Oil Sands Processes Affected Water …
Steve Wiseman
Toxicology Centre
University of Saskatchewan
Problem: How To Deal with the Large
Volumes of OSPW?
 Surface mining industry produces large volumes of OSPW
 OSPW must be reclaimed as viable aquatic habitat
 27 end-pit lakes planned
 First (Base mine lake) will be filled this year with OSPW from WIP (Syncrude)
 EPLs will eventually flow into the natural system
Can We Accelerate Detoxification of OSPW ?
 Toxicity of 1st EPLs are predicted to persist until 2070
 Need to accelerate the detoxification process
Ozonation Decreases NAs in OSPW?
OSPW After Ozonation with 80
mg O3/L
OSPW NAs Before Ozonation
Relative Intensity
O3
6
4
Relative Intensity
8
8
6
4
2
2
12
6
0
7 8 91011
0Rings
1213141516
17181920
2122
Carbon number
0
1…
6
7 8 91011
0Rings
1213141516
17181920
Carbon number 2122
Attenuation of Embryotoxicity
aa
Pericardial
Edema
Pericardial
Edema
aa
aa
100
bb
80
60
40
20
ha
SP
W
SP
W
cc
80
60
40
20
O
bb
aa
bb
ND
0
FW
3-
O
O
rc
oa
l-O
SP
W
FW
0
Percent embryos with pericardial
edema (168 hpf)
120
C
Fathead minnow embryo survival
(168 hpf)
Survival
Survival
arc
Ch
oa
S
l-O
PW
PW
OS
W
SP
O
O3
Attenuation of Developmental Toxicity
100
A
Mean Wet Weight
0.010
A
0.008
0.006
B
0.004
0.002
0.000
Freshwater
OSPW
O3-OSPW
Percent Adult Emergence
0.012
A
A
80
60
40
20
B
0
Freshwater
OSPW
O3-OSPW
No Effect on Estrogenicity of OSPW – in vitro
Estrogenic response
3.0
b
 Ozonation neither attenuated nor
potentiated estrogenicity of OSPW.
b
2.5
 Chemical(s) in OSPW and
ozonated OSPW bind to the ER.
2.0
1.5
a
a
a
1.0
0.5
0.0
ia
d
Me
W
ICI
ICI
P
PW
S
S
+
+
O
-O
W
W
3
P
P
O
S
OS
-O
3
O
Rowland et al., 2011
No Effect on Estrogenicity of OSPW – in vivo
Egg Envelope Proteins- Males
Egg Envelope Proteins - Females
1.5
8
a
a
a
1.0
0.5
0.0
b b
VTG
b b
b b
CHG L
CHG H
mRNA Abundance
(Relative to Freshwater)
mRNA Abundance
(Relative to Freshwater)
2.0
b
b
6
Freshwater
OSPW
O3-OSPW
b
4
a
a
2
a
a
a
a
0
VTG
CHG-L
CHG-H
 Could explain the decreased fecundity in female minnows (Kavanagh et al., 2011)
and less prominent male secondary sexual characteristics in male minnows.
Problem: How do we monitor for exposure to
and effects of OSPW?
Need: Determine the Critical Mechanism(s) of
Toxicity of Oil Sands Process
Affected Water
If we know critical mechanisms of action then we can design assays to monitor
for exposure to OSPW
Mechanism(s) of Toxicity of OSPW
 Because NAs are surfactants, OSPW might have toxicity via narcosis.
Control
CL
CS
NA
OSPW
O3-OSPW
AC-OSPW
ug chol/million
cells
40
30
20
10
0
Membrane cholesterol
Transcriptomics
Given the complexity of OSPW there might be multiple mechanisms of toxicity.
Adverse outcome pathways
•Processes that lead to toxicity are often initiated at the molecular level
+
Chemical
Direct interaction
with receptor
Molecular event
(ie. transcriptional response)
Cellular processes
Population level effect
Organism level effect
Quantify abundances of transcripts in the livers of male fathead minnows exposed
to OSPW might provide some insight into potential mechanisms of toxicity.
Results 1 : Global Gene Expression
Freshwater -vs- Untreated OSPW
Down
(95)
UP
(109)
Functional annotation using GO terms and
KEGG mapping to identify process
indicative of effects of OSPW.
Biotransformation
OSPW-OC
Transcript
Fold Change
CYP1A
CYP2k19
CYP2k6
CYP2N
CYP2AD2
UGT 5B4
UGT 5F1
Sulfotransferase 1,3
GST (mitochondrial)
GST (cytosolic)
MDR-2
Aldehyde oxidase 1
Aldehyde dehydrogenase
Monoamine oxidase
Epoxide hydrolase
2.1
11.3
10.1
2.7
2.2
6.3
-4.3
1.8
4.5
>23.3
3.3
3.1
3.6
3.2
2.0
Phase I
Phase II
AhR
CAR
PXR
CYP1A
GST
MDR
UGT
CYP2
CYP3
GST
MDR
ST
Phase III
Oxidative
metabolism
Are organics in OSPW being
metabolized?
Effect on toxicity?
Oxidative Stress - I
Transcript
Fold Change
Glutathione synthase
Glutathione reductase
Glutathione peroxidase
Transketolase
6-phosphogluconate dehydrogenase
Glucose-6-phosphate dehydrogenase
Nuclear factor like 2
3.1
3.2
1.7
2.4
10.1
2.7
1.8
ROS
Transcription factor
GSH
NADP
NADPH
H202
Glutathione
Peroxidase
Glutathione
Reductase
G-6-PDH
6-PGDH
Transketolase
GST UGT MDR
Pentose-phosphate
shunt
GSH Synthase
NRF2
AO MOA AlDH EH
Glutathione metabolism
GSSG
H20 + 02
Oxidative Stress - II
Transcript
Fold Change
NADH dehydrogenase 1 beta
subcomplex subunit 1
Acyl carrier (mitochondrial precursor)
Cytochrome b-c1 complex subunit 9
Cytochrome b561 domain 2
Cytochrome b5a
1.8
Complex I
1.5
1.5
3.3
8.8
ROS
http://en.wikipedia.org/wiki/File:Mitochondrial_electron_transport_chain%E2%80%94Etc4.svg
Complex III
Complex I and III are
major sites of production
of ROS
Apoptosis
Transcript
Fold Change
Apoptosis-inducing factor 3
4.3
Apoptosis-inducing factor mitochondrial associated-2
4.1
Poly [ADP-ribose] polymerase
4.8
Programmed cell death 4a
1.5
DNA damage-regulated autophagy modulator protein 2
> 23.3
Cathepsin b
1.5
BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 -1.8
Forkhead box transcription factor O3A
-3.3
AIF
PARP
ROS
AIF
Cathepsin b
AIF
Mechanism of Toxicity
OSPW-OC
CAR
AhR
mitochondria
Complex I
Complex III
PXR
nucleus
GST
UGT
MDR
CYP1A
CYP2K
CYP2AD
CYP2N
GST
UGT
MDR
AO
MOA
AlDH
EH
nrf2
ROS
OSPW-OC
Apoptosis
Development of Deformities
Hemorrhage
Pericardial edema
Malformation of spine
Effects are similar to those caused by dioxins and dioxin-like chemicals (PAHs).
Biotransformation Enzymes
Fold-change in abundance
of transcript
3.0
Freshwater
Untreated OSPW
03-OSPW
AC-OSPW
b
2.5
2.0
a
a
1.5
1.0
a
0.5
0.0
cyp1a
cyp3a
 No change in transcript abundance of cyp1a
• No activation of Aryl-hydrocarbon Receptor (AhR) signaling
• No PAHs in OSPW.
 Greater transcript abundance of cyp3a
• Activation of Pregnane-X-Receptor (PXR)
 Ozonation & activated charcoal treatment attenuated the effects
Oxidative stress
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Oxidative stress responsive genes
a
a
n
Co
l
tro
OS
PW
Freshwater
Untreated OSPW
03-OSPW
AC-OSPW
5
b
a
W
W
SP
SP
O
O
AC
O3
Fold-change in abundance
of transcript
Concentration of ROS
Reactive oxygen species
b
4
b
3
b
2
1
a
aa
aa
a
a a
a
0
gst
sod
cat
 Greater conc. of ROS
 Greater transcript abundance of gst & sod
 Ozonation & activated charcoal treatment attenuated the effects
Apoptosis
Fold-change in abundance
of transcript
5
b
4
3
b
2
a
a a
1
0
Freshwater
Untreated OSPW
03-OSPW
AC-OSPW
b
aa
a
a
aa
p3 asp9 opIn
En
s
p
a
o
c
c
ap
ap
ba
x
p5
3
 Caspase-activated apoptotic cell death induced by oxidative stress
 Ozonation & activated charcoal treatment attenuated the effects
Conclusion
The toxicity of OSPW due to dissolved organic compounds.
The mechanisms of the effects of the dissolved organic compounds are unknown.
The identities of the compounds that cause effects are unknown.
OSPW
Toxicity
(250,000 chemicals?)
Characterization of
Biological Effects
Development of
Appropriate Bioassays
Characterization of
Chemical Content
Effects
Chemical and biological
characterization of
OSPW will lead to
strategies to deal with
OSPW
Biomarkers
John Giesy
Yuhe He
Julie Anderson
Rishi Mandinky
Markus Hecker
Paul Jones
Sarah Peterson
Warren Zubot
Jon Martin
Mohamed Gamal El-Din
Nan Wang
Leo Perez-Estrada