Application of a Fluorescence-Based Instrument Prototype for Chlorophyll Measurements and Its Utility in an Herbicide Algal Ecotoxicity Assay

Abstract

Project Aquafluosense was designed to develop prototypes for a modular fluorescence-based instrumental setup for in situ measurement of major water quality parameters. A fluorometer was developed for algal density estimation based on the fluorescent excitation of chlorophyll. The appropriate type of sample holder microplate was determined, along with the need for dark acclimation, prior to the measurements during the instrument’s development. Model species of green (Raphidocelis subcapitata) and blue-green alga (Microcystis aeruginosa) were applied in forms of pure monocultures and their mixtures, and improved analytical limits of detection were achieved (3.70 × 103 cell/mL and 1.13 × 105 for R. subcapitata and M. aeruginosa, respectively). The fluorescence-based determination of algal density was validated by conventional methods, such as cell counting in a Bürker chamber, optical density measurement, and chlorophyll extraction with ethanol. The signals obtained by the fluorometer correlated well with the conventional methods. Pearson r coefficients (applied where the correlation was linear) were ≥0.988 and Spearman ρ coefficients (applied where the correlation was not linear) were >0.976, indicating a strong and positive correlation. The applicability of the developed fluorometer was demonstrated in a growth inhibition ecotoxicity assay on R. subcapitata using the herbicide active ingredient isoxaflutole. During the assay, light intensity (continuous, 104.9 ± 14.9 µE/m2/s), temperature (22 ± 2 °C), pH of algal media (pH = 6–7 for Zehnder and Allen media, as well), and intensity of stirring (continuous, 100 rpm) were controlled. The results indicated that the FluoroMeter Module is applicable for screening algal toxicity: the observed ratio of fluorescence decrease determined by fluorescence induction provided significantly lower toxicity values (EC50: 0.015 ± 0.001 µg/mL) compared to values determined by the optical density (EC50: 0.034 ± 0.004 µg/mL) and chlorophyll a content (EC50: 0.033 ± 0.000 µg/mL).