The in vitro endocrine disruptive effects of untreated and ozone treated oil sands process water Y. He, S. B. Wiseman, X. Zhang, M. Hecker, P. D. Jones, M. G. El-Din, J. W. Martin, J. P. Giesy.

Oil Sand Process Water (OSPW) The extraction process results in the production of large volumes of wastewater, commonly referred to oil sands process water (OSPW) Sands, clay, metals, unrecoverable bitumen Polycyclic aromatic Hydrocarbons (PAHs) Naphthenic acids (NAs) NAs are thought to be a / the key contributor to OSPW toxicity. In the surface mining oil sands industry, the extraction of bitumen from oil sands involves the Clarke hot water extraction method. Firstly, Hot water and caustic soda added to sand. And then the Resulting slurry is piped to the extraction plant It is agitated and the oil skimmed from the top Finally, Combination of hot water and agitation releases bitumen from the oil sand This process results in the production of large volumes of wastewater. This wastewater is commonly referred to oil sands process water (OSPW). It contains sands, clay, metals, unrecoverable bitumen, hydrocarbons, and a water soluble organic acid fraction known commonly as naphthenic acids (NAs).

Reclamation Large quantity of OSPW Reclamation Zero discharge policy Growing volume of stored OSPW Over 109 m3 stored in various basins Reclamation Aging reduces acute toxicity of OSPW Chronic toxicity remains Long half-life for natural degradation: ~12 yr Need more aggressive efforts: ozonation ? OSPW is not intentionally released in accordance with a zero discharge policy, and in 2006 it was estimated that greater than 109 m3 OSPW were currently being stored on site in various basins, and it is still growing. Eventually, OSPW needs to be remediated and released back to the environment.

Need to be careful we don’t create new toxic species… Need to be careful we don’t create new toxic species…. Potentially endocrine disruptors? This is good! O3 (20 ppm) Any low MW NAs formed during ozonation, or hydroxylated NAs resembling steroids, may become more bioavailable and consequently adversely affect aquatic organisms. This may not be good!

Endocrine Disruption in the Literature Exposures to tailing pond OSPW Reduce in vitro production of E2 & T by ovarian and testicular tissue (slimy sculpin ) (Tetreault et al., 2003) Reduce plasma sex hormones in yellow perch (van de Heauvel et al., 1999) & goldfish (Lister et al., 2008) Cause longer time to produce first clutch of eggs, and fewer number of clutches of eggs (fathead minnow) (Siwik et al., 2000) Reduce plasma T and 11-keto-T in male, and reduce spawning in female fathead minnow (Kavanagh, et al., 2010)

Objectives To assess the endocrine disruptive effects of OSPW To determine the effects of ozonation on the endocrine disruptive properties of OSPW

Sex Hormone Production and Action HPG Axis Site of Hormone Action Hypothalamus GnRH Pituitary LH Testes Ovaries Testosterone Estradiol

Study 1: Hormone Production Ozonation attenuates the steroidogenic disruptive effects of sediment free oil sands process water in the H295R cell line Y. He, S. B. Wiseman, X. Zhang, M. Hecker, P. D. Jones, M. G. El-Din, J. W. Martin, J. P. Giesy. 2010. Ozonation attenuates the endocrine disruptive effects of sediment-free oil sands process water in the H295R cell line. Chemosphere. 80: 578-584.

The H295R Cell Line Cholesterol Pregnenolone 17α-OH-Pregnenolone DHEA CYP11A CYP17 CYP17 Pregnenolone 17α-OH-Pregnenolone DHEA 3β-HSD 3β-HSD 3β-HSD CYP17 CYP17 Progesterone 17α-OH-Progesterone Androstenedione 17β-HSD CYP19 CYP21 CYP21 Testosterone Estrone 11-Deoxy- Corticosterone 11-Deoxycortisol CYP19 17β-HSD CYP11B1 17β-Estradiol CYP11B2 Cortisol Corticosterone CYP11B2 Aldosterone

Results: T & E2 production Effects of OSPW on Sex Hormone Production Generally, untreated OSPW decreased production of T and increased the production of E2 after 48 h exposure (Figure 1). T production was significantly decreased by 0.45-, 0.42-, 0.32-, and 0.26-fold with exposure to full-strength untreated, 24% and 85% ozonated OSPW, and 10-fold diluted untreated OSPW, respectively (Figure 1A). Significantly greater E2 production was detected when cells were exposed to full-strength untreated (2.0-fold) and 24% ozonated OSPW (1.7-fold), whereas exposure to full-strength 85% ozonated OSPW did not affect E2 production at any dose (Figure 1B). E2 production was also significantly increased in cells exposed to 24% ozonated OSPW diluted 10000-fold, however, this result is likely an experimental anomaly as the effect was small and no significant effects were observed at dilutions of 1000-, 100- and 10-fold (Figure 1B). Linear regression analysis of hormone concentrations released by cells exposed to full-strength untreated, 24% ozonated or 85% ozonated OSPW indicated a significant relationship between degree of ozonation and the decrease in T production (r2 = 0.578). A significant relationship between degree of ozonation and the increase in E2 production (r2 = 0.964) was also observed. T production decreased E2 production increased Ozonation attenuates the effects

Results: Aromatase & E2 metabolism Effects of OSPW on Cyp19 mRNA Expression and Aromatase Enzyme Activity Exposure to full-strength untreated OSPW significantly increased CYP19a mRNA expression after 2 h, 4 h, and 8 h by 1.8-, 2.0-, and 3.0-fold, respectively, post-exposure. Exposure to either full-strength 24% ozonated OSPW for 4 h or 85% ozonated OSPW for 8 h also resulted in a significant increase in CYP19a mRNA expression by 1.9- and 1.5-fold, respectively. In all exposure groups mRNA abundance returned to control levels by 24 h post-exposure (Figure 3). Exposure to 10-fold diluted and full-strength untreated OSPW, full-strength 24% ozonated OSPW, and full-strength 85% ozonated OSPW increased aromatase activity 1.9-, 2.5-, 2.5-, and 2.2-fold, respectively, at 24 h post-exposure (Figure 4). Aromatase activity increased E2 metabolism decreased Ozonation eliminates the adverse effects at 85%

Conclusion – Study 1 Ozonation attenuated the adverse effects of OSPW T and E2 production E2 metabolism Aromatase enzyme activity Inhibition of E2 breakdown is the major reason for increased E2 production Effects of Ozone Treatment Generally, ozonation attenuated the adverse effects of exposure to OSPW on T and E2 production. The significant decrease in T production in cells exposed to 10-fold diluted OSPW was abolished by ozonation. In cells exposed to full-strength OSPW T production remained significantly decreased in both the 24% and 85% ozonated OSPW groups; but the extent of the decrease appeared to be diminished with increasing ozonation. The increase in E2 levels observed in cells exposed to full-strength untreated OSPW was only detected in cells exposed to full-strength and 24% ozonated OSPW, but exposure to 85% ozonated OSPW did not affect E2 levels. As was observed in the untreated OSPW exposure groups, aromatase mRNA abundance and enzyme activity remained significantly elevated, and ozonation delayed the maximal expression of CYP19a gene expression, and increased ozone treatment further increases the time required for maximal induction. Ozonation also impacted the effect of OSPW on the metabolism of E2. This attenuation was most pronounced in the cells exposed to 85% ozonated OSPW, as no significant inhibition of E2 metabolism was detected in the exposure groups. In addition, when diluted sufficiently, 24% ozonated OSPW abolished the effects on E2 metabolism. Overall, these results suggest that the inhibition of E2 breakdown is the major reason for increased E2 levels. Furthermore, these results suggest that ozonation may abolish the attenuating effects of OSPW on E2 production. No changes to aromatase mRNA expression or activity, or E2 metabolism, were observed in cells where OSPW did not affect hormone production. These results suggest that ozonation of OSPW does not inroduce new or enhanced steroidogenic altering properties on OSPW. Exposure to ozone-treated OSPW resulted in lesser effects on E2 and T production than did exposure to full-strength OSPW. There was an inverse relationship between degree of ozonation and related decreases in NAs and effects on both T and E2 production. Together, these results suggest that greater ozonation decreases the impact of OSPW on T and E2 production. Again, these results support the hypothesis that ozonation of OSPW does not impart new or enhanced steroidogenic altering properties on OSPW.

Study 2: Receptor signaling To determine whether OSPW affects androgenicity and estrogenicity through receptor mediated signaling pathways in vitro To evaluate the effects of ozonation of OSPW on the receptor mediated signaling pathway

Background Information Androgen receptor (AR) and Estrogen receptor (ER) Play important roles in sexual differentiation of the reproductive tract, accessory reproductive organs, and other tissues during fetal development Test systems used are rapid, sensitive, and convenient assays for detecting AR/ER agonist/antagonist : MDA-kb2: Transformed with an androgen-responsive luciferase reporter plasmid T47D-kbluc: Transformed with a luciferase reporter gene containing ERE Androgens and Estrogens, through interactions with the androgen receptor (AR) and estrogen receptor (ER), respectively, play decisive roles in sexual differentiation of the male reproductive tract, accessory reproductive organs, and other tissues during fetal development. The assays using two stable cell lines, MDA-kb2 and T47D-kbluc are rapid, sensitive and data-reproducible.

OSPW shows anti-androgenicity and estrogenicity Henry: You need to include stats in the figure. I can see the attenuation of the effect in the AR assay. In the ER assay it just seems that there is no effect of ozonation. This is still good as we want to accomplish two things with ozonation: Decrease effects Not increase any effects. OSPW shows anti-androgenicity, and ozonation attenuates the effect OSPW shows estrogenicity, and ozonation does not attenuate the effect

Anti-androgenicity OSPW is weak AR antagonist OSPW potentiates the effect of T at lower concentrations..

Estrogenicity Co-exposure with OSPW & E2 shows additive effects on ER activity Ozonation does not attenuate the effect

Estrogenicity is receptor mediated a, b & c show significant different within groups OSPW, ozonated OSPW, and commercial NAs are estrogenic The estrogenicity act through estrogen receptor-mediated pathway as the effect can be inhibited by a strong antagonist (ICI 182,780) Ozonation does not attenuate the effect

Conclusion – Study 2 Both full-strength and ozonated OSPW can disrupt receptor mediated androgen and estrogen signaling Both full-strength and ozonated OSPW show anti-androgenicity through weak competitor binding Both full-strength and ozonated OSPW show estrogenicity Ozonation attenuates the effect of OSPW on anti- androgenicity, but not on estrogenicity

Summary OSPW affects sex hormones production (T & E2 ) Ozonation attenuates the adverse effects of OSPW T and E2 production E2 metabolism Aromatase enzyme activity OSPW affects receptor mediated signaling (AR & ER ) Ozonation attenuates the effect of OSPW on anti- androgenicity, but not on estrogenicity Future studies on fathead minnow Gene expression profile Circulating hormones level Reproduction and embryo development

Acknowledgements A research grant from the Alberta Water Research Institute. A Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. A research grant from Western Economic Diversification Canada, J.P.G. is supported by the Canada Research Chair Program The authors would like to thank Warren Zubot of Syncrude Canada Inc for supplying the OSPW.