Toxicology Centre
Predicting acute toxicity of oil sands
process affected waters
Garrett Morandi
Ph.D. student
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Objectives:
• Develop a method to assess the acute aquatic toxicity of the dissolved
organic fraction of OSPW based on instrumental analysis (WQC)
General approach:
Toxicity
assessment
Extraction
Sample
SETAC
NA 2015
Qualifying
exam
Title or place of presentation
Thermo Scientific™ Q
Exactive™ Hybrid
Quadrupole-Orbitrap
Mass Spectrometer
Spreadsheet model
Date of presentation
November
42thnd, ,2015
September
2015
Toxicology Centre
Some assumptions in model development:
• All chemicals contributing to toxicity can be
detected
Water
concentration
• Mode of action – Narcosis
• Toxicity of mixture follows concentration addition
• Toxic units
1.
2.
3.
Identify
SETAC NA 2015
Title or place of presentation
Toxicity
Date of presentation
November
4th, 2015
Toxicology Centre
Our approach:
1. Identify chemical species in sample using Orbitrap mass
spectrometry (i)
Orbitrap Mass
Spectrometer
2. Calculate aqueous concentrations
• Assume response factor of one for all chemical species
• Concentration ~ Relative intensity
[M]i =
(RIi ∗𝑴𝑴𝑴𝑴)
Molecular mass i
3. Assessment of chemical species toxicity
• Little information on toxicity and structure of chemicals
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
3. Predicting toxicity of chemical species
• Target Lipid Model (TLM) has been developed to estimate
the 96-hr LC50 of narcotic chemicals by use of KOW
TLM:
Log (LC50)i = -0.945 · log (KOW)i + Log Cbb
SETAC NA 2015
Title or place of presentation
Figure. Log(LC50) versus log(kow) for
Pimephales promelas for chemicals
acting by a narcosis mode of action (Di
Toro et al., 2000).
Date of presentation
November
4th, 2015
Toxicology Centre
Test of the Model:
Concentration:
Identify
(RIi ∗𝑴𝑴𝑴𝑴)
[M]i = Molecular
mass i
TU Calculated: (Concentration /
Toxicity:
Tox.)
Log (LC50)i = -0.945log (KOW)i
+ Log Cbb
TU ≥ 1
Expect LC50
or greater
Sum TU and
predict
toxicity
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Preliminary results presented last year:
8
7
Predicted
y = 2.8446x + 12.239
R² = 0.881
6
Probits
5
4
3
2
1
0
-3.6
-3.4
-3.2
-3
-2.8
-2.6
-2.4
-2.2
-2
Concentration of OSPW (Log M)
Figure. Comparison of model predicted LC50 to observed LC50 for
embryos of fathead minnow exposed to the F1-Pool sample of
OSPW for 96 hr.
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Since then…
+ F1-Pool
+ F2-Pool
+ F3-Pool
SETAC NA 2015
Title or place of presentation
Figure. Overall fractionation schematic of OSPW sample. Taken from
Morandi et al. (2015).
96-hr embryo-lethality assay
Pimephales promelas
Date of presentation
November
4th, 2015
Toxicology Centre
No. samples
With
observed
LC50
No
observed
LC50
SETAC NA 2015
8
Total
10
+ F1-Pool
+ F2-Pool
+ F3-Pool
Title or place of presentation
Acute toxicity
(LC50)
96-hr embryo-lethality assay
Pimephales promelas
Date of presentation
November
4th, 2015
Toxicology Centre
Results:
Predicted LC50 (mg/L)
1e+5
F1-AE
1e+4
F3-NE2a
F2-NE1
>
1e+3
1e+2
Model I
>
F1-BE
1e+1
1e+0
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
Observed LC50 (mg/L)
SETAC NA 2015
Title or place of presentation
Figure. Comparison of model predicted LC50 to observed LC50 for embryos of
fathead minnow exposed to samples of OSPW for 96 hr.
Date of presentation
November
4th, 2015
Toxicology Centre
F3-NE2a
F1-AE
100
Observed mortality (%)
Model I
80
60
40
F2-NE1
20
0
0.001
SETAC NA 2015
Title or place of presentation
0.01
0.1
1
10
Predicted TU
Figure. Comparison of model predicted TU to observed mortality for embryos of FHM
exposed to samples of OSPW.
Date of presentation
November
4th, 2015
Toxicology Centre
Model performance:
Fold difference from Observed LC50
Model I Results (n=8)
2 – fold
50%
4 – fold
75%
> 10 - fold
25%
Potential deficiencies of model:
• Most chemicals in OSPW are polar
• Does KOW best describe their accumulation potential?
• Thus, are TLM predictions using KOW appropriate?
Taken from Van Wezel et al., 1995
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Membrane affinity assay:
• Solid supported lipid membranes (SSLM) mimic interaction of polar
compounds with cell membrane
• Analogous to KOW partitioning between media and SSLM can be
derived (KMembrane affinity)
TRANSIL® bead
• KMA
• Mimics cell membrane
SETAC NA 2015
Title or place of presentation
KMA = 𝐶𝐶𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚
𝐶𝐶𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤
Date of presentation
November
4th, 2015
Toxicology Centre
log MA
4
3
2
O2+
SO+
NO+
NO2+
O2SO2-
1
0
-5
-4
-3
-2
-1
0
1
log KPDMS
2
3
4
Figure. Measured membrane affinity and Poly(dimethyl)siloxane partition
coefficients for dissolved chemical species in OSPW.
Title or place of presentation
Date of presentation
Toxicology Centre
Development of Model II:
• When a chemical species i has a measured KMA and KOW
• KMA will be used if > KOW
Therefore…
For
Chemical
species i
SETAC NA 2015
Title or place of presentation
KOWi
KMAi
if
KMAi > KOWi
TLM
Log (LC50)i = -0.945log (BP)i + Log Cbb
BPi = KOW or
KMA
Date of presentation
November
4th, 2015
Toxicology Centre
Test of the Model:
Concentration:
Identify
(RIi ∗𝑴𝑴𝑴𝑴)
[M]i = Molecular
mass i
TU Calculated: (Concentration /
Toxicity:
Tox.)
Log (LC50)i = -0.945log (BP)i
+ Log Cbb
TU ≥ 1
Expect LC50
or greater
BPi = KOW
or KMA
SETAC
NA 2015
Qualifying
exam
Title or place of presentation
Sum TU and
predict
toxicity
Date of presentation
November
4th, 2015
Toxicology Centre
Model results:
Predicted LC50 (mg/L)
1e+5
F1-AE
1e+4
>
1e+3
1e+2
F3-NE2a
F3-NE2a
SETAC NA 2015
Model I
Model II
>
1e+1
1e+0
1e+0
Title or place of presentation
F1-AE
1e+1
1e+2
1e+3
1e+4
1e+5
Observed LC50 (mg/L)
Figure. Comparison of model predicted LC50 to observed LC50 for embryos of
fathead minnow exposed to samples of OSPW for 96 hr.
Date of presentation
November
4th, 2015
Toxicology Centre
F3-NE2a
F1-AE
Observed mortality (%)
100
80
Model I
Model II
60
40
F2-NE1
20
0
0.001
0.01
0.1
1
10
Predicted TU
SETAC NA 2015
Title or place of presentation
Figure. Comparison of model predicted TU to observed mortality for embryos of FHM exposed
to samples of OSPW.
Date of presentation
th
November 4 , 2015
Toxicology Centre
Fold difference
from Observed
LC50
Model I
(n=8)
2 – fold
50%
50%
4 – fold
75%
75%
> 10 - fold
25%
0%
Model II
(n = 8)
Median Residual
Mean Residual
LOG LC50 residual (mg/L)
Model performance:
5
4
Model I
Model II
3
2
1
0
-1
5
LOG LC50 residual (mg/L)
MAD
RMSD
4
3
2
1
0
-1
Model I
Model II
Model
Model I
Model II
Model
Table. Calculated mean residual, median residual, mean absolute
deviation (MAD) and root mean square deviation (RMSD)
between predicted and observed LC50 for Model I and II.
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Summary:
• Developed a model to predict the acute lethality of dissolved organic
chemicals in OSPW to embryos of Fathead minnow (Within 10- fold of
observed LC50)
• Explicit consideration of accumulation potential into polar lipids
(phospholipids) improved toxicity predictions
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
•
•
•
•
Steve Wiseman
John P. Giesy
Rishi Mankidy
Hattan Alharbi
• Jonathan Martin
• Alberto Dos Santos Pereira
• Kun Zhang
• Warren Zubot
SETAC NA 2015
Title or place of presentation
Funding:
Funding: NSERC CRD with Syncrude
Date of presentation
November
4th, 2015
Toxicology Centre
Happy to take any questions…
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
What chemicals are most potent?
+ F1-Pool
96-hr embryo-lethality assay
Pimephales promelas
+ F2-Pool
+ F3-Pool
15- min IC50
Vibiro fisheri
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Chemical characterization:
*
1e+7
F3-NE2a
F3-NE2b
*
Intensity
8e+6
6e+6
4e+6
2e+6
*
*
*
*
*
O
O
2
O
3
O
4
O
5
O
6
O
OS
2
OS
3
OS
4S
O
ON
2
ON
3
ON
4
ON
O NS
2N
S
O
O
2
O
3
O
4
O
5
O
6
O
OS
2
OS
3
OS
4S
O
ON
2
ON
3
ON
4
ON
O NS
2N
S
0
Heteroatom class
Heteroatom class
Figure. Total abundances of species by heteroatom class based on the sum of the peak areas in the
chromatograms of tertiary fractions of BML-OSPW in A) ESI+ and B) ESI-.
• Highlighted contribution of O+/-, O2+/-, SO+, NO+ and SO2chemical classes in causing the acute lethality of OSPW.
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Their predicted contribution to toxicity:
O+
4%
SO29%
[CATEGORY
NAME]
[PERCENTAGE]
[CATEGORY
NAME]
[PERCENTAGE]
[CATEGORY
NAME] 0.01%
[CATEGORY
NAME]
[PERCENTAGE]
[CATEGORY
NAME]
[PERCENTAGE]
Figure. Percent contribution to total TU of the
F1-Pool sample of chemical classes.
SETAC NA 2015
Title or place of presentation
Figure. Percent contribution to total TU of the F1Pool sample of chemical classes plotted as a function
of relative intensity.
• These seven chemical classes account for >97% of TU
and < 43% of total mass spectral response
Date of presentation
November
4th, 2015
Toxicology Centre
Are there differences in potencies?
TU / Unit Relative Intensity
10
8
• Further highlights chemical classes
based on their potency
• SO+ are most potent and identified as
most biaoccumulative chemical class in
OSPW (Zhang et al., 2015).
6
4
2
0
O-
O2-
SO2-
O+
O2+
SO+
NO+
Chemical Class
SETAC NA 2015
Title or place of presentation
Figure. Relative potencies of chemical classes calculated as the
total TU of a chemical class over its percent relative response.
Date of presentation
November
4th, 2015
Toxicology Centre
Deficiencies of KOW:
• KOW best models accumulation of chemicals into neutral lipids
• What about accumulation into polar lipids?
• Does using an explicit assessment of accumulation into
polar lipids improve model predictions?
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Extending the Model:
1./2. Chemical characterization,
quantification and empirical
toxicity data
+
3. Bioaccumulation estimates
(KOW)
SETAC NA 2015
Title or place of presentation
…Predict sample toxicity
Date of presentation
November
4th, 2015
Toxicology Centre
Some background:
Theory of concentration addition and the
Toxic Unit approach (TU)
Qualifying exam
Title or place of presentation
Date of presentation
September
2nd, 2015
Toxicology Centre
An example: Individual assessment of
chemicals A + B
Table 1. Measured concentration and toxicity of individual chemicals A and B:
Qualifying exam
Title or place of presentation
Chemical
Measured
concentration
(ppm)
WQC
threshold
value
(LC50)
Concern?
A
5
10
B
15
20
No concern
using WQC
for individual
chemicals
September
2nd, 2015
Date of presentation
Toxicology Centre
What if we look at A + B together?
• Assume concentration addition of A + B
• Normalize water concentration by inherent toxicity (LC50)
Table 2. Predicted toxicity of mixture of chemicals A and B:
Chemical
Measured
concentration
(ci , ppm)
WQC
threshold
value
(ECxi , LC50)
Calculated
TUi
A
5
10
0.5
B
15
20
0.75
Total
TU
(mixture)
+ = 1.25
Concern
YES- Expect
greater than
50% lethality
When…
TU ≥ 1
Expect your
endpoint
• Considering additive toxicity, we expect to see an LC50
Qualifying exam
Title or place of presentation
Date of presentation
September
2nd, 2015
Toxicology Centre
Information for a model:
1. Identify
2. Water
concentration
1.
2.
3. Toxicity
3.
Table 2. Predicted toxicity of mixture of chemicals A and B:
Chemical
Measured
concentration
(ppm)
WQC
threshold
value
(LC50)
Calculated
TU
(ind.)
A
5
10
0.5
B
15
20
0.75
Total
TU
(mixture)
+ = 1.25
Effect
Expect
greater than
50% lethality
• Looking at mixture effect we expect to see a LC50
Qualifying exam
Title or place of presentation
Date of presentation
September
2nd, 2015
Toxicology Centre
Effect of KOW vs. KMA on toxicity predictions?
• To investigate the effect of partition coefficient on toxicity predictions, a matrix approach was
taken to develop models, therefore 3 models were tested
• Goodness-of-fit statistics were compared to assess model performance
Table 3. Bioaccumulation estimates used for Model development.
Model
PDMS (KOW)
TRANSIL® (KMA)
I
XXX
---
II
---
XXX
III
XXX
XXX*
*TRANSIL membrane affinity data used in preference of PDMS
XXX indicates partition coefficient used for toxicity predictions
Qualifying exam
Title or place of presentation
Date of presentation
September
2nd, 2015
Toxicology Centre
Compare model results:
1e+5
Predicted LC50 (mg/L)
1e+4
1e+3
1e+2
Model I
Model II
Model III
• Blue = 2-fold error
• Predicted toxicity of
fractions
• n = 10 per model
1e+1
1e+0
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
Observed LC50 (mg/L)
Figure. Model predicted and observed toxicity (LC50) for embryos of Fathead minnow exposed to
fractions of OSPW. Black solid line is line of agreement and blue lines represent 2-fold error.
Qualifying exam
Title or place of presentation
Date of presentation
September
2nd, 2015
Toxicology Centre
LOG LC50 residual (mg/L)
Model Performance
Median Residual
Mean Residual
2
Model I
Model II
Model III
1
0
-1
LOG LC50 residual (mg/L)
MAD
RMSD
2
1
0
-1
Model I
Model II
Model
Qualifying exam
Title or place of presentation
Model III
Model I
Model II
Model III
Model
Figure. Calculated mean residual, median residual, mean absolute deviation (MAD) and root
mean square error (RMSD) of Model I, II and III between predicted and observed LC50.
Date of presentation
September
2nd, 2015
Toxicology Centre
Complex mixture of organic chemicals
• Suggested to contain in excess of 200,000 chemicals (Hao et al., 2005)
• Organic fraction contains many classes of compounds (S, Ox, N, etc.) as well as PAHs,
DBTPs etc.
• Structures predominantly unknown
4.00E+07
B
A
B
Peak area
3.00E+07
2.00E+07
OSPW
1.00E+07
0.00E+00
Title
or place
presentation
Qualifying
exam
SETAC
NAof2015
Figure 1. Heteroatom class distribution of whole OSPW in positive mode (A) and the carbon
number, double bond equivalency distribution of O2+ compounds (B) identified by use of
Orbitrap ultra-high resolution mass spectrometry.
nd
Date of presentation
December
2014
September
, 2015
November
4th1,,22015
Toxicology Centre
What causes acute toxicity of OSPW?
• Mixture is complex:
• Metals (V, Fe, Pb etc.)
• High salinity
• Complex organic fraction
• Majority of acute toxicity is due to organic fraction
Nanjing
University
Committee
SETAC
NA
2015
meeting
Qualifying
exam
990 Seminar
Title or place of presentation
nd
June
13,
2014
Date of
presentation
November
, ,2015
September
2015
December
1,42th2014
Toxicology Centre
Chemicals responsible for toxicity:
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015
Toxicology Centre
Development of Model II:
• Explicitly consider the accumulation of chemical species to both
neutral (PDMS) and polar (TRANSIL) lipids
• Assume toxicity is related to the concentration in phospholipids and
storage lipids
Polar lipid
Chemical species i
KMAi
Neutral lipid
KOWi
Toxicity assessment
SETAC NA 2015
Title or place of presentation
Date of presentation
November
4th, 2015