Dissolved oxygen regulates density-driven accumulation of waterborne L-DOPA and physiological homeostasis in turbot recirculating aquaculture systems

High stocking density in recirculating aquaculture systems (RAS) can promote the accumulation of endogenous bioactive metabolites, yet the environmental factors controlling their formation remain unclear. L-3,4-dihydroxyphenylalanine (L-DOPA) has recently linked to growth inhabitation in crowded turbot ( Scophthalmus maximus ) culture. Here, we used a 2 × 2 factorial design with two dissolved oxygen (DO) levels (HDO, 10 mg/L; NDO, 7 mg/L) and two stocking densities (HD, 9.39 kg/m 2 ; LD, 4.70 kg/m 2 ) to examine whether oxygen availability regulates waterborne L-DOPA accumulation and associated biological effects in turbot RAS. After 60 days, fish under normoxic conditions and high density (NDO-HD) showed marked growth inhibition, whereas HDO restored growth to levels comparable with low-density treatments. Waterborne L-DOPA in the NDO-HD group increased to approximately twice that in the other groups, while HDO prevented this density-associated accumulation. Elevated L-DOPA coincided with increased blood methemoglobin, lactate and cortisol, suggesting impaired oxygen transport, enhanced anaerobic metabolism and systemic stress. It was also associated with sustained activation of HIF-1 signaling pathway, including upregulating of hypoxia-inducible factor-1α ( hif-1α ), erythropoietin ( epoa ) and EGL nine homolog 1( egln1a ), and with disrupted L-DOPA metabolism, characterized by increased tyrosine hydroxylase ( th ) and reduced phenylalanine hydroxylase ( pah ) and dopa decarboxylase ( ddc ) expression. Transcriptomic analysis further indicated that oxygen supplementation reduced stress-related transcriptional responses and supported pathways related to energy metabolism and anabolic growth. These findings identify DO as a key environmental regulator of endogenous L-DOPA accumulation and suggest that oxygen management can reduce endogenous bioactive metabolite buildup in intensive RAS.