FLOW CYTOMETRIC ANALYSIS OF THE PENTACHLOROPHENOL EXPOSED GREEN ALGA CHLORELLA FUSCA PUBLISHEDC-M PETRESCU, I. STANA, C-V MIHALI, V. TURCUȘ, D. BRATOSIN
Pentachlorophenol is a synthetic substance, made from other chemicals, and does not occur naturally in the environment Pentachlorophenol is released to the air by evaporation from treated wood surfaces and factory disposal. It enters surface water and groundwater from factories, wood-treatment facilities, and hazardous waste sites. It also enters the soil as a result of spills, disposal at hazardous waste sites, and its use as a pesticide. The disturbance of aquatic ecosystems provoked by heavy metals pollution from industrial and domestic sources, has as consequence the loss of biological diversity, as well as increased bioaccumulation and magnification of toxicants in the food chain. The aim of this study was to track the induced changes of Chlorella fusca microalgae after exposure to various concentrations of pentachlorophenol (PCP) using flow cytometry, optical and ESEM microscopy. In particular, we investigated the effect of PCP on the induction of ROS and the metabolic activity based on the level of esterases activity assessed with Calcein-AM. Results indicated that cell size, cell granularity and internal complexity were influenced by Pentachlorophenol, confirming earlier findings on ultrastructural changes on exposed microalgae. Our results show a high toxicity of pentachlorophenol which translates by the existence of very small algal colonies and many chlorotic cells, or even of some solitary algae in the extracellular matrix, showing strong influence of PCP's on photosynthesis and cellular metabolism. These changes were observed by FL3/FSC system flow cytometric analysis. The morphological changes observed by ESEM scanning microscopy at different CFP concentrations and different times of incubation, lead to the occurrence of a particular phenotype at a concentration of 0.079 g/L (0.3 mM PCP/L), where the cells have the appearance of boiling cells, phenotype characteristic of eukaryotic animal cells in apoptosis, and described by us under the influence of heavy metals in the scientific literature. The action of PCP's induces in algal cells production of ROS influenced by the concentrations and times of incubation. The metabolic activity of the algae Chlorella fusca determined with Calcein-AM is deeply affected, being maximal inhibited after 6 days of incubation, when the morphological changes were the deeper. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.
pentachlorophenol, algae, flow cytometry, toxicity.