Effects of Ozone-Treated and
Untreated Sediment-Free Oil
Sand Processed Water on In
Vitro Steroidogenesis
Yuhe He, Steve B. Wiseman,, Xiowei Zhang, Markus Hecker , Paul D.
Jones, Mohamed Gamel El-Din, Jonathan W. Martin, John P Giesy
Alberta’s Oil Sands
 Athabasca
Basin, Alberta, Canada
 One
of the largest reserve of
petroleum
•
•
173.2 billion barrels of bitumen
1/3 of known global oil reserves
 Global
demand for oil
 Production
growing
604,700 barrels per day
in 2000
• 1,184,000 barrels per day
in 2008
•
Oil Sand Process Water (OSPW)
 Clarke
•
2.5 barrels of water per barrel of oil produced
 Oil
•
sand process water (OSPW)
Sands, clay, unrecoverable bitumen, hydrocarbons (PAHs), and
naphthenic acids
 Large
•
•
•
•
Hot Water Extraction
quantity
No discharge policy
Growing
Over 109 m3 stored in various basins
Remediation and release
Naphthenic Acids (NAs)
 CnH2n+ZO2
•
Z is related to the number of rings
 Primary
•
•
toxic constituent
Microbial action
Toxicity mechanism: narcosis?
 Reclamation
•
•
•
Chronic toxicity
Long half-life for natural degradation: ~12 yr
Need more aggressive efforts: ozonation
Ozonation
Relative Intensity
Relative Intensity
10
8
6
4
2
0
7
9
10
8
6
4
2
0
7
11
13
15
17
Carbon number
3
2
1 Rings
0
19
O- + H+
Parent NA
O2 +
O-
13
15
17
Estrogen
Mimic?
OH
HO
11
Carbon number
OH
O3
O3
O3
9
New NA ?
3
2
1
Rings
0
19
Objective
 To
assess the steroidogenic effects of untreated
sediment-free OSPW on sex hormone production
using the H295R cell line
 To
determine if these baseline effects are
modulated by ozone treatment
H295R cell line
Cholesterol
CYP11A
CYP17
Pregnenolone
17α-OH-Pregnenolone
3β-HSD
Progesterone
CYP21
11-DeoxyCorticosterone
CYP17
DHEA
3β-HSD
3β-HSD
CYP17
17α-OH-Progesterone
CYP21
11-Deoxycortisol
CYP17
Androstenedione
17β-HSD
Testosterone
CYP19
CYP19
Estrone
17β-HSD
CYP11B1
CYP11B2
Cortisol
Corticosterone
CYP11B2
Aldosterone
17β-Estradiol
Method
Estradiol metabolism
assay
Incubate 24h
Seed Plate
Real Time PCR
assay for CYP19a
Aromatase assay
 Treatment
•
•
•
1, 2, 4,
8, 24 h
[3H]-E2, 1 h
Exposure to
treatment media
48 h
24 h
media prepared by
Full-strength OSPW (77 mg/ml NAs)
24% ozonated OSPW (24% NAs depleted)
85% ozonated OSPW (85% NAs depleted)
Estradiol &
Testosterone
ELISA measurement
Results: T & E2 production
T
production decreased
 E2 production increased
 Ozonation attenuates the effects
Results: CYP19a & aromatase

CYP19a mRNA expression increased
 mRNA abundance returned to control levels by 24 h
 Aromatase activity increased
Results: E2 metabolism
 E2
metabolism decreased
 Ozonation eliminates the adverse effects at 85%
Discussion
 Effects
•
•
•
•
•
•
•
•
of OSPW on Steroidogenesis
Both T and E2 productions decrease in previous studies
Decreased T production: consistent
Increased E2 production: contrast
Decreased E2 metabolism
Increased CYP19a gene expression & aromatase activity
Samples difference: higher NAs conc. than previous studies
Systems difference: cells vs tissues/organism
Species difference: mammalian vs fish
Discussion
 Ozonation
•
•
•
attenuated the adverse effects of OSPW
T and E2 production
E2 metabolism
Aromatase mRNA abundance & enzyme activity
 Inhibition
of E2 breakdown is the major reason for
increased E2
 Inverse
relationship between degree of ozonation
and NAs conc. / adversely steroidogenic effects
Conclusion
 Exposure
to untreated OSPW from an active
settling basin can alter the steroidogenic pathway
in the H295R cell line
 All
the adverse effects are mitigated or even
recovered by ozonation treatment
 Further
study is needed to more fully determine the
in vivo effects of OSPW and ozonation of OSPW
on steroidogenesis
Acknowledgement
 Alberta Water
Research Institute
 Discovery Grant from the National Science
 Engineering Research Council of Canada
 Western Economic Diversification Canada
 Canada Foundation for Infrastructure
 Syncrude
Androgenicity
MDA-kb2 cell line
 Treatment media prepared with OSPW
and Ozonated OSPW

Cell exposure to treatment media to test
androgenicity
 Cell exposure to treatment media with
androgen to test anti-androgenicity

Estrogenicity
MCF-7 transgenic cell line
 Treatment media prepared with OSPW
and Ozonated OSPW

Cell exposure to treatment media to test
estrogenicity
 Cell exposure to treatment media with
androgen to test anti-estrogenicity

In vivo exposure
Control
OSPW
Ozonated OSPW

Fathead minnow, 96 hr exposure

3 exposure groups (OSPW, Ozonated OSPW, control)

2 tanks per group

12 fish in each tank

50% water renewal per day
RNA sequencing
RNA-seq technique
 RNA samples isolated from target organs
(liver, kidney, gonad, etc.)
 Pool samples
 Gene expression profile comparison
(control, ospw, ozonated ospw)
 Susceptible gene determined for further
study
