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Development of reproducible culture methods to produce cyanotoxins to be used to generate acute and chronic endpoints for freshwater organisms.
Citation:
Lazorchak, Jim, S. DeCelles, A. Kascak, W. Thoeny, N. Dugan, T. Sanan, I. Struewing, Joel 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 Europe 32nd Annual Meeting, Cophenhagen, N/A, DENMARK, May 15 - 19, 2022.
Impact/Purpose:
This presentation will present the progress to date in culturing cyanobacteria to generate toxins to be used in ecotoxicity tests under SSWR 4.1.3
Description:
There is a lack of information to estimate safe exposure levels for aquatic life to natural toxins produced by cyanobacteria. The uncertainty in concentrations and purity of standards for cyanotoxins, and their high cost, challenge their use for acute and chronic toxicity tests. An alternative approach was tested, using cultures of cyanobacteria and algae to generate toxins. In this study, we have established laboratory cultures of toxin producers Microcystis aeruginosa lab cultured environmental strain (microcystin), Aphanizomenon flos-aquae PCC7905 (cylindrospermopsin), and Dolichospermum circinale CS-337 (saxitoxin) as well as nontoxin strain of A. flos-aquae. Early tests were conducted with microcystins obtained by removing cells from culture media and lysing them. Microcystin concentrations varied if cell density and time since inoculation (~150 days) of culture were tracked, with 1.75 X 106 cells/mL resulting in a microcystin concentration of 885 µg/L, while 4.16 X 106 cells/mL yielded 37 µg/L. Acute tests conducted with Ceriodaphnia dubia, Neocloeon triangulifer, Hyalella azteca, and larval Pimephales promelas using microcystins, did not cause any lethality different from the control at concentrations as high as 74 µg/L. However, the non-toxin-producing strain of A. flos-aquae caused mortality greater than the controls to N. triangulifer. For chronic tests IC25 for total microcystins 346 #SETACCopenhagen 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 getting consistent intracellular toxin levels, an approach was taken to harvest M. aeruginosa cultures during stationary phase to quantify microcystin concentrations by ELISA. Reproducible toxin concentrations (varied < 20%) were attained in both 500-mL and 1000-mL cultures, however, lysed cell extracts were too low to use for testing. Th ese cultures were maintained at 25 +/- 1°C under a 16:8 h light/dark cycle at 15± 1 ?mol m-2 s -1 . A culture of this same species under lower temperature and illumination resulted in higher cell lysate concentrations after 6 to 12 months. These culture conditions were repeated, and the lysate concentrations were tested. Results of the new culture methods used for M. aeruginosa and acute and chronic toxicity results will be presented.
