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Toxicology Centre
Date of presentation Title or place of presentation
Characterizing the toxicity of oil sands process affected waters (OSPW)
Garrett Morandi PhD student
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
• 4 year NSERC Collaborative Research and Development grant • Awarded to Dr. John Giesy, Dr. Jon Martin, Syncrude Canada ltd.
• Focused on characterizing the toxicity of the dissolved organic fraction of OSPW collected from Base Mine Lake (BML)
SETAC 2016 November 10th, 2016
Project overview. Toxicity Identification and Evaluation study
BML, established December 2012
Toxicology Centre
Date of presentation Title or place of presentation
State of the science (2012).
• Toxic effects of OSPW • Range of toxic effects have been observed across a number of species
• Chemical culprits • Dissolved organic fraction is responsible for the majority of toxicity
• Very complex mixture ~ 20,000 chemicals • Polar organic acids and semivolatile organic compounds (neutrals) are
responsible for majority of toxicity (Mackinnon and Boerger, 1986 and Verbeek et al., 1993)
• Long believed to be Naphthenic acids but never definitively demonstrated • Mode of action is narcosis (Frank et al., 2009)
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Project overview.
1. What heteroatom classes contribute to the acute toxicity of the dissolved organic fraction of OSPW?
2. Do heteroatom atom classes in OSPW have the potential to bioaccumulate and can we predict their toxicity?
3. Do these heteroatom classes have reproductive toxicity?
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
1. Identifying causative chemicals of toxicity.
SETAC 2016 November 10th, 2016
Approach. • Bioassay-effects directed analysis
• Sequential fractionation and toxicity testing
15- min IC50 Vibrio fisheri
96-hr embryolethality assay Pimephales promelas
Toxicity assays.
Chemical analysis. • HPLC-Orbitrap uHRMS
Toxicology Centre
Date of presentation Title or place of presentation
What we did.
F1
F2
F3 20 mg/L 14 mg/L
Committee meeting November 25, 2014
Sequential solvent extraction
Sequential liquid- liquid wash
Ion exchange column
990 Seminar December 1, 2014 Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
Fraction 96-hr LC50 (X ± 95% CI)
15 min IC50 (X ± 95% CI)
F3-NE2a 0.73 (0.97) 1.23 (0.34) F3-NE2b 2.18 (0.36) 5.05 (2.47)
Figure 1. Toxicity of fractions of OSPW to embryos of Fathead minnow.
• Dose response of F3-NE2a indicative of a narcosis MOA
• F3-NE2b MOA unknown
Table 1. Toxicity of fractions of OSPW to embryos of Fathead minnow and Vibrio fisheri.
Fraction concentration (x)
0 2 4 6 8 10
Mor
talit
y (%
)
0
20
40
60
80
100
F3-NE2aF3-NE2bF3-Pool
Toxicity results.
Heteroatom class
O+O2+
O3+O4+
O5+O6+
OS+O2S+
O3S+O4S+ON+
O2N+O3N+
O4N+ONS+
O2NS+
Inte
nsity
0.0
0.2
0.4
0.6
0.8
Heteroatom class
O-O2-
O3-O4-
O5-O6-
OS-O2S-
O3S-O4S- ON-
O2N-O3N-
O4N-ONS-
O2NS-0.0
0.2
0.4
0.6
0.8
F3-NE2aF3-NE2b
• Evidence suggests that NA (O2-) are among the most potent chemicals in OSPW
SETAC 2016 November 10th, 2016
• But Other heteroatom classes contribute as well • O+, O2
+, O3+, SO+, NO+
Chemical characterization of tertiary fractions.
Toxicology Centre
Date of presentation Title or place of presentation
2. Assessing bioaccumulation potential of chemical species in OSPW and development of a predictive
acute toxicity model
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Biomimetic approaches to assess accumulation.
TRANSIL® bead • Dmembrane/water
• Mimics cell membrane • Ionic compounds
DMW = 𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝐶𝐶𝑤𝑤𝑚𝑚𝑤𝑤𝑚𝑚𝑚𝑚
Dow = 𝐶𝐶𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙
𝐶𝐶𝑤𝑤𝑚𝑚𝑤𝑤𝑚𝑚𝑚𝑚 ~
PDMS stirbar • KPDMS
• Neutral surrogate lipid • Neutral compounds
KPDMS = 𝐶𝐶𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝐶𝐶𝑤𝑤𝑚𝑚𝑤𝑤𝑚𝑚𝑚𝑚
• Can assess accumulation potential of chemical species in the whole mixture by use of lipid mimicking materials.
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Results.
• Deviation from linear relationship of DOW and DMW • Suggests differing potentials to accumulate of chemical species by use of the two
methods
Figure taken from Zhang et al., 2016
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
• All chemicals contributing to toxicity can be detected- Orbitrap MS- ESI+/-
Development of a predictive toxicity model.
Water concentration
Toxicity endpoint
1. 2.
3.
• Mode of action – Narcosis • Target lipid model can be used to predict toxicity by
use of accumulation estimates (DOW/ DMW)
SETAC NA 2015 November 4th, 2015
• Toxicity of mixture follows concentration addition • Toxic units • Normalize aqueous concentration to
hazard (LC50)
CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Identify
Toxicology Centre
Date of presentation Title or place of presentation
Spreadsheet Model. Concentration: Toxicity:
TU Calculated: (Concentration / Tox.)
[M]i = (RIi ∗𝑴𝑴𝑴𝑴)Molecular mass i
Log (LC50)i = -0.945log (DOW/DMW)i + Log Cbb
Sum TU and predict toxicity
Identify
If TU ≥ 1 Expect LC50 or greater
SETAC NA 2015 November 4th, 2015 CEW 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
96-hr embryo-lethality assay Pimephales promelas
SETAC NA 2015 November 4th, 2015
Test of model.
CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
With observed
LC50
No observed
LC50
+ F1-Pool
+ F2-Pool
+ F3-Pool
No. samples
Acute toxicity (LC50)
8
Total 10
96-hr embryo-lethality assay Pimephales promelas
SETAC NA 2015 November 4th, 2015
Test of model.
CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Observed LC50 (mg/L)
1e+0 1e+1 1e+2 1e+3 1e+4 1e+5
Pred
icte
d LC
50 (m
g/L)
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
Model results.
November 4th, 2015 September 26th, 2016
Fold difference from Observed
LC50
Model (n = 8)
2 – fold 50% 4 – fold 75%
> 10 - fold 0%
Table 2. Percent of samples greater than X-fold different from observed.
• All LC50s predicted within 10-fold of observed.
Figure 2. Model predicted LC50 v. observed LC50, blue-line is a 5-fold difference from observed.
November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Chemical
Class
Percent TU (%) of dissolved organic fraction
of OSPW
C5-15 C16-20 C21-25 C26-30
SO+ 5.79 20.3 2.59 3.25
SO2- 0.85 7.75 0.15 <0.01
NO+ 8.33 7.40 1.24 <0.01
O2- 4.42 11.9 0.91 <0.01
O2+ 7.05 7.93 6.12 0.03
O+ 2.41 1.43 0.20 <0.01
O- <4.60E-4 <4.60E-4 <4.60E-4 <4.60E-4
Total TU 29% 57% 11.2% 2.8%
Table 3. Contribution of chemical class, carbon number ranges to toxicity of the F1-Pool sample.
CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Chemical
Class
Percent TU (%) of dissolved organic fraction
of OSPW
C5-15 C16-20 C21-25 C26-30
SO+ 5.79 20.3 2.59 3.25
SO2- 0.85 7.75 0.15 <0.01
NO+ 8.33 7.40 1.24 <0.01
O2- 4.42 11.9 0.91 <0.01
O2+ 7.05 7.93 6.12 0.03
O+ 2.41 1.43 0.20 <0.01
O- <4.60E-4 <4.60E-4 <4.60E-4 <4.60E-4
Total TU 29% 57% 11.2% 2.8%
Table 3. Contribution of chemical class, carbon number ranges to toxicity of the F1-Pool sample.
• O2+/- and SO+ chemical
classes contribute most of predicted toxicity (~70%)
• Carbon number range C5-
20 contribute > 85% of predicted toxicity
• C16-20 predominate (>57%)
CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
3. Reproductive toxicity of dissolved organic chemicals in OSPW
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
F1
F2
F3 20 mg/L 14 mg/L
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation Heteroatom class
O+O2+
O3+O4+
O5+O6+
OS+O2S+
O3S+O4S+ON+
O2N+O3N+
O4N+ONS+
O2NS+
Inte
nsity
0.0
0.2
0.4
0.6
0.8
Heteroatom class
O-O2-
O3-O4-
O5-O6-
OS-O2S-
O3S-O4S- ON-
O2N-O3N-
O4N-ONS-
O2NS-0.0
0.2
0.4
0.6
0.8
F3-NE2aF3-NE2b
SETAC 2016 November 10th, 2016
Fraction concentration (x)
0 2 4 6 8 10
Mor
talit
y (%
)
0
20
40
60
80
100
Fraction concentration (x)
0 2 4 6 8 10
Mor
talit
y (%
)
0
20
40
60
80
100
• Fractions have differing chemical profiles and MOA
F3-NE2a F3-NE2b
Toxicology Centre
Date of presentation Title or place of presentation
Fathead minnow 21-day reproductive bioassay
Treatments
Control
S. Control (<5E-3% EtOH)
25% OSPW
25% eqv. F3-NE2a
25% eqv. F3-NE2b
10- liter aquaria
1 Male 2 Female
Endpoints
Survival
Total fecundity
Fertilisation
Time to Hatch
Morphometric indices
Circulating plasma hormone conc.
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Results.
Time (Days)0 5 10 15 20C
umul
ativ
e no
. of e
ggs/
fem
ale/
day
0
5
10
15
20
25ControlS. ControlOSPWF3-NE2aF3-NE2b
Figure. Cumulative egg production.
Experimental set-up, 21-day reproductive assay
Embryos of fathead minnow
SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
*
Male
Sample
Control
S. ControlOSPW
F3-NE2a
F3-NE2bGon
ado-
som
atic
inde
x (%
)
0
1
2
3
4
5Male
Sample
Control
S. ControlOSPW
F3-NE2a
F3-NE2b
Con
ditio
n fa
ctor
0.0
0.5
1.0
1.5
2.0
2.5
3.0 Male
Sample
Control
S. ControlOSPW
F3-NE2a
F3-NE2bHep
ato-
som
atic
inde
x (%
)
0
1
2
3
4
5
Female
Sample
Control
S. ControlOSPW
F3-NE2a
F3-NE2b
Con
ditio
n fa
ctor
0.0
0.5
1.0
1.5
2.0
2.5
3.0 Female
Sample
Control
S. ControlOSPW
F3-NE2a
F3-NE2bHep
ato-
som
atic
inde
x (%
)
0
1
2
3
4
5
6 Female
Sample
Control
S. ControlOSPW
F3-NE2a
F3-NE2bGon
ado-
som
atic
inde
x (%
)
02468
10121416
Condition factor GSI HSI
Male
Female
*
SETAC 2016
Toxicology Centre
Date of presentation Title or place of presentation
2. Development of a acute aquatic toxicity model • Developed a model to predict acute toxicity of chemicals in OSPW to embryos of Fathead
minnow within 10-fold of observed toxicity • Consideration of accumulation into polar lipids helps improve toxicity predictions • Improved our understanding of heteroatom class contributions to toxicity
Overall conclusions.
SETAC 2016 November 10th, 2016
3. Assessing reproductive toxicity of dissolved organic chemicals in OSPW • Whole OSPW (25% v/v) and fractions of OSPW did not demonstrate effects on the
reproductive performance of Fathead minnow • Male Fathead minnow exposed to OSPW had significantly higher HSI
1. Identification of heteroatom classes contributing to acute toxicity • Naphthenic acids (O2
-) do contribute to toxicity but other chemical classes do as well (i.e. O+, O2
+, SO+, NO+)
Toxicology Centre
Date of presentation Title or place of presentation Funding:
• Steve Wiseman • John P. Giesy • Rishi Mankidy • Hattan Alharbi
• Jonathan Martin • Alberto Dos Santos Pereira • Kun Zhang • Chenxing Sun
• Warren Zubot
Funding: NSERC CRD with Syncrude SETAC NA 2015 November 4th, 2015 CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Happy to take any questions…
SETAC NA 2015 November 4th, 2015 CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016 [email protected]
Toxicology Centre
Date of presentation Title or place of presentation
Acute toxicity tests:
15- min IC50 Vibrio fisheri
96-hr embryolethality assay Pimephales promelas
Nanjing University June 13, 2014 Committee meeting November 25, 2014 990 Seminar December 1, 2014 Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Development of a predictive toxicity model. • Assess the acute aquatic toxicity of the dissolved organic fraction of
OSPW based on instrumental analysis (WQC)
General schematic:
Thermo Scientific™ Q Exactive™ Hybrid
Quadrupole-Orbitrap Mass Spectrometer
Sample
Toxicity assessment
Qualifying exam September 2nd, 2015
Extraction
Spreadsheet model
SETAC NA 2015 November 4th, 2015 CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
What we found…
Microtox results
F1-AE, F1-NE > F1-BE Microtox results
F2-NE2 > F2-NE1
Microtox results
F3-NE2a > F3-NE2b
F1 Results: F2 Results: F3 Results:
F2 Results: Fractionate F2-NE2
F3 Results: F3-NE2a > F3-NE2b
Both are toxic Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
F1 Results: Fractionate F1-NE
Toxicology Centre
Date of presentation Title or place of presentation
Next step… Round 2 fractionation
F1
F2
F3 20 mg/L 14 mg/L 10.8% 7.6%
Committee meeting
Sequential solvent extraction
Sequential liquid- liquid wash
Ion exchange column
990 Seminar Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
What we found…Round 2
Microtox results (1/IC50)
F1-AE, F1-NE > F1-BE Microtox results (1/IC50)
F2-NE2 > F2-NE1
Microtox results
F3-NE2a > F3-NE2b
F1 Results: F3 Results:
F3 Results: F3-NE2a > F3-NE2b
Both are toxic
F2 Results: Fractionate F2-NE2
Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
F1 Results: Fractionate F1-NE
Toxicology Centre
Date of presentation Title or place of presentation
One last time…
F1
F2
F3
Sequential solvent extraction
Sequential liquid- liquid wash
Ion exchange column
Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Microtox results (1/IC50)
F1-AE, F1-NE > F1-BE Microtox results (1/IC50)
F2-NE2 > F2-NE1
Microtox results (1/IC50)
F3-NE2a > F3-NE2b
F1 Results: F2 Results:
F3 Results: F3-NE2a > F3-NE2b
Both are toxic
F1 Results: Fractionate F1-NE
F2 Results: Fractionate F2-NE2
What we found…Round 3
Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Predicting toxicity of narcotic chemicals.
• Target Lipid Model (TLM)- developed to estimate the 96-hr LC50 of narcotic chemicals by use of KOW
Figure. Log(LC50) versus log(kow) for Pimephales promelas for chemicals acting by a narcosis mode of action (Di Toro et al., 2000).
TLM: Log (LC50)i = -0.945 · log (KOW)i + Log Cbb
SETAC NA 2015 November 4th, 2015
• With an understanding of chemical species accumulation potential (i.e. Kow) we can predict toxicity of narcotic chemicals
CEW 2016 September 26th, 2016 SETAC 2016 November 10th, 2016
Toxicology Centre
Date of presentation Title or place of presentation
Embryo viability.
Figure. Percent fertilization of collected embryos.
Sample
Control
S. ControlOSPW
F3-NE2aF3-NE2b
Perc
ent f
ertil
isatio
n (%
)
0
20
40
60
80
100
Sample
Control
S. ControlOSPW
F3-NE2aF3-NE2b
Tim
e to
Hat
ch (D
ays)
0
1
2
3
4
5
6
7Day 7Day 14
N=4, n=560-680 eggs/ female SETAC 2016 N=4, n=10 embryos/ replicate