The modes of action of pollutants are diverse, and a common consequences to pollutant exposure is oxidative stress. This phenomenon is caused by an imbalance or disurption in the control of Reactive Oxygen Species (ROS) resulting in an accumulation of free radicals. Oxidative stress may cause damages to the DNA, phospholipids and proteins, and lead to cell death. Due to the possible contribution of oxidative stress to pollutant toxicity, it is valuable to assess its occurrence, role and mechanism. Detection of oxidative stress at low concentrations soon after the onset of exposure can be a sensitive, general marker for contamination. This study aimed at developing and benchmarking a set of novel fluorescence-based procedures to assess the occurrence of oxidative stress in zebrafish larvae (96 hpf) by measuring the antioxidant glutathione (GSH) and general ROS. Zebrafish larvae were exposed to tert-butyl hydroperoxide (t-BHP). ROS and GSH were made visible by means of specific fluorescent molecular probes in different experimental scenarios. The induction was qualified using microscopy and quantified through photometric measurement. For quantitative assessment, an approach based on homogenized larvae and a non-invasive plate assay were developed. The novel procedures proved suitable for oxidative stress detection. Comparisons of qualitative to quantitative data showed that the orientation of the larvae in the well can influence fluorescence data evaluation. The non-invasive quantitative assay proved robust against any influence of the orientation of the larvae. The developed protocols promise to be useful tools for the detection of oxidative stress in zebrafish larvae.

污染物的作用方式是多种多样的，导致污染物暴露的常见后果是氧化应激。此现象是由于控制活性氧（ROS）的不平衡或干扰引起的，从而导致自由基的积累。氧化应激可能会损坏DNA，磷脂和蛋白质，并导致细胞死亡。由于氧化应激可能会导致污染物毒性，因此评估其发生，作用和机理是很有价值的。暴露后不久立即检测低浓度的氧化应激可能是敏感的，一般的污染标记。这项研究旨在开发和基准化一套新的基于荧光的程序，以通过测量抗氧化剂谷胱甘肽（GSH）和一般ROS来评估斑马鱼幼虫（96 hpf）中氧化应激的发生。斑马鱼的幼虫暴露于叔丁基氢过氧化物（t- BHP）。在不同的实验情况下，可以通过特定的荧光分子探针使ROS和GSH可见。使用显微镜对诱导进行鉴定，并通过光度测量对其进行定量。为了进行定量评估，开发了一种基于均质幼虫和非侵入性平板检测的方法。新方法被证明适用于氧化应激检测。定性数据与定量数据的比较表明，孔中幼虫的方向会影响荧光数据的评估。事实证明，这种非侵入性的定量分析方法能够抵抗幼虫方向的任何影响。所开发的协议有望成为检测斑马鱼幼虫中氧化应激的有用工具。