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Development of reproducible culture methods to produce cyanotoxins to be used to generate acute and chronic endpoints for freshwater organisms. (SETAC 2022)
Citation:
Lazorchak, J., S. Decelles, A. Kascak, W. Thoeny, N. Dugan, T. Sanan, I. Struewing, Hubert Allen, AND J. Lu. Development of reproducible culture methods to produce cyanotoxins to be used to generate acute and chronic endpoints for freshwater organisms. (SETAC 2022). SETAC NA, Pittsburg, PA, November 13 - 17, 2022.
Impact/Purpose:
Presentation of current finding for generating ecotoxicity for Microcystin
Description:
There is a lack of information to estimate safe exposure levels to toxins produced by cyanobacteria for freshwater aquatic life. The uncertainty in concentrations and purity of standards for cyanotoxins, and potentially their cost, challenge their use for conducting acute and chronic toxicity tests. To address this, different approaches to culture cyanobacteria for toxicity assessments were compared. Microcystin-producing Microcystis aeruginosa with confirmed mcy genes by qPCR and RT-qPCR was used in these cultures. Initial tests were conducted using lysates obtained by removing cells from culture media by centrifugation and then lysing the cells by freeze thawing them three times. Early culture efforts resulted in a range of total microcystin concentrations (37-73 µg/L) that varied in cell density (3.73-4.42 x106 cells/mL) and similar age (≈150 days). Another method using a different vessel format (8 flasks of different ages combined into 21 L), similar age of culture (≈150 days), and a cell density of 1.75 x106 cells/mL, resulted in a microcystin concentration of 885 µg/L. This culture vessel format was repeated and resulted in a cell density of 1.38 x106 cells/mL and a lysate concentration of 426 µg/L. Due to the variability in toxin yields, an approach was taken to grow M. aeruginosa cultures up to a stationary phase in 250-mL and 1-L flasks, then test for toxin concentrations using ELISA. However, cell extracts were too low to be suitable for adequate aquatic toxicity testing, with microcystin concentrations of 25 µg/L. Finally, an aerated culture under nutrient replete conditions with lower temperature and illumination in two carboys containing 14 L in each, a 28-day cell density of 9.5 x106 cells/mL, resulted in a lysate microcystin concentration of 517 µg/L. These culture conditions were repeated, and the lysate microcystin concentrations were tested. Acute toxicity tests conducted with Ceriodaphnia dubia, Neocloeon triangulifer, Hyalella azteca, and larval Pimephales promelas using microcystin lysate did not cause significant mortality at concentrations as high as 74 µg/L. For chronic tests, IC25 for total microcystins for C. dubia, P. promelas, H. azteca, N. triangulifer were 8.9, 74.0, 408.9, and 10.1 µg/L, respectively. Results of continued testing of the new culture methods and acute and chronic toxicity tests will be presented.
