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Development of a culture method to produce sufficient amounts of microcystin to conduct multispecies acute and chronic toxicity tests.
Citation:
Lazorchak, Jim, S. DeCelles, A. Kascak, W. Thoeny, N. Dugan, T. Sanan, I. Struewing, Joel Allen, AND J. Lu. Development of a culture method to produce sufficient amounts of microcystin to conduct multispecies acute and chronic toxicity tests. 2022 SETAC North America Annual Meeting, Pittsburg, PA, November 13 - 17, 2022.
Impact/Purpose:
This abstract if accepted will represent a presentation that will be made on our SSWR work on generating ecotoxicity information for cyanotoxins. This will illustrate our approach we have taken to the scientific community in order to get feedback on our research
Description:
There is a lack of information to estimate safe exposure levels for freshwater aquatic life to toxins produced by cyanobacteria. 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. Cultures of an environmental strain of Microcystis aeruginosa that produces microcystin and has been confirmed by qPCR and RT-qPCR methods for amplifying mcy genes were used. Initial tests were conducted using lysates obtained by removing cells from culture media by centrifugation and then lysing the cells by freeze thawing them 3 times. Early culture efforts resulted in a range of microcystin concentrations (37-73 µg/L) that varied in cell density (3.73–4.42 x106 cells/mL) and age (≈150 days) of culture were similar. 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 within the same time frame a cell density of 1.38 x106 cells/mL resulted in a lysate concentration of 426 µg/L. Acute toxicity tests conducted with Ceriodaphnia dubia, Neocloeon triangulifer, Hyalella azteca, and larval Pimephales promelas using microcystin lysate did not cause any 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. 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, initial cell extracts were too low to be suitable for aquatic toxicity testing, with microcystin concentrations of 25 µg/L. An aerated culture of this same species under nutrient replete conditions with lower temperature and illumination in 2 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. Results of the new culture methods used for M. aeruginosa and acute and chronic toxicity results will be presented.
