Non-invasive determination of critical dissolved oxygen thresholds for stress physiology in fish using triple-oxygen stable isotopes and aquatic respirometry

Understanding the critical thresholds of dissolved oxygen (O₂) that trigger adaptive physiological responses in aquatic organisms is long hampered by a lack of robust, non-lethal or non-invasive methodologies. The isotope fractionation of triple O₂ isotopes (¹⁸O/¹⁷O/¹⁶O) during respiration is linked to the amount of oxygen utilised, offering a potential avenue for new insights. Our experimental research involved measuring the oxygen isotope fractionation of dissolved O₂ in closed-system aquatic respirometry experiments with wild sticklebacks (<i>Gasterosteus aculeatus</i>). These fish were either naturally adapted or experimentally acclimated to hypoxic and normoxic conditions. The aim was to observe their oxygen usage and isotope fractionation in response to increasingly severe hypoxia. Initial observations revealed a progressive ¹⁸O enrichment from the preferential uptake of ¹⁶O to a dissolved oxygen threshold of 3–5 mg O₂ L^–1, followed by an apparent reversal in oxygen isotope fractionation, which is mixing of ¹⁶O and ¹⁷O with the remaining O₂ pool across all populations and indicative of a systematic change in oxygen metabolism among the fish. Unexpectedly, sticklebacks adapted to hypoxia but acclimated to normoxia exhibited stronger oxygen isotope fractionation compared to those adapted to normoxia and acclimated to hypoxia, contradicting the hypothesis that hypoxia adaptation would lead to reduced isotope discrimination due to more efficient oxygen uptake. These preliminary experimental results highlight the novel potential of using dissolved O₂ isotopes as a non-invasive, non-lethal method to quantitatively assess metabolic thresholds in aquatic organisms. This approach could significantly improve our understanding of the critical oxygen responses and adaptation mechanisms in fish and other aquatic organisms across different oxygen environments, marking a significant step forward in aquatic ecological and physiological research.

长期以来，由于缺乏可靠、非致死或非侵入性的研究方法，我们对触发水生生物适应性生理反应的溶解氧（O₂）临界阈值的认知一直受到阻碍。呼吸过程中三重氧同位素（¹⁸O/¹⁷O/¹⁶O）的同位素分馏（isotope fractionation）与耗氧量密切相关，为获取全新研究视角提供了潜在途径。本研究通过对野生棘鱼（*Gasterosteus aculeatus*）开展封闭系统水生呼吸代谢（respirometry）实验，测定了其溶解氧的氧同位素分馏特征。这些实验鱼分别经自然适应或实验驯化至低氧（hypoxia）与常氧（normoxia）环境，旨在观测它们在逐步加剧的低氧环境下的耗氧情况与同位素分馏表现。初步观测结果显示，随着对¹⁶O的优先摄取，在溶解氧阈值达到3–5 mg O₂ L⁻¹前，¹⁸O呈现逐步富集；当阈值突破后，氧同位素分馏现象出现明显逆转——此时所有种群的剩余氧库中均发生¹⁶O与¹⁷O的混合，这表明实验鱼的氧代谢模式发生了系统性变化。出乎意料的是，经低氧适应但常氧驯化的棘鱼，其氧同位素分馏强度高于经常氧适应但低氧驯化的个体，这与"低氧适应会因氧气摄取效率提升而降低同位素判别（isotope discrimination）能力"的假说相悖。本初步实验结果凸显了利用溶解氧同位素作为非侵入、非致死手段定量评估水生生物代谢阈值的全新应用潜力。该方法可显著加深我们对不同氧环境下鱼类及其他水生生物的临界氧响应与适应机制的认知，为水生生态与生理研究领域迈出重要一步。