Vargas Methods</head>

Methods

Preparing Retene and Wood leachates

(Figure A) Purified retene was dissolved into seawater and made a saturated solution which is equal to the solubility of retene, roughly ~20 ug/L (Vehnianinen et al. 2003)).

(Figure B) Four wood leachates were prepared (fir bark, hemlock chips, cedar chips, and fir chips). Woods were soaked in seawater for 24 hours and then filtered (concentration is 250 grams of wood/ liter of sewater).

Survival and Growth Protocol

Protocol for survival and growth experiment was done as described by Dinnel et al. (2011) In brief, 15-20, 7-day old larvae were placed in 400 mL beakers with 200 mL of test or control solution (five test concentrations and a seawater control with 4 replicates per treatment). On day 1 of the test, the beakers were reduced to 10 healthy feeding larvae. On days 2, 4, and 6, the beakers were cleaned of old Artemia, the dead larvae were counted and removed and test and control solutions were renewed by replacing 75% of the volume with fresh solution. On day 7, the remaining live larvae in each beaker were counted, anesthetized with MS-222 and preserved in 95% ethyl alcohol until their dry weights could be measured.

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Literature Cited

Dinnel, P., Middaugh, D., Schwarck, N., Farren, H., Haley, R., Hoover, R., Elphick, J., Tobiason, K. and Marshall, R. 2011. Methods for conducting bioassays using embryos and larvae of Pacific herring, Clupea pallasi. Archives of Environmental Contamination Toxicology 60:290-308.

Vehnianinen, E., Hakkinen, J. and Oikari, A. 2003. Photoinduced lethal and sublethal toxicity of retene, a polycyclic aromatic hydrocarbon derived from resin acid, to coregonid larvae. Environmental Toxicology and Chemistry, Vol. 12:2995-3000.
Page Updated 06.12.2012