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Development and novel application of an in vitro fish gill cell assay to elucidate the ichthyotoxic mechanism of the microalga Chattonella marina (Raphidophyceae)


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Dorantes, JJ 2012 , 'Development and novel application of an in vitro fish gill cell assay to elucidate the ichthyotoxic mechanism of the microalga Chattonella marina (Raphidophyceae)', PhD thesis, University of Tasmania.

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Chattonella marina algal blooms have been associated with major farmed and wild
fish mortalities in tropical (Mexico), subtropical (South Australia) and temperate
regions (Japan). However, the precise toxic mechanisms involved remain
incompletely known and disputed. A novel sensitive in vitro assay using the rainbow
trout cell line RTgill-W1 was developed to assess toxicity of lipid extracts, ruptured
cells and intact cultures of 6 strains of C. marina. Chattonella was found to be less
toxic than the haptophyte Prymnesium parvum, but more toxic than the
raphidophytes Heterosigma akashiwo, Fibrocapsa japonica and dinoflagellate
Karenia mikimotoi. Ruptured cells from Australian CMPL01 and Japanese N-118 C.
marina strains were the most toxic, decreasing gill cell viability by 71 and 65%,
respectively. The Mexican CMCV-1 strain was the least toxic (35%), possibly
because it is larger and less fragile than other strains. Chattonella marina is unique
among ichthyotoxic microalgae in its high production of superoxide anion (19 pmol
cell-1 hr-1). Sonicated cultures showed higher levels of superoxide than intact cultures
(19.0 vs 9.5 pmol cell-1 hr-1, respectively). However, O2
- on its own did not appear to
be the main cause of toxicity (only 14% loss of cell viability). Superoxide production
was highest when grown in medium enriched with 1 μM Fe(III) compared to the 5
μM standard medium (25 vs 9.5 pmol cell-1 hr-1), especially after a sudden change
from dark adaptation to light conditions (200 μmol photons m-2 s-1) (up to 37.6), but
lowest (4.1) at 100 nM Fe(III). The three major fatty acids in C. marina were
palmitic (PA), eicosapentaenoic (EPA), and octadecatetraenoic (OTA) acids. Higher
levels of EPA were found in ruptured cells (21.4 – 29.4% of total fatty acids)
compared to intact cells (8.5 – 25.3%). Gill damaging effects from free fatty acid
(FFA) fractions were conclusively demonstrated (LC50 at 1 hr of 0.44 μg mL-1), and
remain candidates of ichthyotoxicity when well-defined toxins such as brevetoxin or
karlotoxin can be ruled out as causative factors. The aldehydes 2E,4E-decadienal and
2E,4E-heptadienal, which may be generated by FFA oxidation, also showed high
impacts on gill cell viability, with LC50 (at 1 hr) of 0.34 and 0.36 μg mL-1,
respectively. In conclusion, C. marina was more toxic after cell disruption and when
switching from dark to light conditions, associated with higher EPA levels and a higher production of superoxide anion. Implications of this work for finfish
aquaculture management and mitigation are discussed.

Item Type: Thesis - PhD
Authors/Creators:Dorantes, JJ
Keywords: Chattonella marina, ichthyotoxicity,phytoplankton, algal blooms, in vitro assay, gill cells, RTgill-W1
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Copyright 2012 the Author

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