Master of Science (MSc)
Faculty of Science
An investigation of cause was started in the 1990s at a bleached Kraft mill (BKM), located in Saint John NB, Canada. The 5th effect chemical recovery condensates waste stream (derived from wastewater generated during the pulping process) was identified as having the greatest potential for affecting mummichog (Fundulus heteroclitus), compared to the rest of the inmill waste streams. A solid phase extraction (SPE) protocol was utilized to isolate the causative chemicals in the condensate stream. Goldfish testicular androgen receptors (ARs) were exposed to five extracts: filter paper non-polar/polar, solid phase extraction nonpolar/polar, and residual condensates. The filter paper non-polar (FP-NP) extract contained a large quantity of ligands for the AR. A normal phase high performance liquid chromatography (HPLC) method was then developed to fractionate the FP-NP extract based on polarity, further isolating the androgenic compounds present in the condensates. Most of the androgenic activity (p≤0.05) was isolated in one HPLC fraction, with a smaller amount of activity identified in 3 additional fractions, demonstrating the presence of multiple ligands. Gas chromatography mass spectrometry (GC-MS) analysis of the active HPLC fractions identified four different families of diterpenoid compounds probably responsible for most of the androgenic activity. The naturally occurring cyclic diterpene manool was identified as a novel androgenic compound. Manool accounted for 25 and 14 % of the androgenicity in the FP-NP extract and the main HPLC fraction, respectively. To investigate if the AR ligands identified in condensates were present in mill discharges, final effluent from the Saint John mill and 10 other mills from Canada (3), Brazil (5) and New Zealand (2) were analyzed for manool and androgenic activity. It was determined that final effluent from all mills exhibited androgenic activity in vitro. The highest levels of activity were consistently associated with solid phase extraction non-polar (SPE-NP) extracts of each effluent (with the exception of two mills), which ranged from ~100-800 ng testosterone /L condensate (equivalent). Of the two excepted mills, one was a thermomechanical pulp mill that treated effluent with activated sludge, while the other was a kraft pulp mill, which utilized reverse osmosis (RO) to treat effluent. All other mills used biological treatment to treat effluent. The highest androgenic activity detected was 3500 ng/L, from the Saint John mill, which was also the only mill effluent that had detectable levels of manool (115 ng/L).
Scott, Philip David, "Isolation and Characterization of Ligands for the Goldfish Testicular Androgen Receptor from Kraft Mill Chemical Recovery Condensates" (2010). Theses and Dissertations (Comprehensive). 982.