposition are also proposed to be possible factors contributing to the increased Fe loading in freshwater environments. Ferrocene is useful in the modern organometallic chemistry industry due to its versatile applications. In this study, the toxicity potential and related toxicity mechanisms of acute ferrocene exposure as well as the protective potential of borax supplementation against ferrocene were investigated in rainbow trout during 96 h under semi-static conditions. In target tissues multiplexed endpoints of hematological indices, genotoxicity, oxidative stress response, DNA damage and apoptosis levels, as well as tumor necrosis factor alpha, and interleukin-6 activities were assessed in blood tissue. In liver tissue, in addition to the parameters studied in blood tissue (except cortisol), the nuclear factor erythroid-2, which regulates the expression of detoxification enzymes, was investigated. When the results obtained from blood analyzes were examined, ferrocen treatment caused different reactions (increase/decrease) in blood indexes, and these findings were confirmed by MN tests. In ferrocene-induced hematoxicite, the healing effect of borax application has been observed to increase inhibited values and decrease in indexes with increasing tendencies. Besides, this hematoxicity was also supported by cortisol increases. Our findings showed that ferrocene inhibited antioxidant enzyme activities and increased lipid peroxidation, 8-OH-dG, caspase 3, TNF-α, and IL-6 levels in both blood and liver tissues. Similarly, cortisol level (in blood tissue) and Nrf-2 level (in liver tissue) increased with ferrocene application. In the ferrocen+borax group, the MDA level decreased 11 % at the end of the 96th hour compared to the 48th hour, and the Nrf2 level increased 9 %. In general, enzyme inhibitions in blood and liver tissues have shown that ferrocen-mediated toxicity occurs in induced ROS, DNA damage, apoptos activity, and BX applications have a positive effect on the correction of toxicity in the direction of hormesis. In a conclusion, the present study suggested that borax migt exhibite ameliorative potential against ferrocene-induced toxicity in O. mykiss blood and liver via regulating the ROS/TNF-α/Nrf-2 pathway.