Aguilera_2025_Mar.Pollut.Bull_220_118407

Zooplankton, particularly copepods, play a crucial role in marine ecosystems, making it critical to understand the impact of abiotic stressors such as pollutants and climate change on their physiology. Biochemical markers are considered effective tools for assessing the organism's response to contaminant exposure; in the case of copepods and other small zooplankton, however, the application of standard biochemical methodologies is compromised due to their small size and low basal enzymatic activities. Thus, this study aimed to optimize biomarker analytical protocols, including buffer selection, extraction method and enzymatic assay conditions for application to copepods and similarly-sized organisms. The goal is to enhance sensitivity and reduce sample size requirements, while allowing measurements of a broader range of assays from a single sample to address multiple physiological endpoints. The inclusion of NaCl in a phosphate buffer improved the biomarker's stability, and all enzymatic activities peaked at pH 8.0. We also compared the efficacy of two extraction methods (sonication vs. tissue lyser) and the effects of adding the detergent Triton X-100. The final choice of method and Triton X-100 concentration may depend on the targeted biomarkers and the equipment available. The use of the anaesthetic tricaine methane sulfonate allowed more efficient handling of copepods without interfering with enzymatic acetylcholinesterase activity. Minimum copepod density tests confirmed reliable enzymatic measurements with small sample sizes. By refining and adapting biomarker methodologies, this study contributes to advancing the application of ecotoxicological testing for small zooplankton like copepods.