Download - Characterizing the toxicity of oil sands process affected ...s New... · 1. What heteroatom classes contribute to the acute toxicity of the dissolved organic fraction of OSPW? 2

$: Characterizing the toxicity of oil sands process affected ...s New... · 1. What heteroatom classes contribute to the acute toxicity of the dissolved organic fraction of OSPW? 2$
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


• 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)


Project overview. Toxicity Identification and Evaluation study

BML, established December 2012

Toxicology Centre


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)


Toxicology Centre


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?


Toxicology Centre


1. Identifying causative chemicals of toxicity.


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


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


• But Other heteroatom classes contribute as well • O+, O2

+, O3+, SO+, NO+

Chemical characterization of tertiary fractions.

Toxicology Centre


2. Assessing bioaccumulation potential of chemical species in OSPW and development of a predictive

acute toxicity model


Toxicology Centre


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.


Toxicology Centre


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


Toxicology Centre


• 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


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


96-hr embryo-lethality assay Pimephales promelas


Test of model.


Toxicology Centre


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


Test of model.


Toxicology Centre


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


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.


Toxicology Centre


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%)


Toxicology Centre


3. Reproductive toxicity of dissolved organic chemicals in OSPW


Toxicology Centre


F1

F2

F3 20 mg/L 14 mg/L


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



0 2 4 6 8 10

Mor

talit

y (%

)

0

20

40

60

80

100


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


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.


Toxicology Centre


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


Toxicology Centre


*

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


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.


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


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


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


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


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


Next step… Round 2 fractionation

F1

F2

F3 20 mg/L 14 mg/L 10.8% 7.6%

Committee meeting



Ion exchange column

990 Seminar Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016

Toxicology Centre


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:


Both are toxic


Qualifying exam September 2nd, 2015 SETAC 2016 November 10th, 2016


Toxicology Centre


One last time…

F1

F2

F3



Ion exchange column


Toxicology Centre



F1-AE, F1-NE > F1-BE Microtox results (1/IC50)

F2-NE2 > F2-NE1


F3-NE2a > F3-NE2b

F1 Results: F2 Results:


Both are toxic



What we found…Round 3


Toxicology Centre


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


• With an understanding of chemical species accumulation potential (i.e. Kow) we can predict toxicity of narcotic chemicals


Toxicology Centre


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

Download - Characterizing the toxicity of oil sands process affected ...s New... · 1. What heteroatom classes contribute to the acute toxicity of the dissolved organic fraction of OSPW? 2

Top Related