Toxicity of Untreated and Ozone-treated Oil Sands Process-Affected Water to Early Life Stages of the
Fathead Minnow (Pimephales promelas)
Patterson, Sarah1, He, Yuhe1, Hecker, Markus1,2, Martin, Jonathan W.3, El-Din, Mohamed Gamal4, Giesy, John P.1,5,6, Wiseman, Steve B.¹
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 2 School of Environment and Sustainability, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
3 Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Chemistry, University of Alberta, Edmonton, Alberta, Canada, 4 Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta, Canada,
5 Department of Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada6 Department of Zoology, Center for Integrative Toxicology, Michigan State University, E. Lansing, MI, USA
1
! Toxicity of OSPW is mostly attributable to water soluble organic
compounds, in particular naphthenic acids (NAs).
! Ozonation of OSPW is one possible method for remediation
because it decreases the concentrations of NAs, and it
attenuates endocrine disrupting and immuno-toxic effects.
Embryo Lethality
RESULTS 2 – GENE EXPRESSION
Metabolism of Xenobiotics
a
a
100
3
a
!  Exposure to untreated OSPW caused
significantly greater lethality.
!  Ozonation of OSPW and treatment of
OSPW with activated carbon
attenuated the effects, with ozonation
being more effective than activated
carbon.
75
b
50
25
! Additional research was required to ensure that ozonation of
OSPW does not create toxic by-products.
Fold change of transcript abundance
! Large volumes of oil sands process-affected water (OSPW) are
produced during the surface mining of oil sands in Alberta,
Canada. The volume of OSPW currently stored in tailings is
greater than 1 billion m³.
RESULTS 1 – MORTALITY AND DEFORMITIES
Embryo Survival (%)
BACKGROUND
b
2.5
2
a
1.5
a
a
1
0.5
0
0
Figure 1: Aerial view of an oil sands
extraction facility adjacent to the
Athabasca river. Some freshwater is
drawn from the river for use in the
extraction process. A no-release policy
requires that OSPW be stored and
cannot be released to the natural
environment.
(image from nationalgeographic.com)
CTRL
OSPW
O3-OSPW
AC-OSPW
CYP1A
CYP3A
! OSPW did not affect CYP1A expression but
significantly induced CYP3A. Therefore, dioxin-like
compounds such as polyaromatic hydrocarbons
can be excluded as the causative agents.
! Expression of CYP3A is regulated by the
pregnane x receptor (PXR) suggesting that
agonists of the PXR are present in OSPW.
! CYP3A might metabolize organics in OSPW.
! Ozonation of OSPW or treatment of OSPW with
activated carbon attenuate the effects on the
expression of CYP3A.
Figure 2: Effects of untreated oil sands process affected water (OSPW), ozonetreated OSPW (O3-OSPW) and activated carbon stripped OSPW (AC-OSPW) on
survival of embryos of fathead minnows. The duration of the exposure was 168h.
Bars represent the mean ± SEM of 8 independent exposures. Different letters
represent statistically significant differences (one-way ANOVA with Tukey’s posthoc test, p < 0.05).
50
b
b
50
40
40
30
30
20
20
ac
c
Water Samples
b
60
50
40
!  168h exposure with 50% (2 mL) water renewal daily.
!  6 well plates with 10-15 eggs per well.
!  Macroscopic examination of lethality, spontaneous embryo
movement and malformations (hemorrhage, spinal curvature,
pericardial edema).
Embryo Exposure – Gene Expression
!  96h exposure with 50% (2 mL) water renewal daily.
!  6 well plates with 10-15 eggs per well.
!  Determine abundances of transcripts of genes involved in
metabolism of xenobiotics, the response to oxidative stress,
and apoptosis.
20
a
a
a
0
3
b
2.5
2
a
a
1.5
a
a
a
a
1
0.5
GST
(1)
ROS
(2)
CYP3A
!  Abundances of transcripts of GST and SOD
were significantly greater in embryos exposed
to untreated OSPW.
!  Induction of SOD indicates that OSPW caused
oxidative stress.
!  Ozonation of OSPW or treatment of OSPW with
activated carbon attenuated the effects on the
abundances of transcripts of GST and SOD.
SOD
(3)
(1)
mitochondrion
nucleus
Caspase
dependent
apoptosis
Figure 7: Proposed mechanisms of toxicity of untreated OSPW
to embryos of fathead minnows: (1) Organic constituents in
OSPW bind to the pregnane-x-receptor (PXR) and cause
expression of cytochrome P450 3A (CYP3A); (2) CYP3A
metabolizes organic compounds in OSPW causing generation of
excess reactive oxygen species (ROS); and (3) ROS stimulate
caspase enzyme dependent apoptosis via the mitochondrial
pathway.!
Cat
a
C) Pericardial Edema
10
3.5
a
0
30
Embryo Exposure – Lethality and
Teratogenicity
b
a
0
% of embryos with
pericardial edema
!  Concentration of NAs in AC-OSPW was 6.4 mg/L.
a
70
4
CTRL
OSPW
O3-OSPW
AC-OSPW
0
10
10
!  Fresh OSPW was collected from the West In Pit during 2010.
!  Concentration of NAs in untreated OSPW was 19.7 mg/L (HPLC/
HRMS).
!  Ozone was bubbled into the untreated OSPW at the University
of Alberta (O3-OSPW).
!  Concentration of NAs in O3-OSPW decreased approximately
90% to 1.9 mg/L (HPLC/HRMS).
!  Untreated OSPW was mixed with activated carbon to reduce
the concentrations of the organic constituents (AC-OSPW).
Fold change of transcript abundance
60
4.5
!  T h e r e w e r e s i g n i f i c a n t l y m o r e
malformations among embryos exposed
to OSPW.
!  These types of malformations are
consistent with those observed due to
exposure to dioxin-like chemicals that
exert their effects via the arylhydrocarbon receptor (AhR).
!  Ozonation of OSPW and treatment with
activated carbon attenuated the effects.
Figure 5: Effects of untreated oil sands process affected water (OSPW), ozonetreated OSPW (O3-OSPW) and activated carbon stripped OSPW (AC-OSPW) on
abundances of transcripts of glutathione-S-transferase (GST), superoxide dismutase
(SOD), and catalase (Cat).
Bars represent the mean ± SEM of 8 independent exposures. Different letters
represent statistically significant differences (one-way ANOVA with Tukey’s post-hoc
test, p < 0.05).
Regulation of Apoptosis
Fold change of transcript abundance
METHODS
OSPW-OC
Figure 4: Effects of untreated oil sands process affected water (OSPW), ozonetreated OSPW (O3-OSPW) and activated carbon stripped OSPW (AC-OSPW) on
abundances of transcripts of cytochrome P4501A1 (CYP1A) and cytochrome
P4503A (CYP3A).
Bars represent the mean ± SEM of 8 independent exposures. Different letters
represent statistically significant differences (one-way ANOVA with Tukey’s post-hoc
test, p < 0.05).
B) Spinal Curvature
% of embryos with
spinal curvature
! To investigate mechanisms of toxicity by quantifying
abundances of transcripts of target genes in embryos exposed
to OSPW.
A) Hemorrhage
% of embryos with
hemorrhage
! To determine the toxicity of untreated and ozone-treated
OSPW towards embryos of the fathead minnow.
•  Because ozonation and treatment with activated carbon
decreased concentrations of dissolved organic compounds,
including NAs, in OSPW, the effects of untreated OSPW are
caused by the organic fraction of OSPW.
PXR
Response to Oxidative Stress
OBJECTIVES
CONCLUSIONS
4.5
4
CTRL
OSPW
O3-OSPW
AC-OSPW
b
3.5
3
b
2.5
a
2
a
a
1.5
a
a
1
0.5
0
Casp3
Casp9
ApoIn5
a
! Abundances of transcripts of genes involved with
processes indicative of apoptosis (Casp9 and
Apoen) were significantly greater in embryos
exposed to untreated OSPW.
! The results suggest that exposure to untreated
OSPW has the potential to cause apoptosis.
! Ozonation of OSPW or treatment of OSPW with
activated carbon attenuated the effects on the
abundances of transcripts of Casp9 and Apoen.
ACKNOWLEDGEMENTS
Research grant from the Alberta Water Research Institute to
J.P.G, J.W.M and M.G.E.-D (Project # C4288)
Discovery Grant from the Natural Science and Engineering
Research Council of Canada Project # 326415-07)
Grants from Western Economic Diversification Canada (Project
# 6578 and 6807) to J.P.G.
Instrumentation grant from the Canada Foundation for
Innovation (J.P.G.)
NSERC Industrial Research Chair in Oil Sands tailings Water
Treatment (M.G.E.-D.)
Helmholtz-Alberta Initiative (M.G.E.-D. and J.W.M.)
NSERC Research Tools Program (M.G.E.-D).
Discovery Grant from the Natural Science and Engineering
Research Council of Canada To M.H.
Warren Zubot of Syncrude Canada Inc. for supplying the
OSPW.
ApoEn
A
Figure 3: Effects of untreated oil sands process affected water (OSPW), ozonetreated OSPW (O3-OSPW) and activated carbon stripped OSPW (AC-OSPW) on
incidences of malformations in embryos of fathead minnows. The duration of the
exposure was 168h. Bars represent the mean ± SEM of 8 independent
exposures. Different letters represent statistically significant differences (one-way
ANOVA with Tukey’s post-hoc test, p < 0.05).
Figure 6: Effects of untreated oil sands process affected water (OSPW), ozonetreated OSPW (03-OSPW) and activated carbon stripped OSPW (AC-OSPW) on
abundances of transcripts of caspase3 (Casp3), caspase9 (Casp9), apoptosis
inhibitor-5 (Apoin5) and apoptosis enhancing nuclease (Apoen). Bars represent the
mean ± SEM of 8 independent exposures. Different letters represent statistically
significant differences (one-way ANOVA with Tukey’s post-hoc test, p < 0.05).
a