Ecotoxicity of 2,4-Dichlorophenol to Microsorium pteropus by High Spatial Resolution Mapping of Stoma Oxygen Emission

The toxicity of emerging organic pollutants to photosystems of aquatic plants is still not well clarified. This study aimed to develop a novel ecotoxicological experimental protocol based on nanoscale electrochemical mapping of photosynthetic oxygen evolution of aquatic plants by scanning electrochemical microscopy (SECM). The protocol was also checked by confocal laser scanning microscopy (CLSM), the traditional Clark oxygen electrode method, and the chlorophyll fluorescence technique. The typical persistent organic pollutant 2,4-dichlorophenol (2,4-DCP) in a water environment and the common aquatic Microsorium pteropus (M. pteropus) were chosen as the model organic pollutant and tested plant, respectively. It was found that the SECM method could discriminate the responses of stoma micromorphology and spatial pattens of photosynthetic oxygen evolution on single stoma well. The shape of stoma blurred with increasing 2,4-DCP concentration, which was in good agreement with the CLSM images. The dose–response curves and IC50 values obtained from the SECM data were verified by the data measured by the traditional Clark oxygen electrode method and chlorophyll fluorescence test. The IC50 value of single-stoma oxygen emission of plant leaves exposed for 24 h, which was derived from the SECM current data (32,535 μg L−1), was close to those calculated from the maximum photosynthetic efficiency (Fv/Fm) measured by the chlorophyll fluorescence test (33,963 μg L−1) and the Clark oxygen electrode method photosynthetic oxygen evolution rate (32,375 μg L−1). The 72 h and 96 h 2,4-DCP exposure data further confirmed the reliability of the nanoscale stoma oxygen emission mapping methodology for ecotoxicological assessment. In this protocol, the procedures for how to collect effective electrochemical data and how to extract useful information from the single-stoma oxygen emission pattern were well established. This study showed that SECM is a feasible and reliable ecotoxicological tool for evaluation of toxicity of organic pollutants to higher plants with a unique nanoscale visualization advantage over the conventional methods.