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Environmental applications of luminescence

Bioluminescence tests using Pyrocystis lunula (Schutt, 1896) under effect of anionic surfactants contamination

Andrade, F. G. . B. W. C. C. M. F. C. F. S. M. S. G. V. S. T. P. A. D. S. F. M. P. L. . O. A. &. . B. E.s.1

1University of São Paulo, Oceanographic Institute, Brazil

E-mail: gabriellaa@usp.br

The bioluminescence of the dinoflagellate Pyrocystis lunula is an excellent indicator for observing the effects of different contaminants on organisms in the marine environment. In this study, standardized tests were applied in P. lunula from the Aidar & Kutner Microorganism Bank of the Oceanographic Institute of the University of São Paulo. The cultivation medium was Guillard F/2 medium. The culture was exposed to photoperiod of 12h night/12h day. The anionic surfactants used were sodium dodecyl sulfate (C12H25OSO3Na – SDS) and sodium dodecylbenzene sulfonate (CH12)11C6H4SO3Na - LAS). These components commonly used in detergents and differ by the presence of benzene. The mother culture had around 11,700 cells mL-1 and the experimental design followed ABNT standards for ecotoxicological testing with marine microalgae (ABNT). In the bioassay, 250 mL Erlenmeyer flasks with 50 mL of the mother culture in 100 mL of Guillard medium formed 150 mL of solution with a concentration of 4,000 cells.mL-1. Three different treatments were performed: i) dodecyl sulfate sodium (SDS), ii) sodium dodecylbenzene sulfonate (LAS) and iii) both. Different concentrations were used besides the control. They are: 5 mg, 10 mg, 25 mg and 50 mg of surfactants. The exposure lasted 24h and 48h. The Tecan Infinite® luminometer was used to measure bioluminescence. Cell density was measured in a Sedgewick-Rafter. Cells were counted under Leica® optical microscope model DME with 40x magnification. Chlorophyll was measured by spectrophotometry. Exposure to SDS was lethal to cells, with the inhibition of light emission from the dosage of 25 mg.L⁻¹, representing their mortality. High emission values of light in 24 and 48h, at a dosage of 5 mg.L⁻¹, indicate a first degree of damage to cells, as they are associated with the rupture of scintillon membranes. These organelles contain the luciferin-luciferase enzyme complex and its breakdown results in an increase in light emission, the increases observed in dosages of 5 mg.L⁻¹, and subsequent decrease at 10 mg.L⁻¹ of exposure may be related to the denaturation of this membrane. A lethality was observed at 25 and 50 mg.L⁻¹ was corroborated by observation of dead cells in the Sedgewick-Rafter chamber presented anomalies, such as the encysted and extroverted nucleus and the cellular membrane damaged associated with the entry of surfactant into the cell. In turn, exposure to LAS was characterized as less harmful. In low dosage (5 mg.L⁻¹), light emission was greater than in the control, even with greater light emission even after 48 hours, also related to the rupture of the scintillons. At dosage of 10 mg.L⁻¹, the light emission were lower compared to samples exposed to SDS at the same dose. From 25 mg.L⁻¹, inhibition was observed of bioluminescence, with values close to zero at 50 mg.L⁻¹.

Keywords: Bioluminescence, dinoflagellate, surfactants, toxicity

Acknowledgments: Acknowledgement to FAPESP 2023/14215-0, CNPq 440893/2023-6 and 308916/2022-0.

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