Oil Sands Processes Affected Water …

Steve WisemanToxicology Centre

University of Saskatchewan

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

Problem: How To Deal with the Large Volumes of OSPW?

Toxicity of 1st EPLs are predicted to persist until 2070

Need to accelerate the detoxification process

Can We Accelerate Detoxification of OSPW ?

Ozonation Decreases NAs in OSPW?

7 8 9 10

11

12

13

14

15

16

17

18

19

20

21

22

0

2

4

6

8

024681012

Carbon number

Rel

ativ

e In

tens

ity

Rings

O3

OSPW NAs Before Ozonation OSPW After Ozonation with 80 mg O3/L

7 8 9 10

11

12

13

14

15

16

17

18

19

20

21

22

0

2

4

6

8

024681012

Carbon numberR

elat

ive

Inte

nsity

Rings

Attenuation of EmbryotoxicityFa

thea

d m

inno

w e

mbr

yo s

urvi

val

(168

hpf

)

0

20

40

60

80

100

120

Survival

a a a

b

a a a

b

Survival

Perc

ent e

mbr

yos w

ith p

eric

ardi

al e

dem

a (1

68 h

pf)

0

20

40

60

80

ND

Pericardial Edema

bb

a

c

bb

a

cPericardial Edema

Attenuation of Developmental Toxicity

Freshwater OSPW O3-OSPW

Mean

Wet

Weig

ht

0.000

0.002

0.004

0.006

0.008

0.010

0.012A

A

B

Freshwater OSPW O3-OSPW

Perce

nt A

dult

Emer

genc

e

0

20

40

60

80

100 A A

B

No Effect on Estrogenicity of OSPW – in vitro

Estro

geni

c re

spon

se

MediaOSPW

OSPW + ICIO3-OSPW

O3-OSPW + ICI0.0

0.5

1.0

1.5

2.0

2.5

3.0

a a a

bb Ozonation neither attenuated nor potentiated estrogenicity of OSPW.

Chemical(s) in OSPW and ozonated OSPW bind to the ER.

Rowland et al., 2011

VTG CHG L CHG H

mRNA

Abu

ndan

ce(R

elativ

e to

Fres

hwat

er)

0.0

0.5

1.0

1.5

2.0Egg Envelope Proteins - Females

aa

a

b b b b b b

Could explain the decreased fecundity in female minnows (Kavanagh et al., 2011) and less prominent male secondary sexual characteristics in male minnows.

Egg Envelope Proteins- Males

VTG CHG-L CHG-HmR

NA A

bund

ance

(Rela

tive t

o Fr

eshw

ater

)

0

2

4

6

8FreshwaterOSPWO3-OSPW

aa

b

aa

a a

b

b

No Effect on Estrogenicity of OSPW – in vivo

Need: Determine the Critical Mechanism(s) of Toxicity of Oil Sands ProcessAffected Water

If we know critical mechanisms of action then we can design assays to monitorfor exposure to OSPW

Problem: How do we monitor for exposure to and effects of OSPW?

Mechanism(s) of Toxicity of OSPW Because NAs are surfactants, OSPW might have toxicity via narcosis.

CLControl CS NA

OSPW O3-OSPW AC-OSPW

Control CLCS (1hr)

CS (2hr)OSPW

O3-OSPWAC-OSPW NA

05

1015202530

Membrane cholesterol

ug c

hol/m

illio

n ce

lls

TranscriptomicsGiven 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

Organism level effect

Population level effect

Quantify abundances of transcripts in the livers of male fathead minnows exposedto OSPW might provide some insight into potential mechanisms of toxicity.

Results 1 : Global Gene Expression

UP(109)

Down(95)

Freshwater -vs- Untreated OSPW

Functional annotation using GO terms and KEGG mapping to identify process indicative of effects of OSPW.

BiotransformationTranscript Fold ChangeCYP1A 2.1CYP2k19 11.3CYP2k6 10.1CYP2N 2.7CYP2AD2 2.2UGT 5B4 6.3UGT 5F1 -4.3Sulfotransferase 1,3 1.8GST (mitochondrial) 4.5GST (cytosolic) >23.3MDR-2 3.3Aldehyde oxidase 1 3.1Aldehyde dehydrogenase

3.6

Monoamine oxidase 3.2Epoxide hydrolase 2.0

Phase I

Phase II

Phase III

Oxidative metabolism

AhR PXRCAR

OSPW-OC

CYP1AGSTMDRUGT

CYP2 CYP3GSTMDRST

Are organics in OSPW beingmetabolized?

Effect on toxicity?

Transcript Fold Change

Glutathione synthase 3.1Glutathione reductase 3.2Glutathione peroxidase 1.7Transketolase 2.46-phosphogluconate dehydrogenase 10.1Glucose-6-phosphate dehydrogenase 2.7Nuclear factor like 2 1.8

Oxidative Stress - I

Glutathione metabolism

Pentose-phosphate shunt

Glutathione

Reductase

GSH

GSSG

GSH Synthase

Glutathione Peroxidase

NADP

NADPH

G-6-PDH6-PGDH

Transketolase

H202

H20 + 02

NRF2

AO MOA AlDH EH

GST UGT MDR

ROS

Transcription factor

Oxidative Stress - IITranscript Fold

ChangeNADH dehydrogenase 1 beta subcomplex subunit 1

1.8

Acyl carrier (mitochondrial precursor)

1.5

Cytochrome b-c1 complex subunit 9

1.5

Cytochrome b561 domain 2 3.3Cytochrome b5a 8.8

Complex I

Complex III

Complex I and III are major sites of productionof ROS

http://en.wikipedia.org/wiki/File:Mitochondrial_electron_transport_chain%E2%80%94Etc4.svg

ROS

ApoptosisTranscript Fold ChangeApoptosis-inducing factor 3 4.3Apoptosis-inducing factor mitochondrial associated-2

4.1

Poly [ADP-ribose] polymerase 4.8Programmed cell death 4a 1.5DNA damage-regulated autophagy modulator protein 2

> 23.3

Cathepsin b 1.5BCL2/adenovirus E1B 19 kDa protein-interacting protein 3

-1.8

Forkhead box transcription factor O3A -3.3AIF

PARP

Cathepsin b

AIF AIF

ROS

AhR PXR

CAR

OSPW-OC

CYP1ACYP2KCYP2ADCYP2N

OSPW-OC

nucleus

nrf2AOMOAAlDHEH ROS

Complex IComplex III

Mechanism of Toxicity

GSTUGTMDR

Apoptosis

GSTUGTMDR

mitochondria

Hemorrhage Pericardial edema Malformation of spine

Effects are similar to those caused by dioxins and dioxin-like chemicals (PAHs).

Development of Deformities

Biotransformation Enzymes

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

cyp1a cyp3a

Fold-

chan

ge in

abun

danc

e o

f tra

nscri

pt

0.0

0.5

1.0

1.5

2.0

2.5

3.0 Freshwater Untreated OSPW03-OSPWAC-OSPW

aa

a

b

Oxidative stressReactive oxygen species

Greater conc. of ROS Greater transcript abundance of gst & sod Ozonation & activated charcoal treatment attenuated the

effects

Oxidative stress responsive genes

aaa

baa

a

b

aa

a

b

Control OSPWO3-OSPW

AC-OSPW

Conc

entra

tion

of R

OS

0.00.20.40.60.81.01.21.41.61.82.0

gst sod catFo

ld-ch

ange

in ab

unda

nce

of tr

ansc

ript

0

1

2

3

4

5Freshwater Untreated OSPW03-OSPWAC-OSPW

aaa

b

Apoptosis

Caspase-activated apoptotic cell death induced by oxidative stress

Ozonation & activated charcoal treatment attenuated the effects

Fold-

chan

ge in

abun

danc

eof

tran

scrip

t

0

1

2

3

4

5 Freshwater Untreated OSPW03-OSPWAC-OSPW

aa

a

b

b

b

a a

aaaa

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.

Conclusion

OSPW

Characterization of Chemical Content

Characterization of Biological Effects

Toxicity(250,000

chemicals?)

Development of Appropriate Bioassays

Effects

BiomarkersChemical and

biological characterization of OSPW will lead to strategies to deal

with OSPW

Jon MartinMohamed Gamal El-DinNan WangLeo Perez-Estrada

John GiesyYuhe HeJulie AndersonRishi MandinkyMarkus HeckerPaul JonesSarah Peterson

Warren Zubot